ASTM D6503-99(2005)
(Test Method)Standard Test Method for Enterococci in Water Using Enterolert
Standard Test Method for Enterococci in Water Using Enterolert
SIGNIFICANCE AND USE
This test provides an easy and reliable method for the detection of enterococci in water within 24 h. For recreational water (fresh and marine) testing is performed to insure areas are safe for swimming. Enterolert also can be used for testing bottled water and drinking water.
SCOPE
1.1 This test method covers a simple procedure for the detection of enterococci in water and wastewater. It is based on IDEXX's patented Defined Substrate Technology (DST). This product, Enterolert, utilizes a nutrient indicator that fluoresces when metabolized. It can detect these bacteria at one colony forming unit (CFU)/100 mL within 24 h. The presence of this microorganism in water is an indication of fecal contamination and the possible presence of enteric pathogens.
1.2 This test method can be used successfully with drinking water, source water, recreational (fresh and marine) water, and bottled water. It is the user's responsibility to ensure the validity of this test method for waters of untested matrices.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D 6503 – 99 (Reapproved 2005)
Standard Test Method for
Enterococci in Water Using EnterolertY
This standard is issued under the fixed designation D 6503; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3.2.3 presence-absence, n—a term used to indicate if en-
terococci is present in a water sample. It is a qualitative value,
1.1 This test method covers a simple procedure for the
“yes” or “no” for reporting results.
detection of enterococci in water and wastewater. It is based on
3.2.4 quanti-trayy, n—a system for the quantification of
IDEXX’s patented Defined Substrate Technologyt (DSTy).
enterococci. It consists of a sealer and trays which have
This product, Enterolert, utilizes a nutrient indicator that
multi-wells and can enumerate up to 2000 CFU/100 mL
fluoresceswhenmetabolized.Itcandetectthesebacteriaatone
without dilution.
colony forming unit (CFU)/100 mL within 24 h. The presence
3.2.5 snap pack, n—apackagecontainingEnterolertreagent
of this microorganism in water is an indication of fecal
for testing 100-mL sample either in the P/A format or quanti-
contamination and the possible presence of enteric pathogens.
tatively, that is, Quanti-Trayy system).
1.2 This test method can be used successfully with drinking
water, source water, recreational (fresh and marine) water, and
4. Summary of Test Method
bottled water. It is the user’s responsibility to ensure the
4.1 Thistestmethodisusedforthedetectionofenterococci,
validity of this test method for waters of untested matrices.
such as E. faecium, E. faecalis in drinking water, source water,
1.3 This standard does not purport to address all of the
recreationalwaters(marinewaterandfresh),andbottledwater.
safety concerns, if any, associated with its use. It is the
When the reagent is added to the sample and incubated at 41 6
responsibility of the user of this standard to establish appro-
0.5°C for 24 h, Enterolert can detect these bacteria at 1
priate safety and health practices and determine the applica-
CFU/100 mL. Fluorescence is produced when enterococci
bility of regulatory limitations prior to use.
metabolizes the nutrient indicator. Enterolert can be used as a
2. Referenced Documents presence-absence test or for quantification (5-tube, 10-tube
MPN, 15-tube serial dilution or the Quanti-Tray system).
2.1 ASTM Standards:
D 1129 Terminology Relating to Water
5. Significance and Use
D 1193 Specification for Reagent Water
5.1 This test provides an easy and reliable method for the
D 3370 Practices for SamplingWater from Closed Conduits
detection of enterococci in water within 24 h. For recreational
3. Terminology water (fresh and marine) testing is performed to insure areas
are safe for swimming. Enterolert also can be used for testing
3.1 Definitions—For definitions of terms used in this test
bottled water and drinking water.
method, refer to Terminology D 1129.
3.2 Definitions of Terms Specific to This Standard:
6. Interferences
3.2.1 enterococci, n—a gram positive bacteria possessing
6.1 The presence of Bacillus spp. can interfere with the
the enzyme b-D-glucosidase, which cleaves the nutrient indi-
testing of marine water samples. To eliminate interference, a
cator and produces fluorescence under a long wave length (366
1:10 dilution is required with sterile water (deionized or
nm) ultraviolet (UV) light.
distilled).
3.2.2 most probable number (MPN), n—a statistical method
for determining bacterial density based on the Poisson distri-
7. Apparatus
bution.
7.1 Ultraviolet Lamp, 6-watt long wavelength (366 nm).
7.2 41°C Incubator (60.5°C), air or water bath.
