Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC

Assessment of cell viability using methods which do not require cell culture is essential in the field of aquatic microbiology, since many bacteria known to be present in aquatic environments cannot be grown in culture. The study of bacterial
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  Assessment of  E. coli  and  Salmonella  Viabilityand Starvation by Confocal Laser Microscopy and FlowCytometry Using Rhodamine 123, DiBAC4(3),Propidium Iodide, and CTC R. Lo´pez-Amoro´s, 1 S. Castel, 2  J. Comas-Riu, 2 and J. Vives-Rego 1 * 1 Departament de Microbiologia, Facultad de Biologia, Universitat de Barcelona, Barcelona, Spain 2 Servei Cientı´fico-Te`cnic, Universitat de Barcelona, Barcelona, Spain Received 25 October 1996; Accepted 14 July 1997 Assessment of cell viability using methods whichdo not require cell culture is essential in the field ofaquatic microbiology, since many bacteria known tobe present in aquatic environments cannot be grownin culture. The study of bacterial biofilms, whichpreviously needed an epifluorescent microscope,has recently been enhanced by the use of flowcytometry and confocal scanning laser microscopy(CSLM). A method based on the combination ofseveral membrane potential related dyes, a mem-brane integrity dye and a redox probe was used tomeasure cell viability by flow cytometry and confo-cal laser microscopy. Rhodamine–propidium iodide(PI) double staining was used to discriminate viablefrom nonviable cells in CSLM observations. Mem-brane depolarization during  E. coli  and  Salmonella starvation measured by DiBAC4(3) incorporation(flow cytometry and CSLM) was found to be inconcordance with respiratory activity as detected bya tetrazolium salt (CTC) reduction. Cytometry 29:298–305, 1997.   1997 Wiley-Liss, Inc. Key terms: starvation; rhodamine 123; bis-oxonol;propidium iodide; CTC; flowcytometry; laser micros-copy;  E.coli; Salmonella Assessment of cell viability is one of the main require-ments in several areas of microbiology, from environmen-tal research to industrial applications. The need to deter-mine bacterial viability is particularly important in aquaticenvironmental microbiology (16,23,31); this has led to thedevelopment of direct methods that are faster and moreaccurate than culture methods (4,20,25). Advances in digitalimaging and flow cytometrynow allow us to fullyexploit thepotentialsofepifluorescentmicroscopy(8,28,37).The most widely used probes developed for assessmentof viability involve lipophilic cationic dyes (rhodamine123, cyanines) that tendto accumulate on the inner side of energized membranes and have been used for epifluores-cence (24,41) and flow cytometry (7,22). In addition, thelatest reports of membrane potential determinations byflow cytometry involve the use of the negatively chargedoxonols that enter depolarized cells and bind to lipid-richcomponents (5,12,18). The limited applicability of rhoda-mine 123 in aquatic systems (28) has led to the use of oxonols for viability assessments. The maintenance of membrane integrity is also a cell viability indicator. DNAintercalators (propidium iodide, ethidium monoazide) thatcannot cross unaltered cell membranes are also often usedas viabilitystains (12,18,21,39).Another group of probes consists of redox dyes, derivedfrom the nonfluorescent INT (2-(p-iodo-phenil)-3-(p-nitro-phenil)-5-phenil tetrazolium chloride), that are capable of determining cellular respiratory activity in both starvedand vegetative bacteria (36,40,42). The redox fluorescentCTC (5-cyano-2,3-ditolyl tetrazolium chloride) has beenused to detect microscopically active bacteria in samplesfrom sewage water, seawater, drinking water, and soil(29,30,32,41) and it has also been used in flow cytometricdetection of active bacteria (15,16,19). The introduction of confocal laser microscopy has established a new standard of analytical precision by virtually eliminating interference fromout-of-focus particles and providing the ability to quantifymicroorganismsastheyoccurin anondestructive analysis(3).The aim of this study was to evaluate the possibility of using double staining protocols, developed in previous Contract grant sponsor: Ministerio de Educacio´n y Ciencia, Spain;Contract grant number: CICYT AMB05-0049; Contract grant sponsor:Eloise, European Union; Contract grant number: MAST III (1994-1998) PL950439.