THE INFLUENCE OF BACTERIOPHAGE, ANTIBIOTICS, AND Eh ON THE LACTIC FERMENTATION OF CUCUMBERS THE ILTLUEtTCE 01 BACTEBIOPHAGE, ANTIBIOTICS, AND % ON THE LACTIC JEEKENTATION OP CUCUMBERS by LOUIS WILLIAM EAVILLE A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partiel fulfillment of the requirements for the degree of DOCTOE 0? PHILOSOPHY Department of Bacteriology and Public Health ish e ACKNOWLEDGMEHT The author wishes to express his sincere appreciation to Dr. F. W. Fabian, Professor of Bacteriology and Public Health, under whose ahle guidance this work was done, for his unfailing interest throughout the course of the work and for his interest and criticisms during the preparation of this manuscript. TABLE Of COCT SETS List of T a b l e s ............................... .. Page tii List of F i g u r e s ......................................... itr Introduction.......................................... .. i 1 Literature Review 3 ................. ............ .. Id eiit ification Studies E xp e ri m en t al ...................................... Results anc! Discussion .......................... TLe Occurrence of Bacteriophage Paces Specific for the Lactobacilli Concerned with the Cucumber fermentation Literature . ....................................... Experimental ....................................... Results and Discussion ............................ The Antagonistic Action of Aerobic Sporogenic Soil Organisms toward the Lactohacilli Concerned with the fermentation of Cucumbers Literature . ..................................... Experimental ........ .. Results and Discussion ................... The Relationship between the Metabolic Activity of Aerobic Sporogenic Bacteria and their Ability to Grow in Cucumber Brines L i t e r a t u r e ................... .................. .. 1. Determination of normal time-potential curves of 16° and h0° salometer. Experimental ............................... Results and Discussion ..................... 2. The Z etermination of time-potential curves of some of the organisms from cucumber brines Experimental . ........................... Results and Discussion . . . . . . . . . . . 3. The effect of sodium chloride and lactic acid on the time-potential curves of aerobic sporogenic bacteria Experimental . . . . . . . . . ............. Results and Discussion . . ................. U. The influence of the oxidetion-reduetion potential of the medium on the sensitivity of spore-formers to salt Experimental . . . ......................... Results and Discussion . . . ............... i U 6 10 12 16 17 IS 20 25 27 29 37 38 Ui Uif TAEL35 OF C0ETJ3TS (Continued) General Lis cues ion Summary Page ...................................50 ................................................ 5& Literature C i t e d * . ............................ ii 5^ LIST OF TABLES Teble 1 2 3 U 5 6 Page Results of analyses of "brines from spoiled pickles ............................................ Characteristics of aerobic sporogenic bacteria isolated from spoiled cucumber pickle brines . . . 13 Analyses of brines from spoiled processed dills and salt stock cucumbers ............................ lU Analyses of brine samples from 12 vats of spoiled genuine d i l l s ...................................... lU The antagonistic action of aerobic sporogenic organ­ isms toward strains of lactic acid bacteria iso­ lated from sources other than pickle brines. . . . Showing the effect of the composition of the medium on the elaboration of antibiotic substances against the lactic acid organisms isolated from cucumber brines. ................... S The effect of the oxidetion-reduction potential of the medium on the sensitivity of B. cereus to sodium chloride.. . . . . .................... 10 7 Lactobacilli from sources other than fermentinr cucumbers ................................... 7 9 5 • 21 U5 The effect of oxide tion-reduct ion potentials on the sensitivity of spore-formers to sodium chloride in nutrient broth .......................... The effect of oxidation-reduetion potentials on the sensitivity of spore-formers to sodium chloride in cucumber extract ........................... ill V7 ^5 LIST OF FIGURES Figure 1 2 3 1+ 5 6 7 8 9 10 Page Apparatus used for determining time-potential curves of fargentine- cucumbers.................... 28 Normal oxidation-reduetion potential, pH, and peptonicer curves of cucumbers salted at Lo° sel onset e r ......................................... 30 Normal oxidation-reduetion potential, pH* end peptonizer curves of cucumbers salted at 16° salometer, ...................................... 3^ Apparatus for determining time-potential curves of pure c u l t u r e s ..................................... 33 Time-potential curves of B. cereus, B. subtilIs, 3. mesentericus fus c u b , and B. vulgatusin (a) cucumber extract and (b) nutrient broth •. . 35 Time-potential curves of B, megatherium, 3. pumllus, L. plantarum, and A, aerogenes in (a) cucumber extract and (b) nutrient broth . . ............... The The The jG effect of salt on the time-potential curves of aerobic snorogenic bacteria isolated from spoiled pickle b r i n e s ............................. 39 effect of salt on t*e time-potential curves of aerobic sporogenic bacteria isolated from spoiled pickle brines ...................... .. *40 effect oflactic acid on the time-potential curves of B. cereus.................. , . . , . *42 The combined effects of lactic acid and salt on the tirae-potenticl curves of 3, c e r e u s , ............ iv ^3 XNTBODUCTIOK In many commercial fermentations contamination can be controlled ■by sterilization of the raw materials and "by inoculation with pure cul­ tures of the fermenting* agents. The cucumber fermentation, on the other hand, depends upon the addition of relatively large amounts of salt to inhibit the development of undesirable micro-organisms. These undesirable micro-organisms, of which members of the genus Bacillus are probably the most Important, are normal inhabitants of the soil and gain access to the fermentation vats through the small particles of soil adhering to the cucumbers at the time they are salted. Each year the pickle industry suffers considerable losses due to spoilage. Usually this spoilage occurs in the latter stages of the fermentation, although in some instances spoilage is evident within the first few weeks after the cucumbers have been salted. During the past two years numerous samples of brine from spoiled salted cucumbers from all parts of the United States and Canada have been analyzed in this laboratory. The preponderance of aerobic spore-formers in many of these brines has suggested the need for further investigations. Although a certain amount of attention hap been devoted to the study of this particular group of organisms, there has been no attempt to define the conditions which determine their ability to overgrow the normal lactic acid organisms in certain vats and yet fail to develop in others under apparently similar corditions. The purpose of this study is to investigate some of the factors which might explain the failure of the lactic fermentation of cucumbers to proceed normally and thus permit these aerobic sporogenic bacteria to grow and in some cases cause spoilage. 2 LITERATURE REVIEW Bacteriological invest Igat ions of spoilage in cucumber fermentatlone to date have dealt primarily with the isolation of a limited number of orgenisms from the brines of softened picles and with studies of their tolerance to salt and their abilities to cause softening of pickles. As early as 1899 Aderhold (l) isolated spore-forming bacteria and Bacterium coll from softened pickles. This investigator, however, erroneously concluded that Bacterium coll was the cause of the softening and attached no significance to the presence of the spore-formers. Kossowics (30), in I90S, similarly studied the softening of pickles and reported that the Bacillus mesenterlcus group was concerned with soft pickles and not, as reported by Aderhold, Bacterium coll. Eahn (37), In I 913, conducted one of the earliest investigations of spoilage of pickles in this country. tributing to spoilage of salt stock. He recorded various factors con­ Acid was found to be effective in inhibiting the growth of proteolytic bacteria while salt alone was of little value. LeEevre (31) studied the ability of 50 organisms to attack vegetable matter and found that 16 of this number were capable of soften­ ing cucumbers. Only two organisms (B. vulgatus and B. mesenterlcus fus- cus) were capable of growing in hi#i salt concentrations and only four (B. vulgatua, B. carotovorus, B. cereus, and B. lactic aerogenes) could grow over a range of pH 7.0 to pH b.0. LeEevre concluded that softening was caused by a variety of organisms and that 7-8 per cent salt was the critical point in the relationship of salt to the preservation of cucumbers. Joslyn (27)* In 1928, investigated the softening of dill pickles and agreed with previous workers that this type of spoilage was caused by