&EPA
United States
Environmental Protection
Agency
Method 1106.1: Enterococci in Water by
Membrane Filtration Using membrane-
Enterococcus-Esculin Iron Agar
(mE-EIA)
July 2006
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-06-008
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Acknowledgments
The following laboratories are gratefully acknowledged for their participation in the validation study for
this method in disinfected wastewater effluents which was conducted in 2004:
Volunteer Research Laboratories
• EPA Office of Research and Development, National Risk Management Research Lab: Mark C.
Meckes
• U.S. Army Corps of Engineers, Washington Aqueduct: Elizabeth A. Turner, Michael L. Chicoine,
and Lisa Neal
Volunteer Verification Laboratory
• Orange County Sanitation District, Environmental Sciences Laboratory: Charles McGee, Michael
von Winckelmann, Kim Patton, Linda Kirchner, James Campbell, Arturo Diaz, and Lisa McMath
Volunteer Participant Laboratories
• American Interplex: John Overbey, Steve Bradford, and Jessica Young
• County Sanitation Districts of Los Angeles County, Joint Water Pollution Control Plant (JWPCP):
Kathy Walker, Michele Padilla, and Albert Soof
• East Bay Municipal Utility District: Bill Ellgas and Daniel Mills
• Environmental Associates (EA): Susan Boutros and Madelyn Glase
• Hampton Roads Sanitation District (HRSD): Anna Rule, Paula Hogg, and Bob Maunz
• Hoosier Microbiological Laboratories (HML): Don Hendrickson, Katy Bilger, Keri Nixon, and
Lindsey Shelton
• Massachusetts Water Resources Authority (MWRA): Steve Rhode and Mariya Gofshteyn
• San Francisco Water: Phil Caskey, Paul McGregor, and Bonnie Bompart
• University of Iowa Hygienic Laboratory: Nancy Hall and Cathy Lord
• Wisconsin State Laboratory of Hygiene (WSLH): Sharon Kluender, Linda Peterson, and Jeremy
Olstadt
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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 CSC 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. 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 2
4.0 Interferences and Contamination 2
5.0 Safety 2
6.0 Equipment and Supplies 2
7.0 Reagents and Standards 4
8.0 Sample Collection, Preservation, and Storage 7
9.0 Quality Control 8
10.0 Calibration and Standardization 11
11.0 Procedure 11
12.0 Verification Procedure 13
13.0 Data Analysis and Calculations 14
14.0 Sample Spiking Procedure 14
15.0 Method Performance 19
16.0 Pollution Prevention 23
17.0 Waste Management 23
18.0 References 24
IV
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List of Appendices
Appendices A and B are taken from Microbiological Methods for Monitoring the Environment: Water
and Wastes (Reference 18.7).
Appendix A: Part II (General Operations), Section A (Sample Collection, Preservation, and Storage)
Appendix B: Part II (General Operations), Sections C.3.5 (Counting Colonies) and C.3.6
(Calculation of Results)
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Method 1106.1: Enterococci in Water by Membrane Filtration Using
membrane-Enterococcus-Esculin Iron Agar (mE-EIA)
July 2006
1.0 Scope and Application
1.1 Method 1106.1 describes a membrane filter (MF) procedure for the detection and enumeration of
the enterococci bacteria in ambient water. The enterococci bacteria 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.
1.2 The enterococci test is recommended as a measure of ambient fresh and marine 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 enterococci in recreational fresh or marine water
samples is the direct relationship between the density of enterococci and the risk of
gastrointestinal illness associated with swimming in the water (References 18.1 and 18.2).
1.3 For method application please refer to Title 40 Code of Federal Regulation Part 13 6 (40 CFR Part 136).
1.4 Method 1106.1 was submitted to interlaboratory validation in disinfected wastewater matrices.
Based on the 60.5% false negative confirmation rate for unspiked disinfected wastewater, an
estimated 14.1% of total colonies observed during the study would have been incorrectly
identified as non-enterococci colonies. As a result, Method 1106.1 is not approved for the
analysis of disinfected wastewater. A summary of false positive and negative results are provided
in Section 15.4.2. Detailed study results are provided in the validation study report (Reference
18.3). Laboratories wishing to test for enterococci in wastewater using a membrane filtration
method are referred to EPA Method 1600.
2.0 Summary of Method
2.1 Method 1106.1 provides a direct count of bacteria in water based on the development of colonies
on the surface of the membrane filter (Reference 18.4). A water sample is filtered through the
membrane which retains the bacteria. Following filtration, the membrane is placed on a selective
medium, mE agar, and incubated at 41°C ± 0.5°C for 48 ± 3 hours. The filter is then transferred
to a differential medium, EIA, and incubated at 41°C ± 0.5°C for 20-30 minutes. Pink to red
enterococci colonies on mE will develop a black or reddish-brown precipitate on the underside of
the filter after transfer to EIA. A fluorescent lamp with a magnifying lens is used for counting to
give maximum visibility of colonies.
July 2006
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Method 1106.1
3.0 Definitions
3.1 In Method 1106.1, enterococci are those bacteria which produce pink to red colonies after
incubation on mE agar and that form a black or reddish-brown precipitate after subsequent
transfer to EIA medium. Enterococci include Enterococcus faecalis (E. faecalis), E. faecium, E.
avium, E. gallinarium and their variants. The genus Enterococcus includes the enterococci
formerly assigned to the Group D fecal streptococci.
4.0 Interferences and Contamination
4.1 Water samples containing colloidal or suspended particulate materials can clog the membrane
filter and prevent filtration, or cause spreading of bacterial colonies which could interfere with
enumeration and identification of target colonies.
5.0 Safety
5.1 The analyst 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 The selective medium (mE) and azide-dextrose broth used in this method contain sodium azide as
well as other potentially toxic components. Caution must be exercised during the preparation,
use, and disposal of these media to prevent inhalation or contact with the medium or reagents.
5.3 This method does not address all of the safety issues associated with its use. It is the
responsibility of the laboratory to establish appropriate safety and health practices prior to use of
this method. A reference file of material safety data sheets (MSDSs) should be available to all
personnel involved in Method 1106.1 analyses.
5.4 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 Sterile graduated cylinders, 100-1000 mL, covered with aluminum foil or kraft paper
6.7 Sterile membrane filtration units (filter base and funnel), glass, plastic or stainless steel, wrapped
with aluminum foil or kraft paper
July 2006 2
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Method 1106.1
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 Flask, filter, vacuum, usually 1 L, with appropriate tubing
6.11 Filter manifold to hold a number of filter bases (optional)
6.12 Flask for safety trap placed between the filter flask and the vacuum source
6.13 Forceps, straight or curved, with smooth tips to handle filters without damage
6.14 Ethanol, methanol or isopropanol in a small, wide-mouth container, for flame-sterilizing forceps
6.15 Burner, Bunsen or Fisher type, or electric incinerator unit for sterilizing loops and needles
6.16 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
6.17 Petri dishes, sterile, plastic, 9x50 mm with tight-fitting lids; or 15 x 60 mm with loose fitting
lids; and 15 x 100 mm with loose fitting lids
6.18 Bottles, milk dilution, borosilicate glass, screw cap with neoprene liners, 125 mL volume
6.19 Flasks, borosilicate glass, screw cap, 250-2000 mL volume
6.20 Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 (im pore size
6.21 Platinum wire inoculation loops, at least 3 mm diameter in suitable holders; or sterile plastic
loops
6.22 Incubator maintained at 41°C ± 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 Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes
6.26 Test tubes, screw cap, borosilicate glass, 16 x 125 mm or other appropriate size
6.27 Autoclave or steam sterilizer capable of achieving 121°C [15 Ib pressure per square inch (PSI)]
for 15 minutes
July 2006
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Method 1106.1
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-grade water conforming to specifications in: Standard Methods for the
Examination of Water and Wastewater (latest edition approved by EPA in 40 CFR Part 136 or
141, as applicable), Section 9020 (Reference 18.6).
