xvEPA
United States
Environmental Protection
Agency
Method 1609.1: Enterococci in Water
by TaqMan® Quantitative
Polymerase Chain Reaction (qPCR)
with Internal Amplification Control
(IAC) Assay
April 2015
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-820-R-15-099
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Method 1609.1
Acknowledgments
This method was developed under the direction of Rich Haugland, Kevin Oshima and Alfred P. Dufour of
the U.S. Environmental Protection Agency's (EPA) Human Exposure Research Division, National
Exposure Research Laboratory, Cincinnati, Ohio.
The following laboratories are gratefully acknowledged for their participation in the multi-laboratory
validation of this method in fresh and marine waters:
Participant Laboratories
Marine ambient water multi-laboratory validation:
• BioVir: Rick Danielson and Rosie Newton
• Hampton Roads Sanitation District: Robin Parnell and Tiffany Elston
• Mycometrics: King-Teh Lin and Rose Lee
• Orange County Public Health Laboratory: Richard Alexander and Joe Guzman
• Orange County Water District: Donald Phipps and Menu Leddy
• San Francisco Public Water Utilities: Eunice Chern
• University of South Florida: Jody Harwood and Zachary Staley
Fresh ambient water multi-laboratory validation:
• BioVir: Rick Danielson and Rosie Newton
• Hampton Roads Sanitation District: Robin Parnell and Tiffany Elston
• New York State Department of Health: Ellen Braun-Howland and Stacey Chmura
• Orange County Water District: Donald Phipps and Menu Leddy
• Scientific Methods Incorporated: Fu-Chih Hsu and Rebecca Wong
• Texas A&M University - College Station: Suresh Pillai and Charlotte Rambo
• University of Iowa Hygienic Lab: Nancy Hall, Greg Gingerich, and Lucy DesJardin
• Wisconsin State Lab of Hygiene: Sharon Kluender and Jeremy Olstadt
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Method 1609.1
Disclaimer
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
apparatus, product, or process discussed in this method, or represents that its use by such a 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 or OSTCWAMethods@epa.gov
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Method 1609.1
Introduction
Enterococci are commonly found in the feces of humans and other warm-blooded animals. Although
these organisms can be persistent in the environment, the presence of enterococci in water is an indication
of fecal pollution and the possible presence of enteric pathogens. Epidemiological studies have led to the
development of criteria for promulgating recreational water standards based on established relationships
between the measured density of enterococci colony forming units (CPUs) in the water by culture based
methods and the risk of gastrointestinal illness associated with swimming in the water (References 16.3,
16.8, 16.9, 16.10, and 16.11).
Method 1609.1 describes a quantitative polymerase chain reaction (qPCR) procedure for the detection of
DNA from enterococci bacteria in ambient water matrices based on the amplification and detection of a
specific region of the large subunit ribosomal ribonucleic acid (RNA) gene (IsrRNA, 23 S rRNA) from
these organisms. The advantage of Method 1609.1 over currently accepted culture methods that require
24 - 48 hours to obtain results is its relative rapidity. Results can be obtained by Method 1609.1 in 3 - 4
hours, allowing same-day notification of recreational water quality.
In Method 1609.1, water samples are filtered to collect enterococci on polycarbonate membrane filters.
Following filtration, total deoxyribonucleic acid (DNA) is solubilized from the filter retentate using a
bead beater. Enterococci target DNA sequences present in the clarified homogenate are detected by the
real-time quantitative polymerase chain reaction (qPCR) technique using TaqMan® Environmental master
mix PCR reagent and the TaqMan® probe system. The TaqMan® system signals the formation of PCR
products by a process involving enzymatic hydrolysis of a fluorogenically labeled oligonucleotide probe
when it hybridizes to the target sequence.
Method 1609.1 uses an arithmetic formula, the comparative cycle threshold (CT) method, to calculate the
ratio of Enterococcus IsrRNA gene copies (target sequences) recovered in total DNA extracts from water
samples relative to those in similarly prepared extracts of calibrator samples containing a known quantity
of Enterococcus cells. The target sequence ratio can be multiplied by the number of Enterococcus cells in
the calibrator sample to obtain estimates of calibrator cell equivalents (CCE) in the water samples. The
absolute quantity of target sequences in the calibrator sample extracts can also be determined and used in
the comparative CT method to obtain estimates of the target sequence copies or, together with
Enterococcus cell numbers in the calibrator sample, to obtain CCE estimates with associated defined
target sequences per CCE in the water samples. CT values for sample processing control (SPC) sequences
added in equal quantities to both the water filtrate and calibrator samples before DNA extraction are used
to normalize results for potential differences in DNA recovery or to signal inhibition or fluorescence
quenching of the PCR analysis caused by a sample matrix component or possible technical error. In
addition, an internal amplification control (IAC) is added to each qPCR analysis for Enterococcus DNA
and is co-amplified simultaneously with the target sequence; to specifically identify polymerase inhibition
in the reactions.
in
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Method 1609.1
Table of Contents
1.0 Scope and Application 1
2.0 Summary of Method 1
3.0 Definitions 2
4.0 Interferences 3
5.0 Safety 3
6.0 Equipment and Supplies 3
7.0 Reagents and Standards 5
8.0 Sample Collection, Handling, and Storage 11
9.0 Quality Control 12
10.0 Calibration and Standardization of Method-Related Instruments 21
11.0 Procedure 21
12.0 Data Analysis and Calculations 34
13.0 Method Performance 37
14.0 Pollution Prevention 38
15.0 Waste Management 38
16.0 References 39
17.0 Acronyms 40
IV
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Method 1609.1
List of Appendices
Appendix A: Part II (General Operations), Section A (Sample Collection, Preservation, and Storage)
Appendix B: Life Technologies StepOnePlus™, Applied Biosystems (AB) 7900, and AB 7500 Real-
Time PCR System Operation
Appendix C: Cepheid Smart Cycler® Operation
Appendix D: Protocol for MPN Estimation of DNA Standard Concentrations
Appendix E: How to Obtain DNA Standards from EPA
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Method 1609.1
Method 1609.1: Enterococci in Water by Quantitative Polymerase
Chain Reaction (qPCR) with Internal Amplification Control (IAC)
Assay
April 2015
1.0 Scope and Application
1.1 Method 1609.1 describes a qPCR procedure for the measurement of large subunit ribosomal RNA
(IsrRNA, 23S rRNA) target gene sequences (target sequences) from all known species of
enterococci bacteria in water. This method is based on the collection of enterococci on
membrane filters, extraction of total DNA using a bead beater, and detection of enterococci target
sequences in the supernatant by real-time polymerase chain reaction (PCR) using TaqMan®
Environmental master mix PCR reagent and the TaqMan® probe system. The TaqMan® system
signals the formation of PCR products by a process involving the enzymatic hydrolysis of a
labeled fluorogenic probe that hybridizes to the target sequence.
1.2 The Method 1609.1 test is recommended as a measure of ambient marine and fresh recreational
water quality. The significance of finding Enterococcus DNA target sequences in recreational
water samples stems from the association between the density of these sequences and the reported
occurrence of gastrointestinal (GI) illness associated with swimming observed in recent
epidemiological studies (References 16.11, 16.13, and 16.14).
1.3 The variable recoveries observed during the validation studies should be taken into consideration
when analyzing results from Method 1609.1. Guidance is provided in Section 9 of this document
on how laboratories can monitor the initial and ongoing performance of the method in their
hands. It is expected and has been observed that the performance of the method will improve as
laboratories gain additional experience with it through continued practice.
1.4 This method assumes the use of an Applied Biosystems (AB) Sequence Detector as the default
platform. (Note: Applied Biosystems is now Life Technologies). The Cepheid Smart Cycler®
may also be used. The user should refer to the platform specific instructions for these instruments
in the appendices. Users should thoroughly read the method in its entirety before preparation of
reagents and commencement of the method to identify differences in protocols for different
platforms.
2.0 Summary of Method
The method is initiated by filtering a water sample through a membrane filter. Following
filtration, the membrane containing the bacterial cells and DNA is placed in a microcentrifuge
tube with glass beads and buffer, and then shaken at high speed to extract the DNA into solution.
The supernatant is used for PCR amplification and detection of target sequences using the
TaqMan® Environmental master mix PCR reagent and probe system.
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Method 1609.1
3.0 Definitions
3.1 Enterococci: all species of the genus Enterococcus for which IsrRNA gene nucleotide sequences
were reported in the GenBank database (http://www.ncbi.nlm.nih/gov/Genbank') at the time of
method development.
3.2 Target sequence: An approximately 94 base pairs (bp) segment of the Enterococcus IsrRNA gene
containing nucleotide sequences that are homologous to both the primers and probe used in the
Enterococcus qPCR assay and that is common only to species within this genus.
3.3 Sample processing control (SPC) sequence: A 77 bp segment of the ribosomal RNA gene operon,
internal transcribed spacer region 2 of chum salmon, Oncorhynchus keta (O. ketd) and other
salmon spp., containing nucleotide sequences that are homologous to the primers and probe used
in the SPC qPCR assay. SPC sequences are added as part of a total salmon DNA solution in
equal quantities to all water sample filtrate and calibrator samples prior to extracting DNA from
the samples. The purpose of this control is to determine whether the sample was processed
correctly and/or to identify and correct for sample matrix effects on total DNA recovery (as
described in Section 9.12).
3.4 DNA standard: A purified, RNA-free and quantified and characterized Enterococcus faecalis (E.
faecalis) strain ATCC® 29212™ genomic DNA preparation. Note: DNA standards are used to
generate standard curves for determination of performance characteristics of the qPCR assays and
instrument with different preparations of master mixes containing TaqMan® reagent, primers and
probe as described in Section 9.10. Also used for quantifying target sequences in calibrator
sample extracts as described in Section 12.4.
3.5 Calibrator sample: Samples containing defined added quantities of E. faecalis strain ATCC®
29212™ cells and SPC sequences that are extracted and analyzed in the same manner as water
sample filtrates. Calibrator sample analysis results are used as positive controls for the
Enterococcus target sequence and SPC qPCR assays and as the basis for target sequence
quantification in water sample filtrates using the AACT comparative cycle threshold calculation
method (AACT method) as described in Section 12.5. Analysis results of these samples provide
corrections for potential daily or weekly method-related variations in Enterococcus cell lysis,
target sequence recovery and PCR efficiency. qPCR analyses for SPC sequences from these
samples are also used to correct for variations in total DNA recovery in the extracts of water
sample filtrates that can be caused by contaminants in these filtrates, as described in Section 12.4,
and/or to signal potentially significant PCR inhibition caused by these contaminants as described
in Section 9.8.
3.6 AACT method: A calculation method derived by AB (Reference 16.1) for calculating the ratios of
target sequences in two DNA samples (e.g., a calibrator and water filtrate sample) that normalizes
for differences in total DNA recovery from these samples using qPCR analysis CT (cycle
threshold) values for a reference (SPC) sequence that is initially present in equal quantities prior
to DNA extraction.
3.7 Amplification factor (AF): A measure of the average efficiency at which target or SPC sequences
are copied and detected by their respective primer and probe assays during each thermal cycle of
the qPCR reaction that is used in the AACT method. AF values can range from 1 (0% of
sequences copied and detected) to 2 (100% of sequences copied and detected) and are calculated
from a standard curve as described in Section 12.3.
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Method 1609.1
3.8 Internal amplification control (IAC): A non-target DNA sequence that is used to help specifically
identify false negative reactions or reduced amplification efficiency due to Taq DNA polymerase
inhibition. For an explanation of this type of PCR interference, see Sections 4.0 and 9.13.
4.0 Interferences
Water samples containing colloidal or suspended particulate materials can clog the membrane
filter and prevent filtration. the PCR analysis by
inhibiting the enzymatic activity of the Taq DNA polymerase and/or by interfering with the
annealing of the primer and probe oligonucleotides to sample target DNA, enzyme and/or by the
quenching of hydrolyzed probe fluorescence and/or by reducing (or potentially facilitating) total
5.0 Safety
5.1 The analyst/technician must know and observe the normal safety procedures required in a
microbiology and/or molecular biology laboratory while preparing, using, and disposing of
cultures, reagents, and materials, and while operating sterilization equipment.
5.2 Where possible, facial masks should be worn to prevent sample contamination.
5.3 Mouth-pipetting is prohibited.
6.0 Equipment and Supplies
6.1 Separated, and dedicated workstations for reagent preparation and for sample preparation,
preferably with high efficiency particulate air (HEPA)-filtered laminar flow hoods and an
Ultraviolet (UV) light source, each having separate supplies (e.g., pipettors, tips, gloves, etc.).
Note: While not recommended, the same workstation may be used for the entire procedure
provided that it has been cleaned with bleach and UV sterilized as specified in Section 11.8.1
between reagent and sample preparation. Under recommended conditions, the two dedicated
workstations should be in separate rooms with unidirectional workflow (i.e., all reagents should
be prepared before sample preparation and the reagent preparation room should not be re-entered
after moving to the sample preparation room.
6.2 Balance capable of accuracy to 0.01 g
6.3 Extraction tubes: semi-conical, screw cap microcentriruge tubes, 2.0 mL (e.g., Sarstedt
D-51588 or equivalent)
6.4 Glass beads, acid washed, 212 - 300 um (e.g., Sigma G-1277 or equivalent)
6.4.1 May also be purchased in extraction tubes from various commercial vendors (e.g., Gene-
Rite S0205-50 or equivalent)
6.5 Autoclave, capable of achieving and maintaining 121°C [15 Ib pounds per square inch (PSI)] for
minimally 15 minutes (optional, Note: Avoid use of autoclaves that are routinely used for
sterilizing biological materials. These autoclaves may contain target DNA contamination that has
been generated by aerosolization of DNA from the previous materials. This DNA is not
necessarily totally destroyed by the autoclaving process, and can contaminate your newly
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Method 1609.1
sterilized materials. If a dedicated autoclave for sterilizing non-biological materials is not
available, it may be preferable to either use disposable materials or sterilize reusable items with
bleach and UV light treatment.)
6.6 Workstation for water filtrations, preferably a HEPA-filtered laminar flow hood with a UV light
source. This can be the same as used for sample preparation, Section 6.1.
6.7 Sterile bottles/containers for sample collection
6.8 Membrane filtration units (filter base and funnel) for 47 mm diameter filters, sterile glass, plastic
stainless steel, or disposable plastic (e.g., Pall German 4242, Nalgene CN 130-4045,CN 145-
0045, or equivalent), cleaned and bleach treated (rinsed with 10% v/v bleach, then 3 rinses with
reagent-grade water), covered with aluminum foil or Kraft paper, and autoclaved or UV-
sterilized if non-disposable
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
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, 2 pairs
6.14 Polycarbonate membrane filters, sterile, white, 47 mm diameter, with 0.45 um pore size
(e.g., Millipore HTTP04700 or equivalent)
6.14.1 May also be purchased in sterile single use membrane filter units from various
commercial vendors (e.g., Pall FMFNL 1050 or equivalent)
6.15 Graduated cylinders, 100 - 1000 mL, cleaned and bleach treated (rinsed with 10% v/v bleach,
then 3 rinses with reagent-grade water), covered with aluminum foil or Kraft paper and
autoclaved or UV-sterilized
6.16 Petri dishes, sterile, plastic or glass, 100 x 15 mm with loose fitting lids
6.17 Disposable loops, 1 uL and 10 uL
6.18 Permanent ink marking pen for labeling tubes
6.19 Visible wavelength spectrophotometer capable of measuring at 595 nm
6.20 Single or multi-place bead beater (e.g., Biospec Products Inc. 3110BX or equivalent).
6.21 Microcentrifuge capable of 12,000 x g
6.22 Micropipettors with 10, 20, 200 and 1000 uL capacity. Under ideal conditions, each workstation
should have a dedicated set of micropipettors (one micropipettor set for pipetting reagents not
containing cells or DNA and one set for reagents containing DNA and test samples).
6.23 Micropipettor tips with aerosol barrier for 10, 20, 200 and 1000 uL capacity micropipettors.
Note: All micropipetting should be done with aerosol barrier tips. The tips used for reagents not
containing DNA should be separate from those used for reagents containing DNA and test
samples. Each workstation should have a dedicated supply of tips.
6.24 Microcentrifuge tubes, low retention, clear, 1.7 mL (e.g., GENE MATE C-3228-1 or equivalent)
6.25 Test tube rack for microcentrifuge tubes
6.26 Graduated conical centrifuge tubes, sterile, screw cap, 50 mL
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Method 1609.1
6.27 Test tubes, screw cap, borosilicate glass, 16 x 125 mm
6.28 Pipet containers, stainless steel, aluminum or borosilicate glass, for glass pipets
6.29 Pipets, sterile, T.D. bacteriological or Mohr, disposable glass or plastic, of appropriate volume
(disposable pipets preferable)
6.30 Vortex mixer (ideally one for each work station)
6.31 Dedicated lab coats for each work station
6.32 Disposable powder-free gloves for each work station
6.33 Refrigerator, 4°C (ideally one for reagents and one for DNA samples)
6.34 Freezer, -20°C and/or -80°C (ideally, one for reagents and one for DNA samples)
6.35 Ice, crushed or cubes for temporary preservation of samples and reagents
6.36 Printer (optional)
6.37 Data archiving system (e.g., flash drive or other data storage system)
6.38 UV spectrophotometer capable of measuring wavelengths of 260 and 280 nm using small volume
capacity (e.g., 0.1 mL) cuvettes orNanoDrop® (ND-2000) spectrophotometer (or equivalent)
capable of the same measurements at 2 uL sample volumes
6.39 Life Technologies StepOnePlus™, or AB 7500 Real-Time PCR System
6.39.1 Optical 96 well PCR reaction tray (e.g., Life Technologies MicroAmp™ 4346906 or
equivalent)
6.39.2 Optical adhesive PCR reaction tray tape (e.g., Life Technologies MicroAmp™ 4311971
or equivalent) or MicroAmp™ caps (e.g., Applied Biosystems N8010534 or equivalent)
6.39.3 Life Technologies StepOnePlus™, AB 7900, or AB 7500 Real-Time PCR System
6.40 Cepheid Smart Cycler® (optional to Section 6.39)
6.40.1 Smart Cycler® 25 uL PCR reaction tubes (e.g., Cepheid 900-0085 or equivalent)
6.40.2 Rack and microcentrifuge for Smart Cycler® PCR reaction tubes. Note: Racks and
microcentrifuge are provided with the Smart Cycler® thermocycler
6.40.3 Cepheid Smart Cycler® System thermocycler
7.0 Reagents and Standards
Note: The E.faecalis stock culture (Section 7.8), Salmon DNA/extraction buffer (Section 7.12),
and DNA extraction tubes (Section 7.18), may be prepared in advance.
7.1 Purity of Reagents: Use molecular grade reagents and chemicals in all tests
7.2 Control Culture
• E. faecalis ATCC® 29212™
May also be purchased in a quantified form from commercial vendors (e.g., bioMerieux 56005)
7.3 SPC DNA (source of SPC control sequences)
Salmon testes DNA (e.g., Sigma D1626 or equivalent)
7.4 Phosphate Buffered Saline (PBS)
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Method 1609.1
7.4.1 Composition:
Component Amount
Monosodium phosphate (NaH2PO4) 0.58 g
Disodium phosphate (Na2HPO4) 2.5 0 g
Sodium chloride 8.5 0 g
Reagent-grade water l.OL
7.4.2 Dissolve reagents in 1 L of reagent-grade 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 PSI) for 15 minutes. Final pH
should be 7.4 ± 0.2.
7.4.3 May also be purchased pre-made from various commercial vendors (e.g., Fisher
Scientific BP2438-4 or equivalent)
7.5 Brain heart infusion broth (BHIB)
7.5.1 Composition:
Component Amount
Calf brains, infusion from 200.0 g 7.7 g
Beef heart, infusion from 250.0 g 9.8 g
Proteose peptone 10. Og
Sodium chloride 5.0g
Disodium phosphate (Na2HPO4) 2.5 g
Dextrose 2.0 g
Reagent-grade water l.OL
7.5.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
completely. Dispense 10 mL volumes in screw cap 16 x 125 mm tubes and autoclave at
121°C (15 PSI) for 15 minutes. Final pH should be 7.4 ± 0.2.
