ORIGINAL PAPER

Effect of different fluorescent dyes on thermal stability of DNAand cell viability of the hyperthermophilic archaeon Aeropyrumpernix

Miha Crnigoj Æ Rok Kostanjsek Æ Gonul Kaletunc ÆNatasa Poklar Ulrih

Received: 11 September 2007 / Accepted: 4 March 2008 / Published online: 18 March 2008� Springer Science+Business Media B.V. 2008

Abstract The interactions of DNA-binding dyes (Hoechst33258, DAPI, acridine orange) and DiBAC4(3) withhyperthermophilic archaeon Aeropyrum pernix cells wereinvestigated by the combination of calorimetric, spectro-scopic and microscopic techniques. All of the dyes, studiedhere, affect the thermal stability of DNA in vivo andin vitro. Hoechst 33258 is highly DNA-specific probe,which does not affect the thermal transitions of othercellular components as can be detected by differentialscanning calorimetry (DSC). Due to this unique property, itcan be used as a potential DNA marker for in vivo DSCstudies. The localization of the dyes in the cells and via-bility assay was revealed by fluorescence microscopy.Hoechst 33258, DAPI and acridine orange did not distin-guish between viable and non-viable cells of Aeropyrumpernix. Only with the commercially available Live/DeadBacLightTM kit we were able to discriminate viable andnon-viable Aeropyrum pernix cells.

Keywords Differential scanning calorimetry �Fluorescence microscopy � Hoechst 33258 � DAPI �Acridine orange � DiBAC4(3) � Live/Dead BacLightTM kit

Introduction

The hyperthermophilic Archaea and Bacteria with optimalgrowth temperatures above 80�C exist at the upper tem-perature border of life (Beck and Huber 1997; Stetter1999). Aeropyrum pernix is a thermophile that was isolatedfrom a coastal solfataric thermal vent on Kodakara-JimaIsland in Japan (Sako et al. 1996). It is the first reportedobligately aerobic and neutrophilic hyperthermophilicarchaeon, with an optimum growth temperature between90�C and 95�C, and it was classified as a member of theCrenarchaeota.

The rigidity of the cell membrane of extremophilesarises from ether-linked lipids and unique cell-wall com-ponents. This can lead to a lower permeability of theirmembranes to some dyes, and consequently this mightcause problems with viability detection by differentiationbetween viable and non-viable cells. A variety of fluores-cent dyes are used as indicators for the detection of cellviability (Huber et al. 1985; Haidinger et al. 2003). Beckand Huber (1997) tested a number of fluorescent dyes ondifferent Archaea with the purpose of developing a uni-versal approach for rapid discrimination of viable and non-viable hyperthermophiles using fluorescent dyes and theapplication of this approach for the isolation of livingsingle cells using a laser microscope. They showed thatDiBAC4(3) was the most suitable dye to differentiatebetween living and dead cells using fluorescence micros-copy. As DiBAC4(3) is known to selectively staindepolarized cells only, it was considered to be a universal

M. Crnigoj � N. Poklar Ulrih (&)Department of Food Science and Technology, BiotechnicalFaculty, University of Ljubljana, Jamnikarjeva 101,1000 Ljubljana, Sloveniae-mail: natasa.poklar@bf.uni-lj.si

R. KostanjsekDepartment of Biology, Biotechnical Faculty,University of Ljubljana, Vecna pot 111, 1000 Ljubljana,Slovenia

G. KaletuncDepartment of Food, Agricultural and Biological Engineering,The Ohio State University, 590 Woody Hayes Drive, Columbus,OH 43210-1057, USA 123World J Microbiol Biotechnol (2008) 24:2115–2123DOI 10.1007/s11274-008-9717-3

dye that should be suitable for the detection of cell viabilityfor hyperthermophiles (de Poorter et al. 2001; Alakomiet al. 2005). Additionally, the commercially available Live/Dead BacLightTM kit has also been successfully used fordetection of extremophilic Archaea and the visualization ofmicroorganisms in hypersaline environmental samples(Leuko et al. 2004).

