Chapter 11 Agar Plate Culture and Lederberg-Style Replica Plating of Mammalian Cells

Abstract

This chapter describes a technique for obtaining colonies of most established mammalian cells on the surface of agar plates in the absence of overlay liquid medium. This technique is closely analogous to that for culture of bacteria. Agar plate culture has the following advantages: (1) it is easy to prepare agar plates and isolate clones, (2) the plating efficiency is comparable to that of already established cloning techniques, (3) it can be used for assay of transformation, and (4) it is applicable to Lederberg style replica plating. In the original replica plating technique of Lederberg, colonies growing on the master plate are imprinted on pile fabrics and then transferred to a series of replica plates. In a similar way, colonies of mammalian cells on an agar plate can be transferred to identical positions on replica plates. In our method, velveteen (cotton) or velvet (silk or synthetic fabric) are used for imprinting. A sterilized 15-cm square of fabric is placed, nap upward, on a cylindrical stainless steel support (8.5 cm in diameter for a 90-mm petri dish) and fixed firmly in place with a metal hoop. Master plate carrying 30-40 well isolated colonies on its surface is inverted over the fabric with slight digital pressure to transfer the colonies. Then, the fabric is used for imprinting replica-inocula on a series of replica plates by impression in the same way. The replica plates are incubated in a CO 2 -incubator for 1 week.