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Monitoring andcontrol of drinkingwater qualityInventory and evaluation of monitoringtechnologies for key-parameters

TechneauOctober 2008


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2008 TECHNEAUTECHNEAU is an Integrated Project Funded by the European Commission under the Sixth FrameworkProgramme, Sustainable Development, Global Change and Ecosystems Thematic Priority Area(contractnumber 018320). All rights reserved. No part of this book may be reproduced, stored in a databaseor retrieval system, or published, in any form or in any way, electronically, mechanically, by print,

photoprint, microfilm or any other means without prior written permission from the publisher

TechneauOctober 2008

Monitoring and

control of drinkingwater qualityInventory and evaluation of monitoringtechnologies for key-parameters


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This report is:

PU= Public

Colofon

Title

Monitoring and control of drinking water quality Inventory andevaluation of monitoring technologies for key-parameters

Author(s)Margreet Mons (Ed.), all WA3 partners

Quality AssuranceAll WA 3 partners

Deliverable numberD 3.1.3


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Monitoring and control of drinking water quality

TECHNEAU - 4 - October 2008

Contents

Contents 4

1 Introduction 9

2 Method 10

3 Microbiological parameters 14

3.1 E. coli and coliform bacteria 143.1.1 Monitoring technology nr 1: Lactose TTC Tergitol Method (ISO 9308-1) 143.1.2 Monitoring technology nr 2: Colilert-18/Quanti-Tray (IDExx, UK National Standard

Method W 18) 16

3.1.3 Monitoring technology nr 3: Membrane Lauryl Sulphate Agar (MLSA) (NEN 6553) 183.1.4 Monitoring technology nr 4: Membrane Lauryl Suphate Broth (MLSB) (UK National

Standard Method W 2) 203.1.5 Monitoring technology nr 5: Chromocult Coliform Agar (Merck) 22

3.2 Intestinal enterococci 253.2.1 Monitoring technology nr 1: ISO method 7899-1 253.2.2 Monitoring technology nr 2: ISO method 7899-2 27

3.3 Clostridium perfringens 293.3.1 Monitoring technology nr. 1: Guideline according to Council Directive 98/83/EC -

membrane filtration and cultivation on m-CP agar 293.3.2 Monitoring technology nr. 2: Membrane filtration and cultivation on TSC agar,

subsequent confirmation tests (draft of ISO 6461 CD part 2) 313.3.3 Monitoring technology nr 3: Membrane filtration and cultivation on fluorogenic TSC

agar 343.3.4 Confirmation technology nr. 1: C. perfringens Detection System (Biotecon

Diagnostics) 363.3.5 Confirmation technology nr. 2: API 32 A (Biomerieux) 36

3.4 Heterotrophic Plate Counts 383.4.1 Monitoring technology nr 1: ISO 6222 383.4.2 Monitoring technology nr 2: Plating on R2A medium 40

3.5 Enteroviruses 433.5.1 Monitoring technology nr 1: Cell culture methods 433.5.2 Monitoring technology nr 2: RT-PCR 44

3.6 Giardia/Cryptosporidium 463.6.1 Monitoring technology nr 1: Method 1622 473.6.2 Monitoring technology nr 2: UK method 483.6.3 Monitoring Technology nr. 3: method 1623 483.6.4 Monitoring technology nr 4: ISO 15553 483.6.5 Monitoring technology nr 5: cross flow ultrafiltration 48

3.7 Thermotolerant Campylobacterspecies 523.7.1 Monitoring technology nr 1: Detection and enumeration of thermotolerant

Campylobacter species (ISO 17995:2005) 52

3.8 Legionellaand Legionella pneumophila 553.8.1 Monitoring technology nr 1: ISO method 11731:1998 553.8.2 Monitoring technology nr 2: Q-PCR for Legionella pneumophila 56


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3.9 Pseudomonas aeruginosa 583.9.1 Monitoring technology nr. 1: Filtration and cultivation according to EN ISO 16266 583.9.2 Monitoring technology nr. 2: VIT-Pseudomonas aeruginosa 603.9.3 Confirmation technology nr. 1: API-Test 20E 62

3.10 Aeromonas 633.10.1 M onitoring technology nr 1: EPA Method 1605 633.10.2 Monitoring technology nr 2: MALDI 653.10.3 Monitoring technology nr 3: PCR 67

3.11 Bacteriophages 703.11.1 Monitoring technology nr 1: ISO method 10705-1 703.11.2 Monitoring technology nr 2: ISO method 10705-2 723.11.3 Monitoring technology nr 3: ISO method 10705-4 (modified) 74

3.12 Aerobic spore forming bacteria 773.12.1 Monitoring technology nr 1: HPC counts 773.12.2 Monitoring technology nr 2: Membrane filtration 79

3.12.3 Monitoring technology nr: 3. Assays, based on cell constituents 803.12.4 Monitoring technology nr 4: PCR 84

3.13 Biofilmformation rate (BFR) 873.13.1 Monitoring technology nr 1: ATP measurement of biofilm 87

3.14 Total cell counts 903.14.1 Monitoring technology nr 1: Direct total microbial count (based on fluorescence

microscopy) 903.14.2 Monitoring technology nr 2: Direct total microbial count (based on flow cytometry) 92

3.15 Cultivation free viability analysis 953.15.1 Monitoring technology nr 1: Cultivation free viability analysis (based on flow

cytometry or fluorescence microscopy) 95

3.15.2 Monitoring technology nr 2: Analysis of bacterial ATP 98

4 Chemical parameters 100

4.1 Metals: Antimony, arsenic, boron, cadmium, chromium, copper, lead, mercury,nickel, selenium, sodium, calcium, magnesium, aluminum, iron, manganese 100

4.1.1 Monitoring technology nr 1: AAS (Atomic adsorption spectroscopy) 1014.1.2 Monitoring technology nr 2: AFS (Atomic fluorescence spectroscopy) 1024.1.3 Monitoring technology nr 3: ICP-OES: Inductively-coupled plasma with optical

emission spectroscopy 1034.1.4 Monitoring technology nr 4: ICP-MS: Inductively-coupled plasma with mass

spectrometry 104

4.2 Benzene 1054.2.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-FID or GC-MS 1054.2.2 Monitoring technology nr 2: Headspace, GC-FID or GC-MS 1064.2.3 Monitoring technology nr 3: Purge&trap, GC-FID or GC-MS 1074.2.4 Monitoring technology nr 4: Solid-phase micro extraction (SPME), GC-FID or GC-MS108

4.3 Benzo(a)pyrene and other PAHs 1104.3.1 Monitoring technology nr 1: Liquid-liquid extraction, HPLC/FLD 1104.3.2 Monitoring technology nr 2: Liquid-liquid extraction, GC/MS 111

4.4 Bromate 1134.4.1 Monitoring technology nr 1: Ion chromatography with conductivity detection

(IC/CD) 113

4.4.2 Monitoring technology nr 2: Ion chromatography with UV detection (IC/UV) 1144.4.3 Monitoring technology nr 3: Ion chromatography with fluorescence detection

(IC/FLD) 115


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4.4.4 Monitoring technology nr 4: Ion chromatography with inductively coupled plasmamass spectrometry detection (IC/ICP-MS) 116

4.5 Cyanides 1184.5.1 Monitoring technology nr 1: Photometric method (batch mode) 118

4.5.2 Monitoring technology nr 2: Continuous flow analysis 1194.6 1,2-Dichloroethane 1214.6.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-ECD(-ECD) 1224.6.2 Monitoring technology nr 2: Headspace, GC-ECD-(ECD) 1234.6.3 Monitoring technology nr 3: purge&trap, GC-MS 124

4.7 Fluoride 1254.7.1 Monitoring technology nr 1: Ion-selective electrode (ISE) 1254.7.2 Monitoring technology nr 2: Ion chromatography with conductivity detection

(IC/CD) 126

4.8 Nitrite 1274.8.1 Monitoring technology nr 1: Ion Chromatography 127

4.8.2 Monitoring technology nr 2: Wet chemical analysis Colorimetric method 1284.8.3 Monitoring technology nr 3: Spectrometric method, derivative spectroscopy 130

4.9 Nitrate 1324.9.1 Monitoring technology nr 1: Wet Chemical Analysis 1324.9.2 Monitoring technology nr 2: Electrochemical method 1344.9.3 Monitoring technology nr 3: Spectrometric method, single wavelength 1354.9.4 Monitoring technology nr 4: Spectrometric method, derivative spectroscopy 137

4.10 Polycyclic aromatic hydrocarbons 139

4.11 Pesticides 139

4.12 Tetra- and trichloroethene 140

4.12.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-ECD(-ECD) 1404.12.2 Monitoring technology nr 2: Headspace, GC-ECD-(ECD) 1414.12.3 Monitoring technology nr 3: purge&trap, GC-MS 142

