EXPERIMENTS ON THE NUTRITION OFSTREPTOCOCCI

S. H. HUTNER

Laboratory of Bacteriology, College of Agriculture, Cornell University,Ithaca, New York

Received for publication September 24, 1937

The identification of the indispensable nutritional requirementsof the streptococci has been the object of few investigations, de-spite the importance of the group and a number of clear-cutearly advances. Hosoya and Kuroya (1923) discovered that apyogenic streptococcus needed something accompanying vitaminB1 in an alcoholic extract of rice bran. This factor was stableto heat and acid; destroyed by drastic alkali treatment; ad-sorbed by fuller's earth; and precipitated by phosphotungsticacid. Freedman and Funk (1922), using the same hemolyticstrain studied by Mueller, obtained similar data. Beef-heartinfusion decolorized by norite charcoal allowed no growth, butwas reactivated by the addition of peptone. Peptone alone didnot permit growth. A streptococcus factor removed from auto-lyzed yeast by fuller's earth could be eluted with Ba(OH)2.Mueller (1922) found that norite-inactivated meat infusion wasreactivated by the addition of acid-hydrolyzed casein as well as bypeptone. One of the essential amino acids in the hydrolysate wasidentified as methionine. Another, found in the "monoamino"fraction of the Dakin butyl alcohol separation, and precipitatedby HgCl2 in 5 per cent H2SO4 and by Ag2SO4, was not identified.Some additional properties of the hemolytic streptococcus factorwere recorded: presence in spinach and blood; unextractibilityby ether; stability toward oxidants and reductants.

Orla-Jensen and associates (1936a, b) recently reported theirextensive study of the nutrition of saprophytic streptococci.They found that the active material in skim milk, adsorbed onto

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fuller's earth or norite, was eluted by a pyridine-methanol mix-ture. Deproteinized milk was made a better medium by theaddition of casein. Riboflavin was viewed as essential. Severalamino acids were found favorable, although ammonium saltssufficed as sole nitrogen source. Evaluation of these data isrendered difficult by the absence of details of the methods usedfor minimizing contamination of experimental media with ma-terial carried over from stock cultures. The present brief in-vestigation developed from attempts to extend the work fromOrla-Jensen's laboratory, and was as much aimed at finding thebest species for experimental work as at identifying some of thegrowth essentials.

CULTURAL TECHNIQUE

The basic medium consisted of inorganic salts (NH4Cl 0.05 percent; MgSO4.7H20 0.05 per cent; K2HPO4 0.033 per cent;KH2PO4 0.017 per cent; FeSO4 7H2O approximately 0.001 percent) plus Na citrate 11/2 H20 0.5 per cent and sucrose 2.0per cent. The citrate served as buffer and to minimize heavymetal toxicity. Sucrose was chosen because it can be autoclavedwithout decomposition. Only sucrose-fermenting streptococciwere used. The initial pH of all media was adjusted to 7.0to 7.3. Cultures were maintained in 125 cc. Pyrex Erlen-meyer flasks containing 35 cc. of medium. Concentrations ofnutrients were calculated, however, on a basis of a 25 cc. volumeof medium so that all culture fluids were more dilute by the fac-tor 5/7. Sterilization was by autoclaving for 10 minutes at115'C. No evidence was found of destruction of growth essen-tials by this treatment. Growth was measured by the number ofcubic centimeters of 0.1 N NaOH necessary to titrate a culture topinkness with phenolphthalein after subtraction of the alkaliexpended on an uninoculated duplicate flask. Cultures wereincubated at least 24 hours at 370C. - The stock broth used inmost of this work had the following composition: Difco beefextract 0.3 per cent; Difco peptonized milk 0.5 per cent; Difcoyeast extract 0.5 per cent; Difco tryptone 0.1 per cent; glucose0.2 per cent; and CaCO3 in excess. The slight, or no, growth in

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control flasks made up of the basal solution plus adequate aminoacids in the form of acid-hydrolyzed casein indicated that grosscarry-over effects from the one-drop inocula were negligible.Eastman organic chemicals were used.

CHOICE OF ORGANISMS

Streptococcus liquefaciens was selected as our initial experimentalobject because of its good growth in peptone and exceptional resis-tance for a streptococcus to extreme physical conditions. Un-fortunately the strain used, No. 805, produced so much alkali indeproteinized milk or yeast media that titration figures did notmatch growth. A strain of the closely related Streptococcuszymogenes (No. Inl) was found free of this objection but wasstudied only at the close of the investigation. The followingvigorously-growing forms were selected:

Streptococcus bovis No. 11. Isolated by Mrs. C. N. Stark fromthe mouth of cattle.

