requirements - jb.asm.org · studies on cultural requirements of bacteria v. thediphtheriabacillus...

STUDIES ON CULTURAL REQUIREMENTS OFBACTERIA

V. THE DIPHTHERIA BACILLUS

J. HOWARD MUELLERDepartment of Bacteriology and Immunology, Harvard University Medical

School, Boston'

Received for publication, November 16, 1934

In a preliminary report (Mueller et al., 1933), evidence has beenpresented as to the nature of certain substances required forsuccessful cultivation of a strain of diphtheria bacillus underinvestigation. It was there shown that a medium composed ofmeat extract, the amino acids formed on complete acid hydrolysisof casein, with tryptophane added, and a mixture of inorganicsalts, permitted satisfactory growth. Omission of tryptophaneresulted in failure to obtain growth; this amino acid was thereforeessential for our strain or organism. Separation of the otheramino acids by means of Dakin's (1920) butyl alcohol extractionmethod indicated that the unextracted residue as well as the "pro-line" fraction were essential. The only constituent of the formerfraction shown at that time to be required, was cystine, which,when added alone to suitable control solutions, resulted in as goodgrowth as when the whole crude fraction was used. Pure prolinecould not replace the crude proline fraction, so that another com-pound was inferred. Various attempts to isolate active, purematerials from meat extract failed, but it was shown that a non-basic substance, active in small amounts was involved.

1 The writer is indebted to Professor E. W. H. Cruickshank of the Departmentof Physiology, Professor N. B. Dreyer of the Department of Pharmacology andProfessor R. P. Smith of the Department of Pathology, of Dalhousie University,

. Halifax, for their courtesy in extending him the hospitality of their laboratoriesduring the summers of 1933 and 1934, where a part of this work was carried out.He also acknowledges with thanks the cooperation of the Difco Laboratories,Detroit, Michigan which have from time to time supplied some of the materialrequired.

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It is now possible to report more fully on the amino acid require-ments, and it will be shown that excellent growth may be ob-tained by the use of a few pure amino acids together with meatextract and inorganic salts and some enriching substance. It isproposed immediately to undertake a re-examination of the meatextract component with the better methods now available and tobe described here. A part of the findings to be detailed have beenthe subject of a brief preliminary note.2

GENERAL CONSIDERATIONS

Preparation of mediaMedia are prepared by dissolving the specified substances in

water, (or in the case of cystine, in dilute HQl), and mixing in theproper proportions. Phenol red is added, in amounts of one dropof 0.2 per cent solution to each 10 cc. of completed medium, andthe pH brought to 7.4 to 7.6 by addition of NaOH. Water isadded until the desired quantities are contained in exactly 10 cc.of solution, and the media are tubed and sterilized at 10 poundspressure for ten minutes.

Culture usedThe same strain of diphtheria bacillus used in the earlier work

has been maintained by daily transfer on meat infusion-"pro-teose" peptone broth, and kept at 360 to 37°. A small portion ofthe pellicle from such a culture after from eighteen to twenty-fourhours is transferred to each tube of medium to be tested by meansof a 3 mm. loop. It has not proved possible to inoculate moreevenly by using a dilute emulsion of the organism, for growth fre-quently fails or is slow in becoming initiated. Moreover, growthon duplicate tubes is quite uniform with this method of inocula-tion. No attempt has been made to carry the organisms throughrepeated subculture on these simplified media, but naturally thiswill be done in due time. For the moment, the objective is stronggrowth in the first generation.

2 Mueller, J. H.: Proc. Soc. Exp. Biol. and Med., 1934, 32, 318.

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STUDIES ON CULTURAL REQUIREMENTS OF BACTERIA 517

Method of determining amount of growthIn our earlier work this was done by attempting to evaluate the

thickness of the pellicle formed in twenty-four and forty-eighthours, and the results were recorded as - or i to ++ +-+ +.It became increasingly clear to us that this method was entirelyunsuitable. With a series of media to be examn ed the difficultiesof using such a rough, subjective method are obvious, and more-over, it is completely impossible to compare results over a periodof time. Because of the nature of the mixtures used-commercialmeat extract, and crude protein hydrolysates-cleanly negativecontrols could practically never be secured. It was felt that theamount of growth produced must be accurately determined, thusrendering possible the differentiations which it was necessary tomake, in such a manner that results could be strictly comparableeven over a period of months.The method finally settled upon was a micro-kjeldahl deter-

