file :i* includes ! orig9al illu/rations4 ,see #bggac-;h4htm or #bggac-;h4zip3 7https3_/_/www4gut5b]g4org_/dirs_/#b_/g_ /g_/a_/bggac_/bggac-;h_/#bggac-;h4htm7 or 7https3_/_/www4gut5b]g4org_/dirs_/#b_/g_ /g_/a_/bggac_/bggac-;h4zip7 ,transcrib]'s note3 ,text enclos$ by tilde m>ks 0 9 bold face 9 ! orig9al 7bold74 ,text enclos$ by und]score m>ks is 9 italics 7italics74 ,! italic designation = s+le italiz$ lett]s 7su* z v>iables 9 equations7 & =eign abbreviations has be5 omitt$ = ease ( read+4 ,9 numb]s1 equations1 & *emical =mulas1 an und]score indicates t ! foll[+ t]m enclos$ )9 curly brackets is a subscript4 ,examples3 ,,co#b1 ,h#b,,s#d4 ,a c>at "* indicates t ! foll[+ t]m enclos$ )9 curly brackets is a sup]script4 ,= example1 #aa4ac is #aa4a to ! ?ird p[]4 ,m9or typographical errors h be5 correct$4 ,,! ,,elem5ts ,,( ,,bact]iological ,,te*nique ,a ,laboratory ,guide = ,m$ical1 ,d5tal1 & ,te*nical ,/ud5ts by ,j4 ,w4 ,h4 ,,eyre1 ,m4,d41 ,m4,s41 ,f4,r4,s4 7,,ed947 ,director ( ! ,bact]iological ,dep>tm5t ( ,guy's ,hospital1 ,london1 & ,lectur] on ,bact]iology 9 ! ,m$ical & ,d5tal ,s*ools2 =m]ly ,lectur] on ,bact]iology at ,*>+ ,cross ,hospital ,m$ical ,s*ool1 & ,bact]iologi/ to ,*>+ ,cross ,hospital2 sometime ,hunt]ian ,pr(essor1 ,royal ,college ( ,surgeons1 ,engl& ,second ,edition ,rewritt5 & ,enl>g$ ,philadelphia & ,london ,w4 ,b4 ,saund]s ,company #aiac ,copyright1 #aijb1 by ,w4 ,b4 ,saund]s & ,company ,revis$1 entirely reset1 repr9t$1 & recopyright$ ,july1 #aiac ,copyright1 #aiac1 by ,w4 ,b4 ,saund]s ,company ,regi/]$ at ,/ation]s' ,hall1 ,london1 ,engl& ,pr9t$ 9 ,am]ica ,press ( ,w4 ,b4 ,saund]s ,company ,philadelphia ,,to ,,! ,,memory ,,( ,,john ,,wi*5=d ,,wa%b\rn1 ,c4,m4,g41 ,m4,d41 ,f4,r4,c4,p4 ,physician to ,guy's ,hospital & ,lectur] on ,bact]iology 9 ! ,m$ical ,s*ool1 & ,physician to ! ,london ,fev] ,hospital ,,my ,,tea*]1 ,,fri5d1 ,,& ,,co-,,work] ,,preface ,,to ,,! ,,second ,,edition ,bact]iology is ess5tially a practical /udy1 & ev5 ! elem5ts ( its te*nique c only be taught by p]sonal in/ruction 9 ! laboratory4 ,?is is a self-evid5t proposition t ne$s no emphasis1 yet ,i v5ture to believe t ! =m] collection ( tri$ & prov$ me?ods has already be5 ( "s utility1 n only to ! /ud5t 9 ! abs5ce ( 8 tea*]1 b also to isolat$ work]s 9 laboratories f> remov$ f c5tres ( in/ruction1 rem9d+ !m ( =gott5 details 9 me?ods already acquir$4 ,if ?is assumption is bas$ on fact no fur!r apology is ne$$ = ! pres5t revis$ edition 9 :i* ! *anges are *iefly 9 ! nature ( additions--r5d]$ necess>y by ! introduction ( new me?ods dur+ rec5t ye>s4 ,i take ?is opportunity ( express+ my deep s5se ( obligation to my confr]e 9 ! ,physiological ,dep>tm5t ( our m$ical s*ool--,mr4 ,j4 ,h4 ,ryffel1 ,b4 ,c41 ,b4 ,sc4--:o has revis$ ^? pages deal+ ) ! analysis ( ! metabolic products ( bact]ial life2 to successive colleagues 9 ! ,bact]iological ,dep>tm5t ( ,guy's ,hospital1 = _! ready co-op]ation 9 work+ out or 9 te/+ new me?ods2 & f9ally to my ,*ief ,laboratory ,assi/ant1 ,mr4 ,j4 ,c4 ,turn] ^: assi/ance & exp]i5ce h be5 ( ! utmo/ value to me 9 ! prep>ation ( ?is volume4 ,i h also to ?ank ,mrs4 ,con/ant ,pond] = _m ( ! new l9e draw+s & = r$raw+ a numb] ( ! orig9al cuts4 ,,john ,w4 ,h4 ,,eyre4 ,,guy',s ,,hospital1 ,s4 ,e4 ,july1 #aiac4 ,,preface ,,to ,,! ,,fir/ ,,edition ,9 ! foll[+ pages ,i h endeav\r$ to arrange briefly & concisely ! v>i\s me?ods at pres5t 9 use = ! /udy ( bact]ia1 & ! elucidation ( su* po9ts 9 _! life-hi/ories z are debatable or /ill undet]m9$4 ,( ^! me?ods1 "s are new1 o!rs are n2 b all are reliable1 only su* hav+ be5 includ$ z are capable ( giv+ satisfactory results ev5 9 ! h&s ( beg9n]s4 ,9 fact1 ! bulk ( ! matt] is simply an elaboration ( ! typewritt5 notes di/ribut$ to "s ( my laboratory classes 9 practical & appli$ bact]iology2 consequ5tly an attempt has be5 made to pres5t ! elem5ts ( bact]iological te*nique 9 _! logical sequ5ce4 ,i make no apology = ! space devot$ to illu/rations1 ne>ly all ( :i* h be5 prep>$ especially = ?is volume2 = a picture1 if good1 possesses a high] educational value & conveys a m accurate impression ?an a page ( pr9t2 & ev5 sket*es ( app>atus s]ve a di/9ct purpose 9 sugge/+ to ! /ud5t ^? alt]ations & modifications :i* may be r5d]$ necess>y or advisable by ! "* ( 8 laboratory equipm5t4 ,! excell5t & appropriate t]m9ology introduc$ by ,*e/] 9 8 rec5t "w on ,det]m9ative ,bact]iology ,i h adopt$ 9 its entirety1 = ,i consid] x only ne$s to be us$ to conv9ce "o ( its extreme utility1 :il/ its inclusion 9 an elem5t>y manual is calculat$ to induce 9 ! /ud5t habits ( accurate obs]vation & concise description4 ,) ! exception ( ,section ,,xvii--,outl9es = ! ,/udy ( ,pa?og5ic ,bact]ia--introduc$ ) ! idea ( complet+ ! volume f ! po9t ( view ( ! m$ical & d5tal /ud5t1 ! "w has be5 arrang$ to all[ ( its use z a laboratory guide by ! te*nical /ud5t g5]ally1 :e!r ( brew+1 dairy+1 or agriculture4 ,s alive am ,i to its _m inp]fections t x appe>s almo/ sup]flu\s to /ate t ! book is 9 no s5se int5d$ z a rival to ! _m & excell5t manuals ( bact]iology at pres5t 9 use1 b aims only at supplem5t+ ! usually scanty details ( te*nique1 & at in/ruct+ ! /ud5t h[ to fit up & adapt app>atus = 8 daily "w1 & h[ to c>ry out ?or\ghly & sy/ematically ! v>i\s bact]ioscopical analyses t are daily dem&$ ( ! bact]iologi/ by ! hygi5i/4 ,f9ally1 x is ) mu* pleasure t ,i ackn[l$ge ! valuable assi/ance receiv$ f my late assi/ant1 ,mr4 ,j4 ,b4 ,gall1 ,a4 ,i4 ,c41 9 ! prep>ation ( ! section deal+ ) ! *emical products ( bact]ial life1 & :i* has be5 bas$ ^u ! "w ( ,lehmann4 ,,john ,w4 ,h4 ,,eyre4 ,,guy',s ,,hospital1 ,s4 ,e4 ,,cont5ts ,,page ,i4 ,,laboratory ,,regulations #a ,,ii4 ,,glass ,,app>atus ,,9 ,,common ,,use #c ,! ,selection1 ,prep>ation1 & ,c>e ( ,glassw>e1 #h--;,clean+ ( ,glass ,app>atus1 #ah--,plugg+ ,te/-tubes & ,flasks1 #bd4 ,,iii4 ,,me?ods ,,( ,,/]ilisation #bf ,/]ilis+ ,ag5ts1 #bf--,me?ods ( ,application1 #bg--;,electric ,signal ,tim+ ,clock1 #ch4 ,,iv4 ,,! ,,microscope #di ,ess5tials1 #di--;,accessories1 #eg--,me?ods ( ,micrometry1 #fa4 ,v4 ,,microscopical ,,exam9ation ,,( ,,bact]ia ,,& ,,o!r ,,micro-,,fungi #fi ,app>atus & ,reag5ts us$ 9 ,ord9>y ,microscopical ,exam9ation1 #fi--,me?ods ( ,exam9ation1 #gd4 ,,vi4 ,,/a9+ ,,me?ods #ij ,bact]ia ,/a9s1 #ij--;,contra/ ,/a9s1 #ic--,tissue ,/a9s1 #ie--;,blood ,/a9s1 #ig--,me?ods ( ,demon/rat+ ,/ructure ( ,bact]ia1 #ii--;,diff]5tial ,me?ods ( ,/a9+1 #ajh4 ,,vii4 ,,me?ods ,,( ,,demon/rat+ ,,bact]ia ,,9 ,,tissues #aad ,freez+ ,me?od1 #aae--,p>aff9 ,me?od1 #aag--,special ,/a9+ ,me?ods = ,sections1 #aba4 ,,viii4 ,,classification ,,( ,,fungi #abf ,morphology ( ! ,hyphomycetes1 #abf--,morphology ( ! ,bla/omycetes1 #abi4 ,,ix4 ,,s*izomycetes #aca ,anatomy1 #acd--,physiology1 #acf--;,bio*emi/ry1 #add4 ,x4 ,,nutri5t ,,m$ia #adf ,meat ,extract1 #adh--,/&>disation ( ,m$ia1 #aed--,! ,filtration ( ,m$ia1 #aef--,/or+ ,m$ia 9 ,bulk1 #aei--,tub+ ,nutri5t ,m$ia1 #afj4 ,,xi4 ,,ord9>y ,,or ,,/ock ,,culture ,,m$ia #afc ,,xii4 ,,special ,,m$ia #ahb ,,xiii4 ,,incubators #baf ,,xiv4 ,,me?ods ,,( ,,cultivation #bba ,a]obic1 #bbb--;,ana]obic1 #bcf4 ,,xv4 ,,me?ods ,,( ,,isolation #bdh ,,xvi4 ,,me?ods ,,( ,,id5tification ,,& ,,/udy #bei ,s*eme ( ,/udy1 #bei--,macroscopical ,exam9ation ( ,cultivations1 #bfa--,microscopical ,me?ods1 #bgb--;,bio*emical ,me?ods1 #bgf--,physical ,me?ods1 #bie--;,inoculation ,me?ods1 #cae--;,immunisation1 #cba--;,active ,immunisation1 #cbb--,! ,prep>ation ( ,haemolytic ,s]um1 #cbg--,! ,titration ( ,haemolytic ,s]um1 #cbh--,/orage ( ,haemolys91 #cca4 ,,xvii4 ,,exp]im5tal ,,inoculation ,,( ,,animals #ccb ,selection & ,c>e ( ,animals1 #cce--,me?ods ( ,inoculation1 #ceb4 ,,xviii4 ,,! ,,/udy ,,( ,,exp]im5tal ,,infections ,,dur+ ,,life #cgj ,g5]al ,obs]vations1 #cga--;,blood ,exam9ations1 #cgc--,s]ological ,inve/igations1 #cgh--;,agglut991 #cha--,opson91 #chg--;,immune ,body1 #cic4 ,,xix4 ,,po/-,,mortem ,,exam9ation ,,( ,,exp]im5tal ,,animals #cif ,,xx4 ,,! ,,/udy ,,( ,,! ,,pa?og5ic ,,bact]ia #djh ,,xxi4 ,,bact]iological ,,analyses #dae ,bact]iological ,exam9ation ( ,wat]1 #daf--;,exam9ation ( ,milk1 #dda--;,ice ,cream1 #deg--;,exam9ation ( ,cream & ,butt]1 #deg--;,exam9ation ( ,uns\nd ,meats1 #dfj--;,exam9ation ( ,oy/]s & ,o!r ,%ellfi%1 #dfc--;,exam9ation ( ,sewage & ,sewage ,efflu5ts1 #dff--;,exam9ation ( ,air1 #dfh--;,exam9ation ( ,soil1 #dgj--,te/+ ,filt]s1 #dgh--,te/+ ( ,dis9fectants1 #dhj4 ,,app5dix #dib ,,index #eje ,illu/ration ,,bact]iological ,,te*nique4 ,i4 ,,laboratory ,,regulations4 ,! foll[+ regulations are laid d[n = obs]vance 9 ! ,bact]iological ,laboratories "u ! direction ( ! au?or4 ,simil> regulations %\ld be en=c$ 9 all laboratories :]e pa?og5ic bact]ia are /udi$4 ,guy's ,hospital4 ,,bact]iological ,,dep>tm5t4 ,,h&l+ ,,( ,,infective ,,mat]ials4 ,! foll[+ ,regulations h be5 drawn up 9 ! int]e/ ( ^? work+ 9 ! ,laboratory z well z ! public at l>ge1 & w be /rictly en=c$4 ,_! object is to avoid ! dang]s ( infection :i* may arise f neglect ( necess>y precautions or f c>elessness4 ,ev]yone mu/ note t by neglect+ ! g5]al rules laid d[n he n only runs grave risk himself1 b is a dang] to o!rs4 ,,regulations4 #a4 ,ea* work] mu/ we> a g[n or ov]all1 provid$ at 8 own exp5se1 :i* mu/ be kept 9 ! ,laboratory4 #b4 ,! h&s mu/ be dis9fect$ ) lysol #b p] c5t4 solution1 c>bolic acid #e p] c5t4 solution1 or corrosive sublimate #a p] mille solution1 aft] deal+ ) infecti\s mat]ial1 & be=e us+ t[els4 #c4 ,on no acc\nt mu/ ,laboratory t[els or du/]s be us$ = wip+ up infecti\s mat]ial1 & if su* t[els or du/]s d become soil$1 !y mu/ be imm$iately /]ilis$ by boil+4 #d4 ,special pails conta9+ dis9fectant are provid$ to receive any wa/e mat]ial1 & no?+ mu/ be ?r[n on ! floor4 #e4 ,all in/rum5ts mu/ be flam$1 boil$1 or o!rwise dis9fect$ imm$iately aft] use4 #f4 ,labels mu/ be moi/5$ ) wat]1 & n by ! m\?4 #g4 ,all disus$ cov]-glasses1 slides1 & pipettes aft] use 9 h&l+ infecti\s mat]ial1 etc41 mu/ be plac$ 9 #b p] c5t4 lysol solution4 ,a vessel is suppli$ on ea* b5* = ?is purpose4 #h4 ,all plate & tube cultures ( pa?og5ic organisms :5 done )1 mu/ be plac$ = imm$iate dis9fection 9 ! boxes provid$ = ! purpose4 #i4 ,no fluids are to be dis*>g$ into s9ks or dra9s unless previ\sly dis9fect$4 #aj4 ,animals are to be dissect$ only aft] be+ nail$ out on ! wood5 bo>ds1 & _! sk9 ?or\ghly wa%$ ) dis9fectant solution4 #aa4 ,imm$iately aft] ! po/-mortem exam9ation is complet$ ea* cadav] mu/ be plac$ 9 ! z9c animal-box--)\t remov+ ! c>case f ! po/-mortem bo>d--& ! cov] ( ! box replac$1 ready = c>riage to ! de/ructor4 #ab4 ,dead animals1 :5 done )1 are cremat$ 9 ! de/ructor1 & ! laboratory att5dant mu/ be notifi$ :5 ! bodies are ready = cremation4 #ac4 ,none ( ! work]s 9 ! laboratory are all[$ to ent] ! animal h\ses unless accompani$ by ! special att5dant 9 *>ge1 :o mu/ scrupul\sly obs]ve ! same directions reg>d+ p]sonal dis9fection z ! work]s 9 ! laboratories4 #ad4 ,no cultures are to be tak5 out ( ! laboratory )\t ! p]mission ( ! head ( ! ,dep>tm5t4 #ae4 ,all accid5ts1 su* z spill+ infect$ mat]ial1 cutt+ or prick+ ! f+]s1 mu/ be at once report$ to ! bact]iologi/ 9 *>ge4 ,,ii4 ,,glass ,,app>atus ,,9 ,,common ,,use4 ,! equipm5t ( ! bact]iological laboratory1 s f> z ! glass app>atus is conc]n$1 diff]s b little f t ( a *emical laboratory1 & ! cleanl9ess ( ! app>atus is equally important4 ,! glassw>e compris$ 9 ! foll[+ li/1 9 addition to be+ clean1 mu/ be /or$ 9 a /]ile or g]m-free condition4 ,te/-tubes4--,x is conv5i5t to keep sev]al sizes ( te/-tubes 9 /ock1 to meet special requirem5ts1 viz43 #a4 #ah x #a4e cm41 to conta9 m$ia = ord9>y tube cultivations4 #b4 #ah x #a4c cm41 to conta9 m$ia us$ = p\r+ plate cultivations1 & also = hold+ /]ile swabs4 #c4 #ah x #b cm41 to conta9 w$ges ( potato1 beetroot1 or o!r vegetable m$ia4 #d4 #ac x #a4e cm41 to conta9 inspissat$ blood-s]um4 ,! tubes %\ld be made f ! be/ ,g]man pota% glass1 blue-l9$1 /\t & heavy1 ) ! edge ( ! m\? ( ! tube slightly turn$ ov]1 b n to su* an ext5t z to =m a def9ite rim4 7,co/ ab\t #a4ej1 or #f %ill+s p] gross47 ,su* tubes are exp5sive x is true1 b !y are suffici5tly /\t to resi/ r\gh h&l+1 d n usually break if accid5tally all[$ to drop 7a po9t ( "s mom5t :5 deal+ ) cultures ( pa?og5ic bact]ia71 c be clean$1 /]ilis$1 & us$ ov] & ov] aga91 & by _! l5g? ( life fully ju/ify _! initial exp5se4 ,a po9t be not$ is t ! manufactur]s r>ely turn out su* tubes z ^! absolutely uni=m 9 calibre1 & a bat* ( #ah by #a4e cm4 tubes usually conta9s su* extreme sizes z #ah by #b cm4 & #ah by #a4c cm4 ,consequ5tly1 if a set ( /&>d tubes is kept = comp>ison or callip]s are us$ ea* new supply ( s-call$ #ah by #a4e cm4 tubes may be easily sort$ out into ^! ?ree sizes1 & s simplify ord]+4 #e4 #e x #j4g cm41 = use 9 ! inv]t$ position inside ! tubes conta9+ c>bohydrate m$ia1 z gas-collect+ tubes4 ,^! tubes1 unrimm$1 may be ( common ?9 glass z less ?an two p] c5t4 are fit = use a second "t4 ,illu/ration3 ,,fig4 #a4--;,bohemian flask4 ,illu/ration3 ,,fig4 #b4--,pe>-%ap$ flask4 ,illu/ration3 ,,fig4 #c4--;,erl5mey] flask 7n>r[ neck74 ,bohemian ,flasks 7,fig4 #a74--,^! are ! ord9>y flasks ( ! *emical laboratory4 ,a good v>iety1 rang+ 9 capacity f #bej to #cjjj c4c41 %\ld be kept on h&4 ,a modifi$ =m1 kn[n z ! pe>-%ap$ 7,fig4 #b71 is pref]able = ! small] sizes--i4 e41 #bej & #ejj c4c4 ,erl5mey]'s ,flasks 7,fig4 #c74--;,erl5mey]'s flasks ( #ge1 #ajj1 & #bej c4c4 capacity are extremely useful4 ,= use z culture flasks c>e %\ld be tak5 to select only su* z h a n>r[ neck ( ab\t #b cm4 9 l5g?4 ,kolle's ,culture ,flasks 7,fig4 #d74--,^! ?91 flat flasks 7to conta9 ag> or gelat9e1 :i* is all[$ to solidify 9 a lay] on "o side7 are extremely useful on acc\nt ( ! l>ge nutri5t surface available = gr[?4 ,a surface cultivation 9 "o ( ^! w yield z mu* gr[? z t5 or twelve oblique tube cultures4 ,! wide m\?1 h[ev]1 is a disadvantage1 & = _m purposes ?91 flat culture bottles kn[n z ,r\x's bottles 7,fig4 #e7 are to be pref]r$4 ,illu/ration3 ,,fig4 #d4--,kolle's culture flask4 ,illu/ration3 ,,fig4 #e4--,r\x's culture bottle4 ,illu/ration3 ,,fig4 #f4--;,guy's culture bottle4 ,illu/ration3 ,,fig4 #g4--;,filt] flask4 ,an ev5 m conv5i5t patt]n is t us$ 9 ! au?or's laboratory 7,fig4 #f71 z ow+ to ! great] dep? ( m$ium :i* x is possible to obta9 9 ^! flasks an exce$+ly luxuriant gr[? is possible2 ! n>r[ neck r$uces ! *ance ( accid5tal contam9ation to a m9imum & ! g5]al %ape p]mits ! flasks to be /ack$ "o ^u ! o!r4 ,filt] ,flasks or ,kitasato's ,s]um ,flasks 7,fig4 #g74--,v>i\s sizes1 f #bej to #bjjj c4c4 capacity4 ,^! mu/ be ( /\t glass1 to resi/ ! pressure to :i* !y are subject$1 b at ! same "t mu/ be ?or\ghly well anneal$1 9 ord] to )/& ! temp]ature necess>y = /]ilisation4 ,all flasks %\ld be ei!r ( ,j5a glass or ! almo/ equally well-kn[n ,resi/ance or ,r glass1 ! extra initial exp5se be+ ju/ifi$ by ! comp>ative immunity ( ! glass f breakage4 ,petri's ,di%es or ,plates 7,fig4 #h1 a74--,^! h n[ completely replac$ ! rectangul> %eets ( glass introduc$ by ,ko* = ! plate me?od ( cultivation4 ,ea* plate consi/s ( a pair ( circul> discs ( glass ) %>ply upturn$ edges1 ?us =m+ %all[ di%es1 "o ( slightly great] diamet] ?an ! o!r1 & s1 :5 inv]t$1 =m+ a cov] or cap = ! small]4 ,plates hav+ an outside diamet] ( #aj cm4 & a height ( #a4e cm4 are ! mo/ g5]ally useful4 ,a bat* ( eighte5 su* plates is /]ilis$ & /or$ 9 a cyl9drical copp] box 7#cj cm4 high by #ab cm4 diamet]7 provid$ ) a pull-(f lid4 ,inside ea* box is a copp] /irrup ) a circul> bottom1 ^u :i* ! plates re/1 & by means ( :i* ea* c be rais$ 9 turn to ! m\? ( ! box 7,fig4 #i7 = removal4 ,capsules 7,fig4 #h1 b & c74--,^! are ,petri's di%es ( small] diamet] b great] dep? ?an ^? t]m$ plates4 ,two sizes w be f\nd especially useful--viz41 #d cm4 diamet] by #b cm4 high1 capacity ab\t #ad c4c42 & #e cm4 diamet] by #b cm4 high1 capacity ab\t #be c4c4 ,^! are /or$ 9 copp] cyl9d]s ( simil> con/ruction to ^? us$ = plates1 b measur+ #bj by #f cm4 & #bj by #g cm41 respectively4 ,illu/ration3 ,,fig4 #h4--,petri di% 7a71 & capsules 7b1 c74 ,illu/ration3 ,,fig4 #i4--,plate box ) /irrup4 ,graduat$ ,pipettes4--,sev]al v>ieties ( ^! are requir$1 viz43 #a4 ,pipettes ( #a c4c4 capacity graduat$ 9 #j4a c4c4 #b4 ,pipettes ( #a c4c4 capacity graduat$ 9 #j4ja c4c4 7,fig4 #aj1 a74 #c4 ,pipettes ( #aj c4c4 capacity graduat$ 9 #j4a c4c4 7,fig4 #aj1 b74 ,^! %\ld be ab\t #cj cm4 9 l5g? 7#a & #b ( fairly n>r[ bore71 graduat$ to ! extreme po9t1 & hav+ at lea/ a #aj cm4 l5g? ( cle> space betwe5 ! fir/ graduation & ! upp] end2 ! op5 m\? %\ld be plugg$ ) cotton-wool4 ,ea* v>iety %\ld be /]ilis$ & /or$ 9 a sep>ate cyl9drical copp] case "s #cf by #f cm41 ) pull-(f lid1 ^u :i* is /amp$1 9 pla9 figures1 ! capacity ( ! conta9$ pipettes4 ,illu/ration3 ,,fig4 #aj4--,measur+ pipettes1 a & b4 ,! laboratory %\ld also be provid$ ) a complete set ( ,/&>d graduat$ pipettes1 ea* pipette 9 ! set be+ /amp$ & au!nticat$ by a c]tificate f "o ( ! recognis$ ,physical ,measurem5t ,laboratories1 su* z ,*>lott5burg4 ,^! in/rum5ts are exp5sive & %\ld be res]v$ solely = /&>dis+ ! pipettes 9 ord9>y use1 & = calibrat+ small pipettes manufactur$ 9 ! laboratory4 ,su* a set %\ld comprise1 at lea/1 pipettes deliv]+ #aj c4c41 #e c4c41 #b4e c4c41 #b c4c41 #a c4c41 #j4e c4c41 #j4be c4c41 #j4b c4c41 #j4a c4c41 #j4je c4c41 & #j4ja c4c41 respectively4 ,9 ! imm$iately foll[+ sections are describ$ small pieces ( glass app>atus :i* %\ld be prep>$ 9 ! laboratory f glass tub+ ( v>i\s sizes4 ,9 _! prep>ation ?ree articles are ess5tial2 fir/ a ?ree-squ>e h>d-/eel file or pref]ably a glass-work]'s knife ( h>d ,?ur+ian /eel = cutt+ glass tubes etc42 next a bl[pipe flame1 = al?\gh mu* c be done ) ! ord9>y ,buns5 burn]1 a bl[pipe flame makes = rapid "w2 & la/ly a bat's-w+ burn]4 ,illu/ration3 ,,fig4 #aa4--;,glass-cutt+ knife4 a4 h&le4 b4 d\ble edg$ blade4 c4 %aft4 d4 lock+ nut4 e4 spann] = nut4 #a4 ,! glass-cutt+ knife4 ,?is article is sold 9 two =ms1 a b5* knife 7,fig4 #aa7 & a pocket knife4 ,! =m] is provid$ ) a blade "s #h cm4 9 l5g? & hav+ two cutt+ edges4 ,! cutt+ edge :5 exam9$ 9 a /rong light is se5 to be compos$ ( small closely set tee?1 simil> to ^? 9 a saw4 ,! knife %\ld be kept %>p by frequ5t /ropp+s on a s&/one hone4 ,! pocket =m1 ab\t #f-;cm4 long ov] all1 consi/s ( a small spr+ blade ) "o cutt+ edge m\nt$ 9 scales l an ord9>y pocket knife4 #b4 ,= real conv5i5ce ( "w ! bl[pipe %\ld be m\nt$ on a special table connect$ up ) cyl9drical bell[s op]at$ by a p$al4 ,t figur$ 7,fig4 #ab7 is made by m\nt+ a teak top #fj cm4 squ>e ^u ! uprights ( an enclos$ d\ble-action conc]t9a bell[s 7,enf]'s7 & provid$ ) a ,flet*]'s ,univ]sal gas bl[pipe4 #c4 ,an ord9>y bat's-w+ gas-burn] m\nt$ at ! f> corn] ( ! table top is invaluable 9 ! prep>ation ( tubul> app>atus ) %>p curves1 & = coat+ newly-made glass app>atus ) a lay] ( soot to prev5t too rapid cool+1 & its usually associat$ result--crack+4 ,illu/ration3 ,,fig4 #ab4--;,glass bl[]'s table ) ,enf]'s foot bell[s4 #f4 ,s$im5tation tubes #ex#j4e cm41 = s$im5tation reactions1 etc41 & = conta9+ small quantities ( fluid to be c5trifugalis$ 9 ! haematocrit4 ,^! are made by tak+ #ad-;cm4 l5g?s ( /\t glass tub+ ( ! requisite diamet] & heat+ ! c5tre 9 ! ,buns5 or bl[pipe flame4 ,:5 ! c5tral portion is q s(t draw ! ends quickly ap>t & !n r\nd (f ! po9t$ ends ( ! two te/-tubes ?us =m$4 ,) ! glass-cutt+ knife cut (f :atev] may be necess>y f ! op5 ends to make ! tubes ! requir$ l5g?4 ,a rectangul> block ( pla/ic9e 7modell+ clay7 into :i* ! conical ends c be ?ru/ makes a v conv5i5t /& = ^! small tubes4 ,capill>y ,pipettes or ,pa/eur's ,pipettes 7,fig4 #ac a74--,^! little in/rum5ts are invaluable1 & a goodly supply %\ld be kept on h&4 ,!y are prep>$ f s(t-glass tub+ ( v>i\s-siz$ calibre 7! mo/ g5]ally useful size be+ #h mm4 diamet]7 9 ! foll[+ mann]3 ,hold a #aj cm4 l5g? ( glass tube by ea* end1 & :il/ rotat+ x heat ! c5tral portion 9 ! ,buns5 flame or ! bl[pipe bla/-flame until ! glass is r$ hot & s(t4 ,n[ remove x f ! flame & /eadily pull ! ends ap>t1 s draw+ ! heat$ portion out into a roomy capill>y tube2 break ! capill>y portion at its c5tre1 seal ! brok5 ends 9 ! flame1 & r\nd (f ! edges ( ! op5 end ( ea* pipette4 ,a loose plug ( cotton-wool 9 ! op5 m\? completes ! capill>y pipette4 ,aft] a numb] h be5 prep>$1 !y are /]ilis$ & /or$ 9 bat*es1 ei!r 9 metal cases simil> to ^? us$ = ! graduat$ pipettes or 9 l>ge-siz$ te/-tubes--seal$ ends d[nw>d & plugg$ ends t[>d ! m\? ( ! case4 ,illu/ration3 ,,fig4 #ac4--;,capill>y pipettes4 a1 b1 c4 ,! fill+ & empty+ ( ! capill>y pipette is mo/ satisfactorily accompli%$ by slipp+ a small rubb] teat 7simil> to t on a baby's fe$+ bottle b n p]=at$7 on ! upp] end1 aft] cutt+ or snapp+ (f ! seal$ po9t ( ! capill>y portion4 ,if pressure is n[ ex]t$ ^u ! ela/ic bulb by a f+] & ?umb :il/ ! capill>y end is bel[ ! surface ( ! fluid to be tak5 up1 "s ( ! conta9$ air w be driv5 out1 & subsequ5t relaxation ( t pressure 7result+ 9 ! =mation ( a p>tial vacuum7 w cause ! fluid to asc5d ! capill>y tube4 ,subsequ5t compression ( ! bulb w naturally result 9 ! complete expulsion ( ! fluid f ! pipette 7,fig4 #ad74 ,illu/ration3 ,,fig4 #ad4--;,fill+ ! capill>y teat-pipette4 ,a modification ( ?is pipette1 9 :i* a con/riction or %ort l5g? ( capill>y tube is introduc$ j bel[ ! plugg$ m\? 7,fig4 #ac1 b71 w also be f\nd extremely useful 9 ! collection & /orage ( morbid exudations4 ,a ?ird =m1 :]e ! capill>y portion is ab\t #d or #e cm4 long & only =ms a small fraction ( ! entire l5g? ( ! pipette 7,fig4 #ac1 c71 w also be f\nd useful4 ,blood ,pipettes 7,fig #ae74--,special pipettes = ! collection ( fairly l>ge quantities ( blood 7z sugge/$ by ,pakes7 %\ld also be prep>$4 ,^! are made f s(t glass tub+ ( #a cm4 bore1 9 a simil> mann] to ! ,pa/eur pipettes1 except t ! po9t ( ! bl[pipe flame mu/ be us$ 9 ord] to obta9 ! %>p %\ld] at ei!r end ( ! c5tral bulb4 ,! t]m9al tubes mu/ reta9 a diamet] ( at lea/ #a mm41 9 ord] to avoid capill>y action dur+ ! collection ( ! fluid4 ,illu/ration3 ,,fig4 #ae4--;,blood pipettes & hair-lip p9 9 a te/-tube4 ,illu/ration3 ,,fig4 #af4--;,blood-pipette 9 metal !rmomet] case4 ,= /]ilisation & /orage ea* pipette is plac$ inside a te/-tube1 re/+ on a wad ( cotton-wool1 & ! tube plugg$ 9 ! ord9>y mann]4 ,z ^! tubes are us$ almo/ exclusively = blood "w1 x is usual to place a lance-head$ h>e-lip p9 or a ,no4 #i flat ,hag$orn ne$le inside ! tube s t ! entire outfit may be /]ilis$ at "o "t4 ,= ! collection ( small quantities ( blood = agglut9ation reactions & ! l1 _m pref] a %ort /raight piece ( n>r[ glass tub+ drawn out at ei!r extremity to almo/ capill>y dim5sions4 ,su* pipettes1 ab\t #h cm4 9 l5g? ov] all1 are mo/ conv5i5tly /]iliz$ 9 ord9>y metal !rmomet] cases 7,fig4 #af74 ,graduat$ ,capill>y ,pipettes 7,fig4 #ag74--,^! %\ld also be made 9 ! laboratory--f manomet] tub+--( simple1 conv5i5t %ape1 & graduat$ by ! aid ( /&>d pipettes 79 hundr$?s7 to conta9 su* quantities z #aj1 #ej1 & #ij c4 mm41 & c>efully m>k$ ) a writ+ diamond4 ,^!1 previ\sly /]ilis$ 9 l>ge te/-tubes1 w be f\nd extremely useful 9 prep>+ accurate p]c5tage solutions1 :5 only m9ute quantities ( fluid are available4 ,illu/ration3 ,,fig4 #ag4--;,capill>y graduat$ pipettes4 ,automatic 7,?rottle7 ,pipettes4--,^! +5i\s pipettes1 introduc$ by ,wright1 c easily be calibrat$ 9 ! laboratory & are exce$+ly useful = graduat+ small pipettes1 = measur+ small quantities ( fluids1 9 prep>+ dilutions ( s]um = agglut9ation reactions1 etc4 ,!y are usually made f ! ,capill>y ,pa/eur pipettes 7,fig4 #ac1 a74 ,! foll[+ description ( ! manufacture ( a #e c4 mm4 pipette w s]ve to %[ h[ ! small automatic pipettes are calibrat$4 #a4 ,select a pipette ! capill>y portion ( :i* is fairly roomy 9 bore & possesses regul> ev5 walls1 & remove ! cotton-wool plug f ! op5 end4 #b4 ,heat ! capill>y portion ne> ! free extremity 9 ! by-pass flame ( ! buns5 burn] & draw x out into a v f9e hair-l tube & break ?is across4 ,?is hair-l extremity w p]mit ! passage ( air b is too f9e = metallic m]cury to pass4 #c4 ,f a /&>d graduat$ pipette deliv] #e c4 mm4 clean m]cury into ! upp] wide portion ( ! pipette4 #d4 ,adju/ a rubb] teat to ! pipette & by pressure on ! bulb gradually drive ! m]cury 9 an unbrok5 column d[n ! capill>y tube until x is /opp$ by ! fili=m extremity4 #e4 ,cut (f ! capill>y tube exactly at ! upp] level ( ! column ( m]cury1 inv]t x & all[ ! m]cury to run out4 #f4 ,snap (f ! rema9d] ( ! capill>y tube f ! broad upp] portion ( ! pipette :i* is n[ de/9$ to =m ! cov]+ tube or air *amb]1 or :at we may t]m ! b>rel4 ,?is b>rel n[ has ! l[] end 9 ! =m ( a truncat$ cone1 ! upp] end be+ cut squ>e4 ,remove ! teat4 #g4 ,introduce ! capill>y tube into ?is b>rel ) ! fili=m extremity upp]mo/1 & ! squ>e cut end project+ ab\t #j4e cm4 beyond ! tap]+ end ( ! b>rel4 ,illu/ration3 ,,fig4 #ah4--,?rottle pipette--small capacity4 #h4 ,drop a small pellet ( seal+ wax into ! b>rel by ! side ( ! capill>y tube & !n w>m ! tube at ! gas flame until ! wax becomes s(t5$ & makes an air-tight jo9t betwe5 ! capill>y tube & ! end ( ! b>rel4 #i4 ,fit a rubb] teat to ! op5 end ( ! b>rel1 & s complete a pipette :i* c be dep5d$ ^u to always aspirate & deliv] exactly #e cm4 ( fluid4 ,slight modification ( ?is proc$ure is necess>y 9 mak+ tubes to measure l>g] volumes ?an say #ge c4 mm4 ,?us to make a ?rottle pipette to measure #ajj c4 mm43 #a4 ,take a %ort l5g? ( quill tub+ & draw out "o end into a roomy capill>y /em1 & aga9 draw out ! extremity into a f9e hair po9t1 ?us =m+ a small ,pa/eur pipette ) a hair-l capill>y extremity4 #b4 ,) a /&>d pipette fill #ajj c4 mm4 into ! neck ( ?is pipette1 & make a scrat* ) a writ+ diamond at ! upp] level 7a7 ( ! m]cury m5iscus 7,fig4 #ai1 ,a74 ,illu/ration3 ,,fig4 #ai4--,mak+ ?rottle pipettes--l>ge capacity ,n[ =ce ! m]cury d[n into ! capill>y /em z f> z x w g1 s z to leave ! upp] "p ( ! tube 9 ! region ( ! diamond scrat* empty 7,fig4 #ai1 ,b74 #c4 ,heat ! tube 9 ! region ( ! diamond scrat* 9 ! bl[pipe flame1 & remov+ ! tube f ! flame draw x out s t ! diamond scrat* n[ occupies a position some:]e ne> ! c5tre ( ?is new capill>y portion 7,fig4 #ai1 ,c74 #d4 ,heat ! tube 9 ?is position 9 ! peep flame ( ! ,buns5 burn]1 & draw x out into a hair-l extremity4 ,snap (f ! glass tube1 leav+ ab\t #e mm4 ( hair-l extremity atta*$ to ! upp] capill>y portion 7,fig4 #ai1 ,d74 ,all[ ! glass to cool4 #e4 ,lift up ! bulb by ! long capill>y /em & all[ ! m]cury to return to its orig9al position--an op]ation :i* w be facilitat$ by snapp+ (f ! hair-l extremity f ! long piece ( capill>y tub+4 #f4 ,m>k on ! capill>y /em ) a grease p5cil ! position ( ! end ( ! column ( m]cury 7,fig4 #ai1 ,e47 #g4 ,w>m ! capill>y tub+ at ?is spot 9 ! peep flame ( ! ,buns5 burn]1 & draw x out v slightly s t :5 cut at ?is position a po9t$ extremity w be obta9$4 #h4 ,) a glass-cutt+ knife cut ! capill>y tube ?r\gh at ! po9t b1 & all[ ! m]cury to run out4 #i4 ,n[ apply a ?ick lay] ( seal+ wax to ! neck ( ! bulb4 #aj4 ,take a piece ( #e mm4 bore glass tub+ & draw x out z if mak+ an ord9>y ,pa/eur pipette4 #aa4 ,break ! capill>y portion (f s z to leave a cov]+ tube simil> to t already us$ = ! small] graduat$ pipettes4 ,into ?is cov]+ tube drop ! graduat$ bulb & draw ! capill>y /em d[n ?r\gh ! conical extremity until fur!r progress is /opp$ by ! lay] ( seal+ wax4 #ab4 ,w>m ! pipette 9 ! gas flame s z to melt ! seal+ wax & make an air-tight jo9t4 #ac4 ,fit an india-rubb] teat ov] ! op5 end ( ! cov]+ tube1 & ! automatic pipette is ready = use 7,fig4 #ai1 ,f74 ,s$im5tation ,pipettes 7,fig4 #bj74--,^! are prep>$ f #aj cm4 l5g?s ( n>r[ glass tub+ by seal+ "o extremity1 bl[+ a small bulb at ! c5tre1 & plugg+ ! op5 end ) cotton-wool2 aft] /]ilisation ! op5 end is provid$ ) a %ort piece ( rubb] tub+ & a glass m\?piece4 ,:5 x is necess>y to obs]ve s$im5tation reactions 9 v small quantities ( fluid1 ^! tubes w be f\nd mu* m conv5i5t ?an ! #e by #j4e cm4 te/-tubes previ\sly m5tion$4 ,illu/ration3 ,,fig4 #bj4--,s$im5tation pipette4 ,pa/eur pipettes fitt$ ) india-rubb] teats w also be f\nd useful = s$im5tation te/s :5 deal+ ) m9ute quantities ( s]um1 etc4 ,illu/ration3 ,,fig4 #ba4--;,f]m5tation tubes4 ,f]m5tation ,tubes 7,fig4 #ba74--,^! are us$ = ! collection & analysis ( ! gases lib]at$ f ! m$ia dur+ ! gr[? ( "s v>ieties ( bact]ia & may be ei!r pla9 7a7 or graduat$ 7b74 ,a simple =m 7,fig4 #ba1 c7 may be made f #ad cm4 l5g?s ( s(t glass tub+ ( #a4e cm4 diamet]4 ,! ,buns5 flame is appli$ to a spot "s #e cm4 f "o end ( su* a piece ( tub+ & ! tube slightly drawn out to =m a con/riction1 ! con/rict$ "p is b5t 9 ! bat's-w+ flame1 to an acute angle1 & ! op5 extremity ( ! long arm seal$ (f 9 ! bl[pipe flame4 ,! op5 end ( ! %ort arm is r\nd$ (f & !n plugg$ ) cotton-wool1 & ! tube is ready = /]ilisation4 ,,clean+ ,,( ,,glass ,,app>atus4 ,all glassw>e us$ 9 ! bact]iological laboratory mu/ be ?or\ghly clean$ be=e use1 & ?is rule applies z =cibly to new z to old app>atus1 al?\gh ! me?ods employ$ may v>y slightly4 ,to ,clean ,new ,te/-tubes4-- #a4 ,place ! tubes 9 a bucket or o!r conv5i5t receptacle1 fill ) wat] & add a h&ful ( ,sapon or o!r soap p[d]4 ,see t ! tubes are full & subm]g$4 #b4 ,fix ! bucket ov] a l>ge ,buns5 flame & boil = ?irty m9utes--or boil 9 ! autoclave = a simil> p]iod4 #c4 ,cleanse ! int]ior ( ! tubes ) ! aid ( te/-tube bru%es1 & r9se ?or\ghly 9 cold wat]4 #d4 ,inv]t ! tubes & all[ !m to dra9 completely4 #e4 ,dry ! tubes & poli% ! glass inside & out ) a s(t clo?1 su* z selvyt4 ,new flasks1 plates1 & capsules mu/ be clean$ 9 a simil> mann]4 ,to ,clean ,new ,graduat$ ,pipettes4-- #a4 ,place ! pipettes 9 a conv5i5t receptacle1 fill$ ) wat] to :i* soap p[d] has be5 add$4 #b4 ,boil ! wat] vigor\sly = tw5ty m9utes ov] a ,buns5 flame4 #c4 ,r9se ! pipettes 9 runn+ wat] & dra94 #d4 ,run di/ill$ wat] ?r\gh ! pipettes & dra94 #e4 ,run rectifi$ spirits ?r\gh ! pipette & dra9 z completely z possible4 #f4 ,place ! pipettes 9 ! hot-air ov5 7vide page #ca71 close ! door1 op5 ! v5tilat+ slide1 & run ! temp]ature sl[ly up to ab\t #hj deg4 ,c4 ,turn (f ! gas & all[ ! ov5 to cool4 ,or #f;a4 ,atta* ea* pipette 9 turn to ! rubb] tube ( ! foot bell[s1 or bl[pipe air-bla/1 & bl[ air ?r\gh ! pipette until ! int]ior is dry4 ,glassw>e t has already be5 us$ is reg>d$ z infect$1 & is treat$ 9 a slightly diff]5t mann]4 ,infect$ ,te/-tubes4-- #a4 ,pack ! tubes 9 ! wire basket ( ! autoclave 7hav+ previ\sly remov$ ! cotton-wool plugs1 caps1 etc471 9 ! v]tical position1 & be=e replac+ ! basket see t !re is a suffici5cy ( wat] 9 ! bottom ( ! boil]4 ,n[ atta* a piece ( rubb] tub+ to ! ne>e/ wat] tap1 & by means ( ?is fill ea* tube ) wat]4 #b4 ,dis9fect completely by expos+ ! tubes1 etc41 to a temp]ature ( #abj deg4 ,c4 = tw5ty m9utes 7vide page #cg74 7,if an autoclave is n available1 ! tubes mu/ be plac$ 9 a dige/]1 or ev5 a l>ge pan or pail ) a tightly fitt+ cov]1 & boil$ vigor\sly = "s ?irty to =ty-five m9utes to ensure dis9fection47 #c4 ,:il/ /ill hot1 empty ea* tube 9 turn & r\ghly clean its int]ior ) a /iff te/-tube bru%4 #d4 ,place ! tubes 9 a bucket or o!r conv5i5t receptacle1 fill ) wat] & add a h&ful ( ,sapon or o!r soap p[d]4 ,see t ! tubes are full & subm]g$4 #e4 ,fix ! bucket ov] a l>ge ,buns5 flame & boil = ?irty m9utes4 #f4 ,cleanse ! int]ior ( ! tubes ) ! aid ( te/-tube bru%es1 & r9se ?or\ghly 9 cold wat]4 #g4 ,dra9 (f ! wat] & imm]se tubes 9 a l>ge j> conta9+ wat] acidulat$ ) #b to #e p] c5t4 hydro*loric acid4 ,all[ !m to rema9 !re = ab\t fifte5 m9utes4 #h4 ,remove f ! acid j>1 dra91 r9se ?or\ghly 9 runn+ wat]1 !n ) di/ill$ wat]4 #i4 ,inv]t ! tubes & all[ !m to dra9 completely4 ,dry ! tubes & poli% ! glass inside & out ) a s(t clo?1 su* z selvyt4 ,infect$ flasks1 plates1 & capsules mu/ be treat$ 9 a simil> mann]4 ,flasks :i* h be5 us$ only 9 ! prep>ation ( m$ia mu/ be clean$ imm$iately !y are f9i%$ )4 ,fill ea* flask ) wat] to :i* "s soap p[d] & a few cry/als ( potassium p]manganate h be5 add$1 & let boil ov] ! nak$ flame4 ,! int]ior ( ! flask c !n usually be p]fectly clean$ ) ! aid ( a flask bru%1 b 9 "s cases wat] acidulat$ ) #e p] c5t4 nitric acid1 or a l>ge wad ( wet cotton-wool previ\sly roll$ 9 silv] s&1 mu/ be %ak5 ar\nd ! int]ior ( ! flask1 aft] :i* r9se ?or\ghly ) clean wat]1 dry1 & poli%4 ,infect$ ,pipettes4-- #a4 ,plunge infect$ pipettes imm$iately aft] use into tall glass cyl9d]s conta9+ a #b p] c5t4 solution ( lysol1 & all[ !m to rema9 !re9 = "s days4 #b4 ,remove f ! j> & dra94 ,boil 9 wat] to :i* a little soap has be5 add$1 = ?irty m9utes4 #c4 ,r9se ?or\ghly 9 cold wat]4 #d4 ,imm]se 9 #e p] c5t4 nitric acid = an h\r or two4 #e4 ,r9se aga9 9 runn+ wat] to remove all traces ( acid4 #f4 ,complete ! clean+ z describ$ "u new pipettes4 ,:5 deal+ ) graduat$ capill>y pipettes employ$ = blood or s]um "w 7:e!r new or infect$71 mu* "t is consum$ 9 ! v>i\s /eps f #e onw>d1 & ! cleans+ process c be mat]ially ha/5$ if ! foll[+ device is adopt$4 ,fit up a l>ge-siz$ ,kitasato's filt] flask to a ,spr5gel's suction pump or a ,g]yk air pump 7see page #dc74 ,to ! side tubulure ( ! filt] flask atta* a #bj cm4 l5g? ( rubb] pressure tub+ hav+ a calibre suffici5tly l>ge to admit ! ends ( ! pipettes4 ,next fill a small beak] ) di/ill$ wat]4 ,atta* ! fir/ pipette to ! free end ( ! rubb] tub+1 place ! pipette po9t d[nw>d 9 ! beak] ( wat] & />t ! pump 7,fig4 #bb74 ,illu/ration3 ,,fig4 #bb4--;,clean+ blood pipettes4 ,:5 all ! wat] has be5 aspirat$ ?r\gh ! pipette into ! filt] flask1 fill ! beak] ) rectifi$ _s & :5 ?is is exhau/$ refill ) e!r4 ,deta* ! pipette & dry 9 ! hot-air ov54 ,slides & cov]-slips 7,fig4 #bc71 :5 fir/ pur*as$1 h greasy surfaces1 ^u :i* wat] ga!rs 9 m9ute drops & effectually prev5ts ! spread+ ( ?91 ev5 films4 ,microscopical ,slides4--,! slides 9 g5]al use are ^? kn[n z ?ree by "o slips 7measur+ #c in*es by #a in*1 or #gf by #bf mm471 & %\ld be ( good :ite cr[n glass1 ) gr\nd edges4 ,new slides %\ld be all[$ to rema9 9 alcohol acidulat$ ) #e p] c5t4 hydro*loric acid = "s h\rs1 r9s$ 9 runn+ wat]1 r\ghly dra9$ on a t[el1 dri$1 & f9ally poli%$ ) a selvyt clo?4 ,illu/ration3 ,,fig4 #bc4--,slides & cov]-slips1 actual size4 ,if only a few slides are requir$ = imm$iate use a good plan is to rub ! surface ) jewel]'s em]y pap] 7,hub]t's #jj74 ,a piece ( h>d wood #gfx#bfx#bf mm4 ) a piece ( ?is em]y pap] gumm$ tightly ar\nd x is an exce$+ly useful article on ! microscope b5*4 ,cov]-slips4--,! mo/ useful sizes are ! #ai mm4 squ>es = ord9>y cov]-glass film prep>ations1 & #ch by #ai mm4 rectangles = blood films & s]ial sections2 bo? v>ieties mu/ be ( ,no4 #a ?ickness1 :i* v>ies betwe5 #j4ae & #j4bb mm41 t !y may be available = use ) ! high-p[] imm]sion l5ses4 ,cov]-slips %\ld be clean$ 9 ! foll[+ mann]3 #a4 ,drop ! cov]-slips "o by "o into an enamell$ iron pot or tall glass beak]1 conta9+ a #aj p] c5t4 solution ( *romic acid4 #b4 ,heat ov] a ,buns5 flame & all[ ! acid to boil g5tly = tw5ty m9utes4 ,,note4--,a few pieces ( pipe-clay or pumice may be plac$ 9 ! beak] to prev5t ! spurt+ ( ! *romic acid4 #c4 ,turn ! cov]-slips out into a flat glass di% & wa% 9 runn+ wat] "u ! tap until all trace ( yell[ col\r has disappe>$4 ,dur+ ! wa%+ keep ! cov]-slips 9 motion by imp>t+ a rotatory movem5t to ! di%4 #d4 ,wa% 9 di/ill$ wat] 9 a simil> mann]4 #e4 ,wa% 9 rectifi$ _s4 #f4 ,transf] ! cov]-slips1 by means ( a pair ( clean =ceps1 previ\sly heat$ 9 ! ,buns5 flame to de/roy any trace ( grease1 to a small beak] ( absolute alcohol4 ,dra9 (f ! alcohol & transf] ! cov]-slips1 by means ( ! =ceps1 to a wide-m\!d glass pot1 conta9+ absolute alcohol1 9 :i* !y are to be /or$1 & /opp] tightly4 ,,note4--,aft] once be+ plac$ 9 ! *romic acid1 ! cov]-slips mu/ on no acc\nt be t\*$ by ! f+]s4 ,us$ ,slides & ,cov]-slips4--,us$ slides ) ! m\nt$ cov]-slip prep>ations1 & cov]-slips us$ = hang+-drop m\nts1 %\ld1 :5 disc>d$1 be ?r[n into a pot conta9+ a #b p] c5t4 solution ( lysol4 ,aft] imm]sion !re9 = a week or s1 ev5 ! cov]-slips m\nt$ ) ,canada balsam c be readily deta*$ f _! slides4 ,slides4-- #a4 ,wa% ! slides ?or\ghly 9 runn+ wat]4 #b4 ,boil ! slides 9 wat] to :i* sapon has be5 add$1 = half an h\r4 #c4 ,r9se ?or\ghly 9 cold wat]4 #d4 ,dry & poli% ) a dry clo?4 ,cov]-slips4-- #a4 ,wa% ! cov]-slips ?or\ghly 9 runn+ wat]4 #b4 ,boil ! cov]-slips 9 #aj p] c5t4 solution ( *romic acid1 z = new cov]-slips4 #c4 ,wa% ?or\ghly 9 runn+ wat]4 #d4 ,pick out ^? cov]-slips :i* %[ mu* adh]5t dirty matt]1 & rub !m betwe5 ?umb & =ef+] "u ! wat] tap4 ,! dirt usually rubs (f easily1 z x has become friable f contact ) ! *romic acid4 #e4 ,return all ! cov]-slips to ! beak]1 fill 9 fre% *romic acid solution1 & treat z new cov]-slips4 ,,note4--,te/-tubes1 plates1 capsules1 etc41 :i*1 f long use1 h become scrat*$ & hazy1 or :i* _c be clean$ 9 any o!r way1 may be dealt ) by imm]s+ !m 9 an enamell$ iron ba?1 conta9+ wat] acidulat$ to #a p] c5t4 ) hydr(luoric acid1 = t5 m9utes1 r9s+ ?or\ghly 9 wat]1 dry+1 & poli%+4 ,,plugg+ ,,te/-,,tubes ,,& ,,flasks4 ,be=e /]ilisation all te/-tubes & flasks mu/ be c>efully plugg$ ) cotton-wool1 & = ?is purpose be/ absorb5t cotton-wool 7pref]ably t put up 9 cyl9drical "o-p\nd packets & int]leav$ ) tissue pap]--kn[n z surgeons' wool7 %\ld be employ$4 #a4 ,= a te/-tube or a small flask1 te> a /rip ( cotton-wool "s #aj cm4 long by #b cm4 wide f ! roll4 #b4 ,turn 9 ! ends neatly & roll ! /rip ( wool lightly betwe5 ! ?umb & f+]s ( bo? h&s to =m a long cyl9d]4 #c4 ,d\ble ?is at ! c5tre & introduce ! n[ r\nd$ end into ! op5 m\? ( ! tube or flask4 #d4 ,n[1 :il/ support+ ! wool betwe5 ! ?umb & f+]s ( ! "r h&1 rotate ! te/-tube betwe5 ^? ( ! left1 & gradually screw ! plug ( wool into its m\? = a di/ance ( ab\t #b4e cm41 leav+ ab\t ! same l5g? ( wool project+4 ,illu/ration3 ,,fig #bd44--,plugg+ te/-tubes3 a1 cyl9d] ( wool be+ roll$2 b1 cyl9d] ( wool be+ d\bl$2 c1 cyl9d] ( wool be+ ins]t$ 9 tube4 ,! plug mu/ be firm & fit ! tube or flask fairly tightly1 suffici5tly tightly 9 fact to be> ! weight ( ! glass plus ! am\nt ( m$ium ! vessel is int5d$ to conta91 b n s tightly z to prev5t x f be+ easily remov$ by a screw+ motion :5 grasp$ betwe5 ! f\r?1 or ?ird & f\r?1 f+]s1 & ! palm ( ! h&4 ,= a l>ge flask a simil> b l>g] /rip ( wool mu/ be tak52 ! me?od ( mak+ & ins]t+ ! plug is id5tical4 ,,iii4 ,,me?ods ,,( ,,/]ilisation4 ,,/]ilis+ ,,ag5ts4 ,/]ilisation--i4 e41 ! removal or ! de/ruction ( g]m life--may be effect$ by ! use ( v>i\s ag5ts4 ,z appli$ to ! practical requirem5ts ( ! bact]iological laboratory1 _m ( ^! ag5ts1 su* z electricity1 sunlight1 etc41 are ( little value1 o!rs are limit$ 9 _! applications2 o!rs aga9 are s well suit$ to p>ticul> purposes t _! use is almo/ entirely re/rict$ to su*4 ,! /]ilis+ ag5ts 9 common use are3 ,*emical ,reag5ts4--,dis9fectants 7= ! dis9fection ( glass & metal app>atus & ( morbid tissues74 ,physical ,ag5ts4 ,,heat4--7a7 ,dry ,heat3 #a4 ,nak$ flame 7= ! /]ilisation ( plat9um ne$les1 etc474 #b4 ,muffle furnace 7= ! /]ilisation ( filt] c&les1 & = ! de/ruction ( morbid tissues74 #c4 ,hot air 7= ! /]ilisation ( all glassw>e & ( metal app>atus74 7b7 ,moi/ ,heat3 #a4 ,wat] at #ef deg4 ,c4 7= ! /]ilisation ( c]ta9 album9\s fluids74 #b4 ,wat] at #ajj deg4 ,c4 7= ! /]ilisation ( surgical in/rum5ts1 rubb] tub+1 & /opp]s1 etc474 #c4 ,/ream+ /eam at #ajj deg4 ,c4 7= ! /]ilisation ( m$ia74 #d4 ,sup]heat$ /eam at #aae deg4 ,c4 or #abj deg4 ,c4 7= ! dis9fection ( contam9at$ articles & ! de/ruction ( old cultivations ( bact]ia74 ,,filtration4-- #a4 ,cotton-wool filt]s 7= ! /]ilisation ( air & gases74 #b4 ,porcela9 filt]s 7= ! /]ilisation ( v>i\s liquids74 ,,me?ods ,,( ,,application4 ,*emical ,reag5ts1 su* z belong to ! class kn[n z antiseptics 7i4 e41 sub/ances :i* inhibit ! gr[? (1 b d n de/roy1 bact]ial life71 are obvi\sly useless4 ,dis9fectants or g]micides 7i4 e41 sub/ances :i* de/roy bact]ial life71 on ! o!r h&1 are ( value 9 ! dis9fection ( morbid mat]ial1 & also ( v>i\s pieces ( app>atus1 su* z pipettes1 p5d+ _! cleans+ & complete /]ilisation by o!r processes4 ,to ?is class 79 ord] ( g5]al utility7 belong3 ,lysol1 #b p] c5t4 solution2 ,p]*loride ( m]cury1 #j4a p] c5t4 solution2 ,c>bolic acid1 #e p] c5t4 solution2 ,absolute alcohol2 ,e!r2 ,*lor(orm2 ,camphor2 ,?ymol2 ,toluol2 ,volatile oils1 su* z oil ( mu/>d1 oil ( g>lic4 ,=maldehyde is a p[]ful g]micide1 b its p5etrat+ vapor re/ricts its use4 ,^! dis9fectants are b little us$ 9 ! f9al /]ilisation ( app>atus1 *iefly on acc\nt ( ! difficulty ( effect+ _! complete removal1 = ! pres5ce ( ev5 traces ( ^! *emicals is suffici5t to s inhibit or alt] ! gr[? ( bact]ia z to vitiate subsequ5t exp]im5ts conduct$ by ! aid ( app>atus /]ilis$ 9 ?is mann]4 ,,note4--,tubes1 flasks1 filt] flasks1 pipettes1 glass tub+1 etc41 may be rapidly /]ilis$1 9 case ( em]g5cy1 by wa%+1 9 turn1 ) di/ill$ wat]1 p]*loride ( m]cury solution1 alcohol1 & e!r1 dra9+1 & f9ally g5tly heat+ ov] a gas flame to completely drive (f ! e!r vapor4 ,*lor(orm or o!r volatile dis9fectants may be add$ to v>i\s fluids 9 ord] to effect ! de/ruction ( conta9$ bact]ia1 & :5 ?is has be5 done1 may be completely driv5 (f f ! fluid by ! application ( g5tle heat4 ,dry ,heat4--,! nak$ flame ( ! ,buns5 burn] is inv>iably us$ = /]ilis+ ! plat9um ne$les 7:i* are heat$ to r$ness7 & may be employ$ = /]ilis+ ! po9ts ( =ceps1 or o!r small in/rum5ts1 cov]-glasses1 pipettes1 etc41 a v %ort exposure to ?is heat be+ suffici5t4 ,e!r ,flame4--,9 an em]g5cy small in/rum5ts1 ne$les1 etc41 may be /]ilis$ by dipp+ !m 9 e!r & aft] removal light+ ! adh]5t fluid & all[+ x to burn (f ! surface ( ! in/rum5ts4 ,repeat ! process twice4 ,x may !n be safely assum$ t ! app>atus s treat$ is /]ile4 ,illu/ration3 ,,fig4 #be4--,muffle furnace4 ,muffle ,furnace 7,fig4 #be74--;,al?\gh ?is =m ( heat is *iefly us$ = ! de/ruction ( ! dead bodies ( small infect$ animals1 morbid tissues1 etc41 x is also employ$ = ! /]ilisation ( porcela9 filt] c&les 7vide p4 #db74 ,filt] c&les are dis9fect$ imm$iately aft] use by boil+ 9 a beak] ( wat] = "s fifte5 or tw5ty m9utes4 ,?is treatm5t1 h[ev]1 leaves ! dead bodies ( ! bact]ia ^u ! surface & block+ ! int]/ices ( ! filt]4 ,to de/roy ! organic matt] & prep>e ! filt] c&le = fur!r use proce$ z foll[s3 #a4 ,roll ea* b\gie up 9 a piece ( asbe/os clo?1 secure ! ends ( ! clo? ) a few turns ( copp] wire1 & place inside ! muffle 7a small muffle #gfx#hhx#afc mm4 w hold p]haps f\r small filt] c&les74 #b4 ,light ! gas & raise ! cont5ts ( ! muffle to a :ite heat2 ma9ta9 ?is temp]ature = five m9utes4 #c4 ,ext+ui% ! gas1 & :5 ! muffle has become q cold remove ! filt] c&les1 & /ore !m 7)\t remov+ ! asbe/os wrapp+s7 9 /]ile metal boxes4 ,,note4--,! too rapid cool+ ( ! c&les1 su* z takes place if !y are remov$ f ! muffle be=e x has cool$ d[n to ! room temp]ature1 may give rise to microscopic cracks & flaws :i* w effectually de/roy _! effici5cy4 ,hot ,air4--,hot air at #aej deg4 ,c4 de/roys all bact]ia1 spores1 etc31 9 ab\t ?irty m9utes2 a mom5t>y exposure to a temp]ature ( #age deg4 to #ahj deg4 ,c4 w effect ! same result & (f]s ! m conv5i5t me?od ( /]ilisation4 ,?is me?od is only applicable to glass & metallic sub/ances1 & ! small bulk ( cotton-wool compris$ 9 ! te/-tube plugs1 etc4 ,l>ge masses ( fabric are n effectually /]ilis$ by dry heat--%ort ( *>r+--z its p[] ( p5etration is n great4 ,/]ilisation by hot air is effect$ 9 ! hot-air ov5 7,fig4 #ah74 ,?is is a rectangul>1 d\ble-wall$ metal box1 m\nt$ on a /& & heat$ f bel[ by a l>ge ,buns5 burn]4 ,! int]ior ( ! ov5 is provid$ ) loose %elves ^u :i* ! articles to be /]ilis$ are arrang$1 ei!r s+ly or pack$ 9 squ>e wire baskets or crates1 kept specially = ?is purpose4 ,"o ( ! sides is h+$ to =m a door4 ,! c5tral portion ( ! metal bottom1 on :i* ! ,buns5 flame w\ld play1 is cut away1 & replac$ by firebrick plates1 :i* slide 9 metal grooves & are easily replac$ :5 brok5 or worn out4 ,! top ( ! ov5 is provid$ ) a p]=at$ v5tilator slide & two tubulures1 ! "o = ! reception ( a c5tigrade !rmomet] graduat$ to #bjj deg4 or #bej deg4 ,c41 ! o!r = a !rmo-regulator4 ,an ord9>y m]curial !rmo-regulator may be us$ b x is pref]able to employ a regulat+ capsule ( ! ,he>son type 7see p4 #bai7 ) a spr+ arm adju/$ to ! lev] s t :5 ! boil+-po9t ( ! capsule 7e4 g41 #age deg4 ,c47 is rea*$ ! gas supply is absolutely cut (f & ! jet _c aga9 be light$ until ! spr+-arm has be5 readju/$ by h&4 ,! !rmo-regulator is by no means a necessity1 & may be replac$ by a l>ge bore !rmomet] ) a slid+ plat9um po9t1 connect$ ) an electric bell1 :i* c be easily adju/$ to r+ at any giv5 temp]ature4 ,ev5 if ! /]ilis] is provid$ ) ! capsule regulator above describ$ ! contact !rmomet] %\ld also be fitt$4 ,illu/ration3 ,,fig4 #bf4--;,hot-air ov54 ,,to ,,use ,,! ,,hot-,,air ,,ov54-- #a4 ,place ! crates ( te/-tubes1 metal cases conta9+ plates & pipettes1 loose app>atus1 etc41 inside ! ov51 tak+ p>ticul> c>e t none ( ! cotton-wool plugs are 9 contact ) ! walls1 o!rwise ! heat transmitt$ by ! metal w *> or ev5 flame !m4 ,to prep>e a wire crate = ! reception ( te/-tubes1 etc41 cov] ! bottom ) a lay] ( ?ick asbe/os clo?2 or take "s asbe/os fibre1 moi/5 x ) a little wat] & knead x into a pa/e2 pla/] ! pa/e ov] ! bottom ( ! crate1 work+ x into ! me%es & smoo?+ ! surface by means ( a pe/le4 ,:5 sev]al crates h be5 ?us treat$1 place !m inside ! hot-air ov51 close ! door1 op5 ! v5tilat+ slide1 light ! gas1 & run ! temp]ature ( ! int]ior up to ab\t #afj deg4 ,c4 ,aft] an int]val ( t5 m9utes ext+ui% ! gas1 op5 ! ov5 door1 & all[ ! cont5ts to cool4 ,! asbe/os n[ =ms a smoo?1 dry1 spongy lay] ov] ! bottom1 :i* w la/ _m mon?s be=e ne$+ r5ewal1 & w consid]ably dim9i% ! loss ( tubes f breakage4 ,copp] cyl9d]s & l>ge te/-tubes int5d$ = ! reception ( pipettes are prep>$ 9 a simil> mann]1 9 ord] to protect ! po9ts ( ^! articles f injury4 #b4 ,close ! ov5 door1 & op5 ! v5tilat+ slide1 9 ord] t any moi/ure left 9 ! tubes1 etc41 may escape2 light ! gas bel[2 set ! electric al>m to r+ at #ajj deg4 ,c4 #c4 ,:5 ! temp]ature ( ! ov5 has rea*$ #ajj deg4 ,c41 close ! v5tilat+ slide2 reset ! al>m to r+ at #age deg4 ,c4 #d4 ,run ! temp]ature up to #age deg4 ,c4 #e4 ,ext+ui% ! gas at once1 & all[ ! app>atus to cool4 #f4 ,:5 ! temp]ature ( ! int]ior1 z record$ by ! !rmomet]1 has fall5 to #fj deg4 ,c4--b n be=e--! door may be op5$ & ! /]ile articles remov$ & /or$ away4 ,,note4--,neglect ( ?is precaution>y cool+ ( ! ov5 to #fj deg4 ,c4 w result 9 num]\s crack$ & brok5 tubes4 ,on removal f ! ov51 ! cotton-wool plugs w probably be slightly br[n 9 col\r4 ,metal in/rum5ts1 su* z knives1 scissors1 & =ceps1 may be /]ilis$ 9 ! hot-air ov5 z describ$ above1 b exposure to #age deg4 ,c4 is likely to s]i\sly affect ! temp] ( ! /eel & c]ta9ly blunts ! cutt+ edges4 ,if1 h[ev]1 x is desir$ to /]ilise surgical in/rum5ts by hot air1 !y %\ld be pack$ 9 a metal box1 or boxes1 & heat$ to #acj deg4 ,c4 & reta9$ at t temp]ature = ab\t ?irty m9utes4 ,moi/ ,heat4--,wat] at #ef deg4 ,c4--,?is temp]ature1 if ma9ta9$ = ?irty m9utes1 is suffici5t to de/roy ! vegetative =ms ( bact]ia1 b has practically no effect on spores4 ,its use is limit$ to ! /]ilisation ( su* album9\s fluid m$ia z w\ld coagulate at a high] temp]ature4 ,,me?od4-- #a4 ,fit up a wat]-ba?1 heat$ by a ,buns5 flame :i* is controll$ by a !rmo-regulator1 s t ! temp]ature ( ! wat] rema9s at #ef deg4 ,c4 #b4 ,imm]se ! tubes or flasks conta9+ ! album9\s fluid 9 ! wat]-ba? s t ! upp] level ( su* fluid is at lea/ #b cm4 bel[ ! level ( ! wat]4 7,! temp]ature ( ! ba? w n[ fall some:at1 b aft] a few m9utes w aga9 rise to #ef deg4 ,c74 #c4 ,aft] ?irty m9utes' exposure to #ef deg4 ,c1 ext+ui% ! gas1 remove ! tubes or flasks f ! ba?1 & subject !m to ! action ( runn+ wat] s t _! cont5ts are rapidly cool$4 #d4 ,! vegetative =ms ( bact]ia pres5t 9 ! liquid be+ kill$1 /& x = tw5ty-f\r h\rs 9 a cool1 d>k place2 at ! end ( t "t "s at lea/ ( su* spores z may be pres5t w h g]m9at$ & assum$ ! vegetative =m4 #e4 ,de/roy ^! new vegetative =ms by a simil> exposure to #ef deg4 ,c4 on ! second day1 :il/ o!rs1 ( sl[] g]m9ation1 may be caught on ! ?ird day1 & s on4 #f4 ,9 ord] to ensure ?or\gh /]ilisation1 repeat ! process on ea* ( six successive days4 ,?is me?od ( expos+ liquids to a temp]ature ( #ef deg4 ,c4 9 a wat]-ba? = half an h\r on ea* ( six successive days is t]m$ fractional /]ilisation4 ,wat] at #ajj deg4 ,c4 de/roys ! vegetative =ms ( bact]ia almo/ in/antane\sly1 & spores 9 f five to fifte5 m9utes4 ,?is me?od ( /]ilisation is applicable to ! metal in/rum5ts1 su* z knives1 =ceps1 etc41 us$ 9 animal exp]im5ts2 syr+es1 rubb] corks1 rubb] & glass tub+1 & o!r small app>atus1 & is effect$ 9 :at is usually spok5 ( z ! wat] /]ilis] 7,fig4 #bg74 ,illu/ration3 ,,fig4 #bg4--,wat] /]iliz]4 ,?is is a rectangul> copp] box1 #bf cm4 long1 #ah cm4 wide1 & #ab cm4 deep1 m\nt$ on legs1 heat$ f bel[ by a ,buns5 or radial gas burn]1 & conta9+ a movable copp] wire tray1 #b cm4 small] 9 e dim5sion ?an ! /]ilis] itself1 & provid$ ) h&les4 ,! top ( ! /]ilis] is h+$ to =m a lid4 ,,me?od4-- #a4 ,place ! in/rum5ts1 etc41 to be /]ilis$ inside ! copp] basket1 & replace ! basket 9 ! /]ilis]4 #b4 ,p\r a suffici5t quantity ( wat] into ! /]ilis]1 %ut d[n ! lid1 & light ! gas bel[4 ,illu/ration3 ,,fig4 #bh4--,ko*'s /]ilis]4 ,illu/ration3 ,,fig4 #bi4--;,arnold's /]ilis]4 #c4 ,aft] ! wat] has boil$ & /eam has be5 issu+ f b5ea? ! lid = at lea/ t5 m9utes1 ext+ui% ! gas1 op5 ! lid1 & lift out ! wire basket by its h&les & re/ x diagonally on ! walls ( ! /]ilis]2 ! conta9$ in/rum5ts1 etc41 are n[ /]ile & ready = use4 #d4 ,aft] use1 or :5 accid5tally contam9at$1 replace ! in/rum5ts 9 ! basket & return t to ! /]ilis]2 completely dis9fect by a fur!r boil+ = fifte5 m9utes4 #e4 ,aft] dis9fection1 & :il/ /ill hot1 take out ! in/rum5ts1 dry c>efully & at once1 & return !m to _! /ore cases4 ,/ream+ /eam--i4 e41 /eam at #ajj deg4 ,c4--de/roys ! vegetative =ms ( bact]ia 9 f fifte5 to tw5ty m9utes1 & ! spor+ =ms 9 f "o to two h\rs4 ,?is me?od is *iefly us$ = ! /]ilisation ( ! v>i\s nutri5t m$ia int5d$ = ! cultivation ( bact]ia1 & is c>ri$ out 9 a /eam kettle ( special con/ruction1 kn[n z ,ko*'s /eam /]ilis] 7,fig4 #bh7 or 9 "o ( its _m modifications1 ! mo/ effici5t ( :i* is ,arnold's 7,fig4 #bi74 ,! /eam /]ilis] 9 its simple/ =m consi/s ( a tall t9n$-iron or copp] cyl9drical vessel1 divid$ into two unequal p>ts by a movable p]=at$ metal diaphragm1 ! l[]1 small] portion s]v+ = a wat] res]voir1 & ! upp] "p = ! reception ( wire baskets conta9+ ! articles to be /]ilis$4 ,! vessel is clos$ by a loose conical lid1 provid$ ) h&les1 & p]=at$ at its apex by a tubulure2 x is m\nt$ on a tripod /& & heat$ f bel[ by a ,buns5 burn]4 ,! m elaborate /]ilis] is cas$ ) felt or asbe/os bo>d1 & provid$ ) a wat] gauge1 also a tap = empty+ ! wat] comp>tm5t4 ,,to ,,use ,,! ,,/eam ,,/]ilis]4-- #a4 ,fill ! wat] comp>tm5t to ! level ( ! p]=at$ diaphragm1 place ! lid 9 position1 & light ! ,buns5 burn]4 #b4 ,aft] ! wat] has boil$1 all[ suffici5t "t to elapse = /eam to replace ! air 9 ! /]ilis+ comp>tm5t1 z %[n by ! /eam issu+ 9 a /eady1 cont9u\s /ream f ! tubulure 9 ! lid4 #c4 ,remove ! lid1 quickly l[] ! wire basket conta9+ m$ia tubes1 etc41 into ! /]ilis+ comp>tm5t until x re/s on ! diaphragm1 & replace ! lid4 #d4 ,aft] an int]val ( tw5ty m9utes 9 ! case ( fluid m$ia1 or ?irty m9utes 9 ! case ( solid m$ia1 take (f ! lid & remove ! basket ) its cont5ts4 #e4 ,n[1 b n be=e1 ext+ui% ! gas4 ,,note4--,aft] remov+ tubes1 flasks1 etc41 f ! /eam /]ilis]1 !y %\ld be at once sep>at$ freely 9 ord] to prev5t moi/ure cond5s+ ^u ! cotton-wool plugs & soak+ ?r\gh into ! int]ior ( ! tubes4 ,?is treatm5t w de/roy any vegetative =ms ( bact]ia2 dur+ ! h\rs ( cool+ any spores pres5t w g]m9ate1 & ! "y organisms w be de/roy$ by repeat+ ! process tw5ty-f\r h\rs lat]2 a ?ird /]ilisation aft] a simil> int]val makes assurance d\bly sure4 ,! me?od ( /]ilis+ by exposure to /ream+ /eam at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree consecutive days is t]m$ discont9u\s or int]mitt5t /]ilisation4 ,exposure to /eam at #ajj deg4 ,c4 = a p]iod ( "o or two h\rs1 or cont9u\s /]ilisation1 _c always be dep5d$ ^u & is !re=e n to be recomm5d$4 ,sup]heat$ /eam--i4 e41 /eam "u pressure 7see ,pressure-temp]ature table1 ,app5dix1 page #ejj7 9 seal$ vessels at a temp]ature ( #aae deg4 ,c4--w de/roy bo? ! vegetative & ! spor+ =ms ( bact]ia )9 fifte5 m9utes2 if ! pressure is increas$1 & ! temp]ature rais$ to #abj deg4 ,c41 ! same end is atta9$ 9 t5 m9utes4 ,?is me?od 0 =m]ly employ$ = ! /]ilisation ( m$ia 7& inde$ is s us$ 9 "s laboratories /ill71 b mo/ work]s n[ realise t m$ia subject$ to ?is high temp]ature und]go hydrolytic *anges :i* r5d] !m unsuitable = ! cultivation ( ! m delicate micro-organisms4 ,! use ( sup]heat$ /eam %\ld be re/rict$ almo/ entirely to ! dis9fection ( su* contam9at$ articles1 old cultivations1 etc41 z _c be dealt ) by dry heat or ! actual furnace4 ,/]ilisation by means ( sup]heat$ /eam is c>ri$ out 9 a special boil]--,*amb]l&'s autoclave 7,fig4 #cj74 ,! autoclave consi/s ( a /\t copp] cyl9d]1 provid$ ) a copp] or gun-metal lid1 :i* is secur$ 9 place by means ( bolts & ?umbscrews1 ! jo9t betwe5 ! cyl9d] & its lid be+ h]metically seal$ by ! int]position ( a rubb] wa%]4 ,! cov] is p]=at$ = a bran*$ tube c>ry+ a v5t cock1 a manomet]1 & a safety valve4 ,! copp] boil] is m\nt$ 9 ! upp] half ( a cyl9drical %eet-iron case--two conc5tric circul> r[s ( ,buns5 burn]s1 ea* circle hav+ an indep5d5t gas-supply1 occupy+ ! l[] half4 ,9 ! int]ior ( ! boil] is a l>ge movable wire basket1 m\nt$ on legs1 = ! reception ( ! articles to be /]ilis$4 ,,to ,,use ,,! ,,autoclave4-- #a4 ,pack ! articles to be /]ilis$ 9 ! wire basket4 #b4 ,run wat] into ! boil] to ! level ( ! bottom ( ! basket2 also fill ! conta9$ flasks & tubes ) wat]4 #c4 ,see t ! rubb] wa%] is 9 position1 !n replace ! cov] & fa/5 x tightly on to ! autoclave by means ( ! ?umbscrews4 #d4 ,op5 ! v5t cock & light bo? r+s ( burn]s4 #e4 ,:5 /eam is issu+ 9 a /eady1 cont9u\s /ream f ! v5t tube1 %ut (f ! v5t cock & ext+ui% ! out] r+ ( gas burn]s4 #f4 ,wait until ! index ( ! manomet] records a temp]ature ( #abj deg4 ,c41 !n regulate ! gas & ! spr+ safety valve 9 su* a mann] t ?is temp]ature is j ma9ta9$1 & leave x ?us = tw5ty m9utes4 ,9 ! m exp5sive patt]ns ( autoclave ?is regulation ( ! safety valve is c>ri$ out automatically1 ! manomet] be+ fitt$ ) an adju/able po9t] :i* c be set to any requir$ pressure-temp]ature & s arrang$ t :5 ! index ( ! manomet] co9cides ) ! adju/able h& ! safety valve is op5$4 #g4 ,ext+ui% ! gas & all[ ! manomet] index to fall to z]o4 ,illu/ration3 ,,fig4 #cj4--;,*amb]l&'s ,autoclave4 #h4 ,n[ op5 ! v5t cock sl[ly1 & all[ ! int]nal pressure to adju/ itself to t ( ! atmosph]e4 #i4 ,remove ! cov] & take out ! /]ilis$ cont5ts4 ,/]ilisation ,p]iods4--,an exce$+ly useful device = ! tim+ ( /]ilisation p]iods 7& inde$ = _m o!r op]ations 9 ! laboratory7 is ! ,,electric ,,signal ,,tim+ ,,clock4 ,?is is a clock ( ,am]ican type 9 :i* ! face is surr\nd$ by a metal plate hav+ a s]ies ( #fj holes at equal di/ances ap>t1 correspond+ to ! m9utes on ! dial4 ,?is plate is connect$ ) "o ( ! poles ( a dry batt]y1 ! o!r pole ( :i* is connect$ to ! metal case ( ! clock = ! purpose ( actuat+ an ord9>y magnet al>m bell4 ,9 ! c5tre ( ea* ( ! holes 9 ! plate a metal rod is fix$1 :i* !n passes ?r\gh an insulat+ r+ & projects inside ! clock face1 :]e x makes contact ) ! h\r h&4 ,! clock is m\nt$ on a heavy base1 ) a key-bo>d conta9+ #bj numb]$ plugs4 ,if "o ( ! plugs is ins]t$ 9 a hole 9 ! plate x makes contact ) ! rod1 & :5 ! h\r h& ( ! clock t\*es ! o!r end ! circuit is complet$ & ! bell />ts r++4 ,! p]iod ( ?is friction contact is approximately #bj seconds4 ,! clock c !re=e be us$ = electrically not+ ! p]iods ( "t f "o m9ute by multiples ( "o m9ute up to "o h\r4 ,illu/ration3 ,,fig4 #ca4--;,electric signal tim+ clock4 ,filtration4--7a7 ,cotton-wool ,filt]4--,practically ! only me?od 9 use 9 ! laboratory = ! /]ilisation ( air or ( a gas is by filtration ?r\gh dry cotton-wool or glass-wool1 ! fibres ( :i* entangle ! micro-organisms & prev5t _! passage4 ,p]haps ! be/ example ( su* a filt] is ! cotton-wool plug :i* closes ! m\? ( a culture tube4 ,n only does ord9>y diffusion take place ?r\gh x1 b if a tube plugg$ 9 ! usual mann] ) cotton-wool is remov$ f ! hot incubator1 ! temp]ature ( ! conta9$ air rapidly falls to t ( ! laboratory1 & a p>tial vacuum is =m$2 air passes into ! tube1 ?r\gh ! cotton-wool plug1 to re/ore ! equilibrium1 &1 s long z ! plug rema9s dry1 9 a g]m-free condition4 ,if1 h[ev]1 ! plug becomes moi/1 ei!r by absorption f ! atmosph]e1 or f liquids com+ into contact ) x1 micro-organisms 7especially ! m\ld fungi7 comm5ce to multiply1 & ! long ?read =ms rapidly p5etrate ! sub/ance ( ! plug1 & ga9 access to & contam9ate ! int]ior ( ! tube4 ,illu/ration3 ,,fig4 #cb4--;,cotton-wool air filt]4 ,,me?od4-- ,if x is desir$ to /]ilise gases be=e admission to a vessel conta9+ a pure cultivation ( a micro-organism1 z1 = in/ance1 :5 =c+ a curr5t ( oxyg5 ov] or ?r\gh a bro? cultivation ( ! diph!ria bacillus1 ?is c be readily effect$ z foll[s3 #a4 ,take a l5g? ( glass tub+ (1 say1 #a4e cm4 diamet]1 9 ! c5tre ( :i* a bulb has be5 bl[n1 fill ! bulb ) dry cotton-wool 7,fig4 #cb71 wrap a lay] ( cotton-wool ar\nd ea* end ( ! tube1 & secure 9 position ) a turn ( ?9 copp] wire or /r+2 !n /]ilise ! piece ( app>atus 9 ! hot-air ov54 #b4 ,prep>e ! cultivation 9 a ,ruff] or ,woodhead flask 7,fig4 #cc7 ! inlet tube ( :i* has its free extremity envelop$ 9 a lay] ( cotton-wool1 secur$ by ?read or wire1 :il/ ! exit tube is plugg$ 9 ! usual mann]4 ,illu/ration3 ,,fig4 #cc4--,ruff]'s flask4 #c4 ,/]ilise a %ort l5g? ( rubb] tub+ by boil+4 ,transf] x f ! boil+ wat] to a beak] ( absolute alcohol4 #d4 ,:5 all is ready remove ! rubb] tube f ! alcohol by means ( a pair ( =ceps1 dra9 x ?or\ghly1 & pass ?r\gh ! flame ( a ,buns5 burn] to burn (f ! la/ traces ( alcohol4 #e4 ,remove ! cotton-wool wraps f ! entry tube ( ! flask & f "o end ( ! filt] tube & rapidly c\ple !m up by means ( ! /]ile rubb] tub+4 #f4 ,connect ! o!r end ( ! bulb tube ) ! deliv]y tube f ! gas res]voir4 ,! gas 9 its passage ?r\gh ! dry /]ile cotton-wool 9 ! bulb ( ! filt] tube w be fre$ f any conta9$ micro-organisms & w ent] ! flask 9 a /]ile condition4 7b7 ,porcela9 ,filt]4--,! /]ilisation ( liquids by filtration is effect$ by pass+ !m ?r\gh a cyl9drical vessel1 clos$ at "o end l a te/-tube1 & made ei!r ( por\s biscuit porcela91 h>d-burnt & unglaz$ 7,*amb]l& sy/em71 or ( ,kieselguhr1 a f9e diatomace\s e>? 7,b]kefeld sy/em71 & t]m$ a b\gie or c&le 7,fig4 #cd74 ,,note4--,9 select+ c&les = use 9 ! laboratory avoid ^? ) metal fitt+s1 s9ce dur+ /]ilisation cracks develop at ! junction ( ! metal & ! silice\s mat]ial ow+ to ! unequal expansion4 ,9 ?is me?od ! bact]ia are reta9$ 9 ! pores ( ! filt] :ile ! liquid passes ?r\gh 9 a g]m-free condition4 ,x is obvi\s t to be effective ! pores ( ! filt] mu/ be extremely m9ute1 & !re=e ! rate ( filtration w usually be sl[4 ,*amb]l& filt] c&les possess f9] *annels ?an ,b]kefeld c&les & consequ5tly filt] mu* m sl[ly4 ,to ov]come ?is disadvantage1 ei!r aspiration or pressure1 or a comb9ation ( ^! two =ces1 may be employ$ to ha/5 ! process4 ,d\ltons :ite porcela9 filt]s x may be not$ are z effici5t z ! ,*amb]l& c&les & filt] r m rapidly4 ,app>atus ,requir$4-- #a4 ,sep>atory funnel conta9+ ! unfilt]$ fluid4 #b4 ,/]ile filt] c&le 7,fig4 #cd71 ! op5 end fitt$ ) a rubb] /opp] 7,fig4 #cd1 a7 p]=at$ to receive ! deliv]y tube ( ! sep>atory funnel1 & its neck pass$ ?r\gh a l>ge rubb] wa%] 7,fig4 #cd1 b7 :i* fits ! m\? ( ! filt] flask4 #c4 ,/]ile filt] flask ( suitable size1 = ! reception ( ! filt]$ fluid1 its m\? clos$ by a cotton-wool plug4 #d4 ,wat] injector ,spr5gel 7see ,fig4 #ch1 c7 pump1 or ,g]yk's pump 7an air pump on ! hydraulic pr9ciple1 seal$ by means ( l[ vapor-t5sion oil1 ,fig4 #ce74 ,if ?is latt] is employ$1 a ,wulff's bottle1 fitt$ z a wa%-bottle & conta9+ sulphuric acid1 mu/ be int]pos$ betwe5 ! filt] flask & ! pump1 9 ord] to prev5t moi/ air rea*+ ! oil 9 ! pump4 #e4 ,air filt] 7vide page #dj7 /]ilis$4 #f4 ,pressure tub+4 #g4 ,screw clamps 7,fig4 #cf74 ,,me?od4-- #a4 ,c\ple ! exhau/ pipe ( ! suction pump ) ! lat]al tube ( ! filt] flask 7fir/ remov+ ! cotton-wool plug f ?is latt]71 by means ( pressure tub+1 int]pos+1 if necess>y1 ! wa%-bottle ( sulphuric acid4 ,illu/ration3 ,,fig4 #cd4--,porcela9 filt] c&le4 ,illu/ration3 ,,fig4 #ce4--;,g]yk air pump4 #b4 ,remove ! cotton-wool plug f ! neck ( ! filt] flask & adju/ ! porcela9 c&le 9 its place4 ,illu/ration3 ,,fig4 #cf4--,screw clamps4 #c4 ,atta* ! nozzle ( ! sep>atory funnel to ! filt] c&le by means ( ! p]=at$ rubb] /opp] 7,fig4 #cg74 ,illu/ration3 ,,fig4 #cg4--;,app>atus arrang$ = filt]+--aspiration4 #d4 ,op5 ! tap ( ! funnel1 & exhau/ ! air f ! filt] flask & wa%-bottle2 ma9ta9 ! vacuum until ! filtration is complete4 #e4 ,:5 ! filtration is complet$ close ! tap ( ! funnel2 adju/ a screw clamp to ! pressure tub+ atta*$ to ! lat]al bran* ( ! filt] flask2 screw x up tightly1 & disconnect ! acid wa%-bottle4 #f4 ,atta* ! air filt] to ! op5 end ( ! pressure tub+2 op5 ! screw clamp gradually1 & all[ filt]$ air to ent] ! flask1 to aboli% ! negative pressure4 #g4 ,deta* ! rubb] tub+ f ! lat]al bran* ( ! flask1 flame ! end ( ! bran* 9 ! ,buns51 & plug its orifice ) /]ile cotton-wool4 #h4 ,remove ! filt] c&le f ! m\? ( ! flask1 flame ! m\?1 & plug ! neck ) /]ile cotton-wool4 #i4 ,dis9fect ! filt] c&le & sep>atory funnel by boil+4 ,if x is f\nd necess>y to employ pressure 9 addition to or 9 place ( suction1 ins]t a p]=at$ rubb] /opp] into ! m\? ( ! sep>atory funnel & secure 9 position ) copp] wire2 next fit a piece ( glass tub+ ?r\gh ! /opp]1 & connect ! ext]nal orifice ) an air-pressure pump ( "s k9d 7an ord9>y foot pump su* z is employ$ = inflat+ bicycle tyres is "o ( ! mo/ g5]ally useful1 = ?is purpose7 or ) a cyl9d] ( compress$ air or o!r gas4 ,9 ord] to filt] a l>ge bulk ( fluid v rapidly x is necess>y to use a high] pressure ?an glass w\ld /&1 & 9 ^! cases ! metal receptacle design$ by ,pakes 7,fig4 #ch1 a71 to hold ! filt] c&le itself z well z ! fluid to be filt]$1 %\ld be employ$4 7,a vacuum mu/ also be ma9ta9$ 9 ! filt] flask1 by means ( an exhau/ pump1 dur+ ! entire process47 ,?is piece ( app>atus consi/s ( a brass cyl9d]1 capacity #bejj c4c41 ) two %\ld]s2 & an op5+ 9 ! neck at ea* end1 provid$ ) screw ?reads4 ,a nut c>ry+ a pressure gauge fits into ! top screw2 & into ! bottom is fitt$ a brass cyl9d] c>ry+ ! filt] c&le & prolong$ d[nw>ds into a deliv]y tube4 ,leakage is prev5t$ by means ( rubb] wa%]s4 ,into ! top %\ld] a tube is ins]t$1 b5t at "r angles & provid$ ) a tap4 ,all ! brass-"w is t9n$ inside 7,fig4 #ch1 a74 ,9 use ! res]voir is g5]ally m\nt$ on a tripod /&4 ,to ,/]ilise4-- #a4 ,ins]t ! filt] c&le into its cyl9d] & screw ?is loosely on4 ,illu/ration3 ,,fig4 #ch4--,pakes' filt]+ res]voir--pressure & aspiration4 #b4 ,wrap a lay] ( cotton-wool ar\nd ! deliv]y tube & fa/5 9 position4 #c4 ,remove ! nut c>ry+ ! pressure gauge & plug ! neck ) cotton-wool4 #d4 ,heat ! :ole app>atus 9 ! autoclave at #abj deg4 ,c4 = tw5ty m9utes4 ,,me?od4-- #a4 ,remove ! app>atus f ! autoclave1 & all[ x to cool4 #b4 ,screw home ! box c>ry+ ! b\gie4 #c4 ,set ! app>atus up 9 position1 ) its deliv]y tube 7f :i* ! cotton-wool wrapp+ has be5 remov$7 pass+ ?r\gh a p]=at$ rubb] /opp] 9 ! neck ( a filt] flask4 ,illu/ration3 ,,fig4 #ci4--;,clos$ c&le arrang$ = filt]+4 #d4 ,fill ! fluid to be filt]$ into ! cyl9d] & screw on ! nut c>ry+ ! pressure gauge4 7,?is nut %\ld be imm]s$ 9 boil+ wat] = a few m9utes previ\s to screw+ on1 9 ord] to /]ilise x47 #e4 ,connect ! horizontal arm ( ! entry tube ) a cyl9d] ( compress$ oxyg5 7or c>bon dioxide1 ,fig4 #ch1 b71 by means ( pressure tub+4 #f4 ,connect ! lat]al arm ( ! filt] flask ) ! exhau/ pump 7,fig4 #ch1 c7 & />t ! latt] work+4 #g4 ,op5 ! tap ( ! gas cyl9d]2 !n op5 ! tap on ! entry tube ( ! filt] cyl9d] & raise ! pressure 9 its int]ior until ! desir$ po9t is record$ on ! manomet]4 ,ma9ta9 ?is pressure1 usually "o or "o & a half atmosph]es1 until filtration is complet$1 by regulat+ ! tap on ! entry tube4 ,"s =ms ( filt] c&le are made ) ! op5 end contract$ into a deliv]y nozzle1 :i* is glaz$4 ,9 ?is case ! app>atus is fitt$ up 9 a slightly diff]5t mann]2 ! fluid to be filt]$ is conta9$ 9 an op5 cyl9d] into :i* ! c&le is plung$1 :ile its deliv]y nozzle is connect$ ) ! filt] flask by means ( a piece ( flexible pressure tub+ 7previ\sly /]ilis$ by boil+71 z 9 figure #ci4 ,,iv4 ,,! ,,microscope4 ,! ess5tials ( a microscope = bact]iological "w may be briefly summ$ up z foll[s3 ,illu/ration3 ,,fig4 #dj4--,microscope /&4 ,! in/rum5t1 ( ! monocul> type1 mu/ be ( good workman%ip & well f9i%$1 rigid1 firm1 & free f vibration1 n only :5 upright1 b also :5 incl9$ to an angle or 9 ! horizontal position4 ,! v>i\s jo9ts & movem5ts mu/ "w smoo?ly & precisely1 equally free f ! defects ( loss ( "t & slipp+4 ,all screws1 etc41 %\ld con=m to ! ,royal ,microscopical ,society's /&>d4 ,x mu/ also be provid$ ) good l5ses & a suffici5tly l>ge /age4 ,! details ( its compon5t p>ts1 to :i* att5tion mu/ be specially direct$1 are z foll[s3 ,illu/ration3 ,,fig4 #da4--;,foot1 ?ree types4 #a4 ,! ,base or ,foot 7,fig4 #dj1 a74--,two elem5t>y =ms--! tripod 7,fig4 #da1 a7 & ! v]tical column set into a plate kn[n z ! horse-%oe 7,fig4 #da1 b7--s]ve z ! patt]ns = c\ntless modifications 9 %ape & size ( ?is portion ( ! /&4 ,! *ief desid]ata--/ability & ease ( manipulation--are atta9$ 9 ! fir/ by means ( ! spread ( ! ?ree feet1 :i* are usually %od ) cork2 9 ! second1 by ! dead weight ( ! foot-plate4 ,! tripod is me*anically ! m correct =m1 & = practical use is mu* to be pref]r$4 ,its *ief rival1 ! ,jackson foot 7,fig4 #da1 c71 is bas$ ^u ! same pr9ciple1 & on ! score ( appe>ance has mu* to recomm5d x4 #b4 ,! body tube 7,fig4 #dj1 b7 may be ei!r t kn[n z ! long or ,engli% 7l5g? #bej mm471 or ! %ort or ,cont95tal 7l5g? #afj mm474 ,nei!r l5g? appe>s to possess any mat]ial advantage ov] ! o!r1 b x is absolutely necess>y to secure objectives :i* h be5 manufactur$ = ! p>ticul> tube l5g? *os54 ,9 ! high-class microscope ( ! pres5t day ! body tube is usually %ort] ?an ! ,cont95tal1 b is provid$ ) a draw tube :i*1 :5 fully ext5d$1 gives a tube l5g? great] ?an ! ,engli%1 ?us p]mitt+ ! use ( ei!r =m ( objective4 ,illu/ration3 ,,fig4 #db4--;,co>se adju/m5t4 ,illu/ration3 ,,fig4 #dc4--;,f9e adju/m5t4 ,= practical purposes ! tube l5g? di/ance f ! end ( ! nosepiece to ! eyeglass ( ! ocul>4 ,?is is ! measurem5t ref]r$ to 9 speak+ ( long or %ort tube4 #c4 ,! co>se adju/m5t 7,fig4 #dj1 c7 %\ld be a rack-&-p9ion movem5t1 /ead9ess & smoo?ness ( action be+ secur$ by means ( accurately fitt+ dovetail$ be>+s & p]fect correspond5ce betwe5 ! tee? ( ! rack & ! leaves ( ! p9ion 7,fig4 #db74 ,also provision %\ld be made = tak+ up ! slack 7z by ! screws ,,aa1 ,fig4 #db74 #d4 ,! f9e adju/m5t 7,fig4 #dj1 d7 %\ld on no acc\nt dep5d ^u ! direct action ( spr+s1 b %\ld be ( ! lev] patt]n1 pref]ably ! ,nelson 7,fig4 #dc74 ,9 ?is =m ! unequal l5g? ( ! arms ( ! lev] secures v delicate movem5t1 &1 moreov]1 only a small portion ( ! weight ( ! body tube is transmitt$ to ! ?read ( ! v]tical screw actuat+ ! movem5t4 ,illu/ration3 ,,fig4 #dd4--,sp9dle head to f9e adju/m5t4 ,a sp9dle mill$ head 7,fig4 #dd7 w be f\nd a v useful device to h fitt$ 9 place ( ! ord9>y mill$ head controll+ ! f9e adju/m5t4 ,9 ?is contrivance ! axis ( ! mill$ head is prolong$ upw>d 9 a %ort column1 ! diamet] ( :i* is "o-six? ( t ( ! head4 ,! sp9dle c be rapidly rotat$ betwe5 ! f+]s = m$ium p[] adju/m5ts :ile ! l>g] mill$ head c be sl[ly mov$ :5 focuss+ high p[]s4 #e4 ,! /age 7,fig4 #dj1 e7 %\ld be squ>e 9 %ape & l>ge 9 area--at lea/ #ab cm4--flat & rigid1 9 ord] to af=d a safe support = ! ,petri di% us$ = plate cultivations2 & %\ld be suppli$ ) spr+ clips 7removable at w7 to secure ! #c by #a glass slides4 ,a me*anical /age mu/ be class$ z a necessity r ?an a luxury s f> z ! bact]iologi/ is conc]n$1 z :5 work+ ) high p[]s1 & especially :5 exam9+ hang+-drop specim5s1 x is almo/ impossible to execute suffici5tly delicate movem5ts ) ! f+]s4 ,9 select+ a me*anical /age1 pref]5ce %\ld be giv5 to "o :i* =ms an integral "p ( ! in/rum5t 7,fig4 #de7 r ?an "o :i* ne$s to be clamp$ on to an ord9>y pla9 /age e "t x is requir$1 & its trav]s+ movem5ts %\ld be controll$ by /ation>y mill$ heads 7,fig4 #de1 ,,aa'74 ,! %ape ( ! ap]ture is a n unimportant po9t2 x %\ld be squ>e to all[ ( free movem5t ov] ! sub/age cond5s]4 ,! me*anical /age %\ld be tapp$ = ?ree 7removable7 screw /uds to be us$ 9 place ( ! slid+ b>1 s t if desir$ ! ,v]ni] f9d] 7,fig4 #de1 ,,bb'71 su* z is usually fitt$ to ?is class ( /age1 or a ,maltwood f9d]1 may be employ$4 ,illu/ration3 ,,fig4 #de4--,me*anical /age4 ,illu/ration3 ,,fig4 #df4--;,iris diaphragm4 #f4 ,diaphragm4--,sep>ate s+le diaphragms mu/ be avoid$2 a revolv+ plate pi]c$ ) diff]5t siz$ ap]tures & secur$ bel[ ! /age is pref]able1 b und\bt$ly ! be/ =m is ! iris diaphragm 7,fig4 #df7 :i* ent]s into ! con/ruction ( ! sub/age cond5s]4 #g4 ,! sub/age cond5s] is a necess>y "p ( ! optical outfit4 ,its purpose is to collect ! beam ( p>allel rays ( light reflect$ by ! plane mirror1 by virtue ( a %ort focus sy/em ( l5ses1 into a cone ( l>ge ap]ture 7r$ucible at w by means ( an iris diaphragm m\nt$ z a "p ( ! cond5s]71 :i* c be accurately focuss$ on ! plane ( ! object4 ,?is focuss+ mu/ be p]=m$ anew = ea* object1 on acc\nt ( ! v>iation 9 ! ?ickness ( ! slides4 ,! =m 9 mo/ g5]al use is t kn[n z ! ,abbe 7,fig4 #dg7 & consi/s ( a plano-convex l5s m\nt$ above a biconvex l5s4 ,?is comb9ation is c>ri$ 9 a screw-c5t]+ hold] kn[n z ! sub/age bel[ ! /age ( ! microscope 7,fig4 #dj f71 & mu/ be accurately adju/$ s t its optical axis co9cides ) t ( ! objective4 ,v]tical movem5t ( ! entire sub/age app>atus effect$ by means ( a rack & p9ion is a decid$ advantage1 & "s means %\ld be provid$ = tempor>ily remov+ ! cond5s] f ! optical axis ( ! microscope4 ,illu/ration3 ,,fig4 #dg--,optical "p ( ,abbe illum9ator4 ,) ! oil imm]sion objective1 h[ev]1 an a*romatic cond5s]1 giv+ an illum9at+ cone ( ab\t #j4i1 %\ld be us$ if ! full value ( ! l5s is to be obta9$4 ,x is g5]ally assum$ t a good objective requires an illum9at+ cone equival5t to two-?irds ( its num]ical ap]ture4 ,! be/ ,abbe cond5s] transmits a cone ( ab\t 4#de :il/ ! ap]ture ( ! #a_/ab in* imm]sion l5ses ( diff]5t mak]s v>ies f #a4j to #a4d1 h5ce1 ! effici5cy ( ^! l5ses is mu* curtail$ if ! cond5s] is m]ely ! ,abbe4 ,^! improv$ cond5s]s mu/ be absolutely c5t]$ to ! objective & capable ( v accurate focuss+ o!rwise mu* ( _! value is lo/4 #h4 ,mirrors4--,bel[ ! sub/age cond5s] is atta*$ a gymbal c>ry+ a rev]sible circul> frame ) a plane mirror on "o side & a concave mirror on ! o!r 7,fig4 #dj1 g74 ,! plane mirror is t usually employ$1 b occasionally1 z = example :5 us+ l[ p[]s & ) ! cond5s] rack$ d[n & ?r[n out ( ! optical axis1 ! concave mirror is us$4 #i4 ,ocul>s1 or ,eyepieces4--,^? kn[n z ! ,huygh5ian ocul>s 7,fig4 #dh7 w be suffici5t = all ord9>y "w )\t resort+ to ! m exp5sive comp5sation ocul>s4 ,two or ?ree1 magnify+ ! real image 7=m$ by ! objective7 f\r1 six1 or eight times respectively1 =m a useful equipm5t4 ,z an accessory ,ehrli*'s ,eyepiece is a v useful piece ( app>atus :5 ! enum]ation ( cells or bact]ia has to be c>ri$ out4 ,?is is an ord9>y eyepiece fitt$ ) an adju/able squ>e diaphragm op]at$ by a lev] project+ f ! side ( ! m\nt4 ,?ree not*es are made 9 "o ( ! sides ( ! squ>e & by mov+ ! lev] squ>e ap]ture c be r$uc$ to ?ree-qu>t]s1 "o-half or "o-qu>t] ( ! orig9al size4 #aj4 ,objectives4--,?ree objectives are necess>y3 "o = l[-p[] "w--e4 g41 #a in*1 #b_/c in*1 or #a_/b in*2 "o = high-p[] "w--e4 g41 #a_/ab in* oil imm]sion l5s2 & an int]m$iate m$ium-p[] l5s--e4 g41 #a_/f in* or #a_/h in* 7dry74 ,^! l5ses mu/ be c>efully select$1 especial att5tion be+ paid to ! foll[+ po9ts3 7a7 ,correction ( ,sph]ical ,ab]ration4--,sph]ical ab]ration gives rise to an ill-def9$ image1 due to ! c5tral & p]iph]al rays focuss+ at diff]5t po9ts4 7b7 ,correction ( ,*romatic ,ab]ration4--,*romatic ab]ration gives rise to a col\r$ fr+e ar\nd ! edges ( objects due to ! fact t ! diff]5t-col\r$ rays ( ! spectrum possess v>y+ refrangibilities & t a simple l5s acts t[>d !m z a prism4 7c7 ,flatness ( ,field4--,! ideal visual field w\ld be l>ge &1 above all1 flat2 9 o!r words1 objects at ! p]iph]y ( ! field w\ld be z di/9ctly 9 focus z ^? 9 ! c5tre4 ,un=tunately1 h[ev]1 ?is is an optical impossibility & ! field is always sph]ical 9 %ape4 ,"s mak]s succe$ 9 giv+ a l>g] c5tral area t is 9 focus at "o "t ?an o!rs1 & al?\gh ?is may !oretically cause an inf9itesimal sacrifice ( o!r qualities1 x %\ld always be s\ght =4 ,successive zones & ! entire p]iph]al r+ %\ld come into focus ) ! alt]ation ( ! f9e adju/m5t4 ,?is simultane\s %>pness ( ! entire circle is an indication ( ! p]fect c5t]+ ( ! :ole ( ! l5ses 9 ! objective4 ,illu/ration3 ,,fig4 #dh4--;,huygh5ian eyepiece4 7d7 ,good ,def9ition4--,actual magnification is1 )9 limits1 ( c\rse1 ( less value ?an cle> def9ition & high resolv+ p[]1 = x is ^u ^! prop]ties we dep5d = our k ( ! detail$ /ructure ( ! objects exam9$4 7e7 ,num]ical ,ap]ture 7,n4 ,a474--,! num]ical ap]ture may be def9$1 9 g5]al t]ms1 z ! ratio ( ! effective diamet] ( ! back l5s ( ! objective to its equival5t focal l5g?4 ,! det]m9ation ( ?is po9t is a process requir+ consid]able te*nical skill & ma!matical ability1 & is completely beyond ! p[]s ( ! av]age microscopi/4#a ,al?\gh ) ! increase 9 p[] x is correspond+ly difficult to comb9e all ^! corrections 9 "o objective1 !y are br\ght to a high pit* ( excell5ce 9 ! pres5t-day a*romatic objectives1 & s remove ! necessity = ! use ( ! high] pric$ & less durable apo*romatic l5ses4 ,9 select+ objectives ! be/ te/ objects to employ are3 #a4 ,a ?9 7"o cell lay]71 ev5 #a1 #b_/c1 #a_/b3 blood film1 /a9$ ) ,j5n]'s = #a_/f1 #a_/h or ,roman[sky's /a94 #a_/ab oil #b4 ,a ?9 cov]-slip prep>ation ( a "y cultivation ( #a_/h dry ,b4 diph!riae 7%[+ = segm5tation7 /a9$ ) #a_/ab oil me?yl5e-blue4 ,accessories4--,eye ,%ade 7,fig4 #di74--,?is piece ( app>atus consi/s ( a pe>-%ap$ piece ( black5$ metal or ebonite1 h+$ to a coll> :i* rotates on ! upp] "p ( ! body tube ( ! microscope4 ,x c be us$ to %ut out ! image ( surr\nd+ objects f ! unoccupi$ eye1 & :5 c>ry+ out prolong$ obs]vations w be f\nd ( real s]vice4 ,nosepiece4--,p]haps ! mo/ useful accessory is a nosepiece to c>ry two ( ! objectives 7,fig4 #ej71 or1 bett] /ill1 all ?ree 7,fig4 #ea74 ,?is nosepiece1 pref]ably con/ruct$ ( alum9ium1 mu/ be ( ! cov]$-9 type1 consi/+ ( a curv$ plate atta*$ to ! l[] end ( ! body tube--a circul> ap]ture be+ cut to correspond to ! lum5 ( t tube4 ,to ! "u surface ( ?is plate is pivot$ a simil>ly curv$ plate1 fitt$ ) ?ree tubulures1 ea* ( :i* c>ries an objective4 ,by rotat+ ! l[] plate ea* ( ! objectives c be br\ght successively 9 to ! optical axis ( ! microscope4 ,illu/ration3 ,,fig4 #di4--;,eye %ade4 ,= critical "w & p>ticul>ly = photo-micrography1 h[ev]1 ! int]*angeable nosepiece is by no means p]fect z x is next to impossible to secure accurate c5tre+ ( ea* l5s 9 ! optical axis4 ,= special purposes1 !re=e1 x is necess>y to employ a special nosepiece su* z t made by ,zeiss or ,leitz into :i* ea* objective slides on its own c>ri] & ^u :i* x is accurately c5tr$4 ,illu/ration3 ,,fig4 #ej4--;,d\ble nosepiece4 ,illu/ration3 ,,fig4 #ea4--,triple nosepiece4 ,w>m ,/age 7,fig4 #eb74--,?is is a flat metal case conta9+ a sy/em ( tubes ?r\gh ! int]ior ( :i* wat] ( any requir$ temp]ature c be circulat$4 ,x is made to clamp on to ! /age ( ! microscope by ! screws ,a ,a'1 & is p]=at$ ) a l>ge hole co9cid+ ) ! optical axis ( ! microscope2 a %ort tube ,b1 project+ f "o end ( ! w>m /age p]mits wat] ( ! desir$ temp]ature to be conduct$ f a res]voir ?r\gh a l5g? ( rubb] tub+ to ! int]ior ( ! /age & a simil> tube at ! o!r end ,b' ( ! /age all[s exit to ! wa/e wat]4 ,by rais+ ! temp]ature ( hang+-drop prep>ations1 etc41 plac$ ^u x1 above t ( ! surr\nd+ atmosph]e1 ! w>m /age r5d]s possible exact obs]vations on spore g]m9ation1 hang+-drop cultivations1 etc4 ,illu/ration3 ,,fig4 #eb4--,w>m /age4 ,a bett] =m is ! electrical hot /age design$ by ,lorra9 ,smi?2#b x requires ! addition ( a lamp resi/ance & slid+ rheo/at1 also a delicate ammet] read+ to 4#ja ( an amp]e4 ,x consi/s ( a wood5 frame support+ a flat glass bulb ) a long neck b5t upw>d at an obtuse angle 7,fig4 #ec74 ,! bulb is fill$ ) liquid p>aff91 :i* rises 9 ! op5 neck :5 exp&$ by heat4 ,! neck also accommodates ! !rmomet]4 ,two coils ( mangan9 wire run 9 ! p>aff9 at opposite sides ( ! bulb 7outside ! field ( vision71 c\pl$ to brass t]m9als on ! wood5 frame by plat9um wire fus$ into ! glass4 ,! resi/ance ( ! two coils 9 s]ies is ab\t #aj ohms4 ,a curr5t ( #b-a_/b amp]es is ne$$1 & is conduct$ to ! coils 9 ! /age ?r\gh ! rheo/at4 ,) ! help ( ! ammet] any desir$ temp]ature c be obta9$ & ma9ta9$1 up to ab\t #bjj deg4 ,c4 ,if imm]sion oil contact is made betwe5 ! top l5s ( ! cond5s] & ! l[] surface ( ! bulb1 ?is /age works v well inde$ ) ! #a_/ab-;in* oil imm]sion l5s4 ,illu/ration3 ,,fig4 #ec4--,lorra9 ,smi?'s w>m /age4 ,d>k ,gr\nd or ,p>aboloid ,cond5s]4--,?is is an imm]sion sub/age cond5s] ( high ap]ture by means ( :i* un/a9$ objects su* z bact]ia c be %[n z bright :ite p>ticles ^u a d5se black backgr\nd4 ,! c5tral rays ( light are block$ out by means ( an opaque /op :ile ! p]iph]al rays are reflect$ f ! p>aboloidal sides ( ! cond5s] & refract$ by ! object view$4 ,to obta9 ! be/ results ) ?is type ( cond5s] a p[]ful illum9ant--su* z a small arc lamp or an inc&esc5t gas lamp--is ne$$1 toge!r ) pick$ slides ( a c]ta9 ?ickness 7specifi$ = ! p>ticul> make ( cond5s] b g5]ally #a mm47 & specially ?9 cov]-glasses 7n m ?an #j4ag mm47 ,! objective mu/ n h a high] ,,na ?an #a4j1 consequ5tly imm]sion l5ses mu/ be fitt$ ) an int]nal /op to cut d[n ! ap]ture4 ,micromet]4--,"s =m ( micromet] = ! purpose ( measur+ bact]ia & o!r objects is also ess5tial4 ,details ( ^? 9 g5]al use w be f\nd 9 ! foll[+ pages4 ,illu/ration3 ,,fig4 #ed--;,diamond ,object m>k]4 ,object ,m>k] 7,fig4 #ed74--,?is is an exce$+ly useful piece ( app>atus4 ,made 9 ! =m ( an objective1 ! l5ses are replac$ by a diamond po9t1 set slightly out ( ! c5tre1 :i* c be rotat$ by means ( a mill$ plate4 ,screw$ on to ! nosepiece 9 place ( ! objective1 rotation ( ! diamond po9t w rule a small circle on ! object slide to p]man5tly record ! position ( an int]e/+ portion ( ! specim54 ,! diamond is m\nt$ on a spr+ :i* regulates ! pressure1 & ! size ( ! circle c be adju/$ by means ( a lat]al screw4 ,,me?ods ,,( ,,micrometry4 ,! unit ( l5g? z appli$ to ! measurem5t ( microscopical objects is ! "o-?\s&? "p ( a millimetre 7#j4jja mm471 d5om9at$ a micron 7sometimes1 & errone\sly1 ref]r$ to z a micro-millimetre71 & indicat$ 9 writ+ by ! ,greek lett] mu4 ,( ! _m me?ods 9 use = ! measurem5t ( bact]ia1 ?ree only w be "h describ$1 viz43 7a7 ,by means ( ! ,cam]a ,lucida4 7b7 ,by means ( ! ocul> or ,eyepiece ,micromet]4 7c7 ,by means ( ! ,fil> ,micromet] 7,ramsd5's micromet] eyepiece74 ,= ea* ( ^! me?ods a /age micromet] is necess>y4 ,?is is a #c by #a in* glass slip hav+ engrav$ on x a scale divid$ to hundr$?s ( a millimetre 7#j4ja mm471 e t5? l9e be+ made long] ?an ! int]v5+ ones1 to facilitate c\nt+2 & f ^! engrav$ l9es ! measurem5t 9 e case is evaluat$4 ,a cov]-glass is cem5t$ ov] ! scale to protect x f injury4 ,illu/ration3 ,,fig4 #ee4--;,cam]a lucida1 ,abbe patt]n4 7a7 ,by means ( ! ,cam]a ,lucida4 #a4 ,atta* a cam]a lucida 7( ! ,wolla/on1 ,beale1 or ,abbe patt]n7 7,fig4 #ee7 to ! eyepiece ( ! microscope4 #b4 ,adju/ ! micromet] on ! /age ( ! microscope & accurately focus ! divisions4 #c4 ,project ! scale ( ! /age micromet] on to a piece ( pap] & ) p5 or p5cil sket* 9 ! magnifi$ image1 ea* division ( :i* corresponds to #aj mu4 ,m>k on ! pap] ! optical comb9ation 7ocul> objective & tube l5g?7 employ$ to produce ?is p>ticul> magnification4 #d4 ,repeat ?is proc$ure = ea* ( ! possible comb9ations ( ocul>s & objectives fitt$ to ! microscope suppli$1 & c>efully pres]ve ! scales ?us obta9$4 ,to measure an object by ?is me?od simply project ! image on to ! scale correspond+ to ! p>ticul> optical comb9ation 9 use at ! mom5t4 ,read (f ! numb] ( divisions x occupies & express !m z micra4 ,9 place ( pres]v+ a scale = ea* optical comb9ation1 ! object to be measur$ & ! micromet] scale may be project$ & sket*$1 9 turn1 on ! same piece ( pap]1 tak+ p>ticul> c>e t ! c5tre ( ! eyepiece is #be cm4 f ! pap] on :i* ! divisions are drawn4 ,illu/ration3 ,,fig4 #ef4--;,eyepiece micromet]1 ord9>y4 ,illu/ration3 ,,fig4 #eg4--;,eyepiece micromet]1 net4 7b7 ,by means ( ! ,eyepiece ,micromet]4 ,! eyepiece micromet] is a circul> glass disc hav+ engrav$ on x a scale divid$ to t5?s ( a millimetre 7#j4a mm47 7,fig4 #ef71 or ! entire surface rul$ 9 #j4a mm4 squ>es 7! net micromet]7 7,fig4 #eg74 ,x c be fitt$ inside ! m\nt ( any ocul> j above ! ap]ture ( ! diaphragm & mu/ be adju/$ exactly 9 ! focus ( ! eye l5s4 ,"s mak]s m\nt ! glass disc toge!r ) a circul> cov]-glass 9 su* a way t :5 plac$ 9 position 9 any ,huygh5ian eyepiece ( _! own manufacture1 ! scale is exactly 9 focus = normal vision4 ,special eyepieces are also obta9able hav+ a sl$g+ adju/m5t to ! eye l5s = focuss+ ! micromet]4 ,! value ( "o division ( ! micromet] scale mu/ fir/ be asc]ta9$ = ea* optical comb9ation by ! aid ( ! /age micromet]1 ?us3 #a4 ,ins]t ! eyepiece micromet] inside ! ocul> & adju/ ! /age micromet] on ! /age ( ! microscope4 #b4 ,focus ! scale ( ! /age micromet] accurately2 ! l9es w appe> to be imm$iately bel[ ^? ( ! eyepiece micromet]4 ,make ! l9es on ! two micromet]s p>allel by rotat+ ! ocul>4 #c4 ,make two ( ! l9es on ! ocul> micromet] co9cide ) ^? b\nd+ "o division ( ! /age micromet]2 ?is is effect$ by increas+ or dim9i%+ ! tube l5g?2 & note ! numb] ( includ$ divisions4 #d4 ,calculate ! value ( ea* division ( ! eyepiece micromet] 9 t]ms ( mu1 by means ( ! foll[+ =mula3 x #aj y4 ,:]e x ! numb] ( includ$ divisions ( ! eyepiece micromet]4 y ! numb] ( includ$ divisions ( ! /age micromet]4 #e4 ,note ! optical comb9ation employ$ 9 ?is exp]im5t & record x ) ! calculat$ micromet] value4 ,repeat ?is process = ea* ( ! o!r comb9ations4 ,c>efully record ! results4 ,to measure an object by ?is me?od read (f ! numb] ( divisions ( ! eyepiece micromet] x occupies & express ! result 9 micra by a ref]5ce to ! /&>d value = ! p>ticul> optical comb9ation employ$4 ,zeiss prep>es a comp5sat+ eyepiece micromet] = use ) 8 apo*romatic objectives1 ! divisions ( :i* are s comput$ t 7) a tube l5g? ( #afj mm47 ! value ( ea* is equival5t to z _m micra z !re are millimetres 9 ! focal l5g? ( ! objective employ$4 ,wright's ,eikonomet] is really a modification ( ! eyepiece micromet] = rapidly measur+ microscopical objects by direct inspection1 hav+ previ\sly det]m9$ ! magnify+ p[] ( ! p>ticul> optical comb9ation employ$4 ,x is a small piece ( app>atus resembl+ an eyepiece1 ) a slid+ eye l5s1 :i* c be accurately focuss$ on a micromet] scale fix$ )9 ! in/rum5t4 ,:5 plac$ ov] ! microscope ocul> ! divisions ( ?is scale measure ! actual size ( ! virtual image 9 millimetres4 ,9 ord] to use ?is in/rum5t = direct measurem5t1 x is fir/ necess>y to det]m9e ! magnify+ p[] ( ea* comb9ation ( ocul>1 tube l5g? & objective4 ,place a /age micromet] divid$ into hundr$?s ( a millimetre on ! microscope /age & focus accurately4 ,re/ ! eikonomet] on ! eyepiece4 ,obs]vation ?r\gh ! eikonomet] %[s its micromet] scale sup]pos$ on ! image ( ! /age micromet]4 ,rotate ! eikonomet] until ! l9es on ! two scales are p>allel1 & make ! v>i\s adju/m5ts to ensure t two l9es on ! eikonomet] scale co9cide ) two l9es on ! /age micromet]4 ,= ! sake ( illu/ration x may be assum$ t five ( ! divisions on ! /age micromet] accurately fill "o ( ! divisions ( ! eikonomet] scale2 ?is indicates a magnify+ p[] ( #ejj z ! con/ant = t p>ticul> optical comb9ation1 & a record %\ld be made ( ! fact4 ,! magnification con/ants ( ! v>i\s o!r optical comb9ations %\ld be simil>ly made & record$4 ,to measure any object subsequ5tly x %\ld be fir/ focuss$ c>efully 9 ! ord9>y way4 ,! eikonomet] %\ld !n be appli$ to ! eyepiece & ! size ( ! object read (f on ! eikonomet] scale z millimetres1 & ! actual size calculat$ by divid+ ! obs]v$ size by ! magnification con/ant = ! p>ticul> optical comb9ation employ$ 9 ! obs]vation4 7c7 ,by means ( ! fil> micromet]4 ,illu/ration3 ,,fig4 #eh4--,ramsd5's ,fil> micromet]4 ,illu/ration3 ,,fig4 #ei4--,ramsd5's micromet] field1 a1 fix$ wire2 b1 ref]5ce wire 7fix$72 c1 travell+ wire4 ,! ,fil> or cobweb ,micromet] 7,ramsd5's micromet]7 eyepiece 7,fig4 #eh7 consi/s ( an ocul> hav+ a f9e fix$ wire /ret*+ horizontally across ! field 7,fig4 #ei71 a v]tical ref]5ce wire--fix$--adju/$ at "r angles to ! fir/2 & a f9e wire1 p>allel to ! ref]5ce wire1 :i* c be mov$ across ! field by ! action ( a micromet] screw2 ! drum head is divid$ into "o hundr$ p>ts1 :i* successively pass a fix$ index z ! head is turn$4 ,9 ! l[] "p ( ! field is a comb ) ! int]vals betwe5 its tee? correspond+ to "o complete revolution ( ?is screw-head4 ,z 9 ! previ\s me?od1 ! value ( ea* division ( ! micromet] scale 7i4 e41 ! comb7 mu/ fir/ be det]m9$ = ea* optical comb9ation4 ,?is is effect$ z foll[s3 #a4 ,place ! fil> micromet] & ! /age micromet] 9 _! respective positions4 #b4 ,rotate ! screw ( ! fil> micromet] until ! movable wire co9cides ) ! fix$ "o1 & ! index m>ks z]o on ! drum head4 7,if :5 ! drum head is at z]o ! two wires d n exactly co9cide !y mu/ be adju/$ by loos5+ ! drum screw & resett+ ! drum47 #c4 ,focus ! scale ( ea* micromet] accurately1 & make ! l9es on !m p>allel4 #d4 ,rotate ! head ( ! micromet] screw until ! movable l9e has transv]s$ "o division ( ! /age micromet]4 ,note ! numb] ( complete revolutions 7by means ( ! record+ comb7 & ! fractions ( a revolution 7by means ( scale on ! head ( ! micromet] screw71 :i* are requir$ to measure ! #j4ja mm4 #e4 ,make sev]al su* e/imations & av]age ! results4 #f4 ,note ! optical comb9ation employ$ 9 ?is exp]im5t & record x c>efully1 toge!r ) ! micromet] value 9 t]ms ( mu4 #g4 ,repeat ?is process = ea* ( ! diff]5t optical comb9ations & record ! results4 ,to measure an object by ?is me?od1 simply note ! numb] ( revolutions & fractions ( a revolution ( ! screw-head requir$ to trav]se su* object f edge to edge1 & express ! result z micra by ref]5ce to ! record$ values = t p>ticul> optical comb9ation4 ,microscope ,illum9ant4--,9 tropical & subtropical regions diffuse daylight is ! be/ illum9ant4 ,9 temp]ate climes h[ev] daylight ( ! desirable quantity is n always available1 & rec\rse mu/ be _h to oil lamps1 gas lamps--pref]ably ^? ) inc&esc5t mantles--& electricity2 & ( ^! ! la/ is und\bt$ly ! be/4 ,a h&y lamp hold] :i* c be manufactur$ 9 ! laboratory is %[n 9 ,fig4 #fj4 ,x consi/s ( a base bo>d weight$ ) lead to :i* is atta*$ ! ord9>y dome/ic lamp hold]1 & beh9d ?is is fa/5$ a curv$ %eet-iron reflector4 ,an obscur$ metal filam5t lamp ( ab\t #af c&le p[] gives ! mo/ suitable light1 & if mono*romatic light is ne$$1 ! blue grease p5cil is /reak$ ov] ! side ( ! lamp ne>e/ ! microscope2 ! curr5t is swit*$ on & :5 ! glass bulb is w>m1 rubb+ ) a wad ( cotton-wool w readily di/ribute ! blue greasy mat]ial 9 an ev5 film ov] ! gr\nd glass4 ,illu/ration3 ,,fig4 #fj4--;,electric microscope lamp4 ,,footnotes3 #a ,its importance w be realis$1 h[ev]1 :5 x is /at$ 9 ! words ( ! late ,pr(essor ,abbe3 ,! num]ical ap]ture ( a l5s det]m9es all its ess5tial qualities2 ! brightness ( ! image increases ) a giv5 magnification & o!r ?