Heat Resistance of Clostridium perfringensl

CANADA, JAMES C. (University of Wisconsin, Madison), AND DOROTHY H. STRONG. Effect of animal alimentary passage on the heat resistance of Clostridium perfringens. Appl. Microbiol. 13:788-792. 1965.-The resistance to heat, as measured by D values and phantom thermal death time curves, was observed to increase for one of three strains of Clostridium perfringens type A subsequent to animal passage. Animal passage was accomplished by the force-feeding of germ-free mice with bacterial suspensions of the organism, followed by the force-feeding of additional gnotobiotic mice with the contaminated feces. For the one strain in which an increase in heat resistance was noted, the result could not be attributed to mouse feces per se, since the presence of sterile germ-free mouse feces in a suspending medium did not protect C. perfringens spores from elevated temperature destruction. Results from separate research studies have led to two apparently contradictory conclusions concerning the basis for different degrees of heat resistance which have been observed among strains of Clostridium perfringens. (i) Foodpoisoning strains of C. perfringens are more heatresistant soon after their isolation from mammalian feces than after an extended maintenance on laboratory media (Hobbs et al., 1953), and (ii) the resistance to the lethal effects of heat is genetically controlled for each strain of the organism and is not an adaptive mechanism (Collee, Knowlden, and Hobbs, 1961). Because of the uncertainty suggested by these reports, it appeared further investigation was justified. Experiments were designed to determine whether the heat resistance of laboratory strains of C. perfringens type A would be increased after the strains were associated with feces through animal passage. MATERIALS AND METHODS Three strains of C. perfringens type A were used in the study: S-45 (Hall et al., 1963), 214D (Strong and Canada, 1964), and 65 (Canada, Strong, and Scott, 1964; Strong and Canada, 1964). These 1 Published with the permission of the Director of the Wisconsin Agricultural Experiment Station, Madison. This paper is part of a thesis submitted by the senior author to the Graduate School of the University of Wisconsin in fulfillment of the requirements for the Ph.D. degree. 2Present address: Gerber Products Co., Fremont, Mich. strains of the organism had been carried in veal broth for several months. The research plan was as follows. A standardized bacterial suspension (I) was prepared with a stock culture inoculum. The resistance that the spores of this suspension displayed toward elevated temperature stress was measured. Bacterial suspension I was also used to contaminate orally a germ-free mouse (Ha/ICR, Schmidt). After an incubation period of 48 hr, the feces from this mouse were used to contaminate orally another germ-free mouse. After a total of six such alimentary passages through mice, the organism was recovered from feces in Thioglycollate Medium (Difco). Another standardized bacterial suspension (II) was prepared with the fecesinoculated Thiogylcollate culture as inoculum. The resistance of the spores of bacterial suspension II to elevated temperature stress was measured, and the resistance values were compared with those obtained from suspension I. Bacterial suspension I was prepared by inoculating a tube containing 20 ml of Fluid Thioglycollate Medium (Difco) with 1 ml of stock culture grown in veal broth. Bacterial suspension II differed only in being seeded with a single dropping from a C. perfringens-exposed mouse after six transfers. After 24 hr of incubation at 37 C, 1 ml of the Fluid Thiogylcollate Medium culture was transferred to 10 ml of tubed Thioglycollate Medium without sugar. After incubating for 4 hr at 37 C, 1 ml of this second culture was transferred to each of two tubes containing 10 ml of SEC sporulating broth (Hall et al., 1963). The SEC broth cultures were allowed to incubate for 40 hr at 37 C. They were then combined and diluted in cold SEC broth to an optical density (OD) of 0.06 788 on N ovem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom HEAT RESISTANCE OF C. PERFRINGENS TABLE 1. Scheme for the identification and treatrnent of thermal death time tubes IdentificaTreatment tion no. 1 Entered into germ-free mouse isolator 2 Entered into germ-free mouse isolator 3 Not pasteurized (total count) 4 Not pasteurized (total count) 5 Not pasteurized (total count) 6 Pasteurized (spore count) 7 Pasteurized (spore count) 8 Pasteurized (spore count) 9 Heated at 80 C for 20 mi 10 Heated at 80 C for 25 min 11 Heated at 80 C for 30 miii 12 Heated at 85 C for 7 min 13 Heated at 85 C for 15 min 14 Heated at 85 C for 25 min 15 Heated at 90 C for 2 min 16 Heated at 90 C for 5 min 17 Heated at 90 C for 10 min 18 Heated at 95 C for 1 min 19 Heated at 95 C for 5 min 20 Heated at 95 C for 10 min (660 my), as measured by a Spectronic-20 colorimeter (Bausch & Lomb, Inc.). This OD value approximated 10,000,000 viable cells per milliliter. The diluted bacterial suspension was then chilled in an ice-water bath. A 1-ml amount of the chilled suspension was injected into each of 20 thermal death time (TDT) tubes (Canada et al., 1964). The tubes were sealed with flame, numbered, and immersed in ice water. A scheme, identifying each TDT tube and its treatment, is presented in Table 1. TDT tubes numbered 1 and 2 were transported to the supplier of the germ-free mice. The exterior of the sealed tubes was sterilized with peracetic acid, and theni the tubes were aseptically entered into a film isolator containiing seven 6-week-old, female, germ-free mice. Other sterile material provided in the isolator included forceps, pliers, 1ml syringes, needles, cloth toweling, screw-capped tubes of Fluid Thiogylcollate Medium, a vial containiing 70% ethyl alcohol, mouse cages, drinking water, feed (Purina 5010 I)iet; Ralston Purina Co.), and bedding. The first of the seven mice was orally inoculated with the bacterial suspension. The process was accomplished by snipping off one end of a TDT tube with pliers. A sample (0.5 ml) of the suspensioni was withdrawn with a needle and syringe, and a portion of this sample was then force-fed to