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Botulism and Heat-Processed Seafoods

JOSEPH J. L1CCIARDELLO

IntroductionFrom 1899 to 1973 there have been688 reported outbreaks of botulism inthe United States, involving 1,784cases and 978 deaths (Center for Disease Control , 1974a). Seventy-twopercent of the outbreaks were associated with home-canned foods, where

as commercially processed productswere involved in only 9 percent of theoutbreaks. In Japan, Canada, Sweden, Denmark, and Russia, the majority of outbreaks have been due toimproper home preservation of fishand fish products. Since 1940 therehas been a decline in the number ofbotulism cases in the United States.This is believed attributable to an improvement in canning proceduresboth in the home and industry.When one considers the total number of cans of commercially processedfood produced in this country sincethe inception of the canning industry,on the whole the overall safety recordpertaining to botulism is remarkable.Out of 775 billion cans of food soldbetween 1926 and 1971, only threecans have produced (four) fatal casesof botulism (Borgeson, 1971).Most botulism cases associatedwith commercially processed foodshave not involved fully sterilizedproducts, but semi-preserved products (i.e., smoked, brined, pickled,pasteurized) which must be refrigerated and which rely upon salt content,pH, etc. , for semi-preservation. InThe author is with the Gloucester Laboratory,Northeast Fisheries Center, National MarineFisheries Service, NOAA, Emerson Avenue,Gloucester, MA 01930.

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such products, the commensal microbial flora has been either reduced orinhibited, whereas the spores of C.botulinum may be unaffected. Undersuitable conditions, germination andoutgrowth may occur without competition from other bacterial types, producing toxins-and sometimes withno outward sign of spoilage.Nevertheless, when botulism fromcommercially produced foods doesoccur, the sensational publicity generated by the insidious nature of thedisease arouses great concern andaccusations which diminish publicconfidence in the food industry involved. This causes serious economicloss to the affected industry. Rebuilding public confidence in such situations may take 2 years or more(Anonymous, 1978).

Commercial SeafoodProducts InvolvedThe principal habitat of C. botulin-um is in soil; therefore, it is not surprising that vegetables have been implicated in the majority (17.4 percent)

of U.S. outbreaks (Center for DiseaseControl , 1974a). Fish and fish products have been implicated in 4.4 percent of all outbreaks, whereas meatproducts were involved in 1.7 percent.However, in 66.7 percent of all outbreaks, the vehicle for the intoxication was either not known or not reported (Center for Disease Control,1974a).Botulism associated with commercially prepared fish products in theUnited States has involved cannedclams, clam juice, crab, salmon, saraines, sprat , tuna, and smoked orvacuum-packed ciscoes and whitefish

(Meyer and Eddie, 1965; Center forDisease Cont rol, 1974a). Most ofthese products, however, are usuallyconsumed directly from the containerat ambient temperature with no further culinary preparation which couldinactivate any toxins.Distribution and

Occurrence in FishThere are seven known strains ortypes of C. botulinum, designatedtype A to G, which produce pharmacologically identical but serologicallydistinct toxins. Types A, B, E, and Fare responsible for human botulism,whereas types C and D are usuallyassociated with birds and animals(Smith and Holdeman, 1968). Therole of type G has not yet been established.The predominant strain in fish

related botulism has been type E,although types A and B have beenoccasionally implicated. Type E C.botulinum is ubiquitously distributedin nature and has been isolated fromterrestrial soils, rivers and freshwaterlakes, marine muds and waters, warmwaters (i.e., Gul f of Mexico), andcold waters (i.e., Baltic Sea) (Graikoski, 1971; Hobbs, 1976). It hasbeen found in such principal U.S.fishing areas as the Great Lakes, Gulfof Mexico, and the Atlant ic and Pacific coasts (Dolman, 1957; Bott etaI., 1966; Ward and Carroll, 1965;Nickerson et aI., 1967; Presnell et a\.,1967; Ward et a\., 1967; Craig et a\.,1968; Eklund and Poysky, 1970;Laycock and Longard, 1972).Type E C. botulinum seems to haveits greatest concentrat ions in bays,

