REVIEW
published: 08 August 2016
doi: 10.3389/fmicb.2016.01225
Frontiers in Microbiology | www.frontiersin.org 1 August 2016 | Volume 7 | Article 1225
Edited by:
Christophe Nguyen-The,
Institut National de la Recherche
Agronomique, France
Reviewed by:
Kiiyukia Matthews Ciira,
Mount Kenya University, Kenya
Marielle Bouix,
Agro ParisTech, France
*Correspondence:
Nadia Oulahal
nadia.oulahal@univ-lyon1.fr
Specialty section:
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 17 May 2016
Accepted: 22 July 2016
Published: 08 August 2016
Citation:
Léonard L, Bouarab Chibane L, Ouled
Bouhedda B, Degraeve P and
Oulahal N (2016) Recent Advances on
Multi-Parameter Flow Cytometry to
Characterize Antimicrobial Treatments.
Front. Microbiol. 7:1225.
doi: 10.3389/fmicb.2016.01225
Recent Advances on Multi-Parameter
Flow Cytometry to Characterize
Antimicrobial Treatments
Lucie Léonard, Lynda Bouarab Chibane, Balkis Ouled Bouhedda, Pascal Degraeve and
Nadia Oulahal
*
Univ Lyon, Université Claude Bernard Lyon 1, ISARA Lyon, BioDyMIA (Bioingénierie et Dynamique Microbienne aux
Interfaces Alimentaires), Equipe Mixte d’Accueil n
◦
3733, IU T Lyon 1, Bourg en Bresse, France
The investigation on antimicrobial mechanisms is a challenging and crucial issue in the
fields of food or clinical microbiology, as it constitutes a prerequisite to the development
of new antimicrobial processes or compounds, as well as to anticipate phenomenon
of microbial resistance. Nowadays it is accepted that a cells population exposed to a
stress can cause the appearance of different cell populations and in particular sub-lethally
compromised cells which could be defined as viable but non-culturable (VBNC). Recent
advances on flow cytometry (FCM) and especially on multi-parameter flow cytometry
(MP-FCM) provide the opportunity to obtain high-speed information at real time on
damage at single-cell level. This review gathers MP-FCM methodologies based on
individual and simultaneous staining of microbial cells employed to investigate their
physiological state following different physical and chemical antimicrobial treatments.
Special attention will be paid to recent studies exploiting the possibility to corroborate
MP-FCM results with additional techniques (plate counting, microscopy, spectroscopy,
molecular biology techniques, membrane modeling) in order to elucidate the antimicrobial
mechanism of action of a given antimicrobial treatment or compound. The combination
of MP-FCM methodologies with these additional methods is namely a promising
and increasingly used approach to give further insight in differences in microbial
sub-population evolutions in response to antimicrobial treatments.
Keywords: multi-parameter flow cytometry, microorganisms, antimicrobial treatment, double-staining,
antimicrobial mechanism, viability, culturability
INTRODUCTION
Inactivation of microorganisms by physical treatments [heat, Ultra-Violet (UV) light irradiation,
supercritical carbon dioxide, high hydrostatic pressure,.. . ] or by the action of antimicrobial
compounds (biocides, organic acids, peptides, essential oils,. . . ) can result from several
mechanisms: inhibition of cell wall synthesis, disruption of the cytoplasmic membrane, binding
to DNA, inhibition of protein synthesis, or anti-metabolite activity (
Lee et al., 2015). To develop
new antimicrobial processes or compounds in food or medical microbiology, an understanding of
their mechanisms of action is vital in order to apply them best and to anticipate microbial resistance
phenomenon.