4.13 Disinfection byproducts (trihalomethanes) 1434.13.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-ECD(-ECD) 1434.13.2 Monitoring technology nr 2: Headspace, GC-ECD-(ECD) 1444.13.3 Monitoring technology nr 3: purge&trap, GC-MS 145

4.14 Radioactivity 1464.14.1 Semiconductor Detectors 1464.14.2 Liquid Scintillation Counting 1474.14.3 Inductively Coupled Plasma Mass Spectrometry 148

4.15 Endocrine disruption chemicals 1504.15.1 Introduction 1514.15.2 Analysis of water using bioassay 1514.15.3 Analysis using bioassays 1524.15.4 General evaluation of the bioassays 153

4.16 Genotoxicity 1574.16.1 Introduction 1574.16.2 Mechanisms of genotoxicity 1574.16.3 Tests to detect genotoxicity 1584.16.4 General review 1584.16.5 Sensitivity and specificity 160

4.16.6 Robustness 1614.16.7 Time to result 1614.16.8 Ease of use and instrumentation 161


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4.17 Acute toxicity 1624.17.1 Standard toxicity tests 1624.17.2 Monitoring technology nr 2: Daphnia toximeter 1644.17.3 Monitoring technology nr 3: Fish toximeter 1664.17.4 Monitoring technology nr 4: Combined Fish and Daphnia toximeter 170

4.17.5 Monitoring technology nr 5: Algae toximeter 1724.17.6 Monitoring technology nr 6:Luminiscent bacteria 1744.17.7 Monitoring technology nr 7: Mussel monitor 175

4.18 Algae toxins 1784.18.1 Monitoring technology nr 1-4: LC-DAD, LC-MS/MS, ELISA, PPIA 179

4.19 Pesticides, pharmaceuticals, industrial chemicals and other organic micropollutants1834.19.1 Monitoring technology nr 1: Gas Chromatography-Mass Spectrometry 1834.19.2 Monitoring technology nr 2: High-Performance Liquid Chromatography-UltraViolet

Diode Array Detection 1844.19.3 Monitoring technology nr 3: High-Performance Liquid Chromatography-Mass

Spectrometry 185

4.20 pH 187

4.21 Monitoring technology nr 1: pH indicator 1874.21.1 Monitoring technology nr 2: pH meter 189

4.22 Chloride/nitrate/sulphate 1914.22.1 Monitoring technology nr 1: Ion chromatography with conductivity detection

(IC/CD) 191

4.23 Conductivity 1924.23.1 Monitoring technology nr 1: Conductimeter 192

4.24 Calcium & magnesium 194

4.25 Sulphate 1944.26 Aluminium 194

4.27 Ammonium 1954.27.1 Monitoring technology nr 1: Ion Selective Electrode 1954.27.2 Monitoring technology nr 2: Ion Chromatography 1964.27.3 Monitoring technology nr 3: Photometric test kit 1974.27.4 Monitoring technology nr 4: Automatic Analyser 198

4.28 Iron 200

4.29 Manganese 200

4.30 Taste & Odor 2014.30.1 Monitoring technology nr 1: Sensory panel 2024.30.2 Monitoring technology nr 2: GC- MS 2034.30.3 Monitoring technology nr 3: Electronic Nose & Tongue 204

4.31 Colour 2074.31.1 Monitoring technology nr 1: Visual Comparison method 2074.31.2 Monitoring technology nr 2: Colorimetric analysis 2094.31.3 Monitoring technology nr 3: Online spectrophotometric measurement 210

4.32 Turbidity 2134.32.1 Monitoring technology nr 1: Analysis using a laboratory colorimeter 2134.32.2 Monitoring technology nr 2: Online turbidity meter 2144.32.3 Monitoring technology nr 3: Online spectrophotometric measurement 216

4.33 AOC 2184.33.1 Monitoring technology nr. 1: The original van der Kooij assay 218


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4.33.2 Monitoring technology nr. 2: The Werner and Hambsch assay 2204.33.3 Monitoring technology nr. 3: The Stanfield and Jago ATP-based assay 2224.33.4 Monitoring technology nr. 4: The LeChevallier assay 2244.33.5 Monitoring technology nr.5: The Eawag-AOC assay 226

4.34 DOC/TOC 2284.34.1 Monitoring technology nr 1: High temperature combustion 2294.34.2 Monitoring technology nr 2: Persulfate oxidation 2304.34.3 Monitoring technology nr 3: UV - oxidation / conductivity 2324.34.4 Monitoring technology nr 4: UV-spectroscopy 233

4.35 UV absorbing organic constituents 2354.35.1 Monitoring technology nr 1: Single Wavelength absorption measurement 2354.35.2 Monitoring technology nr 2: Full spectral analysis 236

4.36 Particle counts 2394.36.1 Monitoring technology nr 1: Particle counting systems 239

4.37 Oxygen 243

4.37.1 Monitoring technology nr 1: Titration 2434.37.2 Monitoring technology nr 2: Electrochemical Measurement 2444.37.3 Monitoring technology nr 3: Optical Measurement 246

Annex I. Individual evaluation forms for endocrine disrupting effects 249

Annex II. Individual evaluation form for genotoxic effects 281


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1 Introduction

Monitoring and control technologies are indispensable for the production of

safe drinking water. They allow for the surveillance of source water qualityand the detection of biological and chemical threats, thus defining theboundary conditions for the subsequent treatment and providing early-warning in case of unexpected contaminations. They are mandatory for thepermanent control of the treatment process and the efficacy of each singletreatment step, and they safeguard the high quality of finished water.Furthermore, appropriate analytical techniques are indispensable for thedetection of changes in water quality during distribution and for monitoringdrinking water quality at consumers tap. Reliable monitoring technologiescontribute to a large extent to the consumers trust in a high drinking waterquality.

Following the overall objective of the TECHNEAU project, the majorobjective of WA 3 is to provide a set of analytical techniques and methodsthat ensure the provision of safe high quality drinking water that has the trustof the consumers. In WP 3.1 existing monitoring technologies are evaluatedaccording to their suitability for application in controlling water quality in thewhole drinking water production process. This evaluation includes not onlybasic analytical techniques, but also new and innovative monitoringtechnologies like effect-related DNA-arrays or electronic nose technology.

A first report resulting from WP 3.1 dealt with selection of key-parameters toassess water quality. It described the different locations and purposes inwhich monitoring and control technologies need to be applied. The respectivebiological and chemical water quality parameters that provide essentialinformation for water suppliers (so-called 'key-parameters') are identified andlisted.The current report is a follow-up and describes the results of a survey onmonitoring technologies for the selected key-parameters. The existingmonitoring technologies are identified and evaluated based on informationon e.g. ease-of-use, maintenance requirements, cists, and technicalspecifications. Also the suitability of the techniques for use in small-scale-

systems (3S) is evaluated.

This report can be used as reference when deciding on the analytical chemicaland biological techniques to be used for monitoring water quality from sourceto tap.


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2 Method

All evaluations of the monitoring technologies have been made by the

participants in WA3. Although there is a large expertise among the WA3partners, it should be kept in mind that these evaluations are based onpersonal experience and judgement and should not be taken as absolute.Evaluations focus on the methods for the selected key-parameters, not on thesuitability or accuracy of instruments from different suppliers.

The basis for this report is the table that was prepared in the TECHNEAUreport Monitoring and control of drinking water quality - Selection of key-parameters (Mons et al., 2007, see www.techneau.org). This table is presentedin table 1.To prepare evaluations in a uniform format, and make sure that evaluationsfrom different partners include information on similar aspects, a standardevaluation form was prepared. The basic evaluation form is shown in Fig 1.As most expertise within the WA3 partners lies in the field of chemicaland/or microbiological techniques, the process parameters (includinginhibitors) were left out of this evaluation. Also for radioactivity there was nogood option for a partner to create an evaluation. This problem was solved byincluding an evaluation retrieved from the literature.

