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Further studies on the bacterial flora of the `Kingston cheese’ Kelly, Clifford Darton 1923-05-30

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of i& e iS^^fw. Qj]**fr  '-h OS? 0 ttilfp llmttpraitg of Irittafj (Eolumbia DEPARTMENT O F DAIRYIN G VANCOUVER, CANAD A May 5th,1924, John Ridington, Esq., Librarian, The University of British Columbia. .Dear Sir:-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 Clifford Darton Kelly,3.3.i. A Thesis submitted for the Jegree o: Master of doienoe in Agriculture aone in the department of dairying. THE UNIVERSITY OF BRITISH COLUMBIA April 1924. 4**80*1* 8 YTABLE O F CONTENTS . 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 other than Streptococcus lactis(Lister; 22 Observations. 26 Summary  8 References 33 Plates \ FURTHER 3TUJIE3 ON THE BACTERIA! FLORA OS THE »ZING3TQN GHEE3E.' C. D. KELLY, B. 3. A. DJTROQUCTICH. The process of manufacture of the 'Kingston Cheese* was worked out in England by Alec. Todd and Wilfrid 3adler (19) to meet the demand for a cheese of the hard pressed variety, a cheese not too large for the average family and one which couli be used with little or nc waste. ^Jhile this cheese is made on the hard pressed principle, the system and procedures employed are such as to give a cheese that will ripen in ten days. The ripe cheese is usually one pound in weight, has a flavour of its own and a soft granular texture, rich ana buttery. For the past three years this cheese his been manufactured in the laboratories of the University of British Columbia. In a Thesis presented by me in April 1923 (11) detailed refer ences were made to the system on which the cheese is nanu-ractured, and complete records of the cheese p.ade during a -2-large part of the year were presented. The Thesis included, moreover, the results of bacteriological examinations of certain of the cheese referred to immediately above. As far as the bacteriological examinations were concerned, the re sults were very incomplete, yet the technique worked ouJ at that time, and the data obtained, have served as an invaluable preliminary to carrying on the work reported herein. The work was so planned that quantitative bacteriological analyses were to be made of a cheese when one day old, and of a cheese of the sane day's make when ready for market, also the organisms occurring with the great est frequency were to be isolated from the plates of each cheese examined. It has been anticipated that the data thus secured might lead to some information being obtained on the risening processes of this cheese. BACT2HICI0GICAL SALII!: a~ICTS OF 7R 3 '2I1IG3T 71 CII32S3 MEDIA 2KPLGY3D. Beef-peptono-agar, Jtandara Methods (l6> Glucose-agar 0.5 per cent glucose added to beef-peptone-agar. Lactose-agar 0.5 per cent lactose added to beef-peptone-agar. Milk- agar, Ayers and Mudge(l) -3-MacConkeyTs neutral-red-bile-salt-lactose-agar (15) for specific reactions of the organisms which ferment lactose to acid and gas. MacConkeyTs neutral-red-bile-salt-lactose-broth (15J for detecting the presence of the organisms which ferment lactose to acid and gas. Neutrient-agar to which specific carbohydrates were added as desired, using brom-crssol-purple as indicator.(4)(l8). Heutrient-broth to which specific carbohydrates v-ere added as desired, using brom-thymol-blue as indicator.(l6). Peptone-medium for Methyl ftel and Toges-Proskuuer de terminations. Standard Methods (l(>  ). Peptone-medium for Indol Production. Standard. Methods (l£ /. Hit rate-agar. Manual of Methods (18). Glucoee-agar and glucose-gelatin were used through out for the quantitative plate counts. For comparative purposes lactose-agar and milk-agarf1) were used in addition to the two media mentioned above, ""he dilution method of quantitative analyses was employed when using litmus-milk and the Yoges-Oroskauor broth. Reaction of Media. All media, excepting bile-salt media, were adjusted according to hydrogen-ion concentration. nhe final reaction was p.B. - 7 using brom-thymol-blue as indicator(l6) EXAMIHATiai AID T3CHNIiU3. The same technique was used for the -4-bacteriological examinations of the cheese as was worked out during the previous studies on the 'Kingston Cheese.'(11) On arrival at the laboratory the one pound cheese was cut in quarters and the necessary precautions being observed, six samples were taken throughout the cheese, the whole approximating as near as possible one gram. The material was placed in a small weighed aluminium dish with a tight fitting cover. The dish was again weighed, and the weight of the cheese determined. The sample was then ground with sand in a sterile glass mortar,and with the aid of a glass rod fitted at one end with a rubber policeman, was washed into a large-mouthed bottle using a known quantity of sterile water. As before (11) an electrically-driven hor izontal shaker was used for the mixing of the dilution. The mixing occupied five minutes, and to provide against organ isms being carried down by sand, shaking by hand was done for another five minutes. The higher dilutions were then made in water blanks, and it was found that dilutions of l-j?0Q,000 and 1-Z, 000,000 gave in practice the most sat isfactory plates. Throughout the work the greatest care was taken to keeo the cheese free from contamination while in the laboratory. All knives with which the cheese was cut were first placed in alcohol and then flamed, again placed in alcohol and again flamed each time before using. The dishes, mortars and pestles were wrapped in paper and steril ized with dry heat for at least two hours at 170°C. or for thirty minutes at 15 pounds pressure in the autoclave, "est tubes were filled with sand plugged with cotton wool and autoclaved. The large-mouthed bottles were plugged with cotton wool and sterilized, while the rubber stoppers for the bottles, arid the glass red