6. Potassium carbonate.
7. Silver nitrate.
8. Formaldehyde.
2.23 Analysis of LAM
Glycosyl and Fatty Acyl
Compositions
1. Glass vials with Teflon seals.
2. Air or nitrogen stream.
3. Gas-chromatograph fitted with a TG1MS fused-silica capillary
column (30 m  0.25 mm) and connected to an ISQ™ single
quadrupole mass spectrometer with electron energy of 70 eV.
4. Trifluoroacetic acid (TFA).
5. HCl.
6. NaOH.
7. Pyridine (see Note 2).
8. Hexamethyldisilazane.
9. Trimethylchlorosilane.
10. BF 3 in methanol (see Note 2).
11. Arabinose.
12. Mannose.
13. Mannoheptose.
14. Palmitic acid.
15. Pentadecanoic acid.
3 Methods
3.1 Extraction of
“Free Lipids”
1. Grow cells as surface pellicles (see Note 1) in appropriate media
in glass flasks [41, 49].
2. Harvest cells in the Biosafety laboratory (see Note 1) by pouring off the growth medium.
3. Add 30 mL of methanol followed by 15 mL of chloroform to
obtain a one-phase solvent system.
4. Shake the mixture gently to dissociate the clumps and let stand
at room temperature (RT) for 48 h (see Note 6).
5. Transfer the supernatant organic extract in a round-bottom
flask (250 mL) through a funnel containing a pre-weighed filter
paper circle (diameter 110 cm) (see Note 2).
6. Add 25 mL of methanol to cell residues followed by 25 mL of
chloroform.
7. Shake gently and let stand at RT overnight.
8. Repeat step 5.
Lipid and Lipoarabinomannan
123
7. Silver nitrate.
8. Formaldehyde.
2.23 Analysis of LAM
Glycosyl and Fatty Acyl
Compositions
1. Glass vials with Teflon seals.
2. Air or nitrogen stream.
3. Gas-chromatograph fitted with a TG1MS fused-silica capillary
column (30 m  0.25 mm) and connected to an ISQ™ single
quadrupole mass spectrometer with electron energy of 70 eV.
4. Trifluoroacetic acid (TFA).
5. HCl.
6. NaOH.
7. Pyridine (see Note 2).
8. Hexamethyldisilazane.
9. Trimethylchlorosilane.
10. BF 3 in methanol (see Note 2).
11. Arabinose.
12. Mannose.
13. Mannoheptose.
14. Palmitic acid.
15. Pentadecanoic acid.
3 Methods
3.1 Extraction of
“Free Lipids”
1. Grow cells as surface pellicles (see Note 1) in appropriate media
in glass flasks [41, 49].
2. Harvest cells in the Biosafety laboratory (see Note 1) by pouring off the growth medium.
3. Add 30 mL of methanol followed by 15 mL of chloroform to
obtain a one-phase solvent system.
4. Shake the mixture gently to dissociate the clumps and let stand
at room temperature (RT) for 48 h (see Note 6).
5. Transfer the supernatant organic extract in a round-bottom
flask (250 mL) through a funnel containing a pre-weighed filter
paper circle (diameter 110 cm) (see Note 2).
6. Add 25 mL of methanol to cell residues followed by 25 mL of
chloroform.
7. Shake gently and let stand at RT overnight.
8. Repeat step 5.
Lipid and Lipoarabinomannan
123