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1. Introduction
The Enterolert*-DW test, based on Defined Substrate Technology* (DST*), is designed to detect enterococci in drinking water samples. As enterococci grow in the liquefied medium, the organisms’ enzymes metabolize a substrate specific for β -glucosidase, changing the medium’s color from blue to green and confirming the presence of enterococci in water samples.
The Enterolert-DW and Quanti-Tray* system (Enterolert-DW/Quanti-Tray) is used for the detection and enumeration of enterococci in water samples. A blister pack of Enterolert-DW reagent is added to a sterile bottle containing the water sample. After the powder reagent is added, the vessel is shaken to dissolve the powder. The dissolved sample/medium mixture is added to a Quanti-Tray or Quanti-Tray 2000, and the tray is sealed with the Quanti-Tray sealer. The tray is incubated at
41 ± 0.5ºC for 24 hours. The quantification of confirmed enterococci in the sample is determined by counting the number of green colored wells on the Quanti-Tray device using the Most Probable
Number (MPN) statistics.
2. Scope of application of Enterolert-DW/Quanti-Tray system
The Enterolert-DW/Quanti-Tray system is intended for the analysis of drinking water samples. It is designed to be comparable in performance to ISO 7899-2:2000 Water Quality – Detection and enumeration of intestinal enterococci – Part 2: Membrane filtration method (ISO 7899-2) and The
Microbiology of Drinking Water (2006) – Part 5: The isolation and enumeration of enterococci by membrane filtration (MoDW 2006, Part 5) in drinking water applications. The Enterolert-DW/Quanti-
Tray system provides quantitative, confirmed results for the detection of enterococci in drinking water samples within 24 hours
3. Target organism identification (ISO/TR 13843 sections 10.2.1 and 9.2)
The Enterolert-DW/Quanti-Tray system detects enterococci based on their β -glucosidase activity using the Defined Substrate Technology. When enterococci are present in the test sample, the medium changes color from blue to green. The Quanti-Tray system provides quantitative results based on the MPN statistics.
3.1 Pure culture challenge (ISO/TR 13843 section 10.2.1)
Typical reactions of the Enterolert-DW/Quanti-Tray system were characterized by challenging the medium using pure cultures of reference strains from the American Type Culture Collection (ATCC).
The target enterococcus bacteria, as well as nontarget Gram-negative and Gram-positive bacteria, were tested. Typical results for these reference strains are listed in Table 3.1. These strains are used by IDEXX for routine quality control of the Enterolert-DW/Quanti-Tray system.
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Table 3.1 Reference strains of enterococcus bacteria and nontarget Gram-negative and Gram-positive bacteria used for confirming typical positive and negative reactions in the Enterolert-DW/Quanti-
Tray system
2BU
Bacterium
Enterococcus faecium
Enterococcus faecalis
Enterococcus hirae
Serratia marcescens**
Staphylococcus aureus***
1BU
Strain
ATCC 35667
ATCC 33186
ATCC 8043
ATCC 43862
ATCC 6538
** Up to 10
6
CFU/100 mL *** Up to 10
4
CFU/100 mL
3BU
Typical reaction in Enterolert-DW
Green
Green
Green
Blue (no color change)
Blue (no color change)
3.2 Sensitivity, specificity, false positive/negative rates, efficiency and selectivity studies
(ISO/TR 13843 section 9.2)
In terms of microbiological methods these characteristics are defined by ISO/TR 13843 as: a) number of presumptive positives found positive (true positives) b) number of presumptive negatives found positive (false negatives) c) number of presumptive positives found negative (false positives) d) number of presumptive negatives found negative (true negatives)
1) sensitivity = a/(a + b) , the fraction of the total positives correctly assigned in the presumptive count
2) specificity = d/(c + d) , the fraction of the total negatives correctly assigned in the presumptive count
3) false positive rate = c/(a + c) , the fraction of the observed positives wrongly assigned
4) false negative rate = b/(b + d) , the fraction of the observed negatives wrongly assigned
5) Efficiency (E) = (a + d)/n
6) Selectivity (F) = lg [ (a + c)/n ]
For the Enterolert-DW/Quanti-Tray system, these characteristics relate to the number of wells which were positive for enterococci that actually contained target bacteria and the number of negative wells that were truly negative for the target bacteria.
The assessment of sensitivity, specificity and selectivity of the Enterolert-DW/Quanti-Tray system was achieved through the identification of bacterial isolates from positive and negative wells of tested samples. The samples were prepared by seeding drinking water samples with either chlorinated or nonchlorinated sewage effluent samples.
In the present validation, false positives and false negatives of the Enterolert-DW/Quanti-Tray system were determined by the selective isolation and bacterial identification from 163 positive wells and 186 negative wells selected at random from samples collected in 49 locations in the U.S., U.K.,
Italy, Belgium and Spain. The VITEK  II biochemical identification system (bioMérieux, Inc. Durham,
NC, U.S.A.) was used. Results are detailed in Appendix I and Appendix II, respectively.
The performance characteristics of the Enterolert-DW/Quanti-Tray system, as defined by ISO/TR
13843, are listed in Table 3.2.1
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Table 3.2.1 Enterolert-DW/Quanti-Tray system sensitivity, specificity, false positive/negative rates, efficiency and selectivity
Characteristic
Sensitivity
Specificity
False-positive rate
False-negative rate
Efficiency
Selectivity
Results
92%
98%
2%
7%
95%
-0.3
Bacterial isolates from four (4) of 163 positive wells were identified as nonenterococcus species and thirteen (13) isolates from the 186 negative wells were actually found to be enterococcus species.
The bacterial species giving rise to false-positive and false-negative results in this validation are detailed in Tables 3.2.2 and 3.2.3 respectively.
Table 3.2.2 False-positive bacterial identification using VITEK II
False-positive ID
Lactococus raffinolactis
Elizabethkingia meningoseptica
U
Occurrences
(out of 163 positives)
1
3
Table 3.2.3 False-negative bacterial identification using VITEK II
False-negative ID
Enterococcus hirae
Enterococcus faecium
Occurrences
(out of 186 negatives)
2
4
Enterococcus avium
Enterococcus faecalis
Enterococcus casseliflavus
5
1
1
The outcomes of this validation indicate that the Enterolert-DW/Quanti-Tray system is sensitive and specific (92% and 98%, respectively). The method is also selective, having a value of -0.3, which is considerably better than the guidance value of -1 suggested by ISO/TR 13843 for colony-count methods. With a calculated efficiency value of 95%, the data suggest that the method is highly efficient. The identification data indicate that the Enterolert-DW/Quanti-Tray system recovers a broad range of enterococcus species.
4. Counting uncertainty (ISO 13843 section 10.2.1 and Annex B)
Repeatability and reproducibility are two estimates of the reliability of a method that can be achieved analytically. Repeatability (r) and reproducibility (R) are defined as follows:
Repeatability: Closeness of the agreement between the results of successive measurements of the same measure carried out under the same conditions of measurement
Reproducibility: Closeness of the agreement between the results of measurements of the same measure carried out under changed conditions of measurement
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When applied to counting of microbiological colonies on an agar plate, or positive reactions in an
MPN test, the analyst is the ‘condition’ that either remains the same or is changed. Thus, in this context, repeatability is the agreement in counts obtained by repeated counting events by the same analyst, and reproducibility is the agreement between subsequent counts obtained by two or more analysts.
ISO/TR 13843 Annex B provides guidance on the assessment of counting repeatability and reproducibility using the relative standard deviations (RSDc) of repeated counts. It also recommends that, when using pure cultures, RSDc should ideally be < 0.02 (i.e., not more than 2% deviation).
Counting uncertainty studies were conducted by IDEXX analysts, and the resulting data are presented in Appendix III. The counting was performed such that each analyst was unaware of other analysts’ counts, and the resulting MPN values were recorded as whole integers. The calculated RSDs for MPN values for the Enterolert-DW/Quanti-Tray system, inoculated with
Enterococcus faecalis ATCC 19433, are listed in Table 4.1.
Table 4.1 Enterolert-DW repeatability and reproducibility
U
Counting uncertainty
Repeatability (r)
Reproducibility (R)
U
Relative standard deviation
(RSDc)
0.006
0.005
The calculated values for typical enterococci are much less than the guidance value of < 0.02
(ISO/TR 13843 section B1), indicating that reliable MPN counting of enterococci can be achieved with the Enterolert-DW/Quanti-Tray method.
5. Robustness (ISO/TR 13843 sections 10.2.2 and B.4)
The aspect of robustness that is relevant to the Enterolert-DW/Quanti-Tray method is the incubation period of 24 hours with a valid result up to 28 hours, as described in the method. The results of triplicate counts of suspensions of reference cultures of E. faecium, E. faecalis, E. durans, E. hirae,
E. mundtii and E. gallinarum analysed using the same batch of Enterolert-DW at both the 24-hour and 28-hour incubation periods are presented in Appendix IV.
There was no change in the counts observed for 6 of the 7 type strains validated. The E. gallinarum
ATCC 49573 counts increased by one positive well during the 24- to 28-hour read window. This strain of E. gallinarum ATCC 49573 appears to show a relatively slower growth rate or lower enzyme activity compared to the other bacterial strains tested.
The data suggest that the recommended incubation period of a minimum of 24 hours and up to 28 hours is robust. However, for some strains of enterococci that either grow slowly or have reduced
β
glucosidase activity, there may be a marginal increase in counts at 28 hours of incubation, compared to those at 24 hours of incubation.
An increase in counts over a prescribed incubation period (e.g., 40–48 hours for typical membrane filtration methods, such as ISO 7899-2 or MoDW 2006, part 5) is a well recognized phenomenon, particularly with environmental enterococci. However, provided that the increase in counts after an extended incubation is not excessive, for practical and operational considerations, counts from the
Enterolert-DW/Quanti-Tray system at either 24 or 28 hours incubation can be considered acceptable.
6. Upper working limits (ISO/TR 13843 sections 10.2.4 and 6.3.3)
The Enterolert-DW/Quanti-Tray system is an MPN-based method with an arbitrary counting range of
0 to 201 for Quanti-Tray and 0 to 2,420 for Quanti-Tray/2000, determined by the number of wells in the Quanti-Tray. According to ISO/TR 13843, section 6.3.3, no upper limit can be set for MPN
IDEXX Laboratories, Inc. 4

