Oxgall Medium for Identification of Pasteurella Pestis.

with 5% glucose, but without agar, was used in all growth and inhibition studies. Davis and Mingioli (J. Bacteriol. 60:17, 1950) synthetic liquid medium supplemented with 0.4% sodium glutamate was used for inoculum preparation. Inocula were prepared from cells of single colonies. Aerobic growth studies were carried out in a water-bath shaker at 35 C and anaerobic studies in Brewer jars. Comparative sensitivity studies of various agents were done by the serial dilution technique. Serially diluted tubes were read after 20 hr of incubation at 35 C. RD mutants were obtained by growing the wild-type yeast in a liquid medium containing 10 ,ug of acriflavine per ml for several successive transfers. The tetrazolium agar overlay technique of Ogur et al. (Science 125:928, 1957) and Nagai (Science 130:1188, 1959) was used to detect RD mutants. Respiration deficiency was confirmed by the glucose-limiting method of Ogur and St. John (J. Bacteriol. 72:500, 1956). The acriflavine-treated yeast culture, when plated on acriflavine-free medium, gave rise only to RD colonies. However, the RD colony population was of two main types distinguishable by colony size. More than 99% of the colonies were uniformly small or "petite" in size, measuring 1 mm in diameter. The few remaining colonies were 2 mm in diameter and designated as large RD mutants. Wild-type colonies on control plates measured about 4 mm in diameter. When platings were done of each of the RD types, the resulting clones were all respiration deficient, with more than 99% retaining their characteristic colonial size. A characteristic small colony "petite" and large colony "large RD" were selected for further study. Microscopic INCUBATION TIME (HOURS)