REVIEW ARTICLE
The potential of flow cytometry in the studyof Bacillus cereusU.P. Cronin and M.G. Wilkinson
Department of Life Sciences, University of Limerick, Castletroy, Co. Limerick, Ireland
Introduction
The Gram-positive spore-former Bacillus cereus is the
aetiological agent of both a food-borne infection (the
diarrhoeal syndrome) and an intoxication (the emetic
syndrome) as well as a number of clinical illnesses such
as endophthalmitis (Wolf and Barker 1968; Arnesen et al.
2008; Miller et al. 2008). While neither of these illnesses
could be considered to be a grave threat to public health
and symptoms do not usually persist beyond 24 h (Top-
ley and Wilson 1998; Dahl 1999), recent changes in eating
habits and the ever-increasing reliance by consumers on
convenience of pre-prepared cooked chilled foods [also
known as refrigerated processed foods of extended dura-
bility (REPFEDs)] have led researchers in the area of food
safety microbiology to suggest that, in the future, inci-
dents and outbreaks of B. cereus food poisoning will arise
with greater frequency (Arnesen et al. 2008). Considering
the earlier information and given B. cereus’ ubiquity in
nature (Claus and Berkeley 1986) and in tested foodstuffs
(Choma et al. 2000), it would be of benefit to develop
novel, rapid and reliable methods for the study of this
organism in various modern food products. Flow cyto-
metry (FCM) offers the possibility of doing so.
In FCM, light scattered or emitted by cells is measured
by a number of detectors as they pass by an interrogation
point (usually one or more lasers) in a fluid stream
(Davey et al. 1999; Sincock and Robinson 2001). The
intensity of light scattered by cells yields information on
cell size, shape and cytoplasmic content (Gunasekera et al.
2003). Intrinsic fluorescence or that produced by fluoro-
chrome staining may be used to derive information on
specific components or physiological states, e.g. DNA
content, protein content, cell membrane integrity or cyto-
plasmic esterase activity (Shapiro 2003). FCM allows
the taking of multiparametric data (from two scatter
detectors and, commonly, six fluorescence detectors)
from a large number of individual cells at high speeds
Keywords
Bacillus cereus, detection, flow cytometry,
fluorescence, staining.
Correspondence
Martin G. Wilkinson, Department of Life
Sciences, Schrodinger Building, University of
Limerick, Castletroy, Co. Limerick, Ireland.
E-mail: [email protected]
2008 ⁄ 2097: received 08 December 2008,
revised 02 March 2009 and accepted 12 April
2009
doi:10.1111/j.1365-2672.2009.04370.x
Summary
Flow cytometry (FCM) is a rapid method allowing the acquisition of multi-
parametric data from thousands of individual cells within a sample. As well as
measuring the intrinsic light scattering properties of cells, a plethora of fluores-
cent dyes may be employed to yield information on macromolecule content,
surface antigens present or physiological status. Despite FCM’s indispensability
within other fields e.g. immunology, it is underutilized within microbiological
research. In this review, a strong case is presented for the potential of FCM in
the study of Gram-positive spore-former, Bacillus cereus. Previous reports
where FCM was successfully used in the study of B. cereus are reviewed along
with relevant studies involving other members of the genus. Under headings
reflecting common research themes associated with B. cereus, specific instances
where FCM has generated novel data, providing a unique insight into the
organism, are discussed. Further applications are posited, based on the authors’
own research with FCM and B. cereus and work extant in the broader field of
microbial cytometry. The authors conclude that, while the expense of equip-
ment and reagents is an undeniable disadvantage, FCM is a technique capable
of generating significantly novel data and allows the design and execution of
experiments that are not possible with any other technique.
Journal of Applied Microbiology ISSN 1364-5072
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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16 1
(>1000 cells s)1; Nebe-von-Caron et al. 2000; Shapiro
1995). The technique allows for the measurement of
heterogeneity within a population as opposed to
obtaining an average value for that population using
conventional techniques such as microtitre plate assays
(Davey and Kell 1996; Edwards et al. 1996).
Compared with the vast literature generated on the
application of FCM to the study of mammalian cells,
especially those of the human immune system, relatively
little work has been reported on the application of FCM
to microbiology. Few of the published microbial FCM
studies have focussed on B. cereus or indeed many topics
of direct relevance to the broad field of food safety micro-
biology. However, FCM has enormous potential applica-
tions in the area of food safety microbiology, and
especially as an integral component of the methodology
used to study B. cereus.
The current research into B. cereus is divided into a
number of broad categories, and a short summary of the
current methods as applied to each topic is provided. In
the case of each topic, an examination of the possible
application of FCM to questions within the area is then
presented. Applications presented are based on published
work carried out by the current authors on B. cereus,
work by other authors on B. cereus itself or other mem-
bers of the genus, relevant studies dealing with micro-
organisms other than Bacillus spp., or are based on
potential uses for FCM in the field of microbiology sug-
gested by authors such as Robinson (2000) and Winson
and Davey (2000).
