NUTRITIONAL PATTERNS OF SOME ANAEROBIC STREPTOCOCCI1

STEPHAN E. MERGENHAGEN AND HENRY W. SCHERPDepartment of Bacteriology, School of Medicine and Dentistry, University of Rochester, Rochester, New York

Received for publication June 20, 1957

Various obligatorily anaerobic streptococciare reported to be indigenous to the human oralcavity and vagina. They occasionally producestubborn putrefactive wound infections, some-times in synergism with staphylococci or sundryother bacteria. Bergey's Manual of DeterminativeBacteriology (Breed et al., 1948) recognizes 8species of anaerobic streptococci on the basis ofgas formation, fermentation reactions, andmorphology, as suggested by Pr6vot. However,the criteria for separation into species are notwell defined; data on cultural conditions, preva-lence, biochemical activities, and antigenic com-position are scarce. Stone (1940), using biochemi-cal and serological reactions, could not correlatethe two methods into any workable taxonomicscheme. Due to serological cross-reactions, hesuggested that anaerobic streptococci may havesome relationship with Lancefield groups A, B,and C streptococci.

Knight (1955) has stated that the nutritionalpatterns of microorganisms may be among thecharacters well worth considering for use inclassification. The nutritional requirements ofmembers of the family Lactobacteriaceae are ingeneral very complex (Tittsler et al., 1952).Nutritional observations on the anaerobicstreptococci, however, are as yet incomplete.The present investigation was therefore made ofthe nutritional requirements of 8 strains ofanaerobic streptococci in order to reveal anydefinitive differences or similarities between themand other members of the genus Streptococcus.

MATERIALS AND METHODS

Eight strains of obligatorily anaerobic strep-tococci were obtained from the following sources:

1 The data in this paper were taken from a thesissubmitted by Stephan E. Mergenhagen in partialfulfillment of the requirements for the degree ofDoctor of Philosophy at the University of Roches-ter. This research was aided by a contract, DA-49-007-MD-460, between the Office of the SurgeonGeneral, Department of the Army, and the Uni-versitv of Rochester.

SFA and SFB, from normal vaginal culturestaken by members of the Gynecology Depart-ment, Strong i\Iemorial Hospital, Rochester,N. Y.; SM, from a normal human gingival cul-ture; 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 suppurativeperiodontitis by Dr. J. B. ]\Iacdonald, Divisionof Dental Research, Faculty of Dentistry,University of Toronto, Toronto, Canada.

All strains failed to grow on the surface ofsheep-blood agar plates incubated aerobically butgrew well on such plates incubated in a Breweranaerobic jar at 37 C for 72 hr, at which timethey formed nonhemolytic pinpoint colonies.Stock cultures were maintained by weekly trans-fer in fluid thioglycolate medium (Difco), inwhich all strains grew as strongly gram-positivecocci in chains (figure 1). Strains SFA, SFB,SFC, SPU, and I-V3 produced a fetid odor andabundant gas in liquid media, whereas the oralstrains, JR4, JS9, and SM, produced neither gasnor odor. Other biochemical cbaracteristics ofJR4 and JS9 were determined by AMacdonaldet al. (1954). Table 1 summarizes the speciesidentification of the 8 strains according to thekey for anaerobic streptococci in Bergey's Manualof Determinative Bacteriology (Breed et al., 1948).

Prevot (1925) distinguished 2 species, Strep-tococcus foetidus and Streptococcus anaerobius, onthe basis that S. foetidus formed large spheresnormally in short chains, whereas S. anaerobiusformed average sized spheres normally in longchains. We designated strains SFA, SFB, andSFC as S. foetidus, since on primary isolationthey grew in short chains. Upon continued sub-culture or culture in various media, however, thechain lengths of these strains fluctuated widely.Furthermore, none of them fermented maltose, acharacteristic that identifies them as S. foetidusaccording to Pr6vot's key in Bergey's Manual ofDeterminative Bacteriology. Dack (1940) alsocould not distinguish between the 2 species, S.

74)

MERGENHAGEN AND SCHERP

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. Photo-graphed at 980 X magnification.

TABLE 1Characteristics of various strains of anaerobic streptococci

Strain Gas and Fetid Growth in Broth Maltose coagulated H2S Characterized Species

SFA + Clumping colonies _ + S. foetidusSFB + Clumping colonies _ + S. foetidusSFC + Clumping colonies - + S. foetidusSPU + General turbidity + + S. putridusI-V3 + General turbidity + + S. putridusSM _ General turbidity _ + _ S. intermediusJR4 _ General turbidity _ _JS9 General turbidity -

foetidus and S. anaerobius. He observed in addi-tion that the distinctive characteristics of Strep-tococcus putridus were the production of hydrogensulfide and, after about 10 days, blackening inblood broth cultures. These reactions, along withthe others noted in table 1, identified our strainsSPU and I-V3 as S. putridus.

