navigation bar

 

 

 


 

CONTENTS

Station and sample list

bulletA*
bulletChlorophyll Samples
bulletProductivity C14 Samples
bulletC14 Stock Prep
bulletN15
bulletNutrient Samples
bulletPAR
bulletParticulate Organic Material
bulletParticulate Organic Carbon
bulletPhytoplankton Samples
bulletPRR
bulletQuick Reference Chart
bulletWipe Test

 

Point Lobos CTD Cruises
Methods and Protocols

Tim Pennington 1/96

Point Lobos Time Series cruises occur every 3 weeks aboard R/V Point LobosCruise schedule.  For information regarding these cruises please see Tim Pennington x1715


Station and Sample List

POINT LOBOS MAJOR STATIONS: C1, Mooring1, Mooring2

major_stations.gif (5030 bytes)

SAMPLE LIST
1. Secchi disk
2. Salinity Samples
a) After cast fill another salinity bottle from the 200 m Niskin.
3. Chlorophyll
a) 11 samples down to 200 m; 280 mls thru GFF, into scintillation vial + 10 ml 90% acetone, store into dark freezer (0, 5, 10, 20, 30, 40, 60, 80, 100, 150, 200m).
b) Two 100-ml samples at 0 m  for 1 and 5 um fractionation; use correct filters; treat as chlorophylls above.
4. Productivity Incubations
a) 7 incubations (100, 50, 30, 15, 5, 1, 0.1% light); take water from estimated light depth; add 100 ml C14 stock; into correct light tube in incubator.
b) One 100% light bottle for 1.0 and 5.0 um fractionation; treat as above.
c) One bottle each from 100% and 0.1% light levels for T0; innoculate 100 ml C14; filter immediately thru GFF; store dry in freezer with chl’s.
5. 0 m nutrient sample. Fill plastic scintillation vial 1/2–2/3 full; freeze upright in freezer.
6. 0 m quantitative phytoplankton sample. Fill gluteraldehyde-containing bottle using intermediate bottle
(keep away from rosette and van).
7. 0 m Particulate Organic Carbon (POC; 500 ml). Filter onto combusted GFF; store in glassine in freezer with chl’s.
8. 0 m flow cytometry sample. Fill cryovial; add 2 drops fix from toxics cooler; freeze in N2.

ADDITIONAL SAMPLES FOR MOORING1:

1. From 0, 10, 20, 40 m take 500 ml each for A* analysis thru uncombusted GFF, into cryovial, freeze in N2.
2. POM. Filter 1 L 0-m water thru combusted GFF; store in glassine.
3. Nutrient samples taken all depths; 1/2–2/3 full in plastic scintillation vial freeze upright in freezer.
4. N15 incubations for Dugdale (7 light levels+NO3&NH4 SAT).
5. Flow cytometry profile—all depths.

WIPE TESTS OF VAN (front half only):
The purpose of the wipe test is for radiation contamination analysis.  Using GFF filters perform a wipe at each of the following.  Store filters in scintillation vials.
1. Starboard counter
2. Floor
3. Port counter
4. Sink CHLOROPHYLL (to 200 m)
top


CHLOROPHYLL SAMPLE COLLECTION

SHIPBOARD:
1. Rinse bottle and lid 3X and fill to the top from GoFlo's.
2. Filter sample through Whatman GFF (glass fiber filters) at 5–7 um Hg pressure (plastic filter setups are fastest).
3. With forceps, place the filter in a labeled scintillation vial.
4. Add 10 mls of 90% acetone with repipette (if no acetone, can freeze until end of cruise).
5. Chl fractionations. Filter 280 ml of surface water through 1 and 5 um nuclepore filters. Handle as above.
6. Extract samples in freezer and keep in dark 24–48 hrs.

