NUTRITIONAL PATTERNS OF SOME ANAEROBIC STREPTOCOCCI1

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1 NUTRITIONAL PATTERNS OF SOME ANAEROBIC STREPTOCOCCI1 STEPHAN E. MERGENHAGEN AND HENRY W. SCHERP Department of Bacteriology, School of Medicine and Dentistry, University of Rochester, Rochester, New York Received for publication June 2, 1957 Various obligatorily anaerobic streptococci are reported to be indigenous to the human oral cavity and vagina. They occasionally produce stubborn putrefactive wound infections, sometimes in synergism with staphylococci or sundry other bacteria. Bergey's Manual of Determinative Bacteriology (Breed et al., 1948) recognizes 8 species of anaerobic streptococci on the basis of gas formation, fermentation reactions, and morphology, as suggested by Pr6vot. However, the criteria for separation into species are not well defined; data on cultural conditions, prevalence, biochemical activities, and antigenic composition are scarce. Stone (194), using biochemical and serological reactions, could not correlate the two methods into any workable taxonomic scheme. Due to serological cross-reactions, he suggested that anaerobic streptococci may have some relationship with Lancefield groups A, B, and C streptococci. Knight (1955) has stated that the nutritional patterns of microorganisms may be among the characters well worth considering for use in classification. The nutritional requirements of members of the family Lactobacteriaceae are in general very complex (Tittsler et al., 1952). Nutritional observations on the anaerobic streptococci, however, are as yet incomplete. The present investigation was therefore made of the nutritional requirements of 8 strains of anaerobic streptococci in order to reveal any definitive differences or similarities between them and other members of the genus Streptococcus. MATERIALS AND METHODS Eight strains of obligatorily anaerobic streptococci were obtained from the following sources: 1 The data in this paper were taken from a thesis submitted by Stephan E. Mergenhagen in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Rochester. This research was aided by a contract, DA-49-7-MD-46, between the Office of the Surgeon General, Department of the Army, and the Universitv of Rochester. SFA and SFB, from normal vaginal cultures taken by members of the Gynecology Department, Strong i\iemorial Hospital, Rochester, N. Y.; SM, from a normal human gingival culture; SFC and SPU, from Dr. A. R. Prevot, Institut Pasteur, Paris, France; I-V3, from Dr. C. G. A. Thomas, Department of Bacteriology, St. Thomas Hospital Medical School, London, England; JR4 and JS9, from suppurative periodontitis by Dr. J. B. ]\Iacdonald, Division of Dental Research, Faculty of Dentistry, University of Toronto, Toronto, Canada. All strains failed to grow on the surface of sheep-blood agar plates incubated aerobically but grew well on such plates incubated in a Brewer anaerobic jar at 37 C for 72 hr, at which time they formed nonhemolytic pinpoint colonies. Stock cultures were maintained by weekly transfer in fluid thioglycolate medium (Difco), in which all strains grew as strongly gram-positive cocci in chains (figure 1). Strains SFA, SFB, SFC, SPU, and I-V3 produced a fetid odor and abundant gas in liquid media, whereas the oral strains, JR4, JS9, and SM, produced neither gas nor odor. Other biochemical cbaracteristics of JR4 and JS9 were determined by AMacdonald et al. (1954). Table 1 summarizes the species identification of the 8 strains according to the key for anaerobic streptococci in Bergey's Manual of Determinative Bacteriology (Breed et al., 1948). Prevot (1925) distinguished 2 species, Streptococcus foetidus and Streptococcus anaerobius, on the basis that S. foetidus formed large spheres normally in short chains, whereas S. anaerobius formed average sized spheres normally in long chains. We designated strains SFA, SFB, and SFC as S. foetidus, since on primary isolation they grew in short chains. Upon continued subculture or culture in various media, however, the chain lengths of these strains fluctuated widely. Furthermore, none of them fermented maltose, a characteristic that identifies them as S. foetidus according to Pr6vot's key in Bergey's Manual of Determinative Bacteriology. Dack (194) also could not distinguish between the 2 species, S. 74)

