THE ROLES OF BIOTIN AND CARBON DIOXIDE IN THE CULTIVATION OFMYCOBACTERIUM TUBERCULOSIS, 2

WERNER B. SCHAEFER, MAURICE L. COHN, AND GARDNER MIDDLEBROOKDepartment of Research and Laboratories, National Jeish Hospital, and Department of Microbiology,

University of Colorado School of Medicine, Denver, Colorado

Received for publication December 10, 1954

During a study of the growth of tuberclebacilli on primary isolation on oleic acid-albuminagar and on the egg medium of the AmericanTrudeau Society (ATS), the observation wasmade that a number of strains, which gavegrowth on ATS medium, did not grow, or grewvery poorly, on oleic acid-albumin agar medium(Middlebrook, Cohn, and Schaefer, 1954).In an attempt to improve this latter medium

various growth factors were added, and it wasfound that biotin was either markedly stimu-lating or even absolutely required for the arti-ficial cultivation of several strains (Middlebrook,Cohn, and Schaefer, 1954). Similarly, incuba-tion under an atmosphere with increased COscontent also stimulated the growth of thesestrains markedly. One strain has been observedfor which biotin would not support growth at37 C, and which was permitted to grow only byincubation under an atmosphere of air containingadded C02.The purpose of this paper is to report our

observations on the roles of biotin and carbondioxide as growth factors for tubercle bacilli.

MATERIALS AND METHODS

The basic oleic acid-albumin agar mediumused in these experiments had the followingcomposition: KH2PO4, 1 g; Na,HPO4 (an-hydrous), 2.5 g; Nas citrate 2H20, 0.1 g;MgSO4.7H20, 0.05 g; CaCl2-2H,O, 0.0005 g;ZnSO4-7H,O, 0.0005 g; CuSO4.5H20, 0.0005 g;ferric ammon. citrate, 0.05 g; iglutate(sodium salt), 0.5 g; vitamin-free casein hy-drolyzate, 0.1 g; (NH4)VS04, 0.5 g; glucose, 2 g;

I Supported in part by grants from the DazianFoundation.

2Presented in part at the General Meeting ofthe Society of American Bacteriologists in Pitts-burgh on May 7, 1954.

' This medium was proved to contain less than0.001 pg biotin/ml by microbiological assay witha biotin requiring strain of Lactobacillus arabinosus.

glycerol (reagent grade), 2 ml; oleic acid-al-bumin complex, 50 ml; distilled water, ad 1,000ml; agar, 15 g.The pH was adjusted before autoclaving to

6.8 (unless otherwise indicated). After auto-claving, its value was found to be 0.15 to 0.2pH units lower. Before autoclaving, malachitegreen (Coleman and Bell), in a final concentra-tion of 0.5 to 1.0 ,ug/ml, was added in order toreduce the incidence of contamination. Thisconcentration was found to have no significanteffect on the growth of tubercle bacilli in thismedium. Glucose and oleic acid-albumin com-plex, prepared as described by Dubos andMiddlebrook (1947), were added after auto-claving. The medium was poured into petridishes. For the inoculation of the plates, thefollowing procedures were used.

Clinical specimens were treated before inocula-tion by the concentration and deconinationprocedures previously described (Middlebrooket al., 1954). When cultures in "Tween-albumin"liquid medium were to be used as inocula, theywere ground in a Teflon grinder (Pierce, Dubos,and Schaefer, 1953) and filtered through anM-type sintered glass filter in order to obtaina suspension consisting predominantly of iso-lated bacterial celLs (Cohn et al., 1954). Thesuspension was diluted to 10- or 10-6 in sterilesolution of 0.2 per cent bovine serum albumin indistilled water, and 0.1 ml of each dilution wasspread over the agar surface with a gla#s spreaderwhile the plate was revolving on a turtable.The plates were placed in polyethylene plasticbags in order to protect them from desiccationand incubated at 37 C. For the study of theeffect of incubation under an atmosphere of airwith higher CO content, the plates were placedin glass desiccators which were evacuated to50 mm Hg pressure and then filled to atmosphericpressure with a gas mixture of air containing5 per cent CO2 obtained from a commerciallyavailable cylinder. For the routine use of incuba-

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tion under increased CO2 tension in the diagnosticlaboratory, a large metal chamber with hermeticclosure was employed, into which a gas mixtureof 10 per cent C02 in air was appropriatelyintroduced to give a final C02 concentration ofapproximately 2 to 5 per cent. The chamber was

refilled with the gas mixture each time afteropening.

