Method A: Enterococci in Water by 

TaqMan® Quantitative Polymerase Chain 

Reaction (qPCR) Assay 

 April 2010 

Note that this method will be updated following it’s validation in marine 

and fresh ambient waters. 
U.S. Environmental Protection Agency 

Office of Water (4303T) 

1200 Pennsylvania Avenue, NW 

Washington, DC 20460 

EPA-821-R-10-004
iii

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 single laboratory 

validation of this method in fresh and marine waters: 

Participant Laboratories

• New York State Department of Health, Environmental Biology Laboratory: Ellen Braun-Howland 

and Stacey Chmura 

• Mycometrics, LLC: King-Teh Lin and Pi-shiang Lai 
iv

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 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 

 
v

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 (CFU) in the water by culture based 

methods and the risk of gastrointestinal illness (GI) associated with swimming in the water (References 

17.3, 17.6, 17.7, and 17.8). 

Method A 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 RNA gene (lsrRNA, 23S rRNA) from these organisms. The 

advantage of this method over currently accepted culture methods that require 24-48 hr to obtain results is 

its relative rapidity. Results can be obtained by this method in 3-4 hr, allowing same-day notifications of 

recreational water quality. While measurements of enterococci DNA by Method A and enterococci CFU’s 

by culture methods such as EPA Method 1600 are performed for essentially the same purpose, i.e., to 

indicate fecal pollution, the results of these two approaches may not always be correlated with each other 

due to potential differences in the ratios of viable and non-viable bacteria in different water environments. 

Never-the-less, more recent epidemiological studies conducted at freshwater recreational beaches 

(Reference 17.3) have demonstrated similar or improved positive correlations between enterococci DNA 

measurements by this method and swimming-associated GI illness rates compared with those established 

for enterococci CFU measurements. 

In Method A, water samples are filtered to collect enterococci on membranes. Following filtration, total 

DNA is solubilized from the retentate using a bead beater. Enterococci target DNA sequences present in 

the clarified homogenate are detected by the real time polymerase chain reaction technique using 

TaqMan® Universal 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 fluorogenicallylabeled oligonucleotide probe when it hybridizes to the target sequence. 

Method A uses an arithmetic formula, the comparative cycle threshold (CT) method, to calculate the ratio 

of Enterococcus lsrRNA gene target sequences (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. Mean estimates of the absolute quantities of target sequences in the 

calibrator sample extracts are then used to determine the absolute quantities of target sequences 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. 
vi

Table of Contents

1.0 Scope and Application .....................................................................................................................1 

2.0 Summary of Method ........................................................................................................................1 

3.0 Definitions .......................................................................................................................................1 

4.0 Interferences.....................................................................................................................................2 

5.0 Safety ...............................................................................................................................................3 

6.0 Equipment and Supplies ..................................................................................................................3 

7.0 Reagents and Standards ...................................................................................................................5 

8.0 Sample Collection, Handling, and Storage ......................................................................................8 

9.0 Quality Control ................................................................................................................................9 

10.0 Calibration and Standardization of Method-Related Instruments..................................................11 

11.0 Procedure .......................................................................................................................................11 

12.0 Data Analysis and Calculations .....................................................................................................20 

13.0 Sample Spiking Procedure.............................................................................................................23 

14.0 Method Performance......................................................................................................................23 

15.0 Pollution Prevention ......................................................................................................................24 

16.0 Waste Management........................................................................................................................24 

17.0 References.....................................................................................................................................25 

18.0 Acronyms 
vii

List of Appendices 

Appendix A: ABI 7900 and ABI 7500 Sequence Detector Operation 

Appendix B: Cepheid Smart Cycler® Operation 
1

Method A: Enterococci in Water by Quantitative Polymerase Chain 

Reaction (qPCR) Assay 

April 2010 

1.0 Scope and Application

1.1 Method A describes a quantitative polymerase chain reaction (qPCR) procedure for the 

measurement of large subunit ribosomal RNA (lsrRNA, 23S rRNA) 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 using TaqMan® Universal 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 Enterococci are commonly found in the feces of humans and other warm-blooded animals. 

Although DNA from these organisms can be persistent in the environment, its presence in water 

is an indication of fecal pollution and the possible presence of enteric pathogens. 

1.3 The Method A test is recommended as a measure of ambient fresh and marine recreational water 

quality. Epidemiological studies have been conducted at freshwater and marine water beaches 

has been conducted that may lead to the potential development of criteria that can be used to 

promulgate recreational water standards based on established relationships between health effects 

and water quality measurements by this method. The significance of finding Enterococcus DNA 

target sequences in recreational water samples stems from the direct relationship between the 

density of these sequences and the risk of gastrointestinal illness associated with swimming in the 

water that have been observed thus far in the epidemiological studies (Reference and 17.9). 

1.4 This method assumes the use of an ABI Sequence Detector as the default platform. 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 agitated to extract the DNA into solution. The 

supernatant is used for PCR amplification and detection of target sequences using the TaqMan® 

Universal Master Mix PCR reagent and probe system. 

3.0 Definitions

3.1 Enterococci: all species of the genus Enterococcus for which lsrRNA gene nucleotide sequences 

were reported in the GenBank database (http://www.ncbi.nlm.nih/gov/Genbank) at the time of 

method development. 
Method A 

2

3.2 Target sequence: A segment of the Enterococcus lsrRNA gene containing nucleotide sequences 

that are homologous to both the primers and probe used in the Enterococcus qPCR assay and that 

are common only to species within this genus. 

3.3 Sample processing control (SPC) sequence (may also be referred to as reference sequence): A 

segment of the ribosomal RNA gene operon, internal transcribed spacer region 2 of chum salmon,

Oncorhynchus keta, 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. 

3.4 DNA standard: A purified, RNA-free and spectrophotometrically quantified and characterized E. 

faecalis strain ATCC #29212 genomic DNA preparation. 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 9.6. Also used for quantifying target sequences in calibrator sample extracts as described 

in Section 12.2. 

3.5 Calibrator sample: Samples containing constant 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 ΔΔCT comparative cycle threshold calculation 

method as described in Section 12.4. 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 ΔΔCT comparative cycle threshold calculation method (ΔΔCT method): A calculation method 

derived by Applied Biosystems (Reference 17.1) 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 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 comparative cycle threshold calculation methods. 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 12.2. 

4.0 Interferences

Water samples containing colloidal or suspended particulate materials can clog the membrane 

filter and prevent filtration. These materials can also interfere with DNA recovery and with the 

PCR analysis by inhibiting the enzymatic activity of the Taq DNA polymerase, and/or inhibiting 

the annealing of the primer and probe oligonucleotides to sample target DNA, enzyme or 

quenching of hydrolyzed probe fluorescence. 
Method A 

3

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 HEPA-filtered laminar flow hoods and an Ultraviolet (UV) light source, each 

having separate supplies (e.g., pipettors, tips, gloves, etc.). Note: 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 11.6.1 between reagent and sample preparation. Under ideal conditions, two 

dedicated workstations are used for sample preparation: one for preparing samples with high 

target sequence DNA concentrations (e.g., DNA standards and calibrator samples) and one for 

preparing samples with expected low target sequence DNA concentrations (e.g., filter blanks and 

ambient water samples). 

6.2 Balance capable of accuracy to 0.01 g 

6.3 Extraction tubes: semi-conical, screw cap microcentrifuge tubes, 2.0-mL (e.g., PGC 506-636 or 

equivalent) 

6.4 Glass beads, acid washed, 212 - 300 μm (e.g., Sigma G-1277 or equivalent) 

6.5 Autoclave, capable of achieving and maintaining 121°C [15 lb pressure per square inch (PSI)] for 

minimally 15 minutes (optional) 

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 

(e.g. Pall Gelman 4242, or equivalent), stainless steel, or disposable plastic (e.g., Nalgene CN 

130-4045 or 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 Fm pore size 

(e.g., GE Osmotics Inc. 04CP04700 or equivalent) 
Method A 

4

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 × 15 mm with loose fitting lids 

6.17 Disposable loops, 1 and 10 μL 

6.18 Permanent ink marking pen for labeling tubes 

6.19 Visible wavelength spectrophotometer capable of measuring at 595 nm 

6.20 Single or 8-place mini bead beater (e.g., Biospec Products Inc. # 3110BX or equivalent). 

6.21 Microcentrifuge capable of 12,000 × g 

6.22 Micropipettors with 10, 20, 200 and 1000 μL capacity. Under ideal conditions, each workstation 

should have a dedicated set of micropipettors (one micropipettor set for pipetting reagents not 

containing cells or reference DNA and one set for reagents containing reference DNA and for test 

samples). 

