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

with a Bunsen burner. The sections were then melted out at 1 °C in filtered (GF/F,
Whatman) and sterilized (120°C for 20 minutes in autoclave) lake water from the lake of
origin, to prevent osmotic shock when the microbes were defrosted. The inoculation
process was the same as for lake water. The plates were incubated at +17°C for 1 week,
after which the plates were sub-cultured using a sterile streak plate technique, so that
individual isolates (determined by colony morphology) were plated on to the media upon
which they were originally grown. Each isolate was numbered, recorded in a database
and then incubated at +17°C for 1 week. Original sample plates were stored at 1 °C until
verification of isolate growth was made from sub-cultures. Once sub-cultures were grown
and verified, they were Gram stained (refer to Gram stain - section 2.6). All Gram stain
results were recorded into a database along with basic observations of cellular and colony
morphology (Appendix B 1).
2.5 -
Preservation and transportation of the microbial collection.
In order to maintain pure isolates and to transport them between Antarctica and
England, it was necessary to place the cultures in cryogenic stasis. The isolates were
cultured in their respective liquid media containing 10% glycerol (Appendix A 1). Each
isolate was inoculated into 500µl of medium, which had been aliquoted individually into
sterile 1.5m1 flip top Eppendorf tubes (autoclaved 121 °C, 20 minutes). Once inoculated,
the tubes were incubated at +15°C for 1 week. Following incubation, the tubes were
frozen at -20°C for 1 week. After one week of storage, plating them out onto solid media
tested the viability of each of the cryo-cultures. Once viability and correct isolate
identification (via colony morphology) had been assessed, the original plated cultures
were destroyed. All isolates were duplicated and putative AFP active isolates were
quadruplicated.
2.6 -
Gram stain technique
All isolates were Gram stained for basic phenetic classification using a basic
Gram method (Rodina, 1972). Mid log phase growth cultures provide the best results
with the Gram stain as variability can occur in very young or very old cells (Singleton &
Sainsbury, 1997), therefore cultures incubated for up to 1 week were used. The isolates
were tested using the following procedure. A heat-fixed smear of bacterial cells was
stained for approximately I minute, using Crystal Violet dye (triphenylmethane dye 88%,
38