118 027 THS COMPARISON OF A V-B MEDIUM WITH OTHER MEDIA FOR THE DETERMINATION OF ACID-PRODUCING BACTERIA Thesis for The Bags-co of M. S. MICHIGAN STATE 3 LLEGE Jcsaph Murray Marriciz I952 This is to certify that the thesis entitled Gospel-on of Y-O medium with Other “edits tn- the Datamation at Acid-Promoting hot one. presented by Joseph tin-ray lea-risk has been accepted towards fulfillment of the requirements for AL degree ill—3m run to halt): 6" 5 f If” I In: " . "l . ' 4 ; . F-e .' - up -'I‘M<.+‘f' a ' fi 1"? ‘_ ‘w' I ;.\.-‘A.A ‘- .“.'. I \L- - - fi‘s 8“ .8‘ ,- V ‘ _ 1‘. .143,“ 1." ~ 0 “'V' ~ . e' Tessa)" \e " .’: A ru’ COMDARISON OF A v-8 MEDIUM WITH OTHER MEDIA FOR THE DETERMINATION OF ACIDbPRCDUCING BACTERIA BY Joseph Murray herrick A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fu1fillment of the requirements for the degree of MASTER OF SCIENCE jDepartment of Bacteriology and Public Health Year 1952 i. _‘\ (Rs) \ ACKNOWLEDGEMENT The author wishes to express his sincere appreciation to Dr. F. W. Fabian for his encouragement and helpful suggest- iens given during the course of this study. He is indebted to Sam Rosen whose im- measurable assistance and encouraging manner have added greatly to the completion of this thesis. He else wishes to eXpress his sincere gratitude to Rel Fulde for his many suggestions and for the taking of pictures. TABLE INTRODUCTION . . e e e . . REVIEW OF THE LITERATURE e .EXPEBIMENTAL e e . e . e e IRESULTS AND DISCUSSION . e SW. 0 O O O O O O O O IEEBLIOGRAPHY . e e e e e e OF CONTENTS Page 12 29 30 ~——_ -——7-‘__I 4 INTRODUCTION The lactic acid bacteria, a ubiquitous group of erganisms, have been found in the alimentary tract, in soil, and in foods. They are responsible for the fermenp tation in.many kinds of food such as cheese and other fenmentod.milk products, cabbage and cucumbers. They also may cause spoilage in canned foods, in wines and in breweries. Since these organisms are important, it is necessary to isolate and study them if we are to understand their activity. In order to do this, it is desirable to have a medium.capable of detecting and differentiating them from.ether organisms. Many media have been suggested for the isolation and enumeration of lactic acid bacteria. It has been felt that the media now in use have not been specific enough for the detection of these organisms. Many allow other microorganisms to grow abundantly, completely ever- growing the lactic acid bacteria in a short time. Some media will support growth of the lactic acid bacteria, but the colenies are often too small to count. In View ef these difficulties, an attempt was made to develop a better medium, to isolate and enumerate lactic acid organisms. This medium consisted of a V-8 vegetable Juice as a base plus tryptose, lactose, beef extract and 0.1 gram per liter of brom cresol green indicator. This medium.was used at a pH ef 5.7. The purpose of this study was to compare the above mentioned V-B medium.with other media that have been suggested for the isolation and enumeration of lactic acid bacteria. REVIEW OF THE LITERATURE Because of the importance of studying the lactic acid bacteria, many investigators have attempted the preparation of nutrient media for cultivating these organisms. In 1920, Ayers and.Mudge (1) prepared a milk-powder medium.fer the determination of bacteria in milk. This medium gave the total count and also enumerated the strong and weak acid-producers, the alkali-forming, the inert and the peptonizing bacteria. Fabian, Bryan and Etehells (9) found the milk-powder agar of Ayers and.Mudge (l) to be an excellent medium for growing the bacteria found in fermenting pickle brine and also that it differentiated the bacteria into four main groups as mentioned above. Rettger and Kulp (20) tried to find a