A Year in the Lab: Perfecting PFGE and Collecting Water Samples in Lincoln

Monday 3rd February 2014

I began the week by collecting the equipment I would need for water sample collection in Lincoln:

  • 2 autoclaved 500ml Duran Flasks
  • Extendable water collection rod
  • Gloves

I also decided to set up the water filtration system ready for when I got back from water collection. I then headed out to North Hykeham in order to take 2 water samples from the stretch of the river Witham where effluent is disposed from the nearby waste water treatment plant (WWTP). After my River Witham walk I am very familiar with this particular stretch of river and therefore it only took around an hour to travel there from the Science Building at Lincoln University, walk round to site, collect samples and return.

Once back I began filtering 100μl of my collected samples through individual 0.45 micron filter membranes, before using forceps to carefully place each filter side up onto MLSA plates, incubating overnight at 44ºC. MLSA, 0.45 micron filters and incubation at 44ºC were chosen to encourage selective growth of Escherichia coli. 

IMG_5171

I decided to set up and run another PFGE gel in order to continue perfecting the settings for Escherichia coli plugs. After observing my previous gel from last Friday I decided on trying the following settings:

  • 14ºC
  • 1% Agarose Gel
  • 0.5x TAE Buffer
  • 24 Hours
  • 3V/cm
  • 50-110 Second Switch Intervals

I choose these settings in order to test if a change in V/cm would result in clearer and more separated bands, as well as helping to prevent my samples and ladder running off the bottom of the gel. I boiled and poured my gel then filled the wells with cut CHEF DNA marker ladder and Escherichia coli plugs before running the gel overnight.

Finally I made, autoclaved and poured another stack of MLSA plates in preparation for streak plating out isolated Escherichia coli to confirm identification.

Tuesday 4th February 2014

I started the day by removing and observing the MLSA plates which were inoculated with water sample filter membranes. All MLSA plates showed characteristic abundant yellow colonies which indicated the presence of Escherichia coli. Each individual colony was placed into 10ml of nutrient broth before incubation overnight at 44ºC.

IMG_5188IMG_5191

Once finished I then removed the PFGE gel I set up the day before, draining the unit, staining with ethidium bromide and viewing under UV light. The gel was very disappointing, as movement through the gel was significantly reduced with no banding visible from the ladder used. Dark wells made it clear that DNA within the plugs hadn’t managed to move out and into the gel enough to demonstrate bands. It was decided to perform a fresh restriction digest on different plugs from the original first batch, in order to determine if the problems were with the plugs or with the change to 3V/cm.

PFGE Gel 4

I therefore incubated each plug in NotI restriction enzyme and buffer overnight at 37ºC. I then made some more nutrient broth, decanting 10ml into each glass universal before autoclaving.

Wednesday 5th February 2014

Today began by removing the inoculated nutrient broths from the incubator and streak plating each onto MLSA in order to confirm that each were pure Escherichia coli isolates. These plates were then incubated overnight at 44ºC.

IMG_5201

I also inoculated nutrient agar slopes/slants with a disposable loop dipped into overnight inoculated nutrient broth to ensure that I have slow growing colonies continuously available. I used all the nutrient agar slopes/slants that I had, so I decided to produce more for further inoculations. I combined nutrient agar with distilled water before boiling, decanting 10ml into glass universals, autoclaving and tilting to produce sloped/slanted surface when set.

IMG_5196

I removed the newly incubated plugs from restriction digest before washing with wash buffer and equilibrating in 0.5x TAE buffer while I set up a new PFGE gel. I boiled and poured my gel, then filled the wells with cut CHEF DNA marker ladder and newly digested Escherichia coli plugs before running the gel overnight, using the following settings:

  • 14ºC
  • 1% Agarose Gel
  • 0.5x TAE Buffer
  • 24 Hours
  • 6V/cm
  • 50-110 Second Switch Intervals

I decided to use 6V/cm as it gave successful results last week and would hopefully allow me to observe whether the problems were due to the change in voltage or the plugs.

Thursday 6th February 2014

I began the day by removing the streak plated MLSA plates from incubation and observing/photographing each on an illuminated colony counter. All plates had grown colonies – mostly yellow Escherichia coli, however some plates demonstrated pink colonies which indicated a mixed organism sample. A singly colony was removed from each plate and inoculated into tryptone peptone water and incubated overnight at 44ºC.

IMG_5248Escherichia coli isolate streak plated onto MLSA

Once it had finished I removed and stained my latest gel with ethidium bromide, viewing under UV light. This gel was the best I have produced so far! The CHEF DNA marker ladder demonstrated clear bands and the Escherichia coli plugs had begun to show visible bands amongst some blurring. This confirmed that the change in voltage may have affected the previous gel and that so far 6V/cm has given better results in terms of separation. One thing that was noted was that bands seen from Escherichia coli plugs were still fairly blurry, which may be due to insignificant amount of cell lysis during lysozyme, proteinase K and washing stages. It was therefore decided to make a second batch of plugs using more identified Escherichia coli isolates, modifying the cell lysis and washing procedure to create cleaner and clearer bands during PFGE.

PFGE Gel 5

It was also decided to try destaining the gel after the initial ethidium bromide stain, in order to clean up the background and blurred staining surrounding the bands. Time also remains a problem with the samples still running off the bottom of the gel, it was therefore decided to reduce the overall run time to 22 hours in attempts to reduce this. This will however have to be conducted next week due to the time it takes for plug production to be completed.

Friday 7th February 2014

Today started with a supervisor meeting from 10am until 12pm. Once finished I removed the inoculated tryptone peptone waters before adding 5 drops of Kovac’s reagent to each and observing any colour change. As before, the presence of a cherry red ring indicates an indole positive organism, in this case Escherichia coli while a yellow/lack of colour change indicates an indole negative organism. This allowed me to determine that the majority of isolated organisms were Escherichia coli, however several were indole negative and were therefore removed from the collection. This collection will be further supplemented with Escherichia coli isolates from the original bacterial isolate collection I identified in order to give around 50 isolates.

IMG_5252

Next week…

This week was very successful, resulting in the isolation of a full collection of Escherichia coli isolates ready for antibiotic resistance testing and the production of my best PFGE gel to date! Next week will involve continuing work to perfect PFGE of Escherichia coli isolate plugs, starting with the modification of their production. It will also involve antibiotic resistance testing of Lincoln Escherichia coli isolate collection, as well as beginning work on identification of CTX by PCR.

A Year in the Lab…

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