JOURNAL OF BACTERIOLOGY, Dec., 1965Copyright © 1965 American Society for Microbiology

Vol. 90, No. 6Printed in U.S.A.

Differential Media for the Identification ofBacillus anthracis

RALPH F. KNISELYU.S. Army Biological Laboratories, Fort Detrick, Frederick, Maryland

Received for publication 21 August 1965

ABSTRACT

KNISELY, RALPH F. (U.S. Army Biological Laboratories, Fort Detrick, Frederick,Md.). Differential media for the identification of Bacillus anthracis. J. Bacteriol.90:1778-1783. 1965.-Two new differential media for the identification of Bacillus anthra-cis are described. Phenethyl alcohol and chloral hydrate were used as selective ingre-dients. These media should aid in the differentiation of B. anthracis from B. cereus and,if used in conjunction with other differential tests, they should also differentiate B.anthracis from the majority of other aerobic sporeforming bacilli. These media mightalso be useful in the classification of Bacillus species.

The differentiation of Bacillus anthracis fromother aerobic sporeformers, particularly B.cereus, is difficult to carry out in small labora-tories that do not possess animal facilities. Separ-ation of B. anthracis from B. cereus by serologicalagglutination tests was unsuccessful, as reportedby Lamanna and Eisler (1960) and Lamannaand Jones (1961). Leise et al. (1959) reported the"string-of-pearls" reaction of Jensen and Klee-meyer (1953) and sensitivity to y bacteriophageas sufficient to identify B. anthracis, with absenceof motility and hemolysis, along with a char-acteristic morphology on nutrient agar, as sup-porting evidence. Differentiation of B. anthracisand B. cereus by toxin production was reportedby Bonventre and Eckert (1963).The distinction between virulent and avirulent

B. anthracis and B. cereus is still a problem andsome authors have considered B. anthracis as apathogenic variety of B. cereus (Smith, Gordon,and Clark, 1952; Brown et al., 1958).Two new differential media, one containing

phenethyl alcohol (PEA) and the other chloralhydrate (CH), are described in this report. Thesemedia should aid in the differentiation of bothvirulent and avirulent strains of B. anthracisfrom B. cereus. These media should also differ-entiate B. anthracis from the majority of otheraerobic sporeforming bacilli, if used in conjunc-tion with other differential tests, such as hemo-lytic activity and motility, as advocated by Bur-don (1956) and Leise et al. (1959).

MATERIALS AND METHODS

Strains. The strains tested were: 21 B. anthracis,9 B. cereus, 5 B. cereus var. mycoides, and 20 mis-

cellaneous Bacillus species (Tables 1 to 3). All B.anthracis strains were lysed by y phage (Brownand Cherry, 1955).

Preparation of cells. Growth from 24-hr HeartInfusion Agar (HIA; Difco) slants was removedand suspended in 10 ml of 0.06 M phosphate buffer(pH 7.3). Triplicate plates of the test media wereinoculated with 0.1 ml of a dilution containing ap-proximately 5 X 102 organisms per milliliter andwere incubated at 37 C for 18 hr.PEA medium. PEA (2-Phenylethanol; Mathe-

son,Coleman and Bell, Cincinnati, Ohio) was addedto HIA in a final concentration of 0.3%.CH medium. A 10% solution of CH (USP) was

freshly prepared, seitz-filtered, and added to HIAin a final concentration of 0.25%.

Control medium. HIA was used as a 100% re-covery control.

Hemolysis. The hemolytic activity of the or-ganisms was determined by streaking cultures onHIA containing 2% fresh, defibrinated, washedsheep cells.

Reduction of methylene blue. The reduction ofmethylene blue was done according to Burdon(1956). A soft agar medium (pH 7.3) containing 1%methylene blue was inoculated by a single stabwith a straight needle. The cultures were incuba-ted at 37 C for 24 hr and observed for growth andfor reduction of the dye.

Litmus milk. The characteristics of growth inLitmus Milk (BBL) were observed and recorded.