Streptococcus inulinaceous. Ditto.Streptococcus asalignus (nina). Originally isolated by Prof.

Frost.Streptococcus mastitidis No. iP1. Isolated by Dr. F. R.

Smith from normal human feces and giving the precipitin testfor Lancefield Group B.The Dochez "N. Y. 5" strain of "pyogenes" was chosen in

preference to the typical "pyogenes" described by Evans (1936)because of its greater hardiness and more abundant growth bothin skim milk and yeast media.

Titration values for the forms enumerated were a satisfactoryindex of growth.

AMINO ACID REQUIREMENTS

The ease and thoroughness with which the protein can be re-moved from skim milk, leaving very little protein behind, madeskim milk thus treated efficient in detecting amino acid require-ments. The good growth most streptococci make in skim milkimply it to be a good source of their vitamins. Furthermore it ischeap and available, and its non-protein bulk consists largely of

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readily separable ash and lactose. Following Orla-Jensen'slead, skim milk fractions were made the basis of experimentalwork. Okuda and Zeller's (1921) directions for maximal pro-tein removal were followed: a suspension of skim milk powder inwater at pH 4.5 was steamed 10 minutes at 100'C. The proteincoagulum was filtered off and washed with acidulated hot water.The combined filtrate and washings were brought to neutralitywith NaOH and the calcium phosphate precipitate removed.The resulting deproteinized skim milk (PFM) was preserved withchloroform, as were all other putrescible solutions. No loss inactivity was detected in the preparation after several months atroom temperature in darkness.

TABLE 1Growth of S. pyogenes in deproteinized milk and hydrolyzed casein

NaOH

cc.PFM 1.0 per cent............................................. 0.9PFM 1.0 per cent plus AHC 0.5 per ceit...................... 3.3PFM 2.0 per cent............................................. 0.4PFM 2.0 per cent plus AHC 0.5 per cent...................... 4.4

A sulfuric acid hydrolysate of casein (ARC) was prepared inthe usual manner from Pfanstiehl's "vitamin-free" product.

S. pyogenes makes a relatively poor growth in PFM plus ARCas compared with its growth in unaltered skim milk. A likelyexplanation is that in the process of protein removal seriousamounts of growth factors are lost by adsorption on the protein.Freedman and Funk (1922) in fact noted that hydrolysates ofordinary unpurified proteins allowed growth while hydrolysatesof purified proteins did not. Tests with PFM clearly showeddependence upon amino acids (table 1).The poor and somewhat irregular growth of this streptococcus

in these media led to a comparison between PFM and yeastextract (Difco) as vitamin sources, all cultures being suppliedwith AHC at the 0.5 per cent level. The results are given intable 2.

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EXPERIMENTS ON NUTRITION OF STREPTOCOCCI 433

There was clear evidence of complementary action-an indica-tion of multiple factors. Other experiments proved that trypto-phane was not a factor in the stimulation by yeast. Because ofthis complexity of growth factors, other streptococci growingfar more vigorously in deproteinized milk were studied. It wasalways easy to elicit the casein stimulation (table 3).

TABLE 2Growth of S. pyogenes in deproteinized milk and yeast extract

NaOH

cc.

AHC alone................................................... 0.0PFM 0.5 per cent ............................................. 1.1PFM 1.0 per cent............................................. 1.0Yeast 0.1 per cent............................................. 0.5Yeast 0.5 per cent............................................. 0.2PFM 0.5 per cent plus yeast 0.1 per cent...................... 3.0

TABLE 3Growth of streptococci in deproteinized milk, yeast, and hydrolyzed casein

NaOH

S. asa. S. bow. S. inu. S. mag.

CC. CC. cC. Cc.PFM 2.0 per cent....................... 0.3 0.5 1.3 0.3PFM 2.0 per cent plus AHC 0.5 per cent. . 8.4 6.3 7.7 8.4PFM 2.0 per cent plus yeast 0.25 per centplusAHC 0.25 per cent............... 8.8 9.1 12.3 8.8

Yeast 0.25 per cent plus AHC 0.25 percent............................... 1.4 10.3 7.0 1.7

It will be noted that S. mastitidis and S. asalignus grow poorlyin yeast. This is in marked contrast to S. bovis and the closelyrelated S. inulinaceous, the first of which grows as luxuriantly asS. liquefaciens in milk or yeast media.A similar effect of hydrolyzed casein was found for Strepto-

coccus liquefaciens, Streptococcus zymogenes, Streptococcus duransand Streptococcus salivarius.