substitute for whey broth and whey agar, because those required exacting technique to obtain a clear medium. They tried galactose agar as a substitute. The galactose agar contained 0.5 to 1 percent of the carbohydrate and although colonies of lactic acid organisms were smaller and did not grow as rapidly as in the whey agar, they were more readily iden- tifiable by their 'weoly or hairy appearance". The same authors in a later paper found that casein and Klim digest media were superior for cultivating Lactobacillus acidophilus and Lactobacillus bulgaricus and that galactose appeared to be the most favorable carbohydrate (15). Kulp (14) found that a tomato juice medium containing peptone, agar, distilled water and adjusted to pH 7.0 gave comparative results with whey-galactose agar and a digest galactose medium and was more easily prepared. In an attempt to isolate lactobacilli from soil and grain, Hunt and Rettger (12) had difficulty in using an acid enrichment broth containing a fermentable carbohydrate since fungi readily overgrew the lactobacilli. To obtain satisfactory results, they used a.medium containing corn flour, peptone, and water with the pH adjusted to 3.5 to h.0. They inoculated broth tubes with the test material and then put a layer of sterile mineral oil 10 to 15 mm thick over the mash. Plates poured from these tubes seldom contained molds and often contained only lactobaeilli in the higher dilutions. In maintaining stock cultures they used a yeast-water broth and agar containing glucose and peptone. They also used the tomato juice broth devised by’Kulp (1h) and found it to be almost as good as yeast- 'wator for some strains and better for others. Weiss and Rettger (23) used a tomato broth medium Ifor isolating Lactobacillus bifidus. This was prepared by taking the contents of a large can of tomatoes and :filtoring the material through coarse filter paper. The Ifiltrato was then diluted with distilled water. To this 'Ehoy added peptonized milk (Difco), peptone (Difco), yeast extract (Difco), and two-percent granular agar. They found this medium to be particularly good for isolating intestinal lactobacilli, since there was a large amount of carbohydrate present. Thus, enough acid could be produced to lower the pH to 3.6 to h.0 which could suppress other intestinal organisms. Barber and Frazier (2), in a study of the dissociants of lactobacilli, used a modification of the carrot liver agar described by Garey, Foster and Frazier (10). They used United states flake agar in place of washed agar and also an infusion of ground beef liver instead of the liver extract. McLaughin (17) reported a medium which gave much better results than the commercial whey and tomato juice agars. This medium was called 'trypticaso sugar agar” and contained pancreatic digest of casein, lactose, glucose, sucrose, gelatin and agar. The pH was adjusted to 6.0. He found more and larger colonies on this medium.and was also able to count them.aftor h8 hours. ‘Wado, Smiley, and Smith (22) suggested a.method of obtaining the ratio of acid-forming to non-acid-forming bacteria. This medium, a modification of the one suggested by Garey, Rittschoff, Stone, and Boruff (11), consisted of filtered tomato juice, yeast extract, glucose, and a buffer. To this was added CaCO3 and an alcoholic solution of brom oresel purple. Strong acid-producing colonies changed the indicator from purple to yellow and also dissolved the GaCOB, resulting in a clear zone around the colonies. Weak acid-producers changed the indicator but did not dissolve the CaCOB. Neutral colonies caused no change while the alkali-forming colonies imparted a deeper purple color to the agar. A medium which reduced the number of common contam- inants found when isolating lactobacilli from oral, vaginal and fecal