AFB Smears from Liquid Culture

Once an MGIT tube is positive by fluorescence or visual observation, prepare a smear and stain with carbol fuchsin stain. 

Procedure:

1. Use a clean slide. 

2. Mix the broth by vortexing, and then remove an aliquot using a sterile pipette. Place 1-2 drops on the slide and spread over a small area (approx. 1½ x 1 cm). 

3. Let the smear air dry.

4. Heat-fix the smear by passing it over a flame a few times or using an electric warmer at 65ºC - 70ºC for 2 hours to overnight. Do not leave the smear openly exposed to the UV light of the safety cabinet. 

5. Stain the smear with Ziehl-Neelsen.

6. Air dry, but do not blot dry.

7. Place a drop of oil on the stained and completely dried smear and screen under a low-power objective to locate stained bacteria. Then, switch to an oil immersion objective lens for detailed observation.

8. If the broth appears turbid or contaminated, irrespective of AFB smear results, subculture on a blood or chocolate agar, or TSI, to rule out the presence of contaminating bacteria. 

9. If the smear is negative for AFB and the tube does not appear to be contaminated, i.e. broth is clear, re-enter the tube into the instrument for further monitoring. Repeat AFB smears after 1-3 days.

   Resource

Mycobacteriology Laboratory Manual 

Assessment