Zeihl Neelson Staining (ZN Staining)
The Ziehl-Neelsen stain, also known as the acid-fast stain
Aim
To demonstrates acid fast bacilli in given clinical sample.
Principle
Acid fast organism contain mycolic acid lipids in their cell wall which are hydrophobic & make the cell impermeable to routine stains. The Phenol present in the carbon & heating which is employed during Z.N Staining procedure help in dissolution of the mycolic acid & lipid creating pores in the Cell wall through which the primary stain. (carbon fuchsin) enters. Once stained there organism resist decolourisation by dilute mineral acids.
Requirements
- Clinical Sample - sputum / CSF/P.F/urine
- New glass slide
- Wooden Stick
- whatmen no 1 filter paper..
Reagent
1) Primary stain - Carbon fuchsin (contain basic fuchsin, phenol, ethanol, & distilled water).
2) Decolouriser - 20%. H2SO4 or 3%. Acid Alchol
3) Counter stain - Methylene / malachite green / brilliant green.
Procedure
Smeare Preparation
1) Take a new unscratched Slide & made on oval shaped mark (2cmx3cm) at the center by using a glass marker.
2)Make a smear from blood-tinged & yellow purulent portion of the sputum living a stick in marked area.
3)Allow the smear to air dry for 15-30 min.
4) fix the smear by passing the slide over the flame 3-4 times.
Staining
1)Take a heat fixed smear & place it on the stanning rack.
2) Take carbon fuchsin in a test-tube & heat the upper portion untill vapours starts rising.
3) Pour the stain on the smear.
4) leave the carbol fuchsin on the slide for 5-8 min.
5) Decolorize with 20% H2SO4 for 2-3 min then, wash with tap water... Repeat this step till smear become colourless.
6) Counterstrain with methylene blue for 30 sec, wash again with tap water.
7) Air dry the slide & observe under oil immersion objective (Examine either whole smear or min. 100. field / spend 10-15 min in each side I examine the smear in zig-zag manner.
Microscopic visualisation of the acid-fast bacteria Mycobacterium tuberculosis (top) and Mycobacterium leprae (bottom) and background cellular material in blue using the Ziehl–Neelsen stain
Observation & result
Bright red Bacilli seen against a blue back ground.
reported as fast bacilli seen.
RNTCP Grading
No bacilli/100 0IF - Negative
1-9 bacilli/ 100 OIF- scanty
10-99 bacili /100 OIF - 1+
1-9 bacilli/OlF - 2+
More than 10 bacilli /OIF- 3+
Precautions
1) New glass slide should be used.
2)Smear should be prepared from purulent part of sputum.
3) lens should be cleaned after observing any slide.
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