RESULTS

(1) A strain requiring biotin or CO2. The effectof various concentrations of biotin and of incuba-tion under C02 enriched air on a strain requiringeither biotin or carbon dioxide is represented intable 1. It can be seen from this table that on

the agar plates, which had been incubated inatmospheric air, no growth was obtained in theabsence, partial growth in the presence of 0.02Ag/ml, and full growth in the presence of 0.1,ug/ml or higher concentrations of biotin. On theplates which were incubated in an atmosphere of5 per cent C02 in air, full growth occurred in theabsence as well as in the presence of biotin. Inliquid Tween-albumin medium incubated inatmospheric air, the rate and total amount ofgrowth increased with increasing biotin concen-

tration in the medium. Desthiobiotin had thesame growth stimulating effect as biotin. Pimelicacid, diamino-pimelic acid,4 asparagine, asparticacid, oxalacetic acid, acetic acid, and argininehad no effect. Oleic acid and pyruvic acid alsodid not replace biotin but stimulated growthin the presence of biotin. Variations of the pHor a decrease of the incubation temperaturefrom 37 to 34 C had no effect on the biotinrequirement of this strain. Experiments ofincubation in air with different concentrationsof C02 indicated that a concentration of 1 percent C02 in air was sufficient to support growthin the absence of biotin. The described strainwas resistant to 500 ,ug/ml of streptomycin andto 30 ,ug/ml of isoniazid. It maintained its re-

quirements for biotin or C02 after many transfersin biotin containing Tween-albumin medium.Occasionally, however, rare colonies were re-

covered from biotin-free agar media incubatedunder atmospheric air, and these colonies provedto be biotin-independent mutants.

4 Kindly supplied by Dr. L. D. Wright, Directorof Research in Microbiological Chemistry, Re-search Division, Sharp & Dohme, Inc., WestPoint, Pa.

