FLOW CYTOMETRY An Innovative Approach to Assessing Water Quality Dr. Ian Barnabas 21/09/2016
WATER QUALITY MICROBIOLOGY TRADITIONAL METHODS VS FLOW CYTOMETRY 2 Images courtesy of Pall, IDEXX & BD
CELL STAINING AND FLUORESCENCE SYBR Green 1 (SG1) Enters into all cells - living and dead. Fluoresces at 520nm (Green light). Differentiates bacteria from background noise. Gives us our Total Cell Count (TCC). Propidium iodide (Pi) Only enters cells with damaged membranes. Damaged membrane is indicative of cell death. Fluoresces at 617nm (Red light). When combined with SG1 differentiates between dead and alive cells. Gives us our Intact Cell Count (ICC). 3
BD ACCURI C6 Validation Reproducibility <5% Repeatability <5% Limit of detection 2000 cells/ml Accuracy R2 = 0.99 Sensitivity 0.05% Stain stability 1hr Sample stability 3 days Carryover <1% 4
Red fluorescence (FL3) DOT PLOTS Staining with SYBR Green 1 Dead cells P1 Total Cell Count (TCC) Contains both living and dead cells. Dual staining with SYBR Green 1 and Propidium iodide Background Noise Bacteria Intact Cell Count (ICC) Contains only living cells 5 Green fluorescence (FL1)
WATER TREATMENT WORKS (WTW) EFFICIENCY - GUNNERTON Gunnerton Raw Water Gunnerton Final Reduction TCC: 96.76% ICC : 99.91% 2,603,400 TCC/ml 1,990,700 ICC/ml 84320 TCC/ml 1,820 ICC/ml 6
19/05/16 06/09/2016 16/06/16 23/06/2016 30/06/2016 07/07/2016 14/07/2016 21/07/16 28/07/16 04/08/16 11/08/16 19/05/16 14/06/16 21/06/2016 27/06/2016 01/07/2016 07/07/2016 13/07/2016 18/07/2016 22/07/16 29/07/16 04/08/16 11/08/16 16/08/16 % Reduction % Reduction 19/05/16 06/09/2016 16/06/16 23/06/2016 30/06/2016 07/07/2016 14/07/2016 21/07/16 28/07/16 04/08/16 11/08/16 19/05/16 06/09/2016 16/06/16 23/06/2016 30/06/2016 07/07/2016 14/07/2016 21/07/16 28/07/16 04/08/16 11/08/16 % Reduction % Reduction MONITORING PERFORMANCE (GUNNERTON WTW) Overall Performance Clarifier Performance 100.00 99.98 99.96 99.94 99.92 99.90 99.88 99.86 99.84 99.82 ICC 85.00 80.00 75.00 70.00 65.00 60.00 55.00 50.00 ICC 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 Filter performance ICC 100.50 100.00 99.50 99.00 98.50 98.00 97.50 97.00 96.50 96.00 95.50 Chlorination Efficiency ICC 7
CELL COUNTS AND WATER QUALITY? Gunnerton Raw Water Gunnerton Final 2,603,400 TCC/ml 84,320 ICC/ml 1,907,000 TCC/ml 1,820 ICC/ml 8
HOW MANY IS TOO MANY? 6.5 billion Lactobacillus casei 9 Images courtesy of PhoneSoap at https://www.phonesoap.com and Yakult at http://www.yakult.co.uk/
Cell count POPULATION PROFILES LNA HNA High Nucleic Acid (HNA) Our Large approach amount of has DNA been within to cell. quantify Different these A greater population waters binding produce of profiles SYBR Green in distinctly terms 1 of different Produces histograms. greater fluorescence. how likely they are to be culturable/pathogenic contains the potentially pathogenic by assigning bacteria. it a Has Water More been Quality likely referred to be Risk culturable. to Score as the (WQRS). population Sensitive to fingerprint. chlorine. Represents the actively growing population and Green fluorescence Low Nucleic acid (LNA) This Waters allows with us similar to ICC can be very Contain less DNA. different Differentiate in terms waters of microbial of similar quality. water quality. Less binding of SYBR Green1. Monitor and identify significant changes in Lower fluorescence. Non-culturable water quality. and non pathogenic. Resistant Communicate to chlorine. results with a better understanding of risk. 10
WATER QUALITY RISK SCORE (WQRS) TCC 45,120 cells/ml ICC 34,260 cells/ml WQRS 7.56 TCC 44,000 cells/ml ICC 34,260 cells/ml WQRS 9.02 11
Colony Count Colony count RISK SCORES AND TVC 22 o C TVC 37 o C TVC 300 300 250 250 200 200 150 150 100 100 50 50 0 8.00 8.50 9.00 9.50 10.00 WQRS 0 8.00 8.50 9.00 9.50 10.00 10.50 11.00 WQRS 12
Colony Counts WQRS AND CULTURE METHODS 1000 900 800 700 600 500 400 300 200 100 0 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 WQRS 13
19/05/16 06/09/2016 16/06/16 23/06/2016 30/06/2016 07/07/2016 14/07/2016 21/07/16 28/07/16 04/08/16 11/08/16 19/05/16 06/09/2016 16/06/16 23/06/2016 30/06/2016 07/07/2016 14/07/2016 21/07/16 28/07/16 04/08/16 11/08/16 Cell count WQRS % Reduction MONITORING WATER QUALITY AND PERFORMANCE Final Water WTW Performance 1,000,000.00 8.00 101.00 100,000.00 10,000.00 1,000.00 100.00 10.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 100.00 99.00 98.00 97.00 96.00 95.00 1.00 0.00 94.00 TCC ICC HNA WQrs TCC ICC HNA WQrs 14
Free Chlorine (mg/l) FREE CHLORINE AND WATER QUALITY 1.4 1.2 1 0.8 0.6 0.4 0.2 0 4 5 6 7 8 9 10 WQRS 15
Gunnerton (WTW) Keepwick fell Warden Hill Page Croft Huntercrook Moorfield Gilsland Plenmeller Free Chlorine WQRS FREE CHLORINE AND WATER QUALITY Service Reservoirs from Gunnerton WTW 1.2 10.00 1 0.8 9.00 8.00 0.6 0.4 0.2 7.00 6.00 Cl2 WQrs 0 5.00 Service Reservoirs in order of distance from Gunnerton WTW 16
ROV SPOT SAMPLING Relationship between free chlorine, cell counts, and water quality. How reservoir design, operation, maintenance, and sampling can affect water quality, and how it is reported. 17
ON GOING WORK Continue to establish WTW and network baseline performance Monitor changes in performance across our assets Further profiling of clarifiers and filters Interrogate escada for contributing factors Monitor re-growth potential in the distribution network ROV profiling of service reservoirs Optimise treatment/network practices Focus efforts to achieve the best return in performance improvement for our investment Changing minds/changing language 18
THANK YOU FOR LISTENING QUESTIONS? 19