Monday 10th February
This week actually began Sunday evening, with the inoculation of three nutrient broths with single colonies from individual Escherichia coli nutrient agar slopes. These were then incubated overnight at 37ºC, for 16 hours to obtain maximum growth for PFGE plug production.
After 16 hours incubation the broths were removed and the optical density (OD) read using a bench top spectrometer at 595nm. Appropriate OD for PFGE plug production is between 0.8-1, as this ensures enough bacterial cells are present to result in a significant amount of DNA after lysis. Each broth varies slightly due to individual differences, so it is important to note down the individual OD.
Each bacterial broth was then processed as before, however with an increase amount of lysozyme and proteinase K added to each plug, as well as an increase in incubation. This was conducted in attempts to increase cell lysis, as mentioned last week, in order to reduce the smearing seen during PFGE of plugs.
I then reviewed the database I produced during phenotypic analysis of the original Lincoln bacterial isolate collection, in order to select isolates which were Escherichia coli and had tested positive for CTX-M. These glycerol stock samples were then spread plated onto MLSA and incubated overnight at 44°C.
More samples were still required to produce a large collection of Escherichia coli isolates to represent Lincoln, therefore it was decided to filter more of the waste water treatment plant effluent onto MLSA. This was conducted in the same way as previously, and incubated overnight at 44°C.
Tuesday 11th February
Once plugs had finished incubation in proteinase K, each plug was washed to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same, and plugs were then stored in 1x wash buffer at 4ºC. I did want to conduct a restriction digest on this new batch of plugs overnight ready for Wednesday, however my order of fresh NotI had not come yet and therefore the plugs were stored until the restriction enzyme arrived.
The overnight spread plated Escherichia coli isolates were then removed from the incubator and a single colony inoculated into tryptone peptone water and incubated overnight at 44°C, to allow indole testing. Another single colony was removed from each plate and inoculated onto individual nutrient agar slopes to allow slow bench growth and continuous access to single colonies.
Finally, stocks were produced as before for the following: MLSA plates, Nutrient broth, Tryptone peptone water and Nutrient agar slopes.
Wednesday 12th – Friday 14th February
Today began with indole testing of overnight tryptone peptone waters inoculated with Escherichia coli isolates. This was conducted as before, by adding a few drops of Kovac’s reagent and observing for a colour change.
As my order of fresh NotI restriction enzyme and antibiotic susceptibility discs still hadn’t arrived I spent the rest of the week waiting for their arrival as well as completing paperwork, conducting further research for the next stage of the project – PCR and preparing a protocol/list of required reagent and materials.
The work I had planned to do this week was definitely halted due to my lack of fresh restriction enzyme and antibiotic susceptibility discs. However, I do now have a fresh batch of plugs ready to be digested, plenty of stocks and a complete Escherichia coli bacterial collection ready for genotypic identification. Next week I hope to be able to run my fresh batch of Escherichia coli plugs, conduct antibiotic susceptibility testing and begin preparation for PCR of samples.
A Year in the Lab…