6.23 Micropipettor tips with aerosol barrier for 10, 20, 200 and 1000 μL 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, use a separate rack for each set of tubes 

6.26 Conical centrifuge tubes, sterile, screw cap, 50-mL 

6.27 Test tubes, screw cap, borosilicate glass, 16 × 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 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 or NanoDrop® (ND-1000) spectrophotometer (or equivalent) 

capable of the same measurements at 2 μL sample volumes 

6.39 ABI 7900 Sequence Detector

6.39.1 Optical 96 well PCR reaction tray (e.g., Applied Biosystems N801-0560 or equivalent) 

6.39.2 Optical adhesive PCR reaction tray tape (e.g., Applied Biosystems 4311971 or 

equivalent) or MicroAmpTM caps (e.g., Applied Biosystems N8010534 or equivalent) 
Method A 

5

6.39.3 ABI 7900 sequence detector 

6.40 Cepheid Smart Cycler® (optional to ABI) 

6.40.1 Smart Cycler® 25 μL 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: Molecular grade reagents and chemicals shall be used in all tests 

7.2 Control Culture 

• Enterococcus faecalis (E. faecalis) ATCC #29212 

7.3 Sample Processing Control (SPC) DNA (source of SPC control sequences) 

• Salmon testes DNA (e.g., Sigma D1626 or equivalent) 

7.4 Phosphate Buffered Saline (PBS) 

7.4.1 Composition: 

Monosodium phosphate (NaH2PO4) 0.58 g 

Disodium phosphate (Na2HPO4) 2.50 g 

Sodium chloride 8.50 g 

Reagent-grade water 1.0 L 

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 lb pressure per square inch 

(PSI)] for 15 minutes. Final pH should be 7.4 ± 0.2. 

7.5 Brain heart infusion broth (BHIB) 

7.5.1 Composition: 

Calf brains, infusion from 200.0 g 7.7 g 

Beef heart, infusion from 250.0 g 9.8 g 

Proteose peptone 10.0 g 

Sodium chloride 5.0 g 

Disodium phosphate (Na2HPO4) 2.5 g 

Dextrose 2.0 g 

Reagent-grade water 1.0 L 

7.5.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve 

completely. Dispense 10 mL volumes in screw cap 16 × 125 mm tubes and autoclave at 

121°C (15 PSI) for 15 minutes. Final pH should be 7.4 ± 0.2. 
Method A 

6

7.6 Brain heart infusion agar (BHIA) 

7.6.1 Composition: 

BHI agar 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 × 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 37EC for 24 hours). Centrifuge to create 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 to 

determine CFU concentration as described in section 11.1. Note: 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 water from VWR EM-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 

 0.5 mM EDTA (Ethylenediaminetetraacetic acid) 

7.12 Salmon DNA/extraction buffer 

7.12.1 Composition: 

Stock Salmon testes DNA (10 μg/mL) (Section 7.3) 

AE Buffer (Section 7.11) 

7.12.2 Preparation of stock Salmon testes DNA: Each bottle of Salmon DNA contains a 

differing number of units. Note the units. Add an equal volume of PCR grade water to 

dissolve the Salmon testes DNA, and stir, using a magnetic stir bar at low to medium 

speed, until dissolved (2-4 hours if necessary). The solution at this point will be 

equivalent to 50 μg Salmon testes DNA/mL. Dilute using PCR grade water to a 

concentration of ~ 10 μg/mL. Determine concentration of Salmon testes DNA stock by 

OD260 reading in a spectrophotometer. An OD260 of 1 is approximately equal to 50 

μg/mL (one Unit). This is your Salmon testes DNA stock solution. Unused portion may 

be aliquoted and frozen at !20°C. 

 Note: For example, if the bottle contains 250 mg of DNA, using sterilized scissors and 

sterilized forceps, cut a piece of DNA to weigh approximately 20 mg (approximately 304 

Units), and place in a sterile weigh boat. After weighing, place the DNA into a sterile 50 

mL tube and add 20 mL PCR grade water. Cap tightly, and resuspend by 2-4 hours of 

gentle rocking. The concentration should be about 1 mg/mL. Remove three, 10 μL 

aliquots and dilute each to 1 mL with PCR grade water. Check absorbance (OD260) and 

calculate DNA using the assumption 1 Unit DNA is equal to 1 OD260, which is then 
Method A 

7

equivalent to 50 μg/mL DNA. Adjust this stock to 10 μg/mL based on calculated initial 

concentration of 1 mg/mL by diluting with PCR grade water. Aliquot portions of the 

adjusted DNA stock and freeze. 

7.12.3 Dilute Salmon testes DNA stock with AE buffer to make 0.2 μg/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 μL) × total number of samples to be analyzed 

including controls, water samples, and calibrator samples. For example, for 18 samples, 

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 μL 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 μL × 25 tubes = 15,000 μL. 

Dilute the Salmon testes DNA working stock 1:50, for a total volume needed (15,000 μL) 

÷ 50 = 300 μL of 10 μg/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 μL - 300 μL = 14,700 μL 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® Universal PCR Master Mix (e.g., Applied Biosystems 4304437 or equivalent) 

 Composition: 

 AmpliTaq Gold® DNA Polymerase 

 AmpErase® UNG 

 dNTPs with dUTP 

 Passive Reference 1 (ROXTM fluorescent dye) 

 Optimized buffer components (KCl, Tris, EDTA, MgCl2) 

7.16 Bovine serum albumin (BSA), fraction V powder (e.g., Sigma B-4287 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 purified. 

7.17.1 Enterococcus primer and probe set (Reference 17.5): 

Forward primer: 5'-GAGAAATTCCAAACGAACTTG 

Reverse primer: 5'-CAGTGCTCTACCTCCATCATT 

TaqMan® probe: 5'-FAM-TGGTTCTCTCCGAAATAGCTTTAGGGCTA-TAMRA 

7.17.2 Salmon DNA primer and probe set (Reference 17.4): 

Forward primer: 5'-GGTTTCCGCAGCTGGG 

Reverse primer (Sketa 22): 5'-CCGAGCCGTCCTGGTC 
Method A 

8

TaqMan® probe: 5'-FAM-AGTCGCAGGCGGCCACCGT-TAMRA 

7.17.3 Preparation of primer/probes: Using a micropipettor with aerosol barrier tips, add PCR 

grade water to the lyophilized primers and probe from the vendor to create stock 

solutions of 500 μM primer and 100 μM probe and dissolve by extensive vortexing. 

Pulse centrifuge to coalesce droplets. 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. 

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. 

Autoclave tubes at 121°C (15 PSI) for 15 minutes. 

7.19 Purified, RNA-free quantified and characterized E. faecalis genomic DNA preparations for use as 

standards used to generate a standard curve (see Section 11.2). 

7.20 RNase A (e.g., Sigma Chemical #R-6513) 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 min. 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 μg/μL. 

7.21 DNA extraction kit (Gene-Rite #K102-02C-50 DNA-EZ® RW02 or equivalent) 

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 
Method A 

9

temperature. Take care 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 A 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 17.2. 

9.2 Media sterility check — The laboratory should test media sterility by incubating one unit (tube 

or plate) from each batch of medium (BHIA/BHIB) as appropriate and observing for growth. 

Absence of growth indicates media sterility. On an ongoing basis, the laboratory should perform 

a media sterility check every day that samples are analyzed. 

9.3 Method blank (water sample filtration blank) — Filter a 50-mL volume of sterile PBS before 

beginning the sample filtrations. Remove the funnel 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 7.20. Extract as in Section 11.5. Absence 

of fluorescence growth curve during PCR analysis of these samples (reported as “0" on 

SmartCycler and “undetermined” on ABI model 7900) indicates the absence of contaminant 

target DNA (see Data Quality Acceptance, below). Prepare one method blank filter for every 

six samples.

9.4 Positive controls — The laboratory should analyze positive controls to ensure that the method is 

performing properly. Fluorescence growth curve (PCR amplification trace) with an appropriate 

cycle threshold (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. 

9.4.1 Calibrator samples will serve as the positive control. Analyze as described in Section 

11.0. Note: Calibrator samples contain the same amount of extraction buffer and starting 

amount of Salmon DNA as the test samples; hence E. faecalis calibrator DNA extracts 

(Section 11.3) will be used as a positive control for both Enterococcus and SPC qPCR 

assays. 

9.4.2 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 

to exhibit the appropriate fluorescence growth curve response, prepare new calibrator 

samples and reanalyze (see Section 9.7). 

