CDC Safety Protocols and Gram Stain Procedure

CDC Safety Protocols

• Follow CDC protocols for PPE and equipment usage.
• Safety requirements like goggles and gloves vary by lab based on risk assessment.
• Follow CDC protocols for disposal of sharps and biohazard waste.

Gram Stain Technique

• Purpose: Differentiate bacterial species into gram-positive and gram-negative.
• Basis: Chemical and physical properties of cell walls.

Reagents Used in Gram Stain

1. Crystal violet (primary stain)
2. Iodine solution (mordant)
3. Decolorizer (usually 50-50 acetone/alcohol mixture)
4. Safranine or Carbolfuxin (counterstain)

Gram Stain Results

• Gram-positive: Retain crystal violet, appear purple.
• Gram-negative: Do not retain crystal violet, appear pink after counterstaining.

Steps in Gram Stain Procedure

1. Crystal Violet
   • Apply to heat-fixed smear evenly.
   • Wait for 15 seconds.
   • Rinse with water.
2. Iodine Solution
   • Apply to slide.
   • Wait for 15 seconds.
   • Rinse with water.
3. Decolorizer
   • Apply while holding slide at an angle until runoff is clear.
   • Immediate rinse with water to stop decolorization.
   • Note: Timing may vary; ensure not to over or under decolorize.
4. Counterstain (Safranine/Carbolfuxin)
   • Apply to slide.
   • Wait for 30 seconds.
   • Rinse with water.
   • Air dry slide.

Observations Under Microscope

• Gram-positive organisms (e.g., Staphylococcus aureus) appear purple.
• Gram-negative organisms (e.g., E. coli) appear pink.

Equipment Needed

• Personal protective equipment (PPE)
• Clean water source
• Slide rack
• Timer
• Reagents: Crystal violet, Gram's iodine, decolorizer, safranine/carbolfuxin
• Thin, heat-fixed bacterial smear

Important Notes

• Procedure follows ASM protocols but may vary: follow manufacturer's instructions.
• Reagents usually placed beside the sink; slide rack over sink for ease of rinsing.
• Ensure proper timing and application to achieve accurate results.