STUDIES OH ma HUTRMGNAL REQUEREMENTS OF BACILLUS COMM: Thais fior i‘ho Dew @5 Ph. D. MICHIGAN STATE UNIVERSITY Thomas Wilfiiam Humphrey: 1956- This is to certifg that the means cnuueu STUDIES ON THE NUTRITIONAL REQUIREEENTS OF BACILLUS COAGULANS presented by THOMAS WILLIAM HUMPHREY S has been accepted towards fulfillment of the requirements for Ph. D. degree in Microbiology 8: Po Health / WWW" 'j' Major professor Date October 22, 1956 0-169 STUDIES ON THE NUTRITIONAL REQUIREMENTS OF BACILLUS COAGULANS By THOMAS WILLIAM HUMPHREYS A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health \ 1956 1 5 93/57 ? A237 STUDIES ON THE NUTRITIONAL REQUIHEKENTS OF BACILLUS CCAGULANS ABSTRACT .A.study was made of 177 spore-fOrming bacteria isolated from the wash waters of Canadian tomato juice packing plants. Only about 30 percent of these were able to produce flat-sour spoilage in tomato juice both aerobically and anaerobically. The remaining 70 percent grew only aerobically, generally producing thick cheesy pellicles and alkaline reactions. Since all organisms grew well on thermoacidurans agar (Difco) (DTA), it was concluded that routine bacteria counts on this medium do not provide a true index of spoilage possibilities. Results indicated that the addition of 0.00h percent bromcresol green indicator to DTA, and increasing its dextrose concentration to 1.0 percent, rendered it more valuable as a plating medium. The significant acid- producing types were readily distinguished in this modified medium. Taxonomic studies showed that Bacillus coagulans predominated among those organisms producing acid whereas those producing a more alkaline reaction in tomato juice resembled Bacillus subtilis. The vitamin and amino acid requirements of 15 of the canning plant isolates identified as g. coagglans and 8 other authentic strains of this organism were studied at 37°C. In a medium comprising vitamin-free acid hydrolyzed casein, yeast extract, glucose, and mineral salts, growth occurred earlier at pH 5.5 than at 7.5. Substitution of malate, succinate, fumarate or citrate for glucose in this medium indicated that citrate was not utilized and response to the other three was feeble. Sporadic results obtained in this medium when vitamins were substituted for yeast extract did not result from a deficiency of cysteine or tryptophan. The variation was eliminated by substituting enzymic casein hydrolysate fbr the acid hydrolysate. Biotin and thiamin were required by all strains in this semi-synthetic medium. Niacin, also, was required by one strain, and folic acid by another. An additional requirement for folic acid (or PARA) was noted for most strains in a synthetic medium containing 12 amino acids. This was the most important difference between results obtained using the semi-synthetic medium and the synthetic. The amino acid requirements were generally non-specific at 37°C. However, glutamic acid appeared essential for a few strains and stimulated others. The enhanced growth generally afforded by the enzvmic casein hydrolysate was largely a reflection of buffering action resulting from its high acetate content. Phosphate buffer appeared more favorable for B. coagulans since one strain proved acetate sensitive. Geiwm m Ralph\N. Costilow Associate Professor {mi—#— TABLE OF CONTENTS LIST OF FIGURES............................... LIST OF TABLES................................ ACKNOWLEDGEMENTS.............................. INTRODUCTION.................................. REVIEW OF LITERATURE.......................... General................................. Nutrition............................... EXPERIMENTAL METHODS AND MATERIALS............ Identification and Methods of Handling cultureSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Methods of Cleaning Glassware........... Methods of Estimating Growth............ media and ReagentSOOOOOOOOOO0.0.0.000... RESULTSOIOOOOOOO...0....OOOOOOOOOOOOOOOOCOCOOO Studies of the Spore-forming Bacteria Isolated from Tomato Wash Water....... Preliminary Nutritional Studies......... Study of Vitamin Requirements........... Study of Amino Acid Requirements........ Studies on Stimulation.................. DISCUSSION.................................... SUMMARY....................................... BIBLIOGRAPBX.................................. APPENDmooooooooooooooooooooooooooooooooooooo. Page iii iv v 1 3 3 7 12 12 16 16 17 18 18 21 33 39 4o 51 56 59 63 i” ‘_ _.¢:'i LIST OF FIGURES Figure Page No. 1. Distribution of spore-forming cultures from tomato wash water with respect to the unit change in pH produced in tomato juice00000000000.000000000000000...0000.0 2O 2. Effect of initial pH on the growth of B. coggglanSOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOO 23 3. Growth of’B, coagulans in synthetic vs. semi-synthetic mediaooooo0000000000000... 32 4. Titration curves for semi-synthetic and synthetic media with and without additional bufferSOOOOOOO00.00.000.000... 48 5. Comparison of modified trypsin hydrolysates and synthetic media with N.B.C. casein hYdrOJ-ysateOO0......OOOOOOODOOOOOOOOOOOO. 49 Table No. 1. LIST OF TABLES The vitamin and/or amino acid requirements of B, goagglans as noted by various workerBOOOOO0.00.00000000000000COOOOO... Growth of B, gagglens using different carbon sourOGSOOOOOOOOO00.000.000.000... Growth of _B_. W in enzymic vs. acid hydrolysates of.casein.;................ Growth of B, ggagnlgng upon deletion of vitamins from semi-synthetic medium..... Growth response of Q. coagglans to minimal vitamin mixtures in semi-synthetic ”dine...00000000000000.0000...cooeeeeo Growth of B, coagulans upon deletion of vitamins from synthetic medium.......... Growth of B, coagglans upon deletion of amino acids from synthetic medium....... Effect of increased concentration of DL- amino acids on the growth of B, coagglans in synthetic medium..................... Effect of trypsin and non-dialyzable casein hydrolysate on the growth of B, WOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOO Page 11 24 28 35 36 38 41 43 45 ACKNOWLEDGEMENTS The encouragement and counsel of Dr. Ralph N. Costilow, Department of Microbiology and Public Health, Michigan State University, during the conduction of the work and the preparation of this thesis is greatly appreciated. Also, the co- operation of the Bacteriology Division, Science Service, Department of Agriculture, Ottawa, Canada, is gratefully acknowledged. In this connection, the technical advise and criticisms of Dr. H. Katznelson, Chief of this Division, and the technical assistance of Miss Agnes MacIntosh were most helpful. STUDIES ON THE NUTRITIONAL REQUIREMENTS OF BACILLUS COAGULANS INTRODUCTION Recently, considerable effort has been made to determine the vitamin and amino acid requirements of figcillus coagulans (synonyms Bacillus thermogcidurgns), the organism believed to cause flat-sour spoilage of tomato juice. Most reports on the nutritional requirements of this organism have shown a lack of agreement among results for which there has been no concrete explanation. The overall picture is confusing in that different workers tested different strains using various methods and incubation temperatures. In general, most investigations have involved too few strains to provide sufficient information on variation within the species. Recent, more comprehensive studies indicate that variation within the species, at least with respect to vitamin requirements, may be somewhat less than indicated by earlier, less complete reports. The present investigation was undertaken in the ‘hOPe that the examination of a variety of strains under similar conditions would provide a more comp]-ate Picture of the species and perhaps explain some of the contIOVersy. Strains of a. coggglans isolated from canning plants and some examined previously by other workers were included in this study. REVIEW OF LITERATURE general Bacillus coagglgns was first isolated and described by Hammer (1915) as the cause of an outbreak of coagulation in evaporated milk. Hussong and Hammer (1928) described another organism, Bacillus calidolacticus, which coagulated milk by producing lactic acid and small amounts of volatile acid. Later, Series and Hammer (1932) noted that B. coagulgns pro- duced large quantities of lactic, acetic, and propionic acids from carbohydrate. Although the organism showed no visible evidence of proteolysis, it increased greatly both.the soluble and amino nitrogen in skim.and evaporated milks. Berry (1933) described an apparently new species, Bacillus thermogcidurgns, as the cause of off-flavor in commercially packed tomato juice. Still another new species, Bacillus dextrolgcticus, was described by Andersen and Werkman (1940) which.was related to B. coggplans but differed from it by producing consider- ably more lactic acid, forming acid from arabinose and sorbitol, and reducing nitrate to nitrite. Reliable methods for classifying the aerobic, mesophilic, sporeforming bacteria were published by Smith.