Preparing Your Slides

• 1). Place a small drop of sterile deionized water in the center of the slide. With proper aseptic technique, use the inoculating loop to smear a small amount of the culture into the drop.
  
  
• 2). Allow the slide to air dry. This may take awhile, depending on how much water you put on the slide and how thin you smeared the culture. The smear should be completely dry before proceeding.
  
  
• 3). Heat fix the slide by passing it over the flame two or three times. This kills the cells on the slide.
  
  

Gram Staining

• 1). Flood the heat-fixed slide with crystal violet. Let it stand for 60 seconds, then rinse gently with tap water.
  
  
• 2). Flood the slide with the iodine and let sit for 60 seconds, then rinse with tap water.
  
  
• 3). Decolorize with ethanol or acetone. Hold the slide at a slant and squirt with the decolorizing agent for three seconds, letting the decolorizer and excess dye run off the slide. Do not over-decolorize. Rinse immediately with water.
  
  
• 4). Counterstain by flooding the slide with safranin for two minutes. Rinse with water and blot dry with the tissue paper.
  
  When the cells are viewed with the microscope, ascospores will be Gram-negative (pink) and the vegetative Saccharomyces cells will be Gram-positive (violet).