Gram staining

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

This method is used to differentiate between Gram Negative and Gram Positive bacteria. The method exploits the differing structures of the cell wall in both types of bacteria. In Gram positive bacteria, named like it is because it responds "positively" to staining, it is surrounded by a thick peptidoglycan layer, which offsets the need for it to have an additional outer membrane. This therefore sees a Gram positive bacterial cell wall, easily penetratable by the crystal violet stain used in Gram staining; it hence produces a purple stained bacteria as a result of the stain being retained by the thick peptidoglycan. In Gram negative bacteria, there is a reduced presence of peptidoglycan layers. It however has an additional outer membrane. This membrane stops the crystal violet stain from penetrating the cell wall and hence does not become purple in response to the crystal violet stain (also due to the lack of peptidoglycan, meaning the stain cannot be retained), but instead, once counterstained with safranin, turns pink/red colour, 

It's also useful to note there is a third group of bacteria, that do not respond to Gram staining. This group of bacteria can be categorised as Gram Neutral. 

Method

• Heat fix the bacterial sample to the slide, so that it 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 wash 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.