+s be+ equal1 z ! squ>e ( ! ap]ture2 ! resolv+ & def9+ p[]s are directly relat$ to x1 ! focal dep? ( diff]5tiation ( dep?s v>ies inv]sely z ! ap]ture1 & s =?4 #b ,made by ,mr4 ,otto ,baumba*1 #aj1 ,lime ,grove1 ,man*e/]4 ,v4 ,,microscopical ,,exam9ation ,,( ,,bact]ia ,,& ,,o!r ,,micro-,,fungi4 ,,app>atus ,,& ,,reag5ts ,,us$ ,,9 ,,ord9>y ,,microscopical ,,exam9ation4 ,! foll[+ comprises ! ess5tial app>atus & reag5ts = r\t9e "w ) :i* ea* /ud5t %\ld be provid$4 #a4 ,india-rubb] *ange-mat ^u :i* cov]-glasses may be re/$ dur+ ! process ( /a9+4 #b4 ,squ>es ( blott+ pap] ab\t #aj cm41 = dry+ cov]-slips & slides4 7,! filt] pap] kn[n z ,g]man l9$--a highly absorb5t1 closely wov5 pap]1 hav+ an ev5 surface & no loose fluff to adh]e to ! specim5s--is ! mo/ useful = ?is purpose47 ,illu/ration3 ,,fig4 #fa4--;,dis9fectant ,j>4 #c4 ,glass j> fill$ ) #b p] c5t4 lysol solution = ! reception ( infect$ cov]-glasses & infect$ pipettes1 etc4 #d4 ,a squ>e glaz$ e>!nw>e box ) a loose l9+ conta9+ #b p] c5t4 lysol solution = ! reception ( infect$ mat]ial & us$ slides4 ,! bottom ( ! l9+ is p]=at$ s t :5 full ! l9+ & its cont5ts c be lift$ bodily out ( ! box1 :5 ! dis9fectant solution dra9s away & ! slides1 etc41 c easily be empti$ out4 ,! empty l9+ is !n return$ to ! box ) its dis9fectant solution 7,fig4 #fa74 #e4 ,buns5 burn] provid$ ) peep-flame by-pass4 #f4 ,porcela9 tr\gh hold+ five or six hang+-drop slides 7,fig4 #fb74 ,illu/ration3 ,,fig4 #fb4--;,hang+-drop slides3 a1 ,d\ble cell se5 f above2 b1 s+le cell se5 f ! side4 ,! be/ =m ( hang+-drop slide is a modification ( ,boett*]'s glass r+ slide1 & is prep>$ by cem5t+ a circul> cell ( t91 #ac to #ae mm4 diamet]1 & #a to #b mm4 9 height1 to ! c5tre ( a #c by #a slip by means ( ,canada balsam4 ,x is (t5 extremely conv5i5t to h two ( ^! cells cem5t$ close toge!r on "o slide 7,fig4 #fb1 a74 ,ano!r =m ( hang+-drop slide is made 9 :i* a circul> or oval concavity or cell is gr\nd out ( ! c5tre ( a #c by #a slip4 ,^! are m exp5sive1 less conv5i5t to "w )1 & are m easily contam9at$ by drops ( mat]ial "u exam9ation1 & %\ld be c>efully avoid$4 #g4 ,?ree alum9ium rods 7,fig4 #fc71 ea* ab\t #be cm4 long & c>ry+ a piece ( #j4jae gauge plat9o-iridium wire #g4e cm4 9 l5g?4 ,! end ( "o ( ! wires is b5t r\nd to =m an oval loop1 ( ab\t #a mm4 9 its %ort diamet]1 & is t]m$ a loop or an oese2 ! t]m9al #c or #d mm4 ( ano!r wire is flatt5$ out by hamm]+ x on a smoo? iron surface to =m a spatula2 ! ?ird is left unt\*$ or is po9t$ by ! aid ( a file4 ,^! in/rum5ts are us$ = inoculat+ culture tubes & prep>+ specim5s = microscopical exam9ation4 ,illu/ration3 ,,fig4 #fc4--;,ends ( plat9um rods4 a1 loop2 b1 spatula2 c1 ne$le4 ,! me?od ( m\nt+ ^! wires may be describ$ z foll[s3 ,take a piece ( alum9ium wire #be cm4 long & ab\t #j4be cm4 9 diamet]1 & drill a f9e hole completely ?r\gh ! wire ab\t a c5timetre f "o end4 ,s9k a /raight n>r[ *annel along "o side ( ! wire1 9 its long axis1 f ! hole to ! ne>e/ end1 %all[ at fir/1 b gradually becom+ deep]4 ,on ! opposite side ( ! wire make a %ort cut1 #b mm4 9 l5g?1 lead+ f ! hole 9 ! same direction4 ,! use ( a f9e d5tal drill & small circul> saw1 work$ by a d5tal motor facilitates ! manufacture ( ^! alum9ium h&l$ in/rum5ts4 ,n[ pass "o end ( ! plat9um wire ?r\gh ! hole1 turn up ab\t #b mm4 at "r angles & press ! %ort piece into ! %ort cut4 ,turn ! long end ( ! wire %>ply1 also at "r angles1 & s9k x into ! long *annel s t x em]ges f ab\t ! c5tre ( ! cut end ( ! alum9ium wire 7,fig4 #fc74 ,a few %>p taps ) a wat* mak]'s hamm] w n[ close 9 ! sides ( ! two *annels ov] ! wire & hold x securely4 ,illu/ration3 ,,fig4 #fd4--,plat9um rod 9 alum9ium h&le--me?od ( m\nt+4 ,! plat9um wire may be fus$ into ! end ( a piece ( glass rod1 b su* a h&le is va/ly inf]ior to alum9ium & is n to be recomm5d$4 #h4 ,two pairs ( %>p-po9t$ spr+ =ceps 7#aj cm4 long71 "o ( :i* mu/ be kept p]fectly clean & res]v$ = h&l+ clean cov]-slips1 ! o!r be+ = use dur+ /a9+ op]ations4 #i4 ,a box ( clean #c by #a glass slips4 #aj4 ,a glass capsule ) tightly fitt+ 7gr\nd on7 glass lid1 conta9+ clean cov]-slips 9 absolute alcohol4 #aa4 ,"o ( ,fab]'s grease p5cils 7yell[1 r$1 or blue7 = writ+ on glass4 #ab4 ,a wood5 rack 7,fig4 #fe7 ) twelve drop-bottles 7,fig4 #ff7 ea* #fj c4c4 capacity1 conta9+ ,anil9e wat]4 ,g5tian violet1 saturat$ alcoholic solution4 ,lugol's 7,gram's7 iod9e4 ,absolute alcohol4 ,me?yl5e-blue1 ,fu*s91 basic1 saturat$ alcoholic solution4 ,neutral r$1 #a p] c5t4 aque\s solution4 ,lei%man's modifi$ ,roman[sky /a94 ,c>bolic acid1 #e p] c5t4 aque\s solution4 ,acetic acid1 #a p] c5t4 solution4 ,sulphuric acid1 #be p] c5t4 solution4 ,xylol4 ,illu/ration3 ,,fig4 #fe4--,/a9+ rack1 rubb] *ange mat & lysol pot4 ,illu/ration3 ,,fig4 #ff4--;,drop bottle4 ,illu/ration3 ,,fig4 #fg4--;,canada balsam pot4 ,& two pots ) air-tight glass caps 7,fig4 #fg71 ea* provid$ ) a piece ( glass rod & fill$ respectively ) ,canada balsam dissolv$ 9 xylol1 & /]ile vasel9e4 ,,me?ods ,,( ,,exam9ation4 ,bact]ia1 etc41 are exam9$ microscopically4 #a4 ,9 ! liv+ /ate1 un/a9$1 or /a9$4 #b4 ,9 ! fix$ condition 7i4 e41 fix$1 kill$1 & /a9$ by suitable me?ods74 ,! prep>ation ( a specim5 f a tube cultivation = exam9ation by ^! me?ods may be describ$ z foll[s3 #a4 ,liv+1 ,un/a9$4--7a7 ,fre% ,prep>ation4-- #a4 ,clean & dry a #c by #a glass slip & place x on "o ( ! squ>es ( filt] pap]4 ,deposit a drop ( wat] 7pref]ably di/ill$7 or a drop ( #a p] c5t4 solution ( cau/ic pota%1 on ! c5tre ( ! slip1 by means ( ! plat9um loop4 ,illu/ration3 ,,fig4 #fh4--;,hold+ tubes = remov+ bact]ial gr[?1 z se5 f ! front4 ,,te*nique ,,( ,,op5+ ,,& ,,clos+ ,a ,,culture ,,tube4 #b4 ,remove ! tube cultivation f its rack or j> ) ! left h& & ignite ! cotton-wool plug by hold+ x to ! flame ( ! ,buns5 burn]4 ,ext+ui% ! flame by bl[+ on ! plug1 :il/ rotat+ ! tube on its long axis1 its m\? direct$ v]tically upw>d1 betwe5 ! ?umb & f+]s4 7,?is op]ation is t]m$ flam+ ! plug1 & is int5d$ to de/roy any micro-organisms t may h become entangl$ 9 ! loose fibres ( ! cotton-wool1 & :i*1 if n ?us de/roy$1 might fall into ! tube :5 ! plug is remov$ & s accid5tally contam9ate ! cultivation47 #c4 ,hold ! tube at or ne> its c5tre betwe5 ! ends ( ! ?umb & fir/ two f+]s ( ! left h&1 & all[ ! seal$ end to re/ ^u ! back ( ! h& betwe5 ! ?umb & =ef+]1 ! plug po9t+ to ! "r4 ,keep ! tube z ne>ly 9 ! horizontal position z is consi/5t ) safety1 to dim9i% ! risk ( ! accid5tal entry ( organisms 7,fig4 #fh74 #d4 ,take ! h&le ( ! loop betwe5 ! ?umb & =ef+] ( ! "r h&1 hold+ ! in/rum5t 9 a position simil> to t occupi$ by a p5 or a pa9t-bru%1 & /]ilise ! plat9um portion by hold+ x 9 ! flame ( a ,buns5 burn] until x is r$ hot4 ,/]ilise ! adjac5t portion ( ! alum9ium h&le by pass+ x rapidly twice or ?rice ?r\gh ! flame4 ,aft] /]ilis+ x1 ! loop mu/ n be all[$ to leave ! h& or to t\* aga9/ any?+ b ! mat]ial x is int5d$ to exam9e1 until x is f9i%$ ) & has be5 aga9 /]ilis$4 #e4 ,grasp ! cotton-wool plug ( ! te/-tube betwe5 ! little f+] & ! palm ( ! "r h& 7:il/ /ill hold+ ! loop z direct$ 9 /ep #d71 & remove x f ! m\? ( ! tube by a screw+ motion ( ! "r h&4 #f4 ,introduce ! plat9um loop into ! tube & hold x 9 ?is position until satisfi$ t x is q cool4 7,! cool+ may be ha/5$ by t\*+ ! loop on "o ( ! drops ( moi/ure :i* are usually to be f\nd cond5s$ on ! int]ior ( ! glass tube1 or by dipp+ x into ! cond5sation wat] at ! bottom2 at ! same "t c>e mu/ be tak5 9 ! case ( cultures on solid m$ia to avoid t\*+ ei!r ! m$ium or ! gr[?47 #g4 ,remove a small portion ( ! gr[? by tak+ up a drop ( liquid1 9 ! case ( a fluid culture1 9 ! loop2 or by t\*+ ! loop on ! surface ( ! gr[? :5 ! culture is on solid m$ium2 & )draw ! loop f ! tube )\t aga9 t\*+ ! m$ium or ! glass sides ( ! tube4 #h4 ,replace ! cotton-wool plug 9 ! m\? ( ! tube4 #i4 ,replace ! tube cultivation 9 its rack or j>4 #aj4 ,mix ! cont5ts ( ! loop ?or\ghly ) ! drop ( wat] on ! #c by #a slide4 #aa4 ,aga9 /]ilise ! loop z direct$ 9 /ep #d1 & replace x 9 its /&4 #ab4 ,remove a cov]-slip f ! glass capsule by means ( ! cov]-slip =ceps1 re/ x = a mom5t on its edge1 on a piece ( filt] pap] to remove ! excess ( alcohol1 !n pass x ?r\gh ! flame ( ! ,buns5 burn]4 ,?is burns (f ! rema9d] ( ! alcohol1 & ! cov]-slip s flam$ is n[ clean1 dry1 & /]ile4 #ac4 ,l[] ! cov]-slip1 /ill held 9 ! =ceps1 on to ! surface ( ! drop ( fluid on ! #c by #a slip1 c>efully & g5tly1 to avoid ! inclusion ( air bubbles4 #ad4 ,exam9e microscopically 7vide infra74 ,dur+ ! microscopical exam9ation1 /a9s & o!r reag5ts may be run 9 "u a cov]-slip by ! simple me?od ( plac+ a drop ( ! reag5t 9 contact ) "o edge ( ! cov]-glass & apply+ ! torn edge ( a piece ( blott+ pap] to ! opposite side4 ,! reag5t may !n be obs]v$ to fl[ across ! field & come into contact ) su* ( ! micro-organisms z lie 9 its pa?4 ,! non-toxic basic dyes mo/ g5]ally employ$ = ! intra-vitam /a9+ ( bact]ia are ,neutral r$1 ,qu9ole9e blue ,me?yl5e gre5 9 #j4e p] c5t4 aque\s solutions4 ,vesuv91 ,negative ,/a9 7,burri74--,by ?is me?od ( demon/ration ! appe>ances pres5t$ by d>k gr\nd illum9ation 7by means ( a p>aboloid cond5s]7 are closely simulat$1 s9ce m9ute p>ticles1 bact]ia1 blood or pus cells etc4 /& out z brilliantly :ite or col\rless bodies on a d>k grey-br[n backgr\nd4 ,reag5t requir$3 ,any "o ( ! liquid wat]pro( black draw+ inks 7,*9-*91 ,pelican1 etc474 ,?is is prep>$ = use z foll[s3 ,measure out & mix3 ,liquid black ink1 #be c4c4 ,t9cture ( iod9e #a c4c4 ,all[ ! mixture to /& #bd h\rs1 c5trifugalise ?or\ghly1 pipette (f ! sup]natant liquid to a clean bottle & !n add a cry/al ( ?ymol or "o drop ( =mal9 z a pres]vative4 ,,me?od4-- #a4 ,) ! /]ilis$ loop deposit "o drop ( ! liquid ink close to "o end ( a #c by #a slide4 #b4 ,) ! /]ilis$ loop deposit a drop ( ! fluid culture 7or ( an emulsion f a solid culture7 by ! side ( ! drop ( ink 7,fig4 #fi1 a72 mix ! two drops ?or\ghly by ! aid ( ! loop4 #c4 ,/]ilise ! loop4 #d4 ,hold ! slide firmly on ! b5* ) ! ?umb & =ef+] ( ! left h& appli$ to ! end ne>e/ ! drop ( fluid4 #e4 ,take ano!r clean #c by #a slide 9 ! "r h& & l[] its %ort end obliquely 7at an angle ( ab\t #fj deg47 transv]sely on to ! mix$ ink & culture on ! fir/ slide1 & all[ ! fluid to spread across ! slide & fill ! angle ( incid5ce4 #f4 ,ma9ta9+ ! orig9al angle1 draw ! second slide firmly & ev5ly along ! fir/ t[>d ! end f>!/ f ! left h& 7,fig4 #fi1 b74 #g4 ,?r[ ! second slide into a pot ( dis9fectant2 all[ ! fir/ slide to dry 9 ! air4 ,illu/ration3 ,,fig4 #fi4--,spread+ negative film4 #h4 ,place a drop ( imm]sion oil on ! c5tre ( ! film1 l[] ! #a_/ab-;in* objective into ! oil & exam9e microscopically )\t ! int]v5tion ( a cov]-slip4 7,! film ( ink may be cov]$ ) a long cov]-glass & xylol balsam z a p]man5t prep>ation47 7b7 ,hang+-drop ,prep>ation4-- #a4 ,sme> a lay] ( /]ile vasel9e on ! upp] surface ( ! r+ cell ( a hang+-drop slide by means ( ! glass rod provid$ ) ! vasel9e bottle1 & place ! slide on a piece ( filt] pap]4 #b4 ,flame a cov]-slip & place x on ! filt] pap] by ! side ( ! hang+-drop slide4 #c4 ,place a drop ( wat] on ! c5tre ( ! cov]-slip by means ( ! plat9um loop4 #d4 ,obta9 a small quantity ( ! mat]ial x is desir$ to exam9e1 9 ! mann] detail$ above 7pages #gd-gf1 /eps #b to #aa mu/ be foll[$ 9 _! entirety & ) ! /ricte/ exactitude :5ev] tube cont5ts are be+ h&l$71 & mix x ) ! drop ( wat] on ! cov]-slip4 #e4 ,raise ! cov]-slip 9 ! po9ts ( ! =ceps & rapidly inv]t x on to ! r+ cell ( ! hang+-drop slide1 s t ! drop ( fluid occupies ! c5tre ( ! r+4 7,c>efully avoid contact betwe5 ! drop ( fluid & ei!r ! r+ cell or ! lay] ( vasel9e4 ,%\ld ?is happ51 ! n[ infect$ hang+-drop slide & its cov]-slip mu/ be dropp$ into ! pot ( lysol & a new prep>ation made47 #f4 ,press ! cov]-slip firmly d[n into ! vasel9e on to ! top ( ! r+ cell4 7,?is spreads out ! vasel9e into a ?9 lay]1 & besides ensur+ ! adhesion ( ! cov]-slip1 seals ! cells & s ret>ds evaporation47 #g4 ,exam9e microscopically4 ,! exam9ation ( a fre% specim5 or a hang+-drop prep>ation is direct$ to ! det]m9ation ( ! foll[+ data3 #a4 ,! nature ( ! bact]ia pres5t--e4 g41 cocci1 bacilli1 etc4 #b4 ,! purity ( ! cultivation2 ?is c only be det]m9$ :5 gross morphological diff]5ces exi/ betwe5 ! organisms pres5t4 #c4 ,! pres5ce or abs5ce ( spores2 :5 pres5t1 spores %[ _! typical refrangibility exce$+ly well by ?is me?od4 #d4 ,! pres5ce or abs5ce ( mobility4 ,9 a hang+-drop specim5 "s =m ( movem5t c practically always be obs]v$1 & its "* mu/ be c>efully det]m9$ by not+ ! relative positions ( adjac5t micro-organisms4 7a7 ,br[nian or molecul> movem5t4 ,m9ute p>ticles ( solid matt] 7includ+ bact]ia71 :5 susp5d$ 9 a fluid1 w always %[ a vibratory movem5t affect+ ! entire field1 b nev] alt]+ ! relative positions ( ! bact]ia4 7,cocci exhibit ?is movem5t1 b ) ! exception ( ! ,micrococcus agilis1 ! cocci are non-motile47 7b7 ,/ream+ movem5t4 ,?is is due to curr5ts set up 9 ! hang+ drop z a result ( j>r+ ( ! specim5 or ( evaporation1 or to ! fact t ! cov]-slip is n p]fectly level1 & al?\gh ! relative positions ( ! bact]ia may v>y1 /ill ! fl[+ movem5t ( l>ge numb]s ( organisms 9 "s "o direction w usually be suffici5t to demon/rate ! nature ( ?is motion4 7c7 ,locomotive movem5t1 or true motility1 is det]m9$ by obs]v+ "s "o p>ticul> bacillus *ang+ its position 9 ! field indep5d5tly (1 & 9 a direction contr>y to1 o!r organisms pres5t4 ,:5 ! exam9ation is complet$ & ! specim5 f9i%$ )1 ! fre% specim5--i4 e41 ! slide ) ! cov]-slip atta*$--mu/ be dropp$ into ! lysol pot4 ,9 ! hang+-drop specim51 h[ev]1 ! cov]-slip only is infect$1 & ?is may be rais$ f ! r+ cell by means ( =ceps & dropp$ into ! dis9fectant4 ,p]man5t ,/a9+ ( ! ,hang+-drop ,specim54--,occasionally x is necess>y to fix & /a9 a hang+-drop prep>ation4 ,?is may be done z foll[s3 #a4 ,remove ! cov]-slip f ! cell by ! aid ( ! =ceps4 #b4 ,if ! drop is small1 fix x by dropp+ x face d[nw>d1 :il/ /ill wet1 on to ! surface ( "s ,gull&'s solution or corrosive sublimate solution 7vide page #hb7 9 a wat*-glass4 ,if ! drop is l>ge1 place x face upw>d on ! rubb] mat1 cov] x ) an inv]t$ wat*-glass1 & all[ x to dry4 ,!n fix x 9 ! alcohol & e!r solution 7vide1 page #hb74 #c4 ,dip ! cov]-glass into a beak] conta9+ hot wat] 9 ord] to remove "s ( ! vasel9e adh]+ to x4 #d4 ,wa% successively 9 alcohol1 xylol1 e!r1 & alcohol1 to remove ! la/ traces ( grease4 #e4 ,wa% 9 wat]4 #f4 ,/a91 wa%1 dry1 & m\nt z = an ord9>y cov]-slip film prep>ation 7vide pages #hc-he74 #b4 ,kill$1 ,/a9$4--,9 ?is me?od ?ree di/9ct processes are necess>y3 ,prep>+ & fix+ ! film4 ,/a9+4 ,m\nt+4 ,prep>+ ! ,film4-- #a4 ,flame a cov]-slip & place x on a piece ( filt] pap]4 #b4 ,place a drop ( wat] on ! c5tre ( ! cov]-slip by means ( plat9um loop4 #c4 ,obta9 a small quantity ( ! mat]ial to be exam9$ ^u a /]ilis$ plat9um loop 7see pages #gd-gf1 /eps #b to #aa7 & mix x ) ! drops ( wat] on ! cov]-slip4 #d4 ,spread ! drop ( emulsion ev5ly ov] ! cov]-slip 9 ! =m ( a squ>e film to )9 #a mm4 ( ea* edge ( ! cov]-slip4 #e4 ,all[ x to dry completely 9 ! air4 ,fix+4--,fix by pass+ ! cov]-slip1 held 9 ! f+]s1 ?ree or f\r times ?r\gh ! flame ( a ,buns5 burn]4 ,9 "s in/ances 7e4 g41 :5 ! films aft] /a9+ are int5d$ = micrometric obs]vations7 x is almo/ ess5tial to fix by exposure to a uni=m temp]ature ( #aae deg4 ,c41 = tw5ty m9utes4 ,?is is be/ done 9 a c>efully regulat$ hot-air ov54 ,fixation may also be effect$ by imm]s+ 9 "s fixative fluid1 su* z "o ( ! foll[+3 #a4 ,absolute alcohol1 = five to fifte5 m9utes4 equal p>ts1 = five to ?irty #b4 ,absolute alcohol1 m9utes 7e4 g41 = blood or ,e!r1 milk74 #c4 ,osmic acid1 #a p] c5t4 aque\s solution1 = ?irty seconds4 #d4 ,corrosive sublimate1 saturat$ aque\s solution1 = five m9utes4 #e4 ,corrosive sublimate 7,lang71 = five m9utes4 ,?is solution is prep>$ by dissolv+3 ,sodium *loride #j4ge gramme ,hydr>g4 p]*loride #ab4jj grammes ,acetic acid #e4jj grammes ,9 di/ill$ wat] #ajj4jj c4c4 ,filt]4 #f4 ,gull&'s solution1 = five m9utes4 ,?is solution is prep>$ by mix+3 ,absolute alcohol #be4j c4c4 ,e!r #be4j c4c4 ,corrosive sublimate1 #bj p] c5t4 alcoholic solution #j4d c4c4 #g4 ,=mal9 #aj p] c5t4 aque\s solution 7 #d p] c5t4 aque\s solution ( =maldehyde s9ce =mal9 is a #dj p] c5t4 solution ( ! gas 9 wat]74 ,ei!r ( ^! me?ods ( fixation coagulates ! album9\s mat]ial & ensures p]fect adhesion ( ! film to ! cov]-slip4 ,cle>+4--,wa% ! cov]-slip ?or\ghly 9 runn+ wat] & proce$ ) ! /a9+4 ,if ! film has be5 prep>$ f bro?1 liquefi$ gelat9e1 or pus or o!r morbid exudations1 saturate ! film aft] fixation ) acetic acid #b p] c5t4 & all[ x to act = two m9utes4 ,wa% ) alcohol1 !n let ! alcohol rema9 on ! cov]-slip = two m9utes4 7,?is w cle> ! gr\ndwork & give a mu* %>p] & clean] film ?an w\ld o!rwise be obta9$47 ,if ! film has be5 prep>$ f blood or blood/a9$ fluid1 treat ) acetic acid #b p] c5t4 = two m9utes aft] fixation4 ,wa% ) wat]1 dry1 & proce$ ) ! /a9+4 7,?is w remove ! haemoglob9 & facilitate exam9ation47 ,/a9+4-- #a4 ,re/ ! cov]-slip1 film side upp]mo/1 on ! rubb] mat4 #b4 ,by means ( a drop-bottle1 cov] ! film side ( ! cov]-slip ) ! select$ /a91 all[ x to act = a few m9utes1 !n wa% (f ! excess 9 runn+ wat]4 ,! p5etrat+ p[] ( /a9s is increas$ by 7a7 physical means--e4 g41 heat+ ! /a92 7b7 *emical means--e4 g41 by ! addition ( c>bolic acid1 #e p] c5t4 aque\s solution2 cau/ic alkalies1 #b p] c5t4 aque\s solutions2 wat] saturat$ ) anil9e oil2 borax1 #j4e p] c5t4 aque\s solution4 ,! mo/ commonly us$ dyes = cov]-slip film prep>ations are ! anil9e dyes4 7,a7 ,basic3 7a7 ,me?yl5e-blue4 7b7 ,g5tian violet4 7c7 ,fu*s94 ,^! dyes are kept 9 saturat$ alcoholic 7#ij p] c5t47 solutions s t decomposition may be ret>d$4 ,two or ?ree drops ( alcoholic solution ( ^! dyes to1 say1 #d c4c4 wat]1 usually makes a suffici5tly /rong /a9+ fluid = cov]-slip film prep>ations4 ,c>bolic me?yl5e-blue 7,c4,m4,b47 & c>bol fu*s9 7,c4,f47 are prep>$ by cov]+ ! cov]-slip ) #e p] c5t4 solution ( c>bolic acid & add+ a few drops ( ! saturat$ alcoholic solution ( me?yl5e-blue or fu*s9 respectively to x4 ,= anil9e g5tian violet 7,a4,g4,v47 ! /a9 is add$ to a saturat$ solution ( anil9e oil 9 wat]4 7d7 ,?ion9e blue4 7e7 ,bism>ck br[n4 7f7 ,neutral r$4 7,b7 ,acid3 7a7 ,eos91 aque\s yell[i%4 7b7 ,safran9e4 ,^! dyes are kept 9 #a p] c5t4 aque\s solution to :i* is add$ #e p] c5t4 ( alcohol1 z a pres]vative4 ,!y are g5]ally us$ 9 ?is =m4 ,a few nucle> /a9s 7c>m9e1 haematoxyl97 are occasionally us$ m especially 9 section "w4 ,decol\risation4--,aft] ov]/a9+1 films may be decol\ris$ by wa%+ = a long] or %ort] "t 9 "o ( ! foll[+ reag5ts arrang$ 9 asc5d+ ord] ( p[] #a4 ,wat]4 #b4 ,*lor(orm4 #c4 ,acetic acid1 #a p] c5t4 #d4 ,alcohol4 #e4 ,alcohol absolute1 equal p>ts4 ,acetic acid1 #a p] c5t41 ,hydro*loric1 #a p] c5t4 aque\s solution4 ,hydro*loric1 #a p] c5t4 ,alcoholic 7#ij p] c5t47 solution4 #f4 ,m9]al acids3 ,sulphuric1 #be p] c5t4 aque\s solution4 ,nitric1 #cc p] c5t4 aque\s solution4 ,c\nt]/a9+4--,use col\rs :i* w contra/ ) ! fir/ /a92 e4 g41 ,vesuv91 ,neutral r$1 = films /a9$ by me?yl5e-blue or ,eos91 ,gram's me?od4 ,fu*s91 ,me?yl5e-blue1 = films /a9$ by fu*s94 ,g5tian violet1 #h4 ,m\nt+4-- #a4 ,wa% ! film c>efully 9 runn+ wat]4 #b4 ,blot (f ! sup]flu\s wat] ) ! filt] pap]1 or dry m completely betwe5 two folds ( blott+ pap]4 #c4 ,complete ! dry+ 9 ! air1 or by hold+ ! cov]-slip 9 ! f+]s at a safe di/ance above ! flame ( ! ,buns5 burn]4 #d4 ,place a drop ( xylol balsam on ! c5tre ( a clean #c by #a glass slide & inv]t ! cov]-slip ov] ! balsam1 & l[] x c>efully to avoid ! inclusion ( air bubbles4 ,,note4--,xylol is us$ 9 pref]5ce to *lor(orm to dissolve ,canada balsam1 z x does n decol\rise ! specim54 ,impression films 7,klats*praep>at7 are prep>$ f isolat$ colonies ( bact]ia 9 ord] t _! *>act]i/ic =mation may be exam9$ by high] p[]s ?an c be br\ght to be> on ! liv+ cultivation4 ,!y are prep>$ f plate cultivations 7vide page #bcj7 9 ! foll[+ mann]4 #a4 ,remove a clean cov]-slip f ! alcohol pot ) /]ile =ceps & burn (f ! _s4 #b4 ,op5 ! plate & re/ "o edge ( ! cov]-slip on ! surface ( ! m$ium a little to "o side ( ! select$ colony4 ,l[] x cauti\sly ov] ! colony until horizontal4 ,avoid any lat]al movem5t or ! inclusion ( bubbles ( air4 #c4 ,make g5tle v]tical pressure on ! c5tre ( ! cov]-slip ) ! po9ts ( ! =ceps to ensure p]fect contact ) ! colony4 #d4 ,/eady "o edge ( ! cov]-slip ) ! =ceps & pass ! po9t ( a m\nt$ ne$le j "u ! opposite edge & raise ! cov]-slip c>efully2 ! colony w be adh]5t to x4 ,:5 ne>ly v]tical1 grasp ! cov]-slip ) ! =ceps & remove x f ! plate4 ,re-cov] ! plate4 #e4 ,place ! cov]-slip1 film upp]mo/1 on ! rubb] mat1 & cov] x ) an inv]t$ wat*-glass until dry4 #f4 ,fix by imm]s+ 9 "o ( ! fix+ fluids previ\sly m5tion$ 7vide page #hb74 #g4 ,cle> ) acetic acid & alcohol4 #h4 ,/a9 & m\nt z an ord9>y cov]-slip film prep>ation1 be+ c>eful to p]=m all wa%+ op]ations ) extreme g5tl5ess4 ,microscopical ,exam9ation ( ! ,un/a9$ ,specim5s4-- #a4 ,place ! body tube ( ! microscope 9 ! v]tical position4 #b4 ,arrange ! hang+-drop slide on ! microscope /age s t ! drop ( fluid is 9 ! optical axis ( ! in/rum5t1 & secure x 9 t position by means ( ! spr+ clips4 #c4 ,use ! #a_/f-;in* objective1 rack d[n ! body tube until ! front l5s ( ! objective is almo/ 9 contact ) ! cov]-slip--t is1 well )9 its focal di/ance4 ,?is is be/ done :il/ b5d+ d[n ! head to "o side ( ! microscope1 s t ! eyes are on a level ) ! /age4 #d4 ,apply ! eye to ! ocul> & adju/ ! plane mirror to ! position :i* secures ! be/ illum9ation4 #e4 ,rack ! cond5s] d[n slightly & cut d[n ! ap]ture ( ! iris diaphragm s t ! light1 al?\gh ev51 is dim4 #f4 ,rack up ! body tube by means ( ! co>se adju/m5t until ! bact]ia come into view2 !n focus exactly by means ( ! f9e adju/m5t4 ,"s difficulty is (t5 exp]i5c$ at fir/ 9 f9d+ ! hang+ drop1 & if ! fir/ attempt is unsuccessful1 ! /ud5t mu/ n on any acc\nt1 :il/ /ill apply+ 8 eye to ! ocul>1 rack ! body tube d[n 7= by s do+ !re is e likelihood ( ! front l5s ( ! objective be+ =c$ ?r\gh ! cov]-glass1 & n only spoil+ ! specim51 b also contam9at+ ! objective72 b1 on ! contr>y1 )draw 8 eye1 rack ! tube up1 & comm5ce aga9 f /ep #b4 ,d>k ,gr\nd ,illum9ation4-- #a4 ,set up ! microscope /& 9 ! v]tical position & ins]t ! highe/ eyepiece available4 #b4 ,remove ! nosepiece f ! microscope tube & fit ! #b_/c in* objective 9 place4 #c4 ,remove ! sub/age cond5s] & replace x by ! d>k gr\nd cond5s]4 #d4 ,fit up ! s\rce ( illum9ation "s #cj-ej cm4 di/ant f ! microscope4 7,?is %\ld be ! ,liliput ,arc ,lamp 7,leitz71 ,n]n/ ,lamp or inc&esc5t gas lamp2 if ei!r ( ! two latt] are employ$1 a bull's eye cond5s] to produce p>allel rays mu/ be int]pos$ betwe5 light & microscope72 & adju/ illum9ant & microscope s t ! sub/age plane mirror is completely fill$ ) light4 #e4 ,focus ! two conc5tric r+s engrav$ ^u ! upp] surface ( ! cond5s] & c5tre !m accurately by means ( ! c5tr+ screws4 #f4 ,prep>e a fre% specim5 7see pages #gd-gf7 ( ! mat]ial x is desir$ to obs]ve1 us+ select$1 new1 #c by #a glass slips ( less ?an #a mm4 ?ickness1 & ,no4 #a cov]-glasses 7#j4ag mm4 ?ick71 :i* %\ld be clean$ ) a piece ( s(t wa%lea!r & n ) ! em]y pap]1 z scrat*es on ! glass produce haz9ess 9 ! prep>ation4 #g4 ,deposit a l>ge drop ( imm]sion oil 7or pure wat]7 on ! upp] surface ( ! cond5s] & rack x d[n a few millimetres4 #h4 ,adju/ ! fre% prep>ation on ! microscope /age & fa/5 x 9 position ) ! /age clips4 #i4 ,rack up ! cond5s] until ! imm]sion fluid makes contact ) ! "u surface ( ! slide2 avoid ! =mation ( air bubbles4 #aj4 ,adju/ ! sub/age mirror s t ! light is reflect$ upw>d4 ,a bright spot w be se5 on ! fre% prep>ation ne> ! c5tre ( ! field4 #aa4 ,replace ! #b_/c-;in* objective by ! #a_/ab-;in* oil imm]sion l5s :i* has be5 fitt$ ) ! special /op to r$uce its ,n4 ,a42 place a drop ( imm]sion oil ^u ! c5tre ( ! cov]-glasses ( ! fre% prep>ation & l[] ! microscope tube until ! front l5s ( ! objective has ent]$ ! oil drop4 #ab4 ,focus ! bright spot ref]r$ to 9 /ep #aj4 ,if x no long] occupies ! c5tre ( ! field1 alt] ! angle ( ! sub/age mirror until x does4 #ac4 ,n[ focus ! l5s accurately on ! film1 cauti\sly v>y ! height ( ! d>k gr\nd cond5s] until ! be/ position is f\nd4 ,! int5sely illum9at$ bact]ia w /& out 9 vivid contra/ to ! d>k backgr\nd4 ,illu/ration3 ,,fig4 #gj4--;,imm]sion oil bottle4 ,microscopical ,exam9ation ( ! ,/a9$ ,specim54--7,! body tube ( ! microscope may be v]tical or incl9$ to an angle47 #a4 ,secure ! slide on ! /age ( ! microscope by means ( ! spr+ clips4 #b4 ,place a drop ( c$>wood oil on ! c5tre ( ! cov]-slip4 ,! imm]sion oil is pure c$>wood oil1 & is kept 9 a small bottle ( /\t glass 7,fig4 #gj71 ! cavity ( :i* is %ap$ l an inv]t$ cone1 & is provid$ ) a safety funnel 7s t ! oil does n escape if ! bottle is accid5tally ov]turn$7 & a du/ cap ( boxwood fitt$ ) a wood5 rod ) :i* ! drop ( oil is appli$ to ! cov]-glass or l5s4 #c4 ,use ! #a_/ab-;in* oil imm]sion l5s ( ! microscope4 ,rack d[n ! body tube till ! front l5s ( ! objective is 9 contact ) ! oil & ne>ly t\*+ ! cov]-slip4 #d4 ,rack up ! cond5s] until x is 9 contact ) ! "u surface ( ! slide4 #e4 ,apply ! eye to ! ocul> & arrange ! plane mirror s z to obta9 ! greate/ possible am\nt ( light4 #f4 ,rack up ! body tube until ! /a9$ film comes into view4 #g4 ,focus ! cond5s] accurately on ! film4 #h4 ,focus ! film accurately by means ( ! f9e adju/m5t4 ,,vi4 ,,/a9+ ,,me?ods4 ,9 ! foll[+ pages are collect$ ! v>i\s /ock /a9s 9 ev]yday use 9 ! bact]iological laboratory1 toge!r ) a selection ( ! mo/ conv5i5t & g5]ally useful /a9+ me?ods = demon/rat+ p>ticul> /ructures or diff]5tiat+ gr\ps ( bact]ia4 ,! /a9s employ$ %\ld ei!r be ^? prep>$ by ,gruebl]1 ( ,leipzig1 or ,m]ck1 ( ,d>m/adt4 ,! me?ods pr9t$ 9 ord9>y type are ^? :i* a long exp]i5ce has %[n to be ! mo/ reliable1 & to give ! be/ results--^? relegat$ to small type comprise su* z are n s g5]ally useful1 b give excell5t results 9 ! h&s ( ! exp]i5c$ work]4 ,,bact]ia ,,/a9s4 ,me?yl5e-blue4-- #a4 ,saturat$ ,aque\s ,solution4 ,weigh out ,me?yl5e-blue #a4e grammes ,place 9 a /opp]$ bottle hav+ a capacity ( f #aej to #bjj c4c4 & add ,di/ill$ wat] #ajj4j c4c4 ,all[ ! wat] to rema9 9 contact ) ! dye = two weeks1 %ak+ ! cont5ts ( ! bottle vig\r\sly = a few mom5ts e day4 ,filt]4 #b4 ,saturat$ ,alcoholic ,solution4 ,weigh out ,me?yl5e-blue #a4e grammes ,place 9 a /opp]$ bottle ( #aej c4c4 capacity & add ,alcohol1 #ij p] c5t #ajj4j c4c4 ,all[ ! alcohol to rema9 9 contact ) ! dye = two h\rs1 %ak+ vig\r\sly e few m9utes4 ,filt]4 #c4 ,c>bolic ,me?yl5e-blue 7,kuehne74 ,weigh out ,me?yl5e-blue #a4e grammes ,c>bolic acid #e4j grammes & dissolve 9 ,di/ill$ wat] #ajj4j c4c4 & add ,absolute alcohol #aj4j c4c4 ,filt]4 #d4 ,alkal9e ,me?yl5e-blue 7,loeffl]74 ,measure out & mix ,me?yl5e-blue1 saturat$ alcoholic solution #cj4j c4c4 ,cau/ic pota%1 #j4a p] c5t4 aque\s solution #ajj4j c4c4 ,filt]4 ,g5tian ,violet4-- #e4 ,saturat$ ,aque\s ,solution4 ,weigh out ,g5tian violet #b4be grammes & proce$ z 9 prep>+ ! correspond+ solution ( me?yl5e-blue4 #f4 ,saturat$ ,alcoholic ,solution4 ,weigh out ,g5tian violet #e4j grammes & proce$ z 9 prep>+ ! correspond+ solution ( me?yl5e-blue4 #g4 ,c>bolic ,g5tian ,violet 7,nicolle74 ,measure out & mix ,g5tian violet1 saturat$ alcoholic solution #aj4j c4c4 ,c>bolic acid1 #a p] c5t4 aque\s solution #ajj4j c4c4 ,filt]4 #h4 ,anil9 ,wat] ,solution 7,ko*-,ehrli*74 ,measure out ,di/ill$ wat] #ajj c4c4 ,add anil9 oil drop by drop 7%ak+ well aft] ! addition ( ea* drop7 until ! solution is opaque4 ,filt] until cle>4 & add ,absolute alcohol #aj c4c4 ,saturat$ alcoholic solution g5tian violet #aa c4c4 ,filt]4 ,,note4--,?is solution w n keep long] ?an #ad days4 ,?ion9e ,blue 7or ,lau?'s ,violet74-- #i4 ,c>bolic ,?ion9e ,blue 7,nicolle74 ,weigh out ,?ion9e blue #a4j gramme ,c>bolic acid #b4e grammes & dissolve 9 ,di/ill$ wat] #ajj4j c4c4 ,filt]4 ,be=e use dilute ) equal quantity ( di/ill$ wat] & aga9 filt]4 ,fu*s9 7,basic74-- #aj4 ,saturat$ ,aque\s ,solution4 ,weigh out ,basic fu*s9 #a4e grammes & proce$ z 9 prep>+ ! correspond+ solution ( me?yl5e-blue 7q4 v474 #aa4 ,saturat$ ,alcoholic ,solution4 ,weigh out ,basic fu*s9 #c4e grammes & proce$ z 9 prep>+ ! correspond+ solution ( me?yl5e-blue4 #ab4 ,c>bolic ,fu*s9 7,ziehl74 ,weigh out ,basic fu*s9 #a4j gramme ,c>bolic acid #e4j grammes dissolve 9 ,di/ill$ wat] #ajj4j c4c4 & add ,absolute alcohol #aj4j c4c4 ,filt]4 ,,contra/ ,,/a9s4 ,eos94--,!re are sev]al comm]cial v>ieties ( eos91 :i*1 f ! bact]iological po9t ( view1 possess v diff]5t values4 ,gruebl] li/s f\r v>ieties1 ( :i* two only are useful = bact]iological "w3 ,eos91 aque\s yell[i%4 ,eos91 aque\s blui%4 #ac4 ,eos9 ,aque\s ,solution 7,yell[i% or ,blui% ,%ade71 #a p] c5t4 ,weigh out ,eos91 aque\s #a4j gramme dissolve 9 ,di/ill$ wat] #ajj4j c4c4 & add ,absolute alcohol #e4j c4c4 ,filt]4 #ad4 ,eos9 ,alcoholic ,solution1 #j4e p] c5t4 ,weigh out ,eos91 alcoholic #j4e gramme & dissolve 9 ,alcohol 7#gj p] c5t47 #ajj4j c4c4 ,filt]4 ,safran9e4-- #ae4 ,aque\s ,solution4 ,weigh out4 ,safran9e #j4e gramme & dissolve 9 ,di/ill$ wat] #ajj4j c4c4 ,filt]4 ,neutral ,r$4-- #af4 ,aque\s ,solution4 ,weigh out ,neutral r$ #a4j gramme & dissolve 9 ,di/ill$ wat] #ajj4j c4c4 ,filt]4 ,vesuv9 7or ,bism>ck ,br[n74-- #ag4 ,saturat$ ,aque\s ,solution4 ,weigh out ,vesuv9 #j4e gramme & dissolve 9 ,di/ill$ wat] #ajj4j c4c4 ,filt]4 ,,tissue ,,/a9s4 ,anil9e ,g5tian ,violet 7,= ,weig]t's ,fibr9 ,/a974-- ,weigh out ,g5tian violet #a4j gramme & dissolve 9 ,absolute alcohol #ae4j c4c4 ,di/ill$ wat] #hj4j c4c4 !n add ,anil9e oil #c4j c4c4 ,%ake well & filt] be=e use4 ,haematoxyl9 7,ehrli*74-- #a4 ,weigh out ,haematoxyl9 #b4j grammes & dissolve 9 ,absolute alcohol #ajj4j c4c4 #b4 ,weigh out ,ammonium alum #b4j grammes & dissolve 9 ,di/ill$ wat] #ajj4j c4c4 #c4 ,mix #a & #b1 all[ ! mixture to /& =ty-eight h\rs1 !n filt]4 #d4 ,add ,glyc]9e #he4j c4c4 ,acetic acid1 glacial #aj4j c4c4 #e4 ,all[ ! /a9 to /& = "o mon? expos$ to light2 !n filt] aga9 ready = use4 ,haemat9 7,may]'s74-- ,a4 ,weigh out ,haemat9 #a4j gramme & dissolve 9 ,alcohol #ij p] c5t4 7w>m$ to #cg deg4 ,c47 #ej c4c4 ,b4 ,weigh out ,pota% alum #ej grammes & dissolve 9 ,di/ill$ wat] #ajj c4c4 ,prep>e ^! two solutions 9 sep>ate flasks4 ,take a clean flask ( #bej c4c4 capacity & ins]t a l>ge funnel 9 its neck4 ,p\r ! solutions ,a & ,b simultane\sly & sl[ly into ! funnel to mix ?or\ghly4 ,/ore = future use4 ,,note4--,if acid haemat9 is requir$1 introduce glacial acetic acid 7#c c4c47 into ! mix+ flask be=e add+ ! solutions ,a & ,b4 ,alum ,c>m9e 7,may]74-- ,weigh out ,alum #b4e grammes ,c>m9e #a4j gramme & place 9 a glass beak]4 ,measure out 9 a measur+ cyl9d]1 ,di/ill$ wat] #ajj4j c4c4 ,place ! beak] on a s&-ba?1 add ! wat] 9 successive small quantities1 & keep ! mixture boil+ = tw5ty m9utes4 ,measure ! solution & make up to #ajj c4c4 by ! addition ( di/ill$ wat]4 ,filt]4 ,li?ium ,c>m9e 7,or?74-- ,weigh out ,c>m9e #b4e grammes & dissolve 9 ,li?ium c>bonate1 cold saturat$ solution #ajj4j c4c4 ,filt]4 ,picroc>m9e4-- ,weigh out ,picroc>m9e #b4j grammes & dissolve 9 ,di/ill$ wat] #ajj4j c4c4 ,,blood ,,/a9s ,:5 wat]y solutions ( m$ic9al me?yl5e blue & wat] soluble eos9s are mix$ a precipitate is =m$ :i* is soluble only 9 alcohol1 & solutions ( ?is precipitate imp>t a peculi> r$di%-purple col\r to *romat94 ,?is comp\nd 0 fir/ us$ by ,roman[sky to demon/rate mal>ial p>asites1 b v>i\s modifications are n[ employ$ = /a9+ blood films g5]ally1 & also = bact]ia & protozoa4 ,! be/ modifications ( ! orig9al ,roman[sky are ^? ( ,j5n] & ,lei%man--,j5n] be+ mo/ suitable = ! hi/ological /udy ( ! blood1 & ,lei%man = ! demon/ration ( protozoa4 ,j5n]'s ,/a94-- ,a4 ,weigh out3 ,eos9 aque\s yell[ #f4j grammes ,dissolve 9 ,di/ill$ wat] 7non-alkal9e7 #bej c4c4 ,?is w make a ?ick solution4 ,b4 ,weigh out3 ,me?yl5e blue 7m$ic9ally pure7 ,hoe*/ #e4j grammes ,dissolve 9 ,di/ill$ wat] 7non-alkal9e7 #bej c4c4 #a4 ,add ,b to ,a v sl[ly1 /irr+ all ! "t4 ,a visc\s precipitate =ms :i* frequ5tly loses its viscosity :5 heat is appli$4 7,?is expla9s ! necessity ( mix+ sl[ly74 #b4 ,evaporate sl[ly 9 a porcela9 bas91 /irr+ occasionally1 on a wat] ba? at #ee deg4 ,c4 ,:5 a pa/e beg9s to =m scrape & break up occasionally4 7,on no acc\nt mu/ ! pa/e be all[$ to fuse47 #c4 ,gr9d ! result+ mass into an amorph\s p[d]4 #d4 ,weigh out3 ,amorph\s p[d] #j4e grammes ,dissolve 9 ,me?ylic alcohol 7,m]ck's puriss1 = analysis7 #ajj c4c4 ,all[ "t = true solution4 7,ab\t ?ree days is suffici5t47 ,,me?od4-- #a4 ,prep>e film1 dry1 b d n fix4 #b4 ,flood ! unfix$ film ) ! /a91 all[ x to act = #c m9utes 7! me?ylic alcohol ( ! /a9 fixes ! film74 #c4 ,p\r (f ! /a9 & wa% 9 di/ill$ wat] until ! film pres5ts a p9k col\r4 #d4 ,dry & m\nt4 ,lei%man's ,/a94-- ,a4 ,weigh out3 ,me?yl5e blue 7m$ic9al7 #a gramme ,dissolve 9 ,sodium c>bonate1 #j4e p] c5t4 aque\s solution #ajj c4c4 ,keep at #fe deg4 ,c4 = #ab h\rs 9 ei!r a hot incubator or a wat]-ba?2 !n /& 9 d>k place at room temp]ature 7#bj deg4 ,c47 = t5 days4 ,b4 ,weigh out3 ,eos91 extra ,b4 ,a4 #j4a gramme ,dissolve 9 ,di/ill$ wat] #ajj c4c4 #a4 ,mix ! two solutions ,a & ,b 9 equal volumes1 & all[ ! mixture to /& = #ab h\rs ) occasional /irr+4 #b4 ,filt]1 & collect precipitate on filt] pap]4 #c4 ,wa% precipitate ?or\ghly ) di/ill$ wat]1 & dry4 #d4 ,weigh out #j4ae gramme ( ! dri$ precipitate2 rub up 9 a mort> ) #e c4c4 ( me?ylic alcohol 7,m]ck's puriss1 = analysis74 ,all[ undissolv$ p[d] to settle1 !n decant ! sup]natant fluid to a clean #ajj c4c4 measur+ cyl9d]4 #e4 ,add fur!r #e c4c4 alcohol to s$im5t 9 mort> & repeat ! process1 & s on until all ! s$im5t has be5 dissolv$4 #f4 ,n[ make up ! fluid 9 ! measur+ cyl9d] to #ajj c4c4 by ! addition ( m me?ylic alcohol4 ,,me?od4-- #a4 ,prep>e film1 dry1 b d n fix4 #b4 ,flood ! unfix$ film ) /a91 all[ x to act #cj seconds4 #c4 ,add d\ble ! volume ( di/ill$ wat] to ! /a9 on ! film1 & mix ) glass rod or plat9um loop4 #d4 ,all[ ?is dilut$ /a9 to act five m9utes4 #e4 ,wa% (f ) di/ill$ wat]4 #f4 ,leave "s wat] on film = ?irty seconds to int5sify ! col\r contra/s4 #g4 ,dry & m\nt4 ,,me?ods ,,( ,,demon/rat+ ,,/ructure ,,( ,,bact]ia1 ,,etc4 ,to ,demon/rate ,capsules4 #a4 ,mac,conkey4-- ,/a94-- ,weigh out ,dahlia #j4e gramme ,me?yl gre5 7#jj cry/als7 #a4e grammes rub up 9 a mort> ) ,di/ill$ wat] #ajj4j c4c4 ,add ,fu*s91 saturat$ alcoholic solution #aj4j c4c4 & make up to #bjj c4c4 by ! addition ( ,di/ill$ wat] #ij4j c4c4 ,filt]4 ,all[ ! /a9 to /& = two weeks be=e use2 keep 9 a d>k place or 9 an amb] glass bottle4 ,ow+ to ! un/able "* ( ! me?yl gre51 ?is /a9 det]iorates aft] ab\t six mon?s4 ,,me?od4-- #a4 ,prep>e & fix film 9 ! usual mann]4 #b4 ,flood ! cov]-slip ) ! /a9 & all[ x to act = five to t5 m9utes4 #c4 ,wa% v ?or\ghly 9 wat]2 if necess>y1 direct a p[]ful /ream ( wat] on ! film f a wa%-bottle4 #d4 ,dry & m\nt4 #b4 ,muir's ,me?od4-- #a4 ,prep>e1 dry & fix film 9 ! ord9>y mann]4 #b4 ,flood ! film ) c>bolic fu*s91 w>m until /eam beg9s to rise4 ,all[ ! /a9 to act = ?irty seconds4 #c4 ,wa% quickly ) me?ylat$ _s4 #d4 ,wa% ?or\ghly ) wat]4 #e4 ,subject ! film to ! action ( ! foll[+ mordant = five seconds3 ,corrosive sublimate1 saturat$ aque\s solution #b c4c4 ,tannic acid1 #bj p] c5t4 aque\s solution #b c4c4 ,pota% alum saturat$ aque\s solution #e c4c4 #f4 ,wa% ?or\ghly 9 wat]4 #g4 ,treat ) me?ylat$ _s = ab\t sixty seconds4 7,! prep>ation %\ld n[ be pale r$47 #h4 ,wa% ?or\ghly 9 wat]4 #i4 ,c\nt]/a9 9 me?yl5e blue1 aque\s solution ?irty seconds4 #aj4 ,wa% 9 wat]4 #aa4 ,dehydrate 9 alcohol4 #ab4 ,cle> 9 xylol & m\nt 9 xylol balsam4 #c4 ,wel*'s ,me?od4-- #a4 ,prep>e & fix film 9 ! usual mann]4 #b4 ,flood ! slide ) acetic acid #b p] c5t42 all[ ! acid to rema9 9 contact ) ! film = two m9utes4 ,?is swells up & fixes ! capsule & enables x to take ! /a94 #c4 ,bl[ (f ! acetic acid by ! aid ( a pipette4 #d4 ,imm]se 9 anil9e g5tian violet1 five to ?irty seconds4 #e4 ,wa% 9 wat]4 #f4 ,dry & m\nt4 #d4 ,ribb]t's ,me?od4-- ,/a94-- ,measure out & mix3 ,acetic acid1 glacial #ab4e c4c4 ,alcohol1 absolute #ej4j c4c4 ,di/ill$ wat] #ajj4j c4c4 ,w>m to #cf deg4 ,c4 7e4 g41 9 ! hot incubator7 & saturate ) dahlia4 ,filt]4 ,,me?od4-- #a4 ,prep>e & fix films 9 ! usual mann]4 #b4 ,cov] ! film ) ! /a9 & all[ x to act = "o or two seconds only4 #c4 ,wa% ?or\ghly 9 wat]4 #d4 ,dry & m\nt4 ,to ,demon/rate ,flagella4 #a4 ,muir's ,modifi$ ,pitfield4--,?is is ! be/ me?od & gives ! mo/ reliable results1 = n only is ! p]c5tage ( successful prep>ations high] ?an ) any o!r1 b ! bacilli & flagella reta9 _! relative proportions4 7a7 ,mordant4-- ,tannic acid1 #aj p] c5t4 aque\s solution #aj c4c4 ,corrosive sublimate1 saturat$ aque\s solution #e c4c4 ,alum1 saturat$ aque\s solution #e c4c4 ,c>bolic fu*s9 7,ziehl7 #e c4c4 ,mix ?or\ghly4 ,a precipitate =ms :i* mu/ be all[$ to settle = a few h\rs4 ,decant (f ! cle> fluid into tubes & c5trifugalise ?or\ghly4 ,?is solution is at its be/ "s f\r or five days aft] manufacture2 x keeps = ab\t a c\ple ( weeks1 b mu/ be re-c5trifugalis$ ea* "t1 be=e use4 7b7 ,/a94-- ,alum1 saturat$ aque\s solution #be c4c4 ,g5tian violet1 saturat$ alcoholic solution #e c4c4 ,filt]4 ,?is /a9 mu/ be fre%ly prep>$4 ,,me?od4--,! cultivations employ$ %\ld be sme> ag> cultures1 twelve to eighte5 h\rs old if incubat$ at #cg deg4 ,c1 tw5ty-f\r to ?irty h\rs if incubat$ at #bb deg4 ,c4 #a4 ,remove a v small quantity ( ! gr[? by means ( ! plat9um spatula4 #b4 ,emulsify x ) a few cubic c5timetres ( di/ill$ wat] 9 a wat*-glass1 by g5tly mov+ ! spatula to & fro 9 ! wat]4 ,d n rub up ! gr[? on ! side ( ! wat*-glass4 ,"s work]s pref] to use tap wat]1 o!rs employ normal sal9e solution1 b di/ill$ wat] gives ! be/ emulsion4 #c4 ,spread a ?9 film ( ! emulsion on a newly flam$ cov]-slip1 us+ no =ce1 b r lead+ ! drop ov] ! cov]-slip ) ! plat9um loop4 #d4 ,all[ ! film to dry 9 ! air1 prop]ly protect$ f fall+ du/4 #e4 ,fix by pass+ ?rice ?r\gh ! ,buns5 flame1 hold+ ! cov]-slip :il/ do+ s by "o corn] betwe5 ! f+] & ?umb4 #f4 ,p\r on ! film z mu* ( ! mordant z ! cov]-glass w hold4 ,grasp ! cov]-slip ) ! =ceps & hold x1 high above ! flame1 until /eam rises4 ,all[ ! /eam+ mordant to rema9 9 contact ) ! film two m9utes4 #g4 ,wa% well 9 wat] & dry c>efully4 #h4 ,p\r on ! film z mu* ( ! /a9 z ! cov]-glass w hold4 ,/eam ov] ! flame z be=e = two m9utes4 #i4 ,wa% well 9 wat]4 #aj4 ,dry & m\nt4 #b4 ,pitfield ,orig9al ,me?od4-- 7a7 ,mordant4-- ,tannic acid #a gramme ,wat] #aj c4c4 7b7 ,/a94-- ,saturat$ aque\s solution ( alum #aj c4c4 ,saturat$ alcoholic solution ( g5tian violet #a c4c4 ,di/ill$ wat] #e c4c4 ,mix equal p>ts ( a & b be=e us+4 #a4 ,prep>e & fix ! film 9 ! mann] describ$ above4 #b4 ,boil ! mixture & imm]se ! cov]-slip 9 x1 :il/ /ill hot1 = "o m9ute4 #c4 ,wa% 9 wat]4 #d4 ,exam9e 9 wat]2 if satisfactory1 dry & m\nt 9 ,canada balsam4 #c4 ,mac,crorrie's ,me?od4-- ,mordant-,/a94-- ,measure out & mix4 ,night blue1 saturat$ alcoholic solution #aj c4c4 ,pota% alum1 saturat$ aque\s solution #aj c4c4 ,tann91 #aj p] c5t4 aque\s solution #aj c4c4 ,,note4--,! addition ( gallic acid1 #j4a to #j4b gramme1 may improve ! solution1 b is n necess>y4 ,,me?od4-- #a4 ,prep>e & fix ! films z above4 #b4 ,p\r "s ( ! mordant-/a9 on ! film & w>m g5tly1 high above ! flame1 = two m9utes 7or place 9 ! hot incubator = a l p]iod74 #c4 ,wa% ?or\ghly 9 wat]4 #d4 ,dry & m\nt4 #d4 ,loeffl]'s ,me?od4-- 7a7 ,mordant4-- ,tannic acid1 #bj p] c5t4 aque\s solution #aj c4c4 ,f]r\s sulphate1 saturat$ aque\s solution #e c4c4 ,haematoxyl9 solution #c c4c4 ,c>bolic acid1 #a p] c5t4 aque\s solution #d c4c4 ,?is solution mu/ be fre%ly prep>$4 ,haematoxyl9 solution is prep>$ by boil+ #a gramme logwood ) #h c4c4 di/ill$ wat]1 filt]+ & replac+ ! loss f evaporation4 ,alt]native ,mordant 7,bunge's ,mordant74-- ,tannic acid1 #bj p] c5t4 aque\s solution #aj c4c4 ,f]r\s sulphate1 saturat$ aque\s solution #e c4c4 ,fu*s91 saturat$ alcoholic solution #a c4c4 7b7 ,/a94-- ,weigh out ,me?yl5e-blue ,or me?yl5e-violet #d grammes ,or fu*s9 & dissolve 9 ,anil9e wat]1 fre%ly saturat$ & filt]$ #ajj c4c4 ,,me?od4-- #a4 ,prep>e & fix films z above4 #b4 ,p\r ! mordant on to ! film & w>m cauti\sly ov] ! flame till /eam rises2 keep ! mordant g5tly /eam+ = "o m9ute4 #c4 ,wa% well 9 di/ill$ wat] till no m col\r is dis*>g$2 if necess>y1 wa% c>efully ) absolute alcohol4 #d4 ,filt] a few drops ( ! /a9 on to ! film1 w>m z be=e1 & all[ ! /eam+ /a9 to act = "o m9ute4 #e4 ,wa% well 9 di/ill$ wat]4 #f4 ,dry & m\nt4 ,,note4--,! flagella ( "s organisms c be demon/rat$ bett] by means ( an alkal9e /a9 or an acid /a9--a po9t to be det]m9$ = ea*4 ,speak+ g5]ally1 ^? bacilli :i* give rise to an acid reaction 9 ! culture m$ium require an alkali2 ^? :i* =m alkali 9 cultivation require an acid4 ,accord+ to requirem5ts1 !re=e1 ,loeffl] recomm5ds ! addition ( sodium hydrate1 #a p] c5t4 aque\s solution1 #a c4c42 or an equal quantity ( an exactly comp>able solution ( sulphuric acid4 #e4 ,van ,erm5gem's ,me?od4--,?is me?od1 be+ m]ely a precipitation ( a silv] salt on ! micro-organisms & n a true /a91 creates a false impression z to ! relative proportions ( bact]ia & flagella4 7a7 ,fix+ ,fluid4-- ,osmic acid1 #b p] c5t4 aque\s solution #aj c4c4 ,tannic acid1 #bj p] c5t4 aque\s solution #bj c4c4 ,acetic acid1 glacial #a c4c4 ,! fix+ fluid %\ld be prep>$ "s days be=e use & filt]$ z requir$4 ,9 col\r x %\ld be di/9ctly violet4 7b7 ,s5sitis+ ,solution4-- ,silv] nitrate1 #j4e p] c5t4 aque\s solution4 ,?is solution mu/ be kept 9 a d>k blue glass bottle or 9 a d>k cupbo>d4 ,filt] imm$iately be=e use4 7c7 ,r$uc+ ,solution4-- ,weigh out ,gallic acid #e grammes ,tannic acid #c grammes ,potassium acetate1 fus$ #aj grammes & dissolve 9 ,di/ill$ wat] #cej c4c4 ,filt]4 ,?is solution w keep active = sev]al days1 b fre% solution mu/ be us$ = ea* prep>ation4 ,,me?od4-- #a4 ,prep>e emulsion1 make & fix films z above 9 ! prec$+ me?od1 /eps #a to #d4 #b4 ,p\r on ! film z mu* ( ! fix+ solution z ! cov]-glass w hold1 heat c>efully ov] ! flame till /eam rises1 & all[ ! /eam+ fix+ fluid to act = five m9utes4 #c4 ,wa% well 9 wat]4 #d4 ,wa% 9 absolute alcohol4 #e4 ,wa% 9 di/ill$ wat]4 #f4 ,p\r "s ( ! s5sitis+ solution on ! film & all[ x to act = f ?irty seconds to "o m9ute2 blot (f ! excess ( fluid ) filt] pap]4 #g4 ,)\t wa%+1 transf] ! film to a wat*-glass conta9+ ! r$uc+ solution & all[ x to rema9 !re9 = f ?irty seconds to "o m9ute2 blot (f ! excess ( fluid ) filt] pap]4 #h4 ,)\t wa%+1 aga9 treat ! film ) ! s5sitis+ solution1 ?is "t until ! film comm5ces to turn black4 #i4 ,wa% 9 di/ill$ wat]4 #aj4 ,dry & m\nt4 ,to ,/a9 ,nuclei ( ,yea/ ,cells4 #a4 ,prep>e & fix film 9 ! usual mann]4 #b4 ,soak 9 f]ric ammonia sulphate #c p] c5t4 aque\s solution = two h\rs4 #c4 ,wa% ?or\ghly 9 wat]4 #d4 ,/a9 9 haematoxyl9 solution 7see page #ie7 = ?irty m9utes4 #e4 ,wa% 9 wat]4 #f4 ,diff]5tiate 9 f]ric ammonia sulphate solution = #a-a_/b-b m9utes1 exam9+ wet "u microscope dur+ ! process4 ,to ,/a9 ,spores4 #a4 ,s+le ,/a94-- #a4 ,prep>e cov]-slip film 9 ! usual way4 #b4 ,9 fix+1 pass ! cov]-slip film fifte5 or ?irty times ?r\gh ! flame in/ead ( only ?ree4 ,?is de/roys ! resi/+ p[] ( ! spore membrane & all[s ! /a9 to rea* ! int]ior4 #c4 ,/a9 9 ! usual way ) me?yl5e-blue or fu*s94 #d4 ,wa% 9 wat]4 #e4 ,dry & m\nt4 #b4 ,d\ble ,/a94-- #a4 ,prep>e & fix film 9 ! usual way--i4 e41 pass ?ree times ?r\gh flame to fix4 #b4 ,cov] ! film ) hot c>bol-fu*s9 & hold 9 ! =ceps above a small flame until ! fluid beg9s to /eam4 ,set ! cov]-slip d[n & all[ x to cool4 ,repeat ! process :5 ! /a9 ceases to /eam & cont9ue to repeat until ! /a9 has be5 9 contact ) ! film = tw5ty m9utes4 7,?is /a9s bo? spores & bact]ia47 #c4 ,wa% 9 wat]4 #d4 ,decol\rise 9 alcohol1 #b p>ts2 acetic acid1 #a p] c5t41 #a "p4 7,?is removes ! /a9 f ev]y?+ b ! spores47 #e4 ,wa% 9 wat]4 #f4 ,m\nt ! cov]-slip 9 wat] & exam9e microscopically ) ! #a_/f-;in* objective4 7,spores %\ld be r$1 & ! re/ ( ! film col\rless or a v light p9k47 ,if satisfactory1 pass on to section #g2 if unsatisfactory1 repeat /eps #b to #e4 #g4 ,c\nt]/a9 9 weak me?yl5e-blue4 7,n[ spores r$1 bacilli blue47 #h4 ,wa% 9 wat]4 #i4 ,dry & m\nt4 ,! spores ( diff]5t bacilli diff] greatly 9 _! resi/ance to decol\ris+ reag5ts2 ev5 ! spores ( ! same species ( organisms v>y accord+ to _! age4 ,"y spores are m easily decol\ris$ ?an ^? m mature4 ,sulphuric acid1 #a p] c5t4 aque\s solution1 & hydro*loric acid1 #j4e p] c5t4 alcoholic 7#ij p] c5t47 solution1 are useful decol\ris+ reag5ts4 #c4 ,moell]'s ,me?od4-- #a4 ,prep>e & fix films 9 ! usual mann]4 #b4 ,imm]se 9 absolute alcohol = two m9utes1 !n 9 *lor(orm = two m9utes2 wa% 9 wat]4 ,?is dissolves out any fat or cry/als t might o!rwise reta9 ! spore /a94 #c4 ,imm]se 9 *romic acid1 #e p] c5t4 aque\s solution1 = "o m9ute2 wa% 9 wat]4 #d4 ,p\r ,ziehl's c>bolic fu*s9 on ! film1 w>m z 9 previ\s me?ods1 & all[ x to act = t5 m9utes4 #e4 ,wa% 9 wat]4 #f4 ,decol\rise 9 sulphuric acid1 #e p] c5t4 aque\s solution1 = five seconds4 #g4 ,wa% 9 wat]4 #h4 ,c\nt]/a9 ) ,kuehne's c>bolic me?yl5e-blue = "o or two m9utes4 #i4 ,wa% 9 wat]4 #aj4 ,dry & m\nt4 7,spores r$1 bacilli blue47 #d4 ,abbott's ,me?od4-- #a4 ,prep>e & fix films 9 ! usual mann]4 #b4 ,p\r ,loeffl]'s alkal9e me?yl5e-blue on ! film2 w>m cauti\sly ov] ! flame till /eam rises & all[ ! hot /eam to act = "o to five m9utes4 #c4 ,wa% ?or\ghly 9 wat]4 #d4 ,decol\rise 9 nitric acid1 #b p] c5t4 alcoholic 7alcohol #hj p] c5t47 solution4 #e4 ,wa% ?or\ghly 9 wat]4 #f4 ,c\nt]/a9 9 eos91 #a p] c5t4 aque\s solution4 #g4 ,wa%4 #h4 ,dry & m\nt4 7,spores blue1 bacilli r$47 ,,diff]5tial ,,me?ods ,,( ,,/a9+4 ,gram's ,me?od4--,?is me?od dep5ds ^u ! fact t ! protoplasm ( "s bact]ia p]mits anil9e g5tian violet & ,lugol's iod9e solution1 :5 appli$ consecutively1 to ent] into a *emical comb9ation :i* results 9 ! =mation ( a new blue-black pigm5t1 only v sp>+ly soluble 9 absolute alcohol4 ,su* organisms are said to /a9 by ,gram1 or to be ,gram positive4 #a4 ,prep>e a cov]-slip film & fix 9 ! usual way4 #b4 ,/a9 9 anil9e g5tian violet ?ree to five m9utes4 ,filt] z mu* anil9e wat] on to ! cov]-slip z x w hold2 !n add ! smalle/ quantity ( alcoholic solution ( g5tian violet :i* suffices to saturate ! anil9e wat] & =m a bronze scum ^u its surface--if too mu* ( ! alcoholic g5tian violet is add$ ! alcohol pres5t r$issolves ?is scum4 ,to prep>e anil9e wat]1 p\r #d or #e c4c4 anil9e oil into a /opp]$ bottle & add di/ill$ wat]1 #ajj c4c4 ,%ake vig\r\sly & filt] imm$iately be=e use4 ,! excess ( oil s9ks to ! bottom ( ! bottle & may be us$ aga94 #c4 ,wa% 9 wat]4 #d4 ,treat ) ,lugol's iod9e solution until ! film is black or d>k br[n4 ,to d ?is treat ) iod9e solution = a few seconds1 wa% 9 wat]1 & exam9e ! film ov] a piece ( :ite filt] pap]4 ,note ! col\r4 ,repeat ?is process until ! film ceases to d>k5 ) ! fre% application ( iod9e solution4 ,lugol's solution is prep>$ by dissolv+ ,iod9e #a gramme ,iodide ( potassium #c grammes ,9 di/ill$ wat] #cjj c4c4 #e4 ,wa% 9 wat]4 #f4 ,wa% ) alcohol until no m col\r is dis*>g$ & ! alcohol runs away cle> & col\rless4 ,! foll[+ mixture may be sub/itut$ = absolute alcohol z a decol\ris] ,acetone #aj c4c4 ,absolute alcohol #ajj c4c4 #g4 ,wa% 9 wat]4 #h4 ,c\nt]/a9 v lightly ) aque\s solution ( ,neutral ,r$4 ,o!r c\nt]/a9s may be us$ su* z dilute eos91 dilute fu*s91 or vesuv94 ,,note4--,?is section may be omitt$ :5 deal+ ) films prep>$ f pure cultivations4 #i4 ,wa% 9 wat]4 #aj4 ,dry & m\nt4 ,gram-,claudius ,me?od4-- #a4 ,prep>e a cov]-slip film & fix 9 ! usual way4 #b4 ,/a9 9 me?yl violet1 #a p] c5t4 aque\s solution = ?ree to five m9utes4 #c4 ,treat ) two lots picric acid1 saturat$ aque\s solution4 #d4 ,wa% 9 wat] & dry4 #e4 ,decol\rise ) clove oil4 #f4 ,wa% (f clove oil ) xylol4 #g4 ,m\nt 9 xylol balsam4 ,gram-,weig]t ,me?od4-- #a-e4 ,proce$ z = ! correspond+ sections ( ,gram's me?od 7quod vide74 #f4 ,dry 9 ! air4 #g4 ,wa% 9 anil9e oil1 #a "p1 xylol1 #b p>ts1 until no m col\r is dis*>g$4 #h4 ,wa% 9 xylol4 #i4 ,m\nt 9 xylol balsam4 ,modifi$ ,gram-,weig]t ,me?od4--7,to demon/rate tri*ophyta 9 hair47 #a4 ,soak ! hairs 9 e!r = t5 m9utes to remove ! fat4 #b4 ,/a9 ?irty m9utes 9 a t>-l solution ( anil9e g5tian violet 7prep>$ by add+ #ae drops ( ! alcoholic solution ( g5tian violet to #c drops ( anil9e wat]74 #c4 ,dry ! hairs betwe5 pieces ( blott+ pap]4 #d4 ,treat ) p]fectly fre% iod9e solution4 #e4 ,aga9 dry betwe5 blott+ pap]4 #f4 ,treat ) anil9e oil to remove excess ( /a94 7,if necess>y1 add a drop or two ( nitric acid to ! oil47 #g4 ,aga9 treat ) anil9e oil4 #h4 ,treat ) anil9e oil & xylol1 equal p>ts4 #i4 ,cle> ) xylol4 #aj4 ,m\nt 9 xylol balsam4 ,to obta9 ! be/ diff]5tiation ! prep>ation %\ld be repeat$ly exam9$ microscopically 7) a #a_/f-;in* objective7 betwe5 /eps #e & #i1 z ! actual "t involv$ v>ies ) diff]5t specim5s4 ,ziehl-,neels5's ,me?od4--7,to demon/rate tub]cle & o!r acid-fa/ bacilli47 #a4 ,sme> a ?91 ev5 film ( ! specim5 on ! cov]-slip by means ( ! plat9um loop4 7,9 ! case ( sputum1 if x is a v wat]y specim51 all[ ! film to dry1 !n spread a second & ev5 a ?ird lay] ov] ! fir/47 #b4 ,fix by pass+ ?ree times ?r\gh ! flame4 #c4 ,/a9 9 hot c>bol-fu*s9 7z 9 /a9+ = spores7 = five to t5 m9utes4 7,?is /a9s ev]y?+ on ! film47 ,avoid ov]-heat+4 #d4 ,decol\rise by dipp+ 9 sulphuric acid1 #be p] c5t4 7,?is removes /a9 f ev]y?+ b acid-fa/ bacilli2 e4 g41 tub]cle1 leprosy1 & smegma bacilli & ! film turns yell[47 #e4 ,wa% 9 wat]4 7,a pale r$ col\r returns to ! film74 #f4 ,wa% 9 alcohol till no m col\r is dis*>g$4 7,?is (t51 b n inv>iably1 removes ! /a9 f acid-fa/ bacilli o!r ?an tub]cle2 e4 g41 smegma bacillus47 #g4 ,wa% 9 wat]4 #h4 ,c\nt]/a9 9 weak me?yl5e-blue4 7,/a9s non-acid-fa/ bacilli1 leucocytes1 epi!lial cells1 etc47 #i4 ,wa% 9 wat]1 dry1 & m\nt4 ,papp5heim's ,me?od4-- ,?is me?od is suppos$ to diff]5tiate betwe5 ,b4 tub]culosis & o!r acid-fa/ micro-organisms4 #a4 ,prep>e & fix film 9 ! usual way4 #b4 ,/a9 9 c>bol-fu*s9 )\t heat = ?ree m9utes4 #c4 ,)\t previ\sly wa%+ 9 wat] treat ! film ) ?ree or f\r successive applications ( corall9 7,rosolic acid7 solution4 ,corall9 #a gramme ,me?yl5e-blue 7saturat$ alcoholic solution7 #ajj c4c4 ,glyc]9e #bj c4c4 #d4 ,wa% 9 wat]4 #e4 ,dry & m\nt4 ,neiss]'s ,me?od--,modifi$4--7,to demon/rate diph!roid bacilli47 ,/a9 ,i4-- ,measure out & mix ,me?yl5e-blue1 saturat$ alcoholic solution #d4j c4c4 ,acetic acid1 #e p] c5t4 aque\s solution #if4j c4c4 ,filt]4 ,/a9 ,,ii4-- ,weigh out ,neutral r$ #b4e grammes & dissolve 9 ,di/ill$ wat] #ajjj c4c4 ,filt]4 ,,me?od4-- #a4 ,prep>e & fix films 9 ! usual way4 #b4 ,p\r /a9 ,i on ! film & all[ x to act = two m9utes4 #c4 ,wa% ?or\ghly 9 wat]4 #d4 ,treat ) ,lugol's iod9e = t5 seconds4 #e4 ,wa% ?or\ghly 9 wat]4 #f4 ,p\r /a9 ,,ii on to ! film & all[ x to act = ?irty seconds4 #g4 ,wa% ?or\ghly 9 wat]4 #h4 ,dry & m\nt4 ,,note4--,! cultivation f :i* ! films are prep>$ mu/ be ^u blood-s]um :i* has be5 incubat$ at #cg deg4 ,c4 = f n9e to eighte5 h\rs4 ,! bacilli are /a9$ a light r$ by ! neutral r$1 :i* contra/s well ) ! two or ?ree black spots1 situat$ at ! poles & occasionally "o 9 ! c5tre repres5t+ protoplasmic aggregations 78 meta*romatic granules7 /a9$ by ! acid me?yl5e-blue4 ,:eal & ,*[n 7,ox=d7 ,me?od4--7,to demon/rate act9omyces47 #a4 ,/a9 briefly ) ,ehrli*'s haematoxyl9 7until nuclei are fa9t blue aft] wa%+ ) tap wat]74 #b4 ,wa% 9 tap wat]4 #c4 ,/a9 9 hot c>bol-fu*s9 7z = tub]cle bacilli7 = five to t5 m9utes4 #d4 ,wa% 9 tap wat]4 #e4 ,decol\rise ) ,sp5gl]'s picric acid alcohol4 ,?is is prep>$ by mix+3 ,alcohol1 absolute #bj c4c4 ,picric acid1 saturat$ aque\s solution #aj c4c4 ,di/ill$ wat] #aj c4c4 ,dur+ ! progress ( /eps #a-e ! prep>ation mu/ be repeat$ly exam9$ microscopically ) ! #a_/f-;in* objective4 ,:5 prop]ly diff]5tiat$ ! clubs appe> brilliant r$ on gre5i% gr\nd4 #f4 ,dehydrate 9 alcohol4 #g4 ,cle> 9 xylol4 #h4 ,m\nt 9 xylol balsam4 ,?is me?od s]ves equally well = films & = sections4 ,,vii4 ,,me?ods ,,( ,,demon/rat+ ,,bact]ia ,,9 ,,tissues4 ,= bact]iological purposes1 sections ( tissue are mo/ conv5i5tly prep>$ by ei!r ! freez+ me?od or ! p>aff9 me?od4 ,! latt] is decid$ly pref]able1 b z x is ( great] importance to demon/rate ! bact]ia1 if su* are pres5t1 ?an to pres]ve ! tissue elem5ts unalt]$1 ! froz5 sections are (t5 ( value4 ,:i*ev] me?od is select$1 x is necess>y to take small pieces ( ! tissue = section+1--#b to #e mm4 cubes :5 possible1 b 9 any case n exce$+ half a c5timetre 9 ?ickness4 ,po/-mortem mat]ial %\ld be secur$ z soon aft] ! dea? ( ! animal z possible4 ,! tissue is prep>$ = cutt+ by-- 7a7 ,fixation2 t is1 by caus+ ! dea? ( ! cellul> elem5ts 9 su* a mann] t !y reta9 _! *>act]i/ic %ape & =m4 ,! fix+ fluids 9 g5]al use are3 ,absolute alcohol2 corrosive sublimate1 saturat$ aque\s solution2 corrosive sublimate1 ,lang's solution 7vide page #hb72 =maldehyde1 #d p] c5t4 aque\s solution4 7,( ^!1 ,lang's corrosive sublimate solution is decid$ly ! be/ all-r\nd fixative47 7b7 ,h>d5+2 t is1 by r5d]+ ! tissue ( suffici5t consi/5cy to admit ( ?9 slices or sections be+ cut f x4 ,?is is effect$ by pass+ ! tissue successively ?r\gh alcohols ( gradually increas+ /r5g?3 #cj p] c5t4 alcohol1 #ej p] c5t4 alcohol1 #ge p] c5t4 alcohol1 #ij p] c5t4 alcohol1 absolute alcohol4 ,9 bo? ^! processes a l>ge excess ( fluid %\ld always be us$4 ,,freez+ ,,me?od4 #a4 ,fixation4 ,place ! pieces ( tissue 9 a wide-m\!d glass bottle & fill ) absolute alcohol4 ,all[ ! tissues to rema9 !re9 = tw5ty-f\r h\rs4 #b4 ,h>d5+4 ,remove ! alcohol 7no long] absolute1 z x has tak5 up wat] f ! tissues7 f ! bottle & replace x ) fre% absolute alcohol4 ,all[ ! tissues to rema9 !re9 = tw5ty-f\r h\rs4 ,illu/ration3 ,,fig4 #ga4--,wa%+ tissues4 ,,note4--,if n ne$$ = cutt+ imm$iately1 ! h>d5$ tissues c be /or$ 9 #ge p] c5t4 alcohol4 #c4 ,remove ! alcohol f ! tissues by soak+ 9 wat] f "o to two h\rs4 ,remove ! /opp] f ! bottle2 re/ a glass funnel 9 ! op5 m\? & place "u a tap ( runn+ wat]4 ,! wat] ( c\rse1 ov]fl[s1 b ! tissues rema9 9 ! bottle 7,fig4 #ga74 #d4 ,impregnate ! tissues ) mucilage = twelve to tw5ty-f\r h\rs1 accord+ to size4 ,transf] ! pieces ( tissue to a bottle conta9+ /]ilis$ gum mixture4 ,=mula4-- ,gum arabic #e grammes ,sac*>ose #a gramme ,boric acid #a gramme ,wat] #ajj c4c4 #e4 ,place ! tissue on ! plate ( a freez+ microtome 7,ca?c>t's is p]haps ! be/ =m71 cov] & surr\nd ) fre% gum mixture2 freeze ) e!r1 or = pref]5ce1 c>bon dioxide1 & cut sections4 #f4 ,float ! sections (f ! knife into a glass di% conta9+ tepid wat] & all[ !m to rema9 !re9 = ab\t an h\r to dissolve out ! gum4 7,if n requir$ at once1 /ore 9 #ij p] c5t4 alcohol47 #g4 ,transf] to a glass capsule conta9+ ! select$ /a9+ fluid1 by means ( a section lift]4 #h4 ,transf] ! sections 9 turn to a capsule conta9+ absolute alcohol 7to dehydrate7 & to "o conta9+ xylol or oil ( cloves 7to cle>74 #i4 ,m\nt 9 xylol balsam4 ,alt]native ,rapid ,me?od4-- #a4 ,cut v small blocks ( ! tissue4 #b4 ,fix 9 =mal9 #aj p] c5t4 aque\s solution 7fixation fluid ,no4 #g1 page #hb7 = #bd h\rs4 #c4 ,transf] block to plate ( freez+ microtome & freeze ) c>bon dioxide vap\r4 #d4 ,float ! sections (f ! knife into a glass di% ( tepid wat]4 #e4 ,/a9 ! sections 9 glass capsules conta9+ select$ /a9s4 #f4 ,place ! /a9$ section 9 a di% ( clean wat] & introduce a glass slide obliquely b5ea? ! section2 ) a m\nt$ ne$le draw ! section on to ! slide & hold x !re2 g5tly remove ! slide f ! wat]1 tak+ c>e t any folds 9 ! section are float$ out be=e ! slide is f9ally remov$ f ! wat]4 #g4 ,dra9 away z mu* wat] z possible f ! section4 ,drop absolute alcohol on to ! section f a drop bottle1 to dehydrate x4 #h4 ,d\ble a piece ( blott+ pap] & g5tly press x on ! section to dry x4 #i4 ,drop on xylol to cle> ! section4 #aj4 ,place a l>ge drop ( xylol balsam on ! section & c>efully l[] a cov]-glass on to ! balsam4 ,,p>aff9 ,,me?od4 #a4 ,fixation4 ,place ! pieces ( tissue1 re/+ on cotton-wool1 9 a wide-m\!d glass bottle4 ,p\r on a suffici5t quantity ( ! corrosive sublimate fix+ fluid2 all[ ! tissue to rema9 !re9 = twelve to tw5ty-f\r h\rs accord+ to size4 #b4 ,p\r (f ! fix+ fluid & wa% ?or\ghly 9 runn+ wat] = tw5ty m9utes to half an h\r to remove ! excess ( corrosive sublimate4 ,illu/ration3 ,,fig4 #gb4--,l-%ap$ brass m\lds4 ,illu/ration3 ,,fig4 #gc4--,p>aff9 kettle4 #c4 ,h>d5+4 ,place ! tissues 9 ea* ( ! foll[+ /r5g?s ( alcohol 9 turn = f twelve to tw5ty-f\r h\rs3 #ej p] c5t41 #ge p] c5t41 #ij p] c5t41 absolute4 #d4 ,dehydration is effect$ by transf]r+ ! tissues to fre% absolute alcohol4 #e4 ,cle>+4 ,half fill a wide-m\!d bottle ) *lor(orm4 ,on ! surface ( ! *lor(orm float a lay] ( absolute alcohol ab\t five to t5 millimetres 9 dep?4 ,place ! pieces ( tissue 9 ! lay] ( alcohol & :5 !y h sunk ?r\gh ?is lay]1 transf] !m to pure *lor(orm = f six to tw5ty-f\r h\rs accord+ to ! size ( ! pieces4 ,:5 cle>$1 ! tissue becomes m or less transp>5t4 #f4 ,infiltration4 ,place ! cle>$ tissues 9 fre% *lor(orm ) sev]al pieces ( p>aff9 wax & /& 9 a w>m place1 su* z on ! top ( ! w>m incubator4 ,! w>m? gradually melts ! p>aff9 & ! tissues %\ld rema9 9 ! mixture ab\t tw5ty-f\r h\rs4 #g4 ,transf] ! tissues to a vessel conta9+ pure melt$ p>aff94 ,place ?is vessel 9 a p>aff9 wat]-ba? regulat$ = #b deg4 ,c4 above ! melt+-po9t ( ! p>aff9 us$1 & all[ ! tissues to soak = "s f\r to six h\rs to ensure complete impregnation4 ,! p>aff9 us$ %\ld h a melt+-po9t ( n m ?an #eh deg4 ,c4 ,= all ord9>y purposes #ed deg4 ,c4 w be f\nd q high en\gh4 #h4 ,imb$ 9 fre% p>aff9 9 a metal 7or pap]7 m\ld4 7a7 ,arrange a pair ( ,l-%ap$ pieces ( metal on a plate ( glass to =m a rectangul> tr\gh 7,fig4 #gb74 7b7 ,p\r fre% melt$ p>aff9 into ! m\ld f a special vessel 7,fig4 #gc74 7c7 ,lift ! piece ( tissue f ! p>aff9 ba? & arrange x 9 ! m\ld4 7d7 ,bl[ g5tly on ! surface ( ! p>aff9 9 ! m\ld1 & z soon z a film ( solid p>aff9 has =m$1 c>efully lift ! glass plate on :i* ! m\ld is set & l[] plate & m\ld toge!r into a bas9 ( cold wat]4 7e7 ,:5 ! block is cold1 break (f ! metal ,l's2 trim (f ! excess ( p>aff9 f ar\nd ! tissue ) a knife1 tak+ c>e to reta9 ! rectangul> %ape1 & /ore ! block 9 a pill-box4 ,:5 sev]al pieces ( tissue h to be imb$d$ at "o "t1 %apes ( /\t copp]1 #aj cm41 #e cm41 & #b4e cm4 squ>e respectively1 & #j4ge cm4 deep 7,fig4 #gd7 w be f\nd extremely useful4 ,^! plac$ ^u plates ( glass replace ! pair ( ,l's 9 ! above process4 ,:5 ! p>aff9 has set firmly ! screw a %\ld be loos5$ to all[ ! two halves ( ! flange b to sep>ate slightly--?is facilitates removal ( ! p>aff9 block4 ,illu/ration3 ,,fig4 #gd4--,p>aff9 m\ld4 #h4 ,cem5t ! block on ! c>ri] ( a p>aff9 microtome 7! ,m9ot1 ! ,jung1 or ! ,cambridge ,rock]7 ) a little melt$ p>aff94 ,great] security is obta9$ if ! p>aff9 ar\nd ! base ( ! block is melt$ by means ( a hot metal or glass rod4 #i4 ,cut sections--?91 & if possible 9 ribb&s4 ,m\nt+ ,p>aff9 ,sections4-- #a4 ,place a l>ge drop ( #cj p] c5t4 alcohol on ! c5tre ( a slide 7or cov]-slip7 & float ! section on to ! surface ( ! drop1 f a section lift]4 #b4 ,hold ! slide 9 ! f+]s ( "o h& & w>m cauti\sly ov] ! flame ( a ,buns5 burn]1 t\*+ ! "u surface ( ! glass f "t to "t on ! back ( ! o!r h&4 ,z soon z ! slide feels di/9ctly w>m to ! sk91 ! p>aff9 section w flatt5 out & all wr9kles disappe>4 7,! slide ) ! section float+ on x may be re/$ on ! top ( ! p>aff9 ba? = two or ?ree m9utes1 in/ead ( w>m+ ov] ! flame z "h describ$47 #c4 ,cauti\sly tilt up ! slide & blot (f ! excess ( _s ) blott+ pap]1 leav+ ! section atta*$ to ! c5tre ( ! slide4 #d4 ,place ! slide 9 a wire rack 7,fig4 #ge71 section d[nw>d1 9 ! hot incubator = twelve to tw5ty-f\r h\rs4 ,at ! end ( ?is "t ! section is firmly adh]5t to ! glass1 & is treat$ dur+ ! subsequ5t /eps z a fix$ cov]-glass film prep>ation4 ,,note4--,if l>ge1 ?ick sections h to be manipulat$1 or if "t is ( importance or acids are us$ dur+ ! /a9+ process1 x is (t5 advisable to add a trace ( ,may]'s album9 to ! alcohol be=e float+ out ! section4 ,if ?is sub/ance is employ$1 a soj\rn ( tw5ty m9utes to half an h\r 9 ! hot incubator w be f\nd ample to ensure firm adhesion ( ! section to ! slide4 ,! album9\s fluid is prep>$ z foll[s3 ,illu/ration3 ,,fig4 #ge4--,section rack4 ,may]'s ,album94-- ,weigh out ,salicylate ( soda #a gramme & dissolve 9 ,glyc]9e #ej c4c4 ,add ,:ite ( egg #ej c4c4 ,mix ?or\ghly by means ( an egg :isk4 ,filt] into a clean bottle4 ,z an alt]native me?od pa9t a ?9 lay] ( ,s*allibaum's solution on ! slide ) a camel's hair p5cil2 lay ! section c>efully on ?is film & heat g5tly to fix ! section4 ,s*allibaum's solution3 ,clove oil #cj c4c4 ,collodion #aj c4c4 ,keep 9 a d>k blue bottle 9 a cool place4 ,/a9+ ,p>aff9 ,sections4-- #a4 ,w>m p>aff9 section ov] ! ,buns5 flame to s(t5 7b n to melt7 ! p>aff91 !n dissolve out ! wax ) xylol p\r$ on f a drop bottle4 #b4 ,remove xylol by flu%+ ! section ) alcohol4 #c4 ,if ! tissue 0 orig9ally fix$ 9 a corrosive sublimate solution1 ! section mu/ n[ be treat$ ) ,lugol's iod9e solution = two m9utes & subsequ5tly imm]s$ 9 #ij p] c5t4 alcohol to remove all traces ( yell[ /a9+4 #d4 ,wa% 9 wat]4 #e4 ,/a9 deeply1 if us+ a s+le /a91 z ! subsequ5t processes decol\rise4 #f4 ,wa% 9 wat]1 decol\rise if necess>y4 #g4 ,flood ) sev]al *anges ( absolute alcohol to dehydrate ! section4 #h4 ,cle> 9 xylol4 7,oil ( cloves is n usually employ$1 z x decol\rises ! section47 #i4 ,m\nt 9 xylol balsam4 ,,special ,,/a9+ ,,me?ods ,,= ,,sections4 ,d\ble-/a9+ ,c>m9e & ,gram-,weig]t4-- #a4 ,prep>e ! section = /a9+ z above1 sections #a to #c4 #b4 ,/a9 9 li?ium c>m9e 7,or?'s7 or picroc>m9e = t5 to ?irty m9utes1 9 a porcela9 /a9+ pot 7,fig4 #gf74 #c4 ,wa% 9 picric acid solution until yell[4 ,at ?is /age cell nuclei are r$1 protoplasm is yell[1 & bact]ia are col\rless4 ,picric acid solution is prep>$ by mix+ ,picric acid1 saturat$ aque\s solution #dj c4c4 ,hydro*loric acid #a c4c4 ,alcohol 7#ij p] c5t47 #afj c4c4 #d4 ,wa% 9 wat]4 #e4 ,wa% 9 alcohol4 #f4 ,/a9 9 anil9e g5tian violet4 #g4 ,wa% 9 iod9e solution till d>k br[n or black4 #h4 ,wa% 9 wat]4 #i4 ,dip 9 absolute alcohol = a second4 #aj4 ,decol\rise ) anil9e oil till no m col\r is dis*>g$4 ,illu/ration3 ,,fig4 #gf4--,/a9+ pot4 #aa4 ,wa% ) anil9e oil1 #b p>ts1 xylol1 #a "p4 #ab4 ,cle> ) xylol4 #ac4 ,m\nt 9 xylol balsam4 ,alt]native ,gram-,weig]t ,me?od = ,sections4-- #a4 ,fix p>aff9 section on slide & prep>e = /a9+ 9 ! usual mann]4 #b4 ,/a9 9 alum c>m9e = ab\t fifte5 m9utes4 #c4 ,wa% ?or\ghly 9 wat]4 #d4 ,filt] anil9e g5tian violet solution on to ! section on ! slide & all[ to /a9 ab\t tw5ty-five m9utes4 #e4 ,wa% ?or\ghly 9 wat]4 #f4 ,treat ) ,lugol's iod9e until section ceases to become any black]4 #g4 ,wa% ?or\ghly 9 wat]4 #h4 ,treat ) a mixture ( equal p>ts ( anil9e oil & xylol until no m col\r comes away4 #i4 ,wa% ?or\ghly ) xylol4 #aj4 ,decol\rise & dehydrate rapidly ) absolute alcohol until !re rema9s only a v fa9t blui% t9t4 #aa4 ,cle> ) xylol4 #ab4 ,m\nt 9 xylol balsam4 7,!n fibr9 & hyal9e tissue are /a9$ deep blue1 :il/ bact]ia :i* /a9 ,gram appe> ( a deep blue-violet col\r47 ,unna-,papp5heim ,me?od4-- ,/a94-- ,weigh out & mix ,me?yl5e gre5 #j4ae gramme ,pyron9 #j4be gramme & dissolve 9 ,c>bolic acid #j4e p] c5t4 aque\s solution #gh c4c4 ,measure out ,alcohol #b4e c4c4 ,glyc]9e #bj4j c4c4 & add to ! /a94 ,me?od4-- #a4 ,place tissue 9 ! above /a9 = t5 m9utes4 #b4 ,diff]5tiate & dehydrate ) absolute alcohol4 #c4 ,cle> 9 xylol4 #d4 ,m\nt 9 xylol balsam4 ,to ,demon/rate ,capsules4-- #a4 ,mac,conkey's ,me?od4--,/a9 precisely z = cov]-slip films 7vide page #ajj74 #b4 ,fri$la5d]'s ,me?od4-- ,/a94-- ,g5tian violet1 saturat$ alcoholic solution #ej c4c4 ,acetic acid1 glacial #aj c4c4 ,di/ill$ wat] #ajj c4c4 ,,me?od4-- #a4 ,prep>e ! sections = /a9+1 secundum artem4 #b4 ,/a9 sections 9 ! w>m 7e4 g41 9 ! hot incubator7 = tw5ty-f\r h\rs4 #c4 ,wa% ) wat]4 #d4 ,decol\rise lightly ) acetic acid1 #a p] c5t4 #e4 ,dehydrate rapidly ) absolute alcohol4 #f4 ,cle> ) xylol4 #g4 ,m\nt 9 xylol balsam4 ,to ,demon/rate ,acid-fa/ ,bacilli4-- #a4 ,prep>e ! sections = /a9+ 9 ! usual way4 #b4 ,/a9 ) haemat9 solution t5 to tw5ty seconds1 to obta9 a pure nucle> /a92 !n wa% 9 wat]4 #c4 ,/a9 ) c>bolic fu*s9 tw5ty to ?irty m9utes at #dg deg4 ,c42 !n wa% 9 wat]4 #d4 ,treat ) anil9e hydro*lorate1 #b p] c5t4 aque\s solution1 = two to five seconds4 #e4 ,decol\rise 9 #ge p] c5t4 alcohol till section appe>s free f /a9--fifte5 to ?irty m9utes4 #f4 ,dehydrate ) absolute alcohol4 #g4 ,cle> v rapidly ) xylol4 #h4 ,m\nt 9 xylol balsam4 ,to ,demon/rate ,spiro*aetes 9 ,tissues4 ,pirid9 ,me?od 7,levaditi74-- #a4 ,cut slices ( tissue #a mm4 ?ick4 #b4 ,fix 9 #aj p] c5t4 =mal9 solution = tw5ty-f\r h\rs4 #c4 ,wa% 9 wat] = "o h\r4 #d4 ,place 9 #if p] c5t4 alcohol = tw5ty-f\r h\rs4 #e4 ,measure into a d>k gre5 or amb] bottle #ajj c4c4 silv] nitrate solution #a p] c5t41 & #aj grammes pyrid9 puriss4 ,transf] slices ( tissue to ?is4 ,/opp] & keep at room temp]ature ?ree h\rs1 !n 9 !rmo/at at #ej deg4 ,c4 = f\r to six h\rs4 #f4 ,wa% quickly 9 #aj p] c5t4 pyrid9 solution4 #g4 ,r$uce silv] by transf]r+ slices ( tissue to foll[+ solution = =ty-eight h\rs4 ,pyrogallic acid #d grammes ,acetone #aj c4c4 ,pyrid9 puriss #ae grammes ,di/ill$ wat] #ajj c4c4 #h4 ,wa% well 9 wat]4 ,take ?r\gh alcohols ( increas+ /r5g? up to absolute1 keep+ 9 ea* /r5g? = tw5ty-f\r h\rs4 #i4 ,cle>1 emb$1 cut v ?9 sections1 m\nt1 remove p>aff91 aga9 cle> & m\nt 9 xylol balsam4 ,! spiro*aetes if pres5t are black & %[ up aga9/ ! pale yell[ color ( ! backgr\nd4 ,weak c>bol fu*s91 neutral r$ or toluid9 blue c also be us$ to /a9 ! backgr\nd if desir$1 aft] ! removal ( ! p>aff9 9 /ep #i4 ,to ,demon/rate ,protozoa 9 ,sections 7,lei%man74-- ,reag5ts requir$3 ,lei%man's ,poly*rome /a94 ,acetic acid #a 9 #aejj aque\s solution4 ,cau/ic soda #a 9 #gjjj aque\s solution4 ,di/ill$ wat]4 #a4 ,m\nt section1 remove p>aff9 & take into di/ill$ wat] z usual 7vide page #aba74 #b4 ,dra9 (f ! excess ( wat]4 #c4 ,cov] ! section ) dilut$ ,lei%man 7#a "p /a91 #b p>ts di/ill$ wat]7 & all[ to act = five to t5 m9utes 7until tissue appe>s a deep blue74 #d4 ,decol\rise ) acetic acid solution until only ! nuclei appe> blue 7exam9e ! section wet1 ) l[ p[] objective74 #e4 ,if ! eos9 col\r is too well m>k$ treat ) ! cau/ic soda solution until ! desir$ t9t is obta9$ 7z se5 ) ! #a_/f-;in* objective74 #f4 ,wa% ) di/ill$ wat]4 #g4 ,rapidly dehydrate ) alcohol4 #h4 ,cle> ) xylol4 #i4 ,m\nt 9 xylol balsam4 ,,viii4 ,,classification ,,( ,,fungi4 ,= practical purposes ,,fungi may be divid$ into3 #a4 ,hym5omycetes 7includ+ ! mu%rooms1 etc474 #b4 ,hyphomycetes 7m\lds74 #c4 ,bla/omycetes 7yea/s & torulae74 #d4 ,s*izomycetes 7bact]ia74 ,,note4--,=m]ly myxomycetes 7 includ$ 9 ! fungi2 !y are n[ recogniz$ z belong+ to ! animal k+dom1 & are t]m$ mycetozoa4 ,,morphology ,,( ,,! ,,hyphomycetes4 ,at ! comm5cem5t ( 8 /udies1 ! att5tion ( ! /ud5t is direct$ to ! v>i\s non-pa?og5ic m\lds & yea/s1 n only t he may ga9 ! necess>y te*nique :il/ h&l+ cultivations ( h>mless organisms1 b also because ^! v species are among/ ! commone/ ( ^? t may accid5tally contam9ate 8 future prep>ations4 ,! hyphomycetes are compos$ ( a mycelium ( %ort jo9t$ rods or hyphae spr++ f an axis or g]m9al tube :i* develops f ! spore4 ,hyphae are-- 7a7 ,nutritive or subm]g$4 7b7 ,reproductive or a]ial4 ,! protoplasm ( ^! cells conta9s granules1 pigm5t1 oil globules1 & sometimes cry/als ( calcium oxalate4 ,reproduction4--,apical spore =mation--asexual2 zoospores--sexual4 ,mucor9ae4--,mucor 7,fig4 #gg74--,note ! bran*+ filam5ts--mycelium 7a71 hyphae 7b74 ,note ! asexual reproduction4 #a4 ,a filam5t gr[s upw>d4 ,at its apex a septum =ms1 !n a globul> swell+ appe>s--sporagium 7d74 ,?is possesses a def9ite membrane4 #b4 ,f ! septum gr[s a club-%ap$ mass ( protoplasm--columella 7c74 ,illu/ration3 ,,fig4 #gg4--,mucor muc$o4 ,illu/ration3 ,,fig4 #gh4--;,asp]gillus #c4 ,! re/ ( ! conta9$ protoplasm breaks up into sw>m spores 7e74 ,f9ally ! membrane ruptures & spores escape4 ,p]isporaceae4--,asp]gillus 7,fig4 #gh74--,note ! bran*+ filam5ts--mycelium 7a74 ,illu/ration3 ,,fig4 #gi4--,p5icillium4 ,note ! asexual reproduction4 #a4 ,a filam5t 7b7 gr[s upw>d1 its t]m9ation becomes clubb$2 on ! clubb$ extremity flask-%ap$ cells appe>--/]igmata 7c74 #b4 ,at free end ( ea* /]igma is =m$ an oval body--a spore or gonidium 7d71 :i*1 :5 ripe1 is ?r[n (f f ! /]igma4 ,two or m gonidia may be support$ ^u ea* /]igma4 ,p5icillium 7,fig4 #gi74--,note ! bran*+ filam5ts--mycelium 7a7 7frequ5tly conta9+ globules74 ,note ! asexual reproduction4 #a4 ,a filam5t gr[s upw>d--goniodophore 7b7--& its apex divides up into sev]al bran*es--basidia 7c74 #b4 ,at ! apex ( ea* basidium a flask-%ap$ cell1 /]igma 7d71 appe>s4 #c4 ,at ! apex ( ea* /]igma appe>s a r[ ( oval cells--spores or conidia 7e74 ,^!1 :5 ripe1 are ca/ (f f ! /]igmata4 ,illu/ration3 ,,fig4 #hj4--,oidium4 ,ascomycetae4--,oidium 7,fig4 #hj74--7,?is family is p]haps z ne>ly relat$ to ! bla/omycetes z x is to ! hyphomycetes47 ,note ! bran*+ filam5ts--pseudomycelium 7a74 ,"h & !re filam5ts are brok5 up at _! ends into oval or rod-%ap$ segm5ts1 oidia1 & behave z spores4 ,note ! asexual reproduction4 ,f ! pseudomycelium arise true hyphae 7b71 ea* ( :i* 9 turn ends 9 a *a9 ( spores 7c74 ,,morphology ,,( ,,! ,,bla/omycetes4 ,! bla/omycetes are compos$ ( sph]ical or oval cells 7#h to #i4e mu 9 diamet]71 :i*1 :5 rapidly multiply+ by budd+1 may =m a spuri\s mycelium4 ,a ?9 cell-wall encloses ! granul> protoplasm1 9 :i* vacuoles & sometimes a nucleus may be not$4 ,?is latt] is be/ se5 :5 /a9$ ) haematoxyl9 7see page #aje74 ,dur+ _! gr[? & multiplication ! bla/omycetes split up solutions conta9+ sug> into alcohol & ,,co#b4 ,sac*>omyces 7,fig4 #ha74--,note ! r\nd or oval cells ( granul> protoplasm 7a7 conta9+ solid p>ticles & vacuoles 7c71 & surr\nd$ by a def9ite envelope4 ,reproduction4--,budd+2 ascospores--asexual4 ,note ! asexual reproduction4 #a4 ,gemmation--t is1 ! budd+ out ( daught] cells 7b7 f v>i\s p>ts ( ! gradually enl>g+ "m cell4 ,^! are ev5tually ca/ (f & 9 turn become "m cells & =m fre% gr\ps ( buds4 ,illu/ration3 ,,fig4 #ha4--,sac*>omyces ) ascospores4 ,illu/ration3 ,,fig4 #hb4--,torula4 #b4 ,spore =mation--ascospores 7e74 ,^! are =m$ at def9ite temp]atures & )9 well-def9$ p]iods2 e4 g41 ,sac*>omyces c]evisiae1 ?irty h\rs at #be deg4 to #cg deg4 ,c41 or t5 days at #ab deg4 ,c4 ,torulae 7,fig4 #hb74--,torulae1 :il/ resembl+ yea/s 9 almo/ e o!r respect1 nev] =m endo-spores4 ,note ! elongat$1 sausage-%ap$ cells 7a7 ! l>g] oval cells 7b7 & ! globul> cells 7c7 ! =m] two (t5 int]lac+ & gr[+ z a film4 ,note ! abs5ce ( ascospore =mation4 ,,ix4 ,,s*izomycetes4 ,classification & ,morphology4--,bact]ia are (t5 classifi$1 9 g5]al t]ms1 accord+ to _! life functions1 into-- ,saprog5ic1 or putrefactive bact]ia2 ,zymog5ic1 or f]m5tative bact]ia2 ,pa?og5ic1 or disease-produc+ bact]ia2 or accord+ to _! food requirem5ts into-- ,prototrophic1 requir+ no organic food 7e4 g41 nitrify+ bact]ia72 ,metatrophic1 requir+ organic food 7e4 g41 saprophytes & facultative p>asites72 ,p>atrophic1 requir+ liv+ food 7obligate p>asites72 or accord+ to _! metabolic products into-- ,*romog5ic1 or pigm5t-produc+ bact]ia2 ,photog5ic1 or light-produc+ bact]ia2 ,a]og5ic1 or gas-produc+ bact]ia2 & s on4 ,su* broad gr\p+s z ^! h1 h[ev]1 b little practical value :5 appli$ to ! sy/ematic /udy ( ! fission fungi4 ,on ! o!r h&1 no really sci5tific classification ( ! s*izomycetes has yet be5 drawn up1 & ! v>y+ morphological appe>ances ( ! memb]s ( ! family are /ill utilis$ z a basis = classification1 z "u-- #a4 ,cocci4 7,fig4 #hc74--,r\nd$ or oval cells1 subdivid$ accord+ to ! arrangem5t ( ! individuals aft] fission1 into-- ,diplococci & ,/reptococci1 :]e division takes place 9 "o plane only1 & ! individuals rema9 atta*$ 7a7 9 pairs or 7b7 9 *a9s4 ,tetrads1 ,m]ismop$ia1 or ,p$iococci1 :]e division takes place alt]nately 9 two planes at "r angles to ea* o!r1 & ! individuals rema9 atta*$ 9 flat tablets ( f\r1 or its multiples4 ,illu/ration3 ,,fig4 #hc4--,types ( bact]ia--cocci3 #a1 ,diagram ( sph]e indicat+ planes ( fission2 #b1 diplococci2 #c1 /reptococci2 #d1 tetrads2 #e1 s>c9ae2 #f1 /aphylococci4 ,s>c9ae1 :]e division takes place 9 ?ree planes successively1 & ! individuals rema9 atta*$ 9 cubical packets ( eight & its multiples4 ,illu/ration3 ,,fig4 #hd4--,types ( bact]ia--bacilli1 etc43 #a1 ,bacilli2 #b1 diplobacilli2 #c /reptobacilli2 #d1 spirilla2 #e1 vibrios2 #f1 spiro*aetae4 ,micrococci or ,/aphylococci1 :]e division takes place 9 ?ree planes1 b ) no def9ite sequ5ce2 consequ5tly ! individuals rema9 atta*$ 9 pairs1 %ort *a9s1 plates ( f\r1 cubical packets ( eight1 & irregul> masses conta9+ num]\s cocci4 #b4 ,bacilli 7,fig4 #hd1 #a to #c74--,rod-%ap$ cells4 ,a bacillus1 h[ev] %ort1 c usually be di/+ui%$ f a coccus 9 t two sides are p>allel4 ,"s bacilli aft] fission reta9 a *>act]i/ic arrangem5t & may be spok5 ( z ,diplobacilli or ,/reptobacilli4 ,lepto?rix is a t]m t 9 ! pa/ has be5 loosely us$ to signify a long ?read1 b is n[ re/rict$ to su* =ms z belong to ! lepto?riciae 7vide infra74 #c4 ,spirilla 7,fig4 #hd1 #d to #f74--;,curv$ & twi/$ filam5ts4 ,classifi$1 accord+ to %ape1 into-- ,spirillum4 ,vibrio 7comma74 ,spiro*aeta4 ,_m ,spiro*aetes appe> to belong to ! animal k+dom & are gr\p$ "u protozoa2 o!r organisms to :i* ?is "n has be5 giv5 are und\bt$ly bact]ia4 ,high] =ms ( bact]ia are also met )1 :i* possess ! foll[+ *>act]i/ics3 ,!y are atta*$1 unbran*$1 filam5t\s =ms1 %[+-- 7a7 ,diff]5tiation betwe5 base & apex2 7b7 ,gr[? app>5tly apical2 7c7 ,exagg]at$ pleomorphism2 7d7 ,pseudo-bran*+ f apposition ( cells2 & are classifi$ into-- #a4 ,beggiotoa4 ,free swimm+ =ms1 :i* #b4 ,?io?rix4 conta9 sulphur granules4 #c4 ,cr5o?rix4 #d4 ,clado?rix4 ,^! =ms d n conta9 #e4 ,lepto?rix4 sulphur granules4 #f4 ,/repto?rix4 ,a gr\p :i* exhibits true b n di*otom\s bran*+1 & conta9s "s pa?og5ic species4 ,! morphology ( ! same bact]ium may v>y greatly "u diff]5t conditions4 ,= example1 "u "o set ( conditions ! exam9ation ( a pure cultivation ( a bacillus may %[ a %ort oval rod z ! pr$om9ant =m1 :il/ ano!r culture ( ! same bacillus1 b gr[n "u diff]5t conditions1 may consi/ almo/ entirely ( long filam5ts or ?reads4 ,?is v>iation 9 morphology is kn[n z pleomorphism4 ,"s ( ! factors influ5c+ pleomorphism are3 #a4 ,! composition1 reaction1 etc41 ( ! nutri5t m$ium 9 :i* ! organism is gr[+4 #b4 ,! atmosph]e 9 :i* x is cultivat$4 #c4 ,! temp]ature at :i* x is incubat$4 #d4 ,exposure to or protection f light4 ,! v>i\s po9ts 9 ! anatomy morphology & physiology ( bact]ia ^u :i* /ress is laid 9 ! foll[+ pages %\ld be /udi$ z closely z is possible 9 prep>ations ( ! micro-organisms nam$ 9 connection ) ea*4 ,,anatomy4 #a4 ,capsule 7,fig4 #he1 b74--,a gelat9\s envelope 7probably ak9 to muc9 9 composition7 surr\nd+ ea* individual organism1 & prev5t+ absolute contact betwe5 any two4 ,9 "s species ! capsule 7e4 g41 ,b4 pneumoniae7 is well m>k$1 b x _c be demon/rat$ 9 all4 ,9 v well m>k$ cases ( gelat9isation ( ! cell wall1 ! individual cells are cem5t$ toge!r 9 a coh]5t mass1 to :i* ! t]m zoogloea is appli$ 7e4 g41 ,/reptococcus mes5t]oides74 ,9 "s species col\r+ matt] or f]ric oxide is /or$ 9 ! capsule4 #b4 ,cell ,wall 7,fig4 #he1 c74--,a protective diff]5tiation ( ! out] lay] ( ! cell protoplasm2 difficult to demon/rate1 b treatm5t ) iod9e or salt solution sometimes causes %r9kage ( ! cell cont5ts--plasmolysis--& s r5d]s ! cell wall app>5t 7e4 g41 ,b4 mega!rium7 9 ! mann] %[n 9 figure #he4 ,/a9$ bacilli1 :5 exam9$ ) ! pol>is+ microscope1 (t5 %[ a d\bly refractile cell wall 7e4 g41 ,b4 tub]culosis & ,b4 an?racis74 ,9 "s ( ! high] bact]ia ! cell wall exhibits ?is diff]5tiation to a m>k$ degree & =ms a h>d %ea? )9 :i* ! cell protoplasm is freely movable2 & dur+ ! process ( reproduction ! cell protoplasm may be extrud$1 leav+ ! empty tube unalt]$ 9 %ape4 ,illu/ration3 ,,fig4 #he4--;,dragrammatic sket* ( composite bact]ium to illu/rate details ( anatomical /ructure4 ,illu/ration3 ,,fig4 #hf4--,plasmolysis4 #c4 ,cell ,cont5ts4--,protoplasm 7mycoprote97 conta9s a high p]c5tage ( nitrog51 b is said to diff] f proteid 9 t x is n precipitat$ by ,c#b;,h#f,o4 ,x is usually homog5e\s 9 appe>ance--sometimes granul>--& may conta9 oil globules or sap vacuoles 7,fig4 #he1 d71 *romat9 granules1 & ev5 sulphur granules4 ,sap vacuoles mu/ be di/+ui%$ f spores1 on ! "o h&1 & ! vacuolat$ appe>ance due to plasmolysis1 on ! o!r4 ,! cell cont5ts may sometimes be diff]5tiat$ into a p>ietal lay]1 & a c5tral body 7e4 g41 beggiotoa7 :5 /a9$ by haematoxyl94 #d4 ,nucleus4--,?is /ructure has n be5 conclusively prov$ to exi/1 b 9 "s bact]ia *romat9 p>ticles h be5 obs]v$ ne> ! c5tre ( ! bact]ial cell & d5s] masses ( protoplasm situat$ at ! poles :i* exhibit a m m>k$ aff9ity ?an ! re/ ( ! cell protoplasm = anil9e dyes4 ,^! latt] are t]m$ pol> granules or ,polko]n] 7,fig4 #he1 e74 ,occasionally ^! aggregations ( protoplasm alt] ! col\r ( ! dye !y take up4 ,!y are !n kn[n z meta*romatic bodies or ,ern/s*5 ,ko]n] 7e4 g41 ,b4 diph!riae74 #e4 ,flagella 7,organs ( ,locomotion1 ,fig4 #he1 a74--,^! are gelat9\s elongations ( ! cell protoplasm 7or m probably ( ! capsule71 occurr+ ei!r at "o pole1 at bo? poles1 or scatt]$ ar\nd ! entire p]iph]y4 ,flagella are n pseudopodia4 ,! possession ( flagella 0 at "o "t sugge/$ z a basis = a sy/em ( classification1 :5 ! foll[+ types ( ciliation 7 diff]5tiat$ 7,fig4 #hg73 ,illu/ration3 ,,fig4 #hg4--,types ( ciliation4 #a4 ,pol>3 7a7 ,monotri*\s 7a s+le flagellum situat$ at "o pole2 e4 g41 ,b4 pyocyaneus74 7b7 ,amphitri*\s 7a s+le flagellum at ea* pole2 e4 g41 ,spirillum volutans74 7c7 ,lophotri*\s 7a tuft or bun* ( flagella situat$ at ea* pole2 e4 g41 ,b4 cyanog5us74 #b4 ,diffuse3 ,p]itri*\s 7flagella scatt]$ ar\nd ! entire p]iph]y e4 g41 ,b4 typhosus74 ,,physiology4 ,reproduction4--,active ,/age4--,vegetative1 i4 e41 by ! division ( cells1 or fission4 #a4 ,! cell becomes elongat$ & ! protoplasm aggregat$ at opposite poles4 #b4 ,a circul> con/riction ( ! organism takes place midway betwe5 ^! aggregations1 & a septum is =m$ 9 ! int]ior ( ! cell at "r angles to its l5g?4 #c4 ,! division deep5s1 ! septum divides into two lamellae1 & f9ally two cells are =m$4 ,illu/ration3 ,,fig4 #hh4--;,fission ( cocci4 ,illu/ration3 ,,fig4 #hi4--;,fission ( bact]ia4 #d4 ,! daught] cells may rema9 unit$ by ! gelat9\s envelope = a v>iable "t4 ,ev5tually !y sep>ate & !mselves subdivide4 ,cultures on artificial m$ia1 aft] gr[+ 9 ! same m$ium = "s "t--i4 e41 :5 ! pabulum is exhau/$--%[ involution =ms 7,fig4 #ij71 well exemplifi$ 9 cultures ( ,b4 pe/is on ag> two days old1 ,b4 diph!riae on potato f\r to six days old4 ,illu/ration3 ,,fig4 #ij4--;,involution =ms4 ,!y are ( two classes1 viz43 7a7 ,involution =ms *>act]is$ by alt]ations ( %ape 7,fig4 #ij74 7,n necess>ily dead47 7b7 ,involution =ms *>act]is$ by loss ( /a9+ p[]4 7,always dead47 ,re/+ ,/age4--,spore ,=mation4--,conditions influ5c+ spore =mation3 ,9 an old culture no?+ may be left b spores4 ,x us$ to be suppos$ t spores 7 always =m$1 s t ! species might n become ext9ct1 :5 7a7 ,! supply ( nutri5t 0 exhau/$4 7b7 ,! m$ium became toxic f ! accumulation ( metabolic products4 7c7 ,! environm5t became unfav\rable2 e4 g41 *ange ( temp]ature4 ,?is is n altoge!r correct2 e4 g41 ! temp]ature at :i* spores are be/ =m$ is con/ant = ea* bact]ium1 b v>ies ) diff]5t species2 aga91 a]obes require oxyg5 = sporulation1 b ana]obes w n spore 9 its pres5ce4 7,a7 ,ar?rog5\s3 ,not$ only 9 ! micrococci4 ,"o complete elem5t result+ f ord9>y fission becomes diff]5tiat$ = ! purpose1 enl>ges1 & develops a d5se cell wall4 ,"o or m ( ! cells 9 a s]ies may und]go ?is alt]ation4 ,?is process is probably n real spore =mation1 b m]ely relative increase ( resi/ance4 ,^! s-call$ ar?rospores h nev] be5 obs]v$ to g]m9ate1 nor is _! resi/ance v m>k$1 z !y fail to initiate new cultures1 aft] hav+ be5 expos$ to a temp]ature ( #hj deg4 ,c4 = t5 m9utes4 7,b7 ,endog5\s3 ,! cell protoplasm becomes diff]5tiat$ & cond5s$ into a sph]ical or oval mass 7v r>ely cyl9drical74 ,aft] fur!r contraction ! out] lay]s ( ! mass become /ill m highly diff]5tiat$ & =m a di/9ct spore membrane1 & ! spore itself is n[ highly refractile4 ,x has be5 sugge/$1 & app>5tly on good gr\nds1 t ! spore membrane consi/s ( two lay]s1 ! exosporium & ! endosporium4 ,ea* cell =ms "o spore only1 usually 9 ! middle1 occasionally at "o end 7"s exceptions1 h[ev]1 are record$2 e4 g41 ,b4 inflatus74 ,! %ape ( ! p>5t cell may be unalt]$1 z 9 ! an?rax bacillus1 or alt]$1 z 9 ! tetanus bacillus1 & ^! po9ts s]ve z ! basis = a classification ( spore-be>+ bacilli1 z foll[s3 7,a7 ,cell body ( ! p>5t bacillus unalt]$ 9 %ape 7,fig4 #ia1 a74 7,b7 ,cell ( ! p>5t bacillus alt]$ 9 %ape4 #a4 ,clo/ridium 7,fig4 #ia1 b73 ,rod swoll5 at ! c5tre & att5uat$ at ! poles2 sp9dle %ape2 e4 g41 ,b4 butyricus4 #b4 ,cuneate 7,fig4 #ia1 c73 ,rods swoll5 slightly at "o pole & m or less po9t$ at ! o!r2 w$ge-%ap$4 ,illu/ration3 ,,fig4 #ia--,types ( spore-be>+ bacilli4 #c4 ,clavate 7,fig4 #ia1 d73 ,rods swoll5 at "o pole & cyl9drical 7unalt]$7 at ! o!r2 keyhole-%ap$2 e4 g41 ,b4 *auvei4 #d4 ,capitate 7,fig4 #ia1 e73 ,rods ) a sph]ical enl>gem5t at "o pole2 drum/ick-%ap$2 e4 g41 ,b4 tetani4 ,! endo-spores rema9 )9 ! p>5t cell = a v>iable "t 79 "o case x is /at$ t g]m9ation ( ! spore occurs )9 ! int]ior ( ! p>5t cell--endo-g]m9ation71 b are ev5tually set free1 z a result ( ! swell+ up & solution ( ! cell membrane ( ! p>5t bacillus 9 ! surr\nd+ liquid1 or ( ! rupture ( t membrane4 ,!y !n pres5t ! foll[+ *>act]i/ics3 #a4 ,well-=m$1 d5se cell membranes1 :i* r5d]s !m extremely difficult to /a91 b :5 once /a9$ equally difficult to decol\rise4 #b4 ,high refractility1 :i* di/+ui%$ !m f vacuoles4 #c4 ,high] resi/ance ?an ! p>5t organism to su* le?al ag5ts z heat1 desiccation1 />vation1 "t1 etc41 ?is resi/ance be+ due to 7a7 ,l[ wat] cont5ts ( plasma ( ! spore4 7b7 ,l[ heat-conduct+ p[] ( ! spore 7c7 ,l[ p]meability membrane4 ,?is resi/ance v>ies some:at ) ! p>ticul> species--e4 g41 "s spores may resi/ boil+ = a few m9utes--b practically all are kill$ if ! boil+ is cont9u$ = t5 m9utes4 ,g]m9ation4--,:5 transplant$ to suitable m$ia & plac$ "u fav\rable conditions1 ! spores g]m9ate1 usually )9 tw5ty-f\r to ?irty-six h\rs1 & successively und]go ! foll[+ *anges :i* may be foll[$ 9 hang+-drop cultures on a w>m /age3 #a4 ,swell up sl[ly & enl>ge1 ?r\gh ! absorption ( wat]4 #b4 ,lose _! refrangibility4 #c4 ,at ?is /age "o ( ?ree processes 7b ! p>ticul> process is always con/ant = ! same species7 may be obs]v$3 7a7 ,! spore gr[s out into ! new bacillus )\t disc>d+ ! spore membrane 7:i* 9 ?is case n[ becomes ! cell membrane72 e4 g41 ,b4 leptosporus4 7b7 ,x loses its spore membrane by solution2 e4 g41 ,b4 an?racis4 7c7 ,x loses its spore membrane by rupture4 ,9 ?is process ! rupture may be ei!r pol> 7at "o pole only e4 g41 ,b4 butyricus71 or bipol> 7e4 g41 ,b4 sessile71 or equatorial2 7e4 g41 ,b4 subtilis74 ,9 ^? cases :]e ! spore membrane is disc>d$ ! cell membrane ( ! new bacillus may ei!r be =m$ f-- 7a7 ,! inn] lay] ( ! spore membrane1 :i* has und]gone a prelim9>y splitt+ into p>ietal & visc]al lay]s2 e4 g41 ,b4 butyricus4 7b7 ,! out] lay]s ( ! cell protoplasm1 :i* become diff]5tiat$ = t purpose2 e4 g41 ,b4 mega!rium4 ,! new bacillus n[ increases 9 size1 elongates1 & takes on a vegetative gr[?--i4 e41 und]goes fission--! bacilli result+ f :i* may 9 _! turn give rise to spores4 ,illu/ration3 ,,fig4 #ib4 ,simple4 ,illu/ration3 ,,fig4 #ic4 ,solution4 ,illu/ration3 ,,fig4 #id4 ,pol>4 ,illu/ration3 ,,fig4 #ie4 ,bipol>4 ,illu/ration3 ,,fig4 #if4 ,equatorial4 ,food ,/uffs4--#a4 ,organic ,foods4-- 7a7 ,! pure p>asites 7e4 g41 ,b4 leprae7 w n live outside ! liv+ body4 7b7 ,bo? saprophytic & facultative p>asitic bact]ia agree 9 requir+ non-conc5trat$ food4 7c7 ,! facultative p>asites ne$ highly organis$ foods2 e4 g41 proteids or o!r s\rces ( nitrog5 & c>bon1 & salts4 7d7 ,! saprophytic bact]ia are m easily cultivat$2 e4 g41 #a4 ,"s bact]ia w gr[ 9 almo/ pure di/ill$ wat]4 #b4 ,"s bact]ia w gr[ 9 pure solutions ( ! c>bohydrates4 #c4 ,wat] is absolutely ess5tial to ! gr[? ( bact]ia4 ,food ( a def9ite reaction is ne$$ = ! gr[? ( bact]ia4 ,z a g5]al rule gr[? is mo/ active 9 m$ia :i* react slightly acid to ph5olph?ale9--t is1 neutral or fa9tly alkal9e to litmus4 ,m\ld gr[?1 on ! o!r h&1 is mo/ vig\r\s 9 m$ia t are /rongly acid to ph5olph?ale94 ,environm5t4--,! influ5ce ( physical ag5ts ^u bact]ial life & gr[? is /rongly m>k$4 #a4 ,atmosph]e4--,! pres5ce ( oxyg5 is necess>y = ! gr[? ( "s bact]ia1 & dea? foll[s :5 ! supply is cut (f4 ,su* organisms are t]m$ obligate a]obes4 ,"s bact]ia appe> to ?rive equally well :e!r suppli$ ) or depriv$ ( oxyg54 ,^! are t]m$ facultative ana]obes4 ,a ?ird class w only live & multiply :5 ! access ( free oxyg5 is completely exclud$4 ,^! are t]m$ obligate ana]obes4 #b4 ,temp]ature4--,practically no bact]ial gr[? occurs bel[ #e deg4 ,c1 & v little above #dj deg4 ,c4 #cj deg4 ,c4 to #cg deg4 ,c is ! mo/ favorable = ! l>ge majority ( micro-organisms4 ,! maximum & m9imum temp]atures at :i* gr[? takes place1 z well z ! optimum1 are fairly con/ant = ea* bact]ium4 ,bact]ia h be5 classifi$1 accord+ to _! optimum temp]ature1 into-- ,,m94 ,,opt4 ,,max4 #a4 ,psy*rophilic bact]ia 7*iefly wat] organisms7 #j deg4 ,c4 #ae deg4 ,c4 #cj deg4 ,c4 #b4 ,mesophilic bact]ia 7includes pa?og5ic bact]ia7 #ae deg4 ,c4 #cg deg4 ,c4 #de deg4 ,c4 #c4 ,!rmophilic bact]ia #de deg4 ,c4 #ee deg4 ,c4 #gj deg4 ,c4 ,! !rmal dea?-po9t ( an organism is ano!r biological con/ant2 & is t temp]ature :i* causes ! dea? ( ! vegetative =ms :5 ! exposure is cont9u$ = a p]iod ( t5 m9utes 7see pages #bih-cja74 #c4 ,light4--,_m organisms are indiff]5t to ! pres5ce ( light4 ,on ! o!r h&1 light frequ5tly imp$es gr[?1 & alt]s to a great] or less] ext5t ! bio*emical *>act]s ( ! organisms--e4 g41 *romog5icity or p[] ( liquefaction4 ,pa?og5ic bact]ia und]go a progressive loss ( virul5ce :5 cultivat$ 9 ! pres5ce ( light4 #d4 ,movem5ts4--,movem5ts1 if slight & simply ( a fl[+ "*1 d n appe> to injuri\sly affect ! gr[? ( bact]ia2 b viol5t agitation1 su* z %ak+1 absolutely kills !m4 ,a condition ( p]fect re/ w\ld seem to be t mo/ conducive to bact]ial gr[?4 ,! ,metabolic ,products ( ,bact]ia4--,pigm5t ,production4--,_m micro-organisms produce "o or m vivid pigm5ts--yell[1 orange1 r$1 violet1 fluoresc5t1 etc4--dur+ ! c\rse ( _! life & gr[?4 ,! col\r+ matt] usually exi/s z an int]cellul> excrem5titi\s sub/ance4 ,occasionally1 h[ev]1 x appe>s to be /or$ actually )9 ! bodies ( ! bact]ia4 ,! *romog5ic bact]ia are !re=e classifi$1 9 accordance ) ! f9al de/9ation ( ! col\r+ matt] !y elaborate1 into-- ,*romop>\s ,bact]ia3 9 :i* ! pigm5t is diffus$ out ^u & into ! surr\nd+ m$ium4 ,*romophor\s ,bact]ia3 9 :i* ! pigm5t is /or$ 9 ! cell protoplasm ( ! organism4 ,p>a*romophor\s ,bact]ia3 9 :i* ! pigm5t is /or$ 9 ! cell wall ( ! organism4 ,diff]5t species ( *romog5ic bact]ia diff] 9 _! requirem5ts z to environm5t1 = ! production ( _! *>act]i/ic pigm5ts2 e4 g41 "s ne$ oxyg51 light1 or high temp]ature2 o!rs aga9 favor ! conv]se ( ^! conditions4 ,light ,production4--,"s bact]ia1 & usually ^? orig9ally d]iv$ f wat]1 :e!r fre% or salt1 exhibit m>k$ phosphoresc5ce :5 cultivat$ "u suitable conditions4 ,^! are class$ z photog5ic4 ,enzyme ,production4--,_m bact]ia produce soluble f]m5ts or enzymes dur+ ! c\rse ( _! gr[?1 z evid5c$ by ! liquefaction ( gelat9e1 ! clott+ ( milk1 etc4 ,^! f]m5ts may belong to ei!r ( ! foll[+ well-recognis$ classes3 proteolytic1 dia/atic1 inv]t91 r5net4 ,tox9 ,production4--,a l>ge numb]1 especially ( ! pa?og5ic bact]ia1 elaborate or secrete poison\s sub/ances conc]n+ :i* b little exact k is available1 al?\gh _m w\ld appe> to be enzymic 9 _! action4 ,^! tox9s are usually diff]5tiat$ into-- ,extracellul> 7or ,soluble7 ,tox9s3 ^? :i* are diffus$ into1 & held 9 solution by1 ! surr\nd+ m$ium4 ,intracellul> 7or ,insep>ate7 ,tox9s3 ^? :i* are s closely b\nd up ) ! cell protoplasm ( ! bact]ia elaborat+ !m t up to ! pres5t "t no means has be5 devis$ = _! sep>ation or extraction4 ,end-products ( ,metabolism4--,"u ?is head+ are includ$-- ,organic ,acids 7e4 g41 lactic1 butyric1 etc474 ,alkalies 7e4 g41 ammonia74 ,aromatic ,comp\nds 7e4 g41 indol1 ph5ol74 ,r$uc+ ,sub/ances 7e4 g41 ^? r$uc+ nitrates to nitrites74 ,gases 7e4 g41 sulphurett$ hydrog51 c>bon dioxide1 etc474 ,& :ile ! discussion ( _! =mation1 etc41 is beyond ! scope ( a laboratory h&book1 ! me?ods 9 use = _! detection & sep>ation come into ! ord9>y r\t9e "w & w !re=e be describ$ 7vide page #bgf et seq474 ,x4 ,,nutri5t ,,m$ia4 ,9 ord] t ! life & gr[? ( bact]ia may be accurately obs]v$ 9 ! laboratory1 x is necess>y-- #a4 ,to isolate individual memb]s ( ! diff]5t v>ieties ( micro-organisms4 #b4 ,to cultivate organisms1 ?us isolat$1 ap>t f o!r associat$ or contam9at+ bact]ia--i4 e41 9 pure culture4 ,= ! successful a*ievem5t ( ^! objects x is necess>y to provide nutrim5t 9 a =m suit$ to ! ne$s ( ! p>ticul> bact]ium or bact]ia "u obs]vation1 & 9 a g5]al way x may be said t ! nutri5t mat]ials %\ld approximate z closely z possible1 9 composition & "*1 to ! natural pabulum ( ! organism4 ,! g5]al requirem5ts ( bact]ia z to _! food-supply h already be5 indicat$ 7page #adb7 & _m comb9ations ( proteid & ( c>bohydrate h be5 devis$1 f "t to "t1 on ^? l9es4 ,^!1 toge!r ) v>i\s vegetable tissues1 physiological or pa?ological fluid secretions1 etc41 are collectively spok5 ( z nutri5t m$ia or culture m$ia4 ,! great] numb] ( ^! m$ia are prim>ily fluid1 b1 on acc\nt ( ! rapidity ) :i* bact]ial gr[? diffuses itself ?r\gh a liquid1 x is impossible to /udy !re9 ! *>act]i/ics ( individual organisms4 ,_m su* m$ia are1 !re=e1 subsequ5tly r5d]$ solid by ! addition ( sub/ances l gelat9e or ag>1 9 v>y+ proportions1 ! proportions ( su* add$ mat]ial be+ g5]ally m5tion$ :5 ref]r+ to ! m$ia2 e4 g41 #aj p] c5t4 gelat9e1 #b p] c5t4 ag>4 ,gelat9e is employ$ = ! solidification ( ^? m$ia x is int5d$ to use 9 ! cultivation ( bact]ia at ! room temp]ature or 9 ! cold incubator4 ,9 ! p]c5tages usually employ$1 gelat9e m$ia become fluid at #be deg4 ,c42 high] p]c5tages rema9 solid at some:at high] temp]atures1 b ! difficulty ( filt]+ /rong solutions ( gelat9e militates aga9/ _! g5]al use4 ,m$ia1 on ! o!r h& :i* h be5 solidifi$ by ! addition ( ag>1 only become liquid :5 expos$ to #ij deg4 ,c4 = ab\t t5 m9utes1 & aga9 solidify :5 ! temp]ature falls to #dj deg4 ,c4 ,:5 x becomes necess>y to r5d] ^! m$ia fluid1 heat is appli$1 ^u ! )drawal ( :i* !y aga9 assume _! solid condition4 ,su* m$ia %\ld be ref]r$ to z liquefiable m$ia2 9 po9t ( fact1 h[ev]1 !y are usually gr\p$ toge!r ) ! solid m$ia4 ,,note4--,x mu/ "h be /at$ t ! designation #aj p] c5t4 gelat9e or #b p] c5t4 ag> ref]s only to ! quantity ( ^? sub/ances actually add$ 9 ! process ( manufacture1 & n to ! p]c5tage ( gelat9e or ag>1 z ! case may be1 pres5t 9 ! f9i%$ m$ium2 ! explanation be+ t ! comm]cial products employ$ conta9 a l>ge proportion ( insoluble mat]ial :i* is sep>at$ (f by filtration dur+ ! prep>ation ( ! liquefiable m$ia4 ,o!r m$ia1 aga9--e4 g41 potato1 coagulat$ blood-s]um1 etc4--_c be aga9 liquefi$ by physical means1 & ^! are spok5 ( z solid m$ia4 ,! foll[+ pages detail ! me?od ( prep>+ ! v>i\s nutri5t m$ia1 9 ord9>y use 7see also ,*apt] ,,xi71 ^? :i* are only occasionally requir$ = m highly specialis$ "w are gr\p$ toge!r 9 ,*apt] ,,xii4 ,x mu/ be premis$ t scrupul\s cleanl9ess is to be obs]v$ ) reg>d to all app>atus1 vessels1 funnels1 etc41 employ$ 9 ! prep>ation ( m$ia2 al?