Growing On Agar

Replicate onto several different media

mEDIUM WITH ANTIBIOTIC

mEDIUM LACKING SINGLE NUTRIENT

cONTROL PLATE (COMPLETE MEDIUM WITHOUT ANTIBIOTIC)

mEDIUM WITH ANTIBIOTIC

mEDIUM LACKING SINGLE NUTRIENT

cONTROL PLATE (COMPLETE MEDIUM WITHOUT ANTIBIOTIC)

Replica plating allows screening for mutants that fail to grow under the test conditions.

tetrazolium dyes, respond to oxidation reduction reactions. They may be used to monitor the ability of bacteria to oxidize certain growth substrates.

More specific indicators, such as substrates for individual enzymes, may also be used. For example, X-gal is a substrate for b-galactosidase that releases a blue dye when split by this enzyme (see Ch. 7). Bacterial colonies expressing significant levels of b-galac-tosidase turn blue in the presence of X-gal. One of the most cheerful indicators is the use of selenium salts. Bacteria that can reduce selenate or selenite accumulate granules of elemental selenium that are bright red. In this case several steps of a specific pathway, rather than just one enzyme, are involved. Note that for an indicator system to work well, the colored product must be insoluble, otherwise it will diffuse through the agar and the color will no longer be localized to the colony that performed the reaction.

Replica plating is another widely used form of phenotypic screening. It is particularly useful when searching for mutants that have lost the ability to grow under certain conditions. The same mutagenized bacterial colonies are tested for growth on a variety of media and colonies that fail to grow on the medium of interest are kept. For example, media with different carbon sources, growth supplements or growth inhibitors may be used. Since it is not possible to subculture a colony that failed to grow on a test medium, the mutagenized colonies are first grown on normal (i.e. non-selective) medium. Filter paper or velvet pads are then pressed onto the colonies and pick up bacteria from each original colony. Bacteria corresponding to each colony are then transferred to assorted test media by pressing the filter paper or velvet pads onto the surface of the fresh medium (Fig. 13.27). This technique preserves the arrangement of the colonies on the agar and allows colonies missing on the test medium to be retrieved from the original master plates.

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