The BactiQuant is a new patented method that provides water companies and the industry with a rapid method to estimate total numbers of bacteria in water samples. The method is well suited for HACCP and Water Safety Plans in the industry and water distribution. With the BactiQuant, bacterial contaminants can be concentrated from large volumes of water allowing low analysis time (10-30 minutes) even at relatively low bacterial concentrations. The use of larger sample volumes (100-5000 ml) also ensures that samples are representative for the water system to be tested. The BactiQuant can be applied to a range of liquid samples including: Potable water, processed water, CIP (cleaning in place) and recreational water. Other liquids include: Liquid extracts of a solid material or slurries, suspended or solubilized surface samples and liquid industrial products.

1. Bactiquant®-water
Total bacterial analysis in minutes

2. Outline of the presentation
Bactiquant® water
How it works
Documentation
Application cases
Questions
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3. Third party verification
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US-EPA Verified Technology
U.S. Environmental Protection AgencyU.S. Environmental Protection Agency

4. Laborious
Skilled lab personel
Long analysis time
Industry Challenges:

5. Problem
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You do not get timely results

6. Rapid results
Maintain control
Reduce downtime
Industry Requirements:
Save time and money

7. Total bacterial analysis in minutes

8. Bactiquant®-water
Total bacteria
From days to minutes
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9. Sampling
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10. Enzyme targeted analysis
+
Bacteria-enzyme
Enzyme substrate Fluorescent compound released

11. Enzyme targeted analysis
Enzyme substrate
Fluorophore

12. Robust concept
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•Naturally occuring enzyme activity
•High specificity for bacteria
•Amplifying signal – adjustable sensitivity

13. Simple interpretation
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The more fluorescense the more bacteria is
present in the sample

14. Linearity with sample volume and
reaction time
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y = 17,745x
R² = 0,999
0
200
400
600
800
0 10 20 30 40
Flourescence
Reaction time (minutes)
Bactiquant®-water test on 500 ml tap water. Fluorescence
measured as function of reaction time. Conducted at 26oC
y = 0,7709x
R² = 0,9959
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100
Flourescence
ml tap water sample
Bactiquant®-water test on tap water samples.
Fluorescense measured as function of sample size.
Conducted at 25oC and with 65 minutes reaction time.
R2 Agar showed 28 CFU/ml in tap water.
Calculation of Bactiquant Value (BQV) for this trial.
1 BQV = 0.5 CFU/ml

15. The BQ value
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250 ml water sample
30 minutes reaction
23 C (73,4 F)
The Bactiquant value is universal

16. Calculating the BQ value
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LOD = 10 fluorescence units
(Reading-blank) (vol / 250)* * (Reaction time / 30) * (Temperature adjustment)

17. Simple protocol
•Concentrate bacteria on a sterile filter using
syringe/vacuum manifold
•Concentration from as large a volume as possible
•Can be sampled directly from sample points
•Use the filter as the reaction vehicle
•Saturate filter with substrate
•React in filter for a set time
•Typically 10 – 30 min incubation
•Flush out cleaved fluorophore into cuvette
•Measure using hand-held fluorometer

18. Bactiquant®- Advantages
Exclusion of a cultivation step
Bacteria does not have to reproduce prior to analysis
Reduction in performance time from days to minutes
Fluorescent substrate analogues
No permeabilization of cells necessary
Robust chemistry
Concentration step
Increases sensitivity
Reduction in detection time
Minimal human intervention
Closed filtration unit
No handling of filter membrane necessary
Robust and repeatable

19. Detecting all bacteria
Traditional plate counts do not measure particle bound bacteria
Particle bound bacteria are important for water quality
Bactiquant®-water
BactiQuant®Water detects and quantifies suspended
bacteria as well as bacteria located on particles or in aggregates.
Why is this important ?