*Correspondence to: J. Vives-Rego, Departament de Microbiologia,Universitat de Barcelona, Av. Diagonal 645, 08028, Barcelona, Spain.E-mail:   1997 Wiley-Liss, Inc. Cytometry 29:298–305 (1997)  studies using flow cytometry, to observe by laser micros-copy cells retained on a filter. These double-stainingprotocols were obtained by combining membrane poten-tial-related dyes (rhodamine 123, bis-oxonol) and a mem- brane integrity indicator dye (PI). The use of bis-oxonolDiBAC4(3) to monitor the viability of starving bacteria inseawater was also combined with the determination of metabolic activity by CTC reduction with both flowcytometryand confocal scanning laser microscopy. MATERIALS AND METHODSStrains, Cultures, and Starvation Conditions All experiments were conducted with  Escherichia coli 536, an isolate from a urinary tract infection (2), and Salmonella typhimurium  ATCC 14028 (American TissueCulture Collection, Rockville, MD). In order to obtainstarved cultures of these strains, cells were incubatedovernight in Luria broth (LB) medium at 30°C, harvested,washed twice by centrifugation at 6,000  g  for 5 min insterile artificial or natural seawater, and resuspended inthe same medium. Both natural and artificial seawaterwere filtered (0.2 µm cellulose acetate, GSWP, Millipore;Bedford, MD) and sterilized by autoclaving. Appropriatevolumes of these resuspensions were used to inoculateflasks containing 250 ml of sterile artificial (ADSA-Micro,Barcelona, Spain) or naturalseawater, in order to obtain aninitial population of 10 6 to 10 7 cells per ml. Flasks wereincubated for up to 60 days at 20°C in the dark withagitation (100 rpm). Viable cells were calculated from thenumber of CFU (colony forming units) on tryptone soyaagar plates (TSA) incubated at 30°C for 5 days. Staining ProtocolsMembrane Potential and Membrane Integrity Re-lated Dyes.  Rhodamine 123 (Sigma-Aldrich Quı´mica S.A.,Barcelona, Spain), a lipophilic cationic dye, was added to afinal concentration of 2 µg/ml from a stock solution of 1mg/ml in ethanol. Anionic bis-oxonol DiBAC4(3) (Molecu-lar Probes, Eugene, OR) was used at 1 µM from a 250 µMstock solution in methanol. Staining of starved cultures inseawater with DiBAC4(3) was performed while the cellswere in the starvation medium, whereas the rhodamine123 staining required replacement of the starvation me-dium with a 0.9%NaCl solution prior to staining. In orderto permeabilize the bacterial outer membrane, ethyleneglycol-bis( β -aminoethyl ether)-N,N,N’,N’-tetraacetic acid(EGTA) (Sigma-Aldrich Quı´mica S.A.) was added prior tothe dyes to a final concentration of 1 mM. The DNAintercalator propidium iodide (PI, Molecular Probes) wasused at a final concentration of 10 µg/ml. Respiratory Activity Three concentrations of the redox probe CTC (5-cyano-2,3-ditolyl tetrazolium chloride) (Polyscience, Warrington,PA) were used to evaluate the kinetics of reduction by  E.coli  and  Salmonella  according to culture conditions andnutrient supplementation. These studies were performedwith cultures of the above-mentioned strains grown in LB(30°C, 12 h) washed and harvested in NaCl solution(0.9%). The experiments were conducted with and with-out the addition of TSB (Tryptone Soya Broth) at 0.03 and0.3 mg/ml. A CTC stock solution