\gh 9 ! prelim9>y /ages ( ! prep>ation ( mo/ m$ia absolute /]ility ( ! app>atus us$ is n ess5tial4 ,,meat ,,extract4 ,a wat]y solution ( ! extractives1 etc41 ( lean meat 7usually beef7 =ms ! basis ( sev]al nutri5t m$ia4 ,?is solution is t]m$ meat extract & x has be5 det]m9$ empirically t its prep>ation %all be c>ri$ out by extract+ half a kilo ( moi/ meat ) "o litre ( wat]4 ,= _m purposes1 h[ev]1 x is m conv5i5t to h a m conc5trat$ extract2 "o kilo ( meat %\ld !re=e be extract$ ) "o litre ( wat]1 to =m ,d\ble ,/r5g? meat extract4 ,x 0 cu/om>y at "o "t1 & is ev5 n[ 9 "s laboratories to use ei!r %9 ( beef or beef-/eak--bo? conta9 muscle sug> :i* (t5 ne$s to be remov$ be=e ! nutri5t m$ium c be complet$4 ,he>t muscle 7bullock's he>t or %eep's he>t7 is mu* to be pref]r$ & f ! po9t ( economy1 ease & cleanl9ess ( manipulation1 & extractive value1 ! import$ froz5 bullock's he>ts provide ! be/ extract4 ,meat extract 7,fleis*wass]7 is prep>$ z foll[s3 #a4 ,measure #ajjj c4c4 ( di/ill$ wat] into a l>ge flask 7or glass beak]1 or enamell$ iron pot7 & add #ajjj grammes 7r\ghly1 #b-a_/b p\nds7 ( fre% lean meat--e4 g41 bullock's he>t--f9ely m9c$ 9 a m9c+ ma*9e4 #b4 ,heat ! mixture g5tly 9 a wat]-ba?1 tak+ c>e t ! temp]ature ( ! cont5ts ( ! flask does n exce$ #dj deg4 ,c4 = ! fir/ tw5ty m9utes4 7,?is dissolves out ! soluble proteids1 extractives1 salts1 etc47 #c4 ,n[ raise ! temp]ature ( ! mixture to ! boil+-po9t1 & ma9ta9 at ?is temp]ature = t5 m9utes4 7,?is precipitates "s ( ! album9s1 ! haemoglob91 etc41 f ! solution47 #d4 ,/ra9 ! mixture ?r\gh /]ile butt] musl9 or a p]=at$ porcela9 funnel1 !n filt] ! liquid ?r\gh ,sw$i% filt] pap] into a /]ile normal litre flask1 & :5 cold make up to #ajjj c4c4 by ! addition ( di/ill$ wat]--to replace ! loss f evaporation4 #e4 ,if n ne$$ at once1 /]ilise ! meat extract 9 bulk 9 ! /eam /]ilis] = tw5ty m9utes on ea* ( ?ree consecutive days4 ,calf1 %eep1 or *ick5 fle% is occasionally sub/itut$ = ! beef2 or ! meat extract may be prep>$ f animal visc]a1 su* z bra91 sple51 liv]1 or kidneys4 ,,note4--,z an alt]native me?od1 #e c4c4 ( ,br&'s meat juice or #c grammes ( ,wye?'s beef juice1 or #aj grammes ,liebig's extract ( meat 7,lemco7 may be dissolv$ 9 #ajjj c4c4 di/ill$ wat]1 & heat$ & filt]$ z above to =m ord9>y or s+le /r5g? meat extract4 ,m$ia1 prep>$ f su* meat extracts are1 h[ev]1 em95tly unsatisfactory :5 us$ = ! cultivation ( ! m highly p>asitic bact]ia2 al?\gh :5 work+ 9 tropical & subtropical regions _! use is well-nigh compulsory4 ,reaction ( ,meat ,extract4--,meat extract ?us prep>$ is acid 9 its reaction1 ow+ to ! pres5ce ( acid phosphates ( potassium & sodium1 weak acids ( ! glycolic s]ies1 & organic comp\nds 9 :i* ! acid "* pr$om9ates4 ,ow+ to ! nature ( ! sub/ances f :i* x d]ives its reaction1 ! total acidity ( meat extract c only be e/imat$ accurately :5 ! solution is at ! boil+-po9t4 ,moreov]1 x has be5 obs]v$ t prolong$ boil+ 7su* z is involv$ 9 ! prep>ation ( nutri5t m$ia7 causes x to und]go hydrolytic *anges :i* increase its acidity1 & ! meat extract only becomes /able 9 ?is respect aft] x has be5 ma9ta9$ at ! boil+-po9t = =ty-five m9utes4 ,al?\gh meat extract always reacts acid to ph5olph?ale91 x occasionally reacts neutral or ev5 alkal9e to litmus2 & aga91 meat extract t has be5 r5d]$ exactly neutral to litmus /ill reacts acid to ph5olph?ale94 ,?is peculi> behavi\r dep5ds ^u two factors3 #a4 ,litmus is ins5sitive to _m weak organic acids ! pres5ce ( :i* is readily indicat$ by ph5olph?ale94 #b4 ,dibasic sodium phosphate :i* is =m$ dur+ ! process ( neutralisation is a salt :i* reacts alkal9e to litmus1 b neutral to ph5olph?ale94 ,9 ord]1 !re=e1 to obta9 an accurate e/imation ( ! reaction ( any giv5 sample ( meat extract1 x is ess5tial t-- #a4 ,! meat extract be previ\sly expos$ to a temp]ature ( #ajj deg4 ,c4 = =ty-five m9utes4 #b4 ,! e/imation be p]=m$ at ! boil+-po9t4 #c4 ,ph5olph?ale9 be us$ z ! indicator4 ,! e/imation is c>ri$ out by means ( titration exp]im5ts aga9/ /&>d solutions ( cau/ic soda1 9 ! foll[+ mann]3 ,me?od ( ,e/imat+ ! ,reaction4-- ,app>atus ,requir$3 ,solutions ,requir$3 #a4 #be c4c4 burette graduat$ #a4 #aj,n ,na,o,h1 accurately 9 t5?s ( a c5timetre4 /&>dis$4 #b4 #a c4c4 pipette graduat$ 9 #b4 n_/#a ,na,o,h1 accurately hundr$?s1 & provid$ /&>dis$ ) rubb] tube1 p9*-cock1 & deliv]y nozzle4 #c4 #be c4c4 measure 7cyl9d] or #c4 n_/#aj ,na,o,h1 accurately pipette1 calibrat$ = /&>dis$4 #ih deg4 ,c4--n #ae deg4 ,c74 #d4 ,sev]al #fj c4c4 conical #d4 #j4e p] c5t4 solution ( beak]s or ,erl5mey] ph5olph?ale9 9 #ej p] flasks4 c5t4 alcohol4 #e4 ,:ite porcela9 evaporat+ bas91 fill$ ) boil+ wat] & arrang$ ov] a gas flame z a wat]-ba?4 #f4 ,bohemian glass flask1 fitt$ z a wa%-bottle1 & fill$ ) di/ill$ wat]1 :i* is kept boil+ on a tripod /&4 ,,me?od4--,arrange ! app>atus z indicat$ 9 figure #ig4 7,a7 #a4 ,fill ! burette ) n_/#aj ,na,o,h4 #b4 ,fill ! pipette ) n_/#a ,na,o,h4 ,illu/ration3 ,,fig4 #ig4--;,arrangem5t ( app>atus = titrat+ m$ia4 #c4 ,measure #be c4c4 ( ! meat extract 7previ\sly heat$ 9 ! /eam] at #ajj deg4 ,c4 = =ty-five m9utes7 into "o ( ! beak]s by means ( ! measure2 r9se out ! measure ) a v small quantity ( boil+ di/ill$ wat] f ! wa%-bottle1 & !n add ?is r9se wat] to ! meat extract already 9 ! beak]4 #d4 ,run 9 ab\t #j4e c4c4 ( ! ph5olph?ale9 solution & imm]se ! beak] 9 ! wat]-ba?1 & raise to ! boil4 #e4 ,to ! m$ium 9 ! beak] run 9 n_/#aj ,na,o,h cauti\sly f ! burette until ! end-po9t is rea*$1 z indicat$ by ! developm5t ( a p9ki% t+e1 %[n 9 figure #ih 7b74 ,note ! am\nt ( dec9ormal soda solution us$ 9 ! process4 ,,note4--,j be=e ! end-po9t is rea*$1 a v slight opalesc5ce may be not$ 9 ! fluid1 due to ! precipitation ( dibasic phosphates4 ,aft] ! true end-po9t is rea*$1 ! fur!r addition ( ab\t #j4e c4c4 ( ! dec9ormal soda solution w produce a deep mag5ta col\r 7,fig4 #ih1 c71 :i* is ! s-call$ end-po9t ( ! ,am]ican ,committee ( ,bact]iologi/s4 ,illu/ration3 ,,fig4 #ih4--;a1 ,sample ( filt]$ meat extract or nutri5t gelat9e to :i* ph5olph?ale9 has be5 add$4 ,! m$ium is acid1 z evid5c$ by ! unalt]$ col\r ( ! sample4 b1 ,! same neutralis$ by ! addition ( n_/#aj ,na,o,h4 ,! production ( ?is fa9t rose-p9k col\r indicates t ! end-po9t1 or neutral po9t to ph5olph?ale91 has be5 rea*$4 ,if su* a sample is cool$ d[n to say #cj deg4 or #bj deg4 ,c41 ! col\r w be f\nd to become m di/9ct & decid$ly deep] & bright]1 resembl+ t %[n 9 c4 c1 ,also if1 aft] ! end-po9t is rea*$1 a fur!r #j4e c4c4 or #a4j c4c4 n_/#aj ,na,o,h be add$ to ! sample1 ! m>k$ alkal9ity is evid5c$ by ! deep col\r "h %[n4 7,b7 ,p]=m a control titration 7occasionally two controls may be necess>y71 z foll[s3 #a4 ,measure #be c4c4 ( ! meat extract into "o ( ! beak]s1 wa% out ! measure ) boil+ wat]1 & add ! ph5olph?ale9 z 9 ! fir/ e/imation4 #b4 ,run 9 n_/#a ,na,o,h f ! pipette1 j %ort ( ! equival5t ( ! am\nt ( deci-normal soda solution requir$ to neutralise ! #be c4c4 ( m$ium4 7,= example1 if 9 ! fir/ e/imation #e c4c4 ( n_/#aj ,na,o,h 7 requir$ to r5d] #be c4c4 ( m$ium neutral to ph5olph?ale91 only add #j4dh c4c4 ( n_/#a ,na,o,h47 ,imm]se ! beak] 9 ! wat]-ba?4 #c4 ,complete ! titration by ! aid ( ! n_/#aj ,na,o,h4 #d4 ,note ! am\nt ( n_/#aj ,na,o,h solution requir$ to complete ! titration1 & add x to ! equival5t ( ! n_/#a ,na,o,h solution previ\sly run 94 ,take ! total z ! correct e/imation4 ,me?od ( ,express+ ! ,reaction4-- ,! reaction or titre ( meat extract1 m$ium1 or any solution e/imat$ 9 ! =ego+ mann]1 is mo/ conv5i5tly express$ by indicat+ ! numb] ( cubic c5timetres ( normal alkali 7or normal acid7 t w\ld be requir$ to r5d] "o litre ( ! solution exactly neutral to ph5olph?ale94 ,illu/ration3 ,,fig4 #ii4--,/ock bottle = dekanormal soda solution4 ,! sign 7plus7 is prefix$ to ?is numb] if ! orig9al solution reacts acid1 & ! sign - 7m9us7 if x reacts alkal9e4 ,= example1 meat extract #aj1 indicates a sample ( meat extract :i* reacts acid to ph5olph?ale91 & w\ld require ! addition ( #aj c4c4 ( normal ,na,o,h p] litre1 to neutralise x4 ,,note4--,su* a solution w\ld probably react alkal9e to litmus4 ,conv]sely1 if z ! result ( our titration exp]im5ts we f9d t #be c4c4 ( meat extract require ! addition ( #e c4c4 n_/#aj ,na,o,h to neutralise1 !n #ajjj c4c4 ( meat extract w require ! addition ( #bjj c4c4 n_/#aj ,na,o,h #bj c4c4 n_/#a ,na,o,h4 ,& ?is la/ figure1 #bj1 prec$$ by ! sign 7i4 e41 #bj71 to signify t x is acid1 indicates ! reaction ( ! meat extract4 ,,note4--,! /&>d soda solutions %\ld be prep>$ by accurate measur+ op]ations1 controll$ by titrations1 f a /ock solution ( #aj,n ,na,o,h1 :i* %\ld be v c>efully /&>dis$4 ,if a l>ge supply is made or ! consumption is small ?is /ock solution mu/ be kept 9 an aspirator bottle to :i* air c only ga9 access aft] x has be5 dri$ & r5d]$ free f ,,co#b4 ,?is may be done by fir/ lead+ x ov] ,h#b,,s#d & soda lime1 or soda lime alone1 by "s su* arrangem5t z is %[n 9 figure #ii1 :i* also %[s a con/ant burette arrangem5t = ! deliv]y ( small measur$ quantities ( ! dekanormal soda solution4 ,,/&>disation ,,( ,,m$ia4 ,diff]5ces 9 ! reaction ( ! m$ium 9 :i* x is gr[n w provoke n only diff]5ces 9 ! rate ( gr[? ( any giv5 bact]ium1 b also well-m>k$ diff]5ces 9 its cultural & morphological *>act]s2 & ne>ly e organism w be f\nd to affect a def9ite optimum reaction--a po9t to be c>efully det]m9$ = ea*4 ,= mo/ bact]ia1 h[ev]1 ! optimum usually approximates fairly closely to #aj2 & z exp]im5t has %[n t ?is reaction is ! mo/ g5]ally useful = r\t9e laboratory "w1 x is ! "o :i* may be adopt$ z ! /&>d = all nutri5t m$ia d]iv$ f meat extract4 ,briefly1 ! me?od ( /&>dis+ a litre ( m$ia to #aj consi/s 9 subtract+ #aj f ! initial titre ( ! m$ium mass2 ! rema9d] indicates ! numb] ( cubic c5timetres ( normal soda solution t mu/ be add$ to ! m$ium1 p] litre1 to r5d] ! reaction #aj4 ,/&>dis+ ,nutri5t ,b\illon4--,= example1 #ajjj c4c4 b\illon are prep>$2 at ! fir/ titration x is f\nd #a4 #be c4c4 require ! addition ( #e4ej c4c4 n_/#aj ,na,o,h to neutralise4 ,two controls give ! foll[+ results3 #b4 #be c4c4 require ! addition ( #e4gj c4c4 n_/#aj ,na,o,h to neutralise4 #c4 #be c4c4 require ! addition ( #e4fj c4c4 n_/#aj ,na,o,h to neutralise4 ,av]ag+ ^! two controls1 #be c4c4 require ! addition ( #e4fe c4c4 n_/#aj ,na,o,h to neutralise1 & !re=e #ajjj c4c4 require ! addition ( #bbf c4c4 n_/#aj ,na,o,h1 or #bb4fj c4c4 n_/#a ,na,o,h1 or #b4bf c4c4 n_/#aj ,na,o,h4 ,initial titre ( ! b\illon #bb4f1 & z su* requires ! addition ( 7#bb4f c4c4 - #aj c4c47 #ab4f c4c4 ( n_/#a ,na,o,h p] litre to leave its f9i%$ reaction #aj4 ,b ! ?ree titrations1 ea* on #be c4c4 ( m$ium1 h r$uc$ ! orig9al bulk ( b\illon to 7#ajjj - #ge c4c47 #ibe c4c4 ,! am\nt ( n_/#a ,na,o,h requir$ to r5d] ! reaction ( ?is quantity ( m$ium #aj may be d$uc$ ?us3 #ajjj c4c43#ibe c4c433#ab4f c4c43x4 ,!n x #aa4fe c4c4 n_/#a ,na,o,h4 ,:5ev] possible1 h[ev]1 ! requir$ reaction is produc$ by ! addition ( dekanormal soda solution1 on acc\nt ( ! m9ute increase x causes 9 ! bulk1 & ! consequ5t insignificant di/urbance ( ! p]c5tage composition ( ! m$ium4 ,by means ( a pipette graduat$ to #j4ja c4c4 x is possible to deliv] v small quantities2 b if ! calculat$ am\nt runs into ?\s&? p>ts ( a cubic c5timetre1 ^! are replac$ by correspond+ quantities ( normal or ev5 dec9ormal soda4 ,9 ! above example x is necess>y to add #aa4fe c4c4 normal ,na,o,h or its equival5t1 #a4afe c4c4 dekanormal ,na,o,h4 ,! fir/ be+ too bulky a quantity1 & ! second inconv5i5tly small = exact measurem5t1 ! total weight ( soda is obta9$ by sub/itut+ #a4af c4c4 dekanormal soda solution1 & ei!r #j4je c4c4 ( normal soda solution or #j4e c4c4 ( dec9ormal soda solution4 ,/&>dis+ ,nutri5t ,ag> & ,gelat9e4--,! me?od ( /&>dis+ ag> & gelat9e is precisely simil> to t describ$ "u b\illon4 ,,! ,,filtration ,,( ,,m$ia4 ,fluid m$ia are usually filt]$ ?r\gh /\t ,sw$i% filt] pap] 7occasionally ?r\gh a porcela9 filt] c&le71 & 9 ord] to accel]ate ! rate ( filtration ! filt] pap] %\ld be fold$ 9 t =m :i* is kn[n z ! physiological filt]1 n 9 ! ord9>y quadrant %ape1 z by ?is means a l>ge surface is available = filtration & a small] area 9 contact ) ! glass funnel support+ x4 ,to fold ! filt] proce$ ?us3 #a4 ,take a circul> piece ( filt] pap] & fold x exactly ?r\gh its c5tre to =m a semicircle 7,fig4 #ajj1 a74 #b4 ,fold ! semicircle exactly 9 half to =m a quadrant2 make ! crease #b1 di/9ct by runn+ ! ?umbnail along x1 !n op5 ! filt] out to a semicircle aga94 #c4 ,fold ea* end ( ! semicircle 9 to ! c5tre & s =m ano!r quadrant2 smoo? d[n ! two new creases #c & #c;a1 ?us =m$ & aga9 op5 out to a semicircle4 #d4 ,! semicircle n[ appe>s z 9 figure #ajj1 a1 ! d>k l9es indicat+ ! creases already =m$4 #e4 ,fold ! po9t #a ov] to ! po9t #c1 & #a;a to #c;a1 to =m ! creases #d & #d;a1 indicat$ 9 ! diagram by ! light l9es4 ,fold po9t #a ov] to #c;a1 & #a;a to #c1 to =m ! creases #e & #e;a4 ,illu/ration3 ,,fig4 #ajj4--;,filt] fold+3 a1 ,filt] fold$ 9 half1 %[+ creases2 b1 appe>ance ( filt] on completion ( fold+2 c1 filt] op5$ out ready = use4 #f4 ,?us f> ! creases h all be5 made on ! same side ( ! pap]4 ,n[ subdivide ea* ( ! eight sectors by a crease ?r\gh its c5tre on ! opposite side ( ! pap]1 indicat$ by ! fa9t brok5 l9es 9 ! diagram4 ,fold up ! filt] gradually z ea* crease is made1 & :5 f9i%$ ! filt] has assum$ ! %ape ( a w$ge1 z 9 figure #ajj1 b4 ,:5 op5$ out ! filt] assumes ! %ape repres5t$ 9 figure #ajj1 c4 ,! fold$ filt] is next plac$ inside a glass funnel support$ on a retort /&1 & moi/5$ ) hot di/ill$ wat] be=e ! filtration ( ! m$ium is comm5c$4 ,liquefiable solid m$ia are filt]$ ?r\gh a specially made filt] pap]--papi] ,*>d9--:i* is sold 9 boxes ( tw5ty-five ready-fold$ filt]s4 ,illu/ration3 ,,fig4 #aja4--;,hot-wat] filt] funnel & r+ burn]4 ,gelat9e1 :5 prop]ly made1 filt]s ?r\gh ?is pap] z quickly z b\illon does ?r\gh ! ,sw$i% filt] pap]1 & does n require ! use ( ! hot-wat] funnel4 ,ag>1 likewise1 if prop]ly made1 filt]s readily1 al?\gh n at s rapid a rate z gelat9e4 ,if badly egg$1 & also dur+ ! w9t] mon?s1 x is necess>y to surr\nd ! glass funnel1 9 :i* ! filtration ( ! ag> is c>ri$ on1 by a hot-wat] jacket4 ,?is is done by plac+ ! glass funnel inside a d\ble-wall$ copp] funnel--! space betwe5 ! walls be+ fill$ ) wat] at ab\t #ij deg4 ,c4--& support+ ! latt] on a r+ gas burn] fix$ to a retort /& 7,fig4 #aja74 ,! gas is light$ & ! wat] jacket ma9ta9$ at a high temp]ature until filtration is complet$4 ,if ! /eam /]ilis] ( ! laboratory is suffici5tly l>ge1 x is sometimes m conv5i5t to place ! flask & filt]+ funnel bodily inside1 close ! /]ilis] & all[ filtration to proce$ 9 an atmosph]e ( live /eam1 ?an to use ! gas r+ & hot-wat] funnel4 ,,/or+ ,,m$ia ,,9 ,,bulk4 ,aft] filtration fill ! m$ium into /]ile litre flasks ) cotton-wool plugs & /]ilise 9 ! /eam] = tw5ty m9utes on ea* ( ?ree consecutive days4 ,aft] ! ?ird /]ilisation1 & :5 ! flasks & cont5ts are cool1 cut (f ! top ( ! cotton-wool plug squ>e ) ! m\? ( ! flask2 pu% ! plug a %ort di/ance d[n into ! neck ( ! flask & fill 9 ) melt$ p>aff9 wax to ! level ( ! m\?4 ,:5 ! wax has set ! flasks are /or$ 9 a cool d>k cupbo>d = future use4 ,illu/ration3 ,,fig4 #ajb4--,rubb] cap clos+ /ore bottle4 a1 be=e1 & b1 aft] /]iliz+4 ,?is plan is n absolutely satisfactory1 al?\gh v g5]ally employ$ on occasion1 & x is pref]able to fill ! m$ium into long-neck$ fl9t glass bottles 7! qu>t size1 hold+ ne>ly #ajjj c4c41 su* z ^? 9 :i* ,pa/euris$ milk is retail$7 & to close ! neck ( ! bottle by a special rubb] cap4#c ,?is cap is made ( s(t rubb]1 ! l[] "p1 dome-%ap$ ) ?9 walls1 be+ slipp$ ov] ! neck ( ! bottle 7,fig4 #ajb1 a74 ,! upp] "p is solid1 b ) a %>p clean-cut 7made ) a cat>act or t5otomy knife7 runn+ completely ?r\gh its axis f ! c5tre ( ! disc to ! top ( ! dome4 ,dur+ /]ilisation ! air 9 ! neck ( ! bottle1 exp&$ by ! heat1 is driv5 out ?r\gh ! valvul> ap]ture 9 ! solid portion ( ! /opp]4 ,on remov+ ! bottle f ! /eam *amb]1 ! liquid contracts z x cools1 & ! pressure ( ! ext]nal air drives ! solid piece ( rubb] d[n into ! neck ( ! bottle1 & =ces toge!r ! lips ( ! slit 7,fig4 #ajb1 b74 ,?us seal$1 ! bottle w pres]ve its cont5ts /]ile = an indef9ite p]iod )\t loss f evaporation4 ,,tub+ ,,nutri5t ,,m$ia4 ,aft] ! f9al filtration1 ! nutri5t m$ium is usually tub$--i4 e41 fill$ into /]ile tubes 9 def9ite measur$ quantities1 usually #aj c4c4 ,?is process is sometimes c>ri$ out by means ( a l>ge sep>ator funnel fitt$ ) a ?ree-way tap :i* communicates ) a small graduat$ tube 7capacity #bj c4c4 & graduat$ 9 cubic c5timetres7 atta*$ to ! side4 ,! %ape ( ?is piece ( app>atus1 kn[n z ,tresk['s funnel1 r5d]s x p>ticul>ly liable to damage4 ,x is bett]1 !re=e1 to arrange a less exp5sive piece ( app>atus :i* w s]ve ! purpose equally well 7,fig4 #ajc74 ,a ,geissl]'s ?ree-way /op-cock has ! tube on "o side ( ! tap gr\nd obliquely at its extremity1 & ! tube on ! opposite side cut (f )9 #c cm4 ( ! tap4 ,! %ort tube is connect$ by means ( a p]=at$ rubb] cork ) a #aj cm4 l5g? ( /\t glass tub+ 7#a4e cm4 bore74 ,! ?ird *annel ( ! ?ree-way tap is connect$1 by means ( rubb] tub+1 ) ! nozzle ( an ord9>y sep>ator funnel4 ,f9ally1 ! receiv+ cyl9d] above ! ?ree-way tap is graduat$ 9 cubic c5timetres up to #bj1 by p\r+ into x measur$ quantities ( wat] & m>k+ ! v>i\s levels on ! outside ) a writ+ diamond4 ,fluid m$ia conta9+ c>bohydrates are fill$ into f]m5tation tubes 7vide ,fig4 #ba72 or into ord9>y m$ia tubes :i* already h small] tubes1 inv]t$1 inside !m 7,fig4 #ajd71 to collect ! products ( gr[? ( gas-=m+ bact]ia4 ,:5 fir/ fill$1 ! small tubes float on ! surface ( ! m$ium aft] ! fir/ /]ilisation ne>ly all ! air is replac$ by ! m$ium1 & aft] ! f9al /]ilisation ! gas tubes w be subm]g$ & completely fill$ ) ! m$ium4 ,illu/ration3 ,,fig4 #ajc4--,sep>atory funnel & ?ree-way tap arrang$ = tub+ m$ia4 ,illu/ration3 ,,fig4 #ajd4--;,gas tube 7,durham74 ,/or+ ,tub$ ,m$ia4--,m$ia aft] be+ tub$ are be/ /or$ by pack+1 9 ! v]tical position1 9 oblong boxes hav+ an int]nal measurem5t ( #cg cm4 long by #ab cm4 wide by #aj cm4 deep4 ,ea* box 7,fig4 #aje7 has a movable p>tition =m$ by ! v]tical face ( a weight$ triangul> block ( wood1 slid+ free on ! bottom 7,fig4 #aje1 ,a72 or by a flat piece ( wood slid+ 9 a metal groove 9 ! bottom ( ! box1 :i* c be fix$ at any spot by tight5+ ! ?umbscrew ( a brass guide rod :i* transfixes ! p>tition 7,fig4 #aje1 ,b74 ,! front ( ! box is provid$ ) a h&le & a celluloid label = ! "n ( ! conta9$ m$ium4 ,^! boxes are arrang$ ^u %elves 9 a d>k cupbo>d--or pref]ably an iron safe--:i* %\ld be r5d]$ z ne>ly air-tight z possible1 & %\ld h ! words m$ia /ores pa9t$ on its doors4 ,illu/ration3 ,,fig4 #aje4--,m$ium box1 %[+ alt]native p>titions ,a & ,b4 ,,footnotes3 #c ,?is rubb] cap has be5 made = me by ! ,holborn ,surgical ,in/rum5t ,co41 ,?avies ,inn1 ,london1 ,w4 ,c4 ,,xi4 ,,culture ,,m$ia4 ,,ord9>y ,,or ,,/ock ,,m$ia4 ,nutri5t ,b\illon4-- #a4 ,measure out d\ble /r5g? meat extract1 #ejj c4c41 into a litre flask & add #cjj c4c4 di/ill$ wat]4 #b4 ,weigh out ,witte's peptone1 #aj grammes 7 #a p] c5t471 salt1 #e grammes 7 #j4e p] c5t471 & mix into a smoo? pa/e ) #bjj c4c4 ( di/ill$ wat] previ\sly heat$ to #fj deg4 ,c4 7,be c>eful to leave no unbrok5 globul> masses ( peptone47 #c4 ,add ! peptone emulsion to ! meat extract 9 ! flask & heat 9 ! /eam] = =ty-five m9utes 7to completely dissolve ! peptone1 & to r5d] ! acidity ( ! meat extract /able74 #d4 ,e/imate ! reaction ( ! m$ium2 control ! result2 r5d] ! reaction ( ! f9i%$ m$ium #aj 7vide page #aee74 #e4 ,heat = half an h\r 9 ! /eam] at #ajj deg4 ,c4 7to complete ! precipitation ( ! phosphates1 etc474 #f4 ,filt] ?r\gh ,sw$i% filt] pap] into a /]ile flask4 #g4 ,fill into /]ile tubes 7#aj c4c4 9 ea* tube74 #h4 ,/]ilise 9 ! /eam] = tw5ty m9utes on ea* ( ?ree consecutive days--i4 e41 by ! discont9u\s me?od 7vide page #ce74 ,,note4--,z an alt]native me?od :5 nei!r fre% nor froz5 meat is available nutri5t b\illon may be prep>$ f a comm]cial meat extract1 z foll[s3 ,lemco ,bro?4-- #a4 ,measure out #bej c4c4 di/ill$ wat] into a litre flask4 #b4 ,weigh out #aj grammes ,liebig's ,lemco ,meat ,extract on a piece ( clean filt] pap] & add to ! wat] 9 ! flask4 ,%ake ! flask well to make an ev5 emulsion ( ! meat extract4 #c4 ,weigh out ,witte's peptone 7#aj grammes71 salt 7#e grammes74 ,mix into smoo? pa/e ) #ajj c4c4 di/ill$ wat] previ\sly heat$ to #fj deg4 ,c4 #d4 ,add ! peptone salt emulsion to ! meat extract emulsion 9 ! flask & add #fej c4c4 di/ill$ wat]4 ,heat 9 ! /eam] = =ty-five m9utes4 #e4 ,/&>dise ! m$ium & complete z = nutri5t b\illon4 ,nutri5t ,gelat9e4-- #a4 ,weigh a #b-litre flask on a trip balance 7,fig4 #ajf7 & note ! weight1 or c\nt]poise c>efully4 ,illu/ration3 ,,fig4 #ajf4--,trip balance4 ,an extremely useful c\nt]poise is a small %eet-brass cyl9d] ab\t #ch mm4 high & #ch mm4 9 diamet]1 ) a funnel-%ap$ top & provid$ ) a side tube by :i* its cont5ts1 f9e du/ %ot1 may be empti$ out 7,fig4 #ajg74 ,illu/ration3 ,,fig4 #ajg4--;,c\nt]poise2 weight :5 empty1 #ce grammes2 :5 full ( du/ %ot1 #bjj grammes4 #b4 ,measure out d\ble /r5g? meat extract1 #ejj c4c41 into ! t>$ flask4 #c4 ,weigh out & mix #aj grammes ( peptone1 #e grammes ( salt1 & make into a ?ick pa/e ) #aej c4c4 di/ill$ wat]2 !n add ! emulsion to ! meat extract 9 ! flask2 also add #ajj grammes %eet gelat9e cut into small pieces2 place ! flask 9 ! wat]-ba? & raise to ! boil4 ,illu/ration3 ,,fig4 #ajh4--;,arrangem5t ( /eam c & wat]-ba? = ! prep>ation ( m$ia4 #d4 ,arrange a #e-litre t9 c 7) copp] bottom1 su* z is us$ 9 ! prep>ation ( di/ill$ wat]7 by ! side ( ! wat] ba?1 fill ! c ) boil+ wat] & place a light$ ,buns5 burn] "u x4 ,fit a long safety tube to ! neck ( ! c & also a deliv]y tube1 b5t twice at "r angles2 adju/ ! tube to rea* to ! bottom ( ! int]ior ( ! flask conta9+ ! gelat9e1 etc4 7,fig4 #ajh74 #e4 ,keep ! wat] 9 ! /eam c vig\r\sly boil+1 & s /eam at #ajj deg4 ,c1 bubbl+ ?r\gh ! m$ium mass1 = t5 m9utes1 by :i* "t complete solution ( ! gelat9e is effect$4 ,a c]ta9 am\nt ( /eam w cond5se z wat] 9 ! m$ium flask dur+ ?is process--h5ce ! necessity = ! use ( d\ble /r5g? meat extract--b if ! wat] ba? is kept boil+ ?is cond5sation w n exce$ #ajj c4c4 #f4 ,weigh ! flask & its cont5ts2 !n 7#aaaed grammes weight ( ! flask7 m9us 7weight ( ! flask & its cont5ts7 equals ! weight ( wat] requir$ to make up ! bulk to #a litre4 ,! addition ( ! requisite quantity ( wat] is c>ri$ out z foll[s3 ,9 "o pan ( ! trip balance place ! c\nt]poise ( ! t>$ flask 7or its equival5t 9 weights7 toge!r ) ! weights mak+ up ! calculat$ m$ium weight4 ,9 ! opposite pan place ! flask conta9+ ! m$ium mass4 ,n[ add boil+ di/ill$ wat] f a wa% bottle until ! two pans are exactly balanc$4 #g4 ,titrate & e/imate ! reaction ( ! m$ium mass2 control ! result4 ,calculate ! am\nt ( soda solution requir$ to make ! reaction ( ! m$ium mass #aj 7i4 e41 calculate = #ajjj c4c41 less ! quantity us$ = ! titrations74 #h4 ,add ! necess>y am\nt ( soda solution & heat 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes1 to precipitate ! phosphates1 etc4 #i4 ,all[ ! m$ium mass to cool to #fj deg4 ,c4 ,well :ip ! :ites ( two eggs1 add to ! cont5ts ( ! flask & replace 9 ! /eam] at #ajj deg4 ,c4 = ab\t half an h\r 7until ! egg-album5 has coagulat$ & =m$ l>ge1 firm masses float+ on & 9 cle> gelat9e74 #aj4 ,filt] ?r\gh papi] ,*>d9 into a /]ile flask4 #aa4 ,tube 9 quantities ( #aj c4c4 #ab4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree consecutive days--i4 e41 by ! discont9u\s me?od4 ,nutri5t ,ag>-ag>4-- #a4 ,weigh a #b-litre flask & note ! weight--or c\nt]poise exactly4 #b4 ,measure out d\ble /r5g? meat extract1 #ejj c4c41 into ! t>$ flask4 #c4 ,weigh out & mix #aj grammes ( peptone1 #e grammes ( salt1 & #bj grammes ( p[d]$ ag>1 & make into a ?ick pa/e ) #aej c4c4 di/ill$ wat]1 & add to ! meat extract 9 ! flask2 place ! flask 9 a wat]-ba?4 #d4 ,arrange ! /eam c & wat]-ba? z already direct$ 7= ! prep>ation ( gelat9e7 & figur$4 #e4 ,bubble live /eam 7at #ajj deg4 ,c47 ?r\gh ! m$ium mass1 = tw5ty-five m9utes1 by :i* "t complete solution ( ! ag> is effect$4 #f4 ,n[ weigh ! flask & its cont5ts2 !n 7#ajcee grammes weight ( flask7 m9us 7weight ( flask & its cont5ts7 equals ! weight ( wat] requir$ to make up ! bulk ( ! m$ium to #a litre4 ,add ! requisite am\nt 7see prep>ation ( gelat9e1 page #aff1 /ep #f74 #g4 ,titrate1 & e/imate ! reaction ( ! m$ium mass2 control ! result4 ,calculate ! am\nt ( soda solution requir$ to make ! reaction ( ! m$ium mass #aj 7i4 e41 calculat$ = #ajjj c4c41 less ! quantity us$ = ! titrations74 #h4 ,add ! necess>y am\nt ( soda solution & replace 9 ! /eam] = tw5ty m9utes 7to complete ! precipitation ( ! phosphates1 etc474 #i4 ,all[ ! m$ium mass to cool to #fj deg4 ,c4 ,well :ip ! :ites ( two eggs1 add to ! cont5ts ( ! flask1 & replace 9 ! /eam] at #ajj deg4 ,c4 = ab\t "o h\r 7until ! egg-album5 has coagulat$ & =m$ l>ge1 firm masses float+ on & 9 cle> ag>47 #aj4 ,filt] ?r\gh papi] ,*>d91 by ! aid ( a hot-wat] funnel1 if necess>y 7,fig4 #aja71 into a /]ile flask4 #aa4 ,tube 9 quantities ( #aj c4c4 or #ae c4c4 #ab4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes on ea* ( ?ree consecutive days--i4 e41 by ! discont9u\s me?od4 ,blood-s]um 7,inspissat$74-- #a4 ,/]ilise cyl9drical glass j> 7,fig4 #aji7 & its cov] by dry heat1 or by wa%+ fir/ ) e!r & !n ) alcohol & dry+4 #b4 ,collect blood at ! slaught] h\se f ox or %eep 9 ! /]ile cyl9d]4 #c4 ,all[ ! vessel to /& = fifte5 m9utes = ! blood to coagulate4 7,?is mu/ be done be=e leav+ ! slaught]h\se1 o!rwise ! s]um w be /a9$ ) haemoglob947 #d4 ,sep>ate ! clot f ! sides ( ! vessel by means ( a /]ile glass rod 7! yield ( s]um is mu* small] :5 ?is is n done71 & place ! cyl9d] 9 ! ice-*e/ = tw5ty-f\r h\rs4 #e4 ,remove ! s]um ) /]ile pipettes1 or syphon x (f1 & fill into /]ile tubes 7#e c4c4 9 ea*7 or flasks4 #f4 ,heat tubes conta9+ s]um to #ef deg4 ,c4 9 a wat]-ba? = half an h\r on ea* ( two successive days4 #g4 ,on ! ?ird day1 heat ! tubes1 9 a slop+ position1 9 a s]um inspissator to ab\t #gb deg4 ,c4 7,a coagulum is =m$ at ?is temp]ature :i* is fairly transp>5t2 above #gb deg4 ,c41 a ?ick turbid coagulum is =m$47 ,illu/ration3 ,,fig4 #aji4--;,blood-s]um j> ) wick] basket = transport4 ,! s]um inspissator 7,fig4 #aaj7 9 its simple/ =m is a d\ble-wall$ rectangul> copp] box1 clos$ 9 by a loose glass lid1 & cas$ 9 felt or asbe/os--! space betwe5 ! walls is fill$ ) wat]4 ,! inspissator is support$ on adju/able legs s t ! s]um may be solidifi$ at any desir$ slant1 & is heat$ f bel[ by a ,buns5 burn] controll$ by a !rmo-regulator4 ,! m elaborate =ms resemble ! hot-air ov5 7,fig4 #bf7 9 %ape & are provid$ ) adju/able %elves s t any desir$ obliquity ( ! s]um slope c be obta9$4 #h4 ,place ! tubes 9 ! incubator at #cg deg4 ,c4 = =ty-eight h\rs 9 ord] to elim9ate ^? t h be5 contam9at$4 ,/ore ! rema9d] 9 a cool place = future use4 ,alt]native ,me?od4 ,/eps #a-e z above4 #f4 ,/]ilise ! s]um by ! fractional me?od--t is1 by exposure 9 a wat]-ba? to a temp]ature ( #ef deg4 ,c4 = half an h\r on ea* ( six consecutive days2 /ore 9 ! fluid condition4 #g4 ,coagulate 9 ! inspissator :5 ne$$4 ,illu/ration3 ,,fig4 #aaj4--,s]um inspissator4 ,s]um ,wat]4-- ,?is =ms ! basis ( _m useful m$ia1 & is prep>$ z foll[s3 #a4 ,collect blood 9 ! slaught]h\se 7see page #afh7 & :5 firmly clott$ collect all ! express$ s]um & measure 9 a graduat$ cyl9d]4 #b4 ,= e #ajj c4c4 ( s]um add #cjj c4c4 di/ill$ wat] & mix 9 a flask4 #c4 ,heat ! mixture 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes4 7,?is de/roys any dia/atic f]m5t pres5t 9 ! s]um & p>tially /]ilises ! fluid47 #d4 ,filt] if turbid4 #e4 ,if n ne$$ at once complete ! /]ilisation ( ! s]um wat] by two subsequ5t /eam+s at #ajj deg4 ,c4 = tw5ty m9utes at tw5ty-f\r h\r int]vals4 ,citrat$ ,blood ,ag>4 ,guy's4-- #a4 ,kill a small rabbit ) *lor(orm vap\r1 & nail x out on a bo>d 7z = a necropsy72 moi/5 ! hair ?or\ghly ) #b p] c5t4 solution ( lysol4 #b4 ,/]ilise sev]al pairs ( =ceps1 scissors1 etc4 by boil+4 #c4 ,reflect ! sk9 ov] ! ?orax ) /]ile in/rum5ts4 #d4 ,op5 ! ?oracic cavity by ! aid ( a fre% set ( /]ile in/rum5ts4 #e4 ,op5 ! p]ic>dium ) ano!r set ( /]ile in/rum5ts4 #f4 ,se> ! surface ( ! left v5tricle ) a r$-hot iron4 #g4 ,take a /]ile capill>y pipette 7,fig4 #ac1 c72 break (f ! seal$ extremity ) a pair ( /]ile =ceps4 #h4 ,/eady ! he>t 9 a pair ( =ceps & ?ru/ ! po9t ( ! pipette ?r\gh ! wall ( ! v5tricle & ?r\gh ! se>$ area1 apply suction to ! plugg$ end ( ! pipette & fill x ) blood4 #i4 ,transf] ! entire quantity ( blood collect$ f ! rabbit's he>t to a small ,erl5mey] flask conta9+ a numb] ( /]ile glass beads & #e c4c4 conc5trat$ sod4 citrate solution4 7,see page #cgh47 #aj4 ,agitate ?or\ghly & set aside = a c\ple ( h\rs4 #aa4 ,melt up sev]al tubes ( nutri5t ag> 7see page #afg7 & cool to #db deg4 ,c4 #ab4 ,) a /]ile #aj c4c4 graduat$ pipette transf] #a c4c4 citrat$ blood f ! ,erl5mey] flask to ea* tube ( liquefi$ ag>4 ,rotate ! tube betwe5 ! h&s 9 ord] to diffuse ! citrat$ blood ev5ly ?r\gh\t ! ag>4 #ac4 ,place ! tubes 9 a slop+ position & all[ ! m$ium to set4 #ad4 ,place tubes ( blood ag> = =ty-eight h\rs 9 ! incubator at #cg deg4 ,c4 & at ! end ( t "t elim9ate any contam9at$ tubes4 #ae4 ,/ore su* tubes z rema9 /]ile = future use4 ,milk4-- #a4 ,p\r #a litre ( fre% c['s or goat's milk into a l>ge sep>at+ funnel1 & heat 9 ! /eam] at #ajj deg4 ,c4 = "o h\r4 #b4 ,remove f ! /eam] & e/imate ! reaction ( ! milk 7normal c[s' milk av]ages #ag74 ,if ( high] acidity ?an #bj1 or l[] ?an #aj1 reject ?is sample ( milk & proce$ ) ano!r supply ( milk f a diff]5t s\rce4 ,reject milk to :i* antiseptics h be5 add$ z pres]vatives4 #c4 ,all[ ! milk to cool1 :5 ! fat or cream w rise to ! surface & =m a ?ick lay]4 #d4 ,draw (f ! subnatant fat-free milk into /]ile tubes 7#aj c4c4 9 ea*74 #e4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( five successive days4 #f4 ,incubate at #cg deg4 ,c4 = =ty-eight h\rs & elim9ate any contam9at$ tubes4 ,/ore ! rema9d] = future use4 ,litmus ,milk4-- #a4 ,prep>e milk z describ$ above1 sections #a to #c4 #b4 ,draw (f ! subnatant fat-free milk into a flask4 #c4 ,add /]ile litmus solution1 suffici5t to col\r ! milk a deep lav5d]4 #d4 ,tube1 /]ilise1 etc41 z = milk4 ,nutrose ,ag> 7,eyre74-- 7,?is is a modification ( ! well kn[n ,drigalski-,conradi m$ium orig9ally introduc$ = ! isolation ( ,b4 typhosus74 #a4 ,collect #bej c4c4 p]fectly fre% ox s]um 7vide ,blood ,s]um1 page #afh1 /eps #a to #e7 & add to x #dej c4c4 /]ile di/ill$ wat]4 #b4 ,weigh out ag> p[d]1 #bj grammes1 & emulsify x ) #bej c4c4 ( ! cold s]um wat]4 #c4 ,weigh out ,witte's peptone #aj grammes ,sodium *loride #e grammes ,nutrose #aj grammes & dissolve 9 #bjj c4c4 ( s]um wat] heat$ to #hj deg4 ,c4 #d4 ,mix ! ag> emulsion & ! peptone-nutrose solution 9 a t>$ flask ( #b-litre capacity & add a fur!r #ajj c4c4 s]um wat]4 #e4 ,complete ! solution ( ! v>i\s +r$i5ts by bubbl+ live /eam ?r\gh ! flask z 9 mak+ nutri5t ag>4 #f4 ,add fur!r #bej c4c4 s]um wat]4 #g4 ,weigh ! flask & its cont5ts3 !n 7#ajde grammes weight ( flask7 m9us 7weight ( flask & its pres5t cont5ts7 weight ( fluid requir$ to make up ! bulk ( ! m$ium to #a litre4 ,add ! requisite am\nt ( /]ile di/ill$ wat]4 #h4 ,titrate & e/imate ! reaction ( ! m$ium mass4 ,!n /&>dise to reaction ( #b4e4 #i4 ,cl>ify ) egg1 & filt] z = nutri5t ag>4 7,9 cl>ify+1 aft] ! addition ( ! egg :ite ! mixture %\ld be 9 ! /eam] = full two h\rs47 #aj4 ,aft] filtration is complete measure ! filtrate1 & to e #aej c4c4 ( ! m$ium add3 ,litmus solution 7,kahlbaum7 #bj c4c4 ,kry/al violet aque\s solution 7#a3ajjj7 7,b4 ,hoe*/7 #a4e c4c4 ,lactose #a4e grammes #aa4 ,tube 9 quantities ( #ae c4c4 #ab4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes on ea* ( ?ree successive days--i4 e41 by ! discont9u\s me?od = ?ree days4 ,egg ,m$ium 7,dorset74-- #a4 ,prep>e #ajjj c4c4 ( a #j4he p] c5t4 solution ( sodium *loride 9 a /\t #b-litre flask4 #b4 ,/]ilise 9 ! autoclave at #abj deg4 ,c4 = tw5ty m9utes4 ,cool to #bj deg4 ,c4 #c4 ,take #ab fre% eggs2 wa% ! %ells fir/ ) wat] !n ) undilut$ =mal93 all[ ! %ells to dry4 #d4 ,break ! eggs into a /]ile graduat$ cyl9d] & measure ! total volume ( ! mix$ :ites & yolks4 ,add "o "p /]ile sal9e solution to ?ree p>ts mix$ eggs4 #e4 ,transf] ?is mixture to a l>ge wide-m\!d /opp]$ bottle previ\sly /]ilis$4 ,add /]ile glass beads & %ake ?or\ghly 9 a me*anical %ak] = ab\t ?irty m9utes1 or :ip ) an egg-:isk4 #f4 ,filt] ?r\gh co>se butt] musl9 into a /]ile flask4 ,,note4--,a few drops ( alcoholic solution ( basic fu*s9 7suffici5t to give a def9ite p9k col\r71 or a few drops ( wat]pro( ,*9ese ink add$ to ! m$ium at ?is /age facilitates ! subsequ5t fi%+ ( colonies4 #g4 ,tube 9 quantities ( #aj c4c4 #h4 ,solidify 9 ! slop+ position 9 ! inspissator at #ge deg4 ,c4 = "o h\r4 #i4 ,place ! tubes = =ty-eight h\rs 9 ! incubator at #cg deg4 ,c41 & elim9ate any contam9at$ tubes4 ,to prev5t dry+1 #j4e c4c4 glyc]9e b\illon 7see page #bji7 may be add$ to ea* tube betwe5 /eps #h & #i4 #aj4 ,cap ^? tubes ( m$ia :i* rema9 /]ile ) india-rubb] caps & /ore = future use4 ,potato4-- #a4 ,*oose fairly l>ge potatoes1 wa% !m well1 & scrub ! peel ) a /iff nail-bru%4 #b4 ,peel & take out ! eyes4 #c4 ,remove cyl9d]s f ! longe/ diamet] ( ea* potato by means ( an apple-cor] or a l>ge cork-bor] 7i4 e41 "o ( ab\t #a4d cm4 diamet]74 ,! reaction ( ! fre% potato is /rongly acid to ph5olph?ale94 ,if1 !re=e1 ! potatoes are requir$ to approximate #aj1 z = ! cultivation ( "s ( ! vibrios1 ! cyl9d]s %\ld be soak$ 9 a #a p] c5t4 solution ( sodium c>bonate = ?irty m9utes4 #d4 ,cut ea* cyl9d] obliquely f end to end1 =m+ two w$ge-%ap$ portions4 #e4 ,place a small piece ( /]ilis$ cotton-wool1 moi/5$ ) /]ile wat]1 at ! bottom ( a /]ile te/-tube2 ins]t ! potato w$ge into ! tube s t its base re/s ^u ! cotton-wool4 ,n[ plug ! tube ) cotton-wool 7,fig4 #aaa74 #f4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( five consecutive days4 ,illu/ration3 ,,fig4 #aaa4--,potato tube4 ,,note4--,! cork bor] res]v$ = cutt+ ! potato cyl9d]s %\ld be silv] electro-plat$ bo? inside & out1 & ! knife us$ = divid+ ! cyl9d]s %\ld be ( silv] or silv] plat$4 ,:5 ^! precautions are adopt$ ! potato w$ges w reta9 _! :ite color & w n %[ ! discoloration s (t5 obs]v$ :5 /eel in/rum5ts are employ$4 ,be] ,wort4--,wort is *iefly us$ z a m$ium = ! cultivation ( yea/s1 m\lds1 etc41 bo? 9 its fluid =m & also :5 made solid by ! addition ( gelat9e or ag>4 ,! wort is prep>$ z foll[s3 #a4 ,weigh out #bej grammes cru%$ malt & place 9 a #b-litre flask4 #b4 ,add #ajjj c4c4 di/ill$ wat]1 heat$ to #gj deg4 ,c41 & close ! flask ) a rubb] /opp]4 #c4 ,place ! flask 9 a wat]-ba? regulat$ to #fj deg4 ,c4 & all[ ! mac]ation to cont9ue = "o h\r4 #d4 ,/ra9 ?r\gh butt] musl9 into a clean flask & heat 9 ! /eam] = ?irty m9utes4 #e4 ,filt] ?r\gh ,sw$i% filt] pap]4 #f4 ,tube 9 quantities ( #aj c4c4 or /ore 9 flasks4 #g4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree consecutive days4 ,! natural reaction ( ! wort %\ld n be int]f]$ )4 ,,note4--,x is sometimes m conv5i5t to obta9 unhopp$#f be] wort direct f ! brew]y4 ,9 ?is case x is dilut$ ) an equal quantity ( di/ill$ wat]1 /eam$ = an h\r1 filt]$1 fill$ into /]ile flasks or tubes1 & /]ilis$ by ! discont9u\s me?od4 ,wort ,gelat9e4-- #a4 ,measure out wort 7prep>$ z above71 #ijj c4c41 into a /]ile flask4 #b4 ,weigh out gelat9e1 #ajj grammes 7 #aj p] c5t471 & add x to ! wort 9 ! flask4 #c4 ,bubble live /eam ?r\gh ! mixture = t5 m9utes1 to dissolve ! gelat9e4 #d4 ,cool to #fj deg4 ,c42 cl>ify ) egg z = nutri5t gelat9e 7vide page #afd74 #e4 ,filt] ?r\gh papi] ,*>d94 #f4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,wort ,ag>4-- #a4 ,measure out wort 7z above71 #gjj c4c41 into a /]ile flask4 #b4 ,weigh out p[d]$ ag>1 #bj grammes2 mix into a smoo? pa/e ) #bjj c4c4 ( cold wort & add to ! wort 9 ! flask4 #c4 ,bubble live /eam ?r\gh ! mixture = tw5ty m9utes1 to dissolve ! ag>4 #d4 ,cool to #fj deg4 ,c42 cl>ify ) egg z = nutri5t ag> 7vide page #afg74 #e4 ,filt] ?r\gh papi] ,*>d91 us+ ! hot-wat] funnel4 #f4 ,tube1 & /]ilise z = nutri5t ag>4 ,peptone ,wat] 7,dunham74-- #a4 ,weigh out ,witte's peptone1 #aj grammes1 & salt1 #e grammes1 & emulsify ) ab\t #bej c4c4 ( di/ill$ wat] previ\sly heat$ to #fj deg4 ,c4 #b4 ,p\r ! emulsion into a litre flask & make up to #ajjj c4c4 by ! addition ( di/ill$ wat]4 #c4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes4 #d4 ,filt] ?r\gh ,sw$i% filt] pap]4 #e4 ,tube 9 quantities ( #aj c4c4 ea*4 #f4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree consecutive days4 ,sug> or ,c>bohydrate ,m$ia4-- ,=m]ly ! ability ( bact]ia to induce hydrolytic *anges 9 c>bohydrate sub/ances 0 obs]v$ only 9 connection ) a few well-def9$ sug>s1 b ( rec5t ye>s x has be5 %[n t :5 us+ litmus z an indicator ^! s-call$ f]m5tation reactions facilitate ! diff]5tiation ( closely alli$ species1 & ! li/ ( sub/ances employ$ 9 ?is connection has be5 consid]ably ext5d$4 ,! m$ia prep>$ ) !m are n[ no long] reg>d$ z special1 b are compris$ 9 ! /ock m$ia ( ! laboratory4 ,! *ief ( ^! sub/ances are ! foll[+1 arrang$ 9 accordance ) _! *emical con/itution3 ,monosac*>ides ,dextrose 7glucose71 laevulose1 galactose1 mannose1 arab9ose1 xylose4 ,disac*>ides ,maltose1 lactose1 sac*>ose4 ,trisac*>ides ,raff9ose 7mellitose74 ,polysac*>ides ,dextr91 inul91 />*1 glycog51 amidon4 ,glucosides ,amygdal91 conif]91 salic91 helic91 phlorrhiz94 ,polyatomic alcohols ,trihydric1 ,glyc]94 ,tetrahydric1 ,ery?rite4 ,p5tahydric1 ,adonite4 ,hexahydric1 ,dulcite1 7dulcitol or melampirite71 isodulcite 7rhamnose71 mannite 7mannitol71 sorbite 7sorbitol71 inosite4 ,^! sub/ances %\ld be obta9$ f ,kahlbaum 7( ,b]l972 9 ! pure =m1 & :5 possible z l>ge cry/als1 & ! me?od ( prep>+ a m$ium conta9+ ei!r ( !m may be exemplifi$ by describ+ ,dextrose ,solution4 ,dextrose ,solution4-- #a4 ,weigh out ,peptone #bj grammes ,glucose #aj grammes & gr9d toge!r 9 a mort>2 !n emulsify 9 #ajj c4c4 ( di/ill$ wat] heat$ to #fj deg4 ,c4 #b4 ,place 9 a flask & add ,di/ill$ wat] #hej c4c4 #c4 ,/eam 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes to dissolve ! peptone & glucose4 #d4 ,add ,kubel-,tiemann litmus solution 7,kahlbaum7 #ej c4c4 7,! sub/ances enum]at$ above react acid to ph5olph?ale91 b v>i\sly t[>d ! neutral litmus solution4 ,to su* z react acid1 add v cauti\sly n_/#a sodium hydrate solution to ! m$ium 9 bulk until ! neutral t9t has return$74 #e4 ,fill into tubes 9 :i* h previ\sly be5 plac$ ! inv]t$ ,durham's gas tubes4 #f4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree successive days4 ,,note4--,on no acc\nt %\ld ^! m$ia be /]ilis$ 9 ! autoclave1 z temp]atures above #ajj deg4 ,c4 !mselves induce hydrolytic *anges 9 ! sub/ances 9 "q4 ,x is equally important t ! tw5ty m9utes %\ld n be exce$$ 9 /]ilisation1 z neglect ( ?is precaution may discol\r ! litmus or lead to ! production ( yell[i% t9ts :5 ! tubes are subsequ5tly inoculat$ ) acid-=m+ bact]ia4 ,neutral ,litmus ,solution4 ,! mo/ satisfactory is ! ,kubel-,tiemann1 prep>$ by ,kahlbaum4 ,x c h[ev] be made 9 ! laboratory z foll[s3 #a4 ,weigh out ,comm]cial litmus #ej grammes1 & place 9 a well /opp]$ #ejj c4c4 bottle2 measure out & add #cjj c4c4 alcohol #ie p] c5t4 #b4 ,%ake well at lea/ once a day = sev5 days--! alcohol acquires a gre5 col\r4 #c4 ,decant (f ! gre5 alcohol & fill a fur!r #cjj c4c4 #ie p] c5t4 alcohol into ! bottle & repeat ! %ak+4 #d4 ,repeat ?is process until on add+ fre% alcohol ! fluid only becomes t+$ ) violet4 #e4 ,p\r (f ! alcohol1 leav+ ! litmus z dry z possible4 ,connect up ! bottle to an air pump & evaporate (f ! la/ traces ( alcohol4 #f4 ,transf] ! dry litmus to a litre flask1 measure 9 #fjj c4c4 di/ill$ wat] & all[ to rema9 9 contact #bd h\rs ) frequ5t %ak+s4 #g4 ,filt] ! solution into a clean flask & add "o or two drops ( pure conc5trat$ sulphuric acid until ! litmus solution is di/9ctly w9e-r$ 9 col\r4 #h4 ,add excess ( pure solid b>yta & all[ to /& until ! reaction is aga9 alkal9e4 #i4 ,filt]4 #aj4 ,bubble ,,co#b ?r\gh ! solution until reaction is def9itely acid4 #aa4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes on ea* ( ?ree consecutive days4 ,?is /]ilises ! solution & also drives (f ! c>bon dioxide1 leav+ ! solution neutral4 ,m$ia = ana]obic cultures4 ,9 addition to ! =ego+ m$ia1 all ( :i* c be1 & are employ$ 9 ! cultivation ( ana]obic bact]ia1 c]ta9 special m$ia conta9+ readily oxidis$ sub/ances are commonly us$ = ?is purpose4 ,! pr9cipal ( ^! are z foll[s3 ,bile ,salt ,bro? 7,mac,conkey74-- #a4 ,weigh out ,witte's peptone1 #bj grammes 7 #b p] c5t471 & emulsify ) #bjj c4c4 di/ill$ wat] previ\sly w>m$ to #fj deg4 ,c4 #b4 ,weigh out sodium tauro*olate 7comm]cial71 #e grammes 7 #j4e p] c5t471 & glucose1 #e grammes 7 #j4e p] c5t471 & dissolve 9 ! peptone emulsion4 #c4 ,wa% ! peptone emulsion into a flask ) #hjj c4c4 di/ill$ wat]1 & heat 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes4 #d4 ,filt] ?r\gh ,sw$i% filt] pap] into a /]ile flask4 #e4 ,add /]ile litmus solution suffici5t to col\r ! m$ium to a deep purple1 usually #ac p] c5t4 requir$4 #f4 ,fill1 9 quantities ( #aj c4c41 into tubes conta9+ small gas tubes 7vide ,fig4 #ajd1 page #afa74 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree consecutive days4 ,glucose ,=mate ,b\illon 7,kitasato74-- #a4 ,measure out nutri5t b\illon1 #ajjj c4c4 7vide page #afc1 sections #a to #f74 #b4 ,weigh out glucose1 #bj grammes 7 #b p] c5t471 sodium =mate1 #d grammes 7 #j4d p] c5t471 & dissolve 9 ! fluid4 #c4 ,tube1 & /]ilise z = b\illon4 ,glucose ,=mate ,gelat9e 7,kitasato74-- #a4 ,prep>e nutri5t gelat9e 7vide page #afd1 sections #a to #g7 & measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b p] c5t471 & sodium =mate1 #d grammes 7 #j4d p] c5t471 & dissolve 9 ! hot gelat9e4 #c4 ,filt] ?r\gh papi] ,*>d94 #d4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,glucose ,=mate ,ag> 7,kitasato74-- #a4 ,prep>e nutri5t ag> 7vide page #afg1 sections #a to #h74 ,measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b p] c5t471 sodium =mate1 #d grammes 7 #j4d p] c5t471 & dissolve 9 ! ag>4 #c4 ,tube1 & /]ilise z = nutri5t ag>4 ,sulph9digotate ,b\illon 7,weyl74-- #a4 ,measure out nutri5t b\illon 7vide page #afc1 sections #a to #f #ajjj c4c474 #b4 ,weigh out glucose1 #bj grammes 7 #b p] c5t471 sodium sulph9digotate1 #a gramme 7 #j4a p] c5t471 & dissolve 9 ! fluid4 #c4 ,tube1 & /]ilise z = b\illon4 ,sulph9digotate ,gelat9e 7,weyl74-- #a4 ,prep>e nutri5t gelat9e 7vide page #afd1 sections #a to #g74 ,measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b p] c5t471 & sodium sulph9digotate1 #a gramme 7 #j4a p] c5t471 & dissolve 9 ! hot gelat9e4 #c4 ,filt] ?r\gh papi] ,*>d94 #d4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,sulph9digotate ,ag>4-- #a4 ,prep>e nutri5t ag> 7vide page #afg1 sections #a to #h74 ,measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b p] c5t471 sodium sulph9digotate1 #a gramme 7 #j4a p] c5t471 & dissolve 9 ! hot ag>4 #c4 ,tube1 & /]ilise z = nutri5t ag>4 ,,note4--,! ,sulph9digotate m$ia are ( a blue col\r1 :i* dur+ ! gr[? ( ana]obic bact]ia is oxidis$ & decol\ris$ to a light yell[4 ,,footnotes3 #d ,?is figure is obta9$ by add+ toge!r #a litre wat]1 #ajjj grammes2 #aj p] c5t4 gelat9e1 #ajj grammes2 #a p] c5t4 peptone1 #aj grammes2 #j4e p] c5t4 salt1 #e grammes2 total1 #aaae grammes4 ,modifications ( ! above process1 z to quantities & p]c5tages1 w require correspond+ alt]ations ( ! figures4 ,! av]age weight ( a measur$ litre ( #aj p] c5t4 nutri5t gelat9e :5 prep>$ 9 ?is way aft] filtration is #ajhj grammes4 #e ,?is figure is obta9$ by add+ toge!r #a litre ( wat] 7meat extract71 #ajjj grammes2 #b p] c5t4 ag>1 #bj grammes2 #a p] c5t4 peptone1 #aj grammes2 #j4e p] c5t4 salt1 #e grammes--total #ajce grammes4 ,modifications ( ! process z to quantities or p]c5tages w necessitate correspond+ alt]ations 9 ! calculat$ m$ium figure4 ,! av]age weight ( a measur$ litre ( #b p] c5t4 ag> :5 prep>$ 9 ?is way1 aft] filtration1 is #ajaj4e grammes4 #f ,hopp$ wort ex]ts a toxic effect ^u _m bact]ia1 includ+ ! lactic acid bact]ia4 ,,xii4 ,,special ,,m$ia4 ,9 ?is *apt] are collect$ a numb] ( m$ia :i* h be5 elaborat$ by v>i\s work]s = special purposes1 gr\p$ toge!r "u head+s :i* indicate _! *ief utility4 ,9 _m in/ances ! "n ( ! orig9ator ( ! m$ium is giv51 b )\t ref]5ce to 8 orig9al in/ructions1 s9ce ^! are 9 _m cases inadequate to ! requirem5ts ( ! isolat$ work]1 :o w\ld probably fail to reproduce ! m$ium 9 a =m giv+ ! results attribut$ to x by its au?or4 ,su* modifications h !re=e be5 introduc$ z make = uni=mity betwe5 ! diff]5t bat*es ( m$ia4 ,a consid]able numb] ( col\r$ m$ia1 *iefly int5d$ = "w ) inte/9al bact]ia1 h be5 includ$2 b beyond ! fact t ! au?or's modification ( ! ,drigalski-,conradi m$ium has be5 includ$ among/ ! r\t9e m$ia ( ! laboratory1 no comm5t has be5 made ^u _! relative values1 s9ce only by obs]vation & practice c ! skill necess>y to utilise _! full value be acquir$4 ,! in/ructions z to /]ilisation are r>ely giv5 9 full2 ! r\t9e me?od ( exposure 9 ! /eam /]ilis] at #ajj deg4 ,c4 7)\t pressure7 = tw5ty m9utes on ea* ( ?ree successive days = all fluid m$ia1 & ?irty m9utes on ea* ( ?ree successive days = all liquefiable or solid m$ia mu/ be c>ri$ out2 & only :5 ^! g5]al rules are to be dep>t$ f are fur!r details giv54 ,m$ia = ! ,/udy ( ! ,*emical ,composition ( ,bact]ia4 ,asp>ag9 ,m$ium 7,us*9sky74-- #a4 ,weigh out & mix ,asp>ag9 #c4d grammes ,ammonium lactate #aj4j grammes ,sodium *loride #e4j grammes ,magnesium sulphate #j4b gramme ,calcium *loride #j4a gramme ,acid potassium phosphate 7,,kh#b,,po#d7 #a4j gramme #b4 ,dissolve ! mixture 9 di/ill$ wat] #ajjj c4c4 #c4 ,add glyc]9e1 #dj c4c4 #d4 ,tube1 & /]ilise z = nutri5t b\illon4 ,asp>ag9 ,m$ium 7,frankel & ,voges74-- #a4 ,weigh out & mix ,asp>ag9 #d grammes ,sodium phosphate1 7,na#b;,,hpo#d7 #ab,,oh #b grammes ,ammonium lactate #f grammes ,sodium *loride #e grammes & dissolve 9 ,di/ill$ wat] #ajjj c4c4 #b4 ,tube1 & /]ilise z = nutri5t b\illon4 ,,note4--,ei!r ( ! above asp>ag9 m$ia1 aft] ! addition ( #aj p] c5t4 gelat9e or #a4e p] c5t4 ag>1 may be advantage\sly employ$ 9 ! solid condition4 ,proteid ,free ,bro? 7,us*9sky74-- #a4 ,weigh out & mix ,calcium *loride #j4a gramme ,magnesium sulphate #j4b gramme ,acid potassium phosphate 7,,kh#b,,po#d7 #b4j grammes ,potassium asp>tate #c4j grammes ,sodium *loride #e4j grammes ,ammonium lactate #f4j grammes #b4 ,dissolve ! mixture 9 di/ill$ wat] #ajjj c4c4 #c4 ,add glyc]9e #cj c4c4 #d4 ,tube & /]ilise z = nutri5t bro?4 ,m$ia = ! ,/udy ( ,bio*emical ,reaction4 ,inosite-free ,m$ia--,b\illon 7,durham74-- #a4 ,prep>e meat extract1 #ajjj c4c4 7vide page #adh71 f bullock's he>t :i* has be5 hung = a c\ple ( days4 #b4 ,prep>e nutri5t b\illon 7#aj71 #ajjj c4c4 7vide1 page #afa71 f ! meat extract1 & /ore 9 #a-litre flask4 #c4 ,inoculate ! b\illon f a pure cultivation ( ! ,b4 lactis a]og5es1 & incubate at #cg deg4 ,c4 = =ty-eight h\rs4 #d4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes to de/roy ! bacilli & "s ( _! products4 #e4 ,e/imate ! reaction ( ! m$ium & if necess>y re/ore to #aj4 #f4 ,inoculate ! b\illon f a pure cultivation ( ! ,b4 coli communis & incubate at #cg deg4 ,c4 = =ty-eight h\rs4 #g4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes4 ,n[ fill two f]m5tation tubes ) ! b\illon1 t9t ) litmus solution1 & /]ilise2 inoculate ) ,b4 lactis a]og5es4 ,if no acid or gas is =m$1 ! b\illon is 9 a sug>-free condition2 b if acid or gas is pres5t1 aga9 make ! b\illon 9 ! flask #aj1 re9oculate ) "o or o!r ( ! above-m5tion$ bact]ia1 & incubate2 !n te/ aga94 ,repeat ?is till nei!r acid nor gas appe>s 9 ! m$ium :5 us$ = ! cultivation ( ei!r ( ! bacilli ref]r$ to above4 #h4 ,aft] ! f9al heat+1 /& ! flask 9 a cool place & all[ ! gr[? to s$im5t4 ,filt] ! sup]natant bro? ?r\gh ,sw$i% filt] pap]4 ,if ! filtrate is cl\dy1 filt] ?r\gh a porcela9 filt] c&le4 #i4 ,tube1 & /]ilise z = b\illon4 ,b\illon prep>$ 9 ! above-describ$ mann] w prove to be absolutely sug>-free2 & f x may be prep>$ nutri5t sug>-free gelat9e or ag>1 by dissolv+ 9 x ! requir$ p]c5tage ( gelat9e or ag> respectively & complet+ ! m$ium accord+ to directions giv5 on pages #aff & #afg4 ,! mo/ important application ( inosite-free b\illon is its use 9 ! prep>ation ( sug> b\illons1 :e!r glucose1 maltose1 lactose1 or sac*>ose1 ( exact p]c5tage composition4 ,sug> 7,dextrose7 ,b\illon4-- #a4 ,measure out nutri5t b\illon1 #ajjj c4c4 7vide page #afc1 sections #a to #f7 or sug>-free b\illon 7vide supra74 #b4 ,weigh out glucose 7anhydr\s71 #bj grammes 7 #b p] c5t471 & dissolve 9 ! fluid4 #c4 ,tube1 & /]ilise z = b\illon4 ,ord9>y comm]cial glucose s]ves ! purpose equally well1 b is n recomm5d$1 z dur+ ! process ( /]ilisation x causes ! m$ium to gradually deep5 9 col\r4 ,,note4--,9 c]ta9 cases a correspond+ p]c5tage ( lactose1 maltose1 or sac*>ose is sub/itut$ = glucose4 ,sug> ,gelat9e4-- #a4 ,prep>e nutri5t gelat9e 7vide page #afd1 sections #a to #g74 ,measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b p] c5t471 & dissolve 9 ! hot gelat9e4 #c4 ,filt] ?r\gh papi] ,*>d94 #d4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,sug> ,ag>4-- #a4 ,prep>e nutri5t ag> 7vide page #afg1 sections #a to #h74 ,measure out #ajjj c4c4 #b4 ,weigh out glucose1 #bj grammes 7 #b p] c5t471 & dissolve 9 ! cle> ag>4 #c4 ,tube1 & /]ilise z = nutri5t ag>4 ,,note4--,o!r sug> m$ia are prep>$ by sub/itut+ a correspond+ p]c5tage ( lactose1 maltose 7or any o!r ( ! sub/ances ref]r$ to "u ,sug> ,m$ia1 page #agg7 = ! glucose4 ,iron ,b\illon4-- #a4 ,measure out nutri5t b\illon1 #ajjj c4c4 7vide page #ada1 sections #a to #f74 #b4 ,weigh out f]ric t>trate1 #a gramme 7 #j4a p] c5t471 & dissolve x 9 ! b\illon4 #c4 ,tube1 & /]ilise z = b\illon4 ,,note4--,! lactate ( iron may be sub/itut$ = ! t>trate4 ,lead ,b\illon4-- #a4 ,measure out nutri5t b\illon1 #ajjj c4c4 7vide page #afc1 sections #a to #f74 #b4 ,weigh out lead acetate1 #a gramme 7 #j4a p] c5t471 & dissolve x 9 ! b\illon4 #c4 ,tube1 & /]ilise z = b\illon4 ,nitrate ,b\illon4-- #a4 ,measure out nutri5t b\illon1 #ajjj c4c4 7vide page #afc1 sections #a to #f74 #b4 ,weigh out potassium nitrate1 #e grammes 7 #j4e p] c5t471 & dissolve x 9 ! b\illon4 #c4 ,tube1 & /]ilise z = b\illon4 ,,note4--,! nitrate ( sodium or ammonium may be sub/itut$ = t ( potassium1 or ! salt may be add$ 9 ! proportion ( f #j4a to #a p] c5t4 to meet special requirem5ts4 ,iron ,peptone ,solution 7,pakes74-- #a4 ,weigh out peptone1 #cj grammes1 & emulsify x ) #bjj c4c4 tap wat]1 previ\sly heat$ to ab\t #fj deg4 ,c4 #b4 ,wa% ! emulsion into a litre flask ) #hjj c4c4 tap wat]4 #c4 ,weigh out salt1 #e grammes1 & sodium phosphate1 #c grammes1 & dissolve 9 ! mixture 9 ! flask4 #d4 ,heat ! mixture 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes1 to complete ! solution ( ! peptone1 & filt] into a clean flask4 #e4 ,fill into tubes 9 quantities ( #aj c4c4 ea*4 #f4 ,add to ea* tube #j4a c4c4 ( a #b p] c5t4 neutral solution ( f]ric t>trate4 7,a yell[i%-:ite precipitate =ms47 #g4 ,/]ilise z = nutri5t b\illon4 ,lead ,peptone ,solution4-- ,prep>e z = iron peptone solution b 9 /ep #f sub/itute #j4a c4c4 ( a #a p] c5t4 neutral aque\s solution ( lead acetate4 ,nitrate ,peptone ,solution 7,pakes74-- #a4 ,weigh out ,witte's peptone1 #aj grammes1 & emulsify x ) #bjj c4c4 ammonia-free di/ill$ wat] previ\sly heat$ to #fj deg4 ,c4 #b4 ,wa% ! emulsion into a flask & make up to #ajjj c4c41 ) ammonia-free di/ill$ wat]4 #c4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes4 #d4 ,weigh out sodium nitrate1 #a gramme1 & dissolve 9 ! cont5ts ( ! flask4 #e4 ,filt] ?r\gh ,sw$i% filt] pap]4 #f4 ,tube1 & /]ilise z = nutri5t b\illon4 ,litmus ,b\illon4-- #a4 ,measure out nutri5t b\illon1 #ajjj c4c4 7vide page #afc1 sections #a to #f74 #b4 ,add suffici5t /]ile litmus solution to t9t ! m$ium a d>k lav5d] col\r4 7,m$ia r5d]$ #aj w usually react v fa9tly alkal9e or occasionally neutral to litmus47 #c4 ,tube1 & /]ilise z = b\illon4 ,rosolic ,acid ,peptone ,solution4-- #a4 ,weigh out rosolic acid 7corall971 #j4e gramme1 & dissolve x 9 #hj p] c5t4 alcohol1 #ajj c4c4 ,keep ?is z a /ock solution4 #b4 ,measure out peptone wat] 7,dunham71 #ajj c4c41 & rosolic acid solution1 #b c4c41 & mix4 #c4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes4 #d4 ,filt] ?r\gh ,sw$i% filt] pap]4 #e4 ,tube1 & /]ilise z = nutri5t b\illon4 ,capaldi-,proskau] ,m$ium1 ,no4 ,i4-- #a4 ,weigh out & mix ,sodium *loride #b4j grammes ,magnesium sulphate #j4a gramme ,calcium *loride #j4b gramme ,monopotassium phosphate #b4j grammes #b4 ,dissolve 9 wat] #ajjj c4c4 9 a #b-litre flask #c4 ,weigh out & mix ,asp>ag9 #b grammes ,mannite #b grammes & add to cont5ts ( flask4 #d4 ,measure out #be c4c4 ( ! solution & titrate x aga9/ dec9ormal sodic hydrate1 us+ litmus z ! indicator4 ,control ! result & e/imate ! am\nt ( sodic hydrate necess>y to be add$ to r5d] ! rema9d] ( ! solution neutral to litmus4 ,add ?is quantity ( sodic hydrate4 #e4 ,filt]4 #f4 ,add litmus solution #dg4e c4c4 7 #e p] c5t474 #g4 ,tube1 & /]ilise z = nutri5t b\illon4 ,capaldi-,proskau] ,m$ium ,no4 ,,ii4-- #a4 ,weigh out & mix ,peptone #bj grammes ,mannite #a gramme #b4 ,dissolve 9 wat] #ajjj c4c4 9 a #b-litre flask4 #c4 ,neutralise to litmus z 9 ,no4 ,i 7vide supra1 ,/ep #d74 #d4 ,filt]4 #e4 ,add litmus solution #dg4e c4c4 7 #e p] c5t474 #f4 ,tube1 & /]ilise z = nutri5t b\illon4 ,ur9e ,m$ia4 ,b\illon4-- #a4 ,collect fre%ly pass$ ur9e 9 /]ile flask4 #b4 ,place ! flask 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes4 #c4 ,filt] ?r\gh two ?icknesses ( ,sw$i% filt] pap]4 #d4 ,tube1 & /]ilise z = nutri5t b\illon4 7,leave ! reaction unalt]$47 ,ur9e ,gelat9e4-- #a4 ,collect fre%ly pass$ ur9e 9 /]ile flask4 #b4 ,take ! specific gravity1 &1 if above #ajaj1 dilute ) /]ile wat] until t gravity is rea*$4 #c4 ,e/imate 7) control7 at ! boil+-po9t1 & note ! reaction ( ! ur9e4 #d4 ,weigh out gelat9e1 #aj p] c5t41 & add to ! ur9e 9 ! flask4 #e4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = "o h\r to dissolve ! gelat9e4 #f4 ,e/imate ! reaction & add suffici5t cau/ic soda solution to re/ore ! reaction ( ! m$ium mass to ! equival5t ( ! orig9al ur9e4 #g4 ,cool to #fj deg4 ,c4 & cl>ify ) egg z = nutri5t gelat9e 7vide page #aff74 #h4 ,filt] ?r\gh papi] ,*>d94 #i4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,ur9e ,gelat9e 7,hell]74-- #a4 ,collect fre%ly pass$ ur9e 9 /]ile flask4 #b4 ,filt] ?r\gh animal *>coal to remove "p ( ! col\r+ matt]4 #c4 ,take ! specific gravity1 & if above #ajaj1 dilute ) /]ile wat] till ?is gravity is rea*$4 #d4 ,add ,witte's peptone1 #a p] c5t42 salt1 #j4e p] c5t42 gelat9e1 #aj p] c5t4 #e4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = "o h\r1 to dissolve ! gelat9e1 etc4 #f4 ,add normal cau/ic soda solution 9 successive small quantities1 & te/ ! reaction f "t to "t ) litmus pap]1 until ! fluid reacts fa9tly alkal9e4 #g4 ,cool to #fj deg4 ,c4 & cl>ify ) egg z = nutri5t gelat9e 7vide page #aff74 #h4 ,filt] ?r\gh papi] ,*>d94 #i4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,ur9e ,ag>4-- #a4 ,collect fre%ly pass$ ur9e 9 /]ile flask4 #b4 ,take ! specific gravity & if above #ajaj1 dilute ) /]ile wat] till ?is gravity is rea*$4 #c4 ,weigh out #a4e p] c5t4 or #b p] c5t4 p[d]$ ag>1 & add x to ! ur9e4 #d4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = n9ety m9utes to dissolve ! ag>4 #e4 ,cool to #fj deg4 ,c4 & cl>ify ) egg z = nutri5t ag> 7vide page #afh74 #f4 ,filt] ?r\gh papi] ,*>d91 us+ ! hot-wat] funnel4 #g4 ,tube1 & /]ilise z = nutri5t ag>4 7,leave ! reaction unalt]$47 ,s]um ,sug> ,m$ia 7,hiss74-- ,9 ^! m$ia ! f]m5tation ( c>bohydrate sub/ance by bact]ial action is indicat$ by ! coagulation ( ! s]um proteids 9 addition to ! production ( an acid reaction4 ,s]um ,dextrose ,wat] 7,hiss74-- #a4 ,measure out into a litre flask ,s]um wat] 7,see page #agj7 #ajjj c4c4 #b4 ,weigh out ,dextrose #aj grammes & dissolve 9 ! s]um wat]4 #c4 ,filt] ?r\gh ,sw$i% filt] pap]4 #d4 ,measure out & add to ! m$ium ,litmus solution 7,kahlbaum7 #ej c4c4 #e4 ,tube 9 quantities ( #aj c4c4 & /]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree successive days4 ,laevulose1 galactose1 maltose1 lactose1 etc41 c be sub/itut$ 9 simil> am\nts = dextrose & ! m$ium complet$ z above4 ,omeliansky's ,nutri5t ,fluid 7,= ,cellulose ,f]m5t]s74-- #a4 ,weigh out & mix ,potassium phosphate #d4j grammes ,magnesium sulphate #b4j grammes ,ammonium sulphate #d4j grammes ,sodium *loride #j4be gramme #b4 ,dissolve 9 di/ill$ wat] #djjj c4c4 #c4 ,flask 9 quantities ( #bej c4c4 #d4 ,weigh out & add #e grammes precipitat$ *alk to ea* flask4 #e4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree successive days4 ,m$ia = ! ,/udy ( ,*romog5ic ,bact]ia4 ,milk ,rice 7,eis5b]g74-- #a4 ,measure out nutri5t b\illon1 #gj c4c41 & milk1 #baj c4c41 & mix ?or\ghly4 #b4 ,weigh out rice p[d]1 #ajj grammes1 & rub x up 9 a mort> ) ! milk & bro? mixture4 #c4 ,fill ! pa/e into /]ile capsules1 spread+ x out s z to =m a lay] ab\t #j4e cm4 ?ick1 ov] ! bottom ( ea*4 #d4 ,heat ov] a wat]-ba? at #ajj deg4 ,c4 until ! mixture solidifies4 #e4 ,replace ! lids ( ! capsules4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes on ea* ( ?ree consecutive days4 7,a solid m$ium ( ! col\r ( cafe au lait is ?us produc$47 ,milk ,rice 7,soyka74-- #a4 ,measure out nutri5t b\illon1 #ej c4c41 & milk1 #aej c4c41 & mix ?or\ghly4 #b4 ,weigh out rice p[d]1 #ajj grammes1 & rub x up 9 a mort> ) ! milk & bro? mixture4 #c4 ,fill ! pa/e into /]ile capsules1 to =m a lay] ov] ! bottom ( ea*4 #d4 ,replace ! lids ( ! capsules4 #e4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes on ea* ( ?ree consecutive days4 7,a pure :ite1 opaque m$ium is ?us =m$47 ,m$ia = ! ,/udy ( ,phosphoresc5t & ,photog5ic ,bact]ia4 ,fi% ,b\illon4-- #a4 ,weigh out h]r+1 mack]el1 or cod1 #ejj grammes1 & place 9 a l>ge porcela9 beak] 7or enamell$ iron pot74 #b4 ,weigh out sodium *loride1 #bf4e grammes2 potassium *loride1 #j4ge gramme2 magnesium *loride1 #c4be grammes2 & dissolve 9 #ejj c4c4 di/ill$ wat]4 ,add ! solution to ! fi% 9 ! beak]4 #c4 ,place ! beak] 9 a wat]-ba? & proce$ z 9 prep>+ meat extract--i4 e41 heat g5tly at #dj deg4 ,c4 = tw5ty m9utes1 !n rapidly raise ! temp]ature to1 & ma9ta9 at1 ! boil+-po9t = t5 m9utes4 #d4 ,/ra9 ! mixture ?r\gh butt] musl9 into a clean flask4 #e4 ,weigh out peptone1 #e grammes1 & emulsify ) ab\t #bjj c4c4 ( ! hot fi% wat]2 incorporate ?or\ghly ) ! rema9d] ( ! fi% wat] 9 ! flask4 #f4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes to complete ! solution ( ! peptone4 #g4 ,filt] ?r\gh ,sw$i% filt] pap]4 #h4 ,:5 ! fi% b\illon is cold1 if x is to be us$ z fluid m$ium1 make up to #ajjj c4c4 by ! addition ( di/ill$ wat]4 ,if1 h[ev]1 x is to be us$ z ! basis = ag> or gelat9e m$ia /ore x 9 ! ,d\ble ,/r5g? condition4 #i4 ,tube & /]ilise z = nutri5t b\illon4 ,z an alt]native me?od ,m>vis fi% food 7#af grammes7 may be sub/itut$ = ! #ejj grammes ( fre% fi%4 ,fi% ,gelat9e4-- #a4 ,measure out d\ble /r5g? fi% b\illon1 #ejj c4c41 into a t>$ #b-litre flask4 #b4 ,add %eet gelat9e1 #ajj grammes1 cut into small pieces4 #c4 ,bubble live /eam ?r\gh ! mixture = fifte5 m9utes to dissolve ! gelat9e4 #d4 ,weigh ! flask & its cont5ts2 adju/ ! weight to ! calculat$ figure = "o litre ( m$ium 7#aace4e grammes7 by ! addition ( di/ill$ wat] at #ajj deg4 ,c4 7vide page #aff74 #e4 ,cool to bel[ #fj deg4 ,c41 & cl>ify ) egg4 #f4 ,filt] ?r\gh papi] ,*>d94 #g4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,%ake well aft] ! f9al /]ilisation1 to a]ate ! m$ium4 ,fi% ,gelat9e-,ag>4-- #a4 ,weigh out p[d]$ ag>1 #e grammes1 & emulsify x ) #bjj c4c4 d\ble /r5g? fi% b\illon4 #b4 ,wa% ! emulsion into a t>$ #b-litre flask ) #cjj c4c4 fi% b\illon4 #c4 ,weigh out %eet gelat9e1 #gj grammes1 cut x into small pieces & add x to ! cont5ts ( ! flask4 #d4 ,bubble live /eam ?r\gh ! mixture to dissolve ! gelat9e & ag>4 #e4 ,weigh ! flask & cont5ts4 ,adju/ ! weight to ! calculat$ figure = "o litre ( m$ium 7#aaaj4e grammes7 by ! addition ( di/ill$ wat] at #ajj deg4 ,c4 7vide page #aff74 #f4 ,cool to bel[ #fj deg4 ,c4 & cl>ify ) egg4 #g4 ,filt] ?r\gh papi] ,*>d94 #h4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,%ake well aft] ! f9al /]ilisation1 to a]ate ! m$ium4 ,m$ia = ! ,/udy ( ,yea/s & ,m\lds4 ,pa/eur's ,solution4-- 7,reaction alkal9e74 #a4 ,weigh out & mix ! a% f #aj grammes ( yea/2 ammonium t>trate1 #aj grammes2 cane sug>1 #ajj grammes4 #b4 ,dissolve ! mixture 9 di/ill$ wat]1 #ajjj c4c4 #c4 ,tube or flask1 & /]ilise z = nutri5t b\illon4 ,yea/ ,wat] 7,pa/eur74-- #a4 ,weigh out press$ yea/1 #ge grammes2 place 9 a #b-litre flask & add #ajjj c4c4 di/ill$ wat]4 #b4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes4 #c4 ,filt] ?r\gh papi] ,*>d94 #d4 ,tube or flask1 & /]ilise z = nutri5t b\illon4 ,cohn's ,solution4-- #a4 ,weigh out & mix ,acid potassium phosphate 7,,kh#b,,po#d7 #e4j grammes ,calcium phosphate #j4e gramme ,magnesium sulphate #e4j grammes ,ammonium t>trate #aj4j grammes & dissolve 9 ,di/ill$ wat] #ajjj c4c4 #b4 ,tube1 or flask & /]ilise z = nutri5t b\illon4 ,naegeli's ,solution4-- #a4 ,weigh out & mix ,dibasic potassium phosphate 7,k#b;,,hpo#d7 #a4j gramme ,magnesium sulphate #j4b gramme ,calcium *loride #j4a gramme ,ammonium t>trate #aj4j grammes & dissolve 9 ,di/ill$ wat] #ajjj c4c4 #b4 ,tube or flask2 /]ilise z = nutri5t b\illon4 ,pla/]-(-,p>is ,discs4-- #a4 ,take l>ge corks1 #b4e cm4 diamet]1 & roll a piece ( /iff note-pap] r\nd ea*1 s t ab\t a c5timetre projects z a ridge above ! upp] surface ( ! cork1 & secure 9 position ) a p9 7,fig4 #aab74 #b4 ,mix pla/]-(-,p>is into a /iff pa/e ) di/ill$ wat]1 & fill ea* ( ! cork m\lds ) ! pa/e4 #c4 ,:5 ! pla/] has set1 remove ! pap] f ! corks1 & raise ! pla/] discs4 #d4 ,place ! pla/] discs on a piece ( asbe/os bo>d & /]ilise by expos+ 9 ! hot-air ov5 to #aej deg4 ,c4 = half an h\r4 ,illu/ration3 ,fig4 #aab4--;,cork & pap] m\ld = pla/]-(-,p>is disc4 #e4 ,remove ! /]ile discs f ! ov5 by means ( /]ile =ceps1 place ea* inside a /]ile capsule1 & moi/5 ) a little /]ile wat]4 #f4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes on ea* ( ?ree consecutive days4 ,gypsum ,blocks 7,engel & ,hans574-- ,^! are 9 ! =m ( truncat$ cones & = _! prep>ation small t9 m\lds are requir$1 ea* hav+ a diamet] ( #e4e cm4 at ! base & #d cm4 at ! truncat$ apex4 ,! height 7or dep?7 ( a m\ld is #d4e to #e cm4 #a4 ,mix p[d]$ calc9$ gypsum into a /iff pa/e ) di/ill$ wat]4 #b4 ,fill ! pa/e into ! m\lds & all[ x to set & dry by exposure to air4 #c4 ,remove ! block f ! m\ld & transf] x to a d\ble glass di% ( adequate size 7#g cm4 diamet] x #g cm4 high74 #d4 ,/]ilise block 9 its di% = "o h\r 9 ! hot-air ov5 at #aae deg4 ,c4 #e4 ,c>efully op5 ! di% & add /]ile di/ill$ wat] to moi/5 ! block & =m a lay] 9 ! bottom ( ! di% #a cm4 deep4 ,w9e ,mu/4--7,w9e mu/ is obta9$ f ,sicily1 9 h]metically seal$ t9s1 9 a highly conc5trat$ =m--z a ?ick syrup--b n /]ilis$47 #a4 ,weigh out w9e mu/1 #bjj grammes1 place 9 a #b-litre flask & add di/ill$ wat]1 #hjj c4c4 #b4 ,weigh out ammonium t>trate1 #e grammes1 & add to ! dilute mu/4 #c4 ,place ! flask 9 a wat]-ba? regulat$ to #fj deg4 ,c4 = "o h\r & incorporate ! mixture ?or\ghly by frequ5t %ak+4 #d4 ,filt] ?r\gh papi] ,*>d94 #e4 ,tube1 & /]ilise z = nutri5t b\illon4 ,:eat ,b\illon 7,gasp]9i74-- #a4 ,weigh out & mix :eat fl\r1 #aej grammes2 magnesium sulphate1 #j4e gramme2 potassium nitrate1 #a gramme2 glucose1 #ae grammes4 #b4 ,dissolve ! mixture 9 #ajjj c4c4 ( wat] heat$ to #ajj deg4 ,c4 #c4 ,filt] ?r\gh papi] ,*>d94 #d4 ,tube1 & /]ilise z = nutri5t b\illon4 ,bread ,pa/e4-- #a4 ,grate /ale bread f9ely on a bread-grat]4 #b4 ,di/ribute ! crumbs 9 /]ile ,erl5mey] flasks1 suffici5t to =m a lay] ab\t "o c5timetre ?ick ov] ! bottom ( ea*4 #c4 ,add z mu* di/ill$ wat] z ! crumbs w soak up1 b n en\gh to cov] ! bread4 #d4 ,plug ! flasks & /]ilise 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes on ea* ( f\r consecutive days4 ,m$ia = ! ,/udy ( ,p>asitic ,m\lds4 ,fr5* ,pro( ,ag> 7,sab\raud74-- #a4 ,weigh out ,*assa+'s peptone1 #aj grammes1 & emulsify x ) #bjj c4c4 di/ill$ wat] previ\sly heat$ to #fj deg4 ,c4 #b4 ,weigh out p[d]$ ag>1 #ac grammes1 & emulsify ) #bjj c4c4 cold di/ill$ wat]4 #c4 ,mix ! two emulsions & wa% into a t>$ #b-litre flask ) #fjj c4c4 di/ill$ wat]4 #d4 ,bubble live /eam ?r\gh ! mixture = tw5ty m9utes1 to dissolve ! ag>4 #e4 ,cool to #fj deg4 ,c4 & cl>ify ) egg z = nutri5t ag> 7vide page #afh74 #f4 ,filt] ?r\gh ,papi] ,*>d91 us+ ! hot-wat] funnel4 #g4 ,weigh out ,fr5* maltose1 #dj grammes1 & dissolve 9 ! ag>4 #h4 ,tube1 & /]ilise z = nutri5t ag>4 ,engli% ,pro( ,ag> 7,blaxall74--,sub/itute ,witte's peptone = t ( ,*assa+1 & proce$ z = ,fr5* pro( ag>4 ,fr5* ,mannite ,ag>1 ,sab\raud4--7,= cultivation ( ,favus47 ,proce$ exactly z 9 prep>+ ,fr5* ,pro( ag> vide supra sub/itut+ ,mannite 7#ch grammes7 = maltose4 ,m$ia = ! ,/udy ( ,milk ,bact]ia4 ,gelat9e ,ag>4--,?is m$ium is prep>$ by add+ to nutri5t gelat9e suffici5t ag> to ensure ! solidity ( ! m$ium :5 incubat$ at temp]atures above #bb deg4 ,c4 ,if x is int5d$ to employ an incubat+ temp]ature ( #cj deg4 ,c41 #aj p] c5t4 gelat9e & #j4e p] c5t4 ag> mu/ be dissolv$ 9 ! meat extract be=e ! addition ( ! peptone & salt2 :ile = incubat+ at #cg deg4 ,c41 #ab p] c5t4 gelat9e & #j4ge p] c5t4 ag> mu/ be us$4 ,avoid ! addition ( m ag> ?an is absolutely necess>y1 o!rwise ! action ^u ! m$ium ( su* organisms z elaborate a liquefy+ f]m5t may be ret>d$ or completely abs5t4 #a4 ,measure out #djj c4c4 d\ble /r5g? meat extract into a t>$ #b-litre flask1 & add to x gelat9e1 #ajj grammes4 #b4 ,weigh out p[d]$ ag>1 #e grammes1 emulsify ) #ajj c4c41 cold di/ill$ wat] & add to ! cont5ts ( ! flask4 #c4 ,dissolve ! ag> & gelat9e by bubbl+ live /eam ?r\gh ! flask = tw5ty m9utes4 #d4 ,weigh out peptone1 #aj grammes2 salt1 #e grammes2 emulsify ) #ajj c4c4 d\ble /r5g? meat extract previ\sly heat$ to #fj deg4 ,c41 & add to ! cont5ts ( ! flask4 #e4 ,replace 9 ! /eam] = fifte5 m9utes4 ,!n adju/ ! weight to ! calculat$ figure = "o litre 79 ?is in/ance #aabj grammes7 by ! addition ( di/ill$ wat] at #ajj deg4 ,c4 #f4 ,e/imate ! reaction2 control ! result4 ,!n add suffici5t cau/ic soda solution to r5d] ! reaction #aj4 #g4 ,replace 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes4 #h4 ,cool to #fj deg4 ,c4 ,cl>ify ) egg z = nutri5t ag>4 #i4 ,filt] ?r\gh papi] ,*>d91 us+ ! hot-wat] funnel4 #aj4 ,tube1 & /]ilise z = nutri5t ag>4 ,ag> ,gelat9e 7,gu>ni>i74-- #a4 ,measure out d\ble /r5g? meat extract1 #djj c4c41 into a t>$ #b-litre flask1 & add to x gelat9e1 #ej grammes4 #b4 ,weigh out p[d]$ ag>1 #c grammes2 emulsify ) cold di/ill$ wat]1 #ej c4c41 & add to ! cont5ts ( ! flask4 #c4 ,dissolve ! ag> & gelat9e by bubbl+ live /eam ?r\gh ! flask = tw5ty m9utes4 #d4 ,weigh out ,witte's peptone1 #be grammes2 salt1 #e grammes1 & emulsify ) #ajj c4c4 d\ble /r5g? meat extract previ\sly heat$ to #fj deg4 ,c41 & add to ! cont5ts ( ! flask4 #e4 ,replace 9 ! /eam] = fifte5 m9utes4 #f4 ,weigh ! flask & make up ! m$ium mass to ! calculat$ figure = "o litre 7#ajhc grammes7 by ! addition ( di/ill$ wat] at #ajj deg4 ,c4 #g4 ,neutralise c>efully to litmus pap] by ! successive additions ( small quantities ( normal soda solution4 #h4 ,replace 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes4 #i4 ,cool to #fj deg4 ,c4 ,cl>ify ) egg z = nutri5t ag>4 #aj4 ,filt] ?r\gh papi] ,*>d91 us+ ! hot-wat] funnel4 #aa4 ,tube1 & /]ilise z = nutri5t ag>4 ,:ey ,gelat9e4-- #a4 ,curdle fre% milk by w>m+ to #fj deg4 ,c41 & add+ r5net2 filt] (f ! :ey into a /]ile t>$ flask4 #b4 ,e/imate & note ! reaction ( ! :ey4 #c4 ,weigh out gelat9e1 #aj p] c5t41 & add x to ! :ey 9 ! flask4 #d4 ,bubble live /eam ?r\gh ! mixture fifte5 m9utes to dissolve ! gelat9e2 & weigh4 #e4 ,e/imate ! reaction ( ! m$ium mass2 !n add suffici5t cau/ic soda solution to re/ore ! reaction ( ! m$ium mass 7i4 e41 total weight m9us weight ( flask7 to ! equival5t ( ! orig9al :ey4 #f4 ,cool to #fj deg4 ,c4 & cl>ify ) egg z = nutri5t gelat9e 7vide page #aff74 #g4 ,filt] ?r\gh papi] ,*>d94 #h4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,:ey ,ag>4-- #a4 ,curdle fre% milk by w>m+ to #fj deg4 ,c41 & add+ r5net2 filt] (f ! :ey into a /]ile flask4 #b4 ,weigh out ag>1 #a4e or #b p] c5t41 & add x to ! :ey 9 ! flask4 #c4 ,bubble live /eam ?r\gh ! mixture = tw5ty m9utes1 to dissolve ! ag>4 #d4 ,cool to #fj deg4 ,c42 cl>ify ) egg z = nutri5t ag> 7vide page #afh74 #e4 ,filt] ?r\gh papi] ,*>d91 us+ ! hot-wat] funnel4 #f4 ,tube1 & /]ilise z = nutri5t ag>4 ,litmus ,:ey4-- #a4 ,curdle fre% milk by w>m+ to #fj deg4 ,c4 & add+ r5net4 #b4 ,filt] (f ! :ey ?r\gh butt] musl9 into a /]ile flask4 #c4 ,neutralise to litmus by ! cauti\s addition ( citric acid solution #d p] c5t4 7,d n neutralise ) m9]al acid47 #d4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = "o h\r to coagulate all ! proteid4 7,if ! :ey is cl\dy :5 remov$ f ! /eam] all[ x to /& = =ty-eight h\rs 9 ! ice *e/ & !n decant (f ! cle> fluid--or filt] ?r\gh a ,b]kefeld filt] c&le47 #e4 ,filt] into a /]ile flask4 #f4 ,t9t ! :ey ) litmus solution to a deep purple r$4 #g4 ,tube1 & /]ilise z = milk4 ,litmus ,:ey 7,petrus*ky74-- #a4 ,measure out into a flask ,fre% milk #ajjj c4c4 #b4 ,add ,hydro*loric acid 7or glacial acetic acid7 #a4e c4c4 & boil4 #c4 ,filt] (f coagulat$ case94 #d4 ,neutralise to litmus by ! addition ( n_/#a cau/ic soda solution & boil4 ,:ey n[ cl\dy & acid aga94 #e4 ,aga9 neutralise to litmus by addition ( n_/#aj cau/ic soda solution4 #f4 ,filt]4 #g4 ,t9t ! :ey ) neutral litmus solution to a deep purple col\r4 #h4 ,tube & /]ilise z = milk4 ,litmus ,:ey ,gelat9e4-- #a4 ,measure out milk #ajjj c4c4 into a t>$ #b-litre flask4 #b4 ,add hydro*loric acid 7or glacial acetic acid7 #a4e c4c4 & boil = five m9utes4 #c4 ,filt] (f ! case91 & make ! :ey fa9tly alkal9e to litmus4 #d4 ,weigh out ,peptone #aj grammes & emulsify 9 a few cubic c5timet]s ( ! :ey & return to ! flask4 #e4 ,weigh out ,gelat9e #ej grammes add x to ! :ey 9 ! flask & incorporate ! mixture by bubbl+ ?r\gh live /eam4 #f4 ,cle> ) egg & filt]4 #g4 ,make ! weight ( ! m$ium mass to ! calculat$ figure = "o litre 7#ajfj grammes7 by ! addition ( di/ill$ wat]4 #h4 ,weigh out ,dextrose #ae grammes & dissolve 9 ! fluid :ey gelat9e4 #i4 ,add /]ile litmus solution to ! requir$ t9t4 #aj4 ,tube & /]ilise = tw5ty m9utes 9 /eam] at #ajj deg4 ,c4 on ea* ( five successive days4 ,?is m$ium w rema9 semi-fluid at ! room temp]ature1 & may be us$ = cultures 9 ! cool or hot incubator4 ,litmus ,:ey ,ag> is prep>$ 9 a simil> mann] to ,:ey ,gelat9e1 ) ! sub/itution ( #ae grammes ( ag> = ! gelat9e4 ,malt ,extract ,solution 7,h]s*ell74-- #a4 ,measure into a flask di/ill$ wat] #ajjj c4c4 #b4 ,weigh out ,extractum malti 7malt extract7 #be grammes & add to di/ill$ wat] 9 flask4 #c4 ,boil = five m9utes1 all[ to /&1 & decant (f cle> fluid f s$im5t4 #d4 ,tube & /]ilise z = nutri5t b\illon4 ,m$ia = ! ,/udy ( ,e>? ,bact]ia1 ,nitrog5 ,fix]s4 ,e>?y ,salts ,ag> 7,lipman & ,br[n74--7,= ! enum]ation ( soil organisms47 #a4 ,measure out ,ag> #bj grammes4 ,emulsify 9 #bjj c4c4 di/ill$ wat]4 #b4 ,wa% ! ag> emulsion into a t>$ #b-litre flask ) #djj c4c4 di/ill$ wat]4 #c4 ,weigh out ,peptone #j4e gramme4 ,emulsify 9 #ej c4c4 di/ill$ wat] & add to ! cont5ts ( ! flask4 #d4 ,bubble live /eam ?r\gh ! mixture = tw5ty m9utes to dissolve ! ag>4 #e4 ,weigh out & mix ,dextrose #aj4j grammes4 ,potassium phosphate #j4e gramme4 ,magnesium sulphate #j4b gramme4 ,potassium nitrate #j4jf gramme4 & add to ! cont5ts ( ! flask4 #f4 ,adju/ ! weight ( ! m$ium mass to ! calculat$ figure = "o litre 7#ajbe grammes7 by ! addition ( di/ill$ wat] at #ajj deg4 ,c4 #g4 ,titrate ! m$ium mass & adju/ ! reaction to #e4 #h4 ,cool to #fj deg4 ,c4 ,cl>ify ) egg & filt]4 #i4 ,tube 9 quantities ( #aj c4c4 & /]ilise z = nutri5t ag>4 ,beyr9ck's ,solution4 ,i4--7,= ! cultivation ( nitrog5 fix+ organisms47 #a4 ,weigh out & mix #a gramme potassium hydrog5 phosphate1 #j4b gramme magnesium sulphate1 & #j4jb gramme sodium *loride4 #b4 ,dissolve 9 wat] #ajjj c4c41 9 a #b-litre flask4 #c4 ,add #a c4c4 ( a "o p] ?\s& aque\s solution ( f]r\s sulphate4 #d4 ,add #a c4c4 ( a "o p] ?\s& solution manganese sulphate4 #e4 ,weigh out #bj grammes dextrose & add to ! cont5ts ( ! flask 7dextrose up to #dj grammes may be us$ = ! diff]5t organisms74 #f4 ,/eam = tw5ty m9utes1 filt]4 #g4 ,tube1 & /]ilise z = nutri5t b\illon4 ,beyr9ck's ,solution4 ,,ii4--7,= gr[? ( ,azobact]47 ,proce$ z 9 prep>+ solution ,no4 ,i1 sub/itut+ mannite = dextrose 9 /ep #e4 ,w9ogradsky's ,solution 7= ,nitric ,organisms74-- #a4 ,weigh out & mix4 ,potassium phosphate #a4j gramme ,magnesium sulphate #j4e gramme ,calcium *loride #j4ja gramme ,sodium *loride #b4j grammes & dissolve 9 ,di/ill$ wat] #ajjj c4c4 #b4 ,fill into flasks1 9 quantities ( #bj c4c4 & add to ea* a small quantity ( fre%ly wa%$ magnesium c>bonate4 #c4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree consecutive days4 #d4 ,add to ea* flask conta9+ #bj c4c4 solution1 #b c4c4 ( a /]ile #b p] c5t4 solution ( ammonium sulphate4 #e4 ,incubate at #cg deg4 ,c4 = =ty-eight h\rs & elim9ate any contam9at$ culture flasks4 ,/ore ! rema9d] = future use4 ,w9ogradsky's ,solution 7= ,nitr\s ,organisms74-- #a4 ,weigh out & mix ,ammonium sulphate #a gramme ,potassium sulphate #a gramme & dissolve 9 ,di/ill$ wat] #ajjj c4c4 #b4 ,add #e to #aj grammes basic magnesium c>bonate1 previ\sly /]ilis$ by boil+4 #c4 ,fill into flasks & /]ilise1 etc41 z = previ\s solution4 ,silicate ,jelly 7,w9ogradsky74-- #a4 ,weigh out & mix ,ammonium sulphate #j4dj gramme ,magnesium sulphate #j4je gramme ,calcium *loride #j4ja gramme & dissolve 9 ,di/ill$ wat] #ej c4c4 ,label--,solution ,a4 #b4 ,weigh out & mix ,potassium phosphate #j4aj gramme ,sodium c>bonate #j4fj gramme & dissolve 9 ,di/ill$ wat] #ej c4c4 ,label--,solution ,b4 #c4 ,weigh out ,silicic acid #c4d grammes & dissolve 9 ,di/ill$ wat] #ajj c4c4 #d4 ,p\r ! silicic acid solution into a l>ge porcela9 bas94 #e4 ,mix equal quantities ( ! solutions ,a & ,b2 !n add successive small quantities ( ! mix$ salts to ! silicic acid solution1 /irr+ cont9u\sly ) a glass rod1 until a jelly ( suffici5tly firm consi/5ce has be5 =m$4 #f4 ,spread a lay] ( ?is jelly ov] ! bottom ( ea* ( sev]al l>ge capsules or plates4 #g4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes on ea* ( ?ree consecutive days4 ,m$ia = ! ,/udy ( ,wat] ,bact]ia4 ,naehr/(f ,ag> 7,hesse & ,ni$n]74--7,= enum]ation ( wat] organisms47 #a4 ,weigh out3 ag>1 #ab4e grammes & emulsify 9 #bej c4c4 di/ill$ wat]4 #b4 ,wa% ! ag> emulsion into a t>$ #b-litre flask ) a fur!r #bej c4c4 di/ill$ wat]4 #c4 ,dissolve by bubbl+ live /eam ?r\gh ! mixture4 #d4 ,emulsify ,naehr/(f-,heyd5 7albumose7 #g4e grammes 9 #bjj c4c4 cold di/ill$ wat] & add to melt$ ag>4 #e4 ,adju/ weight ( m$ium mass to ! calculat$ figure = "o litre 7#ajbj grammes7 by addition ( di/ill$ wat] at #ajj deg4 ,c4 #f4 ,cl>ify ) :ite ( egg & filt]4 #g4 ,tube 9 quantities ( #aj c4c4 & /]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree successive days4 ,bile ,salt ,bro?--,d\ble ,/r5g?4-- #a4 ,weigh out ,witte's peptone1 #dj grammes1 & emulsify ) #cjj c4c4 di/ill$ wat] previ\sly w>m$ to #fj deg4 ,c4 #b4 ,wa% ! peptone emulsion into a litre flask ) #fjj c4c4 di/ill$ wat]4 #c4 ,weigh out sodium tauro*olate1 #aj grammes1 & glucose1 #aj grammes2 dissolve 9 #ajj c4c4 di/ill$ wat] & add to ! peptone emulsion 9 ! flask4 #d4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes4 #e4 ,filt] ?r\gh ,sw$i% filt] pap] into a /]ile flask4 #f4 ,add /]ile neutral litmus solution suffici5t to col\r ! m$ium to a deep purple4 #g4 ,fill into small ,erl5mey] flasks 9 quantities ( #be c4c4 #h4 ,/]ilise z = nutri5t b\illon4 ,m$ia = ! ,/udy ( ,plant ,bact]ia4 ,beetroot4-- ,c>rot4-- are prep>$ tubes & /]ilis$ 9 a mann] ,turnip4-- precisely simil> to t describ$ = potato4 ,p>snip4-- ,hay ,infusion4-- #a4 ,weigh out dri$ hay1 #aj grammes1 *op x up into f9e p>ticles & place 9 a flask4 #b4 ,add #ajjj c4c4 di/ill$ wat]1 heat$ to #gj deg4 ,c42 close ! flask ) a solid rubb] /opp]4 #c4 ,mac]ate 9 a wat]-ba? at #fj deg4 ,c4 = ?ree h\rs4 #d4 ,replace ! /opp] by a cotton-wool plug1 & heat 9 ! /eam] at #ajj deg4 ,c4 = "o h\r4 #e4 ,filt] ?r\gh ,sw$i% filt] pap]4 #f4 ,tube1 & /]ilise z = nutri5t b\illon4 ,h>icot ,b\illon4--7,= cultivation ( bact]ia f tub]cles ( ,legumes47 #a4 ,measure out #ajjj c4c4 di/ill$ wat] into a #b-litre flask4 #b4 ,weigh out #bej grammes h>icot beans & add to ! wat] 9 ! flask4 #c4 ,weigh out #aj grammes sodium *loride & add to ! cont5ts ( ! flask4 #d4 ,add #a c4c4 ( a #a p] c5t4 solution ( sodium bic>bonate4 #e4 ,place 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes4 #f4 ,filt]4 #g4 ,weigh out #bj grammes sac*>ose & add to ! filtrate4 #h4 ,tube1 & /]ilise z = nutri5t b\illon4 ,h>icot ,ag>4-- #a4 ,measure out #djj c4c4 di/ill$ wat] into a t>$ #b-litre flask4 #b4 ,weigh out #ae grammes ag> & mix into a ?ick pa/e ) #ajj c4c4 cold di/ill$ wat]1 & add to ! flask4 #c4 ,dissolve ! ag> by bubbl+ live /eam ?r\gh ! mixture z 9 mak+ nutri5t ag>4 #d4 ,weigh out #bej grammes h>icot beans1 place 9 ! flask ) ! ag> mixture4 #e4 ,add #a c4c4 ( #a p] c5t4 aque\s solution sodium bic>bonate4 #f4 ,weigh out #aj grammes sodium *loride & add to ! cont5ts ( ! flask4 #g4 ,place 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes4 #h4 ,adju/ ! weight ( ! m$ium mass to #ajcj grammes 7! figure p] litre obta9$ exp]im5tally7 by ! addition ( di/ill$ wat] at #ajj deg4 ,c4 #i4 ,cool to #fj deg4 ,c41 cl>ify ) egg & filt]4 #aj4 ,weigh out #bj grammes sac*>ose & add to ! cont5ts ( ! flask4 #aa4 ,tube1 & /]ilise z = nutri5t ag>4 ,wood ,a% ,ag>4-- #a4 ,measure #djj c4c4 di/ill$ wat] into a t>$ #b-litre flask4 #b4 ,weigh out #aj grammes ag> & make into a ?ick pa/e ) #ajj c4c4 cold di/ill$ wat]4 #c4 ,add ?is ag> pa/e to ! di/ill$ wat] 9 ! flask4 #d4 ,dissolve ! ag> by pass+ live /eam ?r\gh x1 z 9 prep>+ nutri5t ag>4 #e4 ,weigh out #e grammes clean wood a% & place 9 a second flask conta9+ #bjj c4c4 di/ill$ wat] ) "s /]ile glass beads3 %ake ?or\ghly 9 a me*anical %ak] = t5 m9utes4 #f4 ,heat 9 /eam] at #ajj deg4 ,c41 = ?irty m9utes4 #g4 ,aft] removal f ! /eam] dry ! outside ( ! flask ?or\ghly1 place x ov] a ,buns5 flame & boil = "o m9ute4 #h4 ,filt] directly into ! flask conta9+ ! melt$ ag> mixture4 #i4 ,weigh out #d grammes maltose4 ,add to ! cont5ts ( ! flask4 #aj4 ,adju/ ! weight ( ! m$ium mass to ! calculat$ figure = "o litre 7#ajai grammes7 by ! addition ( di/ill$ wat] at #ajj deg4 ,c4 #aa4 ,replace ! flask 9 ! /eam] = tw5ty m9utes1 cool to #fj deg4 ,c41 & cl>ify ) egg & filt]4 #ab4 ,tube1 & /]ilise z = nutri5t ag>4 ,m$ia = ! ,/udy ( ,special ,bacilli4 ,b4 ,acnes4 ,oleic ,acid ,ag> 7,flem+74-- #a4 ,measure out into a /]ile /\t glass bottle :i* already conta9s ab\t #aj /]ile glass beads ,ascitic fluid #bej c4c4 #b4 ,weigh out ,oleic acid #be grammes & add x to ! ascitic fluid 9 ! bottle4 #c4 ,emulsify ev5ly by %ak+ 7ei!r by h& or 9 a %ak+ ma*9e7 = t5 m9utes4 #d4 ,liquefy & measure out into a flask ,nutri5t ag> #gej c4c4 !n cool to #ee deg4 ,c4 #e4 ,mix ! oleic acid emulsion ) ! ag>4 #f4 ,add #aj c4c4 /]ile neutral r$1 #a p] c5t4 aque\s solution4 #g4 ,tube 9 quantities ( #aj c4c41 slant1 & all[ to set4 #h4 ,incubate = =ty-eight h\rs at #cg deg4 ,c4 & reject any contam9at$ tubes4 ,/ore ! /]ile tubes = future use4 ,coli-typhoid ,gr\p4 ,p>ietti's ,b\illon4-- #a4 ,measure out pure hydro*loric acid1 #d c4c41 & add to x c>bolic acid solution 7#e p] c5t471 #ajj c4c4 ,all[ ! solution to /& at lea/ a few days be=e use4 #b4 ,?is solution is add$ 9 quantities ( #j4a1 #j4b4 & #j4c c4c4 7deliv]$ by means ( a /]ile graduat$ pipette7 to tubes ea* conta9+ #aj c4c4 ( previ\sly /]ilis$ nutri5t b\illon 7vide page #afc74 #c4 ,incubate at #cg deg4 ,c4 = =ty-eight h\rs to elim9ate contam9at$ tubes4 ,/ore ! rema9d] = future use4 ,c>bolis$ ,b\illon4-- #a4 ,prep>e nutri5t b\illon 7vide page #afc1 sections #a to #f74 ,measure out #ajjj c4c4 #b4 ,weigh out c>bolic acid1 #a gramme 7#b4e or #e grammes may be ne$$ = special purposes71 & dissolve x 9 ! m$ium4 #c4 ,tube1 & /]ilise z = b\illon4 ,c>bolis$ ,gelat9e4-- #a4 ,prep>e nutri5t gelat9e 7vide page #afd1 sections #a to #g74 ,measure out #ajjj c4c4 #b4 ,weigh out c>bolic acid1 #e grammes 7 #j4e p] c5t471 & dissolve x 9 ! gelat9e4 #c4 ,filt] if necess>y ?r\gh papi] ,*>d94 #d4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,"o or #b4e grammes ( c>bolic acid 7 #j4a p] c5t4 or #j4be p] c5t47 are occasionally us$ 9 place ( ! #e grammes to meet special requirem5ts4 ,c>bolis$ ,ag>4-- #a4 ,prep>e nutri5t ag> 7vide page #afg1 sections #a to #h74 ,measure out #ajjj c4c4 #b4 ,weigh out #a gramme pure ph5ol & dissolve 9 ! m$ium4 #c4 ,filt] if necess>y ?r\gh papi] ,*>d94 #d4 ,tube1 & /]ilise z = nutri5t ag>4 ,litmus ,gelat9e4-- #a4 ,prep>e nutri5t gelat9e 7vide page #afd1 sections #a to #h74 #b4 ,add /]ile litmus solution1 suffici5t to t9t ! m$ium a deep lav5d] col\r4 #c4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,lactose ,litmus ,b\illon 7,lakmus ,molke74-- #a4 ,weigh out peptone1 #d grammes1 & emulsify x ) #bjj c4c4 meat extract 7vide page #adh71 previ\sly heat$ to #fj deg4 ,c4 #b4 ,weigh out salt1 #b grammes1 & lactose1 #bj grammes1 & mix ) ! emulsion4 #c4 ,wa% ! mixture into a /]ile litre flask ) #bjj c4c4 meat extract & add #fjj c4c4 di/ill$ wat]4 #d4 ,heat 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes1 to completely dissolve ! peptone1 etc4 #e4 ,neutralise c>efully to litmus pap] by ! successive additions ( small quantities ( dec9ormal soda solution4 #f4 ,replace 9 ! /eam] = tw5ty m9utes to precipitate phosphates1 etc4 #g4 ,filt] ?r\gh two ?icknesses ( ,sw$i% filt] pap]4 #h4 ,add /]ile litmus solution1 suffici5t to col\r ! m$ium a deep purple4 #i4 ,tube1 & /]ilise z = b\illon4 ,lactose ,litmus ,gelat9e 7,wurtz74-- #a4 ,prep>e nutri5t gelat9e 7vide page #afd1 sections #a to #d74 #b4 ,r5d] ! reaction ( ! m$ium mass -#e4 #c4 ,replace 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes4 #d4 ,cl>ify ) egg z = gelat9e4 #e4 ,weigh out lactose1 #bj grammes 7 #b p] c5t471 & dissolve x 9 ! m$ium4 #f4 ,filt] ?r\gh papi] ,*>d94 #g4 ,add suffici5t /]ile litmus solution to col\r ! m$ium pale lav5d]4 #h4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,lactose ,litmus ,ag> 7,wurtz74-- #a4 ,prep>e nutri5t ag> 7vide page #afg1 sections #a to #d74 #b4 ,r5d] ! reaction ( ! m$ium mass -#e4 #c4 ,replace 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes4 #d4 ,cool to #fj deg4 ,c4 & cl>ify ) egg z = nutri5t ag>4 #e4 ,weigh out lactose1 #bj grammes 7 #b p] c5t471 & dissolve x 9 ! m$ium4 #f4 ,filt] ?r\gh papi] ,*>d91 us+ ! hot-wat] funnel4 #g4 ,add /]ile litmus solution1 suffici5t to col\r ! m$ium a pale lav5d]4 #h4 ,tube1 & /]ilise z = nutri5t ag>4 ,glyc]9e ,potato ,b\illon4-- #a4 ,take #a kilo ( potatoes1 wa% ?or\ghly 9 wat]1 peel1 & grate f9ely on a bread-grat]4 #b4 ,weigh ! potato grat+s1 place !m 9 a #b-litre flask1 & add di/ill$ wat] 9 ! proportion ( #a c4c4 = e gramme weight ( potato4 ,all[ ! flask to /& 9 ! ice-*e/ = twelve h\rs4 #c4 ,/ra9 ! mixture ?r\gh butt] musl9 & filt] ?r\gh ,sw$i% filt] pap] into a graduat$ cyl9d]4 ,note ! am\nt ( ! filtrate4 #d4 ,place ! filtrate 9 a flask1 add an equal quantity ( di/ill$ wat]1 & heat 9 ! /eam /]ilis] = sixty m9utes4 #e4 ,add glyc]9e1 #d p] c5t41 mix ?or\ghly1 & aga9 filt]4 #f4 ,tube & /]ilise z = nutri5t b\illon4 ,potato ,gelat9e 7,elsn]74-- #a4 ,take #a kilo ( potatoes1 wa% ?or\ghly 9 wat]1 peel1 & f9ally grate f9ely on a bread-grat]4 #b4 ,weigh ! potato grat+s1 place !m 9 a #b-litre flask1 & add di/ill$ wat] 9 ! proportion ( #a c4c4 = e gramme weight ( potato4 ,all[ ! flask to /& 9 ! ice-*e/ = twelve h\rs4 #c4 ,/ra9 ! mixture ?r\gh butt] musl91 & filt] ?r\gh ,sw$i% filt] pap] into a graduat$ cyl9d]4 #d4 ,add #ae p] c5t4 gelat9e to ! potato decoction & bubble live /eam ?r\gh ! mixture = t5 m9utes4 #e4 ,e/imate ! reaction2 adju/ ! reaction ( ! m$ium mass to #be4 #f4 ,cool ! m$ium to bel[ #fj deg4 ,c42 cl>ify ) egg z = nutri5t gelat9e 7vide page #aff74 #g4 ,add #a p] c5t4 potassium iodide 7p[d]$7 to ! m$ium4 #h4 ,filt] ?r\gh papi] ,*>d94 #i4 ,tube & /]ilise z = nutri5t gelat9e4 ,aescul9 ,ag>4--7,b4 coli & alli$ organisms give black colonies surr\nd$ by black halo47 #a4 ,measure out #djj c4c4 di/ill$ wat] into a t>$ #b-litre flask4 #b4 ,weigh out ,ag> #ae grammes ,peptone #aj grammes ,sodium tauro*olate #e grammes & make into a ?ick pa/e ) #aej c4c4 di/ill$ wat]4 #c4 ,add ?is pa/e to ! di/ill$ wat] 9 ! flask4 #d4 ,dissolve ! +r$i5ts by bubbl+ live /eam ?r\gh ! mixture4 #e4 ,weigh out ,aescul9 #a4j gramme ,f]ric citrate #j4e gramme & dissolve 9 a second flask conta9+ #ajj c4c4 di/ill$ wat]4 #f4 ,mix ! cont5ts ( ! two flasks--adju/ ! weight to ! calculat$ m$ium figure 79 ?is case #ajca4e grammes7 by ! addition ( di/ill$ wat] at #ajj deg4 ,c4 #g4 ,cl>ify ) egg & filt]4 #h4 ,tube & /]ilise z = nutri5t ag>4 ,bile ,salt ,ag> 7,mac,conkey74-- #a4 ,weigh out p[d]$ ag>1 #ae grammes 7 #a4e4 p] c5t471 & emulsify ) #bjj c4c4 cold tap wat]4 #b4 ,weigh out peptone1 #bj grammes 7 #b p] c5t471 & emulsify ) #bjj c4c4 tap wat] previ\sly w>m$ to #fj deg4 ,c4 #c4 ,mix ! peptone & ag> emulsions ?or\ghly4 #d4 ,weigh out sodium tauro*olate1 #e grammes 7 #j4e p] c5t471 dissolve x 9 #cjj c4c4 tap wat]1 & use ! solution to wa% ! ag>-peptone emulsion into a t>$ #b-litre flask4 #e4 ,bubble live /eam ?r\gh ! mixture = tw5ty m9utes4 #f4 ,adju/ ! weight ( ! m$ium mass to ! calculat$ figure = "o litre 7#ajdj grammes74 #g4 ,cool to #fj deg4 ,c4 & cl>ify ) egg z = nutri5t ag> 7vide page #afh74 #h4 ,filt] ?r\gh papi] ,*>d91 us+ ! hot-wat] funnel4 #i4 ,weigh out lactose1 #aj grammes 7 #a p] c5t471 & dissolve x 9 ! ag>4 ,if desir$1 add #e c4c4 ( a #a p] c5t4 7 #j4e p] c5t47 aque\s solution ( neutral r$4 #aj4 ,tube1 & /]ilise z = nutri5t ag>4 ,litmus ,nutrose ,ag> 7,drigalski-,conradi74-- ,?is m$ium %\ld be prep>$ 9 precisely ! same mann] z ! ,nutrose ag> describ$ on page #agb sub/itut+ meat extract = s]um wat]1 & increas+ ! p]c5tage ( ag> add$ p] litre to #c p] c5t4 ,fu*s9 ,ag> 7,braun74-- #a4 ,liquefy & measure out into a /]ile flask3 ,nutri5t ag> #ajjj c4c4 #b4 ,weigh out3 lactose #aj grammes & dissolve 9 ! fluid ag>4 #c4 ,adju/ ! reaction to -#e & filt]4 #d4 ,measure out & mix ?or\ghly ) ag>3 ,fu*s91 alcoholic solution #e c4c4 ,! fu*s9 solution is prep>$ by mix+3 ,fu*s9 7basic7 #c grammes4 ,absolute alcohol #fj c4c4 ,all[ to /& tw5ty-f\r h\rs1 !n c5trifugalise ?or\ghly & decant ! sup]natant fluid into a well-/opp]$ bottle4 #e4 ,measure out & add to ! nutri5t ag>1 sodium sulphite1 #aj p] c5t4 aque\s solution1 fre%ly prep>$ #be c4c4 #f4 ,tube & /]ilise z = nutri5t ag>4 #g4 ,/ore 9 a d>k cupbo>d4 ,fu*s9 ,sulphite ,ag> 7,endo74-- #a4 ,liquefy & measure out into a /]ile flask3 ,nutri5t ag> #ajjj c4c4 #b4 ,weigh out ,lactose #aj grammes4 & dissolve 9 ! fluid ag>4 #c4 ,adju/ ! reaction to #c & filt]4 #d4 ,measure out & mix ?or\ghly ) ! fluid ag>4 ,fu*s91 alcoholic solution 7vide supra7 #e c4c4 #e4 ,measure out & add to ! m$ium ,sodium sulphite1 #aj p] c5t4 aque\s solution #be c4c4 #f4 ,tube & /]ilise z = nutri5t ag>4 ,brilliant ,gre5 ,ag> 7,conradi74-- #a4 ,liquefy & measure out into a /]ile flask ,nutri5t ag> #ajjj c4c4 #b4 ,adju/ reaction to #cj by ! addition ( normal phosphoric acid2 & filt]4 #c4 ,measure out & mix ?or\ghly ) ! fluid m$ium ,brilliant gre5 7,hoe*/7 #a p] ?\s& aque\s solution #f4e c4c4 #d4 ,measure out & add to ! m$ium ,picric acid 7,gruebl]71 #a p] c5t4 aque\s solution #f4e c4c4 #e4 ,tube & /]ilise z = nutri5t ag>4 ,brilliant ,gre5 ,bile ,salt ,ag> 7,fawcus74-- #a4 ,weigh out ag> #bj grammes & emulsify 9 #ajj c4c4 cold di/ill$ wat]4 #b4 ,wa% ! emulsion into a t>$ #b-litre flask ) #ejj c4c4 di/ill$ wat]4 #c4 ,dissolve ! ag> by bubbl+ live /eam ?r\gh ! flask4 #d4 ,cool1 cl>ify ) egg & filt]4 #e4 ,weigh out ,sodium tauro*olate #e grammes ,peptone #bj grammes & add to ! m$ium 9 ! flask4 #f4 ,weigh out ,lactose #e grammes & add to ! m$ium 9 ! flask4 #g4 ,adju/ reaction to #ae & filt] if necess>y4 #h4 ,measure out ,brilliant gre51 #a p] ?