§§,§l. (1946). A description of B. coggglags was included but was amended in a later report by Gordon 4 and Smith (1949). Knight and Proom (1950) identified 15 new isolates as B. coagulans in substantiating the methods of Smith g3 31. (1946). They further noted that these isolates were urease and lecithinase negative and would grow in 4 percent sodium chloride broth. In comparing B, thermOgcidurans Berry with.B. coggglans Hammer (1915), Becker and Pederson (1950) 1 concluded that they were essentially alike and that the latter name had priority. A second publication by Smith.g§,g;. (1952) which superseded the first (1946), included the amended description of B. coggglgns by Gordon and Smith (1949). These workers reported that B, calidolacticus Hussong and Hammer, B. thermoacidurang Berry, and B. dextrolgcticus Andersen and‘Werkman, were synonymous with.B. coggulans Hammer. Therefore, all cultures designated by these names will be considered to be B, coggglgns in this study. The standard description given by these workers is as follows: "Iegetgtive gggg - 0.6n to 1.Qp by 2.5u to 5.Qu; not in chains; uniformly stained; motile; Gram positive. Bpgggg - 0.9u to l.Qn by 1.2u to 1.2u; oval; thin-walled; sub-terminal to terminal. Bpgrgngig - definitely swollen in some cases; not swollen in others. Cglonies - not distinctive. Nutrient 35g; BBQBBB - growth variable. Bag; 9393B - no growth.in 5 percent con- centration of NaCl. glucose-nutrient agay glgpyg - growth soft; moist; more abundant than on nutrient agar. gtiligation g; citrate - negative. Potato - growth erratic. Soybean.§ggg‘§1gp§§ - growth usually heavier than on nutrient agar. Prgteose-peptone agig,§gay,§lgggg - good growth. Byggg-culture Egg; slagyg - growth as good as or better than on nutrient agar. glucose-asparagine ggag §;gpy§ - growth scant if any. ggggyggygggg m;;§ - curdled in l to 3 days at 45°C. we: item - positive. Production 9; gcetylmethylcarbinol - usually pesitive. 2B 2; glucose 2323B - 5.0 or less at 7 days; acid without gas from glucose. Hydrolysis g; gelatin - negative or weak. Hydrolysis g; gggg;p — negative or weak. Beductign g; nitrgtg 32 pitritg - usually negative. Agaggobic productigp g; gag ggpm nityatg - negative. Agaeropic groyyh,;g glucose Byoth - positive; pH below 5.2. 6 Temperature £9; ggowth - good growth from 33° to 45°C.; maximum temperature for majority of strains 55° to 60°C.; no growth at 65°C.; poor growth 1: any at 28°C." Ford g5 31. (1952) studied 43 strains of spore- forming bacteria associated with flat-sour spoilage of tomato Juice. Although variable with respect to gelatin hydrolysis, growth in 5 percent sodium.chloride, and acetylmethylcarbinol production, the majority of the strains constituted a homogeneous group. The above review shows that a reliable des- cription of’B, coagulggs is of comparatively recent origin. Minor discrepancies noted probably reflect strain variation. A special proteose-peptone acid agar medium was recommended for the detection and cultivation of B. thermogcidurags by Stern gg‘gl. (1942). Sporulation on this medium was apparently greater than on nutrient agar. Also, the medium is of value in differentiating between.B. ggagglggg and Bacillus stearothermophilus (Smith.§§ gl., 1952). Cameron and Bohrer (1948) reported that proteose-peptone acid agar "promotes the growth of the flat sour types of bacteria while inhibiting the growth of types not capable of spoilage of tomato Juice. The count on this medium represents the number of Bacillgs thermogcidurgps and other acid tolerant types present and is an index of spoilage possibilities”. This medium is commercially avail- able in dehydrated form from.Difco Laboratories, Detroit, Michigan, under the trade name thermo- acidurans agar. Nutrition .Reports on the nutritional requirements of B. ggggglggg are someWhat controversial and few data are available on its nutritional requirements at 37°C. This temperature was suggested by Berry (1933) to be the optimum temperature for flat-sour spoilage of tomato juice. Some reports (Campbell and Williams, l953a,b; Bhat and Bilimoria, 1955) indicate that growth temperatures may influence the nutritional requirements of this organism. The absence of numerical data in most reports makes it difficult to assess the quality of the substrates employed and impossible to determine the quantitative effects of various modifications on growth. Andersen and Workman (1940) found that ngillgg dextrolacticus reQuired either thiamin or riboflavin and an unidentified component found in an ether soluble fraction of acidified yeast extract and in hydrolyzed casein. Requirement for this latter factor was satisfied by 3 amino acids; y;3., glutamic acid, cystine, and either of arginine or threonine. A single strain of B. coagglans (N.R.S. No. 27) studied by Cleverdon Lt alo (1949) grew at 37° and 55°C. in a basal medium containing L-cystine, DL-tryptophan, thiamin, biotin, and niacin. Growth and sporulation were better at the lower temperature. In contrast to these reports, Allen (1953) found that 3 strains of B. ggggglggg grew in a simple basal medium containing glucose and monosodium glutamate. The addition of casein hydrolysate permitted an additional 2 strains to grow, whereas another 2 strains preferred a mixture of carbon sources and a richer mineral complement. Allen reported that a large excess of calcium ions replaced the biotin and riboflavin requirements for a number of thermophiles. In a more comprehensive study, Knight and Proom (1950) investigated 15 new isolates of B. cgagylang along with.a reference strain (A.T.C.C. 7050) of this organism. They used an incubation temperature of 45°C. The nitrogen requirements were satisfied by casein digest but not by ammonia. Although thiamin and biotin were usually required, some strains, or selected variants thereof, became self-sufficient with respect to one or the other of these vitamins upon subculture. In further studies, Proom and Knight (1955) found this group to be nutritionally homogeneous with.respect to their requirements for biotin and thiamin in a l4-amino acid medium. The amino acid demands were considered complex in the light of the absence of growth in a 7-amino acid medium. The authors could not explain differences in their results from those of Cleverdon gt al. (1949) and of Allen (1953). Baker gt 3;. (1953) mentioned that a strain of a. coggglgns grew in a basal medium supplemented with either glycerol or carbohydrate. Later, Baker 2; 5;. (1955) suggested employing a strain of a. goggglgng for folic acid assay at 55°C. in an unsterilized substrate. Campbell and'Williams (19533) noted that the vitamin and amino acid requirements of a. coagglgng (N.C.A. strains 2, 12, 32, 1039) may vary with the temperature of incubation. Folic acid was required by all strains at 45° and 55°C but not necessarily at 36°. In another publication (1953b), they reported on variation with.respect to the biotin requirement of 6 strains of this organism (N.C.A. strains 16, 18, 32, 43P, 831, and 1039). Whereas, oxybiotin fulfilled the biotin requirements of all strains at different temperatures of incubation, desthiobiotin, pimelic acid, aspartic acid, oleic acid, and aspartic plus oleic acid varied in this respect. None of the tricarboxylic acid cycle intermediates replaced biotin 10 at 36°, 45°, or 55°C. Bhat and Bilimoria (1955) found that a. W failed to grow in a medium containing inorganic nitrogen at 37° and 55°C. in the presence or absence of biotin. Studies with 2 strains at 55°C. showed that both required sulphur-containing amino acids and one strain, also, required thiamin and histidine. However, neither strain required these supplements at 37°C. A summary of the nutritional requirements of a. coggulans found in the literature is given in Table 1. Although the vitamin and amino acid require- ments of this organism are probably not numerous, much conflicting evidence indicates that there are many unexplained factors . 