Point Whitney Hatchery and Lab Information

Last Revised: 20181030 SJ Gurr

Lab Need to Know:

• Dry bench (1st bench, closest to light set switches) should stay dry, do not move wet/liquid materials since electronics including laptops are kept here.

• Record ALL data and notes in the designated lab notebook. Write clear enough for others to read the notes and understand the thought process. Label each page with the page number in the top right-hand corner of the notebook page. Write your initials and the date next to each entry or form of data you record.

Daily Tasks:

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First thing in the morning:

1. Check the CO₂ tank to make sure the black tick mark is just below 1000 PSI. The farthest scale to the left (directly horizontal) is reading the levels of the CO₂ tank. The scales above each CO₂ line is reading the level of CO₂ in each line directly below it.

   • If the tank is low on CO₂, contact Hollie @ hputnam.uri.edu asap so more can be ordered. It can take up to 1 week to arrive.

2. Check Apex Fusionfor current and overnight tank conditions. Make sure these data match with what the desired conditions/fluctuations are set to for each tank. Note: the Apex Fusion app on you phone is very useful and will send you text alerts at “alarm” conditions (e.g. C02 tan runs dry - apex sends text alert when pH rises above target threshold)

• **Tris_Calibration**
  • With a falcon tube 1/2 full of tris solution (1-3 days old), measure pH (mV) and temperature (°C) across a range below your lowest temperature (in your experimental tanks) and above your highest at ~0.4°C increments. (Hint: warm the tube with your hand make sure to lightly rotate/mix while recording data)
  • Save a new CSV file named today’s date (yyyymmdd.csv) and save to the “pH_Calibration_Files” folder found in Geoduck_Conditioning/RAnalysis/Data/pH_Calibration_Files.
  • Run the R Script titled “pH_Tris_Curve.R” on RStudio. You will need to change the name of your file to call. Check the R^2 value and do not move on until the R^2 is equal to or above 0.98. This is often due to measurement error (e.g. rapid temp increase), but if this reoccurs you may need to replace tris buffer.
  • NOTE: tris calibration must be completed everyday. It is most convenient to do this in the algae room where small batch cultures are stored. There is a small refridgerator here to reuse an ice pack and the cold room prevents large temperature spikes. Refill falcon tube with tris buffer every 3 days
• **Measure** pH, temperature, and salinity of each tank in use.
  • Take these measurements: a.) once a day for long-term experiments b.) 2-3 times daily for short-term experiments
  • If measuring in a larval tank use the falcon tube with 153µm mesh, the plastic round bin and turkey baster to collect water sample so as not to interfere with larvae or carry them over on the probe.
  • Remember to rinse falcon tube, plastic round bin, and turkey baster in between measurements of tanks with larvae.
  • Update “Daily_Temp_pH_Sal.csv”.
• Take **water_samples** immediately as/after probe measurements are taken from each tank.
  • Take water samples 1 time daily unless the experiment is short (1-2 days) and then take multiple water samples each day along with multiple probe measurements.
  • If measuring TA same day, use a filter to take water samples to avoid confounds due to algae cells (Note: iso, pavlova, tet genera are ~5-12um)
• Measure and calculate **flow_rates** for all tanks.
  • Update “Flow_Rates.csv”.
  • Flow rate (if no flow meter) can be measured with stopwatch and graduated cylinder
• Run Titration_Alkalinity Water Samples.
  • Plan time accordingly, 9 samples will take 1.5 number of hours with BioMin_URI method.
  • NOTE: if you are completing TA everyday, it is most effective to complete the daily set-up routine first thing in the morning (titrant purge for air bubbles, pH calibration, and CRMs).

Daily Data Logging and Backup

• Take detailed notes every day in the notebook. Make sure to include introduction and summary sentences, not just list of tanks or samples.
• Upload to the shared google drive the photos of the
  • pH calibration log, daily measurements log,
  • tris calibration log,
  • wet lab notebook pages, and
  • TA notebook pages.
  • Make sure the corner of each page is numbered and initials are written next to entries.
  • Name each image as the type of calibration followed by the page number. (e.g.: “Tris_Calibration_page3”).

Every Couple Days:

• Re_calibrate pH and conductivity probes It is best to do this at least every 3 days. Both meter probes (used for daily discrete measurements) and APEX probes must be calibrated every three days with corresponding solutions (Apex uses solutions from Neptune Systems - handheld Mettler Toledo (pH) and Thermoscience (conductivity) probes use buffers from TA and probe-specific solutions, respectively).
  • Update your notebook with the date and time of calibration.

