Week 03 of the Spring 2023 Quarter Summary

The colonies gathered from the previous two weeks of the quarter have not showed significant signs of adapting to the recirculating seawater system at UWT. As such, I have developed an alternative protocol that will involve the isolation of epithelial tissue directly from colonies immediately removed from the field. This protocol will be carried out for the first time on 04-17-2023.

Planning for 04-17-2023:

  • Collect 6 colonies of Botryllus schlosseri that are about 100 mm^2 and adhered to mussels. Ideally colonies are at blastogenic stage D however for this first run of this protocol any stage will do.
  • Bring animals back to UWT lab. Record the following:
    • Number of star systems
    • Approximate area in mm^2 of colony
    • Location of sampling
    • Date
    • Blastogenic stage

  • Subdivide colony and tie one half to a 3’’ x 2’’ x 1.2 mm glass slide with cotton thread as described in this notebook post.

  • Immerse the other half of the colony that is still attached to the mussel in 2x ASW-PSA. Take a photo of the colony and under the stereomiscroscope dissect out buds using a pair of 28-31 G syringe needles.

  • Transfer groups of 10 buds to a 70-100 um cell strainer and rinse the excised buds with 70% ethanol for 30 seconds.

  • Follow this with a triplicate 30 mL rinse of 2x ASW-G.

  • With clean and sharp foreceps, transfer the group of 10 buds to a 15 mL conical tube filled with 5 mL of 2x ASW-PSA. (Buds can stay in tube while you finish processing your other colonies).

  • Once all the tissue has been collected transfer all conical tubes to biosafety cabinet

  • Pour out contents of each tube into seperate wells on a six well plate.

  • Remove excess 2x ASW-PSA and replace with 2 mL of TCM (2x PSA mix).

  • Incubate cells at 20 C