\s& aque\s solution #bj c4c4 & mix ?or\ghly ) ! fluid ag>4 #i4 ,measure out & add to ! m$ium ,picric acid1 #a p] c5t4 aque\s solution #bj c4c4 #aj4 ,tube & /]ilise z = nutri5t ag>4 ,*9a ,gre5 ,ag> 7,w]bitski74-- #a4 ,liquefy & measure out into a /]ile flask ,nutri5t ag> #ajjj c4c4 #b4 ,adju/ ! reaction accurately to #ac & filt]4 #c4 ,measure out & mix ?or\ghly ) ! fluid ag> ,*9a gre5 #j4b p] c5t4 aque\s solution #ae c4c4 #d4 ,tube & /]ilise z = nutri5t ag>4 ,mala*ite ,gre5 ,ag> 7,loeffl]74-- #a4 ,liquefy & measure out into a /]ile flask ,nutri5t ag> #ajjj c4c4 #b4 ,weigh out ,dextrose #aj grammes4 & dissolve 9 nutri5t ag>4 #c4 ,adju/ ! reaction to #c1 & filt]4 #d4 ,measure out & mix ?or\ghly 9 ! fluid ag> ,mala*ite gre51 #j4a p] c5t4 aque\s solution #af c4c4 = weak m$ium4 #d;a4 ,to ! filt]$ ag> add ,mala*ite gre51 #b p] c5t4 aque\s solution #be c4c4 = /rong m$ium4 #e4 ,tube & /]ilise z = nutri5t ag>4 ,d\ble ,sug> ,ag> 7,russell74-- #a4 ,liquefy & measure out into a /]ile flask ,nutri5t ag> #ajjj c4c4 #b4 ,add #ajj c4c4 litmus solution to ! fluid ag>4 #c4 ,weigh out & dissolve 9 ! fluid ag>4 ,lactose #aj grammes ,dextrose #aj grammes4 #d4 ,r5d] ! reaction ( ! m$ium neutral to litmus pap] by ! cauti\s addition ( normal cau/ic soda4 #e4 ,tube 9 quantities ( #aj c4c4 & /]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree successive days4 #f4 ,/ore = use 9 a cool d>k place4 ,b4 ,diph!riae4 ,glyc]9e ,blood-s]um4-- #a4 ,prep>e blood-s]um z describ$1 page #afh1 sections #a to #d4 #b4 ,add #e p] c5t4 pure glyc]9e4 #c4 ,complete z describ$ above = ord9>y blood-s]um1 sections #e to #g4 ,,note4--,diff]5t p]c5tages ( glyc]9e--f #d p] c5t4 to #h p] c5t4--are us$ = special purposes4 ,five p] c5t4 is t usually employ$4 ,blood-s]um 7,loeffl]74-- #a4 ,prep>e nutri5t b\illon 7vide page #afc71 us+ meat extract made f veal in/ead ( beef4 #b4 ,add #a p] c5t4 glucose to ! b\illon1 & all[ x to dissolve completely4 #c4 ,n[ add #cjj c4c4 cle> blood-s]um 7vide page #afh1 sections #a to #d7 to e #ajj c4c4 ( ?is b\illon4 #d4 ,fill into /]ile tubes & complete z = ord9>y blood-s]um4 ,blood-s]um 7,lorra9 ,smi?74-- #a4 ,collect blood-s]um 7vide page #afh1 sections #a to #d71 z free f haemoglob9 z possible4 #b4 ,weigh out #j4ae p] c5t4 sodium hydrate & dissolve x 9 ! fluid 7or add #j4cge c4c4 ( dekanormal soda solution = e #ajj c4c4 ( s]um74 #c4 ,tube1 & /iff5 at #ajj deg4 ,c4 9 ! s]um inspissator4 #d4 ,incubate at #cg deg4 ,c4 = =ty-eight h\rs to elim9ate any contam9at$ tubes4 ,/ore ! rema9d] = future use4 ,blood ,s]um 7,c\ncilman & ,mallory74-- #a4 ,collect blood s]um 9 slaught]h\se1 coagulate1 remove s]um & tube 7vide page #afh74 ,great c>e mu/ be tak5 to avoid ! inclusion ( air bubbles--inde$ if only a few tubes are fill$ at "o "t1 x is a good plan to /& !m upright 9 ! receiv] ( an air pump & to exhau/ z completely z possible be=e transf]r+ to ! s]um inspissator4 #b4 ,heat ! tubes 9 a slant+ position 9 hot-air /]ilis] at #ij deg4 ,c4 till firmly coagulat$1 say half an h\r4 #c4 ,/]ilise 9 /eam /]ilis] at #ajj deg4 ,c4 = #bj m9utes on ea* ( ?ree successive days4 ,result+ m$ium n transluc5t1 b opaque & firm4 ,b4 ,tub]culosis4 ,egg ,m$ium 7,lub5au74-- ,?is modification ( ,dorset's egg m$ium 7quod vide page #agd7 is pref]r$ by "s = ! gr[? ( ! tub]cle bacillus ( ! human type4 ,x consi/s 9 ! addition ( "o "p ( #f p] c5t4 glyc]9e 9 normal sal9e solution1 to ! egg mixture betwe5 /eps #d & #e4 ,glyc]9e ,b\illon4-- #a4 ,measure out nutri5t b\illon1 #ajjj c4c4 7vide page #afc1 sections #a to #f74 #b4 ,measure out glyc]9e1 #fj c4c4 7 #f p] c5t471 & add to ! b\illon4 #c4 ,tube1 & /]ilise z = b\illon4 ,glyc]9e ,ag>4-- #a4 ,prep>e nutri5t ag> 7vide page #afg1 sections #a to #h74 ,measure out #ajjj c4c4 #b4 ,measure out pure glyc]9e1 #fj c4c4 7 #f p] c5t471 & add to ! ag>4 #c4 ,tube1 & /]ilise z = nutri5t ag>4 ,glyc]9e ,blood-s]um4-- #a4 ,prep>e blood-s]um z describ$1 page #afh1 sections #a to #d4 #b4 ,add #e p] c5t4 pure glyc]9e4 #c4 ,complete z describ$ above = ord9>y blood-s]um1 sections #e to #g4 ,,note4--,diff]5t p]c5tages ( glyc]9e--f #d p] c5t4 to #h p] c5t4--are us$ = special purposes4 ,five p] c5t4 is t usually employ$4 ,glyc]9at$ ,potato4-- #a4 ,prep>e ord9>y potato w$ges 7vide page #agd1 sections #a to #d74 #b4 ,soak ! w$ges 9 #be p] c5t4 solution ( glyc]9e = fifte5 m9utes4 #c4 ,moi/5 ! cotton-wool pads at ! bottom ( ! potato tubes ) a #be p] c5t4 solution ( glyc]9e4 #d4 ,ins]t a w$ge ( potato 9 ea* tube & replug ! tubes4 #e4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( five consecutive days4 ,animal ,tissue ,m$ia 7,frugoni74-- #a4 ,take a numb] ( /]ile te/-tubes #af x #c or #d cm41 plugg$ ) cotton wool1 & into ea* ins]t a #b cm4 l5g? ( /\t glass tub+ 7ab\t #a cm4 diamet]72 fill 9 glyc]9e 7#f p] c5t47 b\illon to ! upp] level ( ! piece ( glass tub+4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree successive days4 #b4 ,kill a small rabbit by means ( *lor(orm vap\r4 #c4 ,"u /rictly aseptic precautions remove ! lungs1 liv] & o!r solid organs & transf] !m to a /]ile d\ble glass di%4 #d4 ,) ! help ( /]ile scissors & =ceps divide ! organs into r\ghly rectangul> blocks #c x #a4e x #a cm4 #e4 ,p\r into ! di% a suffici5t quantity ( /]ile glyc]9e solution 7#f p] c5t4 9 normal sal9e71 cov]1 & all[ to /& = "o h\r4 #f4 ,introduce a block ( tissue into ea* tube s t x re/s ^u ! upp] end ( ! piece ( glass tub+4 7,! surface ( ! tissue w n[ be kept moi/ by capill>y attraction & cond5sation74 #g4 ,/]ilise 9 ! autoclave at #abj deg4 ,c4 = ?irty m9utes4 #h4 ,cap ! tubes & /ore !m 9 ! ice *e/ = future use4 ,tissues obta9$ at po/mortems c also be us$ aft] prelim9>y /]ilisation by boil+ or autoclav+4 ,m$ia = ! ,/udy ( ,special ,cocci4 ,diplococcus ,gonorrhoeae4 ,ascitic ,b\illon 7,s]um ,b\illon74-- #a4 ,collect ascitic fluid 7pleuritic fluid1 hydrocele fluid1 etc471 by aspiration directly into /]ile flasks1 "u /rictly aseptic precautions4 #b4 ,mix ! s]um ) twice its bulk ( /]ile nutri5t b\illon 7vide page #afc74 #c4 ,if consid]$ necess>y 7on acc\nt ( ! pres5ce ( blood1 cry/als1 etc471 filt] ! s]um b\illon ?r\gh porcela9 filt] c&le4 #d4 ,tube1 & /]ilise 9 ! wat] ba? at #ef deg4 ,c4 = half an h\r on ea* ( five consecutive days4 #e4 ,incubate at #cg deg4 ,c4 = =ty-eight h\rs & elim9ate contam9at$ tubes4 ,/ore ! rema9d] = future use4 ,s]um ,ag> 7,heiman74-- #a4 ,prep>e nutri5t ag> 7vide page #afg71 to foll[+ =mula3 ,ag> #b4j p] c5t4 ,peptone #a4e p] c5t4 ,salt #j4e p] c5t4 ,meat extract quantum sufficit4 #b4 ,make reaction ( m$ium #aj4 #c4 ,filt]2 tube 9 quantities ( #f c4c4 #d4 ,/]ilise z = nutri5t ag>4 #e4 ,aft] ! ?ird /]ilisation cool ! tubes to #db deg4 ,c41 & add to ea* #c c4c4 ( /]ile hydrocele fluid1 ascitic fluid1 or pleuritic effusion 7previ\sly /]ilis$1 if necess>y1 by ! fractional me?od72 all[ ! tubes to solidify 9 a slop+ position4 #f4 ,:5 solid1 incubate at #cg deg4 ,c4 = =ty-eight h\rs1 & elim9ate any contam9at$ tubes4 ,/ore ! rema9d] = future use4 ,s]um ,ag> 7,w]!im]74-- #a4 ,prep>e nutri5t ag> 7vide page #afg71 to ! foll[+ =mula3 ,ag> #b4j p] c5t4 ,peptone #b4j p] c5t4 ,salt #j4e p] c5t4 ,meat extract quantum sufficit4 #b4 ,make reaction ( m$ium #aj4 #c4 ,filt]2 tube 9 quantities ( #e c4c4 #d4 ,/]ilise z = nutri5t ag>4 #e4 ,aft] ! la/ /]ilisation cool to #db deg4 ,c41 !n add #e c4c4 /]ile blood-s]um f human plac5ta 7/]ilis$1 if necess>y1 by ! fractional me?od7 to ea* tube2 slope ! tubes4 #f4 ,:5 solid1 incubate at #cg deg4 ,c4 = =ty-eight h\rs1 & elim9ate any contam9at$ tubes4 ,/ore ! rema9d] = future use4 ,s]um ,ag> 7,kan?ack & ,/ev5s74-- #a4 ,collect ascitic1 pleuritic1 or hydrocele fluid 9 /]ile flasks & all[ to /& 9 ! ice-*e/ = twelve h\rs to s$im5t4 #b4 ,decant #ajjj c4c4 ( ! cle> fluid into a measur+ cyl9d] & transf] to /]ile litre flask4 #c4 ,add #j4e c4c4 dekanormal ,na,o,h solution = e #ajj c4c4 s]um 7i4 e41 #e4j c4c471 & mix ?or\ghly4 #d4 ,heat 9 ! /eam] = tw5ty m9utes4 #e4 ,weigh out #ae grammes ag>1 emulsify 9 a sep>ate vessel ) #bjj c4c4 ( ! alkal9e fluid previ\sly cool$ to ab\t #bj deg4 ,c41 & !n add to ! rema9d] ( ! fluid 9 ! flask4 #f4 ,bubble live /eam ?r\gh ! mixture = tw5ty m9utes to dissolve ! ag>4 #g4 ,filt] ?r\gh papi] ,*>d91 us+ a hot-wat] funnel4 #h4 ,weigh out glucose #aj grammes 7 #a p] c5t471 & dissolve x 9 ! cle> ag>4 #h;a4 ,if desir$1 add glyc]9e1 #e p] c5t41 to ! cle> ag>4 #i4 ,tube1 & /]ilise z = nutri5t ag>4 ,s]um ,ag> 7,libman74-- #a4 ,prep>e nutri5t ag> 7vide1 page #afg7 us+1 h[ev]1 #a4e p] c5t4 peptone 7t is #ae grammes p] litre in/ead ( #aj grammes74 #b4 ,adju/ ! reaction to #j 7i4 e41 neutral to ph5olph?ale974 #c4 ,filt] & transf] #ajjj c4c4 liquefi$ m$ium to a /]ile flask4 #d4 ,weigh out dextrose #bj grammes & dissolve 9 ! fluid ag>4 #e4 ,tube 9 quantities ( #f c4c42 & /]ilise 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes on ea* ( ?ree consecutive days4 #f4 ,aft] ! ?ird /]ilisation cool to #db deg4 ,c4 & add to ea* tube #c c4c4 ( /]ile hydrocele fluid1 ascitic fluid or pleuritic effusion 7previ\sly /]ilis$1 if necess>y1 by ! fractional me?od72 all[ ! tubes to solidify 9 a slop+ position4 #g4 ,:5 solid1 incubate at #cg deg4 ,c4 = =ty-eight h\rs1 & elim9ate any contam9at$ tubes4 ,/ore ! rema9d] = future use4 ,egg-album51 ,inspissat$4-- #a4 ,break sev]al fre% eggs 7h5s'1 ducks'1 or turkeys' eggs71 & collect ! :ites 9 a graduat$ cyl9d]1 tak+ c>e to avoid admixture ) ! yolks4 #b4 ,add #dj p] c5t4 di/ill$ wat]1 & incorporate ! mixture ?or\ghly by ! aid ( an egg-:isk4 #c4 ,weigh out #j4ae p] c5t4 sodium hydrate & dissolve x 9 ! fluid 7or add ! am\nt ( dekanormal cau/ic soda solution calculat$ to yield ! requir$ p]c5tage ( soda 9 ! total bulk ( ! fluid--i4 e41 #j4cge c4c4 ( dekanormal ,na,o,h solution p] #ajj c4c4 ( ! mixture74 #c;a4 ,glucose to ! ext5t ( #a to #b p] c5t4 may n[ be add$1 if desir$4 #d4 ,/ra9 ! mixture ?r\gh butt] musl9 & filt] ?r\gh a porcela9 filt] c&le into a /]ile filt] flask4 #e4 ,tube1 & /iff5 at #ajj deg4 ,c4 9 ! s]um inspissator4 #f4 ,incubate at #cg deg4 ,c4 = =ty-eight h\rs & elim9ate any contam9at$ tubes2 /ore ! rema9d] = future use4 ,egg-album5 7,t>*an(f & ,kolesnik(f74-- #a4 ,place unbrok5 h5s' eggs 9 dekanormal cau/ic soda solution = t5 days4 7,aft] ?is "t ! :ite becomes firm l gelat9e47 #b4 ,c>efully remove ! %ell & cut ! egg into f9e slices4 #c4 ,wa% = two h\rs 9 runn+ wat]4 #d4 ,place ! egg slices 9 a l>ge beak] & /]ilise 9 ! /eam] at #ajj deg4 ,c4 = "o h\r4 #e4 ,transf] ea* slice ( egg by means ( a pair ( /]ilis$ =ceps to a ,petri di% or l>ge capsule4 #f4 ,/]ilise 9 ! /eam] at #ajj deg4 ,c4 = tw5ty m9utes on ea* ( ?ree consecutive days4 ,egg ,album9 ,bro? 7,lips*uetz74-- #a4 ,weigh out ,egg album9 7extra f9e p[d]1 ,m]ck74 #d grammes & place 9 a #b-litre flask ) a numb] ( /]ile glass beads4 #b4 ,measure out di/ill$ wat] #bjj c4c4 into a half-litre flask & w>m to #cg deg4 ,c4 9 ! incubator4 #c4 ,add ! wat] to ! flask conta9+ ! album9 & beads & dissolve by %ak+4 #d4 ,add n_/#aj-,na,o,h1 #dj c4c4 ,all[ ! mixture to /& = ?irty m9utes ) frequ5t %ak+4 #e4 ,filt] ?r\gh ,sw$i% filt] pap]4 #f4 ,/]ilise by boil+ two or ?ree times at int]vals ( two h\rs4 #g4 ,add ord9>y nutri5t b\illon #fjj c4c4 #h4 ,fill into small ,erl5mey] flasks 9 quantities ( #ej c4c4 #i4 ,incubate = =ty-eight h\rs at #cg deg4 ,c4--disc>d any contam9at$ flasks & /ore ! rema9d] = future use4 ,egg ,album9 ,ag>4-- #a4 ,prep>e egg album9 solution z above #a-f4 #b4 ,liquefy & measure out ord9>y nutri5t ag> #fjj c4c4 & add to ! egg album9 solution 79 place ( ! nutri5t bro?74 #c4 ,complete z above #h-i4 ,diplococcus ,m5+itidis ,intracellul>is4 ,ascitic ,fluid ,ag> 7,wass]mann7 ,synonym ,n-z-g> 7,m]vyn ,gordon74 #a4 ,liquefy & measure out into a /]ile flask3 ,nutri5t ag> #fjj c4c4 #b4 ,measure out into a half litre flask ,di/ill$ wat] #baj c4c4 & add to x ,ascitic fluid #ij c4c4 ,nutrose #f grammes #c4 ,heat ov] a buns5 flame1 %ak+ con/antly until ! fluid boils1 & ! nutrose is dissolv$4 #d4 ,add ! nutrose ascitic solution to ! fluid ag>4 #e4 ,heat 9 ! /eam] = ?irty m9utes1 !n filt]4 #f4 ,tube & /]ilise z = nutri5t ag>4 ,,note4--,! f9i%$ m$ium 9 ?is case measures #ijj c4c4 only s9ce inconv5i5t fractions w\ld be introduc$ 9 mak+ up to "o litre exactly4 ,diplococcus ,pneumoniae4 ,blood ,ag> 7,wa%b\rn74-- #a4 ,melt up sev]al tubes ( nutri5t ag> 7vide page #afg7 & all[ !m to solidify 9 ! oblique position4 #b4 ,place ! tubes1 9 ! horizontal position1 9 ! hot incubator = =ty-eight h\rs1 to evaporate (f "s ( ! cond5sation wat]4 #c4 ,kill a small rabbit ) *lor(orm & nail x out on a bo>d 7z = a necropsy74 ,moi/5 ! hair ?or\ghly ) #b p] c5t4 solution ( lysol4 #d4 ,/]ilise sev]al pairs ( =ceps1 scissors1 etc41 by boil+4 #e4 ,reflect ! sk9 ov] ! ?orax ) /]ile in/rum5ts4 #f4 ,op5 ! ?oracic cavity by ! aid ( a fre% set ( /]ile in/rum5ts4 #g4 ,op5 ! p]ic>dium ) ano!r set ( /]ile in/rum5ts4 #h4 ,se> ! surface ( ! left v5tricle ) a r$-hot iron & remove fluid blood f ! he>t by means ( /]ile pipettes 7e4 g41 ^? %[n 9 ,fig4 #ac1 c74 #i4 ,deliv] a small quantity ( ! blood on ! slant$ surface ( ! ag> 9 ea* ( ! tubes1 & all[ x to run ov] ! entire surface ( ! m$ium4 #aj4 ,place ! tubes 9 ! slant+ position & all[ ! blood to coagulate4 #aa4 ,return ! blood ag> to ! hot incubator = =ty-eight h\rs & elim9ate any contam9at$ tubes4 ,/ore ! rema9d] = future use4 ,m$ia = ! ,/udy ( ,m\? ,bact]ia ,g5]ally4 ,potato ,gelat9e 7,goadby74-- #a4 ,prep>e glyc]9e potato bro? 7see page #bjc1 sections #a to #e74 #b4 ,add #aj p] c5t4 gelat9e to ! potato decoction & bubble live /eam ?r\gh ! mixture = t5 m9utes4 #c4 ,e/imate ! reaction2 adju/ ! reaction ( ! m$ium to #e4 #d4 ,cool ! m$ium to bel[ #fj deg4 ,c41 cl>ify ) egg z = nutri5t gelat9e4 #e4 ,filt] ?r\gh papi] ,*>d94 #f4 ,tube1 & /]ilise z = nutri5t gelat9e4 ,m$ia = ! ,/udy ( ,protozoa4 ,tissue ,m$ium 7,nogu*i74--,= spiro*aetes 7cultivations mu/ be gr[n ana]obically74 #a4 ,plug & /]ilise te/-tubes #bj x #b cm4 #b4 ,kill a small rabbit ) *lor(orm vap\r4 ,op5 ! abdom5 ) all aseptic precautions1 remove kidneys & te/icles & transf] to a /]ile glass di%4 ,cut up ! organs ) /]ile scissors into small pieces--say #d millimetre cubes4 ,! f\r organs %\ld yield f #be to #cj pieces ( tissue4 #c4 ,drop a small piece ( /]ile tissue into ! bottom ( ea* /]ilis$ tube4 #d4 ,take a flask conta9+ ab\t #djj c4c4 nutri5t ag> 7#aj reaction71 liquefy ! m$ium by heat & cool 9 a wat] ba? to #ej deg4 ,c4 #e4 ,add #bjj c4c4 ascitic or hydrocele fluid 7horse or %eep s]um may be employ$1 b is n s good7 to ! liquid ag> & mix c>efully to avoid =mation ( air bubbles4 #f4 ,fill ab\t #bj c4c4 ( ! ascitic ag> into ea* ( ! /]ilis$ tubes :i* already conta9s a piece ( /]ile rabbit's tissue1 /& all ! tubes upright 9 racks or a j>1 & all[ ag> to set4 #g4 ,aft] solidification p\r /]ile p>aff9 oil on ! surface ( ! m$ium 9 ea* tube to ! dep? ( #c c5timetres4 #h4 ,incubate tubes at #cg deg4 ,c4 = sev]al days & disc>d any :i* prove to be contam9at$4 #i4 ,/ore su* tubes z are /]ile = future use4 ,,xiii4 ,,incubators4 ,illu/ration3 ,,fig4 #aac4--;,incubator4 ,an incubator 7,fig4 #aac7 consi/s ess5tially ( a *amb] = ! reception ( cultivations1 etc41 surr\nd$ by a wat] jacket1 ! walls ( :i* are ( metal1 usually copp]1 & outside all an asbe/os or felt jacket1 or wood5 cas+4 ,! wat] 9 ! jacket is heat$ by gas or electricity & ma9ta9$ at "s con/ant temp]ature by a !rmo-regulator4 ,! cellul> incubator 7,fig4 #aad7 :i* 0 made = me#g "s ye>s ago is ( ! greate/ practical utility4 ,"h ! c5tral cavity is subdivid$ by five d\ble-wall$ p>titions 79 :i* wat] circulates 9 connection ) ! wat] tanks at ! top & base ( ! incubator7 & aga9 by iron %elves to =m tw5ty-f\r pigeon holes4 ,into ea* ( ^! slides an iron draw] #ce cm4 long x #ab cm4 wide x #bb cm4 high =m+ a self-conta9$ incubator4 ,! draw] is fitt$ ) a wood5 =m to :i* is fix$ a h&le & a numb]$ label4 ,! !rmo-regulat+ app>atus is ! well-kn[n ,he>son capsule4 ,illu/ration3 ,,fig4 #aad4--;,cellul> incubator4 ,two incubators at lea/ are requir$ 9 ! laboratory1 = ! cultivation ( bact]ia ! "o regulat$ to ma9ta9 a temp]ature ( #cg deg4 ,c41 & kn[n z ! hot incubator2 ! o!r1 #bj deg4 ,c4 to #bb deg4 ,c41 & kn[n z ! cool or cold incubator4 ,two o!r incubators1 regulat$ to #db deg4 ,c4 & #fj deg4 ,c4 respectively1 :il/ n absolutely1 necess>y v soon ju/ify _! pur*ase4 ,!rmo-regulators4--,! !rmo-regulator is ! mo/ ess5tial portion ( ! incubator1 z ^u its effici5t work+ dep5ds ! ma9t5ance ( a con/ant temp]ature 9 ! cultivation *amb]4 ,x is also us$ 9 ! fitt+ up ( wat] & p>aff9 ba?s1 & = _m o!r purposes4 ,illu/ration3 ,,fig4 #aae4--,rei*]t's !rmo-regulator4 ,( ! _m =ms & v>ieties ( !rmo-regulator 7o!r ?an electrical71 two only are ( suffici5tly g5]al use to ne$ m5tion4 ,9 "o ( ^! ! fl[ ( gas to ! gas-jet is controll$ by ! expansion or contraction ( m]cury )9 a glass bulb2 9 ! o!r1 by alt]ations 9 ! position ( ! walls ( a metallic capsule conta9+ a fluid1 ! boil+-po9t ( :i* corresponds to ! temp]ature at :i* ! incubator is int5d$ to act4 ,!y are3 7a7 ,rei*]t's 7,fig4 #aae71 consi/s ( a bulb conta9+ m]cury :i* is to be susp5d$ 9 ! m$ium1 :e!r air or wat]1 ! temp]ature ( :i* x is desir$ to regulate4 ,gas ent]s at ,a1 & passes out to ! jet by ,b4 ,z ! temp]ature rises ! m]cury exp&s & cuts (f ! ma9 gas supply4 ,z ! temp]ature falls ! m]cury contracts & reop5s ! n>r[ tube ,c4 ,by means ( a ?umbscrew ,d 7:i* me*anically raises or l[]s ! column ( m]cury irrespective ( ! temp]ature7 & ! aid ( a !rmomet] ! app>atus c be set to keep ! incubator at any desir$ temp]ature4 ,) ?is =m a special gas burn] is requir$1 ) sep>ate supply ( gas to a pilot jet at ! side4 7b7 ,he>son's capsule regulator consi/s ( a metal capsule h]metically seal$ & fill$ ) a liquid :i* boils at ! requir$ temp]ature1 ?is is adju/$ 9 ! int]ior ( ! incubator4 ,sold]$ to ! upp] side ( ! capsule is a ?ick piece ( metal hav+ a c5tral cup to receive ! l[] end ( a rigid rod1 ?r\gh :i* ! movem5ts ( ! walls ( ! capsule are transmitt$ to ! gas valve fix$ outside ! incubator4 ,! gas valve or gov]nor is %[n 9 figure #aaf4 ,a is ! inlet = gas1 ,c ! outlet to burn] heat+ ! wat] jacket1 ,b ,d a lev] pivot$ to /&>ds at ,g1 & act$ ^u by ! capsule1 ?r\gh ! rigid rod :i* ent]s ! socket bel[ ! screw ,p4 ,illu/ration3 ,,fig4 #aaf4--;,capsule !rmo-regulator4 ,! con/ruction ( ! valve is su* t1 :5ev] ! %ort arm ( ! lev] ,b ,d presses on ! disc bel[ ! end ,b1 ! ma9 supply ( gas is entirely cut (f4 ,at su* times1 h[ev]1 a v small portion ( gas passes f ,a to ,c1 ?r\gh an ap]ture inside ! valve1 ! size ( :i* ap]ture c be adju/$ by ! screw ne$le ,s1 h5ce ! gas flame bel[ ! incubator is nev] ext+ui%$4 ,! expansion ( ! metal walls ( ! capsule1 :i* takes place ^u ! boil+ ( its cont5ts1 provides ! motive =ce1 transmitt$ ?r\gh ! rigid rod to raise ! long arm ( ! lev] ,b ,d1 & z ?is expansion only takes place at a pr$et]m9$ temp]ature1 ! lev] w only be act$ ^u :5 ! critical temp]ature is rea*$1 no s5sible effect be+ produc$ at ev5 #a deg4 ,c4 bel[ t at :i* ! capsule is de/9$ to act4 ,w is a weight slid+ on ! lev] rod ,d2 by increas+ ! di/ance betwe5 ! weight & ! fulcrum ( ! l[] increas$ pressure is br\ght to be> ^u ! walls ( ! capsule ) ! result t ! boil+-po9t ( ! liquid 9 ! capsule is slightly rais$1 & a range ( ab\t two degrees c ?us be obta9$ ) any p>ticul> capsule4 ,,footnotes3 #g ,made by ! firm ( ,*as4 ,he>son ,co41 #bce ,reg5t ,/41 ,london1 ,w4 ,,xiv4 ,,me?ods ,,( ,,cultivation4 ,cultivations ( micro-organisms are usually prep>$ 9 ! laboratory 9 "o ( ?ree ways3 ,tube cultures4 ,plate cultures4 ,hang+-drop cultures4 ,^! may be incubat$ ei!r a]obically 7i4 e41 9 ! pres5ce ( oxyg57 or ana]obically 7i4 e41 9 ! abs5ce ( oxyg51 or 9 ! pres5ce ( an indiff]5t gas1 su* z hydrog51 nitrog51 or c>bon dioxide74 ,) reg>d to ! temp]ature at :i* ! cultivations are gr[n1 x may be /at$ z a g5]al rule t all m$ia r5d]$ solid by ! addition ( gelat9e are incubat$ at #bj deg4 ,c41 or at any rate at a temp]ature n exce$+ #bb deg4 ,c4 7t is1 9 ! cold incubator72 :il/ fluid m$ia & all o!r solid m$ia are incubat$ at #cg deg4 ,c4 7t is1 9 ! hot incubator74 ,exceptions to ?is rule are num]\s4 ,= in/ance1 9 /udy+ ! gr[? ( ! psy*rophylic bact]ia1 ! yea/s & ! m\lds1 ! cold incubator is employ$ = all m$ia4 ,tube cultivations are usually pack$ 9 ! incubator 9 small t9 cyl9d]s1 su* z ^? 9 :i* ,am]ican cig>ettes are sold1 or 9 squ>e t9 boxes4 ,beak]s or tumbl]s may be us$ = ! same purpose1 b be+ fragile are n s conv5i5t4 ,metal te/-tube racks1 long en\gh to j fit into ! int]ior ( ! incubator & ea* accommodat+ two r[s ( tubes1 are also exce$+ly useful4 ,,a]obic4 ,! ,prep>ation ( ,tube ,cultivations4 ,! prep>ation ( a tube cultivation consi/s 93 7a7 ,inoculat+ a tube ( /]ile nutri5t m$ium ) a portion ( ! mat]ial to be exam9$4 7b7 ,incubat+ ! inoculat$ tube at a suitable temp]ature4 ,! details ( ! fir/ ( ^! processes mu/ be v>i$ some:at accord+ to :e!r ! tubes ( nutri5t m$ia are inoculat$ or plant$ f-- #a4 ,pre-exi/+ cultivations4 #b4 ,morbid mat]ial previ\sly collect$ 7vide page #cgc74 #c4 ,fluids1 tissues1 etc41 or f ! animal body direct4 ,! me?od ( prep>+ tube cultivations f pre-exi/+ cultivations is z foll[s3 ,illu/ration3 ,,fig4 #aag4--;,inoculat+ tubes1 se5 f ! front4 #a4 ,fluid ,m$ia 7e4 g41 ,nutri5t ,b\illon74-- #a4 ,flame ! cotton-wool plug ( ! tube conta9+ ! cultivation & also t ( ! tube ( /]ile b\illon4 #b4 ,hold ! two tubes1 side by side1 betwe5 ! left ?umb & ! fir/ & ?ird f+]s1 all[+ ! seal$ ends to re/ on ! dorsum ( ! h&1 & sep>at+ ! m\?s ( ! tubes 7:i* are po9t$ to ! "r7 by ! tip ( ! second f+]4 ,keep ! tubes z ne>ly horizontal z is possible )\t all[+ ! fluid 9 ! b\illon tube to rea* ! cotton-wool plug 7,fig4 #aag74 #c4 ,/]ilise ! plat9um loop & all[ x to cool4#h #d4 ,grasp ! plug ( ! tube conta9+ ! cultivation betwe5 ! little f+] & palm ( ! h& & remove x f ! tube4 #e4 ,grasp ! plug ( ! b\illon tube betwe5 ! f\r? f+] & ! ball ( ! ?umb & remove x f ! tube4 #f4 ,pass ! plat9um loop into ! tube conta9+ ! culture--d n all[ ! loop to t\* ! sides ( ! tube1 or ! h&le to t\* ! m$ium--& remove a small portion ( ! gr[?2 )draw ! loop f ! tube1 keep+ ! infect$ side ( ! loop d[nw>d4 #g4 ,pass ! loop into ! b\illon tube almo/ d[n to ! level ( ! fluid1 rev]se ! loop s t ! infect$ side faces upw>d1 emulsify ! portion ( ! gr[? 9 ! moi/ure adh]+ to ! side ( ! tube :i* is upp]mo/4 ,)draw ! loop4 #h4 ,replug bo? tubes4 #i4 ,/]ilise ! plat9um loop4 #aj4 ,label ! b\illon tube ) 7a7 ! "n ( ! organism & 7b7 ! date ( inoculation4 #aa4 ,incubate4 #b4 ,solid ,m$ia4--,solid m$ia are /or$ 9 tubes 9 "o ( two ways3 #a4 ,oblique tube or slant$ tube 7,fig4 #aah71 9 :i* ! m$ium has be5 all[$ to solidify :il/ ! tube 0 reta9$ 9 an incl9$ position1 s =m+ an ext5sive surface ( m$ium ext5d+ f ! bottom ( ! tube almo/ to its m\?4 ,?is is employ$ = /reak or sme> cultivations 7,/ri*cultur74 #b4 ,/raight tube 7,fig4 #aai71 9 :i* ! m$ium =ms a cyl9drical mass 9 ! l[] portion ( ! tube & pres5ts an upp] surface :i* is at "r angles to ! long axis ( ! tube4 ,?is is employ$ = /ab or /ick cultivations 7,/i*cultur71 or by inoculat+ ! m$ium :il/ fluid1 & all[+ to solidify 9 ?is position1 = %ake cultivations4 ,/reak ,culture4-- #a4 ,flame ! plugs1 /]ilise ! plat9um loop 7or spatula74 ,op5 ! tubes & *>ge ! loop z 9 previ\s inoculation4 #b4 ,pass ! infect$ loop to ! bottom ( ! tube to be inoculat$ & draw x1 z lightly z possible1 along ! c5tre ( ! surface ( ! m$ium1 t]m9at+ ! /reak ov] ! ?9 lay] ( m$ium ne> ! m\? ( ! tube4 #c4 ,replug ! tubes1 /]ilise ! plat9um loop4 #d4 ,label ! newly inoculat$ tube & incubate4 ,sme> ,culture4--,proce$ g5]ally z 9 /reak culture1 b rub ! infect$ loop all ov] ! surface ( ! m$ium1 in/ead ( re/rict+ ! inoculation to a n>r[ l9e4 ,,note4--,gelat9e & ag> oblique tubes %\ld be fre%ly slant$ be=e use4 ,/ab ,culture4-- #a4 ,flame ! plugs1 op5 ! tubes1 /]ilise ! plat9um ne$le & *>ge x ) ! inoculum z 9 ! previ\s cultivations4 #b4 ,pass ! plat9um ne$le into ! tube to be inoculat$ until x t\*es ! c5tre ( ! surface ( ! m$ium4 ,n[ ?ru/ x deeply into ! sub/ance ( ! m$ium1 keep+ ! ne$le z ne>ly z possible 9 ! axis ( ! cyl9d] ( m$ium4 ,!n )draw ! ne$le4 #c4 ,replug ! tubes4 ,/]ilise ! plat9um ne$le4 #d4 ,label ! newly plant$ tube & incubate4 ,,note4--,:5 gelat9e is /or$ = "s "t ! upp] surface ( ! cyl9d] becomes concave ow+ to evaporation4 ,tubes %[+ ?is appe>ance %\ld be liquefi$ & aga9 all[$ to set be=e use = /ab culture1 o!rwise :5 ! ne$le ent]s ! m$ium1 ! surface t5sion w cause ! gelat9e cyl9d] to split4 ,illu/ration3 ,,fig4 #aah4--,slop$ or slant$ m$ium = /reak or sme> culture4 ,illu/ration3 ,,fig4 #aai4--,/raight tube4 ,%ake ,culture4-- #a4 ,liquefy a tube ( nutri5t gelat9e 7or ag>1 or o!r simil> m$ium71 by heat+ 9 a wat]-ba? 7,fig4 #aba74 #b4 ,inoculate ! liquefi$ m$ium & label x1 etc41 precisely z if deal+ ) a tube ( b\illon4 #c4 ,place ! newly plant$ tube 9 ! upright position 7e4 g41 9 a te/-tube rack7 & all[ x to solidify4 #d4 ,label ! tube2 :5 solid1 incubate4 ,esm>*'s ,roll ,cultivation4-- #a4 ,liquefy ?ree tubes ( gelat9e by heat4 #b4 ,prep>e ?ree dilutions ( ! inoculum 7z describ$ = plate cultivations1 page #bbh1 /eps #d to #g74 #c4 ,roll ! tubes1 held almo/ horizontally1 9 a groove made 9 a block ( ice1 until ! gelat9e has set 9 a ?9 film on ! inn] surface ( tube 7,fig4 #abj72 or "u ! cold-wat] tap4 ,illu/ration3 ,,fig4 #abj4 ,esm>*'s roll culture on block ( ice4 ,9 ord] t ! m$ium may adh]e firmly to ! glass1 ! ag> us$ = roll cultivation %\ld h #a p] c5t4 gelat9e or #a p] c5t4 gum arabic add$ to x be=e /]ilisation4 ,roll cultivations1 :i* s]v$ a mo/ important purpose 9 ! days be=e ! introduction ( ,petri di%es = plate cultivations1 are n[ obsolete 9 mod]n laboratories & are m]ely m5tion$ = ! b5efit ( /ud5ts1 s9ce exam9]s :o are int]e/$ 9 ! academic & hi/orical aspects ( bact]iology sometimes expect c&idates to be acqua9t$ ) ! me?od ( prep>+ !m4 ,! ,prep>ation ( ,plate ,cultures4 ,if a small numb] ( bact]ia are susp5d$ 9 liquefi$ gelat9e1 ag>1 or o!r simil> m$ium1 & ! infect$ m$ium spread out 9 an ev5 lay] ov] a flat surface & all[$ to solidify1 ea* individual micro-organism becomes fix$ to a c]ta9 spot & its fur!r developm5t is re/rict$ to ! vic9ity ( ?is spot4 ,aft] a v>iable int]val ! gr[? ( ?is organism becomes visible to ! nak$ eye z a colony4 ,?is is ! pr9ciple ^u :i* ! me?od ( plate cultivation is bas$ & its practice enables ! bact]iologi/ to /udy ! p>ticul> mann] ( developm5t affect$ by ea* species ( microbe :5 gr[+ 7a7 unre/rict$ ^u ! surface ( ! m$ium1 7b7 9 ! dep?s ( ! m$ium4 ,! me?od itself is z foll[s3 ,app>atus ,requir$4-- #a4 ,tripod levell+ /&4 #b4 ,l>ge %all[ glass di%1 ) a squ>e %eet ( plate glass to cov] x4 #c4 ,_s level4 #d4 ,case ( /]ile ,petri di%es4 #e4 ,tubes ( /]ile nutri5t m$ia1 gelat9e 7or ag>7 previ\sly liquefi$ by heat+ 9 ! wat]-ba? & cool$ to #db deg4 ,c41 o!rwise ! heat ( ! m$ium w\ld de/roy _m1 if n all1 ( ! bact]ia introduc$4 #f4 ,tube ( cultivation to be plant$ f4 #g4 ,plat9um loop4 #h4 ,buns5 burn]4 #i4 ,grease p5cil4 ,illu/ration3 ,,fig4 #aba4--;,h&y =m ( wat]-ba? = melt+ tubes ( ag> & gelat9e previ\s to slant+ !m2 or to mak+ %ake cultures or p\r+ plates4 ,me?od ( ,p\r+ ,plates4-- #a4 ,place ! glass di% on ! levell+ tripod 7,figs4 #abb1 #abc72 if gelat9e plates are to be p\r$ fill ! di% ) ice wat]--gelat9e solidifies s sl[ly t x is necess>y to ha/5 ! process2 if ag> is to be us$ fill ) wat] at #ej deg4 ,c4--ag> sets almo/ imm$iately at ! room temp]ature & by slightly ret>d+ ! process lump9ess is avoid$2 cov] ! di% ) ! squ>e %eet ( glass4 #b4 ,place ! _s level on ! %eet ( glass & by means ( ! levell+ screws adju/ ! surface ( ! glass to ! horizontal4 ,?is level+ is an important matt] s9ce ! developm5t ( a colony is to "s ext5t proportionate to ! supply ( m$ium available = its nutrition4 ,?us 9 a sme> on slop$ tube culture1 ! colonies at ! upp] "p ( ! m$ium are /unt$ & small b increase 9 size & luxuriance ( gr[? ! ne>] !y approa* to ! bottom ( ! tube1 :]e !re is ! greate/ dep? ( m$ium4 ,illu/ration3 ,,fig4 #abb4--,plate-levell+ /&4 #c4 ,place ?ree /]ile ,petri di%es 9 a r[ on ! surface ( ! glass plate & numb] !m #a1 #b1 & #c1 f left to "r4 ,illu/ration3 ,,fig4 #abc4--,plate-levell+ /&1 side view4 #d4 ,numb] ! previ\sly liquefi$ tubes ( nutri5t m$ia #a1 #b1 & #c4 ,flame ! plugs & see t ea* plug c be readily remov$ f ! m\? ( its tube4 #e4 ,add "o loopful ( ! inoculum to tube ,no4 #a1 treat+ ! liquefi$ m$ium z b\illon4 ,aft] replugg+1 grasp ! tube ne> its m\? by ! ?umb & fir/ f+] ( ! "r h&1 & ) an ev5 circul> movem5t ( ! :ole arm1 diffuse ! inoculum ?r\gh\t ! m$ium2 avoid j]ky movem5ts1 z ^! cause bubbles ( air to =m 9 ! m$ium4 ,illu/ration3 ,,fig4 #abd4--,mix+ emulsion = plates4 ,! knack ( mix+ ev5ly )\t produc+ air bubbles1 is n always easily acquir$1 by ?is me?od4 ,an alt]native plan is to hold ! inoculat$ tube v]tically upright betwe5 ! oppos$ palms & to rotate x betwe5 !m by rapid backw>d & =w>d movem5ts ( ! two h&s 7,fig4 #abd74 ,illu/ration3 ,,fig4 #abe4--,p\r+ plates4 #f4 ,/]ilise ! plat9um loop1 & add two loopfuls ( dilut$ inoculum to tube ,no4 #b1 & mix z be=e4 #g4 ,9 a simil> mann] transf] ?ree loopfuls ( liquefi$ m$ium f tube ,no4 #b to tube ,no4 #c1 & mix ?or\ghly4 #h4 ,flame ! plug ( tube ,no4 #a1 remove x1 !n flame ! lips ( ! tube2 slightly raise ! cov] ( ,petri di% ,no4 #a1 introduce ! m\? ( ! tube2 !n1 elevat+ ! bottom ( ! tube1 p\r ! liquefi$ m$ium into ! ,petri di%1 to =m a ?9 lay]4 ,remove ! m\? ( ! tube & close ! plate4 ,if ! m$ium has fail$ to fl[ ev5ly ov] ! bottom ( ! plate1 raise ! plate f ! levell+ plat=m & by tilt+ 9 diff]5t directions rectify ! fault4 #i4 ,p\r plates ,no4 #b & ,no4 #c1 9 a simil> mann]1 f tubes ,nos4 #b & #c4 #aj4 ,label ! plates ) ! di/9ctive "n or numb] ( ! inoculum1 also ! date2 ! numb] ( ! dilution hav+ be5 previ\sly indicat$ 7/ep #c74 #aa4 ,place 9 ! cool incubator = ?ree or m days1 z may be necess>y4 ,9 ?is way colonies may be obta9$ q pure & sep>ate f ea* o!r4 ,9 plate ,no4 #a1 probably1 ! colonies w be s num]\s & cr[d$1 & !re=e s small1 z to r5d] x useless4 ,9 plate ,no4 #b !y w be m widely sep>at$1 b usually ,no4 #c is ! plate res]v$ = c>eful exam9ation1 z 9 ?is ! colonies are usually widely sep>at$1 few 9 numb]1 & l>ge 9 size4 ,ag> plates are p\r$ 9 a simil> mann]1 b ! ag> mu/ be melt$ 9 boil+ wat] & !n all[$ to cool to #de deg4 ,c4 or #db deg4 ,c4 9 a c>efully regulat$ wat]-ba? be=e be+ inoculat$1 & ! entire process mu/ be c>ri$ out v rapidly1 o!rwise ! ag> w h solidifi$ be=e ! op]ation is complet$4 ,,note4--,9 p\r+ plates1 s9ce tube ,no4 #a 7= ! fir/ dilution7 r>ely gives a plate t is ( any practical value x is frequ5tly replac$ by a tube ( b\illon or /]ile salt solution1 & 9 su* case plate ,no4 #a is n p\r$4 ,surface ,plates4-- ,?is me?od ( p\r+ :at may be t]m$ :ole plates 7s9ce colonies may appe> bo? on ! surface & 9 ! dep?s ( ! m$ium7 is ess5tial to ! accurate /udy ( ! =mation ( colonies "u v>i\s conditions1 b :5 ! ma9 object ( ! sep>ation ( ! bact]ia is to obta9 subcultivations f a numb] ( individual bact]ia1 surface plates mu/ be prep>$1 s9ce "h colony =mation is re/rict$ to ! surface ( ! m$ium4 ,! me?od adopt$ v>ies slightly accord+ to :e!r ! m$ium employ$ is gelat9e or ag>1 or "o ( ! d]ivatives or v>iants ( ! latt]4 7a7 ,gelat9e ,surface ,plates4-- #a4 ,liquefy ?ree tubes ( nutri5t gelat9e4 #b4 ,p\r ea* tube into a sep>ate ,petri di% & all[ x to solidify4 ,!n turn ea* plate & its cov] upside d[n4 ,illu/ration3 ,,fig4 #abf4--,surface plate spread]4 #c4 ,:5 q cold raise ! bottom ( plate #a1 rev]t x & deposit a drop ( ! inoculum 7:e!r a fluid culture or an emulsion f solid culture7 ^u ! surface ( ! gelat9e ) a plat9um loop--close to "o side ( ! plate2 replace ! bottom half ( ! ,petri di% 9 its cov]4 #d4 ,take a piece ( ?9 glass rod1 /\t plat9um wire or be/ ( all a piece ( alum9ium wire 7say #b mm4 diamet]7 ab\t #bh cm4 long4 ,b5d ! t]m9al #d cm4 at "r angles to ! rema9d]1 mak+ an ,l-%ap$ rod 7,fig4 #abf74 ,/]ilise ! %ort arm & adjac5t portion ( ! long arm1 9 ! ,buns5 flame1 & all[ x to cool4 #e4 ,n[ raise ! bottom ( ! ,petri di% 9 ! left h&1 leav+ ! cov] on ! laboratory b5*1 & hold+ x v]tically1 sme> ! drop ( inoculum all ov] ! surface ( ! gelat9e ) ! %ort arm ( ! spread] by a rotatory motion1 7,fig4 #abg74 ,replace ! di% 9 its cov]4 #f4 ,raise ! bottom ( plate #b & rub ! infect$ spread] all ov] ! surface ( ! gelat9e--!n g on 9 l mann] to ! ?ird plate 9 ! s]ies4 #g4 ,/]ilise ! spread]4 #h4 ,label & incubate ! plates4 ,illu/ration3 ,,fig4 #abg4--,spread+ surface plate4 ,aft] incubation1 plate ,no4 #a w probably yield an enorm\s numb] ( colonies2 plate #b w %[ few] colonies1 s9ce only ^? bact]ia adh]+ to ! rod aft] rubb+ ov] plate #a w\ld be deposit$ on its surface1 & by ! "t ! rod rea*$ plate #c b v few organisms %\ld rema9 ^u x4 ,s t ! ?ird plate z a rule w only %[ a v few scatt]$ colonies1 em95tly suitable = detail$ /udy4 7b7 ,ag> ,surface ,plates4-- #a4 ,liquefy ?ree tubes ( nutri5t ag>--nutrose ag> or ! l4 #b4 ,p\r ea* tube into a sep>ate ,petri di% & all[ x to solidify4 #c4 ,:5 q solid inv]t ea* di%1 raise ! bottom half & re/ x obliquely on its inv]t$ cov] 7,fig4 #abh7 & place x 9 ?is position 9 an incubator at #fj deg4 ,c4 = =ty-five m9utes 7or 9 an incubator at #db deg4 ,c4 = two h\rs74 ,?is evaporates ! wat] ( cond5sation & gives ! m$ium a firm1 dry surface4 #d4 ,on remov+ ! plates f ! incubator close ea* di% & place x--/ill upside d[n--on ! laboratory b5*4 ,illu/ration3 ,,fig4 #abh4--;,dry+ surface plate ( ag>4 #e4 ,inoculate ! plates 9 s]ies ( ?ree1 z describ$ = gelat9e surface plates #c-h4 ,hang+-drop ,cultivation4 ,app>atus ,requir$4-- ,hang+-drop slides4 ,cov]-slips4 ,section rack 7,fig4 #ge74 ,blott+ pap]4 ,bell glass to cov] slides4 ,orig9al culture4 ,tubes ( bro?1 or liquefi$ gelat9e or ag>4 ,=ceps4 ,plat9um loop4 ,buns5 burn]4 ,grease p5cil4 ,/]ile vasel9e4 ,lysol4 7a7 ,fluid ,m$ia4-- #a4 ,prep>e fir/ & second dilutions ( ! inoculum z direct$ = plate cultivations 7vide pages #bbh-bbi1 sections #d to #f71 sub/itut+ tubes ( nutri5t bro? = ! liquefi$ gelat9e4 #b4 ,/]ilise a hang+-drop slide by wa%+ ?or\ghly 9 wat] & dry+1 !n plung+ x into a beak] ( absolute alcohol1 dra9+ (f ! great] "p ( ! _s1 grasp+ ! slide 9 a pair ( =ceps1 & burn+ (f ! rema9d] ( ! alcohol 9 ! flame4 #c4 ,place ! hang+-drop slide on a piece ( blott+ pap] moi/5$ ) #b p] c5t4 lysol solution & cov] x ) a small bell glass t has be5 r9s$ out ) ! same solution & n dri$4 #d4 ,raise ! bell glass slightly & sme> /]ile vasel9e ar\nd ! rim ( ! metal cell by means ( a /]ile spatula ( /\t plat9um wire4 #e4 ,remove a clean cov]-slip f ! alcohol pot ) /]ile =ceps & burn (f ! alcohol2 aga9 raise ! bell glass & place ! /]ile cov]-slip on ! blott+ pap] by ! side ( ! hang+-drop slide4 #f4 ,remove a drop ( ! bro? f ! second dilution tube ) a l>ge plat9um loop2 raise ! bell glass & deposit ! drop on ! c5tre ( ! cov]-slip4 ,/]ilise ! loop4 #g4 ,raise ! bell glass suffici5tly to all[ ( ! cov]-slip be+ grasp$ ) =ceps1 inv]t$1 & adju/$ ov] ! cell ( ! hang+-drop slide4 ,remove ! bell glass altoge!r & press ! cov]-slip firmly on to ! cell4 #h4 ,ei!r incubate & exam9e at def9ite int]vals1 or obs]ve cont9u\sly ) ! microscope1 us+ a w>m /age if necess>y 7,fig4 #ec74 7b7 ,solid ,m$ia4--,obs]v+ precisely simil> te*nique1 a few drops ( liquefi$ gelat9e or ag> f ! second dilution tube may be run ov] ! surface ( ! /]ile cov]-slip & a hang+-drop plate cultivation !reby prep>$4 ,?is me?od is extremely useful 9 connection ) ! /udy ( yea/s1 :5 ! circul> cell on ! hang+-drop slide %\ld be replac$ by a rectangul> cell "s #ch by #ai mm41 & ! gelat9e spread ov] a cov]-slip ( simil> size4 ,aft] seal+ d[n ! prep>ation1 ! upp] surface ( ! cov]-slip may be rul$ into squ>es by ! aid ( ! grease p5cil or a writ+ diamond & numb]$ to facilitate ! subsequ5t id5tification ( ! colonies :i* are obs]v$ to develop f solit>y g]ms4 ,hang+-block ,culture 7,hill74-- ,app>atus requir$3 ,z = hang+-drop cultivation ) ! addition ( a scalpel4 ,c>ry out ! me?od z f> z possible "u cov] ( a bell glass1 to avoid a]ial contam9ation4 #a4 ,liquefy a tube ( nutri5t ag> 7or gelat9e7 & p\r into a ,petri di% to ! dep? ( ab\t #d mm4 & all[ to set4 #b4 ,) a %>p scalpel cut out a block "s #h mm4 squ>e1 f ! entire ?ickness ( ! ag> lay]4 #c4 ,raise ! ag> block on ! blade ( ! scalpel & transf] x1 "u side d[n1 to ! c5tre ( a /]ile slide4 #d4 ,spread a drop ( fluid cultivation 7or an emulsion ( gr[? f a solid m$ium7 ov] ! upp] surface ( ! ag> block z if mak+ a cov]-slip film4 #e4 ,place ! slide & block cov]$ by ! bell glass 9 ! incubator at #cg deg4 ,c4 = t5 m9utes to dry slightly4 #f4 ,take a clean dry /]ile cov]-slip 9 a pair ( =ceps1 & ) ! help ( a second pair ( =ceps l[] x c>efully on ! inoculat$ surface ( ! ag> 7avoid+ air bubbles71 s z to leave a cle> m>g9 ( cov]-slip ov]lapp+ ! ag> block4 #g4 ,inv]t ! prep>ation & ) ! blade ( ! scalpel remove ! slide f ! ag> block4 #h4 ,) a plat9um loop run a drop or two ( melt$ ag> ar\nd ! edges ( ! block4 ,?is solidifies at once & seals ! block to ! cov]-slip4 #i4 ,prep>e a /]ile hang+-drop slide1 & sme> h>d vasel9e or melt$ :ite wax on ! rim ( ! metal cell4 #aj4 ,inv]t ! cov]-slip ) ! block atta*$ on to ! hang+-drop slide1 & seal ! cov]-slip firmly 9 place4 #aa4 ,obs]ve z = hang+-drop cultivations4 ,,ana]obic ,,cultivations4 ,num]\s me?ods h be5 devis$ = ! cultivation ( ana]obic bact]ia1 ! majority requir+ ! employm5t ( special app>atus4 ,! pr9ciple ^u :i* any me?od is bas$ & ^u :i* x dep5ds = its success falls "u "o or ano!r ( ! foll[+ head+s3 7a7 ,exclusion ( air f ! cultivation4 7b7 ,exhau/ion ( air f ! vessel conta9+ ! cultivation by means ( an air pump--i4 e41 cultivation 9 vacuo4 7c7 ,absorption ( oxyg5 f ! air 9 contact ) ! cultivation by means ( pyrogallic acid r5d]$ alkal9e ) cau/ic soda--i4 e41 cultivation 9 an atmosph]e ( nitrog54 7d7 ,displacem5t ( air by an indiff]5t gas1 su* z hydrog5 or coal gas--i4 e41 cultivation 9 an atmosph]e ( hydrog54 7e7 ,a comb9ation ( two or m ( ! above me?ods4 ,a selection ( ! simple/ & mo/ g5]ally useful me?ods is giv5 "h4 ,:5ev] possible1 ! nutri5t m$ia t are employ$ 9 any ( ! processes %\ld conta9 "s easily oxidisable sub/ance1 su* z sodium =mate 7#j4d p] c5t47 or sodium sulph9digotate 7#j4a p] c5t471 :i* w absorb all ! available oxyg5 held 9 solution by ! m$ium4 ,! fur!r addition ( glucose1 #b p] c5t41 favors ! gr[? ( ana]obic bact]ia 7vide1 pages #ahi-aij74 ,fur!r1 x is advisable to seal all jo9ts betwe5 india-rubb] /opp]s & tubulures or ! m\?s ( ! tubes ) melt$ p>aff92 glass /opp]s & taps %\ld be lubricat$ ) res9 o9tm5t or a mixture ( beeswax #a "p1 olive oil #d p>ts4 7,a7 ,me?od ,i 7,hesse's ,me?od74-- #a4 ,make a /ab culture 9 gelat9e or ag>1 *oos+ = ! purpose a /raight tube conta9+ a deep column ( m$ium1 & ?ru/+ ! inoculat+ ne$le to ! bottom ( ! tube4 #b4 ,p\r a lay] ( /]ilis$ oil 7olive oil1 vasel9e1 or petroleum71 #a or #b cm4 deep1 ^u ! surface ( ! m$ium4 #c4 ,incubate4 ,me?od ,,ii4--,?is me?od is only available :5 deal+ ) pure cultivations4 #a4 ,liquefy a tube ( gelat9e 7or ag>7 by heat1 p\r x into a ,petri di%1 & all[ x to solidify4 #b4 ,inoculate ! surface ( ! m$ium 9 "o spot only4 #c4 ,remove a cov]-slip f ! pot ( absolute alcohol ) /]ile =ceps2 burn (f ! alcohol 9 ! gas flame4 #d4 ,l[] ! n[ /]ile cov]-slip c>efully on to ! inoculat$ surface ( ! m$ium1 c>efully exclud+ air bubbles1 & press x d[n firmly ) ! po9ts ( ! =ceps4 7,a /]ile disc ( mica may be sub/itut$ = ! cov]-slip47 #e4 ,incubate4 ,me?od ,,iii 7,r\x's ,physical ,me?od74-- #a4 ,prep>e tube cultures ( fluid m$ia 7or solid m$ia r5d]$ fluid by heat7 9 ! usual way4 #b4 ,aspirate "s ( ! inoculat$ m$ia into capill>y pipettes4 #c4 ,seal bo? ends ( ea* pipette 9 ! bl[pipe flame4 #d4 ,incubate4 ,me?od ,,iv 7,r\x's ,biological ,me?od74-- #a4 ,plant a deep /ab1 z 9 me?od ,i4 #b4 ,p\r a lay]1 #a or #b cm4 deep1 ( bro? cultivation ( a vig\r\s a]obe--e4 g41 ,b4 aquatilis sulcatus or ,b4 prodigiosus--^u ! surface ( ! m$ium2 or an equal dep? ( liquefi$ gelat9e1 :i* is !n inoculat$ ) ! a]obic organism4 #c4 ,incubate4 ,! gr[? ( ! a]obe w use up all ! oxyg5 t rea*es x & w n all[ any to pass ?r\gh to ! m$ium bel[1 :i* w consequ5tly rema9 9 an ana]obic condition4 7,b7 ,me?od ,v4-- #a4 ,prep>e tube or flask cultivations 9 ! usual way4 #b4 ,replace ! cotton-wool plug by an india-rubb] /opp] p]=at$ ) "o hole & fitt$ ) a l5g? ( glass tub+ :i* has a con/riction ab\t #c cm4 above ! /opp] & is !n b5t at "r angles 7,fig4 #abi74 ,! /opp] & glass tub+ are /]ilis$ by be+ boil$ 9 a beak] ( wat] = five m9utes4 ,illu/ration3 ,,fig4 #abi4--,vacuum culture4 #c4 ,connect ! tube lead+ f ! culture vessel ) a wat] or air pump1 int]pos+ a ,wulff's bottle fitt$ z a wa%-bottle & conta9+ sulphuric acid4 #d4 ,exhau/ ! air f ! culture vessel4 #e4 ,be=e disconnect+ ! app>atus1 seal ! glass tube f ! culture vessel at ! con/riction1 us+ ! bl[pipe flame4 #f4 ,incubate4 7,c7 ,me?od ,,vi 7,bu*n]'s ,me?od74 ,app>atus & ,solutions ,requir$4-- ,bu*n]'s tube 7a /\t glass te/-tube #bc cm4 long & #d cm4 9 diamet]1 fitt$ ) india-rubb] /opp]1 ,fig4 #acj74 ,pyrogallic acid 9 compress$ tablets ea* conta9+ #a gram4 ,dekanormal solution ( cau/ic soda4 ,,me?od4-- #a4 ,prep>e ! tube cultivation 9 ! usual way4 #b4 ,moi/5 ! india-rubb] /opp] ( ! ,bu*n]'s tube ) wat] & see t x fits ! m\? ( ! tube accurately4 #c4 ,remove ! /opp] f ! cau/ic soda bottle4 #d4 ,drop "o ( ! pyrogallic acid tablets#i into ! ,bu*n]'s tube 7r\ghly1 use #a gramme pyrogallic acid = e #ajj c4c4 air capacity ( ! receiv+ vessel74 #e4 ,add ab\t #a c4c4 ( ! soda solution4 #f4 ,place ! inoculat$ tube inside ! ,bu*n]'s tube4 ,! pyrogallic tablet acts z a buff] & prev5ts damage to ei!r ! inoculat$ tube or ! ,bu*n]'s tube ev5 %\ld x be slipp$ 9 hurri$ly4 #g4 ,fit ! india-rubb] /opp] tightly into ! m\? ( ! ,bu*n]'s tube4 ,illu/ration3 ,,fig4 #acj4--;,bu*n]'s tube4 ,! pyrogallic acid tablet dissolves sl[ly 9 ! soda solution & its oxidation proce$s v sl[ly at fir/ s t ample "t is available :5 ?is me?od is adopt$4 #h4 ,re/opp] ! cau/ic soda bottle4 #i4 ,place ,bu*n]'s tube 9 a wire support1 & incubate4 ,me?od ,,vii 7,wright's ,me?od74-- #a4 ,prep>e tube cultivation 9 ! usual way4 #b4 ,cut (f t portion ( ! cotton-wool plug project+ above ! m\? ( ! tube ) scissors1 !n pu% ! plug into ! tube = a di/ance ( #b or #c cm4 #c4 ,by means ( a pipette drop ab\t #a c4c4 ( pyrogallic acid #aj p] c5t4 aque\s solution on to ! plug4 ,x w imm$iately be absorb$ by ! cotton-wool4 #d4 ,) ano!r pipette run 9 an equal quantity ( ! cau/ic soda solution4 #e4 ,quickly close ! m\? ( ! tube ) a tightly fitt+ india-rubb] /opp]4 #f4 ,incubate4 ,illu/ration3 ,,fig4 #aca4--,mc,leod's ana]obic plate base ) half petri di% inv]t$ 9 situ ,me?od ,,viii 7,mc,leod's ,me?od74-- ,app>atus & ,solutions ,requir$4-- ,mc,leod's plate base 7a holl[ glaz$ e>!nw>e disc #i cm4 9 diamet] & #b cm4 deep3 ! upp] surface is pi]c$ by a c5tral hole1 #b cm4 9 diamet]1 giv+ access to ! int]ior1 ! l[] "p ( :i* is divid$ into two by a l[ p>tition4 ,a %all[ groove encircles ! upp] surface ne> to ! edge74 ,pla/ic9e4 ,pyrogallic acid 7#a gramme7 compress$ tablets4 ,sodic hydroxide 7#j4d gramme7 compress$ tablets4 ,wa% bottle ( di/ill$ wat]4 ,surface plates ( "o or o!r ag> m$ium 79 petri di%es ( #h cm4 diamet]74 ,surface plate spread]4 ,,me?od4-- #a4 ,roll out a long cyl9d] ( pla/ic9e & fit x into ! groove on ! upp] surface ( ! e>!nw>e base4 #b4 ,place a tablet ( pyrogallic acid 9 "o division ( ! int]ior ( ! plate base1 & two tablets ( sodic hydroxide 9 ! o!r4 #c4 ,prep>e surface plate culture ( ! organism to be cultivat$4 #d4 ,run a few cubic c5timetres ( di/ill$ wat] into t division ( ! plate base conta9+ ! sodic hydroxide4 #e4 ,inv]t ! bottom half ( ! surface plate ov] ! plate base & press its edges firmly d[n into ! pla/ic9e fill+ ! groove4 #f4 ,label & incubate4 7,d7 ,me?od ,,ix4-- ,app>atus ,requir$4-- ,small ,ruff]'s or ,woodhead's flask 7,fig4 #cc74 ,/]ile india-rubb] /opp]4 ,india-rubb] tub+4 ,glass tub+4 ,metal screw clips4 ,cyl9d] ( compress$ hydrog52 or hydrog5 gas app>atus ,,me?od4-- #a4 ,/]ilise a glass vessel1 %ap$ z 9 a ,ruff]'s or ,woodhead's flask1 9 ! hot-air ov54 7,! tubulure & ! side tubes are plugg$ ) cotton-wool47 ,aft] /]ilisation1 fix a %ort piece ( rubb] tub+ occlud$ by a metal clip to ea* side tube4 #b4 ,inoculate a l>ge quantity 7e4 g41 #bjj c4c47 ( ! m$ium4 ,:]e solid m$ia are employ$ !y mu/ fir/ be liquefi$ by heat4 #c4 ,remove ! cotton-wool plug f ! tubulure & p\r ! inoculat$ m$ium into ! glass vessel4 #d4 ,close ! tubulure by means ( an india-rubb] /opp] previ\sly /]ilis$ by boil+ 9 a beak] ( wat]4 ,illu/ration3 ,,fig4 #acb4--,kipp's hydrog5 app>atus1 7a7 connect$ up to two wa%+ bottles conta9+ 7b7 lead acetate #aj p] c5t4 solution1 to remove ,h#b,s & 7c7 silv] nitrate solution to remove ,as,h#c4 ,a ?ird wa%+ bottle conta9+ pyrogallic acid #aj p] c5t4 solution1 r5d]$ alkal9e1 to remove any trace ( oxyg51 is sometimes introduc$4 ,illu/ration3 ,,fig4 #acc4--;,improv$ gas app>atus2 ! metal is conta9$ 9 a p]=at$ glass tube :i* is subm]g$ 9 acid :5 ! triangul> bottle is upright 7a71 b is above ! level ( ! liquid :5 ! bottle is turn$ on its side 7b74 #e4 ,connect up ! india-rubb] tub+ on "o ( ! side tubes ) a cyl9d] ( compress$ hydrog5 7or ! deliv]y tube ( a ,kipp's ,fig4 #acb or o!r hydrog5 app>atus1 ,fig4 #acc71 int]pos+ a %ort piece ( glass tub+2 & 9 l mann] connect a long piece ( rubb] tub+ :i* %\ld be l$ into a bas9 ( wat]1 to ! opposite side tube4 #f4 ,op5 bo? metal clips & pass hydrog5 ?r\gh ! vessel until ! atmosph]ic air is replac$ by hydrog54 ,?is is det]m9$ by collect+ "s ( ! gas :i* bubbles ?r\gh ! wat] 9 ! bas9 9 a te/-tube & te/+ x by means ( a light$ tap]4 #g4 ,close ! metal clip on ! tube ?r\gh :i* ! gas is ent]+2 close ! clip on ! exit tube4 #h4 ,disconnect ! gas app>atus4 #i4 ,incubate4 ,me?od ,x 7,botk9's ,me?od74-- ,app>atus ,requir$4-- ,l>ge glass di% #bj cm4 diamet] & #h cm4 deep4 ,flat lead5 cross slightly %ort] ?an ! int]nal diamet] ( ! glass di%4 ,bell glass ab\t #ae cm4 diamet] & #bj to #be cm4 high4 ,metal frame = plate cultivations4 ,or1 glass batt]y j> = tube cultivations4 ,cyl9d] ( compress$ hydrog54 ,rubb] tub+4 ,two pieces ( ,u-%ap$ glass tub+ 7ea* arm #h cm4 9 l5g?74 ,half a litre ( glyc]9e 7or metallic m]cury74 ,,me?od4-- #a4 ,place ! lead5 cross inside ! glass di%1 re/+ on ! bottom4 #b4 ,prep>e ! cultivations 9 ! usual way4 #c4 ,place ! tube cultivations 9 a glass batt]y j> 7or ! plate cultivations on a metal frame71 re/+ on ! c5tre ( ! lead5 cross4 #d4 ,cov] ! cultivations ) ! bell j>4 #e4 ,adju/ ! ,u-%ap$ pieces ( glass tub+ 9 a v]tical position on opposite sides ( ! bell j>1 "o arm ( ea* inside ! j>1 ! o!r outside4 ,^! tubes are be/ held 9 position by emb$d+ ! ,u-%ap$ b5ds 9 two lumps ( pla/]9e /uck on ! bottom ( ! glass di%4 ,fix a %ort l5g? ( rubb] tub+ clamp$ ) a metal clip to ea* ( ! outside arms 7,fig4 #acd74 #f4 ,fill ! glass di% ) glyc]9e or metallic m]cury to a dep? ( ab\t #e cm4 ,illu/ration3 ,,fig4 #acd4--;,botk9's app>atus4 #g4 ,connect up "o ,u-%ap$ tube ) ! hydrog5 cyl9d] 7or gas app>atus7 by means ( rubb] tub+4 ,replace ! atmosph]ic air by hydrog51 z 9 me?od ,,ix4 #h4 ,clamp ! tubes & disconnect ! gas app>atus4 #i4 ,incubate4 ,me?od ,,xi 7,novy's ,me?od74-- ,app>atus ,requir$4-- ,j> = plate cultivations 7,fig4 #ace74 ,or1 j> = tube cultivations 7,fig4 #acf74 ,lubricant = /opp] ( j>4 ,rubb] tub+4 ,cyl9d] ( compress$ hydrog54 ,,me?od4-- #a4 ,prep>e cultivations 9 ! usual way4 #b4 ,place ^! inside ! j>4 #c4 ,lubricate ! /opp] & ins]t x 9 ! m\? ( ! j>1 ) ! h&le 9 a l9e ) ! two side tubes4 #d4 ,connect up ! deliv]y tube a ) ! hydrog5 gas supply by means ( rubb] tub+4 ,illu/ration3 ,,fig4 #ace4--,novy's j> = plate cultivations4 ,illu/ration3 ,,fig4 #acf4--,novy's j> = tube cultivations4 #e4 ,atta* a piece ( rubb] tub+ to ! exit tube b & collect samples ( ! issu+ gas 7ov] wat]7 & te/ f "t to "t4 #f4 ,:5 ! air is completely displac$ by hydrog51 turn ! h&le ( ! /opp] at "r angles to ! l9e ( entry & exit tubes2 ?is seals ! orifice ( bo? tubes4 #g4 ,disconnect ! gas app>atus & incubate4 7,e7 ,me?od ,,xii 7,bullo*'s ,me?od74-- ,app>atus ,requir$4-- ,bullo*'s j>4 ,pot ( res9 o9tm5t4 ,small glass di% #ad cm4 diamet] by #e cm4 deep4 ,vessel = tube cultures or metal rack = plate cultures4 ,pyrogallic acid tablets4 ,cyl9d] ( compress$ hydrog54 ,g]yk or o!r air pump4 ,rubb] pressure tub+4 #aj c4c4 pipette4 ,glass tub+4 ,dry granulat$ cau/ic soda or compress$ tablets ea*1 conta9+ #j4d grammes sodic hydroxide4 ,small beak] ( wat]4 ,,me?od4-- #a4 ,prep>e ! cultivations 9 ! usual way4 #b4 ,place ! glass di% 9 ! c5tre ( ! glass slab1 & /& ! cultivations inside ?is4 #c4 ,place a suffici5t numb] ( pyrogallic acid tablets at "o side ( ! glass di% 7i4 e41 #a tablet = ea* #ajj cubic c5timet]s air capacity ( ! bell j>74 ,place a small heap ( dry granulat$ soda 7or half a doz5 tablets ( sodic hydroxide7 by ! side ( ! pyro tablets4 #d4 ,sme> ! flange ( ! bell j> ) res9 o9tm5t & apply ! j> firmly to ! glass slab1 cov]+ ! cultivations--s arrang$ t ! long tube passes ) its l[] end into ! glass di% at a po9t directly opposite to ! pyrogallic acid tablets4 ,lubricate ! two /op-cocks ) res9 o9tm5t 7,fig4 #acg74 #e4 ,connect up ! %ort tube a ) ! gas-supply by means ( rubb] pressure tub+ & op5 bo? /op-cocks4 #f4 ,connect a long1 /raight piece ( glass tub+ to ! long tube b by means ( a piece ( rubb] tub+ int]pos+ a screw clamp3 & collect samples ( ! issu+ gas f "t to "t & te/4 #g4 ,:5 ! air is displac$1 %ut (f ! /op-cock ( ! entry tube1 !n t ( ! exit tube b4 ,screw d[n ! clamp & remove ! glass tube f ! rubb] connection & connect up ! %ort tube a to ! air pump by means ( pressure tub+4 #h4 ,op5 ! /op-cock ( tube a & ) two or ?ree /rokes ( ! air pump1 aspirate a small quantity ( gas1 s creat+ a slight vacuum4 ,!n %ut (f ! /op-cock & disconnect ! air pump4 #i4 ,fill ! #aj c4c4 bulb pipette ) wat]2 ins]t its po9t into ! rubb] tub+ on ! long tube b z f> z ! screw clamp4 ,op5 ! screw clamp & run 9 wat] until /opp$ by ! int]nal pressure4 ,%ut (f /op-cock4 7,! wat] dissolves ! soda & pyrogallic acid conv]t+ ! latt] into alkal9e pyro4 & s br++ its lat5t capacity = oxyg5 into action74 ,illu/ration3 ,,fig4 #acg4--;,bullo*'s j>4 #aj4 ,rev]se ! tubes f ! tubulures s t !y meet1 out ( h>m's way1 ov] ! top ( ! bell glass2 aga9 see t all jo9ts are tight & transf] ! app>atus to ! incubator4 ,?is la/ me?od is ! mo/ satisfactory = ana]obic cultivations1 z by its means complete ana]obiosis c be obta9$ ) ! lea/ exp5diture ( "t & tr\ble4 ,,footnotes3 #h ,see also me?od ( op5+ & clos+ culture tubes1 pages #gd-gf4 #i ,if compress$ tablets ( pyrogallic acid _c be obta9$ make up a /ock acid pyro solution ,pyrogallic acid1 #aj grammes ,hydro*loric acid1 #a4e c4c4 ,di/ill$ wat]1 #ajj c4c4 & at /ep #d1 run 9 #aj c4c4 ( ! solution4 ,,xv4 ,,me?ods ,,( ,,isolation4 ,! "w 9 ! prec$+ sections1 arrang$ to demon/rate ! *ief biological *>act]s ( bact]ia 9 g5]al1 is int5d$ to be c>ri$ out by means ( cultivations ( v>i\s organisms previ\sly isolat$ & id5tifi$ & suppli$ to ! /ud5t 9 a /ate ( purity4 ,a cultivation :i* comprises ! prog5y ( a s+le cell is t]m$ a pure culture2 "o :i* conta9s repres5tatives ( two or m species ( bact]ia is spok5 ( z an impure1 or mix$ cultivation1 & x n[ becomes necess>y to indicate ! *ief me?ods by :i* "o or m organisms may be isolat$ 9 a /ate ( purity f a mixture2 :e!r t mixture exi/s z an impure laboratory cultivation1 or is conta9$ 9 pus & o!r morbid exudations1 infect$ tissues1 or wat] or food-/uffs4 ,illu/ration3 ,,fig4 #ach4--;,haematocytomet] cell1 %[+1 a1 section ?r\gh ! c5tre ( ! cell1 & b1 a magnifi$ image ( ! cell rul+s4 ,be=e ! introduction ( solid m$ia ! only me?od ( obta9+ pure cultivations 0 by dilution--by no means a reliable me?od4 ,dilution consi/$ 9 e/imat+ approximately ! numb] ( bact]ia pres5t 9 a giv5 volume ( fluid 7by means ( a graduat$-cell$ slide resembl+ a haematocytomet]1 ,fig4 #ach71 & dilut+ ! fluid by ! addition ( /]ile wat] or b\illon until a giv5 volume 7usually #a c4c47 ( ! dilution conta9$ b "o organism4 ,by plant+ ?is volume ( ! fluid into sev]al tubes or flasks ( nutri5t m$ia1 x occasionally happ5$ t ! result+ gr[? 0 ! product ( "o individual microbe4 ,a me?od s unc]ta9 is n[ =tunately replac$ by _m o!rs1 m reliable & conv5i5t1 & 9 ^? me?ods select$ = description "h1 ! segregation & isolation ( ! requir$ bact]ia may be effect$-- ,a4 ,by ,me*anical ,sep>ation4 #a4 ,by surface plate cultivation3 7a7 ,gelat9e4 7b7 ,ag>4 7c7 ,s]um ag>4 7d7 ,blood ag>4 7e7 ,hang+-drop or block4 #b4 ,by ,esm>*'s roll cultivation3 ,?is ar*aic me?od 7see page #bbf7 is no long] employ$ = ! isolation ( bact]ia4 #c4 ,by s]ial cultivation4 ,b4 ,by ,biological ,diff]5tiation4 #d4 ,by diff]5tial m$ia4 7a7 ,selective4 7b7 ,det]r5t4 #e4 ,by diff]5tial incubation4 #f4 ,by diff]5tial /]ilisation4 #g4 ,by diff]5tial atmosph]e cultivation4 #h4 ,by animal inoculation4 ,! selection ( ! me?od to be employ$ 9 any specific in/ance w dep5d ^u a v>iety ( circum/ances1 & (t5 a comb9ation ( two or m w ensure a quick] & m reliable result ?an a rigid adh]5ce to any "o me?od4 ,exp]i5ce is ! only reliable guide1 b z a g5]al rule ! use ( ei!r ! fir/ or ! ?ird me?od w be f\nd mo/ conv5i5t1 af=d+ z ea* ( !m does an opportunity = ! simultane\s isolation ( sev]al or all ( ! v>ieties ( bact]ia pres5t 9 a mixture4 #a4 ,surface ,plate ,cultivations4-- 7a7 ,gelat9e 7vide page #afd74 7b7 ,ag> 7vide page #afg74 7c7 ,alkal9e s]um ag> 7vide page #baa74 ,^! plates are prep>$ 9 a mann] precisely simil> to t adopt$ = nutri5t gelat9e & ag> surface plates 7vide pages #bca-bcc74 7d7 ,s]um ,ag>4-- #a4 ,melt ?ree tubes ( nutri5t ag>1 label !m #a1 #b1 & #c1 & place !m1 ) ?ree tubes ( /]ile fluid s]um1 also labell$ #a;a1 #b;a1 & #c;a1 9 a wat]-ba? regulat$ at #de deg4 ,c42 all[ suffici5t "t to elapse = ! temp]ature ( ! cont5ts ( ea* tube to rea* t ( ! wat]-ba?4 #b4 ,take s]um tube ,no4 #a;a & ag> tube ,no4 #a4 ,flame ! plugs & remove !m f ! tubes 7reta9+ ! plug ( ! ag> tube 9 ! h&72 flame ! m\?s ( ! tubes1 p\r ! s]um into ! tube ( liquefi$ ag> & replace ! plug ( ! ag> tube4 #c4 ,mix ?or\ghly & p\r plate ,no4 #a secundum artem4 #d4 ,treat ! rema9+ tube ( ag> & s]um 9 a simil> fa%ion1 & p\r plates ,nos4 #b & #c4 #e4 ,dry ! s]um ag> plates 9 ! incubator runn+ at #fj deg4 ,c4 = "o h\r 7see page #bcb74 #f4 ,inoculate ! plates 9 s]ies z describ$ = gelat9e surface plates 7page #bca74 7e7 ,blood ,ag>1 ,human4-- #a4 ,melt a tube ( /]ile ag> & p\r x into a /]ile plate2 let x set4 #b4 ,collect a few drops ( human blood1 "u all aseptic conditions1 9 a /]ile capill>y teat pipette4 #c4 ,raise ! cov] ( ! ,petri di% v slightly1 ins]t ! extremity ( ! capill>y pipette1 & deposit ! blood on ! c5tre ( ! ag> surface4 ,close ! di%4 #d4 ,*>ge a plat9um loop ) a small quantity ( ! inoculum4 ,raise ! cov] ( ! plate1 introduce ! loop1 mix its cont5ts ) ! drop ( blood1 remove ! loop1 close ! di% & /]ilise ! loop4 #e4 ,f9ally sme> ! mixture ov] ! surface ( ! ag> ) a /]ilis$ ,l-%ap$ rod4 #f4 ,label & incubate4 7,if consid]$ necess>y1 two1 ?ree1 or m simil> plates may be inoculat$ 9 s]ies47 7f7 ,blood ,ag>1 ,animal4-- ,:5 prep>+ citrat$ blood ag> 7page #aga7 x is always advisable to p\r sev]al blood ag> tubes into plates1 :i* c be /or$ 9 ! ice *e/ ready = use at any mom5t = surface plate cultures4 7g7 ,hang+-drop or block culture1 7vide page #bcc74 #c4 ,s]ial ,cultivations4--,^! are usually made ^u ag> or blood-s]um1 al?\gh gelat9e may also be us$4 ,! me?od is z foll[s3 #a4 ,take at lea/ f\r slant$ tubes ( m$ia & numb] !m consecutively4 #b4 ,flame all ! plugs & see t ea* c be readily remov$4 #c4 ,*>ge ! plat9um loop ) a small quantity ( ! inoculum1 obs]v+ ! usual r\t9e1 & plant tube ,no4 #a1 sme>+ ?or\ghly all ov] ! surface4 ,if any wat] ( cond5sation has collect$ at ! bottom ( ! tube1 use ?is z a dilu5t be=e sme>+ ! cont5ts ( ! loop ov] ! surface ( ! m$ium4 #d4 ,)\t /]ilis+ or re*>g+ ! loop1 inoculate tube ,no4 #b1 by mak+ ?ree p>allel /reaks f end to end ( ! slant$ surface4 #e4 ,plant ! rema9d] ( ! tubes 9 ! s]ies z sme>s l tube ,no4 #a4 #f4 ,label ) di/9ctive "n or numb]1 & date2 incubate4 ,! gr[? t ensues 9 ! fir/ two or ?ree tubes ( ! s]ies w probably be s cr[d$ z to be useless4 ,t[>d ! end ( ! s]ies1 h[ev]1 discrete colonies w be f\nd1 ea* ( :i* c be transf]r$ to a fre% tube ( nutri5t m$ium )\t risk ( contam9ation f ! neighb\r+ colonies4 ,work+ up ,plates4-- ,hav+ succe$$ 9 obta9+ a plate 7or tube cultivation7 9 :i* ! colonies are well gr[n & suffici5tly sep>at$ f ea* o!r1 ! process ( work+ up1 prick+ out1 or fi%+ ! colonies 9 ord] to obta9 subcultures 9 a /ate ( purity f ea* ( ! diff]5t bact]ia pres5t mu/ n[ be proce$$ )4 ,occasionally x happ5s t ?is is q a simple matt]4 ,= example1 ! orig9al mix$ cultivation :5 exam9$ microscopically 0 f\nd to conta9 a ,gram positive micrococcus1 a ,gram positive /raight bacillus & a ,gram negative %ort bacillus4 ,! ?ird gelat9e plate prep>$ f ?is mixture1 on inspection aft] f\r day's incubation1 %[$ tw5ty-five colonies--sev5 moi/ yell[ colonies1 ea* s9k+ into a %all[ pit ( liquefi$ gelat9e1 f\rte5 flat irridesc5t filmy colonies1 & f\r rais$ :ite slimy colonies4 ,a film prep>ation 7/a9$ ,gram7 f ea* v>iety exam9$ microscopically %[$ t ! yell[ liquefy+ colony 0 compos$ ( ,gram positive micrococci2 ! flat colony ( ,gram positive bacilli & ! :ite colony ( gram negative bacilli4 ,"o ( ea* ( ^! v>ieties ( colonies w\ld be transf]r$ by means ( ! /]ilis$ loop to a fre% gelat9e culture tube1 & aft] incubation ! gr[? 9 ea* subculture w\ld correspond culturally & microscopically ) t ( ! plate colony f :i* x 0 d]iv$1--! object aim$ at w\ld !re=e be a*iev$4 ,usually1 h[ev]1 ! colonies _c be ?us readily diff]5tiat$1 & unless !y are work$ up 9 an ord]ly & sy/ematic mann] mu* lab\r w be va9ly exp5d$ & valuable "t wa/$4 ,! foll[+ me?od m9imises ! difficulties involv$4 7,a7 ,inspection4 a4 ,)\t op5+ ! plate c>efully /udy ! v>i\s colonies ) ! nak$ eye1 ) ! assi/ance ( a wat*mak]'s l5s or by inv]t+ ! plate on ! /age ( ! microscope & view+ ) ! #a-;in* objective ?r\gh ! bottom ( ! plate & ! lay] ( m$ium4 b4 ,if gross diff]5ces c be detect$ m>k a small circle on ! bottom ( ! plate ar\nd ! site ( ea* ( ! select$ colonies1 ) ! grease p5cil4 c4 ,if no obvi\s diff]5ces c be made out *oose n9e colonies haphaz>d & indicate _! positions by p5cil m>ks on ! bottom ( ! plate4 7,b7 ,fi%+ ,colonies4-- a4 ,take a /]ile ,petri di% & inv]t x ^u ! laboratory b5*4 ,rule two p>allel l9es on ! bottom ( ! di% ) a grease p5cil1 & two m p>allel l9es at "r angles to ! fir/ pair--s divid+ ! area ( ! di% into n9e portions4 ,numb] ! top "r-h& portion #a1 & ! c5tral bottom portion #h 7,fig4 #aci74 ,rev]t ! di%4 ,! numb]s #a & #h c be readily recognis$ ?r\gh ! glass & by _! positions enable any ( ! o!r divisions to be localis$ by numb]4 ,?is is ! /ock di%4 b4 ,slightly raise ! cov] ( ! di%1 & ) a /]ile teat-pipette deposit a small drop ( /]ile wat] 9 ! c5tre ( ea* ( ! n9e divisions4 c4 ,) ! /]ilis$ plat9um spatula raise "o ( ! m>k$ colonies f ! plate #c & transf] x to ! fir/ division 9 ! rul$ plate & emulsify x 9 ! drop ( wat] await+ x4 ,repeat ?is process ) ! rema9+ colonies1 emulsify+ a sep>ate colony 9 ea* drop ( wat]4 7,c7 ,prelim9>y ,diff]5tiation ( ,bact]ia4-- a4 ,prep>e a cov]-slip film prep>ation f ea* drop ( emulsion 9 ! /ock di% & numb] to correspond to ! division f :i* x 0 tak54 ,/a9 by ,gram's me?od4 b4 ,exam9e microscopically1 us+ ! oil imm]sion l5s & note ! numb]s ( ^? cov]-slips :i* morphologically & by ,gram results appe> to be compos$ ( diff]5t species ( bact]ia4 ,illu/ration3 ,,fig4 #aci4--;,diagram = /ock plate4 7,d7 ,prep>+ ,isolation ,subcultures4-- a4 ,inoculate an ag> slope & a bro? tube f ! emulsion 9 ! /ock di% correspond+ to ea* ( ^! specially select$ numb]s4 b4 ,asc]ta9 :e!r ! cov]-slips f ! n9e emulsions 9 ! /ock di% include all ! v>ieties repres5t$ 9 ! cov]-slip film prep>ation made f ! orig9al mixture be=e plat+4 c4 ,if "s v>ieties are miss+ prep>e a second /ock di% f o!r colonies on plate #c1 & repeat ! process until ea* morphological =m or t9ctorial v>iety has be5 secur$ 9 subculture4 d4 ,place ! /ock di%es 9 ! ice *e/ to await ! results ( incubation4 7,if any ( ! subcultures fail1 fur!r mat]ial c be obta9$ f ! correspond+ emulsion2 or if x has dri$1 by moi/5+ x ) a fur!r drop ( /]ile di/ill$ wat]47 e4 ,incubate all ! subcultures & id5tify ! organisms pick$ out4 #d4 ,diff]5tial ,m$ia4-- 7a7 ,selective4--,"s v>ieties ( m$ia are specially suitable = c]ta9 species ( bact]ia & enable !m to ov]gr[ & f9ally *oke out o!r v>ieties2 e4 g41 wort is ! mo/ suitable m$ium-base = ! gr[? ( torulae & yea/s & %\ld be employ$ :5 p\r+ plates = ! isolation ( ^! organisms4 ,to obta9 a pure cultivation ( yea/ f a mixture conta9+ bact]ia z well1 x is (t5 suffici5t to inoculate wort f ! mixture & incubate at #cg deg4 ,c4 = tw5ty-f\r h\rs4 ,plant a fre% tube ( wort f ! result+ gr[? & incubate4 ,repeat ! process once m1 & f ! gr[? 9 ?is ?ird tube plant a /reak on wort gelat9e1 & incubate at #bj deg4 ,c4 ,! result+ gr[? w almo/ c]ta9ly be a pure culture ( ! yea/4 7b7 ,det]r5t4--,! conv]se ( ! above also obta9s4 ,c]ta9 m$ia possess ! p[] ( inhibit+ ! gr[? ( a great] or less numb] ( species4 ,= in/ance1 m$ia conta9+ c>bolic acid to ! am\nt ( #a p] c5t4 w inhibit ! gr[? ( practically ev]y?+ b ! ,bacillus coli communis4 #e4 ,diff]5tial ,incubation4-- ,9 isolat+ c]ta9 bact]ia1 advantage is tak5 ( ! fact t diff]5t species v>y 9 _! optimum temp]ature4 ,a mixture conta9+ ! ,bacillus typhosus & ! ,bacillus aquatilis sulcatus1 = example1 may be plant$ on two slant$ ag> tubes1 ! "o incubat$ at #dj deg4 ,c41 & ! o!r at #ab deg4 ,c4 ,aft] tw5ty-f\r h\rs' incubation ! fir/ w %[ a pure cultivation ( ! ,bacillus typhosus1 :il/ ! second w be an almo/ pure culture ( ! ,bacillus aquatilis4 #f4 ,diff]5tial ,/]ilisation4-- 7a7 ,non-spor+ ,bact]ia4--,simil>ly1 advantage may be tak5 ( ! v>y+ !rmal dea?-po9ts ( bact]ia4 ,f a mixture ( two organisms ^: !rmal dea?-po9ts diff] by1 say1 #d deg4 ,c4--e4 g41 ,bacillus pyocyaneus1 !rmal dea?-po9t #ee deg4 ,c41 & ,bacillus mes5t]icus vulgatus1 !rmal dea?-po9t #fj deg4 ,c4--a pure cultivation ( ! latt] may be obta9$ by heat+ ! mixture 9 a wat]-ba? to #eh deg4 ,c4 & keep+ x at t po9t = t5 m9utes4 ,! mixture is !n plant$ on to fre% m$ia & incubat$1 :5 ! result+ gr[? w be f\nd to consi/ entirely ( ! ,b4 mes5t]icus4 7b7 ,spor+ ,bact]ia4--,?is me?od f9ds its *ief practical application 9 ! diff]5tiation ( a spore-be>+ organism f "o :i* does n =m spores4 ,9 ?is case ! mixture is heat$ 9 a wat]-ba? at #hj deg4 ,c4 = fifte5 to tw5ty m9utes4 ,at ! end ( ?is "t ! non-spor+ bact]ia are dead1 & cultivations made f ! mixture w yield a gr[? result+ f ! g]m9ation ( ! spores only4 ,diff]5tial /]ilisation at #hj deg4 ,c4 is mo/ conv5i5tly c>ri$ out 9 a wat]-ba? ( special con/ruction1 design$ by ,balf\r ,/ew>t 7,fig4 #adj74 ,x consi/s ( a d\ble-wall$ copp] vessel m\nt$ on legs1 & provid$ ) a tubulure communicat+ ) ! space betwe5 ! walls4 ,?is space is ne>ly fill$ ) b5zole 7boil+-po9t #hj deg4 ,c42 pure b5zole1 free f ?ioph5e mu/ be employ$ = ! purpose1 o!rwise ! boil+-po9t gradually & p]ceptibly rises 9 ! c\rse ( "t71 & to ! tubulure is fitt$ a long glass tube1 "s #b metres long & ab\t #j4ge cm4 diamet]1 s]v+ z a cond5s+ tube 7a tube half ?is l5g? if provid$ ) a cond5s+ bulb at ! c5tre w be equally effici5t74 ,! int]ior ( ! vessel is p>tly fill$ ) wat] & cov]$ ) a lid :i* is p]=at$ = a !rmomet]4 ,?is latt] dips into ! wat] & records its temp]ature4 ,a v small ,buns5 flame "u ! app>atus suffices to keep ! b5zole boil+ & ! wat] )9 at a con/ant temp]ature ( #hj deg4 ,c4 ,! ba? is ?us always ready = use4 ,,me?od4--,to use ! app>atus4 #a4 ,place "s ( ! mixture itself1 if fluid1 conta9+ ! spores1 or an emulsion ( ! same if d]iv$ f solid mat]ial1 9 a te/-tube4 #b4 ,imm]se ! te/-tube 9 ! wat] conta9$ 9 ! b5zole ba?1 tak+ c>e t ! upp] level ( ! liquid 9 ! tube is at lea/ #b cm4 b5ea? ! surface ( ! wat] 9 ! copp] vessel4 #c4 ,! temp]ature ( ! wat]1 ( c\rse1 falls a few degrees aft] op5+ ! ba? & introduc+ a tube ( cold] liquid1 b aft] a few m9utes ! temp]ature w h aga9 rea*$ #hj deg4 ,c4 #d4 ,:5 ! !rmomet] aga9 records #hj deg4 ,c41 note ! "t1 & fifte5 m9utes lat] remove ! tube conta9+ ! mixture f ! ba?4 #e4 ,make cultures ^u suitable m$ia2 incubate4 ,illu/ration3 ,,fig4 #adj4--;,b5zole ba?4 #g4 ,diff]5tial ,atmosph]e ,cultivation4-- 7a7 ,by adapt+ ! atmosph]ic conditions to ! p>ticul> organism x is desir$ to isolate1 x is comp>atively easy to sep>ate a /rict a]obe f a /rict ana]obe1 & vice v]sa4 ,9 ! fir/ case1 h[ev]1 x is important t ! cultivations %\ld be made ^u solid m$ia1 = if c>ri$ out 9 fluid m$ia ! a]obes multiply+ 9 ! upp] lay]s ( fluid r5d] ! dep?s completely ana]obic1 & "u ^! conditions ! gr[? ( ! ana]obes w cont9ue un*eck$4 7b7 ,:5 x is desir$ to sep>ate a facultative ana]obe f a /rict ana]obe1 x is g5]ally suffici5t to plant ! mixture ^u ! slop$ surface ag>1 incubate a]obically at #cg deg4 ,c41 & exam9e c>efully at frequ5t int]vals4 ,at ! fir/ sign ( gr[?1 subcultivations mu/ be prep>$ & treat$ 9 a simil> mann]4 ,z a result ( ^! rapid subcultures1 ! facultative ana]obe w be secur$ 9 pure culture at ab\t ! ?ird or f\r? g5]ation4 7c7 ,if1 on ! o!r h&1 ! /rict ana]obe is ! organism requir$ f a mixture ( facultative & /rict ana]obes1 p\r plates ( glucose =mate ag> 7or gelat9e7 9 ! usual mann]1 place !m 9 a ,bullo*'s or ,novy's j>1 & incubate at a suitable temp]ature4 ,pick (f ! colonies ( ! requir$ organism :5 ! gr[? appe>s1 & transf] to tubes ( ! v>i\s m$ia4 ,incubate "u suitable conditions z to temp]ature & atmosph]e4 #h4 ,animal ,inoculation4-- ,f9ally1 :5 deal+ ) pa?og5ic organisms1 x is (t5 advisable to inoculate "s ( ! impure culture 7or ev5 "s ( ! orig9al mat]ies morbi7 into an animal specially *os5 on acc\nt ( its susceptibility to ! p>ticul> pa?og5ic organism x is desir$ to inoculate4 ,inde$1 ) "s ( ! m s5sitive & /rictly p>asitic bact]ia ?is me?od ( animal inoculation is practically ! only me?od t w yield a satisfactory result4 ,,xvi4 ,,me?ods ,,( ,,id5tification ,,& ,,/udy4 ,9 ord] to id5tify an organism aft] isolation1 tube1 plate1 & o!r cultivations mu/ be prep>$1 incubat$ "u suitable conditions z to temp]ature & environm5t1 & exam9$ f "t to "t 7a7 macroscopically1 7b7 by microscopical me?ods1 7c7 by *emical me?ods1 7d7 by physical me?ods1 7e7 by inoculation me?ods1 & ! results ( ^! exam9ations duly record$4 ,x mu/ be /at$ def9itely t no micro-organism c be id5tifi$ by any "o "* or prop]ty1 :e!r microscopical1 biological or *emical1 b t on ! contr>y its entire life hi/ory mu/ be c>efully /udi$ & !n its id5tity e/abli%$ f a consid]ation ( ! sum total ( ^! obs]vations4 ,9 ord] to give to ! record$ results _! maximum value x is ess5tial t !y %\ld be exact & sy/ematic1 !re=e "s su* s*eme z ! foll[+ %\ld be adh]$ to2 & especially is ?is necess>y 9 describ+ an organism n previ\sly isolat$ & /udi$4 ,,s*eme ,,( ,,/udy4 ,designation3 ,orig9ally isolat$ by 7obs]v]'s "n7 9 7date71 f 7s\rce ( organism74 #a4 ,cultural ,*>act]s4--7,vide ,macroscopical ,exam9ation ( ,cultivation1 page #bfa47 ,gelat9e plates1 ,gelat9e /reak1 at #bj deg4 ,c4 ,gelat9e /ab1 ,gelat9e %ake1 ,ag> plates1 ,ag> /reak or sme>1 ,ag> /ab1 ,inspissat$ blood-s]um1 at #bj deg4 ,c4 & #cg deg4 ,c4 ,b\illon1 ,litmus milk1 ,potato1 ,special m$ia = ! purpose ( demon/rat+ *>act]i/ic appe>ances4 #b4 ,morphology4--7,vide ,microscopical ,exam9ation ( ,cultivations1 page #bgb47 ,vegetative =ms3 ,%ape4 ,size4 ,motility4 ,flagella 7if pres5t74 ,capsule 7if pres5t74 ,involution =ms4 ,pleomorphism 7if obs]v$74 ,spor+ =ms 7if obs]v$74 ,( :i* class8 ,/a9+ reactions4 #c4 ,*emical ,products ( ,gr[?4--7,vide ,*emical ,exam9ation ( ,cultivations1 page #bgf47 ,*romog5esis4 ,photog5esis4 ,enzyme =mation4 ,f]m5tation ( c>bohydrates3 ,acid =mation4 ,alkali =mation4 ,indol =mation4 ,ph5ol =mation4 ,r$uc+ & oxidis+ sub/ances4 ,gas =mation4 #d4 ,biology4--7,vide ,physical ,exam9ation ( ,cultures1 page #bie47 ,atmosph]e4 ,temp]ature4 ,reaction ( nutri5t m$ia4 ,resi/ance to le?al ag5ts3 ,physical3 ,desiccation4 ,light4 ,col\rs4 ,*emical g]micides4 ,vitality4 #e4 ,pa?og5icity3 ,susceptible animals1 subsequ5tly arrang$ 9 ord] ( susceptibility4 ,immune animals4 ,exp]im5tal inoculation1 symptoms ( disease4 ,po/-mortem appe>ances4 ,virul5ce3 ,l5g? ( "t ma9ta9$4 ,optimum m$ium8 ,m9imal le?al dose4 ,exaltation & att5uation ( virul5ce8 ,tox9 =mation4 ,,macroscopical ,,exam9ation ,,( ,,cultivations4 ,9 describ+ ! nak$-eye & l[-p[] appe>ances ( ! bact]ial gr[? ! descriptive t]ms introduc$ by ,*e/] 7& includ$ 9 ! foll[+ s*eme7 %\ld be employ$4 ,,solid ,,m$ia4 ,plate ,cultures4-- ,gelat9e4--,note ! pres5ce or abs5ce ( liquefaction ( ! surr\nd+ m$ium4 ,if liquefaction is pres5t1 note %ape & "* 7vide page #bfi1 /ab cultures74 ,ag>4--,no liquefaction takes place 9 ?is m$ium4 ,! liquid f\nd on ! surface ( ! ag> 7or at ! bottom ( ! tube 9 ag> tube cultures7 is m]ely wat] :i* has be5 express$ dur+ ! rapid solidification ( ! m$ium & has subsequ5tly cond5s$4 ,gelat9e & ,ag>4--,exam9e ! colonies at int]vals ( tw5ty-f\r h\rs4 7a7 ,) ! nak$ eye4 7b7 ,) a h& l5s or wat*mak]'s glass4 7c7 ,"u a l[ p[] 7#a in*7 ( ! microscope1 or by means ( a small dissect+ microscope4 ,di/+ui% sup]ficial f deep colonies & note ! *>act]s ( ! individual colonies4 7,a7 ,size4--,! diamet] 9 millimetres1 at ! v>i\s ages4 7,b7 ,%ape4-- ,puncti=m3 ,dim5sions too slight = def9+ =m by nak$ eye2 m9ute1 rais$1 hemisph]ical4 ,r\nd3 ,( a m or less circul> outl9e4 ,elliptical3 ,( a m or less oval outl9e4 ,irregul>3 ,outl9es n con=m+ to any recognis$ %ape4 ,fusi=m3 ,sp9dle-%ap$1 tap]+ at ea* end4 ,co*leate3 ,spiral or twi/$ l a snail %ell 7,fig4 #ada1 a74 ,illu/ration3 ,,fig4 #ada4--,types ( colonies3 a1 ,co*leate2 b1 amoeboid2 c1 mycelioid4 ,amoeboid3 ,v irregul>1 /ream+ 7,fig4 #ada1 b74 ,mycelioid3 ,a filam5t\s colony1 ) ! radiate "* ( a m\ld 7,fig4 #ada1 c74 ,filam5t\s3 ,an irregul> mass ( loosely wov5 filam5ts 7,fig4 #adb1 a74 ,floccose3 ,( a d5se woolly /ructure4 ,rhizoid3 ,( an irregul>1 bran*$1 root-l "* 7,fig4 #adb1 b74 ,conglom]ate3 ,an aggregate ( colonies ( simil> size & =m 7,fig4 #adb1 c74 ,toruloid3 ,an aggregate ( colonies1 l ! budd+ ( ! yea/ plant 7,fig4 #adb1 d74 ,rosulate3 ,%ap$ l a rosette4 ,illu/ration3 ,,fig4 #adb4--,types ( colonies3 a1 ,filam5t\s2 b1 rhizoid2 c1 conglom]ate2 d1 toruloid4 7,c7 ,surface ,elevation4-- #a4 ,g5]al ,"* ( ,surface z a ,:ole3 ,flat3 ,?91 leafy1 spread+ ov] ! surface 7,fig4 #adc1 a74 ,effus$3 ,spread ov] ! surface z a ?91 veily lay]1 m delicate ?an ! prec$+4 ,rais$3 ,gr[? ?ick1 ) abrupt t]rac$ edges 7,fig4 #adc1 b74 ,convex3 ,surface ! segm5t ( a circle1 b v flatly convex 7,fig4 #adc1 c74 ,pulv9ate3 ,surface ! segm5t ( a circle1 b decid$ly convex 7,fig4 #adc1 d74 ,capitate3 ,surface hemisph]ical 7,fig4 #adc1 e74 ,umbilicate3 ,hav+ a c5tral pit or depression 7,fig4 #adc1 f74 ,conical3 ,cone ) r\nd$ apex 7,fig4 #adc1 g74 ,umbonate3 ,hav+ a c5tral convex nipple-l elevation 7,fig4 #adc1 h74 #b4 ,detail$ ,*>act]s ( ,surface3 ,smoo?3 ,surface ev51 )\t any ( ! foll[+ di/9ctive *>act]s4 ,alveolate3 ,m>k$ by depressions sep>at$ by ?9 walls s z to resemble a honeycomb 7,fig4 #add74 ,punctate3 ,dott$ ) punctures l p9-pricks4 ,bullate3 ,l a bli/]$ surface1 ris+ 9 convex prom95ces1 r co>se4 ,vesicul>3 ,m or less cov]$ ) m9ute vesicles due to gas =mation2 m m9ute ?an bullate4 ,illu/ration3 ,,fig4 #adc4--,surface elevation ( colonies3 a1 ,flat2 b1 rais$2 c1 convex2 d1 pulv9ate2 e1 capitate2 f1 umbilicate2 g1 conical2 h1 umbonate4 ,illu/ration3 ,,fig4 #add4--,types ( colonies--alveolate4 ,v]rucose3 ,w>t-l1 be>+ w>t-l prom95ces4 ,squamose3 ,scaly1 cov]$ ) scales4 ,e*9ate3 ,beset ) po9t$ prom95ces4 ,papillate3 ,beset ) nipple or mamma-l processes4 ,rugose3 ,%ort irregul> folds1 due to %r9kage ( surface gr[?4 ,corrugat$3 ,9 long folds1 due to %r9kage4 ,cont\r$3 ,an irregul> b smoo?ly undulat+ surface1 resembl+ ! surface ( a relief map4 ,rimose3 ,ab\nd+ 9 *9ks1 clefts1 or cracks4 7,d7 ,int]nal ,/ructure ( ,colony 7,microscopical74-- ,refraction ,weak3 ,outl9e & surface ( relief n /rongly def9$4 ,refraction ,/rong3 ,outl9e & surface ( relief /rongly def9$2 d5se1 n filam5t\s colonies4 ,illu/ration3 ,,fig4 #ade4--,types ( colonies3 a1 ,grumose2 b1 moruloid2 c1 cl\d$4 #a4 ,g5]al3 ,amorph\s3 ,)\t any def9ite /ructure1 su* z is specifi$ bel[4 ,hyal9e3 ,cle> & col\rless4 ,homog5e\s3 ,/ructure uni=m ?r\gh\t all p>ts ( ! colony4 ,homo*rom\s3 ,col\r uni=m ?r\gh\t4 #b4 ,granulations or ,blot*+s3 ,f9ely granul>4 ,co>sely granul>4 ,grumose3 ,co>s] ?an ! prec$+1 ) a clott$ appe>ance1 & p>ticles 9 clu/]$ gra9s 7,fig4 #ade1 a74 ,moruloid3 ,hav+ ! "* ( a mulb]ry1 segm5t$1 by :i* ! colony is divid$ 9 m or less regul> segm5ts 7,fig4 #ade1 b74 ,cl\d$3 ,hav+ a pale gr\nd1 ) ill-def9$ pat*es ( a deep] t9t 7,fig4 #ade1 c74 ,illu/ration3 ,,fig4 #adf4--,types ( colonies3 a1 ,reticulate2 b1 gyrose2 c1 m>morat$4 #c4 ,colony ,m>k+ or ,/rip+3 ,reticulate3 ,9 ! =m ( a network1 l ! ve9s ( a leaf 7,fig4 #adf1 a74 ,areolate3 ,divid$ into r irregul>1 or angul>1 spaces by m or less def9ite b\nd>ies4 ,gyrose3 ,m>k$ by wavy l9es1 indef9itely plac$ 7,fig4 #adf1 b74 ,m>morat$3 ,%[+ fa9t1 irregul> /ripes1 or trav]s$ by ve9-l m>k+s1 z 9 m>ble 7,fig4 #adf1 c74 ,rivulose3 ,m>k$ by l9es l ! riv]s ( a map4 ,rimose3 ,%[+ *9ks1 cracks1 or clefts4 ,illu/ration3 ,,fig4 #adg4--,types ( colonies--curl$4 #d4 ,filam5t\s ,colonies3 ,filam5t\s3 ,z already def9$4 ,floccose3 ,compos$ ( filam5ts1 d5sely plac$4 ,curl$3 ,filam5ts 9 p>allel /r&s1 l locks or r+lets 7,fig4 #adg74 7,e7 ,edges ( ,colonies4-- ,entire3 ,)\t too?+ or division 7,fig4 #adh1 a74 ,undulate3 ,wavy 7,fig4 #adh1 b74 ,rep&3 ,l ! bord] ( an op5 umbrella 7,fig4 #adh1 c74 ,erose3 ,z if gnaw$1 irregul>ly too!d 7,fig4 #adh1 d74 ,illu/ration3 ,,fig4 #adh4--;,edges ( colonies3 a1 ,entire2 b1 undulate2 c1 rep&2 d1 erose4 ,lobate4 ,lobulate3 ,m9utely lobate 7,fig4 #adi1 e74 ,auriculate3 ,) e>-l lobes 7,fig4 #adi1 f74 ,lac]ate3 ,irregul>ly cleft1 z if torn 7,fig4 #adi1 g74 ,fimbriate3 ,fr+$ 7,fig4 #adi1 h74 ,ciliate3 ,hair-l ext5sions1 radiately plac$ 7,fig4 #adi1 j74 ,tuft$4 ,filam5t\s3 ,z already def9$4 ,curl$3 ,z already def9$4 ,illu/ration3 ,,fig4 #adi4--;,edges ( colonies3 e1 ,lob>-lobulate2 f1 auriculate2 g1 lac]ate2 h1 fimbriate2 i1 ciliate4 7,f7 ,optical ,*>act]s 7aft] ,%uttlewor?74-- #a4 ,g5]al ,*>act]s3 ,transp>5t3 ,transmitt+ light4 ,vitre\s3 ,transp>5t & col\rless4 ,oleag9\s3 ,transp>5t & yell[2 olive to l9se$-oil col\r$4 ,res9\s3 ,transp>5t & br[n1 v>ni% or res9-col\r$4 ,transluc5t3 ,fa9tly transp>5t4 ,porcelane\s3 ,transluc5t & :ite4 ,opalesc5t3 ,transluc5t2 greyi%-:ite by reflect$ light4 ,nacre\s3 ,transluc5t1 greyi%-:ite1 ) pe>ly lu/re4 ,sebace\s3 ,transluc5t1 yell[i% or greyi%-:ite4 ,butyr\s3 ,transluc5t & yell[4 ,c]ace\s3 ,transluc5t & wax-col\r$4 ,opaque4 ,cretace\s3 ,opaque & :ite1 *alky4 ,dull3 ,)\t lu/re4 ,gli/5+3 ,%9+4 ,fluoresc5t4 ,iridesc5t4 #b4 ,*romog5icity3 ,col\r ( pigm5t4 ,pigm5t re/rict$ to colonies4 ,pigm5t re/rict$ to m$ium surr\nd+ colonies4 ,pigm5t pres5t 9 colonies & 9 m$ium4 ,/reak or ,sme> ,cultures4-- ,gelat9e & ,ag>4--,note g5]al po9ts z indicat$ "u plate cultivations4 ,inspissat$ ,blood-s]um4--,note ! pres5ce or abs5ce ( liquefaction ( ! m$ium4 7,! pres5ce ( cond5sation wat] at ! bottom ( ! tube mu/ n be conf\nd$ ) liquefaction ( ! m$ium47 ,all ,oblique ,tube ,cultures4-- #a4 ,colonies ,discrete3 ,size1 %ape1 etc41 z = plate cultivations 7vide page #bfa74 #b4 ,colonies ,conflu5t3 ,surface elevation & "* ( edge1 z = plate cultivations 7vide page #bfc74 ,*romog5icity3 ,z = plate cultures4 ,gelat9e ,/ab ,cultures4-- 7,a7 ,surface ,gr[?4--,z = individual colonies 9 plate cultures 7vide page #bfa74 ,illu/ration3 ,,fig4 #aej4--,/ab cultivations--types ( gr[?3 a1 ,fili=m2 b1 bead$2 c1 e*9ate2 d1 vill\s2 e1 arboresc5t4 7,b7 ,l9e ( ,puncture4-- ,fili=m3 ,uni=m gr[?1 )\t special *>act]s 7,fig4 #aej1 a74 ,nodose3 ,consi/+ ( closely aggregat$ colonies4 ,bead$3 ,consi/+ ( loosely plac$ or disjo9t$ colonies 7,fig4 #aej1 b74 ,papillate3 ,beset ) papillate ext5sions4 ,e*9ate3 ,beset ) acicul> ext5sions 7,fig4 #aej1 c74 ,vill\s3 ,beset ) %ort1 undivid$1 hair-l ext5sions 7,fig4 #aej1 d74 ,plumose3 ,a delicate fea!ry gr[?4 ,illu/ration3 ,,fig4 #aea4--,/ab cultivations--types ( gr[?3 f1 ,crat]i=m2 g1 saccate2 h1 infundibuli=m2 j1 napi=m2 k1 fusi=m2 l1 /rati=m4 ,arboresc5t3 ,bran*$ or tree-l1 beset ) bran*$ hair-l ext5sions 7,fig4 #aej1 e74 7,c7 ,area ( ,liquefaction 7if pres5t74-- ,crat]i=m3 ,a sauc]-%ap$ liquefaction ( ! gelat9e 7,fig4 #aea1 f74 ,saccate3 ,%ape ( an elongat$ sack1 tubul> cyl9drical 7,fig4 #aea1 g74 ,infundibuli=m3 ,%ape ( a funnel1 conical 7,fig4 #aea1 h74 ,napi=m3 ,%ape ( a turnip 7,fig4 #aea1 j74 ,fusi=m3 ,outl9e ( a p>snip1 n>r[ at ei!r end1 broade/ bel[ ! surface 7,fig4 #aea1 k74 ,/rati=m3 ,liquefaction ext5d+ to ! walls ( ! tube & d[nw>d horizontally 7,fig4 #aea1 l74 7,d7 ,"* ( ! ,liquefi$ ,gelat9e4-- #a4 ,pellicle on surface4 #b4 ,uni=mly turbid4 #c4 ,granul>4 #d4 ,ma9ly cle>1 b conta9+ flocculi4 #e4 ,deposit at apex ( liquefi$ portion4 7,e7 ,production ( ,gas ,bubbles4 ,%ake ,cultures4-- #a4 ,pres5ce or abs5ce ( liquefaction4 #b4 ,production ( gas bubbles4 #c4 ,bulk ( gr[? at ! surface--a]obic4 #d4 ,bulk ( gr[? 9 dep?s--ana]obic4 ,fluid ,m$ia4 #a4 ,surface ( ! ,liquid4-- ,pres5ce or abs5ce ( fro? due to gas bubbles4 ,pres5ce or abs5ce ( pellicle =mation4 ,"* ( pellicle4 #b4 ,body ( ! ,liquid4-- ,uni=mly turbid4 ,flocculi 9 susp5sion4 ,granules 9 susp5sion4 ,cle>1 ) precipitate at bottom ( tube4 ,col\ration ( fluid1 pres5ce or abs5ce (4 #c4 ,precipitate4-- ,"*4 ,am\nt4 ,col\r4 ,c>bohydrate ,m$ia4-- ,gr[?4 ,reaction4 ,gas =mation4 ,coagulation or n ( s]um album5 7:5 s]um wat] m$ia are employ$74 ,litmus ,milk ,cultivations4-- ,unalt]$4 #a4 ,reaction3 ,acid4 ,alkal9e4 #b4 ,od\r4 #c4 ,=mation ( gas4 ,unalt]$4 #d4 ,consi/5cy3 ,peptonis$ 7"* ( solution74 ,coagulat$4 h>d3 solid4 #e4 ,clot3 ,"* s(t3 flocul5t4 ragg$ & brok5 up by gas bubbles4 7a7 ,coagulum undissolv$4 7b7 ,coagulum f9ally peptonis$1 completely3 incompletely4 ,result+ solution1 cle>3 turbid4 ,abundant4 ,scanty4 #f4 ,:ey3 ,cle>4 ,turbid4 ,coagulat$ by boil+1 or n4 ,,by ,,microscopical ,,me?ods4 ,z a c\ncil ( p]fection prep>ations mu/ be made f pure cultivations #d1 #f1 #h1 #ab1 #ah1 & #bd h\rs2 & subsequ5tly at int]vals (1 say1 tw5ty-f\r h\rs1 dur+ ! entire p]iod !y are "u obs]vation1 & exam9$-- 7,a7 ,liv+4--#a4 ,9 hang+ drop1 to det]m9e motility or non-motility4 ,9 ?is connection x mu/ be rememb]$ t "u c]ta9 conditions z to environm5t 7e4 g41 :5 exam9$ 9 an unsuitable m$ium1 atmosph]e1 temp]ature1 etc47 motile bacilli may fail to exhibit activity4 ,no organism1 !re=e1 %\ld be record$ z non-motile f "o obs]vation only2 a s]ies ( obs]vations at diff]5t ages & "u v>y+ conditions %\ld =m ! basis ( an op9ion z to ! abs5ce ( true locomotion4 ,size4--,9 ! case ( non-motile or sluggi%ly motile organisms1 endeav\r to measure sev]al individuals 9 ea* hang+ drop by means ( ! eyepiece micromet] or ! eikonomet] 7vide page #fc71 & av]age ! results4 ,if ! organism is "o :i* =ms spores1 obs]ve-- 7a7 ,spore ,=mation4--,prep>e hang+-drop cultivations 7vide page #gh7 f vegetative =ms ( ! organism1 add+ a trace ( mag5ta solution 7#j4e p] c5t47 or o!r intra vitam /a9 7see page #gg7 to ! drop1 on ! po9t ( ! plat9um ne$le1 to facilitate ! obs]vation ( ! ph5om5on by r5d]+ ! bacilli m di/9ct4 ,place ! prep>ation on ! /age ( ! microscope2 if necess>y1 us+ a w>m /age4 ,arrange illum9ation1 etc41 & select a solit>y bacillus = obs]vation1 by ! help ( ! #a_/f-;in* l5s4 ,sub/itute ! #a_/ab-;in* oil-imm]sion l5s = ! six?1 & obs]ve ! =mation ( ! spore2 if possible1 measure any alt]ation 9 size :i* may occur by means ( ! ,ramsd5 micromet]4 7b7 ,spore ,g]m9ation4--,prep>e hang+-drop cultivations f old cultivations 9 :i* no liv+ vegetative =ms are pres5t1 & obs]ve ! process ( g]m9ation 9 a simil> mann]4 ,! com=t ( ! microscopi/ is l>gely enhanc$ 9 ^? cases :]e ! p]iod ( obs]vation is at all l5g?y1 by use ( "s =m ( eye scre5 be=e ! unemploy$ eye1 su* z is figur$ on page #eh 7,fig4 #di74 ,if x is impossible to c>ry out ! me?od sugge/$ above1 proce$ z foll[s3 7a7 ,spore ,=mation4--,plant ! organism 9 bro? & incubate "u optimum conditions4 ,at regul> int]vals1 say e ?irty m9utes1 remove a loopful ( ! cultivation & prep>e a cov]-slip film prep>ation4 ,fix1 :ile /ill wet1 9 ! corrosive sublimate fix+ solution4 ,/a9 ) anil9e g5tian violet1 & p>tially decol\rise ) #b p] c5t4 acetic acid4 ,m\nt & numb] consecutively2 !n exam9e4 7b7 ,spore ,g]m9ation4--,expose a ?ick emulsion ( ! spores to a temp]ature ( #hj deg4 ,c4 = t5 m9utes 9 ! diff]5tial /]ilis] 7vide page #beg74 ,transf] ! emulsion to a tube ( /]ile nutri5t bro? & incubate4 ,remove specim5s f ! tube culture at int]vals (1 say1 five m9utes4 ,fix1 /a91 etc41 wet1 z "u 7a71 & exam9e4 7,b7 ,fix$4--#b4 ,9 /a9$ prep>ations4 7a7 ,to det]m9e po9ts 9 morphology3 ,%ape 7vide classification1 page #aca74 ,size3 7a7 ,prep>e cov]-slip film prep>ations at ! v>i\s ages1 & fix by exposure to a temp]ature ( #aae deg4 ,c4 = tw5ty m9utes 9 hot-air ov54 7b7 ,/a9 ! prep>ations by ,gram's me?od 7if applicable7 or ) dilute c>bol-fu*s91 & m\nt 9 ! usual way4 7c7 ,measure 7vide page #ff7 "s tw5ty-five individuals 9 ea* film by means ( ! ,ramsd5's or ! /age micromet] & av]age ! result4 ,pleomorphism2 ,if not$1 record-- ,! pr$om9ant "* ( ! v>iant =ms4 ,on :at m$ium or m$ia !y are obs]v$4 ,at :at p]iod ( developm5t4 7b7 ,to demon/rate details ( /ructure3 ,flagella3 ,if not$1 record-- ,me?od ( /a9+ 7vide page #aja74 ,position & arrangem5t 7vide page #acf74 ,numb]4 ,spores3 ,if not$1 record-- ,me?od ( /a9+4 ,%ape4 ,size4 ,position )9 ! p>5t cell4 ,condition1 z to %ape1 ( ! p>5t cell 7vide page #aci74 ,optimum m$ium & temp]ature4 ,age ( cultivation4 ,conditions ( environm5t z to temp]ature1 atmosph]e4 ,me?od ( g]m9ation 7vide page #adj74 ,involution ,=ms3 ,if not$1 record-- ,me?od ( /a9+4 ,"* 7e4 g41 if liv+ or dead74 ,%ape4 ,on :at m$ium !y are obs]v$4 ,age ( m$ium4 ,environm5t4 ,meta*romatic ,granules3 ,if not$1 record-- ,me?od ( /a9+4 ,"* ( granules4 ,numb] ( granules4 ,col\r ( granules4 #c4 ,/a9+ ,reactions4-- #a4 ,gram's ,me?od4--,positive or negative4 #b4 ,neiss]'s ,me?od4--,if granules are not$1 record-- #a4 ,position4 #b4 ,numb]4 #c4 ,ziehl-,neels5's ,me?od4--,acid-fa/ or decol\ris$4 #d4 ,simple ,anil9e ,dyes4--7,not+ ^? giv+ ! be/ results1 ) details ( /a9+ processes47 ,me?yl5e-blue ,fu*s9 & _! modifications4 ,g5tian violet ,?ion9e blue ,,by ,,bio*emical ,,me?ods4 ,te/ cultivations ( ! organism = ! pres5ce (-- ,soluble enzymes--proteolytic1 dia/atic1 inv]tase4 ,organic acids--7a7 quantitatively--i4 e41 e/imate ! total acid production2 7b7 qualitatively = =mic1 acetic1 propionic1 butyric1 lactic4 ,ammonia4 ,neutral volatile sub/ances--e?yl alcohol1 aldehyde1 acetone4 ,aromatic products--indol1 ph5ol4 ,soluble pigm5ts4 ,te/ ! p[] ( r$uc+ 7a7 col\r+ matt]s1 7b7 nitrates to nitrites4 ,inve/igate ! gas production--,h#b,s1 ,,co#b1 ,h#b4 ,e/imate ! ratio betwe5 ! la/ two gases4 ,prep>e all cultivations = ^! me?ods ( exam9ation "u optimum conditions1 previ\sly det]m9$ = ea* ( ! organisms x is int5d$ to inve/igate1 z to 7a7 ,reaction ( m$ium2 7b7 ,incubation temp]ature2 7c7 ,atmosph]ic environm5t2 & keep c>eful records ( ^! po9ts1 & also ( ! age ( ! cultivation us$ 9 ! f9al exam9ation4 ,exam9e ! cultivations = ! v>i\s products ( bact]ial metabolism aft] =ty-eight h\rs' gr[?1 & nev] omit to exam9e control 7un9oculat$7 tube or flask ( m$ium f ! same bat*1 kept = a simil> p]iod "u id5tical conditions4 ,if ! results are negative1 te/ fur!r cultivations at ?ree days1 five days1 & t5 days4 #a4 ,enzyme ,production4-- 7,a7 ,proteolytic ,enzymes4--7,conv]t prote9s into proteose1 peptone & fur!r products ( hydrolysis2 e4 g41 ,b4 pyocyaneus47 ,m$ia ,requir$3 ,blood-s]um & milk-s]um :i* h be5 c>efully filt]$ ?r\gh a porcela9 c&le4 ,reag5ts ,requir$3 ,ammonium sulphate4 ,?irty p] c5t4 cau/ic soda solution4 ,copp] sulphate1 #j4e p] c5t4 aque\s solution4 ,"o p] c5t4 acetic acid solution4 ,millon's reag5t4 ,glyoxylic acid solution4 ,conc5trat$ sulphuric acid4 ,,me?od4-- #a4 ,prep>e cultivations 9 bulk 7#ej c4c47 9 a flask & incubate4 #b4 ,make ! liquid fa9tly acid ) acetic acid1 !n boil4 7,?is precipitates ! unalt]$ prote9s47 #c4 ,filt]4 #d4 ,take #aj c4c4 ( ! filtrate 9 a te/-tube & add #a c4c4 ( ! cau/ic soda1 !n add ! copp] sulphate drop by drop4 ,p9k col\r :i* becomes violet ) m copp] sulphate proteose & peptone4 #e4 ,saturate ! re/ ( ! filtrate ) ammonium sulphate4 ,precipitate proteose4 #f4 ,filt] & divide ! filtrate into ?ree p>ts a1 b & c4 a4 ,repeat ! copp] sulphate te/1 us+ excess ( cau/ic soda to displace ! ammonia f ! ammonium sulphate4 ,p9k col\r peptone4 b4 ,boil ) ,millon's reag5t4 ,r$ col\r tyros9e4 c4 ,add glyoxylic acid solution & run 9 conc5trat$ sulphuric acid4 ,violet r+ at upp] level ( acid tryptophane4 ,bo? ! tyros9e & tryptophane may be ei!r 9 ! free /ate or 9 comb9ation z polypeptid or peptone4 7,b7 ,dia/ase4--7,conv]ts />* into sug>2 e4 g41 ,b4 subtilis47 ,m$ium ,requir$3 ,inosite-free b\illon4 ,reag5ts ,requir$3 ,/>*4 ,?ymol4 ,fehl+'s solution4 ,,me?od4-- #a4 ,prep>e tube cultivation & incubate4 #b4 ,prep>e a ?9 />* pa/e & add #b p] c5t4 ?ymol to x4 #c4 ,mix equal p>ts ( ! cultivation to be te/$ & ! />* pa/e1 & place 9 ! incubator at #cg deg4 ,c4 = six to eight h\rs4 #d4 ,filt]4 ,te/ ! filtrate = sug>4 ,boil "s ( ! ,fehl+'s solution 9 a te/-tube4 ,add ! filtrate drop by drop until1 if necess>y1 a quantity has be5 add$ equal 9 am\nt to ! ,fehl+'s solution employ$1 keep+ ! mixture at ! boil+-po9t dur+ ! process4 ,yell[ or orange precipitate sug>4 7,c7 ,inv]tase4--7,conv]t sac*>ose into a mixture ( dextrose & laevulose e4 g41 ,b4 fluoresc5s liquefaci5s47 ,m$ium ,requir$3 ,inosite-free b\illon4 ,reag5ts ,requir$3 ,cane sug>1 #b p] c5t4 aque\s solution4 ,c>bolic acid4 ,,me?od4-- #a4 ,prep>e tube cultivations & incubate4 #b4 ,add #b p] c5t4 ( c>bolic acid to ! sug> solution4 #c4 ,mix equal quantities ( ! c>bolis$ sug> solution & ! cultivation 9 a te/-tube2 all[ ! mixture to /& = sev]al h\rs4 #d4 ,filt]4 ,te/ ! filtrate = r$uc+ sug> z 9 ! prec$+ section4 7,d7 ,r5n9 & ,lab ,enzymes4--7,coagulate milk indep5d5tly ( ! action ( acids2 e4 g41 ,b4 prodigiosus47 ,m$ia ,requir$3 ,inosite-free b\illon4 ,litmus milk4 ,,me?od4-- #a4 ,prep>e tube cultivations & incubate4 #b4 ,aft] incubation heat ! cultivation to #ee deg4 ,c4 = half an h\r1 to /]ilise4 #c4 ,by means ( a /]ile pipette run #e c4c4 ( ! cultivation into ea* ( ?ree tubes ( litmus milk4 #d4 ,place 9 ! cold incubator at #bb deg4 ,c4 & exam9e ea* day = t5 days4 ,abs5ce ( coagulation at ! end ( t p]iod w indicate abs5ce ( r5n9 f]m5t =mation4 ,f]m5tation ,reactions4 ,z te/$ ^u c>bohydrate sub/ances & organic salts4 ,m$ia ,requir$3 ,peptone wat] conta9+ v>i\s p]c5tages 7g5]ally #b p] c5t47 ( ea* ( ! sub/ances ref]r$ to "u sug> m$ia 7page #agg71 also tubes ( peptone wat] conta9+ #a p] c5t4 respectively ( ea* ( ! foll[+3 ,organic salts3 ,sodium citrate1 =mate1 lactate1 malate1 t>trate4 ,,me?od4-- #a4 ,prep>e tube cultivations 9 ea* ( ! above m$ia4 #b4 ,obs]ve f day to day up to ! expiration ( t5 days if necess>y4 #c4 ,note gr[?1 reaction1 gas production4 #b4 ,acid ,production4 7a7 ,quantitative4-- ,m$ium ,requir$3 ,sug> 7glucose7 b\illon ( kn[n optimum reaction4 ,app>atus & ,reag5ts ,requir$3 ,z = e/imat+ reaction ( m$ia 7vide page #aej74 ,,me?od4-- #a4 ,prep>e cultivation 9 bulk 7#ajj c4c47 9 a flask2 also control flask ( m$ium f same bat*4 #b4 ,aft] suitable incubation1 heat bo? flasks 9 ! /eam] at #ajj deg4 ,c4 = ?irty m9utes to /]ilise4 #c4 ,det]m9e ! titre ( ! m$ium 9 inoculat$ & control flasks z describ$ 9 ! prep>ation ( nutri5t m$ia 7vide page #aea74 #d4 ,! diff]5ce betwe5 ! titre ( ! m$ium 9 ! two flasks gives ! total acid production ( ! bact]ium "u obs]vation 9 t]ms ( normal ,na,o,h4 ,,note4--,if ! gr[? is v heavy x may be a difficult matt] to det]m9e ! end-po9t4 ,! cultivation %\ld !n be filt]$ ?r\gh a ,b]kefeld filt] c&le previ\s to /ep #b1 & ! filtrate employ$ 9 ! titration4 7b7 ,qualitative 7( all ! organic acids pres5t74-- ,m$ium ,requir$3 ,sug> 7glucose or lactose7 b\illon z 9 quantitative exam9ation4 ,reag5ts ,requir$3 ,hydro*loric acid1 conc5trat$4 ,hydro*loric acid1 #be p] c5t4 ,sulphuric acid1 conc5trat$ 7pure74 ,phosphoric acid1 conc5trat$ solution4 ,ammonia4 ,ammonium sulphate4 ,b>yta wat]4 ,sodium c>bonate1 saturat$ aque\s solution4 ,absolute alcohol4 ,e!r4 ,calcium *loride4 ,calcium *loride solution4 ,z9c c>bonate4 ,copp] sulphate saturat$ aque\s solution4 ,alcoholic ?ioph5e solution 7#j4ae c4c4 9 #ajj c4c474 ,animal *>coal4 ,five p] c5t4 sodium nitroprusside solution4 ,potassium bi*romate4 ,s*iff's reag5t4 ,ars5i\s oxide4 ,f]ric *loride1 #d p] c5t4 aque\s solution4 ,silv] nitrate1 #a p] c5t4 aque\s solution4 ,lugol's iod9e4 ,t5 p] c5t4 cau/ic soda solution4 ,h>d p>aff9 wax 7melt+-po9t ab\t #eb deg4 ,c474 ,,me?od4-- #a4 ,prep>e cultivation 9 bulk 7#ejj c4c47 9 a litre flask & add /]ilis$ precipitat$ *alk1 #aj grammes4 ,incubate at ! optimum temp]ature4 #b4 ,aft] incubation ?r[ a piece ( p>aff9 wax 7ab\t a c5timetre cube7 into ! cultivation & connect up ! flask ) a cond5s]4 ,! p>aff91 :i* liquefies & =ms a ?9 lay] on ! surface ( ! fluid1 is necess>y to prev5t ! cultivation fro?