141 / 276
Annexure -1
Ziehl-neelsen staining procedure
1. A new unscratched slide is selected and the slide is labelled with the Laboratory Serial
Number with a diamond marking pencil.
2. A smear is made from yellow purulent portion of the sputum using a broom stick. A good
smear is spread evenly, 2 cms x 3 cms in size and is neither too thick nor too thin. The
optimum thickness of the smear can be assessed by placing the smear on a printed
matter. The print should be readable through the smear. Smear preparation should be
done near a flame. This is required, as six inches around the flame is considered as a
sterile zone which coagulates the aerosol raised during smear preparation.
3. The slide is allowed to air dry for 15–30 minutes.
4. The slide is fixed by passing it over a flame 3–5 times for 3–4 seconds each time.
5. 1% filtered carbol fuchsin is poured to cover the entire slide.
6. The slide is gently heated with carbol fuchsin on it, until vapours rise. Do not boil.
7. Carbol fuchsin is left on the slide for 5 minutes.
8. The slide is gently rinsed with tap water until all free carbol fuchsin stain is washed
away. At this point, the smear on the slide looks red in colour.
9. 25% sulphuric acid is poured onto the slide and allowed to stand for 2–4 minutes.
10.The slide is gently rinsed with tap water and tilted to drain off the water.
11.A properly decolourised slide appears light pink in color .If the slide is still red, sulphuric
acid is reapplied for 1–3 minutes and then rinsed gently with tap water.The back of the
slide is wiped clean with a swab dipped in sulphuric acid,
12.0.1% methylene blue is poured onto the slide and left for 30 seconds. Then theslide is
rinsed gently with tap water and allowed to dry.
13.The slide is examined under the binocular microscope using x40 lens to select the
suitable area and then examined under x100 lens using a drop of immersion oil.
14.The results are recorded in the Laboratory Form and the Laboratory Register.
15.The slides are inverted on a tissue paper till the immersion oil is completelyabsorbed.
Xylene is not to be used for cleaning the slides, as it may give falseresults at repeat
examination after storage.
16.All positive and negative slides are stored serially in the same slide-box untilinstructed
by the supervisor.
17.All contaminated materials are disinfected as per guidelines before discarding.
134