11 TABLE 1 The vitamin and/or amino acid requirements of E. ggagglgng as noted by various workers Reference Requirements noted Allen (1953) Andersen and Werkman (1940) Baker e 3;. (as? Bhat and Bilimoria (1955) Campbell and Williams (1953a) Cleverdon.§§,gl. (1949) Knight and Proom (1950) Proom and Knight (1955) Specific requirements not clearly defined. Thiamin.gg riboflavin; arginine g; threonine; glutamic acid; cystine. Folic acid. Strain No. 3084 recommended for assay of this vitamin with.incubation at 55°C. Thiamin, S-containing amino acids6 and histidine required at 55 but not at 37°C. Thiamin and biotin required at 36°, 45°, 55°C. Requirement for folic acid, niacin, valine, histidine, leucine, methionine, and tryptophan varied with incubation temperature. Thiamin, biotin, niacin, cystine, and tryptophan required at 37° and 55 . Thiamin and biotin required at 45°C. Amino acids also required, but individual demands not shown. EXPERIMENTAL METHODS AND MATERIALS . Identification gnd Methods 9; gandling Cultures Cultures of Bacillus coggglang were selected ' from a collection of spore-forming bacteria isolated from wash waters in Canadian tomato juice processing plants (Johns, 1953). This involved the screening of 177 cultures and the final identification of the a. coggulans strains. Cultures were purified by plating at least three times on thermoacidurans agar (Difco) (DTA), pH 5.0, in conjunction with microscopic . observations. The organisms were then screened to determine their spoilage potentialities and to secure information on the selectivity of DTA. The ability of the spores and of the vegetative cells of each organism to produce flat-sour spoilage in 5.0 ml. of tomato Juice,pH 4.4,when incubated for 21 days at 37°C. was determined both aerobically and anaerobically. To fulfill the latter condition for the vegetative cells, tubes of tomato Juice sterilized for 10 minutes under 15 lb. steam pressure were boiled to exhaust the dissolved oxygen, rapidly cooled in cold water (5°C), and inoculated with a 3.0 mm. loopful of washed cells from 24éhour DTA slant cultures grown at 37°C. These tubes were sealed immediately with sterile vaspar. For anaerobic inoculation with spores, saline suspensions were similarly prepared from DTA slopes after sporulation 13 was confirmed microscopically. Here, tubes of tomato Juice inoculated with.two 3.0 mm. loopfuls of suspension were heated for 10 minutes at 180°F. to destroy vegetative cells and exhaust the dissolved oxygen; subsequently, they were rapidly cooled and immediately sealed with vaspar. For aerobic studies, inoculated tubes remained unsealed. The following authentic strains of E. coggglansl reference purposes: 43F, C2253, 711, and 7050. were included for Following incubation, tubes were generally examined macroscopically, microscopically, and pH determinations made with.a Beckman model.HZ, glass electrode, pH meter. The type of reaction produced by each organism in tomato Juice was compared with its reaction at 37°C. on DTA containing 0.004 percent bromcresol green indicator. Thirty-seven cultures representative of the varied reactions obtained in tomato Juice were selected and identified according to the methods of Smith 21; 5;. (1952) with the following modifications:- (a) Yeast milk - tomato Juice was excluded from this medium because of the variable results noted by Smith gt g1. in its presence. l. Supplied by Dr. 0.8. Pederson, New York State Experimental Farm, Geneva, N.Y. 14 (b) Proteose peptone acid agar - DTA was substituted here since it is essentially the same in composition. (c) Starch hydrolysis - Plates were flooded with Lugol's iodine solution instead of alcohol. (d) pH determinations - the potentiometric method was substituted for the colorimetric method. (a) Citrate utilization - Koser citrate medium (Difco) supplemented with 15 gm. agar and 0.08 gm. bromthymol blue indicator per liter was used in place of the modification of this medium employed by Smith gt 3;. ‘ The following authentic cultures of the genus Bacillus were included for reference purposes during the procedure of identifications g. subtilig, a. polymyxg, g. macerans, Q. cereus, E. cereug var. gycoides, a. lus, a. stearothermophilus, and B. coagulgns. Fifteen cultures identified as E. ggagglang were selected from the above group of cultures along with strains 43F, C2253, 711, and 7050 for nutritional studies. The following authentic strains were included in the study after their identity was confirmed by the methods of Smith.gt 31.: strain 1604 supplied by Dr. A.H. Jones, Department of Agriculture, Ottawa, Canada, who isolated it from commercial tomato Juice showing evidence of flat-sour spoilage; strain.B-36 15 obtained from the National Research Council, Ottawa, Canada. Strain 12245 obtained from the American Type Culture Collection. At first, cultures were carried on normal DTA. This medium proved unsatisfactory for certain.cultures as evidenced by their sporadic response after storage for one month.at 5°C. The difficulty was attributed to their frequent failure to sporulate on this medium.and the subsequent dying off of the vegetative cells during storage. Consequently, cultures were carried routinely on nutrient agar (Difco) fortified with 0.25 percent yeast extract and 0.1 percent dextrose for nutritional studies (pH 6.8). This medium was a modification of one suggested by Becker and Pederson (1950) which resulted in earlier and more complete sporulation of’fi. spagulagg than occurred on protease-peptone acid agar. Cells to be used for inoculum in nutritional studies were obtained from 20-24 hour broth.cultures. In the preliminary work, cells were harvested by centrifugation, washed 3 times in 0.9 percent saline and resuspended in saline to give a final light transmittance of 75 percent at 545 mu in a Luxtrol photoelectric calorimeter. In the determination of the vitamin requirements in semi- synthetic media, cells were washed in distilled water. Cells washed in l/20 phosphate buffer were employed in the determination of vitamin and amino acid requirements 16 in synthetic media and in the subsequent work on stimulation. A volume of 0.05 ml. was used to inoculate test media. An incubation temperature of 37°C. was used in all instances. Methods 9; Cleaning Glassware Pipettes were soaked overnight in chromic acid cleaning solution, rinsed in running tap water in an automatic pipette washer for one day, rinsed thoroughly in distilled water, autoclaved in distilled water, and dried in a hot-air oven. In the early work, pyrex test tubes and flasks were cleaned in hot H2304 containing KNO , rinsed in 3 running tap water until neutral to bromthymol blue indicator, rinsed thoroughly in distilled water, auto- claved in distilled water, and dried in a hot-air oven. In later work, owing to the large number required, test 1 tubes were detergent-cleaned with Alconox in a manner similar to that described by Campbell and Williams (1953a). Methods 9; Estimating Growth Growth.was recorded as percent transmittance after 72 hours incubation using a Luxtrol photoelectric colorimeter at 545 mu wavelength. In preliminary studies l. Manufactured by Alconox Inc., Jersey City, N.J. 9;, FIT—’#~_':' — s .- 17 on the influence of carbon source and initial pH, the incubation temperature was extended to 96 hours. In one case (Appendix VII), growth in semi-synthetic and synthetic media was also compared by titration with 15/10 NaOH to the phenolphthalein endpoint. Media £39. Reagent; Vitamins, amino acids, trypsin 1—500, and vitamin-free enzymically-hydrolysed casein were obtained from Nutritional Biochemicals Inc., Cleveland, Ohio; vitamin-free casamino acids, micro inoculum broth, yeast extract, nutrient agar, and thermoacidurans agar were obtained from Difco Laboratories Inc., Detroit, Michigan; inorganic chemicals, dextrose, etc. were obtained from Fisher Scientific Company, Montreal, Quebec; vitamin and fat-free casein used in stimulation studies was a product of the British Drug House, Toronto, Ontario. Metabolites and reagents were incorporated into media from stock solutions of appropriate strength prior to adJusting the final volume. Except in the pre- liminary studies on the influence of the initial pH level on development, all media were adJusted to pH 6.8-7.0 prior to sterilization. The pH was checked color- imetrically after sterilization using bromthymol blue. ,3; g,- Awm I.\I.MI"_' QWWK ' (‘4.