Prepared by

ROY ELLIS

IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011

Principle

Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These are ß-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 elusion (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.

Technical Points: Include a control

Method

1. Place the working solution in a coplin jar and pre-heat in 58 -60 ºC water bath for 10 minutes.
2. Deparaffinize sections, bring to water.
3. Stain in the pre-heated working solution in the water bath for 15 minutes.
4. Place the COPLIN JAR containing the slides into running cold tap water for 2 minutes.
5. Remove the slides from the coplin jar and wash in running water for 1 minute.
6. Differentiate in acid alcohol until no more color runs from the slide.
7. Wash briefly in water to remove the acid alcohol
8. Counterstain with methylene blue for 15 to 30 seconds.
9. Wash in water, dehydrate, clear and mount in DPX.

Results

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

Reagent Formulae

1. Staining solution:

Stock Solution A (stable for 6 months)

                        L.O.C. High Suds (Amway) ............ 0.6 ml

                        Distilled water .......................... 100 ml

Stock Solution B

                        Basic fuchsin ............................ 1 g

                        Absolute ethyl alcohol .................. 10 ml

            The two solutions can be kept as stock solution and mixed before use.

2. Working Solution (stable for 1 month)

                    Mix 50ml of A with 5 ml of B.

3.  acid alcohol - 3% hydrochloric acid in 95% ethyl alcohol

                        Absolute ethyl alcohol .............. 95ml

                        Distilled water ......................  2 ml

                        Concentrated hydrochloric acid ... 3 ml

            Make up the alcohol solution then add the concentrated acid. Use extreme care when

            handling concentrated acid.

4.  0.25% methylene blue in 1% acetic acid

Methylene blue ........................  0.25 g

Distilled water ......................... 99 ml

Acetic acid ............................. 1 ml

References

1. Lillie, R.D. (1977). H.J. Conn’s biological stains, 9th edition. William’s and Wilkins.

2. Lyon, H (1991), theorey and strategy in histochemistry. Springer - Verlag.

3. Neelsen, P. (1883). Zentralblatt fur de Medizinischen Wissenschafen, V21, pg497.

4. Ziehl F. (1882) Zur Farbung des Tuberkelbacillum. Deutsche Medizinische Wochenschrift, V8, pg 451.

5. Ellis, R.C., Zabrowarny L.A. (1993) J. Clinical Pathology. V46, pg 559-560