U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2011–5150 

Prepared in cooperation with Ohio Water Development Authority and City of Delphos, Ohio 

Quantifying Viruses and Bacteria in Wastewater—Results, 

Interpretation Methods, and Quality Control 
Cover: Report coauthor Brian Mailot collecting a post-preliminary wastewater sample at one of the study sites. 

(Photograph by Donna Francy, U.S. Geological Survey.) 
Quantifying Viruses and Bacteria in 

Wastewater—Results, Interpretation 

Methods, and Quality Control 

By Donna S. Francy, Erin A. Stelzer, Rebecca N. Bushon, Amie M.G. Brady, 

Brian E. Mailot, Susan K. Spencer, Mark A. Borchardt, Ashley G. Elber, 

Kimberly R. Riddell, and Terry M. Gellner 

Prepared in cooperation with Ohio Water Development Authority and City of Delphos, Ohio 

Scientific Investigations Report 2011–5150

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2011 

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reproduce any copyrighted materials contained within this report.

Suggested citation:

Francy, D.S., Stelzer, E.A., Bushon, R.N., Brady, A.M.G., Mailot, B.E., Spencer, S.K., Borchardt, M.A., Elber, A.G., 

Riddell, K.R., and Gellner, T.M., 2011, Quantifying viruses and bacteria in wastewater—Results, interpretation 

methods, and quality control: U.S. Geological Survey Scientific Investigations Report 2011–5150, 44 p. 
iii

Acknowledgments

We gratefully acknowledge the wastewater-treatment operators and managers for granting 

access to their plants, providing advice on sampling locations, and providing in-kind services 

during sampling. In addition to the cooperating agencies, Ohio Environmental Protection Agency 

(OEPA) and the U.S. Environmental Protection Agency (USEPA) provided in-kind services, and 

Kubota and Ovivo provided financial support for this study. We thank Stefan Wuertz (University 

of California, Davis) for help with quantifying viruses and Fu-Chih Hsu (Scientific Methods, 

Granger, Ind.) for providing reagents and advice on the EasyPhage method. We also thank 

the peer-review team for their help and advice throughout the study: Paul Novak and Mike 

McCullough (OEPA), Mark Meckes (USEPA), Jim Porteous and Douglas Bucher (Ovivo), and 

Minoru Fijitani (Kubota Membrane USA).

Finally, we thank two U.S. Geological Survey colleagues: Hon Ip (USGS, National Wildlife Health 

Center, Madison, Wis.), for advice on interpreting virus data and manuscript review; and Sandra 

Embrey (USGS Washington Water Science Center), for manuscript review. 
iv

Contents

Acknowledgments........................................................................................................................................iii

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................2

Purpose and Scope ..............................................................................................................................3

Enteric Viruses and Microbiological Indicators of Fecal Contamination....................................3

Methods of Study...........................................................................................................................................4

Sampling Frequency and Procedures...............................................................................................6

Microbiological Analyses....................................................................................................................7

Initial Processing of Samples ....................................................................................................7

Enteric Viruses by Cell Culture ..................................................................................................7

DNA Extraction and Enteric Viruses by qPCR or qRT-PCR ...................................................8

Standard Curves for Enteric Viruses by qPCR or qRT-PCR...................................................8

Assay Limits of Quantification and Sample Reporting Limits for Enteric Viruses 

by qPCR, qRT-PCR, and Cell Culture............................................................................8

Indicator Bacteria and Coliphage...........................................................................................11

Quality Assurance and Quality Control ...........................................................................................11

Indicator Bacteria and Coliphage...........................................................................................12

Enteric Viruses ...........................................................................................................................12

Indicator Bacteria, Coliphage, and Viruses in Wastewater.................................................................12

Enumeration Considerations for Enteric Viruses by qPCR and qRT-PCR..................................12

Quality-Control Considerations for Interpreting Bacterial-Indicator, Coliphage, 

and Enteric-Virus Data .........................................................................................................17

Quality-Control Samples for Bacterial Indicators and Coliphage .....................................17

Quality-Control Samples for Enteric Viruses.........................................................................19

Concentrations of Bacterial Indicators and Coliphage in Wastewater.....................................21

Concentrations of Enteric Viruses in Wastewater........................................................................27

Summary........................................................................................................................................................29

References Cited..........................................................................................................................................30

Glossary.........................................................................................................................................................43
v

Figures

1. Schematic showing processes involved in wastewater treatment for 

conventional and Membrane bioreactors (MBR) facilities, and sampling 

points for the 2008–10 study........................................................................................................5

2. Photograph showing equipment setup for filtering wastewater samples 

for enteric viruses.........................................................................................................................7

3. Schematic showing determination of effective final concentrated sample 

volumes (FCSVs) for quantitative polymerase chain reaction (qPCR) for 

adenovirus or quantitative reverse transcriptase PCR (qRT-PCR) for 

enterovirus or norovirus GI.......................................................................................................10

4. Graphs showing quality-control replicate samples for concentrations in 

wastewater. A, bacterial indicators. B, coliphage................................................................18

5. Graphs showing concentrations found by use of the single agar layer 

and EasyPhage methods in wastewater samples, 2008. A, F-specific. 

B, somatic coliphage..................................................................................................................20

6. Graph showing quality-control replicate samples for concentrations of 

viruses in wastewater, 2008–10................................................................................................21

7A. Graphs showing concentrations in wastewater samples, 2008–10: 

Escherichia coli...........................................................................................................................22

7B. Graphs showing concentrations in wastewater samples, 2008–10: 

enterococci..................................................................................................................................23

7C. Graphs showing concentrations in wastewater samples, 2008–10: 

fecal coliforms.............................................................................................................................24

7D. Graphs showing concentrations in wastewater samples, 2008–10: 

somatic coliphage ......................................................................................................................25

7E. Graphs showing concentrations in wastewater samples, 2008–10: 

F-specific coliphage...................................................................................................................26
vi

Tables

1. Membrane bioreactor (MBR) and conventional wastewater-treatment plants, 

2008–10 ...........................................................................................................................................4

2. Primers and probes for enteric viruses ....................................................................................9

3. Water-quality, bacterial indicator, and coliphage results from wastewater 

samples, 2008–10 ........................................................................................................................34

4. Virus concentrations in wastewater samples, 2008–10.......................................................38

5. Quality-control equipment and field-blank data for bacterial indicators, 

coliphage, and viruses in wastewater, 2008–10 ....................................................................13

6. Quality-control concurrent replicate data for bacterial indicators and 

coliphage in wastewater, 2008–10...........................................................................................14

7. Quality-control method test samples for coliphage in wastewater, 2008–10...................15

8. Quality-control concurrent replicate data for enteric viruses in wastewater, 

2008–10 .........................................................................................................................................16

9. Performance characteristics of quantitative polymerase chain reaction 

(qPCR) and reverse transcriptase qPCR (qRT-PCR) standard curves for 

each assay...................................................................................................................................17

10. Summary statistics for viruses by quantitative polymerase chain reaction 

(qPCR) and reverse transcriptase qPCR (qRT-PCR) and cell culture in 

wastewater samples, 2008–10..................................................................................................28
vii

Conversion Factors and Abbreviated Water Quality Units

Multiply By To obtain

Length

millimeter (mm) 0.03937 inch (in)

micrometer (μm) 1,000 millimeter (mm)

Volume

liter (L) 0.2642 gallon (gal)

milliliter (mL) 0.06102 cubic inch (in3

)

microliter (μL) 1,000 milliliter (mL)

Flow rate

million gallons per day (Mgal/d) 0.04381 cubic meter per second (m3/s) 

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: 

°F=(1.8×°C)+32

Concentrations of bacterial indicators are given in colony-forming units per 100 milliliters 

(CFU/100 mL).

Concentrations of viruses are given in genomic copies per liter (gc/L).

Concentrations of coliphage are given in plaque-forming units per 100 milliliters (PFU/100 mL).

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius 

(μS/cm at 25°C). 

Quantifying Viruses and Bacteria in Wastewater—

Results, Interpretation Methods, and Quality Control

By Donna S. Francy,1 Erin A. Stelzer,1 Rebecca N. Bushon,1 Amie M.G. Brady,1 Brian E. Mailot,1 Susan K. 

Spencer,2

 Mark A. Borchardt,3 Ashley G. Elber,4 Kimberly R. Riddell,5 and Terry M. Gellner 6 

Abstract

Membrane bioreactors (MBR), used for wastewater treatment in Ohio and elsewhere in the United States, have pore 

sizes small enough to theoretically reduce concentrations of 

protozoa and bacteria, but not viruses. Sampling for viruses 

in wastewater is seldom done and not required. Instead, 

the bacterial indicators Escherichia coli (E. coli) and fecal 

coliforms are the required microbial measures of effluents for 

wastewater-discharge permits. Information is needed on the 

effectiveness of MBRs in removing human enteric viruses 

from wastewaters, particularly as compared to conventional 

wastewater treatment before and after disinfection. 

A total of 73 regular and 28 quality-control (QC) samples 

were collected at three MBR and two conventional wastewater plants in Ohio during 23 regular and 3 QC sampling 

trips in 2008–10. Samples were collected at various stages in 

the treatment processes and analyzed for bacterial indicators 

E. coli, fecal coliforms, and enterococci by membrane filtration; somatic and F-specific coliphage by the single agar layer 

(SAL) method; adenovirus, enterovirus, norovirus GI and GII, 

rotavirus, and hepatitis A virus by molecular methods; and 

viruses by cell culture. While addressing the main objective of 

the study—comparing removal of viruses and bacterial indicators in MBR and conventional plants—it was realized that 

work was needed to identify data analysis and quantification 

methods for interpreting enteric virus and QC data. Therefore, 

methods for quantifying viruses, qualifying results, and applying QC data to interpretations are described in this report. 

During each regular sampling trip, samples were collected (1) before conventional or MBR treatment (post-preliminary), (2) after secondary or MBR treatment (post-secondary 

or post-MBR), (3) after tertiary treatment (one conventional 

plant only), and (4) after disinfection (post-disinfection). 

Glass-wool fiber filtration was used to concentrate enteric 

viruses from large volumes, and small volume grab samples 

were collected for direct-plating analyses for bacterial indicators and coliphage. After filtration, the viruses were eluted 

from the filter and further concentrated. The final concentrated 

sample volume (FCSV) was used for enteric virus analysis 

by use of two methods—cell culture and a molecular method, 

polymerase chain reaction (PCR). Quantitative PCR (qPCR) 

for DNA viruses and quantitative reverse-transcriptase PCR 

(qRT-PCR) for RNA viruses were used in this study. 

To support data interpretations, the assay limit of detection (ALOD) was set for each virus assay and used to determine 

sample reporting limits (SRLs). For qPCR and qRT-PCR the 

ALOD was an estimated value because it was not established 

according to established method detection limit procedures. 

The SRLs were different for each sample because effective 

sample volumes (the volume of the original sample that was 

actually used in each analysis) were different for each sample. 

Effective sample volumes were much less than the original 

sample volumes because of reductions from processing steps 

and (or) from when dilutions were made to minimize the 

effects from PCR-inhibiting substances. Codes were used to 

further qualify the virus data and indicate the level of uncertainty associated with each measurement. 

Quality-control samples were used to support data interpretations. Field and laboratory blanks for bacteria, coliphage, 

and enteric viruses were all below detection, indicating that it 

was unlikely that samples were contaminated from equipment 

or processing procedures. The absolute value log differences 

(AVLDs) between concurrent replicate pairs were calculated 

to identify the variability associated with each measurement. 

For bacterial indicators and coliphage, the AVLD results 

indicated that concentrations <10 colony-forming units or 

plaque-forming units per 100 mL can differ between replicates by as much as 1 log, whereas higher concentrations can 

differ by as much as 0.3 log. The AVLD results for viruses 

1

U.S. Geological Survey, Ohio Water Science Center, Columbus, Ohio.

2

U.S. Department of Agriculture/U.S. Geological Survey Laboratory, 

Marshfield, Wisconsin.

3

U.S. Department of Agriculture, Agricultural Research Service, 

Marshfield, Wis.

4

CT Consultants, Mentor, Ohio.

5

(Formerly) City of Delphos, Ohio.

6

CT Consultants, Akron, Ohio.
2 Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control

indicated that differences between replicates can be as great as 

1.2 log genomic copies per liter, regardless of the concentration of virus. Relatively large differences in molecular results 

for viruses between replicate pairs were likely due to lack of 

precision for samples with small effective volumes. 

Concentrations of E. coli, fecal coliforms, enterococci, 

and somatic and F-specific coliphage in post-secondary and 

post-tertiary samples in conventional plants were higher than 

those in post-MBR samples. In post-MBR and post-secondary 

samples, concentrations of somatic coliphage were higher than 

F-specific coliphage. In post-disinfection samples from two 

MBR plants (the third MBR plant had operational issues) and 

the ultraviolet conventional plant, concentrations for all bacterial indicators and coliphage were near or below detection; 

from the chlorine conventional plant, concentrations in postdisinfection samples were in the single or double digits. All of 

the plants met the National Pollutant Discharge Elimination 

System required effluent limits established for fecal coliforms. 

Norovirus GII and hepatitis A virus were not detected 

in any samples, and rotavirus was detected in one sample but 

could not be quantified. Adenovirus was found in 100 percent, 

enterovirus in over one-half, and norovirus GI in about onehalf of post-preliminary wastewater samples. Adenovirus and 

enterovirus were detected throughout the treatment processes, 

and norovirus GI was detected less often than the other two 

enteric viruses. Culturable viruses were detected in post-preliminary samples and in only two post-treatment samples from 

the plant with operational issues. 

Introduction 

Bacterial indicators, such as Escherichia coli (E. coli) 

and fecal coliforms, are the required microbial measures of 

effluents for wastewater-discharge permits in Ohio and elsewhere in the United States. Most bacterial indicators do not 

cause disease but are used to indicate the possible presence of 

pathogens transmitted via the fecal-oral route. Sampling for 

viruses and other pathogens in wastewaters is seldom done 

and is not required. Bacteria, viruses, and protozoa are three 

types of microorganisms that can be found in wastewater. 

Waterborne bacterial pathogens in the United States include 

species of Salmonella, Shigella, Vibrio, Campylobacter, and 

pathogen strains of E. coli. Waterborne protozoan pathogens 

include Cryptosporidium and Giardia. Human enteric viruses, 

which cause a wide range of diseases and symptoms and are 

excreted in high numbers in the feces of infected individuals, 

include enteroviruses, adenoviruses, noroviruses, rotaviruses, 

and hepatitis A virus. Many wastewater-treatment processes 

can remove microorganisms, but no process removes all 

microorganisms from wastewater. 

Membrane bioreactors (MBRs) are a relatively new 

wastewater-treatment technology in which conventional 

secondary treatment or secondary with tertiary treatment are 

replaced by a membrane separation process. The use of this 

technology is increasing in the United States; for example, in 

Ohio, there are 15 operational municipal MBR wastewatertreatment plants and 5 operational industrial MBR facilities. 

This process is accomplished by submerging membranes in 

the conventional activated sludge tank to physically separate 

the solids from the liquid and filter the wastewater through the 

membrane. The microfiltration membranes used in Ohio and 

elsewhere have an effective pore size of 0.10 to 0.4 mm, small 

enough to reduce concentrations of protozoa (4 to 15 mm) and 

bacteria (0.5 to 3 mm) but too large to theoretically provide 

reliable reductions of viruses (0.02 to 0.08 mm). There is little 

information, however, on the effectiveness of MBRs in removing human enteric viruses from wastewaters, particularly as 

compared to conventional secondary or secondary with tertiary wastewater treatment before and after disinfection. There 

is also little information comparing virus removal to bacteria 

and virus indicator (coliphage) removal in these plants. 

Methods for quantifying bacterial indicators and coliphage in water and wastewater and interpreting results are 

well established; however, corresponding methods are not 

well established for enteric viruses. In order to quantify enteric 

virus removal in wastewaters, compare virus removal to 

bacteria and virus indicator removal, and determine the need 

for disinfection in MBR plants as compared to conventional 

plants, virus quantification and data interpretation methods 

are needed. Methods for quantifying viruses in water and 

wastewater include cell culture and molecular methods. Cell 

culture methods have been used for years, and data interpretation methods are standardized; however, molecular methods, 

particularly quantitative molecular methods, are relatively new 

and require data interpretation procedures that are not well 

documented. Few studies describe how to interpret enteric 

virus concentration and quality-control data for solving problems, answering practical questions, and informing the public. 

