Lab 2:

Gram Stain

Information gathered by:
Elizabeth Rigg


The gram stain is one of several laboratory procedures which can be used to narrow down the identities of unknown bacteria.  Although the gram stain was originally developed in 1844 by the Danish physician Hans Christian Gram for the purpose of discerning the difference between two types of pneumonia, it is still used to identify bacteria today.

The gram stain procedure separates all bacteria into one of two groups - into gram-negative bacteria which do not stain purple and into gram-positive cells which do stain purple.  In structural terms, the ability of a cell to become stained during the gram stain procedure is due to the chemical makeup of the cell wall.  

Gram positive and gram negative

The gram staining procedure consists of fixing a colony of bacteria onto a slide and then flooding the colony with various chemicals.  First, crystal violet dye is dropped onto the bacterial cells, staining gram-positive cells purple.  Iodine is added to fix the violet dye into place and then ethanol is used to wash the dye off the unstained cells.  Finally, a red dye called safranin is used to stain any gram-negative cells present so that they will be visible.  Once the gram stain procedure is complete, the gram-positive bacteria appear purple under a microscope while gram-negative cells appear pink or red. 


Laboratory Procedure

1. First, isolate a colony of the bacteria to be studied. 

When you perform the gram stain as an operator, you will already have a colony of bacteria which you wish to study.  For this lab, you can create a colony of bacteria by taking a sample of water or wastewater and placing a few drops of the sample water into a petri dish.  Incubate the petri dish until a colony of bacteria develops.  A good sample culture will be about 18 to 24 hours old when the gram stain test is performed. 

2. Prepare the slide by rinsing it with cleansing solution and blotting it dry with bibulous paper. 

3. Transfer a small amount of the bacteria colony onto the slide. 

First, add a drop of water to the slide.  Then use the inoculation loop to transfer a very small amount of bacteria into the water droplet.  Still using the inoculation loop, spread the solution of bacteria and water out into a thin smear on the surface of the slide.  The finished smear should be a circle about the size of a dime.  Finally, allow the smear to dry thoroughly before proceeding to the next step. 

Make sure that you have all of the reagents on hand before moving on to the next step since the rest of the procedure must be done quickly.  The procedure can be messy, so you should wear a lab coat and work near the sink.

4. Cover the smear completely with crystal violet dye.  Let the dye stand on the slide for 20 seconds (or up to 60 seconds if the smear is thick.)

5. Rinse the smear with water for approximately 5 seconds, being careful to remove only the dye and not the smear.   You can either use a plastic water bottle or a slow stream of water from the faucet for this step.  After washing, the smear should appear to be blue-violet in color. 

6. Cover the smear completely with iodine.  Let the iodine stand on the slide for 20 seconds (or up to 60 seconds if the smear is thick.) 

7. Rinse the smear with water for approximately 5 seconds.  The smear should still appear blue-violet. 

8. Add drops of ethanol to the slide so that they run over the smear.  This step should be performed quickly, but you must be careful not to add too much ethanol or it will leach the color from gram-positive cells and make them appear to be gram-negative.  You should stop dropping ethanol onto the slide when the ethanol running off the smear first becomes colorless, which should occur within about 20 seconds. 

9. Rinse the smear with water for approximately 5 seconds.  The smear should now be a paler violet color or clear (depending on whether the bacteria are gram positive or gram negative.)

10. Cover the smear completely with safranin.  Allow the dye to stand on the slide for 60 seconds. 

12. Rinse the smear with water for approximately 5 seconds.  The smear may appear violet or pink. 

13.  Let the smear dry at room temperature or blot the smear with bibulous paper.

14.  Observe the slide under the microscope and determine whether the bacteria are gram-positive (stained purple) or gram-negative (stained red.)

15.  Attempt to identify your bacterial species.  The flow chart below can be used to identify some species found in wastewater. 

Dichotomous Key