file which includes the original illustrations4 ,see #bggac-;h4htm or #bggac-;h4zip3 7https3_/_/www4gutenberg4org_/dirs_/#b_/ g_/g_/a_/bggac_/bggac-;h_/#bggac-;h4htm7 or 7https3_/_/www4gutenberg4org_/dirs_/#b_/ g_/g_/a_/bggac_/bggac-;h4zip7 ,transcriber's note3 ,text enclosed by tilde marks was in bold face in the original 7bold74 ,text enclosed by underscore marks is in italics 7italics74 ,the italic designation for single italized letters 7such as variables in equations7 and foreign abbreviations has been omitted for ease of reading4 ,in numbers1 equations1 and chemical formulas1 an underscore indicates that the following term enclosed within curly brackets is a subscript4 ,examples3 ,,co#b1 ,h#b,,so#d4 ,a carat character indicates that the following term enclosed within curly brackets is a superscript4 ,for example1 #aa4ac is #aa4a to the third power4 ,minor typographical errors have been corrected4 ,,the ,,elements ,,of ,,bacteriological ,,technique ,a ,laboratory ,guide for ,medical1 ,dental1 and ,technical ,students by ,j4 ,w4 ,h4 ,,eyre1 ,m4,d41 ,m4,s41 ,f4,r4,s4 7,,edin47 ,director of the ,bacteriological ,department of ,guy's ,hospital1 ,london1 and ,lecturer on ,bacteriology in the ,medical and ,dental ,schools2 formerly ,lecturer on ,bacteriology at ,charing ,cross ,hospital ,medical ,school1 and ,bacteriologist to ,charing ,cross ,hospital2 sometime ,hunterian ,professor1 ,royal ,college of ,surgeons1 ,england ,second ,edition ,rewritten and ,enlarged ,philadelphia and ,london ,w4 ,b4 ,saunders ,company #aiac ,copyright1 #aijb1 by ,w4 ,b4 ,saunders and ,company ,revised1 entirely reset1 reprinted1 and recopyrighted ,july1 #aiac ,copyright1 #aiac1 by ,w4 ,b4 ,saunders ,company ,registered at ,stationers' ,hall1 ,london1 ,england ,printed in ,america ,press of ,w4 ,b4 ,saunders ,company ,philadelphia ,,to ,,the ,,memory ,,of ,,john ,,wichenford ,,washbourn1 ,c4,m4,g41 ,m4,d41 ,f4,r4,c4,p4 ,physician to ,guy's ,hospital and ,lecturer on ,bacteriology in the ,medical ,school1 and ,physician to the ,london ,fever ,hospital ,,my ,,teacher1 ,,friend1 ,,and ,,co-,,worker ,,preface ,,to ,,the ,,second ,,edition ,bacteriology is essentially a practical study1 and even the elements of its technique can only be taught by personal instruction in the laboratory4 ,this is a self-evident proposition that needs no emphasis1 yet ,i venture to believe that the former collection of tried and proved methods has already been of some utility1 not only to the student in the absence of his teacher1 but also to isolated workers in laboratories far removed from centres of instruction1 reminding them of forgotten details in methods already acquired4 ,if this assumption is based on fact no further apology is needed for the present revised edition in which the changes are chiefly in the nature of additions--rendered necessary by the introduction of new methods during recent years4 ,i take this opportunity of expressing my deep sense of obligation to my confrere in the ,physiological ,department of our medical school--,mr4 ,j4 ,h4 ,ryffel1 ,b4 ,c41 ,b4 ,sc4--who has revised those pages dealing with the analysis of the metabolic products of bacterial life2 to successive colleagues in the ,bacteriological ,department of ,guy's ,hospital1 for their ready co-operation in working out or in testing new methods2 and finally to my ,chief ,laboratory ,assistant1 ,mr4 ,j4 ,c4 ,turner whose assistance and experience have been of the utmost value to me in the preparation of this volume4 ,i have also to thank ,mrs4 ,constant ,ponder for many of the new line drawings and for redrawing a number of the original cuts4 ,,john ,w4 ,h4 ,,eyre4 ,,guy',s ,,hospital1 ,s4 ,e4 ,july1 #aiac4 ,,preface ,,to ,,the ,,first ,,edition ,in the following pages ,i have endeavoured to arrange briefly and concisely the various methods at present in use for the study of bacteria1 and the elucidation of such points in their life-histories as are debatable or still undetermined4 ,of these methods1 some are new1 others are not2 but all are reliable1 only such having been included as are capable of giving satisfactory results even in the hands of beginners4 ,in fact1 the bulk of the matter is simply an elaboration of the typewritten notes distributed to some of my laboratory classes in practical and applied bacteriology2 consequently an attempt has been made to present the elements of bacteriological technique in their logical sequence4 ,i make no apology for the space devoted to illustrations1 nearly all of which have been prepared especially for this volume2 for a picture1 if good1 possesses a higher educational value and conveys a more accurate impression than a page of print2 and even sketches of apparatus serve a distinct purpose in suggesting to the student those alterations and modifications which may be rendered necessary or advisable by the character of his laboratory equipment4 ,the excellent and appropriate terminology introduced by ,chester in his recent work on ,determinative ,bacteriology ,i have adopted in its entirety1 for ,i consider it only needs to be used to convince one of its extreme utility1 whilst its inclusion in an elementary manual is calculated to induce in the student habits of accurate observation and concise description4 ,with the exception of ,section ,,xvii--,outlines for the ,study of ,pathogenic ,bacteria--introduced with the idea of completing the volume from the point of view of the medical and dental student1 the work has been arranged to allow of its use as a laboratory guide by the technical student generally1 whether of brewing1 dairying1 or agriculture4 ,so alive am ,i to its many inperfections that it appears almost superfluous to state that the book is in no sense intended as a rival to the many and excellent manuals of bacteriology at present in use1 but aims only at supplementing the usually scanty details of technique1 and at instructing the student how to fit up and adapt apparatus for his daily work1 and how to carry out thoroughly and systematically the various bacterioscopical analyses that are daily demanded of the bacteriologist by the hygienist4 ,finally1 it is with much pleasure that ,i acknowledge the valuable assistance received from my late assistant1 ,mr4 ,j4 ,b4 ,gall1 ,a4 ,i4 ,c41 in the preparation of the section dealing with the chemical products of bacterial life1 and which has been based upon the work of ,lehmann4 ,,john ,w4 ,h4 ,,eyre4 ,,guy',s ,,hospital1 ,s4 ,e4 ,,contents ,,page ,i4 ,,laboratory ,,regulations #a ,,ii4 ,,glass ,,apparatus ,,in ,,common ,,use #c ,the ,selection1 ,preparation1 and ,care of ,glassware1 #h--;,cleaning of ,glass ,apparatus1 #ah--,plugging ,test-tubes and ,flasks1 #bd4 ,,iii4 ,,methods ,,of ,,sterilisation #bf ,sterilising ,agents1 #bf--,methods of ,application1 #bg--;,electric ,signal ,timing ,clock1 #ch4 ,,iv4 ,,the ,,microscope #di ,essentials1 #di--;,accessories1 #eg--,methods of ,micrometry1 #fa4 ,v4 ,,microscopical ,,examination ,,of ,,bacteria ,,and ,,other ,,micro-,,fungi #fi ,apparatus and ,reagents used in ,ordinary ,microscopical ,examination1 #fi--,methods of ,examination1 #gd4 ,,vi4 ,,staining ,,methods #ij ,bacteria ,stains1 #ij--;,contrast ,stains1 #ic--,tissue ,stains1 #ie--;,blood ,stains1 #ig--,methods of ,demonstrating ,structure of ,bacteria1 #ii--;,differential ,methods of ,staining1 #ajh4 ,,vii4 ,,methods ,,of ,,demonstrating ,,bacteria ,,in ,,tissues #aad ,freezing ,method1 #aae--,paraffin ,method1 #aag--,special ,staining ,methods for ,sections1 #aba4 ,,viii4 ,,classification ,,of ,,fungi #abf ,morphology of the ,hyphomycetes1 #abf--,morphology of the ,blastomycetes1 #abi4 ,,ix4 ,,schizomycetes #aca ,anatomy1 #acd--,physiology1 #acf--;,biochemistry1 #add4 ,x4 ,,nutrient ,,media #adf ,meat ,extract1 #adh--,standardisation of ,media1 #aed--,the ,filtration of ,media1 #aef--,storing ,media in ,bulk1 #aei--,tubing ,nutrient ,media1 #afj4 ,,xi4 ,,ordinary ,,or ,,stock ,,culture ,,media #afc ,,xii4 ,,special ,,media #ahb ,,xiii4 ,,incubators #baf ,,xiv4 ,,methods ,,of ,,cultivation #bba ,aerobic1 #bbb--;,anaerobic1 #bcf4 ,,xv4 ,,methods ,,of ,,isolation #bdh ,,xvi4 ,,methods ,,of ,,identification ,,and ,,study #bei ,scheme of ,study1 #bei--,macroscopical ,examination of ,cultivations1 #bfa--,microscopical ,methods1 #bgb--;,biochemical ,methods1 #bgf--,physical ,methods1 #bie--;,inoculation ,methods1 #cae--;,immunisation1 #cba--;,active ,immunisation1 #cbb--,the ,preparation of ,haemolytic ,serum1 #cbg--,the ,titration of ,haemolytic ,serum1 #cbh--,storage of ,haemolysin1 #cca4 ,,xvii4 ,,experimental ,,inoculation ,,of ,,animals #ccb ,selection and ,care of ,animals1 #cce--,methods of ,inoculation1 #ceb4 ,,xviii4 ,,the ,,study ,,of ,,experimental ,,infections ,,during ,,life #cgj ,general ,observations1 #cga--;,blood ,examinations1 #cgc--,serological ,investigations1 #cgh--;,agglutinin1 #cha--,opsonin1 #chg--;,immune ,body1 #cic4 ,,xix4 ,,post-,,mortem ,,examination ,,of ,,experimental ,,animals #cif ,,xx4 ,,the ,,study ,,of ,,the ,,pathogenic ,,bacteria #djh ,,xxi4 ,,bacteriological ,,analyses #dae ,bacteriological ,examination of ,water1 #daf--;,examination of ,milk1 #dda--;,ice ,cream1 #deg--;,examination of ,cream and ,butter1 #deg--;,examination of ,unsound ,meats1 #dfj--;,examination of ,oysters and ,other ,shellfish1 #dfc--;,examination of ,sewage and ,sewage ,effluents1 #dff--;,examination of ,air1 #dfh--;,examination of ,soil1 #dgj--,testing ,filters1 #dgh--,testing of ,disinfectants1 #dhj4 ,,appendix #dib ,,index #eje ,illustration ,,bacteriological ,,technique4 ,i4 ,,laboratory ,,regulations4 ,the following regulations are laid down for observance in the ,bacteriological ,laboratories under the direction of the author4 ,similar regulations should be enforced in all laboratories where pathogenic bacteria are studied4 ,guy's ,hospital4 ,,bacteriological ,,department4 ,,handling ,,of ,,infective ,,materials4 ,the following ,regulations have been drawn up in the interest of those working in the ,laboratory as well as the public at large1 and will be strictly enforced4 ,their object is to avoid the dangers of infection which may arise from neglect of necessary precautions or from carelessness4 ,everyone must note that by neglecting the general rules laid down he not only runs grave risk himself1 but is a danger to others4 ,,regulations4 #a4 ,each worker must wear a gown or overall1 provided at his own expense1 which must be kept in the ,laboratory4 #b4 ,the hands must be disinfected with lysol #b per cent4 solution1 carbolic acid #e per cent4 solution1 or corrosive sublimate #a per mille solution1 after dealing with infectious material1 and before using towels4 #c4 ,on no account must ,laboratory towels or dusters be used for wiping up infectious material1 and if such towels or dusters do become soiled1 they must be immediately sterilised by boiling4 #d4 ,special pails containing disinfectant are provided to receive any waste material1 and nothing must be thrown on the floor4 #e4 ,all instruments must be flamed1 boiled1 or otherwise disinfected immediately after use4 #f4 ,labels must be moistened with water1 and not by the mouth4 #g4 ,all disused cover-glasses1 slides1 and pipettes after use in handling infectious material1 etc41 must be placed in #b per cent4 lysol solution4 ,a vessel is supplied on each bench for this purpose4 #h4 ,all plate and tube cultures of pathogenic organisms when done with1 must be placed for immediate disinfection in the boxes provided for the purpose4 #i4 ,no fluids are to be discharged into sinks or drains unless previously disinfected4 #aj4 ,animals are to be dissected only after being nailed out on the wooden boards1 and their skin thoroughly washed with disinfectant solution4 #aa4 ,immediately after the post-mortem examination is completed each cadaver must be placed in the zinc animal-box--without removing the carcase from the post-mortem board--and the cover of the box replaced1 ready for carriage to the destructor4 #ab4 ,dead animals1 when done with1 are cremated in the destructor1 and the laboratory attendant must be notified when the bodies are ready for cremation4 #ac4 ,none of the workers in the laboratory are allowed to enter the animal houses unless accompanied by the special attendant in charge1 who must scrupulously observe the same directions regarding personal disinfection as the workers in the laboratories4 #ad4 ,no cultures are to be taken out of the laboratory without the permission of the head of the ,department4 #ae4 ,all accidents1 such as spilling infected material1 cutting or pricking the fingers1 must be at once reported to the bacteriologist in charge4 ,,ii4 ,,glass ,,apparatus ,,in ,,common ,,use4 ,the equipment of the bacteriological laboratory1 so far as the glass apparatus is concerned1 differs but little from that of a chemical laboratory1 and the cleanliness of the apparatus is equally important4 ,the glassware comprised in the following list1 in addition to being clean1 must be stored in a sterile or germ-free condition4 ,test-tubes4--,it is convenient to keep several sizes of test-tubes in stock1 to meet special requirements1 viz43 #a4 #ah x #a4e cm41 to contain media for ordinary tube cultivations4 #b4 #ah x #a4c cm41 to contain media used for pouring plate cultivations1 and also for holding sterile swabs4 #c4 #ah x #b cm41 to contain wedges of potato1 beetroot1 or other vegetable media4 #d4 #ac x #a4e cm41 to contain inspissated blood-serum4 ,the tubes should be made from the best ,german potash glass1 blue-lined1 stout and heavy1 with the edge of the mouth of the tube slightly turned over1 but not to such an extent as to form a definite rim4 7,cost about #a4ej1 or #f shillings per gross47 ,such tubes are expensive it is true1 but they are sufficiently stout to resist rough handling1 do not usually break if accidentally allowed to drop 7a point of some moment when dealing with cultures of pathogenic bacteria71 can be cleaned1 sterilised1 and used over and over again1 and by their length of life fully justify their initial expense4 ,a point be noted is that the manufacturers rarely turn out such tubes as these absolutely uniform in calibre1 and a batch of #ah by #a4e cm4 tubes usually contains such extreme sizes as #ah by #b cm4 and #ah by #a4c cm4 ,consequently1 if a set of standard tubes is kept for comparison or callipers are used each new supply of so-called #ah by #a4e cm4 tubes may be easily sorted out into these three sizes1 and so simplify ordering4 #e4 #e x #j4g cm41 for use in the inverted position inside the tubes containing carbohydrate media1 as gas-collecting tubes4 ,these tubes1 unrimmed1 may be of common thin glass as less than two per cent4 are fit for use a second time4 ,illustration3 ,,fig4 #a4--;,bohemian flask4 ,illustration3 ,,fig4 #b4--,pear-shaped flask4 ,illustration3 ,,fig4 #c4--;,erlenmeyer flask 7narrow neck74 ,bohemian ,flasks 7,fig4 #a74--,these are the ordinary flasks of the chemical laboratory4 ,a good variety1 ranging in capacity from #bej to #cjjj c4c41 should be kept on hand4 ,a modified form1 known as the pear-shaped 7,fig4 #b71 is preferable for the smaller sizes--i4 e41 #bej and #ejj c4c4 ,erlenmeyer's ,flasks 7,fig4 #c74--;,erlenmeyer's flasks of #ge1 #ajj1 and #bej c4c4 capacity are extremely useful4 ,for use as culture flasks care should be taken to select only such as have a narrow neck of about #b cm4 in length4 ,kolle's ,culture ,flasks 7,fig4 #d74--,these thin1 flat flasks 7to contain agar or gelatine1 which is allowed to solidify in a layer on one side7 are extremely useful on account of the large nutrient surface available for growth4 ,a surface cultivation in one of these will yield as much growth as ten or twelve oblique tube cultures4 ,the wide mouth1 however1 is a disadvantage1 and for many purposes thin1 flat culture bottles known as ,roux's bottles 7,fig4 #e7 are to be preferred4 ,illustration3 ,,fig4 #d4--,kolle's culture flask4 ,illustration3 ,,fig4 #e4--,roux's culture bottle4 ,illustration3 ,,fig4 #f4--;,guy's culture bottle4 ,illustration3 ,,fig4 #g4--;,filter flask4 ,an even more convenient pattern is that used in the author's laboratory 7,fig4 #f71 as owing to the greater depth of medium which it is possible to obtain in these flasks an exceedingly luxuriant growth is possible2 the narrow neck reduces the chance of accidental contamination to a minimum and the general shape permits the flasks to be stacked one upon the other4 ,filter ,flasks or ,kitasato's ,serum ,flasks 7,fig4 #g74--,various sizes1 from #bej to #bjjj c4c4 capacity4 ,these must be of stout glass1 to resist the pressure to which they are subjected1 but at the same time must be thoroughly well annealed1 in order to withstand the temperature necessary for sterilisation4 ,all flasks should be either of ,jena glass or the almost equally well-known ,resistance or ,r glass1 the extra initial expense being justified by the comparative immunity of the glass from breakage4 ,petri's ,dishes or ,plates 7,fig4 #h1 a74--,these have now completely replaced the rectangular sheets of glass introduced by ,koch for the plate method of cultivation4 ,each plate consists of a pair of circular discs of glass with sharply upturned edges1 thus forming shallow dishes1 one of slightly greater diameter than the other1 and so1 when inverted1 forming a cover or cap for the smaller4 ,plates having an outside diameter of #aj cm4 and a height of #a4e cm4 are the most generally useful4 ,a batch of eighteen such plates is sterilised and stored in a cylindrical copper box 7#cj cm4 high by #ab cm4 diameter7 provided with a pull-off lid4 ,inside each box is a copper stirrup with a circular bottom1 upon which the plates rest1 and by means of which each can be raised in turn to the mouth of the box 7,fig4 #i7 for removal4 ,capsules 7,fig4 #h1 b and c74--,these are ,petri's dishes of smaller diameter but greater depth than those termed plates4 ,two sizes will be found especially useful--viz41 #d cm4 diameter by #b cm4 high1 capacity about #ad c4c42 and #e cm4 diameter by #b cm4 high1 capacity about #be c4c4 ,these are stored in copper cylinders of similar construction to those used for plates1 but measuring #bj by #f cm4 and #bj by #g cm41 respectively4 ,illustration3 ,,fig4 #h4--,petri dish 7a71 and capsules 7b1 c74 ,illustration3 ,,fig4 #i4--,plate box with stirrup4 ,graduated ,pipettes4--,several varieties of these are required1 viz43 #a4 ,pipettes of #a c4c4 capacity graduated in #j4a c4c4 #b4 ,pipettes of #a c4c4 capacity graduated in #j4ja c4c4 7,fig4 #aj1 a74 #c4 ,pipettes of #aj c4c4 capacity graduated in #j4a c4c4 7,fig4 #aj1 b74 ,these should be about #cj cm4 in length 7#a and #b of fairly narrow bore71 graduated to the extreme point1 and having at least a #aj cm4 length of clear space between the first graduation and the upper end2 the open mouth should be plugged with cotton-wool4 ,each variety should be sterilised and stored in a separate cylindrical copper case some #cf by #f cm41 with pull-off lid1 upon which is stamped1 in plain figures1 the capacity of the contained pipettes4 ,illustration3 ,,fig4 #aj4--,measuring pipettes1 a and b4 ,the laboratory should also be provided with a complete set of ,standard graduated pipettes1 each pipette in the set being stamped and authenticated by a certificate from one of the recognised ,physical ,measurement ,laboratories1 such as ,charlottenburg4 ,these instruments are expensive and should be reserved solely for standardising the pipettes in ordinary use1 and for calibrating small pipettes manufactured in the laboratory4 ,such a set should comprise1 at least1 pipettes delivering #aj c4c41 #e c4c41 #b4e c4c41 #b c4c41 #a c4c41 #j4e c4c41 #j4be c4c41 #j4b c4c41 #j4a c4c41 #j4je c4c41 and #j4ja c4c41 respectively4 ,in the immediately following sections are described small pieces of glass apparatus which should be prepared in the laboratory from glass tubing of various sizes4 ,in their preparation three articles are essential2 first a three-square hard-steel file or preferably a glass-worker's knife of hard ,thuringian steel for cutting glass tubes etc42 next a blowpipe flame1 for although much can be done with the ordinary ,bunsen burner1 a blowpipe flame makes for rapid work2 and lastly a bat's-wing burner4 ,illustration3 ,,fig4 #aa4--;,glass-cutting knife4 a4 handle4 b4 double edged blade4 c4 shaft4 d4 locking nut4 e4 spanner for nut4 #a4 ,the glass-cutting knife4 ,this article is sold in two forms1 a bench knife 7,fig4 #aa7 and a pocket knife4 ,the former is provided with a blade some #h cm4 in length and having two cutting edges4 ,the cutting edge when examined in a strong light is seen to be composed of small closely set teeth1 similar to those in a saw4 ,the knife should be kept sharp by frequent stroppings on a sandstone hone4 ,the pocket form1 about #f-;cm4 long over all1 consists of a small spring blade with one cutting edge mounted in scales like an ordinary pocket knife4 #b4 ,for real convenience of work the blowpipe should be mounted on a special table connected up with cylindrical bellows operated by a pedal4 ,that figured 7,fig4 #ab7 is made by mounting a teak top #fj cm4 square upon the uprights of an enclosed double-action concertina bellows 7,enfer's7 and provided with a ,fletcher's ,universal gas blowpipe4 #c4 ,an ordinary bat's-wing gas-burner mounted at the far corner of the table top is invaluable in the preparation of tubular apparatus with sharp curves1 and for coating newly-made glass apparatus with a layer of soot to prevent too rapid cooling1 and its usually associated result--cracking4 ,illustration3 ,,fig4 #ab4--;,glass blower's table with ,enfer's foot bellows4 #f4 ,sedimentation tubes #ex#j4e cm41 for sedimentation reactions1 etc41 and for containing small quantities of fluid to be centrifugalised in the haematocrit4 ,these are made by taking #ad-;cm4 lengths of stout glass tubing of the requisite diameter and heating the centre in the ,bunsen or blowpipe flame4 ,when the central portion is quite soft draw the ends quickly apart and then round off the pointed ends of the two test-tubes thus formed4 ,with the glass-cutting knife cut off whatever may be necessary from the open ends to make the tubes the required length4 ,a rectangular block of plasticine 7modelling clay7 into which the conical ends can be thrust makes a very convenient stand for these small tubes4 ,capillary ,pipettes or ,pasteur's ,pipettes 7,fig4 #ac a74--,these little instruments are invaluable1 and a goodly supply should be kept on hand4 ,they are prepared from soft-glass tubing of various-sized calibre 7the most generally useful size being #h mm4 diameter7 in the following manner3 ,hold a #aj cm4 length of glass tube by each end1 and whilst rotating it heat the central portion in the ,bunsen flame or the blowpipe blast-flame until the glass is red hot and soft4 ,now remove it from the flame and steadily pull the ends apart1 so drawing the heated portion out into a roomy capillary tube2 break the capillary portion at its centre1 seal the broken ends in the flame1 and round off the edges of the open end of each pipette4 ,a loose plug of cotton-wool in the open mouth completes the capillary pipette4 ,after a number have been prepared1 they are sterilised and stored in batches1 either in metal cases similar to those used for the graduated pipettes or in large-sized test-tubes--sealed ends downward and plugged ends toward the mouth of the case4 ,illustration3 ,,fig4 #ac4--;,capillary pipettes4 a1 b1 c4 ,the filling and emptying of the capillary pipette is most satisfactorily accomplished by slipping a small rubber teat 7similar to that on a baby's feeding bottle but not perforated7 on the upper end1 after cutting or snapping off the sealed point of the capillary portion4 ,if pressure is now exerted upon the elastic bulb by a finger and thumb whilst the capillary end is below the surface of the fluid to be taken up1 some of the contained air will be driven out1 and subsequent relaxation of that pressure 7resulting in the formation of a partial vacuum7 will cause the fluid to ascend the capillary tube4 ,subsequent compression of the bulb will naturally result in the complete expulsion of the fluid from the pipette 7,fig4 #ad74 ,illustration3 ,,fig4 #ad4--;,filling the capillary teat-pipette4 ,a modification of this pipette1 in which a constriction or short length of capillary tube is introduced just below the plugged mouth 7,fig4 #ac1 b71 will also be found extremely useful in the collection and storage of morbid exudations4 ,a third form1 where the capillary portion is about #d or #e cm4 long and only forms a small fraction of the entire length of the pipette 7,fig4 #ac1 c71 will also be found useful4 ,blood ,pipettes 7,fig #ae74--,special pipettes for the collection of fairly large quantities of blood 7as suggested by ,pakes7 should also be prepared4 ,these are made from soft glass tubing of #a cm4 bore1 in a similar manner to the ,pasteur pipettes1 except that the point of the blowpipe flame must be used in order to obtain the sharp shoulder at either end of the central bulb4 ,the terminal tubes must retain a diameter of at least #a mm41 in order to avoid capillary action during the collection of the fluid4 ,illustration3 ,,fig4 #ae4--;,blood pipettes and hair-lip pin in a test-tube4 ,illustration3 ,,fig4 #af4--;,blood-pipette in metal thermometer case4 ,for sterilisation and storage each pipette is placed inside a test-tube1 resting on a wad of cotton-wool1 and the tube plugged in the ordinary manner4 ,as these tubes are used almost exclusively for blood work1 it is usual to place a lance-headed hare-lip pin or a ,no4 #i flat ,hagedorn needle inside the tube so that the entire outfit may be sterilised at one time4 ,for the collection of small quantities of blood for agglutination reactions and the like1 many prefer a short straight piece of narrow glass tubing drawn out at either extremity to almost capillary dimensions4 ,such pipettes1 about #h cm4 in length over all1 are most conveniently sterilized in ordinary metal thermometer cases 7,fig4 #af74 ,graduated ,capillary ,pipettes 7,fig4 #ag74--,these should also be made in the laboratory--from manometer tubing--of simple1 convenient shape1 and graduated by the aid of standard pipettes 7in hundredths7 to contain such quantities as #aj1 #ej1 and #ij c4 mm41 and carefully marked with a writing diamond4 ,these1 previously sterilised in large test-tubes1 will be found extremely useful in preparing accurate percentage solutions1 when only minute quantities of fluid are available4 ,illustration3 ,,fig4 #ag4--;,capillary graduated pipettes4 ,automatic 7,throttle7 ,pipettes4--,these ingenious pipettes1 introduced by ,wright1 can easily be calibrated in the laboratory and are exceedingly useful for graduating small pipettes1 for measuring small quantities of fluids1 in preparing dilutions of serum for agglutination reactions1 etc4 ,they are usually made from the ,capillary ,pasteur pipettes 7,fig4 #ac1 a74 ,the following description of the manufacture of a #e c4 mm4 pipette will serve to show how the small automatic pipettes are calibrated4 #a4 ,select a pipette the capillary portion of which is fairly roomy in bore and possesses regular even walls1 and remove the cotton-wool plug from the open end4 #b4 ,heat the capillary portion near the free extremity in the by-pass flame of the bunsen burner and draw it out into a very fine hair-like tube and break this across4 ,this hair-like extremity will permit the passage of air but is too fine for metallic mercury to pass4 #c4 ,from a standard graduated pipette deliver #e c4 mm4 clean mercury into the upper wide portion of the pipette4 #d4 ,adjust a rubber teat to the pipette and by pressure on the bulb gradually drive the mercury in an unbroken column down the capillary tube until it is stopped by the filiform extremity4 #e4 ,cut off the capillary tube exactly at the upper level of the column of mercury1 invert it and allow the mercury to run out4 #f4 ,snap off the remainder of the capillary tube from the broad upper portion of the pipette which is now destined to form the covering tube or air chamber1 or what we may term the barrel4 ,this barrel now has the lower end in the form of a truncated cone1 the upper end being cut square4 ,remove the teat4 #g4 ,introduce the capillary tube into this barrel with the filiform extremity uppermost1 and the square cut end projecting about #j4e cm4 beyond the tapering end of the barrel4 ,illustration3 ,,fig4 #ah4--,throttle pipette--small capacity4 #h4 ,drop a small pellet of sealing wax into the barrel by the side of the capillary tube and then warm the tube at the gas flame until the wax becomes softened and makes an air-tight joint between the capillary tube and the end of the barrel4 #i4 ,fit a rubber teat to the open end of the barrel1 and so complete a pipette which can be depended upon to always aspirate and deliver exactly #e cm4 of fluid4 ,slight modification of this procedure is necessary in making tubes to measure larger volumes than say #ge c4 mm4 ,thus to make a throttle pipette to measure #ajj c4 mm43 #a4 ,take a short length of quill tubing and draw out one end into a roomy capillary stem1 and again draw out the extremity into a fine hair point1 thus forming a small ,pasteur pipette with a hair-like capillary extremity4 #b4 ,with a standard pipette fill #ajj c4 mm4 into the neck of this pipette1 and make a scratch with a writing diamond at the upper level 7a7 of the mercury meniscus 7,fig4 #ai1 ,a74 ,illustration3 ,,fig4 #ai4--,making throttle pipettes--large capacity ,now force the mercury down into the capillary stem as far as it will go1 so as to leave the upper part of the tube in the region of the diamond scratch empty 7,fig4 #ai1 ,b74 #c4 ,heat the tube in the region of the diamond scratch in the blowpipe flame1 and removing the tube from the flame draw it out so that the diamond scratch now occupies a position somewhere near the centre of this new capillary portion 7,fig4 #ai1 ,c74 #d4 ,heat the tube in this position in the peep flame of the ,bunsen burner1 and draw it out into a hair-like extremity4 ,snap off the glass tube1 leaving about #e mm4 of hair-like extremity attached to the upper capillary portion 7,fig4 #ai1 ,d74 ,allow the glass to cool4 #e4 ,lift up the bulb by the long capillary stem and allow the mercury to return to its original position--an operation which will be facilitated by snapping off the hair-like extremity from the long piece of capillary tubing4 #f4 ,mark on the capillary stem with a grease pencil the position of the end of the column of mercury 7,fig4 #ai1 ,e47 #g4 ,warm the capillary tubing at this spot in the peep flame of the ,bunsen burner1 and draw it out very slightly so that when cut at this position a pointed extremity will be obtained4 #h4 ,with a glass-cutting knife cut the capillary tube through at the point b1 and allow the mercury to run out4 #i4 ,now apply a thick layer of sealing wax to the neck of the bulb4 #aj4 ,take a piece of #e mm4 bore glass tubing and draw it out as if making an ordinary ,pasteur pipette4 #aa4 ,break the capillary portion off so as to leave a covering tube similar to that already used for the smaller graduated pipettes4 ,into this covering tube drop the graduated bulb and draw the capillary stem down through the conical extremity until further progress is stopped by the layer of sealing wax4 #ab4 ,warm the pipette in the gas flame so as to melt the sealing wax and make an air-tight joint4 #ac4 ,fit an india-rubber teat over the open end of the covering tube1 and the automatic pipette is ready for use 7,fig4 #ai1 ,f74 ,sedimentation ,pipettes 7,fig4 #bj74--,these are prepared from #aj cm4 lengths of narrow glass tubing by sealing one extremity1 blowing a small bulb at the centre1 and plugging the open end with cotton-wool2 after sterilisation the open end is provided with a short piece of rubber tubing and a glass mouthpiece4 ,when it is necessary to observe sedimentation reactions in very small quantities of fluid1 these tubes will be found much more convenient than the #e by #j4e cm4 test-tubes previously mentioned4 ,illustration3 ,,fig4 #bj4--,sedimentation pipette4 ,pasteur pipettes fitted with india-rubber teats will also be found useful for sedimentation tests when dealing with minute quantities of serum1 etc4 ,illustration3 ,,fig4 #ba4--;,fermentation tubes4 ,fermentation ,tubes 7,fig4 #ba74--,these are used for the collection and analysis of the gases liberated from the media during the growth of some varieties of bacteria and may be either plain 7a7 or graduated 7b74 ,a simple form 7,fig4 #ba1 c7 may be made from #ad cm4 lengths of soft glass tubing of #a4e cm4 diameter4 ,the ,bunsen flame is applied to a spot some #e cm4 from one end of such a piece of tubing and the tube slightly drawn out to form a constriction1 the constricted part is bent in the bat's-wing flame1 to an acute angle1 and the open extremity of the long arm sealed off in the blowpipe flame4 ,the open end of the short arm is rounded off and then plugged with cotton-wool1 and the tube is ready for sterilisation4 ,,cleaning ,,of ,,glass ,,apparatus4 ,all glassware used in the bacteriological laboratory must be thoroughly cleaned before use1 and this rule applies as forcibly to new as to old apparatus1 although the methods employed may vary slightly4 ,to ,clean ,new ,test-tubes4-- #a4 ,place the tubes in a bucket or other convenient receptacle1 fill with water and add a handful of ,sapon or other soap powder4 ,see that the tubes are full and submerged4 #b4 ,fix the bucket over a large ,bunsen flame and boil for thirty minutes--or boil in the autoclave for a similar period4 #c4 ,cleanse the interior of the tubes with the aid of test-tube brushes1 and rinse thoroughly in cold water4 #d4 ,invert the tubes and allow them to drain completely4 #e4 ,dry the tubes and polish the glass inside and out with a soft cloth1 such as selvyt4 ,new flasks1 plates1 and capsules must be cleaned in a similar manner4 ,to ,clean ,new ,graduated ,pipettes4-- #a4 ,place the pipettes in a convenient receptacle1 filled with water to which soap powder has been added4 #b4 ,boil the water vigorously for twenty minutes over a ,bunsen flame4 #c4 ,rinse the pipettes in running water and drain4 #d4 ,run distilled water through the pipettes and drain4 #e4 ,run rectified spirits through the pipette and drain as completely as possible4 #f4 ,place the pipettes in the hot-air oven 7vide page #ca71 close the door1 open the ventilating slide1 and run the temperature slowly up to about #hj deg4 ,c4 ,turn off the gas and allow the oven to cool4 ,or #f;a4 ,attach each pipette in turn to the rubber tube of the foot bellows1 or blowpipe air-blast1 and blow air through the pipette until the interior is dry4 ,glassware that has already been used is regarded as infected1 and is treated in a slightly different manner4 ,infected ,test-tubes4-- #a4 ,pack the tubes in the wire basket of the autoclave 7having previously removed the cotton-wool plugs1 caps1 etc471 in the vertical position1 and before replacing the basket see that there is a sufficiency of water in the bottom of the boiler4 ,now attach a piece of rubber tubing to the nearest water tap1 and by means of this fill each tube with water4 #b4 ,disinfect completely by exposing the tubes1 etc41 to a temperature of #abj deg4 ,c4 for twenty minutes 7vide page #cg74 7,if an autoclave is not available1 the tubes must be placed in a digester1 or even a large pan or pail with a tightly fitting cover1 and boiled vigorously for some thirty to forty-five minutes to ensure disinfection47 #c4 ,whilst still hot1 empty each tube in turn and roughly clean its interior with a stiff test-tube brush4 #d4 ,place the tubes in a bucket or other convenient receptacle1 fill with water and add a handful of ,sapon or other soap powder4 ,see that the tubes are full and submerged4 #e4 ,fix the bucket over a large ,bunsen flame and boil for thirty minutes4 #f4 ,cleanse the interior of the tubes with the aid of test-tube brushes1 and rinse thoroughly in cold water4 #g4 ,drain off the water and immerse tubes in a large jar containing water acidulated with #b to #e per cent4 hydrochloric acid4 ,allow them to remain there for about fifteen minutes4 #h4 ,remove from the acid jar1 drain1 rinse thoroughly in running water1 then with distilled water4 #i4 ,invert the tubes and allow them to drain completely4 ,dry the tubes and polish the glass inside and out with a soft cloth1 such as selvyt4 ,infected flasks1 plates1 and capsules must be treated in a similar manner4 ,flasks which have been used only in the preparation of media must be cleaned immediately they are finished with4 ,fill each flask with water to which some soap powder and a few crystals of potassium permanganate have been added1 and let boil over the naked flame4 ,the interior of the flask can then usually be perfectly cleaned with the aid of a flask brush1 but in some cases water acidulated with #e per cent4 nitric acid1 or a large wad of wet cotton-wool previously rolled in silver sand1 must be shaken around the interior of the flask1 after which rinse thoroughly with clean water1 dry1 and polish4 ,infected ,pipettes4-- #a4 ,plunge infected pipettes immediately after use into tall glass cylinders containing a #b per cent4 solution of lysol1 and allow them to remain therein for some days4 #b4 ,remove from the jar and drain4 ,boil in water to which a little soap has been added1 for thirty minutes4 #c4 ,rinse thoroughly in cold water4 #d4 ,immerse in #e per cent4 nitric acid for an hour or two4 #e4 ,rinse again in running water to remove all traces of acid4 #f4 ,complete the cleaning as described under new pipettes4 ,when dealing with graduated capillary pipettes employed for blood or serum work 7whether new or infected71 much time is consumed in the various steps from #e onward1 and the cleansing process can be materially hastened if the following device is adopted4 ,fit up a large-sized ,kitasato's filter flask to a ,sprengel's suction pump or a ,geryk air pump 7see page #dc74 ,to the side tubulure of the filter flask attach a #bj cm4 length of rubber pressure tubing having a calibre sufficiently large to admit the ends of the pipettes4 ,next fill a small beaker with distilled water4 ,attach the first pipette to the free end of the rubber tubing1 place the pipette point downward in the beaker of water and start the pump 7,fig4 #bb74 ,illustration3 ,,fig4 #bb4--;,cleaning blood pipettes4 ,when all the water has been aspirated through the pipette into the filter flask1 fill the beaker with rectified spirit and when this is exhausted refill with ether4 ,detach the pipette and dry in the hot-air oven4 ,slides and cover-slips 7,fig4 #bc71 when first purchased1 have greasy surfaces1 upon which water gathers in minute drops and effectually prevents the spreading of thin1 even films4 ,microscopical ,slides4--,the slides in general use are those known as three by one slips 7measuring #c inches by #a inch1 or #gf by #bf mm471 and should be of good white crown glass1 with ground edges4 ,new slides should be allowed to remain in alcohol acidulated with #e per cent4 hydrochloric acid for some hours1 rinsed in running water1 roughly drained on a towel1 dried1 and finally polished with a selvyt cloth4 ,illustration3 ,,fig4 #bc4--,slides and cover-slips1 actual size4 ,if only a few slides are required for immediate use a good plan is to rub the surface with jeweler's emery paper 7,hubert's #jj74 ,a piece of hard wood #gfx#bfx#bf mm4 with a piece of this emery paper gummed tightly around it is an exceedingly useful article on the microscope bench4 ,cover-slips4--,the most useful sizes are the #ai mm4 squares for ordinary cover-glass film preparations1 and #ch by #ai mm4 rectangles for blood films and serial sections2 both varieties must be of ,no4 #a thickness1 which varies between #j4ae and #j4bb mm41 that they may be available for use with the high-power immersion lenses4 ,cover-slips should be cleaned in the following manner3 #a4 ,drop the cover-slips one by one into an enamelled iron pot or tall glass beaker1 containing a #aj per cent4 solution of chromic acid4 #b4 ,heat over a ,bunsen flame and allow the acid to boil gently for twenty minutes4 ,,note4--,a few pieces of pipe-clay or pumice may be placed in the beaker to prevent the spurting of the chromic acid4 #c4 ,turn the cover-slips out into a flat glass dish and wash in running water under the tap until all trace of yellow colour has disappeared4 ,during the washing keep the cover-slips in motion by imparting a rotatory movement to the dish4 #d4 ,wash in distilled water in a similar manner4 #e4 ,wash in rectified spirit4 #f4 ,transfer the cover-slips1 by means of a pair of clean forceps1 previously heated in the ,bunsen flame to destroy any trace of grease1 to a small beaker of absolute alcohol4 ,drain off the alcohol and transfer the cover-slips1 by means of the forceps1 to a wide-mouthed glass pot1 containing absolute alcohol1 in which they are to be stored1 and stopper tightly4 ,,note4--,after once being placed in the chromic acid1 the cover-slips must on no account be touched by the fingers4 ,used ,slides and ,cover-slips4--,used slides with the mounted cover-slip preparations1 and cover-slips used for hanging-drop mounts1 should1 when discarded1 be thrown into a pot containing a #b per cent4 solution of lysol4 ,after immersion therein for a week or so1 even the cover-slips mounted with ,canada balsam can be readily detached from their slides4 ,slides4-- #a4 ,wash the slides thoroughly in running water4 #b4 ,boil the slides in water to which sapon has been added1 for half an hour4 #c4 ,rinse thoroughly in cold water4 #d4 ,dry and polish with a dry cloth4 ,cover-slips4-- #a4 ,wash the cover-slips thoroughly in running water4 #b4 ,boil the cover-slips in #aj per cent4 solution of chromic acid1 as for new cover-slips4 #c4 ,wash thoroughly in running water4 #d4 ,pick out those cover-slips which show much adherent dirty matter1 and rub them between thumb and forefinger under the water tap4 ,the dirt usually rubs off easily1 as it has become friable from contact with the chromic acid4 #e4 ,return all the cover-slips to the beaker1 fill in fresh chromic acid solution1 and treat as new cover-slips4 ,,note4--,test-tubes1 plates1 capsules1 etc41 which1 from long use1 have become scratched and hazy1 or which cannot be cleaned in any other way1 may be dealt with by immersing them in an enamelled iron bath1 containing water acidulated to #a per cent4 with hydrofluoric acid1 for ten minutes1 rinsing thoroughly in water1 drying1 and polishing4 ,,plugging ,,test-,,tubes ,,and ,,flasks4 ,before sterilisation all test-tubes and flasks must be carefully plugged with cotton-wool1 and for this purpose best absorbent cotton-wool 7preferably that put up in cylindrical one-pound packets and interleaved with tissue paper--known as surgeons' wool7 should be employed4 #a4 ,for a test-tube or a small flask1 tear a strip of cotton-wool some #aj cm4 long by #b cm4 wide from the roll4 #b4 ,turn in the ends neatly and roll the strip of wool lightly between the thumb and fingers of both hands to form a long cylinder4 #c4 ,double this at the centre and introduce the now rounded end into the open mouth of the tube or flask4 #d4 ,now1 whilst supporting the wool between the thumb and fingers of the right hand1 rotate the test-tube between those of the left1 and gradually screw the plug of wool into its mouth for a distance of about #b4e cm41 leaving about the same length of wool projecting4 ,illustration3 ,,fig #bd44--,plugging test-tubes3 a1 cylinder of wool being rolled2 b1 cylinder of wool being doubled2 c1 cylinder of wool being inserted in tube4 ,the plug must be firm and fit the tube or flask fairly tightly1 sufficiently tightly in fact to bear the weight of the glass plus the amount of medium the vessel is intended to contain1 but not so tightly as to prevent it from being easily removed by a screwing motion when grasped between the fourth1 or third and fourth1 fingers1 and the palm of the hand4 ,for a large flask a similar but larger strip of wool must be taken2 the method of making and inserting the plug is identical4 ,,iii4 ,,methods ,,of ,,sterilisation4 ,,sterilising ,,agents4 ,sterilisation--i4 e41 the removal or the destruction of germ life--may be effected by the use of various agents4 ,as applied to the practical requirements of the bacteriological laboratory1 many of these agents1 such as electricity1 sunlight1 etc41 are of little value1 others are limited in their applications2 others again are so well suited to particular purposes that their use is almost entirely restricted to such4 ,the sterilising agents in common use are3 ,chemical ,reagents4--,disinfectants 7for the disinfection of glass and metal apparatus and of morbid tissues74 ,physical ,agents4 ,,heat4--7a7 ,dry ,heat3 #a4 ,naked flame 7for the sterilisation of platinum needles1 etc474 #b4 ,muffle furnace 7for the sterilisation of filter candles1 and for the destruction of morbid tissues74 #c4 ,hot air 7for the sterilisation of all glassware and of metal apparatus74 7b7 ,moist ,heat3 #a4 ,water at #ef deg4 ,c4 7for the sterilisation of certain albuminous fluids74 #b4 ,water at #ajj deg4 ,c4 7for the sterilisation of surgical instruments1 rubber tubing1 and stoppers1 etc474 #c4 ,streaming steam at #ajj deg4 ,c4 7for the sterilisation of media74 #d4 ,superheated steam at #aae deg4 ,c4 or #abj deg4 ,c4 7for the disinfection of contaminated articles and the destruction of old cultivations of bacteria74 ,,filtration4-- #a4 ,cotton-wool filters 7for the sterilisation of air and gases74 #b4 ,porcelain filters 7for the sterilisation of various liquids74 ,,methods ,,of ,,application4 ,chemical ,reagents1 such as belong to the class known as antiseptics 7i4 e41 substances which inhibit the growth of1 but do not destroy1 bacterial life71 are obviously useless4 ,disinfectants or germicides 7i4 e41 substances which destroy bacterial life71 on the other hand1 are of value in the disinfection of morbid material1 and also of various pieces of apparatus1 such as pipettes1 pending their cleansing and complete sterilisation by other processes4 ,to this class 7in order of general utility7 belong3 ,lysol1 #b per cent4 solution2 ,perchloride of mercury1 #j4a per cent4 solution2 ,carbolic acid1 #e per cent4 solution2 ,absolute alcohol2 ,ether2 ,chloroform2 ,camphor2 ,thymol2 ,toluol2 ,volatile oils1 such as oil of mustard1 oil of garlic4 ,formaldehyde is a powerful germicide1 but its penetrating vapor restricts its use4 ,these disinfectants are but little used in the final sterilisation of apparatus1 chiefly on account of the difficulty of effecting their complete removal1 for the presence of even traces of these chemicals is sufficient to so inhibit or alter the growth of bacteria as to vitiate subsequent experiments conducted by the aid of apparatus sterilised in this manner4 ,,note4--,tubes1 flasks1 filter flasks1 pipettes1 glass tubing1 etc41 may be rapidly sterilised1 in case of emergency1 by washing1 in turn1 with distilled water1 perchloride of mercury solution1 alcohol1 and ether1 draining1 and finally gently heating over a gas flame to completely drive off the ether vapor4 ,chloroform or other volatile disinfectants may be added to various fluids in order to effect the destruction of contained bacteria1 and when this has been done1 may be completely driven off from the fluid by the application of gentle heat4 ,dry ,heat4--,the naked flame of the ,bunsen burner is invariably used for sterilising the platinum needles 7which are heated to redness7 and may be employed for sterilising the points of forceps1 or other small instruments1 cover-glasses1 pipettes1 etc41 a very short exposure to this heat being sufficient4 ,ether ,flame4--,in an emergency small instruments1 needles1 etc41 may be sterilised by dipping them in ether and after removal lighting the adherent fluid and allowing it to burn off the surface of the instruments4 ,repeat the process twice4 ,it may then be safely assumed that the apparatus so treated is sterile4 ,illustration3 ,,fig4 #be4--,muffle furnace4 ,muffle ,furnace 7,fig4 #be74--;,although this form of heat is chiefly used for the destruction of the dead bodies of small infected animals1 morbid tissues1 etc41 it is also employed for the sterilisation of porcelain filter candles 7vide p4 #db74 ,filter candles are disinfected immediately after use by boiling in a beaker of water for some fifteen or twenty minutes4 ,this treatment1 however1 leaves the dead bodies of the bacteria upon the surface and blocking the interstices of the filter4 ,to destroy the organic matter and prepare the filter candle for further use proceed as follows3 #a4 ,roll each bougie up in a piece of asbestos cloth1 secure the ends of the cloth with a few turns of copper wire1 and place inside the muffle 7a small muffle #gfx#hhx#afc mm4 will hold perhaps four small filter candles74 #b4 ,light the gas and raise the contents of the muffle to a white heat2 maintain this temperature for five minutes4 #c4 ,extinguish the gas1 and when the muffle has become quite cold remove the filter candles1 and store them 7without removing the asbestos wrappings7 in sterile metal boxes4 ,,note4--,the too rapid cooling of the candles1 such as takes place if they are removed from the muffle before it has cooled down to the room temperature1 may give rise to microscopic cracks and flaws which will effectually destroy their efficiency4 ,hot ,air4--,hot air at #aej deg4 ,c4 destroys all bacteria1 spores1 etc31 in about thirty minutes2 a momentary exposure to a temperature of #age deg4 to #ahj deg4 ,c4 will effect the same result and offers the more convenient method of sterilisation4 ,this method is only applicable to glass and metallic substances1 and the small bulk of cotton-wool comprised in the test-tube plugs1 etc4 ,large masses of fabric are not effectually sterilised by dry heat--short of charring--as its power of penetration is not great4 ,sterilisation by hot air is effected in the hot-air oven 7,fig4 #ah74 ,this is a rectangular1 double-walled metal box1 mounted on a stand and heated from below by a large ,bunsen burner4 ,the interior of the oven is provided with loose shelves upon which the articles to be sterilised are arranged1 either singly or packed in square wire baskets or crates1 kept specially for this purpose4 ,one of the sides is hinged to form a door4 ,the central portion of the metal bottom1 on which the ,bunsen flame would play1 is cut away1 and replaced by firebrick plates1 which slide in metal grooves and are easily replaced when broken or worn out4 ,the top of the oven is provided with a perforated ventilator slide and two tubulures1 the one for the reception of a centigrade thermometer graduated to #bjj deg4 or #bej deg4 ,c41 the other for a thermo-regulator4 ,an ordinary mercurial thermo-regulator may be used but it is preferable to employ a regulating capsule of the ,hearson type 7see p4 #bai7 with a spring arm adjusted to the lever so that when the boiling-point of the capsule 7e4 g41 #age deg4 ,c47 is reached the gas supply is absolutely cut off and the jet cannot again be lighted until the spring-arm has been readjusted by hand4 ,the thermo-regulator is by no means a necessity1 and may be replaced by a large bore thermometer with a sliding platinum point1 connected with an electric bell1 which can be easily adjusted to ring at any given temperature4 ,even if the steriliser is provided with the capsule regulator above described the contact thermometer should also be fitted4 ,illustration3 ,,fig4 #bf4--;,hot-air oven4 ,,to ,,use ,,the ,,hot-,,air ,,oven4-- #a4 ,place the crates of test-tubes1 metal cases containing plates and pipettes1 loose apparatus1 etc41 inside the oven1 taking particular care that none of the cotton-wool plugs are in contact with the walls1 otherwise the heat transmitted by the metal will char or even flame them4 ,to prepare a wire crate for the reception of test-tubes1 etc41 cover the bottom with a layer of thick asbestos cloth2 or take some asbestos fibre1 moisten it with a little water and knead it into a paste2 plaster the paste over the bottom of the crate1 working it into the meshes and smoothing the surface by means of a pestle4 ,when several crates have been thus treated1 place them inside the hot-air oven1 close the door1 open the ventilating slide1 light the gas1 and run the temperature of the interior up to about #afj deg4 ,c4 ,after an interval of ten minutes extinguish the gas1 open the oven door1 and allow the contents to cool4 ,the asbestos now forms a smooth1 dry1 spongy layer over the bottom1 which will last many months before needing renewal1 and will considerably diminish the loss of tubes from breakage4 ,copper cylinders and large test-tubes intended for the reception of pipettes are prepared in a similar manner1 in order to protect the points of these articles from injury4 #b4 ,close the oven door1 and open the ventilating slide1 in order that any moisture left in the tubes1 etc41 may escape2 light the gas below2 set the electric alarm to ring at #ajj deg4 ,c4 #c4 ,when the temperature of the oven has reached #ajj deg4 ,c41 close the ventilating slide2 reset the alarm to ring at #age deg4 ,c4 #d4 ,run the temperature up to #age deg4 ,c4 #e4 ,extinguish the gas at once1 and allow the apparatus to cool4 #f4 ,when the temperature of the interior1 as recorded by the thermometer1 has fallen to #fj deg4 ,c4--but not before--the door may be opened and the sterile articles removed and stored away4 ,,note4--,neglect of this precautionary cooling of the oven to #fj deg4 ,c4 will result in numerous cracked and broken tubes4 ,on removal from the oven1 the cotton-wool plugs will probably be slightly brown in colour4 ,metal instruments1 such as knives1 scissors1 and forceps1 may be sterilised in the hot-air oven as described above1 but exposure to #age deg4 ,c4 is likely to seriously affect the temper of the steel and certainly blunts the cutting edges4 ,if1 however1 it is desired to sterilise surgical instruments by hot air1 they should be packed in a metal box1 or boxes1 and heated to #acj deg4 ,c4 and retained at that temperature for about thirty minutes4 ,moist ,heat4--,water at #ef deg4 ,c4--,this temperature1 if maintained for thirty minutes1 is sufficient to destroy the vegetative forms of bacteria1 but has practically no effect on spores4 ,its use is limited to the sterilisation of such albuminous fluid media as would coagulate at a higher temperature4 ,,method4-- #a4 ,fit up a water-bath1 heated by a ,bunsen flame which is controlled by a thermo-regulator1 so that the temperature of the water remains at #ef deg4 ,c4 #b4 ,immerse the tubes or flasks containing the albuminous fluid in the water-bath so that the upper level of such fluid is at least #b cm4 below the level of the water4 7,the temperature of the bath will now fall somewhat1 but after a few minutes will again rise to #ef deg4 ,c74 #c4 ,after thirty minutes' exposure to #ef deg4 ,c1 extinguish the gas1 remove the tubes or flasks from the bath1 and subject them to the action of running water so that their contents are rapidly cooled4 #d4 ,the vegetative forms of bacteria present in the liquid being killed1 stand it for twenty-four hours in a cool1 dark place2 at the end of that time some at least of such spores as may be present will have germinated and assumed the vegetative form4 #e4 ,destroy these new vegetative forms by a similar exposure to #ef deg4 ,c4 on the second day1 whilst others1 of slower germination1 may be caught on the third day1 and so on4 #f4 ,in order to ensure thorough sterilisation1 repeat the process on each of six successive days4 ,this method of exposing liquids to a temperature of #ef deg4 ,c4 in a water-bath for half an hour on each of six successive days is termed fractional sterilisation4 ,water at #ajj deg4 ,c4 destroys the vegetative forms of bacteria almost instantaneously1 and spores in from five to fifteen minutes4 ,this method of sterilisation is applicable to the metal instruments1 such as knives1 forceps1 etc41 used in animal experiments2 syringes1 rubber corks1 rubber and glass tubing1 and other small apparatus1 and is effected in what is usually spoken of as the water steriliser 7,fig4 #bg74 ,illustration3 ,,fig4 #bg4--,water sterilizer4 ,this is a rectangular copper box1 #bf cm4 long1 #ah cm4 wide1 and #ab cm4 deep1 mounted on legs1 heated from below by a ,bunsen or radial gas burner1 and containing a movable copper wire tray1 #b cm4 smaller in every dimension than the steriliser itself1 and provided with handles4 ,the top of the steriliser is hinged to form a lid4 ,,method4-- #a4 ,place the instruments1 etc41 to be sterilised inside the copper basket1 and replace the basket in the steriliser4 #b4 ,pour a sufficient quantity of water into the steriliser1 shut down the lid1 and light the gas below4 ,illustration3 ,,fig4 #bh4--,koch's steriliser4 ,illustration3 ,,fig4 #bi4--;,arnold's steriliser4 #c4 ,after the water has boiled and steam has been issuing from beneath the lid for at least ten minutes1 extinguish the gas1 open the lid1 and lift out the wire basket by its handles and rest it diagonally on the walls of the steriliser2 the contained instruments1 etc41 are now sterile and ready for use4 #d4 ,after use1 or when accidentally contaminated1 replace the instruments in the basket and return that to the steriliser2 completely disinfect by a further boiling for fifteen minutes4 #e4 ,after disinfection1 and whilst still hot1 take out the instruments1 dry carefully and at once1 and return them to their store cases4 ,streaming steam--i4 e41 steam at #ajj deg4 ,c4--destroys the vegetative forms of bacteria in from fifteen to twenty minutes1 and the sporing forms in from one to two hours4 ,this method is chiefly used for the sterilisation of the various nutrient media intended for the cultivation of bacteria1 and is carried out in a steam kettle of special construction1 known as ,koch's steam steriliser 7,fig4 #bh7 or in one of its many modifications1 the most efficient of which is ,arnold's 7,fig4 #bi74 ,the steam steriliser in its simplest form consists of a tall tinned-iron or copper cylindrical vessel1 divided into two unequal parts by a movable perforated metal diaphragm1 the lower1 smaller portion serving for a water reservoir1 and the upper part for the reception of wire baskets containing the articles to be sterilised4 ,the vessel is closed by a loose conical lid1 provided with handles1 and perforated at its apex by a tubulure2 it is mounted on a tripod stand and heated from below by a ,bunsen burner4 ,the more elaborate steriliser is cased with felt or asbestos board1 and provided with a water gauge1 also a tap for emptying the water compartment4 ,,to ,,use ,,the ,,steam ,,steriliser4-- #a4 ,fill the water compartment to the level of the perforated diaphragm1 place the lid in position1 and light the ,bunsen burner4 #b4 ,after the water has boiled1 allow sufficient time to elapse for steam to replace the air in the sterilising compartment1 as shown by the steam issuing in a steady1 continuous stream from the tubulure in the lid4 #c4 ,remove the lid1 quickly lower the wire basket containing media tubes1 etc41 into the sterilising compartment until it rests on the diaphragm1 and replace the lid4 #d4 ,after an interval of twenty minutes in the case of fluid media1 or thirty minutes in the case of solid media1 take off the lid and remove the basket with its contents4 #e4 ,now1 but not before1 extinguish the gas4 ,,note4--,after removing tubes1 flasks1 etc41 from the steam steriliser1 they should be at once separated freely in order to prevent moisture condensing upon the cotton-wool plugs and soaking through into the interior of the tubes4 ,this treatment will destroy any vegetative forms of bacteria2 during the hours of cooling any spores present will germinate1 and the young organisms will be destroyed by repeating the process twenty-four hours later2 a third sterilisation after a similar interval makes assurance doubly sure4 ,the method of sterilising by exposure to streaming steam at #ajj deg4 ,c4 for twenty minutes on each of three consecutive days is termed discontinuous or intermittent sterilisation4 ,exposure to steam at #ajj deg4 ,c4 for a period of one or two hours1 or continuous sterilisation1 cannot always be depended upon and is therefore not to be recommended4 ,superheated steam--i4 e41 steam under pressure 7see ,pressure-temperature table1 ,appendix1 page #ejj7 in sealed vessels at a temperature of #aae deg4 ,c4--will destroy both the vegetative and the sporing forms of bacteria within fifteen minutes2 if the pressure is increased1 and the temperature raised to #abj deg4 ,c41 the same end is attained in ten minutes4 ,this method was formerly employed for the sterilisation of media 7and indeed is so used in some laboratories still71 but most workers now realise that media subjected to this high temperature undergo hydrolytic changes which render them unsuitable for the cultivation of the more delicate micro-organisms4 ,the use of superheated steam should be restricted almost entirely to the disinfection of such contaminated articles1 old cultivations1 etc41 as cannot be dealt with by dry heat or the actual furnace4 ,sterilisation by means of superheated steam is carried out in a special boiler--,chamberland's autoclave 7,fig4 #cj74 ,the autoclave consists of a stout copper cylinder1 provided with a copper or gun-metal lid1 which is secured in place by means of bolts and thumbscrews1 the joint between the cylinder and its lid being hermetically sealed by the interposition of a rubber washer4 ,the cover is perforated for a branched tube carrying a vent cock1 a manometer1 and a safety valve4 ,the copper boiler is mounted in the upper half of a cylindrical sheet-iron case--two concentric circular rows of ,bunsen burners1 each circle having an independent gas-supply1 occupying the lower half4 ,in the interior of the boiler is a large movable wire basket1 mounted on legs1 for the reception of the articles to be sterilised4 ,,to ,,use ,,the ,,autoclave4-- #a4 ,pack the articles to be sterilised in the wire basket4 #b4 ,run water into the boiler to the level of the bottom of the basket2 also fill the contained flasks and tubes with water4 #c4 ,see that the rubber washer is in position1 then replace the cover and fasten it tightly on to the autoclave by means of the thumbscrews4 #d4 ,open the vent cock and light both rings of burners4 #e4 ,when steam is issuing in a steady1 continuous stream from the vent tube1 shut off the vent cock and extinguish the outer ring of gas burners4 #f4 ,wait until the index of the manometer records a temperature of #abj deg4 ,c41 then regulate the gas and the spring safety valve in such a manner that this temperature is just maintained1 and leave it thus for twenty minutes4 ,in the more expensive patterns of autoclave this regulation of the safety valve is carried out automatically1 the manometer being fitted with an adjustable pointer which can be set to any required pressure-temperature and so arranged that when the index of the manometer coincides with the adjustable hand the safety valve is opened4 #g4 ,extinguish the gas and allow the manometer index to fall to zero4 ,illustration3 ,,fig4 #cj4--;,chamberland's ,autoclave4 #h4 ,now open the vent cock slowly1 and allow the internal pressure to adjust itself to that of the atmosphere4 #i4 ,remove the cover and take out the sterilised contents4 ,sterilisation ,periods4--,an exceedingly useful device for the timing of sterilisation periods 7and indeed for many other operations in the laboratory7 is the ,,electric ,,signal ,,timing ,,clock4 ,this is a clock of ,american type in which the face is surrounded by a metal plate having a series of #fj holes at equal distances apart1 corresponding to the minutes on the dial4 ,this plate is connected with one of the poles of a dry battery1 the other pole of which is connected to the metal case of the clock for the purpose of actuating an ordinary magnet alarm bell4 ,in the centre of each of the holes in the plate a metal rod is fixed1 which then passes through an insulating ring and projects inside the clock face1 where it makes contact with the hour hand4 ,the clock is mounted on a heavy base1 with a key-board containing #bj numbered plugs4 ,if one of the plugs is inserted in a hole in the plate it makes contact with the rod1 and when the hour hand of the clock touches the other end the circuit is completed and the bell starts ringing4 ,the period of this friction contact is approximately #bj seconds4 ,the clock can therefore be used for electrically noting the periods of time from one minute by multiples of one minute up to one hour4 ,illustration3 ,,fig4 #ca4--;,electric signal timing clock4 ,filtration4--7a7 ,cotton-wool ,filter4--,practically the only method in use in the laboratory for the sterilisation of air or of a gas is by filtration through dry cotton-wool or glass-wool1 the fibres of which entangle the micro-organisms and prevent their passage4 ,perhaps the best example of such a filter is the cotton-wool plug which closes the mouth of a culture tube4 ,not only does ordinary diffusion take place through it1 but if a tube plugged in the usual manner with cotton-wool is removed from the hot incubator1 the temperature of the contained air rapidly falls to that of the laboratory1 and a partial vacuum is formed2 air passes into the tube1 through the cotton-wool plug1 to restore the equilibrium1 and1 so long as the plug remains dry1 in a germ-free condition4 ,if1 however1 the plug becomes moist1 either by absorption from the atmosphere1 or from liquids coming into contact with it1 micro-organisms 7especially the mould fungi7 commence to multiply1 and the long thread forms rapidly penetrate the substance of the plug1 and gain access to and contaminate the interior of the tube4 ,illustration3 ,,fig4 #cb4--;,cotton-wool air filter4 ,,method4-- ,if it is desired to sterilise gases before admission to a vessel containing a pure cultivation of a micro-organism1 as1 for instance1 when forcing a current of oxygen over or through a broth cultivation of the diphtheria bacillus1 this can be readily effected as follows3 #a4 ,take a length of glass tubing of1 say1 #a4e cm4 diameter1 in the centre of which a bulb has been blown1 fill the bulb with dry cotton-wool 7,fig4 #cb71 wrap a layer of cotton-wool around each end of the tube1 and secure in position with a turn of thin copper wire or string2 then sterilise the piece of apparatus in the hot-air oven4 #b4 ,prepare the cultivation in a ,ruffer or ,woodhead flask 7,fig4 #cc7 the inlet tube of which has its free extremity enveloped in a layer of cotton-wool1 secured by thread or wire1 whilst the exit tube is plugged in the usual manner4 ,illustration3 ,,fig4 #cc4--,ruffer's flask4 #c4 ,sterilise a short length of rubber tubing by boiling4 ,transfer it from the boiling water to a beaker of absolute alcohol4 #d4 ,when all is ready remove the rubber tube from the alcohol by means of a pair of forceps1 drain it thoroughly1 and pass through the flame of a ,bunsen burner to burn off the last traces of alcohol4 #e4 ,remove the cotton-wool wraps from the entry tube of the flask and from one end of the filter tube and rapidly couple them up by means of the sterile rubber tubing4 #f4 ,connect the other end of the bulb tube with the delivery tube from the gas reservoir4 ,the gas in its passage through the dry sterile cotton-wool in the bulb of the filter tube will be freed from any contained micro-organisms and will enter the flask in a sterile condition4 7b7 ,porcelain ,filter4--,the sterilisation of liquids by filtration is effected by passing them through a cylindrical vessel1 closed at one end like a test-tube1 and made either of porous biscuit porcelain1 hard-burnt and unglazed 7,chamberland system71 or of ,kieselguhr1 a fine diatomaceous earth 7,berkefeld system71 and termed a bougie or candle 7,fig4 #cd74 ,,note4--,in selecting candles for use in the laboratory avoid those with metal fittings1 since during sterilisation cracks develop at the junction of the metal and the siliceous material owing to the unequal expansion4 ,in this method the bacteria are retained in the pores of the filter while the liquid passes through in a germ-free condition4 ,it is obvious that to be effective the pores of the filter must be extremely minute1 and therefore the rate of filtration will usually be slow4 ,chamberland filter candles possess finer channels than ,berkefeld candles and consequently filter much more slowly4 ,to overcome this disadvantage1 either aspiration or pressure1 or a combination of these two forces1 may be employed to hasten the process4 ,doultons white porcelain filters it may be noted are as efficient as the ,chamberland candles and filter rather more rapidly4 ,apparatus ,required4-- #a4 ,separatory funnel containing the unfiltered fluid4 #b4 ,sterile filter candle 7,fig4 #cd71 the open end fitted with a rubber stopper 7,fig4 #cd1 a7 perforated to receive the delivery tube of the separatory funnel1 and its neck passed through a large rubber washer 7,fig4 #cd1 b7 which fits the mouth of the filter flask4 #c4 ,sterile filter flask of suitable size1 for the reception of the filtered fluid1 its mouth closed by a cotton-wool plug4 #d4 ,water injector ,sprengel 7see ,fig4 #ch1 c7 pump1 or ,geryk's pump 7an air pump on the hydraulic principle1 sealed by means of low vapor-tension oil1 ,fig4 #ce74 ,if this latter is employed1 a ,wulff's bottle1 fitted as a wash-bottle and containing sulphuric acid1 must be interposed between the filter flask and the pump1 in order to prevent moist air reaching the oil in the pump4 #e4 ,air filter 7vide page #dj7 sterilised4 #f4 ,pressure tubing4 #g4 ,screw clamps 7,fig4 #cf74 ,,method4-- #a4 ,couple the exhaust pipe of the suction pump with the lateral tube of the filter flask 7first removing the cotton-wool plug from this latter71 by means of pressure tubing1 interposing1 if necessary1 the wash-bottle of sulphuric acid4 ,illustration3 ,,fig4 #cd4--,porcelain filter candle4 ,illustration3 ,,fig4 #ce4--;,geryk air pump4 #b4 ,remove the cotton-wool plug from the neck of the filter flask and adjust the porcelain candle in its place4 ,illustration3 ,,fig4 #cf4--,screw clamps4 #c4 ,attach the nozzle of the separatory funnel to the filter candle by means of the perforated rubber stopper 7,fig4 #cg74 ,illustration3 ,,fig4 #cg4--;,apparatus arranged for filtering--aspiration4 #d4 ,open the tap of the funnel1 and exhaust the air from the filter flask and wash-bottle2 maintain the vacuum until the filtration is complete4 #e4 ,when the filtration is completed close the tap of the funnel2 adjust a screw clamp to the pressure tubing attached to the lateral branch of the filter flask2 screw it up tightly1 and disconnect the acid wash-bottle4 #f4 ,attach the air filter to the open end of the pressure tubing2 open the screw clamp gradually1 and allow filtered air to enter the flask1 to abolish the negative pressure4 #g4 ,detach the rubber tubing from the lateral branch of the flask1 flame the end of the branch in the ,bunsen1 and plug its orifice with sterile cotton-wool4 #h4 ,remove the filter candle from the mouth of the flask1 flame the mouth1 and plug the neck with sterile cotton-wool4 #i4 ,disinfect the filter candle and separatory funnel by boiling4 ,if it is found necessary to employ pressure in addition to or in place of suction1 insert a perforated rubber stopper into the mouth of the separatory funnel and secure in position with copper wire2 next fit a piece of glass tubing through the stopper1 and connect the external orifice with an air-pressure pump of some kind 7an ordinary foot pump such as is employed for inflating bicycle tyres is one of the most generally useful1 for this purpose7 or with a cylinder of compressed air or other gas4 ,in order to filter a large bulk of fluid very rapidly it is necessary to use a higher pressure than glass would stand1 and in these cases the metal receptacle designed by ,pakes 7,fig4 #ch1 a71 to hold the filter candle itself as well as the fluid to be filtered1 should be employed4 7,a vacuum must also be maintained in the filter flask1 by means of an exhaust pump1 during the entire process47 ,this piece of apparatus consists of a brass cylinder1 capacity #bejj c4c41 with two shoulders2 and an opening in the neck at each end1 provided with screw threads4 ,a nut carrying a pressure gauge fits into the top screw2 and into the bottom is fitted a brass cylinder carrying the filter candle and prolonged downwards into a delivery tube4 ,leakage is prevented by means of rubber washers4 ,into the top shoulder a tube is inserted1 bent at right angles and provided with a tap4 ,all the brass-work is tinned inside 7,fig4 #ch1 a74 ,in use the reservoir is generally mounted on a tripod stand4 ,to ,sterilise4-- #a4 ,insert the filter candle into its cylinder and screw this loosely on4 ,illustration3 ,,fig4 #ch4--,pakes' filtering reservoir--pressure and aspiration4 #b4 ,wrap a layer of cotton-wool around the delivery tube and fasten in position4 #c4 ,remove the nut carrying the pressure gauge and plug the neck with cotton-wool4 #d4 ,heat the whole apparatus in the autoclave at #abj deg4 ,c4 for twenty minutes4 ,,method4-- #a4 ,remove the apparatus from the autoclave1 and allow it to cool4 #b4 ,screw home the box carrying the bougie4 #c4 ,set the apparatus up in position1 with its delivery tube 7from which the cotton-wool wrapping has been removed7 passing through a perforated rubber stopper in the neck of a filter flask4 ,illustration3 ,,fig4 #ci4--;,closed candle arranged for filtering4 #d4 ,fill the fluid to be filtered into the cylinder and screw on the nut carrying the pressure gauge4 7,this nut should be immersed in boiling water for a few minutes previous to screwing on1 in order to sterilise it47 #e4 ,connect the horizontal arm of the entry tube with a cylinder of compressed oxygen 7or carbon dioxide1 ,fig4 #ch1 b71 by means of pressure tubing4 #f4 ,connect the lateral arm of the filter flask with the exhaust pump 7,fig4 #ch1 c7 and start the latter working4 #g4 ,open the tap of the gas cylinder2 then open the tap on the entry tube of the filter cylinder and raise the pressure in its interior until the desired point is recorded on the manometer4 ,maintain this pressure1 usually one or one and a half atmospheres1 until filtration is completed1 by regulating the tap on the entry tube4 ,some forms of filter candle are made with the open end contracted into a delivery nozzle1 which is glazed4 ,in this case the apparatus is fitted up in a slightly different manner2 the fluid to be filtered is contained in an open cylinder into which the candle is plunged1 while its delivery nozzle is connected with the filter flask by means of a piece of flexible pressure tubing 7previously sterilised by boiling71 as in figure #ci4 ,,iv4 ,,the ,,microscope4 ,the essentials of a microscope for bacteriological work may be briefly summed up as follows3 ,illustration3 ,,fig4 #dj4--,microscope stand4 ,the instrument1 of the monocular type1 must be of good workmanship and well finished1 rigid1 firm1 and free from vibration1 not only when upright1 but also when inclined to an angle or in the horizontal position4 ,the various joints and movements must work smoothly and precisely1 equally free from the defects of loss of time and slipping4 ,all screws1 etc41 should conform to the ,royal ,microscopical ,society's standard4 ,it must also be provided with good lenses and a sufficiently large stage4 ,the details of its component parts1 to which attention must be specially directed1 are as follows3 ,illustration3 ,,fig4 #da4--;,foot1 three types4 #a4 ,the ,base or ,foot 7,fig4 #dj1 a74--,two elementary forms--the tripod 7,fig4 #da1 a7 and the vertical column set into a plate known as the horse-shoe 7,fig4 #da1 b7--serve as the patterns for countless modifications in shape and size of this portion of the stand4 ,the chief desiderata--stability and ease of manipulation--are attained in the first by means of the spread of the three feet1 which are usually shod with cork2 in the second1 by the dead weight of the foot-plate4 ,the tripod is mechanically the more correct form1 and for practical use is much to be preferred4 ,its chief rival1 the ,jackson foot 7,fig4 #da1 c71 is based upon the same principle1 and on the score of appearance has much to recommend it4 #b4 ,the body tube 7,fig4 #dj1 b7 may be either that known as the long or ,english 7length #bej mm471 or the short or ,continental 7length #afj mm474 ,neither length appears to possess any material advantage over the other1 but it is absolutely necessary to secure objectives which have been manufactured for the particular tube length chosen4 ,in the high-class microscope of the present day the body tube is usually shorter than the ,continental1 but is provided with a draw tube which1 when fully extended1 gives a tube length greater than the ,english1 thus permitting the use of either form of objective4 ,illustration3 ,,fig4 #db4--;,coarse adjustment4 ,illustration3 ,,fig4 #dc4--;,fine adjustment4 ,for practical purposes the tube length distance from the end of the nosepiece to the eyeglass of the ocular4 ,this is the measurement referred to in speaking of long or short tube4 #c4 ,the coarse adjustment 7,fig4 #dj1 c7 should be a rack-and-pinion movement1 steadiness and smoothness of action being secured by means of accurately fitting dovetailed bearings and perfect correspondence between the teeth of the rack and the leaves of the pinion 7,fig4 #db74 ,also provision should be made for taking up the slack 7as by the screws ,,aa1 ,fig4 #db74 #d4 ,the fine adjustment 7,fig4 #dj1 d7 should on no account depend upon the direct action of springs1 but should be of the lever pattern1 preferably the ,nelson 7,fig4 #dc74 ,in this form the unequal length of the arms of the lever secures very delicate movement1 and1 moreover1 only a small portion of the weight of the body tube is transmitted to the thread of the vertical screw actuating the movement4 ,illustration3 ,,fig4 #dd4--,spindle head to fine adjustment4 ,a spindle milled head 7,fig4 #dd7 will be found a very useful device to have fitted in place of the ordinary milled head controlling the fine adjustment4 ,in this contrivance the axis of the milled head is prolonged upward in a short column1 the diameter of which is one-sixth of that of the head4 ,the spindle can be rapidly rotated between the fingers for medium power adjustments while the larger milled head can be slowly moved when focussing high powers4 #e4 ,the stage 7,fig4 #dj1 e7 should be square in shape and large in area--at least #ab cm4--flat and rigid1 in order to afford a safe support for the ,petri dish used for plate cultivations2 and should be supplied with spring clips 7removable at will7 to secure the #c by #a glass slides4 ,a mechanical stage must be classed as a necessity rather than a luxury so far as the bacteriologist is concerned1 as when working with high powers1 and especially when examining hanging-drop specimens1 it is almost impossible to execute sufficiently delicate movements with the fingers4 ,in selecting a mechanical stage1 preference should be given to one which forms an integral part of the instrument 7,fig4 #de7 rather than one which needs to be clamped on to an ordinary plain stage every time it is required1 and its traversing movements should be controlled by stationary milled heads 7,fig4 #de1 ,,aa'74 ,the shape of the aperture is a not unimportant point2 it should be square to allow of free movement over the substage condenser4 ,the mechanical stage should be tapped for three 7removable7 screw studs to be used in place of the sliding bar1 so that if desired the ,vernier finder 7,fig4 #de1 ,,bb'71 such as is usually fitted to this class of stage1 or a ,maltwood finder1 may be employed4 ,illustration3 ,,fig4 #de4--,mechanical stage4 ,illustration3 ,,fig4 #df4--;,iris diaphragm4 #f4 ,diaphragm4--,separate single diaphragms must be avoided2 a revolving plate pierced with different sized apertures and secured below the stage is preferable1 but undoubtedly the best form is the iris diaphragm 7,fig4 #df7 which enters into the construction of the substage condenser4 #g4 ,the substage condenser is a necessary part of the optical outfit4 ,its purpose is to collect the beam of parallel rays of light reflected by the plane mirror1 by virtue of a short focus system of lenses1 into a cone of large aperture 7reducible at will by means of an iris diaphragm mounted as a part of the condenser71 which can be accurately focussed on the plane of the object4 ,this focussing must be performed anew for each object1 on account of the variation in the thickness of the slides4 ,the form in most general use is that known as the ,abbe 7,fig4 #dg7 and consists of a plano-convex lens mounted above a biconvex lens4 ,this combination is carried in a screw-centering holder known as the substage below the stage of the microscope 7,fig4 #dj f71 and must be accurately adjusted so that its optical axis coincides with that of the objective4 ,vertical movement of the entire substage apparatus effected by means of a rack and pinion is a decided advantage1 and some means should be provided for temporarily removing the condenser from the optical axis of the microscope4 ,illustration3 ,,fig4 #dg--,optical part of ,abbe illuminator4 ,with the oil immersion objective1 however1 an achromatic condenser1 giving an illuminating cone of about #j4i1 should be used if the full value of the lens is to be obtained4 ,it is generally assumed that a good objective requires an illuminating cone equivalent to two-thirds of its numerical aperture4 ,the best ,abbe condenser transmits a cone of about 4#de whilst the aperture of the #a_/ab inch immersion lenses of different makers varies from #a4j to #a4d1 hence1 the efficiency of these lenses is much curtailed if the condenser is merely the ,abbe4 ,these improved condensers must be absolutely centered to the objective and capable of very accurate focussing otherwise much of their value is lost4 #h4 ,mirrors4--,below the substage condenser is attached a gymbal carrying a reversible circular frame with a plane mirror on one side and a concave mirror on the other 7,fig4 #dj1 g74 ,the plane mirror is that usually employed1 but occasionally1 as for example when using low powers and with the condenser racked down and thrown out of the optical axis1 the concave mirror is used4 #i4 ,oculars1 or ,eyepieces4--,those known as the ,huyghenian oculars 7,fig4 #dh7 will be sufficient for all ordinary work without resorting to the more expensive compensation oculars4 ,two or three1 magnifying the real image 7formed by the objective7 four1 six1 or eight times respectively1 form a useful equipment4 ,as an accessory ,ehrlich's ,eyepiece is a very useful piece of apparatus when the enumeration of cells or bacteria has to be carried out4 ,this is an ordinary eyepiece fitted with an adjustable square diaphragm operated by a lever projecting from the side of the mount4 ,three notches are made in one of the sides of the square and by moving the lever square aperture can be reduced to three-quarters1 one-half or one-quarter of the original size4 #aj4 ,objectives4--,three objectives are necessary3 one for low-power work--e4 g41 #a inch1 #b_/c inch1 or #a_/b inch2 one for high-power work--e4 g41 #a_/ab inch oil immersion lens2 and an intermediate medium-power lens--e4 g41 #a_/f inch or #a_/h inch 7dry74 ,these lenses must be carefully selected1 especial attention being paid to the following points3 7a7 ,correction of ,spherical ,aberration4--,spherical aberration gives rise to an ill-defined image1 due to the central and peripheral rays focussing at different points4 7b7 ,correction of ,chromatic ,aberration4--,chromatic aberration gives rise to a coloured fringe around the edges of objects due to the fact that the different-coloured rays of the spectrum possess varying refrangibilities and that a simple lens acts toward them as a prism4 7c7 ,flatness of ,field4--,the ideal visual field would be large and1 above all1 flat2 in other words1 objects at the periphery of the field would be as distinctly in focus as those in the centre4 ,unfortunately1 however1 this is an optical impossibility and the field is always spherical in shape4 ,some makers succeed in giving a larger central area that is in focus at one time than others1 and although this may theoretically cause an infinitesimal sacrifice of other qualities1 it should always be sought for4 ,successive zones and the entire peripheral ring should come into focus with the alteration of the fine adjustment4 ,this simultaneous sharpness of the entire circle is an indication of the perfect centering of the whole of the lenses in the objective4 ,illustration3 ,,fig4 #dh4--;,huyghenian eyepiece4 7d7 ,good ,definition4--,actual magnification is1 within limits1 of course1 of less value than clear definition and high resolving power1 for it is upon these properties we depend for our knowledge of the detailed structure of the objects examined4 7e7 ,numerical ,aperture 7,n4 ,a474--,the numerical aperture may be defined1 in general terms1 as the ratio of the effective diameter of the back lens of the objective to its equivalent focal length4 ,the determination of this point is a process requiring considerable technical skill and mathematical ability1 and is completely beyond the powers of the average microscopist4#a ,although with the increase in power it is correspondingly difficult to combine all these corrections in one objective1 they are brought to a high pitch of excellence in the present-day achromatic objectives1 and so remove the necessity for the use of the higher priced and less durable apochromatic lenses4 ,in selecting objectives the best test objects to employ are3 #a4 ,a thin 7one cell layer71 even #a1 #b_/c1 #a_/b3 blood film1 stained with ,jenner's for #a_/f1 #a_/h or ,romanowsky's stain4 #a_/ab oil #b4 ,a thin cover-slip preparation of a young cultivation of #a_/h dry ,b4 diphtheriae 7showing for segmentation7 stained with #a_/ab oil methylene-blue4 ,accessories4--,eye ,shade 7,fig4 #di74--,this piece of apparatus consists of a pear-shaped piece of blackened metal or ebonite1 hinged to a collar which rotates on the upper part of the body tube of the microscope4 ,it can be used to shut out the image of surrounding objects from the unoccupied eye1 and when carrying out prolonged observations will be found of real service4 ,nosepiece4--,perhaps the most useful accessory is a nosepiece to carry two of the objectives 7,fig4 #ej71 or1 better still1 all three 7,fig4 #ea74 ,this nosepiece1 preferably constructed of aluminium1 must be of the covered-in type1 consisting of a curved plate attached to the lower end of the body tube--a circular aperture being cut to correspond to the lumen of that tube4 ,to the under surface of this plate is pivoted a similarly curved plate1 fitted with three tubulures1 each of which carries an objective4 ,by rotating the lower plate each of the objectives can be brought successively in to the optical axis of the microscope4 ,illustration3 ,,fig4 #di4--;,eye shade4 ,for critical work and particularly for photo-micrography1 however1 the interchangeable nosepiece is by no means perfect as it is next to impossible to secure accurate centreing of each lens in the optical axis4 ,for special purposes1 therefore1 it is necessary to employ a special nosepiece such as that made by ,zeiss or ,leitz into which each objective slides on its own carrier and upon which it is accurately centred4 ,illustration3 ,,fig4 #ej4--;,double nosepiece4 ,illustration3 ,,fig4 #ea4--,triple nosepiece4 ,warm ,stage 7,fig4 #eb74--,this is a flat metal case containing a system of tubes through the interior of which water of any required temperature can be circulated4 ,it is made to clamp on to the stage of the microscope by the screws ,a ,a'1 and is perforated with a large hole coinciding with the optical axis of the microscope2 a short tube ,b1 projecting from one end of the warm stage permits water of the desired temperature to be conducted from a reservoir through a length of rubber tubing to the interior of the stage and a similar tube at the other end ,b' of the stage allows exit to the waste water4 ,by raising the temperature of hanging-drop preparations1 etc41 placed upon it1 above that of the surrounding atmosphere1 the warm stage renders possible exact observations on spore germination1 hanging-drop cultivations1 etc4 ,illustration3 ,,fig4 #eb4--,warm stage4 ,a better form is the electrical hot stage designed by ,lorrain ,smith2#b it requires the addition of a lamp resistance and sliding rheostat1 also a delicate ammeter reading to 4#ja of an ampere4 ,it consists of a wooden frame supporting a flat glass bulb with a long neck bent upward at an obtuse angle 7,fig4 #ec74 ,the bulb is filled with liquid paraffin1 which rises in the open neck when expanded by heat4 ,the neck also accommodates the thermometer4 ,two coils of manganin wire run in the paraffin at opposite sides of the bulb 7outside the field of vision71 coupled to brass terminals on the wooden frame by platinum wire fused into the glass4 ,the resistance of the two coils in series is about #aj ohms4 ,a current of #b-a_/b amperes is needed1 and is conducted to the coils in the stage through the rheostat4 ,with the help of the ammeter any desired temperature can be obtained and maintained1 up to about #bjj deg4 ,c4 ,if immersion oil contact is made between the top lens of the condenser and the lower surface of the bulb1 this stage works very well indeed with the #a_/ab-;inch oil immersion lens4 ,illustration3 ,,fig4 #ec4--,lorrain ,smith's warm stage4 ,dark ,ground or ,paraboloid ,condenser4--,this is an immersion substage condenser of high aperture by means of which unstained objects such as bacteria can be shown as bright white particles upon a dense black background4 ,the central rays of light are blocked out by means of an opaque stop while the peripheral rays are reflected from the paraboloidal sides of the condenser and refracted by the object viewed4 ,to obtain the best results with this type of condenser a powerful illuminant--such as a small arc lamp or an incandescent gas lamp--is needed1 together with picked slides of a certain thickness 7specified for the particular make of condenser but generally #a mm47 and specially thin cover-glasses 7not more than #j4ag mm47 ,the objective must not have a higher ,,na than #a4j1 consequently immersion lenses must be fitted with an internal stop to cut down the aperture4 ,micrometer4--,some form of micrometer for the purpose of measuring bacteria and other objects is also essential4 ,details of those in general use will be found in the following pages4 ,illustration3 ,,fig4 #ed--;,diamond ,object marker4 ,object ,marker 7,fig4 #ed74--,this is an exceedingly useful piece of apparatus4 ,made in the form of an objective1 the lenses are replaced by a diamond point1 set slightly out of the centre1 which can be rotated by means of a milled plate4 ,screwed on to the nosepiece in place of the objective1 rotation of the diamond point will rule a small circle on the object slide to permanently record the position of an interesting portion of the specimen4 ,the diamond is mounted on a spring which regulates the pressure1 and the size of the circle can be adjusted by means of a lateral screw4 ,,methods ,,of ,,micrometry4 ,the unit of length as applied to the measurement of microscopical objects is the one-thousandth part of a millimetre 7#j4jja mm471 denominated a micron 7sometimes1 and erroneously1 referred to as a micro-millimetre71 and indicated in writing by the ,greek letter mu4 ,of the many methods in use for the measurement of bacteria1 three only will be here described1 viz43 7a7 ,by means of the ,camera ,lucida4 7b7 ,by means of the ocular or ,eyepiece ,micrometer4 7c7 ,by means of the ,filar ,micrometer 7,ramsden's micrometer eyepiece74 ,for each of these methods a stage micrometer is necessary4 ,this is a #c by #a inch glass slip having engraved on it a scale divided to hundredths of a millimetre 7#j4ja mm471 every tenth line being made longer than the intervening ones1 to facilitate counting2 and from these engraved lines the measurement in every case is evaluated4 ,a cover-glass is cemented over the scale to protect it from injury4 ,illustration3 ,,fig4 #ee4--;,camera lucida1 ,abbe pattern4 7a7 ,by means of the ,camera ,lucida4 #a4 ,attach a camera lucida 7of the ,wollaston1 ,beale1 or ,abbe pattern7 7,fig4 #ee7 to the eyepiece of the microscope4 #b4 ,adjust the micrometer on the stage of the microscope and accurately focus the divisions4 #c4 ,project the scale of the stage micrometer on to a piece of paper and with pen or pencil sketch in the magnified image1 each division of which corresponds to #aj mu4 ,mark on the paper the optical combination 7ocular objective and tube length7 employed to produce this particular magnification4 #d4 ,repeat this procedure for each of the possible combinations of oculars and objectives fitted to the microscope supplied1 and carefully preserve the scales thus obtained4 ,to measure an object by this method simply project the image on to the scale corresponding to the particular optical combination in use at the moment4 ,read off the number of divisions it occupies and express them as micra4 ,in place of preserving a scale for each optical combination1 the object to be measured and the micrometer scale may be projected and sketched1 in turn1 on the same piece of paper1 taking particular care that the centre of the eyepiece is #be cm4 from the paper on which the divisions are drawn4 ,illustration3 ,,fig4 #ef4--;,eyepiece micrometer1 ordinary4 ,illustration3 ,,fig4 #eg4--;,eyepiece micrometer1 net4 7b7 ,by means of the ,eyepiece ,micrometer4 ,the eyepiece micrometer is a circular glass disc having engraved on it a scale divided to tenths of a millimetre 7#j4a mm47 7,fig4 #ef71 or the entire surface ruled in #j4a mm4 squares 7the net micrometer7 7,fig4 #eg74 ,it can be fitted inside the mount of any ocular just above the aperture of the diaphragm and must be adjusted exactly in the focus of the eye lens4 ,some makers mount the glass disc together with a circular cover-glass in such a way that when placed in position in any ,huyghenian eyepiece of their own manufacture1 the scale is exactly in focus for normal vision4 ,special eyepieces are also obtainable having a sledging adjustment to the eye lens for focussing the micrometer4 ,the value of one division of the micrometer scale must first be ascertained for each optical combination by the aid of the stage micrometer1 thus3 #a4 ,insert the eyepiece micrometer inside the ocular and adjust the stage micrometer on the stage of the microscope4 #b4 ,focus the scale of the stage micrometer accurately2 the lines will appear to be immediately below those of the eyepiece micrometer4 ,make the lines on the two micrometers parallel by rotating the ocular4 #c4 ,make two of the lines on the ocular micrometer coincide with those bounding one division of the stage micrometer2 this is effected by increasing or diminishing the tube length2 and note the number of included divisions4 #d4 ,calculate the value of each division of the eyepiece micrometer in terms of mu1 by means of the following formula3 x #aj y4 ,where x the number of included divisions of the eyepiece micrometer4 y the number of included divisions of the stage micrometer4 #e4 ,note the optical combination employed in this experiment and record it with the calculated micrometer value4 ,repeat this process for each of the other combinations4 ,carefully record the results4 ,to measure an object by this method read off the number of divisions of the eyepiece micrometer it occupies and express the result in micra by a reference to the standard value for the particular optical combination employed4 ,zeiss prepares a compensating eyepiece micrometer for use with his apochromatic objectives1 the divisions of which are so computed that 7with a tube length of #afj mm47 the value of each is equivalent to as many micra as there are millimetres in the focal length of the objective employed4 ,wright's ,eikonometer is really a modification of the eyepiece micrometer for rapidly measuring microscopical objects by direct inspection1 having previously determined the magnifying power of the particular optical combination employed4 ,it is a small piece of apparatus resembling an eyepiece1 with a sliding eye lens1 which can be accurately focussed on a micrometer scale fixed within the instrument4 ,when placed over the microscope ocular the divisions of this scale measure the actual size of the virtual image in millimetres4 ,in order to use this instrument for direct measurement1 it is first necessary to determine the magnifying power of each combination of ocular1 tube length and objective4 ,place a stage micrometer divided into hundredths of a millimetre on the microscope stage and focus accurately4 ,rest the eikonometer on the eyepiece4 ,observation through the eikonometer shows its micrometer scale superposed on the image of the stage micrometer4 ,rotate the eikonometer until the lines on the two scales are parallel1 and make the various adjustments to ensure that two lines on the eikonometer scale coincide with two lines on the stage micrometer4 ,for the sake of illustration it may be assumed that five of the divisions on the stage micrometer accurately fill one of the divisions of the eikonometer scale2 this indicates a magnifying power of #ejj as the constant for that particular optical combination1 and a record should be made of the fact4 ,the magnification constants of the various other optical combinations should be similarly made and recorded4 ,to measure any object subsequently it should be first focussed carefully in the ordinary way4 ,the eikonometer should then be applied to the eyepiece and the size of the object read off on the eikonometer scale as millimetres1 and the actual size calculated by dividing the observed size by the magnification constant for the particular optical combination employed in the observation4 7c7 ,by means of the filar micrometer4 ,illustration3 ,,fig4 #eh4--,ramsden's ,filar micrometer4 ,illustration3 ,,fig4 #ei4--,ramsden's micrometer field1 a1 fixed wire2 b1 reference wire 7fixed72 c1 travelling wire4 ,the ,filar or cobweb ,micrometer 7,ramsden's micrometer7 eyepiece 7,fig4 #eh7 consists of an ocular having a fine fixed wire stretching horizontally across the field 7,fig4 #ei71 a vertical reference wire--fixed--adjusted at right angles to the first2 and a fine wire1 parallel to the reference wire1 which can be