TABLE 1Effect of biotin and CO2 on growth of a biotin

requiring strain of Mycobacteriumtuberculosis

Oleic Acid-Albumin Tween-Albu-Solid Agar Medium* mint Liquid(Growth after 17 days) MediumBiotin _____ Biotin (Optical

- ~~~~~~densityAir 5% air after 12

in air days)pg/ml pg/ml0.5 +++ +++ 0.05 0.3000.1 +++ +++ 0.025 0.3280.02 + +++ 0.012 0.2440.004 0 +++ 0.006 0.1990 0 +++ 0.003 0.164

0 0.089

* Inoculum: 0.1 ml of 10-6 dilution of culturein Tween-albumin medium.

t Inoculum: 1/100 dilution of culture in Tween-albumin medium, at pH 6.8.+++ - full growth; + = sparse growth.

This strain was reisolated from the patient atintervals of several months without showing anychange of its characteristic properties.

(2) Strains requring biotin or CO2 only onoleic acid-albumin medium of acid reaction.Several strains of tubercle bacilli have beenencountered which showed a requirement forbiotin or carbon dioxide on oleic acid-albumin

TABLE 2Effects of pH and of oleic acid on the biotin require-ment of a strain of Mycobacterium tuberculosiswhich requires biotin only at acid reactions

(Incubation at 37 C for 3 weeks underatmospheric air.)

pHMedium Biotin

6.5 6.9 7.3

pg/miAlbumin agar 0.5 400* 500 450

+ + :1=-0 400 500 450

+ + Ai

Albumin agar plus 0.5 387 473 4120.005% oleic acid +++ ++ +

0 140 466 412

* Numbers of colonies. The crosses indicatethe relative sizes of the colonies: +++ = 2-3 mmdiameter; i - just visible, grossly.

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TABLE 3Reversal of the inhibiting effect of 4-(imidazolidone-2) caproic acid

by biotin and by increased CO2 tesion

4-(Imidazolidon-2) Caproic Acid (jug/ml)Biotin pg/mi)

0 1 0.08 A0.4 2.0 10.0 50.0 100.0

Incubation under atmospheric air (0.03% C02)

0 +++* + 0 0 0 0 00.01 +++ +++ + 0 0 0 00.05 +++ +++ +++ +++ +++ +++ +++

Incubation under air with 1% C02

0 +++ +++ +++ +++ 0 0 00.01 +++ +++ +++ +++ +++ + 00.05 +++ +++ +++ +++ +++ +++ +++

Incubation under air with 10% C02

0 +++ +++ +++ +++ 0 0 00.01 +++ +++ +++ +++ +++ + 00.05 +++ +++ +++ +++ +++ +++ +++

* The crosses indicate the relative amounts of growth after 3 weeks of incubation.

medium of more or les acid reaction, but not onmedia of neutral or alkaline reaction. On albuminagar medium without oleic acid, these strainswere able to grow in the absence of biotin evenwhen the reaction of the medium was acid. Anexample of the effect of biotin on the growth ofsuch a strain on oleic acid-albumin agar and onalbumin agar media of various pH's is repre-sented in table 2. This group included isoniazidand streptomycin-sensitive strains as well asstrains resistant to isoniazid alone or to bothdrugs. Their requirement for biotin or C02 underthe above described conditions remained thesame after repeated subcultures in vitro and onrepeated isolation from the specific patient'ssputa.

(3) The inhibiting effect of the biotin analogue4-(imidazolidone-2) caproic acid and its reversalby increased CO2 tension. Having shown thatincubation under increased C02 tension sub-stitutes for a requirement for biotin, it seemedof interest to investigate whether increased C02tension actually eliminates the need for biotinas a necessary growth factor or only decreasesthe amount of biotin required for C02 assimila-tion. For this study the biotin analogue 4-(imi-dazolidone-2) caproic acid" was used because

6 Kindly supplied by Dr. J. A. Aeschlimann,Director of Chemical Research, Hoffmann-LaRoche, Inc., Nutley, New Jersey.

it is known to inhibit growth of tubercle bacilliby antagonizing endogenous biotin (Pope, 1952).The effect of increased C02 tension and of biotinon this inhibition was studied. The experimentswere made with a nonbiotin requiring strain.Their results are recorded in table 3.

It can be seen from this table that underincreased C02 tension about 50-fold higherconcentrations of the biotin antagonist werenecessary for inhibition of growth than underatmospheric air. The inhibiting effect of still largerconcentrations of the inhibitor was reversed bythe addition of large amounts of biotin but notby an increase of the C02 tension. These resultssuggest that minimal amounts of endogenousbiotin are required for the utilization of C02 forgrowth.

