The Use of Flow Cytometry to Study the Germination of Bacillus cereus Endospores

The Use of Flow Cytometry to Study theGermination of Bacillus cereus Endospores

Ultan P. Cronin and Martin G. Wilkinson*Department of Life Sciences, University of Limerick, Castletroy, Co. Limerick, Ireland

Received 19 July 2006; Revision Received 22 November 2006; Accepted 28 November 2006

Background: At present the study of endospore germina-tion is conducted using microbiological methods which areslow and yield data based on the means of large heteroge-neous populations. Flow cytometry (FCM) offers the poten-tial to rapidly quantify and identify germination and out-growth events for large numbers of individual endospores.Methods: Standard methods were employed to arrest thegermination of Bacillus cereus endospores at definedstages. Endospores were then stained with SYTO 9 aloneor carboxyfluorescein diacetate (CFDA) together withHoechst 33342 and analysed using FCM. Comparisonswere made between FCM as a method to measure germi-nation rate and standard microbiological techniques.Results: Germinating endospores displayed increases inpermeability to SYTO 9 and hydrolysis of CFDA comparedwith controls. Statistically significant correlations were

found between the standard plate count method and bothFCM methods for measuring the percentage of germinat-ing and outgrowing endospores up to 75 min after addi-tion of germinant.Conclusions: Using FCM, the percentage of germinatingor outgrowing endospores at various time points duringgermination and/or outgrowth can be quantified. FCMwith CFDA/Hoechst 33342 staining may be used to esti-mate overall germination rate, whereas FCM with SYTO 9staining may be used to quantify ungerminated, germinat-ing and outgrowing endospores. q 2007 International Society

for Analytical Cytology

Key terms: Bacillus cereus; endospores; flow cytometry;germination; outgrowth

When presented with a sample of endospores of thefood-poisoning bacterium Bacillus cereus from a foodproduct or a culture suspension, it is important to ascer-tain both the number of spores present and their physio-logical state. Although numerous methods exist which pro-vide information on the physiological state of endospores(1–3), most of these, e.g. the fluorescent b-glucosidaseassay (4), generate a mean value for a sample containingseveral million individual endospores, but yield little dataconcerning the heterogeneity of that population. In con-trast, flow cytometry (FCM) can measure a number of pa-rameters for each individual cell in a population and istherefore a useful method for rapidly determining both thephysiological status of individual endospores and the heter-ogeneity of a bacterial population within a sample (5).

Endospores of species within the genus Bacillus havebeen the subject of a wide diversity of FCM-based physio-logical studies. The membrane potential of gamma-irra-diated endospores of B. globigii was examined usingDiBAC4 (3), a dye that is excluded from cells that exhibit amembrane potential (6). Both irradiated and control pre-parations of endospores were positive for the dye, indicat-ing that it could not discriminate between viable and non-viable endospores. In a later study (7), it was shown that,DiOC6 (3), a dye which accumulates on hyperpolarized

membranes, could be used as an early membrane poten-tial indicator in germinating endospores of B. cereus andB. subtilis var. niger. Reis et al. (8) used FCM to study thepopulation dynamics within continuous cultures of B.

licheniformis and the subsequent physiological responsesto either starvation or a glucose pulse. The dyes usedwere DiOC6 (3) and a nucleic acid binding dye, propidiumiodide (PI). These workers reported the differentiation offour sub-populations of cells on the basis of their fluores-cent staining, including endospores with depolarized cyto-plasmic membranes, which failed to accumulate eitherstain. A spore-forming organism Paenibacillus polymyxa

(formerly B. polymyxa), was the subject of a flow cyto-metric investigation by Comas and Vives-Rego (9). Forwardscattered light (FSC) and fluorescent intensity profiles ofendospores stained with SYTO 13 (a nucleic acid-binding

*Correspondence to: Martin G. Wilkinson, Department of Life Sciences,

Schr€odinger Building, University of Limerick, Castletroy, Co. Limerick, Ireland.

E-mail: [email protected]

Grant sponsor: Safefood–the Food Safety Promotion Board, Ireland.

Published online 2 January 2007 in Wiley InterScience (www.

interscience.wiley.com).

DOI: 10.1002/cyto.a.20368

q 2007 International Society for Analytical Cytology Cytometry Part A 71A:143–153 (2007)

dye) and PI were used to identify a number of sub-popula-tions in liquid cultures, and a protocol was developed forthe determination of the percentage of viable endosporesby quantifying the number residing in each of the sub-populations. Stopa (10) demonstrated that live and deadendospores of B. anthracis could be differentiated on thebasis of FSC and side scattered light (SSC) profiles.

