FSD 17932

Bio-Rad Laboratories Water Testing
Water Safety
Guide for Bio-Rad Products in Water Testing
Quality Indicators Testing
Water Safety
Guide for Bio-Rad Products in Water Testing
Total flora
Contents EN ISO 6222 (07/1999)
Water quality
Part 1 Quality Indicators Testing Enumeration of culturable micro-organisms.
Colony-count by inoculation
Total flora EN ISO standard 3 in a nutrient agar culture medium.
E. coli EN ISO miniaturized MPN standard 4 Pour-Plate method
E. coli Automated alternative method: XplOrer64 System 5

Water sample
E. coli/Coliforms NF EN ISO standards 6 Ordering Information
(preparation)
Catalog# Description
E. coli/Coliforms RAPIDE.coli 2 alternative method 7
PCA (Plate Count Agar)
Enterococci EN ISO miniaturized MPN standard 8 without Dextrose/Agar
3554437 200 ml 6 bottles
Enterococci Automated alternative method: XplOrer64 System

9 3564474 500 g
Enterococci EN ISO standard 10
Enterococci RAPIDEnterococcus alternative method 11
Part 2 Pathogen Testing

PCA without Dextrose
Sulfite-Reducing Anaerobes (Clostridium) NF EN ISO standards 12-13
Legionella spp. & Legionella pneumophila NF EN ISO standard 14-15

68 hrs 44 hrs
Legionella spp. and Legionella pneumophila Extraction for real-time PCR method 16 22C 36C
Legionella spp. and Legionella pneumophila Alternative real-time PCR method 17 Reading/ Reading/
enumeration enumeration
Legionella detection in aerosols with CIP10-M Apparatus Bio-Rad solution 18
Legionella detection in biofilms, Amoeba Bio-Rad technical notes 19
Pseudomonas aeruginosa EN ISO standard 20
Pseudomonas aeruginosa RAPIDP.aeruginosa alternative method 21
Salmonella spp. ISO standard 22-23
Staphylococcus aureus NF standard 24-25

This booklet is only an indication provided by Bio-Rad for the use of Bio-Rad products.
It is not a contractual document.
Guide for Bio-Rad Products in Water Testing | | 3

Ordering Information E. coli - EN ISO miniaturized MPN standard E. coli - Automated alternative method: Ordering Information
(with Fluoplate 2 Software) XplOrer64 System Catalog# Description
DSM + Bromophenol Blue
EN ISO 9308-3 (03/1999) Automated real-time and continuous quantification XplOrer64 System
3554282 18 ml 60 tubes
Water quality of Escherichia coli in bathing Full Configuration (220/110V)
DSM (Dehydrated Special Detection and enumeration of Escherichia coli (coastal sea and inland water) and waste waters XplOrer64 System for up to 64 samples,
Microplates Diluent) and coliforms bacteria delivered with computer, UPS, color
3553784 100 g Part 3: Miniaturized method (Most Probable printer, CheckNSafe temperature control
Number) for the detection and enumeration Samples poor in Samples with high Non filterable water cells, cleaning box and 4 racks
EDS (Distilled Water for of E. coli in surface and residual water suspended matters (SM) SM level samples
Microplates) 100 ml protocol 10 ml protocol 1 ml protocol XplOrer64 System
3554283 18 ml 60 tubes Bathing water (sea, inland) Treated or untreated Untreated Configuration w/o PC (220V)
Water sample Treated waste water waste water waste water XplOrer64 System 220V for up to 64
Fluoplate 2 Software (dilutions in DSM or EDS)
Drinking waters samples, CheckNSafe temperature
Data management software for the control cells, cleaning box and 4 racks
statistical estimation of MPN and its Water sample Water sample Pipetting of
confidence interval filtration filtration 1 ml of water XplOrer64 System
Please contact your local representative sample Configuration w/o PC (110V)
XplOrer64 System 110V for up to 64
MUG/EC E. coli Microplates Kit samples, CheckNSafe temperature
3553785 25 opaque white plates control cells, cleaning box and 4 racks
with stickers MUG/EC (microplates) Please contact your local representative
3553782 25 transparent plates
with stickers CheckNSafe E.coli Kit
3554720 9 ml 60 tests
Long-Wave UV Lamp, Compact
Portable CheckNSafe Screw Caps
Membrane inserting Membrane inserting
1660500 1 lamp (366 nm, requires 3672 hrs 3593457 Sterile caps (1 bag of 30 units)
into into Direct inoculation
4 AA batteries) 44C
CheckNSafe CheckNSafe CheckNSafe
Portable UV Observation Chamber E.coli E.coli E.coli
Reading
(without Wood Lamp)
( = 366 nm UV light)
3550717 1 chamber
UV Lamp
Square Petri Dishes
3550304 120 mm 252 units
UV Observation Chamber
Key Benefits
(Wood Lamp) Automated detection or quantification with Easy-to-use and automated
3550718 1 UV tube (366 nm) XplOrer64 System methodto quantify E. coli in
Total E. coli realtime, without confirmation
analysis cycle: Random access for up to 64
- bathing water: 8 hrs samples analyzed simultaneously
Calculation and interpretation - waste water: 12 hrs Fast Results:
- drinking water: 12 hrs 2.000 E. coli/100 ml detected
Fluoplate 2 Software in 4 hrs 20 min
Key Benefits 500 E. coli/100 ml detected
Automated real-time results analysis within 5 hrs
Statistical control oftheinputvalues and interpretation with Automatic expression of results
Creation of a database easily according to 76/160/EEC or
exportable into Excel *
or Access
* XplOrer64 Software
2006/7/EC European Directive
Multi-language software and reports: Accurate alternative method
English, French, Spanish, Italian and compared to miniaturized
German MUG/EC Microplates
*Excel is a trademark of Microsoft
*Access is a trademark of Access Systems
4 | | Guide for Bio-Rad Products in Water Testing Guide for Bio-Rad Products in Water Testing | | 5
E. coli/Coliforms - Alternative method:

