The document summarizes the PAS staining technique and Ziehl-Neelsen staining technique for acid-fast bacteria. The PAS stain involves oxidizing carbohydrates using periodic acid, then treating with Schiff's reagent to produce a magenta color. The Ziehl-Neelsen stain takes advantage of mycobacteria's lipid-rich cell wall, using a carbol fuchsin solution which is retained after differentiation in acid or alcohol, allowing visualization of acid-fast bacilli. Both techniques are described in detail, outlining the required solutions and step-by-step procedures.

1. Principle:
The PAS stain is a histochemical reaction in
that the periodic acid oxidizes the carbon to carbon
bond forming aldehyde which react to the fuchsin-
sulfurous acid which form the magenta color.
Glucose + Periodic acid - aldehyde
(Carbohydrate)
Aldehyde + Schiff’s reagent - magenta
colored compound

2. PAS Technique:
- Periodic Acid Solution
Periodic acid - 1 gm
Distilled water - 200 ml
- Schiff Reagent
Dissolve 1gm of basic fuchsin in 200 ml of
boiling distilled water. Allow the solution to cool 50 °c,
and add 2-gm potassium metabisulphite with mixing.
Allow to cool to room temperature then add 2 ml conc.
Hydrochloric acid, mix, add 2 gm activated charcoal and
leave overnight in the dark at room temperature.
Filter, the solution should be either clear or pale yellow
color. Store in a dark container at 4°c.

3. Procedure:
o Dewax sections and bring to distilled water.
o Treat with periodic acid, 10 mins.
o Wash well with several changes of distilled water.
o Cover with schiff’s reagent, 10-20 min.
o Wash in running tap water, 5-10 min.
o Stain nuclei with Harris’s haematoxylin or an iron
haematoxylin. (Do not use Ehrlich’s haematoxylin,
which will also stain some PAS positive compounds.)
o Differentiate in acid-alcohol .
o Wash in water, dehydrate in alcohol, clear in xylene
and mount as desired.

14. Ziehl-Neelsen (ZN) Stain for Mycobacterium
Bacillus
Mycobacteria are difficult to demonstrate by the Gram
technique because they possess a capsule containing a long
chain fatty acids that make them hydrophobic.
Phenolic acid or heat may be used to reduce the surface
tension and increase the porosity.

15.  PRINCIPLE
Mycobacteria tuberculosis (tubercle bacilli) have a lipid-rich cell
wall which is capable of taking up strong phenol dye solutions
(eg. carbol fuchsin solution) such that the dye is retained upon
subsequent differentiation in acid or alcohol ie. they are said to
be acid and alcohol fast (AAFB = acid and alcohol fast bacilli).

16. Solutions
 Carbol – fuchsin
1 g basic fuchsin dissolved in 10 ml of absolute
alcohol; add 100 ml of 5% aqueous phenol. Mix
well and filter before use.
 Acidified methylene blue
0.25% methylene blue in 1 % acetic alcohol.
 Malachite green
Blue green counterstain used

17. Method
1. Deparaffinize and rehydrate through graded alcohols to
distilled water.
2. Flood sections with freshly filtered carbol-fuchsin and
heat to steaming with intermittent flaming, 15 minutes,
or stain in Coplin jar at 56°-60°C, 30 min.
3. Wash well in tap water.
4. Differentiate in 1 % acid alcohol, 10 min.
5. Wash well in tap water.
6. Counterstain in methylene blue solution, 30 seconds.
7. Blot and differentiate by alternate dehydration and
rehydration until the background is a delicate pale blue.
8. Dehydrate, clear and mount.

18. PROCEDURE FOLLOWED IN OUR
HISTOPATHOLOGY LAB

26. Wash in water, dehydrate in alcohol, clear in
xylene and mount as desired

27.  Mycobacterium
 Nocardia
 Legionella Micdadei
 Smegma bacilli
 Rhodococcus
 Isospora
 Spermatic head
 Cryptococcal cyst

28. Mycobacterium tuberculosis in lung, Ziehl-Neelsen acid fast stain.

30. 1.Deparaffinize in 2 changes of xylene peanut oil ,6 min each
2.Drain slides vertically on paper towel and wash in warm,
running tap water for 3 min
3.Stain in carbol fuschin at room temperature for 3 min
4.Wash in warm running tap water for 3 min and drain excess
water
5.Decolorize with 5% sulphuric acid and 25% alcohol two
changes of 90 seconds each
6.Wash in warm running tap water for 5 min
7.Counterstain in working methylene blue one quick dip
8.Wash in warm running tap water for 5 min
9.Blot sections and dry in 50-55 C oven for 5 min
10.Once dry one quick dip in xylene then mount

34. Thank you