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 and certain other pathogens.
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, cerebrospinal fluid) are concentrated (eg, by centrifugation) before examination.
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)
Identifies polymorphonuclear leukocytes, indicating bacterial infection rather than colonization
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. Finding many squamous cells in a sputum specimen suggests that the specimen is contaminated with saliva and thus is of limited diagnostic usefulness.
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).
These stains are used to identify the following:
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 allow detection at lower concentrations (< 1 × 104 cells/mL). Examples are
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.
Wet mounts of unstained samples can be used to detect the following via darkfield microscopy:
Visibility of fungi can be increased by applying 10% potassium hydroxide (KOH) to dissolve surrounding tissues and nonfungal organisms.
India ink stain is used to detect mainly Cryptococcus neoformans and other encapsulated fungi in a cell suspension (eg, cerebrospinal fluid sediment). The background field, rather than the organism itself, is stained, making any capsule around the organism visible as a halo. In cerebrospinal fluid, the test is not as sensitive as cryptococcal antigen. Specificity is also limited; leukocytes may appear encapsulated.
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.