Among the DNA-binding fluorescent dyes we studiedhere, Hoechst 33258 and DAPI bind in the minor groove ofDNA with high sequence specificities for adenine and thy-mine (Breusegem et al. 2002; Kiser et al. 2005); acridineorange interacts with DNA and RNA by intercalation andelectrostatic attractions (Kapuscinski and Darzynkiewicz1990). DiBAC4(3) was included in this study as an anionicmembrane-potential-sensitive probe, which is distributedacross membranes according to the Nernst-type equilibrium(Brauner et al. 1984). The DNA-Hoechst 33258 complexalso has a higher thermal stability than DNA alone in vitro.Lee and Kaletunc (2005) demonstrated that this thermalstability increase can be used to identify DNA transition in acomplex whole-cell thermogram where the transitions of theindividual cellular components are difficult to identifywithout comparisons with differential scanning calorimetry(DSC) transitions of the isolated components. In our previ-ous study (Milek et al. 2007), we used DSC to analyse therelative thermal stabilities of cellular components of Aero-pyrum pernix in vivo. We found that DNA, which is one ofthe most stable macromolecules in bacteria, is the least stablemacromolecule in the hyperthermophilic archaeon A. pernix.To further investigate DNA stability inside the cell, we usedthree commercially available DNA-binding fluorescentprobes, Hoechst 33258, DAPI and acridine orange, whichallowed us to identify the DNA peak in DSC thermogramsand to visualise the DNA molecules inside cells using fluo-rescence microscopy.

Materials and methods

Growth conditions and preparation of cells

The Aeropyrum pernix K1 strain was purchased from theJapanese Collection of Microorganisms (JCM number9820, Wako-shi, Japan). A. pernix cells were cultivated in a1.0 l, heavy-walled, glass-flask, bioreactor system withoutany steel parts, as previously described (Milek et al. 2005).The Archaeon was grown at 92�C in medium containing37.4 g/l Marine Broth 2216 media (DifcoTM, Becton,Dickinson, Sparks, USA), 1.0 g/l Na2S2O3 9 5H2O(Alkaloid, Skopje, Macedonia) and 4.766 g/l HEPES(Sigma), with the pH adjusted to pH 7.0 with 4.0 M NaOHbefore autoclaving. The cells were grown at 92�C undermaximal aeration until the mid-to-late exponential phase

(*15 h) (Milek et al. 2005). Cell growth was monitored byoptical density at 650 nm (OD650).

After cultivation, the cells were harvested by centrifu-gation (Beckman, JA-14 rotor; 8,000 rpm, 10 min forDSC; Eppendorf centrifuge 5415 C; 12,000 rpm for fluo-rimetry), and washed twice in wash buffer: 10 mMHEPES, pH 7.0, 260 mM NaCl (or 513 mM NaCl formicroscopy). After washing, the fluorescent dyes wereadded according to the cell mass (for DSC) or per cellsuspension turbidity determined at 650 nm (for fluorimetryand microscopy). The starting concentrations of the cellsuspensions were adjusted to an OD650 of 1.0, and diluted afurther 10-fold for fluorimetry. The biomass concentrationsof A. pernix were obtained using the correlation developedpreviously (Milek et al. 2005).

The numbers of cells in liquid cultures were determinedby cell counting in a Hausser Scientific PartnershipCounting Chamber (Cat. No 3900) under differentialinterference contrast (DIC) microscopy. The depth ofcounting chamber (0.02 mm) was greater than the radius ofthe A. pernix cells, so an instant sequence of six to10 micrographs was taken. These were later united into onemicrograph (stacking) using the Helicon Focus software (http://HeliconFocus.com). Altogether, 60 squares werecounted and approximate values were calculated. Onaverage, there were 81.5 cells per 5 9 10-5 ll (volume ofthe field calculated from 50 9 50 9 20 lm), whichequates to 1.6 9 109 cells per ml (less than 1% of the cellswere dead).

