THE NUTRITION OF STAPHYLOCOCCUS AUREUS

THE INFLUENCE OF BIOTIN, Bios IIB AND VITAMIN H ON THEGROWTH OF SEVERAL STRAINS

J. R. PORTER AND MICHAEL J. PELCZAR, JR.

Department of Bacteriology, College of Medicine, State University of Iowa,Iowa City

Received for publication June 12, 1940

Results of recent investigations have made possible the cultiva-tion of Staphylococcus aureus in a chemically-defined medium.In the past it has been necessary to add a minute amount of anunidentified supplement to a medium composed of amino acids,glucose and inorganic salts before aerobic growth would occur.The necessity for the supplement was first shown by Hughes(1932), who prepared a fraction from meat extract capable ofsupporting growth in a medium in which most of the ingredientswere known entities. Later, Knight (1935) obtained a highlypurified fraction from autolyzed yeast extract (marmite) whichallowed growth to take place when as little as 0.004 microgramper milliliter was present in a deficient basal medium consistingof acid-hydrolyzed gelatin, glucose and salts. Subsequentlyit was found possible (Fildes, Richardson, Knight and Gladstone1936, Gladstone 1937) to replace the gelatin hydrolysate in thebasal medium with a mixture of 16 amino acids. Following thisKnight (1937a, b) resolved the active marmite fraction intothree parts, two being the pyrimidine and thiazole componentsof vitamin B1 and the third being nicotinamide, a component ofWarburg's coenzymes. Vitamin B1 was adequate in 0.002 micro-gram per milliliter and nicotinic acid or its amide in 0.2 microgramper milliliter of the amino acid-glucose medium. The specificityof the components of vitamin B1 and related derivatives, as wellas several pyridine compounds closely related to nicotinic acidor its amide, was the subject of a careful study by Knight and

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McIlwain (1938). Their results emphasize that vitamin B1and nicotinamide are highly specific for the growth of this or-ganism. Only very limited departures from these structures arepermissible if growth activity is to be maintained. For theanaerobic growth of Staphylococcus aureus, Richardson (1936)has demonstrated that uracil must be added to the medium inaddition to the above compounds.

Following these reports a study was undertaken in our labora-tory to determine whether the quantity of growth produced byStaphylococcus aureus in the chemically-defined medium ofGladstone (1937) was equal to that obtainable in standard glucosemeat-infusion broth. Preliminary results revealed that thechemically-defined medium was inferior to glucose meat-infusionbroth. More interesting, however, was the fact that a few strainscould not initiate growth on continued subculture in the syntheticmedium.

In an attempt to increase the quantity of growth of those strainscapable of growing in the basic synthetic medium, several acces-sory factors of known nutritional importance were incorporatedin the basal medium. At the same time it was hoped that thesubstance (or substances) required for the growth of the more"fastidious" strains might be found. The addition of thesesubstances, separately or in various combinations, resulted inlittle if any increase in growth beyond that obtained in the un-supplemented basal synthetic medium. Furthermore, the moreexacting strains failed to grow on continued subculture, indicatingthe lack of some nutrient or vitamin-like substance. At thistime extracts from both plant and animal tissues were prepared,subjected to various chemical treatments and tested for theirgrowth-promoting properties. The results indicated that theessential growth substance required by the "fastidious" strainsin a synthetic medium was biotin or some closely related com-pound. Furthermore, when biotin was added to the basal syn-thetic medium, at no time did the growth equal or exceed thatobtained in meat-infusion broth.

Biotin was first prepared by Kogl and T6nnis (1936), who, byan ingenious technique, obtained 1.1 mgm. of crystalline material

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from 250 kgm. of dried egg yolks. Their preparation was fullyactive for yeast at 0.000025 microgram per milliliter of medium,one of the lowest concentrations yet recorded at which anysubstance is physiologically active. Like some of the othergrowth factors and vitamins, biotin seems to have ubiquitousdistribution in nature. For example, Kogl and van Hasselt(1936) found it to be present in many organs and tissues of dogs,cattle and hens. By bio-assay, dogs were found to have anaverage of 0.004 microgram, cows 0.007 microgram, and hens0.02 microgram per gram of tissue. Similarly, Kogl and Haagen-Smit (1936) tested the biotin content of 35 varieties of seeds andconcluded that this substance must be considered an activephytohormone since it is stored in the cotyledon and affects theembryo during germination. More recently, the biotin contentof several materials has been assayed by Robbins and Schmidt(1939), and Snell, Eakin and Williams (1940).