2 75() MERGENHAGEN AND SCHERP [VOL. 74 4AL A~~~~~~~~~~~~~ Figure 1. (Left) Oral anaerobic streptococcus, strain SM; (Right) Vaginal anaerobic streptococcus, Streptococcus foetidus, strain SFA. Crystal violet stains from 48-hr fluid thioglyceolate cuiltures. Photographed at 98 X magnification. TABLE 1 Characteristics of various strains of anaerobic streptococci Strain Gas and Fetid Growth in Broth Maltose coagulated H2S Characterized Species SFA Clumping colonies _ S. foetidus SFB Clumping colonies _ S. foetidus SFC Clumping colonies - S. foetidus SPU General turbidity S. putridus I-V3 General turbidity S. putridus SM _ General turbidity S. intermedius JR4 _ General turbidity JS9 General turbidity - foetidus and S. anaerobius. He observed in addition that the distinctive characteristics of Streptococcus putridus were the production of hydrogen sulfide and, after about 1 days, blackening in blood broth cultures. These reactions, along with the others noted in table 1, identified our strains SPU and I-V3 as S. putridus. Oral strains JR4 and JS9 did not correspond to any of the recognized species that produce neither odor nor gas, owing to the great variability of their biochemical reactions. Our own oral isolate, SMi, resembled most closely the description of Streptococcus intermedius. Hare et al. (1952) made the same identification of all of the anaerobic streptococci that they isolated from the respiratory tract. For the preparation of inocula, an artificial medium containing 3 mg casamino acids (Difco), 1 mg yeast extract (Difco), 1 mg glucose, 1 mg cystine, and 1 mg tryptophan per 1 ml was used for 6 of the strains. Strains I-V3 and SPU grew poorly in this medium but grew satisfactorily in fluid thioglycolate medium minus agar: trypticase (Baltimore Biological Laboratory), 15 mg; cystine, 5 mg; glucose, 5 mg; yeast extract (Difco), 5 mg; sodium chloride, 25 mg; and sodium thioglycolate, 5 mg, per 1 ml. These media were adjusted to ph 7.2 with sodium hydroxide and sterilized by autoclaving at 12 C for 2 min. Tubes of these media were inoculated from the stock cultures in fluid thioglycolate medium and incubated anaerobically for 48 hr at 37 C. The cells were then collected by centrifugation, washed twice with.85 per cent sodium chloride solution ("saline"), and resuspended in saline to a density equal to that of

3 19571 NUTRITION OF ANAEROBIC STREPTOCOCCI 751 the McFarland standard no. 1, as measured by a Klett-Summerson photoelectric colorimeter with red filter no. 66. Since neither direct nor viable counts of the anaerobic streptococci were feasible, owing to the character of their growth, cell concentrations were expressed with reference to the conventional figure of 3 X 18 cells per ml for standard no. 1 (Kolmer et al., 1951). In most of the experiments,.1 ml of a 1:1 dilution of this suspension in saline was used to incculate 4.9 ml of the test medium, giving a final concentration of 6, cells per ml. ct a: - 24 SFC * SM 2 -x JR4 16 u 12 o K9812 *Kh Eir so 8 X o ph Figure 2. Optimum ph for initiation of growth of 5 strains of anaerobic streptococci. z w CZ w w -J 15 1_ 5 o FLUID THIOGLYCOLATE (TITRATABLE ACIDITY). of (TURBIDITY) SYNTHETIC (TITRATABLE ACIDITY) * (TURBIDITY) o It HOURS Figure 3. Growth curves of Streptococcusfoetidus in fluid thioglycolate and in the synthetic medium as determined turbidimetrically and acidimetrically TABLE 2 A complete synthetic medium for Streptococcus foetidus Component mg/s ml Component mg/s ntl Glucose 5 DL-Isoleucine 2. K2HPO4 2.5 L-Leucine 1. KH2PO4 2.5 DL-Methionine 2. MgSO4*7H2 1. DL-Phenylalanine 2. FeSO4-7H2.5 L-Proline 1. MnSO4*7H2.5 DL-Serine 2. NaCl.5 DL-Threonine 2. Adenine S4.5 DL-Tryptophan 2. Guanine*HCl.5 L-Tyrosine 1. Uracil.5 DL-Valine 2. Sodium thio- 5. sg/5 glycolate nil Agar (purified) 5. p-aminobenzoic 2. acid DL-Alanine 2.5 Calcium panto- 2. thenate Glycine 1. L-Aspartic acid 2.5 Nicotinic acid 2. L-Glutamic acid 2.5 Pyridoxal-HCl 1. L-Arginine HCl 2. Riboflavin 2. L-Lysine HCl 2. Thiamin HCI 1. L-Cystine 3. Biotin.1 L-Histidine