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

digital camera (Fig. 4.3 & 4.4). After several pictures were taken, the plate was placed
back on the cold block in case the digital pictures were not satisfactory.
4.1.2.3 -
Advantages and disadvantages of the assay
The HTAP assay is much cheaper than the `SPLAT' assay as the only pieces of
specialised equipment necessary were the freeze plate (CamLab, Fryka KP 281) and
-70°C freezer (which is common place in most well equipped laboratories). Apart from
consumables (sucrose, microtitre plates, etc. ) and the equipment involved in the protein
extraction methodology, there are no further costs. The `SPLAT' assay required
expensive CO2 cylinders, solvents (2,2,4 - trimethyl pentane) and microscope objectives
(EF L 20/0.30 160/0-2). Although the -70°C
freezer was an advantage in the HTAP assay,
tests using a -20°C freezer proved successful at establishing activity, although the test
was not as discriminatory as when using -70°C to supercool the samples.
Identification of AFP activity within the protein samples was sometimes hindered
by colouration, caused by carotenoid pigments (found extensively in Antarctic bacteria as
protection against UV damage; Vincent & Quesada, 1994, George et al., 2001), which
were not removed by the protein extraction protocol. Cultured Antarctic bacterial samples
within this study showed high levels of carotenoid pigmentation. Colouration does not
affect a `SPLAT' assay because the sample used is so thin and analysis is done at the
microscopic level, but with a HTAP assay the large volume of sample required (50 µL)
means that coloration can make it very difficult to differentiate between an active or non-
active sample. Normally a darkened sample, i. e. one which has greater opacity, would be
considered positive, but, if a sample has colouration, it is difficult to ascertain the level of
refraction. This artefact meant that samples with refraction inhibitory colouration were
recorded as positive for AFP activity and then assessed using the `SPLAT' assay at a
later date. Due to the large sample size and uncertainty of location of pigment within the
cell, fractionation of the solution to remove pigmentation was not performed. Despite the
fact that a large number of false positive are incurred using this protocol. it is still more
advantageous as a first step field screen for reducing the number of isolates which require
' SPLAT' analysis. It therefore provides more time for field analysis and bacterial
isolation and prevents the need for costly and cumbersome `SPLAT' analysis in the field.
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