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:
Option A
1. Pour carbol-auramine-rhodamine onto each section until the slide is full.
2. Grip a cotton ball in long forceps, and dip into absolute ethanol.
3. Cover all inflammable fluids, then ignite the cotton ball.
4. Move the flame under the sections as evenly as possible.
5. Heat until the carbol-auramine-rhodamine steams.
6. Leave 10 minutes.
7. Repeat the heating and leave for a further 10 minutes.
Option B
1. Put some carbol-auramine-rhodamine in a small Erlenmeyer flask.
2. Heat on a hot plate until it almost boils.
3. Pour onto each slide, ensuring each section is covered.
4. Leave 10 minutes.
5. 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.

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.

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

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.