To fill this gap and provide practical information on how 

to interpret enteric virus data, the U.S. Geological Survey 

(USGS) and CT Consultants, in cooperation with Ohio 

Water Development Authority, the City of Delphos, and the 

U.S. Department of Agriculture/USGS Marshfield (Wisconsin) Laboratory, studied the removal of viruses and indicators 

throughout the wastewater-treatment process in conventional 

and MBR plants. The wastewater plants were located in northern Ohio, where disinfection of effluents is required during 

the recreational season from May through October. During 

26 sampling trips over the recreational seasons of 2008–10, 

73 regular and 28 quality-control (QC) samples were collected at three MBR and two conventional Ohio wastewatertreatment plants at various stages in the treatment processes. 

Samples were analyzed for enteric viruses, bacterial indicators, and coliphage. 

The goal of the study was to determine whether disinfection is needed after MBR treatment to provide the same level 

of protection of public health as that found for conventional 

systems that use disinfection after secondary treatment. While 

working towards this goal, the study team realized that work 

was needed to identify data analysis methods for interpreting 

enteric virus and quality-control data. 
Introduction 3

Purpose and Scope

The purpose of this report is to document procedures for 

quantifying and qualifying data on concentrations of enteric 

viruses by use of molecular methods and for applying qualitycontrol sample results. These data-analysis procedures can be 

used by others who are struggling to interpret and use microbiological data to answer water-resource questions. Data from 

samples for enteric viruses, bacterial indicators, and coliphage 

are presented in this report. Quality-control data associated 

with bacterial indicator, coliphage, and enteric virus data also 

are discussed to aid in data interpretations. 

Current plans (August 2011) are to address the main 

study goal in companion reports that (1) present concentrations and log removals of bacterial indicators, coliphage, 

and enteric viruses through wastewater-treatment processes 

and (2) discuss treatment effectiveness in terms of National 

Pollutant Discharge Elimination System (NPDES) permit 

requirements.

Enteric Viruses and Microbiological Indicators 

of Fecal Contamination

Enteric viruses include waterborne human pathogenic 

viruses transmitted through the fecal-oral route. Five groups of 

enteric viruses were targeted during the study—enteroviruses, 

adenoviruses, noroviruses, rotaviruses, and hepatitis A virus. 

Human enteroviruses are among the most commonly detected 

enteric viruses in contaminated waters (Gregory and others, 

2006). More than 60 types of enteroviruses cause a variety of 

illnesses from mild respiratory infections to serious conditions 

such as myocarditis. In a study in Milwaukee, enteroviruses in 

clinical cases and in water samples were found to occur more 

frequently from July through October than other times of the 

year (Sedmak and others, 2005). Adenoviruses, the only DNA 

enteric viruses included in the present study (all other human 

enteric viruses in this study were RNA viruses), can survive 

for prolonged periods in environmental waters (He and Jiang, 

2005). Although many infections caused by adenoviruses 

are asymptomatic, adenoviruses have been recognized as 

important etiological agents of respiratory and gastrointestinal 

illnesses. Adenoviruses have been proposed as indicators of 

fecal viral contamination because of their consistent prevalence and stability in human sewage (Bofill-Mas and others, 

2006). Noroviruses are one of the most common etiological 

agents of gastrointestinal illness outbreaks and are highly contagious; as few as 10 viral particles may be sufficient to infect 

an individual (Centers for Disease Control and Prevention, 

2010). Although norovirus infections occur throughout the 

year, they are expected to be highest during the winter months 

(da Silva and others, 2007). Rotavirus is the leading cause of 

severe diarrhea in infants and children worldwide (Centers 

for Disease Control and Prevention, 2010). Hepatitis A virus 

causes inflammation of the liver; this disease is self-limiting, 

and rates in the United States are the lowest they have been in 

40 years (Centers for Disease Control and Prevention, 2011). 

Cell culture and a molecular method, polymerase chain 

reaction (PCR), were used in the present study to quantify 

viruses in wastewater. The cell culture method provides information on the infectivity of the viruses but requires weeks for 

confirmatory results and does not by itself identify specific 

viruses. For the cell culture method, a flask containing a layer 

of mammalian cells is inoculated with sample and examined 

microscopically for the appearance of cytopathic effects 

(CPE, cell disintegration or changes in cell morphology). 

Not all enteric viruses grow in cell culture or exhibit CPE, 

and no single cell-culture system can be used for all human 

enteric viruses. Polymerase chain reaction for DNA viruses 

or reverse-transcriptase PCR (RT-PCR) for RNA viruses has 

been used to detect specific viruses in different water matrices 

(Ando and others, 1995; da Silva and others, 2007; De Leon 

and others, 1990; He and Jiang, 2005; Kuo and others, 2010; 

Rajal and others, 2007). For both methods, specific polymerase enzymes are used to amplify, to detectable levels, 

viral nucleic acid sequences that are present in low concentrations in water. The PCR method provides results within a day 

and can identify specific viruses, but it does not determine 

the infectivity of the viruses (the viruses do not have to be 

intact or active to be detected). Although traditional PCR is 

a presence/absence method, quantitative PCR (qPCR) and 

quantitative RT-PCR (qRT-PCR) are newer techniques that can 

provide data on the level of specific viruses in a water sample 

within 3 hours. The qPCR and qRT-PCR rely on the detection 

of a fluorescent signal produced during the amplification reaction, and the pattern of signal generation can be used to infer 

the amount of starting material (DNA or RNA) in the sample. 

Many hurdles remain for the accurate quantification of 

viruses from water matrices by use of qPCR and qRT-PCR 

(Gregory and others, 2006). Because viruses are generally 

found in low numbers in treated wastewater, large volumes of 

sample must first be filtered and concentrated. Recovery by 

filtration is variable and should be evaluated for each water 

matrix and virus type (Lambertini and others, 2008). Another 

obstacle to accurate quantification is the presence of substances that inhibit the PCR, which may be purified along with 

the viruses. Inhibitory compounds include polysaccharides, 

divalent cations, and humic, fulvic, or tannic acids present 

in natural waters. In addition, quantification procedures rely 

on establishing a robust and reproducible standard curve for 

each assay and identifying sample-specific detection limits for 

quantification, none of which are established or standardized. 

Quantitative PCR and qRT-PCR assays have been used 

to determine concentrations of human enteric viruses in water 

and wastewater. Studies include those that describe concentrations of adenovirus (He and Jiang, 2005; Biofill-Mas and others, 2006; Haramoto and others, 2007; Kuo and others, 2010) 

and less frequently, concentrations of norovirus (da Silva and 

others, 2007) or enterovirus (Rutjes, 2005) in different types 

of water and wastewater samples. Rajal and others (2007) 
4 Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control

describe the quantification of enteroviruses and adenoviruses 

in storm-water samples. Uncommon in the literature, the 

equations that were used to determine final virus quantification 

numbers and sample-specific limits of detection were clearly 

defined in Rajal and others (2007). 

Methods for bacterial indicators and coliphage were used 

in the present study because they are used to satisfy permitting requirements or are the basis for water-quality standards 

for recreational or drinking waters. Unlike virus quantification methods, methods to detect fecal-indicator bacteria (fecal 

coliforms, E. coli, and enterococci) in water and wastewater 

are well established and yield highly reproducible results 

(American Public Health Association (APHA) and others, 

2005). However, concentrations of fecal-indicator bacteria 

do not necessarily correlate with the presence or concentration of enteric viruses in environmental samples. Researchers 

sometimes use coliphages as surrogates for the transport and 

survival of enteric viruses in the environment. Coliphages are 

viruses that infect and replicate in coliform bacteria, such as 

E. coli, and do not infect or harm humans. They are similar 

to enteric viruses in size and shape, transport characteristics, 

and resistance to disinfection processes. The two main groups 

of coliphages used in water-quality studies are somatic and 

F-specific coliphages, and they are both present in appreciable numbers in sewage (Sobsey and others, 1995). Somatic 

coliphages infect coliform bacteria by attachment to the outer 

cell membrane or cell wall; they are widely distributed in both 

fecal-contaminated and uncontaminated waters and may not 

be consistent indicators of fecal contamination (Sobsey and 

others, 1995). F-specific coliphages infect coliform bacteria 

only by attachment to the F-pilus, a structure made only by 

bacteria grown at higher temperatures. F-specific coliphage 

found in environmental samples, therefore, presumably come 

from warmblooded animals or sewage and are generally present at low levels in uncontaminated environmental samples 

(Long and Sobsey, 2004). A method to enumerate coliphage 

in water is established and documented (U.S. Environmental 

Protection Agency, 2001a). 

Methods of Study

The five small- to medium-sized wastewater-treatment 

plants included in this study were three Kubota® Membrane 

Systems by Ovivo MBR Process system plants with ultraviolet 

(UV) disinfection, one conventional treatment plant with UV 

disinfection and tertiary treatment, and one conventional plant 

with chlorine disinfection (table 1). Plants 1–3 were included 

in the original study design, and plants 4 and 5 were added 

when additional funding was secured; sampling at plant 2 was 

discontinued in 2010 because of operational issues and an 

effluent contamination that was identified and eliminated after 

sampling. The MBR plants selected for this study all used the 

same manufacturer and design, with a pore size of 0.4 mm. In 

this report, a conventional plant refers to an activated sludge 

biological process consisting of aeration tanks and secondary 

tanks or clarifiers (fig. 1). Air is introduced to the wastewater 

in the aeration tank whereby the organics are reduced to cell 

matter (solids) and stabilized before removing the solids by 

gravity in the secondary settling tanks. In some conventional 

plants a tertiary treatment process is used. Tertiary treatment 

removes more solids than secondary treatment does, and it 

typically targets the removal of suspended solids and nutrients 

such as phosphorus and nitrogen. A common tertiary method 

is filtration, whereby sand or activated carbon is used to filter 

the wastewater. In this study, plant 3 provides tertiary treatment through sand filtration. 

Table 1. Membrane bioreactor (MBR) and conventional wastewater-treatment plants, 2008–10. 

[Mgal/d, million gallons per day; QC, quality-control samples]

Plant 

number Treatment system Year built

Design 

capacity 

(Mgal/d)

Average 

daily flow 

(Mgal/d)

Sample numbers 

(in parentheses) 

and dates

1 MBR with ultraviolet 

disinfection

2006 12 3.8 2008–(1) Aug. 12, (5) Oct. 21

2009–(6) May 12, (8) June 16, 

(11) Aug. 4, (13) Oct. 14

2010–May 5 (QC only)

2 MBR with ultraviolet 

disinfection

2006 .5 .25 2008–(2) Sept. 9, (4) Oct. 7

2009–(7) May 26, (10) July 20

3 Conventional tertiary (sand 

filtration) with ultraviolet 

disinfection

1985; upgraded late 

1990s

1.6 .5 2008–(3) Sept. 22

2009–(9) June 29, (12) Sept. 14

2010–(18) Aug. 2

4 MBR with ultraviolet 

disinfection

2008 6 3 2010–April 13 (QC only), (15) June 23, 

(17) July 21, (19) Aug. 17, (21) Sept. 14, 

(23) Oct. 5

5 Conventional secondary with 

chlorine disinfection

1916; upgraded in 

1953, 1967, 1986, and 

2004

10 5 2010–April 13 (QC only), (14) June 1, 

(16) July 5, (20) Aug. 31, (22) Sept. 20
Methods of Study 5

CONVENTIONAL 

SECONDARY TREATMENT

*TERTIARY

TREATMENT

POSTPRELIMINARY

POSTPRELIMINARY

AERATION

TANK CLARIFIERSETTLER POSTSECONDARY

POSTTERTIARY

DISINFECTION POSTDISINFECTION

STREAM

RECYCLE SLUDGE

WASTE SLUDGE

*Plant No. 5 does not employ Tertiary treatment

MBR SECONDARY TREATMENT

IMMERSED MEMBRANE MODULE

ANOXIC

TANK

AERATION

TANK

PERMEATE 

PUMP

POSTMBR

DISINFECTION POSTDISINFECTION

STREAM

AIR

RECIRCULATION SLUDGE

Indicates sampling points

EXPLANATION

[Modified from www.newworldencyclopedia.com and wastewaterengineering.com]

Figure 1. Processes involved in wastewater treatment for conventional and Membrane bioreactors (MBR) facilities, and sampling 

points for the 2008–10 study. 
6 Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control

Sampling Frequency and Procedures

Twenty-three regular sampling trips and three qualitycontrol trips were made during the recreational seasons 

(May 1–October 31) of 2008–10 (table 1). During each regular 

sampling trip, one wastewater plant was visited, and 3 to 

4 samples were collected at different points within the treatment stream (fig. 1):

• Before conventional or MBR secondary treatment 

(post-preliminary) 

• After secondary or MBR treatment (post-secondary or 

post-MBR) 

• After tertiary, but before disinfection (post-tertiary)—

Plant 3 only

• After disinfection (post-disinfection)

Compensation was made for detention time between 

sample points within the treatment plant to ensure the approximate same slug of water was collected. Both filtered samples 

and fixed-interval grab samples were collected, as described 

below. 

Glass-wool fiber filtration (Lambertini and others, 2008) 

was done to concentrate enteric viruses from large-volume 

samples. Filters were prepared by order (USDA/USGS Laboratory, Marshfield, Wis.) and stored in the refrigerator for up to 

a month before use. Glass-wool filtration was also used in the 

early part of the study to compare concentrations of coliphage 

after filtration to those obtained by direct plating of a grab 

sample. Filtration was done in the field, with the exception of 

the post-preliminary wastewater samples, which were filtered 

at the USGS Ohio Water Microbiology Laboratory (OWML) 

in Columbus, Ohio. Sample volumes for enteric viruses were 

established on the basis of initial experiments in the OWML 

(unpublished data). The following sample volumes were filtered for enteric viruses and coliphage:

• Post-preliminary—4 L 

• Post-secondary—100 L or 3.5 hours filtration, whichever came first

• Post-MBR, post-tertiary, and post-disinfection—500 L 

or 3.5 hours filtration, whichever came first

For field filtration, the sampling apparatus consisted of an 

inlet tubing line, a Masterflex® peristaltic pump (Cole Parmer, 

Vernon Hills, Ill.), glass-wool filter, and a discharge tubing line 

(fig. 2). The peristaltic pump was adjusted to achieve a flow 

rate of 2–3 L/min. If needed, a second peristaltic pump was 

used to meter 0.5 N hydrochloric acid (HCl) through smalldiameter plastic tubing into the inlet line (1 N HCl was used 

the first year of the study) to adjust the sample pH to 6.5–7.0, 

in accordance with the glass-wool filter protocol (Lambertini 

and others, 2008). Before filtering the wastewater through the 

filter, the sample line was rinsed with wastewater and the pH 

was measured from the discharge tubing by using a pH probe 

in a glass beaker, in accordance with standard USGS protocols 

(Wilde, 2005). If the pH was greater than 7.0, the HCl metering pump was started. Over the next few minutes, the flow 

rate of HCl was adjusted to achieve the target pH. After the 

target pH was established, the glass-wool filter was connected 

to the line from the peristaltic pump. The pH was measured 

again and additional adjustments to the flow rate of HCl were 

made as needed. The pH and flow rate were measured subsequently at least every 30 minutes for all samples, even if the 

acid metering pump was not used. Flow rate was measured 

manually for 1 minute by collecting discharge wastewater 

into a graduated cylinder or marked carboy. Disposal of the 

discharged post-disinfection wastewater was via the effluent 

of the treatment system. All other wastewater was discharged 

into a drain or other point that flowed back through the treatment system. After the required volume was collected or the 

required time frame was achieved, the pumps were turned off, 

the filter was disconnected and placed in a plastic bag, and the 

inlet and outlet lines were sealed. Original sample volume was 

calculated from the flow rate and sampling duration. 

Grab samples were collected for analyses of bacterial 

indicators and coliphage and for lab filtration of post-preliminary wastewater samples. This was done by collecting slightly 

less than 1 L every 10 minutes for 1 hour (5–6 L of postpreliminary sample for filtration) or 0.5 L every 10 minutes for 

1 hour (3 L for bacterial indicators and coliphage). Subsamples were collected by use of 1-L sterile polypropylene bottles 

and composited into sterile 3-L bottles. Bacterial indicators 

and most coliphage analyses were done by direct plating of 

grab samples. 

