Use of the cold-finger apparatus to determine selective entrapment of cells in ice. This protocol is designed to use with temperature-sensitive psychrophilic cells. Control treatment will consist in a solution of microbeads. Length of experiment has been limited to 1h to reduce effects of cell reproduction during the experiment.
- Culture 250 mL 34H in 1/2x2216 at 8°C to mid exponential phase.
- Perform 3 replicas of each for each treatment:
- only ASW (no culture)
- 34H culture - with direct melts
- 34H culture - with saline melts
Day 1. Preliminary work
- Pre-weight containers covered with foil (Beaker 200 mL (with stirrer), beaker 300 mL)
- 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
- Prepare 250 ppt Sea salt brine for isohaline melt
- Check availability of pre-chilled sterile ASW for cell spinning and resuspension (at least 400mL per experiment)
- Label 1.5 mL microcentrifuge tubes to fix cell samples (ini / fin_ice, fin_sol)
Day 2. Cold-finger experiments Cold-finger experiments take place in a 8 °C cold room using aseptic technique.
8:00 - 10:00 am
- Bring to the cold room: 300mL sterile beaker, temperature probe, ethanol flask, kimwipes, NaCl solution, ice cubes
- Add NaCl solution and ice cubes to the external water bath
- Clean cold finger with ethanol
- Set apparatus to -10 °C to generate a thin layer of ice by deposition (should take aprox 1h).
- Once the thin ice layer has been generated on the cold finger, set cold-finger to target temperature.
- Check salinity of sterile ASW with refractometer (record as "ini")
- Measure OD of culture
- Perform initial harvest:
- Centrifuge 200mL culture (12,000g, 10 min, 4 °C)
- Discard media
- Rinse with sterile, pre-chilled ASW and spin (3 times, 12,000g, 10 min, 4 °C)
- Resuspend cells in 25 mL sterile, pre-chilled ASW
- Working in an ice bath under the laminar flow hood, bring cells to a final volume of 200 mL of pre-chilled ASW in sterile 200 mL beaker (with stirrer) for a final concentration of 10^7 cells mL
- Take a 1.5 mL aliquot in micro-centrifuge tube and fix with formaline for a final concentration of 2% (label "ini")
- Cover beaker with foil, wipe exterior, weight, move in ice bath to cold room
- Working in an ice bath under the laminar flow hood, resuspend beads in 200 mL of pre-chilled ASW in sterile 200 mL beaker (with stirrer) for a final concentration of 10^7 beads mL
- Check bath temperature. Should be at freezing point of seawater.
- Place 200 mL beaker with pre-chilled sample in external bath.
- Clean temperature probe with ethanol and kimwipes
- Check temperature of the solution
- When temperature of the solution is close to 0 °C, start experiment
- Register initial conditions
- Temperature of the solution
- Temperature of the bath
- After 1 hour of freezing, move coldfinger away from the solution to a sterile 300 mL beaker outside of the water bath. As the ice is being separated from the liquid fraction, it may release drops of brine. For uniformity, the first drop 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 foil the beakers containing the ice and liquid fraction
- Weight each beaker and bring to laminar flow hood
In the flask holding the solution:
- Take a 1.5 mL aliquot in micro-centrifuge tube and fix with formaline for a final concentration of 2% (label "sol")
- Check salinity with refractometer (record as "sol")
- Discard remaining solution
Use spreadsheet to calculate brine needed to achieve isohaline melt, based on weight (ini, sol, ice) and salinity (ini, sol).
In the flask holding the ice:
- Add volume of pre-chilled brine
- Shake lightly until melted
- Stir to get an homogeneous solution
- Take a 1.5 mL aliquot in micro-centrifuge tube and fix with formaline for a final concentration of 2% (label "ice")
- Check salinity with refractometer (record as "ice")
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.