Hello Everyone! Sorry I Haven’t done a blog in a while… But i will start doing them now!
So here is a basic low down on what is going on in my project!
First I had been extracting E.coli from the Brayford and growing them on MSLA agar plates. These are the red plates and they will only grow the Ecoli from my sample as yellow, well rounded colonies. With the whole streak plate/broth/agar situation.. I have never really had much practice with it.. But now i know how to do it all! 🙂 (Thanks BioMeds)
In the last couple of days I have filtered 100ml of Brayford Water onto MSLA plates containing 100ug Ampicillin. This is a very high concentration of antibiotic, therefore the colonies I obtained on these plates were very resistant and pure. I then proceeded to make several LB broths with Ampicillin with the colonies from the plates. Today I used the QIAcube with my plasmid broths, and I also did alkaline lysis by hand and ran the samples side by side on a gel to observe the differences/similarity between the different methods and to see whether I had any plasmids and to interpret the banding patterns seen on the gel.
I found that in 2 out of my 6 samples I had a very distinct interesting band pattern occuring, so I will keep these and do futher work with them later and all the rest showed nothing so they have been disposed of!
I will now start making agar plates with different antibiotics on – starting with Ceftriaxone.
Once I have gathered all of this information from the different antibiotics I will see the range and frequency of plasmid stability systems present in plasmids conferring antibiotic resistance in the Brayford allowing understanding of their persistence in the environment!
This will also enable me to have an understanding of plasmids from E.coli that confer antibiotic resistance, focussing on the CTX-M ESBL gene which is found in E.coli which are resistant to third generation cephalosporins such as Ceftriaxone and cefotaxime. This is why I will be doing the above procedure just with these antibiotics on the plates and in the broths!
See you all tomorrow 🙂 Xxx