CHLOROPHYLL DATA PRODUCTION—TURNER FLUOROMETER
ASHORE:
1. Before reading, remove samples from freezer, shake gently, and allow to warm 30–60 min in dark or at least dim light. Also turn on and allow fluorometer to warm up >20 min.
2. Zero the fluorometer with 90% acetone, with sensitivity at 100 x 31.6.
3a. Pour supernatant from vial into cuvette; read total fluorescence (Fo). Set the sensitivity so that readings are between 3.2 and 10 on top scale. Always use top scale.
3b. Record data manually on Log Sheets
4a. Add 3 drops of 10% HCl to cuvette, and re-read at same sensitivity setting (Fa). (Acid destroys the Chl, so your reading is now phaeophytin, which is included in the first reading). Do not change sensitivity.
4b. Rinse cuvette with acetone
5. When finished, discard acetone down northeastern sink in wet lab; vials go in regular trash.
6. Enter data in "CRUZ" program, see "BOG User's Guide"

NOTE: The amounts of these compounds are produced by the CRUZ program in ACCESS, but the calculation is as follows:

Chl (ug/l) = F*Ve((Fo-Fa)/S)/Vf

and

Phaeo (ug/l) = F*Ve[((FoFaCal*Fa) - Fo)/S]/Vf

where:
F= fluorometer calibration factor
Fo = total fluorescence
Fa = fluorescence after acid
Ve = extract volume (acetone extract; 10 mls)
Vf = filtration volume (volume s.w. filtered in liters; often .28 L)
S = sensitivity
FoFaCal = Fo/Fa = calib. factor for phaeopigments --- ratio of Fo:Fa in the absence of
phaeopigments during calibration with spinach chla.

2.1 = ratio of Fo:Fa in the absence of phaeopigments

NOTE on CRUZ calculations: Calibration of Turner’s is done with standard chls from spinach, with maximum starting concentrations of about 1.2 ug/ml. But CRUZ wants to express chl vals as ug/L (=mg/m3), so we need to multiply the answer by 1000. This is done by CRUZ by using Ve in mls (usually 10), and Vf in liters (often 0.28) --- a confusing and dodgy but corrrect method.
top


Productivity— C14

SHIPBOARD:

1. Productivity samples are taken from 100, 50, 30, 15, 5, 1 and 0.1% light depths. Determine the secchi depth using the secchi disk. Use wall chart to determine GoFlo depth closest to the above light levels.

[Or, calculate depths from which each sample should be taken as depth=(In(Id/Io) x secchi depth)/1.7 where Id/Io=fraction light (i.e., 50%=0.5). If a secchi depth cannot be determined (e.g., night) use depths from the previous station.]
2. Rinse polycarbonate sample bottles three times with sample water, then fill to the neck from correct GoFlo's. One bottle per each of the 7 light levels.
3. Fill 2 extra bottles from light depths 100% and 0.1% for time zero (T0) uptake.
4. Fill another (one) bottle (100% light depth) for C14 fractionations.
5. Inoculate all but T0 bottles with 100 ul C-14 stock. Wear gloves and do not spill or spray. Make sure Eppindorph repeating pipette set on ‘2’ and using correct pipette tips (?2 ml).
6. Put the sample bottles in their appropriate light tube, cap, place in the incubator, and record the time.
7. Inoculate T0 bottles with 100 ul C-14. Filter immediately through GFF. Remove filter with forceps and place in a labeled scintillation vial and freeze. Upon return to Moss Landing in the evening, add 1 ml 0.5 N HCl to the vials and let sit open in hood overnight. Next day, add 10 mls scintillation cocktail. (These samples the amount of C14 adsorbed by POC, and caught on filter—i.e., non phytoplankton uptake).

