Gram Staining - Introduction, Purpose, Reagent, Principle, Procedure, Result

Introduction, Purpose of Gram Staining

Gram staining is a differential staining technique widely used to separate bacteria into two major groups- gram-positive bacteria and gram-negative bacteria. It is an essential method used for the classification and differentiation of bacteria.

Reagents for Gram Staining

The reagents used for gram staining include:

• Primary stain: Crystal violet

• Mordant: Gram's Iodine

• Decolorizing agent: Ethyl alcohol (95%)

• Counterstain: Safranin

Principle of Gram Staining

Bacterial cells contain pores through which stains can enter and give a prominent color, making their morphology and arrangement visible.

Primary stain

Crystal violet is used as a primary stain for gram staining. This stain colors all cells purple.

Mordant

Gram's Iodine serves as a mordant for gram staining that binds firmly with the primary stain i.e. crystal violet present resulting in the formation of crystal violet - Iodine (CV-I) complex. This complex intensifies the color of the stain and cells appear purple-black.

In cases of gram-positive organisms, the CV-I complex binds with the magnesium-ribonucleic acid of the cell wall forming magnesium-ribonucleic acid-crystal violet-Iodine complex (Mg-RNA-CV-I). Since Mg-RNA-CV-I is larger than just CV-I, it is more difficult to remove from bacterial cells.

Decolorizing agent

The decolorizing agent ethyl alcohol works both as a protein-dehydrating agent and a lipid solvent in gram staining. In gram-positive microorganisms, the cell walls have low lipid concentration while in gram-negative microorganisms, the cell walls have high lipid concentration.

When ethyl alcohol is used, it dissolves the lipid present in the bacterial cell walls resulting in the formation of new cell wall pores. Such pores are larger in gram-negative bacteria than in Gram-positive bacteria, which suffer minute pores, due to high cell wall lipid concentration in Gram-negative microorganisms.

Moreover, small pores of gram-positive bacteria are closed off due to dehydrating effect of alcohol. But in the case of gram-negative bacteria, pores are not able to be closed off by alcohol's dehydrating effect. As a result, the CV-I complex escapes from gram-negative bacteria while the Mg-RNA-CV-I complex remains in gram-positive microorganisms.

Counterstain

Safranin is the dye of choice in the counterstaining procedure of gram staining. The decolorized cells, that have lost the CV-I complex absorb the counterstain changing its color to red.

Gram-positive bacteria still hold Mg-RNA-CV-I complex from the primary stain during the decolorizing procedure and are unable to take in the safranin that has been used as a counterstain. Thus, they appear purple in color.

Procedure for Gram Staining

The procedure followed for gram staining is as follows:

1. Take a clean, grease-free slide

2. Prepare a bacterial smear of the test organism

3. Allow the smear to air dry

4. Heat fix the dried smear by quickly passing the smear through the bunsen burner 2-3 times

5. Flood the smear with primary stain (crystal violet) for 30 seconds to 1 minute

6. Wash the smear with slow-running water

7. Flood the smear with gram's iodine (mordant) for 1 minute

8. Wash the smear with slow-running water

9. Pour decolorizing agent (95% ethyl alcohol) on the smear drop by drop for 10-20 seconds

   * the purple color of crystal violet is removed during this procedure

10. Wash the smear with slow-running water

11. Flood with counterstain (safranin) for 45 seconds

12. Gently tap the slide with blotting paper to remove the remaining water/dye

13. Examine under a microscope (oil immersion at 100X)

Heat fixation

Heat fixation is done in most staining procedures (except capsule staining, and negative staining) because, during the washing steps, the microbial smear could wash away. During heat fixation, the bacterial proteins are coagulated and fixed to the glass slide surface.

It is done by rapidly passing the air-dried smear 2-3 times over a bunsen burner's flame.

* Instead of heat fixation, several methods of heat fixation have been recognized. One such commonly used procedure is the alcohol-based fixative. Ethanol or methanol can also be used as they also function by precipitating proteins.

Result of Gram Staining

The organisms after gram staining which have retained deep violet color from crystal violet are Gram-positive microorganisms.

The organisms after gram staining that have lost crystal violet and taken safranin to appear red or pink in color are gram-negative microorganisms.