In this video, we will prepare the rhodamine dye standards used for the calibration of the EXO Total Algae sensor for the chlorophyll, phycocyanin, and phycoerythrin channels.
While the dye is not toxic, it can stain easily. So it’s a good idea to wear your safety glasses, your gloves, and a lab coat. You will need three one-liter volumetric flasks, a high-accuracy pipette that can measure 5 mL, a high-accuracy pipette that can measure 200 µL, three labeled amber bottles for storage, deionized water, a transfer pipet, and rhodamine dye. Make sure you have purchased Rhodamine WT as a 2.5% solution. This is the stock rhodamine dye. You can purchase this from Kingscote Chemicals, which is referenced in the EXO manual.
First, prepare a working solution of the Rhodamine dye. This will be a 125 mg/L dilution that you will use to prepare further dilutions. Make sure you use good lab techniques, especially when pipetting, to minimize errors in transferring liquids. Using a clean one-liter volumetric flask, fill it to about 90 to 95% of the volumetric mark with deionized water. This does not have to be exact. Using a micropipette, or other high-accuracy pipette, transfer 5.00 mL of the 2.5% Rhodamine dye into the one-liter flask. Deposit the dye directly in the water and rinse the pipet tip to get all of the dye in the solution.
Now fill the flask up to the volumetric mark to make one liter of solution. Use a transfer pipet to get an accurate measurement. Cap the flask and invert three times to mix. This is now your working dye solution of 125 mg/L. Store this solution in an amber bottle in the refrigerator to minimize degradation. You can come back to this working solution to make your standard dilutions for up to two years.
Now, we will prepare the 625 µg/L concentration that we’ll use for the calibration of both the Chlorophyll and Phycocyanin channels of the Total Algae sensor. First, we will take a clean one-liter volumetric flask and fill it to about 90 to 95% of the volumetric mark with DI water. Again, doesn’t have to be exact. Now, use a micropipette to transfer 5.00 mL of the 125 mg/L solution to the flask. And rinse the pipette tip to make sure all of the dye transfers.
Then, fill the flask up to the volumetric mark to make one liter of 625 µg/L solution. And use a pipet to get an accurate volume. Cap the flask and invert three times to mix. I’ll transfer this solution to a labeled bottle. And now we have our 625 µg/L standards to use for calibration of the Total Algae sensor for both Chlorophyll and Phycocyanin. Now we will make the 25 µg/L standards used to calibrate the Phycoerythrin channel of a Total Algae – Phycoerythrin sensor.
We will take a clean one-liter volumetric flask and fill it to about 90 to 95% of the volumetric mark with DI water. Then we use the micropipette to transfer 200 µL of our 125 mg/L working solution. And rinse the pipette tip to get all of the dye in the solution. Finally, we fill the flask to the one-liter mark with DI water. Use a transfer pipet to get right to the line. Cap the flask and invert three times to mix. I’ll transfer this to a labeled bottle. And we now have our 25 µg/l standards for Phycoerythrin calibration. Keep in mind, these prepared standards can be stored in the refrigerator at 4 degrees Celsius and should be used within 30 days.
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