ASHORE:

1. After 24 hours incubation on dock, remove samples from incubator at correct time.
2. Prepare "TOTALS" from the 100% and 0.1% light depths as follows: using a 1000 ul pipette (blue tips), take a 1000 ul sample from the incubated bottle and inject it into a scintillation vial, filled to the neck (20 mls) with scintillation cocktail. Shake and label this as a "TOTAL" (these estimate total activity of C14 originally added to the bottles—no filtering).
3. Filter samples through GFF filters; remove filters with forceps; place in labelled scintillation vials, acidify with 0.5 ml 0.5 N HCl for 12–24 hrs in hood. Also fume T0 samples!
4. Fractionations. From the 100% light fractionation bottle, filter 100 ml each through 5 and 1 um nucleopore or poretics filters, and acidify.
5. 12–24 hrs later, add 10 ml scintillation cocktail; shake. Samples are now stable.
6. Run LS using ‘user 1’ program for both totals and samples, at least 24 hrs after adding fluor to samples.
7. Wipe tests: After all incubs have been filtered, wipe a 10 cm square area on each bench, fluor, sink and hood with a GFF each, as a wipe test. Put filters in vials, add fluor, and run in LS with samples.
top


C14 DATA PRODUCTION—-LIQUID SCINTILLATION COUNTER

1. Keep C14 room locked at night, on weekends, and when not in use. No food or drinks.
2. LS should be on; check to see printer is on line.
2b. Run samples >1 day following addition of fluor.
3. Run standards behind AUTOCALIBRATE flag; do not change their order only the first one is important.
4. Run TOTALS and samples in racks behind USER 1 flag.
For C1, Mooring1, Mooring2:
Positions 1–7: 100–0.1% light samples
Positions 8–9: 5.0 and 1.0 um fractionation samples
Positions 10–11: 100 and 0.1% light T0 samples

5. Run WIPE tests from the Point Lobos, the Point Lobos Van, and the Moss Landing Chem Lab. Also include a clean filter in fluor as a blank. These data need to be filed in the front office Radioactive Safety file, after every cruise.
6. Terminate run with HALT flag in red rack.
7. Begin run by pressing "auto count" on front of LS.
8. Enter data in "CRUZ" program, See "BOG User's Guide".
9. Periodically dispose of samples/vials in radioactive waste at Moss Landing.
[Note: LS takes 5+ minutes per sample.]
top


Nutrient Samples
(surface at all stations; all depths at H3/Mooring1)

1. Obtain 20+ treated plastic scintillation vials from Carole. She prepares them by seasoning in DI water for a week or so.
2. Fill plastic scintillation vials 1/2–2/3 full (not more!) from the GoFlo's.
3. Freeze upright in scintillation vial flat with the Chlorophylls.

Quantative Phytoplankton Samples
(surface; all stations)

PRECUISE:
Wrap a phytoplankton sample bottle with duct tape and label with the date, station name and depth the sample was taken from. Rinse bottles in FW to remove dust. Use the glutaraldehyde repipet, add 5 ml glutaraldehyde. This step is generally done in lab prior to cruise.

SHIPBOARD:

1. Use an empty sample bottle to get the sample from the surface niskin.
2. Then fill french square containing glut to shoulder. Do not take these glut bottles into van or fill  them directly from Niskins!!!
3. Store samples in little Toxics cooler.

Preparation of Phytoplankton Slides for Epifluorescence
(Kurt Buck generally does this)

1. Work in fume hood under dim light.
2a. Use a filter setup for glut, and a 0.2 um Nucleopore or Poretics membrane filter (shiny side up) on top of a 0.45 um Gelman or Millipore filter.
2b. Wet base filter with water, and then suck membrane filter onto base without bubbles before putting on funnel.
3. If already preserved, use full scintillation vial as measure of 25 mls sample from french square. Invert french square 10X before pouring.
4. Filter until dry.
5. Remove filter with vacuum on, center on labelled slide, add 1 drop immersion oil, cover with cover slip.
6. Put slide in slide box; put box in freezer.
7. Wash filter equipment in water; discard dry glut trash.
top


Particulate Organic Material (POC)
(surface samples at Major Stations)

1. Rinse and fill a 500 ml bottle with surface water at each of the major stations.
2. Filter sample through a combusted GFF.
3. With forceps, fold filter and place in a glassine envelope labelled with cruise date, station name, volume filtered.
4. After cruise dry filters in a 50 C oven.
5. Store filters in dessicator until packed in a CHN wheel.

