Figure 6-3 Gram stain procedures and principles. A, Gram-positive bacteria observed under oil immersion appear purple. B, Gramnegative bacteria observed under oil immersion appear pink. (Modified from Atlas RM: Principles of microbiology, St Louis, 1995, Mosby.)
number of bacteria for any normally sterile body site and to describe the quantity as rare or few based on microscopic observation may be understating their significance in a clinical specimen. On the other hand, noting the relative amounts seen on direct smear may be useful laboratory information to correlate smear results with the amount of growth observed subsequently from cultures.
Although Gram stain evaluation of direct smears is routinely used as an aid in the diagnosis of bacterial infections, unexpected but significant findings of other infectious etiologies may be detected and cannot be ignored. For example, fungal cells and elements generally stain gram-positive, but they may take up the crystal violet poorly and appear gram-variable (e.g., both pink and purple) or gram-negative. Because infectious agents besides bacteria may be detected by Gram stain, any unusual cells or structures observed on the smear should be evaluated further before being dismissed as unimportant (Figure 6-7).
Gram Stain of Bacteria Grown in Culture. The Gram stain also plays a key role in the identification of bacteria grown in culture. Similar to direct smears, smears prepared from bacterial growth are evaluated for the bacterial cells' Gram reactions, morphologies, and arrangements (see Figure 6-4). If growth from
Figure 6-4 Examples of common bacterial cellular morphologies, Gram staining reactions, and cellular arrangements.
more than one specimen is to be stained on the same slide, a wax pencil may be used to create divisions. Drawing a "map" of such a slide so that different Gram stain results can be recorded in an organized fashion is helpful (Figure 6-8). The smear results will be used to determine subsequent testing for identifying and characterizing the organisms isolated from the patient specimen.
The acid-fast stain is the other commonly used stain for light-microscopic examination of bacteria.
Principle. The acid-fast stain is specifically designed for a subset of bacteria whose cell walls contain long-chain fatty (mycolic) acids. Mycolic acids render the cells resistant to decolorization, even with acid alcohol decolorizers. Thus, these bacteria are referred to as being acid-fast. Although these organisms may stain slightly or poorly as gram-positive, the acid-fast stain takes full advantage of the waxy content of the cell walls to
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