were sterilized in water for at least one hour at 15 pounds pressure in the autoclave. The sterile water blanks were prepared in ground glass stoppered bottles, the tops being covered with cot4on wool. The grinding of the cheese and the washing of it into the large-mouthed bottle were done in a sterile glass case, the door of which was raised just enough to admit the hand3. The case was previously washed with a 1:1,000 solution of mercuric bi-choloride. Hands and arms were washed with soap and water and rinsed in lysol. COMPARATIVE VALUES Of CERTAIN MEDIA. In the work on the ' Kingston Cheese* report ed in 1^21  (11) the Indications were that glucose agar would give better results than lactose agar. At that time there was not sufficient data on which to form any definite conclusions. During the work herein recorded a comparison was made of glucose agar with lactose agar; and, later of -b-glucose agar with the milk agar of Ayers and Mudge (1). Glucose agar proved to he the most satisfactory mod' m (See Table I.) Glucose gelatin plates were also made, and as a rule, higher counts ??ere obtained than when usi glucose agar. On hot days, however, it was very difficult to keep the gelatin from melting. As is stated on page 3, the dilution method was employed when using litmus milk and the 7oges-Proskauer broth respectively. In many in stances growth in higher dilutions was observed in the lit mus milk than was the case with the Voges-Proskauer medium. QUANTITATIVE BACTERIOLOGICAL ANALYSES. All quantitative examinations were made in triplicate and the results given below and in Plates I and II are averages of these three counts. The gelatin plates were incubated for five days at room temperature. The bile-salt broth tubes and bile-salt-agar plates wore incubated for forty-eight hours at 57.3°C. All other media were incubated for five days at 22°C. In order to form some idea of the percentage of acid-formers present on the plates, litmus and brom-cresol-purple were used as indicators in a number of giucose-agar-plates. A3 was experienced before (11) in many instances the indicator in the whole of the medium was chai gel. On plates where the acid-formers could -7 be differentiated, the number in the majority of cases was a hundred per cent of the count and the proportion was not below ninety-seven per cent in any examination. The quantitative analyses were made of a specific cheese of a certain day's'make' at the time when the cheese was about twenty-four hours old. Another cheese of the same '-make' was examined when ripe, about ten days later, hence, as cheese made on ten different days were examined., the determinations on a total of twenty cheese have been recorded. As has been exolained in a previous report (11; it was not possible to examine each cheese more than once, because being so small it had to be cut up com pletely. As yet,no attempt has been made to determine the variations in numbers of bacteria present 5n individual cheeses on the same daya' 'make.' The work on Cheddar cheese, however, by Harrison and Conne]l(7> has shown that the variations in bacterial count of different parts o°  the same cheese were not greater than thirty per cent. In every ca3e, excepting the cheese made October the eleventh, a considerable reduction was shown in the bacterial count of the ripe 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 different cheese examined. One cheese a day old gave a count as high as 4,000,000,000 micro-organisms per gram; and another, also a day old, had a count as low as 9,000,000 micro-organisms per gram. The counts in the ripe cheese varied from 147,000,000 per gram to 12,000,000 per <• ram. Though there wa3 this great difference in the number of organisms found in the different cheeses, there was no great difference in the quality of the cheese as judged for market. More detailed results will be found on Plates I and II. Shere was a greater number of bacteria present in the ripe cheese made October the eleventh than in the green cheese of the same date. In s )ite of this apoarent discrepancy the number of bacteria oresent in the cheese eleven days old was very close to the average of th? counts of all the ripe cheeses. The starter U3ed for this cheese waa not very vigorous, when the cheese went to ire3s the acidity was quite low and the green cheese when examined in the laboratory was not typical; or as the cheese-maker would say, was on the 'sweet side.' After eleven days.however, the flavour was quite typical though the"texture was a little soft. One example is not enough on which to base any con clusions, but it would seem to indicate that even though the cheese was vatted when quite sweet the bacteria continued to multiply; and that with this increase, there was an in crease in acidity till at eleven lays the cheese was normal. fhie increase in acidity as the bacteria continued to multiply -9-18 in accord with the findings of Baker et al (2). As in the examinations of tv;c rears ago there has been no atter, "t to make anaerobic examinations of the cheese. It is fully realized that this par* of the work should be undertaken; but it was not possible simultan eously to do both aerobic ana anaerobic examinations, and it was decided for the time being to concentrate on work under aerobic conditions. It is to be desired that an en quiry into the anaerobic flora of the cheese shall be in stituted. An effort was made to conduct histological ex aminations of the cheese in order to get some i iea of the grouping of the organisms; and to secure counts of the bacteria by the microscopic method as recommended by Hucker (9)» Thus far, time has not permitted the doing of other than preliminary work on this phase of the investigation. At a later date it is to be desired that the examination of histological speoimens shall also receive eonsiiera4 ion. QUALITATIVE 3AC?3HI ^LOGICAL AITAIY333. Colonies which ap?eare.l