Ordering Information E. coli/Coliforms - NF EN ISO standards RAPIDE.coli 2 for Water Testing
EN ISO 9308-1 (09/2000) NF T90-413 (10/1985) EN ISO 9308-1:2014 RAPIDE.coli 2 for Water Testing Ordering Information
BCP (Bromocresol Purple) Water quality Water testing Water quality Enumeration Chromogenic agar medium for the Catalog# Description
Lactose/Broth Detection and enumeration of Detection and enumeration of of Escherichia coli and direct detection and enumeration RAPIDE.coli 2 for Water
3553414 10 ml 25 tubes Escherichia coli and coliforms bacteria coliforms and thermotolerant coliform bacteria Part 1: of E. coli and coliforms, by Testing, Kit
Part 1: Membrane-filtration method coliforms Membrane filtration method membranefiltration 3555296 6 bottles of agar + 6 bottles
BGBLB (Brilliant Green Bile General method by culture in for waters with low bacterial of supplement
Lactose Broth) liquid media (MPN) background flora
3578024 10 ml 25 tubes
Kovacs Reagent
+
BRD 07/2003/11
WATER ANALYSIS METHODS
3555313 15 ml 2 bottles
www.nf-validation.afnor.org/en
Peptone Water (without Indole)/
Broth Water sample filtration Water sample (dilutions) Water sample filtration Water sample filtration
3554180 9 ml 25 tubes
3564334 500 g RAPIDE.coli 2 Agar
for Water Testing
Schubert/Broth BCP Lactose Broth 3563982 55 mm 20 dishes
3569634 500 g or
Tryptose Lauryl Sulfate Ready to use (to be supplemented)
Tergitol 7 (0.2%)/Supplement
Broth 3555297 200 ml 6 bottles
3562632 50 ml 1 bottle 3555299 100 ml 6 bottles
3562635 5 ml 1 vial Membrane
2448 hrs
30C Dehydrated base
TTC (0.05%)/Supplement
3564024 500 g
3562652 50 ml 1 bottle Standard test Rapid test
3562655 5 ml 1 vial
Trouble and gas+ Supplement
TTC Tergitol 7 TSA Membrane Membrane
3555298 6 vials of supplement
TTC Tergitol 7/Agar
BaseMedium 24 hrs 24 hrs
36C 36C
3554687 200 ml 6 bottles for E. coli and for E. coli and
3564454 500 g 4-5 hrs BGBLB BGBLB
coliforms coliforms
36C or
TTC Tergitol 7/Agar 21 hrs
(+21 hrs) Schubert
Complete Medium
36C
3563706 55 mm 10 dishes
3563980 55 mm 100 dishes RAPIDE.coli 2 Agar
(10) yellow colonies Transfer the CCA
48 hrs 2448 hrs for Water Testing
Trypto-Casein-Soy (TSA)/Agar with yellow area membrane on TBA 37C 44C
3563884 90 mm 20 dishes RAPIDE.coli 2
3555944 8 ml 25 slanted tubes 1920 hrs
44C Trouble Trouble
3564554 500 g
gas + gas +
Tryptophan Broth TSA Tryptophan Membrane on Total Thermotolerant
3554194 3 ml 25 tubes 10 tubes Broth indole paper coliforms coliforms Key Benefits
10 tubes + UV
Tryptose Lauryl Sulfate Broth
3564774 500 g Certified by NF VALIDATION
Enumeration of typical Enumeration of typical One single plate for simultaneous
21 hrs 21 hrs 1030
Oxidase Disks 36C 44C min colonies colonies E.coli and coliforms testing
3553834 2 vials of 50 oxidase disks Indole test Pink colonies = Coliforms Green colonies = Complete results within 21 3 hrs
(Kovacs Reagent Violet colonies = E. coli Coliforms at just one temperature of 36C
Oxidase test Indole test Red Confirmation: Grey blue to violet Good colony coloration contrast
Oxidase Kovacs colonies in Schubert)
Oxidase on coliforms colonies = E. coli fora rapid reading
Disks Reagent E. coli No confirmation step required
Enterococci - EN ISO miniaturized MPN standard Enterococci - Automated alternative method: Ordering Information
Ordering Information
(with Fluoplate 2 Software) XplOrer64 System Catalog# Description
EN ISO 7899-1 (03/1999) Automated real-time and XplOrer64 System
DSM + Bromophenol Blue
Water quality continuous quantification of Full Configuration (220/110V)
3554282 18 ml 60 tubes
Detection and enumeration of Enterococci intestinal Enterococci in bathing XplOrer64 System for up to 64 samples,
DSM (Dehydrated Special in surface and waste water water (coastal sea and inland BRD 07/19 - 11/09 delivered with computer, UPS, color
MicroplatesDiluent) water) and waste waters WATER ANALYSIS METHODS printer, CheckNSafe temperature control
Miniaturized method (Most Probable Number) www.nf-validation.afnor.org/en
3553784 100 g by inoculation in liquid medium cells, cleaning box and 4 racks
EDS (Distilled Water for Samples poor in Samples with high Non filterable water XplOrer64 System
Microplates) suspended matters (SM) SM level samples Configuration w/o PC (220V)
100 ml protocol 10 ml protocol 1 ml protocol XplOrer64 System 220V for up to 64
3554283 18 ml 60 tubes
Water sample Bathing water (sea, inland) Treated or untreated Untreated samples, CheckNSafe temperature
Fluoplate 2 Software
Treated waste water waste water waste water control cells, cleaning box and 4 racks
(dilutions in DSM or EDS)
Data management software for the
statistical estimation of MPN and its
XplOrer64 System
Water sample Water sample Pipetting of Configuration w/o PC (110V)
confidence interval
Please contact your local representative
filtration filtration 1 ml of water XplOrer64 System 110V for up to 64
sample samples, CheckNSafe temperature

