vvEPA
United States
Environmental Protection
Agency
Method 1603: Escherichia coli (E. coli) in
Water by Membrane Filtration Using
Modified membrane-Thermotolerant
Escherichia coli Agar (Modified mTEC)
September 2002
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-02-023
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Disclaimer
The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and
approved this report for publication. The Office of Science and Technology directed, managed, and
reviewed the work of DynCorp in preparing this report. Neither the United States Government nor any of
its employees, contractors, or their employees make any warranty, expressed or implied, or assumes any
legal liability or responsibility for any third party's use of or the results of such use of any information,
apparatus, product, or process discussed in this report, or represents that its use by such party would not
infringe on privately owned rights. This document combines the information previously published in Test
Methods for Escherichia coli and Enterococci in Water by the Membrane Filter Procedure (EPA-600/4-
85-076) (Reference 18.6 and Improved Enumeration Methods for the Recreational Water Quality
Indicators: Enterococci and Escherichia coli (EPA/821/R-97/004) (Reference 18.4. Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.
Questions concerning this method or its application should be addressed to:
Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S. EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro.robin@epa.gov
202-566-1075
202-566-1053 (facsimile)
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Table of Contents
1.0 Scope and Application 1
2.0 Summary of Method 1
3.0 Definitions 1
4.0 Interferences and Contamination 1
5.0 Safety 1
6.0 Equipment and Supplies 2
7.0 Reagents and Standards 3
8.0 Sample Collection, Preservation, and Storage 6
9.0 Quality Control 6
10.0 Calibration and Standardization 6
11.0 Procedure 6
12.0 Data Analysis and Calculations 7
13.0 Method Performance 7
14.0 Reporting Results 7
15.0 Verification Procedure 7
16.0 Pollution Prevention 8
17.0 Waste Management 9
18.0 References 9
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Method 1603: Escherichia coll (E. coll) in Water by Membrane
Filtration Using Modified membrane- Thermotolerant Escherichia coll
Agar (modified mTEC)
September 2002
1.0 Scope and Application
1.1 This method describes a revised Escherichia coll membrane filter (MF) procedure, a single-step
method that uses one medium, modified mTEC Agar, and does not require the transfer of the
membrane filter to another medium or other substrate. The modified medium contains a
chromogen (5-bromo-6-chloro-3-indolyl-« «D-glucuronide), which is catabolized to glucuronic
acid and a red- or magenta-colored compound by E. coll that produce the enzyme • *D-
glucuronidase. The apparatus and equipment, and sampling, filtration, and verification procedures
for this modified mTEC method are identical to those of the original mTEC method. Because the
bacterium is a natural inhabitant only of the intestinal tract of warm-blooded animals, its presence
in water samples is an indication of fecal pollution and the possible presence of enteric pathogens.
1.2 The E. coll test is recommended as a measure of recreational fresh water quality.
Epidemiological studies have led to the development of criteria which can be used to promulgate
recreational water standards based on established relationships between health effects and water
quality. The significance of finding E. coll in recreational fresh water samples is the direct
relationship between the density of E. coll and the risk of gastrointestinal illness associated with
swimming in the water (Reference 18.3.
1.3 The test for E. coll can be applied to fresh, estuarine, and marine waters.
1.4 Since a wide range of sample volumes or dilutions can be analyzed by the MF technique, a wide
range of E. coll levels in water can be detected and enumerated.
2.0 Summary of Method
2.1 The MF method provides a direct count of E. coll in water based on the development of colonies
that grow on the surface of a membrane filter. A water sample is filtered through the membrane,
which retains the bacteria. After filtration, the membrane containing the bacteria is placed on a
selective and differential medium, modified mTEC Agar, incubated at35±0.5°C for 2 h to
resuscitate the injured or stressed bacteria, and then incubated at 44.5 ± 0.2°C for 22 h. The
modified method eliminates the transfer of the membrane filter to another substrate. The target
colonies on modified mTEC agar are red or magenta in color after the incubation period.