Besides being an active accessory factor for common yeast,biotin seems to be very important in the nutrition of other fungi.Ashbya gossypii, Lophodermium pinastri, Melanospora destruens,Debaryomyces mucosus and others, require biotin for growth in aninorganic medium plus glucose (K6gl and Fries 1937, Robbinsand Schmidt 1939, Hawker 1939, Melin and Lindeberg 1939 andSartory, Meyer and Netter 1939a, b).The first report on the use of biotin in bacterial nutrition studies

was that by Kogl and van Wagtendonk (1938). When as littleas 5 X 10-6 microgram of the methyl ester of biotin was addedper milliliter of the basic medium (Knight 1935) the growth ofStaphylococcus aureus was increased. The optimum concentra-tion of biotin for this organism, however, was 0.005 microgramper milliliter of medium. Apparently, only one strain of Staphy-lococcus aureus was used in this study and it was able to grow inthe basic medium without biotin. More recently, Sartory,Meyer and Netter (1939a, b) have studied the effect of purifiedbiotin preparations on the growth of Staphylococcus aureus andreported that their material was stimulative in an amount of0.098 microgram per milliliter of an otherwise synthetic medium.It is of interest to note that they obtained the best growth of this

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organism when the medium was supplemented with an ether-alcohol soluble sterol in addition to biotin. However, their testorganism was capable of growing in the basal medium lackingbiotin as was the case with the culture used by Kogl and vanWagtendonk. Moller (1939) used a highly purified biotin prepa-ration in his nutrition studies on several lactic acid bacteria andfound it to be essential for optimum growth. One of the essentialgrowth factors for certain species of the genus Rhizobium isbiotin. Hoover and Allison (1935) designated this substance ascoenzyme R, and more recent studies by West and Wilson (1939a,b, 1940), Nilsson, Bjailfve and Burstr6m (1939), and Gyorgy,Melville Burk and du Vigneaud (1940) have shown that co-enzyme R and biotin are closely related, if not identical. In-vestigation of the specific nutritive requirements of Clodtridiumacetobutylicum led Weizmann and Rosenfeld (1939) to the con-clusion that biotin was an essential growth factor for this or-ganism. This fact was confirmed by Oxford, Lampen andPeterson (1940). Snell and Williams (1939) have reported biotinto be the active growth factor for the butyl-alcohol-producinganaerobe, Clostridium butylicum. A concentration as low as0.0000133 microgram of the pure methyl ester of biotin permilliliter of medium permitted growth, while larger amountswere more stimulative. Evidence was presented to show thatbiotin is the only growth factor required for the butyl-alcohol-producing clostridia. Evans, Handley and Happold (1939)reported that biotin was not an essential growth factor for Coryne-bacterium diphtheriae.Although few chemical data are available at this time, biotin

seems to be closely related to "bios IIB" (Miller, Eastcott andSparling 1932, Miller 1937), "coenzyme R" (Hoover and Allison1935) and vitamin H (Gy6rgy, Melville, Burk and du Vigneaud1940). All four substances are dialyzable, heat-stable and re-sistant to treatment with acid and alkali. They are soluble inwater and alcohol but insoluble in chloroform, ether and pe-troleum ether. All are adsorbed readily on charcoal. Noneis precipitated by lead acetate. Treatment of vitamin H orbiotin with benzoyl chloride in pyridine results in an inactiveproduct. Nitrous acid brings about inactivation of both sub-

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stances. Reports on the electrophoretic behavior of coenzyme Rcoincide closely with observations on vitamin H. On the basisof analytical results Kogl (1937) reported the empirical formulaof the methyl ester of biotin to be C11H1803N2S, and Rainbow andBishop (1939) estimated the empirical formula of "bios IIB"as C7H12N203. Further interesting chemical data will be foundin the papers just cited and in those by Drumel and Hubert(1938) and by Snell, Eakin and Williams (1940).The present work describes the preparation and nature of the

essential growth factor for certain strains of Staphylococcus aureus,and the effect of the active material on the growth of severalother strains of the same species which are capable of growingin Gladstone's chemically-defined medium.

TECHNIQUE

Bacteriological methods and culturesThe technical details followed in this study were similar to

those suggested by Fildes, Richardson, Knight and Gladstone(1936), and Evans, Happold and Handley (1939) for bacterialnutrition studies. Special precautions were taken to excludecontaminating constituents from the media and the purest chemi-cals obtainable were used at all times.The strains of Staphylococcus aureus employed were kindly

supplied by Dr. 0. N. Fellowes of our laboratory. All of thecultures were typical with respect to morphology, pigment forma-tion and physiological characteristics. The strains designatedas X3 and Y2 were extremely mucoid when cultured on bloodagar media. The source of each culture is given in table 1.

Preliminary experiments were usually carried out in test tubescontaining 10 ml. of media, while for the quantitative data theorganisms were cultivated in 50 ml. of media in 250 ml. Erlen-meyer flasks. Unless otherwise stated the cultures were incu-bated at 370C. for 24 hours.