methods for practical and statistical reasons, “because precision does not depend in a simple way on the number of particles introduced in the detection set.”
7. Precision (ISO/TR 13843 sections 10.2.5 and 9.5.5)
The precision of an MPN method is described by the 95% confidence intervals (CIs) calculated for each MPN value (ISO/TR 13843, section 9.5.5). The CIs for counts from the Enterolert-DW/Quanti-
Tray system, calculated using the program available online from the U.S. Food and Drug
Administration (FDA) Bacteriological Analytical Manual (BAM) (at http://www.cfsan.fda.gov/~ebam/bam-a2.html#excl), are given in Appendix V.
The theoretical repeatability of each MPN value can be derived from the MPN and its CIs. Relative uncertainty values for each MPN value (log10SD, calculated from the MPN 95% CI values) and repeatability (relative standard uncertainty), given as coefficients of variance (CV%), are also presented in Appendix VI. These Poisson values can be used as minimum precision values that can be combined with any appropriate data a laboratory may have (e.g., on variation in decanting the test volume or counting uncertainty) to derive an overall estimate of repeatability for each MPN value.
8. Relative recoveries (ISO/TR 13843 section 10.2.6 and ISO 17994)
The relative recovery of enterococcus spp. by the Enterolert-DW/Quanti-Tray system was compared to that of the reference methods of ISO 7899-2 and MoDW 2006, part 5, following the requirements of ISO 17994:2004 Water Quality – Criteria for establishing equivalence between microbiological methods (ISO 17994).
A total of 403 drinking water samples from geographically diverse locations within the United
Kingdom, Italy, Spain, Belgium and the U.S. were used in this validation. Split samples were used in each of the three methods under comparison. Among those, 198 samples were chlorinated and 205 were not chlorinated.
Paired confirmed count data for the two methods (either the Enterolert-DW/Quanti-Tray system and
ISO 7899-2 or the Enterolert-DW/Quanti-Tray system and MoDW part 5) were transformed using natural logarithms (ln), and the relative difference in counts for each pair (xi) was calculated using the formula [ln(ai) – ln(bi)] x 100% (where ai and bi are the paired confirmed counts for the trial and reference methods respectively).
Note: If one of the counts is zero, then 1 is added to both counts prior to log-transformation.
The mean relative difference and the standard deviation were calculated from the data. Evaluation of equivalence was based upon the mean relative difference and the expanded uncertainty (U)
(calculated from the standard deviation of the mean value [U = 2s/ √ n]), from which the lower (xL) and higher (xH) limits of the confidence interval are calculated. The outcomes of the studies are listed in Tables 8.1 and 8.2.
The analysis showed that the Enterolert-DW/Quanti-Tray method gives overall higher recoveries in comparison with ISO 7899-2. This result was largely driven by the relative differences obtained using chlorinated sample matrices. For the nonchlorinated samples, the validation indicated
“Methods not different” between the two methods.
The overall conclusion was “Methods not different” when comparing the Enterolert-DW/Quanti-Tray system to MoDW 2006, Part 5, with results from the Enterolert-DW/Quanti-Tray system being higher in the chlorinated samples and showing no difference in the nonchlorinated samples.
The observed differences in recoveries between the Enterolert-DW/Quanti-Tray system and the two standard methods were most likely driven by the performance degradation of the standard methods when chlorinated samples were used. This is further supported by the low false-positive rate (2%) and high specificity (98%) determined for the Enterolert-DW/Quanti-Tray system. Since it was a split-sample validation, the results indicated that the standard methods were unable to detect some
IDEXX Laboratories, Inc. 5

