Grams Staining Technique

AIM: The aim of the experiment is to differentiate the gram positive bacteria from the gram negative bacteria by gram staining method

PRINCIPLE:

 Differential staining requires use of one or more chemical reagents .The most important differential stain used in bacteriology is gram staining .It is named after Dr Hans Christian’s gram. The description of the reagent and mechanism are as follows:

Primary Stain:

Crystal Violet:

            It stains the entire cell in violet colour.

Mordant: Gram’s Iodine

            Mordant or fixative is the substance which form the insoluble complex by binding to the primary stain

            The resultant crystal violet iodine complex intensify the colour of the stain and the cell appears violet or purple at the region .In the gram positive cells CVI complex binds to the mgRNA  component of the cell wall resulting in the mgRNA CVI complex, which tightly bind to the cell wall and it is more difficult to remove.

Decolorizing Agent:

            Ethyl alcohol 95% can act as a liquid solvent and protein dehydrating agent .The cell wall of the gram positive cells possess low lipid content .So it retains the mgRNA CVI complex.

            When decolorized, it forms minute cell wall pores which are closed by alcohol dehydration effect .As a result in gram positive bacteria it tightly bound the primary stain which is difficult to remove and the cell wall remains violet. The cell wall of the gram negative cells has high lipid content which are dissolved by alcohol which creates a large pores on the cell wall and are not enclosed by alcohol dehydration effect of the cell wall protein. This facilitates the release of CVI complex and the cell become colorless and unstained.

Counter stain:

            Gram negative organisms that are primarily decolorized absorb the counter stain and appear pink in colour and gram positive organism that retains the primary stain so it appears violet in color.

MATERIALS REQUIRED:

 Slide

Inoculation loop

Bunsen burner

Bacterial Culture

Gram staining reagent

PROCEDURE:

1.A bacterial smear was made in a clean grease free slide and it is fixed by air drying.

2.Crystal violet was applied to the smear and allowed for one minute and then wash the slide with tap water.

3. Iodine was added and it was allowed for one minute and then washes the slide with tap water.

4.The slide was then treated with alcohol through out the smear.

5.The counter stain saffranin was poured and allow it for 2 min and then wash the slide with tap water

6.The slide was allowed to dry and then observe the slide under oil immersion objective of the microscope.