moved across the field by the action of a micrometer screw2 the drum head is divided into one hundred parts1 which successively pass a fixed index as the head is turned4 ,in the lower part of the field is a comb with the intervals between its teeth corresponding to one complete revolution of this screw-head4 ,as in the previous method1 the value of each division of the micrometer scale 7i4 e41 the comb7 must first be determined for each optical combination4 ,this is effected as follows3 #a4 ,place the filar micrometer and the stage micrometer in their respective positions4 #b4 ,rotate the screw of the filar micrometer until the movable wire coincides with the fixed one1 and the index marks zero on the drum head4 7,if when the drum head is at zero the two wires do not exactly coincide they must be adjusted by loosening the drum screw and resetting the drum47 #c4 ,focus the scale of each micrometer accurately1 and make the lines on them parallel4 #d4 ,rotate the head of the micrometer screw until the movable line has transversed one division of the stage micrometer4 ,note the number of complete revolutions 7by means of the recording comb7 and the fractions of a revolution 7by means of scale on the head of the micrometer screw71 which are required to measure the #j4ja mm4 #e4 ,make several such estimations and average the results4 #f4 ,note the optical combination employed in this experiment and record it carefully1 together with the micrometer value in terms of mu4 #g4 ,repeat this process for each of the different optical combinations and record the results4 ,to measure an object by this method1 simply note the number of revolutions and fractions of a revolution of the screw-head required to traverse such object from edge to edge1 and express the result as micra by reference to the recorded values for that particular optical combination4 ,microscope ,illuminant4--,in tropical and subtropical regions diffuse daylight is the best illuminant4 ,in temperate climes however daylight of the desirable quantity is not always available1 and recourse must be had to oil lamps1 gas lamps--preferably those with incandescent mantles--and electricity2 and of these the last is undoubtedly the best4 ,a handy lamp holder which can be manufactured in the laboratory is shown in ,fig4 #fj4 ,it consists of a base board weighted with lead to which is attached the ordinary domestic lamp holder1 and behind this is fastened a curved sheet-iron reflector4 ,an obscured metal filament lamp of about #af candle power gives the most suitable light1 and if monochromatic light is needed1 the blue grease pencil is streaked over the side of the lamp nearest the microscope2 the current is switched on and when the glass bulb is warm1 rubbing with a wad of cotton-wool will readily distribute the blue greasy material in an even film over the ground glass4 ,illustration3 ,,fig4 #fj4--;,electric microscope lamp4 ,,footnotes3 #a ,its importance will be realised1 however1 when it is stated in the words of the late ,professor ,abbe3 ,the numerical aperture of a lens determines all its essential qualities2 the brightness of the image increases with a given magnification and other things being equal1 as the square of the aperture2 the resolving and defining powers are directly related to it1 the focal depth of differentiation of depths varies inversely as the aperture1 and so forth4 #b ,made by ,mr4 ,otto ,baumbach1 #aj1 ,lime ,grove1 ,manchester4 ,v4 ,,microscopical ,,examination ,,of ,,bacteria ,,and ,,other ,,micro-,,fungi4 ,,apparatus ,,and ,,reagents ,,used ,,in ,,ordinary ,,microscopical ,,examination4 ,the following comprises the essential apparatus and reagents for routine work with which each student should be provided4 #a4 ,india-rubber change-mat upon which cover-glasses may be rested during the process of staining4 #b4 ,squares of blotting paper about #aj cm41 for drying cover-slips and slides4 7,the filter paper known as ,german lined--a highly absorbent1 closely woven paper1 having an even surface and no loose fluff to adhere to the specimens--is the most useful for this purpose47 ,illustration3 ,,fig4 #fa4--;,disinfectant ,jar4 #c4 ,glass jar filled with #b per cent4 lysol solution for the reception of infected cover-glasses and infected pipettes1 etc4 #d4 ,a square glazed earthenware box with a loose lining containing #b per cent4 lysol solution for the reception of infected material and used slides4 ,the bottom of the lining is perforated so that when full the lining and its contents can be lifted bodily out of the box1 when the disinfectant solution drains away and the slides1 etc41 can easily be emptied out4 ,the empty lining is then returned to the box with its disinfectant solution 7,fig4 #fa74 #e4 ,bunsen burner provided with peep-flame by-pass4 #f4 ,porcelain trough holding five or six hanging-drop slides 7,fig4 #fb74 ,illustration3 ,,fig4 #fb4--;,hanging-drop slides3 a1 ,double cell seen from above2 b1 single cell seen from the side4 ,the best form of hanging-drop slide is a modification of ,boettcher's glass ring slide1 and is prepared by cementing a circular cell of tin1 #ac to #ae mm4 diameter1 and #a to #b mm4 in height1 to the centre of a #c by #a slip by means of ,canada balsam4 ,it is often extremely convenient to have two of these cells cemented close together on one slide 7,fig4 #fb1 a74 ,another form of hanging-drop slide is made in which a circular or oval concavity or cell is ground out of the centre of a #c by #a slip4 ,these are more expensive1 less convenient to work with1 and are more easily contaminated by drops of material under examination1 and should be carefully avoided4 #g4 ,three aluminium rods 7,fig4 #fc71 each about #be cm4 long and carrying a piece of #j4jae gauge platino-iridium wire #g4e cm4 in length4 ,the end of one of the wires is bent round to form an oval loop1 of about #a mm4 in its short diameter1 and is termed a loop or an oese2 the terminal #c or #d mm4 of another wire is flattened out by hammering it on a smooth iron surface to form a spatula2 the third is left untouched or is pointed by the aid of a file4 ,these instruments are used for inoculating culture tubes and preparing specimens for microscopical examination4 ,illustration3 ,,fig4 #fc4--;,ends of platinum rods4 a1 loop2 b1 spatula2 c1 needle4 ,the method of mounting these wires may be described as follows3 ,take a piece of aluminium wire #be cm4 long and about #j4be cm4 in diameter1 and drill a fine hole completely through the wire about a centimetre from one end4 ,sink a straight narrow channel along one side of the wire1 in its long axis1 from the hole to the nearest end1 shallow at first1 but gradually becoming deeper4 ,on the opposite side of the wire make a short cut1 #b mm4 in length1 leading from the hole in the same direction4 ,the use of a fine dental drill and small circular saw1 worked by a dental motor facilitates the manufacture of these aluminium handled instruments4 ,now pass one end of the platinum wire through the hole1 turn up about #b mm4 at right angles and press the short piece into the short cut4 ,turn the long end of the wire sharply1 also at right angles1 and sink it into the long channel so that it emerges from about the centre of the cut end of the aluminium wire 7,fig4 #fc74 ,a few sharp taps with a watch maker's hammer will now close in the sides of the two channels over the wire and hold it securely4 ,illustration3 ,,fig4 #fd4--,platinum rod in aluminium handle--method of mounting4 ,the platinum wire may be fused into the end of a piece of glass rod1 but such a handle is vastly inferior to aluminium and is not to be recommended4 #h4 ,two pairs of sharp-pointed spring forceps 7#aj cm4 long71 one of which must be kept perfectly clean and reserved for handling clean cover-slips1 the other being for use during staining operations4 #i4 ,a box of clean #c by #a glass slips4 #aj4 ,a glass capsule with tightly fitting 7ground on7 glass lid1 containing clean cover-slips in absolute alcohol4 #aa4 ,one of ,faber's grease pencils 7yellow1 red1 or blue7 for writing on glass4 #ab4 ,a wooden rack 7,fig4 #fe7 with twelve drop-bottles 7,fig4 #ff7 each #fj c4c4 capacity1 containing ,aniline water4 ,gentian violet1 saturated alcoholic solution4 ,lugol's 7,gram's7 iodine4 ,absolute alcohol4 ,methylene-blue1 ,fuchsin1 basic1 saturated alcoholic solution4 ,neutral red1 #a per cent4 aqueous solution4 ,leishman's modified ,romanowsky stain4 ,carbolic acid1 #e per cent4 aqueous solution4 ,acetic acid1 #a per cent4 solution4 ,sulphuric acid1 #be per cent4 solution4 ,xylol4 ,illustration3 ,,fig4 #fe4--,staining rack1 rubber change mat and lysol pot4 ,illustration3 ,,fig4 #ff4--;,drop bottle4 ,illustration3 ,,fig4 #fg4--;,canada balsam pot4 ,and two pots with air-tight glass caps 7,fig4 #fg71 each provided with a piece of glass rod and filled respectively with ,canada balsam dissolved in xylol1 and sterile vaseline4 ,,methods ,,of ,,examination4 ,bacteria1 etc41 are examined microscopically4 #a4 ,in the living state1 unstained1 or stained4 #b4 ,in the fixed condition 7i4 e41 fixed1 killed1 and stained by suitable methods74 ,the preparation of a specimen from a tube cultivation for examination by these methods may be described as follows3 #a4 ,living1 ,unstained4--7a7 ,fresh ,preparation4-- #a4 ,clean and dry a #c by #a glass slip and place it on one of the squares of filter paper4 ,deposit a drop of water 7preferably distilled7 or a drop of #a per cent4 solution of caustic potash1 on the centre of the slip1 by means of the platinum loop4 ,illustration3 ,,fig4 #fh4--;,holding tubes for removing bacterial growth1 as seen from the front4 ,,technique ,,of ,,opening ,,and ,,closing ,a ,,culture ,,tube4 #b4 ,remove the tube cultivation from its rack or jar with the left hand and ignite the cotton-wool plug by holding it to the flame of the ,bunsen burner4 ,extinguish the flame by blowing on the plug1 whilst rotating the tube on its long axis1 its mouth directed vertically upward1 between the thumb and fingers4 7,this operation is termed flaming the plug1 and is intended to destroy any micro-organisms that may have become entangled in the loose fibres of the cotton-wool1 and which1 if not thus destroyed1 might fall into the tube when the plug is removed and so accidentally contaminate the cultivation47 #c4 ,hold the tube at or near its centre between the ends of the thumb and first two fingers of the left hand1 and allow the sealed end to rest upon the back of the hand between the thumb and forefinger1 the plug pointing to the right4 ,keep the tube as nearly in the horizontal position as is consistent with safety1 to diminish the risk of the accidental entry of organisms 7,fig4 #fh74 #d4 ,take the handle of the loop between the thumb and forefinger of the right hand1 holding the instrument in a position similar to that occupied by a pen or a paint-brush1 and sterilise the platinum portion by holding it in the flame of a ,bunsen burner until it is red hot4 ,sterilise the adjacent portion of the aluminium handle by passing it rapidly twice or thrice through the flame4 ,after sterilising it1 the loop must not be allowed to leave the hand or to touch against anything but the material it is intended to examine1 until it is finished with and has been again sterilised4 #e4 ,grasp the cotton-wool plug of the test-tube between the little finger and the palm of the right hand 7whilst still holding the loop as directed in step #d71 and remove it from the mouth of the tube by a screwing motion of the right hand4 #f4 ,introduce the platinum loop into the tube and hold it in this position until satisfied that it is quite cool4 7,the cooling may be hastened by touching the loop on one of the drops of moisture which are usually to be found condensed on the interior of the glass tube1 or by dipping it into the condensation water at the bottom2 at the same time care must be taken in the case of cultures on solid media to avoid touching either the medium or the growth47 #g4 ,remove a small portion of the growth by taking up a drop of liquid1 in the case of a fluid culture1 in the loop2 or by touching the loop on the surface of the growth when the culture is on solid medium2 and withdraw the loop from the tube without again touching the medium or the glass sides of the tube4 #h4 ,replace the cotton-wool plug in the mouth of the tube4 #i4 ,replace the tube cultivation in its rack or jar4 #aj4 ,mix the contents of the loop thoroughly with the drop of water on the #c by #a slide4 #aa4 ,again sterilise the loop as directed in step #d1 and replace it in its stand4 #ab4 ,remove a cover-slip from the glass capsule by means of the cover-slip forceps1 rest it for a moment on its edge1 on a piece of filter paper to remove the excess of alcohol1 then pass it through the flame of the ,bunsen burner4 ,this burns off the remainder of the alcohol1 and the cover-slip so flamed is now clean1 dry1 and sterile4 #ac4 ,lower the cover-slip1 still held in the forceps1 on to the surface of the drop of fluid on the #c by #a slip1 carefully and gently1 to avoid the inclusion of air bubbles4 #ad4 ,examine microscopically 7vide infra74 ,during the microscopical examination1 stains and other reagents may be run in under a cover-slip by the simple method of placing a drop of the reagent in contact with one edge of the cover-glass and applying the torn edge of a piece of blotting paper to the opposite side4 ,the reagent may then be observed to flow across the field and come into contact with such of the micro-organisms as lie in its path4 ,the non-toxic basic dyes most generally employed for the intra-vitam staining of bacteria are ,neutral red1 ,quinoleine blue ,methylene green in #j4e per cent4 aqueous solutions4 ,vesuvin1 ,negative ,stain 7,burri74--,by this method of demonstration the appearances presented by dark ground illumination 7by means of a paraboloid condenser7 are closely simulated1 since minute particles1 bacteria1 blood or pus cells etc4 stand out as brilliantly white or colourless bodies on a dark grey-brown background4 ,reagent required3 ,any one of the liquid waterproof black drawing inks 7,chin-chin1 ,pelican1 etc474 ,this is prepared for use as follows3 ,measure out and mix3 ,liquid black ink1 #be c4c4 ,tincture of iodine #a c4c4 ,allow the mixture to stand #bd hours1 centrifugalise thoroughly1 pipette off the supernatant liquid to a clean bottle and then add a crystal of thymol or one drop of formalin as a preservative4 ,,method4-- #a4 ,with the sterilised loop deposit one drop of the liquid ink close to one end of a #c by #a slide4 #b4 ,with the sterilised loop deposit a drop of the fluid culture 7or of an emulsion from a solid culture7 by the side of the drop of ink 7,fig4 #fi1 a72 mix the two drops thoroughly by the aid of the loop4 #c4 ,sterilise the loop4 #d4 ,hold the slide firmly on the bench with the thumb and forefinger of the left hand applied to the end nearest the drop of fluid4 #e4 ,take another clean #c by #a slide in the right hand and lower its short end obliquely 7at an angle of about #fj deg47 transversely on to the mixed ink and culture on the first slide1 and allow the fluid to spread across the slide and fill the angle of incidence4 #f4 ,maintaining the original angle1 draw the second slide firmly and evenly along the first toward the end farthest from the left hand 7,fig4 #fi1 b74 #g4 ,throw the second slide into a pot of disinfectant2 allow the first slide to dry in the air4 ,illustration3 ,,fig4 #fi4--,spreading negative film4 #h4 ,place a drop of immersion oil on the centre of the film1 lower the #a_/ab-;inch objective into the oil and examine microscopically without the intervention of a cover-slip4 7,the film of ink may be covered with a long cover-glass and xylol balsam as a permanent preparation47 7b7 ,hanging-drop ,preparation4-- #a4 ,smear a layer of sterile vaseline on the upper surface of the ring cell of a hanging-drop slide by means of the glass rod provided with the vaseline bottle1 and place the slide on a piece of filter paper4 #b4 ,flame a cover-slip and place it on the filter paper by the side of the hanging-drop slide4 #c4 ,place a drop of water on the centre of the cover-slip by means of the platinum loop4 #d4 ,obtain a small quantity of the material it is desired to examine1 in the manner detailed above 7pages #gd-gf1 steps #b to #aa must be followed in their entirety and with the strictest exactitude whenever tube contents are being handled71 and mix it with the drop of water on the cover-slip4 #e4 ,raise the cover-slip in the points of the forceps and rapidly invert it on to the ring cell of the hanging-drop slide1 so that the drop of fluid occupies the centre of the ring4 7,carefully avoid contact between the drop of fluid and either the ring cell or the layer of vaseline4 ,should this happen1 the now infected hanging-drop slide and its cover-slip must be dropped into the pot of lysol and a new preparation made47 #f4 ,press the cover-slip firmly down into the vaseline on to the top of the ring cell4 7,this spreads out the vaseline into a thin layer1 and besides ensuring the adhesion of the cover-slip1 seals the cells and so retards evaporation47 #g4 ,examine microscopically4 ,the examination of a fresh specimen or a hanging-drop preparation is directed to the determination of the following data3 #a4 ,the nature of the bacteria present--e4 g41 cocci1 bacilli1 etc4 #b4 ,the purity of the cultivation2 this can only be determined when gross morphological differences exist between the organisms present4 #c4 ,the presence or absence of spores2 when present1 spores show their typical refrangibility exceedingly well by this method4 #d4 ,the presence or absence of mobility4 ,in a hanging-drop specimen some form of movement can practically always be observed1 and its character must be carefully determined by noting the relative positions of adjacent micro-organisms4 7a7 ,brownian or molecular movement4 ,minute particles of solid matter 7including bacteria71 when suspended in a fluid1 will always show a vibratory movement affecting the entire field1 but never altering the relative positions of the bacteria4 7,cocci exhibit this movement1 but with the exception of the ,micrococcus agilis1 the cocci are non-motile47 7b7 ,streaming movement4 ,this is due to currents set up in the hanging drop as a result of jarring of the specimen or of evaporation1 or to the fact that the cover-slip is not perfectly level1 and although the relative positions of the bacteria may vary1 still the flowing movement of large numbers of organisms in some one direction will usually be sufficient to demonstrate the nature of this motion4 7c7 ,locomotive movement1 or true motility1 is determined by observing some one particular bacillus changing its position in the field independently of1 and in a direction contrary to1 other organisms present4 ,when the examination is completed and the specimen finished with1 the fresh specimen--i4 e41 the slide with the cover-slip attached--must be dropped into the lysol pot4 ,in the hanging-drop specimen1 however1 the cover-slip only is infected1 and this may be raised from the ring cell by means of forceps and dropped into the disinfectant4 ,permanent ,staining of the ,hanging-drop ,specimen4--,occasionally it is necessary to fix and stain a hanging-drop preparation4 ,this may be done as follows3 #a4 ,remove the cover-slip from the cell by the aid of the forceps4 #b4 ,if the drop is small1 fix it by dropping it face downward1 whilst still wet1 on to the surface of some ,gulland's solution or corrosive sublimate solution 7vide page #hb7 in a watch-glass4 ,if the drop is large1 place it face upward on the rubber mat1 cover it with an inverted watch-glass1 and allow it to dry4 ,then fix it in the alcohol and ether solution 7vide1 page #hb74 #c4 ,dip the cover-glass into a beaker containing hot water in order to remove some of the vaseline adhering to it4 #d4 ,wash successively in alcohol1 xylol1 ether1 and alcohol1 to remove the last traces of grease4 #e4 ,wash in water4 #f4 ,stain1 wash1 dry1 and mount as for an ordinary cover-slip film preparation 7vide pages #hc-he74 #b4 ,killed1 ,stained4--,in this method three distinct processes are necessary3 ,preparing and fixing the film4 ,staining4 ,mounting4 ,preparing the ,film4-- #a4 ,flame a cover-slip and place it on a piece of filter paper4 #b4 ,place a drop of water on the centre of the cover-slip by means of platinum loop4 #c4 ,obtain a small quantity of the material to be examined upon a sterilised platinum loop 7see pages #gd-gf1 steps #b to #aa7 and mix it with the drops of water on the cover-slip4 #d4 ,spread the drop of emulsion evenly over the cover-slip in the form of a square film to within #a mm4 of each edge of the cover-slip4 #e4 ,allow it to dry completely in the air4 ,fixing4--,fix by passing the cover-slip1 held in the fingers1 three or four times through the flame of a ,bunsen burner4 ,in some instances 7e4 g41 when the films after staining are intended for micrometric observations7 it is almost essential to fix by exposure to a uniform temperature of #aae deg4 ,c41 for twenty minutes4 ,this is best done in a carefully regulated hot-air oven4 ,fixation may also be effected by immersing in some fixative fluid1 such as one of the following3 #a4 ,absolute alcohol1 for five to fifteen minutes4 equal parts1 for five to thirty #b4 ,absolute alcohol1 minutes 7e4 g41 for blood or ,ether1 milk74 #c4 ,osmic acid1 #a per cent4 aqueous solution1 for thirty seconds4 #d4 ,corrosive sublimate1 saturated aqueous solution1 for five minutes4 #e4 ,corrosive sublimate 7,lang71 for five minutes4 ,this solution is prepared by dissolving3 ,sodium chloride #j4ge gramme ,hydrarg4 perchloride #ab4jj grammes ,acetic acid #e4jj grammes ,in distilled water #ajj4jj c4c4 ,filter4 #f4 ,gulland's solution1 for five minutes4 ,this solution is prepared by mixing3 ,absolute alcohol #be4j c4c4 ,ether #be4j c4c4 ,corrosive sublimate1 #bj per cent4 alcoholic solution #j4d c4c4 #g4 ,formalin #aj per cent4 aqueous solution 7 #d per cent4 aqueous solution of formaldehyde since formalin is a #dj per cent4 solution of the gas in water74 ,either of these methods of fixation coagulates the albuminous material and ensures perfect adhesion of the film to the cover-slip4 ,clearing4--,wash the cover-slip thoroughly in running water and proceed with the staining4 ,if the film has been prepared from broth1 liquefied gelatine1 or pus or other morbid exudations1 saturate the film after fixation with acetic acid #b per cent4 and allow it to act for two minutes4 ,wash with alcohol1 then let the alcohol remain on the cover-slip for two minutes4 7,this will clear the groundwork and give a much sharper and cleaner film than would otherwise be obtained47 ,if the film has been prepared from blood or bloodstained fluid1 treat with acetic acid #b per cent4 for two minutes after fixation4 ,wash with water1 dry1 and proceed with the staining4 7,this will remove the haemoglobin and facilitate examination47 ,staining4-- #a4 ,rest the cover-slip1 film side uppermost1 on the rubber mat4 #b4 ,by means of a drop-bottle1 cover the film side of the cover-slip with the selected stain1 allow it to act for a few minutes1 then wash off the excess in running water4 ,the penetrating power of stains is increased by 7a7 physical means--e4 g41 heating the stain2 7b7 chemical means--e4 g41 by the addition of carbolic acid1 #e per cent4 aqueous solution2 caustic alkalies1 #b per cent4 aqueous solutions2 water saturated with aniline oil2 borax1 #j4e per cent4 aqueous solution4 ,the most commonly used dyes for cover-slip film preparations are the aniline dyes4 7,a7 ,basic3 7a7 ,methylene-blue4 7b7 ,gentian violet4 7c7 ,fuchsin4 ,these dyes are kept in saturated alcoholic 7#ij per cent47 solutions so that decomposition may be retarded4 ,two or three drops of alcoholic solution of these dyes to1 say1 #d c4c4 water1 usually makes a sufficiently strong staining fluid for cover-slip film preparations4 ,carbolic methylene-blue 7,c4,m4,b47 and carbol fuchsin 7,c4,f47 are prepared by covering the cover-slip with #e per cent4 solution of carbolic acid and adding a few drops of the saturated alcoholic solution of methylene-blue or fuchsin respectively to it4 ,for aniline gentian violet 7,a4,g4,v47 the stain is added to a saturated solution of aniline oil in water4 7d7 ,thionine blue4 7e7 ,bismarck brown4 7f7 ,neutral red4 7,b7 ,acid3 7a7 ,eosin1 aqueous yellowish4 7b7 ,safranine4 ,these dyes are kept in #a per cent4 aqueous solution to which is added #e per cent4 of alcohol1 as a preservative4 ,they are generally used in this form4 ,a few nuclear stains 7carmine1 haematoxylin7 are occasionally used more especially in section work4 ,decolourisation4--,after overstaining1 films may be decolourised by washing for a longer or shorter time in one of the following reagents arranged in ascending order of power #a4 ,water4 #b4 ,chloroform4 #c4 ,acetic acid1 #a per cent4 #d4 ,alcohol4 #e4 ,alcohol absolute1 equal parts4 ,acetic acid1 #a per cent41 ,hydrochloric1 #a per cent4 aqueous solution4 ,hydrochloric1 #a per cent4 ,alcoholic 7#ij per cent47 solution4 #f4 ,mineral acids3 ,sulphuric1 #be per cent4 aqueous solution4 ,nitric1 #cc per cent4 aqueous solution4 ,counterstaining4--,use colours which will contrast with the first stain2 e4 g41 ,vesuvin1 ,neutral red1 for films stained by methylene-blue or ,eosin1 ,gram's method4 ,fuchsin1 ,methylene-blue1 for films stained by fuchsin4 ,gentian violet1 #h4 ,mounting4-- #a4 ,wash the film carefully in running water4 #b4 ,blot off the superfluous water with the filter paper1 or dry more completely between two folds of blotting paper4 #c4 ,complete the drying in the air1 or by holding the cover-slip in the fingers at a safe distance above the flame of the ,bunsen burner4 #d4 ,place a drop of xylol balsam on the centre of a clean #c by #a glass slide and invert the cover-slip over the balsam1 and lower it carefully to avoid the inclusion of air bubbles4 ,,note4--,xylol is used in preference to chloroform to dissolve ,canada balsam1 as it does not decolourise the specimen4 ,impression films 7,klatschpraeparat7 are prepared from isolated colonies of bacteria in order that their characteristic formation may be examined by higher powers than can be brought to bear on the living cultivation4 ,they are prepared from plate cultivations 7vide page #bcj7 in the following manner4 #a4 ,remove a clean cover-slip from the alcohol pot with sterile forceps and burn off the spirit4 #b4 ,open the plate and rest one edge of the cover-slip on the surface of the medium a little to one side of the selected colony4 ,lower it cautiously over the colony until horizontal4 ,avoid any lateral movement or the inclusion of bubbles of air4 #c4 ,make gentle vertical pressure on the centre of the cover-slip with the points of the forceps to ensure perfect contact with the colony4 #d4 ,steady one edge of the cover-slip with the forceps and pass the point of a mounted needle just under the opposite edge and raise the cover-slip carefully2 the colony will be adherent to it4 ,when nearly vertical1 grasp the cover-slip with the forceps and remove it from the plate4 ,re-cover the plate4 #e4 ,place the cover-slip1 film uppermost1 on the rubber mat1 and cover it with an inverted watch-glass until dry4 #f4 ,fix by immersing in one of the fixing fluids previously mentioned 7vide page #hb74 #g4 ,clear with acetic acid and alcohol4 #h4 ,stain and mount as an ordinary cover-slip film preparation1 being careful to perform all washing operations with extreme gentleness4 ,microscopical ,examination of the ,unstained ,specimens4-- #a4 ,place the body tube of the microscope in the vertical position4 #b4 ,arrange the hanging-drop slide on the microscope stage so that the drop of fluid is in the optical axis of the instrument1 and secure it in that position by means of the spring clips4 #c4 ,use the #a_/f-;inch objective1 rack down the body tube until the front lens of the objective is almost in contact with the cover-slip--that is1 well within its focal distance4 ,this is best done whilst bending down the head to one side of the microscope1 so that the eyes are on a level with the stage4 #d4 ,apply the eye to the ocular and adjust the plane mirror to the position which secures the best illumination4 #e4 ,rack the condenser down slightly and cut down the aperture of the iris diaphragm so that the light1 although even1 is dim4 #f4 ,rack up the body tube by means of the coarse adjustment until the bacteria come into view2 then focus exactly by means of the fine adjustment4 ,some difficulty is often experienced at first in finding the hanging drop1 and if the first attempt is unsuccessful1 the student must not on any account1 whilst still applying his eye to the ocular1 rack the body tube down 7for by so doing there is every likelihood of the front lens of the objective being forced through the cover-glass1 and not only spoiling the specimen1 but also contaminating the objective72 but1 on the contrary1 withdraw his eye1 rack the tube up1 and commence again from step #b4 ,dark ,ground ,illumination4-- #a4 ,set up the microscope stand in the vertical position and insert the highest eyepiece available4 #b4 ,remove the nosepiece from the microscope tube and fit the #b_/c inch objective in place4 #c4 ,remove the substage condenser and replace it by the dark ground condenser4 #d4 ,fit up the source of illumination some #cj-ej cm4 distant from the microscope4 7,this should be the ,liliput ,arc ,lamp 7,leitz71 ,nernst ,lamp or incandescent gas lamp2 if either of the two latter are employed1 a bull's eye condenser to produce parallel rays must be interposed between light and microscope72 and adjust illuminant and microscope so that the substage plane mirror is completely filled with light4 #e4 ,focus the two concentric rings engraved upon the upper surface of the condenser and centre them accurately by means of the centring screws4 #f4 ,prepare a fresh specimen 7see pages #gd-gf7 of the material it is desired to observe1 using selected1 new1 #c by #a glass slips of less than #a mm4 thickness1 and ,no4 #a cover-glasses 7#j4ag mm4 thick71 which should be cleaned with a piece of soft washleather and not with the emery paper1 as scratches on the glass produce haziness in the preparation4 #g4 ,deposit a large drop of immersion oil 7or pure water7 on the upper surface of the condenser and rack it down a few millimetres4 #h4 ,adjust the fresh preparation on the microscope stage and fasten it in position with the stage clips4 #i4 ,rack up the condenser until the immersion fluid makes contact with the under surface of the slide2 avoid the formation of air bubbles4 #aj4 ,adjust the substage mirror so that the light is reflected upward4 ,a bright spot will be seen on the fresh preparation near the centre of the field4 #aa4 ,replace the #b_/c-;inch objective by the #a_/ab-;inch oil immersion lens which has been fitted with the special stop to reduce its ,n4 ,a42 place a drop of immersion oil upon the centre of the cover-glasses of the fresh preparation and lower the microscope tube until the front lens of the objective has entered the oil drop4 #ab4 ,focus the bright spot referred to in step #aj4 ,if it no longer occupies the centre of the field1 alter the angle of the substage mirror until it does4 #ac4 ,now focus the lens accurately on the film1 cautiously vary the height of the dark ground condenser until the best position is found4 ,the intensely illuminated bacteria will stand out in vivid contrast to the dark background4 ,illustration3 ,,fig4 #gj4--;,immersion oil bottle4 ,microscopical ,examination of the ,stained ,specimen4--7,the body tube of the microscope may be vertical or inclined to an angle47 #a4 ,secure the slide on the stage of the microscope by means of the spring clips4 #b4 ,place a drop of cedarwood oil on the centre of the cover-slip4 ,the immersion oil is pure cedarwood oil1 and is kept in a small bottle of stout glass 7,fig4 #gj71 the cavity of which is shaped like an inverted cone1 and is provided with a safety funnel 7so that the oil does not escape if the bottle is accidentally overturned7 and a dust cap of boxwood fitted with a wooden rod with which the drop of oil is applied to the cover-glass or lens4 #c4 ,use the #a_/ab-;inch oil immersion lens of the microscope4 ,rack down the body tube till the front lens of the objective is in contact with the oil and nearly touching the cover-slip4 #d4 ,rack up the condenser until it is in contact with the under surface of the slide4 #e4 ,apply the eye to the ocular and arrange the plane mirror so as to obtain the greatest possible amount of light4 #f4 ,rack up the body tube until the stained film comes into view4 #g4 ,focus the condenser accurately on the film4 #h4 ,focus the film accurately by means of the fine adjustment4 ,,vi4 ,,staining ,,methods4 ,in the following pages are collected the various stock stains in everyday use in the bacteriological laboratory1 together with a selection of the most convenient and generally useful staining methods for demonstrating particular structures or differentiating groups of bacteria4 ,the stains employed should either be those prepared by ,gruebler1 of ,leipzig1 or ,merck1 of ,darmstadt4 ,the methods printed in ordinary type are those which a long experience has shown to be the most reliable1 and to give the best results--those relegated to small type comprise such as are not so generally useful1 but give excellent results in the hands of the experienced worker4 ,,bacteria ,,stains4 ,methylene-blue4-- #a4 ,saturated ,aqueous ,solution4 ,weigh out ,methylene-blue #a4e grammes ,place in a stoppered bottle having a capacity of from #aej to #bjj c4c4 and add ,distilled water #ajj4j c4c4 ,allow the water to remain in contact with the dye for two weeks1 shaking the contents of the bottle vigourously for a few moments every day4 ,filter4 #b4 ,saturated ,alcoholic ,solution4 ,weigh out ,methylene-blue #a4e grammes ,place in a stoppered bottle of #aej c4c4 capacity and add ,alcohol1 #ij per cent #ajj4j c4c4 ,allow the alcohol to remain in contact with the dye for two hours1 shaking vigourously every few minutes4 ,filter4 #c4 ,carbolic ,methylene-blue 7,kuehne74 ,weigh out ,methylene-blue #a4e grammes ,carbolic acid #e4j grammes and dissolve in ,distilled water #ajj4j c4c4 and add ,absolute alcohol #aj4j c4c4 ,filter4 #d4 ,alkaline ,methylene-blue 7,loeffler74 ,measure out and mix ,methylene-blue1 saturated alcoholic solution #cj4j c4c4 ,caustic potash1 #j4a per cent4 aqueous solution #ajj4j c4c4 ,filter4 ,gentian ,violet4-- #e4 ,saturated ,aqueous ,solution4 ,weigh out ,gentian violet #b4be grammes and proceed as in preparing the corresponding solution of methylene-blue4 #f4 ,saturated ,alcoholic ,solution4 ,weigh out ,gentian violet #e4j grammes and proceed as in preparing the corresponding solution of methylene-blue4 #g4 ,carbolic ,gentian ,violet 7,nicolle74 ,measure out and mix ,gentian violet1 saturated alcoholic solution #aj4j c4c4 ,carbolic acid1 #a per cent4 aqueous solution #ajj4j c4c4 ,filter4 #h4 ,anilin ,water ,solution 7,koch-,ehrlich74 ,measure out ,distilled water #ajj c4c4 ,add anilin oil drop by drop 7shaking well after the addition of each drop7 until the solution is opaque4 ,filter until clear4 and add ,absolute alcohol #aj c4c4 ,saturated alcoholic solution gentian violet #aa c4c4 ,filter4 ,,note4--,this solution will not keep longer than #ad days4 ,thionine ,blue 7or ,lauth's ,violet74-- #i4 ,carbolic ,thionine ,blue 7,nicolle74 ,weigh out ,thionine blue #a4j gramme ,carbolic acid #b4e grammes and dissolve in ,distilled water #ajj4j c4c4 ,filter4 ,before use dilute with equal quantity of distilled water and again filter4 ,fuchsin 7,basic74-- #aj4 ,saturated ,aqueous ,solution4 ,weigh out ,basic fuchsin #a4e grammes and proceed as in preparing the corresponding solution of methylene-blue 7q4 v474 #aa4 ,saturated ,alcoholic ,solution4 ,weigh out ,basic fuchsin #c4e grammes and proceed as in preparing the corresponding solution of methylene-blue4 #ab4 ,carbolic ,fuchsin 7,ziehl74 ,weigh out ,basic fuchsin #a4j gramme ,carbolic acid #e4j grammes dissolve in ,distilled water #ajj4j c4c4 and add ,absolute alcohol #aj4j c4c4 ,filter4 ,,contrast ,,stains4 ,eosin4--,there are several commercial varieties of eosin1 which1 from the bacteriological point of view1 possess very different values4 ,gruebler lists four varieties1 of which two only are useful for bacteriological work3 ,eosin1 aqueous yellowish4 ,eosin1 aqueous bluish4 #ac4 ,eosin ,aqueous ,solution 7,yellowish or ,bluish ,shade71 #a per cent4 ,weigh out ,eosin1 aqueous #a4j gramme dissolve in ,distilled water #ajj4j c4c4 and add ,absolute alcohol #e4j c4c4 ,filter4 #ad4 ,eosin ,alcoholic ,solution1 #j4e per cent4 ,weigh out ,eosin1 alcoholic #j4e gramme and dissolve in ,alcohol 7#gj per cent47 #ajj4j c4c4 ,filter4 ,safranine4-- #ae4 ,aqueous ,solution4 ,weigh out4 ,safranine #j4e gramme and dissolve in ,distilled water #ajj4j c4c4 ,filter4 ,neutral ,red4-- #af4 ,aqueous ,solution4 ,weigh out ,neutral red #a4j gramme and dissolve in ,distilled water #ajj4j c4c4 ,filter4 ,vesuvin 7or ,bismarck ,brown74-- #ag4 ,saturated ,aqueous ,solution4 ,weigh out ,vesuvin #j4e gramme and dissolve in ,distilled water #ajj4j c4c4 ,filter4 ,,tissue ,,stains4 ,aniline ,gentian ,violet 7,for ,weigert's ,fibrin ,stain74-- ,weigh out ,gentian violet #a4j gramme and dissolve in ,absolute alcohol #ae4j c4c4 ,distilled water #hj4j c4c4 then add ,aniline oil #c4j c4c4 ,shake well and filter before use4 ,haematoxylin 7,ehrlich74-- #a4 ,weigh out ,haematoxylin #b4j grammes and dissolve in ,absolute alcohol #ajj4j c4c4 #b4 ,weigh out ,ammonium alum #b4j grammes and dissolve in ,distilled water #ajj4j c4c4 #c4 ,mix #a and #b1 allow the mixture to stand forty-eight hours1 then filter4 #d4 ,add ,glycerine #he4j c4c4 ,acetic acid1 glacial #aj4j c4c4 #e4 ,allow the stain to stand for one month exposed to light2 then filter again ready for use4 ,haematin 7,mayer's74-- ,a4 ,weigh out ,haematin #a4j gramme and dissolve in ,alcohol #ij per cent4 7warmed to #cg deg4 ,c47 #ej c4c4 ,b4 ,weigh out ,potash alum #ej grammes and dissolve in ,distilled water #ajj c4c4 ,prepare these two solutions in separate flasks4 ,take a clean flask of #bej c4c4 capacity and insert a large funnel in its neck4 ,pour the solutions ,a and ,b simultaneously and slowly into the funnel to mix thoroughly4 ,store for future use4 ,,note4--,if acid haematin is required1 introduce glacial acetic acid 7#c c4c47 into the mixing flask before adding the solutions ,a and ,b4 ,alum ,carmine 7,mayer74-- ,weigh out ,alum #b4e grammes ,carmine #a4j gramme and place in a glass beaker4 ,measure out in a measuring cylinder1 ,distilled water #ajj4j c4c4 ,place the beaker on a sand-bath1 add the water in successive small quantities1 and keep the mixture boiling for twenty minutes4 ,measure the solution and make up to #ajj c4c4 by the addition of distilled water4 ,filter4 ,lithium ,carmine 7,orth74-- ,weigh out ,carmine #b4e grammes and dissolve in ,lithium carbonate1 cold saturated solution #ajj4j c4c4 ,filter4 ,picrocarmine4-- ,weigh out ,picrocarmine #b4j grammes and dissolve in ,distilled water #ajj4j c4c4 ,,blood ,,stains ,when watery solutions of medicinal methylene blue and water soluble eosins are mixed a precipitate is formed which is soluble only in alcohol1 and solutions of this precipitate impart a peculiar reddish-purple colour to chromatin4 ,this compound was first used by ,romanowsky to demonstrate malarial parasites1 but various modifications are now employed for staining blood films generally1 and also for bacteria and protozoa4 ,the best modifications of the original ,romanowsky are those of ,jenner and ,leishman--,jenner being most suitable for the histological study of the blood1 and ,leishman for the demonstration of protozoa4 ,jenner's ,stain4-- ,a4 ,weigh out3 ,eosin aqueous yellow #f4j grammes ,dissolve in ,distilled water 7non-alkaline7 #bej c4c4 ,this will make a thick solution4 ,b4 ,weigh out3 ,methylene blue 7medicinally pure7 ,hoechst #e4j grammes ,dissolve in ,distilled water 7non-alkaline7 #bej c4c4 #a4 ,add ,b to ,a very slowly1 stirring all the time4 ,a viscous precipitate forms which frequently loses its viscosity when heat is applied4 7,this explains the necessity of mixing slowly74 #b4 ,evaporate slowly in a porcelain basin1 stirring occasionally1 on a water bath at #ee deg4 ,c4 ,when a paste begins to form scrape and break up occasionally4 7,on no account must the paste be allowed to fuse47 #c4 ,grind the resulting mass into an amorphous powder4 #d4 ,weigh out3 ,amorphous powder #j4e grammes ,dissolve in ,methylic alcohol 7,merck's puriss1 for analysis7 #ajj c4c4 ,allow time for true solution4 7,about three days is sufficient47 ,,method4-- #a4 ,prepare film1 dry1 but do not fix4 #b4 ,flood the unfixed film with the stain1 allow it to act for #c minutes 7the methylic alcohol of the stain fixes the film74 #c4 ,pour off the stain and wash in distilled water until the film presents a pink colour4 #d4 ,dry and mount4 ,leishman's ,stain4-- ,a4 ,weigh out3 ,methylene blue 7medicinal7 #a gramme ,dissolve in ,sodium carbonate1 #j4e per cent4 aqueous solution #ajj c4c4 ,keep at #fe deg4 ,c4 for #ab hours in either a hot incubator or a water-bath2 then stand in dark place at room temperature 7#bj deg4 ,c47 for ten days4 ,b4 ,weigh out3 ,eosin1 extra ,b4 ,a4 #j4a gramme ,dissolve in ,distilled water #ajj c4c4 #a4 ,mix the two solutions ,a and ,b in equal volumes1 and allow the mixture to stand for #ab hours with occasional stirring4 #b4 ,filter1 and collect precipitate on filter paper4 #c4 ,wash precipitate thoroughly with distilled water1 and dry4 #d4 ,weigh out #j4ae gramme of the dried precipitate2 rub up in a mortar with #e c4c4 of methylic alcohol 7,merck's puriss1 for analysis74 ,allow undissolved powder to settle1 then decant the supernatant fluid to a clean #ajj c4c4 measuring cylinder4 #e4 ,add further #e c4c4 alcohol to sediment in mortar and repeat the process1 and so on until all the sediment has been dissolved4 #f4 ,now make up the fluid in the measuring cylinder to #ajj c4c4 by the addition of more methylic alcohol4 ,,method4-- #a4 ,prepare film1 dry1 but do not fix4 #b4 ,flood the unfixed film with stain1 allow it to act #cj seconds4 #c4 ,add double the volume of distilled water to the stain on the film1 and mix with glass rod or platinum loop4 #d4 ,allow this diluted stain to act five minutes4 #e4 ,wash off with distilled water4 #f4 ,leave some water on film for thirty seconds to intensify the colour contrasts4 #g4 ,dry and mount4 ,,methods ,,of ,,demonstrating ,,structure ,,of ,,bacteria1 ,,etc4 ,to ,demonstrate ,capsules4 #a4 ,mac,conkey4-- ,stain4-- ,weigh out ,dahlia #j4e gramme ,methyl green 7#jj crystals7 #a4e grammes rub up in a mortar with ,distilled water #ajj4j c4c4 ,add ,fuchsin1 saturated alcoholic solution #aj4j c4c4 and make up to #bjj c4c4 by the addition of ,distilled water #ij4j c4c4 ,filter4 ,allow the stain to stand for two weeks before use2 keep in a dark place or in an amber glass bottle4 ,owing to the unstable character of the methyl green1 this stain deteriorates after about six months4 ,,method4-- #a4 ,prepare and fix film in the usual manner4 #b4 ,flood the cover-slip with the stain and allow it to act for five to ten minutes4 #c4 ,wash very thoroughly in water2 if necessary1 direct a powerful stream of water on the film from a wash-bottle4 #d4 ,dry and mount4 #b4 ,muir's ,method4-- #a4 ,prepare1 dry and fix film in the ordinary manner4 #b4 ,flood the film with carbolic fuchsin1 warm until steam begins to rise4 ,allow the stain to act for thirty seconds4 #c4 ,wash quickly with methylated spirit4 #d4 ,wash thoroughly with water4 #e4 ,subject the film to the action of the following mordant for five seconds3 ,corrosive sublimate1 saturated aqueous solution #b c4c4 ,tannic acid1 #bj per cent4 aqueous solution #b c4c4 ,potash alum saturated aqueous solution #e c4c4 #f4 ,wash thoroughly in water4 #g4 ,treat with methylated spirit for about sixty seconds4 7,the preparation should now be pale red47 #h4 ,wash thoroughly in water4 #i4 ,counterstain in methylene blue1 aqueous solution thirty seconds4 #aj4 ,wash in water4 #aa4 ,dehydrate in alcohol4 #ab4 ,clear in xylol and mount in xylol balsam4 #c4 ,welch's ,method4-- #a4 ,prepare and fix film in the usual manner4 #b4 ,flood the slide with acetic acid #b per cent42 allow the acid to remain in contact with the film for two minutes4 ,this swells up and fixes the capsule and enables it to take the stain4 #c4 ,blow off the acetic acid by the aid of a pipette4 #d4 ,immerse in aniline gentian violet1 five to thirty seconds4 #e4 ,wash in water4 #f4 ,dry and mount4 #d4 ,ribbert's ,method4-- ,stain4-- ,measure out and mix3 ,acetic acid1 glacial #ab4e c4c4 ,alcohol1 absolute #ej4j c4c4 ,distilled water #ajj4j c4c4 ,warm to #cf deg4 ,c4 7e4 g41 in the hot incubator7 and saturate with dahlia4 ,filter4 ,,method4-- #a4 ,prepare and fix films in the usual manner4 #b4 ,cover the film with the stain and allow it to act for one or two seconds only4 #c4 ,wash thoroughly in water4 #d4 ,dry and mount4 ,to ,demonstrate ,flagella4 #a4 ,muir's ,modified ,pitfield4--,this is the best method and gives the most reliable results1 for not only is the percentage of successful preparations higher than with any other1 but the bacilli and flagella retain their relative proportions4 7a7 ,mordant4-- ,tannic acid1 #aj per cent4 aqueous solution #aj c4c4 ,corrosive sublimate1 saturated aqueous solution #e c4c4 ,alum1 saturated aqueous solution #e c4c4 ,carbolic fuchsin 7,ziehl7 #e c4c4 ,mix thoroughly4 ,a precipitate forms which must be allowed to settle for a few hours4 ,decant off the clear fluid into tubes and centrifugalise thoroughly4 ,this solution is at its best some four or five days after manufacture2 it keeps for about a couple of weeks1 but must be re-centrifugalised each time1 before use4 7b7 ,stain4-- ,alum1 saturated aqueous solution #be c4c4 ,gentian violet1 saturated alcoholic solution #e c4c4 ,filter4 ,this stain must be freshly prepared4 ,,method4--,the cultivations employed should be smear agar cultures1 twelve to eighteen hours old if incubated at #cg deg4 ,c1 twenty-four to thirty hours if incubated at #bb deg4 ,c4 #a4 ,remove a very small quantity of the growth by means of the platinum spatula4 #b4 ,emulsify it with a few cubic centimetres of distilled water in a watch-glass1 by gently moving the spatula to and fro in the water4 ,do not rub up the growth on the side of the watch-glass4 ,some workers prefer to use tap water1 others employ normal saline solution1 but distilled water gives the best emulsion4 #c4 ,spread a thin film of the emulsion on a newly flamed cover-slip1 using no force1 but rather leading the drop over the cover-slip with the platinum loop4 #d4 ,allow the film to dry in the air1 properly protected from falling dust4 #e4 ,fix by passing thrice through the ,bunsen flame1 holding the cover-slip whilst doing so by one corner between the finger and thumb4 #f4 ,pour on the film as much of the mordant as the cover-glass will hold4 ,grasp the cover-slip with the forceps and hold it1 high above the flame1 until steam rises4 ,allow the steaming mordant to remain in contact with the film two minutes4 #g4 ,wash well in water and dry carefully4 #h4 ,pour on the film as much of the stain as the cover-glass will hold4 ,steam over the flame as before for two minutes4 #i4 ,wash well in water4 #aj4 ,dry and mount4 #b4 ,pitfield ,original ,method4-- 7a7 ,mordant4-- ,tannic acid #a gramme ,water #aj c4c4 7b7 ,stain4-- ,saturated aqueous solution of alum #aj c4c4 ,saturated alcoholic solution of gentian violet #a c4c4 ,distilled water #e c4c4 ,mix equal parts of a and b before using4 #a4 ,prepare and fix the film in the manner described above4 #b4 ,boil the mixture and immerse the cover-slip in it1 whilst still hot1 for one minute4 #c4 ,wash in water4 #d4 ,examine in water2 if satisfactory1 dry and mount in ,canada balsam4 #c4 ,mac,crorrie's ,method4-- ,mordant-,stain4-- ,measure out and mix4 ,night blue1 saturated alcoholic solution #aj c4c4 ,potash alum1 saturated aqueous solution #aj c4c4 ,tannin1 #aj per cent4 aqueous solution #aj c4c4 ,,note4--,the addition of gallic acid1 #j4a to #j4b gramme1 may improve the solution1 but is not necessary4 ,,method4-- #a4 ,prepare and fix the films as above4 #b4 ,pour some of the mordant-stain on the film and warm gently1 high above the flame1 for two minutes 7or place in the hot incubator for a like period74 #c4 ,wash thoroughly in water4 #d4 ,dry and mount4 #d4 ,loeffler's ,method4-- 7a7 ,mordant4-- ,tannic acid1 #bj per cent4 aqueous solution #aj c4c4 ,ferrous sulphate1 saturated aqueous solution #e c4c4 ,haematoxylin solution #c c4c4 ,carbolic acid1 #a per cent4 aqueous solution #d c4c4 ,this solution must be freshly prepared4 ,haematoxylin solution is prepared by boiling #a gramme logwood with #h c4c4 distilled water1 filtering and replacing the loss from evaporation4 ,alternative ,mordant 7,bunge's ,mordant74-- ,tannic acid1 #bj per cent4 aqueous solution #aj c4c4 ,ferrous sulphate1 saturated aqueous solution #e c4c4 ,fuchsin1 saturated alcoholic solution #a c4c4 7b7 ,stain4-- ,weigh out ,methylene-blue ,or methylene-violet #d grammes ,or fuchsin and dissolve in ,aniline water1 freshly saturated and filtered #ajj c4c4 ,,method4-- #a4 ,prepare and fix films as above4 #b4 ,pour the mordant on to the film and warm cautiously over the flame till steam rises2 keep the mordant gently steaming for one minute4 #c4 ,wash well in distilled water till no more colour is discharged2 if necessary1 wash carefully with absolute alcohol4 #d4 ,filter a few drops of the stain on to the film1 warm as before1 and allow the steaming stain to act for one minute4 #e4 ,wash well in distilled water4 #f4 ,dry and mount4 ,,note4--,the flagella of some organisms can be demonstrated better by means of an alkaline stain or an acid stain--a point to be determined for each4 ,speaking generally1 those bacilli which give rise to an acid reaction in the culture medium require an alkali2 those which form alkali in cultivation require an acid4 ,according to requirements1 therefore1 ,loeffler recommends the addition of sodium hydrate1 #a per cent4 aqueous solution1 #a c4c42 or an equal quantity of an exactly comparable solution of sulphuric acid4 #e4 ,van ,ermengem's ,method4--,this method1 being merely a precipitation of a silver salt on the micro-organisms and not a true stain1 creates a false impression as to the relative proportions of bacteria and flagella4 7a7 ,fixing ,fluid4-- ,osmic acid1 #b per cent4 aqueous solution #aj c4c4 ,tannic acid1 #bj per cent4 aqueous solution #bj c4c4 ,acetic acid1 glacial #a c4c4 ,the fixing fluid should be prepared some days before use and filtered as required4 ,in colour it should be distinctly violet4 7b7 ,sensitising ,solution4-- ,silver nitrate1 #j4e per cent4 aqueous solution4 ,this solution must be kept in a dark blue glass bottle or in a dark cupboard4 ,filter immediately before use4 7c7 ,reducing ,solution4-- ,weigh out ,gallic acid #e grammes ,tannic acid #c grammes ,potassium acetate1 fused #aj grammes and dissolve in ,distilled water #cej c4c4 ,filter4 ,this solution will keep active for several days1 but fresh solution must be used for each preparation4 ,,method4-- #a4 ,prepare emulsion1 make and fix films as above in the preceding method1 steps #a to #d4 #b4 ,pour on the film as much of the fixing solution as the cover-glass will hold1 heat carefully over the flame till steam rises1 and allow the steaming fixing fluid to act for five minutes4 #c4 ,wash well in water4 #d4 ,wash in absolute alcohol4 #e4 ,wash in distilled water4 #f4 ,pour some of the sensitising solution on the film and allow it to act for from thirty seconds to one minute2 blot off the excess of fluid with filter paper4 #g4 ,without washing1 transfer the film to a watch-glass containing the reducing solution and allow it to remain therein for from thirty seconds to one minute2 blot off the excess of fluid with filter paper4 #h4 ,without washing1 again treat the film with the sensitising solution1 this time until the film commences to turn black4 #i4 ,wash in distilled water4 #aj4 ,dry and mount4 ,to ,stain ,nuclei of ,yeast ,cells4 #a4 ,prepare and fix film in the usual manner4 #b4 ,soak in ferric ammonia sulphate #c per cent4 aqueous solution for two hours4 #c4 ,wash thoroughly in water4 #d4 ,stain in haematoxylin solution 7see page #ie7 for thirty minutes4 #e4 ,wash in water4 #f4 ,differentiate in ferric ammonia sulphate solution for #a-a_/b-b minutes1 examining wet under microscope during the process4 ,to ,stain ,spores4 #a4 ,single ,stain4-- #a4 ,prepare cover-slip film in the usual way4 #b4 ,in fixing1 pass the cover-slip film fifteen or thirty times through the flame instead of only three4 ,this destroys the resisting power of the spore membrane and allows the stain to reach the interior4 #c4 ,stain in the usual way with methylene-blue or fuchsin4 #d4 ,wash in water4 #e4 ,dry and mount4 #b4 ,double ,stain4-- #a4 ,prepare and fix film in the usual way--i4 e41 pass three times through flame to fix4 #b4 ,cover the film with hot carbol-fuchsin and hold in the forceps above a small flame until the fluid begins to steam4 ,set the cover-slip down and allow it to cool4 ,repeat the process when the stain ceases to steam and continue to repeat until the stain has been in contact with the film for twenty minutes4 7,this stains both spores and bacteria47 #c4 ,wash in water4 #d4 ,decolourise in alcohol1 #b parts2 acetic acid1 #a per cent41 #a part4 7,this removes the stain from everything but the spores47 #e4 ,wash in water4 #f4 ,mount the cover-slip in water and examine microscopically with the #a_/f-;inch objective4 7,spores should be red1 and the rest of the film colourless or a very light pink47 ,if satisfactory1 pass on to section #g2 if unsatisfactory1 repeat steps #b to #e4 #g4 ,counterstain in weak methylene-blue4 7,now spores red1 bacilli blue47 #h4 ,wash in water4 #i4 ,dry and mount4 ,the spores of different bacilli differ greatly in their resistance to decolourising reagents2 even the spores of the same species of organisms vary according to their age4 ,young spores are more easily decolourised than those more mature4 ,sulphuric acid1 #a per cent4 aqueous solution1 and hydrochloric acid1 #j4e per cent4 alcoholic 7#ij per cent47 solution1 are useful decolourising reagents4 #c4 ,moeller's ,method4-- #a4 ,prepare and fix films in the usual manner4 #b4 ,immerse in absolute alcohol for two minutes1 then in chloroform for two minutes2 wash in water4 ,this dissolves out any fat or crystals that might otherwise retain the spore stain4 #c4 ,immerse in chromic acid1 #e per cent4 aqueous solution1 for one minute2 wash in water4 #d4 ,pour ,ziehl's carbolic fuchsin on the film1 warm as in previous methods1 and allow it to act for ten minutes4 #e4 ,wash in water4 #f4 ,decolourise in sulphuric acid1 #e per cent4 aqueous solution1 for five seconds4 #g4 ,wash in water4 #h4 ,counterstain with ,kuehne's carbolic methylene-blue for one or two minutes4 #i4 ,wash in water4 #aj4 ,dry and mount4 7,spores red1 bacilli blue47 #d4 ,abbott's ,method4-- #a4 ,prepare and fix films in the usual manner4 #b4 ,pour ,loeffler's alkaline methylene-blue on the film2 warm cautiously over the flame till steam rises and allow the hot steam to act for one to five minutes4 #c4 ,wash thoroughly in water4 #d4 ,decolourise in nitric acid1 #b per cent4 alcoholic 7alcohol #hj per cent47 solution4 #e4 ,wash thoroughly in water4 #f4 ,counterstain in eosin1 #a per cent4 aqueous solution4 #g4 ,wash4 #h4 ,dry and mount4 7,spores blue1 bacilli red47 ,,differential ,,methods ,,of ,,staining4 ,gram's ,method4--,this method depends upon the fact that the protoplasm of some bacteria permits aniline gentian violet and ,lugol's iodine solution1 when applied consecutively1 to enter into a chemical combination which results in the formation of a new blue-black pigment1 only very sparingly soluble in absolute alcohol4 ,such organisms are said to stain by ,gram1 or to be ,gram positive4 #a4 ,prepare a cover-slip film and fix in the usual way4 #b4 ,stain in aniline gentian violet three to five minutes4 ,filter as much aniline water on to the cover-slip as it will hold2 then add the smallest quantity of alcoholic solution of gentian violet which suffices to saturate the aniline water and form a bronze scum upon its surface--if too much of the alcoholic gentian violet is added the alcohol present redissolves this scum4 ,to prepare aniline water1 pour #d or #e c4c4 aniline oil into a stoppered bottle and add distilled water1 #ajj c4c4 ,shake vigourously and filter immediately before use4 ,the excess of oil sinks to the bottom of the bottle and may be used again4 #c4 ,wash in water4 #d4 ,treat with ,lugol's iodine solution until the film is black or dark brown4 ,to do this treat with iodine solution for a few seconds1 wash in water1 and examine the film over a piece of white filter paper4 ,note the colour4 ,repeat this process until the film ceases to darken with the fresh application of iodine solution4 ,lugol's solution is prepared by dissolving ,iodine #a gramme ,iodide of potassium #c grammes ,in distilled water #cjj c4c4 #e4 ,wash in water4 #f4 ,wash with alcohol until no more colour is discharged and the alcohol runs away clear and colourless4 ,the following mixture may be substituted for absolute alcohol as a decolouriser ,acetone #aj c4c4 ,absolute alcohol #ajj c4c4 #g4 ,wash in water4 #h4 ,counterstain very lightly with aqueous solution of ,neutral ,red4 ,other counterstains may be used such as dilute eosin1 dilute fuchsin1 or vesuvin4 ,,note4--,this section may be omitted when dealing with films prepared from pure cultivations4 #i4 ,wash in water4 #aj4 ,dry and mount4 ,gram-,claudius ,method4-- #a4 ,prepare a cover-slip film and fix in the usual way4 #b4 ,stain in methyl violet1 #a per cent4 aqueous solution for three to five minutes4 #c4 ,treat with two lots picric acid1 saturated aqueous solution4 #d4 ,wash in water and dry4 #e4 ,decolourise with clove oil4 #f4 ,wash off clove oil with xylol4 #g4 ,mount in xylol balsam4 ,gram-,weigert ,method4-- #a-e4 ,proceed as for the corresponding sections of ,gram's method 7quod vide74 #f4 ,dry in the air4 #g4 ,wash in aniline oil1 #a part1 xylol1 #b parts1 until no more colour is discharged4 #h4 ,wash in xylol4 #i4 ,mount in xylol balsam4 ,modified ,gram-,weigert ,method4--7,to demonstrate trichophyta in hair47 #a4 ,soak the hairs in ether for ten minutes to remove the fat4 #b4 ,stain thirty minutes in a tar-like solution of aniline gentian violet 7prepared by adding #ae drops of the alcoholic solution of gentian violet to #c drops of aniline water74 #c4 ,dry the hairs between pieces of blotting paper4 #d4 ,treat with perfectly fresh iodine solution4 #e4 ,again dry between blotting paper4 #f4 ,treat with aniline oil to remove excess of stain4 7,if necessary1 add a drop or two of nitric acid to the oil47 #g4 ,again treat with aniline oil4 #h4 ,treat with aniline oil and xylol1 equal parts4 #i4 ,clear with xylol4 #aj4 ,mount in xylol balsam4 ,to obtain the best differentiation the preparation should be repeatedly examined microscopically 7with a #a_/f-;inch objective7 between steps #e and #i1 as the actual time involved varies with different specimens4 ,ziehl-,neelsen's ,method4--7,to demonstrate tubercle and other acid-fast bacilli47 #a4 ,smear a thin1 even film of the specimen on the cover-slip by means of the platinum loop4 7,in the case of sputum1 if it is a very watery specimen1 allow the film to dry1 then spread a second and even a third layer over the first47 #b4 ,fix by passing three times through the flame4 #c4 ,stain in hot carbol-fuchsin 7as in staining for spores7 for five to ten minutes4 7,this stains everything on the film47 ,avoid over-heating4 #d4 ,decolourise by dipping in sulphuric acid1 #be per cent4 7,this removes stain from everything but acid-fast bacilli2 e4 g41 tubercle1 leprosy1 and smegma bacilli and the film turns yellow47 #e4 ,wash in water4 7,a pale red colour returns to the film74 #f4 ,wash in alcohol till no more colour is discharged4 7,this often1 but not invariably1 removes the stain from acid-fast bacilli other than tubercle2 e4 g41 smegma bacillus47 #g4 ,wash in water4 #h4 ,counterstain in weak methylene-blue4 7,stains non-acid-fast bacilli1 leucocytes1 epithelial cells1 etc47 #i4 ,wash in water1 dry1 and mount4 ,pappenheim's ,method4-- ,this method is supposed to differentiate between ,b4 tuberculosis and other acid-fast micro-organisms4 #a4 ,prepare and fix film in the usual way4 #b4 ,stain in carbol-fuchsin without heat for three minutes4 #c4 ,without previously washing in water treat the film with three or four successive applications of corallin 7,rosolic acid7 solution4 ,corallin #a gramme ,methylene-blue 7saturated alcoholic solution7 #ajj c4c4 ,glycerine #bj c4c4 #d4 ,wash in water4 #e4 ,dry and mount4 ,neisser's ,method--,modified4--7,to demonstrate diphtheroid bacilli47 ,stain ,i4-- ,measure out and mix ,methylene-blue1 saturated alcoholic solution #d4j c4c4 ,acetic acid1 #e per cent4 aqueous solution #if4j c4c4 ,filter4 ,stain ,,ii4-- ,weigh out ,neutral red #b4e grammes and dissolve in ,distilled water #ajjj c4c4 ,filter4 ,,method4-- #a4 ,prepare and fix films in the usual way4 #b4 ,pour stain ,i on the film and allow it to act for two minutes4 #c4 ,wash thoroughly in water4 #d4 ,treat with ,lugol's iodine for ten seconds4 #e4 ,wash thoroughly in water4 #f4 ,pour stain ,,ii on to the film and allow it to act for thirty seconds4 #g4 ,wash thoroughly in water4 #h4 ,dry and mount4 ,,note4--,the cultivation from which the films are prepared must be upon blood-serum which has been incubated at #cg deg4 ,c4 for from nine to eighteen hours4 ,the bacilli are stained a light red by the neutral red1 which contrasts well with the two or three black spots1 situated at the poles and occasionally one in the centre representing protoplasmic aggregations 78 metachromatic granules7 stained by the acid methylene-blue4 ,wheal and ,chown 7,oxford7 ,method4--7,to demonstrate actinomyces47 #a4 ,stain briefly with ,ehrlich's haematoxylin 7until nuclei are faint blue after washing with tap water74 #b4 ,wash in tap water4 #c4 ,stain in hot carbol-fuchsin 7as for tubercle bacilli7 for five to ten minutes4 #d4 ,wash in tap water4 #e4 ,decolourise with ,spengler's picric acid alcohol4 ,this is prepared by mixing3 ,alcohol1 absolute #bj c4c4 ,picric acid1 saturated aqueous solution #aj c4c4 ,distilled water #aj c4c4 ,during the progress of steps #a-e the preparation must be repeatedly examined microscopically with the #a_/f-;inch objective4 ,when properly differentiated the clubs appear brilliant red on greenish ground4 #f4 ,dehydrate in alcohol4 #g4 ,clear in xylol4 #h4 ,mount in xylol balsam4 ,this method serves equally well for films and for sections4 ,,vii4 ,,methods ,,of ,,demonstrating ,,bacteria ,,in ,,tissues4 ,for bacteriological purposes1 sections of tissue are most conveniently prepared by either the freezing method or the paraffin method4 ,the latter is decidedly preferable1 but as it is of greater importance to demonstrate the bacteria1 if such are present1 than to preserve the tissue elements unaltered1 the frozen sections are often of value4 ,whichever method is selected1 it is necessary to take small pieces of the tissue for sectioning1--#b to #e mm4 cubes when possible1 but in any case not exceeding half a centimetre in thickness4 ,post-mortem material should be secured as soon after the death of the animal as possible4 ,the tissue is prepared for cutting by-- 7a7 ,fixation2 that is1 by causing the death of the cellular elements in such a manner that they retain their characteristic shape and form4 ,the fixing fluids in general use are3 ,absolute alcohol2 corrosive sublimate1 saturated aqueous solution2 corrosive sublimate1 ,lang's solution 7vide page #hb72 formaldehyde1 #d per cent4 aqueous solution4 7,of these1 ,lang's corrosive sublimate solution is decidedly the best all-round fixative47 7b7 ,hardening2 that is1 by rendering the tissue of sufficient consistency to admit of thin slices or sections being cut from it4 ,this is effected by passing the tissue successively through alcohols of gradually increasing strength3 #cj per cent4 alcohol1 #ej per cent4 alcohol1 #ge per cent4 alcohol1 #ij per cent4 alcohol1 absolute alcohol4 ,in both these processes a large excess of fluid should always be used4 ,,freezing ,,method4 #a4 ,fixation4 ,place the pieces of tissue in a wide-mouthed glass bottle and fill with absolute alcohol4 ,allow the tissues to remain therein for twenty-four hours4 #b4 ,hardening4 ,remove the alcohol 7no longer absolute1 as it has taken up water from the tissues7 from the bottle and replace it with fresh absolute alcohol4 ,allow the tissues to remain therein for twenty-four hours4 ,illustration3 ,,fig4 #ga4--,washing tissues4 ,,note4--,if not needed for cutting immediately1 the hardened tissues can be stored in #ge per cent4 alcohol4 #c4 ,remove the alcohol from the tissues by soaking in water from one to two hours4 ,remove the stopper from the bottle2 rest a glass funnel in the open mouth and place under a tap of running water4 ,the water of course1 overflows1 but the tissues remain in the bottle 7,fig4 #ga74 #d4 ,impregnate the tissues with mucilage for twelve to twenty-four hours1 according to size4 ,transfer the pieces of tissue to a bottle containing sterilised gum mixture4 ,formula4-- ,gum arabic #e grammes ,saccharose #a gramme ,boric acid #a gramme ,water #ajj c4c4 #e4 ,place the tissue on the plate of a freezing microtome 7,cathcart's is perhaps the best form71 cover and surround with fresh gum mixture2 freeze with ether1 or for preference1 carbon dioxide1 and cut sections4 #f4 ,float the sections off the knife into a glass dish containing tepid water and allow them to remain therein for about an hour to dissolve out the gum4 7,if not required at once1 store in #ij per cent4 alcohol47 #g4 ,transfer to a glass capsule containing the selected staining fluid1 by means of a section lifter4 #h4 ,transfer the sections in turn to a capsule containing absolute alcohol 7to dehydrate7 and to one containing xylol or oil of cloves 7to clear74 #i4 ,mount in xylol balsam4 ,alternative ,rapid ,method4-- #a4 ,cut very small blocks of the tissue4 #b4 ,fix in formalin #aj per cent4 aqueous solution 7fixation fluid ,no4 #g1 page #hb7 for #bd hours4 #c4 ,transfer block to plate of freezing microtome and freeze with carbon dioxide vapour4 #d4 ,float the sections off the knife into a glass dish of tepid water4 #e4 ,stain the sections in glass capsules containing selected stains4 #f4 ,place the stained section in a dish of clean water and introduce a glass slide obliquely beneath the section2 with a mounted needle draw the section on to the slide and hold it there2 gently remove the slide from the water1 taking care that any folds in the section are floated out before the slide is finally removed from the water4 #g4 ,drain away as much water as possible from the section4 ,drop absolute alcohol on to the section from a drop bottle1 to dehydrate it4 #h4 ,double a piece of blotting paper and gently press it on the section to dry it4 #i4 ,drop on xylol to clear the section4 #aj4 ,place a large drop of xylol balsam on the section and carefully lower a cover-glass on to the balsam4 ,,paraffin ,,method4 #a4 ,fixation4 ,place the pieces of tissue1 resting on cotton-wool1 in a wide-mouthed glass bottle4 ,pour on a sufficient quantity of the corrosive sublimate fixing fluid2 allow the tissue to remain therein for twelve to twenty-four hours according to size4 #b4 ,pour off the fixing fluid and wash thoroughly in running water for twenty minutes to half an hour to remove the excess of corrosive sublimate4 ,illustration3 ,,fig4 #gb4--,l-shaped brass moulds4 ,illustration3 ,,fig4 #gc4--,paraffin kettle4 #c4 ,hardening4 ,place the tissues in each of the following strengths of alcohol in turn for from twelve to twenty-four hours3 #ej per cent41 #ge per cent41 #ij per cent41 absolute4 #d4 ,dehydration is effected by transferring the tissues to fresh absolute alcohol4 #e4 ,clearing4 ,half fill a wide-mouthed bottle with chloroform4 ,on the surface of the chloroform float a layer of absolute alcohol about five to ten millimetres in depth4 ,place the pieces of tissue in the layer of alcohol and when they have sunk through this layer1 transfer them to pure chloroform for from six to twenty-four hours according to the size of the pieces4 ,when cleared1 the tissue becomes more or less transparent4 #f4 ,infiltration4 ,place the cleared tissues in fresh chloroform with several pieces of paraffin wax and stand in a warm place1 such as on the top of the warm incubator4 ,the warmth gradually melts the paraffin and the tissues should remain in the mixture about twenty-four hours4 #g4 ,transfer the tissues to a vessel containing pure melted paraffin4 ,place this vessel in a paraffin water-bath regulated for #b deg4 ,c4 above the melting-point of the paraffin used1 and allow the tissues to soak for some four to six hours to ensure complete impregnation4 ,the paraffin used should have a melting-point of not more than #eh deg4 ,c4 ,for all ordinary purposes #ed deg4 ,c4 will be found quite high enough4 #h4 ,imbed in fresh paraffin in a metal 7or paper7 mould4 7a7 ,arrange a pair of ,l-shaped pieces of metal on a plate of glass to form a rectangular trough 7,fig4 #gb74 7b7 ,pour fresh melted paraffin into the mould from a special vessel 7,fig4 #gc74 7c7 ,lift the piece of tissue from the paraffin bath and arrange it in the mould4 7d7 ,blow gently on the surface of the paraffin in the mould1 and as soon as a film of solid paraffin has formed1 carefully lift the glass plate on which the mould is set and lower plate and mould together into a basin of cold water4 7e7 ,when the block is cold1 break off the metal ,l's2 trim off the excess of paraffin from around the tissue with a knife1 taking care to retain the rectangular shape1 and store the block in a pill-box4 ,when several pieces of tissue have to be imbedded at one time1 shapes of stout copper1 #aj cm41 #e cm41 and #b4e cm4 square respectively1 and #j4ge cm4 deep 7,fig4 #gd7 will be found extremely useful4 ,these placed upon plates of glass replace the pair of ,l's in the above process4 ,when the paraffin has set firmly the screw a should be loosened to allow the two halves of the flange b to separate slightly--this facilitates removal of the paraffin block4 ,illustration3 ,,fig4 #gd4--,paraffin mould4 #h4 ,cement the block on the carrier of a paraffin microtome 7the ,minot1 the ,jung1 or the ,cambridge ,rocker7 with a little melted paraffin4 ,greater security is obtained if the paraffin around the base of the block is melted by means of a hot metal or glass rod4 #i4 ,cut sections--thin1 and if possible in ribbands4 ,mounting ,paraffin ,sections4-- #a4 ,place a large drop of #cj per cent4 alcohol on the centre of a slide 7or cover-slip7 and float the section on to the surface of the drop1 from a section lifter4 #b4 ,hold the slide in the fingers of one hand and warm cautiously over the flame of a ,bunsen burner1 touching the under surface of the glass from time to time on the back of the other hand4 ,as soon as the slide feels distinctly warm to the skin1 the paraffin section will flatten out and all wrinkles disappear4 7,the slide with the section floating on it may be rested on the top of the paraffin bath for two or three minutes1 instead of warming over the flame as here described47 #c4 ,cautiously tilt up the slide and blot off the excess of spirit with blotting paper1 leaving the section attached to the centre of the slide4 #d4 ,place the slide in a wire rack 7,fig4 #ge71 section downward1 in the hot incubator for twelve to twenty-four hours4 ,at the end of this time the section is firmly adherent to the glass1 and is treated during the subsequent steps as a fixed cover-glass film preparation4 ,,note4--,if large1 thick sections have to be manipulated1 or if time is of importance or acids are used during the staining process1 it is often advisable to add a trace of ,mayer's albumin to the alcohol before floating out the section4 ,if this substance is employed1 a sojourn of twenty minutes to half an hour in the hot incubator will be found ample to ensure firm adhesion of the section to the slide4 ,the albuminous fluid is prepared as follows3 ,illustration3 ,,fig4 #ge4--,section rack4 ,mayer's ,albumin4-- ,weigh out ,salicylate of soda #a gramme and dissolve in ,glycerine #ej c4c4 ,add ,white of egg #ej c4c4 ,mix thoroughly by means of an egg whisk4 ,filter into a clean bottle4 ,as an alternative method paint a thin layer of ,schallibaum's solution on the slide with a camel's hair pencil2 lay the section carefully on this film and heat gently to fix the section4 ,schallibaum's solution3 ,clove oil #cj c4c4 ,collodion #aj c4c4 ,keep in a dark blue bottle in a cool place4 ,staining ,paraffin ,sections4-- #a4 ,warm paraffin section over the ,bunsen flame to soften 7but not to melt7 the paraffin1 then dissolve out the wax with xylol poured on from a drop bottle4 #b4 ,remove xylol by flushing the section with alcohol4 #c4 ,if the tissue was originally fixed in a corrosive sublimate solution1 the section must now be treated with ,lugol's iodine solution for two minutes and subsequently immersed in #ij per cent4 alcohol to remove all traces of yellow staining4 #d4 ,wash in water4 #e4 ,stain deeply1 if using a single stain1 as the subsequent processes decolourise4 #f4 ,wash in water1 decolourise if necessary4 #g4 ,flood with several changes of absolute alcohol to dehydrate the section4 #h4 ,clear in xylol4 7,oil of cloves is not usually employed1 as it decolourises the section47 #i4 ,mount in xylol balsam4 ,,special ,,staining ,,methods ,,for ,,sections4 ,double-staining ,carmine and ,gram-,weigert4-- #a4 ,prepare the section for staining as above1 sections #a to #c4 #b4 ,stain in lithium carmine 7,orth's7 or picrocarmine for ten to thirty minutes1 in a porcelain staining pot 7,fig4 #gf74 #c4 ,wash in picric acid solution until yellow4 ,at this stage cell nuclei are red1 protoplasm is yellow1 and bacteria are colourless4 ,picric acid solution is prepared by mixing ,picric acid1 saturated aqueous solution #dj c4c4 ,hydrochloric acid #a c4c4 ,alcohol 7#ij per cent47 #afj c4c4 #d4 ,wash in water4 #e4 ,wash in alcohol4 #f4 ,stain in aniline gentian violet4 #g4 ,wash in iodine solution till dark brown or black4 #h4 ,wash in water4 #i4 ,dip in absolute alcohol for a second4 #aj4 ,decolourise with aniline oil till no more colour is discharged4 ,illustration3 ,,fig4 #gf4--,staining pot4 #aa4 ,wash with aniline oil1 #b parts1 xylol1 #a part4 #ab4 ,clear with xylol4 #ac4 ,mount in xylol balsam4 ,alternative ,gram-,weigert ,method for ,sections4-- #a4 ,fix paraffin section on slide and prepare for staining in the usual manner4 #b4 ,stain in alum carmine for about fifteen minutes4 #c4 ,wash thoroughly in water4 #d4 ,filter aniline gentian violet solution on to the section on the slide and allow to stain about twenty-five minutes4 #e4 ,wash thoroughly in water4 #f4 ,treat with ,lugol's iodine until section ceases to become any blacker4 #g4 ,wash thoroughly in water4 #h4 ,treat with a mixture of equal parts of aniline oil and xylol until no more colour comes away4 #i4 ,wash thoroughly with xylol4 #aj4 ,decolourise and dehydrate rapidly with absolute alcohol until there remains only a very faint bluish tint4 #aa4 ,clear with xylol4 #ab4 ,mount in xylol balsam4 7,then fibrin and hyaline tissue are stained deep blue1 whilst bacteria which stain ,gram appear of a deep blue-violet colour47 ,unna-,pappenheim ,method4-- ,stain4-- ,weigh out and mix ,methylene green #j4ae gramme ,pyronin #j4be gramme and dissolve in ,carbolic acid #j4e per cent4 aqueous solution #gh c4c4 ,measure out ,alcohol #b4e c4c4 ,glycerine #bj4j c4c4 and add to the stain4 ,method4-- #a4 ,place tissue in the above stain for ten minutes4 #b4 ,differentiate and dehydrate with absolute alcohol4 #c4 ,clear in xylol4 #d4 ,mount in xylol balsam4 ,to ,demonstrate ,capsules4-- #a4 ,mac,conkey's ,method4--,stain precisely as for cover-slip films 7vide page #ajj74 #b4 ,friedlaender's ,method4-- ,stain4-- ,gentian violet1 saturated alcoholic solution #ej c4c4 ,acetic acid1 glacial #aj c4c4 ,distilled water #ajj c4c4 ,,method4-- #a4 ,prepare the sections for staining1 secundum artem4 #b4 ,stain sections in the warm 7e4 g41 in the hot incubator7 for twenty-four hours4 #c4 ,wash with water4 #d4 ,decolourise lightly with acetic acid1 #a per cent4 #e4 ,dehydrate rapidly with absolute alcohol4 #f4 ,clear with xylol4 #g4 ,mount in xylol balsam4 ,to ,demonstrate ,acid-fast ,bacilli4-- #a4 ,prepare the sections for staining in the usual way4 #b4 ,stain with haematin solution ten to twenty seconds1 to obtain a pure nuclear stain2 then wash in water4 #c4 ,stain with carbolic fuchsin twenty to thirty minutes at #dg deg4 ,c42 then wash in water4 #d4 ,treat with aniline hydrochlorate1 #b per cent4 aqueous solution1 for two to five seconds4 #e4 ,decolourise in #ge per cent4 alcohol till section appears free from stain--fifteen to thirty minutes4 #f4 ,dehydrate with absolute alcohol4 #g4 ,clear very rapidly with xylol4 #h4 ,mount in xylol balsam4 ,to ,demonstrate ,spirochaetes in ,tissues4 ,piridin ,method 7,levaditi74-- #a4 ,cut slices of tissue #a mm4 thick4 #b4 ,fix in #aj per cent4 formalin solution for twenty-four hours4 #c4 ,wash in water for one hour4 #d4 ,place in #if per cent4 alcohol for twenty-four hours4 #e4 ,measure into a dark green or amber bottle #ajj c4c4 silver nitrate solution #a per cent41 and #aj grammes pyridin puriss4 ,transfer slices of tissue to this4 ,stopper and keep at room temperature three hours1 then in thermostat at #ej deg4 ,c4 for four to six hours4 #f4 ,wash quickly in #aj per cent4 pyridin solution4 #g4 ,reduce silver by transferring slices of tissue to following solution for forty-eight hours4 ,pyrogallic acid #d grammes ,acetone #aj c4c4 ,pyridin puriss #ae grammes ,distilled water #ajj c4c4 #h4 ,wash well in water4 ,take through alcohols of increasing strength up to absolute1 keeping in each strength for twenty-four hours4 #i4 ,clear1 embed1 cut very thin sections1 mount1 remove paraffin1 again clear and mount in xylol balsam4 ,the spirochaetes if present are black and show up against the pale yellow color of the background4 ,weak carbol fuchsin1 neutral red or toluidin blue can also be used to stain the background if desired1 after the removal of the paraffin in step #i4 ,to ,demonstrate ,protozoa in ,sections 7,leishman74-- ,reagents required3 ,leishman's ,polychrome stain4 ,acetic acid #a in #aejj aqueous solution4 ,caustic soda #a in #gjjj aqueous solution4 ,distilled water4 #a4 ,mount section1 remove paraffin and take into distilled water as usual 7vide page #aba74 #b4 ,drain off the excess of water4 #c4 ,cover the section with diluted ,leishman 7#a part stain1 #b parts distilled water7 and allow to act for five to ten minutes 7until tissue appears a deep blue74 #d4 ,decolourise with acetic acid solution until only the nuclei appear blue 7examine the section wet1 with low power objective74 #e4 ,if the eosin colour is too well marked treat with the caustic soda solution until the desired tint is obtained 7as seen with the #a_/f-;inch objective74 #f4 ,wash with distilled water4 #g4 ,rapidly dehydrate with alcohol4 #h4 ,clear with xylol4 #i4 ,mount in xylol balsam4 ,,viii4 ,,classification ,,of ,,fungi4 ,for practical purposes ,,fungi may be divided into3 #a4 ,hymenomycetes 7including the mushrooms1 etc474 #b4 ,hyphomycetes 7moulds74 #c4 ,blastomycetes 7yeasts and torulae74 #d4 ,schizomycetes 7bacteria74 ,,note4--,formerly myxomycetes were included in the fungi2 they are now recognized as belonging to the animal kingdom1 and are termed mycetozoa4 ,,morphology ,,of ,,the ,,hyphomycetes4 ,at the commencement of his studies1 the attention of the student is directed to the various non-pathogenic moulds and yeasts1 not only that he may gain the necessary technique whilst handling cultivations of harmless organisms1 but also because these very species are amongst the commonest of those that may accidentally contaminate his future preparations4 ,the hyphomycetes are composed of a mycelium of short jointed rods or hyphae springing from an axis or germinal tube which develops from the spore4 ,hyphae are-- 7a7 ,nutritive or submerged4 7b7 ,reproductive or aerial4 ,the protoplasm of these cells contains granules1 pigment1 oil globules1 and sometimes crystals of calcium oxalate4 ,reproduction4--,apical spore formation--asexual2 zoospores--sexual4 ,mucorinae4--,mucor 7,fig4 #gg74--,note the branching filaments--mycelium 7a71 hyphae 7b74 ,note the asexual reproduction4 #a4 ,a filament grows upward4 ,at its apex a septum forms1 then a globular swelling appears--sporagium 7d74 ,this possesses a definite membrane4 #b4 ,from the septum grows a club-shaped mass of protoplasm--columella 7c74 ,illustration3 ,,fig4 #gg4--,mucor mucedo4 ,illustration3 ,,fig4 #gh4--;,aspergillus #c4 ,the rest of the contained protoplasm breaks up into swarm spores 7e74 ,finally the membrane ruptures and spores escape4 ,perisporaceae4--,aspergillus 7,fig4 #gh74--,note the branching filaments--mycelium 7a74 ,illustration3 ,,fig4 #gi4--,penicillium4 ,note the asexual reproduction4 #a4 ,a filament 7b7 grows upward1 its termination becomes clubbed2 on the clubbed extremity flask-shaped cells appear--sterigmata 7c74 #b4 ,at free end of each sterigma is formed an oval body--a spore or gonidium 7d71 which1 when ripe1 is thrown off from the sterigma4 ,two or more gonidia may be supported upon each sterigma4 ,penicillium 7,fig4 #gi74--,note the branching filaments--mycelium 7a7 7frequently containing globules74 ,note the asexual reproduction4 #a4 ,a filament grows upward--goniodophore 7b7--and its apex divides up into several branches--basidia 7c74 #b4 ,at the apex of each basidium a flask-shaped cell1 sterigma 7d71 appears4 #c4 ,at the apex of each sterigma appears a row of oval cells--spores or conidia 7e74 ,these1 when ripe1 are cast off from the sterigmata4 ,illustration3 ,,fig4 #hj4--,oidium4 ,ascomycetae4--,oidium 7,fig4 #hj74--7,this family is perhaps as nearly related to the blastomycetes as it is to the hyphomycetes47 ,note the branching filaments--pseudomycelium 7a74 ,here and there filaments are broken up at their ends into oval or rod-shaped segments1 oidia1 and behave as spores4 ,note the asexual reproduction4 ,from the pseudomycelium arise true hyphae 7b71 each of which in turn ends in a chain of spores 7c74 ,,morphology ,,of ,,the ,,blastomycetes4 ,the blastomycetes are composed of spherical or oval cells 7#h to #i4e mu in diameter71 which1 when rapidly multiplying by budding1 may form a spurious mycelium4 ,a thin cell-wall encloses the granular protoplasm1 in which vacuoles and sometimes a nucleus may be noted4 ,this latter is best seen when stained with haematoxylin 7see page #aje74 ,during their growth and multiplication the blastomycetes split up solutions containing sugar into alcohol and ,,co#b4 ,saccharomyces 7,fig4 #ha74--,note the round or oval cells of granular protoplasm 7a7 containing solid particles and vacuoles 7c71 and surrounded by a definite envelope4 ,reproduction4--,budding2 ascospores--asexual4 ,note the asexual reproduction4 #a4 ,gemmation--that is1 the budding out of daughter cells 7b7 from various parts of the gradually enlarging mother cell4 ,these are eventually cast off and in turn become mother cells and form fresh groups of buds4 ,illustration3 ,,fig4 #ha4--,saccharomyces with ascospores4 ,illustration3 ,,fig4 #hb4--,torula4 #b4 ,spore formation--ascospores 7e74 ,these are formed at definite temperatures and within well-defined periods2 e4 g41 ,saccharomyces cerevisiae1 thirty hours at #be deg4 to #cg deg4 ,c41 or ten days at #ab deg4 ,c4 ,torulae 7,fig4 #hb74--,torulae1 whilst resembling yeasts in almost every other respect1 never form endo-spores4 ,note the elongated1 sausage-shaped cells 7a7 the larger oval cells 7b7 and the globular cells 7c7 the former two often interlacing and growing as a film4 ,note the absence of ascospore formation4 ,,ix4 ,,schizomycetes4 ,classification and ,morphology4--,bacteria are often classified1 in general terms1 according to their life functions1 into-- ,saprogenic1 or putrefactive bacteria2 ,zymogenic1 or fermentative bacteria2 ,pathogenic1 or disease-producing bacteria2 or according to their food requirements into-- ,prototrophic1 requiring no organic food 7e4 g41 nitrifying bacteria72 ,metatrophic1 requiring organic food 7e4 g41 saprophytes and facultative parasites72 ,paratrophic1 requiring living food 7obligate parasites72 or according to their metabolic products into-- ,chromogenic1 or pigment-producing bacteria2 ,photogenic1 or light-producing bacteria2 ,aerogenic1 or gas-producing bacteria2 and so on4 ,such broad groupings as these have1 however1 but little practical value when applied to the systematic study of the fission fungi4 ,on the other hand1 no really scientific classification of the schizomycetes has yet been drawn up1 and the varying morphological appearances of the members of the family are still utilised as a basis for classification1 as under-- #a4 ,cocci4 7,fig4 #hc74--,rounded or oval cells1 subdivided according to the arrangement of the individuals after fission1 into-- ,diplococci and ,streptococci1 where division takes place in one plane only1 and the individuals remain attached 7a7 in pairs or 7b7 in chains4 ,tetrads1 ,merismopedia1 or ,pediococci1 where division takes place alternately in two planes at right angles to each other1 and the individuals remain attached in flat tablets of four1 or its multiples4 ,illustration3 ,,fig4 #hc4--,types of bacteria--cocci3 #a1 ,diagram of sphere indicating planes of fission2 #b1 diplococci2 #c1 streptococci2 #d1 tetrads2 #e1 sarcinae2 #f1 staphylococci4 ,sarcinae1 where division takes place in three planes successively1 and the individuals remain attached in cubical packets of eight and its multiples4 ,illustration3 ,,fig4 #hd4--,types of bacteria--bacilli1 etc43 #a1 ,bacilli2 #b1 diplobacilli2 #c streptobacilli2 #d1 spirilla2 #e1 vibrios2 #f1 spirochaetae4 ,micrococci or ,staphylococci1 where division takes place in three planes1 but with no definite sequence2 consequently the individuals remain attached in pairs1 short chains1 plates of four1 cubical packets of eight1 and irregular masses containing numerous cocci4 #b4 ,bacilli 7,fig4 #hd1 #a to #c74--,rod-shaped cells4 ,a bacillus1 however short1 can usually be distinguished from a coccus in that two sides are parallel4 ,some bacilli after fission retain a characteristic arrangement and may be spoken of as ,diplobacilli or ,streptobacilli4 ,leptothrix is a term that in the past has been loosely used to signify a long thread1 but is now restricted to such forms as belong to the leptothriciae 7vide infra74 #c4 ,spirilla 7,fig4 #hd1 #d to #f74--;,curved and twisted filaments4 ,classified1 according to shape1 into-- ,spirillum4 ,vibrio 7comma74 ,spirochaeta4 ,many ,spirochaetes appear to belong to the animal kingdom and are grouped under protozoa2 other organisms to which this name has been given are undoubtedly bacteria4 ,higher forms of bacteria are also met with1 which possess the following characteristics3 ,they are attached1 unbranched1 filamentous forms1 showing-- 7a7 ,differentiation between base and apex2 7b7 ,growth apparently apical2 7c7 ,exaggerated pleomorphism2 7d7 ,pseudo-branching from apposition of cells2 and are classified into-- #a4 ,beggiotoa4 ,free swimming forms1 which #b4 ,thiothrix4 contain sulphur granules4 #c4 ,crenothrix4 #d4 ,cladothrix4 ,these forms do not contain #e4 ,leptothrix4 sulphur granules4 #f4 ,streptothrix4 ,a group which exhibits true but not dichotomous branching1 and contains some pathogenic species4 ,the morphology of the same bacterium may vary greatly under different conditions4 ,for example1 under one set of conditions the examination of a pure cultivation of a bacillus may show a short oval rod as the predominant form1 whilst another culture of the same bacillus1 but grown under different conditions1 may consist almost entirely of long filaments or threads4 ,this variation in morphology is known as pleomorphism4 ,some of the factors influencing pleomorphism are3 #a4 ,the composition1 reaction1 etc41 of the nutrient medium in which the organism is growing4 #b4 ,the atmosphere in which it is cultivated4 #c4 ,the temperature at which it is incubated4 #d4 ,exposure to or protection from light4 ,the various points in the anatomy morphology and physiology of bacteria upon which stress is laid in the following pages should be studied as closely as is possible in preparations of the micro-organisms named in connection with each4 ,,anatomy4 #a4 ,capsule 7,fig4 #he1 b74--,a gelatinous envelope 7probably akin to mucin in composition7 surrounding each individual organism1 and preventing absolute contact between any two4 ,in some species the capsule 7e4 g41 ,b4 pneumoniae7 is well marked1 but it cannot be demonstrated in all4 ,in very well marked cases of gelatinisation of the cell wall1 the individual cells are cemented together in a coherent mass1 to which the term zoogloea is applied 7e4 g41 ,streptococcus mesenteroides74 ,in some species colouring matter or ferric oxide is stored in the capsule4 #b4 ,cell ,wall 7,fig4 #he1 c74--,a protective differentiation of the outer layer of the cell protoplasm2 difficult to demonstrate1 but treatment with iodine or salt solution sometimes causes shrinkage of the cell contents--plasmolysis--and so renders the cell wall apparent 7e4 g41 ,b4 megatherium7 in the manner shown in figure #he4 ,stained bacilli1 when examined with the polarising microscope1 often show a doubly refractile cell wall 7e4 g41 ,b4 tuberculosis and ,b4 anthracis74 ,in some of the higher bacteria the cell wall exhibits this differentiation to a marked degree and forms a hard sheath within which the cell protoplasm is freely movable2 and during the process of reproduction the cell protoplasm may be extruded1 leaving the empty tube unaltered in shape4 ,illustration3 ,,fig4 #he4--;,dragrammatic sketch of composite bacterium to illustrate details of anatomical structure4 ,illustration3 ,,fig4 #hf4--,plasmolysis4 #c4 ,cell ,contents4--,protoplasm 7mycoprotein7 contains a high percentage of nitrogen1 but is said to differ from proteid in that it is not precipitated by ,c#b;,h#f,o4 ,it is usually homogeneous in appearance--sometimes granular--and may contain oil globules or sap vacuoles 7,fig4 #he1 d71 chromatin granules1 and even sulphur granules4 ,sap vacuoles must be distinguished from spores1 on the one hand1 and the vacuolated appearance due to plasmolysis1 on the other4 ,the cell contents may sometimes be differentiated into a parietal layer1 and a central body 7e4 g41 beggiotoa7 when stained by haematoxylin4 #d4 ,nucleus4--,this structure has not been conclusively proved to exist1 but in some bacteria chromatin particles have been observed near the centre of the bacterial cell and denser masses of protoplasm situated at the poles which exhibit a more marked affinity than the rest of the cell protoplasm for aniline dyes4 ,these latter are termed polar granules or ,polkoerner 7,fig4 #he1 e74 ,occasionally these aggregations of protoplasm alter the colour of the dye they take up4 ,they are then known as metachromatic bodies or ,ernstschen ,koerner 7e4 g41 ,b4 diphtheriae74 #e4 ,flagella 7,organs of ,locomotion1 ,fig4 #he1 a74--,these are gelatinous elongations of the cell protoplasm 7or more probably of the capsule71 occurring either at one pole1 at both poles1 or scattered around the entire periphery4 ,flagella are not pseudopodia4 ,the possession of flagella was at one time suggested as a basis for a system of classification1 when the following types of ciliation were differentiated 7,fig4 #hg73 ,illustration3 ,,fig4 #hg4--,types of ciliation4 #a4 ,polar3 7a7 ,monotrichous 7a single flagellum situated at one pole2 e4 g41 ,b4 pyocyaneus74 7b7 ,amphitrichous 7a single flagellum at each pole2 e4 g41 ,spirillum volutans74 7c7 ,lophotrichous 7a tuft or bunch of flagella situated at each pole2 e4 g41 ,b4 cyanogenus74 #b4 ,diffuse3 ,peritrichous 7flagella scattered around the entire periphery e4 g41 ,b4 typhosus74 ,,physiology4 ,reproduction4--,active ,stage4--,vegetative1 i4 e41 by the division of cells1 or fission4 #a4 ,the cell becomes elongated and the protoplasm aggregated at opposite poles4 #b4 ,a circular constriction of the organism takes place midway between these aggregations1 and a septum is formed in the interior of the cell at right angles to its length4 #c4 ,the division deepens1 the septum divides into two lamellae1 and finally two cells are formed4 ,illustration3 ,,fig4 #hh4--;,fission of cocci4 ,illustration3 ,,fig4 #hi4--;,fission of bacteria4 #d4 ,the daughter cells may remain united by the gelatinous envelope for a variable time4 ,eventually they separate and themselves subdivide4 ,cultures on artificial media1 after growing in the same medium for some time--i4 e41 when the pabulum is exhausted--show involution forms 7,fig4 #ij71 well exemplified in cultures of ,b4 pestis on agar two days old1 ,b4 diphtheriae on potato four to six days old4 ,illustration3 ,,fig4 #ij4--;,involution forms4 ,they are of two classes1 viz43 7a7 ,involution forms characterised by alterations of shape 7,fig4 #ij74 7,not necessarily dead47 7b7 ,involution forms characterised by loss of staining power4 7,always dead47 ,resting ,stage4--,spore ,formation4--,conditions influencing spore formation3 ,in an old culture nothing may be left but spores4 ,it used to be supposed that spores were always formed1 so that the species might not become extinct1 when 7a7 ,the supply of nutrient was exhausted4 7b7 ,the medium became toxic from the accumulation of metabolic products4 7c7 ,the environment became unfavourable2 e4 g41 change of temperature4 ,this is not altogether correct2 e4 g41 the temperature at which spores are best formed is constant for each bacterium1 but varies with different species2 again1 aerobes require oxygen for sporulation1 but anaerobes will not spore in its presence4 7,a7 ,arthrogenous3 ,noted only in the micrococci4 ,one complete element resulting from ordinary fission becomes differentiated for the purpose1 enlarges1 and develops a dense cell wall4 ,one or more of the cells in a series may undergo this alteration4 ,this process is probably not real spore formation1 but merely relative increase of resistance4 ,these so-called arthrospores have never been observed to germinate1 nor is their resistance very marked1 as they fail to initiate new cultures1 after having been exposed to a temperature of #hj deg4 ,c4 for ten minutes4 7,b7 ,endogenous3 ,the cell protoplasm becomes differentiated and condensed into a spherical or oval mass 7very rarely cylindrical74 ,after further contraction the outer layers of the mass become still more highly differentiated and form a distinct spore membrane1 and the spore itself is now highly refractile4 ,it has been suggested1 and apparently on good grounds1 that the spore membrane consists of two layers1 the exosporium and the endosporium4 ,each cell forms one spore only1 usually in the middle1 occasionally at one end 7some exceptions1 however1 are recorded2 e4 g41 ,b4 inflatus74 ,the shape of the parent cell may be unaltered1 as in the anthrax bacillus1 or altered1 as in the tetanus bacillus1 and these points serve as the basis for a classification of spore-bearing bacilli1 as follows3 7,a7 ,cell body of the parent bacillus unaltered in shape 7,fig4 #ia1 a74 7,b7 ,cell of the parent bacillus altered in shape4 #a4 ,clostridium 7,fig4 #ia1 b73 ,rod swollen at the centre and attenuated at the poles2 spindle shape2 e4 g41 ,b4 butyricus4 #b4 ,cuneate 7,fig4 #ia1 c73 ,rods swollen slightly at one pole and more or less pointed at the other2 wedge-shaped4 ,illustration3 ,,fig4 #ia--,types of spore-bearing bacilli4 #c4 ,clavate 7,fig4 #ia1 d73 ,rods swollen at one pole and cylindrical 7unaltered7 at the other2 keyhole-shaped2 e4 g41 ,b4 chauvei4 #d4 ,capitate 7,fig4 #ia1 e73 ,rods with a spherical enlargement at one pole2 drumstick-shaped2 e4 g41 ,b4 tetani4 ,the endo-spores remain within the parent cell for a variable time 7in one case it is stated that germination of the spore occurs within the interior of the parent cell--endo-germination71 but are eventually set free1 as a result of the swelling up and solution of the cell membrane of the parent bacillus in the surrounding liquid1 or of the rupture of that membrane4 ,they then present the following characteristics3 #a4 ,well-formed1 dense cell membranes1 which renders them extremely difficult to stain1 but when once stained equally difficult to decolourise4 #b4 ,high refractility1 which distinguished them from vacuoles4 #c4 ,higher resistance than the parent organism to such lethal agents as heat1 desiccation1 starvation1 time1 etc41 this resistance being due to 7a7 ,low water contents of plasma of the spore4 7b7 ,low heat-conducting power of the spore 7c7 ,low permeability membrane4 ,this resistance varies somewhat with the particular species--e4 g41 some spores may resist boiling for a few minutes--but practically all are killed if the boiling is continued for ten minutes4 ,germination4--,when transplanted to suitable media and placed under favourable conditions1 the spores germinate1 usually within twenty-four to thirty-six hours1 and successively undergo the following changes which may be followed in hanging-drop cultures on a warm stage3 #a4 ,swell up slowly and enlarge1 through the absorption of water4 #b4 ,lose their refrangibility4 #c4 ,at this stage one of three processes 7but the particular process is always constant for the same species7 may be observed3 7a7 ,the spore grows out into the new bacillus without discarding the spore membrane 7which in this case now becomes the cell membrane72 e4 g41 ,b4 leptosporus4 7b7 ,it loses its spore membrane by solution2 e4 g41 ,b4 anthracis4 7c7 ,it loses its spore membrane by rupture4 ,in this process the rupture may be either polar 7at one pole only e4 g41 ,b4 butyricus71 or bipolar 7e4 g41 ,b4 sessile71 or equatorial2 7e4 g41 ,b4 subtilis74 ,in those cases where the spore membrane is discarded the cell membrane of the new bacillus may either be formed from-- 7a7 ,the inner layer of the spore membrane1 which has undergone a preliminary splitting into parietal and visceral layers2 e4 g41 ,b4 butyricus4 7b7 ,the outer layers of the cell protoplasm1 which become differentiated for that purpose2 e4 g41 ,b4 