Microbiology: THE Gram Staining Technique

This article is geared towards acquainting the reader with the usefulness of the Gram staining technique, the underlying principles, as well as the procedure for carrying out Gram stain.

The Gram Staining Technique

Materials Required:

·         Crystal violet dye

·         Lugol’s iodine

·         Acetone or 95% ethanol

·         Safranin or neutral red (0.1%w/v)

Note: Acetone or 95% ethanol can be used alone. A mixture of both is however recommended because it decolorizes more rapidly and is less likely to over-decolorize smears.


1.      Create a smear by emulsifying a colony of bacteria in a drop of distilled water on a glass slide, using a flamed and cooled wire loop (the wire loop must be sterile).

2.      Leave the slide in a safe place for the smear to air-dry, protected from dust, flies, ants, and direct sunlight. Use gentle heat to dry quickly if urgent staining is required.

3.      Rapidly pass the slide, smear uppermost, 3 times through the flame of a Bunsen burner to fix, and then allow to cool. This is called heat fixation.

4.       Cover the fixed smear with crystal violet dye for 30-60 seconds

5.      Rapidly wash off the stain with clean water, tip off the water, and cover the smear with Lugol’s iodine for 30-60 seconds.

6.      Wash off the iodine with clean water and rapidly decolorize with acetone or ethanol (or a mixture of both) for a few seconds. Wash immediately with clean water.

7.      Cover the smear with safranin or neutral red stain for 2 minutes, and wash off the stain with clean water.

8.      Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry.

9.      Examine the smear under a microscope, first using the 40X objective to check the staining and to see the distribution of material, and then with the oil immersion objective to report the bacteria and cells.


Gram-positive bacteria – Dark purple

Gram-negative bacteria – Pale to dark red or pink

Reporting Gram Reaction

The report should include the following:

1.      Number of bacteria present, whether many, moderate, few or scanty.

2.      The color observed i.e. the Gram reaction of the organism, whether positive or negative.

3.      The morphology of the bacteria, whether cocci (spherical), bacilli (rod-shaped), diplococci (2 spherical cells joined together), streptococci (a chain of spherical cells), or cocco-bacilli (a combination of cocci and bacilli). Also state whether the organisms are intacellular.

Examples of Gram-negative rod-shaped bacteria are: Escherichia coli, Pseudomonas aeruginosa, and Salmonella spp. Bacillus subtilis is a Gram-positive rod-shaped bacteria. Staphylococcus spp. and Streptococcus spp. are Gram-positive spherical bacteria.

Gram negative cocci in pus from eye. (Photo credit: Nathan Reading)

microscopic image of a Gram stain of mixed Gram-positive cocci (Staphylococcus aureus ATCC 25923, purple) and Gram-negative bacilli (Escherichia coli ATCC 11775, red). Magnification:1,000. (Photo credit: Wikipedia)

microscopic image of Pseudomonas aeruginosa (ATCC 27853). Gram staining, magnification:1,000 (Photo credit: Wikipedia)

Note: Gram-positive organisms may lose their ability to retain crystal violet and Gram-negative due to:

1.      Cell wall damage as a result of excessive heat fixation of the smear.

2.      Over-decolorization of the smear.

3.      Use of an iodine solution which is too old (i.e. yellow instead of brown in color)

4.      Preparation of slide from an old culture.

Gram- negative bacteria may not be fully decolorized if the smear is too thick, and so they may appear as Gram-positive. To eliminate any possible sources of errors, a control experiment has to be set up, using a smear containing known Gram-positive and Gram-negative organisms.


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One Response to “Microbiology: THE Gram Staining Technique”
  1. Emmett Says...

    On April 26, 2012 at 2:28 pm

    This article was really helpful. I enjoyed reading it and love the simplicity. I am a student of microbiology and reading your article has helped to broaden my understanding of the \’Gram stain\’ concept. Thanks a lot!

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