VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

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