MUD/SF Enterococci control cells, cleaning box and 4 racks

MicroplatesKit Please contact your local representative
3553786 25 opaque white plates MUD/SF (microplates)
with stickers CheckNSafe Enterococci Kit
3553783 25 transparent plates 3554721 10 ml 60 tests
with stickers
Membrane inserting Membrane inserting CheckNSafe Screw Caps
Long-Wave UV Lamp, into into Direct inoculation 3593458 Sterile caps (1 bag of 30 units)
CompactPortable 3672 hrs CheckNSafe CheckNSafe CheckNSafe
1660500 1 lamp (366 nm, requires 44C Enterococci Enterococci Enterococci
4 AA batteries)
Reading
Portable UV Observation Chamber
( = 366 nm - UV light)
(without Wood Lamp)
UV Lamp Key Benefits
3550717 1 chamber
Square Petri Dishes First alternative method certified
3550304 120 mm 252 units Automated detection or quantification with NFVALIDATION for bathing and
waste water monitoring
UV Observation Chamber XplOrer64 System
Easy-to-use and automated
(WoodLamp) Total Enterococci methodto quantify in real-time,
3550718 1 UV tube (366 nm) analysis cycle: without confirmation
- bathing water: 12.5 hrs Random access for up to 64
- waste water: 16 hrs samples analyzed simultaneously
Fast Results:
Calculation and interpretation 400 Enterococci/100 ml
Key Benefits Fluoplate 2 Software detected in 8 hrs 30 min
250 Enterococci/100 ml
Statistical control oftheinputvalues Automated real-time results analysis detected within 9 hrs
Creation of a database easily and interpretation with Automatic expression of results
exportable into Excel * or Access *

XplOrer64 Software according to 76/160/EEC or
Multi-language software and reports: 2006/7/EC European Directive
English, French, Spanish, Italian and Accurate alternative method
German compared to miniaturized
*Excel is a trademark of Microsoft MUD/SF microplates
*Access is a trademark of Access Systems
Enterococci - RAPIDEnterococcus
Ordering Information Enterococci - EN ISO standard alternative method Ordering Information
Catalog# Description Catalog# Description
NF EN ISO 7899-2 (08/2000) RAPIDEnterococcus
BEA/Agar Water quality RAPIDEnterococcus
Chromogenic agar for
3563994 90 mm 20 dishes Detection and enumeration the direct detection and enumeration 3554409 100 ml 6 bottles
3564184 500 g of intestinal Enterococci of intestinal Enterococci,
TTC (0.2%)/Supplement Part 2: Membrane-filtration method by membrane-filtration
3562665 5 ml 1 vial
Slanetz and Bartley/Agar
BaseMedium
3554819 100 ml 6 bottles
3564934 500 g
Water sample filtration Water sample filtration
Slanetz and Bartley/Agar
CompleteMedium
3563716 55 mm 10 dishes
3563981 55 mm 100 dishes
Membrane
Membrane
Slanetz and Bartley
RAPIDEnterococcus
810 ml per plate

( = 55 mm)
44 hrs
36C
44 hrs 4 hrs
Reading 44C
Transfer the membrane on BEA
2 hrs Key Benefits

44C Enumeration of typical colonies
Blue colonies = Enterococcus
Direct identification and
Reading/enumeration enumeration of Enterococci
No confirmation step required
Ready-to-use medium
Save time and material
Pathogen Testing
Ordering Information Sulfite-Reducing Anaerobes (Clostridium) - NF EN ISO standards

NF T90-415 (10/1985) NF EN 26461-1 (07/1993) ISO/DIS 6461-2 (06/2005)
Columbia/Agar
Water testing Water quality (ISO 6461-2:1986 review)
3564674 500 g
Detection and enumeration of spores of Detection and enumeration of Water quality
3564678 5 kg
sulfito-reducing anaerobic bacteria spores of sulfito-reducing anaerobic Detection and enumeration of the spores of
Meat-Liver Sulfite Iron/Agar and of Clostridium micro-organisms (Clostridia) sulfitereducing anaerobes (Clostridia).
Complete Medium General method by incorporation of agar Part 1: method by enrichment Part 2: Membrane-filtration method
3554777 20 ml 25 tubes in deep tubes in a liquid medium
3554794 7.5 ml 25 tubes
3569654 500 g
TSC without D-Cycloserine/
Agar Base Medium Water sample (preparation) Water sample (preparation) Water sample (preparation)
3554419 100 ml 6 bottles
3569644 500 g 15 min
60C
10 min 15 min
80C 75C Water sample filtration
Meat-Liver Sulfite Iron DRCM
Anaerobic 24 hrs Anaerobic 44 hrs