7.5.3 Dry reagents may also be purchased pre-mixed from various commercial vendors (e.g.,
Bacto™ Brain Heart Infusion, Difco-BBL 237400 or equivalent) and dissolved with
water and sterilized as in Section 7.5.2.
7.6 Brain heart infusion agar (BHIA)
7.6.1 Composition:
BHIA contains the same components as BHIB with the addition of 15.0 g agar per liter of
BHI broth.
7.6.2 Add agar to formula for BHIB provided above. Prepare as in Section 7.5.2. After
sterilization, dispense 12-15 mL into 100 x 15 mm petri dishes. Final pH should be
7.4 ±0.2.
7.7 Sterile glycerol (used for preparation of E. faecalis stock culture as described in Section 7.8)
7.8 Preparation of E. faecalis (ATCC® 29212™) stock culture
Rehydrate lyophilized E. faecalis per manufacturer's instructions (for ATCC® stocks, suspend in
5-6 mL of sterile BHIB and incubate at 37°C for 24 hours). Centrifuge for 5 minutes at
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Method 1609.1
6000 x g to create a pellet. Using a sterile pipet, discard supernatant. Resuspend pellet in 10 mL
of fresh sterile BHI broth containing 15% glycerol and dispense in 1.5 mL aliquots in
microcentrifuge tubes. Freeze at -20°C (short term storage) or -80°C (long term storage). Note:
Aliquots of suspension may be plated (Section 11.1) to determine colony forming unit (CPU)
concentration. It is advisable to verify the E. faecalis culture as described, for example, in
Section 15 of EPA Method 1600.
7.9 PCR-grade water (e.g., OmniPur 9602, EMD Chemicals 9610 or equivalent). Water must be
DNA/DNase free.
7.10 Isopropanol or ethanol, 95%, for flame-sterilization
7.11 AE Buffer, pH 9.0 (e.g., Qiagen 19077 or equivalent) (Note: pH 8.0 is acceptable)
Composition:
10 mM Tris-Cl (Tris-chloride)
0.5 mM EDTA (Ethylenediaminetetraacetic acid)
7.12 Salmon DNA/extraction buffer
7.12.1 Composition:
Stock Salmon testes DNA (10 ug/mL) (Section 7.3)
AE Buffer (Section 7.11)
7.12.2 Preparation of stock Salmon testes DNA: The recommended source of Salmon DNA is
provided as a dried aggregate of purified high molecular weight genomic DNA. This
material can be dissolved either in its entirety or a desired mass can be separated from the
aggregate and dissolved. Add a volume in milliliters of AE buffer equal to the number
of milligrams of DNA to be dissolved and stir, using a magnetic stir bar at low to medium
speed, until dissolved (2-4 hours or longer, if necessary) followed by vigorous vortexing
to obtain a homogeneous DNA solution. Dilute an aliquot of this solution to a
concentration of ~ 10 ug/mL using AE buffer. Determine concentration of this solution
by optical density at 260 (OD26o) reading in a spectrophotometer. An OD26o of 0.2 is
approximately equal to 10 ug/mL. This is your Salmon testes DNA working stock
solution. Unused portions of the 1 mg/mL stock solution may be aliquoted and frozen at
-20°C.
Note: For example, if the bottle contains 250 mg of dried DNA, add 250 mL AE buffer
directly to the bottle. Cap tightly, and dissolve by at least 2-4 hours of gentle stirring
followed by vigorous vortexing. The concentration should be about 1 mg/mL. Remove a
0.5 mL aliquot and dilute to 50 mL with AE buffer. The concentration should be about 10
(ig/mL. Check absorbance (OD26o) and calculate actual DNA concentration using the
assumption that 1 OD26o, unit is equivalent to 50 ug/mL DNA. Adjust this working stock
to 10 ug/mL if necessary, either by further dilution or by adding additional 1 mg/mL
stock, vortexing and rechecking OD260. Aliquot portions of the adjusted working DNA
stock and store at 4°C in refrigerator.
7.12.3 Dilute Salmon testes DNA stock with AE buffer to make 0.2 ug/mL Salmon
DNA/extraction buffer. Extraction buffer may be prepared in advance and stored at 4°C
for a maximum of 1 week.
Note: Determine the total volume of Salmon DNA/extraction buffer required for each
day or week by multiplying volume (600 uL) x total number of samples to be analyzed
including controls, water samples, and calibrator samples. For example, for 18 samples,
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Method 1609.1
prepare enough Salmon/DNA extraction buffer for 24 extraction tubes [(18-^6 = 3,
therefore, 3 extra tubes for water sample filtration blanks (method blanks) and 3 extra
tubes for calibrator samples)]. Note that the number of samples is divided by 6 because
you should conduct one method blank for every 6 samples analyzed. Additionally,
prepare excess volume to allow for accurate dispensing of 600 uL per tube, generally 1
extra tube. Thus, in this example, prepare sufficient Salmon/DNA extraction buffer for
24 tubes plus one extra. The total volume needed is 600 uL x 25 tubes = 15,000 uL.
Dilute the Salmon testes DNA working stock 1:50, for a total volume needed (15,000 uL)
+ 50 = 300 uL of 10 ug/mL Salmon testes DNA working stock. The AE buffer needed is
the difference between the total volume and the Salmon testes DNA working stock. For
this example, 15,000 uL - 300 uL = 14,700 uL AE buffer needed.
7.13 Bleach solution: 10% v/v bleach (or other reagent that hydrolyzes DNA), used for cleaning work
surfaces
7.14 Sterile water (used as rinse water for work surface after bleaching)
7.15 TaqMan® Environmental PCR master mix 2.0 (e.g., Life Technologies 4396838 or equivalent)
7.16 Bovine serum albumin (BSA), fraction V powder (e.g., Sigma A-5611 or equivalent)
Dissolve in PCR-grade water a concentration of 2 mg/mL.
7.17 Primer and probe sets: Primer and probe sets may be purchased from commercial sources.
Primers should be desalted, probes should be HPLC (high-performance liquid chromatography)
purified.
7.17.1 Enterococcus primer and probe set (Reference 16.4):
Forward primer: 5'-GAGAAATTCCAAACGAACTTG
Reverse primer: 5'-CAGTGCTCTACCTCCATCATT
TaqMan® probe: [6-FAM]-5'-TGGTTCTCTCCGAAATAGCTTTAGGGCTA-TAMRA
7.17.2 Salmon DNA primer and probe set (Reference 16.4):
Forward primer: 5'-GGTTTCCGCAGCTGGG
Reverse primer: 5'-CCGAGCCGTCCTGGTC
TaqMan® probe: [6-FAM]-5'-AGTCGCAGGCGGCCACCGT-TAMRA
7.17.3 Internal Amplification Control (IAC) primer and probe set (Reference 16.4)
Primers: same as Enterococcus assay
UClPlTaqMan® probe: [VIC]-5'-CCTGCCGTCTCGTGCTCCTCA-TAMRA
Note: If using a Smart Cycler or other platforms that do not have channels for reading
VIC reporter dye, TET may be substituted for VIC as the reporter dye.
7.17.4 Preparation of primer/probes: Using a micropipettor with aerosol barrier tips, add AE
buffer to the lyophilized primers and probe from the vendor to create stock solutions of
500 uM primer and 100 uM probe and dissolve by extensive vortexing. Pulse centrifuge
to coalesce droplets. (Note: Some vendors, i.e., AB/Life Technologies, provide probes
already in solution at 100 uM concentration, part 450003). Store stock solutions at -20°C.
7.18 DNA extraction tubes
Note: It is recommended that tube preparation be performed in advance of water sampling and
DNA extraction procedures.
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Method 1609.1
Prepare 1 tube for each sample, and 1 extra tube for every 6 samples (i.e., for method blank) and
minimum of 3 tubes per week for calibrator samples. Weigh 0.3 ± 0.01 g of glass beads (Section
6.4) and pour into extraction tube. Seal the tube tightly, checking to make sure there are no beads
on the O-ring of the tube. Check the tube for proper O-ring seating after the tube has been closed.
Optional: Autoclave tubes at 121°C (15 PSI) for 15 minutes.
7.19 Purified, RNA-free quantified and characterized E. faecalis genomic DNA preparations or
commercial synthetic plasmid DNA for use as standards used to generate a standard curve (see
Sections 11.2 and 11.5).
7.20 RNase A (e.g., Sigma Chemical R6513) or equivalent
7.20.1 Composition:
RNase A
Tris-Cl
NaCl
7.20.2 Dissolve 10 mg/mL pancreatic RNase A in 10 mM Tris-Cl (pH 7.5), 15 mM NaCl. Heat
to 100°C for 15 minutes. Allow to cool to room temperature. Dispense into aliquots and
store at -20°C. For working solution, prepare solution in PCR-grade water at
concentration of 5 (ig/(iL.
7.21 DNA extraction kit (Gene-Rite K102-02C-5 0 DNA-EZ® RW02 or equivalent). (Note: This kit is
not required if using BioBalls).
7.22 Restriction enzyme: plasmid vector dependent (e.g., Pvu I for pIDTsmart cloning vector provided
by Integrated DNA Technologies or Hind III for pCR2.1 cloning vector provided by
EurofmsMWG/Operon). Alternative enzyme and/or plasmid vector combinations may be chosen
on the basis that the enzyme must have a single recognition site within the plasmid vector and no
recognition sites within the inserted template sequence.
7.23 IAC (Reference 16.4)
Brief description: The IAC5 template contains interspersed sequences that are homologous to the
forward and reverse primers of several qPCR assays including the Enterococcus qPCR assay used
in Method 1609.1. The Enterococcus assay primer pair produces a 111 bp amplicon from this
template that contains the UC1P1 probe recognition sequence and is slightly longer than the
corresponding native target sequence amplicon (see Section 3.2). The IAC5 template can be
ordered as a custom-synthesized gene from commercial vendors. In this process the template
sequence (see below) is synthesized in vitro, ligated with a plasmid vector chosen by the vendor
(e.g., pIDTSMART-KAN, Integrated DNA Technologies, Coralville IA or pCR2.1,
EurofmsMWG/Operon, Huntsville AL) and cloned into Escherichia coli. The recombinant
plasmid is recovered, purified and lyophilized by the commercial provider.
Template sequencea'b: 5'-
TCATGCAAGTCGAGCGATGGAGAAATTCCAAACGAACTTGGGGGTTCTGAGAGGAA
GGTGGTAGAGCACTGTTTCGGCATCTGAGGAGCACGAGACGGCAGGCTCGAGAAT
GATGGAGGTAGAGCACTGAAAAGGAAGATTAATACCGCATAGAGAATGTTATCACG
GGAGACAAGTAGCGTGAAGGATGACGG
a Enterococcus forward and reverse primer recognition sequences underlined
b UC1P1 probe recognition sequence in bold
-------
Method 1609.1
7.23.1 Calculation of IAC concentrations
o Centrifuge tube briefly and dissolve lyophilized plasmid (IAC) in AE buffer to make
a concentration of 20 (ig/mL (e.g., 2 (ig in 100 (iL). Incubate at room temperature for
30 minutes and then vortex for 20 seconds.
o Check three, 2 (iL aliquots of the dissolved plasmid for A260 and A280 absorbance
using a spectrophotometer against an AE buffer blank and record average
concentration. If the difference in expected and recorded concentrations is greater
than 20% of expected (Table 1), stop protocol and recalculate dilutions provided in
Table 2.
Table 1. Example Calculation of Stock Plasmid Concentration
Molecular weight (from vendor)
Target sequence copy (TSC)/g: (avo # / mol. Wt)
TSC/ng: molecules/g x 1xiQ"9g/ng
Expected stock concentrations (ng/uL)
Expected stock TSC concentrations (TSC/uL)
Mean NanoDrop concentration
1337095
4.50 x
4.50
1017
x108
20
9.01
x109
7.23.2 IAC linearization by restriction enzyme digestion
o Mix restriction enzyme buffer to make appropriate concentration (see Section 7.22
for enzyme choices and follow enzyme manufacturer's instructions for selection and
dilution of appropriate buffer) and 20 units of restriction enzyme and add PCR-grade
water to achieve a final volume of 40 (iL.
o Add 10 \\L (or 200 ng equivalent volume) of dissolved IAC plasmid stock. (Note:
Follow the manufacturer instructions, adjusting AE buffer volumes so that your final
concentration is 2 x 104 TSC/(iL.)
o Mix gently and incubate at 37°C for 1 hour to digest DNA.
o Dilute with AE buffer to bring the lAC/restriction enzyme digest volume up to 90
jiL.
o In appropriately labeled 1.7 mL low retention microcentrifuge tubes, perform serial
dilutions of the digested plasmid stock using AE buffer to achieve final digested
plasmid DNA concentration 2 x 104 TSC/(iL (2 x 10"5 dilution). Store digested
plasmid stock and 2 x 10"1 - 2 x 10'4 dilutions at -80°C. Store the 2 x 10'5 dilution in
the refrigerator.
Table 2. Initial Dilution of Digested IAC Plasmid Stock
Digested stock TSC concentrations:
Dilute 20 ML of stock w/80 ul_ AE (2 x 1 o1 diln)
Dilute 100 ul_of2 x 10'1 diln w/ 900 uLAE (2 x I0'2diln)
Dilute 100 ul_of2 x 10'2 diln w/ 900 uLAE (2 x I0'3diln)
Dilute 100 ul_of2 x 10'3 diln w/ 900 uLAE (2 x I0'4diln)
Dilute 100 ul_of2 x 10'4 diln w/ 900 uLAE (2 x I0'5diln)
IAC TSC/uL
1.0x 109
2.0 x 108
2.0 x 107
2.0 x 10s
2.0 x 105
2.0 x 104
IAC TSC/2 ul_
2.0 x 109
4.0 x 108
4.0 x 107
4.0 x 10s
4.0 x 105
4.0 x 104
7.23.3 Preparation of IAC working stocks
o Perform serial dilutions of the 2 x 105 dilution to prepare working stocks as indicated
in Table 3.
10
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Method 1609.1
Table 3. Example Preparation of IAC Working Stocks
Dilute
Dilute
Dilute
1000 ML
1000 ML
2500 ML
of2x
of2x
of2x
10'5diln
10'6diln
10'7diln
w/ 9000
w/ 9000
w/ 7500
MLAE
MLAE
MLAE
(2x 10'6
(2x 10'7
(8x 10'7
diln)
diln)
diln)
IAC TSC/ML
2
2
5
Ox 103
Ox 102
Ox 101
IAC TSC/2 ML
4.0 >
4.0 >
1.0 >
< 103
< 102
<102
o Prepare Entero/IAC master mix and each of the four dilutions (2 x 10~5, 2 x 10~6,
2 x 10~7, and 8 x 10~7) of IAC using basic recipe shown in Table 4. Note: Prepare
60 (iL of each IAC dilution to allow for three reactions per dilution.
Table 4. Example Preparation of Master Mix
Environmental master mix
BSA (2 mg/mL)
Primer/probe working stock (see Section 11.8.3)
IAC dilutions
Per Rxn
12.5 ML
2.5 ML
3.0 ML
2 ML
Per 3 Rxn
37.5 ML
7.5 ML
9.0 ML
6.0 ML
In PCR plate set up three reactions with each master mix containing 20 (iL of master
mix and 5 \\L of AE buffer and analyze.
Check slope of CT results from the 4 dilutions of IAC in the master mix (see Sections
9.10 and 12.4) and check CT results of reactions containing each dilution to
determine which dilution produces CT values in an acceptable range (CT = 30 - 34).
Dilutions giving CT values in this range are acceptable as IAC working stocks. If
none of the dilutions are within this range or if slope is substantially different from
that of the Enterococcus assay, repeat preparation of IAC plasmid dilutions and/or
prepare new IAC primer/probe working stock and reanalyze.
Split the remainder of the IAC working stock(s) into aliquots (e.g., enough for one
week of analyses each) in non-retentive tubes. Aliquots should be stored at -20°C.
8.0 Sample Collection, Handling, and Storage
8.1 Sampling procedures are briefly described below. Adherence to sample preservation procedures
and holding time limits is critical to the production of valid data. Samples not collected according
to these procedures should not be analyzed.
8.2 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 provide head space for proper mixing before analyses.
8.3 Storage Temperature and Handling Conditions
Ice or refrigerate water 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
11
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Method 1609.1
temperature. Ensure that sample bottles are tightly closed and are not totally immersed in water
during transit.
8.4 Holding Time Limitations
Examine samples as soon as possible after collection. Do not hold samples longer than 6 hours
between collection and initiation of filtration.
9.0 Quality Control
9.1 Each laboratory that uses Method 1609.1 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. Additional recommendations for QA and quality control (QC) procedures for
microbiological laboratories are provided in Reference 16.2.
9.2 The minimum analytical QC requirements for the analysis of samples using Method 1609.1
include an initial demonstration of laboratory capability through performance of initial DNA
standard curve (Section 9.10) and calibrator sample analyses (Section 9.11) followed by initial
precision and recovery (IPR) analyses (Section 9.3). Ongoing demonstration of laboratory
capability should be demonstrated through performance of the ongoing precision and recovery
(OPR) analysis (Section 9.4) and matrix spike (MS) analysis (Section 9.5), and the routine
analysis of positive control (Section 9.6), no template controls (NTCs [Section 9.7]), method
blanks (Section 9.8), media sterility checks (Section 9.9), DNA standard curves (Section 9.10),
calibrator samples (Section 9.11), and SPCs (Section 9.12). For the IPR, OPR and MS analyses,
it is necessary to spike samples with either laboratory-prepared spiking suspensions as described
in Section 11.3 or BioBalls as described in Section 11.4.
9.3 Initial precision and recovery (IPR) — The IPR analyses are used to demonstrate acceptable
method performance (recovery and precision) and should be performed by each laboratory before
the method is used for monitoring field samples. An IPR should be performed by each analyst.
The IPR analyses are performed as follows:
9.3.1 Prepare two or three calibrator samples and at least four reference (PBS) matrix spike
samples with same number of cells as indicated in Section 11.3. Preparation of calibrator
and reference (PBS) matrix spike samples with BioBalls is described in Section 11.4.
Extract calibrator and reference (PBS) matrix spike samples as indicated in Section 11.3
and analyze as indicated in Sections 11.7 - 11.9. Calculate the calibrator cell equivalents
(CCEs) in the reference (PBS) matrix spike samples according to Section 12. Note:
Prepare calibrator samples from the same source of cells used to prepare the reference
(PBS) matrix spike samples, i.e., for samples spiked with laboratory-prepared cell
suspensions, use the same laboratory-prepared cell suspensions used to prepare calibrator
samples; for samples spiked with BioBalls, use the same lot of BioBalls to prepare
calibrator and reference (PBS) matrix spike samples.
9.3.2 Calculate the percent recovery (R) for each IPR sample according to the Method 1609.1
Calculation Excel file.
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.
12
-------
Method 1609.1
9.3.4 Compare the mean recovery and RSD with the corresponding IPR criteria in Table 5. 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 recovery
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 the IPR analyses.
Note: Meeting the criteria established using BioBalls should also be a goal for laboratory
spikes.
Table 5. Calculated IPR Acceptance Criteria at 550 CPU
Spike Type
Lab-prepared
BioBall®
IPR Mean Recovery (%)
Detect -2, 897
22-197
IPR RSD (%)
126
104
OPR Recovery (%)
Detect -3, 064
Detect - 256
9.4 Ongoing precision and recovery (OPR) — To demonstrate ongoing control of the analytical
system, the laboratory should routinely process and analyze reference (PBS) matrix spike
samples. The laboratory should minimally analyze one OPR sample once a week that samples
are analyzed. The OPR analysis is performed as follows:
9.4.1 Prepare one reference (PBS) matrix spike and two or three calibrator samples as indicated
in Sections 9.3.1 and 11.3 (calibrator samples can be same as those used for weekly
analyses). Preparation of calibrator and reference (PBS) matrix spike samples with
BioBalls is described in Section 11.4. Filter and process the OPR sample according to
the procedures in Section 11 and calculate the target sequences in the samples according
to Section 12.
