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: martin.wilkinson@ul.ie

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

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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.

References

Abee, T. and Delves-Broughton, J. (2003) Bacteriocins – nisin.

In Food Preservatives ed. Russel, N.J. and Gould, G.W.

pp. 146–178. New York: Kluwer Academic.

Adams, M. (2005) Detecting sublethally damaged cells. In

Understanding Pathogen Behaviour ed. Griffiths, M.W.

pp. 198–211. New York: CRC Press.

Agata, N., Ohata, M. and Yokoyama, K. (2002) Production of

Bacillus cereus emetic toxin (cereulide) in various foods.

Int J Food Microbiol 73, 23–27.

Akesson, A., Hedstrom, S.A. and Ripa, T. (1991) Bacillus

cereus: a significant pathogen in postoperative and

post-traumatic wounds on orthopaedic wards. Scand

J Infect Dis 23, 71–77.

Albertini, M.C., Accorsi, A., Teodori, L., Pierfelici, L.,

Uguccioni, F., Rocchi, M.B.L., Burattini, S. and Citterio,

B. (2006) Use of multiparameter analysis for Vibrio

alginolyticus viable but nonculturable state determination.

Cytometry 69A, 260–265.

Alvarez-Barrientos, A., Arroyo, J., Canton, R., Nombela, C.

and Sanchez-Perez, M. (2000) Applications of flow cyto-

metry to clinical microbiology. Clin Microbiol Rev 13,

167–195.

Anderson, I., Sorokin, A., Kapatral, V., Reznik, G., Bhattach-

arya, A., Mikhailova, N., Burd, H., Joukov, V. et al. (2005)

Comparative genome analysis of Bacillus cereus group

genomes with Bacillus subtilis. FEMS Microbiol Lett 250,

175–184.

Andersson, M.A., Mikkola, R., Helin, J., Andersson, M.C. and

Salkinoja-Salonen, M.S. (1998) A novel sensitive bioassay

for detection of Bacillus cereus emetic toxin and related dep-

sipeptide ionophores. Appl Environ Microbiol 64, 1338–1343.

Aran, N. (2001) The effect of calcium and sodium lactates on

growth from spores of Bacillus cereus and Clostridium per-

fringens in a ‘sous-vide’ beef goulash under temperature

abuse. Int J Food Microbiol 63, 117–123.

Arnesen, L.P.S., Fagerlund, A. and Granum, P.E. (2008) From

soil to gut: Bacillus cereus and its food poisoning toxins.

FEMS Microbiol Rev 32, 579–606.

Assuncao, P., Antunes, N.T., Rosales, R.S., de la Fe, C., Pove-

da, C., Poveda, J.B. and Davey, H.M. (2006) Flow cyto-

metric method for the assessment of the minimal

inhibitory concentrations of antibacterial agents to Myco-

plasma agalactiae. Cytometry 69A, 1071–1076.

van der Auwera, G.A., Timmery, S., Hoton, F. and Mahillon,

J. (2007) Plasmid exchanges among members of the

Bacillus cereus group in foodstuffs. Int J Food Microbiol

113, 164–172.

Baat, C.A. (1999) Bacillus cereus. In Encyclopedia of Food

Microbiol ed. Robinson, J.P., Batt, C.A. and Patel, P.D.

pp. 119–124. New York: Academic Press.

Banerjee, M. and Sarkar, P.K. (2004) Growth and enterotoxin

production by sporeforming bacterial pathogens from

spices. Food Control 15, 491–496.

Barbesti, S., Citterio, S., Labra, M., Baroni, M.D., Neri, M.G.

and Sgorbati, S. (2000) Two and three-colour fluorescence

flow cytometric analysis of immunoidentified viable

bacteria. Cytometry 40, 214–218.

Barbour, W.M. and Tice, G. (1997) Genetic and immunologic

techniques for detecting foodborne pathogens and toxins.

In Food Microbiology: Fundamentals and Frontiers ed.

Doyle, A., Belichat, B. and Montville, C. pp. 710–727.

Washington: American Society for Microbiology.

Beecher, D.J. and Macmillan, J.D. (1991) Characterization of

the components of hemolysin BL from Bacillus cereus.

Infect Immun 99, 1778–1784.

Beecher, D.J., Olsen, T.W., Somers, E.B. and Wong, C.L.

(2000) Evidence for contribution of tripartite haemolysin

BL, phosphatidylcholine-preferring phospholipase C, and

collagenases to virulence of Bacillus cereus endophthalmitis.

Infect Immun 68, 5269–5276.

Bennet, R.W. and Belay, N. (2001) Bacillus cereus. In Micro-

biological Examination of Foods ed. Downes, F.P. and Ito,

K. pp. 311–316. Washington, DC, USA: American Public

Health Association.

Betz, J.W., Aretz, W. and Hartel, W. (1984) Use of flow

cytometry in industrial microbiology for strain improve-

ment programmes. Cytometry 5, 145–150.

Black, E.P., Koziol-Dube, K., Guan, D., Wei, J., Setlow, B.,

Cortezzo, D.E., Hoover, D.G. and Setlow, P. (2005)

Factors influencing germination of Bacillus subtilis spores

via activation of nutrient receptors by high pressure. Appl

Environ Microbiol 71, 5879–5887.

Black, E.P., Wei, J., Atluri, S., Cortezzo, D.E., Koziol-Dube, K.,

Hoover, D.G. and Setlow, P. (2006) Analysis of factors

influencing the rate of germination of spores of Bacillus

subtilis by very high pressure. J Appl Microbiol 102, 65–76.

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 9

Blake, M.R. and Weimer, B.C. (1997) Immunomagnetic detec-

tion of Bacillus stearothermophilus spores in food and envi-

ronmental samples. Appl Environ Microbiol 63, 1643–1646.

Bonetta, L. (2005) Flow cytometry smaller and better. Nat

Methods 2, 785–795.

Bouwer-Hertzberger, S.A. and Mossel, D.A.A. (1982) Quantita-

tive isolation and identification of Bacillus cereus. In Isola-

tion and Identification Methods for Food Poisoning

Organisms ed Corry, J., Roberts, D. and Skinner, F.A. pp.

255–259. New York: Academic Press Inc.

Braga, P.C., Bovio, C., Culici, M. and dal Sasso, M. (2003)

Flow cytometric assessment of susceptibilities of Strepto-

coccus pyogenes to erythromycin and rokitamycin.