mination of the bacterial nitrogen formed on a given amount ofmedium in a fairly definite time. The method has been alreadydescribed elsewhere3 and need not here be detailed. It is ad-mittedly tedious in examining considerable numbers of cultures,but has so thoroughly proved its worth that we have no hesitationin urging that it be used by others working in this field, even ifonly on the basis that it permits of direct and easy duplication ofresults in various widely separated laboratories. Our tables andgraphs are given entirely in terms of milligrams of total nitrogenpresent in the bacterial growth from exactly 10 cc. of culturemedium, after an incubation period of fifty-five to sixty hours at350C. For the most part, each point plotted is the average ofduplicate determinations. The tubes used are Pyrex test tubeswith conical ends, described in the above citation, and they areincubated in a slanted position to allow adequate aeration andpellicle formation. Ordinary stock Pyrex test tubes of the samedimensions may be employed, but greater care is essential aftercentrifugation in pipetting off the supernatant, to avoid loss ofbacterial substance.

3Mueller, J. H.: Jour. Bact. 29, 383-388.


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J. HOWARD MUELLER

Planning of protocolsOur initial medium, which we wish to equal or improve upon,

contains: Liebigs extract, 0.5 per cent; peptone 1.0 per cent; andNaCi 0.5 per cent.

This was simplified as to the peptone by substituting for thatsubstance, casein acid hydrolysate, 1 per cent; and tryptophane,0.01 per cent.

Tryptophane, normally present in casein and in enzymatichydrolysates, is destroyed by strong acid hydrolysis and must bereplaced. The amount used will later be shown to be adequate.There is no diminution in amount of growth obtained as a resultof this substitution, certainly none for which adequate recompensecannot readily be made. The possibility of peptide linkages orother more complex effects than that of suitable amino acids isthereby practically removed.

Also from this point a salt mixture, with the following formula,is substituted for NaCl, which, in the presence of whole Liebigsextract may be unnecessary, but insures the presence of the com-moner inorganic elements.

Salt mixtureNaCl..................................................... 70.0 gramsK2HPO4.................................................. 2.5 gramsCaCl2............. 0.15 gramMgSO4................................................... 0.15 gramFeC13............. 0.05 gramHC1 (concentrated)....................................... 0.5 cc.H20 to............. 350 cc.

0.25 cc. of this solution is added to each 10 cc. of medium

The twenty-odd amino acids comprising the casein hydrolysatewere then divided roughly into three fractions by means of Da-kin's (1920) butyl alcohol extraction method, viz: I. Monoaminofraction. II. Proline fraction. III. Unextracted residue.

These three fractions added in appropriate amounts in placeof the casein hydrolysate practically reconstitute it, and likewiseduplicate its effect. Of the three, fractions II and III were foundto be essential in our earlier experiments while, fraction I waswithout marked effect. By omitting fraction I, using crude

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fraction II and substituting the various amino acids present infraction III, it has already been shown that cystine is the essentialcomponent, and in a concentration of 0.01 per cent which willlater be shown quantitatively to be adequate, can replace fractionIII.

This, then, was the situation at the outset of the work here tobe described, and our attention was occupied in the attempt toreplace fraction II in the following formula by a known aminoacid or compound.

Liebigs extract......... 0.5 per centTryptophane.........: 0.01 per centCystine........ 0.01 per centSalt mixture(Fraction II.......0O.1 per cent to 0.2 per cent)

This illustrates the method utilized throughout the work,namely to provide all the essentials for growth either pure or incrude form except the fraction under immediate examination.Cultures made on media lacking this fraction (controls), and thesame controls plus various preparations to be examined constitutethe experiment.

THE PROLINE FRACTION (FRACTION II, "P" FRACTION)

Using the above control solution and adding to it various "pro-line" fractions it soon became apparent that not all behaved alike.(It had already been shown that proline itself, alone, was withouteffect.) Some showed marked enhancement of growth over con-trols, others none. At this stage of the investigation quantitativemethods of recording results had not yet been adopted.

