Negative Staining in Microbiology

Overview

• Negative Staining: A simple staining technique where the background is stained, leaving the cell colorless.
• Purpose: Bacteria are less distorted as heat fixation is not used.

Materials Needed

• Two slides
• Nigrosin dye
• Bacterial culture (solid or broth)
• Distilled water
• Inoculating loop
• Bunsen burner

Procedure

For Solid Media Cultures

1. Prepare Slides:
   • Place a small drop of nigrosin at the end of each slide.
   • Add a loopful of distilled water to emulsify culture in the nigrosin drop.
2. Sterilization:
   • Flame the loop before and after use.
   • Remove cap from culture tube and flame the tube.
3. Mixing:
   • Mix a loopful of bacteria with the nigrosin-water drop.
4. Smearing:
   • Use another slide to spread the drop, varying from opaque black to gray.
   • Let the smear air dry (do not heat fix).

For Broth Cultures

1. Prepare Slides:
   • Mix a loopful of broth culture into the nigrosin drop.
2. Sterilization:
   • Flame the loop before and after use.
   • Remove cap from broth tube and flame the tube.
3. Mixing:
   • Mix a loopful of broth with the drop of nigrosin.
4. Smearing:
   • Spread the drop using another slide, varying from opaque black to gray.
   • Let it air dry (do not heat fix).

Examination

• Use a microscope to examine the slide.
• Key Points:
  • Thick stains may appear cracked and are unviewable.
  • Areas with too little stain will not contrast well.
  • Ideal observation shows colorless cells against a black background.

Staining Chemistry

• Nigrosin: Negatively charged dye.
• Mechanism: Repelled by the negatively charged bacterial surface, resulting in a clear cell and capsule against a dark-stained background.

Post-Examination

• Gently blot oil from the slide.
• Store the slide in a slide box for future reference.

Notes

• Ensure all materials are sterilized appropriately throughout the procedure.
• The angle of the spreading slide affects the thickness of the smear, which is crucial for optimal observation.