Preparing the Isolate for Liquid Culture DST

1. Isolates can be grown on solid or liquid media before being tested in the MGIT DST.

•   Need healthy cells in the logarithmic phase of growth

•   Need fairly light inoculum so that drug concentrations are not overwhelmed

2. Growth should be AFB-positive and identified as a pure culture of the MTB complex before further testing. 

Inoculum from the MGIT tube: It is important that the growth is within the following recommended timeframe.

•  The day an MGIT tube is positive by the instrument is considered Day 0.

•  The tube should be kept incubated for at least one more day (Day 1) before being used for the susceptibility testing (it may be incubated in a separate incubator at  37ºC ± 1ºC).

•  A positive tube may be used for drug susceptibility testing up to and including the fifth day (Day 5) after it becomes instrument positive. A tube that has been positive for more than 5 days 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. Use this tube from one to five days of instrument positivity as described  above.

•  If growth in a tube is on Day 1 or Day 2, mix well (vortex) to break up clumps. Leave the tube undisturbed for about 5-10 minutes to let big clumps settle on the bottom. Use the supernatant undiluted for inoculation of the drug set.

• If growth is on Day 3, 4, or 5, mix well to break up the clumps. Let the large clumps settle for 5-10 minutes, and then dilute 1.0 ml of the positive broth with 4.0  ml of sterile saline. This will be a 1:5 dilution. Use this well-mixed diluted culture for inoculation.

Resource

Mycobacteriology Laboratory Manual      

Assessment