INTRO
We’re beginning to gather some preliminary data for a project examining the effects of “stress priming” on _Crassostrea gigas (Pacific oyster). GitHub repo is here. As part of that, Steven recently found out about a protocol to easily evaluate respiration in oysters using resazurin (Wikipedia). According to (González-Pinzón, Haggerty, and Myrold 2012), the reduction of resazurin to resorufin is almost perfectly correlated with respiration in aerobic bacteria and, thus, can be used as a proxy to measure respiration. Offhand, I have not looked at literature detailing its use with eukaryotic cells - I just glanced at the literature cited by Wikipedia. :)
Ideally, using this method would provide a quick, cheap, and wasy way for us and hatcheries to monitor respiration in live oysters, with the potential to use the method as a means to detect stress-inducing events.
For this initial test, I followed the resazurin assay (commit 27441a7) shared by Steven, with the following modifications:
Non-sterile water/sea water was used.
No penn/strep/Fungizone used.
Small oysters were distributed by Steven in two, 12-well (3x4) plates, and covered completely with 2mL of resazurin Assay Media:
unstressed
- Oysters were in all 12 wells.
heat stressed (not sure what the temp or duration was)
- Well C4 (bottom right) contained no oysters.
Two larger oyster which would not fit in the 12-well plates were each placed in a glass Petri dish and covered with 20mL of resazurin Assay Media.
Fluorescence was mesasured on a Victor 1420 (Perkin Elmer) plate reader. Plates were read directly, with the oysters in them. For the Petri dishes, 2mL were transferred from each into wells of a new 12-well plate and measured.
RESULTS
Overall, it seems like there was a difference in fluorescence between resazurin control wells (i.e. resazurin assay media only) and wells which contained oysters. So, it appears that the assay is working. Admittedly, I’m a bit surprised by how dark the assay media is. I’d definitely like to play around with the amount resazurin being used, as I think less resazurin might allow us to visually identify differences, in addition to collecting fluorescence readings.
Output files
All files have been added to https://github.com/RobertsLab/project-gigas-carryover/blob/main/lifestage_carryover/data/resazurin_trial_01/.
20240307-cgig-resazurin-control-bigs-and-two-controls-t2.0.xlsx: Raw data file (Excel workbook) from Victor (Perkin Elmer) Wallac plate reader of fluorescence measurements of 2mL resazurin assay media from oysters in glass petri dishes after two hours. SeeNotescolumn in first sheet for plate layout.20240307-cgig-resazurin-control-t2.0.xlsx: Raw data file (Excel workbook) from Victor (Perkin Elmer) Wallac plate reader of fluorescence measurements from oysters in 12-well after two hour](control_p1.png) (Image). SeeNotescolumn in first sheet for plate layout.20240307-cgig-resazurin-stress-t1.5.xlsx: Raw data file (Excel workbook) from Victor (Perkin Elmer) Wallac plate reader of fluorescence measurements from oysters in 12-well 1.5hrs after an unkown heat stress temp/duration. SeeNotescolumn in first sheet for plate layout.