Transcript A long-term comparison of assessment methodologies for detecting fecal coliform bacteria in natural waters D.W.

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 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 labs
currently utilize
membrane filtration methods
for fecal coliform analysis
using an m-FC broth
requiring a secondary
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 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
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 assessment method (u1= u2)
Sampling sites:
Cumberland
Buckingham
Appomattox
Amelia
Prince Edward
Nottoway
Sampling sites:
(10) Locations at bridges/access points
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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.)
Materials and Methods:
Sample Collections
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:
Membrane Filtration
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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:
Colilert® Defined Substrate
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Performed according to IDEXX
(Westbrtook, ME) directions
IDEXX Colilert® media
– ONPG to detect total coliforms
– MUG to detect E. coli
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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)
Results: Comparison of pooled data
Descriptives:
Group Statistics
Test
E-coli
Fecal Coliform
CCOUNT
N
Mean
633.4235
808.3547
327
327
Std.
Deviation
1739.606
1899.763
Std. Error
Mean
96.20046
105.0571
T-test:
Independent Samples Test
Levene's Test for
Equality of Variances
F
CCOUNT
Equal variances
assumed
Equal variances
not assumed
1.410
Sig.
.235
t-test for Equality of Means
t
df
Sig. (2-tailed)
Mean
Difference
Std. Error
Difference
95% Confidence
Interval of the
Difference
Lower
Upper
-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
Results
E-Coli and FC Counts (+/- SE)
Mean E-Coli and Fecal Coliform Counts Across all Streams
1200
1000
800
600
400
200
0
FC
E-Coli
Test
Results: Comparisons by year and by season
Two-factor ANOVA – Test and Year
Tests of Between-Subjects Effects
Dependent Variable: CCOUNT
Type III Sum
Source
of Squares
df
Mean Square
Corrected Model
89925990.6a
7
12846570.08
Intercept
202921263
1
202921263.3
NTEST
3746551.256
1
3746551.256
NYEAR
82104530.1
3
27368176.70
NTEST * NYEAR
2818209.699
3
939403.233
Error
2.078E+09
646
3217018.932
Total
2.508E+09
654
Corrected Total
2.168E+09
653
a. R Squared = .041 (Adjusted R Squared = .031)
F
3.993
63.077
1.165
8.507
.292
Sig.
.000
.000
.281
.000
.831
Two-factor ANOVA – Test and Season
Tests of Between-Subjects Effects
Dependent Variable: Count all streams
Type III Sum
Source
of Squares
df
Corrected Model
5862650.039a
3
Intercept
195194702
1
NTEST
3915035.483
1
NSEASON
269218.462
1
NTEST * NSEASON
1304391.346
1
Error
2.094E+09
342
Total
2.291E+09
346
Corrected Total
2.100E+09
345
a. R Squared = .003 (Adjusted R Squared = -.006)
Mean Square
1954216.680
195194701.6
3915035.483
269218.462
1304391.346
6124041.175
F
.319
31.874
.639
.044
.213
Sig.
.812
.000
.425
.834
.645
Results
Mean E-Coli and Fecal Coliform Counts per Year
1800
E-Coli and Fecal Coliform
Counts (+/- SE)
1600
E-Coli
FC
1400
1200
1000
800
600
400
200
0
2000
2001
2002
Year
2003
Results
E-Coli and Fecal Coliform
Counts (+/- SE)
1600
Mean E-Coli and Fecal Coliform
Counts per Winter and Summer Seasons
1400
E-Coli
1200
FC
1000
800
600
400
200
0
Dec/Jan/Feb
Jun/Jul/Aug
Results:
t-tests of coliform counts per stream
Independent Samples Test
Levene's Test for
Equality of Variances
F
ANG
APP1
APP2
BUF15
GRE16
SAY5
SAY6
SAY7
SAY8
VAU14
Equal variances
assumed
Equal variances
not assumed
Equal variances
assumed
Equal variances
not assumed
Equal variances
assumed
Equal variances
not assumed
Equal variances
assumed
Equal variances
not assumed
Equal variances
assumed
Equal variances
not assumed
Equal variances
assumed
Equal variances
not assumed
Equal variances
assumed
Equal variances
not assumed
Equal variances
assumed
Equal variances
not assumed
Equal variances
assumed
Equal variances
not assumed
Equal variances
assumed
Equal variances
not assumed
Sig.
1.635
1.646
.019
.100
.025
.041
.288
.026
3.622
.090
.206
.204
.889
.753
.876
.840
.593
.873
.062
.765
t-test for Equality of Means
t
df
Sig. (2-tailed)
Mean
Difference
Std. Error
Difference
95% Confidence
Interval of the
Difference
Lower
Upper
-.615
62
.541
-396.6094
645.21527
-1686.38
893.1576
-.615
56.652
.541
-396.6094
645.21527
-1688.80
895.5837
-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
-.360
66
.720
-132.3824
367.46614
-866.052
601.2875
-.360
63.795
.720
-132.3824
367.46614
-866.526
601.7610
-.683
64
.497
-72.2727
105.74677
-283.526
138.9807
-.683
63.902
.497
-72.2727
105.74677
-283.532
138.9869
-.307
64
.760
-125.7576
409.59868
-944.025
692.5099
-.307
63.999
.760
-125.7576
409.59868
-944.025
692.5100
-.248
64
.805
-151.7576
613.12643
-1376.62
1073.103
-.248
63.761
.805
-151.7576
613.12643
-1376.71
1073.192
-.556
64
.580
-390.7576
702.36769
-1793.90
1012.383
-.556
63.789
.580
-390.7576
702.36769
-1793.99
1012.473
-.278
62
.782
-118.9375
428.00274
-974.503
736.6278
-.278
61.999
.782
-118.9375
428.00274
-974.503
736.6279
-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
-.274
64
.785
-88.2727
322.39632
-732.333
555.7880
-.274
63.176
.785
-88.2727
322.39632
-732.495
555.9498
Results
Mean E-coli and Fecal Coliform Counts per Stream
E-coli and Fecal Coliform
Counts +/- SE
3000
2500
E-coli
FC
2000
1500
1000
500
0
Ang17
App1
App2
Buf15
Gre16
Say5
Stream
Say6
Say7
Say8
Vau14
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.
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
Conclusions:
Versatility
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:
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?