target organisms from the same water sample. The relative recoveries for each method are summarized in Appendix VII.
During this validation, it was observed that there were significant differences in relative recovery between the two standard methods, ISO 79-899-2 and MoDW 2006, Part 5. The results are summarized in Table 8.3.
Table 8.1 Enterolert-DW/Quanti-Tray system vs. ISO 7899-2
Drinking water sample matrix
Mean relative difference (%RD)
Expanded uncertainty LO
(%XL)
Expanded uncertainty HI
(%XH)
All matrices 18 11 26
Conclusion
Enterolert-DW higher
Chlorinated matrix
Nonchlorinated matrix
31
6
19
-3
43
13
Enterolert-DW higher
Methods no difference
Table 8.2 Enterolert-DW/Quanti-Tray system vs. MoDW 2006, Part 5
Drinking water sample matrix
All matrices
Mean relative difference (%RD)
3
Expanded uncertainty LO
(%XL)
-4
Expanded uncertainty HI
(%XH)
10
Conclusion
Methods no difference
Chlorinated matrix
Nonchlorinated matrix
13
-8
3
-15
26
0
Enterolert-DW higher
Inconclusive
IDEXX Laboratories, Inc. 6

Table 8.3 ISO 7899-2 vs. MoDW 2006, Part 5
Drinking water sample matrix
All matrices
Mean relative difference (%RD)
-15
Expanded uncertainty LO
(%XL)
-14
Chlorinated matrix
Nonchlorinated matrix
-17
-13
-15
-12
Expanded uncertainty HI
(%XH)
-16
-19
-15
Conclusion
Methods significantly different
Methods significantly different
Methods significantly different
9. References
 ISO 7899-2:2000 Water Quality – Detection and enumeration of intestinal enterococci - Part
2: Membrane filtration method. Geneva: International Organization for Standardization
 The Microbiology of Drinking Water (2006) - Part 5 - The isolation and enumeration of enterococci by membrane filtration. Nottingham: Environment Agency
 The Microbiology of Drinking Water (2002) - Part 3 - Practices and procedures for laboratories. Nottingham: Environment Agency
 ISO/TR 13843:2000(E) Water Quality – Guidance on validation of microbiological methods .
Geneva: International Organization for Standardization
 ISO 17994:2004 Water Quality – Criteria for establishing equivalence between microbiological methods. Geneva: International Organization for Standardization
IDEXX Laboratories, Inc. 7

Appendix I: Identification of isolates from positive Enterolert-DW/Quanti-Tray wells
35
36
37
38
39
29
30
31
32
33
34
8
9
16
17
18
19
20
21
22
23
10
11
12
13
14
15
U
Sample ID
1
4
5
2
3
6
7
24
25
26
27
28
U
Enterolert-DW reactions
+
U
VITEK ID
E. gallinarum
+
+
+
+
+
E. faecium
E. faecium
E. gallinarum
E. gallinarum
E. faecium
+ E. faecalis
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
E. faecium
E. faecium
E. gallinarum
E. gallinarum
E. faecium
E. hirae
E. faecalis
E. faecium
E. gallinarum
E. faecium
E. gallinarum
E. faecalis
E. avium
E. faecalis
E. durans/hirae
E. faecalis
E. seriolicida
E. faecalis
E. seriolicida
E. seriolicida
E. gallinarum
E. seriolicida/L. lactis ssp lactis
E. faecium
Lactococus raffinolactis
E. gallinarum
E. seriolicida
E. faecium
E. faecium
E. faecium
E. gallinarum
E. faecium
E. faecium
U
ID confidence
99%
97%
99%
99%
99%
99%
99%
99%
96%
99%
99%
97%
99%
99%
98%
99%
98%
93%
99%
92%
99%
Low Dis
99%
99%
99%
99%
95%
98%
Low Dis
99%
90%
99%
99%
99%
99%
99%
99%
99%
99%
IDEXX Laboratories, Inc. 8