A number of authors have assessed B. subtilis endo-spores for their physiological status using fluorescent mi-croscopy by staining with DNA or nucleic acid dyes. It hasbeen reported on a number of occasions that germinat-ing/germinated undamaged endospores stain brightlywith SYTO 9 (a nucleic acid-binding dye) but do not takeup PI. However, nongerminated undamaged endosporesexhibit only peripheral staining with these two dyes,while nonviable endospores and dead germinated endo-spores stain only with PI (11,6,12). In addition, irradiatedendospores (6), peroxynitrite-killed germinated endo-spores, (13) and those germinated with dodecylamine(14) were permeable to PI. Young and Setlow (12) reportno penetration of either SYTO 9 or PI into the core of dor-mant or germinated endospores treated with either of twogeneral decontaminants, Decon or OxoneTM.

The DNA-specific dye, 40,6-diamidino-2-phenylindole(DAPI), has been employed in a number of studies invol-ving the fluorescent microscopy of B. subtilis. The coresof dormant B. subtilis endospores are faintly stained bythis dye, whereas those of germinated endospores stainintensely (15). Different DAPI staining patterns areobserved for either ethanol- or acid-killed endospores.Endospores killed with ozone, Decon, or OxoneTM displaybrighter peripheral staining than intact controls and donot take up dye into their cores in either the dormant orgerminated state (12,16). Setlow et al. (14) found thatdodecylamine-germinated endospores stained with DAPI,with some cells exhibiting stained ring-shaped nucleoids.Other dyes used in the fluorescent microscopy of B. subti-lis include acridine orange (AO), a nucleic acid-bindingdye, which behaves in a similar way to DAPI (15) andCTC, a tetrazolium, which intact germinating endosporesreduced to a fluorescent formazan dye after 1 h of incuba-tion while irradiated cells did not carry out this reaction(6). Ethidium bromide (EB), another nucleic acid bindingdye, has been used in conjunction with confocal scanninglaser microscopy to study the changes in endospore per-meability of B. cereus during germination, with increasedpermeability to the dye being observed to occur over timein germinating endospores (17). Despite the reportedadvances in differential staining, fluorescent microscopyand FCM of Bacillus spp., methodology has not beenestablished which can identify, quantify, and assesschanges in the permeability and metabolism of B. cereusendospores during germination.

In the present study, endospores were stained with ei-ther SYTO 9 or a combination of carboxyfluorescein dia-cetate (CFDA) and Hoechst 33342 (a nonfluorescent com-pound cleaved by nonspecific intracellular esterases toform a fluorescent compound and a DNA-specific bindingdye, respectively). Subsequently, FCM methods were

developed to identify and quantify spores during germina-tion and outgrowth. Comparisons were then madebetween the measurement of germination, using standardmicrobiological methods and FCM methods.

MATERIALS AND METHODSBacterial Strain Used and the Preparation of Pure

Suspensions of Endospores

B. cereus NCTC 7464 was utilized in this study as thetest organism in all experiments. The maintenance of typestrain phenotypic integrity was monitored regularlythrough the use of API 50 CH strips (Biomerieux, Lyon,France) and by plating of colonies on the diagnostic andselective medium, polymyxin pyruvate egg-yolk bro-mothymol blue agar, PEMBA (18). Pure suspensions ofendospores were generated based on a modification ofthe method of Gaillard et al. (19). This procedure yieldedendospore suspensions of greater than 95% purity interms of phase bright bodies when examined using aphase contrast microscope.

Germination, Outgrowth, and the Responseof Endospores to Germinant

The experimental design for this series of experimentsis outlined in Figure 1. The composition of the four solu-tions referred to in the figure are; ‘‘PBS’’—sterile phos-phate buffered saline (100 mM, pH 7.2); ‘‘L-alanine’’—agermination induction solution consisting of sterile PBScontaining 10 mM L-alanine (12); ‘‘D-alanine’’—a germinantinhibitor solution consisting of sterile PBS containing 10 mML-alanine, to which was added 5 min later, to allow a cer-tain proportion of endospores time to commit to germina-tion, 0.9 M D-alanine, which blocks germinant receptors(20); ‘‘EDTA’’ - an outgrowth inhibitor solution consistingof sterile PBS containing 10 mM L-alanine and 0.9 mMethylenediaminetetraacetic acid (EDTA). EDTA was foundby Hansen et al. (21) to completely block vegetativegrowth of B. cereus at the aforementioned concentration,while Varnam and Evans (22) refer to the ability of thiscompound to prevent outgrowth. Activation in Figure 1refers to heating at 80�C for 10 min and lethal heat refersto heating at 90�C for 20 min - a treatment previouslyfound to induce a six log reduction in viability [data notshown]). Each sample of endospores involved densities of�1.0 3 106–7 cfu ml21. For each of this series’ 12 experi-mental conditions, n5 3.

