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SOP

Gram Staining Procedure

SOP for differential Gram staining to classify bacteria as Gram-positive or Gram-negative and assess morphology.

Last verified 1 month ago 2 min read Microbiology Staining Bacteriology Laboratory Techniques #microbiology #Gram Stain #Bacteriology #gram staining #differential stain #gram positive #gram negative
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Document Control
Version
1.0
Effective
Aug 28, 2026
Reviewed
Aug 29, 2026
Department
Microbiology
Principle

Gram staining differentiates bacteria based on cell wall composition. Gram-positive bacteria have thick peptidoglycan layers that retain the crystal violet-iodine complex after decolorization, appearing purple. Gram-negative bacteria have thin peptidoglycan and outer lipid membranes; the decolorizer dissolves the lipid, allowing the crystal violet-iodine complex to wash out, and they take up the counterstain (safranin), appearing pink/red.

Standard Operating Procedure: Gram Staining

1. Purpose and Scope

To perform differential Gram staining for preliminary identification of bacteria based on cell wall characteristics, morphology, and arrangement.

2. Specimen Requirements

  • Type: Bacterial colonies from culture, clinical specimens (sputum, CSF, pus, urine sediment).
  • Volume: Small amount sufficient for smear preparation.
  • Rejection Criteria: Dried or improperly collected specimens.

3. Safety and Precautions

  • Wear appropriate PPE (lab coat, gloves, eye protection).
  • Handle all specimens as potentially infectious.
  • Use Bunsen burner in well-ventilated area.
  • Crystal violet and safranin are irritants.

4. Equipment and Reagents

  • Clean glass slides, inoculating loop, Bunsen burner.
  • Reagents:
  • Primary stain: Crystal violet (1% aqueous or Hucker's)
  • Mordant: Gram's iodine
  • Decolorizer: 95% ethanol or acetone-alcohol
  • Counterstain: Safranin (0.5% aqueous) or basic fuchsin
  • Microscope with 100x oil immersion objective, immersion oil.

5. Step-by-Step Procedure

  1. Smear Preparation:
  • For liquid specimens: Place 1-2 loops on slide, spread to form thin film.
  • For colonies: Emulsify small amount in drop of saline/water on slide.
  • Air dry completely.
  1. Heat Fixation: Pass slide through flame 2-3 times (specimen side up). Do not overheat.
  2. Staining:
  • Flood smear with crystal violet for 1 minute. Rinse gently with water.
  • Flood with Gram's iodine for 1 minute. Rinse with water.
  • Decolorize: Tilt slide, apply 95% ethanol drop by drop for 5-15 seconds until runoff is clear. CRITICAL STEP - immediately rinse with water.
  • Flood with safranin for 30-60 seconds. Rinse with water.
  1. Drying: Air dry or blot gently with bibulous paper.
  2. Microscopy: Examine under 100x oil immersion objective.

6. Interpretation

  • Gram-positive: Purple/violet color (thick peptidoglycan retains crystal violet-iodine complex).
  • Gram-negative: Pink/red color (thin peptidoglycan, decolorized, takes up safranin).
  • Note morphology (cocci, bacilli, spirilla) and arrangement (chains, clusters, pairs).

7. Quality Control

  • Run known Gram-positive (Staphylococcus aureus ATCC 25923) and Gram-negative (Escherichia coli ATCC 25922) controls with each new batch of reagents.
  • Check reagent freshness; replace if contaminated or expired.

8. Reporting Results

  • Report Gram reaction, morphology, and arrangement (e.g., "Gram-positive cocci in clusters," "Gram-negative bacilli").
  • Semi-quantify if from clinical specimen (Rare, Few, Moderate, Many).

Frequently Asked Questions

Over-decolorization, old cultures (>24 hours), cell wall damage, or certain species (e.g., Bacillus, Clostridium) can cause variable staining.
Over-decolorization causes Gram-positives to appear Gram-negative; under-decolorization causes Gram-negatives to appear Gram-positive.