HCl 2. Folic acid.1 In early tests of the efficacy of various media to support the growth of the anaerobic streptococci, acid production was used as a measure of growth. The entire contents of each tube were titrated to ph 7.5 with.1 N sodium hydroxide and the amount of acid was recorded after subtracting the titer of an uninoculated tube. Later experiments showed that the typical clumping colonies of these organisms in semisolid media could be suspended evenly if the tubes were agitated sufficiently. The amount of growth could then be determined turbidimetrically. Data presented subsequently (figure 3) show that acid production and turbidimetric determinations correlated closely. The requirements for amino acids, purines, pyrimidines, and vitamins were determined by testing the effect of omitting them, singly or in groups, from a complete synthetic medium, based on the chemically defined medium of Broquist and Snell (1951) for S. faecalis, supplemented with.1 per cent sodium thioglycolate and.1 per cent methanol-extracted agar (Ley

4 752 MERGENHAGEN AND SCHERP [VOL. 74 and MAueller, 1946) (table 2). Each medium, minus the agar, was made up from stock solutions of the ingredients, adjusted to ph 7.2, and sterilized by filtration through a Selas 2 candle. To the filtrate kept at 6 C in a water bath was added aseptically } 5 of its volume of melted 2.5 per cent agar with a preheated pipette. The medium was then distributed aseptically in 4 by l in tubes in 4.9-ml amounts. If the medium was stored for longer than 48 hr, the tubes were placed in a boiling water bath for 5 min to drive off dissolved oxygen. The completeness of the synthetic media was checked with S. faecalis, ATCC strain 843. All experiments were repeated at least once. WiThen a nutrient or group of nutrients appeared to be dispensable, the observation was confirmed by successful subculture in the respective deficient medium. RESULTS AND DISCUSSION In preliminary experiments, growth of S. foetidus strain SFA was initiated regularly by as few as 6, cells per ml in fluid thioglycolate medium, which was henceforth adopted as the reference standard complete artificial medium. Next, the optimum ph for the initiation of growth of 5 of the strains was determined by inoculating them into this medium at various ph values (strains K98 and K112 were oral anaerobic streptococci obtained from Dr. J. B. Macdonald and were not used in other experiments reported). The results, summarized in figure 2, indicated an optimum ph range from 7. to 7.5. A chemically defined medium similar to that of Broquist and Snell (1951) (table 2, minus thioglycolate and agar) fors. faecalis failed to support the growth of strain SFA, even from inocula of 6 million cells per ml. Addition of.1 per cent sodium thioglycolate to the synthetic medium made possible growth from as few as 6, cells per ml, but the maximum growth was only % that attained in fluid thioglycolate medium. Equimolar concentrations of glutathione and cysteine were substituted for thioglycolate but were ineffective. Additional supplementation with.1 per cent agar, purified by exhaustive extraction successively with water and boiling methanol (Ley and Mueller, 1946), soluble starch, or bovine serum albumin (fraction V, Armour) doubled the amount of growth but ovalbumin and acidwashed charcoal (Norit A) had no effect. Data illustrating the effect of each additive are given in table 3. It seems probable that the S. faecalis medium contains the necessary nutrients for S. foetidus, strain SFA, but that even under anaerobic conditions the latter cannot reduce the oxidation-reduction potential sufficiently without the aid of thioglycolate. If this explanation were correct, then glutathione and cysteine should have been as effective as thioglycolate. However, Peters and Snell (1954) have shown that glutathione inhibits Lactobacillus delbrueckii. The function of the agar is not clear. It may supply a surface where nutrients can accumulate in optimal concentrations. Reed and Orr (1943) observed that twice the concentration of cells of various species of Clostridium was needed to initiate growth in a medium with only thioglycolate added instead of both