55- Larger volumes of cells can be stained by scaling up the quan-
tity of dye used. For example, 1 mL of cells can be stained by
the addition of 5 μL of the CV-AM stock solution and 10 μL
of SG stock solution (20 μM). For this volume of cells, optimal
staining is achieved through incubation at 37 °C for 1 h, whilst
shaking at 220 rpm. The cells are left overnight to fix them
prior to flow cytometry. - An un-stained cell sample is treated similarly to all stained sam-
ples to provide a control. - A fixation time of 30 min has been found to be sufficient for
the sterilisation of M. tuberculosis (at an OD 540 nm of 0.5) to
allow for removal from biosafety containment level 3 for flow
cytometry analyses, i.e., all organisms are expected to be non-
viable. Scientists should validate their own fixation step, locally,
with the medium and procedures that they use. - The published method [ 2 ] uses Summit software version 4.3
for analyses. - A description of each population gate can be found in Fig. 1.
- A worked example of the analysis process from gating strategy
to graphical representation of proportion of cells within each
gate can be found in Fig. 2.
Fig. 1 (a) Cartoon depicting the position of the four population gates on a two parameter dot-plot with each
event representing a cell in terms of its fluorescence in channel FL1 (SG) and FL6 (CV-AM). (b) Table describing
the population gates and the controls used for positioning the gates
A Flow Cytometry Method for Assessing M. tuberculosis Responses to Antibiotics