Common themes in Bacillus cereus research andthe utility of flow cytometry within each area
Bacillus cereus is studied for reasons ranging from its
involvement in clinical illness (Akesson et al. 1991) and
its prevalence in commercial chilled cooked food (Choma
et al. 2000) to the resistance mechanisms of endospores
to heat (Gaillard et al. 1998). Unlike B. subtilis, B. cereus
is generally not utilized as a model for aerobic spore-
forming organisms or prokaryotic differentiation. There-
fore, nothing approaching the immense quantity of basic
research carried out on B. subtilis has been carried out on
B. cereus. Additionally, unlike many members of its
genus, B. cereus is not an industrially important fermenta-
tion organism. However, the fact that B. cereus is increas-
ingly recognised as an important pathogen and spoilage
organism has resulted in a significant amount of basic
and applied research being focussed on this organism. It
is of benefit to workers interested in B. cereus that B. sub-
tilis has been chosen by the scientific community as a
model Gram-positive micro-organism and is now one of
the best characterized micro-organisms. Hence, many
techniques, especially biochemical and nucleic acid based,
originally developed for B. subtilis can be applied to
B. cereus [see Harwood and Archibald (1990)]. Below, a
number of research themes common to the B. cereus liter-
ature are described along with an overview of the current
techniques employed by researchers to study these topics
and the potential application of FCM to that area.
Detection
Traditionally, detection, whether in the context of aca-
demic studies, quality control or public health laborato-
ries, has been carried out exclusively by plate counting.
This is a growth-based method relying on media which
suppress the growth of Gram-negative organisms thereaf-
ter providing a presumptive identification of the organ-
ism based on its ability to hydrolyse lecithins and its
inability to ferment mannitol (Bouwer-Hertzberger and
Mossel 1982; Varnam and Evans 1991). Practically, every
isolation method utilizes one of two selective ⁄ diagnostic
media, namely, polymyxin pyruvate egg yolk mannitol
bromothymol blue agar (PEMBA; Holbrook and Ander-
son 1980) or mannitol egg yolk polymyxin agar (MYP;
Mossell et al. 1967).
Following isolation and presumptive identification on
selective media, a number of tests are normally carried
out on the isolates until it can be stated with a high
degree of confidence that the organism under scrutiny is
indeed B. cereus. In common with national regulatory
agencies, peer-reviewed academic papers normally include
three to four confirmatory procedures followed by the
use of API 50 CHB strips (BioMerieux, Marcy l’Etoile,
France) for the positive identification of a presumptive
B. cereus isolate.
A number of serological and molecular techniques exist
for the detection of B. cereus or other members of the
genus Bacillus (Chen et al. 2001; Hansen and Hendriksen
2001; Uyttendaele and Debevere 2003; Lampel et al. 2004;
Kim et al. 2005; Varughese et al. 2007). These non-
growth-based methods are rapid and provide an unequiv-
ocal identification of the organism without the need to
perform further tests, as is the case with plate counting
(Mathews 2003; Zwirglmaier 2005). Although much less
laborious than plate counts, these methods, being non-
growth-based, have a major disadvantage – lack of dis-
crimination between live and dead cells. The viable but
nonculturable issue within the genus, Bacillus, is not as
contentious as within, e.g. the genus Vibrio (Adams 2005;
Albertini et al. 2006). However, as Bacillus sporulate,
serological and molecular methods such as ELISA, FISH
and PCR tend to overestimate the numbers of cells pres-
ent in a sample compared with the plate count method
(Kaspar and Tartera 1990; O’Connor and Maher 1999).
Study of B. cereus using flow cytometry U.P. Cronin and M.G. Wilkinson
2 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16
ª 2009 The Authors
The efficiency of DNA amplifications ⁄ hybridizations and
antibody–antigen reactions is affected by certain compo-
nents of the food matrix (termed food matrix inhibition;
Barbour and Tice 1997; Mathews 2003); hence, separation
of target cells can be problematic (Zwirglmaier 2005) and,
for low densities of target cells, a concentration step such
as immunomagnetic separation (Blake and Weimer 1997)
or enrichment (Forsythe 2000) may be required.
Prerequisites to allow flow cytometric detection of
B. cereus within food samples include the ability to effi-
ciently separate vegetative cells or endospores from the
food matrix (e.g. using density grade centrifugation or
immunomagnetic separation; Barbour and Tice 1997;
Robinson 1999; Hibi et al. 2006) and the availability of
specific fluorescent tags to label B. cereus vegetative cells
or endospores and differentiate them from other micro-
organisms and debris present (Barbour and Tice 1997;
Forsythe 2000; Mathews 2003). Fluorescent tags can be
antibodies recognizing a component of the surface of cells
or endospores or nucleic acid probes, which specifically
bind to a unique sequence such as that of a 16S rRNA
gene in B. cereus (Phillips and Martin 1988; O’Connor
and Maher 1999; Zoetendal et al. 2002; Zwirglmaier
2005). If detection of both vegetative cells and endospores
is the objective, then two fluorescent antibodies are
probably required as the surfaces of vegetative cells and
endospores are quite dissimilar (Bennet and Belay 2001).
A single nucleic acid probe may be capable of binding to
a target sequence in both vegetative cells and endospores,
although permeabilization and hydration steps would
likely be required for endospores to allow access and
binding of the probe (Leuschner and Lillford 2000; Setlow
2006).
