Lecture on Gram Staining

Introduction

• Presenter: Praveen Kambalkar from TNAO
• Topic: Gram Staining, an essential technique in microbiology
• Purpose: Identify unknown bacteria as gram positive or gram negative
• Developed by: Hans Christian Gram

Overview

• Differential staining method
• Differentiation based on cell wall content
  • Gram positive: More peptidoglycan
  • Gram negative: Less peptidoglycan

Materials Required

1. Cerelyze inoculating loop
2. Clean glass slide
3. Petri plate with bacterial culture
4. Crystal violet (primary stain)
5. Gram's iodine (mordant)
6. 95% ethanol (decolorizer)
7. Saffronine (counter stain)

Procedure

Slide Preparation

• Wash glass slide with 75% ethanol to remove contaminants.
• Mark slide with initials for identification.
• Place a drop of water on the slide.

Bacterial Smear

• Sterilize inoculating loop by flaming until red hot.
• Use loop to pick bacterial colonies and mix with water drop to form a smear.
• Air dry the slide.
• Heat fix by passing slide over flame for 2-3 seconds.

Staining Steps

1. Primary Staining with Crystal Violet

   • Apply crystal violet to smear; wait 30 seconds.
   • Rinse with sterile water to remove excess dye.

2. Mordant Application with Gram's Iodine

   • Apply Gram's iodine; leave for 1 minute.
   • Rinse with sterile water.

3. Decolorization with 95% Ethanol

   • Apply ethanol at 45-degree angle until flow is clear.
   • Rinse with sterile water.

4. Counter Staining with Saffronine

   • Apply saffronine; leave for 1 minute.
   • Rinse with sterile water.

Observations

• Under microscope at 100x Oil Emulsion Objective Lens:
  • Gram positive bacteria appear purple
  • Gram negative bacteria appear red/pink