BISC209: The Gram Stain

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Wellesley College-BISC 209 Microbiology -Spring 2010

Gram's Stain

This staining procedure is used to classify bacteria. It is one of the most common and useful tools in identifying bacteria in the microbiology laboratory. Gram-positive and Gram-negative bacteria are both stained purple by the crystal violet (primary) stain. Addition of the iodine leads to the formation of a crystal violet-iodine complex within the cell wall. The decolorizer extracts lipid from the cell wall of Gram-negative bacteria, so the crystal violet-iodine complex diffuses from these cells and loses color. The crystal violet-iodine complex remains within the Gram-positive bacteria because their cell walls lack the lipid-rich outer membrane of Gram-negative bacteria. Due to the increase in porosity of the Gram negative cells after lipid loss from decolorization, safranin (counterstain) is able to permeate the cell wall of Gram-negative bacteria. Purple (primary stain retaining) indicates Gram positive and red (counter stain uptake) indicates Gram negative. Some organisms and dead or dying cells do not take up or lose the stain appropriately and can not be classified as either Gram positive or Gram negative.


1. Prepare a bacterial smear slide (Find protocol at BISC209: Preparing a bacterial smear slide).


To Gram stain the desired organism(s):
Use the staining trays and sink area. Be sure you evenly cover all the smears on the slide throughout this procedure.

1. Place your smear on the staining tray. It is important that the slide be level during staining so use paper towels under the tray to get it leveled. If you do, it is much easier to prevent dye from running off the slide during the staining process and to be sure that your smears are evenly covered with each reagent.

2. Dispense just enough Crystal Violet solution (0.5% crystal violet, 12% ethanol, 0.1% phenol) to completely cover each smear and stain for 1 minute. (Crystal violet is the primary stain.)

3. Rinse the slide by lifting it at a 45 degree angle (using gloves or a clothes pin or slide holder) in a very gentle stream of water that is directed above the top smear until the waste water coming off the bottom is relatively clear; drain off excess water by touching the edge of the slide to a paper towel.

4. Dispense just enough Gram's Iodine (mordant)to completely cover each smear. Let stand for 1 minute, and rinse thoroughly with a gentle stream of water as in Step 1.

5. Lift the slide at a 45 degree angle and drip Decolorizing Reagent (80% isopropyl alcohol, 20% acetone) down the length of the slide making sure it comes in contact with all three smears. This step is tricky as it is easy to over- or under-decolorize. Do this for 10 seconds and IMMEDIATELY rinse, as in step 3, with a gentle stream of water.

6. Place the slide flat on the staining tray and dispense just enough Counterstain Solution (0.6% safranin in 20% ethanol) to cover each smear. Let stand for 2 minutes; rinse with water as in step 3.

7. Blot dry using the bibulous paper package found in your orange drawer. Do not tear out the pages, just insert your slide and pat it dry.

8. Clean up your area; rise your staining tray. Leave it upside down by the sink on paper towels

9. Observe your stained microbes microscopically following carefully the procedure for using the the oil immersion objective on your compound brightfield microscope BISC209: Microscopy described in the protocol page.

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab 12