A Year in the Lab: Running PFGE and Preparing for Lincoln Water Sample Collection

Monday 27th January 2014

I began the week by conducting a restriction digest on my washed Escherichia coli PFGE plugs from last week. The restriction enzyme NotI ‘cuts’ the bacterial DNA present at a specific sequence of bases resulting in multiple fragments of varying length that can then be visulised by PFGE.

How to conduct a restriction digestion on PFGE plugs

  • Wash each plug for an hour in 1ml of 0.1x wash buffer at room temperature with agitation.
  • Remove wash buffer and add another 1ml of 0.1x wash buffer.

IMG_5078

  • Remove this and add 1ml of 1x NotI buffer for an hour at room temperature.

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  • Remove this buffer and add 300μl 1x NotI and 30U/ml of NotI restriction enzyme, incubating overnight at 37ºC.

During these incubations I researched the antibiotic discs that would be most suitable and useful to use on my collected and isolated organisms next week. As I will be isolating a collection of extended spectrum beta lactamase (ESBL) producing Enterobacteriacae, specifically: Escherichia coli and Klebsiella spp. the most useful resistance to test for would be those which are as a result of ESBLs. ESBLs are hydrolytic enzymes produced by a variety of Gram negative bacteria that make them resistant to cephalosporins (cefuroxime, cefotaxime and ceftazidime), penicillins and monobactams. As these antibiotics are now widely used in many hospitals, resistance poses a major health concern both in the UK and worldwide.

I decided on using a selection of the following:

Ampicillin/Amoxicillin

Ceftazidime (Cefotaxime/Ceftriaxone)

Ciprofloxacin

Gentamicin

Imipenem/Meropenem

Ertapenem

Piperacillin/Tazobactam

Penicillin G

I also recorded information on the references ranges for each antibiotic disc, including measurements for individual organisms: Resistant, Intermediate and Susceptible to antibiotics.

Tuesday 28th January 2014

I started the day by continuing PFGE plug restriction digestion:

  • Remove NotI and buffer, and add 1ml 1x wash buffer to each plug for 30 minutes at room temperature with agitation.
  • Remove wash buffer and equilibrate each plug in 0.5x TAE buffer for an hour until use.

While the plugs were incubating I made and poured a 1% agarose gel in the specific gel box for PFGE, which has a removable base that fits into the CHEF unit. I also made up 6L of 0.5x TAE buffer (PFGE uses a lot more buffer than standard electrophoresis!) which I stored in a large plastic container. I have another of these containers to drain the used buffer into after running, as it can be used again but does have a limited numbers of uses.

IMG_5085

Once ready to run I removed the plugs from buffer and using a marked petri dish to measure easily, I cut off a 3mm section of each plug as well as a Saccharomyces cerevisiae PFGE ladder and low range PFGE ladder to allow comparison. Then, using a small sterilised spatula I gently pushed each into individual wells in the prepared and set 1% agarose gel.

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Once loaded I removed the outer casing of the gel box and placed the gel (Still on top of the base, to prevent plugs falling out) into the CHEF PFGE unit. I then filled the unit with the prepared 0.5x TAE buffer until the gel was covered and turned on the cooling module and pump to get the buffer flowing and cooling.

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I decided to try these plugs with settings that were recommended by a paper attached to the bacterial PFGE plug production kit details:

  • 1% agarose gel
  • 0.5x TAE buffer
  • 22 hours
  • 6V/cm (200V)
  • 5-50 second switch intervals
  • Unsealed plugs
  • 14ºC

I set up the CHEF PFGE unit according to these settings and ran the gel overnight for 22 hours.

Once finished I decided to begin preparing equipment for next week’s collection of Lincoln river Witham water samples. I began by finding and autoclaving 2 500ml Duran flasks ready for water collection using the extendable rod that I used before to collect water from the Brayford Pool. I located 0.45 micron filter membranes for initial water filtration and then made, autoclaved and aseptically poured a stack of MLSA (For initial overnight filter membrane growth) and MH agar plates (Antibiotic disc resistance testing).

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I also made, decanted and autoclaved baskets of 50 10ml tryptone and nutrient broths for indole testing.