7.3 Vessels, sterile, nonfluorescent.
This test method is under the jurisdiction of ASTM Committee D19 on Water
7.4 Quanti-Tray Sealer.
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved June 1, 2005. Published June 2005. Originally
approved in 1999. Last previous edition approved in 1999 as D 6503 – 99.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM This test method is based on Enterolert, a product of IDEXX Laboratories,
Standards volume information, refer to the standard’s Document Summary page on Westbrook, ME 04092.
the ASTM website. Available from IDEXX Laboratories, One Idexx Dr., Westbrook, ME 04092.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D 6503 – 99 (2005)
7.5 Quanti-Tray or Quanti-Tray 2000. 12. Procedure
12.1 Presence/Absence—See package insert.
8. Reagents and Materials
12.1.1 Samples should be brought to room temperature (18
8.1 Purity of Water—Unless otherwise indicated, references
to 30°C).
to water shall be understood to mean reagent water conforming
12.1.2 Carefully separate one snap pack from the strip.
to Specification D 1193, Type IV. Sterilize the water by either
12.1.3 Tap the snap pack to insure that all of the powder is
autoclaving or by sterile filtration (0.22 micron-filtered water).
towards the bottom of the pack.
8.2 Enterolert Test Kit.
12.1.4 Open the pack by snapping back the top of the score
line. Do not touch the opening of pack.
9. Precautions
12.1.5 Add the reagent to a 100-mL water sample, which is
in a sterile, transparent, nonfluorescent vessel.
9.1 The analyst must observe the normal good laboratory
12.1.6 Aseptically cap and seal the vessel.
practices and safety procedures required in a microbiology
12.1.7 Shake until dissolved.
laboratory while preparing, using, and disposing of cultures,
12.1.8 Incubate Enterolert for 24 h at 41 6 0.5°C,
reagents and materials and while operating sterilization equip-
12.1.9 Read results at 24 h. If the sample is inadvertently
ment and other equipment.
incubated over 28 h without observation, the following guide-
lines apply: Lack of fluorescence after 28 h is a valid negative
10. Sampling
test. Fluorescence after 28 h is an invalid result.
10.1 Collect the sample as described in detail in the USEPA
5 12.1.10 Check for fluorescence by placing a 6-W 366-nm
microbiological methods manual and in accordance with
UV light within 5 in. of the sample in a dark environment. Be
Practices D 3370.
sure the light is facing away from your eyes and towards the
10.2 Sample Storage Temperature and Handling
vessel. If fluorescence is observed, the presence of enterococci
Conditions—Ice or refrigerate water samples at a temperature
is confirmed.
of 2 to 8°C during transit to the laboratory. Use insulated
12.2 MPN—Quanti-tray enumeration test procedure for
containers to ensure proper maintenance of storage tempera-
100-mL sample (see package insert).
tures. Take care that sample bottles are not totally immersed in
12.2.1 Follow steps 12.1.1-12.1.7.
water during transit or storage.
12.2.2 Pour the reagent sample into the Quanti-Tray avoid-
10.3 Holding Time Limitations—Examine samples, as soon
ing contact with the foil tab and seal the tray according to the
as possible, after collection. Do not hold samples longer than
Quanti-Tray package insert.
6 h between collection and initiation of analyses.
12.2.3 Incubate for 24 h at 41 6 0.5°C.
12.2.4 Follow the same interpretation instructions from
11. Quality Control Check
12.1.9through12.1.10,andcountthenumberofpositivewells.
11.1 Check and record temperatures in incubators daily to
Refer to the MPN table (see Table 1) provided with the
ensure temperature is within stated limits.
Quanti-Tray to determine the CFU/100 mL.
11.2 Qualitycontrolshouldbeconductedoneachnewlotof
12.3 MPN—5-tube 320mL,10-tube 310mLand15-tube
Enterolert. See package insert for the recommended quality
serial dilution.
control procedure, which consists of the following protocol:
12.3.1 Follow 12.1.1-12.1.7.
11.2.1 For each type of the American Type Culture Collec-
12.3.2 sterile nonfluorescent tubes or transfer 20 mL of the
tion (ATCC) bacterial strain listed below, streak the culture
reagent sample into five sterile nonfluorescent tubes.
onto labeledTSAor blood agar plates and incubate at 35°C for
12.3.3 Incubate for 24 h at 41 6 0.5°C.
18 to 24 h.
12.3.4 Follow 12.1.9 and 12.1.10 for interpretation.
11.2.2 For each bacterial strain, touch a 1-µl loop to a
12.3.5 Refer to the MPN tables (see Tables 2-4) to deter-
colony and use it to inoculate a labeled test tube containing
mine the CFU/100 mL.
