Bacteria and Gram Staining

Gram Staining Process

• Definition: Gram staining is a laboratory method used to differentiate bacterial species into two groups based on their cell wall structure, correlating with antibiotic sensitivity.

Steps to Perform Gram Staining:

1. Preparation of Smear:

   • Place bacteria on a glass slide.
   • Allow it to air dry completely.
   • Heat fix the bacteria to the slide.

2. Primary Stain - Crystal Violet:

   • Apply crystal violet stain.
   • Leave for 1 minute.
   • Rinse with water.

3. Iodine Treatment:

   • Add iodine as a mordant.
   • Rinse with water for 10-30 seconds.

4. Decolorization:

   • Use alcohol or acetone for decolorization.

5. Counterstain - Safranin:

   • Add safranin counterstain.
   • Leave for 1 minute.

6. Final Observations:

   • Rinse the slide again.
   • Place under a microscope using an oil immersion lens to observe.

Color Results:

• Purple: Gram-positive bacteria retain the crystal violet stain - indicative of a thick peptidoglycan layer in the cell wall.
• Pink/Red: Gram-negative bacteria appear due to the safranin counterstain, characterized by a thinner peptidoglycan layer.

Bacterial Shapes

• Bacillus: Rod-like or cylindrical (single or chains).
• Coccus: Spherical or round (single, pairs, chains, or clusters).
• Spirillum: Helical/spiral (single); assessed by the number of twists, thickness, flexibility, and motility.
• Filamentous: Long, thread-like structures forming chains or filaments.

Bacteria vs. Archaebacteria

Bacteria:

• Cell Wall: Composed of peptidoglycan.
• RNA Polymerase: Simple.
• Gene Structure: Similar to eukaryotes regarding protein coding and organization.
• Ribosomes: 70S; differ in protein composition.
• Metabolism:
  • Heterotrophic: Energy from organic molecules.
  • Autotrophic: Energy from photosynthesis or oxidation of inorganic molecules.

Archaebacteria:

• Cell Wall: Lacks peptidoglycan.
• RNA Polymerase: Complex (similar to eukaryotes).
• Gene Structure: More similar to eukaryotes; contains introns.
• Ribosomes: 70S; more similar in structure and function to eukaryotes.

Positive vs. Negative Gram Bacteria

Gram Positive:

• Color: Appears purple.
• Stain Binding: Binds to the crystal violet stain.
• Peptidoglycan Layer: Thick layer.

Gram Negative:

• Color: Appears pink or red.
• Stain Binding: Does not bind to the primary stain.
• Peptidoglycan Layer: Thin layer.

Bacterial Pathogens Overview