Motility. Motility was determined by inoculat-ing tubes of Motility Test Medium (Difco) towhich 0.2 to 0.3% beef extract had been added. Thetubes were incubated at 37 C for 48 to 96 hr.

Sensitivity to penicillin. Tryptose Agar (Difco)slants were prepared to contain 10 units of penicil-lin G (The Upjohn Co., Kalamazoo, Mich.) per ml.The slants were inoculated by a single streak, in-

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IDENTIFICATION OF BACILLUS ANTHRACIS

TABLE 1. Reactions of Bacillus anthracis strainsa

Strain

D 14 (12); D 311 (Sax)...D 297 (15); Vollum B.....Avir Vaccine Str; 1014....HBA 199; HBA 273.......HBA 201.................Ohio; HBA 87; Willard;

D-379; D-380; D-382;D-384; D-390; D-417;VIB; Michigan Vollum;D-53 (NRS 722 orATCC 944)............

PEA CHmediumb mediumb

+000

0

00000

0

HemolysisMethylene

blueagar

±4

Litmus Motilitymilk medium

Penicillinagar

Bicar-bonateme-diumc

MMRMM

M

String-of-pearlstest

+

a Reactions for designated media other than PEA and CH: +, positive; i, partial or incomplete;and -, no reaction or growth.

bRecovery on PEA and CH media compared with HIA (as 100% recovery control) and recordedas follows: ++++, 100% or above; +++, 75 to 99%; ++, 50 to 74%; i, 1 to 24%; and 0, none.

c Colonial growth on bicarbonate agar (20% C02): M, mucoid; R, rough; and S, smooth.d Except D-379.

TABLE 2. Reactions of Bacillus cereus strains*

Methyl- Mo- Peni- Bicar-Strain PEA mediumt CH mediumt Hemo- ene Litmus tility cillin bonatelysis blue milk me-

agrme-

agar dium agr diumt

B. cereusV-4 (At 4509) .................... ++++ ++± ± + + + + RV-2 (At 7004); V-6 ............... ++++ ++++ + i + + + RATCC 7064...................... ++++ ++++ + + + - + RATCC 7483...................... ++ +++ + i + - + RATCC 9620...................... +++ ++++ + + + - + RV-7............................ +++ ++++ - + + - + RHBA 112......................... ++++ ++++ +4 + + - - RHBA 248......................... ++++ ++++ + + + - + R

B. cereus var. mycoidesATCC 6462...................... ++++ ++++ + ± + + + RATCC 6463...................... ++++ ++++ i + + - + RNRS 306......................... ++ ++++ + + + + + RNRS 327......................... ++++ ++++ - + + + + RATCC 10206..................... 0 0 + - _ - : R

* Reactions for designated media other than PEA and CH: ±, positive; 4, partial or incomplete; and-, no reaction or growth.

t Recovery on PEA and CH media compared with HIA (as 100% recovery control) and recordedas follows: ++++, 100% or above; +++, 75 to 99%; ++, 50 to 74%; +, 25 to 49%c; i, 1 to 24%,; and0, nonie.

I Colonial growth on bicarbonate agar (20% C02): R, rough.

cubated at 37 C, and observed for growth duringa 24- to 48-hr period.

String-of-pearls test. (Jensen and Kleemeyer,1953). The growth from 18-hr Tryptose Agar slantswas heavily streaked on Tryptose Agar plates con-taiising 0.05 and 0.5 unit of penicillin G per ml, andwas incubated at 37 C for 3 to 6 hr according toJensen and Kleemeyer (1955). The plates wereexamined by placing a cover slip over the inocu-lated area, and were observed under the high, dryobjective for the presence of round, cellular forms

characteristic of the string-of-pearls reaction.Negative plates were held overnight at room tem-perature and re-examined.

Growth on bicarbonate medium under C02. Nu-trient agar plates (Difco) coistaining 5% sodiumbicarbonate were prepared and heavily iisoculatedby streaking according to Thorne, Gomez, andHousewright (1952) and Chu (1952). The plateswere incubated for 24 to 48 hr at 37 C in a 20% CO,atmosphere and were examined for rough or mu-coid colonies.