to occur with the greatest frequency were taken off the plates and retained in pure culture. A large percentage of these colonies were similar to the small sub-surface and surface colonies-Group 2- reported in the studies undertaken in l rjll (11 J. Great difficulty was found at that time (11) in keeping the -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 -11-recommended by Conn and Hucker (4> and given in the 'Manual of Methods for Pure Culture otudy of Bacteria' (l8j were used for the determination of acid and gas in all the carbo hydrate media employed. It has been noted above that for these de terminations one drop, from a finely-bored pipette, of the Voges-Proskauer glucose solution was used for the inoculation in each case. The possibility of the infinitesimal amount of glucose present in the drop of culture being sufficient to show acidity in the media other than glucose suggested itself. To check this, ten tubes of nutrient agar slants containing indicator but no sugar were inoculated at the same time as the other media. The control tubes remained neutral. Ten tubes of nutrient-agar were not considered as sufficient check, but the fact that a large percentage of the cultures did-not produce acid on lactose-agar was considered as conclusive proof that the minute quantity of glucose present in the drop of inoculating material was not sufficient to give an acid reaction to the media inocul ated. One hundred and seventeen organisms were isolated from plates made of the cheese, and five organisms from plates made of the starter used in the making of the cheese. Cultural and morphological details of these -12-organisms will be found on Plate III. .Vhen the reactions of the cultures to maltose, salicin, glycerol and mannitol were determined, it was found that twenty-three organisms had died. The formation already obtained about them, how ever, was enough to show to which main group they belonged. The one hundred and seventeen organisms divide themselves into five main groups. GROUP I. In this group are found organisms isolated from pin-head colonies growing more particularly under the surface of the media. Gram positive, spherical, occurring in chains, ones and twos and in clumps, Each strain ferments glucose and lactose to acid, produces acid and clot in litmus milk and fails to liquefy gelatin, deventy-nine strains find themselves in this group. V/ithin the group, however, specifio strains vary in the size of the cells and in the action on certain of the carbohydrates other than glucose and lactose. A complete record of the charac teristics of each of the seventy-nine strains is presented on Plate III. On Plate IV the variations in size of cell and in the action on the carbohydrates other than glucose and lactose are recognized and sub-groups are established. Of the seventy-nine strains thus placed in sub-groups, Cultures 115, 121, 212, 136, 135, 172,156 and 152 prove to -13-be representative of the number of strains respectively, as shown in the second column of the plate. Cultures 156 and 152 representing five and two strains respectively, agree in the essential characteristics with the desoription of streptococcus lactls(Lister; according to Bergey's Manual of Jeterminative Bacteriology {H)»  strains repre sented by Cultures 115, 121, 212, 136, 135 and 172, however, oannot be classified as typical forms of jtreptocoocus lactls (Lister; because of the failure to act on salicin, on mannitol, or on both fl7>« It may be observed, Plate III, that of the strains isolated from starter, those included in this group, ferment salicin and mannitol to acid and are apparently identical with Culture 156. It will be noted on Plate III that only three strains coming in this group and isolated from cheese, ferment salicin and mannitol to acia, while three r:iore strains ferment salicin only. It 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 salicin and mannitol, thred strains had lost the power of fermenting mannitol only, while three strains had retained the power of fermenting both these oarbohydrates. It would seem that with forms of itreptococcus lactis(, Lister; attenuation takes place in passing through cheese. Support for this suggestion is found in the work -14-of Iloyd on cheddar cheese(12j. i^'rom well-matured cheese he isolated strains of true lactic acid producing organisms which he defined as Bacillus acidi lactici — now considered as Streptococcus lactis (Lister J {n){lZj --  which failed to clot milk. Further, of the organisms isolated from cheese - dee Plate III - three only of the strains included in this group ferment salicin and mannitol to acia, while three of the strains ferment salicin to acid but fail to act on mannitol. The remainder of those cultures isolated from cheese and included in this group have no action on either salicin or mannitol. Of the organisms comprising Group I, every strain is found to be identical with one or more of the strains isolated by Hucker from cheddar cheese (10;. jiach of his strains is classified as Streptococcus lactis(ListerXlQ)« In studying the organisms placed here in ©roup I, the reactions to salicin, mannitol and maltose, have been determined in addition to the reaction recorded by Hucker for his strains. Consequently, while Hucker (10) classified his organisms as Streptococcus lactis(Lister) the additional reactions recorded for the Cultures - Group I -of this report permit of a more specific classification, following Bergey (17j. Accordingly, Cultures 152 and 156 are placed as typical forms of Streptococcus lactis(Lister) (17;, after Bergey; and Cultures 115, 121, 212, 156, 155, and 172, on account of their failure to ferment mannitol or -15-salicin, or mannitol and salicin, are placed as attenuated forms of Streptococcus lactis(Lister)(17)» GROUP II. Gram positive rod-shaped acid formers. Nineteen cultures represented in Plate IV by Cultures 101, 102, 103, 