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estuarine areas, and the surroundingland masses. These areas act as catchbasins for rivers draining the contiguous land masses and thus maintainreservoirs of the organisms in the immediate environs (Graikoski, 1971).However, type E habitat is not confined to littoral waters. Dolman(1957) isolated the organism frombenthic mud off the coast of BritishColumbia at depths ranging from 153to >765 yards (140 to >700 m). Laycock and Longard (1972) detected itin waters 150 miles (241 km) off thenortheast coast of Newfoundland,and the spores are probably disseminated throughout the marine environment by underwater currents.Since the organism inhabits marineand freshwater environments, it hasbeen detected in or on the associatedfauna. In infected fish or shellfish, itis usually found on the slime or exoskeleton, the gills, and in the intestinaltract (Huss, 1981). Among salmontaken in Alaskan waters (coastal andrivers), the number of positive gillsamples exceeded the number of positive viscera specimens, particularlywith the fish caught in rivers (Houghtby and Kaysner, 1969). The authorsbelieved the gills were acting as filtering agents.While much research has been di-rected toward establishing the qualitative incidence of type E C. botulinumin various fishes, there remains a paucity of information on the concentration of viable cells or spores, particularly in edible portions. A survey byGoldbli th and Nickerson (1965) forthe quantitative contamination bytype E C. botulinum of commerciallyproduced haddock fIllets in Boston,Mass., revealed that about 24 percentof the samples taken from five different processing plants were contaminated, but the highest level found inany of the samples was 17 organismsper 100 g of fillets. No attempt wasmade to differentiate between cell andspore content. The incidence of typeE spores among herring taken fromNorwegian fishing grounds was estimated at

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process which receives a sterilizingvalue equivalent to 2.45 minutes at250of would satisfy the requirementfor a minimum botulinum cook; however, for safety reasons industry commonly considers the "minimum botulinum cook" as equal or equivalent toFm = 3.0. This sterilizing value is applied in the absence of satisfactoryTDT data which would show that alower sterilizing value is appropriate.Another parameter often used inthermal process evaluation is the Dvalue, referred to as the death rateconstant, or decimal reduction time.This is defined as the time required ata given temperature to effect a 90 percent reduction in numbers of the organism during heating, or the time required for the rate-of-destructioncurve to traverse one log cycle whennumbers of survivors are plotted as afunction of heating time on semilogpaper. The D,so for C. botulinumspores in phosphate buffer was determined to be 0.20 minutes (Schmidt,1964). I f the thermal death time valueof 2.45 minutes (at 250F) is dividedby the D,so value of 0.20 minutes, avalue of 12 is obtained. This indicatesthat the 60 billion spores in the studyof Esty and Meyer were reduced by 12log cycles or a factor of 10 -12 afterheating for 2.45 minutes at 250F.Consequently, a sterilizing process designed to effect a minimum botulinumcook is also termed a 12 D process.One perspective for perceiving thesafety factor inherent in a minimumbotulinum cook is to envision 60 million cans of product each arbitrarilycontaining 1,000 C. botulinumspores. Theoretically, after a minimum botulinum heat process, onlyone among all these cans would contain a viable spore. Today commercialheat processes for low-acid cannedfoods are usually not designed principally for the destruction of C. botu-linum, but rather for the spores ofmore heat-resistant nonpathogenssuch as Putrefactive Anaerobe 3679, astrain of Clostridium sporogeneswhich could cause putrefactive spoilage if they survived the process (Lewisand Hall, 1970).Stumbo et al. (1975) recommended

February 1983,45(2)

a sterilizing value (F250 ) of 5-6 forcommercial heat processes for lowacid conduction-heating canned foodsat a retort temperature of 240F toobtain sterility with a moderate degreeof assurance that only minor economic losses from spoilage by sporeforming mesophilic bacteria moreheat resistant than C. botulinumwould result. However, when goodthermal resistance data are available,thermal processes may be based onsterilizing values that are somewhatlower than 5 or 6. Commercial processes today are generally based onthese recommended F values of 5-6.This is twice the lethal requirement ofC. botulinum and thus provides awide margin of safety.One must caution, however, tha tthese sterilizing values are founded onmoist-heat inactivation kinetics wherebacterial spores are relatively heatlabile. Bacterial spores are more resistant with dry heat, i.e., in a hot-airoven or in oil. Lang and Dean (1934)found C. botulinum spores to bemore resistant in fish products packedin oil. Neufeld (1971) recovered viablemicroorganisms from some cans oforganoleptically sound, commerciallyheat-processed fish products, withsurvival attributed to fat protectionsince the isolated cultures were notfound to be heat resistant.Canned foods are not generallyconsidered absolutely sterile butrather "commercially" sterile. Thisterm implies that a few viable or injured cells may be present in a product, but growth and multiplication aresuppressed by environmental conditions in the substrate. In low-acidcanned foods, spores of heat-resistantthermophiles such as B. stearotherm-ophilus are the only ones which areexpected to survive the processesused.Another factor which contributesto the prevention of botulism is theheat lability of the toxin. Simplyheating the food to the boiling temperature before eating should be sufficient for detoxifying a product (Licciardello et aI., 1967). Most outbreakswith commercially packed seafoodsinvolve products which may be served

cold and directly from the can (i.e.,tuna, crab, and sardines).Factors Responsible fora Botulism Hazard

