STUDIES ON THE NUTRITION AND PHYSIOLOGY OF PASTEURELLA PESTIS

I. A CASEIN HYDROLYZATE MEDIUM FOR THE GROWTH OF Pasteurella pestis

KIYOSHI HIGUCHI AND CHARLES E. CARLIN'Fort Detrick, Frederick, Maryland

Received for publication August 6, 1956

The ability of Pasteurella pestis to grow in achemically defined medium is well established(Rao, 1939, 1940; Berkman, 1942; Doudoroff,1943; Herbert, 1949; and Rockenmacher et al.,1952). The culturing of the organism in caseinhydrolyzate media has been described by Smithand Phillips (1943), Seal and Mukherji (1950),Sokhey et al., (1950), and by Englesberg andLevy (1954). However, the amount of growthobtained has been rather low (5 X 108 to 50 X108 cells per ml). When these values were com-pared to those obtained with the Brucella, whereyields of 30 X 109 to 100 X 109 cells per ml havebeen reported (McCullough et al., 1947; Sanderset al., 1953), it was apparent that a better growthmedium for P. pestis might be developed. Thepresent report deals with the factors influencingthe growth of P. pestis in a casein hydrolyzatemedium. Results indicate that viable cell yieldsof 30 X 109 to 100 X 109 per ml in culturesincubated at 27 C can be obtained. The resultsof a similar study on a chemically defined me-dium will be described in a subsequent paper.

MATERIALS AND METHODS

Preparation of media. The principal componentof the medium was a partial hydrolyzate ofcasein. The material was prepared by hydrolysisof 100 g of casein in dilute H2SO4 (33.3 ml concH2SO4 + 500 ml H20) in an autoclave for 2 hr at20 to 22 pounds steam pressure. The hydrolyzatewas treated with an anion-exchange resin (Am-berlite IR-4B) to remove the H2SO4. Approxi-mately 200 g of resin (the amount varied withthe moisture content of the resin) was rinsedseveral times with distilled water and then addedto the acid hydrolyzate. The reaction was al-lowed to proceed for 30 min with continuousstirring. The addition of 20 ml of toluene to themixture aided in preventing excessive foaming.The final pH of the solution was approximately

1 Present address: Wheelabrator Corporation,Mishawaka, Indiana.

3.5. The suspension was then filtered and theresin washed repeatedly. The combined filtrateand washings were made up to a volume of 2 L.To the partially de-acidified hydrolyzate, 10 gof activated charcoal were added and the mixturewas boiled for 5 min. The solution was cooled,filtered, and made up again to 2 L. A pH ofapproximately 3.5 and a "solids" content in therange of 4.5 to 5.0 per cent (determined bydrying an aliquot at 100 C) were desirable. Thismaterial was designated as "5 per cent DCPH"(de-acidified casein partial hydrolyzate). DCPHwas employed in the growth medium at a concen-tration equivalent to 2.5 per cent of casein.Certain variables in the preparation and evalua-tion of DCPH are described later.The salt components of the medium (excepting

Na2S203) were prepared as a single tenfold con-centrated stock solution. The DCPH and the saltmixture were combined, adjusted to pH 7.5 andsterilized by autoclaving for 15 min at 121 C.The carbohydrate and Na2S203 were separatelyautoclaved and added aseptically to the medium.Inoculum and cultures. A number of strains of

P. pestis (both virulent and avirulent, table 1)was included in these studies, but strain A1122(Jawetz and Meyer, 1943) was used for a majorportion of the work. All strains of P. pestisexamined have grown well in the final medium.The inoculum was grown routinely 20 to 30 hrin the DCPH liquid medium after transfer froma stock slant of blood agar base (Difco), andemployed at a concentration of 1 per cent byvolume.