Fluorescent dyes

Hoechst 33258

Pentahydrate bis-benzimide phenol 4-[5-(4-methyl-1-pip-erazinyl)[2,50-bi-1H-benzimidazol]-20-yl]-trihydrochloride(Molecular Probes, Inc., Oregon, USA) is an AT-selective,minor-groove, DNA-binding dye (kex = 352 nm, kem =

461 nm).

DAPI, dilacetate

40,6-Diamidino-2-phenylindole dilacetate (MolecularProbes, Inc., Oregon, USA) is also an AT-selective DNA-binding dye (kex = 358 nm, kem = 461 nm).

Acridine orange

3,6-Acridinediamine N,N,N0,N0-tetramethyl-monohydro-chloride (Molecular Probes, Inc., Oregon, USA) is a DNA-binding dye (kex = 500 nm, kem = 526 nm).

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DiBAC4(3)

Bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Molec-ular Probes, Inc., Oregon, USA) is a dye that binds proteinsand membranes (kex = 493 nm, kem = 516 nm).

LIVE/DEAD BacLightTM Bacterial Viability Kit

This kit includes pre-prepared solutions of SYTO 9 (kex =

490 nm, kem = 520 nm) and propidium iodide (kex = 490 nm,kem = 635 nm) (http://probes.invitrogen.com/).

All of the dyes used, except for DiBAC4(3), were dissolvedin ultrapure, double-distilled water. DiBAC4(3) was dissolvedin 25% ethanol, which resulted in a final ethanol concentrationof less than 0.1%. The concentrations of the dyes weredetermined spectrophotometrically (HP 8453 spectropho-tometer, Hewlett Packard, Palo Alto, USA) at 25�C, using thefollowing extinction coefficients (e): Hoechst 33258, 40,000M-1 cm-1 at 352 nm; DAPI, 21,000 M-1 cm-1 at 344 nm;acridine orange, 53,000 M-1 cm-1 at 500 nm; andDiBAC4(3), 140,000 M-1 cm-1 at 493 nm (http://probes.invitrogen.com/).

Isolation of genomic DNA

The A. pernix cultures were grown to the late-exponentialphase. The biomass was collected by centrifugation, asdescribed above, and the genomic DNA was isolated aspreviously described (Pitcher et al. 1989). Additionalchloroform–phenol purification of the DNA was needed,until no interphase impurities were seen (up to eight suc-cessive extractions). The A260/A280 ratio was 1.73 ± 0.02.The concentrations of DNA per base pair were determinedspectrophotometrically (HP 8453 spectrophotometer,Hewlett Packard, Palo Alto, USA) at 260 nm and 25�C,using an extinction coefficient of 12,800 M-1 cm-1.

Titration of A. pernix cells with dyes

The A. pernix cells were resuspended in wash buffer andtitrated directly into the cuvette at 25�C and 50�C by addingaliquots of the different dyes. The fluorescence emissionspectra were measured after each addition of Hoechst 33258,DAPI, acridine orange and DiBAC4(3) to 1-cm cuvettescontaining the A. pernix cells (0.027 mg dry cell mass perml) using a Cary Eclipse Fluorescence Spectrophotometer(Varian, Australia). The emission spectra were recorded inthe wavelength range from 400 nm to 600 nm for Hoechstand DAPI, and from 500 nm to 700 nm for acridine orangeand DiBAC4(3). The characteristic excitation wavelength,kex, for each dye was used (see above). The emission spectraof the dyes were corrected for the solvent blank and weremultiplied by the dilution factor. The final spectra were themeans of three individual scans.

Melting profile of genomic DNA

The fluorescence emission intensity of DNA (10 lM perbase pair) in the presence of each of the dyes, and of thedyes in the absence of DNA (base-line) was followed at asingle wavelength versus temperature (melting profile).The characteristic kem for each of the dyes was used (seeabove). The temperature was raised in 1�C increments inthe temperature range from 20�C to 100�C, and the sam-ples were allowed to equilibrate for 1 min at eachtemperature. All of the experiments were carried out at pH7.0 (10 mM HEPES) and at a DNA (per base pair) to dyemolar ratio of 20 to 1, with 0.5 lM final dye concentration.For each optically detected transition, the melting tem-perature (Tm) of the DNA was taken as the transitionmidpoint. The final Tm values given are the means of threeindividual measurements.