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J. BACTERIOL.

FIG. 1. Recovery of a virutilent strain of Bacilluisanthracis (Ohio) (top) and an avirulent vaccinestr-ain (bottom) on (1) phenethyl alcohol agar, (2)chloral hydrate agar, and (3) Heatt Infusion Agarcontrol after 18 hr of incuibation at 37 C.

RESULTSRecovery of B. anthracis strains on differential

miiedia. The 21 B. anthracis strains (Table 1)were generally inhibited on the PEA and CHmedia after 18 hr of incubation at 37 C. Therewere two strains (HBA 112 and HBA 248) thathad been originally received as B. anthracis.These two strains have been reclassified as B.cereus and are listed in Table 2.

FIG. 2. Recoveery of Bacilluis cereus strains on (1)phenethyl alcohol agar, (2) chloral hydrate agar, an(d(3) Heart Infusion Agar control after 24 hr of incut-bation at 37 C. The colonies were easily visible at18 hi, buit the plates were permitted the additionalincuibation time so the colonies could be readily seenin the photograph.

The reactions of the HB3A 112 strain (Table2), insensitivity to -y phage and positive stainingwith both B. anthracis and B. cereus fluorescentantisera, are the bases for reclassification. Simailarreactions for the HBA 248 strain, which did notstain with B. anthracis fluorescent antisera, alsojustify reclassification.

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IDENTIFICATION OF BACILLUS ANTHRACIS

TABLE 3. Reactions of miscellaneous Bacillus species*

Methyl- Mo- Peni Bicar-StrainPEAmediumtCHemo- ene Litmus tility cill bonateStrain PEA mediamt CH mediumt lysis blue milk me- cillin me-

agar dium agar dium++

B. circulansATCC 9966...................... ++++ ++++ i + - - + R

B. megateriumV-9 ........................... ++++ ++++ + + + - + RATCC9885...................... 0 0 + MATCC 11561..................... 0 ++ --+ MATCC6458...................... 0 +++ + + - - + RATCC11561-E................... 0 +++ + + + - + MATCC8245...................... + ++++ + ± + + _R

B. polymyxaATCC 10401......... 0 0 - + + + - S

B. sphaerticusX-2 0 0 - _ - + - SATCC245....................... +++ 0 - + - _ - RATCC4525...................... 0 0 - 4 - + - SATCC7054...................... 0 0 - + - + - S

B. subtilisATCC9466 .............. +++ 0 - _ _ _ -ATCC9524...................... ++++ 0 - ___ - RATCC 9860.......... +++ ++++ + + _ - + RiATCC8480.......... ++ ++++ + - _ - + RATCC6051 ......++++ 0 - + -_ - SATCC943.......... ++++ 0 - i - - + S

B. thuringiensisNRS 996.......... ++++ ++++ i + + - + RNRS 1328..........+ 0 + + + + + R

* Reactions for designated media other than PEA and CH: +, positive; +, partial or incomplete; and-, no reaction or growth.

t Recovery on PEA and CH media compared with HIA (as 100% recovery control) and recorded asfollows: ++++, 100% or above; +++, 75 to 99%; ++, 50 to 74%/o; + 25 to 49%7; i, 1 to 24%7; and 0,none.

I Colonial growth oIn bicarbonate agar (20% CO2): M, mucoid; R, rough; and S, smooth.

TABLE 4. Typical reactions of gram-positive aerobic sporeforming bacilli*

Characteristic B. anthracis B. cereus, B. cereus B. circulans, B. megaterium,var. mtycoides B. subtilis, B. thuringiensis

Recovery on 0.3%O PEA agar in18 hr. 0 to + ++ to ++++ Usually ++ to

++++ on one orboth media

Recovery oI 0.25% CH agar in18 hr. None +++ to ++++

Hemolysis on sheep blood-agarplates in 24 hr................ None or slight Marked Usually marked

Motility medium............... Nonmotile Variable Usually nonmotileGrowth on penicillin agar (10

units/ml) ................ Nonie or slight Good Usually goodColony type on bicarbonatemedia under 20%/,o C02........ Virulent-mucoid; Rough Rough (some strains

avirulent-rough of B. megateriumappear mucoid)

* Symbols: 0, none; +, 25 to 49%; ++, 50 to 74%;+ +++, 100% or above.

