Conditions necessary for the production of thymineless mutants in Gramnegative bacteria by aminopterin treatment were examined. Although and Proteus strains were readily made ‘thymineless’ (i.e. requiring added thymine) no such mutants were obtained with the majority of Aerobacter strains. However, two methods which produced thymineless mutants in all the Aerobacter strains were found; these were: treatment with alkaline EDTA before, or temperature shift down during, incubation with aminopterin. In every case where temperature shift alone resulted in the production of thymineless mutants more were found when it was used in conjunction with EDTA pretreatment. It was also found that the phase and size of inoculum, the method used to sterilize aminopterin and the concentration of thymine in the plating media had profound effects on the production and detection of thymineless mutants.


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