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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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Ziehl–Neelsen Stain For Acid-Fast Organisms – Method and Tips


The Ziehl–Neelsen (ZN) stain is a common standard stain which is readily performed in a majority of histopathology laboratories around the world. It was first described by Dr. Franz Ziehl and Dr Friedrich Neelsen, a German bacteriologist and a German pathologist respectively. The ZN stain is mostly used to identify acid-fast mycobacteria, the most important of which is Mycobacterium Tuberculosis, the organism responsible for tuberculosis (TB). The ZN stain also stains other organisms such as Nocardia.

As Mycobacterium are unable to be visualised on standard haematoxylin and eosin (H+E) and gram stains, the ZN stain was developed. It is based on the tubercle bacilli having a lipid-rich cell wall that takes up phenol-dye solutions (eg. carbol fuchsin, the main dye used in the ZN stain) and after subsequent differentiation, retains the phenol-dye.

Below is the method used by this author.

Solutions

Carbol fuchsin – (1g basic fuchsin in 10ml ethanol) + (5g phenol in 100ml distilled water), then filter.

Methylene Blue – 0.2% methylene blue

Method

1. Take sections to water.

2. Cover section with filtered carbol fuchsin for 20 minutes.

3. Wash well in tap water.

4. Differentiate in 1% acid alcohol until section is a very pale pink.

5. Wash well in tap water.

6. Stain with methylene blue for 1 minute.

7. Dehydrate, clear and mount.

Tips

– Before covering section with carbol fuchsin try covering the section with a little filter paper to reduce precipitate on the slide.

– Some methods still say to the slide to steaming temperature after covering it with carbol fuchsin. This author has found this of no use and is an unnecessary extra step, plus removes the hazard of using a naked flame.

– Before differentiation with acid alcohol wash slide with 70% alcohol for about 1 minute to remove a majority of the stain. This will reduce your differentiation time.

– Blot dry your slide after washing in water after the methylene blue counterstain. This will reduce your dehydration time and therefore result in less leaching of the methylene blue counterstain from the section.

– Some tap water contaminants have been described that stain with carbol fuchsin and are resistant to differentiation. These appear on a different focal plane to true acid-fast organisms within the section.

I welcome any other tips and comments.

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

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