Development of an FCM-based detection method for
B. cereus would have many potential advantages over con-
ventional methods. First, this method would be much
more rapid than growth-based methods, with data gener-
ated after a number of hours rather than the two days
currently required for plate counts. Secondly, the neces-
sity for confirmatory tests would be eliminated if the anti-
bodies or probe used were specific to strains of B. cereus.
Thirdly, the use of physiological dyes in tandem with
specific markers would allow additional information on
the physiological status of detected vegetative cells or
endospores to be generated. Finally, through the use of
nucleic acid probes for e.g. toxin genes, one could con-
ceivably detect subsets of e.g. enterotoxigenic strains
among the detected B. cereus vegetative cells and spores
(Uyttendaele and Debevere 2003). Recently, Schumacher
et al. (2008) reported the development of a two-colour
FCM assay for the simultaneous detection and virulence
determination (via an antiprotective antigen fluorescent
antibody) of B. anthracis endospores.
Growth
Growth of B. cereus in various media and in foods as influ-
enced by conditions of temperature, oxygen tension, pH,
etc. has been the focus of numerous research studies. The
purpose of these studies includes basic research into the
nutritional requirements of B. cereus (Folmsbee et al.
2004), identification of optimum growth temperature or aw
(Lindsay et al. 2002; Haque and Russel 2004), determina-
tion of growth rates in various foods under different condi-
tions (Kimanya et al. 2003; Valero et al. 2003; Banerjee and
Sarkar 2004; Turner et al. 2006) and generation of empiri-
cal growth models (Chorin et al. 1997; Nauta et al. 2003).
Knowledge derived from such studies can be applied to the
risk assessment of certain foods or production processes
(Notermans et al. 1997; Notermans and Batt 1998) and are
essential in the development of strategies to prevent the
contamination and spoilage of foods (Snowdon et al.
2002). Growth is typically assessed using plate counting,
although turbidometry and other indirect methods have
been reported (Chorin et al. 1997). The culturing method
of choice for those involved in the study of the growth of
B. cereus is liquid culture. In a typical report, Fernandez
et al. (1999) isolated pure endospores from solid medium
and proceeded to examine outgrowth at various tempera-
tures through the cultivation of suspensions of endospores
in tryptone soya broth.
FCM can enumerate the number of suspended parti-
cles, including cells, in a volume of sample (Shapiro
2003). Indeed, any growth experiment involving B. cereus
cells in liquid media is amenable to FCM enumeration,
the advantages of which over traditional enumeration
methods include rapidity and ability to acquire additional
information such as DNA content or physiological status
on individual enumerated cells (Nebe-von-Caron et al.
2000). There are many examples of workers utilizing
FCM to enumerate micro-organisms in suspension,
including estimation of growth rates of bacteria under
various conditions and construction of growth curves
(Comas and Vives-Rego 2002; see Cronin and Wilkinson
2008c). Recently, Leser et al. (2008) utilized FCM in con-
junction with the staining of samples with the permeant
nucleic acid dye, SYTO 13, in order to enumerate the
number of endospores and vegetative cells recovered from
the gastrointestinal tract of pigs given direct-fed additives
constituted by a mixture of B. subtilis and B. licheniformis
endospores.
As well as measurement of growth rate, FCM has appli-
cations including the study during growth and ⁄ or differ-
ing growth conditions of cell physiology (Cronin and
Wilkinson 2008c), changes in cell size (reflected by
changes in FSC properties of cells; Robertson and Button
1999), changes in cytoplasmic density (reflected by
U.P. Cronin and M.G. Wilkinson Study of B. cereus using flow cytometry
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16 3
changes in SSC properties of cells; Davey 1994), DNA
(Reardon and Scheper 1993; Stecchini et al. 2001) and
lipid or protein contents (Reardon and Scheper 1993) of
B. cereus. The physiological status of cells may be assessed
by staining with a number of fluorescent dyes to detect
alterations in membrane permeability, enzyme activity,
redox activity, membrane potential, oxidative damage
to DNA and intracellular Ca2+ and pH (Coder 1997;
Nebe-von-Caron et al. 2000; Sincock and Robinson 2001;
Shapiro and Nebe-von-Caron 2004). For example, Reis
et al. (2005) and Lopes da Silva et al. (2005) used a com-
bination of propidium iodide (PI, a nucleic acid dye
that only enters cells with damaged membranes) and
DiOC6(3) (a dye that accumulates in cells with membrane
potential) to monitor the response of B. licheniformis cells
to starvation and a glucose and ⁄ or lactose pulse, and they
reported that higher quantities of stressed cells were pres-
ent during starvation than following the glucose pulse
and that the physiological response of cells differed
depending on the disaccharide supplied.