9.4.2 Calculate the percent recovery (R) for the OPR sample according to the Method 1609.1
Calculation Excel file using the appropriate spreadsheets.
9.4.3 Compare the OPR result (percent recovery) with the corresponding OPR recovery criteria
in Table 5, 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, reagents,
and controls, correct the problem and repeat the OPR analysis.
9.4.4 As part of the laboratory QA program, 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
1609.1 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 Matrix spikes (MS) — MS analyses are performed to determine the effect of a particular matrix
on enterococci recoveries. The laboratory should analyze one MS sample when recreational
water samples are first received from a source from which the laboratory has not previously
analyzed samples. Subsequently, 5% of field samples (1 per 20) from a given source should
include a MS sample. MS samples must be accompanied by the analysis of an unspiked field
sample sequentially collected from the same sampling site or of another unspiked aliquot of the
same field sample. The MS analysis is performed as follows:
9.5.1 Filter two, field samples (See Section 11.6.4) that were sequentially collected from the
same site or different aliquots of the same field sample as described in Section 11.6. One
sample will remain unspiked and will be analyzed to determine the background or
ambient concentration of enterococci for calculating MS recoveries (Section 9.5.3). The
13
-------
Method 1609.1
other sample will serve as the MS sample and will be spiked with E. faecalis ATCC®
29212™ according to the spiking procedure in Section 11.3 or Section 11.4 if using
BioBalls. Note: For matrix spiking, filter the same volume as was filtered for the field
samples.
9.5.2 Extract both the unspiked and spiked field samples according to the procedures in Section
11.7 and analyze in parallel with two or three calibrator sample extracts as described in
Section 11.3 or Section 11.4 if using BioBalls.
9.5.3 For the MS sample, calculate the number of CCE and/or target sequences according to
Section 12 and adjust based on any background enterococci CCE and/or target sequences
calculated in the same manner in the unspiked matrix sample.
9.5.4
9.5.5
9.5.6
Calculate the percent recovery (R) for the MS sample (adjusted based on ambient
enterococci in the unspiked sample) according to the Method 1609.1 Calculation Excel
File using the appropriate spreadsheets. Note: recovery results for sample containing
ambient enterococci in the unspiked sample that are > the spike density should be
excluded from this analysis (Reference 16.12)
Compare the MS result (percent recovery) with the appropriate method performance
criteria in Table 6. If the MS recovery meets the acceptance criteria, and system
performance is acceptable, then analysis of field samples from this source may continue.
If the MS recovery is unacceptable and the OPR sample result associated with this batch
of samples is acceptable, a matrix interference may be causing the poor results. If the MS
recovery is unacceptable, all associated field data should be flagged. Note: Meeting
criteria established using BioBalls should also be a goal for laboratory spikes.
Laboratories should record and maintain a control chart comparing MS recoveries for all
matrices to batch-specific and cumulative OPR sample results analyzed using Method
1609.1. These comparisons should help laboratories recognize matrix effects on method
recovery and may also help to recognize inconsistent or sporadic matrix effects from a
particular source.
Table 6. Calculated MS Precision and Recovery Acceptance Criteria at 550 CPU
Matrix
MW
FW
Spike Type
Lab-prepared
BioBall®
Lab-prepared
BioBall®
MS Recovery (%)
Detect -13,51 3
Detect -181
Detect -6, 268
Detect - 330
MS RPD 1 (%)
243
145
918
275
9.6
Relative percent difference
Positive controls — The laboratory should analyze positive controls to ensure that the method is
performing properly. Fluorescence amplification growth curve (PCR amplification trace) with an
appropriate CT value during PCR indicates proper method performance. On an ongoing basis, the
laboratory should perform positive control analyses every day that samples are analyzed. In
addition, controls should be analyzed when new lots of reagents or filters are used.
9.6.1 Calibrator samples will serve as the positive control. Analyze as described in Section
11.3 or Section 11.4 if using calibrator samples prepared from BioBalls. Note: Calibrator
samples contain the same amount of extraction buffer and starting amount of Salmon
DNA as the test samples; hence E. faecalis calibrator sample extracts (Section 11.3) will
be used as a positive control for both Enterococcus and SPC qPCR assays.
9.6.2 If the positive control fails to exhibit the appropriate fluorescence growth curve response,
check and/or replace the associated reagents, and reanalyze. If positive controls still fail
14
-------
Method 1609.1
to exhibit the appropriate fluorescence growth curve response, prepare new calibrator
samples and reanalyze (see Section 9.11).
9.7 No template control (NTC) — The laboratory should analyze NTCs to ensure that the PCR
master mix reagents are not significantly contaminated. On an ongoing basis, the laboratory
should perform NTC analyses every day (or at least every week) that samples are analyzed. If the
mean CT value of the NTC reactions produces a CSE or CCE value that is greater than the method
limit of detection/quantification (see Section 9.14), the analyses should be repeated with new
master mix working stock preparations. If unacceptable results are persistent, consider
individually replacing each of the components of the reaction mixes (see Table 8) with new lots
or new sources of the components and retesting. Note: While NTC analyses should not produce a
CT value (i.e., reported as "0" on Smart Cycler® and "Undetermined" on AB instruments),
available results from a number of laboratories have suggested that this result can be difficult to
achieve for all NTC analyses. CT values producing CCE values that are less than the method
limit of detection/quantification are therefore considered acceptable for recreational water quality
monitoring in association with the EPA RWQC (Reference 16.11) since the levels of
contaminating target sequences that these values represent should not lead to incorrect beach
management decisions. This relaxed acceptance criterion, however, is based on the provisions
that standard curve slope and intercept parameters follow the general guidelines provided in
Section 9.10 and that no more than a 5-fold dilution of the test sample extracts are analyzed.
9.8 Method blank (water sample filtration blank) — Filter a 20-30 mL volume of sterile PBS before
beginning the sample filtrations. Remove the runnel from the filtration unit. Using two sterile or
flame-sterilized forceps, fold the filter on the base of the filtration unit and place it in an
extraction tube with glass beads as described in Section 7.18. Extract as in Section 11.7. Criteria
for the acceptance of method blank sample analysis results are the same as for NTC analyses (see
Section 9.7). Prepare at least one method blank filter for every PCR run (e.g., a minimum
two method blanks per 96 well plate or 1 per Smart Cycler run - see Sections 11.9 and
11.10). If unacceptable results are persistent, consider replacing PBS (Section 7.4) and/or AE
(Section 7.11) with new lots or new sources of these buffers and retesting. If unacceptable results
continue to persist there may be a contamination problem in the physical environment of the
filtration workstation. If possible, perform filtrations at a new location to confirm. Clean current
filtration workstation and filtration manifold thoroughly with bleach solution.
9.9 Media and PBS contamination check — The laboratory should test media sterility by
incubating one unit (plate or tube) from each batch of medium (BHIA/BHIB) as appropriate and
observing for growth. PBS sterility should be checked by filtering 20 mL of PBS and incubating
the filter on BHIA as appropriate and observing for growth. Absence of growth indicates media
and PBS sterility. On an ongoing basis, the laboratory should perform a PBS sterility with each
new container of PBS, and media sterility checks should be performed with each new batch of
media as needed.
9.10 DNA standards and standard curves — Purified, RNA-free and spectrophotometrically
quantified E. faecalis genomic DNA should be prepared as described in Section 11.2. Based on
reported values for it size, the weight of a single E. faecalis genome can be estimated to be -3.6
fg and there are four IsrRNA gene copies per genome in this species (see the rRNA operon
database at http://rrndb.cme.msu.edu). The concentration of IsrRNA gene copies per uL in the
standard E. faecalis genomic DNA preparation can be determined from this information and from
its spectrophotometrically determined total DNA concentration by the formula:
Concentration of IsrRNA Total DNA concentration (fg/uL) x 4 IsrRNA gene copies
gene copies per uL = 3 6 fg/genome
genome
15
-------
Method 1609.1
A composite standard curve should be generated from estimated log 10 target sequence copy
numbers and CT values of at least four independent analyses of at least four serial dilutions of this
DNA standard (see Sections 11.2 and 12.3) using the Enterococcus primer and probe assay and
subjected to linear regression analysis after removal of any significant outlier CT measurements.
Regressions can be performed using the "regression" Analysis Tool which can be accessed in the
drop down menu when clicking on "Data Analysis" under the "Data" tab in Microsoft Office
Excel 2007 (access may differ in different versions of Excel). This tool provides the mean and
95% confidence ranges for slope and intercept parameters in the initial composite standard curve
as well as R-square values (see example in Figure 1 below). A simplified and recommended tool
for performing this analysis as well as a tool for identifying outlier measurements is also provided
in the Method 1609.1/1611.1 calculation spreadsheet available at:
(http://water.epa.gov/scitech/methods/cwa/bioindicators/biological_index.cfm).
Figure 1. Regression Analysis Standard Curve Data.
Regression Statistics
Multiple R 0.97998503
R Square 0.96037065
Ad j u ste d R Sq u a re 0. 96027784
Standard Error 0.57840384
Observations | 429
ANOVA 1 1
df
SS
MS
ignificance F
Regression
Residual
Total
1
427
428
3461.881244 3461.881
142.8532799 0.334551
3604.734524
10347.84
1.87E-301
Coefficients Standard Error tStat P-value Lower 95% Upper 95%
Intercept 37.2137739 0.070444654 528.2697 0 37.075312 37.3522353
X Variable 1 -3.3402532 0.03283632 -101.724 1.9E-301 -3.404794 -3.2757123
Following general industry recommendations
(http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR/real-time-
pcr/qpcr-education/pcr-understanding-ct-application-note.html), curve slope values should be
within the range of 3.1 - 3.6 (amplification efficiency between 90 and 110%) for each individual
curve used to generate the composite curve. Based on available data from curves generated with
the reagents and thermal cycling conditions specified in this method on a StepOnePlus™
platform and using DNA extracts with most probable number (MPN) estimated target sequence
copy concentrations (Reference 16.6), y-intercept values should fall within the range illustrated in
Figure 2 for a given slope value. (Note: This range can only be provided as a guideline and not as
a requirement, due to the uncertainty in the available MPN-estimated target sequence copy
concentrations; expected intercept values also may differ for other platforms).
Figure 2. Guidelines for Intercept and Slope Values from Standard Curves.
16
-------
Method 1609.1
38.5
38.0
.37.5
ffi
'37.0
36.5
36.0
-3.6
-3.5
-3.4
-3.3
-3.2
-3.1
Slope
9.11
The R square values from regressions of these curves should ideally be 0.99 or greater. A
convenient ANCOVA test for determining similarity of curves is provided in the Method
1609.1/1611.1 calculation spreadsheet. In the event that the combination of slope and intercept
values or R square values from the regressions of one or more individual curves are substantially
outside of the guidelines in Figure 2 or differ significantly from the other curves generated by the
individual laboratory, these curves should be repeated. Amplification efficiency factor (AF)
values can be calculated from the slope of the initial composite standard curve as described in
Section 12.3.
From that point on, it is highly recommended that additional standard curves be generated from
triplicate analyses of these same diluted standard samples with each new lot of TaqMan® master
mix reagents or primers and probes to demonstrate comparable performance by the new reagents.
The R square values from regressions of these curves should ideally be 0.99 or greater after
removal of any outlier CT measurements. Comparable performance is assessed by the slopes of
the new curves which should be within the range of values from the initial curves established by
the individual laboratory. These assessments can be made either empirically or preferably by
using the ANCOVA test in the Method 1609.1/1611.1 calculation spreadsheet.
In the event that the slope value from a subsequent standard curve regression is outside of the
initial range, the standards should be re-analyzed. If this difference persists, new working stocks
of the reagents should be prepared and the same procedure repeated. Failure to meet this criterion
or the observation of a significantly higher intercept value also may be an indication of
deterioration of the DNA standards. New serial dilutions can be prepared from a freezer stock of
the DNA standard (see Section 11.2) or frozen aliquots of the previously prepared standards can
be thawed and analyzed as described above. If the differences continue to persist, a new
composite standard curve should be generated and the AF values recalculated as described above.
Calibrator samples — The cell concentration of each cultured E. faecalis stock suspension used
for the preparation of calibrator sample extracts should be determined as described in Section
11.1. The manufacturer's lot-specific certificate of analysis can be used to identify cell numbers
in BioBalls. Initially, a minimum of four sets of calibrator sample extracts should be prepared
17
-------
Method 1609.1
and analyzed on different days from different freezer-stored aliquots of these stocks or from
different BioBalls (i.e., two or three (three is recommended) frozen cell or BioBall® extracts
prepared per day and analyzed in duplicate on at least four different days) as described in Section
11.3 or 11.4. Dilutions of these calibrator sample extracts equivalent to the anticipated dilutions
of the test samples used for analysis (e.g., 1, 5 and/or 25-fold) should be analyzed induplicate
with different preparations of the Entero/IAC and Salmon DNA (Sketa 22) assay mixes (Section
11.8) on each day. The overall average and standard deviation of the Entero/IAC and Salmon
DNA (Sketa 22) assay ACT values from these analyses should be determined for each relevant
dilution after removal of any extreme measurements (> ± 3 standard deviations). A simple test for
identifying extreme measurements is to exclude any measurements that are suspected of being
extreme from the data set, determine the standard deviation of the remaining measurements and
then determine whether the excluded measurements are within ± 3 standard deviations of the
mean. The mean ACT value for each calibrator sample and the overall mean and standard
deviation of these ACT values should also be calculated to obtain initial laboratory-specific
baseline values. These calculations are automatically performed in the Method 1609.1/1611.1
calculation spreadsheet.
From that point on, fresh calibrator sample extracts should be prepared from an additional frozen
aliquot of the same stock cell suspension or from the same lot of BioBalls at least weekly either
before or with preparation and analyses of each batch of test samples as described in Section 11.3
or 11.4. Dilutions of each new calibrator sample extract equivalent to the initial dilutions (e.g., 1,
5 and/or 25 fold) should be analyzed in duplicate using the Entero/IAC and Sketa22 assays. Each
individual Entero and Sketa22 CT measurement and the mean ACT value from these analyses
should be within ± 3 standard deviations from the laboratory's mean baseline values from
analyses of their initial calibrator sample extracts. Laboratories will be able to use ongoing
calibrator sample results if at least one of the Entero and Sketa22 CT measurements meets this
acceptance criterion. They may, however, wish to adopt more stringent QC requirements such as,
for example, a majority or all of the CT measurements having to meet this criterion. If, as
expected, more than one of the calibrator sample Entero and Sketa22 CT values meets this
criterion, the average of the acceptable CT values from each assay should be taken and the average
Sketa22 CT value should be subtracted from the average Entero CT value to give a mean ACT
value. The mean ACT value also should meet the within 3 standard deviations criterion. Results
from calibrator samples that do not meet the laboratory's criteria should not be used for
calculating test sample results. These analyses are automatically performed in the Method
1609.1/1611.1 calculation spreadsheet. If the calibrator sample measurements do not meet the
laboratory's acceptance criteria, new calibrator extracts should be prepared from another frozen
aliquot of the same stock cell suspension or lot of BioBalls and analyzed in the same manner as
described above. If the results are still not within the acceptance range, the reagents should be
checked by the generation of a standard curve as described in Section 9.10. Calibrator samples
prepared from new cultured E. faecalis cell suspensions or new lots of BioBalls must also meet
the laboratory's ongoing acceptance criteria. If these samples repeatedly fail the criteria, the
above procedures for determining initial calibrator sample ACT measurements should be repeated
with samples prepared from the new cultured E. faecalis cell suspension or lot of BioBalls. Note:
The 25-fold dilution is not applicable with extracts of laboratory-prepared calibrator samples
containing <1000 cells per sample or extracts of BioBall® calibrator samples, as these dilutions
may contain target sequence quantities that are below the method's limit of detection (see also
Sections 9.14, 11.3 and/or 11.4).
9.12 Salmon DNA Sample Processing Control (SPC) sequence analyses — The Salmon DNA SPC
is used as a DNA recovery control. Possibly due to difficulties in obtaining homogenous
suspensions of this high molecular weight DNA or due to lot to lot variations in this product,
some degree of variability in the CT values may be obtained from different initial preparations or
18
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Method 1609.1
diluted batches of this DNA. Available data from the StepOnePlus™ platform suggest that CT
values from undiluted 0.2 (ig/mL salmon DNA extraction buffer s should fall within a range of ~
18-22 units for this assay (values may differ for other platforms). Because it is not used as an
absolute standard in the method, such variation may be acceptable, however, changes in CT
values from new diluted batches of this DNA may necessitate the generation of new initial
calibrator baseline ACT values in the Method 1609.1/1611.1 calculation spreadsheet for ongoing
analyses (see Section 9.11). It is permissible to adjust the concentrations of new diluted batches
of this DNA to obtain CT values that are similar to those of previous diluted batches in order to
avoid having to generate new initial calibrator baseline ACT values.
While not mandatory, it is also good practice to prepare and analyze serial dilutions of the
Salmon DNA extraction buffer as described for the Enterococcus DNA standards in Section 9.10.
To our knowledge, rRNA gene operon copy numbers per genome have not been reported for the
salmon species O. keta. However, log-transformed target sequence values of the Enterococcus
DNA standards can be used for both assays for comparisons. A procedure is provided in a
guidance document from the developers of the AACT calculation (Reference 16.1), for comparing
the slopes of the Sketa22 and Enterococcus assays. (Note: serial dilutions of salmon and
Enterococcus DNA do not necessarily need to be performed in the same tubes for this analysis as
specified in the guidance document but it is recommended that the analyses of these dilutions be
performed on the same plates). Available data indicates that the slopes of the EnterolA and
Sketa22 assays meet the criterion for acceptable similarity specified in the guidance document. If
results from an individual lab do not meet this criterion, it is recommended to make new working
stocks of the Sketa22 assay probe and primers and/or new serial dilutions of the salmon DNA and
repeat the test.
In general, target DNA concentrations in test samples can be calculated as described in Section
12. However, the Salmon DNA PCR assay results for each test sample should be within 3 CT
units of the mean of the similarly diluted calibrator Salmon DNA sample results. Higher CT
values may indicate significant PCR inhibition or poor DNA recovery possibly due to physical,
chemical, or enzymatic degradation. While poor DNA recovery is compensated for by the
calculation method, a salmon DNA assay offset CT value of greater than 3 CT units suggests that
total DNA recovery was < -10% and therefore potentially unacceptably high levels of
enterococci may not be detectable due to the very low recovery. When the Salmon PCR assay CT
value is within the 3 CT unit cutoff, the result from the IAC assay can be used to further determine
the acceptability of the sample result as indicated in Section 9.13.
Note: If salmon DNA assay CT values from analyses of a particular batch of test samples are all
substantially higher or lower (e.g., > ±1 CT unit) than the mean of corresponding diluted
calibrators samples, and this phenomenon is not normally observed for samples from this site, it
is recommended that new calibrator samples be prepared and the test samples and new calibrator
sample be re-analyzed. While consistent salmon DNA assay CT offsets between the test samples
and calibrator samples of > 1 CT unit may be attributable to certain water matrices (Reference
16.4), such results have also been observed to be occasionally attributable to an as yet
unidentified source of error in the analytical method. Therefore the validity of these types of
salmon DNA assay results should be confirmed. Determination of percent recovery of matrix
spikes of these test samples (see Section 9.5) also may be useful in determining whether the
abnormal differences between salmon DNA assay CT values for these test samples and the
calibrators are an artifact of the analytical method or represent true test sample effects on DNA
recovery. Potential mitigation procedures involving the use of higher salmon DNA concentrations
in the extraction buffer for extraction of samples from sites that consistently produces
substantially higher CT measurements than those of corresponding diluted calibrators samples
19
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Method 1609.1
have been proposed (Reference 16.4). Salmon DNA concentrations in the extraction buffer used
to extract calibrator samples should always be equivalent to those used to extract test samples.
