Question

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Answer 1

Propose of 4 reagents -

Gram staining is a bacterial staining procedure which stains bactera on the basis of their cell wall composition. Gram positive cell which is having a thick peptidoglycan cell wall stains purple and gram negative bacteria which is having a thin peptidoglycan stains reddish orange.

The steps and reagents used in this teachnique are -

1. Preparation of bacterial smear on a slide.

2. Heat fix the bacterials cells to the slide.

3. Add crystal violet stain and wait for 1 minute. Wash with water.

4. Add iodine which a mordant and wait for 1 minute. Wash with water.

These 3 steps (3 and 4) leads to the formation of big sized crystal violet iodine complexes which get entrapped in the meshwork of peptidogylcan layer.

Iodine is a mordant or entrapping agent which helps the trapping of crystal violet stain molecules into peptidoglycan.

5. Add alcohol as decolouriser. Wait for 30 seconds. Wash with water.

Decoloriser removes excess stain. Crystal violet iodine complexes are retained by thick peptidoglycan of gram positive bacteria whereas they are washed away from thin peptidoglycan of gram negative bacteria. These big sized complexes could not be hold by thin peptidoglycan of gram negative bacteria.

6. Add counterstain safranine. Wait for 30 seconds. Observe under the microscope.

Counterstain stains the molecules which got destained by decolourizer so, that they also appear reddish orange.

Answer -

1. Crystal violet complexes will not be held by thick peptidoglycan and gram positive will appear reddish too.

2. Gram negative bacteria appears purple too.

3. Gram negative bacteria appears colourless.

4. All bacteria will appear red.

5. Gram positive will also lose crystal violet iodine complexes and will get stained by safranine and will appear red.

6. Gram negative bacteria will also appear purple.

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