United Stales Environmental Monitoring jn«j
Environmental Protection Support Uiboraiory
Agency Cincinnati OH 45268
R«so«rcn and Development EPA-600'4 -85/076
A__m TEST METHODS FOR ESCHERICHIA COLI AND ENTEROCOCCI
&EPA
IN WATER BY THE MEMBRANE FILTER PROCEDURE
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— - DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade names
or commercial products does not constitute endorsement or recommendation for
use. !
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FOREWORD
Environmental measurements are required to determine the quality of
ambient waters and the character of wastewater effluents. The Environmental
Monitoring and Support Laboratory - Cincinnati (EMSL-Cincinnati) conducts
research to:
Develop and evaluate methods to measure the presence and
concentration of physical, chemical, and radiological pollutants in
water, wastewater, bottom sediments, and solid waste.
Investigate methods for the concentration, recovery, and identifi-
cation of viruses, bacteria, and other microbiological organisms in
water; and to determine the responses of aquatic organisms to water
quality.
Develop and operate an Agency-wide quality assurance program to
assure standardization and quality control of systems for monitoring
water and wastewater.
Develop and operate a computerized system for instrument automation
leading to improved data collection, analysis, and quality control.
The methods described in this report can be used to measure the
bacteriological quality of recreational, shellfish growing, ambient, and
potable waters. A direct relationship between the density of enterococci and
E. coll in water and the occurrence of swimming-associated gastroenteritis has
been established through epidemiological studies of marine and fresh water
bathing beaches. These studies have led to the development of criteria which
can be used to establish recreational water standards based on recognized
health effects-water quality relationships.
<^*-&&7
Robert L. Booth, Director '
Environmental Monitoring and Support
Laboratory - Cincinnati
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TABLE OF CONTENTS
Foreword
Acknowledgements
ESCHERICHIA COLI IN HATER BY THE MEMBRANE FILTER PROCEDURE
i
Section |
i
1 Citation
2 Scope and Application
3 Summary
4 Definition
5 Interferences
6 Safety Procedures
7 Apparatus and Equipment
8 Reagents and Materials !
9 Sample Collection, Preservation, and Holding Times
10 Calibration and Standardization
11 Quality Control
12 Procedures
13 Calculation of Results
14 Reporting Results
15 Verification Procedure
16 Precision and Bias
Page
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iii
1
1
1
1
2
2
2
4
7
7
7
7
8
9
9
9
ENTERXOCCI IN WATER BY THE MEMBRANE FILTER PROCEDURE
Section
1 Citation
2 Scope and Application
3 Summary
4 Definition
5 Interferences
6 Safety Precautions
7 Apparatus and Equipment
8 Reagents and Materials
9 Sample Collection, Preservation, and Holding Times
10 Calibration and Standardization
11 Quality Control
12 Procedure
13 Calculation of Results
14 Reporting Results
15 Verification Procedure
16 Precision and Bias.
13
13
13
14
14
14
14
15
18
18
19
19
20
20
20
21
iv
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FIGURES
PrhC1^°nu!^1mates.for Escherlchfa coli in Hater
by the Membrane Filter Procedure
Precision Estimates for Enterococci in Water
by the Membrane Filter Procedure 23
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ACKNOWLEDGEMENTS
This is to acknowledge the major contributions of Alfred Diifour and
.Theodore Er+ck-sen of the Toxicology and Microbiology Division (TMD) Health
Effects Research Laboratory (HERL) in preparation of the original methods and
their assistance and that of Robert Bordner, Biological Methods Branch and
John Winter and Paul Britton, Quality Assurance Branch, Environmental Monitor-
Ing and Support Laboratory - Cincinnati (EMSL-Cincinnati), U.S. Environmental
Protection Agency (USEPA) 1n preparing the final protocol and in completing
the formal method validation stud;ies.
• i
The participation of the follbwing individuals in the interlaboratory test-
ing of these methods is also gratefully acknowledged:
Rebecca Calderon
Dept. of Epidemiology and
Public Health
School of Medicine
Yale University
New Haven, CT
I
Wendy Davis-Hoover
TMD, HERL
USEPA ;
Cincinnati, OH
Rodger Fukjioka
Hater Resources Research Center
University of Hawaii at Manoa
Honolulu, HA
John Haines
Biological Methods Branch
EMSL-Cincinnati
USEPA
Cincinnati OH
Linda Hopkins i
Technion International
Cincinnati, OH
Don Johnstone
Environmental Engineering Department
Washington State University
Pullman, WA
Vincent Olivieri
Dept. of Environmental Health
Johns Hopkins University
Baltimore, MD
Scott Ri ppey
Construction Battalion Center
U.S. Public Health Service
Department of Health and
Human Services
North Kingston, RI
William Randolph
Bionetic Corporation
Cincinnati, OH
George J. Vasconcelos
Region 10 Laboratory
USEPA
Manchester, WA
Larry Vlassoff
Laboratory Branch
Ontario Ministry of Environment
Rexdale, Ontario, Canada
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• TEST METHOD
ESCHERICHIA COLI IN WATER BY THE MEMBRANE FILTER PROCEDURE
~~ " • METHOD 1103.1
1985
1. Citation
, /
2. Scope and Application
• 2.1 This method describes a membrane filter (MF) procedure for the
detection and enumeration of Escherichia coll. Because the bacterium
is a natural inhabitant only or the intesTTnal tract of warm-blooded
animals, Us presence In water samples is an Indication of fecal
pollution and the possible presence of enteric pathogens.
