APPLIED MICROBIOLOGY, Oct. 1974, p. 655-660 Vol. 28, No. 4Copyright © 1974 American Society for Microbiology Printed in U.S.A.

Clostridium perfringens in the Environment'JACK R. MATCHES, JOHN LISTON, AND DONALD CURRAN2

Institute for Food Science and Technology, College of Fisheries, University of Washington,Seattle, Washington 98195

Received for publication 7 February 1974

Clostridium perfringens was isolated from samples collected in Puget Sound inthe state of Washington and areas considered as possible sources of theseorganisms to Puget Sound. The distribution of C. perfringens in the totalClostridium population was determined for fish gut contents and sedimentscollected in highly polluted and less polluted areas, sewage samples, freshwatersediments, and soils. The greatest numbers of C. perfringens were obtained frommarine sediments collected near the sewage outfall at West Point. Fewer isolateswere made from fish collected from less polluted stations, although the number ofC. perfringens remained high in sediments from other Puget Sound stations. Theproportion of C. perfringens in the total Clostridium populations varied between56 and 71% for sewage samples and only 0.4 to 4.1% for freshwater sediments andsoil samples. Only 25 C. perfringens isolates out of 137 from fish guts, or 18%,were identifiable serologically and these fell into 12 groups. C. perfringens werefed to fish and the fish were sacrificed after varying lengths of time. The numberof C. perfringens increased slightly in the gut during the first 24 h and then thenumbers decreased rapidly for the next 120 h.

Clostridium perfringens is more widelyspread than any other pathogenic bacterium.Its principal habitats are the soil and theintestinal contents of man and animals (3, 18).This organism has been recognized since thelate 1800's, when reports linked it with foodpoisoning; however, it was not until 1945 that C.perfringens foodborne illness was reported (9).The illness received considerable attention inGreat Britain in 1953 (7) and has been recog-nized as a very important food poisoning orga-nism in the United States since that time (3, 8).The presence of C. perfringens has been

reported by a number of workers in soils (14, 17,19, 20) and in marine sediments (1, 2, 5, 10, 11,16). This work was done to determine its pres-ence in Puget Sound, an area used both forsports and for commericial fishery and recrea-tion, and also areas which may contributeclostridia to Puget Sound.

MATERIALS AND METHODSSampling: fish stomach and gut contents. Fish

were caught by the College of Fisheries research vesselMV Commando with an otter trawl which collects fishlying on the bottom and swimming within 3 to 4 feet(ca.0.914 to 1.21 m) of the bottom. Immediately afterthe trawl net was retrieved, the fish were removed and

I Contribution no. 403, College of Fisheries, University ofWashington, Seattle, Wash. 98195.

2Present address. Department of Microbiology, OregonState University, Corvallis, Oregon 97331.

the desired species were selected. Fish stomach andgut contents were removed from fish with sterile in-struments. The surfaces of the fish were washed withwater and swabbed with 70% ethanol. The belly wallof the fish was cut with a pair of sterile scissors. Thestomach and gut were removed with scissors andscalpels sterilized by soaking in alcohol and flaming.When large fish like the Pacific dogfish were taken,one gut was large enough for a sample. Smallerfish like English and Dover sole have a small gut, anda composite of contents from five fish was collected insterile bottles. Samples were held in ice until used.

Sediments. Sediments were collected aboard theMV Commando with a gravity geological corer con-taining a 3.8-cm plastic liner. The corer was sterilizedby rinsing with 70% ethanol before use. Approxi-mately equal quantities of water and sediment at theinterface were removed from the plastic sleeve andplaced in sterile jars. Samples collected aboard theresearch vessel were either processed in the laboratoryaboard the vessel immediately after collection oncruises of several days, or returned to the laboratory inthe College of Fisheries for processing on 1-day trips.Samples were refrigerated until processed, usually 4to 6 h.