samples has been devised by Rogosa, Mitchell and Wisoman (21). This medium consisted of trypticaso, yeast extract, KHzPOh, ammonium citrate, a salt solution, glucose, I sorbitan.mono-oleato, sodium acetate hydrate, acetic acid, agar and distilled water. The final pH was 5.h. Emard and Vaughn (6) found that sorbic acid was effective in selectively favoring the growth of the catalaso negative lactic acid bacteria. Twelve-hundredths of one percent of this acid in liver broth permitted the growth of lactic acid bacteria and clostridia but inhibited the catalaso positive actinomycotes, bacteria, molds, and yeasts if the initial pH range of the media was 5.0 to 5.5. In a study of the chemical and bacteriological changes in dill-pickle brinos during fermentation, Jones, Veldhuis and Veerhoff (13) used nutritive caseinate agar (Difco) for the baotorial analysis. The acid-formers showed definite zones of precipitated casein about the colony. They added eight cc of 0.h percent brom cresol purple indicator as a further aid in identifying the acid-forming bacteria. McCloskoy, Faville, and Barnett (16) in studying characteristics of Leuconcstoc mesenteroides isolated from cane juice employed a medium using raw sugar, tryptone, yeast extract, agar, water and adjusted to pH 6.7. The Committee on the Microbiological Examination of Foods (h) suggested a tryptone-glucose-yeast extract agar for the examination of sauerkraut. This medium had yeast extract substituted for beef extract, and is otherwise the sumo as tryptone glucose extract agar (Difco). Murdock, Folinazzo and Troy (18) in evaluating E plating media for citrus concentrates employed a number of I different media to determine maximum counts of several leuconostoc, lactobacilli, and yeasts. They found that the orange serum agar at pH 5.h was a suitable differential medium for the leuconostoc, lactobacilli, and yeasts. The orange serum agar consisted of tryptone, yeast extract, dextrose, KZHPOu, agar, orange serum and distilled water. EXPERIMENTAL The experimental work consisted of a comparison of V68 medium and different media for plating lactic acid organisms, non-lactic acid bacteria and yeasts. The pour plate technique was used and total counts and colony size were taken. The latter part of the experimental work consisted of plating samples from cucumber and kraut fer- mentations in six different media. The following organisms were used in this experiment: Lactic acid organisms.-- Lactobacillus plantarwm (ATCC 8014), Lactobacillus plantarum (NRRL B-227), £225!- bacillus fermenti (NRRL B-585), Lactobacillus delbrueckii (NRRL B-hhS). Lactobacillus‘ggggi (NRRL B-uuz), £22227 bacillus 2223i (NRRL B-hhl), Lactobacillus 2332i.(ATCC 7469), Streptobacterium dextranicum.(NRRL B-lZSh), B222, bacterium vermiforme (NRRL 3-1127), Leuconcstoc mesenter- oides (ATCC 80u2), and Streptococcus faecalis. 'Yoasts.-- Hansenula subpelliculosa (Etchell's No. RY-lBS), Torulopsis caroliniana (Etchell's No. RY-th), Torulopsis holmii (Etchell's No. FFLAY-BOY), zygosaccharo- 5129.3. §p. A. (Etchell's No. YS-S90), Torulaspora _1_-_g_s__e_i (Etchell's No. Rx-a). f, we? r- -H Hon-lactic acid bacteria.-- Sarcina 12333, Micrococcus pyegones var. aureus, Serratia marcescens, Escherichia 221i, Aorobactor cloacae, Bacillus subtilis. As soon as these cultures were obtained, transfers to be used as stock cultures were made as follows: lactic acid organisms - stab culture in micro assay culture agar (Difco) with the exception of Streptococcus faecalis which was treated like the non-lactic acid bacteria; yeasts - dextrose broth (Difco) plus 0.2 percent yeast extract; non-lactic acid bacteria - nutrient agar slants (Difco). When a test was to be made, the organisms were trans- ferred from the stock cultures to the following media: lactic acid organisms - micro inoculum.broth (Difco); yeasts - dextrose broth plus 0.2 percent yeast extract; non-lactic acid bacteria - nutrient broth. The organisms were incubated at the following times and temperatures: Egctic acid organisms.