1. Rapid Detection of E. coli in 8 hr from Beach Water Using KwikCount EC Medium Fu-Chih Hsu and Rebecca Wong

2. Currently available culture methods to detect E. coli in beach water take 18- 24 hrs Same day results (8 hrs) will be helpful for beach monitoring Introduction

3. Validation of new method, KwikCount EC medium to enumerate E. coli in beach water Comparison of a reference method, USEPA Method 1603 (modified mTEC medium) Follow USEPA ATP protocol Purpose of The Study

4. EPA Microbiological Alternate Test Procedure Protocol Drinking, Ambient, Waste water, and Sewage Sludge September 2010 ATP Protocol

5. Contains a fluorogenic/chromogenic enzyme substrate mixture Contains a special nutrients formula to promote the growth of E. coli Membrane filtration Incubation at 41°C for 8 hrs KwikCount EC Medium

6. Blue fluorescent colonies can be observed under 366 nm UV light between 6-12 hrs Blue color colonies under ambient light after 12 hrs KwikCount EC Kit contains broth, petri dishes, filter membranes and pads KwikCount EC Medium

7. Blue Fluorescent Colonies (8 hrs)

8. Blue Colonies (20 hrs)

9. Modified mTEC agar Two steps of incubation 35°C ± 0.5°C for 2 ± 0.5 hours 44.5°C ± 0.2°C for 22 ± 2 hours in water bath Method 1603

10. Red or Magenta Colonies by Modified mTEC

11. Side-by-side methods comparison One source of non-chlorinated secondary sewage effluent is used for spiking 10 sources of fresh beach water from different states in USA Study Plan

12. 1.Michigan: Sliver beach, St. Joseph (Lake Michigan) 2.Indiana: Indiana Dunes National Lakeshore (Lake Michigan) 3.Indiana: Potato Creek State Park (inland lake) 4.Oregon: Blue Lake Regional Park (inland lake) 5.Colorado: Chatfield State Park (Lake) 6.Texas: White Rock Lake (Reservoir) 7.Alabama: Chewacla State Park (Lake Chewacla) 8.Washington: Matthews Beach (Lake Washington) 9.Kansas: Longview Lake (inland lake) 10.Florida: Moss Park (inland lake) 10 Sites for Fresh beach Water

13. Determination of E. coli recovery by both methods Verify 20 typical and atypical colonies for KwikCount EC, and 20 typical colonies for modified mTEC for each sources of fresh beach water Colony verification: oxidase, indole, Simmons citrate test, and EC broth Study Plan

14. Comparison of recovery, precision, and false positive rate Determination of normality by the Shapiro-Wilk test ANOVA (Analysis of Variance) Non-parametric test: Kruskal-Wallis Statistical Analysis

15. Results

16. Source Water Characteristics SourcepHTurbidity (NTU)HPC (CFU/mL) Matthew's Beach6.050.58110 Silver Beach7.156.751000 Potato Creek7.103.801090 Lake Mary (Moss Park)4.893.0914400 Longview Lake5.342.2713100 White Rock Lake8.423.30970 Colorado State Park (Chatfield)7.601.61261 Chewacla Swimming Beach6.954.648800 Blue Lake6.931.641000 Indiana State Park (Sand Dune)7.696.55471000

17. Mean Recovery, Standard Deviation, and Normality KwikCount (%) Mod. mTEC (%) Significant SourceMean STDEV Mean STDEV Normality difference Matthew’s Beach 71.5 10.6 76.1 13.1 Y/YNo Silver Beach 50.9 9.5 46.4 10.3 Y/YNo Potato Creek 56.8 10.4 44.4 8.2 Y/YYes Lake Mary 86.8 13.0 88.4 8.1 Y/YNo Longview Lake 76.1 11.7 81.5 10.6 Y/YNo White Rock Lake 60.5 12.7 49.9 15.2 Y/YYes Chatfield 97.1 17.8 109.2 15.3 Y/YYes Chewacla 88.0 10.7 84.8 11.8 N/YNo Blue Lake 90.9 15.8 84.0 14.5 Y/YNo Sand Dune (IN)106.8 17.5 99.3 15.0 Y/YNo

