Method
We use alizarin red stain, but other chemicals are available. Most notably, calcein is commonly used, but we prefer alizarin since it is visible both to the naked eye (or under brightfield illumination) and via fluorescence microscopy.
Make a plan: You first need to devise a plan for how you will cover corals (what size and type of bag), and then calculate how much alizarin you need to achieve ~5 mg/kg. For example, in our Coral Reefs paper, we used plastic tarps tied around the base of the colony with bungie. This allowed us to adjust the tarp to achieve approximately our target volume of 8 L (this is loosely estimated by eye). We prepared our alizarin by dissolving it in fresh water in 20 mL scintillation vials. Thus, we calculated a concentration of 2 g/L alizarin in these vials (or 40 mg per vial) to achieve our target ~5 mg/kg exposure to the corals. There is inevitably some leakage from the bottom of the bag, so we ultimately estimated the corals were exposed to a range of 3-5 mg/kg. In our Marine Biology paper, we used a similar approach but used 6 mg/kg. Also, be aware that it is best to dissolve the stock alizarin solution in fresh water, so you want to make sure you are not changing the salinity dramatically inside the bag.
Procedure: The clear plastic tarp (or large bag) and bungie technique has worked the best for us. This is easiest in calm weather and for smaller colonies of course. We find it easier to first tie the bungie around the base of the colony (the more mushroom-shaped, the better), and then tuck the plastic under the bungie. Then, pull the bungie enough that you can squeeze a scintillation vial under the plastic, and open the vial by grabbing it from the outside of the plastic. We have found that exposures of 4-6 hours leave bright enough lines to be easily visible by eye. But much shorter exposures also work, even as little as 30 minutes. These may be harder to see by eye, but are very easily visualized under a fluorescence microscope.
Make a plan: You first need to devise a plan for how you will cover corals (what size and type of bag), and then calculate how much alizarin you need to achieve ~5 mg/kg. For example, in our Coral Reefs paper, we used plastic tarps tied around the base of the colony with bungie. This allowed us to adjust the tarp to achieve approximately our target volume of 8 L (this is loosely estimated by eye). We prepared our alizarin by dissolving it in fresh water in 20 mL scintillation vials. Thus, we calculated a concentration of 2 g/L alizarin in these vials (or 40 mg per vial) to achieve our target ~5 mg/kg exposure to the corals. There is inevitably some leakage from the bottom of the bag, so we ultimately estimated the corals were exposed to a range of 3-5 mg/kg. In our Marine Biology paper, we used a similar approach but used 6 mg/kg. Also, be aware that it is best to dissolve the stock alizarin solution in fresh water, so you want to make sure you are not changing the salinity dramatically inside the bag.
Procedure: The clear plastic tarp (or large bag) and bungie technique has worked the best for us. This is easiest in calm weather and for smaller colonies of course. We find it easier to first tie the bungie around the base of the colony (the more mushroom-shaped, the better), and then tuck the plastic under the bungie. Then, pull the bungie enough that you can squeeze a scintillation vial under the plastic, and open the vial by grabbing it from the outside of the plastic. We have found that exposures of 4-6 hours leave bright enough lines to be easily visible by eye. But much shorter exposures also work, even as little as 30 minutes. These may be harder to see by eye, but are very easily visualized under a fluorescence microscope.