VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010)
by incubation of samples with a single antimicrobial
agent (nalidixic acid) and nutrients (yeast extract).
Nalidixic acid acts as a specific inhibitor of DNA
synthesis and prevents cell division without affecting
other cellular metabolic activities, including cell
growth; thus viable cells growth but do not divide,
thus becoming longer/larger than cells unable to grow
(Kogure et al., 1979). Experiments were done with
40mL samples from each microcosms in which the
sample was filtered through 0.45 µm filter (2 and 3)
supplemented with yeast extract (50 mg / L final
concentration), nalidixic acid (20 mg / L final
concentration) (Kogure et al., 1979) and gasoline
(0.5% final concentration); 17 hours before the start
of experiment all samples were kept in an incubator
at a temperature of 30 o C and continuous stirring.
Subsequently samples were incubated under the
conditions previously reported and samples were
harvested each two hours (considering the time To,
T1 –after 2 hours, T2- after 4 hours; T3- after 6 hours,
T4- after 8 hours).
3. Results and Discussions
1. Total count cell (AO+, DAPI+), permeable
(dead) cells (PI+) and (putative) capsulated cells
(AB+)
In experimental microcosms we viewed the
gasoline tolerant/oxidizing bacteria to make a clear
distinction between the total number of cells (stained
with AO, DAPI), the number of encapsulated, active
cells, (AB +), and the number of permeable (PI+) ,
dead (figures 1 and 3).
Fig 1. Comparison between the total cell count (AO+
and DAPI+) and permeable cells density (IP+)
129
As shown in figure 1 the total number of
heterotrophic cells counted using AO or DAPI is
practically the same. Quantification was performed
on samples previously fixed in experimental
microcosms (M1 and M2). Comparing the total
number of bacterial cells obtained with AO and
DAPI stained (20 μL/mL sample) in experimental
microcosms, we have shown that there are no
significant differences in the use of two
fluorochromes on natural samples (M1: 13.719,5
cells ml -1 – SD (±39,7) AO and 13.494,6 cells ml -1 -
SD (±22,2) DAPI, respectively M2: 14.619,1 cells
ml -1 – SD (±28,8) AO and 15.787,4 cells ml -1 SD
(±32,8) DAPI).
Fig 2. Total number of cells obtained using AO and
SG in natural sample
To assess the number of cells obtained after
staining AO and SG, we used unfixed samples
collected from microcosm 1.
As shown in figure 2 there are differences in
total counts obtained by the use of either AO or
SYBR Green 1, the higher count obtained with the
last fluorochrome (47,6%) being due to its higher
fluorescence yield, in agreement with international
literature (Weinbauer et al., 1998; Luna et al., 2002;
Lunau et al., 2005 ), allowing the visualization of
smaller cells.
As shown in figure 1, the number of dead cells
(PI positive) is 12.3% of the total number obtained
with the two fluorochromes, AO and DAPI.
In figure 3 one can see the cell densities of
putative capsulated cells which are 10% from the
density obtained with acridine orange.