‘- :9 RESULTS Studies 2; Egg Spgre-fgrmigg Bacterig Isglgteg from 399539 Wgsh‘Wgter It may be noted in Fig. 1 (Appendix I-V) that only about one-third of the 177 spore-forming cultures isolated from.tomato wash water produced sufficient acid in tomato Juice to lower the pH significantly. This was true when grown under either aerobic or anaerobic conditions. Many of the cultures failed to affect the pH significantly and still others brought about a decided increase. However, those organisms which.increased the pH of tomato Juice failed to do so under anaerobic conditions. This evidence is of partic- ular interest since all of these cultures were able to develop on thermoacidurans agar. This raises Considerable question as to the value of this medium for detecting the presence of flat-sour producing organisms in tomato Juice. The results of this experiment were similar when spores were used for inocula. In the unsealed tubes, growth of the acid producers was manifested macroscopically by a sparse, delicately-flaky pellicle, with small white granules extending about é-inch beneath the surface. Anaerobic growth of these organisms was evidenced microscopically by long, thin, filamentous cells. Acid production was usually accompanied by an off-flavor and odor. 19 Organisms failing to lower the pH of tomato Juice did not grow in sealed tubes. They usually produced thick, cheesy pellicles and alkaline reactions. Extensive proteolysis of the Juice by this latter group was apparent. When the cultures were streaked on DTA containing bromcresol green, the acid producers were readily distinguished within 24 hours at 37°C. These, including the authentic flat-sour strains, rendered the medium yellow while the colonies per se were a distinctive deep yellow-green. Acid-tolerant, apparent non-acid producers either failed to affect the color indicator during growth or rendered the medium alkaline (blue). Increasing the dextrose concentration in DTA from 0.5 to 1.0 percent permitted the acid producers to reach their limiting hydrogen ion concentration; otherwise, they apparently first exhausted the dextrose and then either utilized the organic acids produced, or increased the pH by proteolytic action. The medium usually became more alkaline within 72 hours. Omission of phosphate buffer from the medium resulted in reduced acid production as shown by a slower change in and a reduced final intensity of the color. Based on the methods of Smith _e_t_ 3;. (1952), 19 of the 37 isolates selected for taxonomic study were identified as B. co n , 3 as B. licheniformig and 15 as _B_. gubtilis. PERCENT OF' TOTAL. CULTURES 20 60- ; Hi I AEROBIC U ANAEROBIC [[- -.6I TO ”.4 8 TO -.35TO -.|O TO 9|.l6 TO +3.20 T0 -.49 -.36 -.|5 +.l0 +2.30 +4.53 Figure 1. UNIT CHANGE IN PH Distribution of spore-forming cultures from tomato wash water with respect to the unit change in pH produced in tomato Juice. 21 Preliminary nutritional Studies The existing controversy concerning the nutri- tional requirements of B. coggulgns indicated that a preliminary study would be in order prior to the selection or development of a synthetic medium.in whiCh all strains would flourish consistently. Consequently, the following series of experiments was performed. ngggt 2; pg 9g develgpmen. - Strains 1604, 43F, and 02253 were grown in 5.0 ml. of micro inoculum broth (Difco). Cells were then washed and resuspended in saline and inoculated into a medium of the following composition per 100 ml. (substrate CAI): Casamino acids (Difco) 5.0 gm. Yeast extract 0.5 gm. NaCl 100 mg. KZHPO4 100 mg. KH2P04 100 mg. Salts solution x1 o. 5 ml. Aliquots of this medium were adJusted to pH levels of 7.5, 7.0, 6.0, and 5.5 and dispensed in 10.0 ml. amounts in cotton-plugged, optically- standardized test tubes. Following autoclaving, 0.25 ml. of sterile dextrose solution (0.4 mg/ml.) l. MgSO4.7H20, 10.0 gm.; FeSO4, 0.273 gm.; MnSO4.H20, 0.379 gm.; distilled water 250 ml. (modified from Johnson. 1949.). 22 was added per tube. This gave a final concentration of about 1 percent. It was evident from the results of this work (Figure 2, Appendix VI) that the strains of’B. ggagulang tested had a longer lag phase at pH 7.5 than at the lower pH's. However a greater number of cells was. produced after extended incubation at pH 7.5 and 7.0 than.at the two lower levels. Consequently, a pH of 7.0 was considered appropriate in subsequent studies. Effect 9f; different $11191; sources _o_n gag—Wm - The same experimental procedure described for pH studies was employed in these experiments except that individual aliquots of medium CAY adJusted to pH 7.0 were modified by adding 1.0 percent malate, succinate, fumarate, or citrate in place of glucose as a carbon source. Table 2 shows that of the carbon sources tested, glucose was decidedly superior. Citrate was not utilized. Growth in the other sources was feeble and the cells were extremely difficult to disperse for accurate turbidity readings. Therefore, glucose was used routinely as a carbon source in all further studies. Raglgcement g; yggst extrgct - Good growth of each strain occurred in medium CA! but failed to Therefore, a preliminary occur when yeast extract was omitted. attempt was made to grow a few representative strains in medium CA! with a mixture of ten vitamins of the PH 7'5 PH 7.0 E N U' l U! 0 O STRAIN I604 o STRAIN 43F AST RAIN C2253 'TFRA.nasrwI-r-rh.nic u U! PM 5-5 "16-0 25~ t. 2 ll] 50 U " O I! Ill “'75- I l J I I l l l I 2 3 4. I 2 3 .4 IDIA‘Y s Figure 2. Effect of initial pH on the growth of 2. coagulans. 24 TABLE 2 Growth of.§. ggggulggg using different carbon sources Carbon Incubation Culture source (2:;:) 1604 43p 02253 Percent trgnsmittgnce Glucose 1 58.5 58.2 78.1 " 2 45.5 28.9 48.7 " 3 42.0 26.3 46.0 " 4 39.1 23.9 42.1 Malate 1 93.2 99.3 97.6 n 2 --* -- 91.0 .. 3 -- -- 87.3 n 4 80.3 85.6 80.1 Succinate 1 -- 99.7 97.8 “ 2 -- -- 88.6 n 3 -- -- 83.8 " 4 78.0 81.1 77.5 Fumarate 1 93.7 98.5 97.8 ” 2 -- -- 86.7 " 3 -- -- 83.0 " 4 76.2 78.8 76.0 Citrate 1 99.5 100 100 " 4 99.5 100 100 * t -- Growth difficult to disperse 25 B-complex plus a mixture of pyrimidines and purines replac ing yeast extract. The inoculum was prepared by growing the organisms in medium CAY, and the cells were washed and resuspended in saline. The results appeared promising in that partial replacement of yeast extract occurred with vitamins alone . Pyrimidines and purines usually provided slight stimulation but did not support growth in the absence of vitamins . It now appeared feasible to attempt to determine the specific vitamin requirements of each strain. Medium CAY was modified by replacing yeast extract with the following vitamin complement to form the basal medium (medium CAV) for these tests. Thiamin.HCl Pyridoxine.HCl Pyridoxal.HCl Ca pantothenate Niacin Riboflavin Inositol Choline Paraaminobenzoic acid (PABA) Pyridoxamine.HC1 Folic acid Biotin ConcentratiogllOO mi, 50.0 ugm. 50.0 pgm. 50.0 ugm. 50.0 ugm. 50.0 pgm. 50.0 )1gm. 50.0 pgm. 50.0‘ugm. 50.0 pgm. 10.0 pgm. 0.2 pgm. 0.1 pgm. 26 Also, DL-tryptophan, 10.0 mg. and L-cysteine.HCl, 10.0 mg., were added per 100 m1. of substrate. Tests for individual vitamin requirements were made by deleting individual vitamins from this medium and for pyridoximers by deleting them as a group. Medium GAY supplemented with tryptophan and cysteine with and without yeast extract were run as controls. Although all strains grew as expected in the presence of yeast extract, growth in the CAV medium with the complete vitamin supplement was sporadic. This fact was difficult to account for in the light of the successful preliminary trials. Attempts were made to improve growth in medium CAV by replacing cysteine with cystine and by adding the following substances per 100 m1. of medium individually: CaClZ, 5.0 mg.; sodium oleete, 5.0 mg.; sodium acetate, 50.0 mg.; the ash from 0.5 gm. of yeast extract; biotin, 0.9}1gm. In addition, the influence of the concentration of casamino acids on growth was determined by preparing medium CAV with the following percentages of this component: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, and 2.0. None of these modifications was effective in eliminating, the sporadic results obtained in medium CAV. Cystine appeared more favorable than cysteine in the presence of the vitamin mixture but this effect was 27 not apparent in the presmce of yeast extract. The addition of 011012 or sodium acetate was inconsequen-- tial. More biotin favored one strain. Responses were markedly reduced by oleate and no growth occurred in the medium supplemented with yeast extract ash. Increasing the casamino acids concentration resulted in an increased response in medium CAY which fell off