Fig. 1 Evaluation form


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Table 1. Total list of parameters and where they are/can be used

Parameter

Catchment

Sour

cewater

SW

Sour

cewater

GW

Treatment1

Finishedwater

Distribution

ingress

Distribution

time

related

Customerstap

Microbiological parameters

E. coliEnterococciClostridium perfringensTotal coliformsColony count/HPCEnteric virusesGiardia/CryptosporidiumCampylobacterLegionellaPseudomonas aeruginosaAeromonasF-specific RNA phagesAerobic spore-formingbacteriaBiofilm formationTotal cell countsCultivation-free viabilityanalysis

Chemical parameters

antimonyarsenicbenzenebenzo(a)pyreneboronbromatecadmiumcopperchromiumcyanides1,2-dichloroethanefluoride

leadmercurynickelnitritenitratePAHspesticidesseleniumtetra- & trichloroethenedisinfection byproducts2

radioactivity

EDCsgenotoxicityacute toxicityalgae toxins


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Parameter

Catchment

So

urcewater

SW So

urcewater

GW

Treatment1

Finishedwater

Distribution

in

gress

Distribution

timerelated

Customerstap

pharmaceuticalsindustrial chemicalsorganic micropollutants3

pHchloridealkalinitysaturation index4

sodiumconductivitycalciummagnesiumsulphatealuminumammoniumironmanganesetasteodourcolourturbidityAOC/BDOCDOC/TOC

UV absorptionparticle countsoxygeninhibitors

Process parameters5

head lossfilter velocityresidence timeozone dose, contact time (Ct)ozone concentrationresidual ozone

UV doseoxidant doseresidual oxidant conc.disinfectant doseresidual disinfectant conc.inhibitorssediments (e.g. iron oxides)flow ratetransmembrane pressurepressure dropparticle size distribution

membrane (bio)fouling1. Various parameters are suitable/preferable for different treatment steps. For details see TECHNEAUreport "Monitoring and control of drinking water quality. Selection of key-parameters".


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2 disinfection by-products: chlorination by-products, ozonation by-products, UV/AOP by-products3 general group, consisting of e.g. pharmaceuticals, industrial pollutants etc4 saturation in dex is only a calculation and will not be further evaluated in this report5 process parameters will not be evaluated in this report


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3 Microbiological parameters

3.1 E. coli and coliform bacteria

Prepared by: TZW

Required technical specifications:For the detection and enumeration of E. coli and coliform bacteria in drinkingwater a detection limit of one bacterial cell / 100 mL water sample isrequired. Methods suitable for analyses of drinking waters with higherbacterial numbers or source waters have to provide a high selectivity to avoidinterference with background flora.

Monitoring technologies:1. Lactose TTC Tergitol Method (ISO 9308-1)2. Colilert-18/Quanty-Tray (IDExx, UK National Standard Method W 18)3. Membrane Lauryl Sulphate Agar (MLSA) (NEN 6553)4. Membrane Lauryl Suphate Broth (MLSB) (UK National Standard MethodW 2)5. Chromocult Coliform Agar (Merck)

3.1.1 Monitoring technology nr 1: Lactose TTC Tergitol Method (ISO 9308-1)

Description:The European Drinking Water directive (EU DWD) defines Lactose TTCTergitol Method according to ISO 9308-1 as reference method for thedetection and enumeration of E. coli and coliform bacteria.

Method and mode of actionE. coli and coliform bacteria are detected and enumerated by membranefiltration and subsequent culture on the differential agar medium LactoseTTC Tergitol as described in ISO 9308-1. Lactose is degraded to acid by E. coliand coliform bacteria which is indicated by a colour change of the medium.Tergitol 7 (sodium heptadecylsulfate) and TTC (triphenyl-

tetrazoliumchloride) inhibit the growth of gram positive non-targetorganisms. TTC is also part of the differential system. The reduction of TTCby lactose negative bacteria produces dark red colonies, whereas lactosepositive E. coli and coliform bacteria reduce TTC only weakly resulting inyellow-orange colonies.

Procedure and Evaluation100 mL water sample is filtered through a membrane filter. The membranefilter is transferred to Lactose TTC Tergitol Agar and incubated at 36 2C for21 3 hours. Lactose positive bacteria produce yellow-orange colonies andunder the membrane yellow-orange halos. The count of these typical coloniesis considered to be presumptive coliform bacteria count. For confirmation ofE. coli and coliform bacteria count further subculture of typical colonies on anon selective agar ( e.g. Tryptic Soy Agar) for oxidase test and in Tryptophane


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Broth for indole production is required. Colonies that are oxidase negative areconsidered to be coliform bacteria. Coliform bacteria that form indole fromtryptophane at 44 0.5C within 21 3 hours are considered to be E. coli.Results are obtained after 1 day (negative results) or 2 days.

Equipment and consumablesAs described in ISO 9308-1 (e.g. autoclave, incubator, water bath, filtrationunit, scale, pH-meter, gas burner, glassware, Petri dishes, membrane filter,Lactose TTC Agar with Tergitol 7, Tryptic Soy Agar, Tryptophane Broth,Kovacs-Indole and oxidase reagent etc.).

ReferencesAnon. (2000) ISO 9308-1: Water quality - Detection and enumeration ofEscherichia coli and total coliform bacteria - Part 1: Membrane filtrationmethod (ISO 9308-1:2000). Geneva, Switzerland: International Organisation

for Standardisation.

Evaluation:The membrane filtration method ISO 9308-1 for the detection andenumeration of E. coli and coliform bacteria is suitable for disinfected watersand other drinking waters with low bacterial numbers. Due to the lowselectivity of Lactose TTC Tergitol Agar background growth can interferewith the reliable enumeration of E. coli and coliform bacteria. It is thereforenot recommended for drinking waters with high bacterial numbers or forsource waters. For these waters alternative methods e.g. Colilert-18/Quanti-Tray (see monitoring technology nr 2) or MLSA (see monitoring technology

nr 3) are more suitable. The costs for consumables are low, but experiencedlaboratory staff is required for test performance and evaluation. Time toresult is 1-2 days.


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Monitoring technology nr 1: Lactose TTC Tergitol Agar (ISO 9308-1)Criteria 1 2 3 4 5 CommentsTechnical specifications

sensitivity (A)source water

drinking waterx

xHeavy background growth canoccur , only for waters with lowbacterial numbers (e.g.disinfected drinking water)

robustness (A)operational robustnessselectivity x

xGrowth of non target organismscan occur

time to result 1-2 days

Operational specifications

ease-of-use (B) xmaintenance requirements (C) x

Costs

instrumentation (C) xoperational costs (C)

consumablesmaintenance

xx

Recommendation for use in SSS (D) x

Overall conclusion Inexpensive method, but requires experienced laboratory

staff. Due to low selectivity, it is only recommended forvery clean watersamples(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high(B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good(C): 1 = very high 2 = high 3 = average 4 = low 5 = very low(D): 2 = no 3 = yes 4 = strongSSS: small-scale systems

3.1.2 Monitoring technology nr 2: Colilert-18/Quanti-Tray (IDExx, UK NationalStandard Method W 18)

Description:Colilert-18/Quanti-Tray has been approved as alternative method for thedetection and enumeration of E. coli and coliform bacteria according to theEU DWD in a number of EU countries e.g. Germany, Italy, Czech Republicand Hungary. Colilert-18/Quanti-Tray is included in American and UKStandard Methods.

Method and mode of actionColilert-18/Quanti-Tray is a Most Probable Number test and allowssimultaneous detection of E. coli and coliform bacteria. The method is based

on an enzyme substrate reaction. The major carbon sources are ONPG (o-nitrophenyl- -D-galactopyranoside) and MUG (4-methyl-umbelliferyl--D-glucuronide), which are metabolised by the enzymes -galactosidase


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(coliform bacteria) and -glucuronidase (E. coli). When ONPG is metabolisedby coliform bacteria the medium changes from colourless to yellow. Thedegradation of MUG by E. coli creates fluorescence. Most non-coliforms donot have these enzymes and are unable to grow and interfere. Furthermore,

Colilert uses Defined Substrate Technology (DST) to inhibit growth ofnon-target organisms.

Procedure and evaluation100 mL water sample is transferred to a sterile bottle with antifoam reagent.Colilert reagent is added and sample is mixed until reagent is completelydissolved. Sample/reagent is filled in a sterile Quanti-Tray. The tray issealed and incubated for 18 - 22 h at 36C. Positive wells are counted (yellow= coliform bacteria, yellow and fluorescent (UV light) = E. coli) and thenumbers of E. coli and coliform bacteria are determined from the MPN tables.

Equipment and consumablesAs described e.g. in the UK National Standard Method W 18 (e.g. incubator,water bath, gas burner, Quanti-Tray heat sealer, long wavelength UV lightsource and viewer, Quanti-Trays and reference comparator, Colilertreagent, antifoam reagent, sterile glassware).

ReferencesIDExx Laboratories, Milton Court, Churchfield Road, Chalfont St Peter,Buckinghamshire, SL9 9EW.

Health Protection Agency (2004). Enumeration of coliforms and Escherichia

coli by Idexx (Colilert 18) Quanti-TrayTM

. National Standard Method W 18Issue 2.http://www.hpastandardmethods.org.uk/pdf_sops.asp.

Chromogenic Substrate Coliform Test (9223) (1997) in: Standard Methods forthe Examination of Water and Wastewater, 21st Edition (2005), AmericanPublic Health Association, 1015 Fifteenth Street, NW, Washington, DC 20005.