conditions 37C conditions 37C
Reading Reading/enumeration
Membrane
Anaerobic
48 hrs
conditions 37C
Meat-Liver Sulfite Iron Agar Reading/enumeration TSC without D-Cycloserine
Anaerobic 21hrs 3
conditions 44C 1C
Reading/enumeration
Confirmation
(Columbia Agar)
Pathogen Testing
Ordering Information Legionella spp. & Legionella pneumophila NF EN ISO standard methods
ISO 11731 (05/1998): NF EN ISO 11731-2 (07/2008): NF T90-431 (11/2014):
BCYE Biplate/Agar Water quality Water quality Water quality
3563718 90 mm 20 dishes Detection and enumeration of Legionella Detection and enumeration Detection and enumeration of
BCYE with L-Cysteine/Agar of Legionella Legionella spp. and Legionella pneumophila
Part 2: Direct membrane-filtration method Method by direct inoculation
3563720 90 mm 20 dishes
for water with low bacterial counts and after concentration by membrane filtration
BCYE without L-Cysteine/Agar or centrifugation
3563722 90 mm 20 dishes
Columbia Agar + Horse Blood/Agar Water sample
3563804 90 mm 20 dishes
Direct sample analysis Membrane filtration (0,5 L)
GVPC/Agar
3563717 90 mm 20 dishes
3563719 90 mm 100 dishes
Ultrasonication or raising or centrifugation of the membrane
Legionella pneumophila Latex Kit
3562790 50 tests
Legionella pneumophila and Concentrate
Legionella Species Latex Kit
3562795 50 tests
MonoFluo Kit 0.2 ml 1/10 direct inoculation heat treatment acid treatments
Legionella pneumophila 0.2 ml for dirty water 0.1 ml 0.1 ml 0.2 ml
3532514 24 tests GVPC GVPC GVPC GVPC GVPC 811 days
Nutrient/Agar (for Water Testing) 36C
3563786 90 mm 20 dishes
Portable
1660500 1 lamp (366 nm, requires
4 AA batteries)
Portable UV Observation Chamber Confirmation
(Without Wood Lamp) of Legionella spp.
3550717 1 chamber or
UV Observation Chamber BCYE with BCYE without (or Blood Agar
(WoodLamp)
BCYE Biplate
L-Cysteine
+ L-Cysteine or Nutrient Agar)
48 hrs
36C
3550718 1 UV tube (366 nm)
Confirmation of Legionella pneumophila

MonoFluo Kit or
Legionella pneumophila Latex kit
Note: Legionella Latex Kit Quick Guides are available on request
Pathogen Testing
Legionella spp. and Legionella pneumophila

Ordering Information Legionella DNA Extraction Kit - Bio-Rad solution Alternative real-time PCR method Ordering Information
Catalog# Description Catalog# Description
Extraction step Analysis step
Aquadien Kit
Aquadien DNA Extraction Kit iQ-Check Legionella real-time PCR Kits Reagent Kits
3578121 96 tests iQ-Check Quanti
NF VALIDATION certified according to T90-471 : 2015: Legionellaspp. Kit
Water quality Detection and quantification of Legionella spp. 3578102 96 tests
and/or Legionella pneumophila by concentration and genic
BRD 07/15 12/07 iQ-Check Quanti
amplification by polymerase chain reaction (PCR) BRD 07/16 12/07
WATER ANALYSIS METHODS L.pneumophila Kit
http://nf-validation.afnor.org 3578103 96 tests
Aerosol sample Biofilm sample Water sample iQ-Check Screen

Legionellaspp. Kit
Also available:
3578104 96 tests
Protocol for clogging samples
DNA Extraction Aquadien Kit iQ-Check Screen
Aquadien W2, Wash Solution
L.pneumophila Kit
3578119 48 tests
3578105 96 tests
Aquadien Lysis Solution Instruments and Software
3578125 5 ml 1 bottle, sufficient for
Real-time PCR Cycler
32air samples
3594990 CFX96 Touch Deep Well
Water sample filtration RealTime PCR Detection
System
Software
3593893 CFX Manager Software
Industrial Diagnostic Edition
Documentation
Real-time PCR amplification
Method Validation Guide
Aquadien Kit (NF T90-471)
Lysis of bacteria using thermal shock
95C 15 min
95C
Screening Quantification
Purification of DNA using Ultrafiltration iQ-Check Screen iQ-Check Quanti
Legionella spp. Legionella spp. Key Benefits
iQ-Check Screen iQ-Check Quanti
2 10 min L. pneumophila L. pneumophila Just 4 hours to the result without
confirmation
Detection of Legionella spp. and
Key Benefits L. pneumophila in the same assay
A 96-wells format adaptable to all
High extraction yield Automatic result analysis and interpretation routine throughput
Compatible with all type of Ready-to-use reagents
Elution of DNA for real-time PCR analysis
water (drinking water as well as Fully automated results analysis
coolingtower) Reference Material provided
NF VALIDATION certified Connected to the Standard
Ready-to-use reagents Reference Material
Easy protocol NF VALIDATION certified
Pathogen Testing
Legionella detection in aerosols