Comparison of FCM Methods for Determining theRate of Germination with Standard Methods

The rate of germination of B. cereus endospores wasmeasured using both the standard plate count method andby monitoring the change in optical density at 600 nm(OD600) of endospore suspensions. Data generated usingthese two methods were compared with data obtained bymeasuring the rate using the two FCM staining regimes.The standard plate count method (1,23) works on the prin-ciple that endospores lose their heat resistance as they ger-

144 CRONIN AND WILKINSON

Cytometry Part A DOI 10.1002/cyto.a

minate (24,3). The experimental design for this experi-ment is detailed in Figure 2. For plate counting, suspen-sions were plated out on nutrient agar (NA) supplemented

with 10 mM L-alanine and plates were subsequently incu-bated at 30�C for 24 h. The germination rate of three sepa-rate endospore suspensions was determined for thisexperiment, which was repeated once. Data from all threesuspensions and from both experiments were combinedfor statistical analysis.The OD600 of suspensions of endospores was measured,

using a SynergyTM HT absorbance/fluorescence/biolumi-nescence microplate reader (BIO-TEK� Instruments, VT)together with Costar 96-well sterile, clear, and flat-bot-tomed polystyrene plates (Corning, NY). An analate vol-ume of 300 ll per well was employed in all cases. Micro-plates, pre-heated to 30�C, were run at 30�C over thecourse of 2 h, with readings being taken every 15 min fol-lowing shaking for 5 s. Four randomly-placed blanks perplate consisting of PBS containing L-alanine were includedin each plate as were controls consisting of endosporessuspended only in PBS. Absorbance readings were takenwith a top probe vertical offset of 1 mm. A mean for eachtime-point was derived using the data from three full 96-well plates.For the measurement of germination rate using FCM,

endospores were stained as described later using twostaining regimes, SYTO 9 and CFDA/Hoechst 33342. Allstained samples were vortexed and immediately analyzedusing FCM. The rate of germination of five independentendospore suspensions was determined. The experimentwas repeated once using fresh endospore suspensionsand reagents. The number of events in regions corre-sponding to germinating and outgrowing endosporeswere recorded. Data for all five suspensions and for repeat

FIG. 1. The design of experiments enquiring into the germination, out-growth, and response of endospores of Bacillus cereus to germinant.

FIG. 2. The design of experiments for comparingFCM methods for determining the rate of germinationof Bacillus cereus endospores with standard methodsi.e. plate counting and the measurement of OD600.

145FLOW CYTOMETRIC STUDY OF SPORE GERMINATION


experiments were combined for the purpose of statisticalanalysis.

Staining of Endospores

Two staining regimes were implemented. The firstinvolved the use of SYTO 9 (Molecular Probes, Leiden, theNetherlands), which fluoresces only when bound to eitherdouble-stranded DNA or RNA and which has an emissionmaximum of 520 nm (green). Endospores were stainedwith a final concentration of SYTO 9 of 10 lM. The sec-ond staining regime involved a combination of CFDA(5[and 6]-CFDA ‘‘mixed isomers’’, Molecular Probes, Lei-den, the Netherlands) and Hoechst 33342 (Sigma-Aldrich,Dublin, Ireland). CFDA, which is nonfluorescent, is hydro-lyzed by nonspecific intracellular esterases to form car-boxyfluorescein (CF), which is excited between 490 65 nm and emits at 515 6 5 nm (green). Hoechst 33342binds to DNA and has an excitation maximum at 355 nmand an emission maximum at 464 nm (violet). Endosporesuspensions were stained with a final concentration of50 lM CFDA and 0.27 lM Hoechst 33342. Samples weremixed thoroughly using a vortex both directly after stain-ing and immediately prior to analysis using FCM and epi-fluorescence microscopy.

FCM of Endospores

Flow cytometric analyses were performed on a BD LSR(BD Biosciences, Shannon, Ireland), using the instru-ment’s 20 mW 488 nm argon laser, which generates FSCand SSC signals and four fluorescence signals, and theinstrument’s 8 mW 325 nm HeCd laser, which generatestwo additional fluorescence signals. The sheath fluid usedfor all experiments was FacsflowTM (BD Biosciences, Shan-non, Ireland), filtered through a 0.22 lm Pall Kleenpak fil-ter (VWR International, Ashbourne, Ireland). Instrumentperformance was monitored daily using CalibriteTM three-color calibration beads (BD Biosciences, Shannon, Ireland)and SPHEROTMUltra Rainbow 6-peak calibration particles(Spherotech Inc., Libertyville, IL). The software used fordata acquisition and analysis was CellQuestTM (BD Bios-ciences, Shannon, Ireland). All data were stored as FCS2.0 files. Green fluorescence (from SYTO 9 or CF) wascaptured using the FL1 detector, and violet fluorescence(from Hoechst 33342) using the FL5 detector. The devel-opment of a protocol for the analysis of CFDA/Hoechst33342-stained endospores involved a number of steps. Toarrive at amp gains and photomultiplier tube (PMT) vol-tage settings which positioned the fluorescence of eachindividual stain on-scale, unheated, activated, or lethallyheat-treated endospores stained using only one of thestains and suspended in sterile PBS containing 10 mML-alanine, were analyzed on the flow cytometer whileadjustments were made to both amp and PMT parameters.Next, mixtures of CFDA-only- and Hoechst 33342-only-stained endospores and unstained endospores were ana-lyzed. To exclude ‘‘noise’’ from analyses, dual-stainedendospores positive for both fluorochromes were ana-lyzed, and an FSC threshold level was chosen which