Evaluation:The Colilert-18/Quanti-Tray method is applicable to the enumeration of E.coli and coliform bacteria in drinking water, source water and environmental

water. Due to the presence of -galactosidase in some species which areunable to produce acid from lactose using Colilert-18 Quanti-Tray can resultin higher coliform numbers compared to other culture-based tests (e.g.Lactose TTC Tergitol Method or MLSA). The method is easy to perform anddoes not require further identification tests. It can be performed by lessexperienced laboratory staff and is suitable for small water supplies, buthigher costs for consumables incur. Test results are available after 1 day.


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Monitoring technology nr 2: Colilert-18/Quanti-Tray (IDExx, UK NationalStandard Method W 18)Criteria 1 2 3 4 5 CommentsTechnical specifications


drinking waterxx

No interference in waters withhigher bacterial numbers

robustness (A)operational robustnessselectivity

xx

time to result 1 day



Costsinstrumentation (C) xoperational costs (C)


xx


Overall conclusion Fast and easy to use method. More expensive compared toother culture-based tests due to higher costs forconsumables. Is suitable for drinking water and sourcewater.

(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high(B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good(C): 1 = very high 2 = high 3 = average 4 = low 5 = very low(D): 2 = no 3 = yes 4 = strongSSS: small-scale systems

3.1.3 Monitoring technology nr 3: Membrane Lauryl Sulphate Agar (MLSA) (NEN 6553)

Description:Membrane Lauryl Sulphate Agar (MLSA) has been approved as alternativemethod for the detection and enumeration of E. coli and coliform bacteria

according to the EU DWD in the Netherlands.

Method and mode of actionE. coli and coliform bacteria are simultaneously detected and enumerated bymembrane filtration and subsequent culture on the differential agar mediumMLSA which contains lactose as major carbon source. E. coli and coliformbacteria degrade lactose to acid which is indicated by a change of the colonycolour to yellow. Laurylsulphate inhibits the growth of non-target organisms.Yellow oxidase negative colonies are considered as coliform bacteria andyellow oxidase negative colonies which produce indole from tryptophane areconsidered as E. coli.


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Monitoring technology nr 3: Membrane Lauryl Sulphate Agar (MLSA) (NEN6553)Criteria 1 2 3 4 5 CommentsTechnical specifications


drinking waterx

xrobustness (A)operational robustnessselectivity

xx More selective than Lactose TTC

Tergitol Agartime to result 1-2 days



Costs



xx


Overall conclusion Inexpensive method, but requires experienced laboratorystaff. Late sample delivery can cause inconvenience due tothe two-stage incubation procedure. Is suitable for water

samples with various contamination levels.(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high(B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good(C): 1 = very high 2 = high 3 = average 4 = low 5 = very low(D): 2 = no 3 = yes 4 = strongSSS: small-scale systems

3.1.4 Monitoring technology nr 4: Membrane Lauryl Suphate Broth (MLSB) (UKNational Standard Method W 2)

Description:Membrane Lauryl Sulphate Broth (MLSB) has been approved as alternativemethod for the detection and enumeration of E. coli and coliform bacteriaaccording to the EU DWD in the UK. MLSB is included in the UK NationalStandard Methods.

Method and mode of actionE. coli and coliform bacteria are detected separately and enumerated bymembrane filtration and subsequent culture on an absorbent pad saturatedwith MLSB as differential medium.MLSB contains lactose as major carbon source, which is degraded to acid by

E. coli and coliform bacteria, indicated by a change of the colony colour toyellow. Laurylsulphate inhibits the growth of non-target organisms. Yellow


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colonies are to be confirmed as E. coli and coliform bacteria by furtherconfirmatory tests (acid from lactose, oxidase activity, indole production).

Procedure and evaluation

100 mL of water sample is filtered through a membrane filter (2 filters foreach sample). The 2 membrane filters are transferred to pads soaked withMLSB and incubated (at 30 1 C for 4 1 hours followed by incubation at37C 1 C for 14 1 hours for coliform bacteria and at 30 1 C for 4 1hours followed by incubation at 44 2 C for 14 1 hours for E. coli). Yellowcolonies are counted on each pad as presumptive coliform bacteria (37 C)and presumptive E. coli (44 C). Yellow colonies from both membrane filtersare transferred to Lactose Peptone Water (LPW), MacConkey Agar (MA) andNutrient Agar (NA) for incubation at 37 C for 20 4 hours and to LPW andTrypton Water (TP) for incubation at 44 C for 20 4 hours. Colonies grownon NA are checked for oxidase activity and indole test is done in TP. Coliform

bacteria produce red colonies on MA, are oxidase negative and produce acidfrom lactose in LPW at 37 C. E. coli produces acid from lactose at 44 C, isoxidase negative and indole positive. Results are obtained after 1 day(negative results) or 2-3 days.

Equipment and consumablesAs described in the UK National Standard Method W 2 (e.g. autoclave,incubator, water bath, filtration unit, scale, pH-meter, gas burner, glassware,Petri dishes, membrane filter, Membrane Lauryl Sulphate Broth, NutrientAgar, McConkey Agar, Kovacs-Indole and oxidase reagent etc.).

ReferencesHealth Protection Agency (2007). Enumeration of coliform bacteria andEscherichia coli by membrane filtration. National Standard Method W 2 Issue4. http://www.hpastandardmethods.org.uk/pdf_sops.asp.

Evaluation:The membrane filtration method NSM W 2 for the detection and enumerationof E. coli and coliform bacteria is suitable for drinking waters with variouscontamination levels and for source water. MLSB is like MLSA more selectivefor target organism compared to Lactose TTC Tergitol Agar (ISO 9308-1).However, it is also not recommended for highly contaminated surface water.

The costs for consumables are low, but experienced laboratory staff isrequired for test performance and evaluation. Compared to MLSA the MLSBmethod is more involved and labour-intensive. Late sample delivery cancause inconvenience for the laboratory workflow due to the two-stageincubation procedure. Time to result is 1-3 days.


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Monitoring technology nr 4: Membrane Lauryl Suphate Broth (MLSB) (UKNational Standard Method W 2)Criteria 1 2 3 4 5 CommentsTechnical specifications


drinking waterx

xrobustness (A)operational robustnessselectivity x

xMore selective than Lactose TTCTergitol Agar




Costs



xx


Overall conclusion Inexpensive method, but requires experienced laboratorystaff. Late sample delivery can cause inconvenience due tothe two-stage incubation procedure. Is suitable for water

samples with various contamination levels.(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high(B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good(C): 1 = very high 2 = high 3 = average 4 = low 5 = very low(D): 2 = no 3 = yes 4 = strongSSS: small-scale systems

3.1.5 Monitoring technology nr 5: Chromocult Coliform Agar (Merck)

Description:Chromocult Coliform Agar (Merck) is a selective agar for the simultaneous

detection of total coliforms and E. coli in drinking water and processed foodsamples. The approval of this method by US-EPA is pending. Furthermore, ithas been taken into consideration to develope an ISO standard.

Method and mode of actionE. coli and coliform bacteria are detected and enumerated by membranefiltration and subsequent culture on CCA as selective and differential agarmedium. CCA contains chromogenic substrates which change colour tosalmon-red when degraded by -galactosidase positive coliform colonies andto dark blue-violet when degraded by -galactosidase and -glucuronidasepositive E. coli colonies. Non-target organisms are largely inhibited by

addition of Tergitol 7 and E. coli/Coliform Selective-Supplement. In case ofgrowth colonies of non-target organisms appear colourless or light-blue.


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Procedure and evaluation100 mL of water sample is filtered through a membrane filter. The membranefilter is placed on CCA and incubated at 35-37 C for 24 hours. Salmon to red-galactosidase positive colonies are counted as non E. coli coliforms. No

further confirmatory tests are required for non E. coli coliforms. Dark-blue toviolet -galactosidase and -glucuronidase positive colonies are counted as E.coli and are confirmed by testing for indole production. Results are availableafter 1 day.

Equipment and consumablesUsual laboratory equipment and in addition: autoclave, incubator, waterbath, filtration unit, scale, pH-meter, gas burner, glassware, Petri dishes,membrane filter, Chromocult Coliform Agar (see Merck description Cat.No. 1.10426.0100/500).

ReferencesOssmer, R., Schmidt, W., Mende, U. (1999). Chromocult Coliform Agar -Influence of Membrane Filter Quality on Performance. - xVII Congresso de laSociedad, Granada.

Finney, M., Smullen, J., Foster, H.A., Brokx, S., Storey, D.M. (2003). Evaluationof Chromocult coliform agar for the detection and enumeration ofEnterobacteriaceae from faecal samples from healthy subjects. Journal ofMicrobiological Methods 54: 353-358.

