MODIFICATION OF THE PENICILLIN TECHNIQUE FOR THE SELECTION OF AUXOTROPHIC BACTERIA

With the development of the "penicillin technique" independently by Lederberg and Zinder (1948) and by Davis (1948), truly selective cultural conditions were established for the auxotrophic members of mixed populations. This technique takes advantage of the fact that penicillin sterilizes only growing cells; incubating a mixture of wild type and auxotrophic cells in minimal medium contaiing penicillin greatly increases the proportion of auxotrophs in the surviving population. The present paper describes a modification of the selection technique, the novel feature of which is the use of solid medium throughout all steps; under these conditions, each mutation gives rise to one colony at the end of the procedure. Secondly, by keeping the cells in minimal medium at all times prior to the final enrichment with growth factors and by applying penicillin to microcolonies instead of to single cells, the yield of auxotrophs is raised to a high and reproducible level. Finally, the feature of "delayed enrichment" miimizes the number of wild type colonies among colonies to be picked. With the strain of Eswherichia coli used in most of these experiments over 90 per cent of the picked colonies were stable mutants, representing from 2 per cent to 3 per cent of the ultraviolet survivors.