Since the crude fraction contains traces of monoamino acids,separable only by repeated re-solution in absolute alcohol, addi-tions of monoamino acid were again tried, although in earlierexperiments they appeared evidently unnecessary. Now, how-ever, almost invariable improvement in growth resulted. Theconcomitant presence of the proline fraction frequently but notinvariably gave still better growth. In general, this fraction wasprepared by distilling the butyl alcohol off in vacuum, dissolvingthe residue in hot absolute ethyl alcohol, cooling, and filtering


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J. HOWARD MUELLER

off the insoluble portion. A certain amount of water-insolublesticky material was then removed by distilling off the ethyl alco-hol, dissolving the residue as completely as possible in warm waterfiltering when cold, again distilling to a syrup and redissolvng inabsolute ethyl alcohol. The crude material thus obtained wasordinarily kept on hand dissolved in ethyl alcohol to preventdanger of bacterial contaminations and molds, and for use, thealcohol from a small portion was largely removed by warming ona watch glass over the water bath and redissolving in water.There seemed to be no reason to fear that the traces of alcoholremaining would influence growth, since they were too small tobe inhibitory; however, the point was finally controlled, and itwas quickly found that small quantities of alcohol considerablyincreased the amount of growth, quite evidently to an extentwhich appeared to explain the entire, (and irregular) effect of the"proline" fraction.

It was this finding which emphasized the necessity for a quanti-tative estimation of the amount of growth and the nitrogenmethod was worked out at this point. Table 1 and chart 1 showthe effect of the addition of ethyl alcohol to the control medium.The rationale for the use of glutam c acid in the control solutionwill appear later.

Control solution (quantities given are for 10 cc. medium): 10per cent Liebigs extract, 0.75 cc.; l-cystine, 1.0 mgm.; 1-trypto-phane, 1.0 mgm.; d-glutamic acid hydrochloride, 50.0 mgm.;"mono-amino" fraction, 50.0 mgm.; and salt mixture, 0.25 cc.

THE BUTYL ALCOHOL RESIDUE FRACTION (FRACTION III)

Glutamic acidWith a quantitative method available for estimating the

amount of growth, it was learned that cystine did not account forthe full effect of fraction III of the amino acids obtained by Da-kin's method. Substitution of the pure amino acids other thancystine present in this fraction quickly demonstrated that theadditional growth stimulus was supplied by glutamic acid. Theother amino acids present were without effect in the controls ashere constituted. It will be shown below, that one further com-

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ponent of the fraction, namely histidine, must be supplied formaximum growth with further refinement of the medium.The effect of the addition of glutamic acid in the form of the

hydrochloride is shown in chart 2. In the interest of brevity the

TABLE 1

LOT NUMBER COMPOSITION NITROGEW

cc. mgm.1 Control solution and ethyl alcohol 0.00 0.842 Control solution and ethyl alcohol 0.01 1.363 Control solution and ethyl alcohol 0.025 1.79*4 Control solution and ethyl alcohol 0.05 1.875 Control solution and ethyl alcohol 0.06 1.926 Control solution and ethyl alcohol 0.07 1.847 Control solution and ethyl alcohol 0.08 1.918 Control solution and ethyl alcohol 0.09 1.789 Control solution and ethyl alcohol 0.1 1.5510 Control solution and ethyl alcohol 0.125 0.9511 Control solution and ethyl alcohol 0.250 0.5812 Control solution and ethyl alcohol 0.50 0.35

* From thism nations.

point the figures given are the averages of duplicate deter-

a,

Contro-10%UebIS3 Extract (a75cc

1 Cystine 1.0mg.I-Try pa to0mA.d- Glutamic Acid Ha 50.0mg.

Mo homW tcion5FrorSalt Mixture 0.25cc.

01 0.2 0.5 Q4

c-c ethyl alcohol In lOc.Qmedium.CHART I

actual figures need not be tabulated, but can be read with sufficientaccuracy from the figure.

Alcohol in its optimal concentration is included in this and


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J. HOWARD MUELLER

subsequent media in order to push the maximum growth as highas possible so that the requirements for other factors may be moreclearly demonstrated.

Tryptophane and cystineAlready recognized and reported as essential constituents of the

medium, only qualitative information was available as to theamounts necessary for optimal growth. It was therefore deemedexpedient to demonstrate quantitatively the effect of these twosubstances. The following curves (charts 3 and 4) are self ex-planatory, and indicate that the quantity of 1 mgm. per 10 cc.

.2EoO

c / ~~~~~~~~~10%UeblPExtrac Mmc^/ ~~~~~~~~1-Cy~th~le ILOn"S /~~~~~~~~~ITwpohane lI-OmLO1 a'mndoFho 5ckond,wA * ~~~~~~~~~EthlAkohol 05 rccx, ~~~~~~~~~SortMiixture OL2Scc

0.5

I 20 40 60 80 100 120 KO0 160 180 200W6 SlutaMic acid-NCI in locr. medium

CHART II

used in earlier controls is probably about correct. The result isquite different from that shown by glutamic acid, and one mayinfer that the latter substance is used as a source of energy, where-as tryptophane and cystine probably each contribute an essentialatom grouping, in the case of cystine, perhaps connected with theoxidation-reduction potential of the medium.

