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
1. Include a control
Method
1. Place the working solution in a coplin jar and pre-heat in 58 -60oC waterbath 10 mins
2. Deparaffinise sections, bring to water.
3. Stain in the pre-heated working solution in the water bath 15 mins
4. Place the COPLIN JAR containing the slides into running cold tap water 2 mins
5. Remove the slides from the coplin jar and wash in running water 1 min
6. Differentiate in 3% hydrochloric acid in 95% ethyl alcohol until no more colour runs
from the slide.
7. Wash briefly in water to remove the acid alcohol.
8. Counterstain with 0.25% methylene blue in 1% acetic acid 15 to 30 secs
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. 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