interested in testing time points beyond 7 days, make sure to
plate appropriate dilutions that take into account extra killing
in control cells not exposed to test agent.
23. Even after vigorous mixing, we have observed cells by light
microscopy adhering tightly to the well bottoms of 96-well
plates. Extra care must be taken to ensure cells around the well
perimeters are adequately mixed.
24. This step presents an opportunity to set up the charcoal agar
resazurin assay (CARA) [31, 32]. By semiquantitatively determining the bactericidal impact of compounds on nonreplicating M. tuberculosis, the CARA can help identify the
approximate concentrations of compound and which time
points to use for CFU analysis (Subheading 3.4). Unlike the
standard outgrowth assay, the CARA is less influenced by
compound carryover during the replicating phase of the assay
due to sequestration of compound by activated charcoal. Thus,
in the same step, when 10 μL aliquots are being dispensed into
liquid outgrowth assay plates, another set of 10 μL aliquots can
be spotted onto solid CARA microplates. CARA microplates
are typically incubated for 10–14 postinoculation, after which
bacterial recovery on the solid surface is quantified by developing with resazurin and subsequent fluorescence reading using a
spectrophotometer.
25. The addition of acidified NaNO 2 often results in colonies on
agar plates that appear smaller than those that were not
exposed to NaNO 2 . Make sure after counting colonies to
reincubate the plates another week to ensure all surviving
bacteria had adequate time to grow and be enumerated.
26. If the DMSO-treated cells show multilog 10 killing by day
7 (compared to the inoculum counts) by a CFU assay: (1) confirm the pH of nonreplicating medium was 5.0 using a doubly
calibrated pH meter (using pH 4.0 and 7.0 standards with a
linear range > 97%) because NaNO2 at pH < 5 will produce
significantly more nitric oxide and reactive nitrogen species,
which can kill M. tuberculosis; (2) make sure to use the recommended BSA or OADC; and (3) periodically check the hypoxic
chamber calibration.
27. Another challenge in studying nonreplicating M. tuberculosis is
that they may exist in a state termed DD (differentially detectable [54]) in which they are undetectable as colony-forming
units (CFU) on solid media. Depending on the laboratory or
bacterial species, the DD state is also called “VBNC” (viable
but nonculturable) or “DC” (differentially culturable [55]).
DD M. tuberculosis are quantified by comparing the number of
viable cells recovered by a CFU assay with the number of viable
cells estimated using a limiting dilution assay coupled with a
630
Ben Gold et al.
plate appropriate dilutions that take into account extra killing
in control cells not exposed to test agent.
23. Even after vigorous mixing, we have observed cells by light
microscopy adhering tightly to the well bottoms of 96-well
plates. Extra care must be taken to ensure cells around the well
perimeters are adequately mixed.
24. This step presents an opportunity to set up the charcoal agar
resazurin assay (CARA) [31, 32]. By semiquantitatively determining the bactericidal impact of compounds on nonreplicating M. tuberculosis, the CARA can help identify the
approximate concentrations of compound and which time
points to use for CFU analysis (Subheading 3.4). Unlike the
standard outgrowth assay, the CARA is less influenced by
compound carryover during the replicating phase of the assay
due to sequestration of compound by activated charcoal. Thus,
in the same step, when 10 μL aliquots are being dispensed into
liquid outgrowth assay plates, another set of 10 μL aliquots can
be spotted onto solid CARA microplates. CARA microplates
are typically incubated for 10–14 postinoculation, after which
bacterial recovery on the solid surface is quantified by developing with resazurin and subsequent fluorescence reading using a
spectrophotometer.
25. The addition of acidified NaNO 2 often results in colonies on
agar plates that appear smaller than those that were not
exposed to NaNO 2 . Make sure after counting colonies to
reincubate the plates another week to ensure all surviving
bacteria had adequate time to grow and be enumerated.
26. If the DMSO-treated cells show multilog 10 killing by day
7 (compared to the inoculum counts) by a CFU assay: (1) confirm the pH of nonreplicating medium was 5.0 using a doubly
calibrated pH meter (using pH 4.0 and 7.0 standards with a
linear range > 97%) because NaNO2 at pH < 5 will produce
significantly more nitric oxide and reactive nitrogen species,
which can kill M. tuberculosis; (2) make sure to use the recommended BSA or OADC; and (3) periodically check the hypoxic
chamber calibration.
27. Another challenge in studying nonreplicating M. tuberculosis is
that they may exist in a state termed DD (differentially detectable [54]) in which they are undetectable as colony-forming
units (CFU) on solid media. Depending on the laboratory or
bacterial species, the DD state is also called “VBNC” (viable
but nonculturable) or “DC” (differentially culturable [55]).
DD M. tuberculosis are quantified by comparing the number of
viable cells recovered by a CFU assay with the number of viable
cells estimated using a limiting dilution assay coupled with a
630
Ben Gold et al.