The "monoamino" fractionUsing, now, control media containing the above described fac-

tors, it may readily be shown that the prect Th reasingquantities of the monoamino"fraction causes a marked and pro-gressive increase in growth. This is illustrated in chart 5.

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In order to analyze this effect one must consider the aminoacids known to be present in this fraction. These comprise thefollowing: glycine, alanine, valine, leucine, seine, phenylalanine,methionine, tyrosine and oxyproline. Each of these amino acidswas substituted for the monoamino fraction, but only a single

E

5 GControb-10% Lbb Extrac OJ5mc

* I-Cystine l.Om&w d-CdutaecAcidHCN 510.0m

'MoosmFiacon 5.Orn0c Ethyl Alcohol 0.05c.CI SdtMlxture 25mcc

0 to 2.0 a0 40 50

MS tryptophane in Oc. medium.

CHART III

'S(S

E 5 I .n...h-10%Uebl.ss Extrac 075cc

4) I-Tryptophane to wrs2 to i- GlutemkAciddCI 50Om&_ w~~~~~Monosminaorractin 5Q0.0C ~~~~~~EthylAkohol QOLctcX ~~~~~~SsNMixture 025ct

o to 2.0 3. 4.0 SO

MS cystine In IO0cemedium.CHART IV

one, methionine, showed regularly any accelerating effect, andthis a peculiar one, in that small amounts caused a marked in-crease in growth over the control, whereas, with even slightlygreater quantities, the effect was lost. This fact is brought out inchart 6.A considerable number of experiments such as the above have

JOURNAL OF BACTERIOLOGY, VOL. 29. No. 5


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524 J. HOWARD MUELLER

been carried out, and the result is invariably the same. Nomaterial difference can be observed between the 1- and the dl-forms. The latter is consequently used preferentially to rule outthe possibility of the presence of unrecognized impurities. Thecurve starts up exactly as do those for tryptophane and cystine,but immediately flattens and descends with still a relatively low

S a

E

lT0%Uetbpho.tr Q75c.

1.0 1;ryphane LO0M&d- GlutemicAcdd HCI 50mOngEthyl Alcohol 0.05cc.Salt Mixture 025cc

s03~~~~~~~~~~~~~~~~~0

0 * 20 30 40 50 t00 20o

Mei monoamino fraction in 1Ocmmedlum.CHART V

.2Fig L5 Control:-9 10%Ueblgs Extract a75fcS2 A - Cyfilne 1.04.,

LtO d-GlurnlcAc:dHCI5L0m&X§ \ ~~~~EthylAkoho QOLccQ

0 2 4 6 a lo to 40

M&dl-methlonine In IOcemedlumCHART VI

concentration of methionine, until it approaches the level of thecontrol tubes. With even considerably larger amounts, however,it does not sink below this quantity, thus differing sharply fromthe effect shown by alcohol, which is of course inhibitory inmoderate excess. Moreover, in the case of the addition of thewhole monoamino fraction, which probably contains in the neigh-


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borhood of 10 per cent methionine, it is clear that no such dropin the growth curve takes place, even in the presence of perhaps15 to 20 mgm. of methionine (200 mgm. monoamino acid) in 10cc. of medium. It would appear that methionine acts, in amanner quantitatively similar to tryptophane and cystine, bysupplying a distinctive atom grouping, but only in the presence ofsome other compound, which occurs in traces in the controlmedium, probably in the Liebigs extract, and also in the mono-amino fraction. The attempt to provide such a factor by theaddition of any of the other known components of the monoaminofraction failed, either when these were used simply or in com-binations.

2.0

r_ conftrlt-101 Uebijf Extract (175cc.

1.0 1-Cyatlne1-0rCII- Tryptophane tam&d- Glutahm Acid HN 5Orn5.I- HiatidineKCI tl .0

Ethyl Alcohol 0.05c.cSolt Mixture 025cc.

0 2 4 6 810 20

M& dl-methionine in fOccmmedlum.CHART VII

In various ways, the attempt was made to isolate from themonoamino fraction the substance responsible for this combinedeffect with methionine. It was finally learned that practicallyall of the desired material could be precipitated with silver oxideand baryta. This preparation, freed from silver and barium gavea marked Pauly reaction, and following this clue, histidine wastried, and proved at least partially effective as shown in chart 7.

