Gram Staining Procedure

Overview

• Gram staining is a four-step procedure using two different dyes.
• It is one of the first tests to identify unknown bacterial species.

Materials Needed

• Microscope slide
• Wax pencil
• Metal inoculation loop
• Bunsen burner
• Sterile water
• Gram stain set (Crystal Violet, Gram's iodine, decolorizer, safranin)
• Beakers of water

Steps

Preparing the Slide

1. Labeling:
   • Use a wax pencil to label the slide with your initials.
   • Draw a 1-cm diameter circle on the slide.
2. Sterilizing the Loop:
   • Flame the metal inoculation loop using aseptic technique.
3. Adding Water:
   • Add a drop of sterile water to the loop to form a film.
   • Transfer the water to the circle on the slide.
4. Transferring Bacteria:
   • Re-flame and cool the loop.
   • Lightly touch a bacterial colony on an agar plate and transfer it to the slide.
   • Flame the loop again to sterilize.

Preparing the Bacteria

1. Evaporating Liquid:
   • Let the liquid on the slide dry completely (at least 10 minutes).
2. Heat Fixing:
   • Pass the slide through a flame quickly three times.
   • Ensure the flame contacts the slide for about one second each pass.

Staining Process

Crystal Violet Staining

1. Staining:
   • Add Crystal Violet stain to fill the wax circle.
   • Let it stand for one minute.
2. Rinsing:
   • Dip the slide in water until most stain is removed.

Gram's Iodine Staining

1. Staining:
   • Cover the bacteria with Gram's iodine.
   • Let it stand for one minute.
2. Rinsing:
   • Rinse in water until most iodine is removed.

Decolorizing

1. Decolorizing:
   • Apply decolorizer at a 45-degree angle until the flow is clear.
2. Rinsing:
   • Rinse slide in water to remove decolorizer.

Safranin Staining

1. Staining:
   • Cover bacteria with safranin stain.
   • Let it stand for one minute.
2. Final Rinsing:
   • Rinse slide completely in water.

Final Steps

• Allow the slide to dry completely.
• Observe the sample under a microscope.

Safety Note

• Ensure all Bunsen burners are off before using decolorizer due to its high flammability.