VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

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
l`analise d`images couplee a l`epifluorescence.
Oceanis, 21: 113-124.