9.13 Internal Amplification Control (IAC) sequence analyses — The IAC assay is expected (and
thus far has been demonstrated, Reference 16.4) to be a sensitive indicator of inhibition of the
Enterococcus assay since the two assays utilize the same primer set and because the IAC template
is added just prior to the reaction. A more stringent acceptance criterion has been adopted for this
assay where test sample CT values cannot be offset by >1.5 units compared to negative control
samples with no target DNA (Reference 16.4). The Enterococcus, IAC and salmon DNA PCR
assays can be repeated on any samples that fail this criterion using a 5-fold higher dilution of the
sample extracts. If the IAC assay CT value of the diluted extract is now within the 1.5 CT unit
acceptance range, the corresponding Enterococcus and salmon assay results from this dilution can
now be accepted and used in the comparative CT calculation method (see Section 12) with
Enterococcus and salmon assay CT results from equally diluted calibrator samples. As described
in Section 7.23.3, mean CT values for calibrator and/or control samples with this assay are
considered to be acceptable within a range of-30-34 units for StepOnePlus™ platforms (values
may vary for other platforms). These values have been found to be indicative of a sufficient
quantity of IAC template for the assay to show acceptable reproducibility in measurements while
not causing competitive inhibition of Enterococcus target sequence amplification.
Note: available data have indicated that high concentrations of enterococci target sequences in a
sample (e.g., > 105 - 106 copies/sample or > 103 - 104 copies/reaction) can significantly decrease
IAC assay CT values by an unknown facilitation process in this method. When test samples CT
values indicate that Enterococci target sequence quantities in the reactions are in this range or
greater, the IAC CT values from these reactions may be more appropriately compared with those
from calibrator samples containing laboratory prepared cell suspensions (e.g., ~ 104
cells/calibrator sample, see Sections 11.1 and 11.3) than those from the negative control samples
or from BioBall calibrator samples. Available data indicate that Enterococcus sequence
concentrations that are high enough to have this facilitative effect on the IAC CT value will
indicate unacceptable water quality so the IAC result can be ignored from the standpoint of beach
advisory decisions with no further analysis required. For such samples however, it may be
desirable to either add a higher quantity of IAC templates to the reaction or further dilute the
sample extract to the point where the IAC result passes the 1.5 CT offset criterion in order to
exclude the possibility of partial inhibition by the sample matrix. If a higher IAC template
concentration is used in this repeat analysis and passes the 1.5 CT offset criterion, the
Enterococcus assay CT value from the initial analysis should be used for calculating Enterococci
densities in the test sample. If the sample extract is diluted, the Enterococcus assay Ct value from
the repeat analysis should be used in conjunction with Ct results from equally diluted calibrator
sample extracts for calculating Enterococci densities in the test sample as indicated above.
Use of the multiplex IAC assay currently is not mandatory in this method but is highly
recommended as a convenient surrogate test for identifying potential sample matrix inhibition and
as a general positive control for each sample analysis. Determination of percent recovery of
matrix spikes (see Section 9.5) is recommended to obtain the most reliable information on the
effects of sample matrices on method performance.
9.14 Method Limit of Detection/Quantification — A report on the limit of quantification of the
method is available at:
http://water.epa.gov/scitech/methods/cwa/bioindicators/biological index.cfm. The 33%
coefficient of variation values in this report are acceptable. The reported values are for 5x-diluted
extracts and can be extrapolated to undiluted and 25x-diluted extracts by dividing or multiplying
by 5, respectively. These values can be used for determining acceptable NTC and method blank
results (see Sections 9.7 and 9.8). Limits of detection will be considered as equivalent to these
20
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Method 1609.1
values for the purposes of documenting laboratory capability. Laboratories are encouraged to
document a similar limit of detection capability as described in Section 11.2.23.5. Ability to
detect a 5 target sequence copy/reaction sample as described in this section will provide this
documentation. Ability to detect a 10 target sequence copy/reaction standard as described in
Section 11.2.25 will be considered as adequate.
10.0 Calibration and Standardization of Method-Related Instruments
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 National Institute of Standards and Technology
(NIST) certified thermometer or one that meets the requirements of NIST Monograph SP 250-23.
Check columns for breaks.
10.3 Spectrophotometer should be calibrated each day of use using OD calibration standards between
0.01 - 0.5. Follow manufacturer instructions for calibration.
10.4 Micropipettors should be calibrated at least annually and tested for accuracy on a weekly basis.
Follow manufacturer instructions for calibration.
10.5 Follow manufacturer instructions for calibration of real-time PCR instruments.
11.0 Procedure
Note: Procedures described in Section 11.1 can be omitted if laboratories choose to use
commercially available E. faecalis cells (BioBalls) and procedures described in Section 11.2 can
be omitted if laboratories choose to use DNA standards provided by EPA (see Appendix E).
Preparation of calibrator, reference (PBS) matrix spike and matrix spike samples with BioBalls is
described in Section 11.4. Preparation and use of EPA plasmid DNA standards is described in
Section 11.5. Laboratory-prepared E. faecalis cell suspensions (Section 11.1), DNA standards
(Section 11.2) and preliminary calibrator samples (Section 11.3) should be prepared and analyzed
in advance of any test sample analyses (see Sections 9.10 and 9.11).
11.1 Preparation of laboratory-cultured E. faecalis cell suspensions for DNA standards, calibrator
samples, matrix spike samples and spiked reference (PBS) matrix filters
11.1.1 Thaw an E. faecalis (ATCC® 29212™) stock culture (Section 7.8) and streak for
isolation on BHIA plates. Incubate plates at 37°C ± 0.5°C for 24 ± 2 hours.
11.1.2 Pick an isolated colony of E. faecalis from BHIA plates and suspend in 1 mL of sterile
PBS and vortex.
11.1.3 Use 10 uL of the 1 mL suspension of E. faecalis to inoculate a 10 mL BHIB tube.
Place the inoculated tube and one uninoculated tube (sterility check) on a shaker set at
250 rpm [revolutions per minute] and incubate at 37°C ± 0.5°C for 24 ± 2 hours. Note:
It is advisable to verify that the selected colony is Enterococcus as described, for
example, in Section 12.0 of EPA Method 1600.
11.1.4 After incubation, centrifuge the BHIB containing E. faecalis for 5 minutes at 6000 x g.
11.1.5 Aspirate the supernatant and resuspend the cell pellet in 10 mL PBS.
21
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Method 1609.1
11.1.6 Repeat the two previous steps twice and suspend final E. faecalis pellet in 5 mL of
sterile PBS. Label the tube as E. faecalis undiluted stock cell suspension, noting cell
concentration after determination by the following steps.
11.1.7 Determination of calibrator sample cell concentrations based on the plating method
below.
Note: BHIA plates should be prepared in advance if this option is chosen. For
enumeration of the E. faecalis undiluted cell suspension, dilute and inoculate according
to the following.
A) Mix the E. faecalis undiluted cell suspension by vigorously shaking the 5 mL
tube a minimum of 25 times. Use a sterile pipette to transfer 0.5 mL of the
undiluted cell suspension to 49.5 mL of sterile PBS, cap, and mix by vigorously
shaking the tube a minimum of 25 times. With care, a 50 mL tube may be used.
This is cell suspension dilution "A". A 1.0 mL volume of dilution "A" is 10"2
mL of the original undiluted cell suspension.
B) Use a sterile pipette to transfer 5.0 mL of cell suspension dilution "A" to 45 mL
of sterile PBS, cap, and mix by vigorously shaking the tube a minimum of 25
times. This is cell suspension dilution "B". A 1.0 mL volume of dilution "B" is
10"3 mL of the original undiluted cell suspension.
C) Use a sterile pipette to transfer 5.0 mL of cell suspension dilution "B" to45 mL
of sterile PBS, cap, and mix by vigorously shaking the tube a minimum of 25
times. This is cell suspension dilution "C". A 1.0 mL volume of dilution "C" is
10"4 mL of the original undiluted cell suspension.
D) Use a sterile pipette to transfer 5.0 mL of cell suspension dilution "C" to 45 mL
of sterile PBS, cap, and mix by vigorously shaking the tube a minimum of 25
times. This is cell suspension dilution "D". A 1.0 mL volume of dilution "D" is
10"5 mL of the original undiluted cell suspension.
E) Use a sterile pipette to transfer 5.0 mL of cell suspension dilution "D" to 45 mL
of sterile PBS, cap, and mix by vigorously shaking the tube a minimum of 25
times. This is cell suspension dilution "E". A 1.0 mL volume of dilution "E" is
10"6 mL of the original undiluted cell suspension.
F) Prepare BHIA (Section 7.6). Ensure that agar surface is dry. Note: To ensure
that the agar surface is dry prior to use, plates should be made several days in
advance and stored inverted at room temperature or dried using a laminar-flow
hood.
G) Each of the following will be conducted in triplicate, resulting in the evaluation
of nine spread plates:
• Pipet 0.1 mL of dilution "C" onto surface of BHIA plate [10"5 mL (0.00001)
of the original cell suspension].
• Pipet 0.1 mL of dilution "D" onto surface of BHIA plate [10"6 mL (0.000001)
of the original cell suspension].
• Pipet 0.1 mL of dilution "E" onto surface of BHIA plate [ 10"7 mL
(0.0000001) of the original cell suspension].
H) For each spread plate, use a sterile bent glass rod or spreader to distribute
inoculum over surface of medium by rotating the dish by hand or on a rotating
turntable.
22
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Method 1609.1
I) Allow inoculum to absorb into the medium completely.
J) Invert plates and incubate at 35°C ± 0.5°C for 24 ± 4 hours.
K) Count and record number of colonies per plate. Refer to the equation below for
calculation of undiluted cell suspension concentration.
CFU! + CFU2+ + CFUn
CFU/mLundiluted = „ T , Tr , . ,,
Where:
CPU
V
n
E. faecalis CPU [colony forming units]/mL in undiluted cell
suspension
number of colony forming units from BHIA plates yielding
counts within the ideal range of 30 to 300 CPU per plate
volume of undiluted sample in each BHIA plate yielding
counts within the ideal range of 30 to 300 CPU per plate
number of plates with counts within the ideal range
Table 7. Example Calculations of E. faecalis Undiluted Cell Suspension Concentration
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from BHIA plates
10~5 mL plates
275, 250, 301
TNTCb, TNTC, TNTC
10~6 mL plates
30, 10, 5
TNTC, 299, TNTC
10~7 mL plates
0, 0, 0
12, 109, 32
E. faecalis CPU / mL in undiluted
suspension3
(275+250+30) /(10"5+10-5+10'6) =
5557 (2.1 x 10^) = 26,428,571 =
2.6 x 107CFU/mL
(299+109+32) /(10'6+10-7+10-7) =
440 / (1 .2 x 1 0'6) = 366,666,667 =
3.7 x 108CFU/mL
Cell concentration is calculated using all plates yielding counts within the ideal range of 30 to 300 CPU per plate
3 Too numerous to count
11.1.8 Transfer aliquots of the remaining cell suspension into sterile tubes. Aliquot volumes
should be based on the concentration estimate of the cell suspension determined in
Section 11.7. Aliquots for DNA standard preparations (Section 11.2) should contain an
estimated total of ~109 CPU and aliquots for preparation of sub-stocks for calibrator
and matrix spike samples (see paragraph below) should contain ~108 CPU. So for
example, if the estimated concentration of the cell suspension is 109 CFU/mL, transfer
1 mL aliquots for DNA standard preparations and 0.1 mL aliquots for sub-stocks. Note:
If the concentration estimate of the cell suspension, determined in Section 11.7, is
greater than 109 CFU/mL, dilute either part or all of this cell suspension to
approximately this concentration or lower before proceeding with making the aliquots.
This is so that the 10s CPU aliquot volumes will be no less than 0.1 mL. Prepare as
many aliquots of each as needed and either freeze or discard the remainder of the cell
suspension. Freeze the aliquot tubes at -80°C except one tube containing ~108 CPU.
Note: Cell suspension should be stirred or gently vortexed repeatedly while aliquoting.
To make substocks for calibrator and matrix spike samples (Section 11.3), transfer the
entire contents of the unfrozen tube containing ~108 CPU of E. faecalis stock cell
suspension to a sterile tube or bottle containing -100 mL of PBS buffer (100 mL minus
the volume of the cell suspension) to make a cell suspension containing ~106 CFU/mL
(108 CPU in a final volume of 100 mL). Transfer as many 0.2 ml aliquots of this
suspension as needed to sterile tubes (making sure to gently vortex or shake the tube or
23
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Method 1609.1
bottle repeatedly while aliquoting) and freeze the substock aliquots at -80°C. Note:
Frozen aliquots of the original stock suspension may be thawed one time each to make
additional substocks for calibrator and matrix spike samples as described above.
11.2 Preparation of E. faecalis genomic DNA standards
11.2.1 Remove one tube containing ~109 CPU of E. faecalis cells (Section 11.1.8) from
freezer and thaw completely.
11.2.2 Pellet the cells by centrifugation at 12,000 x g for one minute, draw off and discard
supernatant and resuspend the cell pellet in a final volume of 1 mL AE buffer.
11.2.3 Transfer 0.5 mL aliquots of the cell suspension to two extraction tubes with glass
beads. Tightly close the tubes, making sure that the O-rings are seated properly.
11.2.4 Place the tubes in bead beater and shake for 60 seconds at the maximum rate (5000
rpm).
11.2.5 Remove the tubes from the mini bead beater and centrifuge at 12,000 x g for one
minute to pellet the glass beads and debris.
11.2.6 Using a 200 uL micropipettor, transfer 350 uL of supernatants to sterile 1.7 mL
microcentrifuge tubes. Recover supernatants without disrupting the glass beads at the
tube bottom.
11.2.7 Centrifuge crude supernatants at 12,000 x g for 5 minutes and transfer 300 uL of
clarified supernatant to clean, labeled 1.7 mL low retention microcentrifuge tubes,
taking care not to disturb the pellet.
11.2.8 Add 1 uL of 5 ug/uL RNase A solution to each clarified supernatant, mix by vortexing
and incubate at 37°C for 1 hour.
Note: Steps 11.2.9 - 11.2.15 may be substituted with an optional method if a DNA
purification procedure is chosen other than the DNA-EZ purification kit. In such a
case, manufacturer instructions should be followed rather than these steps, and the
method should be followed beginning at step 11.2.16.
11.2.9 Add 0.6 mL of binding buffer solution from a DNA-EZ purification kit to each of the
RNase A-treated extracts and mix by vortexing. Note: In general, a minimum
concentration of 5 x 1Q8 cells is required for this step.
11.2.10 If using the DNA-EZ purification kit, perform the following steps. Insert one
DNAsure™ column from the DNA-EZ purification kit into a collection tube (also
provided with kit) for each of the two extracts. Transfer the extract and binding buffer
mixtures from Section 11.2.9 to a DNAsure™ column and collection tube assembly
and centrifuge for 1 minute at 12,000 x g.
11.2.11 Transfer each of the DNAsure™ columns to new collection tubes. Discard previous
collection tubes and collected liquid.
11.2.12 Add 500 uL EZ-Wash Buffer from the DNA-EZ purification kit to each of the
DNAsure™ columns and centrifuge at 12,000 x g for 1 minute. Discard the liquid in
the collection tube.
11.2.13 Repeat step 11.2.12.
11.2.14 Transfer each of the DNAsure™ columns to a clean, labeled 1.7 mL low retention
microcentrifuge tube and add 50 uL of DNA elution buffer to each column. Centrifuge
for 30 seconds at 12,000 x g. Repeat this procedure again to obtain a total DNA eluate
volume of-100 uL from each column.
24
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Method 1609.1
11.2.15 Pool the two eluates to make a total volume of approximately 200 \\L.
11.2.16 Transfer entire purified DNA eluate volume from each column to a clean and sterile
microcuvette for UV spectrophotometer and read absorbance at 260 and 280 nm. (Note:
The cuvette should be blanked with DNA elution buffer before reading the sample). If
necessary, the sample may be diluted with elution buffer to reach minimum volume
that can be accurately read by the spectrophotometer (see manufacturer's
recommendation); however, this may reduce the DNA concentration to a level that
cannot be accurately read by the spectrophotometer. If available, readings can be taken
of 2 uL aliquots of the sample with aNanoDrop™ Spectrophotometer.
11.2.17 The sample is acceptable as a standard if ratio of OD26o/OD28o readings is >to 1.75.
11.2.18 Record UV spectrophotometer results.
11.2.19 Calculate total DNA concentration in sample by formula:
OD260 reading x 50 ng/uL DNA/OD260
11.2.20 Transfer sample back to labeled 1.7 mL non-retentive microcentrifuge tube and store at
-20°C.
11.2.21 Using total DNA concentration (Section 11.2.19), calculate E. faecalis PCR target
sequence concentration (TSC) in purified extract (Section 9.10).
Example.
OD260 = 0.7271 Total DNA concentration = OD260 Reading x 50 ng/(iL
0.7271 x 50 = 36.36 ng/^L = 36.36 x 106 fg/jiL = 3.636 x 107 fg/jiL
3.636 x 107fg/^L 4 IsrRNA gene copies . „. 1fl7 • , T
, , ,, . *-^~ x & *— = 4.04 x 10'gene copies/nL
3.6fg/genome genome
11.2.22 Record UV spectrophotometer results and all calculations.
11.2.23 In appropriately labeled 1.7 mL low retention microcentrifuge tubes, perform serial
dilutions using AE buffer to prepare DNA standards of 4 x 104/5 uL, 4 x 103 TSC/5
uL, 4 x 102 TSC/5 uL, 40 TSC/5 uL, and 10 TSC/5 uL (see below).
Note: Perform initial dilution of purified and quantified DNA as required to achieve a
stock concentration of 2 x 10s TSC/5 uL4 x 107 TSC/5 uL with AE buffer prior to
initiating serial dilutions. For the example concentration of 4.04 x 107 TSC/uL (2.02 x
10s TSC/5 uL) shown in Section 11.2.21, this would be a 5.05-fold dilution (e.g., 1 mL:
4.05 mL). It is recommended that DNA stocks (i.e., > 4 x 104 TSC/5 uL) be stored and
manipulated in a different room than that used for routine sample processing in order to
minimize the potential for laboratory contamination.
11.2.23.1 Perform 10-fold serial dilutions of the 4 x 107 TSC/5 uL stock (e.g., 100
uL : 900 uL) with AE buffer to achieve stocks of 4 x 106 TSC/5 uL; and
4 x 105 TSC/5 uL.
11.2.23.2 Perform 10-fold dilution of 4 x 105 TSC /5 uL stock (e.g., 100 uL : 900
uL) with AE buffer to achieve the first standard: 4 x 104 TSC/5 uL.
11.2.23.3 Perform successive 10-fold serial dilutions of the 4 x 104 TSC/5 uL
standard (e.g., 100 uL : 900 uL) to achieve 4 x 103 TSC/5 uL; 4 x 102
TSC/5 uL and 40 TSC/5 uL standards.
11.2.23.4 Perform a 4-fold dilution of 40 TSC/5 uL standard (e.g., 100 uL : 300 uL)
to achieve 10 TSC/5 uL standard.
25
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Method 1609.1
11.2.23.5 (Optional) Perform a final 2-fold dilution of 10 TSC/5 uL standard (e.g., 25
uL : 25 uL) to achieve 5 TSC/5 uL. TVofe: Preparation and analysis of this
dilution is not necessary for determining standard curves but is
recommended for determining limit of detection capability (see Section
9.14). CT values resulting from true positive logarithmic amplification
traces should be obtained from at least 90% of the analyses of this dilution
to document the laboratory's limit of detection capability with the method.
Note: It is also strongly recommended that further dilutions of the 5 TSC/5
uL dilution be analyzed to obtain most probable number estimates of target
sequence concentration as described in Reference 16.6 (a detailed protocol
is provided in Appendix D) when using laboratory prepared standards.
Mean target sequence estimates in the standards, as determined by this
technique, should be consistent with the spectrophotometric based
estimates. If they are not, the use of DNA standards that have been
prepared and tested by the EPA is recommended. This analysis should not
be necessary when using these DNA standards (see Section 11.5).
11.2.24 Store DNA standards in refrigerator at 4°C. For long term storage, freeze aliquots at
80°C.