Sewage. Samples of sewage were collected fromSeattle's West Point sewage disposal plant by plantemployees during their normal daily sample collec-tion. Samples collected from this primary treatmentplant included sludge, unchlorinated and chlorinatedeffluent and sludge, plus chlorinated effluent. Sam-ples were collected in sterile large-mouth bottles andheld refrigerated until processed in the laboratory, aperiod of approximately 4 h.

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Freshwater sediment and soils. Samples of sedi-ment from a freshwater stream and soil from thesurrounding forests and farm land were collected inconjunction with other studies. These samples werecollected from the Snohomish River system, whichempties into Puget Sound at the city of Everett.Samples were collected along approximately 70 miles(ca. 112.6 km) of stream from within 2 miles (ca. 3.2km) of the crest of the Cascades down to Puget Sound.Most of the samples were collected along the sectionof stream not affected by tidal action. Samples ofsediment collected from the river in water greaterthan 12 inches deep (ca. 0.3 m) were collected with amud snapper (Kahl Scientific Instrument Corp., SanDiego, Calif.). In the upper reaches of the stream withless than 12 inches of water, samples were collectedfrom the stream banks, forest floor, and cultivatedland with triers and spoons sterilized by soaking inalcohol and by flaming. Samples were refrigerated assoon after collection as possible and held until re-turned to the laboratory, usually no longer than 6 to 8h.

Sampling stations. Sampling stations in PugetSound and off the coast of Washington were selectedfor the College of Fisheries training cruises aboard theresearch vessel and other sampling programs. TheWest Point sampling station is located off Seattle'sMetro sewage disposal plant, which dumps up to 2.5x 106 gallons (ca. 8.5 x 106 liters) per day, and forthese studies was considered a polluted area. Theremaining stations located in northern Puget Soundwere considered less polluted. These stations wereselected to represent different hydrographic condi-tions and are listed elsewhere (10, 11). Freshwatersediments and soil were collected along the Snohom-ish River system at stations selected for other studies.Sewage samples were collected from the settling tanksat the sewage disposal plant and at sampling portswere collected through the treatment plant.Enumeration and identification. Blood agar

plates were prepared with blood agar base (BBL),made up with 5% outdated whole citrated humanblood. This medium was found to support excellentgrowth of both aerobic and anaerobic organismscollected from both freshwater and marine areas andalso permitted the testing of the hemolysis reactions.Blood agar plates were either prepared and usedimmediately or poured in advance, the surface wasdried, and the plates were prereduced by storage inanaerobic jars. Serial decimal dilutions of sampleswere prepared, using freshly prepared 0.1% peptonewater made up with 50% seawater and 50% distilledwater for the other samples. Earlier studies (12)showed that higher anaerobic counts were obtainedfrom marine sediments using 50% seawater thandistilled water in both diluent and media. One-tenth-milliliter quantities of the appropriate dilutions werespread on the surface of the blood agar with sterileglass rods, and plates were incubated in anaerobic jarsor incubated aerobically exposed to the atmosphere,both at 30 C until colonies were large enough to bepicked easily, usually 5 to 7 days. Clostridium specieswere isolated by either picking all colonies on a plateincubated anaerobically, or randomly selecting a

percentage of the colonies. All isolated colonies werepurified by streaking and were tested for both aerobicand anaerobic growth on McClung and Toabe eggyolk medium (10). Isolates were Gram-stained to testfor. gram-positive rods and the presence of spores.Lecithinase-positive organisms growing only anaero-bically were subjected to further testing in thesestudies for identification of C. perfringens. From theanaerobic populations isolated, the total Clostridiumpopulations were identified and will be described in alater publication.No distinction was made between active vegetative

cells and resting spores in these studies because wewere interested in total numbers present and thepotential hazard, rather than the physiological stateof the organisms.The media and methods used for the identification

