The MSAR Lab has cryogenic capabilities and is fitted with liquid nitrogen tanks, a controlled rate freezer, and long term storage dewars! Cryopreservation is a topic of interest for many of our students. This technique preserves germplasm material (think eggs, sperm, embryos, and larvae) for future use by cooling them to sub-zero temperature.
How does cryogenics work?
The process starts by sourcing broodstock oysters or clams. We work with farmers and fishermen throughout Florida in order to find mature adult shellfish for our research. Broodstock is then conditioned in the recirculating tank system housed in our wet lab facility. The shellfish are fed an enriched algal diet and water quality is monitored to ensure good growing conditions.
Natural spawning and strip spawning methods are used to gather samples necessary for the experiment. This includes sperm, egg, embryo and larvae. Once the samples are gathered they are prepared for the freezing process. This involves adding an extender, which is a salt solution that helps to maintain the viability of samples. Cryoprotectants are then added to protect the cell from damage during the freezing and thawing process.
Samples are then packaged in French straws, before being placed in our controlled rate freezer. The controlled rate freezer is connected to a liquid nitrogen tank and allows the researcher to control the rate at which the samples are cooled. Once samples are cooled, they are placed in liquid nitrogen storage dewars until they are ready to be thawed and used.
Establishing reliable protocols for shellfish germplasm preservation offers a number of potential applications including:
- Preserve natural populations
- Preserve specific lines
- Inbred lines by self-fertilization for genetic research
- Mutant breeding programs
- Preserve triploid and tetraploid oysters
- Assist seed production
- Preserve endangered, vulnerable, and threatened species
Click and scroll through the images to learn more!
Cryogenic Saftey– Cryogenic gloves are an important part of lab safety! They offer thermal protection for hands and forearms when working with liquid nitrogen and ultra freezing temperatures.
Packaging Samples to be Frozen– Samples are packaged in 0.5mL French Straws. A handheld straw sealing device is then used to seal the end of the straw before placing them in goblets. Each goblet holds 5 straws during the freezing process and can hold more during long term storage. Two goblets are placed on a metal rack for easy handling during the freezing and storage process.
Freezing Process– A cylinder full of liquid nitrogen is attached to our controlled rate freezer. The controlled rate freezer can be used manually or via a computer connection. Freezing Profiles can be uploaded to the system. An example looks like:
- Wait at 4ºC:
- This brings the chamber to 4ºC. We are then able to place the samples in the chamber.
- Ramp 10ºC/min to -80ºC
- Once the samples are in the chamber the freezing rate can be selected. In this example, the chamber and sample will go from 4ºC to -80ºC. This will be done by decreasing the temperature 10ºC every minute.
- Hold at -80ºC for 5 minutes
- Once the samples have reached -80ºC they will remain at this temperature for another 5 minutes. This allows the samples to be removed from the controlled rate freezers chamber and placed in a storage dewar.
- Heat back up to 4ºC
- After the samples are removed the controlled rate freezer is brought back to 4ºC and then room temperature before freezing the next batch of samples or shutting down the machine.
Storage Process– Samples are stored in liquid nitrogen dewars for long term storage. The dewars are insulated and have a large storage capacity with 6 canister compartments.
If you have any questions about the process please fill out our contact form.