Happy new year! This is my first lab notebook entry. Since I am now starting the second quarter of my graduate program, I will be summarizing my research activities from the previous quarter.

Drafting Protocol for Primary Cell Cultures

As I was unfamiliar with the practice of primary cell culturing I began the process with reviewing basic cell culture techniques from the textbook titled Culture of Animal Cells by R. Ian Freshney (5th Edition). After, I began combing through the document “NSF proposal grant references” in the Joint NSF Tunicate Google Drive to become up to date with the latest methods in culturing B. schlosseri epithelial cells. I found Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges, 2003 by Rabinowitz & Rinkevich particularly useful. Collaborating with Valerie Dong from the UC Davis (UCD) lab, we began adapting the Rabinowitz & Rinkevich 2003 methods into a standard operating procedure that would become applicable to Aim 1 of the NSF proposal, Optimization of primary cultures of B. schlosseri. This protocol is still a work in progress and will need the addition of methodologies for blood cell isolation.

Visiting Stanford’s Hopkins Marine Station

In November 2022 some members from the Gardell and Kueltz Labs visited Stanford’s Tunicate Lab Hopkins Marine Station (HMS) for a two-day workshop led by Dr. Ayelet Voskoboynik. This workshop covered a broad range of topics such as:

  • General B. schlosseri anatomy and biology
  • Practice identifying the seven stages of cyclical asexual reproduction
  • Best practices in colony care and maintenance
  • Methods in dissection of whole zooids and embryos
  • Use of the B. schlosseri Genome Project browser
  • Feedback regarding each labs respective methods and set-up

Practicing the workflow of primary cell culturing

Prior to the conclusion of the Fall Quarter, I wanted to work through the movements of primary cell isolation protocol Valerie, UCD, and I had put together. This exercise was primarily intended to help me better visualize the workflow and to get a better idea of the materials I needed moving into the next quarter.

This process began with gathering a small number of colonies on which to practice stage identification, maintenance, and zooid dissection. I collected one bivalve covered in ~30 orange colonies from dock C9 and another bivalve that had ~10 colonies on dock C10. The salinity in the field was 29 PPT and the temperature was not measured.

Upon arriving to UWT, colonies were scraped off the bivalves with a new razor and placed on 76.2 mm x 50.8 mm x 1.2 mm glass slides. I subdivided the colonies so that there was only one “flower” per glass slide. The glass slides were then placed on glass racks and placed inside a plastic Tupperware that also contained a wet paper towel to create a humidity chamber. The colonies were left for an hour. Only one colony adhered to the glass slide, likely because it endured the least mechanical stress as I did not subdivide since there weren’t many zooids in the tunic. The colonies that did not adhere were tied to the glass slide with a cotton thread.

The colonies were transferred into the recirculating seawater system immediately and were kept there for 3 days only being removed to take an image of each colony under the microscope.

Overall, the colonies health seemed to be in decline over the course of being kept in the recirculating seawater system as the number of zooids per colony reduced. Regardless, at day 4, I took the colonies and submerged them for 24 hours in a solution of Artificial Seawater and Gentamycin (ASW-G) (Rabinowitz & Rinkevich 2003) to begin the initial steps of cell isolation. The salinity of this solution was too high (75 PPT) as I misinterpreted the methods described (ASW needed at least a 1:1 dilution). After the 24 hour ASW-G treatment all 7 colonies exposed no longer had visible blood circulation. Regardless, I practiced dissecting out the zooids of the colonies using dissection pin needles.

The exercise was useful but will need to be redone with the correct solutions to proceed into the following steps of the protocol.