4 Notes
1. We have established the cut-off for HTS assays as Z
0 of 0.4.
Plates with Z
0 values below this cut-off are rejected. This prevents too many false positives and “missing” a compound due
to false negatives. This Z
0 -factor is a key factor in the primary
assay when the compound is tested at single shot; however, in
dose-response assays we could be more flexible with the cutoff.
2. For compounds of unknown solubility, we use DMSO for
dissolution. Heating the sample or a sonication bath can help
to dissolve them.
3. Use as controls antitubercular drugs like isoniazid or fluoroquinolones (moxifloxacin, ciprofloxacin). Rifampicin and
B-lactams are unstable in the culture media, so it is not a
good control. The cell wall inhibitors could interfere in the
method of readout (resazurin or ATP).
4. Manual liquid handling is an appropriate alternative using multichannel pipettes with 8-, 12-, or 16-channel formats, for 96or 384-well plates.
5. We use a D300 Digital Dispenser (TECAN) to prepare the
assay plates directly from the stock solution. This process minimizes the dead volumes of compounds and eliminates the risk
of cross-contamination. The software is very friendly, and it is
possible to use easily for dose-response, synergy, and other
applications. This dispenser is able to dispense volumes from
picoliters to microliters, so it should be possible to work with
1536-well plates.
6. The concentration of compound depends on the MIC that you
are expecting for each compound, this value has to be in the
middle of the plate, in the middle of the range of concentration. For example, if the highest concentration that you have to
test is 125 μM, you have to add 12.5 mM (due to the 1:100
dilution factor from master plate to assay plate). The volume to
add depends on the number of assay plates that you are to
prepare as well as the volume required to perform the serial
dilutions.
7. An alternative way to test more compounds is to assay a single
concentration. The master plate is prepared in the same way
without performing the serial dilution. Different test compounds are added to each well of the plate (still excluding the
columns that are set aside for controls). Normally this process is
done when you have a lot of compounds with a low probability
of being active. Once the inactive compounds are discarded,
you can perform the dose-response curves with the active
compounds to determine the exact MIC or pIC50.
Antimicrobial Susceptibility Testing
645
1. We have established the cut-off for HTS assays as Z
0 of 0.4.
Plates with Z
0 values below this cut-off are rejected. This prevents too many false positives and “missing” a compound due
to false negatives. This Z
0 -factor is a key factor in the primary
assay when the compound is tested at single shot; however, in
dose-response assays we could be more flexible with the cutoff.
2. For compounds of unknown solubility, we use DMSO for
dissolution. Heating the sample or a sonication bath can help
to dissolve them.
3. Use as controls antitubercular drugs like isoniazid or fluoroquinolones (moxifloxacin, ciprofloxacin). Rifampicin and
B-lactams are unstable in the culture media, so it is not a
good control. The cell wall inhibitors could interfere in the
method of readout (resazurin or ATP).
4. Manual liquid handling is an appropriate alternative using multichannel pipettes with 8-, 12-, or 16-channel formats, for 96or 384-well plates.
5. We use a D300 Digital Dispenser (TECAN) to prepare the
assay plates directly from the stock solution. This process minimizes the dead volumes of compounds and eliminates the risk
of cross-contamination. The software is very friendly, and it is
possible to use easily for dose-response, synergy, and other
applications. This dispenser is able to dispense volumes from
picoliters to microliters, so it should be possible to work with
1536-well plates.
6. The concentration of compound depends on the MIC that you
are expecting for each compound, this value has to be in the
middle of the plate, in the middle of the range of concentration. For example, if the highest concentration that you have to
test is 125 μM, you have to add 12.5 mM (due to the 1:100
dilution factor from master plate to assay plate). The volume to
add depends on the number of assay plates that you are to
prepare as well as the volume required to perform the serial
dilutions.
7. An alternative way to test more compounds is to assay a single
concentration. The master plate is prepared in the same way
without performing the serial dilution. Different test compounds are added to each well of the plate (still excluding the
columns that are set aside for controls). Normally this process is
done when you have a lot of compounds with a low probability
of being active. Once the inactive compounds are discarded,
you can perform the dose-response curves with the active
compounds to determine the exact MIC or pIC50.
Antimicrobial Susceptibility Testing
645