bacterial action. He also observed that slipperiness increased in brines of pH 3.0 to pH 3.1 and suggested that the enzymes produced by spoilage organisms were probably active at a lower pE than that tolerated by the organisms themselves. In 193*5 Fabian and Johnson (13) isolated an organism from the brine of soft pickles which was capable of caueina* slipery pickles in 6-12 hours and mushy pickles in 12-2b hours. This organism which corresponded to B. mesenterlcus fuscus prew in salt concentrations up to and including 9 per cent salt. Identification Studies The primary purpose of this study was to attempt to explain the failure of some cucumber fermentations to develop normally. Therefore, only those samples whose brines, upon analysis, showed abnormally large numbers of aerobic spore—forming organisms were selected for study. Un­ doubtedly much spoilage of this type occurs, however only six such brines have been analyzed in this laboratory over the period of time covered by this work. Although none of the pickles from these samples showed visible signs of softening, spoilage was manifested by off-odors, unus­ ually turbid brines, end improper curing. A summary of the pertinent data of these samples is presented in Table 1: k Table X. Sample number B C E Results of analyses of 'brines from spoiled pickles. pH Per cent salt U.70 3.30 3.39 T G H 3.^7 3.30 Per cent lactic acid 0.53 0.75 5.** 3.8 12.1 16.1 0.53 0.53 3.5 17.0 15.0 11.3 1.00 1U.0 1.10 19.8 3.1 Bacterial count * X X x X X * log 101 10, io* 10-5 Ho. of isolates k 3 5 2 9 6 * Hot availabl e« When examined bacteriologically, these samples appeared to contain only Gran—positive spore-forming bacilli. Microscopic examination of colonial types on solid media suggested that several of these brines were practically pure cultures of a certain organism and several of these colonies were isolated for further study. In the remaining samples, two or three colonial types were reco^iized and several of each type were isolated. In all cases, each colony selected represented many similar colonies on the same plate and a total of 29 colonies were isolated and purified by repeated plating for further study. Microscopic examination uroved all cultures to be aerobic sporogenic Gram—positive rods thereby placing them all In the genus Bacillus. All 29 cultures were then subjected to a series of biochemical studies in order to identify them and thereby eliminate duplication of effort in future work. The biochemical tests used in this study were, for the most uart, those suggested by Smith, Gordon, and Clark (38) as the most satisfactory tests for characterizing members of the genus Bacillus. 5 In addition, the tolerance of each organism to salt was determined. Since these organisms were cultivated for several months prior to this experiment on a medium devoid of salt, all strains were transferred daily in a liquid medium of the following composition: Bacto-proteose-peptone J ,Q g Bacto-beef extract 3,0 g Sodium chloride, C, P, 50.0 g Distilled water 1000.0 ml After daily transfer in this 5 P er cent sodium chloride broth for 10 days, the percentage of salt was Increased by 1 per cent on each trans­ fer until a concentration was reached at which each organism failed to grow. The ability of these organisms to elaborate enzymes capable of softening cucumbers was determined by using the method of Fabian and Johnson (13). The organisms were grown for five days at 3O°0 in 500 ml wide—mouth Erlenmeyer flaslcs containing 35® ral of the beet^sugar molasses medium described by these workers. After this period of incubation, a sound pickle from salt stock was freshened to remove salt, washed in toluene, and placed in the flask in such a manner that the entire pickle was covered with the medium. The medium was then layered with toluene. Incubated at 30°C, and examined at appropriate Intervals for deterioration of the cucumber. Results and Discussion On the basis of biochemical studies, these organisms can be divided Into six separate types or species as shown in Table 2. In work of this type it is difficult to definitely assign a specific name to a 6 Table 2. Characteristics of aerobic sporogenic bacteria isolated from sooiled cucumber -Dickie brines. Types Z II III + — ♦ ♦ ♦ + + — * ♦ Fermentation of j Sucrose Lactose Maltose Glucose Fructose Marinos e Sr lactose Xylose Arr.binose Raffinose Shermo se Mannitol Dextrin • • • .. + Casein hydrolysis «► + + + + t + ¥ *■ ¥ — — — — ♦ — + * Starch hydrolysis ♦ Gelatin hydrolysis IV V VI + ♦ + + — - ¥ ¥ * t - ¥ ¥ ¥ ¥ ¥ ¥ t t + + ♦ * ¥ - — ¥ ¥ ¥ ¥ <- ¥ ¥ * + — - ¥ - ♦ * 4- + ¥ * ITitrate Reduction + ♦ +■ - — - Vo ^es-Pros 'truer Reaction + - + - V Salt tolerance (per cent) 9 9 11 11 10 9 Soften*nr of cucumbers - - ¥ + - - Red. , pept. Red. * oeot. U React! ~n in litmus milk Ihrmter of isolates ¥ •Red., ••pept. 15 ♦Red. — reduced / * * o er> t. at r > e r o to n ic e d . y / 7 Red., pept. 2 1 ¥ R e d . , Red., pent. pept. b 1 member of the genus Bacillus on the "basis of the classification in the 6th edition of the Manuel of Determinative Bacteriology (5)* This classifi­ cation is "based on the work of Smith et el. (38) and Is an attempt to con­ solidate the genus* Tor apparent reasons, this grouping of various species Into a single new species is not always compatible with certain rather important characteristics of the organisms. Tor example, those organisms listed as type III In Tahle P. correspond to the previous descriptions of Bacillus mesenterlcus fuscus even to their ability to soften pickles as However, in the 6th edition of reported "by Tablan and Johnson (13)» Breed's Manual, this organism is listed as a synonym of Bacillus subtllls althou#£i differing In several respects from the present description of Bacillus subtills. The organisms listed as type II In the table, on the other hand, correspond veiy well to the description of Bacillus subtil Is and do not soften pickles. One other Important difference between these two groups of organisms is their tolerance to salt. Type III organisms will grow In salt concentrations up to and including 11 per cent while tyoe II organisms do not tolerate above 9 per cent salt. Those organisms grouped In type I appear to be the most prevalent In these brines. Slightly more than half (15) of the 29 organisms studied were classified as Bacillus cereus. This organism, although predominating, does not decompose pickles and does not grow in salt concentrations above 9 per cent. Of the remaining three groups, only those organisms belonging to type V were classified positively. These organisms, which agree in all respects with Bacillus megatherium, are not responsible for the soften- 8 member of the genus Bacillus on the basis of the classification in the 6th edition of the Manual of Determinative Bacteriology (5). This classifi­ cation is based on the work of Smith jst al. (3*0 and is an attempt to con­ solidate the genus. Tor apparent reasons, this grouping of various species into a single new species is not always compatible with certain rather important characteristics of the organisms. For example, those organisms listed as type III in Table ? correspond to the previous descriptions of Bacillus mesenterlcus fuscue even to their ability to soften pickles as reported by Fabian and Johnson (13)* However, in the 6th edition of Breed's Manual, this organism is listed as a synonym of Bacillus subt ills although differing in several respects from the present description of Bacillus subt ills. The organisms listed as type II in the table, on the other hand, correspond very well to the description of Bacillus subtllls and do not soften pickles. One other important difference between these two groups of organisms Is their tolerance to salt. Type III organisms will grow in salt concentrations up to and including 11 per cent while type II organisms do not tolerate above 9 P en cent salt. Those organisms grouped in type I appear to be the most prevalent in these brines. Slightly more than half (15) of the 2 9 organisms studied were classified as Bacillus cereus. This organism, although predominating, does not decompose pickles and does not grow in salt concentrations above 9 per cent. Of the remaining three groups, only those organisms belonging to type V were classified positively. These organisms, which agree in all respects with Bacillus megatherium, are not responsible for the soften- 8 ing of pickles. Type* IV and VI correspond most nearly to description* of Bacillus vulgatus and Bacillus pumjlus respectively. Three of the four organisms "belonging to type IV proved to he capable of softening cucumbers while the single isolate of Bacillus pumllus did not. Individual members of each of the six groups were selected for use in later studies. the occurrence of bacteriophage races specific for the lactobacllll concerned with the cucumber fermentation. The discovery of bacteriophage by d'Herelle (17) led to many attempts to utilize this lytic agent for therapeutic purposes. More recently, however, it