Gram Stain

The Gram Stain is one of the most valuable tools to identifying bacteria. It uses the differences in the bacterial cell wall in order to seperate bacteria into groups. It is performed by staining the bacteria for a minute with Crystal Violet, rinsing with water, applying Gram's Iodine solution for a minute, rinsing with ethanol for 10 seconds, then with water, and finally applying Safranin for a minute for the counter-stain before rinsing and blot drying. Gram positive bacteria have a thin lipid layer on their cell walls while Gram negative bacteria have a very thick lipid layer. These lipids interfere with the binding of Crystal Violet (even with the iodine complex strengthening). While the dye will bind to them it isn't strong enough to withstand the decoloration stage with ethanol. The final staining will make Gram positives purple and negatives red under a microscope. A word of caution, while this works for most bacteria (and all the ones covered in this guide), there are some types that resist this procedure, or may end up halfway between the two.