above the 1.0 per- cent level but growth remained sporadic in medium CAV. Qgsamipg m n. enzmigallz-hzdgolzged ggsein - Since the vitamin complement of medium CAV was fairly complete, it was believed that some other growth factor(s) must be limiting. Growth-promoting factors have been reported to be present in enzymically- hydrolyzed casein (Kitay and Snell, 1950; Kizer a 91., 1955; Sprince and Woolley, 1945; Wright and Skeggs, 1944). Therefore, it seemed advisable to try substituting this amino acid source for the casamino acids. Media GAY and CAV were prepared along with the usual control medium without vitamins or yeast extract. Similar media were prepared in parallel with O. 5 percent enzymically- hydrolyzed casein (Nutritional Biochemicals Corp.) substituted for casamino acids. The inoculum was pre- pared as before in medium CAY. It was found that the strains tested grew markedly better in the media with enzymic hydrolysate than in those containing acid hydrolysate (Table 3) . Further, the enzymic hydrolysate TABLE 3 28 Growth of’fl, spagglags in enzymic vs. acid.hydrolysates of casein Substrate Culture 1604 711 61 13 88 Enzymic hydrolysate plus yeast extract Acid.hydrolysate plus yeast extract Enzymic hydrolysate plus vitamins Acid.hydrolysate plus vitamins 44.5 44.5 53.5 100 Percent transmittance 14.1 29.0 24.6 100 17.4 19.2 24.7 46.0 37.5 48.5 28.0 24.2 28.0 100 100 100 29 plus known vitamins generally supported better growth than the acid hydrolysate plus yeast extract. No growth was evident in medium CAV in this experiment although varying responses were observed in previous work. Further tests showed that all strains would produce consistently good growth in the media containing the enzymic hydrolysate either with yeast extract or vitamins. Trace growth, which often occurred in control tubes or deficient media, failed to increase on prolonged incubation and gradually disappeared in subculture. This precluded the possibility of adaptation or mutation. Repeated washing of inocula failed to eliminate trace growth. ngglgpment g; a synthetic m - Attempts were made to cultivate each strain in a synthetic medium ‘ containing 24 amino acids in concentrations similar to their average in 0.5 gm. of casein (Katznelson, 1955). These were added to the «semi-synthetic medium used to determine individual vitamin requirements in place of enzymically-hydrolyzed casein. Attempts were also made to grow the organisms in the medium of Campbell _e1:_ 5;. (1953a). Neither of these media supported good growth of all strains and results were inconsistent. Con- sequently, studies were conducted in an effort to formulate a synthetic medium in which all strains would produce consistently good growth. The chemical 30 composition of enzymic casein.hydrolysate was obtained from the manufacturer. From these data, a synthetic medium.was devised which contained amino acids in approximately the same concentrations which would be obtained by the use of 0.5 percent enzymic hydrolysate. The complete medium was of the following composition per 100 ml.: L-lysine DL-isoleucine L-leucine DL-valine L-arginine DL-threonine DL-methionine L-cystine DL-phenylalanine L-histidine L-tryptophan L-glutamic acid Thiamin.HCl Riboflavin Niacin Ca pantothenate Pyridoxine.HCl Pyridoxal.HCl PABA 38.0 mg. 32.0 mg. 52.5 mg. 35.0 mg. 16.5 mg. 20.0 mg. 12.5 mg. 1.5 mg. 22.5 mg. 12.0 mg. 4.5 mg. 102.0 mg. 50.0 pgm. 50.0 ugm. 50.0 ugm. 50.0 ugm. 50.0 pgm. 50.0 pgm. 50.0 pgm. 31 Pyridoxamine.HCl 10.0 pgm. Biotin 1.0 Pgm. Folic acid 2.0 pgm. Glucose 1.0 gm. NH4Cl 100.0 mg. Na acetate 50.0 mg. Nae]. 100.0 mg. KZHPO4 100.0 mg. 5 KH2P04 100.0 mg. c Salts solution X 0.25 ml. I Final pH 6.8-7.0 __ A comparison was made of growth and acid pro- duction by 5 strains of ]_3_. coggulans in the synthetic medium, the enzymic hydrolysate plus yeast extract (medium CAEY), and the enzymic hydrolysate plus vitamins (medium CAEV). The latter medium contained the same vitamin complement added to the synthetic medium when growth in the two media was to be compared. It was found that medium CAEY supported slightly better growth than medium CAEV (Figure 3; Appendix VII). Also, growth in each of these media was better than in the synthetic medium. However, consistent and reproducible results were obtained in growth tests run with the synthetic. Therefore, this medium was used for vitamin and amino acid studies. OPTICAL DENSITY 32 A- CASEIN HYDROLYSATE-l- YEAST EXTRACT 8- CASEIN HYDROLYSATE+ VITAMINS C-SYNTHE TIC ML N/IO NAOH A B C A B C Figure 3. Growth of E. coagulans in synthetic vs. semi-synthetic media. 33 Study 9; Vitgmin Beguirements Studies ;g_semi-synthetic medig - On the basis of the foregoing work, a basal medium was devised for pre- paration of inocula and the testing of vitamin require- ments. This medium had the following composition per 100 ml.: Enzymic casein hydrolysate 0.5 gm. NaCl 100.0 mg. KZHPO4 100.0 mg. KH2P04 100.0 mg. Na acetate 50.0 mg. NH4C1 100.0 mg. Salts solutioan 0.25 ml. Final pH 6.9-7.0 After autoclaving, 0.25 ml. of sterile dextrose solution (0.4gm/ml.) was added per tube. For use in preparing inocula, the basal medium was supplemented with.0.5 percent yeast extract. When used to determine vitamin requirements, the vitamin complement used in medium CAV was added. The biotin level was increased from 0.1 ngm to 1.0 pgm/lOO ml. of medium since this appeared favorable in earlier studies with medium CAV. The vitamin requirements were established by deletion of vitamins from the complete medium. The pyridoximers were deleted as a group. Folic acid and PABA were deleted both individually and together. The r—_-.-._ 34 remaining vitamins were deleted individually. In all cases, the complete semi-synthetic medium served as control. All 22 strains of _B. coagulgns tested were found to require biotin and thiamin for significant growth (Table 4; Appendix VIII and DC). In addition, strain 1604 required niacin and strain 140 either folio acid or PABA. Pantothenate appeared stimulatory for many strains, especially strain 132 which showed much variation in response in its absence. Niacin and choline, also, appeared stimulatory for this strain. PABA and folic acid had little effect on growth when deleted individually; but when deleted together, strain 140 showed a requirement and growth of most strains was reduced. It was concluded that PABA and folio acid were interchangeable. Recently, Baker _e__t 31. (1955) reported this same relationship between folic acid and PABA for a single strain of _B_. coagulans. Tests were then conducted to determine the ability of each strain to grow in the presence of minimal vitamin supplements. The design of this experiment may be noted in Table 5 (Appendix X). While limited growth of most strains was evident in the presence of the minimal vitamin mixtures, growth in general was reduced. No stimulation by pantothenate was noted even with strain 132. It is of interest that growth was obtained with 35 TABLE 4 Growth of B, coggulans upon deletion of vitamins from semi-synthetic medium Vitamin Groupt deleted I II III W Percent transmittance None 37.1 34.9 49.3 Thiamin M“ 284g 91,9 Pyridoximers 38.9 41.3 51.8 Pantothenate 45.2 38.8 55.2 Niacin. 41.9 22‘} 52.2 Riboflavin 38.8 33.4 47.0 Biotin 264g 2642 M Inositol 39. 5 33.7 49.1 Choline 40.3 34.0 52.0 PABA 40.1 35.7 49.1 Folic Acid 39.3 31.0 46.9 PABA, Folic Acid 51.5 29.0 fij&§ 411 mm 22.2 22.2 I Group I - Twenty Cultures (average) Group II - Culture 1604 Group III - Culture 140 All results underlined where a vitamin requirement is evident. TABLE 5 Growth response of _B_. oa to minimal vitamin mixtures in semi-synthetic medium Vitamins Groupt present I II III szcent transmittance All ten 42.} 45.7 46.8 (control) Thiamin , niacin, pant othenate , biotin 57.1 42.5 69.9 Thiamin, niacin, 62.0 47.4 74.8 biotin Thiamin, biotin, 60.9 22,0‘1 76.1 pantothenate None 2.4.2.6 2L2. 21:5. Group I - Twenty cultures (average) Group II - Culture 1604 Group III - Culture 140 All results underlined where definite vitamin deficiencies are evident. 36 '5» r ‘ ~21. 'r-M‘e—fffi.