20. Traditional plate count
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Plate count estimate
Total population

21. A comprehensive measure
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Organism Gram stain Phyllum/ class
Athrobacter sp. Negative Actinobacteria / grp. 20
Bacillus cereus Positive Firmicutes / grp. 18
Bacillus subtilis Positive Firmicutes / grp. 18
Bacillus thuringiensis Positive Firmicutes / grp. 18
Clostridium tetani Positive Firmicutes / clostridia
Desulfovibrio desulfouricans Positive Proteobacteria / δ
Enterobacter sp. Negative Proteobacteria / γ
Enterococcous faecalis Positive Firmicutes / bacilli
Eschericia coli Negative Proteobacteria / γ
Escherichia fergusonii Negative Proteobacteria / γ
Flavobacteria bacterium Negative Bacteroidetes / flavobacteria
Flavobacterium johnsoniae Negative Bacteroidetes / flavobacteria
Klebsiella pneumonia Negative Proteobacteria / γ
Lactobacillus acidophilus Positive Firmicutes / bacilli
Lactobacillus reuteri Positive Firmicutes / bacilli
Methanococcus aeolicus Negative Euryarchaeota / methanococci
Micrococcous sodonensis Positive Actinobacteria / actinobacteria
Pseudomonas aeroginosa Negative Proteobacteria / γ
Rhodobacter sphaeroides Negative Proteobacteria / α
Serratia marcescens Positive Proteobacteria / γ
Staphylococcous aureus Positive Firmicutes / bacilli
Sphingomonas Wittichii Negative Proteobacteria / α
Vibrio sp Negative Proteobacteria / γ
Xanthomonas campestris Positive Proteobacteria / γ
Source: Brenda database

22. Comparing BQ and HPC
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• The result does not depend on the ability of
bacteria to grow on a specific media.
• Bactiquant®-water detects and quantifies particle
associated bacteria as well as suspended bacteria.

23. Comparing Bactiquant with ATP
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Parameter Comments Bactiquant ATP
Rapidity 10-30 min 2 min
Ease of use
Moderately
easy
Very
easy
Reproducibility
Permeabilization of cells
necessary.
No Yes
The robustness of assay
chemistry
High Low
The assay sensitive to water
chemistry
Low High
The overall reproducibility High Low
Specificity to Bacteria High No
US-EPA verification
Yes No

24. Quantification in environmental
samples
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25. Complex Environmental Sample
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0
1
2
3
4
5
0 1 2 3 4 5 6
Bactiquant value
Data: Total counts 35oC, 48 hours, R2A , pour plate procedure– Middle East utility
0
1
2
3
4
5
0 1 2 3 4 5
Bactiquant value
All data Populations
Ground water/desalinated water
Treated surface water
Untreated surface water
TVC(CFU/ml)

26. Simple samples – suspended bacteria
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1
10
100
1000
10000
100000
1000000
10000000
1 10 100 1000 10000 100000 1000000
BactiQuant analysis result (fu/ml)
HPC-DS6222(cfu/ml)

27. High reproducibility
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Source US EPA -2011
P. aeruginosa ATCC 27853
Test Iteration
Adjusted Fluorescence (fu)
8.0 x 103 CFU/mL 4.7 x 103 CFU/mL 2.1 x 103 CFU/mL 8.7 x 102 CFU/mL
1 11392 6128 2799 1249
2 12332 6074 2710 1247
3 11376 6012 2559 1297
4 11513 6508 2768 1437
5 11614 6256 2945 1375
Average 11645 6196 2756 1321
Standard deviation 396 196 140 83
RSD (%) 3.4 3.2 5.1 6.3

28. Repeatability and inter assay reproducibility
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Test Iteration
Adjusted Fluorescence (fu)
Indigenous Bacteria from Lake Water
(3.7 x 102 CFU/mL)
P. aeruginosa ATCC 27853
(4.7 x 103 CFU/mL)
Analyst 1 Analyst 2 Analyst 1 Analyst 2
Average 2363 2225 6888 6691
Standard deviation 152 57 333 93
RSD (%) 6.4 2.6 4.8 1.4
RPD (%) 6.0 2.9
Source US EPA -2011

29. Excellent water quality – drinking water
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BQ-values < 40
• 976 water samples
• 200 sampling sites in Copenhagen
•Plate counts (DS/EN ISO 6222) at 22 and 36oC
•BQ value < 40 less than 1% probability of exceeding 200 cfu

30. Legionella and general hygiene
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0
1
2
3
4
5
6
7
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70
BQ
Legionella pneumophila serotype 2-14
BQ value < 40
Courtesy of Viborg-Kjellerup Hospital, clinical microbiology
institute, 2006, Denmark

31. Calibration curve – BQ Value and CFU/ml in
Ultra-pure Water
31
Courtesy of The Danish Technological Institute
Sensitivity level: 1 liter sample, reaction time 5 hours
BQ- Values
HPC(CFU/ml)

