Histotechnology Technical methods

Stain for Acid Fast Bacilli

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(Ellis and Zabrowarny)

Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These are [fl]-hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species (Lyon H 1991).

Basic fuchsin binds to negatively charged groups in bacteria. The mycolic acid (and other cell wall lipids) present a barrier to dye entry as well as elution (washing out with solvent) and this is partly overcome by adding a lipophilic agent to a concentrated aqueous solution of basic fuchsin and partly by heating.

This method does not use phenol and has been provided by Roy Ellis, Principal Hospital Scientist, Histopathology, The Queen Elizabeth Hospital, Woodville, South Australia.


Tissue fixed in 10% neutral buffered formalin. 3 to 5 micron paraffin sections

known positive material


1 Staining solutions

Solution A

  • Basic fuchsin 1 g
  • Absolute ethyl alcohol 10 ml

Solution B
  • L.O.C. High Suds (Amway) 0.6 ml
  • Distilled water 100 ml

Note: Other pure, liquid organic cleaners work just as well as the Amway product. The two solutions can be kept as stock solution and mixed immediately before use.

2. 3% hydrochloric acid in 95% ethyl alcohol

  • Absolute ethyl alcohol 95ml
  • Distilled wate 2 ml
  • Concentrated hydrochloric acid 3 ml
Make up the alcohol solution then add the concentrated acid.

3. 0.25% methylene blue in 1% acetic acid

  • Methylene blue 0.25 g
  • Distilled water 99 ml
  • Acetic acid 1 ml

  1. Place the staining solution in a coplin jar and pre-heat to 60oC for 10 mins
  2. Deparaffinise sections, bring to water and stain in the pre-heated solution for 15 mins
  3. Place the COPLIN JAR containing the slides into running cold tap water for 2 mins before removing the slides from the coplin jar.
  4. Remove the slides from the coplin jar and wash in running water for 1 min
  5. Differentiate in 3% hydrochloric acid in 95% ethyl alcohol until no more colour runs from the slide
  6. Wash briefly in water to remove the acid alcohol
  7. Counterstain with 0.25% methylene blue in 1% acetic acid for 15 to 30 secs
  8. Wash in water. Dehydrate, clear . Mount sections in DPX

AFBDEC picture

  • Acid fast bacilli Red
  • Nuclei Blue
  • Other tissue constituents Blue


  • Take care when handling basic fuchsin as a powder, it is a potential carcinogen. Acetic acid is a strong irritant and a corrosive. Use a fume hood and wear protective clothing.

  • BASIC FUCHSIN: is a triaminotriphenylmethane dye. It is an inflammable powder and should not be handled close to heat or a naked flame. It is a potential carcinogen and an eye, skin and mucous membrane irritant.

  • ABSOLUTE ETHANOL: is a flammable liquid and should never be handled close to heat or a naked flame. The vapour is heavier than air and can travel for a considerable distance along the ground to a source of ignition and flash back. Ethanol is regarded as one of the safest industrial solvents. Although it possesses narcotic properties, vapour concentrations sufficient to produce narcosis are rarely, if ever reached in a medical laboratory. Ethanol is rapidly oxidised, in the body, to carbon dioxide and water and does not produce permanent damage to the central nervous system.

  • ACETIC ACID: is a corrosive substance and will cause severe damage to exposed skin. Ingestion will cause dental erosion, bronchitis and respiratory difficulties. It is an eye and mucous membrane irritant.

  • HYDROCHLORIC ACID: is a strong irritant and corrosive and must be handled with great care. It will cause severe burns to exposed skin and eyes. The vapour is also a strong irritant and corrosive. Always use a fume hood when handling this acid.

  • ALWAYS add acid to water and NEVER water to acid. Heat is generated by the reaction which can be very violent.

  • L.O.C.: A splash to the eye will cause irritation otherwise the solution is not harmful.

  • METHYLENE BLUE: is a quinone-imine dye of the thiazin sub group. It is highly toxic and an eye irritant. It is also a combustible powder and should never be handled close to heat or a naked flame.

  • MOUNTING MEDIUM: usually xylene or toluene based and the effects are as for xylene.

  • XYLENE: is a moderately flammable liquid which is a mild eye and mucous membrane irritant. It is a primary skin irritant and a central nervous system depressant. It will defat skin and may cause dermatitis.Death has been caused from over exposure due to respiratory failure.


  • Ajax Chemicals (1991). Material Safety Data Sheets. Ajax Chemicals Pty Ltd, Auburn, New South Wales.
  • Lillie, R.D. (1977). H.J.Conn's Biological Stains, 9th edition. Williams and Wilkins.
  • Lyon, H (1991). Theory and Strategy in Histochemistry. Springer-Verlag.
  • Neelsen, P. (1883). Zentralblatt f[cedilla]r de Medizinischen Wissenschafen, V21, p497.
  • Ziehl, F. (1882). Zur Farbung des Tuberkelbacillum. Deutsche Medizinische Wochenschrift, V8, p451.

NOTE: No responsibility is assumed by The University of Nottingham or the Queens Medical Centre NHS Trust for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. It is the users responsibilty to ensure that all procedures are carried out according to appropriate Health and Safety requirements.

© Copyright 1997 University of Nottingham Medical School Division of Histopathology. This page was last built on Mon, May 5, 1997 with Frontier. Thanks for looking in. Comments to James.Lowe@nottingham.ac.uk