Special Stains - Ziehl Nielson

For some specialist bacteria, the gram stain is not effective as it can lead to a false negative, for example Mycobacterium tuberculosis. In this case the Ziehl Neelson stain is performed. Here the carbol fuchsin is forced through the resistant lipid capsule of the mycobacteria using heat. The stain is then removed from other tissue structures by differentiation in acid alcohol, while the mycobacteria retain the stain. The lipid withstands differentiation with acid alcohol as they are acid and alcohol fast. Most other organisms lose the dye and take up the counterstain.

The main method for carrying out this technique is listed below

1. Take sections to distilled water.

2. Filter and heat carbol fuchsin solution in boiling flask in heating mantle until steaming

3. Place carbol fuchsin solution onto slides. Leave for 10 minutes

4. Wash well in distilled water

5. Differentiate using 1% acid alcohol until no further colour can be removed.

6. Wash in distilled water

7. Counterstain in 1% methylene blue dilution 1 in 10 with distilled water for 10 seconds

8. Wash in water

9. Dehydrate, clear and mount.

Result

Acid Alcohol Fast Bacilli	Red
Nuclei	Blue

Photo x10 Ziehl Nielson