e. coli workgroup 1/10/07 meeting single sample maximum to assess compliance
TRANSCRIPT
E. coli Workgroup1/10/07 Meeting
Single Sample Maximum to Assess Compliance
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E. coli Workgroup1/10/07
• Topics for this Discussiono Coliforms
• Total• Fecal
E. colio Occurrence o Methods of Detection
• Membrane Filtration• Colilert®
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Coliforms – Total and Fecal
Coliforms: All of the aerobic, facultative anaerobic, gram negative, non-spore forming, rod shaped bacteria that ferment lactose within 48 hours at 35º C
o Fecal Coliforms – can grow at higher temperatures (45º C); grow in the gut of warm blooded animals and in soil
o Escherichia colio Citrobactero Enterobactero Klebsiella
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Fecal Coliforms
• Fecal Coliforms
o Escherichia coli (E. coli) is the most representative – why chosen to indicate presence of fecal contamination and potential presence of pathogens
o In human and warm blooded animal intestine
o Most E. coli strains do not produce toxins
o Enteropathogenic E. coli cause gastroenteritis and is usually contracted via contaminated food
o E. coli 0157:H7 is a toxigenic strain – found in cattle feces - potential to be in runoff
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E. Coli and Single Sample Maximum
•Unlike chemical constituents, dissolved throughout a sample, biological indicators (i.e., E. coli) do NOT distribute uniformly in a sample
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E. Coli and Single Sample Maximum
Biological organisms group together
E. Coli and Single Sample Maximum
E. Coli Images (below) demonstrate grouping or clumping together – a wastewater monitoring issue because they do NOT uniformly distribute
Example:
Effluent sample #1 gets a big “group” and is >235/100 mL; Effluent sample #2 taken immediately after the 1st sample gets a small “group” and is <235/100 mL.
Which sample is representative of the effluent?
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E. coli Occurrence
• Receiving Streams (Marion County Data)
o Dry Weather >10/100 mL to >2,000/100 mL
o Wet Weather >2,000/100 mL to >100,000/100 mL
• Wastewater (Fecal Coliforms - Metcalf & Eddy)
o WWTP Influent >10,000/100 mL to >100,000/100 mL
o Disinfected Effluent (GOAL) <1/100 mL to < 235/100 mL
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E. coli Occurrence
• Source Water Data (12/2/02 CFR – Approval of Analytical Methods for Microbiological Contaminants)
Sample ID Sample Source
E. coli (cfu/100 mL)
982084A Millbrae, CA 230,000
982305A Millbrae, CA 11,000,000
990025A Jacksonville, FL 700,000
990052A Schaumberg, IL 1,000,000
990217A Mission, KS 4,000,000
990273A Salem, OR 1,000,000
990438A Ames, IA 1,000,000
990442A Mission, KS 3,000,000
990443A Liberty, MO 2,000,000
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E. Coli Detection Methods (1)
o Membrane Filtration
o Sample is filtered thru a porous filter
o Filter placed on a nutrient pad in a Petri dish
o Incubated at 35º C for 2 hours to revive injured or stressed cells and then for 22 hours at 44.5 º C
o After incubation filter is transferred to a filter pad saturated with urea substrate
o After 15 minutes colonies of a specific color (yellow/ yellow brown) counted - result is cfu/100mL
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E. Coli Detection – Membrane Filtration
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E. Coli Detection – Membrane Filtration
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Marion, IN Fecal Coliform MF Data – Split Samples
Date #1-cfu/100 mL #2 - cfu/100 mL Avg. Difference
8/17/06 17 31 24 58%
8/18/06 81 34 58 82%
8/22/06 49 83 66 52%
9/7/06 24 71 48 99%
9/19/06 35 70 53 67%
10/4/06 72 42 57 53%
10/6/06 13 40 27 102%
10/10/06 117 32 75 114%
10/13/06 27 108 68 120%
10/30/06 46 38 42 19%
10/31/06 89 42 66 72%
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Marion, IN Fecal Coliform MF Data – Split Samples
• Summary of data collected by Marion, IN WWTP indicates variability of results from MF procedure
• The two samples were 50 mL each taken from the same sample container
(Note: Bacteriological samples are always grab samples)
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E. Coli Detection Methods (2)
• Colilert®
o Most recently approved procedure – Defined Substrate Technology allows growth of E. coli that will appear as a fluorescent yellow
• Sample is mixed with DST Reagent
• Poured into “tray” with multiple “wells”
• Incubated for 24 hours at 35º C• Yellow fluorescent “wells”
counted; use IDEXX MPN Table to calculate result - MPN/100 mL
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E. Coli Detection - Colilert®
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E. Coli Detection - Colilert®
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E. Coli Detection - Colilert®
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Observations on Detection Procedures
• 2003 Wyoming DEQ/WQD Study Comparing MF Enumeration with Colilert® (On Surface Waters)
o Inaccurate MF counts from:
• Variations in filter quality
• Filtering highly turbid samples
• Insufficient rinsing of the sample aliquot container
• High number of non-coliform bacteria or toxic substance
o ColiLert® not affected by presence of non-coliforms
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Observations on Detection Procedures
2003 Wyoming DEQ/WQD Study Comparing MFEnumeration with Colilert® (Surface Water)
• No significant difference in E. coli results between MF and Colilert®
• Mean and median bacteria densities between 2 methods were essentially equal
• Either method could be used with confidence to enumerate E. coli in treated sewage and ambient surface waters
• Colilert® had several advantages
o Samples processed much faster than MFo Trays require more incubator space but single
temperature eliminates need for 2 incubatorso MF processing steps and limitations are not
required and do not influence results
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E. coli Workgroup1/10/07
References
1. Wastewater Engineering: Treatment, Disposal, and Reuse. Metcalf and Eddy, 3rd Edition, McGraw-Hill, 1991.
2. Operation of Wastewater Treatment Plants: A Field Study Training Program. US EPA, OWP, California State University, Sacremento, 2003.
3. Basic Microbiology for Drinking Water Personnel. Dennis Hill, American Water Works Association, 2001.
4. Modified mTEC Agar, Colilert®, and M-FC Agar-Field Trial Comparison of Bacteria Enumeration Methods in Surface Waters of Eastern Wyoming. Eric Hargett and Lanny Goyn, Wyoming DEQ/WQD, 2003.
5. Notice of Data Availability; National Primary and Secondary Drinking Water Regulations: Approval of Analytical Methods for Chemical and Microbiological Contaminants; Additional Information on the Colitag® Method. Federal Register: December 2, 2002 (Volume 67, Number 231); pages 71520 -71523.
6. Colilert® Test Procedure, Illustrative Brochure. IDEXX Laboratories, Westbrook, ME. www.idexx.com
7. Marion, IN Utilities Unpublished Data, 2006.8. Raw Sewage Overflow Long Term Control Plan and Water
Quality Improvement Report. City of Indianapolis, IN, September 2006.
9. Method 1103.1: Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC). US EPA, Office of Water, EPA 821-R-02-020, September 2002.