-- All organisms except Leuco- ncstoc mesenteroides were incubated at 30° C for h8 hours. Leuconcstoc mesenteroides was incubated at room.temperature for h8 hours. Yeasts.-- Hansenula subpelliculosa, Torulopsis‘gggg- liniana and Torulaspora 5232; were incubated for 2h.hours ‘t 30° C. Torulopsis holmii was incubated for 3 days at 30' C. Brettanomyggg versatilis and.§ygosaccharomyggg'SE.‘5 were incubated for 6 days at 30° C. ’1 10 Non-lactic acid bacteria.-- These organisms were incubated at 30° C for an hours. Appropriate dilutions of the actively growing cultures were made and plated by the pour plate method. Duplicate plates were poured for all the lactic acid organisms. All of the organisms were plated in the media listed in.Tab1o 1. An additional medium was used in the yeast study. This was potato dextrose agar acidified with one ml of a 10 percent solution of tartaric acid per 100 m1 of medium. This treatment lowered the pH to 3.5 3.0.1. This was I assigned code no. 7A. The lactic acid organisms after plating were incubated at temperatures described as optimum.in Bergey's Manual of Detorminative Bacteriology (3). The yeasts and the non- lactic organisms were incubated at 30° C. Colonies were counted on plates showing between 30 and 300 colonies after two, three, and five days. The size of the colony was measured in mm. In the sauerkraut fermentation, samples were taken according to a method described by Fulde (8). In this manner, samples could be obtained under a nitrogen atmos- phere. It was also possible to obtain representative samples from the center of the fermentation. In the cucumber fermentation, daily samples of the brine were collected from a commercial fermentation of salt-3tock pickles by the method of Etehells 22.5; (7). TABLE 1 LIST OF MEDIA STUDIED ll Code pH of no. Medium_ medium Reference 1 V-B 5 .7 a 2 Orange serum agar 5.h 18 3 Lemon sormm agar 5.2 18 h Tryptone glucose extract* 7.0 Difco (5) S Nutritive caseinate“ 6.6 Difco (5) 6 McCleskey's agar 6.5 16 7 Potato dextrose agar 5.6 Difco (5) 8 Tryptone glucose yeast extract* 6.8 19 9 Tomato juice agar 6.1 Difco (5) 10 Sabouraud's dextrose agar 5.6 Difco (5) 11 Thermoacidurans agar 5.0 Difco (5) TModia contained 0.0a g brom cresol purple indicator per liter of medium. Platings of samples from the sauerkraut and cucumbers were made on the following media: V-8, orange serum agar, tryptono glucose extract, nutritive caseinate agar, tomato juice agar, and thermoacidurans agar. The plates were incubated at room temperature for two or three days. RESULTS AND DISCUSSION The results are indicated in Tables 2 to h and Figures 1 to 5. Table 2 indicates the total count of different organisms as grown by the various media. Table 3 gives the diameter of subsurface colonies. In consideration of the media studied, it appeared that the V-8 medium was the best. In most cases, after three days incubation, colonies on the V-8 medium were large and easy to count. A colony was produced having the following characteristics: Size: 0.5 - 1.0 mm in diameter Color: dark green to black, having a slight fuzzy appearance around the edges of the colony Halo: characteristic distinct yellow halos appeared around the colonies in.most cases. The halo was two to three times the size of the colony Colonies on the nutritive caseinate, tryptone glucose extract, and tryptone glucose yeast extract agar did not produce a distinct halo in.many cases. It appeared as if the acid produced by the organisms could diffuse through- out the medium. This would make it difficult to differ- entiate