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Further studies on the bacterial flora of the `Kingston cheese’ 1923

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of i& e iS^^fw . Qj]**fr  '- h OS? 0 ttilfp llmttpraitg of Irittafj (Eolumbia D E P A R T M E N T O F D A I R Y I N G VANCOUVER, CANAD A May 5 t h , 1 9 2 4 , John R i d i n g t o n , E s q . , Librar ian , The U n i v e r s i t y of B r i t i s h Columbia. .Dear S i r : - I submit herewith the report of "Further Studies on the Bacterial -lora of the Kingston Cheese" by Mr. Clifford carton Kelly, B.S.A. This work,done in the Jepartment of Dairying,is submitted by Mr. Kelly as a Thesis in partial fulfilment of the requirements for the Jegree of Master of Science in Igrrculture in the University of British Columbia. I have accepted the Thesis as being satisfactory to the Jepartment of Jairying; and it is ae- sired to place on record, that, in our view, the lvork of Mr. Zelly constitutes an original, and a distinct contribution to knowledge. Xours faithfully, Professor and Head of the Jepartment* WS/KMD. FUHEHKH STUJIS3 Oil THE BACTERIAL FLORA OF THE 'KIIIGSTOi? CHEESE.* By C l i f f o r d Darton K e l l y , 3 . 3 . i . A Thes i s submi t ted f o r t h e J e g r e e o: Master of doienoe in A g r i c u l t u r e aone in t h e depar tment of d a i r y i n g . THE UNIVERSITY OF BRITISH COLUMBIA Apr i l 1924. 4 * * 8 0 * 1 * 8 Y T A B L E O F C O N T E N T S . i E a Introduction 1 Media Empl oyed 2 Examination and Technique 3 Comparative Values of Certain Media 5 quantitative Bacteriological Analyses 6 qualitative Bacteriological Analyses 9 Classification of Organisms...... Group I, -Streptococcus lactis Lister,ity->es 12 Group II,Lactobacillus(3eijerinckj tyoes.. 15 Group III,Gram negative,lactose ferment- ing rods 1 8 Group 17, Spore Bearing Rods 2 1 Group 7 , Coccus forms o t h e r than S t r ep tococcus l a c t i s ( L i s t e r ; 22 O b s e r v a t i o n s . 26 Summary 2 8 References 33 P l a t e s \ FURTHER 3TUJIE3 ON THE BACTERIA! FLORA OS THE »ZING3TQN GHEE3E.' C. D. KELLY, B. 3. A. DJTROQUCTICH. The p r o c e s s of manufacture of t h e 'K ings ton Cheese* was worked out in England by Alec . Todd and W i l f r i d 3 a d l e r (19 ) t o meet t h e demand f o r a cheese of t h e ha rd p r e s s e d v a r i e t y , a cheese no t too l a r g e f o r t h e ave rage f ami ly and one which c o u l i be used with l i t t l e or nc w a s t e . ^Jhile t h i s cheese i s made on t h e ha rd p r e s s e d p r i n c i p l e , t h e system and p rocedure s employed a r e such a s t o g i v e a cheese t h a t w i l l r i p e n i n t en d a y s . The r i p e cheese i s u s u a l l y one pound in w e i g h t , has a f l a v o u r of i t s own and a s o f t g r a n u l a r t e x t u r e , r i c h ana b u t t e r y . For t he p a s t t h r e e y e a r s t h i s cheese h i s been manufactured in t he l a b o r a t o r i e s of t h e U n i v e r s i t y of B r i t i s h Columbia. In a Thes i s p r e s e n t e d by me in Apr i l 1923 (11) d e t a i l e d r e f e r - ences were made t o t h e system on which t h e cheese i s nanu- r a c t u r e d , and complete r e c o r d s of t h e cheese p.ade d u r i n g a - 2 - l a r g e p a r t of the y e a r were p r e s e n t e d . The Thes i s i n c l u d e d , moreover , t h e r e s u l t s of b a c t e r i o l o g i c a l examinat ions of c e r t a i n of t h e cheese r e f e r r e d t o immedia te ly above . As f a r as t h e b a c t e r i o l o g i c a l examina t ions were conce rned , the r e - s u l t s were very i n c o m p l e t e , y e t the t e c h n i q u e worked ouJ a t t h a t t i m e , and t h e d a t a o b t a i n e d , have se rved as an i n v a l u a b l e p r e l i m i n a r y t o c a r r y i n g on the work r e p o r t e d h e r e i n . The work was so planned t h a t q u a n t i t a t i v e b a c t e r i o l o g i c a l a n a l y s e s were t o be made of a cheese when one day o l d , and of a cheese of the sane d a y ' s make when ready f o r m a r k e t , a l s o the organisms o c c u r r i n g wi th the g r e a t - e s t f requency were t o be i s o l a t e d from the p l a t e s of each cheese examined. I t has been a n t i c i p a t e d t h a t the d a t a t hus secured might l ead t o some informat ion b e i n g ob t a ined on t h e r i s e n i n g p r o c e s s e s of t h i s c h e e s e . BACT2HICI0GICAL SALII!: a~ICTS OF 7R 3 '2I1IG3T 71 CII32S3 MEDIA 2KPLGY3D. Bee f -pep tono -aga r , J t a n d a r a Methods ( l 6> Glucose-agar 0.5 per cent glucose added to beef- peptone-agar. L a c t o s e - a g a r 0 .5 p e r cen t l a c t o s e added to beef- p e p t o n e - a g a r . Milk- a g a r , Ayers and Mudge(l) - 3 - MacConkeyTs n e u t r a l - r e d - b i l e - s a l t - l a c t o s e - a g a r (15) for spec i f i c reac t ions of the organisms which ferment l a c t o s e to acid and gas . MacConkeyTs n e u t r a l - r e d - b i l e - s a l t - l a c t o s e - b r o t h (15J for de tec t ing the presence of the organisms which ferment l ac to se to acid and gas . Neut r ien t -agar to which spec i f ic carbohydrates were added as des i red , using brom-crssol- purple as i n d i c a t o r . ( 4 ) ( l 8 ) . Heut r ien t -bro th to which spec i f ic carbohydrates v-ere added as des i red , using brom-thymol-blue as i n d i c a t o r . ( l 6 ) . Peptone-medium for Methyl ftel and Toges-Proskuuer de- te rmina t ions . Standard Methods (l(>  ) . Peptone-medium for Indol Production. Standard. Methods (l£ / . Hit r a t e - a g a r . Manual of Methods ( 1 8 ) . Glucoee-agar and g lucose-ge la t in were used through- out for the quan t i t a t i ve p l a t e counts . For comparative purposes l a c to se -aga r and milk-agarf1) were used in addi t ion to the two media mentioned above, ""he d i lu t i on method of q u a n t i t a t i v e analyses was employed when using l i tmus-milk and the Yoges-Oroskauor bro th . Reaction of Media. All media, excepting b i l e - s a l t media, were adjusted according to hydrogen-ion concen t ra t ion . nhe f i n a l reaction was p .B . - 7 using brom-thymol-blue as i n d i c a t o r ( l 6 ) EXAMIHATiai AID T3CHNIiU3. The same technique was used for the - 4 - b a c t e r i o l o g i c a l examinations of the cheese as was worked out during the previous s tud i e s on the 'Kingston Cheese . ' (11 ) On a r r i v a l a t the labora tory the one pound cheese was cut in qua r t e r s and the necessary precaut ions being observed, s ix samples were taken throughout the cheese, the whole approximating as near as poss ib le one gram. The mater ia l was placed in a small weighed aluminium dish with a t i g h t f i t t i n g cover . The dish was again weighed, and the weight of the cheese determined. The sample was then ground with sand in a s t e r i l e g lass mortar,and with the aid of a g lass rod f i t t e d at one end with a rubber policeman, was washed in to a large-mouthed b o t t l e using a known quant i ty of s t e r i l e water . As before (11) an e l e c t r i c a l l y - d r i v e n hor- i z o n t a l shaker was used for the mixing of the d i l u t i o n . The mixing occupied five minutes , and t o provide against organ- isms being car r ied down by sand, shaking by hand was done for another five minutes . The higher d i l u t i o n s were then made in water blanks, and i t was found tha t d i l u t i o n s of l-j?0Q,000 and 1-Z, 000,000 gave in p r a c t i c e the most s a t - is factory p l a t e s . Throughout the work the g r e a t e s t care was taken t o keeo the cheese free from contamination while in the l abo ra to ry . All knives with which the cheese was cut were f i r s t placed in alcohol and then flamed, again placed in a l c o h o l and aga in flamed each t ime be fo re u s i n g . The d i s h e s , mor t a r s and p e s t l e s were wrapped in pape r and s t e r i l - i z e d wi th dry hea t f o r a t l e a s t two h o u r s a t 170°C. or f o r t h i r t y minu te s a t 15 pounds p r e s s u r e in t h e a u t o c l a v e , " e s t tubes were f i l