ZN staining

Ziehl-Neelsen staining for Acid fast organism

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Ziehl-Neelsen staining technique is a special bacteriological stain to identify Acid Fast organism. This was first described by two German scientist doctor, Franz Ziehl and a pathologist Fredrich Neelsen to identify acid fast organism. The Ziehl-Neelsen staining technique was first introduced by Ziehl and later modified by Neelsen. So named as Ziehl-Neelsen staining technique.

Neelsen in 1883 used Ziehl’s carbol fuchsin and heat then decolorized with an acid alcohol, and counter stain with methylene blue. Thus Ziehl-Neelsen staining technique developed. The aim of this staining is to differentiate Acid fast from non acid fast bacteria.

Acid fast organism:

Acid fastness means once these bacterial cells are stained, these organisms resist “decolorization” by acid and or methanol. These are a common procedure in some staining protocol.

Acid fast organism include-

  1. Mycobacterium species- like M.Tuberculosis, M.Leprae, M.Kansasi, M.Marinum, M.bovis, and M.avium complex. Acid fast organism contain large amount of lipid in their cell wall mainly “Mycolic acid”. These lipids resist ordinary dye like “gram stain” to enter into the cell.
  2. Nocardia.

Principle of Acid fastness and Ziehl-Neelsen staining:

When smears are stained with carbol fuchsin, it solubilizes the lipoidal materials present in bacterial cell wall. If heat is applied, carbol fuchsin penetrates further through lipoidal wall and enters into cytoplasm. Then the all organism component become red.

When decolorizing agent are used, usually used 3% HCL in 97% alcohol, (acid-alcohol), acid fast cell are resistant due to presence of large amount of lipoid materials in their cell wall- which prevent acid penetration.

Non acid fast cell/ organism decolorized, as the acid enter inside cell easily ant it become colorless.

Counter stain are used to stain background substances after decolonization. Only decolorized cell absorb methylene blue or other counter stained dye and appear blue, while the acid fast organism retain red color.

Acid fast stain colour

Primary dye : carbol-fuchsin

            Acid fast organism stains: Red

            Non Acid fast organism stains: Red

After Decolorization with acid alcohol

            Acid fast organism stains: Red

            Non Acid fast organism stains: Colourless

Counter stain with methylene blue:

            Acid fast organism stains: Red

            Non Acid fast organism stains: Blue

 Methods of staining: there are two method

Carbol fuchsin, which include Ziehl-Neelsen stain and Kinyoun method staining. In this method, phenol-carbol fuchsin stains are is heated to enable the dye to penetrate the waxy mycobacterial cell wall.

Fluorescent staining
Fluorescent stainingFluorescent staining

Fluorescent procedure using Auramine-Rhodamine dye, seen under fluorescent microscope. In this method, stains are not heated, but penetration into the cells is achieved by increasing concentration of the basic fuchsin and phenol incorporating a “wettingagent “ chemical.

Smears and staining:

There may be two kinds of smears

  • Direct smears: smears are made from direct sample- pus, sputum other materials.
  • Indirect smears: also called the concentration methods. When sample like urine, effusion fluid are examined for AFB, sample is first centrifuged and smears are made from centrifuged deposit.
  • For details on bacteriological sample preparation CLICK HERE. (LINK)

Reagent:

  • Carbol fuchsin stain:
  • Acid alcohol 3% V/V
  • Malachitegreen/ methylene blue 0.5% w/v

Ziehl-Neelsen staining procedure:

  • Sputum or FNAC aspirates or pus is spreaded over the central area of a clean, grease free glass slide. The recommended area size should be 20mmx10mm
  • Keep the slide with smeared surface upwards and dry the smears in air and it may take about 30 minutes.
  • Fix the smears in heat.
  • Cover the whole smear with carbol fuchsin stain.
  • Heat the smears until vapour begins to rise, this is about 60 degree Celsius. Do not boil or over heat.
  • Keep for 5 minuets
  • Wash with water, preferably running water. As the carbol-fuchsin colour is deep and will not wash out easily. After washing smear will look red/pink-red colour.
  • Decolorize the smears with 3% v/v acid alcohol for about 2-3 Minuits, or until is sufficiently decolourize. Decolourization cause smear colour pale pink. Acid alcohol is flammable, use with care.
  • Wash with clean water. At this stage, slide may look pale or colourless. After decolourization only acid fast particle/ organism will retain pink/red colour. If the tap water is not clean, wash with filtered water or boiled rain water.
  • Then cover the smear with malachite green or new methylene blue for 1-2 minuits. This will stain blue colour to all structure other than acid fast particle, which will retain red colour.
  • Wash with clear running water.
  • Place the coloured smears in a draining rack, and dry it. (Do not Blot Dry)
  • Examine under microscope using 100x oil immersion objective. When possible, use 7x eyepieces, this will give a brighter image.

Note

  1. Heat fixation will not kill Mycobacterium Tuberculosis. Alcohol fixation is bacteriocidal. Considering the MDR tuberculosis and viability of bacteria, better to kill the organism with alcohol fixation.
  2. Acid alcohol is inflammable, therefore use it with care.
  3. Care to be taken during Carbol-fuchsin is heating; so that heat may not spread to other site it may cause fire. Staining rack should be made with heat resistance substances.
  4. In staining rack, gap to be maintains between two slides.
  5. All acid fast organisms are equally acid fast, as for example, among mycobacterium species, M. tuberculosis and M.ulcerans are strongly acid fast, while M. leprae is only weakly acid fast. So, in case of M. tuberculosis and M. ulcerans, 3%v/v acid alcohol is used, while in case of M. leprae, 0.5-1% decolorizing solution is used.
  6. 0.5% acid alcohol or 5% Sulphuric acid is used for atypical mycobacterium, M.Leprae, Nocardis species, as they are much less acid fast than M. Tuberculosis.
  7. 1% sulphuric acid alcohol may be used for actinomycetes, nocardia.
  8. 0.5-1% sulphuric acid alcohol for oocyst of isospora, cyclospora
  9. 0.25-0.5% sulphuric acid alcohol for bacteriological endospores.

Interpretation of Ziehl-Neelsen staining

Acid fast organism: Bright red. M.tuberculosis are straight or slightly curved rod, occurring singly or in group, may appear beaded.

Non-Acid fast organism: Blue

Ziehl-Neelsen staining
Ziehl-Neelsen staining

Reporting

  • When any definite red bacilli are seen, Report the smears as “AFB Positive” and give an overall indication of number of bacilli present in the sample as follows:
    • >10 Acid fast bacilli per field   : report as  +++
    • 1-10 Acid fast per field                        : report as ++
    • 10-100 Acid fast per 100 fields           : report as +
    • 1-9Acid fast per 100 fields      : report the exact number.
    • When no AFB are seen after examining 300 field: report as “No AFB seen”.

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