thioglycolate and agar. The beneficial effects of soluble starch and bovine serum albumin may result from a TABLE 3 Growth of various dilutions of washed cells of Streptococcus foetidus strain SFA in various media Medium No. of Cells Inoculated per Ml* 6 6X12 6X13 6X14 6X15 6X16 6X17 Fluid thioglycolate t Synthetic alone... Synthetic plus thioglycolate Synthetic plus thioglycolate and agar Synthetic plus thioglycolate and starch Synthetic plus thioglycolate and serum albumin * Cells harvested from passage medium by centrifugation, washed twice with.85 per cent saline and diluted in saline. All test media incubated anaerobically in Brewer jar for 48 hr at 37 C. t Growth recorded as acidity titratable with.1 N NaOH per 5 ml of medium.

5 1957] NUTRITION OF ANAEROBIC STREPTOCOCCI 753 TABLE 4 Effect of omitting single amino acids from a synthetic medium for Streptococcus foetidus Amino Acid Omitted Amount of Growth of Strain* SFA SFB SFC None Glutamic Aspartic Cystine Methionine Arginine Lysine... Phenylalanine... 1 Tryptophan Tyrosine... Histidine Proline... Serine... 1 Threonine.. Glycine Alanine... Valine... Leucine... Isoleucine... * Expressed as per cent of tturbidity in complete synthetic medium; 1 per cent growth approximates 6 X 18 cells per ml. Inoculum: 6, cells per ml. Incubation for 48 hr at 37 C anaerobically. detoxification, such as these substances have been shown to perform for various other bacteria. With the smaller inocula, from 6 to 6, cells per ml, the complete synthetic media were at least as effective as fluid thioglycolate medium during the 48-hr period of incubation used. However, separate determinations of the growth curves of strain SFA showed that growth in a complete synthetic medium reached its maximum in 48 hr, whereas in fluid thioglycolate medium, growth continued for 72 hr (figure 3). These data also show the close correlation between the titrimetric and turbidimetric methods of measuring growth. These two procedures have generally been found to yield very similar results with essentially homofermentative bacteria (Roberts and Snell, 1946; Lichstein, 1955). Each of the other 2 strains of S. foetidus and the 3 strains of oral anaerobic streptococci grew well in the complete synthetic medium from an inoculum of 6, cells per ml, which was selected for most subsequent tests on the basis of the data in table 3. However, the strains of S. putridus, I-V3 and SPU, could not initiate growth in this medium even from inocula of 6 million cells per ml. Upon the addition of at least 6 mg of a tryptic digest of casein (trypticase, Baltimore Biological Laboratory) per ml, both strains grew as well from inocula of 6, cells per ml in the complete synthetic medium as they did in fluid thioglycolate medium. Maximum stimulation was provided by 12 mg trypticase per ml. It is probable that S. putridus requires one or more factors similar to those supplied by enzymatic digests of casein and other proteins that stimulate the growth of certain lactic acid bacteria (Tittsler et al., 1952). Analysis of the amino acid requirements of the 3 strains of S. foetidus is summarized in table 4. Glutamic and aspartic acids were dispensable, for the omission of either or both did not materially reduce the amount of growth and all 3 strains could be maintained in subculture in the respective deficient media. In the absence of any one of the other amino acids, however, growth either failed to develop or was greatly reduced. In the latter cases, growth was probably attributable to carry-over from the passage medium, for on subculture into the respective deficient media, growth failed to appear in 48 hr. The fact that gram-positive organisms generally contain an TABLE 5 Effect of omitting vitamins, purines, and pyrimidines from a synthetic medium for Streptococcus foetidus Substance Omitted Amount of Growth of Strain* SFA SFB SFC None p-aminobenzoic acid Calcium pantothenate.... Nicotinic acid Pyridoxal-HCI Riboflavin Thiamin HCI... 4 Biotin Folic acid Adenine Guanine Uracil... * Expressed as per cent of turbidity in complete medium; 1 per cent growth approximates 6 X 18 cells per ml. Inoculum: 6, cells per ml. Incubation for 48 hr at 37 C anaerobically.

