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Sampling Protocols

Plankton Tow | Measuring Salinity | Measuring Temperature | Slide Preparation

Microscope Techniques | Data Sheet Completion | Reporting a Bloom | Sample Preservation

Equipment Purchase

Plankton Tow Protocol

PDF Plankton Tow Protocol

• Perform tow from floating dock, pier, bridge, edge of lake, etc. at your desired sampling location.
• Sample at the same location every week (or biweekly) to monitor changes at that particular site over time.
• Pull plankton net for THREE MINUTES with 20 micron mesh plankton net.
• Take sample to laboratory for identification.
• Store at room temperature (NOT in car or refrigerator) with cap loosened to allow some air flow into sample bottle.

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Measuring Salinity Protocol

PDF Measuring Salinity Protocol

The refractometer is used to measure the amount of dissolved salts in ocean water, or salinity. To ensure accuracy, volunteers will need to perform periodic calibration of their refractometers (at least once per month).

To Calibrate Your Refractometer:

• DO NOT PERFORM CALIBRATION IN THE FIELD!
• Use distilled water to rinse the cover and prism 3 times (remove all salt crystals); wipe clean.
• Fill prism with distilled water and close the cover.
• Look through the eyepiece, the sample should read zero (0) parts per thousand (ppt).
• If the reading is different, remove rubber cap on top of refractometer.
• Turn the calibration screw with the provided screwdriver while looking through the eyepiece until the boundary line falls on "0".

General Use:

• If you are using the same pipette to read salinity for different samples, you must rinse the pipette with the new sample 3 times to remove your previous sample.
• Wipe the prism clean with a lens cloth.
• Hold the refractometer at an angle, so the face of the prism is horizontal.
• Open the cover; fill the prism with the sample solution.
• Close the cover; look through the cover and make sure the sample solution covers the entire prism. If there are bubbles or gaps on the prism, you will not get an accurate reading.
• Hold the refractometer up to the brightest light.
• Look through the eyepiece; if your scale is not in focus, adjust it by turning the focusing ring.
• Read the right side of the scale where the blue and white boundaries meet.
• Record your results in parts per thousand (ppt).
• When each measurement is complete, the sample must be cleaned from the prism using fresh water and lens paper or lens cloth.

Precautions:

• Do not drop or handle roughly.
• Do not hold the refractometer under the faucet or splash with water.
• Do not apply rough or abrasive materials to the prism.

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Measuring Temperature Protocol

PDF Measuring Temperature Protocol

General Information: Your glass thermometer is filled with alcohol or kerosene (usually dyed red) rather than mercury, which can be highly toxic to the environment if the thermometer breaks. The thermometer is just as accurate as a mercury thermometer but is more sensitive to temperature changes. IT IS VERY IMPORTANT TO TAKE YOUR TEMPERATURE READING IMMEDIATELY.

• Immerse the thermometer in the water for at least 60 seconds in the same area of your sample collection.
• If you are collecting a tow 3 feet below the water's surface, make sure you are consistent and get the temperature there as well.
• Do not take a temperature reading from your plankton sample!
• Once you remove the thermometer from the water, the temperature will begin to change as soon as it is collected, so you will need to read the temperature promptly.
• Hold the thermometer by the top (not the base) and bring the thermometer to eye level. This will give you the most accurate reading possible. If you leave the thermometer in the water and try to read, you could get a parallax error (the scale will appear different at an angle).
• Try to get a reading to the nearest 0.5 degree.
• After you have recorded the temperature reading in Celsius, rinse the thermometer with fresh water.

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Slide Preparation Protocol

PDF Slide Preparation Protocol

• Tighten cap on sample bottle and mix slowly by turning bottle upside down and back.
• Squeeze pipette and take sample from the bottom of sample bottle.
• Place two to three drops from pipette onto the middle of the slide.
• Lay cover slip at an angle to avoid air bubbles.
• Helpful Tip: Slide will dry out and form salt crystals from the heat of the light so you may need to clean slide and prepare a new one for further identification.

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Microscope Techniques

PDF Microscope Techniques

• Use 10x /20x objective lens to start; can go up to 100x if needed.
• Make sure to dim the amount of light if needed.
• Make at least two slides of the same sample to ensure that you are observing everything in the sample. If you have a class student can do individual/group slides and combine findings (record the average of species found on the data sheet that you submit).
• Move through the slide using the "lawnmower method" to avoid counting the same species twice. The "lawnmower method": start at the corner of the slide and systematically move up and down, left and right, to cover the entire slide.

