U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2009–5222

In cooperation with the Northeast Ohio Regional Sewer District, 

Cuyahoga County Board of Health, Cuyahoga County Sanitary Engineer, 

and Ohio Water Development Authority

Comparison of the Immunomagnetic Separation/

Adenosine Triphosphate Rapid Method and the 

Modified mTEC Membrane-Filtration Method for 

Enumeration of Escherichia coli

Comparison of the Immunomagnetic Separation/

Adenosine Triphosphate Rapid Method and the 

Modified mTEC Membrane-Filtration Method for 

Enumeration of Escherichia coli

By Amie M.G. Brady, Rebecca N. Bushon, and Erin E. Bertke

In cooperation with the Northeast Ohio Regional Sewer District, 

Cuyahoga County Board of Health, Cuyahoga County Sanitary Engineer, 

and Ohio Water Development Authority

Scientific Investigations Report 2009–5222

U.S. Department of the Interior

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

KEN SALAZAR, Secretary

U.S. Geological Survey

Suzette M. Kimball, Acting Director

U.S. Geological Survey, Reston, Virginia: 2009

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Suggested citation:

Brady, A.M.G., Bushon, R.N., and Bertke, E.E., 2009, Comparison of the immunomagnetic separation/adenosine triphosphate rapid method and the modified mTEC membrane-filtration method for enumeration of Escherichia coli: U.S. 

Geolgogical Survey Scientific Investigations Report 2009–5222, 21 p.
iii

Acknowledgments

The authors thank the following individuals for their technical assistance throughout 

the study discussed in this report:

Northeast Ohio Regional Sewer District—Mark Citriglia, Eva Hatvani, Lester Stumpe, 

Ben Tedrick, Cathy Perciado, Eric Soehnlen, and Steve Lizewski.

Cuyahoga County Board of Health—Jill Lis.

Cuyahoga County Sanitary Engineer—Suzanne Britt, Ann McCready-Gliha, and 

Patricia Boone.

v

Contents

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

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

Purpose and Scope .......................................................................................................................................2

Study Area.......................................................................................................................................................2

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

Sampling Frequencies and Locations ...............................................................................................4

Sample-Collection Methods ...............................................................................................................4

Laboratory Methods.............................................................................................................................4

Culture-Based Method ...............................................................................................................4

Immunomagnetic Separation/Adenosine Triphosphate Rapid Method .............................4

Ancillary Data Collection.....................................................................................................................5

Quality Assurance/Quality Control.....................................................................................................5

Data Analysis and Statistics ..............................................................................................................5

Results .............................................................................................................................................................6

Comparison of Composite Sampling to Average of Multiple-Point Samples..............................6

General Patterns in Escherichia coli, Turbidity, and Rainfall ........................................................8

Relations Between Environmental or Water-Quality Factors and 

E. coli Concentrations...........................................................................................................10

Relations Between Culture-Based and IMS/ATP Methods.........................................................10

Sample Processing Versus Direct Addition of Sample .......................................................10

Evaluation of 2006 and 2007 Data............................................................................................11

Effects of Water-Quality and Environmental Factors ..........................................................15

Future Steps..................................................................................................................................................18

Summary and Conclusions.........................................................................................................................18

References Cited..........................................................................................................................................20

Figures

 1. Map showing study area and beach locations, northeastern Ohio.....................................3

2–7. Graphs showing:

2. Scatterplot of Escherichia coli concentrations from composite samples 

and the arithmetic average of multiple-point samples at three Lake Erie 

beaches, May through September 2006 and May through August 2007....................7

3. Relation between the culture-based Escherichia coli concentrations and 

IMS/ATP luminescence at Edgewater, Cleveland, Ohio, during May 

through September 2006 and June through August 2007 ...........................................11

4. Relation between the culture-based Escherichia coli concentrations and 

IMS/ATP luminescence at Villa Angela, Cleveland, Ohio, during May 

through September 2006 and June through August 2007 ...........................................12

5. Relation between the culture-based Escherichia coli concentrations and 

IMS/ATP luminescence at Huntington Reservation, Bay Village, Ohio, 

during May through September 2006 and June through August 2007......................13
vi

Figures—Continued

6. Number of birds at Edgewater Beach at time of sampling as a function 

of month sampled ..............................................................................................................16

7. Number of birds at Villa Angela Beach at time of sampling as a function 

of month sampled ..............................................................................................................17

Tables

 1. Summary statistics for concentrations of Escherichia coli as determined by 

the culture-based method in samples collected from three northeasternOhio beaches, May through September 2006 and May through August 2007 ...................8

