Preparing Bacterial Smear for Microscopic Staining

Initial Preparation

• Cleaning Slides:
  • Apply cleanser to fingers.
  • Spread cleanser on slides.
  • Rinse slides to remove cleanser.
  • Blot slides dry.
• Labeling Slides:
  • Label slides with the microbe type.
  • Draw a dime-sized circle on the underside of each slide.

Smear Preparation from Liquid Culture

1. Flame Sterilization of Loop:

   • Hold loop in the hottest part of flame until red-hot.
   • Allow to cool for ~30 seconds.
   • Do not touch or place loop on counter directly.

2. Handling Liquid Culture:

   • Gently flick tube to suspend bacteria.
   • Hold tube at an angle, remove cap with ring and pinky finger.
   • Pass tube mouth quickly through flame.

3. Transfer Bacteria:

   • Use loop to pick up a loop full of broth.
   • Flame the tube again and replace cap.
   • Spread broth on the slide within the circle.
   • Repeat to place 2-3 loopfuls on slide.

4. Drying the Smear:

   • Allow smear to air dry completely.
   • Do not blow on or heat the slide.

Smear Preparation from Solid Culture

1. Flame Sterilization:

   • Flame loop, put a loopful of water on the slide.
   • Flame loop and allow to cool.

2. Handling Solid Culture:

   • Gently scrape bacteria from agar surface.
   • Remove cap and flame tube, scrape surface.
   • Add scraped bacteria to water on slide.

3. Creating Emulsion:

   • Spread to form a milky emulsion.

4. Drying the Smear:

   • Let smear air dry completely.

Fixing the Smear

• Purpose:

  • Denatures enzymes to prevent digestion of cells.
  • Preserves size and shape of cells during staining.

• Heat Fixing:

  • Pass dried smear quickly through a flame 2-3 times.

• Chemical Fixing:

  • Cover smear with methanol for 1 minute.
  • Let alcohol run off and allow slide to air dry.

Important: Fix only after the smear has completely air dried.