,transcriber's notes3 ,apart from the following corrected misspellings the text of this book has been preserved as in the original3 xolol -- xylol side -- slide overstraining -- overstaining ,where appropriate1 illustrations and footnotes have been positioned adjacent to the relevant text4 ,,section ,,cutting ,,and ,,staining ,a ,,practical ,,introduction ,,to ,,histological ,,methods ,,for ,,students ,,and ,,practitioners ,,by ,w4 ,s4 ,,colman1 ,m4,d41 ,m4,r4,c4,p4 ,,assistant ,,physician 7,,formerly ,,pathologist7 ,,to ,,the ,,national ,,hospital ,,for ,,the ,,paralysed ,,and ,,epileptic2 ,,and ,,to ,,the ,,hospital ,,for ,,sick ,,children1 ,,great ,,ormond ,,street1 ,,etc4 ,,second ,,edition ,,enlarged ,,and ,,in ,,most ,,part ,,re-,,written ,,london ,h4 ,k4 ,,lewis1 #acf ,,gower ,,street1 ,w4,c4 #ahif ,,printed ,,by ,h4 ,k4 ,,lewis1 #acf ,,gower ,,street1 ,,london1 ,w4,c4 ,,preface ,,to ,,the ,,second ,,edition4 ,in preparing this edition ,i have endeavoured to meet the requirements of students1 and of practitioners who desire to keep up their histological work4 ,those methods are selected which have been found to work well in practice1 and it has been thought better to describe a few in detail rather than give a short account of many similar methods4 ,i have again to express my obligation to the various instrument makers for the illustrations of microtomes1 c42 to ,dr4 ,fearnley1 of ,bradford1 for the description of his method for injecting blood vessels1 and to ,messrs4 ,macmillan and ,co4 for permission to copy figures #aj and #aa4 ,w4 ,s4 ,,colman4 ,wimpole ,street1 ,w4 ,sept41 #ahif4 ,,contents4 ,,page ,,chapter ,i4 ,apparatus ,required #a ,,chapter ,,ii4 ,hardening ,processes #ae ,,chapter ,,iii4 ,section ,cutting #bi ,,chapter ,,iv4 ,section ,mounting #ee ,,chapter ,v4 ,general ,staining ,methods #fg ,,chapter ,,vi4 ,special ,methods for ,staining the ,nerve ,centres #hg ,,chapter ,,vii4 ,special ,methods for ,staining ,micro-,organisms and ,blood #ajc ,,chapter ,,viii4 ,injection of ,blood ,vessels #abj ,,chapter ,,ix4 ,directions for ,preparing ,individual ,tissues #abi ,index #aec ,,section ,,cutting ,,and ,,staining4 ,,chapter ,i4 ,,apparatus ,,required4 ,probably there is nothing more perplexing to a beginner than to decide what apparatus is required4 ,if he consult a price list1 it is difficult for him to tell which articles will be necessary1 and which will be either luxuries1 or required only for special investigation4 ,in the following account of requisites1 those only will be described which it is useful to have always at hand4 ,they will be found sufficient for ordinary work1 but for special investigations a more elaborate equipment will be required4 ,all staining and other reagents should be made as far as possible by the worker himself1 according to the directions given in later chapters4 ,this should at any rate be done at first1 as the knowledge thus gained will prove invaluable4 ,it will also effect a great saving if articles that are used in any quantity1 such as methylated spirit1 distilled water1 c41 are bought by the gallon1 and not in small quantities4 ,almost all the processes described here can be carried out without the use of a fully equipped laboratory1 in fact1 in an ordinary room4 ,the only furniture required is a firm table1 and a cupboard and shelves for storing reagents4 ,the following should also be procured3-- 99,jars99 or 99bottles991 with well fitting stoppers or corks1 to contain the tissues while being hardened4 ,they should not hold less than two ounces4 ,empty drug bottles which can usually be obtained from druggists for a few pence1 serve very well4 ,smaller bottles should also be procured for keeping specimens in spirit after they have been hardened until one is ready to cut sections4 ,after sections have been cut from a portion of the specimen1 the rest should be preserved1 in case it is wanted for further investigation4 ,each specimen must be labelled1 with a name or a number corresponding to a reference in the note-book1 and a large number of specimens may then be kept in the same jar4 ,the best way to label them is to write the name or number on a piece of vegetable parchment in ordinary 8marking ink10 and warm it until the writing is black4 ,the little label should then be fixed to a corner of the piece of tissue with a stitch or by a fine pin1 and it may be identified years afterwards4 ,the importance of keeping tissues1 sections1 slides1 c41 99distinctly labelled99 cannot be too strongly impressed on the beginner4 ,the name1 date1 and other particulars should be invariably written on the label at the time4 ,at first the student will be inclined to neglect this1 as he will recognize his pieces of tissue and sections so readily merely by their shape and general appearance4 ,but as time elapses and similar specimens accumulate1 he will find it most difficult or even impossible to identify one from the other4 ,a number of #a oz4 and #b oz4 99stoppered bottles99 for staining reagents4 ,the stopper of these should be fitted with a rod4 ,this is done by simply heating the lower end of the stopper and the upper end of a piece of glass rod of suitable length in a blow-pipe1 until they are plastic1 and then pressing them together4 99,watch glasses499--,at least a dozen watch-glasses1 in which to perform the operations of staining1 clarifying1 c4 ,those with a flat bottom should be employed as they are less easily upset than the others4 ,plenty of 99filter papers994 ,both coarse ones1 for use in the manufacture of reagents1 and small fine white ones 7#b-a_/b inch7 for filtering the staining fluids immediately before using them1 should be procured4 ,before using them a few drops of alcohol or distilled water should be placed in them to saturate the paper4 ,this not only allows the fluid to pass through more rapidly1 but prevents a portion of it being wasted through being absorbed by the pores of the paper4 ,several 99needles99 mounted in handles4 ,they must be kept very bright and smooth1 and care must be taken that the point does not get turned up4 ,a large and small 99funnel994 ,several 99pipettes99 consisting of pieces of glass tube with an internal diameter of #a_/h and about ten inches long1 drawn out almost to a point at one end4 99,section lifter499--,this instrument is required for transferring sections from one reagent to another1 or from oil of cloves1 c41 to the slide4 ,the most convenient form is ,woodhead's1 made of thin sheet copper1 which allows the blade to be bent at any angle to the stem4 ,the stem or handle is about six inches long1 and continuous with1 and at an angle to it1 a flat blade about #c_/d in4 square with the corners rounded off4 ,larger ones can be obtained for mounting sections of large size1 e4g41 kidney1 medulla oblongata1 c4 ,the surface of the blade should be brightly polished1 and kept scrupulously clean4 ,ordinary dissecting 99forceps994 ,one or two 99scalpels994 ,a pair of fine 99scissors994 ,a 99razor99 or other instrument for cutting sections4 ,a smooth 99oil stone99 for keeping the razors and knives properly sharpened4 ,a 99spirit lamp99 for warming the staining fluids4 ,a few 99test tubes994 ,a 99minim measure994 99,scales99 and small weights4 ,a gross of ground glass slides #c x #a in4 ,half a gross of ground glass slides #c x #a-a_/b in4 ,half an ounce of thinnest coverslips1 #g_/h in4 diameter4 ,quarter of an ounce of thinnest coverslips1 #a-a_/d in4 diameter4 99,microscope499--,this is not the place for a description of the microscope as an optical instrument1 but some hints as to the selection of one may be found useful4 ,showy microscopes with much brass work should be avoided1 simplicity of construction being a great recommendation4 ,the microscope should have a large heavy base1 either of the horse-shoe or tripod pattern1 large enough to afford a firm base when the microscope is tilted4 ,mechanical stages are unnecessary and they add greatly to the expense1 and very little to the utility of the instrument for ordinary histological work4 ,binocular arrangements also are of little use for this purpose4 ,the microscope should be provided with a 99coarse and fine adjustment991 which should be most carefully tested before purchasing the instrument4 ,they should work freely and smoothly1 and the slightest turn in either direction should at once alter the focus4 ,there should be a 99reversible mirror991 one side being concave and the other plane4 ,the concave surface is the one usually employed1 the plane surface being chiefly used in conjunction with the sub-stage condenser for the examination of micro-organisms4 ,there should be an 99eye-piece99 of moderate magnifying power4 ,very powerful eye-pieces do not reveal additional details1 but merely enlarge the image1 and with it any defects that may be produced there by faults in the objective4 ,eye-pieces ,,ii4 and ,,iv4 of most makers will be ample for most requirements4 99,objectives499--,these are the most important parts of the microscope1 and the student will be well advised if he spends a little extra money to secure good lenses4 ,most objectives and stands are now made with a universal thread1 so that any objective will fit any make of stand4 ,many workers provide themselves with a cheap stand such as that supplied by ,leitz1 and then fit it with lenses by ,zeiss1 or other first class maker4 ,the most useful lenses are the #a in4 low power lens1 and #a_/e in4 or #a_/f in4 high power1 or ,no4 #c and ,no4 #g of ,continental makers1 or ,zeiss's ,a and ,d4 ,a #a_/b in4 lens will also be found very useful4 ,for minute work1 such as bacteriology and blood investigations1 higher powers will be required1 #a_/h or #a_/ab 99immersion lenses994 ,these objectives come extremely close to the object1 and very thin cover glasses must be employed4 ,in order to avoid the refraction caused by the rays traversing the air between the coverslip and lens some immersion fluid is placed between the two4 ,with some lenses water is employed1 but usually an oil having the same refractive index as glass is used1 and the one most generally employed is cedar oil 7,zeiss prefers the oil from the species ,juniperus virginiana74 ,a spot of oil is placed with a rod just over the object to be examined and the objective carefully lowered by the coarse adjustment till it comes in contact with the droplet of oil4 ,the focussing should then be managed with the fine adjustment only4 ,when the section has been examined the oil must be removed from the lens4 ,for this purpose a soft silk handkerchief or a special piece of chamois leather may be employed1 and used very gently4 ,if all the oil cannot be removed1 the handkerchief may be moistened with a little absolute alcohol1 and the lens hastily wiped4 ,the alcohol must not be allowed to remain in contact with the lens as it is a solvent of ,canada balsam with which the lenses are often cemented in position4 ,illustration3 ,fig4 #a4--;,double or ,triple ,nose-piece4 99,double or triple nose-piece99 7fig4 #a74--,this mechanical arrangement is placed on the lower end of the tube4 ,two or three objectives of different magnifying power are attached to it4 ,the nose-piece rotates round a central pivot in such a way that the objectives can successively be brought accurately into position above the object on the stage4 ,it is1 therefore1 a moment's work to replace a high power objective by a low power one and vice versa4 ,it is an extremely convenient time-saving appliance1 and by its use the risk of dropping and injuring the objectives when screwing them on and off frequently is avoided4 ,those whose microscopes are not already fitted with this appliance can easily have one fitted on at a cost of about a sovereign4 99,substage condenser499--,this mechanism for concentrating light on the object is a necessity for bacteriological work4 ,the most convenient form is 99,abbe's illuminating apparatus99 7fig4 #b74 ,this consists of a system of short focus lenses which collects the light received by the mirror1 and throws it on the object4 ,the amount of light received from the mirror is controlled by an 8iris diaphragm10 the aperture of which can be dilated or contracted by moving a small lever at the side4 ,it can be fitted on to most microscope stands1 but it is better to get a stand in the first instance which is constructed to carry one4 ,the cost of a microscope varies from two guineas to two hundred4 ,there are many excellent microscopes in the market1 and of these several may be mentioned which the writer has found to work satisfactorily4 ,illustration3 ,fig4 #b4--;,abbe's ,illuminating ,apparatus4 ,of the cheaper student's microscopes the 8,star0 microscope made by ,messrs4 ,r4 and ,j4 ,beck1 of ,cornhill1 ,e4,c41 will be found a safe investment4 ,it may be obtained with coarse and fine adjustment1 nose-piece1 and #a in4 and #a_/d in4 objectives1 for about #e4 ,those who require a better instrument will find ,beck's 8,pathological0 microscope fitted with nose-piece1 ,abbe's illuminator1 c41 for #af1 meet all requirements4 ,leitz of ,jena1 supplies two good and cheap microscopes for #c #ajs4 and #e4 ,they are not1 however1 of uniform excellence1 and they should be carefully tested by some competent judge before the purchase is completed4 ,leitz immersion lenses are cheap1 and often extremely good1 but should be carefully tested beforehand1 as their quality is not quite uniform4 ,the microscopes can be obtained from ,mr4 ,a4 ,frazer1 ,teviot ,place1 ,edinburgh4 ,the 8,bacteriological0 microscope1 made by ,messrs4 ,swift1 of ,tottenham ,court ,road1 is one with which no one can be disappointed4 ,it is sold with ,abbe's condenser1 triple nose-piece1 #a_/f in41 and a #a_/ab in4 immersion objective1 for just under #bj4 ,both stand and lenses are turned out in ,swift's first-class style1 and those who can afford the initial outlay will not regret it4 ,or the stand may be purchased1 and the objectives and accessories added singly from time to time4 ,among ,continental makers1 excellent microscopes for histological work are turned out by many makers4 ,zeiss's lenses stand deservedly high in reputation1 as no faulty ,zeiss lens ever leaves the works1 and their optical properties are nearly perfect4 ,for this guarantee1 however1 the purchaser has to pay somewhat higher prices1 but the money is well invested4 ,zeiss's agency is at #bi ,margaret ,street1 ,regent ,street1 ,w4 ,reichert1 of ,vienna1 sells microscopes and lenses which are modelled on the lines of those of ,zeiss1 and though cheaper are often equal to them in excellence1 but the quality is not quite uniform4 ,his instruments can be obtained through any optician1 but his agent in this country is ,mr4 ,a4 ,frazer1 ,teviot ,place1 ,edinburgh4 ,before buying a microscope the student should obtain an illustrated price list from any of the firms mentioned above1 and1 having selected an instrument1 he should test it very carefully1 or better get some experienced friend to test it for him1 before deciding to purchase it4 ,delicate test objects such as diatoms1 scales of butterfly's wing1 or a stained specimen of micro-organisms should be employed4 ,the coarse and fine adjustments should be tried4 ,they should work freely and smoothly and without any delay4 ,the definition of the lens must be tested with the fine objects mentioned4 ,the field should be quite flat1 i4e41 every part should be in focus at the same time1 and the definition should be perfectly sharp and accurate1 and the test objects without double contour4 ,the field should be totally free from prismatic colours4 ,if there is a halo of colour around the objects it indicates a defect in the optical properties of the objective1 and another should be selected4 ,a microscope must always be treated with the greatest care4 ,jars and falls tend to slightly loosen and shift the lenses1 and to permanently impair its optical properties4 ,dust must be most carefully excluded4 ,this is best effected by keeping the instrument under a glass bell jar when not in use4 ,the lenses should be wiped as little as possible1 and when it is necessary1 very soft chamois leather should be employed4 ,the microscope must be kept in a dry room1 or the brass work will soon tarnish and the steel parts will tend to rust4 ,,chapter ,,ii4 ,,hardening ,,processes4 ,for the satisfactory examination of tissues it is necessary that they should be 8hardened0 in certain fluids4 ,the object of this is to give the specimens greater consistence1 so that thin sections may be more readily obtained and more safely manipulated1 and also to 8fix0 the tissue element as far as possible in the same relative position as in the living body4 ,the hardening process also acts on the protoplasm of the cells1 and prevents their swelling up when placed in water1 and in the various staining fluids4 ,the fluid used must be one which will not itself injure the specimen1 and which can be thoroughly removed by washing1 so that it may not interfere with staining operations4 ,the specimens should be kept while hardening in wide mouthed bottles1 on the bottom of which a little cotton wool or tow has been laid4 ,this allows the hardening fluid to come freely in contact with the under surface of the pieces of tissue1 and prevents their being flattened against the hard glass bottom4 ,the hardening fluid requires changing occasionally4 ,this should always be done at the end of twenty-four hours1 in order to get rid of any deposit of blood1 c41 that may have accumulated4 ,besides this1 the tissue when placed in the fluid contained a good deal of water which will have diluted it and consequently an early change is desirable4 ,afterwards the fluid requires to be changed only as often as it becomes turbid1 or any deposit occurs1 usually about once a week4 ,while hardening1 specimens should be kept in a cool place1 as warmth favours changes in the cells1 c4 ,in manipulating the portions of organs1 forceps should always be used and these with great gentleness4 ,the specimens should never be impaled with needles1 or unsightly holes1 which may even be mistaken for pathological appearances1 will appear when a section is examined under the microscope4 ,it requires some practice to know when the tissue is sufficiently hardened4 ,the object aimed at is to make them not really hard but tough4 ,it is almost unnecessary to add that in testing this with the fingers the utmost gentleness must be observed1 or serious damage may be done to the tissue4 ,when the tissue is sufficiently hardened the hardening fluid must be thoroughly dissolved out4 ,this is most quickly effected by placing the specimen in a basin into which cold water from a tap is constantly running4 ,the tissue may then be removed 7forceps always being used and never the needle7 and placed in an imbedding medium as subsequently directed2 or1 if it is not to be cut at once1 into equal parts of methylated spirit and water1 in which it may be kept indefinitely1 the fluid being changed if it becomes at all cloudy4 ,it is unnecessary for ordinary work to have more than the following hardening fluids3--99,mller's fluid993-- ,potassium ,bichromate #b-a_/d grms4 #c-a_/b drachms4 ,sodium ,sulphate #a grm4 #a-a_/b drachms4 ,water to #ajj c4c4 #a pint4 ,two drachms of carbolic acid are sometimes added to each pint of the fluid but as a rule it is not necessary4 ,mller's fluid is the most generally useful of the various fluids employed1 for the following reasons3-- #a4 ,it causes very little shrinking of the elements of the tissue1 and hence may be employed for most delicate objects1 e4g41 the