.
Welcome to this video-demonstration of drug
susceptibility testing of first line anti-TB
drugs using liquid medium.
Drug susceptibility test is essential for
guiding treatment in TB patients.
Two methods are usually available for TB,
one uses the solid l g medium and the other
uses the liquid medium. The most common method
using liquid medium is DST using mycobacterial
growth indicator tube that is MGIT 960 .
We will now see the demonstration of doing
DST to first line anti TB drugs in the laboratory
.
Steps in doing the DST by MGIT include preparation
of the MGIT tube by addition of growth supplement
and the corresponding drug.
Preparation and addition of inoculum. Loading
of the tubes into the machine and unloading
the tubes at the end of the protocol and interpretation
of results. Let us begin with the preparation
of MGIT tubes inside the laboratory, this
MGIT DST is done only in a bio safety level
3 laboratory.
This is a display of all items required ristelled
distilled water, sterile saline the culture
for which DST is to be set up, 5 MGIT tubes
one for the drug free growth control and one
each for the four drugs, and the growth supplement
in the amber bottle. First the drugs that
are available as lyophilized powder in the
septembiles have to be reconstituted using
4 m l of sterile distilled water, after addition
of distilled water the wireless gently mixed
to ensure uniform mixing.
So, while the drugs are getting dissolved,
0.8 m l of the growth supplement is taken
and added to the 5 MGIT tubes .
This is required as it facilitates growth
of the mycobacterium tuberculosis complex
organisms . So, once all the tubes are added
with the growth supplement, the vial closed.
The next step will be to prepare the inoculum,
that is the culture for which the DST is to
be done.
Is mixed well by vortexing and is allowed
to stand for some time to allow the aerosols
to settle down .
Next point 1 m l of each drug is added to
the corresponding tube except the growth control
which is a drug free tube.
. So, the sequence of the drug containing
tubes will be streptomycin, isoniazid, rifampicin
and ethambutol in that order .
As the next step the culture is diluted one
in 100 in a 15 m l centrifuge tube using sterile
saline, and mix gently. 10 m l of saline is
taken 
to which 100 micro litre of the culture is
added the diluted culture is mixed gently
by inversion so, that the culture suspension
is uniformly distributed.
0.5 m l of the undiluted culture is added
to the drug containing tubes; all the drug
containing tubes receive 500 micro litre.
And 500 micro litre of the diluted culture
is added to the drug free growth control tube.
The tubes are now ready they are mixed using
gentle inversion .
The external surfaces of the tubes are wiped
clean with disinfectant.
he tubes are then placed in a five tube carrier
set with a barcode on one end. The growth
control tube is placed closest to the barcode,
and the remaining tubes are placed in the
order of streptomycin, isoniazid, rifampicin
and ethambutol. Next we will be seeing loading
of the tubes into the MGIT machine .
This is the bactec MGIT 960 system with the
display screen that has user friendly icons;
the system has three racks each capable of
holding up to 320 tubes.
The 5 tube rack containing the inoculated
tubes are now ready for loading into the MGIT
machine.
Here in the MGIT racks one of the racks is
opened and the insert icon is pressed which
activates the scanner.
The barcode on the five rack is scanned, and
immediately the system indicates the preposition
into which the tubes will have to be placed.
The rack is placed in position and the protocols
start button is pressed .
DST protocol extends for 14 days. Now once
the protocol is completed the system indicates
it by a flashing positive icon on the front
.
Here the display shows the DST has been completed
for 6 cultures.
Here the cultures that have completed the
protocol are removed, scanned and the output
is taken as a print out .
This particular printout shows the results
for a single culture. The results are interpreted
only when the growth control tube shows a
growth unit of 400 and the results are interpreted
automatically. Here all the vials or the all
the drugs that have been used are found to
be sensitive and if their found to show more
than 100 growth unit, they are called resistant
which will be indicated in the printout. With
this we come to the end of the video demonstration
of doing DST to first line anti TB drugs by
MGIT.
Thank you .