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Wednesday 29th January 2014

I started the day by removing, staining and observing my first PFGE gel which contained identified Escherichia coli isolates. One thing I was particularly interested in finding out from this first gel was whether my first batch of PFGE plugs contained enough DNA and whether the DNA present had been sufficiently cut by the restriction enzyme used. This is very important as if not enough DNA is present and not enough restriction enzyme used DNA will not be cut into fragments and bands will not be visible on a gel.

Dark wells confirmed that an appropriate amount of DNA was present within the plugs, while the presence of dark smeared bands throughout the gel confirmed that enough restriction enzyme was used to cut the DNA and allow separation of fragments according to size. I therefore decided to conduct another restriction digest on some more of the plugs in the first batch, ready for running after the current plugs were used up.

PFGE Gel 1

However, volts were accidentally set at 20V rather than 6V, therefore the gel could not be used to draw any further conclusions. Modifications were made and another gel was ran overnight for 24 hours. Using the following settings:

  • 1% agarose gel
  • 0.5x TAE buffer
  • 24 hours
  • 6V/cm (200V)
  • 60 – 120 second switch intervals
  • Unsealed plugs
  • 14ºC

Thursday 30th January 2014

I started the day by removing my incubated plugs from restriction digest and incubating in 1ml 1x wash buffer for 30 minutes at room temperature with agitation. I then removed the wash buffer and added 1ml of 0.5x TAE buffer allowing equilibration for an hour.

IMG_5128

During incubation I removed, stained and viewed my overnight gel. When I previously conducted PFGE the cooling module was not working, and this really affected how the gels we produced ran. If a gel becomes too hot during a run this can cause DNA to denature and as a result the bands produced become smeared or abnormal. However, as we now have a working cooling unit the gel and surrounding buffer can be constantly maintained at 14ºC.

IMG_5098

The use of 0.5x TAE buffer worked very well along with using 6V/cm (200V). However, 24 hours appeared too long as samples ran off the bottom of the gel. 60 – 120 second switch intervals was too high to allow significant separation, resulting in smearing and unclear bands. Unsealing the plugs did not seem to affect the DNA within the plugs and it’s movement into the gel. The Saccharomyces cerevisiae PFGE ladder worked very well, offering appropriate bands in regards to fragments produced from cut Escherichia coli DNA. However, the low range PFGE ladder produced bands that were much too small for appropriate comparison to cut Escherichia coli DNA. Therefore it was decided to just use the Saccharomyces cerevisiae PFGE ladder.

PFGE Gel 2

The following settings were tried to prevent samples running off the end of the gel and produce clearer separation of bands:

  • 1% agarose gel
  • 0.5x TAE buffer
  • 22 hours
  • 6V/cm (200V)
  • 50 – 100 second switch intervals
  • Sealed plugs
  • 14ºC

IMG_5132

Friday 31st January 2014 

As I set up my PFGE gel fairly late Thursday I didn’t go into the lab until Friday afternoon. I removed, stained and observed the gel. Although I reduced the run time to prevent samples running off the bottom of the gel, this gel demonstrated that this was still happening. I also reduced the switch interval timings, however I don’t think I reduced them enough to make a significant difference. As it was Friday I couldn’t run another gel, so decided to wait until Monday to try out running for the same time but using a much lower switch interval timing to encourage quicker and clearer band separation.

PFGE Gel 3

Next week…

After modifying my GS3 according to my supervisor meeting last week, next week I plan to collect 2 500ml Duran flasks of WWTP effluent from the river Witham in Lincoln, in order to begin isolating Escherichia coli. After collection 50ml/100ml of water will be filtered through a 0.45 micron filter membrane, applied to an MLSA plate before incubating overnight at 44ºC. Escherichia coli colonies appear pink on MLSA and identification can then be confirmed through broth growth. Broth can then be spread plated onto MH agar before application of appropriate antibiotic disc and incubation 0vernight at 37ºC.

I will also continue to run my Escherichia coli PFGE plugs, perfecting the overall run time and switch interval timings to ensure good and clear band separation.

A Year in the Lab…

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