Gram Stain Principle – A Brief Explanation

Gram Stain Principle - A Brief Explanation

A staining technique that classifies bacteria based on the chemical and physical properties of their cell walls, the Gram stain is one of the most important tools in medical laboratory microbiology.

Bacteria can be very difficult to classify, so it’s necessary to have as many tools as possible at your disposal. Once you understand how the Gram stain works (Gram stain principle), you’ll never look at bacteria the same way again!

Gram Stain Principle

The Gram stain, named after Danish bacteriologist Hans Christian Gram, is a method of staining used to differentiate bacteria into two large groups. The two categories are gram-positive and gram-negative. The bacterial cell wall is composed of peptidoglycan.

As gram-positive bacteria have a thicker cell wall. It takes the crystal violet permanently. Gram-negative bacteria have lipopolysaccharide material external to the peptidoglycan wall in the acetone wash. It is disrupted and allows the crystal violet to differentiate, so the gram-negative bacteria take up the basic fuchsin stain.

What is the purpose of this procedure?

The Gram stain procedure is a vital part of microbiology. It helps identify specific microbes based on cell walls, specifically how they react to iodine and crystal violet. The purpose of it is to distinguish between two main classes of bacteria, gram-positive and gram-negative bacteria.

The presence or absence of an outer membrane and thin layer of peptidoglycan are what distinguish these types, with gram-positive having both features in some form or another and gram-negative not having them at all.

What equipment do I need?

microbiology laboratory - medical laboratory

The Gram stain principle can be implemented with a few basic laboratory instruments, including a microscope and forceps. The procedure should be performed in an area known as a medical laboratory because while it doesn’t pose any danger to human health, certain compounds used in microbial identification can be hazardous if not handled properly.


  • Glass slide
  • Nichrome wireloop
  • Bunsen beuner
  • Ammonium oxalate/crystal violet
  • Lugol’s Iodine
  • Iodine-acetone solution
  • Diluted carbol fuchsin

How do I perform the procedure?


  1. Prepare the thin film of test organism with distilled water on a clean glass slide.
  2. Air dry the slide & fix by heat
  3. Apply ammonium oxalate/crystal violet stain for 30 seconds
  4. Wash off thoroughly with tap water
  5. Apply Lugol’s iodine for 30 seconds
  6. Wash thoroughly with tap water
  7. Apply fresh iodine-acetone for 30 seconds
  8. wash throughly with water
  9. Counter stain with dilute carbol fuchsin for 30 seconds
  10. Wash thoroughly with water and drain or blot dry
  11. Observe under the microscope, oil immersion (100 power)
Observe under the microscope

What is the result of performing this test? – Gram Stain Principle

Gram-positive bacteria:- Purple

Gram Stain Principle - Gram-positive bacteria

Gram-negative bacteria:- Pink

Gram Stain Principle - Gram-negative bacteria

Additional information

The whole slide must be flooded with each reagent and the previous reagent must be completely removed at each stage.

Water can be used as a substitute for reagents in routine laboratories for washing purposes.

The insufficient reagent may result in an uneven staining /decolouration safranin can be used as a counterstain for specific organisms (Ex: Neisseria gonorrhoeae)

Tips for learning about microbiology – Gram Stain Principle

If you’re a student and you’re interested in microbiology (like I am), then there are some really good ways to learn about it. If your school offers courses on microbiology, then start taking them! If they don’t, or if you can’t take classes that often, try joining an extracurricular club. My university has several of these, and it was through one of them that I discovered my interest in biomedical science.

Acinetobacter gram stain results – watch now


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