Evaluation:

Chromocult Coliform Agar should be suitable for the enumeration of E. coliand coliform bacteria in drinking water, source water and environmentalwater. Due to the presence of -galactosidase in some species which areunable to produce acid from lactose CCA can like Colilert-18/Quanti-Trayresult in higher coliform numbers compared to other culture-based tests. Nofurther cultivation step is required for confirmation, hence the method isfaster and can be performed by less experienced laboratory staff than. Testresults are obtained after 1 day. Validation data are not yet available


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3.2 Intestinal enterococci

Prepared by: Kiwa Water Research

Required technical specifications:The concentration range that is normally encountered in water samples is:0 cfu per 100 ml water sample in drinking water and groundwater0 to 1000 cfu per 100 ml in surface water

The required resolution of the method is:1 cfu in 100 ml water sample

Monitoring technologies:1. ISO method 7899-1: MPN cultivation in liquid MUD/SF medium.2. ISO method 7899-2: membrane filtration and cultivation on agar mediumcontaining azide and 2,3,5-triphenyltetrazoliumchloride.

3.2.1 Monitoring technology nr 1: ISO method 7899-1

Description:- The diluted sample is inoculated in a row of microtitre plate wellscontaining dehydrated MUD/SF culture medium. The microtiter plates areexamined under ultraviolet light at 366 nm in the dark after an incubationperiod of between 36 h and 72 h at 44C 0.5 C. The presence of enterococciis indicated by the fluorescence resulting from the hydrolysis of 4-methylumbelliferyl--D-glucoside (MUD). The results are given as MostProbable Number (MPN) per 100 ml.

- To perform ISO method 7899-1 an apparatus for sterilization by dry heat orsteam, a thermostatic incubator, a membrane filtration apparatus, a tunneldrier or vertical laminar air flow cabinet, UV observation chamber (Woodslamp 366 nm), pre-set 8-channel multi-pipette and sterile microtiter plates arerequired.

- The method is one of the two ISO-approved methods for the enumeration ofintestinal enterococci in water.

- Reference: ISO 7899-1.

Evaluation:- In general, an accurate method to determine the number of enterococci insurface water samples, but enumeration is based on MPN, which might beless reliable than the colony count method described below.- The detection limit of the method is 15 bacteria per 100 ml, which is too high

for use with drinking water.- The use of microtiter plates, liquid media and multi-pipette makes themethod more robust than the colony count method described below.


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- Incubation time is rather long, because results are obtained after 1.5 to 3days. However, a fast standard method is not available, although methodsbased on DNA-detection have been published and might come available inthe near future.

-It is known that in some water samples (especially sea-water) other micro-organisms might give false-positive results. However, the other ISO-methoddescribed below has a similar limitation because with that method agar platescan be overgrown with other micro-organisms, preventing reliableenumeration of intestinal enterococci colony types.- A disadvantage compared to monitor technology 2 is that the method doesnot contain confirmation steps.

Monitoring technology nr 1: ISO-method 7899-1

Criteria 1 2 3 4 5 Comments

Technical specificationssensitivity (A)source water

drinking water xx

Detection limit: 15 bacteria in 100ml water


x

time to result x Total incubation time: 36 to 72 h



Costs



x


Overall conclusion Acceptable method to determine intestinal enterococci insurface waters. Not applicable to drinking water, becausethe detection limit of the method is too high (15 bacteria per

100 ml). Some water samples might give false-positiveresults. Quantification is based on MPN. Positive results arenot confirmed by additional reactions. Because of thecultivation step this standard method is rather timeconsuming; a faster standard method is preferred, but notyet available.



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Description:- A specified volume of water sample is filtered through a membrane filterwith a pore size (0.45 m) sufficient to retain the bacteria. The filter is placedon a solid selective medium containing sodium azide (to suppress growth ofGram-negative bacteria) and 2,3,5-triphenyltetrazolium chloride, a colourlessdye, that is reduced to red formazan by intestinal enterococci. Typicalcolonies are raised, with a red, maroon or pink colour, either in the centre ofthe colony or throughout.

If typical colonies are observed, a confirmation step is necessary, by transferof the membrane, with all the colonies, onto bile-aesculin-azide agar,preheated at 44 C. Intestinal enterococci hydrolyse aesculin on this medium

in 2 h. The end-product, 6,7-dihydroxycoumarin, combines with iron(III) ionsto give a tan-coloured to black compound which diffuses into the medium

Confirmed colonies are expressed as colony forming units (cfu) per 100 ml.

- To perform ISO method 7899-2 an apparatus for sterilization by dry heat orsteam, two thermostatic incubators (37C and 44C), a membrane filtrationapparatus, sterile membrane filters (0.45 m) and a water bath (100C) (todissolve the agar medium) are required.

- The method is one of the two ISO-approved methods for the enumeration of

intestinal enterococci in water.


Evaluation:- In general, an accurate method to determine the number of enterococci inwater.- The detection limit of the method is 1 cfu per 100 ml, which makes themethod suitable for determining intestinal enterococci in drinking water.- The use of selective agar media and membrane filtration and the

interpretation of colony characteristics make the method slightly less robustthan the first monitoring technology described above.- Incubation time is rather long, because results are obtained after 2 days.However, a fast standard method is not available, although methods based onDNA-detection have been published and might come available in the nearfuture.-It is known that agar plates can sometimes be overgrown with other micro-organisms, preventing reliable enumeration of intestinal enterococci colonytypes (especially in source surface water). However, the other ISO-methoddescribed above has a similar limitation because with that method somewater samples might give false-positive results.

- An advantage compared to monitor technology 1 is that the methodcontains a confirmation step, making the chance of false-positive resultslower.


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- Because this method is suitable for both source and drinking water, themethod is preferred over monitoring technology nr 1, which was describedabove.


Criteria 1 2 3 4 5 CommentsTechnical specifications


drinking waterxx

Detection limit: 1 bacterium in100 ml water


x

time to result x Total incubation time: 46 h



Costs



x


Overall conclusion Acceptable and ease-to-use method to determine intestinalenterococci. Applicable to both source and drinking water.Surface source water samples might give overgrowth ofnon-target bacteria on the agar plates. Because of thecultivation step this standard method is rather timeconsuming; a faster standard method is preferred, but notyet available.

(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high(B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good(C): 1 = very high 2 = high 3 = average 4 = low 5 = very low(D): 2 = no 3 = yes 4 = strong

SSS: small-scale systems


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3.3 Clostridium perfringens

Prepared by: vermicon AG

C. perfringens is a rod-shaped, gram-positive, endo-spore forming and non-motile bacterium of the genus Clostridium. It is strictly anaerobic, but cansurvive a short exposure to oxygen. The micro-organism can be detected inanaerobic zones of soil, in water, foodstuff and in the intestine of humans andanimals.C. perfringens is selected out of the genus Clostridium to serve asmicrobiological parameter, because it is the most important species out of thesulphite reducing Clostridiaand it is normally present in human and animalfaeces.

Required technical specifications:The limit value for C. perfringens(including spores) according to the DrinkingWater Directive (DWD) is 0 cells/100 ml drinking water. The analysis of thisparameter is only necessary, if the water originates from surface water or isinfluenced by surface water. When the limit value is exceeded, the competentauthority will initiate exploratory research to assure that there is no dangerfor human health, due to the occurrence of a pathogenic micro-organism.Furthermore if no C. perfringensis detected in 100 ml drinking water, it can beassumed that no resistant dormant bodies/cysts of a parasitic protozoa arepresent in the water.For the analysis quantitative detection methods have to be applied. Yet, there

is no approved ISO standard procedure available for the detection ofClostridium perfringens.

Monitoring and confirmation technologies:1. Guideline according to DWD - membrane filtration and cultivation on

m-CP agar2. Membrane filtration and cultivation on TSC agar, subsequent

confirmation tests according to draft of ISO 6461 CD part 23. Membrane filtration and cultivation on fluorogenic TSC agar (Araujo

et al., 2004)4. Clostridium perfringens PCR-based-detection system, Biotecon

Diagnostics, Germany5. API 32A, biochemical screening test, Biomerieux, France

3.3.1 Monitoring technology nr. 1: Guideline according to Council Directive 98/83/EC -membrane filtration and cultivation on m-CP agar

Description:Cultivation-based quantitative method for the enumeration of C. perfringensin water samples. 100 ml water samples is filtrated on a membrane filter.Membrane filtration is followed by an anaerobic incubation of the membrane

on m-CP selective agar at 44 +/- 1 C for 21 +/- 3 hours. Opaque yellowcolonies that turn pink or red after exposure to ammonium hydroxide vaporsare counted.


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Material & method

m-CP medium is a selective, chromogenic medium for the rapid identificationand enumeration of Clostridium perfringensin water samples. The medium isdesigned to improve the differentiation of Clostridium perfringensfrom other

Clostridiaspecies and background flora.Chromogenic compounds within the m-CP medium cause Clostridiumperfringenscolonies to turn yellow (based on their ability to ferment sucrose),thus differentiating them from other Clostridium species, whilst colonies ofbackground flora turn purple (based on their ability, unlike C. perfringens, tohydrolyse indoxyl-b-D-glucoside).An additional confirmation of the result is proposed by exposing the cultureplate to ammonium hydroxide. This highly specific reaction causes acidphosphatase-producing C. perfringens yellow colonies to turn into adistinctive dark pink color.

