Gram Stain Interpretation

General Information

  • The Gram stain is a quick test that is used by the microbiology laboratory to differentiate different types of bacteria based on the biochemical properties of their cell walls

  • Gram staining requires the bacterial culture to be smeared to a glass slide, a violet stain is then added to the slide which stains Gram-positive bacteria violet

  • The slide is then rinsed with a decolorizing agent which washes away the violet stain from Gram-negative bacteria

  • Following this step the slide is counterstained with a red dye which colors the Gram-negative bacteria pink

  • Have a thick cell wall which is made up of peptidoglycan (50-90% of cell wall)

  • This thick peptidoglycan layer allows the organisms to retain the violet stain and prevents it from being washed away during the decolorization step

  • Have a thinner layer of peptidoglycan (about 10% of the cell wall), and they lose the violet stain during the decolorization step but retain the counter stain (pink)

  • It helps us differentiate Gram-positive bacteria from Gram-negative bacteria which can help guide empiric treatment

The microbiology lab also uses the morphology and arrangement of bacteria to further differentiate bacteria:

  • Spherical morphology = cocci

    • Arrangement: chains or clusters
    • Gram-positive cocci in clusters usually indicate Staphylococcus species
    • Gram-positive cocci in chains usually indicate Streptococcus or Enterococcus species
  • Rod-shaped morphology = bacilli

    • Ex. Escherichia, Klebsiella, Enterobacter, Pseudomonas, etc.

  • Some organisms are Gram-variable – they may stain either negative or positive, and some organisms are not susceptible to Gram staining, for example when there is no cell wall structure (Ex. Mycoplasma).

  • Any bacteria detected on Gram stain of a sterile site specimen should be considered significant, although a negative Gram stain does not exclude infection

  • Nonsterile specimens also generally contain human cells such as epithelial cells and white blood cells

    • For example: a high-quality sputum specimen has high numbers of white cells and few epithelial cells; a low-quality specimen has high numbers of epithelial cells and low numbers of white cells. Low-quality sputum specimens may be rejected by the laboratory as unfit for culture

  • How the patient is doing. Clinical status of patient in addition to Gram stain results should guide therapy

  • Presence of a multidrug-resistant organism. Assess patient risk factors, recent antimicrobial therapy and prior microbiology when considering the need to cover for multidrug-resistant organisms

    • For example, MRSA risk factors include: history of MRSA infection or colonization, household contact with a MRSA colonized individual, IV drug use, homelessness, incarcerated persons, recent travel to or residing in an MRSA endemic region or community

More Information

Content derived from: NB Provincial Health Authorities Anti-Infective Stewardship Committee. Gram Stain Interpretation Guideline. 2020-10.