ý.,,: V. ý ýý . - Nottingham eTheses - University of Nottingham

protein (as might be found in a total cellular protein extract). the inhibitory activity was
still present and could be visualised against a negative control. This suggests that while
the mechanism of action is non-colligative, the level of activity and ice crystal
morphology are altered by AFP concentration. Secondly, Proteinase K (20mg mL-1.
Sigma) was added to a fish AFP III sample (incubated at 37°C for 1 hour) to destroy the
protein. After this treatment no diffraction was seen in the sample, indicating that ice-
growth inhibition was a result of the fish AFP III.
False negatives were ruled out based on the fact that positive controls never
appeared negative, unless there was some practical error when running the protocol. To
make sure that there were no errors, the experiment was always duplicated. However, it is
possible to concede that some bacteria that were AFP active may have appeared falsely
negative because of some step of the protein extraction protocol or indeed in the HTAP
analysis itself (this will be discussed more in section 4.3).
In field trials using Antarctic bacterial isolates, 1 mg mL-l fish AFP III was used
as a positive control and as an indicator of the opacity which AFP activity might produce
in the test solutions. The negative control was a sterile solution of 30% sucrose. If the test
solutions were darker/more opaque than the negative control then it was assumed that
these solutions had possible activity. Distinguishing the solutions was often difficult as
activity in the total cellular protein extracts was often extremely low (Fig. 4.4).
The actual methodology for the new protocol, named the high-throughput AFP
analysis protocol (HTAP), is as follows. 50 µL of 60% sucrose and 50 µL of protein
extract were aliquoted into 96 well microtitre plate. Fish AFP III (lmg mL-' with 30%
sucrose) was used as a positive control and 30% sucrose solution as a negative control.
The plates were placed at -70°C (cryo-freezer) for 10 minutes to supercool the samples
(producing embryonic ice crystals). The microtitre plate was then placed on a cold plate
(CamLab, Fryka KP 281) for recrystallisation. The cold plate was pre-cooled to -6°C and
stored in a cold room with an ambient temperature of -6°C, this ensured that there Was no
thermal difference (caused by ambient air temperature) within the solution. The plate was
viewed after 5 days using a light box in the cold room. so observation could occur at -6°C
to prevent temperature change within the sample altering ice-crystal size. The microtitre
plates were placed on a transparent plastic platform 15cm from the surface
118