Page 124 - LECTURE NOTES

7. Turn the scalpel blade until it is at a right angle to the cut.

Using the blunt edge of the blades, scrape firmly two or three times along the
edges and bottom of the cut to collect a sample of tissue juice and cells

8. Transfer the sample to a slide. Make a small circular smear, covering evenly an
area measuring 5-7 mm in diameter.

9. Cover the cut with a small dressing. Instruct the patient to remove the dressing as
soon as the cut has healed

10. Ensure the slide is clearly labeled with the patient’s name and identification number

11. When the smear has dried, gently heat-fix by holding the smear upper most over
the flame of a spirit lamp or the pilot flame of a Bunsen burner for a few seconds.

Do not over – heat because this will interfere with the staining of M.leprae.

5.4.4. 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
0
begins to rise (i.e. about 60 C). 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

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