VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

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Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) e) Natural fluorescence - In figure 17 we describe succesfully separatation with ImageJ of cells by natural fluorescence of photosynthetic gasolinetolerant/oxidant microorganisms isolated from mesothermal sulphurous spring in different chanell – red and green- and then each image were automat counted with CellC, obtaining finally the number of cells red and green separately. Fig 17. Natural fluorescence of (A) photosynthetic gasoline-tolerant/oxidant microorganisms isolated from mesothermal sulphurous spring and digital image analysis of chlorophyll autofluorescence in (B) green channel ; (C) red channel and (D-E) total count analysis of red/green channels using the CellC software. In figure 18 there are presented images showing the natural fluorescence of chlorophyll, as an image of marine oxygenic gasoline tolerant/ oxidant phototrophic microorganisms. Difference is clearly apparent width of filaments of cyanobacteria developed in the experimental microcosms. The measurements were performed with Image J program as shown previous (see point 2). a b Fig 18. Autofluorescence of chlorophyll from oxygenic photosynthetic microorganisms: microcosms 1 (a) and 2 (b). Epifluorescence techniques and image analysis has increasingly been used to determine cell size, bacterial abundance and detection of physiological characteristics like damaged versus intact cell membranes. 135 Throughout the investigations conducted continuously attempted to determine the nature of connections between communities of microorganisms and how and to which condition each. 4. Conclusions The utilization of epifluorescence microscopy and digital image analysis enable us to study some morphological and functional aspects of prokaryotes: 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), capsulated cell (labelled with aniline blue) and chlorophyll containing cells, both in enriched cultures and in natural / microcosms samples. The total number of heterotrophic cells counted using AO or DAPI is practically the same whereas total counts obtained with SYBR Green 1 are 47,6% higher. The number of dead cells (PI positive) and that of (putative) capsulated cells are 12.3% and 10%, respectively of the total number ( AO and DAPI). The image analysis systems presented here was performed for counting and estimating the length of bacteria/cyanobacteria with uniform morphology. The presented methods does not totally exclude the need for manual microscope analyses of water samples, and automated procedures must intermittently be validated by independent manual procedures. Acknowledgment We are grateful to Dr. Tech. Jyrki Selinummi (Department of Signal Processing, Tampere University of Technology, Finland) for very useful and kind advices concerning the use of software CellC and ImageJ. 5. References [1] VAN WAMBEKE F., 1995- Numeration et taille des bacteries planctoniques au moyen de lànalise dìmages couplee a lèpifluorescence. Oceanis, 21: 113-124.
Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) [2] MANINI E. & DANOVARO R., 2006- Synoptic determination of living/dead and active/dormant bacterial fraction in marine sediments. FEMS Microbiol. Ecol, 55: 416-423. [3] FALCIONI T., PAPA S., GASOL J.M., 2008- Evaluating the flow-cytometric nucleic acid double-staining protocol in realistic situations of planktonic bacterial death. Appl. Environ. Microbiol, 74: 1767-1779. [4] KIRCHMAN D.L., 2008- Microbial Ecology of the Oceans. John Wiley & Sons, NY, 593. [5] ARDELEAN I.I., GHIŢĂ S., SARCHIZIAN I., 2009-Epifluorescent method for quantification of planktonic marine prokaryotes, Proceedings of the 2 nd International Symposium “New Research in Biotechnology” serie F: 288-296. [6] ISHII T., ADACHI R., OMORI M., SHIMIZU U., IRIE H., 1987 - The identification, counting, and measurement of phytoplankton by an image-processing system. J. Cons. Ciem., 43:253-260. [7] ESTEP K. W. & MACINTYRE F., 1989 - Counting, sizing, and identification of algae using image analysis. Sarsia, 74: 261-268. [8] EMBLETON K. V., GIBSON C. E., HEANEY S. I., 2003 - Automated counting of phytoplankton by pattern recognition: a comparison with a manual counting method. J. Plankt. Res., 25: 669-681. [9] WALSBY A. E. & AVERY A., 1996 - Measurement of filamentous cyanobacteria by image analysis. J. Microbiol. Meth., 26:11-20. [10] CONGESTRI R., CAPUCCI E., ALBERTANO P., 2003 – Morphometric variability of the genus Nodularia (Cyanobacteria) in Baltic natural communities. Aquat. Microb. Ecol. 32: 251-259. [11] SELINUMMI J., SEPPÄLÄ J., YLI-HARJA O., PUHAKKA J.A., 2005 - Software for quantification of labeled bacteria from digital microscope images by automated image analysis”. BioTechniques, 39: 859–863. [12] SELINUMMI J., 2008- On Algorithms for Two and Three Dimensional High Throughput Light Microscopy, Ph.D Thesis for the degree of Doctor of Technology. [13] RIPPKA R., DERUELLES J., WATERBURY J.B,. HERDMAN M., STANIER R.Y., 1979 - 136 Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of Geneneral Microbiology., 111: 1 - 61. [14] ITURBE R., FLORES R.M., TORRES L.G., 2003- Subsoil polluted by hydrocarbons in an out-of-service oil distribution and storage station in Zacatecas, Mexico. Environ Geol, 44: 608–620. [15] MOLINA-BARAHONA L., RODRI´GUEZ- VA´ZQUEZ R., HERNAŃDEZ- VELA´SCO, C. VEGA-JARQUIN, O. ZAPATA-PE´REZ M., MENDOZA- CANTU´ A., ALBORES A., 2004- Diesel removal from contaminated soils by biostimulation and supplementation with crop residues, Appl Soil Ecol, 27: 165–175. [16] LUNA G.M., MANINI E., DANOVARO R., 2002- High fraction of dead and inactive bacteria in coastal marine sediments: comparison of protocols and ecological significance. Appl. Environ. Microbiol, 68: 3509-3513. [17] LUNAU M., LEMKE A., WALTHER K., MARTENS-HABBENA W., SIMON M., 2005- An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy. Appl. Environ. Microbiol, 7: 961-968. [18] HONG Z., DELAUNEY A.J., VERMA D.P.S., 2001-A cell plate-specific callose synthase and its interaction with phragmoplastin.Plant Cell, 13:755-768 [19] NAKANISHI I., KAZUTSUGU K., SUZUKI T., ISHIKAWA M., BANNO I., SAKANE T., HARADA T.,1976- Demonstration of curdlantype polysaccharide and some other β-1, 3- glucan in microorganisms with aniline blue. J. Gen. Appl. Microbiol., 22: 1-11. [20] MC INTOSH M., STONE B. A.,. STANISICH V. A,, 2005-Curdlan and other bacterial (1-3)-β- D-glucans. Appl Microbiol Biotechnol, 68: 163- 173. [21] HEISSENBERGER A., LEPPARD G.G., HERNDL G.J., 1996 - Relationship between the intracellular integrity and the morphology of the capsular envelope in attached and free-living marine bacteria. Appl. Environ. Microbiol., 62: 4521-4528.
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VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

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