Microbiological Analysis of Water Quality•Looking in to the 21st Century

sigma-aldrich.comJanuary 31, 2012

By Richard GlindonPresented by Klaus Herick

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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

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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

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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

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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

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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.

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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?

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Modern microbiology #2

Ribosomal RNA

PCR quantitative PCR

FISHmicroscope, flow cytometer

Alternative sandwich hybridizationassays, macroscopic scale

LFA

Chromogenic media

Molecular BiologyMethods

ImmunologyMethods

Classical CultivationMethods

Growth based onspecific nutrition

ELISA

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.

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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)

0.5

0.7

0.9

1.1

1.3

1.5

Feb-08 Jul-09 Nov-10 Apr-12

Date

Norm

aliz

ed M

easu

re

Series1lower confidenceUpper Confidence

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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 #2Chromogenic media CP ChromoSelect Agar

• 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

Strains Indole reaction

N

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 #3HybriScan® rapid test system

• HybriScan is a rRNA sandwich hybridisation test

• Colorimetric read out at 450 nm• 96 well microplate format

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4

b e e r sa m p l e

cells

/ml

p la t e c o u n t F a s t S c a n -B a c

water sample

cells

/ml

HybriScan®-Total Waste Water

Comparison of plate count test versus HybriScan®D Total Waste Water

0

1

2

3

4

5

6

7

S4 (10.000) S3 (3.000) S2 (1.000)

sign

al/n

oise

ratio

FastScan-Bac FastScan-E.coli/ShigellaHybriScan®-Total Count

HybriScan®-E. coli

Validation of cfu / Standard calibration

of E. coli

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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