Use of the cold-finger apparatus to determine selective entrapment of solutes or particles in ice.
Day 1. Preliminary work
- Sterilize containers
- Beaker 200 mL with stirrer, covered with foil, to hold solution of interest
- Beaker 300 mL, for melting ice
- Graduated cylinder (100 mL)
- Prepare 3 L NaCl solution 25 g/L for external water bath
- Make NaCl ice cubes with part of the solution prepared above
- Label microcentrifugue tubes for EPS measurements (ini / fin_ice, fin_sol)
- Get cuvettes ready for EPS measurement by the PSA method
Day 2. Cold-finger experiments Cold-finger experiments take place in a 8 °C cold room using aseptic technique.
8:00 - 10:00 am
- Add NaCl solution and half of crushed ice cubes to the external water bath
- Working in an ice bath under the laminar flow hood, pour 200 mL of initial solution in sterile 200 mL beaker (with stirrer)
(Alternatively, pour 200mL of artificial seawater and add 20 mg of polysaccharide of interest)
- Take a 1.5 mL aliquot in micro-centrifuge tube and freeze at -20 C until processing with PSA
- Take a 20 mL aliquot in 50 mL test tube and bring to room temperature to check salinity.
- Cover beaker with foil
- Place 200 mL beaker with pre-chilled sample in external bath.
- Check bath temperature
- Set the cold finger to -10 C to generate a thin layer of ice by deposition.
- Once the thin ice layer has been generated, set cold-finger to target temperature.
- Bring to the cold room: 300mL sterile beaker, chronometer, temperature probe, ethanol flask, kimwipes, waste beaker
- Clean temperature probe with ethanol and kimwipes
- Check temperature of the solution
- When temperature of the solution is close to 0 C, start experiment
- Measure initial conditions
- Temperature of the solution
- Temperature of the bath
- (OPTIONAL) Measure
- Sample temperature
- Bath temperature
- Move away coldfinger from the solution to a sterile 300 mL beaker outside of the water bath. When the ice is separated from the liquid fraction, it may release drops of brine. For uniformity, the first two or three drops will be collected as part of the liquid fraction.
- Increase the temperature of the coldfinger to 5 C until the ice is released
- Cover with parafilm the beakers containing the ice and liquid fraction and store at –1 C until next day
Day 3. Measurements
- Let samples equilibrating at room temperature
- Stir to get an homogeneous solution
- Subsample 1.5 mL of each fraction (ice and solution) and freeze in micro-centrifuge tube at –15 C until processed with PSA
- If there is enough solution, take 20 mL and directly measure salinity with conductivity meter, otherwise, dilute with DW to reach 20mL
- Measure volume of each fraction
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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- Ewert, M., & Deming, J. W. (2011). Selective retention in saline ice of extracellular polysaccharides produced by the cold-adapted marine bacterium Colwellia psychrerythraea strain 34H. Annals of Glaciology, 52(57), 111-117.
- Kuiper, M. J., Lankin, C., Gauthier, S. Y., Walker, V. K., & Davies, P. L. (2003). Purification of antifreeze proteins by adsorption to ice. Biochem. Biophys. Res. Comm., 300(3): 645-648.
- Marcela Ewert has experience with this protocol
or instead, discuss this protocol.