Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 200467678685Original ArticleDesign of new probes for Geodermatophilaceae membersC. Urzì, V. La Cono and E. Stackebrandt
Environmental Microbiology (2004) 6(7), 678–685
doi:10.1111/j.1462-2920.2004.00619.x
Design and application of two oligonucleotide probes for the identification of Geodermatophilaceae strains using fluorescence in situ hybridization (FISH) Clara Urzì,1* Violetta La Cono1 and Erko Stackebrandt2 Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Salita Sperone, 31, I-98166 Messina, Italy. 2 DSMZ – German Collection of Microorganisms and Cell Cultures GmbH, Manscheroder Weg 1b, D-38124, Braunschweig, Germany. 1
Summary Bacteria of the family of Geodermatophilaceae are actively involved in the decay processes [Urzì, C. and Realini, M. (1998) Int Biodeterior Biodegrad 42: 45–54; Urzì, C., Salamone, P., Schumann, P., and Stackebrandt, E. (2000) Int J Syst Evol Microbiol 50: 529–536] of stone monuments. Characterization of isolates includes phenotypic, chemotaxonomic and genetic analysis often requiring long-term procedures. The use of specific probes for members of Geodermatophilaceae family could be useful for the easy detection of those strains colonizing rock surfaces and involved in the biodeterioration. Two 16S rRNAtargeted oligonucleotide probes were designed for the specific detection of members of the family Geodermatophilaceae using fluorescence in situ hybridization (FISH); one probe specific for members of the two genera Geodermatophilus/Blastococcus and the second for members of the genus Modestobacter. Introduction It has become evident in recent years that a great variety of actinomycetes must be considered major colonizers of stone surfaces exposed to outdoor and indoor environments (Urzì et al., 2000; 2003). Among these, up to 90– 100% of strains isolated from various rock samples (Urzì et al., 2001) belong to three genera of the family Geodermatophilaceae (Stackebrandt et al., 1997), i.e. Geodermatophilus (Luedemann, 1968), Blastococcus (Ahrens and Moll, 1970; Urzì et al., 2004) and Modestobacter
Received 7 November, 2003; accepted 4 February, 2004. *For correspondence. E-mail
[email protected]; Tel. (+39) 090 6765196; Fax: (+39) 090 392733.
© 2004 Blackwell Publishing Ltd
(Mevs et al., 2000). These bacteria were also often isolated from decaying building stones and it has been suggested that they are actively involved in the decay processes (Urzì and Realini, 1998; Urzì et al., 2000). All these surfaces were in fact characterized by a high level of alteration, including the formation of grey and/or orange diffused patinas and, moreover, by phenomena of alveolization, detachment, biopitting, crumbling and powdering (Urzì et al., 2001). The characterization of isolates resulting in their generic affiliation include time- and cost consuming phenotypic, chemotaxonomic and genomic analysis (Urzì et al., 2001; 2004) and, if restricted to mainly morphological characterization, were not discriminative. Fluorescence in situ hybridization (FISH), circumventing a lengthy identification process, is a rapid and highly valuable tool for the cultivation-independent identification of individual microbial cells from environmental samples using rRNA-targeted oligonucleotide probes (Amann et al., 1990). Since then, the scientific literature witnesses the description and application of a broad battery of taxonspecific oligonucleotide probes (see Amann et al., 1995; 2001) for rapid determination of organisms which are difficult to either differentiate by traditional criteria or to obtain pure cultures. Design of new probes for specific taxa of microorganisms thus allows the application of a top-to-bottom approach for the characterization of the microbial community structure. In recent years, FISH has also been applied in the field of works of arts to study Eubacteria and Archea involved in the biodeterioration of surfaces (Piñar et al., 2001; Urzì and Albertano, 2001; Urzì et al., 2003). However, as sequences of only a few members of the family of Geodermatophilaceae were determined, no specific probes had been designed. In this study we apply two probes to pure cultures isolated recently (Urzì et al., 2000; 2003) one specific for members of the two genera Geodermatophilus and Blastococcus, the second for members of the genus Modestobacter, in order to obtain a rapid tool for the identification that targets all type strains and genomically related isolates. As a result of the fact that most of these microorganisms live on rocks, especially in dry climates, they tend to cluster in more or less thick aggregates with the consequent reduction of cell permeability and the accessibility
Design of new probes for Geodermatophilaceae members 679 tion the target strains fluoresced strongly, whereas the reference strains and strains moderately related to the target organisms showed no fluorescence; above 37∞C the fluorescence intensity decreased significantly. Whole cell hybridizations performed with the bacterial strains (Table 1) demonstrated the specificities of the probe. The probe hybridized only with the Modestobacter strains, showing that the rRNA was accessible to hybridization (Fig. 2A–D). No false positive signals were observed at these hybridization conditions.
