Blue Notes and Bivalves
Exploring oyster metabolism with a jazzy resazurin twist
Landing page for quick info on where we are with implementing easy resazurin metabolism assays for oysters. Please explore the Github Repo, glimpse real-time activity in from the lab here, and dive deeper with the Canonical Protocol.
Summary
This protocol uses a resazurin-based assay to measure individual metabolic responses to elevated temperature stress over a 5-hour period. Resazurin is a redox-sensitive dye that changes fluorescence and color as it is reduced by cellular respiration, providing a proxy for metabolic activity. Individuals are incubated in a resazurin working solution at a high temperature (e.g., ~42°C), and metabolic activity is monitored through fluorescence measurements taken at hourly intervals. Fluorescence measurements are conducted using an excitation wavelength of 530 nm (or a 528/20 nm bandpass filter) and an emission wavelength of 590 nm (or a 590/20 nm bandpass filter) using a plate reader.
In July 2025 we ran assays at VIMS using C. virginica. The protocol was modified slightly and involved allocating oysters to 24 well plates, taking images for size analysis, adding chilled resazurin (4°C ) solution, taking initial measurements (T0), incubating plates at 40°C, staggering plate readings, taking hourly measurements (T1, T2), chilling plates at 4°C after 2.5 hours of exposure, and taking final measurements (T3, T4).
Highlights to date
- Resazurin fluorescence provides a reliable indicator of oyster metabolic activity from small seed to adult oysters.
- Thermal performance assays reveal metabolic optima and tipping points where metabolic stimulation shifts to depression under temperature stress.
- Resazurin fluorescence detects genetic differences in metabolic rates between oyster family groups.
- Under acute stress, increased metabolism of seed at high temperature predicts short-term mortality. In contrast, metabolic depression is associated with higher survival.
- Resazurin fluorescence assays provide an exciting potential tool to survey oyster performance.
Video Demo
Preparing solutions
To prepare the resazurin assay solutions, first make a stock by combining resazurin salt with water and a small amount of DMSO. Store this stock in a dark refrigerator or freezer. The working solution consists primarily of seawater (about 98.5%), with small proportions of resazurin stock (0.2%), DMSO (0.1%), and an antibiotic/antifungal solution (1%). Adjust the total volume based on the number and size of organisms being tested, ensuring animals are fully submerged. Trials are typically scaled for small (≤7 mm), medium (15–40 mm), or large (>40 mm) seed using appropriately sized containers.
Detailed example
- Stock resazurin solution
To make the resazurin stock solution (10 mL) mix the following. We will use this solution for multiple trials.
10 mL DI water
10 µL DMSO
Store in a dark fridge or freezer.
- Working resazurin solution
First, determine the volume of resazurin required depending on the size of your organism and the container size. Here are some examples that we have used before. We recommend performing preliminary trials to ensure that a change in resazurin fluorescence can be detected over the time scale desired. This protocol was developed to detect a change in resazurin fluorescence within 5 hours in small seed (<7mm) and larger seed (10-30mm).
Small seed (<7mm length): trials conducted in 96 well plates (300uL volume in each well)
Medium seed (15-40 mm length): trials conducted in small plastic cups (20 mL volume)
Large seed/adults (>40 mm length): trials can be conducted in tripour cups, beakers, or plastic cups. Scale volume appropriately. The animals should be fully submerged.
To prepare the working solution of resazurin, prepare the following.
This recipe is to make 150 mL of working stock. To run a single 96 well plate, approximately 35 mL will be required (allowing for extra for re-do’s or errors). For example, if we are running 4 total plates, we will need to prepare 4 x 35 mL = 150 mL of working solution. Increase if more is required.
148 mL seawater (DI water with Instant Ocean adjusted to 23-25 ppt or filtered (<1um) seawater)
333 µL resazurin stock solution as made above in step 1
150 µL DMSO
1.5 mL antibiotic solution 100x Penn/Strep & 100x Fungizone - this should be kept frozen in a dark freezer and thawed before use (thaw in the dark or cover with aluminum foil)
Recent Slidedecks
Podcast ?
While not solely focused on resazurin… a majority of content covers what we are doing in this space.