Putt
for Acid Fast Bacteria
Materials
- Putt’s Carbol new fuchsin
- Methylene blue, 0.5% in absolute ethanol
- Glacial acetic acid, 5% in absolute ethanol
- Lithium carbonate, saturated 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 in carbol-new fuchsin overnight.
- Place in 20% formalin for 5 minutes.
- Decolorise in 2% acid alcohol for 5 minutes.
- Rinse with water.
- Place in 1% potassium permanganate for 2-5 minutes.
- Bleach with 2% oxalic acid for 1 minute.
- Wash with water.
- Counterstain with Harris’ hemalum for 2 minutes.
- Wash with water.
- Stain with Fite’s picro-fuchsin until adequately stained.
- Dehydrate with 95% and absolute ethanols.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Acid fast organisms – red
- Background – blue
Notes
- Discard the lithium carbonate solution when it turns blue.
- This method is stated to be suitable for M. leprae.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Putt, F.A., (1972)
Manual of histopathological staining methods
John Wiley & Sons, New York, NY, USA
