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 Mycobacterium tuberculosis

visualization using the Ziehl–Neelsen

stain.

Ziehl–Neelsen stainFrom Wikipedia, the free encyclopedia

The Ziehl–Nee lsen stain, also known as the acid-fast stain, was first described by two German doctors: the

 bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–1898). It is a special bacteriological

stain used to identify acid-fast organisms, mainly Mycobacteria. Mycobacterium tuberculosis is the most important of 

this group because it is responsible for tuberculosis (TB). Other important Mycobacterium species involved in humandisease are  Mycobacterium kansasii, Mycobacterium marinum, and members of the Mycobacterium avium

complex. Acid fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called

mycolic acids. These acids resist staining by ordinary methods such as a Gram stain.[1] It can also be used to stain a few

other bacteria, such as Nocardia. The reagents used are Ziehl–Neelsen carbolfuchsin, acid alcohol, and methylene blue.

Acid-fast bacilli will be bright red after staining.

A variation on this staining method is used in mycology to differentially stain acid-fast incrustations in the cuticular hyphae

of certain species of fungi in the genus Russula.[2][3] It is also useful in the identification of some protozoa, namely

Cryptosporidium and Isospor a. The Ziehl–Neelsen stain can also hinder diagnosis in the case of paragonimiasis because

the eggs in an ovum and parasite sputum sam ple (OnP) can be dissolved by the stain, and is often used in this clinical setting because signs and symptoms of  paragonimiasis closely resemble those of TB.

Contents

1 Procedure

2 Modifications

3 See also

4 References4.1 Online protocol examples

5 References

6 External links

Procedure

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1. Drop suspension onto slide

2. Air dry slide 10 minutes at 60 °C, heat-fix slide 10 minutes at 90 °C

3. Flood slide with Carbol Fuchsin

4. Hold a flame beneath the slide until steam appears but do not allow it to boil

5. Allow hot slide to sit for 3 to 5 minutes, rinse with tap water 

6. Flood slide with 30% hydrochloric acid in isopropyl alcohol

7. Allow to sit 1 minute, rinse with tap water 

8. Flood slide with Methylene Blue9. Allow to sit 1 minute, rinse with tap water 

10. Blot dry

11. View under oil immersion lens

Studies have shown that an AFB stain without a culture has a poor negative predictive value. An AFB Culture should be performed along with an AFB stain; this

has a much higher negative predictive value.

Modifications

5% sulfuric acid is used for destaining Mycobacterium leprae instead of the 20% used for  Mycobacterium tuberculosis.

Kinyoun modification (or cold Ziehl–Neelsen technique) is also available.

A protocol in which a detergent is substituted for the highly toxic phenol in the fuchsin staining solution. [4]

See also

Kinyoun stain

Lowenstein-Jensen medium

References

"Microbiology with Diseases by Body System", Robert W. Bauman, 2009, Pearson Education, Inc.

Morello, Josephine A., Paul A. Granato, Marion E. Wilson, and Verna Morton. Laboratory Manual and Workbook in Microbiology: Applications to Patient

Car. 10th ed. Boston: McGraw-Hill Higher Education, 2006. Print.

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Online protocol examples

Ziehl–Neelsen (http://www-medlib.med.utah.edu/WebPath/HISTHTML/MANUALS/AFB.PDF) protocol (PDF format).

References

1. ^ Morello, Josephine A., Paul A. Granato, Marion E. Wilson, and Verna Morton. Laboratory Manual and Workbook in Microbiology: Applications to Patient Care.

10th ed. Boston: McGraw-Hill Higher Education, 2006. Print.

2. ^ Romagnesi, H. (1967).  Les Russules d'Europe et d'Afrique du Nord . Bordas. ISBN 0-934454-87-6.

3. ^ Largent, D; D Johnson, R Watling. (1977).  How to identify fungi to genus III: microscopic features. Mad River Press. ISBN 0-916422-09-7. p 25.

4. ^ Ellis, RC; LA Zabrowarny. (1993). "Safer staining method for acid fast bacilli" (http://www .ncbi.nlm.nih.gov/pmc/articles/PMC501296/pdf/jclinpath00207-

0071.pdf) . Journal of Clinical Pathology 46: 559–560. doi:10.1136/jcp.46.6.559 (http://dx.doi.org/10.1136%2Fjcp.46.6.559) . PMC 501296

(//www.ncbi.nlm.nih.gov/pmc/articles/PMC501296) . PMID 7687254 (//www.ncbi.nlm.nih.gov/pubmed/7687254) .

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC501296/pdf/jclinpath00207-0071.pdf.

External links

Media related to Ziehl-Neelsen stain at Wikimedia Commons

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Categories: Acid fast bacilli Microbiology techniques Staining Bacteriology Microscopy

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