l e d w i t h sand p lugged wi th c o t t o n wool and a u t o c l a v e d . The l a rge -mou thed b o t t l e s were plugged with c o t t o n wool and s t e r i l i z e d , w h i l e t he rubbe r s t o p p e r s f o r t h e b o t t l e s , arid t h e g l a s s red were s t e r i l i z e d in water f o r a t l e a s t one hour a t 15 pounds p r e s s u r e in t h e a u t o c l a v e . The s t e r i l e wa te r b lanks were p r e p a r e d in ground g l a s s s t o p p e r e d b o t t l e s , the t o p s being covered wi th co t 4 on wool . The g r i n d i n g of t h e cheese and t h e washing of i t i n t o t h e l a rge -mouthed b o t t l e were done in a s t e r i l e g l a s s c a s e , the door of which was r a i s e d j u s t enough t o admit t h e h a n d 3 . The c a s e was p r e v i o u s l y washed wi th a 1:1,000 s o l u t i o n of mercur ic b i - c h o l o r i d e . Hands and arms were washed wi th soap and wa te r and r i n s e d in l y s o l . COMPARATIVE VALUES Of CERTAIN MEDIA. In t h e work on t h e ' Kingston Cheese* r e p o r t - ed in 1^21  (11) t h e I n d i c a t i o n s were t h a t g l u c o s e agar would g ive b e t t e r r e s u l t s than l a c t o s e a g a r . At t h a t t ime t h e r e was not s u f f i c i e n t d a t a on which to form any d e f i n i t e c o n c l u s i o n s . During the work h e r e i n recorded a comparison was made of g lucose aga r wi th l a c t o s e a g a r ; and, l a t e r of - b - g l u c o s e a g a r with the mi lk agar of Ayers and Mudge ( 1 ) . Glucose a g a r proved t o he t h e most s a t i s f a c t o r y mod' m (See Table I . ) Glucose g e l a t i n p l a t e s were a l s o made, and as a r u l e , h i g h e r coun t s ??ere o b t a i n e d than when usi g l u c o s e a g a r . On h o t d a y s , however, i t was v e r y d i f f i c u l t t o keep the g e l a t i n from m e l t i n g . As i s s t a t e d on page 3 , t h e d i l u t i o n method was employed when u s i n g l i t m u s mi lk and t h e 7oges -Proskauer broth r e s p e c t i v e l y . In many i n - s t a n c e s growth in h ighe r d i l u t i o n s was observed in t h e l i t - mus mi lk than was the c a s e w i t h the Voges-Proskauer medium. QUANTITATIVE BACTERIOLOGICAL ANALYSES. A l l q u a n t i t a t i v e examina t ions were made i n t r i p l i c a t e and t h e r e s u l t s g iven below and in P l a t e s I and I I a r e ave rages of t h e s e t h r e e c o u n t s . The g e l a t i n p l a t e s were incuba ted f o r f i v e days a t room t e m p e r a t u r e . The b i l e - s a l t b ro th tubes and b i l e - s a l t - a g a r p l a t e s wore incubated for f o r t y - e i g h t hours a t 57 .3°C. Al l o t h e r media were i n c u b a t e d fo r f i v e days a t 22°C. In o r d e r t o form some idea of t h e pe rcen tage of a c i d - f o r m e r s p r e s e n t on t he p l a t e s , l i t m u s and b r o m - c r e s o l - p u r p l e were used a s i n d i c a t o r s in a number of g i u c o s e - a g a r - p l a t e s . A3 was expe r i enced before (11) in many i n s t a n c e s t he i n d i c a t o r in t he whole of t he medium was chai g e l . On p l a t e s where t he a c i d - f o r m e r s could -7 be d i f f e r e n t i a t e d , the number in the major i ty of cases was a hundred per cent of the count and the proport ion was not below ninety-seven per cent in any examination. The q u a n t i t a t i v e analyses were made of a spec i f ic cheese of a ce r t a in day ' s 'make ' at the time when the cheese was about twenty-four hours o ld . Another cheese of the same '-make' was examined when r i p e , about ten days l a t e r , hence, as cheese made on ten d i f f e r en t days were examined., the determinations on a t o t a l of twenty cheese have been recorded. As has been exolained in a previous report ( 1 1 ; i t was not poss ib le to examine each cheese more than once, because being so small i t had to be cut up com- p l e t e l y . As ye t ,no attempt has been made to determine the v a r i a t i o n s in numbers of bac te r ia present 5n indiv idual cheeses on the same daya' 'make. ' The work on Cheddar cheese, however, by Harrison and Conne]l(7> has shown that the va r i a t i ons in b a c t e r i a l count of d i f f e r en t p a r t s o°  the same cheese were not g r e a t e r than t h i r t y per cen t . In every ca3e, excepting the cheese made October the e leventh , a considerable reduction was shown in the b a c t e r i a l count of the r ipe cheese when compared with the count of the green cheese of the same days' 'make. ' The number of organisms per gram varied considerably in the d i f f e r en t cheese examined. One cheese a day old gave a count as high as 4,000,000,000 micro-organisms per gram; and another , a l so a day o ld , had a count as low as 9,000,000 micro-organisms per gram. The counts in the r i pe cheese var ied from 147,000,000 per gram to 12,000,000 per <• ram. Though there wa3 t h i s grea t difference in the number of organisms found in the d i f fe ren t cheeses, there was no great di f ference in the q u a l i t y of the cheese as judged for market. More de ta i led r e s u l t s wi l l be found on P l a t e s I and I I . Shere was a g r ea t e r number of b a c t e r i a present in the r i p e cheese made October the eleventh than in the green cheese of the same d a t e . In s ) i t e of t h i s apoarent discrepancy the number of bac t e r i a oresent in the cheese eleven days old was very c lose to the average of th? counts of a l l the r i pe cheeses . The s t a r t e r U3ed for t h i s cheese waa not very vigorous, when the cheese went to ire3s the ac id i t y was qu i te low and the green cheese when examined in the laboratory was not t y p i c a l ; or as the cheese-maker would say, was on the 'sweet s i d e . ' After eleven days.however, the f lavour was quite t yp i ca l though the" t ex tu re was a l i t t l e s o f t . One example i s not enough on which to base any con- c lus ions , but i t would seem to ind ica te tha t even though the cheese was vat ted when qui te sweet the bac t e r i a continued to mul t ip ly ; and t h a t with t h i s increase , there was an i n - crease in ac id i t y t i l l a t eleven lays the cheese was normal. fhie increase in a c i d i t y as the bac te r i a continued t o mul t ip ly -9- 18 i n accord wi th t h e f i n d i n g s of Baker e t a l ( 2 ) . As i n t h e examina t i ons of tv;c r e a r s ago t h e r e h a s been no a t t e r , "t to make a n a e r o b i c examina t ions of t h e c h e e s e . I t i s f u l l y r e a l i z e d t h a t t h i s par* of t he work should be u n d e r t a k e n ; but i t was not p o s s i b l e s i m u l t a n - e o u s l y t o do both a e r o b i c ana a n a e r o b i c e x a m i n a t i o n s , and i t was dec ided for the t ime be ing t o c o n c e n t r a t e on work under a e r o b i c c o n d i t i o n s . I t i s to be d e s i r e d t h a t an en- q u i r y i n t o t h e a n a e r o b i c f l o r a of the c h e e s e s h a l l be i n - s t i t u t e d . An e f f o r t was made t o conduct h i s t o l o g i c a l ex - a m i n a t i o n s of t h e cheese in o rde r t o g e t some i iea of t h e g rouping of t h e o rgan i sms ; and t o s e c u r e coun t s of t h e b a c t e r i a by t h e mic roscop ic method a s recommended by Hucker (9 )» Thus f a r , t ime h a s no t p e r m i t t e d t he doing of o t h e r than p r e l i m i n a r y work on t h i s phase of t h e i n v e s t i g a t i o n . At a l a t e r d a t e i t i s t o be des i r ed t h a t t he examinat ion of h i s t o l o g i c a l speoimens s h a l l a l s o r e c e i v e e o n s i i e r a 4 i on . QUALITATIVE 3 A C ? 