Differential scanning calorimetry of whole cells

DSC was used for collection of the thermograms of wholecells (DSC 111 Setaram, Lyon, France). All of the DSCmeasurements were conducted using fluid-tight, stainlesssteel crucibles. A DSC thermogram with an empty sampleand reference crucibles was collected to record the emptycrucible base line. The suspensions of A. pernix cells withthe dyes in the wash buffer were incubated in the dark at25�C for 2–8 h, or at 50�C for 2 h. After the incubations,80 ± 0.5 mg of the cell pellets (at *80% moisture) wereloaded into the stainless steel crucibles, which were closedfluid-tight with the stainless steel caps and aluminum o-rings before being weighed and put into the DSC instru-ment. A known quantity of wash buffer, similar in mass tothe moisture in the sample (64.0 ± 0.2 mg; i.e. *80% ofthe sample wet mass), was placed in the reference crucible.All of the samples were scanned from 20�C to 180�C at aheating rate of 4�C/min, and then reweighed after the DSCmeasurements to check for potential loss of mass duringheating.

The DNA peaks in the DSC thermograms were notvisible in the first scans. As DNA denaturation in the cell isa reversible process, as seen previously (Milek et al. 2007),we performed heat pre-treatment scans (heating from 20�Cto 100�C ± 1�C, and to 110�C ± 1�C for DAPI) toremove the irreversible peaks overlapping with the DNApeaks in the thermograms.

The DSC of the A. pernix cells in the presence ofHoechst 33258 was taken at different times and tempera-tures of incubation (25�C, up to 8 h; 50�C, up to 2 h) and atdifferent dye concentrations (0.625, 3.125, 6.25 and37.5 nmol/mg dry cell mass). The DSC of the A. pernixcells in the presence of DAPI (50 nmol/mg dry cell mass),acridine orange (3.75 nmol/mg dry cell mass) and

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DiBAC4(3) (0.075 nmol/mg and 0.375 nmol/mg dry cellmass) were taken after incubations of 2 h at 50�C.

Fluorescence imaging

All of the imaging was performed using an Axio ImagerZ.1 (Zeiss) microscope that had been upgraded with anApoTome (Zeiss) system for the optimization of fluores-cence microscopy, along with a charge-couple-device HRccamera (Zeiss). For non-fluorescence microscopy, DIC wasused, while for fluorescence microscopy, sets of filters withultraviolet (filter set FS01), blue (FS09) and green (FS15)excitation light were used, depending on the fluorescentdye. Filter FS01 was used for viewing Hoechst 33258 andDAPI stained cells, a combination of filters FS09 and FS15for acridine orange stained cells, and FS09 for cells stainedwith DiBAC4(3). When using BacLightTM kit (SYTO 9and propidium iodide), the FS09 and FS15 filters wereused.

The A. pernix cells for microscopy were resuspended in10 mM HEPES, 513 mM NaCl, pH 7.0. Dead cells wereobtained by incubation of cells at 130�C for 10 min, aspreviously described (Milek et al. 2007). The concentra-tions of dyes used were 1.5 mM Hoechst 33258, 1.2 mMDAPI, 2.2 mM acridine orange, 1.6 mM DiBAC4(3),1 mM SYTO 9, and 20 mM propidium iodide.

Microscopy staining was performed by adding a smallvolume (10 ll) of dye to the edge of a cover slip, andallowing the dye to flow into the cell suspension by dif-fusion (based on the gradient of the dye concentrationbetween the slide and the cover slip). For Hoechst 33258,no gradient of dye was applied; instead, the cells were pre-incubated with the dye at 50�C to assure better penetrationof the dye into the cells. For the BacLightTM kit, both dyeswere added to cell samples 10 min prior to microscopy, asdescribed in the manufacturer protocol. To determine thekit activity, we also stained Escherichia coli cells (washedwith 10 mM HEPES, 0.9% NaCl, pH 7.0).

