Resazurin metabolic assays are used to measure the metabolic rate of marine organisms. This protocol has been developed for use in oysters, but can be adapted for other organisms.
Contact: ashuff (at) uw (dot) edu
Updated 20250813 by Ariana Huffmyer.
Example data sets, scripts, and resources can be found at our GitHub repository here.
This protocol details general approaches for resazurin metabolic rate measurements in oysters. The protocol can be adapted for various sizes, temperature profiles, or different stress exposures following this base protocol.
To make the resazurin stock solution (10 mL) mix the following. We will use this solution for multiple trials.
Store in a dark fridge or freezer.
Here are some suggested vessels for conducting trials. The animals should be fully submerged.
We recommend performing preliminary trials to ensure that a change in resazurin fluorescence can be detected over the time scale desired.
Plan your sample sizes appropriately depending on your research question. Metabolic rates are variable, so we recommend at least n=20 per experimental group as a minimum (higher replication is best).
When selecting containers and volume of solutions, remember to account for blanks. We recommend if you use plates that you include n=3-8 blanks per plate and if using cups, we recommend n=5-10 blanks per temperature/stress/experimental treatment.
First, determine the volume of resazurin required depending on the size of your organism, the number of blanks and samples, and the container size as described above.
To prepare the working solution of resazurin, prepare the following.
Desired working volume x 0.98666 = ___ mL filtered seawater (DI water with Instant Ocean adjusted to 23-25 ppt or filtered (<1um) seawater)
Desired working volume x 0.00222 = ___ mL resazurin stock solution as made above in step 1
Desired working volume x 0.001 = ___ mL DMSO
Desired working volume x 0.01 = ___ 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)
Store at 4°C in dark fridge until use. We recommend making a fresh batch of working stock within 7 days of use.
For example, here is a recipe for 150 mL of working stock.
Label plates with identifying number (e.g. “Plate 1”, “Plate 2”) or label cups with unique numbers/identifiers.
Conduct measurements at treatments desired. We typically conduct measurements at a control temperature and a high temperature. If multiple treatments are desired over multiple days, be sure to run a control treatment each day as reference. Ensure you account for tank effects or other batch effects by randomizing loading order, position in incubators, etc.
For oysters, we have used the following treatments to detect metabolic responses to stress:
We strongly recommend preliminary testing to determine appropriate temperature treatments for your study system and research question.
Resazurin measurements should be collected on a timescale appropriate for your research question. Typically, we run assays over a 3-6 hour period with measurements collected every 30-60 minutes. For example, here is a typical schedule for a day in which we are conducting 5 hour incubations at a control and high temperature.
08:00-09:00: Load plates with oysters, take size images, and load
resazurin solution
09:00: Time 0 measurement
10:00: Time 1 measurement
11:00: Time 2 measurement
12:00: Time 3 measurement
13:00: Time 4 measurement
14:00: Time 5 measurement 14:00-16:00: Clean up and assess survival (if
needed)
Before starting, set the incubator at the desired temperature or set up your treatments.
An example of photograph for size measurements:
Measure size using ImageJ or other imaging software. We typically use maximum shell length (mm) for size normalization.
YYYYMMDD_TemperatureTreatment_Plate#_T0.xlsx
.Resazurin solution can be washed down the sink and flushed with plenty of water. Rinse containers thoroughly.
Prepare the following data frames (see examples at our repository):
Conduct the following analysis steps (see R scripts available for use in our repository):
Written by Noah Ozguner and Madeliene Baird 20250401.
This protocol is written based on the manufacturer’s instructions.
Test kit was ordered here
After testing, do not pour contents back into the tank. Make sure to rinse test tubes after use.
Ammonia (NH3): Ammonia is highly toxic to fish and should always be 0 ppm.
Nitrite (NO2): Nitrite is also toxic and should be 0 ppm.
Nitrate (NO3): While less toxic than ammonia and nitrite, nitrate can still be harmful to fish and corals at high levels. Aim to keep nitrate levels below 20 ppm, and ideally between 0-5 ppm.