A Year in the Lab: Preparing for PFGE and PCR of Lincoln and Bradford Collections

Tuesday 20th May

In order to confirm that all my isolated water samples are purely Escherichia coli and that my nutrient agar slopes haven’t become contaminated over time I decided to inoculate nutrient agar plates. Instead of streak plating completely I decided to just streak once across the whole plate as I am only concerned with viewing colony growth/morphology. I began with all my isolated Lincoln Escherichia coli samples as well as a reference strain for Escherichia coli ATCC 25922 to allow comparison. These plates were then incubated overnight at 44ºC.

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Wednesday 21st May

Today started with removing my streaked agar plates from incubation and comparing them to my reference strain in order to confirm purity. I used a piece of black cloth as a background in order to examine the colony growth/morphology carefully. The samples which were positively confirmed as being Escherichia coli will be made into bacterial plugs for use in PFGE, as well as for PCR.

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Wednesday 28th May

I began today by preparing, autoclaving and pouring more nutrient agar plates.

I spent the rest of the day writing up a plan for lab work for next month, after my exam facilitation work is finished. I also showed one of our laboratory technicians, who will be completing some research work, how to use the Spiral Plater. I prepared some MLSA plates as well as some Escherichia coli overnight broths, which we diluted before spiral plating and incubating overnight.

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Thursday 29th May

I started today by streaking the nutrient plates I made yesterday with all my isolated Bradford Escherichia coli samples as well as a reference strain of Escherichia coli ATCC 25922 to allow comparison. These plates were then incubated overnight at 44ºC.

Following on from yesterday’s spiral plating work, we viewed the incubated spiral plates and I showed him how to use the automatic plate reader.

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Friday 30th May

I removed my streaked agar plates from incubation and compared them to my reference strain in order to confirm purity. Again I used a piece of black cloth as a background in order to examine the colony growth/morphology carefully. The samples which were positively confirmed as being Escherichia coli will be made into bacterial plugs for use in PFGE, as well as PCR.

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Next week…

Now my exam facilitation work is all finished I will be able to get back into the lab and start producing bacterial plugs from all the samples which were positively confirmed as being Escherichia coli (Both Lincoln and Bradford collections). While I am producing these I will simultaneously be running my PCR primer temperature gradients, as well as beginning to run the newly produced bacterial plugs through PFGE.

A Year in the Lab…

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