As Needed, but at least every 3 days:

• Replace NBS pH calibration solutions. Write the pH value, date opened, and your initials on the side of each tube.
• Replace Tris calibration solution. Write “Tris Buffer”, date opened, batch number, and initials on the side of the tube.

Equipment Information

• Update the google spreadsheet of all equipment used. Each entry needs the accuracy and precision of the tool, model and/or serial numbers, and name of the instrument. Include as many details as possible. This information is essential for methods of manuscripts.

Lab Area Information:

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• DI water is located in the dry lab - keep a bottle of DI and a package of kimwipes with your daily chemistry supplies (ph/cond meter with probes, falcon tubes with calibration and tris solutions).

Protocols:

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Tris_Calibration

The goal of the Tris calibration is to measure pH (mV) across a range of temperatures lower and higher than currently experienced in the tanks. The temperature values and corresponding pH values must have a linear relationship and an R^2 value equal to or above 0.98 in order to calculate the pH values for the daily measurements from the calibration curve.

Measured with A325 Thermo meter and Mettler Toledo probe 51343101 and Control Company Certified traceable Temperature probe

1. Fill a small clean container (preferably falcon tube) with certified Tris standard solution from the Dickson Lab.
   • After opening a new bottle, write “Opened”, the date yyyymmdd, your initials and Putnam Lab - e.g., “HP - Putnam Lab”.
2. Set the Tris solution container on ice to cool the solution down to desired temperature.
   • Note: This value will change based on the range of temperature you expect to be measuring. This will change through time in an experiment where temperature is not held constant* -The range must be 1°C below the observed minimum during measurements and 1°C above the observed maximum. A typical range throughout early summer at Point Whitney might be 14°C to 25°C as the temperature of the tanks increases during the hotter months such as August and September**
3. With the certified temperature probe, measure the Tris solution temperature. Swirl the temperature probe in the solution as it measures to ensure the solution is well mixed.
4. With the Thermo A325 meter with the Mettler Toledo pH probe attached, click “Measure” on the meter when the Tris solution is nearing the first temperature (e.g., 25°C). Swirl the pH probe in the solution as it measures to ensure the solution is well mixed.
5. Wait for the temperature and mV value to stabilize and record values.
6. Warm the Tris Buffer by gently holding the outside of the tube/container. But be careful to not hold the container for too long or too aggressively, otherwise the temperature will spike without the chance to record values across the temperature range.
7. Make temperature and pH measurements across the desired range, with increments of about 0.4°C. Read the temperature value as soon as the pH meter states “ready” instead of “stabilizing”.
8. Save measured values a new CSV file named today’s date (yyyymmdd.csv) and save to “pH_Calibration_Files” folder. Column 1: mVTris (pH values); Column 2: TTris (temperature values).
9. Run the R Script titled “pH_Tris_Curve.R” on RStudio. Check the R^2 value and do not move on until the R^2 is equal to or above 0.98. -Remember: to run the R script, you need to:## -Make sure the Tris file was saved as a .csv ** -Make sure that the data in the excel sheet is correct and the signs (+/-) are also correct. -Check that the directory is set to where the Tris file (yyyymmdd.csv) is.** -Change the date in the R code to the date/name (since the name of the file is just the date) of the Tris file you are trying to run

Back to Daily_Tasks.

Measure pH, temperature, and salinity

This will be done with the new pH meter (A325), conductivity probe and the Mettler pH probe (Mettler-Toledo 51343101).

1. Temperature: Using the certified temperature probe, swirl the metal portion of the probe only in the water, but do not let the plastic light grey section of the probe enter the water.
2. pH and Salinity: Using the A325 meter and Mettler-Toledo probe, click “Channel” until the screen splits to display both the pH (mV) and salinity (psu).
   • If the pH or salinity is displayed in different used than desired, click channel until either pH or salinity is displayed. Click “mode” to change the units the probe measures in.
   • pH probe needs to be swirled, but the conductivity (salinity) probe does not. The conductivity probe has cut-outs on either side of the sensor, these need to be fully submerged.
   • ** Temperature probe should not be in contact with anything but water **
3. Update “Daily_Temp_pH_Sal.csv” file in Geoduck_Conditioning/RAnalysis/Data/Daily_Temp_pH_Sal.csv 4. Back to Daily_Tasks.

Collecting Water Samples for Total Alkalinity

These will be used for Titration Alkalinity titrations. To ensure the sample only contains water from that tank or conical, each bottle will be rinsed three times before collecting the final sample.

