Basic Bacteriological Routines

In experimental bacteriology, bacteria are generally manipulated, stored, and shipped in the form of cultures. Depending on various factors, including strain genotype, storage and shipping methods, and manipulator skills, the culture may contain genetic variants or simply contaminants. It is therefore important to begin an experiment by streaking the culture on an agar plate. Streaking, a technique to disperse bacterial cells on the surface of the agar, serves the purpose of isolating individual colonies. A colony originates from a single cell and is a nearly pure culture. On rich LB medium after 24 h of incubation at 37°C, a colony of Salmonella contains ∼5 × 10⁸ cells (about 29 generations). Streaking is also required in experiments that themselves generate single colonies as a result of selection (e.g., when constructing strains or introducing plasmids). Except in the few instances in which the selection efficiently kills all counter-selected bacteria, colonies growing on the selective plates are contaminated, sometimes heavily, with cells from the bacterial lawn. "Purifying" the colonies arising in such experiments by streaking on selective plates is therefore a mandatory step. Here, we show how this can be conveniently done using simple toothpicks. We also briefly describe the steps involved in inoculating liquid cultures, spreading plates, and replica plating.