Ziehl Neelsen Stain Flashcards
Ziehl-Neelsen stain is used for?
It is used for differential staining of tubercul bacilli and other acidfast.
Ziehl-Neelson method is a modification of……
Ziehl-Neelson method is a modification of Ehrlich’s original method for differential staining of tubercul bacilli and other acidfast bacilli with aniline, gentian violet followed by strong nitric acid .
Principle of Ziehl-Neelson ?
When the smear is stained with carbon fuchsin, a lipid soluble and contains phenols, 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 then take up the counterstain. Only decolourized cells absorb the counter stain and take its colour and appears blue while acid-fast cells retain the red colour.
Mechanism of acid fast stain?
Acidfast is ascribed to the high content of lipids, fatty acid and higher alcohol found in tubercle bacilli.
Myocolic acid is high molecular weight containing carboxylate group is found in acid fast bacilli.
The ordinary Anilene dye solution do not readily penetrate the substance of tubercle bacilli and hence unsuitable for staining.
However, by the use of a powerful staining solution containing phenol and with the application of heat. The dye can be made to penetrate the bacilli.
Once stain the tubercle bacillus will withstand the action of powerful decolourizing agent for a considerable time.
Primary stain?
Strong carbol fuchsin- consists of basic fuchsin and phenol
Decolourizer?
25 % sulphuric acid
Counter stain?
0.1% Loeffler’s Methylene blue
Procedure?
- Prepare bacterial smear on clean and grease free slide.
- Allow the smear to air dry and then heat fix.
- Pour filtered carbol fuchsin.
- pour Filtered carbol fuchsin over the smear & heat gently.
- Do not overheat and allowed it to stand for 5 min.
- Wash off the stain with water.
- Decolourized the slide by putting it in a staining jar containing 25% sulphuric acid.
- Wash off with water. If the slide is still red, reapplied 25% of sulphuric acid for 1-2 mins till no more stain comes out. Now wash with tap water.
- Pour methylene blue what for 30 sec to 1 min and then wash again with water.
- allow it to air dry and examine it under oi immersions lens.
Timing for decolourization?
2-4 min
Interpretation?
Acidfast bacilli will be stained bright red colour, while other organism, tissue cells and debris are stained blue.
AFB appears red straight or slightly curved shape at time having beaded appearance..
For sputum sample to be positive with acid fast bacilli in ZN staining at least …..?
10^4 AFB/ml
Diagnosis for positive acid fast bacilli in sputum?
Pulmonary tuberculosis
Diagnosis for positive acid fast bacilli in urine
Renal tuberculosis
Diagnosis for positive acid fast bacilli in CSF
Meninges tuberculosis
Grade 3+
- more than 10 AFB/ oil immersion (OIF)
- result : positive
- no. Of fields to be examined : 20