Filters and grab samples were kept on ice before processing. Samples not processed onsite and filters were transported 

to the OWML by vehicle for processing or analyses within 

24 hours of collection. In the OWML, specific conductance 

and turbidity were measured by use of a specific conductance 

meter and a turbidimeter, respectively. For post-preliminary 

samples, wastewater was filtered in the laboratory under a 

biosafety cabinet by using the same procedure described above 

for field samples. One difference in the protocol was that the 

pH adjustment for post-preliminary wastewater samples was 

done before glass-wool filtration by slowly adding 1 N HCl 

to the sample while stirring until the pH was between 6.5 and 

7.0. 

Cleaning and decontamination for non-autoclavable 

tubing, connectors, bottles, and other equipment for sampling 

included a 15-minute soak in dilute detergent, a rinse with 

tapwater, sterilization in 0.1 percent sodium hypochlorite for 

30 minutes, dechlorination with 0.05 percent sterile sodium 

thiosulfate for 5 minutes, and a rinse with sterile deionized 

water. Autoclavable equipment was cleaned with detergent, 

rinsed with tapwater and deionized water, and autoclaved for 

15 minutes. 
Methods of Study 7

Aci Acid

Acid metering

pump

Main sampling

pump

Discharge line

Inlet

line

Glass-wool

filter

Figure 2. Equipment setup for filtering wastewater samples for enteric viruses.

Figure 2. Equipment 

setup for filtering 

wastewater samples for 

enteric viruses. 

Microbiological Analyses

Wastewater samples were analyzed for culturable enteric 

viruses; for enterovirus, norovirus GI and GII, rotavirus, and 

hepatitis A virus by qRT-PCR and for adenovirus by qPCR; 

for E. coli, fecal coliforms, and enterococci by membrane 

filtration; and for somatic and F-specific coliphage by the 

U.S. Environmental Protection Agency (USEPA) single agar 

layer method and by a commercially available alternative 

method, EasyPhage (Scientific Methods Inc., Granger, Ind.). 

Processing and analyses of samples for bacterial indicators 

were done in the field vehicle or at the OWML within 6 hours 

of sample collection. Analyses of samples for coliphage 

and processing of filters for enteric viruses were done at the 

OWML within 24 hours of sample collection. 

Initial Processing of Samples

After filtration, the viruses were eluted from the filter by 

use of a beef extract and glycine elution solution, in accordance with a procedure described in Lambertini and others 

(2008). A portion of the eluate was removed for coliphage 

analysis, and the remainder was concentrated for enteric 

virus analysis. Briefly, the eluate (approximately 200 mL) 

was adjusted to pH 7–7.5 with 1 M HCL and flocculated with 

16.0 g polyethylene glycol (PEG) and 2.3 g sodium chloride. 

The mixture was stirred for 1 hour at 4°C, held overnight 

at 4°C, and then centrifuged at 3,300 × g for 1.5 hours. The 

pellet was resuspended in 0.15 M sodium phosphate to bring 

the final concentrated sample volume (FCSV) up to 10 mL for 

post-preliminary samples or 5 mL (2008) or 6 mL (2009–10) 

for other wastewater-sample types. The FCSV was aliquoted 

into several centrifuge tubes: two to four tubes for storage 

in the freezer and one tube with 4–6 mL, depending on the 

sample type, for transport to the USDA/USGS Laboratory in 

Marshfield, Wis. (“Marshfield Laboratory”), for enteric virus 

analysis. The FCSVs were transported to the Marshfield Laboratory in batches on dry ice by overnight delivery. 

Enteric Viruses by Cell Culture

The cell culture method was done at the Marshfield Laboratory, based on procedures described in U.S. Environmental 

Protection Agency (2001b). The cell culture method is a twostep procedure that consists of (1) screening and (2) quantification by most probable number (MPN). To screen for different types of culturable enteric viruses, 0.33 mL of FCSV was 

inoculated into each of three culture flasks containing one of 

three cell lines: buffalo green monkey kidney cells (BGMK), 

rhabdomyosarcoma (RD) cells, and Human Caucasian colon 

adenocarcinoma (Caco-2) cells. The three cell lines were used 

because different human enteric viruses grow preferentially 

on different cell lines (Knipe and others, 2007). The cultures 

were observed for up to 14 days for cytopathic effects (CPE) 

to evaluate on which cell line the viruses were most prolific. A 

portion of the cell lysate was analyzed by qRT-PCR for enterovirus to confirm that an enterovirus was the cause of the CPE. 
8 Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control

If a positive result was obtained through screening, 

then culturable enteric viruses were quantified by MPN by 

using the selected cell line. Thirty wells were inoculated with 

0.04 mL of FCSV: 10 with undiluted FCSV, 10 with FCSV 

diluted 1:5 with sterile 0.15 M sodium phosphate, and 10 with 

FCSV diluted 1:25, for a total inoculum in the 30 wells of 

0.496 mL. Wells were incubated for 14 days and periodically 

examined for CPE. After 14 days of observation, the cells 

were lysed and the supernatant passed into new wells with 

cells to confirm the first passage results. If the initial dilutions 

failed to produce the appropriate mix of virus positive and 

virus negative wells for calculating the MPN, a new set of 

30 wells was inoculated with different FCSV dilutions and the 

incubation process was repeated for another 28 days. MPNs 

were calculated on the basis of CPE. To confirm that the CPE 

was caused by an enterovirus, the cell lysate from one positive 

well was analyzed for enteroviruses by qRT-PCR. 

DNA Extraction and Enteric Viruses by qPCR or 

qRT-PCR

At the Marshfield Laboratory, viral nucleic acids 

were extracted from the FCSV with a QIAamp® DNA Blood 

Mini Extraction Kit (Qiagen, Valencia, Calif., 51104) and AVL 

Buffer (Qiagen, 19073) to yield a suspension of 50 μL in 2008 

and 65 μL in 2009–10. AVL Buffer is a lysis buffer designed 

for viruses. The qPCR/qRT-PCR assays are described in 

Lambertini and others (2008), and the primers and probes for 

enterovirus, adenovirus, norovirus, rotavirus, and hepatitis A 

virus are listed in table 2. Assays for adenovirus groups C, D, 

and F were performed on all samples (Cromeans and others, 

2005). In addition, assays for adenovirus A and B were performed on samples collected in 2009–10 (Susan K. Spencer, 

USDA/USGS Marshfield Laboratory, oral commun., 2009). 

Quantitative PCR was performed on a LightCycler® 480 System (Roche Diagnostics Corporation, Indianapolis, Ind.) with 

LightCycler 480 Software Version 1.5.0. Hepatitis G virus 

(HGV) armored RNA (Asuragen, Inc., Austin, Tex.) was used 

as a control to measure PCR inhibition; sample extracts were 

diluted if HGV results indicated that inhibition occurred. 

Standard Curves for Enteric Viruses by qPCR or 

qRT-PCR 

At the Marshfield Laboratory, standard curves were 

prepared as described in Lambertini and others (2008). Briefly, 

virus stocks were treated with Benzonase® (Novagen, Madison, Wis.) for 30 minutes at 37°C, followed by incubation for 

2 days at 4°C. Benzonase is a genetically engineered endonuclease that attacks and degrades all noncapsulated forms 

of DNA and RNA. Stocks were then extracted, the amount of 

virus RNA or DNA was measured by using RiboGreen® or 

PicoGreen® (Molecular Probes, Eugene, Or.), and the number 

of genomic copies (gc) was calculated. RiboGreen and PicoGreen are dyes that fluoresce when bound to RNA or DNA, 

respectively, and the intensity of fluorescence in a sample 

is proportional to the concentration of RNA or DNA. After 

quantification, viral stocks were serially diluted and seeded 

into a negative FCSV made by filtering a negative tapwater 

sample, eluting with beef extract, and precipitating with PEG. 

Each standard point was extracted in duplicate and then tested 

by qPCR or qRT-PCR in triplicate once a quarter. With every 

qPCR or qRT-PCR run, a reference control (low copy standard 

from the standard curve) was run and compared to the results 

of the quarterly standard curve. The reference control was 

required to be ±1/2 the crossing threshold (Ct) value1 from 

the standard curve line for acceptance (Lambertini and others, 2008). If the reference control was greater than ±1/2 Ct, 

then a new standard curve was created and the samples were 

reanalyzed. 

Assay Limits of Quantification and Sample 

Reporting Limits for Enteric Viruses by qPCR, 

qRT-PCR, and Cell Culture

In water-resource studies, such as the MBR wastewater 

study described in this report, limits of detection or quantification help support a robust data analysis and the ability to effectively interpret results. By calculating the limits of detection or 

quantification, results can be reported as less than an established value per volume instead of reporting nondetections as 

zero. For example, reporting one result as <1 gc/L would be 

quite different than reporting a second result as <500 gc/L. 

Further, limits of detection or quantification are needed to 

qualify the data and thus more accurately report with confidence or lack of confidence when viruses are determined to be 

absent or present in low numbers. 

For qPCR and qRT-PCR, the assay limit of quantification (ALOQ) is defined by Rajal and others (2007) as the lowest 

concentration of virus genomes that remain within the linear 

range of quantification. In Rajal and others (2007), the ALOQ

was determined by preparing tenfold dilutions of the target 

DNA or RNA in deionized water and quantifying by qPCR 

or qRT-PCR, respectively—an approach applicable to TaqMan systems. The Roche system used in the present study is 

based on second-derivative Ct determination and nonlinear-fit 

algorithms for standard curve development. For the data in this 

report, therefore, the ALOQ was determined by constructing a 

linear approximation of six dilutions used to develop the standard curve and extrapolating to 1 gc. For cell culture, the ALOQ

is the lowest concentration of viruses that can be quantified 

by observing CPE. For screening, the ALOQ is 1 virus or CPE. 

For cell-culture MPN analysis, the ALOQ is 2.18 MPN/mL, as 

determined by assuming 1 virus in the undiluted sample and 

zero virus in the 1:5 and 1:25 dilutions (U.S. Environmental 

Protection Agency, 2001b).

1

The Ct value is the point at which fluorescence in a sample crosses an 

established threshold.
Methods of Study 9

Table 2. Primers and probes for enteric viruses. 

Assay Primer or probe

Sequence 

(5’ – 3’)

Concentration 

used 

(nanomoles) Reference

Adenovirus Forward primer GGACGCCTCGGAGTACCTGA 500 Cromeans and others, 2005

Reverse primer CGCTGIGACCIGTCTGTGG 500

TaqMan probe CACCGATACGTACTTCAGCCTGGGT 100

Enterovirus Forward primer CCTCCGGCCCCTGAATG 300 DeLeon and others, 1990; 

Monpoeho and others, 2000 Reverse primer ACCGGATGGCCAATCCAA 900

TaqMan probe CGGAACCGACTACTTTGGGTGTCCGT 100

Hepatitis A Forward primer CAGCACATCAGAAAGGTGAG 700 Schwab and others, 1995

Reverse primer CTCCAGAATCATCTCCAAC 700

TaqMan probe TGCTCCTCTTTATCATGCTATG 100

Hepatitis G Forward primer CGGCCAAAAGGTGGTGGATG 200 Schlueter and others, 1996

Reverse primer CGACGAGCCTGACGTCGGG 200

TaqMan probe AGGTCCCTCTGGCGCTTGTGGCGAG 100 Lambertini and others, 2008

Norovirus GI Forward primer GCCATGTTCCGITGGATG 250 Jothikumar and others, 2005

Reverse primer TCCTTAGACGCCATCATCAT 250

TaqMan probe TGTGGACAGGAGATCGCAATCTC 100

Norovirus GII Forward primer TGTCACGATCTCATCATCACC 250 Ando and others, 1995

Reverse primer TGGAATTCCATCGCCCACTGG 250

TaqMan probe ATGTCAGGGGACAGGTTTGT 100

TaqMan probe ATGTCGGGGCCTAGTCCTGT 100

Rotavirus Forward primer TTGCCACCAAATTCAGAATAC 500 Gentsch and others, 1992

Reverse primer ATTTCGGACCATTTATAACC 500

TaqMan probe AGAGAGCACAAGTTAATGAAG 100

Rajal and others (2007) also determined qPCR or qRTPCR assay limits of detection (ALOD) according to established 

method detection limit (MDL) procedures commonly used 

by chemists (U.S. Environmental Protection Agency, 2010). 

The general approach for determining an MDL is to analyze 

at least seven replicate samples near the expected MDL in 

pure water. The MDL is the minimum concentration of a 

substance that can be measured and reported with 99 percent 

confidence that the concentration is greater than zero. In the 

present study, MDLs were not determined; therefore, for 

qPCR and qRT-PCR results, the ALOQ was used as the ALOD. 

This ALOD was, therefore, an estimated value because it was 

not identified according to established MDL procedures; the 

actual sensitivity will need to be established in the future. For 

cell-culture screening analysis, the ALOQ and ALOD are both 

1 CPE or 1 virus. For cell-culture MPN analysis, the ALOD is 

calculated as 1.08 MPN (2.18 MPN/mL × 0.496 mL), where 

2.18 MPN/mL is the ALOQ and 0.496 mL is the total inoculum 

of sample added into 30 wells. 

Because original sample volumes and dilutions were 

different for each sample in the current study, the ALOD was 

applied on a sample-by-sample basis in order to determine 

sample reporting limits (SRLs). The SRLs are the “less-than 

values,” or the limits of detection for each sample and assay. 

In calculating the SRL, it is important to first understand that 

only a small fraction of the original FCSV used for extraction 

was used for qPCR or qRT-PCR. For example, figure 3 shows 

that only 33.6 μL (2008) or 25.8 μL (2009–10) for qPCR and 

5.78 μL (2008) or 4.45 μL (2009–10) for qRT-PCR of the 

original 280 μL of FCSV used for extraction (and concentrated 

to 50 or 65 μL) was actually used for analysis; this is called 

the effective FCSV. Effective FCSVs were less for enterovirus 

and norovirus GI than for adenovirus because the extracted 

eluate was further reduced through the RT steps. For cell 

culture screening and MPN analysis, the additional preparation steps and reductions in FCSV volumes shown in figure 3 

do not apply; therefore, the effective FCSVs were the amounts 

directly added to cells—0.33 mL for screening and 0.496 mL 

for MPN analysis. 
10 Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control

Sample

2.7 to 500 L

100%

=500 L of sample

Filter

and elute

100% total FCSV

4.67% 280 μL of total FCSV

for extraction

SAMPLE

100% Concentrated to 

50 μL or 65 μL*

FCSV

Adenovirus

qPCR

Enterovirus and

Norovirus GI

qRT-PCR

12% or

9%*

Analyze 

6 μL

= 33.6 μL or

25.8 μL*

effective FCSV

17.2% 8.6 μL

for RT

17.2%

or

13.2%*

50 μL

after RT

2.06%

or

1.59%*

Analyze 

6 μL

= 5.78 μL or 4.45 μL* effective FCSV

Figure 3. Determination of effective final concentrated sample volumes (FCSV) for quantitative 

polymerase chain reaction (qPCR) for adenovirus or quantitative reverse transcriptase PCR (qRT-PCR) 

for enterovirus or norovirus GI. (L, liters; mL, milliliters; μL, microliters.) Values followed by an 

asterisk indicate a change in volume of FCSV analyzed between 2008 (first value) and 2009–10 

(second value). 

5, 6, or 10 mL

Figure 3. Determination of effective final concentrated sample volumes (FCSVs) for quantitative 

polymerase chain reaction (qPCR) for adenovirus or quantitative reverse transcriptase PCR (qRT-PCR) 

for enterovirus or norovirus GI. (L, liter; %, percent; mL, milliliter; μL, microliter; values followed by an 

asterisk indicate a change in volume of FCSV analyzed between 2008 (first value) and 2009–10 (second 

value).) 
Methods of Study 11

Effective sample volume is the actual amount of sample 

that was analyzed. Effective sample volumes were sample 

specific because (1) original sample volumes were not consistent, (2) total FCSV volumes varied among different types of 

wastewaters and between years, and (3) samples were sometimes diluted because of PCR inhibition. Taking into account 

these factors, the following equation was used to calculate the 

effective sample volume (Ves): 

 Ves FCSVeffective

FCSVtotal Vsampledilution factor = * * 1

 (1)

where 

FCSVeffective is the volume of FCSV analyzed, 

FCSVtotal is the total volume of FCSV produced from 

each sample, 

Vsample is the original volume of sample filtered, and 

dilution factor is the dilution factor used for each sample 

extract. 