3 H I ^LOGICAL AITAIY333. Co lon ies which ap?eare . l t o occur w i th t h e g r e a t e s t f requency were taken off the p l a t e s and r e t a i n e d i n pure c u l t u r e . A l a r g e p e r c e n t a g e of t h e s e c o l o n i e s were s i m i l a r to the small s u b - s u r f a c e and s u r f a c e c o l o n i e s - Group 2 - r e p o r t e d in t h e s t u d i e s under t aken i n l rjll (11 J . Grea t d i f f i c u l t y was found a t t h a t t ime (11 ) in keep ing t h e -10- organisms of Group 2 alive long enough to do even a prelimin- ary examination of them. Also, the growth on agar slants was found to be insufficient for inoculation purposes. I n order to avoid the difficulties mentioned above , the follow- ing procedure was evolved and followed with good results throughout the present investigation:- The colonies were fished off gelatin plates in the manner found so advantageous in the later stages of the work reported before (11 J; a small lump of gelatin containing the colony was lifted out of the plate, put into Voges-Proskauer broth and held at 37..5 CC till the gelatin melted, freeing the colony. The tubes of broth were then incubated at 22 °C. Whe n a cloudiness in the broth indicated that growth had taken place, this broth was used to inoculate the desired culture media. A  small quantity of the broth was drawn up into a sterile pipette and all tubes of culture media inoculated with one drop each from the point of the pipette. The glucose broth of the Voges-Proskauer test was used because it was the only liquid medium in which the organ- isms of Group 2(11) could be grown satisfactorily. As a preliminary examination , all cultures were stained by Gram and the reactions to litmus-milk, glucose, lactose and sucrose were recorded. Late r it seemed desirable to determine the reaction to maltose, glycerin, salicin and mannitol. Th e results of the above examinations are recorded on Plates III and IV. Th e agar slants - 1 1 - recommended by Conn and Hucker (4> and given in the 'Manual of Methods for Pure Culture otudy of B a c t e r i a ' ( l 8 j were used for the determination of acid and gas in a l l the carbo- hydrate media employed. I t has been noted above t h a t for these de- terminat ions one drop, from a f ine ly-bored p i p e t t e , of the Voges-Proskauer glucose solut ion was used for the inocu la t ion in each case . The p o s s i b i l i t y of the i n f in i t e s ima l amount of glucose present in the drop of cu l tu re being su f f i c i en t to show a c i d i t y in the media other than glucose suggested i t s e l f . To check t h i s , ten tubes of n u t r i e n t agar s l a n t s containing ind ica to r but no sugar were inoculated at the same time as the other media. The control tubes remained n e u t r a l . Ten tubes of n u t r i e n t - a g a r were not considered as su f f i c i en t check, but the fact t ha t a l a rge percentage of the cu l tu res did-not produce acid on l ac tose -aga r was considered as conclusive proof that the minute quant i ty of glucose present in the drop of inocula t ing ma te r i a l was not su f f i c i en t to give an acid react ion to the media inocu l - a ted . One hundred and seventeen organisms were i s o l a t e d from p l a t e s made of the cheese , and f ive organisms from p l a t e s made of the s t a r t e r used in the making of the cheese. Cul tura l and morphological d e t a i l s of these -12- organisms w i l l be found on P l a t e I I I . .Vhen the r eac t ions of the cu l tu re s to mal tose , s a l i c i n , g lycero l and mannitol were determined, i t was found tha t twenty- three organisms had d ied . The formation a l ready obtained about them, how- ever, was enough t o show to which main group they belonged. The one hundred and seventeen organisms d iv ide themselves in to five main groups. G R O U P I . In t h i s group are found organisms i s o l a t e d from pin-head colonies growing more p a r t i c u l a r l y under the surface of the media. Gram p o s i t i v e , sphe r i ca l , occurring in cha ins , ones and twos and in clumps, Each s t r a in ferments glucose and l a c to se t o acid , produces acid and c lo t in l i tmus milk and f a i l s to l iquefy g e l a t i n , deventy-nine s t r a i n s find themselves in t h i s group. V/ithin the group, however, spec i f io s t r a i n s vary in the s ize of the c e l l s and in the ac t ion on cer ta in of the carbohydrates other than glucose and l a c t o s e . A complete record of the charac- t e r i s t i c s of each of the seventy-nine s t r a i n s i s presented on P l a t e I I I . On P la te IV the v a r i a t i o n s in s i ze of c e l l and in the act ion on the carbohydrates other than glucose and l a c t o s e a re recognized and sub-groups are e s t ab l i shed . Of the seventy-nine s t r a i n s thus placed in sub-groups, Cultures 115, 121, 212, 136, 135, 172,156 and 152 prove to -13- be representative of the number of s t ra ins respect ively, as shown in the second column of the p l a t e . Cultures 156 and 152 representing five and two s t ra ins respect ively, agree in the essential charac ter i s t ics with the desoription of streptococcus l a c t l s ( L i s t e r ; according to Bergey's Manual of Jeterminative Bacteriology {H)»  s t ra ins repre- sented by Cultures 115, 121, 212, 136, 135 and 172, however, oannot be c lass i f ied as typical forms of jtreptocoocus l a c t l s (Lis te r ; because of the fai lure to act on sa l ic in , on mannitol, or on both fl7>« I t may be observed, Plate I I I , that of the strains isolated from s t a r t e r , those included in th i s group, ferment sal icin and mannitol to acid and are apparently identical with Culture 156. I t wil l be noted on Plate I I I that only three s t ra ins coming in this group and isolated from cheese, ferment sal icin and mannitol to acia, while three r:iore s t ra ins ferment sal icin only. I t may be that due to having passed through cheese the majority of the organisms which find themselves in Group I had lost the power of fermenting sa l ic in and mannitol, thred s t r a ins had lost the power of fermenting mannitol only, while three strains had retained the power of fermenting both these oarbohydrates. I t would seem that with forms of itreptococcus lactis(, Lister ; attenuation takes place in passing through cheese. Support for th i s suggestion i s found in the work -14- of Iloyd on cheddar cheese(12j. î 'rom well-matured cheese he isolated s t ra ins of true l ac t i c acid producing organisms which he defined as Bacillus acidi l a c t i c i — now considered as Streptococcus l a c t i s (Lister J {n){lZj  --  which failed to clot milk. Further, of the organisms isolated from cheese - dee Plate I I I - three only of the s t ra ins included in th is group ferment salicin and mannitol to acia, while three of the s trains ferment sal icin to acid but fa i l to act on mannitol. The remainder of those cultures isolated from cheese and included in th is group have no action on ei ther sal ic in or mannitol. Of the organisms comprising Group I , every strain is found to be ident ical with one or more of the strains isolated by Hucker from cheddar cheese (10; . jiach of his s t ra ins is classified as Streptococcus lact is(Lis terXlQ)« In studying the organisms placed here in ©roup I , the reactions to sa l ic in , mannitol and maltose, have been determined in addition to the reaction recorded by Hucker for his s t r a ins . Consequently, while Hucker (10) classif ied his organisms as Streptococcus lac t i s (Lis te r ) the additional reactions recorded for the Cultures - Group I - of this report permit of a more specific c lass i f ica t ion , following Bergey (17j . Accordingly, Cultures 152 and 156 are placed as typical forms of Streptococcus lac t i s (Lis te r ) (17; , af ter Bergey; and Cultures 115, 121, 212, 156, 155, and 172, on account of the i r fa i lure to ferment mannitol or - 1 5 - s a l i c i n , or mannitol and s a l i c i n , are placed as a t tenuated forms of Streptococcus l a c t i s ( L i s t e r ) ( 1 7 ) » G R O U P I I . Gram pos i t ive rod-shaped acid formers . Nineteen c u l t u r e s represented in P la te IV by Cultures 101 , 102, 103, 104, Ml2 and M9 a re Gram p o s i t i v e non-spore bearing rods with round ends, varying in length from l u to lOu long, the majori ty approximating the minimum length r a the