Oral strains JR4 and JS9 did not correspond toany of the recognized species that produce neitherodor nor gas, owing to the great variability oftheir biochemical reactions. Our own oral isolate,SMi, resembled most closely the description ofStreptococcus intermedius. Hare et al. (1952)made the same identification of all of the an-aerobic streptococci that they isolated from therespiratory tract.

For the preparation of inocula, an artificialmedium containing 300 mg casamino acids

(Difco), 100 mg yeast extract (Difco), 100 mgglucose, 10 mg cystine, and 10 mg tryptophanper 10 ml was used for 6 of the strains. StrainsI-V3 and SPU grew poorly in this medium butgrew satisfactorily in fluid thioglycolate mediumminus agar: trypticase (Baltimore BiologicalLaboratory), 150 mg; cystine, 5 mg; glucose, 50mg; yeast extract (Difco), 50 mg; sodium chlo-ride, 25 mg; and sodium thioglycolate, 5 mg, per10 ml. These media were adjusted to pH 7.2 withsodium hydroxide and sterilized by autoclavingat 120 C for 20 min. Tubes of these media wereinoculated from the stock cultures in fluid thio-glycolate medium and incubated anaerobicallyfor 48 hr at 37 C. The cells were then collectedby centrifugation, washed twice with 0.85 percent sodium chloride solution ("saline"), and re-suspended in saline to a density equal to that of

75() [VOL. 74

NUTRITION OF ANAEROBIC STREPTOCOCCI

the McFarland standard no. 1, as measured by aKlett-Summerson photoelectric colorimeter withred filter no. 66. Since neither direct nor viablecounts of the anaerobic streptococci were feasible,owing to the character of their growth, cell con-centrations were expressed with reference to theconventional figure of 3 X 108 cells per ml forstandard no. 1 (Kolmer et al., 1951). In most ofthe experiments, 0.1 ml of a 1:100 dilution of thissuspension in saline was used to incculate 4.9 mlof the test medium, giving a final concentrationof 60,000 cells per ml.

240 - 0 SFC* SM

200 -x JR4

cto K9812*Kh

160a:u 120

E 80ir so X0

0o440 0

4.5 5.0 55 60 6.5 7.0 75 8.0pH

Figure 2. Optimum pH for initiation of growthof 5 strains of anaerobic streptococci.

150

z

w

CZ

w

w

0

-J

0

0

o FLUID THIOGLYCOLATE (TITRATABLE ACIDITY). of (TURBIDITY)

SYNTHETIC (TITRATABLE ACIDITY)* (TURBIDITY) o

0

001_

50

0 It

9

8

7

HOURS

Figure 3. Growth curves of Streptococcusfoetidusin fluid thioglycolate and in the synthetic mediumas determined turbidimetrically and acidimetri-cally.

TABLE 2A complete synthetic medium for Streptococcus

foetidusComponent Component

mg/S mg/Sml ntl

Glucose 50 DL-Isoleucine 2.0K2HPO4 2.5 L-Leucine 1.0KH2PO4 2.5 DL-Methionine 2.0MgSO4*7H20 1.0 DL-Phenylalanine 2.0FeSO4-7H20 0.05 L-Proline 1.0MnSO4*7H20 0.05 DL-Serine 2.0NaCl 0.05 DL-Threonine 2.0Adenine S04 0.05 DL-Tryptophan 2.0Guanine*HCl 0.05 L-Tyrosine 1.0Uracil 0.05 DL-Valine 2.0Sodium thio- 5.0 sg/5

glycolate nilAgar (purified) 5.0 p-Aminobenzoic 2.0

acidDL-Alanine 2.5 Calcium panto- 2.0

thenateGlycine 1.0L-Aspartic acid 2.5 Nicotinic acid 2.0L-Glutamic acid 2.5 Pyridoxal-HCl 1.0L-Arginine HCl 2.0 Riboflavin 2.0L-Lysine HCl 2.0 Thiamin HCI 1.0L-Cystine 3.0 Biotin 0.01L-Histidine HCl 2.0 Folic acid 0.1