retina and embryos4 #b4 ,in consequence of its not making the tissues shrink1 it does not squeeze the blood out of the vessels and where the organ has been congested before death1 we may1 by using ,mller's fluid1 preserve a natural injection of the capillaries4 #c4 ,there is comparatively little danger of over-hardening the tissue and rendering it brittle4 #d4 ,sections of organs hardened in ,mller's fluid are usually firm and easy to manipulate4 ,they do not tend to curl up or adhere to one another as much as those hardened in spirit4 #e4 ,it readily permeates the tissues1 and hence large portions of organs1 or even the entire organ may be satisfactorily hardened in it4 #f4 ,it is very cheap4 ,a gallon can be made up for about eightpence4 ,the fluid has however certain slight drawbacks3-- #a4 ,the hardening process is a slow one occupying four to eight weeks4 #b4 ,the fluid gives a permanent dingy colour to the tissue4 ,this does not cause any inconvenience for microscopic purposes1 but it is a disadvantage when it is intended to preserve the rest of the specimen1 as a naked eye preparation4 ,in such cases the organ should be hardened in spirit1 carbolic acid1 or formal4 ,mller's fluid can be used for almost any tissue4 ,it is especially useful for those which contain a large quantity of fluid1 or of blood1 and is essential for nerve tissues which it is intended to stain by ,pal's method 7p4 #hi74 ,to harden a specimen in it at least twenty times the bulk of fluid must be employed4 ,the fluid must be changed on the third day1 and afterwards about every week as may be required4 99,methylated spirit99 is a very useful hardening agent4 ,it hardens in one to three weeks according to the size of the tissue and the quantity of spirit used4 ,its disadvantages are3-- #a4 ,it is more apt to overharden than ,mller's fluid4 #b4 ,it causes a great deal of shrinking of the tissue and thus squeezes much of the blood out of the vessels4 ,it is most useful in hardening tissues containing much epithelium1 e4g41 kidney1 epithelioma1 c4 ,spirit is also frequently employed to complete the hardening by ,mller's fluid and to preserve tissues after they have been hardened4 ,about ten or fifteen times the bulk of spirit should be used for one of the tissues4 ,the fluid should be changed on the third day and afterwards as required4 99,mller's fluid and spirit499--,this is a useful combination for many purposes4 ,it is made thus3--,mller's fluid1 three parts2 methylated spirit1 one part4 ,the fluid must be allowed to cool after mixing before being used1 and if necessary filtered4 ,it will harden specimens satisfactorily in three weeks4 99,mller's fluid and formal499--,is an extremely useful mixture made by adding one part of formal to nine of ,mller's fluid4 ,it hardens in a much shorter time than ,mller's fluid and causes very little shrinkage4 99,absolute alcohol499--,used as a hardening agent where the tissues are to be examined for micro-organisms1 and for specimens to be stained by ,nissl's method 7p4 #aja74 ,a cheaper and equally effective hardening medium is made by dehydrating methylated spirit by adding one ounce of fused carbonate of potassium to each pint of methylated spirit1 and decanting4 ,small pieces must be used4 ,the depths of the block should not exceed #c_/h inch4 ,the fluid should be changed on the third day4 ,hardening will be completed in about ten days or even earlier4 99,osmic acid499--,for rapidity of action1 and for rapid fixing of all the tissue elements in their natural position osmic acid is one of the best hardening reagents we possess4 ,its disadvantages as a hardening agent are3-- #a4 ,its expense4 #b4 ,its irritating and corrosive vapour4 #c4 ,the fact that only small pieces of tissue can be hardened in it1 since the external surface is very rapidly hardened and thus the fluid is prevented from penetrating into the centre of the lump4 ,it is most frequently used as a hardening agent for very delicate structures1 such as the retina1 or embryos1 or for fresh sections of brain 7p4 #ie74 ,the acid itself may be procured in sealed tubes1 each containing one gramme4 ,these should be broken in a bottle under sufficient distilled water to make a one per cent4 solution4 ,the bottle containing it should be covered with brown paper to exclude the light4 ,for hardening purposes small pieces of the tissue1 not much larger than a pea1 should be placed in the acid1 the one per cent4 solution being diluted with five to ten volumes of distilled water4 ,the tissues may be left in this for from three to five days4 ,they must then be 99thoroughly99 washed in distilled water and may afterwards be preserved in methylated spirit4 ,both the hardening and the subsequent washing must be carried on 99in the dark994 ,osmic acid is also a most valuable staining reagent 7see p4 #ha74 99,carbolic acid99 7#e per cent474--,may be used to harden almost any tissue1 but is particularly useful for hardening nervous tissues such as brain or spinal cord which are afterwards to be preserved as museum specimens4 ,it does not discharge the colour of a specimen so rapidly as spirit4 ,three or four times the bulk of fluid should be used4 ,it requires changing at the end of twenty-four hours1 and again at the end of the first week4 ,saturated aqueous solution of 99corrosive sublimate99 is one of the most convenient hardening reagents for small pieces of delicate tissue1 e4g41 embryos4 ,it hardens them in a few days4 ,when they are sufficiently hardened the mercurial salt should be removed by washing first in methylated spirit for a few hours and then in running water4 99,formal499--,an aqueous solution containing about #ce per cent4 of formaldehyde4 ,it is a rapid hardening agent1 causes very little shrinkage of the tissues1 and does not discharge the colour of the specimens as much as alcohol4 ,for hardening formal should be used as a two to five per cent4 solution in distilled water4 ,it may also be used as a ten per cent4 solution for mounting museum preparations1 but there is some tendency for a cloudy deposit to form on the glass after a time4 ,it is the most suitable hardening agent at our disposal for eyes4 ,it rapidly fixes the tissue elements1 but does not cause much contraction4 ,it may also be used for hardening the brain and spinal cord4 ,large quantities of fluid must be used for the latter purpose and it must be frequently changed4 ,as soon as they are sufficiently hardened they should be transferred to methylated spirit4 99,marchi's fluid499--,this consists of3-- ,mller's fluid #b parts4 ,osmic acid solution 7one per cent47 #a part4 ,it is used for hardening specimens as a preliminary to ,golgi's method for staining nerve cells 7p4 #ig71 and also to complete the hardening of sections of spinal cord1 c41 before employing ,schfer's modification of the ,weigert ,pal hmatoxyline method 7p4 #ia74 ,it is also used as a stain for recently degenerated nerve tracts and fibres1 especially after experimental lesions4 ,the fluid has little penetrating power1 and therefore tissues must be cut into small pieces1 about #c_/h inch cube4 ,it is not necessary to place them in this fluid at once on removal from the body1 but the preliminary hardening must be in ,mller's fluid and not in alcohol1 c4 ,,special ,,hardening ,,reagents ,,for ,,rapid ,,fixation ,,in ,,order ,,to ,,study ,,cell ,,structure4 #a4 99,alcohol499 #b4 99,flemming's solution99 7modified by ,friedmann73-- ,osmic acid 7one per cent47 #c c4c4 7xxx474 ,glacial acetic acid #b c4c4 7xx474 ,chromic acid 7one per cent47 #db c4c4 7j47 ,small pieces should be hardened in this fluid for twelve to twenty-four hours1 and then washed and transferred to alcohol for some days before staining4 #c4 99,nitric acid499--,a ten per cent4 solution in distilled water4 ,it hardens the tissue in three to four hours1 and should be followed by #gj per cent4 alcohol1 the hardening being completed in absolute alcohol4 ,in using any of these methods it is necessary that the tissue be removed from the body during life or immediately after death4 ,they are employed for revealing the changes in the cells and their nuclei in rapidly growing or inflamed tissue1 for studying karyokinesis in cancer cells1 and investigating the appearance of nerve cells and gland cells at rest1 when actively employed and when fatigued2 and they are also most useful in preparing specimens of the 8parasitic bodies0 which have been described in many cancer cells4 ,,decalcifying ,,fluids4 ,used in the preparation of bone1 tooth1 osseous tumours1 c4 ,the two best fluids for general use are3-- 99,chromic and nitric fluid499--,this is made as follows3-- ,chromic acid #a gramme #de grains4 ,nitric acid #b grammes #a-a_/b drachms4 ,water #bjj c4c4 #a pint4 ,if the bone is not very compact the fluid may be used diluted with an equal quantity of water4 ,a large quantity of fluid should be used1 and like all decalcifying fluids1 it should be frequently changed4 ,as soon as the specimen is sufficiently flexible1 it should be thoroughly washed in running water for some hours1 and then transferred to spirit until it is convenient to cut sections4 99,von ,ebner's solution993-- ,hydrochloric acid #a gramme #a-a_/b drachms4 ,common salt #aj grammes #b ounces4 ,water to #ajj c4c4 #a pint4 ,it is a very useful decalcifying agent1 but causes the fibrous elements to swell up rather more than chromic and nitric fluid4 ,a large quantity of the fluid must be used1 and it should be changed daily4 ,it must be very thoroughly washed out in running water when the decalcification is completed4 99,bleaching solution99 7eau de ,javelle74 7#a7 8,chloride of lime0 7bleaching powder7 #bj #a_/b oz4 ,water #ajj #b-a_/b oz4 ,shake up well4 7#b7 ,carbonate of potassium #bj #a_/b oz4 ,water #ajj #b-a_/b oz4 ,mix the two solutions4 ,allow them to stand for an hour and filter4 ,it is used chiefly for clearing vegetable sections but may also be used for sections containing a large quantity of pigment4 ,it is particularly useful in decolourizing sections of 8madura foot0 due to the presence of a black fungus4 ,,chapter ,,iii4 ,,section ,,cutting4 99,embedding of sections499--,before sections are made the tissues require to be embedded in some fluid1 which will permeate their interstices1 and is capable of being rendered firm so as to support the most delicate parts when the knife passes through the tissue4 ,the most generally useful substances are3-- 7#a7 gum1 7#b7 celloidin1 7#c7 paraffin or wax4 99,gum499--,picked colourless gum arabic #b parts1 cold water #c parts4 ,leave with frequent stirring until dissolved4 ,add ten drops of carbolic acid to each ounce of the mucilage4 ,specimens are thoroughly freed from all trace of the hardening fluid by washing in water1 and the tissue is then placed in the gum solution for at least twelve hours1 or if enough carbolic acid be added1 it may be left there for an indefinite time4 ,when frozen1 gum forms a firm non-crystalline mass1 which supports the tissue on all sides4 ,it must not be frozen too deeply1 or it becomes hard and rather brittle and is apt to injure the razor4 ,if this have occurred the surface can be softened sufficiently by breathing gently on it4 ,after cutting in gum1 the sections are gently removed from the knife into distilled water by a soft camel's hair brush1 and left there for an hour or two1 until the medium is entirely dissolved out4 ,they may then be stained and mounted1 or they may be put away in spirit for an indefinite time1 and then stained and mounted4 99,celloidin99 is for many purposes almost an ideal embedding medium4 7#a7 ,it has great penetrating power2 7#b7 it can be made of an admirable consistence for cutting purposes2 7#c7 after sections are made it allows them to be very freely manipulated without fear of injuring them3 7#d7 and being perfectly transparent and homogeneous in thin sections1 it does not require to be removed from a section before mounting4 ,it is insoluble in water1 and in weak spirit2 slightly soluble in alcohol of more than #ij per cent4 strength1 and very readily soluble in ether1 or in a mixture of alcohol and ether4 ,the last solvent is the one commonly employed4 ,the embedding solution is made thus3-- ,take some pure celloidin 78,schering's10 sold in boxes containing an ounce of shavings1 is very good7 and pour on it about eight times its volume of a mixture of equal parts of absolute alcohol and ether4 ,allow this to stand all night until the celloidin is dissolved4 ,the solution should be made about the consistence of ordinary mucilage4 ,it is also convenient to have a thinner solution made by using double the proportion of alcohol and ether4 ,both solutions should be kept in wide mouthed stoppered bottles1 and the stopper should be well greased with vaseline as an additional obstacle to the evaporation of the volatile ether4 ,before embedding a specimen it is necessary to dehydrate it thoroughly for twelve to twenty-four hours in absolute alcohol4 ,it should then be placed in a mixture containing alcohol and ether for an hour or two1 and afterwards transferred to the thin solution of celloidin for twenty-four hours1 and then to the thick solution for the same period4 ,the celloidin penetrates slowly and in the case of nerve tissues and other delicate structures it is wise to give the full allowance of time for the different steps4 ,when the tissue has been thoroughly permeated by the celloidin1 it is gently removed from the celloidin and placed in position on a piece of cork of suitable size for clamping in the holder of the microtome4 ,celloidin is painted round the object so that it is supported on every side4 ,it is then left exposed to the air until the surface has become firm1 when the cork is placed1 with the tissue downwards1 in methylated spirit4 ,the cork floats but the tissue and celloidin remain submerged4 ,at the end of twenty-four hours the celloidin will have become semi-opaque and opalescent1 and of the same consistence as hard boiled white of egg4 ,when it is impossible to wait so long1 rapid hardening of the celloidin may be secured by immersing it in methylated chloroform in place of spirit1 but the slower method gives more uniformly satisfactory results4 ,pieces of tissue embedded in celloidin may also be cut on a freezing microtome4 ,after the celloidin has become firm by immersion in methylated spirit1 the tissue with the celloidin round it may be cut off the cork1 washed in water to remove the alcohol1 and then soaked for an hour or two in gum1 placed on the plate of an ether spray microtome1 frozen and cut in the usual way4 ,subsequent staining operations are conducted in the same way as for sections cut by hand or in gum4 ,as celloidin is only slightly stained by hmatoxylin1 alum carmine1 borax carmine1 c41 it is not necessary to remove it from the sections1 but it exhibits so intense a staining reaction with aniline dyes that it is necessary to remove it by treatment with alcohol and ether either before or after the staining operation4 ,the sections after staining may be mounted in ,farrant's solution 7p4 #ei71 or in ,canada balsam 7p4 #fa74 ,if the latter medium is employed1 the section should be clarified1 after dehydration in alcohol1 by means of oil of bergamot1 or oil of origanum1 instead of oil of cloves1 as the latter dissolves out the celloidin and causes the section to break up4 ,celloidin is most useful for cutting sections of the coats of the eye1 of the internal ear1 and of bone marrow4 ,it should always be used for the ,weigert-,pal hmatoxyline method of staining the nervous centres1 as it protects the section from being injured by the transference from one fluid to another which is repeatedly required during the process4 ,the stain is completely discharged from the celloidin by the decolourising solution used 7p4 #ij74 ,paraffin4--,paraffin is a very convenient embedding medium for delicate structures1 as very thin sections can be obtained and the paraffin need not be removed from the section until the latter is safely on the slide4 ,it is unsuitable for large sections4 ,staining operations are not easily carried out after cutting in paraffin1 and it is better to stain the blocks of tissue in bulk before embedding4 ,the best stains for penetrating are borax carmine 7p4 #ge71 alum carmine 7p4 #gf71 and ,kleinenberg's hmatoxyline 7p4 #gj74 ,the tissue must be left in them for four to ten days4 ,various kinds of paraffin are employed4 ,it is usual to keep two kinds1 one 8soft10 melting at #aaj ,f41 and another 8hard10 melting at #adj ,f4 ,a mixture of two parts of the hard and one of the soft will be found most generally useful4 ,in winter a large proportion of the soft variety and in hot weather a larger proportion of the hard may be required4 ,a paraffin mass which is always available has been suggested recently by ,dr4 ,f4 ,e4 ,batten1 who employs an ordinary white candle1 composed of paraffin and wax4 ,if the mass is found to be too hard1 it can easily be made of a suitable consistence by adding a little paraffin with a low melting point4 ,to prepare a piece of tissue for embedding in paraffin1 it should be stained1 washed in distilled water1 and as much moisture as possible removed by blotting paper4 ,the block is then dehydrated1 first in methylated spirit for several hours1 finally in absolute alcohol4 ,it is taken carefully by means of forceps from the alcohol and placed in xylol for an hour or two according to size4 ,superfluous xylol is removed from the surface1 and the tissue placed in the melted paraffin4 ,this will set round the cold tissue at once1 but soon melts again and must be kept at a temperature just above melting point for one to four hours1 according to size4 ,the tissue is then transferred to a mould 7which can be easily made of paper71 about half an inch cube1 and melted paraffin poured round it until the mould is full4 ,the mould may be made by folding a piece of paper to form a box about half an inch cube1 or a small pill box may be used4 ,another convenient method is to place two ,l-shaped pieces of lead in contact with each other so as to enclose a space of suitable size as in the diagram 7fig4 #c74 ,the tissue is now hermetically sealed1 and can be kept indefinitely if it is not convenient to cut it at the time4 ,to prepare it for cutting1 all superfluous paraffin is trimmed away with a warm knife1 and the block is fixed on a piece of wood1 cut so as to suit the clamp of the microtome1 by melting the lower end of the paraffin block with a hot needle or wire and pressing it down on the wood4 ,illustration3 ,fig4 #c4 ,when sections are cut they may be transferred singly to the slide 7which should be lightly smeared beforehand with a saturated solution of celloidin in oil of cloves71 or they may be cut so that the back of one section of the paraffin block adheres to the front of the next1 and in this way a continuous delicate ribbon of serial sections is obtained4 ,the ribbon is broken up into lengths of about two and a half inches and transferred to the slide1 on which several ribbons may be placed side by side1 and so a large number of sections kept in the order in which they are cut4 ,a mark should be made on the slide to indicate where the series begins1 and each slide should be numbered1 so that the exact position of each section in the series can be recognised at once4 ,before mounting1 the paraffin must be removed from the sections4 ,this is easily done on the slide in the case of single sections and of ribbons4 ,if the sections are curled1 a little warmth will make them unbend and lie flat4 ,the slide is