has been discovered thet bacteriophage plays a deleterious role in some industrial fermentation processes which depend ttpon the action of microorganisms, McCoy (33) discusses this unique environment for the propagation of bacteriophages which Is offerred by the Industrial use of microorganisms. Bacteriophage as the cause of "sluggishness** in the acetone-butanol fermentation is well-known in the patent literature but not In scientific literature. The origin of bacteriophage in an Industrial plant Is usually obscure and various control measures have been proposed. More recently, attention has been directed toward the failure of starters In the cheesemaking Industry. Whitehead and Cox (Ul) isolated bacteriophage from an aerated starter culture which had felled to develop acid. In a similar manner, Johns and Catcnelson (26) in Canada demon­ strated that the sudden stoppage of acid development in several experi­ mental vats of Chedder cheese was due to the activity of a polyvalent streptococcal bacteriophage. Although the starter was a mixture of organisms, stoppage was as abrupt as that in cases where single strain starters are used, Whitehead and Hunter (U2) established the presence of bacteriophage for lactic streptococci in the atmosphere of commercial cheese factories. Finely divided particles of whey emitted from a whey separator and whey- 10 contaminated duet were "believed to "be the principle vehicles for the air"borne phage. Hunter (2?) stated that the amount of the initial infection determined the extent to which the presence of bact erlophege was made evident in the vat, a heavy infection "being necessary to cause complete cessation of acid development. In addition to air-borne contamination, several other sources of infection have been indicated. Whitehead and Hunter (U3) demonstrated that whey-infected milk cans might be the cause of Infected starters since the pasteurizing treatment of the cheese milk was not sufficient to destroy "bacteriophage derived from infection of milk cans. Hunter (23) showed that some partially resistant strains of Streptococcus cremoria were able to carry phage in symbiotic association when subcultured daily in milk over long periods of time. The effects of physical and chemical agents on the viability of "bacteriophages for lactic streptococci have been the subjects of several investigations. Hunter and Whitehead (2h) found that sufficient concen­ trations of hydrogen or hydroxyl ions would inactivate streptococcal "bacteriophage but that their effects between pH values negligible. of U and 7 were Lactic acid in a concentration of 2.5 per cent was shown to destroy phage at room temperature in less than five minutes. A concen­ tration of 1 per cent lactic acid was permanently effective In less than 2U hours. Sutton (39) demonstrated that ultraviolet light effectively destroyed bacteriophage under conditions likely to be encountered in cheese factories. Hunter (21) studied the effect of temperature on the growth of several strains of Streptococcus crenorle and their appropriate bacterio­ phage. Phage races were shown to possess a wider diversity of reaction to temperature conditions than the homologous organisms. failed to multiply at 37°C. Some races Prouty (35) reported that several strains of bacteriophage under observation in the dry state were still active after being held for two years at room temperature. The similarity between the "slow" starters in the cheesemaking industry. the "sluggish” fermentations in the acetone-butanol industry, and the "abnormal" fermentations in the pickle industry has suggested the possibility that bacteriophage might be responsible for the latter abnormality. T o r that reason, the following portion of this study con­ sists of a survey of brine, soil, and water samples for the presence of bacteriophage specific for species of the genus Lactobacillus, particu­ larly for those strains of teJ^plantarum which are responsible for the cucumber fermentetion. The organisms used in this survey consisted of 1U cultures of lactobacilli from various sources and 20 cultures which had been isolated from active cucumber fermentations. The lh cultures from non-pickle sources (Table 3) were obtained from Dr. Carl S. Pederson, New York State Agricultural Experiment Station, Geneva, New York, and represented seven different species. In view of the degree of strain specificity shown by Leuconoetoc mesenteroldes phage (32), 20 additional strains of lactic acid bacteria were isolated from actively fermenting brines and purified by repeated 12 Table 3* Lactobacilli from sources other than fennentlng cucumber*. Organism Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Liactobaclllus Source buchneri brevis plantarum plantarum ver. rudensie plantarum buchneri arabinosus easel delbruckii brevis sp. brevis ver. rudensls buchneri plantarum Tomato catsup Vegetable salad Orla-J ensen Molasses *A. T. C. C. A. T. C. C. Kluyver Cameron Pineapple • Bread dough Flour pasts ♦American Type Culture Collection pistinp. All isolated were Gram-positive non-eporogenic rode which occurred singly, in pairs* or in very short chains. No attempt was made to identify these organisms, hut it was assumed that all strains were probably Lectobaclllus plantarum (Orla-Jensen) Bergey et al. as described by Pederson (3^0* This assumption was based on the work of Xtchells and Jones (12) who identified U 9 cultures of lactic acid bacteria Isolated during the acid fermentation of salt stock cucumbers as this species. The brine samples which were assayed for the presence of bacterio­ phage consisted of 10 samples of processed dill and salt stock brines (Table b) from various sources and 12 semples of dill brine (Table 5) received from a Canadian company which lost practically their entire year’s pack of genuine dills. 13 Table U. Brine no. 1 2 Analyses of brines from spoiled processed dills end salt stock cucumbers. Source of brine Processed dills it N M H H * N Salt stock n 7* * k 5 6 7 V 9 10 Table 5 . pH U.70 3.57 3.% 3.^5 3.32 3.90 3.35 3.55 3.05 3. SO Per Cent salt 5-1* 3.5 3.3 U.S 3 .^ 3.7 3.5 lU.i* 22.0 Per cent lactic acid 0.53 0.75 0.75 0.75 0.30 0.30 0.30 0.90 0.75 0.18 Analyses of brine samples from 1? rats of spoiled p-enulne dills. Brine no. Source of brine CC-1 CC-? C C -3 CC-U cc -5 CC -6 CC-7 CC-g cc -9 cc -10 cc -11 CC-12 Genuine dills H S N « n * « 11 H n pH Per cent salt Per cent lactic acid 3.50 U.l 0.75 J.kZ k.l k.e 0.60 7.52 3.55 3.^0 3.50 3-30 3.60 3.60 ■*.60 3.55 2.90 h c 5.0 U.7 k.k u.e 0.75 0.75 0.90 0 ,6s 0.60 0.90 *•3 0.75 0.75 k.z V.* 0.90 0.68 In order to determine whether bacteriophage, if present at the bes-innln/p of the fermentation, might have been introduced Into the vats by Ik water used in the salting process or by the cucumbers themselves, eight samples of La^ce Ontario water were obtained from the Canadian company and four samples of soil were collected directly from cucumber hills on the Michigan State College Experimental Tarm. Tor the assay of brine and water samples the following technique was employed! Each sample was first filtered through a sterile Pasteur- Chamberland filter (L-5 porosity) and 1 ml of this sterile filtrate added aseptically to each of a series of tubes containing 9 of a medium of the following composition: Glucose-Tryptone-Tsast Extract Broth Bacto-tryotone Bacto-yeast extract Bacto-beef extract Glucose K^HPO^ Distilled water pH 10 g 5€ 3g 1g 1g 1000 ml 6.7 After addition of the filtrates, each tube was inoculated with one loop of a 2h hour culture of one of the lactic acid organisms. tube without the filtrate was inoculated with each organism. were observed after one and two days for lysis. A control The tubes If no growth occurred* the bacteriophage suspension was filtered through a sterile PasteurChamber land filter. Inoculated into another tube of the medium and seeded with the organism. If the suspensionretained four or fiverepetitions of this nrocedure. It its lyticactivity after was consideredpositive for beeteriophage against that particular strain of the organism. A somewhat different techniaue was employed with soil samples. 