Excellent descriptions of the range of dyes available to
the microbiologist are published in a number of reviews
(Davey et al. 1999; Jacobsen and Jakobsen 1999; Alvarez-
Barrientos et al. 2000; Nebe-von-Caron et al. 2000; Sin-
cock and Robinson 2001 Shapiro 2003). DNA content is
estimated by staining of cells with fluorochromes such as
Hoechst 33342 that specifically bind to DNA (Lloyd
1999). Protein content may be measured by staining of
cells with protein-binding fluorochromes such as fluores-
cein (Natarajan and Srienc 2000). Rates of cell division
during growth may be measured using a tracking dye
such as carboxyfluorescein diacetate succinimidyl ester
(CFSE; Cronin and Wilkinson 2008c). The expression of
specific genes by individual cells during growth may be
monitored through FCM measurement of fluorescence
derived from the production of GFP or its derivatives by
transgenic cells (Hawley et al. 2004; Maraha et al. 2004).
The spatio-temporal regulation of gene expression within
B. subtilis biofilms was studied using a number of YFP
reporters under the control of promoters for motility,
matrix-production and sporulation (Vlamakis et al.
2008). This approach allowed almost real-time quantifica-
tion of the degree of heterogeneity in biofilms over the
course of their development.
Resistance and survival of vegetative cells
Under the above title studies can be classified concerning
the resistance and survival of vegetative cells following
treatment with antibiotics (Abee and Delves-Broughton
2003), food processing treatments (Browne and Dowds
2001; Hansen et al. 2001), food preservatives (Ultee et al.
2002; Kwon et al. 2003), process equipment sanitation
regimes (Galeano et al. 2003) and survival in foods of low
pH or high NaCl designed to be either bactericidal or
bacteristatic (Browne and Dowds 2002; Collado et al.
2003a; Valero et al. 2003; Valero and Frances 2006a).
Knowledge derived from such studies is directly relevant
to the food industry, where optimized preservative con-
centrations or sanitization regimes are required to reduce
the risk of B. cereus-mediated spoilage or contamination
of products. The vast majority of studies dealing with the
above topics involve the exposure of vegetative cells,
either growing as pure suspensions or within a spiked
foodstuff, to the stressor under investigation and deter-
mining the effect of the stressor on viability through plate
counting. For example, Browne and Dowds (2001) exam-
ined the survival of B. cereus under various conditions of
heat, sodium chloride, ethanol and hydrogen peroxide
concentrations. The synergistic inhibitory effect of a
nisin–monolaurin combination on species within the
genus, Bacillus, was investigated by Mansour and Milliere
(2001) using bacteria growing as a suspension in skim
milk.
FCM allows the study of the responses of individual
cells to antimicrobial compounds or treatments (Alvarez-
Barrientos et al. 2000; Steen 2000) and, by staining trea-
ted B. cereus cells with any of a number of fluorescent
dyes, the mechanism of action of antimicrobial com-
pounds or treatments may be studied (Davey and Kell
1996). Assuncao et al. (2006) utilized a combination of
SYBR green, and PI to measure the minimum inhibitory
concentrations of seven antimicrobial agents, Martinez
et al. (1982) used the intrinsic light scatter properties of
cells in combination with the DNA-binding stains ethidium
bromide and mithramycin to determine the effect of
b-lactam antibiotics on Escherichia coli, Braga et al.
(2003) utilized SYTO 9 and PI to study the susceptibili-
ties of Streptococcus pyrogenes to erythromycin and rokita-
mycin, and Nguefack et al. (2004) tested the antibacterial
activity of five essential oils against Listeria innocua. Simi-
larly, the effect of a particular treatment on a cellular
component of interest such as the plasma membrane
(Trevors 2003) or a property such as intracellular esterase
activity (Hoefel et al. 2003) may be investigated using
FCM, with responses recorded for every individual cell in
the sample (Mason et al. 1999). Through comparison of
the responses of resistant strains with those of susceptible
strains, multiparameter FCM may assist in elucidating
bacterial resistance mechanisms (Davey and Kell 1996).
Using FCM in conjunction with a range of fluorescent
stains allows the percentages of B. cereus cells surviving a
given treatment to be estimated (Steen 2000). Typically,
stains used in such studies include PI (Barbesti et al.
2000; Tanaka et al. 2000; Nunez et al. 2001; Pianetti et al.
2005), carboxyfluorescein diacetate (CFDA – a marker for
Study of B. cereus using flow cytometry U.P. Cronin and M.G. Wilkinson
4 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16
ª 2009 The Authors
intracellular esterase activity; (Schell et al. 1999; Bunthof
et al. 2000; Tanaka et al. 2000; Nguefack et al. 2004; Flint
et al. 2006), rhodamine 123 (a lipophilic cationic dye that
accumulates in viable cells; (Diaper and Edwards 1994)),
oxonol dyes (lipophilic anions that enter cells lacking
membrane potential; Mason et al. 1995) and CTC
(reduced by cellular respiration to an insoluble highly
fluorescent CTC-formazan; Yaqub et al. 2004). The cur-
rent authors investigated the effects of simulated food
processing or sanitisation treatments on the survival and
physiology of B. cereus vegetative cells using a number of
the dyes described above (Cronin and Wilkinson 2008d).
Good correlations were found between the reductions in
viability as measured using plate counting and decreases
in the percentages of cells showing redox activity, esterase
activity or membrane integrity. By utilizing the above
approaches, FCM can form the cornerstone of a rapid
screening method for novel strains (Betz et al. 1984),
antimicrobial compounds or antimicrobial treatments
(Sincock and Robinson 2001), while simultaneously pro-
viding information on the mechanism of action.
