This post details processing of Hawaii 2023 project samples for larval size and symbiont cell density.

For more information on this project, see the GitHub repo here and my previous notebook posts here.

The samples used for this processing were collected in June/July 2023 across larval development in larvae from previously bleached, non-bleached, or wildtype parents. The samples contained 30-50 larvae each in 200 uL Z-fix. There are 90 total samples.

Sample metadata is available here.

I will update this post as I go through the processing and finish samples.

Data sheets used are here:

• Larval counts
• Larval size
• Cell density

Larval size processing

• Jill previously took images of the fixed larvae with all larvae in the sample contained in the images. The images have a scale bar.
• These images are stored on Google Drive.
• My next step is to measure the planar area of each larvae in all images.
• We will use this for larval size as a response and as a normalizer for cell density.

TO DO: I need to measure size of larvae in the images.

Preparation of samples for cell density

The working protocol for isolation of symbiont cells is as follows:

• Remove as much Z-fix as possible from each tube and discard (~200 uL)
• Add 200 uL of DI water
• Homogenize with Tissue Tearer homogenizer
• Centrifuge for 3 minutes at 3,500rcf
• Remove the liquid host fraction and discard (this sample was fixed, so we are not able to keep it for any other assays)
• Centrifuge again for 3 min at 3,500rcf and remove supernatant
• Freeze pellet at -20°C until processing for cell counts

20241210

Samples completed for this step:

• F1-F35

Cell density

• Resuspend pellet in 100 uL DI water and mix with pipette (record resuspension volume)
• Sonicate with microtip tissue homogenizer for 5 sec
• Count cells on hemocytometer in 6 replicates per sample using the Putnam Lab protocol
• Cell density will be calculated in R and normalized to number of larvae (cells per larvae) and larval size (cells per unit planar surface area).

20241209

Samples completed for this step:

• F1

I trialed cell counts for sample F1 today. I did a set of cell counts before sonication and after sonication. I found that the tissue was very clumped and hard to see cells before sonication and were clear to see afterwards.

Counts before sonication were done on 4 squares per replicate with a mean of 114 cells per replicate. CV was 11%.

Counts after sonication were also done on 4 squares and also had a mean of 114 cells per replicate! CV was 14%.

This indicates that cells were not lost/lysed during sonication. I will keep the sonication step to make the cells easier to see. Data for sample F1 is entered.