Prior to conducting in depth assays, we first needed to identify how our FLNFB would behave in our laboratory. The team approached this by identifying what areas were integral to understanding changes in bacterial behaviour. We identified that we needed to characterise growth in liquid culture and on solid agar. Growth on agar was further divided into colony morphology and average diameter after 24 or 48 hours (species dependent). We also characterised the change in growth rate in the presence of our selected chemoattractant –naringenin – relative to standard growth in LB.

As a measure of our methods, we refer to our Escherichia coli (DH5α) control. This is as E. coli has been very thoroughly studied and so we can refer how our E. coli behaves in relation to the literature.

To assess chemotactic behaviour in response to naringenin, our team adopted 3 approaches that were identified from established literature. These approaches included agar-based, capillary-based and microscopy-based techniques. The aim of all variants was to demonstrate a change in behaviour in the presence of different chemicals; said behaviour would then give a representation of chemotaxis in our species.

As previously mentioned, the Alternative Root proof of concept involves naringenin’s ability to influence the microbiome. Because of this, bacterial behaviour in the presence of naringenin was assessed. Observed behaviour would then be compared to a blank control (typically distilled water or 1.5% ethanol) and, when appropriate, a positive control (malate).