of the targeted rRNA (Urzì et al., 2000; La Cono and Urzì, 2003). The protocol describes treatment of aggregate to obtain a more uniform staining by FISH probes. Results Aggregates treatment Geodermatophilus sp. strain BC503 (DSM 44511), and Modestobacter sp. strains BC498 (DSM44446) and BC506 formed cell aggregates with thick cell walls after 15 days of incubation (Fig. 1A and C), while Blastococcus aggregatus DSM4725 and B. saxobsidens BC444 (DSM44509) did not form aggregates. As shown with strains BC498 and BC503 pretreatment with a pestle disintegrated large aggregates and could improve the sample observation (Fig. 1B and D) when confocal microscope is not available.
Probe S-*-GeoBlasto-0211-a-A-19 For the ‘GeoBlasto’ probe the stringency was determined for hybridization at a concentration of 45% of formamide. All hybridizations were tested in a temperature range between 37 and 46∞C. As no differences were noticed in the signal strength within this temperature range, a hybridization temperature of 37∞C was chosen which was also used for the ‘Modesto’ probe. The ‘GeoBlasto’ probe did not discriminate between strains of the genera Geodermatophilus and Blastococcus. In fact, all attempts failed to develop a more discrim-
Probe S-*-Modesto-096-a-A-15 As judged by the optimal signal strength of the hybridized ‘Modesto’ probe hybridization was performed at 37∞C and at a concentration of 20% formamide. At this concentra-
A
B
C
D
Fig. 1. Effect of pestle treatment: destruction of aggregates formed by strains BC498 and BC503. Strain BC498: 15 days before treatment (A), after pestle treatment (B). Strain BC503: 15 days before treatment (C), after pestle treatment (D). Bar is 10 mm. © 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 678–685
680 C. Urzì, V. La Cono and E. Stackebrandt Table 1. Application of FISH probes. Strains
EUB 338
HGC69a
STREPTO
Geo/Blasto
Modesto
Genera
ATCC 6051 ATCC 25922 DSM 40134 DSM 12652T DSM 43161 DSM 43162 DSM 43168T DSM 44406T DSM 46136 DSM 4725T BC 47 BC 412 BC 444T BC 445 BC 448 BC 452 BC 455 BC 460 BC 470 BC 496 BC 498 BC 499 BC 500 BC 501 BC 502 BC 503 BC 506 BC 507 BC 508 BC 509 BC 510 BC 511 BC 512 BC 513 BC 515 BC 516 BC 517 BC 518 BC 519 BC 520 BC 521 BC 524 BC 525 BC 526 BC 532 BC 541 BC 543 BC 544 BC 545 BC 546 BC 562 BC 604 BC 606 BC 607 BC 617 BC 620 BC 626
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
– – + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
– – + – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – + – – – – –
– – – – + + – – + + – + + + + + + – – – – – – – + + – – + + + + + – + + + + + – + + + + + – + – – + – – – – – + +
– – – – – – – + – – – – – – – – – + – + + + + + – – + + – – – – – + – – – – – + – – – – – – – – + – – – + + + – –
Bacillus subtilis E. coli Streptomyces xanthophaeus Marmoricola aurantiacus G. obscurus subsp. dictyosporus G. obscurus subsp.uthaensis Micromonospora carbonacea Modestobacter multiseptatus Geodermatophilus sp. Blastococcus aggregatus Micrococcus luteus Blastococcus sp.a Blastococcus saxobsidens b Blastococcus sp.c Blastococcus saxobsidens b Blastococcus sp.c Blastococcus sp.c Modestobacter sp.a Micromonospora Modestobacter sp.a Modestobacter sp.a Modestobacter sp.a Modestobacter sp.a Modestobacter sp.a Blastococcus sp.a Geodermatophilus sp.a Modestobacter sp.a Modestobacter sp.a Blastococcus sp. Blastococcus saxobsidens b Blastococcus sp.c Blastococcus sp.c Blastococcus saxobsidens b Modestobacter sp.a Blastococcus sp.c Blastococcus sp.c Blastococcus saxobsidens b Blastococcus saxobsidens b Blastococcus saxobsidens b Modestobacter sp.c Blastococcus sp.a Blastococcus sp.c Blastococcus saxobsidens b Blastococcus sp.a Blastococcus sp.c n.i. Geodermatophilus sp. n.i. Modestobacter sp.a Geodermatophilus sp. Micromonospora sp. Streptomyces sp. Modestobacter sp.c Modestobacter sp.c Modestobacter sp.a Geodermatophilus sp.a Blastococcus sp.a
a. Urzì et al. (2001). b. Urzì et al. (2004). c. ARDRA analysis (data not yet published).