Growth conditions. Adequate aeration wasachieved with 25 ml of medium in a 500-mlErlenmeyer flask placed on a reciprocatingshaker operating at a rate of 100 cycles permin with a 3 inch stroke. Less aeration wasreflected in poorer growth of the culture. Theeffect of temperature on the growth of P. pestiswas determined by incubation of cultures at27 ± 1 C and at 37 + 0.5 C. Although 27 C

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TABLE 1Viable cell yields from several strains of Pasteurellapestis grown in the DCPH medium at 27 4 1 C

P. pestis Strain Viable Cell Yieldper ml, X109

AvirulentA1122 .................... 65Tjiwidej ...................... 84Soemedang.................... 105T.S...................... 30

Virulent139L ..................... ..... 69Alexander..................... 65Poona ....................... 114Charleston .................... 86Washington................... 61Kuma....................... 57Yokohama.................... 57

The DCPH medium (table 2) with D-xylose ascarbohydrate substrate was employed for growingthese cultures.

is believed to approximate the optimal growthtemperature (Girard, 1955), considerations otherthan cell yields (such as production of protectiveantigens) have made it desirable to investigatethe growth of P. pestis at the higher tempera-ture.

Chemical analyses. Inorganic phosphate wasdetermined by a modification of the procedureof Fiske and SubbaRow (1925). Reducing sugarswere measured by the method of Folin andMalmros (1929). The procedures for the deter-mination of bacterial protein and DCPH pep-tides were adapted from the method of Stick-land (1951). The pH values were estimated bymeans of Hydrion pH papers or by incorporationof phenol red indicator (10 ppm) in the growthmedium. The Beckman Model H-2 pH meterwas employed for accurate adjustments of pHin the preparation of media.

Measurement of growth. The Coleman Model 9Nepho-Colorimeter was employed routinelyfor measuring growth of cultures. Increases ingrowth, as measured by viable cell counts, wereaccompanied by parallel increases in nephelo-metric values (figure 1). An arbitrary turbiditystandard was employed and readings were madeon cultures diluted 1:10 in distilled water. (Areading of 50 turbidity units obtained with a

diluted culture in a standard 18-mm pyrex testtube gave an optical density value of 0.55 in a col-

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(0

ri 30 lih o 0

o20-X-~X VIABLE CELLS 100 1

LUui p-tai TURBIDITY

7 0 50

10 20 30 40 50

HOURS OF INCUBATION

Figure 1. Growth curves of Pasteurella pestisin the standard DCPH medium with D-xylose asprincipal carbon source and incubated at 27 4d 1 C.

orimeter employing light of 650 msA wavelength).Viable cell counts were made on the cultures byusing potassium phosphate buffer (0.033 m, pH7.3) to prepare the proper dilutions and platingin blood agar base (Difco) which had been forti-fied with 0.1 per cent of glucose and 0.04 percent of Na2SO3. No blood was required in theagar base.

RESULTS

Effects of extent of hydrolysis and degree of ion-exchange treatment of DCPH on the growth of P.pestis. The effects of the extent of hydrolysis onthe chemical and growth-promoting propertiesof DCPH were studied. Aliquots of a suspensionof casein in dilute H2S04, prepared as describedunder MATERIALS AND METHODS were autoclavedfor varying periods of time. Samples, obtainedat half-hour intervals during a period rangingfrom 1 to 3 hr, were tested in a standard mannerafter de-acidification with Amberlite IR-4B anddecolorization with charcoal. The solids contentsof these preparations were practically identical(4.4 4 0.1 per cent), but as was to be expected,the amino nitrogen contents were higher in thosesamples hydrolyzed for longer periods. Typicalexamples of cell yields obtained in media pre-pared from these materials were 57 billion cellsper ml in a medium containing a 1-hr hydroly-

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TABLE 2Composition of the casein partial hydrolyzate

(DCPH) medium for the growth ofPasteurella pestis at 27 C

Component Concentration

DCPH........ 2.,5%*K2HPO4........ 0.025 MCitric acid........ 0.01 MNa-gluconate.......... 0.01 MMgSO*7H2O.0.0025 MFeSO4 * 7H20 .......0..............0001 MMnS04-H20....................... 0.00001 MNa2S203 (sterilized separately) 0..... .0025 MD-Xylose (sterilized separately) .... 1 .0%t