Attention was then directed to the identification of the essen-

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tial amino acids in the hydrolyzed casein. Cystine (furnishedin slight excess) was found partly to replace hydrolyzed casein(table 4). Cystein hydrochloride (0.01 per cent), sterilized andadded separately to avoid decomposition, was as effective. Trialof cystine was suggested by its sharing the property of Mueller'sunidentified amino acid of being precipitated by acid mercuricreagents. The nature of the amino acid deficiencies coming intoplay after the satisfaction of the cystine requirement was thesubject of a few expements. Supplements of methionine, tyro-sine, leucine and phenylalanine together were entirely ineffectivefor the three streptococci tested: S. asalignus, S. bovis and S.mawstitidis. Occasionally growth with cystine would almost

TABLE 4Effect of replacement of casein hydrolysate by cystine

NaOH

8. a8a. . bow. S. ma.._~~~~~~~Ccc, c. cc

PFM 1.0 per cent.......................0.0 0.2 0.0PFM 1.0 per cent pluscystine........... 0.4 2.3 1.5PFM 1.0 per cent plus AHC 0.25 per cent.. 4.4 4.4 7.2

equal that with the casein hydrolysate but on serial subculturegrowth fell off almost entirely in the cystine flasks, but hardlyat all in those with the hydrolysate.

STREPTOCOCCUS VITAMINS

Mueller (1922) reported disappointing results with heavy metalprecipitation procedures for concentrating the S. pyogenes factor.It was felt that the complexity of the requirements of S. pyogenesmight have been partly responsible, and that work with lessexacting streptococci might yield clearer data. S. asalignus,S. bovis and S. mastitid8 were used.Heavy metal precipitation. One liter of PFM was evaporated

on a steam bath under an electric fan to 250 cc., the temperaturenot rising above 750C. This concentrate was used for each

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precipitation. An untreated portion of concentrate served ascontrol.

(a) Ba(OH)2-ethanol. Fifty cubic centimeters of concentratewere chilled to 10'C. Hot saturated Ba(OH)2 was added in equalvolume with vigorous stirring and, immediately after, an excess(400 cc.) of chilled 95 per cent ethanol. The curdy precipitatewas placed in the refrigerator and shaken from time to time toprevent caking. After six hours the precipitate was filtered offwith the aid of "filter-cel" (also used in the other precipitations)and washed with 95 per cent ethanol. Alcohol was removed fromthe filtrate by evaporation on a steam bath under electric fan.Barium was removed with H2SO4. On evaporation of an aliquotof the regenerated original barium precipitate abundant crystalsof lactose formed.

(b) Lead acetate-Ba(OH)2. Fifty cubic centimeters of con-centrate were treated with saturated basic lead acetate untilno further precipitation occurred, then brought to pH 8.6 by theaddition of cold saturated Ba(OH)2. After standing overnightthe precipitate was filtered off and washed with water. It wasthen decomposed with a slight excess of H2SO4, the PbSO4 andBaSO4 filtered off, and the lead remaining in the filtrate removedwith H2S. The PbS precipitate was washed with boiling water.H2S was removed from the PbS filtrate by bringing the solutionto a boil. The original Pb-Ba filtrate was similarly treated.

(c) HgSO4. Fifty cubic centimeters of chilled concentrate wastreated with 25 cc. of West and Peterson's mercury reagent (asaturated solution of HgSO4 in 10 per cent H2SO4). Warm sat-urated Ba(OH)2 was added in small portions after successivechillings so that room temperature was not exceeded. WhenpH 7.0 was reached the flask was set overnight in the refrigera-tor, the combined mercury and BaSO4 precipitate filtered off andwashed with small portions of ice water. The precipitate andfiltrate were worked up as in the lead precipitation.

(d) Copper-lime. Fifty cubic centimeters of concentrate weretreated with 25 cc. of 25 per cent CuSO4. An excess of Ca(OH)2suspension was added and the resulting bulky precipitate broken

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up into a fine suspension. The precipitate was filtered off andwashed thoroughly with saturated Ca(OH)2 and was then de-composed with a slight excess of oxalic acid. H2S was passedin. The combined calcium oxalate and CuS precipitates werefiltered off and washed with boiling water. The oxalate remain-ing in the filtrate was removed with CaCO3. The original fil-trate was worked up in the same way as the precipitate.