6 754 MERGENHAGEN AND SCHERP [VOL. 74 TABLE 6 Effect of omitting single amino acids frcma synthetic medium for oral anaerobic streptococci Amino Acid Omitted None... Glutamic... Aspartic... Cystine... Methionine. Arginine... Lysine... Phenylalanine... Tryptophan... Tyrosine... Histidine... Proline... Serine... Threonine. Glycine... Alanine... Valine... Leucine... Isoleucine... SM Growth of Strain* JR4 a * Growth measured turbidimetrically after 48 hr incubation at 37 C anaerobically. Inoculu m: TABLE 7 6, cells per ml. Effect of omitting vitamins, purines, and = growth varying from 1.5 X 18 to 3 x pyrimidines from a synthetic medium 18 cells per ml. for oral anaerobic streptococci - = no growth. Growth of Strain* amino acid pool supports this conclusion (Tayllor, 1947). The ability of these particular anaerolkbic streptococci to synthesize amino acids seerms, therefore, to be very limited, resembling that of most of the streptococci and lactobacilli that ha wve been studied (Tittsler et al., 1952). The vitamin, purine, and pyrimidine requiire- (table 5). All 3 strains evidently must be supplilied with guanine and uracil but adenine can be ments of these organisms were analyzed similabrly considered nonessential. The vitamin requiire- ments were quite heterogeneous. Only panto- for thenic and nicotinic acids were indispensable all 3 strains. The observation that riboflavin atnd biotin were not required distinguishes th4,ese strains of S. foetidus from most of the Lancefi( Ald groups of streptococci (Swift, 1952). Snell and Strong (1939), however, determined that 7 of the 11 species of lactobacilli that they invet stiat gated did not require riboflavin and that least 4 of these 7 species synthesized this vitamin when cultured in a riboflavin free medium. Using both microbiological assay with Lactobacillus casei (Snell and Strong, 1939) and fluorometry with a Coleman Electronic Photofluorometer, we have determined that strain SFA 9 synthesizes about.2,g of riboflavin per mg of dry cells when grown in our complete synthetic medium minus riboflavin. In this respect, this organism resembles some of the lactobacilli studied by Snell and Strong (1939). The rationale for the synthesis of riboflavin by an anaerobic streptococcus is not evident, for functions of this vitamin in anaerobic metabolism have not i been defined. The amino acids indispensable for the oral anaerobic streptococci, strains SM, JR4, and JS9, were much fewer (table 6). In the case of strain SM, when an inoculum of 6, cells per ml was used, growth appeared in all amino-acid deficient media except the one lacking isoleucine. When the inoculum was reduced to 6, cells per ml, aspartic acid and histidine also were indispensable. Presumably, the larger inoculum carried over sufficient of the essential amino Substance Omitted SMt JR4 JS9 None... p-aminobenzoic acid... Calcium pantothenate... - Nicotinic acid... Pyridoxal HCl... Riboflavin... - Thiamin- HCl... Biotin... Folic acid... Adenine... Guanine... Uracil. * Growth estimated turbidimetrically after 48 hr incubation at 37 C anaerobically. Inoculum of 6, cells per ml. = growth varying from 1.5 X 18 to 3 X 18 cells per ml. - = no growth. t Grown with aspartic acid, histidine, and isoleucine as the sole amino acids.