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J. BACTERIOL.

Figure 1 shows the typical inhibition of a viru-lent and an avirulent strain of B. anthracis onPEA and CH media.

Recovery of B. cereus strains on differentialmiedia. The recovery of 14 B. cereus strains (Table2) was very good on PEA and CH media, withthe exception of strain ATCC 10206. This strain,however, was readily distinguished by its mor-phology on HIA and marked hemolytic activityon blood-agar. The reactions of B. cereus strainsobtained with most of the other differentialmedia were quite variable and were not signifi-cant unless considered as a groul).

Figure 2 shows the typical recovery of two B.cereus strains on PEA and CH media after 24hr of incubation at 37 C. The colonies were readilyvisible after 18 hr. The colonies on the CH me-dium-i weree usually smaller than on the PEA me-dium, but recovery was often greater than on theHIA control.

Recovery of miiscellaneous Bacilluis species ondifferential media. The recovery of the remainingBacillus species (Table 3) followed a variablelattern, but those strains that could have beenconfused with B. anthracis were recovered oneither the l'EA or CH medium. One strain of B.mecgateriumt (ATCC 9885) that was inhibited onboth PEA and CH media showed smooth colonialgrowth on HIA and would not have beeni mis-taken for B. ant1hracis.

B. polymttyxa and B. sphaericus could be readilydistinguished fromii B. anthracis by colony mor-phology and motility. The results on differentialmedia were recorded for comlparison.The recovery of the B. anthracis strains was

never larger than 38% on PEA medium, and norecovery was obtained on CH medium after 18hr. GErowth of B. cereuis and B. cereus var. mtiy-coides was never less than 90% on at least oneof these media. Quite often the recovery of thelatter strains exceeded the recoverv on the HIAcontrol.

Typical results with gram-positive bacilli aresummarized in Table 4. The first four tests shouldbe adequate for l)reliminary identification, andthe others arc useful confirmatory tests.

DIscussIoNThe selective antibacterial action of PEA was

reported by Lilley and Blrewer (1953). Themarked inhibitory effect of PEA on gram-nega-tive bacteria, compared with a slight effect ongram-positive organisms, was shown. They found0.25% PEA as the most effective concentrationfor this purpose.The inhibition of gram-negative bacteria by

PE,A resulting from the selective and reversiblenihibition of the synthesis of deoxyribonucleic

acid was reported by Berrah and Konetzka(1962). Inhibition of B. cereus occurred at a 0.35%concentration of PEA, but not at 0.25%. Or-ganisms such as B. megaterium and B. subtilisgrew in the presence of 0.35% PEA.

Slepecky (1963) reported the inhibition ofsporulation and germination of B. megateriumby PEA. He reported the inhibition of sporula-tion with 0.25% PEA as well as marked growthinhibition at 0.40 %. Complete inhibition ofgrowth occurred at 0.50% PEA and completeinhibition of germination at 0.05%.

Preliminary results in this laboratory showedthat germination of B. anthracis spores wasinhibited on both PEA and CH media, whereasgermination of B. cereus spores was inhibited onPEA medium only.The two strains (HBA 112 and HBA 248)

that did not show typical growth on PEA orCH medium were originaly received as B. an-thracis strains many years ago. It is not knownwhether the original cultures were misclassified,or whether the lpresent strains are contaminantsof the original cultures. Strain HRBA 248 definitelyhas the characteristics of B. cereas; those of HBA112, although not as specific, also indicate that itshould be classified as B. cereus.The experimental data show that a tentative

identification of B. anthracis can be establishedby certain test criteria. These criteria include(i) the presence of nonhemolytic gram-positivebacilli, (ii) absence of motility, (iii) typicalcolony morphology on control plates, and (iv)marked inhibition of growth on PEA and CHmedia during an 18-hr growth period at, 37 C.Valuable confirmatory tests include lysis with-y phage, inhibition of growth on penicillin agar,and a positive string-of-pearls reaction.The PEA and CH imiedia served as imiportant

differential tests and could be useful in the classi-fication of Bacilluis species.

