APPLIED MICROUOLOGY, Jan. 1970, p. 138-145Copyright @ 1970 American Society for Microbiology

Vol. 19, No. 1Printed in U.S.A.

Differentiation of Clostridium botulinum Types A, B,and E by Pyrolysis-Gas-Liquid Chromatography1

R. D. CONE AND R. V. LECHOWICH

Department of Food Science, Michigan State University, East Lansing, Michigan 48823

Received for publication 18 September 1969

Vegetative cells and spores of 10 strains of Closiridium botulinum representingtypes A, B, and E were grown in Trypticase-peptone-sucrose-yeast extract (TPSY)medium. Five type E strains were also grown in Multipeptone-sucrose-Nutraminoacids (MSN) medium. Lyophilized samples were subjected to pyrolysis-gas-liquidchromatography (PGLC) analysis, and the resulting pyrograms were examined forvariations in elution patterns between spores and vegetative cells of types A, B,and E grown in the TPSY medium and spores and vegetative cells of type E grownin the TPSY medium and spores and vegetative cells of type E grown in TPSY andMSN media. Growth and toxin production of all 10 strains of C. botulinum were in-vestigated by using a modified dialysis sac culture technique. The dialysate super-natant fluid (DSF) obtained after centrifugation of the 5-day-old cultures from thedialysate was also subjected to PGLC analysis. Control samples consisting of (i)noninoculated DSF, (ii) noninoculated DSF plus partially purified toxin, and (iii)1.0 mg of partially purified toxin were also analyzed by PGLC. Differences betweenpyrograms of cultures were suitable for positive identification at the type level but notat the strain level. Pyrograms permitting differentiation were also obtained betweenspores and vegetative cells as well as between the same cultures grown in differentmedia. The dialysis sac technique was useful in detecting growth but not toxin pro-duction of C. botulinum.

The increase in type E botulism outbreaksthroughout the world (2, 3) and in the UnitedStates (1) during the last decade has led to asubstantial increase in studies related to themicroorganism Clostridium botulinum. The studyof the food-borne disease of botulism usuallyinvolves (i) demonstration that the filtrate of asuspected food sample contains type-specifictoxin by use of animal neutralization tests, (ii)similar detection of type-specific toxin in apatient's blood sample, and (iii) cultural proce-dures to isolate and identify the causative micro-organism. Animal toxin assay procedures havedefinite time, cost, and technique limitations.The recently described detection methods thatmay include selective growth on egg yolk medium,fluorescent-antibody staining, polyacrylamidegel electrophoresis, immunofluorescence, electronmicroscopy, and immunological techniques ap-pear promising only as supplementary detectionmethods since their reliability has not beenadequately established.

I Michigan State Agricultural Experiment Station Journal ar-ticle no. 4857.

One of the most promising techniques for theidentification of bacteria appears to be pyrolysis-gas-liquid chromatography (PGLC). Since severalworkers have reported the successful use of thistechnique for rapid identification of other generaand species of bacteria (5-8), it seemed feasiblethat it might also be used for detection andidentification of cultures of C. botulinum. Thismethod would not only detect materials thatwould provide adequate identification of cultures,but it might also decrease the minimum timeof 5 days required for detection and identificationby conventional methods. Preparation of thesample for analysis by growth and lyophilizationis also relatively simple compared to some othermethods.The purpose of this study was to investigate

the uses of PGLC for the detection of growthand toxin production of selected serotypes ofpure cultures of C. botulinum in laboratory media.No food systems were used in these preliminaryefforts. The effects of the growth medium andof per cent sporulation on the resulting elutionpatterns after pyrolysis were also studied.

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VOL. 19, 1970 DIFFERENTIATION OF C. BOTULINUM TYPES A, B, AND E

TABLE 1. Sources of various strains of Clostridiumbotulinum types A, B, and E used in pyrolysis-gas-

liquid chromatography experiments

Type Strain Source

A 62 C. F. Schmidt, ContinentalCan Co., Chicago, Ill.

A 78 C. F. Schmidt, ContinentalCan Co., Chicago, Ill.

B 169 C. F. Schmidt, ContinentalCan Co., Chicago, Ill.

B 213 C. F. Schmidt, ContinentalCan Co., Chicago, Ill.

E Vancouver Her- C. F. Schmidt, Continentalring (VH) Can Co., Chicago, Ill.

E Kalamazoo R. W. Johnston, Food and(Kal) Drug Administration,

Detroit, Mich.E Seattle Forks J. T. Graikoski, Bureau of

(SF) Commercial Fisheries,Ann Arbor, Mich.