megatherium4 ,the new bacillus now increases in size1 elongates1 and takes on a vegetative growth--i4 e41 undergoes fission--the bacilli resulting from which may in their turn give rise to spores4 ,illustration3 ,,fig4 #ib4 ,simple4 ,illustration3 ,,fig4 #ic4 ,solution4 ,illustration3 ,,fig4 #id4 ,polar4 ,illustration3 ,,fig4 #ie4 ,bipolar4 ,illustration3 ,,fig4 #if4 ,equatorial4 ,food ,stuffs4--#a4 ,organic ,foods4-- 7a7 ,the pure parasites 7e4 g41 ,b4 leprae7 will not live outside the living body4 7b7 ,both saprophytic and facultative parasitic bacteria agree in requiring non-concentrated food4 7c7 ,the facultative parasites need highly organised foods2 e4 g41 proteids or other sources of nitrogen and carbon1 and salts4 7d7 ,the saprophytic bacteria are more easily cultivated2 e4 g41 #a4 ,some bacteria will grow in almost pure distilled water4 #b4 ,some bacteria will grow in pure solutions of the carbohydrates4 #c4 ,water is absolutely essential to the growth of bacteria4 ,food of a definite reaction is needed for the growth of bacteria4 ,as a general rule growth is most active in media which react slightly acid to phenolphthalein--that is1 neutral or faintly alkaline to litmus4 ,mould growth1 on the other hand1 is most vigourous in media that are strongly acid to phenolphthalein4 ,environment4--,the influence of physical agents upon bacterial life and growth is strongly marked4 #a4 ,atmosphere4--,the presence of oxygen is necessary for the growth of some bacteria1 and death follows when the supply is cut off4 ,such organisms are termed obligate aerobes4 ,some bacteria appear to thrive equally well whether supplied with or deprived of oxygen4 ,these are termed facultative anaerobes4 ,a third class will only live and multiply when the access of free oxygen is completely excluded4 ,these are termed obligate anaerobes4 #b4 ,temperature4--,practically no bacterial growth occurs below #e deg4 ,c1 and very little above #dj deg4 ,c4 #cj deg4 ,c4 to #cg deg4 ,c is the most favorable for the large majority of micro-organisms4 ,the maximum and minimum temperatures at which growth takes place1 as well as the optimum1 are fairly constant for each bacterium4 ,bacteria have been classified1 according to their optimum temperature1 into-- ,,min4 ,,opt4 ,,max4 #a4 ,psychrophilic bacteria 7chiefly water organisms7 #j deg4 ,c4 #ae deg4 ,c4 #cj deg4 ,c4 #b4 ,mesophilic bacteria 7includes pathogenic bacteria7 #ae deg4 ,c4 #cg deg4 ,c4 #de deg4 ,c4 #c4 ,thermophilic bacteria #de deg4 ,c4 #ee deg4 ,c4 #gj deg4 ,c4 ,the thermal death-point of an organism is another biological constant2 and is that temperature which causes the death of the vegetative forms when the exposure is continued for a period of ten minutes 7see pages #bih-cja74 #c4 ,light4--,many organisms are indifferent to the presence of light4 ,on the other hand1 light frequently impedes growth1 and alters to a greater or lesser extent the biochemical characters of the organisms--e4 g41 chromogenicity or power of liquefaction4 ,pathogenic bacteria undergo a progressive loss of virulence when cultivated in the presence of light4 #d4 ,movements4--,movements1 if slight and simply of a flowing character1 do not appear to injuriously affect the growth of bacteria2 but violent agitation1 such as shaking1 absolutely kills them4 ,a condition of perfect rest would seem to be that most conducive to bacterial growth4 ,the ,metabolic ,products of ,bacteria4--,pigment ,production4--,many micro-organisms produce one or more vivid pigments--yellow1 orange1 red1 violet1 fluorescent1 etc4--during the course of their life and growth4 ,the colouring matter usually exists as an intercellular excrementitious substance4 ,occasionally1 however1 it appears to be stored actually within the bodies of the bacteria4 ,the chromogenic bacteria are therefore classified1 in accordance with the final destination of the colouring matter they elaborate1 into-- ,chromoparous ,bacteria3 in which the pigment is diffused out upon and into the surrounding medium4 ,chromophorous ,bacteria3 in which the pigment is stored in the cell protoplasm of the organism4 ,parachromophorous ,bacteria3 in which the pigment is stored in the cell wall of the organism4 ,different species of chromogenic bacteria differ in their requirements as to environment1 for the production of their characteristic pigments2 e4 g41 some need oxygen1 light1 or high temperature2 others again favor the converse of these conditions4 ,light ,production4--,some bacteria1 and usually those originally derived from water1 whether fresh or salt1 exhibit marked phosphorescence when cultivated under suitable conditions4 ,these are classed as photogenic4 ,enzyme ,production4--,many bacteria produce soluble ferments or enzymes during the course of their growth1 as evidenced by the liquefaction of gelatine1 the clotting of milk1 etc4 ,these ferments may belong to either of the following well-recognised classes3 proteolytic1 diastatic1 invertin1 rennet4 ,toxin ,production4--,a large number1 especially of the pathogenic bacteria1 elaborate or secrete poisonous substances concerning which but little exact knowledge is available1 although many would appear to be enzymic in their action4 ,these toxins are usually differentiated into-- ,extracellular 7or ,soluble7 ,toxins3 those which are diffused into1 and held in solution by1 the surrounding medium4 ,intracellular 7or ,inseparate7 ,toxins3 those which are so closely bound up with the cell protoplasm of the bacteria elaborating them that up to the present time no means has been devised for their separation or extraction4 ,end-products of ,metabolism4--,under this heading are included-- ,organic ,acids 7e4 g41 lactic1 butyric1 etc474 ,alkalies 7e4 g41 ammonia74 ,aromatic ,compounds 7e4 g41 indol1 phenol74 ,reducing ,substances 7e4 g41 those reducing nitrates to nitrites74 ,gases 7e4 g41 sulphuretted hydrogen1 carbon dioxide1 etc474 ,and while the discussion of their formation1 etc41 is beyond the scope of a laboratory handbook1 the methods in use for their detection and separation come into the ordinary routine work and will therefore be described 7vide page #bgf et seq474 ,x4 ,,nutrient ,,media4 ,in order that the life and growth of bacteria may be accurately observed in the laboratory1 it is necessary-- #a4 ,to isolate individual members of the different varieties of micro-organisms4 #b4 ,to cultivate organisms1 thus isolated1 apart from other associated or contaminating bacteria--i4 e41 in pure culture4 ,for the successful achievement of these objects it is necessary to provide nutriment in a form suited to the needs of the particular bacterium or bacteria under observation1 and in a general way it may be said that the nutrient materials should approximate as closely as possible1 in composition and character1 to the natural pabulum of the organism4 ,the general requirements of bacteria as to their food-supply have already been indicated 7page #adb7 and many combinations of proteid and of carbohydrate have been devised1 from time to time1 on those lines4 ,these1 together with various vegetable tissues1 physiological or pathological fluid secretions1 etc41 are collectively spoken of as nutrient media or culture media4 ,the greater number of these media are primarily fluid1 but1 on account of the rapidity with which bacterial growth diffuses itself through a liquid1 it is impossible to study therein the characteristics of individual organisms4 ,many such media are1 therefore1 subsequently rendered solid by the addition of substances like gelatine or agar1 in varying proportions1 the proportions of such added material being generally mentioned when referring to the media2 e4 g41 #aj per cent4 gelatine1 #b per cent4 agar4 ,gelatine is employed for the solidification of those media it is intended to use in the cultivation of bacteria at the room temperature or in the cold incubator4 ,in the percentages usually employed1 gelatine media become fluid at #be deg4 ,c42 higher percentages remain solid at somewhat higher temperatures1 but the difficulty of filtering strong solutions of gelatine militates against their general use4 ,media1 on the other hand which have been solidified by the addition of agar1 only become liquid when exposed to #ij deg4 ,c4 for about ten minutes1 and again solidify when the temperature falls to #dj deg4 ,c4 ,when it becomes necessary to render these media fluid1 heat is applied1 upon the withdrawal of which they again assume their solid condition4 ,such media should be referred to as liquefiable media2 in point of fact1 however1 they are usually grouped together with the solid media4 ,,note4--,it must here be stated that the designation #aj per cent4 gelatine or #b per cent4 agar refers only to the quantity of those substances actually added in the process of manufacture1 and not to the percentage of gelatine or agar1 as the case may be1 present in the finished medium2 the explanation being that the commercial products employed contain a large proportion of insoluble material which is separated off by filtration during the preparation of the liquefiable media4 ,other media1 again--e4 g41 potato1 coagulated blood-serum1 etc4--cannot be again liquefied by physical means1 and these are spoken of as solid media4 ,the following pages detail the method of preparing the various nutrient media1 in ordinary use 7see also ,chapter ,,xi71 those which are only occasionally required for more highly specialised work are grouped together in ,chapter ,,xii4 ,it must be premised that scrupulous cleanliness is to be observed with regard to all apparatus1 vessels1 funnels1 etc41 employed in the preparation of media2 although in the preliminary stages of the preparation of most media absolute sterility of the apparatus used is not essential4 ,,meat ,,extract4 ,a watery solution of the extractives1 etc41 of lean meat 7usually beef7 forms the basis of several nutrient media4 ,this solution is termed meat extract and it has been determined empirically that its preparation shall be carried out by extracting half a kilo of moist meat with one litre of water4 ,for many purposes1 however1 it is more convenient to have a more concentrated extract2 one kilo of meat should therefore be extracted with one litre of water1 to form ,double ,strength meat extract4 ,it was customary at one time1 and is even now in some laboratories to use either shin of beef or beef-steak--both contain muscle sugar which often needs to be removed before the nutrient medium can be completed4 ,heart muscle 7bullock's heart or sheep's heart7 is much to be preferred and from the point of economy1 ease and cleanliness of manipulation1 and extractive value1 the imported frozen bullock's hearts provide the best extract4 ,meat extract 7,fleischwasser7 is prepared as follows3 #a4 ,measure #ajjj c4c4 of distilled water into a large flask 7or glass beaker1 or enamelled iron pot7 and add #ajjj grammes 7roughly1 #b-a_/b pounds7 of fresh lean meat--e4 g41 bullock's heart--finely minced in a mincing machine4 #b4 ,heat the mixture gently in a water-bath1 taking care that the temperature of the contents of the flask does not exceed #dj deg4 ,c4 for the first twenty minutes4 7,this dissolves out the soluble proteids1 extractives1 salts1 etc47 #c4 ,now raise the temperature of the mixture to the boiling-point1 and maintain at this temperature for ten minutes4 7,this precipitates some of the albumins1 the haemoglobin1 etc41 from the solution47 #d4 ,strain the mixture through sterile butter muslin or a perforated porcelain funnel1 then filter the liquid through ,swedish filter paper into a sterile normal litre flask1 and when cold make up to #ajjj c4c4 by the addition of distilled water--to replace the loss from evaporation4 #e4 ,if not needed at once1 sterilise the meat extract in bulk in the steam steriliser for twenty minutes on each of three consecutive days4 ,calf1 sheep1 or chicken flesh is occasionally substituted for the beef2 or the meat extract may be prepared from animal viscera1 such as brain1 spleen1 liver1 or kidneys4 ,,note4--,as an alternative method1 #e c4c4 of ,brand's meat juice or #c grammes of ,wyeth's beef juice1 or #aj grammes ,liebig's extract of meat 7,lemco7 may be dissolved in #ajjj c4c4 distilled water1 and heated and filtered as above to form ordinary or single strength meat extract4 ,media1 prepared from such meat extracts are1 however1 eminently unsatisfactory when used for the cultivation of the more highly parasitic bacteria2 although when working in tropical and subtropical regions their use is well-nigh compulsory4 ,reaction of ,meat ,extract4--,meat extract thus prepared is acid in its reaction1 owing to the presence of acid phosphates of potassium and sodium1 weak acids of the glycolic series1 and organic compounds in which the acid character predominates4 ,owing to the nature of the substances from which it derives its reaction1 the total acidity of meat extract can only be estimated accurately when the solution is at the boiling-point4 ,moreover1 it has been observed that prolonged boiling 7such as is involved in the preparation of nutrient media7 causes it to undergo hydrolytic changes which increase its acidity1 and the meat extract only becomes stable in this respect after it has been maintained at the boiling-point for forty-five minutes4 ,although meat extract always reacts acid to phenolphthalein1 it occasionally reacts neutral or even alkaline to litmus2 and again1 meat extract that has been rendered exactly neutral to litmus still reacts acid to phenolphthalein4 ,this peculiar behaviour depends upon two factors3 #a4 ,litmus is insensitive to many weak organic acids the presence of which is readily indicated by phenolphthalein4 #b4 ,dibasic sodium phosphate which is formed during the process of neutralisation is a salt which reacts alkaline to litmus1 but neutral to phenolphthalein4 ,in order1 therefore1 to obtain an accurate estimation of the reaction of any given sample of meat extract1 it is essential that-- #a4 ,the meat extract be previously exposed to a temperature of #ajj deg4 ,c4 for forty-five minutes4 #b4 ,the estimation be performed at the boiling-point4 #c4 ,phenolphthalein be used as the indicator4 ,the estimation is carried out by means of titration experiments against standard solutions of caustic soda1 in the following manner3 ,method of ,estimating the ,reaction4-- ,apparatus ,required3 ,solutions ,required3 #a4 #be c4c4 burette graduated #a4 #aj,n ,na,o,h1 accurately in tenths of a centimetre4 standardised4 #b4 #a c4c4 pipette graduated in #b4 n_/#a ,na,o,h1 accurately hundredths1 and provided standardised with rubber tube1 pinch-cock1 and delivery nozzle4 #c4 #be c4c4 measure 7cylinder or #c4 n_/#aj ,na,o,h1 accurately pipette1 calibrated for standardised4 #ih deg4 ,c4--not #ae deg4 ,c74 #d4 ,several #fj c4c4 conical #d4 #j4e per cent4 solution of beakers or ,erlenmeyer phenolphthalein in #ej per flasks4 cent4 alcohol4 #e4 ,white porcelain evaporating basin1 filled with boiling water and arranged over a gas flame as a water-bath4 #f4 ,bohemian glass flask1 fitted as a wash-bottle1 and filled with distilled water1 which is kept boiling on a tripod stand4 ,,method4--,arrange the apparatus as indicated in figure #ig4 7,a7 #a4 ,fill the burette with n_/#aj ,na,o,h4 #b4 ,fill the pipette with n_/#a ,na,o,h4 ,illustration3 ,,fig4 #ig4--;,arrangement of apparatus for titrating media4 #c4 ,measure #be c4c4 of the meat extract 7previously heated in the steamer at #ajj deg4 ,c4 for forty-five minutes7 into one of the beakers by means of the measure2 rinse out the measure with a very small quantity of boiling distilled water from the wash-bottle1 and then add this rinse water to the meat extract already in the beaker4 #d4 ,run in about #j4e c4c4 of the phenolphthalein solution and immerse the beaker in the water-bath1 and raise to the boil4 #e4 ,to the medium in the beaker run in n_/#aj ,na,o,h cautiously from the burette until the end-point is reached1 as indicated by the development of a pinkish tinge1 shown in figure #ih 7b74 ,note the amount of decinormal soda solution used in the process4 ,,note4--,just before the end-point is reached1 a very slight opalescence may be noted in the fluid1 due to the precipitation of dibasic phosphates4 ,after the true end-point is reached1 the further addition of about #j4e c4c4 of the decinormal soda solution will produce a deep magenta colour 7,fig4 #ih1 c71 which is the so-called end-point of the ,american ,committee of ,bacteriologists4 ,illustration3 ,,fig4 #ih4--;a1 ,sample of filtered meat extract or nutrient gelatine to which phenolphthalein has been added4 ,the medium is acid1 as evidenced by the unaltered colour of the sample4 b1 ,the same neutralised by the addition of n_/#aj ,na,o,h4 ,the production of this faint rose-pink colour indicates that the end-point1 or neutral point to phenolphthalein1 has been reached4 ,if such a sample is cooled down to say #cj deg4 or #bj deg4 ,c41 the colour will be found to become more distinct and decidedly deeper and brighter1 resembling that shown in c4 c1 ,also if1 after the end-point is reached1 a further #j4e c4c4 or #a4j c4c4 n_/#aj ,na,o,h be added to the sample1 the marked alkalinity is evidenced by the deep colour here shown4 7,b7 ,perform a control titration 7occasionally two controls may be necessary71 as follows3 #a4 ,measure #be c4c4 of the meat extract into one of the beakers1 wash out the measure with boiling water1 and add the phenolphthalein as in the first estimation4 #b4 ,run in n_/#a ,na,o,h from the pipette1 just short of the equivalent of the amount of deci-normal soda solution required to neutralise the #be c4c4 of medium4 7,for example1 if in the first estimation #e c4c4 of n_/#aj ,na,o,h were required to render #be c4c4 of medium neutral to phenolphthalein1 only add #j4dh c4c4 of n_/#a ,na,o,h47 ,immerse the beaker in the water-bath4 #c4 ,complete the titration by the aid of the n_/#aj ,na,o,h4 #d4 ,note the amount of n_/#aj ,na,o,h solution required to complete the titration1 and add it to the equivalent of the n_/#a ,na,o,h solution previously run in4 ,take the total as the correct estimation4 ,method of ,expressing the ,reaction4-- ,the reaction or titre of meat extract1 medium1 or any solution estimated in the foregoing manner1 is most conveniently expressed by indicating the number of cubic centimetres of normal alkali 7or normal acid7 that would be required to render one litre of the solution exactly neutral to phenolphthalein4 ,illustration3 ,,fig4 #ii4--,stock bottle for dekanormal soda solution4 ,the sign 7plus7 is prefixed to this number if the original solution reacts acid1 and the sign - 7minus7 if it reacts alkaline4 ,for example1 meat extract #aj1 indicates a sample of meat extract which reacts acid to phenolphthalein1 and would require the addition of #aj c4c4 of normal ,na,o,h per litre1 to neutralise it4 ,,note4--,such a solution would probably react alkaline to litmus4 ,conversely1 if as the result of our titration experiments we find that #be c4c4 of meat extract require the addition of #e c4c4 n_/#aj ,na,o,h to neutralise1 then #ajjj c4c4 of meat extract will require the addition of #bjj c4c4 n_/#aj ,na,o,h #bj c4c4 n_/#a ,na,o,h4 ,and this last figure1 #bj1 preceded by the sign 7i4 e41 #bj71 to signify that it is acid1 indicates the reaction of the meat extract4 ,,note4--,the standard soda solutions should be prepared by accurate measuring operations1 controlled by titrations1 from a stock solution of #aj,n ,na,o,h1 which should be very carefully standardised4 ,if a large supply is made or the consumption is small this stock solution must be kept in an aspirator bottle to which air can only gain access after it has been dried and rendered free from ,,co#b4 ,this may be done by first leading it over ,h#b,,so#d and soda lime1 or soda lime alone1 by some such arrangement as is shown in figure #ii1 which also shows a constant burette arrangement for the delivery of small measured quantities of the dekanormal soda solution4 ,,standardisation ,,of ,,media4 ,differences in the reaction of the medium in which it is grown will provoke not only differences in the rate of growth of any given bacterium1 but also well-marked differences in its cultural and morphological characters2 and nearly every organism will be found to affect a definite optimum reaction--a point to be carefully determined for each4 ,for most bacteria1 however1 the optimum usually approximates fairly closely to #aj2 and as experiment has shown that this reaction is the most generally useful for routine laboratory work1 it is the one which may be adopted as the standard for all nutrient media derived from meat extract4 ,briefly1 the method of standardising a litre of media to #aj consists in subtracting #aj from the initial titre of the medium mass2 the remainder indicates the number of cubic centimetres of normal soda solution that must be added to the medium1 per litre1 to render the reaction #aj4 ,standardising ,nutrient ,bouillon4--,for example1 #ajjj c4c4 bouillon are prepared2 at the first titration it is found #a4 #be c4c4 require the addition of #e4ej c4c4 n_/#aj ,na,o,h to neutralise4 ,two controls give the following results3 #b4 #be c4c4 require the addition of #e4gj c4c4 n_/#aj ,na,o,h to neutralise4 #c4 #be c4c4 require the addition of #e4fj c4c4 n_/#aj ,na,o,h to neutralise4 ,averaging these two controls1 #be c4c4 require the addition of #e4fe c4c4 n_/#aj ,na,o,h to neutralise1 and therefore #ajjj c4c4 require the addition of #bbf c4c4 n_/#aj ,na,o,h1 or #bb4fj c4c4 n_/#a ,na,o,h1 or #b4bf c4c4 n_/#aj ,na,o,h4 ,initial titre of the bouillon #bb4f1 and as such requires the addition of 7#bb4f c4c4 - #aj c4c47 #ab4f c4c4 of n_/#a ,na,o,h per litre to leave its finished reaction #aj4 ,but the three titrations1 each on #be c4c4 of medium1 have reduced the original bulk of bouillon to 7#ajjj - #ge c4c47 #ibe c4c4 ,the amount of n_/#a ,na,o,h required to render the reaction of this quantity of medium #aj may be deduced thus3 #ajjj c4c43#ibe c4c433#ab4f c4c43x4 ,then x #aa4fe c4c4 n_/#a ,na,o,h4 ,whenever possible1 however1 the required reaction is produced by the addition of dekanormal soda solution1 on account of the minute increase it causes in the bulk1 and the consequent insignificant disturbance of the percentage composition of the medium4 ,by means of a pipette graduated to #j4ja c4c4 it is possible to deliver very small quantities2 but if the calculated amount runs into thousandth parts of a cubic centimetre1 these are replaced by corresponding quantities of normal or even decinormal soda4 ,in the above example it is necessary to add #aa4fe c4c4 normal ,na,o,h or its equivalent1 #a4afe c4c4 dekanormal ,na,o,h4 ,the first being too bulky a quantity1 and the second inconveniently small for exact measurement1 the total weight of soda is obtained by substituting #a4af c4c4 dekanormal soda solution1 and either #j4je c4c4 of normal soda solution or #j4e c4c4 of decinormal soda solution4 ,standardising ,nutrient ,agar and ,gelatine4--,the method of standardising agar and gelatine is precisely similar to that described under bouillon4 ,,the ,,filtration ,,of ,,media4 ,fluid media are usually filtered through stout ,swedish filter paper 7occasionally through a porcelain filter candle71 and in order to accelerate the rate of filtration the filter paper should be folded in that form which is known as the physiological filter1 not in the ordinary quadrant shape1 as by this means a large surface is available for filtration and a smaller area in contact with the glass funnel supporting it4 ,to fold the filter proceed thus3 #a4 ,take a circular piece of filter paper and fold it exactly through its centre to form a semicircle 7,fig4 #ajj1 a74 #b4 ,fold the semicircle exactly in half to form a quadrant2 make the crease #b1 distinct by running the thumbnail along it1 then open the filter out to a semicircle again4 #c4 ,fold each end of the semicircle in to the centre and so form another quadrant2 smooth down the two new creases #c and #c;a1 thus formed and again open out to a semicircle4 #d4 ,the semicircle now appears as in figure #ajj1 a1 the dark lines indicating the creases already formed4 #e4 ,fold the point #a over to the point #c1 and #a;a to #c;a1 to form the creases #d and #d;a1 indicated in the diagram by the light lines4 ,fold point #a over to #c;a1 and #a;a to #c1 to form the creases #e and #e;a4 ,illustration3 ,,fig4 #ajj4--;,filter folding3 a1 ,filter folded in half1 showing creases2 b1 appearance of filter on completion of folding2 c1 filter opened out ready for use4 #f4 ,thus far the creases have all been made on the same side of the paper4 ,now subdivide each of the eight sectors by a crease through its centre on the opposite side of the paper1 indicated by the faint broken lines in the diagram4 ,fold up the filter gradually as each crease is made1 and when finished the filter has assumed the shape of a wedge1 as in figure #ajj1 b4 ,when opened out the filter assumes the shape represented in figure #ajj1 c4 ,the folded filter is next placed inside a glass funnel supported on a retort stand1 and moistened with hot distilled water before the filtration of the medium is commenced4 ,liquefiable solid media are filtered through a specially made filter paper--papier ,chardin--which is sold in boxes of twenty-five ready-folded filters4 ,illustration3 ,,fig4 #aja4--;,hot-water filter funnel and ring burner4 ,gelatine1 when properly made1 filters through this paper as quickly as bouillon does through the ,swedish filter paper1 and does not require the use of the hot-water funnel4 ,agar1 likewise1 if properly made1 filters readily1 although not at so rapid a rate as gelatine4 ,if badly egged1 and also during the winter months1 it is necessary to surround the glass funnel1 in which the filtration of the agar is carried on1 by a hot-water jacket4 ,this is done by placing the glass funnel inside a double-walled copper funnel--the space between the walls being filled with water at about #ij deg4 ,c4--and supporting the latter on a ring gas burner fixed to a retort stand 7,fig4 #aja74 ,the gas is lighted and the water jacket maintained at a high temperature until filtration is completed4 ,if the steam steriliser of the laboratory is sufficiently large1 it is sometimes more convenient to place the flask and filtering funnel bodily inside1 close the steriliser and allow filtration to proceed in an atmosphere of live steam1 than to use the gas ring and hot-water funnel4 ,,storing ,,media ,,in ,,bulk4 ,after filtration fill the medium into sterile litre flasks with cotton-wool plugs and sterilise in the steamer for twenty minutes on each of three consecutive days4 ,after the third sterilisation1 and when the flasks and contents are cool1 cut off the top of the cotton-wool plug square with the mouth of the flask2 push the plug a short distance down into the neck of the flask and fill in with melted paraffin wax to the level of the mouth4 ,when the wax has set the flasks are stored in a cool dark cupboard for future use4 ,illustration3 ,,fig4 #ajb4--,rubber cap closing store bottle4 a1 before1 and b1 after sterilizing4 ,this plan is not absolutely satisfactory1 although very generally employed on occasion1 and it is preferable to fill the medium into long-necked flint glass bottles 7the quart size1 holding nearly #ajjj c4c41 such as those in which ,pasteurised milk is retailed7 and to close the neck of the bottle by a special rubber cap4#c ,this cap is made of soft rubber1 the lower part1 dome-shaped with thin walls1 being slipped over the neck of the bottle 7,fig4 #ajb1 a74 ,the upper part is solid1 but with a sharp clean-cut 7made with a cataract or tenotomy knife7 running completely through its axis from the centre of the disc to the top of the dome4 ,during sterilisation the air in the neck of the bottle1 expanded by the heat1 is driven out through the valvular aperture in the solid portion of the stopper4 ,on removing the bottle from the steam chamber1 the liquid contracts as it cools1 and the pressure of the external air drives the solid piece of rubber down into the neck of the bottle1 and forces together the lips of the slit 7,fig4 #ajb1 b74 ,thus sealed1 the bottle will preserve its contents sterile for an indefinite period without loss from evaporation4 ,,tubing ,,nutrient ,,media4 ,after the final filtration1 the nutrient medium is usually tubed--i4 e41 filled into sterile tubes in definite measured quantities1 usually #aj c4c4 ,this process is sometimes carried out by means of a large separator funnel fitted with a three-way tap which communicates with a small graduated tube 7capacity #bj c4c4 and graduated in cubic centimetres7 attached to the side4 ,the shape of this piece of apparatus1 known as ,treskow's funnel1 renders it particularly liable to damage4 ,it is better1 therefore1 to arrange a less expensive piece of apparatus which will serve the purpose equally well 7,fig4 #ajc74 ,a ,geissler's three-way stop-cock has the tube on one side of the tap ground obliquely at its extremity1 and the tube on the opposite side cut off within #c cm4 of the tap4 ,the short tube is connected by means of a perforated rubber cork with a #aj cm4 length of stout glass tubing 7#a4e cm4 bore74 ,the third channel of the three-way tap is connected1 by means of rubber tubing1 with the nozzle of an ordinary separator funnel4 ,finally1 the receiving cylinder above the three-way tap is graduated in cubic centimetres up to #bj1 by pouring into it measured quantities of water and marking the various levels on the outside with a writing diamond4 ,fluid media containing carbohydrates are filled into fermentation tubes 7vide ,fig4 #ba72 or into ordinary media tubes which already have smaller tubes1 inverted1 inside them 7,fig4 #ajd71 to collect the products of growth of gas-forming bacteria4 ,when first filled1 the small tubes float on the surface of the medium after the first sterilisation nearly all the air is replaced by the medium1 and after the final sterilisation the gas tubes will be submerged and completely filled with the medium4 ,illustration3 ,,fig4 #ajc4--,separatory funnel and three-way tap arranged for tubing media4 ,illustration3 ,,fig4 #ajd4--;,gas tube 7,durham74 ,storing ,tubed ,media4--,media after being tubed are best stored by packing1 in the vertical position1 in oblong boxes having an internal measurement of #cg cm4 long by #ab cm4 wide by #aj cm4 deep4 ,each box 7,fig4 #aje7 has a movable partition formed by the vertical face of a weighted triangular block of wood1 sliding free on the bottom 7,fig4 #aje1 ,a72 or by a flat piece of wood sliding in a metal groove in the bottom of the box1 which can be fixed at any spot by tightening the thumbscrew of a brass guide rod which transfixes the partition 7,fig4 #aje1 ,b74 ,the front of the box is provided with a handle and a celluloid label for the name of the contained medium4 ,these boxes are arranged upon shelves in a dark cupboard--or preferably an iron safe--which should be rendered as nearly air-tight as possible1 and should have the words media stores painted on its doors4 ,illustration3 ,,fig4 #aje4--,medium box1 showing alternative partitions ,a and ,b4 ,,footnotes3 #c ,this rubber cap has been made for me by the ,holborn ,surgical ,instrument ,co41 ,thavies ,inn1 ,london1 ,w4 ,c4 ,,xi4 ,,culture ,,media4 ,,ordinary ,,or ,,stock ,,media4 ,nutrient ,bouillon4-- #a4 ,measure out double strength meat extract1 #ejj c4c41 into a litre flask and add #cjj c4c4 distilled water4 #b4 ,weigh out ,witte's peptone1 #aj grammes 7 #a per cent471 salt1 #e grammes 7 #j4e per cent471 and mix into a smooth paste with #bjj c4c4 of distilled water previously heated to #fj deg4 ,c4 7,be careful to leave no unbroken globular masses of peptone47 #c4 ,add the peptone emulsion to the meat extract in the flask and heat in the steamer for forty-five minutes 7to completely dissolve the peptone1 and to render the acidity of the meat extract stable74 #d4 ,estimate the reaction of the medium2 control the result2 render the reaction of the finished medium #aj 7vide page #aee74 #e4 ,heat for half an hour in the steamer at #ajj deg4 ,c4 7to complete the precipitation of the phosphates1 etc474 #f4 ,filter through ,swedish filter paper into a sterile flask4 #g4 ,fill into sterile tubes 7#aj c4c4 in each tube74 #h4 ,sterilise in the steamer for twenty minutes on each of three consecutive days--i4 e41 by the discontinuous method 7vide page #ce74 ,,note4--,as an alternative method when neither fresh nor frozen meat is available nutrient bouillon may be prepared from a commercial meat extract1 as follows3 ,lemco ,broth4-- #a4 ,measure out #bej c4c4 distilled water into a litre flask4 #b4 ,weigh out #aj grammes ,liebig's ,lemco ,meat ,extract on a piece of clean filter paper and add to the water in the flask4 ,shake the flask well to make an even emulsion of the meat extract4 #c4 ,weigh out ,witte's peptone 7#aj grammes71 salt 7#e grammes74 ,mix into smooth paste with #ajj c4c4 distilled water previously heated to #fj deg4 ,c4 #d4 ,add the peptone salt emulsion to the meat extract emulsion in the flask and add #fej c4c4 distilled water4 ,heat in the steamer for forty-five minutes4 #e4 ,standardise the medium and complete as for nutrient bouillon4 ,nutrient ,gelatine4-- #a4 ,weigh a #b-litre flask on a trip balance 7,fig4 #ajf7 and note the weight1 or counterpoise carefully4 ,illustration3 ,,fig4 #ajf4--,trip balance4 ,an extremely useful counterpoise is a small sheet-brass cylinder about #ch mm4 high and #ch mm4 in diameter1 with a funnel-shaped top and provided with a side tube by which its contents1 fine dust shot1 may be emptied out 7,fig4 #ajg74 ,illustration3 ,,fig4 #ajg4--;,counterpoise2 weight when empty1 #ce grammes2 when full of dust shot1 #bjj grammes4 #b4 ,measure out double strength meat extract1 #ejj c4c41 into the tared flask4 #c4 ,weigh out and mix #aj grammes of peptone1 #e grammes of salt1 and make into a thick paste with #aej c4c4 distilled water2 then add the emulsion to the meat extract in the flask2 also add #ajj grammes sheet gelatine cut into small pieces2 place the flask in the water-bath and raise to the boil4 ,illustration3 ,,fig4 #ajh4--;,arrangement of steam can and water-bath for the preparation of media4 #d4 ,arrange a #e-litre tin can 7with copper bottom1 such as is used in the preparation of distilled water7 by the side of the water bath1 fill the can with boiling water and place a lighted ,bunsen burner under it4 ,fit a long safety tube to the neck of the can and also a delivery tube1 bent twice at right angles2 adjust the tube to reach to the bottom of the interior of the flask containing the gelatine1 etc4 7,fig4 #ajh74 #e4 ,keep the water in the steam can vigourously boiling1 and so steam at #ajj deg4 ,c1 bubbling through the medium mass1 for ten minutes1 by which time complete solution of the gelatine is effected4 ,a certain amount of steam will condense as water in the medium flask during this process--hence the necessity for the use of double strength meat extract--but if the water bath is kept boiling this condensation will not exceed #ajj c4c4 #f4 ,weigh the flask and its contents2 then 7#aaaed grammes weight of the flask7 minus 7weight of the flask and its contents7 equals the weight of water required to make up the bulk to #a litre4 ,the addition of the requisite quantity of water is carried out as follows3 ,in one pan of the trip balance place the counterpoise of the tared flask 7or its equivalent in weights7 together with the weights making up the calculated medium weight4 ,in the opposite pan place the flask containing the medium mass4 ,now add boiling distilled water from a wash bottle until the two pans are exactly balanced4 #g4 ,titrate and estimate the reaction of the medium mass2 control the result4 ,calculate the amount of soda solution required to make the reaction of the medium mass #aj 7i4 e41 calculate for #ajjj c4c41 less the quantity used for the titrations74 #h4 ,add the necessary amount of soda solution and heat in the steamer at #ajj deg4 ,c4 for twenty minutes1 to precipitate the phosphates1 etc4 #i4 ,allow the medium mass to cool to #fj deg4 ,c4 ,well whip the whites of two eggs1 add to the contents of the flask and replace in the steamer at #ajj deg4 ,c4 for about half an hour 7until the egg-albumen has coagulated and formed large1 firm masses floating on and in clear gelatine74 #aj4 ,filter through papier ,chardin into a sterile flask4 #aa4 ,tube in quantities of #aj c4c4 #ab4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three consecutive days--i4 e41 by the discontinuous method4 ,nutrient ,agar-agar4-- #a4 ,weigh a #b-litre flask and note the weight--or counterpoise exactly4 #b4 ,measure out double strength meat extract1 #ejj c4c41 into the tared flask4 #c4 ,weigh out and mix #aj grammes of peptone1 #e grammes of salt1 and #bj grammes of powdered agar1 and make into a thick paste with #aej c4c4 distilled water1 and add to the meat extract in the flask2 place the flask in a water-bath4 #d4 ,arrange the steam can and water-bath as already directed 7for the preparation of gelatine7 and figured4 #e4 ,bubble live steam 7at #ajj deg4 ,c47 through the medium mass1 for twenty-five minutes1 by which time complete solution of the agar is effected4 #f4 ,now weigh the flask and its contents2 then 7#ajcee grammes weight of flask7 minus 7weight of flask and its contents7 equals the weight of water required to make up the bulk of the medium to #a litre4 ,add the requisite amount 7see preparation of gelatine1 page #aff1 step #f74 #g4 ,titrate1 and estimate the reaction of the medium mass2 control the result4 ,calculate the amount of soda solution required to make the reaction of the medium mass #aj 7i4 e41 calculated for #ajjj c4c41 less the quantity used for the titrations74 #h4 ,add the necessary amount of soda solution and replace in the steamer for twenty minutes 7to complete the precipitation of the phosphates1 etc474 #i4 ,allow the medium mass to cool to #fj deg4 ,c4 ,well whip the whites of two eggs1 add to the contents of the flask1 and replace in the steamer at #ajj deg4 ,c4 for about one hour 7until the egg-albumen has coagulated and formed large1 firm masses floating on and in clear agar47 #aj4 ,filter through papier ,chardin1 by the aid of a hot-water funnel1 if necessary 7,fig4 #aja71 into a sterile flask4 #aa4 ,tube in quantities of #aj c4c4 or #ae c4c4 #ab4 ,sterilise in the steamer at #ajj deg4 ,c4 for thirty minutes on each of three consecutive days--i4 e41 by the discontinuous method4 ,blood-serum 7,inspissated74-- #a4 ,sterilise cylindrical glass jar 7,fig4 #aji7 and its cover by dry heat1 or by washing first with ether and then with alcohol and drying4 #b4 ,collect blood at the slaughter house from ox or sheep in the sterile cylinder4 #c4 ,allow the vessel to stand for fifteen minutes for the blood to coagulate4 7,this must be done before leaving the slaughterhouse1 otherwise the serum will be stained with haemoglobin47 #d4 ,separate the clot from the sides of the vessel by means of a sterile glass rod 7the yield of serum is much smaller when this is not done71 and place the cylinder in the ice-chest for twenty-four hours4 #e4 ,remove the serum with sterile pipettes1 or syphon it off1 and fill into sterile tubes 7#e c4c4 in each7 or flasks4 #f4 ,heat tubes containing serum to #ef deg4 ,c4 in a water-bath for half an hour on each of two successive days4 #g4 ,on the third day1 heat the tubes1 in a sloping position1 in a serum inspissator to about #gb deg4 ,c4 7,a coagulum is formed at this temperature which is fairly transparent2 above #gb deg4 ,c41 a thick turbid coagulum is formed47 ,illustration3 ,,fig4 #aji4--;,blood-serum jar with wicker basket for transport4 ,the serum inspissator 7,fig4 #aaj7 in its simplest form is a double-walled rectangular copper box1 closed in by a loose glass lid1 and cased in felt or asbestos--the space between the walls is filled with water4 ,the inspissator is supported on adjustable legs so that the serum may be solidified at any desired slant1 and is heated from below by a ,bunsen burner controlled by a thermo-regulator4 ,the more elaborate forms resemble the hot-air oven 7,fig4 #bf7 in shape and are provided with adjustable shelves so that any desired obliquity of the serum slope can be obtained4 #h4 ,place the tubes in the incubator at #cg deg4 ,c4 for forty-eight hours in order to eliminate those that have been contaminated4 ,store the remainder in a cool place for future use4 ,alternative ,method4 ,steps #a-e as above4 #f4 ,sterilise the serum by the fractional method--that is1 by exposure in a water-bath to a temperature of #ef deg4 ,c4 for half an hour on each of six consecutive days2 store in the fluid condition4 #g4 ,coagulate in the inspissator when needed4 ,illustration3 ,,fig4 #aaj4--,serum inspissator4 ,serum ,water4-- ,this forms the basis of many useful media1 and is prepared as follows3 #a4 ,collect blood in the slaughterhouse 7see page #afh7 and when firmly clotted collect all the expressed serum and measure in a graduated cylinder4 #b4 ,for every #ajj c4c4 of serum add #cjj c4c4 distilled water and mix in a flask4 #c4 ,heat the mixture in the steamer at #ajj deg4 ,c4 for thirty minutes4 7,this destroys any diastatic ferment present in the serum and partially sterilises the fluid47 #d4 ,filter if turbid4 #e4 ,if not needed at once complete the sterilisation of the serum water by two subsequent steamings at #ajj deg4 ,c4 for twenty minutes at twenty-four hour intervals4 ,citrated ,blood ,agar4 ,guy's4-- #a4 ,kill a small rabbit with chloroform vapour1 and nail it out on a board 7as for a necropsy72 moisten the hair thoroughly with #b per cent4 solution of lysol4 #b4 ,sterilise several pairs of forceps1 scissors1 etc4 by boiling4 #c4 ,reflect the skin over the thorax with sterile instruments4 #d4 ,open the thoracic cavity by the aid of a fresh set of sterile instruments4 #e4 ,open the pericardium with another set of sterile instruments4 #f4 ,sear the surface of the left ventricle with a red-hot iron4 #g4 ,take a sterile capillary pipette 7,fig4 #ac1 c72 break off the sealed extremity with a pair of sterile forceps4 #h4 ,steady the heart in a pair of forceps and thrust the point of the pipette through the wall of the ventricle and through the seared area1 apply suction to the plugged end of the pipette and fill it with blood4 #i4 ,transfer the entire quantity of blood collected from the rabbit's heart to a small ,erlenmeyer flask containing a number of sterile glass beads and #e c4c4 concentrated sod4 citrate solution4 7,see page #cgh47 #aj4 ,agitate thoroughly and set aside for a couple of hours4 #aa4 ,melt up several tubes of nutrient agar 7see page #afg7 and cool to #db deg4 ,c4 #ab4 ,with a sterile #aj c4c4 graduated pipette transfer #a c4c4 citrated blood from the ,erlenmeyer flask to each tube of liquefied agar4 ,rotate the tube between the hands in order to diffuse the citrated blood evenly throughout the agar4 #ac4 ,place the tubes in a sloping position and allow the medium to set4 #ad4 ,place tubes of blood agar for forty-eight hours in the incubator at #cg deg4 ,c4 and at the end of that time eliminate any contaminated tubes4 #ae4 ,store such tubes as remain sterile for future use4 ,milk4-- #a4 ,pour #a litre of fresh cow's or goat's milk into a large separating funnel1 and heat in the steamer at #ajj deg4 ,c4 for one hour4 #b4 ,remove from the steamer and estimate the reaction of the milk 7normal cows' milk averages #ag74 ,if of higher acidity than #bj1 or lower than #aj1 reject this sample of milk and proceed with another supply of milk from a different source4 ,reject milk to which antiseptics have been added as preservatives4 #c4 ,allow the milk to cool1 when the fat or cream will rise to the surface and form a thick layer4 #d4 ,draw off the subnatant fat-free milk into sterile tubes 7#aj c4c4 in each74 #e4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of five successive days4 #f4 ,incubate at #cg deg4 ,c4 for forty-eight hours and eliminate any contaminated tubes4 ,store the remainder for future use4 ,litmus ,milk4-- #a4 ,prepare milk as described above1 sections #a to #c4 #b4 ,draw off the subnatant fat-free milk into a flask4 #c4 ,add sterile litmus solution1 sufficient to colour the milk a deep lavender4 #d4 ,tube1 sterilise1 etc41 as for milk4 ,nutrose ,agar 7,eyre74-- 7,this is a modification of the well known ,drigalski-,conradi medium originally introduced for the isolation of ,b4 typhosus74 #a4 ,collect #bej c4c4 perfectly fresh ox serum 7vide ,blood ,serum1 page #afh1 steps #a to #e7 and add to it #dej c4c4 sterile distilled water4 #b4 ,weigh out agar powder1 #bj grammes1 and emulsify it with #bej c4c4 of the cold serum water4 #c4 ,weigh out ,witte's peptone #aj grammes ,sodium chloride #e grammes ,nutrose #aj grammes and dissolve in #bjj c4c4 of serum water heated to #hj deg4 ,c4 #d4 ,mix the agar emulsion and the peptone-nutrose solution in a tared flask of #b-litre capacity and add a further #ajj c4c4 serum water4 #e4 ,complete the solution of the various ingredients by bubbling live steam through the flask as in making nutrient agar4 #f4 ,add further #bej c4c4 serum water4 #g4 ,weigh the flask and its contents3 then 7#ajde grammes weight of flask7 minus 7weight of flask and its present contents7 weight of fluid required to make up the bulk of the medium to #a litre4 ,add the requisite amount of sterile distilled water4 #h4 ,titrate and estimate the reaction of the medium mass4 ,then standardise to reaction of #b4e4 #i4 ,clarify with egg1 and filter as for nutrient agar4 7,in clarifying1 after the addition of the egg white the mixture should be in the steamer for full two hours47 #aj4 ,after filtration is complete measure the filtrate1 and to every #aej c4c4 of the medium add3 ,litmus solution 7,kahlbaum7 #bj c4c4 ,krystal violet aqueous solution 7#a3ajjj7 7,b4 ,hoechst7 #a4e c4c4 ,lactose #a4e grammes #aa4 ,tube in quantities of #ae c4c4 #ab4 ,sterilise in the steamer at #ajj deg4 ,c4 for thirty minutes on each of three successive days--i4 e41 by the discontinuous method for three days4 ,egg ,medium 7,dorset74-- #a4 ,prepare #ajjj c4c4 of a #j4he per cent4 solution of sodium chloride in a stout #b-litre flask4 #b4 ,sterilise in the autoclave at #abj deg4 ,c4 for twenty minutes4 ,cool to #bj deg4 ,c4 #c4 ,take #ab fresh eggs2 wash the shells first with water then with undiluted formalin3 allow the shells to dry4 #d4 ,break the eggs into a sterile graduated cylinder and measure the total volume of the mixed whites and yolks4 ,add one part sterile saline solution to three parts mixed eggs4 #e4 ,transfer this mixture to a large wide-mouthed stoppered bottle previously sterilised4 ,add sterile glass beads and shake thoroughly in a mechanical shaker for about thirty minutes1 or whip with an egg-whisk4 #f4 ,filter through coarse butter muslin into a sterile flask4 ,,note4--,a few drops of alcoholic solution of basic fuchsin 7sufficient to give a definite pink colour71 or a few drops of waterproof ,chinese ink added to the medium at this stage facilitates the subsequent fishing of colonies4 #g4 ,tube in quantities of #aj c4c4 #h4 ,solidify in the sloping position in the inspissator at #ge deg4 ,c4 for one hour4 #i4 ,place the tubes for forty-eight hours in the incubator at #cg deg4 ,c41 and eliminate any contaminated tubes4 ,to prevent drying1 #j4e c4c4 glycerine bouillon 7see page #bji7 may be added to each tube between steps #h and #i4 #aj4 ,cap those tubes of media which remain sterile with india-rubber caps and store for future use4 ,potato4-- #a4 ,choose fairly large potatoes1 wash them well1 and scrub the peel with a stiff nail-brush4 #b4 ,peel and take out the eyes4 #c4 ,remove cylinders from the longest diameter of each potato by means of an apple-corer or a large cork-borer 7i4 e41 one of about #a4d cm4 diameter74 ,the reaction of the fresh potato is strongly acid to phenolphthalein4 ,if1 therefore1 the potatoes are required to approximate #aj1 as for the cultivation of some of the vibrios1 the cylinders should be soaked in a #a per cent4 solution of sodium carbonate for thirty minutes4 #d4 ,cut each cylinder obliquely from end to end1 forming two wedge-shaped portions4 #e4 ,place a small piece of sterilised cotton-wool1 moistened with sterile water1 at the bottom of a sterile test-tube2 insert the potato wedge into the tube so that its base rests upon the cotton-wool4 ,now plug the tube with cotton-wool 7,fig4 #aaa74 #f4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of five consecutive days4 ,illustration3 ,,fig4 #aaa4--,potato tube4 ,,note4--,the cork borer reserved for cutting the potato cylinders should be silver electro-plated both inside and out1 and the knife used for dividing the cylinders should be of silver or silver plated4 ,when these precautions are adopted the potato wedges will retain their white color and will not show the discoloration so often observed when steel instruments are employed4 ,beer ,wort4--,wort is chiefly used as a medium for the cultivation of yeasts1 moulds1 etc41 both in its fluid form and also when made solid by the addition of gelatine or agar4 ,the wort is prepared as follows3 #a4 ,weigh out #bej grammes crushed malt and place in a #b-litre flask4 #b4 ,add #ajjj c4c4 distilled water1 heated to #gj deg4 ,c41 and close the flask with a rubber stopper4 #c4 ,place the flask in a water-bath regulated to #fj deg4 ,c4 and allow the maceration to continue for one hour4 #d4 ,strain through butter muslin into a clean flask and heat in the steamer for thirty minutes4 #e4 ,filter through ,swedish filter paper4 #f4 ,tube in quantities of #aj c4c4 or store in flasks4 #g4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three consecutive days4 ,the natural reaction of the wort should not be interfered with4 ,,note4--,it is sometimes more convenient to obtain unhopped#f beer wort direct from the brewery4 ,in this case it is diluted with an equal quantity of distilled water1 steamed for an hour1 filtered1 filled into sterile flasks or tubes1 and sterilised by the discontinuous method4 ,wort ,gelatine4-- #a4 ,measure out wort 7prepared as above71 #ijj c4c41 into a sterile flask4 #b4 ,weigh out gelatine1 #ajj grammes 7 #aj per cent471 and add it to the wort in the flask4 #c4 ,bubble live steam through the mixture for ten minutes1 to dissolve the gelatine4 #d4 ,cool to #fj deg4 ,c42 clarify with egg as for nutrient gelatine 7vide page #afd74 #e4 ,filter through papier ,chardin4 #f4 ,tube1 and sterilise as for nutrient gelatine4 ,wort ,agar4-- #a4 ,measure out wort 7as above71 #gjj c4c41 into a sterile flask4 #b4 ,weigh out powdered agar1 #bj grammes2 mix into a smooth paste with #bjj c4c4 of cold wort and add to the wort in the flask4 #c4 ,bubble live steam through the mixture for twenty minutes1 to dissolve the agar4 #d4 ,cool to #fj deg4 ,c42 clarify with egg as for nutrient agar 7vide page #afg74 #e4 ,filter through papier ,chardin1 using the hot-water funnel4 #f4 ,tube1 and sterilise as for nutrient agar4 ,peptone ,water 7,dunham74-- #a4 ,weigh out ,witte's peptone1 #aj grammes1 and salt1 #e grammes1 and emulsify with about #bej c4c4 of distilled water previously heated to #fj deg4 ,c4 #b4 ,pour the emulsion into a litre flask and make up to #ajjj c4c4 by the addition of distilled water4 #c4 ,heat in the steamer at #ajj deg4 ,c4 for thirty minutes4 #d4 ,filter through ,swedish filter paper4 #e4 ,tube in quantities of #aj c4c4 each4 #f4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three consecutive days4 ,sugar or ,carbohydrate ,media4-- ,formerly the ability of bacteria to induce hydrolytic changes in carbohydrate substances was observed only in connection with a few well-defined sugars1 but of recent years it has been shown that when using litmus as an indicator these so-called fermentation reactions facilitate the differentiation of closely allied species1 and the list of substances employed in this connection has been considerably extended4 ,the media prepared with them are now no longer regarded as special1 but are comprised in the stock media of the laboratory4 ,the chief of these substances are the following1 arranged in accordance with their chemical constitution3 ,monosaccharides ,dextrose 7glucose71 laevulose1 galactose1 mannose1 arabinose1 xylose4 ,disaccharides ,maltose1 lactose1 saccharose4 ,trisaccharides ,raffinose 7mellitose74 ,polysaccharides ,dextrin1 inulin1 starch1 glycogen1 amidon4 ,glucosides ,amygdalin1 coniferin1 salicin1 helicin1 phlorrhizin4 ,polyatomic alcohols ,trihydric1 ,glycerin4 ,tetrahydric1 ,erythrite4 ,pentahydric1 ,adonite4 ,hexahydric1 ,dulcite1 7dulcitol or melampirite71 isodulcite 7rhamnose71 mannite 7mannitol71 sorbite 7sorbitol71 inosite4 ,these substances should be obtained from ,kahlbaum 7of ,berlin72 in the pure form1 and when possible as large crystals1 and the method of preparing a medium containing either of them may be exemplified by describing ,dextrose ,solution4 ,dextrose ,solution4-- #a4 ,weigh out ,peptone #bj grammes ,glucose #aj grammes and grind together in a mortar2 then emulsify in #ajj c4c4 of distilled water heated to #fj deg4 ,c4 #b4 ,place in a flask and add ,distilled water #hej c4c4 #c4 ,steam in the steamer at #ajj deg4 ,c4 for twenty minutes to dissolve the peptone and glucose4 #d4 ,add ,kubel-,tiemann litmus solution 7,kahlbaum7 #ej c4c4 7,the substances enumerated above react acid to phenolphthalein1 but variously toward the neutral litmus solution4 ,to such as react acid1 add very cautiously n_/#a sodium hydrate solution to the medium in bulk until the neutral tint has returned74 #e4 ,fill into tubes in which have previously been placed the inverted ,durham's gas tubes4 #f4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three successive days4 ,,note4--,on no account should these media be sterilised in the autoclave1 as temperatures above #ajj deg4 ,c4 themselves induce hydrolytic changes in the substances in question4 ,it is equally important that the twenty minutes should not be exceeded in sterilisation1 as neglect of this precaution may discolour the litmus or lead to the production of yellowish tints when the tubes are subsequently inoculated with acid-forming bacteria4 ,neutral ,litmus ,solution4 ,the most satisfactory is the ,kubel-,tiemann1 prepared by ,kahlbaum4 ,it can however be made in the laboratory as follows3 #a4 ,weigh out ,commercial litmus #ej grammes1 and place in a well stoppered #ejj c4c4 bottle2 measure out and add #cjj c4c4 alcohol #ie per cent4 #b4 ,shake well at least once a day for seven days--the alcohol acquires a green colour4 #c4 ,decant off the green alcohol and fill a further #cjj c4c4 #ie per cent4 alcohol into the bottle and repeat the shaking4 #d4 ,repeat this process until on adding fresh alcohol the fluid only becomes tinged with violet4 #e4 ,pour off the alcohol1 leaving the litmus as dry as possible4 ,connect up the bottle to an air pump and evaporate off the last traces of alcohol4 #f4 ,transfer the dry litmus to a litre flask1 measure in #fjj c4c4 distilled water and allow to remain in contact #bd hours with frequent shakings4 #g4 ,filter the solution into a clean flask and add one or two drops of pure concentrated sulphuric acid until the litmus solution is distinctly wine-red in colour4 #h4 ,add excess of pure solid baryta and allow to stand until the reaction is again alkaline4 #i4 ,filter4 #aj4 ,bubble ,,co#b through the solution until reaction is definitely acid4 #aa4 ,sterilise in the steamer at #ajj deg4 ,c4 for thirty minutes on each of three consecutive days4 ,this sterilises the solution and also drives off the carbon dioxide1 leaving the solution neutral4 ,media for anaerobic cultures4 ,in addition to the foregoing media1 all of which can be1 and are employed in the cultivation of anaerobic bacteria1 certain special media containing readily oxidised substances are commonly used for this purpose4 ,the principal of these are as follows3 ,bile ,salt ,broth 7,mac,conkey74-- #a4 ,weigh out ,witte's peptone1 #bj grammes 7 #b per cent471 and emulsify with #bjj c4c4 distilled water previously warmed to #fj deg4 ,c4 #b4 ,weigh out sodium taurocholate 7commercial71 #e grammes 7 #j4e per cent471 and glucose1 #e grammes 7 #j4e per cent471 and dissolve in the peptone emulsion4 #c4 ,wash the peptone emulsion into a flask with #hjj c4c4 distilled water1 and heat in the steamer at #ajj deg4 ,c4 for twenty minutes4 #d4 ,filter through ,swedish filter paper into a sterile flask4 #e4 ,add sterile litmus solution sufficient to colour the medium to a deep purple1 usually #ac per cent4 required4 #f4 ,fill1 in quantities of #aj c4c41 into tubes containing small gas tubes 7vide ,fig4 #ajd1 page #afa74 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three consecutive days4 ,glucose ,formate ,bouillon 7,kitasato74-- #a4 ,measure out nutrient bouillon1 #ajjj c4c4 7vide page #afc1 sections #a to #f74 #b4 ,weigh out glucose1 #bj grammes 7 #b per cent471 sodium formate1 #d grammes 7 #j4d per cent471 and dissolve in the fluid4 #c4 ,tube1 and sterilise as for bouillon4 ,glucose ,formate ,gelatine 7,kitasato74-- #a4 ,prepare nutrient gelatine 7vide page #afd1 sections #a to #g7 and measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b per cent471 and sodium formate1 #d grammes 7 #j4d per cent471 and dissolve in the hot gelatine4 #c4 ,filter through papier ,chardin4 #d4 ,tube1 and sterilise as for nutrient gelatine4 ,glucose ,formate ,agar 7,kitasato74-- #a4 ,prepare nutrient agar 7vide page #afg1 sections #a to #h74 ,measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b per cent471 sodium formate1 #d grammes 7 #j4d per cent471 and dissolve in the agar4 #c4 ,tube1 and sterilise as for nutrient agar4 ,sulphindigotate ,bouillon 7,weyl74-- #a4 ,measure out nutrient bouillon 7vide page #afc1 sections #a to #f #ajjj c4c474 #b4 ,weigh out glucose1 #bj grammes 7 #b per cent471 sodium sulphindigotate1 #a gramme 7 #j4a per cent471 and dissolve in the fluid4 #c4 ,tube1 and sterilise as for bouillon4 ,sulphindigotate ,gelatine 7,weyl74-- #a4 ,prepare nutrient gelatine 7vide page #afd1 sections #a to #g74 ,measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b per cent471 and sodium sulphindigotate1 #a gramme 7 #j4a per cent471 and dissolve in the hot gelatine4 #c4 ,filter through papier ,chardin4 #d4 ,tube1 and sterilise as for nutrient gelatine4 ,sulphindigotate ,agar4-- #a4 ,prepare nutrient agar 7vide page #afg1 sections #a to #h74 ,measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b per cent471 sodium sulphindigotate1 #a gramme 7 #j4a per cent471 and dissolve in the hot agar4 #c4 ,tube1 and sterilise as for nutrient agar4 ,,note4--,the ,sulphindigotate media are of a blue colour1 which during the growth of anaerobic bacteria is oxidised and decolourised to a light yellow4 ,,footnotes3 #d ,this figure is obtained by adding together #a litre water1 #ajjj grammes2 #aj per cent4 gelatine1 #ajj grammes2 #a per cent4 peptone1 #aj grammes2 #j4e per cent4 salt1 #e grammes2 total1 #aaae grammes4 ,modifications of the above process1 as to quantities and percentages1 will require corresponding alterations of the figures4 ,the average weight of a measured litre of #aj per cent4 nutrient gelatine when prepared in this way after filtration is #ajhj grammes4 #e ,this figure is obtained by adding together #a litre of water 7meat extract71 #ajjj grammes2 #b per cent4 agar1 #bj grammes2 #a per cent4 peptone1 #aj grammes2 #j4e per cent4 salt1 #e grammes--total #ajce grammes4 ,modifications of the process as to quantities or percentages will necessitate corresponding alterations in the calculated medium figure4 ,the average weight of a measured litre of #b per cent4 agar when prepared in this way1 after filtration1 is #ajaj4e grammes4 #f ,hopped wort exerts a toxic effect upon many bacteria1 including the lactic acid bacteria4 ,,xii4 ,,special ,,media4 ,in this chapter are collected a number of media which have been elaborated by various workers for special purposes1 grouped together under headings which indicate their chief utility4 ,in many instances the name of the originator of the medium is given1 but without reference to his original instructions1 since these are in many cases inadequate to the requirements of the isolated worker1 who would probably fail to reproduce the medium in a form giving the results attributed to it by its author4 ,such modifications have therefore been introduced as make for uniformity between the different batches of media4 ,a considerable number of coloured media1 chiefly intended for work with intestinal bacteria1 have been included2 but beyond the fact that the author's modification of the ,drigalski-,conradi medium has been included amongst the routine media of the laboratory1 no comment has been made upon their relative values1 since only by observation and practice can the skill necessary to utilise their full value be acquired4 ,the instructions as to sterilisation are rarely given in full2 the routine method of exposure in the steam steriliser at #ajj deg4 ,c4 7without pressure7 for twenty minutes on each of three successive days for all fluid media1 and thirty minutes on each of three successive days for all liquefiable or solid media must be carried out2 and only when these general rules are to be departed from are further details given4 ,media for the ,study of the ,chemical ,composition of ,bacteria4 ,asparagin ,medium 7,uschinsky74-- #a4 ,weigh out and mix ,asparagin #c4d grammes ,ammonium lactate #aj4j grammes ,sodium chloride #e4j grammes ,magnesium sulphate #j4b gramme ,calcium chloride #j4a gramme ,acid potassium phosphate 7,,kh#b,,po#d7 #a4j gramme #b4 ,dissolve the mixture in distilled water #ajjj c4c4 #c4 ,add glycerine1 #dj c4c4 #d4 ,tube1 and sterilise as for nutrient bouillon4 ,asparagin ,medium 7,frankel and ,voges74-- #a4 ,weigh out and mix ,asparagin #d grammes ,sodium phosphate1 7,na#b;,,hpo#d7 #ab,,oh #b grammes ,ammonium lactate #f grammes ,sodium chloride #e grammes and dissolve in ,distilled water #ajjj c4c4 #b4 ,tube1 and sterilise as for nutrient bouillon4 ,,note4--,either of the above asparagin media1 after the addition of #aj per cent4 gelatine or #a4e per cent4 agar1 may be advantageously employed in the solid condition4 ,proteid ,free ,broth 7,uschinsky74-- #a4 ,weigh out and mix ,calcium chloride #j4a gramme ,magnesium sulphate #j4b gramme ,acid potassium phosphate 7,,kh#b,,po#d7 #b4j grammes ,potassium aspartate #c4j grammes ,sodium chloride #e4j grammes ,ammonium lactate #f4j grammes #b4 ,dissolve the mixture in distilled water #ajjj c4c4 #c4 ,add glycerine #cj c4c4 #d4 ,tube and sterilise as for nutrient broth4 ,media for the ,study of ,biochemical ,reaction4 ,inosite-free ,media--,bouillon 7,durham74-- #a4 ,prepare meat extract1 #ajjj c4c4 7vide page #adh71 from bullock's heart which has been hung for a couple of days4 #b4 ,prepare nutrient bouillon 7#aj71 #ajjj c4c4 7vide1 page #afa71 from the meat extract1 and store in #a-litre flask4 #c4 ,inoculate the bouillon from a pure cultivation of the ,b4 lactis aerogenes1 and incubate at #cg deg4 ,c4 for forty-eight hours4 #d4 ,heat in the steamer at #ajj deg4 ,c4 for twenty minutes to destroy the bacilli and some of their products4 #e4 ,estimate the reaction of the medium and if necessary restore to #aj4 #f4 ,inoculate the bouillon from a pure cultivation of the ,b4 coli communis and incubate at #cg deg4 ,c4 for forty-eight hours4 #g4 ,heat in the steamer at #ajj deg4 ,c4 for twenty minutes4 ,now fill two fermentation tubes with the bouillon1 tint with litmus solution1 and sterilise2 inoculate with ,b4 lactis aerogenes4 ,if no acid or gas is formed1 the bouillon is in a sugar-free condition2 but if acid or gas is present1 again make the bouillon in the flask #aj1 reinoculate with one or other of the above-mentioned bacteria1 and incubate2 then test again4 ,repeat this till neither acid nor gas appears in the medium when used for the cultivation of either of the bacilli referred to above4 #h4 ,after the final heating1 stand the flask in a cool place and allow the growth to sediment4 ,filter the supernatant broth through ,swedish filter paper4 ,if the filtrate is cloudy1 filter through a porcelain filter candle4 #i4 ,tube1 and sterilise as for bouillon4 ,bouillon prepared in the above-described manner will prove to be absolutely sugar-free2 and from it may be prepared nutrient sugar-free gelatine or agar1 by dissolving in it the required percentage of gelatine or agar respectively and completing the medium according to directions given on pages #aff and #afg4 ,the most important application of inosite-free bouillon is its use in the preparation of sugar bouillons1 whether glucose1 maltose1 lactose1 or saccharose1 of exact percentage composition4 ,sugar 7,dextrose7 ,bouillon4-- #a4 ,measure out nutrient bouillon1 #ajjj c4c4 7vide page #afc1 sections #a to #f7 or sugar-free bouillon 7vide supra74 #b4 ,weigh out glucose 7anhydrous71 #bj grammes 7 #b per cent471 and dissolve in the fluid4 #c4 ,tube1 and sterilise as for bouillon4 ,ordinary commercial glucose serves the purpose equally well1 but is not recommended1 as during the process of sterilisation it causes the medium to gradually deepen in colour4 ,,note4--,in certain cases a corresponding percentage of lactose1 maltose1 or saccharose is substituted for glucose4 ,sugar ,gelatine4-- #a4 ,prepare nutrient gelatine 7vide page #afd1 sections #a to #g74 ,measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b per cent471 and dissolve in the hot gelatine4 #c4 ,filter through papier ,chardin4 #d4 ,tube1 and sterilise as for nutrient gelatine4 ,sugar ,agar4-- #a4 ,prepare nutrient agar 7vide page #afg1 sections #a to #h74 ,measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b per cent471 and dissolve in the clear agar4 #c4 ,tube1 and sterilise as for nutrient agar4 ,,note4--,other sugar media are prepared by substituting a corresponding percentage of lactose1 maltose 7or any other of the substances referred to under ,sugar ,media1 page #agg7 for the glucose4 ,iron ,bouillon4-- #a4 ,measure out nutrient bouillon1 #ajjj c4c4 7vide page #ada1 sections #a to #f74 #b4 ,weigh out ferric tartrate1 #a gramme 7 #j4a per cent471 and dissolve it in the bouillon4 #c4 ,tube1 and sterilise as for bouillon4 ,,note4--,the lactate of iron may be substituted for the tartrate4 ,lead ,bouillon4-- #a4 ,measure out nutrient bouillon1 #ajjj c4c4 7vide page #afc1 sections #a to #f74 #b4 ,weigh out lead acetate1 #a gramme 7 #j4a per cent471 and dissolve it in the bouillon4 #c4 ,tube1 and sterilise as for bouillon4 ,nitrate ,bouillon4-- #a4 ,measure out nutrient bouillon1 #ajjj c4c4 7vide page #afc1 sections #a to #f74 #b4 ,weigh out potassium nitrate1 #e grammes 7 #j4e per cent471 and dissolve it in the bouillon4 #c4 ,tube1 and sterilise as for bouillon4 ,,note4--,the nitrate of sodium or ammonium may be substituted for that of potassium1 or the salt may be added in the proportion of from #j4a to #a per cent4 to meet special requirements4 ,iron ,peptone ,solution 7,pakes74-- #a4 ,weigh out peptone1 #cj grammes1 and emulsify it with #bjj c4c4 tap water1 previously heated to about #fj deg4 ,c4 #b4 ,wash the emulsion into a litre flask with #hjj c4c4 tap water4 #c4 ,weigh out salt1 #e grammes1 and sodium phosphate1 #c grammes1 and dissolve in the mixture in the flask4 #d4 ,heat the mixture in the steamer at #ajj deg4 ,c4 for thirty minutes1 to complete the solution of the peptone1 and filter into a clean flask4 #e4 ,fill into tubes in quantities of #aj c4c4 each4 #f4 ,add to each tube #j4a c4c4 of a #b per cent4 neutral solution of ferric tartrate4 7,a yellowish-white precipitate forms47 #g4 ,sterilise as for nutrient bouillon4 ,lead ,peptone ,solution4-- ,prepare as for iron peptone solution but in step #f substitute #j4a c4c4 of a #a per cent4 neutral aqueous solution of lead acetate4 ,nitrate ,peptone ,solution 7,pakes74-- #a4 ,weigh out ,witte's peptone1 #aj grammes1 and emulsify it with #bjj c4c4 ammonia-free distilled water previously heated to #fj deg4 ,c4 #b4 ,wash the emulsion into a flask and make up to #ajjj c4c41 with ammonia-free distilled water4 #c4 ,heat in the steamer at #ajj deg4 ,c4 for twenty minutes4 #d4 ,weigh out sodium nitrate1 #a gramme1 and dissolve in the contents of the flask4 #e4 ,filter through ,swedish filter paper4 #f4 ,tube1 and sterilise as for nutrient bouillon4 ,litmus ,bouillon4-- #a4 ,measure out nutrient bouillon1 #ajjj c4c4 7vide page #afc1 sections #a to #f74 #b4 ,add sufficient sterile litmus solution to tint the medium a dark lavender colour4 7,media rendered #aj will usually react very faintly alkaline or occasionally neutral to litmus47 #c4 ,tube1 and sterilise as for bouillon4 ,rosolic ,acid ,peptone ,solution4-- #a4 ,weigh out rosolic acid 7corallin71 #j4e gramme1 and dissolve it in #hj per cent4 alcohol1 #ajj c4c4 ,keep this as a stock solution4 #b4 ,measure out peptone water 7,dunham71 #ajj c4c41 and rosolic acid solution1 #b c4c41 and mix4 #c4 ,heat in the steamer at #ajj deg4 ,c4 for thirty minutes4 #d4 ,filter through ,swedish filter paper4 #e4 ,tube1 and sterilise as for nutrient bouillon4 ,capaldi-,proskauer ,medium1 ,no4 ,i4-- #a4 ,weigh out and mix ,sodium chloride #b4j grammes ,magnesium sulphate #j4a gramme ,calcium chloride #j4b gramme ,monopotassium phosphate #b4j grammes #b4 ,dissolve in water #ajjj c4c4 in a #b-litre flask #c4 ,weigh out and mix ,asparagin #b grammes ,mannite #b grammes and add to contents of flask4 #d4 ,measure out #be c4c4 of the solution and titrate it against decinormal sodic hydrate1 using litmus as the indicator4 ,control the result and estimate the amount of sodic hydrate necessary to be added to render the remainder of the solution neutral to litmus4 ,add this quantity of sodic hydrate4 #e4 ,filter4 #f4 ,add litmus solution #dg4e c4c4 7 #e per cent474 #g4 ,tube1 and sterilise as for nutrient bouillon4 ,capaldi-,proskauer ,medium ,no4 ,,ii4-- #a4 ,weigh out and mix ,peptone #bj grammes ,mannite #a gramme #b4 ,dissolve in water #ajjj c4c4 in a #b-litre flask4 #c4 ,neutralise to litmus as in ,no4 ,i 7vide supra1 ,step #d74 #d4 ,filter4 #e4 ,add litmus solution #dg4e c4c4 7 #e per cent474 #f4 ,tube1 and sterilise as for nutrient bouillon4 ,urine ,media4 ,bouillon4-- #a4 ,collect freshly passed urine in sterile flask4 #b4 ,place the flask in the steamer at #ajj deg4 ,c4 for thirty minutes4 #c4 ,filter through two thicknesses of ,swedish filter paper4 #d4 ,tube1 and sterilise as for nutrient bouillon4 7,leave the reaction unaltered47 ,urine ,gelatine4-- #a4 ,collect freshly passed urine in sterile flask4 #b4 ,take the specific gravity1 and1 if above #ajaj1 dilute with sterile water until that gravity is reached4 #c4 ,estimate 7with control7 at the boiling-point1 and note the reaction of the urine4 #d4 ,weigh out gelatine1 #aj per cent41 and add to the urine in the flask4 #e4 ,heat in the steamer at #ajj deg4 ,c4 for one hour to dissolve the gelatine4 #f4 ,estimate the reaction and add sufficient caustic soda solution to restore the reaction of the medium mass to the equivalent of the original urine4 #g4 ,cool to #fj deg4 ,c4 and clarify with egg as for nutrient gelatine 7vide page #aff74 #h4 ,filter through papier ,chardin4 #i4 ,tube1 and sterilise as for nutrient gelatine4 ,urine ,gelatine 7,heller74-- #a4 ,collect freshly passed urine in sterile flask4 #b4 ,filter through animal charcoal to remove part of the colouring matter4 #c4 ,take the specific gravity1 and if above #ajaj1 dilute with sterile water till this gravity is reached4 #d4 ,add ,witte's peptone1 #a per cent42 salt1 #j4e per cent42 gelatine1 #aj per cent4 #e4 ,heat in the steamer at #ajj deg4 ,c4 for one hour1 to dissolve the gelatine1 etc4 #f4 ,add normal caustic soda solution in successive small quantities1 and test the reaction from time to time with litmus paper1 until the fluid reacts faintly alkaline4 #g4 ,cool to #fj deg4 ,c4 and clarify with egg as for nutrient gelatine 7vide page #aff74 #h4 ,filter through papier ,chardin4 #i4 ,tube1 and sterilise as for nutrient gelatine4 ,urine ,agar4-- #a4 ,collect freshly passed urine in sterile flask4 #b4 ,take the specific gravity and if above #ajaj1 dilute with sterile water till this gravity is reached4 #c4 ,weigh out #a4e per cent4 or #b per cent4 powdered agar1 and add it to the urine4 #d4 ,heat in the steamer at #ajj deg4 ,c4 for ninety minutes to dissolve the agar4 #e4 ,cool to #fj deg4 ,c4 and clarify with egg as for nutrient agar 7vide page #afh74 #f4 ,filter through papier ,chardin1 using the hot-water funnel4 #g4 ,tube1 and sterilise as for nutrient agar4 7,leave the reaction unaltered47 ,serum ,sugar ,media 7,hiss74-- ,in these media the fermentation of carbohydrate substance by bacterial action is indicated by the coagulation of the serum proteids in addition to the production of an acid reaction4 ,serum ,dextrose ,water 7,hiss74-- #a4 ,measure out into a litre flask ,serum water 7,see page #agj7 #ajjj c4c4 #b4 ,weigh out ,dextrose #aj grammes and dissolve in the serum water4 #c4 ,filter through ,swedish filter paper4 #d4 ,measure out and add to the medium ,litmus solution 7,kahlbaum7 #ej c4c4 #e4 ,tube in quantities of #aj c4c4 and sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three successive days4 ,laevulose1 galactose1 maltose1 lactose1 etc41 can be substituted in similar amounts for dextrose and the medium completed as above4 ,omeliansky's ,nutrient ,fluid 7,for ,cellulose ,fermenters74-- #a4 ,weigh out and mix ,potassium phosphate #d4j grammes ,magnesium sulphate #b4j grammes ,ammonium sulphate #d4j grammes ,sodium chloride #j4be gramme #b4 ,dissolve in distilled water #djjj c4c4 #c4 ,flask in quantities of #bej c4c4 #d4 ,weigh out and add #e grammes precipitated chalk to each flask4 #e4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three successive days4 ,media for the ,study of ,chromogenic ,bacteria4 ,milk ,rice 7,eisenberg74-- #a4 ,measure out nutrient bouillon1 #gj c4c41 and milk1 #baj c4c41 and mix thoroughly4 #b4 ,weigh out rice powder1 #ajj grammes1 and rub it up in a mortar with the milk and broth mixture4 #c4 ,fill the paste into sterile capsules1 spreading it out so as to form a layer about #j4e cm4 thick1 over the bottom of each4 #d4 ,heat over a water-bath at #ajj deg4 ,c4 until the mixture solidifies4 #e4 ,replace the lids of the capsules4 ,sterilise in the steamer at #ajj deg4 ,c4 for thirty minutes on each of three consecutive days4 7,a solid medium of the colour of cafe au lait is thus produced47 ,milk ,rice 7,soyka74-- #a4 ,measure out nutrient bouillon1 #ej c4c41 and milk1 #aej c4c41 and mix thoroughly4 #b4 ,weigh out rice powder1 #ajj grammes1 and rub it up in a mortar with the milk and broth mixture4 #c4 ,fill the paste into sterile capsules1 to form a layer over the bottom of each4 #d4 ,replace the lids of the capsules4 #e4 ,sterilise in the steamer at #ajj deg4 ,c4 for thirty minutes on each of three consecutive days4 7,a pure white1 opaque medium is thus formed47 ,media for the ,study of ,phosphorescent and ,photogenic ,bacteria4 ,fish ,bouillon4-- #a4 ,weigh out herring1 mackerel1 or cod1 #ejj grammes1 and place in a large porcelain beaker 7or enamelled iron pot74 #b4 ,weigh out sodium chloride1 #bf4e grammes2 potassium chloride1 #j4ge gramme2 magnesium chloride1 #c4be grammes2 and dissolve in #ejj c4c4 distilled water4 ,add the solution to the fish in the beaker4 #c4 ,place the beaker in a water-bath and proceed as in preparing meat extract--i4 e41 heat gently at #dj deg4 ,c4 for twenty minutes1 then rapidly raise the temperature to1 and maintain at1 the boiling-point for ten minutes4 #d4 ,strain the mixture through butter muslin into a clean flask4 #e4 ,weigh out peptone1 #e grammes1 and emulsify with about #bjj c4c4 of the hot fish water2 incorporate thoroughly with the remainder of the fish water in the flask4 #f4 ,heat in the steamer at #ajj deg4 ,c4 for twenty minutes to complete the solution of the peptone4 #g4 ,filter through ,swedish filter paper4 #h4 ,when the fish bouillon is cold1 if it is to be used as fluid medium1 make up to #ajjj c4c4 by the addition of distilled water4 ,if1 however1 it is to be used as the basis for agar or gelatine media store it in the ,double ,strength condition4 #i4 ,tube and sterilise as for nutrient bouillon4 ,as an alternative method ,marvis fish food 7#af grammes7 may be substituted for the #ejj grammes of fresh fish4 ,fish ,gelatine4-- #a4 ,measure out double strength fish bouillon1 #ejj c4c41 into a tared #b-litre flask4 #b4 ,add sheet gelatine1 #ajj grammes1 cut into small pieces4 #c4 ,bubble live steam through the mixture for fifteen minutes to dissolve the gelatine4 #d4 ,weigh the flask and its contents2 adjust the weight to the calculated figure for one litre of medium 7#aace4e grammes7 by the addition of distilled water at #ajj deg4 ,c4 7vide page #aff74 #e4 ,cool to below #fj deg4 ,c41 and clarify with egg4 #f4 ,filter through papier ,chardin4 #g4 ,tube1 and sterilise as for nutrient gelatine4 ,shake well after the final sterilisation1 to aerate the medium4 ,fish ,gelatine-,agar4-- #a4 ,weigh out powdered agar1 #e grammes1 and emulsify it with #bjj c4c4 double strength fish bouillon4 #b4 ,wash the emulsion into a tared #b-litre flask with #cjj c4c4 fish bouillon4 #c4 ,weigh out sheet gelatine1 #gj grammes1 cut it into small pieces and add it to the contents of the flask4 #d4 ,bubble live steam through the mixture to dissolve the gelatine and agar4 #e4 ,weigh the flask and contents4 ,adjust the weight to the calculated figure for one litre of medium 7#aaaj4e grammes7 by the addition of distilled water at #ajj deg4 ,c4 7vide page #aff74 #f4 ,cool to below #fj deg4 ,c4 and clarify with egg4 #g4 ,filter through papier ,chardin4 #h4 ,tube1 and sterilise as for nutrient gelatine4 ,shake well after the final sterilisation1 to aerate the medium4 ,media for the ,study of ,yeasts and ,moulds4 ,pasteur's ,solution4-- 7,reaction alkaline74 #a4 ,weigh out and mix the ash from #aj grammes of yeast2 ammonium tartrate1 #aj grammes2 cane sugar1 #ajj grammes4 #b4 ,dissolve the mixture in distilled water1 #ajjj c4c4 #c4 ,tube or flask1 and sterilise as for nutrient bouillon4 ,yeast ,water 7,pasteur74-- #a4 ,weigh out pressed yeast1 #ge grammes2 place in a #b-litre flask and add #ajjj c4c4 distilled water4 #b4 ,heat in the steamer at #ajj deg4 ,c4 for thirty minutes4 #c4 ,filter through papier ,chardin4 #d4 ,tube or flask1 and sterilise as for nutrient bouillon4 ,cohn's ,solution4-- #a4 ,weigh out and mix ,acid potassium phosphate 7,,kh#b,,po#d7 #e4j grammes ,calcium phosphate #j4e gramme ,magnesium sulphate #e4j grammes ,ammonium tartrate #aj4j grammes and dissolve in ,distilled water #ajjj c4c4 #b4 ,tube1 or flask and sterilise as for nutrient bouillon4 ,naegeli's ,solution4-- #a4 ,weigh out and mix ,dibasic potassium phosphate 7,k#b;,,hpo#d7 #a4j gramme ,magnesium sulphate #j4b gramme ,calcium chloride #j4a gramme ,ammonium tartrate #aj4j grammes and dissolve in ,distilled water #ajjj c4c4 #b4 ,tube or flask2 sterilise as for nutrient bouillon4 ,plaster-of-,paris ,discs4-- #a4 ,take large corks1 #b4e cm4 diameter1 and roll a piece of stiff note-paper round each1 so that about a centimetre projects as a ridge above the upper surface of the cork1 and secure in position with a pin 7,fig4 #aab74 #b4 ,mix plaster-of-,paris into a stiff paste with distilled water1 and fill each of the cork moulds with the paste4 #c4 ,when the plaster has set1 remove the paper from the corks1 and raise the plaster discs4 #d4 ,place the plaster discs on a piece of asbestos board and sterilise by exposing in the hot-air oven to #aej deg4 ,c4 for half an hour4 ,illustration3 ,fig4 #aab4--;,cork and paper mould for plaster-of-,paris disc4 #e4 ,remove the sterile discs from the oven by means of sterile forceps1 place each inside a sterile capsule1 and moisten with a little sterile water4 #f4 ,sterilise in the steamer at #ajj deg4 ,c4 for thirty minutes on each of three consecutive days4 ,gypsum ,blocks 7,engel and ,hansen74-- ,these are in the form of truncated cones and for their preparation small tin moulds are required1 each having a diameter of #e4e cm4 at the base and #d cm4 at the truncated apex4 ,the height 7or depth7 of a mould is #d4e to #e cm4 #a4 ,mix powdered calcined gypsum into a stiff paste with distilled water4 #b4 ,fill the paste into the moulds and allow it to set and dry by exposure to air4 #c4 ,remove the block from the mould and transfer it to a double glass dish of adequate size 7#g cm4 diameter x #g cm4 high74 #d4 ,sterilise block in its dish for one hour in the hot-air oven at #aae deg4 ,c4 #e4 ,carefully open the dish and add sterile distilled water to moisten the block and form a layer in the bottom of the dish #a cm4 deep4 ,wine ,must4--7,wine must is obtained from ,sicily1 in hermetically sealed tins1 in a highly concentrated form--as a thick syrup--but not sterilised47 #a4 ,weigh out wine must1 #bjj grammes1 place in a #b-litre flask and add distilled water1 #hjj c4c4 #b4 ,weigh out ammonium tartrate1 #e grammes1 and add to the dilute must4 #c4 ,place the flask in a water-bath regulated to #fj deg4 ,c4 for one hour and incorporate the mixture thoroughly by frequent shaking4 #d4 ,filter through papier ,chardin4 #e4 ,tube1 and sterilise as for nutrient bouillon4 ,wheat ,bouillon 7,gasperini74-- #a4 ,weigh out and mix wheat flour1 #aej grammes2 magnesium sulphate1 #j4e gramme2 potassium nitrate1 #a gramme2 glucose1 #ae grammes4 #b4 ,dissolve the mixture in #ajjj c4c4 of water heated to #ajj deg4 ,c4 #c4 ,filter through papier ,chardin4 #d4 ,tube1 and sterilise as for nutrient bouillon4 ,bread ,paste4-- #a4 ,grate stale bread finely on a bread-grater4 #b4 ,distribute the crumbs in sterile ,erlenmeyer flasks1 sufficient to form a layer about one centimetre thick over the bottom of each4 #c4 ,add as much distilled water as the crumbs will soak up1 but not enough to cover the bread4 #d4 ,plug the flasks and sterilise in the steamer at #ajj deg4 ,c4 for thirty minutes on each of four consecutive days4 ,media for the ,study of ,parasitic ,moulds4 ,french ,proof ,agar 7,sabouraud74-- #a4 ,weigh out ,chassaing's peptone1 #aj grammes1 and emulsify it with #bjj c4c4 distilled water previously heated to #fj deg4 ,c4 #b4 ,weigh out powdered agar1 #ac grammes1 and emulsify with #bjj c4c4 cold distilled water4 #c4 ,mix the two emulsions and wash into a tared #b-litre flask with #fjj c4c4 distilled water4 #d4 ,bubble live steam through the mixture for twenty minutes1 to dissolve the agar4 #e4 ,cool to #fj deg4 ,c4 and clarify with egg as for nutrient agar 7vide page #afh74 #f4 ,filter through ,papier ,chardin1 using the hot-water funnel4 #g4 ,weigh out ,french maltose1 #dj grammes1 and dissolve in the agar4 #h4 ,tube1 and sterilise as for nutrient agar4 ,english ,proof ,agar 7,blaxall74--,substitute ,witte's peptone for that of ,chassaing1 and proceed as for ,french proof agar4 ,french ,mannite ,agar1 ,sabouraud4--7,for cultivation of ,favus47 ,proceed exactly as in preparing ,french ,proof agar vide supra substituting ,mannite 7#ch grammes7 for maltose4 ,media for the ,study of ,milk ,bacteria4 ,gelatine ,agar4--,this medium is prepared by adding to nutrient gelatine sufficient agar to ensure the solidity of the medium when incubated at temperatures above #bb deg4 ,c4 ,if it is intended to employ an incubating temperature of #cj deg4 ,c41 #aj per cent4 gelatine and #j4e per cent4 agar must be dissolved in the meat extract before the addition of the peptone and salt2 while for incubating at #cg deg4 ,c41 #ab per cent4 gelatine and #j4ge per cent4 agar must be used4 ,avoid the addition of more agar than is absolutely necessary1 otherwise the action upon the medium of such organisms as elaborate a liquefying ferment may be retarded or completely absent4 #a4 ,measure out #djj c4c4 double strength meat extract into a tared #b-litre flask1 and add to it gelatine1 #ajj grammes4 #b4 ,weigh out powdered agar1 #e grammes1 emulsify with #ajj c4c41 cold distilled water and add to the contents of the flask4 #c4 ,dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes4 #d4 ,weigh out peptone1 #aj grammes2 salt1 #e grammes2 emulsify with #ajj c4c4 double strength meat extract previously heated to #fj deg4 ,c41 and add to the contents of the flask4 #e4 ,replace in the steamer for fifteen minutes4 ,then adjust the weight to the calculated figure for one litre 7in this instance #aabj grammes7 by the addition of distilled water at #ajj deg4 ,c4 #f4 ,estimate the reaction2 control the result4 ,then add sufficient caustic soda solution to render the reaction #aj4 #g4 ,replace in the steamer at #ajj deg4 ,c4 for twenty minutes4 #h4 ,cool to #fj deg4 ,c4 ,clarify with egg as for nutrient agar4 #i4 ,filter through papier ,chardin1 using the hot-water funnel4 #aj4 ,tube1 and sterilise as for nutrient agar4 ,agar ,gelatine 7,guarniari74-- #a4 ,measure out double strength meat extract1 #djj c4c41 into a tared #b-litre flask1 and add to it gelatine1 #ej grammes4 #b4 ,weigh out powdered agar1 #c grammes2 emulsify with cold distilled water1 #ej c4c41 and add to the contents of the flask4 #c4 ,dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes4 #d4 ,weigh out ,witte's peptone1 #be grammes2 salt1 #e grammes1 and emulsify with #ajj c4c4 double strength meat extract previously heated to #fj deg4 ,c41 and add to the contents of the flask4 #e4 ,replace in the steamer for fifteen minutes4 #f4 ,weigh the flask and make up the medium mass to the calculated figure for one litre 7#ajhc grammes7 by the addition of distilled water at #ajj deg4 ,c4 #g4 ,neutralise carefully to litmus paper by the successive additions of small quantities of normal soda solution4 #h4 ,replace in the steamer at #ajj deg4 ,c4 for twenty minutes4 #i4 ,cool to #fj deg4 ,c4 ,clarify with egg as for nutrient agar4 #aj4 ,filter through papier ,chardin1 using the hot-water funnel4 #aa4 ,tube1 and sterilise as for nutrient agar4 ,whey ,gelatine4-- #a4 ,curdle fresh milk by warming to #fj deg4 ,c41 and adding rennet2 filter off the whey into a sterile tared flask4 #b4 ,estimate and note the reaction of the whey4 #c4 ,weigh out gelatine1 #aj per cent41 and add it to the whey in the flask4 #d4 ,bubble live steam through the mixture fifteen minutes to dissolve the gelatine2 and weigh4 #e4 ,estimate the reaction of the medium mass2 then add sufficient caustic soda solution to restore the reaction of the medium mass 7i4 e41 total weight minus weight of flask7 to the equivalent of the original whey4 #f4 ,cool to #fj deg4 ,c4 and clarify with egg as for nutrient gelatine 7vide page #aff74 #g4 ,filter through papier ,chardin4 #h4 ,tube1 and sterilise as for nutrient gelatine4 ,whey ,agar4-- #a4 ,curdle fresh milk by warming to #fj deg4 ,c41 and adding rennet2 filter off the whey into a sterile flask4 #b4 ,weigh out agar1 #a4e or #b per cent41 and add it to the whey in the flask4 #c4 ,bubble live steam through the mixture for twenty minutes1 to dissolve the agar4 #d4 ,cool to #fj deg4 ,c42 clarify with egg as for nutrient agar 7vide page #afh74 #e4 ,filter through papier ,chardin1 using the hot-water funnel4 #f4 ,tube1 and sterilise as for nutrient agar4 ,litmus ,whey4-- #a4 ,curdle fresh milk by warming to #fj deg4 ,c4 and adding rennet4 #b4 ,filter off the whey through butter muslin into a sterile flask4 #c4 ,neutralise to litmus by the cautious addition of citric acid solution #d per cent4 7,do not neutralise with mineral acid47 #d4 ,heat in the steamer at #ajj deg4 ,c4 for one hour to coagulate all the proteid4 7,if the whey is cloudy when removed from the steamer allow it to stand for forty-eight hours in the ice chest and then decant off the clear fluid--or filter through a ,berkefeld filter candle47 #e4 ,filter into a sterile flask4 #f4 ,tint the whey with litmus solution to a deep purple red4 #g4 ,tube1 and sterilise as for milk4 ,litmus ,whey 7,petruschky74-- #a4 ,measure out into a flask ,fresh milk #ajjj c4c4 #b4 ,add ,hydrochloric acid 7or glacial acetic acid7 #a4e c4c4 and boil4 #c4 ,filter off coagulated casein4 #d4 ,neutralise to litmus by the addition of n_/#a caustic soda solution and boil4 ,whey now cloudy and acid again4 #e4 ,again neutralise to litmus by addition of n_/#aj caustic soda solution4 #f4 ,filter4 #g4 ,tint the whey with neutral litmus solution to a deep purple colour4 #h4 ,tube and sterilise as for milk4 ,litmus ,whey ,gelatine4-- #a4 ,measure out milk #ajjj c4c4 into a tared #b-litre flask4 #b4 ,add hydrochloric acid 7or glacial acetic acid7 #a4e c4c4 and boil for five minutes4 #c4 ,filter off the casein1 and make the whey faintly alkaline to litmus4 #d4 ,weigh out ,peptone #aj grammes and emulsify in a few cubic centimeters of the whey and return to the flask4 #e4 ,weigh out ,gelatine #ej grammes add it to the whey in the flask and incorporate the mixture by bubbling through live steam4 #f4 ,clear with egg and filter4 #g4 ,make the weight of the medium mass to the calculated figure for one litre 7#ajfj grammes7 by the addition of distilled water4 #h4 ,weigh out ,dextrose #ae grammes and dissolve in the fluid whey gelatine4 #i4 ,add sterile litmus solution to the required tint4 #aj4 ,tube and sterilise for twenty minutes in steamer at #ajj deg4 ,c4 on each of five successive days4 ,this medium will remain semi-fluid at the room temperature1 and may be used for cultures in the cool or hot incubator4 ,litmus ,whey ,agar is prepared in a similar manner to ,whey ,gelatine1 with the substitution of #ae grammes of agar for the gelatine4 ,malt ,extract ,solution 7,herschell74-- #a4 ,measure into a flask distilled water #ajjj c4c4 #b4 ,weigh out ,extractum malti 7malt extract7 #be grammes and add to distilled water in flask4 #c4 ,boil for five minutes1 allow to stand1 and decant off clear fluid from sediment4 #d4 ,tube and sterilise as for nutrient bouillon4 ,media for the ,study of ,earth ,bacteria1 ,nitrogen ,fixers4 ,earthy ,salts ,agar 7,lipman and ,brown74--7,for the enumeration of soil organisms47 #a4 ,measure out ,agar #bj grammes4 ,emulsify in #bjj c4c4 distilled water4 #b4 ,wash the agar emulsion into a tared #b-litre flask with #djj c4c4 distilled water4 #c4 ,weigh out ,peptone #j4e gramme4 ,emulsify in #ej c4c4 distilled water and add to the contents of the flask4 #d4 ,bubble live steam through the mixture for twenty minutes to dissolve the agar4 #e4 ,weigh out and mix ,dextrose #aj4j grammes4 ,potassium phosphate #j4e gramme4 ,magnesium sulphate #j4b gramme4 ,potassium nitrate #j4jf gramme4 and add to the contents of the flask4 #f4 ,adjust the weight of the medium mass to the calculated figure for one litre 7#ajbe grammes7 by the addition of distilled water at #ajj deg4 ,c4 #g4 ,titrate the medium mass and adjust the reaction to #e4 #h4 ,cool to #fj deg4 ,c4 ,clarify with egg and filter4 #i4 ,tube in quantities of #aj c4c4 and sterilise as for nutrient agar4 ,beyrinck's ,solution4 ,i4--7,for the cultivation of nitrogen fixing organisms47 #a4 ,weigh out and mix #a gramme potassium hydrogen phosphate1 #j4b gramme magnesium sulphate1 and #j4jb gramme sodium chloride4 #b4 ,dissolve in water #ajjj c4c41 in a #b-litre flask4 #c4 ,add #a c4c4 of a one per thousand aqueous solution of ferrous sulphate4 #d4 ,add #a c4c4 of a one per thousand solution manganese sulphate4 #e4 ,weigh out #bj grammes dextrose and add to the contents of the flask 7dextrose up to #dj grammes may be used for the different organisms74 #f4 ,steam for twenty minutes1 filter4 #g4 ,tube1 and sterilise as for nutrient bouillon4 ,beyrinck's ,solution4 ,,ii4--7,for growth of ,azobacter47 ,proceed as in preparing solution ,no4 ,i1 substituting mannite for dextrose in step #e4 ,winogradsky's ,solution 7for ,nitric ,organisms74-- #a4 ,weigh out and mix4 ,potassium phosphate #a4j gramme ,magnesium sulphate #j4e gramme ,calcium chloride #j4ja gramme ,sodium chloride #b4j grammes and dissolve in ,distilled water #ajjj c4c4 #b4 ,fill into flasks1 in quantities of #bj c4c4 and add to each a small quantity of freshly washed magnesium carbonate4 #c4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three consecutive days4 #d4 ,add to each flask containing #bj c4c4 solution1 #b c4c4 of a sterile #b per cent4 solution of ammonium sulphate4 #e4 ,incubate at #cg deg4 ,c4 for forty-eight hours and eliminate any contaminated culture flasks4 ,store the remainder for future use4 ,winogradsky's ,solution 7for ,nitrous ,organisms74-- #a4 ,weigh out and mix ,ammonium sulphate #a gramme ,potassium sulphate #a gramme and dissolve in ,distilled water #ajjj c4c4 #b4 ,add #e to #aj grammes basic magnesium carbonate1 previously sterilised by boiling4 #c4 ,fill into flasks and sterilise1 etc41 as for previous solution4 ,silicate ,jelly 7,winogradsky74-- #a4 ,weigh out and mix ,ammonium sulphate #j4dj gramme ,magnesium sulphate #j4je gramme ,calcium chloride #j4ja gramme and dissolve in ,distilled water #ej c4c4 ,label--,solution ,a4 #b4 ,weigh out and mix ,potassium phosphate #j4aj gramme ,sodium carbonate #j4fj gramme and dissolve in ,distilled water #ej c4c4 ,label--,solution ,b4 #c4 ,weigh out ,silicic acid #c4d grammes and dissolve in ,distilled water #ajj c4c4 #d4 ,pour the silicic acid solution into a large porcelain basin4 #e4 ,mix equal quantities of the solutions ,a and ,b2 then add successive small quantities of the mixed salts to the silicic acid solution1 stirring continuously with a glass rod1 until a jelly of sufficiently firm consistence has been formed4 #f4 ,spread a layer of this jelly over the bottom of each of several large capsules or plates4 #g4 ,sterilise in the steamer at #ajj deg4 ,c4 for thirty minutes on each of three consecutive days4 ,media for the ,study of ,water ,bacteria4 ,naehrstoff ,agar 7,hesse and ,niedner74--7,for enumeration of water organisms47 #a4 ,weigh out3 agar1 #ab4e grammes and emulsify in #bej c4c4 distilled water4 #b4 ,wash the agar emulsion into a tared #b-litre flask with a further #bej c4c4 distilled water4 #c4 ,dissolve by bubbling live steam through the mixture4 #d4 ,emulsify ,naehrstoff-,heyden 7albumose7 #g4e grammes in #bjj c4c4 cold distilled water and add to melted agar4 #e4 ,adjust weight of medium mass to the calculated figure for one litre 7#ajbj grammes7 by addition of distilled water at #ajj deg4 ,c4 #f4 ,clarify with white of egg and filter4 #g4 ,tube in quantities of #aj c4c4 and sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three successive days4 ,bile ,salt ,broth--,double ,strength4-- #a4 ,weigh out ,witte's peptone1 #dj grammes1 and emulsify with #cjj c4c4 distilled water previously warmed to #fj deg4 ,c4 #b4 ,wash the peptone emulsion into a litre flask with #fjj c4c4 distilled water4 #c4 ,weigh out sodium taurocholate1 #aj grammes1 and glucose1 #aj grammes2 dissolve in #ajj c4c4 distilled water and add to the peptone emulsion in the flask4 #d4 ,heat in the steamer at #ajj deg4 ,c4 for twenty minutes4 #e4 ,filter through ,swedish filter paper into a sterile flask4 #f4 ,add sterile neutral litmus solution sufficient to colour the medium to a deep purple4 #g4 ,fill into small ,erlenmeyer flasks in quantities of #be c4c4 #h4 ,sterilise as for nutrient bouillon4 ,media for the ,study of ,plant ,bacteria4 ,beetroot4-- ,carrot4-- are prepared tubes and sterilised in a manner ,turnip4-- precisely similar to that described for potato4 ,parsnip4-- ,hay ,infusion4-- #a4 ,weigh out dried hay1 #aj grammes1 chop it up into fine particles and place in a flask4 #b4 ,add #ajjj c4c4 distilled water1 heated to #gj deg4 ,c42 close the flask with a solid rubber stopper4 #c4 ,macerate in a water-bath at #fj deg4 ,c4 for three hours4 #d4 ,replace the stopper by a cotton-wool plug1 and heat in the steamer at #ajj deg4 ,c4 for one hour4 #e4 ,filter through ,swedish filter paper4 #f4 ,tube1 and sterilise as for nutrient bouillon4 ,haricot ,bouillon4--7,for cultivation of bacteria from tubercles of ,legumes47 #a4 ,measure out #ajjj c4c4 distilled water into a #b-litre flask4 #b4 ,weigh out #bej grammes haricot beans and add to the water in the flask4 #c4 ,weigh out #aj grammes sodium chloride and add to the contents of the flask4 #d4 ,add #a c4c4 of a #a per cent4 solution of sodium bicarbonate4 #e4 ,place in the steamer at #ajj deg4 ,c4 for thirty minutes4 #f4 ,filter4 #g4 ,weigh out #bj grammes saccharose and add to the filtrate4 #h4 ,tube1 and sterilise as for nutrient bouillon4 ,haricot ,agar4-- #a4 ,measure out #djj c4c4 distilled water into a tared #b-litre flask4 #b4 ,weigh out #ae grammes agar and mix into a thick paste with #ajj c4c4 cold distilled water1 and add to the flask4 #c4 ,dissolve the agar by bubbling live steam through the mixture as in making nutrient agar4 #d4 ,weigh out #bej grammes haricot beans1 place in the flask with the agar mixture4 #e4 ,add #a c4c4 of #a per cent4 aqueous solution sodium bicarbonate4 #f4 ,weigh out #aj grammes sodium chloride and add to the contents of the flask4 #g4 ,place in the steamer at #ajj deg4 ,c4 for thirty minutes4 #h4 ,adjust the weight of the medium mass to #ajcj grammes 7the figure per litre obtained experimentally7 by the addition of distilled water at #ajj deg4 ,c4 #i4 ,cool to #fj deg4 ,c41 clarify with egg and filter4 #aj4 ,weigh out #bj grammes saccharose and add to the contents of the flask4 #aa4 ,tube1 and sterilise as for nutrient agar4 ,wood ,ash ,agar4-- #a4 ,measure #djj c4c4 distilled water into a tared #b-litre flask4 #b4 ,weigh out #aj grammes agar and make into a thick paste with #ajj c4c4 cold distilled water4 #c4 ,add this agar paste to the distilled water in the flask4 #d4 ,dissolve the agar by passing live steam through it1 as in preparing nutrient agar4 #e4 ,weigh out #e grammes clean wood ash and place in a second flask containing #bjj c4c4 distilled water with some sterile glass beads3 shake thoroughly in a mechanical shaker for ten minutes4 #f4 ,heat in steamer at #ajj deg4 ,c41 for thirty minutes4 #g4 ,after removal from the steamer dry the outside of the flask thoroughly1 place it over a ,bunsen flame and boil for one minute4 #h4 ,filter directly into the flask containing the melted agar mixture4 #i4 ,weigh out #d grammes maltose4 ,add to the contents of the flask4 #aj4 ,adjust the weight of the medium mass to the calculated figure for one litre 7#ajai grammes7 by the addition of distilled water at #ajj deg4 ,c4 #aa4 ,replace the flask in the steamer for twenty minutes1 cool to #fj deg4 ,c41 and clarify with egg and filter4 #ab4 ,tube1 and sterilise as for nutrient agar4 ,media for the ,study of ,special ,bacilli4 ,b4 ,acnes4 ,oleic ,acid ,agar 7,fleming74-- #a4 ,measure out into a sterile stout glass bottle which already contains about #aj sterile glass beads ,ascitic fluid #bej c4c4 #b4 ,weigh out ,oleic acid #be grammes and add it to the ascitic fluid in the bottle4 #c4 ,emulsify evenly by shaking 7either by hand or