Stained smears

Gram stains

Gram-stained smears of body fluids and exudates can show inflammatory cells as well as bacteria. Clumps of Gram-positive cocci suggest staphylococci or peptococci, chains of Gram-positive cocci suggest streptococci or peptostreptococci, large Gram-positive bacilli suggest Clostridia, large Gram-negative bacilli suggest Enterobacteriaceae, and small Gram-negative bacilli suggest Pseudomonas, Hemophilus, or Bacteroides species. However, bacterial morphology can be highly variable, particularly in material from abscesses or from patients on antimicrobial therapy.

Gram stains of sputum are helpful when inflammatory cells suggest pneumonia; however, if there are oral squamous epithelial cells and a wide variety of organisms, the specimen includes saliva and should not be cultured. Pneumonia caused by Streptococcus pneumoniae, Hemophilus influenzae, Staphylococcus aureus, and Enterobacteriaceae can often be diagnosed with a high degree of accuracy from a sputum Gram stain. Although a Gram stain is easily and rapidly performed, considerable experience and expertise is required as the decolorization time depends on the nature of the material being stained. Adequate quality control of Gram-stained smears must be performed, including staining control smears containing a mixture of Gram-positive cocci and Gram-negative bacilli.

Acid-fast and fluorescent stains

Mycobacteria do not stain with the Gram method, but they resist acid decolorization and therefore can be identified by various acid-fast stains of sputum and other specimens. Both the Ziehl-Neelsen stain using hot carbolfuchsin and the Kinyoun stain using cold concentrated carbolfuchsin will stain mycobacteria red. Auramine will stain mycobacteria a bright yellow under fluorescent illumination.

Fluorescein-tagged fluorescent antibody stains are useful in demonstrating various pathogens, such as Legionella pneumophila and common respiratory viruses. Other staining methods include silver stains of biopsies for fungi and Pneumocystis carinii and Giemsa stains of blood smears for malarial parasites.

All staining procedures, particularly fluorescent stains, require adequate controls. Detection of microbial products

The only commonly available techniques for detecting microbial products directly in specimens are various immunological methods used to identify microbial antigens and direct gene probes to detect various viruses, Neisseria gonorrhoeae, and Chlamydia trachomatis. Enzyme-linked immunosorbent assays (ELISAs), using enzyme-labeled antibodies, have been developed for the detection of bacterial antigens.

Considerable advances have recently been made in the development of amplified gene probes, for example by the polymerase chain reaction ( Murray 1995).

However, ready availability of commercial products has not yet been attained, and these tests are currently very labor intensive, have a considerable turn-round time, and are more suitable for batch testing than for testing individual specimens.

In patients with meningitis, capsular polysaccharide antigens of H. influenzae, Strep. pneumoniae, Neisseria meningitidis, and C. neoformans can be detected in cerebrospinal fluid and sometimes in serum, sputum, urine, and effusion fluids. Antigens can be demonstrated by counterimmunoelectrophoresis, latex agglutination, or staphylococcal coagglutination, but false-positive and false-negative results limit the application of these procedures, which are generally only as sensitive as direct microscopy.

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