acid-producing organisms from non-acid-producing organisms. The tryptone glucose extract and the tryptone glucose yeast extract produced smaller colonies than appeared 13 on the V-8 medium. Orange and lemon serum.agars as well as thermoacidurans agar could support growth of the test organisms after two days of incubation. However, this might not have been the case if these media contained an indicator. Although some of the test organisms did not grow out as rapidly on V-8 agar (probably due to the inhibitory effect of the brom cresol green indicator), the colony count at the end of three and five days was very high. all-"m - .11.“ a sac" _X'I.L 7- It is desirable to have a medium that is inhibitory to organisms that are usually found as active or contamp inating organisms in the early part of lactic fermentations. If non-lactic organisms grow out rapidly in a medium, they would be difficult to differentiate from lactic acid-pro- ducing organisms. In Table h it is shown that the V-8 medium inhibited Sarcina 13223, M, pyogenes var. aureus, and Bacillus subtilis. None of the other media showed this-degree of inhibition. Table 6 shows a comparison of various yeasts which were isolated from cucumber fermentations. No medium proved to be outstanding. Torulepsis caroliniana did not grow on nutritive caseinate agar and Zygosaccharm §p. A did not grow on tryptone glucose extract agar, nutritive caseinate agar, McCleskey's agar, and tryptone glucose yeast extract agar. The effect of pH of the various media must also be considered. Although no special study of this effect was 1h 0 made, it was observed that there was a correlation between the pH of the medium and the growth of non-lactic acid bacteria. In general, the lower the pH, the more inhibitory the medium to these organisms. The medium did not show any special differentiation for tho yeasts and lactic acid bacteria as far as pH was concerned. These organisms grew in media possessing both high and low pH values. There is probably an optimum pH for each medium for any one particular organism. V-8 medium, orange serum agar, tryptone glucose extract agar, nutritive caseinate agar, tomato juice agar and thermoacidurans agar were employed to enumerate organ- isms of the sauerkraut and cucumber formentations in order to show how the media would function under practical con- ditions. The V-8 medium, tryptone glucose extract agar, and nutritive caseinate agar were chosen because they con- tained an indicator capable of detecting acid-producing organisms. Orange serum, thermoacidurans and tomato juice agars were chosen because they gave relatively high counts of acid-producing organisms in pure culture (See Table 2). Tryptone glucose yeast extract and lemon serum agars were not used because of their similarity to tryptone glucose extract and orange serum agars respectively. Figure 1 shows a comparison of how these media enumer- ated total counts in the sauerkraut fermentation. The media showed very little difference in evaluating the total count. Figure 2 indicates the numbers of. acid-producing organisms. (1 ‘u—m ‘flm' l6 acid-producing bacteria. Acidnproducing colonies on the V-8 medium were characteristic as in the sauerkraut fermen- tation. The dark green color and yellow halo greatly facilitated differentiation from other organisms. 