E 517 Donald A. Kautter, Foodand Drug Administra-tion, Washington, D.C.

E A6247 R. W. Johnston, Food andDrug Administration,Detroit, Mich.

E 066B nontoxic Haim M. Solomon, Food(NT) and Drug Administra-

tion, Washington, D.C.

MATERIAIS AND METHODSMicroorganisms. The microorganisms studied in

this experiment included two strains of C. botulinumtype A, two proteolytic strains of C. botulinwn typeB, and six strains of C. botulinwn type E (five toxicstrains and one nontoxic variant). The strain numbersand the source of each strain are shown in Table 1.

Culture media. Spores and cells of all 10 strains ofC. botulinwn investigated were grown in TPSY me-dium consisting of 5.0% Trypticase (BBL), 0.5%peptone (Difco), 0.2% sucrose, 1.0% yeast extract(Difco), and 0.1% sodium thioglycolate adjustedto pH 7.2. In addition, five type E strains were grownin MSN medium consisting of 2.5% Multipeptone(Fisher Scientific Co., Pittsburgh, Pa.), 0.2% sucrose,1.0% Nutramino acids (Fisher Scientific Co.), and0.2% sodium thioglycolate. One-liter Erlenmeyerflasks containing 1 liter of the growth medium (TPSYor MSN) were inoculated with 15 ml of a 16- to 18-hractively growing subculture, prepared in the samemedium for each of the C. botulinum strains, andincubated at 32 C. Duplicate flask cultures wereprepared for each strain.

Toxin production and growth were studied bygrowing each of the 10 strains of C. botulinum in1-liter flasks filled with physiological saline solution(0.85%) into which a dialysis sac filled with 250 mlof TPSY medium was suspended. The ratio of themedium to saline solution was approximately 1:4

(v/v). Carry-over of materials from the inoculationmedium to the saline solution was prevented bycentrifuging the inoculum (15 ml of 16- to 18-hractively growing culture) for 10 min at 1,000 X gby using a Sorvall RC-2 refrigerated centrifuge. Theculture was then resuspended by using 5 ml of steriledistilled water and inoculated into the saline solution.The flasks were incubated at 32C, and duplicateflask cultures were prepared for each strain.

Harvesting vegetative cells and spores. Culturesincubated at 32 C for 8 to 10 hr in TPSY and MSNmedia to obtain vegetative cells were immediatelycooled to 4 C to prevent sporulation, dispensed into250-ml centrifuge bottles, and centrifuged for 15 minat 5,000 X g with the centrifuge cooled to 4 C. Thecells were then washed by centrifugation as aboveand resuspended in 200 ml of sterile distilled wateruntil they appeared clean upon microscopic examina-tion (required five to eight washings). The cells weresuspended in 100 ml of sterile distilled water andstored in sterile glass bottles at 2 C after the finalwashing.

Spore crops of C. botulinwn type E were grown inboth media and harvested after 24 to 30 hr of incuba-tion when spores were free from their sporangia.Longer incubation (30 to 36 hr) was necessary toobtain adequate sporulation of type A and B strains.It was then necessary to treat these latter cultureswith lysozyme (0.75 mg of lysozyme per ml of 0.1 NKCI) for 4 to 6 hr at 32 C to obtain free spores. Allwashing and storage procedures of vegetative cellsand spores were the same.

Microorganisms grown by use of the dialysis sactechnique were separated from the dialysate fluid bycentrifugation at 5,000 X g, and the dialysate super-natant fluid (DSF) was retained for subsequentanalysis. The DSF was filtered through a membranefilter (0.80 Am pore size) to remove any microorgan-isms not removed by centrifugation and stored insterile glass bottles at 2 C.

Direct microscopic counts. Direct microscopiccounts were made on all culture samples by using aPetroff-Hausser counting chamber. A 1-ml amountof each culture was added to either 3 or 7 ml of 30%glycerol solution, depending on the concentrationof the microorganisms. The direct counts gave anindication of the total count as well as the per centsporulation. Refractile bodies were considered to bespores.

Toxin assays. Swiss Webster white mice weighing15 to 20 g were injected intraperitoneally with 0.1, 0.2,and 0.5 ml of serial dilutions of each DSF sample.Before injection, the type E samples were digested for60 min at 37 C with 1% Trypsin (Difco, 1:250) in anequal volume of 0.05 M sodium phosphate buffer(pH 6.0), and dilutions of all toxins were made in0.05 M sodium phosphate buffer. The highest dilutionat which injected mice died was used to calculate theminimal lethal dose (MLD).