of the clostridia were the reactions suggested in theliterature (4, 18) and methods used in our laboratory.Reactions used in identifying C. perfringens were:acid from glucose, lactose, maltose, mannose, sucrose,and trehalose; no acid from mannitol and salicin;gelatin and meat digested, with meat turning pink;stormy fermentation in milk (clot and gas); indolenegative; nitrate reductions in approximately 80% ofcultures; lecithinase positive, lipase negative; betahemolysis of human blood and nonmotile. Althoughsome variations were noted, cells under microscopeexamination were short with squared ends. Whenpresent, spores were subterminal; however, demon-stration of sporulation was not possible with mostisolates. All samples were incubated in Brewer Gas-Pak (H. + CO,) Anaerobic Jars (BBL) to ensureanaerobic conditions.

Feeding studies. Survival of C. perfringens in fishgut was determined by feeding cultures of the testorganism to a test animal, the staghorn sculpin(Leptocotlus armatus), an animal found in the sameenvironment as the fish caught and sampled for gutcontents. Although the animals could be force fed orinjected with cultures of C. perfringens, using asyringe, a natural feeding method was desired toreduce stress on the animal. Small Coho salmon(Oncorhynchus kisutch), 5 to 7 cm in length, wereobtained from a local state fish hatchery. Smallvolumes of C. perfringens cultures containing vegeta-tive cells were injected into the gut area of the deadsalmon. The same number of cells was inoculated intoeach salmon, the inoculated salmon were droppedinto the aquarium, and the staghorn sculpins, beingaggressive and active feeders, quickly consumed theentire inoculated fish and were then transferred toother aquaria. The fish were sacrificed after varyingtime periods and the stomach and gut contents wereweighed and blended in a Waring blender with sterilediluent (0.1% peptone in seawater and distilledwater). The total number of C. perfringens survivingwas calculated. The number of cells in the inoculumwas determined by spreading 0.1-ml quantities ofserial decimal dilutions on blood agar.

RESULTSThe stomach and gut contents of otter trawl-

caught fish were examined for total bacterial

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numbers by incubating both aerobically andanaerobically. Colonies picked from the anaero-bically incubated count plates were purifiedand studied for morphological and biochemicalfeatures. Total plate counts of organisms grow-ing both aerobically and anaerobically on bloodagar at 30 C, the types of fish samples, and thenumber of fish per sample are shown in Table 1.Total numbers of bacteria growing both aerobic-ally and anaerobically varied per gram of gutcontents by several orders of magnitude. Aero-bic counts had a range of 2,000 to 32 million,and the anaerobic counts had a range from1,000 to over 6 million. The averages obtainedfor both aerobic and anaerobic counts from fishcollected from the different sampling areas areof the same magnitude. The average aerobiccounts were 2,600,000 and 1,600,000 orga-nisms/g of gut contents for West Point andother sampling stations, respectively. Averageanaerobic counts for the same two areas were630,000 and 580,000 bacteria/g of gut contents,respectively.Samples of sediment and fish gut contents,

collected from the sampling stations in PugetSound and off the coast of Washington, weretested for the presence of C. perfringens (Table2). The largest percentage was found in gutcontents from fish caught from the West Pointsewage disposal outfall diffuser pipe area. FewerC. perfringens were isolated from fish collectedat other sampling stations some distance fromthe source of contamination. Fish samples col-lected off the coast of Washington out to depthsof 850 m, surprisingly, did not yield C. perfrin-gens. Sediment samples collected from PugetSound stations contained Clostridium popula-tions of which C. perfringens made up 38.3 and26.2% for West Point and other samplingstations, respectively. Sediments collected offthe coast of Washington, like fish gut contents,did not yield C. perfringens. Fish caught at theWest Point station contained a higher percent-age of C. perfringens in their gut contents thandid the sediments collected from the same area.This may be due to the organisms or materialused as feed by fish harboring C. perfringens inlarger numbers as a result of the food chain.