TABLE 5Growth of streptococci in heavy metal fractions of deproteinized milk plus 0.6 per

cent casein hydrolysateNaOH

S. ata. S. bow. S. mae.

cc. cc cc.AHC alone.............................. 1.4 0.3 1.4PFM 0.5 per cent ....................... 6.9 9.4 5.3

Ba filtrate .............................. 1.7 7.2 2.6Ba precipitate ................ 2.8 0.3 4.9Ba 0.5 per cent each together............ 5.4 6.7 5.7

Pb filtrates ............................. 0.3 0.3 3.0Pb precipitate .......................... 2.5 5.1 3.6Pb 0.5 per cent together................ 1.0 8.3 5.7

Hg filtrate .............................. 0.1 2.6 1.9Hg precipitate .......................... 1.0 0.2 3.5Hg 0.5 per cent each together........... 2.9 8.0 4.1

Cu filtrate .............................. 0.3 3.4 1.2Cu precipitate .......................... 6.3 5.0 2.2Cu 0.5 per cent each together........... 5.3 6.1 4.6

The growth tests were conducted with a concentration of eachfraction corresponding to 0.5 per cent of the original deprotein-ized milk, considering only the organic matter content, as in allother experiments. This concentration of PFM allows abouttwo-thirds maximal growth. The results are shown in table 5.The data are clearer for S. bovis than for S. asalignus and S.

mastitidis. The mercury precipitation data point to multiplefactors, one of them a base. Later adsorption experiments sup-

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port the belief that the principal loss of growth factors was byadsorption on the sulfide precipitates rather than by actual de-struction. Fractionation with silver nitrate-Ba(OH)2 was at-tempted but the very low activity of the fractions for S. bovisand S. mastitidis, the only strains tested, discouraged additionalwork.

Fuller's earth adsorption Adsorption experiments with ful-ler's earth gave rise to the belief that all streptococci require atleast one basic factor which may be characteristic for the genus.A large number of experiments were carried out with Eimer andAmend's fuller's earth and "Frankonite KL." Several otherbrands, in contrast, were quite inactive. The following is a

TABLE 6Growth in fuller'8-earth-treated deproteinized milk

NaOH

S. asa. S. bor. S. mas.

CC. C. CC.

PFM untreated ......................... 7.1 8.5 5.4Fuller's earth 0.4 per cent.............. 7.8 6.3 6.7Fuller's earth 2.0 per cent.............. 1.8 1.5 2.9Fuller's earth 10.0 per cent.............. 0.3 1.7 2.7

sample experiment: deproteinized milk was brought to pH 4.3and treated with the following concentrations of Frankonite KL:0.4, 2.0 and 10 per cent. The filtrates were brought back toneutrality and tested in concentrations corresponding, as inprevious experiments, to 0.5 per cent of the original deprotein-ized milk. All cultures were with 0.25 per cent hydrolyzedcasein. The results are shown in table 6.The lowest concentration of Frankonite removed virtually

all the riboflavin judging by the disappearance of the pronouncedyellowish-green color of the untreated deproteinized milk. S.liquefaciens also grows poorly in fuller's earth-treated PFM.Additions of pure vitamin B1 (gift of the Winthrop ChemicalCompany), riboflavin, guanine, uracil, and a hydrolysate ofyeast nucleic acid-the last suggested by Richardson's (1936)

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findings for Staphylococcus aureu&-to the fuller's earth filtratesdid not improve growth.

It was hoped that differences in vitamin requirements might bea convenient supplementary mean for differentiating speciesof streptococci, particularly in the "t'ridans" group. Thirty-five strains of S. bovi8 from Mrs. C. N. Stark's collection wereinoculated into deproteinized milk plus casein hydrolysate, yeastextract, and both together. The widest diversity was revealed:some grew in either; others in one but not the other; others onlyin both together; and still others poorly even in both vitaminsources together. This behaviour was foreshadowed by theirgrowth in litmus milk in which they were maintained where somegrew so poorly as not to curdle the milk while others did so in afew hours. Similar results, though with fewer strains, were ob-tained with S. salivarius.

