REPLICA PLATING AND INDIRECT SELECTION OF BACTERIAL MUTANTS

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Abstract
RP is a method of copying a pattern of microbial growth from one agar plate to a series of others. A sheet of pile fabric (velvet, velveteen) is fastened with a hoop over a cylindrical support. The initial plate is inverted and gently pressed down on the velvet to transfer the pattern of colonies. This is re-impressed on fresh agar plates in the same way. RP is applicable to many routine chores involving repetitive testing of many isolates: detection of nutritional variants, antibiotic sensitivity spectra, multiple sugar fermentation tests, and phage typing. The RPs show a minimum of distortion, allowing 200 to 400 colonies per plate, of which any number of copies can be made on different agar media. RP was applied to the problem of the origin of bacterial variants resistant to streptomycin (sm) or phage. The variants of Escherichia coli occur as clonal clusters in films of growth on plain agar: RP to a series of sm or phage plates demonstrates the resistant variants at superimposable locations, which reveal the sites of the clones oh the initial plain agar plate. Inocula from the indicated sites are enriched about 100X in the proportion of resistant variants, (indirect selection). By repeating indirect selection, the variants were obtained in pure and stable culture, without any exposure of the selected bacteria to the selective agent at any time. This process is akin to sibship or pedigree selection in animal improvement. It is reaffirmed that microbial "adaptation" to deleterious agents results from pre-occurring spontaneous mutation followed by natural selection.