A Year in the Life: Preparing for PCR and Antibiotic Susceptibility Testing

Monday 17th February 2014

I began Monday morning checking to see if my restriction enzyme and antibiotic susceptibility discs had arrived yet. Unfortunately neither had been delivered, so I decided to begin preparing for the next stage of Escherichia coli isolate genotyping – polymerase chain reaction (PCR). I intend to use PCR in order to investigate each isolate for the presence of Bla genes which are associated with CTX-M ESBLs. By using a selection of primers, I can amplify any of the 5 types of CTX-M Bla genes – if they are present. Then run samples through electrophoresis in order to observe which type of CTX-M gene were present and had therefore been amplified. The presence of CTX-M genes is very interesting to me as they correspond to the production of ESBLs which my work is concerned with, especially in terms of antibiotic resistance.

The first stage after isolation of Escherichia coli is alkaline lysis, in order to isolate plasmid DNA from each sample. Several solutions are required for the alkaline lysis, therefore I began by preparing each:

Solution 1 – GTE (Lysis Buffer)

  • Glucose
  • Tris-Cl
  • Ethylenediaminetetraacetic acid (EDTA)

Solution 2 – SDS/NaOH (Lysis Buffer)

  • Sodium dodecyl sulfate (SDS)
  • Sodium hydroxide (NaOH)

Solution 3 – KOAc (Neutralising solution)

  • Potassium acetate
  • Glacial acetic acid

IMG_5348Unfortunately, making so many different solutions at once resulted in the accidental death of my favourite stirrer on our magnetic hotplate (Don’t worry though, he has gone on to be become the new lab fridge magnet!)

Tuesday 18th February 2014

The next stage after plasmid isolation is transformation. Transformation is the uptake of isolated plasmid DNA resistant to an antibiotic (In my research – cefotaxime) into a recipient chromosome of a cell which is in a competent state. In order to perform this I needed to produce competent cells, which required ice cold calcium chloride (CaCl2) and a 3/4 hour culture of Escherichia coli in LB broth. I began by producing a 1M stock of CaCl2, before filtering and storing it at 4ºC. I then prepared 100ml of LB broth by combining LB broth powder with distilled water in a Duran flask and autoclaving it. I also made 300ml of separate LB broth to use as a blank when measuring the OD of the 3/4 hour culture of Escherichia coli.

IMG_5322

My antibiotic susceptibility discs had finally arrived so I then inoculated individual nutrient broths with a single colony from my collection of Escherichia coli Lincoln isolate nutrient agar slopes. As I have quite a large collection of isolates now I decided to incubate 1-45 first and then finish the collection off on Wednesday. I incubated these overnight at 37ºC. Having used all of my nutrient broths, I then prepared, decanted and autoclaved more nutrient broth into glass universals.    

IMG_5331

Wednesday 19th February 2014

I began the day by removing my incubated nutrient broths from the incubator and spread plating each onto an individual Mueller Hinton agar plate. I left each to absorb for around 10 minutes before loading the antibiotic disc plunger with my newly arrived discs (Amoxycillin, Imipenem, Ceftazidime, Piperacillin/tazobactam, Ceftriaxone, Ampicillin, Cefotaxime and Gentamicin) and applying them onto each plate. These plates were then incubated overnight at 37ºC.

IMG_5332

I then inoculated the rest of the Lincoln isolate collection (46 – .140) in individual nutrient broths, and again incubated them overnight at 37ºC. As I was running low on Mueller Hinton agar plates I also decided to prepare, autoclave and pour more plates for use tomorrow.

IMG_5335

Finally, I streak plated a nutrient agar plate with a single colony from my E1 Escherichia coli nutrient agar slope ready for inoculation into LB broth tomorrow – to create competent cells. I incubated this streak plate overnight at 37ºC.

Thursday 20th February 2014

I started today preparing competent cells!

How to produce competent cells

  • Inoculate prepared and autoclaved 100ml LB broth with a single colony from overnight Escherichia coli streak plate.

IMG_5355

  • Incubate this LB broth with agitation at 37ºC for 3 hours before checking the OD at 595nm – an appropriate OD is approximately 0.35.

IMG_5361

  • Once this is reached separate the culture into two 50ml Falcon flasks, chill on ice for 10 minutes then centrifuge at full speed for 10 minutes.
  • Remove the supernatent and add 10ml of ice cold 0.1M CaCl2.
  • Re suspend the pellet by vortexing each flask.
  • Place both flasks on ice for 10 minutes before centrifuging again at full speed for 10 minutes.
  • Remove the supernatent and add 2ml of 0.1M CaCl2.
  • Re suspend the pellet by vortexing each flask and place on ice.
  • If not ready to conduct transformation, store at 4ºC in 2ml 0.1M CaCl2.

 

 

 

 

Once finished I removed my antibiotic susceptibility Mueller Hinton agar plates from incubation and began observation of clearing. Similarly to before I used a ruler to measure the zone of clearing around each antibiotic disc to the nearest mm and recorded it in a prepared table in my lab book.

IMG_5372Escherichia coli isolate 13 – if you look really carefully you can see it smiling…

I then spread plated the second batch of inoculated nutrient broths onto Mueller Hinton agar plates, allowing them to absorb, before applying antibiotic susceptibility discs and incubating overnight at 37ºC.

Friday 21st February 2014

Friday began with a supervisor meeting from 9.30am to 1pm. Once finished, I removed the second batch of antibiotic susceptibility Mueller Hinton agar plates from incubation and finished off observation/recording zone clearing into the table from Thursday.

I then decided to produce antibiotic (Cefotaxime) MLSA plates and LB broth ready for alkaline lysis and transformation next week. I began by producing a stock solution of cefotaxime to add to both agar and broth. I combined cefotaxime powder with distilled water then filtered.

IMG_5385

To prepare the MLSA and LB broth, I combined MLSA powder and technical agar/LB broth powder with distilled water before autoclaving. In order to prevent destruction of the antibiotic I waited until both were cool enough to pour and then I added my desired amount of the prepared cefotaxime stock to each. Each was then gently mixed to combine and poured/decanted aseptically.

Next week…

Similarly to last week, this week began fairly frustratingly with no restriction enzyme or antibiotic susceptibility discs! However, this did mean that I had time to prepare solutions for alkaline lysis, as well as competent cells for transformation and future PCR. I did, of course, manage to complete antibiotic susceptibility testing of all Escherichia coli isolates within my Lincoln collection and can now compare these results to data tables. This will allow me to identify those which are resistant, intermediate and susceptible to the antibiotics I used.

The next stage of my research involves me looking specifically at those isolates which demonstrate susceptibility and resistance to cefotaxime, hence my production of MLSA and LB broth containing cefotaxime. Beginning genotypic analysis of the isolates through PCR, will involve me looking specifically for CTX-M ESBLs by amplifying Bla genes. Hopefully my NotI restriction enzyme will also turn up next week, and I can continue perfecting PFGE Escherichia coli isolate plug production and PFGE running conditions!

A Year in the Life…

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