*Note: Sometimes if there is a lot of material in the water it is difficult to filter 500 mls. Therefore, start off by filtering 200 mls and then add increments of 100 mls depending on how quickly they filter.

Particulate Organic Material (POM)
(for Rau; surface at Mooring1)

1. Rinse and fill a 1-L sample bottle with surface water.
2. Filter through a combusted GFF filter.
3. Process as above.
4. Mail accumulations of filters to:

Greg Rau
MS 239-4
NASA-Ames Research Center
Moffett Field, CA 94305-1000
top


N15 at M1
(for Dugdale)

1. NO3 PROFILE—-7 light levels. You want 0.25 ug-AT/bottle. With 10 ug-AT/ml ampuoles (blue top, silver bottom), add 25 ul N-15 NO3.
2. 50% LPD NO3 SAT—same ampuole as above. To one (1) additional bottle from the 50% light level, want 1.25 ug-AT; so add 125 ul N-15 NO3.
3. 50% LPD NH4 SAT—With 10 ug-AT/ml ampuoles (red top; grey bottom), add 1.25 ug-AT or 125ul.

If have 25 ug-AT/ml (red top; white bottom) add 50 ul N-15.
Following incubation (the same procedure as for the primary productivity incubations) filter each bottle onto a GFF combusted filter. Fold in half, place in labeled glassine envelope, dry at low temperature (45 C) over desiccant and then store over desiccant.

Label glassine envelopes with waterproof marker as follows:

Cruise&Date
Cast No./Sta. Name
Light Level
N03 Prod, or SAT N03 or NH4 (ie, 10ug-at/ml added)

After a few cruises, pack up the filters over a desiccant and mail them with log sheets to :

Dr. Richard Dugdale
San Francisco State University / RTC
P.O.Box 855 3150 Paradise Drive
Tiburon, CA 94920

NOTE: Before Tim's time, we used also to do the following with NH3: For each light level, add 25 ml from the 1 ug-at/ml 15 NH4 ampoule (red top, clear bottom, about 2 ml in each ampoule).
top


A* Samples

1. At mooring1 station, take 0.5 L each from 0, 10, 20, 40 m Niskins.
2. Filter samples through GFF, put in labelled cryovial, freeze in nitrogen. If there is no nitrogen available, dry ice or even regular freezer will have to do. Make sure to write volume filtered on cryovial, along with other info.
3. In lab, run A* samples thru spectrophotometer. HPLC analysis yet to be developed.
top


LICOR PAR SENSOR and LOGGER
[This writeup for new logger received January 1996.]

GENERAL

This sensor serves to log the amount of light received by the on-deck C14 incubations. It needs to be setup at the beginning of the cruise, and taken down at the end of incubations.

PROCEDURE

1. Take red cover off sensor; put sensor in cup.
2. Setup of LOGGER (can do afternoon prior to cruise):

•press ON
•press TIME {Should be corrrect time and date in gmt. If not, reset.}
•press CFG {should say INST}
•press ENTER until says LOG {you are then in the data storage menu}
•press enter thru settings, until get to RESET {Make sure reset time (in GMT) is 10–20 min from the present time; this is when it will start logging; it takes a measurement every 10 min}
•set reset time if neccessary (gmt)
•continue to press ENTER until you get back to ‘1M ...’
•then press OFF {If logger now set to start logging, a dark bar will slowly flash in window. This is neccessary.}
•put lid of tupperware on carefully, and stow logger in a safe place, usually under the incubators.
•make sure the sensor is not in the shade of anything.