Basal mediaThe chemically-defined medium used in this study was essen-

tially that described by Gladstone (1937) and will be referred toas the basal synthetic medium. Briefly, it contained in its com-

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178 J. R. PORTER AND M. J. PELCZAR, JR.

TABLE 1Quantitative comparison of growth of Staphylococcus aureus as measured by bacterial

nitrogen determinations

BACTERIAL NITROGEN (moxO.WNLDANL), M3GM. PM30 ML. MEDIA

WIRAIn SOURCE

Gladstone's Glucose mestsynthetic medium infusion broth

IC Enterotoxogenic strain from 1.82* 4.26shrimp salad 1.93* 4.23

CB Enterotoxogenic strain from 2.10 4.54custard pie 2.06 4.45

C161 Enterotoxogenic strain from 2.13 2.58sandwiches 2.20 2.75

AD Enterotoxogenic strain 1.64 2.551.94 2.52

A130 Post operative cholecystec- 1.58 3.24tomy 1.56 3.22

A115 Amputation stump 1.53 3.371.20 3.16

C166 Enterotoxogenic strain from 2.04 3.58liverwurst 1.89 3.26

Reddish 1.73 2.971.78 2.89

C170 Enterotoxogenic strain 1.25 3.291.52 3.11

A146 Empyema 1.72 3.241.76 2.99

K 1.72 3.301.69

A165 From trachea at autopsy 1.53 3.291.52 3.15

3025 Abscess on hand 1.61 2.451.75 2.57

2527 Tissue abscess in osteomyelitis 1.57 2.151.84 2.64

AlOl Peritonitis 1.08 2.691.09 2.69

X3 Mastoid infection 0.00 2.892.55

Y2 Infected amputation stump 0.00 2.813.22

* Duplicate determinations.

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plete form the following 16 amino acids: glycine, a.p., dl-alanine,dl-valine, dl-leucine, 1-proline, 1-oxyproline, 1-aspartic acid anddl-glutamic acid, each in a final concentration of M/1500; dl-phenylalanine, 1-tyrosine, d-arginine HCl, 1-histidine HOl anddl-lysine, each in a concentration of M/4000; 1-tryptophane(M/20,000), 1-cystine (M/10,000) and dl-methionine (M/50,000).The amino acids were obtained from the Chemistry Departmentat the University of Illinois, the Amino Acid Manufactures,University of California at Los Angeles and from Eastman.They were not subjected to further purification. Each aminoacid was made up and sterilized separately in concentratedsolution, so that the addition of a small volume (0.1 to 10 ml.of media) of each gave the final concentration as required in thecomplete medium. All amino acids were dissolved in waterexcept cystine and tyrosine; these were prepared in M/10 HCl,the pH being corrected after their addition to the medium.Except in the case of methionine and cystine, which were filteredthrough sintered glass filters, all solutions were sterilized in theautoclave at 1200C. for 15 minutes. In addition to the aboveamino acids, the medium contained nicotinamide (10-5M),thiamin (vitamin B1, 10-7M), glucose (0.5 per cent) and inor-ganic salts (KH2PO4, MgSO4. 7H20, FeSO4(NH4)2SO4 6H20).The reaction of the medium prior to inoculation was pH 7.2 to7.4.The non-synthetic medium was glucose meat-infusion broth

consisting of the extract of 1.0 pound of lean beef per liter, 1.0per cent peptone, 0.65 per cent NaCl and 1.0 per cent glucose.The pH was adjusted to 7.2 to 7.4.

Determination of bacterial nitrogenQuantitative measurement of growth was made by determin-

ing the amount of bacterial nitrogen in the washed sedimentedcells from a given volume of medium, using a micro-Kjeldahltechnique.The procedure followed for centrifuging and washing the bac-

terial cells was essentially that employed by Mueller (1935).Preliminary experiments showed that centrifugation for 30minutes, at 3000 to 3500 r.p.m., was adequate for the strains

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used in this study. The supernatant medium was carefullypipetted off, and the sedimented cells washed with 20 ml. ofammonia-free distilled water. A second 30-minute centrifuga-tion was then carried out and the wash fluid drawn off. Theamount of nitrogen in the sedimented bacterial mass was thendetermined.For the actual determination of nitrogen, the micro-Kjeldahl

technique described by Sobel, Yuska and Cohen (1937) was used.For details of the method, the original article should be consulted.The digestion was carried out directly in the pyrex centrifugetubes. It was found advantageous to use 1.0 ml. of a digestionmixture consisting of 300 ml. H2S04, 3.0 grams CuSO4 and 1.0gram K2S04, and to make four additions of three drops each of30 per cent H202 during the course of the digestion. The appa-ratus was arranged so that six determinations could be carriedout simultaneously.

