properties of a buffer formulation for extracting ATP from
bacteria [6]. The signal for cell wall synthesis inhibitors is
higher than the expected since at sub-inhibitory concentrations they facilitate bacterial lysis and ATP release. In this
case, the MIC could be underestimated.
17. It is essential to equilibrate the plates and the BacTiter-Glo™
reagent at room temperature to achieve better sensitivity.
Insufficient equilibration leads to the decay of the luminescent
signal.
18. We have observed variability in the signal between batches of
BacTiter-Glo™ so we advise testing each new batch before
starting a HTS campaign in order.
19. The amount of BacTiter-Glo™ reagent used is optimized to
develop the luminescent signal and save reagent. We use 2.5fold less volume than recommended by the manufacturer
(10 μL instead of 25 μL).
20. Incubation time with the BacTiter-Glo™ for different mycobacteria species should be determined in order to obtain the
best sensitivity. For each strain determine the time at which the
highest level of luminescence is achieved. by running a kinetic
curve of luminescence to determine the optimal
incubation time.
21. The integration time could vary from machine to machine. The
recommendation to set this time in a new instrument is to
perform a ATP titration and measure with a range of integration time, choosing the time that gives you the largest signal/
noise ratio.
22. Bubbles interfere with the readout. If bubbles have been produced, a quick spin can remove them.
23. In the single cell suspension step, after the glass bead treatment, it is important to add more volume than the final
needed. For instance, if you need 5 mL from every tube, add
6 mL and take only 5 mL. This will ensure that the bigger
clumps are discarded and the subsequent centrifugation will
remove the remaining clumps.
24. Different luminometers can be uses. Calibrate the instrument
before use to find the best conditions for the strains used
(H37Rv:luc). This can be done with extracellular bacteria;
run a kinetic measurement to determine for the optimum
time after the addition of the reagents and test different integration times.
For example, EnVision Multilabel Reader from Perkin
Elmer is as follows:
(a) Labels: US LUM 384 (cps) 7000004, measurement
height 0 nm, measurement time 0.1 s, CT 0.
Antimicrobial Susceptibility Testing
647
bacteria [6]. The signal for cell wall synthesis inhibitors is
higher than the expected since at sub-inhibitory concentrations they facilitate bacterial lysis and ATP release. In this
case, the MIC could be underestimated.
17. It is essential to equilibrate the plates and the BacTiter-Glo™
reagent at room temperature to achieve better sensitivity.
Insufficient equilibration leads to the decay of the luminescent
signal.
18. We have observed variability in the signal between batches of
BacTiter-Glo™ so we advise testing each new batch before
starting a HTS campaign in order.
19. The amount of BacTiter-Glo™ reagent used is optimized to
develop the luminescent signal and save reagent. We use 2.5fold less volume than recommended by the manufacturer
(10 μL instead of 25 μL).
20. Incubation time with the BacTiter-Glo™ for different mycobacteria species should be determined in order to obtain the
best sensitivity. For each strain determine the time at which the
highest level of luminescence is achieved. by running a kinetic
curve of luminescence to determine the optimal
incubation time.
21. The integration time could vary from machine to machine. The
recommendation to set this time in a new instrument is to
perform a ATP titration and measure with a range of integration time, choosing the time that gives you the largest signal/
noise ratio.
22. Bubbles interfere with the readout. If bubbles have been produced, a quick spin can remove them.
23. In the single cell suspension step, after the glass bead treatment, it is important to add more volume than the final
needed. For instance, if you need 5 mL from every tube, add
6 mL and take only 5 mL. This will ensure that the bigger
clumps are discarded and the subsequent centrifugation will
remove the remaining clumps.
24. Different luminometers can be uses. Calibrate the instrument
before use to find the best conditions for the strains used
(H37Rv:luc). This can be done with extracellular bacteria;
run a kinetic measurement to determine for the optimum
time after the addition of the reagents and test different integration times.
For example, EnVision Multilabel Reader from Perkin
Elmer is as follows:
(a) Labels: US LUM 384 (cps) 7000004, measurement
height 0 nm, measurement time 0.1 s, CT 0.
Antimicrobial Susceptibility Testing
647