in a shaking machine7 for ten minutes4 #d4 ,liquefy and measure out into a flask ,nutrient agar #gej c4c4 then cool to #ee deg4 ,c4 #e4 ,mix the oleic acid emulsion with the agar4 #f4 ,add #aj c4c4 sterile neutral red1 #a per cent4 aqueous solution4 #g4 ,tube in quantities of #aj c4c41 slant1 and allow to set4 #h4 ,incubate for forty-eight hours at #cg deg4 ,c4 and reject any contaminated tubes4 ,store the sterile tubes for future use4 ,coli-typhoid ,group4 ,parietti's ,bouillon4-- #a4 ,measure out pure hydrochloric acid1 #d c4c41 and add to it carbolic acid solution 7#e per cent471 #ajj c4c4 ,allow the solution to stand at least a few days before use4 #b4 ,this solution is added in quantities of #j4a1 #j4b4 and #j4c c4c4 7delivered by means of a sterile graduated pipette7 to tubes each containing #aj c4c4 of previously sterilised nutrient bouillon 7vide page #afc74 #c4 ,incubate at #cg deg4 ,c4 for forty-eight hours to eliminate contaminated tubes4 ,store the remainder for future use4 ,carbolised ,bouillon4-- #a4 ,prepare nutrient bouillon 7vide page #afc1 sections #a to #f74 ,measure out #ajjj c4c4 #b4 ,weigh out carbolic acid1 #a gramme 7#b4e or #e grammes may be needed for special purposes71 and dissolve it in the medium4 #c4 ,tube1 and sterilise as for bouillon4 ,carbolised ,gelatine4-- #a4 ,prepare nutrient gelatine 7vide page #afd1 sections #a to #g74 ,measure out #ajjj c4c4 #b4 ,weigh out carbolic acid1 #e grammes 7 #j4e per cent471 and dissolve it in the gelatine4 #c4 ,filter if necessary through papier ,chardin4 #d4 ,tube1 and sterilise as for nutrient gelatine4 ,one or #b4e grammes of carbolic acid 7 #j4a per cent4 or #j4be per cent47 are occasionally used in place of the #e grammes to meet special requirements4 ,carbolised ,agar4-- #a4 ,prepare nutrient agar 7vide page #afg1 sections #a to #h74 ,measure out #ajjj c4c4 #b4 ,weigh out #a gramme pure phenol and dissolve in the medium4 #c4 ,filter if necessary through papier ,chardin4 #d4 ,tube1 and sterilise as for nutrient agar4 ,litmus ,gelatine4-- #a4 ,prepare nutrient gelatine 7vide page #afd1 sections #a to #h74 #b4 ,add sterile litmus solution1 sufficient to tint the medium a deep lavender colour4 #c4 ,tube1 and sterilise as for nutrient gelatine4 ,lactose ,litmus ,bouillon 7,lakmus ,molke74-- #a4 ,weigh out peptone1 #d grammes1 and emulsify it with #bjj c4c4 meat extract 7vide page #adh71 previously heated to #fj deg4 ,c4 #b4 ,weigh out salt1 #b grammes1 and lactose1 #bj grammes1 and mix with the emulsion4 #c4 ,wash the mixture into a sterile litre flask with #bjj c4c4 meat extract and add #fjj c4c4 distilled water4 #d4 ,heat in the steamer at #ajj deg4 ,c4 for thirty minutes1 to completely dissolve the peptone1 etc4 #e4 ,neutralise carefully to litmus paper by the successive additions of small quantities of decinormal soda solution4 #f4 ,replace in the steamer for twenty minutes to precipitate phosphates1 etc4 #g4 ,filter through two thicknesses of ,swedish filter paper4 #h4 ,add sterile litmus solution1 sufficient to colour the medium a deep purple4 #i4 ,tube1 and sterilise as for bouillon4 ,lactose ,litmus ,gelatine 7,wurtz74-- #a4 ,prepare nutrient gelatine 7vide page #afd1 sections #a to #d74 #b4 ,render the reaction of the medium mass -#e4 #c4 ,replace in the steamer at #ajj deg4 ,c4 for twenty minutes4 #d4 ,clarify with egg as for gelatine4 #e4 ,weigh out lactose1 #bj grammes 7 #b per cent471 and dissolve it in the medium4 #f4 ,filter through papier ,chardin4 #g4 ,add sufficient sterile litmus solution to colour the medium pale lavender4 #h4 ,tube1 and sterilise as for nutrient gelatine4 ,lactose ,litmus ,agar 7,wurtz74-- #a4 ,prepare nutrient agar 7vide page #afg1 sections #a to #d74 #b4 ,render the reaction of the medium mass -#e4 #c4 ,replace in the steamer at #ajj deg4 ,c4 for twenty minutes4 #d4 ,cool to #fj deg4 ,c4 and clarify with egg as for nutrient agar4 #e4 ,weigh out lactose1 #bj grammes 7 #b per cent471 and dissolve it in the medium4 #f4 ,filter through papier ,chardin1 using the hot-water funnel4 #g4 ,add sterile litmus solution1 sufficient to colour the medium a pale lavender4 #h4 ,tube1 and sterilise as for nutrient agar4 ,glycerine ,potato ,bouillon4-- #a4 ,take #a kilo of potatoes1 wash thoroughly in water1 peel1 and grate finely on a bread-grater4 #b4 ,weigh the potato gratings1 place them in a #b-litre flask1 and add distilled water in the proportion of #a c4c4 for every gramme weight of potato4 ,allow the flask to stand in the ice-chest for twelve hours4 #c4 ,strain the mixture through butter muslin and filter through ,swedish filter paper into a graduated cylinder4 ,note the amount of the filtrate4 #d4 ,place the filtrate in a flask1 add an equal quantity of distilled water1 and heat in the steam steriliser for sixty minutes4 #e4 ,add glycerine1 #d per cent41 mix thoroughly1 and again filter4 #f4 ,tube and sterilise as for nutrient bouillon4 ,potato ,gelatine 7,elsner74-- #a4 ,take #a kilo of potatoes1 wash thoroughly in water1 peel1 and finally grate finely on a bread-grater4 #b4 ,weigh the potato gratings1 place them in a #b-litre flask1 and add distilled water in the proportion of #a c4c4 for every gramme weight of potato4 ,allow the flask to stand in the ice-chest for twelve hours4 #c4 ,strain the mixture through butter muslin1 and filter through ,swedish filter paper into a graduated cylinder4 #d4 ,add #ae per cent4 gelatine to the potato decoction and bubble live steam through the mixture for ten minutes4 #e4 ,estimate the reaction2 adjust the reaction of the medium mass to #be4 #f4 ,cool the medium to below #fj deg4 ,c42 clarify with egg as for nutrient gelatine 7vide page #aff74 #g4 ,add #a per cent4 potassium iodide 7powdered7 to the medium4 #h4 ,filter through papier ,chardin4 #i4 ,tube and sterilise as for nutrient gelatine4 ,aesculin ,agar4--7,b4 coli and allied organisms give black colonies surrounded by black halo47 #a4 ,measure out #djj c4c4 distilled water into a tared #b-litre flask4 #b4 ,weigh out ,agar #ae grammes ,peptone #aj grammes ,sodium taurocholate #e grammes and make into a thick paste with #aej c4c4 distilled water4 #c4 ,add this paste to the distilled water in the flask4 #d4 ,dissolve the ingredients by bubbling live steam through the mixture4 #e4 ,weigh out ,aesculin #a4j gramme ,ferric citrate #j4e gramme and dissolve in a second flask containing #ajj c4c4 distilled water4 #f4 ,mix the contents of the two flasks--adjust the weight to the calculated medium figure 7in this case #ajca4e grammes7 by the addition of distilled water at #ajj deg4 ,c4 #g4 ,clarify with egg and filter4 #h4 ,tube and sterilise as for nutrient agar4 ,bile ,salt ,agar 7,mac,conkey74-- #a4 ,weigh out powdered agar1 #ae grammes 7 #a4e4 per cent471 and emulsify with #bjj c4c4 cold tap water4 #b4 ,weigh out peptone1 #bj grammes 7 #b per cent471 and emulsify with #bjj c4c4 tap water previously warmed to #fj deg4 ,c4 #c4 ,mix the peptone and agar emulsions thoroughly4 #d4 ,weigh out sodium taurocholate1 #e grammes 7 #j4e per cent471 dissolve it in #cjj c4c4 tap water1 and use the solution to wash the agar-peptone emulsion into a tared #b-litre flask4 #e4 ,bubble live steam through the mixture for twenty minutes4 #f4 ,adjust the weight of the medium mass to the calculated figure for one litre 7#ajdj grammes74 #g4 ,cool to #fj deg4 ,c4 and clarify with egg as for nutrient agar 7vide page #afh74 #h4 ,filter through papier ,chardin1 using the hot-water funnel4 #i4 ,weigh out lactose1 #aj grammes 7 #a per cent471 and dissolve it in the agar4 ,if desired1 add #e c4c4 of a #a per cent4 7 #j4e per cent47 aqueous solution of neutral red4 #aj4 ,tube1 and sterilise as for nutrient agar4 ,litmus ,nutrose ,agar 7,drigalski-,conradi74-- ,this medium should be prepared in precisely the same manner as the ,nutrose agar described on page #agb substituting meat extract for serum water1 and increasing the percentage of agar added per litre to #c per cent4 ,fuchsin ,agar 7,braun74-- #a4 ,liquefy and measure out into a sterile flask3 ,nutrient agar #ajjj c4c4 #b4 ,weigh out3 lactose #aj grammes and dissolve in the fluid agar4 #c4 ,adjust the reaction to -#e and filter4 #d4 ,measure out and mix thoroughly with agar3 ,fuchsin1 alcoholic solution #e c4c4 ,the fuchsin solution is prepared by mixing3 ,fuchsin 7basic7 #c grammes4 ,absolute alcohol #fj c4c4 ,allow to stand twenty-four hours1 then centrifugalise thoroughly and decant the supernatant fluid into a well-stoppered bottle4 #e4 ,measure out and add to the nutrient agar1 sodium sulphite1 #aj per cent4 aqueous solution1 freshly prepared #be c4c4 #f4 ,tube and sterilise as for nutrient agar4 #g4 ,store in a dark cupboard4 ,fuchsin ,sulphite ,agar 7,endo74-- #a4 ,liquefy and measure out into a sterile flask3 ,nutrient agar #ajjj c4c4 #b4 ,weigh out ,lactose #aj grammes4 and dissolve in the fluid agar4 #c4 ,adjust the reaction to #c and filter4 #d4 ,measure out and mix thoroughly with the fluid agar4 ,fuchsin1 alcoholic solution 7vide supra7 #e c4c4 #e4 ,measure out and add to the medium ,sodium sulphite1 #aj per cent4 aqueous solution #be c4c4 #f4 ,tube and sterilise as for nutrient agar4 ,brilliant ,green ,agar 7,conradi74-- #a4 ,liquefy and measure out into a sterile flask ,nutrient agar #ajjj c4c4 #b4 ,adjust reaction to #cj by the addition of normal phosphoric acid2 and filter4 #c4 ,measure out and mix thoroughly with the fluid medium ,brilliant green 7,hoechst7 #a per thousand aqueous solution #f4e c4c4 #d4 ,measure out and add to the medium ,picric acid 7,gruebler71 #a per cent4 aqueous solution #f4e c4c4 #e4 ,tube and sterilise as for nutrient agar4 ,brilliant ,green ,bile ,salt ,agar 7,fawcus74-- #a4 ,weigh out agar #bj grammes and emulsify in #ajj c4c4 cold distilled water4 #b4 ,wash the emulsion into a tared #b-litre flask with #ejj c4c4 distilled water4 #c4 ,dissolve the agar by bubbling live steam through the flask4 #d4 ,cool1 clarify with egg and filter4 #e4 ,weigh out ,sodium taurocholate #e grammes ,peptone #bj grammes and add to the medium in the flask4 #f4 ,weigh out ,lactose #e grammes and add to the medium in the flask4 #g4 ,adjust reaction to #ae and filter if necessary4 #h4 ,measure out ,brilliant green1 #a per thousand aqueous solution #bj c4c4 and mix thoroughly with the fluid agar4 #i4 ,measure out and add to the medium ,picric acid1 #a per cent4 aqueous solution #bj c4c4 #aj4 ,tube and sterilise as for nutrient agar4 ,china ,green ,agar 7,werbitski74-- #a4 ,liquefy and measure out into a sterile flask ,nutrient agar #ajjj c4c4 #b4 ,adjust the reaction accurately to #ac and filter4 #c4 ,measure out and mix thoroughly with the fluid agar ,china green #j4b per cent4 aqueous solution #ae c4c4 #d4 ,tube and sterilise as for nutrient agar4 ,malachite ,green ,agar 7,loeffler74-- #a4 ,liquefy and measure out into a sterile flask ,nutrient agar #ajjj c4c4 #b4 ,weigh out ,dextrose #aj grammes4 and dissolve in nutrient agar4 #c4 ,adjust the reaction to #c1 and filter4 #d4 ,measure out and mix thoroughly in the fluid agar ,malachite green1 #j4a per cent4 aqueous solution #af c4c4 for weak medium4 #d;a4 ,to the filtered agar add ,malachite green1 #b per cent4 aqueous solution #be c4c4 for strong medium4 #e4 ,tube and sterilise as for nutrient agar4 ,double ,sugar ,agar 7,russell74-- #a4 ,liquefy and measure out into a sterile flask ,nutrient agar #ajjj c4c4 #b4 ,add #ajj c4c4 litmus solution to the fluid agar4 #c4 ,weigh out and dissolve in the fluid agar4 ,lactose #aj grammes ,dextrose #aj grammes4 #d4 ,render the reaction of the medium neutral to litmus paper by the cautious addition of normal caustic soda4 #e4 ,tube in quantities of #aj c4c4 and sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three successive days4 #f4 ,store for use in a cool dark place4 ,b4 ,diphtheriae4 ,glycerine ,blood-serum4-- #a4 ,prepare blood-serum as described1 page #afh1 sections #a to #d4 #b4 ,add #e per cent4 pure glycerine4 #c4 ,complete as described above for ordinary blood-serum1 sections #e to #g4 ,,note4--,different percentages of glycerine--from #d per cent4 to #h per cent4--are used for special purposes4 ,five per cent4 is that usually employed4 ,blood-serum 7,loeffler74-- #a4 ,prepare nutrient bouillon 7vide page #afc71 using meat extract made from veal instead of beef4 #b4 ,add #a per cent4 glucose to the bouillon1 and allow it to dissolve completely4 #c4 ,now add #cjj c4c4 clear blood-serum 7vide page #afh1 sections #a to #d7 to every #ajj c4c4 of this bouillon4 #d4 ,fill into sterile tubes and complete as for ordinary blood-serum4 ,blood-serum 7,lorrain ,smith74-- #a4 ,collect blood-serum 7vide page #afh1 sections #a to #d71 as free from haemoglobin as possible4 #b4 ,weigh out #j4ae per cent4 sodium hydrate and dissolve it in the fluid 7or add #j4cge c4c4 of dekanormal soda solution for every #ajj c4c4 of serum74 #c4 ,tube1 and stiffen at #ajj deg4 ,c4 in the serum inspissator4 #d4 ,incubate at #cg deg4 ,c4 for forty-eight hours to eliminate any contaminated tubes4 ,store the remainder for future use4 ,blood ,serum 7,councilman and ,mallory74-- #a4 ,collect blood serum in slaughterhouse1 coagulate1 remove serum and tube 7vide page #afh74 ,great care must be taken to avoid the inclusion of air bubbles--indeed if only a few tubes are filled at one time1 it is a good plan to stand them upright in the receiver of an air pump and to exhaust as completely as possible before transferring to the serum inspissator4 #b4 ,heat the tubes in a slanting position in hot-air steriliser at #ij deg4 ,c4 till firmly coagulated1 say half an hour4 #c4 ,sterilise in steam steriliser at #ajj deg4 ,c4 for #bj minutes on each of three successive days4 ,resulting medium not translucent1 but opaque and firm4 ,b4 ,tuberculosis4 ,egg ,medium 7,lubenau74-- ,this modification of ,dorset's egg medium 7quod vide page #agd7 is preferred by some for the growth of the tubercle bacillus of the human type4 ,it consists in the addition of one part of #f per cent4 glycerine in normal saline solution1 to the egg mixture between steps #d and #e4 ,glycerine ,bouillon4-- #a4 ,measure out nutrient bouillon1 #ajjj c4c4 7vide page #afc1 sections #a to #f74 #b4 ,measure out glycerine1 #fj c4c4 7 #f per cent471 and add to the bouillon4 #c4 ,tube1 and sterilise as for bouillon4 ,glycerine ,agar4-- #a4 ,prepare nutrient agar 7vide page #afg1 sections #a to #h74 ,measure out #ajjj c4c4 #b4 ,measure out pure glycerine1 #fj c4c4 7 #f per cent471 and add to the agar4 #c4 ,tube1 and sterilise as for nutrient agar4 ,glycerine ,blood-serum4-- #a4 ,prepare blood-serum as described1 page #afh1 sections #a to #d4 #b4 ,add #e per cent4 pure glycerine4 #c4 ,complete as described above for ordinary blood-serum1 sections #e to #g4 ,,note4--,different percentages of glycerine--from #d per cent4 to #h per cent4--are used for special purposes4 ,five per cent4 is that usually employed4 ,glycerinated ,potato4-- #a4 ,prepare ordinary potato wedges 7vide page #agd1 sections #a to #d74 #b4 ,soak the wedges in #be per cent4 solution of glycerine for fifteen minutes4 #c4 ,moisten the cotton-wool pads at the bottom of the potato tubes with a #be per cent4 solution of glycerine4 #d4 ,insert a wedge of potato in each tube and replug the tubes4 #e4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of five consecutive days4 ,animal ,tissue ,media 7,frugoni74-- #a4 ,take a number of sterile test-tubes #af x #c or #d cm41 plugged with cotton wool1 and into each insert a #b cm4 length of stout glass tubing 7about #a cm4 diameter72 fill in glycerine 7#f per cent47 bouillon to the upper level of the piece of glass tubing4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three successive days4 #b4 ,kill a small rabbit by means of chloroform vapour4 #c4 ,under strictly aseptic precautions remove the lungs1 liver and other solid organs and transfer them to a sterile double glass dish4 #d4 ,with the help of sterile scissors and forceps divide the organs into roughly rectangular blocks #c x #a4e x #a cm4 #e4 ,pour into the dish a sufficient quantity of sterile glycerine solution 7#f per cent4 in normal saline71 cover1 and allow to stand for one hour4 #f4 ,introduce a block of tissue into each tube so that it rests upon the upper end of the piece of glass tubing4 7,the surface of the tissue will now be kept moist by capillary attraction and condensation74 #g4 ,sterilise in the autoclave at #abj deg4 ,c4 for thirty minutes4 #h4 ,cap the tubes and store them in the ice chest for future use4 ,tissues obtained at postmortems can also be used after preliminary sterilisation by boiling or autoclaving4 ,media for the ,study of ,special ,cocci4 ,diplococcus ,gonorrhoeae4 ,ascitic ,bouillon 7,serum ,bouillon74-- #a4 ,collect ascitic fluid 7pleuritic fluid1 hydrocele fluid1 etc471 by aspiration directly into sterile flasks1 under strictly aseptic precautions4 #b4 ,mix the serum with twice its bulk of sterile nutrient bouillon 7vide page #afc74 #c4 ,if considered necessary 7on account of the presence of blood1 crystals1 etc471 filter the serum bouillon through porcelain filter candle4 #d4 ,tube1 and sterilise in the water bath at #ef deg4 ,c4 for half an hour on each of five consecutive days4 #e4 ,incubate at #cg deg4 ,c4 for forty-eight hours and eliminate contaminated tubes4 ,store the remainder for future use4 ,serum ,agar 7,heiman74-- #a4 ,prepare nutrient agar 7vide page #afg71 to following formula3 ,agar #b4j per cent4 ,peptone #a4e per cent4 ,salt #j4e per cent4 ,meat extract quantum sufficit4 #b4 ,make reaction of medium #aj4 #c4 ,filter2 tube in quantities of #f c4c4 #d4 ,sterilise as for nutrient agar4 #e4 ,after the third sterilisation cool the tubes to #db deg4 ,c41 and add to each #c c4c4 of sterile hydrocele fluid1 ascitic fluid1 or pleuritic effusion 7previously sterilised1 if necessary1 by the fractional method72 allow the tubes to solidify in a sloping position4 #f4 ,when solid1 incubate at #cg deg4 ,c4 for forty-eight hours1 and eliminate any contaminated tubes4 ,store the remainder for future use4 ,serum ,agar 7,wertheimer74-- #a4 ,prepare nutrient agar 7vide page #afg71 to the following formula3 ,agar #b4j per cent4 ,peptone #b4j per cent4 ,salt #j4e per cent4 ,meat extract quantum sufficit4 #b4 ,make reaction of medium #aj4 #c4 ,filter2 tube in quantities of #e c4c4 #d4 ,sterilise as for nutrient agar4 #e4 ,after the last sterilisation cool to #db deg4 ,c41 then add #e c4c4 sterile blood-serum from human placenta 7sterilised1 if necessary1 by the fractional method7 to each tube2 slope the tubes4 #f4 ,when solid1 incubate at #cg deg4 ,c4 for forty-eight hours1 and eliminate any contaminated tubes4 ,store the remainder for future use4 ,serum ,agar 7,kanthack and ,stevens74-- #a4 ,collect ascitic1 pleuritic1 or hydrocele fluid in sterile flasks and allow to stand in the ice-chest for twelve hours to sediment4 #b4 ,decant #ajjj c4c4 of the clear fluid into a measuring cylinder and transfer to sterile litre flask4 #c4 ,add #j4e c4c4 dekanormal ,na,o,h solution for every #ajj c4c4 serum 7i4 e41 #e4j c4c471 and mix thoroughly4 #d4 ,heat in the steamer for twenty minutes4 #e4 ,weigh out #ae grammes agar1 emulsify in a separate vessel with #bjj c4c4 of the alkaline fluid previously cooled to about #bj deg4 ,c41 and then add to the remainder of the fluid in the flask4 #f4 ,bubble live steam through the mixture for twenty minutes to dissolve the agar4 #g4 ,filter through papier ,chardin1 using a hot-water funnel4 #h4 ,weigh out glucose #aj grammes 7 #a per cent471 and dissolve it in the clear agar4 #h;a4 ,if desired1 add glycerine1 #e per cent41 to the clear agar4 #i4 ,tube1 and sterilise as for nutrient agar4 ,serum ,agar 7,libman74-- #a4 ,prepare nutrient agar 7vide1 page #afg7 using1 however1 #a4e per cent4 peptone 7that is #ae grammes per litre instead of #aj grammes74 #b4 ,adjust the reaction to #j 7i4 e41 neutral to phenolphthalein74 #c4 ,filter and transfer #ajjj c4c4 liquefied medium to a sterile flask4 #d4 ,weigh out dextrose #bj grammes and dissolve in the fluid agar4 #e4 ,tube in quantities of #f c4c42 and sterilise in the steamer at #ajj deg4 ,c4 for thirty minutes on each of three consecutive days4 #f4 ,after the third sterilisation cool to #db deg4 ,c4 and add to each tube #c c4c4 of sterile hydrocele fluid1 ascitic fluid or pleuritic effusion 7previously sterilised1 if necessary1 by the fractional method72 allow the tubes to solidify in a sloping position4 #g4 ,when solid1 incubate at #cg deg4 ,c4 for forty-eight hours1 and eliminate any contaminated tubes4 ,store the remainder for future use4 ,egg-albumen1 ,inspissated4-- #a4 ,break several fresh eggs 7hens'1 ducks'1 or turkeys' eggs71 and collect the whites in a graduated cylinder1 taking care to avoid admixture with the yolks4 #b4 ,add #dj per cent4 distilled water1 and incorporate the mixture thoroughly by the aid of an egg-whisk4 #c4 ,weigh out #j4ae per cent4 sodium hydrate and dissolve it in the fluid 7or add the amount of dekanormal caustic soda solution calculated to yield the required percentage of soda in the total bulk of the fluid--i4 e41 #j4cge c4c4 of dekanormal ,na,o,h solution per #ajj c4c4 of the mixture74 #c;a4 ,glucose to the extent of #a to #b per cent4 may now be added1 if desired4 #d4 ,strain the mixture through butter muslin and filter through a porcelain filter candle into a sterile filter flask4 #e4 ,tube1 and stiffen at #ajj deg4 ,c4 in the serum inspissator4 #f4 ,incubate at #cg deg4 ,c4 for forty-eight hours and eliminate any contaminated tubes2 store the remainder for future use4 ,egg-albumen 7,tarchanoff and ,kolesnikoff74-- #a4 ,place unbroken hens' eggs in dekanormal caustic soda solution for ten days4 7,after this time the white becomes firm like gelatine47 #b4 ,carefully remove the shell and cut the egg into fine slices4 #c4 ,wash for two hours in running water4 #d4 ,place the egg slices in a large beaker and sterilise in the steamer at #ajj deg4 ,c4 for one hour4 #e4 ,transfer each slice of egg by means of a pair of sterilised forceps to a ,petri dish or large capsule4 #f4 ,sterilise in the steamer at #ajj deg4 ,c4 for twenty minutes on each of three consecutive days4 ,egg ,albumin ,broth 7,lipschuetz74-- #a4 ,weigh out ,egg albumin 7extra fine powder1 ,merck74 #d grammes and place in a #b-litre flask with a number of sterile glass beads4 #b4 ,measure out distilled water #bjj c4c4 into a half-litre flask and warm to #cg deg4 ,c4 in the incubator4 #c4 ,add the water to the flask containing the albumin and beads and dissolve by shaking4 #d4 ,add n_/#aj-,na,o,h1 #dj c4c4 ,allow the mixture to stand for thirty minutes with frequent shaking4 #e4 ,filter through ,swedish filter paper4 #f4 ,sterilise by boiling two or three times at intervals of two hours4 #g4 ,add ordinary nutrient bouillon #fjj c4c4 #h4 ,fill into small ,erlenmeyer flasks in quantities of #ej c4c4 #i4 ,incubate for forty-eight hours at #cg deg4 ,c4--discard any contaminated flasks and store the remainder for future use4 ,egg ,albumin ,agar4-- #a4 ,prepare egg albumin solution as above #a-f4 #b4 ,liquefy and measure out ordinary nutrient agar #fjj c4c4 and add to the egg albumin solution 7in place of the nutrient broth74 #c4 ,complete as above #h-i4 ,diplococcus ,meningitidis ,intracellularis4 ,ascitic ,fluid ,agar 7,wassermann7 ,synonym ,n-as-gar 7,mervyn ,gordon74 #a4 ,liquefy and measure out into a sterile flask3 ,nutrient agar #fjj c4c4 #b4 ,measure out into a half litre flask ,distilled water #baj c4c4 and add to it ,ascitic fluid #ij c4c4 ,nutrose #f grammes #c4 ,heat over a bunsen flame1 shaking constantly until the fluid boils1 and the nutrose is dissolved4 #d4 ,add the nutrose ascitic solution to the fluid agar4 #e4 ,heat in the steamer for thirty minutes1 then filter4 #f4 ,tube and sterilise as for nutrient agar4 ,,note4--,the finished medium in this case measures #ijj c4c4 only since inconvenient fractions would be introduced in making up to one litre exactly4 ,diplococcus ,pneumoniae4 ,blood ,agar 7,washbourn74-- #a4 ,melt up several tubes of nutrient agar 7vide page #afg7 and allow them to solidify in the oblique position4 #b4 ,place the tubes1 in the horizontal position1 in the hot incubator for forty-eight hours1 to evaporate off some of the condensation water4 #c4 ,kill a small rabbit with chloroform and nail it out on a board 7as for a necropsy74 ,moisten the hair thoroughly with #b per cent4 solution of lysol4 #d4 ,sterilise several pairs of forceps1 scissors1 etc41 by boiling4 #e4 ,reflect the skin over the thorax with sterile instruments4 #f4 ,open the thoracic cavity by the aid of a fresh set of sterile instruments4 #g4 ,open the pericardium with another set of sterile instruments4 #h4 ,sear the surface of the left ventricle with a red-hot iron and remove fluid blood from the heart by means of sterile pipettes 7e4 g41 those shown in ,fig4 #ac1 c74 #i4 ,deliver a small quantity of the blood on the slanted surface of the agar in each of the tubes1 and allow it to run over the entire surface of the medium4 #aj4 ,place the tubes in the slanting position and allow the blood to coagulate4 #aa4 ,return the blood agar to the hot incubator for forty-eight hours and eliminate any contaminated tubes4 ,store the remainder for future use4 ,media for the ,study of ,mouth ,bacteria ,generally4 ,potato ,gelatine 7,goadby74-- #a4 ,prepare glycerine potato broth 7see page #bjc1 sections #a to #e74 #b4 ,add #aj per cent4 gelatine to the potato decoction and bubble live steam through the mixture for ten minutes4 #c4 ,estimate the reaction2 adjust the reaction of the medium to #e4 #d4 ,cool the medium to below #fj deg4 ,c41 clarify with egg as for nutrient gelatine4 #e4 ,filter through papier ,chardin4 #f4 ,tube1 and sterilise as for nutrient gelatine4 ,media for the ,study of ,protozoa4 ,tissue ,medium 7,noguchi74--,for spirochaetes 7cultivations must be grown anaerobically74 #a4 ,plug and sterilise test-tubes #bj x #b cm4 #b4 ,kill a small rabbit with chloroform vapour4 ,open the abdomen with all aseptic precautions1 remove kidneys and testicles and transfer to a sterile glass dish4 ,cut up the organs with sterile scissors into small pieces--say #d millimetre cubes4 ,the four organs should yield from #be to #cj pieces of tissue4 #c4 ,drop a small piece of sterile tissue into the bottom of each sterilised tube4 #d4 ,take a flask containing about #djj c4c4 nutrient agar 7#aj reaction71 liquefy the medium by heat and cool in a water bath to #ej deg4 ,c4 #e4 ,add #bjj c4c4 ascitic or hydrocele fluid 7horse or sheep serum may be employed1 but is not so good7 to the liquid agar and mix carefully to avoid formation of air bubbles4 #f4 ,fill about #bj c4c4 of the ascitic agar into each of the sterilised tubes which already contains a piece of sterile rabbit's tissue1 stand all the tubes upright in racks or a jar1 and allow agar to set4 #g4 ,after solidification pour sterile paraffin oil on the surface of the medium in each tube to the depth of #c centimetres4 #h4 ,incubate tubes at #cg deg4 ,c4 for several days and discard any which prove to be contaminated4 #i4 ,store such tubes as are sterile for future use4 ,,xiii4 ,,incubators4 ,illustration3 ,,fig4 #aac4--;,incubator4 ,an incubator 7,fig4 #aac7 consists essentially of a chamber for the reception of cultivations1 etc41 surrounded by a water jacket1 the walls of which are of metal1 usually copper1 and outside all an asbestos or felt jacket1 or wooden casing4 ,the water in the jacket is heated by gas or electricity and maintained at some constant temperature by a thermo-regulator4 ,the cellular incubator 7,fig4 #aad7 which was made for me#g some years ago is of the greatest practical utility4 ,here the central cavity is subdivided by five double-walled partitions 7in which water circulates in connection with the water tanks at the top and base of the incubator7 and again by iron shelves to form twenty-four pigeon holes4 ,into each of these slides an iron drawer #ce cm4 long x #ab cm4 wide x #bb cm4 high forming a self-contained incubator4 ,the drawer is fitted with a wooden form to which is fixed a handle and a numbered label4 ,the thermo-regulating apparatus is the well-known ,hearson capsule4 ,illustration3 ,,fig4 #aad4--;,cellular incubator4 ,two incubators at least are required in the laboratory1 for the cultivation of bacteria the one regulated to maintain a temperature of #cg deg4 ,c41 and known as the hot incubator2 the other1 #bj deg4 ,c4 to #bb deg4 ,c41 and known as the cool or cold incubator4 ,two other incubators1 regulated to #db deg4 ,c4 and #fj deg4 ,c4 respectively1 whilst not absolutely1 necessary very soon justify their purchase4 ,thermo-regulators4--,the thermo-regulator is the most essential portion of the incubator1 as upon its efficient working depends the maintenance of a constant temperature in the cultivation chamber4 ,it is also used in the fitting up of water and paraffin baths1 and for many other purposes4 ,illustration3 ,,fig4 #aae4--,reichert's thermo-regulator4 ,of the many forms and varieties of thermo-regulator 7other than electrical71 two only are of sufficiently general use to need mention4 ,in one of these the flow of gas to the gas-jet is controlled by the expansion or contraction of mercury within a glass bulb2 in the other1 by alterations in the position of the walls of a metallic capsule containing a fluid1 the boiling-point of which corresponds to the temperature at which the incubator is intended to act4 ,they are3 7a7 ,reichert's 7,fig4 #aae71 consists of a bulb containing mercury which is to be suspended in the medium1 whether air or water1 the temperature of which it is desired to regulate4 ,gas enters at ,a1 and passes out to the jet by ,b4 ,as the temperature rises the mercury expands and cuts off the main gas supply4 ,as the temperature falls the mercury contracts and reopens the narrow tube ,c4 ,by means of a thumbscrew ,d 7which mechanically raises or lowers the column of mercury irrespective of the temperature7 and the aid of a thermometer the apparatus can be set to keep the incubator at any desired temperature4 ,with this form a special gas burner is required1 with separate supply of gas to a pilot jet at the side4 7b7 ,hearson's capsule regulator consists of a metal capsule hermetically sealed and filled with a liquid which boils at the required temperature1 this is adjusted in the interior of the incubator4 ,soldered to the upper side of the capsule is a thick piece of metal having a central cup to receive the lower end of a rigid rod1 through which the movements of the walls of the capsule are transmitted to the gas valve fixed outside the incubator4 ,the gas valve or governor is shown in figure #aaf4 ,a is the inlet for gas1 ,c the outlet to burner heating the water jacket1 ,b ,d a lever pivoted to standards at ,g1 and acted upon by the capsule1 through the rigid rod which enters the socket below the screw ,p4 ,illustration3 ,,fig4 #aaf4--;,capsule thermo-regulator4 ,the construction of the valve is such that1 whenever the short arm of the lever ,b ,d presses on the disc below the end ,b1 the main supply of gas is entirely cut off4 ,at such times1 however1 a very small portion of gas passes from ,a to ,c1 through an aperture inside the valve1 the size of which aperture can be adjusted by the screw needle ,s1 hence the gas flame below the incubator is never extinguished4 ,the expansion of the metal walls of the capsule1 which takes place upon the boiling of its contents1 provides the motive force1 transmitted through the rigid rod to raise the long arm of the lever ,b ,d1 and as this expansion only takes place at a predetermined temperature1 the lever will only be acted upon when the critical temperature is reached1 no sensible effect being produced at even #a deg4 ,c4 below that at which the capsule is destined to act4 ,w is a weight sliding on the lever rod ,d2 by increasing the distance between the weight and the fulcrum of the lower increased pressure is brought to bear upon the walls of the capsule with the result that the boiling-point of the liquid in the capsule is slightly raised1 and a range of about two degrees can thus be obtained with any particular capsule4 ,,footnotes3 #g ,made by the firm of ,chas4 ,hearson ,co41 #bce ,regent ,st41 ,london1 ,w4 ,,xiv4 ,,methods ,,of ,,cultivation4 ,cultivations of micro-organisms are usually prepared in the laboratory in one of three ways3 ,tube cultures4 ,plate cultures4 ,hanging-drop cultures4 ,these may be incubated either aerobically 7i4 e41 in the presence of oxygen7 or anaerobically 7i4 e41 in the absence of oxygen1 or in the presence of an indifferent gas1 such as hydrogen1 nitrogen1 or carbon dioxide74 ,with regard to the temperature at which the cultivations are grown1 it may be stated as a general rule that all media rendered solid by the addition of gelatine are incubated at #bj deg4 ,c41 or at any rate at a temperature not exceeding #bb deg4 ,c4 7that is1 in the cold incubator72 whilst fluid media and all other solid media are incubated at #cg deg4 ,c4 7that is1 in the hot incubator74 ,exceptions to this rule are numerous4 ,for instance1 in studying the growth of the psychrophylic bacteria1 the yeasts and the moulds1 the cold incubator is employed for all media4 ,tube cultivations are usually packed in the incubator in small tin cylinders1 such as those in which ,american cigarettes are sold1 or in square tin boxes4 ,beakers or tumblers may be used for the same purpose1 but being fragile are not so convenient4 ,metal test-tube racks1 long enough to just fit into the interior of the incubator and each accommodating two rows of tubes1 are also exceedingly useful4 ,,aerobic4 ,the ,preparation of ,tube ,cultivations4 ,the preparation of a tube cultivation consists in3 7a7 ,inoculating a tube of sterile nutrient medium with a portion of the material to be examined4 7b7 ,incubating the inoculated tube at a suitable temperature4 ,the details of the first of these processes must be varied somewhat according to whether the tubes of nutrient media are inoculated or planted from-- #a4 ,pre-existing cultivations4 #b4 ,morbid material previously collected 7vide page #cgc74 #c4 ,fluids1 tissues1 etc41 or from the animal body direct4 ,the method of preparing tube cultivations from pre-existing cultivations is as follows3 ,illustration3 ,,fig4 #aag4--;,inoculating tubes1 seen from the front4 #a4 ,fluid ,media 7e4 g41 ,nutrient ,bouillon74-- #a4 ,flame the cotton-wool plug of the tube containing the cultivation and also that of the tube of sterile bouillon4 #b4 ,hold the two tubes1 side by side1 between the left thumb and the first and third fingers1 allowing the sealed ends to rest on the dorsum of the hand1 and separating the mouths of the tubes 7which are pointed to the right7 by the tip of the second finger4 ,keep the tubes as nearly horizontal as is possible without allowing the fluid in the bouillon tube to reach the cotton-wool plug 7,fig4 #aag74 #c4 ,sterilise the platinum loop and allow it to cool4#h #d4 ,grasp the plug of the tube containing the cultivation between the little finger and palm of the hand and remove it from the tube4 #e4 ,grasp the plug of the bouillon tube between the fourth finger and the ball of the thumb and remove it from the tube4 #f4 ,pass the platinum loop into the tube containing the culture--do not allow the loop to touch the sides of the tube1 or the handle to touch the medium--and remove a small portion of the growth2 withdraw the loop from the tube1 keeping the infected side of the loop downward4 #g4 ,pass the loop into the bouillon tube almost down to the level of the fluid1 reverse the loop so that the infected side faces upward1 emulsify the portion of the growth in the moisture adhering to the side of the tube which is uppermost4 ,withdraw the loop4 #h4 ,replug both tubes4 #i4 ,sterilise the platinum loop4 #aj4 ,label the bouillon tube with 7a7 the name of the organism and 7b7 the date of inoculation4 #aa4 ,incubate4 #b4 ,solid ,media4--,solid media are stored in tubes in one of two ways3 #a4 ,oblique tube or slanted tube 7,fig4 #aah71 in which the medium has been allowed to solidify whilst the tube was retained in an inclined position1 so forming an extensive surface of medium extending from the bottom of the tube almost to its mouth4 ,this is employed for streak or smear cultivations 7,strichcultur74 #b4 ,straight tube 7,fig4 #aai71 in which the medium forms a cylindrical mass in the lower portion of the tube and presents an upper surface which is at right angles to the long axis of the tube4 ,this is employed for stab or stick cultivations 7,stichcultur71 or by inoculating the medium whilst fluid1 and allowing to solidify in this position1 for shake cultivations4 ,streak ,culture4-- #a4 ,flame the plugs1 sterilise the platinum loop 7or spatula74 ,open the tubes and charge the loop as in previous inoculation4 #b4 ,pass the infected loop to the bottom of the tube to be inoculated and draw it1 as lightly as possible1 along the centre of the surface of the medium1 terminating the streak over the thin layer of medium near the mouth of the tube4 #c4 ,replug the tubes1 sterilise the platinum loop4 #d4 ,label the newly inoculated tube and incubate4 ,smear ,culture4--,proceed generally as in streak culture1 but rub the infected loop all over the surface of the medium1 instead of restricting the inoculation to a narrow line4 ,,note4--,gelatine and agar oblique tubes should be freshly slanted before use4 ,stab ,culture4-- #a4 ,flame the plugs1 open the tubes1 sterilise the platinum needle and charge it with the inoculum as in the previous cultivations4 #b4 ,pass the platinum needle into the tube to be inoculated until it touches the centre of the surface of the medium4 ,now thrust it deeply into the substance of the medium1 keeping the needle as nearly as possible in the axis of the cylinder of medium4 ,then withdraw the needle4 #c4 ,replug the tubes4 ,sterilise the platinum needle4 #d4 ,label the newly planted tube and incubate4 ,,note4--,when gelatine is stored for some time the upper surface of the cylinder becomes concave owing to evaporation4 ,tubes showing this appearance should be liquefied and again allowed to set before use for stab culture1 otherwise when the needle enters the medium1 the surface tension will cause the gelatine cylinder to split4 ,illustration3 ,,fig4 #aah4--,sloped or slanted medium for streak or smear culture4 ,illustration3 ,,fig4 #aai4--,straight tube4 ,shake ,culture4-- #a4 ,liquefy a tube of nutrient gelatine 7or agar1 or other similar medium71 by heating in a water-bath 7,fig4 #aba74 #b4 ,inoculate the liquefied medium and label it1 etc41 precisely as if dealing with a tube of bouillon4 #c4 ,place the newly planted tube in the upright position 7e4 g41 in a test-tube rack7 and allow it to solidify4 #d4 ,label the tube2 when solid1 incubate4 ,esmarch's ,roll ,cultivation4-- #a4 ,liquefy three tubes of gelatine by heat4 #b4 ,prepare three dilutions of the inoculum 7as described for plate cultivations1 page #bbh1 steps #d to #g74 #c4 ,roll the tubes1 held almost horizontally1 in a groove made in a block of ice1 until the gelatine has set in a thin film on the inner surface of tube 7,fig4 #abj72 or under the cold-water tap4 ,illustration3 ,,fig4 #abj4 ,esmarch's roll culture on block of ice4 ,in order that the medium may adhere firmly to the glass1 the agar used for roll cultivation should have #a per cent4 gelatine or #a per cent4 gum arabic added to it before sterilisation4 ,roll cultivations1 which served a most important purpose in the days before the introduction of ,petri dishes for plate cultivations1 are now obsolete in modern laboratories and are merely mentioned for the benefit of students1 since examiners who are interested in the academic and historical aspects of bacteriology sometimes expect candidates to be acquainted with the method of preparing them4 ,the ,preparation of ,plate ,cultures4 ,if a small number of bacteria are suspended in liquefied gelatine1 agar1 or other similar medium1 and the infected medium spread out in an even layer over a flat surface and allowed to solidify1 each individual micro-organism becomes fixed to a certain spot and its further development is restricted to the vicinity of this spot4 ,after a variable interval the growth of this organism becomes visible to the naked eye as a colony4 ,this is the principle upon which the method of plate cultivation is based and its practice enables the bacteriologist to study the particular manner of development affected by each species of microbe when growing 7a7 unrestricted upon the surface of the medium1 7b7 in the depths of the medium4 ,the method itself is as follows3 ,apparatus ,required4-- #a4 ,tripod levelling stand4 #b4 ,large shallow glass dish1 with a square sheet of plate glass to cover it4 #c4 ,spirit level4 #d4 ,case of sterile ,petri dishes4 #e4 ,tubes of sterile nutrient media1 gelatine 7or agar7 previously liquefied by heating in the water-bath and cooled to #db deg4 ,c41 otherwise the heat of the medium would destroy many1 if not all1 of the bacteria introduced4 #f4 ,tube of cultivation to be planted from4 #g4 ,platinum loop4 #h4 ,bunsen burner4 #i4 ,grease pencil4 ,illustration3 ,,fig4 #aba4--;,handy form of water-bath for melting tubes of agar and gelatine previous to slanting them2 or to making shake cultures or pouring plates4 ,method of ,pouring ,plates4-- #a4 ,place the glass dish on the levelling tripod 7,figs4 #abb1 #abc72 if gelatine plates are to be poured fill the dish with ice water--gelatine solidifies so slowly that it is necessary to hasten the process2 if agar is to be used fill with water at #ej deg4 ,c4--agar sets almost immediately at the room temperature and by slightly retarding the process lumpiness is avoided2 cover the dish with the square sheet of glass4 #b4 ,place the spirit level on the sheet of glass and by means of the levelling screws adjust the surface of the glass to the horizontal4 ,this leveling is an important matter since the development of a colony is to some extent proportionate to the supply of medium available for its nutrition4 ,thus in a smear on sloped tube culture1 the colonies at the upper part of the medium are stunted and small but increase in size and luxuriance of growth the nearer they approach to the bottom of the tube1 where there is the greatest depth of medium4 ,illustration3 ,,fig4 #abb4--,plate-levelling stand4 #c4 ,place three sterile ,petri dishes in a row on the surface of the glass plate and number them #a1 #b1 and #c1 from left to right4 ,illustration3 ,,fig4 #abc4--,plate-levelling stand1 side view4 #d4 ,number the previously liquefied tubes of nutrient media #a1 #b1 and #c4 ,flame the plugs and see that each plug can be readily removed from the mouth of its tube4 #e4 ,add one loopful of the inoculum to tube ,no4 #a1 treating the liquefied medium as bouillon4 ,after replugging1 grasp the tube near its mouth by the thumb and first finger of the right hand1 and with an even circular movement of the whole arm1 diffuse the inoculum throughout the medium2 avoid jerky movements1 as these cause bubbles of air to form in the medium4 ,illustration3 ,,fig4 #abd4--,mixing emulsion for plates4 ,the knack of mixing evenly without producing air bubbles1 is not always easily acquired1 by this method4 ,an alternative plan is to hold the inoculated tube vertically upright between the opposed palms and to rotate it between them by rapid backward and forward movements of the two hands 7,fig4 #abd74 ,illustration3 ,,fig4 #abe4--,pouring plates4 #f4 ,sterilise the platinum loop1 and add two loopfuls of diluted inoculum to tube ,no4 #b1 and mix as before4 #g4 ,in a similar manner transfer three loopfuls of liquefied medium from tube ,no4 #b to tube ,no4 #c1 and mix thoroughly4 #h4 ,flame the plug of tube ,no4 #a1 remove it1 then flame the lips of the tube2 slightly raise the cover of ,petri dish ,no4 #a1 introduce the mouth of the tube2 then1 elevating the bottom of the tube1 pour the liquefied medium into the ,petri dish1 to form a thin layer4 ,remove the mouth of the tube and close the plate4 ,if the medium has failed to flow evenly over the bottom of the plate1 raise the plate from the levelling platform and by tilting in different directions rectify the fault4 #i4 ,pour plates ,no4 #b and ,no4 #c1 in a similar manner1 from tubes ,nos4 #b and #c4 #aj4 ,label the plates with the distinctive name or number of the inoculum1 also the date2 the number of the dilution having been previously indicated 7step #c74 #aa4 ,place in the cool incubator for three or more days1 as may be necessary4 ,in this way colonies may be obtained quite pure and separate from each other4 ,in plate ,no4 #a1 probably1 the colonies will be so numerous and crowded1 and therefore so small1 as to render it useless4 ,in plate ,no4 #b they will be more widely separated1 but usually ,no4 #c is the plate reserved for careful examination1 as in this the colonies are usually widely separated1 few in number1 and large in size4 ,agar plates are poured in a similar manner1 but the agar must be melted in boiling water and then allowed to cool to #de deg4 ,c4 or #db deg4 ,c4 in a carefully regulated water-bath before being inoculated1 and the entire process must be carried out very rapidly1 otherwise the agar will have solidified before the operation is completed4 ,,note4--,in pouring plates1 since tube ,no4 #a 7for the first dilution7 rarely gives a plate that is of any practical value it is frequently replaced by a tube of bouillon or sterile salt solution1 and in such case plate ,no4 #a is not poured4 ,surface ,plates4-- ,this method of pouring what may be termed whole plates 7since colonies may appear both on the surface and in the depths of the medium7 is essential to the accurate study of the formation of colonies under various conditions1 but when the main object of the separation of the bacteria is to obtain subcultivations from a number of individual bacteria1 surface plates must be prepared1 since here colony formation is restricted to the surface of the medium4 ,the method adopted varies slightly according to whether the medium employed is gelatine or agar1 or one of the derivatives or variants of the latter4 7a7 ,gelatine ,surface ,plates4-- #a4 ,liquefy three tubes of nutrient gelatine4 #b4 ,pour each tube into a separate ,petri dish and allow it to solidify4 ,then turn each plate and its cover upside down4 ,illustration3 ,,fig4 #abf4--,surface plate spreader4 #c4 ,when quite cold raise the bottom of plate #a1 revert it and deposit a drop of the inoculum 7whether a fluid culture or an emulsion from solid culture7 upon the surface of the gelatine with a platinum loop--close to one side of the plate2 replace the bottom half of the ,petri dish in its cover4 #d4 ,take a piece of thin glass rod1 stout platinum wire or best of all a piece of aluminium wire 7say #b mm4 diameter7 about #bh cm4 long4 ,bend the terminal #d cm4 at right angles to the remainder1 making an ,l-shaped rod 7,fig4 #abf74 ,sterilise the short arm and adjacent portion of the long arm1 in the ,bunsen flame1 and allow it to cool4 #e4 ,now raise the bottom of the ,petri dish in the left hand1 leaving the cover on the laboratory bench1 and holding it vertically1 smear the drop of inoculum all over the surface of the gelatine with the short arm of the spreader by a rotatory motion1 7,fig4 #abg74 ,replace the dish in its cover4 #f4 ,raise the bottom of plate #b and rub the infected spreader all over the surface of the gelatine--then go on in like manner to the third plate in the series4 #g4 ,sterilise the spreader4 #h4 ,label and incubate the plates4 ,illustration3 ,,fig4 #abg4--,spreading surface plate4 ,after incubation1 plate ,no4 #a will probably yield an enormous number of colonies2 plate #b will show fewer colonies1 since only those bacteria adhering to the rod after rubbing over plate #a would be deposited on its surface1 and by the time the rod reached plate #c but very few organisms should remain upon it4 ,so that the third plate as a rule will only show a very few scattered colonies1 eminently suitable for detailed study4 7b7 ,agar ,surface ,plates4-- #a4 ,liquefy three tubes of nutrient agar--nutrose agar or the like4 #b4 ,pour each tube into a separate ,petri dish and allow it to solidify4 #c4 ,when quite solid invert each dish1 raise the bottom half and rest it obliquely on its inverted cover 7,fig4 #abh7 and place it in this position in an incubator at #fj deg4 ,c4 for forty-five minutes 7or in an incubator at #db deg4 ,c4 for two hours74 ,this evaporates the water of condensation and gives the medium a firm1 dry surface4 #d4 ,on removing the plates from the incubator close each dish and place it--still upside down--on the laboratory bench4 ,illustration3 ,,fig4 #abh4--;,drying surface plate of agar4 #e4 ,inoculate the plates in series of three1 as described for gelatine surface plates #c-h4 ,hanging-drop ,cultivation4 ,apparatus ,required4-- ,hanging-drop slides4 ,cover-slips4 ,section rack 7,fig4 #ge74 ,blotting paper4 ,bell glass to cover slides4 ,original culture4 ,tubes of broth1 or liquefied gelatine or agar4 ,forceps4 ,platinum loop4 ,bunsen burner4 ,grease pencil4 ,sterile vaseline4 ,lysol4 7a7 ,fluid ,media4-- #a4 ,prepare first and second dilutions of the inoculum as directed for plate cultivations 7vide pages #bbh-bbi1 sections #d to #f71 substituting tubes of nutrient broth for the liquefied gelatine4 #b4 ,sterilise a hanging-drop slide by washing thoroughly in water and drying1 then plunging it into a beaker of absolute alcohol1 draining off the greater part of the spirit1 grasping the slide in a pair of forceps1 and burning off the remainder of the alcohol in the flame4 #c4 ,place the hanging-drop slide on a piece of blotting paper moistened with #b per cent4 lysol solution and cover it with a small bell glass that has been rinsed out with the same solution and not dried4 #d4 ,raise the bell glass slightly and smear sterile vaseline around the rim of the metal cell by means of a sterile spatula of stout platinum wire4 #e4 ,remove a clean cover-slip from the alcohol pot with sterile forceps and burn off the alcohol2 again raise the bell glass and place the sterile cover-slip on the blotting paper by the side of the hanging-drop slide4 #f4 ,remove a drop of the broth from the second dilution tube with a large platinum loop2 raise the bell glass and deposit the drop on the centre of the cover-slip4 ,sterilise the loop4 #g4 ,raise the bell glass sufficiently to allow of the cover-slip being grasped with forceps1 inverted1 and adjusted over the cell of the hanging-drop slide4 ,remove the bell glass altogether and press the cover-slip firmly on to the cell4 #h4 ,either incubate and examine at definite intervals1 or observe continuously with the microscope1 using a warm stage if necessary 7,fig4 #ec74 7b7 ,solid ,media4--,observing precisely similar technique1 a few drops of liquefied gelatine or agar from the second dilution tube may be run over the surface of the sterile cover-slip and a hanging-drop plate cultivation thereby prepared4 ,this method is extremely useful in connection with the study of yeasts1 when the circular cell on the hanging-drop slide should be replaced by a rectangular cell some #ch by #ai mm41 and the gelatine spread over a cover-slip of similar size4 ,after sealing down the preparation1 the upper surface of the cover-slip may be ruled into squares by the aid of the grease pencil or a writing diamond and numbered to facilitate the subsequent identification of the colonies which are observed to develop from solitary germs4 ,hanging-block ,culture 7,hill74-- ,apparatus required3 ,as for hanging-drop cultivation with the addition of a scalpel4 ,carry out the method as far as possible under cover of a bell glass1 to avoid aerial contamination4 #a4 ,liquefy a tube of nutrient agar 7or gelatine7 and pour into a ,petri dish to the depth of about #d mm4 and allow to set4 #b4 ,with a sharp scalpel cut out a block some #h mm4 square1 from the entire thickness of the agar layer4 #c4 ,raise the agar block on the blade of the scalpel and transfer it1 under side down1 to the centre of a sterile slide4 #d4 ,spread a drop of fluid cultivation 7or an emulsion of growth from a solid medium7 over the upper surface of the agar block as if making a cover-slip film4 #e4 ,place the slide and block covered by the bell glass in the incubator at #cg deg4 ,c4 for ten minutes to dry slightly4 #f4 ,take a clean dry sterile cover-slip in a pair of forceps1 and with the help of a second pair of forceps lower it carefully on the inoculated surface of the agar 7avoiding air bubbles71 so as to leave a clear margin of cover-slip overlapping the agar block4 #g4 ,invert the preparation and with the blade of the scalpel remove the slide from the agar block4 #h4 ,with a platinum loop run a drop or two of melted agar around the edges of the block4 ,this solidifies at once and seals the block to the cover-slip4 #i4 ,prepare a sterile hanging-drop slide1 and smear hard vaseline or melted white wax on the rim of the metal cell4 #aj4 ,invert the cover-slip with the block attached on to the hanging-drop slide1 and seal the cover-slip firmly in place4 #aa4 ,observe as for hanging-drop cultivations4 ,,anaerobic ,,cultivations4 ,numerous methods have been devised for the cultivation of anaerobic bacteria1 the majority requiring the employment of special apparatus4 ,the principle upon which any method is based and upon which it depends for its success falls under one or another of the following headings3 7a7 ,exclusion of air from the cultivation4 7b7 ,exhaustion of air from the vessel containing the cultivation by means of an air pump--i4 e41 cultivation in vacuo4 7c7 ,absorption of oxygen from the air in contact with the cultivation by means of pyrogallic acid rendered alkaline with caustic soda--i4 e41 cultivation in an atmosphere of nitrogen4 7d7 ,displacement of air by an indifferent gas1 such as hydrogen or coal gas--i4 e41 cultivation in an atmosphere of hydrogen4 7e7 ,a combination of two or more of the above methods4 ,a selection of the simplest and most generally useful methods is given here4 ,whenever possible1 the nutrient media that are employed in any of the processes should contain some easily oxidisable substance1 such as sodium formate 7#j4d per cent47 or sodium sulphindigotate 7#j4a per cent471 which will absorb all the available oxygen held in solution by the medium4 ,the further addition of glucose1 #b per cent41 favors the growth of anaerobic bacteria 7vide1 pages #ahi-aij74 ,further1 it is advisable to seal all joints between india-rubber stoppers and tubulures or the mouths of the tubes with melted paraffin2 glass stoppers and taps should be lubricated with resin ointment or a mixture of beeswax #a part1 olive oil #d parts4 7,a7 ,method ,i 7,hesse's ,method74-- #a4 ,make a stab culture in gelatine or agar1 choosing for the purpose a straight tube containing a deep column of medium1 and thrusting the inoculating needle to the bottom of the tube4 #b4 ,pour a layer of sterilised oil 7olive oil1 vaseline1 or petroleum71 #a or #b cm4 deep1 upon the surface of the medium4 #c4 ,incubate4 ,method ,,ii4--,this method is only available when dealing with pure cultivations4 #a4 ,liquefy a tube of gelatine 7or agar7 by heat1 pour it into a ,petri dish1 and allow it to solidify4 #b4 ,inoculate the surface of the medium in one spot only4 #c4 ,remove a cover-slip from the pot of absolute alcohol with sterile forceps2 burn off the alcohol in the gas flame4 #d4 ,lower the now sterile cover-slip carefully on to the inoculated surface of the medium1 carefully excluding air bubbles1 and press it down firmly with the points of the forceps4 7,a sterile disc of mica may be substituted for the cover-slip47 #e4 ,incubate4 ,method ,,iii 7,roux's ,physical ,method74-- #a4 ,prepare tube cultures of fluid media 7or solid media rendered fluid by heat7 in the usual way4 #b4 ,aspirate some of the inoculated media into capillary pipettes4 #c4 ,seal both ends of each pipette in the blowpipe flame4 #d4 ,incubate4 ,method ,,iv 7,roux's ,biological ,method74-- #a4 ,plant a deep stab1 as in method ,i4 #b4 ,pour a layer1 #a or #b cm4 deep1 of broth cultivation of a vigourous aerobe--e4 g41 ,b4 aquatilis sulcatus or ,b4 prodigiosus--upon the surface of the medium2 or an equal depth of liquefied gelatine1 which is then inoculated with the aerobic organism4 #c4 ,incubate4 ,the growth of the aerobe will use up all the oxygen that reaches it and will not allow any to pass through to the medium below1 which will consequently remain in an anaerobic condition4 7,b7 ,method ,v4-- #a4 ,prepare tube or flask cultivations in the usual way4 #b4 ,replace the cotton-wool plug by an india-rubber stopper perforated with one hole and fitted with a length of glass tubing which has a constriction about #c cm4 above the stopper and is then bent at right angles 7,fig4 #abi74 ,the stopper and glass tubing are sterilised by being boiled in a beaker of water for five minutes4 ,illustration3 ,,fig4 #abi4--,vacuum culture4 #c4 ,connect the tube leading from the culture vessel with a water or air pump1 interposing a ,wulff's bottle fitted as a wash-bottle and containing sulphuric acid4 #d4 ,exhaust the air from the culture vessel4 #e4 ,before disconnecting the apparatus1 seal the glass tube from the culture vessel at the constriction1 using the blowpipe flame4 #f4 ,incubate4 7,c7 ,method ,,vi 7,buchner's ,method74 ,apparatus and ,solutions ,required4-- ,buchner's tube 7a stout glass test-tube #bc cm4 long and #d cm4 in diameter1 fitted with india-rubber stopper1 ,fig4 #acj74 ,pyrogallic acid in compressed tablets each containing #a gram4 ,dekanormal solution of caustic soda4 ,,method4-- #a4 ,prepare the tube cultivation in the usual way4 #b4 ,moisten the india-rubber stopper of the ,buchner's tube with water and see that it fits the mouth of the tube accurately4 #c4 ,remove the stopper from the caustic soda bottle4 #d4 ,drop one of the pyrogallic acid tablets#i into the ,buchner's tube 7roughly1 use #a gramme pyrogallic acid for every #ajj c4c4 air capacity of the receiving vessel74 #e4 ,add about #a c4c4 of the soda solution4 #f4 ,place the inoculated tube inside the ,buchner's tube4 ,the pyrogallic tablet acts as a buffer and prevents damage to either the inoculated tube or the ,buchner's tube even should it be slipped in hurriedly4 #g4 ,fit the india-rubber stopper tightly into the mouth of the ,buchner's tube4 ,illustration3 ,,fig4 #acj4--;,buchner's tube4 ,the pyrogallic acid tablet dissolves slowly in the soda solution and its oxidation proceeds very slowly at first so that ample time is available when this method is adopted4 #h4 ,restopper the caustic soda bottle4 #i4 ,place ,buchner's tube in a wire support1 and incubate4 ,method ,,vii 7,wright's ,method74-- #a4 ,prepare tube cultivation in the usual way4 #b4 ,cut off that portion of the cotton-wool plug projecting above the mouth of the tube with scissors1 then push the plug into the tube for a distance of #b or #c cm4 #c4 ,by means of a pipette drop about #a c4c4 of pyrogallic acid #aj per cent4 aqueous solution on to the plug4 ,it will immediately be absorbed by the cotton-wool4 #d4 ,with another pipette run in an equal quantity of the caustic soda solution4 #e4 ,quickly close the mouth of the tube with a tightly fitting india-rubber stopper4 #f4 ,incubate4 ,illustration3 ,,fig4 #aca4--,mc,leod's anaerobic plate base with half petri dish inverted in situ ,method ,,viii 7,mc,leod's ,method74-- ,apparatus and ,solutions ,required4-- ,mc,leod's plate base 7a hollow glazed earthenware disc #i cm4 in diameter and #b cm4 deep3 the upper surface is pierced by a central hole1 #b cm4 in diameter1 giving access to the interior1 the lower part of which is divided into two by a low partition4 ,a shallow groove encircles the upper surface near to the edge74 ,plasticine4 ,pyrogallic acid 7#a gramme7 compressed tablets4 ,sodic hydroxide 7#j4d gramme7 compressed tablets4 ,wash bottle of distilled water4 ,surface plates of one or other agar medium 7in petri dishes of #h cm4 diameter74 ,surface plate spreader4 ,,method4-- #a4 ,roll out a long cylinder of plasticine and fit it into the groove on the upper surface of the earthenware base4 #b4 ,place a tablet of pyrogallic acid in one division of the interior of the plate base1 and two tablets of sodic hydroxide in the other4 #c4 ,prepare surface plate culture of the organism to be cultivated4 #d4 ,run a few cubic centimetres of distilled water into that division of the plate base containing the sodic hydroxide4 #e4 ,invert the bottom half of the surface plate over the plate base and press its edges firmly down into the plasticine filling the groove4 #f4 ,label and incubate4 7,d7 ,method ,,ix4-- ,apparatus ,required4-- ,small ,ruffer's or ,woodhead's flask 7,fig4 #cc74 ,sterile india-rubber stopper4 ,india-rubber tubing4 ,glass tubing4 ,metal screw clips4 ,cylinder of compressed hydrogen2 or hydrogen gas apparatus ,,method4-- #a4 ,sterilise a glass vessel1 shaped as in a ,ruffer's or ,woodhead's flask1 in the hot-air oven4 7,the tubulure and the side tubes are plugged with cotton-wool47 ,after sterilisation1 fix a short piece of rubber tubing occluded by a metal clip to each side tube4 #b4 ,inoculate a large quantity 7e4 g41 #bjj c4c47 of the medium4 ,where solid media are employed they must first be liquefied by heat4 #c4 ,remove the cotton-wool plug from the tubulure and pour the inoculated medium into the glass vessel4 #d4 ,close the tubulure by means of an india-rubber stopper previously sterilised by boiling in a beaker of water4 ,illustration3 ,,fig4 #acb4--,kipp's hydrogen apparatus1 7a7 connected up to two washing bottles containing 7b7 lead acetate #aj per cent4 solution1 to remove ,h#b,s and 7c7 silver nitrate solution to remove ,as,h#c4 ,a third washing bottle containing pyrogallic acid #aj per cent4 solution1 rendered alkaline1 to remove any trace of oxygen1 is sometimes introduced4 ,illustration3 ,,fig4 #acc4--;,improved gas apparatus2 the metal is contained in a perforated glass tube which is submerged in acid when the triangular bottle is upright 7a71 but is above the level of the liquid when the bottle is turned on its side 7b74 #e4 ,connect up the india-rubber tubing on one of the side tubes with a cylinder of compressed hydrogen 7or the delivery tube of a ,kipp's ,fig4 #acb or other hydrogen apparatus1 ,fig4 #acc71 interposing a short piece of glass tubing2 and in like manner connect a long piece of rubber tubing which should be led into a basin of water1 to the opposite side tube4 #f4 ,open both metal clips and pass hydrogen through the vessel until the atmospheric air is replaced by hydrogen4 ,this is determined by collecting some of the gas which bubbles through the water in the basin in a test-tube and testing it by means of a lighted taper4 #g4 ,close the metal clip on the tube through which the gas is entering2 close the clip on the exit tube4 #h4 ,disconnect the gas apparatus4 #i4 ,incubate4 ,method ,x 7,botkin's ,method74-- ,apparatus ,required4-- ,large glass dish #bj cm4 diameter and #h cm4 deep4 ,flat leaden cross slightly shorter than the internal diameter of the glass dish4 ,bell glass about #ae cm4 diameter and #bj to #be cm4 high4 ,metal frame for plate cultivations4 ,or1 glass battery jar for tube cultivations4 ,cylinder of compressed hydrogen4 ,rubber tubing4 ,two pieces of ,u-shaped glass tubing 7each arm #h cm4 in length74 ,half a litre of glycerine 7or metallic mercury74 ,,method4-- #a4 ,place the leaden cross inside the glass dish1 resting on the bottom4 #b4 ,prepare the cultivations in the usual way4 #c4 ,place the tube cultivations in a glass battery jar 7or the plate cultivations on a metal frame71 resting on the centre of the leaden cross4 #d4 ,cover the cultivations with the bell jar4 #e4 ,adjust the ,u-shaped pieces of glass tubing in a vertical position on opposite sides of the bell jar1 one arm of each inside the jar1 the other outside4 ,these tubes are best held in position by embedding the ,u-shaped bends in two lumps of plasterine stuck on the bottom of the glass dish4 ,fix a short length of rubber tubing clamped with a metal clip to each of the outside arms 7,fig4 #acd74 #f4 ,fill the glass dish with glycerine or metallic mercury to a depth of about #e cm4 ,illustration3 ,,fig4 #acd4--;,botkin's apparatus4 #g4 ,connect up one ,u-shaped tube with the hydrogen cylinder 7or gas apparatus7 by means of rubber tubing4 ,replace the atmospheric air by hydrogen1 as in method ,,ix4 #h4 ,clamp the tubes and disconnect the gas apparatus4 #i4 ,incubate4 ,method ,,xi 7,novy's ,method74-- ,apparatus ,required4-- ,jar for plate cultivations 7,fig4 #ace74 ,or1 jar for tube cultivations 7,fig4 #acf74 ,lubricant for stopper of jar4 ,rubber tubing4 ,cylinder of compressed hydrogen4 ,,method4-- #a4 ,prepare cultivations in the usual way4 #b4 ,place these inside the jar4 #c4 ,lubricate the stopper and insert it in the mouth of the jar1 with the handle in a line with the two side tubes4 #d4 ,connect up the delivery tube a with the hydrogen gas supply by means of rubber tubing4 ,illustration3 ,,fig4 #ace4--,novy's jar for plate cultivations4 ,illustration3 ,,fig4 #acf4--,novy's jar for tube cultivations4 #e4 ,attach a piece of rubber tubing to the exit tube b and collect samples of the issuing gas 7over water7 and test from time to time4 #f4 ,when the air is completely displaced by hydrogen1 turn the handle of the stopper at right angles to the line of entry and exit tubes2 this seals the orifice of both tubes4 #g4 ,disconnect the gas apparatus and incubate4 7,e7 ,method ,,xii 7,bulloch's ,method74-- ,apparatus ,required4-- ,bulloch's jar4 ,pot of resin ointment4 ,small glass dish #ad cm4 diameter by #e cm4 deep4 ,vessel for tube cultures or metal rack for plate cultures4 ,pyrogallic acid tablets4 ,cylinder of compressed hydrogen4 ,geryk or other air pump4 ,rubber pressure tubing4 #aj c4c4 pipette4 ,glass tubing4 ,dry granulated caustic soda or compressed tablets each1 containing #j4d grammes sodic hydroxide4 ,small beaker of water4 ,,method4-- #a4 ,prepare the cultivations in the usual way4 #b4 ,place the glass dish in the centre of the glass slab1 and stand the cultivations inside this4 #c4 ,place a sufficient number of pyrogallic acid tablets at one side of the glass dish 7i4 e41 #a tablet for each #ajj cubic centimeters air capacity of the bell jar74 ,place a small heap of dry granulated soda 7or half a dozen tablets of sodic hydroxide7 by the side of the pyro tablets4 #d4 ,smear the flange of the bell jar with resin ointment and apply the jar firmly to the glass slab1 covering the cultivations--so arranged that the long tube passes with its lower end into the glass dish at a point directly opposite to the pyrogallic acid tablets4 ,lubricate the two stop-cocks with resin ointment 7,fig4 #acg74 #e4 ,connect up the short tube a with the gas-supply by means of rubber pressure tubing and open both stop-cocks4 #f4 ,connect a long1 straight piece of glass tubing to the long tube b by means of a piece of rubber tubing interposing a screw clamp3 and collect samples of the issuing gas from time to time and test4 #g4 ,when the air is displaced1 shut off the stop-cock of the entry tube1 then that of the exit tube b4 ,screw down the clamp and remove the glass tube from the rubber connection and connect up the short tube a to the air pump by means of pressure tubing4 #h4 ,open the stop-cock of tube a and with two or three strokes of the air pump1 aspirate a small quantity of gas1 so creating a slight vacuum4 ,then shut off the stop-cock and disconnect the air pump4 #i4 ,fill the #aj c4c4 bulb pipette with water2 insert its point into the rubber tubing on the long tube b as far as the screw clamp4 ,open the screw clamp and run in water until stopped by the internal pressure4 ,shut off stop-cock4 7,the water dissolves the soda and pyrogallic acid converting the latter into alkaline pyro4 and so bringing its latent capacity for oxygen into action74 ,illustration3 ,,fig4 #acg4--;,bulloch's jar4 #aj4 ,reverse the tubes from the tubulures so that they meet1 out of harm's way1 over the top of the bell glass2 again see that all joints are tight and transfer the apparatus to the incubator4 ,this last method is the most satisfactory for anaerobic cultivations1 as by its means complete anaerobiosis can be obtained with the least expenditure of time and trouble4 ,,footnotes3 #h ,see also method of opening and closing culture tubes1 pages #gd-gf4 #i ,if compressed tablets of pyrogallic acid cannot be obtained make up a stock acid pyro solution ,pyrogallic acid1 #aj grammes ,hydrochloric acid1 #a4e c4c4 ,distilled water1 #ajj c4c4 and at step #d1 run in #aj c4c4 of the solution4 ,,xv4 ,,methods ,,of ,,isolation4 ,the work in the preceding sections1 arranged to demonstrate the chief biological characters of bacteria in general1 is intended to be carried out by means of cultivations of various organisms previously isolated and identified and supplied to the student in a state of purity4 ,a cultivation which comprises the progeny of a single cell is termed a pure culture2 one which contains representatives of two or more species of bacteria is spoken of as an impure1 or mixed cultivation1 and it now becomes necessary to indicate the chief methods by which one or more organisms may be isolated in a state of purity from a mixture2 whether that mixture exists as an impure laboratory cultivation1 or is contained in pus and other morbid exudations1 infected tissues1 or water or food-stuffs4 ,illustration3 ,,fig4 #ach4--;,haematocytometer cell1 showing1 a1 section through the centre of the cell1 and b1 a magnified image of the cell rulings4 ,before the introduction of solid media the only method of obtaining pure cultivations was by dilution--by no means a reliable method4 ,dilution consisted in estimating approximately the number of bacteria present in a given volume of fluid 7by means of a graduated-celled slide resembling a haematocytometer1 ,fig4 #ach71 and diluting the fluid by the addition of sterile water or bouillon until a given volume 7usually #a c4c47 of the dilution contained but one organism4 ,by planting this volume of the fluid into several tubes or flasks of nutrient media1 it occasionally happened that the resulting growth was the product of one individual microbe4 ,a method so uncertain is now fortunately replaced by many others1 more reliable and convenient1 and in those methods selected for description here1 the segregation and isolation of the required bacteria may be effected-- ,a4 ,by ,mechanical ,separation4 #a4 ,by surface plate cultivation3 7a7 ,gelatine4 7b7 ,agar4 7c7 ,serum agar4 7d7 ,blood agar4 7e7 ,hanging-drop or block4 #b4 ,by ,esmarch's roll cultivation3 ,this archaic method 7see page #bbf7 is no longer employed for the isolation of bacteria4 #c4 ,by serial cultivation4 ,b4 ,by ,biological ,differentiation4 #d4 ,by differential media4 7a7 ,selective4 7b7 ,deterrent4 #e4 ,by differential incubation4 #f4 ,by differential sterilisation4 #g4 ,by differential atmosphere cultivation4 #h4 ,by animal inoculation4 ,the selection of the method to be employed in any specific instance will depend upon a variety of circumstances1 and often a combination of two or more will ensure a quicker and more reliable result than a rigid adherence to any one method4 ,experience is the only reliable guide1 but as a general rule the use of either the first or the third method will be found most convenient1 affording as each of them does an opportunity for the simultaneous isolation of several or all of the varieties of bacteria present in a mixture4 #a4 ,surface ,plate ,cultivations4-- 7a7 ,gelatine 7vide page #afd74 7b7 ,agar 7vide page #afg74 7c7 ,alkaline serum agar 7vide page #baa74 ,these plates are prepared in a manner precisely similar to that adopted for nutrient gelatine and agar surface plates 7vide pages #bca-bcc74 7d7 ,serum ,agar4-- #a4 ,melt three tubes of nutrient agar1 label them #a1 #b1 and #c1 and place them1 with three tubes of sterile fluid serum1 also labelled #a;a1 #b;a1 and #c;a1 in a water-bath regulated at #de deg4 ,c42 allow sufficient time to elapse for the temperature of the contents of each tube to reach that of the water-bath4 #b4 ,take serum tube ,no4 #a;a and agar tube ,no4 #a4 ,flame the plugs and remove them from the tubes 7retaining the plug of the agar tube in the hand72 flame the mouths of the tubes1 pour the serum into the tube of liquefied agar and replace the plug of the agar tube4 #c4 ,mix thoroughly and pour plate ,no4 #a secundum artem4 #d4 ,treat the remaining tube of agar and serum in a similar fashion1 and pour plates ,nos4 #b and #c4 #e4 ,dry the serum agar plates in the incubator running at #fj deg4 ,c4 for one hour 7see page #bcb74 #f4 ,inoculate the plates in series as described for gelatine surface plates 7page #bca74 7e7 ,blood ,agar1 ,human4-- #a4 ,melt a tube of sterile agar and pour it into a sterile plate2 let it set4 #b4 ,collect a few drops of human blood1 under all aseptic conditions1 in a sterile capillary teat pipette4 #c4 ,raise the cover of the ,petri dish very slightly1 insert the extremity of the capillary pipette1 and deposit the blood on the centre of the agar surface4 ,close the dish4 #d4 ,charge a platinum loop with a small quantity of the inoculum4 ,raise the cover of the plate1 introduce the loop1 mix its contents with the drop of blood1 remove the loop1 close the dish and sterilise the loop4 #e4 ,finally smear the mixture over the surface of the agar with a sterilised ,l-shaped rod4 #f4 ,label and incubate4 7,if considered necessary1 two1 three1 or more similar plates may be inoculated in series47 7f7 ,blood ,agar1 ,animal4-- ,when preparing citrated blood agar 7page #aga7 it is always advisable to pour several blood agar tubes into plates1 which can be stored in the ice chest ready for use at any moment for surface plate cultures4 7g7 ,hanging-drop or block culture1 7vide page #bcc74 #c4 ,serial ,cultivations4--,these are usually made upon agar or blood-serum1 although gelatine may also be used4 ,the method is as follows3 #a4 ,take at least four slanted tubes of media and number them consecutively4 #b4 ,flame all the plugs and see that each can be readily removed4 #c4 ,charge the platinum loop with a small quantity of the inoculum1 observing the usual routine1 and plant tube ,no4 #a1 smearing thoroughly all over the surface4 ,if any water of condensation has collected at the bottom of the tube1 use this as a diluent before smearing the contents of the loop over the surface of the medium4 #d4 ,without sterilising or recharging the loop1 inoculate tube ,no4 #b1 by making three parallel streaks from end to end of the slanted surface4 #e4 ,plant the remainder of the tubes in the series as smears like tube ,no4 #a4 #f4 ,label with distinctive name or number1 and date2 incubate4 ,the growth that ensues in the first two or three tubes of the series will probably be so crowded as to be useless4 ,toward the end of the series1 however1 discrete colonies will be found1 each of which can be transferred to a fresh tube of nutrient medium without risk of contamination from the neighbouring colonies4 ,working up ,plates4-- ,having succeeded in obtaining a plate 7or tube cultivation7 in which the colonies are well grown and sufficiently separated from each other1 the process of working up1 pricking out1 or fishing the colonies in order to obtain subcultures in a state of purity from each of the different bacteria present must now be proceeded with4 ,occasionally it happens that this is quite a simple matter4 ,for example1 the original mixed cultivation when examined microscopically was found to contain a ,gram positive micrococcus1 a ,gram positive straight bacillus and a ,gram negative short bacillus4 ,the third gelatine plate prepared from this mixture1 on inspection after four day's incubation1 showed twenty-five colonies--seven moist yellow colonies1 each sinking into a shallow pit of liquefied gelatine1 fourteen flat irridescent filmy colonies1 and four raised white slimy colonies4 ,a film preparation 7stained ,gram7 from each variety examined microscopically showed that the yellow liquefying colony was composed of ,gram positive micrococci2 the flat colony of ,gram positive bacilli and the white colony of gram negative bacilli4 ,one of each of these varieties of colonies would be transferred by means of the sterilised loop to a fresh gelatine culture tube1 and after incubation the growth in each subculture would correspond culturally and microscopically with that of the plate colony from which it was derived1--the object aimed at would therefore be achieved4 ,usually1 however1 the colonies cannot be thus readily differentiated1 and unless they are worked up in an orderly and systematic manner much labour will be vainly expended and valuable time wasted4 ,the following method minimises the difficulties involved4 7,a7 ,inspection4 a4 ,without opening the plate carefully study the various colonies with the naked eye1 with the assistance of a watchmaker's lens or by inverting the plate on the stage of the microscope and viewing with the #a-;inch objective through the bottom of the plate and the layer of medium4 b4 ,if gross differences can be detected mark a small circle on the bottom of the plate around the site of each of the selected colonies1 with the grease pencil4 c4 ,if no obvious differences can be made out choose nine colonies haphazard and indicate their positions by pencil marks on the bottom of the plate4 7,b7 ,fishing ,colonies4-- a4 ,take a sterile ,petri dish and invert it upon the laboratory bench4 ,rule two parallel lines on the bottom of the dish with a grease pencil1 and two more parallel lines at right angles to the first pair--so dividing the area of the dish into nine portions4 ,number the top right-hand portion #a1 and the central bottom portion #h 7,fig4 #aci74 ,revert the dish4 ,the numbers #a and #h can be readily recognised through the glass and by their positions enable any of the other divisions to be localised by number4 ,this is the stock dish4 b4 ,slightly raise the cover of the dish1 and with a sterile teat-pipette deposit a small drop of sterile water in the centre of each of the nine divisions4 c4 ,with the sterilised platinum spatula raise one of the marked colonies from the plate #c and transfer it to the first division in the ruled plate and emulsify it in the drop of water awaiting it4 ,repeat this process with the remaining colonies1 emulsifying a separate colony in each drop of water4 7,c7 ,preliminary ,differentiation of ,bacteria4-- a4 ,prepare a cover-slip film preparation from each drop of emulsion in the stock dish and number to correspond to the division from which it was taken4 ,stain by ,gram's method4 b4 ,examine microscopically1 using the oil immersion lens and note the numbers of those cover-slips which morphologically and by ,gram results appear to be composed of different species of bacteria4 ,illustration3 ,,fig4 #aci4--;,diagram for stock plate4 7,d7 ,preparing ,isolation ,subcultures4-- a4 ,inoculate an agar slope and a broth tube from the emulsion in the stock dish corresponding to each of these specially selected numbers4 b4 ,ascertain whether the cover-slips from the nine emulsions in the stock dish include all the varieties represented in the cover-slip film preparation made from the original mixture before plating4 c4 ,if some varieties are missing prepare a second stock dish from other colonies on plate #c1 and repeat the process until each morphological form or tinctorial variety has been secured in subculture4 d4 ,place the stock dishes in the ice chest to await the results of incubation4 7,if any of the subcultures fail1 further material can be obtained from the corresponding emulsion2 or if it has dried1 by moistening it with a further drop of sterile distilled water47 e4 ,incubate all the subcultures and identify the organisms picked out4 #d4 ,differential ,media4-- 7a7 ,selective4--,some varieties of media are specially suitable for certain species of bacteria and enable them to overgrow and finally choke out other varieties2 e4 g41 wort is the most suitable medium-base for the growth of torulae and yeasts and should be employed when pouring plates for the isolation of these organisms4 ,to obtain a pure cultivation of yeast from a mixture containing bacteria as well1 it is often sufficient to inoculate wort from the mixture and incubate at #cg deg4 ,c4 for twenty-four hours4 ,plant a fresh tube of wort from the resulting growth and incubate4 ,repeat the process once more1 and from the growth in this third tube plant a streak on wort gelatine1 and incubate at #bj deg4 ,c4 ,the resulting growth will almost certainly be a pure culture of the yeast4 7b7 ,deterrent4--,the converse of the above also obtains4 ,certain media possess the power of inhibiting the growth of a greater or less number of species4 ,for instance1 media containing carbolic acid to the amount of #a per cent4 will inhibit the growth of practically everything but the ,bacillus coli communis4 #e4 ,differential ,incubation4-- ,in isolating certain bacteria1 advantage is taken of the fact that different species vary in their optimum temperature4 ,a mixture containing the ,bacillus typhosus and the ,bacillus aquatilis sulcatus1 for example1 may be planted on two slanted agar tubes1 the one incubated at #dj deg4 ,c41 and the other at #ab deg4 ,c4 ,after twenty-four hours' incubation the first will show a pure cultivation of the ,bacillus typhosus1 whilst the second will be an almost pure culture of the ,bacillus aquatilis4 #f4 ,differential ,sterilisation4-- 7a7 ,non-sporing ,bacteria4--,similarly1 advantage may be taken of the varying thermal death-points of bacteria4 ,from a mixture of two organisms whose thermal death-points differ by1 say1 #d deg4 ,c4--e4 g41 ,bacillus pyocyaneus1 thermal death-point #ee deg4 ,c41 and ,bacillus mesentericus vulgatus1 thermal death-point #fj deg4 ,c4--a pure cultivation of the latter may be obtained by heating the mixture in a water-bath to #eh deg4 ,c4 and keeping it at that point for ten minutes4 ,the mixture is then planted on to fresh media and incubated1 when the resulting growth will be found to consist entirely of the ,b4 mesentericus4 7b7 ,sporing ,bacteria4--,this method finds its chief practical application in the differentiation of a spore-bearing organism from one which does not form spores4 ,in this case the mixture is heated in a water-bath at #hj deg4 ,c4 for fifteen to twenty minutes4 ,at the end of this time the non-sporing bacteria are dead1 and cultivations made from the mixture will yield a growth resulting from the germination of the spores only4 ,differential sterilisation at #hj deg4 ,c4 is most conveniently carried out in a water-bath of special construction1 designed by ,balfour ,stewart 7,fig4 #adj74 ,it consists of a double-walled copper vessel mounted on legs1 and provided with a tubulure communicating with the space between the walls4 ,this space is nearly filled with benzole 7boiling-point #hj deg4 ,c42 pure benzole1 free from thiophene must be employed for the purpose1 otherwise the boiling-point gradually and perceptibly rises in the course of time71 and to the tubulure is fitted a long glass tube1 some #b metres long and about #j4ge cm4 diameter1 serving as a condensing tube 7a tube half this length if provided with a condensing bulb at the centre will be equally efficient74 ,the interior of the vessel is partly filled with water and covered with a lid which is perforated for a thermometer4 ,this latter dips into the water and records its temperature4 ,a very small ,bunsen flame under the apparatus suffices to keep the benzole boiling and the water within at a constant temperature of #hj deg4 ,c4 ,the bath is thus always ready for use4 ,,method4--,to use the apparatus4 #a4 ,place some of the mixture itself1 if fluid1 containing the spores1 or an emulsion of the same if derived from solid material1 in a test-tube4 #b4 ,immerse the test-tube in the water contained in the benzole bath1 taking care that the upper level of the liquid in the tube is at least #b cm4 beneath the surface of the water in the copper vessel4 #c4 ,the temperature of the water1 of course1 falls a few degrees after opening the bath and introducing a tube of colder liquid1 but after a few minutes the temperature will have again reached #hj deg4 ,c4 #d4 ,when the thermometer again records #hj deg4 ,c41 note the time1 and fifteen minutes later remove the tube containing the mixture from the bath4 #e4 ,make cultures upon suitable media2 incubate4 ,illustration3 ,,fig4 #adj4--;,benzole bath4 #g4 ,differential ,atmosphere ,cultivation4-- 7a7 ,by adapting the atmospheric conditions to the particular organism it is desired to isolate1 it is comparatively easy to separate a strict aerobe from a strict anaerobe1 and vice versa4 ,in the first case1 however1 it is important that the cultivations should be made upon solid media1 for if carried out in fluid media the aerobes multiplying in the upper layers of fluid render the depths completely anaerobic1 and under these conditions the growth of the anaerobes will continue unchecked4 7b7 ,when it is desired to separate a facultative anaerobe from a strict anaerobe1 it is generally sufficient to plant the mixture upon the sloped surface agar1 incubate aerobically at #cg deg4 ,c41 and examine carefully at frequent intervals4 ,at the first sign of growth1 subcultivations must be prepared and treated in a similar manner4 ,as a result of these rapid subcultures1 the facultative anaerobe will be secured in pure culture at about the third or fourth generation4 7c7 ,if1 on the other hand1 the strict anaerobe is the organism required from a mixture of facultative and strict anaerobes1 pour plates of glucose formate agar 7or gelatine7 in the usual manner1 place them in a ,bulloch's or ,novy's jar1 and incubate at a suitable temperature4 ,pick off the colonies of the required organism when the growth appears1 and transfer to tubes of the various media4 ,incubate under suitable conditions as to temperature and atmosphere4 #h4 ,animal ,inoculation4-- ,finally1 when dealing with pathogenic organisms1 it is often advisable to inoculate some of the impure culture 7or even some of the original materies morbi7 into an animal specially chosen on account of its susceptibility to the particular pathogenic organism it is desired to inoculate4 ,indeed1 with some of the more sensitive and strictly parasitic bacteria this method of animal inoculation is practically the only method that will yield a satisfactory result4 ,,xvi4 ,,methods ,,of ,,identification ,,and ,,study4 ,in order to identify an organism after isolation1 tube1 plate1 and other cultivations must be prepared1 incubated under suitable conditions as to temperature and environment1 and examined from time to time 7a7 macroscopically1 7b7 by microscopical methods1 7c7 by chemical methods1 7d7 by physical methods1 7e7 by inoculation methods1 and the results of these examinations duly recorded4 ,it must be stated definitely that no micro-organism can be identified by any one character or property1 whether microscopical1 biological or chemical1 but that on the contrary its entire life history must be carefully studied and then its identity established from a consideration of the sum total of these observations4 ,in order to give to the recorded results their maximum value it is essential that they should be exact and systematic1 therefore some such scheme as the following should be adhered to2 and especially is this necessary in describing an organism not previously isolated and studied4 ,,scheme ,,of ,,study4 ,designation3 ,originally isolated by 7observer's name7 in 7date71 from 7source of organism74 #a4 ,cultural ,characters4--7,vide ,macroscopical ,examination of ,cultivation1 page #bfa47 ,gelatine plates1 ,gelatine streak1 at #bj deg4 ,c4 ,gelatine stab1 ,gelatine shake1 ,agar plates1 ,agar streak or smear1 ,agar stab1 ,inspissated blood-serum1 at #bj deg4 ,c4 and #cg deg4 ,c4 ,bouillon1 ,litmus milk1 ,potato1 ,special media for the purpose of demonstrating characteristic appearances4 #b4 ,morphology4--7,vide ,microscopical ,examination of ,cultivations1 page #bgb47 ,vegetative forms3 ,shape4 ,size4 ,motility4 ,flagella 7if present74 ,capsule 7if present74 ,involution forms4 ,pleomorphism 7if observed74 ,sporing forms 7if observed74 ,of which class8 ,staining reactions4 #c4 ,chemical ,products of ,growth4--7,vide ,chemical ,examination of ,cultivations1 page #bgf47 ,chromogenesis4 ,photogenesis4 ,enzyme formation4 ,fermentation of carbohydrates3 ,acid formation4 ,alkali formation4 ,indol formation4 ,phenol formation4 ,reducing and oxidising substances4 ,gas formation4 #d4 ,biology4--7,vide ,physical ,examination of ,cultures1 page #bie47 ,atmosphere4 ,temperature4 ,reaction of nutrient media4 ,resistance to lethal agents3 ,physical3 ,desiccation4 ,light4 ,colours4 ,chemical germicides4 ,vitality4 #e4 ,pathogenicity3 ,susceptible animals1 subsequently arranged in order of susceptibility4 ,immune animals4 ,experimental inoculation1 symptoms of disease4 ,post-mortem appearances4 ,virulence3 ,length of time maintained4 ,optimum medium8 ,minimal lethal dose4 ,exaltation and attenuation of virulence8 ,toxin formation4 ,,macroscopical ,,examination ,,of ,,cultivations4 ,in describing the naked-eye and low-power appearances of the bacterial growth the descriptive terms introduced by ,chester 7and included in the following scheme7 should be employed4 ,,solid ,,media4 ,plate ,cultures4-- ,gelatine4--,note the presence or absence of liquefaction of the surrounding medium4 ,if liquefaction is present1 note shape and character 7vide page #bfi1 stab cultures74 ,agar4--,no liquefaction takes place in this medium4 ,the liquid found on the surface of the agar 7or at the bottom of the tube in agar tube cultures7 is merely water which has been expressed during the rapid solidification of the medium and has subsequently condensed4 ,gelatine and ,agar4--,examine the colonies at intervals of twenty-four hours4 7a7 ,with the naked eye4 7b7 ,with a hand lens or watchmaker's glass4 7c7 ,under a low power 7#a inch7 of the microscope1 or by means of a small dissecting microscope4 ,distinguish superficial from deep colonies and note the characters of the individual colonies4 7,a7 ,size4--,the diameter in millimetres1 at the various ages4 7,b7 ,shape4-- ,punctiform3 ,dimensions too slight for defining form by naked eye2 minute1 raised1 hemispherical4 ,round3 ,of a more or less circular outline4 ,elliptical3 ,of a more or less oval outline4 ,irregular3 ,outlines not conforming to any recognised shape4 ,fusiform3 ,spindle-shaped1 tapering at each end4 ,cochleate3 ,spiral or twisted like a snail shell 7,fig4 #ada1 a74 ,illustration3 ,,fig4 #ada4--,types of colonies3 a1 ,cochleate2 b1 amoeboid2 c1 mycelioid4 ,amoeboid3 ,very irregular1 streaming 7,fig4 #ada1 b74 ,mycelioid3 ,a filamentous colony1 with the radiate character of a mould 7,fig4 #ada1 c74 ,filamentous3 ,an irregular mass of loosely woven filaments 7,fig4 #adb1 a74 ,floccose3 ,of a dense woolly structure4 ,rhizoid3 ,of an irregular1 branched1 root-like character 7,fig4 #adb1 b74 ,conglomerate3 ,an aggregate of colonies of similar size and form 7,fig4 #adb1 c74 ,toruloid3 ,an aggregate of colonies1 like the budding of the yeast plant 7,fig4 #adb1 d74 ,rosulate3 ,shaped like a rosette4 ,illustration3 ,,fig4 #adb4--,types of colonies3 a1 ,filamentous2 b1 rhizoid2 c1 conglomerate2 d1 toruloid4 7,c7 ,surface ,elevation4-- #a4 ,general ,character of ,surface as a ,whole3 ,flat3 ,thin1 leafy1 spreading over the surface 7,fig4 #adc1 a74 ,effused3 ,spread over the surface as a thin1 veily layer1 more delicate than the preceding4 ,raised3 ,growth thick1 with abrupt terraced edges 7,fig4 #adc1 b74 ,convex3 ,surface the segment of a circle1 but very flatly convex 7,fig4 #adc1 c74 ,pulvinate3 ,surface the segment of a circle1 but decidedly convex 7,fig4 #adc1 d74 ,capitate3 ,surface hemispherical 7,fig4 #adc1 e74 ,umbilicate3 ,having a central pit or depression 7,fig4 #adc1 f74 ,conical3 ,cone with rounded apex 7,fig4 #adc1 g74 ,umbonate3 ,having a central convex nipple-like elevation 7,fig4 #adc1 h74 #b4 ,detailed ,characters of ,surface3 ,smooth3 ,surface even1 without any of the following distinctive characters4 ,alveolate3 ,marked by depressions separated by thin walls so as to resemble a honeycomb 7,fig4 #add74 ,punctate3 ,dotted with punctures like pin-pricks4 ,bullate3 ,like a blistered surface1 rising in convex prominences1 rather coarse4 ,vesicular3 ,more or less covered with minute vesicles due to gas formation2 more minute than bullate4 ,illustration3 ,,fig4 #adc4--,surface elevation of colonies3 a1 ,flat2 b1 raised2 c1 convex2 d1 pulvinate2 e1 capitate2 f1 umbilicate2 g1 conical2 h1 umbonate4 ,illustration3 ,,fig4 #add4--,types of colonies--alveolate4 ,verrucose3 ,wart-like1 bearing wart-like prominences4 ,squamose3 ,scaly1 covered with scales4 ,echinate3 ,beset with pointed prominences4 ,papillate3 ,beset with nipple or mamma-like processes4 ,rugose3 ,short irregular folds1 due to shrinkage of surface growth4 ,corrugated3 ,in long folds1 due to shrinkage4 ,contoured3 ,an irregular but smoothly undulating surface1 resembling the surface of a relief map4 ,rimose3 ,abounding in chinks1 clefts1 or cracks4 7,d7 ,internal ,structure of ,colony 7,microscopical74-- ,refraction ,weak3 ,outline and surface of relief not strongly defined4 ,refraction ,strong3 ,outline and surface of relief strongly defined2 dense1 not filamentous colonies4 ,illustration3 ,,fig4 #ade4--,types of colonies3 a1 ,grumose2 b1 moruloid2 c1 clouded4 #a4 ,general3 ,amorphous3 ,without any definite structure1 such as is specified below4 ,hyaline3 ,clear and colourless4 ,homogeneous3 ,structure uniform throughout all parts of the colony4 ,homochromous3 ,colour uniform throughout4 #b4 ,granulations or ,blotchings3 ,finely granular4 ,coarsely granular4 ,grumose3 ,coarser than the preceding1 with a clotted appearance1 and particles in clustered grains 7,fig4 #ade1 a74 ,moruloid3 ,having the character of a mulberry1 segmented1 by which the colony is divided in more or less regular segments 7,fig4 #ade1 b74 ,clouded3 ,having a pale ground1 with ill-defined patches of a deeper tint 7,fig4 #ade1 c74 ,illustration3 ,,fig4 #adf4--,types of colonies3 a1 ,reticulate2 b1 gyrose2 c1 marmorated4 #c4 ,colony ,marking or ,striping3 ,reticulate3 ,in the form of a network1 like the veins of a leaf 7,fig4 #adf1 a74 ,areolate3 ,divided into rather irregular1 or angular1 spaces by more or less definite boundaries4 ,gyrose3 ,marked by wavy lines1 indefinitely placed 7,fig4 #adf1 b74 ,marmorated3 ,showing faint1 irregular stripes1 or traversed by vein-like markings1 as in marble 7,fig4 #adf1 c74 ,rivulose3 ,marked by lines like the rivers of a map4 ,rimose3 ,showing chinks1 cracks1 or clefts4 ,illustration3 ,,fig4 #adg4--,types of colonies--curled4 #d4 ,filamentous ,colonies3 ,filamentous3 ,as already defined4 ,floccose3 ,composed of filaments1 densely placed4 ,curled3 ,filaments in parallel strands1 like locks or ringlets 7,fig4 #adg74 7,e7 ,edges of ,colonies4-- ,entire3 ,without toothing or division 7,fig4 #adh1 a74 ,undulate3 ,wavy 7,fig4 #adh1 b74 ,repand3 ,like the border of an open umbrella 7,fig4 #adh1 c74 ,erose3 ,as if gnawed1 irregularly toothed 7,fig4 #adh1 d74 ,illustration3 ,,fig4 #adh4--;,edges of colonies3 a1 ,entire2 b1 undulate2 c1 repand2 d1 erose4 ,lobate4 ,lobulate3 ,minutely lobate 7,fig4 #adi1 e74 ,auriculate3 ,with ear-like lobes 7,fig4 #adi1 f74 ,lacerate3 ,irregularly cleft1 as if torn 7,fig4 #adi1 g74 ,fimbriate3 ,fringed 7,fig4 #adi1 h74 ,ciliate3 ,hair-like extensions1 radiately placed 7,fig4 #adi1 j74 ,tufted4 ,filamentous3 ,as already defined4 ,curled3 ,as already defined4 ,illustration3 ,,fig4 #adi4--;,edges of colonies3 e1 ,lobar-lobulate2 f1 auriculate2 g1 lacerate2 h1 fimbriate2 i1 ciliate4 7,f7 ,optical ,characters 7after ,shuttleworth74-- #a4 ,general ,characters3 ,transparent3 ,transmitting light4 ,vitreous3 ,transparent and colourless4 ,oleaginous3 ,transparent and yellow2 olive to linseed-oil coloured4 ,resinous3 ,transparent and brown1 varnish or resin-coloured4 ,translucent3 ,faintly transparent4 ,porcelaneous3 ,translucent and white4 ,opalescent3 ,translucent2 greyish-white by reflected light4 ,nacreous3 ,translucent1 greyish-white1 with pearly lustre4 ,sebaceous3 ,translucent1 yellowish or greyish-white4 ,butyrous3 ,translucent and yellow4 ,ceraceous3 ,translucent and wax-coloured4 ,opaque4 ,cretaceous3 ,opaque and white1 chalky4 ,dull3 ,without lustre4 ,glistening3 ,shining4 ,fluorescent4 ,iridescent4 #b4 ,chromogenicity3 ,colour of pigment4 ,pigment restricted to colonies4 ,pigment restricted to medium surrounding colonies4 ,pigment present in colonies and in medium4 ,streak or ,smear ,cultures4-- ,gelatine and ,agar4--,note general points as indicated under plate cultivations4 ,inspissated ,blood-serum4--,note the presence or absence of liquefaction of the medium4 7,the presence of condensation water at the bottom of the tube must not be confounded with liquefaction of the medium47 ,all ,oblique ,tube ,cultures4-- #a4 ,colonies ,discrete3 ,size1 shape1 etc41 as for plate cultivations 7vide page #bfa74 #b4 ,colonies ,confluent3 ,surface elevation and character of edge1 as for plate cultivations 7vide page #bfc74 ,chromogenicity3 ,as for plate cultures4 ,gelatine ,stab ,cultures4-- 7,a7 ,surface ,growth4--,as for individual colonies in plate cultures 7vide page #bfa74 ,illustration3 ,,fig4 #aej4--,stab cultivations--types of growth3 a1 ,filiform2 b1 beaded2 c1 echinate2 d1 villous2 e1 arborescent4 7,b7 ,line of ,puncture4-- ,filiform3 ,uniform growth1 without special characters 7,fig4 #aej1 a74 ,nodose3 ,consisting of closely aggregated colonies4 ,beaded3 ,consisting of loosely placed or disjointed colonies 7,fig4 #aej1 b74 ,papillate3 ,beset with papillate extensions4 ,echinate3 ,beset with acicular extensions 7,fig4 #aej1 c74 ,villous3 ,beset with short1 undivided1 hair-like extensions 7,fig4 #aej1 d74 ,plumose3 ,a delicate feathery growth4 ,illustration3 ,,fig4 #aea4--,stab cultivations--types of growth3 f1 ,crateriform2 g1 saccate2 h1 infundibuliform2 j1 napiform2 k1 fusiform2 l1 stratiform4 ,arborescent3 ,branched or tree-like1 beset with branched hair-like extensions 7,fig4 #aej1 e74 7,c7 ,area of ,liquefaction 7if present74-- ,crateriform3 ,a saucer-shaped liquefaction of the gelatine 7,fig4 #aea1 f74 ,saccate3 ,shape of an elongated sack1 tubular cylindrical 7,fig4 #aea1 g74 ,infundibuliform3 ,shape of a funnel1 conical 7,fig4 #aea1 h74 ,napiform3 ,shape of a turnip 7,fig4 #aea1 j74 ,fusiform3 ,outline of a parsnip1 narrow at either end1 broadest below the surface 7,fig4 #aea1 k74 ,stratiform3 ,liquefaction extending to the walls of the tube and downward horizontally 7,fig4 #aea1 l74 7,d7 ,character of the ,liquefied ,gelatine4-- #a4 ,pellicle on surface4 #b4 ,uniformly turbid4 #c4 ,granular4 #d4 ,mainly clear1 but containing flocculi4 #e4 ,deposit at apex of liquefied portion4 7,e7 ,production of ,gas ,bubbles4 ,shake ,cultures4-- #a4 ,presence or absence of liquefaction4 #b4 ,production of gas bubbles4 #c4 ,bulk of growth at the surface--aerobic4 #d4 ,bulk of growth in depths--anaerobic4 ,fluid ,media4 #a4 ,surface of the ,liquid4-- ,presence or absence of froth due to gas bubbles4 ,presence or absence of pellicle formation4 ,character of pellicle4 #b4 ,body of the ,liquid4-- ,uniformly turbid4 ,flocculi in suspension4 ,granules in suspension4 ,clear1 with precipitate at bottom of tube4 ,colouration of fluid1 presence or absence of4 #c4 ,precipitate4-- ,character4 ,amount4 ,colour4 ,carbohydrate ,media4-- ,growth4 ,reaction4 ,gas formation4 ,coagulation or not of serum albumen 7when serum water media are employed74 ,litmus ,milk ,cultivations4-- ,unaltered4 #a4 ,reaction3 ,acid4 ,alkaline4 #b4 ,odour4 #c4 ,formation of gas4 ,unaltered4 #d4 ,consistency3 ,peptonised 7character of solution74 ,coagulated4 hard3 solid4 #e4 ,clot3 ,character soft3 floculent4 ragged and broken up by gas bubbles4 7a7 ,coagulum undissolved4 7b7 ,coagulum finally peptonised1 completely3 incompletely4 ,resulting solution1 clear3 turbid4 ,abundant4 ,scanty4 #f4 ,whey3 ,clear4 ,turbid4 ,coagulated by boiling1 or not4 ,,by ,,microscopical ,,methods4 ,as a council of perfection preparations must be made from pure cultivations #d1 #f1 #h1 #ab1 #ah1 and #bd hours2 and subsequently at intervals of1 say1 twenty-four hours1 during the entire period they are under observation1 and examined-- 7,a7 ,living4--#a4 ,in hanging drop1 to determine motility or non-motility4 ,in this connection it must be remembered that under certain conditions as to environment 7e4 g41 when examined in an unsuitable medium1 atmosphere1 temperature1 etc47 motile bacilli may fail to exhibit activity4 ,no organism1 therefore1 should be recorded as non-motile from one observation only2 a series of observations at different ages and under varying conditions should form the basis of an opinion as to the absence of true locomotion4 ,size4--,in the case of non-motile or sluggishly motile organisms1 endeavour to measure several individuals in each hanging drop by means of the eyepiece micrometer or the eikonometer 7vide page #fc71 and average the results4 ,if the organism is one which forms spores1 observe-- 7a7 ,spore ,formation4--,prepare hanging-drop cultivations 7vide page #gh7 from vegetative forms of the organism1 adding a trace of magenta solution 7#j4e per cent47 or other intra vitam stain 7see page #gg7 to the drop1 on the point of the platinum needle1 to facilitate the observation of the phenomenon by rendering the bacilli more distinct4 ,place the preparation on the stage of the microscope2 if necessary1 using a warm stage4 ,arrange illumination1 etc41 and select a solitary bacillus for observation1 by the help of the #a_/f-;inch lens4 ,substitute the #a_/ab-;inch oil-immersion lens for the sixth1 and observe the formation of the spore2 if possible1 measure any alteration in size which may occur by means of the ,ramsden micrometer4 7b7 ,spore ,germination4--,prepare hanging-drop cultivations from old cultivations in which no living vegetative forms are present1 and observe the process of germination in a similar manner4 ,the comfort of the microscopist is largely enhanced in those cases where the period of observation is at all lengthy1 by use of some form of eye screen before the unemployed eye1 such as is figured on page #eh 7,fig4 #di74 ,if it is impossible to carry out the method suggested above1 proceed as follows3 7a7 ,spore ,formation4--,plant the organism in broth and incubate under optimum conditions4 ,at regular intervals1 say every thirty minutes1 remove a loopful of the cultivation and prepare a cover-slip film preparation4 ,fix1 while still wet1 in the corrosive sublimate fixing solution4 ,stain with aniline gentian violet1 and partially decolourise with #b per cent4 acetic acid4 ,mount and number consecutively2 then examine4 7b7 ,spore ,germination4--,expose a thick emulsion of the spores to a temperature of #hj deg4 ,c4 for ten minutes in the differential steriliser 7vide page #beg74 ,transfer the emulsion to a tube of sterile nutrient broth and incubate4 ,remove specimens from the tube culture at intervals of1 say1 five minutes4 ,fix1 stain1 etc41 wet1 as under 7a71 and examine4 7,b7 ,fixed4--#b4 ,in stained preparations4 7a7 ,to determine points in morphology3 ,shape 7vide classification1 page #aca74 ,size3 7a7 ,prepare cover-slip film preparations at the various ages1 and fix by exposure to a temperature of #aae deg4 ,c4 for twenty minutes in hot-air oven4 7b7 ,stain the preparations by ,gram's method 7if applicable7 or with dilute carbol-fuchsin1 and mount in the usual way4 7c7 ,measure 7vide page #ff7 some twenty-five individuals in each film by means of the ,ramsden's or the stage micrometer and average the result4 ,pleomorphism2 ,if noted1 record-- ,the predominant character of the variant forms4 ,on what medium or media they are observed4 ,at what period of development4 7b7 ,to demonstrate details of structure3 ,flagella3 ,if noted1 record-- ,method of staining 7vide page #aja74 ,position and arrangement 7vide page #acf74 ,number4 ,spores3 ,if noted1 record-- ,method of staining4 ,shape4 ,size4 ,position within the parent cell4 ,condition1 as to shape1 of the parent cell 7vide page #aci74 ,optimum medium and temperature4 ,age of cultivation4 ,conditions of environment as to temperature1 atmosphere4 ,method of germination 7vide page #adj74 ,involution ,forms3 ,if noted1 record-- ,method of staining4 ,character 7e4 g41 if living or dead74 ,shape4 ,on what medium they are observed4 ,age of medium4 ,environment4 ,metachromatic ,granules3 ,if noted1 record-- ,method of staining4 ,character of granules4 ,number of granules4 ,colour of granules4 #c4 ,staining ,reactions4-- #a4 ,gram's ,method4--,positive or negative4 #b4 ,neisser's ,method4--,if granules are noted1 record-- #a4 ,position4 #b4 ,number4 #c4 ,ziehl-,neelsen's ,method4--,acid-fast or decolourised4 #d4 ,simple ,aniline ,dyes4--7,noting those giving the best results1 with details of staining processes47 ,methylene-blue ,fuchsin and their modifications4 ,gentian violet ,thionine blue ,,by ,,biochemical ,,methods4 ,test cultivations of the organism for the presence of-- ,soluble enzymes--proteolytic1 diastatic1 invertase4 ,organic acids--7a7 quantitatively--i4 e41 estimate the total acid production2 7b7 qualitatively for formic1 acetic1 propionic1 butyric1 lactic4 ,ammonia4 ,neutral volatile substances--ethyl alcohol1 aldehyde1 acetone4 ,aromatic products--indol1 phenol4 ,soluble pigments4 ,test the power of reducing 7a7 colouring matters1 7b7 nitrates to nitrites4 ,investigate the gas production--,h#b,s1 ,,co#b1 ,h#b4 ,estimate the ratio between the last two gases4 ,prepare all cultivations for these methods of examination under optimum conditions1 previously determined for each of the organisms it is intended to investigate1 as to 7a7 ,reaction of medium2 7b7 ,incubation temperature2 7c7 ,atmospheric environment2 and keep careful records of these points1 and also of the age of the cultivation used in the final examination4 ,examine the cultivations for the various products of bacterial metabolism after forty-eight hours' growth1 and never omit to examine control 7uninoculated7 tube or flask of medium from the same batch1 kept for a similar period under identical conditions4 ,if the results are negative1 test further cultivations at three days1 five days1 and ten days4 #a4 ,enzyme ,production4-- 7,a7 ,proteolytic ,enzymes4--7,convert proteins into proteose1 peptone and further products of hydrolysis2 e4 g41 ,b4 pyocyaneus47 ,media ,required3 ,blood-serum and milk-serum which have been carefully filtered through a porcelain candle4 ,reagents ,required3 ,ammonium sulphate4 ,thirty per cent4 caustic soda solution4 ,copper sulphate1 #j4e per cent4 aqueous solution4 ,one per cent4 acetic acid solution4 ,millon's reagent4 ,glyoxylic acid solution4 ,concentrated sulphuric acid4 ,,method4-- #a4 ,prepare cultivations in bulk 7#ej c4c47 in a flask and incubate4 #b4 ,make the liquid faintly acid with acetic acid1 then boil4 7,this precipitates the unaltered proteins47 #c4 ,filter4 #d4 ,take #aj c4c4 of the filtrate in a test-tube and add #a c4c4 of the caustic soda1 then add the copper sulphate drop by drop4 ,pink colour which becomes violet with more copper sulphate proteose and peptone4 #e4 ,saturate the rest of the filtrate with ammonium sulphate4 ,precipitate proteose4 #f4 ,filter and divide the filtrate into three parts a1 b and c4 a4 ,repeat the copper sulphate test1 using excess of caustic soda to displace the ammonia from the ammonium sulphate4 ,pink colour peptone4 b4 ,boil with ,millon's reagent4 ,red colour tyrosine4 c4 ,add glyoxylic acid solution and run in concentrated sulphuric acid4 ,violet ring at upper level of acid tryptophane4 ,both the tyrosine and tryptophane may be either in the free state or in combination as polypeptid or peptone4 7,b7 ,diastase4--7,converts starch into sugar2 e4 g41 ,b4 subtilis47 ,medium ,required3 ,inosite-free bouillon4 ,reagents ,required3 ,starch4 ,thymol4 ,fehling's solution4 ,,method4-- #a4 ,prepare tube cultivation and incubate4 #b4 ,prepare a thin starch paste and add #b per cent4 thymol to it4 #c4 ,mix equal parts of the cultivation to be tested and the starch paste1 and place in the incubator at #cg deg4 ,c4 for six to eight hours4 #d4 ,filter4 ,test the filtrate for sugar4 ,boil some of the ,fehling's solution in a test-tube4 ,add the filtrate drop by drop until1 if necessary1 a quantity has been added equal in amount to the ,fehling's solution employed1 keeping the mixture at the boiling-point during the process4 ,yellow or orange precipitate sugar4 7,c7 ,invertase4--7,convert saccharose into a mixture of dextrose and laevulose e4 g41 ,b4 fluorescens liquefaciens47 ,medium ,required3 ,inosite-free bouillon4 ,reagents ,required3 ,cane sugar1 #b per cent4 aqueous solution4 ,carbolic acid4 ,,method4-- #a4 ,prepare tube cultivations and incubate4 #b4 ,add #b per cent4 of carbolic acid to the sugar solution4 #c4 ,mix equal quantities of the carbolised sugar solution and the cultivation in a test-tube2 allow the mixture to stand for several hours4 #d4 ,filter4 ,test the filtrate for reducing sugar as in the preceding section4 7,d7 ,rennin and ,lab ,enzymes4--7,coagulate milk independently of the action of acids2 e4 g41 ,b4 prodigiosus47 ,media ,required3 ,inosite-free bouillon4 ,litmus milk4 ,,method4-- #a4 ,prepare tube cultivations and incubate4 #b4 ,after incubation heat the cultivation to #ee deg4 ,c4 for half an hour1 to sterilise4 #c4 ,by means of a sterile pipette run #e c4c4 of the cultivation into each of three tubes of litmus milk4 #d4 ,place in the cold incubator at #bb deg4 ,c4 and examine each day for ten days4 ,absence of coagulation at the end of that period will indicate absence of rennin ferment formation4 ,fermentation ,reactions4 ,as tested upon carbohydrate substances and organic salts4 ,media ,required3 ,peptone water containing various percentages 7generally #b per cent47 of each of the substances referred to under sugar media 7page #agg71 also tubes of peptone water containing #a per cent4 respectively of each of the following3 ,organic salts3 ,sodium citrate1 formate1 lactate1 malate1 tartrate4 ,,method4-- #a4 ,prepare tube cultivations in each of the above media4 #b4 ,observe from day to day up to the expiration of ten days if necessary4 #c4 ,note growth1 reaction1 gas production4 #b4 ,acid ,production4 7a7 ,quantitative4-- ,medium ,required3 ,sugar 7glucose7 bouillon of known optimum reaction4 ,apparatus and ,reagents ,required3 ,as for estimating reaction of media 7vide page #aej74 ,,method4-- #a4 ,prepare cultivation in bulk 7#ajj c4c47 in a flask2 also control flask of medium from same batch4 #b4 ,after suitable incubation1 heat both flasks in the steamer at #ajj deg4 ,c4 for thirty minutes to sterilise4 #c4 ,determine the titre of the medium in inoculated and control flasks as described in the preparation of nutrient media 7vide page #aea74 #d4 ,the difference between the titre of the medium in the two flasks gives the total acid production of the bacterium under observation in terms of normal ,na,o,h4 ,,note4--,if the growth is very heavy it may be a difficult matter to determine the end-point4 ,the cultivation should then be filtered through a ,berkefeld filter candle previous to step #b1 and the filtrate employed in the titration4 7b7 ,qualitative 7of all the organic acids present74-- ,medium ,required3 ,sugar 7glucose or lactose7 bouillon as in quantitative examination4 ,reagents ,required3 ,hydrochloric acid1 concentrated4 ,hydrochloric acid1 #be per cent4 ,sulphuric acid1 concentrated 7pure74 ,phosphoric acid1 concentrated solution4 ,ammonia4 ,ammonium sulphate4 ,baryta water4 ,sodium carbonate1 saturated aqueous solution4 ,absolute alcohol4 ,ether4 ,calcium chloride4 ,calcium chloride solution4 ,zinc carbonate4 ,copper sulphate saturated aqueous solution4 ,alcoholic thiophene solution 7#j4ae c4c4 in #ajj c4c474 ,animal charcoal4 ,five per cent4 sodium nitroprusside solution4 ,potassium bichromate4 ,schiff's reagent4 ,arsenious oxide4 ,ferric chloride1 #d per cent4 aqueous solution4 ,silver nitrate1 #a per cent4 aqueous solution4 ,lugol's iodine4 ,ten per cent4 caustic soda solution4 ,hard paraffin wax 7melting-point about #eb deg4 ,c474 ,,method4-- #a4 ,prepare cultivation in bulk 7#ejj c4c47 in a litre flask and add sterilised precipitated chalk1 #aj grammes4 ,incubate at the optimum temperature4 #b4 ,after incubation throw a piece of paraffin wax 7about a centimetre cube7 into the cultivation and connect up the flask with a condenser4 ,the paraffin1 which liquefies and forms a thin layer on the surface of the fluid1 is necessary to prevent the cultivation frothing up and running unaltered through the condenser during the subsequent process of distillation4 #c4 ,distill over #bjj to #cjj c4c4 ,use a rose-top burner to minimise the danger of cracking the flask2 and to the same end1 well agitate the contents of the flask to prevent the chalk settling4 ,the distillate ,a will contain alcohol1 etc4 7vide page #bhe72 the residue a will contain the volatile and fixed acids4 #d4 ,disconnect the flask and filter4 ,the residue a then filtrate ,b and residue b4 ,illustration3 ,,fig4 #aeb4--;,arrangement of distillation apparatus for acids1 etc4 #e4 ,residue b4 ,wash the residue from the filter paper1 dissolve by heating with dilute hydrochloric acid1 and add calcium chloride solution and ammonia until alkaline4 ,white precipitate insoluble in acetic acid oxalic acid4 #f4 ,make up filtrate ,b to #ejj c4c4 with distilled water and divide into two parts4 #g4 ,acidify #bej c4c4 with #bj c4c4 concentrated phosphoric acid 7this liberates the volatile acids7 and distil to small bulk4 ,the distillate ,b may contain formic1 acetic1 propionic1 butyric and benzoic acids4 ,,distillate ,b4 7,volatile ,acids47 #a4 ,add baryta water till alkaline1 and evaporate to dryness4 #b4 ,add #ej c4c4 absolute alcohol and allow to stand1 with frequent stirring1 for two to three hours4 #c4 ,filter and wash with alcohol4 --------------------------------------- ,,filtrate ,,residue may contain barium propionate1 may contain barium acetate1 barium butyrate4 barium formate1 barium benzoate4 #a4 ,evaporate to dryness4 #a4 ,evaporate off alcohol and dissolve up the residue on #b4 ,dissolve residue in #aej the filter in hot water and c4c4 water4 neutralise4 #c4 ,acidify with phosphoric #b4 ,divide the solution into acid and distil4 four portions3 #d4 ,saturate distillate with 7a7 ,add ferric chloride solution4 calcium chloride and distill over a few c4c4 ,brown colour acetic or formic acids4 #e4 ,test distillate for butyric acid3 ,buff ppt4 benzoic acid 7see ether soluble acids74 ,add #c c4c4 alcohol and #d drops concentrated sulphuric acid4 7b7 ,add silver nitrate solution2 then add one drop ,smell of pineapple butyric ammonia water1 and boil4 acid4 ,black precipitate of metallic ,propionic acid in small silver formic acid4 quantities cannot be distinguished from butyric 7c7 ,evaporate to dryness2 mix acid by tests within the with equal quantity of scope of the bacteriological arsenious oxide and heat laboratory4 on platinum foil4 ,unpleasant smell of cacodyl acetic acid4 7d7 ,add a few drops of mercuric chloride solution in test-tube1 and heat to #gj deg4 ,c4 ,precipitate of mercurous chloride which is slowly reduced to mercury formic acid4 #h4 ,if the distillation of ,b is continued as long as acid comes over 7distilled water being occasionally added to the distilling flask7 the distillate can be measured and #ej c4c4 used for titration4 ,this will give the amount of volatile acid formation4 #i4 ,the second part of the filtrate ,b 7see page #bhb7 should be examined for lactic1 oxalic1 succinic1 benzoic1 salicylic1 gallic and tannic acids1 as follows3 ,ether ,soluble ,acids4-- #a4 ,evaporate to a thin syrup1 acidify strongly with phosphoric acid4 #b4 ,extract with five times its volume of ether by agitation in a separatory funnel4 #c4 ,evaporate the ethereal extract to a thin syrup4 #d4 ,add #ajj c4c4 water and mix thoroughly4 #e4 ,to a small portion of this solution add slight excess of sodium carbonate1 evaporate to dryness on the water-bath1 dissolve in #e-aj c4c4 pure sulphuric acid1 add #b drops saturated copper sulphate solution1 place in a test-tube and heat in a boiling water-bath for #b minutes1 cool1 add #b or #c drops of the alcoholic thiophene and warm gently4 ,cherry red colour lactic acid4 ,if a brown colour is produced on the addition of sulphuric acid1 another sample should be taken and boiled with animal charcoal before evaporating4 #f4 ,if lactic acid is definitely present1 prepare zinc lactate by boiling part of the solution of the ether extract with excess of zinc carbonate1 filtering and evaporating to crystallise4 ,the crystals so obtained have a characteristic form1 and if dried at #aaj deg4 ,c1 should contain #bf4hg per cent4 of zinc4 #g4 ,test a portion of the rest of the solution of the ether extract for oxalic acid 7page #bhb1 step #e74 ,carefully neutralise the remainder and add ferric chloride solution4 ,red brown gelatinous precipitate succinic acid4 ,buff precipitate benzoic acid1 and other acids related to benzoic acid4 ,violet colour salicylic acid4 ,inky black colour or precipitate gallic acid or tannic acid4 ,for further identification the melting-points of the crystalline acids1 and the percentage of silver in their silver salts should be determined4 #c4 ,ammonia ,production4-- ,medium ,required3 ,nutrient bouillon4 ,reagent ,required3 ,nessler reagent4 ,,method4-- #a4 ,prepare cultivation in bulk 7#ajj c4c47 in a #bej c4c4 flask and incubate together with a control flask4 ,test the cultivation and the control for ammonia in the following manner3 #b4 ,to each flask add #b grammes of calcined magnesia1 then connect up with condensers and distil4 #c4 ,collect #ej c4c4 distillate1 from each1 in a ,nessler glass4 #d4 ,add #a c4c4 ,nessler reagent to each glass by means of a clean pipette4 ,yellow colour ammonia4 ,the depth of colour is proportionate to the amount present4 #d4 ,alcohol1 etc41 ,production4--,divide the distillate ,a obtained in the course of a previous experiment 7vide page #bhb1 step #c7 into four portions and test for the production of alcohol1 acetaldehyde1 acetone4 #a4 ,add ,lugol's iodine1 then a little ,na,o,h solution1 and stir with a glass rod till the colour of the iodine disappears4 ,pale-yellow crystalline precipitate of iodoform1 with its characteristic smell1 appearing in the cold1 indicates acetaldehyde1 or acetone2 appearing only on warming indicates alcohol4 ,the precipitate may be absent even when the odour is pronounced4 #b4 ,add ,schiff's reagent4 ,violet or red colour aldehyde4 #c4 ,to #aj c4c4 of solution add #b4e c4c41 #be per cent4 sulphuric acid1 and a crystal or two of potassium bichromate and distil4 ,reduction of the bichromate to a green colour and a distillate1 which smells of acetaldehyde and reacts with ,schiff's reagent1 shows the presence of alcohol in the original liquid4 #d4 ,add a few drops of sodium nitroprusside solution1 make alkaline with ammonia1 then saturate with ammonium sulphate crystals4 ,acetone gives little colour on the addition of ammonia1 but after the addition of ammonium sulphate a deep permanganate colour1 which takes ten minutes to reach its full intensity4 ,aldehyde gives a carmine red unaltered by ammonium sulphate4 #e4 ,indol ,production4-- ,media ,required3 ,inosite-free bouillon 7vide page #ahc74 ,or peptone water 7vide page #agg74 ,reagents ,required3 ,potassium persulphate1 saturated aqueous solution4 ,paradimethylamino-benzaldehyde solution4 ,this is prepared by mixing3 ,paradimethylamino-benzaldehyde #d grammes ,absolute alcohol #chj c4c4 ,hydrochloric acid1 concentrated #hj c4c4 ,,method4-- ,prepare several test-tube cultivations of the organism to be tested1 and incubate4 ,test for indol by means of the ,rosindol reaction in the following manner4 7,if the culture has been incubated at #cg deg4 ,c41 it must be allowed to cool to the room temperature before applying the test47 #a4 ,remove #b c4c4 of the cultivation by means of a sterile pipette and transfer to a clean tube1 then1 #b4 ,add #b c4c4 paradimethylamino-benzaldehyde solution4 #c4 ,add #b c4c4 potassium persulphate solution4 ,the presence of indol is indicated by the appearance of a delicate rose-pink colour throughout the mixture which deepens slightly on standing4 ,indol is tested for in many laboratories by the ordinary nitrosoindol reaction which1 however1 is not so delicate a method as that above described4 ,the test is carried out as follows3 #a4 ,remove the cotton-wool plug from the tube1 and run in #a c4c4 pure concentrated sulphuric acid down the side of the tube by means of a sterile pipette4 ,place the tube upright in a rack1 and allow it to stand1 if necessary1 for ten minutes4 ,a rose-pink or red colour at the junction of the two liquids indol 7plus a nitrite74 #b4 ,if the colour of the medium remains unaltered1 add #b c4c4 of a #j4ja per cent4 aqueous solution sodium nitrite1 and again allow the culture to stand for ten minutes4 ,red colouration indol4 ,,note4--,in place of performing the test in two stages as given above1 #b c4c4 concentrated commercial sulphuric1 hydrochloric1 or nitric acid 7all of which hold a trace of nitrite in solution71 may be run into the cultivation4 ,the development of a red colour within twenty minutes will indicate the presence of indol4 #e;a4 ,phenol ,production4-- ,medium ,required3 ,nutrient bouillon4 ,reagents ,required3 ,hydrochloric acid1 concentrated4 ,millon's reagent4 ,ferric chloride1 #a per cent4 aqueous solution4 ,,method4-- #a4 ,prepare cultivation in a ,bohemian flask containing at least #ej c4c4 of medium1 and incubate4 ,test for phenol in the following manner3 #b4 ,add #e c4c41 #be per cent4 sulphuric acid to the cultivation and connect up the flask with a condenser4 #c4 ,distil over #ae to #bj c4c4 ,divide the distillate into three portions a1 b and c4 #d4 ,add to 7a7 #j4e c4c4 ,millon's reagent and boil4 ,red colour phenol4 #e4 ,add to 7b7 about #j4e c4c4 ferric chloride solution4 ,violet colour phenol4 7,if the distillate be acid the reaction will be negative47 #f4 ,add to 7c7 bromine water4 ,crystalline white ppt4 of tribromo-phenol phenol4 ,,note4--,if both indol and phenol appear to be present in cultivations of the same organism1 it is well to separate them before testing4 ,this may be done in the following manner3 #a4 ,prepare inosite-free bouillon cultivation1 say #bjj or #cjj c4c41 in a flask as before4 #b4 ,render definitely acid by the addition of acetic acid and connect up the flask with a condenser4 #c4 ,distil over #ej to #gj c4c4 ,distillate will contain both indol and phenol4 #d4 ,render the distillate strongly alkaline with caustic potash and redistil4 ,distillate will contain indol2 residue will contain phenol4 #e4 ,test the distillate for indol 7vide ante74 #f4 ,saturate the residue1 when cold1 with carbon dioxide and redistil4 #g4 ,test this distillate for phenol 7vide ante74 #f4 ,pigment ,production4-- #a4 ,prepare tube cultivations upon the various media and incubate under varying conditions as to temperature 7at #cg deg4 ,c4 and at #bj deg4 ,c471 atmosphere 7aerobic and anaerobic71 and light 7exposure to and protection from74 ,note the conditions most favorable to pigment formation4 #b4 ,note the solubility of the pigment in various solvents1 such as water 7hot and cold71 alcohol1 ether1 chloroform1 benzol1 carbon bisulphide4 #c4 ,note the effect of acids and alkalies respectively upon the pigmented cultivation1 or upon solutions of the pigment4 #d4 ,note spectroscopic reactions4 #g4 ,reducing ,agent ,formation4-- 7a7 ,colour ,destruction4-- #a4 ,prepare tube cultivations in nutrient bouillon tinted with litmus1 rosolic acid1 neutral red1 and incubate4 #b4 ,examine the cultures each day and note whether any colour change occurs4 7b7 ,nitrates to ,nitrites4-- ,medium ,required3 ,nitrate bouillon 7vide page #ahe74 ,or nitrate peptone solution 7vide page #ahf74 ,reagents ,required3 ,sulphuric acid 7#be per cent474 ,metaphenylene diamine1 #e per cent4 aqueous solution4 ,,method4-- #a4 ,prepare tube cultivations and incubate together with control tubes 7i4 e41 uninoculated tubes of the same medium1 placed under identical conditions as to environment74 ,this precaution is necessary as the medium is liable to take up nitrites from the atmosphere1 and an opinion as to the absence of nitrites in the cultivation is often based upon an equal colouration of the medium in the control tube4 ,test both the culture tube and the control tube for the presence of nitrites4 #b4 ,add a few drops of sulphuric acid to the medium in each of the tubes4 #c4 ,then run in #b or #c c4c4 metaphenylene diamine into each tube4 ,brownish-red colour nitrites4 ,the depth of colour is proportionate to the amount present4 #h4 ,gas ,production4-- 7,a7 ,carbon ,dioxide and ,hydrogen4-- ,apparatus ,required3 ,fermentation tubes 7vide page #afa7 containing sugar bouillon 7glucose1 lactose1 etc474 ,the medium should be prepared from inosite-free bouillon 7vide page #ahc74 ,reagent ,required3 n_/#b caustic soda4 ,,method4-- #a4 ,inoculate the surface of the medium in the bulb of a fermentation tube and incubate4 #b4 ,mark the level of the fluid in the closed branch of the fermentation tube1 at intervals of twenty-four hours1 and when the evolution of gas has ceased1 measure the length of the column of gas with the millimetre scale4 ,express this column of gas as a percentage of the entire length of the closed branch4 #c4 ,to analyse the gas and to determine roughly the relative proportions of ,,co#b and ,h#b1 proceed as follows3 ,fill the bulb of the fermentation tube with caustic soda solution4 ,close the mouth of the bulb with a rubber stopper4 ,alternately invert and revert the tube six or eight times1 to bring the soda solution into intimate contact with the gas4 ,return the residual gas to the end of the closed branch1 and measure4 ,the loss in volume of gas carbon dioxide4 ,the residual gas hydrogen4 ,transfer gas to the bulb of the tube1 and explode it by applying a lighted taper4 7,b7 ,sulphuretted ,hydrogen4-- ,media ,required3 ,iron peptone solution 7vide page #ahe74 ,lead peptone solution4 #a4 ,inoculate tubes of media1 and incubate together with control tubes4 #b4 ,examine from day to day1 at intervals of twenty-four hours4 ,the liberation of the ,h#b,s will cause the yellowish-white precipitate to darken to a brownish-black1 or jet black1 the depth of the colour being proportionate to the amount of sulphuretted hydrogen present4 ,quantitative3 ,for exact quantitative analyses of the gases produced by bacteria from certain media of definite composition1 the methods devised by ,pakes must be employed1 as follows3 ,illustration3 ,,fig4 #aec4--;,gas-collecting apparatus4 ,apparatus ,required3 ,bohemian flask 7#cjj to #aejj c4c4 capacity7 containing from #ajj to #djj c4c4 of the medium4 ,the mouth of the flask is fitted with a perforated rubber stopper1 carrying an ,l-shaped piece of glass tubing 7the short arm passing just through the stopper74 ,to the long arm of the tube is attached a piece of pressure tubing some #h cm4 in length1 plugged at its free end with a piece of cotton-wool4 ,measure accurately the total capacity of the flask and exit tube1 also the amount of medium contained4 ,note the difference4 ,gas receiver4 ,this is a bell jar of stout glass1 #ad cm4 high and #i cm4 in diameter4 ,at its apex a glass tube is fused in4 ,this rises vertically #e cm41 and is then bent at right angles1 the horizontal arm being #aj cm4 in length4 ,a three-way tap is let horizontally into the vertical tube just above its junction with the bell jar4 ,an iron cylinder just large enough to contain the bell jar4 ,about #ae kilos of metallic mercury4 ,melted paraffin4 ,an ,orsat-,lunge working with mercury instead of water1 provided with two gas tubes of extra length 7capacity #abj and #fj c4c4 respectively and graduated throughout1 both being water-jacketed7 or other gas analysis apparatus1 capable of dealing with ,,co#b1 ,o#b1 ,h#b1 and ,n#b4 ,,method4-- #a4 ,inoculate the medium in the flask in the usual manner1 by means of a platinum needle1 taking care that the neck of the flask and the rubber stopper are thoroughly flamed before and after the operation4 ,illustration3 ,,fig4 #aed4--,orsat-,lunge gas analysis apparatus4 #b4 ,fill the iron cylinder with mercury4 #c4 ,place the bell jar mouth downward in the mercury--first seeing that there is free communication between the interior of the jar and the external air--and suck up the mercury into the tap2 then shut off the tap4 #d4 ,plug the open end of the three-way tap with melted wax4 #e4 ,connect up the horizontal arm of the culture flask with that of the gas receiver by means of the pressure tubing 7after removing the cotton-wool plug from the rubber tube71 as shown in ,fig4 #aec4 #f4 ,give the three-way tap half turn to open communication between flask and receiver1 and seal all joints by coating with a film of melted wax4 ,when the tap is turned1 the mercury in the receiver will naturally fall4 #g4 ,place the entire apparatus in the incubator4 7,two hours later1 by which time the temperature of the apparatus is that of the incubator1 mark the height of the mercury on the receiver47 #h4 ,examine the apparatus from day to day and mark the level of the mercury in the receiver at intervals of twenty-four hours4 #i4 ,when the evolution of gas has ceased1 remove the apparatus from the incubator2 clear out the wax from the nozzle of the three-way tap 7first adjusting the tap so that no escape of gas shall take place7 and connect it with the ,orsat4 #aj4 ,remove1 say1 #ajj c4c4 of gas from the receiver1 reverse the tap and force it into the culture flask4 ,remove #ajj c4c4 of mixed gases from the culture flask and replace in the receiver4 ,repeat these processes three or four times to ensure thorough admixture of the contents of flask and receiver4 #aa4 ,now withdraw a sample of the mixed gases into the ,orsat and analyse4 ,in calculating the results be careful to allow for the volume of air contained in the flask at the commencement of the experiment4 ,for the collection of gases formed under anaerobic conditions a slightly different procedure is adopted3 #a4 ,fix a culture flask 7#ejj c4c4 capacity7 with a perforated rubber stopper carrying an ,l-shaped piece of manometer tubing1 each arm #e cm4 in length4 #b4 ,prepare a second ,l-shaped piece of tubing1 the short arm #e cm4 and the long arm #bj cm41 and connect its short arm to the horizontal arm of the tube in the culture flask by means of a length of pressure tubing1 provided with a screw clamp4 #c4 ,fill the culture flask completely with boiling medium and pass the long piece of tubing through the plug of an ,erlenmeyer flask 7#aej c4c4 capacity7 which contains #ajj c4c4 of the same medium4 #d4 ,sterilise these coupled flasks by the discontinuous method1 in the usual manner4 ,immediately the last sterilisation is completed1 screw up the clamp on the pressure tubing which connects them1 and allow them to cool4 ,as the fluid cools and contracts it leaves a vacuum in the neck of the flask below the rubber stopper4 #e4 ,to inoculate the culture flask1 withdraw the long arm of the bent tube from the ,erlenmeyer flask and pass it to the bottom of a test-tube containing a young cultivation 7in a fluid medium similar to that contained in the culture flask7 of the organism it is desired to investigate4 #f4 ,slightly release the clamp on the pressure tubing to allow #d or #e c4c4 of the culture to enter the flask4 #g4 ,clamp the rubber tube tightly2 remove the bent glass tube from the culture tube and plunge it into a flask containing recently boiled and quickly cooled distilled water4 #h4 ,release the clamp again and wash in the remains of the cultivation until the culture flask and tubing are completely filled with water4 #i4 ,clamp the rubber tubing tightly and take away the long-armed glass tubing4 #aj4 ,prepare the gas receiver as in the previous method 7in this case1 however1 the mercury should be warmed slightly7 and fill the horizontal arm of the receiver with hot water4 #aa4 ,connect up the culture flask with the horizontal arm of the gas receiver4 #ab4 ,remove the screw clamp from the rubber tubing1 adjust the three-way tap1 seal all joints with melted wax1 and incubate4 #ac4 ,complete the investigation as described for the previous method4 ,,by ,,physical ,,methods4 ,examine cultivations of the organism with reference to its growth and development under the following headings3 ,atmosphere3 7a7 ,in the presence of oxygen4 7b7 ,in the absence of oxygen4 7c7 ,in the presence of gases other than oxygen4 ,temperature3 7a7 ,range4 7b7 ,optimum4 7c7 ,thermal death-point3 ,moist3 ,vegetative forms4 ,spores4 ,dry3 ,vegetative forms4 ,spores4 ,reaction of medium4 ,resistance to lethal agents3 7a7 ,desiccation4 7b7 ,light3 ,diffuse4 ,direct4 ,primary colours4 7c7 ,heat4 7d7 ,chemical antiseptics and disinfectants4 ,vitality in artificial cultures4 ,i4 ,atmosphere4--,the question as to whether the organism under observation is 7a7 an obligate aerobe1 7b7 a facultative anaerobe1 or 7c7 an obligate anaerobe is roughly decided by the appearance of cultivations in the fermentation tubes4 ,obvious growth in the closed branch as well as in the bulb or in the inverted gas tube as well as in the bulk of the medium will indicate that it is a facultative anaerobe2 whilst growth only occurring in the bulb or in the closed branch shows that it is an obligate aerobe or anaerobe respectively4 ,this method1 however1 is not sufficiently accurate for the present purpose1 and the examination of an organism with respect to its behaviour in the absence of oxygen is carried out as follows3 ,apparatus ,required3 ,buchner's tubes4 ,bulloch's apparatus4 ,exhaust pump4 ,pyrogallic acid4 ,dekanormal caustic soda4 ,media ,required3 ,glucose formate agar4 ,glucose formate gelatine4 ,glucose formate bouillon4 ,,method4-- #a4 ,prepare four sets of cultivations3 7,a7 ,sloped glucose formate agar1 and incubate aerobically at #cg deg4 ,c4 ,sloped glucose formate gelatine1 and incubate aerobically at #bj deg4 ,c4 7,b7 ,sloped glucose agar to incubate anaerobically at #cg deg4 ,c4 ,sloped glucose formate gelatine to incubate anaerobically at #bj deg4 ,c4 7,c7 ,sloped glucose formate agar to incubate anaerobically at #cg deg4 ,c4 ,glucose formate bouillon to incubate anaerobically at #cg deg4 ,c4 7,d7 ,sloped glucose formate gelatine to incubate anaerobically at #bj deg4 ,c4 ,glucose formate bouillon to incubate anaerobically at #bj deg4 ,c4 #b4 ,seal the cultures forming set ,b in ,buchner's tubes 7vide page #bci74 #c4 ,seal the cultures forming set ,c in ,bulloch's apparatus2 exhaust the air by means of a vacuum pump1 and provide for the absorption of any residual oxygen by the introduction of pyrogallic acid and caustic soda in solution 7vide page #bde74 ,treat set ,d in the same way4 #d4 ,observe the cultivations macroscopically and microscopically at intervals of twenty-four hours until the completion1 if necessary1 of seven days' incubation4 #e4 ,control these results4 ,gases ,other than ,oxygen4-- ,apparatus ,required3 ,bulloch's apparatus4 ,sterile gas filter 7vide page #dj74 ,gasometer containing the gas it is desired to test 7,,so#b1 ,n#b,o1 ,,no1 ,,co#b1 etc47 or gas generator for its production4 ,,method4-- #a4 ,prepare at least seven tube cultivations upon solid media and deposit them in ,bulloch's apparatus4 #b4 ,connect up the inlet tube of the ,bulloch's jar with the sterile gas filter1 and this again with the delivery tube of the gasometer or gas generator4 #c4 ,open both stop-cocks of the ,bulloch's apparatus and pass the gas through until it has completely replaced the air in the bell jar as shown by the result of analyses of samples collected from the exit tube4 #d4 ,incubate under optimum conditions as to temperature4 #e4 ,examine the cultivations at intervals of twenty-four hours1 until the completion of seven days4 #f4 ,remove one tube from the interior of the apparatus each day4 ,if no growth is visible1 incubate the tube under optimum conditions as to temperature and atmosphere1 and in this way determine the length of exposure to the action of the gas necessary to kill the organisms under observation4 #g4 ,control these results4 ,,ii4 ,temperature4-- 7,a7 ,range4-- #a4 ,prepare a series of ten tube cultivations1 in fluid media1 of optimum reaction4 #b4 ,arrange a series of incubators at fixed temperatures1 varying #e deg4 ,c4 and including temperatures between #e deg4 ,c4 and #ej deg4 ,c4 7,in the absence of a sufficient number of incubators utilise the water-bath employed in testing the thermal death-point of vegetative forms47 #c4 ,incubate one tube cultivation of the organism aerobically or anaerobically1 as may be necessary1 in each incubator1 and examine at half-hour intervals for from five to eighteen hours4 #d4 ,note that temperature at which growth is first observed macroscopically 7,optimum temperature74 #e4 ,continue the incubation until the completion of seven days4 ,note the extremes of temperature at which growth takes place 7,range of temperature74 #f4 ,control these results--if considered necessary arranging the series of incubators to include each degree centigrade for five degrees beyond each of the extremes previously noted4 7,b7 ,optimum4-- #a4 ,prepare a second series of ten tube cultivations under similar conditions as to reaction of medium4 #b4 ,incubate in a series of incubators in which the temperature is regulated at intervals of #a deg4 ,c4 for five degrees on either side of optimum temperature observed in the previous experiment 7,a1 step #d74 #c4 ,observe again at half-hour intervals and note that temperature at which growth is first visible to the naked eye ,optimum temperature4 7,c7 ,thermal ,death-point 7t4 d4 p47-- ,moist--,vegetative ,forms3 ,the t4 d4 p4 here is that temperature which with certainty kills a watery suspension of the organisms in question after an exposure of #aj minutes4 ,illustration3 ,,fig4 #aee4--;,hearson's water-bath4 ,apparatus ,required3 ,water-bath4 ,for the purpose of observing the thermal death-point a special water-bath is necessary4 ,the temperature of this piece of apparatus is controlled by means of a capsule regulator that can be adjusted for intervals of half a degree centigrade through a range of #cj deg41 from #ej deg4 ,c4 to #hj deg4 ,c4 by means of a spring1 actuated by the handle a1 which increases the pressure in the interior of the capsule4 ,a hole is provided for the reception of the nozzle of a blast pump1 so that a current of air may be blown through the water while the bath is in use1 and thus ensure a uniform temperature of its contents4 ,through a second hole is suspended a certified centigrade thermometer1 the bulb of which although completely immersed in the water is raised at least #b cm4 above the floor of the bath4 ,sterile glass capsules4 ,flask containing #bej c4c4 sterile normal saline solution4 ,case of sterile pipettes1 #aj c4c4 7in tenths of a cubic centimetre74 ,special platinum loop4 ,test-tubes1 #ah by #a4e cm41 of thin ,german glass4 ,case of sterile petri dishes4 ,tubes of agar or gelatine4 ,,method4-- #a4 ,prepare tube cultivations on solid media of optimum reaction2 incubate forty-eight hours under optimum conditions as to temperature and atmosphere4 #b4 ,examine preparations from the cultivation microscopically to determine the absence of spores4 #c4 ,pipette #e c4c4 salt solution into each of twelve capsules4 #d4 ,suspend three loopfuls of the surface growth 7using a special platinum loop1 vide page #caf7 in the normal saline solution by emulcifying evenly against the moist walls of each capsule4 #e4 ,transfer emulsion from each capsule to sterile #bej c4c4 flask1 and mix4 #f4 ,pipette #e c4c4 emulsion into each of twelve sterile test-tubes numbered consecutively4 #g4 ,adjust the first tube in the water-bath1 regulated at #dj deg4 ,c1 by means of two rubber rings around the tube1 one above and the other below the perforated top of the bath1 so that the upper level of the fluid in the tube is about #d cm4 below the surface of the water in the bath1 and the bottom of the tube is a similar distance above the bottom of the bath4 #h4 ,arrange a control test-tube containing #e c4c4 sterile saline solution under similar conditions4 ,plug the tube with cotton-wool and pass a thermometer through the plug so that its bulb is immersed in the water4 #i4 ,close the unoccupied perforations in the lid of the water-bath by means of glass balls4 #aj4 ,watch the thermometer in the test-tube until it records a temperature of #dj deg4 ,c4 ,note the time4 ,ten minutes later remove the tube containing the suspension1 and cool rapidly by immersing its lower end in a stream of running water4 #aa4 ,pour three gelatine 7or agar7 plates containing respectively #j4b1 #j4c1 and #j4e c4c4 of the suspension1 and incubate4 #ab4 ,pipette the remaining #d c4c4 of the suspension into a culture flask containing #bej c4c4 of nutrient bouillon1 and incubate4 #ac4 ,observe these cultivations from day to day4 ,no growth must not be recorded as final until after the completion of seven days' incubation4 #ad4 ,extend these observations to the remaining tubes of the series1 but varying the conditions so that each tube is exposed to a temperature #b deg4 ,c4 higher than the immediately preceding one--i4 e41 #db deg4 ,c41 #dd deg4 ,c41 #df deg4 ,c41 and so on4 #ae4 ,note that temperature1 after exposure to which no growth takes place up to the end of seven days' incubation1 the thermal death-point4 #af4 ,if greater accuracy is desired1 a second series of tubes may be prepared and exposed for ten minutes to fixed temperatures varying only #j4e deg4 ,c41 through a range of #e deg4 ,c4 on either side of the previously observed death-point4 ,moist--,spores3 ,the thermal death-point in the case of spores is that time exposure to a fixed temperature of #ajj deg4 ,c4 necessary to effect the death of all the spores present in a suspension4 ,,note4--,if it is desired to retain the time constant #aj minutes and investigate the temperature necessary to destroy the spores1 varying amounts of calcium chloride must be added to the water in the bath1 when the boiling-point will be raised above #ajj deg4 ,c4 according to the percentage of calcium in solution4 ,in such case use the bath figured on page #bbg2 the bath figured on page #bii can only be used if the capsule is first removed4 ,it is determined in the following manner ,apparatus ,required3 ,steam-can fitted with a delivery tube and a large bore safety-valve tube4 ,water-bath at #ajj deg4 ,c4 ,erlenmeyer flask1 #ejj c4c4 capacity1 containing #adj c4c4 sterile normal saline solution and fitted with rubber stopper perforated with four holes4 ,the rubber stopper is fitted as follows3 7a7 ,thermometer to #abj deg4 ,c41 its bulb immersed in the normal saline4 7b7 ,straight entry tube1 reaching to the bottom of the flask1 the upper end plugged with cotton-wool4 7c7 ,bent syphon tube1 with pipette nozzle attached by means of rubber tubing and fitted with pinch-cock4 ,the nozzle is protected from accidental contamination by passing it through the cotton-wool plug of a small test-tube4 7d7 ,a sickle-shaped piece of glass tubing passing just through the stopper1 plugged with cotton-wool1 to act as a vent for the steam4 ,sterile plates4 ,sterile pipettes4 ,sterile test-tubes graduated to contain #e c4c4 ,media ,required3 ,gelatine or agar4 ,culture flasks containing #bjj c4c4 nutrient bouillon4 ,illustration3 ,,fig4 #aef4--;,apparatus arranged for the determination of the death-point of spores4 ,,method4-- #a4 ,prepare twelve tube cultivations upon the surface 7or two cultures in large flat culture bottles--vide page #e7 of nutrient agar and incubate under the optimum conditions 7previously determined71 for the formation of spores4 ,examine preparations from the cultures microscopically to determine the presence of spores4 #b4 ,pipette #e c4c4 sterile normal saline into each culture tube or #cj c4c4 into each bottle and by means of a sterile platinum spatula emulsify the entire surface growth with the solution4 #c4 ,add the #fj c4c4 emulsion to #adj c4c4 normal saline contained in the fitted ,erlenmeyer flask4 #d4 ,place the flask in the water-bath of boiling water4 #e4 ,connect up the straight tube1 after removing the cotton-wool plug1 with the delivery tube of the steam can2 remove the plug from the vent tube4 #f4 ,when the thermometer reaches #ajj deg4 ,c41 open the spring clip on the syphon1 discard the first cubic centimeter of suspension that syphons over 7i4 e41 the contents of the syphon tube72 collect the next #e c4c4 of the suspension in the sterile graduated test-tube and pour plates and prepare flask cultures therefrom as in the previous experiments4 #g4 ,repeat this process at intervals of twenty-five minutes' steaming4 #h4 ,observe the inoculated plates and flasks up to the completion1 if necessary1 of seven days' incubation4 #i4 ,control these experiments1 but in this instance syphon off portions of the suspension at intervals of one-half to one minute during the five or ten minutes preceding the previously determined death-point4 ,thermal ,death-point4-- ,dry--,vegetative ,forms3 ,the thermal death-point in this case is that temperature which with certainty kills a thin film of the organism in question after a time exposure of ten minutes4 ,apparatus ,required3 ,hot-air oven1 provided with thermo-regulator4 ,sterile cover-slips4 ,flask containing #bej c4c4 sterile normal saline solution4 ,case of sterile pipettes1 #aj c4c4 7in tenths of a cubic centimetre74 ,case of sterile capsules4 ,crucible tongs4 ,,method4-- #a4 ,prepare an emulsion with three loopfuls from an optimum cultivation in #e c4c4 normal saline in a sterile capsule and examine microscopically to determine the absence of spore forms4 #b4 ,make twelve cover-slip films on sterile cover-slips2 place each in a sterile capsule to dry4 #c4 ,expose each capsule in turn in the hot-air oven for ten minutes to a different fixed temperature1 varying #e deg4 ,c4 between #fj deg4 ,c4 and #abj deg4 ,c4 #d4 ,remove each capsule from the oven with crucible tongs immediately after the ten minutes are completed2 remove the cover-glass from its interior with a sterile pair of forceps4 #e4 ,deposit the film in a flask containing #bjj c4c4 nutrient bouillon4 #f4 ,prepare subcultivations from such flasks as show evidence of growth1 to determine that no accidental contamination has taken place but that the organism originally spread on the film is responsible for the growth4 #g4 ,control the result of these experiments4 ,dry--,spores3 ,the thermal death-point in this case is that temperature which with certainty kills the spores of the organism in question when present in a thin film after a time exposure of #aj minutes4 ,apparatus ,required3 ,as for vegetative forms4 ,,method4-- #a4 ,prepare a sloped agar tube cultivation and incubate under optimum conditions as to spore formations4 #b4 ,pipette #e c4c4 sterile normal saline into the culture tube and emulsify the entire surface growth in it4 ,examine microscopically to determine the presence of spores in large numbers4 #c4 ,spread thin even films on twelve sterile cover-slips and place each cover-slip in a separate sterile capsule4 #d4 ,expose each capsule in turn for ten minutes to a different fixed temperature1 varying #e deg4 ,c1 between #ajj deg4 ,c4 and #afj deg4 ,c4 #e4 ,complete the examination as for vegetative forms4 ,,iii4 ,reaction of ,medium4 7,a7 ,range4-- #a4 ,prepare a bouillon culture of the organism and incubate1 under optimum conditions as to temperature and atmosphere1 for twenty-four hours4 #b4 ,pipette #j4a c4c4 of the cultivation into a sterile capsule2 add #i4i c4c4 sterile bouillon and mix thoroughly4 #c4 ,prepare a series of tubes of nutrient bouillon of varying reactions1 from #be to -#cj 7vide page #aee71 viz43 #be1 #bj1 #ae1 #aj1 #e1 neutral1 -#e1 -#aj1 -#ae1 -#bj1 -#be1 -#cj4 #d4 ,inoculate each of the bouillon tubes with #j4a c4c4 of the diluted cultivation by means of a sterile graduated pipette and incubate under optimum conditions4 #e4 ,observe the cultures at half-hourly intervals from the third to the twelfth hours4 ,note the reaction of the tube or tubes in which growth is first visible macroscopically 7probably optimum reaction74 #f4 ,continue the incubation until the completion1 if necessary1 of seven days4 ,note the extremes of acidity and alkalinity in which macroscopical growth has developed 7,range of reaction74 #g4 ,control the result of these observations4 7,b7 ,optimum ,reaction4--,the optimum reaction has already been roughly determined whilst observing the range4 ,it can be fixed within narrower limits by inoculating in a similar manner a series of tubes of bouillon which represent smaller variations in reaction than those previously employed 7say1 #a instead of #e7 for five points on either side of the previously observed optimum4 ,for example1 the optimum reaction observed in the set of experiments to determine the range was #aj4 ,now plant tubes having reactions of #ae1 #ad1 #ac1 #ab1 #aa1 #aj1 #i1 #h1 #g1 #f1 #e1 and observe as before4 ,,iv4 ,resistance to ,lethal ,agents4-- 7,a7 ,desiccation4-- ,apparatus ,required3 ,mueller's desiccator4 ,this consists of a bell glass fitted with an exhaust tube and stop-cock 7d71 which can be secured to a plate-glass base 7c7 by means of wax or grease4 ,it contains a cylindrical vessel of porous clay 7a7 into the top of which pure sulphuric acid is poured whilst the material to be dried is placed within its walls on a glass shelf 7b74 ,the air is exhausted from the interior and the acid rapidly converts the clay vessel into a large absorbing surface 7,fig4 #aeg74 ,exhaust pump4 ,pure concentrated sulphuric acid4 ,sterile cover-slips4 ,sterile forceps4 ,culture flask containing #bjj c4c4 nutrient bouillon4 ,sterile ventilated ,petri dish4 ,this is prepared by bending three short pieces of aluminium wire into ,v shape and hanging these on the edge of the lower dish and resting the lid upon them 7,fig4 #aeh74 ,,method4-- #a4 ,prepare a surface cultivation on nutrient agar in a culture bottle and incubate under optimum conditions for forty-eight hours4 #b4 ,examine preparations from the cultivation1 microscopically1 to determine the absence of spores4 #c4 ,pipette #e c4c4 sterile normal saline solution into the flask and suspend the entire growth in it4 #d4 ,spread the suspension in thin1 even films on sterile cover-slips and deposit inside sterile plates to dry4 #e4 ,as soon as dry1 transfer the cover-slip films to the ventilated ,petri dish by means of sterile forceps4 ,illustration3 ,,fig4 #aeg4--,mueller's desiccator4 #f4 ,place the ,petri dish inside the ,mueller's desiccator2 fill the upper chamber with pure sulphuric acid1 cover with the bell jar1 and exhaust the air from its interior4 ,ten minutes later connect up the desiccator to a sulphuric acid wash-bottle interposing an air filter so that only dry sterile air enters4 ,illustration3 ,,fig4 #aeh4--,petri dish for drying cultivations4 #g4 ,at intervals of five hours open the apparatus1 remove one of the cover-slip films from the ,petri dish1 and transfer it to the interior of a culture flask1 with every precaution against contamination4 ,reseal the desiccator and again exhaust1 and subsequently admit dry sterile air as before4 #h4 ,incubate the culture flask under optimum conditions until the completion of seven days1 if necessary2 and determine the time exposure at which death occurs4 #i4 ,pour plates from those culture flasks which grow1 to determine the absence of contamination4 #aj4 ,repeat these observations at hourly intervals for the five hours preceding and succeeding the death time1 as determined in the first set of experiments4 7,b7 ,light4-- 7a7 ,diffuse ,daylight3 #a4 ,prepare a tube cultivation in nutrient bouillon1 and incubate under optimum conditions1 for forty-eight hours4 ,illustration3 ,,fig4 #aei4--,plate with star for testing effect of light4 #b4 ,pour twenty plate cultivations1 ten of nutrient gelatine and ten of nutrient agar1 each containing #j4a c4c4 of the bouillon culture4 #c4 ,place one agar plate and one gelatine plate into the hot and cold incubators1 respectively1 as controls4 #d4 ,fasten a piece of black paper1 cut the shape of a cross or star1 on the centre of the cover of each of the remaining plates 7,fig4 #aei74 #e4 ,expose these plates to the action of diffuse daylight 7not direct sunlight7 in the laboratory for one1 two1 three1 four1 five1 six1 eight1 ten1 twelve hours4 #f4 ,after exposure to light1 incubate under optimum conditions4 #g4 ,examine the plate cultivations after twenty-four and forty-eight hours' incubation1 and compare with the two controls4 ,record results4 ,if growth is absent from that portion of the plate unprotected by the black paper1 continue the incubation and daily observation until the end of seven days4 #h4 ,control the results4 7b7 ,direct ,sunlight3 #a4 ,prepare plate cultivations precisely as in the former experiments and place the two controls in the incubators4 #b4 ,arrange the remaining plates upon a platform in the direct rays of the sun4 #c4 ,on the top of each plate stand a small glass dish #ad cm4 in diameter and #e cm4 deep4 #d4 ,fill a solution of potash alum 7#b per cent4 in distilled water7 into each dish to the depth of #b cm4 to absorb the heat of the sun's rays and so eliminate possible effects of temperature on the cultivations4 #e4 ,after exposures for periods similar to those employed in the preceding experiment1 incubate and complete the observation as above4 7c7 ,primary ,colours3 ,each colour--violet1 blue1 green and red--must be tested separately4 #a4 ,prepare plate cultivations1 as in the previous light experiments1 and incubate controls4 #b4 ,fasten a strip of black paper1 #c cm4 wide1 across one diameter of the cover of each plate4 #c4 ,coat the remainder of the surface of the cover with a film of pure photographic collodion which contains #b per cent4 of either of the following aniline dyes1 as may be necessary3 ,chrysoidin 7for red74 ,malachite green 7for green74 ,eosin1 bluish 7for blue74 ,methyl violet 7for violet74 #d4 ,expose the plates1 thus prepared1 to bright daylight 7but not direct sunlight7 for varying periods1 and complete the observations as in the preceding experiments4 ,the bactericidal action of light appears to depend upon the more refrangible rays of the violet end of the spectrum and is noted whether the red yellow rays are transmitted or not4 #e4 ,control the results4 ,,note4--,the ultra-violet rays obtained from a quartz mercury vapour lamp destroy bacterial life with great rapidity under laboratory conditions4 7,c7 ,heat4--7,vide ,thermal ,death-point1 page #bih47 7,d7 ,antiseptics and ,disinfectants4--,the resistance exhibited by any given bacterium toward any specified disinfectant or germicide should be investigated with reference to the following points3 7,a7 ,inhibition coefficient--i4 e41 that percentage of the disinfectant present in the nutrient medium which is sufficient to prevent the growth and multiplication of the bacterium4 7,b7 ,inferior lethal coefficient--i4 e41 the time exposure necessary to kill vegetative forms of the bacterium suspended in water at #bj deg4 to #be deg4 ,c1 in which the disinfectant is present in medium concentration 7concentration insufficient to cause plasmolysis74 ,and if the bacterium is one which forms spores1 7,c7 ,superior lethal coefficient--i4 e41 the time exposure necessary to kill the spores of the bacterium under conditions similar to those obtaining in ,b4 ,the example here detailed only specifically refers to certain of the disinfectants3 viz3--,bichloride of mercury2 ,formaldehyde2 ,carbolic acid2 investigated with regard to ,b4 anthracis1 but the technique is practically similar for all other chemical disinfectants4 ,inhibition ,coefficient4-- ,apparatus ,required3 ,case of sterile pipettes1 #aj c4c4 7in tenths74 ,case of sterile pipettes1 #a c4c4 7in tenths74 ,sterile tubes or capsules for dilutions4 ,tubes of nutrient bouillon each containing a measured #aj c4c4 of medium4 ,twenty-four-hour-old agar culture of a recently isolated ,b4 ,anthracis4 ,germicides3 #a4 ,five per cent4 aqueous solution of carbolic acid4 #b4 ,one per cent4 aqueous solution of perchloride of mercury4 #c4 ,one-tenth per cent4 aqueous solution of formaldehyde4 ,,method4-- #a4 ,number six bouillon tubes consecutively #a to #f4 ,inoculate each from the stock cultivation of ,b4 anthracis and at once add varying quantities#aj of the carbolic acid solution1 viz43 ,to tube #a add #b4j c4c4 7 #a3ajj7 ,to tube #b add #a4j c4c4 7 #a3bjj7 ,to tube #c add #j4f c4c4 7 #a3cjj7 ,to tube #d add #j4e c4c4 7 #a3djj7 ,to tube #e add #j4d c4c4 7 #a3ejj7 ,to tube #f add #j4b c4c4 7 #a3a1jjj7 #b4 ,prepare a similar series of tube cultivations numbered consecutively #g to #ab and add varying quantities of the mercuric perchloride solution1 viz43 ,to tube #g add #j4a 7 #a3a1jjj7 ,to tube #h add #j4je 7 #a3b1jjj7 ,to tube #i add #j4jc 7 #a3c1jjj7 ,to tube #aj add #j4jbe 7 #a3d1jjj7 ,to tube #aa add #j4jb 7 #a3e1jjj7 ,to tube #ab add #j4ja 7 #a3aj1jjj7 #c4 ,prepare a similar series of tube cultivations numbered consecutively #ac to #ah and add varying quantities of the formaldehyde solution1 viz43 ,to tube ,no4 #ac add #a4j c4c4 7 #a3a1jjj7 ,to tube ,no4 #ad add #j4d c4c4 7 #a3b1ejj7 ,to tube ,no4 #ae add #j4b c4c4 7 #a3e1jjj7 ,to tube ,no4 #af add #j4a c4c4 7 #a3aj1jjj7 ,to tube ,no4 #ag add #j4jge c4c4 7 #a3ae1jjj7 ,to tube ,no4 #ah add #j4je c4c4 7 #a3bj1jjj7 #d4 ,incubate all three sets of cultivations under optimum conditions as to temperature and atmosphere4 #e4 ,examine each of the culture tubes from day to day1 until the completion of seven days1 and note those tubes1 if any1 in which growth takes place4 #f4 ,from such tubes as show growth prepare subcultivations upon suitable media1 and ascertain that the organism causing the growth is the one originally employed in the test and not an accidental contamination4 ,inferior ,lethal ,coefficient4-- ,apparatus ,required3 ,highly concentrated solutions of the disinfectants4 ,sterile test-tubes in which to make dilutions from the concentrated solutions of the disinfectants4 ,hanging-drop slides4 ,cover-slips4 ,erlenmeyer flask containing #ajj c4c4 sterile distilled water4 ,case of sterile pipettes1 #aj c4c4 7in tenths of a cubic centimetre74 ,case of sterile pipettes1 #a c4c4 7in tenths of a cubic centimetre74 ,,method4-- #a4 ,prepare a surface cultivation of the test organism ,b4 anthracis upon nutrient agar in a culture bottle and incubate under optimum conditions for twenty-four hours2 then examine the cultivation microscopically to determine the absence of spores4 #b4 ,prepare solutions of different percentages of each disinfectant4 #c4 ,make a series of hanging-drop preparations from the agar culture1 using a loopful of disinfectant solution of the different percentages to prepare the emulsion on each cover-slip4 #d4 ,examine microscopically and note the strongest solution which does not cause plasmolysis and the weakest solution which does plasmolyse the organism4 #e4 ,make control preparations of these two solutions and determine the percentage to be tested4 #f4 ,pipette #aj c4c4 sterile water into the culture bottle and suspend the entire surface growth in it4 #g4 ,transfer the suspension to the ,erlenmeyer flask and mix it with the #ij c4c4 of sterile water remaining in the flask4 #h4 ,pipette #aj c4c4 of the diluted suspension into each of ten sterile test-tubes4 #i4 ,label one of the tubes ,control and place it in the incubator at #ah deg4 ,c4 #aj4 ,add to each of the remaining tubes a sufficient quantity#aa of a concentrated solution of the disinfectant to produce the percentage previously determined upon 7vide step #e74 #aa4 ,incubate the tubes at #ah deg4 ,c4 to #bj deg4 ,c4 #ab4 ,at hourly intervals remove the control tube and one of the tubes with added disinfectant from the incubator4 #ac4 ,make a subcultivation from both the control and the test suspension1 upon the surface of nutrient agar2 incubate under optimum conditions4 #ad4 ,observe these culture tubes from day to day until the completion of seven days1 and determine the shortest exposure necessary to cause the death of vegetative forms4 ,superior ,lethal ,coefficient4-- #a4 ,prepare surface cultivations of the test organisms upon nutrient agar in a culture bottle1 and incubate under optimum conditions1 for three days1 for the formation of their spores4 #b4 ,transfer the emulsion to a sterile test-tube and heat in the differential steriliser for ten minutes at #hj deg4 ,c4 to destroy all vegetative forms4 #c4 ,employing that percentage solution of the disinfectant determined in the previous experiment1 and complete the investigations as detailed therein1 steps #g to #ad1 increasing the interval between planting the subcultivations to two1 three1 or five hours if considered advisable4 ,,note4--,where it is necessary to leave the organisms in contact with a strong solution of the disinfectant for lengthy periods1 some means must be adopted to remove every trace of the disinfectant from the bacteria before transferring them to fresh culture media2 otherwise1 although not actually killed1 the presence of the disinfectant may prevent their development1 and so give rise to an erroneous conclusion4 ,consequently it is essential in all germicidal experiments to determine first of all the inhibition coefficient of the germicide employed4 ,under the circumstances referred to above it is usually sufficient to prepare the subcultures in such a volume of fluid nutrient medium as would suffice to reduce the concentration of the germicide to about one hundredth of the inhibition percentage1 assuming that the entire bulk of inoculum was made up of that strength of germicide employed in the test4 ,in some cases it is a simple matter to neutralise the germicide and render it inert by washing the organisms in some non-germicidal solution 7such for example as ammonium sulphide when using mercurial salts as the germicide74 ,when1 however1 it is desired to remove the last traces of germicide proceed as follows3 #a4 ,transfer the suspension of bacteria to sterile centrifugal tubes2 add the required amount of disinfectant1 and allow it to remain in contact with the bacteria for the necessary period4 #b4 ,centrifugalise thoroughly1 pipette off the supernatant fluid2 fill the tube with sterile water and distribute the deposit evenly throughout the fluid4 #c4 ,centrifugalise again1 pipette off the supernatant fluid2 fill the tube with sterile water2 distribute the deposit evenly throughout the fluid1 and transfer the suspension to a litre flask4 #d4 ,make up to a litre by the addition of sterile water2 filter the suspension through a sterile porcelain candle4 #e4 ,emulsify the bacterial residue with #e c4c4 sterile bouillon4 #f4 ,prepare the necessary subcultivations from this emulsion4 ,,pathogenesis4 ,living ,bacteria4-- 7a7 ,psychrophilic ,bacteria3 ,when the organism will only grow at or below #ah deg4 to #bj deg4 ,c41 #a4 ,prepare cultivations in nutrient broth and incubate under optimum conditions4 #b4 ,after seven days' incubation inject that amount of the culture corresponding to #a per cent4 of the body-weight of a healthy frog1 into the reptile's dorsal lymph sac4 #c4 ,observe until death takes place1 or1 in the event of a negative result1 until the completion of twenty-eight days 7vide ,chapter ,,xviii74 #d4 ,if1 and when1 death occurs1 make a careful post-mortem examination 7vide ,chapter ,,xix74 7b7 ,mesophilic ,bacteria3 ,when the organism grows at #ce deg4 to #cg deg4 ,c41 #a4 ,prepare cultivations in nutrient broth and incubate under optimum conditions for forty-eight hours4 #b4 ,select two white mice1 as nearly as possible of the same age1 size1 and weight4 #c4 ,inoculate the first mouse1 subcutaneously at the root of the tail1 with an amount of cultivation equivalent to #a per cent4 of its body-weight4 #d4 ,inoculate the second mouse intraperitoneally with a similar dose4 #e4 ,observe carefully until death occurs1 or until the lapse of twenty-eight days4 #f4 ,if the inoculated animals succumb1 make complete post-mortem examination4 ,if death follows shortly after the injection of cultivations of bacteria1 the inoculation experiments should be repeated two or three times4 ,then1 if the organism under observation invariably exhibits pathogenic effects1 steps should be taken to ascertain1 if possible1 the minimal lethal dose 7vide infra7 of the growth upon solid media for the frog or white mouse respectively4 ,other experimental animals--e4 g41 the white rat1 guinea-pig1 and rabbit--should next be tested in a similar manner4 #g4 ,if the inoculated mice are unaffected1 test the action of the organism in question upon white rats1 guinea-pigs1 rabbits1 etc4 ,minimal ,lethal ,dose 7m4 l4 d472 ,if the purpose of the inoculation is to determine the minimal lethal dose1 a slightly different procedure must be followed4 ,for this and other exact experiments a special platinum loop is manufactured1 some #b4e mm4 by #j4ge mm41 with parallel sides1 and calibrated by careful weighing1 to determine approximately the amount of moist bacterial growth1 the loop will hold when filled4 #a4 ,the cultivation must be prepared on a solid medium of the optimum reaction1 incubated at the optimum temperature1 and injected at the period of greatest activity and vigour1 of the particular organism it is desired to test4 #b4 ,arrange four sterile capsules in a row and label them ,i1 ,,ii1 ,,iii1 and ,,iv4 ,into the first deliver #aj c4c4 sterile bouillon by means of a sterile graduated pipette2 and into each of the remaining three1 #i4i c4c4 #c4 ,remove one loopful of the bacterial growth from the surface of the medium in the culture tube1 observing the usual precautions against contamination1 and emulsify it evenly with the bouillon in the first capsule4 ,each cubic centimetre of the emulsion will now contain one-tenth of the organisms contained in the original loopful 7written shortly #j4a loop74 #d4 ,remove #j4a c4c4 of the emulsion in the first capsule by means of a sterile graduated pipette and transfer it to the second capsule and mix thoroughly4 ,drop the infected pipette into a jar of lysol solution4 ,this makes up the bulk of the fluid in the second capsule to #aj c4c41 and therefore every cubic centimetre of bouillon in capsule ,,ii contains #j4jja loop4 #e4 ,similarly1 #j4a c4c4 of the mixture is transferred from capsule ,,ii to capsule ,,iii 7#a c4c4 of bouillon in capsule ,,iii contains #j4jjjja loop71 and then from capsule ,,iii to capsule ,,iv 7#a c4c4 of bouillon in capsule ,,iv contains #j4jjjjjja loop74 ,the dilutions thus prepared may be summarised in a table2 ,capsule ,i #a loopful #aj c4c4 water 4'4 #a c4c4#j4a loop4 ,capsule ,,ii #j4a c4c4 capsule ,i #i4i c4c4 water 4'4 #a c4c4#j4jja loop4 ,capsule ,,iii #j4a c4c4 capsule ,,ii #i4i c4c4 water 4'4 #a c4c4#j4jjjja loop4 ,capsule ,,iv #j4a c4c4 capsule ,,iii #i4i c4c4 water 4'4 #a c4c4 #j4jjjjjja loop4 #f4 ,with sterile graduated pipettes remove the necessary quantity of bouillon corresponding to the various divisors of ten of the loop from the respective capsules1 and transfer each dose to a separate sterile capsule and label2 and to such doses as are small in bulk1 add the necessary quantity of sterile bouillon to make up to #a c4c4 #g4 ,multiples of the loop are prepared by emulsifying #a1 #b1 #e1 or #aj loops each with #a c4c4 sterile bouillon in separate sterile capsules4 #h4 ,inoculate a series of animals with these measured doses1 filling the syringe first from that capsule containing the smallest dose1 then from the capsule containing the next smallest1 and so on4 ,if care is taken1 it will not be found necessary to sterilise the syringe during the series of inoculations4 #i4 ,plant tubes of gelatine or agar1 liquefied by heat1 from each of the higher dilutions1 say from #j4jjjjjja loop to #j4ja loop2 pour plates and incubate4 ,when growth is visible enumerate the number of organisms present in each1 average up and calculate the number of bacteria present in one loopful of the inoculum4 #aj4 ,the smallest dose which causes the infection and death of the inoculated animal is noted as the minimal lethal dose4 ,toxins4-- ,prepare flask cultivations of the organism under observation in glucose formate broth1 and incubate for fourteen days under optimum conditions4 7a7 ,intracellular or ,insoluble ,toxins3 #a4 ,heat the fluid culture in a water-bath at #fj deg4 ,c4 for thirty minutes4 7,the resulting sterile1 turbid fluid is often spoken of as killed culture17 #b4 ,inoculate a tube of sterile bouillon with a similar quantity1 and incubate under optimum conditions4 ,this control then serves to demonstrate the freedom of the toxin from living bacteria4 ,illustration3 ,,fig4 #afj4--;,apparatus arrange for toxin filtration4 #c4 ,inject intraveneously that amount of the cultivation corresponding to #a per cent4 of the body-weight of the selected animal1 usually one of the small rodents4 #d4 ,observe during life or until the completion of twenty-eight days1 and in the event of death occurring during that period1 make a complete post-mortem examination4 #e4 ,repeat the experiment at least once4 ,in the event of a positive result estimate the minimal lethal dose of killed culture for each of the species of animals experimented upon4 7b7 ,extracellular or ,soluble ,toxins3 #a4 ,filter the cultivation through a porcelain filter candle 7,berkefeld7 into a sterile filter flask1 arranging the apparatus as in the accompanying figure 7,fig4 #afj74 #b4 ,inoculate mice1 rats1 guinea-pigs1 and rabbits subcutaneously with that quantity of toxin corresponding to #a per cent4 of the body-weight of each respectively1 and observe1 if necessary1 until the completion of one month4 #c4 ,inoculate a control tube of bouillon with a similar quantity and incubate1 to determine the freedom of the filtered toxin from living bacteria4 #d4 ,in the event of a fatal termination make complete and careful post-mortem examinations4 #e4 ,repeat the experiments and1 if the results are positive1 ascertain the minimal lethal dose of toxin for each of the susceptible animals4 ,the estimation of the m4 l4 d4 of a toxin is carried out on lines similar to those laid down for living bacteria 7vide page #caf7 merely substituting #a c4c4 of toxin as the unit in place of the unit loopful of living culture4 ,it frequently happens1 during the course of casual investigations that a bouillon-tube culture is available for a toxin test whilst a flask cultivation is not4 ,in such cases1 ,martin's small filter candle and tube 7,fig4 #afa7 specially designed for the filtration of small quantities of fluid1 is invaluable4 ,this consists of a narrow filter flask just large enough to accommodate an ordinary #ah x #b cm4 test-tube4 ,the mouth of the tubular ,chamberland candle #ae x #a4e cm4 is closed by a perforated rubber cork into which fits the end of the stem of a thistle headed funnel1 whilst immediately below the butt of the funnel is situated a rubber cork to close the mouth of the filter flask4 ,when the apparatus is fixed in position and connected to an exhaust pump1 the cultivation is poured into the head of the funnel and owing to the relatively large filtering surface the germ free filtrate is rapidly drawn through into the test-tube receiver4 ,raising the ,virulence of an ,organism4--,if it is desired to raise or exalt the virulence of a feebly pathogenic organism1 special methods of inoculation are necessary1 carefully adjusted to the exigencies of each individual case4 ,among the most important are the following3 #a4 ,passage of ,virus4--,the inoculation of pure cultivations of the organism into highly susceptible animals1 and passing it as rapidly as possible from animal to animal1 always selecting that method of inoculation-e4 g41 intraperitoneal--which places the organism under the most favorable conditions for its growth and multiplication4 ,illustration3 ,,fig4 #afa--,martin's filtering apparatus for small quantities of fluid4 #b4 ,virus ,plus ,virulent ,organisms4--,the inoculation of pure cultivations of the organism together with pure cultivations of some other microbe which in itself is sufficiently virulent to ensure the death of the experimental animal1 either into the same situation or into some other part of the body4 ,by this association the organism of low virulence will frequently acquire a higher degree of virulence1 which may be still further raised by means of passages 7vide supra74 #c4 ,virus ,plus ,toxins4--,the inoculation of pure cultivations of the organism into some selected situation1 together with the subcutaneous1 intraperitoneal1 or intravenous injection of a toxin--e4 g41 one of those elaborated by the proteus group--either simultaneously with1 before1 or immediately after1 the injection of the feeble virus4 ,by this means the natural resistance of the animal is lowered1 and the organism inoculated is enabled to multiply and produce its pathogenic effect1 its virulence being subsequently exalted by means of passages4 ,attenuating the ,virulence of an ,organism4--,attenuating or lowering the virulence of a pathogenic microbe is usually attained with much less difficulty than the exaltation of its virulence1 and is generally effected by varying the environment of the cultivations1 as for example3 #a4 ,cultivating in such media as are unsuitable by reason of their 7a7 composition or 7b7 reaction4 #b4 ,cultivating in suitable media1 but at an unsuitable temperature4 #c4 ,cultivating in suitable media1 but in an unsuitable atmosphere4 #d4 ,cultivation in suitable media1 but under unfavorable conditions as to light1 motion1 etc4 ,attenuation of the virus can also be secured by #e4 ,passage through naturally resistant animals4 #f4 ,exposure to desiccation4 #g4 ,exposure to gaseous disinfectants4 #h4 ,by a combination of two or more of the above methods4 ,,immunisation4 ,the further study of the pathogenetic powers of any particular bacterium involves the active immunisation of one or more previously normal animals4 ,this end may be attained by various means2 but it must be remembered that immunisation is not carried out by any hard and fast rule or by one method alone1 but usually by a combination of methods adapted to the exigencies of each particular case4 ,the ordinary methods include3 ,a4 ,active ,immunisation4 ,i4 ,by inoculation with dead bacteria 7i4 e41 bacteria killed by heat2 the action of ultra-violet rays1 of chemical germicides1 or by autolysis74 ,,ii4 ,by the inoculation of attenuated strains of bacteria4 ,,iii4 ,by the inoculation of living virulent bacteria 7exalted in virulence if necessary74 ,b4 ,combined ,active and ,passive ,immunisation3 ,,iv4 ,by the inoculation of toxin-antitoxin mixtures4 ,,active ,,immunisation4 ,the immunisation of the rabbit against the ,diplococcus pneumoniae may be instanced as an example of the general methods of immunisation of laboratory animals4 #a4 ,take a full grown rabbit weighing not less than #abjj to #aejj grammes 7large rabbits of #bjjj grammes and over are the most suitable for immunising experiments74 ,observe weight and temperature carefully during the few days occupied in the following steps4 #b4 ,inoculate a small rabbit intraperitoneally with one or two loopfuls of a twenty-four-hour-old blood agar cultivation of a virulent strain of ,diplococcus pneumoniae4 ,death should follow within twenty-four hours1 and in any case will not be delayed beyond forty-eight hours4 #c4 ,under aseptic precautions1 at the post-mortem1 transfer a loopful of heart blood to an ,erlenmeyer flask containing #ej c4c4 sterile nutrient broth4 ,incubate at #cg deg4 ,c4 for twenty-four hours4 #d4 ,prepare also several blood agar cultures from the heart blood of the rabbit1 label them all ,o4,c4 7original culture74 ,after twenty-four hours incubation at #cg deg4 ,c4 place an india-rubber cap over the plugged mouth of the tube of all but one of these cultures and paint the cap with ,canada balsam or shellac varnish1 dry1 and replace in the hot incubator4 ,this will prevent evaporation1 and cultures thus sealed will remain unaltered in virulence for a considerable time4 #e4 ,make a fresh subcultivation on blood agar from the uncapped ,o4,c4 cultivation and after twenty-four hours incubation at #cg deg4 ,c4 determine the minimal lethal dose of this strain upon a series of mice 7see page #caf74 #f4 ,suspend the flask containing the twenty-four-hour-old broth culture 7step #c7 in the water-bath at #fj deg4 ,c4 for one hour4 ,cool the flask rapidly under a stream of cold water4 #g4 ,determine the sterility of this 787 killed cultivation by transferring one cubic centimetre to each of several tubes of nutrient broth1 and incubate at #cg deg4 ,c4 for twenty-four hours4 ,if growth of ,diplococcus pneumoniae occurs1 again heat culture in water-bath at #fj deg4 ,c4 for one hour and again test for sterility4 #h4 ,inject the selected rabbit intravenously 7see page #cfc7 with #b c4c4 of the killed cultivation1 and inject a further #aj c4c4 into the peritoneal cavity4 ,during the next few days the animal will lose some weight and perhaps show a certain amount of pyrexia4 #i4 ,when the temperature and weight have again returned to normal--generally about seven days after the inoculation--again inject killed cultivation1 this time giving a dose of #e c4c4 intravenously and #bj c4c4 intraperitoneally4 ,a temperature and weight reaction similar to1 but less marked than that following the first injection will probably be observed1 but after about a week's interval the animal will be ready for the next injection4 #aj4 ,when ready to give the third injection prepare a fresh blood agar subculture from another ,o4,c4 tube and after twenty-four hours incubation prepare a minimal lethal dose 7as determined in #e7 and inject it subcutaneously into the rabbit's abdominal wall4 ,a slight local reaction will probably be observed as well as the weight and temperature reactions4 #aa4 ,a week to ten days later inject a similar minimal lethal dose into the peritoneal cavity4 #ab4 ,observe the weight and temperature of the rabbit very carefully1 and regulating the dates of inoculation by the animal's general condition1 continue to inject living cultivations of the pneumococcus into the peritoneal cavity1 gradually increasing the dose by multiples of ten4 #ac4 ,at intervals of two months samples of blood may be collected from the posterior auricular vein and the serum tested for specific antibodies4 #ad4 ,under favourable conditions it will be found after some six months steady work that the rabbit may be injected intraperitoneally with an entire blood agar cultivation without any ill effects being apparent2 and this characteristic--resistance to the lethal effects of large doses of the virus--is the sole criterion of immunity4 ,further1 the serum separated from blood withdrawn from the animal about a week after an injection1 if used in doses of 4#ja c4c41 will protect a mouse against the lethal effects of at least ten minimal lethal doses of living pneumococci4 ,in the foregoing illustration it has been assumed that complete acquired active immunity has been conferred upon the experimental rabbit in consequence of the formation of antibody1 specific to the diplococcus pneumoniac1 sufficient in amount to ensure the destruction of enormous doses of the living cocci--the antigen 7that is the substance injected in response to which antibody has been elaborated7 in this particular case being the bacterial protoplasm of the pneumococcus with its endo-toxins4 ,but provided death does not immediately follow the injection of the antigen1 specific antibody is always formed in greater or lesser amount2 and in experimental work a sufficient amount of any required antibody can often be obtained without carrying the process of immunisation to its logical termination4 ,for instance1 if the immunisation of a rabbit toward ,bacillus typhosus is commenced on the lines already set out it will often be found1 after a few injections of killed cultivation that the blood serum of the animal 7even when diluted with several hundred times its volume of normal saline7 contains specific agglutinin for ,b4 typhosus--and if the sole object of the experiment has been the preparation of agglutinin the inoculations may well be stopped at this point1 although the animal is not yet immune in the strict meaning of the word4 ,again1 antibodies may be formed in response to antigens other than infective particles--thus the injection into suitable animals of foreign proteins such as egg albumin1 heterologous blood sera or red blood discs from a different species of animal1 will result in the formation of specific antibodies possessing definite affinities for their respective antigens4 ,the most important antibody of this latter type is ,haemolysin1 a substance that makes its appearance in the blood serum of an animal previously injected with washed blood cells from an animal of a different species4 ,the serum from such an animal possesses the power of disintegrating red blood discs of the variety employed as antigen and causing the discharge of their contained haemoglobin1 and is specific in its action to the extent of failing to exert any injurious effect upon the red blood cells of any other species of animal4 ,the action of this serum is due to the presence of two distinct bodies1 complement and haemolysin4 ,complement 7or alexine7 is a thermo-labile readily oxidised body present in variable but unalterable amount in the normal serum of every animal4 ,it is a substance which exerts a lytic effect upon all foreign matter introduced into the blood or tissues2 but by itself is a comparatively inert body1 and is only capable of exerting its maximum lytic effect in the presence of and in combination with a specific antibody1 or immune body4 ,complement is obtained 7unmixed with antibody7 by collecting fresh blood serum from any healthy normal 7that is uninoculated7 animal4 ,guinea-pigs' serum is that most frequently employed for experimental work4 ,haemolysin 7immune body1 copula1 sensitising body1 amboceptor7 is a thermostable antibody formed in response to the injection of red cells which although in itself inert is capable of linking up complement present in the normal serum to the red cells of the variety used as antigen--a combination resulting in haemolysis4 ,haemolysin is obtained by collecting fresh blood serum from a suitably inoculated animal and exposing it to a temperature of #ef deg4 ,c4 7to destroy the thermo-labile complement7 for #ae to #cj minutes before use4 ,it is then referred to as inactivated1 and is reactivated by the addition of fresh normal serum--that is serum containing complement4 ,haemolysin is of importance academically owing to the fact that many of the problems of immunity have been elucidated by its aid2 but its present practical importance lies in the application of the haemolytic system 7that is haemolysin1 corresponding erythrocyte solution and complement7 to certain laboratory methods having for their object either the identification of the infective entity or the diagnosis of the existence of infection4 ,for use in these laboratory methods of diagnosis it is most convenient to prepare haemolytic serum specific for human blood--whether the laboratory is isolated or attached to a large hospital4 ,ox blood1 sheep blood or goat blood if readily obtainable1 may however be used instead1 and although the following method is directed to the preparation of human haemolysin the same procedure serves in all cases4 ,,the ,,preparation ,,of ,,haemolytic ,,serum4 ,apparatus ,required3 ,small centrifuge1 preferably electrically driven1 with two receptacles for tubes1 and enclosed in a safety shield 7,fig4 #afb74 ,sterile centrifuge tubes 7#aj c4c4 capacity71 ,fig4 #afc4 ,sterile pipettes 7#aj c4c4 graduated7 in case4 ,sterile glass capsules 7in case74 ,sterile test-tubes4 ,sterile all glass syringe 7#e c4c4 or #aj c4c4 capacity7 and needle4 ,illustration3 ,,fig4 #afb4--,small electrical centrifuge4 ,illustration3 ,,fig4 #afc4--;,centrifuge tube4 ,reagents ,required3 ,normal saline solution4 #aj per cent4 sodium citrate solution in normal saline4 ,human blood 7vide infra74 ,,method4-- #a4 ,select a healthy full-grown rabbit of not less than #bejj grammes weight in accordance with the directions already given 7page #cbb7 and prepare it for intraperitoneal inoculation4 #b4 ,measure out #b c4c4 citrated human blood 7collected at a surgical operation or a venesection1 or withdrawn by venipuncture from the median basilic or median cephalic vein of a normal adult7 into a centrifuge tube and centrifugalise thoroughly4 #c4 ,wash with three changes of normal saline 7vide also page #chh74 #d4 ,transfer the washed cells to a sterile capsule by means of a sterile pipette4 ,add #e c4c4 of normal saline and mix thoroughly4 #e4 ,take up the mixture of cells and saline in the all-glass syringe and inject into the peritoneal cavity of the rabbit4 #f4 ,seven days later inject intraperitoneally the washed cells from #e c4c4 human blood mixed with #e c4c4 normal saline4 #g4 ,seven days later inject the washed cells from #aj c4c4 human blood mixed with #e c4c4 normal saline4 #h4 ,after a further interval of seven days repeat the injection of washed cells from #aj c4c4 human blood mixed with #e c4c4 normal saline4 ,,note4--,better results are obtained if the second and subsequent injections are made intravenously1 even when smaller quantities of washed red cells are employed4 ,if1 however1 the intravenous route is selected exceeding great care must be exercised to avoid the introduction of air into the vein--an accident which is followed1 within a few minutes1 by the death of the rabbit from pulmonary embolism4 #i4 ,allow five days to elapse1 then collect a preliminary sample of blood1 say about #b c4c41 from the rabbit's ear4 ,allow it to clot1 separate off the serum and transfer to a sterile test-tube4 ,place the test-tube in a water-bath at #ef deg4 ,c4 for fifteen minutes 7to inactivate7 and test the serum quantitatively for haemolytic properties in the following manner3 ,,the ,,titration ,,of ,,haemolytic ,,serum4 ,apparatus ,required3 ,electrical centrifuge4 ,sterile centrifuge tubes4 ,water-bath regulated at #ef deg4 ,c4 ,sterilised pipettes #aj c4c4 graduated in tenths4 ,sterilised pipettes #a c4c4 graduated in tenths4 ,sterile test-tubes1 #af x #b cm4 ,small sterile test-tubes1 #i x #a cm4 ,small test-tube rack1 or roll of plasticine4 ,capillary teat pipettes4 ,stout rubber band or length of small rubber tubing4 ,reagents ,required and ,method of ,preparation3 #a4 ,normal saline solution4 #b4 ,haemolytic serum inactivated by preliminary heating to #ef deg4 ,c4 for #ae minutes 7vide supra7 in test-tube labelled ,h4 ,s4 #c4 ,complement4 ,fresh guinea-pig serum in test-tube labelled ,c4 ,kill a normal guinea-pig with chloroform vapour4 ,open the thorax with all aseptic precautions1 and collect as much blood as possible from the heart with a sterile ,pasteur pipette4 ,transfer it to a sterile centrifuge tube and place the tube in the incubator at #cg deg4 ,c4 ,two hours later separate the clot from the sides of the tube1 and centrifugalise thoroughly4 ,pipette off the clear serum to a clean sterilised test-tube4 #d4 ,erythrocyte solution1 in test-tube labelled ,e4 ,collect and wash human red blood cells 7see page #chh1 #a-h74 ,measure the volume of red cells available and prepare a #b per cent4 suspension in normal saline solution4 ,,method4-- #a4 ,take two test-tubes and number them #a and #b1 and pipette into each #i c4c4 of normal saline solution4 #b4 ,add #a c4c4 of haemolytic rabbit serum to tube ,no4 #a and mix thoroughly3 take up #a c4c4 of the mixture and add it to tube ,no4 #b2 mix thoroughly4 #c4 ,set up ten small test-tubes in test-tube rack or in roll of plasticine1 and number #a to #aj4 #d4 ,pipette into tube ,no4 #a #j4e c4c4 #j4e c4c4 haemolytic serum ,from tube ,pipette into tube ,no4 #b #j4a c4c4 #j4a c4c4 ,h4 ,s4 haemolytic serum ,pipette into tube ,no4 #c #j4e c4c4 #j4je c4c4 haemolytic serum ,pipette into tube ,no4 #d #j4c c4c4 #j4jc c4c4 haemolytic serum ,from ,pipette into tube ,no4 #e #j4b c4c4 #j4jb c4c4 tube #a4 haemolytic serum pipette into tube ,no4 #f #j4a c4c4 #j4ja c4c4 haemolytic serum ,pipette into tube ,no4 #g #j4e c4c4 #j4jje c4c4 haemolytic serum ,pipette into tube ,no4 #h #j4c c4c4 #j4jjc c4c4 haemolytic serum ,from ,pipette into tube ,no4 #i #j4b c4c4 #j4jjb c4c4 tube #b4 haemolytic serum ,pipette into tube ,no4 #aj #j4a c4c4 #j4jja c4c4 haemolytic serum #e4 ,to each tube add #a c4c4 of erythrocyte solution4 #f4 ,when necessary 7that is to say in tubes #b1 #d1 #e1 #f1 #h1 #i and #aj7 add normal saline solution to the mixture in the test-tubes till the column of fluid in each reaches to the same level4 #g4 ,shake each tube in turn1 so as to thoroughly mix its contents4 ,plug the mouth of each tube with cotton wool1 and place entire set in the incubator at #cg deg4 ,c4 for one hour4 #h4 ,remove the tubes from the incubator and into each tube pipette #j4a c4c4 complement 7guinea-pig's serum7 and replace tubes in incubator at #cg deg4 ,c4 for further period of one hour4 #i4 ,remove the tubes from the incubator1 and if complete haemolysis has not taken place in every tube1 stand on one side1 preferably in the ice chest1 for an hour4 #aj4 ,then examine the tubes4 ,complete haemolysis is indicated by a clear red solution1 with no deposit of red cells at the bottom of the test-tube4 ,absence of haemolysis is indicated by a clear or turbid colourless fluid1 with a deposit of red cells at the bottom of the test-tubes4 ,the smallest amount of haemolytic serum that has caused complete haemolysis is known as the minimal haemolytic dose 7,m4 ,h4 ,d47 and if haemolysis has occurred in all the tubes down to ,no4 #g--the m4 h4 d4 of this particular serum is 4#jje c4c4 #bjj minimal haemolytic doses per cubic centimetre4 ,such a serum is strong enough for experimental work2 indeed1 for many purposes1 complete haemolysis down to tube #f will indicate a serum sufficiently strong7 #ajj m4 h4 d4 per cubic centimetre74 ,if1 however1 only the first one or two tubes are completely haemolysed1 this is an indication that the rabbit should receive further injections in order to raise the haemolytic power to a sufficiently high level4 ,,storage ,,of ,,haemolysin4 ,if1 and when the haemolysin content of the rabbit's serum is found to be sufficient1 destroy the animal by chloroform vapour4 ,remove as much of its blood as possible from the heart under aseptic precautions into sterilized centrifuge tubes4 ,transfer the tubes of blood to the incubator at #cg deg4 ,c4 for two hours--then centrifugalize thoroughly4 ,pipette off the clear serum1 and fill in quantities of #a c4c41 into small glass ampoules or pipettes1 and hermetically seal in the blowpipe flame1 care being taken to avoid scorching the serum4 ,place the ampoules when filled with serum and sealed1 in a water-bath at #ef deg4 ,c4 for #cj minutes4 ,this destroys the complement1 i4 e41 inactivates the serum1 and at the same time1 provided the various operations have been carried out under aseptic precautions1 ensures its sterility4 ,a longer exposure reduces the haemolytic power4 ,place the ampoules in a closed metal box and store in the ice chest for future use4 ,,footnotes3 #aj ,the quantities here given are not absolutely correct4 ,if exactitude is essential the student must calculate the amount required by the aid of the ,percentage ,formula1 ,appendix1 page #dif4 #aa ,see ,percentage ,formula1 ,appendix1 page #dif4 ,,xvii4 ,,experimental ,,inoculation ,,of ,,animals4 ,the use of living animals for inoculation experiments may become a necessary procedure in the ,bacteriological ,laboratory for some one or more of the following reasons3 ,a4 ,determination of ,pathogenetic ,properties of ,bacteria already ,isolated in ,pure ,culture 7see page #cae74 ,the exact study of the conditions influencing the virulence 7including its maintenance1 exaltation and attenuation7 of an organism1 and precise observations upon the pathogenic effects produced by its entrance into1 and multiplication within the body tissues can obviously only be carried out by means of experimental inoculation2 whilst many points relating to vitality1 longevity1 etc41 can be most readily elucidated by such experiments4 ,b4 ,isolation of ,pathogenetic ,bacteria4 ,certain highly parasitic bacteria 7which grow with difficulty upon the artificial media of the laboratory7 can only be isolated with considerable difficulty from associated saprophytic bacteria when cultural methods alone are employed2 but if the mixture of parasite and saprophytes is injected into an animal susceptible to the action of the former1 the pathogenic organism can readily be isolated from the tissues of the infected animal4 ,the pneumococcus for example occurs in the sputum of patients suffering from acute lobar pneumonia1 but usually in association with various saprophytes derived from the mouth and pharynx4 ,the optimum medium for the growth of the pneumococcus1 blood agar1 is also an excellent pabulum for the saprophytes of the mouth1 and plate cultures are rapidly overgrown by them to the destruction of the more delicate pneumococcus4 ,but inoculate some of the sputum under the skin of a mouse and three or four days later the pneumococcus will have entered the blood stream 7leaving the saprophytes at the seat of inoculation7 and killed the animal4 ,cultivations made at the post-mortem 7see page #cih7 from the mouse's heart blood will yield a pure growth of the pneumococcus4 ,c4 ,identification of ,pathogenetic ,bacteria4 ,the resemblances1 morphological and cultural1 existing between certain pathogenetic bacteria are in some cases so great as to completely overwhelm the differences2 again the same bacterium may under varying conditions assume appearances so different from those regarded as typical or normal as to throw doubt on its identity4 ,in each case a simple inoculation experiment may decide the point at once4 ,as a concrete example may be instanced an autopsy on an animal dead from an unknown infection4 ,cultivations from the heart blood gave a pure growth of a typical 7capsulated7 pneumococcus4 ,cultivations from the liver gave a pure growth of what appeared to be a typical 7non-capsulated7 ,streptococcus pyogenes longus4 ,the latter inoculated into a rabbit caused the death of the animal from pneumococcic septicaemia1 and cultures from the rabbit's blood gave a pure growth of a typical 7capsulated7 pneumococcus4 ,d4 ,study of the ,problems of ,immunity4 ,it is only by a careful and elaborate study of the behaviour of the animal cell and the body fluids vis-a-vis with the infecting bacterium that it becomes possible to throw light upon the complex problem whereby the cell opposes successful resistance to the diffusion of the invading microbe1 or succeeds in driving out the microbe subsequently to the occurrence of that diffusion4 ,at the moment1 however1 our attention is directed to the first of these broad headings1 for it is by the application of the knowledge acquired in its pursuit that we are able to deal with problems arising under any of the remainder4 ,for whatever purpose the inoculation is performed1 it is essential that the experiment should be planned to secure the maximum amount of information and the minimum of discomfort to the animal used4 ,every care therefore must be taken to ensure that the virus is introduced into the exact tissue or organ selected2 and the operation itself must be carried out with skill and expedition1 and under strictly aseptic conditions4 ,in the course of inoculation studies many instances of natural immunity1 both racial and individual1 will be met with2 but it must be recollected that natural immunity is relative only and never absolute1 and care be taken not to label an organism as non-pathogenic until many different methods of inoculation have been performed upon different species of animals1 combined when necessary with various procedures calculated to overcome any apparent immunity1 and have invariably given negative results4 ,in some countries experiments upon animals are only permitted under direct license from the ,government1 and then only within premises specially licensed for the purpose4 ,in ,england this license is in the grant of the ,home ,secretary1 and confers the permission to experiment upon animals under general anaesthesia1 provided that after the experiment is completed the animal must be destroyed before regaining consciousness4 ,if it is intended to carry out simple hypodermic inoculations and superficial venesections1 ,certificate ,a1 granting this specific permission and dispensing with the necessity for general anaesthesia must be obtained in addition to the license2 whilst if the inoculation entails more extensive operative procedures1 and it is necessary to observe the subsequent course of the infection1 should such occur1 the license must