This same equation was used for qPCR, qRT-PCR, and cell 

culture except that for cell culture calculations, a dilution factor was not applied.

For qPCR and qRT-PCR, the SRL is based on the effective sample volume and ALOD (1 gc), as follows:

SRL gc

Ves = 1 (2)

For cell culture by screening, the SRL is similarly based 

on the effective sample volume and ALOD (1 virus), as follows:

SRL virus

Ves = 1 (3)

For cell culture by MPN, the SRL is based on the ALOD 

(1.08 MPN), as follows:

SRL MPN

Ves = 1 0. 8 (4)

Indicator Bacteria and Coliphage

Analyses of samples for the bacterial indicators E. coli, 

enterococci, and fecal coliforms were done by preparing serial 

dilutions for plating by standard membrane filtration methods. 

Appropriate dilutions were based on sample type and ranged 

from 0.0003 mL for post-preliminary samples to 250 mL for 

post-disinfection samples. For E. coli, the modified mTEC 

agar method (U.S. Environmental Protection Agency, 2006a) 

was used. Modified mTEC agar plates were incubated at 

35°C for 2 hours, followed by incubation at 44.5°C for 

22 hours; magenta colonies were counted as E. coli. The mEI 

agar method is a one-step method for the enumeration of 

enterococci (U.S. Environmental Protection Agency, 2006b). 

Agar plates were incubated at 41°C for 24 hours, and all colonies with a blue halo were recorded as enterococci, regardless 

of colony color. The mFC agar method was used for enumeration of fecal coliforms (Britton and Greeson, 1987). Agar 

plates were incubated at 44.5°C for 22 to 24 hours, and blue 

colonies were counted as fecal coliforms. Results from membrane filtration analyses were recorded as colony forming units 

per 100 milliliters (CFU/100 mL). 

The single agar layer (SAL) procedure was mainly 

used for enumeration of F-specific and somatic coliphage 

(U.S. Environmental Protection Agency, 2001a). Antibioticresistant host-culture strains E. coli CN-13 (resistant to 

nalidixic acid) and E. coli F-amp (resistant to streptomycin 

and ampicillin) were used as hosts for somatic and F-specific 

coliphage, respectively. A 100-mL water sample, eluate, or a 

sample or eluate dilution was combined with magnesium chloride, log-phase host bacteria, and double-strength tryptic soy 

agar. The sample mixture was poured into plates and incubated 

at 36°C for 16–24 hours. Circular lysis zones (plaques) were 

counted and summed for all plates from a single sample. The 

quantity of coliphage in a sample is expressed as plaque forming units per 100 milliliters (PFU/100 mL).

An alternative method, EasyPhage (Scientific Methods 

Inc., Granger, Ind.), was tested on 10 post-preliminary, postMBR, or post-secondary samples in 2008 for enumeration of 

F-specific and somatic coliphage. EasyPhage eliminates the 

need to handle molten agar. For each sample, two bottles of 

EasyPhage agar were preheated in a 35°C incubator for 1 hour, 

one bottle each for F-specific and somatic coliphage. To each 

warmed bottle, 100 mL of sample or sample dilution, 2 mL 

of antibiotic, 0.7 mL of EasyPhage stain, and 3.5 mL of the 

bacterial host were added. Twenty milliliters of each mixture 

was added to each of ten 100 × 15 mm Petri plates for a total 

of 10 plates per sample. After incubation at 36°C for 16–24 

hours, the blue plaques were counted as positive for coliphage. 

Quality Assurance and Quality Control

Standard field and data management quality-assurance 

practices for USGS water-quality activities in Ohio are 

described in Francy and Shaffer (2008). Standard laboratory 

quality-assurance/quality-control (QA/QC) practices for the 

OWML are described in Francy and others (2010). 

To facilitate identification of sampling and analytical 

bias, field personnel collected equipment blanks, field blanks, 

field concurrent replicates, method test samples, and seeded 

matrix controls. The types and numbers of QC samples for 

each group of microorganism are listed in the subsections 

below. General definitions of QC samples are provided in the 

glossary. 
12 Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control

Indicator Bacteria and Coliphage

Laboratory QC samples for bacterial indicators and 

coliphage, with information on reagent and media preparation 

and procedures, are described in Francy and others (2010). 

In addition to the routine QC samples, method test samples 

for coliphage were analyzed. These consisted of filtration test 

samples and EasyPhage test samples. Filtration test samples 

were replicate samples analyzed by both direct plating and 

eluate plating for coliphage. Filtration test samples were collected to provide data on recoveries of coliphage in wastewater by use of glass-wool filters. EasyPhage test samples were 

replicate samples analyzed by both the SAL and EasyPhage 

methods and were done to determine whether the EasyPhage 

method is a suitable alternative to the standard SAL method. 

Field QC samples for bacteria and (or) coliphage consisted of field blanks and concurrent replicates. Field blanks 

were aliquots of sterile buffered water poured into sample 

bottles under actual field conditions, transported and analyzed 

in the same manner as a regular sample. A field concurrent 

replicate was collected by alternating the collection of a second grab sample with the first one and compositing the second 

sample into a separate 3-L bottle. 

Enteric Viruses 

Laboratory analytical QC samples for enteric viruses by 

qPCR or qRT-PCR are listed in Lambertini and others (2008). 

Quality-control samples were processed with each batch of 

samples and included extraction positive and negative controls, as well as separate PCR positive and negative controls. 

Each sample was assayed in duplicate by qPCR or qRT-PCR. 

The laboratory QC samples for enteroviruses by cell culture are described in USEPA (2001b). They consisted of an 

inoculum of sodium phosphate buffer (PBS pH 7.0 –7.5) as the 

negative control and 20 PFU poliovirus Sabin 3 as the positive 

control for each batch of samples. Each sample enumerated by 

cell culture MPN had its own negative control. 

Field QC samples for enteric viruses consisted of an 

equipment blank, field blanks, concurrent replicates, and 

seeded matrix controls. Blank samples consisted of 10 L of 

autoclaved, dechlorinated tapwater treated in the same manner as a regular filtered sample in the OWML (equipment 

blank) or at a sampling location (field blank). Field concurrent 

replicates were collected by one of two methods: (1) alternating the collection of a second grab sample with the first one 

and compositing the second sample in two separate 3-L bottles 

(post-preliminary samples) or (2) filtering a second wastewater 

sample onsite concurrently with a second filtration apparatus. 

Seeded matrix controls were collected from representative wastewater matrixes to determine the recovery of viruses 

through all processing and analytical steps. Samples were 

seeded with Mahoney enterovirus (U.S. Environmental Protection Agency, Cincinnati, Ohio) to achieve target seed amounts 

of 104 viruses, estimated by the OWML by use of a RiboGreen 

assay. Four-liter samples of post-preliminary wastewater were 

seeded in the laboratory and then filtered. For other wastewater samples, all but the last 20 L were filtered onsite; the 20-L 

remaining volume was collected, transported to the laboratory, 

seeded, and filtered with the same filter that was used onsite 

for that sample. Unseeded controls were analyzed in conjunction with a seeded matrix control to determine background 

concentrations of enteroviruses. 

Indicator Bacteria, Coliphage, and 

Viruses in Wastewater

Field personnel collected 73 regular samples (tables 3 

and 4, at back of report) during the recreational seasons of 

2008–10. One equipment blank and four field blanks, each 

analyzed for different groups of microorganisms, were collected (table 5). Other quality-control samples included 

10 bacteria and coliphage concurrent replicates (table 6); 

25 method test samples, 10 of which were also EasyPhage test 

samples (table 7); and 13 enteric virus replicates (tables 4 and 

8). Specific conductance and initial pH were measured at the 

time of sampling and turbidity was measured at the OWML 

(table 3). 

Enumeration Considerations for Enteric Viruses 

by qPCR and qRT-PCR

In order to achieve the objectives of this study, it was 

important to quantify viruses as accurately as possible, identify lower detection limits, and qualify any uncertainties associated with a viral measurement. Using available data from the 

analytical laboratory, standard curves were constructed to estimate the assay limit of quantification (ALOQ ). Sample reporting 

limits (SRLs) were calculated from the effective volume for 

each sample and the A LOQ. On the basis of the SRLs, there was 

a need to further qualify measurements. Codes for qualifying 

the data are those established by the USGS National Water 

Information System for QWDATA (U.S. Geological Survey, 

2010). 

Table 9 lists performance characteristics for each standard 

curve for each assay. The dynamic range of the standard curve 

is the lowest and highest concentrations of standards that were 

used in the standard curve development. The amplification 

efficiency is calculated using the following equation: 

E slope = − ( ) − 10 1 1/ (5)

The efficiency of the PCR should be 90–110 percent meaning doubling of the target sequence at each cycle, which 

corresponds to a slope of −3.1 to −3.6 in the standard curve. 

Because norovirus GII and hepatitis A virus were not detected 

in any samples and rotavirus was detected in only one postpreliminary sample, these standard curves are not presented 

here. 
Indicator Bacteria, Coliphage, and Viruses in Wastewater 13

Table 5. Quality-control equipment and field-blank data for bacterial indicators, coliphage, and viruses in wastewater, 2008–10. 

[qPCR, quantitative polymerase chain reaction; qRT-PCR, quantitative reverse transcriptase PCR; <, less than; --, not done; Equip, equipment; OWML, Ohio 

Water Microbiology Laboratory]

Sample 

number

Type of 

blank 

sample

Plant 

or 

location

Bacterial indicators 

(colony-forming units 

per 100 milliliters)

Coliphage

(plaque-forming 

units 

per 100 milliliters)

Enteric viruses by 

qPCR or qRT-PCR 

(genomic copies per liter)

Culturable 

viruses 

(viruses 

per liter)

Escherichia 

coli 

EnterococciFecal 

coliforms Somatic F-specific

AdenovirusEnterovirusNorovirus 

GI Viruses

3 Field 3 <1 <1 <1 <1 <1 -- -- -- <1.5

3 Equip. OWML -- -- -- -- -- <15 <90 <90 --

6 Field 1 <1 <1 <1 <1 <1 <23 <140 <140 --

10 Field 2 -- -- -- -- -- <23 <140 <140 --

16 Field 5 <1 <1 <1 <1 <1 <23 <140 <140 <2.0

The lowest concentration that was detected in the range 

of each standard curve was in the single digits for each assay; 

therefore, for consistency, the ALOQ was established as 1 gc. 

Data were then qualified on the basis of Ct, ALOQ, and SRL, as 

follows:

• If the Ct was less than the Ct associated with 1 gc, the 

result was reported without any qualifiers. 

• If the Ct was greater than the Ct associated with 1 gc 

but less than 40, the result was reported with an E for 

estimated value and b value qualifier. The b indicates 

that the result was extrapolated at the low end (past the 

upper Ct limit of the standard curve). 

• If the Ct was greater than 40 but less than 45, the result 

was reported as <SRL with an M for “material present 

but not quantified.”

• If the Ct was greater than 45, the result was reported as 

<SRL.

Other qualifiers were included in the reporting of results, 

as follows:

• If one duplicate qPCR or qRT-PCR analysis had a Ct 

greater than 40 and the second duplicate had a Ct less 

than 40, the result associated with the less than 40 Ct 

was reported with an E for estimated value and ~ value 

qualifier. The ~ indicates that the duplicates do not 

agree. 

• If the calculated result (in gc/L) was less than the SRL 

because of a dilution, but still within 1 log of the SRL, 

the result was reported with an E for estimated value 

and b value qualifier. The b indicates that the result 

was extrapolated at the low end. 

• If the calculated result (in gc/L) was at least 1 log 

lower than the SRL, the result was reported as <SRL 

with an M for “material present but not quantified.”
Table 6. Quality-control concurrent replicate data for bacterial indicators and coliphage in wastewater, 2008–10. [E. coli, Escherichia coli; CFU/mL, colony-forming units per 100 milliliters; AVLD is absolute value log10 difference between replicate A and B; prelim, preliminary; --, not done; MBR, membrane bioreactor; <, less than; >, greater than; ND, not determined because there were two values below detection; sec, secondary; dis, disinfection; PFUplaque-forming units per 100 milliliters] Sample number Type of sample E. coli (CFU/100 mL) Enterococci (CFU/100 mL) Fecal coliforms (CFU/100 mL) A B AVLD A B AVLD A B 

9 Post-prelim 930,000 1,100,000 0.07 410,000 390,000 0.02 580,000 1,000,000 

10 Post-prelim 970,000 1,700,000 .24 9,200,000 6,600,000 .14 3,100,000 2,700,000 13 Post-prelim -- -- -- -- -- -- -- -- 

5 Post-MBR 2 2 .00 <1 2 >.30 2 12 7 Post-MBR -- -- -- 340 320 .03 -- -- 

19 Post-MBR <1 <1 ND <1 <1 ND <1 <1 16 Post-sec 7,700 6,400 .08 1,100 1,000 .04 14,000 8,000 18 Post-sec 15,000 11,000 .13 3,000 6,000 .30 26,000 18,000 

8 Post-dis <1 <1 ND <1 <1 ND <1 <1 

21 Post-dis <1 <1 ND 1 <1 >.00 <1 <1 

Sample number Type of sample F-specific (PFU/100 mL) Somatic (PFU/100 mL) A B AVLD A B AVLD 9 Post-prelim 9,500 12,000 0.10 23,000 25,000 0.04 10 Post-prelim 140,000 92,000 .18 4,800,000 4,800,000 .00 13 Post-prelim 120,000 120,000 .00 150,000 150,000 .00 5 Post-MBR 4 1 .60 120 180 .18 7 Post-MBR 4 1 .60 430 380 .05 19 Post-MBR <1 7 >.84 520 490 .03 16 Post-sec 11 14 .10 470 280 .22 18 Post-sec 64 40 .20 880 1,000 .06 8 Post-dis <1 <1 ND <1 <1 ND 21 Post-dis <1 <1 ND 8 9 .05
Table 7. Quality-control method test samples for coliphage in wastewater, 2008–10.[PFU/100 mL, plaque-forming units per 100 milliliters; SAL, single-agar layer method; prelim, preliminary; sec, secondary; <, less than; MBR, membrane bioreactor; NA, not abecause filtered was detected and direct was below detection; tert, tertiary; dis, disinfection; ND, not determined because both filtered and direct values were below detection]

Sample 

number

Type of 

sample

Somatic coliphage (PFU/100 mL) F-specific coliphage (PFU/100 mL)

Filtered 

SAL

Direct 

SAL

Percent 

recovery 

by 

filtration

Direct 

EasyPhage

Filtered 

SAL

Direct 

SAL

Percent 

recovery 

by 

filtration

E

1 Post-prelim 600,000 2,200,000 27.3 1,500,000 28,000 250,000 11.2 22 Post-prelim 35,000 310,000 11.3 240,000 28,000 420,000 6.7 23 Post-prelim 108,000 3,000,000 3.6 2,400,000 4,950 120,000 4.1 4 Post-prelim 7,000 170,000 4.1 250,000 1,600 66,000 2.4 5 Post-prelim 28,000 910,000 3.1 1,800,000 260,000 1,300,000 20.0 5

11 Post-prelim 20,667 210,000 9.8 320,000 1,900,000 16.8

3 Post-sec 108 1,600 6.8 804 1.1 7 15.7 9 Post-sec <.14 9 .0 .028 3 .91 Post-MBR 12 921 1.3 630 .1 2 5.0 2 Post-MBR <.40 80 .0 14 .02 4 .5 4 Post-MBR 9.2 100 9.2 58 .13 <1 NA 5 Post-MBR 1.3 120 1.1 52 .057 4 1.4 5 Post-MBR 1.2 180 .7 .014 1 1.46 Post-MBR 2.6 15 17.3 .045 <1 NA7 Post-MBR 2.5 430 .6 .41 4 10.33 Post-tert 29 940 3.1 .37 7 5.3

