Gram staining

Gram staining refers to a experimental method of placing bacteria into two sub-groups.( Gram positive and Gram negative )

Method

• Heat fix the bacterial sample to the slide, so that is is not washed away when applying further chemicals.
• Apply the Primary stain, Crystal Violet for one minute then rinse with water.
• Cover slide with Gram's Iodine for a further minute.
• Apply acetone to decolourise for no more than 5 seconds and wash with water.
• Apply Safranin as a counterstain for 2 minutes and was again with water.

Results

A Gram positive bacteria should give a purple stain. This is because the thick layer of Peptidoglycan retains the purple crystal violet stain.

A Gram negative bacteria should give a pink stain. This is becaue it does not retain the crystal violet because the peptidoglycan layer is in the periplasm. So it is stained by the counterstain of Safranin.