Ziehl-Neelsen staining

チール・ネールゼン染色法

WordNet

1. the act of spotting or staining something (同)spotting, maculation
2. (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）』「2014/06/01 03:04:00」(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, 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. 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. 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

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 3% hydrochloric acid in isopropyl alcohol
7. Allow to sit 1 minute, rinse with tap water
8. Flood slide with Methylene Blue
9. 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.

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 retains 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.

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.

Online protocol examples

• Ziehl–Neelsen protocol (PDF format).

References

External links

• Media related to Ziehl-Neelsen stain at Wikimedia Commons

UpToDate Contents

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• 1. HIV陰性患者における結核の診断 diagnosis of pulmonary tuberculosis in hiv negative patients
• 2. 非結核性抗酸菌の微生物学 microbiology of nontuberculous mycobacteria
• 3. 結核の微生物学および病因 microbiology and pathogenesis of tuberculosis
• 4. HIV感染患者における結核の疫学、臨床症状、および診断 epidemiology clinical manifestations and diagnosis of tuberculosis in hiv infected patients
• 5. 肺結核の臨床症状および評価 clinical manifestations and evaluation of pulmonary tuberculosis

English Journal

• Definite (microbiologically confirmed) tuberculous meningitis: predictors and prognostic impact.

• Jha SK1, Garg RK2, Jain A3, Malhotra HS1, Verma R1, Sharma PK1.
• Infection.Infection.2015 Dec;43(6):639-45. doi: 10.1007/s15010-015-0756-z. Epub 2015 Feb 28.
• BACKGROUND: Microbiological confirmation cannot be obtained in approximately two-third patients with tuberculous meningitis. In this study, we sought to identify epidemiological, clinical, cerebrospinal fluid, and imaging parameters that could indicate the possibility of microbiological confirmation
• PMID 25724799

• Evaluation of variants of carbol fuchsin solution to stain acid-fast bacilli in-situ by the pot method.

• Das S1, Narang P1, Nagamiah S2, Mishra PS1, Deotale V1, Mendiratta DK3.
• The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.Int J Tuberc Lung Dis.2015 Dec;19(12):1470-5. doi: 10.5588/ijtld.15.0272.
• BACKGROUND: Ziehl-Neelsen (ZN) staining requires heating, and pre-stained smears contain viable bacilli.OBJECTIVE: To evaluate four variants of carbol fuchsin solution by the pot method and compare the results with ZN staining, taking culture as gold standard.METHOD: Five hundred sputum samples from
• PMID 26614188

• Double staining of bacilli and antigen Ag85B improves the accuracy of the pathological diagnosis of pulmonary tuberculosis.

• Che N1, Qu Y1, Zhang C1, Zhang L1, Zhang H1.
• Journal of clinical pathology.J Clin Pathol.2015 Nov 27. pii: jclinpath-2015-203244. doi: 10.1136/jclinpath-2015-203244. [Epub ahead of print]
• BACKGROUND: A pathological examination plays an important role in the confirmation of a diagnosis of tuberculosis, especially for smear- and culture-negative cases. However, conventional Ziehl-Neelsen staining and histological tests lack sensitivity and specificity.OBJECTIVE: To evaluate the diagnos
• PMID 26614787

Japanese Journal

• 脳梗塞後症候性てんかん患者の誤嚥性肺炎の治療中に発症した滲出性結核の剖検例

• 松尾 知平,石川 宏明,田地 広明,吉田 和史,寺本 信嗣
• 日本老年医学会雑誌 51(5), 460-465, 2014
• 症例は，90歳，男性．主訴は，歩行困難．近医に脳梗塞，糖尿病で通院中．201x年x月，歩行困難を認め，頭部単純CT施行するも陳旧性病変を認めるも新たな病変を認めず，脳波検査にて，てんかん発作と診断された．その後もてんかん発作を繰り返すため精査加療目的で当院紹介受診．入院時検査にて，胸部X線で両側肺野に浸潤影を認めた．発熱およびCRP上昇を認め，超高齢者で脳梗塞後であることから誤嚥性肺炎と考えSBT …
• NAID 130004713490

• 尋常性狼瘡の1例

• 加藤 麻衣子,三宅 宗晴,大磯 直毅,川田 暁,露口 一成
• 皮膚の科学 13(3), 172-175, 2014
• … 病理組織の Ziehl-Neelsen 染色で抗酸菌を認めなかった。 …
• NAID 130004688252

• 腹腔鏡下肝部分切除術で診断した非結核性抗酸菌症による肝膿瘍の1例

• 松浦 正徒,波多野 悦朗,石井 隆道,藤本 康弘,水本 雅己,上本 伸二
• 日本臨床外科学会雑誌 75(1), 179-183, 2014
• … 菌同定，感受性確認目的で腹腔鏡下肝部分切除術を施行した．肝表面に白色瘢痕を数箇所認め，3箇所を肝部分切除した．<BR>病理組織学的検査で乾酪壊死を伴った類上皮細胞肉芽腫を認め，Ziehl-Neelsen染色で抗酸菌を検出した．クォンティフェロンTB検査が陰性であり他の培養検査で菌種の同定ができず，非定型抗酸菌症と診断し4剤併用療法で治療した．炎症所見は軽快し，術後4カ月で同種末梢 …
• NAID 130004679184

Related Links

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Acid-fast stain (Ziehl-Neelsen method) is a useful differential staining procedure that specifically stains all members of the genera mycobacteria. The procedure utilizes heat and phenol (carbolic acid) to help the penetration of the dye ...

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Ziehl Neelsen Staining -Principle, Procedure and Interpretations in Microbiology Principle This procedure is used to stain mycobacterium tuberculosis and mycobacterium leprae. These bacteria are also called acid fast bacilli. They ...

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Ziehl-Neelsen staining

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Ziehl-Neelsen染色