7 1957] NUTRITION OF ANAEROBIC STREPTOCOCCI 755 TABLE 8 The requirement for lipoic acid by Streptococcus faecalis and 3 strains of obligate anaerobic streptococci Mediu*nStreptococcus Growtht Anaerobic streptococci SFC SM JR4 acids to initiate growth. The experiments summarized in table 7 show that this strain grew satisfactorily from the smaller inoculum when supplied with aspartic acid, histidine, and isoleucine as the sole amino acids. The amino acid requirements of strains JR4 and JS9 were analyzed likewise with an inoculum of 6, cells per ml. Aspartic acid, arginine, phenylalanine, histidine, threonine, valine, and leucine were indispensable for JR4, whereas JS9 failed to grow if arginine, tryptophan, histidine, threonine, or leucine was omitted from the synthetic medium. Evidently these oral anaerobic streptococci have greater synthetic abilities than the gas and odor producing strains (S. putridus and S. foetidus) discussed previously. In this respect,, they resemble S. salivarius, which requires only seven amino acids: glutamic, leucine, arginine, methionine or cystine, lysine, tyrosine, and isoleucine (Smiley et al., 1943). These oral strains were also less exacting in their requirements for purines, pyrimidines, and vitamins (table 7). Like the viridans streptococci, they can dispense with purines and pyrimidines. Nicotinic and pantothenic acids are essential for all 3 strains. JR4 and JS9 require pyridoxal and SM requires riboflavin. Paradoxically, JR4 and JS9 require p-aminobenzoic acid but not folic acid. This significant nutritional difference from the other streptococci that have been studied indicates that these organisms utilize p-aminobenzoic acid for something other than the synthesis of folic acid. It is of course alternatively possible that these two strains are impermeable to folic acid. Streptococcus faecalis has an absolute requirement for acetate, for which lipoic (thioctic) acid plus thiamin can be substituted. Aerobiosis, adequately depleted inoculum and media, and absence of reducing agents are essential conditions for the demonstration of this requirement. Lipoic acid and thiamin are utilized for the synethesis of lipothiamide pyrophosphate, which functions as an agent for the transfer of acyl groups essential for the operation of the Krebs cycle. Accordingly, obligate anaerobes and facultative organisms growing anaerobically should have no need for lipoic acid. Shockman (1956) has in fact shown that S. faecalis grows anaerobically in the absence of lipoic acid. We have investigated the lipoic acid requirements of three strains of obligate anaerobic strepto- Aero- Anaerobically bically Anaerobically Synthetic I Synthetic lipoate propionate Synthetic lipoate Synthetic pro- - pionate Inoculum of 6, cells per ml. Incubation for 72 hr at 37 C. * Both agar and thioglycolate were omitted from the media for S. faecalis. Lipoic acid,.1,ug per ml; sodium propionate, 5 mg per ml, where indicated. t Growth estimated turbidimetrically. = heavy growth; = moderate growth; - = no growth. cocci, using S. faecalis ATCC strain 843 for control tests (table 8). Since S. faecalis grew under the conditions of our experiments in the complete synthetic medium without added lipoic acid, we added sodium propionate as a competitive inhibitor (Patterson et al., 1954). Our control tests confirm Shockman (1956), i. e., S. faecalis failed to grow aerobically in the presence of propionate, but grew well anaerobically. In an additional control, the addition of synthetic a-lipoic acid reversed the inhibitory effect of propionate. As expected, the anaerobic streptococci grew regardless of the propionate, indicating that they have no requirement for lipoic acid. ACKNOWLEDGMENTS We are indebted to Drs. J. B. Macdonald, C. G. A. Thomas, and A. R. Prevot for cultures of anaerobic streptococci and to Dr. I. C. Gunsalus for synthetic a-lipoic acid. SUMMARY The nutritional requirements of 8 strains of obligate anaerobic streptococci have been analyzed, using a chemically defined medium for Streptococcus faecalis, supplemented with thio-

