California Cooperative Oceanic Fisheries Investigations

1. Chl samples are drawn on all rosette bottles tripped from ~200m to surface; sampling on a standard 20-bottle cast usually starts at #7 (sample vol is ~140mls).  For shallow stations, all the bottles may be sampled; noontime prodo casts may have extra bottles to sample; duplicate depths are usually skipped.  Refer to the electronic sample log to verify which bottles to sample or ask the watchleader.

2.  Drawing from the middle valve, add ~20mls, cap loosely, shake then dump; three rinses.  Double-check the sample bottle number matches the rosette bottle number (often).

3.  Chl samples are volumetric so after rinsing, fill it completely, cap loosely, tap the bottle gently against the rosette frame to dislodge any small bubbles then top-off, cap tightly, invert the bottle – if you see a bubble, top-off and check again. Squeezing the sides of the bottle can change the sample volume and create a persistent bubble; cup the bottle in your palm during the final fill to minimize this problem.

4. Once all the chlorophylls have been drawn, draw the LTER water samples such as HPLCs.  The sample volume will vary with chlorophyll concentration so with the LTER watchleader for bottle size and sample depths.

5. Again, all bottles are rinsed three times then fill completely.

6. Once samples are drawn, fill out sample tags or forms, then initial the sample log.

7. When filtering chlorophylls: take the chlorophyll sample form from the clipboard along with the chlorophyll samples to the chl van to filter ASAP.  The prodo experiment filtrations in the evening may delay the filtrations so ask to be notified when the chl van is available.

• Retrieve an empty nutrient rack from the lab fridge if needed and record the color key on the sample log sheet.
• Tubes are inverted when empty and should not be turned over until filled
• Never touch the inside of the cap or tube since residue from your fingers can contaminate the sample – soap contain phosphates.  If unavoidable – rinse several times.
• Nutrients are normally drawn from the middle valve – fill the tube with ~25mls, cap loosely, shake then dump.  Repeat three times.
• Double check the tube number matches the rosette bottle number.
• Fill the tube completely then flick out several mls so the sample reaches the base of the neck/threads.  Cap tightly.
• Draw one sample per rosette bottle (except on the surface bottle).  If tube 24 is not needed, draw a duplicate surface sample.
• Once all the nutrient samples are drawn:
  • Carefully tip the nutrient rack until you can see the sample through the cap of each tube – if a tube is empty and not inverted, double-check the sample log and fill if necessary.
  • fill out a sample label (from the sample log clipboard) with the time, your initials, total number of samples
• Wrap the label around tube #1 and carefully re-insert into rack – be sure to include the extra surface sample to the sample count by listing it as “+1”
  • return the filled rack to the nutrient fridge right away.
  • add your initials to the bottom of the sample log’s nutrient column

Common mistakes are empty tubes that shouldn’t be; missing duplicate surface sample; contaminated samples; and duplicate draws – two sample tubes filled from the same bottle

•  The sample bottle number should ALWAYS match the rosette bottle number – check and double-check this during the sample drawing process.  If you ever have any doubt about the sample, dump it and start over.
• The sample bottles are stored inverted – they should not be turned over until the sample has been taken.  If you need to step away from sampling for any reason and have not filled the sample bottle, return it to the case inverted.
• The bottles should never be stored empty so as the old sample is dumped, use it to rinse any salt that may have crystallized on the threads, thimble, and cap.
• Salt samples are usually drawn from the bottom valve. Fill the bottle with ~40mls of seawater, cap loosely, shake then dump, rinsing the threads and thimble.  Repeat – you should rinse the bottle 3 times; the dumping of old sample does not count as a rinse.
• The last fill should be done without interruption until overflowing, filling the bottle completely; pour ~10ml out over the thimble, place it firmly in the bottle and cap.  The caps are brittle and should be tightened gently. If they crack, retrieve a replacement from the spares Ziploc, keeping the thimble in place.  See photo for optimal fill height.
• Salts are taken from all closed rosette bottles unless directed otherwise.
• Sample bottles are fragile – carefully place them back in the correct slot.  If you drop one on deck or into the case and crack the glass, replace the cracked bottle from the spares case, adding the bottle number.  Redraw the sample.
• Once all the salts are taken:
  • fill out a sample label (on the sample log clipboard) with the time, your initials, total number of samples Place the label in a plastic sleeve and into the box of samples
  • take into the lab, adding it to the end of the “salt box queue”; bring out an empty case for next station.
  • add your initials to the bottom of the sample log’s salt column.