Over the years, several methods have been developed to measure viability and vitality of
microbes under various stresses: plating, slide culture, vital stains, metabolic activity monitoring,
Léonard et al. MP-FCM to Characterize Antimicrobial Treatments
cell components monitoring , fermentation capacity, acidification
potential, or oxygen uptake ability (Hayouni et al., 2008). Such
methods are time-consuming and labor intensive (
Wilkinson,
2015
). Besides, when classical plate count method is used to
assess survival of a bacterial population after exposure to an
environmental stress, the viability is determined by counting
live cells which are those that managed to replicate under
the particular experimental conditions, while all the others
will be presumed dead (
Hayouni et al., 2008). Nowadays, it
is well-documented that, under stress conditions a population
will exhibit cell subpopulations with phenotypes that most
likely escape t hi s logic. Environmental stresses can trigger the
occurrence of certain cell populations, called viable but non-
culturable cells (VBNC), which were stressed and lost their
ability to grow on agar medium, but still showed metabolic
activity (Ananta et al., 2004). Quantification of injured cells
is a great concern for microbiologists, as this subpopulation
might be critical if cells can recover and re vert to their
physiologically active condition (
Ayari et a l., 2013). For these
reasons, fluorescence techniques combined with direct optical
detection met hods for the rapid assessment of bacterial viability
have been increasingly favored for about 10 years.
Among t hese techniques, multi-parameter flow cytometry
(MP-FCM) has been shown to be a powerful tool for rapidly
analyzing populations on a cell-by-cell basis and can be
applied in many areas of food or medical microbiology
(detection of pathogenic bacteria, monitoring lactic acid bacteria
fermentation, rapid microbiological analysis of drinking water;
Schenk et al., 2011). A flow cytometer can be described as
an “automatic microscope” with the advantages of objectivity,
high analysis rate, preci sion and sensitivity (
Díaz et al., 2010).
The principle is that particles in suspension are pumped
into a narrow flow stream intersected by one or more laser
beams. Single particles, such as microbial cells, are illuminated
individually with the resulting light scatter and fluorescence
emission detected at appropriate wavelengths (Bridier et al.,
2015). A very large number of particles can be measured, 5000
cells per second in common and even up to 100,000 in specialized
instruments, measuring multiple cellular parameters on each cell
simultaneously (Díaz et al., 2010). Each individual cell can be
characterized based on its fluorescence color, the intensity of the
fluorescence signal, as well as the size, shape and granularity
of the particles (
Bridier et al., 2015). This method is highly
compatible with a broad range of fluorescent stains and cell
labeling methods. Díaz et al. (2010) detailed principles and
instrumentation through schematic descriptions in their review
about application of flow cytometry (FCM) to monitor industrial
microbial bioprocess.
Firstly FCM developments occurred in h uman clinical
applications especially for immunological analysis (Wilkinson,
2015). For less than 20 years, FCM has become an indispensable
tool to the complex area of microbiology. In 2000, a set
of publications in the Journal of Microbiological Methods
presented cytometry for bacteria (Nebe-von-Caron et al., 2000;
Shapiro, 2000; Steen, 2000
). More recently, these first data were
supplemented by a review of Tracy et al. (2010). The ability
to use FCM to visualize, enumerate and analyze a population
of cells into subpopulations of varying physiological status is
a valuable aid to understanding this intricate area for the
microbiologists.
Bridier et al. (2015) reviewed the applications
in food microbiology such as study of food bacteria function,
detection of food microbial communities or detection and
persistence of food pathogens. Moreover FCM can be used to
elucidate antimicrobial mechanism in food or health domain.
Recently,
Mathur et al. (2016) published a review about FCM
as a tool to study the effects of bacteriocins on prokaryotic and
eukaryotic cells. The advancement of FCM and the introduction
of novel fluorochromes allow to study the viability of cells,
the membrane structure and its integrity, and the membrane
potential at a single-cell level. In the perspective of elucidation of
the antimicrobial mechanism, FCM should be a very interesting
tool. In this review, we describe some recent studies that use
FCM as a tool to evaluate the effect of antimicrobial treatment
on microbial cells. More specifically, we focus on the use of MP-
FCM with individual and simultaneous stai ning describing the
advantages and the limitations. FCM is by definition a multi-
parametric technique: cells are gated on at least size or complexity
parameters and one pa rameter of fluorescence. Nevertheless, MP-
FCM was defined here, as described in the literature, as FCM
using several fluorochromes in the same study (Tracy et al.,
2010). Based on recently published results, the complementarity
with other methods and particularly plate counts met hods is
discussed.