76
77
78
79
80
81
72
73
74
75
67
68
69
70
71
59
60
61
62
63
64
65
66
55
56
57
58
U
Sample ID
40
41
42
43
44
49
50
51
52
53
54
45
46
47
48
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
U
Enterolert-DW reactions
+
+
E. faecium
E. hirae
U
VITEK ID
+
+
+
+
+
+
+
E. hirae
E. faecium
E. hirae
E. hirae
E. gallinarum
E. faecium
E. faecium
+
+
+
+
+
+
+
+
+
+
E. faecium
E. hirae
E. durans/faecium
E. faecalis
E. gallinarum
E. durans
E. faecium
E. faecium
E. faecalis
E. hirae
E. faecium
E. hirae
E. faecium
E. faecium
E. faecium
E. faecium
E. durans
E. faecium
E. faecalis
E. hirae
E. faecium
E. faecalis
E. faecium
E. casseliflavus
E. faecalis
E. casseliflavus
E. seriolicida
E. seriolicida / lactis
E. durans/faecium/hirae
E. casseliflavus
E. gallinarum
E. casseliflavus
E. durans/hirae
IDEXX Laboratories, Inc. 9
99%
95%
99%
99%
96%
89%
99%
99%
99%
Low Dis
Low Dis
97%
99%
89%
Low Dis
96%
99%
99%
99%
92%
99%
99%
99%
94%
99%
97%
98%
U
ID confidence
98%
95%
99%
98%
95%
99%
99%
99%
99%
99%
99%
Low Dis
99%
93%
98%

113
114
115
116
117
118
119
120
121
U
Sample ID
82
83
84
85
86
91
92
93
94
95
96
87
88
89
90
105
106
107
108
109
110
97
98
99
100
101
102
103
104
111
112
+
+
+
+
+
+
+
+
+
+
+
+
+
+
U
Enterolert-DW reactions
+
+
U
VITEK ID
E. gallinarum
E. gallinarum
+
+
+
+
+
+
+
+
+
+
+
+
+
E. hirae
E. faecalis
E. seriolicida /lactis
E. faecium
E. seriolicida /lactis
E. faecium
E. avium
E. gallinarum
E. faecium
E. gallinarum
E. gallinarum
E. gallinarum
E. faecium
+
+
+
+
+
+
+
+
+
+
+
E. faecium
E. faecium
E. faecalis
E. gallinarum
E. seriolicida /lactis
E. faecium
E. faecium
E. faecium
E. gallinarum
E. faecium
E. durans/gallinarum/hirae
E. gallinarum
E. faecalis
E. faecium
Elizabethkingia meningoseptica
E. gallinarum
Elizabethkingia meningoseptica
E. faecium
E. casseliflavus
E. faecium
E. casseliflavus/gallinarum
E. faecium
E. casseliflavus
E. gallinarum
E. faecium/gallinarum
IDEXX Laboratories, Inc. 10
95%
99%
99%
98%
99%
99%
Low Dis
99%
99%
99%
Low Dis
U
ID confidence
98%
99%
95%
99%
Low Dis
95%
Low Dis
98%
95%
99%
93%
99%
99%
99%
98%
99%
99%
99%
99%
Low Dis
96%
99%
92%
99%
99%
Low Dis
99%
99%
98%

156
157
158
159
160
161
162
149
150
151
152
153
154
155
135
136
137
138
139
140
141
142
143
144
145
146
147
148
128
129
130
131
132
133
134
U
Sample ID
122
123
124
125
126
127
+
+
+
+
+
+
+
+
+
+
+
+
+
+
U
Enterolert-DW reactions
+
+
U
VITEK ID
E. gallinarum
E. hirae
+
+
+
+
E. raffinosus
E. faecium
E. faecalis
E. faecalis
Elizabethkingia meningoseptica
E. hirae
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
E. durans
E. casseliflavus
E. seriolicida / lactis
E. faecium
E. casseliflavus/gallinarum
E. gallinarum
E. casseliflavus/gallinarum
E. faecium
E. hirae
E. faecium
E. gallinarum
E. faecium
E. gallinarum
E. faecium
E. faecium
E. gallinarum
E. durans/faecium
E. faecium
E. faecium
E. durans
E. gallinarum
E. faecium/gallinarum
E. faecium
E. faecium
E. faecium
E. faecalis
E. hirae
E. durans
E. faecium
E. faecium
E. durans
E. faecalis
E. durans
IDEXX Laboratories, Inc. 11
U
ID confidence
98%
99%
91%
99%
85%
99%
91%
94%
99%
98%
98%
99%
92%
85%
99%
Low Dis
99%
94%
99%
99%
99%
99%
95%
93%
Low Dis
86%
Low Dis
98%
Low Dis
99%
99%
98%
99%
98%
99%
99%
99%
98%
Low Dis
99%
99%

191
192
193
194
195
186
187
188
189
190
178
179
180
181
182
183
184
185
196
197
198
199
200
201
202
203
204
U
Sample ID
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
U
Enterolert-DW reactions
+
+
U
VITEK ID
E. faecalis
E. durans/faecium
+
+
+
+
+
+
+
+
+
+
+
+
+
E. faecium
E. faecium
E. faecium
E. gallinarum
E. faecalis
E. casseliflavus/gallinarum
E. durans
E. faecium
E. hirae
E. faecium
E. gallinarum
E. faecium
E. hirae
+
+
+
+
+
+
+
+
+
E. avium
E. gallinarum
E. faecalis
E. gallinarum
E. faecium
E. hirae
E. faecium
E. gallinarum
E. gallinarum
E. gallinarum
E. faecium
E. faecium
E. faecium
E. faecium
E. gallinarum
E. gallinarum
E. faecium
E. faecalis
E. faecium
E. gallinarum
E. faecium
E. faecium
E. durans/faecium
E. faecium
E. faecium
E. faecalis
E. faecalis
IDEXX Laboratories, Inc. 12
99%
99%
93%
99%
93%
99%
99%
99%
99%
99%
90%
98%
99%
99%
99%
99%
95%
98%
99%
99%
98%
98%
Low Dis
91%
99%
99%
99%
U
ID confidence
99%
Low Dis
99%
99%
98%
99%
99%
Low Dis
98%
99%
99%
99%
99%
98%
95%