3.0 Definitions
3.1 In this method, E. coll are those bacteria which produce red or magenta colonies on the modified
mTEC agar.
4.0 Interferences and Contamination
4.1 Water samples containing colloidal or suspended particulate material can clog the membrane
filter and prevent filtration, or cause spreading of bacterial colonies which could interfere with
identification of target colonies.
5.0 Safety
September 2002
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Method 1603
5.1 The analyst/technician must know and observe the normal safety procedures required in a
microbiology laboratory while preparing, using, and disposing of cultures, reagents, and materials
and while operating sterilization equipment.
5.2 Mouth-pipetting is prohibited.
6.0 Equipment and Supplies
6.1 Glass lens with magnification of 2-5x, or stereoscopic microscope.
6.2 Lamp, with a cool, white fluorescent tube.
6.3 Hand tally or electronic counting device.
6.4 Pipet container, stainless steel, aluminum or borosilicate glass, for glass pipets.
6.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, of appropriate volume.
6.6 Graduated cylinders, 100-1000 mL, covered with aluminum foil or kraft paper and sterile.
6.7 Membrane filtration units (filter base and funnel), glass, plastic or stainless steel, wrapped with
aluminum foil or kraft paper and sterilized.
6.8 Ultraviolet unit for sanitization of the filter funnel between filtrations (optional).
6.9 Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source. In an emergency or
in the field, a hand pump or a syringe equipped with a check valve to prevent the return flow of
air, can be used.
6.10 Filter flask, vacuum, usually 1 L, with appropriate tubing. A filter manifold to hold a number of
filter bases is optional.
6.11 Flask for safety trap placed between the filter flask and the vacuum source.
6.12 Forceps, straight or curved, with smooth tips to handle filters without damage.
6.13 Ethanol, methanol or isopropanol in a small, wide-mouth container, for flame-sterilizing forceps.
6.14 Burner, Bunsen or Fisher type, or electric incinerator unit for sterilizing loops and needles.
6.15 Thermometer, checked against a National Institute of Standards and Technology (NIST) certified
thermometer, or one that meets the requirements of NIST Monograph SP 250-23.
6.16 Petri dishes, sterile, plastic, 9x50 mm, with tight-fitting lids; or 15 x 60 mm, glass or plastic,
with loose-fitting lids; or 15 x 100 mm.
6.17 Bottles, milk dilution, borosilicate glass, screw-cap with neoprene liners, marked at 99 mL for
1:100 dilutions. Dilution bottles marked at 90 mL or tubes marked at 9 mL may be used for 1:10
dilutions.
6.18 Flasks, borosilicate glass, screw-cap, 250-2000 mL volume.
6.19 Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 ± 0.02 (im pore size.
September 2002
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Method 1603
6.20 Inoculation loops, at least 3 mm diameter, and needles, nichrome or platinum wire, 26 B & S
gauge, in suitable holders. Sterile disposable applicator sticks or plastic loops are alternatives to
inoculation loops.
Note: A platinum loop is required for the cytochrome oxidase test in the verification procedure.
6.21 Incubator maintained at 35 ± 0.5°C, with approximately 90% humidity if loose-lidded petri dishes
are used.
6.22 Waterbath maintained at 44.5 ± 0.5°C.
6.23 Waterbath maintained at 50°C for tempering agar.
6.24 Test tubes, 20 x 150 mm, borosilicate glass or plastic.
6.25 Test tubes, 10 x 75 mm, borosilicate glass
6.26 Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes.
6.27 Test tubes screw-cap, borosilicate glass, 16 x 125 mm or other appropriate size.
6.28 Filter Paper.
6.29 Whirl-Pak® bags.
7.0 Reagents and Standards
7.1 Purity of Reagents: Reagent grade chemicals shall be used in all tests. Unless otherwise
indicated, reagents shall conform to the specifications of the Committee on Analytical Reagents
of the American Chemical Society (Reference 18.5). The agar used in preparation of culture
media must be of microbiological grade.