* The "5% DCPH" prepared as described inthe text was diluted two-fold in the preparation ofthe final medium.

t More xylose was added as required duringgrowth (see text).

zate, 72 billions in a 2-hr hydrolyzate, and 69billions in a 3-hr hydrolyzate. Highest cell yieldswere obtained most consistently with media pre-pared from two hour hydrolyzates.The effect of the Amberlite IR-4B treatment

on the properties of DCPH was studied by treat-ing aliquots of a 2-hr acid hydrolyzate withvarying amounts of ion-exchange resin. Testswere made over a range of 1 to 8 g of resin perg of casein employed. Optimal ion-exchangetreatment, as reflected in cell yields in mediaprepared from these materials, was obtained inthe region of 2 to 3 g of resin per g of casein.The final pH values of DCPH preparations withan optimal degree of de-acidification were in theneighborhood of 3.5. Solutions equivalent to 5per cent of casein contained approximately 4.5per cent of solids.

Effects of varying the glucose concentration inthe medium. The medium finally adopted is pre-sented in table 2. Experiments on the effect ofvaried glucose concentrations on the growth ofP. pestis were made in the basal medium, exceptfor the substitution of glucose for xylose. Thegrowth and pH data presented in figure 2 illus-trate the dual effects of glucose in the growthmedium. Glucose stimulated growth at low con-centrations (1 to 3 mg per ml), but the additionof 4 mg per ml or more resulted in a drastic re-duction in cell yield. Very low terminal pHvalues were obtained in the inhibited cultures.The results were interpreted to mean that al-

though glucose was well utilized, the oxidativeprocesses consuming the intermediary organicacids were not keeping pace with the processesof acid production (fermentation). This appraisalof the course of glucose metabolism in the growthmedium was supported by an experiment inwhich repeated small increments of glucose (1 to3 mg per ml each) were fed to the growing cul-ture. As much as 30 mg per ml, added in 17 in-crements, were utilized over a 90-hr incubationperiod. Viable cell counts of over 60 X 109 cellsper ml were obtained. This was almost ten timesthe yield obtained in a medium with a singleaddition of glucose (figure 2). Each incrementaladdition of glucose was made with considerationof the fact that too much would result in exces-sive acidity and not enough would starve thecells (and also result in an undesirable rise inculture pH). Observations of the culture pHindicated when and how much glucose was re-quired.

In order to avoid the laborious feeding pro-cedures, the continuous feeding device describedby Soltero and Johnson (1954) has been useful.A further elaboration of such a system wouldbe a pH sensing unit to control the feed rate(McKee, 1955). For routine nutritional studies,

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PERCENT OF GLUCOSE IN MEDIUM

Figure 2. Effects of varied glucose concentra-tions on the growth of Pasteurella pestis in thestandard DCPH medium.

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-i

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0- XYLOSE

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HOURS OF INCUBATION

Figure S. Relation between xylose utilizationand pH of culture in the standard DCPH medium.

however, continuous feeding setups are not con-

venient; therefore, a search was made for carbo-hydrates with lower potentials for acid accumu-

lation in the growth medium.Alternate carbohydrates in the DCPH medium.

Substances which appeared promising as a con-

stituent of the growth medium were dextrin, D-

xylose, and D-mannitol. Excessive acid did notaccumulate when these materials were tested inthe DCPH medium at a concentration of 10 mgper ml. Upon further study, however, considera-tion of dextrin for the medium was abandonedbecause of the lack of uniformity in the com-

mercially available dextrins. The inertness of a

large fraction of the dextrin which necessitatedconcentrations as high as 60 mg per ml (6 percent) was another disadvantage of this material.The most suitable carbohydrate was D-xylose.