Results and discussion

Binding of dyes to A. pernix cells in vivo

The titration curves of the A. pernix cells and Hoechst33258, DAPI, acridine orange and DiBAC4(3) are shown inFig. 1. The binding ratios in nmole dye per mg dry cellmass were: Hoechst 33258, 163 ± 7; DAPI, 456 ± 11;acridine orange, 144 ± 11; and DiBAC4(3), 178 ± 11.The fluorescence titration curves indicated that saturationof the A. pernix cells with these dyes is similar, except forDAPI, which is 2-fold higher. It is likely that the cellenvelope of A. pernix has a low permeability towards

DAPI. The binding ratio of the dye to the A. pernix cellswas seen to be independent of the incubation temperature(25, 50�C), although the kinetics of the binding was tem-perature dependent. Dye saturation of cells at 25�C wasachieved in an 8-h incubation for Hoechst 33258, in 4 h forDAPI, and in 2 h for acridine orange and DiBAC4(3). Thetime needed for Hoechst 33258 to penetrate into the cellswas relatively long (8 h at 25�C) in comparison withacridine orange and DiBAC4(3), (2 h at 25�C). Thedrawback of such long incubation times of Hoechst 33258can be overcome by increase the temperature of incubation,e.g. at 50�C 2 h was sufficient to reach saturation.Recently, we have shown that the permeability of A. pernixmembrane to pyrene increases with temperature and cor-relates with fluidity changes measured by electronparamagnetic resonance spectroscopy (EPR). The sharpincrease in the fraction of the less ordered domain wasobserved in the temperature range between 50�C and 60�C(Poklar Ulrih et al. 2007).

Thermal stability of A. pernix genomic DNA in thepresence of the dyes in vitro and in vivo

The effects of dyes on the thermal stability of genomic DNAisolated from A. pernix is shown in Fig. 2. Inspection ofFig. 2 reveals that at a 1–20 molar ratio of dye to DNA basepair (DNAbp) at pH 7.0 (10 mM HEPES buffer), Hoechst33258 thermally stabilized DNA by 11�C, DAPI by 1�C and

Fig. 1 Titration curves of A. pernix cells titrated with fluorescentdyes at 25�C. Normalized fluorescence intensity (F/Fo) of (j)Hoechst 33258, (d) DAPI, (m) acridine orange and (u) DiBAC4(3),plotted as a function of the dye to cell ratio, R, (in nmol dye per mgdry cell mass). F and Fo (maximal) stand for the fluorescenceemission intensities of the dyes in the presence of the A. pernix cells.The excitation wavelengths were: Hoechst 33258, 352 nm; DAPI,358 nm; acridine orange, 500 nm; and DiBAC4(3), 493 nm. Thefluorescence emission intensities of the dyes were measured at:Hoechst 33258 and DAPI, 461 nm; acridine orange, 526 nm; andDiBAC4(3), 516 nm

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acridine orange by 5�C. At the same molar ratio, a thermaldestabilization of DNA by DiBAC4(3) of approximately -

1�C was seen. The melting temperature, Tm, of the isolated A.pernix genomic DNA at pH 7.0 (in 10 mM HEPES), deter-mined from the UV-melting curve, was 70.5 ± 0.5�C.

The DNA thermal stability and transitions inside cellscan be monitored by differential scanning calorimetry(DSC). The thermogram of whole A. pernix cells at pH 7.0(10 mM HEPES, 260 mM NaCl) is shown in Fig. 3. Twomajor overlapping endotherms with peak temperatures of119�C (peak b) and 133�C (peak c) appears in thermogram(Fig. 3a, line 1), which have been previously attributedpredominantly to the denaturation of ribosomal subunits(Milek et al. 2007). An additional endothermic transition oflow enthalpy (peak a) was also seen at 96.5�C, before peakb, which was ascribed to DNA denaturation. Previously,we demonstrated that cells have to be preheated to see theDNA transition in DSC thermograms clearly (Milek et al.2007). When the cells were preheated to 100�C, peak abecame more pronounced, but shifted from 104�C to96.5�C (Table 1).

