WordNet

(microscopy) a dye or other coloring material that is used in microscopy to make structures visible
color for microscopic study; "The laboratory worker dyed the specimen"
color with a liquid dye or tint; "Stain this table a beautiful walnut color"; "people knew how to stain glass a beautiful blue in the middle ages"
produce or leave stains; "Red wine stains the table cloth"
a soiled or discolored appearance; "the wine left a dark stain" (同)discoloration, discolouration
marked or dyed or discolored with foreign matter; "a badly stained tablecloth"; "tear-stained cheeks"
having a coating of stain or varnish (同)varnished
the act of spotting or staining something (同)spotting, maculation
(histology) the use of a dye to color specimens for microscopic study

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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2015/05/09 19:42:05」(JST)

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Mycobacterium tuberculosis visualization using the Ziehl–Neelsen stain.

The Ziehl–Neelsen 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 human disease are Mycobacterium leprae, Mycobacterium kansasii, Mycobacterium marinum,Mycobacterium bovis,Mycobacterium africanum 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 carbol fuchsin, 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 Isospora. The Ziehl–Neelsen stain can also hinder diagnosis in the case of paragonimiasis because the eggs in an ovum and parasite sputum sample (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.

Procedure

A typical AFB stain procedure involves dropping the cells in suspension onto a slide, then air drying the liquid and heat fixing the cells. The slide is flooded with Carbol Fuchsin, which is then heated to dry and rinsed off in tap water. The slide is then flooded with a mild solution of hydrochloric acid in isopropyl alcohol to destain the Carbol Fuchsin, thus removing the stain from cells that are unprotected by a waxy lipid layer. Thereafter, the cells are stained in methylene blue and viewed on a microscope under oil immersion.

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.

Mechanism explanation

Initially, Carbol Fuchsin stains every cell
When they are destained with acid-alcohol, only non-acid-fast bacteria get destained since they don't have a thick, waxy lipid layer like acid-fast bacteria.
When counter stain is applied, non-acid-fast bacteria pick it up and become blue when viewed under the microscope. Acid-fast bacteria retain Carbol Fuchsin so they appear red.

Modifications

5% sulfuric acid is used for destaining Mycobacterium leprae instead of the 20% used for Mycobacterium tuberculosis.
1% sulfuric acid for actinomycetes, nocardia.
0.5-1% sulfuric acid for oocysts of isospora, cyclospora.
0.25–0.5% sulfuric acid for bacterial endospores.
Brucella differential stain – glacial acetic acid used, no heat applied, secondary stain is loeffler's methylene blue.
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]

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.

Online protocol examples

Ziehl–Neelsen protocol (PDF format).

References

^ 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.
^ Romagnesi, H. (1967). Les Russules d'Europe et d'Afrique du Nord. Bordas. ISBN 0-934454-87-6.
^ 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.
^ Ellis, RC; LA Zabrowarny. (1993). "Safer staining method for acid fast bacilli" (PDF). Journal of Clinical Pathology 46: 559–560. doi:10.1136/jcp.46.6.559. PMC 501296. PMID 7687254.

External links

Media related to Ziehl-Neelsen stain at Wikimedia Commons

UpToDate Contents

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English Journal

Identity and public health potential of Cryptosporidium spp. in water buffalo calves in Egypt.

Amer S, Zidan S, Feng Y, Adamu H, Li N, Xiao L.SourceDivision of Foodborne, Waterborne and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA, USA; Department of Zoology, Faculty of Science, Kafr El sheikh University, Kafe El Sheikh, Egypt.
Veterinary parasitology.Vet Parasitol.2013 Jan 16;191(1-2):123-7. doi: 10.1016/j.vetpar.2012.08.015. Epub 2012 Aug 24.
Little is known about the diversity and public health significance of Cryptosporidium species in water buffaloes. In this study, we examined the distribution of Cryptosporidium spp. in water buffalo calves in Egypt. Rectal fecal specimens from 179 calves and 359 adults were screened microscopically
PMID 22963712

Japanese Journal

Hansens Disease: An Imitator of Cutaneous Sarcoidosis

Chinen Katsuya,Hirano Kazuhiko,Fujioka Yasunori
Internal Medicine 50(19), 2257-2258, 2011
NAID 130001087921

病理検体（ホルマリン固定パラフィン包埋組織切片）を用いた感染症遺伝子診断：結核菌Mycobacterium tuberculosis

塩沢英輔,塩沢朋子,矢持淑子,瀧本雅文,太田秀一,丸山梨詠
昭和医学会雑誌 69(5), 426-431, 2009
… 要約：病理検査におけるZiehl-Neelsen抗酸菌染色法は陽性率が低く，結核菌に特異的ではない．結核腫疑い症例のホルマリン固定パラフィン包埋組織切片からDNAを抽出しnested PCR法で結核菌特異的反復挿入配列（IS6110）を同定した．Ziehl-Neelsen染色の抗酸菌同定率が14%（2/14例）であったのに対し，PCR法では57%（8/14例）の症例で結核菌DNAが検出された．結核菌核酸増 …
NAID 130000852995

「染色法」

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種類	染色法	染色結果
塩基性色素	ヘマトキシリン	青
酸性色素	エオジン	赤

色素			染色部位
電荷	種類	例	種類	電荷	例
正	塩基性色素	ヘマトキシリン、アズール	好塩基性	負	核酸、リボソーム(rRNA)、硝子軟骨
負	酸性色素	エオジン	好酸性	正	膠原線維