11.2.25 Generate DNA standard curves by analyzing 5 uL aliquots of the 40,000, 4000, 400, 40
and 10 TSC/5 uL standards with 3 replicate analyses of each standard per curve
according to Section 11.9 (AB) or 11.10 (Smart Cycler®), as appropriate. (Note: EPA
recommends the generation of at least four independent standard curves (from separate
instrument runs performed on different days and/or with different mixes of reagents)
for determination of a composite standard curve as detailed in Section 9.10. It is
further recommended that initial analyses of calibrator sample extracts, as detailed in
Section 9.11, be performed at the same time (preferably in the same instrument runs if
using 96 well format instruments) as the analyses of these standards). Note: CT values
resulting from true positive logarithmic amplification traces should be obtained from at
least 95% of the analyses of each standard to document each laboratory's limit of
detection capability with the method.
11.3 Preparation and DNA extraction of E. faecalis calibrator, matrix spike and reference (PBS)
matrix spike samples prepared from laboratory-grown cell suspensions. Note: If using BioBalls
to prepare calibrator, matrix spike and reference (PBS) matrix spike samples see Section 11.4.
11.3.1 Either two or three calibrator sample extracts should be prepared and extracted either
daily or at least weekly during water sample analyses. Calibrator samples can be
prepared and extracted either in advance of, or in parallel with water sample filters.
11.3.2 Remove one tube containing a 0.2 mL aliquot of E. faecalis substock cell suspension
(Section 11.1.8) from the freezer and allow to thaw. Dilute the cell suspension 100-
fold with PBS buffer (e.g., dilute the 0.2 mL aliquot with 0.8 ml of PBS buffer and then
transfer this entire 1 mL suspension to a larger tube containing 19 mL PBS) and mix
thoroughly by vortexing. Note: This dilution should produce an estimated
concentration of ~104 CFU/mL (see section 11.8).
11.3.3 Prepare one membrane filtration system with polycarbonate filter as described in
Sections 11.6.1-11.6.2 for each calibrator sample to be prepared (see Section 11.3.1).
11.3.4 Filter 1 mL aliquots of the diluted cell suspension from step 11.3.2 (calibrator samples)
through each membrane filtration system prepared in step 11.3.3. After filtering, wash
26
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Method 1609.1
each membrane with 20 mL of sterile PBS (Section 7.4) and continue filtration until all
liquid has been pulled through the filter. Turn off the vacuum and remove the funnel
from the filter base.
11.3.5 Label extraction tubes with glass beads (Section 7.18) to identify samples. Leaving the
filters on the filtration unit base, fold the filter into a cylinder with the sample side
facing inward, being careful to handle the filters only on the edges, where the filters
have not been exposed to the sample. Insert each folded filter into a labeled extraction
tube with glass beads.
11.3.6 Dispense 600 uL of Salmon DNA/extraction buffer (Section 7.12) into each extraction
tube with glass beads and filter.
11.3.7 Place the tubes with filters in the bead beater and shake for 60 seconds at the maximum
rate (5000 rpm).
11.3.8 Remove the tubes from the bead beater and centrifuge at 12,000 x g for one minute to
pellet the glass beads and debris.
11.3.9 Using a 200 uL micropipettor, transfer the crude supernatant to the corresponding
labeled sterile 1.7 mL microcentrifuge tube. Transfer 400 uL of supernatant taking
care to minimize picking up glass beads at the tube bottom. Note: The filter will
normally remain intact during the bead beating and centrifugation process. Generally,
400 uL of supernatant can be easily collected.
11.3.10 Centrifuge at 12,000 x g for 5 minutes and transfer -350 uL of clarified supernatant to
a clean, labeled 1.7 mL tube, taking care not to disturb the pellet. Note: Cell pellet
may not be visible in calibrator samples.
11.3.11 Label the tubes as undiluted or Ix E. faecalis calibrator extracts. Label additional 1.7
mL tubes for 5 and 25 fold dilutions if necessary. In appropriately labeled tubes, using
a micropipettor, add a 50 uL aliquot of each Ix E. faecalis calibrator extracts and dilute
each with 200 uL AE buffer (Section 7.11) to make 5 fold dilutions if necessary. In
appropriately labeled tubes using a micropipettor, add a 50 uL aliquot of each 5 fold
dilution and dilute each with 200 uL AE buffer to make 25 fold dilutions if necessary.
11.3.12 Refrigerate the extracts until ready to analyze. Calibrator extracts can be held in this
manner and reused for repeated analyses with different batches of water sample
extracts for up to 1 week.
11.3.13 Matrix spike samples:
11.3.13.1 Prepare matrix spike samples for interference testing at frequencies
indicated in Section 9.5. Samples should be spiked with the same diluted
E. faecalis cell suspensions as used for preparing daily or weekly calibrator
samples. Diluted E. faecalis cell suspensions can be held in refrigerator for
up to one week for the latter purpose.
11.3.13.2 Transfer a 1 mL aliquot of the diluted cell suspension from Section 11.3.2
to 100 mL (or 50 mL - see Section 11.6.4) of the water sample selected for
testing to create the matrix spike sample.
11.3.13.3 Filter spiked water sample as described in Section 11.6 (see guidance in
Section 9.5 concerning pairing of spiked and unspiked water sample filters
for interference testing).
27
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Method 1609.1
11.3.13.4 Transfer each filter to labeled extraction tube with glass beads and 600 uL
of Salmon DNA/extraction buffer.
11.3.13.5 Extract matrix spike and corresponding unspiked water sample filters in
same manner as described for calibrator samples in Sections 11.3.9 -
11.3.14. Analyze matrix spike and unspiked water sample and calibrator
sample extracts in parallel if possible (as described in Sections 11.8 and
11.9 or 11.10) the same day the water sample extracts are prepared.
11.3.14 Reference (PBS) matrix spike samples:
11.3.14.1 Prepare reference (PBS) matrix spike samples at frequencies described in
Sections 9.3 or 9.4. Samples should be spiked with the same cell
suspensions as used for preparing daily or weekly calibrator samples.
11.3.14.2 Prepare membrane filtration system with polycarbonate filter(s) as
described in Sections 11.6.1 - 11.6.2.
11.3.14.3 Transfer a 1 mL aliquot of the diluted cell suspension from Section 11.3.2
to 50 or 100 mL of PBS to create the reference (PBS) matrix spike sample.
11.3.14.4 Filter reference (PBS) matrix spike sample as described in Section 11.6.
11.3.14.5 Transfer each filter to labeled extraction tube with glass beads and 600 uL
of Salmon DNA/extraction buffer.
11.3.14.6 Analyze reference (PBS) matrix spike and calibrator sample extracts in
parallel if possible (as described in Sections 11.8 and 11.9 or 11.10) within
one week of their preparation.
11.4 Preparation and DNA extraction of E. faecalis calibrator, matrix spike and reference (PBS)
matrix spike samples from BioBalls
11.4.1 Either two or three calibrator extracts should be prepared and extracted either daily or at
least weekly during water sample analyses. BioBall® calibrator samples can be prepared
and extracted either in advance of, or in parallel with water sample filters.
Note: Upon receipt, BioBalls should be stored at -20°C. Culturing of BioBalls prior to
spiking is not necessary, as they can be spiked directly into a sample once the vial is
opened.
11.4.2 Open one BioBall® vial for each calibrator sample to be prepared. Aseptically add each
E. faecalis BioBall® to 1 mL of PBS and dissolve by gentle vortexing. Note: Each
BioBall® contains -550 CPU of E. faecalis. See manufacturer's lot-specific certificate of
analysis to identify exact cell (i.e., CPU) numbers.
11.4.3 Prepare one membrane filtration system with polycarbonate filter as described in
Sections 11.6.1-11.6.2 for each calibrator sample.
11.4.4 Filter each BioBall® cell suspension (calibrator sample) through a membrane filtration
system with a polycarbonate filter. After filtering, wash each membrane with 20 mL of
sterile PBS (Section 7.4) and continue filtration until all liquid has been pulled through
the filter. Turn off the vacuum and remove the funnel from the filter base.
11.4.5 Label extraction tubes with glass beads (Section 7.18) to identify samples. Leaving the
filter on the filtration unit base, fold into a cylinder with the sample side facing inward,
being careful to handle the filter only on the edges, where the filter has not been exposed
to the sample. Insert the folded filter into the labeled extraction tube with glass beads.
Prepare one filter for each BioBall® sample filtered in this manner.
28
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Method 1609.1
11.4.6 Dispense 600 \\L of Salmon DNA/extraction buffer (Section 7.12) into each extraction
tube with glass beads and filter.
11.4.7 Tightly close the tubes, making sure that the O-ring is seated properly.
11.4.8 Extract BioBall® calibrator sample filters in same manner as described for laboratory-
prepared calibrator samples in Sections 11.3.9 - 11.3.14.
11.4.9 Label the tubes as undiluted or Ix E. faecalis calibrator extracts. Label additional 1.7
mL tubes for 5 fold dilutions if necessary. In appropriately labeled tubes, using a
micropipettor, add a 50 uL aliquot of each Ix E. faecalis BioBall® calibrator extracts
and dilute each with 200 uL AE buffer (Section 7.11) to make 5 fold dilutions if
necessary.
11.4.10 Refrigerate the extracts until ready to analyze. BioBall® calibrator extracts can be held
in this manner and reused for repeated analyses with different batches of water sample
extracts for up to 1 week.
11.4.11 Matrix spike samples:
11.4.11.1 Prepare matrix spike samples for interference testing at frequencies
indicated in Section 9.5. Samples should be spiked with the same lot of
BioBalls as used for preparing daily or weekly calibrator samples.
11.4.11.2 Prepare membrane filtration system with polycarbonate filter(s) as
described in Sections 11.6.1 - 11.6.2.
11.4.11.3 Aseptically transfer one BioBall® to 100 mL (or 50 mL - see Section
11.6.4) of the water sample selected for testing as described in Section
11.4.2 and allow to dissolve to create the matrix spike sample.
11.4.11.4 Filter matrix spike sample as described in Section 11.6 (see guidance in
Section 9.5 concerning pairing of matrix spike and unspiked water sample
filters for interference testing)
11.4.11.5 Transfer each filter to extraction tube with glass beads and 600 uL of
Salmon DNA/extraction buffer.
11.4.11.6 Extract matrix spike and corresponding unspiked water sample filters in
same manner as described for calibrator samples in Sections 11.3.9 -
11.3.14. Analyze spiked and unspiked water sample and calibrator sample
extracts in parallel if possible (as described in Sections 11.8 and 11.9 or
11.10) the same day the water sample extracts are prepared.
11.4.12 Reference (PBS) matrix spike samples:
11.4.12.1 Prepare reference (PBS) matrix spike samples at frequencies described in
Section 9.4. Samples should be spiked with the same lot of BioBalls as
used for preparing daily or weekly calibrator samples.
11.4.12.2 Prepare membrane filtration system with polycarbonate filter(s) as
described in Sections 11.6.1 - 11.6.2.
11.4.12.3 Aseptically transfer one BioBall® to 50 or 100 mL of PBS as described in
Section 11.4.2 and allow to dissolve to create reference (PBS) matrix spike
sample.
11.4.12.4 Filter spiked PBS reference (PBS) matrix sample as described in Section
11.6.
29
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Method 1609.1
11.4.12.5 Transfer each filter to extraction tube with glass beads and 600 uL of
Salmon DNA/extraction buffer and label.
11.4.12.6 Extract reference (PBS) matrix spike sample filters in same manner as
described for calibrator samples in Sections 11.3.9- 11.3.14. Analyze
reference (PBS) matrix spike and calibrator sample extracts in parallel if
possible (as described in Sections 11.8 and 11.9 or 11.10) within one week
of their preparation.
11.5 Use of EPA plasmid DNA standards (see Appendix E for instructions on acquiring these
standards).
11.5.1 DNA standards will be provided as akit/package consisting of 5 components: Dilution 1:
~4*104 copies/5 uL; Dilution 2: ~4*103 copies/5 uL; Dilution 3: ~4*102 copies/5 uL;
Dilution 4: -4*10' copies/5 uL; Dilution 5: -IxlO1 copies/5 uL. Note: Mean
concentration estimates and standard deviations may vary slightly by lot. Lot-specific
concentration information will be provided with each kit.
11.5.2 Upon receipt of the kit, freeze all components at -80°C until ready to use (Note: if-80°C
storage capability is not available, it is recommended that the kit be stored at -20°C and
used as soon as possible as described below).
11.5.3 Thaw kit components and store at 4°C.
11.5.4 Generate DNA standard curves by analyzing 5 uL aliquots of kit components 1-5 with 3
replicate analyses of each component per instrument run according to Section 11.9 (Life
Technologies/AB) or 11.10 (Smart Cycler®), as appropriate. Note: Sufficient volumes of
components 1-5 are provided in each kit to generate at least twelve standard curves in this
manner. EPA recommends the generation of at least four independent standard curves
(from separate instrument runs performed on different days and/or with different mixes of
reagents) for determination of a composite standard curve as detailed in Section 9.10.
EPA further recommends that these analyses be completed within one month of thawing
the kit components. Finally, it is recommended that initial analyses of at least eight
independent calibrator sample extracts, as detailed in Section 9.11, be performed at the
same time (preferably in the same instrument runs if using 96 well format instruments) as
the analyses of these standards. Aliquots of the kit components that are not used for initial
analysis after initial thawing immediately should be transferred to low retention tubes
(see Section 6.24) and re-frozen one time at -80°C for subsequent use. Repeated freezing
and thawing of these components should be avoided.
11.6 Water sample filtration
Note: It is required that at least one water sample filtration blank (method blank) be prepared for
every PCR run (Section 9.8) and analyzed by the same procedure as the water samples.
11.6.1 Assemble manifold (Section 6.11), safety trap (Section 6.12) and vacuum system
(Section 6.9) and connect membrane filtration unit(s) (Section 6.8) to manifold.
11.6.2 Place a polycarbonate filter (Section 6.14) on each filter base, and attach the funnel to the
base so that the membrane filter is now held between the funnel and the base. Note: Most
disposable plastic membrane filtration units (e.g., Nalgene CN 130-4045,CN 145-0045,
or equivalent) are provided with a cellulose nitrate filter that must be removed and
replaced with the polycarbonate filter, do not remove filter support pad provided with the
unit.
11.6.3 Shake the sample bottle vigorously 25 times to distribute the bacteria uniformly, and
measure 100 mL of sample (or 50 mL - see Section 11.6.4) into the funnel. (Note: Use
30
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Method 1609.1
of the graduations in most brands of disposable plastic membrane filtration units is
acceptable for measuring)
11.6.4 Filter 100 mL of water sample. After filtering the sample, rinse the sides of the funnel
with 20 mL of sterile PBS (Section 7.4) and continue filtration until all liquid has been
pulled through the filter. Turn off the vacuum and remove the funnel from the filter base.
Note: If 100 mL water sample volumes from your site can not be routinely filtered within
a reasonable amount of time (e.g., ~15 min), it is permissible to filter a 50 mL water
sample volume (see Section 12.7). This deviation must be noted in the results. For
method blanks, only the above rinse procedure with 20 mL of sterile PBS is necessary.
11.6.5 Label an extraction tube with glass beads (Section 7.18) to identify water sample.
Leaving the filter on the filtration unit base, fold into a cylinder with the sample side
facing inward, being careful to handle the filter only on the edges, where the filter has not
been exposed to the sample. Insert the rolled filter into the labeled extraction tube with
glass beads. Prepare one filter for each sample filtered in this manner.
11.6.6 Cap the extraction tube. Tubes may be frozen at -20°C for up to 1 week or -80°C for up
to 1 year until analysis.
11.7 DNA extraction of water sample filters and method blanks
11.7.1 Using a 1000 uL micropipettor, dispense 600 uL of the Salmon DNA/extraction buffer
(Section 7.12) to each labeled extraction tube with glass beads containing water sample
or method blank filters from Section 11.6.
11.7.2 Tightly close the tubes, making sure that the O-ring is seated properly. Extract water
sample and method blank filters in same manner as calibrator samples (see Section 11.3).
Extract the method blank(s) last.
11.7.3 Label the tubes as undiluted or Ix water sample extracts with sample identification.
These are the water sample filter extracts. Also label tubes for method blanks. Label
additional 1.7 mL tubes for 5 fold dilutions. In appropriately labeled tubes, using a
micropipettor, add a 50 uL aliquot of each Ix water sample extract and dilute each with
200 uL AE buffer (Section 7.11) to make 5 fold dilutions. Dilute the method blank
supernatant last.
11.7.4 Store all diluted and undiluted extracts in refrigerator. Note: Use of undiluted samples
for analysis is currently recommended if only one dilution can be analyzed.
11.7.5 qPCR analysis of calibrator and water sample extracts should be performed in parallel if
possible as described in Sections 11.8 and 11.9 or 11.10. Analyses should be performed
immediately if possible and no later than the same day the water sample extracts are
prepared.
11.8 Preparation of qPCR assay mix
11.8.1 To minimize environmental DNA contamination, routinely treat all work surfaces with a
10% bleach solution, allowing the bleach to contact the work surface for a minimum of
15 minutes prior to rinsing with sterile water. If available, turn on UV light for 15
minutes. After decontamination, discard gloves and replace with new clean pair.
11.8.2 Remove primers and probe stocks from the freezer and verify that they have been diluted
to solutions of 500 uM primer and 100 uM probe.
11.8.3 Prepare working stocks of Entero/IAC multiplex assay primer/probe by adding 10 uL of
each primer stock (forward and reverse) and 4 uL each probe (Entero Probe and UC1P1)
31
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Method 1609.1
to 572 \\L PCR-grade water. Note: If performing Entero simplex assay rather than
Entero/IAC multiplex assay, exclude UC1P1 and add an additional 4 uL of water.
Prepare Sketa22 Salmon DNA primer/probe mix by adding 10 uL of each primer stock
(forward and reverse) and 4 uL of probe stock to 576 uL of PCR-grade water, and vortex.
Pulse centrifuge to create a pellet. Use a micropipettor with aerosol barrier tips for all
liquid transfers. Transfer aliquots of working stocks for single day use to separate tubes
and store at 4°C.
11.8.4 Using a micropipettor, prepare assay mix for the Entero/IAC and Salmon DNA (Sketa
22) reactions in separate, sterile, labeled 1.7 mL microcentrifuge tubes as described in
Table 8.
Tables. PCR Assay Mix Composition
Reagent
PCR-grade water (containing IAC for
Entero/IAC multiplex assays)
BSA
TaqMan master mix
Primer/probe working stock solution
Volume/Sample
(multiply by # reactions to be analyzed
2 extra reactions)
per day + at least
2.0 ul_
2.5 |JL
12.5 ul_
3.0 |JL
Note: This will give a final concentration of 1 uM of each primer and 80 nM of probe in the
reactions. Prepare sufficient quantity of assay mix for the number of reactions to be analyzed per
day including calibrators and negative controls plus at least two extra reactions. Prepare assay
mixes each day in clean area (see Section 6.1) before handling any samples containing DNA.
11.8.5 Vortex the assay mix working stocks; then pulse microcentrifuge to coalesce droplets.
Return the primer/probe working stocks and other reagents to the refrigerator.
11.9 Life Technologies StepOnePlus™, and AB 7500 (all non-Fast mode) qPCR assay preparation
(Reference 16.1)
11.9.1 Transfer 20 uL of Entero/IAC and Sketa 22 master mixes (from Table 8) to wells of a 96
well PCR reaction tray equal to number of samples to be analyzed including calibrator
and negative control samples. Pipette into the center of the wells, taking care to not touch
the well walls with the pipette tip. (Note: The same tip can be used for pipetting multiple
aliquots of the same assay mix as long as it doesn't make contact with anything else).
Example: For the analysis of 18 recreational water samples, 48 wells each will require
the addition of Entero/IAC and Sketa 22 master mixes as follows: 18-19 samples, two
replicates each (36-38 total), 2 method blanks, two replicates each (4 total), 2 NTCs and 2
or 3 calibrators, 2 replicates each (4-6 total) = 96 wells.
