Gram Stain Procedure

The Gram stain is a method for identifying bacteria based on the composition of their cell walls. It was developed by Hans Christian Gram in 1884 and is still widely used today in clinical microbiology laboratories.

Principle

The Gram stain procedure is based on the difference in the cell wall structure between Gram-positive and Gram-negative bacteria. Gram-positive bacteria have a thick peptidoglycan layer in their cell walls, while Gram-negative bacteria have a thinner peptidoglycan layer and an outer lipid bilayer containing lipopolysaccharides.

Reagents

• Crystal violet: a purple dye that stains all bacteria
• Iodine: a mordant that helps the crystal violet penetrate the cell wall
• Acetone or ethanol: a decolorizer that removes excess stain from Gram-negative bacteria
• Safranin: a pink dye that stains Gram-negative bacteria

Procedure

1. A sample of bacteria is applied to a microscope slide and allowed to air dry.
2. The slide is then heat-fixed by passing it through a flame, which kills the bacteria and attaches them to the slide.
3. Cystal violet is applied to the slide and allowed to stain for 1-2 minutes.
4. Iodine is applied to the slide and allowed to act as a mordant for 1 minute.
5. The slide is then decolorized with acetone or ethanol, which removes excess stain from Gram-negative bacteria.
6. Safranin is applied to the slide and allowed to stain for 1-2 minutes, staining Gram-negative bacteria pink.
7. The slide is then rinsed with water and allowed to air dry.

Interpretation

The Gram stain results are interpreted based on the color of the bacteria:

• Gram-positive bacteria: appear purple due to the retention of crystal violet
• Gram-negative bacteria: appear pink due to the staining with safranin

Clinical Significance

The Gram stain is a useful tool in clinical microbiology for identifying bacteria and guiding antibiotic therapy. It can help distinguish between different types of infections, such as pneumonia or urinary tract infections, and guide the selection of appropriate antibiotics.