Introduction

Gram staining is a common laboratory technique used to differentiate and classify bacteria into
two broad categories based on their cell wall composition. The method was developed by the
Danish bacteriologist Hans Christian Gram in 1884.

Gram staining involves a series of steps:

1. A thin smear of a bacterial sample is placed on a microscope slide and heat-fixed to attach the
bacteria to the slide.

2. The smear is then treated with a crystal violet stain, which colors all the bacterial cells purple.

3. Iodine solution is applied to the smear, which forms a complex with the crystal violet and
helps to trap the stain within the cell.

4. The slide is washed with a decolorizing agent, such as alcohol or acetone. This step is critical
as it differentiates between Gram-positive and Gram-negative bacteria based on the structure of
their cell walls. Gram-positive bacteria retain the crystal violet stain and appear purple, while
Gram-negative bacteria lose the stain and appear colorless.

5. The smear is then counterstained with a contrasting red or pink stain, such as safranin, which
colors the Gram-negative bacteria red or pink.

6. The stained slide is then observed under a microscope. Gram-positive bacteria appear purple,
while Gram-negative bacteria appear red or pink.

Gram staining is a valuable tool in microbiology for quickly identifying and categorizing
bacteria, which helps in selecting appropriate antibiotics for treatment and in understanding the
nature of the bacterial infection.