Controls. Control samples consisted of (i) non-inoculated DSF; (ii) noninoculated DSF plus partiallypurified toxin of the VH strain of C. botulinwn typeE, obtained from Alexander Emodi, Department of

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CONE AND LECHOWICH

Food Science, Michigan State University, to give afinal toxin titer of 106 MLD per ml; and (iii) a 1.0-mgsample of the partially purified toxin (5.0 X 106 MLDper mg of N).

Lyophiliation of samples. All cultures as well asthe DSF and control samples were lyophilized byusing a VirTis Freeze Mobile. Approximately 5 mlof each culture or sample was added to a 10-ml freeze-drying ampoule. The contents were then shell-frozenby using a dry ice and alcohol bath and lyophilizedfor 8 to 10 hr under a vacuum of 0.25 mm of Hg.PGLC analysis. A Hewlett-Packard pyrolysis unit

(model 80; F and M Scientific Division, Avondale,Pa.) was used to pyrolyze the samples. This instru-ment utilized an automatic pyrolysis cycle in whichthere was a 60-sec delay after activation of the pyroly-sis switch, allowing the gas chromatograph to stabilize.After the delay period, the jaw-style probe elementwhich contained the lyophilized sample was energizedfor 12 sec to bring about complete pyrolysis at 1,200 C.The probe was fitted directly to the injection port ofa Hewlett-Packard gas chromatograph (model 5750 B;F andM Scientific Division) by using a special adapterwhich allowed the pyrolysis products to be sweptdirectly into the columns by the carrier gas afterpyrolysis. The gas chromatograph was equipped withdual-flame ionization detectors and a Moseley 7128 Adual pen strip chart recorder. The operating param-eters of the gas chromatograph were as follows:columns, 72 by Y6 inch (183 by 0.48 cm) coppertubing; sample size, 0.9 to 1.1 mg for spores and cellsand 1.3 to 1.7 mg for DSF and control samples;coating, 15% high temperature-stabilized ethyleneglycol adipate (Analabs, Hamden, Conn.); support,90 to 100 mesh Anakrom ABS (Analabs); columnconditioning, 240 C for 2 weeks; carrier gas, heliumat a pressure of 60 psig (column A, 50 nl/min flowrate; column B, 67 il/min flow rate); hydrogen, 12psig; air, 33 psig; temperatures, 1,200 C for 12 secfor pyrolysis; 250 C for detectors; 230 C for injectionport; column programmed at 10 C per min from 50to 240 C; upper limit interval, variable; chart speed,0.25 inch (0.64 cm) per min; sensitivity and attenua-tion, 102 X 8 for spores and cells, variable for DSFand control samples.

RESULTS AND DISCUSSIONPyrograms of lyophilized cultures were inter-

preted by visual examination. Retention times,the presence or absence of peaks, and the ratio ofpeak heights with relationship to each other wereparticularly useful. No quantitation or identifica-tion of peaks was attempted in these preliminarystudies.

Duplicate samples were grown for each culture,and two or more replicates of each sample wereexamined by PGLC. For purposes of simplifyingthe material presented in this paper, only repre-sentative pyrograms of each sample are shown,since results were consistent between both dupli-cate cultures and replicate pyrolysis determina-tions.

Considerable qualitative and quantitativedifferences were observed in pyrograms obtainedfrom the various types and strains of C. botulinum.The quantitative differences appeared to becaused, in part, by an inability to accuratelycontrol the amount of sample being pyrolyzed,since the jaw-style probe limited the accuracy inweighing the sample size to the nearest 0.1 mg.The consistency of packing the sample into theprobe also appeared to influence the pyrogram,since the pyrolysis products of samples packedtoo firmly were not swept instantaneously intothe column after pyrolysis and those packed tooloosely were often blown off the probe by thecarrier gas. These variations in sample size canclearly be seen in Fig. 1. The pyrograms of 169Band 213B are essentially the same; however, moresample was pyrolyzed with 213B. The differencesmight also be caused by quantitative differencesin the two strains, but this is impossible to predictwith the type of probe that was employed in thisexperiment. Very accurate measurements of thesample pyrolyzed must be obtained beforequantitative differences such as these can beconsidered.Levy (4) also indicated the importance of the