be coupled with ,certificate ,b--since this certificate removes the compulsion to destroy the animal whilst under the anaesthetic4 ,further special certificates and combinations of certificates are required if cats1 dogs1 horses1 asses or cattle are to be the subjects of experiment4 ,under every certificate it is expressly stipulated that if the animal shows signs of pain it must be destroyed immediately4 ,the animals generally employed in the study of the pathogenic properties of the various micro-organisms are3 ,cold ,blooded4 ,warm ,blooded4 ,hot ,blooded4 ,frog4 ,mouse4 ,fowl4 ,toad4 ,rat4 ,pigeon4 ,lizard4 ,guinea pig4 ,rabbit4 ,monkey4 ,preparation4--,before inoculation1 the experimental animals should be carefully examined1 to avoid the risk of employing such as are already diseased3 since it must be remembered that in a state of nature1 as well as in captivity1 the animals employed for laboratory inoculations are subject to infection by various animal and vegetable parasites1 and in some instances such infection presents no symptoms which are obvious to the casual examination2 the sex should be noted1 the weight recorded1 and the rectal temperature taken4 ,the remaining items of importance are the time of the inoculation1 the material that is inoculated1 and the method of inoculation1 and finally under what authority the experiment is performed4 ,in the author's laboratory these data are entered upon a pink card which forms part of a card index system4 ,the card further provides space for notes on the course of the resulting infection1 and carries on the reverse the weight and temperature chart 7,figs4 #afd and #afe74 ,illustration3 ,fig4 #afd4--;,front of inoculation card4 ,preliminary ,inspection and ,examination4--,the preliminary examination should comprise observation of the animal at rest and in motion2 the appearance of the fur1 feathers or scales1 inspection of the eyes1 and of external orifices of the body2 tactile examination of the body and limbs1 and palpation of the groins and abdomen2 and in many cases the microscopical examination of fresh and stained blood-films4 ,some of the commoner forms of naturally acquired infection may be briefly mentioned1 without however touching upon the various fleas1 lice and ticks which at times infect the ordinary laboratory animals4 ,illustration3 ,,fig4 #afe4--;,back of inoculation card4 ,the ,rabbit1 particularly in captivity1 is subject to attacks of ,psoric ,acari1 and the infection is readily transmitted to rabbits in neighbouring cages and also to guinea pigs1 but not to rats and mice4 ,one species 7,sarcoptes minor var4 cuniculi7 gives rise to the ordinary mange4 ,the infection first shows itself as thick yellowish scales and crusts around the nose1 mouth and eyes1 spreads to the bases and outer surfaces of the ears 7never to the inside of the concha71 to the fore and hind legs and into the groins and around the genitals4 ,the acari can be readily demonstrated microscopically in scrapings of the skin1 treated with liquor potassae4 ,another form of scabies 7due to ,psoroptes communis cuniculi7 commences at the bottom of the concha1 which is filled with whitish-yellow masses consisting of dried crusts1 scales1 faeces1 and dead acari4 ,the base of the ear is hard and swollen1 and lifting the animal by the ears--as is usually done--gives rise to considerable pain2 indeed this symptom may be the one which first attracts attention to an infection1 which causes progressive wasting and terminates in death4 ,a mixed infection--sarcoptic plus psorotic acariasis--is sometimes seen4 ,if it is decided to try and save animals suffering from infection by these parasites1 they must be segregated1 the scabs carefully cleaned from the infected areas and the denuded surfaces washed with #e per cent4 solution of ,potassium persulphate 7a few drops being allowed to run into the concha71 or with a preparation containing equal parts of soft paraffin and vaseline with a few drops of lysol4 ,this treatment should be repeated daily until the acarus is destroyed and the animal has regained its normal condition4 ,the cages should be disinfected and all neighbouring animals carefully examined1 and any which show signs of infection should be treated in a similar manner4 ,favus also attacks the rabbit1 and the typical spots are first noted around the base of the ear4 ,infection by ,coccidium oviforme is very common1 without however presenting any symptoms by which the infection may be recognised4 ,usually the condition is only noted post-mortem1 when the liver is found to be studded with numerous cascating tubercles1 which on examination prove to be cystic areas crowded with coccidia4 ,sometimes too the liver of a rabbit dead from some intentional or accidental bacterial infection is found at the post-mortem to be marked by fine yellowish streaks and small tubercles due to the embryos of ,taenia serrata1 while the cystic form 7,cysticercus pisiformis7 is often noted free in the peritoneal cavity1 or invading the mesentery4 ,abscess formation from infection with ordinary pyogenic bacteria occurs naturally in the rabbit1 and frequently the animal house of a laboratory is decimated by an infective septicaemia due to ,b4 cuniculicida4 ,the ,mouse and ,rat suffer from septicaemia1 and from the cysticercus form of ,taenia murina2 the cystic form 7,cysticercus fasciolaris7 of ,t4 crassicollis has its habitat in their livers4 ,these small rodents are frequently infected with scabies1 but if freely provided with clean straw will clean themselves by rubbing through it4 ,the mouse is also attacked by favus1 and the rat is often infected with ,trypanosoma ,lewisi4 ,the ,guinea pig1 like the rabbit1 suffers from scabies and coccidiosis4 ,in addition it is often naturally infected with ,b4 tuberculosis1 and it is a wise precaution to test animals as soon as they reach the laboratory by injecting ,koch's ,old ,tuberculin--#j4e c4c4 causing death in the tuberculous cavy within #dh hours4 ,the ,monkey is naturally prone to tuberculosis1 and should be injected with #a c4c4 ,old ,tuberculin on arrival in the laboratory4 ,the tissues of the monkey also serve as the habitat for a ,nematode worm parasitic in cattle 7,oesophagostoma inflatum7 resembling the ,anchylostomum1 and this parasite frequently bores through the intestinal wall1 and provokes the formation of small cysts in the immediately adjacent mesentery4 ,the presence of these cysts may give rise to considerable speculation at the post-mortem4 ,the ,pigeon may be infected by ,haemosporidia1 and its blood show the presence of halteridia4 ,this bird may also be the subject of a bacterial infection known as pigeon diphtheria2 while the fowl may be subject to scabies and ringworm1 or suffer from fowl cholera or fowl septicaemia--infections due to members of the haemorrhagic septicaemia group4 ,weighing4--,the larger animals are most conveniently weighed in a decimal scale provided with a metal cage for their reception instead of the ordinary pan 7,fig4 #aff74 ,mice and rats are weighed in a modification of the letter balance1 weighing to #bej grammes1 which has a conical wire cage1 7carefully counterpoised7 substituted for its original pan 7,fig4 #afg74 ,illustration3 ,,fig4 #aff4--,rabbit scales4 ,temperature4--,to take the rectal temperature of any of the laboratory animals1 the animal should be carefully and firmly held by an assistant4 ,introduce the bulb of an ordinary clinical thermometer1 well greased with vaseline1 just within the sphincter ani4 ,allow it to remain in this position for a few seconds1 and then push it on gently and steadily until the entire bulb and part of the stem1 as far as the constriction1 have passed into the rectum4 ,three to five minutes later1 the time varying of course with the sensibility of the thermometer used1 withdraw the instrument and take the reading4 ,the thermometers employed for recording temperature should be verified from time to time by comparison with a standard ,kew certified ,thermometer kept in the laboratory for that purpose4 ,illustration3 ,,fig4 #afg4--,mouse scales ,cages4--,during the period which elapses between inoculation and death1 or complete recovery1 the experimental animals must be kept in suitable receptacles which can easily be kept clean and readily disinfected4 ,the mouse is usually stored in a glass jar 7,fig4 #afh7 #aa cm4 high and #aa cm4 in diameter1 closed by a wire gauze cover which is weighted with lead or fastened to the mouth of the jar by a bayonet catch4 ,a small oblong label1 #e cm4 by #b4e cm41 sand-blasted on the side of the cylinder1 is a very convenient device as notes made upon this with an ordinary lead pencil show up well and only require the use of a damp cloth to remove them 7,fig4 #afh74 ,the rat is kept under observation in a glass jar similar1 but larger1 to that used for the mouse4 ,illustration3 ,,fig4 #afh4--,mouse jar4 ,illustration3 ,,fig4 #afi4--,tripod4 ,a layer of sawdust at the bottom of the jar absorbs any moisture and cotton-wool or paper shavings should be provided for bedding4 ,the food should consist of bran and oats with an occasional feed of bread-and-milk sop4 ,the use of a metal tripod1 on the platform of which are soldered two small cups for the reception of the food1 inside the cage1 prevents waste of food or its contamination with excreta 7,fig4 #afi74 ,after use the jars and tripods are sterilised either by chemical reagents or by autoclaving4 ,the rabbit and the guinea-pig are confined in cages of suitable size1 made entirely of metal 7,fig4 #agj74 ,the sides and top and bottom are of woven wire work2 beneath the cage is a movable metal tray filled with sawdust1 for the reception of the excreta4 ,the cage as a whole is raised from the ground on short legs4 ,the sides1 etc41 are generally hinged so that the cage packs up flat1 for convenience of storing and also of sterilising4 ,the ordinary rat cage1 a rectangular wire-work box1 #cj cm4 from front to back1 #bj cm4 wide1 and #ad cm4 high1 makes an excellent cage for guinea-pigs if fitted with a shallow zinc tray1 #ce cm4 by #bd cm41 for it to stand upon4 ,illustration3 ,,fig4 #agj4--,metal rabbit rage4 ,a plentiful supply of straw should be provided for bedding and the food should consist of fresh vegetables1 cabbage leaves1 carrot and turnip tops and the like for the morning meal and broken animal biscuits for the evening meal4 ,occasionally a little water may be placed in the cage in an earthenware dish4 ,the tray which receives the dejecta should be cleaned out and supplied with fresh sawdust each day1 and the soiled sawdust1 remains of food1 etc41 should be cremated4 ,these cages are sterilised after use either by autoclaving or spraying with formalin4 ,as animal inoculation is purely a surgical operation1 the necessary instruments will be similar to those employed by the surgeon1 and1 like them1 must be sterile4 ,in the performance of the inoculation strict attention must be paid to asepsis1 and suitable precautions adopted to guard against accidental contamination of the material to be introduced into the animal4 ,in addition1 the hands of the operator should be carefully disinfected4 ,the list of apparatus used in animal inoculations given below comprises practically everything needed for any inoculation4 ,needless to remark1 all the apparatus will never be required for any one inoculation4 ,illustration3 ,,fig4 #aga4--;,hypodermic syringe with finger rests4 ,apparatus ,required for ,animal ,inoculation3 #a4 ,water steriliser 7vide page #cc74 ,it is also convenient to have a second water steriliser1 similar but smaller 7#bc by #g by #e cm471 for the sterilisation of the syringes4 #b4 ,injection syringe4 ,the best form is one of the ordinary hypodermic pattern1 #a c4c4 capacity graduated in twentieths of a cubic centimeter 7#j4je c4c471 fitted with finger rests1 but with the leather washers and the packing of the piston replaced by those made of asbestos 7,fig4 #aga74 ,the instrument must be easily taken to pieces1 and spare parts should be kept on hand to replace accidental breakage or loss4 ,other useful syringes are those of #b c4c41 #e c4c41 #aj c4c41 and #bj c4c4 capacity4 ,a good supply of needles must be kept on hand1 both sharp-pointed and with blunt ends4 ,to sterilise the syringe1 fill it with water1 loosen the packing of the piston and all the screw joints1 place it in the steriliser and boil for at least five minutes4 ,disinfect the syringe after use1 in a similar manner4 ,the needles1 which are exceedingly apt to rust after being boiled1 should be stored in a pot of absolute alcohol when not in use4 #c4 ,operating table4 #d4 ,surgical instruments4 ,sterilise these before use by boiling1 and disinfect them after use by the same means4 ,wipe perfectly dry immediately after the disinfection is completed4 ,scissors1 probe and sharp-pointed4 ,dissecting forceps of various patterns4 ,pressure forceps4 ,retractors 7small self retaining ,fig4 #agb74 ,aneurism needles1 sharp and blunt4 ,scalpels1 ,keratomes1 with metal handles4 ,trephines1 ,michel's steel clips and special forceps for applying the same4 ,these small steel clips enable the operator to easily and rapidly close skin incisions and are most satisfactory for animal operations4 ,surgical needles4 ,needle holder4 ,soft rubber catheters1 various sizes4 ,gum elastic oesophageal bougies with connection to fit syringe4 ,illustration3 ,,fig4 #agb4 ,small self retaining retractors4 #e4 ,anaesthetic4 7a7 ,general3 ,the safest general anaesthetic for animals is an ,a4 ,c4 ,e4 mixture1 freshly prepared1 containing by volume alcohol #a part1 chloroform #b parts1 ether #f parts1 and should be administered on a cone formed by twisting up one corner of a towel and placing a wad of cotton-wool inside it1 or from a saturated cotton-wool pad packed into the bottom of a small beaker4 7b7 ,local3 #a4 ,cocaine hydrochloride1 #b per cent4 in adrenalin #a per mille solution4 #b4 ,beta-eucaine1 #b per cent4 in adrenalin1 #a per mille solution4 #c4 ,ethyl chloride jet4 #f4 ,sterile glass capsules of various sizes4 #g4 ,cases of sterile pipettes #aj c4c4 7in tenths of a cubic centimetre74 #a c4c4 7in hundredths of a cubic centimetre74 #h4 ,flasks 7#ge c4c47 containing sterilised normal saline solution 7or sterile bouillon74 #i4 ,sterilised cotton-wool4 ,cotton-wool 7absorbent7 is packed loosely in a copper cylinder similar to that used for storing capsules1 and sterilised in the hot-air oven4 #aj4 ,sterilised gauze4 ,gauze is sterilised in the same way as cotton-wool4 #aa4 ,sterilised silk and catgut for sutures4 ,these are sterilised1 as required1 by boiling for some ten minutes in the water steriliser4 #ab4 ,flexible collodion 7or compound tincture of benzoin74 #ac4 ,grease pencil4 #ad4 ,tie-on celluloid labels1 to affix to the cages4 #ae4 ,razor4 #af4 ,small pot of warm water4 #ag4 ,liquid soap4 ,liquid soap is prepared as follows3 ,measure out #ajj grammes of soft soap and add to #ejj c4c4 of #b per cent4 lysol solution in a large glass beaker2 dissolve by heating in a water-bath at about #ij deg4 ,c4 ,bottle and label ,liquid ,soap4 #ah4 ,in place of the liquid soap and razor it is sometimes convenient to use a ,depilatory powder4 ,barium sulphide #a part ,rice starch #c parts ,dust the powder thickly over the area to be denuded of hair1 sprinkle with water and mix into a thin paste in situ2 allow the paste to act for three minutes1 then scrape off with a bone spatula--the hair comes away with the paste and leaves a perfectly bare patch4 ,this process is preferably carried out1 the day previous to the operation4 ,material ,utilised for ,inoculation4--,the material inoculated may be either-- #a4 ,cultures of bacteria--grown in fluid media1 or on solid media4 #b4 ,metabolic products of bacterial activity--e4 g41 toxins in solution4 #c4 ,pathological products 7fluid secretions and excretions1 solid tissues74 ,the ,preparation of the ,inoculum4-- 7a7 ,cultivations in ,fluid ,media4-- #a4 ,flame the plug of the culture tube4 #b4 ,remove the plug and flame the mouth of the tube4 #c4 ,slightly raise the lid of a sterile capsule1 insert the mouth of the culture tube into the aperture and pour some of the cultivation into the capsule4 #d4 ,remove the mouth of the culture tube from the capsule1 replace the lid of the latter1 flame the mouth of the tube1 and replug4 #e4 ,remove the syringe from the steriliser1 squirt out the water from its interior1 and allow to cool4 #f4 ,raise the lid of the capsule sufficiently to admit the needle of the syringe and draw the required amount of the cultivation into the barrel of the syringe4 7,or1 remove a definite measured quantity of the cultivation directly from the tube or flask by means of a sterile graduated pipette1 discharge the measured amount into a sterile capsule1 and fill into the syringe2 or take up the required quantity of the cultivation directly into the graduated syringe from the tube or flask47 ,illustration3 ,,fig4 #agc4--;,conical separatory funnel1 fitted for injection of fluid cultivations4 ,if it is necessary to introduce a large bulk of fluid into the animal1 the cultivation should be transferred with aseptic precautions1 to a sterile separatory funnel1 preferably of the shape shown in figure #agc1 and graduated if necessary4 ,this is supported on a retort stand and raised sufficiently above the level of the animal to be injected1 so as to secure a good fall4 ,a piece of sterilised rubber tubing of suitable length1 fitted with an injection needle and provided with a screw clamp1 is now attached to the nozzle of the funnel and the operation completed according to the requirements of the particular case4 ,this method is quite satisfactory when the injection is made into the pleural or abdominal cavities or directly into a vein but if the injection has to be made into the subcutaneous tissue the fall may not be sufficient to force the fluid in4 ,in this case it will be necessary to transfer the culture to a sterile wash-bottle and fasten a rubber hand bellows to the air inlet tube 7interposing an air filter7 and attach the tubing with the injection needle to the outlet tube 7,fig4 #agd74 ,by careful use sufficient force can be obtained to drive the injection in4 7b7 ,cultivations on ,solid ,media 7e4 g41 ,sloped ,agar74-- #a4 ,by means of a sterile graduated pipette introduce a suitable small quantity of sterile bouillon 7or sterile normal saline solution7 into the culture tube4 ,illustration3 ,,fig4 #agd4--;,arrangement of pressure injection apparatus4 #b4 ,with a sterile platinum loop or spatula scrape the bacterial growth off the surface of the medium1 and emulsify it with the bouillon4 ,it then becomes to all intents and purposes a fluid inoculum4 #c4 ,pour the emulsion into a sterile capsule and fill the syringe therefrom4 7c7 ,toxins4--,prepared by previously described methods 7vide page #cah71 are manipulated in a similar manner to cultivations in fluid media4 7d7 ,pathological ,products4--,fluid secretions1 excretions1 etc41 such as serous exudation1 pus1 blood1 etc41 are treated as fluid cultivations2 but if the material is very thick or viscous1 a small quantity of sterile bouillon or normal saline solution may be used to dilute it1 and thorough incorporation effected by the help of a sterile platinum rod4 ,solid tissues1 such as spleen1 lymph glands1 etc41 may be divided into small pieces by sterile instruments and rubbed up in a sterilised agate mortar 7using an agate pestle71 with a small quantity of sterile bouillon1 and the syringe filled from the resulting emulsion4 ,illustration3 ,,fig4 #age4--;,holding rabbit for shaving4 ,if it is desired to inoculate tissue en masse1 remove from the material a small cube of #a or #b mm4 and introduce it into a wound made by sterile instruments in a suitable situation1 and occlude the wound by means of ,michel's steel clips and a sealed dressing4 ,method of ,securing ,animals ,during ,inoculation4-- ,for the majority of inoculations1 especially when no anaesthetic is administered1 it is customary to employ an assistant to hold the animal 7see ,fig4 #age74 ,if working single handed ,voge's holder for guinea-pigs1 is a useful piece of apparatus the method of using which is readily seen from the accompanying figures 7,figs4 #agf1 #agg74 ,the instrument itself consists of a hollow copper cylinder1 one end of which is turned over a ring of stout copper wire1 and from this open end a slot is cut extending about half way along one side of the cylinder4 ,the opposite end is closed by a pull-off cap and is perforated around its edge by a row of ventilating holes1 which correspond with holes cut in the rim of the cap4 ,in the event of the animal resisting attempts to remove it from the holder backwards1 this cap is taken off and the holder placed on the table and the guinea-pig allowed to walk out4 ,illustration3 ,,fig4 #agf4--,taking guinea-pig's temperature4 ,to provide for different-sized animals1 two sizes of this holder will be found useful3 #a4 ,length1 #af cm42 breadth1 #f cm42 size of slot1 #h cm4 by #b4e cm4 #b4 ,length1 #bj cm42 breadth1 #h cm42 size of slot1 #aj cm4 by #b4e cm4 ,a convenient holder for mice and even small rats is shown in figure #agh1 the tail being securely held by the spring clip4 ,needless to say1 the holder should be entirely of metal1 and the wire cage detachable and easily renewed4 ,illustration3 ,,fig4 #agg4--,voge's holder4 ,when the animal is anaesthetised1 it is more convenient to secure it firmly to some simple form of operating table1 such as ,tatin's 7,fig4 #agi71 which will accommodate rabbits1 guinea-pigs1 and rats3 or to the more elaborate table devised by the author 7,fig4 #ahj74 ,illustration3 ,,fig4 #agh4--,mouse holder4 ,illustration3 ,,fig4 #agi4--,tatin's operation table4 ,operation ,table4--,this is a table of the aseptic type1 composed of steel tubing1 nickel-plated or enamelled4 ,the table-top frame is sufficiently large to accommodate rabbits1 dogs and monkeys2 and is supported upon telescopic uprights1 so that it is adjustable as to height2 in its long axis it can be inclined 7at either end7 to #de deg4 from the horizontal4 ,further it can be completely rotated about its long axis4 ,the table-top itself is composed of a sheet of copper wire gauze loosely suspended from the long sides of the tubular frame4 ,the slackness of the gauze bed permits of an india rubber hot water bottle1 or an electrotherm being placed under the animal1 and if during the course of an experiment it is necessary to reverse the animal1 the table-top frame is completely rotated1 the device adopted for suspending the gauze is detached and the gauze reversed also1 so that it again supports the animal from below4 ,illustration3 ,,fig4 #ahj4--;,author's operating table#ab4 ,,methods ,,of ,,inoculation4 ,the following methods of inoculation apply more particularly to the rabbit1 but from them it will readily be seen what modifications in technique1 if any1 are necessary in the case of the other experimental animals4 #a4 ,cutaneous ,inoculation4--7,anaesthetic1 none47 #a4 ,have the animal firmly held by an assistant 7or secured to the operating table74 #b4 ,apply the liquid soap to the fur1 over the area selected for inoculation1 with a wad of cotton-wool1 and lather freely by the aid of warm water2 shave carefully and thoroughly2 or apply the depilatory powder4 #c4 ,wash the denuded area of skin thoroughly with #b per cent4 lysol solution4 #d4 ,wash off the lysol with ether and allow the latter to evaporate4 #e4 ,make numerous short1 parallel1 superficial incisions with the point of a sterile scalpel4 #f4 ,when the oozing from the incisions has ceased1 rub the inoculum into the scarifications by means of the flat of a scalpel blade1 or a sterile platinum spatula4 #g4 ,cover the inoculated area with a pad of sterile gauze secured in situ by strips of adhesive plaster or by sealing down the edges of the gauze with collodion4 #h4 ,release the animal1 place it in its cage1 and affix a label upon which is written3 7a7 ,distinctive name or number of the animal4 7b7 ,its weight4 7c7 ,particulars as to source and dose of inoculum4 7d7 ,date of inoculation4 #b4 ,subcutaneous ,inoculation4-- 7a7 ,fluid ,inoculum4--7,anaesthetic1 none47 ,steps #a-d4 ,as for cutaneous inoculation4 #e4 ,pinch up a fold of skin between the forefinger and thumb of the left hand2 take the charged hypodermic syringe in the right hand1 enter the needle into a ridge of skin raised by the left finger and thumb1 and push it steadily onward until about #b cm4 of the needle are lying in the subcutaneous tissue4 ,now release the grasp of the left hand and slowly inject the fluid contained in the syringe4 #f4 ,withdraw the needle1 and at the same moment close the puncture with a wad of cotton wool1 to prevent the escape of any of the inoculum4 ,the injected fluid1 unless large in amount1 will be absorbed within a very short time4 #g4 ,label1 etc4 7b7 ,solid ,inoculum4--7,anaesthetic1 none2 or ,ethyl chloride spray47 ,steps #a-d4 ,as for cutaneous inoculation4 #e4 ,raise a small fold of skin in a pair of forceps1 and make a small incision through the skin with a pair of sharp-pointed scissors or with the point of a scalpel4 #f4 ,insert a probe through the opening and push it steadily onward in the subcutaneous tissue1 and by lateral movements separate the skin from the underlying muscles to form a funnel-shaped pocket with its apex toward the point of entrance4 #g4 ,by means of a pair of fine-pointed forceps introduce a small piece of the inoculum into this pocket and deposit it as far as possible from the point of entrance4 ,illustration3 ,,fig4 #aha4--;,glass tube syringe for subcutaneous solid inoculation4 ,or1 improvise a syringe by sliding a piece of glass rod 7to serve as a piston7 into the lumen of a slightly shorter length of glass tubing and secure in position by a band of rubber tubing4 ,sterilise by boiling4 ,withdraw the rod a few millimetres and deposit the piece of tissue within the orifice of the tube1 by means of sterile forceps4 ,now pass the tube into the depths of the pocket1 push on the glass rod till it projects beyond the end of the tube1 and withdraw the apparatus1 leaving the tissue behind in the wound4 #h4 ,close the wound in the skin with ,michel's clips and a dressing of gauze sealed with collodion 7or ,tinct4 benzoin74 #i4 ,label1 etc4 #c4 ,intramuscular4-- 7a7 ,fluid ,inoculum4--7,anaesthetic1 none47 ,steps #a-d4 ,as for cutaneous inoculation4 #e4 ,steady the skin over the selected muscle or muscles with the slightly separated left forefinger and thumb4 #f4 ,thrust the needle of the injecting syringe boldly into the muscular tissue and inject the inoculum slowly4 #g4 ,label1 etc4 7b7 ,solid ,inoculum4--7,anaesthetic1 ,a4 ,c4 ,e47 #a4 ,secure the animal to the operation table and anaesthetise4 #b4 ,shave and disinfect the skin at the seat of operation4 #c4 ,surround the field of operation by strips of gauze wrung out in #b per cent4 lysol solution4 #d4 ,incise skin1 aponeurosis1 and muscle in turn4 #e4 ,deposit the inoculum in the depths of the incision4 #f4 ,close the wound in the muscle with buried sutures and the cutaneous wound with either continuous or interrupted sutures or with ,michel's steel clips4 #g4 ,apply a sealed dressing of gauze and collodion4 #h4 ,remove the animal from the operating table4 #i4 ,label1 etc4 #d4 ,intraperitoneal4-- 7a7 ,fluid ,inoculum4--7,anaesthetic1 none47 ,steps #a-d4 ,as for cutaneous inoculation4 ,shave a fairly broad transverse area1 stretching from flank to flank4 #e4 ,place the left forefinger on one flank and the thumb on the opposite1 and pinch up the entire thickness of the abdominal parietes in a triangular fold4 ,now1 by slipping the peritoneal surfaces 7which are in apposition7 one over the other1 ascertain that no coils of intestine are included in the fold4 #f4 ,take the syringe in the right hand and with the needle transfix the fold near its base 7,fig4 #ahb74 #g4 ,now release the fold1 but hold the syringe steady2 as the parietes flatten out1 the point of the needle is left free in the peritoneal cavity 7see ,fig4 #ahc74 ,illustration3 ,,fig4 #ahb4--;,intraperitoneal inoculation--fluid4 #h4 ,inject the fluid from the syringe4 #i4 ,label1 etc4 ,illustration3 ,,fig4 #ahc4--,section of abdominal wall1 etc41 showing point of needle lying free in the peritoneal cavity above the coils of intestine4 ,second ,method3 ,steps #a-d4 ,as in the first method4 #e4 ,anaesthetise a small selected area of skin by spraying it with ethyl chloride4 #f4 ,heat platinum searing wire 7#j4e mm4 wire1 twisted to the shape indicated in figure #ahd1 mounted in an aluminium handle7 to redness1 and with it burn a hole through the anaesthetic area of skin and abdominal muscle down to1 but not through1 the visceral peritoneum4 #g4 ,fix a blunt-ended needle on to the charged syringe1 and by pressing the rounded end firmly against the peritoneum it can easily be pushed through into the peritoneal cavity4 #h4 ,inject the fluid from the syringe4 #i4 ,label1 etc4 ,this method is especially useful when it is desired to collect samples of the peritoneal fluid from time to time during the period of observation1 as fluid can be removed from the peritoneal cavity1 at intervals1 through this aperture in the abdominal parietes1 by means of a sterile capillary pipette4 ,illustration3 ,,fig4 #ahd4--,platinum wire for burning hole through parietes4 7b7 ,solid ,inoculum 7or the implantation of capsules containing fluid cultivations74--7,anaesthetic1 ,a4 ,c4 ,e47 #a4 ,anaesthetise the animal and secure it to the operating table4 #b4 ,shave a large area of the abdominal parietes4 #c4 ,make an incision through the skin in the middle line about #b cm4 in length1 midway between the lower end of the sternum and the pubes4 #d4 ,divide the aponeuroses between the recti upon a director4 #e4 ,divide the peritoneum upon a director4 #f4 ,introduce the inoculum into the peritoneal cavity4 #g4 ,close the peritoneal cavity with ,lembert's sutures4 #h4 ,close the skin and aponeurosis incisions together with interrupted sutures or ,michel's steel clips1 and apply a sealed dressing4 #i4 ,release the animal from the operating table4 #aj4 ,label1 etc4 ,suitable sacs may be readily prepared by either of the following methods3 ,a4 ,collodion ,sacs4 #a4 ,dip a small test-tube 7#e by #j4e cm471 bottom downward1 into a beaker of collodion1 and dry in the air2 repeat this process three or four times4 #b4 ,dip the tube1 with its coating of collodion1 alternately into a beaker of alcohol and one of water4 ,this loosens the collodion and allows it to be peeled off in the shape of a small test-tube4 #c4 ,take a #bj cm4 length of glass tubing1 of about the diameter of the test-tube used in forming the sac1 and insert one end into the open mouth of the sac4 #d4 ,suspend the glass tube with attached sac1 inside a larger test-tube1 by packing cotton-wool in the mouth of the test-tube around the glass tubing1 and place in the incubator at #cg deg4 ,c4 for twenty-four hours4 ,when removed from the incubator1 the sac will be firmly adherent to the extremity of the glass tubing4 #e4 ,plug the open end of the glass tubing with cotton-wool1 and sterilise the test-tube and its contents in the hot-air oven4 ,to use the sac1 remove the plug from the glass tubing1 partly fill the sac with cultivation to be inoculated1 by means of a sterile capillary pipette1 and replug the tubing4 ,when the abdominal cavity has been opened1 remove the tubing and attached sac from the protecting test-tube1 close the sac by tying a sterilised silk thread tightly around it a little below the end of the glass tubing1 and separate it from the tubing by cutting through the collodion above the ligature1 and the sac is ready for insertion in the peritoneal cavity4 ,b4 ,celloidin ,sacs 7,harris74 ,materials ,required4 ,quill glass tubing4 ,gelatine capsules such as pharmacists prepare for the exhibition of bulky powders4 ,various grades of celloidin1 thick and thin1 in wide-mouthed bottles4 #a4 ,take a piece of quill glass tubing some #d cm4 long by #e mm4 diameter2 heat one end in the bunsen flame4 #b4 ,thrust the heated end of the tube just through one end of a gelatine capsule and allow it to cool 7,fig4 #ahe74 #c4 ,remove any gelatine from the lumen of the tube with a heated platinum needle2 paint the joint between capsule and tube with moderately thick celloidin and allow to dry4 ,illustration3 ,,fig4 #ahe4--,making celloidin capsules4 #d4 ,dip the capsule into a beaker containing thin celloidin1 beyond the junction with the glass and after removal rotate it in front of the blowpipe air blast to dry it evenly4 ,repeat these manoeuvres until a sufficiently thick coating is obtained4 #e4 ,apply thick celloidin to the tube-capsule joint1 the opposite end of the capsule1 and the line of junction of the capsule with its cap2 dry thoroughly4 #f4 ,with a teat pipette fill the capsule 7through the attached tube7 with hot water1 and stand the capsule in a beaker of boiling water for a few minutes to melt the gelatine4 #g4 ,remove the solution of gelatine from the interior of the celloidin case with a pipette4 #h4 ,fill the sac with nutrient broth and place it1 glass tube downward1 in a tube containing sufficient sterile nutrient broth to cover the sac to the depth of #a cm4 ,plug the tube and sterilise in the steamer in the usual manner4 #i4 ,to prepare the sac for use1 empty it out of the broth tube into a sterile glass dish4 #aj4 ,grasp the tube near its junction with the sac in the jaws of sterile forceps1 and with a teat pipette remove sufficient of the contained broth to leave a small space in the sac4 ,introduce the inoculum in the form of an emulsion by means of another pipette4 #aa4 ,still holding the tube in the forceps1 draw it out and seal off near the sac in the blowpipe flame4 #ab4 ,when cool wash the sac in sterile water1 then transfer to a tube of nutrient broth and incubate over night to determine its impermeability to bacteria4 #ac4 ,if the broth outside the sac remains sterile1 insert the sac in the peritoneal cavity of the experimental animal4 #e4 ,intracranial4--7,anaesthetic1 ,a4 ,c4 ,e47 ,illustration3 ,,fig4 #ahf4--;,guarded trephine4 ,trephines and ,surgical ,engine4--,the most useful instrument for intracranial operations upon animals is the small nasal trephine 7,curtis7 having a tooth cutting circle of #g mm4 ,the addition of an adjustable collar guard--secured by a screw--prevents accidental laceration of the dura mater or brain substance#ac 7,fig4 #ahf74 ,this size is suitable for monkeys1 dogs1 cats and large rabbits4 ,other smaller sizes which will be found useful for guinea pigs and other small animals cut circles of #f and #d mm42 for very small animals--young guinea pigs and rats--a small dental drill or screw will make a sufficiently large hole to admit the syringe needle4 ,the trephine can be set in ordinary metal handles and rotated by hand1 but a surgical engine of some kind is much preferable on the score of rapidity and safety to the animal4 ,the ,guy's electrical ,dental engine#ad 7,fig4 #ahg7 which can be connected to a lamp socket or wall plug1 and is operated by a foot switch1 although inexpensive is eminently satisfactory4 ,,note4--,a fine dental drill attached to the dental engine renders the manufacture of aluminium handles needles 7see page #ga7 quite an easy matter4 7a7 ,subdural4 #a4 ,anaesthetise the animal and secure it to the operating table1 dorsum uppermost4 #b4 ,shave a portion of the scalp immediately in front of the ears4 ,illustration3 ,,fig4 #ahg4--;,guy's electrical dental engine4 #c4 ,mark out with a sharp scalpel a crescentic flap of skin muscle1 etc41 convexity forward1 commencing #j4e cm4 in front of the root of one ear and terminating at a similar spot in front of the other ear4 ,reflect the marked flap4 #d4 ,make a corresponding incision through the periosteum and raise it with a blunt dissector4 #e4 ,with a small trephine 7diameter #f mm47 remove a circular piece of bone from the parietal segment4 ,the centre of the trephine hole should be at the intersection of the median line and a line joining the posterior canthi 7,fig4 #ahh74 #f4 ,introduce the inoculum by means of a hypodermic syringe1 perforating the dura mater with the needle and depositing the material immediately below this membrane1 at the same time taking care to avoid injuring the sinuses4 #g4 ,turn back the flap of skin and secure it in position with ,michel's steel clips4 #h4 ,dress with sterile gauze and wool and seal the dressing with collodion4 #i4 ,label1 etc4 7b7 ,intracerebral4--,this inoculation is performed precisely as for subdural save in step #f the needle after perforating the dura mater is pushed onward into the substance of one or other cerebral hemispheres before the contents are ejected4 ,illustration3 ,,fig4 #ahh4--;,intracranial inoculation of rabbit4 ,the circle indicates the situation of the trephine hole4 #f4 ,intraocular4-- 7a7 ,fluid ,inoculum4--7,anaesthetic1 cocaine47 #a4 ,instil a few drops of a sterile solution of cocaine1 and repeat the instillation in two minutes4 #b4 ,five minutes later have the animal firmly held by an assistant as in intravenous injection 7see ,fig4 #ahi71 the head being steadied by the assistant's hands4 #c4 ,select two needles to accurately fit the same syringe and sterilise4 #d4 ,attach one needle to the syringe and take up the required dose of inoculum and remove the needle4 #e4 ,steady the eye with fixation forceps2 then pierce the cornea with the other syringe needle and allow the aqueous to escape through the needle4 #f4 ,without removing the needle from the cornea attach the syringe and make the injection into the anterior chamber4 #g4 ,irrigate the conjunctival sac with sterile saline solution4 #h4 ,label1 etc4 7b7 ,solid ,inoculum4--7,anaesthetic1 ,a4 ,c4 ,e47 #a4 ,anaesthetise the animal and secure it firmly to the operating table4 #b4 ,irrigate the conjunctival sac thoroughly with sterile saline solution4 #c4 ,make an incision through the upper quadrant of the cornea into the anterior chamber by means of a triangular keratome4 #d4 ,separate the lips of the corneal wound with a flexible silver spatula2 seize the solid inoculum in a pair of iris forceps1 introduce it through the corneal wound1 and deposit it on the anterior surface of the iris2 withdraw the forceps4 #e4 ,again irrigate the sac and the surface of the cornea4 #f4 ,release the animal from the operating table4 #g4 ,label1 etc4 #g4 ,intrapulmonary4-- ,fluid ,inoculum4--7,anaesthetic1 none47 #a4 ,have the animal firmly held by an assistant4 7,in this case the foreleg of the selected side is drawn up by the assistant and held with the ear of that side47 #b4 ,shave carefully in the axillary line and disinfect the denuded skin4 #c4 ,thrust the needle of the syringe boldly through the fifth or sixth intercostal space into the lung tissue4 #d4 ,inject the contents of the syringe slowly4 #e4 ,label1 etc4 #h4 ,intravenous4-- ,fluid ,inoculum4--7,anaesthetic1 none47 ,the site selected for the injection in the rabbit is the posterior auricular vein 7see ,fig4 #aib74 ,although this is smaller than the median vein1 it is firmly bound down to the cartilage of the ear by dense connective tissue1 and is therefore more readily accessible4 7,in the guinea-pig the jugular vein must be utilised1 and in order to perform the inoculation satisfactorily a general anaesthetic must be administered to the animal4 ,in the monkey or the dog1 the internal saphenous vein is the most convenient and before puncturing should be distended or rendered prominent by compressing the vein above the selected site47 ,preparation of the ,inoculum4--,care must be taken in preparing the inoculum1 as the injection of even small fragments may cause fatal embolism4 ,to obviate this risk the fluid should1 if possible1 be filtered through sterile filter paper before filling into the syringe4 ,air bubbles1 when injected into a vein1 frequently cause immediate death4 ,to prevent this1 the syringe after being filled should be held in the vertical position1 needle uppermost4 ,a piece of sterile filter paper is then impaled on the needle and the piston of the syringe pressed upward until all the air is expelled from the barrel and needle4 ,should any drops of the inoculum be forced out1 they will fall on the filter paper1 which should be immediately burned4 #a4 ,have the animal firmly held by an assistant4 ,the selected ear is grasped at its root and stretched forward toward the operator4 #b4 ,shave the posterior border of the dorsum of the ear4 #c4 ,disinfect the skin over the vein1 rubbing it vigourously with cotton-wool soaked in lysol4 ,the friction will make the vein more conspicuous4 ,wash the lysol off with ether and allow the latter to evaporate4 #d4 ,direct the assistant to compress the vein at the root of the ear4 ,this will cause its peripheral portion to swell up and increase in calibre4 #e4 ,hold the syringe as one would a pen and thrust the point of the needle through the skin and the wall of the vein till it enters the lumen of the vein 7,fig4 #ahi74 ,now press it onward in the direction of the blood stream--i4 e41 toward the body of the animal4 #f4 ,direct the assistant to cease compressing the root of the ear1 and slowly inject the inoculum4 7,if the fluid is being forced into the subcutaneous tissue1 a condition which is at once indicated by the swelling that occurs1 the injection must be stopped and another attempt made at a spot closer to the root of the ear or at some point on the corresponding vein on the opposite ear47 #g4 ,withdraw the needle and press a pledget of cotton-wool over the puncture to ensure closure of the aperture in the vein wall4 #h4 ,label1 etc4 ,illustration3 ,,fig4 #ahi4--;,intravenous inoculation4 #i4 ,inhalation4-- 7a7 ,fluid ,inoculum4--7,anaesthetic1 none47 #a4 ,place the animal in a closed metal box4 #b4 ,through a hole in one side introduce the nozzle of some simple spraying apparatus1 such as is used for nasal medicaments4 #c4 ,fill the reservoir of the instrument 7previously sterilised7 with the fluid inoculum1 and having attached the bellows1 spray the inoculum into the interior of the box4 #d4 ,on the completion of the spraying1 open the box1 spray the animal thoroughly with a #aj per cent4 solution of formaldehyde 7to destroy any of the virus that may be adhering to fur or feathers74 #e4 ,transfer the animal to its cage4 #f4 ,label1 etc4 #g4 ,thoroughly disinfect the inhalation chamber4 7b7 ,fluid or ,powdered ,inoculum4--,anaesthetic1 ,a4 ,c4 ,e4 #a4 ,anaesthetise the animal and secure it firmly to the operating table4 ,illustration3 ,,fig4 #aij4--;,gag for rabbits4 #b4 ,prop open the mouth by means of some form of gag2 seize the tongue with a pair of forceps and draw it forward4 ,the most convenient form of gag for the rabbit or cat is that shown in ,fig4 #aij4 ,it is simply a strip of hard wood shaped at the middle and provided with a square orifice through which a tracheal or oesophageal tube can be passed4 #c4 ,pass a previously sterilised glass tube 7#ag cm4 long1 #j4e cm4 diameter1 with its terminal #b cm4 slightly curved7 down through the larynx into the trachea4 #d4 ,connect the straight portion of a ,y-shaped piece of tubing to the upper end of the sterilised tube and couple one branch of the ,y to a separatory funnel containing the fluid inoculum1 or insufflator containing the powdered inoculum1 and the other to a hand bellows4 #e4 ,allow the fluid inoculum to run into the lungs by gravity1 or blow in the powdered inoculum by means of a rubber-ball bellows4 #f4 ,remove the intratracheal tube2 release the animal from the table4 #g4 ,label1 etc4 ,as an alternative method in the case of fairly large animals1 such as rabbits1 etc41 a sterile piece of glass tubing of suitable diameter may be passed through the larynx down the trachea almost to its bifurcation4 ,fluid cultivations may then be literally poured into the lungs1 or cultivations1 dried and powdered1 may be blown into the lung by the aid of a small hand bellows or even a teat pipette4 #aj4 ,intragastric ,inoculation4--,fluid or semi-fluid inoculum4 7,anaesthetic none47 ,the method of performing the operation is varied slightly according to the size of the experimental animal4 ,a4 ,monkey1 ,rabbit1 ,guinea-pig4 #a4 ,secure the animal to the operating table ventral surface uppermost4 #b4 ,prop the mouth open with a gag2 draw the tongue forward with forceps4 #c4 ,sterilise a soft rubber catheter 7,no4 #aj or #h ,english scale1 or ,no4 #ah or #ae ,french7 and lubricate it with sterile glycerine4 #d4 ,pass it to the back of the pharynx1 keeping the end in the middle line4 #e4 ,gently assist the progress of the catheter down the oesophagus until it passes the cardiac orifice of the stomach4 ,do not use any force4 #f4 ,take up the required dose of inoculum into a sterilised pipette4 ,insert the point of the pipette into the open end of the catheter and allow the fluid to run down into the stomach4 ,remove the pipette and drop it into a jar of lysol4 #g4 ,with another sterile pipette run one cubic centimetre of sterile saline solution through the catheter to wash out the last traces of the inoculum4 #h4 ,withdraw the catheter4 #i4 ,label1 etc4 ,b4 ,rats and ,mice 7,mark's ,method74 #a4 ,secure the animal in the vertical position4 7a7 ,rat4--,take a pair of catch sinus forceps about #bb cm4 in length and seize the animal by the loose skin of the head as far forward as possible--fix the forceps1 and holding the instrument vertically upward1 transfer to the left hand of an assistant who secures the animal's tail between the fingers grasping the handle of the forceps4 7,see ,fig4 #aia47 ,illustration3 ,,fig4 #aia4--;,intragastric inoculation of rat4 7b7 ,mouse4--,an assistant grasps the loose skin between the ears as far forwards as possible between the forefinger and thumb of the left hand4 ,he now grasps the tail with the right hand1 draws the mouse straight and passes the tail between the fourth and little fingers of the left hand and secures it there4 #b4 ,the assistant takes a closed pair of thin-bladed forceps in his right hand1 passes the ends into the animal's mouth1 then allows the blades to separate4 ,this opens the animal's jaw and serves as a gag4 #c4 ,moisten the sterilised oesophageal tube with sterile water4 7,this tube is of silk rubber1 #f4e cm4 in length1 with the distal end rounded1 the proximal end mounted in a syringe needle head1 which fits the nozzles of the two sterile syringes to be used47 #d4 ,grasp the tube about its middle and pass it into the animal's mouth1 downwards and a little to one side or the other until its length is lost in the digestive tract and mouth4 ,gentle guidance is alone necessary4 ,do not use any force4 #e4 ,take up the required dose of inoculum into the syringe2 insert the nozzle of the syringe into the needle-mount1 and force the piston down4 #f4 ,steadying the needle-mount with the left hand1 detach the syringe4 #g4 ,draw up some sterile water in the second 7sterile7 syringe1 and inserting its nozzle into the needle-mount force a few drops of water through the tube to wash it out4 #h4 ,with one quick upward movement remove the tube from the animal's mouth4 #i4 ,label1 etc4 ,one other method of inoculation remains to be described1 which does not require operative interference4 #aa4 ,feeding4-- #a4 ,fluid ,inoculum4--,small pieces of sterilised bread or sop 7sterilised in the steamer at #ajj deg4 ,c47 are soaked in the fluid inoculum and offered to the animals in a sterile ,petri dish or capsule4 #b4 ,solid ,inoculum4--,small pieces of tissue are placed in sterile vessels and offered to the animals4 ,,footnotes3 #ab ,this table is made by ,messrs4 ,down ,bros41 ,st4 ,thomas's ,street1 ,london1 ,s4 ,e4 #ac ,this modification is made for the author by ,messrs4 ,down ,bros41 ,st4 ,thomas's ,street1 ,london1 ,s4 ,e4 #ad ,manufactured by ,messrs4 ,francis ,lepper1 #ef1 ,great ,marlborough ,street1 ,london1 ,w4 ,,xviii4 ,,the ,,study ,,of ,,experimental ,,infections ,,during ,,life4 ,the possession of pathogenetic properties by an organism under study is indicated by the infection of the experimental animal--a term which is employed to summarise the condition resulting from the successful invasion of the tissues of the experimental animal by the micro-organisms inoculated and by their multiplication therein4 ,infection is considered to have taken place3 #a4 ,when the death of the animal is produced as a direct consequence of the inoculation4 #b4 ,when without necessarily producing death the inoculation causes local or general changes of a pathological character4 #c4 ,when either with or without death1 or local or general changes occurring1 certain substances make their appearance in the body fluids1 which can be shown 7in vitro or in vivo7 to exert some profound and specific effect when brought into contact with subcultivations of the organism originally inoculated4 ,the important factors in the production of infection are3 ,a4 ,seed4 ,virulence of organism4 ,dose of organism4 ,b4 ,soil4 ,resistance offered by the cells of the experimental animal4 ,the first two factors1 although variable1 are to a certain extent under the control of the experimenter4 ,thus by suitable means the virulence of an organism can be exalted or attenuated1 whilst the size of the dose may be increased or diminished4 ,the third factor also varies1 not only amongst different species of animals1 but also amongst different individuals of the same species4 ,the essential causes of this variation are not so obvious1 so that beyond selecting the animals intended for similar experiments with regard to such points as age1 size or sex1 but little can be done to standardise cell resistance4 ,immediately an animal has been inoculated a period of clinical observation must be entered upon1 which should only terminate with the death of the animal4 ,the general observations should at first and if the infection is an acute one1 be made daily--later1 and if the animal appears to be unaffected or if the infection is chronic1 both general and special observations should be carried out at weekly intervals4 ,if the animal appears to be still unaffected1 it should be killed with chloroform vapour at the end of two or three months and a complete post-mortem carried out4 ,a4 ,the general observations should take cognisance of3 #a4 ,general appearance4 ,the experimental animal should be inspected daily1 not only with a view to detecting symptoms due to the experimental infection1 but also to prevent any intercurrent infection1 naturally acquired1 from escaping notice 7vide page #ccg74 #b4 ,the weight of the inoculated animal should be observed and recorded each day during the course of an experimental infection at precisely the same hour1 preferably just before the morning feed4 #c4 ,the temperature should similarly be recorded daily1 if not more frequently1 during the whole period the animal is under observation1 and carefully charted--individual variations will at once become apparent4 ,it should be borne in mind that the temperature regarded as normal for man 7#cg4e deg4 ,c47 is not the normal average temperature of any of the lower animals save the rat and mouse4 ,the accompanying table of normal averages for the animals usually employed in bacteriological research may be of use in preventing the erroneous assumption that pyrexia is present in an animal1 which merely shows its own normal temperature4 ,,normal ,,averages4 ---------------------------------------- ------------ ,rectal ,pulse4 ,respirations4 ,animal4 ,temp4 ------------------------ deg4 ,c4 ,rate per minute4 ---------------------------------------- ------------ ,frog #h4i-ag4b #hj #ab ,mouse #cg4d #abj 444 ,rat #cg4e 444 #baj ,guinea pig #ch4f #aej #hj ,rabbit #ch4g #ace #ee ,cat #ch4g #acj #bd ,dog #ch4f #ie #ae ,goat #dj4j #ge #af ,ox #ch4h #de 44 ,horse #cg4i #ch #aa ,monkey 7,rhesus7 #ch4d #ajj #ai ,pigeon #dj4i #acf #cj ,fowl #da4f #adj #ab ---------------------------------------- ------------ ,b4 ,special observations comprise some or all of the following1 according to the method of inoculation and the character of the virus4 #a4 ,the site of inoculation should be minutely examined at least at weekly intervals1 and the neighbouring lymphatic glands palpated4 #b4 ,any local reaction at the site of inoculation and any other readily accessible lesion should be carefully investigated4 ,any suppurative process which may occur1 whether in the subcutaneous tissues or in joints1 should be explored and the pus carefully examined both microscopically and culturally4 ,fluid secretions and excretions1 such as pus or serous exudates when accessible are collected direct from the body in sterile capillary pipettes 7vide ,fig4 #ac;a17 in the following manner3 #a4 ,open the case containing the pipettes1 grasp one by the plugged end1 remove it from the case1 and replace the lid of the latter4 #b4 ,attach a rubber teat 7vide page #aj7 to the plugged end of the pipette and use the teat as the handle of the pipette4 #c4 ,pass the entire length of the pipette twice or thrice through the flame of the ,bunsen burner4 #d4 ,snap off the sealed end of the pipette with a pair of sterile forceps4 #e4 ,compress the india-rubber teat1 thrust the point of the pipette into the secretion2 now relax the pressure on the teat and allow the pipette to fill4 #f4 ,remove the point of the pipette from the secretion1 allow the fluid to run a short distance up the capillary stem and seal the point of the pipette in the flame4 7,if using a pipette with a constriction below the plugged mouthpiece 7,fig #ac;b71 this portion of the pipette may also be sealed in the flame47 ,when ready to examine the morbid material snap off the sealed end of the pipette with sterile forceps and eject the contents of the pipette into a sterile capsule4 ,the material can now be utilized for cover-slip preparations1 cultivations and inoculation experiment4 #c4 ,the peripheral blood should be examined from time to time for from this tissue is often obtained the fullest information as to the course and progress of an infection4 a4 ,the histological examination of the blood should be directed chiefly to observations on the number and kind of white cells2 and since but few bacteriologists are at the same time expert comparative haematologists1 some notes on the normal characters of the blood of the commoner laboratory animals1 contrasted with those of man1 are inserted for reference4 ,these have been very kindly compiled for me by my friend and one time colleague ,dr4 ,cecil ,price ,jones4 ,,comparative ,,haemocytology ,,of ,,laboratory ,,animals4 ---------------------------------------- ---------------------------- ,totals ,percentages ---------------------------------------- -------------------- ,animal ,hb1 ,lympho-,large ,poly- ,eosin- ,mast ,red cells ,white per cytes1monos1morph1 oph1 cells1 cellscent4 per per per per per cent4 cent4 cent4cent4 cent4 ---------------------------------------- ---------------------------- ,frog #dij1jjj #h1jjj #eh #dj #aj4j #bb4j #ae #ac ,mouse #h1gjj1jjj #h1jjj #gh #fj #ba4e #ag4j #a4d #j4a ,rat #i1jjj1jjj #i1jjj #he #ed #g4j #cg4e #a4c #j4b ,guinea- pig #e1gjj1jjjaj1jjj #ii #ee #i4j #cb4h #c4j #j4b ,rabbit #f1jjj1jjj #g1jjj #gj #ej #b4j #df4j #j4f #a4d ,rhesus #d1ejj1jjjac1jjj #gg #dc #e4j #ej4j #a4c #j4g ,goat #ad1fjj1jjjae1jjj #eh #ce #f4c #ef4g #a4be #j4ge ,fowl #c1ejj1jjjcj1jjj #ajj #di #c4j #db4j #a4j #e4j ,pigeon #c1ejj1jjjbj1jjj #aja #dc #i4j #dc4j #c4j #b4j ---------------------------------------- ---------------------------- ,man 7adult7 #e1jjj1jjj #g1ejj #ajj #be #e4e #fe #d4j #j4e ,normal 7#d4e-e7 7#g-i77ie- 7#bj-cj7 7#d-h77ee- 7#c-e7 7#j4e-b7 limits4 millions4 thou- #aja7 #fh7 sands4 ---------------------------------------- ---------------------------- ,the above table represents in each case the average of a large number of counts4 ,,remarks4 ,frog4--,the red cells are large oval nucleated 7#bj-be mu by #ab-ae mu7 discs1 the nucleus relatively small and irregularly elongated or oval1 about #aj mu in length4 ,many primitive and developing forms are usually observed--also free nuclei and many cells in various stages of degeneration4 ,haemoglobin estimation is difficult owing to turbidity of the blood after dilution with water4 ,the polymorphonuclear leucocytes are large cells1 about #bj mu2 no definite granules can be observed4 ,the eosinophile cells contain large deeply staining coccal-shaped granules4 ,mouse4--,the granules of the polymorphonuclear leucocytes are usually not stained1 or only very faintly so4 ,the nucleus of the eosinophile cell is ring-shaped or much divided1 and the granules are coccal and stain oxyphile4 ,the remarkable character of the blood is the high percentage of large mononuclear cells4 ,rat4--,the fine rod-shaped granules of the polymorphonuclear leucocytes are usually very faintly stained4 ,the granules of eosinophile cells are well stained and coccal-shaped1 the nucleus is often ring shaped4 ,the basophile granular cells are few--but the granules are large1 and stain deeply basophile4 ,guinea-pig4--,polychromasia and punctate basophilia of red cells are very commonly observed--nucleated red cells are also frequent4 ,the large mononuclear cells often contain vacuoles--,kurlow cells--possibly of a parasitic nature4 ,rabbit4--,it is not uncommon to find nucleated red cells in films from quite healthy animals4 ,the granules of the polymorphonuclear leucocytes stain oxyphile4 ,the coarse granules of the eosinophile cells appear to stain less deeply oxyphile1 probably owing to the basophile staining of the cytoplasm4 ,rhesus monkey4--,the blood cells resemble those met with in human blood4 ,the minute neutrophile granules of the polymorphonuclear leucocytes are often very scanty1 and sometimes apparently absent4 ,the eosinophile cells are not so densely packed with coarse oxpyhile granules as in the human eosinophile1 and the nuclei of these cells are usually much divided1 or polymorphous4 ,goat4--,the red cells are small1 nonnucleated discs1 only about #d4e mu diameter1 not much more than half that of the human red cell4 ,the polymorphonuclear leucocytes have only a few very minute coccal-shaped oxyphile granules1 the nucleus is polymorphous4 ,the eosinophile cells are large cells up to #bj mu1 the cytoplasm is basophile and contains coarse coccal-shaped oxyphile granules1 and the nucleus is often much divided4 ,fowl4--,the red cells are oval nucleated discs about #ab mu by #f mu1 the nucleus being relatively small 7about #d mu long71 irregularly elongated or oval2 round1 more deeply stained cells with round or diffuse nuclei1 also free nuclei and degenerated forms of red cells are often present4 ,the granules of the cells corresponding to the polymorphonuclear leucocytes are rod-shaped1 often beaded or with clubbed ends4 ,the nucleus is not polymorphous1 but usually divided into two1 though it may be single4 ,the cells probably corresponding to eosinophile leucocytes have fine coccal-shaped granules1 faintly staining eosinophile or neutrophile4 ,the basophile granules of the mast cells are coccal-shaped1 of various size--often quite powdery4 ,pigeon4--,red cells resemble those of the fowl1 and similar varieties of appearance may be noted4 ,the granules of those cells which correspond to polymorphonuclear leucocytes are rod-shaped1 but smaller and finer than in the fowl1 and do not show clubbed appearances4 ,the nucleus is not polymorphous1 and only occasionally divided4 ,the coccal-shaped granules of the eosinophile cells are stained more deeply oxyphile than those of the corresponding cells of the fowl4 ,the preparation of dried films for this histological examination of the blood is carried out as follows3 #a4 ,small samples of blood for the preparation of blood films are most conveniently obtained from the veins of the ear in most of the ordinary laboratory animals1 viz41 monkey1 goat1 dog1 cat1 rabbit1 guinea-pig2 in the pigeon and fowl the axillary vein should be punctured2 in the rat and mouse either a vein in the ear or preferably by wounding the tip of the tail2 in the frog1 the web of the foot should be selected4 #b4 ,puncture the selected vein with a sharp needle4 ,a flat ,hagedorn needle 7size ,no4 #h7 with a cutting edge is the most useful for this purpose4 ,if the vein cannot be distended by proximal compression1 vigourous friction with a piece of dry lint may have the desired effect--or a test-tube full of water at about #dj deg4 ,c4 may be placed close to the vein4 ,failing these methods1 a drop or two of xylol may be dropped on the skin just over the vein1 left on for a few seconds and then wiped off with a piece of dry lint4 #c4 ,one of the short ends of a #c by #a glass slip is brought into contact with the exuding drop of blood1 so that it picks up a small drop4 #d4 ,the slide is then lowered transversely on to the surface of a second #c by #a slip1 which rests on the bench near to one end at an angle of about #de deg41 and retained in this position for a few seconds1 while the drop of blood spreads along the whole of the line of contact 7see also ,fig4 #fi74 #e4 ,draw the first slide firmly and evenly along the entire length of the lower slide1 leaving a thin regular film which will probably show the blood cells only one layer thick4 #f4 ,allow the film to dry in the air4 #g4 ,stain with one of the polychrome blood stains 7see page #ig74 #h4 ,examine microscopically4 b4 ,the bacteriological examination of the blood is directed solely to the demonstration of the presence in the circulating blood of the organisms previously injected into the animal4 ,for this purpose several cubic centimetres of blood should be taken in an all-glass syringe from an accessible vein corresponding to one of those suggested as the site of intravenous inoculation--and under similar aseptic precautions4 #a4 ,sterilise an all-glass syringe of suitable size1 and when cool draw into the syringe some sterile sodium citrate solution and moisten the whole of the interior of the barrel2 then eject all the citrate solution if less than #e c4c4 blood are to be withdrawn2 if more than #e c4c4 are required retain about half a cubic centimetre of the fluid in the syringe4 ,this prevents coagulation of the blood4 ,the sodium citrate solution is prepared by dissolving3 ,sodium citrate #aj gramme4 ,sodium chloride #j4ge grammes4 ,in distilled water #ajj c4c4 ,sterilise by boiling4 #b4 ,prepare the animal as for intravenous inoculation 7see page #cfc7 and introduce the syringe needle into the lumen of the selected vein4 #c4 ,slowly withdraw the piston of the syringe4 ,when sufficient blood has been collected direct the assistant to release the proximal compression of the vein2 and withdraw the needle4 #d4 ,remove the needle from the nozzle of the syringe and deliver the citrated blood into a small ,ehlenmeyer flask containing about #bej c4c4 of nutrient broth4 #e4 ,label1 incubate and examine daily until growth occurs or until the expiration of ten days4 c4 ,the serological examination of the blood is directed to the demonstration of the presence of certain specific antibodies in the sera of experimentally infected animals1 and within certain limits to an estimation of their amounts4 ,the chief of these bodies are3 ,antitoxin4 ,agglutinin4 ,precipitin4 ,opsonin4 ,immune body or ,bacteriolysin4 ,none of these substances are capable of isolation in a state of purity apart from the blood serum1 consequently special methods have been elaborated to permit of their recognition4 ,in every instance the behaviour of serum from the experimental animal1 which may be termed specific serum1 is studied in comparison with that of serum from an uninoculated animal of the same species1 and which is termed normal serum4 ,in view of minor differences in constitution exhibited by the serum of various individuals of the same series1 it is usual to employ a mixture of sera obtained from several different normal animals of the same species as the inoculated animal1 under the term pooled serum4 ,the method of collecting blood 7e4 g41 from the rabbit7 for serological tests is as follows3 ,collection of ,serum4 ,apparatus required3 ,razor4 ,liquid soap4 ,cotton-wool4 ,lysol #b per cent4 solution1 in drop bottle4 ,ether in drop bottle4 ,flat ,hagedorn needles4 ,blood pipettes 7,fig4 #af1 page #ab74 ,centrifugal machine4 ,centrifuge tubes4 ,glass cutting knife4 ,bunsen flame4 ,writing diamond or grease pencil4 ,,method4 #a4 ,shave the dorsal surface of the ear over the course of the posterior auricular vein 7see ,fig4 #aib74 #b4 ,sterilise the skin by washing with lysol4 ,the lysol should be applied with sterile cotton-wool and the ear vigourously rubbed1 not only to remove superficial scales of epithelium1 but also to render the ear hyperaemic and the vein prominent4 #c4 ,remove the lysol with ether dropped from a drop bottle1 and allow the ether to evaporate4 #d4 ,puncture the vein with a sterile ,hagedorn needle4 #e4 ,take a small blood-collecting pipette 7,fig4 #afa7 and hold it at an angle to the ear1 one end touching the issuing drop of blood1 the other depressed4 ,the blood will now enter the pipette at first by capillarity2 afterward gravity will also come into play and the pipette can be two-thirds filled without difficulty4 #f4 ,hold the tube by the end containing the blood1 the clean end pointing obliquely upward--warm this end at the bunsen flame to expel some of the contained air2 then seal the clean point in the flame4 ,illustration3 ,,fig4 #aib4--;,collecting blood from rabbit4 #g4 ,place the pipette down on a cool surface 7e4 g41 a glass slide74 ,the rapid cooling of the air in the clean end of the pipette creates a negative pressure1 and the blood is sucked back into the pipette1 leaving the soiled end free from blood4 ,seal this end in the bunsen flame4 #h4 ,mark the distinctive title of the specimen 7e4 g41 animal's number7 upon the pipette with a writing diamond or grease pencil4 #i4 ,when the sealed ends are cold and the blood has clotted1 place the pipette on the centrifuge1 clean end downward2 counterpoise and centrifugalise thoroughly4 ,on removing the pipette from the centrifuge1 the red cells will be collected in a firm mass at one end1 and above them will appear the clear serum4 #aj4 ,by marking the blood pipette above the level of the serum with the glass cutting knife and snapping the tube at that point1 the blood-serum becomes readily accessible for testing purposes4 ,if larger quantities of blood are required1 the animal1 after puncturing the vein1 should be inverted1 an assistant holding it up by the legs4 ,blood to the volume of several cubic centimetres will now drop from the punctured vein1 and should be caught in a tapering centrifuge tube1 the tube transferred to the incubator at #cg deg4 ,c4 for two hours1 then placed in the centrifugal machine1 counterpoised and centrifugalised thoroughly4 ,the three most important of the antibodies referred to which can be demonstrated with a certain amount of facility are agglutinin1 opsonin and bacteriolysin2 and the methods of testing for these bodies will now be considered4 ,,agglutinin4 ,agglutinin is the name given to a substance present in the blood-serum of an animal that has successfully resisted inoculation with a certain micro-organism4 ,this substance possesses the power of collecting together in clumps and masses1 or agglutinating watery suspensions of that particular microbe4 ,dilution of the ,specific ,serum3 ,apparatus required3 ,sterile graduated capillary pipettes to contain #aj c4 mm4 7,fig4 #ag74 ,sterile graduated capillary pipettes to contain #ij c4 mm4 7,fig4 #ag74 ,small sterile test-tubes #e x #j4e cm4 ,normal saline solution in flask or test-tube4 ,pipette of specific serum4 ,glass cutting knife1 or three-square file4 ,glass capsule1 nearly full of dry silver sand1 or roll of plasticine4 ,grease pencil4 ,,method4-- #a4 ,take three sterile test-tubes and number them #a1 #b and #c4 #b4 ,pipette #j4i c4c4 sterile normal saline solution into each tube1 and stand tubes upright in the sand in the capsule1 or in the plasticine block4 #c4 ,make a scratch with the glass cutting knife on the blood pipette above the upper level of the clear serum1 and snap off and discard the empty portion of the tube4 #d4 ,remove #j4a c4c4 of the serum from the blood pipette tube1 and mix it thoroughly with the fluid in tube ,no4 #a2 and label s4s41 7specific serum71 #aj per cent4 #e4 ,remove #j4a c4c4 of the solution from tube ,no4 #a by means of a fresh pipette1 and mix it with the contents of tube ,no4 #b2 and label s4s41 #a per cent4 #f4 ,remove #j4a c4c4 of the solution from tube ,no4 #b by means of a fresh pipette1 and mix it with the contents of tube ,no4 #c2 and label s4s41 #j4a per cent4 ,when the yield of serum from the specimen of blood which has been collected1 or is available1 is small1 the above method of diluting is not practicable1 and the dilution should be carried out by ,wright's method in a capillary teat pipette4 ,dilution of ,serum by ,means of a ,teat ,pipette4 ,materials required3 ,blood pipette containing sample of specific serum after centrifugalisation4 ,capsule of diluting fluid--normal saline solution4 ,supply of ,pasteur pipettes 7,fig4 #ac;a74 ,india-rubber teats4 ,small test-tubes4 ,a block of plasticine to act as a test-tube stand4 ,grease pencil4 ,,method3 #a4 ,mark three small test-tubes #aj per cent41 #a per cent4 and #j4a per cent4 respectively1 and stand them upright in the plasticine block4 #b4 ,take a ,pasteur pipette1 nick the capillary stem just above the sealed end with a glass cutting knife1 and snap off the sealed end with a quick movement so that the fracture is clean cut and at right angles to the long axis of the capillary stem--cut square1 in fact4 ,prepare several1 say a dozen1 in this manner4 #c4 ,fit a rubber teat to the barrel of each of the pipettes4 #d4 ,make a mark with the grease pencil on the stem of one of the pipettes about #b or #c cm4 from the open extremity4 ,illustration3 ,,fig4 #aic4--;,filling the capillary teat pipette4 #e4 ,compress the teat between the finger and thumb 7,fig4 #aic7 to such an extent as to drive out the greater part of the contained air4 #f4 ,maintaining the pressure on the teat pass the stem of the pipette into the capsule holding the saline solution1 until the open end of the pipette is below the level of the fluid4 #g4 ,now cautiously relax the pressure on the teat and let the fluid enter the pipette and rise in the stem until it reaches the level of the grease pencil mark4 ,as soon as this point is reached1 check the movement of the column of fluid by maintaining the pressure on the teat1 neither relaxing nor increasing it4 #h4 ,withdraw the point of the pipette clear of the fluid1 and again relax the pressure on the teat very slightly4 ,the column of saline solution rises higher in the stem1 and a column of air will now enter the pipette and serve as an index to separate the first volume of fluid drawn into the stem from the next succeeding one4 #i4 ,again introduce the end of the pipette into the fluid and draw up a second volume of saline to the level of the grease pencil mark1 and follow this with a second air index4 #aj4 ,in like manner take up seven more equal volumes of saline solution and their following air bubbles4 ,there are now nine equal volumes of normal saline in the pipette4 #aa4 ,now pass the point of the pipette into the blood tube and dip the open end below the surface of the serum4 ,proceeding as before1 aspirate a volume of serum into the capillary stem up to the level of the pencil mark4 #ab4 ,eject the contents of the pipette into the small tube marked #aj per cent4 by compressing the rubber teat between thumb and finger4 #ac4 ,mix the one volume of serum with the nine volumes of saline solution very thoroughly by repeatedly drawing up the whole of the fluid into the pipette and driving it out again into the test-tube4 #ad4 ,now take a clean pipette and proceed precisely as before1 #d to #aj4 #ae4 ,having aspirated nine equal volumes of saline into this second pipette1 now take up one similar volume of the fluid in the #aj per cent4 tube4 #af4 ,eject the contents of this pipette into the second tube marked #a per cent4 and mix thoroughly as before4 #ag4 ,in similar fashion make the #j4a per cent4 solution and transfer to the third tube4 #ah4 ,further dilutions in multiples of ten can be prepared in the same way1 and by varying the number of volumes of diluting fluid or serum any required dilution can be made 7see ,appendix1 ,dilution ,tables74 ,,note4--,the saline diluting fluid must always be taken into the pipette first1 otherwise if the serum contains a very large amount of agglutinin the traces of this serum added to the saline solution may be sufficient to entirely vitiate the subsequent observations--whilst if more than one sample of serum is diluted from the same saline solution serious errors may be introduced into the experiments4 ,the ,microscopical ,reaction3 ,apparatus ,required3 ,five hanging-drop slides 7or preferably two slide71 with two cells mounted side by side on each 7,fig4 #fb1 a71 and one slide with one cell only4 ,vaseline4 ,cover-slips4 ,platinum loop4 ,grease pencil4 ,eighteen to twenty-four-hour-old bouillon cultivation of the organism to be tested 7e4 g41 ,bacillus typhi abdominalis7 ,pipette end with the remainder of the specific serum labelled s4s4 ,tubes containing the three solutions of the specific serum1 #aj1 #a1 and #j4a per cent4 respectively4 ,pipette end with pooled normal serum labelled p4s4 ,,method4-- #a4 ,make five hanging-drop preparations1 thus3 7a7 ,one loopful of bouillon cultivation one loopful pooled serum2 label ,control4 7b7 ,one loopful culture one loopful undiluted specific serum2 label #ej per cent4 ,mount these two cover-slips on a double-celled slide4 7c7 ,one loopful bouillon culture one loopful #aj per cent4 serum2 label #e per cent4 ,mount this on single-cell slide4 7d7 ,one loopful bouillon culture one loopful #a per cent4 serum2 label #j4e per cent4 7e7 ,one loopful bouillon culture one loopful #j4a per cent4 serum2 label #j4je per cent4 ,mount these two cover-slips on a double-celled slide4 #b4 ,note the time3 ,examine the control to determine that the bacilli are motile and uniformly scattered over the field--not collected into masses4 #c4 ,next examine the #ej per cent4 serum preparation4 ,if agglutinin is present and the test is giving a positive reaction1 the bacilli will be collected in large clumps4 ,if the test is giving a negative reaction1 the bacilli may be collected in large clumps owing to the viscosity of the concentrated serum4 #d4 ,observe the #e per cent4 preparation microscopically4 ,if the bacilli are aggregated into clumps1 positive reaction4 ,if the bacilli are not aggregated into clumps1 observe until thirty minutes from the time of preparation before recording a negative reaction4 #e4 ,examine the #j4e and #j4je per cent4 preparations4 ,these may or may not show agglutination when the result of the examination of the #e per cent4 preparation is positive1 according to the potency of the specific serum2 and by the examination of a series of dilutions a quantitative comparison of the valency of specific sera from different sources1 or of serum from the same animal at different periods during the course of active immunisation may be obtained4 ,,note4--,the graduated pipettes supplied with ,thoma's haematocytometer 7intended for the collection of the specimen of blood required for the enumeration of leucocytes71 giving a dilution of #a in #aj--;i4 e41 #aj per cent4--may be substituted for the graduated capillary pipettes referred to above1 if the vessel in which the serum has been separated is of sufficiently large diameter to permit of their use4 ,the ,macroscopical ,reaction3 ,sterile graduated capillary pipettes to contain #ij c4 mm4 ,eighteen to twenty-four-hours-old bouillon cultivation of the organism to be tested4 ,three test-tubes containing the #aj1 #a1 and #j4a per cent4 solutions of specific serum 7about #ij c4 mm4 remaining in each74 ,tube containing #ej per cent4 solution of pooled serum4 ,sedimentation pipettes 7vide page #ag7 or teat pipettes4 ,,method4 #a4 ,pipette #ij c4 mm4 of the bouillon culture into each of the tubes containing the diluted serum2 and the same quantity into the tube containing the pooled serum4 #b4 ,fill a sedimentation tube 7by aspirating7 or a teat pipette from the contents of each tube4 ,seal off the lower ends of the sedimentation tubes in the ,bunsen flame4 #c4 ,label each tube with the dilution of serum that it contains--viz41 #e1 #j4e1 and #j4je per cent4 #d4 ,place the pipettes in a vertical position1 in a beaker1 in the incubator at #cg deg4 ,c41 for one or two hours4 #e4 ,observe the granular precipitate which is thrown down when the reaction is positive1 and the uniform turbidity of the negative reaction as compared with the appearances in the control pooled serum4 ,,opsonin4 ,opsonin is the term applied by ,wright to a substance1 present in the serum of an inoculated animal1 which is able to act upon or sensitise bacteria of the species originally injected1 so as to render them an easy prey to the phagocytic activity of polymorphonuclear leucocytes4 ,in the method for demonstrating opsonin about to be described1 a comparison is made between the opsonic power of the pooled serum and the specific serum4 ,apparatus3 ,small centrifuge and tubes for same 7made from the barrels of broken capillary pipettes by sealing the conical ends in the bunsen flame74 ,capillary ,pasteur pipettes4 ,india-rubber teats4 ,grease pencil4 ,bunsen burner with peep flame4 ,electrical signal clock 7see page #ci7 stop watch1 or watch4 ,rectangular glass box or tray to hold pipettes4 ,incubator regulated at #cg deg4 ,c4 #c x #a slides4 ,piece of light rubber tubing4 ,rectangular block of plasticine4 ,flask of normal saline solution4 ,flask of sodium citrate 7#a4e per cent47 in normal saline solution4 ,materials required1 and their preparation3 ,small tube of washed cells 7red blood discs and leucocytes72 human cells are used in estimating the opsonising power of the serum of experimental animals4 ,small tube of emulsion of bacteria of the species responsible for the infection of the experimental animal4 ,blood pipette containing specific serum4 ,blood pipette containing pooled serum4 ,washed ,cells4-- #a4 ,take a small centrifuge tube and half fill it with sodium citrate solution4 ,mark with the grease pencil the upper limit of the fluid4 #b4 ,cleanse the skin of the distal phalanx of the second finger of the left hand above the root of the nail with lint and ether4 ,wind the rubber tubing tightly round the second phalanx2 puncture with a sterile ,hagedorn needle through the cleansed area of skin4 #c4 ,take up a sufficiency of the issuing blood 7more or less according to the number of tests to be performed7 with a teat pipette1 transfer it to the tube of citrate solution and mix thoroughly4 ,make a second mark on the tube at the upper level of the mixed citrate solution and blood4 #d4 ,place the tube in the centrifuge1 counterpoise accurately and centrifugalise until the blood cells are thrown down in a compact mass occupying approximately the same volume as is included between the two pencil marks4 ,the column of fluid in the tube now shows clear supernatant fluid 7citrate solution and blood plasma7 separated from the sharp cut upper surface of the red deposit of corpuscles by a narrow greyish layer of leucocytes4 #e4 ,remove the supernatant column of citrate solution by means of a teat pipette1 fill normal saline solution into the tube up to the upper pencil mark1 and distribute the blood cells throughout the saline by means of the teat pipette4 ,centrifugalise as before4 #f4 ,again remove the supernatant fluid and fill in a fresh supply of saline solution and centrifugalise once more4 #g4 ,remove the supernatant saline solution as nearly down to the level of the leucocytes as can be safely done without removing any of the leucocytes4 #h4 ,next distribute the leucocytes evenly throughout the mass of red cells by rotating the tube between the palms of the hands--just as is done with a tube of liquefied medium prior to pouring a plate4 #i4 ,set the tube upright in the plasticine block near to one end4 ,bacterial ,emulsion4-- #a4 ,take an #ah- to #bd-;hour culture of the required bacterium 7e4 g41 ,diplococcus pneumoniae7 grown upon sloped blood agar at #cg deg4 ,c4 ,pour over the surface of the medium some #e c4c4 of normal saline solution4 #b4 ,with a platinum loop emulsify the growth from the surface of the medium as evenly as possible in the saline solution4 #c4 ,allow the tube to stand for a few minutes so that the large masses of growth may settle down2 transfer the upper portion of the saline suspension to a centrifuge tube and centrifugalise thoroughly4 #d4 ,examine a drop of the supernatant opalescent emulsion microscopically to determine its freedom from clumps and masses4 ,if unsatisfactory prepare another emulsion1 this time scraping up the surface growth with a platinum spatula1 transferring it to an agate mortar and grinding it up with successive small quantities of normal saline4 ,if satisfactory insert the tube in the plasticine block next to that containing the washed cells4 ,specific ,serum4-- ,pooled ,serum4-- ,these sera are collected and treated as already described 7see page #cgi71 and the portions of the blood pipettes containing them are arranged in the remaining space in plasticine block4 ,illustration3 ,,fig4 #aid4--,plasticine block with materials arranged for opsonin estimations4 ,the plasticine block now presents the appearances shown in ,fig4 #aid4 ,,method ,,for ,,determining ,,the ,,opsonic ,,index4-- #a4 ,take a capillary pipette fitted with a teat1 cut the distal end square and make a pencil mark about #b cm4 from the end4 #b4 ,aspirate into the pipette one volume of washed cells1 air index1 one volume of bacterial emulsion1 air index1 and one volume of specific serum 7see ,fig4 #aie74 ,illustration3 ,,fig4 #aie4 ,opsonin pipette4 #c4 ,mix thoroughly on a #c by #a slide by compressing the teat and ejecting the contents of the pipette on to the surface of the slide1 relaxing the pressure and so drawing the fluid up into the pipette again4 ,these two processes should be repeated several times2 finally take up the mixture in an unbroken column to the central portion of the capillary stem4 #d4 ,seal the point of the pipette in the peep flame of the bunsen burner and remove teat4 #e4 ,mark the pipette 7with the grease pencil7 with the distinctive number of the serum and place it in the glass box or tray4 #f4 ,take another similarly prepared pipette and aspirate into it equal volumes of washed cells1 bacterial emulsion and pooled serum4 ,treat precisely as in #c and #d1 label it control or ,n4,s4 7normal serum7 and place in the box by the side of the specific serum preparation4 #g4 ,place the box with the pipettes in the incubator and set the signal clock to ring at #ae minutes 7or start the stop watch74 #h4 ,at the expiration of the incubation time remove the pipettes from the incubator4 #i4 ,cut off the sealed end of the specific serum preparation4 ,mix its contents thoroughly as in step #c1 and then divide the mixture between two #c by #a slips and carefully spread a blood film 7vide page #cgf7 on each in such a way that only one-half of the surface of each slide is covered with blood--the free edge of the blood film approximating to the longitudinal axis of the slide4 ,allow films to dry and label the slides with writing diamond4 #aj4 ,treat the contents of the control pipette in similar fashion4 #aa4 ,select the better film from each pair for fixing and staining4 #ab4 ,fixing and staining must be carried out under strictly comparable conditions1 and to this end the slides are best handled by placing in a glass staining rack which can be lowered in turn into each of a series of glass troughs containing the various reagents 7,fig4 #aif74 ,place the rack in the first trough which contains the alcoholic solution of ,leishman's stain for two minutes to fix4 ,transfer to the second trough containing the diluted stain for ten minutes4 ,transfer to the third trough containing distilled water1 and holding the trough over a sink1 run in a stream of distilled water until washing is complete4 ,remove slides from the rack and dry4 ,leishman's stain is the best for routine work for all bacteria other than ,b4 tuberculosis4 ,films containing tubercle bacilli must of course be stained by the ,ziehl ,neelsen method4 ,illustration3 ,,fig4 #aif4 ,glass staining trough for blood films4 #ac4 ,examine specific serum slide microscopically with #a_/ab inch oil immersion4 ,find the edge of the blood film--along this the bulk of the leucocytes will be collected4 ,starting at one end of the film move the slide slowly across the microscope stage and as each leucocyte comes into view count and record the number of ingested bacteria4 ,the sum of the contents of the first #ej consecutive polymorphonuclears that are encountered is marked down4 7,the average number of bacilli ingested per leucocyte the phagocytic index47 #ad4 ,in precisely similar manner enumerate the bacteria present in the first #ej cells of the control preparation4 ,this number is recorded as the denominator of a vulgar fraction of which the numerator is the number recorded for the specific serum4 ,this fraction1 expressed as a percentage of unity the opsonic index4 ,,immune ,,body4 ,immune body or amboceptor is the name given to a substance present in the serum of an infected animal that has successfully resisted inoculation with some particular micro-organism1 and which possesses the power of linking the complement normally present in the serum to bacteria of the species used as antigen in such a manner that the micro-organisms are rendered innocuous1 and ultimately destroyed4 ,the presence of the immune body in the serum can be demonstrated in vitro by the reaction elaborated by ,bordet and ,gengou1 known as the complement fixation test1 the existence or the absence of the phenomenon of complement fixation being rendered obvious macroscopically by the absence or presence of haemolysis on the subsequent addition of sensitised red blood corpuscles1 7e4 g41 a mixture of crythrocyte solution and the appropriate haemolysin--two of the three essentials in the haemolytic system1 vide page #cbf74 ,apparatus ,required3 ,sterile pipettes #a c4c41 7graduated in tenths74 #af x #b cm4 test-tubes4 #i x #a cm4 test-tubes4 ,test-tube racks for each size of test-tube4 ,reagents ,required3 ,normal saline solution4 ,erythrocyte solution 7human red cells1 page #cbi7 ,e4 ,haemolytic serum 7for human cells7 ,h4,s4 ,complement 7fresh guinea-pig serum7 ,c4 ,specific serum from inoculated animal1 inactivated ,s4,s4 ,control pooled serum from normal animals of same species1 ,inactivated ,p4,s4 ,antigen 7cultivation upon solid medium of the organism 7e4 g41 ,b4 typhosus7 which has already served as antigen in the inoculation of the experimental animal7 ,a4 ,to prepare the antigen for use1 emulsify the whole of the bacterial growth in #e c4c4 normal saline solution4 ,shake the emulsion in a test-tube with some sterilised glass beads to ensure a homogenous emulsion1 and sterilise by heating to #fj deg4 ,c4 in a water-bath for one hour4 ,,method4-- #a4 ,take five small test-tubes1 and number them #a to #e with a grease pencil4 #b4 ,into tubes ,nos4 #a1 #c1 #d and #e pipette #j4a c4c4 of complement4 #c4 ,into tubes ,nos4 #a and #b pipette #j4b c4c4 of the serum to be tested4 #d4 ,into tube ,no4 #d pipette #j4b c4c4 of control serum4 #e4 ,into tubes ,nos4 #a1 #b1 #c and #d pipette #a c4c4 of the bacterial emulsion which forms the antigen4 #f4 ,place the whole set of tubes in the incubator at #cg deg4 ,c4 for a period of one hour4 #g4 ,remove the tubes from the incubator and pipette #a c4c4 erythrocyte solution and #d minimal haemolytic doses of the corresponding haemolysin into each tube4 #h4 ,mix thoroughly and return the tubes to the incubator at #cg deg4 ,c4 for further period of one hour4 #i4 ,at the expiration of that time transfer the tubes to the ice chest1 and allow them to stand for three hours4 #aj4 ,examine the tubes4 ,tubes #c1 #d and #e should show complete haemolysis2 tube #b should give no evidence whatever of haemolysis4 ,these tubes form the controls to the first tube1 which contains the serum to be tested4 ,in tube ,no4 #a the absence of haemolysis would indicate the presence in the serum of the inoculated animal of a specific antibody to the micro-organism used in the inoculations2 since it shows that the complement has been bound by the immune body to the bacterial antigen1 and none has been left free to enter into the haemolytic system2 on the other hand the presence of haemolysis would show that no appreciable amount of antibody has yet been formed in response to the inoculations4 ,in other words1 there is an absence of infection1 since the complement remained unfixed at the time of the addition of the erythrocyte solution and haemolytic serum1 and was ready to combine with those reagents to complete the haemolytic system4 ,the method may be shown diagramatically as under using the symbols already indicated ,test-tubes4 #a #b #c #d #e #j4a c4c4 ,c4 44444444 #j4a c4c4 ,c4 #j4a c4c4 ,c4 #j4a c4c4 ,c4 #j4b c4c4 ,s4,s4 #j4b c4c4 ,s4,s4 444444444 #j4b c4c4 ,p4,s4 44444444 ,a4 ,a4 ,a4 ,a4 44444444 ---------------------------------------- ---------------------------------- ,incubate at #cg deg4 ,c4 for one hour4 ---------------------------------------- ---------------------------------- #a c4c4 ,e4 #a4 c4c4 ,e4 #a c4c4 ,e4 #a c4c4 ,e4 #a c4c4 ,e4 ,h4,s4#d ,h4,s4#d ,h4,s4#d ,h4,s4#d ,h4,s4#d ---------------------------------------- ---------------------------------- ,incubate at #cg deg4 ,c4 for one hour4 ---------------------------------------- ---------------------------------- 787 ,no haemolysis4 ,haemolysis4 ,,note4--,it is sometimes more convenient to sensitise the erythrocytes just before they are needed4 ,this is done forty-five minutes after the experiment has been started 7page #cid1 step #f71 that is to say1 before the completion of the first period of incubation1 thus3 #a4 ,measure out into a sterile test-tube 7or flask7 five c4c4 of erythrocyte solution4 #b4 ,measure out twenty minimal haemolytic doses of haemolysin1 add to the erythrocyte solution on the test-tube4 #c4 ,allow the erythrocyte and haemolysin to remain in contact for fifteen minutes at room temperature4 ,the red cells are then sensitised and ready for use4 #d4 ,when the tubes are removed from the incubator at the end of the first hour 7i4 e41 step #g7 add #a c4c4 sensitised red cells to each tube by means of a graduated pipette4 #e4 ,mix thoroughly1 return the tubes to the incubator at #cg deg4 ,c4 and complete the experiment as previously described 7steps #h onward74 ,,xix4 ,,post-,,mortem ,,examinations ,,of ,,experimental ,,animals4 ,the post-mortem examination should be carried out as soon as possible after the death of the animal1 for it must be remembered that even in cold weather the tissues are rapidly invaded by numerous bacteria derived from the alimentary tract or the cavities of the body1 and from external sources4 ,the following outlines refer to a complete and exhaustive necropsy1 and in routine work the examination will rarely need to be carried out in its entirety4 ,,note4--,throughout the autopsy the searing irons must be freely employed1 and it must be recollected that one instrument is only to be employed to seize or cut one structure4 ,this done1 it must be regarded as contaminated and a fresh instrument taken for the next step4 ,apparatus ,required3 ,water steriliser4 ,scalpels4 ,surgical instruments3 ,scissors4 ,forceps4 ,bone forceps4 ,spear-headed platinum spatula 7,fig4 #aii74 ,searing irons 7,fig4 #aih74 ,tubes of media--bouillon and sloped agar4 ,surface plates in petri dishes 7of agar or one of its derivatives74 ,platinum loop4 ,aluminium spreader4 ,grease pencil4 ,sterile capillary pipettes 7,fig4 #ac1 a74 ,sterile glass capsules1 large and small4 ,cover-slips or slides4 ,bottles of fixing fluid 7vide page #aad7 for pieces of tissue intended for sectioning4 #a4 ,place the various instruments1 forceps1 scissors1 scalpels1 etc41 needed for the autopsy inside the steriliser and sterilise by boiling for ten minutes2 then open the steriliser1 raise the tray from the interior and rest it crosswise on the edges4 #b4 ,heat the searing irons to redness in a separate gas stove4 ,illustration3 ,,fig4 #aig4--;,apparatus for post-mortem examination1 animal on board4 #c4 ,drench the fur 7or feathers7 with lysol solution1 #b per cent4 ,this serves the twofold purpose of preventing the hairs from flying about and entering the body cavities during the autopsy1 and of rendering innocuous any vermin that may be present on the animal4 ,illustration3 ,,fig4 #aih4--,searing iron4 #d4 ,examine the cadaver carefully4 ,recollect that laboratory animals are not always hardy2 death may be due to exposure to heat or cold1 to starvation or over- or improper feeding or to the attack of rats--and not to the bacterial infection4 #e4 ,fasten the body of the animal1 ventral surface upward 7unless there is some special reason for having the dorsum exposed71 out on a board by means of copper nails driven through the extremities4 #f4 ,with sterile forceps and scalpel incise the skin in the middle line from the top of the sternum to the pubes4 ,make other incisions at right angles to the first out to the axillae and groins1 and reflect the skin in two lateral flaps4 7,place the now infected instruments on the board by the side of the body or support them on a porcelain knife rest47 ,seat of ,inoculation4-- #g4 ,inspect the seat of inoculation4 ,if any local lesion is visible1 sear its exposed surface and with the platinum loop1 remove material from the deeper parts to make tube and surface plate cultivations and cover-slip preparations4 ,collect specimens of pus or other exudation in capillary pipettes for subsequent examination4 #h4 ,inspect the neighbouring lymphatic glands and endeavour to trace the path of the virus4 #i4 ,sear the whole of the exposed surface of the thorax with the searing irons4 ,pleural ,cavity4-- #aj4 ,divide the ribs on either side of the sternum and remove a rectangular portion of the anterior chest wall with sterile scissors and a fresh pair of forceps1 exposing the heart4 ,place the infected instruments by the side of the first set4 #aa4 ,observe the condition of the anterior mediastinal glands1 the thymus and the lungs4 ,collect a quantity of pleuritic effusion1 if such is present1 in a pipette for further examination later4 #ab4 ,raise the pericardial sac in a fresh pair of forceps and burn through this structure with a searing iron4 ,collect a sample of pericardial fluid in a pipette for microscopical and cultural examination4 #ac4 ,grasp the apex of the heart in the forceps and sear the surface of the right ventricle4 #ad4 ,plunge the open point of a capillary pipette through the seared area into the ventricle and fill with blood4 ,make cultivations and cover-slip preparations of the heart blood4 #ae4 ,collect a further sample of blood or serum for subsequent investigation as to the presence of antibodies4 ,peritoneal ,cavity4-- #af4 ,sear a broad track in the middle line of the abdominal wall2 open the peritoneal cavity by an incision in the centre of the seared line4 ,observe the condition of the omentum1 the mesentery1 the viscera and the peritoneal surface of the intestines4 #ag4 ,collect a specimen of the peritoneal fluid 7or pus1 if present7 in a capillary pipette4 ,make cultivations1 tube and surface plate1 and cover-slip preparations from this situation4 #ah4 ,collect a specimen of the urine from the distended bladder in a large pipette 7in the manner indicated for heart blood71 for further examination1 by cultivations1 microscopical preparations1 and chemical analysis4 #ai4 ,collect a specimen of bile from the gall bladder in similar manner4 #bj4 ,excise the spleen and place it in a sterile capsule4 ,later1 sear the surface of this organ2 plunge the spear-headed spatula through the centre of the seared area1 twist it round between the finger and thumb1 and remove it from the organ4 ,sufficient material will be brought away in the eye in its head to make cultivations4 ,a repetition of the process will afford material for cover-slip preparations4 #ba4 ,seize one end of the spleen with sterile forceps4 ,sear a narrow band of tissue1 right around the organ and divide the spleen in this situation with a pair of scissors4 ,holding the piece of spleen in the forceps1 dab the cut surface on to a surface plate in a number of different spots4 #bb4 ,in like manner examine the other organs--liver1 lungs1 kidneys1 lymphatic glands 7mesenteric1 hepatic1 lumbar1 etc71 etc4 ,prepare cultivations and cover-slip preparations4 #bc4 ,dissect out a long bone from one upper and one lower limb and one of the largest ribs4 ,prepare cultures from the bone marrow in each case4 ,set aside these bones for the subsequent preparation of marrow films4 #bd4 ,film preparations of bone marrow are best made by the ,price-,jones method4 ,seize the bone in a pair of pliers and squeeze out some of the marrow2 receive it in a platinum loop1 and transfer to a watch glass of dissociating fluid and emulsify4 ,the dissociating fluid is a neutral #aj per cent4 solution of glycerine prepared as follows3-- ,measure out #aj c4c4 ,price's best glycerine and #ij c4c4 sterile ammonia-free distilled water4 ,mix4 ,titrate against n_/#aj sodic hydrate solution using phenolphthalein as the indicator4 ,the initial reaction is usually #j4a to #j4e2 add the calculated amount of n_/#aj sodic hydrate solution to neutralise4 #be4 ,place a loopful of fresh desiccating fluid on a #c x #a glass slide2 add a similar loopful of the marrow emulsion1 and spread very gently over the surface of the slip4 #bf4 ,allow film to dry in the air 7protected from dust7 without heating4 #bg4 ,stain with ,jenner's polychrome stain 7page #ig7 for two and a half minutes4 #bh4 ,wash with ammonia-free distilled water1 dry thoroughly and mount in xylol balsam4 ,cranial and ,spinal ,cavities4-- #bi4 ,in some instances it may be necessary 7e4 g41 experimental inoculation of rabies7 to examine the cranial cavity or to remove the spinal cord4 ,return the viscera to the abdominal cavity2 draw the flaps of skin together and secure with ,michel's steel clips4 ,draw the copper nails securing the limbs to the board1 reverse the animal and again nail the limbs down--the body now being dorsum uppermost4 #cj4 ,make a longitudinal incision in the