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

oxidized and the sample then converted to CO2 and H2O. A dehumidifier removed the
water and a carrier gas swept the C02 to a Non-Dispersive Infrared (NDIR) Detector,
which measured the carbon concentration based on a Potassium Hydrogen Phthalate
(KHP) standard.
2.8 -
Bacterial and flagellate counts
Samples were stained with DAPI (4', 6-diamidino-2-phenylindole)
prior to
filtration through a 0.2µm polycarbonate filter (Poretics) using a pressure no greater than
5 In Hg vacuum. Bacteria were enumerated under epifluoresence microscopy using UV
excitation at x1200 magnification. Both UV and blue filter sets were used to discriminate
the autoflouresence of chlorophyll in the autotrophic bacteria and flagellates.
For each preparation, ten replicate Whipple grids were counted and mean values
calculated. Flagellates were counted as heterotrophic (no auto-fluorescence) or
autotrophic (via auto-fluorescent) in twenty fields of view from which the mean of each
was calculated.
2.9 -
Molecular typing techniques
To enable assessment of the distribution of AFP activity within the taxa of
isolated bacteria, molecular typing techniques were employed to produce a phenetic tree
of relatedness and a phylogenetic tree to suggest evolutionary relationships between the
bacterial isolates, which in turn provided insights not only into the relationships of AFP
active bacterial strains, but also information to enable future expeditions more readily
identify AFP active species in environmental samples.
2.9.1 -
Chromosomal DNA extraction
The CTAB (Cetyltrimethylammonium
bromide) method (Ausubel, 1999; from
Wilson, 1987) was employed for chromosomal DNA extraction. 50ml of bacterial culture
was grown in a conical flask to produce a turbid culture. The media upon which the
bacteria were originally isolated (TSA, YSA, SWA, 1/2 SWA - section 2.4) were used for
liquid media. The cultures were grown in a static incubator at 15°C. The cells were then
centrifuged for 10 min at 4000 g (6000 rpm in JA-20 rotor). The supernatant was
discarded and the pellet was resuspended in 9.5m1 of TE buffer (Appendix A2). To this
were added 0.5m1 of 10% SDS (Sigma) and 50µl of 20mg/ml proteinase K (Sigma),
which was then mixed. This was incubated for 1h at 37°C. Following incubation, I. 8ml
41