EXPERIMENTAL

Quantitative comparison of growth in basal synthetic and non-synthetic medium

When the test organisms were cultivated in the basal syntheticmedium and glucose meat-infusion broth, a significant differ-ence in the quantity of growth was observed. From the resultspresented in table 1 it will be seen that the growth in the lattermedium was almost double that in the former for practicallyevery culture. More interesting, however, was the fact thatcertain strains (X3 and Y2) were unable to initiate growth oncontinued subculture in Gladstone's medium. Repeated at-tempts to cultivate these strains in this medium met with failure.Thus it became apparent that some additional nutrient or vita-mn-like substance was required by these "fastidious" strains(X3 and Y2) of Staphylococcus aureus.

Activity of other amino acids and accessory growth factorsSince the quantity of growth in the basal synthetic medium

was lower than that obtained in the non-synthetic medium, anattempt was made to increase the yield of bacterial nitrogen inthe former by altering the concentration of the ingredients and

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NUTRITION OF STAPHYLOCOCCUS AUREUS 181

by adding other supplements. At the same time it was thoughtthat the adding of these supplements might render the mediumsuitable for the continued growth of the "fastidious" strains.Three experimental media were prepared as indicated below.

EXPERIMENTAL MEDIA

Medium I16 amino acidsglucose

Basal Medium (chemically defined by Gladstone 1937) vitamin Bj*nicotinic acid~inorganic salts

Basal Medium +

Basal Medium + I

Medium IIConcentration

norleucine................ M/1500asparagine................ M/1500a-amino valeric acid................ M/1500#-amino valeric acid................ M/1500taurine................ M/1500threonine................ M/1500

Microgramsper ml.

diiodotyrosine.............................ornithine...................................

Medium IIIpimelic acid...........................p-alanine.............................riboflavin*...........................glutamine.............................

I + glutathione...........................vitamin B,*...........................inositol...............................pantothenic acid (70%)*..............cocarboxylase* ........................cozymase (40%) *......................

Medium IVuracil..........................guanine........................

Basal Medium + II + III + adenine........................l adenylic acid*..................Adenosine triphosphate*........

100100

1.01.01.01.01.01.010.01.01.01.0

10.010.010.010.05.0

* For these compounds we wish to thank the Research Department of Merckand Co., Dr. Henry Tauber, and Professor Roger J. Williams.

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When these media (II, III, IV) were tested for their growth-promoting properties they were found to be no better than thebasal medium (I). The bacterial nitrogen values were lowerthan those obtained with the non-synthetic medium, and the"fastidious" strains were unable to grow on continued subculture.

Preparation of an active growth factorIn view of the fact that the "fastidious" strains of Staphylococ-

cus aureus failed to grow when the basal chemically-definedmedium was supplemented with additional amino acids and sev-eral important growth factors, an investigation was undertakento determine the nutritional requirements of these particularstrains. Extracts of both plant and animal tissues were pre-pared, subjected to various chemical treatments and tested fortheir growth-promoting properties by incorporating them in anotherwise deficient basal synthetic medium. In each case thesubstance (or substances) required by these organisms was sol-uble in water, acetone and alcohol. It was heat-stable and re-sistant to treatment with sulphuric acid and sodium hydroxide.Treatment with nitrous acid rendered the product inactive. Theactive principle could be readily adsorbed on charcoal and elutedwith acetone containing ammonia. These facts, and others,suggested that we were probably dealing with biotin or someclosely allied substance.

Following the procedure of Kogl and T6nnis (1936) for thepreparation and purification of biotin, a concentrate was ob-tained from dried egg yolks. Twenty-five kilograms of driedegg yolks, furnished by Armour and Co., represented the start-ing material. The steps in the procedure for the concentrationof biotin are schematized in figure 1 and represent only a portionof the method described by Kbgl and T6nnis. For the completeprocedure one should consult the original article. It was thoughtadvisable to stop after the completion of eight steps, since at thispoint such a small amount of the highly active principle remainedthat further purification would result in excessive loss of the sub-stance. The final product, totaling 2.92 grams, consisted of adark-brown resinous material.

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NUTRITION OF STAPHYLOCOCCUS AUREUS 183

The activity of the material at each stage in the concentra-tion process was followed by determining the smallest amount

Dried E99 Yo/kJ(25 kg, 55 Ibs)

(,) ' Cold HZ0 E:xtract. (o-io hr)

() ZO4Baing A0 Extracts. One hour each)Total volume 250 /ter,)

(A) Reduced in vacuum (45 -50'C,) to #.5 L.(3o/ids 12 k9g)

t(3) 50% Aetone Precioito/ion

(-OS L acetone added in 3 portions over 8 hours)Centrifuge

(8g) Supernatant (Sohids 4/5 9.) (C) Precipitale (Soybds 7/3 g.)fieducd in vacuum to /.9 1-

(4) 86 % alcohol Precipitotion(/1.67 L. absolute alcohol added in 3 portions over 8 hours)

Ccntrifiuge

Superbratant (D) Precipitate (Solids 482 9)Redned in vacuum to 4.0 L.