removed non-fluorescent events from FL1 and FL5 chan-nels. Finally, the settings arrived were verified by runningunstained, singly-stained, dual stained, or blank samples(containing no endospores) through the machine. The de-velopment of a protocol for the analysis of SYTO 9-stainedendospores followed a similar procedure as that describedearlier. Samples were acquired using the machine’s ‘‘low’’setting, corresponding to a flow rate of �24 ll min21,with 10,000 events being acquired per sample.

Epifluorescent Microscopy of Endospores

Epifluorescence microscopy was carried out using anOlympus BX60 (Olympus Optical Ltd., Tokyo, Japan), towhich were attached the following objective lenses: a40 3 0.75 Ph2, a 60 3 0.99, and an oil immersion 100 31.3. The instrument’s fluorescent filter cube contained thefollowing units (all supplied by Olympus Optical): U-MWU2 narrow-band UV excitation suitable for Hoechst33342, exciter filter, 360–370 nm BP; barrier filter,420 nm; U-MNNIBA dichroic beam splitter suitable forSYTO 9/CF, exciter filter, 470–490 BP; barrier filter, 515–520 nm. Images were captured by an Olympus DP70video camera (Olympus Optical Ltd., Tokyo, Japan). Theimage analysis software used to acquire and analyzeimages was ‘‘analySIS�FIVE�’’ (Soft Imaging System,GmgH, M€unster, Germany). Five ll of stained endosporesuspensions of each of the treatments described earlierwere transferred to clean microscopy slides for trappingand observation. A number of fields per slide wereobserved and recorded.

Statistical Analysis and Presentation of Data

Data were analyzed using Microsoft� Excel 97 SR-1(Microsoft Corporation, Redmond, WA) and SPSS 12.0.1(SPSS Corporation, Chicago). Figures and graphs were alsoprepared using these software packages. FCM data for allstaining experiments and treatments were analyzed usinggeneral loglinear analysis. The model employed for analy-sis (Constant 1 Region/Quadrant 1 No Heat/Activation/Lethal Heat 1 Chemical Treatment) was tested using twogoodness of fit tests (Likelihood Ratio and Pearson Chi-squared) both of which pointed towards rejection of thenull hypothesis (P < 0.001), signifying that either heat orchemical treatment did affect fluorescence emitted orlight scattered by endospores. Unless otherwise stated,means are presented 6 their 0.95 confidence interval.

RESULTSGermination and Outgrowth Responsesof Endospores as Monitored by FCM

SYTO 9-stained endospores were analyzed using FCM-derived dot plots of green fluorescence versus SSC. Duringdata analysis, four groups of endospores could be distin-guished by comparing profiles of the 12 experimentalconditions and, using the CellQuestTM programme,regions were constructed around these groups to facilitatethe enumeration of events within each group (Fig. 3).Region 1 (R1) corresponded to intact, ungerminated endo-



spores, moderately permeable to SYTO 9 with �50 arbi-trary units [AU] of fluorescence. Intact endospores dis-played a broad range of SSC intensities (�14–26 AU). Forall treatments, except for those endospores receiving le-thal heating, R1 formed the major group during 1 h analy-sis. Region 2 (R2) corresponded to germinating endo-spores with almost two log units more fluorescence thanfor intact endospores (�2,000 AU) and a narrower distri-bution of SSC. This region was populated only in the pre-sence of germinant, but comprised a much reduced per-centage of the profile when D-alanine was present andappeared unaffected by EDTA (Table 1). The proportionof endospores in R2 increased for activated endospores.When the conditions for germination existed, R2 becamepopulated over the course of 1 h. Figure 4 displays thetransition of endospores from R1 over time for the varioustreatments.

Region 3 (R3) was consistently populated to varyingdegrees irrespective of endospore treatment and the pre-sence of germinant (Fig. 3 and Table 1). However, activa-tion or lethal heat-treatment of endospores resulted in anincrease in counts in R3 indicating that it may consist ofdamaged or nonviable endospores. Endospores in R3 alsodisplayed much greater fluorescence than R1 (�800–