of 20 mM in distilledwater was preparedandstoredat 4°C until use; concentra-tions of 0.5, 2.5, and 5 mM were obtained from this stocksolution. The reduction of CTC to the fluorescent forma-zan was monitored by flow cytometry after incubating the bacteriaat 30°C and100 rpm agitation andtaking asampleof the culture every30 min for 7 h. The percentage of CTCreducing cells was also studied in starved cultures innatural and artificial seawater. The CTC assay conditionswere the same as those used in the starvation experiments(20°C, 12 h) in the dark. Reduction of CTC by starvedcultures was also evaluated with and without nutrientsupplementation (0.03 and 0.3 mg/ml). Cephalexine(Sigma-Aldrich Quı´mica S.A.) was added to the sample (20µg/ml) in order to prevent cell division which wouldmodify the rate of reducing cells. Double-staining with bis-oxonol was performed following the single-stainingconditions (2 min at room temperature for oxonol, 12 h at20°C for CTC). Validation and Controls Gramicidin treatment (20 µg/ml, 10 min) was applied tothe double stain rhodamine 123–PI in order to obtaindepolarized cells. This treatment was not applied to thedouble stain bis-oxonol–PI due to the formation of molecu-lar complexes between the lipophilic dye and gramicidin,as reported elsewhere (4). Starved cultures in seawaterwere treated in parallel with two biocides (sodium azide,sodium cyanide) and supplemented with nutrients (0.03and0.3 mg/mlTSB) in order to evaluate the effect oftoxinson CTC reduction and bis-oxonol incorporation. The biocides sodium azide (0.4 and 4 mg/ml), sodium cyanide(0.5 and 0.05%), and the uncoupler CCCP (carbonylcyanide m-chlorophenol hydrazone) (10 µM) were used ascontrols for lack of activity of the electron transportsystem. All were co-incubated with CTC (10 h, 20°C). Flow Cytometric Analysis A Coulter Epics Elite flow cytometer (Coulter Cytome-try, Hialeah, FL) equipped with an air-cooled 488 nmargon-ion laser at 15 mW was set up with the standardconfiguration. Fluorescentbeads(1 µmFluoresbrite carbox-ylate microspheres, Polysciences) were used as an internalstandard for scattering and fluorescence. The green emis-sion from rhodamine and bis-oxonol was collected with a525 nm bandpass filter. The red emission from PI and CTCwas collected with a 675 nm bandpass filter. The forwardscatter detector in the Elite flow cytometer is aphotodiodewhich collects light between 1.5 and 19° from the laseraxis, and is able to discriminate particles   0.5 µm indiameter from backgroundnoise. The side scatter detectoris situated at a 90° angle from the laser axis. Due to thedesign of the quartz flow chamber used, light for both sidescatterandfluorescence iscollectedin an angle widerthan90° using a combination of mirrors and lenses in order to299 CSLM AND FCM IN  E. COLI   AND  SALMONELLA  improve efficiency. Datawere analyzedwith Elite softwareversion 4.1 (Coulter). Confocal Laser Scanning Microscopy Confocal images were obtained with a laser scanningconfocal microscope (TCS 4D; Leica Lasertechnik GmbH,Heidelberg, Germany) using an invertedmicroscope (LeitzDMIRBE, Germany) and a 100x (NA 1.3, oil) Leitz Plan-Fluotar objective. Rhodamine 123, bis-oxonol or CTC andPI were simultaneously excited at wavelengths of 488 and568 nm using an air-cooled krypton-argon laser. Twophotomultipliers were used in parallel. A 488-/568-nmdouble dichroic mirror was used as an excitation beamsplitter anda580-nm shortpass filter dividedgreen andredfluorescent light between the two photomultipliers. A520-nm shortpass emission filter was used for rhodamine123 or bis-oxonol in PMT1, a 590-nm longpass filter for PI,and a 665-nm longpass filter for CTC in PMT2. Images of 512 x 512 pixels were stored on an erasable optical disk,visualized with a true color display screen, then photo-graphed on a color slide