2: a hips-lidsdfirqd 1...." . I TABLE 21 NUMBER OF LACTIC ACID BACTERIA PER ML x 107 WHICH DEVELOPED ON THE VARIOUS MEDIA Media L. plantarum (A) L. plantarum (N) L. fermenti no. Days at 30° 0 Days at 30’ 0‘ Days at h0° C a 3 5 2 3 58 2- 3 5 1 E2.0 E2.0 E2.o , s 2‘ 120.0 18.h 19.2 19.2 2 E3.0 a3.0 E3.0 121.0 126.0 126.0 19.1 20.0 20.0 3 no.0 a2.0 #290 119.0 133.0 133.0 22.0 22.0 22.0 h a 16.9 32.0 98.0 131.0 l3h.0 + +' 3.6 5 E3.o E3.0 a3.o 105.0 129.0 129.0 18.2 19.0 19.0 6 33.0 33.0 33.0 89.0 107.0 120.0 15.3 16.1 16.1 7 39.0 39.0 no.0 + + s- 9.0 12.2 12.2 8 o- 29.8 32.0 a 62.0 116.0 13oh 16.1 16.1 9 39.0 39.0 39.0 . er 9u.o 102.0 17.0 18.h 20.0 10 37.0 39.0 39.0 f- 15.9 35.0 15.0 15.2 1592 11 uh.0 h6.0 h6.0 85.0 112.0 12h.0 23.2 23.2 23.2 * Colonies too small to count e No growth in dilutions used A.L. plantarum (ATCC 801h) N L. plantarum (NRRL B-227) 17 L. casei (N1) no. Days at 40° 0 Days at 30° 0 Days at 30° C 2 3 S 2 3 5 2 3 S l a. 139 140 +— s 90.0 s 50.0 66.0 2 130 130 130 94.0 99.0 99.0 70.0 72.0 72.0 3 140 140 140 70.0 85.0 92.0 61.0 63.0 63.0 4 145 145 145 75.0 97.0 101.0 30.0 38.0 44.0 5 128 130 131 32.0 40.0 72.0 55.0 62.0 62.0 6 129 138 138 50.0 78.0 86.0 41.0 57.0 62.0 7 a 105 116 a a 86.0 +- a 30.0 8 125 125 125 35.0 50.0 78.0 50.0 52.0 64.0 9 131 131 131 30.0 54.0 70.0 54.0 55.0 58.0 10 126 126 126 49.0 81.0 86.0 53.0 58.0 58.0 11 132 132 132 79.0 96.0 102.0 58.0 61.0 63.0 N1 L. casei (NRRL B-442) N2 L. casei (NRRL B-44l) TABLE 2 CONT. W L. casei (A1) 8. Media dextranicum B. vermiformo no. Days at 30° 0 Days at 30’ 0 Days at 30. 0 ‘__f 2 3 S 2 g 3 I11 5 2 3 5 1 s 94.0 110.0 a 7.60 9.30 - 3.80 6.20 2 104.0 109.0 109.0 9.20 10.40 10.40 - 5.40 7.00 3 94.0 96.0 96.0 8.40 . 9.20 9.20 - 5.90 10.00 4 85.0 107.0 107.0 s- 6.60 12.00 - s 7.40 5 108.0 110.0 110.0 0.59 1.00 1.30 - 3.30 6.00 6 107.0 110.0 110.0 9.90 *o- as - 3.80 6.10 7 +— s- 52.0 a 10.20 11.30 - 4.20 7.00 8 60.0 99.0 106.0 a 9.70 13.0 - s 7.20 9 s' 97.0 106.0 9.60 10.60 11.20 - 4.80 6.30 10 59.0 97.0 97.0 a» 7.50 8.30 - a 1.02 11 53.0 88.0 88.0 6.70 10.10 11.30 - 8.70 11.30 small to count dilutions used c Colonies too + No growth in as Coalescing of colonies- - No count made Al L. casei (ATCC 7469) m Media n.. L. mesenteroides Days at rgom temp. Streptococcus faecalis 2 Days at 300 0 3 <3 cn -o o~ 01 I? \» a: bi bi Ia F' c: 22.4 23.2 23.3 16.4 18.4 15.2 4!- '3!- 20.1 11.3 . 16.6 24.0 24.0 23.3 18.2 19.0 16.2 19.0 15.4 20.1 14.3 18.2 24.0 24.0 23.3 19.3 19.0 16.2 21.1 18.2 20.1 14.3 18.2 6.3 6.1 ' 6.3 8.3 8.0 13.0 10.5 18.0 6.1 12.1 13.2 6.8 6.5 6.7 9.0 8.3 14.5 12.0 19.2 7.5 13.6 14.0 7.0 6.9 6.8 9.4 8.4 15.0 12.0 20.0 8.4 14.2 15.1 18 I V I t I I I A II I l I I I III I. I ILI II I.I . l I on I I). u - I n s I I a e I I e o I I _ o e . I I. e e I I e o I g 7, rah]. .u. shris 1.. all. I- e .l .. o I . II. ‘ ' Isa ' I I . ‘ el|v I I II... II I I! t l I I l I l I . I I I I 9 l I I I I II II II I l . II I I I II III) III.II I I I l l I! I. v I I I I I I I r I I I r I I I I I IIIIIII.III I.IIII.IIII.I Apmmum qmmz. sandpaaaq .q z Azaom ooa.o o.o-m.o p H.Huo.o o.aum.o o.a-m.o N.H-m.o e.o-m.o m.o-H.o m.aum.a N.Huo.o ~.oum.o o c.0-m.o ».o-m.o ~.oum.o H.H-~.o o.oum.o m.oum.o m.~-o.a o.a-~.o m.ouo.o m m.oum.o + + o.aum.o o.oum.o m.ouH.o m.onm.o m.o-a.o z : m.ano.o o.a-o.o o.anm.o m.a-o.a o.auo.o m.oam.o o.m-m.H m.auo.a o.a-m.o m m.auo.o m.a-m.o N.H-m.o m.Hno.H m.auo.o o.oum.o m.anm.a ~.H-o.a o.aum.o m m.a-m.o m.aum.o m.aup.o H.H-m.o * * o.mum.a m.a-o.a o.a-m.o H m m m m m m . m m m o co: pa «man u eon pd nhmn ; o com as chum awn”: apnoanow on sz eyn¢mwuaa .qw AvafiahdpndHa .A III! IiIIIII dHomz mDOHm<> zH «Hmmaodm QHO< OHBO zH «Hmm0040 0H0¢ 0H0040-zoz 00 2: 2H 020000 mo mmemz Hm Qmadmmzpzm md 0000 m 0. 00000 H0009 00H K a: mMm mBmNMM . o mqmdfi $4 GI 00 d #4 d) LIV) a! 000 db!) 0% IL- >0 Iv Q5 Tryptone glucose extract agar Nutritive caseinate agar Tomato juice agar LI- 1 I. (I Thermoacidurans agar IIDAYS 7 DAYS . l\\\\\\I}\\\\_\\\I\\\\INI“I“\\\\\\\\I\\I\\\\\\‘ W4 . “mum-A...- 4 DAYS AA--UM- 2.5 DAYS ZDAYS mummy/xxx”- ur- 5.5.6. <30 OFF '90. 