Ordering Information with CIP10-M Air Sampler Bio-Rad solution Legionella detection in biofilms, amoeba Bio-Rad Technical Notes
Reagent CIP10-M
Method for the monitoring of airborne micro-organisms,
Aquadien Lysis Solution Technical note # Description
especially Legionella in aerosols
from cooling towers and droplets 16186 Aerosols Testing
Instruments
CIP10-M* Air Sampler/220V 16995 Biofilms Testing
3593084 1 sampler and accessories 16225 Intra-amoebic Legionella
for3 sampling
Legionella Poster - Ref:PO075 (2011)
CIP10-M* Air Sampler/110V
3593094 1 sampler and accessories
Certification of Real-Time PCR Quantification
for3 sampling Methods for Legionella spp. and Legionella
pneumophila (NF T 90-471 Standard)
* The CIP10-M Air Sampler is a product ot Tecora
Aerosol sampling
CIP10-M Certication of Real-Time PCR Quantication Methods
for Legionella spp. and Legionella pneumophila
(NF T 90-471 Standard)
C. Oger-Duroy , A. Quintanar1, C. Mazure1, J.-P. Facon1, J.-P. Tourniaire1, C. Guillaume2, A. Touron-Bodilis3 et M. Binet3, M. Maux4
1
1 Bio-Rad, Food Science, 3 boulevard Raymond Poincar, 92430 Marnes-la-Coquette, France - 2 Ajilon Engineering, Energie-Environnement, 88ter avenue du Gnral Leclerc, 92100 Boulogne-Billancourt, France
3 EDF, Laboratoire National dHydraulique et Environnement, 6 quai Watier, 78401 Chatou Cedex, France - 4 IPL Sant, Environnement Durables Nord,1 rue du Professeur Calmette, 59046 Lille Cedex, France
Summary Inclusivity/Exclusivity Management of Inhibition with CTW

Performance of probes and primers using the iQ-Check
Rapid detection and quantication of Legionella bacteria Legionella pneumophila method The iQ-Check kits include an inhibition control that makes it
represents a major issue for the management of the health Inclusivity of the method was tested for the 15 serogroups possible to evaluate PCR inhibitions and therefore a possible
risk connected with this microorganism in Sanitary Hot of the species Legionella pneumophila. It presents 100% underestimation of the quantity of targets initially present in
Water (SHW) and Cooling Towers Water (CTW). To respond inclusivity. the water sample. A dilution of the DNA makes possible
to this need, Bio-Rad has developed new generations of kits
Real-time NF T90-471 Standard Culture ISO 11731

Exclusivity was tested for the following strains: L. anisa, dilution of the inhibitors, but also leads to a dilution of the
for (i) DNA extraction and (ii) quantication by real-time PCR target. It can be prejudicial for a good estimation of the risk
L. birminghamensis, L. bozemanii, L. cherrii,
(qPCR) of Legionella spp. and Legionella pneumophila.
L. cincinnatiensis, L. dumofi, L. erythra, L. feeleii , because it affects the detection limit of the method which is
These methods incorporating quantication standards L. gormanii, L. hackeliae, L. jordanis, L. lansingensis, quickly reached. In this part, we present the advantages of
(QS1-QS4) as well as a reference material, both linked to the L. longbeachae, L. maceachernii, L. micdadei, L. oakridgensis, use of W2-Aquadien with CTW that is particularly inhibitory
national primary standard, meet the requirements of the L. parisiensis, L. pneumophila 1 to 15, L. sainthelensi, and clogging. This water comes from a specic sampling on
AFNOR NF T 90-471 standard, guaranteeing required L. tucsonensis, L. wadsworthii ; Aeromonas hydrophila, the EDF network.
performance levels (NF VALIDATION Certication no. Alcaligenes faecalis, Bacillus subtilis, Burkholderia
iQ-Check Legionella spp.: BRD 07/15-12/07; iQ-Check cepacia, Clostridium, Enterobacter aerogenes,
Table 3: The various steps of the Aquadien and
Legionella pneumophila: BRD 07/16-12/07). Escherichia coli, Flavobacterium, Klebsiella oxytoca,
W2-Aquadien protocols
Listeria monocytogenes, Proteus vulgaris, Pseudomonas
In order to confront the diversity of water samples to be aeruginosa, Pseudomonas uorescens, Pseudomonas Steps Aquadien Protocol W2-Aquadien Protocol
analyzed, Aquadien protocol variants are available for putida, Serratia marcescens, Stenotrophomonas maltophila. Concentration of cells
3 3
Legionella DNA Extraction

ltrable and difcult to lter water. While answering to the by ltration
requirements for yields/robustness of extraction of the The method presents 100% exclusivity with respect to Cell lysis 3 3
these strains.
NF T 90-471 standard, a complementary protocol Lysate purication 7 3
(W2- Aquadien) makes possible PCR analysis of CTW DNA purication
3 3
Performance of probes and primers using the iQ-Check and concentration
samples that are difcult to lter and particularly rich in PCR
Aquadien Kit
Legionella spp. method. Z Factor 32 36
inhibitors. This new validated protocol has been tested on
water samples from CTW circuits supplied by EDF (lectricit Inclusivity of the method was tested on the following
de France). strains: L. anisa, L. birminghamensis, L. bozemanii, Tables 4: Analysis of inhibition management and PCR
CIP10-M Air Sampler L. cherrii, L. cincinnatiensis, L. dumofi, L. erythra,