The addition of D-cycloserine and polymyxine B, and an incubationtemperature of 44 C improves selectivity of m-CP agar by inhibiting thegrowth of gram-negative bacteria and Staphylococci.No further verification steps are necessary according to the directive.

Equipment and consumables

M-CP agar Basal medium Membrane filtration manifold Sterile filter funnels graduated to 100 ml

Vacuum pump with moisture trap or protective filter, oralternative vacuum force

Incubator: 44 C +/- 1C Facilities for anaerobic incubation Petri dishes Cellulose ester 0,45 m pore size filters

EvaluationThe fastest method for the detection of C. perfringens is monitoring technologyno. 1. This procedure is based on membrane filtration and subsequentcultivation on m-CP agar. Time to result is 1 day, the handling is quite simple

and it can be performed with standard laboratory equipment on low costbasis. Nevertheless, this approach is criticized by experts regarding the poorsensitivity. Due to this fact another method for the detection of C. perfringenswas established in the draft of ISO 6461 CD part 2.


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Monitoring technology nr. 1: Guideline according to Council Directive98/83/EC - membrane filtration and cultivation on m-CP Medium



drinking water-x

Not yet analysed

robustness (A)operational robustness

selectivityxx

time to result 24 h



Costs x

instrumentation (C) x Standard water laboratoryequipment

operational costs (C)consumablesmaintenance

xx


Overall conclusion Very fast, easy & low cost test, but poor reliability(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high

(B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good(C): 1 = very high 2 = high 3 = average 4 = low 5 = very low(D): 2 = no 3 = yes 4 = strongSSS: small-scale systems

3.3.2 Monitoring technology nr. 2: Membrane filtration and cultivation on TSC agar,subsequent confirmation tests (draft of ISO 6461 CD part 2)

Description:Filtration of 100 ml water sample on a membrane filter (maximal pore size0,45 m, no specification of filter material is given). Subsequent incubation of

the membrane filter under anaerobic conditions on TSC-Agar at 44 +/- 1 Cfor 21 +/- 3 hours. All colonies are counted, that show a black or grey toyellow brown staining of the TSC agar when viewed either from above orbelow the filter. Some colonies may exhibit very faint staining of the medium,but should still be counted. Further confirmatory tests should be performedfor purposes of identification.

Material & method:

The nutrient base provides optimal conditions for the development ofClostridia. Colonies producing hydrogen sulfide are characterized by

blackening due to the reaction with sulfite and iron salt. In TSC Agarcycloserine inhibits the accompanying bacterial flora and causes the colonies,


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Vacuum pump with moisture trap or protective filter, oralternative vacuum force

Incubator: 44 C +/- 1C Facilities for anaerobic incubation

Tryptose sulphite cycloserine agar Buffered Nitrate-Motility medium Nitrate reagent A Nitrate reagent B Lactose-gelatine medium: Blood agar: Columbia agar or any other suitable base with 5 %

horse blood Petri dishes 0,45 m pore size filters

EvaluationMonitoring technology nr. 2 (ISO 6461 CD part 2) contains differentconfirmatory tests. In comparison to monitoring technology no. 1 thisapproach is more time-consuming, but a higher reliability and sensitivity isachieved. This ISO standard is still not approved yet and comprisingevaluation studies are missing.


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Monitoring technology nr. 2: Membrane filtration and subsequent cultivation on TSCagar and subsequent confirmation tests



drinking water xnot analysed


selectivityxx




xCostsinstrumentation (C) x Standard water laboratory

equipmentoperational costs (C)


xx


Overall conclusion Low cost test, time consuming, handling is feasible, notrobust, but reliability is very good


3.3.3 Monitoring technology nr 3: Membrane filtration and cultivation on fluorogenicTSC agar

Description:

Membrane filtration procedure is performed according to Monitoringtechnology no 1 and 2, but confirmatory tests are not necessary. By theaddition of the fluorogenic substrate 4-Methylumbelliferyl-phosphate (MUP)Clostridium perfringenscolonies can be identified by UV light.

MethodD-Cycloserine inhibits the accompanying bacterial flora and causes thecolonies which develop to remain smaller. It also reduces a diffuse and thusdisturbing blackening around the C. perfringens colonies. 4-Methylumbelliferyl-phosphate (MUP) is a fluorogenic substrate for thealcaline and acid phosphatase. The acid phosphatase is a highly specific

indicator for C. perfringens. The acid phosphatase splits the fluorogenicsubstrate MUP forming 4-methylumbelliferone, which can be identified as itfluorescence in long wave UV light. Thus a strong suggestion for the presence


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Monitoring technology nr. 3: Membrane filtration and cultivation onfluorogenic TSC agar



drinking water xnot analysed


selectivityxx

time to result 24 h



xCostsinstrumentation (C) x Standard water laboratory

equipmentoperational costs (C)


xx


Overall conclusion Low cost and fast test, handling is feasible and reliability isgood


3.3.4 Confirmation technology nr. 1: C. perfringens Detection System (BioteconDiagnostics)

Oligonucleotides for the specific detection amplification and detection of C.perfringens DNA by PCR suitable for gel based detection are provided byBiotecon Diagnostics. The application of this detection system indicates only

the presence of C. perfringensDNA, while no indication for the presence ofactive cells is given. Additionally well trained personal is needed for theperformance of this non-quantitative test. Thus this method can only be usedas a confirmatory test.

3.3.5 Confirmation technology nr. 2: API 32 A (Biomerieux)

API-Test 32 A (Vendor: Biomerieux) is a test for the identification of differentanaerobic bacteria. The biochemical test is a confirmatory test for coloniesand identification is possible within 4 hours.For the application of this test a pure culture is needed. Thus the test can be

applied after the subculture of a colony as an additional test for theconfirmation of the result.


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References1. Araujo M. Sueiro R.A . Gmez Garrido M.J. (2004) Enumeration of

Clostridium perfringens in groundwater samples: comparison of sixculture media. Journal of Microbiological Methods, 57 (2), 175-180

2.

Armon P. and Payment P. (1988) A modification of m-CP medium forenumerating Clostridium perfringens from water samples. Can. J.Microbiol. 34 78-79

3. Barthel H. Krger W. Mendel B. Suhr R. Die Trinkwasserverordnung2001 bewhrt oder revisionsbedrftig. Bundesgesundheitsblatt GesundheitsforschungGesundheitsschutz 2007, 50: 265-275, Springer Medizin Verlag 2007

4. Payment P. and Franco E. (1993) Clostridium perfringens and somaticcoliphages as indicators of the efficiency of drinking water treatmentfor viruses and protozoan cycts. Appl. Environ. Microbiol. 59, 2418-2124

5. Sartory D. P. (2005) Validation, verification and comparison: Adoptingnew methods in water microbiology Revised paper. Water SA Vol. 31No.3 July 2005

Guidelines1. Council Directive 98/83/EC of November 1998 on the quality of water

intended for human consumption. Official Journal of the EuropeanCommunities

2. Enumeration of Clostridium perfringens by membrane filtration.Issue no: 3.1 Issue date: 03.05.05 Issued by: Standards Unit,

Evaluations and Standards Laboratory on behalf of the Regional Food,Water and Environmental Microbiologist Forum. www.evaluations-standards.org.uk


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microbiological quality of water. Several European countries have adoptedthe ISO 6222 method as standard method according to the DWD (e.g.,TrinkwV, 2001).

Evaluation:The method has the advantage that it has been used for a long time and a lotof experience and data exists in the peer reviewed literature. The maingeneral disadvantage of all HPC methods is that cultivation only detects asmall percentage (typically ca. 1%) of the total microbial concentration in awater sample, and that the methodology is time and labour consumingmethod. Several authors have suggested that the growth medium, incubationtemperature and incubation time is not optimal to achieve the highest platecount results (Reasoner and Geldreich, 1985; Uhl and Schaule, 2004; Berney etal., 2008).

ReferencesISO, 1998. Water Quality-Enumeration of Culturable Micro-organism-ColonyCount by Inoculation in a Nutrient Agar Culture medium, prEN ISO 6222European Committee for Standardisation, Brussels.Clesceri, L.S., Greenberg, A.E. and Eaton, A.D. (Eds.) (1998) StandardMethods for the examination of water and wastewater; 9215 HeterotrophicPlate Counts. ISBN 0-87553-235-7Reasoner DJ and Geldreich EE (1985). A new medium for the enumerationand subculture of bacteria from potable water. Appl. Environ. Microb. 49: 1-7.Uhl, W. and Schaule, G. (2004) Establishment of HPC (R2A) for regrowthcontrol in non-chlorinated distribution systems. International Journal of Food

Microbiology, 92: 317 325.Berney, M., M. Vital, I. Huelshoff, H.-U. Weilenmann, T. Egli, and F.Hammes. Rapid, cultivation-independent assessment of microbial viability indrinking water.Acceptedfor publication in Water Research, July 2008TrinkwV, 2001. verordnung ber die Qualitt von Wasser fr menschlichenGebrauch (Trinkwasserverordnung - TrinkwV 2001). BGB1 I, Nr. 24, issuedMay 28th 2001, pp.959-980.DWD - Council Directive 98/83/EC of 3 November 1998 on the quality ofwater intended for human consumption.


