11. “Swimming pool” apparatuses, if needed (Fig. 2) (see Note 3).
To construct them, open sterile 6-well plates inside of a biosafety cabinet and place a dollop of cyanoacrylate-based glue
into each well. Using sterile tweezers, place an autoclaved
15-mL centrifuge tube cap upside down on top of the glue.
Allow to dry overnight.
12. Bleach.
13. 70% ethanol.
2.2 Sample
Collection
1. Cold metabolite extraction buffer: freshly prepared solution of
acetonitrile:methanol:water in a ratio of 2:2:1 (v/v/v). Prepare
using LC-MS grade acetonitrile and methanol with fresh MilliQ 18 mΩ water (see Note 4).
2. Dry ice.
3. 2-mL bead-beating tubes (screw cap), filled with 0.1 mm zirconia/silica beads (Biospec) to ~300 μL.
4. Sterile tweezers.
5. Microcentrifuge tube cooling block.
2.3 Metabolite
Extraction
1. Temperature-controlled bead beater/homogenizer (Precellys).
2. Temperature-controlled microcentrifuge.
3. 0.22-μm Spin-X centrifuge filter tubes (VWR).
4. Sterile 1.5-mL microcentrifuge tubes (snap caps).
Fig. 2 Swimming pool plates for M. tuberculosis metabolomics. Inside of a
biosafety cabinet, autoclaved centrifuge tube caps are glued upside down into
the wells of a 6-well plate. After the glue dries, these plates can be used as
reservoirs for liquid medium containing different experimental conditions for
M. tuberculosis-laden filters
Metabolomics of Mycobacterium tuberculosis
583
To construct them, open sterile 6-well plates inside of a biosafety cabinet and place a dollop of cyanoacrylate-based glue
into each well. Using sterile tweezers, place an autoclaved
15-mL centrifuge tube cap upside down on top of the glue.
Allow to dry overnight.
12. Bleach.
13. 70% ethanol.
2.2 Sample
Collection
1. Cold metabolite extraction buffer: freshly prepared solution of
acetonitrile:methanol:water in a ratio of 2:2:1 (v/v/v). Prepare
using LC-MS grade acetonitrile and methanol with fresh MilliQ 18 mΩ water (see Note 4).
2. Dry ice.
3. 2-mL bead-beating tubes (screw cap), filled with 0.1 mm zirconia/silica beads (Biospec) to ~300 μL.
4. Sterile tweezers.
5. Microcentrifuge tube cooling block.
2.3 Metabolite
Extraction
1. Temperature-controlled bead beater/homogenizer (Precellys).
2. Temperature-controlled microcentrifuge.
3. 0.22-μm Spin-X centrifuge filter tubes (VWR).
4. Sterile 1.5-mL microcentrifuge tubes (snap caps).
Fig. 2 Swimming pool plates for M. tuberculosis metabolomics. Inside of a
biosafety cabinet, autoclaved centrifuge tube caps are glued upside down into
the wells of a 6-well plate. After the glue dries, these plates can be used as
reservoirs for liquid medium containing different experimental conditions for
M. tuberculosis-laden filters
Metabolomics of Mycobacterium tuberculosis
583