(5) Neutral and A/kahinevLeod a/lt PrecivitoaionAdd 200 g lead acetate in 300 ml l&O. Centrituge. Precipitate discarded.

Add Concentrated Ammonia until no more lead precioi/ate. Centrifuge.Precipitate discarded. Excess lead precipitated with HZS. Precipitate discarded.

(Z) Filtrate 3.75 L. (Solids 230 g.)

(6) Charcoal /2.5 kg. Moercs U S 1?) Adsorption for / hour.filter with -uction

(f) Filtrate and (o) Alcohol Elution (/) 3 Ace*one N/ti ElutionsAueous tlution (1.6 L. 50 % O/cobol) (2.8 L. reduced in vacuum(Solids 2/2 9J (SoAds 2.14 9.) to/10 L., solids /14 9)

(r) ghosphotweftic Acid PrecipitationsAdd 50 % Z0W/032M, , Z5H0 h7 5X /ZSO4

Plltrr

(I) t/rocrciti~

Remove phop'otungstate with 2 7. Ba(0)2s and the excess barium with tt1304Evaprte to dryness

3wr boftAlcohol(a) warm absolute ffxtractions, 30 ml. each

filter. Precioitates discarded.IJ) fitrates evaporated to dryness (232g9

* Armour: Caoverbloom Dried Egg Yolks. Kindly furnished by #! Contest.Director of Research. Armour and Cao

FIG. 1. PREPARATION OF BIOTIN CONCENTRATES FROM DRY EGG YOLKS*

which would permit growth on continued subculture of the testorgansm (Staphylococcus aureus, X3) in 10 ml. of the inadequatebasal synthetic medium. Results obtained for the activity of

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184 J. R. PORTER AND M. J. PELCZAR, JR.

each fraction are presented in table 2. It will be seen that athousand-fold concentration of the active principle was ef-fected during the course of the process. The final concentrate,when added to the basal synthetic medium in an amount of0.0001 microgram per ml., permitted growth of the "fastidious"strains on continued subculture.

Further experimentation revealed that these strains coulddispense with neither nicotinic acid nor vitamin B1 in the basalmedium. The possibility exists that those strains whose nutri-

TABLE 2Activity* of the various fractions obtained in the concentration of biotin from dry

egg yolks

ACTIVITY IN MICROGRAM PERML. OF SYNTHETIC MEDIUM

FRACTION

Fraction Fractionfurther purified disarded

(A) Hot water extract............................... 0.10(B) Acetone supernatant............................. 0.01(C) Acetone precipitate ..10.0(D) Alcohol precipitate ..10.0(E) Before charcoal adsorption....................... 0.001(F) Aqueous eluate................................1.0(G) Alcohol eluate.................................0.1(H) Acetone eluate.................................. 0.0001(I) Phosphotungstic acid supernatant 0.01(J) Absolute alcohol extract......................... 0.0001

* Smallest amount of material which allowed visible growth on continued sub-culture of Staphylococcus aureus (X3 strain) in 24 hours, at 370C.

tional needs are satisfied by Gladstone's medium may requirebiotin for growth, but are capable of synthesizing this physio-logically active substance from the constituents of the substrate.On the other hand, those strains which fail to grow in the samemedium are evidently unable to effect such a synthesis.

Bio-assay of the biotin, bios IIB and vitamin H concentratesDuring the course of assaying the biotin concentrates, a rela-

tionship was observed between the amount of growth which took

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place and the concentration of biotin in the medium. Also, onfurther investigation, it was found possible to replace the biotinpreparation with either bios IIB or vitamin H concentrates,kindly furnished by Dr. C. N. Frey and Dr. Paul Gy6rgy, re-spectively.The relationship between the quantity of growth and the

concentration of the above substances in the medium was de-termined by cultivating the organisms in 50 ml. of the basalsynthetic medium containing various amounts of the above prep-arations. The quantity of growth was measured by the previ-ously described micro-Kjeldahl technique.The results showing the influence of biotin, bios IIB, and vita-

min H concentrates on the growth of the "fastidious" strains ofStaphylococcus aureus are presented in table 3. The ability toreplace the biotin concentrate with either bios IIB or vitamin Hpoints to the similarity in the physiological function of thesethree substances for the organisms under observation. Thisinformation lends further support to recent work of Gyorgy,Melville, Burk, and du Vigneaud (1940) which suggests thatbiotin and vitamin H, as well as coenzyme R, are possiblyidentical compounds. Further analysis of the data in table 3shows that increasing the concentration of these substances in themedium resulted in an increased amount of growth to a maximumpoint, beyond which, little or no stimulation was observed. Theoptimal concentration of the biotin preparation was approxi-mately 0.1 microgram, bios IIB 0.2 microgram, and vitaminH 0.32 microgram per milliliter of the basal synthetic medium.