12 Post-tert 1.5 600 .3 .15 7 2.1

1 Post-dis <.0066 <1 ND <.0066 <1 ND2 Post-dis .12 52 .2 <.0086 <1 ND3 Post-dis <.01 <1 ND <.01 <1 ND4 Post-dis 11 100 11.0 .11 <1 NA5 Post-dis <.007 <1 ND <.007 <1 ND6 Post-dis <.0004 <1 ND <.0004 <1 ND8 Post-dis <.0004 <1 ND <.0004 <1 ND

10 Post-dis 28 1,100 2.5 .523 19 2.8
Table 8. Quality-control concurrent replicate data for enteric viruses in wastewater, 2008–10. [gc/L, genomic copies per liter; MPN/L, most probable number per liter; AVLD is absolute value log10 difference between replicate A and B; prelim, preliminary; <, less than; Nthere were two values below detection; E, estimated value; ~, PCR duplicates do not match; M, presence of material verified but not quantified; >, greater than; MBR, membrandis, disinfection] Sample number Type of sample Adenovirus (gc/L) Enterovirus (gc/L) Norovirus, G1 (gc/L) 

A B AVLD A B AVLD A B AVLD 

9 Post-prelim 2,100 3,000 0.15 <5,600 <5,600 ND <5,600 <5,600 ND 

10 Post-prelim 120,000 430,000 .55 <11,000 E~ 47,000 >0.63 <11,000 <17,000 ND 13 Post-prelim 46,000 32,000 .16 280,000 300,000 .03 <8,300 <8,300 ND 20 Post-prelim 1,900 300 .80 M <560 M <560 ND 2,200 790 0.44 

5 Post-MBR <5.3 E~ .32 <1.2 <30 M <1.5 ND <30 <1.5 ND 

13 Post-MBR 2.0 2.3 .06 7.5 3.1 .38 <2.7 <2.6 ND 19 Post-MBR <.45 <.47 ND <2.6 <2.7 ND <2.6 E~ 17 >.82 23 Post-MBR <.47 <.46 ND <2.7 <2.7 ND <2.7 <2.7 ND 16 Post-sec E~ 10 E~ 2.8 .55 <9.4 <9.3 ND <9.4 <9.3 ND 18 Post-sec E~ 68 <33 >.31 <190 <190 ND <190 <190 ND 22 Post-sec <1.7 E~ 16 >.97 <9.6 <9.5 ND <9.6 <9.5 ND 

8 Post-dis <.45 <.43 ND <2.6 <2.5 ND <2.6 <2.5 ND 

21 Post-dis M <.46 <.46 ND <2.7 <2.7 ND <2.7 <2.7 ND 
Indicator Bacteria, Coliphage, and Viruses in Wastewater 17

Table 9. Performance characteristics of quantitative polymerase chain reaction (qPCR) and reverse transcriptase 

qPCR (qRT-PCR) standard curves for each assay. 

Assay Year Slope y-intercept R2

Dynamic range

(copies/qPCR reaction)

Amplification 

efficiency

(percent)

Adenovirus

Subgroups C, D, F

2008 −3.481 38.87 0.999 1.42 – 1.42E+05 94

Adenovirus

Subgroups C, D, F

2009 −3.312 38.57 .997 1.10 – 1.10E+05 100

Adenovirus

Subgroups C, D, F

2010 −2.963 40.07 .990 1.50 – 1.50E+06 118

Adenovirus

Subgroup A

Assay was not run by Marshfield in 2008

Adenovirus

Subgroup A

2009 −3.223 39.94 .999 6.42 – 6.42E+05 104

Adenovirus

Subgroup A

2010 −3.009 39.85 .995 1.20 – 1.20E+06 115

Enterovirus 2008 −3.325 39.08 .999 1.74 – 1.74E+05 100

Enterovirus 2009 −3.275 38.43 .999 1.50 – 1.50E+05 102

Enterovirus 2010 −3.177 38.21 .994 2.85 – 2.85E+06 106

Norovirus GI 2008 −3.161 38.42 .995 1.50 – 1.50E+04 107

Norovirus GI 2009 −3.278 38.04 .993 1.35 – 1.35E+04 102

Norovirus GI 2010 −3.051 39.67 .996 1.20 – 1.20E+04 113

Quality-Control Considerations for Interpreting 

Bacterial-Indicator, Coliphage, and EntericVirus Data

Quality-control samples were collected and analyzed for 

all microorganisms to aid in data interpretations. The results 

for bacteria, coliphage, and enteric viruses were below detection for all equipment and field blanks (table 5). Laboratory 

blanks for bacteria, coliphage, and viruses were all negative 

(data not shown). These results indicate that it was unlikely 

that samples were contaminated from equipment or processing 

procedures. 

Quality-Control Samples for Bacterial Indicators 

and Coliphage

Concurrent replicates for bacterial indicators and coliphage are listed in table 6 and shown in figure 4, with the 

absolute value log10 differences (AVLD) calculated for each 

replicate pair when at least one replicate included a detection. The highest AVLDs were associated with average 

concentrations for replicate pairs that were less than 10 CFU 

or PFU/100 mL, shown in the shaded area in figure 4A and 

4B, and were for post-MBR samples. For bacterial indicators with average concentrations less than 10 CFU/100 mL, 

eight AVLD s were not determined because both replicate 

results were <1 CFU/100 mL (table 6). For the four replicates 

pairs with average concentrations <10 CFU/100 mL, the 

AVLDs ranged from 0 to 0.78 log CFU/100 mL. For bacterial indicators with average concentrations >10 CFU/100 mL, 

AVLDs ranged from 0.02 to 0.30 log CFU/100 mL. For 

coliphage with average concentrations <10 PFU/100 mL, three 

AVLDs were not determined because both replicates were 

<1 PFU/100 mL (table 6). For the four replicate pairs with 

average concentrations <10 PFU/100 mL, AVLDs ranged from 

0.05 to >0.84 log PFU/100 mL. For coliphage with average concentrations >10 PFU/100 mL, AVLDs ranged from 

0 to 0.22 log PFU/100 mL. These results indicate that, when 

interpreting removals of bacterial indicators and coliphage in 

wastewater samples for samples with concentrations <10 CFU 

or PFU/100 mL, log removals less than or equal to approximately 1 may not be greater than the analytical variability. 

For samples >10 CFU or PFU/100 mL, log removals less than 

or equal to about 0.3 may not be greater than the analytical 

variability. 
18 Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control

1 1 0 100 1,000 10,000 100,000 1,000,000 10,000,000

Average concentration, in CFU/100 mL

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

Range in concentrations of replicate samples, in CFU/100 mL

A. Bacterial indicators

1 1 0 100 1,000 10,000 100,000 1,000,000 10,000,000

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

Range in concentrations of replicate samples, in PFU/100 mL

(2)

B. Coliphage

Average concentration, in PFU/100 mL

Figure 4. Quality-control replicate samples for concentrations in wastewater. A, bacterial indicators. B, coliphage. 

Average concentrations less than 10 CFU/100 mL or PFU/100 mL are shaded in gray; if more than one replicate pair is represented by a 

symbol, the number of pairs is indicated. Each horizontal line represents a replicate sample volume, and the range for each replicate 

pair is represented by the vertical line connecting the two values. A dotted line indicates one of the replicate pairs was below detection. 

(Abbreviations: CFU/100 mL, colony-forming units per 100 milliliters; PFU/100 mL, plaque-forming units per 100 milliliters.) 
Indicator Bacteria, Coliphage, and Viruses in Wastewater 19

Results from method test samples for coliphage are listed 

in table 7, organized by type of wastewater sample. Concentrations of coliphage found by use of filtration and concentration (filtered SAL) can be compared to those found by use of 

direct plating without filtration (direct SAL). When detected, 

direct plating resulted in higher concentrations for all samples. 

In three coliphage samples, however, direct plating results 

were below detection (<1 PFU/100 mL) whereas filtered 

results were detections of 0.04, 0.11, and 0.13 PFU/100 mL. 

Relative to direct plating, measurable percent recoveries in filtered samples averaged 5.7 percent for somatic coliphage and 

6.7 percent for F-specific coliphage. Direct plating, therefore, 

is the preferred method for enumerating coliphage in wastewater samples.

Concentrations of coliphage found by use of the direct 

plating SAL method can be compared to the direct plating 

EasyPhage method. Results from the two methods were found 

to be highly correlated (r = 0.99, fig. 5). For F-specific coliphage, EasyPhage resulted in higher concentrations than SAL 

in all but one sample (fig. 5A). For somatic coliphage, the SAL 

method tended to yield higher concentrations than EasyPhage in low-concentration samples (fig. 5B). The results of a 

Wilcoxon signed rank test (Helsel and Hirsch, 2002) showed 

that the median difference between paired observations was 

significantly different from zero for F-specific coliphage 

(p = 0.0059) but not for somatic coliphage (p = 0.3223). 

These results indicate that either the SAL or EasyPhage can 

be used to determine relative concentrations of coliphage in 

wastewater studies, if one method is chosen over the other for 

consistency. If actual concentrations of F-specific coliphage 

are needed, however, EasyPhage may be the preferred method 

because it yields significantly higher concentrations than the 

SAL method.

Quality-Control Samples for Enteric Viruses

Nine seeded matrix controls were processed and analyzed 

by qRT-PCR for enterovirus in the same manner as regular 

samples by the Marshfield Laboratory. The original seed was 

analyzed by qRT-PCR to determine the starting concentration 

of enterovirus. The Marshfield Laboratory did not find any 

enteroviruses in the seeded matrix controls above background 

concentrations. This was probably because starting concentrations, ranging from 52 to 1,100 genomic copies, were much 

lower than the 104 estimated concentration from the RiboGreen assay. The source of this error is not known but may 

have been degradation of the virus stock and (or) inaccurate 

quantification. Lambertini and others (2008) found recoveries of human enteric viruses with glass-wool filters ranging 

from 8 to 98 percent in seeded groundwater samples. At the 

OWML, average recoveries from seeded lake water samples 

by use of glass-wool filters were 5.2 percent for adenovirus, 

14.5 percent for norovirus, and 8.4 percent for enterovirus 

(unpublished data). These studies showed that viruses can 

be recovered by glass-wool filtration but that recoveries are 

variable. Recoveries of viruses by glass-wool filtration in 

wastewater samples are not documented. Plans are underway 

to collect and analyze a small set of seeded matrix controls for 

representative wastewater samples and include these results in 

companion reports. 

Concurrent replicates for enteric viruses are listed in 

table 8, with the AVLDs calculated for each replicate pair and 

presented in figure 6. Results for the two post-disinfection 

samples were not determined because either both replicates 

were below detection or material was present but not quantified. Many of the results for enterovirus, norovirus, and culturable viruses were similarly not determined. For some sample 

pairs, viruses were detected and quantified in one replicate 

and not detected in the other replicate. This occurred three 

times for adenovirus by qPCR, one time for enterovirus by 

qRT-PCR, one time for norovirus by qRT-PCR, and twice for 

culturable viruses; these instances correspond to AVLDs that 

are < or > values. Overall, concentrations among replicates 

were similar in magnitude with a few exceptions. In sample 

10 (plant 2, post-preliminary), relatively large concentration 

differences were found between sample pairs for adenovirus 

by qPCR, enterovirus by qRT-PCR, and culturable viruses. 

Sample volumes were different (4.0 and 2.7 L) because the 

filter for replicate B clogged and lab personnel were unable to 

filter the entire 4.0 L (table 4). The differences in molecular 

results between replicate pairs in sample 10 were likely due 

to lack of precision for samples with small effective volumes. 

For example, the qRT-PCR results for enterovirus of <11,000 

and E ~ 47,000 gc/L involved a twentyfold dilution factor that 

resulted in very small effective volumes (0.06 and 0.09 mL). 

These replicates highlight the variability of results with small 

effective volumes and the importance of including sample 

reporting limits and not just zero values in water-resource 

studies. Similarly, small effective volumes likely resulted in 

the lack of precision for culturable virus results in sample 10 

and for adenovirus and norovirus results in sample 20 (plant 5, 

post-preliminary). Overall, the AVLDs for viruses ranged from 

0 to <1.2 or >0.97 log gc/L. Unlike the data for bacterial indicator and coliphage replicates, higher average virus concentrations were not associated with smaller differences in concentrations between replicates (fig. 6). These results indicate that, 

when interpreting removals of viruses in wastewater samples, 

removals less than or equal to about 1.2 log may not be greater 

than the analytical variability. 
20 Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control

Single-agar layer method, in log PFU/100 mL

0

2

4

6

EasyPhage, in log PFU/100 mL

1:1 line

A. F-specific

r = 0.99

p <0.0001

Single-agar layer method, in log PFU/100 mL

1

2

3

4

5

6

EasyPhage, in log PFU/100 mL

r = 0.99

p <0.0001

1:1 line

B. Somatic

0 2 4 6

1 2 3 4 5 6

Figure 5. Concentrations found by use of the single agar layer and EasyPhage methods in wastewater 

samples, 2008. A, F-specific. B, somatic coliphage. (Abbreviations: PFU/100 mL, plaque-forming units per 

100 milliliters; r is the Pearson’s correlation coefficient and p is the significance of the relation.) 
Indicator Bacteria, Coliphage, and Viruses in Wastewater 21

1 1 0 100 1,000 10,000 100,000 1,000,000

Average concentration, in genomic copies per liter

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

Range in concentrations of replicate samples, in genomic copies per liter

Figure 6. Quality-control replicate samples for concentrations of viruses in wastewater, 2008–10. Each 

horizontal line represents a replicate sample volume, and the range for each replicate pair is represented by 

the vertical line connecting the two values. A dotted line indicates that one of the replicate pairs was below 

detection.

Concentrations of Bacterial Indicators and 

Coliphage in Wastewater

Concentrations of E. coli, fecal coliforms, enterococci, 

and somatic and F-specific coliphage in wastewater samples 

are listed in table 3 and presented by plant number and type of 

wastewater sample in figures 7A–E. For each organism, results 

from MBR plants (1, 2, and 4) and conventional plants (3 and 

5) are presented in separate plots. Results from direct plating 

by the SAL method for coliphage were used for data presentations. Average concentrations from concurrent replicates for 

bacterial indicators and coliphage, when analyzed, were also 

used. 

In post-preliminary wastewater samples, concentrations 

of E. coli, fecal coliforms, and enterococci were generally 106

to 107 CFU/100 mL with some outliers (figs. 7A–C); concentrations of somatic and F-specific coliphage showed a wider 

range, from 104 to 107 CFU/100 mL (figs. 7D–E). Among 

MBR plants, bacterial indicator and coliphage concentrations 

in post-MBR samples were highest at plant 2. Median concentrations of bacterial indicators and coliphage in post-secondary 

and post-tertiary samples in conventional plants were higher 

than those in post-MBR samples. Median somatic coliphage 

concentrations were higher than F-specific coliphage concentrations in post-treatment samples, but not in post-preliminary 

samples. Other researchers found higher somatic coliphage 

than F-specific coliphage in treated wastewater samples 

(Zhang and Farahbakhsh, 2007). They hypothesized that 

F-specific coliphage tend to absorb to solid surfaces (membrane surfaces and particulate matter) more than somatic coliphage, resulting in higher removals of F-specific coliphage. In 

post-disinfection samples at plants 1, 3, and 4, concentrations 

of bacterial indicators and coliphage were near or below the 

detection limit. At plants 2 and 5, however, post-disinfection 

sample concentrations were in the single or double digits for 

most samples and in the triple digits for somatic coliphage at 

plant 2. Plant 2 had operational issues and effluent contamination of the UV system, and plant 5 was the only chlorine disinfection system in the study. Through their NPDES permits, the 

wastewater plants are required to meet effluent limits for fecal 

coliforms (daily limit of 2,000 CFU/100 mL) and (or) E. coli

(daily limit from 284 –362 CFU/100 mL). All of the plants 

met the NPDES-required effluent limits on the days sampled 

for the present study. 
110 100 1,000 10,000 100,000 1,000,000 10,000,000 100,000,000

Escherichia coli, in CFU /100 mL

Post-Preliminary Post-MBR Post-Disinfection

Plant 1 (n = 6)Plant 2 (n = 4)Plant 4 (n = 5)

A. Escherchia coli

Detection limit

a

2.8x106 4.2x106

2

<1 <1 <1

24

MBR plants

950,000

74

Post-Preliminary Post-Secondary Post-Tert

Detection limit

4.3x106

8,200

1,700

3.5x106

6,100

Outliers2Upper whisker 175th percentile

Median

1The span of the whiskers is the largest and smallest 

values within 1.5 times the interquartile range.