7.2 Whenever possible, use commercial culture media as a means of quality control.
7.3 Purity of Water: Reagent water conforming to Specification Dl 193, reagent water conforming
Type II, Annual Book of ASTM Standards (Reference 18.1).
7.4 Phosphate Buffered Saline
7.4.1 Composition:
Sodium Dihydrogen Phosphate 0.58 g
Sodium Monohydrogen Phosphate 2.5 g
Sodium Chloride 8.5 g
Reagent-Grade Distilled Water 1.0 L
7.4.2 Preparation: Dissolve the ingredients in 1 L of reagent-grade distilled water in a flask ,
and dispense in appropriate amounts for dilutions in screw-cap bottles or culture tubes,
and/or into containers for use as rinse water. Autoclave at 121°C (15 Ib pressure) for 15
min. Final pH should be 7.4 + 0.2.
7.5 Phosphate Buffered Dilution Water (References 18.2)
September 2002
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Method 1603
7.5.1 Composition of Stock Phosphate Buffer Solution:
Phosphate Dihydrogen Phosphate 34.0 g
Reagent-Grade Distilled Water 5 00.0 mL
Preparation: Adjust the pH of the solution to 7.2 with 1 N NaOH, and bring the volume to
1 L with reagent-grade distilled water. Sterilize by filtration or autoclave at 121°C (15 Ib
pressure) for 15 min.
7.5.2 Preparation of Stock Magnesium Chloride Solution: Add 38 g anhydrous MgCl2 or 81.1
g MgCl2*6H2O to 1 L reagent-grade distilled water. Sterilize by filtration or autoclave at
121°C (15 Ib pressure) for 15 min.
7.5.3 Storage of Stock Solutions: After sterilization, store the stock solutions in the refrigerator
until used. Handle aseptically. If evidence of mold or other contamination appears, the
affected stock solution should be discarded and a fresh solution should be prepared.
7.5.4 Working Phosphate Buffered Dilution Water: Mix 1.25 mL of the stock phosphate buffer
and 5 mL of the MgCl2 stock per liter of reagent-grade distilled water. Dispense in
appropriate amounts for dilutions in screwcap bottles or culture tubes, and/or into
containers for use as rinse water. Autoclave at 121°C (15 Ib pressure) for 15 min. Final
pH should be 7.0 ± 0.2.
7.6 Modified mTEC Agar
7.6.1 Composition:
Protease Peptone #3 5.0 g
Yeast Extract 3.0g
Lactose lO.Og
NaCl 7.5 g
Dipotassium Phosphate 3.3g
Monopotassium Phosphate 1.0 g
Sodium Lauryl Sulfate 0.2 g
Sodium Desoxycholate 0.1 g
Chromogen
(5 -bromo-6-chloro-3 -indolyl-
• 'D-glucuronide) 0.5 g
Agar 15. Og
Reagent-Grade Distilled Water 1.0 L
7.6.2 Preparation: Add dry ingredients to 1 L of reagent-grade distilled water in a flask, and
heat to boiling until the ingredients dissolve. Autoclave at 121°C (15 Ib pressure) for 15
min, and cool in a 50°C waterbath. Pour the medium into each 9x50 mm culture dish to
a 4-5 mm depth (approximately 4-6 mL), and allow to solidify. Final pH should be 7.3 ±
0.2. Store in a refrigerator.
7.7 Nutrient Agar
7.7.1 Composition:
Peptone 5.0g
Beef Extract 3.0g
Agar 15.Og
Reagent-Grade Distilled Water 1.0 L
September 2002
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Method 1603
7.7.2 Preparation: Add dry ingredients to 1 L of reagent-grade distilled water, and mix well.
Heat to boiling to dissolve the agar completely. Dispense in screwcap tubes, and
autoclave at 121°C (15 Ib pressure) for 15 min. Remove the tubes and slant. Final pH
should be 6.8 ± 0.2.
