Week Commencing - 16/07/2018

This week we started our trials for the qualitative chemotaxis assay (v1) on 0.75% LB agar. This was done with Echerichia coli in order to practice technique and identify flaws in the protocol. We did not observe any evidence of chemotactic behaviour, which was as we expected. However, we did also note that the growth rate of the E. coli was substantially slower on the plates containing naringenin than the control. As such, we decreased the naringenin concentration from 100µM to 50µM

We also spent this week identifying other potential avenues through which we could demonstrate chemotaxis.

Week Commencing - 23/07/2018

With help from Dr. Maria Del Carmen Montero-Calasanz, we isolated our Pseudomonas fluorescens and Azospirillum brasilense from freeze-dried pellets and successfully produced multiple agar and liquid cultures. After allowing the colonies on these plates to grow to sufficient size, we produced 30% glycerol stocks for E. coli, P. fluorescens, and A. brasilense.

Week Commencing - 30/07/2018

After obtaining experience in the previous weeks, we performed the qualitative chemotaxis assay again but with A. brasilense. However, the results did not indicate chemotactic behaviour which was not expected. We hypothesised that this was either due to the naringenin concentration originally being too low after the previous reduction or that it was unable to diffuse through the agar with ease and as such the concentration was even further reduced.

We also isolated the other two nitogen fixing bacteria (Azorhizobium caulinodans and Herbaspirillum seropedicae) from freeze-dried pellets and prepared to repeat chemotaxis assays with these species

Week Commencing - 06/08/2018

This week we decided to characterise the behaviour of our bacteria on LB agar. We recorded the colony size after 24 and 48 hours of incubation at their recommended optimal temperatures as well as recording the colony morphology. This included the margin, border, elevation and pigmentation of the colony. Results of this characterisation can be found here: link to be added.

Week Commencing - 13/08/18

During this week we performed the semi-qualitative chemotaxis assay (link) on 0.5% LB agar with our 3 nitrogen fixing bacteria and E. coli. However, results once again did not indicate positive chemotaxis. After this, we attempted the quantitative capillary-based chemotaxis assay for all bacteria species. This method is based on the premise that if a capillary contains a chemoattractant and the end of said capillary is submerged in bacterial solution then the bacteria will move into the capillary at a higher rate than a species that is not attracted.

The results of this experiment were largely unexpected as no agar colonies grew from the capillary contents for 4 species, suggesting no movement at all (even in the controls). Herbaspirillum seropedicae was the only species to show movement into the capillary, however, the difference between the control and naringenin capillaries was not significant (p>0.05).

Week Commencing - 20/08/2018

After multiple issues with many assays not showing any evidence of chemotaxis, we decided that future repeats should include malate as a positive control due to the literature also indicating that this is a chemoattractant for our nitrogen-fixing bacteria [1-3].

While doing said repeats, we also noticed that on lower percentage agars, the 'halo' around the H. seropedicae colony was more constricted on plates containing higher concentrations of naringenin (>100μM). After researching this, we found that naringenin can repress chemotaxis genes at higher concentrations which may provide a reason as to why we have not seen positive chemotaxis in this species.

Week Commencing - 27/08/2018

Thanks to our sponsors ibidi, Cells in Focus, we were able to obtain specialised chemotaxis micrscopy slides. The μ-slideIII 3-in-1 chemotaxis slide allows for real time visualisation of bacterial movement in response to controllable chemical gradients. This equipment will be highly valuable to the Alternative Roots project as we use it to verify results as well as provide visuals to help improve our understanding. We spent this week familiarising ourselves with this product so that we could start using it in a timely manner.

During this week we also began to grow our nitrogen-fixing bacteria and E. coli on Minimal A Salt agar in preparation for the semi-quantitative chemotaxis assay as we were not sure if the agar could support growth of all our bacteria.

We also produced dose-response kill curves for E. coli and A. brasilense for naringenin concentrations between 0 and 150μM, increasing by 10μM intervals. These kill curves were produced by observing changes in absorbance at 600nm over 24 hours by using a plate reader.

Week Commencing - 03/09/2018

After some troubleshooting with support from ibidi, we successfully managed to seed all of our bacteria species onto the μ-slideIII and as such we attempted to observe chemotaxis behaviour microscopically. However, we encountered issues with our microscope that meant that were were unable to focus on our bacteria at an objective which allows the camera to detect the bacteria. As such, we delayed these assays until we could communicate with ibidi to find a solution

Last week we successfully grew our bacteria on Minimal A Salt agar. As such, this week we began to run our 3rd variant of the agar chemotaxis assays using this 0.25% agar. We started with A. brasilense and E. coli (secondary control) and observed positive chemotaxis in A. brasilense towards 100μM naringenin between distances of 5-25mm from the naringenin source. We then opted to continue repeats of this assay at 15mm distance from the source.

Week Commencing - 10/09/2018

The success of the latest agar assay led us to perform it with H. seropedicae and A. caulinodans. The results of these experiments were inconclusive as the colonies grew faster than anticipated thus they grew too large and overlapped. This made it hard to define colonies edges. As such, repeats of these experiments would seek to either have greater distances (although this would impact the concentration of the chemoattractant) or will be incubated for less time.

Week Commencing - 17/09/2018

Repeats of the assays from last week revealed chemoattraction towards naringenin in H. seropedicae. However, A. caulinodans has still failed to show any chemotaxis behaviour. While this may be explained as A. caulinodans not experiencing attraction to naringenin as we first predicted, this was also the result that we obtained for malate. Malate has been shown throughout the literature to be a chemoattractant for A. caulinodans. As such we predicted that our bacteria is not as motile; however, the reason for why is not known.

Our suspicions about the reduced motility of A. caulinodans were confirmed later in the week during unrelated microscopy work. In weeks prior, we had used changes in absorbance over a period of time in order to understand growth rates of our bacteria. This week we decided to develop a cell density:optical density index for each of our bacteria in order to make more sense of the collected data and produce it in a form that was suitable for the 'Community' model.

We did this by utilising a haemocytometer (details can be found here). However, while doing this work we noticed that while H. seropedicae and A. brasilense were motile while counting, A. caulinodans was not. As such, we decided that we would dedicate our remaining time to gather more data for the two motile species rather than trying to demonstrate chemotaxis in A. caulinodans.