The aforementioned applications of FCM to the study
of resistance and survival are essentially unique to this
technique. While fluorescence microscopy and other tech-
niques such as confocal laser scanning microscopy can
study the response of individual cells to treatments using
the same repertoire of dyes as those utilized in FCM
(Raybourne and Tortorello 2003; Gatti et al. 2006), these
techniques are labour intensive and prone to large varia-
tion because of reduced sample size (Shapiro 2000).
Other rapid screening methods based e.g. on the use of
microtitre plates cannot yield information on either the
response of individual cells or the mechanism of action
(Alvarez-Barrientos et al. 2000).
Toxin production
Of fundamental interest to food safety researchers is when
and under what circumstances are the virulence factors
responsible for the symptoms associated with the type of
food poisoning caused by their organism of interest
expressed (Granum and Byrnestad 1999; Arnesen et al.
2008). In the case of B. cereus, which produces numerous
toxins, many of which are poorly characterized, basic
research at the genetic level in order to match sequence
to symptom is one approach being pursued (Granum and
Byrnestad 1999; Hansen et al. 2003; Toh et al. 2004).
Other research approaches deal with investigating the
production of a particular toxin under certain environ-
mental conditions and in certain media ⁄ foodstuffs (Agata
et al. 2002; Finlay et al. 2002; Rowan et al. 2003; Toh
et al. 2004; Rajkovic et al. 2006). Other studies attempt to
relate the structure of toxins to their modes of action
(Beecher and Macmillan 1991; Hergenrother and Martin
1997; Callegan et al. 1999; Gilmore et al. 1999; Beecher
et al. 2000). Depending on the toxin under investigation,
toxin titre can be measured or estimated using a number
of methods ranging from the rabbit ileal loop assay for
any of the diarrhoeal toxins (Notermans and Batt 1998)
to a rat liver mitochondria assay for cereulide (Arnesen
et al. 2008).
Until very recently, FCM was used exclusively for mea-
suring the properties of cells and viruses. However, with
the development of multiplex bead array assays, it is now
possible to simultaneously detect and quantify multiple
proteins within a sample (Morgan et al. 2004; Elshal
and McCoy 2006). These assays depend upon the use of
spectrally discrete microspheres coated with the antibody
specific to the protein of interest to capture dis-
solved ⁄ suspended target from the solution under exami-
nation. The microspheres are then counterstained with a
fluorescent antibody against the target protein, with the
levels of fluorescence of this species directly relating to
the concentration of target. The number of proteins that
can be simultaneously detected and measured per sample
depends on the resolving power of the analytical instru-
mentation, with some assays reportedly capable of simul-
taneous detection of up to 25 proteins (Heijmans-
Antonissen et al. 2006). As both detection instruments
and data analysis software improve, assays for the quanti-
fication of hundreds of proteins may become a reality
(D’Costa et al. 2006).
Although such an assay does not at present exist for
B. cereus, a multiplex bead array assay with the ability to
measure levels of the various toxins produced by the
organism under different environmental conditions could
prove very useful. The ability to simultaneously detect
and measure multiple toxins would be of great benefit in
the study of how the various toxins interact to cause the
symptoms of clinical diseases such as endophthalmitis
and gingival infections (Nguyen-The and Broussolle
2005), where B. cereus is thought to play a major role.
Furthermore, multiplex bead array assays could be used
to uncover the ‘toxinome’ of a given strain. As an aside,
because of the chemical structure of cereulide, this dode-
cadepsipeptide has not been found to be immunogenic
(Andersson et al. 1998; Rajkovic et al. 2006) and therefore
would not be amenable to detection using antibody-based
technology. A more conventional FCM approach for the
study of toxin production in B. cereus carried out by the
present authors involved the parallel measurement by
conventional means of PC-PLC activity and analysis by
FCM of the physiological state of cells (Cronin and
Wilkinson 2008c). Using such an approach, it was possi-
ble to relate changes in the proportions of cells within
cultures exhibiting differing protein contents, membrane
U.P. Cronin and M.G. Wilkinson Study of B. cereus using flow cytometry
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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16 5
permeabilities, intracellular esterase and redox activities to
global PC-PLC activity.
Endospore resistance and physiology
As with vegetative cells, the resistance of B. cereus endo-
spores to sporicidal compounds (McDonnell and Den-
ver-Russell 1999; Cortezzo et al. 2004), food processing
treatments (Gaillard et al. 1998; Fernandez et al. 1999;
Collado et al. 2003b, 2004), food preservatives (Chaibi
et al. 1997) and process equipment sanitation regimes
(Raso et al. 1998; Ryu and Beuchat 2005; Ernst et al.
2006) and the survival and germination of endospores
in foodstuffs (Aran 2001; Coroller et al. 2001; Werner
and Hotchkiss 2002; Clavel et al. 2004) comprise a very
important area of research. Basic physiological studies,
although carried out less extensively on B. cereus than
on B. subtilis, also form a significant body of work
within the field of B. cereus research (Stalheim and
Granum 2001; Setlow 2003; Moir 2006).