r than the maximum. There was a tendency to form short chains in the broth as used for the Voges-Proskauer t e s t and to a somewhat l e s s e r extent on agar . Good growth took place both at 22°C. and a t 40°C. All the c u l t u r e s , with the exception of Cultures 204 and 205 were picked off agar and ge la t in p l a t e s incubated a t 22°C» for l i v e days, while cu l tu re s 204 and 205 were taken off p l a t e s incubated a t 40°C» The s t r a i n s do not l iquefy ge l a t in but form a clean acid c lo t in l i tmus-milk in from fourteen to eighteen days at 40°C. followed by bleaching. Gas i s not formed in any of the carbohydrates and g lucose , l a c t o s e , sucrose , s a l i c i n and ' ra f f inose a re fermented to acid. These cu l t u r e s of Group I I seem to bear a marked resemblance to 0r la-Jensen T s Streptobacterium (15) in t ha t they are Gram p o s i t i v e rods tending to form chains, growing at 22°C.to 40°C. and -16- fermenting s a l i o i n t o acid. Smears made from n u t r i e n t aga r s l a n t s showed a l a r g e number of short rods which might be mistaken almost for d ip lococc i with a  number of rods about lOu long. These smears were almost i i e n t i c a l in appearance with Orla-Jensen '8 Streptobaoterium plantarium number 18, Agar s t reak 1 day a t 30° p l a t e XLIl(13) . According t o Bergey's Manual (17) the c u l t u r a l and morphological c h a r a c t e r i s t i c s of the organisms here under d iscuss ion would place them in the Genus Lac tobac i l lus (BeiJer inckj ( 1 7 ) . I t was impossible to i s o l a t e organisms of t h i s type from the freshly made cheese though several a t tempts were made. All the cu l tu res mentioned above were from cheese eight to ten days old. I t has been known tha t Lac tobac i l l i are found in mature cheddar cheese and i t i s i n t e r e s t i n g t o know, tha t in a cheese which r ipens so qu ick ly , Lac tobac i l l i are present in qu i t e considerable numbers. Cultures 101 ana 1,19 do not ferment mal tose, but ferment mannitol to a c id . The c u l t u r a l and morphological c h a r a c t e r i s t i c s of these two organisms would seem to warrant the c lass i fy ing of them as of the Lactobaci l lus bulgaricua (Grigoroff Jtype(17) and as of Rahe's type "D" ( 1 7 ) . Cultures 102 ana 104 ferment mal tose, manni tol , r a f f inose , dex t r in , arabinose and t r eha lose to ac id , t r eha lose being fermented slowly. The c u l t u r a l c h a r a c t e r i s t i c s of these organisms, and p a r t i c u l a r l y t h e i r -17- aotlao • • tb a oarbotgrdrataa , woal d r*v-ir « tha t tba ? » • o laasl f lad a s La o tobaoll lao oaoanarladanaabar a >(17) . How - •Tar, th a lanft h o f tb a oall a o f Cultara o 10 2 an d 10 4 raria a fro* l a t o Id a whil a tb a lanft h o f tb a oall a rooordo d fo r Laotobaolllaa o a o — r l a l a fro s 1.5 a t o 2a . n t h tb a asoaptloo o f tb a raaotlo n t o trahaloaa , th a worphol->gloa l and ooltara l oharaotariatlo a o f oaltura a 10 2 an 4 10 4 ap»aa r to b a idontloa l wit h thoa a raooraa d fo r Laotobaoll l . a alanta,rl(Orla-Janaan>( 17J . Furtha r thaa a tw o organlaat a and Laotobaollla a plantarl . olo t s i l k , vbll a Laotoba o 11 laa ottoonorla doo a not . Th a natio n o f Laotoba o 11 laa oaouaarl a an daxtrlf l ! • aao h alowa r a a oonparo d wit h l t a raaotlo n to trabalooa , whll a wit h attain s 10 2 an d 104 , th a m i n i la tb a aaaa . Trabaloa a i a th a auga r whloh , aooordln g t o largagX 17) dlwlda a Laotobaollla a oaouaarl a an d Laotobaollla a a lantar l . I t l a obrlooa , tharafora , conaldarln g th a obaraatarlatloo o f Laotobaollla a oaoanarl a an d Laotobaollla a a lantarl raapaot lral / , tha t Coltara a 10 2 an a 10 4 ar s t o b a plaoad wit h oa a o r otha r o f thaa a tw o know n atraln a accord - ing t o tb a ralat lr a iaportano a whlo h I t t o b a attaoba d t o tba farna n tatlon o f trahaloa a o n th a on a hand , an d t o th a a l i a o f oal l a an d th a olott in g o f tsll k o n th a othe r hand . OB tb a whol a followin g Bargay , th a wldano a aubaltta d ap*>aar o to b a l a faroo r o f Laotobaollla a oaoanarla : an d Cultara a lOt an d 10 4 ar a olaaaif la d a a t/pa a o f Laotobaollla a caooamrl a -18- (Eenneberg) a f t e r Bergey (17J . Culture 103 does no t ferment maltose and mannitol . The act ion on the carbohydrates would place Culture 103 as e i t h e r Lactobaci l lus caucasicus (gem J (17) or Lactobaci l lus boas-oppleri(Boas and 0ppler)(17>. As ne i the r Lactobaci l lus caucasicus (17) nor Lac tobac i l lus boas-oppler i w i l l grow on ge l a t in a t 22°C» and as Culture 103 was i so l a t ed from a gela t in p l a t e incubated a t room temperature, i t cannot be c l a s s i f i e d e i t h e r as Lactobaci l lus oaacasicus or Lactobaci l lus boas -opple r i , but i s placeo. within the type species Lactobaci l lus caucasicus(Kernj(17)« Culture M12 ferments mal tose , mannitol and raff inose to ac id . The a b i l i t y of t h i s organism t o ferment raffinoBe without fermenting dext r in does not permit of i t being c l a s s i f i e d by BergeyTs Determinative 3acter io logy(17) other than as being a member of the Genus Lactobaci l lus and coming within the type species Lactobaci l lus caucasicus UernJ(17; . G B 0 U P I I I . Gram negat ive non-spore forming rods , which ferment l ac tose t o acio. and gas . There are s ixteen organisms in t h i s group represented on P la t e IV by Cultures 119, 124, 127 and 2 06. All sre short gram negative rods which ferment glucose -19- and lactose to acid and gas, clot milk, reauce n i t ra tes to n i t r i t e s , but fail to liquefy ge la t in . The character is t ics noted above, and the negative reaction to the Voges-Proskauer tes t would place these s trains in the Genus geoherlohla(17j Of the organisms in th i s group, Culture 119 is a non-motile rod which ferments sucrose, sa l i c in , maltose, glycerol, du lc i to l and nannitol to acid; proauces sliminess on agar, in peptone broth and in milk, but fa i l s to produce ei ther indol or acetyl-methyl-carbinol. V/hen f i r s t isolated th is strain fermented sucrose to acid and gas, but after being kept on a r t i f i c i a l media for same months lost the faculty of producing gas on t h i s carbohydrate. In i t s ab i l i ty to produce sliminess on certain media, to ferment glucose and lactose to acid and gas and i t s inab i l i ty to produce i rdo l , Culture 119 shows a resemblance to 3acterium aerogenes (Sscherich) as described by Buchanan and IIammar(3). Their Bacterium aerogenes is shown as fermenting carbohydrates, other than glucose and lactose, to acid and gas,while th is s train ferments them to acid only. Bacterium aerogenes (Sscherich) becomes Aerobacter aerogenesCBscherichj in Bergey's Determinative Bacteriology (17). Following Bergey (17J Culture 119 cannot be classif ied as Aerobacter aerogenes, for i t f a i l s to produce acetyl-methyl-carbinol, but based on the sum of the character is t ics recorded the strain finds i t se l f within the Genus Escherichia. Of the species in t h i s -20- Genus included in Determinative Bacteriology (17) i t appears that Escherichia astheniae presents the features with which the culture under discussion most closely aligns i t s e l f . This an culture, th eref ore, i s here considered as being/a-typical form of Escherichia astheniae (Dawson)(17). Culture 124 i s a non-motile rod, ferments glucose and lactose to acid and gas, but f a i l s to act on sucrose and sa l ic in . Bat for the fact that Culture 124 i s non-motile, i t could be classif ied as Escherichia paragrunt- hali( Castellani and Chalmers) (17) • T l a i 8 l a c k o f mot i l i ty , however, prohibits i t being classif ied as a true form of Escherichia paragrunthali and i t i s classif ied as of the type Escherichia paragrunthali (Castellani and Chalmers) (17). Culture 143 is a strain of not i le rods which forms indol, ferments sal ic in and dulcitol to acid and gas, but fa i l s to ferment