Laboratory Protocols
Histological Staining Techniques
Ziehl, Neelsen, 1882, 1883.
Do not celloidinise slides. Use a positive control section. Gelatinised
slides may be used.
- Dewax sections, rinse in alcohol, rinse in water.
- Filter carbol fuchsin onto slides on a rack over a sink. Note: If the carbol fuchsin solution has produced a lot of precipitate it should be discarded and a fresh solution prepared.
- Hold a flaming, alcohol-soaked, cotton wool ball on a rod beneath the slides until the solution steams. Do this 3 times and leave for 3.5 minutes after each flaming.
- Wash well in tap water.
- Differentiate in acid alcohol (1% HCl in 70% alcohol) in a coplin jar - 10 minutes.
- Wash in tap water then rinse in distilled water.
- Differentiate again with 25% sulphuric acid - 3 minutes.
- Wash in tap water.
- Stain with Mayer's haematoxylin - 30 seconds.
- Wash and blue in tap water - 5 minutes.
- Rinse in cellosolve (2-ethoxyethanol).
- Stain with saturated tartrazine in cellosolve - 30 seconds.
- Rinse in alcohol.
- Clear and mount.
Results:
Acid fast bacilli - deep magenta red.
Nuclei - dark blue.
Background - yellow.
Red blood cells - pale pink.
Note: The more usual counterstain is 0.25% methylene blue in 1% acetic
acid, for 1 minute, applied after the differentiation and washing steps. This
gives a pale blue background:
Acid fast bacilli - deep magenta red.
Nuclei - blue
Background - pale blue to unstained.
Red blood cells - pale pink.
Carbol fuchsin
Dissolve 1g of *basic fuchsin in 10mls of 100% alcohol and add 100mls
of 5% aqueous phenol. Mix well for several hours and filter before use.
*Synonyms basic fuchsin / basic red 9 / pararosaniline = C.I. 42500