Bacteria will fluoresce bright orange against a green-fluoresting or dark background. The nuclei of host cells may also fluoresce.
common fluorochroming methods used in diagnostic microbiology include acridine orange stain, auraminerhodamine stain, and calcofluor white stain.
Acridine Orange. The fluorochrome acridine orange binds to nucleic acid. This staining method (Procedure 6-1) can be used to confirm the presence of bacteria in blood cultures when Gram stain results are difficult to interpret or when the presence of bacteria is highly suspected but none are detected using light microscopy. Because acridine orange stains all nucleic acids, it is nonspecific. Therefore, all microorganisms and host cells will stain and give a bright orange fluorescence. Although this stain can be used to enhance detection, it does not discriminate between gram-negative and gram-
positive bacteria. The stain is also used for detection of cell wall-deficient bacteria (e.g., mycoplasmas) grown in culture that are incapable of retaining the dyes used in the Gram stain (Figure 6-13).
Auramine-Rhodamine. The waxy mycolic acids in the cell walls of mycobacteria have an affinity for the fluorochromes auramine and rhodamine. As shown in Figure 6-14, these dyes will nonspecifically bind to nearly all mycobacteria. The mycobacterial cells appear bright yellow or orange against a greenish background. This fluorochroming method can be used to enhance detection of mycobacteria directly in patient specimens and for initial characterization of cells grown in culture.
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