warmed over a spirit lamp until the paraffin just melts4 ,the sections will keep their places owing to the celloidin beneath4 ,xylol is then allowed to flow over the slide from a pipette1 until the paraffin has been completely dissolved1 which can be ascertained by glancing at the sections under the low power of the microscope4 ,the slide is placed in an almost vertical position to let the xylol drain off1 excess is wiped off from the edge of the slide with blotting paper1 a drop of ,canada balsam solution 7p4 #fa7 is run on the slide1 and a cover-glass of suitable size is applied4 99,microtomes499--,after a large amount of practice1 persons with a fair amount of manual dexterity may acquire sufficient skill to be able to cut very satisfactory sections of specimens embedded in paraffin1 c41 by hand4 ,in the ,pathological ,laboratory of a large ,german ,university1 until quite recently the use of a microtome was prohibited by the ,professor1 who is himself a most distinguished histologist4 ,the amount of time expended before one acquires the necessary skill1 and the cheapness and great convenience of the modern microtome have combined to throw hand cutting into the background1 and some form of microtome is now almost universally adopted4 ,illustration3 ,fig4 #d4--;,cathcart's ,ether ,spray ,microtome4 ,a1 ,b4 ,wooden frame and supports4 ,c4 ,glass runners4 ,g4 ,screw for raising the zinc plate ,h4 ,j4 ,ether bottle4 ,l4 ,tube from air bellows4 ,of these there are a very large number in the market1 each having special advantages1 and often special drawbacks4 ,a few of the more generally useful only will be described4 ,we have microtomes for cutting in gum frozen by ether spray or ice1 and those intended for cutting in paraffin or celloidin4 ,cathcart's 99ether spray microtome99 7fig4 #d74--,this1 or its more recent modifications 7see later71 is perhaps the most useful and economical microtome for the purposes of the student4 ,its prime cost is low1 it is small and portable as well as being clean and inexpensive to work with4 ,it consists of an oak frame which can be firmly clamped on to a table4 ,on this frame are two narrow parallel supports about two inches high1 which are covered by strips of plate glass1 and serve as smooth rests along which the razor may glide in making sections4 ,between them is a brass well and in this a zinc plate firmly fixed in the horizontal position1 which is almost at the level of the glass runners4 ,it is capable of being raised or lowered through about #c_/h inch by means of a screw with a very fine and accurate thread4 ,this screw is turned by a large milled wheel beneath the microtome4 ,just beneath the zinc plate are two small tubes1 one connected with an india-rubber bellows1 the other with a bottle at the side which contains ether4 ,as the air issues from the first tube1 it passes over the open end of the second1 and thus draws the ether out and makes it play on the zinc plate1 and at the same time causes it rapidly to evaporate1 and so reduces the temperature of the zinc plate4 ,in cutting sections with this microtome the tissue is taken out of the gum and placed on the zinc plate4 ,the bellows are then worked until the gum on the zinc plate is completely frozen4 ,the plate should be lowered by means of the screw until the surface of the piece of tissue is on a level with the glass runners4 ,these and the razor should then be wetted with water4 ,the razor being held firmly in the hand is pushed along the glass runners in a rather oblique direction4 ,the plate should then be raised by turning the screw below through a very small arc1 another section taken off and so on4 ,sections are carefully removed from the razor to a vessel of water by means of a soft wet camel's hair brush4 ,the needle should never be used for this purpose4 ,if the specimen is very delicate1 and likely to be spoiled by being curled up on the knife1 the latter should be kept cold by frequently dipping it in a vessel containing lumps of ice in water4 ,the gum will then remain frozen after cutting1 and support the tissue better4 ,each section should be at once transferred to a glass slide from the knife1 washing it off with a stream of ice water from a pipette4 ,the knife that is used may be an ordinary razor1 with the edge ground straight4 ,it requires to be held steadily with both hands4 ,as this is rather inconvenient1 ,dr4 ,sheridan ,dlpine suggested the employment of an ordinary plane iron such as is used in a carpenter's plane4 ,this only requires one hand1 and the other can be kept on the head of the screw beneath to raise the plate at once after each stroke of the knife4 ,its disadvantages are that it is rather heavy for prolonged working1 and that it is less easy to 8set0 than a razor4 ,a4 ,frazer has recently introduced a valuable improvement in the ,cathcart microtome 7fig4 #e74 ,in this the brass frame carrying the zinc plate and ether spray tubes is surrounded by a brass cylinder1 in which it fits accurately1 and is pushed up as desired by turning the screw beneath the instrument4 ,this brass frame and with it the zinc plate1 c41 can be easily drawn altogether out of the outer tube1 and replaced by a second brass well1 which exactly fits its place and can be raised by the screw as desired4 ,in this is a small toothed clamp which can be screwed up so as to hold a piece of wood carrying a piece of tissue embedded in paraffin4 ,sections can also be cut in celloidin with this instrument1 but as oblique strokes with the knife cannot be made1 it is impossible to get very thin sections4 ,the combined microtome can be obtained for a guinea from ,frazer1 #bb ,teviot ,row1 ,edinburgh4 ,illustration3 ,fig4 #e4--;,frazer's ,modification of ,cathcart's ,microtome4 ,a4 ,microtome arranged for ether spray4 ,b4 ,cylinder with clamp for holding object embedded in celloidin1 c4 to replace ether spray apparatus4 ,there is another modification which is more generally useful1 and at the same time more expensive than the original model4 ,in this1 instead of glass runners to support the knife1 there is a flat glass plate about eight inches square sufficiently large to allow of 8,swift's plough0 7fig4 #f7 being used for the purpose of cutting sections4 ,this instrument consists of a triangular brass frame1 supported on three legs1 each of which is a screw1 tipped with ivory4 ,there is one screw in front and two behind4 ,beneath the plate1 and held in position by the posterior screws in front1 and a little clamp behind1 is a razor with the edge directed forwards4 ,the edge can be raised or depressed by turning the anterior screw1 on which the frame is supported4 ,before sections are cut the edge of the razor should be brought down to the level of the tissue1 taking care that all the legs are equal in length4 ,the plough should then be firmly grasped with both hands1 7the index finger of one hand being left free to turn the anterior screw7 and pushed rather obliquely through the tissue4 ,the edge of the razor is then slightly lowered by turning the screw through a very small angle1 and another section made1 and so on4 ,with a little practice very thin uniform sections may be made with great rapidity4 ,another useful ether spray microtome is that made by ,jung of ,heidelburg4 ,the knife swings round a pivot1 and there is an ingenious ratchet arrangement which works synchronously with each swing of the knife1 to raise the tissue automatically the requisite distance for the next section to be made4 ,the exact thickness of the sections can be graduated with great nicety by a simple contrivance4 ,the instrument can be obtained in this country for about #b4 ,it works satisfactorily1 but1 with practice1 the student will get equally good results with the cheaper 8,cathcart40 99,williams' ice freezing microtome99 7fig4 #f74 ,this consists of a round mahogany water-tight box provided with an exit tube below1 and covered with a strong plate glass lid4 ,firmly fixed in the centre of the floor of the box is a stout brass pillar surmounted by a brass disc which fits into a hole in the centre of the glass lid1 so that its surface is on a level with that of the lid4 ,illustration3 ,fig4 #f4--,williams' ,ice ,freezing ,microtome1 with ,swift's plough4 ,to use it1 the box is filled with alternate layers of pounded ice and salt2 the lid is then put on and fixed by means of a lateral screw4 ,the tissue to be frozen is gently removed from the gum and placed on the brass disc and plenty of gum painted round it4 ,it should then be covered with a tin cap for a few minutes until frozen4 ,sections are made with a ,swift's ,plough 7p4 #dd74 ,illustration3 ,fig4 #g4--,schanze ,microtome 7see text74 99,schanze microtome99 7fig4 #g7 is the pattern used in the ,leipsic laboratories4 ,it consists of a heavy iron frame with a large base4 ,the knife is carried in a clamp which slides along the full length of the instrument1 gliding upon two smooth plates of iron which are arranged at an angle to one another4 ,the knife must be moved very steadily and gently1 as when using a long blade vibrations are easily set up which prevent good sections being obtained4 ,the surfaces of contact must be kept scrupulously free from dust1 and lubricated with equal parts of olive oil and castor oil4 ,there are several object holders1 which can be removed and interchanged1 one connected with an ether spray apparatus1 another suitable for holding an object embedded in paraffin1 and a third for grasping an object embedded in celloidin4 ,when celloidin is employed1 a specially long knife must be used1 and it must be fixed very obliquely in the clamp4 ,the object holder is raised by a fine screw worked by a large brass toothed wheel4 ,there is a ratchet arrangement1 by which the object may be raised automatically any desired distance1 after each stroke of the knife4 ,it gives most satisfactory results with celloidin and paraffin4 7,messrs4 ,r4 and ,j4 ,beck are the agents74 ,its cost is about #e4 99,becker's microtome99 is made on exactly the same principles as the ,schanze4 ,the modifications are that the carrier glides on glass plates instead of iron ones1 and that instead of the whole surface of the carrier being in contact with the plates1 there are a few smooth ivory buttons only4 ,friction is thus reduced to a minimum1 and very uniform sections can be obtained4 ,the price is the same as that of the ,schanze4 ,frazer has introduced a 8student's sliding microtome0 on the same principle as the ,schanze which costs about #c4 99,the ,cambridge ,rocking microtome499--,this instrument1 as made by the ,cambridge ,scientific ,instrument ,company1 or the slightly modified form made by ,messrs4 ,swift 7fig4 #h71 is the best instrument for cutting sections of small objects embedded in paraffin4 ,ribbons of serial sections can be obtained from it with greater ease and certainty than with other microtomes4 ,this microtome differs from those which have been previously described in that the knife is fixed1 while the object is moveable4 ,the microtome consists of an oblong heavy metal stand4 ,a long bar is arranged so that it rides in see-saw fashion on two strong vertical pillars arising from the frame4 ,one end of this bar is hollow1 and receives the piece of wood carrying the tissue embedded in paraffin1 which is firmly clamped in position4 ,this end is depressed by means of a strong spiral spring4 ,in order to raise it there is an arrangement by which the other end of the bar is depressed by a cord which revolves round a pulley4 ,when the handle is turned1 the tissue is raised1 and when the cord is relaxed1 the spring pulls the tissue firmly and steadily down4 ,the razor1 which must have a straight edge1 is fixed firmly by screws1 with its edge upwards at the end of the microtome4 ,the object is then adjusted so that in its descent a thin slice is taken off by the razor4 ,there is an ingenious arrangement by which the depression of the bar to raise the section pushes it a little further in the direction of the razor4 ,the distance can be graduated from #a_/ejj to #a_/cjjj inch4 ,the actual working of the machine is therefore very simple4 ,the position of the block containing the tissue to be cut having been adjusted so that the razor just cuts it1 the free end is depressed by means of the pulley4 ,this also pushes the section a little beyond the razor4 ,the strong spring then draws the tissue steadily past the edge of the razor1 and a thin section is left on the blade4 ,this may be at once transferred to a slide1 or if the paraffin be of the proper consistence1 another cut may be made1 when the two sections should adhere by their edges1 and so by repeating the movement a continuous ribbon may be obtained4 ,if there is difficulty in obtaining a good ribbon1 it will usually be got over by taking a little soft paraffin and attaching it by means of a hot needle to the lower end of the paraffin block4 ,the cost of the instrument is about #e4 ,illustration3 ,fig4 #h4--,swift's ,modification of the ,cambridge ,rocking ,microtome4 99,fresh sections499--,although these are not so satisfactory as hardened specimens for accurate histological work1 it is often very useful to make them both in the post-mortem room where an immediate opinion of the nature of the tumour or diseased organ is desired1 and also in the operating theatre4 ,with a little practice sections may be cut1 stained1 and mounted1 within ten minutes of the removal of the specimen from the body4 ,in this way important information may be afforded to the operating surgeon1 and in not a few cases it has caused the proposed treatment to be entirely altered4 ,thus1 in one case1 a supposed chronic periostitis was shown to be a sarcoma1 and the limb was amputated4 ,in another1 a supposed sarcoma of the thigh was found to be a gumma1 when a portion was removed and microscopically examined4 ,a portion of the specimen should be placed without any preparation on the zinc plate of the freezing microtome1 and some gum painted round it4 ,it is then frozen4 ,the serum in the tissues is not in sufficient mass to injure the knife when it is frozen4 ,the knife should be wetted with1 and sections transferred to1 either pericardial serum1 or #c_/d per cent4 solution 7#gj grains to the pint71 of common salt1 neither of which causes the cells to swell up as plain water does4 ,they should be carefully floated out on a glass slide1 an operation which requires much more patience than in the case of hardened sections1 as fresh sections are less coherent and also more sticky1 so that the edges tend to curl up on the knife1 c4 ,they should then be examined1 one unstained1 simply mounted in salt solution2 another stained with picrocarmine and examined in the saline solution2 and a third stained in picrocarmine1 mounted in ,farrant's solution1 and preserved4 ,the last usually gives the best results1 the picrocarmine staining becoming quite brilliant after a week4 ,the glycerine1 however1 is apt to make the sections shrink a good deal1 and the weight of the cover-glass tends to break up the unhardened section4 ,,chapter ,,iv4 ,,section ,,mounting4 #a4 ,by 99flotation994 ,in this method the section whether stained or unstained is placed in a bowl of water1 or normal salt solution 7p4 #ec74 ,a clean slide is then introduced into the water at an angle of about #fj1 a little more than half of its length being submerged4 ,the section is then brought up by the needle and floated as far as possible into position on the slide4 ,one corner is then fixed by the needle1 and on gently withdrawing the slide the section should lie flat4 ,if any folds are left no attempt should be made to smoothen them out with a needle1 but the slide should be re-immersed until the folded part of the section is under water4 ,it should then be gently withdrawn1 when the fold will disappear4 ,this manuvre must be repeated in different directions until the section lies quite smoothly on the slide4 ,stained and unstained sections are floated out in this way before being mounted in ,farrant's medium1 and unstained sections previous to staining in picrocarmine4 #b4 ,by 99transference99 with a 99section lifter994 ,this method is employed in mounting in ,canada balsam in order to transfer the section from the clarifying agent 7p4 #fc7 to the slide4 ,the lifter is polished1 and insinuated under the section4 ,the section being held in position by the needle is now raised from the fluid1 excess of which is removed by holding the section in position with a mounted needle1 and tilting the lifter so as to allow it to drain off4 99,removal of air bubbles from sections499--,when sections contain many air bubbles1 the best plan is to leave them in methylated spirit for a time4 ,the bubbles then coalesce and escape from the section4 ,for delicate structures and for fresh sections the transference to spirit1 and the subsequent flying out of the section when returned to water are risky1 and the best method of treating these is to put the vessel containing them under the receiver of an air pump1 if one is available1 and slightly exhausting the air4 ,the most frequent cause of air bubbles in mounted specimens1 however1 is the employment of cover-glasses which have not been thoroughly cleansed4 ,proper cleansing is best effected by placing the covers when bought in a shallow wide mouthed stoppered bottle containing strong nitric acid1 and leaving them in this fluid for twenty-four hours4 ,the acid should then be drained off and water run through the vessel from a tap1 until the washings no longer give an acid reaction with litmus paper4 ,the water should then be drained off1 and the glasses covered with absolute alcohol4 ,they can be removed one by one and rapidly dried as required4 ,with cover-glasses properly cleansed in this manner1 not only will air bubbles be avoided1 but the covers will be dried much more easily with the cloth1 and fewer will be broken in the process4 ,another very frequent cause is the transference of air bubbles with the mounting medium on the glass rod4 ,this occurs especially if the rod be fused to the stopper4 ,the proper bottles to use1 both for ,farrant's medium and balsam are 8balsam bottles0 which have no stopper1 but the mouth is closed by a glass cap which fits accurately 7fig4 #i74 ,a short glass rod is attached to the cap1 and is used to transfer the medium to the slide4 ,illustration3 ,fig4 #i4--;,balsam bottle4 99,treatment of folded sections499--,the folding may be due3-- 7#a47 ,to the section having creased through being cut with a knife whose surface was not perfectly smooth4 ,this is best remedied by placing the section in methylated spirit for a minute1 and then transferring it to a bowl of clean water1 when the section will rapidly rise to the top1 and spread itself out flat on the surface of the water1 in consequence of the alcohol rapidly diffusing out at the edges into the surrounding water4 7#b7 ,to the section containing a large amount of 99fat991 as in those of the skin and subcutaneous tissue4 ,the fat may be removed from the fat cells without materially altering the appearance of the section4 ,this is done by dehydrating the section in alcohol1 and then transferring to a watch glass containing ether or chloroform to extract the fat4 ,the tissue should be washed free from ether in the alcohol and then transferred to the bowl of water1 and allowed to float out4 ,this process does not interfere with subsequent staining operations4 ,,mounting ,,media4 99,farrant's solution993-- ,gum ,arabic 7picked1 colourless7 ,glycerine equal parts4 ,water ,in making this solution the