• Common mistakes are duplicate draws; samples returned to the case out of sequence (common when more than one person is drawing salts); missing thimble insert; not enough air (can crack the bottle as it warms and not allow the analyst to load the sample); too much air (can shift the salinity value or not give the analyst enough sample to measure); and cracked or broken bottles (drops).

Oxygen Sampling and Analysis General Information

•  Pickling Oxygen Samples using Carpenter’s Modified Winkler Titration Method:
•  Seawater samples are drawn into a calibrated volumetric flasks using tygon tubing.  The flask is rinsed three times then overflowed with twice the sample volume.  Carefully removing the sampling tube to prevent the influx of bubbles, the sample is then fixed with manganous chloride (MnCl2) and sodium iodate/sodium hydroxide (NaI/NaOH).  A stable precipitate forms.  The flask is stoppered and shaken vigorously to homogenize.  A sample label is filled out and the case is covered. After settling for several minutes, a second shake is performed to insure all the oxygen is fixed.  The case is added to the O2 sample queue.  Autotitrator oxygen samples are drawn and fixed in the same manner but the temperature of the sample is measured and recorded.

• After completion of the net tow, concentrate the plankton sample into the cod end by washing down the net with the deck seawater hose (DO NOT use fresh water).  Be sure to spray the net from the outside to minimize damage of the delicate plankton in the sample.   
• Once the sample is concentrated into the cod end, use a screwdriver to loosen the hose clamp.  Give the cod end a tug to detach it from the PVC coupler.  If the sample will overflow the cod-end when removing it from the coupler, grab a bucket and remove the sample over the bucket. Make sure to wash down any sample remaining in the net into the bucket as all our plankton tows are quantitative (no sample can be lost).
• Take the cod end into the preservation sink area.  Remove the appropriate jar from the sample box (pint jar for Pairovet and Manta, quart jar for Bongo; all boxes are labeled accordingly) and set it into the sink rack.  Be sure the begin-tow time is written on both inside & outside labels, using pencil for inside and Sharpie for outside.  Peel off the outside label backing and stick to jar lid.  Put the inside label into jar.
• Using the filtered seawater hose, concentrate the plankton into the bottom of the cod end.  It is ok to gently spray the inside of the cod end at this point but not so hard you will damage the organisms.  Once the plankton are sufficiently concentrated towards the bottom, invert the cod end into the draining sock (a wooden dowel helps).  There are 2 different mesh sizes of draining sock, always make sure you use the appropriate size mesh for the sample you are preserving (.150 for Pairovet samples, .333 for Manta or Bongo).  The draining sock is helpful because it allows you to use as much seawater as necessary to thoroughly rinse the cod end. Gently spray the cod end with enough seawater to remove all the plankton clinging to it.  It is very important not to lose any part of the sample or it will no longer be quantitative.  Large pieces of kelp or grass or anything obviously not plankton can be rinsed off thoroughly and discarded at this point.  Using a spoon, transfer the plankton into the jar.  Rinse the spoon into the draining sock and then invert the draining sock into the jar.  Wash down any of the remaining plankton into the jar with the rest of the sample, filling the jar with seawater to just below the shoulder.
• Wear eye protection.  Add a squirt of supersaturated sodium borate to the sample for buffering purposes. Formalin is slightly acidic so the borate raises the pH of the sample to neutral.  Add 10 cc. to pint jars, 20 cc. to quart jars.  The syringe is usually marked with a “P” for Pint and a “Q” for Quart.
• To add full strength formalin, unclip the plastic binder attached to the tygon tubing of the formalin rig to allow for flow.  Hold the 60 cc. syringe firmly and pull out the stopper to the desired amount (25 cc. for Pint and 50 cc. for Quart).  Re-clip the plastic binder and dispense the formalin into the jar.  Be careful not to spill any or push so hard the formalin squirts out the back of the syringe.  Formalin is very caustic and extra care should be taken when dispensing it.  If you have any allergies to formalin then you should not help with this step of the preservation process.
• After the formalin is added, secure the lid tightly and invert the jar a few times to ensure proper mixing of sample & chemicals.  Return the sample jar back to the correctly labeled box. 
• Rinse the cod end with the deck seawater hose and invert it back to the original position.  Reattach the cod end to the coupler and tighten the hose clamp securely.

Thank you for all your help, it’s greatly appreciated !!