An initial concentration of 10 mg per ml was

employed, and as this was utilized, further addi-tions were made in 10 mg per ml incrementsuntil a total of 20 to 40 mg per ml was con-

sumed. Because larger additions of xylose were

permissible there was less danger of inadvertentlyfailing to maintain a supply of carbohydrate re-

serve in the growth medium. A typical growthcurve together with some of the accompanyingphysicochemical changes in a, growth mediumcontaining xylose are shown by data presentedin figures 1 and 3 and table 3. The pH changeswhich occurred upon the additions as well as

upon depletions of xylose are illustrated by thedata. Xylose was supplied to the culture at 0,16, and 32 hours. Data on the uptake of inor-ganic-P, utilization of biuret-positive peptides,

and synthesis of bacterial protein are presentedin table 3. Peptide utilization was determinedby measurements of biuret-positive materials inthe supernatant fluids after precipitation of thebacterial proteins with trichloracetic acid.

D-Mannitol was also found to be a suitablesubstrate in the growth medium. As much as 10mg per ml (initial concentration) was readilytolerated. It was therefore superior to glucosewith respect to ease of control of pH, but it wassomewhat less suitable than xylose. Yields ofcells obtained with mannitol were 10 to 20 percent lower than those obtained with xylose.However, mannitol possessed an important ad-vantage because of its stability. It could beautoclaved in the medium whereas it was neces-sary to autoclave xylose separately.

Glycerol was a suitable substrate for only cer-tain strains of P. pestis (Girard, 1955). Two so-called glycerol-fermenting strains (Kuma andYokohama) were grown in a glycerol DCPHmedium and yields of approximately 50 X 109cells per ml were obtained. The initial concentra-

TABLE 3The utilization of peptides, uptake of inorganicphosphate, and production of bacterial proteinin the DCPH growth medium by Pasteurellapestis at 27 C

Culture Peptide* Phosphate-Pt Proteint ViableAge pe opCells

hr mg/ml mg/ml mg/ml Xl09/ml

0 15.3 0.80 0.2 0.34 15.2 0.78 0.7 0.48 13.4 0.74 1.6 1.212 12.4 0.67 4.2 8.316 10.0 0.66 5.9 15.422 8.2 0.56 8.2 39.324 8.5 0.58 9.1 50.828 8.0 0.59 10.0 52.632 7.5 0.58 9.8 53.836 8.0 0.55 10.0 -40 7.5 0.55 10.148 7.3 0.56 10.1 52.3

* Measured as biuret-positive material in thesupernatant fluid after trichloroacetic acid pre-cipitation of cells.

t 0.80 mg per ml of P is equivalent to 0.0257 MK2HPO4 in the growth medium.

$ Measured as the trichloracetic acid pre-cipitable material in the growth medium. Caseinwas employed as a standard in both peptide andprotein determinations.

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HIGUCHI AND CARLIN

tion of 10 mg per ml of glycerol was well toler-ated in these tests, and a second 10 mg per mlwas added and utilized before the culturesreached the stationary phase of growth. Theglycerol fermenting strains, Kuma and Yoko-hama, also grew well in the xylose medium(table 1).

Concentrations of the constituents in the medium.The concentrations of the various componentsof the medium were determined by experimentsin which the effects of graded amounts of eachcomponent were tested in media which werebelieved to be otherwise adequate. The composi-tion of the final medium was a result of thesestudies and is presented in table 2.Varying the DCPH concentration of the me-

dium had a marked effect on viable cell yields.A concentration of approximately 2.5 per centof DCPH appeared to be optimal, yielding ap-proximately 70 billion cells per ml. On the otherhand, 2 per cent and 4 per cent of DCPH yieldedonly about 40 billion cells per ml. Evidence thatDCPH was a particularly favorable material forthe growth of P. pestis was seen when severalsampsubstdiumcent)diumprepaTh

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MOLARITY OF MgSO4

Figure 5. The requirement for additional Mgions for the optimal growth of Pasteurella pestisin the DCPH medium.

