Acid-Fast Stain- Principle, Procedure, Interpretation and Examples 4.3/5 (415)

Acid-Fast Stain- Principle, Procedure, Interpretation and Examples

It is the differential staining techniques which was first developed by Ziehl and later on modified by Neelsen. So this method is also called Ziehl-Neelsen staining techniques. Neelsen in 1883 used Ziehl’s carbol-fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl-Neelsen staining techniques was developed.

The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups.

This method is used for those microorganisms which are not staining by simple or Gram staining method, particularly the member of genus Mycobacterium, are resistant and can only be visualized by acid-fast staining.

Principle of Acid-Fast Stain

When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the Mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through lipoidal wall and enters into cytoplasm. Then after all cell appears red. Then the smear is decolorized with decolorizing agent (3% HCL in 95% alcohol) but the acid fast cells are resistant due to the presence of large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing solution. The non-acid fast organism lack the lipoidal material in their cell wall due to which they are easily decolorized, leaving the cells colorless. Then the smear is stained with counterstain, methylene blue. Only decolorized cells absorb the counter stain and take its color and appears blue while acid-fast cells retain the red color.

Summary of Acid-Fast Stain

Application of


Cell colour

Acid fast

Non-acid fast

Primary dye Carbol fuchsin Red Red
Decolorizer Acid alcohol Red Colorless
Counter stain Methylene blue Red Blue
Procedure of Acid-Fast Stain
  1. Prepare bacterial smear on clean and grease free slide, using sterile technique.
  2. Allow smear to air dry and then heat fix.
    Alcohol-fixation: This is recommended when the smear has not been prepared from sodium hypochlorite (bleach) treated sputum and will not be stained immediately. M. tuberculosis is killed by bleach and during the staining process. Heat-fixation of untreated sputum will not kill M. tuberculosis whereas alcohol-fixation is bactericidal.
  3. Cover the smear with carbol fuchsin stain.
  4. Heat the stain until vapour just begins to rise (i.e. about 60 C). Do not overheat. Allow the heated stain to remain on the slide for 5 minutes.
    Heating the stain: Great care must be taken when heating the carbol fuchsin especially if staining is carried out over a tray or other container in which highly flammable chemicals have collected from previous staining. Only a small flame should be applied under the slides using an ignited swab previously dampened with a few drops of acid alcohol or 70% v/v ethanol or methanol. Do not use a large ethanol soaked swab because this is a fire risk.
  5. Wash off the stain with clean water.
    Note: When the tap water is not clean, wash the smear with filtered water or clean boiled rainwater.
  6. Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i.e. pale pink.
    Caution: Acid alcohol is flammable, therefore use it with care well away from an open flame.
  7. Wash well with clean water.
  8. Cover the smear with malachite green stain for 1–2 minutes, using the longer time when the smear is thin.
  9. Wash off the stain with clean water.
  10. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot dry).
  11. Examine the smear microscopically, using the 100 X oil immersion objective.
Interpretation of Acid-Fast Stain

Interpretation of Acid-Fast Stain

Acid fast: Bright red to intensive purple (B), Red, straight or slightly
curved rods, occurring singly or in small groups, may appear beaded

Non-acid fast: Blue color (A)

Examples of Acid-Fast Stain
Mycobacterium tuberculosis visualization using the Ziehl–Neelsen stain

Mycobacterium tuberculosis visualization using the Ziehl–Neelsen stain.
Source: Wikipedia

Acid-fast: Mycobacterium tuberculosis, Mycobacterium smegmatis.

Non-Mycobacterial bacteria: Nocardia

Coccidian Parasites: Cryptosporidium

Acid-Fast Stain- Principle, Procedure, Interpretation and Examples

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Acid-Fast Stain- Principle, Procedure, Interpretation and Examples

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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 !!!


Add a Comment
  1. What happens if we overheat the slide? it is said that heating should be up to a point that there are fumes but boiling should be avoided .. why is this sir ?


    1. It was indicated. Read principles

    2. Heating simple helps in the enhancement on the penetration of the carbol fuchsin(primary dye) into the cytoplasm. This then makes the organism colour red.. hope this helped

  3. ……….so helpful, i like it

  4. thanks for your interested explanation

  5. I am currently working on an unknown microbe project in my Micro class. I wanted to know what are the confirmation test for Mycobacterium smegmatis after acid-fast stain?

  6. nicely illustrated so impressed by simple and easily fathomable notes

  7. Well nicely quoted
    But are there any precautions to dis experiment ??

  8. Mubarak Saleh Bagwai

    That’s good scientists,we appreciate.Am also a Medical laboratory science student at BUK Kano, Nigeria.Interested in Medical microbiology -Virology

  9. nice explanation Mr. Aryal..!!

  10. Acid fast bacteria absorb carbol fuchsin stain only

  11. i think there has been a mistake
    you said counter stain is methylene blue, then why are you adding malachite green in the procedure.