11.9.2 When all wells are loaded, cover tray loosely with aluminum foil or plastic wrap and
transfer to refrigerator or directly to the PCR preparation station used for handling DNA
samples (Section 6.1). Note: All aliquoting of assay mixes to reaction trays must be
performed each day before handling of DNA samples.
11.9.3 DNA standards - Transfer 5 uL each concentration of the undiluted DNA extracts or
EPA plasmid DNA standards (Section 11.5) to separate wells of the PCR reaction tray
containing Enterococcus assay mix. Note: Record positions of each concentration.
11.9.4 Water samples - Transfer 5 uL each of the undiluted (or 5- or 25-fold diluted) DNA
extracts of method blanks and water samples (Section 11.6), and corresponding dilutions
32
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Method 1609.1
of calibrator samples (Section 11.3.13), to separate wells of the PCR reaction tray
containing Entero/IAC and Sketa 22 master mixes. Note: Record positions of each
sample. Use of undiluted calibrator and water sample extracts for analysis is
recommended in this method if only one dilution can be analyzed. Additional analyses of
5-fold diluted calibrator and water sample extracts is recommended if feasible to confirm
absence of PCR inhibition. If the CCE or target sequence density estimates (see Section
12) from the 5-fold diluted extract analysis are more that 4-fold greater than from the
undiluted extract analysis, the 5-fold diluted extract result should be used. Analyses of
25-fold dilutions may unnecessarily sacrifice sensitivity and may result in inadequate
sensitivity of the method in certain situations.
11.9.5 Transfer 5 uL each of the undiluted (or 5- or 25-fold diluted) DNA extracts of method
blanks and water samples (Section 11.7), and then corresponding dilutions of calibrator
samples (Section 11.3.13), to separate wells of the PCR reaction tray containing
Entero/IAC and Sketa 22 master mixes. Note: Record positions of each sample.
11.9.6 Tightly cap wells of PCR reaction tray containing samples or cover tray and seal tightly
with optical adhesive PCR reaction tray.
11.9.7 Run reactions in AB StepOnePlus™, AB 7900, or AB 7500 (all in non-Fast mode) Real-
Time PCR Systems. For platform-specific operation see Appendix A with the exception
that both FAM (Entero PI) and VIC (IAC) reporters are selected from the menu.
11.10 Smart Cycler® qPCR assay preparation
11.10.1 Label 25 uL Smart Cycler® tubes with sample identifiers and assay mix type (see
Section 11.10.8 for examples) or order tubes in rack by sample number and label rack
with assay mix type. It is recommended that the unloaded open Smart Cycler® tubes be
irradiated under UV light in a PCR cabinet for 15 minutes. Using a micropipettor, add
20 uL of the Enterococcus assay mix (Section 11.8.4) to labeled tubes. Avoid
generating air bubbles, as they may interfere with subsequent movement of the liquid
into the lower reaction chamber. The same tip can be used for pipetting multiple
aliquots of the same assay mix as long as it doesn't make contact with anything else.
Repeat procedure for Salmon DNA (Sketa 22) assay mix.
11.10.2 Add5 uL of AE buffer to NTC tubes and close tubes tightly.
11.10.3 Close the other PCR tubes loosely and transfer to refrigerator or directly to the PCR
preparation station used for handling DNA samples (Section 6.1). Note: All aliquoting
of assay mixes to reaction tubes must be performed each day before handling of DNA
samples.
11.10.4 Transfer 5 uL each of the undiluted (or 5- or 25-fold diluted) DNA extracts of method
blanks and water samples (Section 11.7), and then corresponding dilutions of calibrator
samples (Section 11.3.13) to tubes containing Enterococcus and Salmon DNA (Sketa
22) mixes. Close each tube tightly after adding sample. Load the method blank PCR
assays last. Label the tube tops as appropriate.
11.10.5 When all Smart Cycler® tubes have been loaded, place them in a Smart Cycler®
centrifuge, and spin for 2-4 seconds.
11.10.6 Inspect each tube to verify that the sample has properly filled the lower reaction
chamber. A small concave meniscus may be visible at the top of the lower chamber, but
no air bubbles should be present. (If the lower chamber has not been properly filled,
carefully open and reclose the tube, and re-centrifuge). Transfer the tubes to the
thermocycler.
33
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Method 1609.1
11.10.7 For platform-specific operation see Appendix B.
11.10.8 Suggested sample analysis sequence for Smart Cycler®
Example: For analyses on a single 16-position Smart Cycler®, calibrator samples and
water samples will need to be analyzed in separate runs and a maximum of 6 water
samples (3 samples x 2 replicates) can be analyzed per run, as described in Tables 9 and
10. Note: Since calibrator and water sample extracts cannot be analyzed in parallel in
this instance, CT measurements from a single run of calibrator sample extracts can be
used in conjunction with results from multiple water sample extract runs for up to one
week to calculate Enterococci densities in the water samples.
Table 9. Calibrator PCR Run
Sample Description*
2 or 3 Calibrators x 2 replicates (undiluted or diluted 5, or
25 fold)
2 or 3 Calibrators x 2 replicates (undiluted or diluted 5, or
25 fold)
No template controls (reagent blanks)
Quantity
4-6
4-6
2
PCR Assay Master Mix
Enterococcus/IAC
Salmon DMA
Enterococcus/IAC
* Diluted equivalently to the water samples
Table 10. Water Sample PCR Run - 6 Samples + 1 Method Blank per run
Sample Description*
3 Water samples x 2 replicates (undiluted or diluted 5, or 25 fold)
1 Method blank x 2 replicates (undiluted or diluted 5, or 25 fold)
3 Water samples x 2 replicates (undiluted or diluted 5, or 25 fold)
1 Method blank x 2 replicates (undiluted or diluted 5, or 25 fold)
Quantity
6
1
6
1
PCR Assay Master Mix
Enterococcus/IAC
Enterococcus/IAC
Salmon DMA
Salmon DMA
* Use of undiluted calibrator and water sample extracts for analysis is recommended in this method if only
one dilution can be analyzed. Additional analyses of five-fold diluted calibrator and water sample extracts is
recommended if feasible to confirm absence of PCR inhibition. Analyses of 25 fold dilutions may
unnecessarily sacrifice sensitivity and may result in inadequate sensitivity of the method in certain situations.
12.0 Data Analysis and Calculations
12.1 Overview: This section describes a method for determining the ratio of the target sequence
quantities recovered from a test (water filter) sample compared to those recovered from
identically extracted calibrator samples using an arithmetic formula, referred to as the
comparative CT method. The AACT comparative CT method also normalizes these ratios for
differences in total DNA recovery from the test and calibrator samples using qPCR analysis CT
values for a reference sequence provided by the SPC DNA. These ratios are converted to CCEs
recovered from the test samples. Methods are provided in Sections 12.4 - 12.6 for determining
absolute quantities of total target sequences recovered from calibrator and test sample extracts
and using these values to obtain EPA RWQC-adjusted CCE estimates in water samples. These
methods for determining RWQC-adjusted CCE estimates are recommended based on EPA
estimates of the ratios of target sequences to CCEs used to establish beach action values in the
2012 EPA Recreational Water Quality Criteria (References 16.5 and 16.11). Tools for
performing many of the calculations indicated below are provided in the Method 1609.1/1611.1
calculation spreadsheet available at:
(http://water.epa.gov/scitech/methods/cwa/bioindicators/biological index.cfm).
12.2 Direct Calculation of RWQC-adjusted CCEs in test samples based on cell numbers in
calibrator samples
34
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Method 1609.1
Note: This method can be used if laboratories can document that the average target sequence
recovery per cell in their calibrator sample extracts is 15 as determined by dividing the average
target sequence recovery in calibrator sample extracts (calculated in Section 12.4) by the
estimated number of cells in the calibrator samples or by adjusting the cell number values in their
calibrator samples to obtain this value (see Section 12.6: A minimum of two or three fresh
calibrator samples should be extracted at least on a weekly basis and analyzed in association with
each batch of water sample filtrates. QC analysis of the results from these calibrator extracts
should be performed as described in Section 9.11. CT values from the Enterococcus and salmon
DNA SPC qPCR assays for both the calibrator and test samples are used in the AACT method to
determine the ratios of target sequences in the test and calibrator sample extracts and these ratios
are converted to CCEs recovered from the test samples as specified below and illustrated in
Table 11.
12.2.1 Subtract the mean Sketa22 assay CT value (CT, Sketa) from the mean Entero assay CT
value (CT, Entero) for each calibrator sample extract to obtain ACT values and calculate
the overall average ACT value for all calibrator samples.
12.2.2 Subtract the Sketa22 assay CT value (CT, Sketa) from the Entero assay CT value (CT,
Entero) for each water sample filtrate extract to obtain ACT values for each of these test
samples. Note: If duplicate analyses are performed on these samples, use the mean
Entero and Sketa22 CT values to calculate average ACT values.
12.2.3 Subtract the overall average ACT value for the calibrator samples from the ACT value (or
average ACT value) for each of the test samples to obtain AACT values.
12.2.4 Calculate the ratio of the target sequences in the test and calibrator samples using the
formula: AF ("AACT:I, where AF = amplification factor of the target organism qPCR assay,
determined as described in Section 12.2. Note: An alternative formula for this calculation
is: 10A(AACT IE) where B is the mean slope from the standard curve (Reference 16.7).
12.2.5 Multiply the ratio of the target sequences in the test and calibrator samples by the
estimated count (e.g., CPU) determined in Section 11.1.7 to determine the CCEs for each
of the test samples. Note: This calculation can be applied without modification to the
analyses of diluted extracts if both the test sample and calibrator extracts are equally
diluted and equal volumes of diluted extracts are analyzed.
Table 11. Example Calculations (AF = 1.96)
Estimated
Cell Count 1
120000
Sample
Type
Calibrator
Test
Cl,target
24.31
31.49
CT.SPC
21.68
22.01
ACT
2.63
9.48
MCr
6.85
Ratio calib/test
196-MCT
0.01
CCEs in Test Sample Extract
(1. 96 MCTxEst cell count)
0.01x120000 = 1200
12.3 Generation of CT value vs. target sequence standard curve: Serial dilutions of a DNA
standard should be prepared to give concentrations of ~4 x 104, 4 x 103, 4 x 102, 4 x 101 and ~1 x
101 IsrRNA gene sequences per 5 uL (the standard sample volume added to the PCR reactions) as
described in Section 11.2 or 11.5. Note: A procedure to confirm target sequence concentrations in
laboratory-prepared DNA standards is provided in Section 11.2.23. Guidance is also provided in
Section 9.10 for intercept values that should be expected from the standard curves produced by
certain instruments if the target sequence concentrations in the standards are acceptably accurate.
Preparation and analyses of an additional dilution of the standards containing 5 IsrRNA gene
sequences per 5 uL is suggested to better characterize the method's limit of detection in each
laboratory (see Sections 11.2.23.5 and 9.14) but is not required for generation of standard curves.
35
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Method 1609.1
Aliquots of each of these dilutions should be stored frozen at -80°C in low retention
microcentrifuge tubes. Thawed aliquots should be stored at 4°C and should be analyzed within
one month of thawing. qPCR analyses of these diluted standards using the Enterococcus primer
and probe assay should initially be performed in triplicate on at least four separate instrument
runs on different days and/or with different mixes of reagents. CT values from these composite
analyses should be subjected to regression analysis against the loglO-transformed target sequence
numbers per reaction as described in Section 9.10.
AFs used for subsequent AACT calculations (Section 12.4) can be calculated from the slope value
of this curve by the formula AF = 10A(1 / (-) slope value). An example calculation using the
slope value from the example regression in Figure 1 (see Section 9.10) is shown below:
AF=10A(1 73.4163) = 1.96
12.4 Calculation of average target sequence recovery in calibrator sample extracts: A minimum
of eight calibrator sample extracts should initially be prepared from different freezer-stored
aliquots of each cultured E.faecalis stock suspension or from eight BioBalls as described in
Section 9.11. Dilutions of each of these calibrator sample extracts equivalent to the anticipated
dilutions of the test samples used for analysis (e.g., undiluted and/or, 5- and/or 25-fold) should be
analyzed with the Enterococcus Entero/IAC primer and probe assay in at least four independent
instrument runs. (Note: Sketa22 assay results are not needed for this calculation but this assay
should be performed in parallel to determine ACT values as described in Section 9.11. IAC
results also are not needed for this calculation but it is recommended to perform these analyses
with the same primer/probe working stocks that will be used for ongoing analyses). The average
CT value from these analyses and the slope and intercept values of the composite standard curve
generated from the DNA standards (Section 9.10) should be used to determine the average
number of target sequences per 5 uL of extract used in the reactions. Accurate determination of
laboratory-prepared DNA standard concentrations (see Section 11.2.23.5) or use of EPA plasmid
DNA standards (see Section 11.5) is required. An example calculation using an average calibrator
extract CT value of 24.31 and the standard curve from Figure 1 in Section 9.10 is shown below:
Average calibrator target sequences/5 uL extract = 10A[(24.31-37.2137) / -3.3402] = 7297.1270
Note: The number should be rounded to four decimal places (i.e., 4285.0744). Dividing this
value by 5 gives the average calibrator target sequences/uL extract which can be multiplied by the
total volume of the extract (e.g., 600 uL for undiluted extracts, 3000 uL for 5-fold diluted extracts
or 15000 uL for 25-fold diluted extracts) to determine the average total quantity of target
sequences recovered in the calibrator sample extracts. An example of this calculation using the
average calibrator target sequences/reaction value determined immediately above is shown
below:
(7297.1270/5) x 600 = 875655 target sequences / total extract
Note: A tool for calculating average target sequence recovery in calibrator sample extracts is also
provided in the Method 1609.1/1611.1 calculation spreadsheet available at:
(http://water.epa.gov/scitech/methods/cwa/bioindicators/biological index.cfm).
12.5 Calculation of calibrator target sequence equivalents (CSE) in test samples
12.5.1 Perform calculations as described in Sections 12.2.1 - 12.2.4 above.
12.5.2 Multiply the ratio of the target sequences in the test and calibrator samples by the
estimated average target sequences per total calibrator sample extract determined in
Section 12.4. Note: This calculation can be applied without modification to the analyses
of diluted extracts if both the test sample and calibrator extracts are undiluted or equally
diluted and equal volumes of undiluted or diluted extracts are analyzed.
36
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Method 1609.1
Note: A tool for calculating average calibrator target sequence equivalents (CSE) in test
samples is also provided in the Method 1609.1/1611.1 calculation spreadsheet available
at: (http://water.epa.gov/scitech/methods/cwa/bioindicators/biological_index.cfm).
12.5.3 The geometric mean of the measured target sequences and associated coefficients of
variation in multiple water samples can be determined from individual sample CT values
using the following procedure:
12.5.3.1 Use ACT value for each individual water sample extract and the mean
calibrator ACT value to calculate the measured target sequence numbers in
each water sample extract, as described in Section 12.4.
12.5.3.2 Calculate the logic of the measured target sequence numbers in each water
sample (log N)
12.5.3.3 Calculate the mean (M) and standard deviation (S) from the values of log N
obtained in the previous step for all of the water sample extracts.
12.5.3.4 Calculate the geometric mean as 10M.
12.5.4 The implied coefficient of variation (CV) is calculated, based on the log normal
distribution, as the square root of iov/0434 - 1, where V = S2.
12.6 Calculation of EPA RWQC-standardized CCE in test samples.
12.6.1 Divide estimates of target sequences (CSE) pertest sample from Section 12.5 by 15.
Note: 15 is the mean EPA estimate of target sequences per calibrator cell used to
establish CCE beach action values in the 2012 EPA Recreational Water Quality Criteria
(Reference 16.5 and 16.11). The Method 1609.1/1611.1 calculation spreadsheet available
at: (http://water.epa.gov/scitech/methods/cwa/bioindicators/biological_index.cfm)
automatically performs this calculation.
12.7 Reporting Results: Where possible, duplicate analyses should be performed on each sample and
results based on mean CT values. Report the results as Enterococcus CCEs per 100 mL water
sample filtered as per Section 12.2 or as per Sections 12.4 - 12.6). If CCE results are based on
analyses of non-standard water sample volumes (i.e., 50 mL), multiply the CCE values by two
and note that the reported results are based upon this conversion.
13.0 Method Performance
13.1 Multi-laboratory Validation (MLV) of Method 1609 in Marine and Fresh Water
13.1.1 Marine Water
13.1.1.1 Seven volunteer (marine water) laboratories participated in EPA's MLV
study of EPA Method 1609. The purpose of the study was to characterize
method performance across multiple laboratories and multiple marine water
matrices and to develop QC acceptance criteria. A detailed description of the
study and results are provided in the validation study report (Reference
16.12). Results submitted by laboratories were validated using a
standardized data review process to confirm that results were generated in
accordance with study-specific instructions and the May 2012 draft version
of EPA Method 1609 (1609).
13.1.1.2 Recovery — Method 1609 was characterized by mean laboratory-specific
recoveries of enterococci from marine water samples spiked with BioBalls
ranging from 4% to 142% with an overall mean recovery of 63%.
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Method 1609.1
13.1.1.3 Precision — Method 1609 was characterized by laboratory-specific RSDs
from marine water samples spiked with BioBalls ranging from 15% to 440%
with an overall pooled within-laboratory RSD of 79%.
13.1.2 FreshWater
13.1.2.1 Eight volunteer (fresh water) laboratories participated in EPA's MLV study
of EPA Method 1609. A detailed description of the study and results are
provided in the validation study report (Reference 16.12). Results submitted
by laboratories were validated using a standardized data review process to
confirm that results were generated in accordance with study-specific
instructions and the May 2012 draft version of EPA Method R (1609).
13.1.2.2 Recovery — Method 1609 was characterized by mean laboratory-specific
recoveries of enterococci from fresh water samples spiked with BioBalls
ranging from -48% to 415% with an overall mean recovery of 124%.
13.1.2.3 Precision — Method 1609 was characterized by laboratory-specific RSDs
from fresh water samples spiked with BioBalls ranging from -6000% to
198%, with an overall pooled within-laboratory RSD of 84%.
13.1.3 PBS
13.1.3.1 A total of twelve volunteer laboratories analyzed PBS samples during EPA's
MLV study of EPA Method 1609.
13.1.3.2 Recovery — Method 1609 was characterized by mean laboratory-specific
recoveries of enterococci from PBS samples spiked with BioBalls ranging
from 0.1% to 330% with an overall mean recovery of 119%.
13.1.3.3 Precision — Method 1609 was characterized by laboratory-specific RSDs
from PBS samples spiked with BioBalls ranging from 12% to 200%, with an
overall pooled within-laboratory RSD of 73%.
14.0 Pollution Prevention
14.1 The solutions and reagents used in this method pose little threat to the environment when
recycled and managed properly.
14.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials to be disposed.
15.0 Waste Management
15.1 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.
15.2 Samples, reference materials, and equipment known or suspected to have viable enterococci
attached or contained must be sterilized prior to disposal.
15.3 For further information on waste management, consult "The Waste Management Manual for
Laboratory Personnel" and "Less Is Better: Laboratory Chemical Management for Waste
38
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Method 1609.1
Reduction," both available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.
16.0 References
16.1 Anonymous. 1997. User Bulletin #2. ABI Prism 7700 Sequence Detection System. Foster City,
CA, Applied Biosystems.
16.2 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.
16.3 Cabelli, V. J., A. P. Dufour, M. A. Levin, L. J. McCabe, and P. W. Haberman. 1979.
Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches. Amer. Jour.
Public Health. 69:690-696.
16.4 Haugland, R.A., S. Siefring, J. Lavender, and M.Varma, 2012. Influences of Sample Interference
and Interference Controls on Quantification of Enterococci Fecal Indicator Bacteria in Surface
Water by the qPCR Method. Water Research, 46(18):5989-6001.
16.5 Haugland, R.A., S. Siefring, M. Varma, A.P. Dufour, K.P. Brenner, T.J. Wade, E. Sams, S.
Cochran, S. Braun, and M. Sivaganesan. 2014. Standardization of Enterococci Density Estimates
by EPA qPCR Methods and Comparison of Beach Action Value Exceedances in River Waters
with Culture Methods. J. Microbiol. Methods. 105:59-66.