INFORMATIONS RESULTING FROM
DIRECT ANALYSIS OF MICROBIAL CELLS
BY FLOW CYTOMETRY
Although staining of microbial cells with dyes prior to flow
cytometry analysi s is dominating (and will be presented
thereafter), direct analysis of cells without staining cells already
gives information regarding the morphology of a nalyzed cells.
Even without staining the sample, a cell immersed in
the injected solution already produces signals through the
orthogonal-to-flow laser focused beam. The fraction of light
scattered collected in the same dire ction as the incident light
is known as Forward Angle Light Scatter (FALS) or Forward
Scatter (FS or FSC). This fraction allows an estimation of the
cell size: indeed, the quantity of the scattered light increases wit h
the cell size (
Díaz et al., 2010; Tracy et al., 2010). The fraction
of light scattered laterally and fluorescence are collected and
divided by a lens at 90
◦
from the incidence axis of the laser.
The fraction of light scattered in right angle is known as Right
Angle Light Sc a tter (RALS) or Side Scatter (SS or SSC). This
signal is related to cell complexity described by morphological
characteristics such as cell surf ace roughness, cell membrane,
nucleus and internal granular material, number of organelles
(Díaz et al., 2010). For example, this first information concerning
SSC vs. FSC allowed Kramer and Thielmann (2016) detecting
that bacterial cells aggregated during heat exposure by change of
the scatter signals. The strong increase of SSC and FSC indicated
that cells formed large agglomerates, exhibiting up to 100-times
higher s catter signals than single cells.
Booyens and Thantsha
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Léonard et al. MP-FCM to Characterize Antimicrobial Treatments
(2014) also detected differences in shape and density of the
populations’ scatter patterns after exposure to garlic clove extract
compared to control population for all the tested Bifidobacterium
strains. Their hypothesis was that this change in size and external
morphology was a change from rod to cocc oid shape.
Schenk
et al. (2011) highlighted the same modification for Escherichia coli
cells after UV-C light treatment.
ANALYSIS OF MICROBIAL CELLS BY
FLOW CYTOMETRY AFTER THEIR
STAINING WITH DYES
Additional information can be obtained provided that samples
are st a ined using fluorochromes. Scattering and fluorescence
signals provide information about intrinsic and extrinsic cell
parameters, respectively.
Multi-Parameter Flow Cytometric Analysis:
Individual Use of Dyes
A way to use the flow cytometry to characterize antimicrobial
treatments is to perform a multi-parameter analysis with different
stains in combination. This approach will be presented thereafter
when these stains are used simultaneously (in 3.2. part). However,
several researchers have chosen to use dyes separately (Table 1).
The following paragraphs briefly discuss microbial FCM
dyes commonly employed in recent works to characterize
antimicrobial treatments.
Membrane Integrity
To interrogate membrane integrity, the nucleic acids (NA)
content of individual cells is analyzed and dye exclusion methods
are favored. NA dyes can stain DNA, RNA, or both (
Tracy
et al., 2010). Cells showing intact membranes are impermeable
to multiple charges dyes such as dyes of the Sytox
TM
family
or to cyanines such as TO-PRO
R
3. If cells lose membrane
integrity, these dyes enter i nto the cells emitting fluorescence
upon NA binding. Propidium iodide (PI) is the most commonly
used dye (
Díaz et al., 2010). This dye is usually employed
for dead cells detection and it is suitable for multi-parametric
analysis along with green fluorochromes such as SYTO9
R
. It
contains two positive charges and is normally excluded from
cells due to its divalence (
Kim et al., 2009). Therefore, PI can
only enter permeabilized cytoplasmic membranes. For instance,
the commercial available LIVE/DEAD
R
BacLight
TM
kit from
Molecular Probes is the most used (
Possemiers et al., 2005; Kim
et al., 2009; Muñoz et al., 2009; Martínez-Abad et al., 2012; Choi
et al., 2013; Booyens and Thantsha, 2014; Fernandes et al., 2014;
Manoil et al., 2 014; Pal and Srivastava, 2014; Boda et al., 2015;
Freire et al., 2015; Li H. et al., 2015; Li W. et al., 2015).