Histidine has not been commonly recognized as being presentin the monoamino fraction prepared by the butyl alcohol method,and has not actually been isolated from it in these experiments.Calvery (1931) has, however, demonstrated the presence of smallamounts of histidine in the corresponding fraction prepared from


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J. HOWARD MUELLER

crystalline egg albumin, when extraction was continued as long astwo hundred hours. It can only be stated that a substance inthe "monoamino" fraction precipitable by regents ordinarilyprecipitating histidine, and giving the color reaction characteristicof imidazole compounds (among others) appears to exert a pecul-iar biological effect together with methionine, an effect which isapproximated by histidine. The growth curve, however, evenwith considerable quantities of histidine, still shows a fall, afterthe maximum is reached. It may be stated parenthetically thathistidine alone, without methionine, has absolutely no effect onthe growth obtained from control media.A mixture of the amino acids known to be present in the "mono-

amino" fraction (except methionine and histidine) was next pre-pared, each in approximately the proportion in which it occurs incasein. A total quantity of 50 mgm. of this mixture, added to 10cc. of a control containing 2 mgm. methionine and 1 mgm. histi-dine hydrochloride gave a marked increase in the quantity ofgrowth obtained. When used singly, and in correspondingamounts with the same control solution, the individual aminoacids comprising the mixture gave no significant increases overthe control alone. Evidently here again, more than a single sub-stance was involved and it became necessary to eliminate oneamino acid at a time from the total mixture. In this way seineand oxyproline proved to be without effect, alanine and particu-larly leucine appeared to be somewhat inhibitory, while glycine,valine and phenylalanine, when added together, considerably in-creased the quantity of growth, and tyrosine appeared eitherwithout effect or very slightly accelerating. The action of thisrather troublesome mixture was next investigated quantitatively.The accelerating effect is most marked when all three are usedsimultaneously, and by varying the quantities it appears that 1mgm. of dl-phenylalanine, 5 mgm. of glycine and 10 mgm. dl-valine will yield maximal growth. It has not seemed worth whileto carry out sufficiently extensive experiments to construct curvesdemonstrating this fact, but chart 8 illustrates the effect ofvarying the quantity of methionine in the presence of these addi-tional substances.

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To bring together more clearly the results obtained with me-thionine, histidine, phenylalanine, glycine and valine, curves 6,7 and 8 have been superimposed in chart 9.

toto.

et5 tO%~~~~~~~0%UebitbExtract (L75cxc - Cystine tOns

I - Tryptophane LOmg.2 d- Glutamk AcId HCI 5QOm&

I- HistidlneHCI LOm&- dl- Phenylanine LOmg.

dGlycine 5.mg.Vd- eline 10.0mgs

0.5~~~ ~ ~ ~ ~ ~ ~~~~4

0 2 4 B 8 10 20 40

Mg.dl-methionine In lOccimedium.CHART VIII

E Zl

CD Control A:E l~07w-lebiWsExtrcf Mm5c

e- 15 4sg - Cystine t.O Mg.A 'tU>5^ 1~~~~~~-Tryptophane LOm&

^e d- GlutamilcAcid HCI 50QOm&eg\ zi, ~~~~~~~~~EthlAkohdl 0M5Cc

Salt Mbiiure 0.25cc

X0e~ ~ ~~~~~~~~~~4

0 2 4 S 8 tO 20 40

M5dl-methionine in 10ccmedium.CHART IX

It is obvious that without methionine, the other four aminoacids have little or no effect, since all the curves start at approxi-mately the same level of nitrogen. It is evident, moreover, thatthis mixture of amino acids duplicates very closely the effect of


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J. HOWARD MUELLER

the crude "monoamino" fraction, in which a total of 2.5 mgm.nitrogen is reached with 200 mgm. of amino acids in 10 cc. (seechart 5), whereas the curve using pure amino acids rises sharplyto this same value, flattens, and drops slowly as the quantity ofmethionine increases.

