Ziehl Neelsen Staining or AFB Staining Procedure

· Staining techniques

Smear preparation for AFB:
  • Take a grease free scratch free glass slide to prepare the smear. (A new glass slide should be used for smear preparation, if greasy; you can clean the grease by wiping it with alcohol and use it after its air dry).
  • Label the slide with laboratory code and serial number with a diamond marker on the side of the smear at one end of the slide. (An ordinary lead pencil can be used if frosted-end slides are available).
  • Open the sputum container carefully to avoid aerosol production. Then, take a wooden or bamboo stick applicator, break it and select yellow, purulent particle of sputum with jagged end of the broken wood or bamboo stick applicator.
  • Spread the sputum evenly over the central area of the slide using a continuous rotational movement and prepare the smear of about 2 cm x 1cm size.
  • Air dry the smear and heat fix by holding it with forcep and passing the smear side up over the flame 5 times for about 4 seconds. Do not heat-fix moist slides, and do not overheat.

Staining Procedure for AFB:

  1. Place the fixed slide on the staining rack, smear side up. (Slides should be separated by a 1 cm, and should not touch one another if more than one sputum samples are to be stained).
  2. Cover the slide with carbolfuchsin solution. (A strip of filter paper or even newspaper can be placed on the slide which will hold the staining solution and prevent deposits of fuchsin crystals on the smear).
  3. Heat the slide from underneath with the flame of a Bunsen burner, an alcohol lamp or an alcohol soaked cotton swab until steam starts to rise. Staining solution should never be allowed to boil.
  4. Keep the slide covered with hot, steaming carbolfuchsin for 5 minutes by re-flaming as needed.
  5. Rinse gently with water to remove excess carbolfuchsin from the slide and drain off excess rinsing water from it. (Sputum smear appear red in color after this step).
  6. Cover the slide with 20% sulfuric acid or 3% acid-alcohol solution and allow to stand for 3 minutes, after which the red color should have almost completely disappeared.  Repeat sequence until the red color disappears, but do not over decolorise. 
  7. Gently wash away the sulfuric acid or acid alcohol and the excess stain with water and drain off excess rinsing water from the slide.
  8. Rinse the slide individually with water and drain water off the slide.
  9. Cover the slide with methylene blue or malachite green counter staining solution and allow to stand for 1 minute.
  10. Rinse gently with water to remove excess methylene blue or malachite green from the slide and drain off excess rinsing water from it. (A properly stained smear should show a light blue color due to methylene blue and a newsprint should be readable when held underneath the slide but if dark blue i.e., the smear is too thick  and a newsprint cannot be read when held underneath the slide).

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