Gram Staining: Purpose, Principle, Procedure and Observation

In 1883 (originally published in 1884), Dr. Hans Christian Gram (1853-1938) developed a technique for the classification of bacteria into two broad groups, Gram-positive and Gram-negative. It is the most important staining technique for the classification and differentiation of bacteria.

The Gram stain consist of four reagents; crystal violet (use as a primary dye), Gram's iodine (use as a mordant), ethyl alcohol (use as a decolorizer), and safranin (use as a counterstain). The Gram-negative, on the other hand, lose the primary dye (crystal violet) when decolorized and, thus, take the color of counterstain (safranin).

The Gram-reaction rely upon the chemical nature of the bacterial cell wall, especially the lipids which comprise 11-22% in Gram-negative cell wall and 1-4% in the Gram-positive cell wall. In the Gram-negative cell wall, the amount of lipids is very high, when the cell is dissolved in alcohol, it leads to the formation of large pores in the cell wall. The dehydrating result of alcohol cannot fill these pores which cause the liberate of primary stain making the cell colorless. Such cells take the color or counterstain (safranin). On the contrary, the amount of lipid is very low in the Gram-positive cell wall and easily dissolved by in ethyl alcohol, causing the formation of very small pores. These pores are further closed by the dehydrating effect of alcohol which does not permit the primary dye (crystal violet) to leave the cell.

PURPOSE

Identification, differentiation, and classification of the bacteria.

NEEDS

Specimen

Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria or fungi grown and isolated in culture.

Reagents

1. Crystal violet
2. Gram iodine
3. Ethyl alcohol (95%)
4. Safranin
5. Ceder wood oil

Equipment

1. Bunsen burner
2. Wire loop
3. Glass slides
4. Microscope

PROCEDURE

1. Prepare a smear of the specimen, dry in air and then fix it in low flame.
2. Flood the smear with crystal violet, and keep it for 1 minute. Wash the smear with running tap water.
3. Pour Gram's iodine on smear and after 1 minute, wash it with tap water.
4. Pour alcohol on the smear until the purple color no longer comes from the smear.
5. Pour safranin on the smear and after 45 seconds, wash it with tap water and keep the smear dry in air.
6. Observe the stained smear under oil immersion lens and note down the arrangement, shape, and Gram-reaction of the cells.

OBSERVATION

The Gram-positive bacteria appear in purple color and Gram-negative bacteria appear in pink color.