Acid Fast Bacilli (AFB)/TB Culture Using MGIT (BACTEC) System

Principle:
AFB smears may fail to reveal positive AFB results made from the specimen. As a consequence, culture techniques play a key role in the diagnosis of mycobacterial disease. Both inoculated liquid (MGIT tube) and solid media (LJ agar) are stored inside an incubator at 37°C.
Note: Perform all procedures in a suitable biological safety cabinet in a room dedicated for mycobacterial work.

Procedure:

Primary Isolation

• Label all materials: conical tube, MGIT tube, LJ tube and slide. 
• Pour the specimen into the conical tube. 
• Pour equal amount of Mycoprep or up to the 20mL mark. 
• Mix well, vortex every 5 minutes for a total of 15 minutes. 
• Pour phosphate buffer up to the 40 ml mark. 
• Centrifuge the sample in a refrigerated centrifuge for 15 minutes at 3000 rpm. 
• Decant and resuspend with approximately 3mL of phosphate buffer 
• Aspirate 0.7mL of suspension and transfer 5mL into the MGIT tube, 1mL into the LJ agar and 1mL into the slide. 
• Put the inoculated tubes in BACTEC cabinet, LJ in the incubator and the conical tube with remaining sample in the refrigerator. 
• Dry smear, heat fix and stain with AFB staining. 

Drug Susceptibility Testing (Direct Method)-Done Within 2 days of positivity

• Label all materials: 5 MGIT tubes (GC, S, I, R & E) and 2 PZA tubes (GC & PZA). 
• Aseptically add 0.8mL of SIRE supplement to S, I, R, E tubes and 0.8mL PZA supplement to PZA tube. 
• Introduce 0.1mL of the specified antibiotics to S, I, R, E & PZA tubes. 
• Prepare 2 tubes with 10mL NSS (labeled SIRE-GC) and 4.5mL NSS (PZA-GC). 
• Mix well the positive tube to break the clumps. Aspirate 0.1mL to tube SIRE-GC and 0.5mL to PZA-GC. Mix well. 
• From the diluted sample, introduce 0.5mL sample to respective GC tubes. 
• Aspirate 0.5mL from positive tube and transfer to S, I, R, E & PZA tubes. 
• Place tubes in a carrier set and incubate. 

Drug Susceptibility Testing (Dilution Method)-Done within 3-5 days of positivity 

• Label all materials: 5 MGIT tubes (GC, S, I, R & E) and 2 PZA tubes (GC & PZA). 
• Aseptically add 0.8mL of SIRE supplement to S, I, R, E tubes and 0.8mL PZA supplement to PZA tube. 
• Introduce 0.1mL of the specified antibiotics to S, I, R, E & PZA tubes. 
• Prepare 2 tubes with 10mL NSS (labeled SIRE-GC) and 4.5mL NSS (PZA-GC). 
• Mix well the positive tube to break the clumps. Aspirate 0.1mL to tube SIRE-GC and 0.5mL to PZA-GC. Mix well. 
• From the diluted sample, introduce 0.5mL sample to respective GC tubes. 
• Prepare a tube with 4mL NSS. Mix well the positive tube and aspirate 1mL sample to it. Mix well.
• From the diluted sample, aspirate 0.5mL of sample to S, I, R, E & PZA tubes. 
• Place tubes in a carrier set and incubate. 

Interpretation:
Mycobacterium tuberculosis 

Show positive result on AFB smear with cording appearance in union with no growth on sheep’s blood agar and yellowish colony growth on LJ agar.

Mycobacteria other than tuberculosis (MOTT) or Non-tuberculous mycobacteria (NTM)
Show positive result on AFB smear without cording appearance in union with growth on sheep’s blood agar and yellowish colony growth on LJ agar.