Gram Stain

Norm of Gram Stain

Body fluid, drainage, or wound

Interpretation required

Urine No organisms detected


Usage of Gram Stain

Anthrax meningitis (CSF), bacterial vaginosis, Barrett's esophagus, cough (sputum sample), effusion (abdominal or pleural), empyema, gonorrhea, impetigo, infections from catheters, Legionella, pulmonary nocardiosis, tuberculosis, and wounds.
Cough (productive), fever, infections, and pneumonia.
Cystitis and urethritis.


Description of Gram Stain

Gram staining divides bacteria into two groups according to their staining properties: gram negative and gram positive. The staining involves placing drops of crystal violet dye onto the specimen sample, washing off the violet stain, and flooding the smear with an iodine solution followed by a 95% ethyl alcohol (ethanol) solution. Gram-positive cells remain blue, and gram-negative cells are decolorized by the alcohol. The specimen is then stained with a red dye called “safranin O,” which colors the gram-positive cells red and leaves the gram-negative cells appearing purple. The cell wall structure is the basis of the Gram reaction. Gram staining of specimens aids in decision-making for early, broad-spectrum antibiotic therapy. Gram stain is 67.9% sensitive for detection of bacteria in blood cultures.


Professional Considerations of Gram Stain

Consent form NOT required.

  1. Diagnostic: Obtain a sterile container or swab.
  2. Sputum: Obtain a sterile sputum container or suction tubing, suction source, and sputum trap.
  3. Urine: Obtain a sterile container and clean-catch urine specimen collection kit, or a straight catheter or a syringe and needle if the specimen will be collected from an indwelling catheter.
  4. See Client and Family Teaching.



  1. Diagnostic:
    • a. Obtain the specimen using a sterile technique and a sterile container or swab.
    • b. Avoid contamination of the sample with surrounding tissue.
  2. Sputum:
    • a. Collect an early-morning sputum sample into a sterile sputum container.
    • b. Specimens are of best quality when obtained by direct suctioning into a sputum trap.
    • c. For expelled specimens, have the client sit up, take two or three deep breaths without fully exhaling each breath, and then cough expulsively to mobilize the sputum from the respiratory tract directly into the sterile specimen container.
  3. The clean-catch urine technique must be used to decrease the risk of specimen contamination. See clean-catch collection instructions in the test Body fluid, Routine—Culture.


Postprocedure Care

  1. Write the specimen source, the diagnosis, and recent antibiotic therapy on the laboratory requisition.
  2. Place the specimen in the refrigerator if not delivered to the microbiology area immediately after collection.


Client and Family Teaching

  1. Sputum: Cough the specimen directly into the container and avoid holding the sputum in the mouth. Deep coughs are necessary to produce sputum, rather than saliva. To produce the proper specimen, take several breaths in, without fully exhaling each, and then expel sputum with a “cascade cough.”
  2. Urine: See clean-catch collection instructions in the test Body fluid, Routine—Culture.


Factors That Affect Results

  1. Epithelial cells will appear in the specimen if it is contaminated with mucosal surfaces.
  2. Saliva contamination of sputum specimens invalidates the results.


Other Data

  1. Gram staining is not useful for identifying species of bacteria but can be suggestive of certain broad species.
  2. A culture and sensitivity study of the specimen should also be performed to confirm the diagnosis and proper choice of antibiotic.
  3. Compared to Gram stain of vaginal secretions, the cervical Papanicolaou smear has fair sensitivity (55%) and excellent positive predictive value (96%) in the diagnosis of bacterial vaginosis.