film with a digital camera (FocusGraphics, Foster City, Canada). Co-localization analysiswas performed using the Multi Color software (ver. 2.0,Leica Lasertechnik GmbH, Heidelberg, Germany). Thetwo-dimensional confocal cytofluorograms in Figures 1, 3,and 4 show the correlation of the fluorescence signal fromthe two channels as adiagram: the fluorescence intensitiesof the green and red channels are represented on thex-axis and y-axis, respectively (5). The coincidence label-ing between the two single images is shown as the portionof the cytofluorogram located along the diagonal of thegraph (x   y). The vertical (rhodamine 123) and horizon-tal (PI or CTC) portions of the graph are related to thesingle-labeled regions within the two single images. Stain-ing conditions for the dyes were as described for flowcytometric measurements. Once stained, samples (1 ml)were filtered (0.2 µm polycarbonate, GTBP Millipore,Barcelona, Spain) and filters were air-dried, mounted withlow-fluorescence immersion oil (Olympus, nd 1.518) on aslide and observed within the next 15 min. Dye bleachingoccurred for membrane potential dyes when preparationswere observed later than this. RESULTSDiscrimination Between Viableand Non-viable Cells by CSLM Stationaryculturesof  E.coli  stainedwith rhodamine 123and propidium iodide gave a high percentage of greenfluorescent cells (rhodamine 123), while only a few cellsshowed red fluorescence (PI) (Fig. 1a). When samples of the same culture were treated with the ionophor gramici-din, images indicated a lower incorporation of rhodamineand a higher permeability to PI (Fig. 1b). The cytofluoro-grams of images a andb revealeddifferences in the relativefluorescence intensity of the stained cells and the absenceof double stained cells (Fig. 1c,d). Preparations remainedstable for only 15 min. Longer times resulted in alterationsof rhodamine equilibrium and PI permeability.In order to use amembrane potentialdye to stain cells inthe survival medium, rhodamine 123 was replaced withthe anionic lipophilic bis-oxonol (DiBAC4(3). Figure 2shows the image corresponding to an  E. coli  culturestarvedfor 15 days in seawater, stainedwith bis-oxonol for5 min, filtered (0.2 µm) and mounted for observation. Thedensitogram (graph x-y) displays the fluorescence inten-sity of different cells from the same plane of focus,corresponding to different amounts of accumulated dye. Assessment of Respiratory Activity by FlowCytometry and CSLM; Correspondence WithMembrane Potential Measurements The frequency of CTC reducing cells was dependent onthe probe concentration andnutrient addition. The saturat-ing concentration of CTC for a standard sample processedwere: 2.5 mM (with nutrient addition) and 5 mM (nonutrient addition). The detection of cell depolarization instarvation experiments was combined with determinationof the respiratory activity by CTC reduction. Figure 3shows the images obtained after using the CTC-bis-oxonoldouble stain to discriminate active (those accumulatingred formazan) from depolarized cells (bis-oxonol incorpo-rating) using laser microscopy. Samples taken from sur-vival experiments after periods of 15 days in seawaterwere incubated with CTC and exogenous nutrients (0.03mg/ml TSB) for 8 h at 20°C and then stained with bis-oxonol (5 min, room temperature). These starvedcultures still showed a low CTC-reducing capacity andcontained some depolarized cells that incorporated bis-oxonol (Fig. 3a). The supplementation of the exogenousnutrients with the uncoupler CCCP resulted in a decreasein the number of CTC-reducing cells and an increase indepolarized cells (Fig. 3b). Cytofluorograms (Fig. 3c,d)revealed cells with intermediate fluorescence levels, to-gether with the absence of double staining.The effect of nutrient supplementation was also studied by using the double stain