15 A 500 ml Erlenmeyer flask containing 200 ml of glucoee-tryptone-yeast extract broth was inoculated with one drop of a 2b hour culture of es«h organism to be tested. After an incubation period of 12 hours at 30°0, 10 grams of soil were added end the flask incubated for an additional 12 hours. After this period of preliminary enrichment, the supernatant was first filtered through paper and then through a sterile Seits filter. The filtrates were then tested for the presence of bacteriophage according to the procedure described for brine and water samples. Beeults and Discussion Filtrates from the enrichment cultures of all soil samples sup­ pressed growth of all of the 20 brine isolates of L m plantarum for a period of at least Ug hours. This ability to inhibit these organisms persisted through fiTe filtrations, corresponding to a 1-100,000 dilution of the enrichment culture of a 1-2 ,000.000 dilution of the original soil sample. These dilutions were considered to be sufficient to eliminate any possibility that antibiotic substances originally present in the soil could have been carried over into the first filtrate in sufficient con­ centrations to inhibit growth of these organisms. None of the 22 brine samples or six water samples gave evidence of the presence of bacteriophage. The fact that bacteriophage was not found in these brines is not surprising since only one of the samples had a pH value greater than b.O. These results are in agreement with the findings of Hunter and Whitehead (2b) who showed that streptococcal bac­ teriophage was inactivated by hydrogen ions when the pH value was less then b.O. 16 The antagonistic action of aerobic sporogenic soli organisms ~^owar^ the lactobacllli concerned with the fermentation of encumbers. The antagonism exhibited by certain species of microorganisms for other groups of microorganisms has been recognised by microbiologists for more than sixty years. Garre (15) pointed out as early as 1S5S7 that the presence in the soil of bactericidal substances excreted by certain types of soli bacteria might be responsible for the rapid destruction of patho­ genic organisms when they are added to the soil. On the suggestion of Garre that such substances might be valuable therapeutic agents, many un­ successful attempts were made to isolate these substances. After the discovery of sulfa drugs, Interest in these substances produced by soil organisms was revived. Logically enough* emphases has again been placed on their value as therapeutic agents. Dubos ($) (9) (10) in a series of papers described the Isolation of an alcohol soluble waterinsoluble substance from broth cultures of an aerobic sporulating soil bacillus corresponding to Bacillus brevis. This substance which was called tyrothrycln was separated into two fractions* (a) tyrocidlne, which was bactericidal jln vitro for Gram-positive and Gram-negative organ­ isms; and (b) gramicidin .which wsb effective against only Gram^positive organisms. Arlc and Hunt (2) isolated two soil organisms, one of which was identified as Baclllus vulge tus» while the other, a yellow spore-bearing bacillus, was not Identified. These organisms were found to be antagon­ istic in both liquid and solid media to a number of Gram-positive and 17 Grain-negative organisms including Streptococcus lactia, Leuconostoc megenteroIdes. and Lactobacillus acidophilus. Katcnelson (28) Isolated an aerobic spore-bearing baelllus which produced an antibiotic substance in a potato-dextrose-peptone medium. This substanee was found to inhibit 77 81 species tested. The majority of streptococci, staphylococci, lactobacllli, and clostridia were inhibited by this toxic principle while all Gram-negative organisms were unaffected. Jansen and Hlrschmann (2*) reported that subtil in, an antibiotic substance produced by Bacillus subtil Is on a medium consisting of mineral salts, sucrose, asparagine, and trace elements, was active against Grampositive organisms. Foster and Woodruff ( l M described another antibiotic principle produced by a soil isolate of Bacillus subtil Is. This substance, which they called baclllln, was highly active against both Gram-positive and Gram-negative bacteria. Excellent reviews on this subject have been written by Waksman (Lo), Hotchkiss (20), and Hoogerhelde (19)* These reviews thoroughly cover such aspects es the Isolation, purification, chemistry, and physiological pro­ perties of tyrothrycln, tyrocid ine, and gramicidin. Experimental A simple "euo* method was used for determining the ability of the aerobic suororenic organisms used in this study to elaborate antibiotic substances against lactic acid organisms. tryptone-yeast extract agar was added, to 18 Approximately 18 ml of glucose— r sterile petri dish '-hich had "been seeded with 1 ml of a 1—10*000 dilution of a Ph hour broth culture of the lectic acid organism. After the plates had been mixed end allowed to harden, sterile glass rings (8mm high X 16 ann diameter) were heated slightly in the flame and placed on the surface of the agar* In order to complete the seal between the ring and the agar, a thin layer of sterile 3 per cent agar was poured over the surface. added 0«5 To the cup thus formed was R 7^ hour culture of the spore-former which had previously- been filtered through a sterile Pasteur-Chamberland filter. The plates were incubated upright at 30°® hours. examined at 2U and US The degree of inhibition was recorded as the maximum distance, ex­ pressed in millimeters, which inhibition extended beyond the inner edge of the ring. The lactic acid organisms used in this study were comprised of one strain of Leuconostoc mesenteroides, six species of lactobacilli, all of which had been isolated from sources other than piclcle brines, and ten strains of Lactobacillus plantarum which had been isolated from actively fermenting cucumbers. The influence of the comoos it ion of the medium on the elaboration of antibiotic substances was determined by using four different media for growing the sporeformers. These media were: (a) Proteose-peptone broth consisting of 7 grams of proteose—peptone and J gra~p of beef extract per liter; (b) Glucoee-tryptone-yeast extract broth; (c) Beet sugar molasses broth which was prenared by diluting a mixture of 100 grams of beet sugar molasses, 10 grams urea, and 10 grams of monobasic ammonium phosphate to 19 5 ° Erix ana adjusting to pH 7 1 aa& (d) Cucumber extract which was pre­ pared by mincing 200 grams of peeled cucumber end 1000 ml of distilled water in a Waring Blendor, digesting in an Arnold sterilizer for one hour, and filtering, Results and Discussion Of the seven lactic acid organisms which were isolated from sources other than brines, only three showed any degree of inhibition by any of the filtrates (Table 6), Of these three organisms, Leuconostoc mesenteroides, Lactobacillus plantarum, and Lactobacillus arablnosus, the latter two are now regarded as synonomous and certain strains of L. plantarum are believed to be the principle organisms concerned with the fermentation of cucumbers, L. mesenteroides, on the other hand, is not regarded rb an important factor in the cucumber fermentation, but is responsible for inaugurating the sauerkrSTit fermentation. Entirely different results were obtained when the filtrates were tested against the ten laetic acid organisms isolated from brines (Table 7)* The most consistent results were obtained when the spore-formers were grown in cucumber extract. In this medium only B. mesenterlcua fuscus (C-l, H-l) end B. vulgatus (G-S, H- 6 ) elaborated antibiotic principles which were active against the lactobacllll. Two isolates of each of the organisms were strongly inhibitory to all of the lactic acid organisms. These results are perhaps more significant in view of the fact that these species were the only ones which were capable of softening cucumbers. When grown in glucose-tryptone-yeast extract broth, only two of 20 A Table 6 . The antagonistic action of aerobic snorogenic organisms toward strains of lactic acid “bacteria isolated from sources other than pickle “brines. True II F-l 7-2 cp sel - - ara~blno9U8 - brevis - - - - - - plantarum - - - - - i delbruc^ll - - - - - - - tmchnerl - - - - - - - mesenteroides t i i Type III C-l H-l i t i - t - i Type IV S-g H-S Type V E-l E-5 Tyoe I B-l B-2 i i t - t i - i t - - No inhibition ± ■ Incomplete Inhibition, does not extend beyond ring. Type VI 6-7 Table 7. Showing the effoct of the coaoosition of the medium on the elaboration of antibiotic substances against the lactic acid organleas isolated from cucumber brines. ____________ (a) Cucumber extract Type III Typ e II ?7P« -I B-l B-2 lf- l T F -2 C-l ri-i 5-10 5-16 5-1 5-50 5-25 - 6-18 6-2o - _ 6-25 . - - - - - - mm 6-1 - - - *15 mm 15 15 12 15 "✓ 13 1 r. « - - 12 1r 15 20 20 20 20 20 20 20 20 20 20 Type rv 6-8 e -6 *6 6 U If s 15 20 20 20 20 6 8 15 15 If- 20 6 15 6 20 (b) Glucose-tryptone-yeast extract Tt >e II Type III Type IV Type I v _J B-l B-2 5-10 5-16 5-1 5-30 5-25 6-18 6-26 6-25 6-22 6-1 / See end of table for legend. F-l F-2 C-l E-l 0-8 H-b l7Pe V 2-5 Type VI 0-7 _ m m - — - - - mm ~ ~ - - - - - mm - - Type V 2-1 - 2-5 Type VI 0-7 Table 7. (Continued) (c) Pro teos e-peptone broth Type II Tyne I Type III Type V Type IV B-l B-2 J - l F-2 C-l H-l G-S h -6 E-l E-5 5-io 5-16 - - 4 4 4 4 4 4 4 0 J 4 4 3-1 3-30 > 6-1? 