Negative Stain Technique

Purpose

• Provides a clear and distinct image of bacterial cells.
• Unlike traditional methods, it does not use heat, thus avoiding distortion of cells.

Principle

• Negative Stain: Uses a stain that carries a negative charge.
  • Bacterial cells also have a negative charge.
  • Cells repel the stain, resulting in a stained background while cells appear white against a dark background.
  • Provides excellent contrast.

Materials Needed

• Negatively charged stain (e.g., Nigresin).
• Bacterial culture.
• Inoculating loop.
• Staining tray.
• Two microscope slides.
• Heat source (for sterilization of loop).
• Slide warmer.

Technique Steps

1. Prepare the Slide

   • Place a small drop of nigresin near one end of a slide.
   • Avoid getting stain on fingers.

2. Prepare Bacterial Culture

   • Flame and cool loop to sterilize.
   • Collect a sample of bacteria using the loop.
   • Stir bacteria into the drop of stain (move laterally side to side).
   • Flame and cool loop again to destroy excess bacteria.

3. Spread the Stain

   • Use the second slide as a spreader.
   • Place at a 90-degree angle on the base slide.
   • Lean towards dye drop and pull up to allow dye to adhere.
   • Push away from fingers to spread the dye evenly.
   • Roll off the spreader slide at the end.
   • Dispose of contaminated spreader slide in biohazard bin or bleach water beaker.

4. Dry the Slide

   • Place the prepared slide on a slide warmer.
   • As water evaporates, dye bonds bacterial cells to the slide.

Final Product

• Use bibulous paper to check the final product.
• Ensure not too much nigresin is used to avoid long evaporation times.
• Avoid contaminating fingers with bacteria during the process.

Additional Tips

• Image of bacterial cells will show nice contrast due to the negative staining applied.