best gum arabic must be used1 and only the clearest pieces of this4 8,powdered gum acacia0 should be avoided1 as though it looks white it often yields a brown mucilage1 and besides is frequently adulterated with starch1 c4 ,the glycerine and water should be mixed and the gum arabic added4 ,the mixture should be allowed to stand for some weeks1 with frequent stirring until the whole of the gum is dissolved4 ,then allow it to stand for a week or two longer in order that the dirt may subside1 and the bubbles rise to the top4 ,the scum should be removed and the clear fluid decanted from the sediment into a 8,balsam bottle0 7p4 #eh7 containing a few drops of a saturated solution of arseniate of sodium and a small lump of camphor4 ,if properly made it is an extremely useful mounting reagent4 ,it does not clarify the tissues too much1 and in consequence of its containing gum it dries at the edges and cements the cover-glass more or less firmly in a week or two4 ,if this is not the case the medium contains too much glycerine and more gum must be added to compensate for this4 ,this drying at the edge prevents any further evaporation while the glycerine keeps the section permanently moist4 ,the camphor and arseniate of sodium prevent the formation of fungi4 ,sections preserve their original appearance in this medium for many years4 ,after a long time they are apt to become a little cloudy and granular4 ,unstained sections should always be mounted in ,farrant's medium1 as the ,canada balsam process renders them quite transparent4 ,it is suitable for almost any tissue stained or unstained1 but sections of the nervous centres require to be mounted in ,canada balsam1 owing to the opacity of myelin when mounted in glycerine4 99,canada balsam solution993--,the medium is made thus3-- ,the ordinary ,canada balsam which is of a treacly consistence is heated gently in a water bath for some hours1 to drive off turpentine and other volatile oils4 ,it is then allowed to cool to a yellow vitreous mass4 ,take of ,dried ,canada balsam equal parts4 ,xylol ,leave till dissolved1 stirring occasionally4 ,unless the solution be perfectly clear1 it must be filtered through a very thin paper1 previously wetted with xylol4 ,if the medium be too thick more xylol should be added1 if too thin1 the xylol should be allowed to evaporate until the medium is of the consistence of a thin syrup4 ,if the medium is made too thin much annoyance will be caused by its evaporating at the edge of the cover-glass1 leaving an air-space1 which will increase daily until the section is left quite dry4 ,this should be remedied by putting another drop of balsam at the edge of the coverslip and allowing it to run in and displace the air4 ,a ring of cement should be put on as early as possible afterwards4 ,the bottle in which the balsam is preserved must be very carefully dried before being filled and then rinsed out with absolute alcohol1 and afterwards with xylol4 ,turpentine or benzol are often used instead of xylol in the preparation of the medium1 and in the same proportion1 but the latter is less apt to dissolve out the aniline colours from the sections4 ,to mount sections in ,canada balsam they must be transferred first to a watch glass containing absolute alcohol or an alcoholic solution of some staining reagent1 e4g41 eosine 7p4 #gb7 and left in it1 no attempt being made to spread it out1 until it is perfectly dehydrated1 i4e41 in about two minutes4 ,it should then be transferred to the clarifying oil on a mounted needle1 or on a section lifter1 which must be perfectly dry as any spot of moisture that gets on to the section will resist the clarifying action of the oil1 and will cause unsightly opaque areas when the section is mounted4 ,even breathing on the section on its way to the clarifying agent will prevent uniformity of clearing4 ,should white spots appear in the section while in the oil it must be taken out with as little oil as possible1 and again dehydrated in absolute alcohol4 ,the process of 99clarifying99 must be performed in some medium in which ,canada balsam is readily soluble1 and which is also readily miscible with alcohol4 ,those most frequently employed are oil of cloves1 xylol1 oil of bergamot1 oil of cedar1 and origanum oil4 ,the first named has always been much used because of its agreeable odour1 its cheapness1 and the ease with which it can be obtained4 ,but it has the disadvantage of dissolving out many important staining reagents1 especially eosine and the various aniline colours4 ,in addition as it dissolves celloidin1 sections cut in this medium tend to fall to pieces when transferred to oil of cloves1 and one of the other oils 7which have no solvent action on celloidin7 should always be employed with celloidin sections4 ,oil of bergamot is the most generally useful1 but rather expensive4 ,where there are special reasons for employing other dehydrating agents1 they will be indicated in the special directions for particular staining methods in ,chapters ,,vi4 and ,,vii4 ,as soon as the section is plunged into the oil1 the alcohol rapidly diffuses out1 so that the edges of the section fly out with it1 and the section floats quite flat on the surface of the oil4 ,when it is completely clarified 7in about a minute71 as shown by its sinking in the oil1 it should be transferred to the slide by the section lifter1 and the oil drained off4 ,excess of oil may be removed by pressing gently on the section with a flat piece of filter paper folded several times4 ,if carefully performed this manuvre will not injure the section1 but it requires practice4 ,if the tissue is very delicate1 and likely to be injured by changing from one vessel to another1 or if it is larger than the section lifter will conveniently carry1 it should be floated out on a glass slide1 and1 as much water as possible having been removed by blotting paper1 should be dehydrated by adding a little alcohol from a pipette once or twice4 ,most of the alcohol should then be removed by tilting the slide1 and before the remainder has evaporated1 some oil of cloves or bergamot should be added from another pipette4 ,the section will float on the oil at first1 but the latter will gradually come through and appear on the top of the section4 ,when this occurs the clarification is complete1 and the oil may be run off by tilting the slide and the section mounted in ,canada balsam4 99,cementing of cover-glasses499--,the cover-glasses may be cemented down to prevent their shifting and spoiling the specimen4 ,if the cover-glass be circular1 a ,shadbolt's turntable should be used4 ,it consists simply of a horizontal heavy brass disc1 rotating easily on a pivot4 ,there are a number of circles traced on the disc concentrically4 ,the slide is then fixed on the disc by means of the clips1 so that the circumference of the cover-glass corresponds to one of the circles4 ,the disc is then rotated and the cement applied to the edge of the cover-glass with a brush4 ,many materials are employed4 ,the most suitable are3--7#a7 ,canada balsam1 which is almost colourless and transparent and looks very neat4 7#b7 ,gold size4 7#c7 ,marine glue4 ,when these are dry a finished appearance may be given to the slide by laying on a ring of zinc white4 ,this is made as follows3-- ,oxide of zinc #a_/b drachm4 ,benzole half an ounce ,gum dammar of each4 99,preservation of sections499--,they should be kept flat1 and preserved from both light and dust4 ,very useful cardboard trays are now sold by almost all dealers in boxes made to contain twenty-four dozen slides for about eight shillings1 or suitable cabinets may be constructed by a carpenter4 ,,chapter ,v4 ,,general ,,staining ,,methods4 ,much information may be obtained from unstained sections1 and in most cases one section should be examined unstained1 but the specimens mounted in this way are so transparent that it is difficult to study the details of the tissue4 ,they are therefore usually prepared by treating them with some staining reagent1 not merely to render them less transparent1 but also to 8differentiate0 the elements of the section1 by staining one part more deeply than another1 or of a different colour4 ,thus hmatoxyline stains the nuclei and rapidly growing parts of the tissue1 leaving the formed material1 as a rule1 much more lightly tinted4 ,methyl violet again stains healthy tissues blue1 and parts affected with waxy degeneration a red-violet colour4 ,by combining stains also much differentiation of the tissue elements may be obtained4 ,sections should be stained with several reagents1 as their effect on individual specimens varies a good deal4 ,the following are the most useful stains for general purposes3-- 99,logwood499--,this or its purified principle hmatoxyline is the most useful general stain4 ,the hmatoxyline itself is preferable1 giving more constant results1 and less diffuse staining4 ,for general staining purposes the following formula will be found to give excellent results3--99,hmatoxyline4 ,schuchardt's formula499-- 7a7 ,hmatoxyline #c grms4 #cj grs4 ,absolute alcohol #af c4c4 #b-a_/b drms4 7b7 ,pure alum #c grms4 #cj grs4 ,distilled water #ajj c4c4 #b ozs4 ,add 7a7 to 7b7 drop by drop and with constant agitation4 ,keep for some days exposed to diffuse daylight until its colour is so deep that it will not transmit the light4 ,it should then be filtered1 and a crystal of thymol added4 ,it will not give very satisfactory staining reactions at first1 and should be allowed to ripen at least a month or six weeks before using4 ,it improves as a dye with every month that it is kept4 ,whenever hmatoxyline has been made up with alum as in the above formula1 an abundant reddish-brown precipitate forms after some time4 ,this in no way interferes with the activity of the solution1 but it must always be filtered before being used4 99,barrett's formula499--,introduced by ,dr4 ,w4 ,h4 ,barrett1 of ,belfast4 ,it gives almost as good results as the above4 ,it is made from ordinary ,english extract of logwood1 and is considerably cheaper4 ,the extract should be dried1 and finely powdered1 and then extracted with absolute alcohol for several days4 ,powdered extract of logwood #b grms4 #a-a_/b drms4 ,absolute alcohol #aj c4c4 #a oz4 ,filter and add slowly to ,benzoate of sodium #a grm4 #cf grs4 ,alum #a grm4 #cf grs4 ,distilled water #ajj c4c4 #aj ozs4 ,the strength of the solution will vary with different samples of logwood and must be estimated by trial4 ,this solution is comparatively cheap and is useful for class purposes4 99,ehrlich's hmatoxyline499--,this very useful nuclear stain is made as follows3-- 7a7 ,hmatoxyline #b grms4 #i grs4 ,absolute alcohol #ajj c4c4 #b ozs4 7b7 ,glycerine #ajj c4c4 #b ozs4 ,distilled water #ajj c4c4 #b ozs4 ,alum #abj grms4 #b-a_/b ozs4 ,glacial acetic acid #e c4c4 #bd mins4 ,add 7a7 slowly to 7b7 with constant agitation4 ,allow to ripen in sunlight for two months before using4 ,it may be employed as a rapid stain undiluted but far better results are obtained by using a weak solution1 a few drops to a watch-glass full of distilled water1 and staining slowly for from half an hour to two hours4 ,the solution improves by keeping4 ,if after a time the staining becomes diffuse it is an indication that the acetic acid has evaporated1 and a few drops more should be added4 99,kleinenberg's hmatoxyline499--,this formula differs from the previous one in being an alcoholic solution4 ,the calcium chloride is added because it 8sets up diffusion currents between the alcohol in the material to be stained and the alcoholic staining solution1 so enabling the latter to penetrate more rapidly0 7,squire74 ,it is much used in staining embryonic specimens in bulk before embedding in paraffin1 and was strongly recommended for that purpose by ,foster and ,maitland ,balfour4 ,various formul have been given from time to time4 ,that advised by ,squire 7,methods and ,formul1 p4 #be7 can be accurately made up without much difficulty4 7a7 ,crystallised calcium chloride #bj grs4 #a_/b oz4 ,distilled water #aj c4c4 #b drms4 7b7 ,alum #c grms4 #cb grs4 ,distilled water #af c4c4 #agj mins4 ,mix and add ,rectified spirit #bdj c4c4 #h ozs4 ,allow it to stand and any excess of calcium sulphate1 c41 to separate4 ,filter and add ,hmatoxyline #b-a_/b grms4 #be grs4 ,a little thymol should be added as a preservative4 ,in making up these solutions care must be taken that only distilled water is used1 and that all the vessels employed have been previously rinsed out with it1 otherwise precipitation of the hmatoxyline will occur4 ,should sections be overstained in hmatoxyline1 this may be remedied by washing it in a half per cent4 solution of acetic acid1 until sufficient of the stain is discharged1 but the staining is more diffuse than if the happy mean had been hit in the first instance4 ,hmatoxyline stains the nuclei of the cells a beautiful violet colour1 and also tints1 more or less lightly1 the cell protoplasm and the fibrous elements4 ,it also stains the axis cylinders of nerves1 and is much used in special staining of the nerve centres as will be described later1 7pp4 #hh-ia74 ,the stain is permanent4 ,sections may be mounted either in ,farrant's solution1 or in ,canada balsam1 the latter being preferable4 99,eosine499--,much more satisfactory results are obtained from the commercial eosine 7an amorphous orange powder used in dyeing and in the manufacture of red ink71 than from the pure crystalline form4 ,it may be used as an aqueous solution 7#a_/cj per cent47 or as a solution in absolute alcohol 7#a_/ae per cent474 ,sections stained in the former should be rapidly passed through a one per cent4 solution of acetic acid in order to 8fix0 the stain1 and then washed in distilled water4 ,it is a very transparent stain1 and the most delicate details of a section stained with it are perfectly visible4 ,it stains the nucleus but slightly1 while it stains the cell protoplasm and fibrous tissues and especially muscular tissues a beautiful rose colour4 ,it will be seen1 therefore1 that it stains those parts which are left unstained by hmatoxyline1 and vice vers4 ,this complementary action is applied in the following method4 99,double staining with eosine and hmatoxyline499--,sections having been stained in hmatoxyline in the ordinary way1 are washed in distilled water1 and dehydrated in a solution 7about #a in #aejj7 of eosine in absolute alcohol4 ,they should remain in this for about two minutes1 and then be passed through oil of cloves and mounted in ,canada balsam in the ordinary way4 ,this method gives extremely useful and beautiful results with almost all tissues1 and is superior to picrocarmine for differentiating the tissue elements4 ,thus1 the nuclei are stained violet1 the cell protoplasm a much paler and warmer violet1 the fibrous tissues pink1 and red blood corpuscles orange or brick red4 ,the alcoholic solution of eosine is also used as a contrast stain after staining for micro-organisms with blue or violet dyes4 99,carmine499--,it is made as follows3-- ,carmine 7best7 #b #a drachm4 ,strong ammonia #b #a drachm4 ,distilled water #ajj #f ounces4 ,rub the carmine with a little water in a mortar1 add the ammonia1 when the liquid will turn black4 ,gradually add the rest of the water1 rubbing it up all the time4 ,it should be bottled1 allowed to stand for a few days1 and then filtered1 and a piece of camphor put in the bottle4 99,lithium carmine99 resembles closely ammonia carmine in its staining effects4 ,it is usually a matter of individual preference which is employed4 ,carmine #b-a_/b grms4 #aj-a_/b grs4 ,saturated aqueous solution of lithium carbonate #ajj c4c4 #b ozs4 ,dissolve and filter4 ,sections may be sufficiently stained in either of these fluids in from three to five minutes1 but more satisfactory results are to be obtained by diluting with twenty times the bulk of distilled water1 and leaving sections to stain for twenty-four hours4 ,after staining in carmine the sections must be passed through a half per cent4 solution of acetic acid1 in order to fix the carmine in the tissues1 as otherwise the water will dissolve the stain out4 99,borax carmine99-- 7a7 ,borax #d grms4 #c drachms4 ,carmine #b grms4 #a-a_/b drachms4 ,distilled water #ajj c4c4 #e ounces4 ,dissolve with the aid of heat and add slowly to 7b74 7b7 ,alcohol #gj c4c4 #c-a_/b ounces4 ,distilled water #cj c4c4 #a-a_/b ounce4 ,allow to stand for a fortnight4 ,filter1 and add a lump of camphor4 ,to use it1 place sections1 or the tissue in bulk1 in it for from four to twenty-four hours1 according to size1 and then transfer to alcohol 7seventy per cent47 containing a drop to the ounce of hydrochloric acid1 for twenty-four hours1 and then wash thoroughly in water4 ,the tissue may then be placed in gum if it is to be frozen1 dehydrated in alcohol if paraffin or celloidin is to be employed4 ,its advantage is that it is very diffusible1 and so can be used to stain tissues in bulk4 ,it takes a considerable time to stain sufficiently deeply1 but there is little fear of overstaining4 ,it stains nerve-cells and axis cylinders brightly1 and also the connective tissue1 bringing a sclerosed patch out very prominently4 99,alum carmine993-- ,alum five per cent4 solution in distilled water #ajj c4c4 #a oz4 ,pure carmine #a grm4 #d-a_/b grs4 ,boil for twenty minutes4 ,filter4 ,add a few drops of carbolic acid4 ,in using this reagent it should be filtered into a watch glass1 and the sections placed in it for at least an hour4 ,there is no fear of overstaining1 and they may be left all night4 ,after they have been stained they must be thoroughly washed in water to remove the alum1 otherwise numerous crystals of it will be seen in the field when the section is mounted4 ,sections may be mounted in ,farrant's solution or in ,canada balsam4 ,the staining effect improves very much after the section has been kept a few days4 ,if desired its staining action may be complemented by dehydrating it in an alcoholic solution1 either of eosine 7#a in #aejj7 or of picric acid1 and then clearing up in oil of cloves1 and mounting in ,canada balsam4 ,by itself it gives a stain very like that of hmatoxyline1 only warmer4 ,it picks out the nuclei and axis cylinders of nerves1 stains cell protoplasm slightly1 and the fibrous elements scarcely at all4 ,it may be used for the same purposes as hmatoxyline4 ,the colour is less attractive1 and not so deep as that of the latter1 but as it does not overstain sections1 even when left in it for a week1 it is a very convenient stain for general purposes4 ,it is particularly useful as a contrast stain for sections of brain and spinal cord1 after the ,weigert-,pal hmatoxylin process 7p4 #hh74 99,ammonia-picrocarmine99 was formerly very largely used as a staining reagent4 ,its place has now to a large extent been taken by lithio-picrocarmine4 ,in its preparation the best carmine must be used4 ,it is made as follows3-- ,carmine #a part4 ,liq4 ammon4 fort4 #c parts4 ,distilled water #c parts4 ,dissolve with gentle heat1 and add ,cold saturated aqueous solution of picric acid #bjj parts4 ,bring the mixture to the boiling point1 and then place in a shallow vessel1 covered with a glass plate1 and leave it in full sunlight for a month or more4 ,filter1 bottle1 and add six drops of carbolic acid to each ounce of the mixture4 ,it will keep indefinitely and improves with age4 ,it requires filtering from time to time1 as a gelatinous crimson mud tends to deposit from the solution4 99,lithio-picrocarmine499--,prepared as follows3-- ,carmine #be grms4 #aj grs4 ,saturated solution lithium carbonate #ajj