16.6 Sivaganesan, M., S. Siefring, M. Varma, and R.A. Haugland. 2011. MPN Estimation of qPCR
Target Sequence Recoveries from Whole Cell Calibrator Samples. J. Microbiol. Methods.
87:343-349.
16.7 Sivaganesan, M., S. Siefring, M. Varma, and R.A. Haugland. 2014. Comparison of Enterococcus
quantitative polymerase chain reaction analysis results from midwestern U.S. river samples using
EPA Method 1611 and Method 1609 PCR reagents. J. Microbiol. Methods. 101: 9-17.
16.8 USEPA. 1983. Health Effects Criteria for Marine Recreational Waters. EPA-600/1-80-031.
16.9 USEPA. 1984. Health Effects Criteria for Fresh Recreational Waters. EPA-600/1-84-004.
16.10 USEPA. 1986. Ambient water quality criteria for bacteria- 1986. EPA 440/5-84-002.
16.11 USEPA. 2012. Recreational water quality criteria. EPA, Office of Water 820-F-12-058.
16.12 USEPA. 2013. Results of the Multi-Laboratory Validation of EPA Method 1609 (1609) for
Enterococci in Marine and Fresh Waters by TaqMan® Quantitative Polymerase Chain Reaction
(qPCR) with Internal Amplification Control (IAC) Assay. EPA 820-R-13-007.
16.13 Wade, T.J., RL. Calderon, K.P. Brenner, E. Sams, M. Beach, R. Haugland, L. Wymer, and A.P.
Dufour. 2008. High Sensitivity of Children to Swimming-Associated Gastrointestinal Illness:
Results Using a Rapid Assay of Recreational Water Quality. Epidemiol. 19:375-383.
16.14 Wade, T.J., E. Sams, K.P. Brenner, R. Haugland, E. Chern, M. Beach, L. Wymer, C.C. Rankin,
D. Love, Q. Li, R. Noble and A.P. Dufour. 2010. Rapidly Measured Indicators of Recreational
Water Quality and Swimming-Associated Illness at Marine Beaches: A Prospective Cohort
Study. Environ. Health 9:66.
39
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Method 1609.1
17.0 Acronyms
AACT Comparative cycle threshold calculation method
AB Applied Biosystems (currently Life Technologies)
AF Amplification factor
BHIA Brain heart infusion agar
BHIB Brain heart infusion broth
Bp Base pair
BSA Bovine serum albumin
CCE Calibrator cell equivalent
CPU Colony forming units
CT Cycle threshold
CV Coefficient of variation
DNA Deoxyribonucleic acid
EDTA Ethylenediaminetetraacetic acid
GI Gastrointestinal illness
HEPA High efficiency particulate air
IAC Internal amplification control
IsrRNA Large subunit ribosomal RNA (/.e., 23S rRNA)
MLV Multi-laboratory validation
MS Matrix spike
NTC No template control
OD Optical density
OPR Ongoing precision and recovery
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PSI Pounds per square inch
QA Quality assurance
QC Quality control
qPCR Quantitative polymerase chain reaction
RNA Ribonucleic acid
RPD Relative percent difference
rpm Revolutions per minute
RSD Relative standard deviation
SPC Sample processing control
TNTC Too numerous to count
TSC Target sequence copy
UV Ultraviolet (light)
40
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Method 1609.1
Appendix A:
Part II (General Operations), Section A (Sample Collection,
Preservation, and Storage)
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Method 1609.1
Sample Collection1
1.0 Sample Containers
1.1
1.2
1.3
1.4
1.5
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.
Figure 1. Suggested sample containers
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.
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.
Chelating Agent: A chelating agent should be added to sample bottles used to collect
samples suspected of containing >0.0 1 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.
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 The 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.
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Method 1609.1
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
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Method 1609.1
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.
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Method 1609.1
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.
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
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Method 1609.1
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 stormwater, 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.
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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Method 1609.1
Appendix B:
Life Technologies StepOnePlus™, Applied Biosystems AB 7900, and
AB 7500 Real-Time PCR System Operation
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Method 1609.1
Appendix B: Life Technologies StepOnePlus™, Applied Biosystems
AB 7900, and AB 7500 Real-Time PCR System Operation
1.0 Life Technologies StepOnePlus™
1.0.1 Turn on the StepOnePlus™ and then the computer. Launch the StepOnePlus™ software
program by double clicking on its icon on the computer desktop or from the Computer
Programs menu.
1.0.2 On the StepOnePlus™ home screen select Advanced Set Up.
1.0.3 On the right side of the main screen click in the box Experiment Name, enter identifying
information for the experiment (Name/Date etc such that experiment can be identified).
Then go through the following fields:
• Which instrument is going to be used to run the experiment: StepOnePlus™
Instrument (96 wells) is default (highlighted).
• What experiment do you want to set up: Quantitation-Standard Curve is default
(highlighted).
• Which reagents will be used to detect the target: TaqMan® Reagents is default
(highlighted).
• Which ramp speed do you want to use in the instrument run: Click on Standard-2
hours (Not Default)
1.0.4 Click on Plate Set Up from the navigational pane of the present screen to define the
targets, and then assign them to wells in the reaction plate (Step 1.0.8).
1.0.5 Define target - You can add a new target or use a saved target. By clicking on Add
Saved Target the window with the target library will open.
1.0.6 Select the target(s) for your assay(s) and click on Add Selected Targets. All of the
targets may be selected simultaneously by holding the Ctrl key and highlighting the
desired targets (e.g., Entero, Sketa). The selected targets will then be added on to the
define target and sample screen.
1.0.7 Optional: If a new target is to be added click the Enter Target Name cell and type the
name. From the Reporter dropdown menu, select FAM (default). From the Quencher
dropdown menu, select TAMRA (NFQ-MGB) (default). Leave the default in the color
field.
1.0.8 Click on the tab Assign Targets and Samples to see the screen view of the plate layout
with 96 wells.
• Select the wells, based on the plate set up, by highlighting, one target at a time.
• Select the wells for the first target by checking the box for the desired target in
Assign Target to the selected wells and it will automatically populate the wells for
that target. If more than one target is being used, repeat the process above for each
target.
• The wells can be selected individually or by rows by clicking in the left corner of the
row. In addition, the whole plate may be selected by clicking in the left corner of the
plate.
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Method 1609.1
• To deselect a row or well press Ctrl & click the selected portion one more time and it
will deselect the row or well. Selected wells/rows will be highlighted grey, while
unselected wells/rows will remain white.
1.0.9 Run the Method
1.0.9.1 On the run method screen, review the reaction volume and the thermal profile for
the default run. If needed: The default run method can be edited or replaced with one
from the run method library. Click either the Graphical view (default) or Tabular
View tab.
1.0.9.2 Make sure the reaction volume per well field displays 25 \\L, this is not the
default setting.
1.0.9.3 Set the thermal profile to the following holding and cycling stages: Holding
Stage 1: 50.0°C for 2:00 minutes, Holding Stage 2: 95.0°C for 10:00 minutes,
Cycling Stage: 95.0°C for 0:15 seconds. The second step of the Cycling Stage is
defaulted at 60.0°C for 1 minute. Use instrument default of 40 cycles.
1.0.9.4Note: When using a run from the library click on the tab Open Run Method in
the graphical view. Select Run Method and click ok. It will replace the default run
with the saved run.
1.0.10 Load the plate into the instrument.
1.0.11 Click on the Plate Set Up tab and start the run by clicking the green button in the upper
right hand corner of the screen.
1.0.11.1 Save Experiment dialogue box - Click Save to accept default file name and
location (the name assigned when setting up the experiment). The experiment
is saved by default to the :\ applied Biosystems\\experiment folder.
1.0.11.2 Run progress can be viewed from the touch screen of the instrument. At the
beginning of a run do not leave the instrument or computer until you verify
that the run has started.
1.0.12 Once the run is completed remove the reaction plate and discard.
1.0.13 Analyze the run
1.0.13.1 Click on the tab in the top right corner of screen to Analyze the Run.
1.0.13.2 Highlight all of the sample wells and click on Analysis Settings to get to CT
settings (default CT settings).
1.0.13.3 Highlight all the targets running in the plate; uncheck the default setting for
automatic threshold and set the threshold to 0.03. Keep the Automatic
Baseline. Click on Apply Analysis Settings to save changes. The new
settings can be confirmed by viewing the threshold line on the amplification
plot.
1.0.14 Press the Export tab on the left corner of the view plate layout screen. Click on the
browse button to find the correct place to export the data and click Start Exporting.
After the export is done close export tool.
1.1 AB 7900 Real-Time PCR System Operation
1.1.1 Turn on the AB Model 7900 and then the computer. Launch the SDS 2.2.2 software
program by double clicking on its icon on the computer desktop or from the Computer
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Method 1609.1
Programs menu. The computer will establish communication with the 7900 instrument
and if the connection is successful, the software will display the Connected icon in the
status bar when a plate document is opened.
1.1.2 Under File menu, select New.
1.1.3 In resulting New Document window that appears, change container selection from 384
well clear plate to 96 well clear plate using drop down menu. Accept default selections
of Absolute Quantification and Blank Template. Click OK to display a new plate
document.
1.1.4 Click, hold and drag mouse over all PCR reaction tray wells containing samples in upper
left window. Selected wells will be outlined with a bold line and their position numbers
should appear in the results table in the lower left window. To unselect wells, repeat
above process while holding down control key.
1.1.5 Above right hand window, click on Setup tab.
1.1.6 Before any analyses are performed, a specific detector for the method must be created.
Click on Add Detector button at the bottom of the setup screen.
1.1.7 Click on New in the pop-up window that appears. Another pop-up window will appear.
Under Name, type in a name for the detector that will be used by this method (e.g.,
"Method 1609"). Under Group select Default. Under Reporter select FAM and VIC.
Under Quencher select TAMRA. Click on OK to close second pop-up window. This
step only needs to be performed before the initial analysis run of the method. The
detector that is named is selected in all subsequent analysis runs as indicated in step
1.1.8)
1.1.8 In pop-up window that was opened in step 1.1.7, select the desired detector under Names
menu (e.g., Method 1609) and click on Copy to Plate Document button. Click on Done
button to return to setup screen.
1.1.9 Click on Use box next to FAM (Entero and Sketa 22) VIC (IAC) detectors in right hand
window. These boxes should become marked with an X. Name and color codes for
FAM and VIC detectors should appear in each of the selected well positions in the upper
left window and a data column for this detector should be created for each of the selected
well positions in the results table in the lower left window.
1.1.10 Click on Instrument tab right hand window.
1.1.11 In instrument screen, change sample volume to 25 uL and choose 9600 emulation.
1.1.12 Still in instrument screen, click on Connect, then click on Open/Close button in lower
right hand "Real Time" window to open PCR reaction tray holder door on instrument.
1.1.13 Insert PCR reaction tray with prepared reactions in holder.
1.1.14 Click on Open/Close button to close PCR reaction tray holder door on instrument.
1.1.15 Click on Start button in lower right hand "Real Time" window to start thermal cycling in
instrument.
1.1.16 Name run file at prompt.
1.1.17 At termination of the run, instrument-calculated cycle threshold (CT) values should
automatically appear for each well position and detector entry in the lower left hand
results table window.
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Method 1609.1
1.1.18 At termination of the run success complete, choose Analysis Settings from the toolbar.
In that box enter a value for the Manual CT Threshold (see 1.0.13.3). Click on OK.
Click on Analyze from the toolbar. You should see CT values in the Results Table.
1.1.19 The instrument-selected threshold line is indicated by the bold red line on the plot and the
value is listed below the window. This can be manually adjusted by either click-hold and
dragging the line up or down or entering new values below. Note: Based on results thus
far with the instrument, the threshold value can be adjusted from the default value of 0.2
to 0.03, however, this should be done only if the threshold line remains above the
background values (seen before the growth curves) for all of the samples.
1.1.20 Calculated CT values for each of the sample tray positions in the lower left hand "Results
Table" will automatically be updated following adjustments of the threshold line. Once
the threshold is adjusted to the desired level, select "Print Report" under the "File" menu.
Check or uncheck desired report items by clicking on their associated boxes and the click
on "Print" button. Note: Minimum report should have both detector boxes, i.e., "FAM"
checked which will show CT values for all selected tray positions for this detector.
1.1.21 Export data by clicking on File from the toolbar. From the drop down menu choose
Export. In the box you will see Look in: and here you choose a directory to send the
exported file too. Click on Export. Save changes to document? will appear, click on
Yes. Click OK.
1.2 AB 7500 (non-Fast) Real-Time PCR System Operation
1.2.1 Turn on the AB Model 7500 and then the computer. Launch the software program by
double clicking on its icon on the computer desktop or from the Computer Programs
menu. The computer will establish communication with the 7500 instrument. See "How
to Set Up a New Experiment Using the AB 7500" for screen shots.
1.2.2 Click on either the New Experiment or Advanced Setup button to create a new
experiment, which will pull up the Experiment Menu.
1.2.3 From the Setup menu, select Experimental Properties to select the experiment type and
give the experiment a name. Enter the experiment name in the asterisked box.
1.2.3.1 Click on 7500 (96 Wells) to select the instrument type. Note: This protocol
is not designed for the 7500 Fast.
1.2.3.2 Scroll down to access more experiment properties options. Click on
Quantitation - Standard Curve to select the experiment type.
1.2.3.3 Click on TaqMan® Reagents to select the reagents used.
1.2.4 From the Setup menu, select Plate Setup. Click on the Define Targets and Samples
tab to define the reporter-quencher dye for each target and also to enter the sample
identifications (e.g., Ent 5X).
1.2.4.1 Click on Add New Target and enter the name of the target. Under the
Reporter heading, click on the drop down menu to select the reporter dye
FAM (Entero and Sketa 22) and VIC (IAC). Under the Quencher heading,
click on the drop down menu to select TAMRA as the quencher.
1.2.4.2 Repeat the process in Section 1.2.4.1 to add more than one target, e.g., Sketa
5x.
1.2.4.3 In the Define Samples section, click on Add New Sample and enter the
sample name in the Sample 1 box.
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Method 1609.1
1.2.4.4 Click on the Assign Targets and Sample tab to assign the Target, sample ID
and sample type to the wells.
1.2.4.5 Drag the mouse over the desired cells to assign the Target, Task and Sample.
1.2.4.6 While the cells are highlighted, in the Assign target(s) to the selected wells
section, check the box under Assign for the appropriate Target, then under
Task, select U for unknown, S for standard or N for negative control.
1.2.4.7 In the Assign sample(s) to the selected wells section, check the box under
Assign to label the highlighted cells as Sample 1 (e.g.).
1.2.4.8 Repeat the process in Section 1.2.4.4, dragging the mouse over the
appropriate cells, and then assigning them as Unknowns, Standards or
Negative Controls, as appropriate. Note that for the Standards, in the Assign
target(s) to the selected wells section, if there are 4 different values, each
cell will need to be highlighted separately, and a quantity (e.g., 40000.0)
typed in the Quantity box.
1.2.4.9 Click on Print Report at the top of the screen to print a plate layout for
loading master mix and sample extracts.
1.2.4.9.1 Select Plate Layout option by clicking on the box next to it.
1.2.4.9.2 Click Print Report to print the plate layout.
1.2.5 From the Setup menu, select Run Method to set up the thermo cycling profile.
1.2.5.1 In the Graphical View tab, change the Reaction Volume Per Well from the
default 50 uL to 25 uL by typing in 25.
1.2.5.2 Under Cycling Stage section, change the Number of Cycles to 45.
1.2.5.3 Check that the default settings of Temperature and Time for the two Holding
Stages and the Cycling Stage are correct. Specifically, these should be:
Holding Stage 1: 50.0°C for 1:00 minute, Holding Stage 2: 95.0°C for 10:00
minutes, and Cycling Stage: 95.0°C for 0:15 seconds.
1.2.5.4 The second step of the Cycling Stage is defaulted at 60.0°C for 1 minute.
Note: This sets the detection of the fluorescence signal to occur at the end of
the second step in each cycle.
1.2.5.5 Click on Save at the top of the screen to save the run before actually running
the experiment.
1.2.5.6 The Save screen will open so that you may select the folder to save the Run
Data File. Type in the Experiment Name (if you did not already do so at the
beginning of the Experiment Setup) and click on Save. If you had previously
typed in an Experiment Name and there is no change, click on Save. Note:
You must save to the hard drive (rather than a flash drive).
1.2.6 From the Experiment menu, select Run to monitor the run in real time.
1.2.6.1 The Run Status screen will open - click on Start Run. When the run has
successfully started, the Start Run button will turn change from green to red.
1.2.7 When the run has completed, from the Setup menu, select Analysis to export your
results. See "How to Export Results from the AB 7500 Software" for screen shots.
1.2.7.1 The Analysis Settings for Experiment name screen will open.
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Method 1609.1
1.2.7.2 Under the CT Settings tab, click on the Target desired to be set from Default
to Manual Threshold setting (generally, this would be the Ent).
1.2.7.3 In the CT Settings for ENT section, uncheck the boxes next to Use Default
Settings, and Automatic Threshold. Enter the desired Threshold
(e.g., 0.025). If more than one target is present, highlight the next target
(e.g., Sketa) and repeat this step.
1.2.7.4 Click on Apply Analysis Settings to save the changes.
1.2.8 Click on Export at the top of the screen to save the experiment results data and to save
the selected plots required in the report. Note that the threshold value will have changed
on the Amplification plot curve graphic.
1.2.8.1 The Export Data screen will open. In the Select data to export section,
check all of the boxes (i.e., Sample Setup, Raw Data, Amplification Data,
Results, and Multicomponent Data).
1.2.8.2 For Select one file or separate files, select One file to export all the data
into one file with multiple tabs.
1.2.8.3 For Export File Name, enter the name of the Experimental data file to be
exported, and for File Type, choose *.xls.
1.2.8.4 For Export File Location, click on Browse to select the folder in which you
wish to export your file. Always save your file to the hard drive.
1.2.8.5 Click on Start Export. When this is completed, an Export Completed
screen will open - click on Close Export Tool to complete this task.
1.2.9 How to interpret your results. See "How to Interpret Results from the AB 7500" for
screen shots.
1.2.9.1 After exporting your data, from the Experiment menu, select Analysis.
1.2.9.1.1 From the Analysis menu, select Amplification Plot to view the
results of the selected samples at different plot settings.
1.2.9.1.2 Drag the mouse over the desired cells to select the samples to be
plotted in the amplification plot.
1.2.9.1.3 In the Amplification Plot section, in the Plot Settings tab, select
the Plot Type (from the drop down menu) and the Graph Type
(from the drop down menu).
1.2.9.1.4 If the plot requires the display of the Threshold and Baseline, in
the Options tab, check the boxes next to Threshold and
Baseline. If you want to see the results in tabular format, click
on View Well Table, and scroll down or sideways for more
samples and analysis parameters.
1.2.9.1.5 The toolbar above the graph can be used to make hard copy
prints, saving the file as *.jpg, zooming in and out, and changing
the plot properties.
1.2.9.2 For the Standard Curve, select Standard Curve from the Analysis menu to
view the results of the same sample (assuming they have the same target) and
standards in the plot.
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Method 1609.1
1.2.9.2.1 In the Plot Settings tab, click the drop down menu arrow beside
Target to select a different target (if the standards were analyzed
in more than one target), and click the drop down menu arrow
beside Plot Color to change the plot color.
1.2.9.2.2 The standard curve parameter display below the plot (i.e.,
Target, Slope, Y-Inter(cept), R2, and Eff% (percent efficiency) is
useful for data analysis.
1.2.9.3 For Multicomponent plots, click on Multicomponent Plot from the Analysis
menu to view the variation in the fluorescence of the dyes used in the
experiment over the ascending repeat of the 40 thermo cycles based on well,
target, or dye.
1.2.9.4 Click on the drop down menu arrow next to Plot Color in the Plot Settings
tab to select the parameter for the basis of the graph (e.g., well, target, or
dye).