2Outliers are defined as values outside of the whisker

span.

25th percentileLower whisker1

99 Median value

CFU/100 mL, colony-forPFU/100 mL, plaque-forMBR, membrane biorea

Includes a >32 CFU/100 Includes a >24CFU/100 Includes a >80 CFU/100 

abc

EXPLANATION

Median values are sho

Figure 7A. Concentrations in wastewater samples, 2008–10: Escherichia coli. 
B. Enterococci

Plant 1 (n = 6)Plant 2 (n = 4)Plant 4 (n = 5)

b

2.1x106

18

170

<1

4

<1 Detection limit

MBR plants

3.4x106

2.4x106

<1

Detection limit

1.2x10 

6

620,000

2,900

220

1,400

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

100,000,000

Enterococci, in CFU /100 mL

Post-Preliminary Post-MBR Post-Disinfection Post-Preliminary Post-Secondary Post-Ter

Outliers2Upper whisker 175th percentile

Median

1The span of the whiskers is the largest and smallest 

values within 1.5 times the interquartile range.

2Outliers are defined as values outside of the whisker

span.

25th percentileLower whisker1

99 Median value

CFU/100 mL, colony-forPFU/100 mL, plaque-forMBR, membrane biorea

Includes a >32 CFU/100 Includes a >24CFU/100 Includes a >80 CFU/100 

abc

EXPLANATION

Median values are sho

Figure 7B. Concentrations in wastewater samples, 2008–10: enterococci. 
C. Fecal coliforms Plant 1 (n = 6) Plant 2 (n = 4) Plant 4 (n = 5) c 5.5x10 6 6.7x10 6 6 81 <1 <1 55 Detection limit MBR plants 3.4x10 6 <1 Detection limit 

5.2x10 6 12,000 2,800 8,100 

3.3x10 6 Post-Preliminary Post-MBR Post-Disinfection Post-Preliminary Post-Secondary Post-Tert

110 100 1,000 10,000 100,000 1,000,000 10,000,000 100,000,000

Fecal coliforms, in CFU /100 mL

Outliers2Upper whisker 175th percentile

Median

1The span of the whiskers is the largest and smallest 

values within 1.5 times the interquartile range.

2Outliers are defined as values outside of the whisker

span.

25th percentileLower whisker1

99 Median value

CFU/100 mL, colony-formPFU/100 mL, plaque-formMBR, membrane biorea

Includes a >32 CFU/100 Includes a >24CFU/100 Includes a >80 CFU/100 

abc

EXPLANATION

Median values are sho

Figure 7C. Concentrations in wastewater samples, 2008–10: fecal coliforms. 
D. Somatic coliphage

Plant 1 (n = 6)Plant 2 (n = 4)Plant 4 (n = 5) 180,000

260

290,000

570,000

170

390

260

1

Detection limit <1

MBR plants

Detection limit

210,000

1,000

580

370,000

1,000

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

100,000,000

Somatic coliphage, in PFU /100 mL

Post-Preliminary Post-MBR Post-Disinfection Post-Preliminary Post-Secondary Post-Tert

Outliers2Upper whisker 175th percentile

Median

1The span of the whiskers is the largest and smallest 

values within 1.5 times the interquartile range.

2Outliers are defined as values outside of the whisker

span.

25th percentileLower whisker1

99 Median value

CFU/100 mL, colony-formPFU/100 mL, plaque-forMBR, membrane biorea

Includes a >32 CFU/100 Includes a >24CFU/100 Includes a >80 CFU/100 

abc

EXPLANATION

Median values are sho

Figure 7D. Concentrations in wastewater samples, 2008–10: somatic coliphage. 
E. F-specific coliphage

Detection limit

Plant 1 (n = 6)Plant 2 (n = 4)Plant 4 (n = 5) 625,000

150,000 270,000

1 3

MBR plants

<1

<1

2

<1

Detection limit

450,000

19

7

79,000

23

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

100,000,000

F-specific coliphage, in PFU /100 mL

Post-Preliminary Post-MBR Post-Disinfection Post-Preliminary Post-Secondary Post-Terti

Outliers2Upper whisker 175th percentile

Median

1The span of the whiskers is the largest and smallest 

values within 1.5 times the interquartile range.

2Outliers are defined as values outside of the whisker

span.

25th percentileLower whisker1

99 Median value

CFU/100 mL, colony-formPFU/100 mL, plaque-formMBR, membrane biorea

Includes a >32 CFU/100 Includes a >24CFU/100 Includes a >80 CFU/100 

abc

EXPLANATION

Median values are sho

Figure 7E. Concentrations in wastewater samples, 2008–10: F-specific coliphage. 
Indicator Bacteria, Coliphage, and Viruses in Wastewater 27

Concentrations of Enteric Viruses in 

Wastewater

Concentrations for adenovirus, enterovirus, norovirus 

GI, and culturable viruses in wastewater samples are listed 

in table 4, along with effective sample volumes and the data 

used to calculate these values. Because norovirus GII and 

hepatitis A virus were not detected in any samples, and for 

rotavirus material was present but not quantified in one sample 

(sample 7, plant 2, post-preliminary), they are not included in 

table 4. As shown in equation 1, effective FCSV, total FCSV, 

original sample volume, and dilution factor were used to 

calculate the effective sample volumes. The effective FCSVs 

were specific for each assay (and year) and were calculated 

as shown in figure 3 for qPCR and qRT-PCR; they are listed 

by virus assay on the top of table 4. Original sample volumes 

ranged from 2,700 to 579,700 mL (table 4, column D). Total 

FCSVs (table 4, column E) for post-preliminary samples were 

10 mL throughout the study; however, for other wastewater 

samples, FCSV volumes were 5 mL in 2008 and 6 mL in 2009 

and 2010. Because of inhibition of the PCR reaction, some 

sample extracts were diluted by factors ranging from 5 to 

20 (table 4, column F). Using equation 1 and sample number 2, plant 2, post-MBR as an example (fifth sample listed 

in table 4), effective sample volume for qPCR (column G) is 

calculated as follows:

738 0 0336

5

549 400 1

5

mL mL

mL = mL . * , *

For molecular assays, effective sample volumes ranged 

from 0.06 mL (post-preliminary samples for qRT-PCR with a 

dilution factor of 20) (table 4, column I) to almost 4 L (largevolume samples for qPCR that were not diluted) (table 4, 

column G). 

As shown in equations 2–4, effective sample volumes and 

the ALOD were used to calculate SRLs. For the same example 

shown above, the SRL for qPCR is calculated by using equation 2, as follows:

1 4 1

0 738

. / . gc L gc

L =

The SRLs were used in reporting less-than values in 

which viruses were not detected or in which viruses were 

reported with an “M” qualifier. The M indicates that viruses 

were present, but results cannot be quantified because of great 

uncertainty; this qualifier is used for both molecular and cellculture results. Less uncertainty is associated with another data 

qualifier for molecular results, the “E” for estimated value. 

The E values are quantified results, but there is still some level 

of uncertainty associated with the result because of two factors: the PCR duplicates did not agree (indicated with a ~) and 

(or) the PCR value was extrapolated at the low end (past the 

upper Ct limit of the standard curve) (indicated with a b). 

Table 10 lists summary statistics for sample volumes and 

detections for enteric viruses grouped by plant and type of 

wastewater sample. The plants are combined or segregated on 

the basis of operational similarities and differences. Average 

effective sample volumes were considerably less than average original sample volumes. The smallest average effective volume was for post-preliminary samples at plant 2 for 

enterovirus and norovirus GI by qRT-PCR (0.09 mL). The 

largest average effective volume was for post-MBR samples at 

plants 1 and 4 for culturable viruses (30,000 mL). Adenovirus 

was found in 100 percent of post-preliminary samples and 

was detected throughout the treatment processes. Enterovirus 

was detected in the majority of post-preliminary samples and 

throughout the treatment processes except in post-disinfection 

samples from plant 5, the plant with chlorine disinfection. 

Norovirus was detected less often than the other two enteric 

viruses and, in post-disinfection samples, was detected only 

from plant 5. Culturable viruses were detected in post-preliminary samples and 50 percent (2 samples) of post-MBR 

samples from plant 2. Cell culture results were negative in all 

post-secondary, post-tertiary, and post-disinfection samples. 
Table 10. Summary statistics for viruses by quantitative polymerase chain reaction (qPCR) and reverse transcriptase qPCR (qRT-PCR) and cell cultuwastewater samples, 2008–10. [Plants 1 and 4 are membrane bioreactor plants, plant 2 is a membrane bioreactor plant with operational problems, plant 3 is conventional tertiary with ultraviolet disinfection, aventional secondary with chlorine disinfection; mL, milliliters; MBR, membrane bioreactor; sec, secondary; tert, tertiary; dis, disinfection] Plant Type of sample Number of samples Average original sample volume (mL) Average effective sample volumes (mL) Detections, including present but no

(percent) Adenovirus by qPCR Enterovirus and Norovirus GI by qRT-PCR Culturable virus, screening Adenovirus by qPCR Enterovirus by qRT-PCR Norovirus G

by qRT-PCR 

1, 4 Post-preliminary 12 3,900 6.0 1.1 130 100 92 58 

2 5 3,700 .54 .09 120 100 60 40 

3, 5 10 4,000 6.3 1.1 130 100 60 50 1, 4 Post-MBR 15 520,000 2,300 390 30,000 40 20 13 

2 4 450,000 1,500 260 28,000 75 75 25 

3, 5 Post-sec 11 140,000 420 73 7,800 54 9.1 9.1 

3 Post-tert 4 480,000 900 160 28,000 50 25 0 

1, 4 Post-dis 13 510,000 2,300 390 29,000 31 7.7 0 

2 4 420,000 964 170 26,000 25 25 0 3 4 480,000 117 20 28,000 25 25 0 5 4 500,000 2,200 370 28,000 75 0 50 
Summary 29

Summary

The use of membrane bioreactor (MBR) technology 

for wastewater treatment is increasing in the United States. 

Microfiltration membranes used in wastewater treatment 

have a pore size large enough to theoretically reduce concentrations of protozoa and bacteria, but not viruses. There is 

little information, however, on the effectiveness of MBRs in 

removing human enteric viruses from wastewaters, particularly as compared to conventional wastewater treatment before 

and after disinfection. Sampling for viruses in wastewater is 

seldom done and not required. Instead, the bacterial indicators 

Escherichia coli (E. coli) and fecal coliforms are the required 

microbial measures of effluents for wastewater-discharge 

permits in Ohio and elsewhere in the United States. Bacterial 

indicators are used to indicate the removal of bacteria, viruses, 

and protozoan pathogens in wastewater. 

To study the removal of viruses and indicators throughout 

the wastewater-treatment process, 73 regular and 28 qualitycontrol (QC) samples were collected at three MBR wastewater 

plants (plants 1, 2, and 4) and two conventional wastewater 

plants (plants 3 and 5) in Ohio. Four plants were equipped 

with ultraviolet disinfection, and one plant uses chlorine 

disinfection (plant 5). Samples were collected at various stages 

in the treatment processes and analyzed for enteric viruses, 

bacterial indicators, and coliphage. Samples were collected to 

compare the removal of enteric viruses, bacterial indicators, 

and coliphage in MBR and conventional plants and to identify 

whether disinfection provides further reduction of viruses after 

MBR. In order to address the main objectives of the study, it 

was quickly realized that work was needed to identify data 

analysis methods for interpreting enteric virus and QC data. 

Therefore, methods for quantifying viruses, qualifying results, 

and applying QC data to interpretations are described in this 

report; companion reports are planned to address the main 

objectives of the study. 

During each sampling trip, samples were collected 

(1) before conventional or MBR treatment (post-preliminary), 

(2) after secondary or MBR treatment (post-MBR or postsecondary), (3) after tertiary treatment (post-tertiary, plant 3 

only), and (4) after disinfection (post-disinfection). Glasswool fiber filtration was used to concentrate enteric viruses 

from large volumes—4 Liter (L) for post-preliminary samples, 

100 L for post-secondary, and 500 L for all other wastewater 

types (or 3.5 hours, whichever came first). Grab samples were 

collected for direct-plating analyses for bacterial indicators 

by membrane filtration (E. coli, fecal coliforms, enterococci) 

and somatic and F-specific coliphage by the single agar layer 

(SAL) method. 

After filtration, the viruses were eluted from the filter and 

further concentrated. The final concentrated sample volume 

(FCSV) was distributed into different tubes for storage and for 

subsequent enteric virus analysis by use of two methods—cell 

culture and a molecular method, polymerase chain reaction 

(PCR). The cell culture method provides information on the 

infectivity of the viruses but does not identify specific viruses. 

The PCR method for DNA viruses or reverse-transcriptase 

PCR (RT-PCR) for RNA viruses identifies specific viruses but 

does not determine infectivity. Quantitative PCR (qPCR) and 

quantitative RT-PCR (qRT-PCR) provide data on the level of 

specific viruses in a water sample and were used in this study. 

Samples were analyzed for culturable enteric viruses; for 

enterovirus, norovirus GI and GII, rotavirus, and hepatitis A 

virus (RNA viruses) by qRT-PCR; and for adenovirus (a DNA 

virus) by qPCR. 

To support data interpretations, limits of quantification 

and detection were determined for virus results. The methods 

used to define these limits provide data and data qualifiers that 

more accurately report with confidence or lack of confidence 

when viruses are determined to be absent or present. Calculations for molecular assays were based on those for determining assay limits of quantification (ALOQ) and assay limits of 

detection (ALOD) described in Rajal and others (2007), but 

applied specifically for the present study. In the present study, 

the ALOQ for each molecular virus assay was near the lowest 

concentration that was detected in standard curves and was set 

to 1 genomic copy (gc) for consistency; the ALOQ was used as 

the ALOD. For qPCR and qRT-PCR the ALOD was an estimated 

value, because it was not established according to established 

method detection limit procedures; actual sensitivity will 

need to be established in the future. The ALOD for cell-culture 

screening was 1 virus and for cell-culture most probable 

number (MPN) was 1.08 MPN. The ALODs were further used to 

determine sample reporting limits (SRLs). The SRLs were different for each sample because effective sample volumes (the 

volume of the original sample that was actually used in each 

analysis) were different for each sample. Codes were used 

to further qualify the virus data: (1) the ~ indicated that the 

qPCR or qRT-PCR duplicates do not agree, (2) the b indicated 

that the value was extrapolated at the low end, and (3) the M 

indicated that material was present but not quantified. 

Also to support data interpretations, considerable efforts 

were expended to collect and interpret field QC samples for 

viruses, bacterial indicators, and coliphage. These were in 

addition to the usual laboratory QC samples associated with 

each analytical method. Field QC samples for bacteria and 

coliphage included 3 field blanks and 10 concurrent replicates. 

Method test samples for coliphage were also analyzed. These 

included 25 filtration test samples, analyzed to provide data 

on recoveries of coliphage by use of glass-wool filters, and 

10 EasyPhage test samples, included to test an alternative to 

the standard SAL method. Quality-control samples for viruses 

included an equipment blank, three field blanks, 13 concurrent 

replicates, and 9 seeded matrix controls. The blanks for bacteria, coliphage, and enteric viruses were all below detection, 

indicating that it was unlikely that samples were contaminated 

from equipment or processing procedures. 

Quality-control samples for bacterial indicators and 

coliphage provided insights into measurement variability 

and method differences. The absolute value log differences 

(AVLDs) between concurrent replicate pairs were calculated 

to identify the variability associated with each measurement. 
30 Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control

The AVLD results indicated that for samples with concentrations <10 CFU or PFU/100 mL, log removals less than or 

equal to approximately 1 may not be greater than the analytical variability. For samples >10 CFU or PFU/100 mL, log 

removals less than or equal to about 0.3 may not be greater 

than the analytical variability. Filtration test samples were 

used to compare concentrations of coliphage found by use of 

glass-wool filtration to concentrations found by use of direct 

plating without filtration. Concentrations of coliphage by 

filtration averaged approximately 6 percent of direct plating concentrations. The results from direct plating were used 

for reporting coliphage results in this report. EasyPhage test 

samples were compared to the direct plating SAL method. 

