Chemically Defined Medium

CHAMBERLAIN, ROBERT E. (The National Drug Co., Swiftwater, Pa.). Evaluation of live tularemia vaccine prepared in a chemically defined medium. Appl. Microbiol. 13:232-235. 1965.-A chemically defined medium was prepared which adequately supported growth of a vaccine strain of Pasteurella tularensis. This medium differed from those previously described in: (i) concentration of components, (ii) a requirement for calcium pantothenate to obtain increased growth, and (iii) a low initial pH. Varying the concentration of individual components up to 10 times the standard amount did not increase the viable population or affect dissociation. The vaccine strain grown in this chemically defined medium, although lower in viable population, appears to retain its identity and to be equal in potency to that prepared by the conventional method. This preliminary study indicates the potential utility of this medium as a basis for controlled studies of a live bacterial vaccine in terms of growth characteristics, dissociation, virulence, and immunogenicity. Recently, Eigelsbach and Downs (1961) produced a live tularemia vaccine, which has proved effective in both animals and man. The method of preparation, however, employs three distinct types of media prepared from natural products and six serial transfers. Because of the variations in lots of media and their potential effect upon dissociation, the preparation of the final product requires strict control. In the past few years, certain chemically defined media (Mager, Traub, and Grossowicz, 1954; Traub, Mager, and Grossowicz, 1955; Nagle, Anderson, and Gary, 1960) have been successfully employed for the growth of various strains of Pasteurella tularensis. Use of a medium of this type facilitates controlled study of the factors affecting growth, dissociation, virulence, and immunogenicity. The results obtained in a preliminary study of this possibility are presented. MATERIALS AND METHODS Strains. Lyophilized stock cultures for use in this study were prepared from the live vaccine strain (LVS) kindly supplied by H. T. Eigelsbach. For challenge studies, P. tularensis strain SCHU S4 was employed. The LDS was determined by the method of Reed and Muench (1938). Preparation of media. The standard vaccine media, peptone-cysteine-agar (PCA) and modified casein partial hydrolysate (MCPH), and the gelatin-saline diluent were prepared according to Eigelsbach and Downs (1961). The components of the chemically defined medium (CDM) as finally adopted are listed in Table 1. All components except KH2PO4 and K2HPO4 were prepared as individual solutions. The pH of the complete medium was adjusted to 6.2 to 6.4 with 3 N HCl and sterilized by filtration through an ultrafine sintered-glass filter. CDM agar was prepared by adding agar to CDM liquid to give a final agar concentration of 1%. Preparation of vaccine. Vaccine for the following studies was prepared simultaneously in both complex and chemically defined media according to the method of Eigelsbach and Downs (1961). A lyophilized stock culture was first reconstituted with distilled water. Streak plates were prepared on both PCA and CDM. After 4 days of incubation at 37 C, colonial morphology (Eigelsbach and Downs, 1961) was recorded, and growth was transferred to PCA and CDM slants. Slants were incubated for 24 hr, and transfers were made to a second set of slants. Growth from the second slants was harvested (24 hr later) in 10 ml of gelatin-saline, and 5-ml quantities were transferred to 250-ml Erlenmeyer flasks containing 25 ml of MCPH or CDM liquid media. The flasks were incubated with continuous shaking for 18 hr. Quantities (20 ml) of the first liquid cultures were then transferred to 1-liter flasks containing 200 ml of either medium. These were incubated with shaking for 18 hr, and 25-ml quantities were transferred to 2-liter flasks containing 300 ml of either medium. These were also incubated for 18 hr with continuous shaking. The third liquid cultures were used in the subsequent studies. Viable counts were performed according to Downs et al. (1947). Mouse virulence and immunogenicity. The virulence in mice of vaccine prepared in MCPH 232 on O cber 3, 2017 by gest ht://aem .sm .rg/ D ow nladed fom EVALUATION OF LIVE TULAREMIA VACCINE and in CDM was compared by the following method. Female CFW mice (18 to 20 g) were injected subcutaneously in the inner aspect of the hind leg with serial 10-fold dilutions of vaccine over a range of 102, 104, 106, and 108 viable organisms per 0.2-ml dose. Sixty mice were injected with each dilution, and 60 additional mice received diluent only (gelatin-saline) as control. The number of deaths was recorded daily for a 15-day period. All dead mice were autopsied, their gross pathology was observed, and segments of spleen and liver were removed and streaked on GCB plates. At the end of the observation period, the per cent virulence was calculated for each dose level and the average virulence was determined. The survivors from each dose level were challenged subcutaneously with P. tularensis SCHU S4. The challenged mice were observed for an additional 20 days, after which the per cent protection was determined for each challenge dose level. Guinea pig virulence and skin reaction. The virulence of the vaccines prepared by the two methods was determined by subcutaneous injection of 350-g Hartley strain guinea pigs with 107, 108, and 109 viable organisms per 0.2-ml volume from third broth cultures. The pigs were observed for 15 days after injection. Skin tests were performed by placing two drops of a 1:100 dilution of the third broth culture on the shaved surface of the thigh and lightly puncturing the skin through the vaccine 60 times. The pigs were observed over a 15-day period for typical reaction involving inflammation, followed by pustule formation, scab formation, and sloughing.