5 mL of sterile deionized water. Close cap and shake thor-
oughly.
13. Calculation
11.2.3 For each bacterial strain, take a 1-µl loop from the
13.1 For P/A, there are no calculations. For quantification,
test tube and use it to inoculate a labeled vessel containing
refer to Quanti-Tray MPN tables and for the 5, 10, and 15 tube
100 mL.
test results refer to the respective MPN tables.
11.2.4 Follow the Enterolert presence/absence steps listed
above to test these controls. Compare the test results to the
14. Report
following expected results:
Control ATTC No. Expected Result
14.1 Report as positive or negative for presence/absence
Enterococcus faecium 335667 Fluorescence
testing.
Serratia marcescens (g, –) 43862 No fluorescence
14.2 Reporting of results is based on calculation of entero-
Aerococcus viridians (g, +) 10400 No fluorescence
cocci density determined from the appropriate MPN tables.
Bordner, R.H., Winter, J.A., and Scarpino, P.V., Eds., Microbiological Methods
for Monitoring the Environment, Water, and Wastes, EPA-600/8-78-017. Standard Methods for the Examination of Water and Waste Water,19thEdition.
D 6503 – 99 (2005)
15. Precision and Bias the overall standard deviation (St), and the single operator
standard deviation (so), are indicated in Table 5.
15.1 Precision—A limited collaborative study was con-
15.2 Bias—The mean value obtained for the samples
ducted. Nine technicians from three laboratories tested three
(drinking water, recreational water fresh and marine) from the
different matrixes at three levels following Practice D 2777.
nine technicians for the low-, mid- and high-spiked samples all
Outliers were rejected in accordance with the statistical tests
fall within the 95 % confidence interval (poisson distribution)
outlined in Practice D 2777. All data from one technician was
of the actual values obtained from plating on blood agar.
rejected for recreational water-marine and single values were
15.3 Results of this collaborative study may not be typical
rejected for both recreational water-fresh at the low level and
of results for matrices other than those studied.
for recreational water-marine at the low level.The mean count,
16. Keywords
16.1 bottled water; drinking water; enterococci; Enterolert;
7 most probable number; presence-absence; Quanti-Tray; recre-
Supporting data have been filed at ASTM International Headquarters and may
be obtained by requesting Research Report RR: D19–1167. ational water; source water
D 6503 – 99 (2005)
TABLE 1 51-Well Quanti-TrayY MPN Table
95 % Confidence Limits
No. of Wells Giving
MPN/100-mL Sample
Positive Reaction
Lower Upper
0 <1 0.0 3.7
1 1.0 0.3 5.6
2 2.0 0.6 7.3
3 3.1 1.1 9.0
4 4.2 1.7 10.7
5 5.3 2.3 12.3
6 6.4 3.0 13.9
7 7.5 3.7 15.5
8 8.7 4.5 17.1
9 9.9 5.3 18.8
10 11.1 6.1 20.5
11 12.4 7.0 22.1
12 13.7 7.9 23.9
13 15.0 8.8 25.7
14 16.4 9.8 27.5
15 17.8 10.8 29.4
16 19.2 11.9 31.3
17 20.7 13.0 33.3
18 22.2 14.1 35.2
19 23.8 15.3 37.3
20 25.4 16.5 39.4
21 27.1 17.7 41.6
22 28.8 19.0 43.9
23 30.6 20.4 46.3