L. feeleii , L. gormanii, L. hackeliae, L. jordanis, L. lansingensis,
interpretation with CTW that is particularly clogging and
(See p. 16)
Introduction rich in PCR inhibitors
L. longbeachae, L. maceachernii, L. micdadei, L. oakridgensis,
L. parisiensis, L. pneumophila srogroupes 1 to 15, n=20 PCR Interpretation
The performance characteristics, obtained during AFNOR L. sainthelensi, L. tucsonensis, L. wadsworthii. Z Factor (dilution
DNA extract Protocol Not inhibited Inhibited
validations, for the methods developed by Bio-Rad to of the target)
The method presents 100% inclusivity with respect to Aquadien 1 19 32
quantify Legionella spp. and Legionella pneumophila, are
these strains. Pure
presented here. They answer to the requirement levels of the Aquadien-W2 9 11 36
NF T 90-471 standard, concerning both DNA extraction and Exclusivity was tested for the following strains: Aeromonas
real-time qPCR. hydrophila, Alcaligenes faecalis, Bacillus subtilis, n=20 PCR Interpretation
Burkholderia cepacia, Clostridium, Enterobacter Z Factor (dilution
DNA extract Protocol Not inhibited Inhibited
DNA extraction is a key step in the preparation of samples, aerogenes, Escherichia coli, Flavobacterium, Klebsiella of the target)
the issues being to ensure a high yield of DNA recovery, oxytoca, Listeria monocytogenes, Proteus vulgaris, Aquadien 8 12 320
1/10
while limiting the co-extraction of PCR inhibitors. We show Pseudomonas aeruginosa, Pseudomonas uorescens, Aquadien-W2 20 0 360
below the capacity of the W2-Aquadien protocol to manage Pseudomonas putida, Serratia marcescens,
Bio-Rad, S.N.C. au capital de 50 000 000 Euros, Locataire-Grant, 449 990 712 RCS Nanterre - N Intracommunautaire FR 62 449 990 712 - Siret 449 990 712 00019 PO075 Rev A 12/2011
PCR inhibitors with cooling tower water that is particularly Stenotrophomonas maltophila.
clogging and inhibitory. With the panel of CTW tested, the W2-Aquadien protocol
The method presents 100% exclusivity with respect to these presents better inhibition management. On pure DNA
In this study we also present the performance strains.
extracts, 9/20 samples can be directly analyzed with PCR
characteristics for the DNA extraction yield of the two by the W2-Aquadien protocol, as against 1/20 with the
different Aquadien protocols according to the NF T 90-471 Note: The strains Legionella bozemanii, Legionella dunmoi,
Aquadien protocol (p<0.005). After 1/10 dilution of the DNA
standard, on mineral water, sanitary hot water and cooling Legionella bozemanii, recommended in the standard, were
renamed at the international level as, respectively: extract, no inhibition of the PCR signal remains with the
tower water.
Key Benefits
Fluoribacter bozemanae CIP 103872 (eq ATCC 33217), samples extracted with the W2-Aquadien protocol, while
Fluoribacter dumoi CIP 103876 (eq ATCC 33279), Tatlockia 12/20 of the Aquadien DNA extracts (p<0.001) should be
Performance Characteristics micdadei CIP 103882 (eq ATCC 33218). diluted 1/100 to be analyzable by PCR, which would lead to
a Z factor for dilution of the target of 3,200.
Real-time PCR amplification

Table 1: PCR methods Table 2: Recovery of Aquadien and W2-Aquadien DNA
extraction kits The analysis of yields from the Aquadien protocol with easy
iQ-Check Legionella iQ-Check
Steps Evaluated
NF T 90-471 pneumophila Legionella spp. to extract water samples show a better recovery of the DNA
Requirements iQ-Check Legionella iQ-Check
Validation Results Validation Results
pneumophila Legionella spp. during extraction. The W2-Aquadien protocol is less suited
iQ-Check Legionella
Inclusivity/Exclusivity 100% Inclusivity 100% Inclusivity
of probes and primers
See paragraphs below
100% Exclusivity 100% Exclusivity
Validation Results Validation Results to clean water but becomes the better alternative for
Calibration function Matrix Expected level of recovery Aquadien W2-Aquadien Aquadien W2-Aquadien unlterable water. For complex water samples, it represents
Simulates human breath

PCR efciency 75 to 125% 89% 105.5% (NF T 90-471 requirements) (in %) (in %) (in %) (in %)
(in %) the better compromise between the yield of the extraction
Linear regression ELin Qs1 0.15 log 0.15 log 0.13 log
performance ELin Qs2 0.15 log 0.03 log 0.12 log Sanitary hot
water
25-199 38.90 30.90 45.71 50.12 method and the capacity to purify the DNA.
characteristics: ELin Qs3 0.15 log 0.05 log 0.08 log
Kits
accuracy of linearity (ELin) ELin Qs4 0.15 log 0.07 log 0.07 log Cooling tower 45.71 32.36 63.10 34.67
water
Ergonomic
Reference material Calibration error 0.09 log 0.19 log
< 0.3 log Mineral water 52.48 35.48 46.77 31.62
Conclusion
Limit of quantication: ELoQ 0.15 log ELoQ = 0.117 log ELoQ = 0.101 log Average
45.71 32.36 51.29 38.90
yield
LQ accuracy The complete Bio-Rad solution, with DNA extraction
Limit of detection 90% of positive 28/30 positive: 30/30 positive:
S For each sample and matrices type (N=10), 2 different protocols for clean water and clogging water, and
Detection or quantification
at 5UG/PCR samples 93.33% 100%
Portable
Linking to the primary levels of contamination were tested (1,000 and real-time PCR kits, makes it possible to fulll the
standard with CFX96
Equivalence Calibration error of the 0.07 log 0.00 log 100,000 UG/L). The average yields obtained are requirements of the NF T 90-471 standard. Its speed
of the slopes extremes 0.15 log
presented in this table. and level of performance make it a tool of choice for
Mean calibration error Mean calibration error 0.09 log 0.12 log real-time management of the Legionella bacteria risk
0.15 log
Free of cross contamination (See p. 17)