3. Leave logger on incubator until after the incubations are off, the following day.
4. Downloading instructions (from memory; check):

•plug logger to rs232 25 cable pin on back of Tim’s pc
•start procomm on pc in c:\pl\96\xxx96;
•ALT-P; select 4800 N 8 1 and comm port 2; save to disk
•open log file in procomm with ALT F1; call the file XXX92.par
•turn logger on; get to ‘1M ...’ screen,
•press out; accept baud 48, form H, len 50 for parameters
•will download data onto pc screen
•when done exit procomm w/ ALT-X; check that file intact

5. Clear logger’s memory by:

•turn logger on
•press fct-on
•press up arrow until says ‘clear ram’
•press enter, says ‘ok clr mem? NO’
•press fct-on, says ‘ok clr mem? YES’
•press enter, will clr mem. Can check with mem status.
•Don’t clear mem until sure you’ve got the data file ok.
•Never press ‘RESET INSTR’, or you will lose cals and setup into.
•Set logger back on inst mode so that it doesnt start logging at reset time (even when turned off)
and turn off
•repackage for next use

top

PRR
(this writeup dated; check it; tp 1/96)

ASSEMBLE EQUIPMENT:

1. PRR
2. Cable
3a. Blue deck box
3b. Power suppy to deck box
4. "modem" cable to laptop
5. laptop
6. cable to deck sensor
7. deck sensor; place in sun out on deck

OPERATION

1. Cool the intrument by setting over side for a min or so if it has been sitting in the sun.
Start laptop: c:\ppr\prr-prof
Choose cal file in accordance with the prr’s being used accept display param's F4 to profile
change filename in form of xxx96pm1 for cast at m1; xxx96pm2 for cast at m2 enter hdr material: line 1: xxx96c03; mooring2 lat/lon blank; sky state: clear, cloud, fog, haze; deployment position always ‘2’
F4 again to start
G for graphic display
F5 end downcast
F6 end upcast
F9 to save and end

2. Surface Dark Values. On deck with windows covered, let laptop log 30 sec or so.
3. Profile down. 0.5 m/s. Profile up.
4. Surface dark values again, as before. F9 save and end.
5. Rinse w/ fresh water.
6. Done.
7. Postcruise data handling. Copy data off laptop onto floppy. Put data on Tim’s pc in the correct cruise directory— ie: c:\pl\96\xxx96
top


Preparation of Teflon C14 Stocks used in Incubations

A. MATERIALS:

25 mCi C14 in sodium bicarb solution (Amersham CFA.3 1x25 mCi)
Gold label sodium carbonate
1-L volumetric flask
250 ml volumetric flask
funnel
stir plate
UV photo-oxidation system
filtering apparatus: 1-L vacuum flask, filter funnel, glass-fritted filter bottom, pump
pH paper or meter
0.22-um millipore filter
scintillation vial
Teflon bottles for storing stocks

B. PROCEDURE

0. Rinse glassware with dilute Hcl.

1. Stock Solution:
Weigh 0.3 g sodium carbonate (Na2CO3) into a 1-L volumetric flask.
Rinse weigh paper and funnel with Milli-Q water.
Add Milli-Q to 1-L.
Stir overnight.

2. 100 uCi C14/ml:
a. Read pH of Stock Solution. If not 9.5–10, discard and start over.
b. Rinse 250 ml volumetric flask with Stock; add ~100 ml Stock. Break open C14 vial and add to flask.  Rinse vial using a pipette.  Fill to 250 ml with Stock.
c. Fill quartz UV tubes ~2/3 full and irradiate overnight (>6 hrs).

3. Filtering:
a. Setup the 0.22-um Millipore setup.
b. Using electrical tape, make a sling in top of the flask that will hold a scintillation vial under the filter base.
c. Rinse scintillation vial with Stock, rinse filter into vial 3X with Stock, rinse Teflon bottles with Stock from scintillation vial. Discard rinse how???
d. Pour some C14 into filter funnel, and filter directly into a Teflon bottle until ca. 2/3 full. ~25 ml/bottle, so 10 bottles needed @ 2.5 mCi/botl.
e. Repeat until done; label bottles.

