B. Ziehl-Neelson staining method
Developed by Paul Neelson Ziehl-Neelson stain
Ehrlichin1882, and modified by Ziehl and
Mycobacteria which are hardly stained by gram staining method. Once the Mycobacteria is stained with primary stain it can not be decolorized with acid, so named as acid-fast bacteria.
Reagents required: . Carbol-fuchsin . Acid-Alcohol . Methylene blue/Malachite green
Procedure for Ziehl-Neelson staining method
Prepare the smear from the primary specimen and fix it by passing through the flame and label clearly
Place fixed slide on a staining rack and cover each slide with concentrated carbol fuchsin solution.
Heat the slide from underneath with sprit lamp until vapor rises (do not boil it) and wait for 3-5 minutes.
Wash off the stain with clean water.
Cover the smear with 3% acid-alcohol solution until all color is removed (two minutes).
Wash off the stain and cover the slide with 1% methylene blue.for one minute.
Wash off the stain with clean water and let it air-dry.