Acid-Fast staining
* Acid fast stains are used to differentiate acid fast organisms such Mycobacteria.
* Acid fast bacteria have a high content of mycolic acids in their cell walls.
* Acid- fast bacteria are also known as acid-fast bacilli of simply AFB.
* Stained bacteria that are not decolorized by acid alcohol are called acid fast bacteria.
* They are a group of bacteria sharing the characteristic of acid fastness.
* Acid fastness is a physical property that gives a bacterium the ability to resist decolorization by acids during staining procedures.
* This means that once the bacterium is stained, it cannot be decolorized by acids routinely used in the process.
* Bacteria displaying acid fastness include:
1) Genus Mycobacteria - M. leprae, M. tuberculosis, M. smegmatis etc.
2) Genus Nocardia - N. brasiliensis, N. cyriacigeorgica, N. farcinica etc.
* There are two types of acid fast staining : hot method (e.g. Ziehl-Neelsen method) and cold method (e.g. Kinyoun method)
Principle : (some for both hot cold acid fast staining method )
" The cell wall of acid fast bacteria contains a lipid called Mycolic acid which makes them resistant to simple aqueous stains. Thus, a lipid- soluble stain (primary stain) is required to stain them which is retained even after decolorization step. whereas primary stain is lost from the non acid fast bacteria upon decolorization and cells get stained with counterstain".
Mechanism : (same for both hot and cold acid fast staining method)
* The cell wall of Mycobacterium sps typically contain waxy substance (Mycolic acid) that makes it impermeable to staining by aqueous staining solutions.
* These bacteria can not be stained by simple or even by Gram staining.
* They can however be stained by lipid-soluble stain and one stained can not be readity decolourized by weak mineral acid.
* Hence, these bacteria are called acid fast bacilli and the staining method is called acid fast staining.
Reagents: (same for both hot and cold acid fast staining method)
* Primary stain : 0.3% Carbol-fusion,(Dissolve 50g phenol in 100ml ethanol (95%) or methanol (95%). Dissolve 3g Basic fushsin in the mixture and add distilled water to bring the volume to 1L)
*Decolorization Solution : Add 30ml hydrochloric acid to 1L of 95% denatured alcohol. Cool and mix well before use. Alternate decoloring reagent (without alcohol): slowly add 250ml sulfuric acid (at least 95%) to 750ml distilled water. Cool and mix well before using.
Counterstain: 0.3% methylene blue (Dissolve 3.0g methylene blue in 1L distilled water).
Ziehl - Neelsen method
* Acid fast staining technique was developed in 1882 by Paul Ehrlich.
* It is named for two German dactors who modified the stain : the bacteriologist Franz Ziehl and the pathologist Friedrich Neelsen in 1890.
* Z.N. staining or Ziehl - Neelsen staining is a bacterilogical stain used to identify acid-fast organisms mainly mycobacteria.
* Ziehl - Neelsen is a not method of acid fast staining.
* The primary stain used in acid-fast staining is carbolfuchsin.
* It is lipid - soluble and contains phenol, which helps the stain penetrate the cell wall.
* This is further assisted by the addition of heat.
* The smear is then rinsed with a very strong decolorizer, which removes the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms.
* The decolorized non-acid-fast cells then take up the counterstain.
Procedure:
* Prepare and fix the specimen smear prior to staining.
* Heat-fix the dried smear.
* Flood the smear with carbol fuchsin stain.
* Heat the stain to evaporate; replenish stain as needed. Also, prevent stain from boiling. Do not overheat. Allow the heated stain to remain on the slide for 5 minutes.
*Wash off the stain with clean distilled water. Heated slides must be cooled prior to washing.
* Decolorize the smear with acid alcohol for 5 minutes or until the smear is sufficiently decolorized.
* Wash well with clean water.
* Flood the smear with methylene blue stain for 1-2 minutes.
* Wash off the stain with clean water.
* Wipe the back of the slide clean and place it in a draining rack for the smear to air dry.
* Examine the smear microscopically, using the 100X oil.
Observation:
*Acid fast : Bright red colored
*Non-acid fast: Blue color
* The kinyoun method or kinyoun stain cold method) developed by joseph J. Kinyoun, is a peocedure used to stain acid-fast species of the bacteria.
* It is a variation of a method developed by Robert Koch in 1882.
* It does not require heating.
* In the Ziehl-Neelsen stain, heat acts as physical mordant while phenol (carbol of carbol fuschin) acts as the chemical mordant.
* Since the kinyoun stain is a cold method (no heat applied), the concentration of carbol fuschin used is increased.
