Genetics of Bacteria Help (page 3)

By — McGraw-Hill Professional
Updated on Aug 23, 2011

Isolation of Bacterial Mutants

There are several different mutant bacterial phenotypes that can be used to identify mutations affecting various functional pathways. When the mutant phenotype has an advantage over the wild type under a specified set of conditions, a selection scheme is used to isolate the mutant. For example, it is relatively easy to isolate phage- or drug-resistant mutant strains by plating the bacteria on a medium that contains a selective agent, such as a phage or drug. Only resistant cells will form colonies on such plates. Prototrophic mutants can be isolated from an auxotrophic culture by plating on minimal medium; only prototrophic colonies would grow on such a plate. Isolation of auxotropic mutants is more difficult because they do not have a growth advantage over wild type. Generally, either an enrichment or a screening scheme must be used in this case. There are at least four methods for isolating auxotrophic mutants from prototrophic cultures, each of which is enhanced by first treating the culture with a mutagenic agent to increase the mutation rate.

  1. In the delayed enrichment method, a diluted culture is plated on minimal medium and then covered with an agar layer of the same medium. The plate is incubated and the locations of prototrophic colonies are marked on the plate.Alayer of nutrient medium is then added, and the nutrients from this agar are allowed to diffuse through the minimal agar. After another incubation period, the appearance of any new colonies may represent auxotrophs that could only grow after supplementation.
  2. The limited enrichment method is a simplification of the delayed enrichment method. Bacteria are plated on minimal medium containing a very small amount of nutrient supplementation. Under such conditions, auxotrophic bacteria will undergo limited growth until the supply of nutrients is exhausted, and hence will form small colonies. Prototrophic bacteria will continue to grow and produce large colonies.
  3. Penicillin interferes with the development of the bacterial cell wall only in growing cells, causing them to rupture. In the penicillin enrichment technique, the bacterial culture is exposed to penicillin in minimal medium. Growing prototrophic cells die, whereas auxotrophic cells cannot grow and therefore are not killed. The culture is then plated on nutrient medium without penicillin. Only auxotrophic colonies should form on the plate.
  4. In the replica plating technique (Fig. 10-1), the bacteria are first plated on nutrient agar and allowed to form colonies. A sterile velvet pad is then pressed onto the surface of this "master plate." The nap of the velvet picks up representatives from each colony on the plate. The pad is then pressed onto the sterile surface of one or more "replica plates" containing minimal medium. Only prototrophic colonies grow on the replica plate. Colonies on the master plate that are not represented on the replica plate may be auxotrophs. These colonies can be picked from the master plate to form a "pure" auxotrophic culture.
  5. Isolation of Bacterial Mutants

Practice problems for these concepts can be found at:

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