Antimicrob Agents Chemother 47, 408–412.

Browne, N. and Dowds, B.C.A. (2001) Heat and salt stress in

the food pathogen Bacillus cereus. J Appl Microbiol 91,

1085–1094.

Browne, N. and Dowds, B.C.A. (2002) Acid stress in the food

pathogen Bacillus cereus. J Appl Microbiol 92, 404–414.

Bundy, J.G., Willey, T.L., Castell, R.S., Ellar, D.J. and Brindle,

K.M. (2005) Discrimination of pathogenic clinical isolates

and laboratory strains of Bacillus cereus by NMR-based

metabolomic profiling. FEMS Microbiol Lett 242, 127–136.

Bunthof, C.J., van den Braak, S., Breeuwer, P., Rombouts,

F.M. and Abee, T. (2000) Fluorescence assessment of

Lactococcus lactis viability. Int J Food Microbiol 55, 291–

294.

Callegan, M.C., Jett, B.D., Hancock, L.E. and Gilmore, M.S.

(1999) Role of haemolysin BL in the pathogenesis of extra-

intestinal Bacillus cereus infection assessed in an endo-

phthalmitis model. Infect Immun 67, 3357–3366.

Chaibi, A., Ababouch, L.H., Belasri, K., Boucetta, S. and Busta,

F.F. (1997) Inhibition of germination and vegetative

growth of Bacillus cereus T and Clostridium botulinum 62A

spores by essential oils. Food Microbiol 14, 161–174.

Chan, F.K. and Holmes, K.L. (2004) Flow cytometric analysis

of fluorescent resonance energy transfer. In Methods in

Molecular Biology: Flow Cytometry Protocols ed. Hawley,

T.S. and Hawley, R.G. pp. 281–292. New Jersey, USA:

Humana Press Inc.

Chen, M.L. and Tsen, H.Y. (2002) Discrimination of Bacillus

cereus and Bacillus thuringiensis with 16S rRNA and gyrB

gene based PCR primers and sequencing of their annealing

sites. J Appl Microbiol 92, 912–919.

Chen, C.H., Ding, H.C. and Chang, T.C. (2001) Rapid identifi-

cation of Bacillus cereus based on the detection of a 28.5-

kilodalton cell surface antigen. J Food Prot 64, 348–354.

Cherif, A., Borin, S., Rizzi, A., Ouzari, H., Boudabous, A. and

Daffonchio, D. (2003a) Bacillus anthracis diverges from

related clades of the Bacillus cereus group in 16S-23S ribo-

somal DNA intergenic transcribed spacers containing

tRNA genes. Appl Environ Microbiol 69, 33–40.

Cherif, A., Brusetti, L., Borin, S., Rizzi, A., Boudabous, A.,

Khyami-Horani, H. and Daffonchio, D. (2003b) Genetic

relationship in the ‘‘Bacillus cereus group’’ by rep-PCR

fingerprinting and sequencing of a Bacillus anthracis-spe-

cific rep-PCR fragment. J Appl Microbiol 94, 1108–1119.

Choma, C., Guinebretiere, M.H., Carlin, F., Schmitt, P., Velge,

P., Granum, P.E. and Nguyen-The, C. (2000) Prevalence,

characterization and growth of Bacillus cereus in commer-

cial cooked chilled foods containing vegetables. J Appl

Microbiol 88, 617–625.

Chorin, E., Thault, D., Cleret, J.-J. and Bourgeous, C.M.

(1997) Modelling Bacillus cereus growth. Int J Food Micro-

biol 38, 229–234.

Clarke, R.G. and Pinder, A.C. (1998) Improved detection of

bacteria by flow cytometry using a combination of anti-

body and viability markers. J Appl Microbiol 84, 577–584.

Claus, D. and Berkeley, A.C.L. (1986) Genus Bacillus. In Ber-

gey’s Manual of Systematic Bacteriology ed. Sneath, P. pp.

1105–1139. Baltimore: The Williams and Wilkins Co.

Clavel, T., Carlin, F., Lairon, D., Nguyen-The, C. and Schmitt,

P. (2004) Survival of Bacillus cereus spores and vegetative

cells in acid media simulating human stomach. J Appl

Microbiol 97, 214–219.

de Clerck, E., van Mol, K., Jannes, G., Rossau, R. and de Vos,

P. (2004) Design of a 5’ exonucluease-based real-time PCR

assay for simultaneous detection of Bacillus licheniformis,

members of the ‘‘B. cereus group’’ and B. fumarioli in gela-

tine. Lett Appl Microbiol 39, 109–115.

Clery-Barraud, C., Gaubert, A., Masson, P. and Vidal, D.

(2004) Combined effects of high hydrostatic pressure and

temperature for inactivation of Bacillus anthracis spores.

Appl Environ Microbiol 70, 635–637.

Coder, D.M. (1997) Assessment of cell viability. In Current

Protocols in Cytometry ed Robinson, J.P. pp. 9.2.1–9.2.14.

New York: John Wiley and Sons Inc.

Collado, J., Fernandez, A., Cunha, L.M., Ocio, M.J. and Marti-

nez, A. (2003a) Improved model based on the Weibull dis-

tribution to describe the combined effect of pH and

temperature on the heat resistance of Bacillus cereus in

carrot juice. J Food Prot 66, 978–984.

Collado, J., Fernandez, A., Rodrigo, M., Camats, J. and Marti-

nez Lopez, A. (2003b) Kinetics of deactivation of Bacillus

cereus spores. Food Microbiol 20, 545–548.

Collado, J., Fernandez, A., Rodrigo, M. and Martinez Lopez,

A. (2004) Variation of the spore population of a natural

source strain of Bacillus cereus in the presence of inosine.

J Food Prot 67, 934–938.

Collado, J., Fernandez, A., Rodrigo, M. and Martinez Lopez,

A. (2006) Modelling the effect of a heat shock and germi-

nant concentration on spore germination of a wild strain

of Bacillus cereus. Int J Food Microbiol 106, 85–89.

Comas, J. and Vives-Rego, J. (2002) Cytometric monitoring of

growth, sporogenesis and spore cell sorting in Paenibacillus

polymyxa (formerly Bacillus polymyxa). J Appl Microbiol

92, 475–481.

