Acid fast staining

Acid fast staining is differential staining technique which differentiate bacteria into two group- acid fast bacteria and non acid bacteria. It used to identify acid-fast organisms such as members of the genus Mycobacterium .

1. Acid-fast staining
AnupMuni Bajracharya

2. Acid-fast staining
•is a differential staining
•used to differentiate acid fast and non acid fast bacteria.
•used to identify acid-fast organisms such as members of
the genusMycobacterium.
•also known as Ziehl-Neelsenmethod
•first introduced by Paul Ehrlich later modified by two
German doctors bacteriologist Franz Ziehl(1859–1926)
and the pathologistFriedrich Neelsen(1854–1898).
A.B

3. History
•In 1882 Robert Koch discovered the etiology of tuberculosis.
•Soon after Koch’s discovery, Paul Ehrlich developed a stain for
mycobacterium tuberculosis, called the alum hematoxylinstain.
•Franz Ziehlthen altered Ehrlich’s staining technique by using
carbolic acid as the mordant.
•Friedrich Neelsenkept Ziehl’schoice of mordant but changed the
primary stain to carbolfuchsin.
•Ziehland Neelsen’smodifications together have developed the
Ziehl-Neelsenstain.
•Another acid-fast satin was developed by Joseph Kinyounby using
the Ziehl-Neelsenstaining technique but removing the heating step
from the procedure.
•This new stain from Kinyounwas named the Kinyounstain.
A.B

4. What are acid fast bacteria?
•those which have a high content ofmycolicacidsin
their cell walls.
•are characterized by
•wax-like, nearly impermeable cell walls;
•contain mycolicacid and large amounts of fatty acids,
waxes, and complex lipids.
•are highly resistant to disinfectants and dry conditions.
•Due to their waxy cell wall components,
Mycobacterium are acid fast; that is, they retain the
red dye, carbolfuchsin, after rinsing with acid solvents.
A.B

5. Acid fast organisms
Acid-fast organisms like
Mycobacterium contain large
amounts of lipid substances,mycolic
acids. which resist staining by
ordinary methods such as a Gram
stain.
Require acid-faststaining that
is used to screen for the
Mycobacterium,Nocardia,and
Legionellaspecies in body
tissues and fluids.
Acid-fastness is an uncommon
characteristic shared by the
generaMycobacteriumandNocardia
(weakly acid-fast).
Because of this feature, this stain is
extremely helpful in identification in
diseases caused by acid-fast bacteria,
particularly tuberculosis and leprosy.
A.B

6. Principle
•Because the cell wall containing mycolicacid is so waxy and
resistant to most compounds, acid-fast organisms require a
special staining technique.
•The primary stain used in acid-fast staining, carbolfuchsin,
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 strips 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.
•Acid fast bacteria will be red, while nonacid fast bacteria
will stain green.
A.B

7. A.B

8. Requirements
Reagents
•Primary Stain:Carbol-fuchsin.
•Decoloriser:(HCl+Ethanol)
•Counterstain:Methyleneblue.
Materials
Slides
Sample
Inoculating loop
Bunsen burner
Cotton
Microscope
A.B

9. Preparation of reagents
•Primary Stain:0.3% Carbol-fuchsin.
•Dissolve 50 g phenol in 100 mLethanol (95%).
•Dissolve 3 g Basic fuchsinin the mixture and add
distilled water to bring the volume to 1 L.
DecolorizationSolution:(HCl+Ethanol)
•Add 30 mLhydrochloric acid to 1 L of 95% denatured
alcohol.
•Cool and mix well before use.
Counterstain:0.3% Methyleneblue.
•Dissolve 3 g methyleneblue in 1 L distilled water.
A.B

10. Procedure
•Take a clean grease free slide.
•Prepare the smear from provided sample.
•Air dry and heat fixed the smear.
•Place a small strip of filter paper over the top of smear and place the slide
over a boiling hot water bath on a mesh surface.
•Cover the filter paper with the primary stain, carbolfuchsin.
•Leave the slide on the water bath for 5 minutes or more.
•Note-Continue to apply stain if the filter paper begins to dry.
•Remove the filter paper and rinse the slide with water until the solution
runs clear.
•Run acid-alcohol decolorizerover the slide for approximately 10 to 15
seconds.
•Rinse the slide with water.
•Cover the smear with the counterstain, methyleneblue, for 1 minute.
•Gently rinse the slide with water.
•Blot the slide dry with bibulous paper.
•Observe under microscope. A.B

11. Procedure
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12. Observation under Microscope
Acid fast:Red, straight or
slightly curved rods, occurring
singly or in small groups, may
appear beaded
Non-acid fast:Blue color;In
addition, background material
stain blue.
A.B

13. Organisms stained by acid fast staining
A.B

14. Kinyounstain
•Unlike the(Z-N stain), the Kinyounmethod of staining does
not require heating.
•In the ZN stain, heat acts as a physical mordant while
phenol (carbolof carbolfuschin) acts as the chemical
mordant.
•Since the Kinyounstain is a cold method (no heat applied),
the concentration of carbolfuschinused is increased.
A.B