 3. Summary statistics for rainfall at two Cleveland, Ohio, airports on days 

samples were collected ..............................................................................................................9

 2 Summary statistics for turbidity measurements from samples collected at 

three northeastern-Ohio beaches, May through September 2006 and 

May through August 2007............................................................................................................9

 4. Summary of Kendall’s tau correlations between log10-transformed Escherichia 

coli concentrations in water as measured by the culture-based method and 

environmental or water-quality variables at three northeastern-Ohio beaches 

in 2006 and 2007 ..........................................................................................................................10

 5. Summary of Kendall’s tau correlations between results for the culturebased method and direct-addition variation of the IMS/ATP method at three 

northeastern-Ohio beaches......................................................................................................14

 6. Summary of Kendall’s tau correlations between results for the culturebased method and direct-addition variation of the IMS/ATP method at three 

northeastern-Ohio beaches, by month sampled in 2007......................................................15

 7. Summary statistics for rainfall at two Cleveland, Ohio, airports on days 

samples were collected during individual months, 2007 .....................................................18
vii

Conversion Factors

Multiply By To obtain

Length

inch (in.) 25.4 millimeter (mm)

foot (ft) .3048 meter (m)

mile (mi) 1.609 kilometer (km)

Volume

milliliter (mL) .03381 fluid ounce (oz)

microliter (μL) .001 milliliter (mL)

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

°F=(1.8×°C)+32

Bacteria concentrations are given in colony-forming units per 100 milliliters (CFU/100 mL), 

and bacterial adenosine triphosphate concentrations are given in relative light units per 

100 milliliters (RLU/100 mL).

Pore sizes of filters are given in micrometers (μm).

Comparison of the Immunomagnetic Separation/

Adenosine Triphosphate Rapid Method and the 

Modified mTEC Membrane-Filtration Method for 

Enumeration of Escherichia coli

By Amie M.G. Brady, Rebecca N. Bushon, and Erin E. Bertke

Abstract

Water quality at beaches is monitored for fecal indicator 

bacteria by traditional, culture-based methods that can take 

18 to 24 hours to obtain results. A rapid detection method that 

provides estimated concentrations of fecal indicator bacteria 

within 1 hour from the start of sample processing would allow 

beach managers to post advisories or close the beach when 

the conditions are actually considered unsafe instead of a day 

later, when conditions may have changed. A rapid method 

that couples immunomagnetic separation with adenosine 

triphosphate detection (IMS/ATP rapid method) was evaluated 

through monitoring of Escherichia coli (E. coli) at three Lake 

Erie beaches in Ohio (Edgewater and Villa Angela in Cleveland and Huntington in Bay Village).

Beach water samples were collected between 4 and 

5 days per week during the recreational seasons (May through 

September) of 2006 and 2007. Composite samples were created in the lab from two point samples collected at each beach 

and were shown to be comparable substitutes for analysis of 

two individual samples. E. coli concentrations in composite 

samples, as determined by the culture-based method, ranged 

from 4 to 24,000 colony-forming units per 100 milliliters during this study across all beaches. Turbidity also was measured 

for each sample and ranged from 0.8 to 260 neophelometric 

turbidity ratio units. Environmental variables were noted at 

the time of sampling, including number of birds at the beach 

and wave height. Rainfall amounts were measured at National 

Weather Service stations at local airports. Turbidity, rainfall, 

and wave height were significantly related to the culture-based 

method results each year and for both years combined at each 

beach. The number of birds at the beach was significantly 

related to the culture-based method results only at Edgewater 

during 2006 and during both years combined.

Results of the IMS/ATP method were compared to results 

of the culture-based method for samples by year for each 

beach. The IMS/ATP method underwent several changes and 

refinements during the first year, including changes in reagents 

and antibodies and alterations to the method protocol. Because 

of the changes in the method, results from the two years of 

study could not be combined. Kendall’s tau correlation coefficients for relations between the IMS/ATP and culture-based 

methods were significant except for samples collected during 

2006 at Edgewater and for samples collected during 2007 

at Villa Angela. Further, relations were stronger for samples 

collected in 2006 than for those collected in 2007, except at 

Edgewater where the reverse was observed.

