What is Gram Staining?

This Gram staining lab is a method developed by the Danish Scientist Hans Christian Gram in 1884 to distinguish two types of bacteria based on the structure of their cell wall. Gram-positive bacteria stain purple because of its thick peptidoglycan layer in the cell wall. Gram-negative bacteria will stain pink due to having a very thin peptidoglycan layer. The gram staining characteristic of the bacteria will not only tell us about differences in the cell wall but also differences in the behavior of the two types of bacteria.

Function of Each Gram Staining Chemical

  1. Crystal Violet: Penetrate and stain cell wall and membrane
  2. Iodine: Binds with crystal violet and traps it in the cell to give a purple color
  3. Alcohol: A decolorizer to remove purple stain from cell wall. Gram positive bacteria will retain purple stain due to thick wall while gram negative will lose purple stain due to thin wall
  4. Safranin: A pink counter-stain. This will have no effect on the crystal violet in the gram positive bacteria. However since gram negative bacteria is decolorized, it will stain pink with the safranin


Fixing The Bacteria on the Slide

  1. Collect four clean microscope slides
  2. To the far left of each slide, add your initial using a wax pencil
  3. On that same side of the slide, use the wax pencil to draw a circle in the center of each slide
  4. Flip the slides over to opposite unmarked side
  5. Using a sterile loop, add a drop of water on the center of the circle
  6. Using the same sterile loop, touch a colony of the bacteria on the slide marked “before”, and transfer it to the slide by mixing the tip of the loop in the water
  7. Leave the slide on your bench to air dry
  8. Using a wooden clothespin to hold the slide, pass it gently over a Bunsen burner flame to fix the bacteria on the slide. The slide is now ready for staining.
  9. Choosing another bacteria colony on the same half of the plate (side marked “before”), repeat the above procedure to fix another bacteria colony
  10. Repeat the above procedure to fix two more colonies of bacteria, but this time, from the side of the plate marked “after”

Gram Staining the Bacteria

Note: Make sure that in this step you are staining the side that has the bacteria and not the opposite side with the wax mark. 

  1. Cover the bacteria with crystal violet and hold for 60 seconds
  2. Rinse with distilled water
  3. Cover the bacteria with iodine for 60 seconds
  4. Rinse with distilled water
  5. Decolorize the bacteria by washing with alcohol and promptly rinse with distilled water to avoid excessive decolorization
  6. Add safranin to counter-stain and hold for 60 seconds
  7. Rinse with distilled water
  8. Gently dab slide with Kim wipes to dry
  9. Follow the oil immersion lab procedure to observe the bacteria on all four slides
  10. Make sure to take a picture of each stain

Lab Assessment

  1. Call your instructor to look at your slide to verify that you have it properly focused (5 points)
  2. Take a picture of the image using your cell phone camera (2 points)
  3. Repeat the above steps for at least one more specimen (7 points)
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Courtney Simons
Courtney Simons
Author, Dr. Courtney Simons has a Bachelor of Science in Food Science and a Ph. D. in Cereal Science from North Dakota State University.