mesial line from snout to root of tail1 and four transverse incisions--one joining the roots of the two ears1 one across the body at the level of the spinis of the scapulae1 another at the level of the costal margin and the last across the upper level of the pelvis4 ,reflect these flaps of skin4 #ca4 ,with forceps and scalpel dissect out the muscles lying in the furrow on either side of the spinal processes4 #cb4 ,cut through the bases of the transverse processes with bone forceps4 ,cut away the vault of the skull1 cut through the roots of the nerves and remove the brain and spinal cord1 place in a large glass dish for examination4 ,prepare cultivations from the cerebro-spinal fluid4 ,the removal of the brain and cord is a tedious process and during the dissection it is difficult to avoid injury to these structures4 ,the operation is1 however1 carried out very expeditiously and neatly with the aid of the surgical engine 7vide page #cfa74 ,a small circular saw is fitted to the hand piece4 ,the bones of the skull are cut through and the whole of the vault removed1 exposing the entire vertex of the brain4 ,similarly all the spinous processes can be removed in one string by running the saw down first one side of the spinal column and then the other4 ,in this way ample space for the removal of the nervous tissues is obtained with a minimum of labour4 #cc4 ,having completed the preparation of cultures remove small portions of various organs at leisure and place each in separate bottles of fixing fluid for future sectioning4 ,affix to each bottle a label bearing all necessary details as to its contents4 #cd4 ,if necessary1 remove portions of the organs for preservation and display as museum specimens 7vide page #djd74 #ce4 ,gather up all the infected instruments1 return them to the steriliser1 and disinfect by boiling for ten minutes4 ,illustration3 ,,fig4 #aii4--,spear-headed platinum spatula 7actual size47 #cf4 ,sprinkle dry sawdust into the exposed body cavities to absorb blood and fluid4 ,cover the body with blotting or filter paper1 moistened with #b per cent4 lysol solution4 ,place in a galvanised iron pail1 provided with a lid1 ready for transport to the crematorium4 #cg4 ,cremate the cadaver together with the board upon which it is fixed4 #ch4 ,stain the cover-slip preparations by suitable methods and examine microscopically4 #ci4 ,incubate the cultivations and examine carefully from day to day4 #dj4 ,make full notes of the condition of the various body cavities and of the viscera immediately the autopsy is completed2 and add the result of the microscopical and cultural investigation when available4 ,as part of the card index system in use in the author's laboratory already referred to 7vide page #cce7 there is a special yellow card for ,p-,m notes4 ,on the face of the card are printed headings for various data--some of which are sometimes unintentionally omitted--and on the reverse is a schematic figure which can be utilised for indicating the position of the chief lesions in the cadaver of any of the laboratory animals4 ,,autopsy ,,card ,laboratory ,no4 ,date ,animal ,no4 in ,series ,symbols3 male female ,weight ---------------------------------------- -------------------------------- ,died 7or killed7 o'clock m4 ,autopsy made o'clock m4 ---------------------------------------- -------------------------------- ,notes on ,post ,mortem ,examinations4 ,general4 ,a4 ,seat of ,inoculation4 ,b4 ,thoracic ,cavity4 ,c4 ,abdominal ,cavity4 ,d4 ,cranial ,cavity4 ----------------------- --------------------------------------- ,bacteriological ,histological ,organs ,preserved4 ,examination4 ,examination4 ,a4 ,b4 ,c4 ,d4 ,illustration3 ,,fig4 #bjj4--;,front of post-mortem card4 #da4 ,finally1 the results of the action of the organism or organisms isolated may be correlated with the symptoms observed during life and the observations summarised under the following headings3 ,tissue changes3 #a4 ,local--i4 e41 produced in the neighbourhood of the bacteria4 ,position3 7a7 ,at primary lesion4 7b7 ,at secondary foci4 ,character3 7a7 ,vascular changes and tissue ,acute reactions4 or 7b7 ,degeneration and necrosis4 chronic4 #b4 ,general 7i4 e41 produced at a distance from the bacteria1 by absorption of toxins73 7a7 ,in special tissues--e4 g41 nerve cells and fibres1 secreting cells1 vessel walls1 etc4 7b7 ,general effects of malnutrition1 etc4 ,symptoms3 7a7 ,associated with known tissue changes4 7b7 ,without known tissue changes4 ,illustration3 ,,fig4 #bja4--;,back of post-mortem card4 ,permanent ,preparations--,museum ,specimens4-- ,i4 ,tissues4--,the naked-eye appearances of morbid tissues may be preserved by the following method3 #a4 ,remove the tissue or organ from the cadaver as soon after death as possible1 using great care to avoid distortion or injury4 #b4 ,place it in a wide-mouthed stoppered jar1 large enough to hold it conveniently1 resting on a pad of cotton-wool1 and arrange it in the position it is intended to occupy 7but if it is intended to show a section of the tissue or organ1 do not incise it yet74 #c4 ,cover with the ,kaiserling fixing solution1 and stopper the jar2 allow the tissues to remain in this solution for from forty-eight hours to seven days 7according to size7 to fix4 ,make any necessary sections4 ,kaiserling modified solution is prepared as follows3 ,weigh out ,potassium acetate #cj grammes4 ,potassium nitrate #ae grammes4 and dissolve in ,distilled water #ajjj c4c4 then add ,formalin #aej c4c4 ,filter4 ,this fixing solution can be used repeatedly so long as it remains clear4 ,even when it has become turbid1 if simple filtration is sufficient to render it clear1 the filtrate may be used again4 #d4 ,transfer the tissue to a bath of methylated spirit 7#ie per cent47 for thirty minutes to one hour4 #e4 ,remove to a fresh bath of spirit and watch carefully4 ,when the natural colours show in their original tints1 average time three to six hours1 remove the tissues from the spirit bath1 dry off the spirit from the cut surfaces by mopping with a soft cloth1 then transfer to the mounting solution4 ,jore's mounting solution 7modified7 consists of ,glycerine #ejj c4c4 ,distilled water #gej c4c4 ,formalin #b c4c4 ,equally good but much cheaper is ,frost's mounting solution3 ,potassium acetate #afj grammes4 ,sodium fluoride #hj grammes4 ,chloral hydrate #hj grammes4 ,cane sugar 7,tate's cubes7 #c1ejj grammes4 ,saturated thymol water #h1jjj c4c4 #f4 ,after twenty-four hours in this solution1 or as soon as the tissue sinks1 transfer to a museum jar1 fill with fresh mounting solution1 and seal4 #f;a4 ,or transfer to museum jar and fill with liquefied gelatine1 to which has been added #a per cent4 formalin4 ,cover the jar and allow the gelatine to set4 ,when solid1 seal the cover of the jar in place4 #g4 ,to seal the museum preparation first warm the glass plate which forms the cover4 ,this is most conveniently done by placing the cleaned and polished cover-plate upon a piece of asbestos millboard over a bunsen flame turned low4 #h4 ,smear an even layer of hot cement over the flange of the jar4 ,the cement is prepared as follows3 ,weigh out and mix in an iron ladle ,gutta percha 7pure7 #d parts4 ,asphaltum #e parts4 and melt together over a bunsen flame1 stirring with an iron rod until solution is complete4 #i4 ,invert the glass plate over the jar and press down firmly into the cement4 ,place a piece of asbestos board on the top and on that rest a suitable weight until the cement is cold and has thoroughly set4 #aj4 ,trim off any projecting pieces of cement with an old knife1 burr over the joint between jar and cover-plate with a hot smooth piece of metal 7e4 g41 the searing iron74 #aa4 ,paint a narrow band of ,japan black to finish off1 round the joint1 overlapping on to the cover-plate4 ,,ii4 ,tube ,cultivations of ,bacteria4--,when showing typical appearances these may be preserved1 if not permanently1 at least for many years1 as museum specimens1 by the following method3 #a4 ,take a large glass jar #be cm4 high by #ah cm4 diameter1 with a firm base and a broad flange1 carefully ground1 around the mouth4 ,the jar must be fitted with a disc of plate glass ground on one side1 to serve as a lid4 #b4 ,smear a thick layer of resin ointment 7,b4,p47 on the flange around the mouth of the jar4 #c4 ,cover the bottom of the jar with a layer of cotton-wool and saturate it with formalin4 #d4 ,remove the cotton-wool plug from the culture tubes and place them1 mouth upward1 inside the jar4 7,if water of condensation is present in any of the culture tubes1 it should be removed by means of a capillary pipette before placing the tubes in the formalin chamber47 #e4 ,adjust the glass disc1 ground side downward1 over the mouth of the jar and secure it by pressing it firmly down into the ointment1 with a rotary movement4 #f4 ,remove the tubes from the formalin chamber after the lapse of a week1 and dry the exterior of each4 ,illustration3 ,,fig4 #bjb4--;,bulloch's tubes4 #g4 ,seal the open mouth of each tube in the blowpipe flame and label4 ,if the cultivations are intended for museum purposes when they are first planted1 it is more convenient to employ ,bulloch's tubes4 ,these are slightly longer than the ordinary tubes1 and are provided with a constriction some #b cm4 below the mouth 7,fig4 #bjb7--;a feature which renders sealing in the blowpipe flame an easy matter4 ,,xx4 ,,the ,,study ,,of ,,the ,,pathogenic ,,bacteria4 ,the student1 who has conscientiously worked out the methods1 etc41 previously dealt with1 is in a position to make accurate observations and to write precise descriptions of the results of such observations4 ,he is1 therefore1 now entrusted with pure cultivations of the various pathogenic bacteria1 in order that he may study the life-history of each and record the results of his own observations--to be subsequently corrected or amplified by the demonstrator4 ,in this way he is rendered independent of text-book descriptions1 the statements in which he is otherwise too liable to take for granted1 without personally attempting to verify their accuracy4 ,during the course of this work attention must also be directed1 as occasion arises1 to such other bacteria1 pathogenic or saprophytic1 as are allied to the particular organisms under observation1 or so resemble them as to become possible sources of error1 by working them through on parallel lines--in other words the various bacteria should be studied in groups4 ,in the following pages the grouping in use in the author's elementary classes for medical and dental students and for candidates for the ,public ,health service is adopted1 since a fairly long experience has completely vindicated the value and utility of this arrangement1 and by its means a fund of information is obtained with regard to the resemblances and differences1 morphological and cultural1 of a large number of bacteria4 ,the fact that some bacteria appear in more than one of these groups1 so far from being a disadvantage1 is a positive gain to the student1 since with repetition alone will the necessary familiarity with the cultural characters of important bacteria be acquired4 ,the study of the various groups will of course vary in detail with individual demonstrators1 and with the student's requirements--the general line it should take is indicated briefly in connection with the first group only 7pages #daj-daa74 ,this section should be carefully worked through before the student proceeds to the study of bacterioscopical analysis4 ,it is customary to commence the study of the pathogenic bacteria with the ,organisms of ,suppuration4 ,this is a large group1 for all the pathogenic bacteria possess the power1 under certain conditions1 of initiating purely pyogenic processes in place of or in addition to their specific lesions1 7e4 g41 ,bacillus tuberculosis1 ,streptococcus lanceolatus1 ,bacillus typhosus1 etc474 ,there are1 however1 a certain few organisms which commonly express their pathogenicity in the formation of pus4 ,these are usually grouped together under the title of pyogenic bacteria1 as distinct from those which only occasionally exercise a pyogenic role4 ,the organisms included in this group are3 #a4 ,staphylococcus pyogenes albus4 #b4 ,staphylococcus pyogenes aureus4 #c4 ,staphylococcus pyogenes citreus4 #d4 ,streptococcus pyogenes longus4 #e4 ,micrococcus tetragenus4 #f4 ,bacillus pyocyaneus4 #g4 ,bacillus pneumoniae4 and in certain special tissues #h4 ,micrococcus gonorrhoeae4 #i4 ,micrococcus intracellularis meningitidis 7,meningococcus74 #aj4 ,micrococcus catarrhalis4 #aa4 ,bacillus aegypticus 7,koch-,weeks ,bacillus74 ,the group may with advantage be subdivided as indicated in the following pages3 ,i4 ,pyogenic cocci4 ,staphylococcus pyogenes albus4 ,staphylococcus pyogenes aureus4 ,staphylococcus pyogenes citreus4 to contrast with ,micrococcus candicans4 ,micrococcus agilis4 #a4 ,prepare subcultivations from each3 ,bouillon1 ,agar streak1 ,blood serum1 ,litmus milk4 and incubate at #cg deg4 ,c4 ,agar streak1 ,gelatine stab1 ,potato4 and incubate at #bj deg4 ,c4 ,compare the naked-eye appearances of the cultures from day to day4 ,note ,m4 agilis refuses to grow at #cg deg4 ,c4 #b4 ,make hanging-drop preparations from the bouillon and agar cultivations after twenty-four hours' incubation4 ,examine microscopically and compare4 ,note the locomotive activity of ,m4 agilis and the ,brownian movement of the remaining micrococci4 #c4 ,prepare cover-slip films from the agar cultures1 after twenty-four hours' incubation4 ,stain for flagella by the modified ,pitfield's method4 ,note ,m4 agilis is the only micrococcus showing flagella4 #d4 ,make microscopical preparations of each from all the various media after twenty-four and forty-eight hours and three days' incubation4 ,stain carbolic methylene-blue1 carbolic fuchsin1 and ,gram's method4 ,examine the films microscopically and compare4 ,note in the ,gram preparation1 the ,gram negative character of certain individual cocci in each film prepared from the three days' growth--such cocci are dead4 #e4 ,stain section of kidney tissue provided 7showing abscess formation by ,staphylococcus aureus7 by ,gram's method1 and counterstain with cosin4 #f4 ,stain film preparation of pus from an abscess 7containing ,staphylococcus pyogenes aureus7 with carbolic methylene-blue and also by ,gram's method1 counterstained with cosin4 #g4 ,inoculate#ae a white mouse subcutaneously with three loopfuls of a forty-eight-hour agar cultivation of the ,staphylococcus aureus1 emulsified with #j4b c4c4 sterile broth4 ,observe carefully during life1 and when death occurs make a careful post-mortem examination4 ,,ii4 ,pyogenic cocci4 ,micrococcus gonorrhoeae4 ,micrococcus intracellularis meningitidis 7meningococcus74 ,micrococcus catarrhalis4 ,micrococcus tetragenus4 ,micrococcus paratetragenus4 ,,iii4 ,pyogenic cocci4 ,streptococcus pyogenes longus4 ,streptococcus of bovine mastitis4 ,streptococcus lanceolatus 7,diplococcus pneumoniae or pneumococcus74 to contrast with ,streptococcus brevis4 ,streptococcus lebensis4 ,,iv4 ,pyogenic bacilli4 ,bacillus pneumoniae 7,friedlaender74 ,bacillus rhinoscleromatis4 ,bacillus lactis aerogenes4 ,v4 ,pyogenic bacilli4 ,bacillus pyocyaneus4 to contrast with ,bacillus fluorescens liquefaciens4 ,bacillus fluorescens non-liquefaciens4 ,,vi4 ,pneumonia group4 ,streptococcus lanceolatus 7pneumococcus74 ,bacillus pneumoniae 7,friedlaender74 ,streptococcus pyogenes longus4 ,,vii4 ,diphtheroid group4 ,bacillus diphtheriae 7,klebs-,loeffler74 ,bacillus ,hoffmanni4 ,bacillus xerosis4 ,bacillus septus4 ,,viii4 ,coli-typhoid group4 ,b4 typhi abdominalis 7,b4 typhosus74 ,b4 coli communis4 ,b4 enteritidis 7,gaertner74 to contrast with ,b4 aquatilis sulcatus4 ,,ix4 ,escherich group4 ,b4 coli communis 7,escherich74 ,b4 coli communior4 ,b4 lactis aerogenes4 ,b4 cloacae4 ,x4 ,gaertner group4 ,bacillus enteritidis 7,gaertner74 ,b4 paratyphosus ,a4 ,b4 paratyphosus ,b4 ,bacillus cholerae suum 7,hog ,cholera74 ,b4 psittacosis4 ,,xi4 ,eberth group4 ,b4 typhosus 7,eberth74 ,b4 dysenteriae 7,shiga74 ,b4 dysenteriae 7,flexner74 ,b4 faecalis alcaligines4 ,,xii4 ,spirillum group4 ,vibrio cholerae4 ,vibrio metschnikovi4 to contrast with ,vibrio proteus 7,finkler and ,prior74 ,spirillum rubrum4 ,spirillum rugula4 ,,xiii4 ,anthrax group4 ,bacillus anthracis4 to contrast with ,bacillus subtilis4 ,bacillus mycoides4 ,bacillus mesentericus fuscus4 ,,xiv4 ,acid fast group4 ,bacillus tuberculosis 7human74 7bovine74 7avian74 7fish74 to contrast with ,bacillus phlei 7,timothy grass bacillus74 ,butter bacillus of ,rabinowitch4 ,,xv4 ,plague group4 ,bacillus pestis4 ,b4 septicaemiae haemorrhagicae4 ,b4 suipestifer4 ,,xvi4 ,influenzae group4 ,b4 influenzae4 ,bacillus aegypticus 7,koch-,weeks74 ,bacillus pertussis4 ,,xvii4 ,miscellaneous4 ,bacillus leprae4 ,bacillus mallei4 ,micrococcus melitensis4 ,,xviii4 ,streptothrix group4 ,streptothrix actinomycotica4 ,streptothrix madurae4 to contrast with ,cladothrix nivea4 ,,xix4 ,tetanus group4 ,bacillus tetani4 ,bacillus oedematis maligni4 ,bacillus chauvei 7symptomatic anthrax74 ,,xx4 ,enteritidis sporogenes group4 ,bacillus enteritidis sporogenes4 ,b4 botulinus4 ,b4 butyricus4 ,b4 cadaveris4 ,,footnotes3 #ae ,see note on ,vivisection ,license1 page #ccd4 ,,xxi4 ,,bacteriological ,,analyses4 ,each bacteriological or bacterioscopical analysis of air1 earth1 sewage1 various food-stuffs1 etc41 includes1 as a general rule1 two distinct investigations yielding results of very unequal value3 #a4 ,quantitative4 #b4 ,qualitative4 ,the first is purely quantitative and as such is of minor importance as it aims simply at enumerating 7approximately7 the total number of bacteria present in any given unit of volume irrespective of the nature and character of individual organisms4 ,the second and more important is both qualitative and quantitative in character since it seeks to accurately identify such pathogenic bacteria as may be present while1 incidentally1 the methods advocated are calculated to indicate1 with a fair degree of accuracy1 the numerical frequency of such bacteria1 in the sample under examination4 ,the general principles underlying the bacteriological analyses of water1 sewage1 air and dust1 soil1 milk1 ice cream1 meat1 and other tinned stuffs1 as exemplified by the methods used by the author1 are indicated in the following pages1 together with the methods of testing filters and chemical germicides2 and the technique there set out will be found to be capable of expansion and adaptation to any circumstance or set of circumstances which may confront the student4 ,controls4--,the necessity for the existence of adequate controls in all experimental work cannot be too urgently insisted upon4 ,every batch of plates that is poured should include at least one of the presumably sterile medium2 plate or tube cultures should be made from the various diluting fluids2 every tube of carbohydrate medium that is inoculated should go into the incubator in company with a similar but uninoculated tube1 and so on4 ,,bacteriological ,,examination ,,of ,,water4 ,the bacteria present in the water may comprise not only varieties which have their normal habitat in the water and will consequently develop at #bj deg4 ,c41 but also if the water has been contaminated with excremental matter1 varieties which have been derived from1 or are pathogenic for1 the animal body1 and which will only develop well at a temperature of #cg deg4 ,c4 ,in order to demonstrate the presence of each of these classes it will be necessary to incubate the various cultivations at each of these temperatures4 ,further1 the sample of water may contain moulds1 yeasts1 or torulae1 and the development of these will be best secured by plating in wort gelatine and incubating at #bj deg4 ,c4 #a4 ,quantitative4-- ,collection of the ,sample4--,the most suitable vessels for the reception of the water sample are small glass bottles1 #fj c4c4 capacity1 with narrow necks and overhanging glass stoppers 7to prevent contamination of the bottle necks by falling dust74 ,these must be carefully sterilised in the hot-air steriliser 7vide page #ca74 7a7 ,if the sample is obtained from a tap or pipe1 turn on the water and allow it to run for a few minutes4 ,remove the stopper from the bottle and retain it in the hand whilst the water is allowed to run into the bottle and three parts fill it4 ,replace the stopper and tie it down1 but do not seal it4 7b7 ,if the sample is obtained from a stream1 tank1 or reservoir1 fasten a piece of stout wire around the neck of the bottle1 remove the stopper1 and retain it in the hand4 ,then1 using the wire as a handle1 plunge the bottle into the water1 mouth downward1 until it is well beneath the surface2 then reverse it1 allow it to fill1 and withdraw it from the water4 ,pour out a few cubic centimetres of water from the bottle1 replace the stopper1 and tie it down4 ,illustration3 ,,fig4 #bjc4--;,esmarch's collecting bottle for water samples4 7c7 ,if the sample is obtained from a lake1 river or the sea2 or when it is desired to compare samples taken at varying depths1 the apparatus designed by v4 ,esmarch 7,fig4 #bjc7 is employed4 ,in this the sterilised bottle is enclosed in a weighted metal cage which can be lowered1 by means of a graduated line1 until the required depth is reached4 ,at this point the bottle is opened by a thin wire cord attached to the stopper2 when the bottle is full 7as judged by the air bubbles ceasing to rise7 the pull on the cord is released and the tension of the spiral spring above the stopper again forces it into the neck of the bottle4 ,when the apparatus is taken out of the water1 the small bottles are filled from it1 and packed in the ice-box mentioned below4 ,an inexpensive substitute for ,esmarch's bottle can be made in the laboratory thus3 ,select a wide-mouthed glass stoppered bottle of about #ejj c4c4 capacity 7about #bj cm4 high and #h cm4 in diameter74 ,remove the glass stopper and insert a rubber cork with two perforations in its place4 ,through one perforation pass a piece of glass tubing about #e cm4 long and through the other a piece #bb cm4 long1 reaching to near the bottom of the bottle1 each tube projecting about #b4e cm4 above the rubber stopper4 ,plug the open ends of the tubes with cotton wool4 ,secure the stopper in place with thin copper wire4 ,illustration3 ,,fig4 #bjd4--,thresh's deep water sampling bottle4 ,sterilise the fitted bottle in the autoclave4 ,remove the cotton wool plugs and connect the projecting tubes by a piece of loosely fitting stout rubber pressure tubing about #e cm4 long1 previously sterilised by boiling4 ,take a piece of stout rubber cord about #cc cm4 long1 and of #aj mm4 diameter 7such as is used for door springs7 thread a steel split ring upon it and secure the free ends tightly to the neck of the bottle by cord or catgut4 ,attach the cord used for lowering the bottle into the water to the split ring on the rubber suspender4 ,the best material for this purpose is cotton insulated electric wire knotted at every metre4 ,connect the split ring also with the short piece of rubber tubing uniting the two glass tubes by a piece of catgut 7or thin copper wire7 of such length that when the bottle is suspended there is no pull upon the rubber tube1 but which1 however1 will be easily jerked off when a sharp pull is given to the suspending cord4 ,now wind heavy lead tubing about #a cm4 diameter around the upper part of the bottle1 starting at the neck just above the shoulder4 ,this ensures the sinking of the bottle in the vertical position 7,fig4 #bjd74 ,the apparatus being arranged is lowered to the required depth1 a sharp jerk is then given to the suspending cord1 which detaches the rubber tube and so opens the two glass tubes4 ,water enters through the longer tube and the air is expelled through the shorter tube4 ,the bubbles of air can be seen or heard rising through the water1 until the bottle is nearly full1 a small volume of compressed air remaining in the neck of the bottle4 ,as the apparatus is raised1 the air thus imprisoned expands1 and prevents the entry of more water from nearer the surface4 ,illustration3 ,,fig4 #bje4--;,ice-box for transmission of water samples1 etc4 ,transport of ,sample4--,if the examination of the sample cannot be commenced immediately1 steps must be taken to prevent the multiplication of the bacteria contained in the water during the interval occupied in transit from the place of collection to the laboratory4 ,to this end an ice-box such as that shown 7in ,fig4 #bje7 is essential4 ,it consists of a double-walled metal cylinder into which slides a cylindrical chamber of sufficient capacity to accommodate four of the #fj c4c4 bottles2 this in turn is covered by a metal disc--the three portions being bolted together by thumb screws through the overhanging flanges4 ,when in use1 place the bottles1 rolled in cotton-wool1 in the central chamber1 pack the space between the walls with pounded ice1 securely close the metal box by screwing down the fly nuts1 and place it in a felt-lined wooden case4 7,it has been shown that whilst bacteria will survive exposure to the temperature of melting ice1 practically none will multiply at this temperature47 ,on reaching the laboratory1 the method of examination consists in adding measured quantities of the water sample to several tubes of nutrient media previously liquefied by heat1 pouring plate cultivations from each of these tubes1 incubating at a suitable temperature1 and finally counting the colonies which make their appearance on the plates4 ,apparatus ,required3 ,plate-levelling stand4 ,case of sterile plates4 ,case of sterile pipettes1 #a c4c4 7in tenths of a cubic centimetre74 ,case of sterile pipettes1 #aj c4c4 7in tenths of a cubic centimetre74 ,case of sterile capsules1 #be c4c4 capacity4 ,tubes of nutrient gelatine4 ,tubes of nutrient agar4 ,tubes of wort gelatine4 ,one #bej c4c4 flask of sterile distilled water4 ,tall cylinder containing #b per cent4 lysol solution4 ,bunsen burner4 ,grease pencil4 ,water-bath regulated at #db deg4 ,c4 ,,method4-- #a4 ,arrange the plate-levelling platform with its water compartment filled with water1 at #de deg4 ,c4 #b4 ,number the agar tubes1 consecutively1 #a to #f2 the gelatine tubes1 consecutively1 #a to #f1 and the wort tubes1 #a1 #b1 and #c4 ,flame the plugs and see that they are not adherent to the lips of the tubes4 #c4 ,place the agar tubes in boiling water until the medium is melted1 then transfer them to the water-bath regulated at #db deg4 ,c4 ,liquefy the nutrient gelatine and wort gelatine tubes by immersing them in the same water-bath4 #d4 ,remove the bottle containing the water sample from the ice-box1 distribute the bacterial contents evenly throughout the water by shaking1 cut the string securing the stopper1 and loosen the stopper1 but do not take it out4 ,illustration3 ,,fig4 #bjf4--,withdrawing water from water sample bottle4 #e4 ,remove one of the #a c4c4 pipettes from the case1 holding it by the plain portion of the tube4 ,pass the graduated portion twice through the ,bunsen flame4 ,tilt the bottle containing the water sample on the bench holding the neck between the middle and ring fingers of the left hand2 grasp the head of the stopper between the forefinger and thumb1 and remove it from the bottle4 #f4 ,pass the pipette into the mouth of the bottle1 holding its point well below the surface of the water 7,fig4 #bjf74 ,suck up rather more than #a c4c4 into the pipette and allow the pipette to empty2 this moistens the interior of the pipette and renders accurate measurement possible4 ,now draw up exactly #a c4c4 into the pipette4 ,withdraw the pipette from the bottle1 replace the stopper1 and stand the bottle upright4 #g4 ,take the first melted agar tube in the left hand1 remove the cotton-wool plug1 and add to its contents #j4e c4c4 of the water sample from the pipette2 replug the tube and replace it in the water-bath4 ,in a similar manner add #j4c c4c4 water to the contents of the second tube1 and #j4b c4c4 to the contents of the third4 #h4 ,in a similar manner add #a c4c4 of the sample to the contents of the fourth tube4 #i4 ,similarly1 add #j4e c4c4 and #j4a c4c4 respectively to the contents of the fifth and sixth tubes4 #aj4 ,drop the pipette into the cylinder containing lysol solution4 #aa4 ,mix the water sample with the medium in each tube in the manner described under plate cultivations2 pour a plate from each tube4 ,label each plate with 7a7 the distinctive number of the sample1 7b7 the quantity of water sample it contains1 and 7c7 the date4 #ab4 ,pour the contents of a tube of liquefied agar--not inoculated--into a ,petri dish to act as a control to demonstrate the sterility of the batch of agar employed4 #ac4 ,allow the plates to set1 and incubate at #cg deg4 ,c4 #ad4 ,empty the water chamber of the levelling apparatus and refill it with ice-water4 #ae4 ,by means of the sterile #aj c4c4 pipette deliver #i4i c4c4 sterile distilled water into a sterile glass capsule4 #af4 ,add #j4a c4c4 of the water sample to the #i4i c4c4 sterile water in the capsule4 ,this will give a dilution of #a in #ajj4 #ag4 ,plant the six tubes of nutrient gelatine in the following manner3 ,to the first tube add #j4e c4c4 of the water sample direct from the bottle2 to the second1 #j4c c4c42 and to the third1 #j4b c4c42 and pour a plate of each tube4 ,to the fourth tube add #j4e c4c4 of the diluted water sample from the capsule2 to the fifth1 #j4c c4c42 and to the sixth1 #j4b c4c42 and pour a plate from each4 #ah4 ,label each plate with the quantity of the water sample it contains--that is1 #j4e c4c41 #j4c c4c41 #j4b c4c41 #j4jje c4c41 #j4jjc c4c41 and #j4jjb c4c4 #ai4 ,pour a control 7uninoculated7 gelatine plate4 #bj4 ,allow the plates to set1 and incubate at #bj deg4 ,c4 #ba4 ,to the first tube of liquefied wort gelatine add #j4e c4c4 water sample2 to the second1 #j4c c4c42 and to the third1 #j4b c4c4 #bb4 ,label the plates1 allow them to set1 and incubate at #bj deg4 ,c4 #bc4 ,count and record the number of colonies that have developed upon the agar at #cg deg4 ,c4 after forty-eight hours' incubation4 #bd4 ,note the number of colonies present on each of the gelatine and wort gelatine plates after forty-eight hours' incubation4 #be4 ,replace the gelatine and wort plates in the incubator2 observe again at three days1 four days1 and five days4 #bf4 ,calculate and record the number of organisms present per cubic centimetre of the original water from the average of the six gelatine plates at the latest date possible up to seven days--the presence of liquefying bacteria may render the calculation necessary at an earlier date1 hence the importance of daily observations4 ,method of ,counting4--,the most accurate method of counting the colonies on each of the plates is by means of either ,jeffery's or ,pakes' counting disc4 ,each of these discs consists of a piece of paper1 upon which is printed a dead black disc1 subdivided by concentric circles and radii1 printed in white4 ,in ,jeffery's counter 7,fig4 #bjg71 each subdivision has an area of #a square centimetre2 in ,pakes' counter 7,fig4 #bjh71 radii divide the circle into sixteen equal sectors1 and counting is facilitated by concentric circles equidistant from the centre4 ,illustration3 ,,fig4 #bjg4--;,jeffery's disc1 reduced4 ,illustration3 ,,fig4 #bjh4--,pakes' disc1 reduced4 7a7 ,in the final counting of each plate1 place the plate over the counting disc1 and centre it1 if possible1 making its periphery coincide with one or other of the concentric circles4 7b7 ,remove the cover of the plate1 and by means of a hand lens count the colonies appearing in each of the sectors in turn4 ,make a note of the number present in each4 7c7 ,if the colonies present are fewer than #ejj1 the entire plate should be counted4 ,if1 however1 they exceed this number1 enumerate one-half1 or one-quarter of the plate1 or count a sector here and there1 and from these figures estimate the number of colonies present on the entire plate4 ,in practice it will be found that ,pakes' disc is more suitable for the former class of plate2 ,jeffery's disc for the latter4 ,it should be recollected however that unless the plates have been carefully leveled and the medium is of equal thickness all over it is useless to try and average from small areas--since where the medium is thick all the bacteria will develop1 where the layer is a thin one1 only a few bacteria will find sufficient pabulum for the production of visible colonies4 ,it will be noted that the quantities of water selected for addition to each set of tubes of nutrient media have been carefully chosen in order to yield workable results even when dealing with widely differing samples4 ,plates prepared in agar with #j4a c4c4 and in gelatin with #j4jb c4c4 can be counted even when large numbers of bacteria are present in the sample2 whereas if micro-organisms are relatively few1 agar plate #d and gelatine plate #a will give the most reliable counts4 ,again the counts of the plates in a measure control each other2 for example1 the second and third plates of each gelatine series should together contain as many colonies as the first1 and the second should contain about half as many more than the third and so on4 #b4 ,qualitative ,examination4-- ,collection of ,sample4--,the water sample required for the routine examination1 which it will be convenient to consider first1 amounts to about #aaj c4c4 ,it is collected in the manner previously described 7vide page #daf72 similar bottles are used1 and if four are filled the combined contents1 amounting to about #bdj c4c41 will provide ample material for both the qualitative and quantitative examinations4 ,unless the examination is to be commenced at once1 the ice-box must be employed1 otherwise water bacteria and other saprophytes will probably multiply at the expense of the microbes indicative of pollution1 and so increase the difficulties of the investigation4 ,in the routine examination of water supplies it is customary to limit the qualitative examination to a search for ,a4 ,b4 coli and its near allies4 ,b4 ,streptococci1 organisms which are frequently spoken of as microbes of indication1 as their presence is held to be evidence of pollution of the water by material derived from the mammalian alimentary canal1 and so to constitute a danger signal4 ,c4 ,some observers still attach importance to the presence of ,b4 enteritidis sporogenes1 but as the search for this bacterium1 7relatively scarce in water7 necessitates the collection of a fairly large quantity of water it is not usually included in the routine examination4 ,in the case of water samples examined during the progress of an epidemic1 of new supplies and of unknown waters the search is extended to embrace other members of the coli-typhoid group2 and on occasion the question of the presence or absence of ,vibrio cholerae or 7more rarely7 such bacteria as ,b4 anthracis or ,b4 tetani1 may need investigation4 ,when pathogenic or excremental bacteria are present in water1 their numbers are relatively few1 owing to the dilution they have undergone1 and it is usual in commencing the examination1 to adopt one or other of the following methods3 ,a4 ,enrichment1 in which the harmless non-pathogenic bacteria may be destroyed or their growth inhibited1 whilst the growth of the parasitic bacteria is encouraged4 ,this is attained by so arranging the environment1 7i4 e41 ,media1 incubation temperature1 and atmosphere7 as to favor the growth of the pathogenic organisms at the expense of the harmless saprophytes4 ,b4 ,concentration1 whereby all the bacteria present in the sample of water1 pathogenic or otherwise1 are concentrated in a small bulk of fluid4 ,this is usually effected by filtration of the water sample through a porcelain filter candle1 and the subsequent emulsion of the bacterial residue remaining on the walls of the candle with a small measured quantity of sterile bouillon4 ,a4 ,enrichment ,method4 7,dealing with the demonstration of bacteria of intestinal origin47 ,apparatus ,required 7,preliminary ,stage73 ,incubator running at #db deg4 ,c4 ,case of sterile pipettes1 #a c4c4 graduated in tenths4 ,case of sterile pipettes1 #aj c4c4 graduated in c4c4 ,case of sterile pipettes1 graduated to deliver #be c4c4 ,tubes of bile salt broth 7vide page #ahj74 ,flask of double strength bile salt broth 7vide page #aii74 ,tubes of litmus silk4 ,sterile flasks1 #bej c4c4 capacity4 ,buchner's tubes4 ,tabloids pyrogallic acid4 ,tabloids sodium hydrate4 ,bunsen burner4 ,grease pencil4 7,later stage73 ,incubator running at #cg deg4 ,c4 ,surface plates of nutrose agar 7see page #bcb74 ,aluminium spreader4 ,tubes of various media1 including carbohydrate media4 ,agglutinating sera1 etc4 ,,method4-- #a4 ,number a set of bile salt broth1 tubes #a-e1 and a duplicate set #a;a-#e;a4 #b4 ,number one flask #g and another #h4 #c4 ,to ,tubes ,no4 #a and #a;a add #j4a c4c4 water sample4 ,to ,tubes ,no4 #b and #b;a add #a c4c4 water sample4 ,to ,tubes ,no4 #c and #c;a add #b c4c4 water sample4 ,to ,tubes ,no4 #d and #d;a add #e c4c4 water sample4 ,to ,tubes ,no4 #e and #e;a add #aj c4c4 water sample4 #d4 ,put up all the tubes in ,buchner's tubes and incubate anaerobically at #db deg4 ,c4 ,,note4--,the bile salt medium is particularly suitable for the cultivation of bacteria of intestinal origin1 and at the same time inhibits the growth of bacteria derived from other sources4 ,the anaerobic conditions likewise favor the multiplication of intestinal bacteria1 and also their fermentative activity4 ,the temperature #db deg4 ,c4 destroys ordinary water bacteria and inhibits the growth of many ordinary mesophilic bacteria4 #e4 ,pipette #be c4c4 of double strength bile salt broth into flask #f1 and #ej c4c4 double strength bile salt broth into flask #g4 #f4 ,pipette #be c4c4 water sample into flask #f1 and #ej c4c4 water sample into flask #g4 #g4 ,incubate the two flasks aerobically at #db deg4 ,c4 #h4 ,after twenty-four hours incubation note in each culture3 a4 ,the presence or absence of visible growth4 b4 ,the reaction of the medium as indicated by the colour change1 if any1 the litmus has undergone4 c4 ,the presence or absence of gas formation1 as indicated by a froth on the surface of the medium1 and the collection of gas in the inner gas tube4 #i4 ,replace those tubes which show no signs of growth in the incubator4 ,examine after another period of twenty-four hours 7total forty-eight hours incubation7 with reference to the same points4 #aj4 ,remove culture tubes which show visible growth from the ,buchner's tubes1 whether acid production and gas formation are present or not4 #aa4 ,examine all tubes which show growth by hanging-drop preparations4 ,note such as show the presence of chains of cocci4 #ab4 ,prepare surface plate cultivations upon nutrose agar from each tube that shows growth either macroscopically or microscopically1 and incubate for twenty-four hours aerobically at #cg deg4 ,c4 #ac4 ,examine the growth on the plates either with the naked eye or with the help of a small hand lens4 ,practice will facilitate the recognition of colonies of the coli group1 the typhoid group and the paratyphoid group2 also those due to the growth of streptococci4 ,the investigation from this stage proceeds along two divergent lines of enquiry--the first being concerned with the identity of the bacilli--typhoid bacilli1 the second with that of the cocci4 ,a4 ,b4 ,coli and its allies4 #ad4 ,pick off coliform or typhiform colonies2 make streak or smear subcultivations upon nutrient agar2 incubate aerobically for twenty-four hours at #cg deg4 ,c4 #ae4 ,examine the growth in each tube carefully both macroscopically and microscopically4 ,if the growth is impure1 replate on nutrose agar1 pick off colonies and subcultivate again4 ,when the growth in a tube is pure1 add #e c4c4 sterile normal saline solution or sterile broth1 and emulsify the entire surface growth with it4 #af4 ,utilise the emulsion for the preparation of a series of subcultivations upon the media enumerated below1 using the ordinary loop to make the subcultures upon solid media1 but adding one-tenth of a cubic centimetre of the emulsion to each of the fluid media by means of a sterile pipette4 ,gelatine streak4 ,agar streak4 ,potato4 ,nutrient broth4 ,litmus milk4 ,dextrose peptone solution4 ,laevulose peptone solution4 ,galactose peptone solution4 ,maltose peptone solution4 ,lactose peptone solution4 ,saccharose peptone solution4 ,raffinose peptone solution4 ,dulcite peptone solution4 ,mannite peptone solution4 ,glycerin peptone solution4 ,inulin peptone solution4 ,dextrin peptone solution4 #ag4 ,differentiate the bacilli after isolation by means of their cultural reactions and biological characters into members of3 ,i4 ,the ,escherich ,group4 ,b4 coli communis4 ,b4 coli communior4 ,b4 lactis aerogenes4 ,b4 cloacae4 ,,ii4 ,the ,gaertner ,group4 ,bacillus enteritidis 7of ,gaertner74 ,b4 paratyphosus ,a4 ,b4 paratyphosus ,b4 ,bacillus cholerae suum4 ,,iii4 ,the ,eberth ,group4 ,b4 typhosus4 ,b4 dysenteriae 7,shiga74 ,b4 dysenteriae 7,flexner74 ,b4 faecalis alcaligines4 #ah4 ,confirm these results by testing the organisms isolated against specific agglutinating sera obtained from experimentally inoculated animals4 ,if a positive result is obtained when using this method1 it only needs a simple calculation to determine the smallest quantity 7down to #j4a c4c47 of the sample that contains at least one of the microbes of indication4 ,for instance1 if growth occurs in all the tubes from #d to #aj1 and that growth is subsequently proved to be due to the multiplication of ,b4 coli1 then it follows that at least one colon bacillus is present in every #aj c4c4 of the water sample1 but not in every #e c4c4 ,if1 on the other hand1 the presence of the ,b4 coli can only be proved in flask ,no4 #g1 then the average number of colon bacilli present in the sample is at least one in every #ej c4c4 7i4 e41 twenty per litre71 but not one in every #be c4c4 and so on4 ,the general outline of the method of identifying the members of the coli-typhoid group is given in the form of an analytical schema--whilst the full differential details are set out in tabular form4 ,,analytical ,,scheme ,,for ,,isolation ,,of ,,members ,,of ,,the ,,coli ,,and ,,typhoid ,,groups4 ,nutrose agar4 ----------------------------------- ,red colonies4 ,blue colonies4 ,escherich group4 ,gaertner and ,eberth groups4 --------------- ,lactose peptone solution4 --------------- ,gas4 ,no gas4 ,b4 coli communis and its allies4 ,gaertner and ,eberth groups4 ,acid and gas in glucose peptone solution4 ,acid and coagulation in milk4 ,general turbidity and indol in bouillon4 ,glucose peptone solution4 ,gas4 ,no gas4 ,gaertner group4 ,eberth group4 ---------------- ,litmus milk4 ,peptone solution4 ,litmus milk4 ,peptone solution4 ,acid at first4 ,general turbidity4 ,acid4 ,general turbidity4 ,alkaline later4 ,no indol4 ,no coagulation4 ,no indol4 ,no coagulation4 ,serum reaction4 ,serum reaction4 ,b4 ,streptococci4 #ai4 ,pick off streptococcus colonies and subcultivate upon nutrient agar exactly as directed in steps #ad1 #ae and #af4 #bj4 ,differentiate the streptococci isolated into members of the saprophytic group of short-chained cocci1 or members of the parasitic 7pathogenic7 group of long-chained cocci1 by means of their cultural characters1 and record their numerical frequency in the manner indicated for the members of the coli-typhoid group4 ,,differential ,,table ,,of ,,coli-,,typhoid ,,group ,transcriber's note3 ,table split to fit #hj spaces4 ---------------------------------------- ---------------------------- ,m ,d ,l ,g ,m ,l ,s ,r ,d ,a acid reaction o e a a a a a a e ,g gas formation t x e l l c c f t i t v a t t c f r l r u c o o h i i i o l t s s a n n t s o o e e r o y e s s o s e e s e e ---------------------------------------- ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ---------------------------------------- ---------------------------- ,the ,escherich ,group4 ,b4 coli communis ,o ,b4 coli communior ,b4 lactis aerogenes - ,o ,o ,b4 acidi lactici - ,o ,o ,o ,b4 pneumoniae - ,b cloaceae7,a7 ,the ,gaertner ,group4 ,b4 enteritidis ,o ,o ,o ,o ,b4 paratyphosus ,a ,o ,o ,o ,o ,b4 paratyphosus ,b ,o ,o ,o ,o ,b4 cholerae suum ,o ,o ,o ,b4 suipestifer ,o ,o ,o ,the ,eberth ,group4 ,b4 typhosus ,o ,o ,o ,b4 dysenteriae 7,shiga7 - ,o ,o ,o ,o ,o ,b4 dysenteriae 7,flexner7 - ,o ,o _/- ,o ,b4 faecalis alkaligines ,o ,o ,o ,o ,o ,o ,o ,o ---------------------------------------- ---------------------------- ,table ,notes3 7,b7 7,c7 ---------------------------------------- ----------------------------------- ---------------------------------------- ----------------------------- ,i ,s ,g ,d ,m ,s ,i ,litmus ,milk ,aacid reaction n a l u a o n ,ggas formation u l y l n r d l i c c n b o ---------- i c e i i i l ,early,late n i r t t t n i e e e n ------------------------------ ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ---------------------------------------- ---------------------------- ,the ,escherich ,group ,b4 coli communis ,o ,o ,c ,b4 coli communior ,o ,o ,c ,b4 lactis aerogenes ,o ,o ,o ,o - ,c ,b4 acidi lactici ,o ,o ,o ,c ,b4 pneumoniae ,o ,o - ,c ,b cloaceae,a ,o ,o ,o - ,c ,the ,gaertner ,group4 ,b4 enteritidis ,o ,o ,o - _/- - ,b4 paratyphosus ,a ,o _/- ,o - ,o ,b4 paratyphosus ,b ,o ,o ,o - - ,b4 cholerae suum ,o ,o ,o ,o ,o _/- - ,b4 suipestifer ,o ,o ,o - - ,the ,eberth ,group4 ,b4 typhosus ,o ,o ,o ,o - ,b4 dysenteriae 7,shiga7 ,o ,o ,o ,o ,o ,o - - ,b4 dysenteriae 7,flexner7 ,o ,o ,o ,o ,o _/- - ,b4 faecalis alkaligines ,o ,o ,o ,o ,o ,o - - - ---------------------------------------- ---------------------------- ,table ,notes3 7,d7 7,e7 ---------------------------------------- ---------------------------------- ,table ,notes3 7,a7 99 ,liquefies gelatine4 7,b7 motile4 - non-motile4 7,c7 acid or gas production4 _/- slight acid production4 ,o no change4 7,d7 indol production4 _/- slight indol production4 - no indol formed4 7,e7 acid production4 - alkali production4 ,o no change in reaction4 ,c clot4 #ba4 ,determine the pathogenicity for mice 7subcutaneous inoculation7 and rabbits 7intravenous inoculation7 of the streptococci isolated4 ,on the facing insert page is reproduced a blank from the author's ,laboratory ,water ,analysis ,book1 by means of which an exact record can be kept1 with a minimum of labour1 of every sample examined4 ,b4 ,concentration ,method4 ,the remaining organisms referred to on page #dbf are more conveniently sought for by the concentration method4 ,collection of the ,sample4--,the quantity of water required for this method of examination is about #bjjj c4c41 and the vessel usually chosen for its reception is an ordinary blue glass ,winchester quart bottle1 sterilised in the hot-air oven1 and over this a paper or parchment cap fastened with string4 ,the bottle may be packed in a wooden box or in an ordinary wicker case4 ,the method of collecting the sample is identical with that described under the heading of ,quantitative ,examination2 there is1 however1 not the same imperative necessity to pack the sample in ice for transmission to the laboratory4 ,apparatus required3 ,sterile ,chamberland or ,doulton white porcelain open mouth filter candle1 fitted with rubber washer4 ,rubber cork to fit mouth of the filter candle1 perforated with one hole4 ,kitasato serum flask1 #bejj c4c4 capacity4 ,geryk air pump or water force pump4 ,wulff's bottle1 fitted as wash-bottle1 and containing sulphuric acid 7to act as a safety valve between filter and pump74 ,pressure tubing1 clamps1 pinch-cock4 ,retort stand1 with ring and clamp4 ,rubber cork for the neck of ,winchester quart1 perforated with two holes and fitted with one #f cm4 length of straight glass tubing1 and one ,v-shaped piece of glass tubing1 one arm #cb cm4 in length1 the other #eb cm41 the shorter arm being plugged with cotton-wool4 ,the rubber stopper must be sterilised by boiling and the glass tubing by hot air1 before use4 ,flask containing #bej c4c4 sterile broth4 ,test-tube brush to fit the lumen of the candle1 enclosed in a sterile test-tube 7and previously sterilised by dry heat or by boiling74 ,case of sterile pipettes1 #aj c4c4 in tenths4 ,case of sterile pipettes1 #a c4c4 in tenths4 ,case of sterile pipettes1 #a c4c4 in hundredths4 ,tubes of various nutrient media 7according to requirements74 ,twelve ,buchner's tubes with rubber stoppers4 ,pyrogallic acid tablets4 ,caustic soda tablets4 ,illustration3 ,sample form ,illustration3 ,,fig4 #bji4--,water filtering apparatus4 ,that portion of the figure to the left of the vertical line is drawn to a larger scale than that on the right1 in order to show details of ,sprengel's pump4 ,,method4-- #a4 ,fit up the filtering apparatus as in the accompanying diagram 7,fig4 #bji71 interposing the wash-bottle with sulphuric acid between the filter flask and the force-pump 7in the position occupied in the diagram by the central vertical line71 and placing another screw clamp on the rubber tubing connecting the lateral arm of the filter flask with the wash-bottle4 ,illustration3 ,,fig4 #baj4 ,sterile test-tube brush4 #b4 ,filter the entire #bjjj c4c4 of water through the filter candle4 #c4 ,when the nitration is completed1 screw up the clamps and so occlude the two pieces of pressure tubing4 #d4 ,reverse the position of the glass tubes in the ,wulff's bottle so that the one nearest the air pump now dips into the sulphuric acid4 #e4 ,slowly open the metal clamps and allow air to gradually pass through the acid1 and enter filter flask1 and so restore the pressure4 #f4 ,unship the apparatus1 remove the cork from the mouth of the candle4 #g4 ,pipette #aj c4c4 of sterile broth into the interior of the candle1 and by means of the sterile test-tube brush 7,fig4 #baj7 emulsify the slimy residue which lines the candle1 with the broth4 ,practically all the bacteria contained in the original #bjjj c4c4 of water are now suspended in #aj c4c4 of broth1 so that #a c4c4 of the suspension is equivalent1 so far as the contained organisms are concerned1 to #bjj c4c4 of the original water4 7,some bacteria will of course be left behind on the walls of the filter and in its pores47 ,up to this point the method is identical1 irrespective of the particular organism whose presence it is desired to demonstrate2 but from this point onward the methods must be specially adapted to the isolation of definite groups of organisms or of individual bacteria4 ,the ,coli-,typhoid ,group4-- #a4 ,number nine tubes of bile salt broth 7vide page #ahj71 consecutively from #a to #i4 #b4 ,to ,no #a add #a c4c4 of the original water sample #b add #b c4c4 before the nitration is commenced4 #c add #e c4c4 #c4 ,to the remaining tubes of bile salt broth add varying quantities of the suspension by means of suitably graduated sterile pipettes1 as follows3 ,no4 #d #j4je c4c4 7equivalent to #aj c4c4 of the original water sample74 ,no4 #e #j4abe c4c4 7equivalent to #be c4c4 of the original water sample74 ,no4 #f #j4be c4c4 7equivalent to #ej c4c4 of the original water sample74 ,no4 #g #j4e c4c4 7equivalent to #ajj c4c4 of the original water sample74 ,no4 #h #a4j c4c4 7equivalent to #bjj c4c4 of the original water sample74 ,no4 #i #b4e c4c4 7equivalent to #ejj c4c4 of the original water sample74 #d4 ,put up each tube anaerobically in a ,buchner's tube and incubate at #db deg4 ,c4 #e4 ,the subsequent steps are identical with those described under the ,enrichment method 7see page #dbh to #dca2 ,steps #h to #ah74 ,alternative ,methods4-- ,a few of the older methods for the isolation of the members of the coli-typhoid groups are referred to but they are distinctly inferior to those already described4 7,a7 ,the ,carbolic ,method3 #a4 ,take ten tubes of carbolised bouillon 7vide page #bjb7 and number them consecutively from #a to #aj4 #b4 ,inoculate each tube with a different amount of the water sample or suspension1 as in the previous method4 #c4 ,incubate aerobically at #cg deg4 ,c4 #d4 ,examine the culture tubes after twenty-four hours' incubation4 #e4 ,from those tubes which shows signs of growth1 pour plates in the usual manner1 using carbolised gelatine 7vide page #bjb7 in place of the ordinary gelatine1 and incubate at #bj deg4 ,c4 for three1 four1 or five days as may be necessary4 #f4 ,subcultivate from any colonies that make their appearance1 and determine their identity on the lines laid down in the previous method4 7,b7 ,parietti's ,method3 #a4 ,take nine tubes of ,parietti's bouillon 7vide page #bjb7--;i4 e41 three each of those containing #j4a c4c41 #j4b c4c41 and #j4e c4c4 of ,parietti's solution respectively4 ,mark plainly on the outside of each tube the quantity of ,parietti's solution it contains4 #b4 ,to each tube add a different amount of the original water1 or of the suspension1 and incubate at #cg deg4 ,c4 #c4 ,examine the culture tubes after twenty-four and forty-eight hours' incubation1 and plate in nutrient carbolised or potato gelatine from such as have grown4 #d4 ,pick off suspicious colonies1 if any such appear on the plates1 subcultivate them upon the various media1 and identify them4 7,c7 ,elsner's ,method3 ,this method simply consists in substituting ,elsner's potato gelatine 7vide page #bjd7 for ordinary nutrient gelatine in any of the previously mentioned methods4 7,d7 ,cambier's ,candle ,method3 ,treat a large volume of the water sample by the concentration method 7vide page #dcd74 #a4 ,remove the rubber stopper from the mouth of the filter candle1 introduce #aj c4c4 sterile bouillon into its interior1 and emulsify the bacterial sediment2 replug the mouth of the candle with a wad of sterile cotton-wool4 #b4 ,remove the filter candle from the filter flask and insert it into the mouth of a flask or a glass cylinder containing sterile bouillon sufficient to reach nearly up to the rubber washer on the candle4 #c4 ,incubate for twenty-four to thirty-six hours at #cg deg4 ,c4 #d4 ,from the now turbid bouillon in the glass cylinder pour gelatine plates and incubate at #bj deg4 ,c4 #e4 ,subcultivate and identify any suspicious colonies that appear4 7,the method depends upon the assumption that members of the typhoid and coli groups find their way through the porcelain filter from the interior to the surrounding bouillon at a quicker rate than the associated bacteria47 ,b4 ,enteritidis ,sporogenes4-- #a4 ,transfer #e c4c4 of the emulsion from the filter candle to a sterile test-tube and plug carefully4 #b4 ,place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms 7vide page #beg71 and expose to a temperature of #hj deg4 ,c4 for twenty minutes4 #c4 ,number ten tubes of litmus milk consecutively from #a to #aj4 #d4 ,remove the test-tube from the benzole bath and shake well to distribute the spores evenly through the fluid4 #e4 ,to each tube of litmus milk add a measured quantity of the suspension corresponding to the amounts employed in isolating the coli group 7vide page #dcg74 #f4 ,incubate each tube anaerobically at #cg deg4 ,c4 ,anaerobic conditions can be obtained by putting the cultures up in ,buchner's tubes or in ,bulloch's apparatus4 ,if1 however1 whole milk has been used in making the litmus milk the layer of cream that rises to the surface will be sufficient to ensure anaerobiosis2 whilst if separated milk has been employed it will be sufficient to pour a layer of sterile vaseline or liquid paraffin on the surface of the fluid4 #g4 ,examine after twenty-four hours' incubation4 ,note 7if ,b4 enteritidis sporogenes is present7-- 7a7 ,acid reaction of the medium as indicated by the colour of the litmus or its complete decolourisation4 7b7 ,presence of clotting1 and the separation of clear whey4 7c7 ,presence of gas1 as indicated by fissures and bubbles in the coagulum1 and possibly masses of coagulum driven up the tube almost to the plug4 #h4 ,replace the tubes which show no signs of growth in the incubator for a further period of twenty-four hours and again examine with reference to the same points4 #i4 ,remove those tubes which give evidence of growth from the ,buchner's tubes and carefully pipette off the whey2 examine the whey microscopically4 #aj4 ,inoculate two guinea-pigs each subcutaneously with #j4e c4c4 of the whey and observe the result4 ,vibrio ,cholerae4-- #a4 ,number ten tubes of peptone water consecutively from #a to #aj4 #b4 ,to each of the tubes of peptone water add a measured quantity of the suspension1 corresponding to those amounts employed in isolating the members of the coli group 7vide page #dcg74 #c4 ,incubate aerobically at #cg deg4 ,c4 for twenty-four hours4 ,examine the tubes carefully for visible growth1 especially delicate pellicle formation1 which if present should be examined microscopically for vibrios1 both by stained preparations or by fresh specimens with dark ground illumination4 #d4 ,inoculate fresh tubes of peptone water from such of the tubes as exhibit pellicle formation--from the pellicle itself--and incubate at #cg deg4 ,c4 for twenty-four hours4 #e4 ,test the peptone water itself for the presence of indol and nitrite by the addition of pure concentrated ,h#b,,so#d4 #e4 ,prepare gelatine and agar plates in the usual way from such of these tubes as show pellicle formation4 #f4 ,pick off from the plates any colonies resembling those of the ,vibrio cholerae and subcultivate upon all the ordinary laboratory media4 #g4 ,test the vibrio isolated against the serum of an animal immunised to the ,vibrio cholerae for agglutination4 ,b4 ,anthracis4-- #a4 ,transfer #e c4c4 of the emulsion from the filter candle to a sterile test-tube and plug carefully4 #b4 ,place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms 7vide page #beg71 and expose to a temperature of #hj deg4 ,c4 for twenty minutes4 #c4 ,inoculate a young white rat subcutaneously 7on the inner aspect of one of the hind legs7 with #a c4c4 of the emulsion4 ,observe during life1 and1 if the animal succumbs1 make a complete post-mortem examination4 #d4 ,melt three tubes of nutrient agar in boiling water and cool to #db deg4 ,c4 #e4 ,number the tubes #a1 #b1 and #c4 ,to ,no4 #a add #j4b c4c41 to ,no4 #b add #j4c c4c41 and to ,no4 #c add #j4e c4c4 of the suspension1 and pour plates therefrom4 #f4 ,incubate at #cg deg4 ,c4 for twenty-four or forty-eight hours4 #g4 ,pick off any colonies resembling those of anthrax and subcultivate on all the ordinary laboratory media4 #h4 ,inoculate another young white rat as in #c1 using two loopfuls of the agar subcultivation emulsified with #a c4c4 sterile bouillon4 ,observe during life1 and if the animal succumbs1 make a complete post-mortem examination4 ,b4 ,tetani4-- #a4 ,proceed as detailed above in steps #a and #b for the isolation of the ,b4 anthracis4 #b4 ,add #a c4c4 of the suspension to each of three tubes of glucose formate broth1 and incubate anaerobically in ,buchner's tubes at #cg deg4 ,c4 #c4 ,from such of the tubes as show visible growth 7with or without the production of gas7 after twenty-four hours' incubation inoculate guinea-pigs1 subcutaneously 7under the skin of the abdomen71 using #j4a c4c4 of the bouillon cultivation as a dose4 ,observe carefully during life1 and1 if death occurs1 make a complete post-mortem examination4 #d4 ,from the same tubes pour agar plates and incubate anaerobically in ,bulloch's apparatus1 at #cg deg4 ,c4 #e4 ,subcultivate suspicious colonies on the various media1 incubate anaerobically1 making control cultivations on glucose formate agar1 stab and streak1 to incubate aerobically and carry out further inoculation experiments with the resulting growths4 ,,examination ,,of ,,milk4 ,one-cow or whole milk1 if taken from the apparently healthy animal 7that is1 an animal without any obvious lesion of the udder or teats7 with ordinary precautions as to cleanliness1 avoidance of dust1 etc41 contains but few organisms4 ,in dealing with one-cow milk1 from a suspected1 or an obviously diseased animal1 a complete analysis should include the examination 7both qualitative and quantitative7 of samples of 7a7 fore-milk1 7b7 mid-milk1 7c7 strippings1 and1 if possible1 from each quarter of the udder4 ,mixed milk1 on the other hand1 by the time it leaves the retailer's hands1 usually contains as many micro-organisms as an equal volume of sewage and indeed during the examination it is treated as such4 ,it is possible however to collect and store mixed milk in so cleanly a manner that its germ content does not exceed #ejjj micro-organisms per cubic centimetre4 ,such comparative freedom from extraneous bacteria is usually secured by the purveyor only when he resorts to the process of pasteurisation 7heating the milk to #fe deg4 ,c4 for twenty minutes or to #gg deg4 ,c4 for one minute7 or the simpler plan of adding preservatives to the milk4 ,information regarding the employment of these methods for the destruction of bacteria should always be sought in the case of mixed milk samples1 and in this connection the following tests will be found useful3 #a4 ,raw ,milk 7,saul74 ,to #aj c4c4 milk in a test tube1 add #a c4c4 of a #a per cent4 aqueous solution of ortol 7ortho-methyl-amino-phenol sulphate71 recently prepared and mix4 ,next add #j4b c4c4 of a #c per cent4 peroxide of hydrogen solution4 ,the appearance of a brick red color within #cj seconds indicates raw milk4 ,milk heated to #gd deg4 ,c4 for thirty minutes undergoes no alteration in color2 if heated to #ge deg4 ,c4 for ten minutes only1 the brick red color appears after standing for about two minutes4 #b4 ,boric ,acid4 ,evaporate to dryness1 #ej c4c4 of the milk which has been rendered slightly alkaline to litmus1 then incinerate4 ,dissolve in distilled water1 add slight excess of dilute hydrochloric acid and again evaporate to dryness4 ,dissolve the residue in a small quantity of hot water and moisten a piece of turmeric paper with the solution4 ,dry the turmeric paper4 ,rose or cherry-red color borax or boric acid4 #c4 ,formaldehyde 7,hehner74 ,to #aj c4c4 milk in a test tube add #e c4c4 concentrated commercial sulphuric acid slowly1 so that the two fluids do not mix4 ,hold the tube vertically and agitate very gently4 ,violet zone at the junction of the two liquids formaldehyde4 #d4 ,hydrogen ,peroxide4 ,to #aj c4c4 milk 7diluted with equal quantities of water7 in a test tube add #j4d c4c4 of a #d per cent4 alcoholic solution of benzidine and #j4b c4c4 acetic acid4 ,blue coloration of the mixture hydrogen peroxide4 #e4 ,salicylic ,acid4 ,precipitate the caseinogen by the addition of acetic acid and filter4 ,to the filtrate add a few drops of #a per cent4 aqueous solution of ferric chloride4 ,purple coloration salicylic acid4 #f4 ,sodium ,carbonate or ,bicarbonate4 ,to #aj c4c4 of the milk in a test tube add #aj c4c4 of alcohol and #j4c c4c4 of a #a per cent4 alcoholic solution of rosolic acid4 ,brownish color pure milk2 rose color preserved milk4 ,illustration3 ,,fig4 #baa4--,milk-collecting bottle and dipper in case4 ,quantitative4-- ,collection of ,sample4-- ,the apparatus used for the collection of a retail mixed milk sample consists of a cylindrical copper case1 #af cm4 high and #i cm4 in diameter1 provided with a pull-off lid1 containing a milk dipper1 also made of copper2 and inside this1 again1 a wide-mouthed1 stoppered glass bottle of about #bej c4c4 capacity 7about #ad cm4 high by #g cm4 diameter71 having a tablet for notes1 sand-blasted on the side4 ,the copper cylinder and its contents1 secured from shaking by packing with cotton-wool1 are sterilised in the hot-air oven 7,fig4 #bf74 ,when collecting a sample1 #a4 ,remove the cap from the cylinder4 #b4 ,draw out the cotton-wool4 #c4 ,lift out the bottle and dipper together4 #d4 ,receive the milk in the sterile dipper1 and pour it directly into the sterile bottle4 #e4 ,enter the particulars necessary for the identification of the specimen1 on the tablet1 with a lead pencil1 or pen and ink4 #f4 ,pack the apparatus in the ice-box for transmission to the laboratory in precisely the same manner as an ordinary water sample4 ,whole milk may with advantage be collected in the sterile bottle directly since the mouth is sufficiently wide for the milker to direct the stream of milk into it4 ,condensed milk must be diluted with sterile distilled water in accordance with the directions printed upon the label1 then treated as ordinary milk4 ,apparatus ,required3 ,case of sterile capsules 7#be c4c4 capacity74 ,case of sterile graduated pipettes1 #aj c4c4 7in tenths of a cubic centimetre74 ,case of sterile graduated pipettes1 #a c4c4 7in tenths of a cubic centimetre74 ,flask containing #bej c4c4 sterile bouillon4 ,tall cylinder containing #b per cent4 lysol solution4 ,plate-levelling stand4 ,case of sterile plates4 ,tubes nutrient gelatine or gelatine agar4 ,tubes of wort gelatine4 ,tubes of nutrient agar4 ,water-bath regulated at #db deg4 ,c4 ,bunsen burner4 ,grease pencil4 ,,method4-- #a4 ,arrange four sterile capsules in a row2 number them ,i1 ,,ii1 ,,iii1 and ,,iv4 #b4 ,fill #i c4c4 sterile bouillon into the first1 and #i4i c4c4 bouillon into each of the three remaining capsules4 #c4 ,remove #a c4c4 milk from one of the bottles by means of a sterile pipette and add it to the bouillon in capsule ,i2 mix thoroughly by repeatedly filling and emptying the pipette4 #d4 ,remove #j4a c4c4 of the milky bouillon from capsule ,i1 add it to the contents of capsule ,,ii1 and mix as before4 #e4 ,in like manner add #j4a c4c4 of the contents of capsule ,,ii to capsule ,,iii2 and then #j4a c4c4 of the contents of capsule ,,iii to capsule ,,iv4 ,then #a c4c4 of dilution ,i contains #j4a c4c4 milk sample4 #a c4c4 of dilution ,,ii contains #j4jja c4c4 milk sample4 #a c4c4 of dilution ,,iii contains #j4jjjja c4c4 milk sample4 #a c4c4 of dilution ,,iv contains #j4jjjjjja c4c4 milk sample4 #f4 ,melt the gelatine and the agar tubes in boiling water2 then transfer to the water-bath and cool them down to #db deg4 ,c4 #g4 ,number the gelatine tubes consecutively #a to #ab4 #h4 ,inoculate the tubes with varying quantities of the material as follows3 ,to tube ,no4 #a add #a4j c4c4 of the milk sample4 #b add #j4a c4c4 of the milk sample4 #c add #a4j c4c4 from capsule ,i4 #d add #j4a c4c4 from capsule ,i4 #e add #a4j c4c4 from capsule ,,ii4 #f add #j4a c4c4 from capsule ,,ii4 #g add #j4e c4c4 from capsule ,,iii4 #h add #j4c c4c4 from capsule ,,iii4 #i add #j4b c4c4 from capsule ,,iii4 #aj add #j4e c4c4 from capsule ,,iv4 #aa add #j4c c4c4 from capsule ,,iv4 #ab add #j4b c4c4 from capsule ,,iv4 #i4 ,pour plates from the gelatine tubes2 label1 and incubate at #bj deg4 ,c4 #aj4 ,liquefy five wort gelatine tubes and to them add #a4j c4c4 of the milk sample and a similar quantity of the diluted milk from capsules ,i1 ,,ii1 and ,,iii and ,,iv respectively4 #aa4 ,pour plates from the wort gelatine2 label1 and incubate at #bj deg4 ,c4 #ab4 ,inoculate the liquefied agar tubes as follows3 ,to tube ,no4 #a add #j4a c4c4 of the milk sample4 #b add #j4a c4c4 from capsule ,i4 #c add #j4a c4c4 from capsule ,,ii4 #d add #j4a c4c4 from capsule ,,iii4 #e add #a4j c4c4 from capsule ,,iv4 #f add #j4a c4c4 from capsule ,,iv4 #ac4 ,pour plates from the agar tubes2 label1 and incubate at #cg deg4 ,c4 #ad4 ,after twenty-four hours' incubation inspect1 and after forty-eight hours' incubation1 count the agar plates and estimate the number of organisms growing at #cg deg4 ,c4 present per cubic centimetre of the sample of milk4 #ae4 ,after three1 four1 or five days' incubation1 count the gelatine plates and estimate therefrom the number of organisms growing at #bj deg4 ,c4 present per cubic centimetre of the sample of milk4 #af4 ,after a similar interval count the wort gelatine plates and estimate the number of moulds and yeasts present per cubic centimetre of the sample of milk4 ,,note4--,many observers prefer to employ gelatine agar 7see page #aic7 for the quantitative examination4 ,in this case gelatine-agar plates should be poured from tubes containing the quantities of material indicated in step #h1 incubated at #bh deg4 ,c4 to #cj deg4 ,c4 and after five days the total number of organisms developing at #bh deg4 ,c4 recorded4 ,qualitative4--,the qualitative bacteriological examination of milk is chiefly directed to the detection of the presence of one or more of the following pathogenic bacteria and when present to the estimation of their numerical frequency4 ,members of the ,coli-typhoid group4 ,vibrio cholerae4 ,streptococcus pyogenes longus4 ,micrococcus melitensis4 ,staphylococcus pyogenes aureus4 ,bacillus enteritidis sporogenes4 ,bacillus diphtheriae4 ,bacillus tuberculosis4 ,some of these occur as accidental contaminations1 either from the water supply to the cow farm1 or from the farm employees1 whilst others are derived directly from the cow4 ,in milk1 as in water examinations1 two methods are available1 viz43 ,enrichment and ,concentration--the former is used for the demonstration of bacteria of intestinal origin1 the latter for the isolation of the micro-organisms of diphtheria and tubercle4 ,the first essential in the latter process is the concentration of the bacterial contents of a large volume of the sample into a small compass2 but in the case of milk1 thorough centrifugalisation is substituted for filtration4 ,apparatus ,required3 ,a large centrifugal machine4 ,this machine1 to be of real service in the bacteriological examination of milk1 must conform to the following requirements3 #a4 ,the centrifugal machine must be of such size1 and should carry tubes or bottles of such capacity1 as to enable from #bjj to #ejj c4c4 of milk to be manipulated at one time4 #b4 ,the rate of centrifugalisation should be from #bejj to #cjjj revolutions per minute4 #c4 ,the portion of the machine destined to carry the tubes should be a metal disc1 of sufficient weight to ensure good flank movement1 continuing over a considerable period of time4 ,in other words1 the machine should run down very gradually and slowly after the motive power is removed1 thus obviating any disturbance of the relative positions of particulate matter in the solution that is being centrifugalised4 #d4 ,the machine should preferably be driven by electricity1 or by power1 but in the case of hand-driven machines-- 7a7 ,the gearing should be so arranged that the requisite speed is obtained by not more than forty or fifty revolutions of the crank handle per minute1 so that it may be maintained for periods of twenty or thirty minutes without undue exertion4 7b7 ,the handle employed should be provided with a special fastening 7e4 g41 a clutch similar to that employed for the free wheel of a bicycle71 or should be readily detachable so that1 on ceasing to turn1 the handle should not1 by its weight and air resistance1 act as a brake and stop the machine too suddenly4 ,one of the few satisfactory machines of this class is shown in figure #bab4 ,illustration3 ,,fig4 #bab4--;,electrically driven centrifugal machine1 with flexible 7broken7 spindle encircled by the field magnets of the motor4 ,sterile centrifugal tubes1 of some #fj-gj c4c4 capacity1 tapering to a point at the closed end1 plugged with cotton-wool4 ,small centrifugal machine to run two tubes of #aj c4c4 capacity at #bejj to #cjjj revolutions per minute preferably driven by electricity1 of the type figured on page #cbg 7,fig4 #afb74 ,sterile centrifugal tubes of #aj c4c4 capacity with the distal extremity contracted to a narrow tube and graduated in hundredths of a cubic centimetre 7,fig4 #bac74 ,sterilised cork borer4 ,case of sterile pipettes1 #aj c4c4 7in tenths of a cubic centimetre74 ,case of sterile pipettes1 #a c4c4 7in tenths of a cubic centimetre74 ,sterile teat pipettes4 ,flask of sterile normal saline solution4 ,,method4-- #a4 ,fill #ej c4c4 of the milk sample into each of four tubes1 and replace the cotton-wool plugs by solid rubber stoppers 7sterilised by boiling71 and fit the tubes in the centrifugal machine4 ,,note4--,one or two cubic centimetres of paraffinum liquidum introduced into the buckets of the centrifuge before the glass tubes are inserted will obviate any risk of breakage to the latter4 ,illustration3 ,,fig4 #bac4--,milk sedimenting tubes4 ,illustration3 ,,fig4 #bad4--,milk in centrifuge tube4 #b4 ,centrifugalise the milk sample for thirty minutes at a speed of #bejj revolutions per minute4 #c4 ,remove the motive power and allow the machine to slow down gradually4 #d4 ,remove the tubes of milk from the centrifuge4 ,each tube will now show 7,fig4 #bad73 7a7 ,a superficial layer of cream 7varying in thickness with different samples7 condensed into a semi-solid mass1 which can be shown to contain some organisms and a few leucocytes4 7b7 ,a central layer of separated milk1 thin1 watery1 and opalescent1 and containing extremely few bacteria4 7c7 ,a sediment or deposit consisting of the great majority of the contained bacteria and leucocytes1 together with adventitious matter1 such as dirt1 hair1 epithelial cells1 faecal debris1 etc4 #e4 ,withdraw the rubber stopper and remove a central plug of cream from each tube by means of a sterile cork borer2 place these masses of cream in two sterile capsules4 ,label ,c#a and ,c#b4 #f4 ,remove all but the last one or two c4c4 of separated milk from each tube1 by means of sterile pipettes4 #g4 ,mix the deposits thoroughly with the residual milk1 pipette the mixture from each pair of tubes into one sterile #aj c4c4 tube 7graduated7 by means of sterile teat pipettes1 then fill to the #aj c4c4 mark with sterile normal saline solution and mix together4 ,label ,d#a and ,d#b4 #h4 ,place the two tubes of mixed deposit in the centrifuge1 adjust by the addition or subtraction of saline solution so that they counterpoise exactly1 and centrifugalise for ten minutes4 ,,note4--,each tube now contains the deposit from #ajj c4c4 of the milk sample and the amount can be read off in hundredths of a centimetre4 ,the multiplication of this figure by #ajj will give the amount of ,apparent ,filth1 in parts per million--the usual method of recording this quality of milk4 #i4 ,pipette off all the supernatant fluid and invert the tube to drain on to a pad of sterilised cotton-wool1 contained in a beaker4 7,this wool is subsequently cremated47 #aj4 ,examine both cream 7,c#a7 and deposit 7,d#a7 microscopically-- 7a7 ,in hanging-drop preparations4 7b7 ,in film preparations stained carbolic methylene-blue1 by ,gram's method1 by ,neisser's method1 and by ,ziehl-,neelsen's method4 ,note the presence or absence of altered and unaltered vegetable fibres2 pus cells1 blood discs2 cocci in groups or chains1 diphtheroid bacilli1 ,gram negative bacilli or cocci1 spores and acid fast bacteria4 #aa4 ,adapt the final stages of the investigation to the special requirements of each individual sample1 thus3 #a4 ,members of the ,coli-typhoid ,group4-- #a4 ,emulsify the deposit from the second centrifugal tube 7,d#b7 with #aj c4c4 sterile bouillon and inoculate three tubes of bile salt broth as follows3 ,to ,tube ,no4 #a add #b4e c4c4 milk deposit emulsion 7#be c4c4 original milk47 ,to ,tube ,no4 #b add #a4j c4c4 milk deposit emulsion 7#aj c4c4 original milk47 ,to ,tube ,no4 #c add #j4e c4c4 milk deposit emulsion 7 #e c4c4 original milk47 #b4 ,inoculate tube of bile salt broth ,no4 #d with #a c4c4 of the original milk4 #c4 ,inoculate further tubes of bile salt broth with previously prepared dilutions 7see page #dde7 as follows3 ,to tube ,no4 #e add #a4j c4c4 from capsule ,i4 ,to tube ,no4 #f add #j4a c4c4 from capsule ,i4 ,to tube ,no4 #g add #a4j c4c4 from capsule ,,ii4 ,to tube ,no4 #h add #j4a c4c4 from capsule ,,ii4 ,to tube ,no4 #i add #a4j c4c4 from capsule ,,iii4 ,to tube ,no4 #aj add #j4a c4c4 from capsule ,,iii4 ,to tube ,no4 #aa add #a4j c4c4 from capsule ,,iv4 ,to tube ,no4 #ab add #j4a c4c4 from capsule ,,iv4 and incubate anaerobically 7in ,buchner's tubes7 at #db deg4 ,c4 for a maximum period of forty-eight hours4 #d4 ,if growth occurs complete the investigation as detailed under the corresponding section of water examination 7see pages #dbh to #dca74 ,,note4--,the ,b4 coli communis1 derived from the alvine discharges of the cow1 is almost universally present in large or small numbers1 in retail milk4 ,its detection1 therefore1 unless in enormous numbers1 7when it indicates want of cleanliness71 is of little value4 #b4 ,vibrio ,cholerae4--,inoculate tubes of peptone water by using the same amounts as in the search for members of the ,coli-typhoid groups 7vide ante #a-c72 incubate aerobically at #cg deg4 ,c4 and complete the examination as detailed under the corresponding section of water examination 7see page #dci74 #c4 ,b4 ,enteritidis ,sporogenes4--,inoculate tubes of litmus milk with similar amounts to those used in the previous searches1 omitting tube ,no4 #a 7vide ante #a-c7 place in the differential steriliser at #hj deg4 ,c4 for ten minutes and then incubate anaerobically at #cg deg4 ,c4 for a maximum period of forty-eight hours4 ,complete the investigation as detailed under the corresponding section of water examination 7see page #dch74 #d4 ,b4 ,diphtheriae4-- 7,a7 #a4 ,plant three sets of serial cultivations1 twelve tubes in each set1 from 7a7 cream ,c#b1 7b7 deposit ,d#a upon oblique inspissated blood-serum1 and incubate at #cg deg4 ,c4 #b4 ,pick off any suspicious colonies which may have made their appearance twelve hours after incubation1 examine microscopically and subcultivate upon blood-serum and place in the incubator2 return the original tubes to the incubator4 #c4 ,repeat this after eighteen hours' incubation4 #d4 ,from the resulting growths make cover-slip preparations and stain carbolic methylene-blue1 ,neisser's method1 ,gram's method4 ,subcultivate such as appear to be composed of diphtheria bacilli in glucose peptone solution4 ,note those in which acid production takes place4 #e4 ,inoculate guinea-pigs subcutaneously with one or two cubic centimetres forty-eight-hour-old glucose bouillon cultivation derived from the first subcultivation of each glucose fermenter1 and observe the result4 #f4 ,if death1 apparently from diphtheritic toxaemia1 ensues1 inoculate two more guinea pigs with a similar quantity of the lethal culture4 ,reserve one animal as a control and into the other inject #ajjj units of antidiphtheritic serum4 ,if the control dies and the treated animal survives1 the proof of the identity of the organism isolated with the ,klebs-,loeffler bacillus becomes absolute4 #g4 ,inoculate guinea-pigs subcutaneously with filtered glucose bouillon cultivations 7toxins87 and observe the result4 7,b7 #a4 ,emulsify the remainder of the deposit with #e c4c4 sterile bouillon and inoculate two guinea-pigs1 thus3 guinea-pig a1 subcutaneously with #a c4c4 emulsion2 guinea-pig b1 subcutaneously with #b c4c4 emulsion2 and observe the result4 #b4 ,if either or both of the inoculated animals succumb1 make complete post-mortem examination and endeavour to isolate the pathogenic organisms from the local lesion4 ,confirm their identity as in ,a#e and #f 7vide supra74 #e4 ,bacillus ,tuberculosis4-- 7,a7 #a4 ,inoculate each of three guinea-pigs 7previously tested with tuberculin1 to prove their freedom from spontaneous tuberculosis7 subcutaneously at the inner aspect of the bend of the left knee1 with #a c4c4 of the deposit emulsion remaining in one or other tube 7,d#a or ,d#b74 #b4 ,introduce a small quantity of the cream into a subcutaneous pocket prepared at the inner aspect of the bend of the right knee of each of these three animals4 ,place a sealed dressing on the wound4 #c4 ,observe carefully1 and weigh accurately each day4 #d4 ,kill one guinea-pig at the end of the second week and make a complete post-mortem examination4 #e4 ,if the result of the examination is negative or inconclusive1 kill a second guinea-pig at the end of the third week and examine carefully4 ,illustration3 ,,fig4 #bae4--;,cadaver of guinea-pig experimentally infected with ,b4 tuberculosis4 #f4 ,if still negative or inconclusive1 kill the third guinea-pig at the end of the sixth week4 ,make a careful post-mortem examination4 ,examine material from any caseous glands microscopically and inoculate freely on to ,dorset's egg medium4 ,,note4--,every post-mortem examination of animals infected with tuberculous material should include the naked eye and microscopical examination of the popliteal1 superficial and deep inguinal1 iliac1 lumbar and axillary glands on each side of the body1 also the retrohepatic1 bronchial and sternal glands1 the spleen1 liver and lungs 7,fig4 #bae74 7,b7 #a4 ,intimately mix all the available cream and deposit from the milk sample1 and transfer to a sterile ,erlenmeyer flask4 #b4 ,treat the mixture by the antiformin method 7vide ,appendix1 page #ejb74 #c4 ,inoculate each of two guinea-pigs1 intraperitoneally1 with half of the emulsion thus obtained4 #d4 ,kill one of the guinea-pigs at the end of the first week and examine carefully4 #e4 ,kill the second guinea-pig at the end of the second week and examine carefully4 #f4 ,utilise the remainder of the deposit for microscopical examination and cultivations upon ,dorset's egg medium4 ,,note4--,no value whatever attaches to the result of a microscopical examination for the presence of the ,b4 tuberculosis unless confirmed by the result of inoculation experiments4 #f4 ,streptococcus ,pyogenes ,longus4-- 7,a7 #a4 ,spread serial surface plates upon nutrose agar4 ,also plant serial cultivations upon sloped nutrient agar 7six tubes in series74 #b4 ,if the resulting growth shows colonies which resemble those of the streptococcus1 make subcultivations upon agar and in bouillon1 in the first instance1 and study carefully4 7,b7 #a4 ,plant a large loopful of the deposit ,d#b into each of three tubes of glucose formate bouillon1 and incubate anaerobically 7in ,buchner's tubes7 for twenty-four hours at #cg deg4 ,c4 #b4 ,if the resulting growth resembles that of the streptococcus1 make subcultivations upon nutrient agar4 #c4 ,prepare subcultivations of any suspicious colonies that appear1 upon all the ordinary media1 and study carefully4 ,if the streptococcus is successfully isolated1 inoculate serum bouillon cultivations into the mouse1 guinea-pig1 and rabbit1 to determine its pathogenicity and virulence4 #g4 ,staphylococcus ,pyogenes ,aureus4-- #a4 ,examine carefully the growth upon the serial blood serum cultivations prepared to isolate ,b4 diphtheriae and the serial agar cultivations to isolate streptococci after forty-eight hours' incubation4 #b4 ,pick off any suspicious orange coloured colonies1 plant on sloped agar1 and incubate at #bj deg4 ,c4 ,observe pigment formation4 #c4 ,prepare subcultivations from any suspicious growths upon all the ordinary media1 study carefully and investigate their pathogenicity4 #h4 ,micrococcus ,melitensis4--,the milk from an animal infected with ,m4 melitensis usually contains the organisms in large numbers and but few other bacteria4 #a4 ,spread several sets of surface plates upon nutrose agar1 each from one loopful of the deposit in tube ,d#a or ,d#b4 #b4 ,spread several sets of surface plates upon nutrose agar1 each from one drop of the original milk sample4 #c4 ,incubate aerobically at #cg deg4 ,c4 and examine daily up to the end of ten days4 #d4 ,pick off suspicious colonies1 examine them microscopically and subcultivate upon nutrose agar in tubes2 upon glucose agar and in litmus milk4 #e4 ,test the subsequent growth against the serum of an experimental animal inoculated against ,m4 melitensis to determine its agglutinability4 #f4 ,if apparently ,m4 melitensis1 inoculate growth from a nutrose agar culture after three days incubation intracranially into the guinea-pig4 ,,ice ,,cream4 ,collection of the ,sample4-- #a4 ,remove the sample from the drum in the ladle or spoon with which the vendor retails the ice cream1 and place it at once in a sterile copper capsule1 similar to that employed for earth samples 7vide page #dga74 #b4 ,pack for transmission in the ice-box4 #c4 ,on arrival at the laboratory place the copper capsules containing the ice cream in the incubator at #bj deg4 ,c4 for fifteen minutes--that is1 until at least some of the ice cream has become liquid4 ,qualitative and ,quantitative ,examination4--,treat the fluid ice cream as milk and conduct the examination in precisely the same manner as described for milk 7vide page #ddc74 ,,examination ,,of ,,cream ,,and ,,butter4 ,collection of the ,sample4--,collect1 store1 and transmit samples to the laboratory1 precisely as is done in the case of ice cream4 ,quantitative4-- ,apparatus ,required3 ,sterile test-tube4 ,sterilised spatula4 ,water-bath regulated at #db deg4 ,c4 ,case of sterile plates4 ,case of sterile graduated pipettes1 #a c4c4 7in hundredths74 ,tubes of gelatine-agar 7#aj reaction74 ,plate-levelling stand1 with its water chamber filled with water at #db deg4 ,c4 ,,method4-- #a4 ,transfer a few grammes of the sample to a sterile test-tube by means of the sterilised spatula4 #b4 ,place the tube in the water-bath at #db deg4 ,c4 until the contents are liquid4 #c4 ,liquefy eight tubes of gelatine-agar and place them in the water-bath at #db deg4 ,c1 and cool down to that temperature4 #d4 ,inoculate the gelatine-agar tubes with the following quantities of the sample by the help of a sterile pipette graduated to hundredths of a cubic centimetre--viz41 ,to tube ,no4 #a add #a c4c4 liquefied butter4 #b add #j4e c4c4 liquefied butter4 #c add #j4c c4c4 liquefied butter4 #d add #j4b c4c4 liquefied butter4 #e add #j4a c4c4 liquefied butter4 #f add #j4je c4c4 liquefied butter4 #g add #j4jc c4c4 liquefied butter4 #h add #j4jb c4c4 liquefied butter4 #i add #j4ja c4c4 liquefied butter4 #e4 ,pour a plate cultivation from each of the gelatine-agar tubes and incubate at #bh deg4 ,c4 #f4 ,count the plates after three days' incubation1 and from the figures thus obtained estimate the number of organisms present per cubic centimetre of the sample4 ,qualitative4-- ,apparatus ,required3 ,sterile beaker1 its mouth plugged with sterile cotton-wool4 ,counterpoise for beaker4 ,scales and weights4 ,sterilised spatula4 ,water-bath regulated at #db deg4 ,c4 ,separatory funnel1 #bej c4c4 capacity1 its delivery tube protected against contamination by passing it through a cotton-wool plug into the interior of a small ,erlenmeyer flask which serves to support the funnel4 ,this piece of apparatus is sterilised en masse in the hot-air oven4 ,large centrifugal machine4 ,sterile tubes 7for the centrifuge7 closed with solid rubber stoppers4 ,case of sterile pipettes1 #aj c4c4 ,case of sterile graduated pipettes1 #a c4c4 7in tenths of a cubic centimetre74 ,,method4-- #a4 ,weigh out #ajj grammes of the sample in a sterile beaker4 #b4 ,plug the mouth of the beaker with sterile cotton-wool and immerse the beaker in a water-bath at #db deg4 ,c4 until the contents are completely liquefied4 #c4 ,fill the liquefied butter into the sterile separatory funnel4 #d4 ,transfer the funnel to the incubator at #cg deg4 ,c4 and allow it to remain there for four days4 ,at the end of this time the contents of the funnel will have separated into two distinct strata4 7a7 ,a superficial oily layer1 practically free from bacteria4 7b7 ,a deep watery layer1 turbid and cloudy from the growth of bacteria4 #e4 ,draw off the subnatant turbid layer into sterile centrifugal tubes1 previously warned to about #db deg4 ,c41 and centrifugalise at once4 #f4 ,pipette off the supernatant fluid and fill the tubes with sterile #a per cent4 sodium carbonate solution previously warmed slightly2 stopper the tubes and shake vigourously for a few minutes4 #g4 ,centrifugalise again4 #h4 ,pipette off the supernatant fluid2 filling the tubes with warm sterile bouillon1 shake well1 and again centrifugalise1 to wash the deposit4 #i4 ,pipette off the supernatant fluid4 #aj4 ,prepare cover-slip preparations1 fix and clear as for milk preparations1 stain carbolic methylene-blue1 ,gram's method1 ,ziehl-,neelsen's method1 and examine microscopically with a #a_/ab inch oil-immersion lens4 #aa4 ,proceed with the examination of the deposit as in the case of milk deposit 7see pages #dej et seq474 ,,examination ,,of ,,unsound ,,meats4 7,,including ,,tinned ,,or ,,potted ,,meats1 ,,fish1 ,,etc47 ,the bacterioscopic examination of unsound food is chiefly directed to the detection of those members of the ,coli-typhoid group--,b4 enteritidis of ,gaertner and its allies--which are usually associated with epidemic outbreaks of food poisoning1 and such anaerobic bacteria as initiate putrefactive changes in the food which result in the formation of poisonous ptomaines1 consequently the quantitative examination pure and simple is frequently omitted4 ,a4 ,cultural ,examination4 ,quantitative4-- ,apparatus ,required3 ,sterilised tin opener1 7if necessary47 ,erlenmeyer flask 7#ejj c4c4 capacity7 containing #bjj c4c4 sterile bouillon and fitted with solid rubber stopper4 ,counterpoise4 ,scissors and forceps4 ,scales and weights4 ,water steriliser4 ,hypodermic syringe4 ,syringe with intragastric tube4 ,rat forceps4 ,case of sterile capsules4 ,filtering apparatus as for water analysis4 ,case of sterile plates4 ,case of sterile graduated pipettes1 #aj c4c4 7in tenths of a cubic centimetre74 ,case of sterile graduated pipettes1 #a c4c4 7in tenths of a cubic centimetre74 ,plate-levelling stand4 ,tubes of nutrient gelatine4 ,tubes of nutrient agar4 ,water-bath regulated at #db deg4 ,c4 ,bulloch's apparatus4 ,,method4-- #a4 ,place the flask containing #bjj c4c4 sterile broth on one pan of the scales and counterpoise accurately4 #b4 ,mince a portion of the sample by the aid of sterile scissors and forceps1 and add the minced sample to the bouillon in the flask to the extent of #bj grammes4 #c4 ,make an extract by standing the flask in the incubator running at #db deg4 ,c4 7or in a water-bath regulated to that temperature7 for half an hour1 shaking its contents from time to time4 ,better results are obtained if an electrical shaker is fitted inside the incubator and the flask kept in motion throughout the entire thirty minutes4 ,now every centimetre contains the bacteria washed out from #j4a gramme of the original food4 #d4 ,inoculate tubes of liquefied gelatine as follows3 ,to tube ,no4 #a add #a4j c4c4 of the extract4 #b add #j4e c4c4 of the extract4 #c add #j4c c4c4 of the extract4 #d add #j4b c4c4 of the extract4 #e add #j4a c4c4 of the extract4 ,pour plates from these tubes and incubate at #bj deg4 ,c4 #e4 ,prepare a precisely similar set of agar plates and incubate at #cg deg4 ,c4 #f4 ,pipette #e c4c4 of the extract into a sterile tube1 heat in the differential steriliser at #hj deg4 ,c4 for ten minutes4 #g4 ,from the heated extract prepare duplicate sets of agar and gelatine plates and incubate anaerobically in ,bulloch's apparatus at #cg deg4 ,c4 and #bj deg4 ,c4 respectively4 #h4 ,after three days' incubation examine the agar plates both aerobic and anaerobic and enumerate the colonies developed from spores 7#g71 and from vegetative forms and spores 7#e71 and calculate and record the numbers of each group per gramme of the original food4 #i4 ,after seven days' incubation 7or earlier if compelled by the growth of liquefying colonies7 enumerate the gelatine plates in the same way4 #aj4 ,subcultivate from the colonies that make their appearance and identify the various organisms4 #aa4 ,continue the investigations with reference to the detection of pathogenic organisms as described under water 7page #dbi et seq474 ,qualitative4-- ,i4 ,cultural4 ,the micro-organisms sought for during the examination of unsound foods comprise the following3 ,members of the ,coli-typhoid groups 7chiefly those of the ,gaertner class74 ,b4 anthracis4 ,streptococci ,anaerobic ,bacteria3 ,b4 enteritidis sporogenes4 ,b4 botulinus4 ,b4 cadaveris4 ,the methods by which these organisms if present may be identified and isolated have already been described under the corresponding section of water examination with the exception of those applicable to ,b4 botulinus1 and ,b4 cadaveris4 ,these can only be isolated satisfactorily from the bodies of experimentally inoculated animals4 ,,ii ,experimental4 ,tissue4-- #a4 ,feed rats and mice on portions of the sample and observe the result4 #b4 ,if any of the animals die1 make complete post-mortem examinations and endeavour to isolate the pathogenic organisms4 ,extract4-- #a4 ,introduce various quantities of the bouillon extract into the stomachs of several rats1 mice and guinea-pigs repeatedly over a period of two or three days by the intragastric method of inoculation 7see page #cfg7 and observe the result4 ,guinea-pigs and mice are very susceptible to infection by ,b4 botulinus by this method2 rabbits less so4 #b4 ,inoculate rats1 mice1 and guinea-pigs subcutaneously into deep pockets1 and intraperitoneally with various quantities of the bouillon extract1 and observe the result4 #c4 ,filter some of the extract through a ,chamberland candle and incubate the filtrate to determine the presence of soluble toxins4 #d4 ,if any of the animals succumb to either of these methods of inoculation1 make careful post-mortem examinations and endeavour to isolate the pathogenic organisms4 ,,the ,,examination ,,of ,,oysters ,,and ,,other ,,shellfish4 ,on opening the shell of an oyster a certain amount of fluid termed liquor is found to be present4 ,this varies in amount from a drop to many cubic centimetres 7#j4a c4c4 to #aj c4c47--in the latter case the bulk of the fluid is probably the last quantum of water ingested by the bivalve before closing its shell4 ,in order to obtain a working average of the bacteriological flora of a sample1 ten oysters should be taken and the body1 gastric juice and liquor should be thoroughly mixed before examination4 ,the examination1 as in dealing with other food stuffs1 is directed to the search for members of the ,coli-typhoid group1 sewage streptococci and perhaps also ,b4 enteritidis sporogenes4 ,apparatus ,required3 ,two hard nail brushes4 ,liquid soap4 ,sterile water in aspirator jar with delivery nozzle controlled by a spring clip4 ,sterile oyster knives4 ,sterile glass dish1 with cover1 sufficiently large to accommodate ten oysters4 ,sterile forceps4 ,sterile scissors4 ,sterile towels or large gauze pads4 ,sterile graduated cylinders #ajjj c4c4 capacity1 with either the lid or the bottom of a sterile ,petri dish inverted over the open mouth as a cover4 ,glass rods4 ,corrosive sublimate solution1 #a per mille4 ,bile salt broth tubes4 ,litmus milk tubes4 ,surface plates of nutrose agar4 ,case of sterile pipettes1 #a c4c4 7in tenths of a c4c47 ,case of sterile pipettes1 #aj c4c4 7in tenths of a c4c47 ,case of sterile glass capsules4 ,erlenmeyer flasks1 #bej c4c4 capacity4 ,double strength bile salt broth4 ,,method4-- #a4 ,thoroughly clean the outside of the oyster shells by scrubbing each in turn with liquid soap and nail brush under a tap of running water4 ,then1 holding an oyster shell in a pair of sterile forceps wash every part of the outside of the shell with a stream of sterile water running from an aspirator jar2 deposit the oyster inside the sterile glass dish4 ,repeat the process with each of the remaining oysters4 #b4 ,before proceeding further1 cleanse the hands thoroughly with clean nail brush1 soap and water1 then plunge them in lysol #b per cent4 solution1 and finally in sterile water4 #c4 ,spread a sterile towel on the bench4 #d4 ,remove one of the oysters from the sterile glass dish and place it1 resting on its convex shell1 on the towel4 ,turn a corner of the sterile towel over the upper flat shell to give a firmer grip to the left hand1 which holds the shell in position4 #e4 ,with the sterile oyster knife 7in the right hand7 open the shell and separate the body of the oyster from the inner surface of the upper flat shell4 ,bend back and separate the flat shell1 leaving the body of the oyster in and attached to the concave shell4 ,avoid spilling any of the liquor4 7,some dexterity in opening oysters should be acquired before undertaking these experiments74 #f4 ,cut up the body of the oyster with sterile scissors into small pieces and allow the liquor freed from the body during the process to mix with the liquor previously in the shell4 #g4 ,transfer the comminuted oyster and the liquor to the cylinder4 #h4 ,treat each of the remaining oysters in similar fashion4 #i4 ,mix the contents of the cylinder thoroughly by stirring with a sterile glass rod4 ,the total volume will amount to about #ajj c4c4 #aj4 ,use #j4a c4c4 of the mixed liquor to inseminate each of a series of three nutrose surface plates4 #aa4 ,inoculate #j4a c4c4 of the mixed liquor into each of three tubes of litmus milk4 #ab4 ,add sterile distilled water to the contents of the cylinder up to #ajjj c4c4 and stir thoroughly with a sterile glass rod and allow to settle4 ,the bacterial content of each oyster may be regarded1 for all practical purposes1 as comprised in #ajj c4c4 of fluid4 #ac4 ,arrange four glass capsules in a row and number ,i1 ,,ii1 ,,iii1 ,,iv4 ,pipette #i c4c4 sterile distilled water into each4 #ad4 ,to capsule ,no4 ,i add #a c4c4 of the diluted liquor1 etc4 from the cylinder1 and mix thoroughly4 ,to capsule ,,ii add #a c4c4 of dilution in capsule ,i and mix thoroughly4 ,carry over #a c4c4 of fluid from capsule ,,ii to capsule ,,iii1 afterwards adding #a c4c4 of fluid from capsule ,,iii to capsule ,,iv4 #ae4 ,label tubes of bile salt broth and inoculate with the following amounts of diluted oysters3 ,no4 #f with #aj c4c4 cylinder fluid #j4a oyster4 ,no4 #e with #a c4c4 cylinder fluid #j4ja oyster4 ,no4 #d with #a c4c4 capsule ,i fluid #j4jja oyster4 ,no4 #c with #a c4c4 capsule ,,ii fluid #j4jjja oyster4 ,no4 #b with #a c4c4 capsule ,,iii fluid #j4jjjja oyster4 ,no4 #a with #a c4c4 capsule ,,iv fluid #j4jjjjja oyster4 #af4 ,transfer #ajj c4c4 cylinder fluid 7 #a oyster7 to an ,erlenmeyer flask and add #ej c4c4 double strength bile salt broth1 and label #g4 #ag4 ,duplicate all the above indicated cultures4 #ah4 ,put up the tube cultures in ,buchner's tubes and incubate anaerobically at #db deg4 ,c4 ,if growth occurs in tube #a the organism finally isolated1 e4 g41 ,b4 coli1 must have been present to the extent of one million per oyster4 #ai4 ,complete the examination for members of the ,coli-typhoid group and sewage streptococci1 as directed under ,water ,examination1 page #dbi 7steps #aa-ba74 #bj4 ,inoculate a series of #f tubes of litmus milk with quantities of the material similar to those indicated in step #ae2 heat to #hj deg4 ,c4 for ten minutes1 and incubate under anaerobic conditions at #cg deg4 ,c4 ,examine for the presence of ,b4 enteritidis sporogenes as directed under ,water ,examination1 page #dch 7steps #g-aj74 ,,examination ,,of ,,sewage ,,and ,,sewage ,,effluents4 ,quantitative4-- ,collection of the ,sample4--,as only small quantities of material are needed1 the samples should be collected in a manner similar to that