2.2 The E. coll test Is used as a measure of recreational 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. coli 1n recreational water samples is
the direct relationship between the density of E. coll and the risk
of gastrointestinal Illness associated with swimming In the water (1).
2.3 The test for E. coll can be applied to fresh, estuarlne and marine
WauGPS*
2.4 Since a wide range of sample volumes or dilutions can be analyzed by
the MF technique, a wide range of E. coll levels 1n water can be
detected and enumerated. ~
3. Summary
3.1 The MF method provides a direct count of bacteria In water based on
the development of colonies on the surface of the membrane filter (2)
A water sample 1s filtered through the membrane which retains the
bacteria. After filtration, the membrane containing the bacterial
fel ! 1s ?lnce? on a Select1ve and differential medium, mTEC, incu-
bated at 35°C for 2 h to resuscitate injured or stressed bacteria
and then incubated at 44.5<>C for 22 h. Following incubation, the*
filter is transferred to a filter pad saturated with urea substrate.
After 15 min, yellow or yellow-brown colonies are counted with the
aid of a fluorescent lamp and a magnifying lens.
4. Definition
4.1 In this method, E. coll are those bacteria which produce yellow or
yellow-brown colonies on a filter pad saturated with urea substrate
broth after primary culturing on mTEC medium.
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5. Interferences
5.1 Water samples containing colloidal or suspended participate material
can clog the membrane filter and prevent filtration, or cause spread-
ingTof bacterial colonies which could interfere with identification
of target colonies.
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6. Safety Precautions
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6.1 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.
6.2 Mouth-pipetting is prohibited.
7. Apparatus and Equipment
i
7.1 Glass lens, 2-5X magnification, or stereoscopic microscope.
7.2 Lamp with cool, white fluorescent tube and diffuser.
.
7.3 Hand tally or electronic counting device.
7.4 Pipet container, stainless, steel, aluminum, or borosilicate glass,
for glass pi pets. i
7.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, of
appropriate volume.
7.6 Graduated cylinders, covered with aluminum foil or kraft paper and
sterile. i
7.7 Membrane filtration units j(filter base and funnel.), glass, plastic or
stainless steel, wrapped with aluminum foil or kraft paper and
sterile.
7.8 Ultraviolet unit for sterilizing the filter funnel between
filtrations (optional).
7.9 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 valvjs to prevent the return flow of air, can be
used.
i
7.10 Flask, filter, vacuum, usually 1 L, with appropriate tubing. A
filter manifold to hold a number of filter bases is optional.
i
7.11 Flask for safety trap, placed between the filter flask and the vacuum
source.
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7.12 Forceps, straight or curved, with smooth tips to handle filters
without damage.
7.13 EJttiano.1, methanol or isopropanol in a small, wide-mouth container,
for flame-sterilizing forceps.
7.14 Burner, Bunsen or Fisher type, or electric incinerator unit for
sterilizing inoculation loops.
7.15 Thermometer, checked against a National Bureau of Standards (NBS)
certified thermometer, or one traceable to an NBS thermometer.
7.16 Petri dishes, sterile, plastic, 50 x 12 mm, with tight-fitting lids,
or 60 x 15 mm, glass or plastic, with loose-fitting lids. 100 x 15
mm dishes may also be used.
7.17 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.
7.18 Flasks, borosilicate glass, screw-cap, 250-2000 ml volume.
7.19 Membrane filters, sterile, white, grid marked, 47 mm diameter, with
0.45 +. 0.02 ym pore size.
7.20 Absorbent pads, sterile, 47 mm diameter (usually supplied with
membrane filters).
7.21 Inocutation loops, at least 3 mm diameter, and needles, nichrome and
platinum wire, 26 B & S gauge, in suitable holders. Disposable
applicator sticks or plastic loops are alternatives to inoculation
loops. Note: A platinum loop is required for the cytochrome oxidase
test in 15.3.
7.22 Incubator maintained at 35 +_0.5°C, with approximately 90 percent
humidity if loose-lidded petri dishes are used.
7.23 Waterbath incubator maintained at 44.5 + 0.2°C.
7.24 Waterbath maintained at 44-46°C for tempering agar.
7.25 Test tubes, 150 x 20 mm, borosilicate glass or plastic.
7.26 Test tubes, 75 x 10 mm, borosilicate glass.
7.27 Test tube caps, aluminum or autoclavable plastic, for 20 mm
diameter test tubes.
7.28 Test tubes, screw-cap, 125 x 16 mm or other appropriate size.
7.29 Filter paper.
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8. Reagents and Materials
I
8.1 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 (3). The agar used in preparation of
culture media must be of microbiological grade.
8.2 Whenever possible, use commercial culture media as a means of quality
control. !
• 8.3 Purity of Water: Reagent! water conforming to Specification D1193,
Type II water, ASTM Annual Book of Standards (4).
