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FACTORS NECESSARY FOR MASSIVE GROWTH OFGROUP A HEMOLYTIC STREPTOCOCCUS'

ALAN W. BERNHEIMER AND A. M. PAPPENHEIMER, JR.Department of Bacteriology, School of Medicine, University of Pennsylvania

Received for publication July 7, 1941

A number of non-dialyzable biologically active substances,presumably proteins, are found in the culture filtrates of group Ahemolytic streptococci. We have been particularly interested,in this laboratory, in the nature of hemolysin (streptolysin) andof scarlet fever toxin. In order to facilitate the isolation of thesesubstances in a state approaching purity and in sufficient yield topermit chemical studies, we have felt it important to develop aprotein-free medium of defined composition capable of supportingheavy growth. The medium has been developed specifically forthe C203S strain of group A streptococcus because this strainproduces exceptionally high titers of hemolysin. Althoughworked out for this particular strain, we have found that manyother strains also give heavy growth on the medium.The factors essential for the growth of streptococci have been

investigated recently by Hutner (1938), Rane and Subbarow(1938), Subbarow and Rane (1939), McIlwain and co-workers(1939, 1940), Woolley and Hutchings (1939), Pappenheimer andHottle (1940), Hottle et al. (1941), and Woolley (1941). As aresult of this work, several strains of group A hemolytic strepto-coccus have been grown on media of essentially known composi-tion. Most of these workers, however, have been primarilyinterested in determining the presence or absence of growth underdefined conditions and have expressed the amount of growth inrelative rather than absolute terms. For reasons which willbecome apparent, it seems probable that the actual amount ofgrowth taking place on the defined media studied by them wasrelatively slight.

1 The expenses of this work have been defrayed by a generous grant from theCommonwealth Fund.

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482 ALAN W. BERNHEIMER AND A. M. PAPPENHEIMER, JR.

It has been shown by Friedemann (1939) that the hemolyticstreptococcus forms lactic acid almost quantitatively from glu-cose. The accumulation of lactic acid in cultures grown in thepresence of glucose causes the pH to fall rapidly to 4 to 5, at whichpoint growth ceases. Mueller and Klise (1932) and others haveshown that the yield of acid-producing organisms may be greatlyincreased by periodic neutralization with alkali. It appears prob-able that growth factors which are present in optimal amount forgrowth without neutralization, may become limiting factors underconditions where the pH is kept near its optimal value. In thepresent paper we are reporting a study of the factors necessaryfor continued growth of streptococcus on an essentially definedmedium when the lactic acid produced is periodically neutralized.

METHODS AND MATERIALS

Medium. The medium in use at the start of this investigationwas similar to that previously described (Pappenheimer andHottle, 1940). It consisted of a complete acid hydrolysate ofEastman's "de-ashed" gelatin, supplemented with the aminoacids: cystine, tryptophane, tyrosine, methionine, and glutamine;with uracil and adenine; salts, glucose, and the various membersof the vitamin B group previously shown essential for this strain.Cultures were incubated under 1 per cent CO2 in air. Thechanges which have been made in this medium leading to in-creased growth will be considered in a later paragraph. In thepresent experiments, most of the tests were carried out using 20ml. of medium distributed in 8-inch test tubes.

Materials. Since we have been primarily interested in growingstreptococci in mass culture on a simplified medium of low cost,we have used a technical grade of chemicals wherever we havefelt that this grade will give reproducible results. Technicalcasein was obtained from Eastman's; glucose, unless otherwisespecified, was of the technical grade known as cerelose; the biotinconcentrate was obtained from S.M.A. Corporation, ChagrinFalls, Ohio, and is reported to contain 100 gamma biotin permilliliter. Its purity is given as 0.05 per cent. We are indebtedto Merck and Company for generous gifts of thiamine, riboflavin,


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GROWTH OF GROUP A HEMOLYTIC STREPTOCOCCUS

pyridoxine, and calcium pantothenate. Glutamine was preparedfrom common beets by the method of Vickery, Pucher andClark (1935).