1,400 AU), but could be distinguished from R2 based onhigher SSC intensity (�33–68). Region 4 (R4) was popu-lated only when germinant was present, endospores hadbeen activated and when EDTA was absent. Suspension ofendospores in germinant resulted in an accumulation ofevents in R4 after �30–40 min indicating that R4 con-sisted of outgrowing endospores. Endospores in R4 werecharacterized by green fluorescence intensities of �450AU and SSC intensities of <10 AU. When endospores wereheat-treated, events were mainly focussed in R4 but withsome lesser events in other regions, suggesting that arange of endospore SYTO 9 permeabilities and SSC pro-files may arise from heat damage.CFDA/Hoechst 33342-stained endospores had less com-

plex FCM profiles and scatter plots of either violet orgreen fluorescence versus SSC were not as informative asscatter plots of both fluorescent parameters. Analysis ofscatter plots did not necessitate the designation of specificregions hence, whenever this staining combination wasused, plots were divided into quadrants and data analyzedusing this template (Fig. 5). Endospores in the upper left(UL), upper right (UR), lower left (LL), and lower right(LR) quadrants of scatter plots represent the following cellstain uptake patterns; Hoechst 33342-permeable and es-

FIG. 3. FCM-derived scatter plots ofthe green fluorescence versus sidescatter intensities of SYTO 9-stainedendospores of Bacillus cereus ana-lysed 1 h following (i) suspension in10 mM L-alanine, (ii) activation andsuspension in L-alanine, (iii) activationand suspension in 0.9 mM EDTA andL-alanine, and (iv) lethal heat treat-ment and suspension in 10 mM L-ala-nine. In the case of all plots bar thosefor the lethal heat-treated conditions,R1 contains intact endospores, R2 ger-minating endospores, R3 damagedendospores, and R4 outgrowing endo-spores. For each experimental condi-tion, n 5 3. [Color figure can beviewed in the online issue, which isavailable at www.interscience.wiley.com.]



terase negative, Hoechst 33342-permeable and esterasepositive, Hoechst 33342-impermeable and esterase nega-tive, and Hoechst 33342-impermeable and esterase posi-tive, respectively.

The majority (�90%) of unheated endospores sus-pended in PBS only were located in the LL quadrant andwere assumed to be both intact and dormant. However, asmall percentage (�9%) of endospores, found in the ULquadrant, were permeable to Hoechst 33342, possibly

representing a damaged sub-population. FCM profiles ofactivated and lethally heat-treated endospores in PBS werequite similar to those of the unheated endospores exceptfor a slight increase in the amount of Hoechst 33342-per-meable events (Table 2).Unheated endospores suspended in a solution contain-

ing L-alanine had a similar percentage of events in the UL

quadrant to endospores suspended in PBS only. However,

endospores in germinant solution had an increased per-

centage of endospores displaying esterase activity and a

decreased percentage of intact, dormant endospores. Acti-

vated endospores had a greater percentage of esterase-

positive events when compared with unheated endo-

spores. Lethal heat-treatment of endospores resulted in

higher levels of permeability to Hoechst 33342 and ester-

ase positive events.Unheated endospores treated with D-alanine had a

greater percentage of damaged endospores (�20%) com-pared with either endospores placed in PBS alone or L-al-anine. Esterase-positive events for unheated endosporeswere not influenced by the presence of D-alanine. Acti-vated endospores contained fewer events in the ULquadrant, but were otherwise similar to unheated endo-spores. Lethal heat- and D-alanine-treated endosporeshad similar profiles to unheated endospores placed inPBS except for an increased amount of events in the ULquadrant. EDTA treatment of unheated endosporesresulted in a reduced percentage of esterase-positiveevents compared with unheated endospores placed ingerminant solution. Similarly, activated endospores trea-ted with EDTA had a reduced percentage of esterase-positive events compared with activated endosporesplaced in germinant.

Table 1The Percentage of SYTO 9-Stained Endospores Falling into One of Four Flow Cytometry-Defined Regions on SSC Versus

Green Fluorescent Scatter Plots

Chemical treatment andeffect on spores Heat treatment Region 1 Region 2 Region 3 Region 4

ControlNo germination None 95.586 30.59 0.046 0.01 2.626 0.84 0.19 6 0.06

Activation 90.096 28.83 0.096 0.03 6.726 2.15 0.39 6 0.12Lethal heat 21.706 6.94 1.456 0.46 9.866 3.15 44.85 6 14.08

L-alanineGermination and outgrowth None 95.676 4.51 0.396 0.14 2.246 0.71 0.23 6 0.07

Activation 68.636 8.43 13.13 6 2.78 9.106 3.54 0.98 6 0.44Lethal heat 7.876 2.52 1.356 0.04 7.616 2.46 68.71 6 23.00

D-alanineGermination inhibited None 93.256 29.84 0.136 0.04 2.076 0.66 0.75 6 0.06

Activation 90.416 28.93 0.716 0.23 2.896 0.93 1.05 6 0.01Lethal heat 8.406 2.67 2.916 0.93 10.246 2.28 52.08 6 0.51

EDTAGermination occurs andoutgrowth inhibited

None 94.616 30.28 0.526 0.14 2.906 0.93 0.23 6 0.07Activation 92.946 13.85 3.176 1.01 2.866 0.43 0.76 6 0.23Lethal heat 23.026 7.25 3.916 1.25 14.306 4.58 36.29 6 11.61

The mean percentage of events for each treatment falling into one of four regions defined using gates based on scatter plots of thegreen fluorescence versus side scatter profiles of SYTO 9-stained endospores of Bacillus cereus analyzed using flow cytometry. For alltreatments bar those involving lethal heat, Region 1 contains intact endospores, Region 2 germinating endospores, Region 3 damagedendospores, and Region 4 outgrowing endospores. For each experimental condition, n 5 3.

