Testing Waste Water For Fecal Coliforms And/or E.coli .

Testing Waste Water for Fecal Coliformsand/or E.coli using Colilert and Colilert -18& Quanti-Tray Gil DichterWorld Wide Technical Support Manager, Waterwww.idexx.com/water1FOR ALL OF YOU WHO ARE HAVING A BAD DAY.21

OBJECTIVES¾Define total coliforms, fecal and E.coli¾Colilert & Colilert-18 for WW– Theory– How to test– Reading results– Understanding MPN theory and relationship to MF– QC/QA¾Q&A3Bacteria Hierarchy¾ Family¾ Genus (type ofbacteria)¾ Species¾ Enterobacteriaceae¾ Escherichia,Klebsiella, Citrobacter¾ E.coli, E. hermanii,¾ K. pneumoniae,¾ C. freundii42

Coliform Genera¾Escherichia- human and animal feces¾Enterobacter- environment, feces¾Klebsiella- environment, feces¾Citrobacter- environment¾Serratia- environment5Thermotolerant Coliforms [aka FecalColiforms]¾ It is a subset of total coliforms¾Defined as coliform bacteria that can grow at44.5 C¾Consists of the following coliforms:– E. coli– K. pnuemoniae–Enterobacter spp.63

Escherichia coli¾ Named after the German Scientist who found this bacteriaDr. Escherich¾ A genus of Gram negative bacteria of the familyEnterobacteriaceae¾ A type of thermotolerant coliform bacteria commonly found inthe intestines of warm blooded animals including humans¾ Does not occur naturally in soil and vegetation¾ May occur in soil and water as a result of fecal contamination7E. coli(non-pathogenic)¾ Major inhabitant of gastrointestinal tractin warm-blooded animals, birds andhumans¾ Shed in feces at high levels¾ Thus, indicator of true fecalcontamination¾ Most common E. coli detected in water84

E. coli9Citrobacter freundii105

Coliform Bacteria GroupTotal Coliforms (35 C)FecalColiforms thermotolerantKlebsiella(44.5 C)Total ions of Coliforms & E.coli¾ Standard Methods for the Examination of Waterand Wastewater, 21st Edition– 9223 Enzymatic: The total coliform group is defined as all bacteriapossessing the enzyme β-D galactosidase cleaving thechromogenic substrate resulting in the release of the chromogen.– E.Coli is defined as giving a total coliform response andpossessing the enzyme β-D glucuronidase, cleaving thefluorogenic substrate resulting in the release of the fluorogen.126

Why Test for E.coliAnimal# 97-3Pig1583.56.89.7Average94.5%Source:E.coli:Fecal Coliform A.P. Dufour. Special Technical Publication 65, ASRM. Pp48-58, 197713Standard Bacterial Growth Curve¾(A) Lag Phase¾(B) Log or ExponentialPhase¾(C) Stationary Phase¾(D) Decline (Death) Phase147

Commonly Used Indicator Bacteria forWater Testing¾ Most commonly used:––––Total coliformFecal coliformE. coliEnterococci¾ Total coliform used for 100 years¾ Fecal coliform used for 80 years¾ E. coli 20 years15Indicator vs Specific Pathogen Testing¾ In a perfect world we would test for all thepathogens in real time¾ Technology not available¾ Too expensive¾ Too time consuming to safeguard againstunknown or emerging pathogensUsed with the permission of the WI State Laboratory of Hygiene, University of WI Board of Regents. No endorsement is implied.168

Requirements for an IndicatorOrganism¾Present when pathogens are present¾Absent in uncontaminated waters¾Present in higher numbers than pathogens incontaminated water¾Better survival in water than pathogens¾Easy and Safe to analyze¾Rapid results¾Inexpensive¾Accurate17Guidelines for Analysis of Pollutants; AnalyticalMethods for Biological Waste Water¾ Federal Register Notice- August 16th, 2005– Comments on proposal ended on October 17th,2005– Promulgated -March 26th,2007– Effective Date-April 25th, 2007¾ Testing for E.coli and Enterococci added– Approved methods include Colilert, Colilert-18and Enterolert and several MF methods– E.coli for fresh surface waters and enterococci formarine waters189

Defined SubstrateTechnology- Colilert &Colilert-18192010

ONPG Positive ReactionColilert & Colilert-1821MUG Positive ReactionColilert & Colilert-182211

23Quanti-Tray DemonstrationAdd Colilert to sampleand shake to dissolvePour mixture into aQuanti-Tray2412

Quanti-Tray Demonstrationcont.Seal and then incubate at35 C for 24 hoursCount positive wells andrefer to MPN table25E.coli- Blue Fluorescence- Quanti-Trayunder a 365nm UV Light2613

Estimation of Bacterial Densities by MPN¾Dates back to 1915 when the concept was introduced byM.H. McCrady (J of Infectious Disease Vol 17, 1915*)¾Prior to this novel concept; no means of direct counting– Only had presence-absence of fermentation tubes¾The method is a means for estimating without any directcount, the density of organisms in a liquid.¾Multiple samples of the liquid are taken and incubated insuitable media– Record presence or absence of growth in each sample tube– Ingenious application of probability theory– Estimate the number of organisms from the number of negative tubes*The Numerical Interpretation of Fermentation Tube Results27MPN Theory – a short math lessonBasic assumptions:– The organisms are distributed randomly throughout– the liquid¾ Sample is well shaken (important and often neglected)– A sample will exhibit growth (in the culture media) whenever oneor more of the target organisms is present¾ Media should be selective and sensitive– Requires that at least 1 tube shows no growth (sterile)If n samples, each of volume v mL are taken from a liquid, and s of these are sterile,then an estimate of the organism density d per mL in the original sample is:d -2.303/v log(s/n) [Poisson distribution]d can be shown statistically to have the highest probability of estimatingthe actual organism density – the “Most Probable Number” - MPN2814

