with 1:1 chloroform:methanol before use. Polytetrafluoroethylene (PTFE) caps must be used with screw glass tubes.
Solvents. All solvents should be HPLC-grade and freshly
prepared.
5. Addition of a known quantity of non-mycobacterial lipid standard, such as phosphatidylcholine, for example, to the bacteria
pellet allows controlling the overall lipid extraction process and
to normalize comparative analyses. The intensity of the MS
signal of the chosen standard should be within the intensity
range of the analytes of interest. The determination of the
response factor of the standard in the used LC-MS setup will
determine the suitable amount of standard to be added.
6. The tip of the plastic pipette must not contact the chloroform:
methanol solution when transferring the methanolic bacteria
solution. All following steps of the extraction protocol must be
performed in a chemical hood.
7. When manipulating live virulent M. tuberculosis strains, steps
1–7 are performed in BSL3 facility. Contacting the bacterial
pellet with chloroform:methanol mixture also aims at killing
the bacteria before pursuing the protocol outside of the BSL3
facility. Successful sterilization relies on the ratio wet pellet/
chloroform:methanol. From 50 mL of mycobacterium culture,
recovered wet pellet is typically 1 mL. A ratio of 4 parts
solvent to 1 part wet pellet (v/v) is expected to sterilize the
sample overnight. In any case, when setting up this protocol,
the sterilization conditions must be validated by a controlled
experiment showing the absence of colony forming unit when
plating the so-called “sterile” pellet on Petri dish. Once the
sample is sterilized, spray the outside of the glass container with
tuberculocidal agent and continue the protocol outside of the
BSL3 facility in a chemical hood using glass pipettes or syringes
and rinsed glassware as described in Note 4.
8. About 1–1.5 mg of dry lipids is usually recovered from 50 mL
of log phase culture. If normalization of experiments is later
carried out on the quantity of injected lipid extract, weight
measurement must be as reliable as possible, which requires
the complete evaporation of the solvents. Repeat weighing of
the dried lipid film on a microbalance 20 min later and ensure a
constant weight is reached; otherwise, desiccation time must be
prolonged. Drying under vacuum might be necessary depending on the amount of lipid extract.
9. Best results are obtained with freshly prepared extracts. Lipid
extracts should not be stored in a dry state. Oxidative degradation is slower in solutions (up to 1 year). Dichloromethane is
more stable than chloroform for long-term storage, which
should take place in a flammable storage laboratory freezer.
Air and light should be avoided. To minimize the cleavage of
572
Emilie Layre
Solvents. All solvents should be HPLC-grade and freshly
prepared.
5. Addition of a known quantity of non-mycobacterial lipid standard, such as phosphatidylcholine, for example, to the bacteria
pellet allows controlling the overall lipid extraction process and
to normalize comparative analyses. The intensity of the MS
signal of the chosen standard should be within the intensity
range of the analytes of interest. The determination of the
response factor of the standard in the used LC-MS setup will
determine the suitable amount of standard to be added.
6. The tip of the plastic pipette must not contact the chloroform:
methanol solution when transferring the methanolic bacteria
solution. All following steps of the extraction protocol must be
performed in a chemical hood.
7. When manipulating live virulent M. tuberculosis strains, steps
1–7 are performed in BSL3 facility. Contacting the bacterial
pellet with chloroform:methanol mixture also aims at killing
the bacteria before pursuing the protocol outside of the BSL3
facility. Successful sterilization relies on the ratio wet pellet/
chloroform:methanol. From 50 mL of mycobacterium culture,
recovered wet pellet is typically 1 mL. A ratio of 4 parts
solvent to 1 part wet pellet (v/v) is expected to sterilize the
sample overnight. In any case, when setting up this protocol,
the sterilization conditions must be validated by a controlled
experiment showing the absence of colony forming unit when
plating the so-called “sterile” pellet on Petri dish. Once the
sample is sterilized, spray the outside of the glass container with
tuberculocidal agent and continue the protocol outside of the
BSL3 facility in a chemical hood using glass pipettes or syringes
and rinsed glassware as described in Note 4.
8. About 1–1.5 mg of dry lipids is usually recovered from 50 mL
of log phase culture. If normalization of experiments is later
carried out on the quantity of injected lipid extract, weight
measurement must be as reliable as possible, which requires
the complete evaporation of the solvents. Repeat weighing of
the dried lipid film on a microbalance 20 min later and ensure a
constant weight is reached; otherwise, desiccation time must be
prolonged. Drying under vacuum might be necessary depending on the amount of lipid extract.
9. Best results are obtained with freshly prepared extracts. Lipid
extracts should not be stored in a dry state. Oxidative degradation is slower in solutions (up to 1 year). Dichloromethane is
more stable than chloroform for long-term storage, which
should take place in a flammable storage laboratory freezer.
Air and light should be avoided. To minimize the cleavage of
572
Emilie Layre