9.5 No template controls — The laboratory should analyze “No Template Controls” (NTC) to 

ensure that the Master Mix PCR reagents are not contaminated. On an ongoing basis, the 

laboratory should perform NTC analyses every day that samples are analyzed. If greater than 

one-third of the NTC reactions for a PCR master mix exhibit true positive logarithmic 

amplification traces with CT values below 45 (not from chemical degradation of probe with linear 

kinetics that exhibit rising baseline) or if any one NTC reaction has a CT value lower than 35, the 

analyses should be repeated with new Master Mix working stock preparations. 
Method A 

10

9.6 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 lsrRNA gene copies per genome in this species (see the rRNA operon 

database at http://rrndb.cme.msu.edu/rrndb). The concentration of lsrRNA gene copies per μL 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 lsrRNA Total DNA concentration (fg/μL) 4 lsrRNA gene copies 

gene copies per μL = 

3.6 fg/genome 

x 

 genome 

A composite standard curve should be generated from multiple analyses of serial dilutions of this 

DNA standard using the Enterococcus primer and probe assay and subjected to linear regression 

analysis as described in Section 12.2. From that point on, it is recommended that additional 

standard curves be generated from duplicate 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 these new reagents. The r2

 values from regressions of these curves should ideally 

be 0.99 or greater. Comparable performance is assessed by their slopes and y-intercepts which 

should be consistent with those from the initial composite analyses (e.g., within the 95% 

confidence range of the average values). Note: The 95% confidence ranges for these parameters 

in the initial composite standard curve can be generated using the Regression Analysis Tool 

which can be accessed from the “Data Analysis” selection under the “Tools” menu in Excel. 

Subsequent regressions can be performed by plotting the data using the Chart Wizard in Excel 

and using the “add trend line” selection in the Chart menu and “display equation on chart” 

selection under Options to obtain slope and y-intercept values as illustrated in Section 12.2. 

In the event that the slope value from a subsequent standard curve regression is outside of the 

acceptance range, the diluted standards should be re-analyzed. If this difference persists, new 

working stocks of the reagents should be prepared and the same procedure repeated. If the 

differences still persist, the amplification factor values used for calculations of target cell 

numbers, as described in Section 8, should be modified based on the new slope values. If the 

slopes are within acceptance range but Y-intercepts are not within acceptance range of this 

previous average, new serial dilutions of the DNA standard should be prepared and analyzed as 

described above. 

9.7 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. A minimum of nine calibrator sample extracts should initially be prepared from three 

different freezer-stored aliquots of these stocks as described in Section 11.2. Dilutions of each 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 with the Enterococcus primer and probe 

assay. The average and standard deviation of the CT values from these composite analyses should 

be determined. From that point on, a minimum of three fresh calibrator sample extracts should be 

prepared from an additional frozen aliquot of the same stock cell suspension at least weekly and 

preferably daily before analyses of each batch of test samples. Dilutions of each new calibrator 

sample extract equivalent to the initial composite dilutions (e.g., 1, 5 and/or 25 fold) should be 

analyzed using the Enterococcus primer and probe assay. The average CT value from these 

analyses should not be significantly different from the laboratory's average values from analyses 

of the initial calibrator sample extracts from the same stock cell suspension (i.e., not greater than 
Method A 

11

three standard deviations). If these results are not within this acceptance range, new calibrator 

extracts should be prepared from another frozen aliquot of the same stock cell suspension 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 9.6. 

9.8 Salmon DNA Sample Processing Control (SPC) sequence analyses — While not essential, it 

is good practice to routinely prepare and analyze standard curves from serial dilutions of Salmon 

DNA working stocks in a manner similar to that described for the Enterococcus DNA standards 

in Section 9.6. At this time rRNA gene operon copy numbers per genome have not been reported 

in the literature for the salmon species O. keta. Therefore log-transformed total DNA 

concentration values or dilution factor values can be substituted for target sequence copy numbers 

as the x-axis values in these plots and regression analyses. 

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’s 5 fold dilution should 

be within 3 CT units of the mean of the 5 fold diluted calibrator (and/or method blank) sample 

results. Higher CT values may indicate significant PCR inhibition or poor DNA recovery 

possibly due to physical, chemical, or enzymatic degradation. Repeat the Enterococcus and 

Salmon DNA PCR assays of any samples whose 5 fold dilution exhibits a Salmon DNA PCR 

assay CT value greater than 3 CT units higher than the mean of the calibrator sample results using 

a 5 fold higher dilution (net dilution: 25 fold) of the extracts. The Enterococcus PCR result from 

assaying the original 5-fold dilution of the sample can be accepted if its Salmon DNA assay CT

value is lower than that of the corresponding 25 fold dilution of the sample. This pattern of 

results is indicative of poor recovery of total DNA in the extract rather than PCR inhibition. The 

poor DNA recovery is compensated for by the calculation method. Contrarily, if the Salmon 

PCR assay CT value of the 25-fold dilution of the sample is lower than that of the 5 fold dilution 

of the sample, then the Enterococcus PCR assay result from the 25 fold dilution of the sample is 

considered more accurate. However the Enterococcus PCR results should be reported as 

questionable if the Salmon DNA assay’s result is still not within 3 CT units of the mean CT result 

of the 25 fold dilution of the three calibrators. 

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 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: E. faecalis cell suspensions (Section 11.1), and E. faecalis DNA standards (Section 11.2) 

may be prepared in advance. Calibrator samples (Section 11.3) should be prepared at least 

weekly. 
Method A 

12

11.1 Preparation of E. faecalis cell suspensions for DNA standards and calibrator samples 

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 

phosphate buffered saline (PBS) and vortex. 

11.1.3 Use 10 μL 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 

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 15 of EPA Method 

1600 

11.1.4 After incubation, centrifuge the BHIB containing E. faecalis for 5 minutes at 6000 × g. 

11.1.5 Aspirate the supernatant and resuspend the cell pellet in 10 mL PBS. 

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 with one of the following steps. 

11.1.7 Determination of calibrator sample cell concentrations based on one of the three options 

below. 

• Option 1: Spectrophotometric absorbance – Remove three 0.1-mL aliquots of 

undiluted cell suspension and dilute each with 0.9 mL of PBS (10-1 dilution). Read 

absorbance at 595 nm in spectrophotometer against PBS blank (readings should 

range from 0.05 to 0.3 OD). Calculate cells/mL (Y) in undiluted cell suspension 

using the formula from the standard curve shown below where X is the average 595 

nm spectrophotometer reading. 

Y = (1 x 109

 cells / mL × X) / 0.19 

• Option 2: Hemocytometer counts – Serially dilute 10 μL of undiluted cell suspension 

with PBS to 10-1, 10-2, and 10-3 dilutions and determine cell concentration of 10-2 or 

10-3 dilutions in a hemocytometer or Petroff Hauser counting chamber under 

microscope. 

• Option 3: plating – 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. Note: BHIA plates should be made in advance 

if this option is chosen. 

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 1.0 mL of the 

undiluted cell suspension to 99 mL of sterile PBS, cap, and mix by vigorously 

shaking the bottle a minimum of 25 times. 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 11.0 mL of cell suspension dilution “A” to 99 mL 

of sterile PBS, cap, and mix by vigorously shaking the bottle 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 11.0 mL of cell suspension dilution “B” to 99 

mL of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 
Method A 

13

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 11.0 mL of cell suspension dilution “C” to 99 mL 

of sterile PBS, cap, and mix by vigorously shaking the bottle 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 11.0 mL of cell suspension dilution “D” to 99 mL 

of sterile PBS, cap, and mix by vigorously shaking the bottle 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. 

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. 

 

CFU1 + CFU2 + .....+ CFUn

CFU/mLundiluted = V1 + V2 + ... + Vn 

Where: 

 CFU/mLundiluted = E. faecalis CFU/mL in undiluted cell suspension 

 CFU = number of colony forming units from BHIA plates yielding 

counts within the ideal range of 30 to 300 CFU per plate 

 V = volume of undiluted sample in each BHIA plate yielding 

counts within the ideal range of 30 to 300 CFU per plate 

 n = number of plates with counts within the ideal range 
Method A 

14

Table 1. Example Calculations of E. faecalis Undiluted Cell Suspension Concentration

CFU / plate (triplicate analyses) from 

Examples BHIA plates

10-5 mL plates 10-6 mL plates 10-7 mL plates 

E. faecalis CFU / mL in undiluted 

 suspensiona

Example 1 275, 250, 301 30, 10, 5 0, 0, 0

(275+250+30) / (10-5+10-5+10-6) = 

555 / (2.1 x 10-5) = 26,428,571 = 

2.6 x 107 CFU / mL

Example 2 TNTCb

, TNTC, 

TNTC TNTC, 299, TNTC 12, 109, 32

(299+109+32) / (10-6+10-7+10-7) = 

440 / (1.2 x 10-6) =366,666,667 = 

3.7 x 108

 CFU / mL

a Cell concentration is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per plate 

b

 Too numerous to count 

11.1.8 Divide remainder of undiluted cell suspension (approximately 5 mL) into 6 x 0.5 mL 

aliquots for DNA standard preparations and 100-200 x 0.01 mL (10 FL) aliquots for 

calibrator samples and freeze at !20°C. Note: Cell suspension should be stirred while 

aliquoting. It is also recommended that separate micropipettor tips be used for each 10 

FL aliquot transfer and that the volumes in each tip be checked visually for consistency. 

