as outlining the effect of small concentration changes on ice recrystallization. Values can be derived from these experiments that
describe the concentration at which recrystallization inhibition
activity no longer occurs [59]. One of the main issues in this field
is that researchers are using these different assays to assess recrystallization, and the variance between the assays is not known; thus it
makes direct comparisons of data impossible.
Of the aforementioned assays, the SCA and the SSA are the
most common. In the SCA and SSA, recrystallization is analyzed in
solutions containing various concentrations of salts or sugars. Currently, the splat assay (SCA) and the sucrose assay (SSA) are used
extensively to measure IRI activity [4, 62]. The objective of this
chapter is to outline how each of these assays are performed and to
identify any differences in qualitative and quantitative aspects of the
two assays as well as emphasize the importance in selecting the
proper assay when measuring IRI activity.
1.2 Examples of Ice
Recrystallization
Assays
Two ice recrystallization assays can be used, namely, the splat assay
(SCA) and sucrose assay (SSA). The biggest difference between the
two assays is the solution that the analyte is dissolved in. In the case
of the splat assay (SCA), phosphate buffered saline (PBS) is utilized
[62] while the analyte is dissolved in a 40% sucrose solution in the
sucrose assay (SSA). The difference in ice crystal structure between
the standard SCA and the standard SSA is quite evident even when
using very fast cooling rates in the two assays (see Fig. 1). In the
splat assay, freezing a 10 μL drop of PBS via dropping the solution
onto a block of aluminum pre-cooled to À80
C generated a frozen
wafer. After a 40 min annealing time at À6.4
C [64], the ice
crystals recrystallized to a larger mean grain size (see Fig. 1a) with
a very small unfrozen fraction. In the sucrose assay (SSA), the
frozen wafer is produced by rapid cooling of a 1 μL sample on a
pre-cooled aluminum block (À80
C). We expected the cooling
rate in the splat assay to be very high and thought it unlikely that we
could obtain the same rate using the sucrose assay where the
solution is frozen at a rate of 60
C/min (measured independently
with a thermocouple) (see Note 1).
To enable rapid freezing, the volume of the sample in the
sucrose assay (SSA) was reduced. A 1 μL amount of 40% sucrose
using the SSA method resulted in a slurry containing smaller ice
crystals in a large unfrozen fraction after 40 min of annealing at
À6.4
C (see Fig. 1b). The formation of a slurry in the SSA method
can be attributed to the highly concentrated 40% sucrose present.
As the temperature of the solution decreases, the pure water freezes
leaving the solute to concentrate in the open unfrozen channels
[65]. The increasing concentration of solute in the unfrozen fraction leads to an overall depressed freezing point. The highly concentrated unfrozen fraction also leaves little room for the formation
of large ice crystals as seen in the SCA.
Ice Recrystallization Inhibition Assays
273
describe the concentration at which recrystallization inhibition
activity no longer occurs [59]. One of the main issues in this field
is that researchers are using these different assays to assess recrystallization, and the variance between the assays is not known; thus it
makes direct comparisons of data impossible.
Of the aforementioned assays, the SCA and the SSA are the
most common. In the SCA and SSA, recrystallization is analyzed in
solutions containing various concentrations of salts or sugars. Currently, the splat assay (SCA) and the sucrose assay (SSA) are used
extensively to measure IRI activity [4, 62]. The objective of this
chapter is to outline how each of these assays are performed and to
identify any differences in qualitative and quantitative aspects of the
two assays as well as emphasize the importance in selecting the
proper assay when measuring IRI activity.
1.2 Examples of Ice
Recrystallization
Assays
Two ice recrystallization assays can be used, namely, the splat assay
(SCA) and sucrose assay (SSA). The biggest difference between the
two assays is the solution that the analyte is dissolved in. In the case
of the splat assay (SCA), phosphate buffered saline (PBS) is utilized
[62] while the analyte is dissolved in a 40% sucrose solution in the
sucrose assay (SSA). The difference in ice crystal structure between
the standard SCA and the standard SSA is quite evident even when
using very fast cooling rates in the two assays (see Fig. 1). In the
splat assay, freezing a 10 μL drop of PBS via dropping the solution
onto a block of aluminum pre-cooled to À80
C generated a frozen
wafer. After a 40 min annealing time at À6.4
C [64], the ice
crystals recrystallized to a larger mean grain size (see Fig. 1a) with
a very small unfrozen fraction. In the sucrose assay (SSA), the
frozen wafer is produced by rapid cooling of a 1 μL sample on a
pre-cooled aluminum block (À80
C). We expected the cooling
rate in the splat assay to be very high and thought it unlikely that we
could obtain the same rate using the sucrose assay where the
solution is frozen at a rate of 60
C/min (measured independently
with a thermocouple) (see Note 1).
To enable rapid freezing, the volume of the sample in the
sucrose assay (SSA) was reduced. A 1 μL amount of 40% sucrose
using the SSA method resulted in a slurry containing smaller ice
crystals in a large unfrozen fraction after 40 min of annealing at
À6.4
C (see Fig. 1b). The formation of a slurry in the SSA method
can be attributed to the highly concentrated 40% sucrose present.
As the temperature of the solution decreases, the pure water freezes
leaving the solute to concentrate in the open unfrozen channels
[65]. The increasing concentration of solute in the unfrozen fraction leads to an overall depressed freezing point. The highly concentrated unfrozen fraction also leaves little room for the formation
of large ice crystals as seen in the SCA.
Ice Recrystallization Inhibition Assays
273