Deming:coldfinger cells: Difference between revisions

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(New page: ==Overview== Use of the cold-finger apparatus to determine selective entrapment of cells in ice. ==Materials== ==Procedure== # ## == Detailed Procedure== ''' Day 1. Preliminary w...)
 
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== Detailed Procedure==
== Detailed Procedure==
'''Preliminary work'''
* Culture 250 mL 34H in 1/2x2216 at 8°C to mid exponential phase.
* Perform one experiment as below with only ASW (no culture) to determine approximate bulk salinity of ice under these conditions
''' Day 1. Preliminary work'''
''' Day 1. Preliminary work'''
 
* Measure OD of culture
* Sterilize containers
* Sterilize containers
** Beaker 200 mL with stirrer, covered with foil, to hold solution of interest
** Beaker 200 mL with stirrer, covered with foil, to hold solution of interest
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** Graduated cylinder (100 mL)
** Graduated cylinder (100 mL)
* Prepare 3 L NaCl solution 25 g/L for external water bath
* Prepare 3 L NaCl solution 25 g/L for external water bath
* Prepare 250 ppt Sea salt brine for isohaline melt
* Make NaCl ice cubes with part of the solution prepared above
* Make NaCl ice cubes with part of the solution prepared above
* Label microcentrifugue tubes for EPS measurements (ini / fin_ice, fin_sol)
* Label 1.5 mL microcentrifugue tubes to fix cell samples (ini / fin_ice, fin_sol)
* Get cuvettes ready for EPS measurement by the PSA method


''' Day 2. Cold-finger experiments'''
''' Day 2. Cold-finger experiments'''
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'''8:00 - 10:00 am'''
'''8:00 - 10:00 am'''
* Add NaCl solution and half of crushed ice cubes to the external water bath
* 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.


* Working in an ice bath under the laminar flow hood, pour 200 mL of initial solution in sterile 200 mL beaker (with stirrer)
* Working in an ice bath under the laminar flow hood, pour 200 mL of 34H culture 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 fix with formaline for a final concentration of 2% (label "ini")
* Take a 1.5 mL aliquot in micro-centrifuge tube and freeze at -20 C until processing with PSA
* Check salinity with refractometer (record as "ini")
* Take a 20 mL aliquot in 50 mL test tube and bring to room temperature to check salinity.
* Cover beaker with foil, move in ice bath to cold room
* Cover beaker with foil  


* Place 200 mL beaker with pre-chilled sample in external bath.
* Place 200 mL beaker with pre-chilled sample in external bath.
* Check bath temperature
* Check bath temperature. Should be at freezing point of seawater.
* Set the cold finger to -10 C to generate a thin layer of ice by deposition.
* Once the thin ice layer has been generated on the cold finger, set cold-finger to target temperature.
* Once the thin ice layer has been generated, set cold-finger to target temperature.


'''10:00 am'''
'''10:00 am'''
* Bring to the cold room: 300mL sterile beaker, chronometer, temperature probe, ethanol flask, kimwipes,  waste beaker
* Clean temperature probe with ethanol and kimwipes
* Clean temperature probe with ethanol and kimwipes
* Check temperature of the solution
* Check temperature of the solution
* When temperature of the solution is close to 0 C, start experiment
* When temperature of the solution is close to 0 C, start experiment
* Measure initial conditions
* Register initial conditions
** Time
** Time
** Temperature of the solution
** Temperature of the solution
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'''1:00 pm'''
'''1:00 pm'''
* (OPTIONAL) Measure
* Move away coldfinger 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.
** Time
* Increase the temperature of the coldfinger to 5 °C until the ice is released
** Sample temperature
* Cover with foil the beakers containing the ice and liquid fraction and bring to laminar flow hood
** Bath temperature


'''4 pm'''
In the flask holding the solution:
* 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.
* Take a 1.5 mL aliquot in micro-centrifuge tube and fix with formaline for a final concentration of 2% (label "sol")
* Increase the temperature of the coldfinger to 5 C until the ice is released
* Check salinity with refractometer (record as "sol")
* Cover with parafilm the beakers containing the ice and liquid fraction and store at –1 C until next day
* Measure volume
* Discard remaining solution


''' Day 3. Measurements'''
In the flask holding the ice:
* Let samples equilibrating at room temperature
* Using values from the solution, calculate brine needed to achieve isohaline melt (final salinity = 90 if cold finger at -5°C)
* Add volume of pre-chilled brine
* Shake lightly until melted
* Stir to get an homogeneous solution
* 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
* Take a 1.5 mL aliquot in micro-centrifuge tube and fix with formaline for a final concentration of 2% (label "ice")
* If there is enough solution, take 20 mL and directly measure salinity with conductivity meter, otherwise, dilute with DW to reach 20mL
* Check salinity with refractometer (record as "ice")
* Measure volume of each fraction


==Notes==
==Notes==

Revision as of 20:51, 31 October 2013

Overview

Use of the cold-finger apparatus to determine selective entrapment of cells in ice.

Materials

Procedure

Detailed Procedure

Preliminary work

  • Culture 250 mL 34H in 1/2x2216 at 8°C to mid exponential phase.
  • Perform one experiment as below with only ASW (no culture) to determine approximate bulk salinity of ice under these conditions

Day 1. Preliminary work

  • Measure OD of culture
  • 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
  • Prepare 250 ppt Sea salt brine for isohaline melt
  • Make NaCl ice cubes with part of the solution prepared above
  • Label 1.5 mL microcentrifugue 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.
  • Working in an ice bath under the laminar flow hood, pour 200 mL of 34H culture in sterile 200 mL beaker (with stirrer)
  • Take a 1.5 mL aliquot in micro-centrifuge tube and fix with formaline for a final concentration of 2% (label "ini")
  • Check salinity with refractometer (record as "ini")
  • Cover beaker with foil, move in ice bath to cold room
  • Place 200 mL beaker with pre-chilled sample in external bath.
  • Check bath temperature. Should be at freezing point of seawater.
  • Once the thin ice layer has been generated on the cold finger, set cold-finger to target temperature.

10:00 am

  • 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
    • Time
    • Temperature of the solution
    • Temperature of the bath

1:00 pm

  • Move away coldfinger 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 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")
  • Measure volume
  • Discard remaining solution

In the flask holding the ice:

  • Using values from the solution, calculate brine needed to achieve isohaline melt (final salinity = 90 if cold finger at -5°C)
  • 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")

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!


Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers

  1. 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.

    [ewert_and_deming11]

  2. 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.

    [kuiper_etal03]

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