53- Formaldehyde (Scientific Laboratory Supplies).
- Cyan ADP flow cytometer (Beckman Coulter) (see Note 2).
- Sigmaplot graphical and data analysis software version 13.0
(Systat Inc.) (see Note 3).
3 Methods
- Prepare individual batch cultures by inoculating 5 mL of CMM
Mod2 medium [ 7 ] with cells from a mid-exponential culture
of Mycobacterium tuberculosis to achieve an OD 540 nm of 0.05. - To each culture, add antibiotic to achieve a series of concentra-
tions that range from sub-inhibitory levels to several multiples of
the expected minimum inhibitory concentration (MIC). The
range of concentrations will be dependent on the antibiotic used.
Include a control culture that contains no antibiotic. For isonia-
zid, the published method has used 0 μgmL-1, 0.25 μgmL-1,
0.5 μgmL-1 (MIC), 1 μgmL-1, 2 μgmL-^1 , 4 μgmL-1, 8 μgmL-1,
16 μgmL-1, and 32 μgmL-1. - Incubate the batch cultures at 37 °C with shaking at 200 rpm.
Every 24 h, sample 450 μL of culture for CV-AM/SG staining. - Perform counts of colony forming units alongside the staining,
to assess whether cells could be cultured on solid medium, by
performing serial decimal dilutions and plating onto
Middlebrook 7H10 agar + OADC [ 8 ]. - Adjust the cell sample to an OD540nm of 0.05 by diluting the
cells in the growth medium that has been used in culture. - Stain 100 μL of bacteria with 0.5 μL CV-AM and 1 μL SG
(20 μM) in each well of a 96-well microtiter plate and incubate
at 37 °C for 1 h in the dark (see Notes 4 and 5 ). - After staining, spin the bacteria by centrifugation at 2885 × g
for 2 min and resuspend in 100 μL HBSS containing a final
concentration of 4% formaldehyde (v/v) (see Note 6). - Examine bacteria using a flow cytometer that possesses lasers
with excitatory wavelengths of 488 nm and 405 nm (see Note 2). - Detect SG fluorescence emission (ex/em 488 nm/523 nm) in
channel FL1 [530/40 band pass (BP)], and CV-AM fluores-
cence (ex/em 400/452 nm) in channel FL6 (450/50 BP). - Analyse un-stained control samples to set a population gate
around the bacteria to be analysed by using the forward scatter
versus side scatter parameters. - Adjust voltages in channels FL1 (SG) and FL6 (CV-AM) so
that the fluorescence histogram of the un-stained bacteria
3.1 Drug
Susceptibility
Assessment
3.2 Staining
3.3 Flow Cytometry
A Flow Cytometry Method for Assessing M. tuberculosis Responses to Antibiotics