ZN STAIN
INTRODUCTION
The Ziehl-Neelson stain, also known as the acid stain. Acid fast organisms like Mycobacterium
contain large amounts of lipid substances within their cell walls called mycolic acids. These acids
resist staining by ordinary methods such as a Gram stain. It can also be used to stain a few other
bacteria such as Nocardia.
OBJECTIVES

To examine and identify Mycobacterium species.

To differentiate between acid fast and non-acid fast bacilli.

To identify some fungal species such as Cryptosporidium
PRINCIPLE
The lipoid capsule of the acid-fast organism takes up carbol fuchsin and resists decolorization
with a dilute acid rinse. The lipoid capsule of the mycobacteria is of such high molecular weight
that it is waxy at room temperature and successful penetration by the aqueous based staining
solutions (such as Gram’s) is prevented.
REGENTS REQUIRED
1. Carbol Fuchsin
2. 20% sulphuric acid or acid alcohol
3. Methylene Blue or malachite green
PREPARATION OF REAGENTS
1. Carbol fuchsin

Distilled water
: 100ml

Basic fuchsin
: 1g

Ethyl alcohol (1oo% ethanol) : 10ml

Phenol crystals
: 5ml
2. Acid alcohol (3% hydrochloric acid in 95% ethyl alcohol0

Ethyl alcohol
: 95ml

Distilled water
: 2ml

Concentrated hydrochloric acid
: 3ml
3. 0.25% methylene blue in 1% acetic acid

Methylene blue
: 0.25g

Distilled water
:99ml

Acetic acid
: 1ml
PROCEDURE
1. Prepare a smear on a dry glass slide.
2. Allow it to air dry and fix with gentle heat.
3. Flood the smear with Carbol fuchsin stain.
4. Carefully heat the underside of the slide by passing a flame under the rack until steam
rises (without boiling)
5. Keep the preparation moist with stain and steaming for 5 minutes, repeating the heating
as needed.
(Note: overheating causes spattering of the stain and may crack the slide.)
6. Washing in running tap water.
7. Holding the slide with forceps, wash the slide with the decolorizing solvent. Immediately
wash with tap water. Repeat the decolorizing and washing until the stained smear appears
faintly pink and the fluid washing off the slide runs clear.
8. Flood the smear with methylene blue counterstain for 30 seconds, and wash with tap
water.
9. Gently blot, or air dry the smear.
10. Examine under oil immersion.
RESULT AND INTERPRETATION
Acid-fast bacteria
Red
Non acid-fast bacteria (and other organisms and cellular materials)
Blue

> 10 AFB/high power field
:
+++

1-10 AFB/high power field
:
++

10-100 AFB/100 high power fields :
+

1-9 AFB/100 high power fields
exact number
: