Gram Staining & Its Principle

Gram staining, a differential staining procedure, that divides bacteria into two groups i.e. Gram positive and Gram negative, is developed in the year 1884 by the Danish physician Hans Christian Gram. It is the one of the most widely employed staining technique in bacteriology.

In Gram staining procedure, the smear is first stained with a primary stain i.e. Crystal Violet (a basic dye). This is followed by the treatment with a mordant i.e. Iodine solution. Iodine helps the cell to stain more strongly by increasing the interaction between the two i.e. the dye and the cell. After this, the smear is decolorized by washing with acetone or ethanol and then it is counterstained with Safranin (a basic dye).

In this staining procedure, some organisms retain the color of primary stain (blue/purple) whereas others get decolorized and take up the color of counterstain (red/pink). Those organisms that retain the primary stain are known as Gram positive organisms and those that take the Safranin stain are known as Gram negative organisms. Thus, Gram reaction helps in classifying organisms into either of the two groups.

Some example of Gram positive bacteria are Staphylococcus aureus, Streptococcus pneumonia etc. Escherichia coli, Neisseria meningitides etc. are some examples of Gram negative bacteria.

Principle Of Gram Staining:

The basic principle of Gram staining is due to the difference in the cell walls of Gram positive and Gram negative bacteria. Gram positive bacteria have 20-30 nm thick cell wall that contains 70-80% of peptidoglycan and also it has very low lipid content. On the contrary, Gram negative bacteria have only 8-12 nm thick cell wall which contains only 10-20% of peptidoglycan. Lipid content is higher in Gram negative ones as compare to Gram positive. Also Gram negative bacteria have outer membrane which is absent in Gram positive ones.

        

In the Gram staining procedure when decolorizer (mixture of acetone and ethanol) is added, Gram positive cell walls dehydrate causing the closure of pores in the cell wall which results in the retention of crystal violet stain. On the other hand, the lipid content and thin peptidoglycan layer of Gram negative bacteria gets dissolved by the decolorizer as a result of which the dye gets washed off leaving the unstained bacteria which then takes up the counterstain.