Although results from the two methods were highly correlated, 

concentrations of F-specific coliphage were higher with EasyPhage and concentrations of somatic coliphage were higher 

(for lower concentrations) with the direct plating SAL method. 

These results indicate that either the SAL or EasyPhage can 

be used to determine relative concentrations of coliphage in 

wastewater studies, if one method is chosen over the other for 

consistency. However, if actual concentrations of F-specific 

coliphage are needed, EasyPhage may be the preferred method 

because it yields significantly higher concentrations than the 

SAL method. 

Quality-control samples for viruses were included to 

provide information on measurement variability and recovery 

efficiencies. Seeded matrix controls were collected to determine recovery of viruses through all processing and analytical 

steps. Unfortunately, the virus seed concentration was lower 

than expected, and viruses from the seed were not recovered 

in the FCSVs. Plans are underway to collect and analyze a 

small set of seeded matrix controls and include these results in 

companion reports. Overall, concentrations of viruses among 

concurrent replicate pairs were similar in magnitude with 

a few exceptions. Relatively large differences in molecular 

results for viruses between replicate pairs were likely due to 

lack of precision for samples with small effective volumes. 

These results indicate that, when interpreting removals of 

viruses in wastewater samples, removals less than or equal to 

about 1.2 log may not be greater than the analytical variability. 

Concentrations of E. coli, fecal coliform, enterococci, 

and somatic and F-specific coliphage were listed by sample 

and shown in plots. The plots included data by plant for MBR 

and conventional plants and by type of wastewater sample. 

Bacterial indicator and coliphage concentrations in post-MBR 

samples were highest at plant 2, which had operational issues 

and effluent contamination of the UV system. In post-MBR 

and post-secondary samples, concentrations of somatic 

coliphage were higher than F-specific coliphage. This may be 

because F-specific coliphage tend to absorb to solid surfaces 

more than somatic coliphage, resulting in higher removals of 

F-specific coliphage. In post-disinfection samples for the two 

MBR plants that were operating properly (plants 1 and 4) and 

the UV conventional plant (plant 3), concentrations for all bacterial indicators and coliphage were near or below detection. 

For the chlorine conventional plant (plant 5), they were in the 

single or double digits and for the MBR plant with operational 

issues (plant 2), concentrations in post-disinfection samples 

were as high as 1,000 PFU or CFU/100 mL. All of the plants 

met the NPDES required effluent limits on the days sampled. 

Concentrations of adenovirus, enterovirus, norovirus GI, 

and culturable viruses and ancillary data used to determine 

those concentrations were listed by sample and detections 

were presented as summary statistics. Norovirus GII and hepatitis A virus were not detected in any samples, and rotavirus 

was present but not quantified in only one sample. Effective 

sample volumes (actual amount of sample that was analyzed) 

were much less than the original filtered sample volumes. 

These reductions were due to processing steps and (or) any 

dilutions made to minimize the effects from PCR-inhibiting 

substances. 

Adenovirus and enterovirus were found in 100 percent 

and the majority of post-preliminary samples, respectively, and 

were detected throughout the treatment processes. Norovirus 

GI was detected less often than the other two enteric viruses. 

Culturable viruses were detected in post-preliminary samples 

and in only two post-MBR samples from plant 2. Cell culture 

results were negative in all post-secondary, post-tertiary, and 

post-disinfection samples. 

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Table 3. Water-quality, bacterial indicator, and coliphage results from wastewater samples, 2008–10.—Continued [μS/cm, microsiemens per centimeter; NTRU, nephelometric turbidity reference units; E. coli, Escherichia coli; CFU/mL, colony-forming units per 100 milliliters; PFU/100 mLing units per 100 milliliters; prelim, preliminary; K, based on non-ideal colony count; MBR, membrane bioreactor; dis, disinfection; <, less than; sec, secondary; tert, tertiary; Areplicate values; >, greater than; --, not determined] Sample number Plant Type of sample pH Specific conductance (μS/cm) Turbidity (NTRU) E. coli (CFU/100 mL) Fecal coliforms (CFU/100 mL) Enterococci (CFU/100 mL) Coliphage, male-specific (PFU/100 mL) 

1 1 Post-prelim 6.6 1,268 200 5,900,000 K 16,000,000 K 36,000,000 250,000 1 1 Post-MBR 7.5 993 .39 8 K 62 K 160 2 1 1 Post-dis 7.5 997 .27 <1 <1 K 1 <1 2 2 Post-prelim 7.6 1,500 130 3,000,000 K 7,500,000 1,900,000 420,000 2 2 Post-MBR 7.7 3,300 2.8 <1 K 1 <1 4 2 2 Post-dis 7.7 3,300 1.8 K 1 K 1 <1 <1 3 3 Post-prelim 7.7 853 210 7,800,000 7,700,000 1,400,000 120,000 3 3 Post-sec 7.6 742 5.8 24,000 57,000 K 8,700 7 3 3 Post-ter 7.6 749 1.9 20,000 20,000 5,200 7 3 3 Post-dis 7.7 744 1.8 K 1 K 1 <1 <1 4 2 Post-prelim 7.5 1,503 130 2,500,000 3,500,000 970,000 66,000 4 2 Post-MBR 7.8 1,350 1.4 7 21 K 2 <1 4 2 Post-dis 7.8 1,345 1.8 17 29 12 <1 5 1 Post-prelim 6.2 1,417 180 5,400,000 K 120,000,000 31,000,000 1,300,000 5 1 Post-MBR 7.6 1,280 .18 A,K 2 A 7 A,K 1 A 2 5 1 Post-dis 7.8 1,277 .34 <1 <1 K 2 <1 6 1 Post-prelim 6.4 1,120 85 K 600,000 K 1,100,000 K 3,300,000 38,000 6 1 Post-MBR 7.7 1,021 .31 K 2 K 3 K 3 <1 6 1 Post-dis 7.6 1,014 .22 <1 <1 <1 <1 7 2 Post-prelim 7.8 1,182 140 6,700,000 7,500,000 3,000,000 190,000 7 2 Post-MBR 7.6 1,744 2.2 K 140 140 A,K 330 A 2 7 2 Post-dis 7.6 1,752 2.1 >32 >80 >24 4 8 1 Post-prelim 7.3 -- 92 700,000 2,200,000 400,000 1,000,000 8 1 Post-MBR 8.2 952 .50 K 3 10 K 6 <1 8 1 Post-dis 7.8 978 .50 A <1 A <1 A <1 A <1 9 3 Post-prelim 8.1 860 130 A 1,000,000 A 790,000 A 400,000 A 11,000 9 3 Post-sec 7.0 845 .85 3,400 2,800 K 1,300 3 9 3 Post-ter 7.1 856 2.8 K 1,200 2,200 800 5 9 3 Post-dis 7.1 805 .70 K 1 K 1 K 1 <1 
Table 3. Water-quality, bacterial indicator, and coliphage results from wastewater samples, 2008–10.—Continued[μS/cm, microsiemens per centimeter; NTRU, nephelometric turbidity reference units; E. coli, Escherichia coli; CFU/mL, colony-forming units per 100 milliliters; PFU/100 mLing units per 100 milliliters; prelim, preliminary; K, based on non-ideal colony count; MBR, membrane bioreactor; dis, disinfection; <, less than; sec, secondary; tert, tertiary; Areplicate values; >, greater than; --, not determined]

Sample 

number Plant

Type of 

sample pH

Specific 

conductance 

(μS/cm)

Turbidity 

(NTRU)

E. coli

(CFU/100 mL)

Fecal 

coliforms 

(CFU/100 mL)

Enterococci 

(CFU/100 mL)

Coliphage, 

male-specific 

(PFU/100 mL)

10 2 Post-prelim 7.4 -- 120 A 1,300,000 A 2,900,000 A 7,900,000 A 120,000 A 10 2 Post-MBR 7.8 -- 4.5 600 K 1,500 K 1,000 14 10 2 Post-dis 7.9 -- 6.0 K 850 K 1,000 K 1,400 19 

11 1 Post-prelim 6.8 1,155 110 1,200,000 4,500,000 3,500,000 1,900,000 11 1 Post-MBR 7.5 1,080 .24 K 1 K 2 K 4 14 11 1 Post-dis 7.6 1,083 .54 <1 <1 <1 <1 

12 3 Post-prelim 7.7 1,000 190 6,000,000 4,600,000 3,300,000 38,000 12 3 Post-sec 7.9 780 3.8 K 860 2,500 1,000 31 12 3 Post-ter 8.1 790 1.2 700 1,200 K 350 7 12 3 Post-dis 7.9 780 .84 K 2 K 1 K 1 <1 13 1 Post-prelim 7.0 940 510 700,000 2,000,000 K 640,000 A 120,000 13 1 Post-MBR 7.3 870 .48 K 2 K 5 K 4 <1 13 1 Post-dis 7.3 860 .24 <1 <1 K 1 <1 14 5 Post-prelim 7.4 1,403 140 2,400,000 2,800,000 K 650,000 310,000 14 5 Post-sec 7.0 1,377 .68 1,200 2,200 210 4 14 5 Post-dis 7.5 1,385 .68 K 2 K 6 22 <1 15 4 Post-prelim 7.6 1,762 110 4,300,000 5,600,000 2,100,000 250,000 15 4 Post-MBR 7.2 -- .20 <1 <1 <1 <1 15 4 Post-dis 7.2 -- .20 <1 <1 <1 2 16 5 Post-prelim 7.2 1,249 1.2 4,200,000 K 5,200,000 500,000 440,000 16 5 Post-sec 7.1 1,206 2.4 A 7,000 A 11,000 A 1,000 A 12 16 5 Post-dis 7.6 1,266 .89 K 5 K 6 K 60 2 17 4 Post-prelim 7.6 2,210 150 1,300,000 7,300,000 4,100,000 190,000 17 4 Post-MBR 7.3 1,865 .10 <1 <1 <1 <1 17 4 Post-dis 7.2 1,876 .16 <1 <1 <1 1 18 3 Post-prelim 7.4 860 110 2,500,000 2,000,000 900,000 210,000 18 3 Post-sec 6.5 942 10 A 13,000 A 22,000 A 4,500 A 52 18 3 Post-ter 6.6 950 1.5 11,000 14,000 2,000 20 18 3 Post-dis 6.6 944 1.4 K 1 K 1 K 1 <1 
Table 3. Water-quality, bacterial indicator, and coliphage results from wastewater samples, 2008–10.—Continued [μS/cm, microsiemens per centimeter; NTRU, nephelometric turbidity reference units; E. coli, Escherichia coli; CFU/mL, colony-forming units per 100 milliliters; PFU/100 mLing units per 100 milliliters; prelim, preliminary; K, based on non-ideal colony count; MBR, membrane bioreactor; dis, disinfection; <, less than; sec, secondary; tert, tertiary; Areplicate values; >, greater than; --, not determined] Sample number Plant Type of sample pH Specific conductance (μS/cm) Turbidity (NTRU) E. coli (CFU/100 mL) Fecal coliforms (CFU/100 mL) Enterococci (CFU/100 mL) Coliphage, male-specific (PFU/100 mL) 

19 4 Post-prelim 7.4 2,460 170 7,000,000 7,000,000 K 2,100,000 270,000 19 4 Post-MBR 7.2 1,778 .25 A <1 A <1 A <1 A 4 19 4 Post-dis 7.2 1,758 .22 <1 <1 <1 <1 20 5 Post-prelim 7.1 1,266 160 6,000,000 8,000,000 600,000 460,000 20 5 Post-sec 7.0 1,450 .66 2,000 3,400 K 220 33 20 5 Post-dis 7.6 1,460 .69 10 K 26 16 <1 21 4 Post-prelim 7.6 2,108 150 5,400,000 6,700,000 1,800,000 380,000 21 4 Post-MBR 7.2 1,802 .20 <1 K 1 K 1 <1 21 4 Post-dis 7.1 1,788 .25 A <1 A <1 A 1 A <1 22 5 Post-prelim 7.7 1,440 150 2,900,000 5,300,000 930,000 22,000,000 22 5 Post-sec 6.9 1,415 1.1 1,400 2,300 150 120 22 5 Post-dis 7.8 1,446 .98 K 6 18 K 4 37 23 4 Post-prelim 7.6 2,048 160 4,100,000 5,400,000 K 2,000,000 520,000 23 4 Post-MBR 7.2 1,792 .16 <1 <1 <1 5 23 4 Post-dis 7.2 1,796 .12 <1 <1 K 1 1 

Table 4. Virus concentrations in wastewater samples, 2008–10.—Continued [FCSV, final concentrated sample volume; mL, milliliter; MPN, most probable number; gc/L, genomic copies per liter; L, liter; prelim, preliminary; <, less than; MBR, membraM, material present but not quantified; E, estimated value; ~, PCR duplicates do not agree (Ct values); b, value was extrapolated at the low end (past the upper Ct limit of the sta

A B C D E F G H I J K L 

Effective FCSV Quantitative polymerase chain reaction (qPCR) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) 

0.0336 mL in 2008 0.0258 mL in 2009–10 0.00578 mL in 2008 0.00445 mL in 2009–10 0.33 mL 

Sample number Plant Type of sample Original sample volume (mL) FCSV, total volume (mL) Dilution factor Effective sample volume (mL) Adeno- viruses by qPCR (gc/L) Effective sample volume (mL) Entero- viruses by qRT-PCR (gc/L) Norovirus GI by qRT-PCR (gc/L) Effectiv

sampl

volume

screeni

(mL) 

1 1 Post-prelim 4,000 10 1 13 540 2.3 1,700 <440 132 1 1 Post-MBR 565,000 5 1 3,797 2.0 653 <1.5 <1.5 37,290 1 1 Post-dis 579,000 5 1 3,891 1.6 669 <1.5 <1.5 38,214 2 2 Post-prelim 4,000 10 20 0.67 7,000 0.12 16,000 <8,300 132 2 2 Post-MBR 549,400 5 5 738 <1.4 127 M <7.9 <7.9 36,260 2 2 Post-dis 514,000 5 1 3,454 <0.29 594 <1.7 <1.7 33,924 3 3 Post-prelim 4,000 10 20 0.67 1,700 0.12 E ~b 7,900 <8,300 132 3 3 Post-sec 126,000 5 1 847 <1.2 146 M <6.8 <6.9 8,316 3 3 Post-tert 491,400 5 1 3,302 <0.30 568 E ~b 0.24 <1.8 32,432 3 3 Post-dis 485,100 5 20 162 <6.2 28 M <36 <36 32,017 4 2 Post-prelim 4,000 10 20 0.67 8,300 0.12 E b 2,600 E ~b 2,600 132 4 2 Post-MBR 499,220 5 1 3,355 E ~1.0 577 M <1.7 <1.7 32,949 4 2 Post-dis 536,520 5 20 180 <5.6 31 M <32 <32 35,410 5 1 Post-prelim 4,000 10 1 13 220 2.3 E ~b 240 E ~b 49 132 5 1 Post-MBR 564,300 5 20 190 <5.3 33 <30 <30 37,244 5 1 Post-MBR replicate 579,700 5 1 3,896 E ~0.32 663 M <1.5 <1.5 37,851 5 1 Post-dis 573,500 5 1 3,854 E ~0.75 670 <1.5 <1.5 38,260 6 1 Post-prelim 4,000 10 5 2.1 4,600 0.36 <2,800 4,200 132 6 1 Post-MBR 446,250 6 1 1,919 19 331 <3.0 E~b 2.0 24,544 6 1 Post-dis 483,000 6 1 2,077 <0.48 358 <2.8 <2.8 26,565 7 2 Post-prelim 3,850 10 20 0.50 1,200,000 0.09 <11,000 E~ 9,200 127 7 2 Post-MBR 459,000 6 1 1,974 E~ 13 340 <2.9 E~ 2.3 25,245 7 2 Post-dis 406,000 6 10 175 <5.7 30 <33 <33 22,330 
Table 4. Virus concentrations in wastewater samples, 2008–10.—Continued[FCSV, final concentrated sample volume; mL, milliliter; MPN, most probable number; gc/L, genomic copies per liter; L, liter; prelim, preliminary; <, less than; MBR, membraM, material present but not quantified; E, estimated value; ~, PCR duplicates do not agree (Ct values); b, value was extrapolated at the low end (past the upper Ct limit of the sta

