Microbiology Procedure

Index >> Microbial Genetics >> Replica Plating Techniques

Replica Plating Techniques

	Replica Plating Techniques -Nutritional and various other types of mutants are often detected by replica plating technique, developed by Joshua and Esther Lederberg in 1952 in order to provide direct evidence for the existence of preexisting mutations. Their actual experiment concerned with replicating master plates of sensitive cells to two or more plates containing either streptomycin or T1 phage. When the replicas were grown, they were compared to the locations of colonies on the master plate and any resistant colonies that appeared at the same position on all the replica plates were marked. The area of the master plate corresponding to the marked areas was cut out and the bacteria on it resuspended in liquid medium.
This method has been applied in numerous experiments to identify the occurrence of mutations. Many of the biochemical pathways in microbes were elucidated in this way by using nutritional mutants. Replica plating allows the observation of microbes under a series of growth conditions. In replica plating, a piece of sterile velvet is touched to the surface of an agar plate containing surface bacterial colonies.
The fibres of velvet act as fine inoculating needles, picking up the bacterial cells from the surface of this master plate. The velvet with its attached microbes is then touched to the surface of a sterile agar plate, inoculating it. In this manner, microbes can be repeatedly stamped onto media of differing composition. The distribution of microbial colonies should be exactly replicated on each plate unless the colonies represent different genetic strains. Should a colony that develops on a complete medium fail to develop on a minimal medium that lacks a specific growth factor, the occurrence of a nutritional mutant is indicated. The microbes that do not grow on the minimal medium represent auxotrophic strains.
This technique was specifically developed to answer an important and controversial question did mutations occur spontaneously, or directed by the selective agent? For example in case of penicillin resistance, does penicillin direct the mutation or does it simply select out a naturally occurring spontaneous mutant? It was shown by statistical analysis of mutants that the process is indeed a spontaneous one. This method also allows the isolation of penicillin resistant mutants without ever exposing the cells directly to penicillin

Home | Site map | Submit Article | Resources | Search

Language >> English | Français | Español | Deutsch | Italiano | Portugues | Japanese | Korean | Sim-Chinese