8.4 Buffered Dilution Water
8.4.1 Composition:
Sodium Dihydrogen Phosphate 0.58 g
Sodium Monohydrogen Phosphate 2.50 g
Sodium Chloride 8.50 g
8.4.2 Preparation: Dissolve the ingredients in 1 L of reagent 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 after preparation at 121°C
(15 Ib pressure) for 15 min. Final pH should be 7.4 +^0.2
8.5. mTEC Agar (Difco 0334-15-0)
8.5.1 Composition:
Proteose Peptone 5.0 g
Yeast Extract 3.0 g
Lactose 10.0 g
NaCl | 7.5 g
Dipotassium Phosphate 3.3 g
Honopotassium Phosphate 1.0 g
Sodium Lauryl Sulfate 0.2 g
Sodium Desoxycholate 0.1 g
Brom Cresol Purple 0.08 g
Brom Phenol Red 0.08 g
Agar ' 15.0 g
8.5.2 Preparation: Add 45.26 g of dehydrated mTEC medium to 1 L of
reagent water In a flask and heat to boiling, until Ingred-
ients dissolve. Autoclave at 121°C (15 Ib pressure) for 15
minutes and cool in a 44-46°C waterbath. Pour the medium
Into each 50 x 10 mm culture dish to a 4-5 mm depth (approxi-
mately 4-6 mL) and allow to solidify. Final pH should be 7.3
+ 0.2. Store in airefrigerator.
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8.6 Urea Substrate Medium
8.6.1 Composition:
Urea 2.0 g
Phenol red 0.01 g
8.6.2 Preparation: Add dry ingredients to 100 ml reagent water in a
flaX'ur?t1rT?) d1"olve and adjust to PH 5.0 with a few drops
of IN HC1. The substrate solution should be a straw yellow
color at pH 5.0.
8.7 Nutrient Agar (D1fco 0001-02, BBL 11471)
8.7.1 Composition:
Peptone 5.0 g
Beef Extract 3.0 g
Agar 15.0 g
8.7.2 Preparation: Add 23 g of dehydrated nutrient agar to
1 L of reagent water and mix well. Heat in a boiling
waterbath to dissolve the agar completely. Dispense in
screw-cap tubes and autoclave at 121°C (15 Ib pressure) for
15 min. Remove tubes and slant. The final pH should be 6.8 jf
U • fa • "~*
8.8 Tryptic Soy Broth (Difco 0370-02 Trypticase Soy Broth (BBL 11767)
8.8.1 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
8.8.2 Preparation: Add 30 g of dehydrated tryptic soy broth to 1 L
of reagent water. Warm the broth and mix gently to dissolve
the medium completely. Dispense 1n screw-cap tubes and
autoclave at 121°c (15 Ib pressure) for 15 min. The final
pH should be 7.3 + 0.2.
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8.9 Simmons' Citrate Agar (BBL 11619, Difco 0091-02)
8.9.1 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
8.9.2 Preparation: Add 24.28 g of Simmons' citrate agar to 1 L of
reagent water. Heat in boiling waterbath with mixing for
complete solution. Dispense in screw-cap tubes and sterilize
at 121°C (15 Ib pressure) for 15 min. Cool tubes and
slant. The final pH should be 6.8 +_ 0.2.
8.10 Tryptone IX (Difco 0123-02) Tryptophane Broth (BBL 11920)
8.10.1 Composition:
Tryptone or Trypticase Peptone 10.0 g
Reagent Water 1 L
8.10.2 Preparation: Add ilO g of tryptone or trypticase peptone to
1 L of reagent water and heat with mixing until dissolved.
Dispense in five ml. volumes in tubes and autoclave at 121°C
(15 Ibs. pressure)! for 15 min. The final pH should be 7.2 +
0.2.
8.11 EC Broth (Difco 0314-02) EC Broth {BBL 11187)
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8.11.1 Composition:
Tryptose or Trypticase
Peptone 20.0 g
Lactose 5.0 g
Bile Salts No. 3 or
Bile Salts Mixture 1.5 g
Dipotassium Phosphate 4.0 g
Monopotassiurn Phosphate l.S.g
Sodium Chloride 5.0 g
Final pH: 6.9 + 0.2
8.11.2 Preparation: Add 37 grams of dehydrated EC .radium to 1 L of
reagent water and warm to dissolve completely. Dispense into
fermentation tubes
75 x 10 mm vials).
for 15 min. The final pH should be 6.9 +_ 0.2.
(150 x 20 mm tubes containing inverted
Sterilize at 121°C (15 Ib pressure)
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8.12 Cytdchrome Oxidase Reagent: N, N, N'f N1 tetramethyl-p-
pnenylenediamine dihydrochloride, IS aqueous solution.
8.13 Kovac's Indole Reagent: Dissolve 10 g p-dimethylaminobenzal-
dehyde in 150 mL amyl or isoamyl alcohol and then slowly add
Strut- concentrated hydrochloric acid and mix.
9- Sample Collection. Preservation and Holding Times
9.1 Sampling procedures are described in detail in the USEPA micro-
biology methods manual, Section II, A (5). 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. a
9.1.1 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.
9.1.2 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.
10. Calibration and Standardization
10.1 Check temperatures in incubators daily to insure operation within
stated limits.
10.2 Check thermometers at least annually against an .NBS certified
thermometer or one traceable to NBS. Check mercury columns for
11. Quality Control
11.1 See recommendations on quality control for microbiological analyses
in the USEPA microbiology methods manual, Part IV, C (5).
12. Procedure
12.1 Prepare the mTEC agar and urea substrate as directed in 8.5 and 8.6.
12.2 Mark the petri dishes and report forms with sample identification
and sample volumes.
