Casein Hydrolysate Medium

WESSMAN, G. E. (National Animal Disease Laboratory, Ames, Iowa). Cultivation of Pasteurella haemolytica in a casein hydrolysate medium. Appl. Microbiol. 13:426-431. 1965.-The growth of Pasteurella haemolytica strain H44L was studied under aerobic conditions in a medium of acid-hydrolyzed casein, supplementary cysteine, inorganic salts, vitamins, and a carbon source. The concentration of casein hydrolysate necessary for optimal growth was 1.5 or 2.0%, depending upon the carbon source employed. Essential vitamins were calcium pantothenate, nicotinamide, and thiamine. Concentrations as low as 0.01 ,ug/ml of thiamine monophosphate or thiamine pyrophosphate supported maximal growth, but thiamine hydrochloride or thiamine nitrate were active only at the unusually high levels of 10 to 20 jg/ml. The best carbon sources were D-galactose or sucrose. Maximal growth resulted from an inoculum containing fewer than 10 cells per milliliter of medium. Cellular yields averaged 6 X 109 to 7 X 109 cells per milliliter for the test organism and five other strains of P. haemolytica isolated from cases of bovine respiratory diseases. No studies dealing specifically with the nutritional requirements of Pasteurella haemolytica have been reported, although several studies have been done on the closely related species, P. multocida (Banerji and Mukherjee, 1953; Handa, 1958; Carter and Bain, 1960). Berkman (1942), studying the nutrition of various species of Pasteurella, included a single isolate of P. haemolytica; it grew in a medium of hydrolyzed gelatin, supplementary amino acids, glucose, and inorganic salts. The present report concerns the growth of P. haemolytica in a casein hydrolysate medium, and defines some optimal conditions for its culture. MATERIALS AND METHODS Smooth and nonsmooth variants of P. haemolytica H44L were employed in the major portion of the investigation. The smooth variant was isolated from the lung of an animal that died of bovine shipping fever; the nonsmooth variant appeared upon dissociation of the smooth form in static culture. All except the final experiment were conducted with both variants. Where significant differences in response resulted, data for both types are given; otherwise, only the data for smooth variants are reported. Other strains, isolated from cattle with respiratory disease, were tested to determine their ability to grow in the final medium, and are listed in Table 7. The maintenance of strains as stock cultures was described previously (Wessman, 1964). Growth experiments were conducted with 10or 25-ml amounts of medium contained, respectively, in 50or 250-ml Erlenmeyer flasks. Aeration was accomplished by agitation on a rotary shaker (156 rev/min). The cultures were incubated at 37 C. Numbers of viable cells were estimated by means of plate counts as described previously (Wessman, 1964). Turbidimetric measurements of growth were made by reading optical densities on a Bausch & Lomb Spectronic-20 colorimeter, with the use of light of 575-miA wavelength. Readings were made on cultures diluted 1:5 in distilled water with diluted medium as the blank. The medium was a modification of the deacidified casein partial hydrolysate (DCPH) developed by Higuchi and Carlin (1957) for the growth of P. pestis at 37 C. Magnesium, cysteine, vitamins, and carbon sources were prepared as separate filtersterilized solutions, and were added after autoclaving the rest of the medium. For inocula, cells were transferred from blood-agar plates to Brain Heart Infusion (Difco) broth, and grown for 10 hr, with aeration, at 37 C. The cells were separated from 10 ml of medium by centrifugation, washed, and suspended in 10 ml of 0.85% NaCl. This suspension was employed as the inoculum, 1% by volume being added to the medium. Dry-weight determinations were made after washing cells in distilled water and drying over426 on A uust 4, 2017 by gest ht://aem .sm .rg/ D ow nladed fom CULTIVATION OF PASTEURELLA HAEMOLYTICA night at 110 C. The organism was grown in 25-ml amnounts of medium for these determinations.