11.2 Preparation of E. faecalis genomic DNA standards

11.2.1 Remove two 0.5 mL undiluted E. faecalis cell suspensions (Section 11.1.8) from freezer 

and thaw completely. 

11.2.2 Transfer cell suspensions to extraction tubes with glass. 

11.2.3 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 × g for one minute 

to pellet the glass beads and debris. 

11.2.6 Using a 200 μL micropipettor, transfer 350 μL 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 × g for 5 minutes and transfer 300 μL 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 μL of 5 μg/μL 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. Continue onward 

from 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 108

 cells is required for this step. 
Method A 

15

11.2.10 If using the DNA-EZ purification kit, perform the following steps. Insert one 

DNAsureTM column from the DNA-EZ purification kit into a collection tube (also 

provided with kit) for eac

h of the two extracts. Transfer each of the extract and 

binding buffer mixtures from section 11.2.9 to a DNAsureTM column and collection 

tube assembly and centrifuge for 1 min at 12,000 × g. 

11.2.11 Transfer each of the DNAsureTM columns to new collection tubes. Discard previous 

collection tubes and collected liquid. 

11.2.12 Add 500 μL EZ-Wash Buffer from the DNA-EZ purification kit to each of the 

DNAsureTM columns and centrifuge at 12,000 × 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 DNAsureTM columns to a clean, labeled 1.7-mL low retention 

microcentrifuge tube and add 50 μL of DNA elution buffer to each column. 

Centrifuge for 30 seconds at 12,000 × g. Repeat this procedure again to obtain a total 

DNA eluate volume of ~100 μL from each column. 

11.2.15 Pool the two eluates to make a total volume of approximately 200 μL. 

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 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 can 

not be accurately read by the spectrophotometer. If available, readings can be taken of 

2 μL aliquots of the sample with a NanoDropTM Spectrophotometer. 

11.2.17 Sample is acceptable as a standard if ratio of OD260/OD280 readings is > or = to 1.75. 

11.2.18 Calculate total DNA concentration in sample by formula: 

 OD260 reading x 50 ng/μL DNA/OD260 

11.2.19 Transfer sample back to labeled 1.7-mL non-retentive microcentrifuge tube and store 

at -20°C. 

11.3 Extraction of E. faecalis calibrator samples

11.3.1 A minimum of three calibrator extracts should be prepared during each week of analysis. 

Note: To prevent contamination of water sample filtrates and filter blanks, this procedure 

should be performed at a different time, and, if possible, in a different work station than 

the procedures in Sections 11.1 and 11.2 above and Section 11.5 below. 

11.3.2 Remove one tube containing a 10 μL aliquot of E. faecalis undiluted stock cell 

suspension (Section 11.1.8) from the freezer and allow to thaw completely on ice. 

Note: If using BioBalls for calibrators add a single BioBallTM to each of 3, 100-mL sterile 

PBS blanks, filter (Section 11.4), and extract according to (Section 11.5). 

11.3.3 While cell stock is thawing, using sterile (or flame-sterilized) forceps, place one 

polycarbonate filter (Section 6.14) in an extraction tube with glass beads. Prepare one 

filter for each sample to be extracted in this manner. 
Method A 

16

11.3.4 Dispense 590 μL of Salmon DNA/extraction buffer (Section 7.12) into three extraction 

tubes with glass beads and filters. Prepare one tube for each of the three calibrator 

samples to be extracted in this manner. Label tubes appropriately. 

11.3.5 When E. faecalis suspension has thawed, transfer 990 μL AE buffer (Section 7.11) to the 

10 μL E. faecalis stock cell tube and mix thoroughly by vortexing. Pulse microcentrifuge 

tube briefly (1-2 sec.) to coalesce droplets in tube. 

11.3.6 Immediately after vortexing the E. faecalis suspension, spot 10 μL onto the 

polycarbonate filter in a calibrator sample tube. 

11.3.7 Tightly close the tube, making sure that the O-ring is seated properly. 

11.3.8 Repeat Sections 11.3.6 and 11.3.7 for the other two filters to prepare three calibrator 

samples with E. faecalis. 

11.3.9 Place the tubes in the mini bead beater and shake for 60 seconds at the maximum rate 

(5000 rpm). 

11.3.10 Remove the tubes from the mini bead beater and centrifuge at 12,000 × g for one 

minute to pellet the glass beads and debris. 

11.3.11 Using a 200 μL micropipettor, transfer the crude supernatant to the corresponding 

labeled sterile 1.7-mL microcentrifuge tube. Transfer 400 μL of supernatant without 

disrupting the debris pellet or glass beads at the tube bottom. Note: The filter will 

normally remain intact during the bead beating and centrifugation process. Generally, 

400 μL of supernatant can be easily collected. Collect an absolute minimum of 100 

FL of supernatant. 

11.3.12 Centrifuge at 12,000 × g for 5 minutes and transfer 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. 

11.3.13 Label the tubes as undiluted or 1x E. faecalis calibrator extracts. Label additional 1.7-

mL tubes for 5 and 25 fold dilutions. In appropriately labeled tubes, using a 

micropipettor, add a 50 FL aliquot of each 1x E. faecalis calibrator extracts and dilute 

each with 200 μL AE buffer (Section 7.11) to make 5 fold dilutions. In appropriately 

labeled tubes using a micropipettor, add a 50 FL aliquot of each 5 fold dilution and 

dilute each with 200 μL AE buffer to make 25 fold dilutions. Store all diluted and 

undiluted extracts in refrigerator. 

11.3.14 If the extracts are not analyzed immediately, refrigerate. For long term storage, freeze 

at -20°C or -80°C. 

11.4 Water sample filtration

Note: It is required that one water sample filtration blank (method blank) be prepared for every 6 

water samples (Section 9.3) analyzed by the same procedure. 

11.4.1 Place a polycarbonate filter (Section 6.14) on the filter base, and attach the funnel to 

the base so that the membrane filter is now held between the funnel and the base. 

11.4.2 Shake the sample bottle vigorously 25 times to distribute the bacteria uniformly, and 

measure 100 mL of sample into the funnel. 

11.4.3 Filter 100 mL of water sample. After filtering the sample, rinse the sides of the funnel 

with 20-30 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. 
Method A 

17

11.4.4 Label an extraction tube with glass beads (Section 7.20) 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. 

11.4.5 Cap the extraction tube. Tubes may be frozen at -20°C or -80°C until analysis. 

11.5 DNA extraction of water sample filter extracts and method blanks

11.5.1 Using a 1000 μL micropipettor, dispense 590 μL of the Salmon DNA/extraction buffer 

(Section 7.11) to each labeled extraction tube with glass beads containing water sample 

or method blank filters from Section 11.3.4. Extract the method blank last. 

11.5.2 Tightly close the tubes, making sure that the O-ring is seated properly. 

11.5.3 Place the tubes in the mini bead beater and shake for 60 seconds at the maximum rate 

(5000 rpm). 

11.5.4 Remove the tubes from the mini bead beater and centrifuge at 12,000 × g for 1 minute to 

pellet the glass beads and debris. Note: To further prevent contamination, a new pair of 

gloves may be donned for steps 11.4.5, 11.4.6, and 11.4.7 below. 

11.5.5 Using the 200 μL micropipettor, transfer 400 μL of the supernatant to corresponding 

labeled sterile 1.7-mL microcentrifuge tube, taking care not to pick up glass beads or 

sample debris (pellet). Note: The filter will normally remain intact during the bead 

beating and centrifugation process. Generally, 400 μL of supernatant can easily be 

collected. Collect an absolute minimum of 100 FL of supernatant. Recover the method 

blank supernatant last. 

11.5.6 Centrifuge crude supernatant from Section 11.4.5 for 5 minutes at 12,000 × g. Transfer 

350 μL of the clarified supernatant to another 1.7-mL tube, taking care not to disturb 

pellet. Note: pellet may not be visible in water samples. Recover the method blank 

supernatant last. 