DISCUSSION

All streptococci investigated to date require at least one non-amn o-acid growth factor. Its adsorption on fuller's earth andprecipitation by mercuric salts are properties of a base. If thetheory of the close phylogenetic relation between staphylococciand streptococci be accepted, then Knight's (1937) discoverythat Staphylococcus aureus needs nicotinic acid or its amide be-sides vitamin B1 or its cleavage products, is perhaps applicableto streptococci. The properties of nicotinic acid or of its amideare in keeping with those reported for the hypothetical strepto-coccus vitamin. Unfortunately, time did not permit trial ofthese compounds.The difficulty of concentrating vitamin preparations to the

point where their amino-acid content is negligible precludes anysweeping statements as to the number of amino acids that mightbe required even should one succeed in replacing the casein hy-drolysate with known amino acids. No streptococcus was foundcapable of growing with ammonium salts as a sole source of nitro-gen; as previously mentioned growth would occasionally occur indeproteinized milk in the absence of the casein hydrolysate, butcame to an abrupt end on serial subculture. Orla-Jensen's con-

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clusions as to amino-acid requirements have been criticized onsimilar grounds by Wood, Anderson and Werkman (1937).Ehrismann and Dramburg (1937) in a study of a strain of S.pyogenes mention that cystine was supplied as sulfur source butexperimental evidence in support of this was not given in theirpaper.A stimulating effect of riboflavin was not observed-a finding

opposed to that of Orla-Jensen's.

CONCLUSIONS

By the use of deproteinized skim milk and a casein hydrolysate,amino acid requirements for several species of streptococci maybe demonstrated.

Strains of Streptococcus asalignus, Streptococcus bovis and Strep-tococcus mastitidis were found that required cystine (or cysteine)in addition to at least one more amino acid present in acid-hy-drolyzed casein.Treatment of deproteinized milk with fuller's earth inactivated

it for the above streptococci as well as for Streptococcus lique-faciens.

Streptococcus bovis requires at least two non-amino-acid factors:one precipitated by mercuric sulfate, the other not.A great deal of variability in growth requirements was found

within the species Streptococcus bovis and Streptococcus salivarius.

The writer is indebted to the staff and graduate students ofthe Department of Dairy Industry and Bacteriology for theirconstant cooperation.

REFERENCESEHRISMANN, O., AND DRAMBURG, K. 1937 tMber die Verwertung von Ami-

nosauren durch Streptokokken. I. tJ'ber die Bedeutung von Amino-sauren fur das Wachstum von Streptokokken. Zeit. Hyg., 119, 623-634.

EvANs, ALICE C. 1936 Studies on hemolytic streptococci. II. Streptococcuspyogenes. Jour. Bact., 31, 611-624.

FREEDMAN, L., AND FUNK, C. 1922 Nutritional factors in the growth of yeastsand bacteria. I. Vitamins. II. Protein hydrolysates. Jour. Metab.Res., 1, 457-468; 469-480.

HosOYA, S., AND KUROYA, M. 1923 Water-soluble vitamine and bacterialgrowth, with special reference to the chemical and physical properties

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of vitamin essential' for the growth of hemolytic streptococci. Gov.Inst. Infect. Dis. Tokyo Imp. Univ., 2, 26-304.

KNIGHT, B. C. J. G. 1937 The nutrition of Staphylococcus aureus. The activi-ties of nicotinamide, aneurin (vitamin B,) and related compounds.Biochem. Jour., 31, 966-973.

MUELLER, J. H. 1922 Studies on cultural requirements of bacteria. Jour.Bact., 7, 309-324; 325-338.

OKUDA, Y., AND ZOLLER, H. F. 1921 The relations of hydrogen-ion concentra-tion to the heat coagulation of proteins in Swiss cheese whey. Jour.Ind. Eng. Chem., 13, 515-519.

ORLA-JENSEN, S., OTTE, N. C., AND SNOG-KJAER, A. 1936a Der Vitaminbedarfder Milchsiurebakterien. Centbl. Bakt., II Abt., 94, 434-447.

ORLA-JENSEN, S., OTTE, N. C., AND SNOG-KJAzR, A. 1936b Die StickstoffnAhr-ung der Milchaiurebakterien. Centbl. Bakt., II Abt., 94, 460-477.

RICHARDSON, G. M. 1936 The nutrition of Staphylococcus aureus. Necessityof uracil for anaerobic growth. Biochem. Jour., 30, 2184-2190.

WOOD, H. G., ANDERSON, A. A., AND WERKMAN, C. H. 1937 Growth-factors forpropionic and lactic acid bacteria. Proc. Soc. Exptl. Biol. Med., 36.217-219.

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