date started: 20210727; date last revised: 20210727 HGR post creation

post overview

goal: track symbiodiniaceae surface area before, after and during polyp bailout. rationale: algal cell size can increase precipitously following exposure to hyperosmotic stress (hgr annotated bib on algal response to hyperosmotic stress here)

Materials needed (general)

• compound scope with 40x magnification
• microscope camera attachment (ours is omax a3513u, link to free touplite software download for mac, os, linux)
• hemocytometer (protocol on how to count symbiodiniaceae cells with a hemocytometer by the weis lab here)
• microscope cover coverslip
• tweezers
• 70% ethanol
• p200 pipette
• p20 pipette

Materials needed (for each sample)

• 1.5mL microcentrifuge tube
• 50uL seawater (the salinity of this seawater should match experimental salinity)
• p20 pipette tip

Protocol

1. place ~0.5cm2 Pocillopora acuta fragment in a 1.5mL microcentrifuge tube with ~50uL seawater (salinity should match experimental salinity)
2. rinse tweezers with DI water. then use tweezers to scrape tissue off of fragment.
   • why aren’t we airbrushing? fragment size is too small to airbrush here
3. repeat for all samples, let the scraped sample sit in the microcentrifuge tube while scraping other samples.
4. Before adding sample to hemocytometer, pipette up and down to homogenize scraped tissue (which includes symbiodiniaceae cells)! Add 10uL to the hemocytometer. Keep track of which sample is in which hemocytometer well.
5. Set compound microscope to 40x and connect touplite camera to your computer.
6. Take pictures of symbiont cells inside the grids! NOTES:
   • look for golden brown balls! circled below in green in picture 1
   • make sure that the image includes BOTH SIDES of the hemocytometer grid. this is critical for creating an image reference in imageJ (resources in protocol here)
   • take pictures of grid cells until you acquire ~20 cells/ samples (this might mean 3-5 pictures depending on cell density)
   • don’t count cells that are “clouded over” by other tissue types (fuzzy lines will make it difficult to get a precise reading with the freehand tool in imageJ)
7. save photo on touplite software! make sure trial id, day #, sample id and picture # is included in the name.
8. push to github!
9. analyze cell size with imageJ
   • hemocytometer grid lengths needed for calibrating imageJ size reference

cells that should be counted (circled in green)! hemocytometer grid (blue) that needs to be included in image

cells not to count circled in red (edge of cell is fuzzy)

• link to image1
• link to image 2