Acid Fast Bacilli Ziehl Neelsen staining technique Principle, Procedure and reporting

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Acid-fastness  refers to  a physical property of certain bacterial or group of bacteria and eukaryotic cells, as well as some sub-cellular structures found in a organism, which makes it resistant to decolorization by the acid alcohol during laboratory staining procedures

Acid-fast organisms are characterized by wax-like, nearly impermeable cell walls; they also contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids and  are highly resistant to disinfectants and dry conditions.

Once these structures or organisms are stained as part of a sample e.g sputum,urine,sperm,stools, it can resist the acid and/or ethanol-based decolorization solution hence the name acid-fast.

Also read : Acid Fast organisms not Always Mycobacterium species

The Genus mycobacteria are widespread acid fast microorganisms, typically living in and food sources. Tuberculosis and the leprosy organisms are obligate parasites and are not found as free-living members of the genus. The mycobacteria are aerobic and nonmotile bacteria (except for the species Mycobacterium marinum, which has been seen to be motile within macrophages) that are characteristically acid fast.

These Mycobacteria have outer membrane. With no capsules, and most  of them do not form endospores.

Ziehl neelsen stain is one of those differential stain used in identifying acid fast bacteria such as Mycobacterium tuberculosis and leprae.It makes use of a primary stain (Carbol fuchsin) and methylene blue as counterstain.Acid fast bacteria picks up the primary stain (Pink OR Red) while non acid fast bacteria picks up the blue counterstain.

Principle of Ziehl Neelsen stain

  • Mycobacteria are difficult to stain due to their acid fastness characteristic such as high lipid and wax content in their cell walls.
  • When these bacteria are stained with the primary stain (carbol fuchsin) under gentle heat, acid-fast bacilli retain the color of the primary stain even after treatment with strong decolourizing solutions (Acid alcohol). That is pick up the red color even after counterstaining with loeffler’s methylene blue
  • Whereas the microorganisms susceptible to acid take on the blue colour.
Acid fast bacteria (Red) Non acid fast (Blue)
Acid fast bacteria (Red) Non acid fast (Blue)

Material Required for Ziehl Neelsen stain

  • Wash bottle
  • Distilled water
  • Acid-Fast Decolorizer (Concentrated Hydrochloric acid and 95% Ethyl alcohol)
  • Inoculating loop
  • Spirit lamp / Bunsen burner
  • Carbol Fuchsin
  • Methylene blue
  • Glass slide
  • Suspected bacteria culture or sputum sample
  • Personal Protective Equipment (PPE) such as a pair of glove,lab jacket,safety goggle,face mask etc

Procedure for Ziehl Neelsen stain

Make sure all Personal Protective Equipment are worn appropriate to avoid contamination or health hazard

  • Prepare a smear on a clear, dry glass slide from the sample using the Inoculating loop
  • Allow  the smear  to air dry and fix with gentle heat
  • Flood the entire dried smear with Carbol Fuchsin stain
  • Heat   to steaming for 5 minutes with a low flame.
  • Take note not to boil the stain and do not allow the smear to dry while heating
  • Allow it to stand for 5 minutes without further heating.
  • Wash the slide in running tap water
  • Now Decolourize  the washed slide with Acid Fast Decolourizer (Acid alcohol) for 2 minutes or until no more stain comes off in the washings.
  • Note that,If washing is not thorough, you may get false positive results
  • Wash with tap water
  • Counterstain for 30 seconds with Methylene Blue
  • Wash with tap water, dry in air, then examine under oil immersion objective

Result and Reporting of Ziehl Neelsen after staining

The staining characteristics of organisms observed under microscope using oil immersion lens

Also read  Cryptosporidium parvum laboratory diagnosis


  • Bright red – Acid fast organism
  • Blue – Non Acid Fast organisms and cellular material

Reporting  Acid Fast bacilli Result

  • If acid fast bacilli are present, report the smear as Acid Fast Bacilli Positive (AFB positive)

Also indicate the number of AFB present to determine the severity of the infection

More than 10 AFB/field at least in 20 fields+  +
1-10 AFB/field at least in 50 fields+   +
10-99 AFB/ 100 fields      +
1-9 AFB/100 fieldsreport the exact number
  • If no AFB are seen after examining 300 fields, report the smear as ‘No AFB seen’.
  • If  few AFB are seen i.e. when only 1 or 2 AFB are seen after examining 100 fields, request further specimen to examine.It may be that those AFB might have come from tap water (saprophytic mycobacteria), it  may be a scratch from the glass slide or due to the use of the same piece of blotting paper while drying resulting to cross contamination.

Also Read : List of Acid Fast Organisms


  • Why do they Heat carbol fuchsin slide during  Ziehl Neelsen staining?
  • List specimen  where acid fast organisms can be recovered


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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.


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