Transcript E. coli Workgroup 1/10/07 Meeting Single Sample Maximum to Assess Compliance E. coli Workgroup 1/10/07 • Topics for this Discussion o Coliforms • Total • Fecal  E.

E. coli Workgroup
1/10/07 Meeting
Single Sample Maximum to
Assess Compliance
E. coli Workgroup
1/10/07
• Topics for this Discussion
o Coliforms
• Total
• Fecal
 E. coli
o 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
o
o
o
Escherichia coli
Citrobacter
Enterobacter
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
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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?
E. coli Occurrence
• Receiving
Streams
County Data)
o
o
(Marion
Dry Weather >10/100 mL to
>2,000/100 mL
Wet Weather >2,000/100 mL
to >100,000/100 mL
• Wastewater (Fecal Coliforms Metcalf & Eddy)
o
o
WWTP Influent >10,000/100
mL to >100,000/100 mL
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 noncoliforms
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Observations on Detection Procedures
2003 Wyoming DEQ/WQD Study Comparing MF
Enumeration 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
o
o
Samples processed much faster than MF
Trays require more incubator space but single
temperature eliminates need for 2 incubators
MF processing steps and limitations are not
required and do not influence results
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E. coli Workgroup
1/10/07
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Wastewater Engineering: Treatment, Disposal, and Reuse.
Metcalf and Eddy, 3rd Edition, McGraw-Hill, 1991.
Operation of Wastewater Treatment Plants: A Field Study
Training Program. US EPA, OWP, California State University,
Sacremento, 2003.
Basic Microbiology for Drinking Water Personnel. Dennis Hill,
American Water Works Association, 2001.
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.
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.
Colilert® Test Procedure, Illustrative Brochure. IDEXX
Laboratories, Westbrook, ME. www.idexx.com
Marion, IN Utilities Unpublished Data, 2006.
Raw Sewage Overflow Long Term Control Plan and Water
Quality Improvement Report. City of Indianapolis, IN, September
2006.
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.
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