In early tests of the efficacy of various mediato support the growth of the anaerobic strepto-cocci, acid production was used as a measure ofgrowth. The entire contents of each tube weretitrated to pH 7.5 with 0.01 N sodium hydroxideand the amount of acid was recorded after sub-tracting the titer of an uninoculated tube. Laterexperiments showed that the typical clumpingcolonies of these organisms in semisolid mediacould be suspended evenly if the tubes wereagitated sufficiently. The amount of growthcould then be determined turbidimetrically.Data presented subsequently (figure 3) show thatacid production and turbidimetric determina-tions correlated closely.The requirements for amino acids, purines,

pyrimidines, and vitamins were determined bytesting the effect of omitting them, singly or ingroups, from a complete synthetic medium,based on the chemically defined medium ofBroquist and Snell (1951) for S. faecalis, sup-plemented with 0.1 per cent sodium thioglycolateand 0.1 per cent methanol-extracted agar (Ley

75119571

MERGENHAGEN AND SCHERP

and MAueller, 1946) (table 2). Each medium,minus the agar, was made up from stock solutionsof the ingredients, adjusted to pH 7.2, andsterilized by filtration through a Selas 02 candle.To the filtrate kept at 60 C in a water bath was

added aseptically } 5 of its volume of melted2.5 per cent agar with a preheated pipette. Themedium was then distributed aseptically in 4 byl in tubes in 4.9-ml amounts. If the medium was

stored for longer than 48 hr, the tubes were placedin a boiling water bath for 5 min to drive offdissolved oxygen. The completeness of thesynthetic media was checked with S. faecalis,ATCC strain 8043. All experiments were re-

peated at least once. WiThen a nutrient or group ofnutrients appeared to be dispensable, the obser-vation was confirmed by successful subculture inthe respective deficient medium.

RESULTS AND DISCUSSION

In preliminary experiments, growth of S.foetidus strain SFA was initiated regularly by as

few as 6,000 cells per ml in fluid thioglycolatemedium, which was henceforth adopted as thereference standard complete artificial medium.Next, the optimum pH for the initiation ofgrowth of 5 of the strains was determined byinoculating them into this medium at variouspH values (strains K98 and K112 were oralanaerobic streptococci obtained from Dr. J. B.Macdonald and were not used in other experi-ments reported). The results, summarized infigure 2, indicated an optimum pH range from7.0 to 7.5.A chemically defined medium similar to that of

Broquist and Snell (1951) (table 2, minus thio-

glycolate and agar) forS. faecalis failed to supportthe growth of strain SFA, even from inocula of60 million cells per ml. Addition of 0.1 per centsodium thioglycolate to the synthetic mediummade possible growth from as few as 6,000 cellsper ml, but the maximum growth was only %that attained in fluid thioglycolate medium.Equimolar concentrations of glutathione andcysteine were substituted for thioglycolate butwere ineffective. Additional supplementation with0.1 per cent agar, purified by exhaustive extrac-tion successively with water and boiling methanol(Ley and Mueller, 1946), soluble starch, or bovineserum albumin (fraction V, Armour) doubled theamount of growth but ovalbumin and acid-washed charcoal (Norit A) had no effect. Dataillustrating the effect of each additive are givenin table 3. It seems probable that the S. faecalismedium contains the necessary nutrients for S.foetidus, strain SFA, but that even under ana-

erobic conditions the latter cannot reduce theoxidation-reduction potential sufficiently withoutthe aid of thioglycolate. If this explanation were

correct, then glutathione and cysteine shouldhave been as effective as thioglycolate. However,Peters and Snell (1954) have shown that gluta-thione inhibits Lactobacillus delbrueckii. Thefunction of the agar is not clear. It may supply a

surface where nutrients can accumulate inoptimal concentrations. Reed and Orr (1943)observed that twice the concentration of cells ofvarious species of Clostridium was needed toinitiate growth in a medium with only thio-glycolate added instead of both thioglycolateand agar. The beneficial effects of soluble starchand bovine serum albumin may result from a

TABLE 3Growth of various dilutions of washed cells of Streptococcus foetidus strain SFA in various media

No. of Cells Inoculated per Ml*Medium

60 6X102 6X103 6X104 6X105 6X106 6X107

Fluid thioglycolate................... 0 0 1 .85t 4.55 8.15 9.05 9.55Synthetic alone ....................... 0 0 0 0 0 0 0Synthetic plus thioglycolate .......... 0 0 1.20 1.05 2.30 2.55 2.60Synthetic plus thioglycolate and agar. 0 0.30 3.10 3.10 4.30 4.60 5.95Synthetic plus thioglycolate and starch. 0 1.50 2.35 2.70 4.50 4.50 6.20Synthetic plus thioglycolate and serumalbumin ............................ 0 0 0 5.30 5.90 11.60 11.80

* Cells harvested from passage medium by centrifugation, washed twice with 0.85 per cent salineand diluted in saline. All test media incubated anaerobically in Brewer jar for 48 hr at 37 C.

t Growth recorded as acidity titratable with 0.01 N NaOH per 5 ml of medium.