In contrast to type A and type Bspores, the spores of type E C. botu-linum are relatively heat sensitive(Angelotti, 1970), and would be easilydestroyed by a conventional commercial heat process as designated by theNational Food Processors Association (1966). It has been stated that alloutbreaks of botulism from U.S.commercially canned foods in recentyears have been due to technical errors and not to the recommendedthermal process itself (Lewis andHall, 1970).For example, the presence of C.botulinum in commercially cannedmushrooms was thought to have beencaused by use of a new type of fillingmachine which tended to pack themushrooms into a solid mass in thecontainer, thus altering the heat penetration characteristics. The processorunwittingly continued to maintain thesame process schedule which resultedin an underprocessed product. Processing time should be redeterminedwhenever there is any change in product formulation involving particlesize, drained weight, consistency, etc.,which could affect heat transfer characteristics.The 1963 outbreak in Detroit,Mich., resulted from recontaminationof canned tuna due to a faulty seal(Stersky et aI., 1980). The mechanismby which this can occur is as follows:As soon as steam pressure is releasedin a retort at the end of a thermal process, and while the cans are still hot, atremendous strain is imposed on thecontainer seams and microleakagecan occur, particularly if the can hadbeen sealed by a seaming machine thatwas out of adjustment or had badlyworn rolls. During the cooling period

a negative pressure develops withinthe can, and in a can with a defectiveseal, the cooling medium (air orwater) can be sucked in. Although cansizes of approximately 4 inches diameter and smaller are normally designedto withstand internal pressures which

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develop if the sterilizer steam pressureis suddenly released, can sizes greaterthan 4 inches diameter must be cooledunder pressure to prevent strainingand/or buckling of the conta inerends. Failure to pressure cool properly can lead to leaker spoilage problems with these large diameter cans.Post-process recontamination can bereduced by treatment of the coolingwater with an effective germicide, bygentle handling of filled containers toavoid damage to sealed ends, and byproper sanitation of can runways andequipment (Bee and Hontz , 1980; Itoand Seeger, 1980).

Significance of z Value inDetermining Process TimesThe strains of C. botulinum foundin fish or marine foods are usually ofthe nonproteolytic variety (type E and

some types B and F). This is fortunatein the respect that nonproteolyticstrain spores are less heat resistantthan those of proteolytic strains.However , it is unfortunate becausethe proteolytic strains produce aputrid, recognizable spoilage patternwhich would be a deterrent to theconsumption of a toxic product,whereas spoilage and toxin production induced by the nonproteolytictypes are usually less evident. In addition, nonproteolytic strains arecapable of growing at lower temperatures, e.g., 37.4 _41 of (3 _5 0c)(Hobbs, 1976).

Most type E outbreaks with commercially processed fish, particularlyduring the early 1960's, have beenassociated with inadequately refrigera ted smoked fish. As a result, a codeof practice was instituted for commercial production requiring smoked fish(from the Great Lakes) to be heated atleast 30 minutes to an internal temperature of either 180of (82C) if the saltcontent of the aqueous phase is 3.5percent, or 149of (65C) if the saltconcentration is 5.0 percent (Liston,1980). However , it has been demonstrated that this process schedule maynot be adequate for complete elimination of type E spores from contaminated fish (Christ iansen et aI., 1968;Pace et aI. , 1972).