7.4 Phosphate buffered saline (PBS)
7.4.1 Composition:
Sodium dihydrogen phosphate (NaH2PO4) 0.58 g
Disodium hydrogen phosphate (NajHPO^ 2.5 g
Sodium chloride (NaCl) 8.5 g
Reagent-grade water 1.0 L
7.4.2 Dissolve the reagents in 1 L of reagent-grade water 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 PSI) for 15 minutes. Final pH should be 7.4 ± 0.2.
7.5 mE agar
7.5.1 Composition:
Peptone 10.0 g
Sodium chloride (NaCl) 15.0 g
Yeast extract 30.0 g
Esculin 1.0 g
Actidione (cycloheximide) 0.05 g
Sodium azide 0.15 g
Agar 15.0 g
Reagent-grade water 1.0 L
7.5.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
completely. Autoclave at 121°C (15 PSI) for 15 minutes, and cool in a 50°C waterbath.
7.5.3 After sterilization, add 0.24 g nalidixic acid (sodium salt) and 0.15 g triphenyltetrazolium
chloride (TTC) to the basal medium and mix.
7.5.4 Dispense mE agar into 9 x 50 or 15 x 60 mm petri dishes to a 4-5 mm depth
(approximately 4-6 mL), and allow to solidify. Final pH of medium should be 7.1 ± 0.2.
Store in a refrigerator.
July 2006
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Method 1106.1
7.6 Esculin iron agar (EIA)
7.61 Composition:
Esculin l.Og
Ferric citrate 0.5 g
Agar 15. Og
Reagent-grade water l.OL
7.6.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
completely. Autoclave the medium at 121°C (15 PSI) for 15 minutes, and cool in a 50°C
waterbath. After cooling, pour the medium into 9 x 50 or 15 x 60 mm petri dishes to a
depth of 4-5 mm (approximately 4-6 mL), and allow to solidify. Final pH should be
7.1 ± 0.2. Store in a refrigerator.
7.7 Tryptic soy agar (TSA)
7.7.1 Composition:
Pancreatic digest of casein 15.Og
Enzymatic digest of soybean meal 5.0 g
Sodium chloride (NaCl) 5.0 g
Agar 15.Og
Reagent-grade water l.OL
7.7.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
completely. Autoclave at 121°C (15 PSI) for 15 minutes and cool in a 50°C waterbath.
Pour the medium into each 15 x 60 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.
7.8 Brain heart infusion broth (BHIB)
7.8.1 Composition:
Calf brains, infusion from 200.0 g 7.7 g
Beef heart, infusion from 250.0 g 9.8g
Proteose peptone 10.Og
Sodium chloride (NaCl) 5.0 g
Disodium hydrogen phosphate (NajHPO^ 2.5 g
Dextrose 2.0 g
Reagent-grade water l.OL
7.8.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
completely. Dispense in 10-mL volumes in screw cap tubes, and autoclave at 121°C (15
PSI) for 15 minutes. Final pH should be 7.4 ± 0.2.
7.9 Brain heart infusion broth (BHIB) with 6.5% NaCl
7.9.1 Composition:
BHIB with 6.5% NaCl is the same as BHIB broth (Section 7.8 above), but with additional
NaCl.
5 July 2006
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Method 1106.1
7.9.2 Add NaCl to formula provided in Section 7.8 above, such that the final concentration is
6.5 %(65 g NaCl/L). Typically, for commercial BHIB media, an additional 60.0 g NaCl
per liter of medium will need to be added to the base medium. Prepare as in Section
7.8.2.
7.10 Brain heart infusion agar (BHIA)
7.10.1 Composition:
BHIA contains the same components as BHIB (Section 7.8 above) with the addition of
15.0 g agar per liter of BHIB.
7.10.2 Add agar to formula for BHIB provided in Section 7.8 above. Prepare as in Section
7.8.2. After sterilization, slant until solid. Final pH should be 7.4 ± 0.2.
7.11 Bile esculin agar (BEA)
7.11.1 Composition:
Beef extract 3.0g
Pancreatic digest of gelatin 5.0 g
Oxgall 20.0 g
Esculin l.Og
Ferric citrate 0.5 g
Agar 14.0 g
Reagent-grade water l.OL
7.11.2 Add reagents to 1 L reagent-grade water, heat with frequent mixing, and boil 1 minute to
dissolve completely. Dispense 10-mL volumes in tubes for slants or larger volumes into
flasks for subsequent plating. Autoclave at 121°C (15 PSI) for 15 minutes. Overheating
may cause darkening of the medium. Cool in a 50°C waterbath, and dispense into sterile
petri dishes. Final pH should be 6.8 ± 0.2. Store in a refrigerator.
7.12 Azide dextrose broth (ADB)
7.12.1 Composition:
Beef extract 4.5 g
Pancreatic digest of casein 7.5 g
Proteose peptone No. 3 7.5 g
Dextrose 7.5 g
Sodium chloride (NaCl) 7.5 g
Sodium azide 0.2 g
Reagent-grade water l.OL
7.12.2 Add reagents to 1 L of reagent-grade water and dispense in screw cap bottles. Autoclave
at 12FC (15 PSI) for 15 minutes. Final pH should be 7.2 ± 0.2.
July 2006
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Method 1106.1
7.13 Control Cultures
7.13.1 Positive control and/or spiking organism (either of the following are acceptable):
• Stock cultures of Enterococcus faecalis (E. faecalis) ATCC #19433
• E. faecalis ATCC # 1943 3 BioBalls (BTF Pty, Sydney, Australia)
7.13.2 Negative control organism (either of the following are acceptable):
• Stock cultures of Escherichia coll (E. coll) ATCC #11775
• E. coli ATCC #11775 BioBalls (BTF Pty, Sydney, Australia)
8.0 Sample Collection, Handling, and Storage
8.1 Sampling procedures are briefly described below. Detailed sampling methods can be found in
Reference 18.7 (see Appendix A). Adherence to sample preservation procedures and holding
time limits is critical to the production of valid data. Samples should not be analyzed if these
conditions are not met.
8.1.1 Sampling Techniques
Samples are collected by hand or with a sampling device if the sampling site has difficult
access such as a dock, bridge, or bank adjacent to a surface water. Composite samples
should not be collected, since such samples do not display the range of values found in
individual samples. The sampling depth for surface water samples should be 6-12 inches
below the water surface. Sample containers should be positioned such that the mouth of
the container is pointed away from the sampler or sample point. After removal of the
container from the water, a small portion of the sample should be discarded to allow for
proper mixing before analyses.
8.1.2 Storage Temperature and Handling Conditions
Ice or refrigerate bacteriological samples at a temperature of <10°C during transit to the
laboratory. Do not freeze the samples. 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.
8.1.3 Holding Time Limitations
Sample analysis should begin immediately, preferably within 2 hours of collection. The
maximum transport time to the laboratory is 6 hours, and samples should be processed
within 2 hours of receipt at the laboratory.
July 2006
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Method 1106.1
9.0 Quality Control
9.1 Each laboratory that uses Method 1106.1 is required to operate a formal quality assurance (QA)
program that addresses and documents instrument and equipment maintenance and performance,
reagent quality and performance, analyst training and certification, and records storage and
retrieval. General requirements and recommendations for QA and quality control (QC)
procedures for microbiological laboratories are provided in Reference 18.7 (see Appendix B).