Culture technic. The group A streptococcus employed was asmooth variant of the C203S strain sent to this laboratory byDr. Lancefield some years ago. The organism was maintained,for the most part, on extract-peptone broth, in which mediumit was transplanted approximately every eight hours. The testmedia were usually inoculated with 1/200 their volume of a salinesuspension of organisms (washed twice with saline) from a 6-8-hour broth culture. Test media were inoculated late in the eve-ning-usually about 11:00 p.m. They were incubated at 35°C.until the following morning, when, beginning at about 9:00 a.m.,the acid formed was neutralized with 5N NaOH at intervals ofabout one hour until 11: 00 p.m. The cultures were then returnedto the incubator and the following afternoon the growth wasdetermined by measuring turbidity. In order to check theirpurity, the cultures were frequently streaked on blood agar plates.

Estimation of growth. To determine the amount of growth, theorganisms from a known volume of culture were centrifuged andsuspended in a suitable volume of saline. The growth wasmeasured in a photoelectric colorimeter. The galvanometerdeflection was converted to milligrams bacterial nitrogen byreading from a curve obtained by plotting bacterial nitrogen ofstandard suspensions against the logarithm of the deflection. Ithas been found convenient to express growth in arbitrary units,one such unit being equivalent to 2 mg. bacterial nitrogen perliter of medium. Ten to 20 units are equivalent to what is gen-erally considered good growth in 18-hour broth cultures ofstreptococcus.

EXPERIMENTAL

On the 1 per cent gelatin hydrolysate medium, containing 0.25per cent glucose as previously described (Pappenheimer andHottle, 1940), growth of 10-16 units is obtained. About thesame amount of growth is obtained if 1 per cent casein hydrolysateis used instead of gelatin. The amount of acid produced under

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these conditions suggests that all the glucose has been used upand that the glucose concentration has become a limiting factor.However, when the glucose concentration is increased and thecultures periodically neutralized, growth is only slightly im-proved, i.e. to about 15-20 units. It is clear therefore that otherfactors in addition to glucose and acidity are involved. If amedium similar to the above, containing 1 per cent casein hy-drolysate as a base, is supplemented with 1 per cent Parke-Davispeptone and 4 per cent glucose and the acid produced is periodi-cally neutralized with 5N NaOH, growth of 150-200 units maybe obtained with strain C203S. Such massive growth cor-responds to about 300-400 mgm. bacterial nitrogen per liter ornearly 4 grams dry weight of bacteria per liter. It should bepossible to attain at least as high growth on the chemically definedmedium without peptone, if all growth factors are present inoptimal concentrations. The experiments outlined below haveled to production of growth of the C203S strain on a definedmedium, approaching that quoted above for peptone-containingmedia.

Decrease in sodium chloride concentration. In the unmodifiedmedium, the amino acids are supplied chiefly by gelatin hydro-lyzed with 6N HC1. A considerable portion of the chloride isincorporated into the final medium as sodium chloride. An ex-periment with broth cultures in which the final amount of growthdecreased with increasing amounts of NaCl, suggested that thesalt concentration of the gelatin hydrolysate medium might beunfavorable for continued growth. In view of this observation,gelatin, and later casein, were hydrolysed with 6N H2SO4 insteadof HC1. The sulfate was removed by precipitation with hotbarium hydroxide. Substitution of sulfuric for hydrochloric-acid-hydrolysed gelatin, at this point gave slightly improvedgrowth.

Effect of sodium bicarbonate. It was previously shown (Pap-penheimer and Hottle, 1940) that carbon dioxide is essential forgrowth of hemolytic streptococci. If sodium bicarbonate isadded to the medium, carbon dioxide is liberated during growthby the lactic acid produced. The effect of addition of increasing


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GROWTH OF, GROUP A HEMOLYTIC STREPTOCOCCUS 485

amounts of NaHCO3 (sterilized by filtration) to gelatin hydroly-sate medium containing 2.4 per cent glucose is shown in table 1.The titration indicates an optimal concentration of about 0.25per cent NaHCOs. Higher concentrations retard or inhibitgrowth. The favorable effect of bicarbonate on growth is prob-ably due to the combined effect of buffer action and of CO2liberated during growth.

