E.coli, Fecal Coliform and Enterococci Analysis

1. E.coli, Fecal Coliform and Enterococci Analysis Brett Goodrich Maine DEP MAINE DEPARTMENT OF ENVIRONMENTAL PROTECTION Protecting Maine’s Air, Land and Water

2. Gram stain- Mini Lesson in Microbiology

3. How to gram stain

4. Gram stain continued • Gram positive bacteria retain primary stain and remain purple • Primarily found on human skin (Strep, Staph) • Gram negative bacteria decolorize and retain secondary red dye • Found in intestines of warm blooded animals (E.coli, Salmonella, Shigella)

5. “Identifying” Bacteria • Impractical to identify all organisms in a sample • Instead methods use combinations of temperatures/ inhibitory compounds/ color changing enzymes to classify organisms

6. Visual identification E.coli gram stain Salmonella gram stain

7. The way things used to be

8. Why test effluent for bacteria? • To make sure disinfection process is adequate to minimize risk to environment and human health • Wastewater could contain MANY different potential disease causing organisms • But treatment plants only test for E.coli, Fecal coliforms or Enterococci

9. Indicator Organisms • Testing does not directly detect pathogenic organisms • Impossible to test for every potential pathogen (bacteria, viruses, protozoans, fungi, parasites) • Hard to detect- Present in low numbers compared to non-pathogens • Can be dangerous to handle • Indicator organisms are used to assess the likelihood of disease-causing bacteria being present

10. Characteristics of Good Indicator Organisms • Present in equal to or greater numbers than pathogens of concern • Same or greater survival in environment than pathogens of concern • Can be readily cultured on standard culture media • Should not pose significant health risk to laboratory workers following standard biosafety practices

11. Wastewater indicator organisms • E.coli for freshwater dischargers • Fecal coliform for marine dischargers • Enterococci will be added to marine discharge permits at some point

12. Bacteria classification

13. Why are these used? • E.coli is predominant organism found in animal intestines • Fecal coliform includes E.coli and other thermotolerant coliforms • Enterococci are also found in animal intestines and survive longer in salt water than E.coli

14. Methods for Routine Testing • Culture plates and reagents developed that are able to select for almost any organism • Not all methods approved for every type of sample • Matrix interferences • Background bacteria

15. 2 Basic Ways to Count Microorganisms in water • Membrane Filtration • Enzyme Substrate

16. Membrane Filtration Results in CFU/ 100 ml (Colony forming units)

17. Enzyme Substrate (Colilert) Results in MPN/ 100 ml (Most probable number) Traditional 15 Tube Serial Dilution Scheme

18. CFU vs MPN • Colony Forming Units and Most Probable Number Units are equivalent • If your permit states CFU limits, the MPN limit will be the same

19. Document on bench sheet or in logbook Dates/ times Employee that performed the task Temperature of incubator and/or water bath Method reference Grab samples Dechlorination and vigorous shaking immediately before testing Adequate headspace in container(SM9060A.3 says at least 2.5 cm, not practical to measure every sample) All micro tests require… MAINE DEPARTMENT OF ENVIRONMENTAL PROTECTION www.maine.gov/dep

20. Membrane Filtration (MF) Overview • Sample is filtered and placed on agar plate • Filter/plate is incubated for specified time and temperature • Agar type, incubation time and temperature are specific to organism of concern

21. MF- E.coli Incubation conditions • E.coli by EPA 1603 Media: Modified mTEC agar Pre-Incubation: 35oC +/- 0.5oC for 2 +/- 0.5 hour Final Incubation: 44.5oC +/- 0.2oC for 22 +/- 2 hours Colonies: Red or magenta MAINE DEPARTMENT OF ENVIRONMENTAL PROTECTION www.maine.gov/dep

22. MF- Fecal Coliform Incubation conditions • Fecal Coliform by SM9222D Media:mFC Medium Temperature: 44.5oC +/- 0.5oC Incubation Time: 24 +/- 2 hours Colonies: Fecal coliforms are shades of blue; gray, cream and colorless colonies are not counted

23. MF- Key QC Steps • QC testing of media prior to use • Certificate from manufacturer or internal testing • Pre/ post filtration rinse water sterility checks • Replicate sample testing to measure precision • Yearly DMR-QA to measure accuracy • Positive and negative controls with each run • Many more according to EPA/ Standard Methods…

24. Additional Requirements from EPA 1603 QA program requirements for every lab performing EPA 1603 Initial precision and recovery Ongoing precision and recovery Matrix spike

25. MPN-IDEXX Tests Overview • Reagent added to sample and sealed in tray • Tray is incubated for specified time and temperature • Color and/or fluorescence used to count positives • Reagent, time/ temperature are specific to organism of concern • Wastewater labs should always use Quanitray 2000 trays (detects 1-2,419 MPN/ 100 mL without dilution

26. Colilert- E.coli Incubation Conditions This tray has at least 2 colorless wells that are showing fluorescence, these wells are not positive for E.coli • E.coli by SM9223B Temperature: 35oC +/- 0.5oC Incubation Time: Colilert: 24-28 hours Colilert 18: 18-22 hours Interpretation: Yellow wells equal to or darker than comparator ANDfluorescent under 365 nm UV light are E.coli positive

27. Colilert 18- Fecal Coliform Incubation Conditions • Fecal Coliform by SM9223B Temperature: 44.5oC +/- 0.2oC Incubation Time: 18-22 hours (only Colilert 18 approved for fecal coliform) Interpretation: Yellow wells equal to or darker than comparator (fluorescence does not matter)

28. Enterolert- Enterococci Incubation Conditions • Enterococci by IDEXX Enterolert Temperature: 41.5oC +/- 0.5oC Incubation Time: 24-28 hours Interpretation: Blue fluorescent wells under 365 nm UV light are Enterococci positive (no visible color change)

29. Enterococci and Fecal Coliform Key Differences • Reagent used for testing • Fecal coliform: Colilert 18 • Enterococci: Enterolert (not Enterolert E, Enterolert-DW or Enterolert 250) • Counting positive wells • Fecal coliform: Yellow color • Enterococci: Fluorescence under 365 nm UV light • Incubation temperature • Fecal Coliform: 44.5oC water bath • Enterococci: 41.5oC water bath or incubator

30. Determining Final Result for IDEXX Methods • Use correct MPN Table from IDEXX • Or download MPN generator from IDEXX website • Should have positive determination criteria on bench sheet

31. IDEXX Colilert Key QC Procedures • Check each lot of reagent with +/- controls • Check sealer monthly for proper sealing • IDEXX color comparator within expiration date • Yearly DMR-QA to measure accuracy Influent for positive? Distilled water for negative Add 1-2 drops food coloring to 100 mL distilled water and seal in tray, check for leaking between wells Used to determine results with weak color/ fluorescence reactions Requirement for all methods

32. Summary… • Membrane filtration techniques have very stringent QA/QC requirements • Colilert QC is cost effective and practical to perform • Addition of Enterococci testing may require facilities to purchase additional incubator and 365 nm fluorescent light

33. Any Questions? Contact: Brett.A.Goodrich@maine.gov 207-287-9034