Sampling on 30 June 2009
Locations:
B. Hammond
C. Ilwaco
D. Willapa Bay
Sunny, windy weather conditions consistent throughout the day.
The reasoning behind our sampling dates and locations was due to a colleague’s trip the previous week. They reported a slight red coloration of the water in the Willapa Bay location. Additionally, Myrionecta rubra was observed in the samples collected from the previous week using the Flowcam.
Multiple sampling sites were chosen in order to hopes of comparing the genetic sequence of the Myrionecta rubra in each sample. This study will attempt to use a specific Myrionecta primer (oMR-1) in combination with an ITS primer in order to determine any differences in the variable region between the 18s and 28s segment of the RNA of the specific Myrionecta found at each sampling location.
Participants:
Lydie Herfort (Post-Doc)
Rachel Warnick (REU intern)
Freek-Jan Buijsman (Volunteer)
Samples Taken:
The following samples were all collected on site:
· For nucleic acid analysis, water was immediately filtered through 0.2-um-pore-size Sterivex filter (PES, ESTAR, Millipore) using a peristaltic Geopump. Water was forced out of the Sterivex in order to add 2 mL of fixative (RNAlater, Ambion). Samples were kept on dry ice until returning to the lab where they were stored at -80°C.
· pH and salinity: 50ml Falcon tube filled with sample water
· FISH: Falcon tube filled with 40ml sample water, 1.6mL Formalin (37%) added and fixed for an hour at room temperature before samples were placed on dry ice
· Lugol: 2 50mL Falcon tubes were filled with sample water and 7 drops of Lugol solution was added, samples were kept at room temperature
The following samples were collected using water that had been filtered through the Sterivex filter attached to the pump:
· Nutrient: To determine ammonium concentrations, 2 40 mL of filtrate was collected into a 50mL Falcon tubes. were filled To determine nitrate, nitrite, silicate and phosphorus concentrations a acid-washed polyethylene vial (25 mL) was filled to the shoulder.
· DOC: a 20ml polypropylene bottle was filled to the shoulder a small container was filled with about 30mL
· TDP/N: a small container bottle was filled with exactly 20mL of filtrate (water was first measured using a 50mL Falcon tube and then transferred to the containerbottle)
Using a Millipore 25 mm Glass vacuum filter holder and support and a 150mL Erlenmeyer flask filter set up, with tubing attached to the peristaltic geopump to create the vacuum, the following samples were taken. Sample water was filtered through the setup and thea Glass Fibre filter (GFF) (ø 25 mm, Whatman). After the filter visibly showed a change in coloration, filtering was stopped and the volume of sample water filtered was noted.
· Chlorophyll a: filter was placed in a labelled 2 mL orange screw cap Corning Brand Microcentrifuge Tubes
-cap tube
· HPLC: filter was placed in a labelled 2 mL orange screw cap Corning Brand Microcentrifuge Tubes
· SPM: pre-weighed filter needs to be used and stored in a small petri dish
· POC/N: sterilized muffled filter (sterilized placed at 500oC for 6 hours and stored in muffled aluminium foil) needs to be used
Hammond
Location= at the tip of a rock heap bulging into the Columbia River bayestuary
Sampling time: 10:55
· Ebb tide
Water Temperature: 18oC
Salinity: 5 psuPSU
No data collection or sterivex filtering occurred. Samples were taken in jugs.
Nutrient and Lugol fixed samples were collected
Ilwaco
Location= off the boat dock
Sampling time: 11:55
· Ebb tide
Water Temperature: 16.5 oC
Salinity:
Sterivex:
1 = 1060 mL, 2 mL RNAlater
2 = 950 mL, 2 mL RNAlater
Chlorophyll a: 195mL
HPLC: 95mL
SPM: 100mL
POC/N: 100mL
Willapa Bay
Location= off a small dock near a small boat loading/unloading zone
Sampling time: 15:40
· Flood tide
Water Temperature: 18 oC
Salinity: 28 psuPSU
Sterivex:
1 = 980 mL, 2 mL RNAlater
2 = 970 mL, 2 mL RNAlater
Chlorophyll a: 150mL
HPLC: 150mL
SPM: 150mL
POC/N: 150mL
Tidal Chart for 30 June 2009
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