Coote, P.J., Billon, C.M.-P., Pennell, S., McClure, P.J., Ferdi-

nando, D.P. and Cole, M.B. (1994) The use of confocal

scanning laser microscopy (CSLM) to study the germina-

Study of B. cereus using flow cytometry U.P. Cronin and M.G. Wilkinson

10 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16

ª 2009 The Authors

tion of individual spores of Bacillus cereus. J Microbiol

Meth 21, 193–208.

Coroller, L., Leguerinel, I. and Mafart, P. (2001) Effect of

water activities of heating and recovery media on apparent

heat resistance of Bacillus cereus spores. Appl Environ

Microbiol 67, 317–322.

Cortezzo, D.E., Setlow, B. and Setlow, P. (2004) Analysis of

the action of compounds that inhibit the germination of

spores of Bacillus species. J Appl Microbiol 96, 725–741.

Cowan, S.T. and Steel, A.B. (1974) Manual for the Identifica-

tion of Medical Bacteria. Cambridge: Cambridge University

Press.

Cronin, U.P. and Wilkinson, M.G. (2007) The use of flow

cytometry to study the germination of Bacillus cereus

endospores. Cytometry 71A, 143–153.

Cronin, U.P. and Wilkinson, M.G. (2008a) Bacillus cereus

endospores exhibit a heterogeneous response to heat-treat-

ment and low temperature storage. Food Microbiol 25,

235–243.

Cronin, U.P. and Wilkinson, M.G. (2008b) Monitoring

changes in germination and permeability of Bacillus cereus

endospores following chemical, heat and enzymatic treat-

ments using flow cytometry. J Rapid Meth Aut Microbiol

16, 164–184.

Cronin, U.P. and Wilkinson, M.G. (2008c) Monitoring growth

phase-related changes in phosphatidylcholine-specific

phospholipase C production, adhesion properties and

physiology of Bacillus cereus vegetative cells. J Ind Micro-

biol Biotechnol 35, 1695–1703.

Cronin, U.P. and Wilkinson, M.G. (2008d) Physiological

response of Bacillus cereus vegetative cells to simulated

food processing treatments. J Food Protect. 71, 2168–2176.

D’Costa, S.S., Wilkinson, J.G. and Rabellino, E. (2006) Proteo-

mic strategies to analyze cell-free fractions from activated

peripheral blood mononuclear cultures. In ISAC XXIII

International Conference ed. Robinson, J.P. p. 171. Quebec,

Canada: ISAC.

Dahl, M.K. (1999) Bacillus. In Encyclopedia of Food Microbiol-

ogy ed. Robinson, J.P., Batt, C.A. and Patel, P.D. pp. 113–

158. New York: Academic Press.

Davey, H.M. (1994) . Flow Cytometry of Microorganisms.

Thesis. Institute of Biological Sciences. Aberystwyth:

University of Wales.

Davey, H.M. and Kell, D.B. (1996) Flow cytometry and cell

sorting of heterogeneous microbial populations: the

importance of single-cell analyses. Microbiol Rev 60, 641–

696.

Davey, H.M., Weichart, D.H., Kell, D.B. and Kaprelyants, A.S.

(1999) Estimation of microbial viability using flow cytom-

etry. In Current Protocols in Cytometry ed. Robinson, J.P.

pp. 11.13.11–11.13.20. New York: John Wiley and Sons

Inc.

Diaper, J.P. and Edwards, C. (1994) Flow cytometric detection

of viable bacteria in compost. FEMS Microbiol Ecol 14,

213–220.

Doi, R.H. (1989) Sporulation and germination. In Bacillus ed.

Harwood, C.R. pp. 169–215. New York: Plenum Press.

Dricks, A. (2002) Maximum shields: the assembly and function

of the bacterial spore coat. Trends Microbiol 10, 251–254.

Edwards, C., Diaper, J. and Porter, J. (1996) Flow cytometry

for the targeted analysis of the structure and function of

microbial populations. In Molecular Approaches to Environ-

mental Microbiology ed. Pickup, R.W. and Saunders, J.R.

pp. 137–162. Cambridge: University Press.

Elshal, M.F. and McCoy, J.P. (2006) Multiplex bead array

assays: performance evaluation and comparison of sensitiv-

ity to ELISA. Methods 33, 317–323.

Ernst, C., Schulenburg, J., Jakob, P., Dahms, S., Martinez

Lopez, A., Nychas, G., Werber, D. and Klein, G. (2006)

Efficacy of amphoteric surfactant- and peracetic acid-based

disinfectants on spores of Bacillus cereus in vitro and on

food premises of the German armed forces. J Food Prot 69,

1605–1610.

Fernandez, A., Ocio, M.J., Fernandez, P.S., Rodrigo, M. and

Martinez, A. (1999) Application of nonlinear regression

analysis to the estimation of kinetic parameters for two

enterotoxigenic strains of Bacillus cereus spores. Food

Microbiol 16, 607–613.

Finlay, W.J.J., Logan, N.A. and Sutherland, A.D. (2002) Bacil-

lus cereus emetic toxin production in relation to dissolved

oxygen tension and sporulation. Food Microbiol 19, 423–

430.

Flint, S., Drocourt, J.-L., Walker, K., Stevenson, B., Dwyer, M.,

Clarke, I. and McGill, D. (2006) A rapid, two-hour

method for the enumeration of total viable bacteria in

samples from commercial milk powders and whey protein

concentrate manufacturing plants. Int Dairy J 16, 379–384.

Folmsbee, M.J., McInerney, M.J. and Nagle, D.P. (2004)

Anaerobic growth of Bacillus mojavensis and Bacillus subtil-

is requires deoxyribonucleosides or DNA. Appl Environ

Microbiol 70, 5252–5257.

Forsythe, S.J. (2000) Chapter 6: Methods of detection. In The

Microbiology of Safe Food ed. Forsythe, S.J. Oxford: Black-

well Science Ltd.

Friedrich, U. and Lenke, J. (2006) Improved enumeration of

lactic acid bacteria in mesophilic dairy starter cultures by

using multiplex quantitative real-time PCR and flow

cytometry-fluorescence in situ hybridization. Appl Environ

Microbiol 72, 4163–4171.

Gaillard, S., Leguerinel, I. and Mafart, P. (1998) Modelling

combined effects of temperature and pH on the heat resis-

tance of spores of Bacillus cereus. Food Microbiol 15, 625–

630.

