Volume 3

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

    76/952 (page 56)
    the fluctuation test illustrates, and which runs through all of bacterial genetics, is that the subject of analysis is not the individual progeny typical of eucaryotic genetics but constitutes populations of bacteria. Other methods of demonstrating that bacterial variation is caused by spontaneous mutation have been developed since the fluctuation test was devised. In the test of Newcombe (1949) plates are spread with (for example) bacteria sensitive to a virulent phage. After incubation to allow each bacterium to multiply into a colony, half the plates are rubbed with a spreader to redistribute the bacteria. Both the rubbed and nonrubbed plates are then treated with phage and the number of resistant colonies is counted. If adaptation is responsible for resistance, both types of plate should contain the same number of surviving colonies, for redistribution prior to exposure to the phage cannot influence their response. If spon taneous mutation is responsible, the rubbed plates should contain many more resistant colonies than the nonrubbed plates; for on the undisturbed plates each resistant mutant will have given rise to a single colony, remaining in one position, whereas on the respread plates each of the colonies will have been separated so that each of its members can grow into a separate resistant colony after plating with phage. Any character for which selection can be imposed without disturbing the bacteria (phages can be sprayed on, for example) can be tested; the results show that phage resistance and other characters are produced by spontaneous mutation. Examining bacteria for hereditary characteristics demanded lengthy protocols until the technique of replica plating was introduced by Leder- berg and Lederberg (1952). Previously, each colony had to be analyzed individually for every characteristic by transferring samples to a series of plates containing appropriate selective media. Analyses of many col onies for recombinants involving the inheritance of several characters could thus become limited by the very large number of individual actions required. Replica plating allows the pattern of growth on a master plate to be transferred as a whole to a series of replica plates. The master plate is pressed gently onto a square of sterile velvet, which acts as an orderly array of many short needles; bacteria adhere to the velvet so that it carries an exact representation of the bacterial growth pattern of the master plate. Replica plates, each consisting of an appropriate medium, are then pressed onto the velvet pad to pick up from it a sample of the bacterial population; several such replicas can be printed from each pad. Up to one third of the bacteria of the master plate can be transferred to the pad and up to one third of these are transferred to the replicas. By keeping the orientation of master and replica plates the same, each colony can be