11.5.7 Label the tubes from Section 11.4.6 as undiluted or 1x 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 and 25 fold dilutions. In appropriately 

labeled tubes, using a micropipettor, add a 50 FL aliquot of each 1x water sample extract 

and dilute each with 200 μL AE buffer (Section 7.11) to make 5 fold dilutions. In 

appropriately labeled tubes using a micropipettor, add a 50 FL aliquot of each 5 fold 

dilution and dilute each with 200 μL of AE buffer to make 25 fold dilutions. Dilute the 

method blank supernatant last 

11.5.8 Store all diluted and undiluted extracts in refrigerator. Note: Use of 5 fold diluted 

samples for analysis is currently recommended if only one dilution can be analyzed. 

Analyses of undiluted water sample extracts have been observed to cause a significantly 

higher incidence of PCR inhibition while analyses of 25 fold dilutions may unnecessarily 

sacrifice sensitivity. 

11.5.9 If the extracts are not analyzed immediately, refrigerate. For long term storage, freeze at 

-20°C or -80°C. 

11.6 Preparation of qPCR assay mix

11.6.1 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 
Method A 

18

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. 

11.6.2 Remove primers and probe stocks from the freezer and verify that they have been diluted 

to solutions of 500 μM primer and 100 μM probe. 

11.6.3 Prepare working stocks of Enterococcus, and Salmon DNA (Sketa 22) primer/probe 

mixes by adding 10 μL of each Enterococcus or Salmon DNA (Sketa 22) primer stock 

and 4 μL of respective probe stock to 676 μL 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. 

11.6.4 Using a micropipettor, prepare assay mix of the Enterococcus, and Salmon DNA (Sketa 

22) reactions in separate, sterile, labeled 1.7-mL microcentrifuge tubes as described in 

Table 2. 

Table 2. PCR Assay Mix Composition

Reagent Volume/Sample (multiply by # samples to be analyzed per day)

Sterile H2O 1.5 μL

BSA 2.5 μL

TaqMan® master mix 12.5 μL

Primer/probe working stock solution 3.5 μL

Note: This will give a final concentration of 1 μM of each primer and 80 nM of probe in the 

reactions. Prepare sufficient quantity of assay mix for the number of samples to be analyzed per 

day including calibrators and negative controls plus at least two extra samples. Prepare assay 

mixes each day before handling DNA samples. 

11.6.5 Vortex the assay mix working stocks; then pulse microcentrifuge to coalesce droplets. 

Return the primer/probe working stocks and other reagents to the refrigerator. 

11.7 ABI 7900 & ABI 7500 (non-Fast) qPCR assay preparation (Reference 17.1)

 Transfer 20 μL of mastermix containing Enterococcus primers and probe to wells of a 96-well 

PCR reaction tray equal to number of samples to be analyze including calibrator and negative 

control samples. (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, 51 wells will require the addition of 

assay mix with Enterococcus primers and probe as follows: 18 samples, two replicates each (36), 

3 method blanks, two replicates each (6), 3 no template controls, one replicate each (3), and 3 

calibrators, 2 replicates each (6) = 51 wells. 

11.7.1 Transfer 20 μL of mastermix containing Enterococcus primers and probe 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). 
Method A 

19

11.7.2 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. 

11.7.3 Transfer 5 μL each of the diluted (or undiluted) DNA extracts of method blanks and 

water samples (Section 11.5.7), and then corresponding dilutions of calibrator samples 

(Section 11.3.13), to separate wells of the PCR reaction tray containing Enterococcus 

assay mix. Note: Record positions of each sample. 

11.7.4 Transfer 5 μL each of the diluted (or undiluted) DNA extracts of method blanks and 

water samples (Section 11.5.7), and corresponding diluted calibrator samples (Section 

11.3.13) to separate wells of the PCR reaction tray containing Salmon DNA assay mix. 

Record positions of each sample. 

11.7.5 Transfer 5 μL aliquots of AE buffer to wells of PCR reaction tray containing 

Enterococcus master mix that are designated as no-template controls. Record positions 

of these samples. 

11.7.6 Tightly cap wells of PCR reaction tray containing samples or cover tray and seal tightly 

with optical adhesive PCR reaction tray. 

11.7.7 Run reactions in ABI 7900 & ABI 7500 (non-Fast) sequence detector. For platformspecific operation see Appendix A.

11.8 Smart Cycler® qPCR assay preparation 

11.8.1 Label 25 μL Smart Cycler® tubes with sample identifiers and assay mix type (see 

Section 11.7.8 for examples) or order tubes in rack by sample number and label rack with 

assay mix type. It is recommended that the unloaded open SmartCycler® tubes be 

irradiated under ultraviolet light in a PCR cabinet for 15 minutes. Using a micropipettor, 

add 20 μL of the Enterococcus assay mix (section 11.6.5) 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. 

11.8.2 Add 5 μL of AE buffer to no-template control tubes and close tubes tightly. 

11.8.3 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. 

11.8.4 Transfer 5 μL each of the diluted (or undiluted) DNA extracts of method blanks and 

water samples (Section 11.5.7), 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. 

11.8.5 When all Smart Cycler® tubes have been loaded, place them in a Smart Cycler® 

centrifuge, and spin for 2-4 seconds. 

11.8.6 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. 
Method A 

20

11.8.7 For platform-specific operation see Appendix B. 

11.8.8 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 (or 2 replicates of 3 samples) can be analyzed per run, as described in Tables 3

and 4, below. 

Table 3. Calibrator PCR Run - 14 Samples

Sample Description* Quantity PCR Assay Master Mix

3 Calibrators × 2 replicates (1, 5, or 25 fold dilution ) 6 Enterococcus

3 Calibrators × 2 replicates (1, 5, or 25 fold dilution ) 6 Salmon DNA

No template controls (reagent blanks) 2 Enterococcus

* Diluted equivalently to the water samples 

Table 4. Water Sample PCR Run - 14 Samples

Sample Description* Quantity PCR Assay Master Mix

Water samples, (1, 5, or 25 fold dilution ) 6 Enterococcus

Method blank, (1, 5, or 25 fold dilution ) 1 Enterococcus

Water samples, (1, 5, or 25 fold dilution ) 6 Salmon DNA

Method blank, (1, 5, or 25 fold dilution ) 1 Salmon DNA

* Use of five-fold diluted samples for analysis is currently recommended if only one dilution can be analyzed. 

Analyses of undiluted water sample extracts have been observed to cause a significantly higher incidence of 

PCR inhibition while 25 fold dilutions analyses may unnecessarily sacrifice sensitivity. 

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 filtrate) sample compared to those recovered from 

identically extracted calibrator samples using an arithmetic formula, referred to as the ΔΔCT 

comparative cycle threshold calculation method. The ΔΔCT relative quantitation 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 absolute measurements of total target sequence quantities recovered from the test 

samples by multiplying them by the average total number of target sequences that are normally 

recovered from a constant number of target organisms that are added to all calibrator samples. 

The complete procedure for determining target sequence quantities in water samples is detailed 

below. 

12.2 Generation of CT value vs. target sequence number standard curve: Three replicate serial 

dilutions of a DNA standard, prepared as described in section 9.6 should be prepared to give 

concentrations of 4 x 104

, 4 x 103, 4 x 102, 2 x 102 and 1 x 102 lsrRNA gene sequences per 5 μL 

(the standard sample volume added to the PCR reactions) and the replicates of each dilution 

pooled. Note: a procedure for the determinations of target sequence concentrations in the DNA 

standard is also provided in Section 9.6. 
Method A 

21

 Aliquots of each of these dilutions should be stored at 4oC in low retention microcentrifuge tubes 

and can be reused for repeated qPCR analyses. QPCR analyses of these diluted standards using 

the Enterococcus primer and probe assay should be performed at least three separate times in 

duplicate. CT values from these composite analyses should be subjected to regression analysis 

against the log10-transformed target sequence numbers per reaction as described in Section 9.6 

with example results illustrated in Figure 1. 

y = -3.4777x + 38.422

R2

 = 0.9951

20

22

24

26

28

30

32

34

12345

Log target sequences per reaction

Cycle Threshold (CT)

Figure 1: Example plot and regression analysis of qPCR analysis cycle threshold values vs. log target 

sequences per reaction. 