In all of our samples studied here, the cells were incu-bated for 2 h at 50�C in the presence of the dyes and thenpreheated to 100�C. Fig. 3a shows the effects of Hoechst33258 on the A. pernix thermal profile. Increasing con-centrations of Hoechst 33258 shifted the temperature ofpeak a to higher values, while the temperature of peaks band c changed only slightly (Fig. 3a, Table 1). Theseresults suggest that Hoechst 33258 only binds to the DNA

and does not interact with other cellular components. Atthe Hoechst 33258 concentration of 37.5 nmol/mg dry cellmass, peak a merged with peak b (Fig. 3a, line 5). At thisconcentration the DNA inside the cells becomes too ther-mally stable and it is impossible to follow its transitionindependently.

The A. pernix cells incubated with DAPI (50�C, 2 h)needed higher preheating temperatures to make the DNApeak visible in the DSC thermograms, and therefore aseries of pre-treatment was carried out with temperaturesranging from 100 to 112�C (Fig. 3b). The peak of the DNAin the DSC thermograms became visible if the A. pernixcells were preheated to at least 109�C. The denaturationtemperature of the DNA determined from the thermogramswas 96.5�C. In the presence of 50 nmol DAPI per mg dry

Fig. 2 The melting profiles of A. pernix DNA in the presence of thedifferent dyes. The fD is the molar fraction of denatured DNAmolecules. (–) normalized UV-melting profile of A. pernix DNA inthe absence of the dyes; (– –) DNA and Hoechst 33258 (kex, 352 nm;kem, 461 nm); (�����) DNA and DAPI (kex, 358 nm; kem, 461 nm);(– � –) DNA and acridine orange (kex, 500 nm; kem, 526 nm); (– ��)DNA and DiBAC4(3) (kex, 493 nm; kem, 516 nm). The DNAconcentration was 10 lM per base pair (giving a molar ratio of dyeto DNAbp of 1 to 20). The buffer used was 10 mM HEPES, pH 7.0

Fig. 3 DSC thermograms of A. pernix cells incubated with thedifferent dyes. (A) Hoechst 33258 (50�C for 1 h) at 0.625, 3.125, 6.25and 37.5 nmol per mg dry cell mass (1–5); (B) DAPI (50�C for 1 h) at50 nmol/mg dry cell mass, using different pretreatment temperatures:A. pernix cells alone (1), and (2–5) with DAPI pretreated to 100, 109and 112�C, respectively

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cell mass, the denaturation temperature of DNA was102 ± 0.5�C, and it did not change with pre-heating tem-perature (Fig. 3b, Table 1). However, the effects of thepre-heating temperatures in the presence of DAPI wereseen as changes in the shapes of peaks b and c.

The effect of acridine orange on the DSC thermogram ofA. pernix was similar to those seen for Hoechst 33258 andDAPI. At an acridine orange concentration of 3.75 nmol/mg dry cell mass, the Tm of peak a that corresponded toDNA denaturation was shifted from 96.5�C to 99.6�C. Asimilar increase in thermal stabilization of genomic DNA(DT of 4.5�C) was seen from fluorescence-melting curves(Fig. 2). Furthermore, acridine orange affected the tem-perature of the ribosomal peaks b and c (Table 1). Thedecrease in temperature of ribosomal peak b and theincrease in temperature of peak c in the presence of acri-dine orange are probably due to acridine orange binding toribosomes.

Different effects of DiBAC4(3) on the A. pernix DSCprofile were seen (data not shown). As the DiBAC4(3)concentrations increased, the Tm of the DNA peakdecreased (Table 1). At a concentration of 0.375 nmol

DiBAC4(3) per mg of A. pernix dry cell mass, the Tm of theDNA peak was decreased by 5�C. Although DiBAC4(3) isa membrane-sensitive probe, our results demonstrate that itcan bind to DNA, since it makes the DNA transition visibleby fluorescence emission spectroscopy. Our results fromDSC (data not shown) and the fluorescence melting profilesof purified DNA show that DiBAC4(3) has a destabilizingeffect on the thermal stability of DNA.

