ASTM D6503-99
(Test Method)Standard Test Method for Enterococci in Water Using EnterolertTM
Standard Test Method for Enterococci in Water Using Enterolert<sub>TM</sub>
SCOPE
1.1 This test method describes 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.
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.
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An American National Standard
Designation: D 6503 – 99
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 describes 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
fluoresces when metabolized. It can detect these bacteria at one
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 Sampling Water from Closed Con-
2 detection of enterococci in water within 24 h. For recreational
duits
water (fresh and marine) testing is performed to insure areas
3. Terminology are safe for swimming. Enterolert also can be used for testing
bottled water and drinking water.
3.1 Definitions—For definitions of terms used in this test
method, refer to Terminology D 1129.
6. Interferences
3.2 Definitions of Terms Specific to This Standard:
6.1 The presence of Bacillus spp. can interfere with the
3.2.1 enterococci, n—a gram positive bacteria possessing
testing of marine water samples. To eliminate interference, a
the enzyme b-D-glucosidase, which cleaves the nutrient indi-
1:10 dilution is required with sterile water (deionized or
cator and produces fluorescence under a long wave length (366
distilled).
nm) ultraviolet (UV) light.
3.2.2 most probable number (MPN), n—a statistical method
7. Apparatus
for determining bacterial density based on the Poisson distri-
7.1 Ultraviolet Lamp, 6-watt long wavelength (366 nm).
bution.
7.2 41°C Incubator (6 0.5°C), air or water bath.
7.3 Vessels, sterile, nonfluorescent.
This test method is under the jurisdiction ofASTM Committee D-19 on Water
and is the direct responsibility of Subcommittee D19.24 on Microbiology.
Current edition approved Dec. 10, 1999. Published April 2000. This test method is based on Enterolert, a product of IDEXX Laboratories,
Annual Book of ASTM Standards, Vol 11.01. Westbrook, ME 04092.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D6503–99
7.4 Quanti-Tray Sealer . 12. Procedure
7.5 Quanti-Tray or Quanti-Tray 2000 .
12.1 Presence/Absence—See package insert.
12.1.1 Samples should be brought to room temperature
8. Reagents and Materials
(18–30°C).
8.1 Purity of Water—Unless otherwise indicated, references
12.1.2 Carefully separate one snap pack from the strip.
to water shall be understood to mean reagent water conforming
12.1.3 Tap the snap pack to insure that all of the powder is
to Specification D 1193, Type IV. Sterilize the water by either
towards the bottom of the pack.
autoclaving or by sterile filtration (0.22 micron-filtered water).
12.1.4 Open the pack by snapping back the top of the score
8.2 Enterolert Test Kit .
line. Do not touch the opening of pack.
12.1.5 Add the reagent to a 100-mL water sample, which is
9. Precautions
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 12.1.10 Check for fluorescence by placing a 6-W 366-nm
UV light within 5 in. of the sample in a dark environment. Be
microbiological methods manual and in accordance with
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 6 Quanti-Tray package insert.
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.
Refer to the MPN table (see Table 1) provided with the
11.1 Check and record temperatures in incubators daily to
Quanti-Tray to determine the CFU/100 mL.
insure temperature is within stated limits.
12.3 MPN—5-tube 3 20 mL, 10-tube 3 10 mLand 15-tube
11.2 Qualitycontrolshouldbeconductedoneachnewlotof
Enterolert. See package insert for the recommended quality serial dilution.
12.3.1 Follow 12.1.1-12.1.7.
control procedure, which consists of the following protocol:
11.2.1 For each type of the American Type Culture Collec- 12.3.2 sterile nonfluorescent tubes or transfer 20 mL of the
reagent sample into five sterile nonfluorescent tubes.
tion (ATCC) bacterial strain listed below, streak the culture
onto labeledTSAor blood agar plates and incubate at 35°C for 12.3.3 Incubate for 24 h at 41 6 0.5°C.
12.3.4 Follow 12.1.9 and 12.1.10 for interpretation.
18–24 h.
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-
mine the CFU/100 mL.
colony and use it to inoculate a labeled test tube containing 5
mLof sterile deionized water. Close cap and shake thoroughly.
11.2.3 For each bacterial strain, take a 1-µl loop from the 13. Calculation
test tube and use it to inoculate a labeled vessel containing 100
13.1 For P/A, there are no calculations. For quantification,
mL.
refer to Quanti-Tray MPN tables and for the 5, 10, and 15 tube
11.2.4 Follow the Enterolert presence/absence steps listed 6
test results refer to the respective MPN tables.
above to test these controls. Compare the test results to the
following expected results:
14. Report
Control ATTC No. Expected Result
14.1 Report as positive or negative for presence/absence
Enterococcus faecium 335667 Fluorescence
Serratia marcescens (g, –) 43862 No fluorescence testing.
Aerococcus viridians (g, +) 10400 No fluorescence
14.2 Reporting of results is based on calculation of entero-
cocci density determined from the appropriate MPN tables.
Available from IDEXX Laboratories, One Idexx Dr., Westbrook, ME 04092.
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.
D6503–99
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 for this test method are available from ASTM Headquarters.
Request RR: D 19-1167. ational water; source water
D6503–99
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
D6503–99
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 41.1 42.4 43.8 45.1 46.4 47.8 49.1 50.5 51.9
19 23.3 24.6 25.9 27.2 28.5 29.8 31.1 32.4 33.7 35.0 36.3 37.6 39.0 40.3 41.6 43.0 44.3 45.7 47.1 48.4 49.8 51.2 52.6 54.0
20 24.9 26.2 27.5 28.8 30.1 31.4 32.8 34.1 35.4 36.8 38.1 39.5 40.8 42.2 43.6 44.9 46.3 47.7 49.1 50.5 51.9 53.3 54.7 56.1
21 26.5 27.8 29.2 30.5 31.8 33.2 34.5 35.9
...
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