The 2007 dataset was examined to identify possible reasons for the observed difference in significance of relations by 

year. By dividing the 2007 data set into groups as a function of 

sampling date, relations (Kendall’s tau) between methods were 

observed to be stronger for samples collected earlier in the 

season than for those collected later in the season. At Edgewater and Villa Angela, there were more birds at the beach at 

time of sampling later in the season compared to earlier in the 

season. (The number of birds was not examined at Huntington.) Also, more wet days (when rainfall during the 24 hours 

prior to sampling was greater than 0.05 inch) were sampled 

later in the season compared to earlier in the season. Differences in the dominant fecal source may explain the change in 

the relations between the culture-based and IMS/ATP methods. 
2 Comparison of the IMS/ATP Rapid Method and the Mod. mTEC Membrane-Filtration Method for Enumeration of E. coli

Introduction

The State of Ohio uses Escherichia coli (E. coli) as an 

indicator bacterium to assess recreational water quality (Ohio 

Environmental Protection Agency, 2008). Traditional culturebased methods for determining concentrations of E. coli in 

water require at least 18–24 hours from sample collection 

to availability of results. Water-quality advisories are issued 

when concentrations exceed the recreational water-quality 

standard; however, results are not available until the day after 

the sample is collected. By that time, bacteria concentrations 

in the water may have changed significantly (Boehm and 

others, 2002; Wymer and others, 2005). As a result of the lag 

between sample collection and analytical results, recreational 

users may be at risk of contact with water that has exceeded 

the standard and is not considered safe for recreation. Alternatively, the beach may be declared unsafe when the risk is low, 

resulting in lost recreational use and revenue. The need for 

rapid detection methods for monitoring recreational waters is 

widely recognized (U.S. Congress, 2007; U.S. Environmental 

Protection Agency, 2007). Many enumeration methods have 

become available recently that provide results in less than 

4 hours, thereby yielding same-day feedback on bacterial concentrations in recreational waters (Griffith and others, 2004; 

Noble and Weisberg, 2005; Griffith and Weisberg, 2006).

One rapid method for determining concentrations of 

E. coli was developed by researchers at the University of 

Michigan (Lee and Deininger, 2004) and was further modified and field tested by the U.S. Geological Survey (USGS) 

in the Cuyahoga River (within the Cuyahoga Valley National 

Park) during the recreational seasons of 2004–6 (Brady, 2007; 

Bushon and others, 2007, 2009). The method is based on 

immunomagnetic separation (IMS), which selectively captures 

E. coli through the use of antibodies attached to magnetic 

beads. Adenosine triphosphate (ATP) bioluminescence is then 

measured to quantify the captured E. coli. Advantages of the 

IMS/ATP method include availability of results within 1 hour 

of the start of sample processing, simplicity and affordability 

of equipment and supplies, and portability of equipment in 

the field. Additionally, the IMS/ATP method measures viable 

cells through the detection of ATP, which is an energy-carrying 

molecule found in living organisms. In comparison, culturebased methods only measure culturable organisms and would 

not be able to detect those organisms that have entered a viable 

but not culturable state.

The USGS, in cooperation with the Northeast Ohio 

Regional Sewer District (NEORSD), the Cuyahoga County 

Board of Health, the Office of the Cuyahoga County Sanitary 

Engineer (CCSE), and the Ohio Water Development Authority, evaluated the use of the IMS/ATP rapid method at three 

Lake Erie beaches in northeast Ohio.

Purpose and Scope

This report describes the results of a study to test a rapid 

method at three Lake Erie beaches in an attempt to provide 

beach managers with a tool that can determine E. coli concentrations more rapidly than the culture-based method. Water 

samples were collected during the recreational seasons (May–

September) of 2006 and 2007 at Edgewater, Villa Angela, and 

Huntington beaches in northeast Ohio. Samples were analyzed 

for concentrations of E. coli using the traditional culturebased method and the IMS/ATP method. Sample results were 

compared to determine if a relation exists between the results 

of the two methods. Further, environmental or water-quality 

factors (for example, turbidity, wave height, and local rainfall) 

were collected and analyzed to better understand the differences in results between the two methods.

Study Area

The northeastern-Ohio Lake Erie beaches selected for 

this study (fig. 1) were Edgewater Park and Villa Angela, operated by the Ohio Department of Natural Resources and located 

within the Cleveland metropolitan area; and Huntington 

Reservation, operated by Cleveland Metroparks and located in 

suburban Bay Village to the west of Cleveland. The Edgewater 

beach bathing area is open to the lake, and a breakwall on the 

eastern side of beach impedes longshore currents. The Villa 

Angela beach has four breakwalls built between the beach and 

the lake to stabilize the beach. At Huntington, the beach is 

separated into coves by five piers spaced nearly evenly along 

the beach. 

