Differential Staining Techniques in Microbiology

Overview

• Presenter: Cathy with Level Up RN
• Topics Covered: Gram stain, acid-fast stain, capsule stain, endospore stain
• Focus: General steps, result interpretation, rationale
• Quiz: Included at the end to test understanding

Gram Stain

• Importance: Most important staining technique
• Procedure: Steps
  1. Heat-fix slide
  2. Primary stain: Apply crystal violet (cells turn purple)
  3. Mordant: Apply iodine to set stain (complexes form between crystal violet and iodine)
  4. Decolorizing agent: Apply ethanol/acetone
     • Gram-positive: Thick peptidoglycan layer traps complexes (remains purple)
     • Gram-negative: Thin peptidoglycan layer can't retain complexes (dye washed out)
  5. Counter stain: Apply safranin
     • Gram-positive: Remains purple
     • Gram-negative: Turns pink
• Note: Use fresh bacterial cultures to avoid false negatives

Acid-Fast Stain

• Purpose: Differentiate cells with mycolic acid in cell walls
• Common Method: Ziehl-Neelsen method
• Procedure: Steps
  1. Primary stain: Apply carbol fuchsin and steam slide (penetrates mycolic acid)
  2. Decolorizing agent: Apply acid alcohol
     • Non-acid-fast: Red stain removed
     • Acid-fast: Stain not removed (retains red)
  3. Counter stain: Apply methylene blue
     • Non-acid-fast: Turns blue
     • Acid-fast: Remains red/pink

Capsule Stain

• Purpose: Identify bacterial cells with capsules
• Common Method: Anthony's capsule stain
• Procedure: Steps
  1. Prepare and air dry slide (heat-fixing destroys capsules)
  2. Primary stain: Apply crystal violet (stains cell and capsule)
  3. Decolorizing agent and counter stain: Apply 20% copper sulfate
     • Capsule: Removes crystal violet, absorbs copper sulfate
  4. Air dry slide (don't blot)
  5. Observation: Cells and background violet; capsules white/light-blue halos

Endospore Stain

• Purpose: Visualize endospores in bacterial cells
• Common Method: Schaeffer-Fulton method
• Procedure: Steps
  1. Prepare and heat-fix slide
  2. Primary stain: Apply malachite green and steam slide (penetrates spore wall)
  3. Rinse with water (decolorizes vegetative cells, not spores)
  4. Counter stain: Apply safranin
     • Vegetative cells: Turn pink
     • Endospores: Remain green
  5. Observation: Vegetative cells pink, endospores green

Quiz Questions

1. Gram stain result for gram-negative cells? Pink
2. Acid-fast stain purpose? Differentiate cells with mycolic acid
3. Why not heat-fix before capsule stain? Heat can destroy capsule
4. Capsule stain appearance? White/light-blue halos around cells

Conclusion

• Recommendation: Use Level Up RN microbiology flashcards
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