U
Sample ID
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
U
Enterolert-DW reactions
+
+
U
VITEK ID
E. faecium
E. gallinarum
+
+
+
+
+
+
+
+
+
+
+
+
+
E. faecium
E. faecium
E. gallinarum
E. faecalis
E. faecium
E. casseliflavus/E. gallinarum
E. faecalis
E. faecium
E. durans
E. faecium
E. durans
E. faecalis
E. faecium
+
+
+
+
+
+
+
+
+
+
E. seriolicida /lactis
E. faecium
E. faecium
E. faecalis
E. faecium
E. faecalis
E. faecalis
E. faecalis
E. faecium
E. faecium
U
ID confidence
97%
99%
99%
98%
99%
99%
99%
Low Dis
95%
99%
93%
98%
99%
99%
97%
Low Dis
98%
99%
99%
97%
99%
99%
99%
99%
98%
IDEXX Laboratories, Inc. 13

Appendix II: Identification of isolates from negative Enterolert-DW/Quanti-Tray wells
U
Sample ID
32
33
34
35
28
29
30
31
36
37
38
39
40
23
24
25
26
27
18
19
20
21
22
10
11
12
13
14
15
16
17
7
8
5
6
9
3
4
1
2
None
None
None
None
None
None
None
None
None
None
None
None
None
None
E. faecium
None
None
E. avium
E. avium
None
None
None
None
None
None
E. hirae
E. hirae
None
None
None
None
None
None
None
None
None
None
E. faecium
E. faecium
None
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
U
Enterolert-DW reaction
-
-
-
-
-
-
-
-
-
-
-
-
-
-
U
VITEK ID
U
ID confidence
NA
NA
NA
NA
99%
99%
99%
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
99%
98%
NA
NA
NA
99%
95%
NA
NA
NA
NA
NA
IDEXX Laboratories, Inc. 14

U
Sample ID
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
U
Enterolert-DW reaction
-
-
-
-
-
-
-
-
-
-
-
-
-
72
73
74
75
76
67
68
69
70
71
59
60
61
62
63
64
65
66
77
78
79
80
81
54
55
56
57
58
50
51
52
53
46
47
48
49
41
42
43
44
45
U
VITEK ID
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
E. avium
None
None
None
None
None
None
None
None
None
None
None
None
None
None
IDEXX Laboratories, Inc. 15
U
ID confidence
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
99%
NA
NA
NA
NA
NA
NA
NA
NA
NA

U
Sample ID
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
U
Enterolert-DW reaction
-
-
-
-
-
-
-
-
-
-
-
-
-
113
114
115
116
117
108
109
110
111
112
100
101
102
103
104
105
106
107
118
119
120
121
122
95
96
97
98
99
91
92
93
94
87
88
89
90
82
83
84
85
86
U
VITEK ID
None
None
None
None
None
E. casseliflavus
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
E. avium
None
None
None
None
None
E. faecalis
None
None
None
None
None
None
None
None
None
U
ID confidence
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
99%
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
99%
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
99%
NA
IDEXX Laboratories, Inc. 16

U
Sample ID
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
U
Enterolert-DW reaction
-
-
-
-
-
-
-
-
-
-
-
-
-
154
155
156
157
158
149
150
151
152
153
141
142
143
144
145
146
147
148
159
160
161
162
163
132
133
134
135
136
137
138
139
140
123
124
125
126
127
128
129
130
131
U
VITEK ID
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
E. avium
None
None
None
None
None
None
None
E. faecium
None
None
None
None
None
U
ID confidence
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
99%
NA
NA
NA
NA
NA
NA
NA
98%
IDEXX Laboratories, Inc. 17

173
174
175
176
177
178
179
180
181
182
183
184
185
186
164
165
166
167
168
169
170
171
172
U
Sample ID
U
Enterolert-DW reaction
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
U
VITEK ID
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
U
ID confidence
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
IDEXX Laboratories, Inc. 18

Appendix III: Counting Uncertainty for the Enterolert-DW/Quanti-Tray System
Sample
25
26
27
28
19
20
21
22
23
24
29
30
14
15
16
17
18
5
6
7
8
9
1
2
3
4
10
11
12
13
Enterolert-DW / Quanti-Tray in MPN/100mL Repeatability Reproducibility
Analyst A count 1
70
70
59
56
41
56
50
50
50
56
62
70
31
27
45
45
45
22
27
27
36
25
16
16
21
21
34
38
31
32
Analyst B count
70
70
59
56
41
56
50
50
50
56
62
70
31
27
45
45
45
22
27
27
36
25
16
16
21
21
34
38
31
32
Analyst A count 2
70
70
59
56
43
56
50
50
50
56
62
70
31
27
45
45
45
22
27
27
36
25
16
16
21
21
34
38
31
32
Analyst C count Mean SD RSD sq Mean SD RSD sq
70
70
59
56
43
56
50
50
50
56
62
70
31
27
45
45
45
22
27
27
36
25
16
16
21
21
34
38
31
32
16.00 0.0000 0.0000 16.00 0.0000 0.0000
16.00 0.0000 0.0000 16.00 0.0000 0.0000
21.00 0.0000 0.0000 21.00 0.0000 0.0000
21.00 0.0000 0.0000 21.00 0.0000 0.0000
22.00 0.0000 0.0000 22.00 0.0000 0.0000
27.00 0.0000 0.0000 27.00 0.0000 0.0000
27.00 0.0000 0.0000 27.00 0.0000 0.0000
36.00 0.0000 0.0000 36.00 0.0000 0.0000
25.00 0.0000 0.0000 25.00 0.0000 0.0000
34.00 0.0000 0.0000 34.00 0.0000 0.0000
38.00 0.0000 0.0000 38.00 0.0000 0.0000
31.00 0.0000 0.0000 31.00 0.0000 0.0000
32.00 0.0000 0.0000 32.00 0.0000 0.0000
31.00 0.0000 0.0000 31.00 0.0000 0.0000
27.00 0.0000 0.0000 27.00 0.0000 0.0000
45.00 0.0000 0.0000 45.00 0.0000 0.0000
45.00 0.0000 0.0000 45.00 0.0000 0.0000
45.00 0.0000 0.0000 45.00 0.0000 0.0000
42.00 1.4142 0.0011 41.67 1.1547 0.0008
56.00 0.0000 0.0000 56.00 0.0000 0.0000
50.00 0.0000 0.0000 50.00 0.0000 0.0000
50.00 0.0000 0.0000 50.00 0.0000 0.0000
50.00 0.0000 0.0000 50.00 0.0000 0.0000
56.00 0.0000 0.0000 56.00 0.0000 0.0000
70.00 0.0000 0.0000 70.00 0.0000 0.0000
70.00 0.0000 0.0000 70.00 0.0000 0.0000
59.00 0.0000 0.0000 59.00 0.0000 0.0000
56.00 0.0000 0.0000 56.00 0.0000 0.0000
62.00 0.0000 0.0000 62.00 0.0000 0.0000
70.00 0.0000 0.0000 70.00 0.0000 0.0000
Sum
RSD 0.0011
RSDc 0.0061
Sum
RSD 0.0008
RSDc 0.0051
IDEXX Laboratories, Inc. 19