104, Ml2 and M9 are Gram positive non-spore bearing rods with round ends, varying in length from lu to lOu long, the majority approximating the minimum length rather than the maximum. There was a tendency to form short chains in the broth as used for the Voges-Proskauer test and to a somewhat lesser extent on agar. Good growth took place both at 22°C. and at 40°C. All the cultures, with the exception of Cultures 204 and 205 were picked off agar and gelatin plates incubated at 22°C» for live days, while cultures 204 and 205 were taken off plates incubated at 40°C» The strains do not liquefy gelatin but form a clean acid clot in litmus-milk in from fourteen to eighteen days at 40°C. followed by bleaching. Gas is not formed in any of the carbohydrates and glucose, lactose, sucrose, salicin and'raffinose are fermented to acid. These cultures of Group II seem to bear a marked resemblance to 0rla-JensenTs Streptobacterium (15) in that they are Gram positive rods tending to form chains, growing at 22° 40°C. and -16-fermenting salioin to acid. Smears made from nutrient aga r slants showed a large number of short rods which might be mistaken almost for diplococci with a  number of rods about lOu long. These smears were almost iientical in appearance with Orla-Jensen'8 Streptobaoterium plantarium number 18, Agar streak 1 day at 30° plate XLIl(13). According to Bergey's Manual (17) the cultural and morphological characteristics of the organisms here under discussion would place them in the Genus Lactobacillus(BeiJerinckj (17). It was impossible to isolate organisms of this type from the freshly made cheese though several attempts were made. All the cultures mentioned above were from cheese eight to ten days old. It has been known that Lactobacilli are found in mature cheddar cheese and it is interesting to know, that in a cheese which ripens so quickly, Lactobacilli are present in quite considerable numbers. Cultures 101 ana 1,19 do not ferment maltose, but ferment mannitol to acid. The cultural and morphological characteristics of these two organisms would seem to warrant the classifying of them as of the Lactobacillus bulgaricua (Grigoroff Jtype(17) and as of Rahe's type "D" (17). Cultures 102 ana 104 ferment maltose, mannitol, raffinose, dextrin, arabinose and trehalose to acid, trehalose being fermented slowly. The cultural characteristics of these organisms, and particularly their -17-aotlao •• tb a oarbotgrdrataa , woal d r*v-ir « tha t tba ? » • olaaslflad a s La o tobaolllao 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 whila tb a lanft h o f tb a oall a rooordo d fo r Laotobaolllaa oao—rl a l a fro s 1.5 a t o 2a. nt h tb a asoaptloo o f tb a raaotlo n t o trahaloaa , th a worphol->gloa l and ooltaral 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 Laotobaolllaa plantarl . olo t silk , vbll a Laotoba o 11 laa ottoonorla doo a not. Th a nation o f Laotoba o 11 laa oaouaarl a an daxtrlfl ! • aao h alowa r a a oonparo d with lt a raaotlo n to trabalooa , whll a wit h attain s 10 2 an d 104, th a min 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 alantarl. I t l a obrlooa , tharafora , conaldarln g th a obaraatarlatloo o f Laotobaollla a oaoanarl a an d Laotobaollla a alantarl raapaotlral/ , 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 atralna accord -ing to tb a ralatlra iaportano a whlo h I t t o b a attaobad t o tba farna n tatlon o f trahaloa a o n th a on a hand, an d to th a alia o f oall a an d th a olottin g o f tsll k o n th a othe r hand . OB tba whola followin g Bargay , th a wldano a aubaltta d ap*>aar o to b a la faroo r o f Laotobaollla a oaoanarla : an d Cultara a lOt an d 10 4 ar a olaaaifla d a a t/pa a o f Laotobaollla a caooamrl a -18-(Eenneberg) after Bergey (17J. Culture 103 does not ferment maltose and mannitol. The action on the carbohydrates would place Culture 103 as either Lactobacillus caucasicus (gem J (17) or Lactobacillus boas-oppleri(Boas and 0ppler)(17>. As neither Lactobacillus caucasicus (17) nor Lactobacillus boas-oppleri will grow on gelatin at 22°C» and as Culture 103 was isolated from a gelatin plate incubated at room temperature, it cannot be classified either as Lactobacillus oaacasicus or Lactobacillus boas-oppleri, but is placeo. within the type species Lactobacillus caucasicus(Kernj(17)« Culture M12 ferments maltose, mannitol and raffinose to acid. The ability of this organism to ferment raffinoBe without fermenting dextrin does not permit of it being classified by BergeyTs Determinative 3acteriology(17) other than as being a member of the Genus Lactobacillus and coming within the type species Lactobacillus caucasicus UernJ(17;. G B 0 U P III. Gram negative non-spore forming rods, which ferment lactose to acio. and gas. There are sixteen organisms in this group represented on Plate 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 nitrates to nitrites, but fail to liquefy gelatin. The characteristics noted above, and the negative reaction to the Voges-Proskauer test would place these strains in the Genus geoherlohla(17j Of the organisms in this group, Culture 119 is a non-motile rod which ferments sucrose, salicin, maltose, glycerol, dulcitol and nannitol to acid; proauces sliminess on agar, in peptone broth and in milk, but fails to produce either indol or acetyl-methyl-carbinol. V/hen first isolated this strain fermented sucrose to acid and gas, but after being kept on artificial media for same months lost the faculty of producing gas on this carbohydrate. In its ability to produce sliminess on certain media, to ferment glucose and lactose to acid and gas and its inability to produce irdol, 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 this strain ferments them to acid only. Bacterium aerogenes (Sscherich) becomes Aerobacter aerogenesCBscherichj in Bergey's Determinative Bacteriology (17). Following Bergey (17J Culture 119 cannot be classified