7* - 6-26 - - - G-25 - - — — '™t .t.. rj i - — (d) Tyn e I B-l B-2 5-io 4 5-16 *>-l - 5-30 5-25 6-1S 6-26 6-25 6-22 6-1 - t - - - * - - - t t 4 ♦ 4 4 - 4 4 4 4 4 - - 4 - - 4 9 4 4 4 4 4 4 4 7 — — - k 6 6 - - «•* - 4 4 - - 4 4 4 i. — - - — — - * t 7 7 7i ♦ 6 7 4 4 4 7 6 4 6 4 4 Beet sugar nolr.sses broth IV Typ h-6 E-l E-5 4 - 4 4 4 - - 4 4 _ 4 - 5 4 4 4 4 r 0 4 - - - - - - - 4 4 - - - n j - 0-7 4 6 r- 4 Type VI 4 Q j 4 6 - •M 7 0r 7 j 2 - u 4 - k h - — Tyoe VI 0-7 4 - U Q - - 5 5 5 5 4 4 - 6 2 2 9 - 6 - k 6 4 ± s Inhibition incomplete end only under + a Inhibition only under ring* ring; - r Ho inhibition, ♦ s Denotes Kfainum distance in mm that inhibition extends beyond ring. the organisms, B. subtilIs (7-1, 7-2) and B. cereus (B-l, B-2), gave any indication of antibiotic activity and this was very slight and against only one strain of the lactobacilli. The results obtained with proteose-peptone broth and the beet sugar molasses medium were less clear-cut. All of the spore-formers showed an antagonistic action toward some of the lactobacilli in both of these media, elthough in the beet sugar molasses medium, B. subtllls and B. cereus were active against only one of these organisms. 2k The relationship between the metabolic activity of aerobic sporogenic bacteria and their ability to grow in cucumber brinea. The first observation that certain groups of bacteria were character­ ized by definite reduction potentials exhibited in their cultures was made by Gillespie (16). He noticed the markedly different final potentials which developed in cultures of strict aerobes* as contrasted with thoee of facultative anaerobes, and suggested that these differences may apply generally to the two distinct groups of bacteria, aerobes and anaerobes. Quastel and Stephenson (36) found that the presence of cysteine, or other reducing substances. In tryptic broth would induce good aerobic growth of Clostridium sporogcnee. They suggested that the latent period exhibited by oxygenated Cl. sporogenes cultures was the time of Incubation reouired for the non-proliferating cells to produce a certain minimum quantity of reducing substances in the medium. This minimum quantity of reducing substances. It was believed, was necessary in order to establish a limiting reduction potential for proliferation of the organism. In a medium which maintained a hlvher potential proliferation of the anaerobe could not occur. Allyn and Baldwin (3)» observing the effect of cysteine on the zones of growth of Ehlzobla in agar shalce cultures, arrived at similar conclusions. In addition, they believed that the bacteriostatic effect of certain oxidizing and reducing substances was due in part to a "poising" of the medium at a potential unfavorable for growth. Sustain­ ing this theory was the previous worVr of Csnnan «t al. (6) which showed that halogens lost the greater part of their toxicity when added under 25 conditions which prevented the attainment of oxidation-reduction potentials more positive than those of the indophenols. Allyn and Baldwin (U) measured the time-potential curves of Ehlzobla in two basic media: (a) mean itol-yeast water and (t>) raannitol-aitrate "broth. The former medium was more reducing- in nature and supported growth at higher dilutions of the inoculum, Mannitol-nitrate "broth permitted growth in similar dilutions only after the potential had "been reduced, Knayei and Dutky (29) demonstrated that very low potentials also had an Inhibitory effect on the growth of another aerobic organism. Bacillus megatherium. These workers lowered the potential of a meat in­ fusion broth to -0.?5 volts with 0.27 P ® r cent sodium sulfite and in­ hibited the growth of this organism. The relationship between the bacteriostatic action of certain indi­ cator dyes and their capacities to poise media at definite oxidationreduction potentials has been investigated by Dubos (7) and Wood, Yood, and Baldwin (MO. Dubes, working with pneumococci and human and bovine strains of hemolytic streptococci* found that methylene blue and oxidized indophenols were bacteriostatic while the indigoes, malachite green, and litmus were not toxic. Since methylene blue and the indophenols were no longer bacteriostatic when present in the reduced form, Dubos concluded that the "inhibiting* dyes poised the medium at an oxidation-reduction potential outside of the range in which the inhibited organisms could grow. Vood et al. obtained similar results with Bacillus megatherium. The oxidised forms of indicators more positive than methylene blue inhibited this organism for 2h hours while those dyes possessing more negative potentielB did not. 26 The Influence of potential on the "bacteriostatic action of dye* has suggested a possible explanation for the proliferation of "bacterial cells in high brine concentrations. Hof (18), for example, was able to Increase the salt tolerance of soil organisms from 6 to 18 per cent by addins sterile garden soil to the culture medium. Similarly, Tabian and Johnson (13) found that Bacillus mesenterlcue fuscua grew in a higher concentration of salt when grown in a medium containing beet sugar molasses. Doubtlessly the alteration of some physical property of the medium such as the oxidat Ion—reduction potential enabled these workers to decrease the sensitivity of these organisms to salt. In order to study the Influence of salt upon sporogenic aerobic bacteria and the lactobacilli commonly found during cucumber fermenta­ tions, a series of experiments wes conducted to study the oxidation-reduction potentials of these organisms in the presence of different ouantities of salt at various time intervals* 1. Determination of normal time-potential curves of 16° and 5 0 ^ salometer brines* Washed cucumbers were placed in ei.^ht ^ gallon crocke. Half of the crocks were salted at ho° salometer and the remainder at 16° salometer. Pletinum electrodes, prepared by sealing a bright platinum spiral into glass tubing, were immersed to a depth of approximately 8 Inches below the surface of the brine. These electrodes together with KC1 agar bridges were protected from the cucumbers by means of hollow wooden baffles (Tig. 1) through which numerous holes had been drilled to permit free circulation of the brine. 27 SALT BRIDGE HOLLOW WOODEN S BAFFLE ro O ■ th \ •— •.p p. ^ \-4 , . * ‘ ‘ Fig. 3a,"b,c and d. Normal oxidation-redoction potential. pH, and peptonlzer ourres o f cucumbers salted at 16° salometer. i. OG COUNT COUNT pH 8-12 20 20 6- 6- 5- - - 0.1 - 0.1 - 0.2 0.2 -0.3 12 4- 4 -0.3 16 O 4 16 8 DA V S L OG p H C O U N T J4 + 0.3 8-12 8-12 7- 10 7- 10 6- \ V. - 0.1 0.1 12 -s O O. /A ;• -•. p H ;A A ,I P f >< >0 A R ■ ' N H t 31 I ! L D ‘ T t R *>/ p r JN-' 2 t R A> At any rate, "under the conditions of this experiment, the pre­ dominance of the peptonizers was only transitory and they decreased In numbers as the acidity of the brines increased. 2. The determination of time-potential curves of some of the organisms from cucumber brines. The apparatus which was used for determining time-potential curves of pure culture? In this and following experiments Is shown dlagram&tlcally in Pig. U. The Individual cells consisted of pyrex test tubes (l in. X U In.) eeruipped with three-hole rubber stoppers containing a platinum electrode, salt bridge, and a cotton-plugged tube through which the cells could be Inoculated. A battery of six of these cells were arranged so that electrical contact between the cells and a common KC1 reservoir could he established by means of salt bridges. Contact between the KC 1 reservoir and the potentiometer was made with a large calomel electrode. Each cell contained 2 0 ml of medium end the entire assembly was immersed to the level of the medium in a water bath controlled at 30°®* After sterllsation, the cells were incubated for at least 2 b hours before Inoculation to permit stabilization of the potential. The inoculum was 1 ml of a 1-100 dilution of a £h hour broth culture of the test organism, H e m a l time-potential curves were determined for the six species of snorogenic orgrnisms. In addition to these, one strein each of Lactobacillus olanterum and Aerobacter aerogenes were used. Several attempts were me.de to isolate the strain of Aerobacter reported by Etchells Fig. TO POTEA/TI OME TER U. SALT BRIDGE \ Apparatus for determining pure cultures. L A R G E CALOMEL E L E C T R O D E r- time-potential RESERVOIR curree KCL of Fabian, and Jones (ll) as the causative agent of the early gaseous fernentation of cucumbers. All attempts, however, were unsuccessful. The two media used In this study were proteose-peptone "broth and cucumber extract. Results and Discussion One of the most significant features of the time-potential curves of these organisms is the non-reducing nature of Lactobacillus plantarum (Fig. 6c) as compared to Aerobacter aero genes (Fig. 6d) and the six species of sporogenic organisms (Fig. 5S »^»C Fig. 6a and b). Aerobacter, on the other hand, establishes intense reducing conditions in both media. This observation is particularly interesting since this orgrr'rm is v r r r closely related to Aerobacter cloacae which is responsi­ ble for the early gaseous fermentation. The S-shaped curves obtained in cucumber extract with cultures of B. cereus (Fir-, , -B* P^-llus (Fig. 6b) and, to a leaser extent, B. subtil is (Fig. 5b) indicates an accumulation of peroxide in the culture medium. This is shown by the upward trend of the curves starting when the cultures were approximately IP hours old. The recondary drop in potential, coinciding with the appearance of growth in the medium, could be due t o ! (a) an adaptation of the organisms to the medium, (b) secondary grovrth of variants more capable of producing ee.talase in this medium, or (c) a latent formation of peroxidases. Despite the fact that these three species grew concerntivelv well in cucumber extract, they produced but meager growth on the surface of 3** A Fig. 5a *fc*c and Time-potential curres of B. cereus. B. eubtllls, B. meeenterlcue fuscus. and B. rulgatue in~Ta) cucumber extract and (b) nutrient broth. o. 5 (bj •i> 8 ( L n C S •n * 8 SUBTIL IS 0.4 * O.J I ■•~n\ }\ i V'te Y H' 1 j. 24 * 0.5 r (c I B ML S L I L... as n o i , *’J -v 7 L 24 48 72 96 V 96 nO U R S 4 CU 5 ‘ u 5 * . O.S\ (u) j 8 i- UL G 4 r U S 0.4 +(>.3 '■ * 0.2 +-■>.;r - . •*>,/ O 1 ...l.J 96 48 24 48 •*o u r • 'v '• , . ■M & L R : « J Fig. 6a,'b,e and d. Time-potential curve* of B. megatherium, B, pumllus, pi ant arum, and A. aerog’ene* in (a) cucumber extract and (h) nutrient 'broth* (°-) §_ M E G A T H E R / U M (b) B - P U M / L US 0.3 SI 10 A ,o o - o 0.1 - 0.7 - 0.2 0 0 96 + * 0.5 96 HOURS >0.5 r (c) L PLA H r A RUM ^ A AEROGENES 0.4 <> o <> O.J, ♦ 72 24 ^OUR 5 'V * -o .