Staining Techniques For Fluorescent Microscopy

Based on the composition of the fluorescent stain reagents, fluorescent staining techniques may be divided into two general categories: fluorochroming, in which a fluorescent dye is used alone, and immunofluorescence, in which fluorescent dyes have been linked (conjugated) to specific antibodies. The principal differences between these two methods are diagrammed in Figure 6-12.

Fluorochroming

In fluorochroming a direct chemical interaction occurs between the fluorescent dye and a component of the bacterial cell; this interaction is the same as occurs j with the stains used in light microscopy. The t difference is that use of a fluorescent dye enhances contrast and amplifies the observer's ability to detect j stained cells tenfold greater than would be observed '

by light microscopy. For example, a minimum )

concentration of at least 105 organisms per milliliter ;

of specimen is required for visualization by light . ,' microscopy, whereas by fluorescent microscopy that number decreases to 104 per milliliter. The most

Barrier filter

Fluorescent light

Figure 6-11 Principle of fluorescent microscopy. Microorganisms in a specimen are stained with a fluorescent dye. On exposure to excitation light, organisms are visually detected by the emission of fluorescent light by the dye with which they have been stained (i.e., fluorochroming) or "tagged" (i.e., immunofluorescence).

Light source

Lightwave splitting mirror

Excitation light

filter Excitf°n light

Fluorescent light

Lightwave splitting mirror

Excitation light

filter Excitf°n light

Specimen (Contains microorganisms stained with fluorochrome)

Specimen (Contains microorganisms stained with fluorochrome)

A Fluorochroming

Fluorescent dye

Fluorescent dye

B Immunofluorescence to

Specific antibody

Conjugate

Target bacteria to be stained

Antigens

Staining results

All bacteria stain and fluoresce

Specific fluorescence

Specific fluorescence

Figure 6-12 Principles of fluorochroming and immunofluorescence. Fluorochroming <A) involves nonspecific staining of any bacterial cell with a fluorescent dye. Immunofluorescence (B) uses antibodies labeled with fluorescent dye (i.e., a conjugate) to specifically stain a particular bacterial species.

I Procedure 6-1

Acridine Orange Stain

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Bacterial Vaginosis Facts

Bacterial Vaginosis Facts

This fact sheet is designed to provide you with information on Bacterial Vaginosis. Bacterial vaginosis is an abnormal vaginal condition that is characterized by vaginal discharge and results from an overgrowth of atypical bacteria in the vagina.

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