Ziehl-Neelsen
for Acid Alcohol Fast Bacteria
Materials
- Ziehl’s Carbol fuchsin
- 1% Acid alcohol
- Methylene blue, 0.1% aqueous
Tissue Sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are usually satisfactory.
Protocol
- Bring sections to water via xylene and ethanol.
- Place sections on a staining rack and gently cover each section with filter paper soaked in carbol-fuchsin, then do either of the following:
Option A
- Pour carbol-fuchsin onto each section until the slide is full.
- Grip a cotton ball in long forceps, and dip into absolute ethanol.
- Cover all inflammable fluids, then ignite the cotton ball.
- Move the flame under the sections as evenly as possible.
- Heat until the carbol-fuchsin steams.
- Leave for five minutes.
- Repeat the heating and leave for a further five minutes.
Option B
- Put some carbol fuchsin in a small Erlenmeyer flask.
- Heat on a hot plate until it almost boils.
- Pour onto each slide, ensuring each section is covered.
- Leave for five minutes.
- Repeat, and leave for a further five minutes.
- Wash the carbol-fuchsin off with cold water, removing the filter paper.
- Wipe the back of each slide with an ethanol soaked cotton ball or tissue to remove any carbol-fuchsin deposits.
- Wipe around each section with a folded tissue dipped in ethanol to remove any carbol-fuchsin deposits.
- Pour acid alcohol onto each section to decolorise. Dye will flood away.
- When the initial flood of dye stops, pour fresh acid alcohol onto each section. Leave for 10-20 minutes. The background should be pale pink after this time.
- Wash well with cold water.
- Place in methylene blue solution for 10-15 seconds.
- Wash with cold water until pale blue.
- Dehydrate with ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Acid alcohol fast organisms – red
- Background – blue
Notes
- If using method A with an open flame, do rinse the sink well before lighting the alcohol soaked cotton ball so as to wash away any inflammable materials in the sink.
- If using method B, do not heat the carbol-fuchsin in a test tube with a bunsen burner as it may unexpectedly spurt when it reaches boiling.
- Many of the other carbol-fuchsin variants can be successfully used in this method.
- Some technologists increase the hydrochloric acid content to 3%. This decolorises more quickly.
- Some technologists do the initial decolorising with ethanol alone and, when the dye ceases to flood away, replace it with acid alcohol.
- Some technologists counterstain for 30 seconds with a regressive hemalum and blue as a replacement for methylene blue.
- Some technologists stain briefly with tartrazine, saturated in cellosolve, for a short time to obtain a yellow background, as this is more restful to the eyes when scanning many sections.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co. - Culling, C.F.A., Alison, R.T. and Barr, W.T. (1985)
Cellular Pathology Technique, 4th ed.
Butterworths, London, UK. - Drury, R.A.B. and Wallington, E.A., (1980)
Carleton’s histological technique Ed. 5
Oxford University Press, Oxford, UK.