-Lrfi I. "- 37 culture 140 which previously failed to respond in the absence of folio acid and PABA. Sporadic responses with.this culture also occurred in subsequent studies in synthetic media. Vitamin rgguirements in synthetic mggigg - The procedures used in these experiments were essentially the same as described for the examination of the vitamin requirements in the semi-synthetic medium. The complete synthetic medium served as control in each case. Strain A.T.C.C. 12245, which had been reported to require folic acid (Baker g3,gl., 1955), was included in.this study as an additional reference organism. The results of the studies of vitamin requirements in the synthetic medium were similar to those found with the semi-synthetic in that all strains of’B. ooggglang tested required biotin and thiamin and strain 1604 re- quired niacin (Table 6; Appendix XI and XII). Strain 132, although stimulated by pantothenate, did not require it, and the stimulation afforded many other strains by pantothenate in the semi-synthetic medium was not evident in these results. These results differed greatly from the previous ones in that 19 of the 22 strains, including A.T.C.C. 12245, required folio acid or PABA. These vitamins had little or no effect on strain 1604 but afforded marked stimulation for strain 711 and especially for A.T.C.C. 7050. None of these strains, including '——T?l ‘7 .ng‘& I““‘* a. TABLE 6 38 Growth of B, oagulans upon deletion of vitamins from synthetic medium Vitamin Groupt deleted 4___ I II III Percent trgnsmittgnoe None 51.7 53-6 45.5 Thiamin 9:112“ 2848, 91.1 Riboflavin 51.9 52.8 45.8 Niacin 51.9 26,2 45.0 Pantothenate 52.3 55 .1 44.6 Pyridoximers 52.3 52.0 45.6 Biotin 9_6_,_9 28,5 21,3 Folic acid 51-9 55-9 45.4 PABA 53.6 55.4 47.2 PABA, Folic acid 2448 59.3 68,}; All 254.2 2.812 m t Group I - Nineteen cultures (average) Group II - Culture 1604 Group III - Two cultures (average) All results underlined where a vitamin requirement is evident. —. 1‘. 'wr- .4.- ‘-.2.-- .rwu ““‘M [put-H 39 A.T.C.C. 12245, required folic acid or PABA in the semi-synthetic medium. It was tentatively concluded that the enzymic casein hydrolysate either provided factors which.partially replaced the folic acid or PABA requirement of B. coggylans or precursors from which this organism could synthesize these vitamins. Study 9; Amino Acid Requirements The amino acid requirements were determined in the synthetic medium described previously except that riboflavin was omitted from it. Otherwise, the general procedure was essentially the same as employed in vitamin studies. However, instead of being deleted individually, the amino acids were deleted in groups based on their specific type as follows: Group 1 - DL-valine, L-leucine, DL-isoleucine, and DL-threonine Group 2 - DL-methionine, and L-cystine Group 3 - L-glutamic acid Group 4 - DL-phenylalanine, L-tryptophan, and L-histidine Group 5 - L-arginine and L-lysine (All tests were performed in duplicate on at least two different days. None of the strains developed in synthetic media when the nitrogen equivalent as (NH4)2804 was substituted for amino acids. f?“ “Ifflt. {rd—ft . he 40 The amino acid requirements appeared to be mostly non-specific. Of the 22 strains tested, only 4 required any specific group(s) of amino acids (Table 7; Appendix 11111 and XIV). The deletion of Group 1 (DL-valine, L-leuoine, DL-isoleucine and DL-threonine) from the medium had a pronounced effect on the growth of only one strain (No. 132). Four strains (C2253, 88, 43P, rte and 132) were found to require the presence of glutamic acid. Studies 9n §timulgtign ‘br ii.- v). - ‘ L.1.-l" Effect 9;,DL-gmino agig_ - A search was made for the factor(s) responsible for the relatively poor growth in the synthetic medium as compared to the semi-synthetic. The first idea investigated concerned the concentrations of some of the amino acids. In formulating the synthetic medium, amino acids were incorporated according to their percent occurrence in the enzymic hydrolysate without regard for the availability of D-isomers in racemio mixtures. Consequently, the growth of 4 strains in.the synthetic medium containing twice the concentration of the complement of DL-amino acids used originally, was compared with.growth in.the normal medium, and with the semi-synthetic medium containing the enzymic hydrolysate. This procedure had no significant effect 41 TABLE 7 Growth of B, coggulans upon deletion of amino acids from synthetic medium Amino acids Groupt deleted I II III IV Percent transmittance None 46.3 54.3 57.0 44.0 DL-valine, DL-threonine, 55.0 57.7 86,2“ 55.2 . L-leucine, DL-isoleucine i DL-methionine, 53.4 60.5 64.7 57.4 7" L-cystine L-glutamic acid 59.3 ,fléyg §§&& ngg DL-phenylalanine, 47.4 57.6 72.6 variable L-tryptophan, Lahistidine L-arginine, L-lysine 47.8 57.3 61.5 variable 411 25.1 22.2 2612 282.6. 5 Group I — Eighteen cultures (average) Group II - Two cultures (average) Group III - Culture 132 Group IV - Culture 43? It All results underlined where a definite amino acid requirement is evident. 42 on the growth response of the strains tested (Table 8). ,Effggt g; non-digiyzable enzymic cgsein hydrolysgtg gnd 9; tyypsin - Experiments were then conducted to determine if stimulation could be removed from the enzymic casein hydrolysate by dialysis and if any marked degree of stimulation was provided by trypsin 1-300. ‘Under present conditions, the specific hydrolytic agent employed in preparing the enzymic hydrolysate was not known and unobtainable. However, Kizer 23 gl. (1955) reported that factors concomitant with impure trypsin were stimulatory for certain lactic acid bacteria. The normal synthetic medium and the semi-synthetic medium containing 0.5 percent of the enzymic hydrolysate were prepared as controls. Trypsin 1-300, inactivated by heating at 100°C. for 10 minutes, was added to a portion of the synthetic medium at the rate of 0.25 percent as a check on the possibility that stimulation might be associated with this enzyme preparation. The effect of dialysis was determined as follows: 50 ml. of 5.0 percent enzymic hydrolysate were placed in a cellophane bag of l—inch diameter and dialyzed in 16 liters of distilled water at 5°C. for 24 hours. The water was changed twice during this period and discarded. The semi-synthetic medium.was then prepared with.the non-dialyzable fractiOn of the casein hydrolysate substituted for the normal preparation. Oneéhalf of this 43 TABLE 8 Effect of increased concentration of DL-amino acids on the growth.of B, coagulgns in synthetic medium Substrate Culture 141 1604 10545 7050 Percent trgnsmittance Casein hydrolysate 22.4 25.1 26.7 64.5 plus vitamins Normal synthetic 50.4 49.1 47.1 51.6 substrate Synthetic substrate 58.9 47.0 43.4 58.6 with DL-amino acids doubled 44 ‘nedium.was supplemented with the amino acid complement used in the normal synthetic medium since it was assumed that the bulk of the amino acids were removed from the casein during dialysis. The addition of trypsin to the normal synthetic medium.had no significant effect on the growth of 3 strains of B. coagulans. Growth was markedly reduced in the medium.prepared with the undialyzable fraction of the casein hydrolysate; however, this reduction in growth apparently did not result from a deficiency in amino acids (Table 9). Therefore, it appeared that stimulation was associated with a dialyzable factor(s) other than free amino acids. Effect g; various buffers - In a communication with the Nutritional Biochemicals Corporation, it was learned that the pH of their vitamin-free enzymic casein hydroly- sate was lowered to about 3.0 (found to be about 4.0 as received) with acetic acid after hydrolysis and that the incorporation of this product in culture media generally provided sufficient acetate to satisfy the demands of most organisms employed for vitamin assay. The possibility came to mind that the acetate residue might contribute as an indirect stimulus by augmenting the inherent buffering capacity of the semi-synthetic medium with respect to acid produced from.glucose by responsive strains. Investigations were carried out to check this It” 45 TABLE 9 Effect of trypsin and non-dialyzable casein.hydrolysate on the growth of p, coggulgns Substrate Culture 141 10545 1604 Percent trgnsmittance Normal casein 26.7 28.8 27.? fi hydrolysate h 5 Normal synthetic 51.0 49.4 47.8 f substrate L Synthetic substrate 45.6 49.2 48.7 plus trypsin l-300 Dialyzed casein 64.8 63.0 64.8 hydrolysate Dialyzed casein 53.4 45.5 50.6 hydrolysate plus amino acids 46 possibility. ‘A trypsin hydrolysate of casein was prepared in general according to the methods of Kemmerer and Shapiro (1947) using vitamin and fat-free casein (British.Drug House) and trypsin 1-300. After hydroly- sis, aliquots of the hydrolysate were acidified to pH 4.0 with acetic acid, H3P04, and HCl, respectively. .