2.4 Staining microscopic specimens  (Page 4/11)

Part 3

Viewing Cindy’s specimen under the darkfield microscope has provided the technician with some important clues about the identity of the microbe causing her infection. However, more information is needed to make a conclusive diagnosis. The technician decides to make a Gram stain of the specimen. This technique is commonly used as an early step in identifying pathogenic bacteria. After completing the Gram stain procedure , the technician views the slide under the brightfield microscope and sees purple, grape-like clusters of spherical cells ( [link] ).

• Are these bacteria gram-positive or gram-negative?
• What does this reveal about their cell walls?

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Acid-fast stains

Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool. An acid-fast stain is able to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls, and those that do not. Two different methods for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun technique . Both use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin even after a decolorizing agent (an acid-alcohol solution) is applied. A secondary counterstain, methylene blue, is then applied, which renders non–acid-fast cells blue.

The fundamental difference between the two carbolfuchsin-based methods is whether heat is used during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat. Both techniques are important diagnostic tools because a number of specific diseases are caused by acid-fast bacteria (AFB). If AFB are present in a tissue sample, their red or pink color can be seen clearly against the blue background of the surrounding tissue cells ( [link] ).

Using microscopy to diagnose tuberculosis

Mycobacterium tuberculosis , the bacterium that causes tuberculosis , can be detected in specimens based on the presence of acid-fast bacilli. Often, a smear is prepared from a sample of the patient’s sputum and then stained using the Ziehl-Neelsen technique ( [link] ). If acid-fast bacteria are confirmed, they are generally cultured to make a positive identification. Variations of this approach can be used as a first step in determining whether M. tuberculosis or other acid-fast bacteria are present, though samples from elsewhere in the body (such as urine) may contain other Mycobacterium species.

An alternative approach for determining the presence of M. tuberculosis is immunofluorescence. In this technique, fluorochrome-labeled antibodies bind to M. tuberculosis , if present. Antibody-specific fluorescent dyes can be used to view the mycobacteria with a fluorescence microscope.