as Aerobacter aerogenes, for it fails to produce acetyl-methyl-carbinol, but based on the sum of the characteristics recorded the strain finds itself within the Genus Escherichia. Of the species in this -20-Genus included in Determinative Bacteriology (17) it appears that Escherichia astheniae presents the features with which the culture under discussion most closely aligns itself. This an culture,th eref ore, is here considered as being/a-typical form of Escherichia astheniae (Dawson)(17). Culture 124 is a non-motile rod, ferments glucose and lactose to acid and gas, but fails to act on sucrose and salicin. Bat for the fact that Culture 124 is non-motile, it could be classified as Escherichia paragrunt-hali( Castellani and Chalmers) (17) • Tlai8 lack of motility, however, prohibits it being classified as a true form of Escherichia paragrunthali and it is classified as of the type Escherichia paragrunthali (Castellani and Chalmers) (17). Culture 143 is a strain of notile rods which forms indol, ferments salicin and dulcitol to acid and gas, but fails to ferment sucrose. The ability of this strain to reduce nitrates to nitrites, to ferment glucose, lactose, salicin and aulcitol to acia ana gas, in the inability 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 ability of the culture to ferment sucrose and lactose to -21-acid and gas, the inability to produce indol or give the Voges-Proskauer reaction, and its failure to show motility would suggest that it be placed as Escherichia astheniae (JawsonJ(17)• Escherichia astheniae, however, has no action on salicin, while Culture 206,as noted above, ferments this carbohydrate to acid and gas. This difference in action on salicin would not permit the classifying of this strain as a typical form of Escherichia astheniae and it an is classified here as/a-typical form of Escherichia astheniae ((J3awson;(19J. GROUP IV. Spore Bearing Rods. Only one spore bearing organism,Culture 110, was found in this investigation. The strain is a Gram negative motile rod 2u to 4u in length, the cells ocourring singly ana in pairs. The spores are terminal and the rods are swollen at sporulation giving the cell the appearance of a tadpole. The growth on agar is pal e yellowish white after forty-eight hours, gelatin is not liquefied and acid is formed in milk but no clot is produced. Glucose, lactose, and sucrose are fermented to acia. The cultural and morphological characteristics of Culture 110 appear to be identical with those recorded for Bacillus pseudotetanicus (Kruse)(17) with the exception of the action on the -22-cartohydrates; Bacillus oseuaotetanicus showin g no actio n on the carbohydrates. Culture 11 0 bear s a  marke d resemblanc e also to Bacillus ci rculans( Jordanj( 17) but th e tw o culture s do not agree in the reaction to th e Gra m stain. I t i a suggested that in SMite of the difference noted wit h respect to the action to the Gram stain, Culture 11 0 shall be considered as being of the type Bacillus circulana( Jordan) (17 j. GROUP V. Coccus ?orms. Culture 1.53 is a minute Gram positive coccus about Q.3u. in diameter, which liquefies gelatin, ferments glucose, maltose, salicin and mannitol to acid, but has no action on lactose or sucrose. Bile strain bears a marked resemblance to Micrococcus lactis varians of Conn, 3sten and Jtocking(5). These investigators found that some forms of Micrococcus lactis varians did not ierment lactose and sucrose, characteristics applying equally to Culture 133. Comoaring the el aracteristics as a whole, the strain agrees even :rore closely with Micrococcus lactis varian^Type A)  of Conn, 3sten and stocking (4) both a s to the formation of acid in glucose, and in the curdling ana digesting of milk without the formation of acid. Th e resemblance, however, of the strain to Micrococcus -"•arians (Dyarj Conn(17) is not so pronouroad; yet Micrococcus varians -23-(Dyarj Con n it considere d I n Bergey' a Jeterminativ e Bacteriology a s bein g synonymou * with Mlcrooocon a lactl s yarlane o f Uonn , Sste n an d .stocking . Hence , i t woul a see m that Cultur e 13 3 Is t o b e classified a s o f th e typ e Mlorocooons varian s (Dyar j Con n (17J . Culture 14 8 is a  strai n o f Gra n positiv e coooi growin g i n clumps . I t fail s t o liquef y gelatin , form s a clean aci d clo t i n milk , reduce s nitrate s t o nitrite s and ferment s glucose , lactos e an d sucros e t o acid . H o aotion on maltose, salici n o r inuli n ha s bee n noted . Th e growt h is goo d o n artiflcal medi a an d th e pigmen t o n agar afte r fourteen days , aocordin g t o th e 7/inslows ' chart(20J , l a Ligh t Cadmium Yellow, Chro m III, designate d b y thes e worker s a s 'white.' Th e characteristics recorde d woul a sugges t thu t the strai n b e placed i n th e genu s 3taphylooooous ( 17)• T o take th e classificatio n t o a  more specific stage , th e failur e of th e organis m to liquef y gelati n an d th e abilit y wit h whioh lactos e i s fermente d indioat e a  close resemblanc e o f the strai n t o Staphylococcu s tetragenus(Koch-Oaffky )(17)• The identity o f th e tw o strain s canno t b e accepte d withou t qualification fo r cultur e 14 8 reduce s nitrates t o nitrites , and unde r th e microscop e th e cell s appea r i n clumps ; v.