The purpose of Gram staining is to demonstrate the presence of Gram positive and Gram negative bacteria in clinical specimens using morphology and Gram staining. This test is useful for determining the presence of potentially pathogenic bacteria in clinical specimens based on their morphology and demonstrating the purity of cultures; it also aids in the identification of gonococci and pneumococci. Grams staining is required for all suspected cases of bacterial meningitis. The presence or absence of pus cells and squamous epithelial cells is also determined using Gram stained smears.
Principle of Gram Stain
- Gram’s staining procedure calls for the sequential application of four reagents: a basic dye crystal violet (primary stain), an aqueous solution of iodine, a decolorizing solvent, acetone and ethyl alcohol, and a counter stain, safranin or dilute carbol fuchsin.
- In the bacterial protoplasm and cell wall, the crystal violet dye and iodine combine to form a dark purple compound. This compound is dissociable in the decolorizer, ethyl alcohol, which dissolves and removes the primary stain from the cell wall, the removal being much slower in Gram positive bacteria than in Gram negative bacteria because Gram positive organisms have thicker and denser peptidoglycan layers in their cell walls, making them more permeable to the stain.
- Iodine acts as a mordant and is essential in enhancing this difference.
- Gram-positive bacteria that have been decolorized with alcohol retain the crystal violet dye and appear violet or purplish blue.
- Gram-negative bacteria are unable to retain the crystal violet dye after decolorization and instead absorb the red color of the counter stain dilute carbol fuchsin.
Type of Sample used for Gram stain
Any clinical specimen, tissue biopsy and/or bodily fluids collected for bacterial etiology and investigation.
- Clean, dry, grease free microslides/slide
- Normal saline (0.85% or 0.9%)
- Methanol (for fixing)
- Tissue papers
Gram’s stain reagent
- Crystal violet (0.5%) preferably 1 in 10 working solution
- Gram’s Iodine (1%) preferably 1 in 10 working solution
- Decolorizer : Either Acetone or Methyl alcohol
- Dilute Carbol fuchsin (if 0.5 or 1% – 10 mL)
- Immersion oil/Cedar wood oil
Equipment used in Gram Staining
- Bunsen Burner
- Light Microscope – 100 × magnification
Smear Preparation Procedure
- Mark the area of the smear as a circle on the underside of the slide.
- Label the slide with the specific lab no. or name of the organism.
- Fix the smear by gentle heat by passing the slide 3 or 4 times through the flame of bunsen burner so that the smear does not get washed off during the staining procedure or fix the smear in Methanol (in a coplin jar) for 3 minutes.
Gram’s Staining Procedure
- Cover the smear with crystal violet and leave it for 1 minute.
- Wash the slide with distilled water.
- Cover the slide with Gram’s iodine and leave it for 1 minute.
- Wash the slide with distilled water (for few seconds).
- Decolorize quickly with decolorizer until no further violet color comes out
- Wash with distilled water immediately.
- Counter stain with dilute carbol fuchsin for 1 minute.
- Wash with distilled water and blot dry.
- Observe the stained smear under the oil immersion (100 X) objective.
Interpretation of Results
- Gram positive bacteria appear violet or purplish blue and Gram-negative bacteria appear red or pink.
- Indicate the morphology and arrangement specifically, especially for Gram-positive cocci.
- Mention the coccobacillary forms with regard to gram-negative bacilli if seen.
- Yeast cells appear dark purple in color.
- Pus cells appear pink in color.
- Epithelial cells appear pale pink in color
Quality Control Procedure
When using new batches of stains, make sure they’re reliable by comparing them to known bacteria/strains of bacteria.
Staphylococcus aureus purple in color for Gram positive, Escherichia coli pink in color for Gram negative bacilli.
- Follow all safety precautions when handling biological fluids.
- When transferring and handling the specimen, wear a facemask and disposable gloves.
Potential Sources of Variability
- Process specimens before they dry up
- Over decolorization, old Gram’s iodine, old cultures, or overheat fixation can cause Gram positive organisms to lose their ability to retain the crystal violet stain and appear Gram negative.
Limitations of Gram stain
- Gram staining can tell you whether the bacteria are Gram positive or Gram negative, but it can’t tell you what genus and species they are.
- Background flora contamination of the specimen may interfere with the outcome.
- Grams stain cannot be used to stain or differentiate certain organisms (Acid fast organisms and Legionella pneumophilia).
- Gram’s stain is unreliable for detecting urethral gonococcal infection in the cervical, rectal, pharyngeal, or asymptomatic regions.
- Collee, Dugid, Fraser, Marmion. Laboratory strategy in the diagnosis of infective syndromes. 13th Edition Mackie and McCartney. Practical Medical Microbiology 1989;600-50.