18. Overall recovery KwikCount (%) Mod. mTEC (%) Significant Mean STDEV Mean STDEV Normality difference 78.513.376.412.5N/NNo ___________________________________________________

19. Precision from 10 fresh beach water using KwikCount EC and modified mTEC Source KC-EC KC-EC M mTEC M mTEC (stdev) (S 2 ) __________________________________________________________ Matthew’s Beach 10.6 112.4 13.1 171.6 Silver beach 9.5 90.3 10.3 106.1 Potato Creek 10.4 108.2 8.2 67.2 Lake Mary (Moss) 13.0 169.0 8.1 65.6 Longview Lake 11.7 136.9 10.6 112.4 White Rock Lake 12.7 161.3 15.2 231.0 Chatfield 17.8 316.8 15.3 234.1 Chewacla 10.7 114.511.8 139.2 Blue Lake 15.8 249.7 14.5 210.3 Sand Dune (Indiana) 17.5 306.3 15.0 225.0 Mean 176.5 156.3

20. Comparison of Precision KwikCount Mod. mTEC Mean S 2 176.5156.3 ____________________________________________ F=176.5/156.3 =1.13 Since F does not exceed 1.27, the KwikCount EC precision is equal to the reference method, and the KwikCount EC precision is acceptable.

21. Sensitivity, specificity, false positive and negative from KwikCount EC Independent Standard KwikCount EC +- Total + TPFPTP+FP - FNTNFN+ TN Total TP+FNFP+ TNTP+FN+FP+ TN Sensitivity (%) = TP/ (TP + FN) x 100 F= False Specificity (%) = TN/ (TN + FP) x 100 T=True False positive rate (%) = FP / (TN + FP) x 100 N =Negative False negative rate (%) = FN/ (TP + FN) x 100 P=Positive

22. Sensitivity, specificity, false positive and negative KwikCount EC Independent Standard KwikCount EC +- Total + 200 - 21820 Total 221840 Sensitivity = 20/ (22) x 100= 90.9 % F= False Specificity = 18/ (18) x 100= 100 % T=True False positive rate = 0 / (18) x 100= 0% N =Negative False negative rate = 2 / (22) x 100= 9.1% P=Positive

23. Sensitivity, specificity, false positive and negative from 10 fresh beach water using KwikCount EC Sensitivity Specificity False Positive False Negative Matthew’s Beach 90.9 100.0 0.0 9.1 Silver beach100.0 83.3 16.70 Potato Creek100.0 90.9 9.10 Lake Mary (Moss)100.0 80.0 20.00 Longview Lake 94.1 82.6 17.4 5.9 White Rock Lake100.0 76.9 23.10 Chatfield 90.9 100.0 0 9.1 Chewacla100.0 87.0 13.0 0 Blue Lake 95.2 100.0 0 4.8 Sand Dune (Indiana)100.0 95.2 4.8 0 ________________________________________________________________________ Mean97.1 89.9 10.4 1.4

24. False positive isolates from 200 typical colonies isolated from 10 sources of fresh beach water by KwikCount EC and modified mTEC. SourceKC-ECModified mTEC E. coli non-E. coliE. colinon-E. coli Matthew’s Beach 20 0200 Silver beach 16 4182 Potato Creek 18 2173 Lake Mary (Moss) 15 5173 Longview Lake 16 4164 White Rock Lake 14 6173 Chatfield 20 0200 Chewacla 17 3200 Blue Lake 20 0200 Sand Dune (Indiana) 19 1200 Total 175 25 185 15

25. Conclusions It is feasible to detect E. coli within 8 hr using KwikCount EC medium KwikCount has 2.1% higher recovery (78.5% vs. 76.4%), but no significant difference by statistical analysis

26. Conclusions -Precision by modified mTEC is slightly better than by KwikCount (12.5% vs. 13.3%), but no significant difference is found by statistical analysis. -The false positive rate by KwikCount is 10.4% -Difficult to pick up colonies under UV light -Liquid medium vs agar medium

27. Conclusions The overall false negative rate from 10 sources of fresh beach water is low (1.4%) by KwikCount EC

28. Conclusions There is no evidence that there are statistically significant differences in recovery and precision, between the proposed method (KwikCount EC) and EPA Method 1603. It can be concluded that performance by these two methods is equal.