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Spontaneous Mutations

	Spontaneous Mutations - Definite proof to show that mutations occur a spontaneously in bacteria came in 1943 from experiments conducted by Salvador Luria and Max Delbruck. These workers used an E. coli strain susceptible to a bacteriophage but that would also yield phage resistant variants. The question that Luria and Delbruck asked was whether in this bacterium, resistance to the bacteriophage arose as a result of exposure to the phage or whether such resistant clones are always present in the bacterial population but are selected only in the presence of the Phage.
To verify this, one half of a dilute suspension of E. coli was dispensed in I ml aliquots in individual tubes, while the other half was left in the flask. The cultures were allowed to grow until the cell number had increased to about 108 per ml. Each sample in the tube and the culture in the flask were then exposed to a phage and after a while, one ml aliquot was plated to determine the number of phage resistant mutants (Survivors).They predicted that if resistance development to the bacteriophage occurred after the bacteria were exposed to the phage, then all the samples should contain the same number of resistant cells.
As a consequence, each plate should contain the same number of resistant clones. On the other hand, if resistance to phage was the result of a spontaneous mutation, then the number of resistant colonies on each plate should vary. Luria and Delbruck found that the number of resistant mutants fluctuated from sample to sample indicating that mutants existed in the population prior to exposure to the phage and concluded that mutations in bacteria occur spontaneously. These conclusions were further corroborated by Josua Lederberg and Esther Lederberg by the use of the replica plate technique. The technique consists of first plating a small number of the test bacteria on a master plate and incubating it until growth occurred.
A circular piece of wood of the size equal to the inner diameter of the Petri plate is then covered with a piece of sterile velvet cloth and the Master plate is then applied to the velvet so that the clones on the plate are transferred to the cloth.The velvet pad is then gently in printed on several plates containing an inhibitor or a selective agent to detect resistant mutants.
	It was reasoned that if resistant mutants had developed on the master plate before exposure to the inhibitor, then such resistant colonies should be located at exactly the same position on each of the replica plates, while if mutations occurred as a consequence of exposure to the inhibitor then the resistant colonies should be at different locations on different replica plates. The Lederbergs found precisely the former, proving that spontaneous mutations occur in bacteria in the absence of a selective agent. Since then this replica plate technique has been extensively used as a basic technique for mutant detection.

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