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Figure 3.19 Fluorescent Dyes and Tags (a) Dyes that cause live cells to fluoresce green and dead ones red; (b) Auramine is used to stain Mycobacterium species in a modification of the acid-fast technique; (c) Fluorescent antibodies tag specific molecules. In this case, the antibody binds to a molecule that is unique to Streptococcus pyogenes.

Figure 3.19 Fluorescent Dyes and Tags (a) Dyes that cause live cells to fluoresce green and dead ones red; (b) Auramine is used to stain Mycobacterium species in a modification of the acid-fast technique; (c) Fluorescent antibodies tag specific molecules. In this case, the antibody binds to a molecule that is unique to Streptococcus pyogenes.

Fluorescent Dyes and Tags

Depending on the procedure employed, fluorescence can be used to observe total cells, a subset of cells, or cells with certain proteins on their surface (figure 3.19).

Fluorescent Dyes

Some fluorescent dyes bind to compounds found in all cells. For example, acridine orange binds DNA and is often used to determine the number of total microorganisms in a sample. However, dyes that bind DNA do not discriminate between cells that are alive, or viable, and those that are dead. To detect only viable cells, other dyes can be used. For example, the dye CTC is made fluorescent by cellular proteins involved in respiration. Consequently, CTC only fluoresces when bound to viable cells. ■ respiration, p. 146

Other fluorescent dyes bind to compounds primarily found in certain types of cells. For example, calcofluor white binds to a component of the cell walls of fungi, causing those cells to fluoresce bright blue. The fluorescent dyes auramine and rhodamine bind to a compound found in the cell walls of members of the genus Mycobacterium. These two dyes can be used in a staining procedure analogous to the acid-fast stain; cells of Mycobacterium will emit a bright yellow or orange fluorescence.

Immunofluorescence

Immunofluorescence is a technique used to tag specific proteins of interest with a fluorescent compound. By tagging a protein unique to a given organism, immunofluorescence can be used to detect that specific microbe in a sample containing numerous different bacteria. Immunofluorescence uses an antibody to deliver the fluorescent tag. An antibody is produced by the immune system in response to a foreign compound, usually a protein; it binds specifically to that compound. Immunofluorescence exploits the natural function of antibodies. This important part of the body's immune response will be discussed in chapter 16.

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