+ up & runn+ unalt]$ ?r\gh ! cond5s] dur+ ! subsequ5t process ( di/illation4 #c4 ,di/ill ov] #bjj to #cjj c4c4 ,use a rose-top burn] to m9imise ! dang] ( crack+ ! flask2 & to ! same end1 well agitate ! cont5ts ( ! flask to prev5t ! *alk settl+4 ,! di/illate ,a w conta9 alcohol1 etc4 7vide page #bhe72 ! residue a w conta9 ! volatile & fix$ acids4 #d4 ,disconnect ! flask & filt]4 ,! residue a !n filtrate ,b & residue b4 ,illu/ration3 ,,fig4 #aeb4--;,arrangem5t ( di/illation app>atus = acids1 etc4 #e4 ,residue b4 ,wa% ! residue f ! filt] pap]1 dissolve by heat+ ) dilute hydro*loric acid1 & add calcium *loride solution & ammonia until alkal9e4 ,:ite precipitate insoluble 9 acetic acid oxalic acid4 #f4 ,make up filtrate ,b to #ejj c4c4 ) di/ill$ wat] & divide into two p>ts4 #g4 ,acidify #bej c4c4 ) #bj c4c4 conc5trat$ phosphoric acid 7?is lib]ates ! volatile acids7 & di/il to small bulk4 ,! di/illate ,b may conta9 =mic1 acetic1 propionic1 butyric & b5zoic acids4 ,,di/illate ,b4 7,volatile ,acids47 #a4 ,add b>yta wat] till alkal9e1 & evaporate to dryness4 #b4 ,add #ej c4c4 absolute alcohol & all[ to /&1 ) frequ5t /irr+1 = two to ?ree h\rs4 #c4 ,filt] & wa% ) alcohol4 --------------------------------------- ,,filtrate ,,residue may conta9 b>ium propionate1 may conta9 b>ium acetate1 b>ium butyrate4 b>ium =mate1 b>ium b5zoate4 #a4 ,evaporate to dryness4 #a4 ,evaporate (f alcohol & dissolve up ! residue on #b4 ,dissolve residue 9 #aej ! filt] 9 hot wat] & c4c4 wat]4 neutralise4 #c4 ,acidify ) phosphoric #b4 ,divide ! solution into acid & di/il4 f\r portions3 #d4 ,saturate di/illate ) 7a7 ,add f]ric *loride solution4 calcium *loride & di/ill ov] a few c4c4 ,br[n col\r acetic or =mic acids4 #e4 ,te/ di/illate = butyric acid3 ,buff ppt4 b5zoic acid 7see e!r soluble acids74 ,add #c c4c4 alcohol & #d drops conc5trat$ sulphuric acid4 7b7 ,add silv] nitrate solution2 !n add "o drop ,smell ( p9eapple butyric ammonia wat]1 & boil4 acid4 ,black precipitate ( metallic ,propionic acid 9 small silv] =mic acid4 quantities _c be di/+ui%$ f butyric 7c7 ,evaporate to dryness2 mix acid by te/s )9 ! ) equal quantity ( scope ( ! bact]iological ars5i\s oxide & heat laboratory4 on plat9um foil4 ,unpleasant smell ( cacodyl acetic acid4 7d7 ,add a few drops ( m]curic *loride solution 9 te/-tube1 & heat to #gj deg4 ,c4 ,precipitate ( m]cur\s *loride :i* is sl[ly r$uc$ to m]cury =mic acid4 #h4 ,if ! di/illation ( ,b is cont9u$ z long z acid comes ov] 7di/ill$ wat] be+ occasionally add$ to ! di/ill+ flask7 ! di/illate c be measur$ & #ej c4c4 us$ = titration4 ,?is w give ! am\nt ( volatile acid =mation4 #i4 ,! second "p ( ! filtrate ,b 7see page #bhb7 %\ld be exam9$ = lactic1 oxalic1 succ9ic1 b5zoic1 salicylic1 gallic & tannic acids1 z foll[s3 ,e!r ,soluble ,acids4-- #a4 ,evaporate to a ?9 syrup1 acidify /rongly ) phosphoric acid4 #b4 ,extract ) five times its volume ( e!r by agitation 9 a sep>atory funnel4 #c4 ,evaporate ! e!real extract to a ?9 syrup4 #d4 ,add #ajj c4c4 wat] & mix ?or\ghly4 #e4 ,to a small portion ( ?is solution add slight excess ( sodium c>bonate1 evaporate to dryness on ! wat]-ba?1 dissolve 9 #e-aj c4c4 pure sulphuric acid1 add #b drops saturat$ copp] sulphate solution1 place 9 a te/-tube & heat 9 a boil+ wat]-ba? = #b m9utes1 cool1 add #b or #c drops ( ! alcoholic ?ioph5e & w>m g5tly4 ,*]ry r$ col\r lactic acid4 ,if a br[n col\r is produc$ on ! addition ( sulphuric acid1 ano!r sample %\ld be tak5 & boil$ ) animal *>coal be=e evaporat+4 #f4 ,if lactic acid is def9itely pres5t1 prep>e z9c lactate by boil+ "p ( ! solution ( ! e!r extract ) excess ( z9c c>bonate1 filt]+ & evaporat+ to cry/allise4 ,! cry/als s obta9$ h a *>act]i/ic =m1 & if dri$ at #aaj deg4 ,c1 %\ld conta9 #bf4hg p] c5t4 ( z9c4 #g4 ,te/ a portion ( ! re/ ( ! solution ( ! e!r extract = oxalic acid 7page #bhb1 /ep #e74 ,c>efully neutralise ! rema9d] & add f]ric *loride solution4 ,r$ br[n gelat9\s precipitate succ9ic acid4 ,buff precipitate b5zoic acid1 & o!r acids relat$ to b5zoic acid4 ,violet col\r salicylic acid4 ,inky black col\r or precipitate gallic acid or tannic acid4 ,= fur!r id5tification ! melt+-po9ts ( ! cry/all9e acids1 & ! p]c5tage ( silv] 9 _! silv] salts %\ld be det]m9$4 #c4 ,ammonia ,production4-- ,m$ium ,requir$3 ,nutri5t b\illon4 ,reag5t ,requir$3 ,nessl] reag5t4 ,,me?od4-- #a4 ,prep>e cultivation 9 bulk 7#ajj c4c47 9 a #bej c4c4 flask & incubate toge!r ) a control flask4 ,te/ ! cultivation & ! control = ammonia 9 ! foll[+ mann]3 #b4 ,to ea* flask add #b grammes ( calc9$ magnesia1 !n connect up ) cond5s]s & di/il4 #c4 ,collect #ej c4c4 di/illate1 f ea*1 9 a ,nessl] glass4 #d4 ,add #a c4c4 ,nessl] reag5t to ea* glass by means ( a clean pipette4 ,yell[ col\r ammonia4 ,! dep? ( col\r is proportionate to ! am\nt pres5t4 #d4 ,alcohol1 etc41 ,production4--,divide ! di/illate ,a obta9$ 9 ! c\rse ( a previ\s exp]im5t 7vide page #bhb1 /ep #c7 into f\r portions & te/ = ! production ( alcohol1 acetaldehyde1 acetone4 #a4 ,add ,lugol's iod9e1 !n a little ,na,o,h solution1 & /ir ) a glass rod till ! col\r ( ! iod9e disappe>s4 ,pale-yell[ cry/all9e precipitate ( iod(orm1 ) its *>act]i/ic smell1 appe>+ 9 ! cold1 indicates acetaldehyde1 or acetone2 appe>+ only on w>m+ indicates alcohol4 ,! precipitate may be abs5t ev5 :5 ! od\r is pron\nc$4 #b4 ,add ,s*iff's reag5t4 ,violet or r$ col\r aldehyde4 #c4 ,to #aj c4c4 ( solution add #b4e c4c41 #be p] c5t4 sulphuric acid1 & a cry/al or two ( potassium bi*romate & di/il4 ,r$uction ( ! bi*romate to a gre5 col\r & a di/illate1 :i* smells ( acetaldehyde & reacts ) ,s*iff's reag5t1 %[s ! pres5ce ( alcohol 9 ! orig9al liquid4 #d4 ,add a few drops ( sodium nitroprusside solution1 make alkal9e ) ammonia1 !n saturate ) ammonium sulphate cry/als4 ,acetone gives little col\r on ! addition ( ammonia1 b aft] ! addition ( ammonium sulphate a deep p]manganate col\r1 :i* takes t5 m9utes to rea* its full int5sity4 ,aldehyde gives a c>m9e r$ unalt]$ by ammonium sulphate4 #e4 ,indol ,production4-- ,m$ia ,requir$3 ,inosite-free b\illon 7vide page #ahc74 ,or peptone wat] 7vide page #agg74 ,reag5ts ,requir$3 ,potassium p]sulphate1 saturat$ aque\s solution4 ,p>adime?ylam9o-b5zaldehyde solution4 ,?is is prep>$ by mix+3 ,p>adime?ylam9o-b5zaldehyde #d grammes ,absolute alcohol #chj c4c4 ,hydro*loric acid1 conc5trat$ #hj c4c4 ,,me?od4-- ,prep>e sev]al te/-tube cultivations ( ! organism to be te/$1 & incubate4 ,te/ = indol by means ( ! ,ros9dol reaction 9 ! foll[+ mann]4 7,if ! culture has be5 incubat$ at #cg deg4 ,c41 x mu/ be all[$ to cool to ! room temp]ature be=e apply+ ! te/47 #a4 ,remove #b c4c4 ( ! cultivation by means ( a /]ile pipette & transf] to a clean tube1 !n1 #b4 ,add #b c4c4 p>adime?ylam9o-b5zaldehyde solution4 #c4 ,add #b c4c4 potassium p]sulphate solution4 ,! pres5ce ( indol is indicat$ by ! appe>ance ( a delicate rose-p9k col\r ?r\gh\t ! mixture :i* deep5s slightly on /&+4 ,indol is te/$ = 9 _m laboratories by ! ord9>y nitroso9dol reaction :i*1 h[ev]1 is n s delicate a me?od z t above describ$4 ,! te/ is c>ri$ out z foll[s3 #a4 ,remove ! cotton-wool plug f ! tube1 & run 9 #a c4c4 pure conc5trat$ sulphuric acid d[n ! side ( ! tube by means ( a /]ile pipette4 ,place ! tube upright 9 a rack1 & all[ x to /&1 if necess>y1 = t5 m9utes4 ,a rose-p9k or r$ col\r at ! junction ( ! two liquids indol 7plus a nitrite74 #b4 ,if ! col\r ( ! m$ium rema9s unalt]$1 add #b c4c4 ( a #j4ja p] c5t4 aque\s solution sodium nitrite1 & aga9 all[ ! culture to /& = t5 m9utes4 ,r$ col\ration indol4 ,,note4--,9 place ( p]=m+ ! te/ 9 two /ages z giv5 above1 #b c4c4 conc5trat$ comm]cial sulphuric1 hydro*loric1 or nitric acid 7all ( :i* hold a trace ( nitrite 9 solution71 may be run into ! cultivation4 ,! developm5t ( a r$ col\r )9 tw5ty m9utes w indicate ! pres5ce ( indol4 #e;a4 ,ph5ol ,production4-- ,m$ium ,requir$3 ,nutri5t b\illon4 ,reag5ts ,requir$3 ,hydro*loric acid1 conc5trat$4 ,millon's reag5t4 ,f]ric *loride1 #a p] c5t4 aque\s solution4 ,,me?od4-- #a4 ,prep>e cultivation 9 a ,bohemian flask conta9+ at lea/ #ej c4c4 ( m$ium1 & incubate4 ,te/ = ph5ol 9 ! foll[+ mann]3 #b4 ,add #e c4c41 #be p] c5t4 sulphuric acid to ! cultivation & connect up ! flask ) a cond5s]4 #c4 ,di/il ov] #ae to #bj c4c4 ,divide ! di/illate into ?ree portions a1 b & c4 #d4 ,add to 7a7 #j4e c4c4 ,millon's reag5t & boil4 ,r$ col\r ph5ol4 #e4 ,add to 7b7 ab\t #j4e c4c4 f]ric *loride solution4 ,violet col\r ph5ol4 7,if ! di/illate be acid ! reaction w be negative47 #f4 ,add to 7c7 brom9e wat]4 ,cry/all9e :ite ppt4 ( tribromo-ph5ol ph5ol4 ,,note4--,if bo? indol & ph5ol appe> to be pres5t 9 cultivations ( ! same organism1 x is well to sep>ate !m be=e te/+4 ,?is may be done 9 ! foll[+ mann]3 #a4 ,prep>e inosite-free b\illon cultivation1 say #bjj or #cjj c4c41 9 a flask z be=e4 #b4 ,r5d] def9itely acid by ! addition ( acetic acid & connect up ! flask ) a cond5s]4 #c4 ,di/il ov] #ej to #gj c4c4 ,di/illate w conta9 bo? indol & ph5ol4 #d4 ,r5d] ! di/illate /rongly alkal9e ) cau/ic pota% & r$i/il4 ,di/illate w conta9 indol2 residue w conta9 ph5ol4 #e4 ,te/ ! di/illate = indol 7vide ante74 #f4 ,saturate ! residue1 :5 cold1 ) c>bon dioxide & r$i/il4 #g4 ,te/ ?is di/illate = ph5ol 7vide ante74 #f4 ,pigm5t ,production4-- #a4 ,prep>e tube cultivations ^u ! v>i\s m$ia & incubate "u v>y+ conditions z to temp]ature 7at #cg deg4 ,c4 & at #bj deg4 ,c471 atmosph]e 7a]obic & ana]obic71 & light 7exposure to & protection f74 ,note ! conditions mo/ favorable to pigm5t =mation4 #b4 ,note ! solubility ( ! pigm5t 9 v>i\s solv5ts1 su* z wat] 7hot & cold71 alcohol1 e!r1 *lor(orm1 b5zol1 c>bon bisulphide4 #c4 ,note ! effect ( acids & alkalies respectively ^u ! pigm5t$ cultivation1 or ^u solutions ( ! pigm5t4 #d4 ,note spectroscopic reactions4 #g4 ,r$uc+ ,ag5t ,=mation4-- 7a7 ,col\r ,de/ruction4-- #a4 ,prep>e tube cultivations 9 nutri5t b\illon t9t$ ) litmus1 rosolic acid1 neutral r$1 & incubate4 #b4 ,exam9e ! cultures ea* day & note :e!r any col\r *ange occurs4 7b7 ,nitrates to ,nitrites4-- ,m$ium ,requir$3 ,nitrate b\illon 7vide page #ahe74 ,or nitrate peptone solution 7vide page #ahf74 ,reag5ts ,requir$3 ,sulphuric acid 7#be p] c5t474 ,metaph5yl5e diam9e1 #e p] c5t4 aque\s solution4 ,,me?od4-- #a4 ,prep>e tube cultivations & incubate toge!r ) control tubes 7i4 e41 un9oculat$ tubes ( ! same m$ium1 plac$ "u id5tical conditions z to environm5t74 ,?is precaution is necess>y z ! m$ium is liable to take up nitrites f ! atmosph]e1 & an op9ion z to ! abs5ce ( nitrites 9 ! cultivation is (t5 bas$ ^u an equal col\ration ( ! m$ium 9 ! control tube4 ,te/ bo? ! culture tube & ! control tube = ! pres5ce ( nitrites4 #b4 ,add a few drops ( sulphuric acid to ! m$ium 9 ea* ( ! tubes4 #c4 ,!n run 9 #b or #c c4c4 metaph5yl5e diam9e into ea* tube4 ,br[ni%-r$ col\r nitrites4 ,! dep? ( col\r is proportionate to ! am\nt pres5t4 #h4 ,gas ,production4-- 7,a7 ,c>bon ,dioxide & ,hydrog54-- ,app>atus ,requir$3 ,f]m5tation tubes 7vide page #afa7 conta9+ sug> b\illon 7glucose1 lactose1 etc474 ,! m$ium %\ld be prep>$ f inosite-free b\illon 7vide page #ahc74 ,reag5t ,requir$3 n_/#b cau/ic soda4 ,,me?od4-- #a4 ,inoculate ! surface ( ! m$ium 9 ! bulb ( a f]m5tation tube & incubate4 #b4 ,m>k ! level ( ! fluid 9 ! clos$ bran* ( ! f]m5tation tube1 at int]vals ( tw5ty-f\r h\rs1 & :5 ! evolution ( gas has ceas$1 measure ! l5g? ( ! column ( gas ) ! millimetre scale4 ,express ?is column ( gas z a p]c5tage ( ! entire l5g? ( ! clos$ bran*4 #c4 ,to analyse ! gas & to det]m9e r\ghly ! relative proportions ( ,,co#b & ,h#b1 proce$ z foll[s3 ,fill ! bulb ( ! f]m5tation tube ) cau/ic soda solution4 ,close ! m\? ( ! bulb ) a rubb] /opp]4 ,alt]nately inv]t & rev]t ! tube six or eight times1 to br+ ! soda solution into intimate contact ) ! gas4 ,return ! residual gas to ! end ( ! clos$ bran*1 & measure4 ,! loss 9 volume ( gas c>bon dioxide4 ,! residual gas hydrog54 ,transf] gas to ! bulb ( ! tube1 & explode x by apply+ a light$ tap]4 7,b7 ,sulphurett$ ,hydrog54-- ,m$ia ,requir$3 ,iron peptone solution 7vide page #ahe74 ,lead peptone solution4 #a4 ,inoculate tubes ( m$ia1 & incubate toge!r ) control tubes4 #b4 ,exam9e f day to day1 at int]vals ( tw5ty-f\r h\rs4 ,! lib]ation ( ! ,h#b,s w cause ! yell[i%-:ite precipitate to d>k5 to a br[ni%-black1 or jet black1 ! dep? ( ! col\r be+ proportionate to ! am\nt ( sulphurett$ hydrog5 pres5t4 ,quantitative3 ,= exact quantitative analyses ( ! gases produc$ by bact]ia f c]ta9 m$ia ( def9ite composition1 ! me?ods devis$ by ,pakes mu/ be employ$1 z foll[s3 ,illu/ration3 ,,fig4 #aec4--;,gas-collect+ app>atus4 ,app>atus ,requir$3 ,bohemian flask 7#cjj to #aejj c4c4 capacity7 conta9+ f #ajj to #djj c4c4 ( ! m$ium4 ,! m\? ( ! flask is fitt$ ) a p]=at$ rubb] /opp]1 c>ry+ an ,l-%ap$ piece ( glass tub+ 7! %ort arm pass+ j ?r\gh ! /opp]74 ,to ! long arm ( ! tube is atta*$ a piece ( pressure tub+ "s #h cm4 9 l5g?1 plugg$ at its free end ) a piece ( cotton-wool4 ,measure accurately ! total capacity ( ! flask & exit tube1 also ! am\nt ( m$ium conta9$4 ,note ! diff]5ce4 ,gas receiv]4 ,?is is a bell j> ( /\t glass1 #ad cm4 high & #i cm4 9 diamet]4 ,at its apex a glass tube is fus$ 94 ,?is rises v]tically #e cm41 & is !n b5t at "r angles1 ! horizontal arm be+ #aj cm4 9 l5g?4 ,a ?ree-way tap is let horizontally into ! v]tical tube j above its junction ) ! bell j>4 ,an iron cyl9d] j l>ge en\gh to conta9 ! bell j>4 ,ab\t #ae kilos ( metallic m]cury4 ,melt$ p>aff94 ,an ,orsat-,lunge work+ ) m]cury in/ead ( wat]1 provid$ ) two gas tubes ( extra l5g? 7capacity #abj & #fj c4c4 respectively & graduat$ ?r\gh\t1 bo? be+ wat]-jacket$7 or o!r gas analysis app>atus1 capable ( deal+ ) ,,co#b1 ,o#b1 ,h#b1 & ,n#b4 ,,me?od4-- #a4 ,inoculate ! m$ium 9 ! flask 9 ! usual mann]1 by means ( a plat9um ne$le1 tak+ c>e t ! neck ( ! flask & ! rubb] /opp] are ?or\ghly flam$ be=e & aft] ! op]ation4 ,illu/ration3 ,,fig4 #aed4--,orsat-,lunge gas analysis app>atus4 #b4 ,fill ! iron cyl9d] ) m]cury4 #c4 ,place ! bell j> m\? d[nw>d 9 ! m]cury--fir/ see+ t !re is free communication betwe5 ! int]ior ( ! j> & ! ext]nal air--& suck up ! m]cury into ! tap2 !n %ut (f ! tap4 #d4 ,plug ! op5 end ( ! ?ree-way tap ) melt$ wax4 #e4 ,connect up ! horizontal arm ( ! culture flask ) t ( ! gas receiv] by means ( ! pressure tub+ 7aft] remov+ ! cotton-wool plug f ! rubb] tube71 z %[n 9 ,fig4 #aec4 #f4 ,give ! ?ree-way tap half turn to op5 communication betwe5 flask & receiv]1 & seal all jo9ts by coat+ ) a film ( melt$ wax4 ,:5 ! tap is turn$1 ! m]cury 9 ! receiv] w naturally fall4 #g4 ,place ! entire app>atus 9 ! incubator4 7,two h\rs lat]1 by :i* "t ! temp]ature ( ! app>atus is t ( ! incubator1 m>k ! height ( ! m]cury on ! receiv]47 #h4 ,exam9e ! app>atus f day to day & m>k ! level ( ! m]cury 9 ! receiv] at int]vals ( tw5ty-f\r h\rs4 #i4 ,:5 ! evolution ( gas has ceas$1 remove ! app>atus f ! incubator2 cle> out ! wax f ! nozzle ( ! ?ree-way tap 7fir/ adju/+ ! tap s t no escape ( gas %all take place7 & connect x ) ! ,orsat4 #aj4 ,remove1 say1 #ajj c4c4 ( gas f ! receiv]1 rev]se ! tap & =ce x into ! culture flask4 ,remove #ajj c4c4 ( mix$ gases f ! culture flask & replace 9 ! receiv]4 ,repeat ^! processes ?ree or f\r times to ensure ?or\gh admixture ( ! cont5ts ( flask & receiv]4 #aa4 ,n[ )draw a sample ( ! mix$ gases into ! ,orsat & analyse4 ,9 calculat+ ! results be c>eful to all[ = ! volume ( air conta9$ 9 ! flask at ! comm5cem5t ( ! exp]im5t4 ,= ! collection ( gases =m$ "u ana]obic conditions a slightly diff]5t proc$ure is adopt$3 #a4 ,fix a culture flask 7#ejj c4c4 capacity7 ) a p]=at$ rubb] /opp] c>ry+ an ,l-%ap$ piece ( manomet] tub+1 ea* arm #e cm4 9 l5g?4 #b4 ,prep>e a second ,l-%ap$ piece ( tub+1 ! %ort arm #e cm4 & ! long arm #bj cm41 & connect its %ort arm to ! horizontal arm ( ! tube 9 ! culture flask by means ( a l5g? ( pressure tub+1 provid$ ) a screw clamp4 #c4 ,fill ! culture flask completely ) boil+ m$ium & pass ! long piece ( tub+ ?r\gh ! plug ( an ,erl5mey] flask 7#aej c4c4 capacity7 :i* conta9s #ajj c4c4 ( ! same m$ium4 #d4 ,/]ilise ^! c\pl$ flasks by ! discont9u\s me?od1 9 ! usual mann]4 ,imm$iately ! la/ /]ilisation is complet$1 screw up ! clamp on ! pressure tub+ :i* connects !m1 & all[ !m to cool4 ,z ! fluid cools & contracts x leaves a vacuum 9 ! neck ( ! flask bel[ ! rubb] /opp]4 #e4 ,to inoculate ! culture flask1 )draw ! long arm ( ! b5t tube f ! ,erl5mey] flask & pass x to ! bottom ( a te/-tube conta9+ a "y cultivation 79 a fluid m$ium simil> to t conta9$ 9 ! culture flask7 ( ! organism x is desir$ to inve/igate4 #f4 ,slightly release ! clamp on ! pressure tub+ to all[ #d or #e c4c4 ( ! culture to ent] ! flask4 #g4 ,clamp ! rubb] tube tightly2 remove ! b5t glass tube f ! culture tube & plunge x into a flask conta9+ rec5tly boil$ & quickly cool$ di/ill$ wat]4 #h4 ,release ! clamp aga9 & wa% 9 ! rema9s ( ! cultivation until ! culture flask & tub+ are completely fill$ ) wat]4 #i4 ,clamp ! rubb] tub+ tightly & take away ! long-arm$ glass tub+4 #aj4 ,prep>e ! gas receiv] z 9 ! previ\s me?od 79 ?is case1 h[ev]1 ! m]cury %\ld be w>m$ slightly7 & fill ! horizontal arm ( ! receiv] ) hot wat]4 #aa4 ,connect up ! culture flask ) ! horizontal arm ( ! gas receiv]4 #ab4 ,remove ! screw clamp f ! rubb] tub+1 adju/ ! ?ree-way tap1 seal all jo9ts ) melt$ wax1 & incubate4 #ac4 ,complete ! inve/igation z describ$ = ! previ\s me?od4 ,,by ,,physical ,,me?ods4 ,exam9e cultivations ( ! organism ) ref]5ce to its gr[? & developm5t "u ! foll[+ head+s3 ,atmosph]e3 7a7 ,9 ! pres5ce ( oxyg54 7b7 ,9 ! abs5ce ( oxyg54 7c7 ,9 ! pres5ce ( gases o!r ?an oxyg54 ,temp]ature3 7a7 ,range4 7b7 ,optimum4 7c7 ,!rmal dea?-po9t3 ,moi/3 ,vegetative =ms4 ,spores4 ,dry3 ,vegetative =ms4 ,spores4 ,reaction ( m$ium4 ,resi/ance to le?al ag5ts3 7a7 ,desiccation4 7b7 ,light3 ,diffuse4 ,direct4 ,prim>y col\rs4 7c7 ,heat4 7d7 ,*emical antiseptics & dis9fectants4 ,vitality 9 artificial cultures4 ,i4 ,atmosph]e4--,! "q z to :e!r ! organism "u obs]vation is 7a7 an obligate a]obe1 7b7 a facultative ana]obe1 or 7c7 an obligate ana]obe is r\ghly decid$ by ! appe>ance ( cultivations 9 ! f]m5tation tubes4 ,obvi\s gr[? 9 ! clos$ bran* z well z 9 ! bulb or 9 ! inv]t$ gas tube z well z 9 ! bulk ( ! m$ium w indicate t x is a facultative ana]obe2 :il/ gr[? only occurr+ 9 ! bulb or 9 ! clos$ bran* %[s t x is an obligate a]obe or ana]obe respectively4 ,?is me?od1 h[ev]1 is n suffici5tly accurate = ! pres5t purpose1 & ! exam9ation ( an organism ) respect to its behavi\r 9 ! abs5ce ( oxyg5 is c>ri$ out z foll[s3 ,app>atus ,requir$3 ,bu*n]'s tubes4 ,bullo*'s app>atus4 ,exhau/ pump4 ,pyrogallic acid4 ,dekanormal cau/ic soda4 ,m$ia ,requir$3 ,glucose =mate ag>4 ,glucose =mate gelat9e4 ,glucose =mate b\illon4 ,,me?od4-- #a4 ,prep>e f\r sets ( cultivations3 7,a7 ,slop$ glucose =mate ag>1 & incubate a]obically at #cg deg4 ,c4 ,slop$ glucose =mate gelat9e1 & incubate a]obically at #bj deg4 ,c4 7,b7 ,slop$ glucose ag> to incubate ana]obically at #cg deg4 ,c4 ,slop$ glucose =mate gelat9e to incubate ana]obically at #bj deg4 ,c4 7,c7 ,slop$ glucose =mate ag> to incubate ana]obically at #cg deg4 ,c4 ,glucose =mate b\illon to incubate ana]obically at #cg deg4 ,c4 7,d7 ,slop$ glucose =mate gelat9e to incubate ana]obically at #bj deg4 ,c4 ,glucose =mate b\illon to incubate ana]obically at #bj deg4 ,c4 #b4 ,seal ! cultures =m+ set ,b 9 ,bu*n]'s tubes 7vide page #bci74 #c4 ,seal ! cultures =m+ set ,c 9 ,bullo*'s app>atus2 exhau/ ! air by means ( a vacuum pump1 & provide = ! absorption ( any residual oxyg5 by ! introduction ( pyrogallic acid & cau/ic soda 9 solution 7vide page #bde74 ,treat set ,d 9 ! same way4 #d4 ,obs]ve ! cultivations macroscopically & microscopically at int]vals ( tw5ty-f\r h\rs until ! completion1 if necess>y1 ( sev5 days' incubation4 #e4 ,control ^! results4 ,gases ,o!r ?an ,oxyg54-- ,app>atus ,requir$3 ,bullo*'s app>atus4 ,/]ile gas filt] 7vide page #dj74 ,gasomet] conta9+ ! gas x is desir$ to te/ 7,,s#b1 ,n#b,o1 ,,no1 ,,co#b1 etc47 or gas g5]ator = its production4 ,,me?od4-- #a4 ,prep>e at lea/ sev5 tube cultivations ^u solid m$ia & deposit !m 9 ,bullo*'s app>atus4 #b4 ,connect up ! inlet tube ( ! ,bullo*'s j> ) ! /]ile gas filt]1 & ?is aga9 ) ! deliv]y tube ( ! gasomet] or gas g5]ator4 #c4 ,op5 bo? /op-cocks ( ! ,bullo*'s app>atus & pass ! gas ?r\gh until x has completely replac$ ! air 9 ! bell j> z %[n by ! result ( analyses ( samples collect$ f ! exit tube4 #d4 ,incubate "u optimum conditions z to temp]ature4 #e4 ,exam9e ! cultivations at int]vals ( tw5ty-f\r h\rs1 until ! completion ( sev5 days4 #f4 ,remove "o tube f ! int]ior ( ! app>atus ea* day4 ,if no gr[? is visible1 incubate ! tube "u optimum conditions z to temp]ature & atmosph]e1 & 9 ?is way det]m9e ! l5g? ( exposure to ! action ( ! gas necess>y to kill ! organisms "u obs]vation4 #g4 ,control ^! results4 ,,ii4 ,temp]ature4-- 7,a7 ,range4-- #a4 ,prep>e a s]ies ( t5 tube cultivations1 9 fluid m$ia1 ( optimum reaction4 #b4 ,arrange a s]ies ( incubators at fix$ temp]atures1 v>y+ #e deg4 ,c4 & includ+ temp]atures betwe5 #e deg4 ,c4 & #ej deg4 ,c4 7,9 ! abs5ce ( a suffici5t numb] ( incubators utilise ! wat]-ba? employ$ 9 te/+ ! !rmal dea?-po9t ( vegetative =ms47 #c4 ,incubate "o tube cultivation ( ! organism a]obically or ana]obically1 z may be necess>y1 9 ea* incubator1 & exam9e at half-h\r int]vals = f five to eighte5 h\rs4 #d4 ,note t temp]ature at :i* gr[? is fir/ obs]v$ macroscopically 7,optimum temp]ature74 #e4 ,cont9ue ! incubation until ! completion ( sev5 days4 ,note ! extremes ( temp]ature at :i* gr[? takes place 7,range ( temp]ature74 #f4 ,control ^! results--if consid]$ necess>y arrang+ ! s]ies ( incubators to include ea* degree c5tigrade = five degrees beyond ea* ( ! extremes previ\sly not$4 7,b7 ,optimum4-- #a4 ,prep>e a second s]ies ( t5 tube cultivations "u simil> conditions z to reaction ( m$ium4 #b4 ,incubate 9 a s]ies ( incubators 9 :i* ! temp]ature is regulat$ at int]vals ( #a deg4 ,c4 = five degrees on ei!r side ( optimum temp]ature obs]v$ 9 ! previ\s exp]im5t 7,a1 /ep #d74 #c4 ,obs]ve aga9 at half-h\r int]vals & note t temp]ature at :i* gr[? is fir/ visible to ! nak$ eye ,optimum temp]ature4 7,c7 ,!rmal ,dea?-po9t 7t4 d4 p47-- ,moi/--,vegetative ,=ms3 ,! t4 d4 p4 "h is t temp]ature :i* ) c]ta9ty kills a wat]y susp5sion ( ! organisms 9 "q aft] an exposure ( #aj m9utes4 ,illu/ration3 ,,fig4 #aee4--;,he>son's wat]-ba?4 ,app>atus ,requir$3 ,wat]-ba?4 ,= ! purpose ( obs]v+ ! !rmal dea?-po9t a special wat]-ba? is necess>y4 ,! temp]ature ( ?is piece ( app>atus is controll$ by means ( a capsule regulator t c be adju/$ = int]vals ( half a degree c5tigrade ?r\gh a range ( #cj deg41 f #ej deg4 ,c4 to #hj deg4 ,c4 by means ( a spr+1 actuat$ by ! h&le a1 :i* increases ! pressure 9 ! int]ior ( ! capsule4 ,a hole is provid$ = ! reception ( ! nozzle ( a bla/ pump1 s t a curr5t ( air may be bl[n ?r\gh ! wat] :ile ! ba? is 9 use1 & ?us ensure a uni=m temp]ature ( its cont5ts4 ,?r\gh a second hole is susp5d$ a c]tifi$ c5tigrade !rmomet]1 ! bulb ( :i* al?\gh completely imm]s$ 9 ! wat] is rais$ at lea/ #b cm4 above ! floor ( ! ba?4 ,/]ile glass capsules4 ,flask conta9+ #bej c4c4 /]ile normal sal9e solution4 ,case ( /]ile pipettes1 #aj c4c4 79 t5?s ( a cubic c5timetre74 ,special plat9um loop4 ,te/-tubes1 #ah by #a4e cm41 ( ?9 ,g]man glass4 ,case ( /]ile petri di%es4 ,tubes ( ag> or gelat9e4 ,,me?od4-- #a4 ,prep>e tube cultivations on solid m$ia ( optimum reaction2 incubate =ty-eight h\rs "u optimum conditions z to temp]ature & atmosph]e4 #b4 ,exam9e prep>ations f ! cultivation microscopically to det]m9e ! abs5ce ( spores4 #c4 ,pipette #e c4c4 salt solution into ea* ( twelve capsules4 #d4 ,susp5d ?ree loopfuls ( ! surface gr[? 7us+ a special plat9um loop1 vide page #caf7 9 ! normal sal9e solution by emulcify+ ev5ly aga9/ ! moi/ walls ( ea* capsule4 #e4 ,transf] emulsion f ea* capsule to /]ile #bej c4c4 flask1 & mix4 #f4 ,pipette #e c4c4 emulsion into ea* ( twelve /]ile te/-tubes numb]$ consecutively4 #g4 ,adju/ ! fir/ tube 9 ! wat]-ba?1 regulat$ at #dj deg4 ,c1 by means ( two rubb] r+s ar\nd ! tube1 "o above & ! o!r bel[ ! p]=at$ top ( ! ba?1 s t ! upp] level ( ! fluid 9 ! tube is ab\t #d cm4 bel[ ! surface ( ! wat] 9 ! ba?1 & ! bottom ( ! tube is a simil> di/ance above ! bottom ( ! ba?4 #h4 ,arrange a control te/-tube conta9+ #e c4c4 /]ile sal9e solution "u simil> conditions4 ,plug ! tube ) cotton-wool & pass a !rmomet] ?r\gh ! plug s t its bulb is imm]s$ 9 ! wat]4 #i4 ,close ! unoccupi$ p]=ations 9 ! lid ( ! wat]-ba? by means ( glass balls4 #aj4 ,wat* ! !rmomet] 9 ! te/-tube until x records a temp]ature ( #dj deg4 ,c4 ,note ! "t4 ,t5 m9utes lat] remove ! tube conta9+ ! susp5sion1 & cool rapidly by imm]s+ its l[] end 9 a /ream ( runn+ wat]4 #aa4 ,p\r ?ree gelat9e 7or ag>7 plates conta9+ respectively #j4b1 #j4c1 & #j4e c4c4 ( ! susp5sion1 & incubate4 #ab4 ,pipette ! rema9+ #d c4c4 ( ! susp5sion into a culture flask conta9+ #bej c4c4 ( nutri5t b\illon1 & incubate4 #ac4 ,obs]ve ^! cultivations f day to day4 ,no gr[? mu/ n be record$ z f9al until aft] ! completion ( sev5 days' incubation4 #ad4 ,ext5d ^! obs]vations to ! rema9+ tubes ( ! s]ies1 b v>y+ ! conditions s t ea* tube is expos$ to a temp]ature #b deg4 ,c4 high] ?an ! imm$iately prec$+ "o--i4 e41 #db deg4 ,c41 #dd deg4 ,c41 #df deg4 ,c41 & s on4 #ae4 ,note t temp]ature1 aft] exposure to :i* no gr[? takes place up to ! end ( sev5 days' incubation1 ! !rmal dea?-po9t4 #af4 ,if great] accuracy is desir$1 a second s]ies ( tubes may be prep>$ & expos$ = t5 m9utes to fix$ temp]atures v>y+ only #j4e deg4 ,c41 ?r\gh a range ( #e deg4 ,c4 on ei!r side ( ! previ\sly obs]v$ dea?-po9t4 ,moi/--,spores3 ,! !rmal dea?-po9t 9 ! case ( spores is t "t exposure to a fix$ temp]ature ( #ajj deg4 ,c4 necess>y to effect ! dea? ( all ! spores pres5t 9 a susp5sion4 ,,note4--,if x is desir$ to reta9 ! "t con/ant #aj m9utes & inve/igate ! temp]ature necess>y to de/roy ! spores1 v>y+ am\nts ( calcium *loride mu/ be add$ to ! wat] 9 ! ba?1 :5 ! boil+-po9t w be rais$ above #ajj deg4 ,c4 accord+ to ! p]c5tage ( calcium 9 solution4 ,9 su* case use ! ba? figur$ on page #bbg2 ! ba? figur$ on page #bii c only be us$ if ! capsule is fir/ remov$4 ,x is det]m9$ 9 ! foll[+ mann] ,app>atus ,requir$3 ,/eam-c fitt$ ) a deliv]y tube & a l>ge bore safety-valve tube4 ,wat]-ba? at #ajj deg4 ,c4 ,erl5mey] flask1 #ejj c4c4 capacity1 conta9+ #adj c4c4 /]ile normal sal9e solution & fitt$ ) rubb] /opp] p]=at$ ) f\r holes4 ,! rubb] /opp] is fitt$ z foll[s3 7a7 ,!rmomet] to #abj deg4 ,c41 its bulb imm]s$ 9 ! normal sal9e4 7b7 ,/raight entry tube1 rea*+ to ! bottom ( ! flask1 ! upp] end plugg$ ) cotton-wool4 7c7 ,b5t syphon tube1 ) pipette nozzle atta*$ by means ( rubb] tub+ & fitt$ ) p9*-cock4 ,! nozzle is protect$ f accid5tal contam9ation by pass+ x ?r\gh ! cotton-wool plug ( a small te/-tube4 7d7 ,a sickle-%ap$ piece ( glass tub+ pass+ j ?r\gh ! /opp]1 plugg$ ) cotton-wool1 to act z a v5t = ! /eam4 ,/]ile plates4 ,/]ile pipettes4 ,/]ile te/-tubes graduat$ to conta9 #e c4c4 ,m$ia ,requir$3 ,gelat9e or ag>4 ,culture flasks conta9+ #bjj c4c4 nutri5t b\illon4 ,illu/ration3 ,,fig4 #aef4--;,app>atus arrang$ = ! det]m9ation ( ! dea?-po9t ( spores4 ,,me?od4-- #a4 ,prep>e twelve tube cultivations ^u ! surface 7or two cultures 9 l>ge flat culture bottles--vide page #e7 ( nutri5t ag> & incubate "u ! optimum conditions 7previ\sly det]m9$71 = ! =mation ( spores4 ,exam9e prep>ations f ! cultures microscopically to det]m9e ! pres5ce ( spores4 #b4 ,pipette #e c4c4 /]ile normal sal9e into ea* culture tube or #cj c4c4 into ea* bottle & by means ( a /]ile plat9um spatula emulsify ! entire surface gr[? ) ! solution4 #c4 ,add ! #fj c4c4 emulsion to #adj c4c4 normal sal9e conta9$ 9 ! fitt$ ,erl5mey] flask4 #d4 ,place ! flask 9 ! wat]-ba? ( boil+ wat]4 #e4 ,connect up ! /raight tube1 aft] remov+ ! cotton-wool plug1 ) ! deliv]y tube ( ! /eam c2 remove ! plug f ! v5t tube4 #f4 ,:5 ! !rmomet] rea*es #ajj deg4 ,c41 op5 ! spr+ clip on ! syphon1 disc>d ! fir/ cubic c5timet] ( susp5sion t syphons ov] 7i4 e41 ! cont5ts ( ! syphon tube72 collect ! next #e c4c4 ( ! susp5sion 9 ! /]ile graduat$ te/-tube & p\r plates & prep>e flask cultures !refrom z 9 ! previ\s exp]im5ts4 #g4 ,repeat ?is process at int]vals ( tw5ty-five m9utes' /eam+4 #h4 ,obs]ve ! inoculat$ plates & flasks up to ! completion1 if necess>y1 ( sev5 days' incubation4 #i4 ,control ^! exp]im5ts1 b 9 ?is in/ance syphon (f portions ( ! susp5sion at int]vals ( "o-half to "o m9ute dur+ ! five or t5 m9utes prec$+ ! previ\sly det]m9$ dea?-po9t4 ,!rmal ,dea?-po9t4-- ,dry--,vegetative ,=ms3 ,! !rmal dea?-po9t 9 ?is case is t temp]ature :i* ) c]ta9ty kills a ?9 film ( ! organism 9 "q aft] a "t exposure ( t5 m9utes4 ,app>atus ,requir$3 ,hot-air ov51 provid$ ) !rmo-regulator4 ,/]ile cov]-slips4 ,flask conta9+ #bej c4c4 /]ile normal sal9e solution4 ,case ( /]ile pipettes1 #aj c4c4 79 t5?s ( a cubic c5timetre74 ,case ( /]ile capsules4 ,crucible tongs4 ,,me?od4-- #a4 ,prep>e an emulsion ) ?ree loopfuls f an optimum cultivation 9 #e c4c4 normal sal9e 9 a /]ile capsule & exam9e microscopically to det]m9e ! abs5ce ( spore =ms4 #b4 ,make twelve cov]-slip films on /]ile cov]-slips2 place ea* 9 a /]ile capsule to dry4 #c4 ,expose ea* capsule 9 turn 9 ! hot-air ov5 = t5 m9utes to a diff]5t fix$ temp]ature1 v>y+ #e deg4 ,c4 betwe5 #fj deg4 ,c4 & #abj deg4 ,c4 #d4 ,remove ea* capsule f ! ov5 ) crucible tongs imm$iately aft] ! t5 m9utes are complet$2 remove ! cov]-glass f its int]ior ) a /]ile pair ( =ceps4 #e4 ,deposit ! film 9 a flask conta9+ #bjj c4c4 nutri5t b\illon4 #f4 ,prep>e subcultivations f su* flasks z %[ evid5ce ( gr[?1 to det]m9e t no accid5tal contam9ation has tak5 place b t ! organism orig9ally spread on ! film is responsible = ! gr[?4 #g4 ,control ! result ( ^! exp]im5ts4 ,dry--,spores3 ,! !rmal dea?-po9t 9 ?is case is t temp]ature :i* ) c]ta9ty kills ! spores ( ! organism 9 "q :5 pres5t 9 a ?9 film aft] a "t exposure ( #aj m9utes4 ,app>atus ,requir$3 ,z = vegetative =ms4 ,,me?od4-- #a4 ,prep>e a slop$ ag> tube cultivation & incubate "u optimum conditions z to spore =mations4 #b4 ,pipette #e c4c4 /]ile normal sal9e into ! culture tube & emulsify ! entire surface gr[? 9 x4 ,exam9e microscopically to det]m9e ! pres5ce ( spores 9 l>ge numb]s4 #c4 ,spread ?9 ev5 films on twelve /]ile cov]-slips & place ea* cov]-slip 9 a sep>ate /]ile capsule4 #d4 ,expose ea* capsule 9 turn = t5 m9utes to a diff]5t fix$ temp]ature1 v>y+ #e deg4 ,c1 betwe5 #ajj deg4 ,c4 & #afj deg4 ,c4 #e4 ,complete ! exam9ation z = vegetative =ms4 ,,iii4 ,reaction ( ,m$ium4 7,a7 ,range4-- #a4 ,prep>e a b\illon culture ( ! organism & incubate1 "u optimum conditions z to temp]ature & atmosph]e1 = tw5ty-f\r h\rs4 #b4 ,pipette #j4a c4c4 ( ! cultivation into a /]ile capsule2 add #i4i c4c4 /]ile b\illon & mix ?or\ghly4 #c4 ,prep>e a s]ies ( tubes ( nutri5t b\illon ( v>y+ reactions1 f #be to -#cj 7vide page #aee71 viz43 #be1 #bj1 #ae1 #aj1 #e1 neutral1 -#e1 -#aj1 -#ae1 -#bj1 -#be1 -#cj4 #d4 ,inoculate ea* ( ! b\illon tubes ) #j4a c4c4 ( ! dilut$ cultivation by means ( a /]ile graduat$ pipette & incubate "u optimum conditions4 #e4 ,obs]ve ! cultures at half-h\rly int]vals f ! ?ird to ! twelf? h\rs4 ,note ! reaction ( ! tube or tubes 9 :i* gr[? is fir/ visible macroscopically 7probably optimum reaction74 #f4 ,cont9ue ! incubation until ! completion1 if necess>y1 ( sev5 days4 ,note ! extremes ( acidity & alkal9ity 9 :i* macroscopical gr[? has develop$ 7,range ( reaction74 #g4 ,control ! result ( ^! obs]vations4 7,b7 ,optimum ,reaction4--,! optimum reaction has already be5 r\ghly det]m9$ :il/ obs]v+ ! range4 ,x c be fix$ )9 n>r[] limits by inoculat+ 9 a simil> mann] a s]ies ( tubes ( b\illon :i* repres5t small] v>iations 9 reaction ?an ^? previ\sly employ$ 7say1 #a in/ead ( #e7 = five po9ts on ei!r side ( ! previ\sly obs]v$ optimum4 ,= example1 ! optimum reaction obs]v$ 9 ! set ( exp]im5ts to det]m9e ! range 0 #aj4 ,n[ plant tubes hav+ reactions ( #ae1 #ad1 #ac1 #ab1 #aa1 #aj1 #i1 #h1 #g1 #f1 #e1 & obs]ve z be=e4 ,,iv4 ,resi/ance to ,le?al ,ag5ts4-- 7,a7 ,desiccation4-- ,app>atus ,requir$3 ,muell]'s desiccator4 ,?is consi/s ( a bell glass fitt$ ) an exhau/ tube & /op-cock 7d71 :i* c be secur$ to a plate-glass base 7c7 by means ( wax or grease4 ,x conta9s a cyl9drical vessel ( por\s clay 7a7 into ! top ( :i* pure sulphuric acid is p\r$ :il/ ! mat]ial to be dri$ is plac$ )9 its walls on a glass %elf 7b74 ,! air is exhau/$ f ! int]ior & ! acid rapidly conv]ts ! clay vessel into a l>ge absorb+ surface 7,fig4 #aeg74 ,exhau/ pump4 ,pure conc5trat$ sulphuric acid4 ,/]ile cov]-slips4 ,/]ile =ceps4 ,culture flask conta9+ #bjj c4c4 nutri5t b\illon4 ,/]ile v5tilat$ ,petri di%4 ,?is is prep>$ by b5d+ ?ree %ort pieces ( alum9ium wire into ,v %ape & hang+ ^! on ! edge ( ! l[] di% & re/+ ! lid ^u !m 7,fig4 #aeh74 ,,me?od4-- #a4 ,prep>e a surface cultivation on nutri5t ag> 9 a culture bottle & incubate "u optimum conditions = =ty-eight h\rs4 #b4 ,exam9e prep>ations f ! cultivation1 microscopically1 to det]m9e ! abs5ce ( spores4 #c4 ,pipette #e c4c4 /]ile normal sal9e solution into ! flask & susp5d ! entire gr[? 9 x4 #d4 ,spread ! susp5sion 9 ?91 ev5 films on /]ile cov]-slips & deposit inside /]ile plates to dry4 #e4 ,z soon z dry1 transf] ! cov]-slip films to ! v5tilat$ ,petri di% by means ( /]ile =ceps4 ,illu/ration3 ,,fig4 #aeg4--,muell]'s desiccator4 #f4 ,place ! ,petri di% inside ! ,muell]'s desiccator2 fill ! upp] *amb] ) pure sulphuric acid1 cov] ) ! bell j>1 & exhau/ ! air f its int]ior4 ,t5 m9utes lat] connect up ! desiccator to a sulphuric acid wa%-bottle int]pos+ an air filt] s t only dry /]ile air ent]s4 ,illu/ration3 ,,fig4 #aeh4--,petri di% = dry+ cultivations4 #g4 ,at int]vals ( five h\rs op5 ! app>atus1 remove "o ( ! cov]-slip films f ! ,petri di%1 & transf] x to ! int]ior ( a culture flask1 ) e precaution aga9/ contam9ation4 ,reseal ! desiccator & aga9 exhau/1 & subsequ5tly admit dry /]ile air z be=e4 #h4 ,incubate ! culture flask "u optimum conditions until ! completion ( sev5 days1 if necess>y2 & det]m9e ! "t exposure at :i* dea? occurs4 #i4 ,p\r plates f ^? culture flasks :i* gr[1 to det]m9e ! abs5ce ( contam9ation4 #aj4 ,repeat ^! obs]vations at h\rly int]vals = ! five h\rs prec$+ & succe$+ ! dea? "t1 z det]m9$ 9 ! fir/ set ( exp]im5ts4 7,b7 ,light4-- 7a7 ,diffuse ,daylight3 #a4 ,prep>e a tube cultivation 9 nutri5t b\illon1 & incubate "u optimum conditions1 = =ty-eight h\rs4 ,illu/ration3 ,,fig4 #aei4--,plate ) /> = te/+ effect ( light4 #b4 ,p\r tw5ty plate cultivations1 t5 ( nutri5t gelat9e & t5 ( nutri5t ag>1 ea* conta9+ #j4a c4c4 ( ! b\illon culture4 #c4 ,place "o ag> plate & "o gelat9e plate into ! hot & cold incubators1 respectively1 z controls4 #d4 ,fa/5 a piece ( black pap]1 cut ! %ape ( a cross or />1 on ! c5tre ( ! cov] ( ea* ( ! rema9+ plates 7,fig4 #aei74 #e4 ,expose ^! plates to ! action ( diffuse daylight 7n direct sunlight7 9 ! laboratory = "o1 two1 ?ree1 f\r1 five1 six1 eight1 t51 twelve h\rs4 #f4 ,aft] exposure to light1 incubate "u optimum conditions4 #g4 ,exam9e ! plate cultivations aft] tw5ty-f\r & =ty-eight h\rs' incubation1 & comp>e ) ! two controls4 ,record results4 ,if gr[? is abs5t f t portion ( ! plate unprotect$ by ! black pap]1 cont9ue ! incubation & daily obs]vation until ! end ( sev5 days4 #h4 ,control ! results4 7b7 ,direct ,sunlight3 #a4 ,prep>e plate cultivations precisely z 9 ! =m] exp]im5ts & place ! two controls 9 ! incubators4 #b4 ,arrange ! rema9+ plates ^u a plat=m 9 ! direct rays ( ! sun4 #c4 ,on ! top ( ea* plate /& a small glass di% #ad cm4 9 diamet] & #e cm4 deep4 #d4 ,fill a solution ( pota% alum 7#b p] c5t4 9 di/ill$ wat]7 into ea* di% to ! dep? ( #b cm4 to absorb ! heat ( ! sun's rays & s elim9ate possible effects ( temp]ature on ! cultivations4 #e4 ,aft] exposures = p]iods simil> to ^? employ$ 9 ! prec$+ exp]im5t1 incubate & complete ! obs]vation z above4 7c7 ,prim>y ,col\rs3 ,ea* col\r--violet1 blue1 gre5 & r$--mu/ be te/$ sep>ately4 #a4 ,prep>e plate cultivations1 z 9 ! previ\s light exp]im5ts1 & incubate controls4 #b4 ,fa/5 a /rip ( black pap]1 #c cm4 wide1 across "o diamet] ( ! cov] ( ea* plate4 #c4 ,coat ! rema9d] ( ! surface ( ! cov] ) a film ( pure photographic collodion :i* conta9s #b p] c5t4 ( ei!r ( ! foll[+ anil9e dyes1 z may be necess>y3 ,*rysoid9 7= r$74 ,mala*ite gre5 7= gre574 ,eos91 blui% 7= blue74 ,me?yl violet 7= violet74 #d4 ,expose ! plates1 ?us prep>$1 to bright daylight 7b n direct sunlight7 = v>y+ p]iods1 & complete ! obs]vations z 9 ! prec$+ exp]im5ts4 ,! bact]icidal action ( light appe>s to dep5d ^u ! m refrangible rays ( ! violet end ( ! spectrum & is not$ :e!r ! r$ yell[ rays are transmitt$ or n4 #e4 ,control ! results4 ,,note4--,! ultra-violet rays obta9$ f a qu>tz m]cury vap\r lamp de/roy bact]ial life ) great rapidity "u laboratory conditions4 7,c7 ,heat4--7,vide ,!rmal ,dea?-po9t1 page #bih47 7,d7 ,antiseptics & ,dis9fectants4--,! resi/ance exhibit$ by any giv5 bact]ium t[>d any specifi$ dis9fectant or g]micide %\ld be inve/igat$ ) ref]5ce to ! foll[+ po9ts3 7,a7 ,inhibition coeffici5t--i4 e41 t p]c5tage ( ! dis9fectant pres5t 9 ! nutri5t m$ium :i* is suffici5t to prev5t ! gr[? & multiplication ( ! bact]ium4 7,b7 ,inf]ior le?al coeffici5t--i4 e41 ! "t exposure necess>y to kill vegetative =ms ( ! bact]ium susp5d$ 9 wat] at #bj deg4 to #be deg4 ,c1 9 :i* ! dis9fectant is pres5t 9 m$ium conc5tration 7conc5tration insuffici5t to cause plasmolysis74 ,& if ! bact]ium is "o :i* =ms spores1 7,c7 ,sup]ior le?al coeffici5t--i4 e41 ! "t exposure necess>y to kill ! spores ( ! bact]ium "u conditions simil> to ^? obta9+ 9 ,b4 ,! example "h detail$ only specifically ref]s to c]ta9 ( ! dis9fectants3 viz3--,bi*loride ( m]cury2 ,=maldehyde2 ,c>bolic acid2 inve/igat$ ) reg>d to ,b4 an?racis1 b ! te*nique is practically simil> = all o!r *emical dis9fectants4 ,inhibition ,coeffici5t4-- ,app>atus ,requir$3 ,case ( /]ile pipettes1 #aj c4c4 79 t5?s74 ,case ( /]ile pipettes1 #a c4c4 79 t5?s74 ,/]ile tubes or capsules = dilutions4 ,tubes ( nutri5t b\illon ea* conta9+ a measur$ #aj c4c4 ( m$ium4 ,tw5ty-f\r-h\r-old ag> culture ( a rec5tly isolat$ ,b4 ,an?racis4 ,g]micides3 #a4 ,five p] c5t4 aque\s solution ( c>bolic acid4 #b4 ,"o p] c5t4 aque\s solution ( p]*loride ( m]cury4 #c4 ,"o-t5? p] c5t4 aque\s solution ( =maldehyde4 ,,me?od4-- #a4 ,numb] six b\illon tubes consecutively #a to #f4 ,inoculate ea* f ! /ock cultivation ( ,b4 an?racis & at once add v>y+ quantities#aj ( ! c>bolic acid solution1 viz43 ,to tube #a add #b4j c4c4 7 #a3ajj7 ,to tube #b add #a4j c4c4 7 #a3bjj7 ,to tube #c add #j4f c4c4 7 #a3cjj7 ,to tube #d add #j4e c4c4 7 #a3djj7 ,to tube #e add #j4d c4c4 7 #a3ejj7 ,to tube #f add #j4b c4c4 7 #a3a1jjj7 #b4 ,prep>e a simil> s]ies ( tube cultivations numb]$ consecutively #g to #ab & add v>y+ quantities ( ! m]curic p]*loride solution1 viz43 ,to tube #g add #j4a 7 #a3a1jjj7 ,to tube #h add #j4je 7 #a3b1jjj7 ,to tube #i add #j4jc 7 #a3c1jjj7 ,to tube #aj add #j4jbe 7 #a3d1jjj7 ,to tube #aa add #j4jb 7 #a3e1jjj7 ,to tube #ab add #j4ja 7 #a3aj1jjj7 #c4 ,prep>e a simil> s]ies ( tube cultivations numb]$ consecutively #ac to #ah & add v>y+ quantities ( ! =maldehyde solution1 viz43 ,to tube ,no4 #ac add #a4j c4c4 7 #a3a1jjj7 ,to tube ,no4 #ad add #j4d c4c4 7 #a3b1ejj7 ,to tube ,no4 #ae add #j4b c4c4 7 #a3e1jjj7 ,to tube ,no4 #af add #j4a c4c4 7 #a3aj1jjj7 ,to tube ,no4 #ag add #j4jge c4c4 7 #a3ae1jjj7 ,to tube ,no4 #ah add #j4je c4c4 7 #a3bj1jjj7 #d4 ,incubate all ?ree sets ( cultivations "u optimum conditions z to temp]ature & atmosph]e4 #e4 ,exam9e ea* ( ! culture tubes f day to day1 until ! completion ( sev5 days1 & note ^? tubes1 if any1 9 :i* gr[? takes place4 #f4 ,f su* tubes z %[ gr[? prep>e subcultivations ^u suitable m$ia1 & asc]ta9 t ! organism caus+ ! gr[? is ! "o orig9ally employ$ 9 ! te/ & n an accid5tal contam9ation4 ,inf]ior ,le?al ,coeffici5t4-- ,app>atus ,requir$3 ,highly conc5trat$ solutions ( ! dis9fectants4 ,/]ile te/-tubes 9 :i* to make dilutions f ! conc5trat$ solutions ( ! dis9fectants4 ,hang+-drop slides4 ,cov]-slips4 ,erl5mey] flask conta9+ #ajj c4c4 /]ile di/ill$ wat]4 ,case ( /]ile pipettes1 #aj c4c4 79 t5?s ( a cubic c5timetre74 ,case ( /]ile pipettes1 #a c4c4 79 t5?s ( a cubic c5timetre74 ,,me?od4-- #a4 ,prep>e a surface cultivation ( ! te/ organism ,b4 an?racis ^u nutri5t ag> 9 a culture bottle & incubate "u optimum conditions = tw5ty-f\r h\rs2 !n exam9e ! cultivation microscopically to det]m9e ! abs5ce ( spores4 #b4 ,prep>e solutions ( diff]5t p]c5tages ( ea* dis9fectant4 #c4 ,make a s]ies ( hang+-drop prep>ations f ! ag> culture1 us+ a loopful ( dis9fectant solution ( ! diff]5t p]c5tages to prep>e ! emulsion on ea* cov]-slip4 #d4 ,exam9e microscopically & note ! /ronge/ solution :i* does n cause plasmolysis & ! weake/ solution :i* does plasmolyse ! organism4 #e4 ,make control prep>ations ( ^! two solutions & det]m9e ! p]c5tage to be te/$4 #f4 ,pipette #aj c4c4 /]ile wat] into ! culture bottle & susp5d ! entire surface gr[? 9 x4 #g4 ,transf] ! susp5sion to ! ,erl5mey] flask & mix x ) ! #ij c4c4 ( /]ile wat] rema9+ 9 ! flask4 #h4 ,pipette #aj c4c4 ( ! dilut$ susp5sion into ea* ( t5 /]ile te/-tubes4 #i4 ,label "o ( ! tubes ,control & place x 9 ! incubator at #ah deg4 ,c4 #aj4 ,add to ea* ( ! rema9+ tubes a suffici5t quantity#aa ( a conc5trat$ solution ( ! dis9fectant to produce ! p]c5tage previ\sly det]m9$ ^u 7vide /ep #e74 #aa4 ,incubate ! tubes at #ah deg4 ,c4 to #bj deg4 ,c4 #ab4 ,at h\rly int]vals remove ! control tube & "o ( ! tubes ) add$ dis9fectant f ! incubator4 #ac4 ,make a subcultivation f bo? ! control & ! te/ susp5sion1 ^u ! surface ( nutri5t ag>2 incubate "u optimum conditions4 #ad4 ,obs]ve ^! culture tubes f day to day until ! completion ( sev5 days1 & det]m9e ! %orte/ exposure necess>y to cause ! dea? ( vegetative =ms4 ,sup]ior ,le?al ,coeffici5t4-- #a4 ,prep>e surface cultivations ( ! te/ organisms ^u nutri5t ag> 9 a culture bottle1 & incubate "u optimum conditions1 = ?ree days1 = ! =mation ( _! spores4 #b4 ,transf] ! emulsion to a /]ile te/-tube & heat 9 ! diff]5tial /]ilis] = t5 m9utes at #hj deg4 ,c4 to de/roy all vegetative =ms4 #c4 ,employ+ t p]c5tage solution ( ! dis9fectant det]m9$ 9 ! previ\s exp]im5t1 & complete ! inve/igations z detail$ !re91 /eps #g to #ad1 increas+ ! int]val betwe5 plant+ ! subcultivations to two1 ?ree1 or five h\rs if consid]$ advisable4 ,,note4--,:]e x is necess>y to leave ! organisms 9 contact ) a /rong solution ( ! dis9fectant = l5g?y p]iods1 "s means mu/ be adopt$ to remove e trace ( ! dis9fectant f ! bact]ia be=e transf]r+ !m to fre% culture m$ia2 o!rwise1 al?\gh n actually kill$1 ! pres5ce ( ! dis9fectant may prev5t _! developm5t1 & s give rise to an errone\s conclusion4 ,consequ5tly x is ess5tial 9 all g]micidal exp]im5ts to det]m9e fir/ ( all ! inhibition coeffici5t ( ! g]micide employ$4 ,"u ! circum/ances ref]r$ to above x is usually suffici5t to prep>e ! subcultures 9 su* a volume ( fluid nutri5t m$ium z w\ld suffice to r$uce ! conc5tration ( ! g]micide to ab\t "o hundr$? ( ! inhibition p]c5tage1 assum+ t ! entire bulk ( inoculum 0 made up ( t /r5g? ( g]micide employ$ 9 ! te/4 ,9 "s cases x is a simple matt] to neutralise ! g]micide & r5d] x in]t by wa%+ ! organisms 9 "s non-g]micidal solution 7su* = example z ammonium sulphide :5 us+ m]curial salts z ! g]micide74 ,:51 h[ev]1 x is desir$ to remove ! la/ traces ( g]micide proce$ z foll[s3 #a4 ,transf] ! susp5sion ( bact]ia to /]ile c5trifugal tubes2 add ! requir$ am\nt ( dis9fectant1 & all[ x to rema9 9 contact ) ! bact]ia = ! necess>y p]iod4 #b4 ,c5trifugalise ?or\ghly1 pipette (f ! sup]natant fluid2 fill ! tube ) /]ile wat] & di/ribute ! deposit ev5ly ?r\gh\t ! fluid4 #c4 ,c5trifugalise aga91 pipette (f ! sup]natant fluid2 fill ! tube ) /]ile wat]2 di/ribute ! deposit ev5ly ?r\gh\t ! fluid1 & transf] ! susp5sion to a litre flask4 #d4 ,make up to a litre by ! addition ( /]ile wat]2 filt] ! susp5sion ?r\gh a /]ile porcela9 c&le4 #e4 ,emulsify ! bact]ial residue ) #e c4c4 /]ile b\illon4 #f4 ,prep>e ! necess>y subcultivations f ?is emulsion4 ,,pa?og5esis4 ,liv+ ,bact]ia4-- 7a7 ,psy*rophilic ,bact]ia3 ,:5 ! organism w only gr[ at or bel[ #ah deg4 to #bj deg4 ,c41 #a4 ,prep>e cultivations 9 nutri5t bro? & incubate "u optimum conditions4 #b4 ,aft] sev5 days' incubation inject t am\nt ( ! culture correspond+ to #a p] c5t4 ( ! body-weight ( a heal?y frog1 into ! reptile's dorsal lymph sac4 #c4 ,obs]ve until dea? takes place1 or1 9 ! ev5t ( a negative result1 until ! completion ( tw5ty-eight days 7vide ,*apt] ,,xviii74 #d4 ,if1 & :51 dea? occurs1 make a c>eful po/-mortem exam9ation 7vide ,*apt] ,,xix74 7b7 ,mesophilic ,bact]ia3 ,:5 ! organism gr[s at #ce deg4 to #cg deg4 ,c41 #a4 ,prep>e cultivations 9 nutri5t bro? & incubate "u optimum conditions = =ty-eight h\rs4 #b4 ,select two :ite mice1 z ne>ly z possible ( ! same age1 size1 & weight4 #c4 ,inoculate ! fir/ m\se1 subcutane\sly at ! root ( ! tail1 ) an am\nt ( cultivation equival5t to #a p] c5t4 ( its body-weight4 #d4 ,inoculate ! second m\se intrap]itoneally ) a simil> dose4 #e4 ,obs]ve c>efully until dea? occurs1 or until ! lapse ( tw5ty-eight days4 #f4 ,if ! inoculat$ animals succumb1 make complete po/-mortem exam9ation4 ,if dea? foll[s %ortly aft] ! injection ( cultivations ( bact]ia1 ! inoculation exp]im5ts %\ld be repeat$ two or ?ree times4 ,!n1 if ! organism "u obs]vation inv>iably exhibits pa?og5ic effects1 /eps %\ld be tak5 to asc]ta91 if possible1 ! m9imal le?al dose 7vide infra7 ( ! gr[? ^u solid m$ia = ! frog or :ite m\se respectively4 ,o!r exp]im5tal animals--e4 g41 ! :ite rat1 gu9ea-pig1 & rabbit--%\ld next be te/$ 9 a simil> mann]4 #g4 ,if ! inoculat$ mice are unaffect$1 te/ ! action ( ! organism 9 "q ^u :ite rats1 gu9ea-pigs1 rabbits1 etc4 ,m9imal ,le?al ,dose 7m4 l4 d472 ,if ! purpose ( ! inoculation is to det]m9e ! m9imal le?al dose1 a slightly diff]5t proc$ure mu/ be foll[$4 ,= ?is & o!r exact exp]im5ts a special plat9um loop is manufactur$1 "s #b4e mm4 by #j4ge mm41 ) p>allel sides1 & calibrat$ by c>eful weigh+1 to det]m9e approximately ! am\nt ( moi/ bact]ial gr[?1 ! loop w hold :5 fill$4 #a4 ,! cultivation mu/ be prep>$ on a solid m$ium ( ! optimum reaction1 incubat$ at ! optimum temp]ature1 & inject$ at ! p]iod ( greate/ activity & vig\r1 ( ! p>ticul> organism x is desir$ to te/4 #b4 ,arrange f\r /]ile capsules 9 a r[ & label !m ,i1 ,,ii1 ,,iii1 & ,,iv4 ,into ! fir/ deliv] #aj c4c4 /]ile b\illon by means ( a /]ile graduat$ pipette2 & into ea* ( ! rema9+ ?ree1 #i4i c4c4 #c4 ,remove "o loopful ( ! bact]ial gr[? f ! surface ( ! m$ium 9 ! culture tube1 obs]v+ ! usual precautions aga9/ contam9ation1 & emulsify x ev5ly ) ! b\illon 9 ! fir/ capsule4 ,ea* cubic c5timetre ( ! emulsion w n[ conta9 "o-t5? ( ! organisms conta9$ 9 ! orig9al loopful 7writt5 %ortly #j4a loop74 #d4 ,remove #j4a c4c4 ( ! emulsion 9 ! fir/ capsule by means ( a /]ile graduat$ pipette & transf] x to ! second capsule & mix ?or\ghly4 ,drop ! infect$ pipette into a j> ( lysol solution4 ,?is makes up ! bulk ( ! fluid 9 ! second capsule to #aj c4c41 & !re=e e cubic c5timetre ( b\illon 9 capsule ,,ii conta9s #j4jja loop4 #e4 ,simil>ly1 #j4a c4c4 ( ! mixture is transf]r$ f capsule ,,ii to capsule ,,iii 7#a c4c4 ( b\illon 9 capsule ,,iii conta9s #j4jjjja loop71 & !n f capsule ,,iii to capsule ,,iv 7#a c4c4 ( b\illon 9 capsule ,,iv conta9s #j4jjjjjja loop74 ,! dilutions ?us prep>$ may be summ>is$ 9 a table2 ,capsule ,i #a loopful #aj c4c4 wat] 4'4 #a c4c4#j4a loop4 ,capsule ,,ii #j4a c4c4 capsule ,i #i4i c4c4 wat] 4'4 #a c4c4#j4jja loop4 ,capsule ,,iii #j4a c4c4 capsule ,,ii #i4i c4c4 wat] 4'4 #a c4c4#j4jjjja loop4 ,capsule ,,iv #j4a c4c4 capsule ,,iii #i4i c4c4 wat] 4'4 #a c4c4 #j4jjjjjja loop4 #f4 ,) /]ile graduat$ pipettes remove ! necess>y quantity ( b\illon correspond+ to ! v>i\s divisors ( t5 ( ! loop f ! respective capsules1 & transf] ea* dose to a sep>ate /]ile capsule & label2 & to su* doses z are small 9 bulk1 add ! necess>y quantity ( /]ile b\illon to make up to #a c4c4 #g4 ,multiples ( ! loop are prep>$ by emulsify+ #a1 #b1 #e1 or #aj loops ea* ) #a c4c4 /]ile b\illon 9 sep>ate /]ile capsules4 #h4 ,inoculate a s]ies ( animals ) ^! measur$ doses1 fill+ ! syr+e fir/ f t capsule conta9+ ! smalle/ dose1 !n f ! capsule conta9+ ! next smalle/1 & s on4 ,if c>e is tak51 x w n be f\nd necess>y to /]ilise ! syr+e dur+ ! s]ies ( inoculations4 #i4 ,plant tubes ( gelat9e or ag>1 liquefi$ by heat1 f ea* ( ! high] dilutions1 say f #j4jjjjjja loop to #j4ja loop2 p\r plates & incubate4 ,:5 gr[? is visible enum]ate ! numb] ( organisms pres5t 9 ea*1 av]age up & calculate ! numb] ( bact]ia pres5t 9 "o loopful ( ! inoculum4 #aj4 ,! smalle/ dose :i* causes ! infection & dea? ( ! inoculat$ animal is not$ z ! m9imal le?al dose4 ,tox9s4-- ,prep>e flask cultivations ( ! organism "u obs]vation 9 glucose =mate bro?1 & incubate = f\rte5 days "u optimum conditions4 7a7 ,intracellul> or ,insoluble ,tox9s3 #a4 ,heat ! fluid culture 9 a wat]-ba? at #fj deg4 ,c4 = ?irty m9utes4 7,! result+ /]ile1 turbid fluid is (t5 spok5 ( z kill$ culture17 #b4 ,inoculate a tube ( /]ile b\illon ) a simil> quantity1 & incubate "u optimum conditions4 ,?is control !n s]ves to demon/rate ! fre$om ( ! tox9 f liv+ bact]ia4 ,illu/ration3 ,,fig4 #afj4--;,app>atus arrange = tox9 filtration4 #c4 ,inject intrav5e\sly t am\nt ( ! cultivation correspond+ to #a p] c5t4 ( ! body-weight ( ! select$ animal1 usually "o ( ! small rod5ts4 #d4 ,obs]ve dur+ life or until ! completion ( tw5ty-eight days1 & 9 ! ev5t ( dea? occurr+ dur+ t p]iod1 make a complete po/-mortem exam9ation4 #e4 ,repeat ! exp]im5t at lea/ once4 ,9 ! ev5t ( a positive result e/imate ! m9imal le?al dose ( kill$ culture = ea* ( ! species ( animals exp]im5t$ ^u4 7b7 ,extracellul> or ,soluble ,tox9s3 #a4 ,filt] ! cultivation ?r\gh a porcela9 filt] c&le 7,b]kefeld7 into a /]ile filt] flask1 arrang+ ! app>atus z 9 ! accompany+ figure 7,fig4 #afj74 #b4 ,inoculate mice1 rats1 gu9ea-pigs1 & rabbits subcutane\sly ) t quantity ( tox9 correspond+ to #a p] c5t4 ( ! body-weight ( ea* respectively1 & obs]ve1 if necess>y1 until ! completion ( "o mon?4 #c4 ,inoculate a control tube ( b\illon ) a simil> quantity & incubate1 to det]m9e ! fre$om ( ! filt]$ tox9 f liv+ bact]ia4 #d4 ,9 ! ev5t ( a fatal t]m9ation make complete & c>eful po/-mortem exam9ations4 #e4 ,repeat ! exp]im5ts &1 if ! results are positive1 asc]ta9 ! m9imal le?al dose ( tox9 = ea* ( ! susceptible animals4 ,! e/imation ( ! m4 l4 d4 ( a tox9 is c>ri$ out on l9es simil> to ^? laid d[n = liv+ bact]ia 7vide page #caf7 m]ely sub/itut+ #a c4c4 ( tox9 z ! unit 9 place ( ! unit loopful ( liv+ culture4 ,x frequ5tly happ5s1 dur+ ! c\rse ( casual inve/igations t a b\illon-tube culture is available = a tox9 te/ :il/ a flask cultivation is n4 ,9 su* cases1 ,m>t9's small filt] c&le & tube 7,fig4 #afa7 specially design$ = ! filtration ( small quantities ( fluid1 is invaluable4 ,?is consi/s ( a n>r[ filt] flask j l>ge en\gh to accommodate an ord9>y #ah x #b cm4 te/-tube4 ,! m\? ( ! tubul> ,*amb]l& c&le #ae x #a4e cm4 is clos$ by a p]=at$ rubb] cork into :i* fits ! end ( ! /em ( a ?i/le head$ funnel1 :il/ imm$iately bel[ ! butt ( ! funnel is situat$ a rubb] cork to close ! m\? ( ! filt] flask4 ,:5 ! app>atus is fix$ 9 position & connect$ to an exhau/ pump1 ! cultivation is p\r$ into ! head ( ! funnel & ow+ to ! relatively l>ge filt]+ surface ! g]m free filtrate is rapidly drawn ?r\gh into ! te/-tube receiv]4 ,rais+ ! ,virul5ce ( an ,organism4--,if x is desir$ to raise or exalt ! virul5ce ( a feebly pa?og5ic organism1 special me?ods ( inoculation are necess>y1 c>efully adju/$ to ! exig5cies ( ea* individual case4 ,among ! mo/ important are ! foll[+3 #a4 ,passage ( ,virus4--,! inoculation ( pure cultivations ( ! organism into highly susceptible animals1 & pass+ x z rapidly z possible f animal to animal1 always select+ t me?od ( inoculation-e4 g41 intrap]itoneal--:i* places ! organism "u ! mo/ favorable conditions = its gr[? & multiplication4 ,illu/ration3 ,,fig4 #afa--,m>t9's filt]+ app>atus = small quantities ( fluid4 #b4 ,virus ,plus ,virul5t ,organisms4--,! inoculation ( pure cultivations ( ! organism toge!r ) pure cultivations ( "s o!r microbe :i* 9 itself is suffici5tly virul5t to ensure ! dea? ( ! exp]im5tal animal1 ei!r into ! same situation or into "s o!r "p ( ! body4 ,by ?is association ! organism ( l[ virul5ce w frequ5tly acquire a high] degree ( virul5ce1 :i* may be /ill fur!r rais$ by means ( passages 7vide supra74 #c4 ,virus ,plus ,tox9s4--,! inoculation ( pure cultivations ( ! organism into "s select$ situation1 toge!r ) ! subcutane\s1 intrap]itoneal1 or intrav5\s injection ( a tox9--e4 g41 "o ( ^? elaborat$ by ! proteus gr\p--ei!r simultane\sly )1 be=e1 or imm$iately aft]1 ! injection ( ! feeble virus4 ,by ?is means ! natural resi/ance ( ! animal is l[]$1 & ! organism inoculat$ is enabl$ to multiply & produce its pa?og5ic effect1 its virul5ce be+ subsequ5tly exalt$ by means ( passages4 ,att5uat+ ! ,virul5ce ( an ,organism4--,att5uat+ or l[]+ ! virul5ce ( a pa?og5ic microbe is usually atta9$ ) mu* less difficulty ?an ! exaltation ( its virul5ce1 & is g5]ally effect$ by v>y+ ! environm5t ( ! cultivations1 z = example3 #a4 ,cultivat+ 9 su* m$ia z are unsuitable by reason ( _! 7a7 composition or 7b7 reaction4 #b4 ,cultivat+ 9 suitable m$ia1 b at an unsuitable temp]ature4 #c4 ,cultivat+ 9 suitable m$ia1 b 9 an unsuitable atmosph]e4 #d4 ,cultivation 9 suitable m$ia1 b "u unfavorable conditions z to light1 motion1 etc4 ,att5uation ( ! virus c also be secur$ by #e4 ,passage ?r\gh naturally resi/ant animals4 #f4 ,exposure to desiccation4 #g4 ,exposure to gase\s dis9fectants4 #h4 ,by a comb9ation ( two or m ( ! above me?ods4 ,,immunisation4 ,! fur!r /udy ( ! pa?og5etic p[]s ( any p>ticul> bact]ium involves ! active immunisation ( "o or m previ\sly normal animals4 ,?is end may be atta9$ by v>i\s means2 b x mu/ be rememb]$ t immunisation is n c>ri$ out by any h>d & fa/ rule or by "o me?od alone1 b usually by a comb9ation ( me?ods adapt$ to ! exig5cies ( ea* p>ticul> case4 ,! ord9>y me?ods include3 ,a4 ,active ,immunisation4 ,i4 ,by inoculation ) dead bact]ia 7i4 e41 bact]ia kill$ by heat2 ! action ( ultra-violet rays1 ( *emical g]micides1 or by autolysis74 ,,ii4 ,by ! inoculation ( att5uat$ /ra9s ( bact]ia4 ,,iii4 ,by ! inoculation ( liv+ virul5t bact]ia 7exalt$ 9 virul5ce if necess>y74 ,b4 ,comb9$ ,active & ,passive ,immunisation3 ,,iv4 ,by ! inoculation ( tox9-antitox9 mixtures4 ,,active ,,immunisation4 ,! immunisation ( ! rabbit aga9/ ! ,diplococcus pneumoniae may be in/anc$ z an example ( ! g5]al me?ods ( immunisation ( laboratory animals4 #a4 ,take a full gr[n rabbit weigh+ n less ?an #abjj to #aejj grammes 7l>ge rabbits ( #bjjj grammes & ov] are ! mo/ suitable = immunis+ exp]im5ts74 ,obs]ve weight & temp]ature c>efully dur+ ! few days occupi$ 9 ! foll[+ /eps4 #b4 ,inoculate a small rabbit intrap]itoneally ) "o or two loopfuls ( a tw5ty-f\r-h\r-old blood ag> cultivation ( a virul5t /ra9 ( ,diplococcus pneumoniae4 ,dea? %\ld foll[ )9 tw5ty-f\r h\rs1 & 9 any case w n be delay$ beyond =ty-eight h\rs4 #c4 ,"u aseptic precautions1 at ! po/-mortem1 transf] a loopful ( he>t blood to an ,erl5mey] flask conta9+ #ej c4c4 /]ile nutri5t bro?4 ,incubate at #cg deg4 ,c4 = tw5ty-f\r h\rs4 #d4 ,prep>e also sev]al blood ag> cultures f ! he>t blood ( ! rabbit1 label !m all ,o4,c4 7orig9al culture74 ,aft] tw5ty-f\r h\rs incubation at #cg deg4 ,c4 place an india-rubb] cap ov] ! plugg$ m\? ( ! tube ( all b "o ( ^! cultures & pa9t ! cap ) ,canada balsam or %ellac v>ni%1 dry1 & replace 9 ! hot incubator4 ,?is w prev5t evaporation1 & cultures ?us seal$ w rema9 unalt]$ 9 virul5ce = a consid]able "t4 #e4 ,make a fre% subcultivation on blood ag> f ! uncapp$ ,o4,c4 cultivation & aft] tw5ty-f\r h\rs incubation at #cg deg4 ,c4 det]m9e ! m9imal le?al dose ( ?is /ra9 ^u a s]ies ( mice 7see page #caf74 #f4 ,susp5d ! flask conta9+ ! tw5ty-f\r-h\r-old bro? culture 7/ep #c7 9 ! wat]-ba? at #fj deg4 ,c4 = "o h\r4 ,cool ! flask rapidly "u a /ream ( cold wat]4 #g4 ,det]m9e ! /]ility ( ?is 787 kill$ cultivation by transf]r+ "o cubic c5timetre to ea* ( sev]al tubes ( nutri5t bro?1 & incubate at #cg deg4 ,c4 = tw5ty-f\r h\rs4 ,if gr[? ( ,diplococcus pneumoniae occurs1 aga9 heat culture 9 wat]-ba? at #fj deg4 ,c4 = "o h\r & aga9 te/ = /]ility4 #h4 ,inject ! select$ rabbit intrav5\sly 7see page #cfc7 ) #b c4c4 ( ! kill$ cultivation1 & inject a fur!r #aj c4c4 into ! p]itoneal cavity4 ,dur+ ! next few days ! animal w lose "s weight & p]haps %[ a c]ta9 am\nt ( pyrexia4 #i4 ,:5 ! temp]ature & weight h aga9 return$ to normal--g5]ally ab\t sev5 days aft] ! inoculation--aga9 inject kill$ cultivation1 ?is "t giv+ a dose ( #e c4c4 intrav5\sly & #bj c4c4 intrap]itoneally4 ,a temp]ature & weight reaction simil> to1 b less m>k$ ?an t foll[+ ! fir/ injection w probably be obs]v$1 b aft] ab\t a week's int]val ! animal w be ready = ! next injection4 #aj4 ,:5 ready to give ! ?ird injection prep>e a fre% blood ag> subculture f ano!r ,o4,c4 tube & aft] tw5ty-f\r h\rs incubation prep>e a m9imal le?al dose 7z det]m9$ 9 #e7 & inject x subcutane\sly into ! rabbit's abdom9al wall4 ,a slight local reaction w probably be obs]v$ z well z ! weight & temp]ature reactions4 #aa4 ,a week to t5 days lat] inject a simil> m9imal le?al dose into ! p]itoneal cavity4 #ab4 ,obs]ve ! weight & temp]ature ( ! rabbit v c>efully1 & regulat+ ! dates ( inoculation by ! animal's g5]al condition1 cont9ue to inject liv+ cultivations ( ! pneumococcus into ! p]itoneal cavity1 gradually increas+ ! dose by multiples ( t54 #ac4 ,at int]vals ( two mon?s samples ( blood may be collect$ f ! po/]ior auricul> ve9 & ! s]um te/$ = specific antibodies4 #ad4 ,"u fav\rable conditions x w be f\nd aft] "s six mon?s /eady "w t ! rabbit may be inject$ intrap]itoneally ) an entire blood ag> cultivation )\t any ill effects be+ app>5t2 & ?is *>act]i/ic--resi/ance to ! le?al effects ( l>ge doses ( ! virus--is ! sole crit]ion ( immunity4 ,fur!r1 ! s]um sep>at$ f blood )drawn f ! animal ab\t a week aft] an injection1 if us$ 9 doses ( 4#ja c4c41 w protect a m\se aga9/ ! le?al effects ( at lea/ t5 m9imal le?al doses ( liv+ pneumococci4 ,9 ! =ego+ illu/ration x has be5 assum$ t complete acquir$ active immunity has be5 conf]r$ ^u ! exp]im5tal rabbit 9 consequ5ce ( ! =mation ( antibody1 specific to ! diplococcus pneumoniac1 suffici5t 9 am\nt to ensure ! de/ruction ( enorm\s doses ( ! liv+ cocci--! antig5 7t is ! sub/ance inject$ 9 response to :i* antibody has be5 elaborat$7 9 ?is p>ticul> case be+ ! bact]ial protoplasm ( ! pneumococcus ) its endo-tox9s4 ,b provid$ dea? does n imm$iately foll[ ! injection ( ! antig51 specific antibody is always =m$ 9 great] or less] am\nt2 & 9 exp]im5tal "w a suffici5t am\nt ( any requir$ antibody c (t5 be obta9$ )\t c>ry+ ! process ( immunisation to its logical t]m9ation4 ,= in/ance1 if ! immunisation ( a rabbit t[>d ,bacillus typhosus is comm5c$ on ! l9es already set out x w (t5 be f\nd1 aft] a few injections ( kill$ cultivation t ! blood s]um ( ! animal 7ev5 :5 dilut$ ) sev]al hundr$ times its volume ( normal sal9e7 conta9s specific agglut99 = ,b4 typhosus--& if ! sole object ( ! exp]im5t has be5 ! prep>ation ( agglut99 ! inoculations may well be /opp$ at ?is po9t1 al?\gh ! animal is n yet immune 9 ! /rict mean+ ( ! ^w4 ,aga91 antibodies may be =m$ 9 response to antig5s o!r ?an infective p>ticles--?us ! injection into suitable animals ( =eign prote9s su* z egg album91 het]olog\s blood s]a or r$ blood discs f a diff]5t species ( animal1 w result 9 ! =mation ( specific antibodies possess+ def9ite aff9ities = _! respective antig5s4 ,! mo/ important antibody ( ?is latt] type is ,haemolys91 a sub/ance t makes its appe>ance 9 ! blood s]um ( an animal previ\sly inject$ ) wa%$ blood cells f an animal ( a diff]5t species4 ,! s]um f su* an animal possesses ! p[] ( dis9tegrat+ r$ blood discs ( ! v>iety employ$ z antig5 & caus+ ! dis*>ge ( _! conta9$ haemoglob91 & is specific 9 its action to ! ext5t ( fail+ to ex]t any injuri\s effect ^u ! r$ blood cells ( any o!r species ( animal4 ,! action ( ?is s]um is due to ! pres5ce ( two di/9ct bodies1 complem5t & haemolys94 ,complem5t 7or alex9e7 is a !rmo-labile readily oxidis$ body pres5t 9 v>iable b unalt]able am\nt 9 ! normal s]um ( e animal4 ,x is a sub/ance :i* ex]ts a lytic effect ^u all =eign matt] introduc$ into ! blood or tissues2 b by itself is a comp>atively in]t body1 & is only capable ( ex]t+ its maximum lytic effect 9 ! pres5ce ( & 9 comb9ation ) a specific antibody1 or immune body4 ,complem5t is obta9$ 7unmix$ ) antibody7 by collect+ fre% blood s]um f any heal?y normal 7t is un9oculat$7 animal4 ,gu9ea-pigs' s]um is t mo/ frequ5tly employ$ = exp]im5tal "w4 ,haemolys9 7immune body1 copula1 s5sitis+ body1 amboceptor7 is a !rmo/able antibody =m$ 9 response to ! injection ( r$ cells :i* al?\gh 9 itself in]t is capable ( l9k+ up complem5t pres5t 9 ! normal s]um to ! r$ cells ( ! v>iety us$ z antig5--a comb9ation result+ 9 haemolysis4 ,haemolys9 is obta9$ by collect+ fre% blood s]um f a suitably inoculat$ animal & expos+ x to a temp]ature ( #ef deg4 ,c4 7to de/roy ! !rmo-labile complem5t7 = #ae to #cj m9utes be=e use4 ,x is !n ref]r$ to z inactivat$1 & is reactivat$ by ! addition ( fre% normal s]um--t is s]um conta9+ complem5t4 ,haemolys9 is ( importance academically ow+ to ! fact t _m ( ! problems ( immunity h be5 elucidat$ by its aid2 b its pres5t practical importance lies 9 ! application ( ! haemolytic sy/em 7t is haemolys91 correspond+ ery?rocyte solution & complem5t7 to c]ta9 laboratory me?ods hav+ = _! object ei!r ! id5tification ( ! infective entity or ! diagnosis ( ! exi/5ce ( infection4 ,= use 9 ^! laboratory me?ods ( diagnosis x is mo/ conv5i5t to prep>e haemolytic s]um specific = human blood--:e!r ! laboratory is isolat$ or atta*$ to a l>ge hospital4 ,ox blood1 %eep blood or goat blood if readily obta9able1 may h[ev] be us$ in/ead1 & al?\gh ! foll[+ me?od is direct$ to ! prep>ation ( human haemolys9 ! same proc$ure s]ves 9 all cases4 ,,! ,,prep>ation ,,( ,,haemolytic ,,s]um4 ,app>atus ,requir$3 ,small c5trifuge1 pref]ably electrically driv51 ) two receptacles = tubes1 & enclos$ 9 a safety %ield 7,fig4 #afb74 ,/]ile c5trifuge tubes 7#aj c4c4 capacity71 ,fig4 #afc4 ,/]ile pipettes 7#aj c4c4 graduat$7 9 case4 ,/]ile glass capsules 79 case74 ,/]ile te/-tubes4 ,/]ile all glass syr+e 7#e c4c4 or #aj c4c4 capacity7 & ne$le4 ,illu/ration3 ,,fig4 #afb4--,small electrical c5trifuge4 ,illu/ration3 ,,fig4 #afc4--;,c5trifuge tube4 ,reag5ts ,requir$3 ,normal sal9e solution4 #aj p] c5t4 sodium citrate solution 9 normal sal9e4 ,human blood 7vide infra74 ,,me?od4-- #a4 ,select a heal?y full-gr[n rabbit ( n less ?an #bejj grammes weight 9 accordance ) ! directions already giv5 7page #cbb7 & prep>e x = intrap]itoneal inoculation4 #b4 ,measure out #b c4c4 citrat$ human blood 7collect$ at a surgical op]ation or a v5esection1 or )drawn by v5ipuncture f ! m$ian basilic or m$ian cephalic ve9 ( a normal adult7 into a c5trifuge tube & c5trifugalise ?or\ghly4 #c4 ,wa% ) ?ree *anges ( normal sal9e 7vide also page #chh74 #d4 ,transf] ! wa%$ cells to a /]ile capsule by means ( a /]ile pipette4 ,add #e c4c4 ( normal sal9e & mix ?or\ghly4 #e4 ,take up ! mixture ( cells & sal9e 9 ! all-glass syr+e & inject into ! p]itoneal cavity ( ! rabbit4 #f4 ,sev5 days lat] inject intrap]itoneally ! wa%$ cells f #e c4c4 human blood mix$ ) #e c4c4 normal sal9e4 #g4 ,sev5 days lat] inject ! wa%$ cells f #aj c4c4 human blood mix$ ) #e c4c4 normal sal9e4 #h4 ,aft] a fur!r int]val ( sev5 days repeat ! injection ( wa%$ cells f #aj c4c4 human blood mix$ ) #e c4c4 normal sal9e4 ,,note4--,bett] results are obta9$ if ! second & subsequ5t injections are made intrav5\sly1 ev5 :5 small] quantities ( wa%$ r$ cells are employ$4 ,if1 h[ev]1 ! intrav5\s r\te is select$ exce$+ great c>e mu/ be ex]cis$ to avoid ! introduction ( air into ! ve9--an accid5t :i* is foll[$1 )9 a few m9utes1 by ! dea? ( ! rabbit f pulmon>y embolism4 #i4 ,all[ five days to elapse1 !n collect a prelim9>y sample ( blood1 say ab\t #b c4c41 f ! rabbit's e>4 ,all[ x to clot1 sep>ate (f ! s]um & transf] to a /]ile te/-tube4 ,place ! te/-tube 9 a wat]-ba? at #ef deg4 ,c4 = fifte5 m9utes 7to inactivate7 & te/ ! s]um quantitatively = haemolytic prop]ties 9 ! foll[+ mann]3 ,,! ,,titration ,,( ,,haemolytic ,,s]um4 ,app>atus ,requir$3 ,electrical c5trifuge4 ,/]ile c5trifuge tubes4 ,wat]-ba? regulat$ at #ef deg4 ,c4 ,/]ilis$ pipettes #aj c4c4 graduat$ 9 t5?s4 ,/]ilis$ pipettes #a c4c4 graduat$ 9 t5?s4 ,/]ile te/-tubes1 #af x #b cm4 ,small /]ile te/-tubes1 #i x #a cm4 ,small te/-tube rack1 or roll ( pla/ic9e4 ,capill>y teat pipettes4 ,/\t rubb] b& or l5g? ( small rubb] tub+4 ,reag5ts ,requir$ & ,me?od ( ,prep>ation3 #a4 ,normal sal9e solution4 #b4 ,haemolytic s]um inactivat$ by prelim9>y heat+ to #ef deg4 ,c4 = #ae m9utes 7vide supra7 9 te/-tube labell$ ,h4 ,s4 #c4 ,complem5t4 ,fre% gu9ea-pig s]um 9 te/-tube labell$ ,c4 ,kill a normal gu9ea-pig ) *lor(orm vap\r4 ,op5 ! ?orax ) all aseptic precautions1 & collect z mu* blood z possible f ! he>t ) a /]ile ,pa/eur pipette4 ,transf] x to a /]ile c5trifuge tube & place ! tube 9 ! incubator at #cg deg4 ,c4 ,two h\rs lat] sep>ate ! clot f ! sides ( ! tube1 & c5trifugalise ?or\ghly4 ,pipette (f ! cle> s]um to a clean /]ilis$ te/-tube4 #d4 ,ery?rocyte solution1 9 te/-tube labell$ ,e4 ,collect & wa% human r$ blood cells 7see page #chh1 #a-h74 ,measure ! volume ( r$ cells available & prep>e a #b p] c5t4 susp5sion 9 normal sal9e solution4 ,,me?od4-- #a4 ,take two te/-tubes & numb] !m #a & #b1 & pipette into ea* #i c4c4 ( normal sal9e solution4 #b4 ,add #a c4c4 ( haemolytic rabbit s]um to tube ,no4 #a & mix ?or\ghly3 take up #a c4c4 ( ! mixture & add x to tube ,no4 #b2 mix ?or\ghly4 #c4 ,set up t5 small te/-tubes 9 te/-tube rack or 9 roll ( pla/ic9e1 & numb] #a to #aj4 #d4 ,pipette into tube ,no4 #a #j4e c4c4 #j4e c4c4 haemolytic s]um ,f tube ,pipette into tube ,no4 #b #j4a c4c4 #j4a c4c4 ,h4 ,s4 haemolytic s]um ,pipette into tube ,no4 #c #j4e c4c4 #j4je c4c4 haemolytic s]um ,pipette into tube ,no4 #d #j4c c4c4 #j4jc c4c4 haemolytic s]um ,f ,pipette into tube ,no4 #e #j4b c4c4 #j4jb c4c4 tube #a4 haemolytic s]um pipette into tube ,no4 #f #j4a c4c4 #j4ja c4c4 haemolytic s]um ,pipette into tube ,no4 #g #j4e c4c4 #j4jje c4c4 haemolytic s]um ,pipette into tube ,no4 #h #j4c c4c4 #j4jjc c4c4 haemolytic s]um ,f ,pipette into tube ,no4 #i #j4b c4c4 #j4jjb c4c4 tube #b4 haemolytic s]um ,pipette into tube ,no4 #aj #j4a c4c4 #j4jja c4c4 haemolytic s]um #e4 ,to ea* tube add #a c4c4 ( ery?rocyte solution4 #f4 ,:5 necess>y 7t is to say 9 tubes #b1 #d1 #e1 #f1 #h1 #i & #aj7 add normal sal9e solution to ! mixture 9 ! te/-tubes till ! column ( fluid 9 ea* rea*es to ! same level4 #g4 ,%ake ea* tube 9 turn1 s z to ?or\ghly mix its cont5ts4 ,plug ! m\? ( ea* tube ) cotton wool1 & place entire set 9 ! incubator at #cg deg4 ,c4 = "o h\r4 #h4 ,remove ! tubes f ! incubator & into ea* tube pipette #j4a c4c4 complem5t 7gu9ea-pig's s]um7 & replace tubes 9 incubator at #cg deg4 ,c4 = fur!r p]iod ( "o h\r4 #i4 ,remove ! tubes f ! incubator1 & if complete haemolysis has n tak5 place 9 e tube1 /& on "o side1 pref]ably 9 ! ice *e/1 = an h\r4 #aj4 ,!n exam9e ! tubes4 ,complete haemolysis is indicat$ by a cle> r$ solution1 ) no deposit ( r$ cells at ! bottom ( ! te/-tube4 ,abs5ce ( haemolysis is indicat$ by a cle> or turbid col\rless fluid1 ) a deposit ( r$ cells at ! bottom ( ! te/-tubes4 ,! smalle/ am\nt ( haemolytic s]um t has caus$ complete haemolysis is kn[n z ! m9imal haemolytic dose 7,m4 ,h4 ,d47 & if haemolysis has occurr$ 9 all ! tubes d[n to ,no4 #g--! m4 h4 d4 ( ?is p>ticul> s]um is 4#jje c4c4 #bjj m9imal haemolytic doses p] cubic c5timetre4 ,su* a s]um is /rong en\gh = exp]im5tal "w2 inde$1 = _m purposes1 complete haemolysis d[n to tube #f w indicate a s]um suffici5tly /rong7 #ajj m4 h4 d4 p] cubic c5timetre74 ,if1 h[ev]1 only ! fir/ "o or two tubes are completely haemolys$1 ?is is an indication t ! rabbit %\ld receive fur!r injections 9 ord] to raise ! haemolytic p[] to a suffici5tly high level4 ,,/orage ,,( ,,haemolys94 ,if1 & :5 ! haemolys9 cont5t ( ! rabbit's s]um is f\nd to be suffici5t1 de/roy ! animal by *lor(orm vap\r4 ,remove z mu* ( its blood z possible f ! he>t "u aseptic precautions into /]iliz$ c5trifuge tubes4 ,transf] ! tubes ( blood to ! incubator at #cg deg4 ,c4 = two h\rs--!n c5trifugalize ?or\ghly4 ,pipette (f ! cle> s]um1 & fill 9 quantities ( #a c4c41 into small glass amp\les or pipettes1 & h]metically seal 9 ! bl[pipe flame1 c>e be+ tak5 to avoid scor*+ ! s]um4 ,place ! amp\les :5 fill$ ) s]um & seal$1 9 a wat]-ba? at #ef deg4 ,c4 = #cj m9utes4 ,?is de/roys ! complem5t1 i4 e41 inactivates ! s]um1 & at ! same "t1 provid$ ! v>i\s op]ations h be5 c>ri$ out "u aseptic precautions1 ensures its /]ility4 ,a long] exposure r$uces ! haemolytic p[]4 ,place ! amp\les 9 a clos$ metal box & /ore 9 ! ice *e/ = future use4 ,,footnotes3 #aj ,! quantities "h giv5 are n absolutely correct4 ,if exactitude is ess5tial ! /ud5t mu/ calculate ! am\nt requir$ by ! aid ( ! ,p]c5tage ,=mula1 ,app5dix1 page #dif4 #aa ,see ,p]c5tage ,=mula1 ,app5dix1 page #dif4 ,,xvii4 ,,exp]im5tal ,,inoculation ,,( ,,animals4 ,! use ( liv+ animals = inoculation exp]im5ts may become a necess>y proc$ure 9 ! ,bact]iological ,laboratory = "s "o or m ( ! foll[+ reasons3 ,a4 ,det]m9ation ( ,pa?og5etic ,prop]ties ( ,bact]ia already ,isolat$ 9 ,pure ,culture 7see page #cae74 ,! exact /udy ( ! conditions influ5c+ ! virul5ce 7includ+ its ma9t5ance1 exaltation & att5uation7 ( an organism1 & precise obs]vations ^u ! pa?og5ic effects produc$ by its entrance into1 & multiplication )9 ! body tissues c obvi\sly only be c>ri$ out by means ( exp]im5tal inoculation2 :il/ _m po9ts relat+ to vitality1 longevity1 etc41 c be mo/ readily elucidat$ by su* exp]im5ts4 ,b4 ,isolation ( ,pa?og5etic ,bact]ia4 ,c]ta9 highly p>asitic bact]ia 7:i* gr[ ) difficulty ^u ! artificial m$ia ( ! laboratory7 c only be isolat$ ) consid]able difficulty f associat$ saprophytic bact]ia :5 cultural me?ods alone are employ$2 b if ! mixture ( p>asite & saprophytes is inject$ into an animal susceptible to ! action ( ! =m]1 ! pa?og5ic organism c readily be isolat$ f ! tissues ( ! infect$ animal4 ,! pneumococcus = example occurs 9 ! sputum ( pati5ts suff]+ f acute lob> pneumonia1 b usually 9 association ) v>i\s saprophytes d]iv$ f ! m\? & ph>ynx4 ,! optimum m$ium = ! gr[? ( ! pneumococcus1 blood ag>1 is also an excell5t pabulum = ! saprophytes ( ! m\?1 & plate cultures are rapidly ov]gr[n by !m to ! de/ruction ( ! m delicate pneumococcus4 ,b inoculate "s ( ! sputum "u ! sk9 ( a m\se & ?ree or f\r days lat] ! pneumococcus w h ent]$ ! blood /ream 7leav+ ! saprophytes at ! seat ( inoculation7 & kill$ ! animal4 ,cultivations made at ! po/-mortem 7see page #cih7 f ! m\se's he>t blood w yield a pure gr[? ( ! pneumococcus4 ,c4 ,id5tification ( ,pa?og5etic ,bact]ia4 ,! resemblances1 morphological & cultural1 exi/+ betwe5 c]ta9 pa?og5etic bact]ia are 9 "s cases s great z to completely ov]:elm ! diff]5ces2 aga9 ! same bact]ium may "u v>y+ conditions assume appe>ances s diff]5t f ^? reg>d$ z typical or normal z to ?r[ d\bt on its id5tity4 ,9 ea* case a simple inoculation exp]im5t may decide ! po9t at once4 ,z a concrete example may be in/anc$ an autopsy on an animal dead f an unkn[n infection4 ,cultivations f ! he>t blood gave a pure gr[? ( a typical 7capsulat$7 pneumococcus4 ,cultivations f ! liv] gave a pure gr[? ( :at appe>$ to be a typical 7non-capsulat$7 ,/reptococcus pyog5es longus4 ,! latt] inoculat$ into a rabbit caus$ ! dea? ( ! animal f pneumococcic septicaemia1 & cultures f ! rabbit's blood gave a pure gr[? ( a typical 7capsulat$7 pneumococcus4 ,d4 ,/udy ( ! ,problems ( ,immunity4 ,x is only by a c>eful & elaborate /udy ( ! behavi\r ( ! animal cell & ! body fluids vis-a-vis ) ! infect+ bact]ium t x becomes possible to ?r[ light ^u ! complex problem :]eby ! cell opposes successful resi/ance to ! diffusion ( ! invad+ microbe1 or succe$s 9 driv+ out ! microbe subsequ5tly to ! occurr5ce ( t diffusion4 ,at ! mom5t1 h[ev]1 our att5tion is direct$ to ! fir/ ( ^! broad head+s1 = x is by ! application ( ! k acquir$ 9 its pursuit t we are able to deal ) problems aris+ "u any ( ! rema9d]4 ,= :atev] purpose ! inoculation is p]=m$1 x is ess5tial t ! exp]im5t %\ld be plann$ to secure ! maximum am\nt ( in=mation & ! m9imum ( discom=t to ! animal us$4 ,e c>e !re=e mu/ be tak5 to ensure t ! virus is introduc$ into ! exact tissue or organ select$2 & ! op]ation itself mu/ be c>ri$ out ) skill & exp$ition1 & "u /rictly aseptic conditions4 ,9 ! c\rse ( inoculation /udies _m in/ances ( natural immunity1 bo? racial & individual1 w be met )2 b x mu/ be recollect$ t natural immunity is relative only & nev] absolute1 & c>e be tak5 n to label an organism z non-pa?og5ic until _m diff]5t me?ods ( inoculation h be5 p]=m$ ^u diff]5t species ( animals1 comb9$ :5 necess>y ) v>i\s proc$ures calculat$ to ov]come any app>5t immunity1 & h inv>iably giv5 negative results4 ,9 "s c\ntries exp]im5ts ^u animals are only p]mitt$ "u direct lic5se f ! ,gov]nm5t1 & !n only )9 premises specially lic5s$ = ! purpose4 ,9 ,engl& ?is lic5se is 9 ! grant ( ! ,home ,secret>y1 & conf]s ! p]mission to exp]im5t ^u animals "u g5]al anae/hesia1 provid$ t aft] ! exp]im5t is complet$ ! animal mu/ be de/roy$ be=e rega9+ consci\sness4 ,if x is int5d$ to c>ry out simple hypod]mic inoculations & sup]ficial v5esections1 ,c]tificate ,a1 grant+ ?is specific p]mission & disp5s+ ) ! necessity = g5]al anae/hesia mu/ be obta9$ 9 addition to ! lic5se2 :il/ if ! inoculation entails m ext5sive op]ative proc$ures1 & x is necess>y to obs]ve ! subsequ5t c\rse ( ! infection1 %\ld su* occur1 ! lic5se mu/ be c\pl$ ) ,c]tificate ,b--s9ce ?is c]tificate removes ! compulsion to de/roy ! animal :il/ "u ! anae/hetic4 ,fur!r special c]tificates & comb9ations ( c]tificates are requir$ if cats1 dogs1 horses1 asses or cattle are to be ! subjects ( exp]im5t4 ,"u e c]tificate x is expressly /ipulat$ t if ! animal %[s signs ( pa9 x mu/ be de/roy$ imm$iately4 ,! animals g5]ally employ$ 9 ! /udy ( ! pa?og5ic prop]ties ( ! v>i\s micro-organisms are3 ,cold ,blood$4 ,w>m ,blood$4 ,hot ,blood$4 ,frog4 ,m\se4 ,f[l4 ,toad4 ,rat4 ,pigeon4 ,liz>d4 ,gu9ea pig4 ,rabbit4 ,monkey4 ,prep>ation4--,be=e inoculation1 ! exp]im5tal animals %\ld be c>efully exam9$1 to avoid ! risk ( employ+ su* z are already diseas$3 s9ce x mu/ be rememb]$ t 9 a /ate ( nature1 z well z 9 captivity1 ! animals employ$ = laboratory inoculations are subject to infection by v>i\s animal & vegetable p>asites1 & 9 "s in/ances su* infection pres5ts no symptoms :i* are obvi\s to ! casual exam9ation2 ! sex %\ld be not$1 ! weight record$1 & ! rectal temp]ature tak54 ,! rema9+ items ( importance are ! "t ( ! inoculation1 ! mat]ial t is inoculat$1 & ! me?od ( inoculation1 & f9ally "u :at au?ority ! exp]im5t is p]=m$4 ,9 ! au?or's laboratory ^! data are ent]$ ^u a p9k c>d :i* =ms "p ( a c>d index sy/em4 ,! c>d fur!r provides space = notes on ! c\rse ( ! result+ infection1 & c>ries on ! rev]se ! weight & temp]ature *>t 7,figs4 #afd & #afe74 ,illu/ration3 ,fig4 #afd4--;,front ( inoculation c>d4 ,prelim9>y ,inspection & ,exam9ation4--,! prelim9>y exam9ation %\ld comprise obs]vation ( ! animal at re/ & 9 motion2 ! appe>ance ( ! fur1 fea!rs or scales1 inspection ( ! eyes1 & ( ext]nal orifices ( ! body2 tactile exam9ation ( ! body & limbs1 & palpation ( ! gro9s & abdom52 & 9 _m cases ! microscopical exam9ation ( fre% & /a9$ blood-films4 ,"s ( ! common] =ms ( naturally acquir$ infection may be briefly m5tion$1 )\t h[ev] t\*+ ^u ! v>i\s fleas1 lice & ticks :i* at times infect ! ord9>y laboratory animals4 ,illu/ration3 ,,fig4 #afe4--;,back ( inoculation c>d4 ,! ,rabbit1 p>ticul>ly 9 captivity1 is subject to attacks ( ,psoric ,ac>i1 & ! infection is readily transmitt$ to rabbits 9 neighb\r+ cages & also to gu9ea pigs1 b n to rats & mice4 ,"o species 7,s>coptes m9or v>4 cuniculi7 gives rise to ! ord9>y mange4 ,! infection fir/ %[s itself z ?ick yell[i% scales & cru/s ar\nd ! nose1 m\? & eyes1 spreads to ! bases & out] surfaces ( ! e>s 7nev] to ! inside ( ! con*a71 to ! =e & h9d legs & into ! gro9s & ar\nd ! g5itals4 ,! ac>i c be readily demon/rat$ microscopically 9 scrap+s ( ! sk91 treat$ ) liquor potassae4 ,ano!r =m ( scabies 7due to ,psoroptes communis cuniculi7 comm5ces at ! bottom ( ! con*a1 :i* is fill$ ) :iti%-yell[ masses consi/+ ( dri$ cru/s1 scales1 faeces1 & dead ac>i4 ,! base ( ! e> is h>d & swoll51 & lift+ ! animal by ! e>s--z is usually done--gives rise to consid]able pa92 inde$ ?is symptom may be ! "o :i* fir/ attracts att5tion to an infection1 :i* causes progressive wa/+ & t]m9ates 9 dea?4 ,a mix$ infection--s>coptic plus psorotic ac>iasis--is sometimes se54 ,if x is decid$ to try & save animals suff]+ f infection by ^! p>asites1 !y mu/ be segregat$1 ! scabs c>efully clean$ f ! infect$ areas & ! d5ud$ surfaces wa%$ ) #e p] c5t4 solution ( ,potassium p]sulphate 7a few drops be+ all[$ to run into ! con*a71 or ) a prep>ation conta9+ equal p>ts ( s(t p>aff9 & vasel9e ) a few drops ( lysol4 ,?is treatm5t %\ld be repeat$ daily until ! ac>us is de/roy$ & ! animal has rega9$ its normal condition4 ,! cages %\ld be dis9fect$ & all neighb\r+ animals c>efully exam9$1 & any :i* %[ signs ( infection %\ld be treat$ 9 a simil> mann]4 ,favus also attacks ! rabbit1 & ! typical spots are fir/ not$ ar\nd ! base ( ! e>4 ,infection by ,coccidium ovi=me is v common1 )\t h[ev] pres5t+ any symptoms by :i* ! infection may be recognis$4 ,usually ! condition is only not$ po/-mortem1 :5 ! liv] is f\nd to be /udd$ ) num]\s cascat+ tub]cles1 :i* on exam9ation prove to be cy/ic areas cr[d$ ) coccidia4 ,sometimes too ! liv] ( a rabbit dead f "s int5tional or accid5tal bact]ial infection is f\nd at ! po/-mortem to be m>k$ by f9e yell[i% /reaks & small tub]cles due to ! embryos ( ,ta5ia s]rata1 :ile ! cy/ic =m 7,cy/ic]cus pisi=mis7 is (t5 not$ free 9 ! p]itoneal cavity1 or invad+ ! mes5t]y4 ,abscess =mation f infection ) ord9>y pyog5ic bact]ia occurs naturally 9 ! rabbit1 & frequ5tly ! animal h\se ( a laboratory is decimat$ by an infective septicaemia due to ,b4 cuniculicida4 ,! ,m\se & ,rat suff] f septicaemia1 & f ! cy/ic]cus =m ( ,ta5ia mur9a2 ! cy/ic =m 7,cy/ic]cus fasciol>is7 ( ,t4 crassicollis has its habitat 9 _! liv]s4 ,^! small rod5ts are frequ5tly infect$ ) scabies1 b if freely provid$ ) clean /raw w clean !mselves by rubb+ ?r\gh x4 ,! m\se is also attack$ by favus1 & ! rat is (t5 infect$ ) ,trypanosoma ,lewisi4 ,! ,gu9ea pig1 l ! rabbit1 suff]s f scabies & coccidiosis4 ,9 addition x is (t5 naturally infect$ ) ,b4 tub]culosis1 & x is a wise precaution to te/ animals z soon z !y rea* ! laboratory by inject+ ,ko*'s ,old ,tub]cul9--#j4e c4c4 caus+ dea? 9 ! tub]cul\s cavy )9 #dh h\rs4 ,! ,monkey is naturally prone to tub]culosis1 & %\ld be inject$ ) #a c4c4 ,old ,tub]cul9 on arrival 9 ! laboratory4 ,! tissues ( ! monkey also s]ve z ! habitat = a ,nematode worm p>asitic 9 cattle 7,oesophago/oma inflatum7 resembl+ ! ,an*ylo/omum1 & ?is p>asite frequ5tly bores ?r\gh ! inte/9al wall1 & provokes ! =mation ( small cy/s 9 ! imm$iately adjac5t mes5t]y4 ,! pres5ce ( ^! cy/s may give rise to consid]able speculation at ! po/-mortem4 ,! ,pigeon may be infect$ by ,haemosporidia1 & its blood %[ ! pres5ce ( halt]idia4 ,?is bird may also be ! subject ( a bact]ial infection kn[n z pigeon diph!ria2 :ile ! f[l may be subject to scabies & r+worm1 or suff] f f[l *ol]a or f[l septicaemia--infections due to memb]s ( ! haemorrhagic septicaemia gr\p4 ,weigh+4--,! l>g] animals are mo/ conv5i5tly weigh$ 9 a decimal scale provid$ ) a metal cage = _! reception in/ead ( ! ord9>y pan 7,fig4 #aff74 ,mice & rats are weigh$ 9 a modification ( ! lett] balance1 weigh+ to #bej grammes1 :i* has a conical wire cage1 7c>efully c\nt]pois$7 sub/itut$ = its orig9al pan 7,fig4 #afg74 ,illu/ration3 ,,fig4 #aff4--,rabbit scales4 ,temp]ature4--,to take ! rectal temp]ature ( any ( ! laboratory animals1 ! animal %\ld be c>efully & firmly held by an assi/ant4 ,introduce ! bulb ( an ord9>y cl9ical !rmomet]1 well greas$ ) vasel9e1 j )9 ! sph9ct] ani4 ,all[ x to rema9 9 ?is position = a few seconds1 & !n pu% x on g5tly & /eadily until ! entire bulb & "p ( ! /em1 z f> z ! con/riction1 h pass$ into ! rectum4 ,?ree to five m9utes lat]1 ! "t v>y+ ( c\rse ) ! s5sibility ( ! !rmomet] us$1 )draw ! in/rum5t & take ! read+4 ,! !rmomet]s employ$ = record+ temp]ature %\ld be v]ifi$ f "t to "t by comp>ison ) a /&>d ,kew c]tifi$ ,!rmomet] kept 9 ! laboratory = t purpose4 ,illu/ration3 ,,fig4 #afg4--,m\se scales ,cages4--,dur+ ! p]iod :i* elapses betwe5 inoculation & dea?1 or complete recov]y1 ! exp]im5tal animals mu/ be kept 9 suitable receptacles :i* c easily be kept clean & readily dis9fect$4 ,! m\se is usually /or$ 9 a glass j> 7,fig4 #afh7 #aa cm4 high & #aa cm4 9 diamet]1 clos$ by a wire gauze cov] :i* is weight$ ) lead or fa/5$ to ! m\? ( ! j> by a bayonet cat*4 ,a small oblong label1 #e cm4 by #b4e cm41 s&-bla/$ on ! side ( ! cyl9d]1 is a v conv5i5t device z notes made ^u ?is ) an ord9>y lead p5cil %[ up well & only require ! use ( a damp clo? to remove !m 7,fig4 #afh74 ,! rat is kept "u obs]vation 9 a glass j> simil>1 b l>g]1 to t us$ = ! m\se4 ,illu/ration3 ,,fig4 #afh4--,m\se j>4 ,illu/ration3 ,,fig4 #afi4--,tripod4 ,a lay] ( sawdu/ at ! bottom ( ! j> absorbs any moi/ure & cotton-wool or pap] %av+s %\ld be provid$ = b$d+4 ,! food %\ld consi/ ( bran & oats ) an occasional fe$ ( bread-&-milk sop4 ,! use ( a metal tripod1 on ! plat=m ( :i* are sold]$ two small cups = ! reception ( ! food1 inside ! cage1 prev5ts wa/e ( food or its contam9ation ) excreta 7,fig4 #afi74 ,aft] use ! j>s & tripods are /]ilis$ ei!r by *emical reag5ts or by autoclav+4 ,! rabbit & ! gu9ea-pig are conf9$ 9 cages ( suitable size1 made entirely ( metal 7,fig4 #agj74 ,! sides & top & bottom are ( wov5 wire "w2 b5ea? ! cage is a movable metal tray fill$ ) sawdu/1 = ! reception ( ! excreta4 ,! cage z a :ole is rais$ f ! gr\nd on %ort legs4 ,! sides1 etc41 are g5]ally h+$ s t ! cage packs up flat1 = conv5i5ce ( /or+ & also ( /]ilis+4 ,! ord9>y rat cage1 a rectangul> wire-"w box1 #cj cm4 f front to back1 #bj cm4 wide1 & #ad cm4 high1 makes an excell5t cage = gu9ea-pigs if fitt$ ) a %all[ z9c tray1 #ce cm4 by #bd cm41 = x to /& ^u4 ,illu/ration3 ,,fig4 #agj4--,metal rabbit rage4 ,a pl5tiful supply ( /raw %\ld be provid$ = b$d+ & ! food %\ld consi/ ( fre% vegetables1 cabbage leaves1 c>rot & turnip tops & ! l = ! morn+ meal & brok5 animal biscuits = ! ev5+ meal4 ,occasionally a little wat] may be plac$ 9 ! cage 9 an e>!nw>e di%4 ,! tray :i* receives ! dejecta %\ld be clean$ out & suppli$ ) fre% sawdu/ ea* day1 & ! soil$ sawdu/1 rema9s ( food1 etc41 %\ld be cremat$4 ,^! cages are /]ilis$ aft] use ei!r by autoclav+ or spray+ ) =mal94 ,z animal inoculation is purely a surgical op]ation1 ! necess>y in/rum5ts w be simil> to ^? employ$ by ! surgeon1 &1 l !m1 mu/ be /]ile4 ,9 ! p]=mance ( ! inoculation /rict att5tion mu/ be paid to asepsis1 & suitable precautions adopt$ to gu>d aga9/ accid5tal contam9ation ( ! mat]ial to be introduc$ into ! animal4 ,9 addition1 ! h&s ( ! op]ator %\ld be c>efully dis9fect$4 ,! li/ ( app>atus us$ 9 animal inoculations giv5 bel[ comprises practically ev]y?