Monday 24th March 2014
I started off the week by collecting my autoclaved Duran flasks, extendable rod and gloves, and heading out to the River Witham to collect samples from the same location as previously. Once finished I returned to the lab and began filtering my collected water through filter membranes and applying these to MLSA plates, using the same method as before. I then incubated these overnight at 44ºC.
Once finished I then prepared my newest batch of bacterial plugs for PFGE by first incubating them in 0.1 x wash buffer for 1 hour. This was then removed and replaced with 1 x NotI buffer for another hour with gentle agitation. This was again removed and replaced with 1 x NotI buffer and NotI restriction enzyme, incubating overnight at 37ºC.
Finally I prepared, decanted and autoclaved fresh LB broth ready for inoculation with Escherichia coli colonies tomorrow. Once autoclaved and cooled, CTX antibiotic was added to each individually.
Tuesday 25th March 2014
Today started by removing the incubated MLSA plates and inoculating single colonies from the plates into separate LB broths containing antibiotic. These were then incubated overnight with agitation at 37ºC.
I then collected the bacterial plugs from incubation, removing the buffer and enzyme before incubating the plugs in 1 x wash buffer for an hour with agitation. This was then removed and replaced with 0.5 x TAE buffer. While they were incubating I prepared a 1% agarose gel as before. I then loaded this gel with the plugs as well as an appropriate ladder before running overnight using the following settings:
- 20 hours
- 1% agarose gel
- 0.5x TAE
- 40-100 switch intervals per second
- 6 V/cm
- 30 minute stain, 10 minute destain
As these are new plugs produced from different isolates I decided to use my most successful settings so far for Escherichia coli isolate PFGE separation.
Wednesday 26th March 2014
Today began with removing my overnight PFGE gel, staining for 30 minutes then destaining for 10 minutes before viewing under UV. The resulting gel was fairly disappointing – demonstrating clear and appropriate banding for the ladder used however very unclear blurring for the new Escherichia coli bacterial plugs. This excessive blurring indicated that the plugs contained too much DNA, that hadn’t been properly lysed and therefore were also not appropriately cut during incubation with restriction enzyme. As a result of this it was decided to put PFGE on hold until next week when another batch of Escherichia coli bacterial plugs could be made using less DNA.
I then removed the incubated LB broths and inoculated each into lactose peptone water (LPW) and tryptone water (TW) before incubating overnight at 44ºC. Nutrient agar plates were then streak plated with overnight LB broth in order to obtain single colonies and check that all isolated Escherichia coli samples were pure. These plates were incubated overnight at 44ºC. 10μl of each overnight sample was then spread plated onto mueller hinton agar, allowed to absorb before applying with antibiotic susceptibility discs, as before. These plates were also incubated overnight at 44ºC.
Finally, in preparation for tomorrow I made fresh LPW, TW and nutrient agar slopes – as before.
Thursday 27th March 2014
I started off today with observing overnight LPW and TW broths, as well as mueller hinton antibiotic susceptibility and nutrient agar streak plates. As before, the results of each test was recorded in a table in my lab book.
Once finished I prepared fresh nutrient agar slopes and inoculated each with a single colony from my overnight nutrient agar plates. Finally I finished streak plating nutrient agar plates with overnight LB broth in order to obtain single colonies and check that all isolated Escherichia coli samples were pure. Again these plates were incubated overnight at 44ºC.
Friday 28th March 2014
Friday was a fairly quiet day, I observed my overnight streak plates and then inoculated a single colony onto an individual nutrient agar slope.
I would really like to work with the QIAcube next week, therefore I have scheduled a training session with one of my microbiology/molecular biology technicians. In previous weeks I have conducted plasmid isolation by alkaline lysis, which gives fairly good results, however it is very time consuming and requires a lot of pipetting. The QIAcube can process 12 samples in around 50 minutes and gives less end product than alkaline lysis but the final sample is a lot purer. I would like to re process all my Lincoln collection samples and then re run the resulting plasmid DNA through gel electrophoresis.
After the disappointing results from my new bacterial plugs this week, I really want to re make these plugs ensuring that not as much DNA is present. Hopefully by reducing the 16 hour incubation it will give less blurring and clearer band patterns through PFGE.
A Year in the Lab…