'-1- __ - ~.~~1-- ---ployed in the standard medium (0.025 M) wasiles of commercial peptones and digests were ploed in testanare edim (02Mw as

ituted for the DCPH in the standard me- based on results of experiments which showedat an equivalent concentration (2.5 per higher concentrations to be inhibitory.aThe growth obtained in the DCPH me- The requirement for either cysteine or thiosul-was better than with the commercial fate in the DCPH medium was demonstratedration eteted. thanwiththecommercialby data in figure 4. The amount of sodium thio-Lration tested.

e concentration of phosphate buffer em- sulfate (0.0025 M) employed in the final mediumwas in excess of the requirement. The need for

x_ added magnesium in the growth medium isshown by the results in figure 5. The amountemployed in the final medium (0.0025 M) was,therefore, in excess of the requirement.

In order to demonstrate a requirement for ironX/x in the DCPH medium, extraction of the DCPH

component of the medium with a 0.1 per cent1/ solution of 8-OH-quinoline in CHC13 was neces-

t/ sary (table 4). However, even in media whichX X CYSTEINE had not been extracted, iron deficiency occa-*1-@ 0THIOSULFATE sionally has been observed; therefore the stand-

ard DCPH medium was supplemented with10-4 M added FeSO4 to insure sufficiency. Growthmedia prepared with DCPH which had beenextracted with 8-OH-quinoline did not appear

I I deficient with respect to manganese, but 10-s M0.0005 0.0010 0.0015 0.0020 0.0025 MnSO4 was added routinely as a precaution.

MOLARITY OF SULPHUR COMPOUND A requirement for potassium ions in the me-

ure 4. The requirement for either thiosulfate dium was observed when Na2HPO4 was substi-3teine for the growth of Pasteurella pestis tuted for K2HPO4. On the other hand, a require-DCPH medium. ment for sodium ions was not demonstrable in a

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TABLE 4The requirement for iron for the growth ofPasteurella pestis in an iron-deficient mediumprepared by extraction with a chloroform solutionof 8-OH-quinoline

FeSO4-7H20 Added Cell Yields per ml

X104 M X10o

none 17.20.25 34.41.00 48.05.00 60.1

Only the DCPH component of the standardmedium was extracted with the 8-OH-quinolinesolution in chloroform. Possible iron contamina-tion was further reduced by the omission of citrateand gluconate from the test medium.

Concentrations of FeSO4 above 5 X 104 Mwere not tested because this was presumed to bein great excess.

medium when sodium salts were replaced withpotassium and ammonium salts. The employ-ment of citrate and gluconate in the mediumwas not essential, but citrate aided in preventingprecipitation of the cations and gluconate ap-peared to promote an earlier initiation of growth.(Gluconate was readily oxidized by resting cellsuspensions of P. pestis; citrate was not).

Yields of viable cells of several P. pestis strains.Typical yields obtained with several strains ofP. pestis grown in the standard DCPH mediumupon incubation at 27 a 1 C are presented intable 1. Xylose was the carbohydrate employedin these cultures. The growth characteristics ofthese cultures were similar to those illustratedby data in figures 1 and 3 and table 3. Nodifferences were noted in the growth of the viru-lent and avirulent cultures.

Effects of inoculum size. The size of inoculumemployed in most of these studies was ratherlarge. Approximately 1 X 108 to 5 X 108 cellswere usually inoculated per ml of medium. Itwas found that initiation of growth from ex-tremely small inocula (1 to 10 cells per ml) wasdifficult in the standard DCPH medium. Thereappeared to be inhibition by the concentrationof the casein hydrolyzate employed. When step-wise reductions in only the DCPH concentrationof the medium were made, there was a progres-sive decrease in the time required for appearanceof visible growth from small inocula. It was evi-dent that in order to obtain initiation of growth

from small inocula (1 to 10 cells per ml), it wasnecessary to employ only 0.25 per cent of DCPHin the growth medium even though the finalyield of cells was thereby limited.

