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SOP8 – Live Organisms 10-50 μm
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9.5.2. Viable phytoplankton CMFDA/FDA enumeration & concentration procedure
Prior to beginning preliminary assessment, remove CMFDA & FDA probes from freezer to thaw
completely to room temperature. Probes need to be kept in the dark.
9.5.3. Preliminary Assessment
1.
Start with direct 1 mL sample viewed under transmitted light to determine if approximate
cell density is adequate for a direct count.
2.
Invert sample bottle 5 times to insure random distribution
3.
Use 1000 µL pipette with new tip to extract 1 mL from bottle
4.
Add 1 mL sample to 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 ~1.1 mL 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 mLs are needed)
c.
5.
View slide under transmitted light
a.
If >100 cells on slide, then a direct count can be done and no concentration is
necessary.
b.
If <100 cells on slide, then concentration of the sample will be needed.
Note: At UT, cell density has normally been high enough (sometimes too dense) for a direct
count. At DC, cell density has been much lower and sample concentration has been necessary.
9.5.4. Direct Count
1.
Invert sample bottle 5 times
2.
Extract 1ml from sample with 1000 µL pipette and new tip.
3.
Add this 1mL to new 1ml vial
4.
Add 4 µL of CMFDA probe to each vial using 10 µL pipette and new
sterile
tip for each.
5.
Add 4 µL of FDA probe to each vial using 10 µL 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
6.
Vortex each sample
7.
Incubate at 26°C for 15min
8.
Gently agitate the sample and extract 1 mL with the 1000 µL pipette and new tip
9.
Add this sample to 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 ~1.1 mL 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)
10. Start viewing the slide under transmitted light and when in focus switch over to
fluorescence (turn off or diminish transmitted light as much as possible).