rhodamine 123 - CTC on cellsretained on a filter and observed by CSLM. Figure 4displays the images corresponding to starved cultures (30days in seawater) incubated with CTC (12h, 20°C) andstained with rhodamine 123 (5 min, room temperature).At the lowest nutrient concentration used (0.03 mg/ml),most of the cells incorporated rhodamine but no CTCreduction was observed (Fig. 4a and cytofluorogram 4c).Onlyat 0.3 mg/ml of nutrient concentration didsome cellsactivelyreduce CTC (Fig. 4b and cytofluorogram 4d).A more accurate approximation of respiratory activity(detected by CTC reduction) and membrane energization(detected by bis-oxonol incorporation) was obtainedthrough flow cytometric measurements.  E. coli  starvedcultures in seawater were treated with different biocidesand nutrient supplements, following which cell viabilitywas evaluated by viability counts and dye incorporation.Table 1 compares the percentages (from flow cytometriccounts) of respiring cells (CTC reducing), depolarizedcells (bis-oxonol incorporating), and viable cells (CFU/mlon TSA) in starved cultures treated with Azide (0.4 and 4mg/ml), NaCN (0.005 and 0.05 mg/ml), and nutrient300  LO´PEZ-AMORO´S ET AL.  F IG . 1. Confocal images of the double stain rhodamine 123 (greencolor) and propidium iodide (red color). ( a ) Stationary  E. coli  culture. ( b )Stationary  E. coli  culture after gramicidin treatment. ( c,d ) Confocalcytofluorograms of images (a,b) represent the fluorescence from rhoda-mine 123 and PI. In (c), the cytofluorogram shows that there is no signalfrom PI, in contrast to cytofluorogram (d) where almost all fluorescentcounts come from the second channel (red fluorescence, x-axis). Scale bar: 10 µm.Fig. 2. Confocal image of the bis-oxonol staining of an  E. coli  starvedculture in seawater. The densitogram shows the relative fluorescenceintensityof different cells in the same plane of focus.Fig. 3. Confocal images of the bis-oxonol–CTC staining on a starvedculture of   Salmonella typhimurium . ( a ) Sample supplemented with 0.03mg/ml TSB. ( b ) Sample supplemented with 0.03 mg/ml TSB and theuncoupler CCCP. ( c,d ) Confocal cytofluorograms of images (a,b). In (c)there are few fluorescent counts from both channels (green channel,y-axis corresponding to bis-oxonol and red channel, x-axis correspondingto CTC); in (d) the addition of an uncoupler produces significativenumbers of bis-oxonol stained cells.Fig. 4. Confocalimages of the rhodamine 123–CTCstainingof an  E.coli starved culture in seawater. ( a ) Sample supplemented with 0.03 mg/mlTSB. ( b ) Sample supplemented with 0.3 mg/ml TSB. (c,d) Cytofluoro-grams of images (a,b). The cytofluorogram in (c) shows that all thefluorescence signal is in the first channel (y-axis, rhodamine 123) and thatthere is little CTC signal in the second channel (x-axis, red fluorescence).In (d) the ratio of redfluorescent cells is higher. Yellow pixels in the x  yarea represent cells with double fluorescence. 301 CSLM AND FCM IN  E. COLI   AND  SALMONELLA  supplements (0.03 and 0.33 mg/ml TSB). The addition of nutrients to a starved culture resulted in recovery of themembrane potential (showed by a decrease in bis-oxonolpositive counts) and an increase in both CTC reductionandviabilitycounts in culture media. When the addition of nutrients was accompanied by biocides, effects on themetabolic state were not observed, as indicated by higher bis-oxonol positive counts, lower CTC positive counts,andreducedviabilitycounts. Sodium cyanide at the higherconcentration used (0.05 mg/ml) produced cellular lysis.The cellular debris reduced the forward scatter signal(parameterusedastriggersignal), therefore resultingin unreli-able percentagesofbis-oxonolandCTCpositive cells.Both CTC and bis-oxonol were used in flow cytometricmeasurements to monitor bacterial starvation in seawater.Figure 