 Amplification factors (AF) used for subsequent comparative cycle threshold calculations (Section 

12.4) can be calculated from the slope value of this curve by the formula AF = 10^(1 / (-)slope 

value). An example calculation using the slope value from the example regression is shown 

below: 

 AF = 10^(1 / 3.4777) = 1.94 

12.3 Calculation of average target sequence recovery in calibrator sample extracts: A minimum 

of nine calibrator sample extracts should initially be prepared from at least three different freezerstored aliquots of each cultured E. faecalis stock suspension that is prepared as described in 

Section 11.1. Dilutions of each 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 with 

the Enterococcus primer and probe assay. The average CT value from these analyses should be 

interpolated on the standard curve generated from the DNA standard (Section 12.2) to determine 

the average number of target sequences per 5 μL of extract used in the reactions. An example 

calculation using an average calibrator extract CT value of 25.21 is shown below: 

 Average calibrator target sequences/5 μL extract = 10^((25.21-38.44) / -3.477) = 6382 

 Note: Four places should be kept from this calculation for the following calculation (i.e., 

6382.6983). Dividing this value by 5 gives the average calibrator target sequences/ μL extract 

which can be multiplied by the total volume of the extract at the applicable dilution level (e.g.,

600 μL of original extract volume x 5 = 3000 μL for a 5 fold diluted sample) 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: 
Method A 

22

Average target sequences = 6382 target sequences x 3000 μL total extract volume 

Calibrator extract 5 μL extract 

 = 3,829,619 

12.3.1 Calculation of average target sequence recovery per Enterococcus cell in calibrator 

sample extracts (optional): In previous studies, measurements of recreational water 

quality by the qPCR method have been reported as Enterococcus calibrator cell 

equivalents (References 17.4). Calculations performed to obtain this reporting unit are 

identical to those described in Section 12.4 except that the ratios of target sequences 

obtained as described in Sections 12.4.1 - 12.4.4 are multiplied by the estimated 

quantities of Enterococcus cells added to the calibrator samples rather than by the 

average target sequences recovered per calibrator extract as described in Section 12.4.5. 

While the use of this reporting unit is no longer recommended because of the false 

impression it creates concerning the cell concentration detection limit of the qPCR 

method, it still may be of value for comparing previous results with those of future 

studies. 

A prerequisite for making such comparisons is to determine that the ratio of the numbers 

of target sequences recovered in calibrator sample extracts to the numbers of 

Enterococcus cells added to these samples is consistent in different studies. For the 

purpose of determining this ratio, it is recommended that the cell concentrations of the 

cultured E. faecalis stock suspension used for the preparation of calibrator samples in 

each laboratory be determined by at least two of the three alternative methods described 

in Section 11.1 to establish the degree of agreement between these enumeration methods. 

The recommended quantity of cells that are added to each calibrator sample is 100,000. 

Dividing the average target sequences recovered per calibrator extract (determined as 

described in Section 12.3) by this number provides the ratio of target sequence numbers 

to cell numbers. An example of this calculation using the average target sequences/ 

calibrator extract value determined in Section 12.3 is shown below: 

 Ratio of target sequence numbers to cell numbers = 3,829,619 / 100,000 = 38.29 

12.4 Calculation of target sequence quantities in test samples: A minimum of three fresh calibrator 

samples should be extracted and analyzed at least on a weekly basis and preferably on a daily 

basis in association with each batch of water sample filtrates. QC analysis of the analysis results 

from these calibrator extracts should be performed as described in Section 9.7. CT values from 

the Enterococcus target sequence and salmon DNA Sample Processing Control (SPC) qPCR 

assays for both the calibrator and test samples are used in the ΔΔCT comparative cycle threshold 

calculation method to determine the ratios of target sequences in the test and calibrator sample 

extracts and these ratios are converted to absolute measurements of total target sequence 

quantities recovered from the test samples as specified below and illustrated in Table 5. 

12.4.1 Subtract the SPC assay CT value (CT,SPC) from the target assay CT value (CT,target) for each 

calibrator sample extract to obtain ΔCT value and calculate the average ΔCT value for 

these calibrator samples. 

12.4.2 Subtract the SPC assay CT value (CT,SPC) from the target assay CT value (CT,target) for each 

water sample filtrate extract to obtain ΔCT values for each of these test samples. Note: if 

multiple analyses are performed on these samples, calculate the average ΔCT value. 

12.4.3 Subtract the average ΔCT value for the calibrator samples from the ΔCT value (or average 

ΔCT value) for each of the test samples to obtain ΔΔCT values. 
Method A 

23

12.4.4 Calculate the ratio of the target sequences in the test and calibrator samples using the 

formula: AF^(-ΔΔCT), where AF = amplification factor of the target organism PCR assay, 

determined as described in Section 12.2. 

12.4.5 Multiply the ratio of the target sequences in the test and calibrator samples by the average 

target sequences/calibrator extract, determined as described in Section 12.2, to determine 

absolute numbers of total target sequences/ extract 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 5. Example Calculations (Amplification factor = 1.94)

Target 

sequences 

in Sample

Sample 

Type CT,target CT,SPC ΔCT ΔΔCT

Measured Target Sequences in 

Test Sample Extract 

(1.94 -ΔΔCT× avg. target 

sequences/calibrator)

3,829,619 Calibrator 25.21 30.45 -5.24 ---- ----

Unknown Test 32.53 30.65 1.88 7.12 0.0089 x 3,829,619 = 34,198

12.4.6 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: 

12.4.6.1 Use ΔCT value for each individual water sample extract and the mean 

calibrator ΔCT value to calculate the measured target sequence numbers in 

each water sample extract, as described in Section 12.4. 

12.4.6.2 Calculate the log10 of the measured target sequence numbers in each water 

sample (log N) 

12.4.6.3 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. 

12.4.6.4 Calculate the geometric mean as 10M. 

12.4.6.5 The implied coefficient of variation (CV) is calculated, based on the log 

normal distribution, as the square root of 10V/ 0.434 - 1, where V = S2

. 

12.5 Reporting Results: Where possible, duplicate analyses should be performed on each sample. 

Report the results as Enterococcus (large subunit ribosomal RNA gene) target sequences per 

volume of water sample filtered. 

13.0 Sample Spiking Procedure

[This section will be updated after validation study] 

14.0 Method Performance

14.1 Accuracy (Bias) 

The lsrRNA gene (23S rRNA) of Enterococci, which contains the target sequence amplified and 

detected by the primers and probe of the Enterococcus qPCR assay, is present in multiple copies 

in the genome of the different Enterococcus species. Enterococcus faecalis and E. faecium each 

contain 4 and 6 copies of the gene, respectively. The number of lsrRNA gene copies per genome 
Method A 

24

has not been ascertained for all of the Enterococcus species which the Enterococcus qPCR assay 

can amplify and detect. Hence, the use of E. faecalis cells as a calibrator for relative quantitation 

purposes and E. faecalis DNA as a standard for absolute quantitation purposes creates an inherent 

bias potentially affecting the accuracy of the quantitation depending on the species composition 

of the Enterococcus present in a water sample. 

The Enterococcus qPCR method makes the assumption that the Enterococcus cells present in the 

water sample contain the same number of genomes and lsrRNA gene copies as the E. faecalis

calibrator cells which have been grown in culture media to a late-log or stationary phase in batch 

culture. This assumption has not been validated if untrue may bias the accuracy of the results in a 

systematic manner. Bacterial cells contain more than one complete genome during growth and 

cell division phases of their life cycle. The number of genomic copies depends on their growth 

rate and cell division time. More than one cell division cycle is often required to complete 

replication of the genome during rapid log-phase growth and cell division. The lsrRNA genes are 

replicated early in the cell cycle maximizing the number of lsrRNA gene copies present in cells 

during log phase growth. This facilitates the enhanced ribosome production needed for the high 

level of protein translation needed during rapid cell growth and division. 

[This section will be updated after validation study] 

15.0 Pollution Prevention

15.1 The solutions and reagents used in this method pose little threat to the environment when 

recycled and managed properly. 

15.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize 

the volume of expired materials to be disposed. 

16.0 Waste Management

16.1 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. 

16.2 Samples, reference materials, and equipment known or suspected to have viable enterococci 

attached or contained must be sterilized prior to disposal. 

16.3 For further information on waste management, consult “The Waste Management Manual for 

Laboratory Personnel” and “Less Is Better: Laboratory Chemical Management for Waste 

Reduction,” both available from the American Chemical Society’s Department of Government 

Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036. 
Method A 

25

17.0 References

17.1 Anonymous. 1997. User Bulletin #2. ABI Prism 7700 Sequence Detection System. Foster City, 

CA, Applied Biosystems. 

17.2 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 

17.3 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. 

17.4 Haugland, R.A., S.C. Siefring, L.J. Wymer, K.P. Brenner, and A. P. Dufour. 2005. Comparison 

of Enterococcus Density Measurements by Quantitative Polymerase Chain Reaction and 

Membrane Filter Analysis at Two Freshwater Recreational Beaches. Water Research 39:559-

568. 

17.5 Ludwig, W. and K.-H. Schleifer. 2000. How quantitative is quantitative PCR with respect to cell 

counts? System Appl. Microbiol. 23: 556-562. 