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Data Sheet Completion Protocol

PDF Data Sheet Completion Protocol

• Fill out the date and time that sample was taken from the site.
• Record water temperature (must be measured at sampling site) and salinity (can record this in classroom). Instructions for salinity and temperature are on another SEMPN document.
• Indicate location and area of sampling site in case NOAA scientists need to monitor your site for further testing. Please include the name of the group at the bottom of the data sheet.
• While identifying what is in your sample, place a check next to that species on the species list for every time you see it. (Teachers: Remember to use the average of what each group of students observed to come up with the total class abundance ratio.) Record the total number of individual plankton species on your data sheets.
• To determine abundance ratios: you will use a percentage of area covered by a group of Genus or species of plankton.
  • The area under your microscope cover slip is equal 100%.
  • If you divide the area of your microscope slide into 2 equal sections, then the area will be 50%. Divided into 4 sections, each area is equal to 25%, and so on, so forth.
  • Abundance Ratios are as follows:
    • Zero percent area covered equals NONE.
    • Equal to or less than 10% area covered is labeled PRESENT.
    • Greater than 10% or less than or equal to 40% is labeled COMMON.
    • Greater than 40% or less than or equal to 80% is labeled ABUNDANT.
    • Greater than 80% of area covered is labeled BLOOM.
  • Imagine what the total area of all the individuals of a Genus or species will take up. For example, 1 Nitzschia will take up less than 1% but more than 0% of the total area of the cover slip and therefore is labeled PRESENT. By the same token, because 100 Nitzschia are so small, the area that they take up is still less than 10% and will still get the label PRESENT.
  • Rhizosolenia is a much larger diatom than Nitzschia and 60 individuals of that Genus will cover an area greater than 10% and will get a label of COMMON.
  • If the number of species in your slide covers more than 50%, then it is labeled ABUNDANT.
  • If the number of species in your slide covers more than 80%, then it is a bloom and you need to contact PMN staff immediately to determine if further samples and/or research are needed.

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Reporting a Bloom

PDF Reporting a Bloom Protocol

If you and your group think that your monitoring site may have a phytoplankton bloom, please contact the Southeast Phytoplankton Monitoring Network (PMN) staff immediately via phone and/or email. Make as many observations as you can about the site: weather conditions, water coloration, tides, local flora and fauna, and/or any organism mortality. Record these observations on your data sheet. If you have a camera, we would love pictures of your site during a phytoplankton bloom. Please do not handle/touch any dead organisms found at your site.

Depending on the phytoplankton species, we will let you know if we need a live or preserved sample shipped back to our lab. Make sure you label your samples with the location, date, and time of collection. Please keep all your receipts pertaining to the purchase of shipping materials and fees; we will reimburse you for all your expenses. Contact us before shipping; we will provide you with mailing information so that the shipment does not cost you any money.

Preserved Samples: When preserving a sample, please do not use the plankton bottles that were provided for you to avoid staining and contamination of future samples. Transfer the sample from the plankton bottle into a clean plastic or glass jar, vial, or bottle. Next, add Lugol's solution to the sample for a 1% - 2% dilution (for example, add 1 milliliter of Lugol's to 50 milliliters of the plankton sample). Mix gently once or twice; your preserved sample should resemble tea. Place the preserved sample in a zip lock bag, pack the sample into a padded envelop or box and ship it to us via Federal Express Priority.

Live Sample: If we need a live sample, it is importantly it is shipped immediately after collection (most mailings will take 24 hours, which is the average time a plankton sample with survive outside their normal environment). To collect a live sample, just take a direct water sample with a clean container with a cap. You do not need to perform a plankton tow. If we think the sample has the potential to be toxic, we may request a 2-liter sample. A live sample needs to be shipped in a small cooler with packing ice (blue ice or frozen water/soda bottles). Place the packing ice on the bottom of the cooler. Put a small buffer or space between the ice and the sample (bubble wrap, peanuts, or newspaper). Ship the sample back to us via Federal Express Priority.

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Sample Preservation Protocol

PDF Sample Preservation Protocol

Add about 0.5mL of Lugol's solution to a 25mL sample for preservation. You may have to occasionally add additional Lugol's solution over time to maintain the stain on your phytoplankton. The goal is to stain your sample with Lugol's solution so that it is tea-colored.

Lugol's Solution (MSDS pdf)

• 225mL distilled water
• 25g KI (potassium iodide)
• 0.5g I₂ (iodine)
• 25mL glacial acetic acid

Directions: Dissolve in 225mL of distilled water 25g KI (potassium iodide), then 0.5g I₂ (iodine), and finally add 25mL glacial acetic acid.

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Equipment Purchase

PDF Equipment Purchase

• Plankton Net
  • Sea-Gear Corporation: 700-B1 South John Rhodes Blvd., Melbourne, FL 32904; Phone: 321-728-9116; Fax: 321-722-0351
  • Student Plankton Net, 20cm diameter. 3:1 #20 micron mesh, with cod end collar, hose clamp and 1 each 4 and 8 oz. cod end jar
• Extra bottles
  • Fisher Scientific, Inc.: Phone: 1-800-955-1177
  • Fisherbrand HDPE round bottles, 38mm screw cap, 4oz capacity
• Salt Refractometer
  • Forestry Suppliers, Inc.: 205 West Rankin Street, P.O. Box 8397, Jackson, MS 39284-8397; Phone: 1-800-647-5368; Fax: 800-813-2704
• Thermometers
  • Forestry Suppliers, Inc.: 205 West Rankin Street, P.O. Box 8397, Jackson, MS 39284-8397; Phone: 1-800-647-5368; Fax: 800-813-2704
  • Armored Thermometers, (non-mercury filled), -30 to 50 degrees Celsius
• Digital Microscope
  • Olympus MIC-D (This item has been discontinued)
  • Great for taking pictures and videos of plankton. Need a computer to run the microscope; can project images onto LCD projector.

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