5 shows changes in respiratory activity (CTCreducing cells), membrane energization (bis-oxonol incor-poration), and viability counts (CFU/ml on TSA) duringstarvation of   E. coli  in seawater for periods of up to 60days. As starvation time increased, the direct respiratoryactivity measured by CTC reduction (without nutrientsupplementation) decreasedwhile the bis-oxonolincorpo-ration increased (correlation index, r    -0.98). This factwas shown by the increase in both the mean cell fluores-cence and the percentage of cells with a fluorescenceintensity above the limit channel (according to controlsamples). Correlation between viability counts (CFU/ml)and bis-oxonol or CTC positive counts was not very high(r  -0.73 and r  0.81, respectively).The effect of nutrient addition on respiratory activityand membrane energization demonstrated by CSLM wasquantified by flow cytometric measurements. In samplestaken from starved seawater cultures, the levels of bis-oxonol fluorescent cells were inversely related to those of CTC-reducingcells, both with andwithout nutrient supple-mentation (0.03 mg/ml TSB) (Table 2). Different mem- brane depolarization states were indicated by the levels of  bis-oxonol incorporation; this was consistent with the lowquantity of actively respiring cells (direct CTC reduction)and the slight increase in the number of CTC-reducingcells after the nutrient addition. Figure 6 shows thehistogramscorrespondingto the bis-oxonolstaining(mono-parametric) and the CTC reduction (biparametric) of seawater starved cultures before and after adding 0.03mg/ml TSB. The distribution of the fluorescence in starvedcells stained with bis-oxonol showed some depolarized(nonfluorescent) cells (A), although no CTC reductionactivity was detected (C). Nutrient supplementation pro- Table 1 Validation of the CTCStaining in Relation to Bis-oxonol Incorporation and Viability Counts During  E. coli  Starvation;Flow Cytometry Measurements Starved cellsTSB supplementation (mg/ml)0.30.03 0.3 Sodium azide mg/ml Sodium cyanide mg/mlNo biocide addition 0.4 4 0.005 0.05%CTC a ND 27.3 53.7 1.8 0.1 19.5 4.3%BIS  b 40.2 30.7 21.4 26 29.7 18.3 1.2CFU/ml 1.15 · 10 5 2 · 10 5 6.1 · 10 5 7.5 · 10 4 5 · 10 4 9.25 · 10 4 4.8 · 10 4a Percentage of cells with a fluorescence intensity above channel number 10 (channel number determined by controlsamples). Samples were incubated with CTC (2.5 mM) for 12 hours at 20°C in the dark.  b Percentage of cells with a fluorescence intensityabove the specific channel number (according to results obtainedfrom stainedviable cells). ND, notdetected. F IG . 5. Changes in respiratory activity (CTC reduction) and membraneenergization (bis-oxonol incorporation) during  E. coli  starvation in artifi-cial seawater. Flow cytometric percentages of CTC positive counts ( 0 )and DIBAC4(3) ( N ) related to the total analyzed population (triggered byscatter signals) and percentage of CFU/ml ( M ) in relation to the initialviabilitycounts (24 hours). Table 2 Effect of Nutrient Supplementation on the Percentage of Cellsthat Reduce CTCand Incorporate Bis-oxonol in StarvedCultures of   E. coli  and  Salmonella  in Artificial or NaturalSeawater Bis-oxonolCTC%Stainedcells c Meanfluorescence%Stainedcells%Stainedcells (0.03mg/ml TSB) d Escherichia ASW a 52.7 2.16 5.9 16.3NSW  b 61.5 3.15 1.4 4.3 Salmonella ASW 23.2 0.8 14.9 18.6NSW 93.2 16.2 0.3 2.1Samples were taken from flasks after 30 days of starvation. a Filtered(0.2 µm) andautoclavedartificialseawater.  b Filtered(0.2 µm) andautoclavednaturalseawater. c Percentage of cells from the direct sample with a fluorescenceintensity above the limit channel (limit channel according to controlsamples). d Sample supplemented with 0.03 mg/ml TSB and cephalexine (40µg/ml). 302  LO´PEZ-AMORO´S ET AL.
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