The plate count method is the most commonly used
technique in studies evaluating the survival of endospores
in response to a stressor such as heat (Nicholson and Set-
low 1990; Fernandez et al. 1999; Gaillard et al. 1998; Colla-
do et al. 2003b; Valero et al. 2006b). Turbidometry is
much employed in order to estimate the rate of germina-
tion (Laurent et al. 1999), as are phase contrast micros-
copy and biochemical assays measuring the release of
compounds such as dipicolinic acid, small acid-soluble
proteins or peptidoglycan (Harwood and Archibald 1990;
Cortezzo et al. 2004). Physiological studies often involve
staining of endospores with fluorescent dyes for the
purpose of epifluorescent or confocal microscopy (Coote
et al. 1994). Molecular genetic techniques are being
increasingly used to detect the expression of certain genes
during germination in response to stressors (Dricks 2002).
Very few studies on endospore resistance and physio-
logy (of any bacterial species) have been undertaken using
FCM, perhaps reflecting the reluctance of microbiologists
to accept new nonculture-based technologies (Steen
2000). In general, FCM-based studies and similar ones
undertaken using epifluorescence microscopy or a related
microscopic technique involve challenging endospores
with a sporicidal compound or treatment, staining with a
physiological dye such as membrane impermeant PI (Reis
et al. 2005) or the membrane potential dye, DiOC6(3),
(Laflamme et al. 2005) followed by FCM analysis. The
action of the compound or treatment is then related to
its effect on endospore permeability or membrane poten-
tial. Physiological studies, e.g. those of Black et al. (2005,
2006), often focus on germination, which is understand-
able given that ungerminated endospores do not pose a
threat as either food spoilage or poisoning organisms.
Staining with physiological dyes is not always necessary,
as shown by a study describing an interesting alternative
method for differentiating viable and nonviable B. subtilis
endospores based on measuring green autofluorescence
from UV-excited endospores (Laflamme et al. 2006).
Despite the current lack of widespread uptake of FCM
by the microbiological research community, FCM has
much to offer B. cereus endospore research. FCM allows
the rapid taking of measurements from large quantities of
individual endospores in a manner that no other tech-
nique permits (Veal et al. 2000). The current authors
demonstrated a high degree of heterogeneity among
B. cereus endospores subjected to simulated cooking tem-
peratures ⁄ times using FCM together with SYTO 9 ⁄ PI and
CFDA ⁄ Hoechst 33342 (Cronin and Wilkinson 2008a),
while Mathys et al. (2007), using SYTO 16 ⁄ PI to study
the effect of pressure and thermal processing on B. lichen-
iformis, were able to arrive at a three-step model of inac-
tivation. Potentially, staining techniques could be
developed to relate uptake of a specific dye to the loss of
a certain endospore resistance factor e.g. a permeability
barrier such as the inner membrane which is responsible
for the exclusion of small molecules from the core (Nich-
olson et al. 2000; Setlow 2006), allowing rapid screening
of potentially sporicidal compounds. The feasibility of
such a strategy has been suggested by work carried out
by the current authors (Cronin and Wilkinson 2008b),
where endospores received a number of physico-chemical
treatments that removed specific components or halted
germination at defined points and were then analysed
using FCM. Unique staining profiles were found in the
case of e.g. endospores lacking cortex.
Building on work with generic dyes such as PI, the
more careful application of specific dyes (possibly, even
antibody based) detecting damage to a particular compo-
nent such as the cortex could allow elucidation of mecha-
nisms of action of sporicidal compounds or treatments.
Such work has been carried out recently by Thompson
et al. (Thompson et al. 2007), who measured structural
differences in the exosporium of B. anthracis mutants
lacking the bclB gene by using fluorescent antibodies
raised against the exosporial proteins, BclA and BclB.
FCM could potentially be employed to screen for mutants
displaying e.g. increased or decreased resistance to UV
light, using a fluorochrome detecting oxidative damage to
DNA such as the 8-oxoguanine-binding protein contained
in Biotrin OxyDNA Assay (Biotrin, Dublin, Ireland) and
so assist in the elucidation of genes responsible for resis-
tance. As FCM is a tool par excellence for the study of
heterogeneity within populations, the detection and quan-
tification of heterogeneity within endospore populations
as a response to a challenge such as wet heat could be
studied, and phenomena involving a small proportion of
Study of B. cereus using flow cytometry U.P. Cronin and M.G. Wilkinson
6 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16
ª 2009 The Authors
any given endospore population such as superdormancy
(Clery-Barraud et al. 2004) could be understood more
fully.
Generally, a greater understanding of germination and
outgrowth events would benefit from the application of
FCM methods. Factors influencing the germination of
B. cereus endospores could be rapidly screened for
through the use of dyes such as CFDA (Cronin and
Wilkinson 2007) or tetrazolium, which detect insipient
metabolism without the need for culturing. Because FCM
can generate data from large numbers of individual
events, the heterogeneity of a population in its response
to conditions favouring germination could be determined,
allowing the design of more accurate models of germina-
tion (Collado et al. 2004, 2006). Genetically modified
strains with fluorescent reporter genes, such as GFP, fused
with promoters for genes expressed during germination
and outgrowth could be utilized to fully elucidate the cas-
cade of events leading to outgrowth (Smits et al. 2005;
Veening et al. 2005, 2006). By developing stains specific
for the various stages of germination, mechanisms of
action of compounds that affect germination could be
elucidated, and rapid screening for inhibitors of germina-
tion subsequently performed.
