VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

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Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 UTILIZATION OF EPIFLUORESCENCE MICROSCOPY AND DIGITAL IMAGE ANALYSIS TO STUDY SOME MORPHOLOGICAL AND FUNCTIONAL ASPECTS OF PROKARYOTES Simona GHIŢĂ ** , Iris SARCHIZIAN * , Ioan ARDELEAN *** * Ovidius University of Constanţa, Natural Sciences Faculty, Department of Biology, Mamaia Avenue, No. 124, Constanţa, 900552, Romania, e-mail: ghitasimona@aim.com, irissarchizian@yahoo.com ** Constanta Maritime University, Department of Environmental Engineering, Mircea cel Batrin, No. 104, Constanta, 900663, Romania, e-mail:ghitasimona@aim.com; *** Institute of Biology, Splaiul Independenţei, No. 296, Bucharest, 060031, Romania, email:ioan.ardelean57@yahoo.com __________________________________________________________________________________________ Abstract: The aims of this study is to argue, based on original results, the importance of utilization of epifluorescence microscopy to study some morphological and functional aspects of prokaryotes allowing to perform total cell counts , direct viable count count, count of permeabilised cells, chlorophyll containing cells or putatively capsulated cells. Automated image analysis of the results thus obtained was done using CellC and ImageJ software which allow the quantification of bacterial cells from digital microscope images, automated enumeration of bacterial cells, comparison of total count and specific count images, providing also quantitative estimates of cell morphology. Keywords: epifluorescence, digital image analysis, heterotrophic bacteria, cyanobacteria. __________________________________________________________________________________________ 1. Introduction The use of epifluorescence microscopy to study different aspects of prokaryotes at population and single cell level significantly improved the knowledge concerning which species are present in a given sample, the cell density and the metabolic statues of the population as a whole or of each single prokaryote cell (Van Wambeke, 1995; Manini & Danovaro, 2006; Falcioni et al., 2008; Kirchman, 2008; Ardelean et al., 2009). In the last decades there is also an increase in the development and use of different softwares for automated analysis of the digital images thus obtained (Ishii et al. 1987; Estep & Macintyre 1989; Embleton et al., 2003; Walsby, 1996; Congestri et al. 2003; Selinummi et al., 2005, 2008). The aims of this study is to argue, based on original results, the importance of utilization of epifluorescence microscopy coupled with automated image analysis to study some morphological and functional aspects of prokaryotes allowing to perform total cell counts (acridine orange, DAPI, SYBR Green 1), direct viable count (elongated cell in the presence of nalidixic acid, labelled with acridine orange), count of permeabilised cells (cells permeable to propidium iodide), putatively capsulated cell (labelled with aniline blue) and chlorophyll containing cells both in enriched cultures and in natural (microcosms) samples. 2. Material and Methods A. Study area and sampling. Samples were collected in sterile bottles in October 2008 and May 2009 from sulphurous mesothermal spring (Obanul Mare) placed in north of Mangalia City (43˚49’53.6’’N; 28˚34’05.3’’E). The samples were divided in sub-samples, one being immediately fixed with buffered formaldehyde (2% final concentration) and the second one used to isolate cyanobacteria by inoculation into conical flasks with either BG11 ISSN-1453-1267 © 2010 Ovidius University Press
Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) medium or nitrate - free BG11 medium (BG0) (Rippka et al., 1979). Another series of natural samples were collected from Black Sea (Tomis seaport at 0.5m depth; 44 o 10 ’ 44 ’’ N; 28 o 39 ’ 32 ’’ E) in March 2009. B. Culture conditions. Natural samples inoculated in either BG11 or BG0 media, either solid of liquid, were incubated in culture room at 25 ± 1ºC and illuminated with fluorescent tubes having the photon rate of 50 μmol m –2 s –1 at surface of the culture vessels. C. Microcosms. Taking into account the advantages of microcosms (Iturbe et al., 2003; Molina-Barahona et al., 2004) we used this opportunity as previously (Ardelean et al., 2009). D. Total cell count (AO; DAPI, SYBR Green I) Total bacterial count were performed using acridine orange, DAPI and SYBR Green I (Luna et al., 2002; Lunau et al., 2005; Manini & Danovaro, 2006). For AO and DAPI (5 μg/mL dye final concentrations) subsamples were stained for 5 minutes and were filtred on black Millipore 0,22µm pore size filters. Unlike AO, using DAPI for bacterial visualization and enumeration has the advantages of low background fluorescence and that DAPI stains only DNA. For SG (1µL/10µL sample final concentrations) subsamples were stained for 10 minutes and were filtred on black Millipore 0,22 µm pore size filters. Color filters were washed with 10 ml of 17 ‰ saline solution. SG as a permeant DNA-binding stain and determine the total fraction of cells from natural samples. E. Permeabilized (dead) cells (PI+) PI is a double-charged phenanthridium derivative and is one of the most common stains for dead cells (Luna et al., 2002). PI is thus assumed to be unable to penetrate cell membranes. In our natural samples we used a PI concentration of 5 μL/ml sample. Also stained samples were filtered through black Millipore 0,22 µm pore size filters and then inspected under a epifluorescence microscope. The disruption of planktonic cell aggregates for cell enumeration were done as previously shown (Ardelean et al., 2009). 128 F. Enumeration of (putatively) capsulated cells (AB+). Cell capsule was also inspected using aniline blue (AB) which is a fluorescent dye specific which seems to be specific for 1,3 beta glucans (Hong et al., 2001) found in plants and as capsular material in many microorganisms (Nakanishi et al., 1976; McIntosh et al., 2005). Capsular envelopes are widely distributed in marine free-living and particleassociated bacteria (Heissenberger et al., 1996) and are a signature of active bacteria (Stoderegger & Herndl, 2002). Bacteria with an intact intracellular structure, and therefore potentially active bacteria, are surrounded by a capsular layer, while the vast majority of bacteria with a damaged structure lack such a capsule (Heissenberger et al., 1996). Laboratory experiments indicated that active bacteria are constantly renewing their capsular envelope and releasing a significant fraction of the polysaccharide layer into the ambient water (Stoderegger & Herndl, 2002). The samples were treated with AB (5 µg/mL final concentration) for 5 minutes and then filtered and counted as shown above for AO staining . G. The automatic cell analysis were done with two software ImageJ and CellC, who was applied to digital images of whole cells color-stained bacteria and cyanobacteria. The analysis proceeds few important steps: the background is separated from the objects based on the intra-class variance threshold method; noise and specks of staining color in the image can affect the reliability of the analysis, so those was removed. The removal was done applying mathematical morphology operations to the image; then separation of clustered objects was performed (Selinummi, 2008). The length of cells was determined with ImageJ software using a calibration scale. H. Cyanobacteria (natural fluorescence) Visualization of hydrocarbon tolerant phototrophic microorganisms, also for unicellular or filamentous cyanobacteria from sulphurous mesothermal spring; chlorophyll a in natural environments (either marine or spring) was done using an epifluorescence microscope (N-400FL, lamp Hg 100W, type on the blue filter; Sherr et al., 2001) as previously shown (Ardelean et al., 2009). I. Direct viable count (cells capable of division) is based on the Kogure method developped
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VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

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