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12.3 Place a sterile membrane filter on the filter base, grid-side up and
attach the funnel to the base; the membrane filter is now held
between the funnel and the base.
12.4 Shake_the sample bottle vigorously about 25 times to distribute the
bacteria uniformly and measure the desired volume of sample or
dilution into the funnel.
12.5 For ambient surface waters and wastewaters, select sample volumes
based on previous knowledge of pollution level, to produce 20-80
E. coll colonies on the membranes. Sample volumes of 1-100 nt are
normally tested at half-log intervals, for example 100, 30, 10, 3
ml, etc.
12.6 Smaller sample size or sample dilutions can be used to minimize the
interference of turbidity or high bacterial densities. Multiple
volumes of the same sample or sample dilution may be filtered and
the results combined. i
12.7 Filter the sample and rinse the sides of the funnel at least twice
with 20-30 ml of sterilej buffered rinse water. Turn off the vacuum
and remove the funnel from the filter base.
i
12.8 Use sterile forceps to aseptically remove the membrane filter from
the filter base and roll! it onto the mTEC agar to avoid the forma-
tion of bubbles between the membrane and the agar surface. Reseat
the membrane if bubb.les occur. Close the dish, invert, and incubate
at 35°C for 2 h. , .
12.9 After two hours incubatipn at 35°C, transfer the plates to
Whirl-Pak bags, seal, and place inverted in a 44.5°C waterbath for
22-24 h.
12.10 After 22-24 h, remove the dishes from the waterbath. Place
absorbent pads in new petri dishes or the lids of the same petri
dishes, and saturate with urea broth. Aseptically transfer the
membranes to absorbent pads saturated with urea substrate and hold
at room temperature.
12.11 After 15-20 min incubation on the urea substrate at room temper-
ature, count and record the number of yellow or yellow-brown
colonies on those membrane filters ideally containing 20-80 colonies.
13. Calculation of Results
13.1 Select the membrane filter with the number of colonies within
the acceptable range (20-80) and calculate the count per 100 ml
according to the general! formula:
E. col 1/100 ml 3 No. E. coll colonies counted x iQO ml
Volume In mL of sample filtered
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13.2 See general counting rules in the USEPA microbiology methods manual
Part II, C, 3.5 (5).
14. ReportTng Results
14.1 Report the results as E. coli per 100 ml of sample.
15. Verification Procedure
15.1 Yellow or yellow-brown colonies from the urease test can be verified
as E. coli. Verification of colonies may be required in evidence
gathering, and is also recommended as a QC procedure with initial use
of the test and with changes in sample sites, lots of commercial
media or major ingredients in media compounded in the laboratory.
The verification procedure follows:
15.1.1 Using a sterile inoculation loop, transfer growth from the
centers of at least 10 well-isolated typical colonies to
nutrient agar plates or slants and to trypticase soy broth.
Incubate the agar and broth cultures for 24 h at 35°C.
15.1.2 After incubation remove a generous portion of material from
the nutrient agar with a platinum loop and deposit on the
surface of filter paper that has been saturated with cyto-
chrome oxidase reagent prepared fresh that day. A positive
test is indicated within 15 s by the development of a deep
purple color where the bacteria were deposited.
15.1.3 Transfer growth from the trypticase soy broth to Simmons'
citrate agar, tryptone broth, and EC broth in a fermentation
tube. Incubate the Simmons' citrate agar for 24 h and
tryptone broth for 48 h at 35°C. Incubate the EC broth at
44.5°C in a waterbath for 24 h. The water level must be
above the level of the EC broth in the tube. Add one-half ml
of Kovac's 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. E. coli is EC gas positive, indole positive, oxidase
negative, and "does not grow on citrate medium.
15.1.4 Alternatively, commercially available multi-test identi-
identification systems may be used to verify colonies.
Inoculate the colonies into an Identification system for
Enterobacteriaceae that includes lactose fermentation and/or
0-n1trophenyl» -D-galactopyranoside (ONPG) and cytochrome
oxidase test reactions.
16. Precision and Bias
16.1 Performance Characteristics
16.1.1 Precision - The degree of agreement of repeated measurements
of the same parameter expressed quantitatively as the standard
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deviation or as the 95% confidence limits of the mean computed
from the results of a series of controlled determinations.
The mTEC method precision was found to be fairly representa-
— - — tive of what would be expected from counts with a Poisson
distribution (2).
16.1.2 Bias - The persistent positive or negative deviation of the
average value of the method from the assumed or accepted true
value. The bias of the mTEC method has been reported to be
-2% of the true vallue (2).
/ '•
16.1.3 Specificity - The ability of a method to select and or distin-
guish the target bacteria under test from other bacteria in
the same water sample. The specificity characteristic of a
method is usually reported as the percent of false positive
and false negative results. The false positive rate reported
for mTEC medium averaged 9% for marine and fresh water sam-
ples. Less than 1% of the E. coll colonies observed gave a
false negative reaction (2).
16.1.4 Upper Counting Limit (UCL) - That colony count above which
there is an unacceptable counting error. The error may be due
to overcrowding or antibiosis. The UCL for E. coll on mTEC
medium has been reported as 80 colonies per filter (2).
16.2 Collaborative Study Data
16.2.1 A collaborative study was conducted among eleven volunteer
laboratories, each with two analysts who independently tested
local fresh and marine recreational waters and sewage treatment
plant effluent samples, in duplicate. The data were reported
to the Environmental Monitoring and Support Laboratory -
Cincinnati, U.S. Environmental Protection Agency, for statis-
tical calculations.
