Parafilm can be used as a seal but only as a temporary measure
for a maximum of 30 min outside an anaerobic chamber. It is
imperative for the plate seal that it must be oxygen impermeable, non-reactive, and transparent to allow spectrophotometric readings. The ClearVue seal (Molecular Dimensions, UK)
meets all these requirements.
4. Anaerobic chambers are commonly used clinically and in
research for the growth and maintenance of obligate and facultative anaerobic microorganisms. It provides a reliable and
consistent anaerobic atmosphere for working and incubating
and eliminates a lot of the difficulties around using an anaerobic
jar or nitrogen bag. It also facilitates a more efficient entry into
NRP as the mycobacteria use up the oxygen within the
medium; it is not replenished providing a fully hypoxic culture
throughout.
5. The cholesterol must be added very slowly to the mixture to
dissolve. Add 5 mg of cholesterol to the mixture at a time. Keep
at 65
C in a heating block (if using a microcentrifuge tube,
monitor the lid to ensure the lid does not pop open). Interchange the heating with vortexing for 1 min and place back on
heat for 5 min. Then, mix for 30 s in a sonicating water bath
and place immediately back on the heat block. As soon as the
cholesterol visibly dissolves, add the next lot of 5 mg cholesterol. Once 75% of the cholesterol has dissolved, this process
will slow down significantly.
6. Methylene Blue is sky blue in oxygenated cultures. Conversely,
when the culture is hypoxic, it turns colorless. This visual
indicator of hypoxia is useful for identifying when a culture is
starting to enter NRP I, this should take place around 32–48 h
after being placed in anaerobic conditions. Likewise, if there is
any leak in the seal, the culture will begin to return to a blue
coloration. Methylene blue can also be a preliminary indicator
of cell lysis. If the culture permitted to remain stationary for
approximately 10 min, a pellet of cells will appear at the bottom
of the culture tube. If this pellet is blue, it suggests that the cells
have lysed. This can be due to drug action and is a useful tool to
discover when entry to hypoxia has been too fast (for example,
if the screw cap lid is taken off in the anaerobic cabinet).
7. Leaving the screw cap lids on, but loose, allows a slow exchange
of oxygen which will facilitate a slow shiftdown into the NRP
state. This should take place between 48 and 72 h after entry to
the anaerobic cabinet for the hypoxia-induced rich media NRP
assay.
8. It is imperative to ensure the culture has reached NRP stage I
before treatment with antimicrobials. Generally, if a culture has
been in the anaerobic cabinet for 24 h and the methylene blue
In vitro Drug Screening of Hypoxic NRP Mycobacteria
257
for a maximum of 30 min outside an anaerobic chamber. It is
imperative for the plate seal that it must be oxygen impermeable, non-reactive, and transparent to allow spectrophotometric readings. The ClearVue seal (Molecular Dimensions, UK)
meets all these requirements.
4. Anaerobic chambers are commonly used clinically and in
research for the growth and maintenance of obligate and facultative anaerobic microorganisms. It provides a reliable and
consistent anaerobic atmosphere for working and incubating
and eliminates a lot of the difficulties around using an anaerobic
jar or nitrogen bag. It also facilitates a more efficient entry into
NRP as the mycobacteria use up the oxygen within the
medium; it is not replenished providing a fully hypoxic culture
throughout.
5. The cholesterol must be added very slowly to the mixture to
dissolve. Add 5 mg of cholesterol to the mixture at a time. Keep
at 65
C in a heating block (if using a microcentrifuge tube,
monitor the lid to ensure the lid does not pop open). Interchange the heating with vortexing for 1 min and place back on
heat for 5 min. Then, mix for 30 s in a sonicating water bath
and place immediately back on the heat block. As soon as the
cholesterol visibly dissolves, add the next lot of 5 mg cholesterol. Once 75% of the cholesterol has dissolved, this process
will slow down significantly.
6. Methylene Blue is sky blue in oxygenated cultures. Conversely,
when the culture is hypoxic, it turns colorless. This visual
indicator of hypoxia is useful for identifying when a culture is
starting to enter NRP I, this should take place around 32–48 h
after being placed in anaerobic conditions. Likewise, if there is
any leak in the seal, the culture will begin to return to a blue
coloration. Methylene blue can also be a preliminary indicator
of cell lysis. If the culture permitted to remain stationary for
approximately 10 min, a pellet of cells will appear at the bottom
of the culture tube. If this pellet is blue, it suggests that the cells
have lysed. This can be due to drug action and is a useful tool to
discover when entry to hypoxia has been too fast (for example,
if the screw cap lid is taken off in the anaerobic cabinet).
7. Leaving the screw cap lids on, but loose, allows a slow exchange
of oxygen which will facilitate a slow shiftdown into the NRP
state. This should take place between 48 and 72 h after entry to
the anaerobic cabinet for the hypoxia-induced rich media NRP
assay.
8. It is imperative to ensure the culture has reached NRP stage I
before treatment with antimicrobials. Generally, if a culture has
been in the anaerobic cabinet for 24 h and the methylene blue
In vitro Drug Screening of Hypoxic NRP Mycobacteria
257