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Ecotoxicity assay preparation.
Setting up the algae ecotoxicity assay
Setting up the algae ecotoxicity assay
Materials needed: algae stock culture, 50 mL beaker, culturing media, atrazine solution for preparing the algae ecotoxicity assay, 3x 25 mL buret, ring stand with buret clamp, 4 small beakers (10 - 50 mL), well plate, 3 mL plastic pipets
Materials needed: algae stock culture, 50 mL beaker, culturing media, atrazine solution for preparing the algae ecotoxicity assay, 3x 25 mL buret, ring stand with buret clamp, 4 small beakers (10 - 50 mL), well plate, 3 mL plastic pipets
1. Gently swirl the algae stock bottle to re-suspend the algae, then pour 30 mL of stock algae culture into a 50 mL beaker. Label the beaker as algae solution.
2. You will be using the three burets to dispense precise volumes of the algae stock culture, the culturing media, and the atrazine solution (labelled For Ecotoxicity Assay Only), respectively. Start by rinsing each buret with a small amount of the relevant solution using a plastic Pasteur pipet. Then transfer the relevant solution to each buret, making sure that the tap is closed.
3. Prepare the solutions from the table below in the four small beakers, making sure to dispense the algae stock solution first (the cells will begin to settle if the solution has been sitting for more than 5 minutes in the buret). Don't forget to label the beakers!
4. Dispense approximately 2.8 mL of the control and solutions 1-3 into the wells as shown below using a 3 mL plastic pipet. Use a new pipet for each solution. Use enough solution so that the wells are full but the solution is not spilling out of each well. Be sure to swirl your solution before adding it to the well plates so that the algae do not settle to the bottom of your beaker.
5. Wash the four beakers, and then use them to make solutions 4-7 according to the following table:
6. Dispense approximately 2.8 mL of solutions 4-7 into the wells as shown above using a 3 mL plastic pipet. Use a new pipet for each solution. Use enough solution so that the wells are full but the solution is not spilling out of each well. Be sure to swirl your solution before adding it to the well plates so that the algae do not settle to the bottom of your beaker. Make sure to only use the 8 wells in the center of the well plate.
7. Cover the well plate with a plastic lid. Write your name, your partner's name, and section on a piece of tape and place it on the edge of the well plate as shown in the figure above. Do not tape all four sides of your plate with tape, or your algae will suffocate!
The algae will be allowed to grow for 72 hours. The well plates will be placed under a fluorescent lamp. The well plates will then be frozen and you will get them back the following week. You will then take fluorescence measurements to determine the toxicity of your assigned pesticide.
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