“ A‘M—lql Norit A was added for adsorption and the dialysates Amr- filtered under mild suction through Whatman No. 5 i filter paper in a Buchner funnel. The respective t—“—____ _k . , ‘lu filtrates were diluted to give a final concentration of 5.0 percent of original casein. The semi-synthetic medium was then prepared using each of these hydrolysates as well as the N.B.C. hydrolysate. In addition, the synthetic medium was prepared and aliquots were sup- plemented with acetic acid and H3P04 at the rate they occurred in the media prepared with the corresponding hydrolysates. The normal synthetic medium served as control in parallel with that prepared with the HCl- acidified hydrolysate. Media were dispensed in 5.0 ml. lots, autoclaved, and inoculated with strains 1604, 141, and 7050. In addition, the amount of N/lO HCl required to titrate duplicate 50 ml. portions of each medium from pH 7.0 to pH 3.0 was determined electrometrically. It is of interest that the quantity of acetic acid required to 4? acidity the laboratory-prepared hydrolysate was (equivalent to the concentration of total volatile acids in the N.B.C. hydrolysate as determined by steam distillation and subsequent titration with N/lO NaOH to neutrality. Figure 4 Shows typical titration curves obtained by the gradual addition of N/lO HCl to 50.0 ml. quanti- ties of the modified and unmodified synthetic and semi- synthetic media. Individual titration values appear in Appendix.XVI. It should be kept in mind that growth in these media falls off at about pH 4.3 so that little significance is attached to buffer action below this level under present conditions. Apparently buffer action may play an important part in stimulation since those media showing greater buffering capacity generally support better growth.(Figure 5; Appendix XV). This is particularly evident in the case of strains 141 and 1604 which.produced considerably less growth in the HCl- acidified trypsin.hydrolysate than in the same hydrolysate acidified with either H3P04 or acetic acid, or in.the N.B.C. hydrolysate. Growth.of these strains in the latter three media approaches equivalence. The growth of strain A.T.C.C. 7050 was not enhanced in.the medium containing the N.B.C. hydrolysate as compared with.the normal synthetic medium, and less growth was Obtained in the medium.containing the acetic acid-acidified trypsin.hydrolysate. u A r .5 .mn I“! 1““ 4 PH T I T r I I —' I T SYNTHETWC ACETATE _——— PHOSPHATE L——- ----:----No ADDITIONAL \ BUFFER 6 _ \ SEMI-SYNTHETIC 4 r- : J 1 l_ i I L l IO 20 30 40 ML N/IO HCL Figure 4. Titration curves for semi—synthetic and synthetic media with and without additional buffers. 48 new ’T-V. L"; m '3 ' s. I ‘ -‘41' 49 .AVERAGE orz STRAINS DSTRAIN 7050 60 40 20 PERCENT TRANSMI TTANCE A B C A- N.B.C. HYDROLYSATE B-TRYPSIN HYDROLYSATE+ ACETATE C-TRYPSIN HYDROLYSATE-I- PHOSPHATE D- TRYPSIN HYDROLYSATE+ HCL E- SYNTHETIC MEDIUM F-SYNTHETIC MEDIUM+ACETATE G-stTHETIC MEDIUM+ PHOSPHATE Figure 5. Comparison of modified trypsin hydrolysates and synthetic media with N.B.C. casein hydrolysate. 50 In the synthetic medium, the response of all three strains was markedly enhanced by phosphate. In contrast, only strains 141 and 1604 reacted favorably to the supplement of acetate. In general, the addition of a suitable buffer to the synthetic medium results in growth either equivalent to or approaching that obtained in the corresponding semi-synthetic medium. Attempts were not made to establish optimum buffer concentrations because of inherent strain variation. ‘5- HV-‘f—{J’ " '- DISCUSSION In addition to providing strains of Bacillus ggagglggg for nutritional studies, this study brought to light an apparent weakness in thermoacidurans agar (Difco). Based on their ability to grow on this medium, each of the 177 isolates might be considered acid-tolerant and a potential spoilage hazard in commercial packs of tomato Juice. However, only about 30 percent belonged to this select group. The remainder were of no apparent spoilage significance, especially in view of their inability to grow at a low oxygen tension. Therefore, the ability of bacteria to grow on proteose-peptone acid agar may not necessarily indicate their ability to spoil commercially-packed tomato Juice as was suggested by Stern.gt 3;. (1942). Results of this study suggest that the incorporation of a suitable indicator into thermoacidurans agar and increasing its dextrose concentration may enhance its value as a plating medium. Moreover, these modifica- tions may find application in taxonomic studies on aerobic sporeformers since the ability to grow on this medium.and lower its pH appears of greater value in segregating species than growth alone. The study of the vitamin requirements of g. coggulans in semi-synthetic and synthetic media showed that three strains (1604, 711, and A.T.C.C. 7050) ‘1’" EIH‘ 1:.- ..w . -——-_‘?‘ 52 required only biotin and thiamine These observations agree with those of Knight and Proom (1950) and Proom and Knight (1955) who noted that 16 strains (including A.T.C.C. 7050) of this organism required these same growth.factors in semi-synthetic and synthetic media. Unfortunately, strains requested from Campbell ‘gg‘gl. (1953a) for comparative purposes were unavail- able. However, the results obtained in the synthetic medium and in one instance in the semi-synthetic medium (strain 140) support their observations that many strains may require folic acid in addition to biotin and thiamin. Although three strains (711, 7050, and 1604) did not require folic acid, two of the three were stimulated by this vitamin. This suggests that folic acid is of general importance in the nutrition of g. coagulang. It is of interest that a difference in vitamin requirements in semi-synthetic vs. synthetic media was not observed by Knight and Proom (1950), Proom and Knight (1955). However, this study indicates that the test medium used is an important factor in nutritional studies with.§, coagulgng. This was evidenced by the general requirement for either folic acid or PABA in the synthetic but not in the semi-synthetic medium. Only one strain tested was shown to require niacin in the present study. Except for single strains 1’?“ * ‘w*w.*- -.= 53 reported to require niacin by Campbell and Williams (1953a) and by Cleverdon.g§_g_. (1949), no further reports in this respect were found in the literature. Therefore, the requirement for this growth factor appears rare within the species. The amino acid requirements of E. coggulgns were found to be generally non-specific. However, this “Au-I an;" ‘ V i. is not surprising. Campbell and Williams (1953a) found that specificity in the amino acid requirements - ,. -_Ao~\:1.4 ' shown by p. coagulans at higher temperatures of g incubation (45° and 55°C.) may disappear at a lower i incubation temperature (36°C.). Also, Bhat and Bilimoria (1955) noted that certain metabolites necessary for growth at 55°C., were non-essential at 37°C. Nevertheless, the organisms still required organic nitrogen. Apparently, this species possesses synthesiz- ing mechanisms which function at low temperatures but not at high. This study indicated that glutamic acid may play an important role in the nutrition of g. coagulans, either as an essential metabolite or as a growth supplement. Allen (1953) and Andersen and werkman (1940) noted that glutamic acid may be important in the nutrition of this organism. In this study, most strains of’fi.lgggg2;gg§ grew poorly in synthetic media in the absence of glucose 54 indicating that amino acids serve primarily as a nitrogen source. The lesser growth response obtained in the synthetic medium as compared with enzymatic digest (Nutritional Biochemicals Corp.) apparently did not result from a deficiency in L—amino acids; but, the results indicated that the concentration and pro- t_ portion of DL-amino acids may be important with different strains. Also, stimulation did not appear to be associated with trypsin since its addition to the synthetic medium.had a negligible effect on growth. 