Prepare all media in acid-washed vessels. Stock solutions to be mixed volumetrically in acid-washed glassware and filter sterilized with filters that have been pre-washed with 2 volumes of milli-Q water.  Don’t use metal spatulas on any of the reagents!  All chemicals to be purchased from Sigma, BioUltra quality.

  1. Artificial Sea Water (ASW) Preparation
  2. Nutrient Stocks
  3. Phytoplankton Growth Media Recipes
  4. Medium Chemistry
  5. Titrator Priming Protocol
  6. pH Probe Calibration Protocol

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ASW (Artificial Sea Water)

Mix in acid-washed carboy.

Base Salts Solution

  • Per 20L (dissolve sequentially):
Ingredient Amount
NaCl 568.22 g
KCl 15.78 g
CaCl2*2H2O 31.58 g
MgSO4*7H2O 144.28 g
MgCl2*6H2O 103.6 g
Milli-Q H2O 19.92 L
  • Tare a clean 10 mL volumetric flask on the analytical balance.
  • Carefully fill to exactly the mark with base salts solution.
  • Weigh and record density on carboy in grams per mL.

250X (~0.58125 M) NaHCO3

  • To 200 mL milli-Q H2O in an acid-clean container, add 17 g NaHCO3
  • Add approximately 30 mL milli-Q water to a beaker and weight it.
  • Add 100 uL NaHCO3 stock and weigh again.
  • Determine exact concentration by titration and dilute to 0.58125 mol/kg
  • Filter sterilize

ASW Medium Batch Preparation

  • Prepare ASW media 1 L at a time.
  • Autoclave 1 L of salts solution in an acid-clean polycarbonate bottle.
  • After cooling, add 4 mL 250X NaHCO3.
  • Bubble for at least 24 h to equilibrate carbonate system.
  • Add required nutrients for medium type.

Acid/Base Additions for Ocean Acidification Experiments

125X (~0.02 M) NaHCO3:

  • Add 8.6 mL 250X NaHCO3 stock to a 250 mL volumetric flask and bring to 250 mL with milli-Q water
  • Filter sterilize
  • Add 35 mL of ASW of known alkalinity to a tared beaker and record mass.
  • Add 300 uL of 125X NaHCO3 and record new mass.
  • Titrate and calculate specific concentration of 125X NaHCO3 solution in moles per kilogram.
  • Input this value in the OA_Calculator.xlsx spreadsheet in cell B4.
  • Tare a clean 10 mL volumetric flask on the analytical balance.
  • Carefully fill to exactly the mark with 125X NaHCO3 solution.
  • Weigh and record density in grams per mL in cell B5.

125 X (~0.02 M) HCl:

  • Add 50 mL 0.1N HCl (the titrant fluid) to a 250 mL volumetric flask and bring to 250 mL with milli-Q water
  • Filter sterilize
  • Add 35 mL of ASW of known alkalinity to a tared beaker and record mass.
  • Add 300 uL of 125X HCl and record new mass.
  • Titrate and calculate specific concentration of 125X HCl solution in moles per kilogram.
  • Input this value in the OA_Calculator.xlsx spreadsheet in cell B6.
  • Tare a clean 10 mL volumetric flask on the analytical balance.
  • Carefully fill to exactly the mark with 125X HCl solution.
  • Weigh and record density in grams per mL in cell B7.

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NUTRIENT STOCKS FOR PHYTOPLANKTON GROWTH MEDIA:

0.05 M NaH2PO4:         6 g/L (note this is for the salt with no waters)

0.8 M NH4Cl:         42.8 g/L

0.882 M NaNO3:         75 g/L

0.106 M Na2SiO3*9H2O:         30 g/L

Pro99, SN, and F/2 trace metals: as per Andersen 2005, pp 516, 517, and 507, respectively

F/2 Vitamins: as Andersen 2005, p 507

K2HPO4, Na2EDTA, and Na2CO3 as Andersen p 517
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PHYTOPLANKTON GROWTH MEDIA RECIPES

Add the following to 1 L of prepared ASW:

Chemical Pro99 PEv SN*
SEv F/2 F/50 (FEv)
NaNO3 1.76 mL 36.3 uL 1 mL 36.3 uL
NaH2PO4 1 mL 40 uL  — 40 uL 725 uL 40 uL
K2HPO4  —  — 1 mL  —  —  —
Na2EDTA  —  — 1 mL  —  —  —
Na2CO3  —  — 1 mL  —  —  —
Na2SiO3  — 1 mL 40 uL
NH4Cl 1 mL 40 uL  —
Pro99 TM 1 mL 40 uL  —
SN TM 1 mL 20 uL
L1 TM  — 1 mL 40 uL
F/2 vitamins 1 mL 20 uL 500 uL 20 uL

* SN is modified from Andersen 2005. Nitrate concentration has been lowered to bring them to Redfield ratio with phosphorus concentration.