4. Testing activity: Run Totals.
a. Add 100 ul from each Teflon bottle to 280 ml seawater.
b. Add 1 ml of this dilution to a scintillation vial containing ~20 ml fluor.
c. Run in LS as described above.

Notes:
1. For 10 uCi C14 added in 100 ul water to 280 ml seawater, DPM's should be 78–80,000.
2. Amersham sends 25 mCi or 25,000 uCi, which I dilute to 250 ml, so 100 uCi/ml in stock used on cruises.
3. Teflon vials are 25 ml, so 2.5 mCi or 2500 uCi/vial.
4. 1 incub gets 100 ml, so 10 uCi/incub botl.
5. >99% of this not retained on filters, so filters (ie, waste in scintillation vials) <= 0.1 uCi/filter or vial of waste.

top


Operating Instructions for 911PLUS CTD

T. Pennington, January 1996; ss4217


PREPARATIONS

1. Forklift rosette from Tucker's Room; crane aboard.
2. Shackle rosette to sea cable; use cable tie to secure shackles.
3. Attach ground and pwr/data cable to bullet on sea cable; put dummy from pin in sea cable in safe place.
4. Disconnect soap wash syringe from cond cell.
5. Take aluminum cover of 4pi par sensor. Give transmissometer and fluorometer windows a wipe with a soft tissue.
6. Cock bottles; make sure you are starting at bottle 1 by doing a test trip of bottle 12, manually, by rotating fitting in center of top of rosette.
7. In forpeak run batch file ctd.bat on pc and reboot. This frees enough RAM so seasave can run.
8. Make sure you have enough HD space. Can delete any old cast files. Be careful; software doesnt give you a good (or any?) error message when HD full. Casts can take 500K each.
9. Go to dir ss4216 on pc.
10. Start seasave by running cast.bat— this resets pc time and starts seasave.
11. Select Aquire Realtime data. Choose a cast name and enter under Data File Name. If cast to <=200m ptlo.dsp should be fine for display Dont alter con file. Hit F10 to continue Enter hdr information Hit ESC WAIT HERE UNTIL ROSETTE IN WATER AND DECK BOX FUNCTIONING; NEXT RETURN WILL START CAST DATA LOGGING.
12. Fill out datasheet.

DOWNCAST

1. Deploy rosette to 10 m and hold.
2. Turn on SBE deck box.
3. Check Thumbwh. E for pump turn on. Display will go from 10 to 11 when pump comes on after 1 min delay. You will want to wait for the pump to come on after every bottle trip.
4. After pump comes on bring rosette to surface.
5. Start cast; 30 m/min is slow; 60 m/min fast; I generally go down thru the thermocline slow then speed up.
6. Go to desired depth. DO NOT HIT BOTTOM!!! There is no altimeter on rosette; use fathometer display from bridge and I stay ~25 m off the bottom.
7. Start tripping bottles for upcast. Trip with CNTRL F1; wait ~10 sec for trip to be displayed as 111 briefly on deckbox LED and on pc; wait another 10 sec for rmk/upcast data for that depth to be collected; go to next depth. 8. Check-off each trip on datasheet.
9. Terminate cast at keyboard with cntrl-F1 after bottles tripped. You will may get an insufficient RAM error from the pc. This is because our pc is marginal for this application. You will need to restart seasave.
10. Turn off deck box.
11. Bring rosette aboard.
12. Re-cock bottles.

END OF DAY

1. Rinse cond cell with soapy water or just de-I. Use syringe for this. Leave syringe attached and cond cell full.
2. Rinse whole rosette with fresh water from deck hose. Do not rinse inside of bottles; empty of seawater and leave closed.
3. Put par cover back on.
4. Disconnect ground and power wires; put dummy back on pin in sea cable bullet.
5. Unshackle rosette; put bullet over hook on winch.
6. Shackle lifting eye and cable to rosette.
7. Crane off rosette and take to high bay in Operations building.
8. Copy cast data to tape or floppies.

top