Manchester Hardening project final lab sampling plan

manchester-hardening
experimental design
Planning final sampling of Manchester Hardening experiment oysters
Author

Ariana Huffmyer

Published

August 11, 2026

Overview

This post details the experimental design plans for sampling survival and metabolism of Manchester Hardening experiment oysters.

Note that I will keep updating this post as we get more information on sample size and specific protocols.

Oysters

Oysters will be collected from Manchester on Friday, August 15th and transported to UW campus by Mac.

The oysters will be held in red seed bags (n=20 total bags) that correspond to 10 families from 2 treatment groups (10 x 2 = 20). These are our “family treatment groups”.

Each bag will contain a waterproof paper label with the family and treatment information.

We will see how many oysters are surviving from each family treatment group to update and finalize sample size.

Family treatment groups:

The oysters will be identified by a family + a treatment group from our hardening experiment. This will be something like “YC24-087” (family) and “High” or “Control” (treatment). Note that the treatment refers to the hardening treatment, NOT the treatment for survival or resazurin testing. These assays will run at a stressful temperature for all oysters as in the standard lab protocols.

Sample size:

This will be based on how many oysters are sampled. I will update this as we go. I expect at least n=10 per family group (~200 oysters). Depending on how things go, we will prioritize survival assays first. If things go well and we have time, we can also run resazurin.

Survival (week of Aug 17, extending to week of Aug 24 if needed)

Materials needed:

  • Numbered plastic cups (number will be dependent on sample size)
  • Seawater (Instant Ocean adjusted to ambient levels)
  • Seawater table running for temperature control

Base protocol:

  • Pre heat the water table such that cups in the water table reach 36°C (may need to have the table heater set at 38-40°C)
  • Place oysters into numbered cups, being sure to record which treatment is in each cup (e.g., YC24-087 Control or YC24-087 High). Labels are based on the label in each bag of oysters.
  • Fill cups with seawater and ensure each oyster is alive.
  • Record which oysters are dead in the morning and evening of each day for 3-5 days until ~80-90% of oysters have died.
  • Record the temperature in 5 cups in the water table each time mortality is assessed and write down the temperature readings.
  • Send all data to Ariana.

Data sheets that will need to be recorded:

  1. Temperature recorded in n=5 cups at each sampling point
  2. Alive/dead oysters at each time point
  3. The time of the start of the experiment and time of each sampling time point
  4. A metadata sheet that contains the cup number, family and treatment, and any notes

Resazurin (week of Aug 24)

Materials needed:

  • Numbered plastic cups
  • Seawater (Instant Ocean adjusted to ambient levels)
  • Seawater table running for temperature control
  • Resazurin solution - enough to fill up the desired number of cups
  • 96 well plates for resazurin sampling
  • Plate reader and computer in FTR 213
  • P1000 pipette and tips
  • Gloves
  • Camera/phone
  • Whiteboard surface to take images and write identifiers to include in photos

Base protocol:

  • Prepare the resazurin solution
  • Pre heat the water table such that cups in the water table reach 36°C (may need to have the table heater set at 38-40°C)
  • Place oysters into numbered cups, being sure to record which treatment is in each cup (e.g., YC24-087 Control or YC24-087 High). Labels are based on the label in each bag of oysters.
  • Photograph each oyster for size and include the cup number and date in the image. Follow Sam’s protocols for calculating oyster size automatically.
  • Fill cups with seawater resazurin solution and ensure each oyster is alive. Place in water table.
  • Take an initial sample of resazurin into a 96-well plate. Record the plate location of the sample and use this location at every sampling point.
  • Read the sample on the plate reader and save the data with the date and timepoint in the file name.
  • Repeat the sampling at 1, 2, and 3 hours. We can also sample at 30 min intervals as per the most recent SORMI protocol, but this will be dependent on sample size.
  • After measurements are completed, discard resazurin and oysters.
  • Send all data to Ariana.

Datasheets that will need to be recorded:

  1. Temperature recorded in n=5 cups at each sampling point
  2. The time of the start of the experiment and time of each sampling time point
  3. A metadata sheet that contains the cup number, date, family and treatment, plate well location, and any notes for all oysters
  4. Raw plate reader files for all readings