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 ofReagentCell color
Acid fastNon-acid fast
Primary dyeCarbol fuchsinRedRed
DecolorizerAcid alcoholRedColorless
Counter stainMethylene blueRedBlue

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 fiammable chemicals have collected from previous staining. Only a small fiame 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 fiammable, therefore use it with care well away from an open fiame.
  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

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73 thoughts on “Acid-Fast Stain- Principle, Procedure, Interpretation and Examples”

    • The peptidoglycan membrane in the cell wall, which is responsible for retaining the crystal violet during decolorizarion step is thin. This causes the crystal violet to be washed away. As a result the cell is stained with the counter stain safranin this taking up its color (pink-red)

    • This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water. … Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost.

    • Because Gram negative bacteria is made up of Thin Peptidoglycan layer.So the bacteria
      doesn’t absorbs the stain. So Gram negative bacteria doesn’t retains the crystal violet.
      I think I will give to you answer in convincing way.

      • Gram negative bacteria contains lipopolysaccharide and phospholipid with a thin peptidoglycan wall. This CELL WALL undergoes spheroplast( damage of CELL walls as a result of toxics drugs or antibiotics.
        This makes it resist decolorizer and takes the color of counterstain( safranin or neutral red)

        Note THIS , Gram negative bacteria is toxic to human and it is refers to as endotoxins

    • Because they have thinner layer of peptidoglycan in their cell wall.
      And dew to that, when decolorized by acetone the crystal Violet is washed off leaving the cell colourless and they pick the color of the secondary stain safranin being pick or red.

    • 2. Thin layered peptidoglycan Gram-negative cell as it exposed to ethanol, it loses its outer membrane making it the thin layer of peptidoglycan exposed. Because of the few layers of peptidoglycan, it would result to a leakage of the cell membrane and the large crystal violet-iodine complex is washed off from the cell.

    • Because gram negetive bacteria has thinner layer of peptidoglsycan it is decolirised with decolorizer and stained with courter stain , they both stained with primary stain but negetive on is decolorized in second steps, this is the al about

  1. Hello! our professor has used the same picture for our quiz. The shape of the acid-fast stain is a rod? and what would the morphology be?

  2. Please how is the biochemical composition of acid fat bacteria is used in the identification and diagnosis of a named AFB

  3. If we skipped the heat during carbol fuchsin staining step, what is the end result? Will acid-fast bacteria be clear? And will non-acid fast bacteria be colored blue with methylene blue?

    • The heating process allows the primary stain, cabolfuchsin to penetrate further especially when the lipoidol membrane is large which otherwise could not permit penetration. Omitting the step could lead to false results since the cabolfuchsin might be de-colorized leading to false negative results. Both acid fast and non-acid fast cells could take the counter stain and interfere with the end results.

      • No ,the primary stain penetrates the lipid layer when heated, without heat the primary stain can’t be taken into the cell and therefore washed on decolonization, still if the suspected microorganism is acid fast the counter stain will not be absorbed into the cell hence washed and the cell remains colorless

    • There will be no differentiate bw afb and non-afb, bz, there is no enough penetration of carbol fuschin to the cytoplasm that’s why decolorizer flushes off the fuschin, and there will be counterstaining of methylene blue to both type of bacteria if that is AFB or non-AFB.

  4. Nice explanation of the principle. You just boost my understanding of this topic. But what are the acid-alcohol fast bacilli?

    • These are organisms that resist decolourization by 3%Acid alcohol stain after hearing the strong carbolfuchsin solution during staining.
      These have athuck cell wall .
      Have more mycolic acids and wax

  5. Studying Medical Lab Technician. Lutheran College of Health Yala C R S.
    You did well in your explaination, very helpful.
    the causative agent of T.B Mycobacterium, since its staining meth is for differenciation, and so, is it a gram pos or neg? pls attend to my qn thanks.

  6. Please note that Zn procedure it was early introduced by Paul elich and later it was modified by two German scientists ziehl and Nelsen that’s why the procedure clinch as Zn staining but not developed by ziehl and modified by Nelsen as mentioned by writer of this page

  7. Hello! James Adekeye thanks for your explanation.but we have two containers for AFB sample i.e 1 and 2. which one need to contains more acid fast bacilli?. I am a medical lab technitian in ECWA college of health kagoro, kaduna state of Nigeria

  8. 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 ?

  9. 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

  10. 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?

    • Please what is the function of counter stain in Ziehl Neelsen staining, because I read the function differs from that of gram staining

  11. 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?

  12. 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!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

  13. 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!

    • 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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


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