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Steps involved in Gram’s Staining:

Step 1:

Take a loopful of culture of the bacteria and prepare a smear on a clean grease-free glass slide.

Step 2: 

Air-dry and Heat-fix the smear by passing through the flame (2-3 times)

Step 3:

Add Crystal Violet (Primary Stain). Keep for 1 minute. Drain the stain and wash with water.

Step 4:

Add Gram’s Iodine (Mordant). Keep for 1 minute. Drain the stain and wash with water

Step 5:

Wash with 95% Alcohol strictly for 30-45 seconds only. Air dry.

Step 6:

Add Safranin (Counterstain). Keep for 1 minute. Drain the stain and wash with water.

Step 7:

Add a small drop of Cedar wood Oil and observe under Oil immersion lens of microscope.

EXPLAINATION:

The Gram’s staining (Differential staining) method is responsible for classifying the bacteria into two broad groups i.e. Gram Positive bacteria and Gram Negative bacteria according to the stains they take up during the staining procedure.

The bacteria which take up the crystal violet stain and are stained violet-blue in colour are called as Gram-positive bacteria.

The bacteria which take up the count stain (Basic Fuchsin, Saffrain) and are stained pink or red in colour are called as Gram-negative bacteria

FOR GRAM POSITIVE BACTERIA:

The cell walls of gram positive bacteria are thick and are mainly composed of proteins and mucopeptides.

During Gram’s staining, the crystal violet is the primary stain. After the application of a mordant i.e. Gram’s Iodine, a cell is more strongly stained. When treated with alcohol, it causes dehydration and closure of the cell-wall pores, thereby the Crystal-violet iodine complex (CVI) formed remains inside and here the cell remains violet in colour and does not take up the counter stain.

FOR GRAM NEGATIVE BACTERIA:

The gram negative bacteria cell wall is thin, complex, multilayered and contains relatively large lipid content in addition to proteins and mucopeptides. The high amount of lipids are readily dissolved by alcohol, resulting in the formation of pores in the cell wall which causes the Crystal-violet iodine complex (CVI) to leak out and results in decolourisation of the cell which later takes up the counter stain and appears red or pink in colour. 

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