16.2.2 The results of the study are shown in Figure 1 where S0
equals the pooled standard deviation among replicate counts
from a single analyst for three groupings (counts less than
30, counts from 30 to 50, and counts greater than 50) and SB
equals the pooled standard deviation between means of dupli-
cates from analysts In the same laboratory for the same
groupings. The precision estimates from this study did not
show any difference among the water types analyzed.
16.2.3 By linear regression, the precision of the method can be
generalized as:
S0 * 0.028 count/100 mL + 6.11 (dilution factor) and
SB » 0.233 count/100 mL + 0.82 (dilution factor)
100
where dilution factor = volume Of original sample filtered
10
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16.2.4 Because of the instability of nricrobial populations in water
samples, each laboratory analyzed its own sample series and no
full measure of recovery or bias was possible. However, all
laboratories analyzed a single surrogate sample prepared from
a freeze-dried culture of E. coli. The mean count (x) and the
—- - overall standard deviation of the counts (Sj) (which includes
the variability among laboratories for this standardized E.
coli sample) were 31.6 colonies/membrane and 7.61 colonies/
membrane, respectively.
12
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References
V J . A. P. Dufour, M. A. Levin, L. J. McCabe, and P. W.
Hrman "Relationship of Microblal Indicators to Health Effects at
Hart nTSath?ng leases/ Amer. Jour. Public Health 69 . 690-696 ,1979.
2 Dufour, A., E. Strickland, and V. CabelH., "Membrane Filter Method
for Enumerating Escherichia coli." ADD!, and Environ. Mlcrobrol. 41 ,
1152-1158, 1981.
3 "P«an*r.+ Phgmicals.1' American Chemical Society Specifications, 6th
Edition Am? Chem. Soc.. Washington, b.U, 1*1. tor suggestions on
the teltln! of reagents not listed by the American Chemical Society,
see Reagent rLmicals and Standards, Joseph Rosin, D. Van Nostrand
rT Inc Princeton. U.J.. mi. and the United States Pharmacopeia,
Nineteenth EdiJion! United States Phamacopeial Convention, Inc.,
Rockville, Md., 1974.
4. Annual Book of ASTM Standards. Vol. 11.01 .Water, American Society for
Testing and Materials. Philadelphia. PA., 1985.
R J A. Winter and P. V. Scarpino (eds.), Microbiological
'torga the Environment. Water and Wastes
t U.s! knvironmental Protection Agency, Office of
---t .
Research and Development, Environmental Monitoring and Support
Laboratory - Cincinnati, Cincinnati, Ohio, 1978.
13
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TEST METHOD
ENTEROCOCCI IN WATER BY THE MEMBRANE FILTER PROCEDURE
METHOD 1106.1
1985
1. Citation
2. Scope and Application
i
I
2.1 This method describes a membrane filter (MF) procedure for the
detection and enumeration of the enterococci bacteria in water. The
enterococci are commonly found in the feces of humans and other
warm-blooded animals. Although some strains are ubiquitous and not
related to fecal pollution, the presence of enterococci in water is
an indication of fecal pollution and the possible presence of enteric
pathogens.
2.2 'The enterococci test measures the bacteriological quality of recre-
ational waters. Epidemiological studies have led to the development
of criteria which can be used to promulgate recreational water
standards based on the established relationship between health
effects and water quality. The signficance of finding enterococci in
recreational water samples is the direct relationship between the
density of enterococci in the water and swimming-associated gastro-
enteritis studies of marine and fresh water bathing beaches (1).
2.3 The test for enterococci can be applied to potable, fresh, estuarine,
marine, and shellfish growing waters.
2.4 Since a wide range of sample volumes or dilutions can be analyzed by
the MF technique, a wide;range of enterococci levels in water can be
detected and enumerated.
3. Summary
3.1 The MF method provides a;direct count of bacteria in water based on
the development of colonies on the surface of the membrane filter (2).
A water sample is filtered through the membrane which retains the
bacteria. Following filtration, the membrane containing the bacter-
ial cells is placed on a selective medium, n£ agar, and incubated for
48 h at 41°C. After Incubation, the filter is transferred to EIA
agar and held at 41°C for 20 min. Pink to red enterococci colonies
will develop a black or reddish-brown precipitate on the underside of
the filter. These colonies are counted with a fluorescent lamp and a
magnifying lens.
4. Definition
4.1 In this method, enterococci are those bacteria which produce pink to
red colonies with black 6r reddish-brown precipitate after Incubation
14
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on n£ agar and subsequent transfer to EIA medium. Enterococci
include Streptococcus faecal is. Streptococcus faecium. Streptococcus
avlum. and their varTants. v Coccus
5. Interferences
5.1 Water samples containing colloidal or suspended participate materials
can clog the membrane filter and prevent filtration, or cause spread-
Ing of bacterial colonies which could interfere with Identification
of target colonies.
6. Safety Precautions
6,1 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.
6.2 Mouth-pipetting is prohibited.
7. Apparatus and Equipment
7.1 Glass lens with magnification of 2-5X or stereoscopic microscope,
7.2 Lamp, with a cool, white fluorescent tube.
7.3 Hand tally or electronic counting device.
7.4 Pipet container, stainless steel, aluminum or borosilicate
glass, for glass pi pets.
