Definition and Use
- Indications:
- This test should be routinely performed for certain specimen types submitted to the laboratory for bacterial culture (e.g., lower respiratory, wound, tissue, abscess and drainage, sterile fluids, CSF, genital samples).
- Because Gram stain is less sensitive than culture for detection of bacteria, culture should always be performed with Gram stains with a few possible exceptions. Gram stain without culture may provide accurate detection of vaginal and oropharyngeal candidiasis.
- The Gram stain is used for the direct detection and initial presumptive identification of bacteria and yeast in patient specimens. Specimens should be collected and transported according to instructions for specific specimen types. Patient specimens are used to make smears on glass microscope slides. After fixation, slides are sequentially stained with crystal violet followed by iodine solution. The intracellular crystal violet " “iodine complexes formed are too large to escape through the thick peptidoglycan cell wall of gram-positive organisms by alcohol decolorization, rendering them dark blue. But the crystal violet " “iodine complexes can be rinsed through the thinner, fenestrated cell wall of gram-negative organisms, leaving them colorless. After the rinsing step, gram-negative organisms are counterstained with safranin, resulting in mild to intense pink staining.
- The Gram stain is a differential staining technique. Staining characteristics (e.g., pink or blue) and morphology (e.g., cocci or bacilli) and other characteristics of the primary pathogens are reported. This information may contribute to informed decisions regarding initial empirical therapy.
- The Gram stain may demonstrate host PMNs and other evidence of inflammation. Epithelial cells, derived from mucosal or cutaneous surfaces, predict contamination of the specimen with the patients endogenous flora.
- Turnaround time: <4 hours.
Interpretation
- Expected results:
- Specimens from sterile sites should be negative for microorganisms. Smears from nonsterile sites, such as mucosal surfaces, usually demonstrate organisms of various morphologies typical for the endogenous flora of the site (e.g., respiratory, vaginal, GI).
- PMNs and other signs of an inflammatory reaction are not typical for normal tissue specimens and suggest infection (or other inflammatory condition) at the site of collection.
- Positive results:
- Microorganisms (usually a single morphotype), in moderate or heavy amounts, with PMNs, and other inflammatory markers are typical of pyogenic infections.
- Pathogens present in the specimen in concentrations greater than approximately 103 " “104 organisms per milliliter should be detected by Gram stain and will typically yield moderate or heavy growth in culture. Concentration of CSF and sterile fluid specimens, using techniques such as centrifugation, improves detection of microorganisms by Gram stain.
- Any type of organisms seen by Gram stain should be isolated by culture after appropriate processing. Monitoring the correlation of Gram stain and bacterial culture results, therefore, may be used as an important quality assurance (QA) tool. Detection of an organism by Gram stain for which culture yields no comparable isolate suggests that additional cultures, like anaerobic or mycobacterial, may be required.
- Negative results:
- Infections may be associated with low concentrations of pathogens (<103 organisms/mL). For example, in adults with overwhelming bacteremia and sepsis, the concentration of organisms in the bloodstream is typically approximately 1 " “10 organism/mL, well below the detection level by Gram stain microscopy.
- PMNs and other signs of inflammation may increase suspicion of infection in smears negative for microorganisms.
Limitations
- Some pathogenic microorganisms fail to stain avidly by the Gram stain technique. Special modifications or stains may improve detection, like the use of fuchsin as a counterstain for the Gram stain, or acridine orange as a fluorogenic alternative to the Gram stain. Poor specimen collection, such as not sampling the site of infection, may give false-negative or misleading results.
Suggested Reading
1Winn ‚ WC Jr, Allen ‚ SD, Janda ‚ WM, et al. Konemans Color Atlas and Textbook of Diagnostic Microbiology, 6th ed. Baltimore, MD: Lippincott Williams & Wilkins; 2006.