C. perfringens is not a member of the normalflora of fish; however, these organisms and otherpathogens can contaminate fish caught in pol-luted water (6). Since fish can pick up orga-nisms on the surface of skin and gills and thegut during feeding, the survival of C. perfrin-gens was studied. The results of three experi-ments in which duplicate fish were sacrificed ateach sampling time are shown in Fig. 1. Whensampled 1 h after feeding, the feed had not been

TABLE 1. Total counts obtained from fish gutcontents during 1968

No. Count per single

Date Fish type fish samplesample Aerobic Anaerobic

count count

Fish collected at West Point sampling stationJanuary English Solea 4 1,000,000 25,000January Ratfish' 4 32,000,000 6,300,000February Dover Solec 3 250,000 130,000February Ratfish 4 160,000 20,000April Ratfish 4 100,000 5,000May Pacific Dogfishd 1 8,000 2,500May Ratfish 5 2,000 1,000May Sand Solee 4 25,000 16,000July English Sole 2 16,000 3,200July Pacific Hake' 2 16,000 10,000July Ratfish 3 160,000 40,000July Ratfish 3 2,000,000 2,000,000July Rockfishg 4 3,200,000 800,000August Rockfish 3 100,000 50,000August Sand Sole 6 160,000 100,000Average 2,600,000 630,000

Fish collected at other sampling stationsJanuary English Sole 4 320,000 2,500January Pacific Cod' 4 40,000 3,200January Pacific Dogfish 2 400,000 2,000January English Sole 4 20,000 16,000February Pacific Cod 3 400,000 200,000February Dover Sole 3 250,000 130,000February Ratfish 4 160,000 20,000February Sablefishi 4 16,000,000 4,000,000April Pacific Hake 5 10,000 2,500April Sand Sole 5 8,000 8,000July English Sole 5 2,500,000 2,500,000July English Sole 5 200,000 200,000July Rockfish 4 2,500,000 1,300,000August English Sole 5 25,000 10,000August Rockfish 5 630,000 320,000Average 1,600,000 580,000

a Parophrys vetulus.' Hydrolagus colliei.c Microstomus pacificus.d Squalus acanthias.ePsettichthys melanostictus.'Merluccius productus.' Sebastes species.'Gadus macrocephalus.Anaoplopoma fimbria.

digested. The numbers of C. perfringens in-creased 1 log during the first 24 h and thendeclined rapidly during the next 120 h. Furtherchange was not detected after incubation for 288h. The aquarium temperature was 18.7 C,which is above the minimal growth temperaturefor C. perfringens, making the 1-log increasepossible.

C. perfringens isolates were serotyped at theCenter for Communicable Diseases, Atlanta,Ga. Of a total of 137 cultures serotyped only 25,or 18%, were identified as known serotypes of C.perfringens (Table 3). One hundred cultureswere collected from the West Point sampling

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TABLE 2. Distribution of C. perfringens isolated from the marine environment

No. of C per-No. of samples No. of No. of C. C.iper- Avg no. of C.

Origin of samples samples containing Clostridium perfringens in sperfringenscollected C. per- isolatesa isolated | ilates per gram

fringens

Fish gut contents"West Point 15 15 160 105 66 3.4 x 104Other Puget Sound stations 15 5 168 13 7.7 5.0 x 103Off Washington coast 6 0 15 0 0

SedimentsWest Point 38 38 115 44 38.3 2.9 x 103Other Puget Sound stations 66 10 61 16 26.2 1.7 x 103Off Washington coast 6 0 142 0 0

a A randomly selected portion of the Clostridium flora.h Pooled fish samples (See Table 1).

o Number of C perfringens inoculated intofish

* Number of C perfringefns recovered

96 144Time, hours

288

TABLE 3. Serology of C. perfringens isolated fromPuget Sound sediments and fish gut contents

N. No.

c-C.per- C.per-Sampling stations s fringens fringens No. per

tridia iden- serotypes serotypetested tified

West Point sam- 100 21 PS-20 6pling station PS-50 1

PS-63 3PS-67 1PS-73 4PS-77 1PS-79 1PS-80 1PS-83 1PS-88 1

Other Puget 37 4 PS-40-73 1Sound sam- PS-59 3pling stations PS-63 1

FIG. 1. Numbers of C. perfringens inoculated into was expected because the sewage was composedand recovered from staghom sculpins. largely of domestic waste.

station and only 21 were identified. Of theremaining 37 cultures from the other samplingstations, only 4 fell into identifiable groups.

