Author: Rosie Myers

Hello So my Antibiotic Cefotaxime has arrived I…

Rosie Myers / / Uncategorized

Hello,

So my Antibiotic – Cefotaxime has arrived! 🙂

I am now proceeding to follow all of the methods that I used with Ampicillin, just with the Cefotaxime instead and changing them slightly. I made up agar with 4mg/ml of cefotaxime in, and filtered 100ml of Brayford Water onto 2 of the plates. This morning I only had 3 colonies on just one of the plates. This concentration of Antibiotic is quite high for E.coli to be resistant too, so in a way the 3 colonies are highly resistant which is good, but I may have to start doing more plate sto get more out or maybe the rainy weather yesterday affected the amount of E.coli present.
I have now placed these colonies into broths containing cefotaxime again, and tomorrow I will proceed to do Alkaline Lysis with these samples. Once I have a few more, I will run them on a gel and see what banding patterns are going on 🙂

Hello Today i started producing competant cells and…

Rosie Myers / / Uncategorized

Hello!

Today i started producing competant cells and doing transformation.
Competent cells were made by first creating an LB broth by placing a colonie from a DH5 alpha plate into it, and having it incubated and shaking for around 5 hours! Once my cells were at the right optical density, I could proceed. As it happens they grew really fast and well so thats always a good start! Once I had centrifuged my cells down multiple times, placed them on ice, and added amounts of CaCl2 i ended up with cells which were then competent and capable of taking up plasmid DNA. The competent cells could be used immediately for transformation and (hopefully) uptake my plasmid DNA which i have obtained from my previous weeks in the lab!
I then began transformation where i added my different plasmid DNAs with some competant cells and proceeded to heat, cool, heat, cool etc! It is a very long process! But it enables the cells to recover and the antibiotic resistance of the plasmid to express itself 🙂
Basically with the solutions i then had, I spread 100ul from each one onto LB agar containing ampicillin, and if there are colonies present tomorrow morning then this means that the cells have uptaken my plasmids, and that i do have plasmids stocks that are resistant to amicillin! If i do i will have lots of things to play with 🙂 If not… i shall start all over again. 🙁
I have also spread a positive control, so that if it doesnt work tomorrow this will tell me if it was something in my methodology that was wrong or just that the plasmids in my DNA samples are not resistant to ampicillin, and it was probably just the chromosomes 🙁

Any questions feel free to come and ask! :):)

Hello Everyone Sorry I Haven’t done a blog…

Rosie Myers / / Uncategorized

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

Heya I’m Rosie and I have just finished…

Rosie Myers / / Uncategorized

Heya, I’m Rosie and I have just finished my second year as a Forensic Science Student. I will be starting in the micro lab tomorrow and I am looking forward to meeting you all and getting started! My project is looking at the stability systems of plasmids that confer antibiotic resistance, extracted from E. coli recovered from a natural body of water in Lincoln, (The brayford) and I will have Nicola working with me on the SGM project.

I guess I will be writing on here for help a lot with problems that I am inevitably going to come across, but this is why I am very lucky to have Nicola working with me and such a range of people in the lab. I cannot wait for the challenge! 😀

See you all tomorrow! 🙂