3 Therefore, the factor(s) associated with certain proteolytic enzymes reported to be stimulatory for certain lactic acid bacteria by Kizer gt g1. (1955) did not appear to be in operation here. When.the synthetic medium or the laboratory prepared trypsin hydrolysates were supplemented with a similar concentration of acetate to that found in the N.B.C. hydrolysate, approximately the same degree of stimulation generally resulted for the strains tested. Since acetate could be replaced by phosphate, it is tentatively concluded that buffering action is primarily responsible for stimulation. This may ex- plain why stimulation was removed by dialysis since acetate could readily pass through the membrane. Noteworthy, is the failure of strain A.T.C.C. 55 7050 to show evidence of stimulation in media supplemented with acetate, although excellent growth resulted in its absence, or when media were supple- mented with phosphate. It is hypothesized that the extent of growth of this strain was limited by the increasing amount of undissociated acetic acid accumulating during the gradual drop in pH resulting from glucose fermentation. Thus, it is assumed that variation in strain tolerance exists toward acetic acid, and the same may be true for other organic acids x.‘ n‘-‘~0-—— _ ' it - '7‘... .5 “Frat".zrmw (A. 3'. (Rice and Pederson, 1954). Phosphate appears to be the more suitable buffer in view of its relatively non-toxic character. The sporadic results and frequent poor responses obtained using casamino acids as a nitrogen source, and the failure to obtain good growth of all strains in the medium of Campbell and Williams (1953), are not readily explainable. That the synthetic medium developed herein is considerably higher in total N than that of Campbell and Williams, and further differs in the type and proportion of amino acids present, might account for the different results. This idea is in line with the results of Proom and Knight (1955) who obtained growth of 16 strains of B, ggagulgng in a 14-amino acid medium, but not in a medium containing only 7 amino acids. SUMMARY One—hundred and seventy-seven spore-forming bacteria isolated from the wash waters of Canadian tomato Juice packing plants were screened to deter- mine the ability of their spores and vegetative cells to grow aerobically and anaerobically in tomato Juice. Although.each organism grew well on thermoacidurans agar (Difco) (DTA), about 70 percent were not con- -. 0.- r.— fi .‘r-m4F‘ sidered a commercial spoilage hazard owing to their failure to develop in tomato Juice at a low oxygen tension. Aerobically, these cultures produced alkaline {I‘D-r ' “J‘H--‘n_ A . reactions in tomato juice and on DTA, whereas the remaining 30 percent generally produced acid reactions and could develop at a low oxygen tension. It was con- cluded that routine bacteria counts on DTA do not pro- vide a true index of spoilage possibilities. ‘However, results indicated that the addition of 0.004 percent bromcresol green and increasing the dextrose con- centration to 1.0 percent rendered it more valuable as a plating medium for detecting bacteria capable of causing flat-sour spoilage of tomato Juice. 0f 37 of the canning plant isolates selected for taxonomic study, 19 were identified as Bgcilgus ggagulgng, 3 as Basiliug lichenif r , and 15 as Bacillug guptilig. 57 The vitamin and amino acid requirements of 15 of the canning plant isolates identified as g. coagulgns and 8 authentic strains of this organism were studied at 37°C. In a medium comprising vitamin-free acid hydrolyzed casein, yeast extract, glucose, and mineral salts, growth occurred earlier at pH 5.5 than at 7.5. Substitution of malate, succinate, fumarate or citrate for glucose in this . “Eva—m. . .fl'H‘To medium indicated that citrate was not utilized whereas response to the remainder was feeble. The ‘fl;- -._.—..__4 ~ _ v sporadic results obtained in this medium when a mixture of vitamins was substituted for yeast extract were not due to a deficiency of cysteine or trypto- phan, This condition was corrected by substituting vitamin-free, enzymically-hydrolyzed casein for the acid hydrolysate. Biotin and thiamin were required by all strains in the semi-synthetic medium. In addition, one strain also required niacin and another folic acid. An additional requirement for folic acid (or PABA) was noted for most strains in a synthetic medium containing 12 amino acids. This probably reflected inherent differences in composition between the semi- synthetic and synthetic menstrua. ‘With few exceptions, the amino acid requirements were generally non-specific at 37°C. Glutamic acid 58 appeared essential for a few strains and provided stimulation for others. The enhanced growth generally afforded by enzymic casein hydrolysate (Nutritional Biochemicals Corp.) was largely attributed to buffering action resulting from its high acetate content. Phosphate was considered a more suitable buffer for B. coagulgns since one strain proved acetate sensitive. BIBLIOGRAPHY IAllen, M.B. 1953 The thermophilic aerobic sporeforming bacteria. Bact. Rev. 17, 125-173. Andersen, A.A. and Werkman, C.H. 1940 Description of a dextrolactic acid forming organism of the genus Bacillus. Iowa State College J. Science 14, 187-191. Baker, H., Hutner, S.H. and Sobotka, H. 1955 Estima- tion of folic acid with.a thermophilic bacillus. Proc. Soc. Exptl. Biol. Med. 89, 210-212. Baker, H., Sobotka, H. and Hutner, S.H. 1953 Growth requirements of some thermophilic and mesophilic bacilli. J. Gen. Microbiol. 9, 485-493. Becker, MeE. and Pederson, 0.8. 1950 The physiological characters of Bacillus coggulans (Bgcillus thermo- gcidurgns). J. Bact. 59, 717-725. Berry, R.N. 1933 Some new heat-resistant acid- tolerant organisms causing spoilage in tomato juice. J. Bact. 25, 72-73. Bhat, J.V. and Bilimoria, M.H. 1955 Problems in thermophily. IV. The nutritional requirements of some thermophilic bacilli. J. Indian Inst. Sci. 37, 113-115. Cameron, E.J. and Bohrer, C.W. 1948 Spoilage control procedures for tomato Juice. Information Letter Supplement No. 1170, National Canners Assn. ’1’ l.’ .—;- _ _ ._ _a‘nrfllng’Kfl 60 Campbell, L.L. and Williams, 0.3. 1953a The effect of temperature on the nutritional requirements of facultative and obligate thermophilic bacteria. J. Bact. 65, 141-145. 1953b Observations on the biotin requirement of thermophilic bacteria. J. Bact. 65, 146-147. Cleverdon, R.C., Pelczar, M.J., and Doetsch, R.N. 1949 Vitamin requirements of Bgcillus coggulgn . J. Bact. 58, 113-114. Ford, C.M., Brown, R.W., Steward, A.E., Mason, C.T., and Schmidt, C.F. 1952 Biochemical studies of organisms producing flat-sour spoilage of tomato Juice. Proc. Soc. Ame Bact. 18-19. Gordon, R.E. and Smith, N.R. 1949 Aerobic sporeforming bacteria capable of growth at high temperatures. J. Bact. 58, 327-341. Hammer, B.W. 1915 Bacteriological studies on the coagulation of evaporated milk. Iowa Agr. Expt. Sta. Res. Bull. 19, 129. Hussong, R.V. and Hammer, B.W. 1928 A thermophile coagulating milk under practical conditions. J. Bact. 15, 179-188. Johns, C.K. 1953 ‘Unpublished data. Bacteriology Division, Science Service Bldg., Ottawa, Canada. 1"“ ” 61 thnson, B.C. 1949 Methods of vitamin determination. Burgess Pub. 00., Minneapolis, Minn. Katznelson, H. 1955 Personal communication. Bacteriology Division, Science Service Bldg., Ottawa, Canada. Kemmerer, A.R. and Shapiro, F. 1947 Three sources of amino acids for niacin assay. Anal. Chem. 19, 358-359. Kitay, E. and Snell, E.E. 1950 Some additional nutritional requirements of certain lactic acid bacteria. J. Bact. 60, 49-56. Kizer, D.E., Hankin, L., Speck, M.L., and Aurand, L;W. 1955 Stimulation of lactic acid bacteria by substances concomitant to certain proteolytic enzymes. J. Dairy Sci. 38, 303-309. Knight, B.C.J.G. and Proom, H. 1950 A comparative survey of the nutrition and physiology of mesophilic species in the genus Bacillus. J. Gen. Microbiol. 4, 508-538. Proom, H; and Knight, B.C.J.G. 1955 The minimal nutritional requirements of some species in the genus Bgcillus. J. Gen. Microbiol. 13, 474-480. Rice, A.C. and Pederson, 0.8. 1954 Factors influencing growth of Bgcillus coagulans in canned tomato Juice. II ACidic constituents of tomato Juice and specific organic acids. Food.Res. 19, 124-133. Fifi- 1““ pl .: a...” .4:.¢an-—.——-.T 62 Sarles, W.B. and Hammer, B.W. 1932 Observations on Bgcillus coagglan . J. Bact. 23, 301-314. Smith, N.R., Gordon, 3.3., and Clark, F.E. 1946 Aerobic mesophilic sporeforming bacteria. U.S. Dept. Agr. Misc. Pub. No. 559. 1952 Aerobic sporeforming bacteria. U.S. Dept. Agr.. Monograph No. 16. Sprince, H. and Woolley, D.W. 1945 The occurrence of the growth factor strepogenin in purified proteins. J. Am. Chem. Soc. 67, 1734-1736. Stern, R.Mt, Hegarty, C.P., and Williams, 0.B. 1942 Detection of Bacillus thermoacidurgns (Berry) in tomato Juice, and successful cultivation of the organism in the laboratory. Food Res. 7, 186-191. 'Wright, L.D. and Skeggs, H.R. 1944 The growth factor requirements of certain streptococci. J. 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