24 32.4 21.8 48.7
25 34.4 23.3 51.2
26 36.4 24.7 53.9
27 38.4 26.4 56.6
28 40.6 28.0 59.5
29 42.9 29.7 62.5
30 45.3 31.5 65.6
31 47.8 33.4 69.0
32 50.4 35.4 72.5
33 53.1 37.5 76.2
34 56.0 39.7 80.1
35 59.1 42.0 84.4
36 62.4 44.6 88.8
37 65.9 47.2 93.7
38 69.7 50.0 99.0
39 73.8 53.1 104.8
40 78.2 56.4 111.2
41 83.1 59.9 118.3
42 88.5 63.9 126.2
43 94.5 68.2 135.4
44 101.3 73.1 146.0
45 109.1 78.6 158.7
46 118.4 85.0 174.5
47 129.8 92.7 195.0
48 144.5 102.3 224.1
49 165.2 115.2 272.2
50 200.5 135.8 387.6
51 >200.5 146.1 infinite
D 6503 – 99 (2005)
TABLE 2 IDEXX Quanti-Tray/2000 MPN Table (cfu/100 mL)
No. Large No. Small Wells Positive
Wells
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Positive
0 <1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.1 16.1 17.1 18.1 19.1 20.2 21.2 22.2 23.2
1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.1 9.1 10.1 11.1 12.1 13.2 14.2 15.2 16.2 17.3 18.3 19.3 20.4 21.4 22.4 23.5 24.5
2 2.0 3.0 4.1 5.1 6.1 7.1 8.1 9.2 10.2 11.2 12.2 13.3 14.3 15.3 16.4 17.4 18.5 19.5 20.6 21.6 22.6 23.7 24.8 25.8
3 3.1 4.1 5.1 6.1 7.2 8.2 9.2 10.3 11.3 12.4 13.4 14.4 15.5 16.5 17.6 18.6 19.7 20.8 21.8 22.9 23.9 25.0 26.1 27.1
4 4.1 5.2 6.2 7.2 8.3 9.3 10.4 11.4 12.5 13.5 14.6 15.6 16.7 17.8 18.8 19.9 21.0 22.0 23.1 24.2 25.2 26.3 27.4 28.5
5 5.2 6.3 7.3 8.4 9.4 10.5 11.5 12.6 13.7 14.7 15.8 16.9 17.9 19.0 20.1 21.2 22.2 23.3 24.4 25.5 26.6 27.7 28.8 29.9
6 6.3 7.4 8.4 9.5 10.6 11.6 12.7 13.8 14.9 15.9 17.0 18.1 19.2 20.3 21.4 22.5 23.6 24.7 25.8 26.9 28.0 29.1 30.2 31.3
7 7.4 8.5 9.6 10.7 11.8 12.8 13.9 15.0 16.1 17.2 18.3 19.4 20.5 21.6 22.7 23.8 24.9 26.0 27.1 28.3 29.4 30.5 31.6 32.8
8 8.6 9.7 10.8 11.9 13.0 14.1 15.2 16.3 17.4 18.5 19.6 20.7 21.8 22.9 24.1 25.2 26.3 27.4 28.6 29.7 30.8 32.0 33.1 34.3
9 9.8 10.9 12.0 13.1 14.2 15.3 16.4 17.5 18.7 19.8 20.9 22.0 23.2 24.3 25.4 26.6 27.7 28.9 30.0 31.2 32.3 33.5 34.6 35.8
10 11.0 12.1 13.2 14.3 15.5 16.6 17.7 18.9 20.0 21.1 22.3 23.4 24.6 25.7 26.9 28.0 29.2 30.3 31.5 32.7 33.8 35.0 36.2 37.4
11 12.2 13.4 14.5 15.6 16.8 17.9 19.1 20.2 21.4 22.5 23.7 24.8 26.0 27.2 28.3 29.5 30.7 31.9 33.0 34.2 35.4 36.6 37.8 39.0
12 13.5 14.6 15.8 16.9 18.1 19.3 20.4 21.6 22.7 23.9 25.1 26.3 27.5 28.6 29.8 31.0 32.2 33.4 34.6 35.8 37.0 38.2 39.4 40.7
13 14.8 16.0 17.1 18.3 19.5 20.6 21.8 23.0 24.2 25.4 26.6 27.8 29.0 30.2 31.4 32.6 33.8 35.0 36.2 37.5 38.7 39.9 41.1 42.4
14 16.1 17.3 18.5 19.7 20.9 22.1 23.3 24.4 25.7 26.9 28.1 29.3 30.5 31.7 33.0 34.2 35.4 36.7 37.9 39.1 40.4 41.6 42.9 44.2
15 17.5 18.7 19.9 21.1 22.3 23.5 24.7 25.9 27.2 28.4 29.6 30.9 32.1 33.3 34.6 35.8 37.1 38.4 39.6 40.9 42.2 43.4 44.7 46.0
16 18.9 20.1 21.3 22.6 23.8 25.0 26.2 27.5 28.7 30.0 31.2 32.5 33.7 35.0 36.3 37.5 38.8 40.1 41.4 42.7 44.0 45.3 46.6 47.9
17 20.3 21.6 22.8 24.0 25.3 26.5 27.8 29.1 30.3 31.6 32.9 34.1 35.4 36.7 38.0 39.3 40.6 41.9 43.2 44.5 45.9 47.2 48.5 49.8
18 21.8 23.1 24.3 25.6 26.9 28.1 29.4 30.7 32.0 33.3 34.6 35.9 37.2 38.5 39.8 41.
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