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By Kevin C. Hazen, PhD, Professor of Pathology and Director of Clinical Microbiology, Duke University Health System

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Microscopy can be done quickly, but accuracy depends on the experience of the microscopist and quality of equipment. Regulations often limit physicians’ use of microscopy for diagnostic purposes outside a certified laboratory. Microscopic examination of tissue may be required to distinguish invasive disease from surface colonization—a distinction not easily achieved by culture methods.

Most specimens are treated with stains that color pathogens, causing them to stand out from the background, although wet mounts of unstained samples can be used to detect fungi, parasites (including helminth eggs and larvae), vaginal clue cells, motile organisms (eg, Trichomonas), and syphilis (Treponema spirochetes, via darkfield microscopy). Visibility of fungi can be increased by applying 10% potassium hydroxide (KOH) to dissolve surrounding tissues and nonfungal organisms.

The clinician orders a stain based on the likely pathogens, but no stain is 100% specific. Most samples are treated with Gram stain and, if mycobacteria are suspected, with an acid-fast stain. However, some pathogens are not easily visible using these stains; if these pathogens are suspected, different stains or other identification methods are required. Because microscopic detection usually requires a microbe concentration of at least about 1 ×104-5/mL, most body fluid specimens (eg, CSF) are concentrated (eg, by centrifugation) before examination.

Gram stain

The Gram stain does the following:

  • Classifies bacteria according to whether they retain crystal violet stain (gram-positive—blue) or not (gram-negative—red)

  • Highlights cell morphology (eg, bacilli, cocci) and cell arrangement (eg, clumps, chains, diploids)

Such characteristics can direct antibiotic therapy pending definitive identification. Finding a mixture of microorganisms with multiple morphologies and staining characteristics on Gram stain suggests a contaminated specimen or a polymicrobial bacterial infection.

To do a Gram stain, technicians heat-fix specimen material to a slide and stain it by sequential exposure to Gram crystal violet, iodine, decolorizer, and counterstain (typically safranin).

Acid-fast and modified acid-fast stains

These stains are used to identify the following:

  • Acid-fast organisms (Mycobacterium sp)

  • Moderately acid-fast organisms (primarily Nocardia sp)

  • Rhodococcus and related genera

  • Oocysts of some parasites (eg, Cryptosporidium, Cystoisospora [Isospora] belli)

Although detection of mycobacteria in sputum requires at least 10,000 organisms/mL, mycobacteria are often present in lower levels, so sensitivity is limited. Usually, several mL of sputum are decontaminated with sodium hydroxide and concentrated by centrifugation for acid-fast staining. Specificity is better, although some moderately acid-fast organisms are difficult to distinguish from mycobacteria.

Fluorescent stains

These stains allow detection at lower concentrations (< 1 × 104 cells/mL). Examples are

  • Acridine orange (bacteria and fungi)

  • Auramine-rhodamine and auramine O (mycobacteria)

  • Calcofluor white (fungi, especially dermatophytes)

Coupling a fluorescent dye to an antibody directed at a pathogen (direct or indirect immunofluorescence) should theoretically increase sensitivity and specificity. However, these tests are difficult to read and interpret, and few (eg, Pneumocystis and Legionella direct fluorescent antibody tests) are commercially available and commonly used.

India ink (colloidal carbon) stain

This stain is used to detect mainly Cryptococcus neoformans and other encapsulated fungi in a cell suspension (eg, CSF sediment). The background field, rather than the organism itself, is stained, making any capsule around the organism visible as a halo. In CSF, the test is not as sensitive as cryptococcal antigen. Specificity is also limited; leukocytes may appear encapsulated.

Warthin-Starry stain and Dieterle stain

These silver stains are used to visualize bacteria such as

  • Spirochetes

  • Helicobacter pylori

  • Microsporidia

  • Bartonella henselae (the cause of cat-scratch disease)

Wright stain and Giemsa stain

These stains are used for detection of the following:

  • Parasites in blood

  • Histoplasma capsulatumin phagocytes and tissue cells

  • Intracellular inclusions formed by viruses and chlamydia

  • Trophozoites of Pneumocystis jirovecii

  • Some intracellular bacteria

Trichrome stain (Gomori-Wheatley stain) and iron hematoxylin stain

These stains are used to detect intestinal protozoa.

The Gomori-Wheatley stain is used to detect microsporidia. It may miss helminth eggs and larvae and does not reliably identify Cryptosporidium. Fungi and human cells take up the stain.

The iron hematoxylin stain differentially stains cells, cell inclusions, and nuclei. Helminth eggs may stain too dark to permit identification.