Study of exhausted medium. In spite of the decreased salt con-centration and optimal bicarbonate concentration, growth stillfailed to increase beyond 25-30 units upon neutralization.One liter of sulfuric-acid-hydrolysed gelatin medium containing

2.4 per cent glucose was distributed among four 500 ml. flasks,

TABLE 1Effect of sodium bicarbonate on growth of CS0SS strain

NaHCOs ADDEDD TO 10 ML. MZDIUM (BACTERIAL NITROGZE PER 10 ML.) X 50

mgm. mgm.0 11.810 18.625 23.450 16.0100 6.2200 1.6

inoculated and incubated at 35TC. for 27 hours. The cultureswere pooled and the average growth found to be 25 units. ThepH was brought to 7.3 by the addition of 5N NaOH. The organ-isms were then removed by centrifugation and the supernatantfluid sterilized by filtration through a bacterial sintered glassfilter. As expected, this "exhausted" medium supported onlynegligible growth (2 units). When, however, glutamine, calciumpantothenate, riboflavin, thiamine and sodium thioglycollate wereadded back in their original concentrations, 12-14 units of growthwere obtained. As shown in table 2, the exhausted mediumwas found deficient in both glutamine and pantothenic acid.As expected on the basis of this experiment, a substantial in-

crease in growth was achieved by periodically neutralizing cul-tures containing four times the usual amounts of glutamine and


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calcium pantothenate. The glutamine requirement will be dis-cussed in more detail below.

Substitution of casein for gelatin hydrolysate. Since casein is amore complete protein than gelatin, its use as a source of aminoacids has the advantage that the basal medium need not be sup-plemented with tyrosine and methionine. Both these amino acidshave been shown to be essential for optimal growth of the C203Sstrain. Substitution of casein for gelatin hydrolysate and in-creasing the casein hydrolysate from 1 to 1.5 per cent and 2.0 percent has led to further substantial increase in growth (table 3).Using 2 per cent casein hydrolysate, 3-4 per cent glucose and

TABLE 2Study of exhausted medium

Addition mixture: glutamine, calcium pantothenate, riboflavin, thiamine,thioglycollic acid

FACTOR OMITTED FROM ADDMON MIXTURE (BACTERIAL NITROGEN PER10 ML.) X 50

mgm.None........................................... 13.8Glutamine......................................... 4.4Calcium pantothenate................................ 5.2Riboflavin .......................................... 12.6Thiamine.......................................... 14.0All......................................... 2.4

optimal concentrations of the other factors mentioned above,growth of 70-100 units may be regularly obtained and may reachas high as 125 units (250 mgm. bacterial nitrogen per liter).The effects of the various factors which have led to the productionof this heavy growth on the defined medium have been sum-marized in table 3.

Salt concentration. Since lactic acid is formed in high yieldfrom glucose by streptococci it is to be expected that fairly highconcentrations of sodium lactate will be formed during growthwith neutralization. Indeed, the amount of alkali necessary forneutralization may be taken as a fairly good approximation of theamount of growth. For growth of 100-120 units we have foundabout 40 ml. of 5N NaOH required for neutralization of the acid


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GROWTH OF GROUP A HEMOLYTIC STREPTOCOCCUS 487

produced by one liter of culture. This corresponds to the utiliza-tion of about 18-20 grams glucose per liter and a final concentra-tion of 5 molar sodium lactate. In the presence of peptone,where growth up to 200 units has been attained, we have notedthat more than 30 grams of glucose may be used up per liter anda final concentration of 3 molar lactate formed. It seemed possi-ble that this high salt concentration might be sufficiently in-hibitory to cause cessation of growth quite apart from the exhaus-

TABLE 3Summary of experiments leading to increased growth on defined medium

CULTURE (BACTZE-GELATIN CASEIN SODIUM CAWCIUM INCUBATED PEP1ODI- |(ATHYDROL- HYDIROL- DICARt LUA PANTO- GLUCOSE 1NDERCALLY NU`zMROYSATE YSATE BONATE THENATE 02CEN IHTL )R 10

N0aOHJ ML.) X 50

grams grams grams mgm. mgm. gramsper liter per liter piteler p liter per liter permlm.