Galeano, B., Korff, E. and Nicholson, W.L. (2003) Inactivation

of vegetative cells, but not spores, of Bacillus anthracis,

B. cereus, and B. subtilis on stainless steel surfaces coated

with an antimicrobial silver- and zinc-containing zeolite

formulation. Appl Environ Microbiol 69, 4329–4331.

Gatti, M., Bernini, V., Lazzi, C. and Neviani, E. (2006)

Fluorescence microscopy for studying the viability of

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 11

micro-organisms in natural whey starters. Lett Appl Micro-

biol 42, 338–343.

Ghelardi, E., Celandroni, F., Salvetti, S., Barsotti, C., Baggiani, A.

and Senesi, S. (2002) Identification and characterization of

toxigenic Bacillus cereus isolates responsible for two food-

poisoning outbreaks. FEMS Microbiol Lett 208, 129–134.

Gilmore, M.S., Callegan, M.C. and Jett, B.D. (1999) Entero-

coccus faecalis cytolysin and Bacillus cereus bi- and tri-com-

ponent haemolysins. In The Comprehensive Sourcebook of

Bacterial Protein Toxins ed. Alouf, J.E. and Freer, J.H. pp.

419–434. London: Academic Press.

Goepfert, J.M. (1976) Bacillus cereus. In Compendium of Meth-

ods for the Microbiological Examination of Foods ed. Speck,

M.L. pp. 417–423. Washington: American Public Health

Association.

Gould, G.W. (1999) Bacterial endospores. In Encyclopedia of

Food Microbiol ed. Robinson, R.K., Batt, C.A. and Patel,

P.D. pp. 168–172. New York: Academic Press.

Granum, P.E. and Byrnestad, S. (1999) Bacterial toxins as food

poisons. In The Comprehensive Sourcebook of Bacterial Pro-

tein Toxins ed. Alouf, J.E. and Freer, J.H. pp. 669–681.

London: Academic Press.

Griffiths, M.W. and Schraft, H. (2002) Bacillus cereus food poi-

soning. In Foodborne Diseases ed Cliver, D.O. and Rie-

mann, H.P. pp. 261–270. Amsterdam: Academic Press.

Gunasekera, T.S., Veal, D.A. and Attfield, P.V. (2003) Potential

for broad applications of flow cytometry and fluorescence

techniques in microbiological and somatic cell analysis of

milk. Int J Food Microbiol 85, 269–279.

Hansen, B.M. and Hendriksen, N.B. (2001) Detection of

enterotoxic Bacillus cereus and Bacillus thuringensiensis

strains by PCR analysis. Appl Environ Microbiol 67, 185–

189.

Hansen, L.T., Austin, J.W. and Gill, T.A. (2001) Antibacterial

effect of protamine in combination with EDTA and refrig-

eration. Int J Food Microbiol 66, 149–161.

Hansen, B.M., Hoiby, P.E., Jensen, G.B. and Hendriksen, N.B.

(2003) The Bacillus cereus bceT enterotoxin sequence reap-

praised. FEMS Microbiol Lett 223, 21–24.

Haque, M.A. and Russel, N.J. (2004) Strains of Bacillus cereus

vary in the phenotypic adaptation of their membrane lipid

composition in response to low water activity, reduced

temperature and growth in rice starch. Microbiology 150,

1397–1404.

Harwood, C.R. and Archibald, A.R. (1990) Growth, mainte-

nance and general techniques. In Molecular Biological

Methods for Bacillus ed. Harwood, C.R. and Cutting, S.M.

pp. 1–24. Chichester, UK: John Wiley and Sons Ltd.

Hawley, T.S., Herbert, D.J., Eaker, S.S. and Hawley, R.G.

(2004) Multiparameter flow cytometry of fluorescent pro-

tein reporters. In Methods in Molecular Biology: Flow

cytometry protocols ed. Hawley, T.S. and Hawley, R.G. pp.

219–236. New Jersey, USA: Humana Press Inc.

Heijmans-Antonissen, C., Wesseldijk, F., Munnikes, R.J.M.,

Huygen, J.P.M., van der Meijden, P., Hop, W.C.J.,

Hooijkaas, H. and Zijlstra, F.J. (2006) Multiplex bead array

assay for detection of 25 soluble cytokines in blister fluid

of patients with complex regional pain syndrome type 1.

Mediators Inflamm 1, 28398.

Helgason, E., Tourasse, N., Meisal, R., Caugant, D.A. and Kol-

sto, A.-B. (2004) Multilocus sequence typing scheme for

bacteria of the Bacillus cereus group. Appl Environ Micro-

biol 70, 191–201.

Hergenrother, P.J. and Martin, S.F. (1997) Determination

of the kinetic parameters for phospholipase C (Bacillus

cereus) on different phospholipid substrates using a

chromogenic assay based on the quantitation of inorganic

phosphate. Anal Biochem 251, 45–49.

Hibi, K., Abeb, A., Ohashi, E., Mitsubayashi, K., Ushio, H.,

Hayashi, T., Rena, H. and Endoa, H. (2006) Combination

of immunomagnetic separation with flow cytometry for

detection of Listeria monocytogenes. Anal Chim Acta 573–

574, 158–163.

Hoefel, D., Grooby, W.L., Monis, P.T., Andrews, S. and Saint,

C.P. (2003) A comparative study of carboxyfluorescein

diacetate and carboxyfluorescein diacetate succinimidyl

ester as indicators of bacterial activity. J Microbiol Meth

52, 379–388.

Holbrook, R. and Anderson, J.M. (1980) An improved

selective and diagnostic medium for the isolation and

enumeration of Bacillus cereus in foods. Can J Microbiol

26, 753–759.

Holm, C., Mathiasen, T. and Jespersen, L. (2004) A flow cyto-

metric technique for quantification and differentiation of

bacteria in bulk tank milk. J Appl Microbiol 97, 935–941.

Jacobsen, C.N. and Jakobsen, M. (1999) Flow cytometry. In

Encyclopedia of Food Microbiol ed. Robinson, R.K., Batt,

C.A. and Patel, P.D. pp. 826–834. New York: Academic

Press.

Joung, K.-B. and Cote, J.-C. (2002) Evaluation of ribosomal

RNA gene restriction patterns for the classification of

Bacillus species and related genera. J Appl Microbiol 92,

97–108.

Kalyuzhnaya, M.G., Zabinsky, R., Bowerman, S., Baker, D.R.,

Lidstrom, M.E. and Chistoserdova, L. (2006) Fluorescence

in situ hybridization-flow cytometry-cell sorting-based

method for separation and enrichment of type I and type

II methanotroph populations. Appl Environ Microbiol 72,

4293–4301.