9.2 The minimum analytical QC requirements for the analysis of samples using Method 1106.1
include routine analysis of positive and negative controls (Section 9.5), filter sterility checks
(Section 9.7), method blanks (Section 9.8), and media sterility checks (Section 9.10). Additional
analytical QC for the analysis of samples using Method 1106.1 include an initial demonstration of
laboratory capability through performance of the initial precision and recovery (IPR) analyses
(Section 9.3), ongoing demonstration of laboratory capability through performance of the
ongoing precision and recovery (OPR) analysis (Section 9.4). For the IPR and OPR analyses, it
is necessary to spike PBS samples with either laboratory-prepared spiking suspensions or
BioBalls as described in Section 14.
9.3 Initial precision and recovery (IPR)—The IPR analyses are used to demonstrate acceptable
method performance (recovery and precision) by each laboratory before the method is used for
monitoring field samples. EPA recommends but does not require that IPR analyses be performed
by each analyst. IPR samples should be accompanied by an acceptable method blank (Section
9.8) and appropriate media sterility checks (Section 9.10). The IPR analyses are performed as
follows:
9.3.1 Prepare four, 100-mL samples of PBS and spike each sample with E. faecalis ATCC
#19433 according to the spiking procedure in Section 14. Spiking with laboratory-
prepared suspensions is described in Section 14.2 and spiking with BioBalls is described
in Section 14.3. Filter and process each IPR sample according to the procedures in
Section 11 and calculate the number of enterococci per 100 mL according to Section 13.
9.3.2 Calculate the percent recovery (R) for each IPR sample using the appropriate equation in
Section 14.2.4.2 or 14.3.2 for samples spiked with laboratory-prepared spiking
suspensions or BioBalls, respectively.
9.3.3 Using the percent recoveries of the four analyses, calculate the mean percent recovery
and the relative standard deviation (RSD) of the recoveries. The RSD is the standard
deviation divided by the mean, multiplied by 100.
9.3.4 Compare the mean recovery and RSD with the corresponding IPR criteria in Table 1,
below. If the mean and RSD for recovery of enterococci meet acceptance criteria, system
performance is acceptable and analysis of field samples may begin. If the mean or the
RSD fall outside of the required range for recovery, system performance is unacceptable.
In this event, identify the problem by evaluating each step of the analytical process,
media, reagents, and controls, correct the problem and repeat IPR analyses.
July 2006
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Method 1106.1
Table 1.
Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria
Performance test
Initial precision and recovery (IPR)
Mean percent recovery
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (OPR) as percent
recovery
Lab-prepared spike
acceptance criteria
15% -136%
21%
14% -137%
BioBall™
acceptance criteria
86% -102%
12%
80% -108%
9.4 Ongoing precision and recovery (OPR)—To demonstrate ongoing control of the analytical
system, the laboratory should routinely process and analyze spiked PBS samples. The laboratory
should analyze one OPR sample after every 20 field samples or one per week that samples are
analyzed, whichever occurs more frequently. OPR samples should be accompanied by an
acceptable method blank (Section 9.8) and appropriate media sterility checks (Section 9.10). The
OPR analysis is performed as follows:
9.4.1 Spike a 100-mL PBS sample with E.faecalis ATCC #19433 according to the spiking
procedure in Section 14. Spiking with laboratory-prepared suspensions is described in
Section 14.2 and spiking with BioBalls is described in Section 14.3. Filter and process
each OPR sample according to the procedures in Section 11 and calculate the number of
enterococci per 100 mL according to Section 13.
9.4.2 Calculate the percent recovery (R) for the OPR sample using the appropriate equation in
Section 14.2.4.2 or 14.3.2 for samples spiked with BioBalls or laboratory-prepared
spiking suspensions, respectively.
9.4.3 Compare the OPR results (percent recovery) with the corresponding OPR recovery
criteria in Table 1, above. If the OPR result meets the acceptance criteria for recovery,
method performance is acceptable and analysis of field samples may continue. If the
OPR result falls outside of the acceptance criteria, system performance is unacceptable.
In this event, identify the problem by evaluating each step of the analytical process,
media, reagents, and controls, correct the problem and repeat the OPR analysis.
9.4.4 As part of the laboratory QA program, method recovery results for OPR and IPR samples
should be charted and updated records maintained in order to monitor ongoing method
performance. The laboratory should also develop a statement of accuracy for Method
1106.1 by calculating the average percent recovery (R) and the standard deviation of the
percent recovery (sr). Express the accuracy as a recovery interval from R - 2sr to R + 2sr.
9.5 Culture Controls
9.5.1 Negative controls—The laboratory should analyze negative controls to ensure that the
mE and EIA media are performing properly. Negative controls should be analyzed
whenever a new batch of media or reagents is used. On an ongoing basis, the laboratory
should perform a negative control every day that samples are analyzed.
July 2006
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Method 1106.1
9.5.1.1 Negative controls are conducted by filtering a dilute suspension of viable E.
coll (e.g., ATCC #11775) and analyzing as described in Section 11.
Viability of the negative controls should be demonstrated using a
non-selective media (e.g., nutrient agar or tryptic soy agar).
9.5.1.2 If the negative control fails to exhibit the appropriate response, check and/or
replace the associated media or reagents, and/or the negative control, and
reanalyze the appropriate negative control(s).
9.5.2 Positive controls—The laboratory should analyze positive controls to ensure that the mE
and EIA media are performing properly. Positive controls should be analyzed whenever
a new batch of media or reagents is used. On an ongoing basis, the laboratory should
perform a positive control every day that samples are analyzed. An OPR sample (Section
9.4) may take the place of a positive control.
9.5.2.1 Positive controls are conducted by filtering a dilute suspension of viable E.
faecalis (e.g., ATCC #19433) and analyzing as described in Section 11.
9.5.2.2 If the positive control fails to exhibit the appropriate response, check and/or
replace the associated media or reagents, and/or the positive control, and
reanalyze the appropriate positive control(s).
9.5.3 Controls for verification media—All verification media should be tested with
appropriate positive and negative controls whenever a new batch of media and/or
reagents are used. On an ongoing basis, the laboratory should perform positive and
negative controls on the verification media with each batch of samples submitted to
verification. Examples of appropriate controls for verification media are provided in
Table 2.
Table 2. Verification Controls
Medium
Bile esculin agar (BEA)
Brain heart infusion broth (BHIB) with 6.5% NaCI
Brain heart infusion broth (BHIB) incubated at 45°C
Positive Control
£ faecalis
E. faecalis
E. faecalis
Negative Control
£ co//
£ co//
£. co//
9.6 Colony verification—The laboratory should verify 10 typical colonies (positive) and 10 atypical
colonies (negative) per month or 1 typical colony and 1 atypical colony from 10% of all positive
samples, whichever is greater. Verification procedures are provided in Section 12.0.
9.7 Filter sterility check—Place at least one membrane filter on a TSA plate, and incubate for 24 ± 2
hours at 35°C ± 0.5°C. Absence of growth indicates sterility of the filter. On an ongoing basis,
the laboratory should perform a filter sterility check every day that samples are analyzed.
9.8 Method blank—Filter a 50-mL volume of sterile PBS, place the filter on an mE plate, and
incubate for 48 ± 3 hours at 41°C ± 0.5°C. Absence of growth indicates freedom of
contamination from the target organism. On an ongoing basis, the laboratory should perform a
method blank every day that samples are analyzed.
July 2006 10
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Method 1106.1
9.9 Filtration blank—Filter a 50-mL volume of sterile PBS before beginning sample filtrations.
Place the filter on a TSA plate, and incubate for 24 ±2 hours at 35°C ± 0.5°C. Absence of
growth indicates sterility of the PBS and filtration assembly.