effect of sample size upon pyrolysis products.He observed that secondary reactions betweenradicals and fragments formed upon rupture ofprimary bonds during pyrolysis are affected bythe quantity of material pyrolyzed. He alsoindicated that the rate of decomposition of thematerial pyrolyzed is dependent on the filmthickness of the sample in relationship to thegeometry of the source of heat. Ehrler and Frijouf(digest of a paper presented at the PittsburghConf. of Anal. Chem. and Appl. Spectroscopy,Cleveland, Ohio, 1968) have further indicatedthat sample size as well as final temperature, rateof temperature rise, and residence time in thereactor greatly influence pyrolysis elution pat-terns. The final temperature and rate of tempera-ture rise were readily controlled by the pyrolysisapparatus used in this study; however, residencetime was influenced by sample packing andsample size was difficult to accurately control.Pyrograms obtained upon PGLC analysis of

vegetative cells of four strains of C. botulinumtypes A and B are shown in Fig. 1. Examinationof these elution patterns revealed distinct differ-ences in peaks e, h, o, and r. Strain 62A wasdistinguishable from strain 78A and strains oftype B by the large peak o. The type B strainswere not readily differentiated from each other;however, the larger quantity of material elutedin peaks e, h, and r allowed them to be differ-entiated from type A strains. All other variations,including those in peak d, failed to be consistent

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VOL. 19, 1970 DIFFERENTIATION OF C. BOTULINUM TYPES A, B, AND E

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RETENTION TIME (MINUTES)FIG. 1. Pyrogramsfrorn vegetative cells offour strains of Clostridium botulinum types A and B grown in TPSY

medium. The capital letters denote the type and Att is the attenuation.

enough among duplicate samples as well asamong replicates of the same sample to be usedfor identification purposes.A representative pyrogram of vegetative cells

of C. botulinum type E grown in TPSY mediumis shown in part F of Fig. 2. Only quantitativevariations were noted among the six strains oftype E examined, and these differences appearedto be caused by variability in final populationachieved in each culture. Those cultures givinghigher direct microscopic counts (Table 2) pro-duced larger concentrations of material in severalelution peaks. No differences among type Estrains were sufficiently consistent for identifica-tion purposes. Parts D and F of Fig. 2, however,indicated the ease with which the vegetative cellsof type A could be differentiated from those oftype E. Considerable differences were noted inpeaks a through e, whereas peaks i and n weremuch larger in type A than in type E and anadditional peak was present between peaks r ands in type E. Since type B was somewhat like typeA, similar differences existed between types Band E. Thus, it was possible to differentiate

between vegetative cells of C. botulinum at thetype level; however, only the variation in strain62 of type A was noted at the strain level.

Elution patterns obtained upon PGLC analysisof spores of types A and B are shown in Fig. 3.No consistent differences suitable for identifica-tion purposes occurred at the strain level for typeA and B spores. Strain 78A did appear to yieldpyrograms with larger quantities of materialeluted in peaks g, i, and s. Sporulation of thisculture (Table 3), however, was much betterthan that of the other type A and B strains ex-amined, and these differences appeared to berelated to sporulation. Differences in peaks wand z also failed to be consistent. Differentiationbetween spores of type A and type B was thusnot possible. Spores of type E cultures (part Eof Fig. 2) also failed to show any differences inpyrograms at the strain level suitable for identifi-cation purposes. Comparison of part E withpart C of Fig. 2, which shows the pyrogram for arepresentative strain of type A spores, revealeddistinct differences in elution patterns of type Aand thus type B from type E. Differences which

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FIO. 2. Comparison of pyrograms from Clostridium botulinum spores (A) and vegetative cells (B) of type Egrown in MSN, medium; spores (C) and vegetative cells (D) of type A grown in TPSY medium; and spores (E)and vegetative cells (F) of type £ grown in TPSY medium.

were always consistent and distinct occurred inpeaks g, h, i, n, r, s, x, and z.No studies have previously been undertaken

to differentiate strains and types of cultures bypyrolysis of their spores. Differences in elutionpatterns, however, were expected since thesespores were formed from the same vegetativecells in which differences were observed. Gen-erally, the same type of difference was notedbetween spores of C. botulinum as between vegeta-tive cells. Identification was possible at the typelevel and only occasionally at the strain level.

