Mutant Selection

Even when mutagens are used, mutations that appear in the population are rare. This presents a major challenge to the investigator who wants to isolate a desired mutant. Several clever techniques have simplified this process. As discussed in chapter 4, bacteria can multiply on simple media and produce several billion cells per milliliter of medium in less than 24 hours. In such a large population, every gene should be mutant in at least one cell in the population. The major problem becomes how to find and identify the bacteria containing the desired mutation. Depending on the type of mutant being sought, one of two simple techniques can be used, direct or indirect selection.

Direct Selection

Direct selection involves inoculating cells onto a medium on which the mutant, but not the parent, can grow. For example, mutants resistant to the antibiotic streptomycin can be easily selected directly by inoculating cells onto a medium containing streptomycin. Only the rare resistant cells in the population will form a colony (figure 8.8). Antimicrobial-resistant mutants are usually very easy to isolate by direct selection.

Indirect Selection

Indirect selection is required to isolate an auxotrophic mutant, one that requires a growth factor, such as histidine, which the parent strain does not. On a medium that contains histidine, both the parent and the histidine auxotroph grow. On a medium lacking histidine, the histidine-requiring mutant will not grow, but the parent will. There is no medium on which the mutant will grow and the parent will not.

Streptomycin-resistant cell ^

Streptomycin-sensitive cells

Streptomycin-sensitive cells

Medium containing streptomycin

Medium without streptomycin

Figure 8.8 Direct Selection of Mutants Only the streptomycin-resistant cells will grow on the streptomycin-containing medium. All cells will grow on media without streptomycin and have the same appearance.

Medium containing streptomycin

Medium without streptomycin

Figure 8.8 Direct Selection of Mutants Only the streptomycin-resistant cells will grow on the streptomycin-containing medium. All cells will grow on media without streptomycin and have the same appearance.

Nester-Anderson-Roberts: I. Life and Death of 8. Bacterial Genetics © The McGraw-Hill

Microbiology, A Human Microorganisms Companies, 2003

Perspective, Fourth Edition

200 Chapter 8 Bacterial Genetics

200 Chapter 8 Bacterial Genetics

Master plate with bacteria (enriched complex medium)

Master plate with bacteria (enriched complex medium)

Pressed onto sterile velvet

Pressed onto sterile velvet

Sterile velvet

Sterile velvet

Auxotroph,

Enriched complex medium; all colonies grow

Colonies imprinted on velvet

Position of auxotroph

Glucose-salts medium; auxotrophs do not grow

Figure 8.9 Indirect Selection of Mutants by Replica Plating The procedure shown is the one first used by the Lederbergs and continues to be used today in many laboratories.

Colonies imprinted on velvet

Position of auxotroph

Glucose-salts medium; auxotrophs do not grow

Figure 8.9 Indirect Selection of Mutants by Replica Plating The procedure shown is the one first used by the Lederbergs and continues to be used today in many laboratories.

Replica Plating

An ingenious technique for indirect selection of auxotrophic mutants, replica plating, was devised by the husband-and-wife team of Joshua and Esther Lederberg in the early 1950s (figure 8.9). In this technique, a master plate containing isolated colonies of all cells growing on an enriched medium is pressed onto sterile velvet, a fabric with tiny threads that stand on end like tiny bristles. This operation transfers some cells of every bacterial colony onto the velvet. Next, two sterile plates, one containing a glucose-salts (minimal) medium and the second an enriched, complex medium, are pressed in succession onto the same velvet. This procedure transfers cells imprinted on the velvet from the master plate to both the glucose-salts medium and the enriched medium. All cells that do not have a nutritional requirement will form colonies on both the enriched and the glucose-salts medium, but auxotrophs will only form colonies on the enriched medium. ■ glucose-salts medium, p. 93

By keeping the orientation of the two plates the same as they touch the velvet, any colony on the master plate that can grow on the enriched medium but not on the glucose-salts medium can be identified by the pattern the colonies form on the agar. The cells in such a colony must require a growth factor present in the complex medium. The particular growth factor required can then be determined by adding the various factors to the glucose-salts medium and determining which one promotes cell growth. Before the invention of replica plating, each colony had to be individually transferred from the complex to the glucose-salts medium in order to identify the auxotrophs and determine their growth requirements, a very laborious job indeed. ■ growth factor, p. 91

Penicillin Enrichment

Even using mutagenic agents, the frequency of mutation in a particular gene is low, ranging perhaps from less than one in 1,000 to one in 100 million cells. In cases where the parent cell is sensitive to penicillin, the proportion of auxotrophic mutants in the population can be increased by a technique called penicillin enrichment. Following treatment with a mutagen, the cells are grown in a glucose-salts medium containing penicillin. Since penicillin kills only growing cells, most of the cells that have no growth factor requirements will grow and so will be killed, while the non-multiplying auxotrophs will survive (figure 8.10). The enzyme penicillinase is then added to destroy the penicillin, and the cells are plated on an enriched medium. This plate can then be replica plated onto a glucose-salts medium. Any colonies that grow on the enriched medium but not on the glucose-salts medium must be auxotrophic—that is, require a growth factor. ■ action of penicillin, p. 60

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Responses

  • mickie
    How to identify growth factor of auxotrophs?
    2 years ago
  • phillipp
    What is mutant selection?
    1 year ago
  • Kidane
    What is direct selection of mutants?
    1 year ago

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