Microbiology 1- Techniques

Bibulous paper

Lens paper

• 1. Stain smear for 60 seconds using methylene blue
• 2. Rinse off with tap water
• 3. Blot the smear gently with bibulous paper
• 4. Examine with microscope

• Divides bacteria in 2 groups based on the structure of their cell wall
• Gram-positive or gram-negative

If cell is gram-negative, the peptidoglycan is covered by the outer membrane, so it cannot absorb the stain

• Flood the heat-fixed smear with crystal violet for 1 min- all cells turn purple
• Add iodine solution for 1 min- all cells remain purple
• Decolorize with alcohol for 20 sec - gram-positive cells are purple; gram-negative cells are colourless
• Counterstain with safranin for 1-2 mins- gram positive cells are purple, gram-negative cells are pink-red

• Produced by some gram-positive bacteria as a survival strategy
• Highly resistant to adverse conditions such as extreme temperature, drying, radiations, nutrient depletion, chemicals