Inoculum Preparation from MGIT Positive Culture

Inoculum Preparation for Drug-containing Tube (1-2 days)

1. DST must not be set up on the same day an MGIT tube signals positive (0 Day).

2. If the culture is worked up one or two days after signalling positive, it can be used directly to inoculate the MGIT tubes for DST.

3. Mix well (vortex) the confirmed MTB pure culture isolate to break up the clumps. Leave the tube undisturbed for about 5-10 minutes to let big clumps settle on the bottom.

4. Use the undiluted supernatant for inoculation of the drug tube set.

5. Aseptically add 0.5 ml of the well-mixed culture suspension (inoculum) into each drug-containing tube using a pipette. Do not add to the growth control (GC).

 Inoculum Preparation for Growth Control (GC) Tube (1-2 days)

For the growth control (GC) tube, first, dilute the test culture suspension 1:100 by adding 0.1 ml of the test culture suspension to 10.0 ml of sterile saline. Mix well by inverting the tube 5-6 times. Use this diluted suspension to add 0.5 ml into the growth control tube.

  Inoculum Preparation for Drug-containing Tube (3-5 days)

1. If the culture is used to set up DST between three and five days after signaling positive.

2. Mix well (vortex) the confirmed MTB pure culture isolates to break up the clumps. Leave the tube undisturbed for about 5-10 minutes to let big clumps settle on the bottom.

3. Use the undiluted supernatant for preparation of 1:5 dilution, 1 ml of MGIT broth in 4 ml of sterile saline (1:5 dilution).

4. Use this well-mixed diluted culture for inoculation of the drug tube set.

5. Aseptically add 0.5 ml of the well-mixed culture suspension (inoculum) into each drug-containing tube using a pipette. Do not add to the growth control (GC).

 Inoculum Preparation for Growth Control (GC) Tube (3-5 days)

1. For the growth control (GC) tube, first, dilute the test culture suspension 1:100 from 1:5 diluted MGIT tube. by adding 0.1 ml of the test culture suspension to 10.0 ml of sterile saline. Mix well by inverting the tube 5-6 times. Use this diluted suspension to add 0.5 ml into the growth control tube.

2. A tube that has been positive for more than 5 days should not be subjected to DST. Instead, it should be subcultured in a fresh MGIT tube supplemented with MGIT 960 Growth Supplement and should be tested in an MGIT 960 instrument until it is positive.

3 Use this tube from one to five days of instrument positivity as described above.

Resource

Mycobacteriology Laboratory Manual   

Assessment