Gram staining

Gram stain

Objective

To differentiate between gram +ve and gram -ve bacteria by Gram staining.

Materials required

Culture of bacteria or curd, Crystal violet/Gentian violet, Iodine solution/Gram's iodine, 95% ethyl alcohol, safranin, clean glass slide, glass marker pencil, Bunsen burner, etc.

Principle

Gram staining is named after Christian Gram. It is based on principle of differential staining. It divides bacteria into two groups; gram +ve and gram -ve. When bacteria are stained with primary stain i.e., crystal violet and fixed by mordant i.e., iodine solution all the bacteria appear purple.

• Gram +ve bacteria: Gram +ve bacteria have a thick cell wall of peptidoglycan. When decolorized in alcohol, the call wall shrinks and closes the pores in the cell wall. This does not allow the purple color of crystal violet to leave the cell wall. As the cell wall is already stained, this does not allow crystal violet to react with the cell wall. Hence gram +ve bacteria still appear purple.
• Gram -ve bacteria: Gram -ve bacteria have an outer thick wall of lipid, which also catches the stain of crystal violet-iodine complex. But, when treated with alcohol, this lipid-stain complex washed-off, exposing the middle thin cell wall of peptidoglycan. When conterstained in safranin, it catches the color and appears red.

Structures of gram positive and gram negative bacterial cell wall

Procedure

Take a clean dry grease-free glass slide

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Prepare a smear of culture or liquid phase of curd and mark smear side glass marker pencil

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Let it air-dry and heat-fix on flame of Bunsen burner

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Dip the slide in crystal violet for 01 minute and wash in running water

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Dip the slide in iodine solution for 01 minute and wash in running water

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Dip the slide in 95% alcohol for 10 seconds and wash in running water

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Dip the slide in safranin for 01 minute and wash in running water

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Dry the slide in air and observe under compound microscope

Observation and result

• Gram Positive: Blue/Purple Color
• Gram Negative: Pink/Red Color

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Content first created on 06-01-2023
last updated on 06-01-2023