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Several alternative methods for HPC determination are described andstandardised. For example, Standard Methods (Clesceri et al., 1998) describethree different methods (spread plate method, pour plate method, membranefilter method) and four different agars (Plate Count Agar; m-HPC agar; R2A

agar; NWRI agar). Typical variations on the methods include the use ofalternative growth media (e.g. nutrient agar), different incubationtemperatures and different incubation time periods. In some cases, colonycounting with a magnifying glass or with an automated colony counter hasalso been promoted. The membrane filter method can be used if the celldensity of the sample is too low for direct spread plating. The membranefilters have a diameter of 47 mm and a pore size of 0.2 um. The sample upfrom 10 mL can be filtered and the membrane filter is placed on one agarplate (m-HPC agar is prescribed in this instance) (Clesceri et al., 1998). For anoverview, see Bartram et al., 2003.

Equipment and consumablesEquipment:Incubator; autoclave

Consumables:Sterile Petri dishes and sterile medium (e.g. R2A agar)

Status of the techniqueThe method is used for research but not typically included in drinking waterdirectives/legislation.

Evaluation:The method has similar disadvantages to the ISO 6222 method. However,several users agree that the R2A displayes higher numbers for HPC bacteriawhen drinking water is analysed (Reasoner and Geldreich, 1985; Uhl andSchaule, 2004; Berney et al., 2008)

ReferencesBartram, J., Cotruvo, J., Exner, M., Fricker, C. and Glasmacher, A. (2003)Heterotrophic plate counts and drinking-water safety. London, UK: IWAPublishing on behalf of the World Health Organization.Reasoner DJ and Geldreich EE (1985). A new medium for the enumeration

and subculture of bacteria from potable water. Appl. Environ. Microb. 49: 1-7.Uhl, W. and Schaule, G. (2004) Establishment of HPC (R2A) for regrowthcontrol in non-chlorinated distribution systems. International Journal of FoodMicrobiology, 92: 317 325.Berney, M., M. Vital, I. Huelshoff, H.-U. Weilenmann, T. Egli, and F.Hammes. Rapid, cultivation-independent assessment of microbial viability indrinking water.Acceptedfor publication in Water Research, July 2008


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3.5 Enteroviruses

Prepared by: EAWAG

Required technical specifications:Enteroviruses are widely present in water environments

Levels of contamination are widely unknown, also because molecularmethods, which are increasingly used, are not quantitative.Concentrations in freshwaters (Guillot, 2006):1-100 pfu/L in contaminated surface water1-10 pfu/100 L in less polluted surface water1-10 pfu/1000 L in treated drinking water

Since the infectious dose is very low (1-10 infectious particles), detectionmethods should be very sensitive.

Monitoring technologies:1. Cell culture methods2. RT-PCR

3.5.1 Monitoring technology nr 1: Cell culture methods

Description:Concentration:- Adsorption/elution technique using positively charged filters orelectronegative filters (APHA, 1998)- Ultrafiltration- Flocculation (APHA, 1998)- Tangential flow ultrafiltration (Bigliardi et al., 2004)

Detection:Cell cultures for most enteric viruses are commercially available (Fout et al.,1996). Viruses are grown in cell culture monolayers and form visible plaquesor other visible changes to infected cells in the monolayer.

Evaluation:The cell culture detection method is regarded as the golden standard.However, it is time consuming (6-15 days) and requires a lot of expertise inorder to obtain reliable results.


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stain that allows the assessment of the presence of sporozoites in the oocyst,again as mark of viability. Vital staining (PI (Campbell, Robertson et al. 1992)or Syto59 (Belosevic and Finch 1997)) can be used in combination with theIFA test and gives an indication of cell membrane integrity. These dye

exclusion assays provide some information about viability, but should beused with caution as they can (largely) overestimate viability of oocysts thathave been exposed to stressors such as exposure to UV light (Clancy, Hargyet al. 1998).Another assay to assess the viability of Cryptosporidiumoocysts is cell culture.

SpecificityThe specificity of the immunofluorescence assay is based on the specificity ofthe monoclonal antibody-antigen reaction. Although this is highly specific,non-specific binding is observed in natural samples. Many of the particulatesthat react with the monoclonal antibody can be discriminated from oocysts by

a trained observer, but occasionally particles (algae) occur in samples that arevery difficult to discriminate from oocysts. This may lead to false-positiveresults. The immunofluorescence method is also not specific toCryptosporidium species and genotypes that are infectious to humans, alsospecies that are infectious to animals are detected. Molecular techniques(PCR, genotyping) are rapidly evolving and some laboratories are now usingthese methods for environmental monitoring (Xiao, Alderisio et al. 2001;LeChevallier 2004; Xiao, Bern et al. 2004; Heijnen, Wullings et al. 2005).

References

AMVD (2005). Inspection Guidance Document 'Analysis of themicrobiological safety of drinking water'. Ministry of Public Housing,Spatial Planning and the Environment. [In Dutch].

Anonymous (1999). Water Supply (Water Quality) (Amendment)Regulations, SI 1524. Stationery Office, London, UK.

Anonymous (2000). Water Supply (Water Quality) Regulations, SI 3184.Stationery Office, London UK..

Belosevic, G. M. and G. F. Finch (1997). Int. Symp on WaterborneCryptosporidium, Newport Beach Ca, USA.

Campbell, I., L. J. Robertson, et al. (1992). "Viability of Cryptosporidiumparvum oocysts - correlation of in vitro exystation with inclusion or

exclusion of fluorgenic vital dyes.." Appl. Environ. Microbiol. 58: 3488-3493.Clancy, J. F. and T. M. Hargy (2008). Waterborne: drinking water.

Cryptosporidium and Cryptodiosis. R. Fayer and L. Xiao. Boka RatonLondon New York, CRC Press: 305-326.

Clancy, J. L. (2000). "Sydney's 1998 water quality crisis." J. Am. Water WorksAssoc. 92: 55-66.

Clancy, J. L., T. M. Hargy, et al. (1998). "UV light inactivation ofCryptosporidium oocysts." J. Am. Water Works Assoc. 90(9): 92-102.

Craun, G. F. (1990). Waterborne giardiasis.. Human parasitic diseases. E. A.Meyer. Amsterdam, the Netherlands, Elsevier Science Publ. 3: 267-293.

Heijnen, L., B. Wullings, et al. (2005). "Genetic analysis of Cryptosporidiumoocysts from surface water." Submitted for publication.


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Monitoring technology nr 1,2,3,4,5: Method 1622, 1623, ISO15553




drinking waterx

x

Recovery of the method hasbeen improved over time butstill is low and variable (50%)


selectivityx

x

The method requires well-trained analysts (microscopiccounting and oocystidentification); no differentiationin species/infectivity

time to result The method is time consuming


ease-of-use (B) x Once trained, the method is easy

to perform for analysts.maintenance requirements (C) x Due to the high variability

internal QC is required

Costs

instrumentation (C) x IF Microscopyoperational costs (C)


xx


Overall conclusion Available methods to quantify (oo)cysts in water sampleshave been optimized to an acceptable level of accuracy andreproducibility when pre-cautions such as recoveryassessments are implemented in monitoring. Methods tospecify the observed (oo)cysts and to verify the infectivitythough are more comprehensive and only advisable forresearch objectives rather than routine monitoring.


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Monitoring technology nr 1: ISO 17995:2005



drinking waterxx


selectivityx

xCampylobacter species are verysensitive to adverse conditions

time to result 5 days



Costs



xx


Overall conclusion Method is laborious and requires a well equippedmicrobiological laboratory and experienced and well trainedstaff. Method is suitable for all kinds of waters.