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MEDIUM CHEMISTRY

Preparing for measurements

  1. Turn on the titrator. Open the bottle of titrant and insert the intake tube. Screw on the intake tube housing.
  2. Prime titrator dispenser (See TITRATOR PRIMING PROTOCOL below).
  3. Make sure pH probe hasn’t been sitting out. If it has, condition it in 3M KCl for 20 minutes.
  4. Check to make sure the probe fluid isn’t low. If it is, replenish it.
  5. Open the file “AT_Calculator_2013.xlsx” in our cloud folder. If it has been more than 14 days since the pH probe was calibrated (see cell I15), then run the PROBE CALIBRATION PROTOCOL
  6. ALWAYS, prior to placing pH probe in next solution, spray clean with milli-Q water and blot (not wipe) w. Kimwipe.
  7. Plug in the aquarium pump. Leave the valve connecting to the bubbling pipet closed but open one of the other valves (to keep pressure from building up).
  8. Upstairs, turn on the plate reader. Place a falcon tube of m-cresol purple onto syringe dispenser 1. Under Instrument Control, click Prime and dispense 1 mL from dispenser 1 into the white priming tray.

Measuring salinity and density

  1. Start with a clean 30 mL beaker containing a stir bar. Place it on the scale, record the tare weight, and zero out the reading.
  2. Using a sterile disposable 25 mL serological pipet, collect 30 mL of medium and place it carefully (no splashing) into the beaker.
  3. Record the mass in your notebook. Divide it by 30 mL to get the density of the medium.
  4. Using a disposable 1 mL serological pipet, collect 1 mL of medium and dispense 0.2 mL each into 4 wells of a dust-free flat-bottom transparent 96 well plate. Repeat for another 4 wells (filling an entire column). Set aside.
  5. Put the beaker on a stir plate and set to gentle-medium stirring (no splashing).
  6. Turn on the salinity pen and place it in the beaker. You should be able to let go of it and let it sit there. Wait for the number to stop changing and record the value in your lab notebook as the salinity Sal of the medium. Type this value into cell E8 of the AT_Calculator spreadsheet
  7. Use a thermometer to determine the ambient room temperature. Record this value in cell E11 of the AT_Calculator spreadsheet.

Measuring alkalinity

  1. Re-weigh the beaker. Record the mass as the “alkalinity mass” and type it in cell E6 of the AT_Calculator spreadsheet.
  2. Place sample back on stir plate. Blot pH probe dry and then lower into sample. Make sure pH probe, dispensing tube, and aquarium pipette are all in the sample but not so far as to hit the stir bar. If you need to add volume, use milli-Q water.
  3. Turn on stir bar at gentle-medium velocity (until you just see a “whirlpool” form at the surface of the medium). Position beaker to make stir bar spin properly without hitting the sides of the beaker too much.
  4. On titrator main menu, touch Methods, then scroll down and touch GT Alkalinity.
  5. Touch Start. Enter 50 in the Sample size box, then touch Start. Wait for at least 30 seconds after placing the probe in the sample to touch OK after you get to the screen that says ALKALINITY: Add sample 1/1.
  6. The machine will then begin titrating. Make sure the pH value is going down; if it isn’t, stop and prime the burette again. You’ll need to start over with a new sample unfortunately.
  7. Once the measured pH drops below 4, the titrator goes into a 6-minute waiting mode. During this time, you must bubble the sample vigorously with air using the aquarium pump. Close the vent valve that you left open in step 1, and then open the valve connected to the bubbling pipet until vigorous bubbling is observed, but be careful not to bubble so hard that sample is splashing out of the beaker.
  8. When the Time remaining countdown reaches zero, close the pump valve and open the vent valve again.
  9. The machine will now finish the titration down to pH 2.5. Once it has finished, turn off the stir plate, then touch OK twice, which returns you the main menu.
  10. Input the temperature of the titration into cell E11 on the spreadsheet.
  11. Touch Results, then All results, then Samples, then 1, then Data, then Measured values.
  12. Touch Titration (EP)[1], then scroll down to the bottom. Input the final value in the column Volume [mL] in cell E7 of the Calc_Prep
  13. Touch Back, then Titration (EQP)[2].
  14. Starting at cell A7 and going down, type in the values in the Value [mV] column. The spreadsheet will calculate the pH value corresponding to these mV values in column D; you only have to type values in until you get below pH 3.
  15. Copy and paste the values in columns B and C ranging from pH <= 3.5 to pH >= 3 (calculated pH values shown in column D) into the spreadsheet SOP_3b starting at cell C23. Note: you’ll need to use “paste values” to do this.
  16. Type in the value in column B corresponding to the lowest pH measurement that is > pH 3 into cell E17.
  17. Click on the Data tab, then click Solver, then Solve, then
  18. Record the value in cell M17 as your alkalinity Alk in mol/kg.
  19. Pour out the sample, clean the beaker and probe, and proceed to your next sample.

