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. HartmanDepartment of Natural Sciences

Longwood UniversityFarmville, VA 23909

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)

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.)

VA state-approved labscurrently utilize

membrane filtration methodsfor fecal coliform analysis

using an m-FC brothrequiring a secondary

Confirmatory step

The USEPA has approved the use of Defined Substrates

for coliformanalysis

using the Colilert® systemdoes not require

Confirmatory step

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 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 according

to assessment method (u1= u2)

Ha: Fecal coliform counts of natural 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.)• 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.)

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/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: 24±2 hrs; 44.5°±0.2°C

• Plates incubated in Whirl-Pak bags

• CFU’s counted @ 30X magnification

Materials 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 duplicates• Test series blanks• Incubation: 24±2 hrs; 44.5°±0.2°C• Quanti-cult QA/QC cultures (EC/KP/PA)

Materials and Methods:

Group Statistics

327 633.4235 1739.606 96.20046327 808.3547 1899.763 105.0571

TestE-coliFecal Coliform

CCOUNTN Mean

Std.Deviation

Std. ErrorMean

Independent Samples Test

1.410 .235 -1.228 652 .220 -174.9312 142.44832 -454.644 104.7816

-1.228 647.007 .220 -174.9312 142.44832 -454.648 104.7856

Equal variancesassumedEqual variancesnot assumed

CCOUNTF Sig.

Levene's Test forEquality of Variances

t df Sig. (2-tailed)Mean

DifferenceStd. ErrorDifference Lower Upper

95% ConfidenceInterval of the

Difference

t-test for Equality of Means

Results: Comparison of pooled data

T-test:

Descriptives:

Results

Mean E-Coli and Fecal Coliform Counts Across all Streams

0

200

400

600

800

1000

1200

E-Coli FC

Test

E-C

oli

and

FC

Co

un

ts (

+/-

SE

)

Tests of Between-Subjects Effects

Dependent Variable: CCOUNT

89925990.6a 7 12846570.08 3.993 .000202921263 1 202921263.3 63.077 .000

3746551.256 1 3746551.256 1.165 .28182104530.1 3 27368176.70 8.507 .000

2818209.699 3 939403.233 .292 .8312.078E+09 646 3217018.9322.508E+09 6542.168E+09 653

SourceCorrected ModelInterceptNTESTNYEARNTEST * NYEARErrorTotalCorrected Total

Type III Sumof Squares df Mean Square F Sig.

R Squared = .041 (Adjusted R Squared = .031)a.

Results: Comparisons by year and by seasonTwo-factor ANOVA – Test and Year

Tests of Between-Subjects Effects

Dependent Variable: Count all streams

5862650.039a 3 1954216.680 .319 .812195194702 1 195194701.6 31.874 .000

3915035.483 1 3915035.483 .639 .425269218.462 1 269218.462 .044 .834

1304391.346 1 1304391.346 .213 .6452.094E+09 342 6124041.1752.291E+09 3462.100E+09 345

SourceCorrected ModelInterceptNTESTNSEASONNTEST * NSEASONErrorTotalCorrected Total

Type III Sumof Squares df Mean Square F Sig.

R Squared = .003 (Adjusted R Squared = -.006)a.

Two-factor ANOVA – Test and Season

Results

Mean E-Coli and Fecal Coliform Counts per Year

0

200

400

600

800

1000

1200

1400

1600

1800

2000 2001 2002 2003

Year

E-C

oli

an

d F

ec

al C

olif

orm

Co

un

ts (

+/-

SE

)

E-Coli

FC

ResultsMean E-Coli and Fecal Coliform

Counts per Winter and Summer Seasons

0

200

400

600

800

1000

1200

1400

1600

Dec/Jan/Feb Jun/Jul/Aug

E-C

oli

and

Fec

al C

olif

orm

Co

un

ts (+

/- S

E)

E-Coli

FC

Independent Samples Test

1.635 .206 -.615 62 .541 -396.6094 645.21527 -1686.38 893.1576

-.615 56.652 .541 -396.6094 645.21527 -1688.80 895.5837

1.646 .204 -1.118 62 .268 -169.8125 151.92710 -473.510 133.8854

-1.118 45.592 .270 -169.8125 151.92710 -475.700 136.0748

.019 .889 -.360 66 .720 -132.3824 367.46614 -866.052 601.2875

-.360 63.795 .720 -132.3824 367.46614 -866.526 601.7610

.100 .753 -.683 64 .497 -72.2727 105.74677 -283.526 138.9807

-.683 63.902 .497 -72.2727 105.74677 -283.532 138.9869

.025 .876 -.307 64 .760 -125.7576 409.59868 -944.025 692.5099

-.307 63.999 .760 -125.7576 409.59868 -944.025 692.5100

.041 .840 -.248 64 .805 -151.7576 613.12643 -1376.62 1073.103

-.248 63.761 .805 -151.7576 613.12643 -1376.71 1073.192

.288 .593 -.556 64 .580 -390.7576 702.36769 -1793.90 1012.383

-.556 63.789 .580 -390.7576 702.36769 -1793.99 1012.473

.026 .873 -.278 62 .782 -118.9375 428.00274 -974.503 736.6278

-.278 61.999 .782 -118.9375 428.00274 -974.503 736.6279

3.622 .062 -1.199 62 .235 -75.9375 63.31834 -202.509 50.63406

-1.199 50.613 .236 -75.9375 63.31834 -203.078 51.20299

.090 .765 -.274 64 .785 -88.2727 322.39632 -732.333 555.7880

-.274 63.176 .785 -88.2727 322.39632 -732.495 555.9498

Equal variancesassumedEqual variancesnot assumedEqual variancesassumedEqual variancesnot assumedEqual variancesassumedEqual variancesnot assumedEqual variancesassumedEqual variancesnot assumedEqual variancesassumedEqual variancesnot assumedEqual variancesassumedEqual variancesnot assumedEqual variancesassumedEqual variancesnot assumedEqual variancesassumedEqual variancesnot assumedEqual variancesassumedEqual variancesnot assumedEqual variancesassumedEqual variancesnot assumed

ANG

APP1

APP2

BUF15

GRE16

SAY5

SAY6

SAY7

SAY8

VAU14

F Sig.

Levene's Test forEquality of Variances

t df Sig. (2-tailed)Mean

DifferenceStd. ErrorDifference Lower Upper

95% ConfidenceInterval of the

Difference

t-test for Equality of Means

Results: t-tests of coliform counts per stream

ResultsMean E-coli and Fecal Coliform Counts per Stream

0

500

1000

1500

2000

2500

3000

Ang17 App1 App2 Buf15 Gre16 Say5 Say6 Say7 Say8 Vau14

Stream

E-c

oli a

nd F

ecal C

olifo

rm

Counts

+/- SE

E-coli

FC

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 pneumoniaeEnterobacter 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.

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

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

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

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?