-hll s Staphylococcus tetragenu s (17 J doe s no t reduc e nitrate s and th e cell s appea r i n group s o f four . Winslo w an d /inslo w (20j stat e tha t Albococcu s tetragenu s (Gaffky ) - - albocoocu s -24-being a genus since absorbed in the genus Staphylococcus-is closely related to Albococcus eandidus (Conn) and in describing Albococcus canaidus, Winslow et al (21) make the following statement "The second type in abundance in our "study, and the type found most commonly on the skin after "St.epidermidis by Gordon, Is the form which ferments "lactose but fails to liquefy gelatin, identified by YJinslows "as Albococcus eandidus and now to be called Staphylococcus "eandidus. Our strain, however, reauced nitrates and gener ally clotted milk which Gordon's type did not. Three strains "sent to the museum collection as Micrococcus tetragenus "all belonged to this group. Hone of them reduced nitrates "and results are variable in milk and in r?gard to ammonia "production." The strain recorded here agrees with the a:ove description of Staphylococcus eandidus with respect to the clotting of milk ana the reduction of nitrates to nitrites. In Table 10 of the same report(21) Winslow et al show twenty of their strains as fermenting maltose. Culture 148 fails to ferment this sugar ':ut in all other respects it appears to be identical with their cultures ascited. The organism originally described by Conn was placed by him in the Genus Micrococcus as Micrococcus candidus(20). Later organisms showing similar characteristics to Cohn's Mierococcus were placed by the /inslows (20) within the type -25-candidua (CohnJ(21). The eommittee of the society of American Bacteriologists in Bergey's determinative Bacter iology (17 j have again placed organisms of this type in the Genus Micrococcus, as Micrococcus candidus (Cohnj(17). Though Culture 148 differs from Micrococcus candidus(Gohn) (17) in the action to nitrates,in the main the character istics are identical and Culture 148 is classified here as of the type Micrococcus candidus(Cohnj(17j« Culture 214 is a Gram positive coccus, occurring in irregular groups. The strain liquefies gelatin stratiform, produces a white pigment on agar, ferments glucose and sucrose to acid, but no action on lactose can he noted. This culture died before it could be investigated further. According to the cultural and morphological features determined, however, Culture 214 would be placed in the Genua dtaphylococcus, and of the type species Staphylococcus aureus(;xosenbacbJ(l7)« The inability of the strain to ferment lactose, does not "oermit of it being classified more specifically. V/inslow, Hothberg and Parsons in the 7Tr.ite and Urange staphylococci(21J show ten cultures in their Table 8, which are white pigment formers, liquefy ing gelatin, but failing to ferment lactose. Later the same workers say (21J "The lactose-negative gelatin-positive "type of white pigment producers appears in our study, "as in that of Gordon, to be a rarer one, and this form, -26-"as well as the forms which exhibit miscellaneous fermentative "reactions may best be left for the oresent without specific "names." It is felt that Culture 214 should be included in this group, Culture 214, is, therefore, left for the present as of the type species itaphylococcus aureus (Eosenbachj(17) OBSERVATIONS. The technique evolved when engaged on the work in 1922 (11) has been adopted throughout the present in vestigation with pronounced satisfaction. Further, the present paper confirms the previous findings (11J that for the determinations of the bacterial flora of 'Kingston Cheese' glucose-agar ana glucose-gelatin are the most satisfactory meaia. The total number of organisms ^resent in the 'Kingston Cheesef both in the cheese when newly made and the cheese when ten days old, is founo. to approximate very closely the numbers recorded in the work on Cheddar Cheese. Though the total number of organisms present in the 'Kingston Cheese' ten days old is not constant, and may vary, as this investigation shows from 20,000,000 to 350,0J0,0J0 bacteria per gram, the cheese examined in each case were normal both as to texture and flavor. Though no definite effort was made to determine the percentage of acia formers, the results obtained showed that at least ninety-seven per cent of the flora were acid formers. -27-This paoer would seem to indicate that the organisms which occur with the greatest frequency in the cheese when one day old are those of the streptococcus lactis(Lister) type, and in cheese of the same day's 'make' the when ten days ola are those of/Streptococcus lactis(Listerj type and those of the Lactobacillus(Beijerinckj type. It appears to be well established that the successful ripening of cheddar cheese is very largely deoenaent upon the bacterial flora determined according to the recorded literature. The work herein presented, quite definitely shows that the flora of the '.Kingston Cheese' is very similar, if not identical with the flora recorded for cheddar cheese. let in the case of the 'Kingston Cheese' we have a cheese which is mature in ten days after making, while a cheddar cheese requires from three to six months to arrive at maturity. It would seem, therefore, that in contem-plating the factors determining the successful ripening or maturing of the 'Kingston 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-SUMMARY. A brief resume is given of the system adopted in the making of the 'Kingston Cheese' and the specific characteristics of the cheese are noted. Bacteriological analyses have been made of twenty cheese manufactured on ten different days, ten cheese when one day old and ten cheese of the same day's 'make' when mature at ten days after making. The results of the analyses made of these twenty cheese are recorded on Plates I and II. A list of the media employed is recorded and a description of the nethod of examination of the cheese is given. Several varieties of media were used with the object of determining which were best adapted for the de termination of the bacterial analyses of the 'Kingston Cheese'f glucose-agar and glucose-gelatin proving to be most satis factory. The results of the comparison of these media are given On Plate I. One hundred and seventeen organisms have been isolated from plates made of the cheese and five from plates made of the starter used in the making of the cheese. As far as possible the organisms were those wnich appeared to occur with the greatest degree of frequency. Though eighteen of the one hundred and twenty-two