c4c4 #a oz4 ,dissolve1 and add ,saturated solution picric acid #bej c4c4 #b-a_/b oz4 ,add a few drops of carbolic acid to each ounce4 ,it should stand a day or two in sunlight and then be filtered4 ,it improves by keeping4 ,it should be kept in a stoppered bottle with a glass rod fused into the stopper4 ,when sections are to be stained they are to be floated out on a clean glass slide as described on page #ee4 ,the slide should then be tilted to allow the water to drain off1 and superfluous moisture round the section removed by a soft rag1 or blotting paper4 ,a drop or two of the stain should then be transferred to the slide1 which should be left lying quite flat for about ten minutes4 ,unless the room is very warm it is advisable to heat the slide very gently over a spirit lamp1 as this causes the tissues to stain more brightly and more rapidly4 ,the excess of the picrocarmine should be allowed to run off the slide1 and the latter wiped4 ,some of the stain should1 however1 be left on the section1 as its effects go on increasing1 and are often not fully seen until a few weeks have elapsed4 ,they should be mounted in ,farrant's medium4 ,as a rule those mounted in ,canada balsam do not give such good results4 ,should there be special reasons for using this medium1 as in mounting spinal cord sections1 c41 they should be dehydrated after staining in picrocarmine in an alcoholic solution of picric acid 7one part of a saturated alcoholic solution to five of alcohol71 before clarifying in oil of cloves1 as otherwise the alcohol will dissolve out the picric acid1 and much of the differential staining effect will be lost4 ,the nuclei should be stained a bright crimson1 the protoplasm of the cells yellow1 or a dull pink1 the fibrous elements a bright pink1 red corpuscles green1 and all dead material1 e4g41 caseous matter1 bright yellow4 ,it also stains nerve-cells1 and the axis cylinders of nerve fibres very brightly4 ,it is1 however1 a rather uncertain dye4 ,the results are most brilliant in the case of fresh sections4 99,osmic acid99 is invaluable for staining fatty particles in the cells4 ,for ordinary use the one per cent4 stock solution 7p4 #ba7 should be diluted with ten times its bulk of distilled water1 and sections stained in it all night in a dark cupboard1 or the watch glass containing them may be placed inside a small box4 ,the sections must be washed thoroughly in plenty of water4 ,if desired they may be stained subsequently in picrocarmine or methyl violet if waxy degeneration also be present4 ,sections should be mounted in ,farrant's solution1 as ,canada balsam usually gives disappointing results4 ,it demonstrates the most minute fatty particles in degenerating cells1 c41 staining them black4 ,it may be employed to demonstrate the globules of fat blocking up the vessels in fat embolism4 ,it stains the myelin sheaths of nerves black1 and will be again referred to when speaking of methods of staining the spinal cord4 99,nitrate of silver99 is employed for staining the intercellular cement of epithelial cells4 ,it stains this substance a deep black1 while the rest of the tissue takes on a brown colour4 ,it is used as a half per cent4 solution in 99distilled99 water1 and kept in a stoppered bottle carefully covered up with brown paper4 ,to use it take some epithelial tissue1 e4g41 the omentum from a recently killed animal1 or a section of some epithelial tumour1 immediately after excision4 ,wash thoroughly in distilled water to remove all chlorides1 and then place in a watch glass containing the silver solution4 ,keep this in the dark for half an hour and then wash thoroughly in plenty of water4 ,the section should be mounted in glycerine or ,farrant's medium and kept from the light or it will become too darkly stained4 99,chloride of gold99 is employed to demonstrate the peripheral terminations of nerves4 ,it can only be employed within the first half hour after the tissue has been removed from the living body4 ,the pieces of tissue must be small and may be stained in bulk1 sections being subsequently made4 ,a half per cent4 solution in distilled water is employed4 ,the tissue is transferred to this on its removal from the body1 until it becomes lemon coloured4 ,it is then exposed in a one per cent4 solution of acetic acid to a strong light until it assumes a purplish tinge1 which takes from two hours to two days4 ,sections should be mounted in ,farrant's medium4 ,it stains the cells of the tissue1 and nerve cells reddish purple1 and nerve fibrils1 especially the terminal ones1 rather more violet4 ,this is very well seen in the cornea4 ,it is useful sometimes for clinical purposes to excise a portion of muscular tissue and examine the nerve endings by this method4 ,unfortunately the stain is somewhat uncertain in its action4 ,better results are obtained by ,sihler's chloral hmatoxyline method 7p4 #ib74 99,methyl violet499--,a very satisfactory solution may be obtained ready made in the 8telegraphen tinte10 prepared by ,leonhardi1 of ,dresden1 as recommended by ,woodhead4 ,it may also be used as a one per cent4 solution in distilled water1 a few drops of carbolic acid being added to prevent the growth of fungi4 ,it is a very useful selective stain4 ,it gives two reactions1 red violet1 and blue violet4 ,thus it stains the matrix of hyaline cartilage blue violet1 but the cells red violet4 ,it has also a most important pathological application1 as it picks out any parts which have undergone 8waxy0 or 8lardaceous0 degeneration1 staining them red violet1 but the rest of the section blue violet4 ,about ten drops of a one per cent4 solution should be filtered into a watchglassful of water1 and the sections stained for about five minutes4 ,they must then be passed through a half per cent4 solution of acetic acid and washed 99thoroughly99 for some time in a large quantity of water till no more colour comes away4 ,if these steps are not taken with care1 the dye will diffuse out after the section has been mounted1 blurring all details and spoiling the appearance of the section4 ,sections may be mounted in ,farrant's solution 7to which a spot of formic acid may be added73 if mounted in ,canada balsam the sections must be overstained as both the alcohol and oil of cloves rapidly dissolve out the dye4 99,safranine499--,employed as a freshly made saturated solution in aniline oil water warmed to #fj ,c4 7#adj ,f474 ,filter into a watch glass4 ,stain for not more than a minute4 ,dehydrate in alcohol which will remove much of the stain1 clarify in oil of cloves or origanum oil4 ,mount in balsam4 ,another method is to stain for about ten minutes1 and then leave for a minute in ,gram's iodide solution4 ,the sections are then washed in alcohol1 dehydrated1 clarified in oil of cloves1 and mounted in balsam4 ,by these methods the stain is withdrawn except from certain elements1 e4g41 those undergoing colloid or calcareous degeneration4 ,a quarter per cent4 watery solution is sometimes employed as it stains nucleoli and actively dividing nuclei very brightly1 while the rest of the cell is stained faintly4 ,it may be employed to study karyokinesis in the cells of a rapidly growing cancer4 99,ehrlich-,biondi stain499--,this stain has been much employed for staining specimens of blood1 for studying karyokinesis1 and for investigations on the supposed parasitic bodies found in cancer cells4 ,it is prepared by mixing saturated aqueous solutions of the following aniline dyes1 slowly and with constant agitation3-- ,solution of ,orange ,g #ajj parts ,rubin ,s #bj ,methyl ,green ,,oo #ej finally add ,distilled water #gj ,filter from the copious precipitate which forms4 ,the solution must be made up frequently as it does not keep well4 ,sections may be stained rapidly for half an hour or an hour1 but better results are obtained by diluting the fluid with twenty volumes of water1 and staining all night4 ,sections should be washed in water and then passed rapidly through absolute alcohol and xylol and mounted in ,canada balsam4 ,,chapter ,,vi4 ,,special ,,staining ,,methods4--,,special ,,methods ,,for ,,staining ,,the ,,nerve ,,centres4 #a4 ,for staining 99nerve fibres994 ,three methods 7two of which are modifications of the first7 are employed far more often than any others4 ,by these methods the myelin coating is stained4 ,tissues must have been hardened previously for many weeks in ,mller's fluid1 or some other bichromate solution4 ,they are then overstained in a solution of hmatoxyline1 and the section treated with a suitable bleaching reagent1 when the colour is discharged from all the tissue elements except the nerve fibres4 ,this method displays not merely the nerve fibres in the white matter1 but also the fine network in the grey matter of the brain and spinal cord4 ,degenerated fibres are left unstained and so degenerated tracts shew up as unstained spots on a dark background4 ,the sections may be subsequently stained with alum carmine or eosine to shew the cells and neuroglia4 99,weigert's method499--,the piece of cord to be cut after prolonged hardening in ,mller's fluid is transferred without washing to absolute alcohol and dehydrated preparatory to embedding in celloidin 7p4 #cj74 ,when sections are cut they are transferred at once to ,weigert's hmatoxyline solution3-- ,hmatoxyline #a #d grains4 ,alcohol #aj #de minims4 ,distilled water #ajj #a ounce4 ,they are stained in this for twenty-four hours or longer1 until they are quite black4 ,the staining will take place much more rapidly if the fluid be kept at #ajj ,f4 in the incubator4 ,after staining they are transferred to ,weigert's differentiating solution3-- ,borax #b #afj grs4 ,potassium ferricyanide #be #bjj grs4 ,distilled water #ajj #a pint4 ,they are left in this solution for several hours1 until the ground work becomes nearly decolourised4 ,sections will sometimes stain more satisfactorily if they are treated1 according to ,weigert's original directions1 for a few hours with a half saturated solution of acetate of copper4 ,if the hardening in ,mller's fluid has been sufficiently prolonged1 this step is usually superfluous4 99,pal's modification99 of ,weigert's method4--,by this method quicker and more complete decolouration of the neuroglia1 nerve cells1 c41 is obtained4 ,sections are prepared in exactly the same way as in ,weigert's method and then transferred to ,weigert's hmatoxyline4 ,pal recommends that this solution be diluted to half the strength and a few drops of a saturated solution of lithium carbonate added4 ,the writer finds the results equally good if the ordinary ,weigert solution be employed4 ,when the sections have been thoroughly stained they are washed in distilled water and placed in a three-quarter per cent4 solution of permanganate of potassium4 ,the time required in this solution depends on the time the specimen has been in ,mller's fluid4 ,it should not be less than half a minute1 and in very thoroughly hardened specimens1 five minutes may be allowed with advantage4 ,in this solution the sections will become of an opaque brown colour4 ,they are washed in distilled water1 and transferred to3-- 99,pal's differentiating solution499 ,potassium sulphite #a grm4 #dj gr4 ,oxalic acid #a grm4 #dj gr4 ,distilled water #bjj cc4 #a pint4 ,they are kept in this for one to five minutes1 according to the depth of staining1 until the white and grey matter are clearly defined and the brown colour is completely discharged4 ,if the brown stain does not readily clear up1 the section should be returned to the permanganate solution for about half a minute1 and again treated with 8,pal's solution40 ,this manuvre may be repeated several times4 ,as soon as the sections are thoroughly differentiated they are transferred one by one to a large vessel of water and thoroughly washed4 ,the blue stain of the hmatoxyline becomes brighter during the washing process4 ,the sections may be mounted at once1 but more beautiful results will be obtained if they are stained in alum carmine for #bd hours4 ,they should then be washed1 dehydrated in alcohol1 clarified in oil of bergamot1 and mounted in ,canada balsam4 ,the very prolonged hardening in ,mller's fluid which is a necessary preliminary for this method led to the introduction of3-- 99,schfer's modification99 of ,pal's method4--,in this method hardening in ,mller's fluid for three or four weeks is sufficient4 ,the sections are made exactly as in the previous method1 and transferred to ,marchi's fluid 7p4 #bd7 for six hours4 ,they are washed and stained all night in the following3-- ,hmatoxylin #a #d grs4 ,alcohol #aj #de min4 ,acetic acid 7#b per cent4 aqueous solution7 #ajj #a oz4 ,the subsequent processes of differentiation1 bleaching1 c41 are exactly the same as in ,pal's method4 99,osmic acid499--,employed with fresh and also with hardened specimens to demonstrate the medullary sheath4 ,much the best results are obtained with the former4 ,the nerves1 or small pieces of the central nervous system are placed in half to one per cent4 solution of osmic acid as soon as possible after death and kept in the dark for about a week4 ,the tissue must be very thoroughly washed in running water to remove all traces of osmic acid1 and then stained for a couple of days in borax carmine to demonstrate the nuclei and axis cylinders4 ,sections may be made in gum1 or the tissue may be teased with needles and then mounted in ,farrant4 ,embedding in celloidin1 and mounting in balsam are inadvisable1 because the ether tends to dissolve out myelin1 and the clarifying oil to render it too transparent4 #b4 99,intra-muscular ramifications of nerves993-- 99,sihler's chloral hmatoxyline method499--,this method reveals the intra-muscular nerve-endings1 and also brings into prominence the curious 8muscle spindles0 which ,sherrington has shown to be connected with the posterior nerve roots1 and which are believed by some to be the end organs subserving muscular sense4 ,a piece of muscle is taken as soon as possible after death1 or from an amputated limb1 and slices cut about one-tenth of an inch thick with the freezing microtome4 ,transfer for twenty-four hours to the following solution3-- ,acetic acid #a part4 ,glycerine #a ,one per cent4 aqueous solution of chloral hydrate #f parts4 ,the tissues swell up in this fluid and become translucent and gelatinous in appearance4 ,they are now placed in pure glycerine until saturated as shown by their sinking to the bottom of the dish4 ,this usually takes several days4 ,they may now be stained in the following solution3-- ,ehrlich's hmatoxyline 7p4 #gj7 #a part4 ,glycerine #a ,one per cent4 aqueous solution of chloral hydrate #f parts4 ,they may be left in this from three days to a week with little fear of overstaining4 ,portions may then be teased with needles1 and mounted in glycerine1 or the stained tissue may be pressed out into a sufficiently thin layer by squeezing it forcibly between two glass slides4 99,motor and sensory nerve endings499--,these are best stained by the chloride of gold method 7p4 #hb74 ,specimens must be taken from the body immediately after death4 ,the method is therefore useless for the post-mortem room1 but may be used for tissues removed by operation4 ,small pieces of tissue must be employed and must be stained in bulk1 sections being made subsequently4 ,for motor nerve endings the muscle of a frog or human muscle from a limb just amputated may be taken4 ,specimens should be prepared after staining by teasing in preference to making sections4 ,mount in ,farrant4 ,sensory nerve endings may be conveniently studied in the cornea of a recently killed frog or rabbit1 or in a freshly extirpated human eye4 ,tactile end organs may be studied in the lip or finger tips1 taste buds in the papilla foliata of the rabbit's tongue1 and ,pacini's corpuscles are well seen in the mesentery of a thin cat4 #c4 99,staining nerve cells499 99,bevan ,lewis's aniline blue-black method993--,this method is the best for demonstrating the wealth of nerve cells in the fresh cerebral cortex4 ,the solution of aniline blue-black should be of the strength of #a in #djj1 about a grain to the ounce4 ,a piece of the cerebral cortex with pia mater attached1 should be removed as soon as possible after death by parallel cuts about #a_/h inch apart1 and perpendicular to the surface of the convolution1 placed on the plate of the freezing microtome and just frozen--not too hard or the tissue will be brittle and will also injure the edge of the razor4 ,as soon as a good section is obtained the razor should be plunged into a large bowl of cold water to detach the section1 which is at once floated on a glass slide1 and osmic acid solution1 #a_/d per cent4 allowed to flow over it from a pipette4 ,this will fix the tissue elements in about two minutes4 ,the section is again floated off into the bowl of water and thoroughly washed to free it from the osmic acid4 ,it is then stained either on the slide1 or in a watch-glass1 with the aniline blue-black solution for an hour in the cold1 or half-an-hour if the solution is slightly warmed4 ,the dye is thoroughly washed away with distilled water1 excess of moisture wiped off the slide with blotting paper1 and the section allowed to dry under a glass bell jar4 ,it is not practicable to dehydrate by means of alcohol as it would cause sudden shrinking of the tissues4 ,when the section is dry a drop of ,canada balsam is applied and it is covered with alcohol in the usual way4 ,the nerve cells and their processes are stained a deep slate colour1 as are the nuclei of the connective tissue cells1 while the ground work of the neuroglia is faintly stained and of a neutral grey tint4 ,this method gives beautiful results both with normal and morbid specimens4 ,various other aniline dyes1 indulin1 methylene blue1 gentian violet1 have been employed in the same way1 but none of them give such good or uniform results as aniline blue-black4 ,hardened specimens may also be stained with aniline blue-black1 but the results are not to be compared with those obtained by the fresh method4 ,the stain is usually diffuse1 but this can be improved by placing the sections for #a_/b - #b minutes in a #b per cent4 solution of chloral hydrate in distilled water after staining4 99,golgi's metallic stains99 for nerve cells4 ,golgi introduced the methods of producing a metallic deposit of mercury or silver in the nerve cell1 revealing both the cell and its processes4 ,this method has been very fruitful in discoveries1 especially in the hands of ,ramon y ,cajal1 ,kllicher1 ,van ,gehucten and others4 ,it gives best results with embryonic tissues4 ,to ensure good results it is important that the tissue be removed immediately after death4 ,sections of brains removed some hours after death usually give disappointing results4 ,there are several methods now in vogue1 all slight modifications of ,golgi's original methods4 99,silver nitrate method499--,small pieces not more than a quarter of an inch cube1 are transferred straight from the body to a large quantity of ,marchi's fluid 7p4 #bd7 and kept in it for about a week1 or longer in the case of adult specimens4 ,on removal from ,marchi's solution the tissue should be washed for a few seconds in distilled water1 and then placed in a large quantity of a #c_/d per cent4 solution of nitrate of silver solution in distilled water for at least a week4 ,the lump of tissue becomes of a brick red colour owing to a coating of silver chromate4 ,on removal from the silver solution the tissue should be washed in methylated spirit for a few minutes and the incrustation of silver chromate brushed