Biology Reference
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Figure 9: Cytometric differentiation of picophytoplankton populations.
These different sub-populations were identifi ed
according to their differences in side scatter and both red and orange fl uorescence;
Synechococcus
emit a strong orange
fl uorescence whereas
Prochlorococcus
emit only red fl uorescence when excited by blue light (Goericke and Repeta, 1992).
In addition,
Synechococcus
cells are signifi cantly larger than
Prochlorococcus
cells (ca. 1-0.6 µm in diameter respectively)
(Chisholm, 1992). Pico-eukaryotes were identifi ed by their larger size (SSC) and higher red fl uorescence. Scatter plot of
orange versus red fl uorescence (A) and side scatter versus red fl uorescence (B) observed on Station S2 (i.e. Salinity = 2.44%),
showing: (i) 2 populations of pico-cyanobacteria, one exhibiting fl uorescence and side-scatter characteristics of
Prochlorococcus
sp. (referred as
Prochlorococcus
-like) and the second exhibiting fl uorescence and side-scatter characteristics of
Synechococcus
(referred as
Synechococcus
-like) and (ii) 2 populations of pico-eukaryotes (P-Eu
1
and P-Eu
2
).With the kind permission of
M.
Schapira, School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide SA 5001, Australia & Southern Ocean
Group & Ifremer-LERN, Avenue du Général de Gaulle, 14520 Port en Bessin, France. [Schapira
et al.
Saline Systems
(2010)
6:
2; doi:10.1186/1746-1448-6-2].
viruses that were stained with SBYR Gold. A comparison of the estimates made with SYBR Gold-
stained particles using digitized images gave 1.3 times higher titre in comparison to those based
on direct counting with epifl uorescence microscopy. Direct epifl uorescence counting of particles
stained with fl uorochrome SBYR Gold yielded 1.34 times higher values than those estimated by
TEM. Brussaard (2004) optimized procedures for the enumeration of viruses through fl ow cytometry
where it was recommended fi xation of samples with glutaraldehyde (0.5% fi nal concentration) for
15-30 min followed by storing of samples in liquid nitrogen at -80°C. Samples when required were
thawed, diluted in Tris-EDTA buffer (pH 8.0) and stained with SYBR Green I for 10 min in the dark
at 80°C and cooled before analysis. The counts of various viruses made as per this technique yielded
better results and the use of low storage temperatures prevented loss of up to 50 to 80% total virus
abundance (Fig. 10). The disadvantages of prefi xation with glutaraldehyde or formaldehyde of the
marine viral samples for examination through epifl uorescence microscopy have been highlighted by
Wen
et al
. (2004). Prolongation of post-fi xation period resulted in a decrease of viral abundance by
about 72% in 16 days. They recommended the use of unfi xed samples stained with YO-PRO-1, SBYR
Green I or SBYR Gold for enumeration through epifl uorescence microscopy. Prolonged incubation
periods at -20°C for at least 2 weeks or in case of YO-PRO-1 even for one year did not affect the
cyanophage numbers. Other techniques include most probable number assays (Waterbury and
Valois, 1993), radiotracer analysis (Fuhrman and Noble, 1995; Steward
et al
., 1992a,b, 1996), inhibition