[VOL. 74752

NUTRITION OF ANAEROBIC STREPTOCOCCI

TABLE 4Effect of omitting single amino acids from a

synthetic medium for Streptococcus foetidus

Amount of Growth of Strain*Amino Acid Omitted

SFA SFB SFC

None .................... 100 100 100Glutamic ................. 85 84 44Aspartic ................. 94 102 90Cystine .................. 10 5 0Methionine .............. 13 13 0Arginine ................ 13 0 0Lysine ................... 0 0 0Phenylalanine ........... 10 0 0Tryptophan .............. 13 0 0Tyrosine ................. 0 0 0Histidine ................ 9 24 0Proline .................. 0 0 0Serine ................... 10 0 0Threonine . . 0 0 0Glycine .................. 24 0 42Alanine .................. 0 0 0Valine ................... 0 0 0Leucine .................. 0 0 0Isoleucine................ 0 0 0

* Expressed as per cent of tturbidity in completesynthetic medium; 100 per cent growth approxi-mates 6 X 108 cells per ml. Inoculum: 60,000 cellsper ml. Incubation for 48 hr at 37 C anaerobically.

detoxification, such as these substances havebeen shown to perform for various other bacteria.With the smaller inocula, from 600 to 6,000

cells per ml, the complete synthetic media wereat least as effective as fluid thioglycolate mediumduring the 48-hr period of incubation used. How-ever, separate determinations of the growthcurves of strain SFA showed that growth in acomplete synthetic medium reached its maximumin 48 hr, whereas in fluid thioglycolate medium,growth continued for 72 hr (figure 3). These dataalso show the close correlation between thetitrimetric and turbidimetric methods of meas-uring growth. These two procedures have general-ly been found to yield very similar results withessentially homofermentative bacteria (Robertsand Snell, 1946; Lichstein, 1955).Each of the other 2 strains of S. foetidus and

the 3 strains of oral anaerobic streptococci grewwell in the complete synthetic medium from aninoculum of 60,000 cells per ml, which wasselected for most subsequent tests on the basisof the data in table 3. However, the strains of

S. putridus, I-V3 and SPU, could not initiategrowth in this medium even from inocula of 60million cells per ml. Upon the addition of atleast 6 mg of a tryptic digest of casein (trypticase,Baltimore Biological Laboratory) per ml, bothstrains grew as well from inocula of 60,000 cellsper ml in the complete synthetic medium as theydid in fluid thioglycolate medium. Maximumstimulation was provided by 12 mg trypticaseper ml. It is probable that S. putridus requiresone or more factors similar to those suppliedby enzymatic digests of casein and other proteinsthat stimulate the growth of certain lactic acidbacteria (Tittsler et al., 1952).

Analysis of the amino acid requirements of the3 strains of S. foetidus is summarized in table 4.Glutamic and aspartic acids were dispensable, forthe omission of either or both did not materiallyreduce the amount of growth and all 3 strainscould be maintained in subculture in the respec-tive deficient media. In the absence of any oneof the other amino acids, however, growth eitherfailed to develop or was greatly reduced. In thelatter cases, growth was probably attributableto carry-over from the passage medium, for onsubculture into the respective deficient media,growth failed to appear in 48 hr. The fact thatgram-positive organisms generally contain an

TABLE 5Effect of omitting vitamins, purines, andpyrimidines from a synthetic medium

for Streptococcus foetidusAmount of Growth of Strain*

Substance OmittedSFA SFB SFC

None ................... 100 100 100p-Aminobenzoic acid..... 167 86 102Calcium pantothenate... .0 0 0Nicotinic acid........... 0 0 14Pyridoxal-HCI........... 0 0 61Riboflavin .............. 106 29 88Thiamin HCI............ 0 0 40Biotin .................. 52 52 74Folic acid................ 100 90 0Adenine.52 90 102Guanine .................. 12 0 0Uracil ................... 0 0 0

* Expressed as per cent of turbidity in completemedium; 100 per cent growth approximates 6 X108 cells per ml. Inoculum: 60,000 cells per ml.Incubation for 48 hr at 37 C anaerobically.