10* 0 50 1 10 Yes No 1610* 0 50 1 10 Yes Yes 1510 0 50 1 10 Yes No 2010 2.5 50 1 24 Yes No 2310 2 50 1 15 Yes No 24

10 2 50 1 15 Yes No 3010 2 200 4 40 No Yes 57

20 2 200 4 33t No Yes 6315 2 200 4 40 No Yes 8020 2 200 4 40 No Yes 126

* Hydrolyzed with 6N HCO; others hydrolyzed with 6N HaSO and precipitatedwith barium hydroxide.

t Reagent quality glucose.

tion of a particular nutrient substance or vitamin. The effect ofincreasing sodium chloride, potassium chloride, and sodium lactateconcentration on growth, with neutralization is shown in figure 1.It will be noted from figure 1 that no appreciable growth occurswithin 40 hours when the uni-univalent salt concentration reaches0.35 molar. It is significant that this is the same lactate concen-tration reached in the peptone-containing medium which gave200 units of growth. Salt concentrations above 0.15 molarbecome definitely inhibitory whereas in the neutralized cultureseven lower concentrations reduce growth.


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The chief effect of low concentrations of sodium chloride andsodium lactate appears to be in decreasing the rate of growthrather than its final magnitude. Less than 0.05 molar sodiumchloride or lactate causes a significant decrease in the amount ofgrowth observed after 12-20 hours. In the titrations illustrated

0 0.1 0. 0.3

SALT CNENTRATION (Mol s p er 1 it er)FIG. 1. EFFECT OF VARIOUS SALTs ON GROWTH OF C2038 STRAIN

WITTH NEUTRALZATION0, KCI; OD, NaCl; X, sodium lactate

in figure 1, growth was allowed to continue for 40 hours. Itseems probable that the accumulation of sodium lactate is animportant factor in keeping the rate of growth in neutralizedcultures fairly constant in spite of the greatly increased bacterialpopulation.

It must be stated that it is difficult to obtain reproducible


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GROWTH OF GROUP A- HEMOLYTIC STREPTOCOCCUS 489

titrations of the effect of salt concentration on growth with neu-tralization. It has not been convenient to continue neutralizationafter 24-30 hours growth. Since the effect of salt is on the rateof growth as well as its final magnitude, the actual position andshape of the curves shown in figure 1 should not be considered asfinal since it is unlikely that growth was complete in all cases.

The glutamine requirement. It has been shown by McIlwainet al. (1939) that glutamine is essential for growth of most strai

GLUTAMINE PER LITER s&)FIG. 2. EPPECT oF GLurAMINE ON GRowT oF C2038 STRAN

WITH NEUTRLZATION

of group A hemolytic streptococci. Glutamine cannot be replacedby glutamic acid or by other closely related compounds (1939).In figure 2 is shown the titration of glutamine on the 2 per centcasein hydrolysate medium containing 4 per cent glucose. Lacticacid was periodically neutralized as formed. It will be noted thatrelatively large amounts of this amino acid are necessary for heavygrowth. Even with 400 mgm. glutamine per liter the optimumhas not been reached. Since the amide group of glutamine is


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490 ALIAN W. BERNHEIMER AND A. M. PAPPSNHEIMER, JR.

labile and easily hydrolysed, it seemed important to determineits stability in the medium itself. Glutamine was determined bythe method of Pucher et al. (1935) after incubating in caseinhydrolysate medium at 3700. and pH 7.0 for varying intervals oftime. At the end of 48 hours, the labile amide nitrogen was still72 per cent of its initial value indicating that most of the glutaminewas still present. Since glutamine is even more stable under theacid conditions which prevail in growing cultures, we are led tobelieve that streptococci need and utilize amounts of the mag-nitude indicated in figure 2.

TABLE 4Growth of various strains of group A hemolytic 8treptococci on casein

hydrolysate mediumSTAINBCEALMRGl PER 5N NaOH iFoR NEuTRALInzATO

10 ML.) X 50 OFr 20 ML. CULTuRz

.~~~~~~~~~~~~~~~Cmgm. cc.C203S 70-120 0.6-0.8C203M 71 0.69C203R 0 0NY No. 5 56 0.541048M 57 0.821685M 13 0.131840M 43 0.38594B* 70

* Medium contained 300 mgm. asparagine per liter for this strain.

We have observed, in agreement with Fildes and Gladstone(1939) that after two or more days incubation, particularly underan atmosphere of carbon dioxide in air, some cultures may growout even in the absence of glutamine. Up to the present time,we have been unable to subculture these "trained" organisms onmedia free from glutamine.