1. Label small glass bottles with the tank # and treatment type (paper tape).
2. Fill the bottle with filtered tank/conical water about 1/4 full, close with the cap, and shake the bottle. Pour out the water down the outflow drain. Repeat twice more to fully rinse the bottle.
3. Fill the bottle completely full, avoiding any air pockets throughout the bottle or at the top before closing.
4. Repeat for each tank. To collect a junk sample to use prior to CRMs, sample a second time from a random tank and label the bottle with that tank number in order to keep track of the sample and see how much variability there is between using the first slot of the rondo and after several other samples have been run

Since no Mercuric Chloride is being deposited into the samples and the bottles are light-penetrated, water samples MUST be run as soon as possible in order to be as accurate as possible. When light can reach the water sample and no mercuric chloride is used to kill any bacteria or living organisms in the sample, the potential for the water chemistry to change NOT in response to the experiment in much higher.

Back to Daily_Tasks.

flow_rates
Different flow rates between tanks can greatly affect the tank’s water chemistry due to differences in turnover rate.

1. Flow rate will be determined by volume (mL) divided by time (seconds).
2. Measure the amount of water accumulated in a graduated cylinder over 15 seconds.
3. Record volume, time, and the calculated flow rate (volume in milliliters divided by time in seconds). Update “Flow_Rates.csv” in BioMin_HIS/RAnalysis/Data and email to Hollie at hputnam@uri.edu.
4. Calculate the flow rates for all three header tanks and tanks 10-15. Remember to do the drippers as well since these can get clogged up. If a dripper is slow, replace it with a new one. If it continues to be a problem, after the round bins containing the Montipora c. larvae, put a stopper to the dripper line of all tanks and only have one line per tank with the black spigots running.

** Notes: Flow rates may be measured twice a week during heavy work load weeks **

Titration_Alkalinity See full titration protocol

Calibrate Apex pH probes

Header Tanks:

Never do this without consulting Hollie first

1. Log onto the Apex Fusion app.2.
2. Click on the dashboard icon. Once at the dashboard, EB8_1_L, EB8_1_XL, and EB8_1_A switches will show above each treatment’s 3 measurement (pH, temperature, and salinity) graphs (in orange with white data points).
3. TURN OFF THE TOGGLE TO STOP THE CO₂ FROM BEING INJECTED
4. Click on the Settings icon (grey tire-like icon) in the top right-hand corner of the desired probe. Ex: If calibration is needed for XL Header’s pH probe - “PH_XL” will have a settings icon directly above and to the right of the title.
5. In the settings of the desired probe, two boxes will appear: “Summary” and “Configuration”. Under “Configuration”, click the orange “Automatic Probe Calibration” option.
   • If a graph of the tank’s fluctuation appears, the general probe information was clicked instead of the settings icon. Click the back arrow to return to dashboard and try again.
6. The Apex app will be a guide through the calibration, follow the steps indicated.
   • Press “Calibrate”.
   • Select the lowest solution being used - “pH 4.0”. Click “Next”.
   • Place the probe in the pH solution 4.0, swirl to measure, and wait for all readings (acceptable range, reading has settled, and time left) to stabilize. Click “Next” when stabilized.
   • Select the medium solution being used - “pH 7.0”. Click “Next”.
   • Place the probe in the pH solution 7.0,swirl to measure, and wait for all readings (acceptable range, reading has settled, and time left) to stabilize. Click “Next” when stabilized.
   • Select the medium solution being used - “pH 10.0”. Click “Next”.
     1. Place the probe in the pH solution 10.0,swirl to measure, and wait for all readings (acceptable range, reading has settled, and time left) to stabilize. Click “Next” when stabilized.
     2. Click “Finish” once done.
7. Scroll to top of the screen and click the cloud icon with an arrow pointing upwards. This will push the data back to the Apex “brain”. The calibration will not be saved without clicking this icon.
8. TURN THE TOGGLE TO AUTO TO START THE CO₂ INJECTION AGAIN

Back to Daily_Tasks.

Care for probes and meters

Temperature

1. Rinse only the metal portion with DI water and dry the metal probe.
2. Wipe off the meter box with a rag or kimwipe with 80% ethanol to remove any water or salt.
3. Store the probe dry and in a safe place

pH

1. Rinse the pH probe with DI water.
2. Store the pH probe in the pH electrode storage solution with the glass bulb covered in solution
3. Wipe off the meter box with a rag or kimwipe with 80% ethanol to remove any water or salt.
4. Store the meter and probe dry and in a safe place

Salinity

1. Rinse the salinity probe with DI water.
2. Store the salinity probe in DI water with the conductivity cell covered in DI water
3. Wipe off the meter box with a rag or kimwipe with 80% ethanol to remove any water or salt.
4. Store the meter and probe dry and in a safe place