9.10 Media sterility check—The laboratory should test media sterility by incubating one unit (tube or
plate) from each batch of medium (TSA, mE, EIA, and verification media) as appropriate and
observing for growth. Absence of growth indicates media sterility. On an ongoing basis, the
laboratory should perform a media sterility check every day that samples are analyzed.
9.11 Analyst colony counting variability—Laboratories with two or more analysts should compare
each analyst's colony counts from one positive field sample per month. Colony counts should be
within 10% between analysts. Laboratories with a single analyst should have that analyst
perform duplicate colony counts of a single membrane filter each month. Duplicate colony
counts should be within 5% for a single analyst. If no positive field samples are available, a OPR
sample may be substituted for these determinations.
10.0 Calibration and Standardization
10.1 Check temperatures in incubators twice daily with a minimum of 4 hours between each reading 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.
10.3 Refrigerators used to store media and reagents should be monitored daily to ensure proper
temperature control.
11.0 Procedure
11.1 Prepare mE and EIA media as directed in Section 7.5 and 7.6, respectively.
11.2 Mark the petri dishes and report form with the sample identification and volume.
11.3 Place a sterile membrane filter on 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 enterococci concentration, to produce 20-
60 enterococci colonies on each filter. It is recommended that a minimum of three dilutions be
analyzed to ensure that a countable plate (20-60 enterococci colonies) is obtained.
11.6 Smaller sample volumes 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.
11 July 2006
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Method 1106.1
Note: When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS should be added
to the runnel or an aliquot of sample should be dispensed into a 20-30 mL dilution blank prior to
filtration. This will allow even distribution of the sample on the membrane.
11.7 Filter the sample, and rinse the sides of the funnel at least twice with 20-30 mL of sterile PBS.
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 mE plate 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 outside the
area of filtration, close to the edge of the dish, to be sure that the filter is properly seated on the
agar. Close the dish, invert, and incubate at 41°C ± 0.5°C for 48 ± 3 hours. (See Photo 1)
Note: If mE is prepared in 15 x 60 mm loose lid petri dishes, they should be incubated in a tight
fitting container (e.g., plastic vegetable crisper) containing a moistened paper towel to prevent
dehydration of the membrane filter and medium.
Photo 1. Enterococci produces pink to dark red colonies on mE agar.
11.9 After incubation, transfer the membranes to EIA plates that have been warmed to room
temperature (approximately 15-30 minutes), and incubate at 41°C ± 0.5°C for 20 to 30 minutes.
(See Photo 2)
11.10 After EIA incubation, count and record colonies on those membrane filters containing 20-60 pink
to red colonies with 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.
July 2006
12
-------
Method 1106.1
Photo 2. Enterococci colonies are pink to dark red on mE and produce a reddish-
brown to black precipitate on the underside of the filter when placed on EIA.
12.0 Verification Procedure
12.1 Pink to red colonies on mE that produce a black or reddish-brown precipitate after incubation on
EIA are considered to be "typical" enterococci colonies. Verification of typical and atypical
colonies may be required in evidence gathering and it is also recommended as a means of quality
control. The verification procedure follows.
12.2 Using a sterile inoculating loop or needle, transfer growth from the centers of at least 10 well-
isolated typical and 10 well-isolated atypical colonies into a BHIB tube and onto a BHIA slant.
Incubate broth for 24 ±2 hours and agar slants for 48 ± 3 hours at 35°C ± 0.5°C.
12.3 After a 24 hour incubation, transfer growth from each BHIB tube to BEA, BHIB with 6.5% NaCl,
and BHIB.
12.3.1 Incubate BEA and BHIB with 6.5% NaCl for 48 ± 3 hours at 35°C ± 0.5°C.
12.3.2 Incubate BHIB for 48 ± 3 hours at 45°C ± 0.5°C.
12.4 Observe all verification media for growth.
12.5 After 48 hour incubation, perform a Gram stain using growth from each BHIA slant.
12.6 Gram-positive cocci that grow and hydrolyze esculin on BEA (i.e., produce a black or brown
precipitate), and grow in BHIB with 6.5% NaCl at 35°C ± 0.5°C and BHIB at 45°C ± 0.5°C are
verified as enterococci.
12.7 Alternately, commercially available multi-test identification systems (e.g., Vitek®) may be used
to verify colonies. Such multi-test identification systems should include esculin hydrolysis and
growth in 6.5% NaCl test reactions.
13 July 2006
-------
Method 1106.1
13.0 Data Analysis and Calculations
Use the following general rules to calculate the enterococci count per 100 ml of sample:
13.1 If possible, select a membrane filter with 20-60 pink to red colonies that form a black or reddish-
brown precipitate on the underside of the filter when placed on EIA. Calculate the number of
enterococci per 100 mL according to the following general formula:
Number of enterococci colonies
Enterococci/100 mL = x 100
Volume of sample filtered (mL)
13.2 See general counting rules in Reference 18.7 (see Appendix B).
13.3 Report results as enterococci CPU per 100 mL of sample.
14.0 Sample Spiking Procedure
14.1 Method 1106.1 QC requirements (Section 9) include the preparation and analysis of spiked PBS
samples in order to monitor initial and ongoing method performance. For the IPR (Section 9.3)
and OPR (Section 9.4) analyses, it is necessary to spike samples with either laboratory-prepared
spiking suspensions (Section 14.2) or BioBalls (Section 14.3) as described below.
14.2 Laboratory-Prepared Spiking Suspensions
14.2.1 Preparation
14.2.1.1 Stock Culture. Prepare a stock culture by inoculating a TSA slant (or other
non-selective media) with E. faecalis ATCC #19433 and incubating at 35°C
± 3°C for 20 ± 4 hours. This stock culture may be stored in the dark at room
temperature for up to 30 days.
14.2.1.2 Undiluted Spiking Suspension. Prepare a 1% solution of azide dextrose
broth by combining 99 mL of sterile phosphate buffered saline and 1 mL of
sterile single strength azide dextrose broth in a sterile screw cap bottle or re-
sealable dilution water container. From the stock culture of E. faecalis
ATCC #19433 in Section 14.2.1.1, transfer a small loopful of growth to the 1
% azide dextrose broth solution and vigorously shake a minimum of 25
times. Incubate at 35°C ± 3°C for 20 ± 4 hours. This culture is referred to as
the undiluted spiking suspension and should contain approximately 1.0 * 106
- 1.0 x 107 E. faecalis colony forming units (CPU) per mL of culture.
14.2.1.3 Mix the undiluted spiking suspension thoroughly by shaking the bottle a
minimum of 25 times and prepare a series of dilutions in the following
manner:
July 2006 14
-------
Method 1106.1
14.2.1.3.1 Dilution "A"—Aseptically transfer 1.0 mL of the undiluted
spiking suspension to 99 mL of sterile PBS and mix thoroughly
by shaking the bottle a minimum of 25 times. This is spiking
suspension dilution "A" and 1 mL contains 10~2 mL of the
original undiluted spiking suspension.
14.2.1.3.2 Dilution "B"—Aseptically transfer 1.0 mL of dilution "A" to 99
mL of sterile PBS and mix thoroughly by shaking the bottle a
minimum of 25 times. This is spiking suspension dilution "B"
and 1 mL contains 10~4 mL of the original undiluted spiking
suspension.
14.2.1.3.3 Dilution "C"—Aseptically transfer 11.0 mL of dilution "B" to 99
mL of sterile PBS and mix thoroughly by shaking the bottle a
minimum of 25 times. This is spiking suspension dilution "C"
and 1 mL contains 10~5 mL of the original undiluted spiking
suspension.
