2 Materials
2.1 Standard
Solutions
1. Phosphate buffered saline solution: weigh 8 g of NaCl, 1.44 g
of Na 2 HPO 4 , 0.2 g of KCl, and 0.24 g of KH 2 PO 4 all in a 1 L
volumetric flask. Add 700 mL of distilled, deionized water to
the flask until all solutes are dissolved (heat may be required).
Once dissolved, fill the flask to the 1 L mark. Adjust the pH of
the solution by adding concentrated HCl dropwise until
pH 7.4. Store at room temperature.
2. 40% sucrose solution: weigh 40 g of sucrose in a beaker. Add
100 mL of distilled, deionized water to a beaker, and dissolve
the solute by slowly heating the solution. Make sure solution
has cooled to room temperature before use.
2.2 IRI Sample
Testing
1. Polished aluminum block cooled with dry ice.
2. Cryostage connected to a cooling bath (Digital One RTE
7 from Thermo Scientific NESLAB), a peltier unit (Series
800 from Alpha Omega Instruments), a fish pump (SAX1
from Pen Plax), and a drying tube.
3. Dehumidifier.
4. Digital camera (Nikon Coolpix 5000).
5. Microscope with 20Â magnification.
6. Borosilicate coverslips.
2.3 Image Analysis
1. Computer with ImageJ software.
2. Excel spreadsheet to generate the weighted average and standard error of mean grain size.
3 Methods
3.1 IRI Sample
Preparation
1. All IRI samples were lyophilized prior to IRI testing to ensure
they were devoid of solvent (confirmed by
1
H/
13
C NMR).
2. Prepare a minimum of five concentrations to be tested (using
standard solutions) for each compound ranging from 1 mM to
its maximum solubility. The maximum solubility of a compound is defined by the maximum concentration at which the
solution stays dissolved at room temperature (slight heating
can be applied to facilitate dissolution).
3. Prepare each concentration in triplicate (using three different
vials).
4. Prepare a vial of just the standard solutions without any compound present (i.e., 0 mM).
Ice Recrystallization Inhibition Assays
277
2.1 Standard
Solutions
1. Phosphate buffered saline solution: weigh 8 g of NaCl, 1.44 g
of Na 2 HPO 4 , 0.2 g of KCl, and 0.24 g of KH 2 PO 4 all in a 1 L
volumetric flask. Add 700 mL of distilled, deionized water to
the flask until all solutes are dissolved (heat may be required).
Once dissolved, fill the flask to the 1 L mark. Adjust the pH of
the solution by adding concentrated HCl dropwise until
pH 7.4. Store at room temperature.
2. 40% sucrose solution: weigh 40 g of sucrose in a beaker. Add
100 mL of distilled, deionized water to a beaker, and dissolve
the solute by slowly heating the solution. Make sure solution
has cooled to room temperature before use.
2.2 IRI Sample
Testing
1. Polished aluminum block cooled with dry ice.
2. Cryostage connected to a cooling bath (Digital One RTE
7 from Thermo Scientific NESLAB), a peltier unit (Series
800 from Alpha Omega Instruments), a fish pump (SAX1
from Pen Plax), and a drying tube.
3. Dehumidifier.
4. Digital camera (Nikon Coolpix 5000).
5. Microscope with 20Â magnification.
6. Borosilicate coverslips.
2.3 Image Analysis
1. Computer with ImageJ software.
2. Excel spreadsheet to generate the weighted average and standard error of mean grain size.
3 Methods
3.1 IRI Sample
Preparation
1. All IRI samples were lyophilized prior to IRI testing to ensure
they were devoid of solvent (confirmed by
1
H/
13
C NMR).
2. Prepare a minimum of five concentrations to be tested (using
standard solutions) for each compound ranging from 1 mM to
its maximum solubility. The maximum solubility of a compound is defined by the maximum concentration at which the
solution stays dissolved at room temperature (slight heating
can be applied to facilitate dissolution).
3. Prepare each concentration in triplicate (using three different
vials).
4. Prepare a vial of just the standard solutions without any compound present (i.e., 0 mM).
Ice Recrystallization Inhibition Assays
277