*• ^0.2H 0.2 '■v.2* j.: t . 1 48 36 -4. - 72 Zi 96 cucumber extract agar slants. In order to determine whether this phenom­ enon covild "be correlated with catalase production, all six species of the sporogenic organisms were tested for their ability to elaborate catalaee when grown on nutrient agar end on cucumber extract agar. All six gave strongly positive tests for catalase when 1 per centhydrogen peroxide was added to 2 b hour agar slants. specie* However, when similar test* were conducted using cucumber extract agar, B. cereus, B. pumllus, and B. subtllis gave negative tests for catalase. Aerobacter aerogenes, on the other hand, gave strongly positive tests on both media. J. The effect of sodium chloride and lactic acid on the tlm.e-potentled curves of aerobic sporogenic bacteria. The Influence of salt alone on the time-potentiel curves of the six species of sporogenic organisms was determined. 2 .5 , 5 *0 , 1 » 5 * 10.0 per cent Concentration of salt were obtained by adding aseptically 10 ml of a sterile double-strength salt solution to 10 ml of sterile double-strength proteose-peptone broth in the oxidation-reduction cells. This was done to minimize precipitation of thenitrogenous constituents of the medium at the high salt concentrations. o The cells were incubated 21* hours at 30 ® stabilize the potential and then inoculated with 1 ml of a 1—100 dilution of a 2 h hour broth culture. were made at frequent intervals over a US Potential measurements hour period. The combined effect of salt end lactic acid on the time-potential curve of Bacillus cereus was also determined. 2 .5 , p .O, 7 .3 , Salt concentrations of 0, 10.0 per cent were used in proteose-peptone broth which 77 had "been adjusted to pH values of 1**0, 5*0, 6.0, and 7*0 with lactic acid. Eesults and Discussion From the data obtained (Fie;. d Fig. Sa and h ) , the principle effect of increasing the concentration of salt was a lengthening of the time reouired by the organism to reduce the potential of the medium to a level at which multiplication of the cells could ta-ce place. For example, in the case of Bacillus cereus (Fig, 70) * microscopic examin­ ation indicated a lag phase of four hours or more in those cultures con­ taining no salt and 2*5 par cent salt, and over eight hours at 5*0 per cent salt. The cultures containing 7*5 per cent salt showed no indications of multiplication for more than 2h hours and those with 10 per cent did not grow in five days. However, In all cases by the time the organisms had begun to rrultinly the potential of the medium had been reduced nearly three-tenths of a volt below its original level. These results suggest that at least psrt of the bacteriostatic action of sodium chloride is due to its effect on the reducing enzymes of the organisms. Although the degree of retardation of this potential drop with Increasing sa.lt concentrations varied considerably with the organisms used, there was only one exception to the general rule. Bacillus vul— ~r tns (Fi<~. 7d) was able to establish a lower potential (-0.0? volts) in the presence of 7.5 per cent salt than when no salt was added to the medium. However, with notential after 36 7.5 and 10 per cent salt, the sharp increases in and 2U hours respectively indicate an accumulation of 38 Fig. 7a,h,c and d. The effect of salt on the time-potential curves of aerobic sporogenic "bacteria isolated from spoiled pickle "brines* (CL) a ( b) a S U B T / L / s CEREUS +0 3 (Si IQ a ) ^ - + 0.2 0.1 -0 2 + * 0.5 (c) B M E S E N TE R i C U S 48 24 HOURS 12 (d) B V'ULGA TUS F U SC U S 0.3 W o « ^ c -o ] 0.1 I L 12 36 ■ » •. ,V. - ' 7 V - f U ; 2.5 P E R C E N T S Pt RC E N T S A L T I\ r M / 7 ; 8 --- 9 , >0.0 39 ; SALT- & ------------- 7,5 PE R &ERCENT 24 HOURS SALT. - 36 3Pig. 8a and b. T h e effect of salt on the time-potential curves of aerobic sporogenic bacteria isolated from spoiled pickle brines. + 0.5 (a ) b m e g a t h e r + OS /u m +0.4 + 0.4 > 9 gg 0.1 < + 0.2 12 24 36 48 - 0.1 - 0.2 12 HOURS 24 HOURS O -- O , N O S A L T j • --- •, 2 . 5 P E R C E N T S A L T j O -- <9 , 5 . 0 P E R C E N T S A L T ; 9 --- 9 , T . 5 -P E R ­ CENT S A L T ; 9 -- 9 t / 0 . 0 P E R C E N T -- S A L T . 36 48 peroxide In the medium. This presumably Is due to inpaired catalase activity in the higher concentrations of salt* When lactic acid alone was added to the medium, the effect of in­ creasing concentrations on the time-potential curve of cereus (Fig. 9) was very similar to the effects obtained by increasing the concentretion of salt. When the initial pH of the medium was a justed to values of 7*0 and 6.0 there was very little difference in the reducing activity of the organism. However, at pH 5*0 the potential drop was gradual and growth did not appear until after US hours. At pH U.O there was no decrease in potential or growth. Prom the results obtained with combinations of lactic acid and salt (Fi<-. 10a,b,c, and d ) , it appears that the effects obtained when these compounds were added to the medium individually are additive when both are present. For example, B. cereus grew at pH 6,0 (Fig. 10b) when no salt was present and at a concentration of 7*? P er cent salt when the pH of the medium was 7*^ (Fla*. 10a). However, this omr.iera did not grow in the presence of 7*^ P er cent salt when the medium was adjusted, to pH 6.0 with lactic acid. In general, these results indicate that the salt tolerance of this organism decreases with increasing concentrations of lactic acid. The Influence of the oxidat 1on-reduc 11or. potential of the medium on th<= sensitivity of spore-formers to salt. The ourpoee of this study was to determine whether the tolerance of spore-formers to salt could be changed by altering the potential of the medium in which they were grown. Bacillus cereus was selected for the Ul A Fi<% 9 * + The effect of lactic acid on the tine-potential curres of B. cereus. 0.5 p H + 4 0.3 p H 5 X 0.2 p H 6 p H 7 0.2 j_ 36 J2 H O U R S >42 Fig. I 0a,btc and d. The combined effects of lactic acid and salt on the time-potential curres of B. eereua. fj ** 6 . v O IV V >* l A 24 'O U I 36 P J2 43 r> 24 HOURS 36 VOS O5 ,c > «, f (d) P H 4 0 ‘ v 0 4» O4 J ^ w — <•» •<* » * •» >► v 03 +02 h oj vl, ,< + O 1 -0 1 4___.---L. 02 12 24 36 48 HOURS re ► *. ; V' e. 2 . S V r i4 w , l_ T PERCfAt! ;» «, 7.5 P t H C t H T SALT., P£ R SALT. U3 4 preliminary stifles since it was the predominant organism found in the "brines. It was previously observed that the minimum potential attained "by this organism in broth was approximately -O.lOO volt and that active proliferation of the cells did not occur until the potential of the medium had been considerably reduced. For these reasons, attempts were made to reduce the potential to that limiting value. The use of reducing agents such as sodium sulfite did not prove successful since the concen­ trations recuired were too high and the potential attained with these substances was easily altered by famine- the tubes. Indigo carmine in a concentration of 1,3 X 10“^ molar only slightly reduced the medium as did reducinr sugars such as glucose. However, a combination of the "poising” effect of the dye and the reducing action of 1 per cent glucose produced a satisfactory potential of -0 .l0' a volts at 30°C« In order to study the effect of potentials considerably lower than the limiting potential of the orgcn^sm, a thioglycollate medium prepared by the Baltimore Biological Laboratory was used. At 3®°® this medium had a potential of -0 .20^ volts. The sensitivity of the six different species of bacilli to salt at high and low potentials were compared In both nutrient broth and cu­ cumber extract. Hesults and Discussion The influence of the oxidation-reduct ion potential of the medium on thf salt sensitivity of B. cereus is sho"n in Table 8 . In ordinary nutrient broth (B^ * +0.28^ volts) this organism grew in salt concentra­ te Tatie 3, Per cent HeCl 0 1 2 Medium A Medium B Medium C Medium B t 4f > fi 4- 5 444* f+ 6 7 2 v-r -n The effect of the oxidetlon-reduct Ion potential of the medium on the sensitivity of B, cereus to sodium chloride. 