FIG. 4. The percentage over time of ungerminated SYTO 9-stained endo-spores of Bacillus cereus calculated using regions drawn on FCM-derivedscatter plots of green fluorescence versus side scatter. Endospores were(¤) activated and placed in PBS, (u) received no activation and wereplaced in 10 mM L-alanine, (X) activated, placed in L-alanine for 5 min fol-lowed by placement in 0.9 M D-alanine, (�) activated and placed in 0.9 mMEDTA and L-alanine, and (n) activated and placed in L-alanine. For each ex-perimental condition, n5 3, and error bars have been omitted for clarity.



Comparison of FCM Methods for Determining theRate of Germination with Standard Methods

Comparison of data on the rate of germination meas-ured using the plate count method with data generatedusing the two FCM methods found statistically significantcorrelations between all three methods up as far as T75.The Pearson correlation coefficient (parametric) for therelationship between the percentage of heat sensitivebodies up to 75 min post addition of germinant (T75) andthe percentage of germinating and outgrowing endo-spores (events in R2 and R4 in scatter plots of green fluo-rescence vs. SSC) detected using FCM and SYTO 9 stainingwas 0.947 (P 5 0.004, two-tailed). The Spearman’s corre-lation coefficient (non-parametric) for the relationshipbetween the percentage of heat sensitive bodies detectedup to T75 and the percentage of esterase-positive eventswas 0.886 (P 5 0.019, two-tailed), while the Spearman’scorrelation coefficient for the relationship between bothFCM methods was 0.829 (P 5 0.042, two-tailed). Despitethese statistically significant correlations, the percentageof heat-sensitive bodies as measured using the plate countmethod was greater at each time point than the percent-age of germinating and outgrowing endospores as deter-

mined using FCM using SYTO 9 staining. This in turn wasgreater than the percentage of esterase positive eventsmeasured using FCM by CFDA/Hoechst staining (Fig. 6).A statistically significant relationship was found to exist

between the OD600 of germinating suspensions and thepercentage of esterase positive events as measured usingFCM (Spearman coefficient of 0.943, two-tailed, P < 0.01).However, no significant correlation was found betweenOD600 and the percentage of germinating and outgrowingendospores as determined using FCM and SYTO 9 staining(Fig. 6).

Epifluorescent Microscopy

For all treatments, endospores stained with SYTO 9emitted a strong, uniform green fluorescence (Fig. 7).Moderate Hoechst 33342 fluorescence was only observedfor lethally-heat treated endospores. Endospores for treat-ment groups other than lethal heat displayed little violetfluorescence despite extended exposure or increased sen-sitivity settings. Hoechst 33342 fluorescence was not uni-form within endospores, rather it arose as a narrow haloof lesser fluorescence surrounding a more intenselystained centre. Green fluorescence derived from CF was

FIG. 5. FCM-derived scatter plots ofthe green versus violet fluorescenceintensities of CFDA- and Hoechst33342-stained endospores of Bacilluscereus analyzed 60 min following (i)suspension in 10 mM L-alanine, (ii)activation and suspension in L-ala-nine, (iii) lethal heat treatment andsuspension in L-alanine for 5 min fol-lowed by suspension in 0.9 M D-ala-nine, (iv) activation and suspensionin 0.9 mM EDTA and L-alanine.



generally uniform but not as strong as for SYTO 9, withlong exposures necessary to capture satisfactory images.

DISCUSSION

Germination consists of a series of degradative events,during which various permeability barriers responsible fora significant degree of endospores’ resistance propertiesare broken down. These events result in re-hydration ofthe core, facilitating entry of molecules from the externalenvironment (25,26). In this study, intact ungerminated

endospores were found to be moderately permeable toSYTO 9, with permeability increasing upon germination.Outgrowing endospores showed reduced SYTO 9 fluores-cence relative to germinating endospores, which mayreflect the activity of cellular efflux mechanisms with theonset of metabolism (27,28). Low levels of untreated,ungerminated endospores were permeable to SYTO 9 andhad high SSC; these may be comprised of injured endo-spores. Intact ungerminated endospores displayed a broadrange of SSC intensities showing the extent of variabilityin refractility within a population of endospores of B. cer-eus. Germinating and outgrowing endospores had lowermean SSC than intact endospores, which may correspondto their entry into the ‘‘phase dark’’ stage, when endo-spores swell, imbibe water into the core, and lose refracti-lity (24,3). When lethal heat treatment was applied, thepermeability to SYTO 9 of ungerminated endosporesincreased, with the majority of events showing increasesin mean green fluorescent intensity. However, a smallnumber of endospores are still to be found in the intactdormant region (R1), suggesting that (R1) does not solelyrepresent intact dormant spores.A strong correlation was found between changes in

