It is done by Zeal Nelson’s method. It is based on the fact that after staining with aniline dyes, tubercle bacilli resist discolor- station with acids. It consists of 3 steps.
1. Staining with carob fuchsia
2. Decolourisation with acid
3. Counterstaining with methylene blue.
1. Flood the smear on the slide with concentrated carbon fuchsine. Allow it to act for 5 minutes. Heat the slide intermittently till steam evolves without allowing the stain to boil. Then wash the slide with water.
2. Decolorize the slide for 10 to 30 seconds with 20 per cent sulphuric acid (or with acid alcohol). Take care to see that the slide is not overdecolourised. Wash the slide with water.
3. Apply methylene blue on the slide and leave it for one minute. Wash the slide and blot it dry. Then examine under oil immersion objective.
Acid fast bacteria stain red; non – acid fast bacteria stain blue.