Approvals and Studies of Waste Waters DST Technology Colilert, Colilert18, and Enterolert

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Approvals and Studies of Waste WatersDST
Technology (Colilert, Colilert-18, and
Enterolert)

• Gil Dichter
• World Wide Technical Support ManagerEnvironmental
  
• Idexx Laboratories, Inc.

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Objectives

• History of testing
• Intro to water micro
• Why E.coli, enteorococci are being proposed for
  testing wastewaters
• Regulations
• Methods for testing WW
• WW Studies

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History of Coliform Testing

• 1900 - Coliform Testing, LTB
• 1904 - Fecal Coliform (Thermotolerant-44.5C)
• 1930 - Membrane Filtration
• 1960 - Clarks P/A Broth
• 1989 - Colilert
• 1996 - Colisure

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Coliform Genera

• Escherichia- human and animal feces
• Enterobacter- environment, feces
• Klebsiella- environment, feces
• Citrobacter- environment
• Serratia- environment

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Thermotolerant Coliforms aka Fecal Coliforms

• It is a subset of total coliforms
• Defined as coliform bacteria that can grow at
  44.5C (production of gas and turbidity)
• Consists of the following coliforms
• E. coli
• K. pnuemoniae
• Enterobacter spp.
• Both fecal and environmental strains

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Escherichia coli

• A genus of Gram negative bacteria of the family
  Enterobacteriaceae
• A type of thermotolerant coliform bacteria
  commonly found in the intestines of warm blooded
  animals including humans
• Does not occur naturally in soil and vegetation
• May occur in soil and water as a result of fecal
  contamination

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E.coli bacteria in color
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Why Test for E.coli    
  SourceE.coliFecal Coliform A.P. Dufour.
Special Technical Publication 65, ASRM. Pp48-58,
1977
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History of Enterococcus

• 1903- Thierceleu Joulaud proposed enterococcus
  as a genus
• Gram coccoid shape bacteria, origin is
  intestinal
• 1908 Andrews Hodi suggested a name change to
  Streptococcus and spp. should be faecalis based
  on fecal origin (chains of coccoid shape cells)
• 1984- Slanetz Kippler-Baltz suggested
  S.faecalis S.faecium be transferred to the
  genus Enterococcus based on molecular approaches.
  This was accepted. S.avium S. gallinarium were
  also transferred.

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Enterococcus

• A genus of Gram positive facultatively anaerobic
  coccoid bacteria.
• Subset of the Streptococcus family
• Occurs in the intestine of humans and animals
• Some strains are found in the environment

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Enterococcus vs Fecal Streptococcus

• Enterococci are the key subset of fecal
  streptococcus
• Fecal Streptococcus
• 1) Enterococcus spp.
• E. faecalis, E. faecium, E. gallinarum, E. avium,
  E. durans, E. casseliflavus
• 2) Non-enterococci streptococcus
• S. bovis, S. equinus
• Defined as gram , catalase -, grows in 6.5
  saline, 40 bile salts, and at 10C and 45C.

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Testing Focus

• Focus on testing at present is for indicator
  organisms - TC, E.coli Enterococci, which are
  not considered pathogens
• Infers the microbiological water quality using
  these indicators as proxies of human fecal
  contamination
• It is an indirect measurement of public health
  risk
• The detection is rapid, standardized, inexpensive
  and simple to perform

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Colilert, Colilert-18 History

• Invented by a Yale Medical School professor to
  help a nearby water utility avoid false alarms
  due to Klebsiella pneumoniae
• Used more than all other methods combined in US
  drinking water market
• Used on every continent

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Approvals

• USEPA for drinking waters and surface waters
• Ambient Water Rule (FR July 21,2003)
• AOAC/FDA, IBWA, Standard Methods for Water and
  Wastewater
• State approvals for waste waters and surface
  waters
• Approvals in gt26 countries world-wide

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Government Approvals World WideColilert,
Colilert-18 Colisureas of 5-12-05
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Approvals-Enterolert

• ASTM D-6503-99
• Standard Method for Enterococci in Water using
  Enterolert
• Ambient Water Rule (FR July 21, 2003)
• Approved in US, Australia and New Zealand
• Pending US EPA Approval for WW

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Analytical Methods for Biological Pollutants in
the Ambient Water Rule

• Procedure for enumerating E.coli, enterococci and
  Crypto Giardia in ambient water
• Based on 1986 guidelines for E.coli enterococci
• Methods include Colilert and Colilert-18 for
  E.coli, Enterolert for enterococci and Filtamax
  for Crypto.
• Final rule effective - 8/20/03

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States or regions approving E.coli and Colilert
for Waste Water Testing

• Indiana
• Oregon
• Regions of California
• Virginia
• Arizona
• Tennessee
• Utah
• Maryland
• Pennsylvania
• Kentucky

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Guidelines for Analysis of Pollutants Analytical
Methods for Biological Waste Water

• Federal Register Notice- August 16th, 2005
• Comments on proposal ended on October 17th, 2005
• Promulgation expected by the end of 2006 ?
• Testing for E.coli and for Enterococci
• Approved methods include Colilert, Colilert-18
  and Enterolert, EPA MF methods
• E.coli for fresh surface waters and enterococci
  for marine waters

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Status of WW Regulations in Massachusetts

• It is anticipated that the Secretary of State
  will sign the WW Quality Standards by the end of
  the year.
• However, we will have to wait and see
• Implementation of this Standard is not yet
  defined
• Will it change for each utility based on the
  renewal of the permit?
• Will there be a universal change by a specific
  date?