9 10 n 12 13 1U Medium E + + + + ♦ * + + 44* f 4- 15 16 17 12 Medium A s Butrlent broth (lu = fO.?2Sv) Medium B aEutrient broth ¥ 1 per cent glucose (B^ • +0.202v) Medium C s Butrient broth * 1.2 X10"^indigo carmine Gfc « +0.2TSv) Medium D s Butrient broth v 1 per cent glucose + 1.2 X 10“^ molar indigo carmine (B^ * -O.lO^v) Medium S a thioglycollate medium (E^ « -0.20tar). tions up to and inducing 9 P e-' cent. Seduction of thr potential of this medium to a value of +0*208 volts with 1 per cent glucose had no effect, Eovever, wh en indigo carmine alone (E^ * +0.278 volts) or in combination with 1 per cent glucose (E^ a -0 .101} volts) was added to the medium, it was found that the salt tolerance of this organism was increased to lH per cent. When B. cereus was grown in the thioglycollate medium, growth we*» not attained in salt concentrations above 6 per cent. It was found thut in those tubes where growth did occur, the organisms had raised the potential from the initial value of -0*20^ volts to the limiting potential of that species, that is, approximately —0*100 volt. Seduction of the potential of nutrient broth to -0*10h volts by the eddltion of indie-o carmine and glucose tended to increase the salt tolerance of four of the six species tested (Table 9)» on the two remaining species. no effect B. cereus (B-2) grew in lU per cent salt at the lower potential in contrast to 9 per cent in ordinary nutrient broth. Similarly, J3.. mesenterlcus fuscus (C-l) was increased from 11 to 16 per cent, J3. vulgrtus (G-8) from 10 to 16 per cent, and B. megatherium (E-l) from 10 to 1*! per cent. The salt tolerance of subtil is (E-l) andJB. mimllua (G-7), on the other hand, vas not affected by the lower potential* Both species grew up to and including 9 per cent salt at both high and low potentials. When cucumber extract vas used e-s the basal medium (Table 1C), several significant differences were noted. B. cereus (B-2), B. subtlljs (T-l), and B. pumllus (G-7) failed to grow above 3 1*6 cent salt as eom- Table 9. The effect of oxide tIon"reduction potentials on the sensitivity of snore-formera to sodium chloride in nutrient broth. Per cent ir&ci 0 1 2 i 1 7 S 3 10 11 12 17 in Type III ■ m r - A B Type 17 73^3 A B Type 7 A B A e 71 O^TT 2 ♦ + + + + ♦ + ¥ ♦ + 4+ + 44- 16 17 IS Medium A = Nutrient broth, ■ +0*2SSv) Medium B a Nutrient broth ♦ 1 per cent glucose + l.S X 10"^ molar Indigo carmine, { \ ■ -Q.lQhv). Table 10* The effect of oxlflr tlon-r eduction potent Iris on the sensitivity of Epore-foroers to sodium chloride in cucumber extract. e I Per cent EaCl 0 1 o k 5r 0 r? vj 10 n i.? i’ 1U 15 W + + + + Tyoe II " W T ro e U I (C -l) B A £ + + + ¥ * ¥ ¥ + 4 ¥ ¥ 4 B Tyoe IV Type V T o t ^ r i £ i r B Type VI i o ^ r B A B 4 4 4 f f ¥ ¥ 4* ¥ ¥ ¥ ¥ 4 4 4 4 ♦ f f f f ¥ 4 4 4 4 4 4 4 16 17 IS Medium A z Cucumber extract (ll^ » 40*350) Medium B s Cucumber extract + 1 per cent glucose 4 1,2 X 10“^ Taolar Indigo carmine, (E^ » -O.lOifv). Table 9. The effect of oxidation-reduction potentials on the sensitivity of suore-formere to sodium chloride in nutrient broth. *er cent Tyne I Type II Type III (i-st (f-1) A B (0-1) NaCl 0 1 2 ? \ 5 6 7 s 9 10 A B + 4 4 * 4 4 4 4 4 4 - + 4 4 4 4 4 4 4 4 4 4 4 11 12 1 mm • ■* ih 15 15 17 IS 4 4 4 4 4 4 4 4 4 4 - - M A 4 4 4 4 4 4 4 4 4 4 - - A B 4 4 4 4 4 4 4 4 4 4 4 4 _ • - • - - - - - - >e IV (G-■8) tee V Tyne V (35-1) (8-7) B A B A B A 4 4 4 4 4 4 4 4 4 4 4 + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 - 4 4 - 4 4 J* f 4 _ 4 L 4 4 4 - + • L mm « _ - 4 4 4 4 4 4 4 4 4 4 • Medium A - Nutrient broth, (B^ ■ +0*2fS8v) Medium B * Nutrient broth 4 1 per cent glucose 4 l.S X 10“^ molar Indigo carmine, (l^ * -O.lOl'v), Table 10* The effect of oxldr11on-reduc11on potentials on the sensitivity of spore-formers to sodium chloride in cucumber extract. e I Per cent KeCl 0 1 ? 3 2T Type II " I K T A B A B 4 4 4 4 4 4 4 4 4 4 4 + + + 4 4 Type III (c-i) Type IV Type V te it B 4 4 4 4 4 4 4 4 k 4 5 r 4 4 4 4 0 7 3 9 Type VI to-7) A 10 11 12 1 "? 1U 13 16 17 l$ Medium A r Cucumber extract ( \ - +0.J50) . Medium B a Cucumber extract 4 1 per cent glucose 4 1,S X 10"^ molar indigo carmine, (B^ ■ -O.lQiir). pared to 9 P er cent in nutrient "broth. Moreover, lowering the potential to -O.lOU volt 8 had no effect on the salt tolerance of these organisms. B. megatherium (B-l) grew only up to ? per cent salt at the higher potential "but withstood 12 per cent salt at the reduced potential. The tolerance of B. vulgatus (G-S) and B. mesenterlcus f us cub (C-l) to the preeence of salt in cue umber extract was likewise increased "by reducing the potential. In the case of B. vulgatus, the concentration of salt could "be increased from 9 ^ per cent, while B. mesentericus fuecus grew in 16 per cent salt at the lower potential as compared to 1 2 per cent at the higher potential. GEHEBAl DISCUSS I OS' The more the cucumber fermentation ie studied the more complex it hecome 8 . What at first appears to he a simple lactic acid fermentation has many variations depending upon a variety of conditions. Upon further consideration this is not altogether strange since with the cucumbers there is also introduced into the vats a lar^e number of microorganisms from the soil. The mieroflora of the soil is extremely variegated in­ cluding species of molds, yeasts, actinomycetes, and many bacteria. Generally speaking, the molds and actinomycetes do not find conditions to their liking and ouickly disappear. It is the yeasts and bacterie which persist and which must be reckoned with. Teests such rs Debaromycee and Mycoderma abound on the surface and yeasts such as Torulaspora, Hansenula, and Brettanomyces abound in the depths of the brine. At the beginning of the fermentation two grottos of bacteria are dominant, the aerobic sporogenic bacteria and the lactic acid bacteria. If only water were added to the cucumbers in the vats, they would all spoil and become a putrid mushy ness within ?U to US hours. However, the addition of salt in amounts as low as 2.5 to 3 per cent changes the picture entirely. The aerobic sporogenic bacteria which are responsible for the spoilage are suppressed and the lactic ecid bacteria gain the upper-hand. As soon as this latter group produces lactic acid the conditions for growth of the sporeformlng spoilage bacteria become increasingly unfavorable until they are entirely suppressed or disappear. This is what usually happens in the normel fermentation, otherwise the fickle salter and manufacturer would have to go out of business. 50 However, there ia always the occasional tank that spoils and in some years under abnormal conditions of temperature and rainfall there may be several tanks that spoil for no apparent reason. In checking back over the records there Is absolutely nothing to indicate why any of the tanks should have spoiled since they were all treated similarly and were filled with cucumbers erown In the same region. It was to study the possibilities under which the spoilage bacteria (aerobic sporogenic bacteria) could grow and multiply to the point where they could elaborate sufficient enzymes to cause spoilage that this work was undertaken. In this connection there appeared several possibilities. First, that since bacteriophage was known to lyse or destroy bacteria in other commercial fermentations such as the lactic acid fermentation in the cheese industry and the acetone-butyl alcohol fermentation, there was the possibility that bacteriophage might destroy or suppress the lactic acid organisms to a point where the aerobic sporogenic spoilage bacteria of the B. mes ent erl cus fus cue or B. vulgatus types might grow sufficiently so as to produce enough pectolytic enzymes to soften the cucumbers within a few months time. Since enzymes act as catalysts, small amounts of the pectolytic enzymes acting over a period of several months could easily produce softening. It is seldom that a tank of pickles spoils within a short time after It has been salted. Several months are usually required for the softening to occur. In several cases of spollare of genuine dills the evidence pointed very strongly to this possibility of spoilage h" bacteriophage. 