1957] 753

MERGENHAGEN AND SCHERP

TABLE 6Effect of omitting single amino acids frcma a

synthetic medium for oral anaerobicstreptococci

Amino Acid Omitted

None .....................

Glutamic .................

Aspartic .................

Cystine ..................

Methionine.Arginine ................

Lysine ...................

Phenylalanine ............

Tryptophan ..............

Tyrosine .................

Histidine .................

Proline ................Serine ...................

Threonine.Glycine ..................

Alanine ..................

Valine ...................

Leucine .................

Isoleucine ...............

Growth of Strain*

SM

+

+

+

+

JR4

+

* Growth measured turbidimetrically afterhr incubation at 37 C anaerobically. Inoculu6,000 cells per ml.+ = growth varying from 1.5 X 108 to 3

108 cells per ml.- = no growth.

amino acid pool supports this conclusion (Tayl1947). The ability of these particular anaerolstreptococci to synthesize amino acids seer

therefore, to be very limited, resembling thatmost of the streptococci and lactobacilli that habeen studied (Tittsler et al., 1952).The vitamin, purine, and pyrimidine requi

ments of these organisms were analyzed simila(table 5). All 3 strains evidently must be suppliwith guanine and uracil but adenine can

considered nonessential. The vitamin requiments were quite heterogeneous. Only pan

thenic and nicotinic acids were indispensableall 3 strains. The observation that riboflavin a

biotin were not required distinguishes th4strains of S. foetidus from most of the Lancefi(groups of streptococci (Swift, 1952). Snell aStrong (1939), however, determined that 7the 11 species of lactobacilli that they invetgated did not require riboflavin and that

least 4 of these 7 species synthesized thisvitamin when cultured in a riboflavin freemedium. Using both microbiological assay withLactobacillus casei (Snell and Strong, 1939) andfluorometry with a Coleman Electronic Photo-fluorometer, we have determined that strain SFA

9 synthesizes about 0.02,g of riboflavin per mg

of dry cells when grown in our complete syntheticmedium minus riboflavin. In this respect, thisorganism resembles some of the lactobacillistudied by Snell and Strong (1939). The rationalefor the synthesis of riboflavin by an anaerobicstreptococcus is not evident, for functions ofthis vitamin in anaerobic metabolism have not

i been defined.The amino acids indispensable for the oral

anaerobic streptococci, strains SM, JR4, andJS9, were much fewer (table 6). In the case ofstrain SM, when an inoculum of 60,000 cells per

ml was used, growth appeared in all amino-aciddeficient media except the one lacking isoleucine.WWhen the inoculum was reduced to 6,000 cellsper ml, aspartic acid and histidine also were

indispensable. Presumably, the larger inoculumcarried over sufficient of the essential amino

48m: TABLE 7

Effect of omitting vitamins, purines, andx pyrimidines from a synthetic medium

for oral anaerobic streptococci

lor,kbicms,ofwve

ire-brlyliedbeire-to-fortnd,eseAldndof

sti-at

Growth of Strain*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 48hr incubation at 37 C anaerobically. Inoculum of6,000 cells per ml.+ = growth varying from 1.5 X 108 to 3 X

108 cells per ml.- = no growth.t Grown with aspartic acid, histidine, and

isoleucine as the sole amino acids.

754 [VOL. 74

NUTRITION OF ANAEROBIC STREPTOCOCCI

acids to initiate growth. The experiments sum-

marized in table 7 show that this strain grew

satisfactorily from the smaller inoculum whensupplied with aspartic acid, histidine, and isoleu-cine as the sole amino acids. The amino acidrequirements of strains JR4 and JS9 were ana-

lyzed likewise with an inoculum of 6,000 cells per

ml. Aspartic acid, arginine, phenylalanine,histidine, threonine, valine, and leucine were

indispensable for JR4, whereas JS9 failed to growif arginine, tryptophan, histidine, threonine, or

leucine was omitted from the synthetic medium.Evidently these oral anaerobic streptococci havegreater synthetic abilities than the gas and odorproducing strains (S. putridus and S. foetidus)discussed previously. In this respect,, theyresemble S. salivarius, which requires only seven

amino acids: glutamic, leucine, arginine, methio-nine or cystine, lysine, tyrosine, and isoleucine(Smiley et al., 1943).These oral strains were also less exacting in

their requirements for purines, pyrimidines, andvitamins (table 7). Like the viridans strepto-cocci, they can dispense with purines and pyrimi-dines. Nicotinic and pantothenic acids are essen-

tial for all 3 strains. JR4 and JS9 require pyridoxaland SM requires riboflavin. Paradoxically,JR4 and JS9 require p-aminobenzoic acid butnot folic acid. This significant nutritional differ-ence from the other streptococci that have beenstudied indicates that these organisms utilizep-aminobenzoic acid for something other thanthe synthesis of folic acid. It is of course alterna-tively possible that these two strains are im-permeable to folic acid.