off4 ,sections may be cut in gum and celloidin2 or they may be fixed on a cork with celloidin or spirit varnish and cut without embedding3 very thin sections are not required4 ,dehydrate in alcohol1 clear in xylol1 and mount in balsam4 ,goodall advises a mixture of pyridine and xylol for clearing1 and mounts in strong xylol-dammar solution1 without a cover-glass4 ,very careful attention to details and much practice are required before uniformly good results can be obtained4 ,the results are extremely beautiful and well repay the labour expended on them4 ,the cells and their processes appear black on a yellowish ground4 ,a method has been employed for deepening the colour of the stain1 but the writer has no experience of it4 ,kallus 7,zeitsch4 f4 ,wiss4 ,mikr41 #ahic1 #dgg7 dilutes an ordinary hydrokinone developing solution 7prepared as for developing an ordinary photographic plate7 with about ten times its volume of distilled water4 ,just before using a third part of absolute alcohol is added4 ,sections which have been through the silver process when placed in it become grey or black in a few minutes1 and1 after washing in methylated spirit1 are transferred to a #bj per cent4 aqueous solution of hyposulphite of soda for a couple of minutes and then washed very thoroughly in distilled water for twenty-four hours4 ,they are then dehydrated and mounted in balsam4 99,buckley's modification of the silver method499--,described in ,brain1 ,winter number1 #ahie4 ,the method is applicable to specimens that have been hardened in ,mller's fluid4 ,thin slices are cut in the usual way1 and then immersed in ,bichromate of potassium1 #c per cent4 solution #e parts ,osmic acid1 #a per cent4 solution #a part for three to five days4 ,excess of bichromate is removed from the sections by blotting paper1 and they are transferred to the freshly prepared staining mixture3-- ,phospho-molybdic acid 7#aj per cent47 #a minim #b drops4 ,nitrate of silver 7#a per cent47 #a ounce #fj c4c4 which must not be filtered4 ,stain for several days4 ,the sections should be cut at once after removal from the staining solution4 ,it is claimed that the minute details of structure of the cell processes are better shewn by this method4 99,corrosive sublimate99 method4--,this method is similar in its mode of action to the last1 mercury being deposited in the cell instead of silver4 ,it is rather less certain and requires more practice4 ,it seldom stains uniformly4 ,one cell will be found exquisitely stained while those in its vicinity are unaffected4 ,small pieces of cortex are hardened for several weeks in ,mller's fluid1 or other bichromate solution1 and are then transferred direct to a one-half per cent4 aqueous solution of corrosive sublimate1 in which they should be left from three to six weeks4 ,shorter periods will only give disappointing and inconstant results4 ,sections should be cut1 if possible1 in gum4 ,they may be mounted in ,farrant1 or dehydrated and mounted in balsam4 ,tal has proposed to render the effect sharper by transforming the deposit of mercury into mercuric sulphide1 by treating the sections with a solution of sulphide of sodium1 which he prepares by saturating a ten per cent4 solution of caustic soda with sulphuretted hydrogen and then adding an equal quantity of fresh soda solution4 ,they are stained in this for a few minutes and then thoroughly washed4 ,by this method the pyramidal cells and their delicate processes appear as black opaque objects on a light ground4 ,the neuroglia cells with their fine delicate processes are often also beautifully stained4 99,nissl's aniline method499--,this method is complementary to ,golgi's method4 ,the latter impregnates the cell rendering it opaque and shewing its form with great definiteness4 ,nissl's method stains the protoplasm without greatly reducing its transparency and allows us to study details of cell structure4 ,small portions of tissue1 removed as soon as possible after death1 are hardened in alcohol4 ,sections are then cut1 preferably in gum1 as celloidin is inconvenient owing to its staining so deeply with aniline dyes4 ,sections are transferred from alcohol to a one-half per cent4 aqueous solution of methylene blue1 which is heated in a watch glass till it steams freely1 but short of the boiling point4 ,stain for about a quarter of an hour and allow to cool4 ,transfer the sections to a mixture containing one part of aniline oil and ten of absolute alcohol1 and move them about till no more colour comes away4 ,transfer the section to a slide with a section lifter1 drain1 and dry well by pressing folded filter paper carefully on the section4 ,allow some origanum oil to flow over the section and remove excess of this by pressure with blotting paper4 ,moisten with benzine1#a and add a drop of colophonium resin dissolved in benzine4 ,the slide is warmed cautiously till the benzine is driven off and the colophonium liquefied by heat alone1 and then the cover-glass is applied4 #a ,the student will bear in mind the danger of working with benzine near a naked light4 ,magenta and other aniline dyes may also be employed in a similar manner4 ,,chapter ,,vii4 ,,special ,,methods ,,for ,,staining ,,micro-,,organisms ,,and ,,blood4 ,it is impossible1 within the limits of this work1 to attempt any adequate description of the modern methods of bacteriological investigation4 ,some of these are very lengthy and complicated1 and require much skill and practice before good results can be relied on4 ,but those who do not desire to make a special study of bacteriology may often require to examine for the presence of organisms in sections1 or in various excretions1 and it is hoped that they may find the following short description of special methods sufficient for their purpose4 ,for more elaborate work they must consult one of the many excellent textbooks on the subject4 ,the student should provide himself with the following dyes in powder3-- 99,methylene blue499 99,gentian violet499 99,methyl violet499 99,fuchsine499 99,bismarck brown499 ,the following solutions of these dyes are used3-- #a4 ,saturated alcoholic solutions which may be kept in stoppered bottles4 #b4 ,one per cent4 aqueous solutions4 ,these must be freshly made each time of using4 ,in filtering either alcoholic or aqueous solutions it is well to moisten the filter paper beforehand with alcohol or water as the case may be4 ,the following special solutions will also be wanted3-- 99,lffler's methylene blue499--,in this solution a weak solution of caustic potash is employed as a mordant3-- ,saturated alcoholic solution of methylene blue #c volumes4 ,caustic potash1 aqueous solution #a 3 #aj1jjj #aj volumes4 ,filter4 ,this solution is perhaps the most generally useful stain4 ,it colours most bacilli and micrococci1 and while rapid in its action rarely overstains4 ,it must be made up fresh on each occasion4 ,it is the best counterstain after staining tubercle bacilli1 c41 with fuchsine4 99,ziehl's carbol-fuchsine499 ,carbolic acid 7#e per cent4 aqueous solution7 #ajj volumes4 ,fuchsine 7saturated alcoholic solution7 #aa volumes4 ,the solution must be filtered immediately before being used4 99,gram's iodine solution499--,sections are placed in this solution after being stained with aniline dyes4 ,the iodine in some way fixes the dye in the organisms1 so that they are not decolourised along with the rest of the tissues4 ,it is made thus3-- ,iodine #a grm4 #a-a_/b grains4 ,iodide of potassium #b grms4 #c grains4 ,distilled water #cjj c4c4 #a ounce4 ,ten per cent4 aqueous solutions of 99nitric99 and 99sulphuric99 acids should be prepared and may be kept indefinitely4 ,the following are the general methods of employing these reagents for the purpose of staining organisms in sections4 ,special methods are required for special organisms1 but one or two only can be given4 99,weigert's method499--,the sections must be placed in a freshly made one per cent4 aqueous solution of methyl violet1 gentian violet1 fuchsine1 c4 ,the solution may be kept at the temperature of the body in an incubator4 ,the organisms will often stain more readily if the section be passed through a #a in #bjjj solution of corrosive sublimate before putting it into the staining fluid4 ,after staining the section is washed in distilled water and then in methylated spirit until it appears almost decolourised4 ,some prefer to decolourise the tissues by washing in a half per cent4 solution of acetic acid instead of methylated spirit4 ,practice is required before the correct time for decolourising is accurately estimated4 ,the beginner should float a section rapidly on the slide now and then1 put on a cover-glass and examine it under a low power to see if the decoloration has been carried far enough4 ,a contrast stain may then be used1 such as picrocarmine1 after which the section may be mounted in ,farrant's medium3 or a weak solution of another aniline colour may be used as a counter stain1 after which the section is clarified in xylol1 and mounted in balsam dissolved in xylol4 99,gram's method499--,place some aniline oil in a test tube and add ten times its volume of distilled water4 ,close the end with the thumb and shake very thoroughly4 ,filter ninety drops into another clean test tube1 and add ten drops of a saturated solution of gentian violet or some similar dye4 ,filter the mixture into a watch glass4 ,stain sections in it for from three minutes to half-an-hour according to the temperature1--the shorter time for the incubator at #ajj1 the longer when the sections are stained at the ordinary temperature of the room4 ,wash in distilled water1 and transfer to ,gram's iodine solution until they become black1 usually in a few minutes4 ,they are then decolourised in absolute alcohol4 ,this often takes some time4 ,it may be hastened1 as ,crookshank suggests1 by placing the section in clove oil1 returning to alcohol1 and so on4 99,ehrlich's modification99 of ,gram's method4 ,the contrast stain is here used first4 ,stain the section 7e4g41 that of a mitral valve in a case of ulcerative endocarditis71 in an alcoholic solution of eosine 7#a in #aejj74 ,transfer to a solution of some aniline dye1 such as gentian violet1 dissolved in aniline oil water1 exactly as in ,gram's method4 ,the section floats on the surface and spreads out1 owing to the alcohol diffusing out4 ,stain for about twenty minutes4 ,wash the section in water1 and float out 7p4 #ee7 on a glass slide4 ,allow the water to drain off and add ,gram's iodine slowly from a pipette so as not to disarrange the section4 ,when the section has become quite black pour off the ,gram's solution4 ,remove all superfluous fluid from the slide with blotting paper1 and dry the section by carefully and firmly pressing on it a folded piece of blotting paper4 ,if this is done with care the section need not be injured in the least4 ,decoloration is effected on the slide with aniline oil1 instead of alcohol as in the preceding method4 ,the slide is rocked about so that the colour may be evenly discharged by the aniline4 ,when no more colour comes away1 the aniline oil is poured off1 the section clarified in xylol1 and mounted in ,canada balsam4 ,as soon as the section is decolourised it may be treated with a contrast stain1 the most suitable being alcoholic solutions of eosine or ,bismarck brown if a blue stain has been employed1 or methylene blue if fuchsine has been the first stain used4 ,the following will be found the most useful stains and contrast stains3-- ,,stains4 ,,contrast ,,stains4 ,picrocarmine4 ,gentian violet4 ,eosine4 ,methyl violet4 ,bismarck brown4 ,methylene blue4 ,safranine4 ,magenta4 ,methylene blue4 ,fuchsine4 ,and vice vers4 ,much practice is required in using either of the methods before one can judge accurately how long to leave sections in the staining reagents or decolourising agents1 and the beginner must not be discouraged if at first he is unable to obtain good results although he follows the book directions most minutely4 99,ehrlich method for tubercle bacilli499--,sections are stained for six to twenty-four hours in a one per cent4 solution of gentian violet1 methyl violet1 methyl blue or fuchsine4 ,they will stain more rapidly if the staining fluid be kept in an incubator at the body temperature4 ,they should be removed from the staining fluid1 and washed in distilled water1 and then transferred 7preferably on a glass section lifter7 to a ten per cent4 solution of nitric acid in distilled water until they are nearly decolourised4 ,they should then be very thoroughly washed in distilled water4 ,they may then be treated with some suitable contrast stain and mounted in ,canada balsam4 99,neelsen's stain for tubercle bacilli499--,sections are placed in ,ziehl's carbol-fuchsine solution 7p4 #ajc7 which should be warmed for ten minutes to half-an-hour4 ,they are then decolourised in a solution of sulphuric acid4 ,twenty-five per cent4 is the strength originally recommended1 but a ten per cent4 solution does equally well and injures the section less4 ,they are then very thoroughly washed in a large quantity of water1 and afterwards may be treated with a contrast stain4 99,gibbes' double stain for tubercle bacilli499-- 7#a7 ,rosaniline hydrochlorate #b grms4 #be grs4 ,methyl blue #a grm4 #abe grs4 ,triturate in a glass mortar1 7#b7 ,aniline oil #c c4c4 #cge grs4 ,rectified spirit #ae c4c4 #c-a_/b drms4 ,dissolve and add slowly to 7#a74 7#c7 ,lastly add slowly to the mixture ,distilled water #ae c4c4 #c-a_/b drms4 ,some of the solution is filtered into a watch glass and warmed4 ,the sections are placed in it and left for some hours4 ,they are then washed in methylated spirit till they are sufficiently decolourised1 and then rapidly passed through absolute alcohol and oil of cloves and mounted in balsam and xylol4 ,it is a very useful stain for examining the sputum for tubercle bacilli4 ,in order to stain fluids1 such as blood1 pus1 or sputum1 for organisms1 a very thin layer should be obtained by placing a little of the fluid between two clean cover-glasses and pressing them together4 ,they are then separated and allowed to dry4 ,the film is fixed by holding the cover-glass in a pair of forceps1 and passing it slowly through the flame of a spirit lamp two or three times4 ,films of pus should be #8cleared' after fixing by placing them in a twenty per cent4 solution of acetic acid for three minutes4 ,for clinical purposes it is often necessary to examine urine1 fces or vomited matter for bacilli4 ,films are prepared in the usual way and allowed to evaporate slowly1 and then fixed by passing through the flame1 and then washed in distilled water before staining4 ,in the case of vomited matter and fces this is usually done without difficulty4 ,in the case of urine however it is often difficult to get the urine to evaporate completely4 ,a syrupy layer remains1 and if more heat be applied it decomposes and chars1 and the products cause precipitation of aniline during subsequent staining processes4 ,this may be partly avoided by gently washing the film in distilled water before staining4 ,another plan is to mix the urinary deposit with a little gelatine free from organisms1 such as that in unused culture tubes4 ,the gelatin is liquefied by heat1 and mixed with the deposit4 ,films are made from this mixture1 and allowed to set1 and then thoroughly washed in distilled water4 ,the film is then dried thoroughly1 and the cover-glass laid flat with the film uppermost1 and a few drops of the staining fluid filtered on to it4 ,after it has been stained sufficiently the stain is drained off1 and the slip gently washed4 ,the film may then be stained with some contrast stain in exactly the same way as sections1 again washed1 dried between folds of blotting paper1 and mounted in balsam4 ,it is sometimes difficult to tell which is the side of the cover-glass which bears the film4 ,this is readily done by holding the glass obliquely so that light from a window is reflected from its surface4 ,the side which is coated appears dull2 while the other is smooth and bright4 ,,methods ,,of ,,examining ,,blood4 ,in all these methods blood is obtained by pricking the skin of one of the fingers1 or the lobule of the ear1 preferably the latter4 ,the skin must previously be washed with soap and water or ether1 to remove any grease or epithelial scales4 ,the puncture should be made firmly so that blood may escape freely4 ,the finger or ear must not be squeezed4 ,specimens must be made rapidly before red corpuscles have run into rouleaux4 ,the slides and coverslips employed must be scrupulously clean1 or it is impossible to get really good films4 ,they should be cleaned with nitric acid and alcohol according to the directions on page #eg4 ,fresh specimens should be examined4 ,the coverslip is made just to touch the drop of blood at one edge1 so as to transfer a small quantity only1 and is at once lowered on to the slide with the aid of a mounted needle4 ,if slide and coverslip be perfectly clean the blood will spread out into a thin film1 the corpuscles lying quite flat4 ,if there be any delay1 or if the cover-glass be not quite clean the red corpuscles will run into masses and the specimen will be useless for minute examination4 ,another specimen may be mixed with a little of ,ferrier's solution 7p4 #abi7 before mounting4 ,permanent coverslip films may also be prepared4 ,here again the use of absolutely clean coverslips is essential1 and the blood must be taken immediately it escapes from the puncture4 ,a little blood is taken on a cover-glass which is held horizontally4 ,another cover-glass is lowered on to this and by its weight and by capillary attraction1 the drop of blood quickly becomes transformed into a thin film4 ,the two covers are separated as soon as the film is formed by rapidly sliding them off one another4 ,this manuvre requires a little practice and dexterity4 ,the movement of the slips must be in an exactly parallel direction otherwise the coating left will be uneven1 just as when two pieces of bread and butter are pulled apart4 ,even with practice it is difficult to get more than one good film1 the lower being usually best4 ,there are four ways of fixing the film4 #a4 ,exposure to 99osmic acid vapour994 ,the film while still moist is held over the mouth of a bottle containing at least one per cent4 solution of osmic acid4 ,in a minute or two the fixation will be complete1 and the film becomes of a dirty brown colour4 ,it is then left exposed to the air to get rid of all traces of osmic acid1 and may afterwards be stained as described below4 #b4 ,treatment with 99saturated aqueous solution of corrosive sublimate99 7,muir's method74 ,the cover-glass on which the film has been spread1 is floated before the latter has time to dry1 film downwards on a saturated solution of corrosive sublimate in a watch glass for half an hour4 ,the cover-glass is placed in distilled water and then in alcohol to remove excess of corrosive sublimate1 and then stained4 ,a little care is required when washing the film to prevent it sliding bodily off the cover-glass4 #c4 ,by drying and passing rapidly through the flame of a ,bunsen burner1 exactly as in preparing specimens of sputum1 c4 7p4 #aaa74 ,this method is handy for ordinary clinical purposes4 #d4 ,by keeping the coverslips at a temperature of about #bjj ,f4 7,ehrlich's method74 ,ehrlich uses for this purpose a strip of copper about two inches wide and a foot long which is supported on a retort stand in a horizontal position4 ,one end is heated by a ,bunsen's burner beneath4 ,the point in the copper strip at which the temperature is at boiling point is readily ascertained by dropping a little water on4 ,the point at which a drop of water assumes the spherical state indicates a temperature there of #bab ,f4 ,the coverslips are placed an inch or two further than this point1 and kept there at a temperature of about #bjj ,f4 for some hours4 ,,staining ,,methods4 ,fresh blood may be stained by mixing with ,ferrier's fuchsine solution3-- ,fuchsine #a grm4 ,distilled water #aej c4c4 ,dissolve and add ,alcohol 7#hj per cent47 #ej c4c4 ,neutral glycerine #bjj c4c4 ,a spot of this solution is mixed with the blood on a slide by means of a mounted needle1 and covered with a clean cover-glass4 ,the red corpuscles are slightly stained1 while the nuclei of the white corpuscles are stained a bright crimson1 and the 8blood plates0 a deep pink colour4 ,stained preparations may also be obtained by using 99,toison's fluid991 which serves also for diluting the blood in order to determine the exact number of red and white corpuscles present by means of ,gowers' or the ,thoma-,zeiss hmocytometer4 ,it is prepared thus3-- ,glycerine #cj c4c4 #a oz4 ,sodium sulphate #h grms4 #b drms4 ,sodium chloride #a grm4 #ae grs4 ,methyl violet #be grm4 #d grs4 ,distilled water #afj c4c4 #e oz4 ,it stains the nuclei and blood plates1 but does not alter the shape of the red cells4 ,it requires to be made up fresh occasionally as torul are apt to form and multiply in it4 ,dried films may be stained with hmatoxyline1 picrocarmine1 or any of the general stains4 ,the nuclei of the leucocytes may be stained rapidly in a couple of minutes in a one per cent4 solution of methyl violet1 washing in water1 drying between blotting paper and mounting in balsam4 ,the best method for general purposes is to stain with a saturated aqueous solution of methyl blue for half an hour or longer4 ,wash in water1 and then stain for ten minutes in a half saturated aqueous solution of eosine4 ,in this way the eosinophile granules of the leucocytes and the red corpuscles1 are stained by the eosine1 while the nuclei of the leucocytes are stained by the methyl blue4 ,kanthack and ,drysdale recommend that the film should first be stained with a half per cent4 solution of eosine in #ej per cent4 alcohol1 then washed1 dried and fixed in the flame1 and stained for a short time in ,lffler's solution of methylene blue 7p4 #ajd74 ,these films may be stained for micro-organisms in the way described for cover-glass preparations 7p4 #aab74 ,,chapter ,,viii4 ,,injection ,,of ,,blood ,,vessels4 ,injection of blood vessels may be performed on small animals1 or on individual human organs after removal from the body4 ,the object is to fill the vessels with a coloured fluid which will solidify afterwards4 ,it is possible in the same organ to inject the arteries with a red medium1 the veins blue1 and secretory ducts1 such as bile ducts1 yellow or blue4 ,the most convenient basis for an injection mass is gelatine1 as its solutions liquefy at a temperature of about #ajj ,f41 and solidify a little below that point1 and when solidified cut readily1 and do not tend to become brittle4 ,the various masses are prepared as follows3-- 99,red injection mass99 7,woodhead's formula7 consists of gelatine softened by mixture with water and coloured by carmine4 7#a7 ,carmine #d grms4 ,liq4 ammoni ,b4,p4 #h grms4 ,distilled water #aej c4c4 ,dissolve the carmine in the ammonia in a mortar4 ,pour on the water4 ,mix thoroughly and filter4 7#b7 ,gelatine #aj grms4 ,distilled water #ej c4c4 ,allow it to stand in the cold water until the water is absorbed and the gelatine has become soft4 ,warm 7#a7 almost to boiling point over a ,bunsen burner1 and add the gelatine slowly4 ,stir thoroughly and add a ten per cent4 solution of acetic acid until the solution becomes slightly acid4 ,this will be shewn by the mass assuming a darker and duller colour4 ,a little salicylic acid may be added to preserve it4 99,blue injection mass499--,to the gelatine mass 7#b7 prepared as above1 and liquefied by heat1 add instead of carmine ,soluble ,prussian blue #e grms4 ,distilled water #fj c4c4 ,every trace of alkali must be kept away from the mass during and after the preparation4 ,sections of injected organs should be mounted in ,farrant's solution slightly acidulated with formic or acetic acid4 ,with every care1 however1 the blue colour is apt to fade in the course of time4 99,green injection mass499 ,robin's formula 7modified74 7#a7 ,arseniate of soda 7sat4 sol47 #hj c4c4 ,glycerine #ej 7#b7 ,sulphate of copper 7sat4 sol47 #dj ,glycerine #ej ,mix and add one part to three parts of the gelatine mass made as for the red and blue injections4 99,method of injection499--,in injecting the vessels of tissues it is necessary that the organ or the entire animal1 as the case may be1 shall be kept during injection at a temperature well above that at which the gelatine mass will melt1 otherwise the gelatine will 8set0 in the arteries and will never reach the capillaries4 ,this warming is effected by immersing the animal in a water bath4 ,the liquefied gelatine is forced into the artery by a syringe or by air pressure4 ,it is essential that the pressure be uniform and steady4 ,this is so much more easily managed with air pressure that this method is strongly recommended to the beginner4 ,but1 whatever method be adopted1 perfect results can only be obtained with certainty after long practice4 ,sometimes too high pressure will be employed and the vessels give way1 at others the injection may not reach the capillaries at all4 ,the most scrupulous attention to details is essential4 ,by far the most effective apparatus for injecting is the modification of ,ludwig's constant pressure apparatus devised by ,fearnley4#b ,although the apparatus appears complicated1 the various parts are easily obtained and it would be easy to improvise a substitute for the water bath4 #b 8,practical ,histology10 7,macmillan ,co474 ,the apparatus which is shewn in figures #aj and #aa consists of a bath deep enough to contain the animal1 and a vessel containing the injection fluid4 ,the bath is kept at a temperature of about #aaj by an ordinary ,bunsen burner4 ,a large ,wolff's bottle 7#bj-dj oz47 with three necks1 is fitted with three india-rubber stoppers perforated by glass tubes4 ,through the central stopper a glass tube connected by a rubber tube1 with an ordinary ,higginson's syringe1 passes almost to the bottom of the bottle4 ,from one of the other necks a rubber tube passes to an ordinary mercurial manometer1 while from the third a tube passes to the flask containing the liquefied injection mass1 which is immersed in the water bath4 ,this flask is also firmly stoppered1 and should be about half filled with injection material4 ,the delivery tube from the large ,wolff's bottle should only just come through the cork4 ,another glass tube passes down almost to the bottom of the flask1 and is connected by a rubber tube with the cannula inserted into the artery4 ,it will be evident from figure #aa that when water is pumped by the ,higginson's syringe into the ,wolff's bottle the pressure there will be raised 7as indicated by the manometer74 ,this increase of pressure will equally affect the air inside the bottle containing the injection fluid1 and the fluid will be forced out along the tube and through the cannula into the artery4 ,illustration3 ,fig4 #aj4--;,fearnley's arrangement for injecting blood vessels4 7,reproduced by permission of ,messrs4 ,macmillan1 from ,fearnley's ,practical ,histology74 ,illustration3 ,fig4 #aa4--,scheme shewing distribution of pressure in ,fearnley's ,injection ,apparatus 7from ,fearnley's ,practical ,histology74 ,before using the apparatus a clamp should be placed on the exit tube of the vessel containing the injection fluid1 and the pressure should be raised to see that the apparatus is everywhere air-tight4 ,any leaks should be sealed before the actual injection is commenced4 ,if an isolated organ is to be injected1 a cannula of glass or brass should first be inserted into the artery and securely tied in position4 ,the organs1 if cold1 must be soaked in water at #abj ,f4 for about half an hour and then transferred to a water bath4 ,in the case of injecting an entire animal1 such as a rabbit1 rat1 or guinea pig1 the injection is best made a few minutes after death4 ,the animal may be chloroformed1 and then bled to death by opening a large vein4 ,as soon as death has occurred incise the skin over the thorax in the middle line4 ,cut through the costal cartilages to the right of the sternum1 and through the junction of the manubrium and body of the sternum4 ,these incisions being for most part through non-vascular parts will not lead to escape of fluid during injection4 ,the sternum being forcibly raised towards the left1 the pericardium will be exposed and must be carefully divided4 ,an incision must be made into the left ventricle1 and a cannula passed up into the aorta and firmly secured by a ligature passed round the aorta with the assistance of forceps or an aneurism needle4 ,any blood is cleaned away and the animal is then placed in the water bath for about ten minutes4 ,the tube from the bottle containing the injection fluid is then filled by gentle pressure on the syringe1 and clamped when full4 ,its end is then placed on the cannula and secured there by a ligature4 ,the pressure should be raised by squeezing the syringe until the manometer registers one inch4 ,the clamp should then be removed and the injection commenced4 ,the pressure should be raised very gently and constantly by working the syringe1 and the condition of the gums1 lips1 and eyes of the animal observed4 ,the gums will soon shew a pink tinge4 ,the best indications are obtained by watching the effect on the small vessels of the sclerotic4 ,when these are completely filled1 which will be in about five to ten minutes according to the rate at which the air pressure has been increased1 the injection may be stopped4 ,this result will be obtained1 under good conditions1 before the manometer indicates a pressure of five inches4 ,the aorta should now be ligatured1 and the animal placed in cold water frequently renewed until it is thoroughly cooled4 ,the organs may then be removed and placed in methylated spirit and hardened4 ,sections are afterwards cut and mounted in the usual way4 ,,chapter ,,ix4 ,,directions ,,for ,,preparing ,,individual ,,tissues4 99,normal histology499--,it cannot be too strongly impressed on the beginner that a thorough mastery of the normal appearances of tissues and organs is absolutely necessary before attempting to make an accurate study of morbid changes in them4 ,he should not be satisfied with examining one specimen of an organ but as many as he conveniently can1 in order to be fully acquainted with the many deviations from normal which may exist without actual disease4 ,he should therefore obtain several animals1 such as small dogs1 cats1 rabbits1 frogs1 c41 and remove their organs with all care1 and harden them in the various appropriate fluids4 ,he should also obtain specimens of normal human organs from the post-mortem room4 ,many normal tissues 7skin1 muscle1 tendon1 bone1 c471 can also be prepared from a limb amputated for an accident to a healthy patient4 ,by preparing specimens in this way he will not only become the possessor of a set of slides illustrating normal histology1 but will find also that he has acquired that proficiency in hardening and staining the specimens which practice alone can give4 ,the following account of the method of preparing different tissues is merely intended to indicate the lines on which the beginner should proceed4 ,after some practice he will be quite able to select the modes of hardening and staining which special circumstances or cases may seem to demand4 ,the first part of these directions will refer to the preparation of normal tissues1 the second part to morbid histology4 99,blood499--,for special methods of examination see ,chapter ,,vii4 99,blood crystals--,hmoglobin crystals991 obtained from the blood of an animal1 or enough may be collected at any operation4 ,a little water or a little ether is added to the blood which is allowed to stand for half-an-hour after which a drop is allowed to evaporate slowly on a clean slide4 99,hmatin crystals499--,the student should make himself thoroughly familiar with these1 as their presence affords positive proof of the existence of blood colouring matter in a stain4 ,to obtain them a drop of blood should be allowed to dry on a slide4 ,the dried blood is scraped into a little heap with a small piece of clean glass1 and a drop of glacial acetic acid added4 ,as it evaporates minute reddish-brown acicular crystals will appear4 99,hmatoidin crystals499--,obtained from the site of a bruise1 or an old hmorrhage1 e4g41 a cerebral apoplexy or a hmatocele4 99,simple squamous epithelium499--7,endothelium74 ,carefully strip off the lining of the parietal pericardium or parietal pleura1 of a recently killed animal1 or spread out its omentum on a piece of cork1 and 7#a7 stain the intercellular cement with nitrate of silver 7p4 #hb7 so as to reveal the outlines of the cells4 7#b7 ,stain other specimens with hmatoxyline or alum carmine to reveal the nuclei4 99,stratified squamous epithelium499--,specimens from skin of various parts1 finger1 groin1 lip1 tongue should be prepared4 ,harden in ,mller's fluid4 99,transitional epithelium499--,occurs in the pelvis of the kidney1 ureter and bladder4 ,it is very readily detached1 especially if not hardened immediately after death4 ,remove as early as possible4 ,if the bladder is taken it should be cut open and pinned out as flat as possible4 ,harden in osmic acid1 or ,mller's fluid and spirit4 ,embed preferably in celloidin4 99,simple columnar epithelium499--,occurs in many parts4 ,it may be studied in the salivary ducts1 the intestine1 kidney1 c41 of any mammal4 99,goblet-cells499--,seen abundantly among the columnar cells of the intestinal glands1 and in the mucous glands of the mouth and of the cervix uteri4 99,stratified columnar epithelium499--,occurs only in the urethra4 ,harden the penis of a cat in ,mller's fluid1 and cut transverse sections4 99,ciliated epithelium499--,harden the trachea of a recently killed cat in osmic acid or ,mller's fluid4 ,beautiful specimens may also be obtained from an ordinary nasal polypus1 which should be put into hardening fluid immediately after removal4 ,stain all sections of epithelium in picrocarmine1 and in eosine and hmatoxyline4 99,ordinary areolar tissue499--,difficult to obtain free from fat4 ,it may be studied in the subcutaneous tissue of the section of the cat's penis already made4 ,a fragment of the tissue should also be removed and carefully teased in a drop of picrocarmine4 ,areolar tissue may also be studied in sections of skin1 and in the capsules of the different internal organs4 99,elastic tissue499--,may also be studied in most sections of skin4 ,if the ligamentum nuch of a large quadruped 7horse1 bullock71 c41 is available it yields the best specimens1 or the human ligamenta subflava may be examined4 ,pin a piece out on a piece of wood or wax4 ,harden in ,mller's fluid4 ,stain in picrocarmine4 ,both sections and teased specimens should be prepared4 99,tendon499--,readily obtained from an amputated limb4 ,harden in ,mller's fluid4 ,make transverse and longitudinal sections4 ,stain with eosine and hmatoxyline4 ,a preparation should also be made by teasing a little of the fresh tendon in normal salt solution1 and staining with picrocarmine4 99,retiform99 or 99lymphadenoid tissue994--,seen in lymphatic glands and in the lymphoid follicles scattered along the sub-mucous coat of the alimentary canal4 ,prepare sections in the ordinary way4 ,stain in eosine and hmatoxyline or in picrocarmine4 ,some sections should also be prepared by pencilling 7i4e41 dabbing with a camel's hair brush7 or by shaking sections up in a test tube with water or normal salt solution4 ,by this means the leucocytes are removed1 and the structure of the adenoid tissue itself becomes more evident4 99,fat499--,best studied in sections of skin and subcutaneous tissue1 or in the mesentery of the cat4 ,one specimen should be stained with osmic acid and picrocarmine and mounted in ,farrant's medium1 and another in eosine and hmatoxyline and mounted in ,canada balsam4 99,pigment cells499--,branched cells are best studied in the living foot of the frog1 where amboid movements may be seen in them when the light falling on the retina is made to vary in intensity4 ,permanent preparations are most conveniently made from the pallium of the common snail4 ,the shell is removed1 and the pallium snipped out with the scissors4 ,it is then pinned out flat1 hardened for a day in methylated spirit1 and mounted unstained in ,farrant's medium4 ,they are also well seen in sections of the choroid coat of the eye4 99,hyaline cartilage499--,specimens may be obtained from any joint1 from the costal cartilages of young animals1 or from the thyroid cartilage and tracheal rings4 ,it may be hardened in spirit4 ,stain with picrocarmine1 eosine and hmatoxyline1 and with methyl violet4 99,elastic cartilage499--,prepared from the epiglottis1 or from the cartilages of the ear1 e4g41 of a cat4 ,harden in spirit4 ,stain in picrocarmine or in dilute fuchsin4 99,white fibro-cartilage499--,obtained from intervertebral disc4 ,prepare and stain as for hyaline cartilage4 99,bone993-- 99,unsoftened ,bone499--,cut as thin a section as possible with a fine saw4 ,rub the section with the hand on a dry oil stone until it is as thin as possible4 ,then cement it by ,canada balsam 7liquefied by warming7 to a piece of plate glass and continue the rubbing process with this1 examining it now and then with the low power to see if it is thin enough4 ,as soon as it is thin enough it is washed off the slide with methylated spirit1 and washed to get rid of the fine bone dust4 ,it should then be transferred to turpentine and may be mounted in balsam4 99,softened bone499--,specimens may be obtained from an amputated limb or from the femur of a cat4 ,specimens should be decalcified in chromic and nitric fluid1 and the hardening completed in spirit4 ,in studying the process of ossification1 e4g41 in the head of the humerus of a kitten1 it is best to embed the specimen in celloidin before cutting sections1 as the trabecul of bone are very delicate1 and easily detached4 ,very beautiful double staining effects may be obtained with either picrocarmine1 or eosine and hmatoxyline1 and with eosine and methyl violet4 99,bone marrow499--,to obtain good sections of red bone marrow1 take a piece of the clavicle or a rib1 or of one of the carpal or tarsal bones4 ,decalcify in chromic and nitric fluid4 ,embed in celloidin4 ,stain with eosine and logwood1 eosine and alum carmine1 or alum carmine and picric acid4 ,mount in ,canada balsam4 ,the various cells present in bone marrow may also be studied by squeezing some fresh marrow from a rib1 and making a cover-glass film1 and preparing in exactly the same way as is directed in the case of blood films on page #aaf4 99,tooth499--,best cut in situ from the jaw of a cat4 ,decalcify in chromic and nitric fluid1 and cut both vertical and transverse sections4 ,stain in picrocarmine1 or eosine and hmatoxyline4 99,developing tooth499--,extremely good specimens may be obtained from the jaw of a newly-born kitten or puppy4 ,sections can easily be made shewing a milk tooth and a developing permanent tooth by its side4 ,the enamel is dissolved by decalcifying