The Gram Stain Procedure

The Gram Stain Procedure. This protocol is taken from "Benson's Microbiological Applications: Laboratory Manual in General Microbiology" Eleventh Edition.

Some protocols vary depending on the source. Use the one best suited for your laboratory.

The gram stain follows this procedure:

1. Prep your sample on your slide
2. Heat fix the slide
3. Flood with Crystal Violet for 20 seconds
4. Wash for 2 seconds
5. Flood with Gram's Iodine for 1 minute
6. Decolorize with Grams Alcohol for 10-20 seconds
7. Wash for 2 seconds
8. Flood with Safranin for 1 minute
9.Wash for 2 seconds
10. Blot the slide dry

Gram-Positive bacteria have a thick peptidoglycan layer on their cell membrane which retains the crystal violet-iodine complex and stains the bacteria purple.

Gram-Negative Bacteria have a thin peptidoglycan layer on their cell membrane which does not retains the crystal violet-iodine complex and is instead stained by the Safranin. This will leave the cells stained pink.