DISCUSSION

The nutritional requirements of various strains of diphtheriabacilli are known to differ widely. Braun (1930) speaks of "ans-pruchsvolle" and "anspruchslose" strains of this organism, andhas succeeded in growing some of the latter on very simple media.Wheeler and Wadsworth, (1934) as well as others, have also culti-vated certain strains and even produced toxins of some strengthon synthetic media. It has happened that in our work we haveevidently dealt with a strain having a fairly complicated list ofrequirements. Whether the daily passage for a long period oftime on the usual peptone-infusion broth has gradually broughtthis about, or whether it was true from the first, we are unable tostate. Experiments are now in progress in which the effect ofeach of the substances found of value with our strain is beingtested on a number of freshly isolated strains, as well as on otherold laboratory types. We hope to report shortly the results ofthis work.From a somewhat different standpoint, it is also of interest to

speculate as to the reasons for the various effects which have beenbrought out.The ethyl alcohol is probably used directly as a source of energy.

The two-carbon chain may be, for some reason, readily availableto this particular organism. Thus, methyl and propyl alcoholare without effect, whereas acetic acid can be substituted forC2H5OH. On the other hand, both glycerol and maltose areeffective but not enough work has been done to demonstrate howthese substances compare quantitatively.

Glutamic acid, because of the considerable amount required,probably acts also as a source, either of energy or of nitrogen, orboth. The presence of an associated impurity has not beenexcluded, but seems improbable because of the relative ease with

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which this material is obtained in a high degree of analyticalpurity. It cannot be replaced by aspartic acid or asparagine,which indicates again a selective action on carbon chains of adefinite length. We plan to investigate such similar compoundsas glutamide, glutaric acid, etc.

Cystine, tryptophane, methionine, histidine and phenylalanine,producing their maximal effect at low concentrations, and eachpossessing a rather characteristic atom grouping, probably arerequired because of those groupings. The peculiar fall in thegrowth curve obtained with methionine, prevented to a consider-able degree by histidine, particularly in the presence of, or whenacting with, phenylalanine, glycine and valine, remains com-pletely unexplained. The obvious suggestion is that, as a stageof its metabolism, some inhibitory substance is formed, butagainst this is the fact that growth never sinks below that of con-trol tubes. It is quite probable that with the active investigationnow being made in a number of laboratories of the part playedby methionine and similar sulphur compounds in animal metab-olism, an explanation for the phenomenon may be forthcoming.It is interesting that in animal metabolism methionine may takethe place of cystine in artificial diets, whereas here both arerequired. It is perhaps also significant that methionine was firstisolated during the study of a fraction of amino acids which madegrowth of a strain of hemolytic streptococcus possible, (Muller,1922, 1923), although when purified, no effect could be demon-strated. The peculiar quantitative influence now recognizedmay account for that failure, or its presence may have been merelya coincidence.That the medium as now constituted is at least the equivalent

of a meat infusion-peptone broth is indicated by the fact thattotal bacterial nitrogen yielded by 10 cc. quantities of the latteris from 0.85 to 0.90 mgm., whereas our maximum is nearly threetimes as great, viz., 2.50 mgm. Naturally, an increased yieldcould be obtained from the bouillon if it were enriched by alcoholand glutamic acid. The writer feels, however, that by the useof the amino acids discussed in the present paper, the substitutionfor peptone in the bouillon has been adequately made. The


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J. HOWARD MUELLER

study is being continued in a re-investigation of the complexLiebig's Extract fraction, and it is hoped, by means of quantita-tive methods to arrive at a better understanding of the substancesthere involved than was possible in the preliminary investigations.

SUMMARY

It is shown that excellent growth of a rather fastidious strainof diphtheria bacillus can be obtained on a medium composed ofLiebig's Extract, certain pure amino acids, ethyl alcohol andtin-organic salts. The amino acids include l-cystine, 1-tryptophane,d-glutamic acid, dl-methionine, l-histidine, dl-phenylalanine,glycine and dl-valine. The quantitative relations of these variouscompounds are presented, and their various actions discussed.The work is being extended to a further study of the meat-extractfraction and in other directions.

REFERENCES

BRAUN, H. 1930 Hanb. d. biol. Arb. Meth., Abt. XII, Teil 2, Hft. 1, p. 54.CALVERY, H. 0. 1931 Jour. Biol. Chem., 94, 613.DAKIN, H. D. 1920 Jour. Biol. Chem., 44, 499MUELLER, J. H. 1922 Jour. Bact., 7, 325.

1923 Jour. Biol. Chem., 56, 157.MUELLER, J. H., KLISE, K. S., PORTER, E. F., AND GRAYBIEL, A. 1933 Jour.

Bact., 25, 509.WADSWORTH, A., AND WHEELER, M. W. 1934 Jour. Inf. Dis., 55, 123.

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