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Analytical Methods for WW

• Quantitative methods for testing can include
• total coliforms
• E.coli
• fecal coliforms
• enterococci
• Methods include
• LTB/BGLB/EC Medium (MUG)
• Membrane Filtration Methods such as m-Endo, m-FC
• Enzymatic methods such as Colilert, Colilert-18
  and Enterolert

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Defined Substrate Technology

• Nutrient-indicator (substrate) is targeted to
  organism-specific enzyme
• Nutrient-indicator is the major source of food
• Nutrient-indicator changes color or fluoresces
  when metabolized
• Non-targets are suppressed

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ONPG Positive ReactionColilert
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MUG Positive ReactionColilert
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Enterolert Positive Reaction
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Quanti-Tray SealerModel 2X
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Quanti-Tray Demonstration
Add Colilert to sample and shake to dissolve
Pour mixture into a Quanti-Tray
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Quanti-Tray Demonstrationcont.
Seal and then incubate at 35C for 24 hours
Count positive wells and refer to MPN table
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E.coli Positive Tray
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Enterolert Demonstration
Count fluorescent wells and refer to MPN table
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Which Would You Rather Count?
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Quanti-Tray/2000 Scientific Basis
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Quanti-Tray/2000 vs MTF
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Quanti-Tray Is Easy

• No media preparation
• No dilutions needed for counts under 2,419
• No colony interpreting
• No confirmations
• Easy to train staff
• QC in as little as 20 min/month
• Two tests-total coliforms and E.coli

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Studies

• Numerous studies evaluations since 1990
  world-wide.
• Colilert/Colilert-18 and Enterolert compared
  against MF and MTF methods

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WW/Sludge Studies cont.

• Comparison of E.coli, Total coliform, and Fecal
  Coliform Populations as indicators of Wastewater
  Treatment Efficiency- G. Keith Elmund, Water
  Environment Research, May/June 1999, Vol. 71, 3
• N453 WW samples over 1 year
• Samples tested with Colilert/Quanti-Tray and by
  m-FC
• Quanti-Tray was an effective method for
  quantifying E.coli
• Authors agree that E.coli rather than fecal
  coliform to measure WW disinfection efficiency
  would provide public health protection benefits
  for users of recreational and water supplies

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Wastewater evaluation in Indiana

• Evaluation of Colilert-QuantiTray-2000 vs. m-TEC
  - R.A. Whitehead. Presented at the 65th IWPCA,
  Nov. 2001
• r 0.97, n50 and t test indicated equivalence
  between methods
• HPC interference is eliminated
• Less subjectivity in interpreting results

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Comparison of Colilert-18, Colilert/Quanti-Tray
System against USEPA Method 1603 (modified m-TEC)
for Detecting and Enumerating E. Coli in Waste
Water

• Internal Idexx study of primary wastewater sewage
  effluent from 5 different geographic locations
• Both Colilert-18/Quanti-Tray, Colilert/Quanti-Tray
  were equivalent to the m-TEC method for E.coli
• Mean value (n 16) for m-TEC 50.9/100ml
• Mean value (n 16) for Colilert-18 54.5/100ml
• Mean value (n 16) for Colilert 55.6
• Student t test indicated no significant
  difference between m-TEC, Colilert-18 and
  Colilert p (0.05) 0.19 and 0.15 respectively

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WW/Sludge Studies cont.

• Evaluation of Colilert and Enterolert DSM
  Methodology for Wastewater Applications- G.P.
  Yakub, Water Environment Research, March/April
  2002, Vol 74, 2
• Colilert- total coliform test was evaluated for
  its utility in estimating fecal coliforms by
  incubation at 44.5ºC
• The modified coliform test correlated well to
  m-FC (correlation 0.8)
• Enterolert (enterococci) correlated well to the
  MF method
• The methods performed as well or better than the
  traditional methodology

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WW/Sludge Studies cont.

• Internal Study Colilert-18/Quanti-Tray for Fecal
  Coliforms in Water
• 47 chlorinated final effluent samples from 25
  different geographic sites
• Liner regression analysis (r 0.85) showed
  acceptable results between the 2 methods
  (Colilert-18 and m-FC)

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WW/Sludge Studies cont.

• Enumeration of Coliform Bacteria in Wastewater
  Solids using Defined Substrate Technology- T.A.
  Kramer- Water Environment Research, Nov/Dec 2002,
  Vol. 74, 6
• Data indicates that Colilert/Quanti-Tray system
  is capable of producing equal population
  estimates for coliform and E.coli bacteria in WAS
  as the accepted technique of MTF
• This system offers numerous advantages over MTF,
  including ease of use, lower cost and rapid
  detection time

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WW/Sludge Studies cont.

• A comparison of methods used to enumerate E.coli
  in conventionally treated sewage sludge-J.P.
  Eccles, J of Applied Microbiology 2004, 96,
  375-383
• Three methods tested (Colilert-18/Quanti-Tray,
  MGLA and CEC) gave comparable recoveries and did
  not vary by greater than 1 log
• All methods had a false positive rate of lt3
• Colilert/Quanti-Tray had a false negative rate of
  3.8, compared to 7.75 (CEC) and 35.5 (MLGA)

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Evaluation of the effectiveness of a commercially
available defined substrate medium and
enumeration system for measuring Escherichia coli
numbers in faeces and soil samplesR.W. Muirhead,
R.P. Littlejohn and P.J. Bremer, Letters in
Applied Microbiology 2004, 39, 383-387

• Compared Colilert/QuantiTray system against
  miniaturized MPN method with confirmation
• Concluded that Colilert/Quanti-Tray system was
  significantly more precise than the miniaturized
  system
• Colilert/Quanti-Tray system is suitable for
  measuring E.coli in faeces and soil systems
• Advantages are increased precision and reduction
  in lab analysis time

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