(4) A strain requiring C02 for growth at 87 Cbut not at 84 C. A different type of C02 require-ment, which is apparently not due to a deficiencyof biotin, was found for a highly streptomycinand moderately isoniazid-resistant strain isolatedfrom the sputum of a patient with cavitarytuberculosis under treatment with streptomycinand isoniazid. This strain was unable to growon ATS egg medium and on agar medium evenwhen biotin was added to the medium. Itsculture could only be obtained either when suchmedia were incubated at 37 Cunderan atmospherewith increased C02 content or under atmos-

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pheric air when incubated at 34 C. All attemptsto find a medium suitable for the growth of thisstrain at 37 C in air failed. Variations of the pH,omission of oleic acid, the addition of variousamino acids or adenylic acid in various concen-trations, of yeast extract and various vitaminswere tried without success. This strain was alsounable to grow in liquid Tween-albumin mediumwhen incubated at 37 C in loosely capped tubes.It gave, however, growth when it was incubatedin such tubes at 34 C, and also at 37 C whenthe tubes were tightly closed (Thunberg tubes).In these latter tubes growth occurred, however,only after a lag phase of some 5 days. The growthcurves obtained under these various conditionsare shown in figure 1. Subcultures on agar mediafrom the closed tubes incubated under atmos-pheric air at 37 C, as well as from those incubatedat 34 C, showed that the bacteria grown underthese conditions were still CO2 requiring organ-isms. The delayed growth in the sealed tubesincubated at 37 C, therefore, was due, mostprobably, to the slow accumulation of meta-bolically produced CO2 which could not escapefrom the closed tubes. Large inocula of thisstrain on agar media incubated in atmosphericair yielded rare colonies of bacilli which provedto be C02-independent mutants. The mutationrate of this strain from C02-dependence toC02-independence has not been determined.

PRACTICAL APPLICATIONS

In an attempt to evaluate the practical im-portance of increased CO2 tension for the cultureof tubercle bacilli on primary isolation, tuberclebacilli from some 70 sputa were cultivated onbiotin containing oleic acid-albumin medium byincubation under atmospheric air, on the onehand, and under air with increased CO2 content,on the other hand. It was found that incubationunder increased CO2 tension definitely stimulatedthe growth in more than half of the cases andthat none of the strains encountered was re-tarded oIr inhibited by increased CO2 tension.6

6 It should be mentioned that the stock labora-tory strain, H37Rv, which had been repeatedlysubcultured on Tween-albumin medium, wasinhibited by increased CO2 tension when platedonto oleic acid-albumin agar medium. Thisstrain, on the contrary, was stimulated on thesame medium by increased CO2 tension when aculture, which had been recently animal-passed,was employed.

Figure 1. Effect of gaseous environment andtemperature on the growth in "Tween-albumin"liquid medium of a strain of Mycobacteriumtuberculosis which requires increased CO2 tensionat 37 C, but not at 34 C.

(1) 34 C in atmospheric air; (2) 37 C in 5 percent CO2 air; (3) 37 C in atmospheric air (closedsystem); (4) 37 C in atmospheric air (open system).

The stimulating effect of CO2 was particularlymarked on media of acid pH (6.6).An illustration of the beneficial effect of CO2

for the primary isolation of a tubercle bacillusstrain from a sputum specimen is presented infigure 2.

DISCUSSION

Airequirement of strains of tubercle bacilli forbiotin has not previously been reported in theliterature. The studies of Landy and Dicken(1941) and of Pope and Smith (1946) haveshown, on the contrary, that the two human andone bovine strains studied synthesized biotinduring growth on a synthetic medium. Theexperiments presented here show that strains oftubercle bacilli may manifest a requirement forexogenous biotin for their growth from smallinocula on solid and in liquid synthetic media,and especially when they are cultivated on solidoleic acid-albumin media of acid reaction.The observation that all strains could be

cultivated in the absence of biotin when incu-bated in air with increased CO2 content suggeststhat the function of biotin in the metabolismand growth of tubercle bacilli is to promote theassimilation of CO2.That CO2 is essential for the growth of tubercle

bacilli was indicated by the studies of Wherry

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Figure 2. Growth on primary isolation from sputum of a strain of Mycobacterium tuberculosis on oleicacid-albumin medium at various hydrogen ion concentrations incubated under air (top row) and underair with 5 per cent CO2 (bottom row) at 37 C for 3 weeks.