Sporulation
The sporulation of vegetative cells of B. cereus is of direct
practical relevance to food safety. Studies on this topic
can be divided into those based on plate counting meth-
ods to quantify the degree of sporulation of cultures in
artificial media or foods under a number of conditions
(Raso et al. 1995, 1998; Ryu et al. 2005; Ryu and Beuchat
2005) and those dealing with genetic control of sporula-
tion, which examine the role of specific genes in the dif-
ferentiation of a vegetative cell to an endospore (Piggot
1985; Doi 1989; Oosthuizen et al. 2002).
Should fluorescent dyes specific to various structural
components of endospores such as the exosporium or
protein coat become available, or if one had the means
to produce fluorescent antibodies specific to these com-
ponents, FCM could perform real-time analysis of spor-
ulation – a process that takes up to 6 h to complete
and involves a very specific sequence of events (Piggot
1985; Dricks 2002). Sporulating cultures of B. cereus
could be differentially stained at various time points
during the course of sporulation to determine the exact
timing of the appearance of e.g. cortex. Extensive experi-
ments could be carried out to examine the effect of
environmental factors on sporulation events with far
more alacrity and facility than at present; such experi-
ments are currently carried out using microscopy (Pop-
ham et al. 1996).
Studies on the genetic control of sporulation using
GFP-transformed strains together with FCM are now
beginning to appear in the literature (Veening et al. 2005,
2006; Lulko et al. 2007). Such a strategy allows the real-
time visualization of the expression of the many genes
involved in the differentiation of the mother cell that
results in the production of the endospore. With the new
generation of GFP derivatives and cytometers capable of
taking measurements in 12 channels (Chan and Holmes
2004; Hawley et al. 2004; Bonetta 2005; Wells 2006),
FCM offers the possibility of simultaneously studying the
expression of up to 12 genes. GFP-transformed Bacillus
spp. are also now being used in novel flow cytometric
studies such as that conducted by Kim et al. (2007) on
the utility of CotG fusions for bacterial surface display.
A more mundane use of FCM for the study of sporula-
tion involves quantification of the extent of sporulation
within a culture. Sporulation begins within the mother
cell and, as new compounds begin to be produced, the
process of differentiation commences with alterations in
the refractile properties of the mother cell (Gould 1999;
Stevenson and Segner 2001). The increase in refractive
index is reflected in increased SSC (Stopa 2000; Comas
and Vives-Rego 2002; Holm et al. 2004). FCM can there-
fore be used to rapidly quantify the extent of sporulation
of cultures with far superior throughput than conven-
tional techniques such as phase contrast microscopy or
plate counting (Nicholson and Setlow 1990; Priest and
Grigorova 1990; Leuschner and Lillford 2000; Ryu et al.
2005).
Taxonomy
For a number of years, the taxonomy of B. cereus and
various similar species comprising the B. cereus group has
been the subject of some debate (Wolf and Barker 1968;
Cowan and Steel 1974; Goepfert 1976; Claus and Berkeley
1986; Cherif et al. 2003a; Tourasse et al. 2006; Arnesen
et al. 2008). According to some authors, the B. cereus
group, which includes B. anthracis, B. cereus, B. mycoides,
B. pseudomycoides, B. thuringiensis and B. weihenstephan-
ensis, should be combined as one species (Goepfert 1976;
Claus and Berkeley 1986; Bennet and Belay 2001; Chen
and Tsen 2002), which some authors already refer to as
B. cereus sensu lato (de Clerck et al. 2004; Tourasse et al.
2006; van der Auwera et al. 2007). Hence, the strain
designation of isolates and the phylogeny of the B. cereus
group are now the subject of intense study. Because tradi-
tional serological, biochemical and morphological markers
have not proven useful in discriminating between the
members of the B. cereus group (Baat 1999; Arnesen et al.
2008), a wide range of new DNA-based techniques are
being used to elucidate the relationship between its
U.P. Cronin and M.G. Wilkinson Study of B. cereus using flow cytometry
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16 7
members (Griffiths and Schraft 2002; Cherif et al.
2003a,b; Helgason et al. 2004; Tourasse et al. 2006) and
between the B. cereus group and other members of the
genus (Joung and Cote 2002; Anderson et al. 2005).
While this whole area may not appear very relevant to
food safety microbiologists, useful data, e.g. the toxigenic
(Hansen and Hendriksen 2001; Ghelardi et al. 2002; Yang
et al. 2005) and pathogenic (Bundy et al. 2005; van der
Auwera et al. 2007) potential of various B. cereus strains,
are being generated.
