Download Developments in CSIR`s water microbiology laboratory and

Developments in CSIR's water microbiology
laboratory and the introduction of molecular
research
CSIR
NRE
Neil Leat
CSIR Beyond 60 process
Go beyond routine testing and include more innovative research.
Microbiology laboratory will introduce molecular methods in
research.
Interact with clients to ensure interests are taken care of.
-Continue to offer testing service.
-Refer clients to competent external service provider.
-Involve clients in research.
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Traditional Pour-Plate membrane filtration
method.
-A 100 ml volume of a water sample is drawn through a membrane filter (0.45 µm
pore size).
-Bacterial cells are trapped on the membrane surface.
-The filter is placed on a petri dish containing m-FC agar and incubated for 24
hours at 44.5 °C.
-The elevated temperature suppresses the growth of non-faecal bacteria.
-As the faecal coliform colonies grow they produce an acid (through fermenting
lactose) that reacts with the aniline dye in the agar thus giving the colonies their
blue colour.
-For enumeration faecal coliform colony density should be between 60 and 150
per plate.
-For enumeration of E.coli transfer membrane from m-FC to NA-MUG media.
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Colilert tests for monitoring Total coliforms and E.coli.
-Colilert-18 uses Defined Substrate Technology®
-Coliforms use their β-galactosidase enzyme to metabolize ONPG and change it from
colorless to yellow.
-E.coli use β-glucuronidase to metabolize MUG and create fluorescence.
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Using Colilert tests
Step 1.
Add reagent to sample.
Step 2.
Pour into Quanti-Tray®/2000 (counts 1 to 2,419)
Step 3.
Seal in Quanti-Tray® Sealer and place in
35 °C incubator for 18 hours.
Step 4.
Read results: Yellow wells = total coliforms
Fluorescent wells = E. coli
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Colilert-18 Benefits
Easy
Ease of use
Unit-dosed packaging eliminates media preparation.
No repeat testing due to clogged filters or heterotrophic interference.
Rapid
Under one minute hands-on time.
Detects coliforms and E. coli simultaneously in 18 hours or less.
No glassware cleaning or colony counting.
Colilert is US EPA-approved and is included in Standard Methods for Examination of Water and Wastewater.
Accurate
Identifies E. coli specifically.
Suppresses up to 2 million heterotrophs per 100 mL.
Eliminates the subjective interpretation found in traditional methods.
Detects a single viable coliform or E. coli per sample.
Economical
Minimizes evening and weekend work.
Up to 12-month shelf life at room temperature.
Flexible
A Colilert®-18 Snap Pack can be used for presence/absence (P/A) or quantification testing.
Quanti-Tray®/2000 provides counts to 2,419/100 mL without dilutions.
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Molecular Research
Powerful because it provides access to the vast amount of information
encoded in nucleic acid sequences.
Key areas include the following:
-Direct Pathogen Detection: The rapid identification and quantification of pathogens in water. These
approaches are likely to have the greatest impact for pathogens which are difficult to culture or analyse such as
viruses and protozoan parasites and whose presence is not closely associated with faecal indicator microbes.
-Microbial Source-Tracking of faecal pollution: Tracing sources of faecal contamination and
facilitating targeted and cost effective remediation efforts.
-Microbial Community Analysis: Providing a greater understanding of microbial communities in
wastewater treatment environments by studying rRNA sequence heterogeneity. This provides engineers with
information needed to manipulate treatment processes so as to optimize microbial community structure for
wastewater processing.
-Epidemiological studies: In epidemics involving waterborne diseases, modern molecular techniques
provide tools for tracing infectious agents.
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Faecal Source Tracking
-Faecal source-tracking aims to identify which species contribute to the pollution and to establish the
relative importance of each source.
-Most useful in situations where faecal indicator bacteria have been identified but there is uncertainty
about the significance of contributing sources (humans, gulls and agricultural runoff etc).
-Faecal microbes have genotypic characteristics which reflect their origin.
-Analysis of these characteristics can be used to identify which hosts are contributing to the
pollution.
Emerging source tracking techniques include:
[1] Analysis of F+RNA coliphage groups.
[2] Analysis of 16S rRNA sequences from the order Bacteroidales.
[3] Direct detection of host-specific enteric viruses.
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Faecal Source Tracking
Analysis of F+RNA coliphage serotypes
4 F+RNA coliphages subgroups
human faeces
Subgroups II & III
non-human faeces
Subgroups I & IV
Source-tracking using F+RNA coliphages relies on quantifying the ratio of the
four subgroups in contaminated water samples.
Classical method relies on plaque formation followed by serotyping or
genotyping using specific probes.
Modern culture-independent methods use real-time PCR assays for the
simultaneous quantification of all four subgroups in a single tube (Kirs and
Smith, 2007).
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Faecal Source Tracking
Bacteroidales 16S rRNA analysis:
Bacteroidales represent a major component of human and animal faecal flora
(109 to 1011 per gram of faeces).
Specific host-associated 16S rRNA sequences used for source-tracking
assays.
Assays range from a quantitative assay for the detection of overall faecal
pollution to assays which discriminated between pollution from humans,
horses, dogs, elk, cattle, pigs and avian sources.
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Phylogenetic relationships among Bacteroidales 16S rRNA sequences
Human
Bovine
Canine
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Faecal Source Tracking
Direct detection of host specific enteric viruses:
Host specificity of enteric viruses has made them potential source tracking
targets.
Detection of a given group of viruses is a clear indication of contamination by a
specific host.
An obvious challenge with this approach is that virus concentrations in
contaminated waters are likely to be relatively low.
Conventional nested PCR assays are available for
-25 Human Enteroviruses (HEV)
-47 Human adenoviruses (HAdV)
- 7 Bovine Enteroviruses (BEV)
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Faecal Source Tracking: An Example
Freshwater beach, Hamilton Harbour, Lake Ontario, Canada.
E. coli concentrations highest in ankle-depth water (177,000CFU/100ml).
Two source tracking techniques used.
Rep-PCR
Antimicrobial Resistance
Figure – Two-way assignment of Escherichia coli isolates in Bayfront
Park Beach sand and water samples to bird or wastewater fecal
sources by antimicrobial resistance (left) and rep-PCR DNA
fingerprinting (right) analyses. (Edge and Hill, Water Research: 2007
41: 3585-94.)
© CSIR 2008
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© CSIR 2008
www.csir.co.za
© CSIR 2008
www.csir.co.za
Thank You
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