STREAM EngD Cohort IX
- Start Date September 2017
- Research Area: Environment
- Title: Advancing the use of Flow Cytometry (FCM) in the water sector
- Supervisors: Prof Tom Curtis, Dr Dana Ofiteru, Dr Andrew Filby & Northumbrian Water Group
I graduated with a BSc (Hons) in Food Microbiology from the University of Nottingham in 2014, having been motivated to take this degree by a desire to understand the contribution of microbes to different environmental systems (such as food!). An interest in microbial genetics led me to complete an MRes in Molecular Microbiology, also at the University of Nottingham. My research project included development of a novel method of gene knockout from Clostridium acetobutylicum, an organism of interest to the biofuels industry due to its ability to produce butanol and ethanol from sugar substrates in a fermentation process.
Following this, I spent some time running an agricultural laboratory before joining Severn Trent Water as a bacteriological analyst. Surprised by the antiquated nature of microbiological testing in the UK water industry, I leapt upon a project at STW for water analysis using flow cytometry.
Conventional analysis is based upon growing organisms on specially formulated growth medias, a technique termed culturing. This method is around 100 years old, with developments mainly in protocol and constitutional formulation. It is valued for its simplicity, whereby water samples are filtered on to media, incubated for 24-72 hours and resulting colony growth is counted and subjected to basic biochemical tests if required. As a basic indicator of microbial presence in water, 1mL of sample water is pipetted on to a 'one size fits all' total viable count medium. The test is approximated to grow ~1% of bacteria present in water, therefore providing an insensitive measure of microbiological change. Generally used for trend analysis, there are no fixed guidelines for levels of microbes in potable water, with most drinking water containing >10^4 cells/mL. It should be noted that the large majority of this microbial life is harmless and could be considered similarly to the indigenous bacteria that live on our skin and inside our gut.
Flow cytometry (FC) is a method of cell counting that involves applying specific stains to samples and passing these through a laser in a way that cells are aligned to pass the laser or lasers one by one. A fluorescent nucleic acid can be applied which stains the DNA of all microbes in a water sample, so that calibration of FC lasers results in excitation of bound fluorophores and emission of light from stained microbes, allowing for their detection to be recorded. Additionally, refraction of light from particles passing through the lasers can be used to give a rough approximation of individual cell sizes. The total analysis time of this method is approximately 20 minutes from sample to result, with most of this time taken in the staining procedure as collection of results is available almost instantly on a connected computer. Multiparametric data is gained on each individual cell (i.e. size and fluorescence emission). This data can be used to produce an accurate total count of bacteria in water. Further, specific stains can be used to give a 'live'/dead count, and collation of data from each cell used to produce a 'community fingerprint' of the microbes present.
My EngD research is supported by Northumbrian Water and will continue to explore the potential uses of flow cytometry for analysis of water, including acquisition and application of FC data. I aim to showcase how FC can support water quality analysis and characterise microbial communities, where greater understanding of community dynamics can contribute to new focuses in water treatment and distribution. The additional benefit of being part of the STREAM programme allows increased interaction with other researchers working in water based fields, as well as a close relationship with industry and the opportunity to build and develop key skill areas.