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SOP8 – Live Organisms 10-50 μm
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11. Use ‘lawn-mower’ method to count cells moving back and forth until entire slide is
covered.
Count cells as ‘alive’ when they show obvious bright green fluorescence. Some may be motile
(especially controls at UT). Some are ‘live’ but do not fluoresce as bright as others. Live cells
are quickly measured using ocular micrometer and counted if larger than 10 µm.
1.
Record numbers
2.
When complete for each day the following samples will be finished
a.
UT: Control – 3 counts and Treatment – 3 counts
b.
DC: Control – 3 counts and Treatment – 3 counts
3.
Average mean cell density is calculated from these data
9.5.5. Concentrated Counts
If during preliminary assessment, <100 cells on slide then the sample needs to be concentrated
for counts.
1.
Estimate volume that will need to be concentrated from the preliminary assessment.
a.
On DC, 250ml is routine, if numbers are extremely low up to 500ml can be
concentrated
2.
Set up vacuum filtration apparatus.
3.
Place a 2.0 µm filter on each chamber using filter forceps.
4.
Measure sample volume in 250 mL graduated cylinder.
a.
It is best to filter control and treatment samples separately so that apparatus can be
washed down and cleaned to prevent cross-contamination
5.
Using hand vacuum pump filter the sample keeping vacuum pressure no more than 10 Hg
cm
-1
pressure.
6.
Fill 50 mL centrifuge tubes with 20 mL of the corresponding (control/treatment) filtrate.
7.
Fold the filter in half and place it in tube containing the 20 mL of filtrate.
8.
Using the Vortex stirrer, gently agitate each tube to resuspend the phytoplankton captured
on the filter into the 20 mL of filtrate.
9.
Extract a 1mL sub sample from each tube and place into a new 1mL tube.
Procedure at this point is same as direct count above starting with adding the 4uL CMFDA &
FDA probes
9.5.6. Dilutions
In conditions where large densities (>2000 cells mL
-1
) of phytoplankton are encountered, dilution
of the sample may become necessary for an accurate count. In this case samples prepared for a
direct count can still be used but fractioned out. Analyze 500 mL or 250 mL samples from the
initial vial on a Sedgewick rafter slide with the remaining space filled with sterile seawater (same
as with a direct count but different volumes).
If analyses take over 5 hours before finishing, the oldest samples may have set for too long and a
great deal of autofluoresce may occur. This has been noticed before and accurate sample
analysis is not possible due to the autofluorescence obscuring /overpowering some viable cells.
A second staining/sample preparation should be done.