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Microbiology

Stains

Each stain is designed to highlight specific cellular components or organisms based on their unique properties. Understanding the principle behind each stain is crucial for accurate interpretation and reporting of results

0.1 Gram Stain

  • Principle: Differentiates bacteria based on cell wall structure (peptidoglycan layer thickness). Gram-positive = thick layer, Gram-negative = thin layer
  • Procedure: Crystal violet (primary stain), Gram’s iodine (mordant), alcohol/acetone (decolorizer), safranin (counterstain)
  • Interpretation: Gram-positive = purple, Gram-negative = pink/red. Used to determine cell morphology

0.2 Acid-Fast Stain

  • Principle: Detects bacteria with mycolic acid in their cell walls (e.g., Mycobacterium). Mycolic acid resists decolorization
  • Procedure: Carbolfuchsin (primary stain, often with heat), acid-alcohol (decolorizer), methylene blue (counterstain)
  • Interpretation: Acid-fast = red, non-acid-fast = blue

0.3 Modified Acid-Fast Stain

  • Principle: Detects organisms that are partially acid-fast (e.g., Nocardia, Cryptosporidium). Uses a weaker decolorizer
  • Procedure: Similar to acid-fast, but uses a milder acid solution for decolorization
  • Interpretation: Partially acid-fast = red/pink, non-acid-fast = blue

0.4 KOH and Calcofluor-White

  • KOH Principle: Clears debris in specimens to enhance visualization of fungal elements
  • Calcofluor-White Principle: Fluorescent dye that binds to chitin in fungal cell walls, making them fluoresce under UV light
  • Procedure: KOH added to sample, +/- heat. Calcofluor-white added, viewed under UV microscope
  • Interpretation: Fungal elements (hyphae, spores) are visible with KOH, and fluoresce bright apple-green or blue-white with calcofluor-white

0.5 Trichrome Stain

  • Principle: Differential stain used to visualize intestinal parasites in stool samples
  • Procedure: Fixation, staining with trichrome dye mixture, acetic acid rinse, dehydration, clearing, mounting
  • Interpretation: Protozoan cytoplasm = blue-green or purple-pink, nuclear structures = red/purple-red, background = green

0.6 Giemsa Stain

  • Principle: Differential stain used to visualize blood cells, parasites, and some bacteria. Stains nuclei and cytoplasm different colors
  • Procedure: Fixation, staining with Giemsa solution, rinsing
  • Interpretation: Nuclei = purple, cytoplasm = pink. Helps identify blood cells, blood parasites (e.g., malaria), and certain bacteria

0.7 Acridine Orange

  • Principle: Fluorescent stain that binds to DNA and RNA, allowing rapid detection of bacteria and fungi
  • Procedure: Staining with acridine orange, rinsing, viewing under fluorescence microscope
  • Interpretation: Bacteria and fungi fluoresce bright green

0.8 Modified Trichrome

  • Principle: Enhances visualization of microsporidia spores in stool and other specimens
  • Procedure: Similar to trichrome, but with modifications to the dye concentrations and staining times
  • Interpretation: Microsporidia spores = pinkish-red to red-purple, often with a visible polar filament