14.2.1.3.4 Dilution "D"—Aseptically transfer 11.0 mL of dilution "C" to
99 mL of sterile PBS and mix thoroughly by shaking the bottle a
minimum of 25 times. This is spiking suspension dilution "D"
and 1 mL contains 10~6 mL of the original undiluted spiking
suspension.
14.2.2 Sample spiking
14.2.2.1 Add 3.0 mL of the spiking suspension dilution "D" (Section 14.2.1.3.4) to
100 mL (or appropriate volume) of sample and mix thoroughly by shaking
the bottle a minimum of 25 times. The volume of undiluted spiking
suspension added to each 100 mL sample is 3.0 x 10"6 mL, which is referred
to as V splkedper 100mL sampie in Section 14.2.4.1 below. Filter the spiked sample
and analyze the filter according to the procedures in Section 11.
14.2.3 Enumeration of spiking suspension
14.2.3.1 Prepare TSA spread plates, in triplicate, for spiking suspension dilutions "B",
"C", and "D".
Note: Agar plates must be dry and free from condensation prior to use. To
ensure that the agar surface is dry, plates should be made several days in
advance and stored inverted at room temperature or dried using a
laminar-flow hood.
14.2.3.2 Mix dilution "B" by shaking the bottle a minimum of 25 times. Pipet 0.1 mL
of dilution "B" onto the surface of each TSA plate in triplicate.
14.2.3.3 Mix dilution "C" by shaking the bottle a minimum of 25 times. Pipet 0.1 mL
of dilution "C" onto the surface of each TSA plate in triplicate.
14.2.3.4 Mix dilution "D" by shaking the bottle a minimum of 25 times. Pipet 0.1 mL
of dilution "D" onto the surface of each TSA plate in triplicate.
15 July 2006
-------
Method 1106.1
14.2.3.5 Use a sterile bent glass rod or spreader to distribute the inoculum over the
surface of plates by rotating the dish by hand or on a turntable.
Note: Ensure that the inoculum is evenly distributed over the entire surface
of the plate.
14.2.3.6 Allow the inoculum to absorb into the medium of each plate completely.
Invert plates and incubate at 35°C ± 0.5°C for 20 ± 4 hours.
14.2.3.7 Count and record number of colonies per plate. The number of enterococci
(CPU / mL) in the undiluted spiking suspension will be calculated using all
TSA plates yielding counts within the countable range of 30 to 300 CPU per
plate.
14.2.4 Recovery calculations for samples spiked with laboratory-prepared spiking
suspensions
Calculate the concentration of enterococci (CPU / mL) in the undiluted spiking
suspension (Section 14.2.1.2) according to the following equation. Example calculations
are provided in Table 3, below.
Enterococci undiluted spike = (CPU, + CFU2 + ...+ CFUJ / (V, + V2 + ... + Vn)
Where,
Enterococci ^diluted spike = Enterococci (CPU / mL) in undiluted spiking suspension
CPU = Number of colony forming units from TSA plates yielding counts within the
ideal range of 30 to 300 CPU per plate
V = Volume of undiluted sample on each TSA plate yielding counts within the
countable range of 30 to 300 CPU per plate
n = Number of plates with counts within the countable range of 30 to 300 CPU
per plate
Note: The example calculated numbers provided in the tables below have been rounded
at the end of each step for simplification purposes. Generally, rounding should only
occur after the final calculation.
July 2006 16
-------
Method 1106.1
Table 3. Example Calculations of Laboratory-prepared Enterococci Spiking Concentration
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from
TSA plates
1Q-5 mL plates
94, 106, 89
32, 55, 72
10"6 mL plates
9, 11,28
8, 5, 3
10'7 mL plates
1, 0,4
0, 0,0
Enterococci CPU / mL in undiluted
spiking suspension
(Enterococci undilutedspike)a
(94+106+89) /(10-5+10-5+10-5) =
289 / (3.0 x10-5) = 9,633,333 =
9.6x106CFU/mL
(32+55+72) /(10-5+10-5+10-5) =
1 59 / (3.0 x10-5) = 5,300,000 =
5.3x106CFU/mL
3 Enterococci undi|Uted spike is calculated using all plates yielding counts within the ideal range of 30 to 300
CPU per plate
14.2.4.1 Calculate true concentration of spiked enterococci (CPU /100 mL)
according to the following equation. Example calculations are provided in
Table 4, below.
TSpiked Enterococci = (EllterOCOCCI undiluted spike) X (" spiked per 100 mL sample)
Where,
-*- spiked Enterococci
Enterococci
undiluted spike
* spiked per 100 mL sample
Number of spiked Enterococci (CPU / 100 mL)
Enterococci (CPU / mL) in undiluted spiking suspension
mL of undiluted spiking suspension per 100 mL sample
Table 4. Example Calculations for Determination "True" Spiked Enterococci Concentration
Enterococci undiluted spike
9.6x106CFU/mL
5.3x106CFU/mL
" spiked per 100 mL sample
3.0X10-6mLper100mLof
sample
3.0X10-6mLper100mLof
sample
' spiked Enterococci
(9.6 x 1 06 CPU / mL) x (3.0 x 1 0'6 mL / 1 00 mL) =
28.8 CPU/ 100 mL
(2.8 x 106 CPU / mL) x (3.0 x 10'6 mL / 100 mL) =
8.4 CPU/ 100 mL
17
July 2006
-------
Method 1106.1
14.2.4.2 Calculate percent recovery (R) of spiked enterococci (CPU /100 mL)
according to the following equation. Example calculations are provided in
Table 5, below).
R = 100 x
(Ns - Nu)
Where,
R
Ns
Nu
T
Percent recovery
Enterococci (CPU / 100 mL) in the spiked sample (Section 13)
Enterococci (CPU / 100 mL) in the unspiked sample (Section 13)
True spiked enterococci (CPU /100 mL) in spiked sample
(Section 14.2.4.1)
Table 5.
Exam
ale Percent Recovery Calculations for Lab-prepared Spiked Samples
Ns(CFU/100mL)
42
34
10
Nu(CFU/100mL)
<1
10
<1
Tsoiked Enterococci (CFU/100ml_)
28.8
28.8
8.4
Percent recovery (R)
100 x (42-1) 728.8
= 142%
100 x (34 -10) 728.8
= 83%
100x(10-1)/8.4
= 107%
14.3 BioBall™ Spiking procedure
14.3.1 Aseptically add 1 BioBall™ to 100 mL of PBS and mix by vigorously shaking the
sample bottle a minimum of 25 times. Analyze the spiked sample according to the
procedures in Section 11.
14.3.2 Recovery calculations for samples spiked with BioBalls—Calculate percent recovery (R)
of spiked enterococci (CPU / 100 mL) according to the following equation. Example
calculations are provided in Table 6, below.
July 2006
18
-------
Method 1106.1
R = 100 X
(Ns - Nu)
Where,
R
Ns
Nu
T
Percent recovery
Enterococci (CPU / 100 mL) in the spiked sample (Section 13)
Enterococci (CPU / 100 mL) in the unspiked sample (Section 13)
True spiked enterococci (CPU /100 mL) in spiked sample based on the
lot mean value provided by manufacturer
Table 6.
Exam
ale BioBall™ Percent Recovery Calculations
Ns(CFU/100mL)
24
36
Nu(CFU/100mL)
<1
10
T(CFU/100mL)
11.2
32
Percent recovery (R)
100 x (24-1) 732 = 72%
100 x (36- 10)732 = 81%
15.0 Method Performance
15.1 The performance characteristics of Method 1106.1 (mE-EIA) for enumerating enterococci in
marine waters were initially reported by Levin, et al. (Reference 18.4) and are summarized in
Section 15.2. In a subsequent study, Method 1106.1 precision was evaluated in both fresh and
marine recreational waters and in sewage treatment plant effluents. The results of this study are
summarized in Section 15.3. Based on these study results, Method 1106.1 was approved for
enterococci monitoring of ambient recreational waters. More recently, Method 1106.1 was
evaluated for use in disinfected wastewater matrices. The results of this interlaboratory validation
study (Reference 18.3) are summarized in Section 15.4.