Areas serving as possible sources of C. perfrin-gens, including sewage from the West Pointdisposal plant and freshwater sediments andsoil, were investigated (Table 4). Nine hundredand eighty-six Clostridium strains were isolatedand from this population 254 C. perfringenswere identified. The freshwater sediments andsoil contained very low numbers of C. perfrin-gens. This was even true of soil samples col-lected from farm land used during parts of theyear as pasture for cattle. Sewage samplescontained high levels of C. perfringens which

DISCUSSIONThe size of the aerobic bacterial population in

the gut of fish reported in these studies is withinthe range reported by other investigators (13).Little work has been done, however, with thepopulations growing anaerobically, which in-clude both obligate and facultative anaerobes.The wide range in aerobic and anaerobic countsis not surprising, since feeding fish have beenreported to contain high levels of organisms intheir gut. The fast that normally occurs duringspawning with many species, or other periods ofnonfeeding, would reflect the low bacterialloads in the intestine of some fish.

Studies of the numbers of bacteria in Puget

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TABLE 4. Total number of clostridia studied and number of C. perfringens isolated from freshwater sediments,soils, and sewage

Nof Total no. of No. ofOrigin of samples No. of Clostridium Samples No. of C. C. perfrin-Samples isolates containing C. perfringens gens (%)

perfringens

Stream beds 22 247 1 1 0.4Adjacent banks 15 145 1 6 4.1Forest floor 10 100 1 4 4.0Farm land 12 93 2 2 2.1Unchlorinated effluent 15 68 15 48 70.6Chlorinated effluent 10 75 10 46 61.3Sludge 19 181 19 102 56.4Sludge plus chlorinated effluent 3 77 3 45 58.4

Total 106 986 52 254

Sound sediments at the sediment-water inter-face have been published elsewhere (12) andrange from 0.73 x 10' to 23.5 x 104 and 1.21 x

10' to 16.9 x 10' cells/ml of sediment-waterslurry for anaerobic and aerobic counts, re-

spectively. These data from 110 core samplescollected over a 3-year period show the lack ofseasonal variation. The 2-log variation in anae-

robic counts can probably be accounted for bythe composition and especially the organic con-

tent of the sediments collected. The highestcounts were obtained from reduced mud sam-ples and the lowest counts were from sand.

C. perfringens, an important food poisoningorganism, is ubiquitous. This organism hasbeen isolated from marine sediments by severalauthors from both polluted and nonpollutedareas. In the studies reported here, greaternumbers of C. perfringens were isolated frompolluted areas than from less polluted areas.This is in agreement with the report of Bonde(1) that a close relationship between theamount of pollution and numbers of C. perfrin-gens was always established and, although a fewcolonies generally will be found in unpollutedsamples, the counts are always proportional tothe pollution. In the Puget Sound studies,sediments collected near the sewer outfall con-

tained a Clostridium population composed of38.3% C. perfringens. At the other Puget Soundsampling stations C. perfringens made up 26.2%of the population. The number of C. perfringensisolated from sediments in these studies agreesvery well with some of our other studies in whichC. perfringens made up 37.3% of the Clostri-dium population (12). Samples of sedimentcollected off the coast of Washington just southof the Straits of Juan de Fuca at depths of 880 mwere negative for the presence of C. perfringens.Bonde (1) reported an inverse relationship be-

tween numbers of C. perfringens in sedimentsand depth, with the highest counts (> 10,000/g)in shallow water (0 to 5 m). Counts of as muchas 1,000 to 10,000 at depths of 15 to 20 m, butbelow 20 m only very low counts were found.