inatory 16S rRNA targeting probe as members of the genus Blastococcus branches intermediate the validly described subspecies of Geodermatophilus obscurus (Urzì et al., 2004). Of the 47 strains isolated from stone
surfaces 30 strains, classified as members of either Geodermatophilus or Blastococcus, were detected by the ‘GeoBlasto’ probe. None of Modestobacter strains reacted positively with this probe. © 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 678–685
Design of new probes for Geodermatophilaceae members 681
A
B
C
D
Fig. 2. Specificities of the probes S-*-Modesto-096-a-A-15 and S-*-GeoBlasto-0211-a-A-19 in mixed suspensions. A and B. Mixed bacterial suspension of Blastococcus saxobsidens BC444 and Modestobacter strain BC513 stained with DAPI (A) and hybridized with combined use of ‘GeoBlasto’ probe labelled with FITC (green) and ‘Modesto’ labelled with CY3 (red) (B). C and D. Mixed bacterial suspension of Geodermatophilus strain BC503, Modestobacter strain BC498 and E. coli ATCC 25922. DAPI stain (C), mixed probe hybridization with ‘GeoBlasto probe’ labelled with FITC (green), ‘Modesto probe’ labelled with CY3 (red) and E. coli stained with DAPI (blue) (D). Bar is 10 mm.
The combined use of the newly designed probes plus DAPI allowed distinguishing Geodermatophilus from Modestobacter in the mixed population cells (Fig. 2A and B) which also include E. coli (Fig. 2C and D). The 57 strains tested reacted accordingly to the specificity of the probes used. Discussion The Geo/Blasto probe target differs between members of Geodermatophilus and Blastococcus by four nucleotides at the 5¢-terminus of the rRNA gene. Nevertheless, under the conditions used (37∞C, 45% formamide) the probes was highly selective to target members of these two genera but not those of Modestobacter. No other target site was detected within the16S rDNA genes; it can not be excluded that the probe discriminates by forming stable duplices to alternative sites, e.g. at the © 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 678–685
23S rRNA gene. In fact, the newly designed probes were tested with five recently isolated strains, which had hitherto not been subjected to phylogenetic analyses. Based on morphology, they were tentatively assigned to the family Geodermatophilaceae. While two of them did not react positively against both probes, three strains (BC508, BC543, and BC546) reacted positively with the ‘Geo/ Blasto’ probe. Because of the fact that members of Geodermatophilus and Blastococcus can be easily differentiated from each other on the basis of the peculiar colony colour (see Table 2) two strains (BC543, BC546) could be attributed to the genus Geodermatophilus and strain BC508 to the genus Blastococcus. The development of a black pigment in aged colonies of most members of Geodermatophilus and Modestobacter did not interfere with the fluorescent signals of positively reacting probes. The results presented here expand the range of techniques to study of a group of bacteria often found in desert
682 C. Urzì, V. La Cono and E. Stackebrandt Table 2. Differential characteristics among Modestobacter, Geodermatophilus and Blastococcus strains.