    1. Both are counter stains

  12. Is Cryptosporidium confused with Endolimax nana ?

  13. Thanks for suggestion…!



  15. Mensah pascal Kingsley

    Am pascal studying medical lab in technical university Kumasi question is both carbol fuchsin and methylene blue are basic dyes, so what will happen if we use methylene blue as our primary stain?

  16. Nice method thanx

  17. hey doin’ gud. & i don’t dis days of any sainin’ tecs.after many years of endavour now am floating, flyin’ fly fly fly up to z sky!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

  18. I’m studying Pharmacy at University of Dhaka, historically known as Oxford of the East. I want to know more about staining of endospore. Please help me.

  19. Thank you….

  20. I need clarification on the acid fast stain. First section says to use methylene blue, but next section says malachite green. What am I missing?? Is it for different specimens? Thanks much. Your website is awesome!

    1. Webster mhindirira

      Methylene blue and malachite green are used as background stains, so you can use either one of them not both. Then obviously the colour of your background will depend on the colour of the stain used. Am Webster, medical laboratory technician very desperate to upgrade myself to a scientist

    2. U can use one of them either malachite green or 0.3%methylene blue.

  21. Am studying Microbiology…… Am very passionate on medical affairs …
    Am well pleased….
    Thanks alot

  22. Greetings, I hold an associate degree in Medical laboratory Technology. I am now Interested in doing my Bachelor. Can anyone help in finding an online program to do do?


  23. i am a Medical Technology student at Our lady of fatima university. it’s a great help ! thank you ! please continue doing this 🙂

  24. thanks for the suggestion. im studing BSC in biochemistry & microbiology at university of venda

  25. wonderful and simplified procedure.thanks!

  26. Thank you!I am so Happy….
    I am Studying Medical Technology at Baqai Medical University.(BIMT),Karachi.Pakistan.

  27. Thank you for this site. Helps a lot when you’re trying to review a little bit

    1. What is the concentration of Melachite green and carbol fuchsin in Zn staining ?

  28. Waoooooo days great

  29. Nice piece
    Continue with this great work.

  30. Hey Mr Sagar Aryal, i am from Ethiopia, since i am a Microbiologist i appriciate your effort and ur are on the right track. please contact me at this e-mail b/c we will do some thing together.Thank u

  31. Bravo Aryal

  32. this is very good work. but am wondering why we dont blot at the end before viewing ,?

  33. hi, am adiploma holder in medical laboratory and interested to spcialize in microbiology. how do i go about it? thanks you.

  34. it was discovered by Paul Ehrlich and modified by both Ziehl and neelsen .
    or discovered by Ziehl…..plz…I m confused

    1. It was discovered by ehrlich but modified by ziehl and Neelsen

  35. it is so interesting part of microbiology and i like that because i am studying in this subject in 1st year asansol girls college and i want to be a microbiologist in my future.i am from west Bengal.

    1. you can not use methylene blue and melachite green at the same time,

  36. gddddddddddd

  37. The above discussion had been very much educative and it reminded me of my days in the university especially during my industrial training at Adamawa Hospital Yola, Nigeria. Microbiology is an intersting field of study that touches every aspect of human life.

  38. Iam a student undertaking a course of laboratory technician in south Sudan.However am looking forward for more information regarding laboratory management with the help of overlooking through many Windows, urs. y.Emmy

  39. Amit kumar Yadav


  40. nice one Aryal

  41. Great work Sagar,just take the corrections from James Adekeye and use the Monica Chessebrough. I used this book during my studies and it helped me so greatly.

    1. Hello, Thanks for the suggestion. Procedure has been corrected from the book of Monica Chessebrough.

  42. SarahClara Iloke

    Great work Sagar. You only need to highlight how you prepare the smear. Try these reference materials Microbiology by Monica Chessebrough volume 1and 2. I believe it will be of great help as a microbiologist.

  43. In Mexico, we used this tecnic for identification of M. tuberculosis, it´s a National heath program. The report of obervation is in cross: +, ++, +++.

    How do you do the report?

  44. Niluka Velathanthiri

    Good pictures!

  45. Well done Mr. Aryal. Your explanation of the principle and procedure for Acid fast staining were basically correct.
    In your procedure however, the preparation of the smear needs not be sterile but aseptically done to prevent unwanted materials/organisms on your slide and assuring that only the specimen you intend to examine is on your slide.
    Similarly in your steps 4 and 6, it is advisable you rinse rather than wash the slide to avoid removing the smear itself. Rinsing the slide will remove the excess unattached stains from the smear and the slide

    Finally non- mycobaterial organisms like Nocardia are better stained with mordified acid fast stain using a less
    strong decolorizer than the acid alcohol e.g acetic acid. Thank you

    1. Hello, Thanks for the suggestion. Procedure has been corrected.

  46. I m very happy. becausa i make a member of pharmcology msq

  47. Borhanuzzaman Bilash

    i am interest your information. so, go on boys.
    i am aslo a microbiologist. i currently studying in microbiology at Noakhali Science & Technology University (NSTU),Chittagong, Bangladesh …

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