Kuper and May
for Acid Alcohol Fast Bacteria
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.
Other fixatives are usually satisfactory.
- 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-auramine-rhodamine, then do either of the following:
- Pour carbol-auramine-rhodamine 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-auramine-rhodamine steams.
- Leave 10 minutes.
- Repeat the heating and leave for a further 10 minutes.
- Put some carbol-auramine-rhodamine in a small Erlenmeyer flask.
- Heat on a hot plate until it almost boils.
- Pour onto each slide, ensuring each section is covered.
- Leave 10 minutes.
- Repeat, and leave for a further 10 minutes.
- Wash the carbol-auramine-rhodamine off with cold water, removing the filter paper.
- Wipe off any dye deposits with an alcohol wetted tissue.
- Decolorise with acid alcohol for 2 minutes.
- Wash well with cold water.
- Place in potassium permanganate for 2 minutes.
- Rinse well with cold water.
- Dehydrate rapidly with absolute ethanol.
- Clear with xylene and mount with a fluorescence free, resinous medium.
- Acid alcohol fast organisms – golden fluorescence
- Background – unstained
- If using method B, do not heat the carbol-auramine-rhodamine in a test tube with a bunsen burner
as it may unexpectedly spurt when it reaches boiling.
- Be careful not to over decolorise.
- For M. leprae use 0.5% aqueous hydrochloric acid.
If so, Drury & Wallington recommend avoiding ethanol dehydration and drying sections in an oven,
then coverslipping directly with a fluorescence free, resinous medium.
Drury, R.A.B. and Wallington, E.A., (1980)
Carleton's histological technique Ed. 5
Oxford University Press, Oxford, UK.
Culling, C.F.A., Alison, R.T. and Barr, W.T. (1985)
Cellular Pathology Technique, 4th ed.
Butterworths, London, UK.
Last updated January 2019