MODIFIED ZIEHL- NEELSEN STAINING TECHNIQUES

 

Introduction:

The Modified Ziehl-Neelsen stain is a type of differential bacteriological stain used to identify acid-fast organisms such as Mycobacterium leprae, Actinomycetes, Nocardia and Cryptosporidium parvum. Those Acid fast organisms are capable of retaining the primary stain when treated with an acid. Members of the Actinomycetes, Nocardia sp. (N. brasiliensis and N. asteroides are opportunistic pathogens) are partially branching acid-fast bacteria. Oocysts of coccidian parasites, such as Cryptosporidium and Isospora, are also acid-fast. Hence they can also be detected and identified by Modified Ziehl Neelsen staining procedure.

Modified Ziehl Neelsen staining for Mycobacterium leprae

Purpose:

To demonstration of Mycobacterium leprae in slit skin smear (sss) is a time-tested technique by microscopic examination.  This is acid fast bacilli (AFB) in a skin smear is one of the three cardinal signs of leprosy.

Principle:

Mycobacterium leprae cell wall containing mycolic acid that resist decolourization with 5% sulphuric acid and retain the primary dye (Carbol fuchsin), while the other cells take the counter stain (Methylene blue).

Sample type:

Slit skin smears are prepared from earlobes, nasal mucosa, edges of skin lesion and nerve biopsy material.

Sample collection:

Site is cleaned with sprit and a 5mm long and a 3mm deep incision is made with size 15, Bard-Parker blade, scrape the wound with blade several times so that tissue fluid and pulp collect on the side of the blade.

Procedure:

1.    Slide is labeled with specimen details.

2.    Prepare the smear with collected material on glass slide and it is air dried and heat fixed, made the smear size approximately 8mm.

3.    The smear is treated with strong Carbol -fuchsin  and allowed to react for about 7 mins. 

4.    Excess stain is drained out and slide is washed with water.

5.    The smear is decolorized with 5% sulfuric acid for 2 min or until no more color runs from the slide.

6.    Slide is washed with water and counterstained with methylene blue for 1 minute.

7.     The slide is washed with water and air dried and observed under oil immersion.

Result and Interpretation:

Observe for pink colored bacilli and smears are graded based on number of bacilli in all smears examined and both bacteriological index, morphological index are reported.

Grading of smear

Bacteriological index-totaling number of pluses scored in all the smears and divided by number of smears examined

Morphological index – percentage of solidly stained bacilli out of total number of bacilli counted

Very Numerous	( +6 )	Over 1000 bacilli per oil immersion field.
Numerous	( +5 )	100 to 1000 bacilli per oil immersion field.
Moderate	( +4 )	10 to 100 bacilli per oil immersion field.
Few	( +3 )	1 to 10 bacilli per oil immersion field.
Very few	( +2 )	1 to 10 bacilli per 10 fields.
Rare	( +1 )	1 to 10 bacilli per 100 fields.
None found	( NF )	No AFB seen on entire site.

Modified Ziehl Neelsen staining for parasites and Nocardia

Purpose:

To demonstrate the presence of acid fast parasitic forms in stool specimen and Nocardia from clinical samples by microscopic examination.

Principle:

Cryptosporidium, cyclospora and Isospora species seen in stool samples and Nocardia species resist  decolourization with 1% sulfuric acids and retain the primary dye( carbol fuchsin ) and appears pink color while the others cells take the counter stain. Presence of these organisms most commonly associated with immonocompramised.

Sample type:

Stool, Pus/Exudates, tissue

Procedure:

1.    Slide is labeled with specimen details.

2.    A smear is Prepared on clean grease free slide from sample and it is air dried and heat fixed.

3.    The smear is treated with Carbol fuchsin and allowed to react for about 7 mins.  Excess stain is drained out and slide is washed with water.

4.    The smear is decolorize with 1% sulfuric acid for 2 min or until no more color runs from the slide. Slide is washed with water and counterstained with methylene blue for 1 minute.

5.    The slide is washed with water and air dried and observed under oil immersion.

Result and Interpretation:

Parasites: Observe for pink-colored ocysts in stool samples to report Cryptosporidium, cyclospora Isospora species positive.

Nocardia: Observe for pink-colored branching acid-fast bacilli to report Nocardia species.

 

MODIFIED ZIEHL- NEELSEN STAINING TECHNIQUES 

References:

1.    Practical Medical Microbiology by Mackie & McCartney 14th Edition, Page No-796 – 798.

2.    Koneman’s Color Atlas and textbook of Diagnostic Microbiology, Sixth edition by Washington et al., 2006Page no; 1064-1124.

3.    Bailey and Scott’s Diagnostic microbiology, 12th edition by Betty et al., 2007, Page no: 478-509.