fluids4 ,to study it a specimen of unsoftened tooth should be made1 according to the directions given for bone4 99,striped muscle499--,should be studied in various animals4 ,the leg of an insect such as a cockroach may be hardened in osmic acid4 ,one leg should be hardened in a straight position so as to fix the fibrils in the fully extended position1 another should be bent up so as to get specimens of relaxed fibrils4 ,portions of muscle should be removed1 and teased on a glass slide in some staining fluid such as picrocarmine1 a tenth per cent4 solution of eosine or quarter per cent4 of safranine4 ,sections of amphibian and mammalian muscle should be prepared to show their differences in structure4 ,the most convenient part to select is the tongue1 as a view of the fibres is obtained both in longitudinal and transverse sections4 ,sections should be stained in eosine and hmatoxyline which gives a beautiful effect4 ,for special stains for intra-muscular nerve endings see page #ib4 99,heart muscle499--,a portion should be teased fresh in picrocarmine or eosine1 another portion hardened in ,mller's fluid1 and sections made and stained with eosine and hmatoxyline4 99,unstriped muscle99 may be obtained by teasing a fresh portion of the muscular coat of the small intestine of an animal1 or by sections of the hardened intestine1 bladder or uterus4 ,stain in picrocarmine or preferably eosine and hmatoxyline4 99,nerves499--,the special methods for staining nerve tissues are detailed in ,chapter ,,vi4 ,the student must remember that the ordinary staining methods are also applicable to nervous tissues4 99,nerve terminations993-- 99,meissner's corpuscles499--,take the tip of an index finger immediately after amputation4 ,place part of it at once in chloride of gold solution1 and the rest in ,mller's fluid until it is hardened4 ,sections stained with chloride of gold should be mounted in ,farrant's medium4 ,the other sections may be stained in picrocarmine or eosine and hmatoxyline4 99,pacini's corpuscles499--,may be dissected out on the smaller branches of the digital nerves1 or may be found in the mesentery of the cat4 ,the latter should be spread out on wood1 hardened in ,mller's fluid1 stained in hmatoxyline1 and mounted in balsam4 ,other forms of tactile corpuscles may be studied in the tongues of frogs1 ducks1 or geese4 ,a network of nervous fibrils should be studied in the cornea4 ,take the cornea of a newly killed frog or cat and stain with chloride of gold 7p4 #hb74 ,the end plates in which the nerves terminate in muscle may be studied by placing specimens of living muscle of some cold blooded animal into chloride of gold solution1 and staining rather deeply4 99,arteries499--,take a piece of the aorta1 a piece of some medium artery1 as the renal or radial1 and harden in ,mller's fluid4 ,stain in picrocarmine and always in eosine and hmatoxyline4 ,arterioles are best studied in sections of the various organs4 ,thus they are seen in each ,malpighian body of the spleen1 in the boundary zone of the kidney1 and so on4 ,a longitudinal surface view can also be obtained by staining and examining the pia mater4 99,veins499--,remove1 harden1 and stain in the same way4 99,capillaries499--,may be very well seen in the foot of the frog4 ,stun a frog by striking its head1 or by chloroforming it4 ,fix it on a piece of card with a ,v shaped notch at one end4 ,tie one of the hind feet by means of threads attached to its toes so that the web of the foot is gently stretched over the ,v4 ,the foot can then be readily examined under a #a_/b inch objective4 ,the foot must be brushed from time to time with normal salt solution to keep it moist4 ,the movement of blood in the capillaries1 c41 can then be studied for an hour or two4 ,after death the mesentery should be spread out on a piece of wood1 and hardened for a few days in ,mller's fluid4 ,stain with eosine and hmatoxyline4 99,lymphatics499--,commencement of lymphatics in serous membrane4 ,stain a piece of cat's omentum in nitrate of silver 7p4 #hb7 for some minutes4 ,after washing keep in glycerine for about a week and then stain in hmatoxyline and mount in ,farrant's medium4 99,lymphatic glands499--,the lymphatic glands of the neck of the cat may be used4 ,harden in ,mller's fluid4 ,stain in picrocarmine1 eosine and hmatoxyline4 99,skin and sweat glands499--,sections should be made from pieces taken 7a7 from the sole1 7b7 from the skin of the body1 7c7 from the axilla of an adult to study the pigment4 ,harden in ,mller's fluid4 ,stain in picrocarmine or eosine and hmatoxyline4 99,hairs and sebaceous glands499--,take a portion of the scalp1 or of the skin of a puppy4 ,harden in ,mller's fluid4 ,stain in eosine and hmatoxyline1 and mount others unstained4 ,hairs from various parts of the body should also be soaked for some hours in liq4 potass and mounted unstained in ,farrant's medium4 ,they may be bleached subsequently by treatment with eau de ,javelle 7p4 #bg74 99,brain and spinal cord499--,must be removed from the body with extreme care1 all stretching or squeezing being avoided4 ,harden slowly in ,mller's fluid to which a fourth of its bulk of water may be added4 ,the best staining reagents to employ are eosine and hmatoxyline1 alum carmine or borax carmine1 aniline blue-black1 c4 ,staining methods1 see ,chapter ,,vi4 99,eye499--,harden the eye of a recently killed bullock1 cat1 or other animal in formal 7p4 #bc71 puncturing the sclerotic in places to allow the hardening fluid to penetrate4 ,in about a week make a horizontal section through the eye4 ,the anterior half 7the lens having been removed7 may be satisfactorily cut in gum4 ,sections of the crystalline lens are not very satisfactory4 ,the best way to get specimens of the fibres is to tease a piece of the fresh lens of a fish 7e4g41 a cod7 in a #a_/dj per cent4 aqueous solution of eosine4 ,wash the eosine off the slide with #a_/b per cent4 acetic acid1 and mount in ,farrant's solution4 ,the posterior half of the eye should be embedded in celloidin1 as otherwise it is extremely difficult to get sections of the retina in its proper relation to the other coats4 ,mount some specimens unstained4 ,stain others with the ordinary stains4 99,internal ear499--,decalcifying the temporal bone of a cat1 dog1 guinea pig1 c41 in chromic and nitric fluid4 ,as soon as the bone is decalcified complete the hardening of the soft parts in methylated spirit1 embed in celloidin1 and cut sections in the longitudinal axis of the cochlea4 ,owing to the extreme hardness of the bone in adults it will be found best to use the petrous bone of newly born animals4 ,the semi-circular canals will be most readily studied in the temporal bone of fishes1 or of birds1 e4g41 the common fowl4 ,they also must be cut in celloidin1 and stained in the ordinary way4 99,nose and olfactory epithelium499--,it is difficult to obtain specimens from the human subject1 but very satisfactory preparations may be made from the dog1 or more conveniently in a new born puppy where the bones are still cartilaginous4 ,harden the latter in ,mller's fluid1 decalcify adult specimens in chromic and nitric fluid4 ,specimens of ciliated epithelium1 c41 will be obtained from the lower part1 and of the special olfactory epithelium from the upper part4 ,stain in eosine and hmatoxyline4 99,lungs499--,carefully remove the lungs of a cat without injuring the bronchi or trachea4 ,introduce a cannula into the trachea and gently inflate the trachea with air4 ,ligature the trachea and place the lung in ,mller's fluid1 a weight being attached to keep the organ submerged4 ,harden for about six weeks1 and then make sections of the various parts4 ,to demonstrate the endothelium of the alveoli1 inject instead of air1 nitrate of silver4 ,allow it to remain in for half an hour1 then remove it by washing1 and harden in ,mller's fluid4 ,beautiful casts of the alveoli1 c41 may be obtained by placing a cat's or human lung under the receiver of an air-pump1 and when the air is completely exhausted1 injecting fusible metal into the bronchus4 ,the lung tissue is then removed by corrosion or by maceration4 ,portions of the casts should be removed1 fixed in a glass cell with a spot of ,canada balsam1 and examined by reflected light4 99,thyroid gland499--,best obtained from a young subject either human or an animal4 ,harden in ,mller's fluid4 ,stain in picrocarmine or eosine and hmatoxyline4 ,also stain sections in safranine1 which stains the colloid material1 and also picks out any colloid formation in the cells themselves4 99,thymus499--,remove from a ftus or a very young animal1 and prepare in the usual way4 99,tongue499--,that of the cat or rabbit serves very well4 ,ordinary transverse sections should be made1 and also sections through the circumvallate papill in order to study the 8taste buds40 99,salivary glands499--,those of a cat or dog do very well4 ,sections should be made from each of the three glands4 99,stomach499--,that of the cat or dog should be studied4 ,the organ must be removed immediately after death before any post-mortem digestion of the coats has occurred4 ,the stomach should be opened1 washed gently and pinned out flat1 with as little stretching as possible on a piece of wood1 and hardened in ,mller's fluid4 ,sections should be made 7a7 longitudinally through the cardiac end to show the transition from the sophageal to the gastric mucous membrane1 7b7 from a portion of the greater curvature1 7c7 longitudinally through the pyloric valve4 ,eosine and hmatoxyline form the best stain for the alimentary canal4 99,intestine499--,prepare in the same way as the stomach4 ,make sections from 7a7 the upper part of the duodenum to show ,brunner's glands1 7b7 the ileum1 7c7 a ,peyer's patch1 7d7 the vermiform appendix1 7e7 the colon4 99,liver499--,make an injection of one specimen with carmine and gelatin 7p4 #abj74 ,harden in methylated spirit4 ,others should be hardened in ,mller's fluid and stained in the usual way4 99,kidney1 supra-renal1 and pancreas499--,same preparation as for liver4 99,spleen499--,harden in ,mller's fluid4 ,mount one section unstained4 ,shake another up with water in a test tube to shew the structure of the pulp4 ,stain others in eosine and hmatoxyline4 99,bladder499--,must be removed and pinned out immediately after death1 as otherwise the epithelium will be macerated off4 ,consequently it must be taken from an animal1 as a cat4 ,harden in osmic acid4 ,cut in celloidin as the coats are very apt to become detached4 99,penis99 and 99testis994--,readily obtained from dog1 cat1 or rat4 ,stain with eosine and hmatoxyline4 99,uterus1 ovaries1 and ,fallopian tubes499--,may be obtained from the post-mortem room or from the lower animals4 ,harden in ,mller's fluid1 and make sections from the cervix1 the body of the uterus1 the ,fallopian tube1 and the ovary4 ,stain with eosine and hmatoxyline4 99,embryological specimens499--,for systematic work special manuals should be consulted4 ,specimens should be hardened in osmic acid or in ,mller's fluid1 and cut in celloidin1 or paraffin4 99,cloudy swelling499--,specimens are obtained from organs of subjects who have died in the early stage of some fever4 ,they should be always hardened in ,mller's fluid1 as the appearances alter if the tissue is kept in spirit for any length of time4 99,fatty degeneration499--,prepare from patients who have died of exhausting diseases1 phosphorus poisoning1 c4 ,stain in osmic acid4 ,mount in ,farrant's medium and keep in the dark4 99,mucoid degeneration499--,study in goblet cells of normal intestine or of ovarian cysts4 ,there are no satisfactory selective stains for mucin4 99,colloid degeneration499--,occurs in the thyroid gland1 in the tubules of the kidney in many diseases1 and the prostate of the old4 ,stain in safranine4 99,waxy or lardaceous degeneration499--,best studied in liver1 spleen1 or kidneys4 ,it should be searched for in persons who have died from a long illness1 accompanied by suppuration1 e4g41 phthisis or bone disease4 ,mount one section unstained1 stain another in methyl violet1 a third in a weak solution of iodine1 and examine the latter at once both by transmitted and reflected light4 ,the iodine stain is not permanent4 ,another section should be stained in osmic acid1 followed by methyl violet1 as waxy and fatty degeneration frequently co-exist4 99,hyaline degeneration499--,seen in the arterioles of the spleen in some cases of typhoid and diphtheria4 ,the ordinary staining methods must be used4 99,calcareous degeneration499--,occurs after fatty degeneration in gummata and in atheromatous arteries4 ,it also occurs in the matrix of the costal cartilages after middle life4 ,mount one section unstained and examine if possible with the polariscope4 ,stain others in safranine4 99,pigmentary degeneration499--,may be studied in brown atrophy of heart1 nutmeg liver1 c4 ,it is also seen well in spinal and cerebral nerve cells of the aged4 ,harden in ,mller's fluid and mount sections unstained4 ,it will be unnecessary to recapitulate the methods for hardening the various diseased organs as the directions for the normal organs hold good4 ,if the presence of micro-organisms be suspected1 harden in methylated spirit or absolute alcohol1 but as a rule both for diseased organs and tumours ,mller's fluid will be found the most satisfactory reagent for general use4 ,it sometimes happens1 however1 that it is inconvenient to wait several weeks1 until the ,mller's fluid has hardened the specimen sufficiently1 before making sections4 ,in this case the best plan is to make fresh sections1 or else to cut a slice about one-eighth of an inch thick1 and harden for about three days in plenty of methylated spirit1 or in formal 7p4 #bc74 99,tumours499--,mller's fluid should be employed1 unless a more rapid agent is required4 ,methylated spirit may be used in the case of epithelioma1 adenoma1 c41 but for sarcoma1 myxoma1 tumours containing cysts or much blood1 ,mller's fluid yields by far the best results4 ,,books ,,of ,,reference4 ,methods in ,microscopical ,anatomy--,whitman4 ,practical ,pathology--,woodhead4 ,textbook of ,bacteriology--,crookshank4 ,manual for ,physiological ,laboratory--,harris and ,power4 ,practical ,histology--,fearnley4 ,practical ,pathology and ,histology--,gibbes4 ,journal of ,microscopical ,society4 ,methods and ,formul--,squire4 ,the ,human ,brain--,goodall4 ,practical ,bacteriology--,kanthack and ,drysdale4 ,methods of ,microscopical ,research--,cole4 ,,index4 ,abbe's condenser1 #aa ,absolute alcohol1 #ba ,acetate of copper1 #hi ,air bubbles1 removal of1 #ef ,alum carmine1 #gf hmatoxyline1 #fh1 #gj ,amyloid degeneration1 #adi ,aniline blue-black1 #id oil1 #ajb1 #ajh oil water1 #ajg ,apparatus required1 #a ,areolar tissue1 #acc ,bacteria1 stains for1 #ajc ,balsam bottle1 #eh ,barrett's logwood solution1 #fi ,bevan ,lewis's method1 #id ,bichromate of potassium1 #ag ,bismarck brown1 #ajd ,bleaching solution1 #bg ,blood crystals1 #acj ,blood1 methods of examining1 #aac ,blood-vessels1 injection of1 #abj ,blue injection mass1 #aba ,bone marrow1 #acf ,bone1 sections of1 #acf ,borax carmine1 #ge ,brain1 methods of staining1 #id ,buckley's modification of ,golgi's method1 #ii ,calcareous degeneration1 #adi ,canada balsam solution1 #fa ,carbolic acid1 #bc ,carmine1 #gd injection mass1 #abj ,cathcart microtome1 #ci ,cathcart-,frazer microtome1 #db ,cedar oil1 #h1 #fc ,celloidin1 #cj ,cementing cover-glasses1 #fe ,chloral hmatoxyline1 #ib ,chloride of gold1 #hb1 #id ,chromic and nitric decalcifying fluid1 #bf ,ciliated epithelium1 #acb ,circulation in frog's foot1 #ada ,clarifying sections1 #fc ,clearing agents1 #fc ,cloudy swelling1 #adh ,clove oil1 #fc ,colloid degeneration1 #adi ,columnar epithelium1 #acb ,corrosive sublimate hardening1 #bc staining1 #ajj ,cover-glasses1 cleansing of1 #eg ,cover-glass preparations1 #aaa ,decalcifying solutions1 #bf ,dehydration1 #fc ,eau de ,javelle1 #bg ,ebner's solution1 #bg ,ehrlich-,biondi fluid1 #he ,ehrlich's hmatoxyline1 #gj method of fixing blood-films1 #aaf method for tubercle bacilli1 #aaj ,ehrlich-,gram method for staining bacteria1 #ajh ,elastic cartilage1 #ace tissue1 #acc ,embedding methods1 #bi ,endothelium1 #aca ,eosine1 #gb ,eosine and hmatoxyline1 #gc ,epithelial cement1 #aca ,ether spray microtome1 #ci ,fces1 staining for bacilli1 #aab ,farrant's solution1 #ei ,fat1 removal from sections1 #ei staining of1 #acd ,fatty degeneration1 #adh ,fearnley's injection apparatus1 #abc ,ferrier's fuchsine solution1 #aag ,flemming's solution1 #be ,flotation of sections1 #ee ,folded sections1 treatment of1 #eh ,formal1 #bc ,fresh sections1 #eb ,fuchsine1 #ajd ,gentian violet1 #ajd ,gibbes' stain for tubercle bacilli1 #aaa ,gold chloride1 #hb1 #id ,golgi's silver method1 #if sublimate method1 #ii ,gram's iodide solution1 #aje method for staining bacteria1 #ajg ,green injection mass1 #abb ,gum1 #bi ,hmatin crystals1 #aca ,hmatoidin1 #aca ,hmatoxyline1 ,ehrlich's1 #gj ,kleinenberg's1 #gj ,schuchardt's1 #fh ,sihler's1 #ib ,weigert's1 #hh ,hmoglobin crystals1 #acj ,hardening processes1 #ae ,hyaline cartilage1 #ace degeneration1 #adi ,ice freezing microtome1 #df ,immersion lenses1 #h ,injection of blood-vessels1 #abj pulmonary alveoli1 #ade ,internal ear1 #adc ,intestines1 #adf ,iodine solution1 #aje ,jung's ether spray microtome1 #de ,kleinenberg's hmatoxyline1 #gj ,lardaceous degeneration1 #adi ,lithio-picrocarmine1 #gi ,lithium carmine1 #gd ,liver1 #adg ,lffler's methyl blue1 #ajd ,logwood1 #fh ,lymphoid tissue1 #acd ,marchi's fluid1 #bd ,marrow1 #acf ,methyl blue1 #aja1 #ajd violet1 #hc ,methylated spirit1 #ai ,micro-organisms1 stains for1 #ajc ,microscope1 #f ,microtome1 ,becker1 #di ,cambridge rocking1 #di ,cathcart1 #ci ,cathcart-,frazer1 #db ,jung1 #de ,schanze1 #dg ,swift's1 #di ,williams'1 #df ,mould for paraffin embedding1 #cf ,mounting methods1 #ee ,mucoid degeneration1 #adh ,muscle1 #ach ,muir's method of hardening films1 #aaf ,mller's fluid1 #ag and formal1 #bj and spirit1 #bj ,neelsen's stain for tubercle bacilli1 #aaj ,nerve cells1 stains for1 #id endings1 #aci fibres1 stains for1 #hg ,nissl's aniline method1 #aja ,nitrate of silver1 #hb ,nitric acid as hardening agent1 #be decalcifying agent1 #bf decolourising agent1 #aaj ,normal salt solution1 #ec ,nose piece1 #i ,objectives1 #g ,oil of bergamot1 #fc cedar1 #h1 #fc cloves1 #fc origanum1 #fc ,osmic acid as hardening agent1 #ba staining reagent1 #ha ,pal's method1 #hf solution1 #ij ,paraffin1 #cd ,picrocarmine1 #gh ,pigment cells1 #acd ,pigmentary degeneration1 #aej ,plane iron microtome knife1 #db ,rapid hardening1 #aej ,retina1 #adc ,safranine1 #he ,salivary glands1 #adf ,schfer-,pal method1 #ia ,schanze microtome1 #dg ,schuchardt's hmatoxyline1 #fh ,sihler's chloral hmatoxyline1 #ib ,silver nitrate1 stain for nerve cells1 #if epithelial cement1 #aca ,skin1 #adb ,spinal cord1 #hf1 #adb ,spleen1 #adg ,sputum1 staining of1 #aaa ,squamous epithelium1 #aca ,staining methods1 #fg seq4 ,stomach1 #adf ,striped muscle1 #ach ,sulphuric acid1 #aje ,sweat glands1 #adb ,tendon1 #acc ,testing a microscope1 #ac ,thymus gland1 #ade ,thyroid gland1 #ade ,toison's fluid1 #aah ,tooth1 sections of1 #acg ,transitional epithelium1 #acb ,tubercle bacillus1 stains for1 #aaj ,tumours1 hardening of1 #aej ,unstriped muscle1 #aci ,urine1 examination for bacilli1 #aab ,uterus1 #adh ,von ,ebner's decalcifying solution1 #bg ,waxy degeneration1 #adi ,weigert's hmatoxyline method1 #hh method for staining bacteria1 #ajf ,williams' ice freezing microtome1 #df ,woodhead's injection mass1 #abj ,xylol1 #fa ,ziehl's carbol-fuchsine1 #aje