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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Verhoeff Van Gieson Elastin Special Stain – Method and Tips

Elastin is a connective tissue protein which allows the tissues of the body to return to their original shape after distortion or stretching. Elastin fibres can be of varying size and diameter and are particularly well seen histologically in sites such as the lung, heart, blood vessels and the dermis.

 

Histological demonstration of elastin fibres (or lack of them) are important in diagnostic pathology for conditions such as arteriosclerosis, temporal arteritis and elastosis. Fine elastic fibres are not so easily seen on standard haemtoxylin and eosin (H+E) staining therefore special stains which demonstrate elastin clearly are vital.

There are many elastin special stain techniques such as Weigert-Type, Orcein, Aldehyde-Fuchsin and Verhoeff’s. The most common is Verhoeff’s technique of staining elastin due to its quick method and strong elastin colour result. Below is the author’s favoured method for demonstrating elastin which is a version of the Verhoeff’s.

SOLUTIONS

Verhoeff’s solution – (5ml 5% alcoholic haematoxylin) + (2ml 10% aqueous ferric cholride) + (2ml Lugol’s iodine) MAKE IMMEDIATELY PRIOR TO USE.

Note – Lugol’s iodine = 2g potassium iodine dissolved in ~4ml of distilled water, then dissolve 1g iodine, then make up to 100ml.

2 % aqueous ferric chloride

Van Gieson counterstain = (100ml saturated aqueous picric acid) + (1% aqueous acid fuchsin), boil for 3 mins then filter.

METHOD

1. Take sections to water.

2. Stain with Verhoeff’s solution for 15-20 mins.

3. Wash well in tap water.

4. Differentiate in 2% aqueous ferric chloride until only elastin fibres remain darkly stained.

5. Wash in tap water for 5 mins.

6. Counterstain with Van Gieson for 3 mins.

7. Dehydrate, clear and mount.

TIPS

- aim for slight under-differentiation as the Van Gieson stain will continue the differentiation though more slowly.

- dehydrate quickly as alcohol can leach some of the Van Gieson stain from the section. You can accelerate dehydration by blotting the section with filter paper.

Thanks for reading and I welcome any comments.

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