Growth of P. pestis at 37 C. The optimal tem-perature for the growth of P. pestis is generallybelieved to be in the range of 26 to 30 C (Girard,1955). Increased nutritional requirements havebeen observed in cultures grown at 37 C (Hillsand Spurr, 1952). Nevertheless, the culturing ofP. pestis at 37 C has deserved much attentionfor several reasons. A soluble capsular antigen iselaborated in cultures grown at 37 C but is pro-duced only in insignificant amounts at 27 C.(Chen et al., 1952). Furthermore, the in vivostate of physiology of the organism in a hostanimal probably is more closely approached byculturing at 37 C rather than at 27 C. It wasdesirable, therefore, to investigate the growth ofP. pestis at 37 C in the DCPH medium. Thestandard DCPH medium (table 2) frequentlywas unsatisfactory for the growth of strainAl 122 at 37 C. The modifications made in thestandard medium for more consistent and im-proved growth at 37 * 0.5 C are listed in table5. The unusually high magnesium concentration(0.02 M), though resulting in a rather turbidmedium, promoted the growth of A1122 at 37 C.Glycine, supplemented at a concentration of0.027 M, also was distinctly beneficial. Althoughthe employment of cysteine in place of thiosul-fate was not advantageous, there were occasionalindications that the change was beneficial. Theaddition of biotin and pantothenate is in accord

TABLE 5Modifications of the standard medium for improved

growth of Pasteurella pestis A1122at a temperature of 87 C

Modifications*

Compound Concentration Nature of change

Glycine 0.027 M SupplementMgSO4 * 7H20 0.020 M Instead of

0.0025 MCysteine * HCI 0.004 M To replace

Na2S203Ca-Pantothenate 1.0 jug/ml SupplementThiamin *HCI 1.0 ug/ml SupplementBiotin 0.5 ,g/ml Supplement

* The modifications are applicable to the stand-ard medium (table 2).

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TABLE 6Viable cell yields of several strains of Pasteurella

pestis in the modified DCPH medium incubatedat a temperature of 37 C

Strain Cell Yield per ml

xl09

A1122* ......................... 21, 30Soemedang* .................... 50139L........................ 29Alexander...................... 32Yokohama..................... 20

* Avirulent strains.The growth medium is described in tables

2 and 5.

with the report of Hills and Spurr (1952), al-though the rather complete complement of aminoacids supplied in the medium may have madethe growth factors dispensable. Thiamin wasadded because distinct differences were obtainedin the terminal pH values of unsupplementedcultures which indicated that thiamin may belimiting. The organism appeared to grow well inthe absence of added thiamin but there was atendency for these cultures to become more acid.The various modifications described above

were made in order to improve the growth ofstrain Al 122 at 37 C. Possibly, all of the changeswere not required for the other strains. In anycase, the data presented in table 6 show that themodified medium was suitable for the growth ofseveral P. pestis strains at 37 C. Yields of 20 X109 to 30 X 109 viable cells per ml were usuallyobtained.

DISCUSSION

The results presented indicate that the pri-mary cause of low cell yields of P. pestis obtainedby other workers was the incapacity of the or-ganism to maintain a balance between the pro-duction and utilization of the intermediary acidsarising from glucose. The concentration of glu-cose permissible in the medium was limited toonly a few mg per ml even though the culturewas capable of ultimately utilizing larger amountsand thereby attain correspondingly greatergrowth. This situation is, of course, not uncom-mon in other aerobic microbial cultures.The data showing the beneficial effect of re-

duced DCPH concentration on the initiation ofgrowth from small inocula provide another ex-

ample in support of the observation that a con-centration of nutrient which provides for maxi-mal cell yield is not always consistent withoptimal growth conditions.The improvements in the medium described in

this work may contribute to studies on thephysiology and immunology of the plague or-ganism.

ACKNOWLEDGMENTS

The authors wish to express their appreciationto Dr. L. H. Graf and Dr. A. N. Gorelick fortheir support and encouragement. We also wishto thank Mr. Robert Curran for his technicalassistance.