Spores and vegetative cells of C. botulinumtype E grown in MSN medium also failed toprovide pyrograms suitable for identification atthe strain level. However, comparison of type Espore and vegetative cell pyrograms grown inMSN medium (parts A and B of Fig. 2) withthose of type E grown in TPSY medium (Fig.2E and F) revealed appreciable differencesamong elution patterns of the same culturesgrown in the two different media. Differencesin pyrograms of vegetative cells occurred in peaksc, k, m, n, q, s, and z. Peak i was also absent inMSN pyrograms, whereas there was a largeradditional peak between peaks r and s in the

TPSY pyrograms. Major differences in sporepyrograms occurred in peaks a, e, g, n, r, w, andz. Peak x was absent in spores grown in TPSY,and peak z was much larger in spores from TPSY.Oyama and Carle (5) also indicated that the

growth medium had an influence on the pyro-grams, which they obtained when they grewCandida pulcherrima in malt extract and inTrypticase soy broth. They further observed thatorganisms grown in similar media give similarpyrograms.The two media used in this experiment showed

definite similarities as well as differences in pyro-grams for the same culture. Since both mediaare rich in nitrogen, this miglht account for thesimilarities. The differences were probably causedby the presence of yeast extract as well as varyingconcentrations of other nitrogen-rich materials.A comparison of spores and vegetative cells of

C. botulinum types A and E is provided in Fig. 2.Consistent differences in peaks g, n, and z werecommon among all types studied. Type A (and B,similar to A) showed additional differences inpeaks a through d and peaks w, x, and y. Thesedifferences between spores and cells were ex-pected since they have been shown to be physi-

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DIFFERENTIATION OF C. BOTULINUM TYPES A, B, AND E

TABLE 2. Direct microscopic counts of vegetativecells of Clostridium botulinum types A, B, and E

grown in two different media

Total count (organisms/ml)Type Strain

MSN TPSY

A 62 8.3 X 108A 78 7.3 X 108B 169 5.9 X 108B 213 1.6 X 108E VH 2.4 X 108 5.0 X 108E KAL 1.2 X 108 7.5 X 108E SF 108 12.0 X 108E 517 1.1 X 108 15.0X 108E A6247 0.6 X 108 7.5 X 108E 066BNT 5.5 X 108

ologically different. The presence of such mate-rials as calcium and dipicolinic acid in spores andlysis of the sporangia and formation of new coatlayers in spores made them completely differententities than the vegetative cells from which theywere formed. In addition to sporulation, the ageof the culture may also have an influence on the

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pyrogram obtained. The cultures undergo lysis asthey grow older and many of the cell componentsmay be released. This is particularly true for thosecells that do not sporulate. The data collectedin this experiment could not resolve the differencesin pyrograms as being due to different sporulationpercentages or age of the culture, since spores andcells of each strain were necessarily incubated forvarious times to obtain each cell type and variousnumbers of vegetative cells were present insporulated cultures (Table 3).An attempt was made to detect growth and

toxin production of C. botulinum by using amodification of the dialysis sac techniquedescribed by Vinet and Fredette (9). Since mostof the toxin should have been present in the DSFafter growth and centrifugation of the cultures,it was proposed that this toxin might be detectableby PGLC. The dialysis sac technique was pri-marily used to limit and control the availabilityof nutrients to the cultures, thus preventingpossible interference of these compounds withthe pyrograms subsequently obtained. Because ofthe limited availability of nutrients to the culturesgrown in the dialyzed medium, growth and

RETENTION TIME (MINUTES)

FIG. 3. Pyrograms from spores offour strains of Clostridium botulinum types A and B grown in TPSY medium.The capital letters denote the type and Att is the attenuation.

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CONE AND LECHOWICH APPL. MICROBIOL.

TABLE 3. Direct microscopic counts and per cent sporulation of cultures of Clostridiumbotulinum types A, B, and E grown in two different media

MSN TPSY

Type StrainTotal count Sporulation Total count Sporulation

(organisms/ml) (%) (organisms/ml) (%)

A 62 12.0 X 108 54.8A 78 9.5 X 108 94.8B 169 9.0 X 108 65.6B 213 9.5 X 108 85.4E VH 3.8 X 108 76.3 5.2 X 108 93.5E KAL 2.2 X 108 83.2 5.8 X 108 91.3E SF 4.3 X 108 98.6 5.5 X 108 89.4E 517 2.0 X 108 91.3 2.4 X 108 84.3E A6247 3.3 X 108 72.5 5.8 X 108 75.6E 066BNT 4.9 X 108 78.4

C

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ij

RETENTION TIME (MINUTES)

FIG. 4. Comparison ofpyrograms for samples consisting of (A) uninoculated dialysate supernatant fluid (DSF);(B) uninoculated DSF plus partially purified toxin; (C) 1.0 mg of partially purified toxin; (D) DSF of type EClostridium botulinum; (E) DSF oftype A C. botulinum; and (F) DSF oftype B C. botulinum.

sporulation was not as good as in cultures grownin normal TPSY medium. Toxin titers, however,were of the order 103 to 106 MLD per ml.

