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- Report and record the results on the laboratory request form.
Lab Procedure: Ziehl-Neilson Stain
18D Skills and Training ManualWhen: You have to test sputum for acid-fast bacilli (AFB) such as tuberculosis.
What You Need: A properly collected sputum sample, a glass slide, Bunsen burner, pipets, Kinyoun’s
carbol-fuchsin stain, Kinyoun’s acid-alcohol reagent, Kinyoun’s methylene blue reagent, microscope, lab
request form, and a logbook.
What To Do:
- Prepare a smear of the specimen on a glass slide. Air-dry and fix the smear by passing it briefly
through a flame.
CAUTION: Acid-fast bacteria are highly infectious. Specimens and smears must be handled with
care. Wear facemask and gloves and wash hands immediately after performing the AFB examination.
Perform these procedures in a biosafety cabinet, if available. - Stain the smear.
a. Flood the fixed smear with Kinyoun’s carbol-fuchsin stain for 3 minutes.
b. Wash the smear gently with running water.
c. Decolorize it with Kinyoun’s acid-alcohol reagent until no color appears in the washing. This should
take about 2 minutes.
d. Wash the smear gently with running water.
e. Counterstain with Kinyoun’s methylene blue reagent for 30 seconds.
f. Wash the smear gently with running water.
g. Allow the smear to air-dry. - Examine the smear using the oil immersion objective. Scan the length of the slide at least three times
in the stained area (about 300 fields).
NOTE: Acid-fast organisms will appear as red coccobacilli, rods, or filaments. Some may appear beaded.
The background will be bluish.
Lab Procedure: Giemsa Stain for the Presence of Blood
Parasites
18D Skills and Training ManualWhen: You have to test for blood-borne parasites such as malaria.
What You Need: A properly collected blood specimen, a Cameco Wright-Giemsa stain, distilled water,
glass slides, coverslips, staining dishes, water, lint-free cloth, 95% ethanol, rubber gloves, methanol, timer,
microscope, immersion oil, mounting medium, capillary tubes, the laboratory request form and a logbook.
What To Do:
- Prepare the glass slides by cleaning them in 95% ethanol and wiping them until completely dry.
- Prepare the blood smears.
a. Prepare the thin smear.
(1) Place a small drop of blood at a point midway between the sides of the slide and a short
distance from one end.