7.8 Tryptic Soy Broth; Trypticase Soy Broth
7.8.1 Composition:
Tryptone or Trypticase 17.0 g
Soytone or Phytone 3.0 g
Sodium Chloride 5.0 g
Dextrose 2.5 g
Dipotassium Phosphate 2.5 g
Reagent-Grade Distilled Water 1.0 L
7.8.2 Preparation: Add dry ingredients to 1 L of reagent-grade distilled water. Warm the broth,
and mix gently to dissolve the medium completely. Dispense in screw-cap tubes, and
autoclave at 121°C (15 Ib pressure) for 15 min. Final pH should be 7.3 ± 0.2.
7.9 Simmons Citrate Agar
7.9.1 Composition:
Magnesium Sulfate 0.2 g
Monoammonium Phosphate 1.0 g
Dipotassium Phosphate 1.0 g
Sodium Citrate 2.0 g
Sodium Chloride 5.0 g
Brom Thymol Blue 0.08 g
Agar 15.0 g
Reagent-Grade Distilled Water 1.0 L
7.9.2 Preparation: Add dry ingredients to 1 L of reagent-grade distilled water. Heat to boiling
to dissolve completely. Dispense into screwcap tubes, and autoclave at 121°C (15 Ib
pressure) for 15 min. Cool the tubes and slant. Final pH should be 6.8 ± 0.2.
7.10 Tryptone 1%; Tryptophane Broth
7.10.1 Composition:
Tryptone or Trypticase Peptone 10.0 g
Reagent-Grade Distilled Water 1.0 L
7.10.2 Preparation: Add the tryptone or trypticase peptone to 1 L or reagent-grade distilled
water, and heat, mixing until dissolved. Dispense in 5-mL volumes into tubes, and
autoclave 121°C (15 Ib pressure) for 15 min. Final pH should be 7.2 ± 0.2.
7.11 EC Broth
7.11.1 Composition:
Tryptose or Trypticase Peptone 20.0 g
Lactose 5.0g
Bile Salts no. 3 or Bile Salts Mixture 1.5 g
Dipotassium Phosphate 4.0 g
Monopotassium Phosphate 1.5g
Sodium Chloride 5.0 g
Reagent-Grade Distilled Water 1.0 L
5 September 2002
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Method 1603
7.11.2 Preparation: Add dry ingredients to 1 L of reagent-grade distilled water, and warm to
dissolve completely. Dispense into fermentation tubes (20 x 150 mm tubes containing
inverted 10 x 75 mm vials). Autoclave at 121°C (15 Ib pressure) for 15 min. Final pH
should be 6.9 ± 0.2.
7.12 Oxidase Reagent
7.12.1 Composition:
N, N, N', N'-tetramethyl-« 'phenylenediamine dihydrochloride, 1% aqueous
solution (1 g per 100 mL sterile reagent-grade distilled water).
7.13 Kovacs Indole Reagent
7.13.1 Composition:
• 'dimethylaminobenzaldehyde 10.0 g
Amy 1 or Isoamyl Alcohol 150.0mL
Concentrated (12 M) Hydrochloric Acid 50.0 mL
7.13.2 Preparation: Dissolve • •dimethylaminobenzaldehyde in alcohol, slowly add hydrochloric
acid, and mix.
8.0 Sample Collection, Preservation, and Storage
8.1 Sampling procedures are described in detail in the USEPA microbiology methods manual,
Section II, A (Reference 18.2). Adherence to sample preservation procedures and holding time
limits is critical to the production of valid data. Samples not collected according to these rules
should not be analyzed.
8.1.1 Storage Temperature and Handling Conditions
Ice or refrigerate water samples at a temperature of 1-4°C during transit to the laboratory.