SUMMARY

An improved casein hydrolyzate medium ca-pable of supporting yields as high as 1 X 101Oviable cells per ml has been developed for thegrowth of Pasteurella pestis.A study of the metabolism of carbohydrates

by P. pestis aided in selecting appropriate carbonsources and growth conditions for increased cellyields. D-Xylose, D-mannitol, D-glucose, andglycerol were useful under certain conditions.

Determinations of optimal concentrations ofthe principal components of the growth mediumwere made. Casein hydrolyzate corresponding to2.5 per cent of casein, potassium phosphate at0.025 M, MgSO4 at 0.0025 M, Na2S203 at 0.0025M, and FeSO4 at 0.0001 M; each was at optimal(or noninhibitory excess) concentration.A partial acid hydrolyzate of casein, particu-

larly suitable for the requirements of P. pestis inthe growth medium, has been developed.The differences in the growth of P. pestis at

two temperatures (27 C and 37 C) were investi-gated and a modified medium suitable for growthat 37 C has been developed.

REFERENCES

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DOUDOROFF, M. 1943 Studies on the nutritionand metabolism of Pasteurella pestis. Proc.Soc. Exptl. Biol. Med., 53, 73-75.

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FISKE, C. H. AND SUBBAROW, Y. 1925 Thecolorimetric determination of phosphorus.J. Biol. Chem., 66, 375-400.

FOLIN, 0. AND MALMROS, H. 1929 An improvedform of Folin's micro method for blood sugardeterminations. J. Biol. Chem., 83, 115-120.

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HILLS, G. M. AND SPURR, E. D. 1952 The effectof temperature on the nutritional require-ments of Pasteurella pestis. J. Gen. Micro-biol., 6, 64-73.

JAWETZ, E. AND MEYER, K. F. 1943 Avirulentstrains of Pasteurella pestis. J. InfectiousDiseases, 73, 124-143.

MCCULLOUGH, W. G., MILLS, R. C., HERBST,E. J., ROESSLER, W. G., AND BREWER, C. R.1947 Studies on the nutritional requirementsof Brucella suis. J. Bacteriol., 53, 5-15.

McKLE, M. T. 1955 Bacterial culture withcontrolled pH. Appl. Microbiol., 3, 355-360.

RAO, M. S. 1939 Nutritional requirements of

the plague bacillus. Indian J. Med. Re-search, 27, 75-89.

RAO, M. S. 1940 Further studies on the nutri-tion of the plague bacillus: the role of haematinand other compounds. Indian J. Med. Re-search, 27, 833-846.

ROCKENMACHER, M., JAMES, H. A., AND ELBERG,S. S. 1952 Studies on the nutrition andphysiology of Pasteurella pestis. I. A. chemi-cally defined culture medium for Pasteurellapestis. J. Bacteriol., 63, 785-794.

SANDERS, T. H., HIGUCHI, K., AND BREWER, C. R.1953 Studies on the nutrition of Brucellamelitensis. J. Bacteriol., 66, 294-299.

SEAL, S. C. AND MUKHERJI, S. P. 1950 Hy-drolysate of casein as a fluid medium for thegrowth of Pasteurella pestis. Ann. Biochem.and Exptl. Med. (Calcutta), 10, 79-98.

SMITH, L. D. AND PHILLIPS, R. L. 1943 Growthof Pasteurella pestis on a casein digest me-dium. J. Franklin Inst., 235, 536-545.

SOKHEY, S. S., HABBU, M. K., AND BHARUCHA,K. H. 1950 Hydrolysate of casein for thepreparation of plague and cholera vaccines.Bull. World Health Organization, 3, 25-31.

SOLTERO, F. V. AND JOHNSON, M. J. 1954 Con-tinuous addition of glucose for evaluation ofpenicillin producing cultures. Appl. Micro-biol., 2, 41-44.

STICKLAND, L. H. 1951 The determination ofsmall quantities of bacteria by means of thebiuret reaction. J. Gen. Microbiol., 5, 698-703.

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