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3.8 Legionellaand Legionella pneumophila

Prepared by: Kiwa Water Research

Required technical specifications:0 - 106cfu per 250 ml in drinking water0 - 106cfu per 100 ml in water from cooling towers or surface water

The required resolution of the parameter is:100 cfu in litre drinking water (Dutch legislations)

Monitoring technologies:1. ISO method 11731:1998: Detection and enumeration of Legionellaby directmembrane filtration and cultivation on agar medium;2. Detection and quantification of Legionella pneumophilawith the QuantitativePolymerase Chain Reaction (Q-PCR)

3.8.1 Monitoring technology nr 1: ISO method 11731:1998

Description:Bacteria in a water sample are concentrated by membrane filtration or bycentrifugation. To reduce the growth of unwanted bacteria, a portion of theconcentrated sample is subjected to treatment with acid or heat. Treated anduntreated concentrated sample are then inoculated onto plates of agarmedium (semi)-selective for Legionella. Plates are incubated at 37 C for 7days. After incubation, morphologically characteristic colonies which form onthe selective medium are to be confirmed as Legionella by subculture todemonstrate their growth requirements for L-cysteine and iron. The cultureand subculture of Legionellawill require 10 days before results are available.

To perform the ISO culture method no specific equipment, other thanstandard microbiological laboratory equipment, is needed. The ISO method isthe generally accepted method and agar medium is commercially available.

Evaluation:

The culture method is the standard method generally used to enumerateLegionella in water. It uses semi-selective GVPC or BCYE agar medium toculture Legionella. In water samples containing a high microbiologicalbackground, the agar medium is likely to be overgrown by other bacteriathen Legionella. The method is therefore less useful for water samples fromcooling towers or surface water.


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Evaluation:In general, the method is accurate, specific and robust. The detection resultsare available within a few (4) hours, this in comparison with the 7 days

required for the standard culture method. The quantification if based on aDNA standard. The results are therefore given in DNA copies per litre. Forrisk analysis it can be necessary to know if recent disinfections have beencarried out. Heat disinfection can result in uncultivable but PCR detectableDNA copies. The method is some what sensitive to inhibition of the PCR orloss of DNA during DNA isolation. However, the result of the internalcontrol quantitatively shows the overall performance of the analysis persample.

In a few years, this method will presumably be used as the standard detectionmethod for Legionella pneumophila. The Q-PCR results are available within one

day. If necessary, within 24 hours after sampling the samples can additionallybe analyzed with the culture method.

Monitoring technology nr 2: Q-PCR for Legionella pneumophila



drinking waterx

xrobustness (A)

operational robustnessselectivity

xx

time to result result within 4 hours



Costs


consumablesmaintenance xx

Recommendation for use in SSS (D) x needs skilled personel

Overall conclusion Fast, reliable, and sensitive detection method for thedetection of Legionella pneumophila in water. Themethod detects specific Legionella pneumophila DNA inwater samples, but no differentiation between live anddead cells is possible

(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high(B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good

(C): 1 = very high 2 = high 3 = average 4 = low 5 = very low(D): 2 = no 3 = yes 4 = strongSSS: small-scale systems


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Monitoring technology nr. 1: Filtration and cultivation according to EN ISO16266



drinking water-x

not applied


selectivityxx

Experience is needed

time to result Up to 5 days



Costsinstrumentation (C) x Standard water laboratoryequipment

operational costs (C)consumablesmaintenance

xx


Overall conclusion method is time-consuming when colonies have to beconfirmed


3.9.2 Monitoring technology nr. 2: VIT-Pseudomonas aeruginosa

Description:For the application of VIT-Pseudomonas aeruginosa a pre-enrichment of thewater sample in Malachit green broth is necessary (48 h, incubation at 37 C).

An aliquot (2 ml) of a suspicious broth (turbid, colour change into yellow) iscentrifuged, fixed and analysed with VIT-Pseudomonas aeruginosa.Additionally the kit can be used as a confirmatory test for colonies.

Material & Method5 l of the sample (pre-enrichment or fixed colony) is placed on each well of aslide. The sample is fixed on the slide and the VIT-solution is added to thesample. This solution contains specific oligonucleotide probes for P.aerugionosa, labeled with fluorescent dyes. During an incubation step (90 min,at 46 C) the probes bind specifically to their matching signatures on the

genetic material (16S rRNA). Following this a stringent washing step removesall unbound and surplus oligonucleotide probes from the cells (15 min, 46C). The results are evaluated using a fluorescence microscope, whereby P.


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serotype. After hybridization and washing, bound PCR-product is detectedby chemiluminescence and visualized by exposure to X-ray film.

- To use this method, a PCR apparatus, a miniblotter, a water bath and an X-

ray film are required.- Reference: Vinj, J. et al. 2004. Molecular detection blot hybridization.Appl. Environ. Microbiol. 70:5996-6004.

Evaluation:- In general, an accurate method to determine the number of bacteriophages(as indicator for enteric viruses) in water samples. The best indication of thepresence of enteric viruses is obtained by using this method in combinationwith methods to determine somatic coliphages and bacteriophages that infectBacteriodes (Leclerc et al 2000. Bacteriophages as indicators of enteric viruses

and public health risk in groundwaters. J. Appl Microbiol. 88:5-21).- From literature it is known that F-specific RNA bacteriophages are a betterindicator for the presence of enteric viruses in water than somatic coliphages,but might be a less reliable indicator than bacteriophages that infectBacteriodes.- Plaques are often vague, making them more difficult to count than plaquesof somatic coliphages.- ISO method 10705-1 states that confirmation by added RNAse to themedium should be performed in parallel with the method to enumerate F-specific RNA bacteriophages. In case of low number (1 to 5) of pfu of F-specific RNA bacteriophages it is better to take out the plaque and confirm

inhibition of infection by addition of RNAse for each isolated plaque.- Incubation time is rather long, because results are obtained after 18 to 22hours. However, a fast standard method is not available, although methodsbased on RNA-detection have been published and might come available inthe near future.- Determination of the serotype of isolated F-specific RNA bacteriophages canbe used to determine the source of fecal contamination, because with fewexceptions serotype II and III have been associated with human wasteaffected water. Serotype I and IV are associated with animal waste affectedwater.


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(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high(B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good(C): 1 = very high 2 = high 3 = average 4 = low 5 = very low(D): 2 = no 3 = yes 4 = strong

SSS: small-scale systems


Description:- The sample is mixed with a small volume of semisolid nutrient medium. Aculture of host strain Escherichia coli strain C is added and plated on solidnutrient medium. After this, incubation and reading of agar plates for visible

plaques takes place. The results are expressed as the number of plaqueforming units (pfu) per unit of volume.


sensitivity (A)

source waterdrinking water xx

Detection limit: 1 bacteriophage in 10

ml water. In combination withconcentration of the water sample viaHemoflow, detection limits indrinking water can be 1bacteriophage in 10 L or even 1bacteriophage in 2000 L.


selectivityx

xtime to result Total incubation time: 18 to 22 h



Costs



xx


Overall conclusion Most used method together with the method for somatic

coliphages to determine bacteriophages (as indicator for entericviruses) in water samples. One method is not better over the otherand it is advised to determine F-specific RNA bacteriophages,somatic coliphages and bacteriophages that infect Bacteroides.Serotyping isolated F-specific RNA bacteriophages can be used todetermine the source (human versus animal) of faecalcontamination.


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- To perform ISO method 10705-2 an apparatus for sterilization by dry heat orsteam, a thermostatic incubator, a water bath, a counting apparatus, aspectrophotometer, a pH-meter and a deep freezer (-20C 5C) are required.

- The method is one of the three ISO-approved methods for the enumerationof bacteriophages (as indicators for enteric viruses) in water.


Evaluation:- In general, an accurate method to determine the number of bacteriophages(as indicator for enteric viruses) in water samples. The best indication of thepresence of enteric viruses is obtained by using this method in combinationwith methods to determine somatic coliphages and bacteriophages that infect

Bacteriodes (Leclerc et al 2000. Bacteriophages as indicators of enteric virusesand public health risk in groundwaters. J. Appl Microbiol. 88:5-21).- From literature it is known that somatic coliphages are slightly less reliableas indicator for the presence of enteric viruses in water than F-specific RNAbacteriophages or bacteriophages that infect Bacteriodes.- Plaques are clear, making them easier to count than plaques of F-specificRNA bacteriophages.- Incubation time is rather long, because results are obtained after 18 to 22hours. However, a fast standard method is not available.


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3.11.3 Monitoring technology nr 3: ISO method 10705-4 (modified)

Description:- The sample is mixed with a small volume of semisolid nutrient medium. Aculture of host strain Bacteroides is added and plated on solid nutrientmedium. After this, incubation and reading of agar plates for visible plaquestakes place. The results are expressed as the number of plaque forming units(pfu) per unit of volume.

-The Bacteroides strains that have been used as host strains are Bacteroidesfragilis RYC2056 (detection of bacteriophages from animal and humansources; this strain is mentioned in ISO 10705-4), Bacteroides


sensitivity (A)

source waterdrinking water xx

Detection limit: 1 bacteriophage in

10 ml water. In combination withconcentration of the water samplevia Hemoflow, detection limits indrinking water can be 1bacteriophage in 10 L or even 1bacteriophage in 2000 L.


selectivityx

xtime to result Total incubation time: 18 to 22 h



Costs



xx

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