sucrose. The ab i l i ty of th is s train to reduce n i t ra tes to n i t r i t e s , to ferment glucose, lactose , salicin and aulci tol to acia ana gas, in the inabi l i ty to produce acid in sucrose and to form acetyl-methyl-oarbinol places this organism as a true tyoe of Escherichia coli (Escherich) Castellani and Chalmers(17). Culture 206 is a non-motile rod which fer- ments salicin and sucrose to acid and gas, but aoes not produce indol or give the Voges-Proskauer reaction. The ab i l i ty of the culture t o ferment sucrose and lactose to -21- acid and gas, the inab i l i ty to produce indol or give the Voges-Proskauer reaction, and i t s fai lure to show mot i l i ty would suggest that i t be placed as Escherichia astheniae (JawsonJ(17)• Escherichia astheniae, however, has no action on sa l i c in , while Culture 206,as noted above, ferments th is carbohydrate to acid and gas. This difference in action on sal ic in would not permit the classifying of th is strain as a typical form of Escherichia astheniae and i t an is c lass i f ied here as /a- typical form of Escherichia astheniae ((J3awson;(19J. G R O U P IV. Spore Bearing Rods. Only one spore bearing organism,Culture 110, was found in t h i s invest igat ion. The s t rain is a Gram negative motile rod 2u to 4u in length, the cel ls ocourring singly ana in pa i r s . The spores are terminal and the rods are swollen at sporulation giving the cell the appearance of a tadpole. The growth on agar i s pal e yellowish white af ter forty-eight hours, gelatin i s not liquefied and acid i s formed in milk but no clot i s produced. Glucose, lac tose , and sucrose are fermented to acia. The cul tural and morphological character is t ics of Culture 110 appear to be identical with those recorded for Bacillus pseudotetanicus (Kruse)(17) with the exception of the action on the - 2 2 - ca r tohydra tes ; Bac i l lus oseuaotetanicus showin g n o actio n on th e carbohydra tes . Culture 11 0 bear s a  marke d resemblanc e also to Bac i l lus ci rculans( Jordanj( 17) but th e tw o culture s do not agree in the reac t ion to th e Gra m s ta in . I t i a suggested t ha t in SMite of the d i f ference noted wit h respect to the action to the Gram s t a i n , Culture 11 0 sha l l be considered as being of the type Bac i l lu s c i rculana( Jordan ) (17 j. G R O U P V. Coccus ?orms. Culture 1.53 i s a minute Gram pos i t i ve coccus about Q.3u. in diameter , which l i que f i e s g e l a t i n , ferments g lucose , maltose, s a l i c i n and mannitol to ac id , but has no act ion on l a c t o s e or sucrose . Bile s t r a i n bears a marked resemblance t o Micrococcus l a c t i s var ians of Conn, 3sten and J tock ing(5) . These i nves t i ga to r s found tha t some forms of Micrococcus l a c t i s var ians did not ierment l a c to se and sucrose, c h a r a c t e r i s t i c s applying equally t o Culture 133. Comoaring the el a r a c t e r i s t i c s as a whole, the s t r a i n agrees even :rore c lose ly with Micrococcus l a c t i s varian^Type A)  of Conn, 3sten and stocking (4) both a s to the formation of acid in glucose, and in the curd l ing ana d iges t ing of milk without the formation of acid . Th e resemblance, however, of the s t r a i n to Micrococcus -"•arians (Dyarj Conn(17) i s not so pronouroad; yet Micrococcus var ians -23 - (Dyarj Con n i t considere d I n Bergey' a Jeterminativ e Bacteriology a s bein g synonymou * wit h Mlcrooocon a l a c t l s yarlane o f Uonn , Sste n an d .stocking . Hence , i t woul a see m that Cultur e 13 3 I s t o b e c l a s s i f i e d a s o f th e typ e Mlorocooons varian s (Dyar j Con n (17J . Culture 14 8 i s a  strai n o f Gra n pos i t iv e coooi growin g i n clumps . I t f a i l s t o liquef y g e l a t i n , form s a clea n aci d c lo t i n milk , reduce s n i trate s t o n i t r i t e s and ferment s glucose , lac tos e an d sucros e t o acid . H o aotio n on maltose , s a l i c i n o r inuli n ha s bee n noted . Th e growt h i s goo d o n ar t i f l ca l medi a an d th e pigmen t o n aga r afte r fourteen days , aocordin g t o th e 7/inslows ' chart(20J , l a Ligh t Cadmium Yellow , Chro m I I I , designate d b y thes e worker s a s 'white . ' Th e character i s t i c s recorde d woul a sugges t thu t the strai n b e place d i n th e genu s 3taphylooooous ( 17 )• T o take th e c l a s s i f i c a t i o n t o a  mor e speci f i c stage , th e fa i lur e of th e organis m t o liquef y gelat i n an d th e ab i l i t y wit h whioh lactos e i s fermente d indioat e a  c los e resemblanc e o f the strai n t o Staphylococcu s tetragenus(Koch-Oaffky )(17)• The ident i t y o f th e tw o strain s canno t b e accepte d withou t qual i f icat ion fo r cultur e 14 8 reduce s n i t ra te s t o n i t r i t e s , and unde r th e microscop e th e c e l l s appea r i n clumps ; v.-hll s Staphylococcus tetragenu s (17 J doe s no t reduc e n i tra te s and th e c e l l s appea r i n group s o f four . Winslo w an d /inslo w (20j s tat e tha t Albococcu s tetragenu s (Gaffky ) - - albocoocu s -24 - being a genus s ince absorbed in the genus Staphylococcus- i s c lose ly r e l a t ed to Albococcus eandidus (Conn) and in descr ib ing Albococcus canaidus , Winslow et a l (21) make the following statement "The second type in abundance in our "s tudy, and the type found most commonly on the skin a f t e r "St .ep idermidis by Gordon, I s the form which ferments " l ac tose but f a i l s to l iquefy g e l a t i n , i d e n t i f i e d by YJinslows "as Albococcus eandidus and now t o be ca l l ed Staphylococcus "eandidus. Our s t r a i n , however, reauced n i t r a t e s and gener- a l l y c l o t t e d milk which Gordon's type did no t . Three s t r a i n s "sent t o the museum c o l l e c t i o n as Micrococcus tetragenus " a l l belonged t o t h i s group. Hone of them reduced n i t r a t e s "and r e s u l t s a re va r i ab l e in milk and in r?gard t o ammonia "product ion." The s t r a i n recorded here agrees with the a:ove descr ip t ion of Staphylococcus eandidus with respect to the c l o t t i n g of milk ana the reduction of n i t r a t e s to n i t r i t e s . In Table 10 of the same repor t (21) Winslow et a l show twenty of t h e i r s t r a i n s as fermenting mal tose . Culture 148 f a i l s t o ferment t h i s sugar ':ut in a l l other respects i t appears to be i d e n t i c a l with t h e i r cu l tu res a s c i t e d . The organism o r i g i n a l l y described by Conn was placed by him in the Genus Micrococcus as Micrococcus candidus(20) . Later organisms showing s imi lar c h a r a c t e r i s t i c s t o Cohn's Mierococcus were placed by the /inslows (20) within the type -25 - candidua (CohnJ(21). The eommittee of the society of American B a c t e r i o l o g i s t s in Bergey's determinat ive Bacter- iology (17 j have again placed organisms of t h i s type in the Genus Micrococcus, as Micrococcus candidus (Cohnj(17). Though Culture 148 d i f f e r s from Micrococcus candidus(Gohn) (17) in the act ion to n i t r a t e s , i n the main the cha rac t e r - i s t i c s are i d e n t i c a l and Culture 148 i s c l a s s i f i e d here as of the type Micrococcus candidus(Cohnj(17j« Culture 214 i s a Gram p o s i t i v e coccus, occurr ing in i r r e g u l a r groups. The s t r a i n l i q u e f i e s ge la t in s t r a t i f o r m , produces a white pigment on agar, ferments glucose and sucrose t o acid, but no action on l a c to se can he noted . This cu l tu re died before i t could be i nves t iga t ed fu r t he r . According t o the c u l t u r a l and morphological f ea tu res determined, however, Culture 214 would be placed in the Genua dtaphylococcus, and of the type species Staphylococcus aureus(;xosenbacbJ(l7)« The i n a b i l i t y of the s t r a i n t o ferment l a c t o s e , does not "oermit of i t being c l a s s i f i e d more s p e c i f i c a l l y . V/inslow, Hothberg and Parsons in the 7Tr.ite and Urange staphylococci(21J show ten cu l tu res in t h e i r Table 8, which are white pigment formers, l iquefy- ing g e l