Kaspar, C.W. and Tartera, C. (1990) Methods for detecting

microbial pathogens in food and water. In Techniques in

Microbial Ecology ed. Grigorova, R. and Norris, J.R. pp.

497–531. London: Academic Press.

Kim, K., Seo, J., Wheeler, K., Park, C., Kim, D., Park, S., Kim,

W., Chung, S.-I. et al. (2005) Rapid genotypic detection of

Bacillus anthracis and the Bacillus cereus group by multi-

plex real-time PCR melting curve analysis. FEMS Immunol

Med Microbiol 43, 301–310.

Kim, J.-H., Roh, C., Lee, C.-W., Kyung, D., Choi, S.-K., Jung,

H.-C., Pan, J.-G. and Kim, B.-G. (2007) Bacterial surface

Study of B. cereus using flow cytometry U.P. Cronin and M.G. Wilkinson

12 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16

ª 2009 The Authors

display of GPFuv on Bacillus subtilis spores. J Microbiol

Biotechnol 17, 677–680.

Kimanya, M.E., Mamiro, P.R.S., van Camp, J., Devlieghere, F.,

Opsomer, A., Kolsteren, P. and Debevere, J. (2003)

Growth of Staphylococcus aureus and Bacillus cereus during

germination and drying of finger millet and kidney beans.

Int J Food Sci Technol 38, 19–125.

Kwon, J.A., Yu, C.B. and Park, H.D. (2003) Bacteriocidal

effects and inhibition of cell separation of cinnamic alde-

hyde on Bacillus cereus. Lett Appl Microbiol 37, 61–65.

Laflamme, C., Ho, J.Z., Veillette, M., de Latremoille, M.C.,

Verrault, D., Meriaux, A. and Duchaine, C. (2005) Flow

cytometry analysis of germinating Bacillus spores, using

membrane potential dye. Arch Microbiol 183, 107–112.

Laflamme, C., Verrault, D., Ho, J.Z. and Duchaine, C. (2006)

Flow cytometry sorting protocol of Bacillus spore using

ultraviolet laser and autofluorescence as main sorting crite-

rion. J Fluoresc 16, 733–737.

Lampel, K.A., Dyer, D., Kornegay, L. and Orlandi, P.A. (2004)

Detection of Bacillus spores using PCR and FTA filters.

J Food Prot 67, 1036–1038.

Laurent, Y., Arino, S. and Rosso, L. (1999) A quantitative

approach for studying the effect of heat treatment condi-

tions on resistance and recovery of Bacillus cereus spores.

Int J Food Microbiol 48, 149–157.

Leser, T.D., Knarreborg, A. and Worm, J. (2008) Germination

and outgrowth of Bacillus subtilis and Bacillus licheniformis

spores in the gastrointestinal tract of pigs. J Appl Microbiol

104, 1025–1033.

Leuschner, R.G.K. and Lillford, P.J. (2000) Effects of hydration

on molecular mobility in phase-bright Bacillus subtilis

spores. Microbiology 149, 49–55.

Lindsay, D., Oosthuizen, M.C., Brozel, V.S. and von Holy, A.

(2002) Adaptation of a neutrophilic dairy-associated Bacil-

lus cereus isolate to alkaline pH. J Appl Microbiol 92, 81–

89.

Lloyd, D. (1999) Flow cytometry of yeasts. In Current Protocols

in Cytometry ed. Robinson, J.P. pp. 11.10.11–11.10.18.

New York: John Wiley and Sons Inc.

Lopes da Silva, T., Reis, A., Kent, C.A., Kosseva, M., Roseiro,

J.C. and Hewitt, C.J. (2005) Stress-induced physiological

responses to starvation periods as well as glucose and

lactose pulses in Bacillus licheniformis CCMI 1034

continuous aerobic fermentation processes as measured

by multi-parameter flow cytometry. Biochem Eng J 24,

31–41.

Lulko, A., Veening, J.-W., Buist, G., Smits, W.K., Blom, E.J.,

Beekman, A.C., Bron, S. and Kuipers, O.P. (2007) Produc-

tion and secretion stress caused by overexpression of heter-

ologous a-amylase leads to inhibition of sporulation and a

prolonged motile phase in Bacillus subtilis. Appl Environ

Microbiol 73, 5354–5362.

Mansour, M. and Milliere, J.-B. (2001) An inhibitory synergis-

tic effect of a nisin-monolaurin combination on Bacillus

sp. vegetative cells in milk. Food Microbiol 18, 87–94.

Maraha, N., Backman, A. and Jansson, J.K. (2004) Monitoring

physiological status of GFP-tagged Pseudomonas fluorescens

SBW25 under different nutrient conditions and in soil by

flow cytometry. FEMS Microbiol Ecol 51, 123–135.

Martinez, O.V., Gratzner, H.G., Malinin, T.I. and Ingram, M.

(1982) The effect of some b-lactam antibiotics on Escheri-

chia coli studied by flow cytometry. Cytometry 3, 129–133.

Maskow, T., Muller, S., Losche, A., Harms, H. and Kemp, R.

(2005) Control of continuous polyhydroxybutyrate synthe-

sis using calorimetry and flow cytometry. Biotechnol Bioeng

93, 541–552.

Mason, D.J., Lopez-Amoros, R., Allman, R., Stark, J.M. and

Lloyd, D. (1995) The ability of membrane potential dyes

and calcafluor white to distinguish between viable and

non-viable bacteria. J Appl Bacteriol 78, 309–315.

Mason, D.J., Mortimer, F.C. and Gant, V.A. (1999) Antibiotic

susceptibility testing by flow cytometry. In Current Proto-

cols in Cytometry ed. Robinson, J.P. pp. 1.8.1–11.18.19.

New York: John Wiley and Sons Inc.

Mathews, K.R. (2003) Rapid methods for microbial detection

in minimally processed foods. In Microbial Safety of Mini-

mally-Processed Foods ed. Novak, J.S., Sapers, G.M. and

Juneja, V.K. pp. 151–163. New York: CRC Press.

Mathys, A., Chapman, B., Bull, M., Heinz, V. and Knorr, D.

(2007) Flow cytometric assessment of Bacillus spore

response to high pressure and heat. Innov Food Sci Emerg

Technol 8, 519–527.