65‘ c; «a '0 «0' ’Im/SWSIUBBJO - Jeqmnu mquJBSoq Evaluation of media to determine total organisms of sauerkraut Figure 1. fermentation V-8 agar is [:1 Tryptone glucose extract agar Nutritive caseinate agar J WWI\\\IW I)II)»I))))IIIIIIIIIIIIIIIIII\“\\I\\“\\\\‘ Vllll/ll/lllll/l/ll/I/l/I)”III”fillllllllllmIJ AYS DAYS DAYS DAYS N0 acid-producers ) )) I”) MI))))))))))))))))I)I)) MWWIDMWWW WMIIIIW 'tm/swsxuafiao - Jeqmnu wuqxaefioq 05 p. ”III/III)!”))m)))))l)))»))”I”IImI)II V 46 IO OTIME HRS HRS. C) 58 HRS. 52 HRS. Figure 2. Evaluation of media to determine acid-producers 0f sauerkraut fermentation V-8 agar Orange serum agar “‘v “\4 Tryptone glucose extract agar D [B («<- Nutritive caseinate agar AAAAA Tomato juice agar Thermoacidurans agar ..... A .- A ‘ _ '(((((((((((_(/(l((((l(llll(N((lllllllllll(”II/llllllflflllll/lllllt -vv'vv'vvvvv \AMAAAAAMAA ((\((((((((((((((((((((«(«(U («mm-(1W one-no a-aimh'lsno'qo' mus-00¢ 'IW/SWSIUBBJQ - Jeqwnu mquJ8301 2 DAYS 3 DAYS 4DAYS 6DAYS BDAYS I DAY Evaluation of media to determine total organisms of cucumber fermentation Figure 3. V-8 agar SEES Tryptone glucose extract agar C3 Nutritive caseinate agar “? c 'lllllllIllllllllIlIlllllllllllll III\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\I\\\\I\I“II ’lI!Illl/l/Il/l/l/llllll/l/ll/lllIIlllllllllllllllllllllll MM“)IIIIIIIIIII\IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII») -\\\\\\\\\\\\\\\\\\I\I\I\\\\\\\\\\\\I\II\II\IIIIIIIIIIIIW 0.0.9 '2"- “2°.“- GI F- f~ (D ID I) I) V’ 'IW/SWSIUBSJO - Jeqmnu wqqtasioq 8 DAYS ZDAYS 3DAYS 4DAYS SDAYS l DAY Figure 0. Evaluation of media to determine acid- producers of cucumber fermentation 20 hours h8 hours Figure 5. Colonies of Lactobacillus glantarum on V-8 agar SUMMARY A new medium for the detection of lactic acid bacteria has been compared with ten other media used for the detection or these organisms. This medium contained V-8 vegetable Juice as the main constituent and brom cresol green as an indicator. The V-8 medium compared favorably with the other media. Although in some cases growth was not as rapid in this medium as in some of the others, it had the advantages that the acid-producing bacteria gave characteristic colonies with distinct yellow halos and inhibited certain non-lactic acid bacteria. .7" 1. 2. 3. h. 5. 6. 7. 8. 9. 210. 11. BIBLIOGRAPHY Ayers, S. H. and.Mudge, C. S. Milk-powder agar for the determination of bacteria in milk. J. Bact.. 5:565-588. 1920. Barber, F. W. and Frazier,‘W. C. Dissociants of lactobacilli. J. Bact., 50:637-6h9. 19h5.v Breed, R. 3., Murray, E. D. 0., and Hitchens, A. P. Bergey's manual of determinative bacteriology. 6tflaod.'Williams and'Wilkens Co., Baltimore, Md. l9 . Committee on microbiological examination of foods. Microbiological examination of fermented foods. AI. J. Pub. Health, 33:726-727. 1903. Difco manual of dehydrated culture media. 8th ed. Difco Laboratories, Detroit, Mich. Emard, L. 0. and Vaughn, R. H. Selectivity of sorbic acid for the catalaso negative lactic acid bacteria “1 clostridia. Jo BEGto, 632h87-u9I-Lo 1952. Etchells, J. L. and Jones, I. D. Procedure for bac- teriological examination of brined, salted, and pickled vegetables and vegetable products. Am. J. Pub. Health, 36:1112-1123. l9h6. Fulde, R. c. Unpublished thesis. Fabian, F.‘w., Bryan, C. S., and Etehells, J. L. Experimental work on cucumber fermentation. Mich. Agr. Exp. Sta. Tech. Bull. 126, p. 11. 1932. Carey, J. 0., Foster, E. M., and Frazier,'w. C. The bacteriology of brick cheese. I. Growth and activity .r starter bacteria during manufacture. J. 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V . o a ’ H a . _ I is 2 I o r. I A > o . O 1 ' . A I 9 O 1 m D 0 ex _ 1 1 .. . ' . . m _ . 1 P 9 . 1 i _ . ' . . 1 . _ o . _ a a r. , . O I _ a C _ I b 0 0. fl. ,. I . . 0 I. _ . 1 r. n _ \ _ I. U _ r . 5 d? .. 7H . '53,...12 . J“.:. I? ... .-.-..- . . I A r- . P. S . . O C o - 0 _ . Y\ ' fi f _ .K D. I x D f. ' ‘ O . I. r C r \ I . a. o h I . x U ’ I O r 0 I I U .«11‘11 ‘ ¢_{¢1‘¢l .111. laidi. .14 {L .q1 I