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Negative selection through replica plating to screen for Amp- colonies

In molecular biology and microbiology, replica plating is a technique in which one or more secondary Petri plates containing different solid (agar-based) selective growth media (lacking nutrients or containing chemical growth inhibitors such as antibiotics) are inoculated with the same colonies of microorganisms from a primary plate (or master dish), reproducing the original spatial pattern of colonies. The technique involves pressing a velvet-covered disk, nitrocellulose membrane, or filter paper to a primary plate, and then imprinting secondary plates with cells in colonies removed from the original plate by the material. Generally, large numbers of colonies (roughly 30-300) are replica plated due to the difficulty in streaking each out individually onto a separate plate.

The purpose of replica plating is to be able to compare the master plate and any secondary plates to screen for a selectable phenotype. For example, a colony which appeared on the master plate but failed to appear at the same location on a secondary plate shows that the colony was sensitive to a substance on that particular secondary plate. Common screenable phenotypes include auxotrophy and antibiotic resistance.

Replica plating is especially useful for negative selection. For example, if one wanted to select colonies that were sensitive to ampicillin, the primary plate could be replica plated on a secondary Amp⁺ agar plate . The sensitive colonies on the secondary plate would die but the colonies could still be deduced from the primary plate since the two have the same spatial patterns from ampicillin resistant colonies. The sensitive colonies could then be picked off from the primary plate.

By increasing the variety of secondary plates with different selective growth media, it is possible to rapidly screen a large number of individual isolated colonies for as many phenotypes as there are secondary plates.

This technique was first described^[1] by Joshua Lederberg and Esther Lederberg in 1952.

1. ^ Lederberg, J and Lederberg, EM (1952) Replica plating and indirect selection of bacterial mutants. J Bacteriol. 63: 399–406. (Full text)