and Erwin (1918) and conclusively proven byRockwell and Highberger (1926) and by Davies(1940). The CO2 content of atmospheric air isapparently sufficient for the growth of moststrains of tubercle bacilli on the classical egg

yolk media which contain variable amounts of"free" biotin. Growth on such media is furtherfavored by the customary enclosure of thesemedia in sealed tubes, which permits an ac-

cumulation of metabolically produced CO2. Incontrast, in the case of agar media poured in flatpetri dishes, CO2 escapes more easily, and it isremoved from the medium if the reaction of themedium is acid. The long incubation time neces-

sary for the growth of tubercle bacilli and theslow rate of production of CO2 by the bacteriathemselves prevent the amount of CO2 fromreaching "physiologic" concentrations. Biotin,

under these conditions, compensates for the lowCO2 content of the environment.The experiments reported here have shown

that the biotin Or' C02 requirement of tuberclebacilli on acid culture media is increased by thepresence of oleic acid. The mechanism by whicholeic acid exerts this effect is not clear. Thehypothesis that the ability of the medium toretain CO2 may be decreased in the presence ofoleic acid was tested in manometric experi-ments with the Waarburg apparatus7 and dis-proved. It is possible that under those conditionswhere CO2 is lost from the aqueous environment(acid reactions), oleic acid, which normallystimulates growth (Dubos and Davis, 1946;Schaefer, 1952), may be injurious to the bacterial

We are indebted to Mr. Charles Coleman forperforming these experiments.

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cells because the stimulation of the metabolicreactions produced by oleic acid is not accom-panied by an equally increased rate of CO2uptake. It is evident from the results of studiesdescribed here that fatty acid stimulation ofgrowth of these organisms is most strikinglyseenwhenthe environmental concentration of CO2is elevated above that found in atmospheric air.Experiments on the effect of increased CO,

tension on the inhibition of growth produced bythe biotin antagonist, 4-(imidazolidone-2) caproicacid, have lead the writers to conclude that themetabolic activity of small amounts of endoge-nous biotin is essential for the assimilation ofC02, even when C02 is supplied in high con-centration.The hypothesis that biotin functions as a

coenzyme of CO2 transfer was first proposed byBurk and Winsler (1943) and further supportedby Lardy, Potter, and Elvehjem (1947) whoshowed that the growth of Lacobacillus arabino-sus was stimulated by bicarbonate in a mediumwith high, but not in a medium with low, biotincontent. Proof of the role of biotin in the in-corporation or transformation of CO2 into anessential metabolite was given by Wessman andWerkman (1950) who showed that acetonedried preparations of Micrococcus lysodeikticuawere able to incorporate CO2 labeled with heavycarbon into oxalacetate and that this reactionwas inhibited by avidin, an agent known to bindand render biotin unavailable to bacteria.That factors other than biotin deficiency may

be responsible for a requirement for increasedCO2 tension is indicated by our observations ona strain which requires an increased CO2 tensionfor growth at 37 C even on media containing largeamounts of biotin. This strain, however, was ableto grow at atmospheric CO2 tension, and with-out added biotin, when incubated at 34 C. Asimilar observation has been reported by Borekand Waelsch (1951) for a strain of L. arabinosus.This strain required increased CO2 tension forgrowth at 37 C on a medium lacking phenyl-alanine and tyrosine, and for growth at 39 C ona medium lacking aspartic acid. The fact that therequirement for CO2 at the higher temperaturescould be replaced by specific amino acids wasinterpreted as evidence that the synthesis ofthese amino acids from CO2 was inhibited at thehigher temperatures. In the case of the tuberclebacillus no such replacement for the CO2 re-

quirement was found. The cause of this CO2requirement at higher temperature is unknown.

Soltys et al. (1952) reported that StanleyGriffith isolated a strain of tubercle bacilli whichcould grow in primary culture only in the presenceof added CO2. Soltys has also reported thatgrowth of tubercle bacilli may be enhanced byCO2. Our own observations stress the beneficialeffect of increased CO2 tension and of oleic acidmedium of acid reaction for the primary isolationof tubercle bacilli from their natural sources. Noevidence was obtained from our studies that therequirement of tubercle bacilli for biotin or in-creased CO2 tension is related to drug resistanceto streptomycin or isoniazid.

