INTRO
Steven asked me to perform resazurin assays on “selected” juvenile Ruditapes philippinarum (Manila clam) exposed to 40oC acute heat stress. The goal of the experiment is to determine if the “selected” clams have higher metabolic activity than control clams when exposed to heat stress.
Data and results are available here:
MATERIALS & METHODS
Steven picked 48 clams from the “Selected” group and 48 clams from the “Control” group.
Two plates per treatment were measured: “blue” and “red”. The colors reflect the coloration of the clams.
Blanks were included on each plate, and consisted of wells with resazurin working solution, but no clams. Blanks were in Column 6 of each plate.
Plates were kept in a small, table-top, fan-powered incubator at 40oC.
Resazurin Working Solution
Clams were processed in 24-well plates and submerged in 2.2mL of resazurin working solution; prepared by Sam White 20260330:
- 986.66 mL filtered seawater (4oC filtered sea water from 11/19/2025 by AH)
- 2.22 mL resazurin stock solution (from step 1 above)
- 1.00 mL DMSO
- 10.00 mL antibiotic solution (100x Penn/Strep & 100x Fungizone)
Measurements
Plates were measured every 30mins on a Synergy HTX (Agilent) plate reader. The plate reader was set to measure fluorescence with an excitation wavelength of 530nm and an emission wavelength of 590nm.
RESULTS
Here are line plots of raw fluorescence, by well, across all time points. Black lines correspond to the Blanks.
The S-red plate shows changes in fluorescence in the Blanks, suggesting contamination, or, more likely, accidental splashing/wicking of neighboring wells. The volume in the wells was extremely high, so it’s possible that the resazurin working solution was splashed/wicked into the Blank wells via the lid, which had visible resazurin dropletsduring handling.
The S-blue plate shows the overall highest levels of raw fluorescence. Since these plots do not take into account the size of the clams, it’s difficult to draw many conclusions from these data.
Additionally, since the “Control” and “Selected” clams were not randomly assigned to the “red” and “blue” plates, it’s possible that the differences in fluorescence between the plates are due to plate effects, rather than treatment effects.