Appendix IV: Robustness assessment for Enterolert-DW using typed strains
E. faecium ATCC 35667
E. faecalis ATCC 33186
E. faecalis ATCC 19433
E. durans ATCC 11576
E. hirae ATCC 8043
E. mundtii ATCC 43187
E. gallinarum ATCC 49573
Rep
1
2
3
1
2
3
1
2
3
2
3
3
1
2
3
1
1
2
3
1
2
Number of positive wells
24 hr 28 hr % Change
11
19
17
25
18
30
23
19
24
27
37
21
13
17
13
19
15
10
10
23
21
11
19
17
26
18
30
23
19
24
28
38
21
13
17
13
19
15
10
10
23
21
0%
0%
0%
0%
0%
0%
0%
0%
4%
4%
3%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
IDEXX Laboratories, Inc. 20

Appendix V: MPN Table
# positive wells MPN/100 ml
28
29
30
31
32
33
34
35
22
23
24
25
26
27
36
37
38
39
40
41
42
17
18
19
20
21
10
11
12
13
14
15
16
5
6
7
8
9
0
1
2
3
4
40.6
42.9
45.3
47.8
50.4
53.1
56.0
59.1
28.8
30.6
32.4
34.4
36.4
38.4
62.4
65.9
69.7
73.8
78.2
83.1
88.5
11.1
12.4
13.7
15.0
16.4
17.8
19.2
20.7
22.2
23.8
25.4
27.1
5.3
6.4
7.5
8.7
9.9
<1.0
1.0
2.0
3.1
4.2
28.0
29.7
31.5
33.4
35.4
37.5
39.7
42.0
19.0
20.4
21.8
23.3
24.7
26.4
44.6
47.2
50.0
53.1
56.4
59.9
63.9
6.1
7.0
7.9
8.8
9.8
10.8
11.9
13.0
14.1
15.3
16.5
17.7
95% Confidence Limits
Lower
0.0
0.3
Upper
3.7
5.6
0.6
1.1
1.7
2.3
3.0
3.7
4.5
5.3
7.3
9.0
10.7
12.3
13.9
15.5
17.1
18.8
20.5
22.1
23.9
25.7
27.5
29.4
31.3
33.3
35.2
37.3
39.4
41.6
59.5
62.5
65.6
69.0
72.5
76.2
80.1
84.4
43.9
46.3
48.7
51.2
53.9
56.6
88.8
93.7
99.0
104.8
111.2
118.3
126.2
IDEXX Laboratories, Inc. 21

# positive wells MPN/100 ml
48
49
50
51
43
44
45
46
47
94.5
101.3
129.8
200.5
109.1
118.4
144.5
165.2
> 200.5
95% Confidence Limits
Lower
68.2
73.1
Upper
135.4
146.0
78.6
85.0
92.7
102.3
115.2
135.8
146.1
158.7
174.5
195.0
224.1
272.2
387.6
infinite
IDEXX Laboratories, Inc. 22

Appendix VI: 95% percent confidence intervals and theoretical repeatability for each MPN value for the 51 well Enterolert-DW/Quanti-Tray system
95% CI Theoretical repeatability
# positive wells
0
MPN per 100 ml sample
<1.0
Lower
0.0
Upper
3.7
Repeatability
SD (Log
10
SD)
-
Relative standard uncertainty (CV%)
-
1 1.0 0.3 5.6 0.4343 100.0
2
3
2.0
3.1
0.6
1.1
7.3
9.0
0.3071
0.2508
70.7
57.7
4 4.2 1.7 10.7 0.2172 50.0
5 5.3 2.3 12.3 0.1943 44.7
6 6.4 3.0 13.9 0.1774 40.9
7
8
9
7.5
8.7
9.9
3.7
4.5
5.3
15.5
17.1
18.8
0.1643
0.1537
0.1450
37.8
35.4
33.4
10 11.1 6.1 20.5 0.1376 31.7
11
12
12.4
13.7
7.0
7.9
22.1
23.9
0.1313
0.1257
30.2
29.0
13
14
15.0
16.4
8.8
9.8
25.7
27.5
0.1209
0.1166
27.8
26.8
15 17.8 10.8 29.4 0.1127 26.0
16 19.2 11.9 31.3 0.1092 25.1
17
18
20.7
22.2
13.0
14.1
33.3
35.2
0.1061
0.1032
24.4
23.8
19 23.8 15.3 37.3 0.1005 23.1
20 25.4 16.5 39.4 0.0981 22.6
21 27.1 17.7 41.6 0.0959 22.1
22
23
24
28.8
30.6
32.4
19.0
20.4
21.8
43.9
46.3
48.7
0.0938
0.0919
0.0902
21.6
21.2
20.8
25
26
34.4
36.4
23.3
24.7
51.2
53.9
0.0885
0.0870
20.4
20.0
27 38.4 26.4 56.6 0.0856 19.7
28
29
40.6
42.9
28.0
29.7
59.5
62.5
0.0843
0.0830
19.4
19.1
30 45.3 31.5 65.6 0.0819 18.9
31
32
47.8
50.4
33.4
35.4
69.0
72.5
0.0809
0.0799
18.6
18.4
33 53.1 37.5 76.2 0.0791 18.2
34 56.0 39.7 80.1 0.0783 18.0
35 59.1 42.0 84.4 0.0776 17.9
IDEXX Laboratories, Inc.

