THE DELAYED APPEARANCE OF MUTANTS IN BACTERIAL CULTURES

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Abstract
In an attempt to explain the variable delay in the onset of cell division for UV mutants, 4 strains of Escherichia coli (h-lac-,h-lac+,h+lac+, and h+lac-) were grown in minimal medium + histidine. The irradiation dosage used (120 sec. at 14 cm from a Westinghouse Sterilamp) gave a survival of ca 10-6 bacteria, with a frequency of ca 10-3 h+ mutants among the survivors. Two tenths milliliter of concentrated irradiated culture of h-lac+ or h-lac- bacteria were spread on agar plates devoid of histidine. To half of the h-lac+ plates 0.5 ml of a 10-3 dilution of an irradiated culture of h+lac" bacteria were added, and to half of the h"lac" plates similarly treated h+lac+ bacteria were added, in order to reveal any effect of the lac marker. The plates were incubated at 37[degree]C and removed only for respreading, after which they were reincubated for 3 days before counting the colonies. Any h+ colonies present were tested on Endo agar for their ability to ferment lactose. The growth curve for added h+ bacteria indicated an avg. lag period of about 1.5 hrs. and an average generation time of 36 min., whereas for h+ mutants resulting from irradiation, the lag period ranged from 5 hrs. to 27 hrs. This difference was not due to the lac marker and the hypothesis of segregation lag was rejected as untenable. A type of phenotypic lag was involved, but the delay was more than that required for phenotypic expression. When h-lac+ bacteria, were irradiated for only 20 sec., resulting in a survival of ca 10"3 bacteria, the new mutants showed little if any difference in lag from old mutants. Cultures of h- bacteria irradiated for 120 sec. were removed at intervals from plates devoid of histidine and plated on agar with and without histidine. On plates both with and without histidine the h" cultures showed almost normal growth, whereas the h+ bacteria showed a lag period of 10-17 generations on plates without histidine and a lag period of up to 9 generations on those with histidine. The newly arisen h+ mutants apparently differed from old h+ mutants and their h- parents in that high UV doses induced a marked delay in the onset of their growth. The possible relationship between delay in onset of division and mutation is discussed.

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