BISC209: The Gram Stain
The Gram Stain
Procedure:
1. Prepare a bacterial smear slide (Find protocol at BISC209: Preparing a bacterial smear slide).
To Gram stain the desired organism(s):
2. Flood the slide with Crystal Violet solution (0.5% crystal violet, 12% ethanol, 0.1% phenol) and stain for 1 minute. (Crystal violet is the primary stain.)
3. Rinse the slide in a very gentle stream of water; drain off excess water by touching the edge of the slide to a paper towel.
4. Flood slide with Gram's Iodine (mordant), let stand for 1 minute, and rinse with a gentle stream of water.
5. Quickly, drip Decolorizing Reagent (80% isopropyl alcohol, 20% acetone) down the length of the slide to remove the excess dye. This step is tricky as it is easy to over- or under-decolorize. IMMEDIATELY rinse with a gentle stream of water.
6. Flood the slide with the Counterstain Solution (0.6% safranin in 20% ethanol) for 2 minutes; rinse with water.
7. Blot dry using the bibulous paper package in your benches. Do not tear out the pages, just insert your slide and pat it dry. Then observe your stained microbes microscopically following the correct procedure for using the the oil immersion objective on your compound brightfield microscope BISC209: Microscopy found in the protocol page.
