To assess the frequency of mutations within the cII gene, infected E. coli were diluted in TB1 top agar, spread on ten 100-mm TB1-agar plates and incubated at 24 °C for 48 h. The plaques were enumerated, picked and replated to confirm that they were lytic. This provided the number of mutated phage within the sample. To determine the total number of phage undergoing the lysogenic cycle and the mutation frequency, 10-and-50-fold dilutions of the stock of infected E. coli in TB1 top agar were spread on two 100-mm TB1-agar plates and incubated at 37 °C for 24 h. Incubation at 37 °C switches all phage to the lytic cycle, and therefore allows the total number of replication-competent phage to be assessed. The mutation frequency was then calculated as the number of cII-mutated plaques (24 °C)/total number of plaques (37 °C).
