Use of Safranin O for Characterization of Brucella Species.

Moreira-Jacob (J. Bacteriol. 86:599, 1963) reported that 44 strains of Brucella suis were inhibited by safranin 0 in Brucella Agar (Albimi), whereas nearly all of 320 other strains of Brucella were not inhibited. Brinley-Morgan (World Health Organization, Brucellosis document , 246, 1963) examined the growth of more than 80 cultures on serum dextrose agar with added safranin 0, and found that all B. abortus and B. melitensis cultures grew well, whereas 16 of 18 B. suis cultures did not grow. Although only a limited number of cultures were examined in the present study, several hitherto unreported observations were made. Safranin 0 (National Aniline Div., Allied Chemical Corp., New York, N.Y.) was used at a final concentration of 1:5,000 in Trypticase Soy Agar (BBL). Known numbers of organisms from 10 to 1010 were inoculated on media with and without the dye. Five cultures of B. suis and two cultures of B. neotomae (Stoenner and Lackman, Am. J. Vet. Res. 28:947, 1957) were tested and were inhibited by safranin. No evidence of safraninresistant colonies was apparent in these cultures, even when 1010 organisms were inoculated. We found that B. neotomae has several additional characteristics in common with B. suis. Although B. neotomae is inhibited by the concentrations of thionin and basic fuchsin usually used, it will grow in the presence of 1:150,000 thionin but not 1:150,000 basic fuchsin. Both B. neotomae and B. suis show a lytic reaction with brucella phage at 10,000 X routine test dilution (RTD) but not at RTD. Both B. neotomae and B. suis are inhibited by thionin blue at 1:500,000 concentration and by penicillin at a concentration of 5 units per ml. A total of 35 B. abortus cultures were examined for sensitivity to safranin. All 12 C02-dependent strains of B. abortus biotypes 1, 3, 4, and 5 (Stableforth and Jones, Intern. Bull. Bacteriol. Nomen. Taxon. 13:145, 1963) were able to grow well, whereas biotype 2, a dye-sensitive type, would not grow. In general, C02-independent strains, such as 19, 11, and 45/0, did not grow in the presence of safranin, although safranin-resistant colonies appeared in these cultures at a frequency of about 1 in 10$ organisms. Of 15 C02-independent cultures which resembled B. abortus strain 19 on the basis of thionin blue and penicillin sensitivity, and which had been isolated from the milk of cattle that had been vaccinated with strain 19, 13 resembled strain 19 in safranin sensitivity, but the two remaining cultures were able to grow on safranin. B. abortus strain 2308, a highly virulent CO2 -independent strain, grew in the presence of safranin. Only one of seven B. melitensis cultures tested grew well in the presence of safranin. The remaining six were inhibited, but had a high frequency of resistant mutants. Reports on the sensitivity of B. suis cultures to the dye safranin 0 were confirmed, and two B. neotomae cultures were found to be equally sensitive. In contrast to the findings of others, differences in the sensitivity among B. abortus and B. melitensis cultures were observed. In general, C02-dependent B. abortus cultures grew well in the presence of safranin, whereas C02independent cultures were inhibited.