Streptococcus faecalis has an absolute require-ment for acetate, for which lipoic (thioctic)acid plus thiamin can be substituted. Aerobiosis,adequately depleted inoculum and media, andabsence of reducing agents are essential condi-tions for the demonstration of this requirement.Lipoic acid and thiamin are utilized for thesynethesis of lipothiamide pyrophosphate, whichfunctions as an agent for the transfer of acylgroups essential for the operation of the Krebscycle. Accordingly, obligate anaerobes andfacultative organisms growing anaerobicallyshould have no need for lipoic acid. Shockman(1956) has in fact shown that S. faecalis grows

anaerobically in the absence of lipoic acid. Wehave investigated the lipoic acid requirementsof three strains of obligate anaerobic strepto-

TABLE 8The requirement for lipoic acid by Streptococcus

faecalis and 3 strains of obligate anaerobicstreptococci

Growtht

AnaerobicMediu*nStreptococcus streptococci

SFC SM JR4

Aero- Anaero-bically bically Anaerobically

Synthetic ++ ++ ++ ++I++Synthetic + lipoate + + ++ ++ +++ propionate

Synthetic + lipoate ++ + ++ ++ ++Synthetic + pro- - ++ ++ + ++

pionate

Inoculum of 60,000 cells per ml. Incubation for72 hr at 37 C.

* Both agar and thioglycolate were omittedfrom the media for S. faecalis. Lipoic acid, 0.01,ug per ml; sodium propionate, 5 mg per ml, whereindicated.

t Growth estimated turbidimetrically.++ = heavy growth; + = moderate growth;

- = no growth.

cocci, using S. faecalis ATCC strain 8043 forcontrol tests (table 8). Since S. faecalis grewunder the conditions of our experiments in thecomplete synthetic medium without added lipoicacid, we added sodium propionate as a competi-tive inhibitor (Patterson et al., 1954). Our con-trol tests confirm Shockman (1956), i. e., S.faecalis failed to grow aerobically in the presenceof propionate, but grew well anaerobically. Inan additional control, the addition of synthetica-lipoic acid reversed the inhibitory effect ofpropionate. As expected, the anaerobic strepto-cocci grew regardless of the propionate, indicatingthat 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 ofanaerobic streptococci and to Dr. I. C. Gunsalusfor synthetic a-lipoic acid.

SUMMARY

The nutritional requirements of 8 strains ofobligate anaerobic streptococci have been ana-lyzed, using a chemically defined medium forStreptococcus faecalis, supplemented with thio-

1957] 755

MERGENHAGEN AND SCHERP

glycolate and agar. These organisms can be sep-arated into 3 groups. Two strains of Streptococcusputridus required in addition a factor or factorspresent in a tryptic digest of casein, possibly apeptide. Three strains of Streptococcus foetidusrequired 16 amino acids, 3 or 4 vitamins, guanine,and uracil. Glutamic and aspartic acids, ribo-flavin, and biotin were dispensable. It was shownthat one of these strains synthesized riboflavinwhen grown in a medium free of this growthfactor. Three strains of oral anaerobic strepto-cocci, representative of the non-odor and non-gasproducing strains listed in Bergey's Manual ofDeterminative Bacteriology, resembled the oralviridans streptococci in their nutritional require-ments: they could dispense with any one of 11or more amino acids, with purines and pyrimi-dines, and required 3 or 4 vitamins. Tests of 1strain of S. foetidus and 2 strains of oral anaerobicstreptococci showed that they did not requirelipoic acid.

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KNIGHT, B. C. J. G. 1955 Nutritional char-acters. J. Gen. Microbiol., 12, 348-351.

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ROBERTS, E. C. AND SNELL, E. E. 1946 An im-proved medium for microbiological assayswith Lactobacillus casei. J. Biol. Chem., 163,499-509.

SHOCKMAN, G. D. 1956 Acetate requirement forStreptococcus faecalis. J. Bacteriol., 72, 101-104.

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