McClelland, R.G. and Pinder, A.C. (1994) Detection of low

levels of specific Salmonella species by fluorescent

antibodies and flow cytometry. J Appl Bacteriol 77, 440–

447.

McDonnell, G. and Denver-Russell, A. (1999) Antiseptics and

disinfectants: activity, action and resistance. Clin Microbiol

Rev 12, 147–179.

Miller, J.J., Scott, I.U., Flynn, H.W.J., Smiddy, W.E., Murray,

T.G., Berrocal, A. and Miller, D. (2008) Endophthalmitis

caused by Bacillus species. Am J Opthalmol 145, 883–888.

Moir, A. (2006) How do spores germinate? J Appl Microbiol

101, 526–530.

Morgan, E., Varro, R., Sepulveda, H., Ember, J.A., Apgar, J.,

Wilson, J., Lowe, L., Chen, R. et al. (2004) Cytometric

bead array: a multiplexed assay platform with applications

in various areas of biology. Clin Immunol 110, 252–266.

Mossell, D.A.A., Koopman, M.J. and Jongerius, E. (1967) Enu-

meration of Bacillus cereus in foods. Appl. Microbiol. 15,

650–653.

Muller, S. and Babel, W. (2003) Analysis of bacterial DNA pat-

terns-an approach for controlling biotechnological pro-

cesses. J Microbiol Meth 55, 851–858.

Natarajan, A. and Srienc, F. (2000) Glucose uptake rates of

single E.coli cells grown in glucose-limited chemostat cul-

tures. J Microbiol Meth 42, 87–96.

Nauta, M., Litman, S., Barker, G.C. and Carlin, F. (2003) A

retail and consumer phase model for exposure assessment

of Bacillus cereus. Int J Food Microbiol 83, 205–218.

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 13

Nebe-von-Caron, G., Stephens, P.J., Hewitt, C.J., Powell, J.R.

and Badley, R.A. (2000) Analysis of bacterial function by

multi-colour fluorescence flow cytometry and single cell

sorting. J Microbiol Meth 42, 97–114.

Nguefack, J., Budde, B.B. and Jakobsen, M. (2004) Five essen-

tial oils from aromatic plants of Cameroon: their

antibacterial activity and ability to permeabilise the

cytoplasmic membrane of Listeria innocua examined by

flow cytometry. Lett Appl Microbiol 39, 395–400.

Nguyen-The, C. and Broussolle, V. (2005) Bacillus cereus: fac-

tors affecting virulence. In Understanding Pathogen Behav-

iour ed. Griffiths, M.W. pp. 309–330. New York: CRC

Press.

Nicholson, W.L. and Setlow, P. (1990) Sporulation,

germination and outgrowth. In Molecular Biological

Methods for Bacillus ed. Harwood, C.R. and Cutting,

S.M. pp. 391–450. Chichester, UK: John Wiley and Sons

Ltd.

Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J.

and Setlow, P. (2000) Resistance of Bacillus endospores to

extreme terrestrial and extraterrestrial environments.

Microbiol Mol Biol Rev 64, 548–572.

Notermans, S. and Batt, C.A. (1998) A risk assessment

approach for food-borne Bacillus cereus and its toxins.

J Appl Microbiol 84, 51–61.

Notermans, S., Dufrenne, J., Teunis, P., Beumer, R., te Giffel,

M.C. and Weem, P.P. (1997) A risk assessment study of

Bacillus cereus present in pasteurized milk. Food Microbiol

14, 143–151.

Nunez, R., Kamau, S. and Grimm, F. (2001) Flow cytometric

assessment of drug susceptibility in Leishmania infantum

promastigotes. In Current Protocols in Flow Cytometry ed.

Robinson, J.P. pp. 11.14.11–11.14.19. New York: John

Wiley and Sons Inc.

O’Connor, L. and Maher, M. (1999) Molecular biology in

microbiological analysis – DNA-based methods for the

detection of food-borne pathogens. In Encyclopedia of Food

Microbiol ed. Robinson, R.K., Batt, C.A. and Patel, P.D.

pp. 1475–1481. New York: Academic Press.

Oosthuizen, M.C., Steyn, B., Theron, J., Cosette, P., Lindsay,

D., von Holy, A. and Brozel, V.S. (2002) Proteomic analy-

sis reveals differential protein expression by Bacillus cereus

during biofilm formation. Appl Environ Microbiol 68,

2770–2780.

Phillips, A.P. and Martin, K.L. (1988) Limitations of flow

cytometry for the specific detection of bacteria in mixed

populations. J Immunol Methods 106, 109–117.

Pianetti, A., Falcioni, T., Bruscolini, F., Sabatini, L., Sisti, E.

and Papa, S. (2005) Determination of the viability of

Aeromonas hydrophila in different types of water by flow

cytometry, and comparison with classical methods. Appl

Environ Microbiol 71, 7948–7954.

Piggot, P.J. (1985) Sporulation of Bacillus subtilis. In The

Molecular Biology of the Bacilli. pp. 73–108. New York:

Academic Press.

Popham, D.L., Helin, J., Costello, C.E. and Setlow, P. (1996)

Muramic lactam in peptidoglycan of Bacillus subtilis spores

is required for spore outgrowth but not for spore dehydra-

tion or heat resistance. Proc Natl Acad Sci USA 93, 15405–

15410.

Porter, J. and Pickup, R. (2000) Nucleic acid-based fluorescent

probes in microbial ecology: application of flow cytometry.

J Microbiol Meth 42, 75–79.

Porter, J., Deere, D., Pickup, R. and Edwards, C. (1996) Fluo-

rescent probes and flow cytometry: new insights into envi-

ronmental bacteriology. Cytometry 23, 91–96.

Priest, F.G. and Grigorova, R. (1990) Methods for studying the

ecology of endospore-forming bacteria. In Methods in

Microbiology ed. Grigorova, R. and Norris, J.R. pp. 565–

591. London: Academic Press.

Rajkovic, A., Uyttendaile, M., Ombregt, S.-A., Jaaskelainen,

E.L., Salkinoja-Salonen, M.S. and Debevere, J. (2006) Influ-

ence of type of food on the kinetics and overall production

of Bacillus cereus emetic toxin. J Food Prot 69, 847–852.

Raso, J., Palop, A., Bayarte, M., Condon, S. and Sala, F.J.

(1995) Influence of sporulation temperature on the heat

resistance of a strain of Bacillus licheniformis (Spanish Type

Culture Collection 4523). Food Microbiol 12, 357–361.