Growth of other strains of group A streptococcus. A number ofother strains have been tested on the improved medium in themanner described and the results are summarized in table 4. Inaddition to the above strains we have tested two cultures receivedfrom Dr. A. F. Coburn one of which (Faimen) was isolated froma case of rheumatic fever. The other strain (Deitschmann) was


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GROWTH OF GROUP A HEMOLYTIC STRF4TOCOCCUS 491J

a group B strain isolated from a case of sepsis. One (Deitsch-mann strain) gave rapid heavy growth comparable to C203S; theother (Faimen strain) grew very poorly. Of the 10 strains tested,7 gave excellent growth on the defined medium. The data givenin table 4 are probably not maximal and in any case should onlybe regarded as giving an approximate comparison of growth sincethey were obtained from a turbidometer calibrated for C203S anddo not, therefore, take into account differences in cell size, opticaldensity, etc. of the other strains.

DISCUSSION

Although a number of workers have reported growth of groupA hemolytic streptococci on a medium of essentially known com-position, so far as we are aware, no attempt has been made to ob-tain maximal growth on such a medium. Since the hemolyticstreptococcus produces large amounts of lactic acid, maximalgrowth cannot be attained without frequent neutralization. Un-less the acid produced from glucose is neutralized as it is formed,growth must necessarily be light and in terms of bacterial nitrogenwill only amount to 2-5 per cent of what is generally considerednormal growth of organisms, such as the diphtheria bacillus,which are capable of oxidizing the acids formed from sugar. Wehave felt that growth equivalent to at least 150-250 mgm. strep-tococcal nitrogen per liter should be attained on a defined peptone-free medium before the isolation and characterization of suchbiologically active proteins as hemolysin or erythrogenic toxinmay become feasible or practical. Using the C203S strain, thebest growth which could be obtained on the gelatin hydrolysatemedium previously described (Pappenheimer and Hottle, 1940)was 20-30 mgm. streptococcal nitrogen per liter, even with neu-tralization. The following important changes in that mediumhave made it possible to obtain growth equivalent to 250 mgm.streptococcal nitrogen per liter with the C203S strain and com-parable growth with other group A strains:

1. Increase in glucose concentration to 3-4 per cent.2. Frequent neutralization of the lactic acid formed.3. Reduction in total salt concentration to a minimum.


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492 ALN W. BEUNHEIMER AND A. M. PAPPENHEIMER, JR.

4. Use of 2 per cent of sulfuric-acid-hydrolysed casein in placeof 1 per cent of hydrochloric-acid-hydrolysed gelatin.

5. Increase in calcium pantothenate concentration to 4 mgm.per liter.

6. Increase in glutamine concentration to 200 mgm. per liter.There is no reason to suppose that 250 mgm. bacterial nitrogen

per liter represents the maximum possible growth on the definedmedium. Addition of 4 per cent glucose and 1 per cent Parke-Davis peptone to a 1 per cent casein hydrolysate medium mayyield as much as 400 mgm. bacterial nitrogen per liter using theC203S strain. During this amount of growth at least 30 gramsof glucose are utilized per liter and the final sodium lactate con-centration is about 3 molar. Since the same sodium lactate con-centration is sufficient to inhibit growth almost completely whenadded to fresh medium, it seems probable that 400 mgm. bacterialnitrogen per liter does represent close to maxmal possible growthof the C203S strain. Since we have found, further, that evenaddition of small concentrations of salts such as sodium chlorideor lactate greatly retards the rate of growth, it is extremely im-portant to keep the salt concentration as low as possible. Thesalt effect appears to be mainly one of total osmotic pressurerather than a specific toxic action of sodium or lactate ions, sinceother ions such as potassium, ammonium, and chloride areequally effective inhibitors.Of ten strains of group A hemolytic streptococci tested, seven

gave growth of 100 mgm. bacterial nitrogen per liter or better.The question arises whether any other growth factors may berequired by the streptococcus which unknowingly were added tothe present medium, or to that previously described (Pappen-heimer and Hottle, 1940; Hottle et al. 1941). This question isparticularly pertinent since we have admittedly used technicalcasein hydrolysate and glucose in high concentrations both ofwhich may contain unknown growth factors as impurities. How-ever, heavy growth is still obtained with C203S when Eastman'spurified gelatin hydrolysate supplemented by tyrosine andmethionine is used in place of casein and Merck's reagent qualityglucose is used in place of cerelose (table 3). In the case of the