Pump Activity
Ethidium bromide (EB) is a positively-charged monovalent
compound that is used to evaluate the efflux pump system of
bacteria. It is a membrane-permeant and it enters into intact
cell membranes, but it is actively pumped out of the cell via a
non-specific proton anti-port transport system (
Kim et al., 2009;
Díaz et al., 2010
). When the membrane is damaged and the efflux
pump also malfunctions, EB can stain the intracellular DNA of
the cell (
Kim et al., 2009).
Membrane Potential
Membrane potential is generated due to the different ions content
inside and outside the cell and it varies from 100 to 200 mV. Only
living cells are able to maintain membrane potential: therefore,
it is one of the most used parameters to assess cell viability
(
Díaz et al., 2010). When this difference decreases to zero,
the membrane is structurally damaged, and ions go across the
membrane freely, but if it means a de crease in cell activity, it
does not necessarily imply death. Measurements are carried out
by using lipophilic dyes which go throug h the cell membrane and
accumulate according to their charge. The fluorescence signal
can be directly related to cell energy levels and to test the
reliability of staining, it is recommended to observe if the dye
uptake is sensitive to ionophores such as carbonyl cyanide m-
chlorophenylhydrazone (CCCP; Pianetti et al., 2008; Díaz et al.,
2010; Hammer and Heel, 2012; Li W. et al., 2015).
Cationic dyes, such as carbocyanins, DiOC
n
(3), or Rhodamine
123, accumulate inside polarized cells because viable cells are
permeable to those dyes (Díaz et al., 2010; Tracy et al.,
2010). Nevertheless, Gram negative bacteria outer membranes
can present a barrier to lipophilic dyes uptake. However, a
mild treatment with a chelating agent such as Tris-EDTA
(ethylenediamine tetraacetic acid) can overcome this limitation
(
Tracy et al., 2010; Boda et al., 2015).
Anionic and lipophilic dyes, such as those belonging
to oxonols family accumulate inside non-viable cells and
concentrate by association with intracellular compounds.
Without permeabilization protocols, oxonols uptake is more
related to membrane integrity rather than to membrane
potential and depolarization (Díaz et al., 2010). DiBAC
4
(3)
(bis-(1,3-dibarbituric acid)-trimethine oxonol) or BOX seems
to be the most used recently to detect depolarized cells of
numerous species after antimicrobial treatment (Novo et al.,
2000; Wu et al., 2010a,b; Silva et al., 2011; Caldeira et al., 2013;
Kramer and Muranyi, 2014; Duarte et al., 2015; Lee et al., 2015;
Coronel-León et al., 2016; Grau-Campistany et al., 2016; Kramer
and Thielmann, 2016).
Metabolic Activity
Metabolic activity detection suggests the absence of cell death,
but giving the conclusion of alive cell or dead cell is difficult in
the case of cell damage, dormancy, and starvation (
Nebe-von-
Caron et al., 2000; Díaz et al., 2010). In general, a non-fluorescent
permeant substrate is taken up by the cell by diffusion and
converted inside the cell by intracellular enzymes to a fluorescent
substance which is ideally retained in cells with intact membranes
(
Tracy et al., 2010).
Respiratory activity
Bacterial cells with electron transport system activity or
respiratory activity are able to reduce 5-cyano-2, 3-ditolyl
tetrazolium chloride (CTC) to an insoluble fluorescent CTC-
formazan product that accumulates inside the cells (
Caldeira
et al., 2013; Ferreira et al., 2014; Duarte et al., 2015
). For
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Léonard et al. MP-FCM to Characterize Antimicrobial Treatments
TABLE 1 | Examples of individual uses of dyes to perform a multi-parameter flow cytometry a nalysis in order to characterize antimicrobial mechanisms.