+ ne$$ = any inoculation4 ,ne$less to rem>k1 all ! app>atus w nev] be requir$ = any "o inoculation4 ,illu/ration3 ,,fig4 #aga4--;,hypod]mic syr+e ) f+] re/s4 ,app>atus ,requir$ = ,animal ,inoculation3 #a4 ,wat] /]ilis] 7vide page #cc74 ,x is also conv5i5t to h a second wat] /]ilis]1 simil> b small] 7#bc by #g by #e cm471 = ! /]ilisation ( ! syr+es4 #b4 ,injection syr+e4 ,! be/ =m is "o ( ! ord9>y hypod]mic patt]n1 #a c4c4 capacity graduat$ 9 tw5tie?s ( a cubic c5timet] 7#j4je c4c471 fitt$ ) f+] re/s1 b ) ! lea!r wa%]s & ! pack+ ( ! pi/on replac$ by ^? made ( asbe/os 7,fig4 #aga74 ,! in/rum5t mu/ be easily tak5 to pieces1 & sp>e p>ts %\ld be kept on h& to replace accid5tal breakage or loss4 ,o!r useful syr+es are ^? ( #b c4c41 #e c4c41 #aj c4c41 & #bj c4c4 capacity4 ,a good supply ( ne$les mu/ be kept on h&1 bo? %>p-po9t$ & ) blunt ends4 ,to /]ilise ! syr+e1 fill x ) wat]1 loos5 ! pack+ ( ! pi/on & all ! screw jo9ts1 place x 9 ! /]ilis] & boil = at lea/ five m9utes4 ,dis9fect ! syr+e aft] use1 9 a simil> mann]4 ,! ne$les1 :i* are exce$+ly apt to ru/ aft] be+ boil$1 %\ld be /or$ 9 a pot ( absolute alcohol :5 n 9 use4 #c4 ,op]at+ table4 #d4 ,surgical in/rum5ts4 ,/]ilise ^! be=e use by boil+1 & dis9fect !m aft] use by ! same means4 ,wipe p]fectly dry imm$iately aft] ! dis9fection is complet$4 ,scissors1 probe & %>p-po9t$4 ,dissect+ =ceps ( v>i\s patt]ns4 ,pressure =ceps4 ,retractors 7small self reta9+ ,fig4 #agb74 ,aneurism ne$les1 %>p & blunt4 ,scalpels1 ,k]atomes1 ) metal h&les4 ,treph9es1 ,mi*el's /eel clips & special =ceps = apply+ ! same4 ,^! small /eel clips enable ! op]ator to easily & rapidly close sk9 incisions & are mo/ satisfactory = animal op]ations4 ,surgical ne$les4 ,ne$le hold]4 ,s(t rubb] ca!t]s1 v>i\s sizes4 ,gum ela/ic oesophageal b\gies ) connection to fit syr+e4 ,illu/ration3 ,,fig4 #agb4 ,small self reta9+ retractors4 #e4 ,anae/hetic4 7a7 ,g5]al3 ,! safe/ g5]al anae/hetic = animals is an ,a4 ,c4 ,e4 mixture1 fre%ly prep>$1 conta9+ by volume alcohol #a "p1 *lor(orm #b p>ts1 e!r #f p>ts1 & %\ld be adm9i/]$ on a cone =m$ by twi/+ up "o corn] ( a t[el & plac+ a wad ( cotton-wool inside x1 or f a saturat$ cotton-wool pad pack$ into ! bottom ( a small beak]4 7b7 ,local3 #a4 ,coca9e hydro*loride1 #b p] c5t4 9 adr5al9 #a p] mille solution4 #b4 ,beta-euca9e1 #b p] c5t4 9 adr5al91 #a p] mille solution4 #c4 ,e?yl *loride jet4 #f4 ,/]ile glass capsules ( v>i\s sizes4 #g4 ,cases ( /]ile pipettes #aj c4c4 79 t5?s ( a cubic c5timetre74 #a c4c4 79 hundr$?s ( a cubic c5timetre74 #h4 ,flasks 7#ge c4c47 conta9+ /]ilis$ normal sal9e solution 7or /]ile b\illon74 #i4 ,/]ilis$ cotton-wool4 ,cotton-wool 7absorb5t7 is pack$ loosely 9 a copp] cyl9d] simil> to t us$ = /or+ capsules1 & /]ilis$ 9 ! hot-air ov54 #aj4 ,/]ilis$ gauze4 ,gauze is /]ilis$ 9 ! same way z cotton-wool4 #aa4 ,/]ilis$ silk & catgut = sutures4 ,^! are /]ilis$1 z requir$1 by boil+ = "s t5 m9utes 9 ! wat] /]ilis]4 #ab4 ,flexible collodion 7or comp\nd t9cture ( b5zo974 #ac4 ,grease p5cil4 #ad4 ,tie-on celluloid labels1 to affix to ! cages4 #ae4 ,razor4 #af4 ,small pot ( w>m wat]4 #ag4 ,liquid soap4 ,liquid soap is prep>$ z foll[s3 ,measure out #ajj grammes ( s(t soap & add to #ejj c4c4 ( #b p] c5t4 lysol solution 9 a l>ge glass beak]2 dissolve by heat+ 9 a wat]-ba? at ab\t #ij deg4 ,c4 ,bottle & label ,liquid ,soap4 #ah4 ,9 place ( ! liquid soap & razor x is sometimes conv5i5t to use a ,depilatory p[d]4 ,b>ium sulphide #a "p ,rice />* #c p>ts ,du/ ! p[d] ?ickly ov] ! area to be d5ud$ ( hair1 spr9kle ) wat] & mix into a ?9 pa/e 9 situ2 all[ ! pa/e to act = ?ree m9utes1 !n scrape (f ) a bone spatula--! hair comes away ) ! pa/e & leaves a p]fectly b>e pat*4 ,?is process is pref]ably c>ri$ out1 ! day previ\s to ! op]ation4 ,mat]ial ,utilis$ = ,inoculation4--,! mat]ial inoculat$ may be ei!r-- #a4 ,cultures ( bact]ia--gr[n 9 fluid m$ia1 or on solid m$ia4 #b4 ,metabolic products ( bact]ial activity--e4 g41 tox9s 9 solution4 #c4 ,pa?ological products 7fluid secretions & excretions1 solid tissues74 ,! ,prep>ation ( ! ,inoculum4-- 7a7 ,cultivations 9 ,fluid ,m$ia4-- #a4 ,flame ! plug ( ! culture tube4 #b4 ,remove ! plug & flame ! m\? ( ! tube4 #c4 ,slightly raise ! lid ( a /]ile capsule1 ins]t ! m\? ( ! culture tube into ! ap]ture & p\r "s ( ! cultivation into ! capsule4 #d4 ,remove ! m\? ( ! culture tube f ! capsule1 replace ! lid ( ! latt]1 flame ! m\? ( ! tube1 & replug4 #e4 ,remove ! syr+e f ! /]ilis]1 squirt out ! wat] f its int]ior1 & all[ to cool4 #f4 ,raise ! lid ( ! capsule suffici5tly to admit ! ne$le ( ! syr+e & draw ! requir$ am\nt ( ! cultivation into ! b>rel ( ! syr+e4 7,or1 remove a def9ite measur$ quantity ( ! cultivation directly f ! tube or flask by means ( a /]ile graduat$ pipette1 dis*>ge ! measur$ am\nt into a /]ile capsule1 & fill into ! syr+e2 or take up ! requir$ quantity ( ! cultivation directly into ! graduat$ syr+e f ! tube or flask47 ,illu/ration3 ,,fig4 #agc4--;,conical sep>atory funnel1 fitt$ = injection ( fluid cultivations4 ,if x is necess>y to introduce a l>ge bulk ( fluid into ! animal1 ! cultivation %\ld be transf]r$ ) aseptic precautions1 to a /]ile sep>atory funnel1 pref]ably ( ! %ape %[n 9 figure #agc1 & graduat$ if necess>y4 ,?is is support$ on a retort /& & rais$ suffici5tly above ! level ( ! animal to be inject$1 s z to secure a good fall4 ,a piece ( /]ilis$ rubb] tub+ ( suitable l5g?1 fitt$ ) an injection ne$le & provid$ ) a screw clamp1 is n[ atta*$ to ! nozzle ( ! funnel & ! op]ation complet$ accord+ to ! requirem5ts ( ! p>ticul> case4 ,?is me?od is q satisfactory :5 ! injection is made into ! pleural or abdom9al cavities or directly into a ve9 b if ! injection has to be made into ! subcutane\s tissue ! fall may n be suffici5t to =ce ! fluid 94 ,9 ?is case x w be necess>y to transf] ! culture to a /]ile wa%-bottle & fa/5 a rubb] h& bell[s to ! air inlet tube 7int]pos+ an air filt]7 & atta* ! tub+ ) ! injection ne$le to ! outlet tube 7,fig4 #agd74 ,by c>eful use suffici5t =ce c be obta9$ to drive ! injection 94 7b7 ,cultivations on ,solid ,m$ia 7e4 g41 ,slop$ ,ag>74-- #a4 ,by means ( a /]ile graduat$ pipette introduce a suitable small quantity ( /]ile b\illon 7or /]ile normal sal9e solution7 into ! culture tube4 ,illu/ration3 ,,fig4 #agd4--;,arrangem5t ( pressure injection app>atus4 #b4 ,) a /]ile plat9um loop or spatula scrape ! bact]ial gr[? (f ! surface ( ! m$ium1 & emulsify x ) ! b\illon4 ,x !n becomes to all int5ts & purposes a fluid inoculum4 #c4 ,p\r ! emulsion into a /]ile capsule & fill ! syr+e !refrom4 7c7 ,tox9s4--,prep>$ by previ\sly describ$ me?ods 7vide page #cah71 are manipulat$ 9 a simil> mann] to cultivations 9 fluid m$ia4 7d7 ,pa?ological ,products4--,fluid secretions1 excretions1 etc41 su* z s]\s exudation1 pus1 blood1 etc41 are treat$ z fluid cultivations2 b if ! mat]ial is v ?ick or visc\s1 a small quantity ( /]ile b\illon or normal sal9e solution may be us$ to dilute x1 & ?or\gh incorporation effect$ by ! help ( a /]ile plat9um rod4 ,solid tissues1 su* z sple51 lymph gl&s1 etc41 may be divid$ into small pieces by /]ile in/rum5ts & rubb$ up 9 a /]ilis$ agate mort> 7us+ an agate pe/le71 ) a small quantity ( /]ile b\illon1 & ! syr+e fill$ f ! result+ emulsion4 ,illu/ration3 ,,fig4 #age4--;,hold+ rabbit = %av+4 ,if x is desir$ to inoculate tissue en masse1 remove f ! mat]ial a small cube ( #a or #b mm4 & introduce x into a w\nd made by /]ile in/rum5ts 9 a suitable situation1 & occlude ! w\nd by means ( ,mi*el's /eel clips & a seal$ dress+4 ,me?od ( ,secur+ ,animals ,dur+ ,inoculation4-- ,= ! majority ( inoculations1 especially :5 no anae/hetic is adm9i/]$1 x is cu/om>y to employ an assi/ant to hold ! animal 7see ,fig4 #age74 ,if work+ s+le h&$ ,voge's hold] = gu9ea-pigs1 is a useful piece ( app>atus ! me?od ( us+ :i* is readily se5 f ! accompany+ figures 7,figs4 #agf1 #agg74 ,! in/rum5t itself consi/s ( a holl[ copp] cyl9d]1 "o end ( :i* is turn$ ov] a r+ ( /\t copp] wire1 & f ?is op5 end a slot is cut ext5d+ ab\t half way along "o side ( ! cyl9d]4 ,! opposite end is clos$ by a pull-(f cap & is p]=at$ ar\nd its edge by a r[ ( v5tilat+ holes1 :i* correspond ) holes cut 9 ! rim ( ! cap4 ,9 ! ev5t ( ! animal resi/+ attempts to remove x f ! hold] backw>ds1 ?is cap is tak5 (f & ! hold] plac$ on ! table & ! gu9ea-pig all[$ to walk out4 ,illu/ration3 ,,fig4 #agf4--,tak+ gu9ea-pig's temp]ature4 ,to provide = diff]5t-siz$ animals1 two sizes ( ?is hold] w be f\nd useful3 #a4 ,l5g?1 #af cm42 bread?1 #f cm42 size ( slot1 #h cm4 by #b4e cm4 #b4 ,l5g?1 #bj cm42 bread?1 #h cm42 size ( slot1 #aj cm4 by #b4e cm4 ,a conv5i5t hold] = mice & ev5 small rats is %[n 9 figure #agh1 ! tail be+ securely held by ! spr+ clip4 ,ne$less to say1 ! hold] %\ld be entirely ( metal1 & ! wire cage deta*able & easily r5ew$4 ,illu/ration3 ,,fig4 #agg4--,voge's hold]4 ,:5 ! animal is anae/hetis$1 x is m conv5i5t to secure x firmly to "s simple =m ( op]at+ table1 su* z ,tat9's 7,fig4 #agi71 :i* w accommodate rabbits1 gu9ea-pigs1 & rats3 or to ! m elaborate table devis$ by ! au?or 7,fig4 #ahj74 ,illu/ration3 ,,fig4 #agh4--,m\se hold]4 ,illu/ration3 ,,fig4 #agi4--,tat9's op]ation table4 ,op]ation ,table4--,?is is a table ( ! aseptic type1 compos$ ( /eel tub+1 nickel-plat$ or enamell$4 ,! table-top frame is suffici5tly l>ge to accommodate rabbits1 dogs & monkeys2 & is support$ ^u telescopic uprights1 s t x is adju/able z to height2 9 its long axis x c be incl9$ 7at ei!r end7 to #de deg4 f ! horizontal4 ,fur!r x c be completely rotat$ ab\t its long axis4 ,! table-top itself is compos$ ( a %eet ( copp] wire gauze loosely susp5d$ f ! long sides ( ! tubul> frame4 ,! slackness ( ! gauze b$ p]mits ( an india rubb] hot wat] bottle1 or an electro!rm be+ plac$ "u ! animal1 & if dur+ ! c\rse ( an exp]im5t x is necess>y to rev]se ! animal1 ! table-top frame is completely rotat$1 ! device adopt$ = susp5d+ ! gauze is deta*$ & ! gauze rev]s$ also1 s t x aga9 supports ! animal f bel[4 ,illu/ration3 ,,fig4 #ahj4--;,au?or's op]at+ table#ab4 ,,me?ods ,,( ,,inoculation4 ,! foll[+ me?ods ( inoculation apply m p>ticul>ly to ! rabbit1 b f !m x w readily be se5 :at modifications 9 te*nique1 if any1 are necess>y 9 ! case ( ! o!r exp]im5tal animals4 #a4 ,cutane\s ,inoculation4--7,anae/hetic1 none47 #a4 ,h ! animal firmly held by an assi/ant 7or secur$ to ! op]at+ table74 #b4 ,apply ! liquid soap to ! fur1 ov] ! area select$ = inoculation1 ) a wad ( cotton-wool1 & la!r freely by ! aid ( w>m wat]2 %ave c>efully & ?or\ghly2 or apply ! depilatory p[d]4 #c4 ,wa% ! d5ud$ area ( sk9 ?or\ghly ) #b p] c5t4 lysol solution4 #d4 ,wa% (f ! lysol ) e!r & all[ ! latt] to evaporate4 #e4 ,make num]\s %ort1 p>allel1 sup]ficial incisions ) ! po9t ( a /]ile scalpel4 #f4 ,:5 ! ooz+ f ! incisions has ceas$1 rub ! inoculum into ! sc>ifications by means ( ! flat ( a scalpel blade1 or a /]ile plat9um spatula4 #g4 ,cov] ! inoculat$ area ) a pad ( /]ile gauze secur$ 9 situ by /rips ( adhesive pla/] or by seal+ d[n ! edges ( ! gauze ) collodion4 #h4 ,release ! animal1 place x 9 its cage1 & affix a label ^u :i* is writt53 7a7 ,di/9ctive "n or numb] ( ! animal4 7b7 ,its weight4 7c7 ,p>ticul>s z to s\rce & dose ( inoculum4 7d7 ,date ( inoculation4 #b4 ,subcutane\s ,inoculation4-- 7a7 ,fluid ,inoculum4--7,anae/hetic1 none47 ,/eps #a-d4 ,z = cutane\s inoculation4 #e4 ,p9* up a fold ( sk9 betwe5 ! =ef+] & ?umb ( ! left h&2 take ! *>g$ hypod]mic syr+e 9 ! "r h&1 ent] ! ne$le into a ridge ( sk9 rais$ by ! left f+] & ?umb1 & pu% x /eadily onw>d until ab\t #b cm4 ( ! ne$le are ly+ 9 ! subcutane\s tissue4 ,n[ release ! grasp ( ! left h& & sl[ly inject ! fluid conta9$ 9 ! syr+e4 #f4 ,)draw ! ne$le1 & at ! same mom5t close ! puncture ) a wad ( cotton wool1 to prev5t ! escape ( any ( ! inoculum4 ,! inject$ fluid1 unless l>ge 9 am\nt1 w be absorb$ )9 a v %ort "t4 #g4 ,label1 etc4 7b7 ,solid ,inoculum4--7,anae/hetic1 none2 or ,e?yl *loride spray47 ,/eps #a-d4 ,z = cutane\s inoculation4 #e4 ,raise a small fold ( sk9 9 a pair ( =ceps1 & make a small incision ?r\gh ! sk9 ) a pair ( %>p-po9t$ scissors or ) ! po9t ( a scalpel4 #f4 ,ins]t a probe ?r\gh ! op5+ & pu% x /eadily onw>d 9 ! subcutane\s tissue1 & by lat]al movem5ts sep>ate ! sk9 f ! und]ly+ muscles to =m a funnel-%ap$ pocket ) its apex t[>d ! po9t ( entrance4 #g4 ,by means ( a pair ( f9e-po9t$ =ceps introduce a small piece ( ! inoculum into ?is pocket & deposit x z f> z possible f ! po9t ( entrance4 ,illu/ration3 ,,fig4 #aha4--;,glass tube syr+e = subcutane\s solid inoculation4 ,or1 improvise a syr+e by slid+ a piece ( glass rod 7to s]ve z a pi/on7 into ! lum5 ( a slightly %ort] l5g? ( glass tub+ & secure 9 position by a b& ( rubb] tub+4 ,/]ilise by boil+4 ,)draw ! rod a few millimetres & deposit ! piece ( tissue )9 ! orifice ( ! tube1 by means ( /]ile =ceps4 ,n[ pass ! tube into ! dep?s ( ! pocket1 pu% on ! glass rod till x projects beyond ! end ( ! tube1 & )draw ! app>atus1 leav+ ! tissue beh9d 9 ! w\nd4 #h4 ,close ! w\nd 9 ! sk9 ) ,mi*el's clips & a dress+ ( gauze seal$ ) collodion 7or ,t9ct4 b5zo974 #i4 ,label1 etc4 #c4 ,intramuscul>4-- 7a7 ,fluid ,inoculum4--7,anae/hetic1 none47 ,/eps #a-d4 ,z = cutane\s inoculation4 #e4 ,/eady ! sk9 ov] ! select$ muscle or muscles ) ! slightly sep>at$ left =ef+] & ?umb4 #f4 ,?ru/ ! ne$le ( ! inject+ syr+e boldly into ! muscul> tissue & inject ! inoculum sl[ly4 #g4 ,label1 etc4 7b7 ,solid ,inoculum4--7,anae/hetic1 ,a4 ,c4 ,e47 #a4 ,secure ! animal to ! op]ation table & anae/hetise4 #b4 ,%ave & dis9fect ! sk9 at ! seat ( op]ation4 #c4 ,surr\nd ! field ( op]ation by /rips ( gauze wrung out 9 #b p] c5t4 lysol solution4 #d4 ,incise sk91 aponeurosis1 & muscle 9 turn4 #e4 ,deposit ! inoculum 9 ! dep?s ( ! incision4 #f4 ,close ! w\nd 9 ! muscle ) buri$ sutures & ! cutane\s w\nd ) ei!r cont9u\s or int]rupt$ sutures or ) ,mi*el's /eel clips4 #g4 ,apply a seal$ dress+ ( gauze & collodion4 #h4 ,remove ! animal f ! op]at+ table4 #i4 ,label1 etc4 #d4 ,intrap]itoneal4-- 7a7 ,fluid ,inoculum4--7,anae/hetic1 none47 ,/eps #a-d4 ,z = cutane\s inoculation4 ,%ave a fairly broad transv]se area1 /ret*+ f flank to flank4 #e4 ,place ! left =ef+] on "o flank & ! ?umb on ! opposite1 & p9* up ! entire ?ickness ( ! abdom9al p>ietes 9 a triangul> fold4 ,n[1 by slipp+ ! p]itoneal surfaces 7:i* are 9 apposition7 "o ov] ! o!r1 asc]ta9 t no coils ( inte/9e are includ$ 9 ! fold4 #f4 ,take ! syr+e 9 ! "r h& & ) ! ne$le transfix ! fold ne> its base 7,fig4 #ahb74 #g4 ,n[ release ! fold1 b hold ! syr+e /eady2 z ! p>ietes flatt5 out1 ! po9t ( ! ne$le is left free 9 ! p]itoneal cavity 7see ,fig4 #ahc74 ,illu/ration3 ,,fig4 #ahb4--;,intrap]itoneal inoculation--fluid4 #h4 ,inject ! fluid f ! syr+e4 #i4 ,label1 etc4 ,illu/ration3 ,,fig4 #ahc4--,section ( abdom9al wall1 etc41 %[+ po9t ( ne$le ly+ free 9 ! p]itoneal cavity above ! coils ( inte/9e4 ,second ,me?od3 ,/eps #a-d4 ,z 9 ! fir/ me?od4 #e4 ,anae/hetise a small select$ area ( sk9 by spray+ x ) e?yl *loride4 #f4 ,heat plat9um se>+ wire 7#j4e mm4 wire1 twi/$ to ! %ape indicat$ 9 figure #ahd1 m\nt$ 9 an alum9ium h&le7 to r$ness1 & ) x burn a hole ?r\gh ! anae/hetic area ( sk9 & abdom9al muscle d[n to1 b n ?r\gh1 ! visc]al p]itoneum4 #g4 ,fix a blunt-end$ ne$le on to ! *>g$ syr+e1 & by press+ ! r\nd$ end firmly aga9/ ! p]itoneum x c easily be pu%$ ?r\gh into ! p]itoneal cavity4 #h4 ,inject ! fluid f ! syr+e4 #i4 ,label1 etc4 ,?is me?od is especially useful :5 x is desir$ to collect samples ( ! p]itoneal fluid f "t to "t dur+ ! p]iod ( obs]vation1 z fluid c be remov$ f ! p]itoneal cavity1 at int]vals1 ?r\gh ?is ap]ture 9 ! abdom9al p>ietes1 by means ( a /]ile capill>y pipette4 ,illu/ration3 ,,fig4 #ahd4--,plat9um wire = burn+ hole ?r\gh p>ietes4 7b7 ,solid ,inoculum 7or ! implantation ( capsules conta9+ fluid cultivations74--7,anae/hetic1 ,a4 ,c4 ,e47 #a4 ,anae/hetise ! animal & secure x to ! op]at+ table4 #b4 ,%ave a l>ge area ( ! abdom9al p>ietes4 #c4 ,make an incision ?r\gh ! sk9 9 ! middle l9e ab\t #b cm4 9 l5g?1 midway betwe5 ! l[] end ( ! /]num & ! pubes4 #d4 ,divide ! aponeuroses betwe5 ! recti ^u a director4 #e4 ,divide ! p]itoneum ^u a director4 #f4 ,introduce ! inoculum into ! p]itoneal cavity4 #g4 ,close ! p]itoneal cavity ) ,lemb]t's sutures4 #h4 ,close ! sk9 & aponeurosis incisions toge!r ) int]rupt$ sutures or ,mi*el's /eel clips1 & apply a seal$ dress+4 #i4 ,release ! animal f ! op]at+ table4 #aj4 ,label1 etc4 ,suitable sacs may be readily prep>$ by ei!r ( ! foll[+ me?ods3 ,a4 ,collodion ,sacs4 #a4 ,dip a small te/-tube 7#e by #j4e cm471 bottom d[nw>d1 into a beak] ( collodion1 & dry 9 ! air2 repeat ?is process ?ree or f\r times4 #b4 ,dip ! tube1 ) its coat+ ( collodion1 alt]nately into a beak] ( alcohol & "o ( wat]4 ,?is loos5s ! collodion & all[s x to be peel$ (f 9 ! %ape ( a small te/-tube4 #c4 ,take a #bj cm4 l5g? ( glass tub+1 ( ab\t ! diamet] ( ! te/-tube us$ 9 =m+ ! sac1 & ins]t "o end into ! op5 m\? ( ! sac4 #d4 ,susp5d ! glass tube ) atta*$ sac1 inside a l>g] te/-tube1 by pack+ cotton-wool 9 ! m\? ( ! te/-tube ar\nd ! glass tub+1 & place 9 ! incubator at #cg deg4 ,c4 = tw5ty-f\r h\rs4 ,:5 remov$ f ! incubator1 ! sac w be firmly adh]5t to ! extremity ( ! glass tub+4 #e4 ,plug ! op5 end ( ! glass tub+ ) cotton-wool1 & /]ilise ! te/-tube & its cont5ts 9 ! hot-air ov54 ,to use ! sac1 remove ! plug f ! glass tub+1 p>tly fill ! sac ) cultivation to be inoculat$1 by means ( a /]ile capill>y pipette1 & replug ! tub+4 ,:5 ! abdom9al cavity has be5 op5$1 remove ! tub+ & atta*$ sac f ! protect+ te/-tube1 close ! sac by ty+ a /]ilis$ silk ?read tightly ar\nd x a little bel[ ! end ( ! glass tub+1 & sep>ate x f ! tub+ by cutt+ ?r\gh ! collodion above ! ligature1 & ! sac is ready = ins]tion 9 ! p]itoneal cavity4 ,b4 ,celloid9 ,sacs 7,h>ris74 ,mat]ials ,requir$4 ,quill glass tub+4 ,gelat9e capsules su* z ph>maci/s prep>e = ! exhibition ( bulky p[d]s4 ,v>i\s grades ( celloid91 ?ick & ?91 9 wide-m\!d bottles4 #a4 ,take a piece ( quill glass tub+ "s #d cm4 long by #e mm4 diamet]2 heat "o end 9 ! buns5 flame4 #b4 ,?ru/ ! heat$ end ( ! tube j ?r\gh "o end ( a gelat9e capsule & all[ x to cool 7,fig4 #ahe74 #c4 ,remove any gelat9e f ! lum5 ( ! tube ) a heat$ plat9um ne$le2 pa9t ! jo9t betwe5 capsule & tube ) mod]ately ?ick celloid9 & all[ to dry4 ,illu/ration3 ,,fig4 #ahe4--,mak+ celloid9 capsules4 #d4 ,dip ! capsule into a beak] conta9+ ?9 celloid91 beyond ! junction ) ! glass & aft] removal rotate x 9 front ( ! bl[pipe air bla/ to dry x ev5ly4 ,repeat ^! manoeuvres until a suffici5tly ?ick coat+ is obta9$4 #e4 ,apply ?ick celloid9 to ! tube-capsule jo9t1 ! opposite end ( ! capsule1 & ! l9e ( junction ( ! capsule ) its cap2 dry ?or\ghly4 #f4 ,) a teat pipette fill ! capsule 7?r\gh ! atta*$ tube7 ) hot wat]1 & /& ! capsule 9 a beak] ( boil+ wat] = a few m9utes to melt ! gelat9e4 #g4 ,remove ! solution ( gelat9e f ! int]ior ( ! celloid9 case ) a pipette4 #h4 ,fill ! sac ) nutri5t bro? & place x1 glass tube d[nw>d1 9 a tube conta9+ suffici5t /]ile nutri5t bro? to cov] ! sac to ! dep? ( #a cm4 ,plug ! tube & /]ilise 9 ! /eam] 9 ! usual mann]4 #i4 ,to prep>e ! sac = use1 empty x out ( ! bro? tube into a /]ile glass di%4 #aj4 ,grasp ! tube ne> its junction ) ! sac 9 ! jaws ( /]ile =ceps1 & ) a teat pipette remove suffici5t ( ! conta9$ bro? to leave a small space 9 ! sac4 ,introduce ! inoculum 9 ! =m ( an emulsion by means ( ano!r pipette4 #aa4 ,/ill hold+ ! tube 9 ! =ceps1 draw x out & seal (f ne> ! sac 9 ! bl[pipe flame4 #ab4 ,:5 cool wa% ! sac 9 /]ile wat]1 !n transf] to a tube ( nutri5t bro? & incubate ov] night to det]m9e its imp]meability to bact]ia4 #ac4 ,if ! bro? outside ! sac rema9s /]ile1 ins]t ! sac 9 ! p]itoneal cavity ( ! exp]im5tal animal4 #e4 ,intracranial4--7,anae/hetic1 ,a4 ,c4 ,e47 ,illu/ration3 ,,fig4 #ahf4--;,gu>d$ treph9e4 ,treph9es & ,surgical ,eng9e4--,! mo/ useful in/rum5t = intracranial op]ations ^u animals is ! small nasal treph9e 7,curtis7 hav+ a too? cutt+ circle ( #g mm4 ,! addition ( an adju/able coll> gu>d--secur$ by a screw--prev5ts accid5tal lac]ation ( ! dura mat] or bra9 sub/ance#ac 7,fig4 #ahf74 ,?is size is suitable = monkeys1 dogs1 cats & l>ge rabbits4 ,o!r small] sizes :i* w be f\nd useful = gu9ea pigs & o!r small animals cut circles ( #f & #d mm42 = v small animals--"y gu9ea pigs & rats--a small d5tal drill or screw w make a suffici5tly l>ge hole to admit ! syr+e ne$le4 ,! treph9e c be set 9 ord9>y metal h&les & rotat$ by h&1 b a surgical eng9e ( "s k9d is mu* pref]able on ! score ( rapidity & safety to ! animal4 ,! ,guy's electrical ,d5tal eng9e#ad 7,fig4 #ahg7 :i* c be connect$ to a lamp socket or wall plug1 & is op]at$ by a foot swit*1 al?\gh inexp5sive is em95tly satisfactory4 ,,note4--,a f9e d5tal drill atta*$ to ! d5tal eng9e r5d]s ! manufacture ( alum9ium h&les ne$les 7see page #ga7 q an easy matt]4 7a7 ,subdural4 #a4 ,anae/hetise ! animal & secure x to ! op]at+ table1 dorsum upp]mo/4 #b4 ,%ave a portion ( ! scalp imm$iately 9 front ( ! e>s4 ,illu/ration3 ,,fig4 #ahg4--;,guy's electrical d5tal eng9e4 #c4 ,m>k out ) a %>p scalpel a cresc5tic flap ( sk9 muscle1 etc41 convexity =w>d1 comm5c+ #j4e cm4 9 front ( ! root ( "o e> & t]m9at+ at a simil> spot 9 front ( ! o!r e>4 ,reflect ! m>k$ flap4 #d4 ,make a correspond+ incision ?r\gh ! p]io/eum & raise x ) a blunt dissector4 #e4 ,) a small treph9e 7diamet] #f mm47 remove a circul> piece ( bone f ! p>ietal segm5t4 ,! c5tre ( ! treph9e hole %\ld be at ! int]section ( ! m$ian l9e & a l9e jo9+ ! po/]ior can?i 7,fig4 #ahh74 #f4 ,introduce ! inoculum by means ( a hypod]mic syr+e1 p]=at+ ! dura mat] ) ! ne$le & deposit+ ! mat]ial imm$iately bel[ ?is membrane1 at ! same "t tak+ c>e to avoid injur+ ! s9uses4 #g4 ,turn back ! flap ( sk9 & secure x 9 position ) ,mi*el's /eel clips4 #h4 ,dress ) /]ile gauze & wool & seal ! dress+ ) collodion4 #i4 ,label1 etc4 7b7 ,intrac]ebral4--,?is inoculation is p]=m$ precisely z = subdural save 9 /ep #f ! ne$le aft] p]=at+ ! dura mat] is pu%$ onw>d into ! sub/ance ( "o or o!r c]ebral hemisph]es be=e ! cont5ts are eject$4 ,illu/ration3 ,,fig4 #ahh4--;,intracranial inoculation ( rabbit4 ,! circle indicates ! situation ( ! treph9e hole4 #f4 ,intraocul>4-- 7a7 ,fluid ,inoculum4--7,anae/hetic1 coca9e47 #a4 ,in/il a few drops ( a /]ile solution ( coca9e1 & repeat ! in/illation 9 two m9utes4 #b4 ,five m9utes lat] h ! animal firmly held by an assi/ant z 9 intrav5\s injection 7see ,fig4 #ahi71 ! head be+ /eadi$ by ! assi/ant's h&s4 #c4 ,select two ne$les to accurately fit ! same syr+e & /]ilise4 #d4 ,atta* "o ne$le to ! syr+e & take up ! requir$ dose ( inoculum & remove ! ne$le4 #e4 ,/eady ! eye ) fixation =ceps2 !n pi]ce ! cornea ) ! o!r syr+e ne$le & all[ ! aque\s to escape ?r\gh ! ne$le4 #f4 ,)\t remov+ ! ne$le f ! cornea atta* ! syr+e & make ! injection into ! ant]ior *amb]4 #g4 ,irrigate ! conjunctival sac ) /]ile sal9e solution4 #h4 ,label1 etc4 7b7 ,solid ,inoculum4--7,anae/hetic1 ,a4 ,c4 ,e47 #a4 ,anae/hetise ! animal & secure x firmly to ! op]at+ table4 #b4 ,irrigate ! conjunctival sac ?or\ghly ) /]ile sal9e solution4 #c4 ,make an incision ?r\gh ! upp] quadrant ( ! cornea into ! ant]ior *amb] by means ( a triangul> k]atome4 #d4 ,sep>ate ! lips ( ! corneal w\nd ) a flexible silv] spatula2 seize ! solid inoculum 9 a pair ( iris =ceps1 introduce x ?r\gh ! corneal w\nd1 & deposit x on ! ant]ior surface ( ! iris2 )draw ! =ceps4 #e4 ,aga9 irrigate ! sac & ! surface ( ! cornea4 #f4 ,release ! animal f ! op]at+ table4 #g4 ,label1 etc4 #g4 ,intrapulmon>y4-- ,fluid ,inoculum4--7,anae/hetic1 none47 #a4 ,h ! animal firmly held by an assi/ant4 7,9 ?is case ! =eleg ( ! select$ side is drawn up by ! assi/ant & held ) ! e> ( t side47 #b4 ,%ave c>efully 9 ! axill>y l9e & dis9fect ! d5ud$ sk94 #c4 ,?ru/ ! ne$le ( ! syr+e boldly ?r\gh ! fif? or six? int]co/al space into ! lung tissue4 #d4 ,inject ! cont5ts ( ! syr+e sl[ly4 #e4 ,label1 etc4 #h4 ,intrav5\s4-- ,fluid ,inoculum4--7,anae/hetic1 none47 ,! site select$ = ! injection 9 ! rabbit is ! po/]ior auricul> ve9 7see ,fig4 #aib74 ,al?\gh ?is is small] ?an ! m$ian ve91 x is firmly b\nd d[n to ! c>tilage ( ! e> by d5se connective tissue1 & is !re=e m readily accessible4 7,9 ! gu9ea-pig ! jugul> ve9 mu/ be utilis$1 & 9 ord] to p]=m ! inoculation satisfactorily a g5]al anae/hetic mu/ be adm9i/]$ to ! animal4 ,9 ! monkey or ! dog1 ! int]nal saph5\s ve9 is ! mo/ conv5i5t & be=e punctur+ %\ld be di/5d$ or r5d]$ prom95t by compress+ ! ve9 above ! select$ site47 ,prep>ation ( ! ,inoculum4--,c>e mu/ be tak5 9 prep>+ ! inoculum1 z ! injection ( ev5 small fragm5ts may cause fatal embolism4 ,to obviate ?is risk ! fluid %\ld1 if possible1 be filt]$ ?r\gh /]ile filt] pap] be=e fill+ into ! syr+e4 ,air bubbles1 :5 inject$ into a ve91 frequ5tly cause imm$iate dea?4 ,to prev5t ?is1 ! syr+e aft] be+ fill$ %\ld be held 9 ! v]tical position1 ne$le upp]mo/4 ,a piece ( /]ile filt] pap] is !n impal$ on ! ne$le & ! pi/on ( ! syr+e press$ upw>d until all ! air is expell$ f ! b>rel & ne$le4 ,%\ld any drops ( ! inoculum be =c$ out1 !y w fall on ! filt] pap]1 :i* %\ld be imm$iately burn$4 #a4 ,h ! animal firmly held by an assi/ant4 ,! select$ e> is grasp$ at its root & /ret*$ =w>d t[>d ! op]ator4 #b4 ,%ave ! po/]ior bord] ( ! dorsum ( ! e>4 #c4 ,dis9fect ! sk9 ov] ! ve91 rubb+ x vig\r\sly ) cotton-wool soak$ 9 lysol4 ,! friction w make ! ve9 m conspicu\s4 ,wa% ! lysol (f ) e!r & all[ ! latt] to evaporate4 #d4 ,direct ! assi/ant to compress ! ve9 at ! root ( ! e>4 ,?is w cause its p]iph]al portion to swell up & increase 9 calibre4 #e4 ,hold ! syr+e z "o w\ld a p5 & ?ru/ ! po9t ( ! ne$le ?r\gh ! sk9 & ! wall ( ! ve9 till x ent]s ! lum5 ( ! ve9 7,fig4 #ahi74 ,n[ press x onw>d 9 ! direction ( ! blood /ream--i4 e41 t[>d ! body ( ! animal4 #f4 ,direct ! assi/ant to cease compress+ ! root ( ! e>1 & sl[ly inject ! inoculum4 7,if ! fluid is be+ =c$ into ! subcutane\s tissue1 a condition :i* is at once indicat$ by ! swell+ t occurs1 ! injection mu/ be /opp$ & ano!r attempt made at a spot clos] to ! root ( ! e> or at "s po9t on ! correspond+ ve9 on ! opposite e>47 #g4 ,)draw ! ne$le & press a pl$get ( cotton-wool ov] ! puncture to ensure closure ( ! ap]ture 9 ! ve9 wall4 #h4 ,label1 etc4 ,illu/ration3 ,,fig4 #ahi4--;,intrav5\s inoculation4 #i4 ,inhalation4-- 7a7 ,fluid ,inoculum4--7,anae/hetic1 none47 #a4 ,place ! animal 9 a clos$ metal box4 #b4 ,?r\gh a hole 9 "o side introduce ! nozzle ( "s simple spray+ app>atus1 su* z is us$ = nasal m$icam5ts4 #c4 ,fill ! res]voir ( ! in/rum5t 7previ\sly /]ilis$7 ) ! fluid inoculum1 & hav+ atta*$ ! bell[s1 spray ! inoculum into ! int]ior ( ! box4 #d4 ,on ! completion ( ! spray+1 op5 ! box1 spray ! animal ?or\ghly ) a #aj p] c5t4 solution ( =maldehyde 7to de/roy any ( ! virus t may be adh]+ to fur or fea!rs74 #e4 ,transf] ! animal to its cage4 #f4 ,label1 etc4 #g4 ,?or\ghly dis9fect ! inhalation *amb]4 7b7 ,fluid or ,p[d]$ ,inoculum4--,anae/hetic1 ,a4 ,c4 ,e4 #a4 ,anae/hetise ! animal & secure x firmly to ! op]at+ table4 ,illu/ration3 ,,fig4 #aij4--;,gag = rabbits4 #b4 ,prop op5 ! m\? by means ( "s =m ( gag2 seize ! tongue ) a pair ( =ceps & draw x =w>d4 ,! mo/ conv5i5t =m ( gag = ! rabbit or cat is t %[n 9 ,fig4 #aij4 ,x is simply a /rip ( h>d wood %ap$ at ! middle & provid$ ) a squ>e orifice ?r\gh :i* a tra*eal or oesophageal tube c be pass$4 #c4 ,pass a previ\sly /]ilis$ glass tube 7#ag cm4 long1 #j4e cm4 diamet]1 ) its t]m9al #b cm4 slightly curv$7 d[n ?r\gh ! l>ynx into ! tra*ea4 #d4 ,connect ! /raight portion ( a ,y-%ap$ piece ( tub+ to ! upp] end ( ! /]ilis$ tube & c\ple "o bran* ( ! ,y to a sep>atory funnel conta9+ ! fluid inoculum1 or insufflator conta9+ ! p[d]$ inoculum1 & ! o!r to a h& bell[s4 #e4 ,all[ ! fluid inoculum to run into ! lungs by gravity1 or bl[ 9 ! p[d]$ inoculum by means ( a rubb]-ball bell[s4 #f4 ,remove ! intratra*eal tube2 release ! animal f ! table4 #g4 ,label1 etc4 ,z an alt]native me?od 9 ! case ( fairly l>ge animals1 su* z rabbits1 etc41 a /]ile piece ( glass tub+ ( suitable diamet] may be pass$ ?r\gh ! l>ynx d[n ! tra*ea almo/ to its bifurcation4 ,fluid cultivations may !n be lit]ally p\r$ into ! lungs1 or cultivations1 dri$ & p[d]$1 may be bl[n into ! lung by ! aid ( a small h& bell[s or ev5 a teat pipette4 #aj4 ,intraga/ric ,inoculation4--,fluid or semi-fluid inoculum4 7,anae/hetic none47 ,! me?od ( p]=m+ ! op]ation is v>i$ slightly accord+ to ! size ( ! exp]im5tal animal4 ,a4 ,monkey1 ,rabbit1 ,gu9ea-pig4 #a4 ,secure ! animal to ! op]at+ table v5tral surface upp]mo/4 #b4 ,prop ! m\? op5 ) a gag2 draw ! tongue =w>d ) =ceps4 #c4 ,/]ilise a s(t rubb] ca!t] 7,no4 #aj or #h ,engli% scale1 or ,no4 #ah or #ae ,fr5*7 & lubricate x ) /]ile glyc]9e4 #d4 ,pass x to ! back ( ! ph>ynx1 keep+ ! end 9 ! middle l9e4 #e4 ,g5tly assi/ ! progress ( ! ca!t] d[n ! oesophagus until x passes ! c>diac orifice ( ! /oma*4 ,d n use any =ce4 #f4 ,take up ! requir$ dose ( inoculum into a /]ilis$ pipette4 ,ins]t ! po9t ( ! pipette into ! op5 end ( ! ca!t] & all[ ! fluid to run d[n into ! /oma*4 ,remove ! pipette & drop x into a j> ( lysol4 #g4 ,) ano!r /]ile pipette run "o cubic c5timetre ( /]ile sal9e solution ?r\gh ! ca!t] to wa% out ! la/ traces ( ! inoculum4 #h4 ,)draw ! ca!t]4 #i4 ,label1 etc4 ,b4 ,rats & ,mice 7,m>k's ,me?od74 #a4 ,secure ! animal 9 ! v]tical position4 7a7 ,rat4--,take a pair ( cat* s9us =ceps ab\t #bb cm4 9 l5g? & seize ! animal by ! loose sk9 ( ! head z f> =w>d z possible--fix ! =ceps1 & hold+ ! in/rum5t v]tically upw>d1 transf] to ! left h& ( an assi/ant :o secures ! animal's tail betwe5 ! f+]s grasp+ ! h&le ( ! =ceps4 7,see ,fig4 #aia47 ,illu/ration3 ,,fig4 #aia4--;,intraga/ric inoculation ( rat4 7b7 ,m\se4--,an assi/ant grasps ! loose sk9 betwe5 ! e>s z f> =w>ds z possible betwe5 ! =ef+] & ?umb ( ! left h&4 ,he n[ grasps ! tail ) ! "r h&1 draws ! m\se /raight & passes ! tail betwe5 ! f\r? & little f+]s ( ! left h& & secures x !re4 #b4 ,! assi/ant takes a clos$ pair ( ?9-blad$ =ceps 9 8 "r h&1 passes ! ends into ! animal's m\?1 !n all[s ! blades to sep>ate4 ,?is op5s ! animal's jaw & s]ves z a gag4 #c4 ,moi/5 ! /]ilis$ oesophageal tube ) /]ile wat]4 7,?is tube is ( silk rubb]1 #f4e cm4 9 l5g?1 ) ! di/al end r\nd$1 ! proximal end m\nt$ 9 a syr+e ne$le head1 :i* fits ! nozzles ( ! two /]ile syr+es to be us$47 #d4 ,grasp ! tube ab\t its middle & pass x into ! animal's m\?1 d[nw>ds & a little to "o side or ! o!r until its l5g? is lo/ 9 ! dige/ive tract & m\?4 ,g5tle guidance is alone necess>y4 ,d n use any =ce4 #e4 ,take up ! requir$ dose ( inoculum into ! syr+e2 ins]t ! nozzle ( ! syr+e into ! ne$le-m\nt1 & =ce ! pi/on d[n4 #f4 ,/eady+ ! ne$le-m\nt ) ! left h&1 deta* ! syr+e4 #g4 ,draw up "s /]ile wat] 9 ! second 7/]ile7 syr+e1 & ins]t+ its nozzle into ! ne$le-m\nt =ce a few drops ( wat] ?r\gh ! tube to wa% x out4 #h4 ,) "o quick upw>d movem5t remove ! tube f ! animal's m\?4 #i4 ,label1 etc4 ,"o o!r me?od ( inoculation rema9s to be describ$1 :i* does n require op]ative int]f]5ce4 #aa4 ,fe$+4-- #a4 ,fluid ,inoculum4--,small pieces ( /]ilis$ bread or sop 7/]ilis$ 9 ! /eam] at #ajj deg4 ,c47 are soak$ 9 ! fluid inoculum & (f]$ to ! animals 9 a /]ile ,petri di% or capsule4 #b4 ,solid ,inoculum4--,small pieces ( tissue are plac$ 9 /]ile vessels & (f]$ to ! animals4 ,,footnotes3 #ab ,?is table is made by ,messrs4 ,d[n ,bros41 ,/4 ,?omas's ,/reet1 ,london1 ,s4 ,e4 #ac ,?is modification is made = ! au?or by ,messrs4 ,d[n ,bros41 ,/4 ,?omas's ,/reet1 ,london1 ,s4 ,e4 #ad ,manufactur$ by ,messrs4 ,francis ,lepp]1 #ef1 ,great ,m>lbor\gh ,/reet1 ,london1 ,w4 ,,xviii4 ,,! ,,/udy ,,( ,,exp]im5tal ,,infections ,,dur+ ,,life4 ,! possession ( pa?og5etic prop]ties by an organism "u /udy is indicat$ by ! infection ( ! exp]im5tal animal--a t]m :i* is employ$ to summ>ise ! condition result+ f ! successful invasion ( ! tissues ( ! exp]im5tal animal by ! micro-organisms inoculat$ & by _! multiplication !re94 ,infection is consid]$ to h tak5 place3 #a4 ,:5 ! dea? ( ! animal is produc$ z a direct consequ5ce ( ! inoculation4 #b4 ,:5 )\t necess>ily produc+ dea? ! inoculation causes local or g5]al *anges ( a pa?ological "*4 #c4 ,:5 ei!r ) or )\t dea?1 or local or g5]al *anges occurr+1 c]ta9 sub/ances make _! appe>ance 9 ! body fluids1 :i* c be %[n 79 vitro or 9 vivo7 to ex]t "s pr(\nd & specific effect :5 br\ght into contact ) subcultivations ( ! organism orig9ally inoculat$4 ,! important factors 9 ! production ( infection are3 ,a4 ,se$4 ,virul5ce ( organism4 ,dose ( organism4 ,b4 ,soil4 ,resi/ance (f]$ by ! cells ( ! exp]im5tal animal4 ,! fir/ two factors1 al?\gh v>iable1 are to a c]ta9 ext5t "u ! control ( ! exp]im5t]4 ,?us by suitable means ! virul5ce ( an organism c be exalt$ or att5uat$1 :il/ ! size ( ! dose may be increas$ or dim9i%$4 ,! ?ird factor also v>ies1 n only among/ diff]5t species ( animals1 b also among/ diff]5t individuals ( ! same species4 ,! ess5tial causes ( ?is v>iation are n s obvi\s1 s t beyond select+ ! animals int5d$ = simil> exp]im5ts ) reg>d to su* po9ts z age1 size or sex1 b little c be done to /&>dise cell resi/ance4 ,imm$iately an animal has be5 inoculat$ a p]iod ( cl9ical obs]vation mu/ be ent]$ ^u1 :i* %\ld only t]m9ate ) ! dea? ( ! animal4 ,! g5]al obs]vations %\ld at fir/ & if ! infection is an acute "o1 be made daily--lat]1 & if ! animal appe>s to be unaffect$ or if ! infection is *ronic1 bo? g5]al & special obs]vations %\ld be c>ri$ out at weekly int]vals4 ,if ! animal appe>s to be /ill unaffect$1 x %\ld be kill$ ) *lor(orm vap\r at ! end ( two or ?ree mon?s & a complete po/-mortem c>ri$ out4 ,a4 ,! g5]al obs]vations %\ld take cognisance (3 #a4 ,g5]al appe>ance4 ,! exp]im5tal animal %\ld be inspect$ daily1 n only ) a view to detect+ symptoms due to ! exp]im5tal infection1 b also to prev5t any int]curr5t infection1 naturally acquir$1 f escap+ notice 7vide page #ccg74 #b4 ,! weight ( ! inoculat$ animal %\ld be obs]v$ & record$ ea* day dur+ ! c\rse ( an exp]im5tal infection at precisely ! same h\r1 pref]ably j be=e ! morn+ fe$4 #c4 ,! temp]ature %\ld simil>ly be record$ daily1 if n m frequ5tly1 dur+ ! :ole p]iod ! animal is "u obs]vation1 & c>efully *>t$--individual v>iations w at once become app>5t4 ,x %\ld be borne 9 m9d t ! temp]ature reg>d$ z normal = man 7#cg4e deg4 ,c47 is n ! normal av]age temp]ature ( any ( ! l[] animals save ! rat & m\se4 ,! accompany+ table ( normal av]ages = ! animals usually employ$ 9 bact]iological rese>* may be ( use 9 prev5t+ ! errone\s assumption t pyrexia is pres5t 9 an animal1 :i* m]ely %[s its own normal temp]ature4 ,,normal ,,av]ages4 ---------------------------------------- ------------ ,rectal ,pulse4 ,respirations4 ,animal4 ,temp4 ------------------------ deg4 ,c4 ,rate p] m9ute4 ---------------------------------------- ------------ ,frog #h4i-ag4b #hj #ab ,m\se #cg4d #abj 444 ,rat #cg4e 444 #baj ,gu9ea pig #ch4f #aej #hj ,rabbit #ch4g #ace #ee ,cat #ch4g #acj #bd ,dog #ch4f #ie #ae ,goat #dj4j #ge #af ,ox #ch4h #de 44 ,horse #cg4i #ch #aa ,monkey 7,rhesus7 #ch4d #ajj #ai ,pigeon #dj4i #acf #cj ,f[l #da4f #adj #ab ---------------------------------------- ------------ ,b4 ,special obs]vations comprise "s or all ( ! foll[+1 accord+ to ! me?od ( inoculation & ! "* ( ! virus4 #a4 ,! site ( inoculation %\ld be m9utely exam9$ at lea/ at weekly int]vals1 & ! neighb\r+ lymphatic gl&s palpat$4 #b4 ,any local reaction at ! site ( inoculation & any o!r readily accessible lesion %\ld be c>efully inve/igat$4 ,any suppurative process :i* may occur1 :e!r 9 ! subcutane\s tissues or 9 jo9ts1 %\ld be explor$ & ! pus c>efully exam9$ bo? microscopically & culturally4 ,fluid secretions & excretions1 su* z pus or s]\s exudates :5 accessible are collect$ direct f ! body 9 /]ile capill>y pipettes 7vide ,fig4 #ac;a17 9 ! foll[+ mann]3 #a4 ,op5 ! case conta9+ ! pipettes1 grasp "o by ! plugg$ end1 remove x f ! case1 & replace ! lid ( ! latt]4 #b4 ,atta* a rubb] teat 7vide page #aj7 to ! plugg$ end ( ! pipette & use ! teat z ! h&le ( ! pipette4 #c4 ,pass ! entire l5g? ( ! pipette twice or ?rice ?r\gh ! flame ( ! ,buns5 burn]4 #d4 ,snap (f ! seal$ end ( ! pipette ) a pair ( /]ile =ceps4 #e4 ,compress ! india-rubb] teat1 ?ru/ ! po9t ( ! pipette into ! secretion2 n[ relax ! pressure on ! teat & all[ ! pipette to fill4 #f4 ,remove ! po9t ( ! pipette f ! secretion1 all[ ! fluid to run a %ort di/ance up ! capill>y /em & seal ! po9t ( ! pipette 9 ! flame4 7,if us+ a pipette ) a con/riction bel[ ! plugg$ m\?piece 7,fig #ac;b71 ?is portion ( ! pipette may also be seal$ 9 ! flame47 ,:5 ready to exam9e ! morbid mat]ial snap (f ! seal$ end ( ! pipette ) /]ile =ceps & eject ! cont5ts ( ! pipette into a /]ile capsule4 ,! mat]ial c n[ be utiliz$ = cov]-slip prep>ations1 cultivations & inoculation exp]im5t4 #c4 ,! p]iph]al blood %\ld be exam9$ f "t to "t = f ?is tissue is (t5 obta9$ ! fulle/ in=mation z to ! c\rse & progress ( an infection4 a4 ,! hi/ological exam9ation ( ! blood %\ld be direct$ *iefly to obs]vations on ! numb] & k9d ( :ite cells2 & s9ce b few bact]iologi/s are at ! same "t exp]t comp>ative haematologi/s1 "s notes on ! normal *>act]s ( ! blood ( ! common] laboratory animals1 contra/$ ) ^? ( man1 are ins]t$ = ref]5ce4 ,^! h be5 v k9dly compil$ = me by my fri5d & "o "t colleague ,dr4 ,cecil ,price ,jones4 ,,comp>ative ,,haemocytology ,,( ,,laboratory ,,animals4 ---------------------------------------- ---------------------------- ,totals ,p]c5tages ---------------------------------------- -------------------- ,animal ,hb1 ,lympho-,l>ge ,poly- ,eos9- ,ma/ ,r$ cells ,:ite p] cytes1monos1morph1 oph1 cells1 cellsc5t4 p] p] p] p] p] c5t4 c5t4 c5t4c5t4 c5t4 ---------------------------------------- ---------------------------- ,frog #dij1jjj #h1jjj #eh #dj #aj4j #bb4j #ae #ac ,m\se #h1gjj1jjj #h1jjj #gh #fj #ba4e #ag4j #a4d #j4a ,rat #i1jjj1jjj #i1jjj #he #ed #g4j #cg4e #a4c #j4b ,gu9ea- pig #e1gjj1jjjaj1jjj #ii #ee #i4j #cb4h #c4j #j4b ,rabbit #f1jjj1jjj #g1jjj #gj #ej #b4j #df4j #j4f #a4d ,rhesus #d1ejj1jjjac1jjj #gg #dc #e4j #ej4j #a4c #j4g ,goat #ad1fjj1jjjae1jjj #eh #ce #f4c #ef4g #a4be #j4ge ,f[l #c1ejj1jjjcj1jjj #ajj #di #c4j #db4j #a4j #e4j ,pigeon #c1ejj1jjjbj1jjj #aja #dc #i4j #dc4j #c4j #b4j ---------------------------------------- ---------------------------- ,man 7adult7 #e1jjj1jjj #g1ejj #ajj #be #e4e #fe #d4j #j4e ,normal 7#d4e-e7 7#g-i77ie- 7#bj-cj7 7#d-h77ee- 7#c-e7 7#j4e-b7 limits4 millions4 ?\- #aja7 #fh7 s&s4 ---------------------------------------- ---------------------------- ,! above table repres5ts 9 ea* case ! av]age ( a l>ge numb] ( c\nts4 ,,rem>ks4 ,frog4--,! r$ cells are l>ge oval nucleat$ 7#bj-be mu by #ab-ae mu7 discs1 ! nucleus relatively small & irregul>ly elongat$ or oval1 ab\t #aj mu 9 l5g?4 ,_m primitive & develop+ =ms are usually obs]v$--also free nuclei & _m cells 9 v>i\s /ages ( deg5]ation4 ,haemoglob9 e/imation is difficult ow+ to turbidity ( ! blood aft] dilution ) wat]4 ,! polymorphonucle> leucocytes are l>ge cells1 ab\t #bj mu2 no def9ite granules c be obs]v$4 ,! eos9ophile cells conta9 l>ge deeply /a9+ coccal-%ap$ granules4 ,m\se4--,! granules ( ! polymorphonucle> leucocytes are usually n /a9$1 or only v fa9tly s4 ,! nucleus ( ! eos9ophile cell is r+-%ap$ or mu* divid$1 & ! granules are coccal & /a9 oxyphile4 ,! rem>kable "* ( ! blood is ! high p]c5tage ( l>ge mononucle> cells4 ,rat4--,! f9e rod-%ap$ granules ( ! polymorphonucle> leucocytes are usually v fa9tly /a9$4 ,! granules ( eos9ophile cells are well /a9$ & coccal-%ap$1 ! nucleus is (t5 r+ %ap$4 ,! basophile granul> cells are few--b ! granules are l>ge1 & /a9 deeply basophile4 ,gu9ea-pig4--,poly*romasia & punctate basophilia ( r$ cells are v commonly obs]v$--nucleat$ r$ cells are also frequ5t4 ,! l>ge mononucle> cells (t5 conta9 vacuoles--,kurl[ cells--possibly ( a p>asitic nature4 ,rabbit4--,x is n uncommon to f9d nucleat$ r$ cells 9 films f q heal?y animals4 ,! granules ( ! polymorphonucle> leucocytes /a9 oxyphile4 ,! co>se granules ( ! eos9ophile cells appe> to /a9 less deeply oxyphile1 probably ow+ to ! basophile /a9+ ( ! cytoplasm4 ,rhesus monkey4--,! blood cells resemble ^? met ) 9 human blood4 ,! m9ute neutrophile granules ( ! polymorphonucle> leucocytes are (t5 v scanty1 & sometimes app>5tly abs5t4 ,! eos9ophile cells are n s d5sely pack$ ) co>se oxpyhile granules z 9 ! human eos9ophile1 & ! nuclei ( ^! cells are usually mu* divid$1 or polymorph\s4 ,goat4--,! r$ cells are small1 nonnucleat$ discs1 only ab\t #d4e mu diamet]1 n mu* m ?an half t ( ! human r$ cell4 ,! polymorphonucle> leucocytes h only a few v m9ute coccal-%ap$ oxyphile granules1 ! nucleus is polymorph\s4 ,! eos9ophile cells are l>ge cells up to #bj mu1 ! cytoplasm is basophile & conta9s co>se coccal-%ap$ oxyphile granules1 & ! nucleus is (t5 mu* divid$4 ,f[l4--,! r$ cells are oval nucleat$ discs ab\t #ab mu by #f mu1 ! nucleus be+ relatively small 7ab\t #d mu long71 irregul>ly elongat$ or oval2 r\nd1 m deeply /a9$ cells ) r\nd or diffuse nuclei1 also free nuclei & deg5]at$ =ms ( r$ cells are (t5 pres5t4 ,! granules ( ! cells correspond+ to ! polymorphonucle> leucocytes are rod-%ap$1 (t5 bead$ or ) clubb$ ends4 ,! nucleus is n polymorph\s1 b usually divid$ into two1 ?\gh x may be s+le4 ,! cells probably correspond+ to eos9ophile leucocytes h f9e coccal-%ap$ granules1 fa9tly /a9+ eos9ophile or neutrophile4 ,! basophile granules ( ! ma/ cells are coccal-%ap$1 ( v>i\s size--(t5 q p[d]y4 ,pigeon4--,r$ cells resemble ^? ( ! f[l1 & simil> v>ieties ( appe>ance may be not$4 ,! granules ( ^? cells :i* correspond to polymorphonucle> leucocytes are rod-%ap$1 b small] & f9] ?an 9 ! f[l1 & d n %[ clubb$ appe>ances4 ,! nucleus is n polymorph\s1 & only occasionally divid$4 ,! coccal-%ap$ granules ( ! eos9ophile cells are /a9$ m deeply oxyphile ?an ^? ( ! correspond+ cells ( ! f[l4 ,! prep>ation ( dri$ films = ?is hi/ological exam9ation ( ! blood is c>ri$ out z foll[s3 #a4 ,small samples ( blood = ! prep>ation ( blood films are mo/ conv5i5tly obta9$ f ! ve9s ( ! e> 9 mo/ ( ! ord9>y laboratory animals1 viz41 monkey1 goat1 dog1 cat1 rabbit1 gu9ea-pig2 9 ! pigeon & f[l ! axill>y ve9 %\ld be punctur$2 9 ! rat & m\se ei!r a ve9 9 ! e> or pref]ably by w\nd+ ! tip ( ! tail2 9 ! frog1 ! web ( ! foot %\ld be select$4 #b4 ,puncture ! select$ ve9 ) a %>p ne$le4 ,a flat ,hag$orn ne$le 7size ,no4 #h7 ) a cutt+ edge is ! mo/ useful = ?is purpose4 ,if ! ve9 _c be di/5d$ by proximal compression1 vig\r\s friction ) a piece ( dry l9t may h ! desir$ effect--or a te/-tube full ( wat] at ab\t #dj deg4 ,c4 may be plac$ close to ! ve94 ,fail+ ^! me?ods1 a drop or two ( xylol may be dropp$ on ! sk9 j ov] ! ve91 left on = a few seconds & !n wip$ (f ) a piece ( dry l9t4 #c4 ,"o ( ! %ort ends ( a #c by #a glass slip is br\ght into contact ) ! exud+ drop ( blood1 s t x picks up a small drop4 #d4 ,! slide is !n l[]$ transv]sely on to ! surface ( a second #c by #a slip1 :i* re/s on ! b5* ne> to "o end at an angle ( ab\t #de deg41 & reta9$ 9 ?is position = a few seconds1 :ile ! drop ( blood spreads along ! :ole ( ! l9e ( contact 7see also ,fig4 #fi74 #e4 ,draw ! fir/ slide firmly & ev5ly along ! entire l5g? ( ! l[] slide1 leav+ a ?9 regul> film :i* w probably %[ ! blood cells only "o lay] ?ick4 #f4 ,all[ ! film to dry 9 ! air4 #g4 ,/a9 ) "o ( ! poly*rome blood /a9s 7see page #ig74 #h4 ,exam9e microscopically4 b4 ,! bact]iological exam9ation ( ! blood is direct$ solely to ! demon/ration ( ! pres5ce 9 ! circulat+ blood ( ! organisms previ\sly inject$ into ! animal4 ,= ?is purpose sev]al cubic c5timetres ( blood %\ld be tak5 9 an all-glass syr+e f an accessible ve9 correspond+ to "o ( ^? sugge/$ z ! site ( intrav5\s inoculation--& "u simil> aseptic precautions4 #a4 ,/]ilise an all-glass syr+e ( suitable size1 & :5 cool draw into ! syr+e "s /]ile sodium citrate solution & moi/5 ! :ole ( ! int]ior ( ! b>rel2 !n eject all ! citrate solution if less ?an #e c4c4 blood are to be )drawn2 if m ?an #e c4c4 are requir$ reta9 ab\t half a cubic c5timetre ( ! fluid 9 ! syr+e4 ,?is prev5ts coagulation ( ! blood4 ,! sodium citrate solution is prep>$ by dissolv+3 ,sodium citrate #aj gramme4 ,sodium *loride #j4ge grammes4 ,9 di/ill$ wat] #ajj c4c4 ,/]ilise by boil+4 #b4 ,prep>e ! animal z = intrav5\s inoculation 7see page #cfc7 & introduce ! syr+e ne$le into ! lum5 ( ! select$ ve94 #c4 ,sl[ly )draw ! pi/on ( ! syr+e4 ,:5 suffici5t blood has be5 collect$ direct ! assi/ant to release ! proximal compression ( ! ve92 & )draw ! ne$le4 #d4 ,remove ! ne$le f ! nozzle ( ! syr+e & deliv] ! citrat$ blood into a small ,ehl5mey] flask conta9+ ab\t #bej c4c4 ( nutri5t bro?4 #e4 ,label1 incubate & exam9e daily until gr[? occurs or until ! expiration ( t5 days4 c4 ,! s]ological exam9ation ( ! blood is direct$ to ! demon/ration ( ! pres5ce ( c]ta9 specific antibodies 9 ! s]a ( exp]im5tally infect$ animals1 & )9 c]ta9 limits to an e/imation ( _! am\nts4 ,! *ief ( ^! bodies are3 ,antitox94 ,agglut994 ,precipit94 ,opson94 ,immune body or ,bact]iolys94 ,none ( ^! sub/ances are capable ( isolation 9 a /ate ( purity ap>t f ! blood s]um1 consequ5tly special me?ods h be5 elaborat$ to p]mit ( _! recognition4 ,9 e in/ance ! behavi\r ( s]um f ! exp]im5tal animal1 :i* may be t]m$ specific s]um1 is /udi$ 9 comp>ison ) t ( s]um f an un9oculat$ animal ( ! same species1 & :i* is t]m$ normal s]um4 ,9 view ( m9or diff]5ces 9 con/itution exhibit$ by ! s]um ( v>i\s individuals ( ! same s]ies1 x is usual to employ a mixture ( s]a obta9$ f sev]al diff]5t normal animals ( ! same species z ! inoculat$ animal1 "u ! t]m pool$ s]um4 ,! me?od ( collect+ blood 7e4 g41 f ! rabbit7 = s]ological te/s is z foll[s3 ,collection ( ,s]um4 ,app>atus requir$3 ,razor4 ,liquid soap4 ,cotton-wool4 ,lysol #b p] c5t4 solution1 9 drop bottle4 ,e!r 9 drop bottle4 ,flat ,hag$orn ne$les4 ,blood pipettes 7,fig4 #af1 page #ab74 ,c5trifugal ma*9e4 ,c5trifuge tubes4 ,glass cutt+ knife4 ,buns5 flame4 ,writ+ diamond or grease p5cil4 ,,me?od4 #a4 ,%ave ! dorsal surface ( ! e> ov] ! c\rse ( ! po/]ior auricul> ve9 7see ,fig4 #aib74 #b4 ,/]ilise ! sk9 by wa%+ ) lysol4 ,! lysol %\ld be appli$ ) /]ile cotton-wool & ! e> vig\r\sly rubb$1 n only to remove sup]ficial scales ( epi!lium1 b also to r5d] ! e> hyp]aemic & ! ve9 prom95t4 #c4 ,remove ! lysol ) e!r dropp$ f a drop bottle1 & all[ ! e!r to evaporate4 #d4 ,puncture ! ve9 ) a /]ile ,hag$orn ne$le4 #e4 ,take a small blood-collect+ pipette 7,fig4 #afa7 & hold x at an angle to ! e>1 "o end t\*+ ! issu+ drop ( blood1 ! o!r depress$4 ,! blood w n[ ent] ! pipette at fir/ by capill>ity2 aft]w>d gravity w also come into play & ! pipette c be two-?irds fill$ )\t difficulty4 #f4 ,hold ! tube by ! end conta9+ ! blood1 ! clean end po9t+ obliquely upw>d--w>m ?is end at ! buns5 flame to expel "s ( ! conta9$ air2 !n seal ! clean po9t 9 ! flame4 ,illu/ration3 ,,fig4 #aib4--;,collect+ blood f rabbit4 #g4 ,place ! pipette d[n on a cool surface 7e4 g41 a glass slide74 ,! rapid cool+ ( ! air 9 ! clean end ( ! pipette creates a negative pressure1 & ! blood is suck$ back into ! pipette1 leav+ ! soil$ end free f blood4 ,seal ?is end 9 ! buns5 flame4 #h4 ,m>k ! di/9ctive title ( ! specim5 7e4 g41 animal's numb]7 ^u ! pipette ) a writ+ diamond or grease p5cil4 #i4 ,:5 ! seal$ ends are cold & ! blood has clott$1 place ! pipette on ! c5trifuge1 clean end d[nw>d2 c\nt]poise & c5trifugalise ?or\ghly4 ,on remov+ ! pipette f ! c5trifuge1 ! r$ cells w be collect$ 9 a firm mass at "o end1 & above !m w appe> ! cle> s]um4 #aj4 ,by m>k+ ! blood pipette above ! level ( ! s]um ) ! glass cutt+ knife & snapp+ ! tube at t po9t1 ! blood-s]um becomes readily accessible = te/+ purposes4 ,if l>g] quantities ( blood are requir$1 ! animal1 aft] punctur+ ! ve91 %\ld be inv]t$1 an assi/ant hold+ x up by ! legs4 ,blood to ! volume ( sev]al cubic c5timetres w n[ drop f ! punctur$ ve91 & %\ld be caught 9 a tap]+ c5trifuge tube1 ! tube transf]r$ to ! incubator at #cg deg4 ,c4 = two h\rs1 !n plac$ 9 ! c5trifugal ma*9e1 c\nt]pois$ & c5trifugalis$ ?or\ghly4 ,! ?ree mo/ important ( ! antibodies ref]r$ to :i* c be demon/rat$ ) a c]ta9 am\nt ( facility are agglut991 opson9 & bact]iolys92 & ! me?ods ( te/+ = ^! bodies w n[ be consid]$4 ,,agglut994 ,agglut99 is ! "n giv5 to a sub/ance pres5t 9 ! blood-s]um ( an animal t has successfully resi/$ inoculation ) a c]ta9 micro-organism4 ,?is sub/ance possesses ! p[] ( collect+ toge!r 9 clumps & masses1 or agglut9at+ wat]y susp5sions ( t p>ticul> microbe4 ,dilution ( ! ,specific ,s]um3 ,app>atus requir$3 ,/]ile graduat$ capill>y pipettes to conta9 #aj c4 mm4 7,fig4 #ag74 ,/]ile graduat$ capill>y pipettes to conta9 #ij c4 mm4 7,fig4 #ag74 ,small /]ile te/-tubes #e x #j4e cm4 ,normal sal9e solution 9 flask or te/-tube4 ,pipette ( specific s]um4 ,glass cutt+ knife1 or ?ree-squ>e file4 ,glass capsule1 ne>ly full ( dry silv] s&1 or roll ( pla/ic9e4 ,grease p5cil4 ,,me?od4-- #a4 ,take ?ree /]ile te/-tubes & numb] !m #a1 #b & #c4 #b4 ,pipette #j4i c4c4 /]ile normal sal9e solution into ea* tube1 & /& tubes upright 9 ! s& 9 ! capsule1 or 9 ! pla/ic9e block4 #c4 ,make a scrat* ) ! glass cutt+ knife on ! blood pipette above ! upp] level ( ! cle> s]um1 & snap (f & disc>d ! empty portion ( ! tube4 #d4 ,remove #j4a c4c4 ( ! s]um f ! blood pipette tube1 & mix x ?or\ghly ) ! fluid 9 tube ,no4 #a2 & label s4s41 7specific s]um71 #aj p] c5t4 #e4 ,remove #j4a c4c4 ( ! solution f tube ,no4 #a by means ( a fre% pipette1 & mix x ) ! cont5ts ( tube ,no4 #b2 & label s4s41 #a p] c5t4 #f4 ,remove #j4a c4c4 ( ! solution f tube ,no4 #b by means ( a fre% pipette1 & mix x ) ! cont5ts ( tube ,no4 #c2 & label s4s41 #j4a p] c5t4 ,:5 ! yield ( s]um f ! specim5 ( blood :i* has be5 collect$1 or is available1 is small1 ! above me?od ( dilut+ is n practicable1 & ! dilution %\ld be c>ri$ out by ,wright's me?od 9 a capill>y teat pipette4 ,dilution ( ,s]um by ,means ( a ,teat ,pipette4 ,mat]ials requir$3 ,blood pipette conta9+ sample ( specific s]um aft] c5trifugalisation4 ,capsule ( dilut+ fluid--normal sal9e solution4 ,supply ( ,pa/eur pipettes 7,fig4 #ac;a74 ,india-rubb] teats4 ,small te/-tubes4 ,a block ( pla/ic9e to act z a te/-tube /&4 ,grease p5cil4 ,,me?od3 #a4 ,m>k ?ree small te/-tubes #aj p] c5t41 #a p] c5t4 & #j4a p] c5t4 respectively1 & /& !m upright 9 ! pla/ic9e block4 #b4 ,take a ,pa/eur pipette1 nick ! capill>y /em j above ! seal$ end ) a glass cutt+ knife1 & snap (f ! seal$ end ) a quick movem5t s t ! fracture is clean cut & at "r angles to ! long axis ( ! capill>y /em--cut squ>e1 9 fact4 ,prep>e sev]al1 say a doz51 9 ?is mann]4 #c4 ,fit a rubb] teat to ! b>rel ( ea* ( ! pipettes4 #d4 ,make a m>k ) ! grease p5cil on ! /em ( "o ( ! pipettes ab\t #b or #c cm4 f ! op5 extremity4 ,illu/ration3 ,,fig4 #aic4--;,fill+ ! capill>y teat pipette4 #e4 ,compress ! teat betwe5 ! f+] & ?umb 7,fig4 #aic7 to su* an ext5t z to drive out ! great] "p ( ! conta9$ air4 #f4 ,ma9ta9+ ! pressure on ! teat pass ! /em ( ! pipette into ! capsule hold+ ! sal9e solution1 until ! op5 end ( ! pipette is bel[ ! level ( ! fluid4 #g4 ,n[ cauti\sly relax ! pressure on ! teat & let ! fluid ent] ! pipette & rise 9 ! /em until x rea*es ! level ( ! grease p5cil m>k4 ,z soon z ?is po9t is rea*$1 *eck ! movem5t ( ! column ( fluid by ma9ta9+ ! pressure on ! teat1 nei!r relax+ nor increas+ x4 #h4 ,)draw ! po9t ( ! pipette cle> ( ! fluid1 & aga9 relax ! pressure on ! teat v slightly4 ,! column ( sal9e solution rises high] 9 ! /em1 & a column ( air w n[ ent] ! pipette & s]ve z an index to sep>ate ! fir/ volume ( fluid drawn into ! /em f ! next succe$+ "o4 #i4 ,aga9 introduce ! end ( ! pipette into ! fluid & draw up a second volume ( sal9e to ! level ( ! grease p5cil m>k1 & foll[ ?is ) a second air index4 #aj4 ,9 l mann] take up sev5 m equal volumes ( sal9e solution & _! foll[+ air bubbles4 ,!re are n[ n9e equal volumes ( normal sal9e 9 ! pipette4 #aa4 ,n[ pass ! po9t ( ! pipette into ! blood tube & dip ! op5 end bel[ ! surface ( ! s]um4 ,proce$+ z be=e1 aspirate a volume ( s]um into ! capill>y /em up to ! level ( ! p5cil m>k4 #ab4 ,eject ! cont5ts ( ! pipette into ! small tube m>k$ #aj p] c5t4 by compress+ ! rubb] teat betwe5 ?umb & f+]4 #ac4 ,mix ! "o volume ( s]um ) ! n9e volumes ( sal9e solution v ?or\ghly by repeat$ly draw+ up ! :ole ( ! fluid into ! pipette & driv+ x out aga9 into ! te/-tube4 #ad4 ,n[ take a clean pipette & proce$ precisely z be=e1 #d to #aj4 #ae4 ,hav+ aspirat$ n9e equal volumes ( sal9e into ?is second pipette1 n[ take up "o simil> volume ( ! fluid 9 ! #aj p] c5t4 tube4 #af4 ,eject ! cont5ts ( ?is pipette into ! second tube m>k$ #a p] c5t4 & mix ?or\ghly z be=e4 #ag4 ,9 simil> fa%ion make ! #j4a p] c5t4 solution & transf] to ! ?ird tube4 #ah4 ,fur!r dilutions 9 multiples ( t5 c be prep>$ 9 ! same way1 & by v>y+ ! numb] ( volumes ( dilut+ fluid or s]um any requir$ dilution c be made 7see ,app5dix1 ,dilution ,tables74 ,,note4--,! sal9e dilut+ fluid mu/ always be tak5 into ! pipette fir/1 o!rwise if ! s]um conta9s a v l>ge am\nt ( agglut99 ! traces ( ?is s]um add$ to ! sal9e solution may be suffici5t to entirely vitiate ! subsequ5t obs]vations--:il/ if m ?an "o sample ( s]um is dilut$ f ! same sal9e solution s]i\s errors may be introduc$ into ! exp]im5ts4 ,! ,microscopical ,reaction3 ,app>atus ,requir$3 ,five hang+-drop slides 7or pref]ably two slide71 ) two cells m\nt$ side by side on ea* 7,fig4 #fb1 a71 & "o slide ) "o cell only4 ,vasel9e4 ,cov]-slips4 ,plat9um loop4 ,grease p5cil4 ,eighte5 to tw5ty-f\r-h\r-old b\illon cultivation ( ! organism to be te/$ 7e4 g41 ,bacillus typhi abdom9alis7 ,pipette end ) ! rema9d] ( ! specific s]um labell$ s4s4 ,tubes conta9+ ! ?ree solutions ( ! specific s]um1 #aj1 #a1 & #j4a p] c5t4 respectively4 ,pipette end ) pool$ normal s]um labell$ p4s4 ,,me?od4-- #a4 ,make five hang+-drop prep>ations1 ?us3 7a7 ,"o loopful ( b\illon cultivation "o loopful pool$ s]um2 label ,control4 7b7 ,"o loopful culture "o loopful undilut$ specific s]um2 label #ej p] c5t4 ,m\nt ^! two cov]-slips on a d\ble-cell$ slide4 7c7 ,"o loopful b\illon culture "o loopful #aj p] c5t4 s]um2 label #e p] c5t4 ,m\nt ?is on s+le-cell slide4 7d7 ,"o loopful b\illon culture "o loopful #a p] c5t4 s]um2 label #j4e p] c5t4 7e7 ,"o loopful b\illon culture "o loopful #j4a p] c5t4 s]um2 label #j4je p] c5t4 ,m\nt ^! two cov]-slips on a d\ble-cell$ slide4 #b4 ,note ! "t3 ,exam9e ! control to det]m9e t ! bacilli are motile & uni=mly scatt]$ ov] ! field--n collect$ into masses4 #c4 ,next exam9e ! #ej p] c5t4 s]um prep>ation4 ,if agglut99 is pres5t & ! te/ is giv+ a positive reaction1 ! bacilli w be collect$ 9 l>ge clumps4 ,if ! te/ is giv+ a negative reaction1 ! bacilli may be collect$ 9 l>ge clumps ow+ to ! viscosity ( ! conc5trat$ s]um4 #d4 ,obs]ve ! #e p] c5t4 prep>ation microscopically4 ,if ! bacilli are aggregat$ into clumps1 positive reaction4 ,if ! bacilli are n aggregat$ into clumps1 obs]ve until ?irty m9utes f ! "t ( prep>ation be=e record+ a negative reaction4 #e4 ,exam9e ! #j4e & #j4je p] c5t4 prep>ations4 ,^! may or may n %[ agglut9ation :5 ! result ( ! exam9ation ( ! #e p] c5t4 prep>ation is positive1 accord+ to ! pot5cy ( ! specific s]um2 & by ! exam9ation ( a s]ies ( dilutions a quantitative comp>ison ( ! val5cy ( specific s]a f diff]5t s\rces1 or ( s]um f ! same animal at diff]5t p]iods dur+ ! c\rse ( active immunisation may be obta9$4 ,,note4--,! graduat$ pipettes suppli$ ) ,?oma's haematocytomet] 7int5d$ = ! collection ( ! specim5 ( blood requir$ = ! enum]ation ( leucocytes71 giv+ a dilution ( #a 9 #aj--;i4 e41 #aj p] c5t4--may be sub/itut$ = ! graduat$ capill>y pipettes ref]r$ to above1 if ! vessel 9 :i* ! s]um has be5 sep>at$ is ( suffici5tly l>ge diamet] to p]mit ( _! use4 ,! ,macroscopical ,reaction3 ,/]ile graduat$ capill>y pipettes to conta9 #ij c4 mm4 ,eighte5 to tw5ty-f\r-h\rs-old b\illon cultivation ( ! organism to be te/$4 ,?ree te/-tubes conta9+ ! #aj1 #a1 & #j4a p] c5t4 solutions ( specific s]um 7ab\t #ij c4 mm4 rema9+ 9 ea*74 ,tube conta9+ #ej p] c5t4 solution ( pool$ s]um4 ,s$im5tation pipettes 7vide page #ag7 or teat pipettes4 ,,me?od4 #a4 ,pipette #ij c4 mm4 ( ! b\illon culture into ea* ( ! tubes conta9+ ! dilut$ s]um2 & ! same quantity into ! tube conta9+ ! pool$ s]um4 #b4 ,fill a s$im5tation tube 7by aspirat+7 or a teat pipette f ! cont5ts ( ea* tube4 ,seal (f ! l[] ends ( ! s$im5tation tubes 9 ! ,buns5 flame4 #c4 ,label ea* tube ) ! dilution ( s]um t x conta9s--viz41 #e1 #j4e1 & #j4je p] c5t4 #d4 ,place ! pipettes 9 a v]tical position1 9 a beak]1 9 ! incubator at #cg deg4 ,c41 = "o or two h\rs4 #e4 ,obs]ve ! granul> precipitate :i* is ?r[n d[n :5 ! reaction is positive1 & ! uni=m turbidity ( ! negative reaction z comp>$ ) ! appe>ances 9 ! control pool$ s]um4 ,,opson94 ,opson9 is ! t]m appli$ by ,wright to a sub/ance1 pres5t 9 ! s]um ( an inoculat$ animal1 :i* is able to act ^u or s5sitise bact]ia ( ! species orig9ally inject$1 s z to r5d] !m an easy prey to ! phagocytic activity ( polymorphonucle> leucocytes4 ,9 ! me?od = demon/rat+ opson9 ab\t to be describ$1 a comp>ison is made betwe5 ! opsonic p[] ( ! pool$ s]um & ! specific s]um4 ,app>atus3 ,small c5trifuge & tubes = same 7made f ! b>rels ( brok5 capill>y pipettes by seal+ ! conical ends 9 ! buns5 flame74 ,capill>y ,pa/eur pipettes4 ,india-rubb] teats4 ,grease p5cil4 ,buns5 burn] ) peep flame4 ,electrical signal clock 7see page #ci7 /op wat*1 or wat*4 ,rectangul> glass box or tray to hold pipettes4 ,incubator regulat$ at #cg deg4 ,c4 #c x #a slides4 ,piece ( light rubb] tub+4 ,rectangul> block ( pla/ic9e4 ,flask ( normal sal9e solution4 ,flask ( sodium citrate 7#a4e p] c5t47 9 normal sal9e solution4 ,mat]ials requir$1 & _! prep>ation3 ,small tube ( wa%$ cells 7r$ blood discs & leucocytes72 human cells are us$ 9 e/imat+ ! opsonis+ p[] ( ! s]um ( exp]im5tal animals4 ,small tube ( emulsion ( bact]ia ( ! species responsible = ! infection ( ! exp]im5tal animal4 ,blood pipette conta9+ specific s]um4 ,blood pipette conta9+ pool$ s]um4 ,wa%$ ,cells4-- #a4 ,take a small c5trifuge tube & half fill x ) sodium citrate solution4 ,m>k ) ! grease p5cil ! upp] limit ( ! fluid4 #b4 ,cleanse ! sk9 ( ! di/al phalanx ( ! second f+] ( ! left h& above ! root ( ! nail ) l9t & e!r4 ,w9d ! rubb] tub+ tightly r\nd ! second phalanx2 puncture ) a /]ile ,hag$orn ne$le ?r\gh ! cleans$ area ( sk94 #c4 ,take up a suffici5cy ( ! issu+ blood 7m or less accord+ to ! numb] ( te/s to be p]=m$7 ) a teat pipette1 transf] x to ! tube ( citrate solution & mix ?or\ghly4 ,make a second m>k on ! tube at ! upp] level ( ! mix$ citrate solution & blood4 #d4 ,place ! tube 9 ! c5trifuge1 c\nt]poise accurately & c5trifugalise until ! blood cells are ?r[n d[n 9 a compact mass occupy+ approximately ! same volume z is includ$ betwe5 ! two p5cil m>ks4 ,! column ( fluid 9 ! tube n[ %[s cle> sup]natant fluid 7citrate solution & blood plasma7 sep>at$ f ! %>p cut upp] surface ( ! r$ deposit ( corpuscles by a n>r[ greyi% lay] ( leucocytes4 #e4 ,remove ! sup]natant column ( citrate solution by means ( a teat pipette1 fill normal sal9e solution into ! tube up to ! upp] p5cil m>k1 & di/ribute ! blood cells ?r\gh\t ! sal9e by means ( ! teat pipette4 ,c5trifugalise z be=e4 #f4 ,aga9 remove ! sup]natant fluid & fill 9 a fre% supply ( sal9e solution & c5trifugalise once m4 #g4 ,remove ! sup]natant sal9e solution z ne>ly d[n to ! level ( ! leucocytes z c be safely done )\t remov+ any ( ! leucocytes4 #h4 ,next di/ribute ! leucocytes ev5ly ?r\gh\t ! mass ( r$ cells by rotat+ ! tube betwe5 ! palms ( ! h&s--j z is done ) a tube ( liquefi$ m$ium prior to p\r+ a plate4 #i4 ,set ! tube upright 9 ! pla/ic9e block ne> to "o end4 ,bact]ial ,emulsion4-- #a4 ,take an #ah- to #bd-;h\r culture ( ! requir$ bact]ium 7e4 g41 ,diplococcus pneumoniae7 gr[n ^u slop$ blood ag> at #cg deg4 ,c4 ,p\r ov] ! surface ( ! m$ium "s #e c4c4 ( normal sal9e solution4 #b4 ,) a plat9um loop emulsify ! gr[? f ! surface ( ! m$ium z ev5ly z possible 9 ! sal9e solution4 #c4 ,all[ ! tube to /& = a few m9utes s t ! l>ge masses ( gr[? may settle d[n2 transf] ! upp] portion ( ! sal9e susp5sion to a c5trifuge tube & c5trifugalise ?or\ghly4 #d4 ,exam9e a drop ( ! sup]natant opalesc5t emulsion microscopically to det]m9e its fre$om f clumps & masses4 ,if unsatisfactory prep>e ano!r emulsion1 ?is "t scrap+ up ! surface gr[? ) a plat9um spatula1 transf]r+ x to an agate mort> & gr9d+ x up ) successive small quantities ( normal sal9e4 ,if satisfactory ins]t ! tube 9 ! pla/ic9e block next to t conta9+ ! wa%$ cells4 ,specific ,s]um4-- ,pool$ ,s]um4-- ,^! s]a are collect$ & treat$ z already describ$ 7see page #cgi71 & ! portions ( ! blood pipettes conta9+ !m are arrang$ 9 ! rema9+ space 9 pla/ic9e block4 ,illu/ration3 ,,fig4 #aid4--,pla/ic9e block ) mat]ials arrang$ = opson9 e/imations4 ,! pla/ic9e block n[ pres5ts ! appe>ances %[n 9 ,fig4 #aid4 ,,me?od ,,= ,,det]m9+ ,,! ,,opsonic ,,index4-- #a4 ,take a capill>y pipette fitt$ ) a teat1 cut ! di/al end squ>e & make a p5cil m>k ab\t #b cm4 f ! end4 #b4 ,aspirate into ! pipette "o volume ( wa%$ cells1 air index1 "o volume ( bact]ial emulsion1 air index1 & "o volume ( specific s]um 7see ,fig4 #aie74 ,illu/ration3 ,,fig4 #aie4 ,opson9 pipette4 #c4 ,mix ?or\ghly on a #c by #a slide by compress+ ! teat & eject+ ! cont5ts ( ! pipette on to ! surface ( ! slide1 relax+ ! pressure & s draw+ ! fluid up into ! pipette aga94 ,^! two processes %\ld be repeat$ sev]al times2 f9ally take up ! mixture 9 an unbrok5 column to ! c5tral portion ( ! capill>y /em4 #d4 ,seal ! po9t ( ! pipette 9 ! peep flame ( ! buns5 burn] & remove teat4 #e4 ,m>k ! pipette 7) ! grease p5cil7 ) ! di/9ctive numb] ( ! s]um & place x 9 ! glass box or tray4 #f4 ,take ano!r simil>ly prep>$ pipette & aspirate into x equal volumes ( wa%$ cells1 bact]ial emulsion & pool$ s]um4 ,treat precisely z 9 #c & #d1 label x control or ,n4,s4 7normal s]um7 & place 9 ! box by ! side ( ! specific s]um prep>ation4 #g4 ,place ! box ) ! pipettes 9 ! incubator & set ! signal clock to r+ at #ae m9utes 7or />t ! /op wat*74 #h4 ,at ! expiration ( ! incubation "t remove ! pipettes f ! incubator4 #i4 ,cut (f ! seal$ end ( ! specific s]um prep>ation4 ,mix its cont5ts ?or\ghly z 9 /ep #c1 & !n divide ! mixture betwe5 two #c by #a slips & c>efully spread a blood film 7vide page #cgf7 on ea* 9 su* a way t only "o-half ( ! surface ( ea* slide is cov]$ ) blood--! free edge ( ! blood film approximat+ to ! longitud9al axis ( ! slide4 ,all[ films to dry & label ! slides ) writ+ diamond4 #aj4 ,treat ! cont5ts ( ! control pipette 9 simil> fa%ion4 #aa4 ,select ! bett] film f ea* pair = fix+ & /a9+4 #ab4 ,fix+ & /a9+ mu/ be c>ri$ out "u /rictly comp>able conditions1 & to ?is end ! slides are be/ h&l$ by plac+ 9 a glass /a9+ rack :i* c be l[]$ 9 turn into ea* ( a s]ies ( glass tr\ghs conta9+ ! v>i\s reag5ts 7,fig4 #aif74 ,place ! rack 9 ! fir/ tr\gh :i* conta9s ! alcoholic solution ( ,lei%man's /a9 = two m9utes to fix4 ,transf] to ! second tr\gh conta9+ ! dilut$ /a9 = t5 m9utes4 ,transf] to ! ?ird tr\gh conta9+ di/ill$ wat]1 & hold+ ! tr\gh ov] a s9k1 run 9 a /ream ( di/ill$ wat] until wa%+ is complete4 ,remove slides f ! rack & dry4 ,lei%man's /a9 is ! be/ = r\t9e "w = all bact]ia o!r ?an ,b4 tub]culosis4 ,films conta9+ tub]cle bacilli mu/ ( c\rse be /a9$ by ! ,ziehl ,neels5 me?od4 ,illu/ration3 ,,fig4 #aif4 ,glass /a9+ tr\gh = blood films4 #ac4 ,exam9e specific s]um slide microscopically ) #a_/ab in* oil imm]sion4 ,f9d ! edge ( ! blood film--along ?is ! bulk ( ! leucocytes w be collect$4 ,/>t+ at "o end ( ! film move ! slide sl[ly across ! microscope /age & z ea* leucocyte comes into view c\nt & record ! numb] ( +e/$ bact]ia4 ,! sum ( ! cont5ts ( ! fir/ #ej consecutive polymorphonucle>s t are enc\nt]$ is m>k$ d[n4 7,! av]age numb] ( bacilli +e/$ p] leucocyte ! phagocytic index47 #ad4 ,9 precisely simil> mann] enum]ate ! bact]ia pres5t 9 ! fir/ #ej cells ( ! control prep>ation4 ,?is numb] is record$ z ! d5om9ator ( a vulg> fraction ( :i* ! num]ator is ! numb] record$ = ! specific s]um4 ,?is fraction1 express$ z a p]c5tage ( unity ! opsonic index4 ,,immune ,,body4 ,immune body or amboceptor is ! "n giv5 to a sub/ance pres5t 9 ! s]um ( an infect$ animal t has successfully resi/$ inoculation ) "s p>ticul> micro-organism1 & :i* possesses ! p[] ( l9k+ ! complem5t normally pres5t 9 ! s]um to bact]ia ( ! species us$ z antig5 9 su* a mann] t ! micro-organisms are r5d]$ innocu\s1 & ultimately de/roy$4 ,! pres5ce ( ! immune body 9 ! s]um c be demon/rat$ 9 vitro by ! reaction elaborat$ by ,bordet & ,g5g\1 kn[n z ! complem5t fixation te/1 ! exi/5ce or ! abs5ce ( ! ph5om5on ( complem5t fixation be+ r5d]$ obvi\s macroscopically by ! abs5ce or pres5ce ( haemolysis on ! subsequ5t addition ( s5sitis$ r$ blood corpuscles1 7e4 g41 a mixture ( cry?rocyte solution & ! appropriate haemolys9--two ( ! ?ree ess5tials 9 ! haemolytic sy/em1 vide page #cbf74 ,app>atus ,requir$3 ,/]ile pipettes #a c4c41 7graduat$ 9 t5?s74 #af x #b cm4 te/-tubes4 #i x #a cm4 te/-tubes4 ,te/-tube racks = ea* size ( te/-tube4 ,reag5ts ,requir$3 ,normal sal9e solution4 ,ery?rocyte solution 7human r$ cells1 page #cbi7 ,e4 ,haemolytic s]um 7= human cells7 ,h4,s4 ,complem5t 7fre% gu9ea-pig s]um7 ,c4 ,specific s]um f inoculat$ animal1 inactivat$ ,s4,s4 ,control pool$ s]um f normal animals ( same species1 ,inactivat$ ,p4,s4 ,antig5 7cultivation ^u solid m$ium ( ! organism 7e4 g41 ,b4 typhosus7 :i* has already s]v$ z antig5 9 ! inoculation ( ! exp]im5tal animal7 ,a4 ,to prep>e ! antig5 = use1 emulsify ! :ole ( ! bact]ial gr[? 9 #e c4c4 normal sal9e solution4 ,%ake ! emulsion 9 a te/-tube ) "s /]ilis$ glass beads to ensure a homog5\s emulsion1 & /]ilise by heat+ to #fj deg4 ,c4 9 a wat]-ba? = "o h\r4 ,,me?od4-- #a4 ,take five small te/-tubes1 & numb] !m #a to #e ) a grease p5cil4 #b4 ,into tubes ,nos4 #a1 #c1 #d & #e pipette #j4a c4c4 ( complem5t4 #c4 ,into tubes ,nos4 #a & #b pipette #j4b c4c4 ( ! s]um to be te/$4 #d4 ,into tube ,no4 #d pipette #j4b c4c4 ( control s]um4 #e4 ,into tubes ,nos4 #a1 #b1 #c & #d pipette #a c4c4 ( ! bact]ial emulsion :i* =ms ! antig54 #f4 ,place ! :ole set ( tubes 9 ! incubator at #cg deg4 ,c4 = a p]iod ( "o h\r4 #g4 ,remove ! tubes f ! incubator & pipette #a c4c4 ery?rocyte solution & #d m9imal haemolytic doses ( ! correspond+ haemolys9 into ea* tube4 #h4 ,mix ?or\ghly & return ! tubes to ! incubator at #cg deg4 ,c4 = fur!r p]iod ( "o h\r4 #i4 ,at ! expiration ( t "t transf] ! tubes to ! ice *e/1 & all[ !m to /& = ?ree h\rs4 #aj4 ,exam9e ! tubes4 ,tubes #c1 #d & #e %\ld %[ complete haemolysis2 tube #b %\ld give no evid5ce :atev] ( haemolysis4 ,^! tubes =m ! controls to ! fir/ tube1 :i* conta9s ! s]um to be te/$4 ,9 tube ,no4 #a ! abs5ce ( haemolysis w\ld indicate ! pres5ce 9 ! s]um ( ! inoculat$ animal ( a specific antibody to ! micro-organism us$ 9 ! inoculations2 s9ce x %[s t ! complem5t has be5 b\nd by ! immune body to ! bact]ial antig51 & none has be5 left free to ent] into ! haemolytic sy/em2 on ! o!r h& ! pres5ce ( haemolysis w\ld %[ t no appreciable am\nt ( antibody has yet be5 =m$ 9 response to ! inoculations4 ,9 o!r words1 !re is an abs5ce ( infection1 s9ce ! complem5t rema9$ unfix$ at ! "t ( ! addition ( ! ery?rocyte solution & haemolytic s]um1 & 0 ready to comb9e ) ^? reag5ts to complete ! haemolytic sy/em4 ,! me?od may be %[n diagramatically z "u us+ ! symbols already indicat$ ,te/-tubes4 #a #b #c #d #e #j4a c4c4 ,c4 44444444 #j4a c4c4 ,c4 #j4a c4c4 ,c4 #j4a c4c4 ,c4 #j4b c4c4 ,s4,s4 #j4b c4c4 ,s4,s4 444444444 #j4b c4c4 ,p4,s4 44444444 ,a4 ,a4 ,a4 ,a4 44444444 ---------------------------------------- ---------------------------------- ,incubate at #cg deg4 ,c4 = "o h\r4 ---------------------------------------- ---------------------------------- #a c4c4 ,e4 #a4 c4c4 ,e4 #a c4c4 ,e4 #a c4c4 ,e4 #a c4c4 ,e4 ,h4,s4#d ,h4,s4#d ,h4,s4#d ,h4,s4#d ,h4,s4#d ---------------------------------------- ---------------------------------- ,incubate at #cg deg4 ,c4 = "o h\r4 ---------------------------------------- ---------------------------------- 787 ,no haemolysis4 ,haemolysis4 ,,note4--,x is sometimes m conv5i5t to s5sitise ! ery?rocytes j be=e !y are ne$$4 ,?is is done =ty-five m9utes aft] ! exp]im5t has be5 />t$ 7page #cid1 /ep #f71 t is to say1 be=e ! completion ( ! fir/ p]iod ( incubation1 ?us3 #a4 ,measure out into a /]ile te/-tube 7or flask7 five c4c4 ( ery?rocyte solution4 #b4 ,measure out tw5ty m9imal haemolytic doses ( haemolys91 add to ! ery?rocyte solution on ! te/-tube4 #c4 ,all[ ! ery?rocyte & haemolys9 to rema9 9 contact = fifte5 m9utes at room temp]ature4 ,! r$ cells are !n s5sitis$ & ready = use4 #d4 ,:5 ! tubes are remov$ f ! incubator at ! end ( ! fir/ h\r 7i4 e41 /ep #g7 add #a c4c4 s5sitis$ r$ cells to ea* tube by means ( a graduat$ pipette4 #e4 ,mix ?or\ghly1 return ! tubes to ! incubator at #cg deg4 ,c4 & complete ! exp]im5t z previ\sly describ$ 7/eps #h onw>d74 ,,xix4 ,,po/-,,mortem ,,exam9ations ,,( ,,exp]im5tal ,,animals4 ,! po/-mortem exam9ation %\ld be c>ri$ out z soon z possible aft] ! dea? ( ! animal1 = x mu/ be rememb]$ t ev5 9 cold wea!r ! tissues are rapidly invad$ by num]\s bact]ia d]iv$ f ! alim5t>y tract or ! cavities ( ! body1 & f ext]nal s\rces4 ,! foll[+ outl9es ref] to a complete & exhau/ive necropsy1 & 9 r\t9e "w ! exam9ation w r>ely ne$ to be c>ri$ out 9 its entirety4 ,,note4--,?r\gh\t ! autopsy ! se>+ irons mu/ be freely employ$1 & x mu/ be recollect$ t "o in/rum5t is only to be employ$ to seize or cut "o /ructure4 ,?is done1 x mu/ be reg>d$ z contam9at$ & a fre% in/rum5t tak5 = ! next /ep4 ,app>atus ,requir$3 ,wat] /]ilis]4 ,scalpels4 ,surgical in/rum5ts3 ,scissors4 ,=ceps4 ,bone =ceps4 ,spe>-head$ plat9um spatula 7,fig4 #aii74 ,se>+ irons 7,fig4 #aih74 ,tubes ( m$ia--b\illon & slop$ ag>4 ,surface plates 9 petri di%es 7( ag> or "o ( its d]ivatives74 ,plat9um loop4 ,alum9ium spread]4 ,grease p5cil4 ,/]ile capill>y pipettes 7,fig4 #ac1 a74 ,/]ile glass capsules1 l>ge & small4 ,cov]-slips or slides4 ,bottles ( fix+ fluid 7vide page #aad7 = pieces ( tissue int5d$ = section+4 #a4 ,place ! v>i\s in/rum5ts1 =ceps1 scissors1 scalpels1 etc41 ne$$ = ! autopsy inside ! /]ilis] & /]ilise by boil+ = t5 m9utes2 !n op5 ! /]ilis]1 raise ! tray f ! int]ior & re/ x crosswise on ! edges4 #b4 ,heat ! se>+ irons to r$ness 9 a sep>ate gas /ove4 ,illu/ration3 ,,fig4 #aig4--;,app>atus = po/-mortem exam9ation1 animal on bo>d4 #c4 ,dr5* ! fur 7or fea!rs7 ) lysol solution1 #b p] c5t4 ,?is s]ves ! tw(old purpose ( prev5t+ ! hairs f fly+ ab\t & ent]+ ! body cavities dur+ ! autopsy1 & ( r5d]+ innocu\s any v]m9 t may be pres5t on ! animal4 ,illu/ration3 ,,fig4 #aih4--,se>+ iron4 #d4 ,exam9e ! cadav] c>efully4 ,recollect t laboratory animals are n always h>dy2 dea? may be due to exposure to heat or cold1 to />vation or ov]- or improp] fe$+ or to ! attack ( rats--& n to ! bact]ial infection4 #e4 ,fa/5 ! body ( ! animal1 v5tral surface upw>d 7unless !re is "s special reason = hav+ ! dorsum expos$71 out on a bo>d by means ( copp] nails driv5 ?r\gh ! extremities4 #f4 ,) /]ile =ceps & scalpel incise ! sk9 9 ! middle l9e f ! top ( ! /]num to ! pubes4 ,make o!r incisions at "r angles to ! fir/ out to ! axillae & gro9s1 & reflect ! sk9 9 two lat]al flaps4 7,place ! n[ infect$ in/rum5ts on ! bo>d by ! side ( ! body or support !m on a porcela9 knife re/47 ,seat ( ,inoculation4-- #g4 ,inspect ! seat ( inoculation4 ,if any local lesion is visible1 se> its expos$ surface & ) ! plat9um loop1 remove mat]ial f ! deep] p>ts to make tube & surface plate cultivations & cov]-slip prep>ations4 ,collect specim5s ( pus or o!r exudation 9 capill>y pipettes = subsequ5t exam9ation4 #h4 ,inspect ! neighb\r+ lymphatic gl&s & endeav\r to trace ! pa? ( ! virus4 #i4 ,se> ! :ole ( ! expos$ surface ( ! ?orax ) ! se>+ irons4 ,pleural ,cavity4-- #aj4 ,divide ! ribs on ei!r side ( ! /]num & remove a rectangul> portion ( ! ant]ior *e/ wall ) /]ile scissors & a fre% pair ( =ceps1 expos+ ! he>t4 ,place ! infect$ in/rum5ts by ! side ( ! fir/ set4 #aa4 ,obs]ve ! condition ( ! ant]ior m$ia/9al gl&s1 ! ?ymus & ! lungs4 ,collect a quantity ( pleuritic effusion1 if su* is pres5t1 9 a pipette = fur!r exam9ation lat]4 #ab4 ,raise ! p]ic>dial sac 9 a fre% pair ( =ceps & burn ?r\gh ?is /ructure ) a se>+ iron4 ,collect a sample ( p]ic>dial fluid 9 a pipette = microscopical & cultural exam9ation4 #ac4 ,grasp ! apex ( ! he>t 9 ! =ceps & se> ! surface ( ! "r v5tricle4 #ad4 ,plunge ! op5 po9t ( a capill>y pipette ?r\gh ! se>$ area into ! v5tricle & fill ) blood4 ,make cultivations & cov]-slip prep>ations ( ! he>t blood4 #ae4 ,collect a fur!r sample ( blood or s]um = subsequ5t inve/igation z to ! pres5ce ( antibodies4 ,p]itoneal ,cavity4-- #af4 ,se> a broad track 9 ! middle l9e ( ! abdom9al wall2 op5 ! p]itoneal cavity by an incision 9 ! c5tre ( ! se>$ l9e4 ,obs]ve ! condition ( ! om5tum1 ! mes5t]y1 ! visc]a & ! p]itoneal surface ( ! inte/9es4 #ag4 ,collect a specim5 ( ! p]itoneal fluid 7or pus1 if pres5t7 9 a capill>y pipette4 ,make cultivations1 tube & surface plate1 & cov]-slip prep>ations f ?is situation4 #ah4 ,collect a specim5 ( ! ur9e f ! di/5d$ bladd] 9 a l>ge pipette 79 ! mann] indicat$ = he>t blood71 = fur!r exam9ation1 by cultivations1 microscopical prep>ations1 & *emical analysis4 #ai4 ,collect a specim5 ( bile f ! gall bladd] 9 simil> mann]4 #bj4 ,excise ! sple5 & place x 9 a /]ile capsule4 ,lat]1 se> ! surface ( ?is organ2 plunge ! spe>-head$ spatula ?r\gh ! c5tre ( ! se>$ area1 twi/ x r\nd betwe5 ! f+] & ?umb1 & remove x f ! organ4 ,suffici5t mat]ial w be br\ght away 9 ! eye 9 its head to make cultivations4 ,a repetition ( ! process w af=d mat]ial = cov]-slip prep>ations4 #ba4 ,seize "o end ( ! sple5 ) /]ile =ceps4 ,se> a n>r[ b& ( tissue1 "r ar\nd ! organ & divide ! sple5 9 ?is situation ) a pair ( scissors4 ,hold+ ! piece ( sple5 9 ! =ceps1 dab ! cut surface on to a surface plate 9 a numb] ( diff]5t spots4 #bb4 ,9 l mann] exam9e ! o!r organs--liv]1 lungs1 kidneys1 lymphatic gl&s 7mes5t]ic1 hepatic1 lumb>1 etc71 etc4 ,prep>e cultivations & cov]-slip prep>ations4 #bc4 ,dissect out a long bone f "o upp] & "o l[] limb & "o ( ! l>ge/ ribs4 ,prep>e cultures f ! bone m>r[ 9 ea* case4 ,set aside ^! bones = ! subsequ5t prep>ation ( m>r[ films4 #bd4 ,film prep>ations ( bone m>r[ are be/ made by ! ,price-,jones me?od4 ,seize ! bone 9 a pair ( pli]s & squeeze out "s ( ! m>r[2 receive x 9 a plat9um loop1 & transf] to a wat* glass ( dissociat+ fluid & emulsify4 ,! dissociat+ fluid is a neutral #aj p] c5t4 solution ( glyc]9e prep>$ z foll[s3-- ,measure out #aj c4c4 ,price's be/ glyc]9e & #ij c4c4 /]ile ammonia-free di/ill$ wat]4 ,mix4 ,titrate aga9/ n_/#aj sodic hydrate solution us+ ph5olph?ale9 z ! indicator4 ,! initial reaction is usually #j4a to #j4e2 add ! calculat$ am\nt ( n_/#aj sodic hydrate solution to neutralise4 #be4 ,place a loopful ( fre% desiccat+ fluid on a #c x #a glass slide2 add a simil> loopful ( ! m>r[ emulsion1 & spread v g5tly ov] ! surface ( ! slip4 #bf4 ,all[ film to dry 9 ! air 7protect$ f du/7 )\t heat+4 #bg4 ,/a9 ) ,j5n]'s poly*rome /a9 7page #ig7 = two & a half m9utes4 #bh4 ,wa% ) ammonia-free di/ill$ wat]1 dry ?or\ghly & m\nt 9 xylol balsam4 ,cranial & ,sp9al ,cavities4-- #bi4 ,9 "s in/ances x may be necess>y 7e4 g41 exp]im5tal inoculation ( rabies7 to exam9e ! cranial cavity or to remove ! sp9al cord4 ,return ! visc]a to ! abdom9al cavity2 draw ! flaps ( sk9 toge!r & secure ) ,mi*el's /eel clips4 ,draw ! copp] nails secur+ ! limbs to ! bo>d1 rev]se ! animal & aga9 nail ! limbs d[n--! body n[ be+ dorsum upp]mo/4 #cj4 ,make a longitud9al incision 9 ! mesial l9e f sn\t to root ( tail1 & f\r transv]se incisions--"o jo9+ ! roots ( ! two e>s1 "o across ! body at ! level ( ! sp9is ( ! scapulae1 ano!r at ! level ( ! co/al m>g9 & ! la/ across ! upp] level ( ! pelvis4 ,reflect ^! flaps ( sk94 #ca4 ,) =ceps & scalpel dissect out ! muscles ly+ 9 ! furr[ on ei!r side ( ! sp9al processes4 #cb4 ,cut ?r\gh ! bases ( ! transv]se processes ) bone =ceps4 ,cut away ! vault ( ! skull1 cut ?r\gh ! roots ( ! n]ves & remove ! bra9 & sp9al cord1 place 9 a l>ge glass di% = exam9ation4 ,prep>e cultivations f ! c]ebro-sp9al fluid4 ,! removal ( ! bra9 & cord is a t$i\s process & dur+ ! dissection x is difficult to avoid injury to ^! /ructures4 ,! op]ation is1 h[ev]1 c>ri$ out v exp$iti\sly & neatly ) ! aid ( ! surgical eng9e 7vide page #cfa74 ,a small circul> saw is fitt$ to ! h& piece4 ,! bones ( ! skull are cut ?r\gh & ! :ole ( ! vault remov$1 expos+ ! entire v]tex ( ! bra94 ,simil>ly all ! sp9\s processes c be remov$ 9 "o /r+ by runn+ ! saw d[n fir/ "o side ( ! sp9al column & !n ! o!r4 ,9 ?is way ample space = ! removal ( ! n]v\s tissues is obta9$ ) a m9imum ( lab\r4 #cc4 ,hav+ complet$ ! prep>ation ( cultures remove small portions ( v>i\s organs at leisure & place ea* 9 sep>ate bottles ( fix+ fluid = future section+4 ,affix to ea* bottle a label be>+ all necess>y details z to its cont5ts4 #cd4 ,if necess>y1 remove portions ( ! organs = pres]vation & display z museum specim5s 7vide page #djd74 #ce4 ,ga!r up all ! infect$ in/rum5ts1 return !m to ! /]ilis]1 & dis9fect by boil+ = t5 m9utes4 ,illu/ration3 ,,fig4 #aii4--,spe>-head$ plat9um spatula 7actual size47 #cf4 ,spr9kle dry sawdu/ into ! expos$ body cavities to absorb blood & fluid4 ,cov] ! body ) blott+ or filt] pap]1 moi/5$ ) #b p] c5t4 lysol solution4 ,place 9 a galvanis$ iron pail1 provid$ ) a lid1 ready = transport to ! crematorium4 #cg4 ,cremate ! cadav] toge!r ) ! bo>d ^u :i* x is fix$4 #ch4 ,/a9 ! cov]-slip prep>ations by suitable me?ods & exam9e microscopically4 #ci4 ,incubate ! cultivations & exam9e c>efully f day to day4 #dj4 ,make full notes ( ! condition ( ! v>i\s body cavities & ( ! visc]a imm$iately ! autopsy is complet$2 & add ! result ( ! microscopical & cultural inve/igation :5 available4 ,z "p ( ! c>d index sy/em 9 use 9 ! au?or's laboratory already ref]r$ to 7vide page #cce7 !re is a special yell[ c>d = ,p-,m notes4 ,on ! face ( ! c>d are pr9t$ head+s = v>i\s data--"s ( :i* are sometimes un9t5tionally omitt$--& on ! rev]se is a s*ematic figure :i* c be utilis$ = indicat+ ! position ( ! *ief lesions 9 ! cadav] ( any ( ! laboratory animals4 ,,autopsy ,,c>d ,laboratory ,no4 ,date ,animal ,no4 9 ,s]ies ,symbols3 male female ,weight ---------------------------------------- -------------------------------- ,di$ 7or kill$7 o'clock m4 ,autopsy made o'clock m4 ---------------------------------------- -------------------------------- ,notes on ,po/ ,mortem ,exam9ations4 ,g5]al4 ,a4 ,seat ( ,inoculation4 ,b4 ,?oracic ,cavity4 ,c4 ,abdom9al ,cavity4 ,d4 ,cranial ,cavity4 ----------------------- --------------------------------------- ,bact]iological ,hi/ological ,organs ,pres]v$4 ,exam9ation4 ,exam9ation4 ,a4 ,b4 ,c4 ,d4 ,illu/ration3 ,,fig4 #bjj4--;,front ( po/-mortem c>d4 #da4 ,f9ally1 ! results ( ! action ( ! organism or organisms isolat$ may be correlat$ ) ! symptoms obs]v$ dur+ life & ! obs]vations summ>is$ "u ! foll[+ head+s3 ,tissue *anges3 #a4 ,local--i4 e41 produc$ 9 ! neighb\rhood ( ! bact]ia4 ,position3 7a7 ,at prim>y lesion4 7b7 ,at second>y foci4 ,"*3 7a7 ,vascul> *anges & tissue ,acute reactions4 or 7b7 ,deg5]ation & necrosis4 *ronic4 #b4 ,g5]al 7i4 e41 produc$ at a di/ance f ! bact]ia1 by absorption ( tox9s73 7a7 ,9 special tissues--e4 g41 n]ve cells & fibres1 secret+ cells1 vessel walls1 etc4 7b7 ,g5]al effects ( malnutrition1 etc4 ,symptoms3 7a7 ,associat$ ) kn[n tissue *anges4 7b7 ,)\t kn[n tissue *anges4 ,illu/ration3 ,,fig4 #bja4--;,back ( po/-mortem c>d4 ,p]man5t ,prep>ations--,museum ,specim5s4-- ,i4 ,tissues4--,! nak$-eye appe>ances ( morbid tissues may be pres]v$ by ! foll[+ me?od3 #a4 ,remove ! tissue or organ f ! cadav] z soon aft] dea? z possible1 us+ great c>e to avoid di/ortion or injury4 #b4 ,place x 9 a wide-m\!d /opp]$ j>1 l>ge en\gh to hold x conv5i5tly1 re/+ on a pad ( cotton-wool1 & arrange x 9 ! position x is int5d$ to occupy 7b if x is int5d$ to %[ a section ( ! tissue or organ1 d n incise x yet74 #c4 ,cov] ) ! ,kais]l+ fix+ solution1 & /opp] ! j>2 all[ ! tissues to rema9 9 ?is solution = f =ty-eight h\rs to sev5 days 7accord+ to size7 to fix4 ,make any necess>y sections4 ,kais]l+ modifi$ solution is prep>$ z foll[s3 ,weigh out ,potassium acetate #cj grammes4 ,potassium nitrate #ae grammes4 & dissolve 9 ,di/ill$ wat] #ajjj c4c4 !n add ,=mal9 #aej c4c4 ,filt]4 ,?is fix+ solution c be us$ repeat$ly s long z x rema9s cle>4 ,ev5 :5 x has become turbid1 if simple filtration is suffici5t to r5d] x cle>1 ! filtrate may be us$ aga94 #d4 ,transf] ! tissue to a ba? ( me?ylat$ _s 7#ie p] c5t47 = ?irty m9utes to "o h\r4 #e4 ,remove to a fre% ba? ( _s & wat* c>efully4 ,:5 ! natural col\rs %[ 9 _! orig9al t9ts1 av]age "t ?ree to six h\rs1 remove ! tissues f ! _s ba?1 dry (f ! _s f ! cut surfaces by mopp+ ) a s(t clo?1 !n transf] to ! m\nt+ solution4 ,jore's m\nt+ solution 7modifi$7 consi/s ( ,glyc]9e #ejj c4c4 ,di/ill$ wat] #gej c4c4 ,=mal9 #b c4c4 ,equally good b mu* *eap] is ,fro/'s m\nt+ solution3 ,potassium acetate #afj grammes4 ,sodium fluoride #hj grammes4 ,*loral hydrate #hj grammes4 ,cane sug> 7,tate's cubes7 #c1ejj grammes4 ,saturat$ ?ymol wat] #h1jjj c4c4 #f4 ,aft] tw5ty-f\r h\rs 9 ?is solution1 or z soon z ! tissue s9ks1 transf] to a museum j>1 fill ) fre% m\nt+ solution1 & seal4 #f;a4 ,or transf] to museum j> & fill ) liquefi$ gelat9e1 to :i* has be5 add$ #a p] c5t4 =mal94 ,cov] ! j> & all[ ! gelat9e to set4 ,:5 solid1 seal ! cov] ( ! j> 9 place4 #g4 ,to seal ! museum prep>ation fir/ w>m ! glass plate :i* =ms ! cov]4 ,?is is mo/ conv5i5tly done by plac+ ! clean$ & poli%$ cov]-plate ^u a piece ( asbe/os millbo>d ov] a buns5 flame turn$ l[4 #h4 ,sme> an ev5 lay] ( hot cem5t ov] ! flange ( ! j>4 ,! cem5t is prep>$ z foll[s3 ,weigh out & mix 9 an iron ladle ,gutta p]*a 7pure7 #d p>ts4 ,asphaltum #e p>ts4 & melt toge!r ov] a buns5 flame1 /irr+ ) an iron rod until solution is complete4 #i4 ,inv]t ! glass plate ov] ! j> & press d[n firmly into ! cem5t4 ,place a piece ( asbe/os bo>d on ! top & on t re/ a suitable weight until ! cem5t is cold & has ?or\ghly set4 #aj4 ,trim (f any project+ pieces ( cem5t ) an old knife1 burr ov] ! jo9t betwe5 j> & cov]-plate ) a hot smoo? piece ( metal 7e4 g41 ! se>+ iron74 #aa4 ,pa9t a n>r[ b& ( ,japan black to f9i% (f1 r\nd ! jo9t1 ov]lapp+ on to ! cov]-plate4 ,,ii4 ,tube ,cultivations ( ,bact]ia4--,:5 %[+ typical appe>ances ^! may be pres]v$1 if n p]man5tly1 at lea/ = _m ye>s1 z museum specim5s1 by ! foll[+ me?od3 #a4 ,take a l>ge glass j> #be cm4 high by #ah cm4 diamet]1 ) a firm base & a broad flange1 c>efully gr\nd1 ar\nd ! m\?4 ,! j> mu/ be fitt$ ) a disc ( plate glass gr\nd on "o side1 to s]ve z a lid4 #b4 ,sme> a ?ick lay] ( res9 o9tm5t 7,b4,p47 on ! flange ar\nd ! m\? ( ! j>4 #c4 ,cov] ! bottom ( ! j> ) a lay] ( cotton-wool & saturate x ) =mal94 #d4 ,remove ! cotton-wool plug f ! culture tubes & place !m1 m\? upw>d1 inside ! j>4 7,if wat] ( cond5sation is pres5t 9 any ( ! culture tubes1 x %\ld be remov$ by means ( a capill>y pipette be=e plac+ ! tubes 9 ! =mal9 *amb]47 #e4 ,adju/ ! glass disc1 gr\nd side d[nw>d1 ov] ! m\? ( ! j> & secure x by press+ x firmly d[n into ! o9tm5t1 ) a rot>y movem5t4 #f4 ,remove ! tubes f ! =mal9 *amb] aft] ! lapse ( a week1 & dry ! ext]ior ( ea*4 ,illu/ration3 ,,fig4 #bjb4--;,bullo*'s tubes4 #g4 ,seal ! op5 m\? ( ea* tube 9 ! bl[pipe flame & label4 ,if ! cultivations are int5d$ = museum purposes :5 !y are fir/ plant$1 x is m conv5i5t to employ ,bullo*'s tubes4 ,^! are slightly long] ?an ! ord9>y tubes1 & are provid$ ) a con/riction "s #b cm4 bel[ ! m\? 7,fig4 #bjb7--;a feature :i* r5d]s seal+ 9 ! bl[pipe flame an easy matt]4 ,,xx4 ,,! ,,/udy ,,( ,,! ,,pa?og5ic ,,bact]ia4 ,! /ud5t1 :o has consci5ti\sly work$ out ! me?ods1 etc41 previ\sly dealt )1 is 9 a position to make accurate obs]vations & to write precise descriptions ( ! results ( su* obs]vations4 ,he is1 !re=e1 n[ entru/$ ) pure cultivations ( ! v>i\s pa?og5ic bact]ia1 9 ord] t he may /udy ! life-hi/ory ( ea* & record ! results ( 8 own obs]vations--to be subsequ5tly correct$ or amplifi$ by ! demon/rator4 ,9 ?is way he is r5d]$ indep5d5t ( text-book descriptions1 ! /atem5ts 9 :i* he is o!rwise too liable to take = grant$1 )\t p]sonally attempt+ to v]ify _! accuracy4 ,dur+ ! c\rse ( ?is "w att5tion mu/ also be direct$1 z occasion arises1 to su* o!r bact]ia1 pa?og5ic or saprophytic1 z are alli$ to ! p>ticul> organisms "u obs]vation1 or s resemble !m z to become possible s\rces ( error1 by work+ !m ?r\gh on p>allel l9es--9 o!r words ! v>i\s bact]ia %\ld be /udi$ 9 gr\ps4 ,9 ! foll[+ pages ! gr\p+ 9 use 9 ! au?or's elem5t>y classes = m$ical & d5tal /ud5ts & = c&idates = ! ,public ,heal? s]vice is adopt$1 s9ce a fairly long exp]i5ce has completely v9dicat$ ! value & utility ( ?is arrangem5t1 & by its means a fund ( in=mation is obta9$ ) reg>d to ! resemblances & diff]5ces1 morphological & cultural1 ( a l>ge numb] ( bact]ia4 ,! fact t "s bact]ia appe> 9 m ?an "o ( ^! gr\ps1 s f> f be+ a disadvantage1 is a positive ga9 to ! /ud5t1 s9ce ) repetition alone w ! necess>y famili>ity ) ! cultural *>act]s ( important bact]ia be acquir$4 ,! /udy ( ! v>i\s gr\ps w ( c\rse v>y 9 detail ) individual demon/rators1 & ) ! /ud5t's requirem5ts--! g5]al l9e x %\ld take is indicat$ briefly 9 connection ) ! fir/ gr\p only 7pages #daj-daa74 ,?is section %\ld be c>efully work$ ?r\gh be=e ! /ud5t proce$s to ! /udy ( bact]ioscopical analysis4 ,x is cu/om>y to comm5ce ! /udy ( ! pa?og5ic bact]ia ) ! ,organisms ( ,suppuration4 ,?is is a l>ge gr\p1 = all ! pa?og5ic bact]ia possess ! p[]1 "u c]ta9 conditions1 ( initiat+ purely pyog5ic processes 9 place ( or 9 addition to _! specific lesions1 7e4 g41 ,bacillus tub]culosis1 ,/reptococcus lanceolatus1 ,bacillus typhosus1 etc474 ,!re are1 h[ev]1 a c]ta9 few organisms :i* commonly express _! pa?og5icity 9 ! =mation ( pus4 ,^! are usually gr\p$ toge!r "u ! title ( pyog5ic bact]ia1 z di/9ct f ^? :i* only occasionally ex]cise a pyog5ic role4 ,! organisms includ$ 9 ?is gr\p are3 #a4 ,/aphylococcus pyog5es albus4 #b4 ,/aphylococcus pyog5es aureus4 #c4 ,/aphylococcus pyog5es citreus4 #d4 ,/reptococcus pyog5es longus4 #e4 ,micrococcus tetrag5us4 #f4 ,bacillus pyocyaneus4 #g4 ,bacillus pneumoniae4 & 9 c]ta9 special tissues #h4 ,micrococcus gonorrhoeae4 #i4 ,micrococcus intracellul>is m5+itidis 7,m5+ococcus74 #aj4 ,micrococcus cat>rhalis4 #aa4 ,bacillus aegypticus 7,ko*-,weeks ,bacillus74 ,! gr\p may ) advantage be subdivid$ z indicat$ 9 ! foll[+ pages3 ,i4 ,pyog5ic cocci4 ,/aphylococcus pyog5es albus4 ,/aphylococcus pyog5es aureus4 ,/aphylococcus pyog5es citreus4 to contra/ ) ,micrococcus c&icans4 ,micrococcus agilis4 #a4 ,prep>e subcultivations f ea*3 ,b\illon1 ,ag> /reak1 ,blood s]um1 ,litmus milk4 & incubate at #cg deg4 ,c4 ,ag> /reak1 ,gelat9e /ab1 ,potato4 & incubate at #bj deg4 ,c4 ,comp>e ! nak$-eye appe>ances ( ! cultures f day to day4 ,note ,m4 agilis refuses to gr[ at #cg deg4 ,c4 #b4 ,make hang+-drop prep>ations f ! b\illon & ag> cultivations aft] tw5ty-f\r h\rs' incubation4 ,exam9e microscopically & comp>e4 ,note ! locomotive activity ( ,m4 agilis & ! ,br[nian movem5t ( ! rema9+ micrococci4 #c4 ,prep>e cov]-slip films f ! ag> cultures1 aft] tw5ty-f\r h\rs' incubation4 ,/a9 = flagella by ! modifi$ ,pitfield's me?od4 ,note ,m4 agilis is ! only micrococcus %[+ flagella4 #d4 ,make microscopical prep>ations ( ea* f all ! v>i\s m$ia aft] tw5ty-f\r & =ty-eight h\rs & ?ree days' incubation4 ,/a9 c>bolic me?yl5e-blue1 c>bolic fu*s91 & ,gram's me?od4 ,exam9e ! films microscopically & comp>e4 ,note 9 ! ,gram prep>ation1 ! ,gram negative "* ( c]ta9 individual cocci 9 ea* film prep>$ f ! ?ree days' gr[?