16. This is not the same as one round of filling wells with 60 μL. We
noticed that the larger volume leads to splashing of media and
cells onto the plate surface.
17. The risk of mold contamination increases due to the nature of a
long assay that has a 3-day drug exposure and 7–10 day outgrowth. It helps to periodically wipe tissue culture incubators
with bleach and ethanol, autoclave plate filler cassettes, wipe
outgrowth plate surfaces with 70% ethanol, use a fungicide in
incubator water baths, use 0.2 μm filters on all gas lines, and
employ exceptionally sterile technique, including wiping the
surfaces of all reagents and equipment with 70% ethanol prior
to putting into the biosafety hood. We have also noted a higher
rate of mold contamination using acoustically dispensed assay
ready plates, likely due to our acoustic machine not being
housed in a sterile hood.
18. The use of optical stickers for 384-well plates is not recommended for nonreplicating assays. We found that removing the
sticker to decontaminate the plate draws the M. tuberculosis
culture liquid out of the wells. This is likely related to the liquid
of each well approaching the top of the well. This is a serious
safety concern for those using wild-type M. tuberculosis.
19. Removing the lid, in combination with equilibrating the plate
temperature to room air, allows one to replace the lid on the
microplate without having condensation on the inner side of
the lid. This is essential to read OD 580 . If this is done with wildtype M. tuberculosis, the spectrophotometer has to be in a
glovebox in the event of a spill. For M. tuberculosis
ΔpanCDΔlysA, the spectrophotometer can be in the BSL2+ -
rated laboratory without extra precautions.
20. Sometimes, we have seen that cells do not grow evenly across
the bottom of our 384-well plates and this leads to the formation of a cell layer looking like “doughnuts” on the well bottoms. This is a difficult problem to solve. The best solution is to
ensure you use “tissue culture-treated” microplates, which
helps distribute the cell growth across well bottoms. Also,
sometimes when the replicating outgrowth medium is added
(as 2 Â 30 μL), the 384-well microplate can be rotated 180
after the first 30 μL is added in an effort to help mix the cells
adhering to the well bottoms. It helps to keep stacks as three to
four plates to control media wicking to the well surfaces.
21. Due to the equation used to calculate % inhibition, it is unnecessary to sacrifice a well to blank the plate.
22. In setting up a time-kill experiment, please note that in the
absence of a test agent, significant, multilog 10 kill may occur
after a >7 day incubation under nonreplicating conditions. We
recommend testing times between 0 and 7 days; if you are
Multi-stress Model for Nonreplicating M. tuberculosis
629
noticed that the larger volume leads to splashing of media and
cells onto the plate surface.
17. The risk of mold contamination increases due to the nature of a
long assay that has a 3-day drug exposure and 7–10 day outgrowth. It helps to periodically wipe tissue culture incubators
with bleach and ethanol, autoclave plate filler cassettes, wipe
outgrowth plate surfaces with 70% ethanol, use a fungicide in
incubator water baths, use 0.2 μm filters on all gas lines, and
employ exceptionally sterile technique, including wiping the
surfaces of all reagents and equipment with 70% ethanol prior
to putting into the biosafety hood. We have also noted a higher
rate of mold contamination using acoustically dispensed assay
ready plates, likely due to our acoustic machine not being
housed in a sterile hood.
18. The use of optical stickers for 384-well plates is not recommended for nonreplicating assays. We found that removing the
sticker to decontaminate the plate draws the M. tuberculosis
culture liquid out of the wells. This is likely related to the liquid
of each well approaching the top of the well. This is a serious
safety concern for those using wild-type M. tuberculosis.
19. Removing the lid, in combination with equilibrating the plate
temperature to room air, allows one to replace the lid on the
microplate without having condensation on the inner side of
the lid. This is essential to read OD 580 . If this is done with wildtype M. tuberculosis, the spectrophotometer has to be in a
glovebox in the event of a spill. For M. tuberculosis
ΔpanCDΔlysA, the spectrophotometer can be in the BSL2+ -
rated laboratory without extra precautions.
20. Sometimes, we have seen that cells do not grow evenly across
the bottom of our 384-well plates and this leads to the formation of a cell layer looking like “doughnuts” on the well bottoms. This is a difficult problem to solve. The best solution is to
ensure you use “tissue culture-treated” microplates, which
helps distribute the cell growth across well bottoms. Also,
sometimes when the replicating outgrowth medium is added
(as 2 Â 30 μL), the 384-well microplate can be rotated 180
after the first 30 μL is added in an effort to help mix the cells
adhering to the well bottoms. It helps to keep stacks as three to
four plates to control media wicking to the well surfaces.
21. Due to the equation used to calculate % inhibition, it is unnecessary to sacrifice a well to blank the plate.
22. In setting up a time-kill experiment, please note that in the
absence of a test agent, significant, multilog 10 kill may occur
after a >7 day incubation under nonreplicating conditions. We
recommend testing times between 0 and 7 days; if you are
Multi-stress Model for Nonreplicating M. tuberculosis
629