Typical results showing the stimulation of growth by biotinare presented graphically in figure 2, where the quantity ofgrowth in terms of milligrams of bacterial nitrogen is plottedagainst the concentration of the biotin preparation per milli-liter of medium. This type of stimulation is characteristic forboth the X3 and Y2 strains of Staphylococcus aureus.The manner in which these particular organisms respond to

the biotin, bios IIB and vitamin H concentrates, suggests thepossibility of using them for the bio-assay of these substances.

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186 J. R. PORTER AND M. J. PELCZAR, JR.

Employing such strains in a technique similar to the yeast-growth test of Snell, Eakin and Williams (1940) might be ad-vantageous, since biotin (bios IIB, vitamin H) is required before

TABLE 3Effect of increasing the concentration of biotin on the growth of Staphylococcus aureus

(X: and YS strains) and its replacement with bios IIB and vitamin Hconcentrates

CONCENTRATZ

Biotin...................

Bios 'l.....B. 4.

Vitamin H*.............

MICROGRAMS PERML. OF MEDIUM

0.00.0050.010.0150.020.050.10.150.2

0.00.010.020.050.10.150.20.30.4

0.00.0040.0080.0120.0160.160.32

BACTERIAL NITROGEN (MICRO-KJELDAJE),MGM. PEBR 0 ML. MEDIA

Strain X3 strain Y2

0.00.410.580.780.851.111.251.251.30

0.00.370.520.590.711.211.361.511.50

0.00.270.430.500.571.121.35

0.00.380.500.810.721.161.371.491.21

0.00.300.47

0.751.101.371.361.26

0.00.110.500.500.521.261.18

0.00.280.420.630.750.750.780.890.91

0.00.390.600.891.311.351.421.471.44

0.00.280.570.810.781.261.64

0.00.330.410.600.690.740.870.740.83

0.00.430.630.871.311.471.451.341.58

0.00.360.550.690.74

1.67* One milligram of solids is equivalent to approximately 20 vitamin H units.

any detectable growth will occur. Evaluation of such a tech-nique must naturally await experimental data obtained withcrystalline biotin.

I

1

kl

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INUTRITION OF STAPHYLOCOCCUS AUREUS 187

Effect of biotin, bios IIB, and vitamin H concentrates on the growthof several strains of Staphylococcus aureus

After demonstrating that certain strains of Staphylococcusaureus require biotin for growth, we thought it would be in-teresting to ascertain the growth-stimulating property of this

/4

/13

1. I

/06

_ |r 8 4ofln Concenfrafe Bacteria/- N2t

.7

ar| ,oer M/ Mg 'Per 50m/ofmed1a0.0 0.0

06 .0005 /4

U , as | 0.0/ 0.581 05 0.0/5 o.7C.T ~~~0.02e 0.6.5

0.05 1/

B8a TIN CONCENTRATE(microqram per ml. of medium)

FIG. 2. EFFECT OF INCREASING THE CONCENTRATION OF BIOTIN ON THE GROWTHOF STAPHYLOCOCCUS AUREUS (X3 STRAIN)

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J. R. PORTER AND M. J. PELCZAR, JR.

TABLE 4Effect of biotin, bios IIB and vitamin H concentrates on the quantity of growth of

several strains of Staphylococcus aureus in an otherwisechemically-defined medium

BACrUIAL ITROGUN (McaO-XJMDAM), MGM. PZB 50 ML. BASIC M3DIA

Plum 0.2 'y biotin Plus 40 y bios It, Plus 0.12 unit vitaminpemL Pwml. H per mL

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~2.492.54

2.062.03

2.212.32

2.282.15

1.992.06

2.001.96

2.212.32

2.302.11

1.961.92

2.412.20

2.242.28

2.091.93

2.612.64

2.392.56

1.701.63

1.301.21

0.910.83

2.382.17

2.172.28

1.921.89

1.961.96

1.51

1.371.39

1.921.89

1.911.86

1.281.42

1.631.75

1.681.74

1.581.65

1.681.68

1.771.68

1.401.40

1.591.68

1.671.57

2.37

1.75

1.62

1.82

2.21

2.78

1.50

1.40

188

IC

CB

C161

AD

A130

A115

C166

Reddish

C170

A146

K

A165

3025

2527

A1O0

X3

Y2

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NUTRITION OF STAPHYLOCOCCUS AUREUS

substance for other strains of this species which are capableof growing in the basal chemically-defined medium of Gladstone(1937). At the same time similar experiments were conductedin which bios IIB or vitamin H was substituted for the biotin.In each instance the optimum concentration of the growthsubstance was employed (see table 3). The results of theseexperiments are presented in table 4.