15.2 Performance of mE-EIA Method in Marine Waters
15.2.1 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
mE method was established by Levin et al. (Reference 18.4) who demonstrated that the
method did not exceed the expected limits for counts having the Poisson distribution.
15.2.2 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 mE agar has been
reported to be +2% of the true value (Reference 18.4).
19
July 2006
-------
Method 1106.1
15.2.3 Specificity - The ability of a method to select and/or distinguish the target bacteria 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 specificity
for this medium as reported for various environmental water samples was 10% false
positive and 11.7% false negative (Reference 18.4).
15.3 Collaborative Study Data for Ambient Waters
15.3.1 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.
15.3.2 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 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.
15.3.3 By linear regression, the precision of the method can be generalized as:
S0 = 0.013 count/100 mL + 2.42 (dilution factor) and
SB = 0.152 count/100 mL + 5.16 (dilution factor)
100
Where dilution factor =
Volume of original sample filtered
15.3.4 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 E. faecalis. The mean count (x) and the standard deviation of the
counts (Sr) (including the variability among laboratories for this standardized
enterococci sample) were 32.5 colonies/membrane and 9.42 colonies/membrane,
respectively.
July 2006 20
-------
Method 1106.1
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-------
Method 1106.1
15.4 Intel-laboratory validation of Method 1106.1 in disinfected wastewater
15.4.1 Ten volunteer participant laboratories, an enterococci verification laboratory, and two
research laboratories participated in the U.S. Environmental Protection Agency's (EPA's)
interlaboratory validation study of EPA Method 1106.1. The purposes of the study were
to characterize method performance across multiple laboratories and disinfected
wastewater matrices and to develop quantitative quality control (QC) acceptance criteria.
A detailed description of the of the study and results are provided in the validation study
report (Reference 18.3). The false positive and negative assessments for Method 1106.1
are provided in Tables 7 and 8, respectively.
15.4.2 Method 1106.1 is not approved for the analysis of enterococci in wastewater because of
the high estimated number of false positive and negative colonies observed during the
validation study. Users wishing to test for enterococci in wastewater using a membrane
filtration method are referred to EPA Method 1600.
Table 7. False Positive Assessment of Unspiked Disinfected and Unspiked Secondary
Wastewater Effluents
Matrix
Disinfected
Secondary
Disinfected + Secondary
Total colonies
Typical
877
689
1566
Atypical
267
212
479
False Positive (FP) Assessment
Typical colonies
submitted
107
80
187
No. FP
colonies
0
15
15
FP
confirmation
rate (%) a
0
18.8
8
% of total
colonies that
would have beer
aFPb
0
14.3
6.1
3 False positive confirmation rate = number of false positive colonies / number of typical colonies submitted
b Calculated as follows: (total typical colonies * FP confirmation rate) / (total number of typical and atypical colonies
observed); e.g., (689x(15/80))/(689+212) = 0.1434 = 14.3%
July 2006
22
-------
Method 1106.1
Table 8. False Negative Assessment of Unspiked Disinfected and Unspiked Secondary
Wastewater Effluents
Matrix
Disinfected
Secondary
Disinfected + Secondary
Total colonies
Typical
877
689
1566
Atypical
267
212
479
False Negative (FN) Assessment
Atypical colonies
submitted
38
56
94
No. FN
colonies
23
31
54
FN
confirmation
rate (%) a
60.5
55.4
57.4
% of total
colonies that
would have
been a FN b
14.1
13.0
13.5
a False negative confirmation rate = number of false negative colonies / number of atypical colonies submitted
b Calculated as follows: (total atypical colonies * FN confirmation rate) / (total number of typical and atypical colonies
observed); e.g., (212x(31/56))/(689+212) = 0.1302 = 13.0%
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 enterococci
attached or contained must be sterilized prior to disposal.
17.3 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.
23
July 2006
-------
Method 1106.1
18.0 References
18.1 Cabelli, V. J., A. P. Dufour, M. A. Levin, L. J. McCabe, and P. W. Haberman, 1979. Relationship
ofMicrobial Indicators to Health Effects at Marine Bathing Beaches. American Journal of
Public Health. 69:690-696.
18.2 Dufour, A.P. 1984. Health Effects Criteria for Fresh Recreational Waters, EPA 600/1-84-004.
Office of Research and Development, USEPA.
18.3 USEPA. 2005. Results of the Interlab oratory Validation of EPA Method 1106.1 (mE-EIA) for
Enterococci in Wastewater Effluent. December 2004. EPA-821-R-04-018.
18.4 Levin, M. A., J. R. Fischer and V. J. Cabelli. \915.Membrane Filter Technique for Enumeration
of Enterococci inMarine Waters. Applied and Environmental Microbiology 30:66-71.
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 APF£A. American Public Health Association. 1998. Standard Methods for the Examination of
Water and Wastewater (20th ed.), 1015 Fifteenth Street NW, Washington D.C.
18.7 Bordner, R., J.A. Winter and 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
July 2006 24
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Appendix A:
Part II (General Operations), Section A (Sample Collection,
Preservation, and Storage)
-------
Sample Collection1
1.0 Sample Containers
1.1 Sample Bottles: bottles must be resistant to sterilizing conditions and the solvent action
of water. Wide-mouth borosilicate glass bottles with screw-cap or ground-glass stopper
or heat-resistant plastic bottles may be used if they can be sterilized without producing
toxic materials (see examples A and C in Figure 1). Screw-caps must not produce
bacteriostatic or nutritive compounds upon sterilization.
k; A
Figure 1. Suggested sample containers.
1.2 Selection and Cleaning of Bottles: Samples bottles should be at least 125 mL volume
for adequate sampling and for good mixing. Bottles of 250 mL, 500 mL, and 1000 mL
volume are often used for multiple analyses. Discard bottles which have chips, cracks,
and etched surfaces. Bottle closures must be water-tight. Before use, thoroughly cleanse
bottles and closures with detergent and hot water, followed by a hot water rinse to
remove all trace of detergent. Then rinse them three times with laboratory-pure water.
1.3 Dechlorinating Agent: The agent must be placed in the bottle when water and
wastewater samples containing residual chlorine are anticipated. Add sodium thiosulfate
to the bottle before sterilization at a concentration of 0.1 mL of a 10% solution for each
125 mL sample volume. This concentration will neutralize approximately 15 mg/L of
residue chlorine.
1.4 Chelating Agent: A chelating agent should be added to sample bottles used to collect
samples suspected of containing >0.01 mg/L concentrations of heavy metals such as
copper, nickel or zinc, etc. Add 0.3 mL of a 15% solution of ethylenediaminetetraacetic
acid (EDTA) tetrasodium salt, for each 125 mL sample volume prior to sterilization.
lrThe text is taken from Part II, Section A, of the EPA publication "Microbiological Methods for
Monitoring the Environment" EPA-600/8-78-017, December 1978.
1
-------
1.5 Wrapping Bottles: Protect the tops and necks of glass stoppered bottles from
contamination by covering them before sterilization with aluminum foil or kraft paper.
