Monday 17th March 2014
This week began with preparing overnight cultures of 2 E.coli samples from my Lincoln isolate collection ready for plug preparation. I did this by taking a single colony from the appropriate nutrient agar slope and inoculating into LB broth, incubating overnight at 37ºC with agitation.
Once finished I then continued glycerol stocking my Lincoln bacterial isolate collection by streak plating the rest of the sloped isolates (E 42-.140) onto nutrient agar in order to ensure all samples were purely Escherichia coli and achieve single colonies. These plates were then incubated overnight at 37ºC.
Tuesday 18th March 2014
Today started with removing the overnight streak plates from the incubator and inoculating LB broth with a single colony from each plate. These broths were then incubated overnight at 37ºC.
Once finished I began bacterial plug production by removing the overnight broths and reading the optical density (OD) using a bench top spectrometer at 595nm. Appropriate OD for PFGE plug production is between 0.8-1, as this ensures enough bacterial cells are present to result in a significant amount of DNA after lysis. Each bacterial broth was then processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.
Wednesday 19th March 2014
Once plugs had finished incubation in proteinase K, each plug was washed to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs were then stored in 1x wash buffer at 4ºC.
I then removed the overnight LB broth cultures from incubation and continued glycerol stocking. I pipetted 500μl of each overnight LB broth culture into separate tubes before adding 500μl of 10% glycerol LB broth. These were then carefully labelled and stored in the freezer.
Thursday 20th – Friday 21st March 2014
As I didn’t have enough time left in the week to incubate the new batch of plug in restriction enzyme and then run PFGE I decided to just keep the plugs stored in 1x wash buffer at 4ºC until next week.
I then started preparation for further sample collection next week. As I have been processing my collected water samples they have slowly been whittled down to a much smaller collection than originally started with. Therefore I would really like to collect some more water to ensure I end up with a substantial collection before I begin PCR. I found and autoclaved 2 empty Duran flasks in preparation for water collection, then made, decanted and autoclaved lactose peptone water and tryptone water for E.coli identification.
Now I have a new batch of bacterial plugs made from samples in my Lincoln isolate collection I would like to start the week off by running these through PFGE with my best settings to date to see how the banding patterns differ from each other. Therefore, beginning to visulise species differences between Escherichia isolates and allow comparison. I would also like to collect and process more water samples to ensure I have a substantial amount of samples within the Lincoln collection before I began conducting PCR.
A Year in the Lab…