--su* cocci are dead4 #e4 ,/a9 section ( kidney tissue provid$ 7%[+ abscess =mation by ,/aphylococcus aureus7 by ,gram's me?od1 & c\nt]/a9 ) cos94 #f4 ,/a9 film prep>ation ( pus f an abscess 7conta9+ ,/aphylococcus pyog5es aureus7 ) c>bolic me?yl5e-blue & also by ,gram's me?od1 c\nt]/a9$ ) cos94 #g4 ,inoculate#ae a :ite m\se subcutane\sly ) ?ree loopfuls ( a =ty-eight-h\r ag> cultivation ( ! ,/aphylococcus aureus1 emulsifi$ ) #j4b c4c4 /]ile bro?4 ,obs]ve c>efully dur+ life1 & :5 dea? occurs make a c>eful po/-mortem exam9ation4 ,,ii4 ,pyog5ic cocci4 ,micrococcus gonorrhoeae4 ,micrococcus intracellul>is m5+itidis 7m5+ococcus74 ,micrococcus cat>rhalis4 ,micrococcus tetrag5us4 ,micrococcus p>atetrag5us4 ,,iii4 ,pyog5ic cocci4 ,/reptococcus pyog5es longus4 ,/reptococcus ( bov9e ma/itis4 ,/reptococcus lanceolatus 7,diplococcus pneumoniae or pneumococcus74 to contra/ ) ,/reptococcus brevis4 ,/reptococcus leb5sis4 ,,iv4 ,pyog5ic bacilli4 ,bacillus pneumoniae 7,fri$la5d]74 ,bacillus rh9oscl]omatis4 ,bacillus lactis a]og5es4 ,v4 ,pyog5ic bacilli4 ,bacillus pyocyaneus4 to contra/ ) ,bacillus fluoresc5s liquefaci5s4 ,bacillus fluoresc5s non-liquefaci5s4 ,,vi4 ,pneumonia gr\p4 ,/reptococcus lanceolatus 7pneumococcus74 ,bacillus pneumoniae 7,fri$la5d]74 ,/reptococcus pyog5es longus4 ,,vii4 ,diph!roid gr\p4 ,bacillus diph!riae 7,klebs-,loeffl]74 ,bacillus ,h(fmanni4 ,bacillus x]osis4 ,bacillus septus4 ,,viii4 ,coli-typhoid gr\p4 ,b4 typhi abdom9alis 7,b4 typhosus74 ,b4 coli communis4 ,b4 ent]itidis 7,ga]tn]74 to contra/ ) ,b4 aquatilis sulcatus4 ,,ix4 ,es*]i* gr\p4 ,b4 coli communis 7,es*]i*74 ,b4 coli communior4 ,b4 lactis a]og5es4 ,b4 cloacae4 ,x4 ,ga]tn] gr\p4 ,bacillus ent]itidis 7,ga]tn]74 ,b4 p>atyphosus ,a4 ,b4 p>atyphosus ,b4 ,bacillus *ol]ae suum 7,hog ,*ol]a74 ,b4 psittacosis4 ,,xi4 ,eb]? gr\p4 ,b4 typhosus 7,eb]?74 ,b4 dys5t]iae 7,%iga74 ,b4 dys5t]iae 7,flexn]74 ,b4 faecalis alcalig9es4 ,,xii4 ,spirillum gr\p4 ,vibrio *ol]ae4 ,vibrio mets*nikovi4 to contra/ ) ,vibrio proteus 7,f9kl] & ,prior74 ,spirillum rubrum4 ,spirillum rugula4 ,,xiii4 ,an?rax gr\p4 ,bacillus an?racis4 to contra/ ) ,bacillus subtilis4 ,bacillus mycoides4 ,bacillus mes5t]icus fuscus4 ,,xiv4 ,acid fa/ gr\p4 ,bacillus tub]culosis 7human74 7bov9e74 7avian74 7fi%74 to contra/ ) ,bacillus phlei 7,timo?y grass bacillus74 ,butt] bacillus ( ,rab9[it*4 ,,xv4 ,plague gr\p4 ,bacillus pe/is4 ,b4 septicaemiae haemorrhagicae4 ,b4 suipe/if]4 ,,xvi4 ,influ5zae gr\p4 ,b4 influ5zae4 ,bacillus aegypticus 7,ko*-,weeks74 ,bacillus p]tussis4 ,,xvii4 ,miscellane\s4 ,bacillus leprae4 ,bacillus mallei4 ,micrococcus melit5sis4 ,,xviii4 ,/repto?rix gr\p4 ,/repto?rix act9omycotica4 ,/repto?rix madurae4 to contra/ ) ,clado?rix nivea4 ,,xix4 ,tetanus gr\p4 ,bacillus tetani4 ,bacillus o$ematis maligni4 ,bacillus *auvei 7symptomatic an?rax74 ,,xx4 ,ent]itidis sporog5es gr\p4 ,bacillus ent]itidis sporog5es4 ,b4 botul9us4 ,b4 butyricus4 ,b4 cadav]is4 ,,footnotes3 #ae ,see note on ,vivisection ,lic5se1 page #ccd4 ,,xxi4 ,,bact]iological ,,analyses4 ,ea* bact]iological or bact]ioscopical analysis ( air1 e>?1 sewage1 v>i\s food-/uffs1 etc41 includes1 z a g5]al rule1 two di/9ct inve/igations yield+ results ( v unequal value3 #a4 ,quantitative4 #b4 ,qualitative4 ,! fir/ is purely quantitative & z su* is ( m9or importance z x aims simply at enum]at+ 7approximately7 ! total numb] ( bact]ia pres5t 9 any giv5 unit ( volume irrespective ( ! nature & "* ( individual organisms4 ,! second & m important is bo? qualitative & quantitative 9 "* s9ce x seeks to accurately id5tify su* pa?og5ic bact]ia z may be pres5t :ile1 incid5tally1 ! me?ods advocat$ are calculat$ to indicate1 ) a fair degree ( accuracy1 ! num]ical frequ5cy ( su* bact]ia1 9 ! sample "u exam9ation4 ,! g5]al pr9ciples und]ly+ ! bact]iological analyses ( wat]1 sewage1 air & du/1 soil1 milk1 ice cream1 meat1 & o!r t9n$ /uffs1 z exemplifi$ by ! me?ods us$ by ! au?or1 are indicat$ 9 ! foll[+ pages1 toge!r ) ! me?ods ( te/+ filt]s & *emical g]micides2 & ! te*nique !re set out w be f\nd to be capable ( expansion & adaptation to any circum/ance or set ( circum/ances :i* may confront ! /ud5t4 ,controls4--,! necessity = ! exi/5ce ( adequate controls 9 all exp]im5tal "w _c be too urg5tly insi/$ ^u4 ,e bat* ( plates t is p\r$ %\ld include at lea/ "o ( ! presumably /]ile m$ium2 plate or tube cultures %\ld be made f ! v>i\s dilut+ fluids2 e tube ( c>bohydrate m$ium t is inoculat$ %\ld g into ! incubator 9 company ) a simil> b un9oculat$ tube1 & s on4 ,,bact]iological ,,exam9ation ,,( ,,wat]4 ,! bact]ia pres5t 9 ! wat] may comprise n only v>ieties :i* h _! normal habitat 9 ! wat] & w consequ5tly develop at #bj deg4 ,c41 b also if ! wat] has be5 contam9at$ ) excrem5tal matt]1 v>ieties :i* h be5 d]iv$ f1 or are pa?og5ic =1 ! animal body1 & :i* w only develop well at a temp]ature ( #cg deg4 ,c4 ,9 ord] to demon/rate ! pres5ce ( ea* ( ^! classes x w be necess>y to incubate ! v>i\s cultivations at ea* ( ^! temp]atures4 ,fur!r1 ! sample ( wat] may conta9 m\lds1 yea/s1 or torulae1 & ! developm5t ( ^! w be be/ secur$ by plat+ 9 wort gelat9e & incubat+ at #bj deg4 ,c4 #a4 ,quantitative4-- ,collection ( ! ,sample4--,! mo/ suitable vessels = ! reception ( ! wat] sample are small glass bottles1 #fj c4c4 capacity1 ) n>r[ necks & ov]hang+ glass /opp]s 7to prev5t contam9ation ( ! bottle necks by fall+ du/74 ,^! mu/ be c>efully /]ilis$ 9 ! hot-air /]ilis] 7vide page #ca74 7a7 ,if ! sample is obta9$ f a tap or pipe1 turn on ! wat] & all[ x to run = a few m9utes4 ,remove ! /opp] f ! bottle & reta9 x 9 ! h& :il/ ! wat] is all[$ to run into ! bottle & ?ree p>ts fill x4 ,replace ! /opp] & tie x d[n1 b d n seal x4 7b7 ,if ! sample is obta9$ f a /ream1 tank1 or res]voir1 fa/5 a piece ( /\t wire ar\nd ! neck ( ! bottle1 remove ! /opp]1 & reta9 x 9 ! h&4 ,!n1 us+ ! wire z a h&le1 plunge ! bottle into ! wat]1 m\? d[nw>d1 until x is well b5ea? ! surface2 !n rev]se x1 all[ x to fill1 & )draw x f ! wat]4 ,p\r out a few cubic c5timetres ( wat] f ! bottle1 replace ! /opp]1 & tie x d[n4 ,illu/ration3 ,,fig4 #bjc4--;,esm>*'s collect+ bottle = wat] samples4 7c7 ,if ! sample is obta9$ f a lake1 riv] or ! sea2 or :5 x is desir$ to comp>e samples tak5 at v>y+ dep?s1 ! app>atus design$ by v4 ,esm>* 7,fig4 #bjc7 is employ$4 ,9 ?is ! /]ilis$ bottle is enclos$ 9 a weight$ metal cage :i* c be l[]$1 by means ( a graduat$ l9e1 until ! requir$ dep? is rea*$4 ,at ?is po9t ! bottle is op5$ by a ?9 wire cord atta*$ to ! /opp]2 :5 ! bottle is full 7z judg$ by ! air bubbles ceas+ to rise7 ! pull on ! cord is releas$ & ! t5sion ( ! spiral spr+ above ! /opp] aga9 =ces x into ! neck ( ! bottle4 ,:5 ! app>atus is tak5 out ( ! wat]1 ! small bottles are fill$ f x1 & pack$ 9 ! ice-box m5tion$ bel[4 ,an inexp5sive sub/itute = ,esm>*'s bottle c be made 9 ! laboratory ?us3 ,select a wide-m\!d glass /opp]$ bottle ( ab\t #ejj c4c4 capacity 7ab\t #bj cm4 high & #h cm4 9 diamet]74 ,remove ! glass /opp] & ins]t a rubb] cork ) two p]=ations 9 its place4 ,?r\gh "o p]=ation pass a piece ( glass tub+ ab\t #e cm4 long & ?r\gh ! o!r a piece #bb cm4 long1 rea*+ to ne> ! bottom ( ! bottle1 ea* tube project+ ab\t #b4e cm4 above ! rubb] /opp]4 ,plug ! op5 ends ( ! tubes ) cotton wool4 ,secure ! /opp] 9 place ) ?9 copp] wire4 ,illu/ration3 ,,fig4 #bjd4--,?re%'s deep wat] sampl+ bottle4 ,/]ilise ! fitt$ bottle 9 ! autoclave4 ,remove ! cotton wool plugs & connect ! project+ tubes by a piece ( loosely fitt+ /\t rubb] pressure tub+ ab\t #e cm4 long1 previ\sly /]ilis$ by boil+4 ,take a piece ( /\t rubb] cord ab\t #cc cm4 long1 & ( #aj mm4 diamet] 7su* z is us$ = door spr+s7 ?read a /eel split r+ ^u x & secure ! free ends tightly to ! neck ( ! bottle by cord or catgut4 ,atta* ! cord us$ = l[]+ ! bottle into ! wat] to ! split r+ on ! rubb] susp5d]4 ,! be/ mat]ial = ?is purpose is cotton insulat$ electric wire knott$ at e metre4 ,connect ! split r+ also ) ! %ort piece ( rubb] tub+ unit+ ! two glass tubes by a piece ( catgut 7or ?9 copp] wire7 ( su* l5g? t :5 ! bottle is susp5d$ !re is no pull ^u ! rubb] tube1 b :i*1 h[ev]1 w be easily j]k$ (f :5 a %>p pull is giv5 to ! susp5d+ cord4 ,n[ w9d heavy lead tub+ ab\t #a cm4 diamet] ar\nd ! upp] "p ( ! bottle1 />t+ at ! neck j above ! %\ld]4 ,?is ensures ! s9k+ ( ! bottle 9 ! v]tical position 7,fig4 #bjd74 ,! app>atus be+ arrang$ is l[]$ to ! requir$ dep?1 a %>p j]k is !n giv5 to ! susp5d+ cord1 :i* deta*es ! rubb] tube & s op5s ! two glass tubes4 ,wat] ent]s ?r\gh ! long] tube & ! air is expell$ ?r\gh ! %ort] tube4 ,! bubbles ( air c be se5 or he>d ris+ ?r\gh ! wat]1 until ! bottle is ne>ly full1 a small volume ( compress$ air rema9+ 9 ! neck ( ! bottle4 ,z ! app>atus is rais$1 ! air ?us imprison$ exp&s1 & prev5ts ! entry ( m wat] f ne>] ! surface4 ,illu/ration3 ,,fig4 #bje4--;,ice-box = transmission ( wat] samples1 etc4 ,transport ( ,sample4--,if ! exam9ation ( ! sample _c be comm5c$ imm$iately1 /eps mu/ be tak5 to prev5t ! multiplication ( ! bact]ia conta9$ 9 ! wat] dur+ ! int]val occupi$ 9 transit f ! place ( collection to ! laboratory4 ,to ?is end an ice-box su* z t %[n 79 ,fig4 #bje7 is ess5tial4 ,x consi/s ( a d\ble-wall$ metal cyl9d] into :i* slides a cyl9drical *amb] ( suffici5t capacity to accommodate f\r ( ! #fj c4c4 bottles2 ?is 9 turn is cov]$ by a metal disc--! ?ree portions be+ bolt$ toge!r by ?umb screws ?r\gh ! ov]hang+ flanges4 ,:5 9 use1 place ! bottles1 roll$ 9 cotton-wool1 9 ! c5tral *amb]1 pack ! space betwe5 ! walls ) p\nd$ ice1 securely close ! metal box by screw+ d[n ! fly nuts1 & place x 9 a felt-l9$ wood5 case4 7,x has be5 %[n t :il/ bact]ia w survive exposure to ! temp]ature ( melt+ ice1 practically none w multiply at ?is temp]ature47 ,on rea*+ ! laboratory1 ! me?od ( exam9ation consi/s 9 add+ measur$ quantities ( ! wat] sample to sev]al tubes ( nutri5t m$ia previ\sly liquefi$ by heat1 p\r+ plate cultivations f ea* ( ^! tubes1 incubat+ at a suitable temp]ature1 & f9ally c\nt+ ! colonies :i* make _! appe>ance on ! plates4 ,app>atus ,requir$3 ,plate-levell+ /&4 ,case ( /]ile plates4 ,case ( /]ile pipettes1 #a c4c4 79 t5?s ( a cubic c5timetre74 ,case ( /]ile pipettes1 #aj c4c4 79 t5?s ( a cubic c5timetre74 ,case ( /]ile capsules1 #be c4c4 capacity4 ,tubes ( nutri5t gelat9e4 ,tubes ( nutri5t ag>4 ,tubes ( wort gelat9e4 ,"o #bej c4c4 flask ( /]ile di/ill$ wat]4 ,tall cyl9d] conta9+ #b p] c5t4 lysol solution4 ,buns5 burn]4 ,grease p5cil4 ,wat]-ba? regulat$ at #db deg4 ,c4 ,,me?od4-- #a4 ,arrange ! plate-levell+ plat=m ) its wat] comp>tm5t fill$ ) wat]1 at #de deg4 ,c4 #b4 ,numb] ! ag> tubes1 consecutively1 #a to #f2 ! gelat9e tubes1 consecutively1 #a to #f1 & ! wort tubes1 #a1 #b1 & #c4 ,flame ! plugs & see t !y are n adh]5t to ! lips ( ! tubes4 #c4 ,place ! ag> tubes 9 boil+ wat] until ! m$ium is melt$1 !n transf] !m to ! wat]-ba? regulat$ at #db deg4 ,c4 ,liquefy ! nutri5t gelat9e & wort gelat9e tubes by imm]s+ !m 9 ! same wat]-ba?4 #d4 ,remove ! bottle conta9+ ! wat] sample f ! ice-box1 di/ribute ! bact]ial cont5ts ev5ly ?r\gh\t ! wat] by %ak+1 cut ! /r+ secur+ ! /opp]1 & loos5 ! /opp]1 b d n take x out4 ,illu/ration3 ,,fig4 #bjf4--,)draw+ wat] f wat] sample bottle4 #e4 ,remove "o ( ! #a c4c4 pipettes f ! case1 hold+ x by ! pla9 portion ( ! tube4 ,pass ! graduat$ portion twice ?r\gh ! ,buns5 flame4 ,tilt ! bottle conta9+ ! wat] sample on ! b5* hold+ ! neck betwe5 ! middle & r+ f+]s ( ! left h&2 grasp ! head ( ! /opp] betwe5 ! =ef+] & ?umb1 & remove x f ! bottle4 #f4 ,pass ! pipette into ! m\? ( ! bottle1 hold+ its po9t well bel[ ! surface ( ! wat] 7,fig4 #bjf74 ,suck up r m ?an #a c4c4 into ! pipette & all[ ! pipette to empty2 ?is moi/5s ! int]ior ( ! pipette & r5d]s accurate measurem5t possible4 ,n[ draw up exactly #a c4c4 into ! pipette4 ,)draw ! pipette f ! bottle1 replace ! /opp]1 & /& ! bottle upright4 #g4 ,take ! fir/ melt$ ag> tube 9 ! left h&1 remove ! cotton-wool plug1 & add to its cont5ts #j4e c4c4 ( ! wat] sample f ! pipette2 replug ! tube & replace x 9 ! wat]-ba?4 ,9 a simil> mann] add #j4c c4c4 wat] to ! cont5ts ( ! second tube1 & #j4b c4c4 to ! cont5ts ( ! ?ird4 #h4 ,9 a simil> mann] add #a c4c4 ( ! sample to ! cont5ts ( ! f\r? tube4 #i4 ,simil>ly1 add #j4e c4c4 & #j4a c4c4 respectively to ! cont5ts ( ! fif? & six? tubes4 #aj4 ,drop ! pipette into ! cyl9d] conta9+ lysol solution4 #aa4 ,mix ! wat] sample ) ! m$ium 9 ea* tube 9 ! mann] describ$ "u plate cultivations2 p\r a plate f ea* tube4 ,label ea* plate ) 7a7 ! di/9ctive numb] ( ! sample1 7b7 ! quantity ( wat] sample x conta9s1 & 7c7 ! date4 #ab4 ,p\r ! cont5ts ( a tube ( liquefi$ ag>--n inoculat$--into a ,petri di% to act z a control to demon/rate ! /]ility ( ! bat* ( ag> employ$4 #ac4 ,all[ ! plates to set1 & incubate at #cg deg4 ,c4 #ad4 ,empty ! wat] *amb] ( ! levell+ app>atus & refill x ) ice-wat]4 #ae4 ,by means ( ! /]ile #aj c4c4 pipette deliv] #i4i c4c4 /]ile di/ill$ wat] into a /]ile glass capsule4 #af4 ,add #j4a c4c4 ( ! wat] sample to ! #i4i c4c4 /]ile wat] 9 ! capsule4 ,?is w give a dilution ( #a 9 #ajj4 #ag4 ,plant ! six tubes ( nutri5t gelat9e 9 ! foll[+ mann]3 ,to ! fir/ tube add #j4e c4c4 ( ! wat] sample direct f ! bottle2 to ! second1 #j4c c4c42 & to ! ?ird1 #j4b c4c42 & p\r a plate ( ea* tube4 ,to ! f\r? tube add #j4e c4c4 ( ! dilut$ wat] sample f ! capsule2 to ! fif?1 #j4c c4c42 & to ! six?1 #j4b c4c42 & p\r a plate f ea*4 #ah4 ,label ea* plate ) ! quantity ( ! wat] sample x conta9s--t is1 #j4e c4c41 #j4c c4c41 #j4b c4c41 #j4jje c4c41 #j4jjc c4c41 & #j4jjb c4c4 #ai4 ,p\r a control 7un9oculat$7 gelat9e plate4 #bj4 ,all[ ! plates to set1 & incubate at #bj deg4 ,c4 #ba4 ,to ! fir/ tube ( liquefi$ wort gelat9e add #j4e c4c4 wat] sample2 to ! second1 #j4c c4c42 & to ! ?ird1 #j4b c4c4 #bb4 ,label ! plates1 all[ !m to set1 & incubate at #bj deg4 ,c4 #bc4 ,c\nt & record ! numb] ( colonies t h develop$ ^u ! ag> at #cg deg4 ,c4 aft] =ty-eight h\rs' incubation4 #bd4 ,note ! numb] ( colonies pres5t on ea* ( ! gelat9e & wort gelat9e plates aft] =ty-eight h\rs' incubation4 #be4 ,replace ! gelat9e & wort plates 9 ! incubator2 obs]ve aga9 at ?ree days1 f\r days1 & five days4 #bf4 ,calculate & record ! numb] ( organisms pres5t p] cubic c5timetre ( ! orig9al wat] f ! av]age ( ! six gelat9e plates at ! late/ date possible up to sev5 days--! pres5ce ( liquefy+ bact]ia may r5d] ! calculation necess>y at an e>li] date1 h5ce ! importance ( daily obs]vations4 ,me?od ( ,c\nt+4--,! mo/ accurate me?od ( c\nt+ ! colonies on ea* ( ! plates is by means ( ei!r ,jeff]y's or ,pakes' c\nt+ disc4 ,ea* ( ^! discs consi/s ( a piece ( pap]1 ^u :i* is pr9t$ a dead black disc1 subdivid$ by conc5tric circles & radii1 pr9t$ 9 :ite4 ,9 ,jeff]y's c\nt] 7,fig4 #bjg71 ea* subdivision has an area ( #a squ>e c5timetre2 9 ,pakes' c\nt] 7,fig4 #bjh71 radii divide ! circle into sixte5 equal sectors1 & c\nt+ is facilitat$ by conc5tric circles equidi/ant f ! c5tre4 ,illu/ration3 ,,fig4 #bjg4--;,jeff]y's disc1 r$uc$4 ,illu/ration3 ,,fig4 #bjh4--,pakes' disc1 r$uc$4 7a7 ,9 ! f9al c\nt+ ( ea* plate1 place ! plate ov] ! c\nt+ disc1 & c5tre x1 if possible1 mak+ its p]iph]y co9cide ) "o or o!r ( ! conc5tric circles4 7b7 ,remove ! cov] ( ! plate1 & by means ( a h& l5s c\nt ! colonies appe>+ 9 ea* ( ! sectors 9 turn4 ,make a note ( ! numb] pres5t 9 ea*4 7c7 ,if ! colonies pres5t are few] ?an #ejj1 ! entire plate %\ld be c\nt$4 ,if1 h[ev]1 !y exce$ ?is numb]1 enum]ate "o-half1 or "o-qu>t] ( ! plate1 or c\nt a sector "h & !re1 & f ^! figures e/imate ! numb] ( colonies pres5t on ! entire plate4 ,9 practice x w be f\nd t ,pakes' disc is m suitable = ! =m] class ( plate2 ,jeff]y's disc = ! latt]4 ,x %\ld be recollect$ h[ev] t unless ! plates h be5 c>efully level$ & ! m$ium is ( equal ?ickness all ov] x is useless to try & av]age f small areas--s9ce :]e ! m$ium is ?ick all ! bact]ia w develop1 :]e ! lay] is a ?9 "o1 only a few bact]ia w f9d suffici5t pabulum = ! production ( visible colonies4 ,x w be not$ t ! quantities ( wat] select$ = addition to ea* set ( tubes ( nutri5t m$ia h be5 c>efully *os5 9 ord] to yield workable results ev5 :5 deal+ ) widely diff]+ samples4 ,plates prep>$ 9 ag> ) #j4a c4c4 & 9 gelat9 ) #j4jb c4c4 c be c\nt$ ev5 :5 l>ge numb]s ( bact]ia are pres5t 9 ! sample2 :]eas if micro-organisms are relatively few1 ag> plate #d & gelat9e plate #a w give ! mo/ reliable c\nts4 ,aga9 ! c\nts ( ! plates 9 a measure control ea* o!r2 = example1 ! second & ?ird plates ( ea* gelat9e s]ies %\ld toge!r conta9 z _m colonies z ! fir/1 & ! second %\ld conta9 ab\t half z _m m ?an ! ?ird & s on4 #b4 ,qualitative ,exam9ation4-- ,collection ( ,sample4--,! wat] sample requir$ = ! r\t9e exam9ation1 :i* x w be conv5i5t to consid] fir/1 am\nts to ab\t #aaj c4c4 ,x is collect$ 9 ! mann] previ\sly describ$ 7vide page #daf72 simil> bottles are us$1 & if f\r are fill$ ! comb9$ cont5ts1 am\nt+ to ab\t #bdj c4c41 w provide ample mat]ial = bo? ! qualitative & quantitative exam9ations4 ,unless ! exam9ation is to be comm5c$ at once1 ! ice-box mu/ be employ$1 o!rwise wat] bact]ia & o!r saprophytes w probably multiply at ! exp5se ( ! microbes indicative ( pollution1 & s increase ! difficulties ( ! inve/igation4 ,9 ! r\t9e exam9ation ( wat] supplies x is cu/om>y to limit ! qualitative exam9ation to a se>* = ,a4 ,b4 coli & its ne> allies4 ,b4 ,/reptococci1 organisms :i* are frequ5tly spok5 ( z microbes ( indication1 z _! pres5ce is held to be evid5ce ( pollution ( ! wat] by mat]ial d]iv$ f ! mammalian alim5t>y canal1 & s to con/itute a dang] signal4 ,c4 ,"s obs]v]s /ill atta* importance to ! pres5ce ( ,b4 ent]itidis sporog5es1 b z ! se>* = ?is bact]ium1 7relatively sc>ce 9 wat]7 necessitates ! collection ( a fairly l>ge quantity ( wat] x is n usually includ$ 9 ! r\t9e exam9ation4 ,9 ! case ( wat] samples exam9$ dur+ ! progress ( an epidemic1 ( new supplies & ( unkn[n wat]s ! se>* is ext5d$ to embrace o!r memb]s ( ! coli-typhoid gr\p2 & on occasion ! "q ( ! pres5ce or abs5ce ( ,vibrio *ol]ae or 7m r>ely7 su* bact]ia z ,b4 an?racis or ,b4 tetani1 may ne$ inve/igation4 ,:5 pa?og5ic or excrem5tal bact]ia are pres5t 9 wat]1 _! numb]s are relatively few1 ow+ to ! dilution !y h und]gone1 & x is usual 9 comm5c+ ! exam9ation1 to adopt "o or o!r ( ! foll[+ me?ods3 ,a4 ,enri*m5t1 9 :i* ! h>mless non-pa?og5ic bact]ia may be de/roy$ or _! gr[? inhibit$1 :il/ ! gr[? ( ! p>asitic bact]ia is enc\rag$4 ,?is is atta9$ by s arrang+ ! environm5t1 7i4 e41 ,m$ia1 incubation temp]ature1 & atmosph]e7 z to favor ! gr[? ( ! pa?og5ic organisms at ! exp5se ( ! h>mless saprophytes4 ,b4 ,conc5tration1 :]eby all ! bact]ia pres5t 9 ! sample ( wat]1 pa?og5ic or o!rwise1 are conc5trat$ 9 a small bulk ( fluid4 ,?is is usually effect$ by filtration ( ! wat] sample ?r\gh a porcela9 filt] c&le1 & ! subsequ5t emulsion ( ! bact]ial residue rema9+ on ! walls ( ! c&le ) a small measur$ quantity ( /]ile b\illon4 ,a4 ,enri*m5t ,me?od4 7,deal+ ) ! demon/ration ( bact]ia ( inte/9al orig947 ,app>atus ,requir$ 7,prelim9>y ,/age73 ,incubator runn+ at #db deg4 ,c4 ,case ( /]ile pipettes1 #a c4c4 graduat$ 9 t5?s4 ,case ( /]ile pipettes1 #aj c4c4 graduat$ 9 c4c4 ,case ( /]ile pipettes1 graduat$ to deliv] #be c4c4 ,tubes ( bile salt bro? 7vide page #ahj74 ,flask ( d\ble /r5g? bile salt bro? 7vide page #aii74 ,tubes ( litmus silk4 ,/]ile flasks1 #bej c4c4 capacity4 ,bu*n]'s tubes4 ,tabloids pyrogallic acid4 ,tabloids sodium hydrate4 ,buns5 burn]4 ,grease p5cil4 7,lat] /age73 ,incubator runn+ at #cg deg4 ,c4 ,surface plates ( nutrose ag> 7see page #bcb74 ,alum9ium spread]4 ,tubes ( v>i\s m$ia1 includ+ c>bohydrate m$ia4 ,agglut9at+ s]a1 etc4 ,,me?od4-- #a4 ,numb] a set ( bile salt bro?1 tubes #a-e1 & a duplicate set #a;a-#e;a4 #b4 ,numb] "o flask #g & ano!r #h4 #c4 ,to ,tubes ,no4 #a & #a;a add #j4a c4c4 wat] sample4 ,to ,tubes ,no4 #b & #b;a add #a c4c4 wat] sample4 ,to ,tubes ,no4 #c & #c;a add #b c4c4 wat] sample4 ,to ,tubes ,no4 #d & #d;a add #e c4c4 wat] sample4 ,to ,tubes ,no4 #e & #e;a add #aj c4c4 wat] sample4 #d4 ,put up all ! tubes 9 ,bu*n]'s tubes & incubate ana]obically at #db deg4 ,c4 ,,note4--,! bile salt m$ium is p>ticul>ly suitable = ! cultivation ( bact]ia ( inte/9al orig91 & at ! same "t inhibits ! gr[? ( bact]ia d]iv$ f o!r s\rces4 ,! ana]obic conditions likewise favor ! multiplication ( inte/9al bact]ia1 & also _! f]m5tative activity4 ,! temp]ature #db deg4 ,c4 de/roys ord9>y wat] bact]ia & inhibits ! gr[? ( _m ord9>y mesophilic bact]ia4 #e4 ,pipette #be c4c4 ( d\ble /r5g? bile salt bro? into flask #f1 & #ej c4c4 d\ble /r5g? bile salt bro? into flask #g4 #f4 ,pipette #be c4c4 wat] sample into flask #f1 & #ej c4c4 wat] sample into flask #g4 #g4 ,incubate ! two flasks a]obically at #db deg4 ,c4 #h4 ,aft] tw5ty-f\r h\rs incubation note 9 ea* culture3 a4 ,! pres5ce or abs5ce ( visible gr[?4 b4 ,! reaction ( ! m$ium z indicat$ by ! col\r *ange1 if any1 ! litmus has und]gone4 c4 ,! pres5ce or abs5ce ( gas =mation1 z indicat$ by a fro? on ! surface ( ! m$ium1 & ! collection ( gas 9 ! inn] gas tube4 #i4 ,replace ^? tubes :i* %[ no signs ( gr[? 9 ! incubator4 ,exam9e aft] ano!r p]iod ( tw5ty-f\r h\rs 7total =ty-eight h\rs incubation7 ) ref]5ce to ! same po9ts4 #aj4 ,remove culture tubes :i* %[ visible gr[? f ! ,bu*n]'s tubes1 :e!r acid production & gas =mation are pres5t or n4 #aa4 ,exam9e all tubes :i* %[ gr[? by hang+-drop prep>ations4 ,note su* z %[ ! pres5ce ( *a9s ( cocci4 #ab4 ,prep>e surface plate cultivations ^u nutrose ag> f ea* tube t %[s gr[? ei!r macroscopically or microscopically1 & incubate = tw5ty-f\r h\rs a]obically at #cg deg4 ,c4 #ac4 ,exam9e ! gr[? on ! plates ei!r ) ! nak$ eye or ) ! help ( a small h& l5s4 ,practice w facilitate ! recognition ( colonies ( ! coli gr\p1 ! typhoid gr\p & ! p>atyphoid gr\p2 also ^? due to ! gr[? ( /reptococci4 ,! inve/igation f ?is /age proce$s along two div]g5t l9es ( enquiry--! fir/ be+ conc]n$ ) ! id5tity ( ! bacilli--typhoid bacilli1 ! second ) t ( ! cocci4 ,a4 ,b4 ,coli & its allies4 #ad4 ,pick (f coli=m or typhi=m colonies2 make /reak or sme> subcultivations ^u nutri5t ag>2 incubate a]obically = tw5ty-f\r h\rs at #cg deg4 ,c4 #ae4 ,exam9e ! gr[? 9 ea* tube c>efully bo? macroscopically & microscopically4 ,if ! gr[? is impure1 replate on nutrose ag>1 pick (f colonies & subcultivate aga94 ,:5 ! gr[? 9 a tube is pure1 add #e c4c4 /]ile normal sal9e solution or /]ile bro?1 & emulsify ! entire surface gr[? ) x4 #af4 ,utilise ! emulsion = ! prep>ation ( a s]ies ( subcultivations ^u ! m$ia enum]at$ bel[1 us+ ! ord9>y loop to make ! subcultures ^u solid m$ia1 b add+ "o-t5? ( a cubic c5timetre ( ! emulsion to ea* ( ! fluid m$ia by means ( a /]ile pipette4 ,gelat9e /reak4 ,ag> /reak4 ,potato4 ,nutri5t bro?4 ,litmus milk4 ,dextrose peptone solution4 ,laevulose peptone solution4 ,galactose peptone solution4 ,maltose peptone solution4 ,lactose peptone solution4 ,sac*>ose peptone solution4 ,raff9ose peptone solution4 ,dulcite peptone solution4 ,mannite peptone solution4 ,glyc]9 peptone solution4 ,inul9 peptone solution4 ,dextr9 peptone solution4 #ag4 ,diff]5tiate ! bacilli aft] isolation by means ( _! cultural reactions & biological *>act]s into memb]s (3 ,i4 ,! ,es*]i* ,gr\p4 ,b4 coli communis4 ,b4 coli communior4 ,b4 lactis a]og5es4 ,b4 cloacae4 ,,ii4 ,! ,ga]tn] ,gr\p4 ,bacillus ent]itidis 7( ,ga]tn]74 ,b4 p>atyphosus ,a4 ,b4 p>atyphosus ,b4 ,bacillus *ol]ae suum4 ,,iii4 ,! ,eb]? ,gr\p4 ,b4 typhosus4 ,b4 dys5t]iae 7,%iga74 ,b4 dys5t]iae 7,flexn]74 ,b4 faecalis alcalig9es4 #ah4 ,confirm ^! results by te/+ ! organisms isolat$ aga9/ specific agglut9at+ s]a obta9$ f exp]im5tally inoculat$ animals4 ,if a positive result is obta9$ :5 us+ ?is me?od1 x only ne$s a simple calculation to det]m9e ! smalle/ quantity 7d[n to #j4a c4c47 ( ! sample t conta9s at lea/ "o ( ! microbes ( indication4 ,= in/ance1 if gr[? occurs 9 all ! tubes f #d to #aj1 & t gr[? is subsequ5tly prov$ to be due to ! multiplication ( ,b4 coli1 !n x foll[s t at lea/ "o colon bacillus is pres5t 9 e #aj c4c4 ( ! wat] sample1 b n 9 e #e c4c4 ,if1 on ! o!r h&1 ! pres5ce ( ! ,b4 coli c only be prov$ 9 flask ,no4 #g1 !n ! av]age numb] ( colon bacilli pres5t 9 ! sample is at lea/ "o 9 e #ej c4c4 7i4 e41 tw5ty p] litre71 b n "o 9 e #be c4c4 & s on4 ,! g5]al outl9e ( ! me?od ( id5tify+ ! memb]s ( ! coli-typhoid gr\p is giv5 9 ! =m ( an analytical s*ema--:il/ ! full diff]5tial details are set out 9 tabul> =m4 ,,analytical ,,s*eme ,,= ,,isolation ,,( ,,memb]s ,,( ,,! ,,coli ,,& ,,typhoid ,,gr\ps4 ,nutrose ag>4 ----------------------------------- ,r$ colonies4 ,blue colonies4 ,es*]i* gr\p4 ,ga]tn] & ,eb]? gr\ps4 --------------- ,lactose peptone solution4 --------------- ,gas4 ,no gas4 ,b4 coli communis & its allies4 ,ga]tn] & ,eb]? gr\ps4 ,acid & gas 9 glucose peptone solution4 ,acid & coagulation 9 milk4 ,g5]al turbidity & indol 9 b\illon4 ,glucose peptone solution4 ,gas4 ,no gas4 ,ga]tn] gr\p4 ,eb]? gr\p4 ---------------- ,litmus milk4 ,peptone solution4 ,litmus milk4 ,peptone solution4 ,acid at fir/4 ,g5]al turbidity4 ,acid4 ,g5]al turbidity4 ,alkal9e lat]4 ,no indol4 ,no coagulation4 ,no indol4 ,no coagulation4 ,s]um reaction4 ,s]um reaction4 ,b4 ,/reptococci4 #ai4 ,pick (f /reptococcus colonies & subcultivate ^u nutri5t ag> exactly z direct$ 9 /eps #ad1 #ae & #af4 #bj4 ,diff]5tiate ! /reptococci isolat$ into memb]s ( ! saprophytic gr\p ( %ort-*a9$ cocci1 or memb]s ( ! p>asitic 7pa?og5ic7 gr\p ( long-*a9$ cocci1 by means ( _! cultural *>act]s1 & record _! num]ical frequ5cy 9 ! mann] indicat$ = ! memb]s ( ! coli-typhoid gr\p4 ,,diff]5tial ,,table ,,( ,,coli-,,typhoid ,,gr\p ,transcrib]'s note3 ,table split to fit #hj spaces4 ---------------------------------------- ---------------------------- ,m ,d ,l ,g ,m ,l ,s ,r ,d ,a acid reaction o e a a a a a a e ,g gas =mation t x e l l c c f t i t v a t t c f r l r u c o o h i i i o l t s s a n n t s o o e e r o y e s s o s e e s e e ---------------------------------------- ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ---------------------------------------- ---------------------------- ,! ,es*]i* ,gr\p4 ,b4 coli communis ,o ,b4 coli communior ,b4 lactis a]og5es - ,o ,o ,b4 acidi lactici - ,o ,o ,o ,b4 pneumoniae - ,b cloaceae7,a7 ,! ,ga]tn] ,gr\p4 ,b4 ent]itidis ,o ,o ,o ,o ,b4 p>atyphosus ,a ,o ,o ,o ,o ,b4 p>atyphosus ,b ,o ,o ,o ,o ,b4 *ol]ae suum ,o ,o ,o ,b4 suipe/if] ,o ,o ,o ,! ,eb]? ,gr\p4 ,b4 typhosus ,o ,o ,o ,b4 dys5t]iae 7,%iga7 - ,o ,o ,o ,o ,o ,b4 dys5t]iae 7,flexn]7 - ,o ,o _/- ,o ,b4 faecalis alkalig9es ,o ,o ,o ,o ,o ,o ,o ,o ---------------------------------------- ---------------------------- ,table ,notes3 7,b7 7,c7 ---------------------------------------- ----------------------------------- ---------------------------------------- ----------------------------- ,i ,s ,g ,d ,m ,s ,i ,litmus ,milk ,aacid reaction n a l u a o n ,ggas =mation u l y l n r d l i c c n b o ---------- i c e i i i l ,e>ly,late n i r t t t n i e e e n ------------------------------ ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ,a ,g ---------------------------------------- ---------------------------- ,! ,es*]i* ,gr\p ,b4 coli communis ,o ,o ,c ,b4 coli communior ,o ,o ,c ,b4 lactis a]og5es ,o ,o ,o ,o - ,c ,b4 acidi lactici ,o ,o ,o ,c ,b4 pneumoniae ,o ,o - ,c ,b cloaceae,a ,o ,o ,o - ,c ,! ,ga]tn] ,gr\p4 ,b4 ent]itidis ,o ,o ,o - _/- - ,b4 p>atyphosus ,a ,o _/- ,o - ,o ,b4 p>atyphosus ,b ,o ,o ,o - - ,b4 *ol]ae suum ,o ,o ,o ,o ,o _/- - ,b4 suipe/if] ,o ,o ,o - - ,! ,eb]? ,gr\p4 ,b4 typhosus ,o ,o ,o ,o - ,b4 dys5t]iae 7,%iga7 ,o ,o ,o ,o ,o ,o - - ,b4 dys5t]iae 7,flexn]7 ,o ,o ,o ,o ,o _/- - ,b4 faecalis alkalig9es ,o ,o ,o ,o ,o ,o - - - ---------------------------------------- ---------------------------- ,table ,notes3 7,d7 7,e7 ---------------------------------------- ---------------------------------- ,table ,notes3 7,a7 99 ,liquefies gelat9e4 7,b7 motile4 - non-motile4 7,c7 acid or gas production4 _/- slight acid production4 ,o no *ange4 7,d7 indol production4 _/- slight indol production4 - no indol =m$4 7,e7 acid production4 - alkali production4 ,o no *ange 9 reaction4 ,c clot4 #ba4 ,det]m9e ! pa?og5icity = mice 7subcutane\s inoculation7 & rabbits 7intrav5\s inoculation7 ( ! /reptococci isolat$4 ,on ! fac+ ins]t page is reproduc$ a blank f ! au?or's ,laboratory ,wat] ,analysis ,book1 by means ( :i* an exact record c be kept1 ) a m9imum ( lab\r1 ( e sample exam9$4 ,b4 ,conc5tration ,me?od4 ,! rema9+ organisms ref]r$ to on page #dbf are m conv5i5tly s\ght = by ! conc5tration me?od4 ,collection ( ! ,sample4--,! quantity ( wat] requir$ = ?is me?od ( exam9ation is ab\t #bjjj c4c41 & ! vessel usually *os5 = its reception is an ord9>y blue glass ,w9*e/] qu>t bottle1 /]ilis$ 9 ! hot-air ov51 & ov] ?is a pap] or p>*m5t cap fa/5$ ) /r+4 ,! bottle may be pack$ 9 a wood5 box or 9 an ord9>y wick] case4 ,! me?od ( collect+ ! sample is id5tical ) t describ$ "u ! head+ ( ,quantitative ,exam9ation2 !re is1 h[ev]1 n ! same imp]ative necessity to pack ! sample 9 ice = transmission to ! laboratory4 ,app>atus requir$3 ,/]ile ,*amb]l& or ,d\lton :ite porcela9 op5 m\? filt] c&le1 fitt$ ) rubb] wa%]4 ,rubb] cork to fit m\? ( ! filt] c&le1 p]=at$ ) "o hole4 ,kitasato s]um flask1 #bejj c4c4 capacity4 ,g]yk air pump or wat] =ce pump4 ,wulff's bottle1 fitt$ z wa%-bottle1 & conta9+ sulphuric acid 7to act z a safety valve betwe5 filt] & pump74 ,pressure tub+1 clamps1 p9*-cock4 ,retort /&1 ) r+ & clamp4 ,rubb] cork = ! neck ( ,w9*e/] qu>t1 p]=at$ ) two holes & fitt$ ) "o #f cm4 l5g? ( /raight glass tub+1 & "o ,v-%ap$ piece ( glass tub+1 "o arm #cb cm4 9 l5g?1 ! o!r #eb cm41 ! %ort] arm be+ plugg$ ) cotton-wool4 ,! rubb] /opp] mu/ be /]ilis$ by boil+ & ! glass tub+ by hot air1 be=e use4 ,flask conta9+ #bej c4c4 /]ile bro?4 ,te/-tube bru% to fit ! lum5 ( ! c&le1 enclos$ 9 a /]ile te/-tube 7& previ\sly /]ilis$ by dry heat or by boil+74 ,case ( /]ile pipettes1 #aj c4c4 9 t5?s4 ,case ( /]ile pipettes1 #a c4c4 9 t5?s4 ,case ( /]ile pipettes1 #a c4c4 9 hundr$?s4 ,tubes ( v>i\s nutri5t m$ia 7accord+ to requirem5ts74 ,twelve ,bu*n]'s tubes ) rubb] /opp]s4 ,pyrogallic acid tablets4 ,cau/ic soda tablets4 ,illu/ration3 ,sample =m ,illu/ration3 ,,fig4 #bji4--,wat] filt]+ app>atus4 ,t portion ( ! figure to ! left ( ! v]tical l9e is drawn to a l>g] scale ?an t on ! "r1 9 ord] to %[ details ( ,spr5gel's pump4 ,,me?od4-- #a4 ,fit up ! filt]+ app>atus z 9 ! accompany+ diagram 7,fig4 #bji71 int]pos+ ! wa%-bottle ) sulphuric acid betwe5 ! filt] flask & ! =ce-pump 79 ! position occupi$ 9 ! diagram by ! c5tral v]tical l9e71 & plac+ ano!r screw clamp on ! rubb] tub+ connect+ ! lat]al arm ( ! filt] flask ) ! wa%-bottle4 ,illu/ration3 ,,fig4 #baj4 ,/]ile te/-tube bru%4 #b4 ,filt] ! entire #bjjj c4c4 ( wat] ?r\gh ! filt] c&le4 #c4 ,:5 ! nitration is complet$1 screw up ! clamps & s occlude ! two pieces ( pressure tub+4 #d4 ,rev]se ! position ( ! glass tubes 9 ! ,wulff's bottle s t ! "o ne>e/ ! air pump n[ dips into ! sulphuric acid4 #e4 ,sl[ly op5 ! metal clamps & all[ air to gradually pass ?r\gh ! acid1 & ent] filt] flask1 & s re/ore ! pressure4 #f4 ,un%ip ! app>atus1 remove ! cork f ! m\? ( ! c&le4 #g4 ,pipette #aj c4c4 ( /]ile bro? into ! int]ior ( ! c&le1 & by means ( ! /]ile te/-tube bru% 7,fig4 #baj7 emulsify ! slimy residue :i* l9es ! c&le1 ) ! bro?4 ,practically all ! bact]ia conta9$ 9 ! orig9al #bjjj c4c4 ( wat] are n[ susp5d$ 9 #aj c4c4 ( bro?1 s t #a c4c4 ( ! susp5sion is equival5t1 s f> z ! conta9$ organisms are conc]n$1 to #bjj c4c4 ( ! orig9al wat]4 7,"s bact]ia w ( c\rse be left beh9d on ! walls ( ! filt] & 9 its pores47 ,up to ?is po9t ! me?od is id5tical1 irrespective ( ! p>ticul> organism ^: pres5ce x is desir$ to demon/rate2 b f ?is po9t onw>d ! me?ods mu/ be specially adapt$ to ! isolation ( def9ite gr\ps ( organisms or ( individual bact]ia4 ,! ,coli-,typhoid ,gr\p4-- #a4 ,numb] n9e tubes ( bile salt bro? 7vide page #ahj71 consecutively f #a to #i4 #b4 ,to ,no #a add #a c4c4 ( ! orig9al wat] sample #b add #b c4c4 be=e ! nitration is comm5c$4 #c add #e c4c4 #c4 ,to ! rema9+ tubes ( bile salt bro? add v>y+ quantities ( ! susp5sion by means ( suitably graduat$ /]ile pipettes1 z foll[s3 ,no4 #d #j4je c4c4 7equival5t to #aj c4c4 ( ! orig9al wat] sample74 ,no4 #e #j4abe c4c4 7equival5t to #be c4c4 ( ! orig9al wat] sample74 ,no4 #f #j4be c4c4 7equival5t to #ej c4c4 ( ! orig9al wat] sample74 ,no4 #g #j4e c4c4 7equival5t to #ajj c4c4 ( ! orig9al wat] sample74 ,no4 #h #a4j c4c4 7equival5t to #bjj c4c4 ( ! orig9al wat] sample74 ,no4 #i #b4e c4c4 7equival5t to #ejj c4c4 ( ! orig9al wat] sample74 #d4 ,put up ea* tube ana]obically 9 a ,bu*n]'s tube & incubate at #db deg4 ,c4 #e4 ,! subsequ5t /eps are id5tical ) ^? describ$ "u ! ,enri*m5t me?od 7see page #dbh to #dca2 ,/eps #h to #ah74 ,alt]native ,me?ods4-- ,a few ( ! old] me?ods = ! isolation ( ! memb]s ( ! coli-typhoid gr\ps are ref]r$ to b !y are di/9ctly inf]ior to ^? already describ$4 7,a7 ,! ,c>bolic ,me?od3 #a4 ,take t5 tubes ( c>bolis$ b\illon 7vide page #bjb7 & numb] !m consecutively f #a to #aj4 #b4 ,inoculate ea* tube ) a diff]5t am\nt ( ! wat] sample or susp5sion1 z 9 ! previ\s me?od4 #c4 ,incubate a]obically at #cg deg4 ,c4 #d4 ,exam9e ! culture tubes aft] tw5ty-f\r h\rs' incubation4 #e4 ,f ^? tubes :i* %[s signs ( gr[?1 p\r plates 9 ! usual mann]1 us+ c>bolis$ gelat9e 7vide page #bjb7 9 place ( ! ord9>y gelat9e1 & incubate at #bj deg4 ,c4 = ?ree1 f\r1 or five days z may be necess>y4 #f4 ,subcultivate f any colonies t make _! appe>ance1 & det]m9e _! id5tity on ! l9es laid d[n 9 ! previ\s me?od4 7,b7 ,p>ietti's ,me?od3 #a4 ,take n9e tubes ( ,p>ietti's b\illon 7vide page #bjb7--;i4 e41 ?ree ea* ( ^? conta9+ #j4a c4c41 #j4b c4c41 & #j4e c4c4 ( ,p>ietti's solution respectively4 ,m>k pla9ly on ! outside ( ea* tube ! quantity ( ,p>ietti's solution x conta9s4 #b4 ,to ea* tube add a diff]5t am\nt ( ! orig9al wat]1 or ( ! susp5sion1 & incubate at #cg deg4 ,c4 #c4 ,exam9e ! culture tubes aft] tw5ty-f\r & =ty-eight h\rs' incubation1 & plate 9 nutri5t c>bolis$ or potato gelat9e f su* z h gr[n4 #d4 ,pick (f suspici\s colonies1 if any su* appe> on ! plates1 subcultivate !m ^u ! v>i\s m$ia1 & id5tify !m4 7,c7 ,elsn]'s ,me?od3 ,?is me?od simply consi/s 9 sub/itut+ ,elsn]'s potato gelat9e 7vide page #bjd7 = ord9>y nutri5t gelat9e 9 any ( ! previ\sly m5tion$ me?ods4 7,d7 ,cambi]'s ,c&le ,me?od3 ,treat a l>ge volume ( ! wat] sample by ! conc5tration me?od 7vide page #dcd74 #a4 ,remove ! rubb] /opp] f ! m\? ( ! filt] c&le1 introduce #aj c4c4 /]ile b\illon into its int]ior1 & emulsify ! bact]ial s$im5t2 replug ! m\? ( ! c&le ) a wad ( /]ile cotton-wool4 #b4 ,remove ! filt] c&le f ! filt] flask & ins]t x into ! m\? ( a flask or a glass cyl9d] conta9+ /]ile b\illon suffici5t to rea* ne>ly up to ! rubb] wa%] on ! c&le4 #c4 ,incubate = tw5ty-f\r to ?irty-six h\rs at #cg deg4 ,c4 #d4 ,f ! n[ turbid b\illon 9 ! glass cyl9d] p\r gelat9e plates & incubate at #bj deg4 ,c4 #e4 ,subcultivate & id5tify any suspici\s colonies t appe>4 7,! me?od dep5ds ^u ! assumption t memb]s ( ! typhoid & coli gr\ps f9d _! way ?r\gh ! porcela9 filt] f ! int]ior to ! surr\nd+ b\illon at a quick] rate ?an ! associat$ bact]ia47 ,b4 ,ent]itidis ,sporog5es4-- #a4 ,transf] #e c4c4 ( ! emulsion f ! filt] c&le to a /]ile te/-tube & plug c>efully4 #b4 ,place ! te/-tube 9 ! int]ior ( ! b5zole ba? employ$ 9 sep>at+ out spore-be>+ organisms 7vide page #beg71 & expose to a temp]ature ( #hj deg4 ,c4 = tw5ty m9utes4 #c4 ,numb] t5 tubes ( litmus milk consecutively f #a to #aj4 #d4 ,remove ! te/-tube f ! b5zole ba? & %ake well to di/ribute ! spores ev5ly ?r\gh ! fluid4 #e4 ,to ea* tube ( litmus milk add a measur$ quantity ( ! susp5sion correspond+ to ! am\nts employ$ 9 isolat+ ! coli gr\p 7vide page #dcg74 #f4 ,incubate ea* tube ana]obically at #cg deg4 ,c4 ,ana]obic conditions c be obta9$ by putt+ ! cultures up 9 ,bu*n]'s tubes or 9 ,bullo*'s app>atus4 ,if1 h[ev]1 :ole milk has be5 us$ 9 mak+ ! litmus milk ! lay] ( cream t rises to ! surface w be suffici5t to ensure ana]obiosis2 :il/ if sep>at$ milk has be5 employ$ x w be suffici5t to p\r a lay] ( /]ile vasel9e or liquid p>aff9 on ! surface ( ! fluid4 #g4 ,exam9e aft] tw5ty-f\r h\rs' incubation4 ,note 7if ,b4 ent]itidis sporog5es is pres5t7-- 7a7 ,acid reaction ( ! m$ium z indicat$ by ! col\r ( ! litmus or its complete decol\risation4 7b7 ,pres5ce ( clott+1 & ! sep>ation ( cle> :ey4 7c7 ,pres5ce ( gas1 z indicat$ by fissures & bubbles 9 ! coagulum1 & possibly masses ( coagulum driv5 up ! tube almo/ to ! plug4 #h4 ,replace ! tubes :i* %[ no signs ( gr[? 9 ! incubator = a fur!r p]iod ( tw5ty-f\r h\rs & aga9 exam9e ) ref]5ce to ! same po9ts4 #i4 ,remove ^? tubes :i* give evid5ce ( gr[? f ! ,bu*n]'s tubes & c>efully pipette (f ! :ey2 exam9e ! :ey microscopically4 #aj4 ,inoculate two gu9ea-pigs ea* subcutane\sly ) #j4e c4c4 ( ! :ey & obs]ve ! result4 ,vibrio ,*ol]ae4-- #a4 ,numb] t5 tubes ( peptone wat] consecutively f #a to #aj4 #b4 ,to ea* ( ! tubes ( peptone wat] add a measur$ quantity ( ! susp5sion1 correspond+ to ^? am\nts employ$ 9 isolat+ ! memb]s ( ! coli gr\p 7vide page #dcg74 #c4 ,incubate a]obically at #cg deg4 ,c4 = tw5ty-f\r h\rs4 ,exam9e ! tubes c>efully = visible gr[?1 especially delicate pellicle =mation1 :i* if pres5t %\ld be exam9$ microscopically = vibrios1 bo? by /a9$ prep>ations or by fre% specim5s ) d>k gr\nd illum9ation4 #d4 ,inoculate fre% tubes ( peptone wat] f su* ( ! tubes z exhibit pellicle =mation--f ! pellicle itself--& incubate at #cg deg4 ,c4 = tw5ty-f\r h\rs4 #e4 ,te/ ! peptone wat] itself = ! pres5ce ( indol & nitrite by ! addition ( pure conc5trat$ ,h#b,,s#d4 #e4 ,prep>e gelat9e & ag> plates 9 ! usual way f su* ( ^! tubes z %[ pellicle =mation4 #f4 ,pick (f f ! plates any colonies resembl+ ^? ( ! ,vibrio *ol]ae & subcultivate ^u all ! ord9>y laboratory m$ia4 #g4 ,te/ ! vibrio isolat$ aga9/ ! s]um ( an animal immunis$ to ! ,vibrio *ol]ae = agglut9ation4 ,b4 ,an?racis4-- #a4 ,transf] #e c4c4 ( ! emulsion f ! filt] c&le to a /]ile te/-tube & plug c>efully4 #b4 ,place ! te/-tube 9 ! int]ior ( ! b5zole ba? employ$ 9 sep>at+ out spore-be>+ organisms 7vide page #beg71 & expose to a temp]ature ( #hj deg4 ,c4 = tw5ty m9utes4 #c4 ,inoculate a "y :ite rat subcutane\sly 7on ! inn] aspect ( "o ( ! h9d legs7 ) #a c4c4 ( ! emulsion4 ,obs]ve dur+ life1 &1 if ! animal succumbs1 make a complete po/-mortem exam9ation4 #d4 ,melt ?ree tubes ( nutri5t ag> 9 boil+ wat] & cool to #db deg4 ,c4 #e4 ,numb] ! tubes #a1 #b1 & #c4 ,to ,no4 #a add #j4b c4c41 to ,no4 #b add #j4c c4c41 & to ,no4 #c add #j4e c4c4 ( ! susp5sion1 & p\r plates !refrom4 #f4 ,incubate at #cg deg4 ,c4 = tw5ty-f\r or =ty-eight h\rs4 #g4 ,pick (f any colonies resembl+ ^? ( an?rax & subcultivate on all ! ord9>y laboratory m$ia4 #h4 ,inoculate ano!r "y :ite rat z 9 #c1 us+ two loopfuls ( ! ag> subcultivation emulsifi$ ) #a c4c4 /]ile b\illon4 ,obs]ve dur+ life1 & if ! animal succumbs1 make a complete po/-mortem exam9ation4 ,b4 ,tetani4-- #a4 ,proce$ z detail$ above 9 /eps #a & #b = ! isolation ( ! ,b4 an?racis4 #b4 ,add #a c4c4 ( ! susp5sion to ea* ( ?ree tubes ( glucose =mate bro?1 & incubate ana]obically 9 ,bu*n]'s tubes at #cg deg4 ,c4 #c4 ,f su* ( ! tubes z %[ visible gr[? 7) or )\t ! production ( gas7 aft] tw5ty-f\r h\rs' incubation inoculate gu9ea-pigs1 subcutane\sly 7"u ! sk9 ( ! abdom571 us+ #j4a c4c4 ( ! b\illon cultivation z a dose4 ,obs]ve c>efully dur+ life1 &1 if dea? occurs1 make a complete po/-mortem exam9ation4 #d4 ,f ! same tubes p\r ag> plates & incubate ana]obically 9 ,bullo*'s app>atus1 at #cg deg4 ,c4 #e4 ,subcultivate suspici\s colonies on ! v>i\s m$ia1 incubate ana]obically1 mak+ control cultivations on glucose =mate ag>1 /ab & /reak1 to incubate a]obically & c>ry out fur!r inoculation exp]im5ts ) ! result+ gr[?s4 ,,exam9ation ,,( ,,milk4 ,"o-c[ or :ole milk1 if tak5 f ! app>5tly heal?y animal 7t is1 an animal )\t any obvi\s lesion ( ! udd] or teats7 ) ord9>y precautions z to cleanl9ess1 avoidance ( du/1 etc41 conta9s b few organisms4 ,9 deal+ ) "o-c[ milk1 f a suspect$1 or an obvi\sly diseas$ animal1 a complete analysis %\ld include ! exam9ation 7bo? qualitative & quantitative7 ( samples ( 7a7 =e-milk1 7b7 mid-milk1 7c7 /ripp+s1 &1 if possible1 f ea* qu>t] ( ! udd]4 ,mix$ milk1 on ! o!r h&1 by ! "t x leaves ! retail]'s h&s1 usually conta9s z _m micro-organisms z an equal volume ( sewage & inde$ dur+ ! exam9ation x is treat$ z su*4 ,x is possible h[ev] to collect & /ore mix$ milk 9 s cleanly a mann] t its g]m cont5t does n exce$ #ejjj micro-organisms p] cubic c5timetre4 ,su* comp>ative fre$om f extrane\s bact]ia is usually secur$ by ! purveyor only :5 he resorts to ! process ( pa/eurisation 7heat+ ! milk to #fe deg4 ,c4 = tw5ty m9utes or to #gg deg4 ,c4 = "o m9ute7 or ! simpl] plan ( add+ pres]vatives to ! milk4 ,in=mation reg>d+ ! employm5t ( ^! me?ods = ! de/ruction ( bact]ia %\ld always be s\ght 9 ! case ( mix$ milk samples1 & 9 ?is connection ! foll[+ te/s w be f\nd useful3 #a4 ,raw ,milk 7,saul74 ,to #aj c4c4 milk 9 a te/ tube1 add #a c4c4 ( a #a p] c5t4 aque\s solution ( ortol 7or?o-me?yl-am9o-ph5ol sulphate71 rec5tly prep>$ & mix4 ,next add #j4b c4c4 ( a #c p] c5t4 p]oxide ( hydrog5 solution4 ,! appe>ance ( a brick r$ color )9 #cj seconds indicates raw milk4 ,milk heat$ to #gd deg4 ,c4 = ?irty m9utes und]goes no alt]ation 9 color2 if heat$ to #ge deg4 ,c4 = t5 m9utes only1 ! brick r$ color appe>s aft] /&+ = ab\t two m9utes4 #b4 ,boric ,acid4 ,evaporate to dryness1 #ej c4c4 ( ! milk :i* has be5 r5d]$ slightly alkal9e to litmus1 !n inc9]ate4 ,dissolve 9 di/ill$ wat]1 add slight excess ( dilute hydro*loric acid & aga9 evaporate to dryness4 ,dissolve ! residue 9 a small quantity ( hot wat] & moi/5 a piece ( turm]ic pap] ) ! solution4 ,dry ! turm]ic pap]4 ,rose or *]ry-r$ color borax or boric acid4 #c4 ,=maldehyde 7,hehn]74 ,to #aj c4c4 milk 9 a te/ tube add #e c4c4 conc5trat$ comm]cial sulphuric acid sl[ly1 s t ! two fluids d n mix4 ,hold ! tube v]tically & agitate v g5tly4 ,violet zone at ! junction ( ! two liquids =maldehyde4 #d4 ,hydrog5 ,p]oxide4 ,to #aj c4c4 milk 7dilut$ ) equal quantities ( wat]7 9 a te/ tube add #j4d c4c4 ( a #d p] c5t4 alcoholic solution ( b5zid9e & #j4b c4c4 acetic acid4 ,blue coloration ( ! mixture hydrog5 p]oxide4 #e4 ,salicylic ,acid4 ,precipitate ! case9og5 by ! addition ( acetic acid & filt]4 ,to ! filtrate add a few drops ( #a p] c5t4 aque\s solution ( f]ric *loride4 ,purple coloration salicylic acid4 #f4 ,sodium ,c>bonate or ,bic>bonate4 ,to #aj c4c4 ( ! milk 9 a te/ tube add #aj c4c4 ( alcohol & #j4c c4c4 ( a #a p] c5t4 alcoholic solution ( rosolic acid4 ,br[ni% color pure milk2 rose color pres]v$ milk4 ,illu/ration3 ,,fig4 #baa4--,milk-collect+ bottle & dipp] 9 case4 ,quantitative4-- ,collection ( ,sample4-- ,! app>atus us$ = ! collection ( a retail mix$ milk sample consi/s ( a cyl9drical copp] case1 #af cm4 high & #i cm4 9 diamet]1 provid$ ) a pull-(f lid1 conta9+ a milk dipp]1 also made ( copp]2 & inside ?is1 aga91 a wide-m\!d1 /opp]$ glass bottle ( ab\t #bej c4c4 capacity 7ab\t #ad cm4 high by #g cm4 diamet]71 hav+ a tablet = notes1 s&-bla/$ on ! side4 ,! copp] cyl9d] & its cont5ts1 secur$ f %ak+ by pack+ ) cotton-wool1 are /]ilis$ 9 ! hot-air ov5 7,fig4 #bf74 ,:5 collect+ a sample1 #a4 ,remove ! cap f ! cyl9d]4 #b4 ,draw out ! cotton-wool4 #c4 ,lift out ! bottle & dipp] toge!r4 #d4 ,receive ! milk 9 ! /]ile dipp]1 & p\r x directly into ! /]ile bottle4 #e4 ,ent] ! p>ticul>s necess>y = ! id5tification ( ! specim51 on ! tablet1 ) a lead p5cil1 or p5 & ink4 #f4 ,pack ! app>atus 9 ! ice-box = transmission to ! laboratory 9 precisely ! same mann] z an ord9>y wat] sample4 ,:ole milk may ) advantage be collect$ 9 ! /]ile bottle directly s9ce ! m\? is suffici5tly wide = ! milk] to direct ! /ream ( milk into x4 ,cond5s$ milk mu/ be dilut$ ) /]ile di/ill$ wat] 9 accordance ) ! directions pr9t$ ^u ! label1 !n treat$ z ord9>y milk4 ,app>atus ,requir$3 ,case ( /]ile capsules 7#be c4c4 capacity74 ,case ( /]ile graduat$ pipettes1 #aj c4c4 79 t5?s ( a cubic c5timetre74 ,case ( /]ile graduat$ pipettes1 #a c4c4 79 t5?s ( a cubic c5timetre74 ,flask conta9+ #bej c4c4 /]ile b\illon4 ,tall cyl9d] conta9+ #b p] c5t4 lysol solution4 ,plate-levell+ /&4 ,case ( /]ile plates4 ,tubes nutri5t gelat9e or gelat9e ag>4 ,tubes ( wort gelat9e4 ,tubes ( nutri5t ag>4 ,wat]-ba? regulat$ at #db deg4 ,c4 ,buns5 burn]4 ,grease p5cil4 ,,me?od4-- #a4 ,arrange f\r /]ile capsules 9 a r[2 numb] !m ,i1 ,,ii1 ,,iii1 & ,,iv4 #b4 ,fill #i c4c4 /]ile b\illon into ! fir/1 & #i4i c4c4 b\illon into ea* ( ! ?ree rema9+ capsules4 #c4 ,remove #a c4c4 milk f "o ( ! bottles by means ( a /]ile pipette & add x to ! b\illon 9 capsule ,i2 mix ?or\ghly by repeat$ly fill+ & empty+ ! pipette4 #d4 ,remove #j4a c4c4 ( ! milky b\illon f capsule ,i1 add x to ! cont5ts ( capsule ,,ii1 & mix z be=e4 #e4 ,9 l mann] add #j4a c4c4 ( ! cont5ts ( capsule ,,ii to capsule ,,iii2 & !n #j4a c4c4 ( ! cont5ts ( capsule ,,iii to capsule ,,iv4 ,!n #a c4c4 ( dilution ,i conta9s #j4a c4c4 milk sample4 #a c4c4 ( dilution ,,ii conta9s #j4jja c4c4 milk sample4 #a c4c4 ( dilution ,,iii conta9s #j4jjjja c4c4 milk sample4 #a c4c4 ( dilution ,,iv conta9s #j4jjjjjja c4c4 milk sample4 #f4 ,melt ! gelat9e & ! ag> tubes 9 boil+ wat]2 !n transf] to ! wat]-ba? & cool !m d[n to #db deg4 ,c4 #g4 ,numb] ! gelat9e tubes consecutively #a to #ab4 #h4 ,inoculate ! tubes ) v>y+ quantities ( ! mat]ial z foll[s3 ,to tube ,no4 #a add #a4j c4c4 ( ! milk sample4 #b add #j4a c4c4 ( ! milk sample4 #c add #a4j c4c4 f capsule ,i4 #d add #j4a c4c4 f capsule ,i4 #e add #a4j c4c4 f capsule ,,ii4 #f add #j4a c4c4 f capsule ,,ii4 #g add #j4e c4c4 f capsule ,,iii4 #h add #j4c c4c4 f capsule ,,iii4 #i add #j4b c4c4 f capsule ,,iii4 #aj add #j4e c4c4 f capsule ,,iv4 #aa add #j4c c4c4 f capsule ,,iv4 #ab add #j4b c4c4 f capsule ,,iv4 #i4 ,p\r plates f ! gelat9e tubes2 label1 & incubate at #bj deg4 ,c4 #aj4 ,liquefy five wort gelat9e tubes & to !m add #a4j c4c4 ( ! milk sample & a simil> quantity ( ! dilut$ milk f capsules ,i1 ,,ii1 & ,,iii & ,,iv respectively4 #aa4 ,p\r plates f ! wort gelat9e2 label1 & incubate at #bj deg4 ,c4 #ab4 ,inoculate ! liquefi$ ag> tubes z foll[s3 ,to tube ,no4 #a add #j4a c4c4 ( ! milk sample4 #b add #j4a c4c4 f capsule ,i4 #c add #j4a c4c4 f capsule ,,ii4 #d add #j4a c4c4 f capsule ,,iii4 #e add #a4j c4c4 f capsule ,,iv4 #f add #j4a c4c4 f capsule ,,iv4 #ac4 ,p\r plates f ! ag> tubes2 label1 & incubate at #cg deg4 ,c4 #ad4 ,aft] tw5ty-f\r h\rs' incubation inspect1 & aft] =ty-eight h\rs' incubation1 c\nt ! ag> plates & e/imate ! numb] ( organisms gr[+ at #cg deg4 ,c4 pres5t p] cubic c5timetre ( ! sample ( milk4 #ae4 ,aft] ?ree1 f\r1 or five days' incubation1 c\nt ! gelat9e plates & e/imate !refrom ! numb] ( organisms gr[+ at #bj deg4 ,c4 pres5t p] cubic c5timetre ( ! sample ( milk4 #af4 ,aft] a simil> int]val c\nt ! wort gelat9e plates & e/imate ! numb] ( m\lds & yea/s pres5t p] cubic c5timetre ( ! sample ( milk4 ,,note4--,_m obs]v]s pref] to employ gelat9e ag> 7see page #aic7 = ! quantitative exam9ation4 ,9 ?is case gelat9e-ag> plates %\ld be p\r$ f tubes conta9+ ! quantities ( mat]ial indicat$ 9 /ep #h1 incubat$ at #bh deg4 ,c4 to #cj deg4 ,c4 & aft] five days ! total numb] ( organisms develop+ at #bh deg4 ,c4 record$4 ,qualitative4--,! qualitative bact]iological exam9ation ( milk is *iefly direct$ to ! detection ( ! pres5ce ( "o or m ( ! foll[+ pa?og5ic bact]ia & :5 pres5t to ! e/imation ( _! num]ical frequ5cy4 ,memb]s ( ! ,coli-typhoid gr\p4 ,vibrio *ol]ae4 ,/reptococcus pyog5es longus4 ,micrococcus melit5sis4 ,/aphylococcus pyog5es aureus4 ,bacillus ent]itidis sporog5es4 ,bacillus diph!riae4 ,bacillus tub]culosis4 ,"s ( ^! occur z accid5tal contam9ations1 ei!r f ! wat] supply to ! c[ f>m1 or f ! f>m employees1 :il/ o!rs are d]iv$ directly f ! c[4 ,9 milk1 z 9 wat] exam9ations1 two me?ods are available1 viz43 ,enri*m5t & ,conc5tration--! =m] is us$ = ! demon/ration ( bact]ia ( inte/9al orig91 ! latt] = ! isolation ( ! micro-organisms ( diph!ria & tub]cle4 ,! fir/ ess5tial 9 ! latt] process is ! conc5tration ( ! bact]ial cont5ts ( a l>ge volume ( ! sample into a small compass2 b 9 ! case ( milk1 ?or\gh c5trifugalisation is sub/itut$ = filtration4 ,app>atus ,requir$3 ,a l>ge c5trifugal ma*9e4 ,?is ma*9e1 to be ( real s]vice 9 ! bact]iological exam9ation ( milk1 mu/ con=m to ! foll[+ requirem5ts3 #a4 ,! c5trifugal ma*9e mu/ be ( su* size1 & %\ld c>ry tubes or bottles ( su* capacity1 z to enable f #bjj to #ejj c4c4 ( milk to be manipulat$ at "o "t4 #b4 ,! rate ( c5trifugalisation %\ld be f #bejj to #cjjj revolutions p] m9ute4 #c4 ,! portion ( ! ma*9e de/9$ to c>ry ! tubes %\ld be a metal disc1 ( suffici5t weight to ensure good flank movem5t1 cont9u+ ov] a consid]able p]iod ( "t4 ,9 o!r words1 ! ma*9e %\ld run d[n v gradually & sl[ly aft] ! motive p[] is remov$1 ?us obviat+ any di/urbance ( ! relative positions ( p>ticulate matt] 9 ! solution t is be+ c5trifugalis$4 #d4 ,! ma*9e %\ld pref]ably be driv5 by electricity1 or by p[]1 b 9 ! case ( h&-driv5 ma*9es-- 7a7 ,! ge>+ %\ld be s arrang$ t ! requisite spe$ is obta9$ by n m ?an =ty or fifty revolutions ( ! crank h&le p] m9ute1 s t x may be ma9ta9$ = p]iods ( tw5ty or ?irty m9utes )\t undue ex]tion4 7b7 ,! h&le employ$ %\ld be provid$ ) a special fa/5+ 7e4 g41 a clut* simil> to t employ$ = ! free :eel ( a bicycle71 or %\ld be readily deta*able s t1 on ceas+ to turn1 ! h&le %\ld n1 by its weight & air resi/ance1 act z a brake & /op ! ma*9e too sudd5ly4 ,"o ( ! few satisfactory ma*9es ( ?is class is %[n 9 figure #bab4 ,illu/ration3 ,,fig4 #bab4--;,electrically driv5 c5trifugal ma*9e1 ) flexible 7brok57 sp9dle encircl$ by ! field magnets ( ! motor4 ,/]ile c5trifugal tubes1 ( "s #fj-gj c4c4 capacity1 tap]+ to a po9t at ! clos$ end1 plugg$ ) cotton-wool4 ,small c5trifugal ma*9e to run two tubes ( #aj c4c4 capacity at #bejj to #cjjj revolutions p] m9ute pref]ably driv5 by electricity1 ( ! type figur$ on page #cbg 7,fig4 #afb74 ,/]ile c5trifugal tubes ( #aj c4c4 capacity ) ! di/al extremity contract$ to a n>r[ tube & graduat$ 9 hundr$?s ( a cubic c5timetre 7,fig4 #bac74 ,/]ilis$ cork bor]4 ,case ( /]ile pipettes1 #aj c4c4 79 t5?s ( a cubic c5timetre74 ,case ( /]ile pipettes1 #a c4c4 79 t5?s ( a cubic c5timetre74 ,/]ile teat pipettes4 ,flask ( /]ile normal sal9e solution4 ,,me?od4-- #a4 ,fill #ej c4c4 ( ! milk sample into ea* ( f\r tubes1 & replace ! cotton-wool plugs by solid rubb] /opp]s 7/]ilis$ by boil+71 & fit ! tubes 9 ! c5trifugal ma*9e4 ,,note4--,"o or two cubic c5timetres ( p>aff9um liquidum introduc$ into ! buckets ( ! c5trifuge be=e ! glass tubes are ins]t$ w obviate any risk ( breakage to ! latt]4 ,illu/ration3 ,,fig4 #bac4--,milk s$im5t+ tubes4 ,illu/ration3 ,,fig4 #bad4--,milk 9 c5trifuge tube4 #b4 ,c5trifugalise ! milk sample = ?irty m9utes at a spe$ ( #bejj revolutions p] m9ute4 #c4 ,remove ! motive p[] & all[ ! ma*9e to sl[ d[n gradually4 #d4 ,remove ! tubes ( milk f ! c5trifuge4 ,ea* tube w n[ %[ 7,fig4 #bad73 7a7 ,a sup]ficial lay] ( cream 7v>y+ 9 ?ickness ) diff]5t samples7 cond5s$ into a semi-solid mass1 :i* c be %[n to conta9 "s organisms & a few leucocytes4 7b7 ,a c5tral lay] ( sep>at$ milk1 ?91 wat]y1 & opalesc5t1 & conta9+ extremely few bact]ia4 7c7 ,a s$im5t or deposit consi/+ ( ! great majority ( ! conta9$ bact]ia & leucocytes1 toge!r ) adv5titi\s matt]1 su* z dirt1 hair1 epi!lial cells1 faecal debris1 etc4 #e4 ,)draw ! rubb] /opp] & remove a c5tral plug ( cream f ea* tube by means ( a /]ile cork bor]2 place ^! masses ( cream 9 two /]ile capsules4 ,label ,c#a & ,c#b4 #f4 ,remove all b ! la/ "o or two c4c4 ( sep>at$ milk f ea* tube1 by means ( /]ile pipettes4 #g4 ,mix ! deposits ?or\ghly ) ! residual milk1 pipette ! mixture f ea* pair ( tubes into "o /]ile #aj c4c4 tube 7graduat$7 by means ( /]ile teat pipettes1 !n fill to ! #aj c4c4 m>k ) /]ile normal sal9e solution & mix toge!r4 ,label ,d#a & ,d#b4 #h4 ,place ! two tubes ( mix$ deposit 9 ! c5trifuge1 adju/ by ! addition or subtraction ( sal9e solution s t !y c\nt]poise exactly1 & c5trifugalise = t5 m9utes4 ,,note4--,ea* tube n[ conta9s ! deposit f #ajj c4c4 ( ! milk sample & ! am\nt c be read (f 9 hundr$?s ( a c5timetre4 ,! multiplication ( ?is figure by #ajj w give ! am\nt ( ,app>5t ,fil?1 9 p>ts p] million--! usual me?od ( record+ ?is quality ( milk4 #i4 ,pipette (f all ! sup]natant fluid & inv]t ! tube to dra9 on to a pad ( /]ilis$ cotton-wool1 conta9$ 9 a beak]4 7,?is wool is subsequ5tly cremat$47 #aj4 ,exam9e bo? cream 7,c#a7 & deposit 7,d#a7 microscopically-- 7a7 ,9 hang+-drop prep>ations4 7b7 ,9 film prep>ations /a9$ c>bolic me?yl5e-blue1 by ,gram's me?od1 by ,neiss]'s me?od1 & by ,ziehl-,neels5's me?od4 ,note ! pres5ce or abs5ce ( alt]$ & unalt]$ vegetable fibres2 pus cells1 blood discs2 cocci 9 gr\ps or *a9s1 diph!roid bacilli1 ,gram negative bacilli or cocci1 spores & acid fa/ bact]ia4 #aa4 ,adapt ! f9al /ages ( ! inve/igation to ! special requirem5ts ( ea* individual sample1 ?us3 #a4 ,memb]s ( ! ,coli-typhoid ,gr\p4-- #a4 ,emulsify ! deposit f ! second c5trifugal tube 7,d#b7 ) #aj c4c4 /]ile b\illon & inoculate ?ree tubes ( bile salt bro? z foll[s3 ,to ,tube ,no4 #a add #b4e c4c4 milk deposit emulsion 7#be c4c4 orig9al milk47 ,to ,tube ,no4 #b add #a4j c4c4 milk deposit emulsion 7#aj c4c4 orig9al milk47 ,to ,tube ,no4 #c add #j4e c4c4 milk deposit emulsion 7 #e c4c4 orig9al milk47 #b4 ,inoculate tube ( bile salt bro? ,no4 #d ) #a c4c4 ( ! orig9al milk4 #c4 ,inoculate fur!r tubes ( bile salt bro? ) previ\sly prep>$ dilutions 7see page #dde7 z foll[s3 ,to tube ,no4 #e add #a4j c4c4 f capsule ,i4 ,to tube ,no4 #f add #j4a c4c4 f capsule ,i4 ,to tube ,no4 #g add #a4j c4c4 f capsule ,,ii4 ,to tube ,no4 #h add #j4a c4c4 f capsule ,,ii4 ,to tube ,no4 #i add #a4j c4c4 f capsule ,,iii4 ,to tube ,no4 #aj add #j4a c4c4 f capsule ,,iii4 ,to tube ,no4 #aa add #a4j c4c4 f capsule ,,iv4 ,to tube ,no4 #ab add #j4a c4c4 f capsule ,,iv4 & incubate ana]obically 79 ,bu*n]'s tubes7 at #db deg4 ,c4 = a maximum p]iod ( =ty-eight h\rs4 #d4 ,if gr[? occurs complete ! inve/igation z detail$ "u ! correspond+ section ( wat] exam9ation 7see pages #dbh to #dca74 ,,note4--,! ,b4 coli communis1 d]iv$ f ! alv9e dis*>ges ( ! c[1 is almo/ univ]sally pres5t 9 l>ge or small numb]s1 9 retail milk4 ,its detection1 !re=e1 unless 9 enorm\s numb]s1 7:5 x indicates want ( cleanl9ess71 is ( little value4 #b4 ,vibrio ,*ol]ae4--,inoculate tubes ( peptone wat] by us+ ! same am\nts z 9 ! se>* = memb]s ( ! ,coli-typhoid gr\ps 7vide ante #a-c72 incubate a]obically at #cg deg4 ,c4 & complete ! exam9ation z detail$ "u ! correspond+ section ( wat] exam9ation 7see page #dci74 #c4 ,b4 ,ent]itidis ,sporog5es4--,inoculate tubes ( litmus milk ) simil> am\nts to ^? us$ 9 ! previ\s se>*es1 omitt+ tube ,no4 #a 7vide ante #a-c7 place 9 ! diff]5tial /]ilis] at #hj deg4 ,c4 = t5 m9utes & !n incubate ana]obically at #cg deg4 ,c4 = a maximum p]iod ( =ty-eight h\rs4 ,complete ! inve/igation z detail$ "u ! correspond+ section ( wat] exam9ation 7see page #dch74 #d4 ,b4 ,diph!riae4-- 7,a7 #a4 ,plant ?ree sets ( s]ial cultivations1 twelve tubes 9 ea* set1 f 7a7 cream ,c#b1 7b7 deposit ,d#a ^u oblique inspissat$ blood-s]um1 & incubate at #cg deg4 ,c4 #b4 ,pick (f any suspici\s colonies :i* may h made _! appe>ance twelve h\rs aft] incubation1 exam9e microscopically & subcultivate ^u blood-s]um & place 9 ! incubator2 return ! orig9al tubes to ! incubator4 #c4 ,repeat ?is aft] eighte5 h\rs' incubation4 #d4 ,f ! result+ gr[?s make cov]-slip prep>ations & /a9 c>bolic me?yl5e-blue1 ,neiss]'s me?od1 ,gram's me?od4 ,subcultivate su* z appe> to be compos$ ( diph!ria bacilli 9 glucose peptone solution4 ,note ^? 9 :i* acid production takes place4 #e4 ,inoculate gu9ea-pigs subcutane\sly ) "o or two cubic c5timetres =ty-eight-h\r-old glucose b\illon cultivation d]iv$ f ! fir/ subcultivation ( ea* glucose f]m5t]1 & obs]ve ! result4 #f4 ,if dea?1 app>5tly f diph!ritic toxaemia1 ensues1 inoculate two m gu9ea pigs ) a simil> quantity ( ! le?al culture4 ,res]ve "o animal z a control & into ! o!r inject #ajjj units ( antidiph!ritic s]um4 ,if ! control dies & ! treat$ animal survives1 ! pro( ( ! id5tity ( ! organism isolat$ ) ! ,klebs-,loeffl] bacillus becomes absolute4 #g4 ,inoculate gu9ea-pigs subcutane\sly ) filt]$ glucose b\illon cultivations 7tox9s87 & obs]ve ! result4 7,b7 #a4 ,emulsify ! rema9d] ( ! deposit ) #e c4c4 /]ile b\illon & inoculate two gu9ea-pigs1 ?us3 gu9ea-pig a1 subcutane\sly ) #a c4c4 emulsion2 gu9ea-pig b1 subcutane\sly ) #b c4c4 emulsion2 & obs]ve ! result4 #b4 ,if ei!r or bo? ( ! inoculat$ animals succumb1 make complete po/-mortem exam9ation & endeav\r to isolate ! pa?og5ic organisms f ! local lesion4 ,confirm _! id5tity z 9 ,a#e & #f 7vide supra74 #e4 ,bacillus ,tub]culosis4-- 7,a7 #a4 ,inoculate ea* ( ?ree gu9ea-pigs 7previ\sly te/$ ) tub]cul91 to prove _! fre$om f spontane\s tub]culosis7 subcutane\sly at ! inn] aspect ( ! b5d ( ! left knee1 ) #a c4c4 ( ! deposit emulsion rema9+ 9 "o or o!r tube 7,d#a or ,d#b74 #b4 ,introduce a small quantity ( ! cream into a subcutane\s pocket prep>$ at ! inn] aspect ( ! b5d ( ! "r knee ( ea* ( ^! ?ree animals4 ,place a seal$ dress+ on ! w\nd4 #c4 ,obs]ve c>efully1 & weigh accurately ea* day4 #d4 ,kill "o gu9ea-pig at ! end ( ! second week & make a complete po/-mortem exam9ation4 #e4 ,if ! result ( ! exam9ation is negative or inconclusive1 kill a second gu9ea-pig at ! end ( ! ?ird week & exam9e c>efully4 ,illu/ration3 ,,fig4 #bae4--;,cadav] ( gu9ea-pig exp]im5tally infect$ ) ,b4 tub]culosis4 #f4 ,if /ill negative or inconclusive1 kill ! ?ird gu9ea-pig at ! end ( ! six? week4 ,make a c>eful po/-mortem exam9ation4 ,exam9e mat]ial f any case\s gl&s microscopically & inoculate freely on to ,dorset's egg m$ium4 ,,note4--,e po/-mortem exam9ation ( animals infect$ ) tub]cul\s mat]ial %\ld include ! nak$ eye & microscopical exam9ation ( ! popliteal1 sup]ficial & deep +u9al1 iliac1 lumb> & axill>y gl&s on ea* side ( ! body1 also ! retrohepatic1 bron*ial & /]nal gl&s1 ! sple51 liv] & lungs 7,fig4 #bae74 7,b7 #a4 ,intimately mix all ! available cream & deposit f ! milk sample1 & transf] to a /]ile ,erl5mey] flask4 #b4 ,treat ! mixture by ! anti=m9 me?od 7vide ,app5dix1 page #ejb74 #c4 ,inoculate ea* ( two gu9ea-pigs1 intrap]itoneally1 ) half ( ! emulsion ?us obta9$4 #d4 ,kill "o ( ! gu9ea-pigs at ! end ( ! fir/ week & exam9e c>efully4 #e4 ,kill ! second gu9ea-pig at ! end ( ! second week & exam9e c>efully4 #f4 ,utilise ! rema9d] ( ! deposit = microscopical exam9ation & cultivations ^u ,dorset's egg m$ium4 ,,note4--,no value :atev] atta*es to ! result ( a microscopical exam9ation = ! pres5ce ( ! ,b4 tub]culosis unless confirm$ by ! result ( inoculation exp]im5ts4 #f4 ,/reptococcus ,pyog5es ,longus4-- 7,a7 #a4 ,spread s]ial surface plates ^u nutrose ag>4 ,also plant s]ial cultivations ^u slop$ nutri5t ag> 7six tubes 9 s]ies74 #b4 ,if ! result+ gr[? %[s colonies :i* resemble ^? ( ! /reptococcus1 make subcultivations ^u ag> & 9 b\illon1 9 ! fir/ in/ance1 & /udy c>efully4 7,b7 #a4 ,plant a l>ge loopful ( ! deposit ,d#b into ea* ( ?ree tubes ( glucose =mate b\illon1 & incubate ana]obically 79 ,bu*n]'s tubes7 = tw5ty-f\r h\rs at #cg deg4 ,c4 #b4 ,if ! result+ gr[? resembles t ( ! /reptococcus1 make subcultivations ^u nutri5t ag>4 #c4 ,prep>e subcultivations ( any suspici\s colonies t appe>1 ^u all ! ord9>y m$ia1 & /udy c>efully4 ,if ! /reptococcus is successfully isolat$1 inoculate s]um b\illon cultivations into ! m\se1 gu9ea-pig1 & rabbit1 to det]m9e its pa?og5icity & virul5ce4 #g4 ,/aphylococcus ,pyog5es ,aureus4-- #a4 ,exam9e c>efully ! gr[? ^u ! s]ial blood s]um cultivations prep>$ to isolate ,b4 diph!riae & ! s]ial ag> cultivations to isolate /reptococci aft] =ty-eight h\rs' incubation4 #b4 ,pick (f any suspici\s orange col\r$ colonies1 plant on slop$ ag>1 & incubate at #bj deg4 ,c4 ,obs]ve pigm5t =mation4 #c4 ,prep>e subcultivations f any suspici\s gr[?s ^u all ! ord9>y m$ia1 /udy c>efully & inve/igate _! pa?og5icity4 #h4 ,micrococcus ,melit5sis4--,! milk f an animal infect$ ) ,m4 melit5sis usually conta9s ! organisms 9 l>ge numb]s & b few o!r bact]ia4 #a4 ,spread sev]al sets ( surface plates ^u nutrose ag>1 ea* f "o loopful ( ! deposit 9 tube ,d#a or ,d#b4 #b4 ,spread sev]al sets ( surface plates ^u nutrose ag>1 ea* f "o drop ( ! orig9al milk sample4 #c4 ,incubate a]obically at #cg deg4 ,c4 & exam9e daily up to ! end ( t5 days4 #d4 ,pick (f suspici\s colonies1 exam9e !m microscopically & subcultivate ^u nutrose ag> 9 tubes2 ^u glucose ag> & 9 litmus milk4 #e4 ,te/ ! subsequ5t gr[? aga9/ ! s]um ( an exp]im5tal animal inoculat$ aga9/ ,m4 melit5sis to det]m9e its agglut9ability4 #f4 ,if app>5tly ,m4 melit5sis1 inoculate gr[? f a nutrose ag> culture aft] ?ree days incubation intracranially into ! gu9ea-pig4 ,,ice ,,cream4 ,collection ( ! ,sample4-- #a4 ,remove ! sample f ! drum 9 ! ladle or spoon ) :i* ! v5dor retails ! ice cream1 & place x at once 9 a /]ile copp] capsule1 simil> to t employ$ = e>? samples 7vide page #dga74 #b4 ,pack = transmission 9 ! ice-box4 #c4 ,on arrival at ! laboratory place ! copp] capsules conta9+ ! ice cream 9 ! incubator at #bj deg4 ,c4 = fifte5 m9utes--t is1 until at lea/ "s ( ! ice cream has become liquid4 ,qualitative & ,quantitative ,exam9ation4--,treat ! fluid ice cream z milk & conduct ! exam9ation 9 precisely ! same mann] z describ$ = milk 7vide page #ddc74 ,,exam9ation ,,( ,,cream ,,& ,,butt]4 ,collection ( ! ,sample4--,collect1 /ore1 & transmit samples to ! laboratory1 precisely z is done 9 ! case ( ice cream4 ,quantitative4-- ,app>atus ,requir$3 ,/]ile te/-tube4 ,/]ilis$ spatula4 ,wat]-ba? regulat$ at #db deg4 ,c4 ,case ( /]ile plates4 ,case ( /]ile graduat$ pipettes1 #a c4c4 79 hundr$?s74 ,tubes ( gelat9e-ag> 7#aj reaction74 ,plate-levell+ /&1 ) its wat] *amb] fill$ ) wat] at #db deg4 ,c4 ,,me?od4-- #a4 ,transf] a few grammes ( ! sample to a /]ile te/-tube by means ( ! /]ilis$ spatula4 #b4 ,place ! tube 9 ! wat]-ba? at #db deg4 ,c4 until ! cont5ts are liquid4 #c4 ,liquefy eight tubes ( gelat9e-ag> & place !m 9 ! wat]-ba? at #db deg4 ,c1 & cool d[n to t temp]ature4 #d4 ,inoculate ! gelat9e-ag> tubes ) ! foll[+ quantities ( ! sample by ! help ( a /]ile pipette graduat$ to hundr$?s ( a cubic c5timetre--viz41 ,to tube ,no4 #a add #a c4c4 liquefi$ butt]4 #b add #j4e c4c4 liquefi$ butt]4 #c add #j4c c4c4 liquefi$ butt]4 #d add #j4b c4c4 liquefi$ butt]4 #e add #j4a c4c4 liquefi$ butt]4 #f add #j4je c4c4 liquefi$ butt]4 #g add #j4jc c4c4 liquefi$ butt]4 #h add #j4jb c4c4 liquefi$ butt]4 #i add #j4ja c4c4 liquefi$ butt]4 #e4 ,p\r a plate cultivation f ea* ( ! gelat9e-ag> tubes & incubate at #bh deg4 ,c4 #f4 ,c\nt ! plates aft] ?ree days' incubation1 & f ! figures ?us obta9$ e/imate ! numb] ( organisms pres5t p] cubic c5timetre ( ! sample4 ,qualitative4-- ,app>atus ,requir$3 ,/]ile beak]1 its m\? plugg$ ) /]ile cotton-wool4 ,c\nt]poise = beak]4 ,scales & weights4 ,/]ilis$ spatula4 ,wat]-ba? regulat$ at #db deg4 ,c4 ,sep>atory funnel1 #bej c4c4 capacity1 its deliv]y tube protect$ aga9/ contam9ation by pass+ x ?r\gh a cotton-wool plug into ! int]ior ( a small ,erl5mey] flask :i* s]ves to support ! funnel4 ,?is piece ( app>atus is /]ilis$ en masse 9 ! hot-air ov54 ,l>ge c5trifugal ma*9e4 ,/]ile tubes 7= ! c5trifuge7 clos$ ) solid rubb] /opp]s4 ,case ( /]ile pipettes1 #aj c4c4 ,case ( /]ile graduat$ pipettes1 #a c4c4 79 t5?s ( a cubic c5timetre74 ,,me?od4-- #a4 ,weigh out #ajj grammes ( ! sample 9 a /]ile beak]4 #b4 ,plug ! m\? ( ! beak] ) /]ile cotton-wool & imm]se ! beak] 9 a wat]-ba? at #db deg4 ,c4 until ! cont5ts are completely liquefi$4 #c4 ,fill ! liquefi$ butt] into ! /]ile sep>atory funnel4 #d4 ,transf] ! funnel to ! incubator at #cg deg4 ,c4 & all[ x to rema9 !re = f\r days4 ,at ! end ( ?is "t ! cont5ts ( ! funnel w h sep>at$ into two di/9ct /rata4 7a7 ,a sup]ficial oily lay]1 practically free f bact]ia4 7b7 ,a deep wat]y lay]1 turbid & cl\dy f ! gr[? ( bact]ia4 #e4 ,draw (f ! subnatant turbid lay] into /]ile c5trifugal tubes1 previ\sly w>n$ to ab\t #db deg4 ,c41 & c5trifugalise at once4 #f4 ,pipette (f ! sup]natant fluid & fill ! tubes ) /]ile #a p] c5t4 sodium c>bonate solution previ\sly w>m$ slightly2 /opp] ! tubes & %ake vig\r\sly = a few m9utes4 #g4 ,c5trifugalise aga94 #h4 ,pipette (f ! sup]natant fluid2 fill+ ! tubes ) w>m /]ile b\illon1 %ake well1 & aga9 c5trifugalise1 to wa% ! deposit4 #i4 ,pipette (f ! sup]natant fluid4 #aj4 ,prep>e cov]-slip prep>ations1 fix & cle> z = milk prep>ations1 /a9 c>bolic me?yl5e-blue1 ,gram's me?od1 ,ziehl-,neels5's me?od1 & exam9e microscopically ) a #a_/ab in* oil-imm]sion l5s4 #aa4 ,proce$ ) ! exam9ation ( ! deposit z 9 ! case ( milk deposit 7see pages #dej et seq474 ,,exam9ation ,,( ,,uns\nd ,,meats4 7,,includ+ ,,t9n$ ,,or ,,pott$ ,,meats1 ,,fi%1 ,,etc47 ,! bact]ioscopic exam9ation ( uns\nd food is *iefly direct$ to ! detection ( ^? memb]s ( ! ,coli-typhoid gr\p--,b4 ent]itidis ( ,ga]tn] & its allies--:i* are usually associat$ ) epidemic outbreaks ( food poison+1 & su* ana]obic bact]ia z initiate putrefactive *anges 9 ! food :i* result 9 ! =mation ( poison\s ptoma9es1 consequ5tly ! quantitative exam9ation pure & simple is frequ5tly omitt$4 ,a4 ,cultural ,exam9ation4 ,quantitative4-- ,app>atus ,requir$3 ,/]ilis$ t9 op5]1 7if necess>y47 ,erl5mey] flask 7#ejj c4c4 capacity7 conta9+ #bjj c4c4 /]ile b\illon & fitt$ ) solid rubb] /opp]4 ,c\nt]poise4 ,scissors & =ceps4 ,scales & weights4 ,wat] /]ilis]4 ,hypod]mic syr+e4 ,syr+e ) intraga/ric tube4 ,rat =ceps4 ,case ( /]ile capsules4 ,filt]+ app>atus z = wat] analysis4 ,case ( /]ile plates4 ,case ( /]ile graduat$ pipettes1 #aj c4c4 79 t5?s ( a cubic c5timetre74 ,case ( /]ile graduat$ pipettes1 #a c4c4 79 t5?s ( a cubic c5timetre74 ,plate-levell+ /&4 ,tubes ( nutri5t gelat9e4 ,tubes ( nutri5t ag>4 ,wat]-ba? regulat$ at #db deg4 ,c4 ,bullo*'s app>atus4 ,,me?od4-- #a4 ,place ! flask conta9+ #bjj c4c4 /]ile bro? on "o pan ( ! scales & c\nt]poise accurately4 #b4 ,m9ce a portion ( ! sample by ! aid ( /]ile scissors & =ceps1 & add ! m9c$ sample to ! b\illon 9 ! flask to ! ext5t ( #bj grammes4 #c4 ,make an extract by /&+ ! flask 9 ! incubator runn+ at #db deg4 ,c4 7or 9 a wat]-ba? regulat$ to t temp]ature7 = half an h\r1 %ak+ its cont5ts f "t to "t4 ,bett] results are obta9$ if an electrical %ak] is fitt$ inside ! incubator & ! flask kept 9 motion ?r\gh\t ! entire ?irty m9utes4 ,n[ e c5timetre conta9s ! bact]ia wa%$ out f #j4a gramme ( ! orig9al food4 #d4 ,inoculate tubes ( liquefi$ gelat9e z foll[s3 ,to tube ,no4 #a add #a4j c4c4 ( ! extract4 #b add #j4e c4c4 ( ! extract4 #c add #j4c c4c4 ( ! extract4 #d add #j4b c4c4 ( ! extract4 #e add #j4a c4c4 ( ! extract4 ,p\r plates f ^! tubes & incubate at #bj deg4 ,c4 #e4 ,prep>e a precisely simil> set ( ag> plates & incubate at #cg deg4 ,c4 #f4 ,pipette #e c4c4 ( ! extract into a /]ile tube1 heat 9 ! diff]5tial /]ilis] at #hj deg4 ,c4 = t5 m9utes4 #g4 ,f ! heat$ extract prep>e duplicate sets ( ag> & gelat9e plates & incubate ana]obically 9 ,bullo*'s app>atus at #cg deg4 ,c4 & #bj deg4 ,c4 respectively4 #h4 ,aft] ?ree days' incubation exam9e ! ag> plates bo? a]obic & ana]obic & enum]ate ! colonies develop$ f spores 7#g71 & f vegetative =ms & spores 7#e71 & calculate & record ! numb]s ( ea* gr\p p] gramme ( ! orig9al food4 #i4 ,aft] sev5 days' incubation 7or e>li] if compell$ by ! gr[? ( liquefy+ colonies7 enum]ate ! gelat9e plates 9 ! same way4 #aj4 ,subcultivate f ! colonies t make _! appe>ance & id5tify ! v>i\s organisms4 #aa4 ,cont9ue ! inve/igations ) ref]5ce to ! detection ( pa?og5ic organisms z describ$ "u wat] 7page #dbi et seq474 ,qualitative4-- ,i4 ,cultural4 ,! micro-organisms s\ght = dur+ ! exam9ation ( uns\nd foods comprise ! foll[+3 ,memb]s ( ! ,coli-typhoid gr\ps 7*iefly ^? ( ! ,ga]tn] class74 ,b4 an?racis4 ,/reptococci ,ana]obic ,bact]ia3 ,b4 ent]itidis sporog5es4 ,b4 botul9us4 ,b4 cadav]is4 ,! me?ods by :i* ^! organisms if pres5t may be id5tifi$ & isolat$ h already be5 describ$ "u ! correspond+ section ( wat] exam9ation ) ! exception ( ^? applicable to ,b4 botul9us1 & ,b4 cadav]is4 ,^! c only be isolat$ satisfactorily f ! bodies ( exp]im5tally inoculat$ animals4 ,,ii ,exp]im5tal4 ,tissue4-- #a4 ,fe$ rats & mice on portions ( ! sample & obs]ve ! result4 #b4 ,if any ( ! animals die1 make complete po/-mortem exam9ations & endeav\r to isolate ! pa?og5ic organisms4 ,extract4-- #a4 ,introduce v>i\s quantities ( ! b\illon extract into ! /oma*s ( sev]al rats1 mice & gu9ea-pigs repeat$ly ov] a p]iod ( two or ?ree days by ! intraga/ric me?od ( inoculation 7see page #cfg7 & obs]ve ! result4 ,gu9ea-pigs & mice are v susceptible to infection by ,b4 botul9us by ?is me?od2 rabbits less s4 #b4 ,inoculate rats1 mice1 & gu9ea-pigs subcutane\sly into deep pockets1 & intrap]itoneally ) v>i\s quantities ( ! b\illon extract1 & obs]ve ! result4 #c4 ,filt] "s ( ! extract ?r\gh a ,*amb]l& c&le & incubate ! filtrate to det]m9e ! pres5ce ( soluble tox9s4 #d4 ,if any ( ! animals succumb to ei!r ( ^! me?ods ( inoculation1 make c>eful po/-mortem exam9ations & endeav\r to isolate ! pa?og5ic organisms4 ,,! ,,exam9ation ,,( ,,oy/]s ,,& ,,o!r ,,%ellfi%4 ,on op5+ ! %ell ( an oy/] a c]ta9 am\nt ( fluid t]m$ liquor is f\nd to be pres5t4 ,?is v>ies 9 am\nt f a drop to _m cubic c5timetres 7#j4a c4c4 to #aj c4c47--9 ! latt] case ! bulk ( ! fluid is probably ! la/ quantum ( wat] +e/$ by ! bivalve be=e clos+ its %ell4 ,9 ord] to obta9 a work+ av]age ( ! bact]iological flora ( a sample1 t5 oy/]s %\ld be tak5 & ! body1 ga/ric juice & liquor %\ld be ?or\ghly mix$ be=e exam9ation4 ,! exam9ation1 z 9 deal+ ) o!r food /uffs1 is direct$ to ! se>* = memb]s ( ! ,coli-typhoid gr\p1 sewage /reptococci & p]haps also ,b4 ent]itidis sporog5es4 ,app>atus ,requir$3 ,two h>d nail bru%es4 ,liquid soap4 ,/]ile wat] 9 aspirator j> ) deliv]y nozzle controll$ by a spr+ clip4 ,/]ile oy/] knives4 ,/]ile glass di%1 ) cov]1 suffici5tly l>ge to accommodate t5 oy/]s4 ,/]ile =ceps4 ,/]ile scissors4 ,/]ile t[els or l>ge gauze pads4 ,/]ile graduat$ cyl9d]s #ajjj c4c4 capacity1 ) ei!r ! lid or ! bottom ( a /]ile ,petri di% inv]t$ ov] ! op5 m\? z a cov]4 ,glass rods4 ,corrosive sublimate solution1 #a p] mille4 ,bile salt bro? tubes4 ,litmus milk tubes4 ,surface plates ( nutrose ag>4 ,case ( /]ile pipettes1 #a c4c4 79 t5?s ( a c4c47 ,case ( /]ile pipettes1 #aj c4c4 79 t5?s ( a c4c47 ,case ( /]ile glass capsules4 ,erl5mey] flasks1 #bej c4c4 capacity4 ,d\ble /r5g? bile salt bro?4 ,,me?od4-- #a4 ,?or\ghly clean ! outside ( ! oy/] %ells by scrubb+ ea* 9 turn ) liquid soap & nail bru% "u a tap ( runn+ wat]4 ,!n1 hold+ an oy/] %ell 9 a pair ( /]ile =ceps wa% e "p ( ! outside ( ! %ell ) a /ream ( /]ile wat] runn+ f an aspirator j>2 deposit ! oy/] inside ! /]ile glass di%4 ,repeat ! process ) ea* ( ! rema9+ oy/]s4 #b4 ,be=e proce$+ fur!r1 cleanse ! h&s ?or\ghly ) clean nail bru%1 soap & wat]1 !n plunge !m 9 lysol #b p] c5t4 solution1 & f9ally 9 /]ile wat]4 #c4 ,spread a /]ile t[el on ! b5*4 #d4 ,remove "o ( ! oy/]s f ! /]ile glass di% & place x1 re/+ on its convex %ell1 on ! t[el4 ,turn a corn] ( ! /]ile t[el ov] ! upp] flat %ell to give a firm] grip to ! left h&1 :i* holds ! %ell 9 position4 #e4 ,) ! /]ile oy/] knife 79 ! "r h&7 op5 ! %ell & sep>ate ! body ( ! oy/] f ! inn] surface ( ! upp] flat %ell4 ,b5d back & sep>ate ! flat %ell1 leav+ ! body ( ! oy/] 9 & atta*$ to ! concave %ell4 ,avoid spill+ any ( ! liquor4 7,"s dext]ity 9 op5+ oy/]s %\ld be acquir$ be=e und]tak+ ^! exp]im5ts74 #f4 ,cut up ! body ( ! oy/] ) /]ile scissors into small pieces & all[ ! liquor fre$ f ! body dur+ ! process to mix ) ! liquor previ\sly 9 ! %ell4 #g4 ,transf] ! comm9ut$ oy/] & ! liquor to ! cyl9d]4 #h4 ,treat ea* ( ! rema9+ oy/]s 9 simil> fa%ion4 #i4 ,mix ! cont5ts ( ! cyl9d] ?or\ghly by /irr+ ) a /]ile glass rod4 ,! total volume w am\nt to ab\t #ajj c4c4 #aj4 ,use #j4a c4c4 ( ! mix$ liquor to insem9ate ea* ( a s]ies ( ?ree nutrose surface plates4 #aa4 ,inoculate #j4a c4c4 ( ! mix$ liquor into ea* ( ?ree tubes ( litmus milk4 #ab4 ,add /]ile di/ill$ wat] to ! cont5ts ( ! cyl9d] up to #ajjj c4c4 & /ir ?or\ghly ) a /]ile glass rod & all[ to settle4 ,! bact]ial cont5t ( ea* oy/] may be reg>d$1 = all practical purposes1 z compris$ 9 #ajj c4c4 ( fluid4 #ac4 ,arrange f\r glass capsules 9 a r[ & numb] ,i1 ,,ii1 ,,iii1 ,,iv4 ,pipette #i c4c4 /]ile di/ill$ wat] into ea*4 #ad4 ,to capsule ,no4 ,i add #a c4c4 ( ! dilut$ liquor1 etc4 f ! cyl9d]1 & mix ?or\ghly4 ,to capsule ,,ii add #a c4c4 ( dilution 9 capsule ,i & mix ?or\ghly4 ,c>ry ov] #a c4c4 ( fluid f capsule ,,ii to capsule ,,iii1 aft]w>ds add+ #a c4c4 ( fluid f capsule ,,iii to capsule ,,iv4 #ae4 ,label tubes ( bile salt bro? & inoculate ) ! foll[+ am\nts ( dilut$ oy/]s3 ,no4 #f ) #aj c4c4 cyl9d] fluid #j4a oy/]4 ,no4 #e ) #a c4c4 cyl9d] fluid #j4ja oy/]4 ,no4 #d ) #a c4c4 capsule ,i fluid #j4jja oy/]4 ,no4 #c ) #a c4c4 capsule ,,ii fluid #j4jjja oy/]4 ,no4 #b ) #a c4c4 capsule ,,iii fluid #j4jjjja oy/]4 ,no4 #a ) #a c4c4 capsule ,,iv fluid #j4jjjjja oy/]4 #af4 ,transf] #ajj c4c4 cyl9d] fluid 7 #a oy/]7 to an ,erl5mey] flask & add #ej c4c4 d\ble /r5g? bile salt bro?1 & label #g4 #ag4 ,duplicate all ! above indicat$ cultures4 #ah4 ,put up ! tube cultures 9 ,bu*n]'s tubes & incubate ana]obically at #db deg4 ,c4 ,if gr[? occurs 9 tube #a ! organism f9ally isolat$1 e4 g41 ,b4 coli1 mu/ h be5 pres5t to ! ext5t ( "o million p] oy/]4 #ai4 ,complete ! exam9ation = memb]s ( ! ,coli-typhoid gr\p & sewage /reptococci1 z direct$ "u ,wat] ,exam9ation1 page #dbi 7/eps #aa-ba74 #bj4 ,inoculate a s]ies ( #f tubes ( litmus milk ) quantities ( ! mat]ial simil> to ^? indicat$ 9 /ep #ae2 heat to #hj deg4 ,c4 = t5 m9utes1 & incubate "u ana]obic conditions at #cg deg4 ,c4 ,exam9e = ! pres5ce ( ,b4 ent]itidis sporog5es z direct$ "u ,wat] ,exam9ation1 page #dch 7/eps #g-aj74 ,,exam9ation ,,( ,,sewage ,,& ,,sewage ,,efflu5ts4 ,quantitative4-- ,collection ( ! ,sample4--,z only small quantities ( mat]ial are ne$$1 ! samples %\ld be collect$ 9 a mann] simil> to t describ$ "u wat] = quantitative exam9ation & transmitt$ 9 ! ice app>atus us$ 9 pack+ ^? samples4 ,app>atus ,requir$4--,z = wat] 7vide page #dbj74 ,,me?od4-- #a4 ,arrange f\r /]ile capsules 9 a r[ & numb] !m ,i1 ,,ii1 ,,iii1 ,,iv4 #b4 ,pipette #i c4c4 /]ile b\illon into capsule ,no4 ,i4 #c4 ,pipette #i4i c4c4 /]ile b\illon into capsules ,,ii1 ,,iii1 & ,,iv4 #d4 ,add #a c4c4 ( ! sewage to capsule ,no4 ,i by means ( a /]ile pipette1 & mix ?or\ghly4 #e4 ,take a fre% /]ile pipette & transf] #j4a c4c4 ( ! mixture f ,no4 ,i to ,no4 ,,ii & mix ?or\ghly4 #f4 ,9 l mann] transf] #j4a c4c4 f ,no4 ,,ii to ,no4 ,,iii1 & !n #j4a c4c4 f ,no4 ,,iii to ,no4 ,,iv4 ,n[ #a c4c4 ( dilution ,no4 ,i conta9s #j4a c4c4 ( ! orig9al sewage4 #a c4c4 ( dilution ,no4 ,,ii conta9s #j4jja c4c4 ( ! orig9al sewage4 #a c4c4 ( dilution ,no4 ,,iii conta9s #j4jjjja c4c4 ( ! orig9al sewage4 #a c4c4 ( dilution ,no4 ,,iv conta9s #j4jjjjjja c4c4 ( ! orig9al sewage4 #g4 ,p\r a set ( gelat9e plates f ! cont5ts ( ea* capsule1 ?ree plates 9 a set1 & conta9+ respectively #j4b1 #j4c1 & #j4e c4c4 ( ! dilution4 ,label c>efully2 incubate at #bj deg4 ,c4 = ?ree1 f\r1 or five days4 #h4 ,enum]ate ! organisms pres5t 9 ^? sets ( plates :i* h n liquefi$1 probably ^? f dilution ,,iii or ,,iv1 & calculate !refrom ! numb] pres5t p] cubic c5timetre ( ! orig9al sample ( sewage4 ,qualitative4--,! qualitative exam9ation ( sewage is conc]n$ ) ! id5tification & enum]ation ( ! same bact]ia dealt ) "u ! correspond+ section ( wat] exam9ation2 x is consequ5tly conduct$ on precisely simil> l9es to ^? already indicat$ 7vide pages #dbf to #dda74 ,,exam9ation ,,( ,,air4 ,quantitative4-- ,app>atus ,requir$3 ,aspirator bottle1 #aj litres capacity1 fitt$ ) a deliv]y tube1 & hav+ its m\? clos$ by a p]=at$ rubb] /opp]1 ?r\gh :i* passes a %ort l5g? ( glass tub+4 ,erl5mey] flask1 #bej c4c4 capacity 7hav+ a wide m\? prop]ly plugg$ ) wool71 conta9+ #ej c4c4 /]ile wat]4 ,rubb] /opp] to fit ! m\? ( ! flask1 p]=at$ ) two holes1 & fitt$ z foll[s3 ,take a #i cm4 l5g? ( glass tub+ & b5d up #c cm4 at "o end at "r angles to ! ma9 l5g? ( tub+4 ,pass ! long arm ( ! angle ?r\gh "o ( ! p]=ations 9 ! /opp]2 plug ! op5 end ( ! %ort arm ) cotton-wool4 ,take a glass funnel #e or #f cm4 9 diamet] ) a /em #ab cm4 9 l5g? & b5d ! /em close up to ! apex ( ! funnel1 9 a g5tle curve ?r\gh a qu>t] ( a circle2 pass ! long /em ?r\gh ! o!r p]=ation 9 ! rubb] /opp]4 ,a batt]y j> or a small wat]-ba? to hold ! ,erl5mey] flask :5 pack$ r\nd ) ice4 ,supply ( brok5 ice4 ,rubb] tub+4 ,screw clamps & spr+ clips1 = tub+4 ,wat] /]ilis]4 ,retort /& & clamps4 ,app>atus = plat+ 7z = enum]ation ( wat] organisms1 vide page #dbj74 ,,me?od4-- #a4 ,fill #aj litres ( wat] into ! aspirat+ bottle & atta* a piece ( rubb] tub+ ) a screw clamp to ! deliv]y tube4 ,op5 ! taps fully & regulate ! screw clamp1 by actual exp]im5t1 s t ! tube deliv]s #a c4c4 ( wat] e second4 ,! screw clamp is n t\*$ aga9 dur+ ! exp]im5t4 ,at ?is rate ! aspirator bottle w empty itself 9 j "u ?ree h\rs4 ,%ut (f ! tap & make up ! cont5ts ( ! aspirator bottle to #aj litres aga94 #b4 ,/]ilise ! fitt$ rubb] cork1 ) its funnel & tub+1 by boil+ 9 ! wat] /]ilis] = t5 m9utes4 #c4 ,remove ! cotton-wool plug f ! flask1 & replace x by ! rubb] /opp] ) its fitt+s4 ,make sure t ! end ( ! /em ( ! funnel is imm]s$ 9 ! b\illon4 #d4 ,place ! flask 9 a glass or metal vessel & pack x r\nd ) p\nd$ ice4 ,arrange ! flask ) its ice cas+ j above ! neck ( ! aspirator bottle4 ,illu/ration3 ,,fig4 #baf4--;,arrangem5t ( app>atus = air analysis4 #e4 ,connect up ! free end ( ! glass tube f ! flask--aft] remov+ ! cotton-wool plug--) ! air-entry tube 9 ! m\? ( ! aspirat+ bottle 7,fig4 #baf74 #f4 ,op5 ! tap fully1 & all[ ! wat] to run4 ,repl5i% ! ice f "t to "t if necess>y4 7,9 empty+ itself ! aspirator bottle w aspirate #aj litres ( air sl[ly ?r\gh ! wat] 9 ! ,erl5mey] flask47 #g4 ,:5 ! aspiration is complet$1 disconnect ! flask & remove x f its ice pack+4 #h4 ,liquefy ?ree tubes ( nutri5t gelat9e & add to !m #j4e c4c41 #j4c c4c41 & #j4b c4c41 respectively1 ( ! wat] f ! flask1 by means ( a /]ile graduat$ pipette1 z 9 ! quantitative exam9ation ( wat]4 ,p\r plates4 #i4 ,p\r a second simil> set ( gelat9e plates4 #aj4 ,incubate bo? sets ( plates at #bj deg4 ,c4 #aa4 ,enum]ate ! colonies pres5t 9 ! two sets ( gelat9e plates aft] ?ree1 f\r1 or five days & av]age ! results f ! numb]s s obta9$2 e/imate ! numb] ( micro-organisms pres5t 9 #a c4c41 & !n 9 ! #ej c4c4 ( bro? 9 ! flask4 #ab4 ,! result ( air exam9ation is usually express$ z ! numb] ( bact]ia pres5t p] cubic metre 7i4 e41 kilolitre7 ( air2 & z ! numb] ( organisms pres5t 9 ! #ej c4c4 wat] only repres5t ^? conta9$ 9 #aj litres ( air1 ! result+ figure mu/ be multipli$ by #ajj4 ,qualitative4-- #a4 ,proce$ exactly z 9 ! quantitative exam9ation ( air 7vide supra71 /eps #a to #aj4 #b4 ,p\r plates ( wort ag> ) simil> quantities ( ! air-infect$ wat]1 & incubate at #cg deg4 ,c4 #c4 ,p\r plates ( nutri5t ag> ) simil> quantities ( ! wat] & incubate at #cg deg4 ,c4 #d4 ,p\r simil> plates ( wort gelat9e & incubate at #bj deg4 ,c4 #e4 ,pick (f ! individual colonies t appe> 9 ! sev]al plates1 subcultivate !m on ! v>i\s m$ia1 & id5tify !m4 ,,exam9ation ,,( ,,soil4 ,! bact]iological exam9ation ( soil yields in=mation ( value to ! sanit>ian dur+ ! progress ( ! process ( homog5isation ( made soil 7e4 g41 a dump+ area = ! refuse ( t[n7 & det]m9es ! p]iod at :i* su* an area may ) propriety & safety be utilis$ = build+ purposes2 or to ! agriculturali/ 9 in=m+ him ( ! suitability ( any giv5 area = ! gr[? ( crops4 ,! surface ( ! gr\nd1 expos$ z x is to ! bact]icidal influ5ce ( sunlight & to rapid alt]nations ( heat & cold1 ra9 & w9d1 conta9s b few micro-organisms4 ,aga91 ow+ to ! d5sity ( ! molecules ( deep soil & lack ( a]ation on ! "o h&1 & ! filt]+ action ( ! upp] lay]s ( soil & bact]ial antagonism on ! o!r1 bact]ial life practically ceases at a dep? ( ab\t #b metres4 ,! int]m$iate /ratum ( soil1 situat$ f #be to #ej cm4 bel[ ! surface1 inv>iably yields ! mo/ num]\s & ! mo/ v>i$ bact]ial flora4 ,collection ( ,sample4--,a small copp] capsule #f cm4 high by #f cm4 diamet]1 ) pull-(f cap secur$ by a bayonet cat*1 previ\sly /]ilis$ 9 ! hot-air ov51 is ! mo/ conv5i5t receptacle = samples ( soil4 ,illu/ration3 ,,fig4 #bag4--,soil scoop4 ,! in/rum5t us$ = ! actual removal ( ! soil f its natural position w v>y accord+ to :e!r we require surface samples or soil f v>y+ dep?s4 7a7 ,= surface samples1 use an iron scoop1 %ap$ l a %oe horn1 b provid$ ) a %>p sp9e 7,fig4 #bag74 ,?is is wrapp$ 9 asbe/os clo? & /]ilis$ 9 ! hot-air ov54 ,:5 remov$ f ! ov51 wrap a piece ( oil$ pap]1 silk1 or gutta-p]*a tissue ov] ! asbe/os clo?1 & secure x ) /r+1 z a fur!r protection aga9/ contam9ation4 ,on rea*+ ! spot :5ce ! samples are to be tak51 ! cov]+s ( ! scoop are remov$1 & ! asbe/os clo? employ$ to bru% away loose /ones & debris f ! select$ area4 ,! surface soil is !n brok5 up ) ! po9t ( ! scoop1 scrap$ up & collect$ 9 ! body ( ! scoop1 & transf]r$ to ! /]ile capsule = transmission4 ,illu/ration3 ,,fig4 #bah4--;,fra5kel's bor]4 7b7 ,= deep samples collect$ at v>i\s di/ances f ! surface1 an exp]im5tal tr5* may be cut to ! requir$ dep? & samples collect$ at ! requir$ po9ts on ! face ( ! section4 ,x is1 h[ev]1 pref]able to utilise "s =m ( bor]1 su* z t design$ by ,fra5kel 7,fig4 #bah74 ,fra5kel's ,e>? ,bor]4--,?is in/rum5t consi/s ( a /\t h>d-/eel rod1 #aej cm4 long1 m>k$ 9 c5timetres f ! drill-po9t$ extremity4 ,x is provid$ ) a cross h&le 7adju/able at any po9t along ! l5g? ( ! rod by means ( a screw nut74 ,! t]m9al c5timet]s are ?ick] ?an ! rema9d] ( ! rod1 & on "o side a v]tical cavity ab\t #j4e cm4 deep is cut4 ,?is is cov]$ by a flang$ sleeve s long z ! bor] is driv5 into ! soil clockwise1 & is op5$ = ! reception ( ! sample ( soil1 :5 ! requir$ dep? is rea*$1 by rev]s+ ! screw+ motion1 & aga9 clos$ be=e )drawal ( ! bor] f ! e>? by resum+ ! orig9al direction ( twi/4 ,x c be /]ilis$ 9 a mann] simil> to t adopt$ = ! scoop1 or by repeat$ly fill+ ! cavity ) e!r & burn+ x (f4 ,quantitative4--,f\r di/9ct inve/igations are includ$ 9 ! complete quantitative bact]iological exam9ation ( ! soil3 #a4 ,! enum]ation ( ! a]obic organisms4 #b4 ,! enum]ation ( ! spores ( a]obes4 #c4 ,! enum]ation ( ! ana]obic organisms 7includ+ ! facultative ana]obes74 #d4 ,! enum]ation ( ! spores ( ana]obes4 ,fur!r1 by a comb9ation ( ! results ( ! fir/ & second1 & ( ! ?ird & f\r? ( ^!1 ! ratio ( spores to vegetative =ms is obta9$4 ,app>atus ,requir$3 ,case ( /]ile capsules 7#be c4c4 capacity74 ,case ( /]ile graduat$ pipettes1 #aj c4c4 79 t5?s ( a cubic c5timetre74 ,case ( /]ile graduat$ pipettes1 #a c4c4 79 t5?s ( a cubic c5timetre74 ,flask conta9+ #bej c4c4 /]ile b\illon4 ,tall cyl9d] conta9+ #b p] c5t4 lysol solution4 ,plate-levell+ /&4 #ab /]ile plates4 ,tubes ( nutri5t gelat9e4 ,tubes ( wort gelat9e4 ,tubes ( nutri5t ag>4 ,tubes ( glucose =mate gelat9e4 ,tubes ( glucose =mate ag>4 ,wat]-ba? regulat$ at #db deg4 ,c4 ,buns5 burn]4 ,grease p5cil4 ,/]ile mort> & pe/le 7agate74 ,/]ile wide-m\!d ,erl5mey] flask 7#ejj c4c4 capacity74 ,/]ile metal funnel ) %ort wide bore deliv]y tube to j fit m\? ( flask4 ,solid rubb] /opp] to fit ! flask 7/]ilis$ by boil+74 ,pair ( scales4 ,c\nt]poise 7,fig4 #ajg74 ,/]ile metal 7nickel7 spoon or spatula4 ,fractional /]ilis] 7,fig4 #adj74 ,,me?od4-- #a4 ,arrange f\r /]ile capsules numb]$ ,i1 ,,ii1 ,,iii1 & ,,iv2 pipette #i c4c4 /]ile b\illon into ! fir/ capsule1 & #i4i c4c4 into ea* ( ! rema9+ ?ree4 #b4 ,pipette #ajj c4c4 /]ile b\illon into ! ,erl5mey] flask4 #c4 ,remove ! cotton-wool plug f ! flask & replace x by ! /]ile funnel4 #d4 ,place flask & funnel on "o pan ( ! scales1 & c\nt]poise accurately4 #e4 ,empty ! sample ( soil into ! mort> & triturate ?or\ghly4 #f4 ,by means ( ! /]ile spatula add #aj grammes ( ! e>? sample to ! b\illon 9 ! flask4 ,! f9al results w be m reliable if /eps #b1 #c1 #d1 & #e are p]=m$ "u a hood--to protect f fall+ du/1 etc4 #g4 ,remove ! funnel f ! m\? ( ! flask2 replace x by ! rubb] /opp] & %ake vig\r\sly2 !n all[ ! solid p>ticles to settle = ab\t ?irty m9utes4 ,"o cubic c5timetre ( ! turbid bro? conta9s ! wa%+s f #j4a gramme ( soil4 #h4 ,pipette (f #a c4c4 ( ! sup]natant b\illon1 t]m$ ! soil wat]1 & add x to ! cont5ts ( capsule ,i2 mix ?or\ghly4 #i4 ,remove #j4a c4c4 ( ! infect$ b\illon f capsule ,i & add x to capsule ,,ii1 & mix4 #aj4 ,9 l mann] add #j4a c4c4 ( ! cont5ts ( capsule ,,ii to capsule ,,iii1 & !n #j4a c4c4 ( ! cont5ts ( capsule ,,iii to capsule ,,iv4 ,!n #a c4c4 fluid f capsule ,i conta9s soil wat] f 4#ja gm4 e>?4 ,!n #a c4c4 fluid f capsule ,,ii conta9s soil wat] f 4#jjja gm4 e>?4 ,!n #a c4c4 fluid f capsule ,,iii conta9s soil wat] f 4#jjjjja gm4 e>?4 ,!n #a c4c4 fluid f capsule ,,iv conta9s soil wat] f 4#jjjjjjja gm4 e>?4 7,a7 ,a]obes 7,vegetative ,=ms & ,spores74-- #aa4 ,p\r a set ( gelat9e plates f ! cont5ts ( ea* capsule--two plates 9 a set1 & conta9+ respectively #j4a c4c4 & #j4d c4c4 ( ! dilut$ soil wat]4 ,label & incubate4 #ab4 ,p\r simil> sets ( wort gelat9e plates f ! cont5ts ( capsules ,,ii & ,,iii1 label1 & incubate at #bj deg4 ,c4 #ac4 ,p\r simil> sets ( ag> plates f ! cont5ts ( capsules ,,ii & ,,iii2 label & incubate at #cg deg4 ,c4 #ad4 ,weigh out a second sample ( soil--#aj grammes--dry ov] a wat]-ba? until ( con/ant weight & calculate ! ratio wet soil weight --------------- dry soil weight #ae4 ,c\nt ! plates aft] incubation = ?ree1 f\r1 or five days1 & correct+ ! figures ?us obta9$ by means ( ! wet to dry soil ratio e/imate-- 7a7 ,! numb] ( a]obic micro-organisms pres5t p] gramme ( ! soil4 7b7 ,! numb] ( yea/s & m\lds pres5t p] gramme ( ! soil4 7c7 ,! numb] ( a]obic organisms gr[+ at #cg deg4 ,c4 pres5t p] gramme ( ! soil4 7,b7 ,ana]obes 7,vegetative ,=ms & ,spores74-- #af4 ,p\r simil> sets ( plates 9 glucose =mate gelat9e & ag> & incubate 9 ,bullo*'s ana]obic app>atus4 7,c7 ,a]obes & ,ana]obes 7,spores ,only74-- #ag4 ,pipette #e c4c4 soil wat] into a /]ile tube4 #ah4 ,place 9 ! diff]5tial /]ilis] at #hj deg4 ,c4 = t5 m9utes4 #ai4 ,p\r two sets ( f\r gelat9e plates conta9+ #j4a1 #j4b1 #j4e1 & #a c4c4 respectively ( ! soil wat]2 label & incubate at #bj deg4 ,c41 "o set a]obically1 ! o!r ana]obically 9 ,bullo*'s app>atus4 #bj4 ,c\nt ! plates 7delay ! enum]ation z long z possible7 & e/imate ! numb] ( spores ( a]obes & ana]obes respectively pres5t p] gramme ( ! soil4 #ba4 ,calculate ! ratio exi/+ betwe5 spores & spores vegetative =ms "u ea* ( ! two gr\ps1 a]obic & ana]obic micro-organisms4 ,qualitative ,exam9ation4--,! qualitative exam9ation ( soil is usually direct$ to ! detection ( "o or m ( ! foll[+3 ,memb]s ( ! ,coli-typhoid gr\p4 ,/reptococci4 ,bacillus an?racis4 ,bacillus tetani4 ,bacillus o$ematis maligni4 ,! nitr\s organisms4 ,! nitric organisms4 #a4 ,transf] ! rema9d] ( ! soil wat] 7#hh c4c47 to a /]ile ,erl5mey] flask by means ( a /]ile syphon4 #b4 ,fix up ! filt]+ app>atus z = ! qualitative exam9ation ( wat]1 & filt] ! soil wat]4 #c4 ,susp5d ! bact]ial residue 9 #e c4c4 /]ile b\illon 7te*nique simil> to t describ$ = ! wat] sample1 vide pages #dcd-dcf74 ,e cubic c5timetre ( susp5sion n[ conta9s ! soil wat] f ne>ly #a gramme ( e>?4 ,! me?ods up to ?is po9t are id5tical no matt] :i* organism or gr\p ( organisms x is desir$ to isolate2 b f ?is /age onw>d ! process is v>i$ slightly = ea* p>ticul> bact]ium4 ,i4 ,! ,coli-typhoid ,gr\p4-- ,,ii4 ,/reptococci4-- ,,iii4 ,bacillus ,an?racis4-- ,,iv4 ,bacillus ,tetani4-- ,! me?ods adopt$ = ! isolation ( ^! organisms are id5tical ) ^? already describ$ "u wat] 7page #dcg et seq474 ,v4 ,bacillus ,o$ematis ,maligni4--,me?od precisely simil> to t employ$ = ! ,b4 tetani4 ,,vi4 ,! ,nitr\s ,organisms4-- #a4 ,take t5 tubes ( ,w9ogradsky's solution ,no ,i 7vide page #aih7 & numb] !m consecutively f #a to #aj4 #b4 ,inoculate ea* tube ) v>y+ quantities ( ! mat]ial z foll[s3 ,to tube ,no4 #a add #a4j c4c4 ( ! soil wat]4 ,to tube ,no4 #b add #j4a c4c4 ( ! soil wat]4 ,to tube ,no4 #c add #a4j c4c4 f ,capsule ,i4 ,to tube ,no4 #d add #j4a c4c4 f ,capsule ,i4 ,to tube ,no4 #e add #a4j c4c4 f ,capsule ,,ii4 ,to tube ,no4 #f add #j4a c4c4 f ,capsule ,,ii4 ,to tube ,no4 #g add #a4j c4c4 f ,capsule ,,iii4 ,to tube ,no4 #h add #j4a c4c4 f ,capsule ,,iii4 ,to tube ,no4 #i add #a4j c4c4 f ,capsule ,,iv4 ,to tube ,no4 #aj add #j4a c4c4 f ,capsule ,,iv4 ,label & incubate at #cj deg4 ,c4 ,,vii4 ,! ,nitric ,organisms4-- #c4 ,take t5 tubes ( ,w9ogradsky's solution ,no ,,ii1 numb] !m consecutively f #a to #aj & inoculate ) quantities ( soil wat] simil> to ^? enum]at$ 9 section ,,vi /ep #b4 ,label & incubate at #cj deg4 ,c4 #d4 ,exam9e aft] tw5ty-f\r & =ty-eight h\rs' incubation4 ,f ^? tubes t %[ signs ( gr[? make subcultivations 9 fre% tubes ( ! same m$ium & incubate at #cj deg4 ,c4 #e4 ,make fur!r subcultivations f su* ( ^? tubes z %[ gr[?1 & aga9 incubate4 #f4 ,if gr[? occurs 9 ^! subcultures1 make surface sme>s on plates ( ,w9ogradsky's silicate jelly 7vide page #aih74 #g4 ,pick (f su* colonies z make _! appe>ance & subcultivate 9 ea* ( ^! two m$ia4 ,,te/+ ,,filt]s4 ,porcela9 filt] c&les are exam9$ ) ref]5ce to _! p[] ( hold+ back all ! micro-organisms susp5d$ 9 ! fluids :i* are filt]$ ?r\gh !m1 & p]mitt+ only ! passage ( g]m-free filtrates4 ,9 ord] to det]m9e ! fre$om ( ! filt] f flaws & cracks :i* w\ld p]mit ! passage ( bact]ia no matt] h[ p]fect ! g5]al /ructure ( ! c&le might be1 ! c&le mu/ fir/ be atta*$ by means ( a long piece ( pressure tub+1 to a p[]ful pump1 su* z a foot bicycle pump1 fitt$ ) a manomet]4 ,! c&le is !n imm]s$ 9 a j> ( wat] & held completely subm]g$ :il/ ! int]nal pressure is gradually rais$ to two atmosph]es by ! action ( ! pump4 ,any crack or flaw w at once become obvi\s by reason ( ! /ream ( air bubbles issu+ f x4 ,! exam9ation = p]meability is conduct$ z foll[s3 ,app>atus ,requir$3 ,filt]+ app>atus3 ,! actual filt] c&le t is us$ mu/ be ! "o x is int5d$ to te/ & mu/ be previ\sly c>efully /]ilis$2 ! arrangem5t ( ! app>atus w naturally v>y ) ea* diff]5t =m ( filt]1 "o or o!r ( ^? already describ$ 7vide pages #db-dh74 ,plate-levell+ /&4 ,case ( /]ile plates4 ,case ( /]ile pipettes1 #aj c4c4 79 t5?s74 ,case ( /]ile pipettes1 #a c4c4 79 t5?s74 ,tubes ( nutri5t gelat9e4 ,flask conta9+ /]ile normal sal9e solution4 ,/]ile measur+ flask1 #ajjj c4c4 capacity4 ,,me?od4-- #a4 ,prep>e surface cultivations1 on nutri5t ag> 9 a culture bottle1 ( ! ,bacillus mycoides1 & incubate at #bj deg4 ,c41 = =ty-eight h\rs4 #b4 ,pipette #e c4c4 /]ile normal sal9e into ! culture bottle & emulsify ! entire surface gr[? 9 x4 #c4 ,pipette ! emulsion into ! /]ile measur+ flask & dilute up to #ajjj c4c4 by ! addition ( /]ile wat]4 #d4 ,p\r ! emulsion into ! filt] res]voir & />t ! filtration4 #e4 ,:5 ! filtration is complet$1 p\r six ag> plates ea* conta9+ #a c4c4 ( ! filtrate4 #f4 ,incubate at #cg deg4 ,c4 until1 if necess>y1 ! completion ( sev5 days4 #g4 ,if ! filtrate is n /]ile1 subcultivate ! organism pass$ & det]m9e its id5tity ) ! te/ bact]ium be=e reject+ ! filt]--s9ce ! filtrate may h be5 accid5tally contam9at$4 #h4 ,if ! filtrate is /]ile1 re/]ilise ! c&le & repeat ! te/ n[ sub/itut+ a cultivation ( ,b4 prodigiosus--a bacillus ( small] size4 #i4 ,if ! second te/ is satisfactory1 te/ ! c&le aga9/ a cultivation ( a v small coccus1 e4 g41 ,micrococcus melit5sis1 9 a simil> mann]2 9 ?is in/ance cont9u+ ! incubation ( cultivations f ! filtrate = f\rte5 days4 ,,te/+ ,,( ,,dis9fectants4 ,me?ods h already be5 detail$ 7page #caj7 = ! purpose ( /udy+ ! vital resi/ance (f]$ by micro-organisms to ! le?al effect ( g]micides4 ,b x frequ5tly happ5s t ! bact]iologi/ has to det]m9e ! relative effici5cy ( dis9fectants f ! /&po9ts ( ! sanit>ian & comm]cial man r ?an f ! rese>* work]'s po9t ( view4 ,9 pursu+ ?is l9e ( inve/igation1 x is conv5i5t to comp>e ! effici5cy1 "u laboratory conditions1 ( ! propos$ dis9fectant ) t ( "s /&>d g]micide1 su* z pure ph5ol4 ,9 s do+1 & 9 ord] t ! "w ( diff]5t obs]v]s may be comp>$1 conditions z ne>ly uni=m z possible %\ld be aim$ at4 ,! me?od describ$ is "o t has be5 9 use by ! writ] = _m ye>s pa/1 modifi$ rec5tly by ! adoption ( "s ( ! recomm5dations ( ! ,lancet ,commission on ! ,/&>disation ( ,dis9fectants--p>ticul>ly ( ! calculation = det]m9+ ! ph5ol coeffici5t4 ,?is me?od has _m po9ts 9 common ) t modification ( ! drop me?od kn[n z ! ,rideal-,walk] te/4 ,g5]al ,consid]ations4-- ,^! may be gr\p$ "u ?ree head+s3 ,te/ ,g]m1 ,g]micide1 & ,environm5t4 #a4 ,te/ ,g]m4--,b4 coli4 ,z dis9fectants are te/$ = sanit>y purposes1 x is obvi\s t a memb] ( ! coli-typhoid gr\p %\ld be select$ z ! te/ g]m4 ,b4 coli is select$ on acc\nt ( its relative nonpa?og5icity1 ! ease ) :i* x c be isolat$ & id5tifi$ by diff]5t obs]v]s 9 v>i\s p>ts ( ! _w1 ! /ability ( its fundam5tal *>act]s1 & ev5ness ( its resi/ance :5 utilis$ = ^! te/s2 f9ally s9ce ! colon bacillus is an organism :i* is slightly m resi/ant to ! le?al action ( g]micides ?an ! m pa?og5ic memb]s ( ?is gr\p1 a m>g9 ( safety is introduc$ into ! te/ :i* c]ta9ly enhances its value4 ,b4 coli %\ld be rec5tly isolat$ f a normal /ool1 & plat$ at lea/ twice to ensure ! purity ( ! /ra92 & a /ock ag> culture prep>$ :i* %\ld be us$ ?r\gh\t any p>ticul> te/4 ,= any p>ticul> exp]im5t prep>e a sme> culture on ag> & incubate at #cg deg4 ,c4 = #bd h\rs ana]obically4 ,!n emulsify ! :ole ( ! surface gr[? 9 #aj c4c4 ( /]ile wat]4 ,transf] ! emulsion to a /]ile te/-tube ) "s /]ile glass beads & %ake ?or\ghly to ensure homog5\s emulsion4 ,transf] to a c5trifuge tube & c5trifugalise ! emulsion to ?r[ d[n any masses ( bact]ia :i* may h escap$ ! dis9tegrat+ action ( ! beads4 ,pipette (f ! sup]natant emulsion = use 9 ! te/4 #b4 ,g]micide4-- a4 ,dis9fectant to be te/$4-- ,! fir/ ess5tial po9t is to te/ ! unkn[n dis9fectant1 :i* may be ref]r$ to z g]micide-x1 on ! l9es set out on page #caa to det]m9e its inhibition coeffici5t4 ,?is con/ant hav+ be5 fix$1 prep>e v>i\s solutions ( g]micide-x ) /]ilis$ di/ill$ wat] by accurate volumetric me?ods1 comm5c+ ) a solution some:at /rong] ?an t repres5t+ ! inhibition coeffici5t4 ,! solutions mu/ be prep>$ 9 fairly l>ge bulk1 n less ?an #e c4c4 ( ! dis9fectant be+ utilis$ = ! prep>ation ( any giv5 p]c5tage solution4 b4 ,/&>d ,control4--,ph5ol4 ,! /&>d g]micide us$ = comp>ison %\ld be "o :i* is n subject to v>iation 9 its *emical composition1 & ! "o :i* has obta9$ almo/ univ]sal use is ,ph5ol4 ,! foll[+ table %[s ! effect ( diff]5t p]c5tages ( c>bolic acid ^u ,b4 coli = v>y+ contact times1 compil$ f an exp]im5t conduct$ "u ! /&>d conditions ref]r$ to "u ,environm5t4 ,! results closely correspond to ^? record$ by ! ,lancet ,commission on ,dis9fectants1 #aiji4 ---------------------------------------- ---------------- ,contact "t 9 m9utes4 ,p]c5tage ( ph5ol ---------------------------- #b-a_/b #e #aj #ae #bj #be #cj #ce ---------------------------------------- --------- #a4bj - - - - - - - - #a4aj - - - - - - - - #a4j - - - - - - - #j4i - - - - - - - #j4he - - - - - - #j4hj - - - - - #j4ge - - - #j4g - - #j4fe - ---------------------------------------- --------- - ,no gr[?1 i4 e41 bact]ia kill$4 ,gr[?1 i4 e41 bact]ia /ill liv+4 ,f ?is x w be se5 t ! foll[+ p]c5tage solutions w ne$ to be prep>$1 namely3 #a4a p] c5t41 #a4j p] c5t41 #j4i p] c5t41 #j4ge p] c5t41 #j4g p] c5t41 z controls = ea* exp]im5t4 ,prep>e solutions ( v>y+ p]c5tages by weigh+ out ! quantity ( c>bolic acid requir$ = ea* & dissolv+ 9 #ajj c4c4 ( pure di/ill$ wat] 9 an accurately /&>dis$ measur+ flask4 ,! solutions mu/ be prep>$ fre%ly z requir$ ea* day4 ,environm5t4-- a4 ,g5]al4-- ,close ! w9d[s & doors ( ! laboratory 9 :i* ! inve/igation is c>ri$ out1 to avoid draughts4 ,flu% ov] ! "w b5* & adjac5t floor ) #a3ajjj solution ( corrosive sublimate4 ,caution ! assi/ant1 if "o is employ$1 to avoid unnecess>y movem5t or spee*4 b4 ,contact ,temp]ature1 #ae-ah deg4 ,c4-- ,?is is ! temp]ature at :i* contact betwe5 ! g]micide & ! te/ g]m takes place1 & is ( importance1 s9ce "s g]micides 7e4 g41 ,ph5ol7 appe> to be m p[]ful at high temp]atures4 #ah deg4 ,c4--practically ! ord9>y room temp]ature--is a temp]ature at :i* ! multiplication ( ,b4 coli is a comp>atively sl[ process1 b v>iation ( a degree above ?is temp]ature or ( two or ?ree degrees bel[ is ( no mom5t4 ,if ! room temp]ature is bel[ #ae deg4 ,c4 :5 ! exp]im5ts are 9 progress1 arrange a wat]-ba? regulat$ at #ah deg4 ,c4 = ! reception ( ! tubes conta9+ ! mixture ( g]m & g]micide2 if above #ai deg4 ,c4 imm]se ! tubes 9 cold wat]1 to :i* small pieces ( ice are add$ f "t to "t to prev5t ! temp]ature ris+ above #ah deg4 ,c4 c4 ,relative ,proportional ,bulk ( ,te/ ,g]m & ,g]micide1 #ej3a4-- ,five cubic c5timetres is a conv5i5t am\nt ( g]micidal solution to employ1 & to ?is #j4a c4c4 ( ! emulsion ( te/ g]m %\ld be add$4 d4 ,bulk ( ,sample ,remov$ f ,g]m ,g]micide ,mixture at ,ea* ( ! ,"t ,p]iods1 #j4a c4c4-- ,?is is suffici5t to af=d a fair sample ( ! g]m cont5t ( ! mixture1 & at ! same "t is insuffici5t to ex]t any inhibitory action :5 transf]r$ to ! subculture m$ium4 e4 ,subculture ,m$ium4 ,bile ,salt ,bro?4-- ,a fluid m$ium is ess5tial 9 ord] to obta9 imm$iate dilution ( ! g]micide c>ri$ ov]2 at ! same "t x is advantage\s to employ a selective m$ium :i* fav\rs ! gr[? ( ! te/ g]m to ! exclusion ( organisms likely to contam9ate ! prep>ation1 & if possible "o :i* af=ds *>act]i/ic cultural appe>ances4 ,bile ,salt ,bro? 7page #ahj7 comb9es ^! desid]ata2 x p]mits only ! gr[? ( inte/9al bact]ia1 :il/ ! =mation ( an acid reaction & ! production ( gas 9 subcultures prep>$ f ! g]m-g]micide mixture is fairly complete evid5ce ( ! pres5ce ( liv+ ,b4 coli4 ,! am\nt ( m$ium pres5t 9 ea* te/-tube is a matt] ( importance1 s9ce ! m$ium n only provides pabulum = ! te/ g]m1 b also acts z a dilu5t to ! g]micide1 to r$uce its /r5g? bel[ its inhibition coeffici5t4 ,= r\t9e "w ea* subculture tube conta9s #aj c4c4 ( m$ium1 b x is obvi\s t if g]micide-x possesses an inhibition coeffici5t ( #j4a p] c5t4 ! addition ( #j4a c4c4 ( a #aj p] c5t4 solution to #aj c4c4 ( m$ium w\ld effectually prev5t ! subsequ5t gr[? ( ! te/ g]m aft] a contact p]iod insuffici5t to de/roy its vitality4 ,h5ce ! prelim9>y te/s may 9 "s in/ances indicate ! necessity = ! pres5ce ( #ab c4c41 #ae c4c4 or m ( ! fluid m$ium 9 ! culture tubes4 f4 ,incubation ,temp]ature1 #cg deg4 ,c4-- g4 ,obs]vation ,p]iod ( ! ,subcultivations1 ,sev5 ,days4-- ,9 ord] to det]m9e :e!r or no ! te/ g]ms h be5 de/roy$1 obs]vations mu/ always be cont9u$--:5 gr[? appe>s to be abs5t--up to ! end ( sev5 days be=e record+ no gr[?4 h4 ,id5tification ( ! ,organisms ,develop+ 9 ! ,subcultivations aft] ,contact 9 ! ,g]m ,g]micide ,solution4-- ,?is is bas$ on ! nak$ eye *>act]s ( ! gr[? 