FCM profiles of SYTO 9-stained endospores and standardplate counts for the first 75 min following activation ofendospores and exposure to germinant. However, theextent of germination was much greater as reflected bythe percentages of heat-sensitive bodies measured usingthe plate count method, while the percentage of germinat-ing and outgrowing endospores as measured using FCMand SYTO 9-staining was lower. This suggests that loss ofheat resistance by endospores is not what is causing theincreased permeability of endospores to SYTO 9. Welkos

FIG. 6. The percentage of germinated Bacillus cereus endospores calcu-lated over time following their activation and placement in PBS containing10 mM L-alanine as measured using three methods; (n) the standard platecount method for detecting heat sensitive bodies, (�) a flow cytometricmethod based on the green fluorescent and side scatter profiles of SYTO9-stained endospores and (X), a flow cytometric method based on thegreen and violet fluorescent profiles of CFDA/Hoechst 33342-stainedendospores. Also included in the figure is (}) the OD600 3 100 of germi-nating endospores.

Table 2The Percentage of CFDA and Hoechst 33342-Stained Endospores in Each Flow Cytometry-Defined Quadrant

on Green Versus Violet Fluorescent Scatter Plots

Chemical treatmentand effect on spores

Heattreatment

Upper leftquadrant

Upper rightquadrant

Lower leftquadrant

Lower rightquadrant

ControlNo germination None 8.636 0.28 0.586 0.15 90.71 6 0.25 0.09 6 0.03

Activation 10.93 6 0.05 0.906 0.02 87.92 6 0.09 0.25 6 0.06Lethal heat 11.74 6 2.43 1.096 0.07 86.83 6 2.29 0.34 6 0.08

L-alanineGerminationand outgrowth

None 8.206 3.12 3.356 2.46 85.81 6 5.22 2.64 6 1.23Activation 6.866 3.64 4.226 1.55 83.81 6 4.61 5.11 6 1.39Lethal heat 63.55 6 10.84 13.26 6 2.05 19.96 6 6.98 3.23 6 1.95

D-alanineGermination inhibited None 20.21 6 1.27 5.286 0.89 73.30 6 1.38 1.21 6 0.06

Activation 12.91 6 13.69 4.146 3.93 81.61 6 18.09 1.34 6 0.83Lethal heat 20.77 6 1.12 4.816 0.52 74.12 6 1.66 0.31 6 0.05

EDTAGermination occurs andoutgrowth inhibited

None 7.436 4.47 1.586 0.79 90.22 6 5.78 0.77 6 0.70Activation 4.666 0.60 1.136 0.80 93.35 6 2.32 0.86 6 0.93Lethal heat 14.97 6 10.41 0.736 1.10 84.15 6 11.76 0.16 6 0.25

The mean percentage of events for each treatment falling into quadrants defined using scatter plots of the green versus violet fluores-cence profiles of CFDA- and Hoechst 33342-stained endospores of Bacillus cereus analyzed using flow cytometry. For each experimentalcondition, n5 3. The upper left quadrant corresponds to esterase-negative, Hoechst 33342-permeable endospores, the upper right quad-rant corresponds to esterase-positive, Hoechst 33342-permeable endospores, the lower left quadrant corresponds to esterase-negative,Hoechst 33342-impermeable endospores, and the lower right quadrant corresponds to esterase-positive, Hoechst 33342-impermeableendospores.



et al. (29) found a similar correlation between an increasein permeability to SYTO 9 and the loss of heat resistanceof germinating B. anthracis endospores and, in agreementwith data presented by us in this report, these workersalso attribute increased SYTO 9 permeability to an eventunrelated to the loss of heat sensitivity. Heat sensitivity isone of the first properties acquired by germinating endo-spores and only subsequent to this are the permeabilitybarriers to substances such as SYTO 9 removed (30,31).Epifluorescent microscopy revealed that SYTO 9 fluores-cence originated in all cases from the entirety of the endo-spore without the peripheral staining reported by otherworkers (6,11,12). However, the patterns of permeabilityto SYTO 9 for intact ungerminated, heat-damaged, and ger-minating endospores noted by us broadly agree with theseand other authors (29).