Comparison of these data with those in table 1 shows that thepresence of biotin, bios IIB, or vitamin H resulted in little or nostimulation of growth in strains capable of growing in the basalsynthetic medium. However, other investigators (K6gl andvan Wagtendonk 1938, and Sartory, Meyer and Netter 1939a, b) using strains of Staphylococcus aureus which were capableof growing without the addition of biotin, have noted markedstimulation of growth when their basic media were supplementedwith biotin. Thus it would seem that the organisms belongingto this species can be allocated to three groups on the basis oftheir response to biotin: (1) those which grow on continued sub-culture in the chemically-defined medium of Gladstone and arenot influenced by the addition of biotin, (2) those which willgrow when no biotin is added but are greatly stimulated by itsaddition to the medium (e.s., K6gl and van Wagendonk 1938,and Sartory, Meyer and Netter 1939 a, b), and (3) those whichrequire the addition of biotin to the medium before growth willoccur.

SUMMARY AND CONCLUSIONS

It has been shown that Staphylococcus aureus grows more luxu-riantly in standard glucose meat-infusion broth than in Glad-stone's chemically-defined medium. When bacterial nitrogenwas used as an index of growth the former medium gave valuesalmost twice those obtained with the basal synthetic medium.

Certain strains of Staphylococcus aureus are unable to grow oncontinued subculture in the medium of Gladstone. Evidencehas been presented which indicates that these strains requirethe growth factor, biotin (bios IIB, vitamin H), in addition tonicotinic acid and vitamin B1.

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190 J. R. PORTER AND M. J. PELCZAR, JR.

Growth of several strains, whose nutritional requirements aresatisfied by Gladstone's medium, was not stimulated by theaddition of biotin, bios IIB or vitamin H concentrates.When the basal synthetic medium was supplemented with the

growth factors riboflavin, pimelic acid, glutamine, vitamin Be,inositol, pantothenic acid, cocarboxylase, cozymase, glutathione,uracil, guanine, adenine, adenylic acid, and adenosine triphos-phate, there was no demonstrable influence on growth. Ifthese substances are essential for growth, the organism canapparently synthesize them from the ingredients in the basalmedium.The fact that the highest bacterial nitrogen values were ob-

tained in glucose meat-infusion broth, seems to indicate that thechemically-defined media studied lack one or more chemicalingredients necessary for optimum growth. Possibly, physicaldifferences of the media may also be involved.

REFERENCESDRUMEL, G., AND HUBERT, L. 1938 Quelques charactores chimiques de la

biotine. Arch. intern. physiol., 46, 141-146.EvANs, W. C., HANDLEY, W. R. C., AND HAPPOLD, F. C. 1939 The nutrition of

C. diphtheriae. Pantothenic acid as an essential growth factor forcertain strains of C. diphtheriae graves: The synthesis of some physi-ologically active compounds by C. diphtheriae cultures in syntheticmedia. Brit. J. Exptl. Path., 20, 396-408.

FILDxs, P., RICHARDSON, G. M., KNIGHT, B. C. J. G., AND GoAnsToNu, G. P.1936 A nutrient mixture suitable for the growth of Staphylococcwaureus. Brit. J. Exptl. Path., 17, 481-484.

GLuADsToN, G. P. 1937 The nutrition of Staphylococcus aureus: nitrogenrequirements. Brit. J. Exptl. Path., 18, 322-333.

GY6RGY, P., MELVILLE, D. B., BuRs, D., AND DU VIGNEAUD, V. 1940 Thepossible identity of vitamin H with biotin and coenzyme R. Science,91, 243-245.

HAWKRM, L. E. 1939 Nature of accessory growth factors influencing growthand fruiting of Melanospora destruens Shear, and of other fungi. Ann.Botany, 8, 657-675.

HoovER, S. R., AND ALLISON, F. E. 1935 A growth and respiration factorfor certain Rhizobia. Trans. Third. Intern. Congr. Soil Sci., 1, 158-160.

HUGHES, T. P. 1932 Growth requirements of staphylococci. J. Bact., 22,437-447.

KNIGHT, B. C. J. G. 1935 An essential growth factor for Staphylococcus aureus.Brit. J. Exptl. Path., 16, 315-326.

KNIGHT, B. C. J. G. 1937a The nutrition of Staphylococcus aueus; nicotinicacid and vitamin B,. Biochem. J., 81, 731-737.

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

KNIGHT, B. C. J. G., AND MCILWAIN, H. 1938 The specificity of aneurin andnicotinamide in the growth of Staphylococcus aureus. Biochem. J.,32, 1241-1251.

K6GL, F., AND TONNIS, B. 1936 tlber das Bios-Problem. Darstellung vonkrystallisiertem Biotin aus Eigelb. Z. physiol. Chem., 242, 43-73.