4

When establishing a process schedule, the object ive is usually the destruction of the most heat-resistantstrain of a particular bacterial species.Quite often the thermal resistance ofdifferent bacteria may be comparedeither th rough their thermal deathtimes or D values at a par ticulartemperature. This would be a validcomparison only i f the slopes of theTDT curves were the same (Townsendet aI., 1938). In thermal processingterminology, the slope of a bacterialTDT curve, expressed as the degreesFahrenheit intercepted by the curve intraversing one log cycle, measures thechange in thermal death time as afunction of temperature. This parameter is called the z value. Another concept of the z value is that it representsthe number of Fahrenheit degrees required ~ cause a tenfold change inheating time to achieve the same lethaleffect. In the classic study by Esty andMeyer (1922) referred to previously,the z value in phosphate buffer wasfound to be 18. As this value becomessmaller, the effect of a processingtemperature change becomes moremarked. Thus, two strains of type Espores with different z values couldhave the same heat resistance a t oneparticular temperature, but the sporeswith the smaller z value will be moreresistant at lower temperatures andless resistant at higher temperatures(Perk ins , 1964). A thermal processbased on the heat resistance of thespores with the higher z values in thiscase would result in underprocessing(Perkins et aI., 1975).

The z values for var ious strains oftype E spores in either water, phosphate buffer pH 7, or trypticase peptone glucose broth have been reportedto range from 7.4 to 17 (Ohye andScott , 1957; Schmidt, 1964; Robertsand Ingram, 1965; Crisley et aI., 1968;I to et aI., 1970; Lynt et al. , 1977). Although the substrate is known to affect the z value, variable values werereported even in s itua tions with thesame substrate. There are essentiallytwo different methods for determiningthe z value: 1) From a TDT curve constructed from end-point destructiondata, a nd 2) from a phantom TDT

curve obtained by plotting log D valueas a function of temperature (Perkins,1964).Experimental Comparisonof z Value for C botulinumType E by Two Different Methods

Because of the reported variabilityin z value for type E C. botulinumspores, the following study was conducted to determine the influence ofthe particular me thod by which thevalue was derived. Thermal destruction rates were determined for twostrains of type E C. botulinum spores(8E and Detroit) in eitherM/ 15 phosphate buffer pH 7.0, clam liquor, or ahaddock slurry over a temperatu rerange of 140-180F (60.00-82.2C).The spore suspensions were heated ina thermostatically controlled mineraloil bath in sealed melting-point-determination capillary tubes which allowed rapid heating or cooling to ambient temperature (Licciardello andNickerson, 1962). The number of survivors was counted by asepticallycrushing the acetone/chromic acidcleaned tubes in sterile diluent and inoculating appropriate dilutions intoMiller-Prickett ' tubes containing 0.5ml of 5 percent filter-sterilized sodiumbicarbonate solution and then adding10 ml of a mol ten agar medium (50 gtrypticase, 5 g peptone, 10 g glucose,0.5 g sodium thioglycollate, 0.5 g ferric citrate, 1 g K2HP04, 1 liter distilledwater, pH to 7.2). Tube cultures wereincubated 48 hours a t 85 OF (29.40c)before counting colonies.A typical set of survivor curves isshown in Figure 1 for strain 8E in ahaddock slurry. The nonlinear survival curve with heat-resistant tail exhibited by 8E in haddock slurry also occurred with the other substrates andwith the Detroit strain. Lynt et al.(1977) also repor ted a tailing effectfor thermal destruction of type Espores. The D value was calculatedfor each heating temperature from thereciprocal l inear regression slope ofthe survival data over the first three to'Mention of trade names or commercial firmsdoes not imply endorsement by the NationalMarine Fisheries Service, NOAA.

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Figure I.-Survivor curves for spores of C. botulinumtype E (8E) heated in a haddock slurry. Each point represents the average count of duplicate tubes.Figure 2.-Phantom TDT curves for spores of C. botu-linum type E in various substrates. Each point representsresult of separate trial.

four log cycles of destruction. Although it is acknowledged that thisprocedure for determining D value isvalid only for linear survival curves,in this case the value so derived wouldbe applicable to 99.9-99.99 percent ofthe population, which for all practicalpurposes can be considered to represent the population. The D values obtained in this study support the generalconclusion of others that type E C.botulinum spores are relatively heatsensitive.