Measuring pH

  1. Take the 96-well plate upstairs. Place it into the plate reader and open protocol “MCP_pH_automatic”.
  2. Select the wells containing your samples and run the protocol.
  3. Afterwards the data will be exported to Excel. Copy and paste values into columns J through R of a Spec_PH calculations spreadsheet.
  4. Fill in the ambient room temperature Temp in columns D and E, the medium salinity in column F, and 0.02 (the amount of dye added per injection) in column G.
  5. Fill down the values in columns S through AC.
  6. Calculate the average of the values in column H for each measurement of a given sample, ranging from row x to row y (=average(Hx:Hy)) and the 95% confidence interval (=stdev(Hx:Hy)*tinv(0.05,7)/sqrt(8)). Record these in your notebook as the pH of the sample.

Calculations

  1. Open R on the computer desktop. Type library(seacarb).
  2. Use seacarb in R to calculate how much NaHCO3/HCl or NaOH you need to add to achieve year 2100 or year 2015 conditions, respectively, given the measured alkalinity values of the unamended medium. For year 2100 type y2100<-oa(flag=8, var1= pH, var2= Alk, pCO2f=800, S=Sal, T=Temp); For year 2015 type y2015<-oa(flag=8, var1= 380, var2= Alk, pCO2f=400, S=Sal, T=Temp)
  3. Open the OA_Calculator Input medium density in cell B2, 12.3 into cell B3, and 0.2 into cell B4.
  4. In R, type in y2100$perturbation[5,5]. Input the value in cell B5.
  5. Type in y2015$perturbation[3,3]. Input the value in cell B6.
  6. Write down how much NaHCO3 (cell C11), HCl (cell C12), and H2O (cell C10) in mL is needed per 13 mL CO2+ culture in your lab notebook. Also write down how much H2O (B10) and NaOH (B13) is required for each CO2- culture.
  7. On a piece of label tape, write the medium type, batch #, your initials, the data, density, salinity, and alkalinity, and the required additions for CO2+ and CO2-. Stick it on the media bottle.

Testing amendments

  1. Dispense 12.3 mL of media into two sterile test tubes. They don’t have to be acid-washed.
  2. Mark one tube + and the other -. Amend them accordingly.
  3. Mix by gently vortexing.
  4. Dispense into a 96-well plate to measure pH as described above. Record final pH values in your notebook. They should be between 8.02-8.12 for CO2- and 7.75-7.85 for CO2+.

TITRATOR PRIMING PROTOCOL

  1. Turn titrator on.
  2. Open the bottle of acid titrant and place the siphon tube in. Screw the plastic siphon-holder thing onto the acid bottle.
  3. Make sure there’s a beaker under the dispensing tube to catch the dispensed acid.
  4. Touch Manual, then Burette, then Rinse, then Start. Set Cycles to 3 if it’s not already.
  5. Make sure fluid and not air bubbles moves through the dispensing tube.
  6. If you still see air bubbles on the 3rd rinse, repeat the process until you don’t see any more air bubbles.

PROBE CALIBRATION PROTOCOL

 

  1. Make sure pH probe is full of probe solution. If not, add to the port just below the serial number until it’s full.
  2. Make sure aquarium pump pipet is taped to probe arm and temperature probe is through the appropriate hole in the middle of probe arm. Try to arrange it so that the ends of the pH probe, temperature probe, dispensing tube, and aquarium pump pipet are all about the same level.
  3. Check to see when last calibration was, recorded on the Excel spreadsheet in cell I15. If it’s been less than 1 week since last measurement and you didn’t have to add fluid, you don’t have to calibrate; proceed to step 12.
  4. Rinse a 50 mL beaker with star bar with milli-Q water twice, then wipe clean with a clean Kim-wipe.
  5. Add about 40 mL of pH 7 buffer (yellow) to the beaker. Place on stir plate and turn on stirrer to about setting 8. Position beaker so that stir bar is spinning in the right place.
  6. Blot pH probe and other probes/tubes dry and lower into buffer.
  7. On titrator main menu, touch Methods, then scroll down and touch DG115SC, then Start.
  8. A screen saying “DG1155SC: Add sample 1/3.” pops up. Wait at least 30 seconds after putting the probe in the buffer and then click OK.
  9. Turn off stir plate. Lift probe, spray down with milli-Q, then empty the buffer, spray off the beaker, and wipe clean with a fresh Kim-Wipe. Also blot-dry the probe assemblage.
  10. Add pH 10 buffer (blue) to beaker and repeat steps h and i.
  11. Add pH 4 buffer (pink) to beaker and repeat steps h and i.
  12. Turn off stir plate, empty beaker and spray everything down with milli-Q then blot dry with a fresh Kimwipe.
  13. On the titrator screen, touch OK, then OK again, which takes you back to the main menu. Then touch Results, then All results, then Samples.
  14. Touch 1, then Data, then Raw results. Input the value by ESTMe in cell H20 of the Excel spreadsheet. Touch Back 3 times to get back to the list of samples.
  15. Repeat with sample 2, inputting the ESTMe value in cell H21.
  16. Repeat with sample 3, inputting the ESTMe value in cell H19.
  17. Type today’s date into Cell I15. Click the File tab and then Save. You have completed calibrating the probe.