1.6 Sterilization of Bottles: Autoclave glass or heat-resistant plastic bottles at 121°C for 15
minutes. Alternatively, dry glassware may be sterilized in a hot oven at 170°C for not
less than two hours. Ethylene oxide gas sterilization is acceptable for plastic containers
that are not heat-resistant. Sample bottles sterilized by gas should be stored overnight
before being used to allow the last traces of gas to dissipate.
1.7 Plastic Bags: The commercially available bags (Whirl-pak) (see example B in Figure 1)
are a practical substitute for plastic or glass samples bottles in sampling soil, sediment, or
biosolids. The bags are sealed in manufacture and opened only at time of sampling. The
manufacturer states that such bags are sterilized.
2.0 Sampling Techniques
Samples are collected by hand or with a sampling device if the sampling site has difficult access
such as a bridge or bank adjacent to a surface water.
2.1 Chlorinated Samples: When samples such as treated waters, chlorinated wastewaters or
recreational waters are collected, the sample bottle must contain a dechlorinating agent (see
section 1.3 above).
2.2 Composite Sampling: In no case should a composite sample be collected for bacteriologic
examination. Data from individual samples show a range of values. A composite sample
will not display this range. Individual results will give information about industrial process
variations in flow and composition. Also, one or more portions that make up a composite
sample may contain toxic or nutritive materials and cause erroneous results.
2.3 Surface Sampling by Hand: A grab sample is obtained using a sample bottle prepared as
described in (1) above. Identify the sampling site on the bottle label and on a field log sheet.
Remove the bottle covering and closure and protect from contamination. Grasp the bottle at
the base with one hand and plunge the bottle mouth down into the water to avoid introducing
surface scum (Figure 2). Position the mouth of the bottle into the current away from the
hand of the collector and, if applicable, away from the side of the sampling platform. The
sampling depth should be 15-30 cm (6-12 inches) below the water surface. If the water body
is static, an artificial current can be created, by moving the bottle horizontally in the
direction it is pointed and away from the sampler. Tip the bottle slightly upwards to allow
air to exit and the bottle to fill. After removal of the bottle from the stream, pour out a small
portion of the sample to allow an air space of 2.5-5 cm (1-2 inches) above each sample for
proper mixing of the sample before analyses. Tightly stopper the bottle and place on ice (do
not freeze) for transport to the laboratory.
-------
Figure 2. Grab sampling technique for surface waters.
3.0 Selection of Sampling Sites and Frequency
These will be described for streams, rivers, estuarine, marine, and recreational waters as well as
domestic and industrial wastewaters.
3.1 Stream Sampling: The objectives of the initial survey dictate the location, frequency and
number of samples to be collected.
3.1.1 Selection of Sampling Sites: A typical stream sampling program includes sampling
locations upstream of the area of concern, upstream and downstream of waste
discharges, upstream and downstream from tributary entrances to the river and
upstream of the mouth of the tributary. For more complex situations, where several
waste discharges are involved, sampling includes sites upstream and downstream from
the combined discharge area and samples taken directly from each industrial or
municipal waste discharge. Using available bacteriological, chemical and discharge
rate data, the contribution of each pollution source can be determined.
3.1.2 Small Streams: Small streams should be sampled at background stations upstream of
the pollution sources and at stations downstream from pollution sources. Additional
sampling sites should be located downstream to delineate the zones of pollution.
Avoid sampling areas where stagnation may occur (e.g., backwater of a tributary) and
areas located near the inside bank of a curve in the stream which may not be
representative of the main channel.
-------
3.1.3 Large Streams and Rivers: Large streams are usually not well mixed laterally for long
distances downstream from the pollution sources. Sampling sites below point source
pollution should be established to provide desired downstream travel time and
dispersal as determined by flow rate measurements. Particular care must be taken to
establish the proper sampling points. Occasionally, depth samples are necessary to
determine vertical mixing patterns.
3.2 Estuarine and Marine Sampling: Sampling estuarine and marine waters requires the
consideration of other factors in addition to those usually recognized in fresh water
sampling. They include tidal cycles, current patterns, bottom currents and counter-currents,
stratification, seasonal fluctuations, dispersion of discharges and multi-depth samplings.
The frequency of sampling varies with the objectives. When a sampling program is started,
it may be necessary to sample every hour around the clock to establish pollution loads and
dispersion patterns. The sewage discharges may occur continuously or intermittently.
When the sampling strategy for a survey is planned, data may be available from previous
hydrological studies done by the Coast Guard, Corps of Engineers, National Oceanic and
Atmospheric Administration (NOAA), U.S. Geological Survey, or university and private
research investigations. In a survey, float studies and dye studies are often carried out to
determine surface and undercurrents. Initially depth samples are taken on the bottom and at
five feet increments between surface and bottom. A random grid pattern for selecting
sampling sites is established statistically.
3.2.1 Estuarine Sampling: When a survey is made on an estuary, samples are often taken
from a boat, usually making an end to end traverse of the estuary. Another method
involves taking samples throughout a tidal cycle, every hour or two hours from a
bridge or from an anchored boat at a number of fixed points.
In a large bay or estuary where many square miles of area are involved, a grid or
series of stations may be necessary. Two sets of samples are usually taken from an
area on a given day, one at ebb or flood slack water, and the other three hours earlier,
or later, at the half tidal interval. Sampling is scheduled so that the mid-sampling time
of each run coincides with the calculated occurrence of the tidal condition.
In location sampling sites, one must consider points at which tributary waters enter the
main stream or estuary, location of shellfish beds and bathing beaches. The sampling
stations can be adjusted as data accumulate. For example, if a series of stations half
mile apart consistently show similar values, some of these stations may be dropped
and other stations added in areas where data shows more variability.
Considerable stratification can occur between the salt water from the sea and the fresh
water supplied by a river. It is essential when starting a survey of an unknown estuary
to find out whether there is any marked stratification. This can be done by chloride
determinations at different locations and depths. It is possible for stratification to
occur in one part of an estuary and not in another.
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On a flood tide, the more dense salt water pushing up into the less dense fresh river
water will cause an overlapping with the fresh water flowing on top. A phenomenon
called a salt water wedge can form. As a result, stratification occurs. If the discharge
of pollution is in the salt water layer, the contamination will be concentrated near the
bottom at the flood tide. The flow or velocity of the fresh water will influence the
degree of stratification which occurs. If one is sampling only at the surface, it is
possible that the data will not show the polluted underflowing water which was
contaminated at the point below the fresh water river. Therefore, where stratification
is suspected, samples at different depths will be needed to measure vertical
distribution.
3.2.2 Marine Sampling: In ocean studies, the environmental conditions are most diverse
along the coast where shore, atmosphere and the surf are strong influences. The
shallow coastal waters are particularly susceptible to daily fluctuations in temperature
and seasonal changes.
Sampling during the entire tidal cycle or during a half cycle may be required. Many
ocean studies such as sampling over the continental shelf involve huge areas and no
two areas of water are the same.
Selection of sampling sites and depths are most critical in marine waters. In winter,
cooling of coastal waters can result in water layers which approach 0°C. In summer,
the shallow waters warm much faster than the deeper waters. Despite the higher
temperature, oxygen concentrations are higher in shallow than in deeper waters due to
greater water movement, surf action and photosynthetic activity from macrophytes
and the plankton.
Moving from the shallow waters to the intermediate depths, one observes a
moderation of these shallow water characteristics. In the deeper waters, there is a
marked stabilization of conditions. Water temperatures are lower and more stable.
There is limited turbulence, little penetration of light, sparse vegetation and the ocean
floor is covered with a layer of silts and sediments.