a t i n , but f a i l i n g to ferment l a c t o s e . Later the same workers say (21J "The l ac tose -nega t ive g e l a t i n - p o s i t i v e "type of white pigment producers appears in our s tudy, "as in tha t of Gordon, to be a r a r e r one, and t h i s form, -26- "as well as the forms which exhibi t miscellaneous fermentat ive " reac t ions may best be l e f t for the oresent without spec i f i c "names." I t i s f e l t tha t Culture 214 should be included in t h i s group, Culture 214, i s , t h e r e f o r e , l e f t for the present as of the type species i taphylococcus aureus (Eosenbachj(17) O B S E R V A T I O N S . The technique evolved when engaged on the work in 1922 (11) has been adopted throughout the p resen t i n - ves t iga t ion with pronounced s a t i s f a c t i o n . Fur ther , the present paper confirms the previous f indings (11J tha t for the determinations of the bac t e r i a l f lo ra of 'Kingston Cheese' g lucose-agar ana g lucose-ge la t in are the most s a t i s f a c t o r y meaia. The t o t a l number of organisms ^resent in the 'Kingston Cheese f both in the cheese when newly made and the cheese when ten days old, i s founo. to approximate very c lose ly the numbers recorded in the work on Cheddar Cheese. Though the t o t a l number of organisms present in the 'Kingston Cheese' ten days old i s not cons tan t , and may vary, as t h i s inves t iga t ion shows from 20,000,000 t o 350,0J0,0J0 bac t e r i a per gram, the cheese examined in each case were normal both as to tex ture and f l a v o r . Though no d e f i n i t e e f for t was made to determine the percentage of ac ia formers, the r e s u l t s obtained showed tha t a t l e a s t ninety-seven per cent of the f l o r a were acid formers. -27- This paoer would seem to ind ica t e tha t the organisms which occur with the g r ea t e s t frequency in the cheese when one day old are those of the s treptococcus l a c t i s ( L i s t e r ) type, and in cheese of the same day 's 'make' the when ten days ola are those of/Streptococcus l a c t i s ( L i s t e r j type and those of the Lac tobac i l lus (Bei je r inck j type . I t appears to be well es tabl ished tha t the successful r ipening of cheddar cheese i s very l a rge ly deoenaent upon the b a c t e r i a l f l o r a determined according to the recorded l i t e r a t u r e . The work herein presented, qu i te d e f i n i t e l y shows that the f l o r a of the '.Kingston Cheese' i s very s imi la r , i f not i d e n t i c a l with the f lo ra recorded for cheddar cheese. l e t in the case of the 'Kingston Cheese' we have a cheese which i s mature in ten days a f t e r making, while a cheddar cheese requ i res from th ree to six months t o a r r i v e at maturity. It would seem, therefore, that in contem- plating the factors determining the successful ripening or maturing of the 'Kingsto n Cheese' due regaru. must be paid to the system of manufacture adopted, to the temperature at which ripening takes place and to all the processes associated with the management of the cheese, for as far as this paper can define, the bacterial flora of the 'Kingston Cheese1 is clearly idential with that of the cheddar cheese. -28- S U M M A R Y . A b r i e f resume i s given of the system adopted in the making of the 'Kingston Cheese' and the spec i f i c c h a r a c t e r i s t i c s of the cheese are noted. Bac te r io log ica l analyses have been made of twenty cheese manufactured on ten d i f f e r en t days, ten cheese when one day old and ten cheese of the same day ' s 'make' when mature a t ten days a f t e r making. The r e s u l t s of the analyses made of these twenty cheese are recorded on P la tes I and I I . A l i s t of the media employed i s recorded and a descr ip t ion of the nethod of examination of the cheese i s g iven. Several v a r i e t i e s of media were used with the object of determining which were best adapted for the de- terminat ion of the b a c t e r i a l analyses of the 'Kingston Cheese' f glucose-agar and g lucose-ge la t in proving to be most s a t i s - f ac to ry . The r e s u l t s of the comparison of these media are given On Plate I . One hundred and seventeen organisms have been i so la t ed from p l a t e s made of the cheese and f ive from p la tes made of the s t a r t e r used in the making of the cheese. As fa r as poss ib le the organisms were those wnich appeared to occur with the g r ea t e s t degree of frequency. Though eighteen of the one hundred and twenty-two organisms i so la ted -29 - were of the Genus Escherichia t h i s does not necessa r i ly represent the percentage of organisms of tha t Genus present in the cheese . The s t r a i n s were taken from MaoConkey'a broth tubes and MacConkey's agar p l a t e s in order t o de ter - mine the species of gas formers occurring with the g r e a t e s t frequency. The morphology, c u l t u r a l f ea tu res and physiological reac t ions of organisms i so l a t ed from 'Kingston Cheese' are given on P la t e s I I I and 17. The one hundred and twenty-two organisms i so la t ed are placed in five main groups, and are c l a s s i f i e d according to Bergey's Determinative Bacteriology (17 ) . G B 0 U P I . Streptococcus l a c t i s ( L i s t e r ) t y p e s . Seventy-nine of the one hundred ana seventeen cu l tu res recorded in t h i s paper find themselves in t h i s group, and are c l a s s i f i e d as fol lows. Culture 1^6 ana 152 represent ing in a l l seven s t r a i n s are c l a s s i f i e d as Streptococcus l a c t i s ( L i s t e r Q (17) Cultures 115, 121, 212, 136, 135, and 172, representing- seventy-two s t r a in s are placed as a t tenuated forms of Streptococcus l a c t i s ( L i s t e r ) ( 1 7 ) - 3 0 - G R O U P I I Lac tobac i l lus (Bai jer inck) types (17) Twenty-three s t r a i n s are placed in t h i s main eroup and are c l a s s i f i e d as to spec i e s . Cultures 101 and M9 represent ing four cu l tu re s are c l a s s i f i e d as Lactobaci l lus bulgar icus(Grigoroff) (17J and of the type "D" (Rahe) (17) . Cul tures 132 and 104 represent ing 15 s t r a i n s are placed as a - t y p i c a l forms of Lactobaci l lus cucuraeris (Henneberg) ( 1 7 ) . Cul tures 103 anu M12 represent ing two s t r a i n s each are c l a s s i f i e d as of the type species Lacto- b a c i l l u s cauca3icus( Z e r n ) ( l 7 ) . G R O U P I I I Gram negat ive ,Lactose l a m e n t i n g Rods. In a l l there are s ixteen s t r a i n s in t h i s Group ret>resented by four c u l t u r e s . Sight s t r a i n s represented by Cultures 119 and 206 are c l a s s i f i e d as a - typ ica l forms of Sscherichia astbeniae (Dawson) (17 ) . Three s t r a i n s represented by Culture 124 are considered as a - typ ica l forms of Escherichia -31- paragrun tha l i (Cas t e l l an i and Chalmers)!17) . Five s t r a i n s represented by Culture 143 are c l a s s i f i e d as Escher ichia c o l i ( a s c h e r i c h ) Cas te l l an i and Chalmers(17) G R O U P IV. Siore Bearing Roas. Only one Culture of soore-forninp- rous was found in t h i s i n v e s t i g a t i o n . Culture 110 i s placed as an a - typ ica l form of Baci l lus c i rculans(Jordan,)(17 ) U O U P v. Coccus forms other than Streatococcus l a c t i s ( L i s t e r ) . Three cu l t u r e s find then3elve3 in t h i s group,two of which are Micrococci and one a Staphylococcua. One s t r a i n , C u l t u r e 133, i s c l a s s i f i e d as of the type Micrococcus variansCDyaryConnCl?;» One s t r a i n , Culture 214, i s placed as of the type species Staphylococcus aureus(Rosenbach)( 17) • One s t r a i n , Culture 148, i s considered to be within the type species Micrococcus canaidua( Cohnj(l7) . Observations on the data presented are offered. -32- A C K H 0 W L B D G M E H T 3 . I wish to thank Professor Wilfrid Sadler , a t whose suggestion t h i s work was commenced, with whom I have consulted from time to t ime , and who has kindly read over the manuscr ipt . This i nves t iga t ion has been made poss ib le by labora tory f a c i l i t i e s , which have been placea a t my disposal by the Univers i ty of Br i t i sh Columbia. - 33 - H S I  E H E H C E 3 . 