1.2.9.5 For raw data plots, select Raw Data Plot from the Analysis menu to view
the variation in the emission of fluorescence of the dyes in different filters
present over the ascending repeat of the 40 thermo cycles.
1.2.9.6 In the Options tab, drag the pointer on the cycle scale to the desired cycle
number to view the fluorescence variation through filters of the samples at
that cycle.
1.2.9.7 For quality controls, click on QC Summary from the Analysis menu to view
the analysis summary of the samples.
1.2.9.8 To view all the plots, click on Multiple Plots View from the Analysis menu
to see all of the plots for the samples in a single window. Each plot will have
its own drop down menu.
1.2.10 To print the report, click on Print Report at the top of the screen.
1.2.10.1 Check all of the boxes (i.e., Experimental Summary, Results Summary, Plate
Layout, Amplification Plot (3 boxes), Standard Curves, Results Table, and
QC Summary).
1.2.10.2 Click on Print Report at the bottom of the screen.
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Method 1609.1
Appendix C
Cepheid Smart Cycler® Operation
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Method 1609.1
Appendix C - Cepheid Smart Cycler® Operation
1.0 Smart Cycler® Operation
1.1 This protocol is intended to provide only information about critical instrument settings required to
perform EPA Method 1609. Further details concerning the operation of the instrument can be
obtained from the Smart Cycler® Operation Manual, Cepheid Part DO 190, Rev. D.
1.2 Turn on the Smart Cycler®; then the computer.
1.3 Double-click on the Smart Cycler® icon on the computer desktop.
1.4 The following steps for defining a protocol are only required before the initial run of the
instrument. The protocol that is defined in these steps is used in all subsequent runs of the
instrument. See "How to Set Up a New Experiment Using the Smart Cycler® (Software Version
2.0" for screen shots.
1.4.1 Click on the Define Protocols icon to go to Define Protocols screen.
1.4.2 Click on the New Protocol button to open the Protocol Name? dialog. Enter "EPA
Enterococcus Method 1609" for the new protocol name, and click OK. The protocol
stages are defined in the series of boxes at the bottom of the Define Protocol screen.
Make sure the new protocol is highlighted and begin to choose state settings. To define
Stage 1, click on its drop-down box to display the menu of stage types; then select Hold.
In the Temp column, enter 50.0, and in Sees column, enter 60, leaving the Optics setting
as the default Off setting.
1.4.3 To define Stage 2, click on its drop-down box to display the menu of stage types, and
again select Hold. In the Temp column, enter 95.0, and in Sees column enter 600.
Again, leave the Optics setting on Off.
1.4.4 To define Stage 3, select 2-Temperature Cycle from its drop down menu. For the first
step, enter 95.0 in the first row of the Temp column and 15 in the Sees column, Optics
column Off. For the second step, enter 60.0 in the second row of the Temp column, 60
in the Sees column, and click on the Optics cell to select On from the drop-down menu.
(Note: This sets the detection of the fluorescence signal to occur at the end of the second
step in each cycle.) Enter 40 in the Repeat field at the top of the Stage 3 box to specify
that it should be repeated for 40 cycles. Click the Save Protocol button.
1.4.5 To display primary curve graphs, click Define Graphs. Highlight FAM and VIC in
Graph column. Check the box for Automatically add to new runs. Under Graph
Type choose Optics from the pull down menu. Under Channels check the box for Ch 1
(FAM) and Ch 2 (VIC). Under Show check the boxes for Primary Curve, Threshold
(Vertical) and Threshold (Horizontal). Under Axes check the box for Fluorescence vs.
Cycle. At the bottom of the screen click on Save Graph.
1.5 Click on the Create Run icon to open the Create Run screen. For each new run, enter a unique
name in the Run Name field. (Note: The software does not allow duplicate run names).
1.6 Enter any additional information about the run in the Notes field. Click the arrow in the Dye Set
box to display a drop-down menu of the possible selections. Select FTTC25. (Note: This selects
the dye set: FAM, TET, Tex Red, Cy5, and a 25 uL reaction).
1.7 Click the Add/Remove Sites button. The Select Protocols and Sites... dialog will appear.
Highlight (click on) the "EPA Method 1609" protocol developed prior to the first run (Section
1.4.2) in the Protocols list. In the Sites list, highlight the sites on the instrument to be used with
this protocol in the current run by clicking on them with the shift key held down (Note: Sites refer
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Method 1609.1
to the I-core modules in the Smart Cycler® processing block in which reaction tubes will be
placed; a total of 16 are possible per block. When using multiple blocks, the site numbers will be
preceded by the block letters, e.g., A, B, C); then click the right pointing arrow to transfer the
selected sites and protocol to the Selections table.
1.8 Click on the OK button to save the selections, and return to the Create Run screen.
1.9 Place the loaded Smart Cycler® reaction tubes in the I-core module slots, selected above for
current run. The tubes should snap into place. Either the front or back of the caps can face the
front of the processing block.
1.10 In one of the View menus that is shown, select Analysis Settings. The displayed table includes
one row for each of the four possible dye channels defined in the dye set. Click on the cell in the
FAM row under the Usage column heading, and select Assay from the drop down menu. Set the
Usage cells for VIC in the same manner. (Note: All assays in this protocol use FAM and VIC as
the reporter dye). All other cells in this table should be left at default settings (See Smart Cycler®
Operation Manual).
1.11 In the other View menu that is shown, select the Results Table. Enter the sample identification
information for each site in the Sample ID column (additional information can be entered into the
Notes column.). Leave the other columns as default settings (see Smart Cycler® Operation
Manual.).
1.12 Click on the Start Run button. The orange LEDs on the Smart Cycler® processing block should
turn on, and the software will automatically switch to the View Results screen.
1.13 To display the real time temperature profiles for all sites, click Temperature in either of the
View menus. To display real time growth curves for all samples (i.e., the fluorescence signal vs.
cycle), click FAM and VIC in the other View menu.
1.14 At the end of the run, it is recommended to check the cycle threshold values calculated by the
instrument for each sample by opening the Results Table window by clicking on this selection in
the upper View menu. It is also recommended to inspect the growth curves in the FAM window
and in the VIC window which can be opened in the same manner from the lower View menu.
The default threshold fluorescence value is shown in this window as a single horizontal red line
and the cycle thresholds for each site are shown as vertical red lines. To view the data for
individual sites in this window, click on that site number in the table to the right of the graph. If
the default threshold fluorescence line is well above all of the growth curve lines prior to visible
amplification, the threshold fluorescence value can be changed to a lower value. This is done by
reopening the Analysis Settings window from the upper View menu and entering a new value in
the Manual Thresh Fluor Units cell in the FAM row and VIC row. Conversely if the default
threshold fluorescence line is below any of the growth curve lines prior to visible amplification,
the threshold fluorescence value should be changed to a higher value in the same manner.
Previous studies have indicated that a threshold value of 8 works well for most analyses. Click
on the Update Analysis button to view the new threshold line in the FAM window and VIC
window. The cycle threshold values will be automatically updated in the Results Table.
1.15 Once the threshold fluorescence value is adjusted to an optimal value, click the Save Run button.
(Note: The Smart Cycler® Software does not give a prompt to save changes before printing or
exporting. Therefore, it is possible to make changes to the Results Table or Analysis Settings,
and immediately print or export the data, then close the run without saving the changes. In this
case, the data saved in the Smart Cycler® database will not match the printed or exported data. If
no changes are made in the threshold fluorescence value, the run data is automatically saved as it
is when the program is closed or a new run is created).
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Method 1609.1
1.16 To set up automatic export of raw data, (see "How To Set Up Automatic Export of Raw Data" for
screen shots) from the main menu, click on Setup, then System Defaults, then Export Settings.
From the Export Settings dialog box, check the box beside Results Table and Analysis Settings.
Click the Browse button to select the folder where you want your raw data saved. Click on the
radio button next to Automatic export on run completion, then click the OK button.
1.17 To manually save the Results Table and Analysis setting containing the instrument-calculated
cycle threshold values for each sample (see "How to Manually Export Raw Data" for screen
shots), click the Export button to display the Export Data dialog box. Check the box next to the
heading Export Results Table and Analysis Settings by clicking on it. Leave all other boxes
unchecked. Click on Export. A box labeled Export Data will appear with the run name in the
file name box. Click on the drop down menu to the right of Save In: at the top left of the screen
to change the directory where you want your data saved. Click on Save. Data are exported as
comma-delimited text (*.cvs) files in MS Excel-compatible files to the Export folder in the Smart
Cycler® folder: C:\Smart Cycler®\Export. Always save to the hard drive.
1.18 To archive a run in the Smart Cycler® program, click on Tools. From the drop down menu click
on Data Management and then Archive Runs. Click Proceed. Select the run to be archived by
clicking on its name in the database list. Click OK and then Proceed. A box labeled Archive
Run will appear on the screen. There will be a line labeled Save in: Input which directory you
would like the run to be saved in. There will be a line File Name: Enter a file name. Click
Save. Click OK.
1.19 To print run data (see "How to Print Run Data" for screen shots), after the run is completed, right
click on the graph area, and select Print, then Print Graph with Results Table. An Optics
Graph print preview screen will pop up, click Print. The screen will switch back to the post run
screen. To print the generated report, click Report. A Run Report print preview screen will pop
up, click Print.
1.20 To set up a new analysis (see "How to Set Up a New Run" for screen shots), from the post run
screen, click Create Run at the top left. The results of your just completed will disappear and a
Add/Remove Sites button will appear in the middle of the screen - click it. A Select Protocols
and Sites screen will pop up, select EPA Enterococcus Method 1609 in the Protocols box, and
highlight the amount of sites needed for the assay (A1-A16) in the Sites box. Click the right
arrow to the right of the sites box to move the sights to the Selections box. After double checking
that the correct protocol is highlighted, click OK. Add the Run Name in the Run Name box. The
Site ID, Protocol, Sample ID, Sample Type, Notes (etc.) box will pop up. Enter the Sample ID
for each site. Under this box, find the Usage column and click on Assay to select Unused for
Cy3, TxR and Cy5 (leave Assay for FAM). In the same box, find the Manual Thresh Fluor Units,
and click on the units for FAM to change it from 30.0 to 8.0 (leave the units at 30.0 for Cy3, TxR
and Cy5). After loading the Smart Cycler® with tubes, click Start Run. A red light should
appear on sites that are in use.
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Method 1609.1
Appendix D
Protocol for MPN Estimation of DMA Standard Concentrations
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Method 1609.1
Appendix D: Protocol for MPN Estimation of
DMA Standard Concentrations
Initial qPCR analysis for negative control
1. Prepare master mix containing primer/probe working stock for Entero 1A assay, BSA and 2x ABI
Environmental Master Mix for sufficient reactions and transfer 24 - 20 \\L aliquots to 96 well
reaction plate.
2. Add 5 \\L AE buffer to each well containing master mix and cover plate with optical adhesive
tape
3. Run reactions using ABI instrument default thermal cycling conditions for 45 cycles
4. At completion of run, set instrument fluorescence threshold to 0.03 and inspect results for
frequency of positive wells (wells giving CT values as opposed to "undetermined")
a. Frequency of positive wells should either be zero or very low for MPN analysis. If
frequency of positives is > 25%, there is unacceptable contamination of reagents to
perform MPN analysis. Repeat using a different lot of Environmental Master Mix.
First DNA qPCR analysis
5. Dilute genomic DNA or linearized plasmid stock solution in AE buffer to presumptive
concentration of 4 x 105 copies/5 \\L based on estimated target sequence concentration of the
DNA stock solution determined as described in Section 11.2 (genomic DNA) or Section 7.23
(plasmid DNA).
6. Perform 10-fold serial dilutions of presumptive 4 x 105 copies/5 (iL sample from step 5 with AE
buffer to make DNA standards with presumptive 4 x 104 copies/5 \\L, 4 x 103 copies/5 \\L, 4 x
102 copies/5 \\L and 4 x 101 copies/5 \\L. Note: Perform dilutions in low retention micro-tubes
(see Section 6.24).
7. Prepare master mix containing primer/probe working stock for Entero 1A assay, BSA and 2x ABI
Environmental Master Mix for sufficient reactions and transfer 24 - 20 \\L aliquots to 96 well
reaction plate.
8. Add 5 (iL of presumptive 4 x 104 copies/5 (iL, 4 x 103 copies/5 (iL, 4 x 102 copies/5 (iL, 4 x 101
copies/5 \\L standards and AE buffer to 4 wells each containing master mix and cover plate with
optical adhesive tape.
9. Run reactions using ABI instrument default thermal cycling conditions for 45 cycles.
10. At completion of run, set instrument fluorescence threshold to 0.03 and record CT values.
11. Perform regression analysis of CT values on logio copies/well as described in Section 9.10.
Second DNA qPCR analysis
12. Perform 2-fold serial dilutions of presumptive 4 x 101 copies/5 (iL standard in AE buffer to make
2 x 101 copies/5 jiL, 1 x 101 copies/5 jiL, 5 x 10° copies/5 jiL, 2.5 x 10° copies/5 jiL, 1.25 x 10°
copies/5 \\L and 0.625 x 10° copies/5 \\L. Note: Perform dilutions in low retention micro-tubes
(see Section 6.24).
13. Prepare master mix containing primer/probe working stock for Entero 1A assay, BSA and 2x ABI
Environmental Master Mix for sufficient reactions and transfer 96 - 20 \\L aliquots to two - 96
well reaction plates.
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Method 1609.1
14. Add 5 (iL of presumptive 2 x 101 copies/5 (iL, 1 x 101 copies/5 (iL, 5x10° copies/5 (iL and AE
buffer to 24 wells each of the first plate containing master mix and cover plate with optical
adhesive tape.
15. Add 5 (iL of presumptive 2.5 x 10° copies/5 (iL, 1.25 x 10° copies/5 (iL, 0.625 x 10° copies/5 (iL
and AE buffer to 24 wells each of the second plate containing master mix and cover plate with
optical adhesive tape.
16. Run reactions using ABI instrument default thermal cycling conditions for 45 cycles.
17. At completion of run, set instrument fluorescence threshold to 0.03 and record CT values.
18. Record number of wells giving positive CT values as opposed to "undetermined" for each dilution
of the presumptive 4 x 101 copies/5 \\L standard (and for AE buffer on each plate if applicable).
Note: At least 3 of the dilutions should give a mixture of positive and negative results in the 24
replicate reactions. If this is not the case, it may be necessary to repeat the experiment with even
higher dilutions of the presumptive 4 x 101 copies/5 \\L standard (i.e., if all wells are giving
positive results) or beginning with dilutions from the presumptive 4 x 102 copies/5 (iL standard
(i.e., if all wells are giving negative results)
Statistical analyses
19. Starting with lowest dilution where some wells give negative results, type number of positive
wells in cell C3 of MPN calculation spreadsheet. Note: If any AE wells on same plate are
positive, type following formula in cell C3:
= ((number of positive wells for dilution - number of positive wells for AE) / (24 - number of
positive wells for AE)) x 24
20. Repeat previous step, typing number of positive wells (or formula) from progressively higher
dilutions of the standard in cells C4, C5, C6 etc. up to the highest dilution where any wells are
positive (or outnumber positive AE wells on corresponding plate)
21. Type the value 24 in each row of column B corresponding to rows in column C that contain
values.
22. Calculate the fraction of the volume of the presumptive 4 x 101 copies/5 (iL standard from step 6
that was added to the reactions corresponding to the lowest dilution giving usable data as
described in step 19 (e.g., if usable data started with a two-fold dilution of this standard, the
calculated value would be 0.5; if usable data started with a four-fold dilution of this standard, the
value would be 0.25; if usable data started with an eight-fold dilution of this standard, the value
would be 0.125; etc. Type this initial value in cell A3. Type 0.5 x this value in cell A4; 0.25 x this
value in cell A5, 0.125 x this value in cell A6, etc, corresponding to each row in column C that
contain values.
23. Type the number that then appears in cell J3, into cell G2. Hit Enter key.
24. Then hold down Ctrl key and type "m". Correct MPN (cell G2) logMPN (cell G3), and low and
high 95% confidence limits (cells G7-H8) will now be displayed.
25. Also check value in cell J16 to confirm test assumptions have not been violated (improbability
ratios > l.OOE-04 are considered acceptable in the spreadsheet but available results suggest
improbability ratios should be < l.OE-01)
26. The displayed MPN (cell G2) logMPN (cell G3) values are your corrected estimates of the target
sequences/5 (iL of the presumptive 4 x 101 copies/5 (iL DNA standard from step 6.
D-2
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Method 1609.1
Additional DNA qPCR and statistical analyses
27. To increase confidence in the MPN estimate, it is recommended that the analysis indicated above
should be repeated at least twice (3 replicate runs total). These additional runs only need to be
performed using the three lowest dilutions that all gave both positive and negative results in
different wells in the initial analysis (if lowest such dilution gives close to 100% positives (e.g., >
20/24), avoid this dilution and start with the next dilution to reduce possibility of 100% positives
in some of the replicate runs). EPA data suggests that the coefficient of variation in the MPN
estimates from these multiple runs should not exceed -30%. Assuming similarity of results from
run to run, the MPN estimates from each experiment can be averaged to calculate a final mean
MPN estimate.
28. To characterize uncertainty by statistical analysis, the analysis should be repeated at least 5
additional times (total of at least 6 replicate runs) (Note: Statistical analysis of uncertainty is not
discussed in detail in this guidance. See Reference 16.6 for further guidance).
29. Assuming the standard curve(s) performed in step 9 meet the acceptability criteria for slope
specified in Section 9.10, the final MPN target sequences/5 (iL estimate for the presumptive 4 x
101 copies/5 (iL DNA standard also can be used to obtain corrected concentration estimates of the
other 3 standards prepared in step 6 simply by multiplying by the dilution factors, e.g., lOx, lOOx
and lOOOx.
D-3
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Method 1609.1
Appendix E:
How to Obtain DMA Standards from EPA
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Method 1609.1
Appendix E: How to obtain DMA standards from EPA
A. What we need from you:
1. Please email your request to Robin Oshiro at Oshiro.robin@epa.gov
2. Please put "Request for EPA DNA standards" in the subject line (so that your request does not
inadvertently go into a junk folder).
3. Please provide the following information:
a. Your name, phone number and email address
b. The name, phone number, and email address of the person to whom the DNA standards will
be shipped, if different from (3a)
c. Your institution (e.g., company name)
d. Your courier address (i.e., Federal Express address, which may be different from your U.S.
Postal Service mail delivery address)
4. Please note that:
a. We cannot ship to residential addresses
b. We cannot ship the DNA standards if you do not provide all of the information required (see
[3] above)
c. We are not responsible for incorrect addresses resulting in inability to deliver. If a box is
returned to us, we cannot ship you another box. Therefore, please double check your
information before sending it.
5. Once we have emailed you the Federal Express tracking number, please verify receipt of that
information via email.
B. What we will do:
1. Your request for DNA standards will be acknowledged as soon as possible via email. Once the
verification email is sent to you, with very few exceptions, we cannot change any information for
the shipping label. Therefore, it is vital that you verify your information before sending it to us.
2. We will ship a set of five DNA standards to you on dry ice. Lot-specific concentration
information will be provided with each kit.
3. We expect to ship only on Tuesdays. This is so that if there is any courier holdup, you can still
receive your standards before the end of the week. In most cases, you should expect your box to
arrive the next business day.
a. If Monday or Tuesday is a U.S. Government federal holiday or the federal government in the
DC metro area is closed, your shipment will be made the following week.
4. We use Federal Express. We are not responsible for courier inability to deliver on time due to,
for example, inclement weather or other mishaps.
5. We will email you the tracking number of your box.
C. Restrictions (other than those stated above)
1. The DNA standards will be available until December 2015.
2. Only one set of DNA standards can be sent per laboratory per year (i.e., one set in 2015).
3. The DNA standards can only be sent to addresses in the United States and Territories (all 50
states, American Samoa, District of Columbia, Guam, Northern Mariana Islands, Puerto Rico,
United States Minor Outlying Islands, and United States Virgin Islands). We cannot ship the
standards outside the United States.
E-l
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