4 Notes
1. All the methods herein described take into account safety conditions of work:
Harvest of mycobacterial cells must be conducted in a
biosafety cabinet using appropriate containment: Biosafety
Level 2 for opportunistic pathogens, e.g., M. avium; or Biosafety Level 3 for strict pathogens, e.g., M. tuberculosis. If
centrifugation is required, it must be equipped with aerosolcontainment rotors and tubes. For pathogens such as
M. tuberculosis, growth as surface pellicles on Sauton medium
[49] allows easy collection of cells without centrifugation. The
overnight contact with CHCl 3 /CH 3 OH solvent ensures killing of cells.
2. All experiments using solvents and chemicals must be conducted in a well-ventilated hood.
3. The rotary vacuum is an indispensable tool for lipid studies, but
it presents risks and needs some caution: isolation of the apparatus for protecting workers by means of a screen is an indispensable precaution, and, if possible, the rotary vacuum should
be placed under a well-ventilated hood. Checking and replacement of damaged glass parts have to be regularly done. A
careful examination of the round-bottom flasks before each
use is indispensable. A SpeedVac may usefully replace the rotary
evaporator in the purification of small quantities, e.g., LAM.
4. Due to the chain length (60–100 carbon atoms), mycolic acids
(MA) are insoluble in methanol. This property has been largely
used as a first enrichment step of MA-containing “free lipids”
such as TDM, TMM, GroMM, and MAME.
5. All materials, solvents and buffers used must be pyrogen-free.
Milli-Q water is not per se of sufficient quality. This is of
paramount importance if purified LAM is intended to be used
in functional assays. Removing contaminating LPS from LAM
is virtually impossible (even by the use of polymixin columns).
6. As the 48 h-contact with chloroform/methanol ensures killing
of cells, the subsequent experiments can be conducted outside
the safety laboratory.
7. This constitutes the so-called “free” lipids. The ratio of the
weight of “free” lipids versus that of bacterial delipidated cells
on the filter plus that of “free” lipid represents the percentage
of extractable “free” lipids of the cell dry weight.
8. According to the nature of the lipids to be detected, solvents
with different polarities should be used for TLC or Florisil
column:
142
Marie-Antoinette Lane ´ elle et al.
1. All the methods herein described take into account safety conditions of work:
Harvest of mycobacterial cells must be conducted in a
biosafety cabinet using appropriate containment: Biosafety
Level 2 for opportunistic pathogens, e.g., M. avium; or Biosafety Level 3 for strict pathogens, e.g., M. tuberculosis. If
centrifugation is required, it must be equipped with aerosolcontainment rotors and tubes. For pathogens such as
M. tuberculosis, growth as surface pellicles on Sauton medium
[49] allows easy collection of cells without centrifugation. The
overnight contact with CHCl 3 /CH 3 OH solvent ensures killing of cells.
2. All experiments using solvents and chemicals must be conducted in a well-ventilated hood.
3. The rotary vacuum is an indispensable tool for lipid studies, but
it presents risks and needs some caution: isolation of the apparatus for protecting workers by means of a screen is an indispensable precaution, and, if possible, the rotary vacuum should
be placed under a well-ventilated hood. Checking and replacement of damaged glass parts have to be regularly done. A
careful examination of the round-bottom flasks before each
use is indispensable. A SpeedVac may usefully replace the rotary
evaporator in the purification of small quantities, e.g., LAM.
4. Due to the chain length (60–100 carbon atoms), mycolic acids
(MA) are insoluble in methanol. This property has been largely
used as a first enrichment step of MA-containing “free lipids”
such as TDM, TMM, GroMM, and MAME.
5. All materials, solvents and buffers used must be pyrogen-free.
Milli-Q water is not per se of sufficient quality. This is of
paramount importance if purified LAM is intended to be used
in functional assays. Removing contaminating LPS from LAM
is virtually impossible (even by the use of polymixin columns).
6. As the 48 h-contact with chloroform/methanol ensures killing
of cells, the subsequent experiments can be conducted outside
the safety laboratory.
7. This constitutes the so-called “free” lipids. The ratio of the
weight of “free” lipids versus that of bacterial delipidated cells
on the filter plus that of “free” lipid represents the percentage
of extractable “free” lipids of the cell dry weight.
8. According to the nature of the lipids to be detected, solvents
with different polarities should be used for TLC or Florisil
column:
142
Marie-Antoinette Lane ´ elle et al.