1 . Ayers and Mudge, 1920, Milk Powder Agar , J o u r . of B a c t . V. 6 , p p . 3 6 3 - ^ 8 8 , B a l t i m o r e . 2 . Baker , Brew and Conn, 1919, d e l a t i o n Between L a c t i c Acid P r o d u c t i o n and B a c t e r i a l Growth in + he Souring of M i l k . N.Y. Agr. E x o . S t a . Tech. B u l l . Ho .74 , Geneva. 3 . Buchanan and Hammar, 1 9 1 3 . Slimy and Ropy Mi lk . Agr. E x p . S t a . Iowa S t a t e Co l l ege of Agr. and M e c h . A r t s . Research B u l l . No .22 , p p . 267-270 , Ames. 4 . Conn and Hucker , 1 9 2 1 . The Use of Agar S l a n t s in D e t e c t i n g Fe rmen ta t ion . IT.Y.Agr.Exo . S t a . B u l l . No. 84, Geneva. 5 . Conn, Eaten and S tock ing , 1906. C l a s s i f i c a t i o n of Dai ry B a c t e r i a , Repor t Storrs(Conn^ A g r . E x o . S t a t i o n . 6. F i n a l Rpt . of the Committee of Socy. toier . B a c t . on C h a r a c t e r i z a t i o n and C l a s s i f i c a t i o n of B a c t e r i a l Types, 1920 . J o u r . B a c t e r i o l o g y V . 3 , p p . 191-220 . 7 . H a r r i s o n and C o n n e l l , 1904. A Comparison of t h e B a c t e r i a l Content of Cheese cu red a t d i f f e r e n t Tempera tu res . C e n t r a l l . fUr Bakt . I I ,XI , Bd.po.637-637 , J e n a . 8 . Ha r r i son and Vander leck , 1908 . Aescul in B i l e S a l t Agar fo r Water and Milk A n a l y s i s . Trans .Roy.Socy . C a n . I I I . S e r i e s I I . p p . 103-110 , Ottawa. 9 . Kucker, G. J . 1 9 2 1 . The Microscop ic Stuoy of B a c t e r i a in Cheese . N.Y. Agr. Exp.Sta . Tech. Bull .ITo. 87 .Geneva. 1 0 . Hucker , G . J . 1922. The Types of B a c t e r i a Found in Commercial Cheddar Cheese . N . Y . A g r . E x o . S t a . 3 u l l . No .90 . Geneva. - 3 4 - 1 1 . Kel ly , C. D. 1922. Fur the r Studies on the '.Kingston Cheese, ' with specia l Reference to the Bac t e r i a l Flora of the Cheese. Thesis for degree of 3.S.A. l i b r a r y , Universi ty of B r i t i s h Columbia, Vancouver, B.C. 12. Lloyd, F . J . 1906. Inves t iga t ions i n to the Cause of Flavor in J a i r y Products . Jour . Bath and West and Southern Counties 3ocy. Vol. XVI, Fourth S e r i e s , op. 1-26, Bath. 13 . Jensen-Orla, 3 . 1919. The Lactic Acia Bac te r id . Andr* Fred Host and Son, Kobenhaven. 14. Russel , H.L. l896.The Rise and F a l l of Bacter ia in Cheadar Cheese. Wis. Agr. 3xo. S ta t ion , 13th Ann. a p t . pp. 93-111,Madison. 13. Savaee, W« S. 1906. Bacter ia l Examination of Water Supplies, Lewis, London, p . 2 1 3 . 16. Standard Methods, 1920. Amer. Public Health Assn. , Boston. 17. The Committee on je te rmina t ive Bacteriology of the Society of American B a c t e r i o l o g i s t s , 1923. Bergey's Manual of Determinative Bacter iology, Williams and Villeins Co., Bal t imore. 18. The Committee on Bac ter io log ica l Technic of the Society of American B a c t e r i o l o g i s t s . Manual of Methods for Pure Culture Study of Bac ter ia . Published by the Society, Geneva, II.Y. 19. Todd and Sadler, 1911. The Kingston Cheese. Jour . Board of Agr icu l tu re , Vol. XVIII, H o . 3 . , pp.193-203, Lonaon. 20. Winslow and Winslow, 1908. Systematic Relat ionships of the Coccaceae. Wiley and Sons, II.Y. -35- 2 1 . 7/inslow, Rothberg and Parsons , 1920, Notes on the C l a s s i f i c a t i o n of the White and Orange Staphylococci . Jour . Bact. V. 2 , pp . 145-167. P L A T E I Complete R e s u l t s of Q u a n t i t a t i v e A n a l y s e s . Dates on which cheesa were made. Aug.3/23 Aug.7/23 Aug.10/23 Media on which c o u n t s were made. Gluoose Agar L a c t o s e Agar Glucose G e l a t i n V.P. Broth MacConkey's Agar MacConkey's Broth Glucose Agar Lactose Agar Glucose G e l a t i n V.P .Broth MacConkey's Agar MacConkey's Brot h Glucose Agar Lac tose Agar Glucose G e l a t i n V.P .Broth MacConkey's Agar MacConkey's Broth Green Che Ho.of B a c t e r i a p e r gram. 2 ,175 ,000 ,000 2 ,272 ,000 ,000 3 ,153 ,000 ,000 Growth in 1- 1 ,500 ,000 ,000 2 , 8 0 3 , 0 0 0 , 0 0 0 4 , 5 3 0 , 0 0 0 , 0 0 0 Growth in 1 - 1 ,442 ,000 ,000 131 ,100 ,000 111 ,200 ,000 159,659,000 Growth in 1 -50 ,531 ,000 e s e Ho.of c o l i t y p e s p e r gram. 53 ,000 1,585,000 404,000 Ripe Cheese Jo . of b a c t e r i a p e r gram. 18 ,000 ,000 12 ,000 ,000 2 2 , 0 0 0 , 0 0 0 100 ,000 ,000 116 ,000 ,000 147 ,000 ,000 G 62 ,150 ,000 42 ,000 ,000 83 ,000,000 Growth in 1 - 68 ,846 ,000 Ho.of c o l i t ypes p e r gram No growth No growth 29,000 rowth in 1-95,000 43 ,000 G t h . 1 - 2 2 , 0 0 0 P L A T E ' I ' C O N T I N U E D . Dates on which one8se were made. Media on which coun t s were made. Green Cheese ifo. of b a c t e r i a p e r gram. Mo. of c o l l t y p e s p e r gram N o . o f b a c t e r i a p e r gram Ripe Cheese lo .o f co IT types p e r gram Aug. 15/23 Glucose Agar L a c t o s e Agar V.P.Broth MacConkey's Agar 96 ,700 ,000 61 ,700 ,000 Growth in 1 - 63 ,000,000 131,0 00 Growth in 1- 511.000 Oct . 3/2 3 Glucose Agar Milfe Agar Glucose G e l a t i n V.P .Bro th S t e r i l e Milk tfacConkey's Broth 110 ,970 ,000 92 ,475 ,000 102,750,000 Growth i n 1 - 5 1 , 3 8 0 , 0 0 0 Growth in 1- 82,000 55 ,561 ,000 46 ,893 ,000 64 ,239 ,000 Growth, i n 1 - 183 ,750 ,000 No growth i n 1-117. Oct . 10/23 Glucose Agar Milk Agar Glucose G e l a t i n 7 .P .Bro th S t e r i l e Milk 91 ,274 ,000 91 ,274 ,000 99 ,042 ,000 Growth in 1- 48 ,353 ,000 MacConkey's Agar MacConkeyis Broth 22,165 No.Growth 1 - 15,000 45 ,396 ,000 38 ,844 ,000 73 ,000 ,000 Growth in 1- 58 ,000 ,000 Growth in 1- 58 ,000 ,000 No growth 1- 186 F I A T E ' i ' c 0 N T I N U E D. Datea pn Media Pn which cheese which counts were made were made. Green Cheese . No. cf b a c t e r i a p e r gram No. of c o l i lNo. t y p e s p e r gram p e r gram Ripe Cheese . of b a c t w r i a No. of" c o l i " t y p e s p e r gram Oct.11/23 Glucose Agar Milk Agar Glucose Gelatin V.P.Broth. Sterile Milk MacConkey's Agar MacConkey's Broth 12 ,824 ,000 12 ,824,000 9 ,613,000 Growth in 1 - 200,000,000 Dot.18/23 Glucose Agar Milk Agar Glucose Gelatin V.P.Broth S t e r i l e Milk MacConkey's Broth Oct . 22/2 Glucose Agar Milk Agar Glucose G e l a t i n V.P.Broth 123,200,000 64 ,064 ,000 182,336,000 Growth in 1- 61 ,000 ,000 Growth in 1- 308 ,000 ,000 74 ,830,000 2 7 , l b 6 , 0 0 0 77 ,432 ,000 Growth i n 1- 14.000.0JO 236,000 No growtli in 1-64.000 No growth i n 1-64.000 61 ,000 ,000 36 ,337 ,000 87 ,132 ,000 Growth In 1 - 14 ,000 ,000 Growth i n 1 - 72 ,0u0 ,000 68,665.000 34,635,500 69,675,500 Growth in 1- 50 ,000 ,000 Growth In 1- 5 0 , 0 0 0 , 0 0 0 20 ,521 ,000 48 ,935 ,000 Growth in 1- 65,000,000 No growth i n 1-232 No grpwth i n 1-232 P L A T E'l' C 0 N T I H H I D , Dates on which cheese were made. Media on which counts »ere made Green Cheese No. of bacteria per gram. Ho. of coli types per gram p-g- Ripe Cheese TTo. of "bac te r ia p e r gram No. of coTT types per gram O c t . 2 9 / 2 3 Glucose Agar Glucose G e l a t i n 154,452,000 171 ,435 ,500 29 ,016 ,000 5 0 , 0 7 6 , 0 0 0 j e c . 28 /2 1 Glucose Agar 565 ,984 ,000 353,270,OOX) f 'Green c h e e s e ' r e f e r s to t he cheese not o l u e r than two days from t ime of making. f? ' i t ipe Cheese 1 r e f e r s t o t h e cheese ready f o r m a r k e t , e i g h t t o twelve days frcm t ime of making. • PLATE II. Results of Quantitative Analyses of ten Kingston Cheese Date on whioh cheese was made Dec.12th 1921 Aug. 3rd 1923 Aug. 7th 1923 Aug.10th 1923 Oot. 3rd 1923 Oot.lOth 1923 Oct.11th 1923 Oot.18th 1923 Oct.22nd 1923 Oct.29th 1923 CJounts made on Glucose Agar Number of Bacteria per gram of cheese. # Green Cheese 565,984,000 2,175,000,000 2,803,000,000 131,100,000 110,970,000 91,274,000 12,824,000 123,200,000 74,851,000 154,452,000 ## Ripe 3heese 353,270,000 18,000,000 100,000,000 62,150,000 55,561,000 45,396,000 61,000,000 68,665,000 20,521,000 29,016,000 #'Green Cheese' refers to the cheese not older than two days from time of making. ## 'Rip e Oheese' refers to the cheese ready for market, eight to twelve days from time of making. # PLATE III Morphological and Cultural Details of Organisms. 