Reference Objective Dyes and their function Main result
Antolinos et al., 2 014 Effect of acid shock on cell
viability of Bacillus cereus and
Bacillus weihenstephanensis
vegetative cells
• PI (membrane p ermeability)
• cFDA (esterase activity)
pH 3.4, 3.8, and 4.2 caused membrane disruption and
subsequent bacterial cell death in the first 24 h
exposition to these acidic environments.
Boda et al., 2015 Investigate the generated
reactive oxygen species (ROS)
causing peroxidation of the
membrane lipids and ion channel
proteins, leading to greater
permeabilization of the bacterial
membranes
• DCFH-DA [intracellular indicator of
reactive oxygen species (ROS)]
∼60 and ∼70% reduction was recorded in the survival of
staphylococcal s pecies and Escherichia coli, respectively
at pulsed magnetic field as evaluated by colony forming
unit (CFU) analysis and flow cytometry. A 2–5 fold
increase in intracellular ROS (reactive oxygen species)
levels suggests oxidative stress as the key mediator in
PMF induced bacterial d eath/injury.
Caldeira et al., 2013 Assessment of antibacterial
properties of L-cysteine and
mechanism of action against
Staphylococcus aureus and
Klebsiella pneumoniae
• PI (membrane p ermeability)
• DiBAC
4
(membrane potential)
• CTC (respiratory activity)
The main mechanism of action o f L-Cys on both
bacteria, K. p neumonia, and S. aureus, was the
reduction in metabolic activity, consistent with the results
that confirmed the bacteriostatic effect of L-Cys on both
these bacteria.
Kramer and Muranyi, 2 014 Investigate the effects of a
pulsed light treatment on the
physiological properties of
Listeria innocua and Escherichia
coli
• PI (membrane p ermeability)
• DiBAC
4
(membrane potential)
• EB (efflux pump activity)
• cFDA (esterase activity)
Oxidative s tress with concomitant damage to the DNA
molecule was shown to be directly responsible fo r the
loss of cultivability due to pulsed light rather than a direct
rupture of cell membranes or inactivation of intracellular
enzymes.
Kramer and Thielmann, 2016 Monitoring the live to dead
transition of bacteria during
thermal stress
• PI (membrane permeability)
• DiBAC
4
(membrane potential)
• cFDA (esterase activity)
• 2-NBGD (glucose uptake)
Exposure to moderate heat first of all compromised the
function of the respiration chain and other heat sensitive
proteins of the cell membrane such as efflux pumps.
Membrane rupture and intracellular esterase activity
were less affected and strong differences depending on
the type of bacteria regarding their Gram-staining
behavior were observed.
Lee et al., 2015 Mechanism of action of
scopolendin 2 against E . coli
O157 and Candida albicans
• Sytox Green
• (membrane permeability)
• DiBAC
4
(membrane potential)
• DiSC
3
(membrane potential)
Scopolendin 2 led to the f ormation of pores in microbial
plasma membrane, s ubsequent leakage of cytoplasmic
matrix components and cons equent membrane
depolarization, ultimately resulting in microbial cell d eath.
Morishige et al., 2 015 Analysis of the metabolic
response of H
2
O
2
-treated
Salmonella cells
• CTC (respiratory activity)
• 2-NBGD (glucose uptake)
• EdU-Alexa 488
• (DNA synthesis)
H
2
O
2
-treated Salmonella cells did not lose their
biological activities of living cells all together. Different
subpopulations of Viable But Non-Culturable bacteria
were detected. Cells lost their DNA-synthesis activity
first, then C TC -reducing activity, and finally
glucose-uptake activity to a lesser extent.
Silva et al., 2 011 Coriandrum sativum essential oil
mode of action against C andida
species
• PI (membrane p ermeability)
• DiBAC
4
(membrane potential)
• DRAQ5 ( DN A staining)
Coriander essential oil kills Candida s pp. by damaging
the cytoplasmic membrane, leading to an impairment of
cellular functions.