51 In one case In Canada where several hundred barrels of genuine dills spoiled, there was strong indication that bacteriophage was the cause of the spoilage, but due to the age and acidity of the brines this could not be definitely proven. Several years ago another pickle plant in Michigan had considerable trouble with genuine dill pickles when they changed from a well water to lake water whotential of the medium to r level at ’h^ch nultiplicrtion of the "bacterial cells could trhe place. In other words, the addition of salt increased the lag nhaee of the organism. lor ereanle, when 2?. cereus vas grown ir. media containing either no salt or 2.1” per cent salt, there was a lag phase of aonrorirately four hours before the cells started to proliferate. How­ ever, when the concentration of salt was increased to R per cent there ’■•as a l?.r phase of approximately eight hours. Thus, in this particular fnstar.ee a twofold increa.ee in salt resulted in a doutlinc of the lag -hase and at higher sa.lt concentrations the leg phase was ^reatly length­ ened. lor erauple, in the presence of 7*5 P e** cent salt, the cells did not begin to mult inly for more than ?U hours end had not groi-n at the end of five dry? in the presence of 10 per cent salt. When lactic rcld was added to the medium in the presence of different cone art re t ion*- of salt, the effect was additive. For example, *• cor err- grow at oh o.C without salt an-' at 7.7 per cent salt at pH 7.0, 7U Taut would not grow at pH 6,0 and 7•!? par cent salt. This shows that the salt tolerance of this organ!sr. decreases with increasing amounts of acid. These results indicate ver;^ cl earl .7 the role that sodium chloride and lactic acid plar in the cucumber fermentstion. 55 STJMMAET Bacteriopha-ge races which lysed Lac tobac ill tig plantarum cultures wee isolated fron the soil where cucumbers had grown hut not from water or from genuine dill picicle brines in wv ich spoilage had occurred six to eight months previously-. Aerobic sporogenic bacteria commonly found in the soil and in fermentinr cucumber vats are capable of prodiicinp antibiotic substances which greatly inhibit the growth of Lactobacillus plantarumt the organism chiefly responsible for the fermentation of cucumbers. Bacillus nesenterlcus fuse us and Bacillus vulgatus. the two species capable of producing substances antagonistic to Lactobacillus nlantarum in a cucumber infusion, are also able to elaborate pectin— hydrolysing enzymes which are resuonBible for the softening of cucumber pic’cles. All six species of aerobic sporogenic bacteria isolated from s-ooilcd pichle brines reduced the oxidation-reduction potential of the medium to a considerable degree while the lactobacilli had little or no effect on the =hIncreasinm the sodium chloride content of the medium tended to e place. ifhen lactic acid was added to the medium the effect was similar to that obtained when salt alone was used but --hen lactic acid and sodium chloride vrere added conjointly these effects were additive, thus indicating th8t the selt tolerance of these organisms decreeses with increasing con­ centrations of lactic reid. Most of the spoilare organisms could he induced to grow in signif icantlv higher concentrations of salt if the oxide tion—reduction potential of the medium was artificially reduced to the minimum level established by the organisms during normal growth. 57 LCTET-ATDKE CITED (1) Aderhold , R. Untersuchungen uber das Einsauern ron Fruchten und Genusen. I. Das Elnsauren Gurken. Zen.tr. Bakt. Paras 11enk. » II, 9 , 5II- 51I4. 1 8 9 % (2) Ark, P. A., end Hunt, M. L . , Saprophytes antagonistic to phytopathogenic and other microorganisms. Science, J2j5, 35*+“355* 19^-• (3) Allyn, W. P., and Beldwin, I. L . , The effect of the oxidation-reduction character of the medium on the growth of en aerohic form of "bacterium. J. Bact.» 20, ^17-^37* 1 3 3 0 » (1-) Allyn, W. P., and Baldwin, I. L. Or Ida tion-reduct ion potentials in relation to the growth of an aerohic form of "bacteria. J. Bact., 2J, 369-39®. 1932. (3 ) Breed, R. S., _et. al* Manual of determinative "bacteriology. ed. William and Wilkins, Baltimore, Md. 19*4®. oth (6 ) Cannan, R. 3., Cohen, B., and Clark, W. M. Studies on oxidntionreduction. X. Reduction potentials in cell suspensions. U. S. Pu"b. Health Service, Pub. Health Repts., Suppl., JJ. 1926. (7) Dubos, R. J. The relation of the bacteriostatic action of certain dyes to oxidation—redut ion nrocesses. J. Exptl. Med., b-9, 975-592. 1929. (S) Dubos, R. J. Bacteridicsl effect of an extract of a soil bacillus on Crram-T>ositive cocci. Proe. Soc. Exptl. Biol. Med., *40, 311. I 939. (9) Dubos, E. J. Studies on a bactericidal agent extracted from a soil bacillus. I. Preparation of the agent. Its activity in vitro. J. Exptl. Med., JO, 1-10. 1939(10) Dubos, R. J., and Hotchkiss, R. D . , The production of bactericidal substances by aerobic sporulating bacteria. J. Exptl. Med., 7 3 . 629- 6*40. 19I4I. (11) Etchells, J. 1., Fabian, F. W . , and Jones, I. D. The Aerobacter fermentation of cucumbers during salting. Mich. Agr. Exp. Sta. Tech. Bui. POO. 19*45. (12) Etch ells, J. 1., and Jones, I. D. Characteristics of lactic acid bacteria from commercial cucumber fermentations. J. Bact., ^ 2 . 993-599. 19^ 6 . (13) Fabian, F. W . , and Johnson, E. A. Experimented. wor'c on cucumber fermentations. IX, A bacteriological et'tuly of the cause of soft pidtlee. Mich. Agr. Exp. Sta. Tech. Bui. 157* 193&. (1*0 Foster, J. W. , end Woodruff, E. B. Bacillin. substance fron a soil isole.te of Bacillus 51. 363-369. 19^ 6. A n e w antibiotic subtilis. J. Bact., (IE) Garre, C, Balct., _2, 312* On antagonists among bacteria. Z. 188J* (10) Gillespie, L. J, Reduction potentials of bacterial cultures and of vater-logged soils. Soil Science, 1320. (17) Eerelle, F. d * . The bacteriophage and its bebiavior. Willcins, Baltimore, Me. 1926. Williams and (11) Hof, T. Investigations concerning bacterial life in strong brines. Extrait du F.ecueil des Travaux botanloues neerlandais, J2, 92-173. 1933. (13) Eoogerheide, J. C. Antibiotic substances procLuced by soil bacteria. Botan. Eevs., ^10, 599“6>3-» 19*4*1. (20) Eotchhise, R. D. in Enzymol., Gramicidin, tyrocidin and ty*rothricin. 153-3-93* 19*4*4. Advances (21) Hunter, G. J. E. Bacteriophages for Streptococcus cremorie phr.ge development at various terrperatures. J . IfoaIry Res . , IQ (2), I 36-IU5. I 9U 3. (22) Hunter, G. J. E. The influence of becteriopKetge on the cheesendclng process. J. "Dairy Res., 1^, 2r}b— ‘ ^ 01, 19*4*4. (23) Hunter, G. J. E. Phage-resistant and phage-carrying strains of lactic streptococci. J. Eyg., i5. 307- 3-1 ?. W l . (2h) Hunter, G. J. E . , and Whitehead, E. R. The e.ction of chemical dis­ infectants on bacteriophages for the l a c t i c streptococci. J. Dairy Res. , 1_1, 62-66. 19*40. ( 2 0 Jrnsen, E. ?., and Hirschnnnn, D. J. Subtil In. An antibacterial product of Bacillus subtilis. Arch. Bioelrem,, U» 297-J-09. 19*4*4. (26) Johns, C. S., and Katznelson, H. Studies on "bacteriophage in relation to Cheddar cheesemahiiv*. Car.. J . Res., Sect. C., 1 9 , U 9-5S. I 9UI. (27) Joslyn, M. A. Some observations or. the softening of dill picicles. Fruit Products J., 3, 19? 9» 3-6. 1921. 59 (2£ K a t m e l s o n , H* Inhibition of nicro-orgeni sms by a toxic substance produced “by on aerobic spore-forming ’bacillus. Con. J. Ees. , 20 169. 19h2. , (29 Knaysi, G., and Dut'cy, S. E. The growth of Bacillus megatherium in relation to the oxidation-reduction potential and the oxygen content of the medium. J. Bact., 27, 109-119* 193*+* (70 Kossowiez, A. Bacteriologische TJtersuehungen uber das Weieh werden eingesauerter Gur’cen. 2 . Lnndvirtschaftliche Versuchswesen in Oesterreich, 1!L, 191-197* 1903. Cl LeFevre, £. Bacteriological *=•truly of pic’cle softening. Canr.er, US, 205-20o. I 9I 9. (52 Leive-Quiros, A., and McCles’rey, C. S. The application of "bacterio­ phage and aerology in the differentiation of strains of Leuconostoc mesenteroldes. J. Bact., 5!*. 709- 713. 13H7. (33 McCoy, E. 75. Industrial importance of ’bacteriophage, 19^ 3. The J. Bact., *£• (^ Pederson, C. S. A study of the species Lactobacillus plantarum (Orle-Jensen) Bergey crt al. J. Bact*, ^1, 217-22l£ I 936* (3 Prouty, C. C. starters. (36 Qpastel, J. K . , end Stephenson, M. Experiments on "strict1* enaerobes I. The relationship of B. sporogen.es to oxygen. Biochem. J., 20, 1125-11J7. 1926. (T> i Eahn, 0* Bacteriologicel studies of brine pichles. Dried Fruit Pacher, 'Tov. 20 and ?7 * 19^3* The problem of bacteriophage in relation to cheese Can. Dairy and Ice Cream J., September, U6-U7. 19^-8, The Cnnner and Smith, *T. P.., Gordon, P.. P., and Clarh, F. E.Aerobic nesophilic sporeforminm bacteria. 1T. S. Denit. Agr. Misc. Pub. 559* 19^6* (39 Sutton, W. S. Irradiation of cheese moulds rod bacteriophage by ultra, violet limht, J. Australian Tn^ t. Arr. Science, 67-73* 19^1* (UO Vahsmnn, S. A. Antagonistic relations of microorganisms. F.evs., *, 271-2^1, 19^1 * ri-x Whitehead, H. P., and Cox, G. A."he occurrence ofbacteriophage in cultures of lactic streptococci. New Zealand J. Sci. Tech* 16, 319-320. 1933* 60 Beet. (U2) Whitehead, H. P., end Hunter, G. J. E. Starter cultures for cheese manufacture. Further atterrpts to eliminate failures due to bacteriophsfe. J. Dairy Ees., 12, 63-70. 19^1. (Uj) Whiteheac, H. P., and Hunter, G. J. E. Bacteriophage in cheese marufpicture. Contamination from farm equipment. J. Dairy Ees., 15_, 112-120. I 9U 7 . (UI|) Wood, W. B. , Wood, M. L . , and Baldwin, I. L. Relation of oxldationreduction to g r o w t h of an aerobic microorganism. J. Bact., 30> 553-60?. 1935. 61