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    Acid-Fast Stain (Ziehl-Neelsen Stain)

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    ASHUTOSH KHADANGA
    The Ziehl-Neelsen stain, also called the acid-fast stain, was developed in the late 1800s to identify acid-fast bacteria such as Mycobacterium tuberculosis. The stain uses carbol fuchsin as the primary stain, 20% sulfuric acid as the decolorizer, and methylene blue as the counterstain. Mycobacterium tuberculosis will appear as long, red, beaded acid-fast bacilli against a blue background after staining, allowing it to be identified. Several modifications of the Ziehl-Neelsen stain exist to better identify different acid-fast structures depending on their acid-fast properties.

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    Acid-Fast Stain (Ziehl-Neelsen Stain)

    Uploaded by

    ASHUTOSH KHADANGA
    305 views15 pages
    The Ziehl-Neelsen stain, also called the acid-fast stain, was developed in the late 1800s to identify acid-fast bacteria such as Mycobacterium tuberculosis. The stain uses carbol fuchsin as the primary stain, 20% sulfuric acid as the decolorizer, and methylene blue as the counterstain. Mycobacterium tuberculosis will appear as long, red, beaded acid-fast bacilli against a blue background after staining, allowing it to be identified. Several modifications of the Ziehl-Neelsen stain exist to better identify different acid-fast structures depending on their acid-fast properties.

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    The Ziehl-Neelsen stain, also called the acid-fast stain, was developed in the late 1800s to identify acid-fast bacteria such as Mycobacterium tuberculosis. The stain uses carbol fuchsin as the primary stain, 20% sulfuric acid as the decolorizer, and methylene blue as the counterstain. Mycobacterium tuberculosis will appear as long, red, beaded acid-fast bacilli against a blue background after staining, allowing it to be identified. Several modifications of the Ziehl-Neelsen stain exist to better identify different acid-fast structures depending on their acid-fast properties.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    305 views15 pages

    Acid-Fast Stain (Ziehl-Neelsen Stain)

    Uploaded by

    ASHUTOSH KHADANGA
    The Ziehl-Neelsen stain, also called the acid-fast stain, was developed in the late 1800s to identify acid-fast bacteria such as Mycobacterium tuberculosis. The stain uses carbol fuchsin as the primary stain, 20% sulfuric acid as the decolorizer, and methylene blue as the counterstain. Mycobacterium tuberculosis will appear as long, red, beaded acid-fast bacilli against a blue background after staining, allowing it to be identified. Several modifications of the Ziehl-Neelsen stain exist to better identify different acid-fast structures depending on their acid-fast properties.

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    Acid-fast Stain

    (Ziehl-Neelsen stain )
    INTRODUCTION
     The Ziehl–Neelsen stain, (also called acid-fast
    stain) was first described by two German
    doctors;
    • Franz Ziehl (1859 to 1926), a bacteriologist
    • Friedrich Neelsen (1854 to 1898), a pathologist.
     A differential stain.
     Original method by Ehrlich, later modified &
    improved by Ziehl & Neelsen.
    Mycobacterial cell wall
    REAGENTS
    The reagents used are

    A. Carbol fuchsin- primary stain

    B. 20 % Sulphuric acid – decoloriser

    C. Methylene blue / Malachite green- counter stain

    Acid-fast bacilli will be bright red against a blue


    background after staining.
    REAGENTS
    Carbol fuschin
    Basic fuschin
    Distilled Water
    Absolute alcohol
    Phenol

    Sulphuric acid 20%


    98% sulphuric acid and
    Distilled water

    Methylene blue
    Potassium hydroxide
    Distilled Water
    Methylene blue in alcohol
    PROCEDURE OF STAINING
    Heat fixed smear is taken

    cover it with hot carbol fuchsin(7 ­10 min)

    wash with water

    cover it with 20% H2SO4(3 ­4min)

    wash till no more stain comes off

    counterstain with methylene blue(1­2 min)

    Wash with water

    blot dry & examine under oil immersion


    Z-N stain
    10
    Interpretation
    Mycobacterium tuberculosis appears as
    long slender, beaded, red colored acid-fast
    bacillus.

    other non-acid fast organisms present in


    the smear and the background take up the
    counter stain and appear blue
    Z-N smear evaluation( RNTCP)

    No. of fields
    No. of AFB Result Grading

    >10/F 20 +ve 3+

    1-10/F 50 +ve 2+

    10 - 99/100F 100 +ve 1+

    1-9/100F 100 +ve scanty

    No. of AFB in 100 F 100 -ve


    ACID FAST STRUCTURES
    Very few structures are acid fast; so this makes the stain
    useful for diagnostic purposes.

     All Mycobacteria- M. tuberculosis, M. leprae,


    M. smegmatis and atypical Mycobacterium
     Nocardia spp.
     Head of sperm
     Bacterial spores
     Some coccidian parasites, such as Cryptosporidium
    parvum, Isospora belli and Cyclospora spp. cysts in
    faecal matter.
     Hooklets of hydatid cyst
    MODIFIED Z-N STAINING

     Kinyoun’s modification – instead of hot carbol


    fuchsin, cold carbol fuchsin is used.
     Acid-alcohol can be used as decolorizer
    alternatively.
     Malachite green can be used as counter stain
     Concentration of sulphuric acid may vary
    depending on the acid-fastness of the structure to be
    demonstrated.
     More the content of mycolic acid in the cell wall,
    more is the acid-fastness, hence more is the
    percentage of sulphuric acid needed for
    decolorization
     5% sulphuric acid is used for
    decolorising Mycobacterium leprae.
     1% sulphuric acid for Nocardia,
    Cryptosporidium, Cyclospora, Isospora.,
    hooklets of hydatid cyst
     0.25% sulphuric acid for bacterial
    spores.
     Sperm head-0.5- 1%
     Legionel/a micdadei- 0.5-1%

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