Suppose you are trying to identify an unknown bacterial species. You perform a Gram stain and the slide is a blue/purple color when you are finished. Are the bacteria gram-positive or gram-negative?

GRAM staining is the technique to differentiate bacteria on the basis of stain. In this procedure bacterial cells with blue/purple colour are Gram positive and cells with pink colour are Gram negative.

PROCEDURE

In the first step cells are stained with crystal violet dye which is blue in color.

Then Grams iodine solution is added, it forms a complex between dye and iodine. This complex is insoluble in water.

Next step is addition of ethyl alcohol as a decolorizing agent. It shrinks the peptidoglycan layer by dehydrating it. In gram negative bacteria on the treatment of alcohol this complex( crystal violet- iodine complex) comes out from the outer membrane because of thin peptidoglycan layer. Gram negative bacteria lost color and decolorized. While in Gram positive bacteria because of thick peptidoglycan ,crystal violet-iodine complex trapped in the membrane. And cells don't give violet color of dye.

After this a counterstain safranine is used, which can not color the stained Gram positive bacteria. Gram negative decolorized bacteria stain pink with safranine.