To our knowledge, this is the first report detailing thestaining of endospores of species within the genus, Bacil-lus, using Hoechst 33342 and CFDA and their subsequentflow cytometric analysis. Hoechst 33342 is a DNA-specificdye with a preference for A-T bases, and has been almostexclusively used for the study of mammalian cell physiol-ogy, metabolism, and apoptosis (32). In vegetative B. cer-

eus cells, this dye is poorly retained unless the metabolic

inhibitor, CCCP, is used to prevent efflux (33). Vegetativecells of species within the genus, Bacillus, have been stud-ied using CFDA or a related compound, 5 (6)-carboxyfluor-escein diacetate succinimidyl ester (CFDA-SE). Hornbaeket al. (34), using CFDA-SE and FCM, found correlationsbetween the capacity of B. licheniformis to multiply andintracellular pH, with fluorescence intensity linked to es-terase activity and internal pH. Hoefel et al. (35) reportedthat CFDA, but not CFDA-SE, could differentiate betweenactive and inactive vegetative cells of B. subtilis. Ferenckoet al. (36), using a compound related to CFDA, diacetyl flu-orescein (DAF), measured the esterase activity of B.

anthracis endospores during germination. However, theglass fiber disk format of the assay generated data basedon the mean activities of all of the endospores within12 ll aliquots of endospore suspensions and is not capa-ble of providing information on individual endosporesthat either the aforementioned FCM assays or the data pre-sented in this report have provided. In general, slide-basedand microtitre plate assays do not reveal whether enzymeor substrate is retained within the germinating endospore,whereas any fluorescence detected by a flow cytometerarises from either within a cell/endospore or its outer sur-face.

FIG. 7. Epifluorescence micrographs of the green fluorescence (ii) emitted by Bacillus cereus endospores stained with SYTO 9 and the violet fluores-cence emitted by endospores stained with Hoechst 33342 (iv). A phase contrast image of (i) SYTO 9-stained endospores and a dark-field image of (iii)Hoechst 33342-stained endospores are included. All images acquired though a 1003 objective lens. [Color figure can be viewed in the online issue, whichis available at www.interscience.wiley.com.]



In this study, our evidence suggests that intact ungermi-nated endospores are impermeable to Hoechst 33342 anddo not display esterase activity. In contrast, germinatingendospores possessed esterase activity and could eitherbe permeable or impermeable to Hoechst 33342. This dif-ferential permeability may reflect the fact that endosporesat different stages of germination may be at differentstages of a process leading to the loss of permeability bar-riers (17). Only when endospores were treated with EDTAwas definite separation between green fluorescent-posi-tive and -negative events noted in the FCM scatter profile.An explanation for this may be that events from the parti-cular step inhibited by EDTA (i.e. outgrowth) are normallydetectable in the area between the two distinct regions.Hence, outgrowing endospores stained with CFDA, similarto those stained with SYTO 9, display less green fluores-cence than germinating endospores, again suggesting thatoutgrowing endospores possess efflux pumps.

A strong correlation was found between the percentageof esterase-positive events and both the OD600 over timeof endospore suspensions and the percentage of heat-sen-sitive bodies measured using plate counts in suspensionsof activated endospores up until 75 min post-exposure togerminant. However, in common with SYTO 9-staining,CFDA/Hoechst 33342 may not be detecting the sameevent as plate counts. CFDA/Hoechst 33342 staining, aswith SYTO 9 staining, identified a group of damaged(Hoechst positive/CF negative) endospores among un-treated endospores. This population was larger than thatquantified using SYTO 9 staining (�9% vs. �4%), suggest-ing that different categories of damage are being detectedby the two staining regimes. Exposure to D-alanineappeared to cause an increase in the permeability of endo-spores to Hoechst 33342 whether or not lethal heat treat-ment was used. This was also noted for endosporesstained with SYTO 9. It is difficult to account for thisdamage as no reference to D-alanine as a permeabilisingagent of endospores can be found in the literature. It isintended to deploy cell sorting in the future to clarify thisand other ambiguities raised during the execution of thiswork. In general, Hoechst 33342 fluorescence was veryweak when viewed using epifluorescent microscopy, andthis stain may only be suitable for more sensitive micros-copy systems and FCM. Hoechst 33342 fluorescence wasfound at both the centre and periphery of endospores,indicating that some of the dye may be retained on the en-dospore surface through nonspecific binding by an outercomponent.

As a result of the characteristic SSC intensities and dif-ferential SYTO 9 permeability profiles of germinating andoutgrowing endospores, it appears that it is possible toquantify the percentage of endospores belonging to thesestages at various time points during the germination/out-growth process. FCM in conjunction with CFDA stainingmay be used to estimate the overall rate of germinationrather than the individual stages (germination or out-growth) which staining with SYTO 9 reveals. Using bothstaining methods opens up the possibility of applyingFCM to measure rates of germination and outgrowth for B.

cereus and other spore-formers. FCM also offers the com-bined advantages of rapidity with the simultaneous analy-sis of large numbers of endospores, e.g. 10,000 events persample (37). Practical applications of the methodsdescribed in this report include; the direct examination ofsamples from foodstuffs (provided that endospores can besuccessfully isolated from the surrounding food matrixusing a technique such as density grade centrifugation) tomeasure the germination rate of contaminating endo-spores and the rapid evaluation of sporistatic/sporicidalcompounds for their effectiveness and mechanisms ofaction.

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The Use of Flow Cytometry to Study the Germination of Bacillus cereus Endospores

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