REPLICA PLATING AND INDIRECT SELECTION OF BACTERIAL MUTANTS
JOSHUA LEDERBERG AND ESTHER M. LEDERBERG Department of Genetics,' College of Agriculture, University of Wisconsin, Madison, Wisconsin Received for publication August 31, 1951
Elective enrichment is an indispensable technique in bacterial physiology and genetics (van Niel, 1949). Specific biotypes are most readily isolated by the establishment of cultural conditions that favor their growth or survival. It has been repeatedly questioned, however, whether a selective environment may not only select but also direct adaptive heritable changes. In accord with similar discussions in evolutionary biology (Huxley, 1942), we may denote the concepts of spontaneous mutation and natural selection in contrast to specific induction as "preadaptation" and "directed mutation", respectively. Many lines of evidence have been adduced in support of preadaptation in a variety of systems (Luria and Delbruick, 1943; Lea and Coulson, 1949; Burnet, 1929; Newcombe, 1949; Lewis, 1934; Kristensen, 1944; Novick and Szilard, 1950; Ryan and Schneider, 1949; Demerec, 1948; Welsch, 1950; also reviewed: Braun, 1047; Luria, 1947; Lederberg, 1948, 1949). This paper concerns an approach to this problem that makes use of a replica plating technique which facilitates the handling of large numbers of bacterial clones for classification on a variety of media. METHODS
Replica plating. A frequent chore in bacteriological work is the transfer of isolates from one substrate to other selective or indicator agar media. In place of an inoculating needle, one might imagine a device consisting of many needle tips in fixed array, so that one operation would substitute for repeated transfers with a single needle. The requirements of this design are met by pile fabrics such as velvet or velveteen. The pile provides space in a vertical plane for moisture that might otherwise cause lateral smearing of any impression. (According to Dr. N. Visconti, in a private communication, dampened filter paper may be applicable to some replication problems considered by him independently of the present work.) In our practice, twelve cm squares were cut from velveteen yardage, packed in large petri dishes, and sterilized in the autoclave. A square is placed, nap up, on a cylindrical wood or cork support of nine cm diameter and held firmly in place wvith a metal flange or hoop pushed over the fabric and around the rim of the support. The agar plate carrying the initial colonies is inverted onto the I No. 473. This work has been supported by grants from the Research Committee, Graduate School, University of Wisconsin, with funds supplied by the Wisconsin Alumni Research Foundation, and from the Division of Research Grants and Fellowships, National Microbiological Institute, National Institutes of Health, U. S. Public Health Service. 399
JOSHUA LEDERBERG AND E. M.
fabric with slight digital pressure to transfer the growth. The imprinted fabric then provides the pattern for transferring replica-iniocula to subsequent plates impressed in the same way. Replica plating is used to facilitate routine tests inivolvinlg repetitive inoculations of many isolates on different media. Such tests are frequenitly required in
Figure 1. Replica platinig for the isolation of auxotrophic colonies. A, Initial plate; B, Replica; both on complete agar medium. C, Second replica to minimal agar. The arrows designate the auxotrophic colonies which fail to grow oni minimal medium. The resolution of these replicas is of fair to average quality.
genetic work, but the method should be applicable to other routine practice. Traits which lend themselves to classification by replica plating include antibiotic-sensitivity spectra, responses to bacteriophages (as in phage typing), fermentation characters, nutritionial requirements, or any characteristic for which a selective or iindicator agar medium can be devised. An...
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