17.6 USEPA. 1983. Health Effects Criteria for Marine Recreational Waters. EPA-600/1-80-031. 

17.7 USEPA. 1984. Health Effects Criteria for Fresh Recreational Waters. EPA-600/1-84-004. 

17.8 USEPA . 1986. Ambient water quality criteria for bacteria - 1986. EPA 440/5-84-002. 

17.9 Wade, T.J., Calderon, R.L., Sams, E., Beach, M., Brenner, K.P. & Dufour, A.P. 2006. Rapidly 

measured indicators of recreational water quality are predictive of swimming associated 

gastrointestinal illness. Environmental Health Perspectives 114: 24-28. 
Method A 

26

18.0 Acronyms 

ΔΔCT comparative cycle threshold calculation method 

AF amplification factor 

BHIA brain heart infusion agar 

BHIB brain heart infusion broth 

BSA bovine serum albumin 

CFU colony forming units 

DNA deoxyribonucleic acid 

EDTA ethylenediaminetetraacetic acid 

lsrRNA large subunit ribosomal RNA 

NTC no template control 

PBS phosphate buffered saline 

PCR polymerase chain reaction 

qPCR quantitative polymerase chain reaction 

SPC sample processing control 

TNTC too numerous to count 

UV ultraviolet (light) 

 
Method A

27

Appendix A: 

ABI 7900 and ABI 7500 Sequence Detector Operation
Method A

A-1

Appendix A - ABI 7900 and ABI 7500 (non-Fast) Sequence Detector 

Operation

1.0 ABI 7900 Sequence Detector Operation

1.0.1 Turn on the ABI Model 7900 sequencer 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 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.

1.0.2 Under File menu, select New.

1.0.3 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.

1.0.4 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.

1.0.5 Above right hand window, click on Setup tab.

1.0.6 Click on Add Detector button at the bottom of the setup screen. Note: Before any 

analyses are performed, a specific detector for the method must be created. To do this go 

to step 1.6.1. Once the detector has been created, go to step 1.7.

1.0.7 Click on New in the pop-up window that appears. Another pop-up window will appears. 

Under Name, type in a name for the detector that will be used by this method (e.g., 

“Method 1600"). Under Group select Default. Under Reporter select FAM. 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 1.7)

1.0.8 In pop-up window that was opened in step 1.6, select the desired detector under Names 

menu (e.g., Method 1600) and click on Copy to Plate Document button. Click on Done

button to return to setup screen.

1.0.9 Click on Use box next to FAM detector in right hand window. This box should become 

marked with an X. Name and color code for FAM detector 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.

1.0.10 Click on Instrument tab right hand window.

1.0.11 In instrument screen, change sample volume to 25 μL and choose 9600 emulation.

1.0.12 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.

1.0.13 Insert PCR reaction tray with prepared reactions in holder.

1.0.14 Click on Open/Close button to close PCR reaction tray holder door on instrument.

1.0.15 Click on Start button in lower right hand “Real Time” window to start thermal cycling in 

instrument.
Method A

A-2

1.0.16 Name run file at prompt.

1.0.17 At termination of the run, instrument-calculated cycle threshold values should 

automatically appear for each well position and detector entry in the lower left hand 

results table window.

1.0.18 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 1.18). Click on OK. Click on 

Analyze from the toolbar. You should see Ct values in the Results Table.

1.0.19 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.

1.0.20 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.

1.0.21 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. 

1.1 ABI 7500 (non-Fast) Sequence Detector Operation 

1.1.1 Turn on the ABI Model 7500 sequencer 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 ABI 7500” for screen shots. 

1.1.2 Click on either the New Experiment or Advanced Setup button to create a new 

experiment, which will pull up the Experiment Menu. 

1.1.3 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. 

1.1.3.1 Click on 7500 (96 Wells) to select the instrument type. Note: This protocol is 

not designed for the 7500 Fast. 

1.1.3.2 Scroll down to access more experiment properties options. Click on 

Quantitation – Standard Curve to select the experiment type 

1.1.3.3 Click on TaqMan® Reagents to select the reagents used. 

1.1.4 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). 

1.1.4.1 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. Under the Quencher heading, click on the drop down menu to select 

TAMRA as the quencher. 
Method A

A-3

1.1.4.2 Repeat the process in Section 1.1.4.1 to add more than one target, e.g., Sketa. 

1.1.4.3 In the Define Samples section, click on Add New Sample and enter the 

sample name in the Sample 1 box. 

1.1.4.4 Click on the Assign Targets and Sample tab to assign the Target, sample ID 

and sample type to the wells. 

1.1.4.5 Drag the mouse over the desired cells to assign the Target, Task 

and Sample. 

1.1.4.6 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. 

1.1.4.7 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.). 

1.1.4.8 Repeat the process in Section 1.1.4.4, 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. 

1.1.4.9 Click on Print Report at the top of the screen to print a plate layout for 

loading master mix and sample extracts. 

1.1.4.9.1 Select Plate Layout option by clicking on the box next to it. 

1.1.4.9.2 Click Print Report to print the plate layout. 

1.1.5 From the Setup menu, select Run Method to set up the thermo cycling profile. 

1.1.5.1 In the Graphical View tab, change the Reaction Volume Per Well from the 

default 50μL to 25μL by typing in 25. 

1.1.5.2 Under Cycling Stage section, change the Number of Cycles to 45. 

1.1.5.3 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 2:00 minutes, Holding Stage 2: 95.0°C for 10:00 

minutes, and Cycling Stage: 95.0°C for 0:15 seconds. 

1.1.5.4 The second step of the Cycling Stage is defaulted at 60.0°C for 1 minute – 

change the time to 2:00 minutes. 

1.1.5.5 Click on Save at the top of the screen to save the run before actually running 

the experiment. 

1.1.5.6 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. 

1.1.6 From the Experiment menu, select Run to monitor the run in real time. 

1.1.6.1 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. 
Method A

A-4

1.1.7 When the run has completed, from the Setup menu, select Analysis to export your 

results. See “How to Export Results from the ABI 7500 Software” for screen shots. 

1.1.7.1 The Analysis Settings for Experiment name screen will open. 

1.1.7.2 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). 

1.1.7.3 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. 

1.1.7.4 Click on Apply Analysis Settings to save the changes. 

1.1.8 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 curve graphic. 

1.1.8.1 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). 

1.1.8.2 For Select one file or separate files, select One file to export all the data into 

one file with multiple tabs. 

1.1.8.3 For Export File Name, enter the name of the Experimental data file to be 

exported, and for File Type, choose *.xls. 

1.1.8.4 For Export File Location, click on Browse to select the folder in which you 

wish to export your file. 

1.1.8.5 Click on Start Export. When this is completed, an Export Completed screen 

will open – click on Close Export Tool to complete this task. 

1.1.9 How to interpret your results. See “How to Interpret Results from the ABI 7500” for 

screen shots. 

1.1.9.1 After exporting your data, from the Experiment menu, select Analysis.

1.1.9.1.1 From the Analysis menu, select Amplification Plot to view the 

results of the selected samples at different plot settings.. 

1.1.9.2 Drag the mouse over the desired cells to select the samples to be 

plotted in the amplification plot. 

1.1.9.3 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). 

1.1.9.4 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. 

1.1.9.5 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. 
Method A

A-5

1.1.9.6 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. 

1.1.9.7 The parameter display below the plot (i.e. Target, Slope, YInter(cept), R2

, and Eff% (percent efficiency) is useful for data 

analysis. 

1.1.9.8 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 45 thermo cycles based on the 

wells, target, or dye. 

1.1.9.9 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). 

1.1.9.10 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 45 thermo cycles. 

1.1.9.11 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. 

1.1.9.12 For quality controls, click on QC Summary from the Analysis menu to view 

the analysis summary of the samples. 

1.1.9.13 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 as in Sections 1.1.12-1.1.15. 

1.1.10 To print the report, click on Print Report at the top of the screen. 

1.1.10.1 Check all of the boxes (i.e., Experimental Summary, Results Summary, Plate 

Layout, Amplification Plot (3 boxes), Standard Curves, Results Table, and 

QC Summary). 

1.1.10.2 Click on Print Report at the bottom of the screen. 
Click either button to 

create new experiment

How to Set Up a New Experiment Using the ABI 7
2. Enter the 

Experiment Name

1. Click “Experiment Prope

select the experiment type 

an experiment name
Click the type of instrument 

used - in this case it is the 7500 (96 wells) plate

Scroll down for more experiment properties options
Click to select 

experiment type
Click on TaqMan® Reagents to 

select the reagents used
2. Click “Define Targets and Samples” tab to de

the reporter-quencher dye for each target and aenter sample IDs.