organisms isolated -29-were of the Genus Escherichia this does not necessarily represent the percentage of organisms of that Genus present in the cheese. The strains were taken from MaoConkey'a broth tubes and MacConkey's agar plates in order to deter mine the species of gas formers occurring with the greatest frequency. The morphology, cultural features and physiological reactions of organisms isolated from 'Kingston Cheese' are given on Plates III and 17. The one hundred and twenty-two organisms isolated are placed in five main groups, and are classified according to Bergey's Determinative Bacteriology (17). G B 0 U P I. Streptococcus lactis(Lister)types. Seventy-nine of the one hundred ana seventeen cultures recorded in this paper find themselves in this group, and are classified as follows. Culture 1^6 ana 152 representing in all seven strains are classified as Streptococcus lactis(ListerQ (17) Cultures 115, 121, 212, 136, 135, and 172, representing- seventy-two strains are placed as attenuated forms of Streptococcus lactis (Lister)(17) -30-GROUP II Lactobacillus (Baijerinck) types (17) Twenty-three strains are placed in this main eroup and are classified as to species. Cultures 101 and M9 representing four cultures are classified as Lactobacillus bulgaricus(Grigoroff) (17J and of the type "D" (Rahe) (17). Cultures 132 and 104 representing 15 strains are placed as a-typical forms of Lactobacillus cucuraeris (Henneberg) (17). Cultures 103 anu M12 representing two strains each are classified as of the type species Lacto bacillus cauca3icus( Zern)(l7). GROUP III Gram negative,Lactose lamenting Rods. In all there are sixteen strains in this Group ret>resented by four cultures. Sight strains represented by Cultures 119 and 206 are classified as a-typical forms of Sscherichia astbeniae (Dawson) (17). Three strains represented by Culture 124 are considered as a-typical forms of Escherichia -31-paragrunthali (Castellani and Chalmers)!17). Five strains represented by Culture 143 are classified as Escherichia coli(ascherich) Castellani and Chalmers(17) GROUP IV. Siore Bearing Roas. Only one Culture of soore-forninp- rous was found in this investigation. Culture 110 is placed as an a-typical form of Bacillus circulans(Jordan,)(17) UOUP v. Coccus forms other than Streatococcus lactis(Lister). Three cultures find then3elve3 in this group,two of which are Micrococci and one a Staphylococcua. One strain,Culture 133, is classified as of the type Micrococcus variansCDyaryConnCl?;» One strain, Culture 214, is placed as of the type species Staphylococcus aureus(Rosenbach)( 17) • One strain, Culture 148, is considered to be within the type species Micrococcus canaidua( Cohnj(l7). Observations on the data presented are offered. -32-ACKH0WLBDGMEHT3. I wish to thank Professor Wilfrid Sadler, at whose suggestion this work was commenced, with whom I have consulted from time to time, and who has kindly read over the manuscript. This investigation has been made possible by laboratory facilities, which have been placea at my disposal by the University of British Columbia. -33-H S I E H E H C E 3. 1. Ayers and Mudge, 1920, Milk Powder Agar, Jour. of Bact. V. 6, pp. 363-^88, Baltimore. 2. Baker, Brew and Conn, 1919, delation Between Lactic Acid Production and Bacterial Growth in +he Souring of Milk. N.Y. Agr. Exo.Sta. Tech. Bull. Ho.74, Geneva. 3. Buchanan and Hammar, 1913. Slimy and Ropy Milk. Agr. Exp.Sta. Iowa State College of Agr. and Mech.Arts. Research Bull. No.22, pp. 267-270, Ames. 4. Conn and Hucker, 1921. The Use of Agar Slants in Detecting Fermentation . IT.Y.Agr.Exo .Sta.Bull. No. 84, Geneva. 5. Conn, Eaten and Stocking, 1906. Classification of Dairy Bacteria, Report Storrs(Conn^ Agr.Exo.Station. 6. Final Rpt. of the Committee of Socy.toier. Bact. on Characterization and Classification of Bacterial Types, 1920. Jour. Bacteriology V.3, pp. 191-220. 7. Harrison and Connell, 1904. A Comparison of the Bacterial Content of Cheese cured at different Temperatures. Centrall. fUr Bakt .II ,XI, Bd.po.637-637 , Jena. 8. Harrison and Vanderleck, 1908. Aesculin Bile Salt Agar for Water and Milk Analysis. Trans.Roy.Socy. Can.III. Series II. pp. 103-110, Ottawa. 9. Kucker, G. J. 1921. The Microscopic Stuoy of Bacteria in Cheese. N.Y. Agr. Exp.Sta. Tech. Bull .ITo. 87 .Geneva. 10. Hucker, G.J. 1922. The Types of Bacteria Found in Commercial Cheddar Cheese. N.Y.Agr.Exo.Sta.3ull. No.90. Geneva. -34-11. Kelly, C. D. 1922. Further Studies on the '.Kingston Cheese,' with special Reference to the Bacterial Flora of the Cheese. Thesis for degree of 3.S.A. library, University of British Columbia, Vancouver, B.C. 12. Lloyd, F.J. 1906. Investigations into the Cause of Flavor in Jairy Products. Jour. Bath and West and Southern Counties 3ocy. Vol. XVI, Fourth Series, op. 1-26, Bath. 13. Jensen-Orla, 3. 1919. The Lactic Acia Bacterid. Andr* Fred Host and Son, Kobenhaven. 14. Russel, H.L. l896.The Rise and Fall of Bacteria in Cheadar Cheese. Wis. Agr. 3xo. Station, 13th Ann. apt. pp. 93-111,Madison. 13. Savaee, W« S. 1906. Bacterial Examination of Water Supplies, Lewis, London, p.213. 16. Standard Methods, 1920. Amer. Public Health Assn. , Boston. 17. The Committee on jeterminative Bacteriology of the Society of American Bacteriologists, 1923. Bergey's Manual of Determinative Bacteriology, Williams and Villeins Co., Baltimore. 18. The Committee on Bacteriological Technic of the Society of American Bacteriologists. Manual of Methods for Pure Culture Study of Bacteria. Published by the Society, Geneva, II.Y. 19. Todd and Sadler, 1911. The Kingston Cheese. Jour. Board of Agriculture, Vol. XVIII, Ho.3., pp.193-203, Lonaon. 20. Winslow and Winslow, 1908. Systematic Relationships of the Coccaceae. Wiley and Sons, II.Y. -35-21. 