Fluorescence Imaging

The auto-fluorescence of the A. pernix cells prior to theirtreatment with the dyes was also examined; it was negli-gibly weak with the FS01 UV-filter, and non-existent withthe FS09 blue filter and the FS15 green filter (data notshown). The micrographs of the A. pernix cells incubatedwith Hoechst 33258 at 50�C for 2 h showed that themajority of the cells were intact in the buffer solution used(Fig. 4a, panels I, II, arrowheads 1), although individualdead cells could be seen occasionally.

When using DAPI as a DNA-specific fluorescent dye forthe visualization of cells, a clearly visible centred DNAcoloration was seen in intact cells (Fig. 4b, panel II,arrowheads 1). As can be seen from Fig. 4b (panel IV,arrowheads 2), there was some uniformly distributed col-oration in the presence of dead cells.

Acridine orange coloured the A. pernix cells due toDNA and RNA binding. The micrographs illustrated inFig. 4c (panel II) were generated by combining two pic-tures of the same frame obtained with the different filters(FS9 and FS15). Panels III and IV show separated DNA(III) and RNA (IV). In Fig. 4c (panel II), the central greencoloration is apparent due to DNA localization, with auniformly red coloration due to RNA localization in intactcells (Fig. 4c, arrowheads 1), while a centred DNA color-ation in the dead cells was not seen (Fig. 4c, panel II,arrowheads 2).

DiBAC4(3) showed clear and strong coloration of theintact A. pernix cells (Fig. 4d). As the cells died, theircoloration started to fade and the background fluorescencestarted to grow. Soon it predominated over the cell fluo-rescence signal (Fig. 4d, panel IV). Note that thebackground fluorescence in panel IV was much strongerthan in panel II. DiBAC4(3), which is not suitable for DSCmeasurements, had not been confirmed to clearly distin-guish between viable and non-viable cells of A. pernix asthe Live/Dead BacLightTM kit is known to do.

The Live/Dead BacLightTM kit stained living E. colicells green (arrowheads 1) and dead cells red (arrowheads2), and the coloration was clearly distinguishable whenviewed with the FS09 filter (green and red channel) (Fig. 5,panels I and II). Living A. pernix cells were also stainedgreen (Fig. 5, panels III and IV, arrowheads 1) and dead

Table 1 Effects of the different dyes on the DSC transitions of A.pernix

Treatment conditions Peak a (�C) Peak b (�C) Peak c (�C)

ControlWithout preheating 104.4 ± 0.5 119.0 ± 0.5 132.8 ± 0.5Preheated to100�C 96.5 ± 0.5 117.5 ± 0.5 133.8 ± 0.5

Hoechst 33258Preheated to 100�CConcentrations (nmol/mg dry cell mass):0.625 97.7 ± 0.5 117.3 ± 0.5 132.2 ± 0.53.125 100.4 ± 0.5 118.0 ± 0.5 131.4 ± 0.56.250 102.8 ± 0.5 118.4 ± 0.5 132.2 ± 0.537.500 – 118.0 ± 0.5 131.8 ± 0.5

DAPIPreheated to 100�C – 116.6 ± 0.5 132.0 ± 0.5Preheated to 109�C 102.3 ± 0.5 117.4 ± 0.5 132.5 ± 0.5Preheated to 112�C 101.7 ± 0.5 119.7 ± 0.5 132.2 ± 0.5Concentration: 50 nmol/mg of dry cell mass

Acridine orangePreheated to 100�C 99.6 ± 0.5 118.7 ± 0.5 133.0 ± 0.5Concentration: 3.750 nmol/mg dry cell mass

DiBAC4(3)Preheated to 100�CConcentrations (nmol/mg of dry cell mass):0.075 101.2 ± 0.5 118.3 ± 0.5 132.5 ± 0.50.375 98.9 ± 0.5 118.3 ± 0.5 132.5 ± 0.5