A previous study at Edgewater by Francy and others 

(2006) investigated potential sources of fecal contamination to 

the beach. Remote sources, such as the Cuyahoga and Rocky 

Rivers and Westerly Wastewater Treatment Plant, were shown 

to have little effect on elevated bacteria concentrations in the 

bathing waters at the beach. Local sources, such as runoff 

from parking lots and adjacent areas, reservoirs of E. coli in 

the foreshore sands, and bird feces, were suggested as the 

dominant sources of fecal contamination to the bathing waters 

at the beach.

Potential sources of fecal contamination to Villa Angela 

beach include stormwater and combined-sewer overflows 

from outfalls from streams to the west and Euclid Creek to the 

east. At Huntington, potential sources of fecal contamination 

are largely unknown. At both beaches, birds are also probable 

sources of E. coli.
Study Area 3

Figure 1. Study area and beach locations, northeastern Ohio.

Previous studies at these beaches have been done by the 

USGS, in cooperation with local agencies, to evaluate factors 

related to the concentrations and distribution of E. coli and to 

develop models to predict levels of contamination based on 

easy-to-measure environmental variables (Francy and Darner, 

1998; Francy and others, 2003; Francy and others, 2006; 

Francy and Darner, 2008). Multiple and diffuse sources, natural variability in bacteria concentrations over space and time, 

and dynamic currents, weather patterns, and natural processes 

all contribute to the complexity of estimating bacteria concentrations at these beaches.

� �

�

Euclid

Creek

Ninemile

Creek

Dugway

Brook

Doan

Brook

LAKE ERIE

Cuyahoga

River

Rocky

River

Cahoon

Creek

Creek

Porter

HUNTINGTON

RESERVATION EDGEWATER

PARK

VILLA ANGELA

Bay

Villiage

Cleveland

State Route 2

OHIO

41o30'

41o

35'

81o

50' 81o

45' 81o40' 81o35'

Base map modified from U.S. Geological Survey

Cleveland North 1:24,000, 1963, photorevised

1973; Cleveland South, E. Cleveland, Lakewood

1:24,000, 1963, photorevised 1979; and North

Olmsted 1:24,000, 1963, photorevised 1985

81o55'

0

0

1

1

2 3

2 3 4 MILES

4 KILOMETERS

Westerly

Wastewater Treatment

Plant

Cleveland Burke Lakefront

Airport

Study area
4 Comparison of the IMS/ATP Rapid Method and the Mod. mTEC Membrane-Filtration Method for Enumeration of E. coli

Methods of Study

Sampling Frequencies and Locations

Daily water samples for determining E. coli concentrations by traditional culture-based methods were collected as 

part of established monitoring programs. At Edgewater and 

Villa Angela, water samples were collected by NEORSD five 

times per week at two locations at each beach. At Huntington, 

samples were collected by CCSE at two locations four times 

per week in 2006 and five times per week in 2007. Samples 

were collected at east and west locations at Edgewater and 

Villa Angela and at central and west locations at Huntington. 

Two sampling locations were chosen at each beach so that 

the heterogeneity of bacterial concentrations across the beach 

could be evaluated in sample results. Samples were collected 

during the recreational seasons of 2006 and 2007, starting 

at the end of May or early June through the end of August 

or September.

Sample-Collection Methods

Field personnel used USGS sampling methods to collect 

water samples for analysis by both traditional culture-based 

and IMS/ATP methods. Water samples were collected between 

8 and 9 a.m. in waters ranging from 2.5 to 3 ft in depth by 

means of a grab-sampling technique that minimized contamination of sterile polypropylene bottles (Myers and others, 

2007). Samples were collected approximately 1 ft below 

the surface of the water. When wave heights were greater 

than 2 to 3 ft, field personnel waded out to a safe depth and 

collected the sample or used an extension pole to collect the 

sample near the routine water depth. Samples were immediately capped and placed in coolers with ice for transport to 

the lab.

Laboratory Methods

Samples were kept on ice or refrigerated (0–4°C) until 

analyses could be completed. All samples were analyzed 

within 6 hours of sample collection. During 2006, samples 

from Huntington were analyzed by CCSE using the culturebased method and analyzed by NEORSD using the IMS/

ATP method. CCSE analyzed Huntington samples using both 

methods in 2007. NEORSD analyzed samples from Edgewater 

and Villa Angela using both methods for both years. Composite samples were prepared in the laboratory by adding equal, 

well-mixed volumes of each of the two samples collected from 

the sampling locations at each beach into a clean, sterile bottle. 