ACKNOWLEDGMENT

The authors wish to express their appreciationto Mr. William Jones for his technical assistancein these studies.

SUMMARY

Strains of Mycobacterium tuberculosis are de-scribed which required biotin for their growth onan oleic acid-albumin agar medium of acid re-action (pH 6.3-6.8). Incubation under increasedcarbon dioxide tension (1-5 per cent) abolishedthe requirement of these strains for biotin.Mutants which did not require biotin or in-

creased CO2 tension could be isolated from suchstrains.The inhibitory effect of moderate concentra-

tions of the biotin analogue, 4-(imidazolidone-2)caproic acid, on the growth of biotin-independentstrains was reversed not only by biotin but alsoby increased CO2 tension. However, in thepresence of high concentrations of the inhibitoronly biotin reversed the inhibition, suggestingthat small amounts of biotin are essential forCO2 "fixation".The presence of oleic acid in albumin agar

medium of acid reaction enhanced the require-ment for biotin or carbon dioxide. The majorityof strains obtained on primary culture frompathological materials manifested fastest growthon incubation under 1 to 5 per cent carbondioxide on an oleic acid-albumin agar medium ofpH 6.6.One strain required increased CO2 tension for

growth at 37 C but was able to grow at atmos-pheric CO2 tension, and without biotin, when

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incubated at 34 C. A COrindependent mutantwas readily isolated from this strain.The significance of these findings for the role of

increased C02 tension and biotin in the growth ofM. tuberculosis is discussed, and the advantageof incubation under increased CO2 tension and ofoleic acid-albumin medim of pH 6.6 for theprimary isolation of tubercle bacilli is pointed out.

REFERENCES -BOREK, E., AND W.Aisc, H. 1951 The effect of

temperature on the nutritional requirementsof microorganisms. J. Biol. Chem., 190,191-196.

Buix, D., AND WINZLER, R. F. 1943 Heat-labile, avidin-uncombinable species specificand other vitamers of biotin. Science, 97,57-60.

COHN, M. L., ODA, U., KOVITZ, C., AND MIDDLE-BROOK, G. 1954 Studies on isoniazid andtubercle bacilli. I. The isolation of isoniazid-resistant mutants in vitro. Am. Rev. Tuberc.,70, 465-475.

DAvrms, R. 1940 The effect of carbon dioxideon the growth of the tubercle bacillus. Brit.J. Exptl. Pathol., 21, 243-253.

DUBOs, R. F., AND DAvIs, B. D. 1946 Factorsaffecting the growth of tubercle bacilli inliquid media. J. Exptl. Med., 83, 409.

DuBos, R., AND MIDDLEBROOK, G. 1947 Mediafor tubercle bacilli. Am. Rev. Tubere., 16,334-35.

LANDY, M., AND DICKEN, D. M. 1941 Biotinsynthesis by microorganisms. Proc. Soc.Exptl. Biol. Med., 46, 449-452.

LARDY, H. A., POnmPE, R. L., AND ELvEHJM,C. A. 1947 The role of biotin in bicarbonate

utilization by bacteria. J. Biol. Chem., 169,451-452.

MIDDLEBROOK, G., COHN, M. L., AND SCHAEFER,W. B. 1954 Studies onisoniazidand tuberclebacilli. III. The isolation, drug-suscepti-bility, and catalase-testing of tubercle bacillifrom isoniazid-treated patients. Am. Rev.Tuberc., 70, 852-872.

PIERCE, C. H., DUBOs, R. J., AND SCHAEFER,W. B. 1953 Multiplication and survival oftubercle bacilli in the organs of mice. J.Exptl. Med., 97, 189-206.

POPE, H. 1952 Growth inhibition of tuberclebacilli by analogues of biotin. Am. Rev.Tuberc., 63, 39-45.

POPE, H., AND SMITH, D. T. 1946 Synthesis ofB-complex vitamins by tubercle bacilli whengrown on synthetic media. Am. Rev. Tuberc.,54,559-563.

ROCKWELL, G. E., AND HIGEERGIER, J. H. 1926Carbon dioxide as a factor in the growthof the tubercle bacillus and of other acidfast organisms. J. Infectious Diseases, 38,92-100.

SCHAEFER, W. B. 1952 Growth requirements ofdysgonic and eugonic strains of M. tuberculosisvar. bovis. J. Exptl. Med., 96, 207-219.

SOLTYS, M. A., HILL, C. A. ST., AND AUSELL, F.1952 Tubercle bacillus and laboratory methodsin tuberculosis. E. & S. Livingston Ltd.,Edinburgh & London, 25.

WESSMAN, G. E., AND WZRM AN, C. H. 1950Biotin in the assimilation of heavy carbonin oxalacetate. Arch. Biochem., 26, 214-218.

WHERY, W. B., AND ERWIN, D. M. 1918 Thenecesity of carbon dioxide for the growth ofB. tuberculosis. J. Infectious Diseases, 22,194-197.

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