RSS

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

13 Jun

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.

Follow me on twitter (@skinpathology)

Keep an eye out for my website coming soon (www.skinpathonline.com)

Feel free to email me with any questions or comments on feedback@skinpathonline.com

Advertisements
 

Tags: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

3 responses to “Modified Ziehl–Neelsen Stain For Leprosy Bacilli – Method and Tips

  1. Mitchell Wan

    June 25, 2011 at 7:06 am

    Yes, after counterstaining I leave in hotbox before clearing and mounting.

     
    • Mitchell Wan

      June 25, 2011 at 7:09 am

      I also repeat step #2 if there is excess oil on sections. Blotting on a ‘newer’ part of the blotting paper.
      Also I have used warm water washing after step#2 and it aides in the removal of excess oil.

       
  2. viruspapiloma

    April 28, 2012 at 1:44 pm

    Really, this is a great blog, Thanks!

     

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

 
%d bloggers like this: