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J. Lederberg and E. Lederberg (1952) devised this procedure to demonstrate the spontaneous nature of mutations.
This method is used for detection of biochemical mutants, for the classification of fermentation reactions and for the determination of the spectra of antibiotic sensitivity.
Phage sensitive strain of E.coli on nutrient agar plate is incubated until each cell has formed a clone of a few hundred progeny. On the surface of the agar plate this will appear as a confluent growth. If a few cells of these 108 are spontaneous phage resistant mutants, they will produce phage resistant clones on the agar. The problem is to find out these phage resistant mutants from within the confluent lawn of cells and to isolate it. This is accomplished by replica plating method.
Principle:
Using the threads of velvet or chamios leather which act as inoculating needles, the mutants can be replicated and isolated.
Requirements:
1. Chamios leather/velvet mounted on cylindrical blocks of metal (slightly smaller than the diameter of Petri dish).
2. Petri dishes with bacterial growth.
3. Phage coated agar plates.
4. Inoculating loop/needle.
5. Nutrient agar.
Procedure:
1. Mount a piece of sterile velvet by stretching it on a cylindrical metallic block (slightly smaller than Petri dish).
2. Place the block with velvet side facing upwards.
3. Invert the Petri dish with the lawn of bacterial cells (master plate) and gently press against the velvet. The number of projecting fibres of the velvet (almost 1000/sq. inch) act as inoculating needles sampling every clone of the cells in the lawn.
Remove the Petri dish and press two or more phage coated agar against the velvet in turn.
4. Save the original master plate.
5. Incubate the subsequent phage coated plates.
6. A few colonies appear on the phage coated plates. Some of these may represent mutants that arose during the cell divisions that occurred after replica plating.
7. Colonies found at the identical positions on every replica plate can be presumed to have arisen from an inoculum of phage resistant ones transferred via the velvet from the phage resistant clone on the master plate.
8. Mark the position, pick up the colony with an inoculating needle from the same location from the master plate (let us assume that out of the 105 cells that the needle picks up 2 or 3 alone are phage resistant).
9. Transfer this to a tube of broth, incubate to increase the total cell number.
10. Spread a sample of this over a fresh agar plate (only 105 cells). This time the inoculum is enriched in phage-resistant mutants since they are picked up from the region containing a clone of resistant cells.