9. Extract the bacterial pellet a third time with 50 mL chloroform/methanol 2:1 (v/v). Shake gently and stand at RT
overnight.
10. Repeat step 5, but transfer the entire bacterial cell suspension
through the filter paper funnel.
11. Allow the delipidated bacterial cells to dry at RT in the hood.
12. Weigh the delipidated cells.
13. Pool the organic phases from steps 3, 6, and 9 (see Note 2).
14. Evaporate the solvents using a rotary vacuum evaporator
(or under air or nitrogen stream when volumes of only a few
mL are used) (see Note 3).
15. Dissolve the pooled lipid extract in a minimum of chloroform.
16. Add an equal volume of distilled water.
17. Transfer the lipid extract in a pre-weighed glass tube.
18. Dry under air or nitrogen stream.
19. Weigh the lipid extract that represents total “free lipid” (see
Note 7).
3.2 Characterization
of Free Lipids by TLC
Due to the toxicity of the reagents used, conduct all steps in a wellventilated hood, except for the migration of TLC where the hood
ventilation has to be stopped to avoid turbulence in migration.
1. Dissolve the dried lipid extract (from Subheading 3.1, step 17)
in chloroform at an approximate concentration of 20 mg/mL.
2. Spot around 50 μL on the TLC plate using a Pasteur pipette or
a glass syringe.
3. Prepare solvents with different polarities according to the
nature of the lipids to be detected (see Note 2).
4. Run the TLC with an appropriate solvent (see Note 8) and dry
the plates at RT.
5. Stain the TLC using specific sprays adapted to the lipid to be
detected (see Note 9).
TLCs of representative lipids are shown in Fig. 3C, D, E.
3.3 Preparation of
Fatty Acid Methyl
Esters FAME
and MAME
Most of the mycobacterial fatty acids are found engaged by ester
linkage and their study needs saponification to yield the MA esterifying both complex lipids (e.g., GroMM, TDM, TMM) and arabinogalactan termini, and straight-chain and methyl-branched fatty
acids from “free lipids” e.g., phospholipids, PDIM, SGL, PPT (see
Note 10a).
1. Add 2 mL of a mixture of 40% KOH in water and methoxyethanol (1:7 v/v) to whole cells (surface pellicles from Subheading 3.1, step 2) or delipidated cells (from Subheading 3.1,
step 10).
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overnight.
10. Repeat step 5, but transfer the entire bacterial cell suspension
through the filter paper funnel.
11. Allow the delipidated bacterial cells to dry at RT in the hood.
12. Weigh the delipidated cells.
13. Pool the organic phases from steps 3, 6, and 9 (see Note 2).
14. Evaporate the solvents using a rotary vacuum evaporator
(or under air or nitrogen stream when volumes of only a few
mL are used) (see Note 3).
15. Dissolve the pooled lipid extract in a minimum of chloroform.
16. Add an equal volume of distilled water.
17. Transfer the lipid extract in a pre-weighed glass tube.
18. Dry under air or nitrogen stream.
19. Weigh the lipid extract that represents total “free lipid” (see
Note 7).
3.2 Characterization
of Free Lipids by TLC
Due to the toxicity of the reagents used, conduct all steps in a wellventilated hood, except for the migration of TLC where the hood
ventilation has to be stopped to avoid turbulence in migration.
1. Dissolve the dried lipid extract (from Subheading 3.1, step 17)
in chloroform at an approximate concentration of 20 mg/mL.
2. Spot around 50 μL on the TLC plate using a Pasteur pipette or
a glass syringe.
3. Prepare solvents with different polarities according to the
nature of the lipids to be detected (see Note 2).
4. Run the TLC with an appropriate solvent (see Note 8) and dry
the plates at RT.
5. Stain the TLC using specific sprays adapted to the lipid to be
detected (see Note 9).
TLCs of representative lipids are shown in Fig. 3C, D, E.
3.3 Preparation of
Fatty Acid Methyl
Esters FAME
and MAME
Most of the mycobacterial fatty acids are found engaged by ester
linkage and their study needs saponification to yield the MA esterifying both complex lipids (e.g., GroMM, TDM, TMM) and arabinogalactan termini, and straight-chain and methyl-branched fatty
acids from “free lipids” e.g., phospholipids, PDIM, SGL, PPT (see
Note 10a).
1. Add 2 mL of a mixture of 40% KOH in water and methoxyethanol (1:7 v/v) to whole cells (surface pellicles from Subheading 3.1, step 2) or delipidated cells (from Subheading 3.1,
step 10).
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