Teng et al., 2014 Elucidate further the antimicrobial
mechanism of AvBD103b, an
avian defensin, on the Salmonella
enteritidis CVCC3377 cell
membrane and intracellular DNA
• PI (membrane permeability)
• FITC-labeled AvBD103b
• (AvBD103b permeation of the
membrane)
Antimicrobial target of AvBD103b was the cell
membrane.
PI, Propidium Iodide; cFDA, carboxyFluorescein DiAcetate; DiBAC
4
, Bis-(1,3-DibutylBarbituric ACid) Trimethine Oxonol = bis-oxonol = BOX; CTC , 5-Cyano-2,3-ditolyl Tetrazolium
Chloride; EB, Ethidium Bromide; 2-NBGD, 2-[N-(7-nitrobenz-2-oxa-1,3-Díazol-4-yl) amino]-2-deoxy-Dglucose; EdU: 5-ethynyl-2-deoxyuridine; FITC, fluorescein isothiocyanate; DRAQ5,
1,5-bis{[2-(di-methylamino) ethyl]amino}-4, 8-dihydroxyanthracene-9,10-dione; DiSC
3
, 3,3
′
-Dipropylthiadicarbocyanine Iodide, DCFH-DA, 2
′
,7
′
-dichlorofluorescein-diacetate.
CTC-formazan fluorescence analysis, two regions of CTC-
formazan relative fluorescence were analyzed, depending on the
fluorescence intensity of positive and negative controls (
Ferreira
et al., 2014; Morishige et al., 2015
). Ferreira et al. (2014) and
Díaz et al. (2010) expressed the limit of this stain: CTC staining
allows the detection of the most metabolically active bacteria,
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Léonard et al. MP-FCM to Characterize Antimicrobial Treatments
cells with low respiratory activity may not be detected as CTC-
positive, probably due to the relative toxicity of cellular CTC
accumulation.
Enzymatic activity
Esterase activity is the most common way to evaluate enzymatic
activity (
Díaz et al., 2010), particularly in studies about damages
after antimicrobial treatment (Ananta et al., 2004; Hayouni
et al., 2008; Ananta and Knorr, 2009; Schenk et al., 2011;
Ayari et al., 2013; Thabet et al., 2013; Antolinos et al., 2014;
Kramer and Muranyi, 2014; Surowsky et al., 2014; Hong et al.,
2015; Combarros et al., 2016; Kramer and Thielmann, 2016;
Meng et al., 2016). Fluorescein and fluorescein derivatives have
been used for a wide range of microorganisms as probes for
enzymatic activity measurement (Díaz et al., 2010). Among these,
carboxyfluorescein diacetate (cFDA) is used primarily for the
evaluation of esterase cellular activity (
Ananta et al., 2004). It is
a lipophilic non-fluores ce nt precursor that rapidly diffuses across
the cell membranes. In the intracellular compartment, diacetate
groups of cFDA are hydrolyzed by unspecific esterases into
carboxyfluorescein (cF) which is a polar membrane-permeant
fluorescent compound. The cells only remain fluorescent if their
membranes are intact, thus for cells to be associated as viable,
this probe requires both active intracellular enzymes and intact
membranes (
Hoefel et al., 2003). Moreover, efflux of cF upon
glucose addition could also be used as an additional indicator of
metabolic performance of the cell (Ananta et al., 2004). Schenk
et al. (2011) showed how carefully this staining must be used.
Untreated E. coli, Listeria innocua, and Saccharomyces cerevisiae
stained with cFDA showed a heterogeneous behavior in their
fluorescence labeling properties. Not all cells yielded high green
fluorescence and appeared in expected quadrant in the cytogram.
They formulated two hypotheses: (i) the presence of the outer
membrane with lipopolysaccharides in Gram negative bacteria
or the thick wall of peptidoglycan in Gram positive bacteria
which does not allow cFDA freely diffusing across cytoplasmic
membrane; (ii) the active expulsion of cF outside the cell by
bacteria pumps and consequently the lack of green fluorescence
despite the existence of metabolic activity.