Phantom TDT curves are shown inFigure 2. The z values calculated fromregression slopes were as follows: Forstrain 8E, 9.2 in phosphate buffer, 9.3in c lam liquor, and 9.0 in haddockslurry; for the Detroit strain, 8.2 inclam liquor.Thermal death times were determined by the end-point method forthe Detroit stra in in clam liquor. Aseries of five capillary tubes , each

containing 150 x 10 6 spores washeated for various time intervals a teither 170, 180, 190, or 200F (76.7,82.2, 87.8, or 93.3 0c) and then aseptically subcultured in TPG brothtubes and stratified with vaspar. Following a 2-week incubation period at85 OF (30C), each culture tube wasexamined for viability by phasemicroscopy and for presence of typeE toxin by mouse int raperi toneal injection. A TDT curve was constructedby drawing a straight line between theinterval of heating times for the lastset of toxin-positive tubes and the firstset of five toxin-free tubes for eachtemperature (Fig. 3). From this TDTcurve a z value of 17 was calculated,and this is to be compared with thevalue of 8.2 obtained from the phantom TDT curve. Lynt et al. (1977)similarly computed a higher z valuef rom the TDT curve compared withthe phantom TDT curve for strains of

type E C. botulinum spores in crabmeat. The discrepancy may be due tothe fact that the TDT curve was basedon the thermal resistance of a verysmall proportion of heat resistantspores in the population, whereas thephantom TDT curve was derivedfrom the resistance of the major fraction of the population which wasrelatively heat sensitive. The questionhas long been debated whether the ext reme heat resistance often encountered in a very small proportion of abacterial population is due to I) aphysical phenomenon ( i.e. , clumping), 2) the presence of some driedspores on the inside wall of the TDTtube being subjected to dry ratherthan moist heat, or 3) a var iation inphysiological condition among thespore popula tion . However, in thisstudy and also in another (Graikoskiand Kempe, 1964), it was demonstrated tha t the extreme heat resis-

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500

1000

Figure 3 . -Thermal death times plot for spores of C.botu/mum type E (Detroi t) in clam liquor (+ = toxindeveloped in at least one of five tubes after heating and = toxin did not develop in any of five tubes after heating).

200

Literature CitedAngeloni, R. 1970. The heat resistance of Clo-stridium botulinum type E in food. In M.Herzberg (editor), Proc. First U.S.-JapanConference on Toxic Microorganisms, p.404-409. Published by the UJ R JointPanels on Toxic Microorganisms and theU.S. Department of the Interior.Anonymous. 1978. Botulism-the killer.World Fish. 27(11): 15._--- , - -_. 1982a. Canned salmon inspires another botulism scare in Europe. FoodChern. News 23(49):50.__ .,..--_" 1982b. HHS News. Bull. P82-4.U.S. Dep. Health Human Serv., Wash.,D.C., 2 p.Bee, G. R. , and L. R. Hontz. 1980. Detection

bases a process schedule on a mlntmum botulinum cook, it is not believed that the public health would becompromised. Pflug and Odlaug(1978) have concluded t ha t an Fm of3.0 minutes and a z value of 18 havebeen the standard for the minimumbotulinum cook for more than 40years and there is no epidemiologicalevidence to demonstrate that the standard has failed.

180 190TEMPERATURE (OF)

170

wf -

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with a liquid broth. Finally, enumeration of survivors in the rate of destruction method was based on colonyformation, whereas in the end-pointmethod the criterion for survival wastoxin production. It is not knownwhether all surviving spores werecapable of toxin production. The important question is which of these twomethods is correct. To be realistic,thermal death t imes would probablybe best determined by heating and incubating the spore inoculum in thesame substra te for which the processtime is being determined. This can beaccomplished using TOT cans. It ispossible that heat-injured spores maybe recovered when subcultured into anutrient medium whereas these samespores might not be able to germinateand grow ou t because of an unfavorable environment if lef t in the heatedfood substrate. More work is neededto establish the validity of the twomethodologies reported here.Nevertheless, i f a food processor

tance was not an inherent trait, sincespore crops produced from the resistant survivors possessed the s amethermal resistance as the parent culture. That type E C. botulinum heatresistant spores do exist is evidencedby the recovery by other investigatorsof viable type E spores from chubswhich had been heated in accordancewith the recommended code of practice, i.e., to an internal temperature of180OF for 30 minutes (Pace et aI.,1967; Christiansen et aI., 1968). Dolman and Chang (1953) reported thatsome strains of type E spores withstood 30 minu tes exposure to 212 of(100C). Yet, based on D valuesgeneral ly reported for type E strainsat 180F, 30 minutes should exceed asterility t ime calculated for a 12 Dprocess. In our study the D,so for theDet roi t st rain in c lam l iquor was 0.2minutes. A 12 D process based on thisdatum would require 2.4 minutes at180F. Yet, f rom Figure 3 the TOT at180OF for 150 million spores is shownto be about 65 minutes. Lynt et al.(1977) similarly found that the computed process t ime for type E sporesin crab meat at 185 of (85C) basedon 12 D from a phantom TOT curvewas much less than the value obtainedfrom the regular TOT curve.

