Mutations in E. coli for resistance against phages or antibiotics

Mutations : 2.  Biochemical Level (Biochemical and Microbial Genetics)
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Mutations in E. coli for resistance against phages or antibiotics
Cell counting in suspension
Calculation of mutation rates and frequencies
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Gene sequences and enzyme sequences in biosynthetic pathways
Mutations for resistance can be isolated by spreading large population of bacteria (109 cells) on a plate containing thick layer of phage (e.g., T1) or an antibiotic as the case may be. The surviving colonies would be resistant and represent mutants. S. Luria and M. Delbruck (in 1943) conducted experiments to find the origin of these mutants. There were two possibilities : (i) these could be random genetic changes or (ii) these could arise as physiological adaptation to the presence of phage or antibiotic. In order to test, which of these two alternatives was correct, Luria and Delbruck allowed growth of E. coli cells (using 103 cells per ml) in two sets (i) 20 tubes each with 0.2 ml of medium, and (ii) one tube with 10 ml of medium. After an incubation period, each of the 20 tubes (0.2 ml) were spread on plates with a layer of phage (or antibiotic). Similarly ten 0.2 ml samples from 10 ml tube in (ii) above were drawn and plated on 10 plates. It was shown that individual 0.2 ml cultures showed large variation, while samples drawn from 10 ml bulk culture did not show any variation in the frequency of resistant colonies. The variation in 0.2 ml culture was mainly due to differences in the time of appearance of mutation during multiplication. The experiment proved that mutations appeared as random genetic changes and not as physiological adaptation, because if the latter was true, no variation in mutation frequency in 0.2 ml tubes should be expected.