Use insulated containers to assure proper maintenance of storage temperature. Take care
that sample bottles are not totally immersed in water during transit or storage.
8.1.2 Holding Time Limitations
Examine samples as soon as possible after collection. Do not hold samples longer than
6 h between collection and initiation of analyses.
9.0 Quality Control
9.1 See recommendations on quality control for microbiological analyses in the USEPA
microbiology methods manual, Part IV, C (Reference 18.2).
10.0 Calibration and Standardization
10.1 Check temperatures in incubators daily to ensure operation within stated limits.
10.2 Check thermometers at least annually against a NIST certified thermometer or one that meets the
requirements of NIST Monograph SP 250-23. Check mercury columns for breaks.
11.0 Procedure
11.1 Prepare the modified mTEC Agar as directed in section 7.6.
September 2002 6
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Method 1603
11.2 Mark the petri dish and report form with the sample identification and volume.
11.3 Place a sterile membrane filter on the filter base, grid side up, and attach the funnel to the base so
that the membrane filter is held between the funnel and the base.
11.4 Shake the sample bottle vigorously at least 25 times to distribute the bacteria uniformly, and
measure the desired volume of sample or dilution into the funnel.
11.5 Select sample volumes based on previous knowledge of the pollution level, to produce 20-80 E.
coll colonies on the membranes. Sample volumes of 1-100 mL are normally tested at half-log
intervals (e.g., 100, 30, 10, 3 mL).
11.6 Smaller sample sizes or sample dilutions can be used to minimize the interference of turbidity or
for high bacterial densities. Multiple volumes of the same sample or sample dilutions may be
filtered, and the results may be combined.
11.7 Filter the sample, and rinse the sides of the funnel at least twice with 20-30 mL of sterile buffered
rinse water. Turn off the vacuum, and remove the funnel from the filter base.
11.8 Use sterile forceps to aseptically remove the membrane filter from the filter base, and roll it onto
the modified mTEC Agar to avoid the formation of bubbles between the membrane and the agar
surface. Reseat the membrane if bubbles occur. Run the forceps around the edge of the filter to
be sure that the filter is properly seated on the agar. Close the dish, invert, and incubate 35 ±
0.5°Cfor2h.
11.9 After a 2 h incubation at 35 ± 0.5°C, transfer the plate to a Whirl-Pak® bag, seal the bag, place
the bag with the plate inverted in a test-tube rack, and put the rack in a 44.5 ± 0.2°C waterbath for
22-24 h.
11.10 After 22-24 h, remove the plate from the waterbath, count and record the number of red or
magenta colonies with the aid of an illuminated lens with a 2-5x magnification or a stereoscopic
microscope (See Photo 1).
12.0 Data Analysis and Calculations
Use the following general rules to calculate the E. coll count per 100 mL of sample:
12.1 Select the membrane filter with an acceptable number of magenta or red colonies (20-80), and
calculate the number of E. coll per 100 mL according to the following general formula:
Number of E. coll colonies
E. co///100mL= X 100
Volume of sample filtered (mL)
12.2 See the USEPA microbiology methods manual, Part II, Section C, 3.5, for general counting rules
(Reference 18.2).
12.3 Reporting Results
12.3.1 There should be at least three volumes filtered per sample. Report the results as E. coll
per 100 mL of sample.
13.0 Method Performance
13.1 The false-positive and false-negative rates, reported for various environmental water samples,
were <1% and 4%, respectively.
14.0 Reporting Results
7 September 2002
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Method 1603
14.1 There should be at least three volumes filtered per sample. Report the results as E. coll per 100
mL of sample.
15.0 Verification Procedure
15.1 Red or magenta colonies can be verified as E. coll. Verification of colonies may be required in
evidence gathering and is also recommended as a means of quality control for the initial use of
the test and for changes in sample sites, lots of commercial media, or major ingredients in media
compounded in the laboratory. The verification procedure follows.
Photo 1.