In these studies C. perfringens made up ahigher percentage of the total Clostridium pop-ulation in both fish gut and sediments col-lected at the West Point sampling station thanat the other Puget Sound sampling stations.Also, clostridia made up a higher percentage ofthe population growing anaerobically in sam-ples collected at West Point than at the othersampling stations. It can be reasoned that PugetSound waters, varying from estuarine to subo-ceanic, receive more pollution than waters offthe coast of Washington. Puget Sound is adeep-water port with many seagoing ships andlarge numbers of pleasure boats, and is rapidlybecoming surrounded by cities and housingdevelopments. Although sewage disposal plantsare operating, bacteria can still be isolated inlarge numbers from chlorinated sewage (Table4). Fish feeding near areas receiving pollutionsuch as sewage can pick up enteric bacteria ontheir skin and gills and in their intestines (6).6Although there are little data available on theClostridium flora of fish gut contents, C. per-fringens has been isolated from the gut of fish.Bonde (1) found high numbers of C. perfringensin the gut of plaice, flounder, and mackerel, butsmall to nil numbers in other species.The number of bacteria and C. perfringens in

the gut contents was related to the amount offeed in the fish. The numbers of organisms pergram of gut contents were found to vary be-tween species of fish and even between individ-uals within a species. The numbers of C. per-fringens in the total Clostridium population infish gut were higher than the numbers in

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sediment off the sewer outfall diffuser pipe. Atthe other Puget Sound stations the reverse wastrue. One conclusion that can be drawn fromthese data is that since C. perfringens is abun-dant in human feces (15) and much of theeffluent is domestic waste large numbers of C.perfringens are being dumped into the marineenvironment (Table 4). Fish feeding in thisenvironment, and especially near the bottom,may pick up higher levels of C. perfringens fromthis area than from the other cleaner areas.

Since C. perfringens was found to occur infish gut contents, its survival in this environ-ment was studied. Feeding fish will no doubtcontinue to pick up and discharge organisms.These studies show that one species of fishconsuming C. perfringens during feeding willeliminate these organisms if they do not con-tinue to feed. These data are interesting andfurther studies should be carried out.Primary treatment sewage disposal plants

add high levels of organic matter and bacteriato the environment. Another area contributingbacteria to the marine environment is runofffrom land. The Snohomish River system,chosen in these studies, runs through differentenvironments. Approximately the first 8 miles(ca. 12.9 km) of the stream from the sourcecascades down a timbered canyon through anarea reached only on foot by trail. Below thisarea a few miles the canyon widens and thestream is flanked by farm land. The streamempties into Puget Sound at Everett, Wash.through an industrial area. The numbers of C.perfringens isolated along this stream systemduring both dry and wet weather were lowerthan was expected, and the samples positive forC. perfringens had an average count of 5.9 x102/g. It was felt that farm land used as pasturefor cattle would harbor large numbers of C.perfringens, although Hobbs et al. (7) reportedonly 2 of 113 samples (1.7%) of cattle fecescontained C. perfringens. Sediments collectedfrom the stream bed contained only 0.4% C.perfringens in the total Clostridium population.This level increased to 4.1 and 4.0% in samplescollected along the adjacent stream banks andforest floor. The level dropped to 2.1% in sam-ples of soil collected from farm land.The serotyping of C. perfringens from fish gut

was carried out to determine if there was anycorrelation between these types and the sero-types occurring in food poisoning outbreaks inSeattle. However, only a small proportion (18%)of the strains could be typed and no correlationwas established. Nevertheless the data do indi-cate the possibility of transport of C. perfrin-gens directly into the human food systemthrough fish caught in polluted areas.

