PROCEDURE FOR ISOLATION OF ESCHERICHIA, SALMONELLA, AND PSEUDOMONAS MUTANTS CAPABLE OF GROWTH AT THE REFRACTORY TEMPERATURE OF 54-DEGREES-C

When fully grown cultures of the genetically well characterized genera Escherichia, Salmonella and Psuedomonas were placed at 48-degrees-C for at least 4 days a few (frequency almost-equal-to 10(-8)) mutants able to grow continuously at 48-degrees-C were isolated. None were ever obtained immediately, implying mutation by heat stress but not selection of preexisting mutants. These mutants (thermotolerant or T48 mutants) capable of growth at 48-degrees-C had a generation time of almost-equal-to 35 min at 48-degrees-C. Incubation of the T48 mutants at 54-degrees-C for at least 4 days yielded a few (frequency almost-equal-to 10(-8)) mutants (mesothermophilic or T54 mutants) able to grow continuously with division every 30-40 min at 54-degrees-C. Thus, by the stepwise procedure of prolonged thermal stress, mutants which grow at 54-degrees-C were obtained from these three mesophilic genera. Isolation of mesothermophilic Salmonella (T54) mutants was also achieved directly from mesophilic parents at a frequency of < 10(-10) when the mesophilic strain was incubated for at least 10 days at 54-degrees-C suggesting two stepwise mutational events can take place during a very prolonged exposure to thermal stress. T48 and T54 mutants were found to require cellobiose as an energy source for growth on minimal agar and exhibition of parental auxotrophic genotypes in the thermal environment.