KLGL, F., AND VAN HASSELT, W. 1936 t~ber das Vorkommen von Biotin imtierischen Organismus. Z. physiol. Chem., 243, 189-194.

KOGL, F., AND HAAGEN-SMIT, A. J. 1936 Biotin und Aneurin als Phytohormone.Ein Beitrag zur Physiologie der Keimung. Z. physiol. Chem., 243,209-226.

K6GL, F., AND FRIES, N. 1937 tJber den Einfiluss von Biotin, Aneurin undMeso-Inosit auf das Wachstum verschiedener Pilzarten. Z. physiol.Chem., 249, 93-110.

K6GL, F. 1937 Wirkstoffprinzip und Pflanzenwachstum. Naturwissenschaf-ten, 25, 465-470.

KOGL, F., AND VAN WAGTENDONK, W. J. 1938 tYber die Bedentung von Biotin-fur das Wachstum von Staphylococcu8 pyogenes aureus. Rec. trav.

chim., 57, 747-754.MELIN, E., AND LINDEBERG, G. 1939 The influence of aneurin and biotin on

the growth of some mycorrhizal fungi. Preliminary note. Botan.Notiser, 1939, 241-245. Chem. Abstracts, 34, 1052, 1940.

MILLER, W. L., EASTCOTT, E. V., AND SPARLING, E. M. 1932 The fractionationof "Bios II". Trans. Roy. Soc. Can., III, 26, 165-169.

MILLER, W. L. 1937 Wilder's bios. Purification of bios IIB. Trans. Roy.Soc. Can., III, 31, 159-162.

MULLER, E. F. 1939 Das Wuchsstoffsystem der MilchfAurebakterien. Z.physiol. Chem., 260, 246-256.

MUELLER, J. H. 1935 Studies on cultural requirements of bacteria. IV. Quan-titative estimation of bacterial growth. J. Bact., 29, 383-387.

NILSSON, R., BJALFVE, G., AND BURSTR6M, D. 1939 tVber Zuwachsfaktorenbei Bact. radicicola. V. Lantbruks-Hogskol. Ann. Chemie, 7, 301-331.

OXFORD, A. E., LAMPEN, J. O., AND PETERSON, W. H. 1940 Growth factors forthe acetone-butyl alcohol microorganism, Clostridium acetobutylicum.Abstr. of Papers, 99th Meeting Am. Chem. Soc., Cincinnati, Ohio.

RAINBOW, C., AND BISHOP, L. R. 1939 The concentration and examination ofthe bios required by English brewery top fermentation yeast. J. Inst.Brewing, 45, 593-605.

RICHARDSON, G. M. 1936 The nutrition of Staphylococcus aureus. Necessityfor uracil in anaerobic growth. Biochem. J., 30, 2184-2190.

ROBBINS, W. J., AND SCHMIDT, M. B. 1939 Preliminary experiments on biotin.Bull. Torrey Botan. Club, 66, 139-150.

SARTORY, A., MEYER, J., AND NETTER, A. 1939a Sur l'influence des differentsproduits, ftudi&s comme facteurs energ6tiques de croissance micro-bienne, introduits separ6ment dans les milieux de culture. Bull.acad. m6d., 121, 815-819.

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SARTORY, A., MEYER, J., AND NETTER, A. 1939b etude de l'effet activant dedifferents m6langes de facteurs 6nerg6tiques, combines a la dose op-tima, sur la croissance d'un Staphylocoque et d'une levureabiotique.Compt. rend., X8, 1931-1933.

SNELL, E. E., AND WILLIAMS, R. J. 1939 Biotin as a growth factor for the butylalcohol producing anaerobes. J. Am. Chem. Soc., 61, 3594.

SNELL, E. E., EAKIN, R. E., AND WILLIAMS, R. J. 1940 A quantitative testfor biotin and observations regarding its occurrence and properties.J. Am. Chem. Soc., 62, 175-178.

SOBEL, A. E., YUSKA, H., AND COHEN, J. 1937 A convenient method of deter-mining small amounts of ammonia and other bases by the use of boricacid. J. Biol. Chem., 118, 443-446.

WEIZMANN, C., AND ROSENFELD, B. 1939 The specific nutritive requirementsof Clostridium acetobutylicum (Weizmann). Biochem. J., 33, 1376-1389.

WEST, P. M., AND WILSON, P. W. 1939a The relation of "coenzyme R" to biotin.Science, 89, 607-608.

WEST, P. M., AND WILSON, P. W. 1939b Effect of biotin concentrates on growthof Rhizobium and related species. J. Bact., 38, 110-111.

WEST, P. M., AND WILSON, P. W. 1940 Biotin as a growth stimulant for theroot nodule bacteria. Enzymologia, 8, 152-162.

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