APPL. MICROBIOL.

ACKNOWLEDGMENTSThis research was supported by Public Health Service

grants 5 R01 EF 00882 and 5 R01 FD 00292.

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pearance phenomena of enteric bacteria in the marineenvironment. Rev. Int. Oceanogra. Med. TomeIX:17-44.

3. Bryan, F. L. 1969. What the sanitarium should knowabout Clostridium perfringens foodborne illness. J.Milk Food Technol. 32:381-389.

4. Cato, E. P., C. S. Cummings, L. V. Holdeman, J. L.Johnson, W. E. C. Moore, R. M. Smibert, and L. DS.Smith. 1970. Outline of clinical method in anaerobicbacteriology. The Anaerobe Laboratory, Virginia Poly-technlic Institute and State University, Blacksburg, Va.

5. Davies, J. A. 1967. Isolation and identification of Clos-tridia from North Sea sediments. J. Appl. Bacteriol.32:164-169.

6. Guelin, A. 1952. Bacteriophage et enterobacteries chez lespoissons de mer et le probleme des eaux polluees. Ann.Inst. Pasteur (Paris) 83:45-56.

7. Hobbs, B. C., M. E. Smith, C. L. Oakley, and G. H.Warrack. 1953. Clostridium welchii food poisoning. J.Hyg. 51:75-101.

8. Hobbs, B. C. 1969. Clostridium perfringens and Bacil-lus cereus infections. In H. Rieman (ed.), Food borneinfections and intoxications. Academic Press Inc., NewYork.

9. McClung, L. S. 1945. Human food poisoning due togrowth of Clostridium perfringens (C. welchii) infreshly cooked chickens: preliminary note. J. Bacteriol.50:229-231.

10. McClung, L. S., and R. Toabe. 1947. The egg yolk platereaction for the presumptive diagnosis of Clostridiumsporogenes and certain species of the gangrene andBotulinum groups. J. Bacteriol. 53:139-147.

11. Matches, J. R., and J. Liston. 1973. Methods andtechniques for the isolation and testing of Clostridiafrom the estuarine environment, p. 345-362. In L. H.Stevenson and R. R. Colwell (ed.), Belle W. BaruchLibrary in Marine Science, vol. 1. Estuarine Micro-biol. Ecology. University of South Carolina Press,Columbia.

12. Matches, J. R., and J. Liston. 1974. Mesophilic clostridiain Puget Sound. Can. J. Microbiol. 20(1):1-7.

13. Shewan, J. M. 1961. The microbiology of sea water fish.In G. Borgstrom (ed.), Fish as food, vol. I. AcademicPress Inc., New York.

14. Shinjo, T., and M. Ogata. 1965. Studies on the genusClostridium. H. Clostridial flora in soil. Jap. J. Vet.Sci. 27:305-308.

15. Smith, H. W., and W. E. Crabb. 1961. The faecalbacterial flora of animals and man: its development inthe young. J. Pathol. Bacteriol. 82:53-66.

16. Smith, L. DS. 1968. The clostridial flora of marinesediments from a productive and a non-productivearea. Can. J. Microbiol. 14:1301-1304.

17. Smith, L. DS., and M. V. Gardner. 1949. The occurrenceof vegetative cells of Clostridium perfringens in soil. J.Bacteriol. 58:407-408.

18. Smith, L. DS., and L. V. Holdeman. 1968. The patho-genic anaerobic bacteria. C. C. Thomas Co., FortLauderdale, Fla.

19. Taylor, A. W., and W. S. Gordon. 1940. A survey of thetype of Cl. welchii present in soil and in the intestinalcontents of animal and man. J. Pathol. Bacteriol.50:271-277.

20. Yamagishi, T., S. Ishida, and S. Nishida. 1964. Isolationof toxigenic strains of Clostridium perfringens from thesoil. J. Bacteriol. 88:646-652.

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