Raso, J., Barbosa-Canovas, G. and Swanson, B.G. (1998) Spor-

ulation temperature affects initiation of germination and

inactivation by high hydrostatic pressure of Bacillus cereus.

J Appl Microbiol 85, 17–24.

Raybourne, R.B. and Tortorello, M. (2003) Microscopy tech-

niques: DEFT and flow cytometry. In Detecting Pathogens

in Food ed. McMeekin, T.A. pp. 187–216. New York: CRC

Press.

Reardon, K.F. and Scheper, T.H. (1993) Determination of cell

concentration and characterization of cells. In Biotechnol-

ogy ed. Rehm, H.J., Reed, G., Puhler, A. and Stadler, P.

pp. 181–223. New York: VCH.

Reis, A., Lopes da Silva, T., Kent, C.A., Kosseva, M., Roseiro,

J.C. and Hewitt, C.J. (2005) Monitoring population

dynamics of the thermophilic Bacillus licheniformis

CCMI 1034 in batch and continuous cultures using multi-

parameter flow cytometry. J Biotechnol 115, 199–210.

Robertson, B.R. and Button, D.K. (1999) Determination of

bacterial biomass from flow cytometric measurements of

forward light scatter intensity. In Current Protocols in

Cytometry ed. Robinson, J.P. pp. 11.19.11–11.19.10. New

York: John Wiley and Sons Inc.

Robinson, J.P. (1999) Overview of flow cytometry and micro-

biology. In Current Protocols in Cytometry ed. Robinson,

J.P. pp. 11.11.11–11.11.14. New York: John Wiley and

Sons Inc.

Robinson, J.P. (2000) Microbiological applications. In Current

Protocols in Cytometry ed. Robinson, J.P. pp. 11.10.11–

11.10.12. New York: John Wiley and Sons Inc.

Rowan, N.J., Caldow, G., Gemmell, C.G. and Hunter, I.S.

(2003) Production of diarrhoeal enterotoxins and other

potential virulence factors by veterinary isolates of Bacillus

Study of B. cereus using flow cytometry U.P. Cronin and M.G. Wilkinson

14 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16

ª 2009 The Authors

species associated with nongastrointestinal infections. Appl

Environ Microbiol 69, 2372–2376.

Ryu, J.-H. and Beuchat, L.R. (2005) Biofilm formation and

sporulation by Bacillus cereus on a stainless steel surface

and subsequent resistance of vegetative cells and spores to

chlorine, chlorine dioxide, and a peroxyacetic acid-based

sanitizer. J Food Prot 68, 2614–2622.

Ryu, J.-H., Kim, H. and Beuchat, L.R. (2005) Spore formation

by Bacillus cereus in broth as affected by temperature,

nutrient availability and manganese. J Food Prot 68, 1734–

1738.

Schell, R.F., Moore, A.V., Scott, K.M. and Callister, S.M.

(1999) Mycobacterium tuberculosis susceptibility testing by

flow cytometry. In Current Protocols in Cytometry ed. Rob-

inson, J.P. pp. 11.17.11–11.17.17. New York: John Wiley

and Sons Inc.

Schellenberg, J., Smoragiewicz, W. and Karska-Wysocki, B.

(2006) A rapid method combining immunofluorescence

and flow cytometry for improved understanding of com-

petitive interactions between lactic acid bacteria (LAB)

and methicillin-resistant S. aureus (MRSA) in mixed

culture. J Microbiol Meth 65, 1–9.

Schumacher, W.C., Storozuk, C.A., Dutta, P.K. and Phipps,

A.J. (2008) Identification and characterization of Bacillus

anthracis spores by multiparameter flow cytometry. Appl

Environ Microbiol 74, 5220–5223.

Setlow, P. (2003) Spore germination. Curr Opin Microbiol 6,

550–556.

Setlow, P. (2006) Spores of Bacillus subtilis: their resistance to

and killing by radiation, heat and chemicals. J Appl Micro-

biol 101, 514–525.

Shapiro, H.M. (1995) Practical Flow Cytometry. New York: Wi-

ley-Liss Inc.

Shapiro, H.M. (2000) Microbial analysis at the single-cell level:

tasks and techniques. J Microbiol Meth 42, 3–16.

Shapiro, H.M. (2003) Practical Flow Cytometry. New Jersey:

John Wiley and Sons, Inc.

Shapiro, H.M. and Nebe-von-Caron, G. (2004) Multiparameter

flow cytometry of bacteria. In Methods in Molecular

Biology: flow cytometry protocols ed. Hawley, T.S. and

Hawley, R.G. pp. 33–43. New Jersey, USA: Humana

Press.

Sincock, S.A. and Robinson, J.P. (2001) Flow cytometric

analysis of microorganisms. Methods Cell Biol 64, 511–537.

Smits, W.K., Eschevins, C.C., Susanna, K.A., Bron, S., Kuipers,

O.P. and Hamoen, L.W. (2005) Stripping Bacillus: ComK

auto-stimulation is responsible for the bistable response in

competence development. Mol Microbiol 56, 604–614.

Snowdon, J.A., Buzby, J.C. and Roberts, T. (2002) Epidemiol-

ogy, cost, and risk of foodborne disease. In Foodborne

Diseases ed. Cliver, D.O. and Riemann, H.P. pp. 31–51.

New York: Academic Press.

Stalheim, T. and Granum, P.E. (2001) Characterisation of

spore appendages from Bacillus cereus strains. J Appl

Microbiol 91, 839–845.

Stecchini, M.L., del Torre, M., Donda, S., Maltini, E. and

Pador, S. (2001) Influence of agar content on the growth

parameters of Bacillus cereus. Int J Food Microbiol 64, 81–

88.

Steen, H.B. (2000) Flow cytometry of bacteria: glimpses from

the past with a view to the future. J Microbiol Meth 42,

65–72.

Stevenson, K.E. and Segner, W.P. (2001) Mesophilic aerobic

sporeformers. In Microbiological Examination of Foods ed.

Downes, F.P. and Ito, K. pp. 223–227. Washington, DC,

USA: American Public Health Association.

Stopa, P.J. (2000) The flow cytometry of Bacillus anthracis

spores revisited. Cytometry 41, 237–244.

Tanaka, S., Yamaguchi, N. and Nasu, M. (2000) Viability of

Escherichia coli O157:H7 in natural river water determined

by the use of flow cytometry. J Appl Microbiol 88, 228–

236.