The discrepancy in processing requirement as derived from these twotechniques must result from some unaccountable experimental error inherent in one of the two different methodologies. This is also reflected in thedifferences in z values obtained by thetwo methods. A possible causativefactor could be the differences in incubation time after heating for recovery of viable spores or testing for toxinogenesis. In the TOT method a2-week incubation periodwas allowed,whereas in the rate of destructionmethod the agar tubes could not beincubated beyond 2 days because ofextensive gas formation which precluded counting colonies. The gasresulted from the fermentation of glucose which was considered an essential requirement for maximal recoveryof type E C. botulinum. Also, there isa possibility of differences in recovery, particularly of heat-injuredspores, resulting from subculturing ina solid nutrient medium compared6 Marine Fisheries Review

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and prevention of post processing container handling damage. 1. Food Prot. 43:458-460.Borgeson, L. 1971. So, stop worrying aboutcanned foods. Family Health, Nov., p.22-36.Bott, T. L., 1. S. Deffner, E. McCoy, andE. M. Foster. 1966. Clostridium botulinumtype E in fish from the Great Lakes. 1.Bacteriol. 91 :919-924.Cann, D. c., B. B. Wilson, 1. M. Shewan, andG. Hobbs. 1966. Incidence of Clostridium

botulinum type E in fish products in theUnited Kingdom. Nature (Lond.) 21 I:205.Center for Disease Control. 1974a. Botulismin the United States, 1899-1973. In Handbook for Epidemiologists, Clinicians, andLaboratoryWorkers. DHEW Publ. (CDC)74-8279. U.S. Dep. Hea lth, Educ. , Welfare, Atlanta, Ga._______ . 1974b. Botulinal toxin in anopened can of commercial tuna fish. Morbidity Mortal. Weekly Rep. 23(19): 176.Chris tiansen, L. N., 1. Deffner , E. M. Foster,and H. Sugiyama. 1968. Survival and outgrowth of Clostridium botulinum type Espores in smoked fish. Appl. Microbio l.16:133-137.Corwin, E. 1980. A food poisoning whodunit.FDA Consumer. Nov., p. 144-147.

Craig, 1. M., S. Hayes, and K. S. Pilcher.1968. Incidence of Clostridium botulinumtype E in salmon and other marine fish inthe Pacif ic Northwest . Appl . Microbio l.16:553-557.Crisley, F. D., 1. T. Peeler, R. Angelotti, andH. E. Hall. 1968. Thermal resistance ofspores of five strains of Clostridium botu-linum type E in ground whitefish chubs. 1.Food Sci. 33:411-416.Dack, G. M. 1964. Characteristics of botulismoutbreaks in the United States. In K. H.Lewis and K. C. Cassel, lr . (editors), Botulism, p. 33-38. Public Health Servo Publ.999-FP-1. Public Health Serv., Cincinnati,Ohio.Dolman, C. E. 1957. Type E (fish-borne)botulism: A review. lpn. 1. Med. Sci. BioI.10:383-395.____ , and H. Chang. 1953. Theepidemiology and pathogenesis of type E and fishborne botulism. Can. 1. Public Health. 44:231-244.Dowell, V. R., lr., E. 1. Gangar osa , andR. W. Armstrong. 1970. Clinical and epidemiological aspects of botulism in theUnited States. In M. Herzberg (editor),Pr oc. First U.S.-lapan Conference onToxic Microorganisms, p. 360-364. Publ ished by the U1NR 10int Panels on ToxicMicroorganisms and the U.S. Departmentof the Interior.Eklund, M. W., and F. T. Poysky. 1970. Distribution of Clostridium botulinum on thePacific coast of the United States. In M.Herzberg (editor), First U.S.-lapan Conference on Toxic Microorganisms, p. 304308. Published by the U1NR 10int Panelson Toxic Microorganisms and the U.S. Department of the Interior.Esty, 1. R. , a nd K. F. Meyer. 1922. The heatresistance of the spores of B. botulinus andallied anaerobes. XI. 1. Infect. Dis. 31 :650663.Goldbl it h, S. A., and 1. T. R. Nickerson.1965. The effect of gamma rays on haddock and clams inoculated with Clostridi-um botulinum ty.pe E. Mass. Inst. Tech-

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