Microbiological Analysis of Water Quality

Looking in to the 21st Century

sigma-aldrich.com

January 31, 2012

By Richard Glindon
Presented by Klaus Herick

Content
Introduction
What does the future hold for microbiological water AQC?
How can we meet the demands microbiological water AQC in the future?
What does this mean for microbiology laboratories analysing water?
What does evolving AQC demand of CRM and PT providers?
Modern microbiology
VitroidsTM technology
Features
Applications
VitroidsTM proficiency testing
Summary
Acknowledgements

What does the future hold for microbiological

water AQC?
Where we are today
Still using analytical microbiological methods developed over a century ago
In the UK, good quality water is the expected norm
More than 99.5% of all drinking water achieved required quality standards

What the demands of the next 5 years will be

To maintain and raise standards
Monitor growing environmental pressure
Adapt to the requirements of an increasing demand on water supplies

How can we meet the demands of microbiological

water AQC in the future?
How these demands can be met
Structured, procedural & managed AQC
Laboratory accreditation to ISO 17025
Method validation CRMs from producers accredited to ISO 17025 + ISO Guide 34
UKAS TPS 57, Guidance and Policy on the Selection and Use of Reference
Materials says
Obtaining a reference material from a Reference Material Producer who is accredited to
ISO Guide 34 by a recognised accreditation body will provide the organisation with
confidence that the statements that the Reference Material Producer makes about their
material can be relied upon.

Participation in Proficiency Testing schemes from ISO 17043 accredited providers

Regular Assessment visits from UKAS or other national accreditation bodies

What does this mean for microbiology

laboratories analysing water?
Continue to strive for improvements
More automation and instrument based measurement?
More standardisation of processes?
More regulation e.g. MCERTS for water?

= more reliable results

What does the future demand of CRM and PT

providers?
Must be accredited to ISO Standard 17025, ISO Guide 34 and ISO
Standard 17043
Demanding requirements to demonstrate:
Between and within unit homogeneity
Stability
Reproducibility
Known uncertainty
Need for a wide range of CRMs and PT, both pure and in matrices

Easy (relatively) for chemicals and mixtures of chemicals, but

microbiology depends on living organisms, we must meet this challenge.

Modern microbiology #1
More microbiology supporting 9 billion people worldwide by 2025?
Water and waste water microbiology
Aging infrastructure
Public confidence
Bathing water microbiology
EU Standards

Takes routine microbiology from an applied sciences to metrology?

Modern microbiology #2
PCR
Molecular Biology
Methods

quantitative PCR

FISH
microscope, flow cytometer
Ribosomal RNA
Alternative sandwich hybridization
assays, macroscopic scale

Immunology
Methods

Classical Cultivation
Methods

ELISA

LFA

Growth based on
specific nutrition

Chromogenic media

New methods require proper validation and appropriate AQC

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Modern microbiology #3
Need for CRMs and PT that conform to the required standards
Qualitative, traceable to internationally recognised references.
NCTC, ATCC, etc
Quantitative
Defined CFU
Convenient
Stable, easy to ship, store and use.

Introducing the Vitroids technology

from Sigma-Aldrich
Vitroids Technology
Microbiology CRMs and PT that are as stable, reproducible and precise as
any CRM for chemical analysis

Developed by Dr. A. Codd

Produced to ISO17025 and Guide 34 by Sigma-Aldrich

Certified Reference Materials called Vitroids/RapidCheck

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How VitroidsTM in AQC help maintain high

standards of microbiological water quality
Certified
Qualitative and quantitative microorganisms (ISO17025:2005 + Guide 34:2009)
Proficiency Testing samples to ISO17043:2010
CFU within 2 x SD

Stability
Up to 4 years at 4C for common strains

Convenience
15 minutes rehydration time
Use buffer or add to solid or liquid medium

Multi-purpose
Wide range of organisms and CFU
Constantly being updated
Suitable for many methods

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VitroidsTM: Certified

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VitroidsTM: Stable
Multiple analysis of a manufacturing lot of E. coli
Production date Oct 2007 (target gravimetric value of 1000cfu)
Multiple data points in 2008, 2009, 2010, and 2011
Long term stability graph
Long Term Stability (E. coli)
1.5
Normalized Measure

1.3
Series1

1.1

lower confidence
0.9

Upper Confidence

0.7
0.5
Feb-08

Jul-09

Nov-10
Date

Apr-12
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VitroidsTM: Convenient 15 min reconstitution time

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VitroidsTM applications #1

Membrane Filtration (MF)

Most Probable Number (MPN)
Presence / Absence (P/A)
Traditional culture methods including the use of chromogenic media
Others include the identification, quantification or P/A of an organism which may
require specialised techniques

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VitroidsTM applications #2
Chromogenic media CP ChromoSelect Agar

Strains

Comparison between TSCF agar and the

mCP and CPC (CP ChromoSelect Agar) media for
enumerating strains of C. perfringens in water
samples by membrane filtration

Indole
reaction

C. perfringens

465 (96.3%)

C. tertium

1 (0.2%)

C. botulinum

1 (0.2%)

C. fallax

1 (0.2%)

C. bifermentans

2 (0.4%)

C. sordelli

12 (2.5%)

C. tetani

1 (0.2%)

All green colonies isolated from CP

ChromoSelect Agar and identified with
API system (n=483)

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VitroidsTM applications #3
HybriScan rapid test system
7

HybriScan is a rRNA sandwich

hybridisation test
Colorimetric read out at 450 nm
96 well microplate format

signal/noise ratio

Validation of cfu /
Standard calibration
of E. coli

5
4
3
2
1
0

Comparison of plate count test versus

HybriScanD Total Waste Water

S4 (10.000)

S3 (3.000)

HybriScanTotal Count

S2 (1.000)

HybriScan
FastScan-E.coli/Shigella
E. coli

FastScan-Bac

20000
p la t e c o u n t

FHybriScan
a s t S c a n-Total
- B a cWaste Water

cells/ml
cells/ml

15000
10000
5000
0
1

b water
e e r ssample
a m p le

10

11

12

13

14
17

VitroidsTM proficiency testing

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VitroidsTM product range

Escherichia coli
Bacillus subtilis
Clostridium perfringens
Enterobacter aerogenes
Enterococcus faecalis
Legionella
pneumophilia & bozemanii
Listeria monocytogenes
Pseudomonas aeruginosa
Salmonella typhimurium
Staphylococus aureus
Citrobacter freundii
Aspergillus niger
Candida albicans
CFU levels from 30 to 50,000
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Summary What is needed to meet the water

microbiology demands of the 21st century
Maintain water quality standards and public confidence
Apply chemistry metrology to microbiological AQC
Use new technology and methods to give faster, more reliable results.
Microbiological CRMs from ISO17025 + Guide 34 accredited sources
Microbiological PT from ISO17043 providers
Regulation to standardise microbiological AQC procedure

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Acknowledgements
Dr. Arthur Codd, Newcastle, UK
Jvo Siegrist, Buchs, Switzerland

HybriScan is a trademark of Scanbec GmbH

Vitroids is a registered trademark of Sigma-Aldrich Corporation

For more information see: www.sigma-aldrich.com/microbiology

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