The robustness of the method is demonstrated here and the in SHW and CTW.
S Criteria of expected performance characteristics matrix effect characterized. In particular, the yield is not
(requirements of the NF T 90-471 standard) and substantially affected by the type of water analyzed. All References:
validation results obtained for iQ-Check PCR water types can be tested by using either of the Aquadien or NF T 90-471, Water quality Detection and quantication of Legionella spp. and/or Legionella
Suitable for both culture media

pneumophila by concentration and genic amplication by real-time polymerase chain
Legionella spp. and Legionella pneumophila methods. the W2-Aquadien DNA extraction protocols. reaction (RT PCR).
and real-time PCR analysis
Pathogen Testing
Pseudomonas aeruginosa
Pseudomonas aeruginosa EN ISO standard RAPIDP.aeruginosa alternative method Ordering Information
Catalog # Description
Acetamide/Broth
RAPIDP.aeruginosa
NF EN ISO 16266 (08/2008) 3554355 5 ml 25 tubes
Water quality Chromogenic agar medium for the
CN/Agar
Detection and enumeration of direct detection and enumeration
3563915 55 mm 10 dishes
Pseudomonas aeruginosa BRD 07/21 04/12 of Pseudomonas aeruginosa by
WATER ANALYSIS METHODS 3556034 200 ml 6 bottles
Membrane-filtration method membrane-filtration
www.nf-validation.afnor.org/en 3564899 500 g
Equipment for Fluorescence
Detection
Water sample filtration Water sample filtration 3350717 Portable UV observation
chamber (without wood lamp)
3350718 Wood lamp
Portable
1660500 1 lamp (366 nm, requires
4AAbatteries)
King B/Agar
Membrane
3555278 7 ml 25 tubes
Oxidase Disks
CN Membrane 3553834 2 vials of 50 oxidase disks
RAPIDP.aeruginosa
2230 hrs
36C 3563984 55 mm 20 dishes
3564900 500 g
48 hrs RAPIDP.aeruginosa
37C
Reading/enumeration Reading
( = 366 nm - UV light)
UV Lamp
Nutrient Agar (water testing)

RAPID'P.aeruginosa
48 hrs
37C
Enumeration of typical colonies:
Blue to blue-green colonies
Acetamide Broth Oxidase test
24 hrs Oxidase Disks
37C
Nessler reagent Key Benefits
King B
5 days
37C High specificity for a direct
Reading/enumeration enumeration of Pseudomonas
Reading at = 366 nm
ammonia producing aeruginosa
UV Lamp Complete results within 2230 hrs
colonies
Good colony coloration contrast
for a rapid reading
Enumeration No confirmation step required
fluorescent colonies
Pathogen Testing
Ordering Information Salmonella spp. - ISO standard

Brilliant Green Red Lactose ISO 19250 (07/2010)

Phenol (or Kristensen or Edel and Water quality
Kampelmacher)/Agar Detection and enumeration of
3564464 500 g Salmonella species
Buffered Peptone Water/Broth

3554179 225 ml 6 bottles
3564684 500 g
Kligler-Hajna/Agar
3555378 10 ml 25 slanted tubes Water sample
3564844 500 g Filtration
LDC-ODC-ADH/Broth (if volume > 10 ml)
3553725 5 ml 3 bottles
MKTTn (Muller-Kauffmann No filtration
Tetrathionate Novobiocin)/Broth (if volume < 10 ml)
3556140 10 ml 50 tubes
3564714 500 g
Nutrient Agar (2.1%) without NaCl
3564485 500 g
Membrane
Physiological Sterile Water

(0.85%) Buffered Peptone Water
3554164 9 ml 25 tubes
18 hrs
RVS (Rappaport Vassiliadis Soya)/ 36C
Broth
3555773 10 ml 25 tubes RVS or MKTTn
3564324 500 g
24-48 hrs 24 hrs
TSI (Triple Sugar Iron)/Agar 41.5C 36C
3564384 500 g
First medium Second medium left to choice
XLD (Xylose Lysine XLD Kristensen
Desoxycholate)/Agar
(Brilliant Green/Phenol Red Agar)
3569124 500 g
24 hrs 24 hrs
36C 36C
Nutrient Agar (2.1%)

24 hrs
36C
Biochemical
(TSI, LCD, Kliger-Hajna,
urea agar) and if appropriate
serological confirmation
Refer to Technical Notes
FSD0001/FSD0002/FSD0003
Pathogen Testing
Ordering Information Staphylococcus aureus NF standard

Baird-Parker/Agar XP T90-412 (06/2006):

Base Medium Water quality
3564814 500 g Detection and enumeration
of pathogenic Staphylococci
Baird-Parker/Agar Membrane-filtration method
Complete Medium
3563991 90 mm 20 plates
Baird-Parker for RPF/Agar
Complete Medium Water sample filtration
3563996 90 mm 20 plates
3578618 90 ml 6 bottles
+ 6 vials supplement
BHI (Brain-Heart Infusion)/Broth
3553664 10 ml 25 tubes
3564014 500 g
Chapman Mannitol (for Water Membrane
Testing)/Agar
3563926 55 mm 10 dishes or or
3564824 500 g
Egg yolk with potassium tellurite Chapman Mannitol (Water) Baird-Parker Baird-Parker + RPF
3554201 5 ml 1 vial
3554205 25 ml 1 bottle
Pastorex Staph Plus Kit
3556353 5 50 tests
3556356 50 tests 48 hrs 48 hrs 48 hrs
37C 37C 37C
Rabbit Plasma Kit Enumeration of white Enumeration of black Direct enumeration of black
3556352 Pack for 20 reactions
colonies with yellow colonies with clear or grey colonies surrounded
RPF (Rabbit Plasma Fibrinogen)/ halo/Gram+ halo/Gram+ by a whitish opaque halo/Gram+
Supplement
3564618 10 vials
Sulfamethazine Solution (0.2%)/
Supplement
3562682 2.5 ml 1 vial Transfer
the membrane
Confirmation Confirmation Confirmation