7.5 Pipets, sterile, T.O. bacteriological or Mohr, glass or plastic, of
appropriate volume.
7.6 Graduated cylinders, 100-1000 ml, covered with aluminum foil or kraft
paper and sterile.
7.7 Membrane filtration units (filter base and funnel), glass, plastic or
stainless steel, wrapped with aluminum foil or kraft paper and
sterile.
7.8 Ultraviolet unit for sterilizing the filter funnel between
fUtrations (optional).
7.9 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.
7.10 Flask, filter, vacuum, usually 1 L, with appropriate tubing.
A filter manifold to hold a number of filter bases Is optional.
15
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7.11 Flask for safety trap placed between the filter flask and the vacuum
source.
7.12 Forceps, straight or curved, with smooth tips to handle filters
without damage.
7.13 Ethanol, methanol or isoprppanol in a small, wide-mouth container,
for flame-sterilizing forceps.
7.14 Burner, Bunsen or Fisher type, or electric incinerator unit for
sterilizing loops and needles.
7.15 Thermometer, checked againjst a National Bureau of Standards (NBS)
certified thermometer, or pne traceable to an NBS thermometer.
7.16 Petrl dishes, sterile, plastic, 50 x 12 mm, with tight-fitting lids.
7.17 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.
7.18 Flasks, borosilicate glass, screw-cap, 250-2000 ml volume.
I
/.19 Membrane filters, sterile,! white, grid marked, 47 mm diameter, with
0.45 +_ 0.02 urn pore size.
7.20 Inoculation loops, at least 3-mm diameter, and needles, nichrome or
platinum wire, 26 B & S gaiige, in suitable holders.
i
7.21 Incubator maintained at 41J+ 0.5°C.
7.22 Waterbath maintained at 44^46°C for tempering agar.
i
7.23 Test tubes, 150 x 20 mm, bbrosilicate glass or plastic.
7.24 Caps, aluminum or autoclav|able plastic, for 20 mm diameter
test tubes.
7.25 Test tubes, screw-cap, borosilicate glass, 125 x 16 ram or other
appropriate size.
8. Reagents and Materials |
8.1 Purity of Reagents: Reagent grade chemicals shall be used 1n all
tests. Unless otherwise indicated, reagents shall conform to the
specifications of the Committee on Analytical Reagents of the
American Chemical Society (3). The agar used 1n preparation of
culture media must be of microbiological grade.
i
8.2 Whenever possible, use commercial culture media as a means of quality
control.
16
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8.3 Purity of Water: Reagent water conforming to Specification D1193,
Type II, Annual Book of ASTM Standards (4).
8.4 Btrffered Dilution Water
8.4.1 Composition:
Sodium Dihydrogen Phosphate 0.58 g
Sodium Monohydrogen Phosphate 2.50 g
Sodium Chloride 8.50 g
8.4.2 Preparation: Dissolve the Ingredients in 1 L of reagent 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 after preparation at
121°C {15 Ib pressure) for 15 min. The final pH should be
7.4 +0.2.
8,5 n£ Agar (Difco 0333-15-1)
8.5.1 Composition of Basal Medium:
Peptone 10.0 g
Sodium Chloride 15.0 g
Yeast Extract 30.0 g
Esculin 1.0 g
Actidione 0.05 g
Sodium Azide 0.15 g
Agar 15.0 g
8.5.2 Preparation of basal medium: Add 71.2 g of dehydrated mE
basal medium to 1 L of reagent grade water in a flask and heat
to boiling until ingredients dissolve. Autoclave at 121°C
and 15 Ib pressure for 15 min and cool in a 44-46°C water
bath.
8.5.3 Reagents added after sterilization: Mix 0.25 g nalidixic acid
in 5 ml reagent grade water, add 0.2 ml of 10 N NaOH to
dissolve, and add to the basal medium. Add 0.15 g triphenyl
tetrazolium chloride separately to the basal medium and mix.
8.5.4 Preparation of mE Agar: Pour the mE agar into 50 mm petri
dishes to a 4-5 mm depth (approximately 4-6 ml), and allow to
solidify. The final pH of medium should be 7.1 + 0.2. Store
in a refrigerator.
17
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8.6 EIA Substrate Agar (Difcot 0488-15-4)
8.6.1 Composition:
— - Esculin
Ferric Citrate:
Agar
1.0 g
0.5 g
15.0 g
Preparation: Add 16.5 g of dehydrated EIA medium to 1 L of
reagent grade water in a flask and heat to boiling until
ingredients are dissolved. Autoclave the EIA medium at
121°C and 15 Ib pressure for 15 min and cool 1n a 44-46°C
water bath. After[cooling, pour the medium into 50-mm petri
dishes to a depth of 4-5 mm (approximately 4-6 ml) and allow
to solidify. The final pH should be 7.1 j^ 0.2 before
autoclaving. Store 1n a refrigerator.
i
8.7 Brain Heart Infusion (BHI) (Difco 0037-02, BBL 11058)
8.7.1 Composition:
Infusion
Infusion
Calf Brain
Beef Heart
Peptone !
