3. Activate vacuum and sterilize filtration apparatus using bleach
followed by 70% ethanol. Allow to dry completely.
4. Place filter on holder and slowly pipette 1 mL of liquid bacterial
culture onto filter. Liquid medium is drawn through the filter,
leaving bacterial cells deposited in a uniform layer on the filter
surface (see Note 6).
5. Remove filter from apparatus with sterile tweezers, and place
on a 7H10 plate with the bacterial layer facing away from the
agar. Three filters can be grown per agar plate.
6. Grow at 37
C for 5 days to accrue sufficient biomass for
metabolite detection (see Note 7).
7. After 5 days of growth, use sterile tweezers to transfer filters
from agar plates to swimming pool caps filled with liquid
medium (with the bacterial lawn facing up).
8. Incubate for the desired period of time before collecting
samples.
Fig. 3 Filtration apparatus for M. tuberculosis metabolomics. A vacuum flask is
attached to a filter stand to provide the base for the inoculation of bacteria onto
filters. Once the apparatus is sterilized, a filter is placed on the stand, and 1 mL
of bacterial culture is slowly pipetted on top of the filter. Liquid medium is drawn
through the filter and into the flask, leaving a lawn of M. tuberculosis deposited
on the surface. Bacteria-laden filters are then transferred onto 7H10 agar plates
for growth
Metabolomics of Mycobacterium tuberculosis
585
followed by 70% ethanol. Allow to dry completely.
4. Place filter on holder and slowly pipette 1 mL of liquid bacterial
culture onto filter. Liquid medium is drawn through the filter,
leaving bacterial cells deposited in a uniform layer on the filter
surface (see Note 6).
5. Remove filter from apparatus with sterile tweezers, and place
on a 7H10 plate with the bacterial layer facing away from the
agar. Three filters can be grown per agar plate.
6. Grow at 37
C for 5 days to accrue sufficient biomass for
metabolite detection (see Note 7).
7. After 5 days of growth, use sterile tweezers to transfer filters
from agar plates to swimming pool caps filled with liquid
medium (with the bacterial lawn facing up).
8. Incubate for the desired period of time before collecting
samples.
Fig. 3 Filtration apparatus for M. tuberculosis metabolomics. A vacuum flask is
attached to a filter stand to provide the base for the inoculation of bacteria onto
filters. Once the apparatus is sterilized, a filter is placed on the stand, and 1 mL
of bacterial culture is slowly pipetted on top of the filter. Liquid medium is drawn
through the filter and into the flask, leaving a lawn of M. tuberculosis deposited
on the surface. Bacteria-laden filters are then transferred onto 7H10 agar plates
for growth
Metabolomics of Mycobacterium tuberculosis
585