Latex Pastorex Staph+ Brain Heart Infusion Baird-Parker + RPF
48 hrs 48 hrs
37C 37C
Coagulation reading: Enumeration of black
Rabbit Plasma or grey colonies surrounded
by a whitish opaque halo/
Gram+
Summary
Micro-organisms generally detected and methods the most commonly used according to the origin of water sample
Treated Water Non-Treated Water
Main types Industrial Superficial

Network Tap Artificial Surface Bottled Bathing Waste Cooling Towers Aerosols
of water Process (Surface)
Residual raw
Treated cooling Spa, hydrotherapy, Coastal sea, Drops from
Natural mineral waters, sludge, Non treated
Origins of water Drinking water towers, process, swimming pool, Lakes, rivers estuarine, inland cooling towers,
and source water soil, treatment cooling towers
sanitary water whirlpools water tap water
plant effluents
Turbidity + + + + +++ +++ +++ ++++ ++
Microbial load + + + + ++ ++ +++ +++ +++
MF/MPN MPN/PCR
Typical methods MF (EUR) P/A (US) MF MF MF MF/MPN MPN/PCR PCR
Impedance Impedance
Total flora Total flora Total flora Total flora Total flora Total flora
Total flora
Typical quality E. coli E. coli E. coli E. coli E. coli E. coli
Coliforms Total flora Total flora
indicators Coliforms Coliforms Coliforms Coliforms Coliforms Coliforms
(E. coli)
Enterococci Enterococci Enterococci Enterococci Enterococci Enterococci
Legionella
Legionella Legionella
P. aeruginosa Legionella
Typical P. aeruginosa P. aeruginosa Salmonella Legionella
Staph. aureus P. aeruginosa - Legionella Legionella
pathogens Salmonella Salmonella SRA Salmonella
Salmonella
SRA SRA
SRA
Legends:
SRA = Sulfite-Reducing Anaerobes
MF = Membrane Filtration
MPN = Most Probable Number
P/A = Presence/Absence
PCR = Polymerase Chain Reaction
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 03 710 53 00 Brazil 55 31 3689 6600
Life Science Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00
France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300
Group Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Malaysia 60 3 2117 5260 Mexico 52 555 488 7670
The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700
Russia 7 495 721 14 04 Singapore 65 6415 3170 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700
Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 Thailand 66 2 6518311 United Kingdom 020 8328 2000
Bio-Rad 10 17932 Rev H 2015

FSD 17932

Hochgeladen von

Dokumentinformationen

Originaltitel

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

FSD 17932

Hochgeladen von

Copyright:

Verfügbare Formate

Bio-Rad Laboratories Water Testing

E. coli Automated alternative method: XplOrer64 System 5

Enterococci EN ISO standard 10

Enterococci RAPIDEnterococcus alternative method 11

Part 2 Pathogen Testing

Legionella spp. & Legionella pneumophila NF EN ISO standard 14-15

Legionella detection in biofilms, Amoeba Bio-Rad technical notes 19

Pseudomonas aeruginosa EN ISO standard 20

Pseudomonas aeruginosa RAPIDP.aeruginosa alternative method 21

Salmonella spp. ISO standard 22-23

Staphylococcus aureus NF standard 24-25

Guide for Bio-Rad Products in Water Testing | | 3

E. coli/Coliforms - Alternative method:

MUD/SF Enterococci control cells, cleaning box and 4 racks

Slanetz and Bartley

810 ml per plate

2 hrs Key Benefits

Ordering Information Sulfite-Reducing Anaerobes (Clostridium) - NF EN ISO standards

Meat-Liver Sulfite Iron DRCM

Anaerobic 24 hrs Anaerobic 44 hrs

Meat-Liver Sulfite Iron Agar Reading/enumeration TSC without D-Cycloserine

Confirmation of Legionella pneumophila

Note: Legionella Latex Kit Quick Guides are available on request

Legionella spp. and Legionella pneumophila

Aerosol sample Biofilm sample Water sample iQ-Check Screen

Legionella detection in aerosols

* The CIP10-M Air Sampler is a product ot Tecora

Summary Inclusivity/Exclusivity Management of Inhibition with CTW

Real-time NF T90-471 Standard Culture ISO 11731

Legionella DNA Extraction

CIP10-M Air Sampler L. cherrii, L. cincinnatiensis, L. dumofi, L. erythra,

Real-time PCR amplification

Simulates human breath

Free of cross contamination (See p. 17)

Suitable for both culture media

and real-time PCR analysis

Nutrient Agar (water testing)

Ordering Information Salmonella spp. - ISO standard

Brilliant Green Red Lactose ISO 19250 (07/2010)

Buffered Peptone Water/Broth

Physiological Sterile Water

Nutrient Agar (2.1%)

Ordering Information Staphylococcus aureus NF standard

Baird-Parker/Agar XP T90-412 (06/2006):

Confirmation Confirmation Confirmation

Main types Industrial Superficial

Bio-Rad 10 17932 Rev H 2015

Das könnte Ihnen auch gefallen