Characteristics Cell shape Aggregate cell Cell diameter Bud formation Motility Flagellar insertion Colony colour Oxygen requirement NaCl tolerance 3% 5% 6% Range of T (C∞) Range pH Major menaquinone Hydrolysis of gelatin Casein Carbon sources: Ethanol Glucose NO3 reduction to NO2
Blastococcus aggregatus DSM4725T
Blastococcus strains
G. obscurus DSM 43160T
Geodermatophilus strains
Modestobacter multiseptatus DSM44406T
Modestobacter strains
C, R vibrios + 0.3–1.5 + + Subpolar P Aerobic/Micr
C/R + 0.7–1.7 –/+ –/+ n.d. P-P/O Aerobic
Cocci to cuboids + 0.5–2.0 –/+ –/+ Polar Br/B Aerobic
C-C/R + 1.4–1.7 + n.d. n.d. P/B Aerobic
C, R – 1.0–1.7 + + n.d. Pale pink Aerobic
C-C/R + 0.5–1.7 + 0. n.d. P-P/B Aerobic
+ + – 3–40 n.d. MK-9(H4) – n.d.
–/+ –/+ – 15–37 5–10.5 MK-9,9(H4) –/+ –/+
– – – 18–37 6–9.5 MK-9(H4) +– –
– – – 26–37 5–10.5 MK-9(H4) – –
+ + + 0–28 4.0–9.5 MK-9(H4) – –
–/+ –/+ – 10–37 5.0–10 MK-9(H4) – –/+
w – w
n.d. + –
– + w
n.d. + –
– + –
n.d. + –/+
Legend: B = Black; Br = Brown; P = Pink; O = Orange; Micr = Microaerophilic; C = Cocci; R = Rods.
soil and rock as well as colonizers of decayed stone monuments which, however, are rarely studied in detail because of cultivation difficulties. On the other hand, these organisms are worthwhile studying as they could be used as models for studying life in extreme environments, including in the search of life on other planets. The use of the two newly designed probes in combination is an efficient tool for the rapid genetic affiliation of strains with macro- and micromorphological properties resembling members of Geodermatophilaceae. The study of Gurtner et al. (2000) indicates that the habitat of strains of Geodermatophilaceae is broader than described hitherto as they were also found in humid surfaces and in caves. The application of FISH probes will allow an unambiguous dissection of morphologically similar strains, contributing to unraveling the size and dynamics in natural population. As demonstrated recently (La Cono and Urzì, 2003), in addition to the classical application for visualizing FISH probes (membranes or wells) these probes can be successful applied directly on samples collected with adhesive tapes providing a mirror image of the sampled area and thus giving reliable and direct information of the distribution, the relationships and occurrence of members of Geodermatophilaceae in a complex microbial biofilm. Experimental procedures Bacterial strains Forty-seven strains, isolated from stone monuments, were
obtained from the culture collection of the Department of Microbiological, Genetic and Molecular Sciences of the University of Messina. Ten reference strains were obtained from the American Type Culture Collection (ATCC) and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Table 3). Bacillus subtilis (ATCC 6051) and E. coli (ATCC 25922) were grown in TSA (Tryptic Soy Agar) medium (BBL, USA) for 2 days at 37∞C. The other strains were grown in agarized Luedemann medium (Luedemann, 1968) for 5 days at 28∞C. Further some representative strains (BC444, BC498, BC503, BC506, DSM4725) were incubated for 15 days at 28∞C to eventually allow cell aggregations.