32. Plate counts can be predicted by the rapid method
Good accuracy with ultra-pure water
Results show plate counts of 20 CFU/ml
corresponds to a Bactiquant®-value of 1
Ultra pure water

33. Bactiquant® Applications
Source water/Raw water quality
Water plant (operations/process control)
Distribution systems (finished water)
Industrial process water
Food and beverage production
Tracking pollution events
HACCP (hazard analysis and critical control points)
Technical water (electronics, cooling towers)
Effluents
Recreational waters

34. Application Cases
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35. Monitoring critical control points
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R åvand
16650
Tils ætning af
FeCl 3 og
H2SO 4
5307
Efter filter
209
Efter ozon -
Behandling
70
Afgang
Vandv ærk
10
GAC filter
187
Actifloc
691
Surface
Water
Intake
Sand-
filtration
Ozonization
UVGAC
After
sediment.
Flocullation
Al, Fe
Outlet
Distribution
16650 5300
690 210
70
190 160 BD
Chlorination

36. Advantages of process control
Fewer resources are used on correcting
problems after they have occurred
Enables the end user to be proactive

37. How do we translate analyses results into
operation and optimization of processes ?
Data Handling

38. Data handling software
Establish baselines based on frequent long term monitoring
at Critical Control Points
Establish operational limits

39. Tracking a pollutant

40. Tracking a polution overview
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outletoutlet
O2
Sand filter
Ground water
Boreholes
Water work
12
12
800

41. Tracking a pollutant

42. Tracking a pollution
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Early warning
Rapid adjustment
Maintain control
Value to the customer

44. Cooling water
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45. Cooling water
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0
100
200
300
400
500
600
700
800
900
Leaking cans
Leaking cans
Repair conveyer belt
Leaking cans
Disinfection over weekend
Chlorine pump defect over night
BQvalue
Date

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Early warning
Rapid Adjustment
Maintain control
Value to the customer

47. 11/06/2013 Direction or Department name 47
0
500
1000
1500
2000
2500
1808
2068
1270
934
Bactiquant®value
Bottled and tap water survey,
Malaysia
Courtesy of TECHKOR, Korea

48. Optimising chlorination
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Replicate
Raw Sample
Reaction time
(minutes)
Volume
(ml)
BQV
(FU)
RSD %
1 15 15 10766
2 15 15 10495
3 15 15 10296
4 15 15 10296
5 15 25 10264
Average 10423 2,00 %
NaOCL – 15
minutes
1 15 15 989
2 15 15 708
3 15 15 613
4 15 50 518
Average 707 28 ,70 %
Fracking data
2012
Courtesy of Mycometer Inc., USA

49. Verifying ultrafiltration
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8767
217
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
before filtration after filtration Recirculation 10 days
BQ-value
Courtesy of BIN-X, Denmark
22

50. Bactiquant® Value to industry
Timely results
Respond quickly
Maintain control
Optimize water treatments
Increase over all system reliability

51. Return on investment
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Optimise operating costs
Reduce downtime
Prevent lost production
Extend equipment life

52. QUICK FACTS:
 Duration: 3-5 hours.
 Held at the customer’s location
COURSE OUTLINE:
 Proficiency in performing the
analysis
 Applications, theory, and
precautions
 Implementation options
COURSE OUTCOME:
This course is designed to provide
the customer with the skills and
expertise to use the Bactiquant
technology
Training and certification

53. Quality Assurance Program
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What is a Quality Assurance Program
• The Quality Assurance Program has been
developed for our experienced customers
• The Quality Assurance Program enables the
customer to perform in house Proficiency
testing and verification of instrument function.
How is the quality assurance carried out?
• Customer measures the fluorescence signal
of the samples according to the quality
assurance protocols.
• The results are sent to Mycometer’s testing
laboratory who issues a quality test report
for the proficiency test and the instrument
verification.

54. Flexible
Sampling
Rapid
Analysis
Easy
Interpretation
To wrap it up

55. US-EPA (Environmental Protection Agency)
verified technology
Rapid results – on location
Robust analysis method
Reproducible and repeatable
Data handling software
Training and certification program
Comprehensive product package

56. Whats next ?
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Vittorio Figurato
Marketing Department
ELGA Process Water
vittorio.figurato@veoliawater.com
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