In between helping the UW Friday Harbor Labs, I have been busily prepping for the last stage of my experiment: the infection stage. For the last four weeks, my eelgrass has been growing in some outdoor tanks. Two of the tanks had isopods and snails but the other four have been growing in (hopefully) stress-free conditions. Today, all the eelgrass was taken out, scraped of diatoms, cut, and placed into petri dishes. Tomorrow, at the start of Week Six, we will infect half of these pieces with Labyrinthula spp. by clipping them to inoculated pieces of eelgrass. The other half are going to be clipped to eelgrass pieces covered with sterile seawater.
To prepare for the inoculation, I have been growing the Labyrinthula spp. cultures in both SSA broth and agar media. On Thursday, I counted the number of cells in the SSA broth using a hemocytometer. A hemocytometer is a special microscope slide that has a set volume, so by counting the number of cells, one can calculate the concentration. The same number of cells has to be used in each inoculation to ensure that no piece of eelgrass is more exposed to Labyrinthula than another.
Tomorrow, I need to centrifuge the vials and remove the SSA broth so that Labyrinthula infects the eelgrass instead of eating the broth. After that, I need to replace the SSA broth with sterilized sea water. This is where the hemocytometer comes in. By knowing the concentration of cells in the broth, I can calculate how much sterilized sea water to put in so that every vial has the same concentration of Labyrinthula.
I did a practice run of this procedure on Friday and so far a couple of eelgrass pieces show Labyrinthula which is a good sign. Hopefully everything will run smoothly tomorrow and my eelgrass will be successfully inoculated!
|144 petri dishes of eelgrass waiting to be inoculated in the culture box|