Single dilution – Maximum counting rangeNumber of Tubes MaximumMPN/100mLNumber of Tubes 25807532430102100461d -2.303/v log(s/n)n 50, v 2 mL, s 1d -2.303/2 log (1/50) 1.956 MPN/mL 195.6 MPN/100 mL29Uncertainty of MPN¾Skewed distribution & Uncertainty of Results– For microbiological tests, because of the complications of the skeweddistribution, the estimate of uncertainty from a well-designed precisionexperiment will be regarded as a reasonable estimate of the uncertaintyof results¾When MPN tables are used to obtain results, the 95% CL inthe table will be regarded as a reasonable estimate of theuncertainty of the results¾ Labs are encourage to identify and question unusualcombinations of positive tubes in the tables.–15 tube MPN combination of tubes»1-2-3»3-5-43015

Standard Methods MPN Table 15 Tube31Overcoming the tube numbers problem¾ To increase counting range need more tubes¾ For 100mL sample could use 50 tubes x 2mL¾ Would give counting range of 200MPN/100mL– Big headache for lab – lots of tubes – lots of pipettes -lots ofwashing! Not really practical.– Microtitre plate (96 wells) – MPN range 438/100mL3216

Quanti-Tray ¾ Use multiple well concept but automate thesealing51 well Quanti-TrayTotal sample volume 100mLd -2.303/v log(s/n)n 51, v 1.96 mL, s 1d -2.303/1.96 log (1/51) 2.006 MPN/mL 200.6 MPN/100 mL33Multiple Dilutions¾Not practical to extend the counting range by increasing the number ofwells beyond a certain point.¾Sample serially diluted and each dilution is inoculated into a similarnumber of tubes. Commonly used configurations:¾–3 tubes x 3 decimal dilutions – food industry–5 tubes x 3 decimal dilutions – food, water wastewater (Max MPN 1600/100mL)With multiple dilutions the statistical calculation of the MPN valuebecomes more complex. Thomas provided an approximation that couldbe used for any combination of tubesMPN/100mL # of positive tubes x 100 (# of mL in negative tubes) x (# of mL in all tubes)3417

Quanti-Tray 2000 ¾ Innovative approach to provide a multiple dilutionsystem without adding dilution water¾ Small wells are 1/10th the volume of the large wells– Effectively a 49 x 48 tube serial dilutionUsing the Thomas formula:(MPN is an approximate, deviates fromthe MPN table and is not significant)Number of ve tubes 96Volume of –ve tubes 0.186mLVolume in all tubes 100mLMPNmax 96 x100 (0.186) x (100) 2232/100mL vs 241935Confidence Limits¾Distribution of MPN estimates is approximately log normal (distribution).¾Confidence limits can be calculated (95% limits /- 1.96σ)¾Precision is low when the number of tubes is small.¾For 10 x 10mL tubes with 9 positive: MPN 28/100mL¾the 95% confidence limits are: 11.5 – 68.1 MPN/100mL¾ Confidence limits for 51 well Quanti-tray¾When increased to 51 x 1.96mL with 22 positive: MPN 28.8/100mL¾The 95% confidence limits are: 19.0 – 44.3 MPN/100mL¾Interesting fact: If had 100 x 1mL with 25 positive: MPN 29/100mL¾The 95% confidence limits are: 19.6 – 43.0 MPN/100mL¾Not much improvement – 50 tubes is about optimum3618

3795% Confidence Limit MPN/100mLCFU/100mLMPN/100mLValue CLValue CLValue CLValue CL5.3 2.3-12.35.2 1.8-10.84.5 0.79-155 1.6-7.217.8 10.8-29.417.9 10.7-28.217 6.0-4017 9.9-27.220.7 13-33.320.6 12.7-31.820 6.8-4020 12.2-30.850.4 35.4-72.550.4 35.0-69.149 15-15078.2 56.4-111.278.0 55.6-103.879 22-2203819

QA/QC¾ On line Quality Control Certificate for each product by lotnumber can be obtained from the web atwww.idexx.com/water¾ Read results at 24 hours. Window to 28 hours for ColilertFor Colilert-18 it is 18 hours up to 22 hours¾ Be consistent with time to read results and reading of Y/Fwells¾ Use Comparator as indicated as indicated in the packageinsert¾ Proficiency Testing394020

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43IDEXX Support1-800-321-0207- #1 Customer Support- #2 Technical Service-#3 Select extension-www.idexx.com/water4422

That’s all folks!Thank YouQuestions4523

Quanti-Tray Demonstration Add Colilert to sample and shake to dissolve Pour mixture into a Quanti-Tray. 13 25 Quanti-Tray Demonstration cont. Seal and then incubate at 35 C for 24 hours Count positive wells and refer to MPN table 26 E.coli- Blue Fluorescence- Quanti-Tray under a 365nm UV Light. 14 27File Size: 1MBPage Count: 23Explore furtherFecal coliform and E. coli Analysis in wastewater and .ohiowea.orgAddressing Total Coliform Positive or E.coli Positive .www.epa.govMethod 1604: Total Coliforms and Escherichia coli in Water .www.epa.gov5.11 Fecal Bacteria Monitoring & Assessment US EPAarchive.epa.govMost probable number (MPN) method for counting coliform .www.onlinebiologynotes.comRecommended to you based on what's popular Feedback