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SOP8 – Live Organisms 10-50 μm
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9.8.2. Sample Processing
1.
Samples filtered through 50um sieve to remove macro-organisms (IMO specifications)
2.
View 1mL of filtered water with Sedgewick rafter under transmitted light BF microscope
to determine approximate cell density.
o
Cell density should be 100 cells mL
-1
or higher
o
Concentration of the sample may be needed if cell density is too low
(follow concentration procedures used for regular CMFDA/FDA analysis testing)
3.
3 sub-samples will be taken from each bottle/replicate and 1mL samples will be placed in
micro-centrifuge tubes for further processing
o
3 – live (control: no additional processing)
o
3 – dead heated (treatment 1)
§
1mL sample in microcentrifuge tubes placed in water bath @ 50C for 10
min. then cooled to room temperature before staining
o
3 – dead frozen (treatment 2 – optional)
§
1mL sample in microcentrifuge tubes placed in -20C freezer for 1 hr and
then thawed to room temperature before staining
9.9. Staining with CMFDA/FDA
(From MERC Phytoplankton SOP)
Direct Count
1.
Add this 1ml to new 1ml vial
2.
Add 4uL of CMFDA probe to each vial using 10uL (yellow) pipette and new
sterile
tip for
each.
3.
Add 4uL of FDA probe to each vial using 10uL (yellow) pipette and new
sterile
tip for
each.
a.
(Make sure both CMFDA & FDA probes are completely thawed before using. Only
sterile unused tips should touch the CMFDA/FDA probe)
4.
Vortex each sample
5.
Incubate at 26°C for 15min
6.
Gently agitate the sample and extract 1ml with the 1000uL pipette and new tip
7.
Add this sample to gridded (1-mm2) Sedgewick rafter slide
a.
To prevent air bubbles, set the cover glass on slide at an angle, and insert the
sample under one corner allowing capillary action to draw the sample under the
slide.
b.
The slide holds ~1.1ml so top off the rest of the slide with filter sterilized
seawater. (Filtrate from concentration procedure of same sample or filter some
through 0.22 syringe filter. Only a few ml will be needed)
(Analysis is slightly different than standard)
8.
For each subsample, one row of the Sedgewick rafter grid will be selected randomly
9.
Each field will be observed with both BF and FL
10.
For each field all organisms (10-50 um) will be scored Moving/Non-Moving
(M+, M-)
and Fluorescing/Non-Fluorescing
(F+, F-)
11.
*For these trials any cell with fluorescence visible will be scored as F+