Sodium Chloride
Disodium Phosphate
Dextrose
200.0 g
250.0 g
10.0 g
5.0 g
2.5 g
2.0 g
8.7.2
Preparation: Dissolve 37 g of dehydrated brain heart infusion
in 1 L of reagent grade water. Dispense in 8-10 ml volumes in
screw-cap tubes and autoclave at 121°C (15 Ib pressure) for
15 min. If the medium is not used the same day as prepared
and sterilized, heat 1n boiling water bath for several min to
remove absorbed oxygen, and cool quickly without agitation,
just prior to inoculation. The final pH should be 7.4 ^0.2.
8.8 Brain Heart Infusion (BHI) Broth with 6.5% NaCl
8.8.1 Composition: Brain heart Infusion broth with 6.5% NaCl is
the same as BHI broth in 8.7 with additional NaCl.
\
8.8.2 Preparation: Add 60.0 g NaCl per liter of medium. Since most
commercially available dehydrated media contain sodium
chloride, this amount Is taken into consideration in
determining the final NaCl percentage above.
8.9 Brain Heart Infusion Agar! (Difco 0418-02, BBL 11064)
8.9.1 Composition: Braih heart infusion agar contains the same
components as BHI (see 8.7) with the addition of 15.0 g of
agar per L of BHI broth.
18
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8.9.2 Preparation: Heat to boiling until ingredients are dissolved
Dispense 10-12 ml of medium in screw-cap test tubes and steril
lize for 15 win at 121<>c {15 Ib pressure). Slant after
__ _ sterilization. The final pH should be 7.4 + 0.2.
8.10 Bile Esculln Agar (BEA) (Difco 0879)
8.10.1 Composition:
Bacto Beef Extract 3 o g
Bacto Peptone 5.0 g
Bacto Oxgall 40 0 g
Bacto Esculin 1.0 g
Ferric Citrate 0*5 g
Bacto Agar 15.*0 g
8.10.2 Preparation: Add 64.5 g of dehydrated BEA to 1 L reagent
oa?nrjnd ,heat to boni"9 to dissolve completely. Dispense in
8-10 ml volumes in tubes for slants or into flasks for
subsequent plating. Autoclave at 121°C at 15 Ib pressure
may cause dark*ning of the medium.
fni nu cdlspe!!se 1n*> sterile petri dishes. The
final pH should be 6.6 + 0.2. Store in a refrigerator.
Sample Collection. Preservation and Holding Times
9.1 Sampling procedures are described 1n detail in the USEPA micro-
dS ma!!Ual° Sect1on "• A (5)* Adherence to sample
Pro«dures and holding time limits Is critical to the
9.1.1 Storage Temperature and Handling Conditions
iIC!o^r/errl9erateJbacter1ol°91<:al samples at a temperature of
1-40C during transit to the laboratory. Use Insulated con-
tainers to assure proper maintenance of storage temperature.
Take care that sample bottles are not totally Immersed in
water during transit or storage.
9.1.2 Holding Time Limitations
/
Examine samples as soon as possible after collection. Do not
hold samples longer than 6 h between collection and initiation
or analyses.
Calibration and Standardization
1041 statedtl!SustUreS 10 1ncubators da11* to insure operation within
19
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10.2 Check thermometers at least annually against an NBS certified
thermometer or one traceable to NBS. Check mercury columns for
breaks.
,11. Quality_£Qntrol
11.1 See recommendations on quality control for microbiological analyses
1n the USEPA microbiology methods manual, Part IV, C (5).
12. Procedure
i
12.1 Prepare the n£ agar as directed in 8.5.
12.2 Mark the petri dishes and report forms with sample identification
and sample volumes. ,
12.3 Place a sterile membrane filter on the filter base, grid-side up and
attach the funnel to the base; the membrane filter is now held
between the funnel and the base.
12.4 Shake the sample bottle [vigorously about 25 times to distribute the
bacteria uniformly, and 'measure the desired volume of sample or
dilution into the funnel;.
12.5 For ambient surface waters and wastewaters, select sample volumes
based on previous knowledge of pollution level, to produce 20-60
enterococci colonies on 'membranes. Sample volumes of 1-100 ml are
normally tested at half ilbg intervals, for example 100, 30, 10, 3mL,
etc.
12.6 Smaller sample size or sample dilution can be used to minimize the
interference of turbidity or high bacterial densities. Multiple
volumes of the same sample or dilution of sample may be filtered and
the results combined.
12.7 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.
12.8 Use sterile forceps to aseptically remove the membrane filter from
the filter base and roll it onto the ME agar to avoid the formation
of bubbles between the membrane and the agar surface. Reseat the
membrane if bubbles occur. Close the dish, invert, and incubate at
41 i 0.5°C for 48 h.
12.9 After Incubation, transfer the membranes to EIA agar plates which
have been at room temperjature for 20-30 min, and incubate at 41 °C
for 20 min.
