A procedure is described for identifying and enumerating Clostridium perfringens in foods by means of a simplified agar plating method, followed by confirmation of black colonies in tubes of motility-nitrate medium and sporulation broth. The test is routinely completed within 48 hr. Under experimental conditions, the procedure has been used to quantitatively recover various levels of C. perfringens contamination in a variety of foods and has recovered as few as ten C. perfringens per g without interference from food constituents and associated flora. Under practical conditions of field application, the method has been used to investigate five food-poisoning outbreaks, and C. perfringens was implicated as the etiological agent in two of these outbreaks. Clostridium perfjringens is recognized as the principal cause of gas gangrene in man and as the specific causal agent of lamb dysentery; lamb, sheep, and newborn calf enterotoxemia; "struck" in sheep; and an acute and fatal disease of very young piglets. However, outside of Great Britain, relatively little attention has been given to the possibility that this organism is also capable of causing gastroenteritis in man. C. perfringens is one of the principal causes of foodborne illness in the British Isles, and practically all of the outbreaks described in the literature involve meat dishes. Usually the meat was cooked the day before serving and allowed to cool slowly, either in its own juices or separately. Apparently, the two factors of precooking and lack of immediate refrigeration allow the development of the extremely large numbers of C. perfringens associated with this type of food poisoning. English studies (Knox and 1\IacDonald, 1943; Duncan, 1944; Hobbs et al.; 19.53; Dische and Elek, 1957) reveal C. perfringens food poisoning is caused by atypical type A strains of the organisms. They produce heat-resistant spores, produce relatively small amounts of the a-toxin, and rarely produce any 0-toxin. In the outbreaks reported by McClung (1945) and by Hart, Sherwood, and Wilson (1960) in the United States, the clinical picture resembled that of the English outbreaks, and strains of C. perfringens, type A, were isolated. At the present time, only a few laboratories examine foods incriminated in foodborne disease outbreaks for C. perfringens because of the lack of quantitative methods and the difficulties associated with anaerobic cultivation. To facilitate examination of foods for C. perfringens, investigations have been in progress in this laboratory on the simplification of methods for identification and enumeration of this organism. A procedure has been developed and is presented below. MATERIALS AND MIETHODS Cultures. Included in this study were members of each of the six recognized types of C. perfringens (A through F) obtained from both the American Type Culture Collection and National Collection of Type Cultures (Great Britain). Also included were those strains most frequently encountered in food-poisoning outbreaks in England and designated as Hobbs' strain...

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England, Europe, and Asia, associated with foodpoisoning outbreaks; 28 from the United States, associated with outbreaks or contaminated foods; and 25 from natural or pathological sources-have been studied to determine their serological relationships, sporulation and heat-resistance of spores, and their hemolytic activity on mammalian bloods. A comparison of the results obtained with these three groups of strains reveals that the Eurasian group is characterized by serological typability, poor sporulation with the production of heat-resistant spores, and a hemolytic activity limited to the production of partial hemolysis on horse, ox, and sheep bloods, whereas the strains from natural and pathological sources in this country are not serologically typable, sporulate well but the spores are not heat-resistant, and are hemolytically active, producing both partial and complete hemolysis on horse, ox, and sheep bloods. The American food-poisoning strains have a wide variety of characteristics. Some strains resemble the Eurasian in their serological typability and the production of heatresistant spores, but sporulation and hemolytic activity are more like the strains from classical sources. On the basis of these data, it appears unlikely that C. perfringens food-poisoning out

RECOVERY of coagulase-positive Staphylo¬ coccus aureus from food implicated in a foodpoisoning outbreak is only circumstantial evi¬ dence that the correct etiological agent has been found. Even the determination by animal studies that the strain isolated is capable of pro¬ ducing enterotoxin does not prove that it did so in the food. Positive proof depends on dem¬ onstration of the enterotoxin in the food itself.

Numerous investigations have been conducted to fill the need for simple, reliable, bacteriological tests to determine quantitatively the sanitary quality of food contact surfaces. They have resulted in the development of various swabbing (3, 4, 5, 7, l o ) , rinsing (2, l l ) , and agar contact methods (6,8,9,12). Some methods are better suited for examining flat surfaces than for examining food containers, eating utensils, or processing equipment, etc. ; in fact, there is no one method, presently available, that is applicable to the diversified surfaces encountered in processing plants, restaurants, and related food handling establishments. The plant sanitarian is therefore confronted with the problem of deciding which of the available methods will meet his requirements most satisfactorily. Moreover, information is not available on two important factors to be considered in making such a decision ; namely, the average proportion of the total contamination that a particular method recovers and the reliability of its results.Recently a direct surface agar plate laboratory method (DSAP) (1) has been developed which, based on triplicate determinations, is capable of detecting 88.5% to 9 . 3 % of the bacterial spore contamination on nonporous surfaces in 95 trials out of 100. This 95% confidence interval is based on experiments in which triplicate plate counts of a contaminating suspension were used as the bases for these percentage recoveries. In addition, the coefficient of variation of individual DSAP counts was shown to be 7.1% which is equivalent to the precision of the conventional agar pour plate method of enumerating bacteria. Although this method is probably too complicated for practical application routinely, it has provided an excellent reference procedure for quantitative comparison of surface recovery techniques in terms of the proportion of contamination detected and the precision of the results.Employing the DSAP method as the reference procedure a comparison was made of representative recovery methods and the results evaluated statistically. EXPERIMENTAL PROCEDUREInoculation of test surfaces 1. iCTicrocorciis pyogciies var. aiireiis 209. Maintenance of stock cultures and preparation of milk suspensious were identical to the procedures previously described (1). The fii:al su\pensions contained approximately 10,000 to 13,000 organisms per ml. as determined by triplicate plate counts on tryptone glucose extract agar incubated at 35" C. for 48 hours.Vitreous china salad dishes were selected as test surfaces, because they are readily availablc, can be easily manipulated, and their smooth nonporous surfaces are typical of