Modified Ziehl–Neelsen Stain For Leprosy Bacilli – Method and Tips

Leprosy bacilli in comparison with tubercle bacilli are much less acid- and alcohol-fast. The leprosy bacilli’s lipid envelope is also much more affected by the fat solvents traditionally used to dewax sections (i.e. Xylene). Due to these factors a modification on the standard Ziehl-Neelsen technique is used for the demonstration of leprosy bacilli. 

 

Below is the author’s preferred technique. 

SOLUTIONS  

Dewaxing solution – equal parts of liquid paraffin and rectified turpentine 

Carbol Fuchsin – as per standard Ziehl-Neelsen technique

 

Methylene blue counterstain – as per standard Ziehl-Neelsen technique

 

10% sulphuric acid 

METHOD 

1. Dewax in ‘dewaxing solution’ described above for 30 minutes. 

2. Blot dry and wash in running water for approximately 10 minutes. 

3. Stain with filtered Carbol Fuchsin for 30 minutes at room temperature. 

4. Wash well in tap water. 

5. Differentiate in 10% sulphuric acid until section is pale pink. 

6. Wash well in tap water.

7. Counterstain with Methylene Blue for 15 seconds. 

8. Wash well in tap water. 

9. Blot dry, clear and mount. 

TIPS 

- This author always puts sections on ‘sticky’ slides to prevent any floating off. 

- There are many variations on the ‘softer dewaxing solution’ for the modified Ziehl-Neelsen technique for leprosy bacilli including:

- Two parts xylene to one part vegetable oil / clove oil / groundnut oil / olive oil / cottonseed oil. 

- Residual oil on the section after washing prevents shrinkage of the section. 

- Place slides directly from heater into dewaxing solution as this helps quicken the dewaxing.

- Some methods use a weaker acid-alcohol solution for differentiation, but this author prefers 10% sulphuric acid as it is quicker. 

- Don’t be alarmed when the section is placed into the sulphuric acid as it will turn a black colour. It will return to a pink colour when placed back in water.

- Ensure the counterstain colour isn’t too intense as this can mask some leprosy bacilli and even turn them a purple colour. 

- This is author lets the sections dry after washing after counterstaining and then directly mounts them.

I welcome any other tips and comments.

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