8 756 MERGENHAGEN AND SCHERP [VOL. 74 glycolate and agar. These organisms can be separated into 3 groups. Two strains of Streptococcus putridus required in addition a factor or factors present in a tryptic digest of casein, possibly a peptide. Three strains of Streptococcus foetidus required 16 amino acids, 3 or 4 vitamins, guanine, and uracil. Glutamic and aspartic acids, riboflavin, and biotin were dispensable. It was shown that one of these strains synthesized riboflavin when grown in a medium free of this growth factor. Three strains of oral anaerobic streptococci, representative of the non-odor and non-gas producing strains listed in Bergey's Manual of Determinative Bacteriology, resembled the oral viridans streptococci in their nutritional requirements: they could dispense with any one of 11 or more amino acids, with purines and pyrimidines, and required 3 or 4 vitamins. Tests of 1 strain of S. foetidus and 2 strains of oral anaerobic streptococci showed that they did not require lipoic acid. REFERENCES BREED, R. S., MURRAY, E. G. D., AND HITCHENS, A. P Bergey's manual of determinative bacteriology, 6th ed. The Williams & Wilkins Co., Baltimore, Md. BROQUIST, H. P. AND SNELL, E. E Biotin and bacterial growth. I. Relation to aspartate, oleate, and carbon dioxide. J. Biol. Chem., 188, DACK, G. M. 194 Non-sporeforming anaerobic bacteria of medical importance. Bacteriol. Revs., 4, HARE, R., WILDY, P., BILLETT, F. S., AND TWORT, D. N The anaerobic cocci: gas formation, fermentation reactions, sensitivity to antibiotics and sulphonamides. Classification. J. Hyg., 5, KNIGHT, B. C. J. G Nutritional characters. J. Gen. Microbiol., 12, KOLMER, J. A., SPAULDING, E. H., AND RoBINSON, H. W In Approved laboratory technic, 5th ed. Appleton-Century-Crofts, Inc., New York. LEY, H. L., JR., AND MUELLER, J. H On the isolation from agar of an inhibitor for Neisseria gonorrhoeae. J. Bacteriol., 52, LICHSTEIN, H. C The biotin requirements of the genus Propionibacterium. Arch. Biochem. and Biophys., 58, MACDONALD, J. B., SUTTON, R. M., AND KNOLL, M. L The production of fusospirochetal infections in guinea pigs with recombined pure cultures. J. Infectious Diseases, 95, PATTERSON, E. L., PIERCE, J. V., STOKSTAD, E. L. R., HOFFMAN, C. E., BROCKMAN, J. A., JR., DAY, F. P., MACCHI, M. E., AND JUKES, T. N The isolation of protogen. J. Am. Chem. Soc., 76, PETERS, V. J. AND SNELL, E. E Peptides and bacterial growth. VI. The nutritional requirements of Lactobacillus delbrueckii. J. Bacteriol., 67, PREVOT, A. R Les streptocoques anaerobies. Ann. inst. Pasteur, 39, REED, G. B. AND ORR, J. H Cultivation of anaerobes and oxidation-reduction potentials. J. Bacteriol., 45, ROBERTS, E. C. AND SNELL, E. E An improved medium for microbiological assays with Lactobacillus casei. J. Biol. Chem., 163, SHOCKMAN, G. D Acetate requirement for Streptococcus faecalis. J. Bacteriol., 72, SMILEY, K. L., NIVEN, C. F., JR., AND SHERMAN, J. M Nutrition of Streptococcus salivarius. J. Bacteriol., 45, SNELL, E. E. AND STRONG, F. M The effect of riboflavin and of certain synthetic flavins on the growth of lactic acid bacteria. Enzymologia, 6, STONE, M. L. 194 Studies on the anaerobic streptococcus. J. Bacteriol., 39, SWIFT, H. F The streptococci. In Bacterial and mycotic infections of man. 2nd ed. Edited by R. J. Dubos. J. B. Lippincott Co.. Philadelphia. TAYLOR, E. S The assimilation of amino acids by bacteria. J. Gen. Microbiol., 1, TITTSLER, R. P., PEDERSON, C. S., SNELL, E. E., HENDLIN, D., AND NIVEN, C. F., JR Symposium on the lactic acid bacteria. Bacteriol. Revs., 16,