ACKNOWLEDGMENTSI wish to thanik Franicis J. Weirether for supply-

inig some of the B. anthracis strainis anid bacterio-phages, Donald T. Disquie for performinig thefluiorescenit-antibody tests, Chester L. Redmoind,Jr., for his techniical assistanice, anid the GraphicArts Branch, Fort Detrick, for the photographs.

LITERIATURE CITEDBERRAH, G., AND W. A. KONETZKA. 1962. Selective

anid reversible inhibition of the synthesis ofbacterial deoxyribontucleic acid by phenethylalcohol. J. Bacteriol. 83:738-744.

BONVENTRE, P. F., AND N. J. ECKERT. 1963. ToxiInproduction as a criterion for differentiating Ba-cilluis cereus and Bacilluls anthracis. J. Bacteriol.85:490-491.

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VOL. 90, 1965 IDENTIFICATION OF B

BROWN, E. R., AND W. B. CHERRY. 1955. Specificidentification of Bacillus anthracis by means ofa variant bacteriophage. J. Infect. Diseases96 :34-39.

BROWN, E. R., M. D. MOODY, E. L. TREECE, ANDC. W. SMITH. 1958. Differential diagnosis ofBacillus cereus, Bacillus anthracis, and Bacilluscereus var. mycoides. J. Bacteriol. 75:499-509.

BURDON, K. L. 1956. Useful criteria for the iden-tification of Bacillus anthiacis and related spe-cies. J. Bacteriol. 71:25-42.

CHU, H. P. 1952. Variation of Bacillus anthraciswith special reference to the non-capsulatedavirulent variant. J. Hyg. 50:433-444.

JENSEN, J., AND H. KLEEMEYER. 1953. Die Bak-teriell Differentialdiagnose des Anthrax mittelseines neuen spezifischen Tests (Perischurtest).Zentr. Bakteriol. Parasitenk. Abt. I Orig. 159:494-500.

LAMANNA, C., AND D. EISLER. 1960. Comparativestudy of the agglutinogens of the endospores ofBacillus anthracis and Bacillus cereus. J. Bac-teriol. 79:435-441.

ACILLUS ANTHRACIS 1783

LAMANNA, C., AND L. JONES. 1961. Antigeniic rela-tionship of the endospores of Bacillus cereus-likeinsect pathogens to Bacillus cereus and Bacilliusanthracis. J. Bacteriol. 81:622-625.

LEISE, J. M., C. H. CARTER, H. FRIEDLANDERt,AND S. W. FREED. 1959. Criteria for the idenitifi-cation of Bacillus anthracis. J. Bacteriol. 77:655-660.

LILLEY, B. D., AND J. H. BREWER. 1953. The selec-tive antibacterial action of phenylethyl alcohol.J. Am. Pharm. Assoc. 62:6-8.

SLEPECKY, R. A. 1963. Inhibition of sporulatioiiand germination of Bacillus megaterium byphenethyl alcohol. Biochem. Biophys. Res.Commun. 12:369-373.

SMITH, H. R., IR. E. GORDON, AND F. E. CLARK.1952. Aerobic sporeforming bacteria. U.S. Dept.Agr. Monograph No. 16.

THORNE, C. B., C. A. GOMEZ, AND R. D. HOUSE-WRIGHT. 1952. Synithesis of glutamic acid andglutamyl polypeptide by Bacillus anthracis. II.The effect of carbon dioxide on peptide produc-tion on solid media. J. Bacteriol. 63:363-368.

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