With the use of the correct nucleic acid probes, fluores-
cent in situ hybridization (FISH)-FCM can be used to
assign strains to taxa with a very high degree of certainty
(Porter et al. 1996; Sincock and Robinson 2001; Zoeten-
dal et al. 2002; Zwirglmaier 2005) and could potentially
address some of the phylogenetic controversies mentioned
above. However, FISH-FCM, which is technically
demanding and expensive to perform, is best suited to
detection and ecological studies, where the physiological
status as well as identity of microbes present need to be
determined (Clarke and Pinder 1998; Porter and Pickup
2000; Tang et al. 2005; Friedrich and Lenke 2006;
Kalyuzhnaya et al. 2006; Schellenberg et al. 2006). While
PCR and other non-FISH techniques appear to be the
techniques of choice for phylogenetic studies of B. cereus
(Tourasse et al. 2006), FCM still has a role to play in tax-
onomic studies of the organism apart from genetic analy-
ses. Any technique capable of simultaneously measuring
up to 12 parameters per cell (such as FCM) is bound to
enhance the collection of phenotypic data that can be
then used to further characterize and categorize strains.
Phenotypic data that may be collected using FCM include
enzyme activities, antibiotic resistance, cell size, refractive
index, presence of para-sporal crystals, numbers of cells
comprising each CFU and genome size (Reardon and
Scheper 1993; Porter et al. 1996; Jacobsen and Jakobsen
1999; Nebe-von-Caron et al. 2000). FCM can also be used
to perform serotyping, another helpful technique in the
classification of micro-organisms (McClelland and Pinder
1994; Edwards et al. 1996; Porter et al. 1996; Barbesti
et al. 2000; Sincock and Robinson 2001). The main
advantage in using FCM to acquire phenotypic and sero-
logical data for taxonomic studies is rapidity and high
throughput – many strains may be analysed in a short
space of time.
Disadvantages associated with flow cytometry
The main disadvantage of any FCM-based method is cost.
In addition to the capital investment needed for the pur-
chase of the instrument and the funds required for the
instrument’s service and maintenance, reagents such as
fluorogenic dyes, enumeration beads and, especially, fluo-
rescence-labelled antibodies and nucleic acid probes are
quite expensive (Raybourne and Tortorello 2003). Because
of financial considerations, the use of FCM is an option
unavailable to many workers, especially those in develop-
ing countries. Even in laboratories equipped with a flow
cytometer, the cost of reagents can be a key factor in the
decision as to whether this particular experimental
approach is used or not. However, in some cases, the cost
associated with a particular FCM method is offset by the
technique’s advantages, above all, rapidity: an FCM detec-
tion method could be justified in certain scenarios where
a quick result is needed such as the investigation into the
outbreaks of food poisoning, the diagnosis of ocular
infections and the rapid screening of foodstuffs for their
early release into the market (Flint et al. 2006). Addition-
ally, for situations such as the online measurement of
industrial fermentations (Muller and Babel 2003; Maskow
et al. 2005), where cost of analyses is again outweighed by
the need for rapidity, the application of FCM to measure
growth rates may become a standard practice.
Another consideration with FCM is technical difficulty.
Considerable experience and expertise is required to
extract quality data from FCM analyses. For microbial
cytometrists in particular, limited technical support is
available from manufacturers, suppliers or the wider com-
munity of cytometrists. Additionally, there is a lack of
reagents specific for microbial applications such as suit-
able calibration beads, monoclonal antibodies, ready-
to-use kits and sample preparation devices. The type and
technical capability of each cytometer has a major influ-
ence on the ability to perform microbiological analysis –
many commercially available flow cytometers are incapa-
ble of resolving vegetative cells and endospores on the
basis of their light scatter properties. The inability to
satisfactorily analyse bacteria without a high degree of
‘tinkering’ is a common feature of many commercially
available flow cytometers, the majority of which were
designed for the analysis of much larger eukaryotic cells.
Until the market for instruments specifically intended for
the analysis of bacteria develops, this will remain the case.
Conclusions
Bacillus cereus represents a hazard to consumer and food
industry alike, and much work must be carried out in
order to gain a more comprehensive understanding of
many aspects of this complex spore-former, especially
endospore resistance mechanisms, the effects of sublethal
treatments on both vegetative cells and endospores and
the effect of storage on the recovery of damaged endo-
spores. The utility of FCM in the field of microbiology is
continually being demonstrated through the publication
of an increasing number of diverse and groundbreaking
Study of B. cereus using flow cytometry U.P. Cronin and M.G. Wilkinson
8 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16
ª 2009 The Authors
studies. Already, a respectable body of flow cytometric
studies on B. cereus and other members of the genus,
Bacillus, is extant. As demonstrated above, FCM is a tech-
nique capable of generating significantly novel data and
allows the design and execution of experiments not possi-
ble with any other technique e.g. real-time analysis of
gene expression in a large number of individual cells.
While further development is required to elevate the
overall area of microbial cytometry to the levels of sophis-
tication seen in clinical cytometric studies, it is clear that
FCM has the potential to greatly enhance and render the
study of B. cereus more efficient. With the recent com-
mercial availability of a new generation of less costly
cytometers (e.g. Acccuri’s C6 Flow Cytometer� System or
Partec’s CyFlow� SL), FCM is a technique that will find
itself being applied in more and more laboratories. It is
the responsibility of current advocates of microbial FCM
to pass robust and useful applications, which have been
shown to perform as well as or better than traditional
techniques, on to the new generation of users.
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