How to Gram Stain

Materials:

• Bunsen Burner

• Inoculation Loops

• Microscope Slides

• Microscope

• Crystal Violet (primary stain)

• Gram’s Iodine Solution

• Ethanol (decolorizer)

• Safranin (counterstain)

• Tray For Slides and Gram Stain Waste

• Water

  
  

How To Gram Stain:

1. Heat fix sample on slide

   1. IF SAMPLE IS FROM A PLATE

      1. Place a drop of water on a clean slide (use ethanol to clean the slide).

      2. Using a sterile inoculation loop, grab a small sample of bacteria and smear into the drop of water on the slide to spread out the sample.

   2. IF SAMPLE IS FROM A BROTH:

      1. Vortex the sample to make sure none of the material has settled in the tube.

      2. Using a sterile inoculation loop, grab a small drop of broth and smear on to a clean slide (use ethanol to clean the slide).

   3. Let the sample completely air dry.

   4. Once it has air dried, pass it carefully over the Bunsen burner a few times to fix the sample on the slide and prepare for gram staining.

2. Gram staining

   1. Flood the slide with Crystal Violet and let sit for 1 minute. Then rinse the slide with water.

   2. Cover the slide with Gram’s Iodine to fix the stain and let sit for 1 minute. Then rinse the slide with water.

   3. Tilt the slide at an angle and use Ethanol to decolorize the slide.

      1. THIS IS VERY IMPORTANT: the time for decolorization differs every time. Start with a few drops of ethanol at a time.ii. Keep adding ethanol for about 10 seconds OR until the purple dye stops running.

   4. Flush with water after decolorization.

   5. Flood the slide with Safranin and let sit for 1 minute. Then rise the slide with water.

   6. Blot the slide dry with bibulous paper, avoiding any rubbing.

   7. Observe slide under the microscope.