The Gram stain is a critical test for the presumptive diagnosis of infectious agents.
It allows lab technologists to visualize and categorize bacteria and yeast based on their reaction to the stain, morphology, size, and cellular arrangement directly from clinical specimens using a light microscope, which can be promptly communicated to health care providers.
Gram stains can also be used to visualize bacteria or yeast that have been grown and isolated from liquid media (broth suspension) or from solid media (colonies grown on agar plates), which can be helpful to confirm that the infectious agent grown in the lab is the same as what was initially visualized in the clinical specimen.
The gram stain also provides key information on the quality of specimen collection. For example, the visualization of many epithelial cells (surface skin cells) and few white blood cells (WBCs) from a wound specimen may suggest a superficial specimen. Bacteria visualized on the Gram stain of this specimen may represent colonizing skin flora, which would provide inaccurate information as per the cause of an infection.
· Direct smear
· Gram smear
· Gram’s method
Limits of the Gram Stain
Bacteria that are too small, or that lack a cell wall are unable to be effectively visualized using the Gram stain. Such bacteria include Mycoplasma, Treponema, Chlamydia, Rickettsia, Legionella, Francisella and Brucella species. Molecular or serology methods are utilized in the PHML, or our reference labs, to either directly or indirectly detect these bacteria if suspected.
Other bacteria such as Mycobacterium and Nocardia species have a high lipid content in their cell walls, making it difficult to visualize using the Gram stain method. Acid-fast stains such as Kinyoun or Auramine-rhodamine are utilized at the PHML to determine the presence of acid-fast bacilli (AFB) in clinical specimens.
Bacteria under the effects of antibiotics are under stress and may change their characteristics making interpretation from the Gram stain difficult.
The Gram stain is not recommended to microscopically examine parasites, fungi or viruses.
Gram stains are routinely performed from sterile body sites, tissues, wounds and certain discharge such as abscesses or aspirates.
It is not recommended to perform a Gram stain on non-sterile sites such as throat swabs, nasal swabs, urine, or stool due to the inherent presence of normal bacterial and yeast flora present at the sites where these specimens are collected.
|Specimen||Gram Stain Performed? (Yes/No)|
|Blood culture (before initial incubation)||No|
|Blood culture (after initial incubation and growth detected)||Yes|
|Tissue biopsies or sections||Yes|
|Abscess fluid, exudates, aspirates from sterile sites||Yes|
|Eye lesions or corneal scraping||Yes|
|Fecal matter (stool)||No|
|Sputum from cystic fibrosis or bronchiectasis patients||Yes|
|BAL or tracheal aspirates||Yes|
|Non-human specimens (i.e. prosthetic devices or water)||No|
The Gram stain is part of the initial work up when culture and sensitivity (C&S) is ordered on certain clinical specimens. Please consult individual guide to service pages for rejection criteria for specific clinical specimens where C&S is requested.
Common reasons for rejection of specimens submitted for C&S include, but are not limited to, specimen not transported to the lab in a timely manner, specimen collected in an inappropriate container, age of the specimen (too old), or specimen not stored at the appropriate temperature. Such practices can lead to either overgrowth of clinically insignificant organisms, or lead to death of clinically important organisms, thus providing inaccurate Gram stain results to heath care providers.
Clinically important information that is communicated to health care providers from the Gram stain may include, but is not limited to, the following:
- Bacteria classified as either Gram positive (dark purple stain) or Gram negative (pink/red stain)
- Bacteria classified as bacillus (rod)-shaped, cocci (spherical)-shaped in clusters, chains or singles
- Presence of yeast cells with/without pseudohyphae, which stain Gram positive
- Presence and amount of white blood cells (WBCs)
- Presence and amount of epithelial cells (surface skin or mucous membrane cells)
Information from the Gram stain is the initial report physicians receive from the lab, and can help guide appropriate antibiotic therapy by offering a presumptive ID before subsequent culture and antibiotic susceptibility test results are available from the lab usually 24-48 hours later.
American Society for Microbiology & Jorgensen, J. (2015). Manual of Clinical Microbiology (11th ed). Washington, DC: ASM Press.
American Society for Microbiology & Leber, A. (2016). Clinical Microbiology Procedures Handbook (4th ed). Washington, DC: ASM Press.
|Status||Day(s) and Time(s) Test Performed||Maximum Laboratory Time||Specimen Retention|
|Routine||Daily, 24/7||< 1 hour||1 week for clinical specimens
3 days for isolates grown on solid or liquid growth medium, or isolates from blood cultures
The Gram stain reflects underlying differences in the bacterial cell wall.
A microscope slide containing the clinical specimen is fixed and flooded with Crystal Violet dye followed by Gram’s iodine. The slide is decolorized before Safranin counterstain is added to the slide. Gram positive bacteria retain the initial Crystal Violet stain making them appear purple, whereas Gram negative bacteria lose the Crystal violet dye at the decolorization step, but retain the safranin counterstain, making them appear pink or red.