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,Discovery of causal agentsa history,1850s
2、 correlations between enteric disease and contaminated water (Snow and Budd) 1880s discovery of microbial disease agents (Koch) 1880s use of “Bacillus coli” as indicator for fecal contamination (Escherich),assessment methodologiesa background,1904 assays for E. coli using glucose broths (Eijkman)192
3、0s multiple tube fermentation with lactose broths (Leiter)1951 membrane filtration developed (Goetz & Tsuneishi)1988 defined substrates developed (Edberg et al.),VA state-approved labs currently utilize membrane filtration methods for fecal coliform analysis using an m-FC broth requiring a secondary
4、 Confirmatory step,The USEPA has approved the use of Defined Substrates for coliform analysis using the Colilert system does not require Confirmatory step,Objectives,To compare a Membrane Filtration (MF) method witha Defined Substrate (DS) method for assaying coliformsin a long-term studyComparisons
5、:numerical counts overallnumerical counts by streamnumerical counts by season (cold vs warm)including:accuracy of identificationstime costs,Experimental hypothesis:,Ho: Fecal coliform counts of natural water samples do not differ accordingto assessment method (u1= u2)Ha: Fecal coliform counts of nat
6、ural water samples differ according to assess- ment method (u1= u2),Prince Edward,Buckingham,Cumberland,Amelia,Nottoway,Appomattox,Sampling sites:,Sampling sites: (10) Locations at bridges/access points,Ang17 = Angola Creek Rt 673 (Cumberland Co.) App1 = Appomattox River Rt 609(Buckingham/PE Co.) Ap
7、p2 = Appomattox River Rt 45 (PE/Cumberland Co.) Buf15 = Buffalo Creek Rt 648 (PE Co.) Gre16 = Green Creek Rt 600 (Cumberland Co.) Say5 = Little Saylers Creek Rt 620 (PE Co.) Say6 = Little Saylers Creek Rt 600 (PE Co.) Say7 = Big Saylers Creek Rt 617 (Amelia Co.) Say8 = Big Saylers Creek Rt 620 (Nott
8、oway Co.) Vau 14 = Vaughans Creek Rt 609 (PE Co.),On the 3rd 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,Performed according to sections 9222B/
9、D of Standard Methods (19th 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: 242 hrs; 44.50.2C Plates incubated in Whirl-Pak bags CFUs counted 30X magnification,Materi
10、als and Methods:Membrane Filtration,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 duplic
11、ates Test series blanks Incubation: 242 hrs; 44.50.2C Quanti-cult QA/QC cultures(EC/KP/PA),Materials and Methods:,Results: Comparison of pooled data,T-test:,Descriptives:,Results,Results: Comparisons by year and by season,Two-factor ANOVA Test and Year,Two-factor ANOVA Test and Season,Results,Result
12、s,Results: t-tests of coliform counts per stream,Results,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:Klebsiel
13、la pneumoniaeEnterobacter spp.Citrobacter spp. *Confirmatory tests included 1) indole test, and 2) reaction on MacConkeys agar. Pure culture isolates further identified using BDL Crystal multi-test system.,Discussion: Colilert benefits,Specificity Simultaneous enumeration of both Total Coliforms and
14、 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,Discussion: Colilert liabilities,Cost comparison $5.60+/sample
15、vs $1.75+/sample Possible false positives with turbid samples Reduced reliability for assays of brackish or saline waters Restricted enumeration of Fecal Coliforms,Colilert provides similar fecal coliform counts in freshwater samples as compared with membrane filtration methods over a range of envir
16、onmental conditions including:quality of stream water variations in temperaturevariations in streamflow (not shown here),Conclusions:Versatility,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,Questions?,