12.10 After incubation, count and record colonies on those membrane
filters containing, 1f practical, 20-60 pink-to-red colonies with
20
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F
black or reddish-brown precipitate on the underside of the
membrane. Use magnification for counting and a small fluorescent
lamp to give maximum visibility of colonies. "orescent
13. Calculation" of Results
inn I,he J0110*.1^ 9eneral rules to calculate the enterococci count per
luu mL of sample: •
i «wbrtne$ with Ideally 20-60 pink to red colonies
with black or reddish-brown precipitate on the underside. Calculate
the final value using the formula: ".m«e
Enterococci/100 ml = No. of enterococci colonies ylnn
Volume of sample filtered (ni)
13.2 See the USEPA microbiology manual, Part II, Section C, 3.5 for
general counting rules.5
14. Reporting Results
14.1 Report the results as enterococci per 100 nt of sample.
15. Verification Procedure
15.1 Pink to red colonies with black or reddish-brown precipitate after
incubation on EIA agar can be verified as enterococci. Verification
of colonies may be required in evidence gathering, and is also
recommended as a QC procedure upon initial use of the test and with
changes in sample sites, lots of commercial media, or major
ingredients in media compounded in the laboratory. The verification
procedure follows:
15.2 Using a sterile inoculating needle, transfer cells from the centers
if typical Colon1« Into a brain heart
infusion broth (BHI) tube and onto a BHI slant. Incubate broth
tubes for 24 h and slants for 48 h at 35 + 0.5°C.
15.3 After 24 h incubation, transfer a loopful of material from each BHI
broth tube to:
a. Bile Esculin Agar (BEA) and incubate at 35 + 0.5<>C
•"
b. BHI Broth and incubate at 45 + 0.50C for 48 h.
c. BHI Broth with 6.5% NaCl and Incubate at 35 + 0 5<>c
for 48 h. -
15.4 Observe for growth.
21
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15.5 After 48 h incubation, apply a gram stain to growth from each BHI
agar slant.
15.6 Gram positive cocci which grow in BEA, BHI Broth at 45°C, and BHI
•Broth + 6. 55 NaCl, and hydrolyze esculin, are verified as
enterococci .
16. Precision and Bias
16.1 Performance Characteristics
i
I
16.1.1 Precision - The degree of agreement of repeated measurements
of the same parameter expressed quantitatively as the standard
deviation or as the 95% confidence limits of the mean computed
from the results of a series of controlled determinations.
Precision of the m£ method was established by Levin et al . (2)
who indicated that the method did not exceed the expected
limits for counts having the Poisson distribution.
16.1
.2 Bias - The persistent positive or negative deviation of the
results from the assumed or accepted true value. The bias of
the enterococci MF method with n£ Agar has been reported to be
"" of the true value (2).
+2i
16.1.3 Specificity - The 'ability of a method to select and/or distin-
guish the target bacteria from other bacteria 1n the same
water sample. The specificity characteristic of a method is
usually reported as the percent of false positive and false
negative results. The specificity for this medium as reported
for various environmental water samples was 105 false positive
and 11.75 false negative (2).
16.2 Collaborative Study Data
16.2.1 A collaborative study was conducted among eleven volunteer
laboratories, each with two analysts who Independently tested
local fresh and marine recreational waters and sewage treatment
plant effluent samples, in duplicate. The data were reported
to the Environmental Monitoring and Support Laboratory -
Cincinnati, U.S. Environmental Protection Agency,, for
statistical analyses.
16.2.2 The results of the study are shown 1n Figure 1 where S0
equals the pooled standard deviation among replicate counts
from a single analyst for three groupings (counts less than
30, counts from 30 to 50, and counts greater than 50) and SB
equals the pooled standard deviation between means of
duplicates from analysts In the same laboratory for the same
groupings. The precision estimates from this study did not
differ with the water types tested.
22
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16.2.3 By linear regression, the precision of the method can be
generalized as:
S0 * 0.103 count/100 ml + 2.42 (dilution factor) and
SB = 0.152 count/100 ml + 5.16 (dilution factor)
where dilution factor - " 10°
volume of original samplt filtered
16.2.4 Because of the instability of microbial populations in water
samples, each laboratory analyzed its own sample series and no
full measure of recovery or bias was possible. However, all
laboratories analyzed a single surrogate sample prepared from
a freeze-dried culture of Streptococcus faecal is. The mean
count (x) and the standard deviation of the counts (Sj)
(including the variability among laboratories for this
standardized enterococci sample) were 32.5 colonies/ membrane
and 9.42 colonies/membrane, respectively.
23
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References
1. CabVm, V. J., A. P. Dufour, M. A. Levin, L. J. McCabe, and P. W.
Haberman, "Relationship of Microbial Indicators to Health Effects at
Marine Bathing Beaches," Amer. Jour. Public Health, 69, 690-696, 1979.
2. Levin, M. A., J. R. Fischer and V. J. Cabelli, "Membrane Filter
Technique for Enumeration of Enterococci in Marine Haters," Appl.
Microbiol. 30, 66-71, 1975. -CL-
3. "Reagent Chemicals," American Chemical Society Specifications 6th
Edition, Am. Chem. Soc., Washington, D.C., 1981. For suggestions of
the testing of reagents not listed by the American Chemical Society,
see Reagent Chemicals and Standards. Joseph Rosin, D. Van Nostrand,
Co., Inc., Princeton, N.J., 1967, and the United States Pharmacopeia.
Nineteenth Edition, United States Pharmacopeial Convention, Inc.,~
Rockville, MD, 1974.
4. Annual Book of ASTM Standards, Vol. 11.01, Water, American Society for
Testing and Materials, Philadelphia, PA., 1985.
5. Bordner, R., J. A. Winter and P. V. Scarpino (eds.), "Microbiological
Methods for Monitoring the Environment, Water and WastelT""
EPA-600/8-78-017, U.S. Environmental Protection Agency, Office of
Research and Development, Environmental Monitoring and Support
Laboratory - Cincinnati, Cincinnati, Ohio, 1978.
25
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