1. Click on “Plate Setup”
1. Click to Add 

New Target

2. Enter the Target Name

3. Select the Reporter dye FAM from the drop down menu

4. Select the QuenchTAMRA from the drodown menu
You can add more than one target- usually 

per target DNA (ENT, SKETA)
Select to Add a New Samp

Enter the nam

sampl
Click to designate the target, sample 

ID and sample type to the wells
Drag the mouse over t

desired cells to assign Target, Task and Sam
While the cells are highligh

check the desired Target andesignate whether the samU (Unknown), S (Standard) (Negative Control)

Assign the sample thais to be analyzed in thhighlighted cell(s)
To designate standards, 

highlight the desired cells, add the target and select the “S” for Standard
Type in a Quantity for each 

standard - if there are 4 different values, highlight each cell independently and fill in the quantity
2. Click on “Print Report” to print a 

layout for loading master mix and sextracts
1. Select “Plate Layout” option by 

clicking in the box next to it 

2. Click “Print Repto print the plate layout.
Select “Run Method” to set 

up the thermo cycling profile
1. Change the Reaction Volume Pe

Well from the default of 50uL to 25u

2. Change the Number of Cycles to 45

4. In the data cchange the timthe default 1 mminutes

3. Confirm that the default Temperature and Time settings in both Holding Stages and in step 1 of the Cycling Stage are as displayed – if not, reset them
1. Click to save the run 

before starting the run

2. Select the folder for saving the Run Data file

3. Enter the experiment name if not entered beginning of the experiment setup and clicor if already have the appropriate experimewithout making any changes click “Save”
Click “Run” to monitor the run in real-time

Click to start the run. The green box will turn red when the run has been started successfully
Click on the “Analysis” tab in the Experim

to access the “Analysis Settings”

How to Export Results from the ABI 7500 Softwa
1. To change the threshold setting from 

Automatic to Manual, click on “Analysis Settings”

2. Under the “C

T Settings” tab, 

click on the Target desired to be set from Default to Manual Threshold setting

3. Uncheck the box nex“Use Default Settings”
Uncheck the box next to 

“Automatic Threshold” to enter the desired threshold
Enter the desired Threshold.

*If more than one target is present, highlight the next target and repeat this process
Select “Apply Analysis Settings” to save 

the changes
Click on “Export” to save experiment results 

data and to save the selected plots required in the report

Note that the threshold value has changed on the Amplification 
Check the USE boxes nex

Check the boxes to the desired resexport
From the drop-down m

“One File” to export all single file with multiple 
Enter the name of the 

experiment data file to exported
Select *.xls (Excel) from the “File Type”

drop-down menu
Click on “Browse” to select the 

folder / location to export the File
Click on “Start Export”

to export the data to the determined location
A pop-up window “Export Completed” appears

once the exporting of the file is completed.

Click on “Close Export Tool” to close the export window once data file exporting is completed.
Select “Amplification Plot” to view the res

the selected samples at different plot setti

How to Interpret Results From the ABI 7500
2. Select the 

“Plot Type”from the dropdown menu

3. From the “Graph Type”drop down menu select eiLog or Linear

1. Drag the mouse desired cells to sesamples to be ploamplification plot

Analysis Menu- Amplification Plot
If the plot requires 

the display of the threshold and baseline select by checking the “USE”boxes in “Show”next to Threshold and Baseline

Select “View Well Table” to view the results in a tabular format

Scroll down or sidewaysfor more samples and analysis parameters

Analysis Menu- Amplification Plot
Select “Standard Curve” to view the results of 

the samples (if same target) and standards in the plot

Analysis Menu- Standard Curve
Standard curve 

parameters may be useful in data analysisClick here to select a different Target (if the standards were analyzed in more than one target) or change the Plot Color

Analysis Menu – Standard Curve
Select “Multicomponent Plot” to view the variation in the 

fluorescence of the wells, targets or dyes used over theascending repeat of the 45 thermo cyclesClick on the “Plot Color” drop down menu to select the parameter based on which the graph required to be plotted

Analysis Menu – Multicomponent Plot
Select “Raw Data Plot” to view the variation in the emission of 

fluorescence of the dyes in different filters present over the ascending repeat of the 45 thermo cycles

Drag the pointecycle scale to tcycle number variation in the through filters samples at tha

Analysis Menu – Raw Data Plot
Select “QC Summary” to view the 

analysis summary of the samples in the experiment

Analysis Menu – QC Summary
Select “Multiple Plot View” to view all the plots for the 

samples in one window

Each plot has menu located access variourequired to dialso has the twith various oprinting, saviparameters

Analysis Menu – Multiple Plots View
Click on “Print Report” to make a ha

copy of the results with required plo

Analysis – To Print a Data Report
1. Select the required data an

plots to be printed by checkithe boxes next to the data anplot types in which results caprinted

2. Click “Print Repoprint the required d
Method A

Appendix B: 

Cepheid Smart Cycler® Operation
Method A

B-1

Appendix B - 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 A. Further details concerning the operation of the instrument can be 

obtained from the Smart Cycler® Operation Manual, Cepheid Part #D0190, 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 TaqMan Method" 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 Secs column, enter 120, leaving the Optics setting 

as the default Off setting. (Note: This stage is performed to eliminate potential PCR 

carryover products in the reactions using the Amp-Erase® UNG enzyme provided in the 

TaqMan® Universal PCR Master Mix. Note that the optical reading cannot be 

programmed during a Hold stage.).

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 Secs column enter 600. Again, 

leave the Optics setting on Off. (Note: This stage is performed to inactivate the AmpErase® UNG enzyme). 

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 Secs column, Optics

column Off. For the second step, enter 60.0 in the second row of the Temp column, 120 

in the Secs column, and click on the Optics cell to select On from the drop-down menu. 

This sets the detection of the fluorescence signal to occur at the end of the second step in 

each cycle. Enter 45 in the Repeat field at the top of the Stage 3 box to specify that it 

should be repeated for 45 cycles. Click the Save Protocol button.

1.4.5 To display primary curve graphs, click Define Graphs. Highlight FAM 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). 

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 μL reaction).
Method A

B-2

1.7 Click the Add/Remove Sites button. The Select Protocols and Sites... dialog will appear. 

Highlight (click on) the “EPA Method A" 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 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 all other dyes to Unused in the same manner. (Note: All assays in this protocol 

use FAM 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 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 

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. 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. 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, 
Method A

B-3

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).

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. Analysts will late save the file as an XLS file.

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 TaqMan Method 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. 
1. Click on “New Protocol”

2. This screen will appear - type in the name of the 

How to Set Up a New Experiment Using the Smart Cycler (So

3. Click on OK w
1. New Protocol name will appear in list

Make sure new protocol is highlighted and begin to choose stage settings

2. Set the Temperature in the “HOLD” stage to 50.0 degrees a120 seconds. NOTE optical readings cannot be programmeduring a HOLD stage
In Stage 2, set the temperature to 95.0 

and 600 seconds
1. In Stage 3, set the drop down 

menu at “2-Temperature Cycle”to repeat 45 times (cycles). 

2. For the first temp cycle set the temp at 95 degrees for 15 seconds with optics OFF.

3. In the seconcycle, set the tedegrees for 120 with the optics 

4. Clicwhen 
To set up automatic export of R

into computer, click on “SETUP“System Defaults” then “Export 

How To Set Up Automatic Export of Raw Data
1. Check Results Table and Analysis settings box

3. Be sure that Automatic export on run completion is check

2. Choose select a fothe raw dato

4. Click OK 
How To Manually Export Raw Data

Once run is finished, click E
Check the Export Results Table and Analysis Settings 

then click “Export”
Choose the appropriate file folder to save the Raw Data to 

and click SAVE. NOTE: All raw data will export in the form of a CSV file. Analysts will later save file as an “XLS” file.
Right click on graph area and select “Print” t

“Print Graph with Results Table”

How to Print Run Data
This sheet 

will pop up. Click “Print” to print file
To print the generated report, click on “Report”
Click “Print”

to print out the generated run data report
Click “Create Run”

How to Set Up a New Run
Click “Add/Remove” Sites
1. Be sure the c

protocol is higbefore selectin

2. Highlight the sites needed foA16)
Click “right” ar

move sites to t“Selections” co
Sites have been moved to 

selections column. Double check that the correct protocol is selected and click on “OK”
The Sample IDs and run name can be en

point
Under the “Usage” column, select “Unused

drop down menu for Cy3, TxR and Cy5. Le“Assay” on the FAM line for this particular 
1. Set the Manual Threshold 

Fluorescence Units to 8.0 on the FAM line

2. When smart cycler tubes have been appropriate sites, click “Start Run” - a should appear on sites that are in use