Tang, Y.Z., Gin, K.Y.H. and Lim, T.H. (2005) High-tempera-

ture fluorescent in situ hybridization for detecting Escheri-

chia coli in seawater samples, using rRNA-targeted

oligonucleotide probes and flow cytometry. Appl Environ

Microbiol 71, 8157–8164.

Thompson, B.M., Waller, N.L., Fox, K.F., Fox, A. and Stewart,

G.C. (2007) The BclB glycoprotein of Bacillus anthracis is

involved in exosporium integrity. J Bacteriol 189, 6704–

6713.

Toh, M., Moffitt, M.C., Henrichsen, L., Raftery, M., Barrow,

K., Cox, J.M., Marquis, C.P. and Neilan, B.A. (2004)

Cereulide, the emetic toxin of Bacillus cereus, is putatively

a product of nonribosomal peptide synthesis. J Appl Micro-

biol 97, 992–1000.

Topley, R.T. and Wilson, D.J. (1998) Systematic bacteriology.

In Microbiology and Microbial Infections. London: Oxford

University Press.

Tourasse, N., Helgason, E., Okstad, O.A., Hegna, I.K. and Kol-

sto, A.-B. (2006) The Bacillus cereus group: novel aspects

of population structure and genome dynamics. J Appl

Microbiol 101, 579–593.

Trevors, J.T. (2003) Fluorescent probes for bacterial cytoplas-

mic membrane research. J Biochem Biophys Methods 57,

87–103.

Turner, N.J., Whythe, R., Hudson, J.A. and Kaltovei, S.I.

(2006) Presence and growth of Bacillus cereus in dehydrated

potato flakes and hot-held, ready-to-eat potato products

purchased in New Zealand. J Food Prot 69, 1173–1177.

Ultee, A., Bennik, M.H.J. and Moezelaar, R. (2002) The phe-

nolic hydroxyl group of carvacrol is essential for action

against the food-borne pathogen Bacillus cereus. Appl Envi-

ron Microbiol 68, 1561–1568.

Uyttendaele, M. and Debevere, J. (2003) The use of applied

systematics to identify foodborne pathogens. In Detecting

Pathogens in Food ed. McMeekin, T.A. pp. 332–359. New

York: CRC Press.

Valero, M. and Frances, E. (2006a) Synergistic bactericidal

effect of carvacrol, cinnamaldehyde or thymol and

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 15

refrigeration to inhibit Bacillus cereus in carrot broth. Food

Microbiol 23, 68–73.

Valero, M., Fernandez, P.S. and Salmeron, M.C. (2003) Influ-

ence of pH and temperature on growth of Bacillus cereus

in vegetable substrates. Int J Food Microbiol 82, 71–79.

Valero, M., Sarrias, J.A., Alvarez, D. and Salmeron, M.C.

(2006b) Modeling the influence of electron beam irradia-

tion on the heat resistance of Bacillus cereus spores. Food

Microbiol 23, 367–371.

Varnam, A.H. and Evans, M.G. (1991) Bacillus. In Foodborne

Pathogens: an Illustrated Text ed. Varnam, A.H. and Evans,

M.G. pp. 267–288. London: Wolfe Publishing Ltd.

Varughese, E.A., Wymer, L.J. and Haugland, R.A. (2007) An

integrated culture and real-time PCR method to assess via-

bility of disinfectant treated Bacillus spores using robotics

and the MPN quantification method. J Microbiol Meth 71,

66–70.

Veal, D.A., Deere, D., Ferraria, B., Piperb, J. and Attfield, P.V.

(2000) Fluorescence staining and flow cytometry for moni-

toring microbial cells. J Immunol Methods 243, 191–210.

Veening, J.-W., Hamoen, L.W. and Kuipers, O.P. (2005) Phos-

phatases modulate the bistable sporulation gene expression

pattern in Bacillus subtilis. Mol Microbiol 56, 1481–1494.

Veening, J.-W., Smits, W.K., Hamoen, L.W. and Kuipers, O.P.

(2006) Single cell analysis of gene expression patterns of

competence development and initiation of sporulation in

Bacillus subtilis grown on chemically defined media. J Appl

Microbiol 101, 531–541.

Vlamakis, H., Aguilar, C., Losick, R. and Kolter, R. (2008)

Control of cell fate by the formation of an architecturally

complex bacterial community. Genes Dev 22, 945–953.

Wells, M. (2006) Advances in optical detection strategies for

reporter signal measurements. Curr Opin Biotechnol 17,

28–33.

Werner, B.G. and Hotchkiss, J.H. (2002) Effect of carbon diox-

ide on the growth of Bacillus cereus spores in milk during

storage. J Dairy Sci 85, 15–18.

Winson, M.K. and Davey, H.M. (2000) Flow cytometric analy-

sis of microorganisms. Methods 21, 231–240.

Wolf, J. and Barker, A.N. (1968) The genus Bacillus: aids to

the identification of its species. In Identification Methods

for Microbiologists ed. Gibs, B.M. and Shapton, D.A. pp.

93–109. London: Academic Press.

Yang, I.C., Shih, D.Y.C., Huang, T.P., Huang, Y.P., Wang, J.Y.

and Pan, T.M. (2005) Establishment of a novel multiplex

PCR assay and detection of toxigenic strains of the

species in the Bacillus cereus group. J Food Prot 68,

2123–2130.

Yaqub, S., Anderson, J.G., MacGregor, S.J. and Rowan, N.J.

(2004) Use of a fluorescent viability stain to assess lethal

and sublethal injury in food-borne bacteria exposed to

high-intensity pulsed electric fields. Lett Appl Microbiol 69,

246–251.

Zoetendal, E.G., Ben Amor, K., Harmsen, H.J.M., Schut, F.,

Akkermans, A.D.L. and deVos, W.M. (2002) Quantifica-

tion of uncultured Ruminococcus obeum-like bacteria in

human faecal samples by fluorescent in situ hybridization

and flow cytometry using 16S rRNA-targetd probes. Appl

Environ Microbiol 68, 4225–4232.

Zwirglmaier, K. (2005) Fluorescence in situ hybridisation

(FISH) – the next generation. FEMS Microbiol Lett 246,

151–158.

Study of B. cereus using flow cytometry U.P. Cronin and M.G. Wilkinson

16 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1–16

ª 2009 The Authors