Environmental Engineering Reference
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as well as cell numbers. Isolates recovered from these experiments in initial
experiments included two species of Chryseobacterium, three of Pseudomonas,
three of Acinetobacter, two of Bacillus and a Paenibacillus species [39]. Again,
a number of the recovered strains had interesting properties.
2.3.3 Screens for Ice Nucleation, Ice Recrystallization, Inhibition
and AFP Activity
Although not selective techniques, these methods have been developed as tools to
screen isolates for these properties. Ice nucleation screens are set up essentially as
previously described ice nucleation assays [31, 40, 41]. Briefly, small samples are
loaded onto a polarizing filter, placed in the cryocycler chamber, and illuminated
from below with a fiber-optic source. As the chamber temperature is lowered
from -1 to -158C (at 0.1-0.28C/min), software-controlled digital photographs are
captured every 60 sec through a crossed polarizing filter. After freezing the
samples become visible due to the rotation of the plane of polarization by the
ice crystals, and these images are automatically analyzed and transferred to a
spread sheet. The temperature at which 90% of the samples freeze is taken as the
ice nucleation temperature of the most active fraction of the sample. Groups of
cultures containing ice nucleators normally freeze at temperatures above -88C,
with control, samples freezing at about -128C. This method was used to identify
P. borealis isolates with ice nucleation activities [39] and to optimize the growth
conditions for the production of INPs in P. syringae, P. borealis and recombinant
cultures (S. Wu, D.N. Miao and V.K. Walker, unpublished).
The average ice nucleation temperature of any isolate or lysate can be
determined using a thermal analyzer. This apparatus records temperatures
from thermistors placed in 2-3 ml samples that are chilled from approximately
5 to -158Cat18C/min. The heat of fusion of first-order freezing and thawing
phase transitions causes temperature changes in the sample which are used to
record freezing and thawing [42]. Because of the larger volumes, samples
containing ice nucleators normally freeze above -88C, with controls freezing
at this point or lower. A typical profile from the thermal analyzer shows that
P. borealis, identified using ice-affinity, has ice nucleation activity (Fig. 2.4).
Another technique that has not been used by our group, but which has been
successfully employed to screen bacteria obtained from Antarctic lakes [7]
detects isolates that inhibit IR. Crude lysates from individual isolates are placed
into 96-well microtiter plates, brought to 30% sucrose (vol/vol) and quickly
frozen at -708C. After incubation at -68C for many hours (typically overnight),
frozen isolates with IR inhibition activity can be recognized since they appear
more opaque than the transparent lysates with no activity. IR inhibition
is correlated with AFP activity since at temperatures close to melting. AFPs
prevent the formation of large crystals, which have a relatively clear appear-
ance. In our experience, we have found crossed polarizing filters to be helpful
