52
and binds to DNA. It will not cross intact membranes, but will eas-
ily penetrate compromised membranes that are characteristic of
dead cells. It exhibits more than a 500-fold fluorescence enhance-
ment upon binding nucleic acids after being excited with the blue
laser (detected in channel FL1). These dyes have been used to dual-
stain M. tuberculosis that has been exposed to a range of antibiotics
with different modes of action at different concentrations over time
[ 2 ]. Unlike colony counts that only capture information about bac-
teria that can be cultured on solid media, the flow cytometry analy-
ses potentially capture information about non-growing populations.
Recently, a combination of CV-AM and SYTOX-red was compared
with other dye pairs for their ability to visualize and quantify live/
dead populations during the first phase of bioadhesion in the for-
mation of oral bacterial biofilms in a study by Tawakoli et al. [ 3 ].
More traditional plating methods were unable to quantify viable
but non-culturable oral bacteria; using current approaches, over
50% of oral bacterial microbiome was un-culturable and CV-AM
allowed for a clear distinction between the different susceptibility
phenotypes within the biofilms [ 4 , 5 ]. Another recent study has
also shown that CV-AM combined with a microfluidic approach is
a useful tool for gaining insights into the metabolic activity of grow-
ing and non-growing Corynebacterium glutamicum [ 6 ]. The flow
cytometry approach has an additional advantage in that it provides
insight into the mode of action of the drug; antibiotics targeting
the cell wall give a distinctive flow cytometry profile compared to
those inhibiting intracellular processes.
This rapid drug susceptibility method could identify more
effective antimycobacterial agents, provide information about their
mode of action, and aid the acceleration through the drug devel-
opment pathway into the clinic.2 Materials
- CV-AM (Invitrogen, Life Technologies). Gently centrifuge
each tube of lyophilized CV-AM, received from the manufac-
turer to pellet the dye. Dissolve the pellet in 25 μL of DMSO.
Minimize exposure to UV light by wrapping the tube in foil.
Use freshly dissolved CV-AM on each occasion (see Note 1). - DMSO (Sigma Aldrich) (see Note 1).
- SG (Invitrogen, Life Technologies) (see Note 1) Dilute each
tube of SG stock solution received from the manufacturer from
the concentration of 5 mM to a working solution of 20 μM in
DMSO. Aliquot this and store in opaque vials at − 20 °C for up
to 6 months. Each vial used cannot be refrozen. - Hanks Balanced Salt Solution buffer (HBSS; ThermoFisher
Scientific).
2.1 Reagents for
Staining
Charlotte L. Hendon-Dunn et al.