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SOP8 – Live Organisms 10-50 μm
61
9.4
Equipment
Materials
Type
Notes
Filtration apparatus
-
hand pump
-
bottles
-
bottle top filter
holders
Millipore filters
2.0 µm
Pipette and tips
10, 100 and 1000
µL
Filter forceps
Reusable plastic centrifuge
tubes
50 mL
8 total
Unused vials or
microcentrifuge tubes
1 mL
Sedgewick rafter counting
slide
4 Types
-Live (can be used for Control samples)
-Treatment Only
-Dead (Lugol’s or Formaldehyde)
-Gridded (for point counts)
Graduated cylinder
250 mL
Pipettes and pump
25 mL
Vortex stirrer
9.4.1. Microscopes
Primary
-An
Olympus IX-51
inverted phase/fluorescent microscope using a broadband FITC
(Chroma 41001 FITC/Bodipy/ Fluo 3/ Dio
)
filter set and 100W Xenon arc lamp is used for
analysis on most samples.
Secondary/Field/Backup
– A
Leitz Laborlux S
(modified/equipped) with Optiquip fluorescent
vertical illuminator, FITC
(Chroma 41001 FITC/Bodipy/ Fluo 3/ Dio
)
filter set and 100W Xenon
arc lamp.
9.4.2. Calibration & Measurements
Microscopes used in measurements are equipped with a ocular micrometer reticle. Calibration of
micrometers on all microscopes are done once a year using a NIST standard stage micrometer
slide. (Karlmann Rulings Inc. SN# 12611 Test# 021312-03
Documentation available on
request
)
9.5. Sample Analysis
9.5.1. Live phytoplankton viability via CMFDA/FDA
Samples are analyzed via dual staining method (Steinburg 2011) using both CMFDA and FDA
fluorescein vitality probes. Samples are returned to the lab as quickly as possible once all other
operations are completed. Analysis typically starts ~3-4 hours after sample collection.