3.3 Recreational Waters (Bathing Beaches'): Sampling sites at bathing beaches or other
recreational areas should include upstream or peripheral areas and locations adjacent to
natural drains that would discharge storm water, or run-off areas draining septic wastes from
restaurants, boat marinas, or garbage collection areas. Samples of bathing beach water
should be collected at locations and times of heaviest use. Daily sampling, preferably in the
afternoon, is the optimum frequency during the season. Weekends and holidays which are
periods of highest use must be included in the sampling program. Samples of estuarine
bathing waters should be obtained at high tide, ebb tide and low tide in order to determine
the cyclic water quality and deterioration that must be monitored during the swimming
season.
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3.4 Domestic and Industrial Waste Discharges: It is often necessary to sample secondary and
tertiary wastes from municipal waste treatment plants and various industrial waste treatment
operations. In situations where the plant treatment efficiency varies considerably, grab
samples are collected around the clock at selected intervals for a three to five day period. If
it is known that the process displays little variation, fewer samples are needed. In no case
should a composite sample be collected for bacteriological examination. The National
Pollution Discharge Elimination System (NPDES) has established wastewater treatment
plant effluent limits for all dischargers. These are often based on maximum and mean
values. A sufficient number of samples must be collected to satisfy the permit and/or to
provide statistically sound data and give a fair representation of the bacteriological quality of
the discharge.
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Appendix B:
Part II (General Operations), Sections C.3.5 (Counting Colonies)
and C.3.6 (Calculation of Results)
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Counting Colonies1
1.0 Counting Colonies
Colonies should be counted using a fluorescent lamp with a magnifying lens. The flourescent lamp
should be nearly perpendicular to the membrane filter. Count colonies individually, even if they
are in contact with each other. The technician must learn to recognize the difference between two
or more colonies which have grown into contact with each other and single, irregularly shaped
colonies which sometimes develop on membrane filters. The latter colonies are usually associated
with a fiber or particulate material and the colonies conform to the shape and size of the fiber or
particulates. Colonies which have grown together almost invariably show a very fine line of
contact.
2.0 Calculation of Results
2.1 Select the membrane filter with the number of colonies in the acceptable range and calculate
count per 100 mL according to the general formula:
Count per 100 mL = (No. of colonies counted/Volume of sample filtered, in mL) x 100
2.2 Counts Within the Acceptable Limits
The acceptable range of colonies that are countable on a membrane is a function of the
method. Different methods may have varying acceptable count ranges. All examples in this
appemdix assume that the acceptable range of counts is between 20-80 colonies per
membrane.
For example, assume that filtration of volumes of 50, 15, 5, 1.5, and 0.5 mL produced
colony counts of 200, 110, 40, 10, and 5, respectively.
An analyst would not actually count the colonies on all filters. By inspection the analyst
would select the membrane filter with the acceptable range of target colonies, as defined by
the method, and then limit the actual counting to such membranes.
After selecting the best membrane filter for counting, the analyst counts colonies and applies
the general formula as in section 2.1 above to calculate the count/100 mL.
2.3 More Than One Acceptable Count
2.3.1 If there are acceptable counts on replicate plates, carry counts independently to final
reporting units, then calculate the arithmetic mean of these counts to obtain the final
reporting value.
lrThe text is largely taken from Part II, Section C, of the EPA publication "Microbiological
Methods for Monitoring the Environment" EPA-600/8-78-017, December 1978. Some examples were
kindly provided by Kristen Brenner, US EPA.
1
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Example, if the counts are 24 and 36 for replicate plates of 100 mL each, then the arithmetic
mean is calculated as follows:
(24 CFU/100 mL + 36 CFU/100 mL)
= 30 CPU/100 mL
2.3.2 If there is more than one dilution having an acceptable range of counts, independently
carry counts to final reporting units, then average for final reported value.
For example, if volumes of 100, 10, 1 and 0.1 mL produced colony counts of Too
Numerous To Count (TNTC), 75, 30, and 1, respectively, then two volumes, 10 mL and 1
mL, produced colonies in the acceptable counting range.
Independently carry each MF count to a count per 100 mL:
75
x100 =750CFU/100mL
10
and
30
x100 =3000 CPU/100 mL
Calculate the arithmetic mean as in section 2.3.1 above:
(750 CFU/100 mL + 3000 CFU/100 mL)
= 1875CFU/100mL
Report this as 1875 CFU/100 mL.
2.4 If all MF counts are below the lower acceptable count limit, select the most nearly
acceptable count.
2.4.1 For example, sample volumes of 100, 10 and 1 mL produced colony counts of 17, 1
and 0, respectively.
Here, no colony count falls within recommended limits. Calculate on the basis of the
most nearly acceptable plate count, 17, and report as 17 CFU/100 mL.
Note that in this case, because no calculations were done (i.e. this is the count for 100
mL), the count is reported as 17 CFU/100 mL rather than an "estimated count of
17 CFU/100 mL"
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2.4.2 As a second example, assume a count in which sample volumes of 10 and 1 mL
produced colony counts of 18 and 0, respectively.
Here, no colony count falls within recommended limits. Calculate on the basis of the
most nearly acceptable plate count, 18, and calculate as in section 2.3.2 above.
18
x-ioo =180 CPU/100mL
10
Report this as an estimated count of 180 CFU/100 mL.
2.5 If counts from all membranes are zero, calculate using count from largest filtration volume.
For example, sample volumes of 25, 10, and 2 mL produced colony counts of 0, 0, and 0,
respectively, and no actual calculation is possible, even as an estimated report. Calculate the
number of colonies per 100 mL that would have been reported if there had been one colony
on the filter representing the largest filtration volume. In this example, the largest volume
filtered was 25 mL and thus the calculation would be:
x100 =4 CPU 7100 mL
25
Report this as < (less than) 4 CFU/100 mL.
2.6 If all membrane counts are above the upper acceptable limit, calculate count using the
smallest volume filtered.
For example, assume that the volumes 1, 0.3, and 0.01 mL produced colony counts of
TNTC, 150, and 110 colonies, respectively. Since all colony counts are above the
acceptable limit, use the colony count from the smallest sample volume filtered and estimate
the count as:
110
x-ioo =1,100,000 CPU/100mL
0.01
Report this as estimated count 1.1 x 106 CFU/100 mL
2.7 If typical colonies are too numerous to count (TNTC), use upper limit count with smallest
filtration volume.
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For example, assume that the volumes 1, 0.3, and 0.01 mL all produced too many typical
colonies, and that the laboratory bench record indicated TNTC.
Use the upper acceptable count for the method (80 colonies in this example) as the basis of
calculation with the smallest filtration volume and estimate the count as:
80
x 100 = 800,000 CPU /100 mL
0.01
Report this as > (greater than) 8 x 105 CPU/100 mL
2.8 If colonies are both above and below the upper and lower acceptable limits (i.e., no counts
are within the acceptable limits), select the most nearly acceptable count.
2.8.1 For example, sample volumes of 100, 10 and 1 mL produced colony counts of 84, 8
and 0, respectively.
Here, no colony count falls within recommended limits. Calculate on the basis of the
most nearly acceptable plate count, 84, and report as 84 CFU/100 mL.
Note that in this case, because no calculations were done (i.e. this is the count for 100
mL), the count is reported as 84 CFU/100 mL rather than an "estimated count of
84 CFU/100 mL"
2.8.2 As a second example, assume a count in which sample volumes of 100, 10 and 1 mL
produced colony counts of 98, 18, and 0, respectively.
Here, no colony count falls within recommended limits. Calculate on the basis of the
most nearly acceptable plate count, 18, and calculate as in section 2.3.2 above.
18
x-ioo =180 CPU/100mL
10
Report this as estimated count 180 CFU/100 mL.
2.9 If there is no result because of a confluent growth, > 200 atypical colonies (TNTC), lab
accident, etc., report as No Data and specify the reason.
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