New Researches in Biotechnology - Facultatea de Biotehnologii ...

Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011The microcosms are as follows: Black Sea natural sample- control (M3); controlsupplemented with gasoline (1% v/w) (M2), control supplemented with gasoline (1% v/w)and nutrients (ammonium nitrate 0.005% w/w) (M1), control supplemented with gasoline(1% v/w) and selected population (1 mL) (M4) and control supplemented with gasoline(1% v/w), nutrients (ammonium nitrate 0.005% w/w) and selected population – 1 mL (M5).Sampling was done sequentially at 1, 6, 11, 18, 39 and 53 days from the start of theexperiment.Direct viable countsThe method of direct enumeration of viable cells has followed the original protocol(Kogure et al., 1979), technique as adapted by Ghiţă et al., 2010. The seawater samples (40mL) containing nutrients (yeast extract, 50 mg / L final concentration) and antibiotic(nalidixic acid, 20 mg / L final concentration) were kept at a constant temperature (18°C)incubated in light. Subsequently samples were harvested each two hours (considering thetime T o , T 1 –after 2 hours, T 2 - after 4 hours; T 3 - after 6 hours, T 4 - after 8 hours, T 5 - after 10hours, T 6 - after 24 hours). Then samples were stained with acridine orange (5µg/mL) andvisualized by epifluorescence microscopy.Epifluorescence microscopySamples were viewed immediately by epifluorescence microscopy (N-400FL, lamp Hg 100W, type on the blue filter- 450-480 nm) with immersion 100X objective and 10X eyepieces.Transmission electron microscopy using negative stainingTo determine cell size at the beginning (T o ) and at the end of the experiment (T 6 )incubation of samples in the presence of inhibitor and nutrients in the five microcosms, weused the TEM technique by negative staining with 1% acid fosfotungstic (pH 6.8-7.4),measuring 150 cells. Negative staining electron microscopy is used in one of the mostwidely used methods in the study of microorganisms. Contact time between biologicalpreparation and dye (1% acid fosfotungstic) is 15 seconds.Samples of microcosm 4 were processed and analyzed by Jastrow method (Gundersen etal., 1988; Jastrow et al., 1997) to see some adjustments in the ultrastructure of prokaryoticcell elongation from cultured cells in the presence of yeast extract and nalidixic acid.Capsules that include biological preparation are pyramid sectioned and subjected to cuttingprocess ultramicrotom (Leica Ultracut R) to a sectional dimension of 600 Å. Observationgrids was made on microscope CM Phillips 120ST.Nile blue stain for PHBSamples from microcosms were used for making smears which were subsequently driedwith thermal fixing and stained with Nile blue solution 1% at 55 o C for 10 minutes (Ostle &Holt, 1982).The automatic cell analysis was performed using the Image J and CellC software.Fluorescence microscopy digital images were analyzed and the object has differentintensity than the background. The CellC automatic quantification program presents a set offiles where stored processing data are. Generally removes all detected objects that aresmaller than 1 / 10 the average size of all objects (Selinummi, 2008). Also, we used Image Jsoftware to measure cell length and to obtain statistical values with the size in pixels,selected by user.141