1. A long-term comparison of assessment methodologies for detecting fecal coliform bacteria in natural waters D.W. Buckalew, M.M. Hafez, K.E. Jones, G.A. Grimsley, P. Dirks, and L.J. Hartman Department of Natural Sciences Longwood University Farmville, VA 23909

2. Discovery of causal agents…a history 1850’s – correlations between enteric disease and contaminated water (Snow and Budd) 1880’s – discovery of microbial disease agents (Koch) 1880’s – use of “Bacillus coli” as indicator for fecal contamination (Escherich)

3. assessment methodologies…a background 1904 – assays for E. coli using glucose broths (Eijkman) 1920’s – multiple tube fermentation with lactose broths (Leiter) 1951 – membrane filtration developed (Goetz & Tsuneishi) 1988 – defined substrates developed (Edberg et al.)

4. VA state-approved labs currently utilize membrane filtration methods for fecal coliform analysis using an m-FC broth requiring a secondary Confirmatory step

5. The USEPA has approved the use of Defined Substrates for coliform analysis using the Colilert ® system does not require Confirmatory step

6. Objectives To compare a Membrane Filtration (MF) method with a Defined Substrate (DS) method for assaying coliforms in a long-term study Comparisons: numerical counts overall numerical counts by stream numerical counts by season (cold vs warm) including: accuracy of identifications time costs

7. Experimental hypothesis: Ho: Fecal coliform counts of natural water samples do not differ according to assessment method (u 1 = u 2 ) Ha: Fecal coliform counts of natural water samples differ according to assess- ment method (u 1 = u 2 )

8. Prince Edward Buckingham Cumberland Amelia Nottoway Appomattox Sampling sites:

9. Sampling sites: (10) Locations at bridges/access points Ang17 = Angola Creek @ Rt 673 (Cumberland Co.) App1 = Appomattox River @ Rt 609(Buckingham/PE Co.) App2 = Appomattox River @ Rt 45 (PE/Cumberland Co.) Buf15 = Buffalo Creek @ Rt 648 (PE Co.) Gre16 = Green Creek @ Rt 600 (Cumberland Co.) Say5 = Little Sayler’s Creek @ Rt 620 (PE Co.) Say6 = Little Sayler’s Creek @ Rt 600 (PE Co.) Say7 = Big Sayler’s Creek @ Rt 617 (Amelia Co.) Say8 = Big Sayler’s Creek @ Rt 620 (Nottoway Co.) Vau 14 = Vaughan’s Creek @ Rt 609 (PE Co.)

10. On the 3 rd Tuesday of each month since May 2000… samples obtained via sterile Whirl-Pak bags Caught mid-channel At/near center of stream 50 ft upstream of bridge crossings Materials and Methods: Sample Collections

11. Performed according to sections 9222B/D of Standard Methods (19 th Ed.,1995) 0.45 um Millipore ® membrane filter Sterile ampules – mFC broth Samples added @ 10% dilution (10 ml sample: 90 ml sbw) Field and Sample duplicates Test series blanks Incubation: 24±2 hrs; 44.5°±0.2°C Plates incubated in Whirl-Pak bags CFU’s counted @ 30X magnification Materials and Methods: Membrane Filtration

12. Colilert ® Defined Substrate Performed according to IDEXX (Westbrtook, ME) directions IDEXX Colilert® media –ONPG to detect total coliforms –MUG to detect E. coli Quanti-Tray 2000 envelopes Samples added at 10% dilution (10 ml sample: 90 ml sbw) Field and Sample duplicates Test series blanks Incubation: 24±2 hrs; 44.5°±0.2°C Quanti-cult QA/QC cultures (EC/KP/PA) Materials and Methods:

13. Results: Comparison of pooled data T-test: Descriptives:

14. Results

15. Results: Comparisons by year and by season Two-factor ANOVA – Test and Year Two-factor ANOVA – Test and Season

16. Results

18. Results: t-tests of coliform counts per stream

19. Results

20. Confirmatory* results For the Colilert test, all (100%) of MUG + wells contained culturable E. coli. For the Membrane Filtration test using m-FC broth, most (98.7%) of blue pigmented colonies tested as E. coli. Other colonies tested as: Klebsiella pneumoniae Enterobacter spp. Citrobacter spp. * Confirmatory tests included 1) indole test, and 2) reaction on MacConkey’s agar. Pure culture isolates further identified using BDL Crystal multi-test system.

21. Discussion: Colilert ® benefits Specificity Simultaneous enumeration of both Total Coliforms and E. coli Time savings –less time for Presumptive setup –Confirmatory test not required EC is a reliable indicator for Fecal Coliforms Reduced chance for accidental contamination Extended shelf life of medium Overall ease of interpretation

22. Discussion: Colilert ® liabilities Cost comparison –~$5.60+/sample vs ~$1.75+/sample Possible false positives with turbid samples Reduced reliability for assays of brackish or saline waters Restricted enumeration of Fecal Coliforms

23. Colilert ® provides similar fecal coliform counts in freshwater samples as compared with membrane filtration methods over a range of environmental conditions including: quality of stream water variations in temperature variations in streamflow (not shown here) Conclusions: Versatility

24. Conclusions: Ease of Use No additional tests needed Reduced labor costs Immediate results could eliminate delay in delivery of samples to commercial labs No special equipment to set up and aseptically maintain

25. Questions?