VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010)
spring and heterotrophic and phototrophic bacteria
from marine environment.
The fluorochrome DAPI is the most commonly
bacterial stain for a wide range of sample types.
DAPI is a nonintercalating, DNA-specific stain which
fluoresces blue or bluish-white (at or above 390 nm)
when bound to DNA and excited with light at a
wavelength of 365 nm (Kepner & Pratt, 1994). When
unbound, or bound to non-DNA material, it may
fluoresce over a range of yellow colors (see
figure13). DAPI-stained filaments of cyanobacteria
isolated from sulphurous spring Obanul Mare
(Mangalia) reveal heterogeneous cells as can be seen
in Fig.13a.
a
b
Fig 13. DAPI-stained cyanobacteria isolated from
sulphurous spring (a), arrows indicates septa between
cells ; bacteria/cyanobacteria isolated from marine
environment (b); both a and b treated with Image J
and CellC software.
Fig 14 . Cyanobacteria and heterotrophic cells in
microcosm supplemented with gasoline/M2 - DAPI
stain
134
c) Aniline blue is highly specific for staining
type polysaccharide. Use of aniline blue is a good
method not only for detection of production of
exocellular β-1,3-glucan, but also for detection of
some β-glucan in the cell wall (Nakanishi et al.,
1976). In figure 15 is apparent the AB stained
heterotrophic cells from microcosm supplemented
with gasoline and cyanobacteria cells from
microcosms and sulphurous spring samples.
a b c
Fig 15 . Visualisation of encapsulated bacteria and
cyanobacteria after aniline-blue staining on M2 (a),
M1 (b) and from sulphurous spring samples (c).
d) PI staining is generally used for the
evaluation of plasma membrane integrity by
fluorescence. Literature mentions that molecular
weighs PI is 668,4 and is thus assumed to be unable
to penetrate cell membrane (Manini & Danovaro,
2006). In figure16 living bacteria appeared green due
to the excitation of the AO dye with which the cells
have been stained and the samples stained with PI
and appear red fluorescent cells; the bacteria were
counted under blue excitation.
a b
c
Fig 16. Marine bacteria examined using
epifluorescence microscopy (magnification x1000),
Fig (a) illustrate bacteria stained with propidium
iodide (dead cells) in microcosms 1 and (b)
respectively microcosms 2; total count analysis using
the CellC software (c).