E. coli Workgroup 1/10/07 Meeting Single Sample Maximum to Assess Compliance

2 E. coli Workgroup 1/10/07 Topics for this Discussion oColiforms Total Fecal  E. coli oOccurrence oMethods of Detection Membrane Filtration Colilert®

3 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 coli o Citrobacter o Enterobacter o Klebsiella

4 Fecal Coliforms oEscherichia coli (E. coli) is the most representative – why chosen to indicate presence of fecal contamination and potential presence of pathogens oIn human and warm blooded animal intestine oMost E. coli strains do not produce toxins oEnteropathogenic E. coli cause gastroenteritis and is usually contracted via contaminated food oE. coli 0157:H7 is a toxigenic strain – found in cattle feces - potential to be in runoff

5 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

6 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 1 st sample gets a small “group” and is <235/100 mL. Which sample is representative of the effluent?

8 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

9 E. coli Occurrence Source Water Data (12/2/02 CFR – Approval of Analytical Methods for Microbiological Contaminants) Sample IDSample Source E. coli (cfu/100 mL) AMillbrae, CA230, AMillbrae, CA11,000, AJacksonville, FL700, ASchaumberg, IL1,000, AMission, KS4,000, ASalem, OR1,000, AAmes, IA1,000, AMission, KS3,000, ALiberty, MO2,000,000

10 E. Coli Detection Methods (1) oMembrane Filtration oSample is filtered thru a porous filter oFilter placed on a nutrient pad in a Petri dish oIncubated at 35º C for 2 hours to revive injured or stressed cells and then for 22 hours at 44.5 º C oAfter incubation filter is transferred to a filter pad saturated with urea substrate oAfter 15 minutes colonies of a specific color (yellow/ yellow brown) counted - result is cfu/100mL

11 E. Coli Detection – Membrane Filtration

12 E. Coli Detection – Membrane Filtration

13 Marion, IN Fecal Coliform MF Data – Split Samples Date #1-cfu/100 mL #2 - cfu/100 mLAvg.Difference 8/17/ % 8/18/ % 8/22/ % 9/7/ % 9/19/ % 10/4/ % 10/6/ % 10/10/ % 10/13/ % 10/30/ % 10/31/ %

14 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)

15 E. Coli Detection Methods (2) Colilert® oMost 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

16 E. Coli Detection - Colilert®

17 E. Coli Detection - Colilert®

18 E. Coli Detection - Colilert®

19 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

20 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 Samples processed much faster than MF o Trays require more incubator space but single temperature eliminates need for 2 incubators o MF processing steps and limitations are not required and do not influence results

21 E. coli Workgroup 1/10/07 References 1.Wastewater Engineering: Treatment, Disposal, and Reuse. Metcalf and Eddy, 3 rd Edition, McGraw-Hill, Operation of Wastewater Treatment Plants: A Field Study Training Program. US EPA, OWP, California State University, Sacremento, Basic Microbiology for Drinking Water Personnel. Dennis Hill, American Water Works Association, 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, 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 Colilert® Test Procedure, Illustrative Brochure. IDEXX Laboratories, Westbrook, ME. 7.Marion, IN Utilities Unpublished Data, Raw Sewage Overflow Long Term Control Plan and Water Quality Improvement Report. City of Indianapolis, IN, September Method : Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC). US EPA, Office of Water, EPA 821-R , September 2002.