Gram Stain

Material from infected area

Smear (air-dried) made at bedside or immediately after collection is preferred or one made in laboratory from swab or clinical material.

Clean glass slides, swab in transport or clinical material in sterile container

Determine the presence of microörganisms and to evaluate the type of specimen by type of cells seen (eg, PMN, epithelial)

Organism isolation and identification will usually be performed only if culture is requested. Request for Gram stain will not lead to stain for mycobacteria (TB). For detection of tubercle bacilli, an acid-fast stain must also be requested. Certain organisms do not stain or do not stain well with Gram stain (eg, Legionella pneumophila). As many as 30% of cases of bacterial meningitis have a negative Gram stain. Gram stain is not reliable for diagnosis of cervical, rectal, pharyngeal, or asymptomatic urethral gonococcal infection. In acute bacterial meningitis in adults, the most frequent error was misidentification of Listeria as Streptococcus pneumoniae in smears.¹

Depends on site of specimen

Gram stain is recommended (at an additional charge) with all anaerobic cultures, lower respiratory specimens, wound specimens, tissue specimens, and sterile body fluids. In addition, a Gram stain may be useful in demonstrating Neisseria gonorrhoeae or Mobiluncus with genital specimens. Gram stains are usually scanned for the presence or absence of white blood cells (indicative of infection) and squamous epithelial cells (indicative of mucosal contamination). A sputum specimen showing >25 squamous epithelial cells/lpf, regardless of the number of white blood cells, is indicative that the specimen is grossly contaminated with saliva and the culture results cannot be properly interpreted. Additional sputum specimens should be submitted to the laboratory if evidence of contamination by saliva is revealed.

The Gram stain can be a reliable indicator to guide initial antibiotic therapy in community-acquired pneumonia. It is imperative that a valid sputum specimen be obtained for Gram stain. In a well designed trial, valid expectorated sputum was obtained in 41% (59 of 144) of patients. The Gram stain is reliable but not infallible. Its principal limitations in the diagnosis of pulmonary infections are in detection of H influenzae and in differentiating polymicrobic pneumonia from background contamination of the specimen by oropharyngeal flora.² Although mycobacteria have classically been considered to be gram-positive or faintly gram-positive, they are more correctly characterized as “gram-neutral” on routine stains.³ Culture for mycobacteria should be undertaken when purulent sputum without stainable organisms is encountered.

Gram stain is the most valuable diagnostic test in bacterial meningitis that is immediately available.⁴ Organisms are detectable in 60% to 80% of patients who have not been treated, and in 40% to 60% of those who have been given antibiotics.⁴ Its sensitivity relates to the number of organisms present. The sensitivity of the Gram stain is greater in gram-positive infections, and is only positive in 50% of the instances of gram-negative meningitis. It is positive even less frequently with listeriosis meningitis or with anaerobic infections.⁴ Culture and Gram stain should have priority over antigen detection methods if only a small volume of CSF is available.⁵