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GROWTH OF GROUP A HEMOLYTIC STREPTOCOCCUS

three strains which failed to show good growth on the mediumother factors are probably required. We feel, however, that thepresent medium is complete for the other seven strains. Woolley(1941) has recently described a non-dialysable growth factorwhich is essential for several Group A strains of streptococcus,among them the C203 and 594 strains. We are unable to explainwhy our cultures of C203 and 594 do not appear to require thisfactor. Aside from differences in concentration of the growthfactors added, the only substance actually lacking from Woolley'smedium and present in our own, is sodium bicarbonate.2

In agreement with Mueller and Klise (1932) we have found thatafter prolonged neutralization many of the streptococci stainedgram-negative but remained viable. No change in the colonyform has been noticed after plating out. Some autolysis gen-erally occurs after about twelve hours of continuous neutralization(24 to 30 hours growth).

SUMMARY

A study has been made of the factors necessary for massivegrowth of the C203S strain of hemolytic streptococcus. Growthequivalent to 200-250 mgm. bacterial nitrogen per liter has beenattained on a medium of defined chemical composition using theC203S strain. Several other strains of Group A streptococcushave also given massive growth on the medium.

REFERENCESFILDES, P., AND GLADSTONE, G. P. 1939 Glutamine and the growth of bacteria.

Brit. J. Exptl. Path., 20, 330.FRIEDEMANN, T. E. 1939 The carbohydrate metabolism of streptococci. J.

Biol. Chem., 130, 757-761.HOTTLE, G. A., LAMPEN, J. O., AND PAPPENHEIMER, A. M., JR. 1941 Biotin as a

growth factor for C203S strain of hemolytic streptococcus, Group A.J. Biol. Chem., 137, 457-458.

HUTNER, S. H. 1938 Experiments on the nutrition of streptococci. J. Bact.,35, 429-440.

2 Woolley reports that his factor is easily absorbed by barium sulfate. Weshould expect that if the factor were present in the technical casein used, it wouldhave been eliminated during removal of the sulfuric acid used for hydrolysis. Itmust also be pointed out that we have obtained about twenty times heavier growththan that reported by Woolley as judged by the amount of lactic acid formed.

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McILwAIN, H. 1939 The specificity of glutamine for growth of Streptococcushemolyticus. Biochem. J., 33, 1942-1946.

McILwAIN, H. 1940 The nutrition of Streptococcus hemolyticus. Growth in achemically defined mixture. Brit. J. Exptl. Path., 21, 25.

McILwAIN, H., FILDES, P., GLADSTONE, G. P., AND KNIGHT, B. C. J. G. 1939Glutamine and the growth of Streptococcus hemolyticus. Biochem J.,83, 223.

MUELLER, J. H., AND KisE, K. S. 1932 Mass cultures of Streptococcus hemo-lyticus in broth. Proc. Soc. Exptl. Biol. Med., 29, 454-455.

PAPPENHEIMER, A. M., JR., AND HOTTLE, G. A. 1940 Effect of certain purines,and C02 on growth of strain of Group A hemolytic streptococcus.Proc. Soc. Exptl. Biol. Med., 44, 645-649.

PUCHER, G. W., VICKERY, H. B., AND LEAVENWORTH, C. S. 1935 Determinationof ammonia and of amide nitrogen in plant tissue. Ind. Eng. Chem.,Anal. Ed., 7, 152-156.

RANE, L., AND SUBBAROW, Y. 1938 Studies on the nutritional requirements ofhemolytic streptococci. I. Effect of various substances isolated fromliver extract on hemolytic streptococci. Proc. Soc. Exptl. Biol. Med.,38, 837-839.

SUBBAROW, Y., AND RANE, L. 1939 Pantothenic acid as a growth factor for theDochez NY5 strain of hemolytic streptococcus. J. Am. Chem. Soc.,61, 1616.

VICKERY, H. B., PUCHER, G. W., AND CLARK, H. E. 1935 The preparation ofglutamine. J. Biol. Chem., 109, 39.

WOOLLEY, D. W. 1941 A new growth factor required by certain hemolyticstreptococci. J. Exptl. Med., 73, 487-492.

WOOLLEY, D. W., AND HUTCHINGS, B. L. 1939 Some growth factors for hemo-lytic streptococci. J. Bact., 38, 285-292.


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