Gram Staining FAQs and MCQs

Gram staining is a differential bacterial staining technique used to classify bacteria into Gram-positive and Gram-negative based on their cell wall composition.

Danish bacteriologist Hans Christian Gram in 1882.

It helps quickly identify bacterial infections, guides antibiotic treatment, and is the first step in bacterial identification.

Gram-positive (purple) and Gram-negative (pink/red).

Differences in bacterial cell wall structure (peptidoglycan thickness and lipid content) determine stain retention.

No, it doesn’t work for bacteria without cell walls (e.g., Mycoplasma) or very small bacteria (e.g., Chlamydia).

False results due to over-/under-decolorization, old cultures, or thick smears; cannot identify specific species.

Crystal violet (primary stain), Gram’s iodine (mordant), decolorizer (ethanol/acetone), and safranin/carbol fuchsin (counterstain).

It forms a complex with crystal violet, trapping it in Gram-positive cell walls.

It dissolves lipids in Gram-negative cell walls, washing out the primary stain.

Typically 30–60 seconds per step, but decolorization requires careful timing (5–10 seconds).

To stain decolorized Gram-negative bacteria pink/red for visibility.

Yes, it’s an alternative counterstain, especially for faintly staining Gram-negative bacteria.

Spread a thin bacterial sample on a slide, air-dry, and heat-fix to adhere cells.

It may retain excess stain, leading to false Gram-positive results.

It kills bacteria and adheres them to the slide to prevent washing off during staining.

100× oil immersion lens after initial 10× and 40× checks.

Gram-positive: Purple/blue; Gram-negative: Pink/red.

Bacteria that stain irregularly (mix of pink/purple) due to cell wall variability.

Staphylococcus, Streptococcus, and Enterococcus.

E. coli, Pseudomonas, Klebsiella, and Proteus.

Possibly Neisseria gonorrhoeae or N. meningitidis.

Staphylococcus: Clusters; Streptococcus: Chains.

Mycobacterium spp. (require Ziehl-Neelsen stain).

Over-decolorization, old cultures, or antibiotic treatment.

Likely an active bacterial infection.

For suspected bacterial infections (e.g., UTIs, pneumonia, meningitis).

Pneumonia (S. pneumoniae), UTIs (E. coli), gonorrhea (Neisseria), etc.

Yes, yeasts (e.g., Candida) may appear Gram-positive.

Gram-positive vs. Gram-negative results help narrow antibiotic choices (e.g., vancomycin for Gram-positive).

It provides results in minutes/hours, while cultures take days.

Sputum, blood, urine, CSF, wound swabs, and bodily fluids.

Midstream urine is centrifuged, and the sediment is smeared.

Bacterial meningitis (e.g., S. pneumoniae or N. meningitidis).

Over-decolorization or old Gram-positive cultures.

Under-decolorization or thick smears.

Low bacterial load, prior antibiotics, or improper sample collection.

It may alter bacterial cell walls, leading to atypical staining.

Campylobacter or Brucella may require carbol fuchsin for better visibility.

Squamous epithelial cells suggest poor sample collection (e.g., saliva in sputum).

Mixed with ammonium oxalate and ethanol

Yes, crystal violet may form precipitates; filtering is recommended.

To improve resolution under the 100× objective lens.

Acetone alone or ethanol (95%), but 50:50 acetone-ethanol is common.

Air-dried and kept away from light to prevent fading.

Thick peptidoglycan in Gram-positive bacteria traps the CV-I complex.

Their thin peptidoglycan and outer membrane are disrupted by decolorizer.

Burke’s (aniline-oil decolorizer) and Atkin’s methods.

Its waxy cell wall requires acid-fast staining (e.g., Ziehl-Neelsen).

Yes, Bacillus and Clostridium spores may appear as clear ovals within stained cells.