A B C D E F G H I J K L 

Effective FCSV

Quantitative 

polymerase chain 

reaction (qPCR)

Quantitative reverse transcriptase 

polymerase chain reaction 

(qRT-PCR) 

0.0336 mL in 2008 

0.0258 mL in 2009–10

0.00578 mL in 2008

0.00445 mL in 2009–10

0.33 mL 

Sample 

number Plant

Type of 

sample

Original 

sample volume 

(mL)

FCSV, 

total 

volume 

(mL)

Dilution 

factor 

Effective 

sample volume 

(mL)

Adenoviruses 

by qPCR 

(gc/L)

Effective 

sample volume 

(mL)

Enteroviruses 

by 

qRT-PCR 

(gc/L)

Norovirus GI 

by 

qRT-PCR 

(gc/L)

Effectiv

sampl

volume

screeni

(mL)

8 1 Post-prelim 4,000 10 20 0.52 180,000 0.09 E ~55,000 <11,000 132 8 1 Post-MBR 529,000 6 1 2,275 E~ 6.0 392 <2.6 <2.6 29,095 8 1 Post-dis 516,000 6 1 2,219 <0.45 383 <2.6 <2.6 28,380 8 1 Post-dis 

replicate

543,000 6 1 2,335 <0.43 403 <2.5 <2.5 29,865 

9 3 Post-prelim 4,000 10 10 1.0 2,100 0.18 <5,600 <5,600 132 9 3 Post-prelim 

replicate

4,000 10 10 1.0 3,000 0.18 <5,600 <5,600 132 

9 3 Post-sec 143,000 6 20 31 E~ 120 5.3 <190 <190 7,865 9 3 Post-tert 504,000 6 20 108 E~b 6.3 19 <53 <53 27,720 9 3 Post-dis 502,000 6 20 108 <9.3 19 <53 <53 27,610 

10 2 Post-prelim 4,000 10 20 0.52 120,000 0.09 <11,000 <11,000 132 10 2 Post-prelim 

replicate 

a2,700 10 20 0.35 430,000 0.06 E ~47,000 <17,000 89 

10 2 Post-MBR 302,000 6 20 65 1,300 11 400 <91 16,610 10 2 Post-dis 214,000 6 20 46 Eb 12 7.9 <130 <130 11,770 

11 1 Post-prelim 3,000 10 20 0.39 33,000 0.07 M <14,000 <14,000 99 11 1 Post-MBR 500,890 6 1 2,154 <0.46 371 <2.7 <2.7 27,549 11 1 Post-dis 501,000 6 1 2,154 <0.46 372 <2.7 <2.7 27,555 

12 3 Post-prelim 4,000 10 20 0.52 17,000 0.09 E ~b 5,700 E~b 6,700 132 12 3 Post-sec 107,000 6 20 23 <43 4.0 <250 <250 5,885 12 3 Post-tert 436,000 6 20 94 <11 16 <63 <63 23,980 12 3 Post-dis 505,000 6 20 109 <9.2 19 <53 <53 27,775 
Table 4. Virus concentrations in wastewater samples, 2008–10.—Continued [FCSV, final concentrated sample volume; mL, milliliter; MPN, most probable number; gc/L, genomic copies per liter; L, liter; prelim, preliminary; <, less than; MBR, membraM, material present but not quantified; E, estimated value; ~, PCR duplicates do not agree (Ct values); b, value was extrapolated at the low end (past the upper Ct limit of the sta

A B C D E F G H I J K L 

Effective FCSV Quantitative polymerase chain reaction (qPCR) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) 

0.0336 mL in 2008 0.0258 mL in 2009–10 0.00578 mL in 2008 0.00445 mL in 2009–10 0.33 mL 

Sample number Plant Type of sample Original sample volume (mL) FCSV, total volume (mL) Dilution factor Effective sample volume (mL) Adeno- viruses by qPCR (gc/L) Effective sample volume (mL) Entero- viruses by qRT-PCR (gc/L) Norovirus GI by qRT-PCR (gc/L) Effectiv

sampl

volume

screeni

(mL) 

13 1 Post-prelim 4,000 10 15 0.69 46,000 0.12 280,000 <8,300 132 13 1 Post-prelim replicate 4,000 10 15 0.69 32,000 0.12 300,000 <8,300 132 13 1 Post-MBR 502,000 6 1 2,159 2.0 372 7.5 <2.7 27,610 13 1 Post-MBR replicate 518,000 6 1 2,227 2.3 384 3.1 <2.6 28,490 13 1 Post-dis 458,000 6 1 1,969 1.7 340 M <2.9 <2.9 25,190 14 5 Post-prelim 4,000 10 1 10 1,500 1.8 <550 <550 132 14 5 Post-sec 153,000 6 1 658 E~ 3.6 113 <8.8 <8.8 8,415 14 5 Post-dis 502,000 6 1 2,159 <0.46 372 <2.7 <2.7 27,610 15 4 Post-prelim 4,000 10 1 10 2,200 1.8 950 E~ 390 132 15 4 Post-MBR 500,000 6 1 2,150 <0.47 371 <2.7 <2.7 27,500 15 4 Post-dis 501,000 6 1 2,154 <0.46 372 <2.7 <2.7 27,555 16 5 Post-prelim 4,000 10 1 10 7,800 1.8 E ~ 470 M <560 132 16 5 Post-sec 143,000 6 1 615 E~ 10 106 <9.4 <9.4 7,865 16 5 Post-sec replicate 145,600 6 1 626 E~ 2.8 108 <9.3 <9.3 8,008 16 5 Post-dis 502,000 6 1 2,159 17 372 <2.7 <2.7 27,610 17 4 Post-prelim 4,000 10 1 10 1,200 1.8 2,300 2,600 132 17 4 Post-MBR 540,000 6 1 2,322 <0.43 400 <2.5 <2.5 29,700 17 4 Post-dis 501,000 6 20 108 <9.3 19 <53 <53 27,555 
Table 4. Virus concentrations in wastewater samples, 2008–10.—Continued[FCSV, final concentrated sample volume; mL, milliliter; MPN, most probable number; gc/L, genomic copies per liter; L, liter; prelim, preliminary; <, less than; MBR, membraM, material present but not quantified; E, estimated value; ~, PCR duplicates do not agree (Ct values); b, value was extrapolated at the low end (past the upper Ct limit of the sta

A B C D E F G H I J K L 

Effective FCSV

Quantitative 

polymerase chain 

reaction (qPCR)

Quantitative reverse transcriptase 

polymerase chain reaction 

(qRT-PCR) 

0.0336 mL in 2008 

0.0258 mL in 2009–10

0.00578 mL in 2008

0.00445 mL in 2009–10

0.33 mL 

Sample 

number Plant

Type of 

sample

Original 

sample volume 

(mL)

FCSV, 

total 

volume 

(mL)

Dilution 

factor 

Effective 

sample volume 

(mL)

Adenoviruses 

by qPCR 

(gc/L)

Effective 

sample volume 

(mL)

Enteroviruses 

by 

qRT-PCR 

(gc/L)

Norovirus GI 

by 

qRT-PCR 

(gc/L)

Effectiv

sampl

volume

screeni

(mL)

18 3 Post-prelim 4,000 10 1 10 560 1.8 2,200 <560 132 18 3 Post-sec 140,000 6 20 30 E~ 68 5.2 <190 <190 7,700 18 3 Post-sec 

replicate

141,500 6 20 30 <33 5.2 <190 <190 7,783 

18 3 Post-tert 502,000 6 20 108 E~ 17 19 <53 <53 27,610 18 3 Post-dis 412,000 6 20 89 E~ 39 15 <67 <67 22,660 19 4 Post-prelim 3,480 10 20 0.45 E~ 21,000 0.08 27,000 18,000 115 19 4 Post-MBR 520,000 6 1 2,236 <0.45 386 <2.6 <2.6 28,600 19 4 Post-MBR 

replicate

500,000 6 1 2,150 <0.47 371 <2.7 E~ 17 27,500 

19 4 Post-dis 502,000 6 1 2,159 <0.46 372 <2.7 <2.7 27,610 20 5 Post-prelim 4,000 10 1 10 1,900 1.8 M <560 2,200 132 20 5 Post-prelim 

replicate

4,000 10 1 10 300 1.8 M <560 790 132 

20 5 Post-sec 140,400 6 1 604 M <1.7 104 <9.6 E ~10 7,722 20 5 Post-dis 500,000 6 1 2,150 E ~b 0.2 371 <2.7 E~b 1.8 27,500 21 4 Post-prelim 4,000 10 1 10 2,300 1.8 18,000 E~ 4,000 132 21 4 Post-MBR 501,700 6 1 2,157 <0.46 372 <2.7 <2.7 27,594 21 4 Post-dis 501,700 6 1 2,157 M <0.46 372 <2.7 <2.7 27,594 21 4 Post-dis 

replicate

501,700 6 1 2,157 <0.46 372 <2.7 <2.7 27,594 
Table 4. Virus concentrations in wastewater samples, 2008–10.—Continued [FCSV, final concentrated sample volume; mL, milliliter; MPN, most probable number; gc/L, genomic copies per liter; L, liter; prelim, preliminary; <, less than; MBR, membraM, material present but not quantified; E, estimated value; ~, PCR duplicates do not agree (Ct values); b, value was extrapolated at the low end (past the upper Ct limit of the sta

A B C D E F G H I J K L 

Effective FCSV Quantitative polymerase chain reaction (qPCR) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) 

0.0336 mL in 2008 0.0258 mL in 2009–10 0.00578 mL in 2008 0.00445 mL in 2009–10 0.33 mL 

Sample number Plant Type of sample Original sample volume (mL) FCSV, total volume (mL) Dilution factor Effective sample volume (mL) Adeno- viruses by qPCR (gc/L) Effective sample volume (mL) Entero- viruses by qRT-PCR (gc/L) Norovirus GI by qRT-PCR (gc/L) Effectiv

sampl

volume

screeni

(mL) 

22 5 Post-prelim 4,000 10 1 10 670 1.8 <560 E~b 230 132 22 5 Post-sec 140,400 6 1 604 <1.7 104 <9.6 <9.6 7,722 22 5 Post-sec replicate 142,000 6 1 611 E~ 16 105 <9.5 <9.5 7,810 22 5 Post-dis 501,500 6 1 2,156 E~ 3 372 <2.7 M <2.7 27,583 23 4 Post-prelim 4,000 10 1 10 2,200 1.8 6,800 E ~ 2,200 132 23 4 Post-MBR 500,000 6 1 2,150 <0.47 371 <2.7 <2.7 27,500 23 4 Post-MBR replicate 501,700 6 1 2,157 <0.46 372 <2.7 <2.7 27,594 23 4 Post-dis 502,000 6 1 2,159 <0.46 372 <2.7 <2.7 27,610 

a Only 2,700 mL was filtered because the filter clogged and was no longer able to operate.
Glossary 43

Glossary

The terms included in this section were specifically defined for use in this report.

Absolute value log difference (AVLD) The AVLD was 

determined by calculating the absolute value of the difference 

between concentration results for two replicate wastewater 

samples that were log10 transformed. 

Assay limit of detection (ALOD) The ALOD is the lowest 

concentration of virus that can reasonably be detected with 

some certainty. For the present study, the ALOD was based 

on the ALOQ and set at 1 genomic copy (gc) for molecular 

assays, 1 virus for cell-culture screening analysis, and 1.08 

most probable number (MPN) for cell culture MPN analysis.

Assay limit of quantification (ALOQ) The ALOQ is the 

lowest concentration of virus that can reasonably be quantified with some certainty. For the present study, the ALOQ 

was set at 1 genomic copy (gc) for molecular assays (based 

on extrapolating standard curves), 1 virus for the cell-culture 

screening assay (equivalent to one cytopathic effect), and 2.18 

most probable number (MPN) for the cell-culture MPN assay 

(calculated from MPN tables). 

Cell culture A technique used to detect infectious enteric 

viruses in water samples. Mammalian cells are inoculated with 

sample and then observed for cytological changes that indicate 

virus growth. 

Crossing threshold (Ct) The point at which a fluorescent 

signal crosses an established threshold during qPCR.

Cytopathic effect (CPE) Cell disintegration or changes in cell 

morphology that result in a positive result in the cell culture 

method for enteric viruses.

Deoxyribose nucleic acid (DNA) Molecule that contains the 

genetic instructions for most living organisms. In this report, 

DNA is the genetic material for adenovirus. 

Effective sample volume (Veffective) The actual amount of 

original sample volume that was used for each virus assay. 

Effective sample volumes were a small proportion of the 

original sample volumes and were limited by processing steps 

for each assay.

Equipment blank A blank solution used to determine 

potential contamination from the cleaning and sterilization 

processes. Equipment blanks are processed in a controlled 

environment, such as in the laboratory. 

Field blank A blank solution used to determine potential 

contamination during all steps of sample collection, transport, 

and processing. Field blanks are similar to equipment blanks 

except they are processed under actual field conditions and 

transported to the lab along with environmental samples. 

Final concentrated sample volume (FCSV) The resultant 

volume of sample analyzed for viruses after filtration, elution, 

and concentration. 

Field concurrent replicates Environmental samples collected and analyzed in a manner such that the samples are 

thought to be virtually identical in composition. Concurrent 

replicates are collected to determine sampling and (or) analytical variability.

Laboratory blank A blank solution used to determine potential contamination from equipment and processing procedures 

in the laboratory.

Membrane bioreactor (MBR) A wastewater-treatment 

technology in which secondary treatment is accomplished by 

submerging membranes in the conventional activated sludge 

tank to physically separate the solids from the liquid and filter 

the wastewater through the membrane. 

Method test samples Replicate samples analyzed by use of 

two or more different processing or analytical methods. 

Most probable number (MPN) A method for obtaining quantitative data from presence/absence results. Statistical MPN 

tables were used to determine MPNs of viruses in cell culture. 

Post-disinfection Samples of final effluents collected after 

ultraviolet disinfection or after chlorination and dechlorination, but before being discharged to the receiving stream.

Post-MBR Samples at membrane bioreactor (MBR) plants 

collected after the water is filtered through the membrane 

filtration equipment, but before disinfection.

Post-preliminary Samples after preliminary treatment, which 

may include treatment processes such as influent pumping, 

screening, primary treatment, grit and grease removal, and (or) 

fine screening. 

Post-secondary Samples at the conventional plants that were 

collected after the gravity separation accomplished by the final 

clarifiers. 
44 Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control

Post-tertiary Samples of the effluent of tertiary treatment. 

For this study, only one plant used tertiary treatment (in this 

case, sand filtration). 

Quantitative polymerase chain reaction (qPCR) A technique 

used to amplify DNA many orders of magnitude so that it can 

be detected and quantified. This method was used to detect 

adenovirus in wastewater samples.

Quantitative reverse transcriptase polymerase chain reaction 

(qRT-PCR) A technique used to convert RNA to DNA and 

then magnify DNA many orders of magnitude so that it can 

be detected and quantified. This method was used to detect 

enterovirus, norovirus, rotavirus, and hepatitis A virus in 

wastewater samples.

Ribose nucleic acid (RNA) Molecule that contains the 

genetic instructions for some viruses, including the enteroviruses and noroviruses included in this study. 

Sample reporting limit (SRL) The SRLs are the “less-than 

values” or the limits of detection for each sample and assay. 

For viruses, the SRLs were sample- and assay-specific because 

original sample volumes and dilutions were different for each 

sample and processing steps for different for each assay. 

Seeded matrix controls Wastewater samples seeded with 

target organisms before processing or analysis. Seeded matrix 

controls are collected to determine recoveries of organisms 

through processing and analytical steps.

Single agar layer method (SAL) A method used to enumerate somatic and F-specific coliphage in water by adding the 

sample to a bacterial culture with molten agar and observing 

plaque formation after 24 hours of incubation. A plaque is a 

clearing in the lawn of cells where coliphages are present.

Francy and others—Quantifying Viruses and Bacteria in Wastewater—Results, Interpretation Methods, and Quality Control—Scientific Investigations Report 2011–5150

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