Design and labelling of probes Two new probes complementary to the 16S rRNA of the genera Geodermatophilus/Blastococcus and Modestobacter, respectively, were designed after sequence alignment of the 16S rRNA gene sequences of 55 strains to those of members of the class Actinobacteria, obtained from NCBI/Nucleotide (http://www.ncbi.nlm.nih.gov/) databases. The sequences were aligned by using the ae2 software (Maidak et al., 2001) and probes targeting these sequences determined by eye were designed (Table 4). By the use of the standardized oligonucleotide nomenclature defined by Alm et al. (1996), the probes names are S-*-GeoBlasto-0211-a-A-19 and S-*-Modesto-096-a-A-15. Table 4 also lists additional probes used in this study (for Eubacteria, Gram-positive bacteria with high content G + C and Streptomyces). Visual comparison of aligned 16S rRNA reference sequences initially revealed another potential probe (position 210–229) for Modestobacter genus to be applied in the in situ identification of Geodermatophilaceae © 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 678–685
Design of new probes for Geodermatophilaceae members 683 Table 3. Strains considered in this study. Strain
Genus
Strain
Genus
ATCC 6051 ATCC 25922 DSM 40134 DSM 12652T DSM 43161 DSM 43162 DSM 43168T DSM 44406T DSM 46136 DSM 4725T BC 47 DSM 44517, BC 412 DSM 44509T, BC 444 BC 445 BC 448 BC 452 BC 455 DSM 44446, BC 460 BC 470 DSM 44447, BC 496 DSM 44446, BC 498 DSM 44510, BC 499 BC 500 DSM 44449, BC 501 BC 502 DSM 44511, BC 503 BC 506 BC 507 BC 508
Bacillus subtilis E. coli Streptomyces xanthophaeus Marmoricola aurantiacus G. obscurus subsp. dictyosporus G. obscurus subsp.uthaensis Micromonospora carb. Modestobacter multiseptatus Geodermatophilus sp. Blastococcus aggregatus Micrococcus luteus Blastococcus sp.a Blastococcus saxobsidens b Blastococcus sp.c Blastococcus saxobsidens b Blastococcus sp.c Blastococcus sp.c Modestobacter sp.a Micromonospora sp. Modestobacter sp.a Modestobacter sp.a Modestobacter sp.a Modestobacter sp.a Modestobacter sp.a Blastococcus sp.a Geodermatophilus sp.a Modestobacter sp.a Modestobacter sp.a Blastococcus sp.¢
BC 509 BC 510 BC 511 BC 512 BC 513 BC 515 BC 516 BC 517 BC 518 BC 519 BC 520 DSM 44518, BC 521 BC 524 BC 525 BC 526 BC 532 BC 541 BC 543 BC 544 BC 545 BC 546 BC 562 BC 604 BC 606 BC 607 BC 617 BC 620 BC 626
Blastococcus saxobsidensb Blastococcus sp.c Blastococcus sp.c Blastococcus saxobsidens b Modestobacter sp.a Blastococcus sp.c Blastococcus sp.c Blastococcus saxobsidens b Blastococcus saxobsidens b Blastococcus saxobsidens b Modestobacter sp.c Blastococcus sp.a Blastococcus sp.c Blastococcus saxobsidens b Blastococcus sp.a Blastococcus sp.c n.i. Geodermatophilus sp.¢ n.i. Modestobacter sp.a Geodermatophilus sp.¢ Micromonospora sp. Streptomyces sp. Modestobacter sp.c Modestobacter sp.c Modestobacter sp.a Geodermatophilus sp.a Blastococcus sp.a
a. Urzì et al. (2001). b. Urzì et al. (2004). c. ARDRA analysis (data not yet published).
strains. However, this probe was rejected because of lack of specificity. Probe binding profiles were performed using hybridization buffers containing 0% to 60% formamide at 5% increments on fixed type strains of species (Manz et al., 1992; Liu and Seviour, 2001). The criteria of Weller et al. (2000) were used to determine the formamide concentrations for stringency of all the probes described in this study (Liu et al., 2001). Optimal hybridization conditions were established by monitoring the hybridization of tested bacteria (both target and nontarget bacteria). Recommended stringency was reached
when the target bacteria were detected with a bright signal and data were compared to the empiric stringency obtained by the Ribosomal Database Project II online analysis (Cole et al., 2003). Oligonucleotides were labeled with different fluorochromes such as FITC (Fluoresceine isothiocyanate), Cy3 (Indocarbocianine) or TRITC (Tetramethylrhodamine isothiocyanate) (Biosense S.r.l., Italy).
Pretreatment and fixation of cells Cells were harvested and washed three times in PBS
Table 4. Oligonucleotide probes used in this study.