described under water for quantitative examination and transmitted in the ice apparatus used in packing those samples4 ,apparatus ,required4--,as for water 7vide page #dbj74 ,,method4-- #a4 ,arrange four sterile capsules in a row and number them ,i1 ,,ii1 ,,iii1 ,,iv4 #b4 ,pipette #i c4c4 sterile bouillon into capsule ,no4 ,i4 #c4 ,pipette #i4i c4c4 sterile bouillon into capsules ,,ii1 ,,iii1 and ,,iv4 #d4 ,add #a c4c4 of the sewage to capsule ,no4 ,i by means of a sterile pipette1 and mix thoroughly4 #e4 ,take a fresh sterile pipette and transfer #j4a c4c4 of the mixture from ,no4 ,i to ,no4 ,,ii and mix thoroughly4 #f4 ,in like manner transfer #j4a c4c4 from ,no4 ,,ii to ,no4 ,,iii1 and then #j4a c4c4 from ,no4 ,,iii to ,no4 ,,iv4 ,now #a c4c4 of dilution ,no4 ,i contains #j4a c4c4 of the original sewage4 #a c4c4 of dilution ,no4 ,,ii contains #j4jja c4c4 of the original sewage4 #a c4c4 of dilution ,no4 ,,iii contains #j4jjjja c4c4 of the original sewage4 #a c4c4 of dilution ,no4 ,,iv contains #j4jjjjjja c4c4 of the original sewage4 #g4 ,pour a set of gelatine plates from the contents of each capsule1 three plates in a set1 and containing respectively #j4b1 #j4c1 and #j4e c4c4 of the dilution4 ,label carefully2 incubate at #bj deg4 ,c4 for three1 four1 or five days4 #h4 ,enumerate the organisms present in those sets of plates which have not liquefied1 probably those from dilution ,,iii or ,,iv1 and calculate therefrom the number present per cubic centimetre of the original sample of sewage4 ,qualitative4--,the qualitative examination of sewage is concerned with the identification and enumeration of the same bacteria dealt with under the corresponding section of water examination2 it is consequently conducted on precisely similar lines to those already indicated 7vide pages #dbf to #dda74 ,,examination ,,of ,,air4 ,quantitative4-- ,apparatus ,required3 ,aspirator bottle1 #aj litres capacity1 fitted with a delivery tube1 and having its mouth closed by a perforated rubber stopper1 through which passes a short length of glass tubing4 ,erlenmeyer flask1 #bej c4c4 capacity 7having a wide mouth properly plugged with wool71 containing #ej c4c4 sterile water4 ,rubber stopper to fit the mouth of the flask1 perforated with two holes1 and fitted as follows3 ,take a #i cm4 length of glass tubing and bend up #c cm4 at one end at right angles to the main length of tubing4 ,pass the long arm of the angle through one of the perforations in the stopper2 plug the open end of the short arm with cotton-wool4 ,take a glass funnel #e or #f cm4 in diameter with a stem #ab cm4 in length and bend the stem close up to the apex of the funnel1 in a gentle curve through a quarter of a circle2 pass the long stem through the other perforation in the rubber stopper4 ,a battery jar or a small water-bath to hold the ,erlenmeyer flask when packed round with ice4 ,supply of broken ice4 ,rubber tubing4 ,screw clamps and spring clips1 for tubing4 ,water steriliser4 ,retort stand and clamps4 ,apparatus for plating 7as for enumeration of water organisms1 vide page #dbj74 ,,method4-- #a4 ,fill #aj litres of water into the aspirating bottle and attach a piece of rubber tubing with a screw clamp to the delivery tube4 ,open the taps fully and regulate the screw clamp1 by actual experiment1 so that the tube delivers #a c4c4 of water every second4 ,the screw clamp is not touched again during the experiment4 ,at this rate the aspirator bottle will empty itself in just under three hours4 ,shut off the tap and make up the contents of the aspirator bottle to #aj litres again4 #b4 ,sterilise the fitted rubber cork1 with its funnel and tubing1 by boiling in the water steriliser for ten minutes4 #c4 ,remove the cotton-wool plug from the flask1 and replace it by the rubber stopper with its fittings4 ,make sure that the end of the stem of the funnel is immersed in the bouillon4 #d4 ,place the flask in a glass or metal vessel and pack it round with pounded ice4 ,arrange the flask with its ice casing just above the neck of the aspirator bottle4 ,illustration3 ,,fig4 #baf4--;,arrangement of apparatus for air analysis4 #e4 ,connect up the free end of the glass tube from the flask--after removing the cotton-wool plug--with the air-entry tube in the mouth of the aspirating bottle 7,fig4 #baf74 #f4 ,open the tap fully1 and allow the water to run4 ,replenish the ice from time to time if necessary4 7,in emptying itself the aspirator bottle will aspirate #aj litres of air slowly through the water in the ,erlenmeyer flask47 #g4 ,when the aspiration is completed1 disconnect the flask and remove it from its ice packing4 #h4 ,liquefy three tubes of nutrient gelatine and add to them #j4e c4c41 #j4c c4c41 and #j4b c4c41 respectively1 of the water from the flask1 by means of a sterile graduated pipette1 as in the quantitative examination of water4 ,pour plates4 #i4 ,pour a second similar set of gelatine plates4 #aj4 ,incubate both sets of plates at #bj deg4 ,c4 #aa4 ,enumerate the colonies present in the two sets of gelatine plates after three1 four1 or five days and average the results from the numbers so obtained2 estimate the number of micro-organisms present in #a c4c41 and then in the #ej c4c4 of broth in the flask4 #ab4 ,the result of air examination is usually expressed as the number of bacteria present per cubic metre 7i4 e41 kilolitre7 of air2 and as the number of organisms present in the #ej c4c4 water only represent those contained in #aj litres of air1 the resulting figure must be multiplied by #ajj4 ,qualitative4-- #a4 ,proceed exactly as in the quantitative examination of air 7vide supra71 steps #a to #aj4 #b4 ,pour plates of wort agar with similar quantities of the air-infected water1 and incubate at #cg deg4 ,c4 #c4 ,pour plates of nutrient agar with similar quantities of the water and incubate at #cg deg4 ,c4 #d4 ,pour similar plates of wort gelatine and incubate at #bj deg4 ,c4 #e4 ,pick off the individual colonies that appear in the several plates1 subcultivate them on the various media1 and identify them4 ,,examination ,,of ,,soil4 ,the bacteriological examination of soil yields information of value to the sanitarian during the progress of the process of homogenisation of made soil 7e4 g41 a dumping area for the refuse of town7 and determines the period at which such an area may with propriety and safety be utilised for building purposes2 or to the agriculturalist in informing him of the suitability of any given area for the growth of crops4 ,the surface of the ground1 exposed as it is to the bactericidal influence of sunlight and to rapid alternations of heat and cold1 rain and wind1 contains but few micro-organisms4 ,again1 owing to the density of the molecules of deep soil and lack of aeration on the one hand1 and the filtering action of the upper layers of soil and bacterial antagonism on the other1 bacterial life practically ceases at a depth of about #b metres4 ,the intermediate stratum of soil1 situated from #be to #ej cm4 below the surface1 invariably yields the most numerous and the most varied bacterial flora4 ,collection of ,sample4--,a small copper capsule #f cm4 high by #f cm4 diameter1 with pull-off cap secured by a bayonet catch1 previously sterilised in the hot-air oven1 is the most convenient receptacle for samples of soil4 ,illustration3 ,,fig4 #bag4--,soil scoop4 ,the instrument used for the actual removal of the soil from its natural position will vary according to whether we require surface samples or soil from varying depths4 7a7 ,for surface samples1 use an iron scoop1 shaped like a shoe horn1 but provided with a sharp spine 7,fig4 #bag74 ,this is wrapped in asbestos cloth and sterilised in the hot-air oven4 ,when removed from the oven1 wrap a piece of oiled paper1 silk1 or gutta-percha tissue over the asbestos cloth1 and secure it with string1 as a further protection against contamination4 ,on reaching the spot whence the samples are to be taken1 the coverings of the scoop are removed1 and the asbestos cloth employed to brush away loose stones and debris from the selected area4 ,the surface soil is then broken up with the point of the scoop1 scraped up and collected in the body of the scoop1 and transferred to the sterile capsule for transmission4 ,illustration3 ,,fig4 #bah4--;,fraenkel's borer4 7b7 ,for deep samples collected at various distances from the surface1 an experimental trench may be cut to the required depth and samples collected at the required points on the face of the section4 ,it is1 however1 preferable to utilise some form of borer1 such as that designed by ,fraenkel 7,fig4 #bah74 ,fraenkel's ,earth ,borer4--,this instrument consists of a stout hard-steel rod1 #aej cm4 long1 marked in centimetres from the drill-pointed extremity4 ,it is provided with a cross handle 7adjustable at any point along the length of the rod by means of a screw nut74 ,the terminal centimeters are thicker than the remainder of the rod1 and on one side a vertical cavity about #j4e cm4 deep is cut4 ,this is covered by a flanged sleeve so long as the borer is driven into the soil clockwise1 and is opened for the reception of the sample of soil1 when the required depth is reached1 by reversing the screwing motion1 and again closed before withdrawal of the borer from the earth by resuming the original direction of twist4 ,it can be sterilised in a manner similar to that adopted for the scoop1 or by repeatedly filling the cavity with ether and burning it off4 ,quantitative4--,four distinct investigations are included in the complete quantitative bacteriological examination of the soil3 #a4 ,the enumeration of the aerobic organisms4 #b4 ,the enumeration of the spores of aerobes4 #c4 ,the enumeration of the anaerobic organisms 7including the facultative anaerobes74 #d4 ,the enumeration of the spores of anaerobes4 ,further1 by a combination of the results of the first and second1 and of the third and fourth of these1 the ratio of spores to vegetative forms is obtained4 ,apparatus ,required3 ,case of sterile capsules 7#be c4c4 capacity74 ,case of sterile graduated pipettes1 #aj c4c4 7in tenths of a cubic centimetre74 ,case of sterile graduated pipettes1 #a c4c4 7in tenths of a cubic centimetre74 ,flask containing #bej c4c4 sterile bouillon4 ,tall cylinder containing #b per cent4 lysol solution4 ,plate-levelling stand4 #ab sterile plates4 ,tubes of nutrient gelatine4 ,tubes of wort gelatine4 ,tubes of nutrient agar4 ,tubes of glucose formate gelatine4 ,tubes of glucose formate agar4 ,water-bath regulated at #db deg4 ,c4 ,bunsen burner4 ,grease pencil4 ,sterile mortar and pestle 7agate74 ,sterile wide-mouthed ,erlenmeyer flask 7#ejj c4c4 capacity74 ,sterile metal funnel with short wide bore delivery tube to just fit mouth of flask4 ,solid rubber stopper to fit the flask 7sterilised by boiling74 ,pair of scales4 ,counterpoise 7,fig4 #ajg74 ,sterile metal 7nickel7 spoon or spatula4 ,fractional steriliser 7,fig4 #adj74 ,,method4-- #a4 ,arrange four sterile capsules numbered ,i1 ,,ii1 ,,iii1 and ,,iv2 pipette #i c4c4 sterile bouillon into the first capsule1 and #i4i c4c4 into each of the remaining three4 #b4 ,pipette #ajj c4c4 sterile bouillon into the ,erlenmeyer flask4 #c4 ,remove the cotton-wool plug from the flask and replace it by the sterile funnel4 #d4 ,place flask and funnel on one pan of the scales1 and counterpoise accurately4 #e4 ,empty the sample of soil into the mortar and triturate thoroughly4 #f4 ,by means of the sterile spatula add #aj grammes of the earth sample to the bouillon in the flask4 ,the final results will be more reliable if steps #b1 #c1 #d1 and #e are performed under a hood--to protect from falling dust1 etc4 #g4 ,remove the funnel from the mouth of the flask2 replace it by the rubber stopper and shake vigourously2 then allow the solid particles to settle for about thirty minutes4 ,one cubic centimetre of the turbid broth contains the washings from #j4a gramme of soil4 #h4 ,pipette off #a c4c4 of the supernatant bouillon1 termed the soil water1 and add it to the contents of capsule ,i2 mix thoroughly4 #i4 ,remove #j4a c4c4 of the infected bouillon from capsule ,i and add it to capsule ,,ii1 and mix4 #aj4 ,in like manner add #j4a c4c4 of the contents of capsule ,,ii to capsule ,,iii1 and then #j4a c4c4 of the contents of capsule ,,iii to capsule ,,iv4 ,then #a c4c4 fluid from capsule ,i contains soil water from 4#ja gm4 earth4 ,then #a c4c4 fluid from capsule ,,ii contains soil water from 4#jjja gm4 earth4 ,then #a c4c4 fluid from capsule ,,iii contains soil water from 4#jjjjja gm4 earth4 ,then #a c4c4 fluid from capsule ,,iv contains soil water from 4#jjjjjjja gm4 earth4 7,a7 ,aerobes 7,vegetative ,forms and ,spores74-- #aa4 ,pour a set of gelatine plates from the contents of each capsule--two plates in a set1 and containing respectively #j4a c4c4 and #j4d c4c4 of the diluted soil water4 ,label and incubate4 #ab4 ,pour similar sets of wort gelatine plates from the contents of capsules ,,ii and ,,iii1 label1 and incubate at #bj deg4 ,c4 #ac4 ,pour similar sets of agar plates from the contents of capsules ,,ii and ,,iii2 label and incubate at #cg deg4 ,c4 #ad4 ,weigh out a second sample of soil--#aj grammes--dry over a water-bath until of constant weight and calculate the ratio wet soil weight --------------- dry soil weight #ae4 ,count the plates after incubation for three1 four1 or five days1 and correcting the figures thus obtained by means of the wet to dry soil ratio estimate-- 7a7 ,the number of aerobic micro-organisms present per gramme of the soil4 7b7 ,the number of yeasts and moulds present per gramme of the soil4 7c7 ,the number of aerobic organisms growing at #cg deg4 ,c4 present per gramme of the soil4 7,b7 ,anaerobes 7,vegetative ,forms and ,spores74-- #af4 ,pour similar sets of plates in glucose formate gelatine and agar and incubate in ,bulloch's anaerobic apparatus4 7,c7 ,aerobes and ,anaerobes 7,spores ,only74-- #ag4 ,pipette #e c4c4 soil water into a sterile tube4 #ah4 ,place in the differential steriliser at #hj deg4 ,c4 for ten minutes4 #ai4 ,pour two sets of four gelatine plates containing #j4a1 #j4b1 #j4e1 and #a c4c4 respectively of the soil water2 label and incubate at #bj deg4 ,c41 one set aerobically1 the other anaerobically in ,bulloch's apparatus4 #bj4 ,count the plates 7delay the enumeration as long as possible7 and estimate the number of spores of aerobes and anaerobes respectively present per gramme of the soil4 #ba4 ,calculate the ratio existing between spores and spores vegetative forms under each of the two groups1 aerobic and anaerobic micro-organisms4 ,qualitative ,examination4--,the qualitative examination of soil is usually directed to the detection of one or more of the following3 ,members of the ,coli-typhoid group4 ,streptococci4 ,bacillus anthracis4 ,bacillus tetani4 ,bacillus oedematis maligni4 ,the nitrous organisms4 ,the nitric organisms4 #a4 ,transfer the remainder of the soil water 7#hh c4c47 to a sterile ,erlenmeyer flask by means of a sterile syphon4 #b4 ,fix up the filtering apparatus as for the qualitative examination of water1 and filter the soil water4 #c4 ,suspend the bacterial residue in #e c4c4 sterile bouillon 7technique similar to that described for the water sample1 vide pages #dcd-dcf74 ,every cubic centimetre of suspension now contains the soil water from nearly #a gramme of earth4 ,the methods up to this point are identical no matter which organism or group of organisms it is desired to isolate2 but from this stage onward the process is varied slightly for each particular bacterium4 ,i4 ,the ,coli-typhoid ,group4-- ,,ii4 ,streptococci4-- ,,iii4 ,bacillus ,anthracis4-- ,,iv4 ,bacillus ,tetani4-- ,the methods adopted for the isolation of these organisms are identical with those already described under water 7page #dcg et seq474 ,v4 ,bacillus ,oedematis ,maligni4--,method precisely similar to that employed for the ,b4 tetani4 ,,vi4 ,the ,nitrous ,organisms4-- #a4 ,take ten tubes of ,winogradsky's solution ,no ,i 7vide page #aih7 and number them consecutively from #a to #aj4 #b4 ,inoculate each tube with varying quantities of the material as follows3 ,to tube ,no4 #a add #a4j c4c4 of the soil water4 ,to tube ,no4 #b add #j4a c4c4 of the soil water4 ,to tube ,no4 #c add #a4j c4c4 from ,capsule ,i4 ,to tube ,no4 #d add #j4a c4c4 from ,capsule ,i4 ,to tube ,no4 #e add #a4j c4c4 from ,capsule ,,ii4 ,to tube ,no4 #f add #j4a c4c4 from ,capsule ,,ii4 ,to tube ,no4 #g add #a4j c4c4 from ,capsule ,,iii4 ,to tube ,no4 #h add #j4a c4c4 from ,capsule ,,iii4 ,to tube ,no4 #i add #a4j c4c4 from ,capsule ,,iv4 ,to tube ,no4 #aj add #j4a c4c4 from ,capsule ,,iv4 ,label and incubate at #cj deg4 ,c4 ,,vii4 ,the ,nitric ,organisms4-- #c4 ,take ten tubes of ,winogradsky's solution ,no ,,ii1 number them consecutively from #a to #aj and inoculate with quantities of soil water similar to those enumerated in section ,,vi step #b4 ,label and incubate at #cj deg4 ,c4 #d4 ,examine after twenty-four and forty-eight hours' incubation4 ,from those tubes that show signs of growth make subcultivations in fresh tubes of the same medium and incubate at #cj deg4 ,c4 #e4 ,make further subcultivations from such of those tubes as show growth1 and again incubate4 #f4 ,if growth occurs in these subcultures1 make surface smears on plates of ,winogradsky's silicate jelly 7vide page #aih74 #g4 ,pick off such colonies as make their appearance and subcultivate in each of these two media4 ,,testing ,,filters4 ,porcelain filter candles are examined with reference to their power of holding back all the micro-organisms suspended in the fluids which are filtered through them1 and permitting only the passage of germ-free filtrates4 ,in order to determine the freedom of the filter from flaws and cracks which would permit the passage of bacteria no matter how perfect the general structure of the candle might be1 the candle must first be attached by means of a long piece of pressure tubing1 to a powerful pump1 such as a foot bicycle pump1 fitted with a manometer4 ,the candle is then immersed in a jar of water and held completely submerged whilst the internal pressure is gradually raised to two atmospheres by the action of the pump4 ,any crack or flaw will at once become obvious by reason of the stream of air bubbles issuing from it4 ,the examination for permeability is conducted as follows3 ,apparatus ,required3 ,filtering apparatus3 ,the actual filter candle that is used must be the one it is intended to test and must be previously carefully sterilised2 the arrangement of the apparatus will naturally vary with each different form of filter1 one or other of those already described 7vide pages #db-dh74 ,plate-levelling stand4 ,case of sterile plates4 ,case of sterile pipettes1 #aj c4c4 7in tenths74 ,case of sterile pipettes1 #a c4c4 7in tenths74 ,tubes of nutrient gelatine4 ,flask containing sterile normal saline solution4 ,sterile measuring flask1 #ajjj c4c4 capacity4 ,,method4-- #a4 ,prepare surface cultivations1 on nutrient agar in a culture bottle1 of the ,bacillus mycoides1 and incubate at #bj deg4 ,c41 for forty-eight hours4 #b4 ,pipette #e c4c4 sterile normal saline into the culture bottle and emulsify the entire surface growth in it4 #c4 ,pipette the emulsion into the sterile measuring flask and dilute up to #ajjj c4c4 by the addition of sterile water4 #d4 ,pour the emulsion into the filter reservoir and start the filtration4 #e4 ,when the filtration is completed1 pour six agar plates each containing #a c4c4 of the filtrate4 #f4 ,incubate at #cg deg4 ,c4 until1 if necessary1 the completion of seven days4 #g4 ,if the filtrate is not sterile1 subcultivate the organism passed and determine its identity with the test bacterium before rejecting the filter--since the filtrate may have been accidentally contaminated4 #h4 ,if the filtrate is sterile1 resterilise the candle and repeat the test now substituting a cultivation of ,b4 prodigiosus--a bacillus of smaller size4 #i4 ,if the second test is satisfactory1 test the candle against a cultivation of a very small coccus1 e4 g41 ,micrococcus melitensis1 in a similar manner2 in this instance continuing the incubation of cultivations from the filtrate for fourteen days4 ,,testing ,,of ,,disinfectants4 ,methods have already been detailed 7page #caj7 for the purpose of studying the vital resistance offered by micro-organisms to the lethal effect of germicides4 ,but it frequently happens that the bacteriologist has to determine the relative efficiency of disinfectants from the standpoints of the sanitarian and commercial man rather than from the research worker's point of view4 ,in pursuing this line of investigation1 it is convenient to compare the efficiency1 under laboratory conditions1 of the proposed disinfectant with that of some standard germicide1 such as pure phenol4 ,in so doing1 and in order that the work of different observers may be compared1 conditions as nearly uniform as possible should be aimed at4 ,the method described is one that has been in use by the writer for many years past1 modified recently by the adoption of some of the recommendations of the ,lancet ,commission on the ,standardisation of ,disinfectants--particularly of the calculation for determining the phenol coefficient4 ,this method has many points in common with that modification of the drop method known as the ,rideal-,walker test4 ,general ,considerations4-- ,these may be grouped under three headings3 ,test ,germ1 ,germicide1 and ,environment4 #a4 ,test ,germ4--,b4 coli4 ,as disinfectants are tested for sanitary purposes1 it is obvious that a member of the coli-typhoid group should be selected as the test germ4 ,b4 coli is selected on account of its relative nonpathogenicity1 the ease with which it can be isolated and identified by different observers in various parts of the world1 the stability of its fundamental characters1 and evenness of its resistance when utilised for these tests2 finally since the colon bacillus is an organism which is slightly more resistant to the lethal action of germicides than the more pathogenic members of this group1 a margin of safety is introduced into the test which certainly enhances its value4 ,b4 coli should be recently isolated from a normal stool1 and plated at least twice to ensure the purity of the strain2 and a stock agar culture prepared which should be used throughout any particular test4 ,for any particular experiment prepare a smear culture on agar and incubate at #cg deg4 ,c4 for #bd hours anaerobically4 ,then emulsify the whole of the surface growth in #aj c4c4 of sterile water4 ,transfer the emulsion to a sterile test-tube with some sterile glass beads and shake thoroughly to ensure homogenous emulsion4 ,transfer to a centrifuge tube and centrifugalise the emulsion to throw down any masses of bacteria which may have escaped the disintegrating action of the beads4 ,pipette off the supernatant emulsion for use in the test4 #b4 ,germicide4-- a4 ,disinfectant to be tested4-- ,the first essential point is to test the unknown disinfectant1 which may be referred to as germicide-x1 on the lines set out on page #caa to determine its inhibition coefficient4 ,this constant having been fixed1 prepare various solutions of germicide-x with sterilised distilled water by accurate volumetric methods1 commencing with a solution somewhat stronger than that representing the inhibition coefficient4 ,the solutions must be prepared in fairly large bulk1 not less than #e c4c4 of the disinfectant being utilised for the preparation of any given percentage solution4 b4 ,standard ,control4--,phenol4 ,the standard germicide used for comparison should be one which is not subject to variation in its chemical composition1 and the one which has obtained almost universal use is ,phenol4 ,the following table shows the effect of different percentages of carbolic acid upon ,b4 coli for varying contact times1 compiled from an experiment conducted under the standard conditions referred to under ,environment4 ,the results closely correspond to those recorded by the ,lancet ,commission on ,disinfectants1 #aiji4 ---------------------------------------- ---------------- ,contact time in minutes4 ,percentage of phenol ---------------------------- #b-a_/b #e #aj #ae #bj #be #cj #ce ---------------------------------------- --------- #a4bj - - - - - - - - #a4aj - - - - - - - - #a4j - - - - - - - #j4i - - - - - - - #j4he - - - - - - #j4hj - - - - - #j4ge - - - #j4g - - #j4fe - ---------------------------------------- --------- - ,no growth1 i4 e41 bacteria killed4 ,growth1 i4 e41 bacteria still living4 ,from this it will be seen that the following percentage solutions will need to be prepared1 namely3 #a4a per cent41 #a4j per cent41 #j4i per cent41 #j4ge per cent41 #j4g per cent41 as controls for each experiment4 ,prepare solutions of varying percentages by weighing out the quantity of carbolic acid required for each and dissolving in #ajj c4c4 of pure distilled water in an accurately standardised measuring flask4 ,the solutions must be prepared freshly as required each day4 ,environment4-- a4 ,general4-- ,close the windows and doors of the laboratory in which the investigation is carried out1 to avoid draughts4 ,flush over the work bench and adjacent floor with #a3ajjj solution of corrosive sublimate4 ,caution the assistant1 if one is employed1 to avoid unnecessary movement or speech4 b4 ,contact ,temperature1 #ae-ah deg4 ,c4-- ,this is the temperature at which contact between the germicide and the test germ takes place1 and is of importance1 since some germicides 7e4 g41 ,phenol7 appear to be more powerful at high temperatures4 #ah deg4 ,c4--practically the ordinary room temperature--is a temperature at which the multiplication of ,b4 coli is a comparatively slow process1 but variation of a degree above this temperature or of two or three degrees below is of no moment4 ,if the room temperature is below #ae deg4 ,c4 when the experiments are in progress1 arrange a water-bath regulated at #ah deg4 ,c4 for the reception of the tubes containing the mixture of germ and germicide2 if above #ai deg4 ,c4 immerse the tubes in cold water1 to which small pieces of ice are added from time to time to prevent the temperature rising above #ah deg4 ,c4 c4 ,relative ,proportional ,bulk of ,test ,germ and ,germicide1 #ej3a4-- ,five cubic centimetres is a convenient amount of germicidal solution to employ1 and to this #j4a c4c4 of the emulsion of test germ should be added4 d4 ,bulk of ,sample ,removed from ,germ ,germicide ,mixture at ,each of the ,time ,periods1 #j4a c4c4-- ,this is sufficient to afford a fair sample of the germ content of the mixture1 and at the same time is insufficient to exert any inhibitory action when transferred to the subculture medium4 e4 ,subculture ,medium4 ,bile ,salt ,broth4-- ,a fluid medium is essential in order to obtain immediate dilution of the germicide carried over2 at the same time it is advantageous to employ a selective medium which favours the growth of the test germ to the exclusion of organisms likely to contaminate the preparation1 and if possible one which affords characteristic cultural appearances4 ,bile ,salt ,broth 7page #ahj7 combines these desiderata2 it permits only the growth of intestinal bacteria1 whilst the formation of an acid reaction and the production of gas in subcultures prepared from the germ-germicide mixture is fairly complete evidence of the presence of living ,b4 coli4 ,the amount of medium present in each test-tube is a matter of importance1 since the medium not only provides pabulum for the test germ1 but also acts as a diluent to the germicide1 to reduce its strength below its inhibition coefficient4 ,for routine work each subculture tube contains #aj c4c4 of medium1 but it is obvious that if germicide-x possesses an inhibition coefficient of #j4a per cent4 the addition of #j4a c4c4 of a #aj per cent4 solution to #aj c4c4 of medium would effectually prevent the subsequent growth of the test germ after a contact period insufficient to destroy its vitality4 ,hence the preliminary tests may in some instances indicate the necessity for the presence of #ab c4c41 #ae c4c4 or more of the fluid medium in the culture tubes4 f4 ,incubation ,temperature1 #cg deg4 ,c4-- g4 ,observation ,period of the ,subcultivations1 ,seven ,days4-- ,in order to determine whether or no the test germs have been destroyed1 observations must always be continued--when growth appears to be absent--up to the end of seven days before recording no growth4 h4 ,identification of the ,organisms ,developing in the ,subcultivations after ,contact in the ,germ ,germicide ,solution4-- ,this is based on the naked eye characters of the growth in the bile salt broth1 supplemented where necessary by plating methods1 further subcultivations upon carbohydrate media and agglutination experiments4 ,the sign 77 is used to indicate that growth of the test organism occurred in the subcultivations1 and the sign 7-7 to indicate that the test germs have been destroyed and no subsequent growth has taken place4 ,,method4-- ,apparatus ,required3 ,sterile test-tubes 7narrow1 not exceeding #a4c cm4 diameter74 ,test-tube rack 7,fig4 #bai74 ,sterile graduated pipettes in case1 #a c4c4 7in tenths74 ,sterile graduated pipettes in case1 #e c4c4 7in c4c474 ,circular rubber washers1 #b4e cm4 diameter with central hole1 sterilised by boiling immediately before use1 then transferred to sterilised glass double dish4 ,electric signal clock or stop watch4 ,sterile forceps4 ,sterilised glass beads4 ,shaking machine4 ,grease pencil4 ,material ,required3 ,percentage solutions of germicide-x 7vide page #dha74 ,percentage solutions of pure phenol 7vide page #dhb74 ,aqueous emulsion of ,b4 coli 7vide page #dha74 ,tubes of bile salt broth4 ,preliminary ,tests4-- a4 ,inhibition ,coefficient4-- ,determine the lowest percentage of germicide-x which inhibits growth of ,b4 coli in the bile salt broth1 and the highest percentage which fails to inhibit 7page #caa74 ,on the result of this experiment determine the bulk of medium required in the subculture tubes and the percentage solutions to be employed in the trial trip4 ,assuming the inhibition coefficient to be #a3ajjj1 it will be quite safe to employ the ordinary culture tubes containing #aj c4c4 medium in the subsequent experiments4 b4 ,trial ,trip4-- ,determine the lethal effect of a series of five solutions of germicide-x 7say #a3ajj1 #a3bej1 #a3cjj1 #a3ejj1 #a3fjj7 at contact times of #b-a_/b1 #e1 #be and #cj minutes in the following manner3 #a4 ,arrange five test-tubes marked ,a to ,e in the lower tier of the test-tube rack4 #b4 ,into tube ,a pipette #e c4c4 germicide-x #a3ajj solution4 ,into tube ,b pipette #e c4c4 germicide-x #a3bjj solution4 ,into tube ,c pipette #e c4c4 germicide-x #a3cjj solution4 ,into tube ,d pipette #e c4c4 germicide-x #a3ejj solution4 ,into tube ,e pipette #e c4c4 germicide-x #a3fjj solution4 #c4 ,arrange #bj tubes of bile salt broth in the upper tier of the test-tube rack in two rows1 those in the front row numbered consecutively from left to right #a-aj1 those in the back row #aa-bj4 #d4 ,place a square wire basket of about #ej tubes capacity close to the left of the test-tube rack1 for the reception of the inoculated tubes4 #e4 ,take a sterile #a c4c4 pipette from the case1 pick up a sterile rubber washer with forceps and push the point of the pipette into the central hole4 #f4 ,put down the forceps on the bench with the sterile points projecting over the edge4 ,without taking the tube from the rack remove the cotton-wool plug from tube ,a1 and lower the pipette1 with the rubber washer affixed1 on to the open mouth of the tube2 with the help of the forceps to steady the washer1 push the pipette on through the hole until the point of the pipette has reached to within a few millimetres of the bottom of the tube 7see fig4 #bai74 #g4 ,adjust in the same way a pipette and a washer in the mouth of each of the other tubes1 ,b1 ,c1 ,d and ,e4 #h4 ,set the electric signal clock to ring for the commencement of the experiment and at subsequent intervals of #b-a_/b1 #e1 #be and #cj minutes4 #i4 ,take up #j4e c4c4 of ,b4 coli emulsion in sterile pipette graduated in tenths of a cubic centimetre and stand by4 #aj4 ,as soon as the bell rings lift the pipette from tube ,a with the left hand and from the charged pipette held in the right hand deliver #j4a c4c4 of ,b4 coli emulsion into the #a3ajj solution4 ,then replace the pipette and washer4 ,illustration3 ,,fig4 #bai4--,test-tube rack4 #aa4 ,raise the tube with the left hand and shake it to mix germ and germicide1 whilst returning the delivery pipette in the right hand4 #ab4 ,repeat the process with tubes ,b1 ,c1 ,d and ,e2 then drop the infected delivery pipette in the lysol jar4 ,the inoculation of the five tubes can be carried out very expeditiously1 but a period of #aj seconds must be allowed for each tube4 #ac4 ,when the bell rings at #b-a_/b minutes blow through the pipette in tube ,a 7this agitates the germ germicide mixture and ensures the collection of a fair sample72 allow the mixture to enter the pipette1 and as the column of fluid extends well above the terminal graduation1 the right forefinger adjusted over the butt-end of the pipette before it is lifted will retain more than #j4a c4c4 of the mixture within the bore when the point of the pipette is clear of the fluid in the tube4 ,touch the point of the pipette on the inner wall of the tube1 and allow any excess of fluid to escape1 only retaining #j4a c4c4 in the pipette4 #ad4 ,at the same time1 with the left hand remove ,bile ,salt ,tube ,no4 #a from the upper tier of the rack1 take out the cotton-wool plug with the hand already holding the pipette 7the relative positions of pipette1 plug and culture tubes being practically the same as those of platinum loop1 plug and culture tube shown in ,fig4 #fh1 page #gd74 #ae4 ,insert the point of the pipette into the subculture tube1 and blow out the mixture into the medium--replug the tube and drop it into the wire basket4 ,replace the washer-pipette in tube ,a4 ,as soon as the point of the pipette has entered the mouth of tube ,a it may be released1 since it has already been so adjusted that it just clears the bottom of the test-tube1 and the elastic washer will prevent any damage to the tube4 ,steps #ac1 #ad and #ae occupy on an average #aj seconds4 #af4 ,repeat steps #ac1 #ad and #ae with each of the other tubes ,b1 ,c1 ,d and ,e4 #ag4 ,repeat these various steps #ac-af when the bell rings at #e1 #be and #cj minutes4 #ah4 ,place all the inoculated tubes in the incubator at #cg deg4 ,c4 #ai4 ,examine the tubes at intervals of #bd hours1 and record the results in tabular form as shown in ,table page #dia 7the figures in the squares indicate the number of hours at which the changes in the medium due to the growth of ,b4 coli first appeared74 #bj4 ,if a consideration of the tabulated results indicates strengths of ,germicide-x lethal at #b-a_/b and #cj minutes the final test can be arranged1 but if this result has not been attained1 sufficient evidence will probably be available to enable a second trial test to be planned which will give the required information4 ,final ,test4-- c4 ,determination of ,phenol ,coefficient4-- ,x-,disinfectant4--,this comprises two distinct tests1 one of the ,germicide-x1 the other of the standard phenol4 #a4 ,arrange five test-tubes clearly marked in the lower tier of the rack4 #b4 ,pipette into each #e c4c4 respectively of the five percentage solutions of x-disinfectant which the trial run has already shown will include those affording lethal values at #b-a_/b and #cj minutes4 #c4 ,arrange #bj tubes of bile salt broth in the upper tier of the test-tube rack in two rows1 those in the front row numbered consecutively from left to right #a-aj1 those in the back row #aa-bj4 #d4 ,arrange further #bj tubes of bile salt broth numbered #ba-dj in two rows in a second smaller rack which can be stood on the upper tier of the rack as soon as the first #bj tubes have been inoculated4 #e4 ,place a square wire basket of about #ej tube capacity close to the left of the test-tube rack1 for the reception of the inoculated tubes4 #f4 ,adjust a sterile #a c4c4 pipette in the mouth of each of the tubes1 ,a1 ,b1 ,c1 ,d and ,e1 by means of a washer1 as previously described4 #g4 ,set the electric signal clock to ring for the commencement of the experiment and subsequently at #b-a_/b1 #e1 #aj1 #ae1 #bj1 #be1 #cj and #ce minutes4 #h4 ,complete precisely as indicated in ,trial ,runs1 steps #i-ai4 ,control ,phenol4-- ,immediately the subculture tube from the #cj-minute contact period have been inoculated1 carry out a precisely similar experiment1 in which five percentage strengths of ,phenol1 7e4 g41 #a4a1 #a4j1 #j4i1 #j4ge1 #j4g7 are arranged in the lower tier of the test-tube rack in place of the five strengths of ,germicide-x4 ,calculate the phenol coefficient by the following method3 7a7 ,divide the figure representing the percentage strength of the weakest lethal dilution of the carbolic acid control at the #b-a_/b-minute contact period by the figure representing the percentage strength of the weakest lethal dilution of the x-disinfectant at the same period4 ,the quotient phenol coefficient at #b-a_/b minutes4 7b7 ,similarly obtain the phenol coefficient at #cj minutes contact period4 7c7 ,record the mean of the two coefficients obtained in 7a7 and 7b7 as the mean phenol coefficient1 or simply as the ,phenol ,coefficient4 ,the details of the ,final ,test of an actual determination are set out in the accompanying table4 ,,table #bg ,organism employed1 ,b4 ,coli ,communis4 ,culture ,medium1 ,nutrient ,agar 7#aj74 ,age1 #bd hrs4 ,temp4 of ,incubation1 #cg deg4 ,c4 ,quantities used ,culture ,emulsion #j4a c4c4 #e c4c4 ,germicide4 ,emulsion ,room ,temperature during ,experiments1 #ag deg4 ,c4 ,germicide ,strength ,time of exposure ,incubation #b-a_/b #e #aj #ae #bj #be #cj #ce ,time ,temp4 #a ,germicide-x #d -- -- -- -- -- -- -- -- #g days4 #cg deg4 ,c4 #b ,germicide-x #c #dh -- -- -- -- -- -- -- #g days4 #cg deg4 ,c4 #c ,germicide-x #b #bd #bd #bd #bd #dh #gb #g days4 #cg deg4 ,c4 #d ,germicide-x #a #bd #bd #bd #bd #gb #bd #gb #g days4 #cg deg4 ,c4 #e ,germicide-x #j4e #bd #bd #bd #bd #bd #bd #bd #bd #bd hours4 #cg deg4 ,c4 #a ,phenol #a4aj -- -- -- -- -- -- -- -- #g days4 #cg deg4 ,c4 #b ,phenol #a4jj #bd #g days4 #cg deg4 ,c4 #c ,phenol #j4ge #bd #bd #bd #bd #dh #g days4 #cg deg4 ,c4 #d ,phenol #j4gj #bd #bd #bd #bd #bd #gb #g days4 #cg deg4 ,c4 #e ,phenol #j4fe #bd #bd #bd #bd #bd #dh #bd #bd #b days4 #cg deg4 ,c4 77#a4aj_/d4jj7 7#j4g_/b4j77 #j4bg #j4ce 4#fb ,phenol ,coefficient ------------------------ ----------- --- #j4ca #b #b #b ,,appendix4 ,,metric ,,and ,,imperial ,,systems ,,of ,,weights ,,and ,,measures4 ,the initial unit of the metric system is the ,metre 7m47 or unit of length1 representing one-fourth-millionth part of the circumference of the earth round the poles4 ,the unit of mass is the ,gramme 7g471 and represents the weight of one cubic centimetre of water at its maximum density 7viz4 #d deg4 ,c4 and #gfj mm4 mercury pressure74 ,the unit of the measure of capacity is the ,litre 7l471 and represents the volume of a kilogramme of distilled water at its maximum density4 ,the decimal subdivisions of each of the units are designated by the ,latin prefixes milli #a_/ajjj2 centi #a_/ajj2 deci #a_/aj2 the multiples of each unit by the ,greek prefixes deka #aj2 hecto #ajj2 kilo #ajjj2 myria #aj1jjj4 ,for a comparison of the values of some of the more frequently employed expressions of the ,metric ,system and the ,imperial ,system1 the following may be found convenient for reference3 ,length3 #a millimetre 7 #a mm47 #a_/be of an inch4 #a centimetre 7 #a cm47 #b_/e of an inch4 #a inch 7#a7 #be millimetres or #b-a_/b centimetres4 ,mass3 #a milligramme 7 #a mg47 #j4jaedc grain 7or approximately #a_/fd grain74 #a gramme 7 #a g47 #ae4dcbc grains4 #a kilo or kilogramme 7 #a kgm47 #b pounds1 #c-a_/d ounces avoirdupois4 #a pound avoirdupois 7 #a lb47 #dec4eib grammes4 #a ounce avoirdupois 7 #a oz47 #bh4ce grammes4 #a grain #j4jfdh gramme or #fd4h milligrammes4 ,capacity3 #a cubic centimetre 7 #a c4c47 #af4i minims imperial measure4 #a litre 7 #a l47 #ce4aif fluid ounces imperial measure4 #a fluid ounce imperial measure 7 #a ,symbol3 ounce7 #bh4db cubic centimetres4 #a pint imperial measure 7 #a ,o47 #efh4cd cubic centimetres4 #a gallon imperial measure 7 #a ,c47 #d4edf litres1 or #aj pounds avoirdupois1 of pure water at #fb deg4 ,f4 and under an atmospheric pressure of #cj inches of mercury4 ,,factors ,,for ,,converting ,,from ,,one ,,system ,,to ,,the ,,other4 ,to convert grammes into grains x #ae4dcb4 ,to convert grammes into ounces avoirdupois x #j4jcebg4 ,to convert kilogrammes into pounds x #b4bjdf4 ,to convert cubic centimetres into fluid ounces imperial x #j4jceb4 ,to convert litres into fluid ounces imperial x #ce4b4 ,to convert metres into inches x #ci4cg4 ,to convert grains into grammes x #j4jfdh4 ,to convert avoirdupois ounces into grammes x #bh4ce4 ,to convert troy ounces into grammes x #ca4ajd4 ,to convert fluid ounces into cubic centimetres x #bh4db4 ,to convert pints into litres x #j4efh4 ,to convert inches into metres x #j4jbed4 ,,table ,,for ,,the ,,conversion ,,of ,,degrees ,,centigrade ,,into ,,degrees ,,fahrenheit4 ,x4 deg4 ,c4 77#ix_/#e7 #cb7 deg4 ,f4 ,cent4 ,faht4 ,cent4 ,faht4 ,cent4 ,faht4 #j #cb4j #cd #ic4b #fh #aed4d #a #cc4h #ce #ie4j #fi #aef4b #b #ce4f #cf #if4h #gj #aeh4j #c #cg4d #cg #ih4f #ga #aei4h #d #ci4b #ch #ajj4d #gb #afa4f #e #da4j #ci #ajb4b #gc #afc4d #f #db4h #dj #ajd4j #gd #afe4b #g #dd4f #da #aje4h #ge #afg4j #h #df4d #db #ajg4f #gf #afh4h #i #dh4b #dc #aji4d #gg #agj4f #aj #ej4j #dd #aaa4b #gh #agb4d #aa #ea4h #de #aac4j #gi #agd4b #ab #ec4f #df #aad4h #hj #agf4j #ac #ee4d #dg #aaf4f #ha #agg4h #ad #eg4b #dh #aah4d #hb #agi4f #ae #ei4j #di #abj4b #hc #aha4d #af #fj4h #ej #abb4j #hd #ahc4b #ag #fb4f #ea #abc4h #he #ahe4j #ah #fd4d #eb #abe4f #hf #ahf4h #ai #ff4b #ec #abg4d #hg #ahh4f #bj #fh4j #ed #abi4b #hh #aij4d #ba #fi4h #ee #aca4j #hi #aib4b #bb #ga4f #ef #acb4h #ij #aid4j #bc #gc4d #eg #acd4f #ia #aie4h #bd #ge4b #eh #acf4d #ib #aig4f #be #gg4j #ei #ach4b #ic #aii4d #bf #gh4h #fj #adj4j #id #bja4b #bg #hj4f #fa #ada4h #ie #bjc4j #bh #hb4d #fb #adc4f #if #bjd4h #bi #hd4b #fc #ade4d #ig #bjf4f #cj #hf4j #fd #adg4b #ih #bjh4d #ca #hg4h #fe #adi4j #ii #baj4b #cb #hi4f #ff #aej4h #ajj #bab4j #cc #ia4d #fg #aeb4f ,,table ,,for ,,the ,,conversion ,,of ,,degrees ,,fahrenheit ,,into ,,degrees ,,centigrade4 ,x deg4 ,f4 7#e7x - #cb77_/i deg4 ,c4 ,faht4 ,cent4 ,faht4 ,cent4 ,faht4,cent4 ,faht4 ,cent4 ,faht4 ,cent4 #cb #j4 #fh #bj4j #ajd #dj4j #adj #fj4j #agf #hj4j #cc #j4f #fi #bj4f #aje #dj4f #ada #fj4f #agg #hj4f #cd #a4a #gj #ba4a #ajf #da4a #adb #fa4a #agh #ha4a #ce #a4g #ga #ba4g #ajg #da4g #adc #fa4g #agi #ha4g #cf #b4b #gb #bb4b #ajh #db4b #add #fb4b #ahj #hb4b #cg #b4h #gc #bb4h #aji #db4h #ade #fb4h #aha #hb4h #ch #c4c #gd #bc4c #aaj #dc4c #adf #fc4c #ahb #hc4c #ci #c4i #ge #bc4i #aaa #dc4i #adg #fc4i #ahc #hc4i #dj #d4d #gf #bd4d #aab #dd4d #adh #fd4d #ahd #hd4d #da #e4j #gg #be4j #aac #de4j #adi #fe4j #ahe #he4j #db #e4f #gh #be4f #aad #de4f #aej #fe4f #ahf #he4f #dc #f4a #gi #bf4a #aae #df4a #aea #ff4a #ahg #hf4a #dd #f4g #hj #bf4g #aaf #df4g #aeb #ff4g #ahh #hf4g #de #g4b #ha #bg4b #aag #dg4b #aec #fg4b #ahi #hg4b #df #g4h #hb #bg4h #aah #dg4h #aed #fg4h #aij #hg4h #dg #h4c #hc #bh4c #aai #dh4c #aee #fh4c #aia #hh4c #dh #h4i #hd #bh4i #abj #dh4i #aef #fh4i #aib #hh4i #di #i4d #he #bi4d #aba #di4d #aeg #fi4d #aic #hi4d #ej #aj4j #hf #cj4j #abb #ej4j #aeh #gj4j #aid #ij4j #ea #aj4f #hg #cj4f #abc #ej4f #aei #gj4f #aie #ij4f #eb #aa4a #hh #ca4a #abd #ea4a #afj #ga4a #aif #ia4a #ec #aa4g #hi #ca4g #abe #ea4g #afa #ga4g #aig #ia4g #ed #ab4b #ij #cb4b #abf #eb4b #afb #gb4b #aih #ib4b #ee #ab4h #ia #cb4h #abg #eb4h #afc #gb4h #aii #ib4h #ef #ac4c #ib #cc4c #abh #ec4c #afd #gc4c #bjj #ic4c #eg #ac4i #ic #cc4i #abi #ec4i #afe #gc4i #bja #ic4i #eh #ad4d #id #cd4d #acj #ed4d #aff #gd4d #bjb #id4d #ei #ae4j #ie #ce4j #aca #ee4j #afg #ge4j #bjc #ie4j #fj #ae4f #if #ce4f #acb #ee4f #afh #ge4f #bjd #ie4f #fa #af4a #ig #cf4a #acc #ef4a #afi #gf4a #bje #if4a #fb #af4g #ih #cf4g #acd #ef4g #agj #gf4g #bjf #if4g #fc #ag4b #ii #cg4b #ace #eg4b #aga #gg4b #bjg #ig4b #fd #ag4h #ajj #cg4h #acf #eg4h #agb #gg4h #bjh #ig4h #fe #ah4c #aja #ch4c #acg #eh4c #agc #gh4c #bji #ih4c #ff #ah4i #ajb #ch4i #ach #eh4i #agd #gh4i #baj #ih4i #fg #ai4d #ajc #ci4d #aci #ei4d #age #gi4d #baa #ii4d #bab #ajj4j ,percentage ,formula for addition of salts1 etc41 to completed media4 ,formula for preparing any desired percentage of a given salt1 etc41 in tubed media2 e4 g41 to make #d per cent4 solution of ,,kno#c in a series of tubes of broth each containing #aj c4c4 of medium1 when there is already available a #be per cent4 stock aqueous solution of potassium nitrate4 7,n ,x7 ,y ,a 7,x7 --------------- ---------- #ajj #ajj ,n number of cubic centimetres contained in each tube4 ,x amount of stock solution to be added to each tube4 ,y percentage required in the medium4 ,a percentage of stock solution4 ,then 7#aj ,x7 #d #be ,x ------------ ------ #ajj #ajj ,therefore1 #dj #d,x #be,x4 ,therefore1 #ba,x #dj4 ,x #a4i c4c4 ,this allows for solution added to the original bulk of medium4 ,therefore1 #aj c4c4 broth #a4i c4c4 of a #be per cent4 aqueous solution ,,kno#c makes #aa4i c4c4 medium containing #d per cent4 ,,kno#c4 ,,tables ,,for ,,preparing ,,dilutions 7of ,serum1 ,disinfectants or other substances47 ,in estimating the agglutinin content or titre of a serum1 testing disinfectants and for many other purposes1 it becomes necessary to prepare a series of dilutions of the material under examination1 and in order to avoid unnecessary expenditure of labour it is convenient to adhere to some definite scale of increment1 such for example as the following3 ,from dilutions of #a3aj to #a3hj rise by increments of #e4 ,from dilutions of #a3hj to #a3bjj rise by increments of #aj4 ,from dilutions of #a3bjj to #a3djj rise by increments of #be4 ,from dilutions of #a3djj to #a3ejj rise by increments of #ej4 ,from dilutions of #a3ejj to #a3ajjj rise by increments of #ajj4 ,from dilutions of #a3 #ajjj to #a3ejjj rise by increments of #bej4 ,from dilutions of #a3 #ejjj to #a3aj1jjj rise by increments of #ajjj4 ,from dilutions of #a3aj1jjj to #a3ajj1jjj rise by increments of #ejjj4 ,from dilutions of #a3ajj1jjj to #a3a1jjj1jjj rise by increments of #ajj1jjj4 ,when dealing with a substance of unknown powers--and this is especially true with regard to agglutinating sera--it is customary to run a preliminary test1 using a few widely separated dilutions such as may be obtained in the following manner3 ,,first ,,dilution--,i4 #a c4c4 serum #i c4c4 normal saline solution #aj per cent4 solution or #a3 #aj dilution 7of which #a c4c4 contains #j4a c4c4 of the original serum74 ,when dealing with fluids other than serum the diluent is usually distilled water2 whilst if the original substance is a solid the instructions would read3 #a gram o4s4 #aj c4c4 distilled water #aj per cent4 solution1 etc4 ,,second ,,dilution--,,ii4 #a c4c4 first dilution #i c4c4 normal saline solution #a per cent4 solution or #a3 #ajj dilution4 ,,third ,,dilution--,,iii4 #a c4c4 second dilution #i c4c4 normal saline solution #a per mille solution or #a3 #ajjj dilution4 ,,fourth ,,dilution--,,iv4 #a c4c4 second dilution #i c4c4 normal saline solution #j4a per mille solution or #a3 #aj1jjj dilution4 ,the following tables showing the secondary dilutions that can readily be prepared from each of these four primary dilutions for use in the subsequent determination of the exact titre will probably be found of service by those who are not ready mathematicians4 ,,tables ,,for ,,preparing ,,dilutions4 ---------------------------------------- ----------------------------- ,,table ,i ,,table ,,ii ,using #aj stock solution ,using #a stock solution ,first ,second dilution ,diluent dilution ,diluent ---------------------------------------- ----------------------------- #a3 #aj #a c4c4 #j c4c4 #a3 #ajj #a c4c4 #j c4c4 #a3 #ae #a c4c4 #j4e c4c4 #a3 #aaj #a c4c4 #j4a c4c4 #a3 #bj #a c4c4 #a4j c4c4 #a3 #abj #a c4c4 #j4b c4c4 #a3 #be #a c4c4 #a4e c4c4 #a3 #abe #a c4c4 #j4be c4c4 #a3 #cj #a c4c4 #b4j c4c4 #a3 #acj #a c4c4 #j4c c4c4 #a3 #ce #a c4c4 #b4e c4c4 #a3 #adj #a c4c4 #j4d c4c4 #a3 #dj #a c4c4 #c4j c4c4 #a3 #aej #a c4c4 #j4e c4c4 #a3 #de #a c4c4 #c4e c4c4 #a3 #afj #a c4c4 #j4f c4c4 #a3 #ej #a c4c4 #d4j c4c4 #a3 #agj #a c4c4 #j4g c4c4 #a3 #ee #a c4c4 #d4e c4c4 #a3 #age #a c4c4 #j4ge c4c4 #a3 #fj #a c4c4 #e4j c4c4 #a3 #ahj #a c4c4 #j4h c4c4 #a3 #fe #a c4c4 #e4e c4c4 #a3 #aij #a c4c4 #j4i c4c4 #a3 #gj #a c4c4 #f4j c4c4 #a3 #bjj #a c4c4 #a4j c4c4 #a3 #ge #a c4c4 #f4e c4c4 --------------------------------- #a3 #hj #a c4c4 #g4j c4c4 #a3 #bjj #a c4c4 #a4j c4c4 ------------------------------ #a3 #bbe #a c4c4 #a4be c4c4 #a3 #hj #a c4c4 #g4j c4c4 #a3 #bej #a c4c4 #a4e c4c4 #a3 #ij #a c4c4 #h4j c4c4 #a3 #bge #a c4c4 #a4ge c4c4 #a3 #ajj #a c4c4 #i4jj c4c4 #a3 #cjj #a c4c4 #b4j c4c4 #a3 #aaj #a c4c4 #aj4j c4c4 #a3 #cbe #a c4c4 #b4be c4c4 #a3 #abj #a c4c4 #aa4j c4c4 #a3 #cej #a c4c4 #b4e c4c4 #a3 #abe #a c4c4 #aa4e c4c4 #a3 #cge #a c4c4 #b4ge c4c4 #a3 #acj #a c4c4 #ab4j c4c4 #a3 #djj #a c4c4 #c4j c4c4 #a3 #adj #a c4c4 #ac4j c4c4 --------------------------------- #a3 #aej #a c4c4 #ad4j c4c4 #a3 #djj #a c4c4 #c4j c4c4 #a3 #afj #a c4c4 #ae4j c4c4 #a3 #dej #a c4c4 #c4e c4c4 #a3 #agj #a c4c4 #af4j c4c4 #a3 #ejj #a c4c4 #d4j c4c4 #a3 #age #a c4c4 -#af4e c4c4 --------------------------------- #a3 #ahj #a c4c4 #ag4j c4c4 #a3 #ejj #a c4c4 #d4j c4c4 #a3 #aij #a c4c4 #ah4j c4c4 #a3 #fjj #a c4c4 #e4j c4c4 #a3 #bjj #a c4c4 #ai4j c4c4 #a3 #gjj #a c4c4 #f4j c4c4 ----------------- ------------ #a3 #gej #a c4c4 #f4e c4c4 #a3 #bjj #a c4c4 #ai4j c4c4 #a3 #hjj #a c4c4 #g4j c4c4 #a3 #bbe #a c4c4 #ba4e c4c4 #a3 #ijj #a c4c4 #h4j c4c4 #a3 #bej #a c4c4 #bd4j c4c4 #a3 #ajjj #a c4c4 #i4j c4c4 #a3 #bge #a c4c4 #bf4e c4c4 -------------------------------- #a3 #cjj #a c4c4 #bi4j c4c4 #a3 #ajjj #a c4c4 #i4j c4c4 #a3 #cbe #a c4c4 -#ca4e c4c4 #a3 #bjjj #a c4c4 #ai4j c4c4 #a3 #cej #a c4c4 #cd4j c4c4 #a3 #cjjj #a c4c4 #bi4j c4c4 #a3 #cge #a c4c4 #cf4e c4c4 #a3 #djjj #a c4c4 #ci4j c4c4 #a3 #djj #a c4c4 #ci4j c4c4 #a3 #ejjj #a c4c4 #di4j c4c4 ---------------------------------------- ---------------------- #a3 #djj #a c4c4 #ci4j c4c4 #a3 #dej #a c4c4 #dd4e c4c4 #a3 #ejj #a c4c4 #di4j c4c4 ---------------------------------------- ------------------------ ,,table ,,iii ,,table ,,iv ,using #j4a stock solution ,using #j4ja stock solution ,third ,fourth dilution ,diluent ,dilution ,diluent ---------------------------------------- ------------------------ #a3 #ajjj #a c4c4 #j c4c4 #a3 #aj1jjj #a c4c4 #j c4c4 #a3 #abej #a c4c4 #j4be c4c4 #a3 #ae1jjj #a c4c4 #j4e c4c4 #a3 #aejj #a c4c4 #j4e c4c4 #a3 #bj1jjj #a c4c4 #a4j c4c4 #a3 #agej #a c4c4 #j4ge c4c4 #a3 #be1jjj #a c4c4 #a4e c4c4 #a3 #bjjj #a c4c4 #a4j c4c4 #a3 #cj1jjj #a c4c4 #b4j c4c4 #a3 #bbej #a c4c4 #a4be c4c4 #a3 #ce1jjj #a c4c4 #b4e c4c4 #a3 #bejj #a c4c4 #a4e c4c4 #a3 #dj1jjj #a c4c4 #c4j c4c4 #a3 #bgej #a c4c4 #a4ge c4c4 #a3 #de1jjj #a c4c4 #c4e c4c4 #a3 #cjjj #a c4c4 #b4j c4c4 #a3 #ej1jjj #a c4c4 #d4j c4c4 #a3 #cbej #a c4c4 #b4be c4c4 #a3 #ee1jjj #a c4c4 #d4e c4c4 #a3 #cejj #a c4c4 #b4e c4c4 #a3 #fj1jjj #a c4c4 #e4j c4c4 #a3 #cgej #a c4c4 #b4ge c4c4 #a3 #fe1jjj #a c4c4 #e4e c4c4 #a3 #djjj #a c4c4 #c4j c4c4 #a3 #gj1jjj #a c4c4 #f4j c4c4 #a3 #dbej #a c4c4 #c4be c4c4 #a3 #ge1jjj #a c4c4 #f4e c4c4 #a3 #dejj #a c4c4 #c4e c4c4 #a3 #hj1jjj #a c4c4 #g4j c4c4 #a3 #dgej #a c4c4 #c4ge c4c4 #a3 #he1jjj #a c4c4 #g4e c4c4 #a3 #ejjj #a c4c4 #d4j c4c4 #a3 #ij1jjj #a c4c4 #h4j c4c4 -------------------------------- #a3 #ie1jjj #a c4c4 #h4e c4c4 #a3 #ejjj #a c4c4 #d4j c4c4 #a3 #ajj1jjj #a c4c4 #i4j c4c4 #a3 #fjjj #a c4c4 #e4j c4c4 ----------------------------------- #a3 #gjjj #a c4c4 #f4j c4c4 #a3 #ajj1jjj #j4a c4c4 #j4i c4c4 #a3 #gejj #a c4c4 #f4e c4c4 #a3 #bjj1jjj #j4a c4c4 #a4i c4c4 #a3 #hjjj #a c4c4 #g4j c4c4 #a3 #bej1jjj #j4a c4c4 #b4d c4c4 #a3 #ijjj #a c4c4 #h4j c4c4 #a3 #cjj1jjj #j4a c4c4 #b4i c4c4 #a3 #aj1jjj #a c4c4 #i4j c4c4 #a3 #djj1jjj #j4a c4c4 #c4i c4c4 ------------------------------- #a3 #ejj1jjj #j4a c4c4 #d4i c4c4 #a3 #aj1jjj #a c4c4 #i4j c4c4 ----------------------------------- #a3 #ae1jjj #a c4c4 #ad4j c4c4 #a3 #ejj1jjj #j4a c4c4 #d4i c4c4 #a3 #bj1jjj #a c4c4 #ai4j c4c4 #a3 #fjj1jjj #j4a c4c4 #e4i c4c4 #a3 #be1jjj #a c4c4 #bd4j c4c4 #a3 #gjj1jjj #j4a c4c4 #f4i c4c4 #a3 #cj1jjj #a c4c4 #bi4j c4c4 #a3 #gej1jjj #j4a c4c4 #g4d c4c4 -------------------------------- #a3 #hjj1jjj #j4a c4c4 #g4i c4c4 #a3 #ijj1jjj #j4a c4c4 #h4i c4c4 #a3a1jjj1jjj #j4a c4c4 #i4i c4c4 -------------------------------------- ,,temperature ,,pressure ,,table4 ---------------------------------------- ----------------------- ,temperature ,pounds per sq4 in4 ,centigrade ,mm4 of ,hg4 absolute pressure ,atmospheres ---------------------------------------- ----------------------- #ih deg4 #gjg4a #ac4g #j4ic #ii deg4 #gcc4a #ad4b #j4if #ajj deg4 #gfj4j #ad4g #a4jj #aja deg4 #ghg4h #ae4b #a4jc #ajb deg4 #haf4j #ae4h #a4jg #ajc deg4 #hde4b #af4c #a4aa #ajd deg4 #hge4d #af4i #a4ae #aje deg4 #ijf4d #ag4e #a4ai #ajf deg4 #ich4c #ah4a #a4bc #ajg deg4 #iga4a #ah4h #a4bg #ajh deg4 #ajjd4i #ai4d #a4cb #aji deg4 #ajci4f #bj4a #a4cf #aaj deg4 #ajge4c #bj4h #a4da #aaa deg4 #aaab4j #ba4e #a4df #aab deg4 #aadi4h #bb4b #a4ea #aac deg4 #aahh4f #bb4i #a4ef #aad deg4 #abbh4d #bc4g #a4fa #aae deg4 #abfi4d #bd4e #a4fg #aaf deg4 #acaa4d #be4c #a4gb #aag deg4 #aced4f #bf4b #a4gh #aah deg4 #acii4j #bg4j #a4hd #aai deg4 #addd4e #bg4i #a4ij #abj deg4 #adia4b #bh4h #a4if #aba deg4 #aeci4b #bi4g #b4jb #abb deg4 #aehh4d #cj4g #b4ji #abc deg4 #afch4i #ca4g #b4ae #abd deg4 #afij4g #cb4g #b4bb #abe deg4 #agdc4h #cc4g #b4bi ---------------------------------------- ----------------------- ,,table ,,for ,,desiccation ,,at ,,low ,,temperatures ,,in ,,vacuo4 -------------------------- ,temperature ,centigrade ,mm4 of ,hg4 ------------------------- #ba deg4 #ah4d #bb deg4 #ai4f #bc deg4 #bj4h #bd deg4 #bb4a #be deg4 #bc4e #bf deg4 #bd4i #bg deg4 #bf4d #bh deg4 #bh4j #bi deg4 #bi4g #cj deg4 #ca4e #ca deg4 #cc4c #cb deg4 #ce4c #cc deg4 #cg4c #cd deg4 #ci4e #ce deg4 #da4g #cf deg4 #dd4a #cg deg4 #df4f #ch deg4 #di4b #ci deg4 #ea4i #dj deg4 #ed4h #da deg4 #eg4h #db deg4 #fa4j #dc deg4 #fd4c #dd deg4 #fg4g #de deg4 #ga4c #df deg4 #ge4a #dg deg4 #gi4j #dh deg4 #hc4a #di deg4 #hg4d #ej deg4 #ia4i ------------------------- ,,antiformin ,,method ,for the detection of ,b4 ,tuberculosis4 ,antiformin was introduced into bacteriological technique by ,uhlenhuth in #aijh for the purpose of demonstrating tubercle bacilli when present in small numbers1 in sputum or other material4 ,it is a powerful oxidising agent and rapidly destroys most bacteria1 but tubercle and other acid-fast organisms resist its lethal action for considerable periods1 and upon this fact the method is based4 ,to prepare ,antiformin measure out and mix3-- ,eau de ,javelle 7,liquor sodae chlorinatae--,b4,p47 #ej c4c4 ,sodic hydrate #ae per cent4 aqueous solution #ej c4c4 ,,method4 #a4 ,introduce the sputum or other material 7e4 g4 milk deposit and cream2 pus2 minced gland or other organ2 caseous material2 broken down foci1 etc47 into a sterile tube and then add an equal volume of antiformin4 #b4 ,close the tube with a rubber cork and shake vigorously 7a sample of antiformin that does not foam at this stage is of little use74 ,disintegration of the material at once starts1 associated bacteria are destroyed and the mixture rapidly becomes a homogenous but turbid fluid--a process which may be hastened by3-- #c4 ,placing the tube in the incubator at #cg deg4 ,c4 for #cj minutes--shaking from time to time4 #d4 ,centrifugalise the fluid thoroughly1 at high speed4 #e4 ,pipette off the supernatant fluid1 fill up with sterile distilled water1 cork the tube and shake to distribute the deposit throughout the water4 ,again centrifugalise4 #f4 ,repeat steps #d and #e twice more4 #g4 ,employ one portion of the final deposit to inoculate guinea pigs4 #h4 ,plant the remainder of the deposit freely on ,dorset's ,egg medium2 cap and incubate at #cg deg4 ,c4 ,,note4--,if only microscopical films are needed1 fill up the centrifuge tube with ,ligroin 7a petroleum ether7 in place of sterile distilled water in step #e and prepare the films from the surface of the fluid1 to stain by the ,ziehl-,neelsen process4 ,,index ,abbe's condenser1 #g ,abbott's stain for spores1 #ajg ,aberration1 chromatic1 #ef spherical1 #ee ,absolute alcohol as a fixative1 #hb as an antiseptic1 #bg ,absorbent paper for drying cover-slips1 #fi ,a4 ,c4 ,e4 mixture1 #cde ,acetic acid for clearing films1 #hb ,achromatic condenser1 #ed ,acid haematin1 #if production1 analysis table1 #bhc by bacteria1 #ade investigation of1 #bhj qualitative examination1 #bhc1 #bhd quantitative examination1 #bhj ,acid-fast bacilli in tissues1 to stain1 #abd ,action of various gases on bacteria1 #bie ,active immunisation1 illustrative example1 #cbb ,adjustable water bath1 #bii ,aerobic cultures1 #bba ,aerogenic bacteria1 #aca ,aesculin agar1 #bjd ,agar gelatine 7guarniari71 #aid methods of preparation1 #afg surface plates1 #bcb ,agar-agar1 preparation of1 #afg ,agglutination reaction1 macroscopical1 #chf microscopical1 #che ,agglutinin1 #cha ,air1 analysis of1 #dfh filter1 #dj pump1 ,geryk1 #dc ,albumin solution1 ,mayer's1 #abj ,alcohol production1 test for1 #bhe ,alkaline pyro1 #bci ,alum carmine1 #if ,ammonia production test for1 #bhe ,amphitrichous bacteria1 #acf ,anaerobic cultures1 #bcf ,botkin's method1 #bdc ,buchner's method1 #bch ,bulloch's method1 #bde ,hesse's method1 #bcg ,mc,leod's method1 #bdj media1 #ahj ,novy's method1 #bdd ,anaerobic cultures1 ,roux's biological method1 #bcg physical method1 #bcg vacuum method1 #bch ,wright's method1 #bci ,anaesthetics1 #cde ,analysis of air1 apparatus for1 #dfi method of1 #dfh qualitative bacteriological1 #dgj quantitative bacteriological1 #dfh of butter1 qualitative bacteriological1 #deh quantitative bacteriological1 #deg of cream1 qualitative bacteriological1 #deh quantitative bacteriological1 #deg of fish1 #dfj of ice cream1 qualitative bacteriological1 #deg of meat1 apparatus for1 #dfj method of1 #dfj qualitative bacteriological1 #dfb of milk1 apparatus for1 #ddd collection of samples1 #dda method of1 #dda qualitative bacteriological1 #ddf4 quantitative bacteriological1 #ddd of oysters1 #dfc of sewage1 qualitative bacteriological1 #dfg quantitative bacteriological1 #dff of shellfish1 #dfc of soil1 apparatus for1 #dgc collection of samples1 #dga method of1 #dgj qualitative bacteriological1 #dgf quantitative bacteriological1 #dgc of water1 apparatus for1 #dbj1 #dbg collection of samples1 #daf method of1 #daf qualitative bacteriological1 #dbf ,analysis of water1 quantitative bacteriological1 #dbj ,aniline dyes1 #hc ,gentian violet1 #ie water1 to prepare1 #ajh ,animal tissue media 7,frugoni71 #baj ,animals1 natural infections of1 #ccg ,antiformin method for ,b4 tuberculosis1 #ejb ,antigen1 definition of1 #cbd ,antiseptics1 #bg action of1 #caj 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water1 preparation of1 #agj ,sewage1 analysis of1 qualitative1 #dfg quantitative1 #dff ,shake cultivations1 #bbe description of1 #bga ,shape of colonies1 #bfb ,shaving experimental animals1 #cdi ,shellfish1 analysis of1 #dfc ,silicate jelly 7,winogradsky71 #aih ,single stain for spores1 #ajf ,size of colonies1 #bfb ,slanted tube cultures1 #bbc ,slides1 to clean new1 #bb used1 #bc ,smear culture1 #bbd description of1 #bfh ,soap liquid1 #cdf ,soda solution1 storage of stock1 #aed ,sodium bicarbonate in milk1 test for1 #ddc ,soil1 analysis of1 qualitative1 #dgf quantitative1 #dgc collection of samples1 #dga ,solid media1 #adg ,soluble toxins1 #add ,soyka's milk rice1 #ahi ,spear-headed spatula1 #djb ,special media1 #ahb ,specific serum1 #cgi dilution of1 #chb ,spherical aberration1 #ee ,spirillum1 morphology of1 #acc ,spirochaeta1 morphology of1 #acc ,spirochaetes in tissues1 to stain1 #abd ,spleen extract1 #adi ,sporangium1 #abg ,spore formation1 arthrogenous1 #ach endogenous1 #ach method of1 #ach1 #bgc germination1 method of1 #adj1 #bgd observation of1 #adj1 #bgc ,spores1 characters of1 #aci classification of1 #aci double stain for1 #ajf to stain1 #ajf ,stab culture1 #bbd description of1 #bfe ,stage micrometer1 #fb of microscope1 #eb ,staining methods1 #ij paraffin sections1 #aba reactions of bacteria1 #bgd ,stains intra-vitam1 #gg negative 7,burri71 #gg rack for1 #gb ,standard graduated pipettes1 #g soda solution1 #aed ,standardisation of media1 #aed ,standardising bouillon1 #aee ,staphylococci1 morphology1 #acb ,staphylococcus in milk1 #def ,steam steriliser1 ,arnold1 #ce ,koch1 #ce to use1 #ce streaming1 #ce ,sterigma1 #abg ,sterilisation by chemicals1 #bg by dry heat1 #bh by filters1 #dj by moist heat1 #cb by streaming steam1 #ce by superheated steam1 #cf of albuminous liquids1 #cb of gases1 #dj ,sterilising agents1 #bf ,stichcultur1 #bbd ,stock dilutions1 #dig nutrient media1 #afc plate for isolation work1 #bec ,storage of media in bulk1 #aei of tubed media1 #afa ,store boxes for media1 #afa ,streak culture1 #bbd description of1 #bfh ,streaming movement1 #hj steam1 #ce ,streptobacilli1 morphology1 #acc ,streptococci in soil1 #dgg in water1 detection of1 #dcb morphology of1 #acb ,streptococcus pyogenes longus in milk1 #dee ,streptothrix1 morphology of1 #acc ,strichcultur1 #bbc ,structure1 internal1 of colonies1 #bfe ,study of pathogenic bacteria1 #djh ,subcutaneous inoculation1 #cec ,subdural inoculation1 #cfa ,substage condenser1 #ed ,sugar agar1 #ahe dextrose bouillon1 #ahd gelatine1 #ahd media1 preparation of1 #agg ,sulphindigotate agar1 #aha bouillon 7,weyl71 #aha gelatine 7,weyl71 #aha ,sulphuretted hydrogen in cultures1 test for1 #bij ,sunlight1 action of1 #cji ,superheated steam1 #cf ,superior lethal coefficient1 #caj1 #cac ,suppuration1 organisms of1 #dji ,surface characters of colonies1 #bfd plates1 #bcj ,surgical motor1 electric1 #cfj ,swarm spores1 #abg ,syringe for subcutaneous inoculation of solid material1 #ced hypodermic1 #cdd ,tatin's operating table1 #cea ,taxonomy1 #bfb ,teat-pipettes1 #aj ,temperature1 action of1 #bii optimum1 #bih pressure table1 #ejj range1 #bih taking1 #cdj ,test objects for objectives1 #eg ,testing filters1 #dgh ,test-tubes1 #c to clean infected1 #ai new1 #ah to plug1 #bd to sterilise1 #ca ,tetracocci1 morphology of1 #acb ,thermal death-point1 #adc determination of1 #bih of spores1 #cja1 #cjd of vegetative forms1 #bih1 #cjc ,thermophilic bacteria1 #adc ,thermo-regulators1 ,hearson's capsule1 #bah ,reichert's1 #bah ,thionine blue1 #ib ,thiothrix1 morphology of1 #acc ,thresh's water collecting bottle1 #dah ,throttle pipettes1 #ac ,tinned meat1 analysis of1 #dfj ,tissue medium 7,noguchi71 #bad stains1 #ie ,tissues for sectioning1 fixing1 #aad freezing1 #aaf hardening1 #aad imbedding1 #aah preparation of1 #aad washing1 #aae ,titration of media1 #aej ,torulae1 differentiation from saccharomyces1 #acj ,total acidity1 #bhj ,toxins1 testing of1 #cah ,trephines1 #cfj ,triple nosepiece1 #eh ,true motility1 #hj ,tube cultures1 preparation of1 #bbb length1 #ej ,tubercle bacillus in milk1 #dec to stain1 #aaj1 #abd ,tuberculous guinea-pig1 cadaver of1 #ded ,tubing nutrient media1 #afj ,turnip media1 #bjj ,unna-,pappenheim's stain for sections1 #abc ,unsound meat1 analysis of1 #dfj ,urine agar1 #ahh gelatine1 #ahg 7,heller71 #ahh media bouillon1 #ahg ,uschinsky's solution1 #ahc ,valency of specific sera1 #chf ,van ,ermengem's flagella stain1 #ajd ,vegetative stage of bacteria1 #acf ,vesuvin1 #id ,vibrio cholerae in milk1 #deb in water1 #dci morphology of1 #acc ,virulence1 attenuating1 #cba of organisms1 #cbj raising1 #cbj ,vivisection license1 #ccd ,voges holder1 #cej ,volatile oils as disinfectants1 #bg ,warm stage1 #eh ,washing red blood cells1 #chh tissues1 #aae ,water1 analysis of1 qualitative1 #dbf quantitative1 #daf steriliser1 #cc ,weighing animals1 #cdj ,welch's capsule stain1 #aja ,wertheimer's serum agar1 #baa ,wheat bouillon 7,gasperini71 #aic ,whey agar1 #aie gelatine1 #aie ,wine must1 #aib ,winogradsky's solution ,i1 #aih ,,ii1 #aih ,wire crates for test-tubes1 #ca ,wood ash agar1 #bja ,working up plates1 #beb ,wort agar1 #agf gelatine1 #agf ,wright's anaerobic method1 #bci ,yeast water 7,pasteur71 #aia ,ziehl-,neelsen's stain1 #aaj ,zoogloea1 #acd ,zymogenic bacteria1 #aca ,,saunders' ,,books on ,pathology1 ,physiology ,histology1 ,embryology ,bacteriology1 ,biology 99 99 99 99 99 ,w4 ,b4 ,,saunders ,,company ,,west ,,washington ,,square ,,philadelphia #i1 ,,henrietta ,,street ,,covent ,,garden1 ,,london 99 99 99 99 99 ,,literary ,,superiority ,the excellent judgment displayed in the publications of the house at the very beginning of its career1 and the success of the modern business methods employed by it1 at once attracted the attention of leading men in the profession1 and many of the most prominent writers of ,america offered their books for publication4 ,thus1 there were produced in rapid succession a number of works that immediately placed the house in the front rank of ,medical ,publishers4 ,one need only cite such instances as ,musser and ,kelly's ,treatment1 ,keen's ,surgery1 ,kelly and ,noble's ,gynecology and ,abdominal ,surgery1 ,cabot's ,differential ,diagnosis1 ,de ,lee's ,obstetrics1 ,mumford's ,surgery1 ,cotton's ,dislocations and ,joint ,fractures1 ,crandon and ,ehrenfried's ,surgical ,after-treatment1 ,sisson's ,veterinary ,anatomy1 ,anders and ,boston's ,medical ,diagnosis1 ,gant's ,constipation and ,obstruction1 ,jordan's ,bacteriology1 and ,kemp on ,stomach1 ,intestines1 and ,pancreas4 ,these books have made for themselves places among the best works on their respective subjects4 ,a ,complete ,catalogue of our ,publications will be ,sent upon ,request ,mallory's ,pathologic ,histology ,pathologic ,histology4 ,by ,,frank ,b4 ,,mallory1 ,m4 ,d41 ,associate ,professor of ,pathology1 ,harvard ,university ,medical ,school4 ,octavo of #fgg pages1 with #dig figures containing #fhc original illustrations1 #abd in colors4 ,cloth1 #e4ej net2 ,half ,morocco1 #g4jj net4 ,,reprinted ,,in ,,three ,,months ,dr4 ,mallory here presents pathology from the morphologic point of view4 ,he presents his subject biologically1 first by ascertaining the cellular elements out of which the various lesions are built up2 then he traces the development of the lesions from the simplest to the most complex4 ,he so presents pathology that you are able to trace backward from any given end-result1 such as sclerosis of an organ 7cirrhosis of the liver1 for example71 through all the various acute lesions that may terminate in that particular end-result to the primal cause of the lesion4 ,the illustrations are most beautiful4 ,dr4 ,w4 ,g4 ,mac,callum1 ,columbia ,university ,i have looked over the book and think the plan is admirably carried out and that the book supplies a need we have felt very much4 ,i shall be very glad to recommend it4 99 99 99 99 99 ,howell's ,physiology ,a ,text-,book of ,physiology4 ,by ,,william ,h4 ,,howell1 ,,ph4,d41 ,m4 ,d41 ,professor of ,physiology in the ,johns ,hopkins ,university1 ,baltimore1 ,md4 ,octavo of #ajbj pages1 #cjf illustrations4 ,cloth1 #d4jj net4 ,,the ,,new 7#eth7 ,,edition ,dr4 ,howell has had many years of experience as a teacher of physiology in several of the leading medical schools1 and is therefore exceedingly well fitted to write a text-book on this subject4 ,main emphasis has been laid upon those facts and views which will be directly helpful in the practical branches of medicine4 ,at the same time1 however1 sufficient consideration has been given to the experimental side of the science4 ,the entire literature of physiology has been thoroughly digested by ,dr4 ,howell1 and the important views and conclusions introduced into his work4 ,illustrations have been most freely used4 ,the ,lancet1 ,london ,this is one of the best recent text-books on physiology1 and we warmly commend it to the attention of students who desire to obtain by reading a general1 all-round1 yet concise survey of the scope1 facts1 theories1 and speculations that make up its subject matter4 ,mallory and ,wright's ,pathologic ,technique ,fifth ,edition ,pathologic ,technique4 ,a ,practical ,manual for ,workers in ,pathologic ,histology1 including ,directions for the ,performance of ,autopsies and for ,clinical ,diagnosis by ,laboratory ,methods4 ,by ,,frank ,b4 ,,mallory1 ,m4 ,d41 ,associate ,professor of ,pathology1 ,harvard ,university2 and ,,james ,h4 ,,wright1 ,m4 ,d41 ,director of the ,pathologic ,laboratory1 ,massachusetts ,general ,hospital4 ,octavo of #ejj pages1 with #aeb illustrations4 ,cloth1 #c4jj net4 ,in revising the book for the new edition the authors have kept in view the needs of the laboratory worker1 whether student1 practitioner1 or pathologist1 for a practical manual of histologic and bacteriologic methods in the study of pathologic material4 ,many parts have been rewritten1 many new methods have been added1 and the number of illustrations has been considerably increased4 ,boston ,medical and ,surgical ,journal ,this manual1 since its first appearance1 has been recognized as the standard guide in pathological technique1 and has become well-nigh indispensable to the laboratory worker4 99 99 99 99 99 ,eyre's ,bacteriologic ,technic ,bacteriologic ,technic4 ,a ,laboratory ,guide for the ,medical1 ,dental1 and ,technical ,student4 ,by ,j4 ,w4 ,h4 ,,eyre1 ,m4 ,d41 ,f4 ,r4 ,s4 ,edin41 ,director of the ,bacteriologic ,department of ,guy's ,hospital1 ,london4 ,octavo of #ebj pages1 #bai illustrations4 ,cloth1 #c4jj net4 ,,just ,,ready--,,new 7#b;d7 ,,edition1 ,,rewritten ,dr4 ,eyre has subjected his work to a most searching revision4 ,indeed1 so thorough was his revision that the entire book1 enlarged by some #aej pages and #ej illustrations1 had to be reset from cover to cover4 ,he has included all the latest technic in every division of the subject4 ,his thoroughness1 his accuracy1 his attention to detail make his work an important one4 ,he gives clearly the technic for the bacteriologic examination of water1 sewage1 air1 soil1 milk and its products1 meats1 etc4 ,and he gives you good technic--methods attested by his own large experience4 ,to any one interested in this line of endeavor the new edition of ,dr4 ,eyre's work is indispensable4 ,the illustrations are as practical as the text4 ,mc,farland's ,pathology ,a ,text-,book of ,pathology4 ,by ,,joseph ,,mcfarland1 ,m4 ,d41 ,professor of ,pathology and ,bacteriology in the ,medico-,chirurgical ,college of ,philadelphia4 ,octavo of #hef pages1 with #dcg illustrations1 many in colors4 ,cloth1 #e4jj net2 ,half ,morocco1 #f4ej net4 ,,the ,,new 7#b;d7 ,,edition ,you cannot successfully treat disease unless you have a practical1 clinical knowledge of the pathologic changes produced by disease4 ,for this purpose ,dr4 ,mc,farland's work is well fitted4 ,it was written with just such an end in view--to furnish a ready means of acquiring a thorough training in the subject1 a training such as would be of daily help in your practice4 ,for this edition every page has been gone over most carefully1 correcting1 omitting the obsolete1 and adding the new4 ,some sections have been entirely rewritten4 ,you will find it a book well worth consulting1 for it is the work of an authority4 ,st4 ,paul ,medical ,journal ,it is safe to say that there are few who are better qualified to give a resume of the modern views on this subject than ,mc,farland4 ,the subject-matter is thoroughly up to date4 ,boston ,medical and ,surgical ,journal ,it contains a great mass of well-classified facts4 ,one of the best sections is that on the special pathology of the blood4 99 99 99 99 99 ,mc,farland's ,biology3 ,medical and ,general ,biology3 ,medical and ,general--,by ,,joseph ,,mcfarland1 ,m4 ,d41 ,professor of ,pathology and ,bacteriology in the ,medico-,chirurgical ,college of ,phila4 #abmo1 #deg pages1 #afj illustrations4 ,cloth1 #a4ge net4 ,,just ,,ready--,,new 7#b;d7 ,,edition ,this work is both a general and medical biology4 ,the former because it discusses the peculiar nature and reactions of living substance generally2 the latter because particular emphasis is laid on those subjects of special interest and value in the study and practice of medicine4 ,the illustrations will be found of great assistance4 ,frederic ,p4 ,gorham1 ,a4 ,m41 ,brown ,university4 ,i am greatly pleased with it4 ,perhaps the highest praise which ,i can give the book is to say that it more nearly approaches the course ,i am now giving in general biology than any other work4 ,mc,farland's ,pathogenic ,bacteria and ,protozoa ,pathogenic ,bacteria and ,protozoa4 ,by ,,joseph ,,mcfarland1 ,m4 ,d41 ,professor of ,pathology and ,bacteriology in the ,medico-,chirurgical ,college of ,philadelphia4 ,octavo of #hgh pages1 finely illustrated4 ,cloth1 #c4ej net4 ,,new 7#gth7 ,,edition1 ,,enlarged ,dr4 ,mc,farland has subjected his book to a most vigorous revision1 bringing this edition right down to the minute4 ,important new additions have increased it in size some #ahj pages4 ,by far the most important addition is the inclusion of an entirely new section on ,pathogenic ,protozoa4 ,this section considers every protozoan pathogenic to man2 and in that same clean-cut1 definite way that won for ,mc,farland's work a place in the very front of medical bacteriologies4 ,the illustrations are the best the world affords1 and are beautifully executed4 ,h4 ,b4 ,anderson1 ,m4 ,d41 ,professor of ,pathology and ,bacteriology1 ,trinity ,medical ,college1 ,toronto4 ,the book is a satisfactory one1 and ,i shall take pleasure in recommending it to the students of ,trinity ,college4 ,the ,lancet1 ,london ,it is excellently adapted for the medical students and practitioners for whom it is avowedly written4444 ,the descriptions given are accurate and readable4 99 99 99 99 99 ,hill's ,histology and ,organography ,a ,manual of ,histology and ,organography4 ,by ,,charles ,,hill1 ,m4 ,d41 formerly ,assistant ,professor of ,histology and ,embryology1 ,northwestern ,university1 ,chicago4 #abmo of #dfh pages1 #ccg illustrations4 ,flexible leather1 #b4jj net4 ,,the ,,new 7#b;d7 ,,edition ,dr4 ,hill's work is characterized by a completeness of discussion rarely met in a book of this size4 ,particular consideration is given the mouth and teeth4 ,pennsylvania ,medical ,journal ,it is arranged in such a manner as to be easy of access and comprehension4 ,to any contemplating the study of histology and organography we would commend this work4 99 99 99 99 99 ,,get ,,the ,,new ,,the ,,best ,,standard ,american ,illustrated ,dictionary ,new 7#gth7 ,edition--#ejjj ,sold in ,two ,months ,the ,american ,illustrated ,medical ,dictionary4 ,a new and complete dictionary of the terms used in ,medicine1 ,surgery1 ,dentistry1 ,pharmacy1 ,chemistry1 ,veterinary ,science1 ,nursing1 and kindred branches2 with over #ajj new and elaborate tables and many handsome illustrations4 ,by ,w4 ,a4 ,,newman ,,dorland1 ,m4,d41 ,editor of ,the ,american ,pocket ,medical ,dictionary4 ,large octavo1 #aajg pages1 bound in full flexible leather4 ,price1 #d4ej net2 with thumb index1 #e4jj net4 ,,it ,,defines ,,all ,,the ,,new ,,words--,,it ,,is ,,up ,,to ,,date ,the ,american ,illustrated ,medical ,dictionary defines hundreds of the newest terms not defined in any other dictionary--bar none4 ,these new terms are live1 active words1 taken right from modern medical literature4 ,it gives the capitalization and pronunciation of all words4 ,it makes a feature of the derivation or etymology of the words4 ,in some dictionaries the etymology occupies only a secondary place1 in many cases no derivation being given at all4 ,in the ,american ,illustrated practically every word is given its derivation4 ,every word has a separate paragraph1 thus making it easy to find a word quickly4 ,the tables of arteries1 muscles1 nerves1 veins1 etc41 are of the greatest help in assembling anatomic facts4 ,in them are classified for quick study all the necessary information about the various structures4 ,every word is given its definition--a definition that defines in the fewest possible words4 ,in some dictionaries hundreds of words are not defined at all1 referring the reader to some other source for the information he wants at once4 ,howard ,a1 ,kelly1 ,m4 ,d41 ,johns ,hopkins ,university1 ,baltimore4 ,the ,american ,illustrated ,dictionary is admirable4 ,it is so well gotten up and of such convenient size4 ,no errors have been found in my use of it4 ,j4 ,collins ,warren1 ,m4 ,d41 ,,ll4,d41 ,f4,r4,c4,s4 7,hon471 ,harvard ,medical ,school ,i regard it as a valuable aid to my medical literary work4 ,it is very complete and of convenient size to handle comfortably4 ,i use it in preference to any other4 99 99 99 99 99 ,stengel's ,text-,book of ,pathology ,fifth ,edition ,a ,text-,book of ,pathology4 ,by ,,alfred ,,stengel1 ,m4 ,d41 ,professor of ,medicine in the ,university of ,pennsylvania4 ,octavo volume of #igi pages1 with #djj text-illustrations1 many in colors1 and #g full-page colored plates4 ,cloth1 #e4jj net2 ,sheep or ,half ,morocco1 #f4ej net4 ,,with #djj ,,text-,,cuts1 ,,many ,,in ,,colors1 ,,and #g ,,colored ,,plates ,in this work the practical application of pathologic facts to clinical medicine is considered more fully than is customary in works on pathology4 ,while the subject of pathology is treated in the broadest way consistent with the size of the book1 an effort has been made to present the subject from the point of view of the clinician4 ,in the second part of the work the pathology of individual organs and tissues is treated systematically and quite fully under subheadings that clearly indicate the subject-matter to be found on each page4 ,in this edition the section dealing with ,general ,pathology has been most extensively revised1 several of the important chapters having been practically rewritten4 ,the ,lancet1 ,london ,this volume is intended to present the subject of pathology in as practical a form as possible1 and more especially from the point of view of the 'clinical pathologist4' ,these objects have been faithfully carried out1 and a valuable text-book is the result4 ,we can most favorably recommend it to our readers as a thoroughly practical work on clinical pathology4 99 99 99 99 99 ,stiles' ,nutritional ,physiology ,nutritional ,physiology4 ,by ,,percy ,,goldthwait ,,stiles1 ,assistant ,professor of ,physiology at ,simmons ,college1 ,boston4 #abmo of #bie pages1 illustrated4 ,cloth1 #a4be net4 ,,illustrated ,this new work expresses the most advanced views on this important subject4 ,it discusses in a concise way the processes of digestion and metabolism4 ,the key-word of the book throughout is energy--its source and its conservation4 ,it is remarkable for the fineness of its diction and for its clear presentation of the subject1 relieved here and there by a quaintly humorous turn of phrase that is altogether delightful4--,colin ,c4 ,stewart1 ,ph4 ,d41 ,dartmouth ,college4 99 99 99 99 99 ,jordan's ,general ,bacteriology ,a ,text-,book of ,general ,bacteriology4 ,by ,,edwin ,o4 ,,jordan1 ,,ph4,d41 ,professor of ,bacteriology in the ,university of ,chicago and in ,rush ,medical ,college4 ,octavo of #fbc pages1 illustrated4 ,cloth1 #c4jj net4 ,,new 7#c;d7 ,,edition ,professor ,jordan's work embraces the entire field of bacteriology1 the non-pathogenic as well as the pathogenic bacteria being considered1 giving greater emphasis1 of course1 to the latter4 ,there are extensive chapters on methods of studying bacteria1 including staining1 biochemical tests1 cultures1 etc42 on the development and composition of bacteria2 on enzymes and fermentation-products2 on the bacterial production of pigment1 acid and alkali2 and on ptomaines and toxins4 ,especially complete is the presentation of the serum treatment of gonorrhea1 diphtheria1 dysentery1 and tetanus4 ,the relation of bovine to human tuberculosis and the ocular tuberculin reaction receive extensive consideration4 ,this work will also appeal to academic and scientific students4 ,it contains chapters on the bacteriology of plants1 milk and milk-products1 air1 agriculture1 water1 food preservatives1 the processes of leather tanning1 tobacco curing1 and vinegar making2 the relation of bacteriology to household administration and to sanitary engineering1 etc4 ,prof4 ,severance ,burrage1 ,associate ,professor of ,sanitary ,science1 ,purdue ,university4 ,i am much impressed with the completeness and accuracy of the book4 ,it certainly covers the ground more completely than any other ,american book that ,i have seen4 99 99 99 99 99 ,buchanan's ,veterinary ,bacteriology ,veterinary ,bacteriology4 ,by ,,robert ,e4 ,,buchanan1 ,,ph4,d41 ,professor of ,bacteriology in the ,iowa ,state ,college of ,agriculture and ,mechanic ,arts4 ,octavo1 #eaf pages1 #bad illustrations4 ,cloth1 #c4jj net4 ,,the ,,best ,,published ,professor ,buchanan discusses thoroughly all bacteria causing diseases of the domestic animals4 ,he goes minutely into the consideration of immunity1 opsonic index1 reproduction1 sterilization1 antiseptics1 biochemic tests1 culture-media1 isolation of cultures1 the manufacture of the various toxins1 antitoxins1 tuberculins1 and vaccines that have proved of diagnostic or therapeutic value4 ,then1 in addition to bacteria and protozoa proper1 he considers molds1 mildews1 smuts1 rusts1 toadstools1 puff-balls1 and the other fungi pathogenic for animals4 ,b4 ,f4 ,kaupp1 ,d4 ,v4 ,s41 ,state ,agricultural ,college1 ,fort ,collins4 ,it is the best in print on the subject4 ,what pleases me most is that it contains all the late results of research4 ,it fills a long felt want4 ,heisler's ,embryology ,a ,text-,book of ,embryology4 ,by ,,john ,c4 ,,heisler1 ,m4,d41 ,professor of ,anatomy in the ,medico-,chirurgical ,college1 ,philadelphia4 ,octavo volume of #dce pages1 with #bab illustrations1 #cb of them in colors4 ,cloth1 #c4jj net4 ,,third ,,edition--,,with #bab ,,illustrations1 #cb ,,in ,,colors ,this edition represents all the advances recently made in the science of embryology4 ,many portions have been entirely rewritten1 and a great deal of new and important matter added4 ,a number of new illustrations have also been introduced and these will prove very valuable4 ,heisler's ,embryology has become a standard work4 ,g4 ,carl ,huber1 ,m4,d41 ,professor of ,embryology at the ,wistar ,institute1 ,university of ,pennsylania4 ,i find this edition of ',a ,text-,book of ,embryology1' by ,dr4 ,heisler1 an improvement on the former one4 ,the figures added increase greatly the value of the work4 ,i am again recommending it to our students4 99 99 99 99 99 ,boehm1 ,davidoff1 and ,huber's ,histology ,a ,text-,book of ,human ,histology4 ,including ,microscopic ,technic4 ,by ,,dr4 ,a4 ,a4 ,,boehm and ,,dr4 ,m4 ,,von ,,davidoff1 of ,munich1 and ,g4 ,,carl ,,huber1 ,m4,d41 ,professor of ,embryology at the ,wistar ,institute1 ,university of ,pennsylvania4 ,handsome octavo of #ebh pages1 with #cfa beautiful original illustrations4 ,flexible cloth1 #c4ej net4 ,,second ,,edition1 ,,enlarged ,the work of ,drs4 ,boehm and ,davidoff is well known in the ,german edition1 and has been considered one of the most practically useful books on the subject of ,human ,histology4 ,this second edition has been in great part rewritten and very much enlarged by ,dr4 ,huber1 who has also added over one hundred original illustrations4 ,dr4 ,huber's extensive additions have rendered the work the most complete students' text-book on ,histology in existence4 ,boston ,medical and ,surgical ,journal ,is unquestionably a text-book of the first rank1 having been carefully written by thorough masters of the subject1 and in certain directions it is much superior to any other histological manual4 99 99 99 99 99 ,wells' ,chemical ,pathology ,chemical ,pathology4--,being a ,discussion of ,general ,pathology from the ,standpoint of the ,chemical ,processes ,involved4 ,by ,h4 ,,gideon ,,wells1 ,,ph4,d41 ,m4,d41 ,assistant ,professor of ,pathology in the ,university of ,chicago4 ,octavo of #faf pages4 ,cloth1 #c4be net4 ,,just ,,ready--,,new 7#b;d7 ,,edition ,dr4 ,wells' work is written for the physician1 for those engaged in research in pathology and physiologic chemistry1 and for the medical student4 ,in the introductory chapter are discussed the chemistry and physics of the animal cell1 giving the essential facts of ionization1 diffusion1 osmotic pressure1 etc41 and the relation of these facts to cellular activities4 ,special chapters are devoted to ,diabetes and to ,uric-acid ,metabolism and ,gout4 ,wm4 ,h4 ,welch1 ,m4,d4 ,professor of ,pathology1 ,johns ,hopkins ,university4 ,the work fills a real need in the ,english literature of a very important subject1 and ,i shall be glad to recommend it to my students4 99 99 99 99 99 ,lusk's ,elements of ,nutrition ,elements of the ,science of ,nutrition4 ,by ,,graham ,,lusk1 ,,ph4,d41 ,professor of ,physiology at ,cornell ,medical ,school4 ,octavo volume of #cjb pages4 ,cloth1 #c4jj net4 ,,the ,,new 7#b;d7 ,,edition--,,translated ,,into ,,german ,prof4 ,lusk presents the scientific foundations upon which rests our knowledge of nutrition and metabolism1 both in health and in disease4 ,there are special chapters on the metabolism of diabetes and fever1 and on purin metabolism4 ,the work will also prove valuable to students of animal dietetics at agricultural stations4 ,lewellys ,f4 ,barker1 ,m4 ,d4 ,professor of the ,principles and ,practice of ,medicine1 ,johns ,hopkins ,university4 ,i shall recommend it highly to my students4 ,it is a comfort to have such a discussion of the subject in ,english4 ,daugherty's ,economic ,zoology ,economic ,zoology4 ,by ,l4 ,s4 ,,daugherty1 ,m4 ,s41 ,,ph4 ,d41 ,professor of ,zoology1 ,state ,normal ,school1 ,kirksville1 ,mo41 and ,m4 ,c4 ,,daugherty1 author with ,jackson of ,agriculture ,through the ,laboratory and ,school ,garden4 ,part ,i3 ,field and ,laboratory ,guide4 #abmo of #bcg pages1 interleaved4 ,cloth1 #a4be net4 ,part ,,ii3 ,principles4 #abmo of #djf pages1 illustrated4 ,cloth1 #b4jj net4 ,,illustrated ,there is no other book just like this4 ,not only does it give the salient facts of structural zoology and the development of the various branches of animals1 but also the natural history--the life and habits--thus showing the interrelations of structure1 habit1 and environment4 ,in a word1 it gives the principles of zoology and their actual application4 ,the economic phase is emphasized4 ,part ,i--the ,field and ,laboratory ,guide--is designed for practical instruction in the field and laboratory4 ,to enhance its value for this purpose blank pages are inserted for notes4 99 99 99 99 99 ,drew's ,invertebrate ,zoology ,a ,laboratory ,manual of ,invertebrate ,zoology4 ,by ,,gilman ,a4 ,,drew1 ,,ph4 ,d41 ,assistant ,director at ,marine ,biological ,laboratory1 ,woods ,hole1 ,mass4 ,with the aid of ,former and ,present ,members of the ,zoological ,staff of ,instructors4 #abmo of #bac pages4 ,cloth1 #a4be net4 ,,just ,,ready--,,new 7#b;d7 ,,edition ,the subject is presented in a logical way1 and the type method of study has been followed1 as this method has been the prevailing one for many years4 ,prof4 ,allison ,a4 ,smyth1 ,jr41 ,virginia ,polytechnic ,institute ,i think it is the best laboratory manual of zoology ,i have yet seen4 ,the large number of forms dealt with makes the work applicable to almost any locality4 99 99 99 99 99 ,norris' ,cardiac ,pathology ,studies in ,cardiac ,pathology4 ,by ,,george ,w4 ,,norris1 ,m4,d41 ,associate in ,medicine at the ,university of ,pennsylvania4 ,large octavo of #bce pages1 with #he superb illustrations4 ,cloth1 #e4jj net4 ,,superb ,,illustrations ,the wide interest being manifested in heart lesions makes this book particularly opportune4 ,the illustrations are superb and are faithful reproductions of the specimens photographed4 ,each illustration is accompanied by a detailed description2 besides1 there is ample letter press supplementing the pictures4 ,considerable matter of a diagnostic and therapeutic nature has been interwoven4 ,boston ,medical and ,surgical ,journal ,the illustrations are arranged in such a way as to illustrate all the common and many of the rare cardiac lesions1 and the accompanying descriptive text constitutes a fairly continuous didactic treatise4 99 99 99 99 99 ,mc,connell's ,pathology ,a ,manual of ,pathology4 ,by ,,guthrie ,,mcconnell1 ,m4,d41 ,professor of ,bacteriology and ,pathology at ,temple ,university1 ,philadelphia4 #abmo of #ebc pages1 with #agj illustrations4 ,flexible leather1 #b4ej net4 ,,new 7#b;d7 ,,edition ,dr4 ,mc,connell has discussed his subject with a clearness and precision of style that make the work of great assistance to both student and practitioner4 ,the illustrations have been introduced for their practical value4 ,new ,york ,state ,journal of ,medicine ,the book treats the subject of pathology with a thoroughness lacking in many works of greater pretension4 ,the illustrations--many of them original--are profuse and of exceptional excellence4 99 99 99 99 99 ,hektoen and ,riesman's ,pathology ,,american ,,text-,,book ,,of ,,pathology4 ,edited by ,,ludvig ,,hektoen1 ,m4,d41 ,professor of ,pathology1 ,rush ,medical ,college1 ,chicago2 and ,,david ,,riesman1 ,m4,d41 ,professor of ,clinical ,medicine1 ,philadelphia ,polyclinic4 ,octavo of #abde ,pages1 #ddc illustrations1 #ff in colors4 ,cloth1 #g4ej net2 ,half ,morocco1 #i4jj net4 ,duerck and ,hektoen's ,special ,pathologic ,histology ,atlas and ,epitome of ,special ,pathologic ,histology4 ,by ,,dr4 ,h4 ,,duerck1 of ,munich4 ,edited1 with additions1 by ,,ludvig ,,hektoen1 ,m4 ,d41 ,professor of ,pathology1 ,rush ,medical ,college1 ,chicago4 ,in two parts4 ,part ,i4--,circulatory1 ,respiratory1 and ,gastro-intestinal ,tracts4 #abj colored figures on #fb plates1 and #aeh pages of text4 ,part ,,ii4--,liver1 ,urinary and ,sexual ,organs1 ,nervous ,system1 ,skin1 ,muscles1 and ,bones4 #abc colored figures on #fj plates1 and #aib pages of text4 ,per part3 ,cloth1 #c4jj net4 ,in ,saunders' ,hand-,atlas ,series4 ,the great value of these plates is that they represent in the exact colors the effect of the stains1 which is of such great importance for the differentiation of tissue4 ,the text portion of the book is admirable1 and1 while brief1 it is entirely satisfactory in that the leading facts are stated1 and so stated that the reader feels he has grasped the subject extensively4 ,william ,h4 ,welch1 ,m4,d41 ,professor of ,pathology1 ,johns ,hopkins ,university1 ,baltimore4 ,i consider ,duerck's ',atlas of ,special ,pathologic ,histology1' edited by ,hektoen1 a very useful book for students and others4 ,the plates are admirable4 99 99 99 99 99 ,sobotta and ,huber's ,human ,histology ,atlas and ,epitome of ,human ,histology4 ,by ,,privatdocent ,,dr4 ,j4 ,,sobotta1 of ,wuerzburg4 ,edited1 with additions1 by ,g4 ,,carl ,,huber1 ,m4 ,d41 ,professor of ,histology and ,embryology in the ,university of ,michigan1 ,ann ,arbor4 ,with #bad colored figures on #hj plates1 #fh text-illustrations1 and #bdh pages of text4 ,cloth1 #d4ej net4 ,in ,saunders' ,hand-,atlas ,series4 ,,including ,,microscopic ,,anatomy ,the work combines an abundance of well-chosen and most accurate illustrations1 with a concise text1 and in such a manner as to make it both atlas and text-book4 ,the great majority of the illustrations were made from sections prepared from human tissues1 and always from fresh and in every respect normal specimens4 ,the colored lithographic plates have been produced with the aid of over thirty colors4 ,boston ,medical and ,surgical ,journal ,in color and proportion they are characterized by gratifying accuracy and lithographic beauty4 ,bosanquet on ,spirochaetes ,spirochaetes3 ,a ,review of ,recent ,work1 with ,some ,original ,observations4 ,by ,w4 ,,cecil ,,bosanquet1 ,m4,d41 ,fellow of the ,royal ,college of ,physicians1 ,london4 ,octavo of #aeb pages1 illustrated4 #b4ej net4 ,,illustrated ,this is a complete and authoritative monograph on the spirochaetes1 giving morphology1 pathogenesis1 classification1 staining1 etc4 ,pseudospirochaetes are also considered1 and the entire text well illustrated4 ,the high standing of ,dr4 ,bosanquet in this field of study makes this new work particularly valuable4 99 99 99 99 99 ,levy and ,klemperer's ,clinical ,bacteriology ,the ,elements of ,clinical ,bacteriology4 ,by ,,drs4 ,,ernst ,,levy and ,,felix ,,klemperer1 of the ,university of ,strasburg4 ,translated and edited by ,,augustus ,a4 ,,eshner1 ,m4 ,d41 ,professor of ,clinical ,medicine1 ,philadelphia ,polyclinic4 ,octavo volume of #ddj pages1 fully illustrated4 ,cloth1 #b4ej net4 ,s4 ,solis-,cohen1 ,m4,d41 ,professor of ,clinical ,medicine1 ,jefferson ,medical ,college1 ,philadelphia4 ,i consider it an excellent book4 ,i have recommended it in speaking to my students4 99 99 99 99 99 ,lehmann1 ,neumann1 and ,weaver's ,bacteriology ,atlas and ,epitome of ,bacteriology3 ,,including ,a ,,text-,,book ,,of ,,special ,,bacteriologic ,,diagnosis4 ,by ,,prof4 ,,dr4 ,k4 ,b4 ,,lehmann and ,,dr4 ,r4 ,o4 ,,neumann1 of ,wuerzburg4 ,from the ,second ,revised and ,enlarged ,german ,edition4 ,edited1 with additions1 by ,g4 ,h4 ,,weaver1 ,m4 ,d41 ,assistant ,professor of ,pathology and ,bacteriology1 ,rush ,medical ,college1 ,chicago4 ,in two parts4 ,part ,i4--#fcb colored figures on #fi lithographic plates4 ,part ,,ii4--#eaa pages of text1 illustrated4 ,per part3 ,cloth1 #b4ej net4 ,in ,saunders' ,hand-,atlas ,series4 99 99 99 99 99 ,duerck and ,hektoen's ,general ,pathologic ,histology ,,atlas ,,and ,,epitome ,,of ,,general ,,pathologic ,,histology4 ,by ,,pr4 ,,dr4 ,h4 ,,duerck1 of ,munich4 ,edited1 with additions1 by ,,ludvig ,,hektoen1 ,m4 ,d41 ,professor of ,pathology in ,rush ,medical ,college1 ,chicago4 #agb colored figures on #gg lithographic plates1 #cf text-cuts1 many in colors1 and #cec pages4 ,cloth1 #e4jj net4 ,in ,saunders' ,hand ,atlas ,series4 ,american ,text-,book of ,physiology ,second ,edition ,,american ,,text-,,book ,,of ,,physiology4 ,in two volumes4 ,edited by ,,william ,h4 ,,howell1 ,,ph4 ,d41 ,m4,d41 ,professor of ,physiology in the ,johns ,hopkins ,university1 ,baltimore1 ,md4 ,two royal octavos of about #fjj pages each1 illustrated4 ,per volume3 ,cloth1 #c4jj net2 ,half ,morocco1 #d4be net4 ,the work will stand as a work of reference on physiology4 ,to him who desires to know the status of modern physiology1 who expects to obtain suggestions as to further physiologic inquiry1 we know of none in ,english which so eminently meets such a demand4--,the ,medical ,news4 ,warren's ,pathology and ,therapeutics ,second ,edition ,,surgical ,,pathology ,,and ,,therapeutics4 ,by ,,john ,,collins ,,warren1 ,m4 ,d41 ,,ll4 ,d41 ,f4 ,r4 ,c4 ,s4 7,hon471 ,professor of ,surgery1 ,harvard ,medical ,school4 ,octavo1 #hgc pages1 #acf relief and lithographic illustrations1 #cc in colors4 ,with an ,appendix on ,scientific ,aids to ,surgical ,diagnosis and a series of articles on ,regional ,bacteriology4 ,cloth1 #e4jj net2 ,half ,morocco1 #f4ej net4 ,gorham's ,bacteriology ,a ,,laboratory ,,course ,,in ,,bacteriology4 ,for the ,use of ,medical1 ,agricultural1 and ,industrial ,students4 ,by ,,frederic ,p4 ,,gorham1 ,a4 ,m41 ,associate ,professor of ,biology in ,brown ,university1 ,providence1 ,r4 ,i41 etc4 #abmo of #aib pages1 with #ig illustrations4 ,cloth1 #a4be net4 ,one of the best students' laboratory guides to the study of bacteriology on the market4444 ,the technic is thoroughly modern and amply sufficient for all practical purposes4--,american ,journal of the ,medical ,sciences4 ,raymond's ,physiology ,new 7#c;d7 ,edition ,,human ,,physiology4 ,by ,,joseph ,h4 ,,raymond1 ,a4 ,m41 ,m4 ,d41 ,professor of ,physiology and ,hygiene1 ,long ,island ,college ,hospital1 ,new ,york4 ,octavo of #fhe pages1 with #ddd illustrations4 ,cloth1 #c4ej net4 ,the book is well gotten up and well printed1 and may be regarded as a trustworthy guide for the student and a useful work of reference for the general practitioner4 ,the illustrations are numerous and are well executed4--,the ,lancet1 ,london4 ,ball's ,bacteriology ,seventh ,edition1 ,revised ,,essentials ,,of ,,bacteriology3 being a concise and systematic introduction to the ,study of ,micro-organisms4 ,by ,m4 ,v4 ,,ball1 ,m4 ,d41 ,late ,bacteriologist to ,st4 ,agnes' ,hospital1 ,philadelphia4 #abmo of #bhi pages1 with #ace illustrations1 some in colors4 ,cloth1 #a4jj net4 ,in ,saunders' ,question-,compend ,series4 ,the technic with regard to media1 staining1 mounting1 and the like is culled from the latest authoritative works4--,the ,medical ,times1 ,new ,york4 ,budgett's ,physiology ,new 7#c;d7 ,edition ,,essentials ,,of ,,physiology4 ,prepared especially for ,students of ,medicine1 and arranged with questions following each chapter4 ,by ,,sidney ,p4 ,,budgett1 ,m4 ,d41 formerly ,professor of ,physiology1 ,washington ,university1 ,st4 ,louis4 ,revised by ,,havan ,,emerson1 ,m4 ,d41 ,demonstrator of ,physiology1 ,columbia ,university4 #abmo volume of #bej pages1 illustrated4 ,cloth1 #a4jj net4 ,saunders' ,question-,compend ,series4 ,he has an excellent conception of his subject4444 ,it is one of the most satisfactory books of this class--,university of ,pennsylvania ,medical ,bulletin4 ,leroy's ,histology ,new 7#dth7 ,edition ,,essentials ,,of ,,histology4 ,by ,,louis ,,leroy1 ,m4 ,d41 ,professor of ,histology and ,pathology1 ,vanderbilt ,university1 ,nashville1 ,tennessee4 #abmo1 #bfc pages1 with #ib original illustrations4 ,cloth1 #a4jj net4 ,in ,saunders' ,question-,compend ,series4 ,the work in its present form stands as a model of what a student's aid should be2 and we unhesitatingly say that the practitioner as well would find a glance through the book of lasting benefit4--,the ,medical ,world1 ,philadelphia4 ,barton and ,wells' ,medical ,thesaurus ,a ,,thesaurus ,,of ,,medical ,,words ,,and ,,phrases4 ,by ,,wilfred ,m4 ,,barton1 ,m4 ,d41 ,assistant ,professor of ,materia ,medica and ,therapeutics1 and ,,walter ,a4 ,,wells1 ,m4 ,d41 ,demonstrator of ,laryngology1 ,georgetown ,university1 ,washington1 ,d4,c4 #abmo1 #ecd pages4 ,flexible leather1 #b4ej net2 thumb indexed1 #c4jj net4 ,american ,pocket ,dictionary ,new 7#hth7 ,edition ,,dorland',s ,,pocket ,,medical ,,dictionary4 ,edited by ,w4 ,a4 ,,newman ,,dorland1 ,m4 ,d41 ,editor ,american ,illustrated ,medical ,dictionary4 ,containing the pronunciation and definition of the principal words used in medicine and kindred sciences1 with #fd extensive tables4 #fgg pages4 ,flexible leather1 with gold edges1 #a4jj net2 with patent thumb index1 #a4be net4 ,i can recommend it to our students without reserve4--,j4 ,h4 ,,holland1 ,m4,d41 of the ,jefferson ,medical ,college1 ,philadelphia4