7/inslow, Rothberg and Parsons, 1920, Notes on the Classification of the White and Orange Staphylococci. Jour. Bact. V. 2, pp. 145-167. PLATE I Complete Results of Quantitative Analyses. Dates on which cheesa were made. Aug.3/23 Aug.7/23 Aug.10/23 Media on which counts were made. Gluoose Agar Lactose Agar Glucose Gelatin V.P. Broth MacConkey's Agar MacConkey's Broth Glucose Agar Lactose Agar Glucose Gelatin V.P .Broth MacConkey's Agar MacConkey's Brot h Glucose Agar Lactose Agar Glucose Gelatin V.P.Broth MacConkey's Agar MacConkey's Broth Green Che Ho.of Bacteria per gram. 2,175,000,000 2,272,000,000 3,153,000,000 Growth in 1-1,500,000,000 2,803,000,000 4,530,000,000 Growth in 1-1,442,000,000 131,100,000 111,200,000 159,659,000 Growth in 1 -50,531,000 ese Ho.of coli types per gram. 53,000 1,585,000 404,000 Ripe Cheese Jo. of bacteria per gram. 18,000,000 12,000,000 22,000,000 100,000,000 116,000,000 147,000,000 G 62,150,000 42,000,000 83,000,000 Growth in 1-68,846,000 Ho.of coli types per gram No growth No growth 29,000 rowth in 1-95,000 43,000 Gth.1-22,000 PLAT E'I'C ONTINUED. Dates on which one8se were made. Media on which counts were made. Green Cheese ifo. of bacteria per gram. Mo. of coll types per gram No.ofbacteria per gram Ripe Cheese lo.of coIT types per gram Aug. 15/23 Glucose Agar Lactose 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 Gelatin V.P.Broth Sterile Milk tfacConkey's Broth 110,970,000 92,475,000 102,750,000 Growth in 1-51,380,000 Growth in 1-82,000 55,561,000 46,893,000 64,239,000 Growth, in 1-183,750,000 No growth in 1-117. Oct. 10/23 Glucose Agar Milk Agar Glucose Gelatin 7.P.Broth Sterile 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 bacteria per gram No. of coli lNo. types per gram per gram Ripe Cheese. of bactwria No. of" coli " types per 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 Sterile Milk MacConkey's Broth Oct. 22/2 Glucose Agar Milk Agar Glucose Gelatin 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 27,lb6,000 77,432,000 Growth in 1-14.000.0JO 236,000 No growtli in 1-64.000 No growth in 1-64.000 61,000,000 36,337,000 87,132,000 Growth In 1-14,000,000 Growth in 1-72,0u0,000 68,665.000 34,635,500 69,675,500 Growth in 1-50,000,000 Growth In 1-50,000,000 20,521,000 48,935,000 Growth in 1-65,000,000 No growth in 1-232 No grpwth in 1-232 PLAT E'l' C 0 N T I H HID, 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 "bacteria per gram No. of coTT types per gram Oct. 29/23 Glucose Agar Glucose Gelatin 154,452,000 171,435,500 29,016,000 50,076,000 jec. 28/2 1 Glucose Agar 565,984,000 353,270,OOX) f 'Green cheese' refers to the cheese not oluer than two days from time of making. f? 'itipe Cheese1 refers to the cheese ready for market, eight to twelve days frcm time 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 III 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 ct A ct 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 ct (» >TS ct 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 ct •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 ct A ct-u CJ o O O (-( + 1 + 1 + 1 1 1 + 1 1 1 1 1 1 1 + 1 + 1 M OJ CO ct • •i ct o CJ O Ci 0 HH + 1 + 1 1 1 1 1 1 1 1 + 1 + 1 CO ct •1 • ct-o o o o Q K + i + i + i i i + + i i i i i + i + i M 1 3 CO ct H <* X) ct-O 0 o a o K + i + i + i i i + i i i i i • i + i + ' M O CO ct 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 ct A et (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 ct 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 Culture JS umber T Morphology Habitat Gram Stai n Gelatin Liquefied Glucose Lactose Sucrose ilaltOBB dalicin Glycerol Mannitol Acid Gas Clot Pepton-izing fe 1 ! 1-3 H l—I _ J —< H-l " c i Culture Number. 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 Pi ft3 CO co o et 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 ct 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 ct + 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 ct » ct O o o o a K + i + + i i i i i i i i i i + i +• i to H CO ct H a •d ct O a o o o K + i + i i i i + i i i i i i + i + i ro o to CO ct *t a <B ct O Q o o o + 1 + 1 + 1 1 + 1 1 1 1 1 1 + 1 + 1 to o 00 CO 3" o •i ct M o p< CO t~* 1 1 + + + + + + + + + + + + + + + + + 1 ro a> -a CO 3* o ct w o p> 00 K i i + + + + + + + + + + + + + + + + i ro o CT> CO cr o ct W o p< CO M 1 1 + + + + + + + + + + + + + + -+ + + 1 Culture number Morphology Habitat Gram Stain Gelatin Liquefied Glucose Lactose sucrose Maltose Salicin Glycerol Mannitoi Acid ' Gas Clot ^eptdH-xzing. K M n H M i— w O - ^ " t ) PLATE IP! Kercha ,/,  *  • tunc and  /'/< y •>/,/, y^'.W Reaction*  c(  Ory*ni*m&  fsc/jttd  t  rem %njsltn  Cheese' II 1 tip*. 5 II5\6 IZl\l6 m\ i 136 14 35 p imp Mb ,52 101 PI V03 \(H fie i-fl iii i'4 eot c /33 kM /fi 9 Cocci -15-111 iniitm (fj^i'-/5A'""5-/<i Cocci 1 !-5K-75-IU. Cocci 15-1 Mi)< di*m Coaih/5K-7S~/u Coccil /•SX'TS-fin LC**I -15-1u indiam Cocci 1 I-5X-75 In. 3 | foia tMxI-fOa 1 R  h  uAl-WU Z |/5Ww uxl-IOu \4 [ ff(?-ti)/Al t  l-u  U J 71 / '/fc'i/t. X /-/LAX bkvi-t fctii 3 \  Snort fiWfi ,5 j Uorf RoJ* 0 Mart  RoJi 1 ^fjort  /ortniny  lloci^ i | Micrococci 1 \%Ufkyh  o<rc  i "l. ncOtCi -a + +-+ 4 I 3-. 1 j + + r t-+ 4-+ --~~ h + +-_ " + -f '7 I -g 1  \ <0 _* VJ «1 £3 __, | — ^W/ety Mlh Wfute -------i + -y ^> if + -+ -+ -f-4~ h-. -+•--1 -H-H -4— + -+ -+ + + + + + 4-t-4— +• -t--+ -7 '• vj + -+ -+ -+ -t— +-+ -4— + -4— + -+ — + -+ -+ +• + + + + ++-r----------1 r-f + +• + h + -+ -----K + -T + ----f " -1 " \— -+ ~ + t + f 4 + +--r--— -1 t-^ -+ --I -+ — +•-+ -+--•f-+ +• + +• + + + -+--— 5^ -+- -+ -+ -f-+ +- -+ -4~ + +-+ + + + 4— 4--+•-4j --+ -f -I--f-K--+ -1- -f --+ -+ + + + + + 4— +--+•-+ -7 ft: + + -I--+ --t -+ -1 n i + --5 •f-+ -h-hi f-+•--i -(--4----•~i +• -.•3 + ---H- 1--1 + K5 — 4-f ~5 --t-i-i ~ _ -£ i -r h 1-1 JL Mill + -t-+ +• h f-+ 4 T + h 1 + 1 r + + 1 1-J ------------T l -+ + +-+ + 4-+ + + + 1 -1-i i + h _L 1 1 + 1 1 --------" • " 1 L /reactions t  ptlittvt  ,  -negative  •  tt icil  <tni  qis;  +- acid, -  no  arlion. 


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