95% CI
# positive wells
MPN per 100 ml sample
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
62.4
65.9
69.7
73.8
78.2
83.1
88.5
94.5
101.3
109.1
118.4
129.8
144.5
165.2
200.5
> 200.5
Lower
44.6
47.2
50.0
53.1
56.4
59.9
63.9
68.2
73.1
78.6
85.0
92.7
102.3
115.2
135.8
146.1
Upper
88.8
93.7
99.0
104.8
111.2
118.3
126.2
135.4
146.0
158.7
174.5
195.0
224.1
272.2
387.6 infiniti
Theoretical repeatability
Repeatability
SD (Log
10
SD)
0.0770
0.0765
0.0761
0.0758
0.0756
0.0756
0.0757
0.0761
0.0768
0.0778
0.0794
0.0819
0.0859
0.0929
0.1094
-
Relative standard uncertainty (CV%)
17.7
17.6
17.5
17.4
17.4
17.4
17.4
17.5
17.7
17.9
18.3
18.9
19.8
21.4
25.2
-
IDEXX Laboratories, Inc.

Sample number
33
34
35
36
29
30
31
32
37
38
39
40
22
23
24
25
26
27
28
18
19
20
21
14
15
16
17
3
4
5
6
1
2
10
11
12
13
7
8
9
Appendix VII: Enterolert-DW vs. ISO 7899-2 and MoDW 2006, part 5, relative recovery results
Chlorinated Sample Matrix
Enterolert-DW
(MPN/100mL)
ISO 7899-2
(CFU/100mL)
MoDW 2006, part 5
(CFU/100mL)
16
96
183
101
123
56
201
173
62
41
0
12
83
56
124
34
62
119
74
64
53
70
13
57
18
45
109
23
69
7
54
79
116
10
52
68
39
47
9
58
4
37
172
60
129
31
203
141
154
26
0
3
67
22
75
26
63
66
77
43
82
73
62
58
2
48
29
80
70
7
78
96
90
6
79
61
48
43
10
53
7
50
181
68
153
38
208
153
167
33
0
8
71
25
89
29
73
72
78
53
87
78
72
70
2
54
43
95
86
11
87
106
100
10
95
72
59
52
13
60
IDEXX Laboratories, Inc. 25

Sample number
58
59
60
61
54
55
56
57
62
63
64
65
50
51
52
53
46
47
48
49
41
42
43
44
45
77
78
79
80
81
73
74
75
76
66
67
68
69
70
71
72
Chlorinated Sample Matrix
Enterolert-DW
(MPN/100mL)
ISO 7899-2
(CFU/100mL)
MoDW 2006, part 5
(CFU/100mL)
40
24
25
51
45
201
159
39
35
35
59
49
142
13
5
21
74
165
74
118
95
13
68
9
201
64
65
27
35
70
56
40
56
16
14
50
61
28
63
56
30
5
6
19
22
3
123
97
79
16
16
56
28
79
139
4
0
17
114
17
55
85
3
74
2
156
84
104
6
31
22
54
95
45
9
5
40
45
19
50
57
48
6
7
25
25
6
124
103
103
18
18
57
31
114
149
6
0
18
130
29
72
116
7
95
3
161
55
102
50
12
90
105
8
39
28
5
44
49
20
54
63
52
IDEXX Laboratories, Inc. 26

Sample number
99
100
101
102
95
96
97
98
103
104
105
106
91
92
93
94
87
88
89
90
82
83
84
85
86
114
115
116
117
118
119
120
121
122
107
108
109
110
111
112
113
Chlorinated Sample Matrix
Enterolert-DW
(MPN/100mL)
ISO 7899-2
(CFU/100mL)
MoDW 2006, part 5
(CFU/100mL)
43
59
16
8
75
123
24
74
66
38
38
53
8
1
78
57
63
58
66
55
15
78
49
144
5
86
106
68
55
78
165
165
106
119
37
44
91
91
48
5
95
48
29
16
1
48
22
0
5
43
45
52
38
0
0
13
27
72
87
36
12
17
46
72
44
2
40
123
103
109
105
34
41
39
30
22
39
42
48
19
3
41
50
35
18
1
53
34
0
7
46
46
54
41
0
0
15
35
81
96
37
14
22
48
87
53
3
48
132
112
118
119
45
56
47
32
24
41
51
51
21
5
51
IDEXX Laboratories, Inc. 27

Sample number
136
137
138
139
140
141
142
143
144
145
146
147
128
129
130
131
132
133
134
135
123
124
125
126
127
155
156
157
158
159
160
161
162
163
148
149
150
151
152
153
154
Chlorinated Sample Matrix
Enterolert-DW
(MPN/100mL)
ISO 7899-2
(CFU/100mL)
MoDW 2006, part 5
(CFU/100mL)
44
37
37
21
70
44
37
49
50
42
31
26
114
98
70
53
61
11
137
98
159
31
137
106
96
48
63
50
116
130
137
106
94
47
17
31
23
17
48
31
22
36
28
29
4
65
70
22
25
22
24
22
20
71
9
70
67
79
76
73
73
121
24
110
103
90
80
82
82
78
31
36
41
42
98
46
43
48
61
53
25
42
37
34
35
5
78
76
28
30
27
33
30
24
77
13
80
77
86
93
85
82
133
31
124
117
94
86
86
85
81
38
44
51
49
103
52
55
54
70
61
32
48
IDEXX Laboratories, Inc. 28