Escherichia coll colonies on modified mTEC agar. E. coll colonies are red to magenta.
15.2 Using a sterile inoculation loop, transfer growth from the centers of at least 10 well-isolated
colonies to Nutrient Agar plates or slants and to Trypticase Soy Broth. Incubate the agar and
broth cultures for 24 h at 35 ± 0.5°C.
15.3 After incubation, remove a loopful of growth from the Nutrient Agar slant with a platinum loop,
and deposit it on the surface of a piece of filter paper that has been saturated with freshly prepared
Cytochrome Oxidase Reagent. If the spot where the bacteria were deposited turns deep purple
within 15 seconds, the test is positive.
15.4 Transfer growth from the Trypticase Soy Broth tube to Simmons Citrate Agar, Tryptone Broth,
and an EC Broth fermentation tube.
15.4.1 Incubate the Simmons Citrate Agar and Tryptone Broth for 48 h at 35 ± 0.5°C.
15.4.2 Incubate the EC Broth at 44.5 ± 0.2°C in a waterbath for 24 h. The water level must be
above the level of the EC Broth in the tube.
15.4.3 Add 0.5 mL of Kovacs Indole Reagent to the 48 h Tryptone Broth culture, and shake the
tube gently. A positive test for indole is indicated by a deep red color which develops in
the alcohol layer on top of the broth.
15.4.4 E. coll is EC gas-positive, indole-positive, and oxidase-negative, and does not utilize
citrate (i.e., the medium remains green).
15.5 Alternately, commercially available multi-test identification systems may be used to verify
colonies. Inoculate the colonies into an identification system for Enterobacteriaceae that
includes lactose fermentation, • 'nitrophenyl-* «D-galactopyranoside (ONPG), and cytochrome
oxidase test reactions.
September 2002
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Method 1603
16.0 Pollution Prevention
16.1 The solutions and reagents used in this method pose little threat to the environment when
recycled and managed properly.
16.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials to be disposed.
17.0 Waste Management
17.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations
governing waste management, particularly the biohazard and hazardous waste identification rules
and land disposal restrictions, and to protect the air, water, and land by minimizing and
controlling all releases from fume hoods and bench operations. Compliance with all sewage
discharge permits and regulations is also required.
17.2 Samples, reference materials, and equipment known or suspected to have viable E. coll attached
or contained must be sterilized prior to disposal.
17.3 Samples preserved with HC1 to pH <2 are hazardous and must be neutralized before being
disposed, or must be handled as hazardous waste.
17.4 For further information on waste management, consult "The Waste Management Manual for
Laboratory Personnel" and "Less Is Better: Laboratory Chemical Management for Waste
Reduction," both available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.
18.0 References
18.1 Annual Book of ASTM Standards, Vol. 11.01, American Society for Testing and Materials,
Philadelphia, PA 19103.
18.2 BordnerR., J.A. Winter, P.V. Scarpino (eds.). 1978. Microbiological methods for Monitoring
the Environment: Water and Wastes, EPA-600/8-78-017. Office of Research and Development,
USEPA.
18.3 Cabelli, V.J., A.P. Dufour, M.A. Levin, L.J. McCabe, and P.W. Haberman. 1979. Relationship of
Microbial Indicators to Health Effects at Marine Bathing Beaches. Am. J. Public Health. 69:
690-696.
18.4 Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and
Escherichia coll. 2000. EPA/82 l/R-97/004. Office of Science and Technology, Washington D.C.
18.5 Reagent Chemicals, American Chemical Society Specifications, American Chemical Society,
Washington, DC. For suggestions of the testing of reagents not listed by the American Chemical
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, UK and the
United States Pharmacopeia.
18.6 Test methods for Escherichia coll and Enterococci in Water by the Membrane Filter Procedure.
1985. EPA-600/4-85/076. Environmental Monitoring and Support Laboratory, Cincinnati,
USEPA.
September 2002
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