Stain

Stain

1. Gram Stain

   • 1. Make an emulsion of bacteria and heat fix the bacteria to the slide (see above). Cover the bacteria with 2. Crystal Violet - this is the primary stain. Allow it to stain for 1 minute. Rinse with DI water. Cover the slide with Iodine - this is the mordant. This creates a Crystal Violet-Iodine complex. Allow it to stain for 1 minute then rinse with DI water. Decolorization is what differentiates between Gram-positive and Gram-negative cells. Allow the decolorizer (Isopropanol) to trickle down the slide until the run-off is clear. Rinse with DI Water. Counterstain with safranin for 1 minute. This will stain the now clear Gram-negative bacteria. Rinse with DI Water . Blot with bibulous paper and then after it is dry you can observe under a microscope

2. Simple Stain

   • Apply the stain - covering the entire area. Be very careful - this will also stain clothes and hands. Allow the stain to sit for 30 seconds - 1 minute. Carefully rinse the slide with water. Gently blot the slid with bibulous paper. Do not rub the slide. When dry, observe under oil immersion.

3. Acid-Fast Stain

   • 1. Make an emulsion and heat fix the bacteria (see above for procedures). 2. Place the slide on a slide holder over steam. 3. Cover the smear with a strip of filter paper and flood with carbol-fuchsin. Allow the stain to sit for 5-10 minutes - add more stain as needed so that the slide does not dry out. Carefully discard the filter paper and rinse slide with DI water. Decolorize the smear with acid-alcohol until the runoff is colorless. Gently rinse the smear with DI water. Apply the methylene blue stain for 2 minutes. Rinse with DI water and dry with bibulous paper.