9 ! bile salt bro?1 supplem5t$ :]e necess>y by plat+ me?ods1 fur!r subcultivations ^u c>bohydrate m$ia & agglut9ation exp]im5ts4 ,! sign 77 is us$ to indicate t gr[? ( ! te/ organism occurr$ 9 ! subcultivations1 & ! sign 7-7 to indicate t ! te/ g]ms h be5 de/roy$ & no subsequ5t gr[? has tak5 place4 ,,me?od4-- ,app>atus ,requir$3 ,/]ile te/-tubes 7n>r[1 n exce$+ #a4c cm4 diamet]74 ,te/-tube rack 7,fig4 #bai74 ,/]ile graduat$ pipettes 9 case1 #a c4c4 79 t5?s74 ,/]ile graduat$ pipettes 9 case1 #e c4c4 79 c4c474 ,circul> rubb] wa%]s1 #b4e cm4 diamet] ) c5tral hole1 /]ilis$ by boil+ imm$iately be=e use1 !n transf]r$ to /]ilis$ glass d\ble di%4 ,electric signal clock or /op wat*4 ,/]ile =ceps4 ,/]ilis$ glass beads4 ,%ak+ ma*9e4 ,grease p5cil4 ,mat]ial ,requir$3 ,p]c5tage solutions ( g]micide-x 7vide page #dha74 ,p]c5tage solutions ( pure ph5ol 7vide page #dhb74 ,aque\s emulsion ( ,b4 coli 7vide page #dha74 ,tubes ( bile salt bro?4 ,prelim9>y ,te/s4-- a4 ,inhibition ,coeffici5t4-- ,det]m9e ! l[e/ p]c5tage ( g]micide-x :i* inhibits gr[? ( ,b4 coli 9 ! bile salt bro?1 & ! highe/ p]c5tage :i* fails to inhibit 7page #caa74 ,on ! result ( ?is exp]im5t det]m9e ! bulk ( m$ium requir$ 9 ! subculture tubes & ! p]c5tage solutions to be employ$ 9 ! trial trip4 ,assum+ ! inhibition coeffici5t to be #a3ajjj1 x w be q safe to employ ! ord9>y culture tubes conta9+ #aj c4c4 m$ium 9 ! subsequ5t exp]im5ts4 b4 ,trial ,trip4-- ,det]m9e ! le?al effect ( a s]ies ( five solutions ( g]micide-x 7say #a3ajj1 #a3bej1 #a3cjj1 #a3ejj1 #a3fjj7 at contact times ( #b-a_/b1 #e1 #be & #cj m9utes 9 ! foll[+ mann]3 #a4 ,arrange five te/-tubes m>k$ ,a to ,e 9 ! l[] ti] ( ! te/-tube rack4 #b4 ,into tube ,a pipette #e c4c4 g]micide-x #a3ajj solution4 ,into tube ,b pipette #e c4c4 g]micide-x #a3bjj solution4 ,into tube ,c pipette #e c4c4 g]micide-x #a3cjj solution4 ,into tube ,d pipette #e c4c4 g]micide-x #a3ejj solution4 ,into tube ,e pipette #e c4c4 g]micide-x #a3fjj solution4 #c4 ,arrange #bj tubes ( bile salt bro? 9 ! upp] ti] ( ! te/-tube rack 9 two r[s1 ^? 9 ! front r[ numb]$ consecutively f left to "r #a-aj1 ^? 9 ! back r[ #aa-bj4 #d4 ,place a squ>e wire basket ( ab\t #ej tubes capacity close to ! left ( ! te/-tube rack1 = ! reception ( ! inoculat$ tubes4 #e4 ,take a /]ile #a c4c4 pipette f ! case1 pick up a /]ile rubb] wa%] ) =ceps & pu% ! po9t ( ! pipette into ! c5tral hole4 #f4 ,put d[n ! =ceps on ! b5* ) ! /]ile po9ts project+ ov] ! edge4 ,)\t tak+ ! tube f ! rack remove ! cotton-wool plug f tube ,a1 & l[] ! pipette1 ) ! rubb] wa%] affix$1 on to ! op5 m\? ( ! tube2 ) ! help ( ! =ceps to /eady ! wa%]1 pu% ! pipette on ?r\gh ! hole until ! po9t ( ! pipette has rea*$ to )9 a few millimetres ( ! bottom ( ! tube 7see fig4 #bai74 #g4 ,adju/ 9 ! same way a pipette & a wa%] 9 ! m\? ( ea* ( ! o!r tubes1 ,b1 ,c1 ,d & ,e4 #h4 ,set ! electric signal clock to r+ = ! comm5cem5t ( ! exp]im5t & at subsequ5t int]vals ( #b-a_/b1 #e1 #be & #cj m9utes4 #i4 ,take up #j4e c4c4 ( ,b4 coli emulsion 9 /]ile pipette graduat$ 9 t5?s ( a cubic c5timetre & /& by4 #aj4 ,z soon z ! bell r+s lift ! pipette f tube ,a ) ! left h& & f ! *>g$ pipette held 9 ! "r h& deliv] #j4a c4c4 ( ,b4 coli emulsion into ! #a3ajj solution4 ,!n replace ! pipette & wa%]4 ,illu/ration3 ,,fig4 #bai4--,te/-tube rack4 #aa4 ,raise ! tube ) ! left h& & %ake x to mix g]m & g]micide1 :il/ return+ ! deliv]y pipette 9 ! "r h&4 #ab4 ,repeat ! process ) tubes ,b1 ,c1 ,d & ,e2 !n drop ! infect$ deliv]y pipette 9 ! lysol j>4 ,! inoculation ( ! five tubes c be c>ri$ out v exp$iti\sly1 b a p]iod ( #aj seconds mu/ be all[$ = ea* tube4 #ac4 ,:5 ! bell r+s at #b-a_/b m9utes bl[ ?r\gh ! pipette 9 tube ,a 7?is agitates ! g]m g]micide mixture & ensures ! collection ( a fair sample72 all[ ! mixture to ent] ! pipette1 & z ! column ( fluid ext5ds well above ! t]m9al graduation1 ! "r =ef+] adju/$ ov] ! butt-end ( ! pipette be=e x is lift$ w reta9 m ?an #j4a c4c4 ( ! mixture )9 ! bore :5 ! po9t ( ! pipette is cle> ( ! fluid 9 ! tube4 ,t\* ! po9t ( ! pipette on ! inn] wall ( ! tube1 & all[ any excess ( fluid to escape1 only reta9+ #j4a c4c4 9 ! pipette4 #ad4 ,at ! same "t1 ) ! left h& remove ,bile ,salt ,tube ,no4 #a f ! upp] ti] ( ! rack1 take out ! cotton-wool plug ) ! h& already hold+ ! pipette 7! relative positions ( pipette1 plug & culture tubes be+ practically ! same z ^? ( plat9um loop1 plug & culture tube %[n 9 ,fig4 #fh1 page #gd74 #ae4 ,ins]t ! po9t ( ! pipette into ! subculture tube1 & bl[ out ! mixture into ! m$ium--replug ! tube & drop x into ! wire basket4 ,replace ! wa%]-pipette 9 tube ,a4 ,z soon z ! po9t ( ! pipette has ent]$ ! m\? ( tube ,a x may be releas$1 s9ce x has already be5 s adju/$ t x j cle>s ! bottom ( ! te/-tube1 & ! ela/ic wa%] w prev5t any damage to ! tube4 ,/eps #ac1 #ad & #ae occupy on an av]age #aj seconds4 #af4 ,repeat /eps #ac1 #ad & #ae ) ea* ( ! o!r tubes ,b1 ,c1 ,d & ,e4 #ag4 ,repeat ^! v>i\s /eps #ac-af :5 ! bell r+s at #e1 #be & #cj m9utes4 #ah4 ,place all ! inoculat$ tubes 9 ! incubator at #cg deg4 ,c4 #ai4 ,exam9e ! tubes at int]vals ( #bd h\rs1 & record ! results 9 tabul> =m z %[n 9 ,table page #dia 7! figures 9 ! squ>es indicate ! numb] ( h\rs at :i* ! *anges 9 ! m$ium due to ! gr[? ( ,b4 coli fir/ appe>$74 #bj4 ,if a consid]ation ( ! tabulat$ results indicates /r5g?s ( ,g]micide-x le?al at #b-a_/b & #cj m9utes ! f9al te/ c be arrang$1 b if ?is result has n be5 atta9$1 suffici5t evid5ce w probably be available to enable a second trial te/ to be plann$ :i* w give ! requir$ in=mation4 ,f9al ,te/4-- c4 ,det]m9ation ( ,ph5ol ,coeffici5t4-- ,x-,dis9fectant4--,?is comprises two di/9ct te/s1 "o ( ! ,g]micide-x1 ! o!r ( ! /&>d ph5ol4 #a4 ,arrange five te/-tubes cle>ly m>k$ 9 ! l[] ti] ( ! rack4 #b4 ,pipette into ea* #e c4c4 respectively ( ! five p]c5tage solutions ( x-dis9fectant :i* ! trial run has already %[n w include ^? af=d+ le?al values at #b-a_/b & #cj m9utes4 #c4 ,arrange #bj tubes ( bile salt bro? 9 ! upp] ti] ( ! te/-tube rack 9 two r[s1 ^? 9 ! front r[ numb]$ consecutively f left to "r #a-aj1 ^? 9 ! back r[ #aa-bj4 #d4 ,arrange fur!r #bj tubes ( bile salt bro? numb]$ #ba-dj 9 two r[s 9 a second small] rack :i* c be /ood on ! upp] ti] ( ! rack z soon z ! fir/ #bj tubes h be5 inoculat$4 #e4 ,place a squ>e wire basket ( ab\t #ej tube capacity close to ! left ( ! te/-tube rack1 = ! reception ( ! inoculat$ tubes4 #f4 ,adju/ a /]ile #a c4c4 pipette 9 ! m\? ( ea* ( ! tubes1 ,a1 ,b1 ,c1 ,d & ,e1 by means ( a wa%]1 z previ\sly describ$4 #g4 ,set ! electric signal clock to r+ = ! comm5cem5t ( ! exp]im5t & subsequ5tly at #b-a_/b1 #e1 #aj1 #ae1 #bj1 #be1 #cj & #ce m9utes4 #h4 ,complete precisely z indicat$ 9 ,trial ,runs1 /eps #i-ai4 ,control ,ph5ol4-- ,imm$iately ! subculture tube f ! #cj-m9ute contact p]iod h be5 inoculat$1 c>ry out a precisely simil> exp]im5t1 9 :i* five p]c5tage /r5g?s ( ,ph5ol1 7e4 g41 #a4a1 #a4j1 #j4i1 #j4ge1 #j4g7 are arrang$ 9 ! l[] ti] ( ! te/-tube rack 9 place ( ! five /r5g?s ( ,g]micide-x4 ,calculate ! ph5ol coeffici5t by ! foll[+ me?od3 7a7 ,divide ! figure repres5t+ ! p]c5tage /r5g? ( ! weake/ le?al dilution ( ! c>bolic acid control at ! #b-a_/b-m9ute contact p]iod by ! figure repres5t+ ! p]c5tage /r5g? ( ! weake/ le?al dilution ( ! x-dis9fectant at ! same p]iod4 ,! quoti5t ph5ol coeffici5t at #b-a_/b m9utes4 7b7 ,simil>ly obta9 ! ph5ol coeffici5t at #cj m9utes contact p]iod4 7c7 ,record ! mean ( ! two coeffici5ts obta9$ 9 7a7 & 7b7 z ! mean ph5ol coeffici5t1 or simply z ! ,ph5ol ,coeffici5t4 ,! details ( ! ,f9al ,te/ ( an actual det]m9ation are set out 9 ! accompany+ table4 ,,table #bg ,organism employ$1 ,b4 ,coli ,communis4 ,culture ,m$ium1 ,nutri5t ,ag> 7#aj74 ,age1 #bd hrs4 ,temp4 ( ,incubation1 #cg deg4 ,c4 ,quantities us$ ,culture ,emulsion #j4a c4c4 #e c4c4 ,g]micide4 ,emulsion ,room ,temp]ature dur+ ,exp]im5ts1 #ag deg4 ,c4 ,g]micide ,/r5g? ,"t ( exposure ,incubation #b-a_/b #e #aj #ae #bj #be #cj #ce ,"t ,temp4 #a ,g]micide-x #d -- -- -- -- -- -- -- -- #g days4 #cg deg4 ,c4 #b ,g]micide-x #c #dh -- -- -- -- -- -- -- #g days4 #cg deg4 ,c4 #c ,g]micide-x #b #bd #bd #bd #bd #dh #gb #g days4 #cg deg4 ,c4 #d ,g]micide-x #a #bd #bd #bd #bd #gb #bd #gb #g days4 #cg deg4 ,c4 #e ,g]micide-x #j4e #bd #bd #bd #bd #bd #bd #bd #bd #bd h\rs4 #cg deg4 ,c4 #a ,ph5ol #a4aj -- -- -- -- -- -- -- -- #g days4 #cg deg4 ,c4 #b ,ph5ol #a4jj #bd #g days4 #cg deg4 ,c4 #c ,ph5ol #j4ge #bd #bd #bd #bd #dh #g days4 #cg deg4 ,c4 #d ,ph5ol #j4gj #bd #bd #bd #bd #bd #gb #g days4 #cg deg4 ,c4 #e ,ph5ol #j4fe #bd #bd #bd #bd #bd #dh #bd #bd #b days4 #cg deg4 ,c4 77#a4aj_/d4jj7 7#j4g_/b4j77 #j4bg #j4ce 4#fb ,ph5ol ,coeffici5t ------------------------ ----------- --- #j4ca #b #b #b ,,app5dix4 ,,metric ,,& ,,imp]ial ,,sy/ems ,,( ,,weights ,,& ,,measures4 ,! initial unit ( ! metric sy/em is ! ,metre 7m47 or unit ( l5g?1 repres5t+ "o-f\r?-million? "p ( ! circumf]5ce ( ! e>? r\nd ! poles4 ,! unit ( mass is ! ,gramme 7g471 & repres5ts ! weight ( "o cubic c5timetre ( wat] at its maximum d5sity 7viz4 #d deg4 ,c4 & #gfj mm4 m]cury pressure74 ,! unit ( ! measure ( capacity is ! ,litre 7l471 & repres5ts ! volume ( a kilogramme ( di/ill$ wat] at its maximum d5sity4 ,! decimal subdivisions ( ea* ( ! units are designat$ by ! ,lat9 prefixes milli #a_/ajjj2 c5ti #a_/ajj2 deci #a_/aj2 ! multiples ( ea* unit by ! ,greek prefixes deka #aj2 hecto #ajj2 kilo #ajjj2 myria #aj1jjj4 ,= a comp>ison ( ! values ( "s ( ! m frequ5tly employ$ expressions ( ! ,metric ,sy/em & ! ,imp]ial ,sy/em1 ! foll[+ may be f\nd conv5i5t = ref]5ce3 ,l5g?3 #a millimetre 7 #a mm47 #a_/be ( an in*4 #a c5timetre 7 #a cm47 #b_/e ( an in*4 #a in* 7#a7 #be millimetres or #b-a_/b c5timetres4 ,mass3 #a milligramme 7 #a mg47 #j4jaedc gra9 7or approximately #a_/fd gra974 #a gramme 7 #a g47 #ae4dcbc gra9s4 #a kilo or kilogramme 7 #a kgm47 #b p\nds1 #c-a_/d ounces avoirdupois4 #a p\nd avoirdupois 7 #a lb47 #dec4eib grammes4 #a ounce avoirdupois 7 #a oz47 #bh4ce grammes4 #a gra9 #j4jfdh gramme or #fd4h milligrammes4 ,capacity3 #a cubic c5timetre 7 #a c4c47 #af4i m9ims imp]ial measure4 #a litre 7 #a l47 #ce4aif fluid ounces imp]ial measure4 #a fluid ounce imp]ial measure 7 #a ,symbol3 ounce7 #bh4db cubic c5timetres4 #a p9t imp]ial measure 7 #a ,o47 #efh4cd cubic c5timetres4 #a gallon imp]ial measure 7 #a ,c47 #d4edf litres1 or #aj p\nds avoirdupois1 ( pure wat] at #fb deg4 ,f4 & "u an atmosph]ic pressure ( #cj in*es ( m]cury4 ,,factors ,,= ,,conv]t+ ,,f ,,"o ,,sy/em ,,to ,,! ,,o!r4 ,to conv]t grammes into gra9s x #ae4dcb4 ,to conv]t grammes into ounces avoirdupois x #j4jcebg4 ,to conv]t kilogrammes into p\nds x #b4bjdf4 ,to conv]t cubic c5timetres into fluid ounces imp]ial x #j4jceb4 ,to conv]t litres into fluid ounces imp]ial x #ce4b4 ,to conv]t metres into in*es x #ci4cg4 ,to conv]t gra9s into grammes x #j4jfdh4 ,to conv]t avoirdupois ounces into grammes x #bh4ce4 ,to conv]t troy ounces into grammes x #ca4ajd4 ,to conv]t fluid ounces into cubic c5timetres x #bh4db4 ,to conv]t p9ts into litres x #j4efh4 ,to conv]t in*es into metres x #j4jbed4 ,,table ,,= ,,! ,,conv]sion ,,( ,,degrees ,,c5tigrade ,,into ,,degrees ,,fahr5heit4 ,x4 deg4 ,c4 77#ix_/#e7 #cb7 deg4 ,f4 ,c5t4 ,faht4 ,c5t4 ,faht4 ,c5t4 ,faht4 #j #cb4j #cd #ic4b #fh #aed4d #a #cc4h #ce #ie4j #fi #aef4b #b #ce4f #cf #if4h #gj #aeh4j #c #cg4d #cg #ih4f #ga #aei4h #d #ci4b #ch #ajj4d #gb #afa4f #e #da4j #ci #ajb4b #gc #afc4d #f #db4h #dj #ajd4j #gd #afe4b #g #dd4f #da #aje4h #ge #afg4j #h #df4d #db #ajg4f #gf #afh4h #i #dh4b #dc #aji4d #gg #agj4f #aj #ej4j #dd #aaa4b #gh #agb4d #aa #ea4h #de #aac4j #gi #agd4b #ab #ec4f #df #aad4h #hj #agf4j #ac #ee4d #dg #aaf4f #ha #agg4h #ad #eg4b #dh #aah4d #hb #agi4f #ae #ei4j #di #abj4b #hc #aha4d #af #fj4h #ej #abb4j #hd #ahc4b #ag #fb4f #ea #abc4h #he #ahe4j #ah #fd4d #eb #abe4f #hf #ahf4h #ai #ff4b #ec #abg4d #hg #ahh4f #bj #fh4j #ed #abi4b #hh #aij4d #ba #fi4h #ee #aca4j #hi #aib4b #bb #ga4f #ef #acb4h #ij #aid4j #bc #gc4d #eg #acd4f #ia #aie4h #bd #ge4b #eh #acf4d #ib #aig4f #be #gg4j #ei #ach4b #ic #aii4d #bf #gh4h #fj #adj4j #id #bja4b #bg #hj4f #fa #ada4h #ie #bjc4j #bh #hb4d #fb #adc4f #if #bjd4h #bi #hd4b #fc #ade4d #ig #bjf4f #cj #hf4j #fd #adg4b #ih #bjh4d #ca #hg4h #fe #adi4j #ii #baj4b #cb #hi4f #ff #aej4h #ajj #bab4j #cc #ia4d #fg #aeb4f ,,table ,,= ,,! ,,conv]sion ,,( ,,degrees ,,fahr5heit ,,into ,,degrees ,,c5tigrade4 ,x deg4 ,f4 7#e7x - #cb77_/i deg4 ,c4 ,faht4 ,c5t4 ,faht4 ,c5t4 ,faht4,c5t4 ,faht4 ,c5t4 ,faht4 ,c5t4 #cb #j4 #fh #bj4j #ajd #dj4j #adj #fj4j #agf #hj4j #cc #j4f #fi #bj4f #aje #dj4f #ada #fj4f #agg #hj4f #cd #a4a #gj #ba4a #ajf #da4a #adb #fa4a #agh #ha4a #ce #a4g #ga #ba4g #ajg #da4g #adc #fa4g #agi #ha4g #cf #b4b #gb #bb4b #ajh #db4b #add #fb4b #ahj #hb4b #cg #b4h #gc #bb4h #aji #db4h #ade #fb4h #aha #hb4h #ch #c4c #gd #bc4c #aaj #dc4c #adf #fc4c #ahb #hc4c #ci #c4i #ge #bc4i #aaa #dc4i #adg #fc4i #ahc #hc4i #dj #d4d #gf #bd4d #aab #dd4d #adh #fd4d #ahd #hd4d #da #e4j #gg #be4j #aac #de4j #adi #fe4j #ahe #he4j #db #e4f #gh #be4f #aad #de4f #aej #fe4f #ahf #he4f #dc #f4a #gi #bf4a #aae #df4a #aea #ff4a #ahg #hf4a #dd #f4g #hj #bf4g #aaf #df4g #aeb #ff4g #ahh #hf4g #de #g4b #ha #bg4b #aag #dg4b #aec #fg4b #ahi #hg4b #df #g4h #hb #bg4h #aah #dg4h #aed #fg4h #aij #hg4h #dg #h4c #hc #bh4c #aai #dh4c #aee #fh4c #aia #hh4c #dh #h4i #hd #bh4i #abj #dh4i #aef #fh4i #aib #hh4i #di #i4d #he #bi4d #aba #di4d #aeg #fi4d #aic #hi4d #ej #aj4j #hf #cj4j #abb #ej4j #aeh #gj4j #aid #ij4j #ea #aj4f #hg #cj4f #abc #ej4f #aei #gj4f #aie #ij4f #eb #aa4a #hh #ca4a #abd #ea4a #afj #ga4a #aif #ia4a #ec #aa4g #hi #ca4g #abe #ea4g #afa #ga4g #aig #ia4g #ed #ab4b #ij #cb4b #abf #eb4b #afb #gb4b #aih #ib4b #ee #ab4h #ia #cb4h #abg #eb4h #afc #gb4h #aii #ib4h #ef #ac4c #ib #cc4c #abh #ec4c #afd #gc4c #bjj #ic4c #eg #ac4i #ic #cc4i #abi #ec4i #afe #gc4i #bja #ic4i #eh #ad4d #id #cd4d #acj #ed4d #aff #gd4d #bjb #id4d #ei #ae4j #ie #ce4j #aca #ee4j #afg #ge4j #bjc #ie4j #fj #ae4f #if #ce4f #acb #ee4f #afh #ge4f #bjd #ie4f #fa #af4a #ig #cf4a #acc #ef4a #afi #gf4a #bje #if4a #fb #af4g #ih #cf4g #acd #ef4g #agj #gf4g #bjf #if4g #fc #ag4b #ii #cg4b #ace #eg4b #aga #gg4b #bjg #ig4b #fd #ag4h #ajj #cg4h #acf #eg4h #agb #gg4h #bjh #ig4h #fe #ah4c #aja #ch4c #acg #eh4c #agc #gh4c #bji #ih4c #ff #ah4i #ajb #ch4i #ach #eh4i #agd #gh4i #baj #ih4i #fg #ai4d #ajc #ci4d #aci #ei4d #age #gi4d #baa #ii4d #bab #ajj4j ,p]c5tage ,=mula = addition ( salts1 etc41 to complet$ m$ia4 ,=mula = prep>+ any desir$ p]c5tage ( a giv5 salt1 etc41 9 tub$ m$ia2 e4 g41 to make #d p] c5t4 solution ( ,,kno#c 9 a s]ies ( tubes ( bro? ea* conta9+ #aj c4c4 ( m$ium1 :5 !re is already available a #be p] c5t4 /ock aque\s solution ( potassium nitrate4 7,n ,x7 ,y ,a 7,x7 --------------- ---------- #ajj #ajj ,n numb] ( cubic c5timetres conta9$ 9 ea* tube4 ,x am\nt ( /ock solution to be add$ to ea* tube4 ,y p]c5tage requir$ 9 ! m$ium4 ,a p]c5tage ( /ock solution4 ,!n 7#aj ,x7 #d #be ,x ------------ ------ #ajj #ajj ,!re=e1 #dj #d,x #be,x4 ,!re=e1 #ba,x #dj4 ,x #a4i c4c4 ,?is all[s = solution add$ to ! orig9al bulk ( m$ium4 ,!re=e1 #aj c4c4 bro? #a4i c4c4 ( a #be p] c5t4 aque\s solution ,,kno#c makes #aa4i c4c4 m$ium conta9+ #d p] c5t4 ,,kno#c4 ,,tables ,,= ,,prep>+ ,,dilutions 7( ,s]um1 ,dis9fectants or o!r sub/ances47 ,9 e/imat+ ! agglut99 cont5t or titre ( a s]um1 te/+ dis9fectants & = _m o!r purposes1 x becomes necess>y to prep>e a s]ies ( dilutions ( ! mat]ial "u exam9ation1 & 9 ord] to avoid unnecess>y exp5diture ( lab\r x is conv5i5t to adh]e to "s def9ite scale ( increm5t1 su* = example z ! foll[+3 ,f dilutions ( #a3aj to #a3hj rise by increm5ts ( #e4 ,f dilutions ( #a3hj to #a3bjj rise by increm5ts ( #aj4 ,f dilutions ( #a3bjj to #a3djj rise by increm5ts ( #be4 ,f dilutions ( #a3djj to #a3ejj rise by increm5ts ( #ej4 ,f dilutions ( #a3ejj to #a3ajjj rise by increm5ts ( #ajj4 ,f dilutions ( #a3 #ajjj to #a3ejjj rise by increm5ts ( #bej4 ,f dilutions ( #a3 #ejjj to #a3aj1jjj rise by increm5ts ( #ajjj4 ,f dilutions ( #a3aj1jjj to #a3ajj1jjj rise by increm5ts ( #ejjj4 ,f dilutions ( #a3ajj1jjj to #a3a1jjj1jjj rise by increm5ts ( #ajj1jjj4 ,:5 deal+ ) a sub/ance ( unkn[n p[]s--& ?is is especially true ) reg>d to agglut9at+ s]a--x is cu/om>y to run a prelim9>y te/1 us+ a few widely sep>at$ dilutions su* z may be obta9$ 9 ! foll[+ mann]3 ,,fir/ ,,dilution--,i4 #a c4c4 s]um #i c4c4 normal sal9e solution #aj p] c5t4 solution or #a3 #aj dilution 7( :i* #a c4c4 conta9s #j4a c4c4 ( ! orig9al s]um74 ,:5 deal+ ) fluids o!r ?an s]um ! dilu5t is usually di/ill$ wat]2 :il/ if ! orig9al sub/ance is a solid ! in/ructions w\ld read3 #a gram o4s4 #aj c4c4 di/ill$ wat] #aj p] c5t4 solution1 etc4 ,,second ,,dilution--,,ii4 #a c4c4 fir/ dilution #i c4c4 normal sal9e solution #a p] c5t4 solution or #a3 #ajj dilution4 ,,?ird ,,dilution--,,iii4 #a c4c4 second dilution #i c4c4 normal sal9e solution #a p] mille solution or #a3 #ajjj dilution4 ,,f\r? ,,dilution--,,iv4 #a c4c4 second dilution #i c4c4 normal sal9e solution #j4a p] mille solution or #a3 #aj1jjj dilution4 ,! foll[+ tables %[+ ! second>y dilutions t c readily be prep>$ f ea* ( ^! f\r prim>y dilutions = use 9 ! subsequ5t det]m9ation ( ! exact titre w probably be f\nd ( s]vice by ^? :o are n ready ma!maticians4 ,,tables ,,= ,,prep>+ ,,dilutions4 ---------------------------------------- ----------------------------- ,,table ,i ,,table ,,ii ,us+ #aj /ock solution ,us+ #a /ock solution ,fir/ ,second dilution ,dilu5t dilution ,dilu5t ---------------------------------------- ----------------------------- #a3 #aj #a c4c4 #j c4c4 #a3 #ajj #a c4c4 #j c4c4 #a3 #ae #a c4c4 #j4e c4c4 #a3 #aaj #a c4c4 #j4a c4c4 #a3 #bj #a c4c4 #a4j c4c4 #a3 #abj #a c4c4 #j4b c4c4 #a3 #be #a c4c4 #a4e c4c4 #a3 #abe #a c4c4 #j4be c4c4 #a3 #cj #a c4c4 #b4j c4c4 #a3 #acj #a c4c4 #j4c c4c4 #a3 #ce #a c4c4 #b4e c4c4 #a3 #adj #a c4c4 #j4d c4c4 #a3 #dj #a c4c4 #c4j c4c4 #a3 #aej #a c4c4 #j4e c4c4 #a3 #de #a c4c4 #c4e c4c4 #a3 #afj #a c4c4 #j4f c4c4 #a3 #ej #a c4c4 #d4j c4c4 #a3 #agj #a c4c4 #j4g c4c4 #a3 #ee #a c4c4 #d4e c4c4 #a3 #age #a c4c4 #j4ge c4c4 #a3 #fj #a c4c4 #e4j c4c4 #a3 #ahj #a c4c4 #j4h c4c4 #a3 #fe #a c4c4 #e4e c4c4 #a3 #aij #a c4c4 #j4i c4c4 #a3 #gj #a c4c4 #f4j c4c4 #a3 #bjj #a c4c4 #a4j c4c4 #a3 #ge #a c4c4 #f4e c4c4 --------------------------------- #a3 #hj #a c4c4 #g4j c4c4 #a3 #bjj #a c4c4 #a4j c4c4 ------------------------------ #a3 #bbe #a c4c4 #a4be c4c4 #a3 #hj #a c4c4 #g4j c4c4 #a3 #bej #a c4c4 #a4e c4c4 #a3 #ij #a c4c4 #h4j c4c4 #a3 #bge #a c4c4 #a4ge c4c4 #a3 #ajj #a c4c4 #i4jj c4c4 #a3 #cjj #a c4c4 #b4j c4c4 #a3 #aaj #a c4c4 #aj4j c4c4 #a3 #cbe #a c4c4 #b4be c4c4 #a3 #abj #a c4c4 #aa4j c4c4 #a3 #cej #a c4c4 #b4e c4c4 #a3 #abe #a c4c4 #aa4e c4c4 #a3 #cge #a c4c4 #b4ge c4c4 #a3 #acj #a c4c4 #ab4j c4c4 #a3 #djj #a c4c4 #c4j c4c4 #a3 #adj #a c4c4 #ac4j c4c4 --------------------------------- #a3 #aej #a c4c4 #ad4j c4c4 #a3 #djj #a c4c4 #c4j c4c4 #a3 #afj #a c4c4 #ae4j c4c4 #a3 #dej #a c4c4 #c4e c4c4 #a3 #agj #a c4c4 #af4j c4c4 #a3 #ejj #a c4c4 #d4j c4c4 #a3 #age #a c4c4 -#af4e c4c4 --------------------------------- #a3 #ahj #a c4c4 #ag4j c4c4 #a3 #ejj #a c4c4 #d4j c4c4 #a3 #aij #a c4c4 #ah4j c4c4 #a3 #fjj #a c4c4 #e4j c4c4 #a3 #bjj #a c4c4 #ai4j c4c4 #a3 #gjj #a c4c4 #f4j c4c4 ----------------- ------------ #a3 #gej #a c4c4 #f4e c4c4 #a3 #bjj #a c4c4 #ai4j c4c4 #a3 #hjj #a c4c4 #g4j c4c4 #a3 #bbe #a c4c4 #ba4e c4c4 #a3 #ijj #a c4c4 #h4j c4c4 #a3 #bej #a c4c4 #bd4j c4c4 #a3 #ajjj #a c4c4 #i4j c4c4 #a3 #bge #a c4c4 #bf4e c4c4 -------------------------------- #a3 #cjj #a c4c4 #bi4j c4c4 #a3 #ajjj #a c4c4 #i4j c4c4 #a3 #cbe #a c4c4 -#ca4e c4c4 #a3 #bjjj #a c4c4 #ai4j c4c4 #a3 #cej #a c4c4 #cd4j c4c4 #a3 #cjjj #a c4c4 #bi4j c4c4 #a3 #cge #a c4c4 #cf4e c4c4 #a3 #djjj #a c4c4 #ci4j c4c4 #a3 #djj #a c4c4 #ci4j c4c4 #a3 #ejjj #a c4c4 #di4j c4c4 ---------------------------------------- ---------------------- #a3 #djj #a c4c4 #ci4j c4c4 #a3 #dej #a c4c4 #dd4e c4c4 #a3 #ejj #a c4c4 #di4j c4c4 ---------------------------------------- ------------------------ ,,table ,,iii ,,table ,,iv ,us+ #j4a /ock solution ,us+ #j4ja /ock solution ,?ird ,f\r? dilution ,dilu5t ,dilution ,dilu5t ---------------------------------------- ------------------------ #a3 #ajjj #a c4c4 #j c4c4 #a3 #aj1jjj #a c4c4 #j c4c4 #a3 #abej #a c4c4 #j4be c4c4 #a3 #ae1jjj #a c4c4 #j4e c4c4 #a3 #aejj #a c4c4 #j4e c4c4 #a3 #bj1jjj #a c4c4 #a4j c4c4 #a3 #agej #a c4c4 #j4ge c4c4 #a3 #be1jjj #a c4c4 #a4e c4c4 #a3 #bjjj #a c4c4 #a4j c4c4 #a3 #cj1jjj #a c4c4 #b4j c4c4 #a3 #bbej #a c4c4 #a4be c4c4 #a3 #ce1jjj #a c4c4 #b4e c4c4 #a3 #bejj #a c4c4 #a4e c4c4 #a3 #dj1jjj #a c4c4 #c4j c4c4 #a3 #bgej #a c4c4 #a4ge c4c4 #a3 #de1jjj #a c4c4 #c4e c4c4 #a3 #cjjj #a c4c4 #b4j c4c4 #a3 #ej1jjj #a c4c4 #d4j c4c4 #a3 #cbej #a c4c4 #b4be c4c4 #a3 #ee1jjj #a c4c4 #d4e c4c4 #a3 #cejj #a c4c4 #b4e c4c4 #a3 #fj1jjj #a c4c4 #e4j c4c4 #a3 #cgej #a c4c4 #b4ge c4c4 #a3 #fe1jjj #a c4c4 #e4e c4c4 #a3 #djjj #a c4c4 #c4j c4c4 #a3 #gj1jjj #a c4c4 #f4j c4c4 #a3 #dbej #a c4c4 #c4be c4c4 #a3 #ge1jjj #a c4c4 #f4e c4c4 #a3 #dejj #a c4c4 #c4e c4c4 #a3 #hj1jjj #a c4c4 #g4j c4c4 #a3 #dgej #a c4c4 #c4ge c4c4 #a3 #he1jjj #a c4c4 #g4e c4c4 #a3 #ejjj #a c4c4 #d4j c4c4 #a3 #ij1jjj #a c4c4 #h4j c4c4 -------------------------------- #a3 #ie1jjj #a c4c4 #h4e c4c4 #a3 #ejjj #a c4c4 #d4j c4c4 #a3 #ajj1jjj #a c4c4 #i4j c4c4 #a3 #fjjj #a c4c4 #e4j c4c4 ----------------------------------- #a3 #gjjj #a c4c4 #f4j c4c4 #a3 #ajj1jjj #j4a c4c4 #j4i c4c4 #a3 #gejj #a c4c4 #f4e c4c4 #a3 #bjj1jjj #j4a c4c4 #a4i c4c4 #a3 #hjjj #a c4c4 #g4j c4c4 #a3 #bej1jjj #j4a c4c4 #b4d c4c4 #a3 #ijjj #a c4c4 #h4j c4c4 #a3 #cjj1jjj #j4a c4c4 #b4i c4c4 #a3 #aj1jjj #a c4c4 #i4j c4c4 #a3 #djj1jjj #j4a c4c4 #c4i c4c4 ------------------------------- #a3 #ejj1jjj #j4a c4c4 #d4i c4c4 #a3 #aj1jjj #a c4c4 #i4j c4c4 ----------------------------------- #a3 #ae1jjj #a c4c4 #ad4j c4c4 #a3 #ejj1jjj #j4a c4c4 #d4i c4c4 #a3 #bj1jjj #a c4c4 #ai4j c4c4 #a3 #fjj1jjj #j4a c4c4 #e4i c4c4 #a3 #be1jjj #a c4c4 #bd4j c4c4 #a3 #gjj1jjj #j4a c4c4 #f4i c4c4 #a3 #cj1jjj #a c4c4 #bi4j c4c4 #a3 #gej1jjj #j4a c4c4 #g4d c4c4 -------------------------------- #a3 #hjj1jjj #j4a c4c4 #g4i c4c4 #a3 #ijj1jjj #j4a c4c4 #h4i c4c4 #a3a1jjj1jjj #j4a c4c4 #i4i c4c4 -------------------------------------- ,,temp]ature ,,pressure ,,table4 ---------------------------------------- ----------------------- ,temp]ature ,p\nds p] sq4 94 ,c5tigrade ,mm4 ( ,hg4 absolute pressure ,atmosph]es ---------------------------------------- ----------------------- #ih deg4 #gjg4a #ac4g #j4ic #ii deg4 #gcc4a #ad4b #j4if #ajj deg4 #gfj4j #ad4g #a4jj #aja deg4 #ghg4h #ae4b #a4jc #ajb deg4 #haf4j #ae4h #a4jg #ajc deg4 #hde4b #af4c #a4aa #ajd deg4 #hge4d #af4i #a4ae #aje deg4 #ijf4d #ag4e #a4ai #ajf deg4 #ich4c #ah4a #a4bc #ajg deg4 #iga4a #ah4h #a4bg #ajh deg4 #ajjd4i #ai4d #a4cb #aji deg4 #ajci4f #bj4a #a4cf #aaj deg4 #ajge4c #bj4h #a4da #aaa deg4 #aaab4j #ba4e #a4df #aab deg4 #aadi4h #bb4b #a4ea #aac deg4 #aahh4f #bb4i #a4ef #aad deg4 #abbh4d #bc4g #a4fa #aae deg4 #abfi4d #bd4e #a4fg #aaf deg4 #acaa4d #be4c #a4gb #aag deg4 #aced4f #bf4b #a4gh #aah deg4 #acii4j #bg4j #a4hd #aai deg4 #addd4e #bg4i #a4ij #abj deg4 #adia4b #bh4h #a4if #aba deg4 #aeci4b #bi4g #b4jb #abb deg4 #aehh4d #cj4g #b4ji #abc deg4 #afch4i #ca4g #b4ae #abd deg4 #afij4g #cb4g #b4bb #abe deg4 #agdc4h #cc4g #b4bi ---------------------------------------- ----------------------- ,,table ,,= ,,desiccation ,,at ,,l[ ,,temp]atures ,,9 ,,vacuo4 -------------------------- ,temp]ature ,c5tigrade ,mm4 ( ,hg4 ------------------------- #ba deg4 #ah4d #bb deg4 #ai4f #bc deg4 #bj4h #bd deg4 #bb4a #be deg4 #bc4e #bf deg4 #bd4i #bg deg4 #bf4d #bh deg4 #bh4j #bi deg4 #bi4g #cj deg4 #ca4e #ca deg4 #cc4c #cb deg4 #ce4c #cc deg4 #cg4c #cd deg4 #ci4e #ce deg4 #da4g #cf deg4 #dd4a #cg deg4 #df4f #ch deg4 #di4b #ci deg4 #ea4i #dj deg4 #ed4h #da deg4 #eg4h #db deg4 #fa4j #dc deg4 #fd4c #dd deg4 #fg4g #de deg4 #ga4c #df deg4 #ge4a #dg deg4 #gi4j #dh deg4 #hc4a #di deg4 #hg4d #ej deg4 #ia4i ------------------------- ,,anti=m9 ,,me?od ,= ! detection ( ,b4 ,tub]culosis4 ,anti=m9 0 introduc$ into bact]iological te*nique by ,uhl5hu? 9 #aijh = ! purpose ( demon/rat+ tub]cle bacilli :5 pres5t 9 small numb]s1 9 sputum or o!r mat]ial4 ,x is a p[]ful oxidis+ ag5t & rapidly de/roys mo/ bact]ia1 b tub]cle & o!r acid-fa/ organisms resi/ its le?al action = consid]able p]iods1 & ^u ?is fact ! me?od is bas$4 ,to prep>e ,anti=m9 measure out & mix3-- ,eau de ,javelle 7,liquor sodae *lor9atae--,b4,p47 #ej c4c4 ,sodic hydrate #ae p] c5t4 aque\s solution #ej c4c4 ,,me?od4 #a4 ,introduce ! sputum or o!r mat]ial 7e4 g4 milk deposit & cream2 pus2 m9c$ gl& or o!r organ2 case\s mat]ial2 brok5 d[n foci1 etc47 into a /]ile tube & !n add an equal volume ( anti=m94 #b4 ,close ! tube ) a rubb] cork & %ake vigor\sly 7a sample ( anti=m9 t does n foam at ?is /age is ( little use74 ,dis9tegration ( ! mat]ial at once />ts1 associat$ bact]ia are de/roy$ & ! mixture rapidly becomes a homog5\s b turbid fluid--a process :i* may be ha/5$ by3-- #c4 ,plac+ ! tube 9 ! incubator at #cg deg4 ,c4 = #cj m9utes--%ak+ f "t to "t4 #d4 ,c5trifugalise ! fluid ?or\ghly1 at high spe$4 #e4 ,pipette (f ! sup]natant fluid1 fill up ) /]ile di/ill$ wat]1 cork ! tube & %ake to di/ribute ! deposit ?r\gh\t ! wat]4 ,aga9 c5trifugalise4 #f4 ,repeat /eps #d & #e twice m4 #g4 ,employ "o portion ( ! f9al deposit to inoculate gu9ea pigs4 #h4 ,plant ! rema9d] ( ! deposit freely on ,dorset's ,egg m$ium2 cap & incubate at #cg deg4 ,c4 ,,note4--,if only microscopical films are ne$$1 fill up ! c5trifuge tube ) ,ligro9 7a petroleum e!r7 9 place ( /]ile di/ill$ wat] 9 /ep #e & prep>e ! films f ! surface ( ! fluid1 to /a9 by ! ,ziehl-,neels5 process4 ,,index ,abbe's cond5s]1 #g ,abbott's /a9 = spores1 #ajg ,ab]ration1 *romatic1 #ef sph]ical1 #ee ,absolute alcohol z a fixative1 #hb z an antiseptic1 #bg ,absorb5t pap] = dry+ cov]-slips1 #fi ,a4 ,c4 ,e4 mixture1 #cde ,acetic acid = cle>+ films1 #hb ,a*romatic cond5s]1 #ed ,acid haemat91 #if production1 analysis table1 #bhc by bact]ia1 #ade inve/igation (1 #bhj qualitative exam9ation1 #bhc1 #bhd quantitative exam9ation1 #bhj ,acid-fa/ bacilli 9 tissues1 to /a91 #abd ,action ( v>i\s gases on bact]ia1 #bie ,active immunisation1 illu/rative example1 #cbb ,adju/able wat] ba?1 #bii ,a]obic cultures1 #bba ,a]og5ic bact]ia1 #aca ,aescul9 ag>1 #bjd ,ag> gelat9e 7gu>ni>i71 #aid me?ods ( prep>ation1 #afg surface plates1 #bcb ,ag>-ag>1 prep>ation (1 #afg ,agglut9ation reaction1 macroscopical1 #chf microscopical1 #che ,agglut991 #cha ,air1 analysis (1 #dfh filt]1 #dj pump1 ,g]yk1 #dc ,album9 solution1 ,may]'s1 #abj ,alcohol production1 te/ =1 #bhe ,alkal9e pyro1 #bci ,alum c>m9e1 #if ,ammonia production te/ =1 #bhe ,amphitri*\s bact]ia1 #acf ,ana]obic cultures1 #bcf ,botk9's me?od1 #bdc ,bu*n]'s me?od1 #bch ,bullo*'s me?od1 #bde ,hesse's me?od1 #bcg ,mc,leod's me?od1 #bdj m$ia1 #ahj ,novy's me?od1 #bdd ,ana]obic cultures1 ,r\x's biological me?od1 #bcg physical me?od1 #bcg vacuum me?od1 #bch ,wright's me?od1 #bci ,anae/hetics1 #cde ,analysis ( air1 app>atus =1 #dfi me?od (1 #dfh qualitative bact]iological1 #dgj quantitative bact]iological1 #dfh ( butt]1 qualitative bact]iological1 #deh quantitative bact]iological1 #deg ( cream1 qualitative bact]iological1 #deh quantitative bact]iological1 #deg ( fi%1 #dfj ( ice cream1 qualitative bact]iological1 #deg ( meat1 app>atus =1 #dfj me?od (1 #dfj qualitative bact]iological1 #dfb ( milk1 app>atus =1 #ddd collection ( samples1 #dda me?od (1 #dda qualitative bact]iological1 #ddf4 quantitative bact]iological1 #ddd ( oy/]s1 #dfc ( sewage1 qualitative bact]iological1 #dfg quantitative bact]iological1 #dff ( %ellfi%1 #dfc ( soil1 app>atus =1 #dgc collection ( samples1 #dga me?od (1 #dgj qualitative bact]iological1 #dgf quantitative bact]iological1 #dgc ( wat]1 app>atus =1 #dbj1 #dbg collection ( samples1 #daf me?od (1 #daf qualitative bact]iological1 #dbf ,analysis ( wat]1 quantitative bact]iological1 #dbj ,anil9e dyes1 #hc ,g5tian violet1 #ie wat]1 to prep>e1 #ajh ,animal tissue m$ia 7,frugoni71 #baj ,animals1 natural infections (1 #ccg ,anti=m9 me?od = ,b4 tub]culosis1 #ejb ,antig51 def9ition (1 #cbd ,antiseptics1 #bg action (1 #caj ,app>5t fil? 9 milk1 #dej ,arnold's /eam /]ilis]1 #cd ,ar?rog5\s spores1 #ach ,ascitic b\illon1 #baj fluid ag> 7,wass]mann71 #bac ,ascomycetae1 #abh ,ascopores1 #abi ,asp>ag9 ,m$ia 7,frankel & ,voges71 #ahc 7,us*9sky71 #ahc ,asp]gillus1 #abg ,atmosph]ic conditions1 #bie ,att5uat+ ! virul5ce ( organisms1 #cba ,autoclave1 #cg to use1 #cg ,automatic pipettes1 #ac ,autopsies1 #cif ,autopsy1 c>d index =1 #djb ,bacilli1 morphology (1 #acb ,bacillus an?racis 9 soil1 #dgg 9 wat]1 #ddj coli 9 wat]1 detection (1 #dbi diph!riae 9 milk1 #deb ent]itidis 9 wat]1 #dcg sporog5es 9 milk1 #deb 9 wat]1 #dch o$ematis maligni 9 soil1 #dgg tetani 9 soil1 #dgg 9 wat]1 #dda tub]culosis 9 milk1 #dec anti=m9 me?od1 #ejb typhosus 9 wat]1 #dda ,bact]ia1 anatomy (1 #acd classification (1 #aca gr\p+ (1 = /udy1 #daj 9 tissues1 demon/ration (1 #aad influ5ce ( environm5t on1 #adb metabolic products (1 #adc me?ods ( id5tification1 #bei microscopical exam9ation (1 /a9$1 #ha un/a9$1 #gd physiology (1 #acf ,bact]ia1 simple /a9s =1 #ij ,bact]ial emulsion1 prep>ation (1 #chi enzymes1 #add1 #bgg f]m5ts1 #add food /uffs1 #adb tox9s1 #add ,bact]iological analyses1 g5]al consid]ations1 #dae exam9ation ( blood1 #cgg ,base ( microscope1 #ej ,basidium1 #abh ,be] wort1 prep>ation (1 #age ,beetroot m$ia1 #bjj ,beggiotoa1 morphology (1 #acc ,b5zole ba?1 #bef ,b]kefeld filt]1 #db ,beyr9ck's solution ,i1 #aig ,,ii1 #aih ,bile salt ag> 7,mac,conkey71 #bje bro?1 d\ble /r5g?1 #aii 7,mac,conkey71 #ahj ,bio*emical exam9ation ( cultures1 #bgf ,bio*emi/ry ( bact]ia1 #bgf ,biological diff]5tiation ( bact]ia1 #bdi ,bipol> g]m9ation1 #adj ,bism>ck br[n1 #id ,bla/omycetes1 morphology (1 #abi ,blood ag>1 #aga1 #bad plates1 animal1 #bea human1 #bej 7,wa%b\rn71 #bad bact]iological exam9ation (1 #cgg cells1 wa%+ (1 #chh collection (1 = s]ological exam9ation1 #cgi films1 prep>ations (1 #cgf /a9+ (1 #ig hi/ological exam9ation (1 #cgc pipettes1 #aa s]ological exam9ation (1 #cgh /a9s1 #ig ,blood-s]um 7,c\ncilman & ,mallory71 #bjh inspissat$1 #afh 7,loeffl]71 #bjh 7,lorra9 ,smi?71 #bjh ,bl[pipe table1 #i ,body tube ( microscope1 #ej ,bohemian flask1 #d ,boil+ wat]1 #cc ,bone m>r[1 films1 prep>ation (1 #djj ,bordet-,g5g\ reaction1 #cic ,boric acid 9 milk1 te/ =1 #ddb ,botk9's ana]obic me?od1 #bdc ,b\illon1 prep>ation (1 #afc ,bra9 extract1 #adi ,bread pa/e1 #aic ,brilliant gre5 ag> 7,conradi71 #bjf bile salt ag> 7,fawcus71 #bjf ,br[nian movem5t1 #gi ,bu*n]'s ana]obic me?od1 #bch ,bullo*'s ana]obic me?od1 #bde tubes = p]man5t prep>ations1 #djg ,bunge's mordant1 #ajd ,burri's ,*9ese ink /a91 #gg ,butt]1 analysis (1 #deg qualitative analysis (1 #deh quantitative analysis (1 #deg ,cadav]1 prep>ation (1 = autopsy1 #cig ,cages = gu9ea-pigs1 #cdc = laboratory animals1 #cda = mice1 #cdb = rabbits1 #cdc = rats1 #cdb ,calculat$ figure = weight ( m$ium mass1 #aff1 #afg ,cambi]'s c&le me?od ( isolat+ coli-typhoid gr\ps1 #dch ,cam]a lucida1 #fb ,capaldi-,proskau] m$ium1 ,no ,i1 #ahf ,no ,,ii1 #ahg ,capill>y pipettes1 #aj graduat$1 #ac ,capitate bacilli1 #aci ,capsule =mation1 #acd ( bact]ia1 #acd !rmo-regulator1 #bah ,capsules1 collodion1 inoculation (1 #ceg prep>ation (1 #ceg glass1 #f to clean infect$1 #bj new1 #ah to /a91 #ii to /]ilise1 #ca ,c>bohydrate m$ia1 prep>ation (1 #agg ,c>bolic acid z a g]micide1 #bg1 #dha me?od ( isolat+ coli-typhoid gr\p1 #dcg ,c>bolis$ ag>1 #bjb b\illon1 #bjb gelat9e1 #bjb ,c>bon dioxide 9 cultures1 te/ =1 #bhi ,c>d index1 #ccf1 #djb ,c>rot m$ia1 #bjj ,c$>wood oil = imm]sion l5s1 #hh ,cell wall ( bact]ia1 #acd ,celloid9 sacs1 manufacture (1 #ceh ,cellul> incubator1 #baf ,c5trifugal ma*9e = blood & s]um "w1 #cbg = milk "w1 #ddg ,c5trifugalis$ milk1 #ddi ,c5trigade degrees1 conv]sion (1 #did ,*emical products ( bact]ia1 #ade ,*9a gre5 ag> 7,w]bitski71 #bjg ,*lor(orm z an antiseptic1 #bg ,*romatic ab]ration1 #ef ,*romog5ic bact]ia1 #aca ,*romop>\s bact]ia1 #add ,*romophor\s bact]ia1 #add ,citrat$ blood ag>1 #aia ,clado?rix1 morphology1 #aic ,classification ( bact]ia1 #aca ( fungi1 #abf ,clavate bacilli1 #aci ,cle>+ films ) acetic acid1 #hb ,clo/ridium1 #aci ,co>se adju/m5t1 #ea ,cobweb micromet]1 #ff ,coca9e1 #cde ,cocci1 morphology (1 #aca ,coccidium infection1 #cci ,coeffici5t1 inf]ior le?al1 #cab ( inhibition1 #caa ph5ol1 #dhi sup]ior le?al1 #cac ,cohn's solution1 #aia ,cold incubator1 #bag ,coli-typhoid gr\p1 diff]5tial table1 #dcc 9 milk1 #dea 9 soil1 #dgg isolation (1 #dcb memb]s (1 #dcj ,collection ( blood = bact]iological exam9ation1 #cgh = m$ia mak+1 #afh ( milk samples1 #ddc ( pa?ological mat]ial dur+ life1 #cgc ( pus1 #cgc ( soil sample1 #dga ( wat] samples1 #daf ,collodion capsules1 #ceg sacs1 manufacture (1 #ceg ,colonies ( bact]ia1 edges1 #bfg ,col\r$ light1 action (1 #cji ,columella1 #abg ,comp>ative haemocytology1 #cgd ,complem5t1 def9ition (1 #cbe fixation te/1 #cic ,conc5tration me?od 9 wat]1 analysis1 #dcd ,cond5s] a*romatic1 #ed d>k gr\nd1 #fj p>aboloid1 #fj sub/age1 #ed ,condidium1 #abh ,cont9u\s /]ilisation1 #cf ,contra/ /a9s1 #ic ,corrosive sublimate 7,lang71 #hb ,cotton-wool filt]1 #dj ,c\nt]/a9+ films1 #hd ,c\nt+ plate colonies1 #dbc ,cov]-slip films1 #ha to clean new1 #bb us$1 #bd ,crates = te/-tubes1 #ca ,cream1 analysis (1 #deg qualitative analysis (1 #deh quantitative analysis (1 #deg ,cr5o?rix morphology1 #acc ,crit]ia ( infection1 #cgj ,crit]ion ( immunity1 #cbd ,cultural *>act]s1 macroscopical exam9ation1 #bfa ,culture flask1 ,guy's1 #e ,kolle1 #d ,r\x1 #e ,cuneate bacilli1 #aci ,cutane\s inoculation1 #ceb ,d>k gr\nd cond5s]1 #fj illum9ation1 #hg ,daught] cells1 #abi ,daylight1 diffuse1 action (1 #cjh ,decimal scales1 #cdj ,decol\ris+ ag5ts1 #hd ,def9ition ( objective1 #ef ,depilatory p[d]1 #cdf ,description ( plate culture1 #bfa ,descriptive t]ms1 #bfa ,desiccation1 effects (1 #cjf table1 #eja ,desiccator1 ,muell]'s1 #cjg ,dextrose solution1 prep>ation (1 #agh ,diaphragm1 iris1 #ec ,dia/atic enzymes1 te/s =1 #bgh ,diff]5tial atmosph]e cultivation1 #beg incubation1 #bee m$ia1 #bee /a9+1 #ajh /]ilisation1 #bef ,dilut+ *amb]1 #bdh ,dilution by teat pipette1 #chc ( s]um1 #chb tables1 #dih ,dilutions1 prep>ations (1 #dif ,diph!ria1 bacillus (1 9 milk1 #deb ,diplobacilli1 morphology (1 #acc ,diplococci1 morphology (1 #acc ,diplococcus pneumoniae1 immunisation aga9/1 #cbb ,discont9u\s /]ilisation1 #cf ,discs ( pla/]-(-,p>is1 #aib ,dis9fectants1 action (1 #caj *emical1 #bg te/+ (1 #dhj ,dissociat+ fluid1 ,price ,jones2 #djj ,dosage ( inoculum1 #caf ,d\ble nosepiece1 #eh /a9s = spores1 #ajf sug> ag> 7,russell71 #bjg ,drop-bottle1 #gc ,dry heat1 #bh ,dunham's solution1 #agg ,dyes1 anil9e1 #hc ,e>!nw>e box = dirty slides1 #gj ,e>?y salts ag> 7,lipman & ,br[n71 #aig ,edge ( individual colonies1 *>act]s (1 #bfg ,egg album9 ag>1 #bac bro?1 7,lips*uetz71 #bac m$ia 7,dorset71 prep>ation (1 #agd inspissat$1 #bab 7,lub5au71 #bji 7,t>*an(f & ,kolesnik(f71 #bab to cle> nutri5t m$ia )1 #aff ,ehrli*'s eyepiece1 #ee ,eikonomet]1 #fe ,eis5b]g's milk-rice m$ium1 #ahi ,electric d5tal eng9e1 #cfj signal clock1 #ch w>m /age1 #ei ,elevation ( colonies1 #bfc ,eisn]'s gelat9e1 #bjd me?od ( isolat+ coli3 typhoid gr\p1 #dch ,endog5\s spores1 #ach v>ieties (1 #aci ,endo-g]m9ation1 #aci ,engli% pro( ag>1 ,blaxall1 #aic ,enum]at+ colonies on plates1 #dbc discs1 ,jeff]'s1 #dbd ,pakes'1 #dbd ,enri*m5t me?od 9 wat] analysis1 #dbg ,enum]ation ( micro-organisms1 #dbc ,environm5tal conditions1 #adb ,enzyme production1 inve/igation (1 #bgg ,eos91 #ic ,equatorial g]m9ation1 #adj ,erl5mey] flask1 #d ,ern/s*5 ,ko]n]1 #acf ,esm>*'s roll culture1 #bbf wat] collect+ bottle1 #dag ,e/imation ( reaction ( m$ia1 #bhj ,e!r flame1 #bh soluble acids1 #bhd ,euca9e1 #cde ,exalt+ virul5ce ( organisms1 #cbj ,exam9ation ( milk1 #dda ,exp]im5tal infections1 /udy (1 dur+ life1 #cgj inoculation ( animals1 #ccb ,extracellul> tox9s1 #add ,eyepiece1 ,ehrli*1 #ee micromet]1 #fc ,eyepieces1 #ee ,eye-%ade1 #eg ,fahr5heit degrees1 conv]sion (1 #die ,fe$+ exp]im5ts1 #cfi ,f]m5tation reactions1 #bgi tubes1 #ag ,field ( objective1 #ef ,fil> micromet]1 #ff ,fill+ tubes1 etc41 ) m$ium1 #afj ,film prep>ations1 #ha fix+1 #ha mak+1 #ha m\nt+1 #he /a9+1 #hc ,filt] c&le1 clos$1 #dg op51 #dc te/+ effici5cy (1 #dgh to dis9fect1 #bh to /]ilise1 #bi flask1 #f pap]s1 to fold1 #aef ,filt]s1 cotton-wool1 #dj porcela91 #db te/+ (1 #dgh ,filtration1 #dj by aspiration1 #db ( m$ia1 #aef "u pressure1 #de ,f9e adju/m5t1 #ea sp9dle head1 #eb ,fi%1 analysis (1 #dfj b\illon1 #aij ,fi% gelat9e1 #aij gelat9e-ag>1 #aij ,fi%+ colonies1 #bec ,fission1 reproduction by1 #acf ,fixation1 #ha by heat1 #ha ( tissues1 #aad ,fix+ fluids1 = films1 #hb ,flagella1 classification ( bacilli by1 #acf to /a91 #aja ,flask ,bohemian1 #d ,erl5mey]1 #d filt]1 #f ,kitasato'a s]um1 #f ,kolle's culture1 #d ,flasks & te/ tubes1 to plug1 #bd to clean dirty1 #bj new1 #ah to /]ilise1 #ca ,fleis*wass]1 #adh ,fluid cultures1 description (1 #bga m$ia1 #adf ,foot ( microscope1 #ej ,=maldehyde 9 milk1 ,hehn]'s te/ =1 #ddb ,=mal9 me?od ( pres]v+ cultures1 #djg tissues1 #djd ,fractional /]ilisation1 #cc ,fra5kel & ,voge's solution1 #ahc ,fra5kel's e>? bor]1 #dgb ,freez+ me?od = sections1 #aae ,fr5* ,mannite ,ag> 7,sab\raud71 #aic pro( ag> 7,sab\raud71 #aic ,fre% prep>ations ( bact]ia1 #gd ,fri$la5d]'s capsule /a9 = sections1 #abc ,fro/'s m\nt+ fluid1 #djf ,froz5 sections1 rapid me?od1 #aaf ,fu*s91 #ib ag> 7,braun71 #bje sulphite ag> 7,endo71 #bjf ,gas analysis1 qualitative1 #bij quantitative1 #bij collect+ app>atus1 #bia g5]ators1 #bdb production by bact]ia1 #bhi tubes = m$ia1 #afa ,gasp]9i's solution1 #aic ,gelat9 ag>1 #aic prep>ation (1 #afd surface plates1 #bca ,g5]al anae/hetics1 #cde ,g5tian violet1 #ia ,g]man l9$ pap]1 #fi ,g]micides1 #bg te/+ p[] (1 #dhj ,g]m9ation1 #adj ,g]yk air-pump1 #dc ,glass app>atus 9 common use1 #c to clean1 #ah ,glass-cutt+ knife1 #h ,glucose =mate ag> 7,kitasato71 #ahj b\illon 7,kitasato71 #ahj gelat9e 7,kitasato71 #ahj ,glyc]9at$ potato1 #bji ,glyc]9e ag>1 #bji blood-s]um1 #bjh b\illon1 #bji potato b\illon1 #bjc bro?1 #bjc ,goadby's gelat9e1 #bad ,gonidium1 #abh ,goniodophore1 #abh ,graduat$ capill>y pipettes1 #ac pipettes1 #f ,gram-,claudius' diff]5tial /a91 #aji ,gram's diff]5tial /a91 #ajh ,gram-,weig]t = sections1 #aba1 #abb ,gram-,weig]t's diff]5tial /a91 #aji modifi$1 #aaj ,grease p5cils1 #gb ,gr\p+ ( bact]ia = /udy1 #daj ,gu>d$ trep9e1 #cfj ,gu>ni>i's ag> gelat9e1 #aid ,gu9ea-pig cages1 #cdc hold]1 #cej ,gull&'s solution1 #hb ,gum solution1 prep>ation (1 #aaf ,guy's culture bottle1 #e ,gypsum blocks 7,engel & ,hans571 #aib ,haemat91 #ie ,haematocytomet]1 #bdh ,haematoxil91 #ie ,haemolys91 def9ition (1 #cbf prep>ation (1 #cbg /orage (1 #cca ,haemolytic s]um1 titration (1 #cbh ,hang+-block culture 7,hill71 #bce ,hang+-drop cultures1 #bcc exam9ation (1 #hf1 #gi prep>ation (1 #gh p]man5t /a9+ (1 #hj slides1 #gj ,h>d5+ tissues1 #aad ,h>icot ag>1 #bjj b\illon1 #bjj ,hay infusion1 #bjj ,he>son's wat] ba?1 #bii ,heat effect (1 #bii ,hehn]'s te/1 #ddb ,heiman's s]um ag>1 #baj ,hesse's ana]obic culture me?od1 #bcg ,hi/ological exam9ation ( blood1 #cgc ,hold] = gu9ea-pigs1 #cej ,hot air1 #bi /]ilis]1 #cj to use1 #ca incubator1 #bag ,hot-wat] funnel1 #aeh ,human blood ag> plates1 #bej ,huygh5ian eyepiece1 #ee ,hydrog51 g5]at+ app>atus1 #bdb 9 culture1 te/ =1 #bhi p]oxide 9 milk1 te/ =1 #ddb ,hyphomycetes1 morphology (1 #abf reproduction (1 #abf ,ice-box1 = wat] samples1 #dai ,ice cream1 analysis (1 #deg ,illum9ant = microscope1 #fg ,immune body1 #cic ,immunisation1 me?ods (1 #cba ,imp]ial sy/em1 #dib factors = conv]t+1 #dic ,impression films1 #he ,incubators1 #baf ,index c>ds1 #ccf1 #djc ,indol1 te/ =1 #bhf ,infection1 def9ition (1 #cgj g5]al obs]vations dur+ life1 #cga results (1 #djd ,influ5ce ( environm5t on bact]ial gr[?1 #adb ,inhalation1 fluid inoculum1 #cfe p[d]$ inoculum1 #cff ,inhibition coeffici5t1 #caj1 #caa ,inoculation c>d index1 #ccf cutane\s1 #ceb intracranial1 #cfj intramuscul>1 #cee intraocul>1 #cfb intrap]itoneal1 #cee intrapulmon>y1 #cfc intrav5\s1 #cfc ( collodion capsules1 #ceg subcutane\s1 #cec syr+e1 #cdd ,inoculum1 "* (1 #cdf prep>ation (1 #cdf ,inosite-free m$ia--b\illon 7,durham71 #ahc ,insep>ate tox9s1 #add ,int]mitt5t /]ilisation1 #cf ,intracellul> tox9s1 #add ,intrac]ebral inoculation1 #cfb ,intracranial inoculation1 #cfj ,intraga/ric inoculation1 l>ge animals1 #cfg ,m>ks me?od1 #cfg ,intramuscul> inoculation1 #cee ,intraocul> inoculation1 #cfb ,intrap]itoneal inoculation1 #cee ,intrapulmon>y inoculation1 #cfc ,intrav5\s inoculation1 #cfc ,9 vacuo ana]obia cultures1 #bhi ,inv]t9 enzymes1 te/s =1 #bgi ,involution =ms1 #acg ,iod9e solution1 #ajh ,iron b\illon1 #ahe peptone solution 7,pakes71 #ahe ,isolation by animal exp]im5ts1 #beh by diff]5tial atmosph]e1 #beg incubation1 #bee m$ia1 #bee /]ilisation1 #bef by dilution1 #bdh by plate cultures1 #bej subcultures1 prep>ation (1 #bed ,jeff]'s c\nt+ disc1 #dbd ,j5n]'s /a91 #ig ,jores' m\nt+ fluid1 #dje ,kais]l+ fix+ solution1 #dje ,kan?ack's s]um ag>1 #baa ,kill$ cultivations1 #cah ,kipp's hydrog5 app>atus1 #bdb ,kitasato's s]um flask1 #f ,klebs-,loeffl] bacillus 9 milk1 #deb ,ko*'s /eam /]ilis]1 #cd ,kohle's culture flask1 #d ,lab enzymes1 te/ =1 #bgi ,laboratory animals1 #cce comp>ative haematocytology (1 #cgd normal temp]ature1 #cgb regulations1 #a ,lactose litmus ag> 7,wurtz71 #bjc b\illon1 #bjc gelat9e 7,wurtz71 #bjc ,lakmus ,molke1 #bjc ,lang's solution1 #hb ,lead b\illon1 #ahe peptone solution1 #ahf ,lei%man's /a91 #ih = sections1 #abe ,lemco bro?1 #afc ,lepto?rix1 morphology1 #acc ,le?al dose1 m9imal1 #caf ,leviditi's /a9+ me?od1 #abd ,light1 action (1 #cjh ,liquefiable m$ia1 #adg ,liquid soap1 #cdf ,li?ium c>m9e1 #if ,litmus b\illon1 #ahf gelat9e1 #bjb milk cultures1 description (1 #bgb prep>ation (1 #agb nutrose ag> 7,drigalski-,conradi71 #bje :ey1 #aie ag>1 #aif gelat9e1 #aif 7,petrus*ky71 #aie ,local anae/hetics1 #cde reaction to infection1 #cgb ,locomotive movem5t1 #hj ,loeffl]'s capsule /a91 #ajc s]um1 #bjh ,lophotri*\s bacilli1 #acf ,lorra9 ,smi? electric w>m /age1 #ei s]um1 #bjh ,lugol's solution1 to prep>e1 #ajh ,lysol1 #bg ,mac,conkey's capsule /a91 #ii m$ia1 #ahj1 #aii1 #bje ,mac,crorrie's capsule /a91 #ajc ,macroscopical exam9ation ( cultures1 #bfa ,mala*ite gre5 ag> 7,loeffl]71 #bjg ,malt extract solution 7,h]s*ell71 #aif ,m>g9 ( individual colonies1 #bfg ,m>t9's filt]+ app>atus1 #cbj ,mat]ial = inoculation1 #cdf ,may]'s album91 #abj ,mean ph5ol coeffici5t1 #dij ,measur+ bact]ia1 #fa ,meat1 bact]iological analysis (1 #dfj extract prep>ation (1 #adh reaction (1 #adi ,me*anical sep>ation ( bact]ia1 #bdi /age1 #eb ,m$ia1 filtration (1 #aef prep>ation (1 #afc a]obic culture1 #bbb aescul9 ag>1 #bjd ag>-ag>1 #afg ag> gelat9e 7,gu>ni>i71 #aid ,m$ia1 prep>ation ( ana]obic culture1 #ahj animal tissue 7,frugoni71 #baj ascitic b\illon1 #baj fluid ag> 7,wass]mann71 #bac asp>ag9 7,fra5kel & ,voge's71 #ahc 7,us*9sky71 #ahc be] wort1 #age beetroot1 #bjj ,beyr9ck's solution ,i1 #aig ,,ii1 #aih bile salt ag> 7,mac,conkey71 #bje bro? 7,mac,conkey71 #ahj d\ble /r5g?1 #aii blood ag> 7,wa%b\rn71 #bad blood-s]um1 #afh 7,c\ncilman & ,mallory71 #bjh 7,loeffl]71 #bjh 7,lorra9 ,smi?71 #bjh b\illon1 #afc bread pa/e1 #aic brilliant gre5 ag> 7,conradi71 #bjf bile salt ag> 7,fawcus71 #bjf ,capaldi-,proskau]1 ,no4 ,i1 #ahf ,no4 ,,ii1 #ahg c>bohydrate1 #agg c>bolis$ ag>1 #bjb b\illon1 #bjb gelat9e1 #bjb c>rot1 #bjj ,*9a gre5 ag> 7,w]bitski71 #bjg citrat$ blood ag>1 #aga ,cohn's solution1 #aia dextrose solution1 #agh d\ble sug> ag> 7,russell71 #bjg e>?y salt ag> 7,lipman & ,br[n71 #aig egg ,dorset1 #agd ,lub5au1 #bji egg-album51 inspissat$1 #bab 7,t>*an(f & ,kolesnik(f71 #bab egg-album9 ag>1 #bac bro? 7,lips*uetz71 #bac ,engli% pro( ag> 7,blaxall71 #aic fi% b\illon1 #aij gelat9e1 #aij ag>1 #aij fluid1 #adf ,fr5* mannite ag> 7,sab\raud71 #aic ,m$ia1 prep>ation ( ,fr5* pro( ag> 7,sab\raud71 #aic ,fu*s9 ag> 7,braun71 #bje sulphite ag> 7,endo71 #bjf gelat9e1 #aic ag>1 #aic glucose =mate ag> 7,kitasato71 #ahj b\illon 7,kitasato71 #ahj gelat9e 7,kitasato71 #ahj glyc]9at$ bro?1 #bji potato1 #bji glyc]9e ag>1 #bji blood-s]um1 #bjh1 #bji b\illon1 #bji potato b\illon1 #bjc gypsum blocks 7,engel & ,hans571 #aib h>icot ag>1 #bjj b\illon1 #bjj hay infusion1 #bjj inosite free-b\illon 7,durham71 #ahc iron b\illon1 #ahe peptone solution 7,pakes71 #ahe lactose litmus ag> 7,wurtz71 #bjc b\illon1 #bjc gelat9e 7,wurtz71 #bjc lakmus molke1 #bjc lead b\illon1 #ahe peptone solution1 #ahf lemco bro?1 #afc liquefiable1 #adg litmus b\illon1 #ahf gelat9e1 #bjb milk1 #agb nutrose ag> 7,drigalski-,conradi71 #bje :ey1 #aie ag>1 #aif gelat9e1 #aif 7,petrus*ky71 #aie mala*ite gre5 ag> 7,loeffl]71 #bjg malt extract solution 7,h]s*ell71 #aif milk1 #agb rice 7,eis5b]g71 #ahi 7,soyka71 #ahi ,naegeli's solution1 #aia ,naehr/(f ag> 7,hesse & ,ni$n]71 #aii neutral litmus solution1 #agi nitrate b\illon1 #ahe peptone solution 7,pakes71 #ahf nutri5t1 #adf ag>-ag>1 #afg ,m$ia1 prep>ation ( nutri5t b\illon1 #afc gelat9e1 #afd nutrose ag> 7,eyre71 #agb oleic acid ag> 7,flem+71 #bja ,omeliansky's nutri5t fluid1 #ahi ,p>ietti's b\illon1 #bjb p>snip1 #bjj ,pa/eur's solution1 #aia peptone rosolic acid wat]1 #ahf wat] 7,dunham71 #agg pla/]-(-,p>is discs1 #aib potato1 #agd gelat9e 7,elsn]71 #bjd 7,goadby71 #bad proteid free bro? 7,us*9sky71 #ahc rosolic acid peptone solutions1 #ahf s]um1 b\illon1 #baj dextrose wat]1 7,hiss71 #ahh sug>1 7,hiss71 #ahh wat]1 #agj s]um-ag> 7,heiman71 #baj 7,kan?ack & ,/ev5s71 #baa 7,libman71 #bab 7,w]!im]71 #baa silicate jelly 7,w9ogradsky71 #aih solid1 #adg special1 #ahb /ock nutri5t1 #afc sug>1 #agg ag>1 #ahe 7dextrose7 b\illon1 #ahd gelat9e1 #ahd sulph9digotate ag>1 #aha b\illon 7,weyl71 #aha gelat9e 7,weyl71 #aha tissue 7,nogu*i71 #bad turnip1 #bjj ur9e ag>1 #ahh b\illon1 #ahg gelat9e1 #ahg 7,hell]71 #ahh :eat b\illon 7,gasp]9i71 #aic :ey ag>1 #aie gelat9e1 #aie w9e mu/1 #aib ,w9ogradsky's solution 7= nitric organisms71 #aih 7= nitr\s organisms71 #aih wood a% ag>1 #bja wort ag>1 #agf gelat9e1 #agf ,m$ia1 prep>ation ( yea/ wat] 7,pa/eur71 #aia /&>disation (1 #aed /orage (1 9 bulk1 #aei /or+ tubes (1 #afa sore boxes1 #afb titration (1 #aej tub+ ( nutri5t1 #afj ,m]ismop$ia1 morphology (1 #acb ,mesophilic bact]ia1 #adc pa?og5ic effects1 #cae ,metabolic end-products1 #ade ,meta*romatic granules1 #acf ,metal in/rum5ts1 to /]ilise1 #bh ,metatrophic bact]ia1 #aca ,me?ods ( cultivation1 #bba ( id5tification ( bact]ia1 #bei ( inoculation1 #ceb ( isolation1 #bdh ( /]ilisation1 #bf ,me?yl5e-blue1 #ij ,metric sy/em1 #dib factors = conv]t+1 #dic ,mey]'s c>m9e1 #if ,microbes ( indication1 #dbf ,micrococci1 morphology1 #acb ,micrococcus1 melit5sis 9 milk1 #def ,micromet]1 fil>1 #ff net1 #fc ocul>1 #fc /age1 #fb ,micrometry1 me?ods (1 #fa ,micron1 #fa ,microscope1 #di ,microscopical exam9ation ( bact]ia1 #hf /a9$1 #hh un/a9$1 #hf obs]vations ( cultures1 #bgb ,milk1 analysis (1 qualitative1 #ddf quantitative1 #ddd cond5s$1 analysis (1 #ddd m$ia1 #aic prep>ation (1 #agb rice 7,eis5b]g71 #aic 7,soyka71 #ahi samples1 collection (1 #ddc s$im5t+ tubes1 #ddi ,m9imal le?al dose1 #caf ,mirror = microscope1 #ee ,moell]'s /a9 = spores1 #ajg ,moi/ heat1 #cb ,molecul> movem5t1 #gi ,monotri*\s bacilli1 #acf ,motility1 exam9ation =1 #gi true1 #hj ,m\lds1 exam9ation (1 #abf = p>aff9 imb$d+1 #aag1 #aai ,m\nt+ film prep>ations1 #he p>aff9 sections1 #aai ,m\se cages1 #cdb hold]1 #cea scales1 #cda ,mucor muc$o1 #abf ,mucor9ae1 #abf ,muell]'s desiccator1 #cjg ,muffle furnace1 #bh ,muirs's capsule /a91 #ajj flagella /a91 #aja ,museum prep>ations ( bact]ia1 #djg ( tissues1 #djd seal+ (1 #djf ,mycelium1 #abf ,mycoprote91 #ace ,naegeli's solution1 #aia ,naehr/(f ag> 7,hesse & ,ni$n]71 #aii ,nak$ flame1 #bh ,neiss]'s /a9 modifi$1 #aaa ,net micromet]1 #fc ,neutral litmus solution1 prep>ation (1 #agi r$1 #id ,nitrate b\illon1 #ahe peptone solution 7,pakes71 #ahf ,nitric organisms 9 soil1 #dgh ,nitroso9dol reaction1 #bhg ,nitr\s organisms 9 soil1 #dgg ,normal av]ages 7t4p4r471 #cgb s]um1 #cge ,nosepiece1 #eg d\ble1 #eh triple1 #eh ,navy's ana]obic me?od1 #bdd j>s1 #bde ,nuclei1 to /a91 #aje ,nucleus ( bact]ia1 #ace ,num]ical ap]ture1 #ef ,nutri5t m$ia1 #adf ,nutrose ag> 7,eyre71 prep>ation (1 #agb ,object m>k]1 #fa ,objectives1 #ee ,oblique tube cultures1 #bbc ,ocul> micromet]1 #fc ,ocul>s1 #ee ,oese1 plat9um1 #ga ,oidium1 #abh ,oil ( g>lic1 #bg ( mu/>d1 #bg ,oleic acid ag> 7,flem+71 #bja ,omeliansky's nutri5t fluid1 #ahi ,op]ation tables 7,eyre's71 #ceb 7,tat9's71 #cea ,opsonic index1 #cic ,opsonic index1 det]m9ation (1 #cij ,opson91 #chg ,optical *>act]s ( colonies1 #bfg ,optimum reaction ( m$ium1 det]m9ation (1 #cje temp]ature1 det]m9ation (1 #bih ,organisms ( suppuration1 #dji ,orsat-,lunge gas app>atus1 #bib ,or?'s c>m9e1 #if ,ox=d /a9 = ,act9omyces1 #aab ,oy/]s1 analysis (1 #dfc ,pakes' c\nt+ disc1 #dbd filt] res]voir1 #de ,papi] *>d91 #aeh ,papp5heim's /a91 #aaa ,p>aboloid cond5s]1 #fj ,p>a*romophor\s bact]ia1 #add ,p>aff9 me?od = sections1 #aag sections1 m\nt+ (1 #aai to /a91 #aba ,p>atrophic bact]ia1 #aca ,p>ietti's b\illon1 #bjb me?od ( isolat+ coli-typhoid gr\p1 #dcg ,p>snip m$ium1 #bjj ,passages ( virus1 #cbj ,pa/eur-,*amb]l& filt]1 #db ,pa/eur's pipettes1 #aj solution1 #aia ,pa?og5esis1 inve/igation (1 #cae ,pa?og5ic bact]ia1 #aca /udy (1 #djh ,p$iococci1 morphology (1 #acb ,p5icillium1 #abh ,peptone rosolic acid wat]1 #ahf wat] 7,dunham71 prep>ation (1 #agg ,p]c5tage =mula1 #dif ,p]*loride ( m]cury1 #bg ,p]isporaceae1 #abg ,p]itri*\s bacilli1 #acf ,p]man5t prep>ations ( bact]ia1 #djg ( tissues1 #djd ,petri's di%es1 #f ,phagocytic index1 #cib ,ph5ol coeffici5t1 #dhi production1 te/ =1 #bhg ,photog5ic bact]ia1 #aca1 #add ,physiological filt]1 #aef ,picric acid solution1 #aba 7,sp5gl]'s71 #aab ,picroc>m9e1 #ig ,pigm5t production1 obs]vations on1 #bhh ,pipettes1 automatic1 #ac blood1 #aa capill>y1 #aj cases =1 #g graduat$1 #f capill>y1 #ac ,pa/eur's1 #aj s$im5tation1 #af /&>d graduat$1 #g teat1 #aj ?rottle1 #ac to clean infect$1 #bj new1 #ah to /]ilise1 #ca ,pirid9 me?od ( /a9+ spiro*aetes1 #abd ,pitfield's flagella /a91 #ajc ,plasmolysis1 #ace ,pla/]-(-,p>is discs1 #aib ,plate box1 #g cultures1 description (1 #bfa prep>ation (1 #bbf levell+ /&1 #bbh ,plates1 ,petri's1 #f to clean infect$1 #bj new1 #ah to /]ilise1 #ca ,plat9um ne$les1 #ga me?od ( m\nt+1 #ga ,pleomorphism1 #acc ,pol> g]m9ation1 #adj granules1 #acf ,polko]n]1 #acf ,poly*rome blood /a9s1 #ig ,pool$ s]um1 #cgi ,porcela9 filt]1 #db ,b]kefeld1 #db ,*amb]l&1 #db ,d\lton1 #db ,po/-mortem exam9ation ( exp]im5tal animals1 #cif ,potato gelat9e 7,eisn]71 #bjd 7,goadby71 #bad m$ium1 prep>ation (1 #agd ,pott$ meat1 analysis (1 #dfj ,p\r+ plates1 #bbg ,prep>ation ( exp]im5tal animals1 #cce ,pres]vatives 9 milk1 #ddb ,pressure temp]ature table1 #ejj ,prim>y col\rs1 action (1 #cji ,proteid free bro? 7,us*9sky71 #ahc ,proteolytic enzymes1 te/s =1 #bgg ,prototrophic bact]ia1 #aca ,psy*rophilic bact]ia1 #adc pa?og5ic effects1 #cae ,pus1 collection (1 #cgc ,pyrogallic acid solution1 #bic ,qualitative analysis ( air1 #dgj ( milk1 #ddf ( sewage1 #dfg ( soil1 #dgf ( uns\nd meat1 #dfb ( wat]1 #dbf ,quantitative analysis ( air1 #dfh ( milk1 #ddd ( sewage1 #dff ( soil1 #dgc ( uns\nd meat1 #dfj ,rabbit cages1 #cdc scabies1 treatm5t (1 #cch scales1 #cdj ,rais+ virul5ce ( organisms1 #cbj ,ramsd5's micromet]1 #ff ,range ( m$ium reaction1 measurem5t (1 #cje ( temp]ature1 measurem5t (1 #bih ,rat cages1 #cdb ,raw milk1 ,saul's te/ =1 #ddb ,reaction ( m$ium1 #cje optimum1 #cje range (1 #cje scale1 #aec ,r$uc$ pressure & temp]ature table1 #eja ,r$uc+ ag5ts1 production1 #chi te/s =1 #bhi ,r$uction ( nitrates1 #chi ,rei*]t's !rmo-regulator1 #bah ,relation ( bact]ia to environm5t1 #adb ,removal ( mat]ial f culture tubes1 #gd ,r5n9 enzymes1 te/s =1 #bgi ,reproduction ( bact]ia1 #acf ,resi/ance glass1 #f to le?al ag5ts1 #cjf ,re/+ /age ( bact]ia1 #acg ,re/rictions ^u exp]im5tal inoculations1 #ccd ,ribb]t's capsule /a91 #aja ,roll cultures1 #bbf ,rosolic acid peptone solution1 #ahf ,ros9dol reaction1 #bhf ,r\x's ana]obic culture me?od1 #bcg culture bottle1 #e ,sab\raud's m$ium1 #aic ,sac*>omyces1 morphology (1 #abi ,safran9e1 #id ,salicylic acid 9 milk1 te/ =1 #ddc ,saprog5ic bact]ia1 #aca ,s>c9ae1 morphology (1 #acb ,saul's te/1 #ddb ,scales1 decimal1 #cdj trip1 #afd ,scalpels1 to /]ilise1 #cb1 #cc ,s*allibaum's solution1 #aba ,s*eme = /udy ( bact]ia1 #bei ,s*izomycetes1 classification (1 #aca morphology (1 #aca ,scissors1 to /]ilise1 #cb ,seal+ museum j>s1 #djf ,se>+ iron1 #cig ,sections1 special /a9+ me?ods =1 #aba ,s$im5tation pipettes1 #af tubes1 #i ,select+ objectives1 #eg ,s5sitis+ r$ blood cells1 #cie ,s]ial cultivations1 #bea ,s]ological exam9ation ( blood1 #cgh ,s]um ag> 7,heiman71 #baj 7,kan?ack & ,/ev5s71 #baa 7,libman71 #bab plates1 #bej 7,w]!im]71 #baa b\illon1 #baj collection (1 #cgi dextrose wat] 7,hiss71 #ahh inspissator1 #afi sug> m$ia 7,hiss71 #ahh wat]1 prep>ation (1 #agj ,sewage1 analysis (1 qualitative1 #dfg quantitative1 #dff ,%ake cultivations1 #bbe description (1 #bga ,%ape ( colonies1 #bfb ,%av+ exp]im5tal animals1 #cdi ,%ellfi%1 analysis (1 #dfc ,silicate jelly 7,w9ogradsky71 #aih ,s+le /a9 = spores1 #ajf ,size ( colonies1 #bfb ,slant$ tube cultures1 #bbc ,slides1 to clean new1 #bb us$1 #bc ,sme> culture1 #bbd description (1 #bfh ,soap liquid1 #cdf ,soda solution1 /orage ( /ock1 #aed ,sodium bic>bonate 9 milk1 te/ =1 #ddc ,soil1 analysis (1 qualitative1 #dgf quantitative1 #dgc collection ( samples1 #dga ,solid m$ia1 #adg ,soluble tox9s1 #add ,soyka's milk rice1 #ahi ,spe>-head$ spatula1 #djb ,special m$ia1 #ahb ,specific s]um1 #cgi dilution (1 #chb ,sph]ical ab]ration1 #ee ,spirillum1 morphology (1 #acc ,spiro*aeta1 morphology (1 #acc ,spiro*aetes 9 tissues1 to /a91 #abd ,sple5 extract1 #adi ,sporangium1 #abg ,spore =mation1 ar?rog5\s1 #ach endog5\s1 #ach me?od (1 #ach1 #bgc g]m9ation1 me?od (1 #adj1 #bgd obs]vation (1 #adj1 #bgc ,spores1 *>act]s (1 #aci classification (1 #aci d\ble /a9 =1 #ajf to /a91 #ajf ,/ab culture1 #bbd description (1 #bfe ,/age micromet]1 #fb ( microscope1 #eb ,/a9+ me?ods1 #ij p>aff9 sections1 #aba reactions ( bact]ia1 #bgd ,/a9s intra-vitam1 #gg negative 7,burri71 #gg rack =1 #gb ,/&>d graduat$ pipettes1 #g soda solution1 #aed ,/&>disation ( m$ia1 #aed ,/&>dis+ b\illon1 #aee ,/aphylococci1 morphology1 #acb ,/aphylococcus 9 milk1 #def ,/eam /]ilis]1 ,arnold1 #ce ,ko*1 #ce to use1 #ce /ream+1 #ce ,/]igma1 #abg ,/]ilisation by *emicals1 #bg by dry heat1 #bh by filt]s1 #dj by moi/ heat1 #cb by /ream+ /eam1 #ce by sup]heat$ /eam1 #cf ( album9\s liquids1 #cb ( gases1 #dj ,/]ilis+ ag5ts1 #bf ,/i*cultur1 #bbd ,/ock dilutions1 #dig nutri5t m$ia1 #afc plate = isolation "w1 #bec ,/orage ( m$ia 9 bulk1 #aei ( tub$ m$ia1 #afa ,/ore boxes = m$ia1 #afa ,/reak culture1 #bbd description (1 #bfh ,/ream+ movem5t1 #hj /eam1 #ce ,/reptobacilli1 morphology1 #acc ,/reptococci 9 soil1 #dgg 9 wat]1 detection (1 #dcb morphology (1 #acb ,/reptococcus pyog5es longus 9 milk1 #dee ,/repto?rix1 morphology (1 #acc ,/ri*cultur1 #bbc ,/ructure1 int]nal1 ( colonies1 #bfe ,/udy ( pa?og5ic bact]ia1 #djh ,subcutane\s inoculation1 #cec ,subdural inoculation1 #cfa ,sub/age cond5s]1 #ed ,sug> ag>1 #ahe dextrose b\illon1 #ahd gelat9e1 #ahd m$ia1 prep>ation (1 #agg ,sulph9digotate ag>1 #aha b\illon 7,weyl71 #aha gelat9e 7,weyl71 #aha ,sulphurett$ hydrog5 9 cultures1 te/ =1 #bij ,sunlight1 action (1 #cji ,sup]heat$ /eam1 #cf ,sup]ior le?al coeffici5t1 #caj1 #cac ,suppuration1 organisms (1 #dji ,surface *>act]s ( colonies1 #bfd plates1 #bcj ,surgical motor1 electric1 #cfj ,sw>m spores1 #abg ,syr+e = subcutane\s inoculation ( solid mat]ial1 #ced hypod]mic1 #cdd ,tat9's op]at+ table1 #cea ,taxonomy1 #bfb ,teat-pipettes1 #aj ,temp]ature1 action (1 #bii optimum1 #bih pressure table1 #ejj range1 #bih tak+1 #cdj ,te/ objects = objectives1 #eg ,te/+ filt]s1 #dgh ,te/-tubes1 #c to clean infect$1 #ai new1 #ah to plug1 #bd to /]ilise1 #ca ,tetracocci1 morphology (1 #acb ,!rmal dea?-po9t1 #adc det]m9ation (1 #bih ( spores1 #cja1 #cjd ( vegetative =ms1 #bih1 #cjc ,!rmophilic bact]ia1 #adc ,!rmo-regulators1 ,he>son's capsule1 #bah ,rei*]t's1 #bah ,?ion9e blue1 #ib ,?io?rix1 morphology (1 #acc ,?re%'s wat] collect+ bottle1 #dah ,?rottle pipettes1 #ac ,t9n$ meat1 analysis (1 #dfj ,tissue m$ium 7,nogu*i71 #bad /a9s1 #ie ,tissues = section+1 fix+1 #aad freez+1 #aaf h>d5+1 #aad imb$d+1 #aah prep>ation (1 #aad wa%+1 #aae ,titration ( m$ia1 #aej ,torulae1 diff]5tiation f sac*>omyces1 #acj ,total acidity1 #bhj ,tox9s1 te/+ (1 #cah ,treph9es1 #cfj ,triple nosepiece1 #eh ,true motility1 #hj ,tube cultures1 prep>ation (1 #bbb l5g?1 #ej ,tub]cle bacillus 9 milk1 #dec to /a91 #aaj1 #abd ,tub]cul\s gu9ea-pig1 cadav] (1 #ded ,tub+ nutri5t m$ia1 #afj ,turnip m$ia1 #bjj ,unna-,papp5heim's /a9 = sections1 #abc ,uns\nd meat1 analysis (1 #dfj ,ur9e ag>1 #ahh gelat9e1 #ahg 7,hell]71 #ahh m$ia b\illon1 #ahg ,us*9sky's solution1 #ahc ,val5cy ( specific s]a1 #chf ,van ,erm5gem's flagella /a91 #ajd ,vegetative /age ( bact]ia1 #acf ,vesuv91 #id ,vibrio *ol]ae 9 milk1 #deb 9 wat]1 #dci morphology (1 #acc ,virul5ce1 att5uat+1 #cba ( organisms1 #cbj rais+1 #cbj ,vivisection lic5se1 #ccd ,voges hold]1 #cej ,volatile oils z dis9fectants1 #bg ,w>m /age1 #eh ,wa%+ r$ blood cells1 #chh tissues1 #aae ,wat]1 analysis (1 qualitative1 #dbf quantitative1 #daf /]ilis]1 #cc ,weigh+ animals1 #cdj ,wel*'s capsule /a91 #aja ,w]!im]'s s]um ag>1 #baa ,:eat b\illon 7,gasp]9i71 #aic ,:ey ag>1 #aie gelat9e1 #aie ,w9e mu/1 #aib ,w9ogradsky's solution ,i1 #aih ,,ii1 #aih ,wire crates = te/-tubes1 #ca ,wood a% ag>1 #bja ,work+ up plates1 #beb ,wort ag>1 #agf gelat9e1 #agf ,wright's ana]obic me?od1 #bci ,yea/ wat] 7,pa/eur71 #aia ,ziehl-,neels5's /a91 #aaj ,zoogloea1 #acd ,zymog5ic bact]ia1 #aca ,,saund]s' ,,books on ,pa?ology1 ,physiology ,hi/ology1 ,embryology ,bact]iology1 ,biology 99 99 99 99 99 ,w4 ,b4 ,,saund]s ,,company ,,we/ ,,wa%+ton ,,squ>e ,,philadelphia #i1 ,,h5rietta ,,/reet ,,cov5t ,,g>d51 ,,london 99 99 99 99 99 ,,lit]>y ,,sup]iority ,! excell5t judgm5t display$ 9 ! publications ( ! h\se at ! v beg9n+ ( its c>e]1 & ! success ( ! mod]n bus9ess me?ods employ$ by x1 at once attract$ ! att5tion ( lead+ m5 9 ! pr(ession1 & _m ( ! mo/ prom95t writ]s ( ,am]ica (f]$ _! books = publication4 ,?us1 !re 7 produc$ 9 rapid succession a numb] ( works t imm$iately plac$ ! h\se 9 ! front rank ( ,m$ical ,publi%]s4 ,"o ne$ only cite su* in/ances z ,muss] & ,kelly's ,treatm5t1 ,ke5's ,surg]y1 ,kelly & ,noble's ,gynecology & ,abdom9al ,surg]y1 ,cabot's ,diff]5tial ,diagnosis1 ,de ,lee's ,ob/etrics1 ,mum=d's ,surg]y1 ,cotton's ,dislocations & ,jo9t ,fractures1 ,cr&on & ,ehr5fri$'s ,surgical ,aft]-treatm5t1 ,sisson's ,vet]9>y ,anatomy1 ,&]s & ,bo/on's ,m$ical ,diagnosis1 ,gant's ,con/ipation & ,ob/ruction1 ,jordan's ,bact]iology1 & ,kemp on ,/oma*1 ,inte/9es1 & ,pancreas4 ,^! books h made = !mselves places among ! be/ works on _! respective subjects4 ,a ,complete ,catalogue ( our ,publications w be ,s5t ^u ,reque/ ,mallory's ,pa?ologic ,hi/ology ,pa?ologic ,hi/ology4 ,by ,,frank ,b4 ,,mallory1 ,m4 ,d41 ,associate ,pr(essor ( ,pa?ology1 ,h>v>d ,univ]sity ,m$ical ,s*ool4 ,octavo ( #fgg pages1 ) #dig figures conta9+ #fhc orig9al illu/rations1 #abd 9 colors4 ,clo?1 #e4ej net2 ,half ,morocco1 #g4jj net4 ,,repr9t$ ,,9 ,,?ree ,,mon?s ,dr4 ,mallory "h pres5ts pa?ology f ! morphologic po9t ( view4 ,he pres5ts 8 subject biologically1 fir/ by asc]ta9+ ! cellul> elem5ts out ( :i* ! v>i\s lesions are built up2 !n he traces ! developm5t ( ! lesions f ! simple/ to ! mo/ complex4 ,he s pres5ts pa?ology t y are able to trace backw>d f any giv5 end-result1 su* z scl]osis ( an organ 7cirrhosis ( ! liv]1 = example71 ?r\gh all ! v>i\s acute lesions t may t]m9ate 9 t p>ticul> end-result to ! primal cause ( ! lesion4 ,! illu/rations are mo/ beautiful4 ,dr4 ,w4 ,g4 ,mac,callum1 ,columbia ,univ]sity ,i h look$ ov] ! book & ?9k ! plan is admirably c>ri$ out & t ! book supplies a ne$ we h felt v mu*4 ,i %all be v glad to recomm5d x4 99 99 99 99 99 ,h[ell's ,physiology ,a ,text-,book ( ,physiology4 ,by ,,william ,h4 ,,h[ell1 ,,ph4,d41 ,m4 ,d41 ,pr(essor ( ,physiology 9 ! ,johns ,hopk9s ,univ]sity1 ,baltimore1 ,md4 ,octavo ( #ajbj pages1 #cjf illu/rations4 ,clo?1 #d4jj net4 ,,! ,,new 7#e?7 ,,edition ,dr4 ,h[ell has _h _m ye>s ( exp]i5ce z a tea*] ( physiology 9 sev]al ( ! lead+ m$ical s*ools1 & is !re=e exce$+ly well fitt$ to write a text-book on ?is subject4 ,ma9 emphasis has be5 laid ^u ^? facts & views :i* w be directly helpful 9 ! practical bran*es ( m$ic9e4 ,at ! same "t1 h[ev]1 suffici5t consid]ation has be5 giv5 to ! exp]im5tal side ( ! sci5ce4 ,! entire lit]ature ( physiology has be5 ?or\ghly dige/$ by ,dr4 ,h[ell1 & ! important views & conclusions introduc$ into 8 "w4 ,illu/rations h be5 mo/ freely us$4 ,! ,lancet1 ,london ,?is is "o ( ! be/ rec5t text-books on physiology1 & we w>mly comm5d x to ! att5tion ( /ud5ts :o desire to obta9 by read+ a g5]al1 all-r\nd1 yet concise survey ( ! scope1 facts1 !ories1 & speculations t make up its subject matt]4 ,mallory & ,wright's ,pa?ologic ,te*nique ,fif? ,edition ,pa?ologic ,te*nique4 ,a ,practical ,manual = ,work]s 9 ,pa?ologic ,hi/ology1 includ+ ,directions = ! ,p]=mance ( ,autopsies & = ,cl9ical ,diagnosis by ,laboratory ,me?ods4 ,by ,,frank ,b4 ,,mallory1 ,m4 ,d41 ,associate ,pr(essor ( ,pa?ology1 ,h>v>d ,univ]sity2 & ,,james ,h4 ,,wright1 ,m4 ,d41 ,director ( ! ,pa?ologic ,laboratory1 ,massa*usetts ,g5]al ,hospital4 ,octavo ( #ejj pages1 ) #aeb illu/rations4 ,clo?1 #c4jj net4 ,9 revis+ ! book = ! new edition ! au?ors h kept 9 view ! ne$s ( ! laboratory work]1 :e!r /ud5t1 practition]1 or pa?ologi/1 = a practical manual ( hi/ologic & bact]iologic me?ods 9 ! /udy ( pa?ologic mat]ial4 ,_m p>ts h be5 rewritt51 _m new me?ods h be5 add$1 & ! numb] ( illu/rations has be5 consid]ably increas$4 ,bo/on ,m$ical & ,surgical ,j\rnal ,?is manual1 s9ce its fir/ appe>ance1 has be5 recogniz$ z ! /&>d guide 9 pa?ological te*nique1 & has become well-nigh indisp5sable to ! laboratory work]4 99 99 99 99 99 ,eyre's ,bact]iologic ,te*nic ,bact]iologic ,te*nic4 ,a ,laboratory ,guide = ! ,m$ical1 ,d5tal1 & ,te*nical ,/ud5t4 ,by ,j4 ,w4 ,h4 ,,eyre1 ,m4 ,d41 ,f4 ,r4 ,s4 ,ed941 ,director ( ! ,bact]iologic ,dep>tm5t ( ,guy's ,hospital1 ,london4 ,octavo ( #ebj pages1 #bai illu/rations4 ,clo?1 #c4jj net4 ,,j ,,ready--,,new 7#b;d7 ,,edition1 ,,rewritt5 ,dr4 ,eyre has subject$ 8 "w to a mo/ se>*+ revision4 ,inde$1 s ?or\gh 0 8 revision t ! entire book1 enl>g$ by "s #aej pages & #ej illu/rations1 _h to be reset f cov] to cov]4 ,he has includ$ all ! late/ te*nic 9 e division ( ! subject4 ,8 ?or\ghness1 8 accuracy1 8 att5tion to detail make 8 "w an important "o4 ,he gives cle>ly ! te*nic = ! bact]iologic exam9ation ( wat]1 sewage1 air1 soil1 milk & its products1 meats1 etc4 ,& he gives y good te*nic--me?ods atte/$ by 8 own l>ge exp]i5ce4 ,to any "o int]e/$ 9 ?is l9e ( endeavor ! new edition ( ,dr4 ,eyre's "w is indisp5sable4 ,! illu/rations are z practical z ! text4 ,mc,farland's ,pa?ology ,a ,text-,book ( ,pa?ology4 ,by ,,joseph ,,mcf>l&1 ,m4 ,d41 ,pr(essor ( ,pa?ology & ,bact]iology 9 ! ,m$ico-,*irurgical ,college ( ,philadelphia4 ,octavo ( #hef pages1 ) #dcg illu/rations1 _m 9 colors4 ,clo?1 #e4jj net2 ,half ,morocco1 #f4ej net4 ,,! ,,new 7#b;d7 ,,edition ,y _c successfully treat disease unless y h a practical1 cl9ical k ( ! pa?ologic *anges produc$ by disease4 ,= ?is purpose ,dr4 ,mc,farland's "w is well fitt$4 ,x 0 writt5 ) j su* an end 9 view--to furni% a ready means ( acquir+ a ?or\gh tra9+ 9 ! subject1 a tra9+ su* z w\ld be ( daily help 9 y\r practice4 ,= ?is edition e page has be5 gone ov] mo/ c>efully1 correct+1 omitt+ ! obsolete1 & add+ ! new4 ,"s sections h be5 entirely rewritt54 ,y w f9d x a book well wor? consult+1 = x is ! "w ( an au?ority4 ,/4 ,paul ,m$ical ,j\rnal ,x is safe to say t !re are few :o are bett] qualifi$ to give a resume ( ! mod]n views on ?is subject ?an ,mc,farland4 ,! subject-matt] is ?or\ghly up to date4 ,bo/on ,m$ical & ,surgical ,j\rnal ,x conta9s a great mass ( well-classifi$ facts4 ,"o ( ! be/ sections is t on ! special pa?ology ( ! blood4 99 99 99 99 99 ,mc,farland's ,biology3 ,m$ical & ,g5]al ,biology3 ,m$ical & ,g5]al--,by ,,joseph ,,mcf>l&1 ,m4 ,d41 ,pr(essor ( ,pa?ology & ,bact]iology 9 ! ,m$ico-,*irurgical ,college ( ,phila4 #abmo1 #deg pages1 #afj illu/rations4 ,clo?1 #a4ge net4 ,,j ,,ready--,,new 7#b;d7 ,,edition ,?is "w is bo? a g5]al & m$ical biology4 ,! =m] because x discusses ! peculi> nature & reactions ( liv+ sub/ance g5]ally2 ! latt] because p>ticul> emphasis is laid on ^? subjects ( special int]e/ & value 9 ! /udy & practice ( m$ic9e4 ,! illu/rations w be f\nd ( great assi/ance4 ,fr$]ic ,p4 ,gorham1 ,a4 ,m41 ,br[n ,univ]sity4 ,i am greatly pleas$ ) x4 ,p]haps ! highe/ praise :i* ,i c give ! book is to say t x m ne>ly approa*es ! c\rse ,i am n[ giv+ 9 g5]al biology ?an any o!r "w4 ,mc,farland's ,pa?og5ic ,bact]ia & ,protozoa ,pa?og5ic ,bact]ia & ,protozoa4 ,by ,,joseph ,,mcf>l&1 ,m4 ,d41 ,pr(essor ( ,pa?ology & ,bact]iology 9 ! ,m$ico-,*irurgical ,college ( ,philadelphia4 ,octavo ( #hgh pages1 f9ely illu/rat$4 ,clo?1 #c4ej net4 ,,new 7#g?7 ,,edition1 ,,enl>g$ ,dr4 ,mc,farland has subject$ 8 book to a mo/ vigor\s revision1 br++ ?is edition "r d[n to ! m9ute4 ,important new additions h increas$ x 9 size "s #ahj pages4 ,by f> ! mo/ important addition is ! inclusion ( an entirely new section on ,pa?og5ic ,protozoa4 ,?is section consid]s e protozoan pa?og5ic to man2 & 9 t same clean-cut1 def9ite way t won = ,mc,farland's "w a place 9 ! v front ( m$ical bact]iologies4 ,! illu/rations are ! be/ ! _w af=ds1 & are beautifully execut$4 ,h4 ,b4 ,&]son1 ,m4 ,d41 ,pr(essor ( ,pa?ology & ,bact]iology1 ,tr9ity ,m$ical ,college1 ,toronto4 ,! book is a satisfactory "o1 & ,i %all take pleasure 9 recomm5d+ x to ! /ud5ts ( ,tr9ity ,college4 ,! ,lancet1 ,london ,x is excell5tly adapt$ = ! m$ical /ud5ts & practition]s = :om x is av[$ly writt54444 ,! descriptions giv5 are accurate & readable4 99 99 99 99 99 ,hill's ,hi/ology & ,organography ,a ,manual ( ,hi/ology & ,organography4 ,by ,,*>les ,,hill1 ,m4 ,d41 =m]ly ,assi/ant ,pr(essor ( ,hi/ology & ,embryology1 ,nor?we/]n ,univ]sity1 ,*icago4 #abmo ( #dfh pages1 #ccg illu/rations4 ,flexible lea!r1 #b4jj net4 ,,! ,,new 7#b;d7 ,,edition ,dr4 ,hill's "w is *>act]iz$ by a complet5ess ( discussion r>ely met 9 a book ( ?is size4 ,p>ticul> consid]ation is giv5 ! m\? & tee?4 ,p5nsylvania ,m$ical ,j\rnal ,x is arrang$ 9 su* a mann] z to be easy ( access & compreh5sion4 ,to any contemplat+ ! /udy ( hi/ology & organography we w\ld comm5d ?is "w4 99 99 99 99 99 ,,get ,,! ,,new ,,! ,,be/ ,,/&>d ,am]ican ,illu/rat$ ,diction>y ,new 7#g?7 ,edition--#ejjj ,sold 9 ,two ,mon?s ,! ,am]ican ,illu/rat$ ,m$ical ,diction>y4 ,a new & complete diction>y ( ! t]ms us$ 9 ,m$ic9e1 ,surg]y1 ,d5ti/ry1 ,ph>macy1 ,*emi/ry1 ,vet]9>y ,sci5ce1 ,nurs+1 & k9dr$ bran*es2 ) ov] #ajj new & elaborate tables & _m h&some illu/rations4 ,by ,w4 ,a4 ,,newman ,,dorl&1 ,m4,d41 ,editor ( ,! ,am]ican ,pocket ,m$ical ,diction>y4 ,l>ge octavo1 #aajg pages1 b\nd 9 full flexible lea!r4 ,price1 #d4ej net2 ) ?umb index1 #e4jj net4 ,,x ,,def9es ,,all ,,! ,,new ,,words--,,x ,,is ,,up ,,to ,,date ,! ,am]ican ,illu/rat$ ,m$ical ,diction>y def9es hundr$s ( ! newe/ t]ms n def9$ 9 any o!r diction>y--b> none4 ,^! new t]ms are live1 active words1 tak5 "r f mod]n m$ical lit]ature4 ,x gives ! capitalization & pronunciation ( all words4 ,x makes a feature ( ! d]ivation or etymology ( ! words4 ,9 "s diction>ies ! etymology occupies only a second>y place1 9 _m cases no d]ivation be+ giv5 at all4 ,9 ! ,am]ican ,illu/rat$ practically e ^w is giv5 its d]ivation4 ,e ^w has a sep>ate p>agraph1 ?us mak+ x easy to f9d a ^w quickly4 ,! tables ( art]ies1 muscles1 n]ves1 ve9s1 etc41 are ( ! greate/ help 9 assembl+ anatomic facts4 ,9 !m are classifi$ = quick /udy all ! necess>y in=mation ab\t ! v>i\s /ructures4 ,e ^w is giv5 its def9ition--a def9ition t def9es 9 ! fewe/ possible words4 ,9 "s diction>ies hundr$s ( words are n def9$ at all1 ref]r+ ! read] to "s o!r s\rce = ! in=mation he wants at once4 ,h[>d ,a1 ,kelly1 ,m4 ,d41 ,johns ,hopk9s ,univ]sity1 ,baltimore4 ,! ,am]ican ,illu/rat$ ,diction>y is admirable4 ,x is s well gott5 up & ( su* conv5i5t size4 ,no errors h be5 f\nd 9 my use ( x4 ,j4 ,coll9s ,w>r51 ,m4 ,d41 ,,ll4,d41 ,f4,r4,c4,s4 7,hon471 ,h>v>d ,m$ical ,s*ool ,i reg>d x z a valuable aid to my m$ical lit]>y "w4 ,x is v complete & ( conv5i5t size to h&le com=tably4 ,i use x 9 pref]5ce to any o!r4 99 99 99 99 99 ,/5gel's ,text-,book ( ,pa?ology ,fif? ,edition ,a ,text-,book ( ,pa?ology4 ,by ,,alfr$ ,,/5gel1 ,m4 ,d41 ,pr(essor ( ,m$ic9e 9 ! ,univ]sity ( ,p5nsylvania4 ,octavo volume ( #igi pages1 ) #djj text-illu/rations1 _m 9 colors1 & #g full-page color$ plates4 ,clo?1 #e4jj net2 ,%eep or ,half ,morocco1 #f4ej net4 ,,) #djj ,,text-,,cuts1 ,,_m ,,9 ,,colors1 ,,& #g ,,color$ ,,plates ,9 ?is "w ! practical application ( pa?ologic facts to cl9ical m$ic9e is consid]$ m fully ?an is cu/om>y 9 works on pa?ology4 ,:ile ! subject ( pa?ology is treat$ 9 ! broade/ way consi/5t ) ! size ( ! book1 an ef=t has be5 made to pres5t ! subject f ! po9t ( view ( ! cl9ician4 ,9 ! second "p ( ! "w ! pa?ology ( individual organs & tissues is treat$ sy/ematically & q fully "u subhead+s t cle>ly indicate ! subject-matt] to be f\nd on ea* page4 ,9 ?is edition ! section deal+ ) ,g5]al ,pa?ology has be5 mo/ ext5sively revis$1 sev]al ( ! important *apt]s hav+ be5 practically rewritt54 ,! ,lancet1 ,london ,?is volume is int5d$ to pres5t ! subject ( pa?ology 9 z practical a =m z possible1 & m especially f ! po9t ( view ( ! 'cl9ical pa?ologi/4' ,^! objects h be5 fai?fully c>ri$ out1 & a valuable text-book is ! result4 ,we c mo/ favorably recomm5d x to our read]s z a ?or\ghly practical "w on cl9ical pa?ology4 99 99 99 99 99 ,/iles' ,nutritional ,physiology ,nutritional ,physiology4 ,by ,,p]cy ,,gold?wait ,,/iles1 ,assi/ant ,pr(essor ( ,physiology at ,simmons ,college1 ,bo/on4 #abmo ( #bie pages1 illu/rat$4 ,clo?1 #a4be net4 ,,illu/rat$ ,?is new "w expresses ! mo/ advanc$ views on ?is important subject4 ,x discusses 9 a concise way ! processes ( dige/ion & metabolism4 ,! key-^w ( ! book ?r\gh\t is en]gy--its s\rce & its cons]vation4 ,x is rem>kable = ! f95ess ( its diction & = its cle> pres5tation ( ! subject1 reliev$ "h & !re by a qua9tly humor\s turn ( phrase t is altoge!r delightful4--,col9 ,c4 ,/ew>t1 ,ph4 ,d41 ,d>tm\? ,college4 99 99 99 99 99 ,jordan's ,g5]al ,bact]iology ,a ,text-,book ( ,g5]al ,bact]iology4 ,by ,,edw9 ,o4 ,,jordan1 ,,ph4,d41 ,pr(essor ( ,bact]iology 9 ! ,univ]sity ( ,*icago & 9 ,ru% ,m$ical ,college4 ,octavo ( #fbc pages1 illu/rat$4 ,clo?1 #c4jj net4 ,,new 7#c;d7 ,,edition ,pr(essor ,jordan's "w embraces ! entire field ( bact]iology1 ! non-pa?og5ic z well z ! pa?og5ic bact]ia be+ consid]$1 giv+ great] emphasis1 ( c\rse1 to ! latt]4 ,!re are ext5sive *apt]s on me?ods ( /udy+ bact]ia1 includ+ /a9+1 bio*emical te/s1 cultures1 etc42 on ! developm5t & composition ( bact]ia2 on enzymes & f]m5tation-products2 on ! bact]ial production ( pigm5t1 acid & alkali2 & on ptoma9es & tox9s4 ,especially complete is ! pres5tation ( ! s]um treatm5t ( gonorrhea1 diph!ria1 dys5t]y1 & tetanus4 ,! relation ( bov9e to human tub]culosis & ! ocul> tub]cul9 reaction receive ext5sive consid]ation4 ,?is "w w also appeal to academic & sci5tific /ud5ts4 ,x conta9s *apt]s on ! bact]iology ( plants1 milk & milk-products1 air1 agriculture1 wat]1 food pres]vatives1 ! processes ( lea!r tann+1 tobacco cur+1 & v9eg> mak+2 ! relation ( bact]iology to h\sehold adm9i/ration & to sanit>y eng9e]+1 etc4 ,pr(4 ,sev]ance ,burrage1 ,associate ,pr(essor ( ,sanit>y ,sci5ce1 ,purdue ,univ]sity4 ,i am mu* impress$ ) ! complet5ess & accuracy ( ! book4 ,x c]ta9ly cov]s ! gr\nd m completely ?an any o!r ,am]ican book t ,i h se54 99 99 99 99 99 ,bu*anan's ,vet]9>y ,bact]iology ,vet]9>y ,bact]iology4 ,by ,,rob]t ,e4 ,,bu*anan1 ,,ph4,d41 ,pr(essor ( ,bact]iology 9 ! ,i[a ,/ate ,college ( ,agriculture & ,me*anic ,arts4 ,octavo1 #eaf pages1 #bad illu/rations4 ,clo?1 #c4jj net4 ,,! ,,be/ ,,publi%$ ,pr(essor ,bu*anan discusses ?or\ghly all bact]ia caus+ diseases ( ! dome/ic animals4 ,he goes m9utely into ! consid]ation ( immunity1 opsonic index1 reproduction1 /]ilization1 antiseptics1 bio*emic te/s1 culture-m$ia1 isolation ( cultures1 ! manufacture ( ! v>i\s tox9s1 antitox9s1 tub]cul9s1 & vacc9es t h prov$ ( diagno/ic or !rapeutic value4 ,!n1 9 addition to bact]ia & protozoa prop]1 he consid]s molds1 mildews1 smuts1 ru/s1 toad/ools1 puff-balls1 & ! o!r fungi pa?og5ic = animals4 ,b4 ,f4 ,kaupp1 ,d4 ,v4 ,s41 ,/ate ,agricultural ,college1 ,=t ,coll9s4 ,x is ! be/ 9 pr9t on ! subject4 ,:at pleases me mo/ is t x conta9s all ! late results ( rese>*4 ,x fills a long felt want4 ,heisl]'s ,embryology ,a ,text-,book ( ,embryology4 ,by ,,john ,c4 ,,heisl]1 ,m4,d41 ,pr(essor ( ,anatomy 9 ! ,m$ico-,*irurgical ,college1 ,philadelphia4 ,octavo volume ( #dce pages1 ) #bab illu/rations1 #cb ( !m 9 colors4 ,clo?1 #c4jj net4 ,,?ird ,,edition--,,) #bab ,,illu/rations1 #cb ,,9 ,,colors ,?is edition repres5ts all ! advances rec5tly made 9 ! sci5ce ( embryology4 ,_m portions h be5 entirely rewritt51 & a great deal ( new & important matt] add$4 ,a numb] ( new illu/rations h also be5 introduc$ & ^! w prove v valuable4 ,heisl]'s ,embryology has become a /&>d "w4 ,g4 ,c>l ,hub]1 ,m4,d41 ,pr(essor ( ,embryology at ! ,wi/> ,in/itute1 ,univ]sity ( ,p5nsylania4 ,i f9d ?is edition ( ',a ,text-,book ( ,embryology1' by ,dr4 ,heisl]1 an improvem5t on ! =m] "o4 ,! figures add$ increase greatly ! value ( ! "w4 ,i am aga9 recomm5d+ x to our /ud5ts4 99 99 99 99 99 ,boehm1 ,david(f1 & ,hub]'s ,hi/ology ,a ,text-,book ( ,human ,hi/ology4 ,includ+ ,microscopic ,te*nic4 ,by ,,dr4 ,a4 ,a4 ,,boehm & ,,dr4 ,m4 ,,von ,,david(f1 ( ,muni*1 & ,g4 ,,c>l ,,hub]1 ,m4,d41 ,pr(essor ( ,embryology at ! ,wi/> ,in/itute1 ,univ]sity ( ,p5nsylvania4 ,h&some octavo ( #ebh pages1 ) #cfa beautiful orig9al illu/rations4 ,flexible clo?1 #c4ej net4 ,,second ,,edition1 ,,enl>g$ ,! "w ( ,drs4 ,boehm & ,david(f is well kn[n 9 ! ,g]man edition1 & has be5 consid]$ "o ( ! mo/ practically useful books on ! subject ( ,human ,hi/ology4 ,?is second edition has be5 9 great "p rewritt5 & v mu* enl>g$ by ,dr4 ,hub]1 :o has also add$ ov] "o hundr$ orig9al illu/rations4 ,dr4 ,hub]'s ext5sive additions h r5d]$ ! "w ! mo/ complete /ud5ts' text-book on ,hi/ology 9 exi/5ce4 ,bo/on ,m$ical & ,surgical ,j\rnal ,is unque/ionably a text-book ( ! fir/ rank1 hav+ be5 c>efully writt5 by ?or\gh ma/]s ( ! subject1 & 9 c]ta9 directions x is mu* sup]ior to any o!r hi/ological manual4 99 99 99 99 99 ,wells' ,*emical ,pa?ology ,*emical ,pa?ology4--,be+ a ,discussion ( ,g5]al ,pa?ology f ! ,/&po9t ( ! ,*emical ,processes ,involv$4 ,by ,h4 ,,gideon ,,wells1 ,,ph4,d41 ,m4,d41 ,assi/ant ,pr(essor ( ,pa?ology 9 ! ,univ]sity ( ,*icago4 ,octavo ( #faf pages4 ,clo?1 #c4be net4 ,,j ,,ready--,,new 7#b;d7 ,,edition ,dr4 ,wells' "w is writt5 = ! physician1 = ^? engag$ 9 rese>* 9 pa?ology & physiologic *emi/ry1 & = ! m$ical /ud5t4 ,9 ! introductory *apt] are discuss$ ! *emi/ry & physics ( ! animal cell1 giv+ ! ess5tial facts ( ionization1 diffusion1 osmotic pressure1 etc41 & ! relation ( ^! facts to cellul> activities4 ,special *apt]s are devot$ to ,diabetes & to ,uric-acid ,metabolism & ,g\t4 ,wm4 ,h4 ,wel*1 ,m4,d4 ,pr(essor ( ,pa?ology1 ,johns ,hopk9s ,univ]sity4 ,! "w fills a real ne$ 9 ! ,engli% lit]ature ( a v important subject1 & ,i %all be glad to recomm5d x to my /ud5ts4 99 99 99 99 99 ,lusk's ,elem5ts ( ,nutrition ,elem5ts ( ! ,sci5ce ( ,nutrition4 ,by ,,graham ,,lusk1 ,,ph4,d41 ,pr(essor ( ,physiology at ,cornell ,m$ical ,s*ool4 ,octavo volume ( #cjb pages4 ,clo?1 #c4jj net4 ,,! ,,new 7#b;d7 ,,edition--,,translat$ ,,into ,,g]man ,pr(4 ,lusk pres5ts ! sci5tific f\ndations ^u :i* re/s our k ( nutrition & metabolism1 bo? 9 heal? & 9 disease4 ,!re are special *apt]s on ! metabolism ( diabetes & fev]1 & on pur9 metabolism4 ,! "w w also prove valuable to /ud5ts ( animal dietetics at agricultural /ations4 ,lewellys ,f4 ,b>k]1 ,m4 ,d4 ,pr(essor ( ! ,pr9ciples & ,practice ( ,m$ic9e1 ,johns ,hopk9s ,univ]sity4 ,i %all recomm5d x highly to my /ud5ts4 ,x is a com=t to h su* a discussion ( ! subject 9 ,engli%4 ,daugh]ty's ,economic ,zoology ,economic ,zoology4 ,by ,l4 ,s4 ,,daugh]ty1 ,m4 ,s41 ,,ph4 ,d41 ,pr(essor ( ,zoology1 ,/ate ,normal ,s*ool1 ,kirksville1 ,mo41 & ,m4 ,c4 ,,daugh]ty1 au?or ) ,jackson ( ,agriculture ,?r\gh ! ,laboratory & ,s*ool ,g>d54 ,"p ,i3 ,field & ,laboratory ,guide4 #abmo ( #bcg pages1 int]leav$4 ,clo?1 #a4be net4 ,"p ,,ii3 ,pr9ciples4 #abmo ( #djf pages1 illu/rat$4 ,clo?1 #b4jj net4 ,,illu/rat$ ,!re is no o!r book j l ?is4 ,n only does x give ! sali5t facts ( /ructural zoology & ! developm5t ( ! v>i\s bran*es ( animals1 b also ! natural hi/ory--! life & habits--?us %[+ ! int]relations ( /ructure1 habit1 & environm5t4 ,9 a ^w1 x gives ! pr9ciples ( zoology & _! actual application4 ,! economic phase is emphasiz$4 ,"p ,i--! ,field & ,laboratory ,guide--is design$ = practical in/ruction 9 ! field & laboratory4 ,to enhance its value = ?is purpose blank pages are ins]t$ = notes4 99 99 99 99 99 ,drew's ,inv]tebrate ,zoology ,a ,laboratory ,manual ( ,inv]tebrate ,zoology4 ,by ,,gilman ,a4 ,,drew1 ,,ph4 ,d41 ,assi/ant ,director at ,m>9e ,biological ,laboratory1 ,woods ,hole1 ,mass4 ,) ! aid ( ,=m] & ,pres5t ,memb]s ( ! ,zoological ,/aff ( ,in/ructors4 #abmo ( #bac pages4 ,clo?1 #a4be net4 ,,j ,,ready--,,new 7#b;d7 ,,edition ,! subject is pres5t$ 9 a logical way1 & ! type me?od ( /udy has be5 foll[$1 z ?is me?od has be5 ! prevail+ "o = _m ye>s4 ,pr(4 ,allison ,a4 ,smy?1 ,jr41 ,virg9ia ,polyte*nic ,in/itute ,i ?9k x is ! be/ laboratory manual ( zoology ,i h yet se54 ,! l>ge numb] ( =ms dealt ) makes ! "w applicable to almo/ any locality4 99 99 99 99 99 ,norris' ,c>diac ,pa?ology ,/udies 9 ,c>diac ,pa?ology4 ,by ,,george ,w4 ,,norris1 ,m4,d41 ,associate 9 ,m$ic9e at ! ,univ]sity ( ,p5nsylvania4 ,l>ge octavo ( #bce pages1 ) #he sup]b illu/rations4 ,clo?1 #e4jj net4 ,,sup]b ,,illu/rations ,! wide int]e/ be+ manife/$ 9 he>t lesions makes ?is book p>ticul>ly opportune4 ,! illu/rations are sup]b & are fai?ful reproductions ( ! specim5s photograph$4 ,ea* illu/ration is accompani$ by a detail$ description2 besides1 !re is ample lett] press supplem5t+ ! pictures4 ,consid]able matt] ( a diagno/ic & !rapeutic nature has be5 int]wov54 ,bo/on ,m$ical & ,surgical ,j\rnal ,! illu/rations are arrang$ 9 su* a way z to illu/rate all ! common & _m ( ! r>e c>diac lesions1 & ! accompany+ descriptive text con/itutes a fairly cont9u\s didactic treatise4 99 99 99 99 99 ,mc,connell's ,pa?ology ,a ,manual ( ,pa?ology4 ,by ,,gu?rie ,,mcconnell1 ,m4,d41 ,pr(essor ( ,bact]iology & ,pa?ology at ,temple ,univ]sity1 ,philadelphia4 #abmo ( #ebc pages1 ) #agj illu/rations4 ,flexible lea!r1 #b4ej net4 ,,new 7#b;d7 ,,edition ,dr4 ,mc,connell has discuss$ 8 subject ) a cle>ness & precision ( /yle t make ! "w ( great assi/ance to bo? /ud5t & practition]4 ,! illu/rations h be5 introduc$ = _! practical value4 ,new ,york ,/ate ,j\rnal ( ,m$ic9e ,! book treats ! subject ( pa?ology ) a ?or\ghness lack+ 9 _m works ( great] pret5sion4 ,! illu/rations--_m ( !m orig9al--are pr(use & ( exceptional excell5ce4 99 99 99 99 99 ,hekto5 & ,riesman's ,pa?ology ,,am]ican ,,text-,,book ,,( ,,pa?ology4 ,edit$ by ,,ludvig ,,hekto51 ,m4,d41 ,pr(essor ( ,pa?ology1 ,ru% ,m$ical ,college1 ,*icago2 & ,,david ,,riesman1 ,m4,d41 ,pr(essor ( ,cl9ical ,m$ic9e1 ,philadelphia ,polycl9ic4 ,octavo ( #abde ,pages1 #ddc illu/rations1 #ff 9 colors4 ,clo?1 #g4ej net2 ,half ,morocco1 #i4jj net4 ,du]ck & ,hekto5's ,special ,pa?ologic ,hi/ology ,atlas & ,epitome ( ,special ,pa?ologic ,hi/ology4 ,by ,,dr4 ,h4 ,,du]ck1 ( ,muni*4 ,edit$1 ) additions1 by ,,ludvig ,,hekto51 ,m4 ,d41 ,pr(essor ( ,pa?ology1 ,ru% ,m$ical ,college1 ,*icago4 ,9 two p>ts4 ,"p ,i4--,circulatory1 ,respiratory1 & ,ga/ro-inte/9al ,tracts4 #abj color$ figures on #fb plates1 & #aeh pages ( text4 ,"p ,,ii4--,liv]1 ,ur9>y & ,sexual ,organs1 ,n]v\s ,sy/em1 ,sk91 ,muscles1 & ,bones4 #abc color$ figures on #fj plates1 & #aib pages ( text4 ,p] "p3 ,clo?1 #c4jj net4 ,9 ,saund]s' ,h&-,atlas ,s]ies4 ,! great value ( ^! plates is t !y repres5t 9 ! exact colors ! effect ( ! /a9s1 :i* is ( su* great importance = ! diff]5tiation ( tissue4 ,! text portion ( ! book is admirable1 &1 :ile brief1 x is entirely satisfactory 9 t ! lead+ facts are /at$1 & s /at$ t ! read] feels he has grasp$ ! subject ext5sively4 ,william ,h4 ,wel*1 ,m4,d41 ,pr(essor ( ,pa?ology1 ,johns ,hopk9s ,univ]sity1 ,baltimore4 ,i consid] ,du]ck's ',atlas ( ,special ,pa?ologic ,hi/ology1' edit$ by ,hekto51 a v useful book = /ud5ts & o!rs4 ,! plates are admirable4 99 99 99 99 99 ,sobotta & ,hub]'s ,human ,hi/ology ,atlas & ,epitome ( ,human ,hi/ology4 ,by ,,privatdoc5t ,,dr4 ,j4 ,,sobotta1 ( ,wu]zburg4 ,edit$1 ) additions1 by ,g4 ,,c>l ,,hub]1 ,m4 ,d41 ,pr(essor ( ,hi/ology & ,embryology 9 ! ,univ]sity ( ,mi*igan1 ,ann ,arbor4 ,) #bad color$ figures on #hj plates1 #fh text-illu/rations1 & #bdh pages ( text4 ,clo?1 #d4ej net4 ,9 ,saund]s' ,h&-,atlas ,s]ies4 ,,includ+ ,,microscopic ,,anatomy ,! "w comb9es an abundance ( well-*os5 & mo/ accurate illu/rations1 ) a concise text1 & 9 su* a mann] z to make x bo? atlas & text-book4 ,! great majority ( ! illu/rations 7 made f sections prep>$ f human tissues1 & always f fre% & 9 e respect normal specim5s4 ,! color$ li?ographic plates h be5 produc$ ) ! aid ( ov] ?irty colors4 ,bo/on ,m$ical & ,surgical ,j\rnal ,9 color & proportion !y are *>act]iz$ by gratify+ accuracy & li?ographic beauty4 ,bosanquet on ,spiro*aetes ,spiro*aetes3 ,a ,review ( ,rec5t ,"w1 ) ,"s ,orig9al ,obs]vations4 ,by ,w4 ,,cecil ,,bosanquet1 ,m4,d41 ,fell[ ( ! ,royal ,college ( ,physicians1 ,london4 ,octavo ( #aeb pages1 illu/rat$4 #b4ej net4 ,,illu/rat$ ,?is is a complete & au?oritative monograph on ! spiro*aetes1 giv+ morphology1 pa?og5esis1 classification1 /a9+1 etc4 ,pseudospiro*aetes are also consid]$1 & ! entire text well illu/rat$4 ,! high /&+ ( ,dr4 ,bosanquet 9 ?is field ( /udy makes ?is new "w p>ticul>ly valuable4 99 99 99 99 99 ,levy & ,klemp]]'s ,cl9ical ,bact]iology ,! ,elem5ts ( ,cl9ical ,bact]iology4 ,by ,,drs4 ,,ern/ ,,levy & ,,felix ,,klemp]]1 ( ! ,univ]sity ( ,/rasburg4 ,translat$ & edit$ by ,,augu/us ,a4 ,,e%n]1 ,m4 ,d41 ,pr(essor ( ,cl9ical ,m$ic9e1 ,philadelphia ,polycl9ic4 ,octavo volume ( #ddj pages1 fully illu/rat$4 ,clo?1 #b4ej net4 ,s4 ,solis-,coh51 ,m4,d41 ,pr(essor ( ,cl9ical ,m$ic9e1 ,jeff]son ,m$ical ,college1 ,philadelphia4 ,i consid] x an excell5t book4 ,i h recomm5d$ x 9 speak+ to my /ud5ts4 99 99 99 99 99 ,lehmann1 ,neumann1 & ,weav]'s ,bact]iology ,atlas & ,epitome ( ,bact]iology3 ,,includ+ ,a ,,text-,,book ,,( ,,special ,,bact]iologic ,,diagnosis4 ,by ,,pr(4 ,,dr4 ,k4 ,b4 ,,lehmann & ,,dr4 ,r4 ,o4 ,,neumann1 ( ,wu]zburg4 ,f ! ,second ,revis$ & ,enl>g$ ,g]man ,edition4 ,edit$1 ) additions1 by ,g4 ,h4 ,,weav]1 ,m4 ,d41 ,assi/ant ,pr(essor ( ,pa?ology & ,bact]iology1 ,ru% ,m$ical ,college1 ,*icago4 ,9 two p>ts4 ,"p ,i4--#fcb color$ figures on #fi li?ographic plates4 ,"p ,,ii4--#eaa pages ( text1 illu/rat$4 ,p] "p3 ,clo?1 #b4ej net4 ,9 ,saund]s' ,h&-,atlas ,s]ies4 99 99 99 99 99 ,du]ck & ,hekto5's ,g5]al ,pa?ologic ,hi/ology ,,atlas ,,& ,,epitome ,,( ,,g5]al ,,pa?ologic ,,hi/ology4 ,by ,,pr4 ,,dr4 ,h4 ,,du]ck1 ( ,muni*4 ,edit$1 ) additions1 by ,,ludvig ,,hekto51 ,m4 ,d41 ,pr(essor ( ,pa?ology 9 ,ru% ,m$ical ,college1 ,*icago4 #agb color$ figures on #gg li?ographic plates1 #cf text-cuts1 _m 9 colors1 & #cec pages4 ,clo?1 #e4jj net4 ,9 ,saund]s' ,h& ,atlas ,s]ies4 ,am]ican ,text-,book ( ,physiology ,second ,edition ,,am]ican ,,text-,,book ,,( ,,physiology4 ,9 two volumes4 ,edit$ by ,,william ,h4 ,,h[ell1 ,,ph4 ,d41 ,m4,d41 ,pr(essor ( ,physiology 9 ! ,johns ,hopk9s ,univ]sity1 ,baltimore1 ,md4 ,two royal octavos ( ab\t #fjj pages ea*1 illu/rat$4 ,p] volume3 ,clo?1 #c4jj net2 ,half ,morocco1 #d4be net4 ,! "w w /& z a "w ( ref]5ce on physiology4 ,to him :o desires to "k ! /atus ( mod]n physiology1 :o expects to obta9 sugge/ions z to fur!r physiologic inquiry1 we "k ( none 9 ,engli% :i* s em95tly meets su* a dem&4--,! ,m$ical ,news4 ,w>r5's ,pa?ology & ,!rapeutics ,second ,edition ,,surgical ,,pa?ology ,,& ,,!rapeutics4 ,by ,,john ,,coll9s ,,w>r51 ,m4 ,d41 ,,ll4 ,d41 ,f4 ,r4 ,c4 ,s4 7,hon471 ,pr(essor ( ,surg]y1 ,h>v>d ,m$ical ,s*ool4 ,octavo1 #hgc pages1 #acf relief & li?ographic illu/rations1 #cc 9 colors4 ,) an ,app5dix on ,sci5tific ,aids to ,surgical ,diagnosis & a s]ies ( articles on ,regional ,bact]iology4 ,clo?1 #e4jj net2 ,half ,morocco1 #f4ej net4 ,gorham's ,bact]iology ,a ,,laboratory ,,c\rse ,,9 ,,bact]iology4 ,= ! ,use ( ,m$ical1 ,agricultural1 & ,indu/rial ,/ud5ts4 ,by ,,fr$]ic ,p4 ,,gorham1 ,a4 ,m41 ,associate ,pr(essor ( ,biology 9 ,br[n ,univ]sity1 ,provid5ce1 ,r4 ,i41 etc4 #abmo ( #aib pages1 ) #ig illu/rations4 ,clo?1 #a4be net4 ,"o ( ! be/ /ud5ts' laboratory guides to ! /udy ( bact]iology on ! m>ket4444 ,! te*nic is ?or\ghly mod]n & amply suffici5t = all practical purposes4--,am]ican ,j\rnal ( ! ,m$ical ,sci5ces4 ,raymond's ,physiology ,new 7#c;d7 ,edition ,,human ,,physiology4 ,by ,,joseph ,h4 ,,raymond1 ,a4 ,m41 ,m4 ,d41 ,pr(essor ( ,physiology & ,hygi5e1 ,long ,isl& ,college ,hospital1 ,new ,york4 ,octavo ( #fhe pages1 ) #ddd illu/rations4 ,clo?1 #c4ej net4 ,! book is well gott5 up & well pr9t$1 & may be reg>d$ z a tru/wor?y guide = ! /ud5t & a useful "w ( ref]5ce = ! g5]al practition]4 ,! illu/rations are num]\s & are well execut$4--,! ,lancet1 ,london4 ,ball's ,bact]iology ,sev5? ,edition1 ,revis$ ,,ess5tials ,,( ,,bact]iology3 be+ a concise & sy/ematic introduction to ! ,/udy ( ,micro-organisms4 ,by ,m4 ,v4 ,,ball1 ,m4 ,d41 ,late ,bact]iologi/ to ,/4 ,agnes' ,hospital1 ,philadelphia4 #abmo ( #bhi pages1 ) #ace illu/rations1 "s 9 colors4 ,clo?1 #a4jj net4 ,9 ,saund]s' ,"q-,comp5d ,s]ies4 ,! te*nic ) reg>d to m$ia1 /a9+1 m\nt+1 & ! l is cull$ f ! late/ au?oritative works4--,! ,m$ical ,times1 ,new ,york4 ,budgett's ,physiology ,new 7#c;d7 ,edition ,,ess5tials ,,( ,,physiology4 ,prep>$ especially = ,/ud5ts ( ,m$ic9e1 & arrang$ ) que/ions foll[+ ea* *apt]4 ,by ,,sidney ,p4 ,,budgett1 ,m4 ,d41 =m]ly ,pr(essor ( ,physiology1 ,wa%+ton ,univ]sity1 ,/4 ,l\is4 ,revis$ by ,,havan ,,em]son1 ,m4 ,d41 ,demon/rator ( ,physiology1 ,columbia ,univ]sity4 #abmo volume ( #bej pages1 illu/rat$4 ,clo?1 #a4jj net4 ,saund]s' ,"q-,comp5d ,s]ies4 ,he has an excell5t conception ( 8 subject4444 ,x is "o ( ! mo/ satisfactory books ( ?is class--,univ]sity ( ,p5nsylvania ,m$ical ,bullet94 ,l]oy's ,hi/ology ,new 7#d?7 ,edition ,,ess5tials ,,( ,,hi/ology4 ,by ,,l\is ,,l]oy1 ,m4 ,d41 ,pr(essor ( ,hi/ology & ,pa?ology1 ,v&]bilt ,univ]sity1 ,na%ville1 ,t5nessee4 #abmo1 #bfc pages1 ) #ib orig9al illu/rations4 ,clo?1 #a4jj net4 ,9 ,saund]s' ,"q-,comp5d ,s]ies4 ,! "w 9 its pres5t =m /&s z a model ( :at a /ud5t's aid %\ld be2 & we unhesitat+ly say t ! practition] z well w\ld f9d a glance ?r\gh ! book ( la/+ b5efit4--,! ,m$ical ,_w1 ,philadelphia4 ,b>ton & ,wells' ,m$ical ,!saurus ,a ,,!saurus ,,( ,,m$ical ,,words ,,& ,,phrases4 ,by ,,wilfr$ ,m4 ,,b>ton1 ,m4 ,d41 ,assi/ant ,pr(essor ( ,mat]ia ,m$ica & ,!rapeutics1 & ,,walt] ,a4 ,,wells1 ,m4 ,d41 ,demon/rator ( ,l>yngology1 ,georget[n ,univ]sity1 ,wa%+ton1 ,d4,c4 #abmo1 #ecd pages4 ,flexible lea!r1 #b4ej net2 ?umb index$1 #c4jj net4 ,am]ican ,pocket ,diction>y ,new 7#h?7 ,edition ,,dorl&',s ,,pocket ,,m$ical ,,diction>y4 ,edit$ by ,w4 ,a4 ,,newman ,,dorl&1 ,m4 ,d41 ,editor ,am]ican ,illu/rat$ ,m$ical ,diction>y4 ,conta9+ ! pronunciation & def9ition ( ! pr9cipal words us$ 9 m$ic9e & k9dr$ sci5ces1 ) #fd ext5sive tables4 #fgg pages4 ,flexible lea!r1 ) gold edges1 #a4jj net2 ) pat5t ?umb index1 #a4be net4 ,i c recomm5d x to our /ud5ts )\t res]ve4--,j4 ,h4 ,,holl&1 ,m4,d41 ( ! ,jeff]son ,m$ical ,college1 ,philadelphia4