0) . •M c z 101 102 103 104 105 106 107 168 109 110 111 112 113 114 115 116 117 118 119 o o -1 u o Long a Short Rods Long & Short Hods Long & Short Rods Long & Short Rods Streptococci Streptococci Streptococci Streptococci Streptococci Spore-forming ROd Streptococci Streptococci Streptococci Streptococci Streptococci Short rods Short rods Short rods Short rods •p ) •p •H J M M u M I I I I I I I I I I I I I I T ! •H a) -p W u Ci> + + + + + + + + + - + + + + + - - - - id <D C'H •P (D 3 ; i i  .- i - - - - - - - - - - - - - - - - - - - a ra o o H + - +- + - +- +- + - +- + - + - + - +- +- +- +- + - + + + + + + ++ ) 03 a •p o +- +- +- +- + - + - + - + - + » +- +- +- +- +- +- + + ++ ++ ++ m o u o a + - +- +- +- -- — — — — +- -- «•«• — — — +- +- +- +- » O •P H 1 •21 + - + - + - + - + - + - +- + - + - + - + - + - + - + - •H O •H J tn + - — +- +- — -- — — — +- — — +- •-. +- +- H o o >» H C + - -- + - + - -- + - + - +- +- +- H o •H c 1 + - +- + - -- -- + - + - + - + - + - Milt •H O + + + + + + + + + - + + + + + + + + + to e - - - - - - - - - - - - - - - - - - - +» a rH c + + + + + + + + + - + + + + + + + + + i C j •PC CLfH - - - - - - - - - - - - - - - - - - - PLATE III 301TIHUEI) h C 2 3 120 121 122 123 124 125 126 127 128 129 130 131 132 133 135 136 137 138 139 140 141 ft o H O * t Short rods Streptoooooi Streptoooooi Streptoooooi Short rode Short rods Short rods Short rods Streptoooooi Streptoooooi Streptoooooi Streptoooooi Streptoooooi Mioroooool Streptooo ;oi Streptoooooi Streptoooooi - •H •3 i i i i T I I I 1^ I M M M I I I I Streptooooi'i I Long & Shorl Rods Streptoooooj Lonf 1 Shorl Hods M M M ! • • I - • + + - - - - + + + + + + + + + + + •f + 1 -> : ^ 1 1 •3- i - - - - - - - - - - - - - + - - - - - - J • + • + - + - +- + + ++ + + ++ + - + - + - + - + - +- + - + - +- +- •f- +- •- ) 3 3 + • + - + - + - ++ ++ ++ ++ + - + - + - + - + - - - + - + - + - + - + - + - • - > > 8 9 I 1 4 - -- -- -- — -- -- -- -- + - -- -- -- -- -- + - ™ — ' + - 4 4 •-+ •f-f •+ ++ +- + - +- +- + - +- +- +- -- +- -- ; D - t I + - + + + + ++ + + -- -- + - + - + - -- -- -- • - -- • f - ? •• + - + + + + + + + + -- -- + - -- + - -- •¥- -- + - r< O + - + • + + + + + + -- -- • f - + - + - -- -- •« -- -- +- 1 1 • • 4 + | + + + • • + • ' " • + • + • • • Milk • - - - - - - - • - - — - - H - - - - - - - > • • • • • + • • • •f + 4 •f - + • • ••• • • • I, i f • - - - - - - - - - m - - + - - • tt - - - PLATE II I SOFTimJED . rf © n 142 143 144 145 146 147 146 149 L50 L51 L52 L53 m. 155 156 157 118 159 160 161 162 • H O I 8 Strepto303G: Short Rods Short Rods Short Rods Long & Shor Rods Long & Short Rods Streptocoa3: Strepto30G3i Str©pto3033i Long & Shorl Rods Streptoooooi StreptoooG3J Streptoooooi StreptocoGoi StreptoGOGcj Streptooooo: Streptoooooj Streptooooo: Shoet Rods Streptooooo: Streptooooc: 4» 4» •H fit 1 .M M M M 1 M M . M M M M I I I I S s s s s I I B 3 n * - - - I P© 85 3 to- tals - - - - + + + + + + + + + + + + + + - + + - - - - - - - - - « - - - - . • 8 I 4- 44 44 44 4- 4- + - 4- 4- 4- 4- +- 4- 4- 4- 4- 4- ++ 4- 4- © O 3 +- ++ ++ ++ +- +- +- +- +- +- +- +- +- +- +- +- +- ++ +- +- © • | © 1 o 4» H 1 1 4- - — u+ • -- _- 4- 4- +- — — 4- 4- — =» • 4- 4- 4- 4- +4 -- 44 44 — 4- — 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- ++ V a H O H B *» •» ++ H O M • o am «k 44 + + 3 + + 1 44 4 4 4- 4- -- — — 4- 4- i _ , w » 4- 4- 4- +- 44 4- 4- 4- +- — — - — ++ -- <a» • ++ 44 44 4- 4- + - + - 4- -- • » +- 4- 4- + - ++ tlilk ' 5 + + F + 4 4 4 + 4 + 4 4 4 + + + 4 + 4 + + • I i - 1 4 • + - " - - - - - - — *» - - — 4 - - Q H O + -f • + 4 + + + + + + + + + + + 4 4 4 4 4 1 lia - - - - - - - - - - - - - - - - - - - M CD 03 CO ct 4 • •a ct- O 0 o o (J K + i + + i i + i i i i i + . + i P CD co ct *1 A ct- O a o Q Q h-1 + 1 + 1 + 1 1 1 1 + 1 + 1 P CD M CO c t A c t a a o o a i—i + 1 + + i i i i i + i + i CD O CO ct A ct O CJ o o a K + • + i + i i i i i + i + i CO c t (» >TS c t O O o Q CJ g + 1 + t + 1 1 1 + 1 1 1 1 1 + . + 1 1-1 CD CO ct • •9 ct a o 0 o Q —̂' + 1 + 1 + 1 1 1 + 1 1 1 1 1 1 + 1 + 1 p - J - 4 CO ct- A ct O CJ o O o g + 1 + 1 + 1 1 1 1 + 1 + 1 M - J CT> CO ct- • ct- O Q O CJ o HH + 1 •f 1 + 1 1 1 + 1 1 1 1 1 1 1 + 1 + 1 CO c t •1 A •3 ct O o o o M + 1 + 1 + 1 1 1 + 1 1 1 1 1 1 1 + 1 + 1 ilk CO c t A ct- u CJ o O O (-( + 1 + 1 + 1 1 1 + 1 1 1 1 1 1 1 + 1 + 1 M OJ CO c t • •i ct o CJ O Ci 0 HH + 1 + 1 1 1 1 1 1 1 1 + 1 + 1 CO c t • 1 • ct- o o o o Q K + i + i + i i i + + i i i i i + i + i M 1 3 CO c t H <* X) ct- O 0 o a o K + i + i + i i i + i i i i i • i + i + ' M O CO c t A •fl ct o a o Q o K + i + + i i i + i i • { i i + i + i CO ct 4 a ct a o o a K + i + i < i i + i i i i i i i + i + i H cr> CO c t A e t (J O a a o M + 1 + 1 + 1 1 1 1 1 + • + 1 a> ~3 CO c+ A •5 c+ O (J o Q Q M + 1 + 1 + 1 1 1 + 1 1 1 1 1 1 1 + 1 + 1 cr> CO ct- A ct- o CJ 0 o o M + 1 + 1 + 1 1 1 1 1 + 1 + 1 H CO c t A O o o o M + 1 + 1 + 1 1 1 + 1 1 1 1 1 1 + 1 + 1 H CO ct- H A « ct O o o o o l-H + 1 + 1 + 1 1 1 1 + 1 + 1 w CO ct- A •d ct- o Ci o o CJ l-H + 1 + 1 + 1 1 1 1 + 1 + 1 C u l t u r e JS umber T Morphology H a b i t a t Gram S t a i n G e l a t i n L i q u e f i e d Glucose Lac tose Sucrose i la l tOBB d a l i c i n G l y c e r o l M a n n i t o l A c i d Gas C l o t P e p t o n - i z i n g fe 1 ! 1-3 H l—I _ J — < H-l " c i C u l t u r e  N u m b e r .  184 185 186 187 L88 L89 L90 L91 L92 L93 L95 L96 L97 L98 L99 200 801 202 203 204 205 PLATE III o t-i o t o ft Strepto ^ocoi Streptococci Streptococci Streptoiooci Streptococci Streptococci Streptococci Streptococci Streptococci Streptococci Streptococci Streptococci Streptococci Streptococci Streptococci Streptococci Streptococci Streptococci Streptooooci Long & Short Hods Long & Short Hods 4» a} +» •H & ai W M H M M I I I I M M M I I I I M II :: M M r a •H CO +» to C5 i + + + + + + + + + + + + + + + + + + + + + **? rt<H p 0 3 H a* ©•H - - - - - - - - - - - - - - - - - - - - - CO O o a M O + - + - + - + - + - + - + - + - + - + - +- +- + - + - + - + - + - + - + - + - + - lONTINUEL <D CO o •p o 3 + - + - +- + - +- +- + - +- +- +- +- +- +- +- + - +- +- +- +- +- +- 09 OS o u o i — mm -- -- -- mm -- -- -- -- — -- -- — -- -- — -- + - + - CO o •p iH cS + - +- +- + - +- +- + - + - +- +- +- +- +- +- +- +- +- +- a O a to + - -- -- -- -- + - -- — -- — -- -- -- — -- — -- +- +- H O N © O >> 3 - - - - - - - - — - - — - - - - — — - - - - - - — - - + - + - H O H a — -- — — -- -- -- -- -- — -- -- -- mm -- -- — + - + - Milk "H O < + + + + + + + + + + + + + + + + + + + + + 1 - - - - - - - - - - - - - - - - - - - - - O M O + + + + + + + + + + + + + + + + + + + + + DM 1««H - - - - - - - - - - - - - - - - - - - - - -o w<S o P i ft3 CO co o e t g + • + 1 + 1 + 1 + 1 + 1 1 + 1 + . + 1 IS o 3 o P 8 ° CO co 3- 0 ct K + 1 + 1 + 1 + 1 + + 1 1 1 + 1 + 1 + 1 o o 3 UJC*J o P< 8° CO co o >-; ct m + i + + i + i + i + i + i + • + i + i m O 3 o P 8 ° to CO 3* O N c t K + ' + + i + 1 i + i i i + i + i + i H 03 o 3 o P>8° CO CO 3" o K + i + i + i + i + i + i + i + i + i + i O wn>) o P< 8° CO CO 3* o ct + 1 + 1 + 1 + 1 1 + 1 + 1 + 1 + 1 + 1 H o 3 o p*R° CO co B o c t + 1 + 1 + 1 + 1 + 1 + 1 + 1 + + . + 1 o 3 WHO O p , go CO 02 o ct g + 1 + 1 + 1 + 1 + + 1 + 1 + 1 + 1 + 1 to CO ct-I 3* «0 H o Q o a o •* H + ¥ + 1 1 1 + 1 1 1 + 1 H ro co c t » ct O o o o a K + i + + i i i i i i i i i i + i +• i to H CO c t H a •d c t O a o o o K + i + i i i i + i i i i i i + i + i ro o to CO c t *t a <B c t O Q o o o + 1 + 1 + 1 1 + 1 1 1 1 1 1 + 1 + 1 to o 00 CO 3" o •i c t M o p< CO t~* 1 1 + + + + + + + + + + + + + + + + + 1 ro a> -a CO 3* o c t w o p> 00 K i i + + + + + + + + + + + + + + + + i ro o CT> CO cr o c t W o p< CO M 1 1 + + + + + + + + + + + + + + - + + + 1 C u l t u r e number M o r p h o l o g y H a b i t a t Gram S t a i n G e l a t i n L i q u e f i e d G l u c o s e L a c t o s e s u c r o s e M a l t o s e S a l i c i n G l y c e r o l Mannitoi A c i d ' Gas C l o t ^ e p t d H - x z i n g . 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