Gram Staining: Principle, Procedure, Interpretation, Examples and Animation

Gram Staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884. This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations of microorganisms.

Principle of Gram Staining

When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in colour.

In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. Then when again stained with safranin, they take the stain and appears red in color.

Reagents Used in Gram Staining
  • Crystal Violet, the primary stain
  • Iodine, the mordant
  • A decolorizer made of acetone and alcohol (95%) 
  • Safranin, the counterstain

Procedure of Gram Staining

  1. Take a clean, grease free slide.
  2. Prepare the smear of suspension on the clean slide with a loopful of sample.
  3. Air dry and heat fix
  4. Crystal Violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.
  5. Flood the gram’s iodine for 1 minute and wash with water.
  6. Then ,wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.
  7. Add safranin  for about 1 minute and wash with water.
  8. Air dry, Blot dry and Observe under Microscope.
Interpretation

Gram Positive: Blue/Purple Color
Gram Negative: Red Color

Gram Staining Interpretation

Examples

Gram Positive Bacteria: Actinomyces, Bacillus, Clostridium, Corynebacterium, Enterococcus, Gardnerella, Lactobacillus, Listeria, Mycoplasma, Nocardia, Staphylococcus, Streptococcus, Streptomyces ,etc.
Gram Negative Bacteria: Escherichia coli (E. coli), Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas,Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella etc

Animation

Download Animation from Below:
Gram Staining Animation

15 thoughts on “Gram Staining: Principle, Procedure, Interpretation, Examples and Animation”

    • The role of iodine is that it act as grams mordant and increases the interaction between bacterial cell wall and the dye (crystal violet) so that the dye is more tightly bound on the cell and is more stained.

  1. Hi
    Very much appreciated
    Would be great if u also add some points about the modifications and variations and also gram variable bacteria and some general info about why gram staining is needed and where it is not indicated !!
    Because in medicine what accompanies practicals are viva and these questions can be very important
    Cheers
    Gud luck

  2. Greatly impressed by this article. Painstakingly put together in a simple language. I studied microbiology at Polytechnic(HND) and presently a 300 level student of Lead City University,Ibadan, Nigeria studying Biology Education.This piece has really helped in solving my Assignment on Biological Techniques(BIO 315)

  3. All enterobcteriaceae are gram negative bacteria but not all gram negative are enterobcteriaceae to distinguish the enterobcteriaceae you have to do oxidase test because enterobcteriaceae are oxidase negative bacteria

  4. Great!
    But if i may ask, which step in Gram staining tech can be omited without affecting the final result?
    Mr Len artifacts results from poor methods of rinsing of slides.
    heat fixing of smear blackened and disolve the lipoid cpnt of the cell as well as it schring of cell cpnt.
    Beside, what is the color of safranin?

  5. Try fixing with 100% Methanol for one minute and letting the slide dry, rather than heat fixing..preserves cellular integrity and reduces artifact caused by heat fixation. Good for for CSF’s, pus and epithelial cells. Also, use acetone alcohol – 50% acetone and 50% of 95% ETOH to decolorize. More of a controlled decolorization.

    Len Fligg, ASCP cm

  6. If Gram positive organisms have such complex cell wall that could defy decolorisation,why then is a mordant used?

    • the function of the mordant is to fix (attach) the crystal violet to the cell wall (peptidoglycan cell wall)so as not to be washed away easily thereby forming a complex as stated earlier. Moreover, the test procedure is performed to differentiate between the two groups of bacteria.

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