How to Gram Stain

Left image is an example of a gram-negative bacteria under the microscope. Right image is an example of a gram-positive bacteria under the microscope.

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

Procedure

Tips

Clean slide with ethanol. Heat fix sample on slide. IF SAMPLE IS FROM A PLATE: Place a drop of water on a clean slide. 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. IF SAMPLE IS FROM A PLATE: Vortex the sample to make sure none of the material has settled in the tube. Using a sterile inoculation loop, grab a small drop of broth and smear onto a clean slide. Let sample completely air dry. Once it has air dried, pass if carefully over the Bunsen burner a few time to heat fix the sample on the slide and prepare for Gram staining. Gram staining: Flood the slide with crystal violet and let sit for 1 minute. Then rinse the slide with water. Cover the slide with Gram's iodine to fix the stain and let sit for 1 minute. Then rinse the slide with water. Tilt the slide at an angle and use Ethanol to decolorize the slide. THIS IS VERY IMPORTANT: the time for decolorization differs every time. Start with a few drops of ethanol as a time. Keep adding ethanol for about 10 seconds OR until the purple dye stops running. Flush with water after decolorization. Flood the slide with safranin and let sit for 1 minute. Then rinse the slide with water. Blot the slide dry with bibulous paper, avoiding any rubbing. Observe the slide under the microscope.

To sterilize an inoculation loop, place it over the bunsen burner for until it turns red hot and let cool for 30 seconds - 1 minute. This is important because you don't want to kill any of the bacteria you are dealing with. It is important to allow slide samples to air dry, so make sure to use a small drop of water. DO NOT dry slides over bunsen burner. Tilt the slide to a 45 degree angle when rinsing.

Gram Staining Procedure