Target group Bacteria Negative control complementary to EUB338 Gram-positive bacteria with high G + C content Geodermatophilus/Blastococcus group Modestobacter genus Streptomyces genus
Probe sequence (5¢ to 3¢)
Target sitea (rRNA position)
% FAb
Probe acronym
Reference
GCTGCCTCCCGTAGGAGT ACTCCTACGGGAGGCAGC
16S (338) 16S (338)
35 35
EUB338 NOT EUB338
Amann et al. (1995) Amann et al. (1995)
TATAGTTACCACCGCCGT
23S (1901)
35
Roller et al. (1992)
CCATCCCCAGCCGGAAACC
16S (211)
45
GGC69a HGC69A (HGC) Geo/Blasto
TTCGCCGCTAGGGCA GCGTCGAATTAAGCCACA
16S (96) 16S (924)c
20 50
Modesto Strepto
In this study Stackebrandt et al. (1991
a. E. coli numbering (Brosius et al., 1981). b. Percentage of formamide (FA) in hybridization buffer. c. Streptomyces ambofaciens position. © 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 678–685
In this study
684 C. Urzì, V. La Cono and E. Stackebrandt (130 mM NaCl, 10 mM sodium phosphate, pH 7.2). Three volumes of fresh fixation buffer (4% paraformaldehyde in PBS, pH 7.2) were added to one volume of sample and incubated for 3 h at 4∞C. Following fixation cells were centrifuged and washed in PBS, harvested by centrifugation, resuspended in 1:1 (v/v) PBS/ethanol at concentration of 109 cell ml-1 (OD 1 at 550 nm) and stored at -20∞C. Strains incubated for 15 days (BC444, BC498, BC503, BC506 and DSM4725), were pretreated before fixation with Pellet pestle (Sigma) for 10 s, in order to disrupt the aggregate structure.
Whole-cell hybridization Pure cultures. The hybridization was performed in 12well Teflon-coated slides (Electron Microscope Science, USA). The slides were cleaned by soaking in ethanolic KOH (10% KOH in ethanol) and coated with gelatin (0.1 gelatin, 0.01% CrK(SO4)2·12 H2O). Each strain was tested with all probes listed in Table 3. Three microlitres of each fixed sample was applied to the wells. The slide were air-dried and dehydrated by successive passages through 50%, 80% and 95% ethanol washes (3 min each). After dehydration the cells were treated per 30 min at room temperature with 1 mg ml-1 of lysozyme in Tris-HCl 100 mM + EDTA 5 mM buffer (Hahn et al., 1993) and dehydrated as described above. For hybridization, 9 ml of hybridization solution (0.9 M NaCl, 20 mM Tris-HCl, 0.1% SDS, formamide as given in Table 3) were mixed with 1 ml (50 ng) of each probe. The slides were incubated at 37–46∞C in a moisture chamber for at least 2 h. After hybridization, the slides were washed in wash solution (180 mM NaCl, 20 mM TrisHCl (pH = 7.2), 0.1% SDS, 5 mM EDTA) for 30 min at 38– 48∞C and rinsed with distilled water and air-dried. Mixed suspension A suspension of Geodermatophilus sp. (BC 503), Modestobacter sp. (BC 498) and E. coli (ATCC 25922) was obtained by mixing 1 ml of each suspension. The hybridization procedure used was described above, and samples were hybridized with two probes simultaneously. The probe with the highest concentration of formamide was hybridized first, whereas the probe with less concentrated formamide was hybridized after washing and rinsing the first probe. Fluorescent staining was performed with 1 mg ml-1 of 4¢,6diamidino-2-phenylindole dihydrochloride (DAPI) for 10 min, rinsed with distilled water and air dried. The slides were mounted with SlowFade® Light Antifade kit (Molecular Probe, Eugene, Oregon, USA).
Image capture and digital image processing Slides were observed with a Leica DMR/HCS epifluorescence microscope equipped with a 50 W mercury lamp HBO
and a different combination of filters. Image caption was carried out with a photocamera using a Kodak Gold ultra film 400ASA or with a Leica DC 300F CCD CAMERA and the images were processed using image analysis software (QFluoro Leica Pro, ver. 1.2.0).
Acknowledgements The authors acknowledge the financial support of European Community through contract nos. EVK4-1999–20001 ‘COALITION’ and EVK4-2000–00028 ‘CATS’, of Consiglio Nazionale delle Ricerche (C.N.R.) through contract no. 01.00654.PF36 and P.R.A. 2000/2002.
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