Representative pyrograms of the DSF of typesE, A, and B of C. botulinum investigated are

shown in parts D, E, and F of Fig. 4. Pyrogramswere essentially the same among strains of eachtype, but differences were noted at the type level.Types A and B were distinguishable on the basisof peaks f, j, o, and p, whereas pyrograms of type

144

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VOL. 19, 1970 DIFFERENTIATION OF C. BOTULINUM TYPES A, B, AND E

E were entirely different from those of type A ortype B after peak f. These differences wereprobably caused by physiological differences inthe three types. Since type A and type B wereproteolytic, they assimilated and dissimilateddifferent compounds than type E which wasnonproteolytic and primarily saccharolytic. TypesA and B are also markedly less saccharolytic thantype E.

Parts A, B, and C of Fig. 4 illustrate pyro-grams of control samples which consisted of (i)noninoculated DSF, (ii) noninoculated DSFplus partially purified toxin, and (iii) 1.0 mg ofpartially purified toxin, respectively.Comparison of the pyrograms obtained by

PGLC of the control samples with those of theDSF from the cultures provided rather interestingresults. Toxin production was not detectable,since practically all of the toxin was eluted insmall peaks during the first 15 min (part C, Fig.4). The toxin peaks appeared to be hidden bypeaks produced by other pyrolytic products.The toxin plus DSF control failed to be of anysignificant differentiating value, since the presenceof toxin seemed to cause little variation in theelution pattern (part B, Fig. 4). Detection ofgrowth, however, was possible with this modifieddialysis sac technique. The pyrogram for thenoninoculated DSF control (part A, Fig. 4) wasconsiderably different from those of the threetypes of C. botulinum. Peaks k and m of the typeE pyrogram (part D, Fig. 4) were consistentlydifferent from those in the control, and thepyrograms of types A and B were completelydifferent from the control after peak f.

Generally, PGLC appeared to show promiseas an analytical tool for identification of C.botulinum at the type level. Further studies that

include other species of Clostridium and otherbacterial genera as well should be conducted.The investigation of other column materials andother operating parameters, however, is alsonecessary to determine the usefulness of thistechnique at the strain level since this study re-vealed only a few reliable differences. PGLC mayalso be extremely valuable for detection of growthof C. botulinum. Further studies must be con-ducted, however, to determine whether growth inactual food products can be detected.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Serviceresearch grant UI-00173-03 and Public Health Service traininggrant 5-TOI-Ul-01031 from the Consumer Protection and En-vironmental Health Service, National Center for Urban and In-dustrial Health.

LITERATURE CITED

1. Anonymous. 1964. Botulism outbreak from smoked whitefish.Food Technol. 18:71-74.

2. Dolman, C. E., and H. Iida. 1963. Type E botulism: its epi-demiology, prevention, and specific treatment. Can. J. PublicHealth 54:293-308.

3. lida, H., Y. Nakamura, L. Nakagawa, and T. Karashimada.1958. Additional type E outbreaks in Hokkaido, Japan. J.Med. Sci. Biol. 11:215-222.

4. Levy, R. L. 1967. Trends and advances in design of pyrolysisunits for gas chromatography. J. Gas Chromatogr. 5:107-113.

5. Oyama, V. I., and G. C. Carle. 1967. Pyrolysis gas chroma-tography application to life detection and chemotaxonomy.

J. Gas Chromatogr. 5:151-154.6. Reiner, E. 1965. Identification of bacterial strains by pyrolysis-

gas-liquid chromatography. Nature (London) 206:1272-1274.

7. Reiner, E. 1967. Studies on differentiation of microorganismsby pyrolysis-gas-liquid chromatography. J. Gas Chromatogr.5:65-67.

8. Reiner, E., and W. H. Ewing. 1968. Chemotaxonomic studiesof some gram-negative bacteria by means of pyrolysis-gas-liquid chromatography. Nature (London) 217:191-194.

9. Vinet, G., and V. Fredette. 1951. Apparatus for the culture ofbacteria in cellophane tubes. Science 114:549-550.

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