Gram Staining: Principle, Procedure, Interpretation, Examples and Animation 4.37/5 (237)

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

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

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

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The Author

Sagar Aryal

I am Sagar Aryal, a passionate Microbiologist and the Scientific Blogger. I did my Master's Degree in Medical Microbiology and currently working as a Lecturer at Department of Microbiology, St. Xavier's College, Kathmandu, Nepal. I am particularly interested in research related to Medical Microbiology and Virology. Find me on Facebook, Twitter or Linkedin !!!

56 Comments

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  1. Poonam kumar sonkar

    very nice & easy

  2. Great.it’s helpful

  3. Very well articulated.

  4. Very well articulated..

  5. Thanks it really helped me

  6. Kailash Singh raturi

    Great helpful thanks

  7. nice article… this help me out for my practical work….thank you☺☺

  8. bobo nyo

  9. This is really good. Am having practicals this morning and you’ve helped a great deal. Thank you

  10. Upcoming microbiologist,a class of 2019 from university of kabianga…am now on a research on POSSIBILITY OF BEDBUGS TO SPREAD HEPATITIS B AND HIV/AIDS. Any support will be highly appreciated from university of kabianga- kericho kenya.

  11. wow,great and understandable.thanks
    University of Nairobi

    1. Very nice and easier to understand..

  12. Mohamed Ibrahim Soojeede

    Nice presentation

  13. 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

  14. Osward Hamanyuma Jnr

    simplified and easy to read,continue with your good work

  15. I have a practical exam tomorrow from Gram’s stain to biochemical tests and identification of bacteria and this website helped me a lot! I only discovered it now! Thank you!

  16. very nice and easy thank you

  17. Rev. Oyetayo Oluwatoyin

    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)

  18. very nice and easier to understand. am also student at Kenya medical training college

  19. 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

  20. This information very nice

  21. Good one

  22. M also done MSc.in Microbiology,and m impressed at research work

  23. siddhartha (DMLT)

    thank you

  24. were kalasa dennis

    very good scientific information that has rejuvenated my efforts towards working out scientific projects, am impressed personally.

  25. Ahimbisibwe joseph

    this is so good, orderly and helpful to us. God bless you!

  26. Very easy to understand. Thankyou very much

  27. Thank you very much for this article it helped me in my assignment… God bless you for the good work

  28. Thank you so much.I easily understood this principle after seeing this.

  29. Amazing…great article & great animated video…easy to grasp the concept..particularly essential for student’s demonstration..awesome job..keep it up & gud luck…

  30. Amazing…great literature & great animated video…easy to grasp the concept..particularly essential for student’s demonstration..awesome job..keep it up & gud luck…

  31. Amazing issues here. I am very glad to look your article.
    Thank you a lot and I’m looking forward to touch you.
    Will you please drop me a mail?

  32. Though Gram Staining is the most important and most crucial step of Microbial Identification, since ages there has been no steps to improve or standardise the method. After completed my Graduation, I was very keen to explore the options for automation in Gram staining and in Microbiology. My search ended with bioMerieux- a 118 year old company started by Dr. Marcel Merieux, a student and Co worker of Dr. Louis Pasteur.
    I am happy to be a part of this Organization today. This is not a promotion but for all the upcoming talented microbiologists of the future, do visit the website http://www.biomerieux.com
    You will be enthrilled to know about the level of Automation in Microbiology in the current working scenario. What paricularly excited me was the automated Gram Staining system wherein no heat fixation required so no chances of alteration of the morphology of a microbial cell or no over and under decolorization. And 30 slides can be stained in a go in 6 sec.

  33. 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?

  34. This is very. Informative and easily understood.
    Thanks

  35. Hi Sagar
    i would like to do the phd in microbiology subject and which topic is best like a virology or some other please guide me thank u

  36. Job well done. Thanks for enlightening us. Its a standard procedure.

  37. amazing and compelling gold standard lab method of identification 🙂

    1. Correct Fixation is a sensitive method !

  38. Sir can u send me soft copy mail to my email address

  39. 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

  40. Easy n nice to have info abt this so can u plzz provide info abt albert staining

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

    1. 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.

  42. Easy to understand thanq

  43. What is other type of counter stain ?

    And what about over decolorization

    1. Neutral red another type of counter stain

  44. Thnx for the information… 🙂

  45. Very easy to understand

  46. simply understand all

  47. simply understand for all

  48. Priyadarshini Vankani

    Thank uhh very much.. I am in S.Y BSc. I’m from Ahmedabad, Gujarat… I really want to know at least 6 gram negative and gram positive bacterial species names and I got them here… Thank u very much

  49. Good technical Information for Vets.

  50. I liked it. Thank you. Plz help me out. Plz send me mail in iamsunildotel@gmail.com

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