Ziehl-Neelsen Technique For Staining M. Leprae
Required
- Carbolfuchsin stain (Filtered)
- 1% acid alcohol
- Malachite green, 5 g/l (0.5% W/v)
* If preferred, methylene blue 5g/l may be used instead of malachite green.
Method:
1. Cover the smear with filtered carbolfuchsin stain. Heat the stain until vapor just begins to rise (i.e. about 600C). Do not overheat. Allow the heated stain to remain on the slide for 10-15 minutes (ensure the stain does not dry on the smear).
2. Wash off the stain with clean water. When the tap water is not clean, use boiled filtered rainwater.
3. Decolorize the smear rapidly (about 5 seconds) by rinsing it with 1 %( v/v) acid alcohol.
Caution: Acid alcohol is flammable; therefore use it with care well away from an open flame.
4. Wash carefully with clean water. Then cover the smear with malachite green stain (or methylene blue) for 1-2 minutes
5. Wash off the stain with clean water; wipe the back of the dry slide (do not blotdry). Protect it from direct sun light.
6. Examine the smear microscopically, first with the 40x objective to see the distribution of material and then with the oil immersion to look for acid-fast bacilli.
Result
M. leprae …………………………….. Red solid bacilli or beaded forms, occurring singly or in masses
Macrophage cells ……………………… green*
*Blue if methylene blue counter stain has been used
- Reporting M.leprae smear;
Report the smear as ‘positive’ if M.Leprae bacteria are seen or ‘Negative’ if no bacteria are seen after examining entire smear or at least 100 high power microscope fields.
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