Controls were performed with A. pernix cells in 10 mM HEPES,260 mM NaCl, pH 7.0

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cells were stained red (Fig. 5, panels III and IV, arrow-heads 2). However, the red coloration of A. pernix cells wasnot as intense as that seen for E. coli cells. A. pernix cellsproduced a green-orange picture when viewed with theFS09 filter (green and red channel). When the cells wereviewed with the FS15 filter (red channel), dead cells wereeasier to see as the green colour of living cells was sup-pressed (Fig. 5). The most suitable probe for the detectionof cell viability of A. pernix cells is the commerciallyavailable Live/Dead BacLightTM kit, although the differ-entiation between the living and dead cells of A. pernix wasnot as good as for E. coli.

Conclusions

Protocols to study microbial cell structures have beendesigned mainly for mesophilic organisms. Therefore,these methods have to be adapted in order to be applicableto organisms such as Aeropyrum pernix that has optimumgrowth above 80�C.

Here we reported the effect of four different dyes on thethermal stability of archaeon A. pernix DNA. Based on thedata obtained in this study, we can conclude that at thesame molar ratios of DNAbp to dye, Hoechst 33258 has thehighest thermal stabilization effect on DNA, while DAPI

Fig. 4 Micrographs of A. pernix cells in the presence of the dyes. (a)Hoechst 33258; (b) DAPI; (c) acridine orange; and (d) DiBAC4(3), at25�C. (a, b, d) panel I: DIC micrographs; panel II: correspondingfluorescence micrographs of intact A. pernix cells with the selecteddye in 10 mM HEPES, 516 mM NaCl, pH 7.0; panel III: DICmicrograph of cells previously inactivated with 10 min at 130�C; andpanel IV: fluorescence micrographs of the same cells with thecorresponding dye. Arrowheads 1, living cells; arrowheads 2, dead

cells. (c) Panel I: DIC micrograph; panel II: DNA and RNAfluorescence micrographs (2 micrographs merged); panel III: fluores-cence micrograph of DNA; and panel IV: fluorescence micrograph ofRNA. The FS01 filter was used for Hoechst 33258 and DAPI, thecombination of FS09 and FS15 for acridine orange (DNA appearsgreen or yellow, while RNA is red), and FS09 for DiBAC4(3). Forfurther details, see Materials and methods

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has the lowest, in vivo and in vitro. Furthermore, DAPI isnot a suitable dye for DSC studies, as it requires a higherpre-heating temperature than 110�C for visualization of theDNA peak in DSC thermograms. On the other hand,Hoechst 33258 thermally stabilizes the DNA inside thecells, does not affect the thermal transitions of other cel-lular components (Table 1), and requires a lower pre-heating temperature (100�C). Based on these characteris-tics, Hoechst 33258 would be a good DNA marker in vivofor DSC studies. However, DAPI, Hoechst 33258 andacridine orange are not suitable probes for fluorescencemicroscopy, as they cannot clearly distuinguish betweenliving and dead A. pernix cells. The commercially availableLive/Dead BacLightTM kit was the most suitable probe fordetection of cell viability of A. pernix cells using fluores-cence microscopy. DiBAC4(3) cannot be used for DSCmeasurements and we have not confirmed that it canclearly distuinguish between viable and non-viable cells ofA. pernix, although it has been reported to be suitable forrapid discrimination of viable and non-viable hypertherm-ophiles (Beck and Huber 1997). The reasons for differentbehaviours might lie in the lipids of A. pernix, which aredifferent from those of anaerobic sulphur-dependent hy-perthermophyles in terms of the lack of tetraether lipids

and the direct linkages of the inositol and sugar moieties(Morii et al. 1999). Furthermore, A. pernix is composed ofC25,25-arheol-based lipids. The thickness of the A. pernixmembrane is assumed to be 20% greater than the mem-brane of the C20,20-arheol-based lipids of other achaea(Tindal 1985; DeRosa et al. 1983).

Acknowledgments Our thanks go to the Slovenian ResearchAgency (J1-6487-0481-04) and to a NATO Collaborative LinkageGrant (LST.GLC: 978884), for financial support.

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