Anyway, concentrations of probes, incubation periods,
cytometric setting, and control should be assessed for each
studied bacteria individually (
Kramer and Thielmann, 2016),
as performed by Nexmann Jacobsen et al. (1997) for Listeria
monocytogenes. They compared t h e use of L IVE/DEAD
R
BacLight
TM
kit, Rhodamine 123, 2
′
,7
′
-bis(2-carboxyethyl)-
5(6)-carboxyfluorescein acetoxymethylester (BCECF-AM),
Chemchrome B and cFDA. In conclusion, they determined that
only cFDA and Chemchrome B were suitable for rapid, almost
real time counting of pure cultures of L. monocytogenes by flow
cytometry, after 6−24 h incubat ion in selective enrichment
media.
FCM with fluorescent dyes can thus be considered as a
suitable tool for the assessment of structural and/or functional
microbial cell properties such as metabolic activity, membrane
potential, and integrity. The combination of dyes is a pertinent
multi-method approach to reveal the presence of intermediate
physiological states between life and cell death , showing t h e
heterogeneities of microbial populations.
Nebe-von-Caron et al.
(2000) classified cells according to some active functions or
the integrity of cell structures. These authors distinguished
among reproductively viable, met abolically active, intact, and
permeabilized cells.
A multi-method approach is a suitable tool to monitor
the impact of inactivation t reatments on bacteria, providing
information about the mode of action, the heterogeneity of
populations, species-specific differences to stressors and valuable
insight in vital functions beyond pure culturability (
Kramer and
Thielmann, 2016).
The results obtained by FCM are in the form of graphical
visualization of scattering and fluorescence cell parameters,
which are being analyzed and stored for further analysis
(Díaz et al., 2010). Acquired data are identified as events,
as the number of cells showing a desired physical property
or probe. In the case of single-staining cells, two types of
graphical results could be obtained (e.g., Figure 1). A mono-
parametric histogram presents the number of cells (y-axis) vs.
the scattering or fluorescence intensity ( x-axis; e.g., Figure 1-I).
Lee et al. (2015) detected membrane permeabilization of
Candida albicans by Sytox Green fluorescence. Cells treated
by scolopendin 2 and melittin indicated a signific a nt increase
in fluores cence intensity. These results show that the fungal
cell membranes were permeabilized by the peptides. A bi-
parametric histogram represents the intensity of the signals
corresponding to different parameters in each axis (e.g.,
Figure 1-II: scattering intensity (y-axis) vs. CTC-fluorescence
activity). Each dot represents a single cell and different regions
can be defined into the cytogram to des cribe cells physiological
state.
The publication of
Silva et al. (2011) showed well the value
of this approach in determining the mechanism of coriander
essential oil against Candida spp. with three dyes: PI (membrane
permeability), DiBAC
4
(membrane potential), DRAQ5 (DNA
staining). Firstly, the percentage of PI-positive cells seemed
to depend on essential oil concentration: higher essential
oil concentration caused higher membrane permeability. The
structure of the cell membrane was disrupted by the essential
oil. Permeation to PI, particularly following short incubation
period, such as 30 min, indicated that the mode of action of
the essential oil involved a lesion of the cell membrane that
resulted from direct damage to the cell membrane instead
of a metabolic impairment le ading to secondary membrane
damage. Secondly, the percentage of depolarized cells was also
essential oil concentration dependent. Thirdly, DNA distribution
histograms were very similar in the control and in essential oil-
treated cells, which could indicate that coriander essential oil
did not interfere wit h DNA synthesis. However, at ½Minimal
Inhibitory Concentration (MIC), fluorescence intensity values
were slightly different: essential oil-treated cells showed higher
fluorescence intensity values than control cells. This could
indicate that in response to cell damage by coriander essential
oil, cells were synthesizing more DNA in order to repair
damage functions. Moreover, at 1 MIC, lowest fluorescence
intensity values were observed, probably indicating DNA leakage
from the cells. In conclusion, these three staining procedures
Frontiers in Microbiology | www.frontiersin.org 5 August 2016 | Volume 7 | Article 1225