GRAM'S STAINING - PRINCIPLE , PROCEDURE,OBSERVATION

GRAM'S STAINING

INTRODUCTION  :

HANS CHRISTIAN GRAM

- The gram stain was developed in 1884 by danish bacteriologist HANS CHRISTIAN GRAM .It is very important differential staining because it separates bacteria into two broad categories namely GRAM POSITIVE  and GRAM NEGATIVE.

- this staining require 4 reagent 

    1).  primary stain - crystal violet 

    2). second reagent - gram's iodine 

    3). third reagent  - 95% alcohol or acetone  as a decolorizing agent 

    4). fourth reagent - secondary stain or counterstain (safranin stain )

PRINCIPLE 

When the bacteria are stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram-positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram-positive bacteria and appears blue or purple in color.

In the case of gram-negative bacteria, the cell wall also takes up the CV-Iodine complex but due to the thin layer of peptidoglycan and a thick outer layer which is formed of lipids, the CV-Iodine complex gets washed off. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. Then when again stained with safranin, they take the stain and appear red in color.

REQUIREMENT :

1). young culture of the tested organism

2).crystal violet

3).gram's iodine

4).95% alcohol or acetone

5).safranin stain

PROCEDURE :

1. Take a clean, grease-free slide.
2. Prepare the smear of suspension on the clean slide with a loopful of the sample.
3. Air dry and heat fix
4. Crystal Violet was poured and kept for about 30 seconds to 1 minute and rinse with water.
5. Flood the gram’s iodine for 1 minute and wash with water.
6. Then, wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.
7. Add safranin for about 1 minute and wash with water.
8. Air dry, Blot dry, and Observe under Microscope.

OBSERVATION:

Gram-Positive: Blue/Purple Color
Gram-Negative: Red Color