Microbiological water analysis

Coliforms Enterococci Clostridium Cryptosporidium ??? Total cell counts

Water sample

•Target cell components,antibodies or nucleic acids with fluorescently tagged stain

1

•Draw sample into flow cytometer, where allparticles are aligned to pass a laser sequentially

2

•Forward light scatter, side light scatter and fluorescence emission are detected and recorded as electronic pulses

3

•Pulses are converted to digital signals and processedfor analysis by a computer4

MLGA plate TSC plate Immunoassay & microscopic identification

Flow cytometer

1 432 65

What’s in the water?• Potable water typically has bacterial concentration

of 103-106 cells/mL • Microbiological water quality is routinely tested

using traditional laborious methods (1-5 in Figure 1).

• The heterotrophic plate count (method 5) is a regulatory requirement, but only 0.01-1% bacteria grow this way, so a very low quality method

• Agar methods take 16-72h from analysis to result

Flow cytometry as a rapid method• Multiparametric method: forward scattered light, side

scattered light, fluorescence emission data for every cell (Figure 2)

• 20 minute analysis to result time

• Opportunity to use different dyes for advanced analysis

• Nucleic acid staining used for total (bacterial) cell count

Figure 2. Simplified process of flow cytometry analysis and an internal diagram of a flow cytometer

mENT plate HPC plate

Figure 1. 1-4, Indicator organisms used as water quality measures, and common analytical techniques: MLGA= membrane lactose glucoronide agar; mENT= mEnteroccous agar; TSC= tryptose sulphite cycloserine agar; 5 & 6, methods for measuring bacteriological quality, HPC= heterotrophic plate count.

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Figure 3. Two flow cytometry dot plots displaying the division of high nucleic acid (HNA) and low nucleic acid (LNA) in aquatic bacterial DNA. A: clustered division. B: dual peak histogram of green fluorescence emission, point division at 2.104 a. u. (Prest et al., 2013)

Research questions• How do we interpret flow cytometry data in terms of water quality: out of the 103-106cells/mL often

found in potable water, the majority are primarily indigenous water bacteria, not pathogens

• Nucleic acid staining of environmental bacteria can be used to divide bacteria into high nucleic acid (HNA) and low nucleic acid (LNA) groupings by relative fluorescence emission (Figure 3): does this have any significance for water quality measures?

• Can flow cytometry also be used to measure viral abundance in drinking water?

• Would other cell component, immuno- or nucleic acid sequence based stains provide useful information on water quality?

Advancing flow cytometry

in the water sector

Lindsey Furness1, Aidan Marsh2, Andrew

Filby2, Dana Ofiteru1, Tom P. Curtis1

For further information: L.Furness2@ncl.ac.ukPostal Address: Newcastle University, School of Engineering, Cassie Reception, Newcastle upon Tyne, NE1 7RU

ReferencePrest, E. I. et al. (2013) ‘Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method’, Water Research, 47(19), pp. 7131–7142.

StreamThe Industrial Doctorate Centre for the

Water Sector