Effect of Temperature on Growth and Alpha Toxin Production by Clostridium perfringens

Abstract

Growth and alpha toxin production by a strain of Clostridium perfringens was determined in Thioglycollate medium, beef broth with ground beef, and beef broth with ground beef and soy protein. Incubation temperatures ranged from 1S to SO C. In Thioglycollate medium, maximum alpha toxin production occurred at 3S C and was 40 times greater than that observed at 4S C. However, generation time and maximum population were approximately the same at 3S and 4S C. At l S C, a two log cycle reduction in viable counts occurred within 6 h. Irrespective of incubation temperature, alpha toxin levels in Thioglycollate medium declined as the incubation period was extended beyond the stationary growth phase. In the beef broth with ground beef system which was studied at 3S Conly, the organism grew slower and produced less toxin than in Thioglycollate medium. The amount of alpha toxin detected was influenced to a greater extent by the incubation time and temperature, the holding time beyond the stationary growth phase, and the growth medium than by the population level of C. perfringens. It has been suggested that quantification of alpha toxin produced by Clostridium perfringens can be used as an index of growth of the organism (8). Results obtained by the alpha toxin procedure may be influenced by several factors. For example, a wide variation has been demonstrated between strains of C. perfringens with respect to alpha toxin production (6,11,15). Temperature and substrate are known to influence biological activity of all microorganisms and would also likely affect the growth and alpha toxin production of C. perfringens. This paper reports on the effects of incubation time and temperature as well as on effect of the nature of the substrate on estimation of numbers of C. perfringens by alpha toxin assay. MATERIALS AND METHODS Test organism The strain (OSU 1) of C. perfringens used in the experiment was obtained from the Department of Animal Science, The Ohio State University, Columbus, Ohio. The organism was propagated in Thioglycollate medium. Test media The test media included Brewer's Thioglycollate medium (Difco) and beef broth (RJR Foods, Inc .. Winston-Salem. North Carolina) with added ground beef or a mixture of ground beef and Promine-D (Central Soya, Chicago, Illinois). The beef broth was blended with 20% 'Approved as Journal Series Article IOI-78, Ohio Agricultural Research and Development Center, Wooster. ground beef, or 14% ground beef and 6% Promine-D and sterilized (121 C for 1S min) in the blender jars. These media were blended well before use and distributed into previously sterilized SO-ml screw-capped Erlenmeyer flasks. No attempt was made to control final Eh of the beef/soy protein systems. Measurement of growth and alpha toxin production One tenth milliliter of an overnight broth culture of C. perfringens was inoculated into 40 ml of the test medium contained in an Erlenmeyer flask. For the Thioglycollate medium, incubation was carried out at 1S, 2S, 3S, 4S, and SOC in a temperature-controlled water bath. However, when the beef broth with ground meat and/or soy protein were used as the test media, optimum incubation temperature (3S C) was used. Samples were taken at selected time intervals in sterile test tubes (12 x 100 mm) for the measurement of viable counts, production and pH. The viable cell population was estimated by pour plates prepared with SFP agar (17) without addition of Polymyxin B sulfate and Kanamycin sulfate. Alpha toxin production was measured by the Hemolysin Indicator (HI) plate test of Duncan and Harmon (5) with modifications suggested by Park and Mikolajcik (12). The pH was measured using a PHM 62 Standard pH Meter (Radiometer, Copenhagen). Growth curves were constructed by plotting the logarithm of the colony-forming units (CFU) versus incubation time. The generation time was calculated by the formula: Gt = t/n = t/(3.3 log10 b/a) where Gt (generation time) is equal to the time, t = the elapsed time between measurement of a, the initial population, and b, the final population, divided by the number of generations, n (number of generations being equal to 3.3log 10 b/a. RESULTS AND DISCUSSION Growth and alpha toxin production in Thioglycollate medium. Results for cell growth, toxin production and pH changes at 25, 35, 45 and 50 C are shown in Fig. 1-4. Data at 15 C are not presented because the organisms failed to grow and exhibited a two-log reduction in counts within 6 h of incubation. In general, increases in alpha toxin activity closely paralleled population increases and a population of at least 400,000/ml was required before alpha toxin activity could be detected. These data are in agreement with those found by Harmon and Kautter (7) who showed that there is a relationship between C. perfringens population in a food sample and alpha toxin activity. It was observed that the incubation temperature strongly influenced the rate of alpha toxin production and maximum yield of alpha toxin. The optimum temperature for alpha toxin production was 35 C (Fig. 2). The rate of alpha toxin production was closely related to the growth rate,