Sample number
177
178
179
180
181
182
183
184
185
186
187
188
169
170
171
172
173
174
175
176
164
165
166
167
168
189
190
191
192
193
194
195
196
197
198
Chlorinated Sample Matrix
Enterolert-DW
(MPN/100mL)
ISO 7899-2
(CFU/100mL)
MoDW 2006, part 5
(CFU/100mL)
9
7
10
13
22
58
48
6
5
2
32
29
5
5
11
37
28
19
21
27
39
130
159
103
12
26
26
33
29
1
159
155
64
65
22
29
25
16
23
12
51
59
30
23
0
38
33
0
7
2
18
36
41
34
33
44
49
69
52
41
40
44
37
37
3
122
131
29
42
74
36
31
22
29
17
58
68
36
32
0
49
40
0
8
2
22
43
45
40
34
51
56
78
57
44
49
49
39
39
4
127
138
40
48
91
IDEXX Laboratories, Inc. 29

Sample number
14
15
16
17
18
10
11
12
13
5
6
7
8
9
1
2
3
4
27
28
29
30
31
32
33
23
24
25
26
19
20
21
22
38
39
40
41
42
34
35
36
37
43
44
45
46
47
48
Nonchlorinated Sample Matrix
Enterolert-DW
(MPN/100mL)
ISO 7899-2
(CFU/100mL)
MoDW 2006, part 5
(CFU/100mL)
24
21
21
17
29
20
201
21
17
17
23
15
17
23
15
19
8
20
28
44
25
31
39
35
34
35
41
34
25
21
29
25
17
17
32
76
72
72
30
42
36
35
61
44
59
48
63
33
20
22
22
21
18
23
24
20
25
21
29
17
19
20
21
20
21
18
26
21
21
53
50
50
51
29
21
29
20
23
27
29
21
64
57
53
44
49
51
51
51
48
51
42
45
51
49
58
23
29
25
25
23
25
29
26
29
27
35
18
23
24
23
25
22
27
30
25
23
54
51
51
52
32
25
31
22
33
30
32
22
65
58
55
44
53
52
54
54
49
57
51
50
70
63
65
IDEXX Laboratories, Inc. 30

Sample number
79
80
81
82
83
84
85
86
87
88
89
70
71
72
73
74
66
67
68
69
75
76
77
78
94
95
96
97
90
91
92
93
53
54
55
56
49
50
51
52
57
58
59
60
61
62
63
64
65
Nonchlorinated Sample Matrix
Enterolert-DW
(MPN/100mL)
ISO 7899-2
(CFU/100mL)
MoDW 2006, part 5
(CFU/100mL)
25
33
30
38
32
35
34
47
21
37
51
32
21
19
19
18
33
21
39
44
19
23
17
23
41
32
32
10
31
36
38
118
26
28
34
47
23
28
23
30
35
19
33
41
42
52
45
34
49
29
26
34
39
39
31
33
32
29
40
37
23
26
19
23
21
35
21
32
25
22
21
16
17
28
30
33
34
35
36
28
17
38
35
34
32
47
29
35
44
37
35
40
28
37
34
34
36
33
32
32
36
41
47
38
40
37
34
48
42
25
29
22
28
26
37
23
38
32
29
26
17
24
31
36
39
36
37
38
30
18
41
40
37
35
52
30
37
44
39
37
41
30
40
37
38
40
36
IDEXX Laboratories, Inc. 31

Sample number
128
129
130
131
132
133
134
135
136
137
138
115
116
117
118
119
120
121
122
123
124
125
126
127
139
140
141
142
143
144
145
146
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
Nonchlorinated Sample Matrix
Enterolert-DW
(MPN/100mL)
ISO 7899-2
(CFU/100mL)
MoDW 2006, part 5
(CFU/100mL)
5
2
40
43
40
38
30
24
33
38
35
49
4
4
5
5
47
50
67
29
2
1
2
2
12
15
11
11
41
12
11
15
201
29
65
165
35
52
173
44
44
183
144
183
42
53
44
4
32
4
3
39
31
27
32
28
31
29
36
39
21
3
4
6
4
15
13
12
18
3
2
4
4
14
9
15
9
31
10
8
15
26
20
37
38
29
30
31
35
36
36
37
31
19
14
18
11
15
5
3
46
37
34
37
36
35
43
46
44
25
4
5
6
4
18
19
19
22
4
2
4
5
16
10
16
10
39
12
14
17
26
22
38
39
30
31
33
37
39
40
39
33
24
15
26
14
20
IDEXX Laboratories, Inc. 32

Sample number
177
178
179
180
181
182
183
184
185
186
187
164
165
166
167
168
169
170
171
172
173
174
175
176
188
189
190
191
192
193
194
195
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
Nonchlorinated Sample Matrix
Enterolert-DW
(MPN/100mL)
ISO 7899-2
(CFU/100mL)
MoDW 2006, part 5
(CFU/100mL)
34
40
26
28
28
25
29
33
22
33
31
34
24
35
50
32
21
25
21
24
35
38
29
27
26
29
37
29
31
35
37
30
22
21
15
44
14
11
15
5
36
25
28
35
32
26
34
31
29
28
33
35
38
33
38
32
30
33
60
43
28
35
32
25
33
36
26
38
23
36
37
33
28
46
36
41
37
50
48
41
44
33
30
35
34
13
11
12
35
29
32
30
30
28
41
28
30
30
35
41
45
42
38
41
36
30
39
61
51
30
38
42
35
40
39
31
39
28
44
41
41
35
47
40
46
42
55
52
46
48
40
36
40
39
15
13
13
42
35
37
33
34
32
45
32
35
32
IDEXX Laboratories, Inc. 33

Sample number
196
197
198
199
200
201
202
203
204
205
Nonchlorinated Sample Matrix
Enterolert-DW
(MPN/100mL)
ISO 7899-2
(CFU/100mL)
MoDW 2006, part 5
(CFU/100mL)
37
43
36
35
31
27
30
22
15
17
40
38
38
39
36
39
34
36
25
25
43
41
40
41
42
44
39
43
28
26
IDEXX Laboratories, Inc. 34