Turbidity was measured by the sample-collecting agency 

from the prepared composite sample and reported in nephelometric turbidity ratio units (NTRU). At least two aliquots from 

each sample were measured until two consecutive measurements agreed within 10 percent or within 1 NTRU if readings 

were less than 10 NTRU.

The Ohio bathing-water single-sample standard for 

E. coli of 235 CFU/100 mL (Ohio Environmental Protection 

Agency, 2008) was used as a benchmark to evaluate water 

quality in this study and will henceforth be referred to as “the 

water-quality standard” (WQS). 

Culture-Based Method

Individual and composite samples were analyzed for 

E. coli using the traditional culture-based method of membrane filtration on modified mTEC media (U.S. Environmental 

Protection Agency, 2006). A series of sample volumes were 

filtered through 0.45-μm-pore-size filters, placed on agar 

plates, and incubated for 2 hours at 35°C and then 22 hours at 

44.5°C. Any colonies that were red or magenta were counted 

as positive for E. coli. Concentrations were calculated as 

described by Myers and others (2007) and reported in colonyforming units per 100 milliliters (CFU/100 mL). 

Both individual and composite samples were analyzed 

using the traditional culture-based method to evaluate whether 

the result of a composite sample yields an E. coli concentration that is comparable to the current practice of averaging the 

results of the individual samples at each beach.

Immunomagnetic Separation/

Adenosine Triphosphate Rapid Method

The IMS/ATP method for E. coli was originally described 

in Lee and Deininger (2004), with further modifications 

described in Bushon and others (2009a and 2009b). Variations of the method were tested and optimized throughout 

the project as described below. Because of funding restraints, 

only the composite sample for each beach was used for IMS/

ATP analyses. 

For samples collected in 2006, two variations of the IMS/

ATP method were tested for each sample. One variation that 

included preliminary sample processing steps (filtration and 

elution) was compared to a second variation where samples 

were analyzed directly with no filtration (direct addition). 

For the filtration and elution variation, 50 mL of sample were 

filtered through a 0.45-μm-pore-size filter to capture target 

bacteria. The bacteria were then eluted from the filter by 

transferring the filter to a 50-mL centrifuge tube containing 

phosphate-buffered saline (PBS) with 0.05 percent Tween 

20 (Sigma-Aldrich, St. Louis, Mo.) and mixing by hand for 

1 minute. The eluate was used for subsequent processing 

steps. For the direct-addition variation, 5 to 30 mL of sample 

was added directly to a 50-mL centrifuge tube; for volumes 

less than 30 mL, the total volume was brought up to 30 mL 

using PBS.
Methods of Study 5

Ancillary Data Collection

Wave height was measured by use of a graduated rod 

at the time of sample collection. The rod was placed at the 

sampling location, and the minimum and maximum water 

heights were noted over the course of 1 minute. The number of 

birds at the beach were counted before sampling at Edgewater 

and Villa Angela. Rainfall information was compiled for the 

National Weather Service stations at local airports (Cleveland Hopkins International1

 and Cleveland Burke Lakefront) 

(http://www.erh.noaa.gov). Hourly rainfall amounts were 

totaled for the 24 hours prior to 9 a.m. on the reported date. 

Rainfall data from Cleveland Hopkins International Airport 

were used for data analysis for Edgewater and Huntington, 

and rainfall data from Cleveland Burke Lakefront Airport were 

used for data analysis for Villa Angela.

Quality Assurance/Quality Control

Quality-assurance and quality-control laboratory and 

field procedures (Myers and others, 2007; Francy and others, 

2008) were followed throughout the study. No problems were 

observed for quality-control samples analyzed during this 

study. Filter blanks and method blanks were analyzed daily for 

the culture-based and IMS/ATP methods, respectively. Filter 

blanks consisted of approximately 50 mL of sterile buffer filtered and plated on modified mTEC agar prior to filtering the 

sample. Method blanks consisted of 30 mL (2006) or 25 mL 

(2007) of PBS treated in the same manner as a regular sample 

and analyzed by means of the IMS/ATP method. Replicate 

environmental samples were not processed in 2006 because 

each sample was analyzed by using the filtration and elution 

variation as well as the direct-addition variation of the IMS/

ATP rapid method. In 2007, a total of 27 replicate samples 

were collected and analyzed by the direct-addition variation 

of the rapid method (13 replicates processed by NEORSD and 

14 replicates processed by CCSE). To examine the variability 

in the replicates, the absolute value of the relative percent difference (ARPD) was calculated by taking the absolute value 

of the difference between the two replicates, dividing by the 

average, and multiplying by 100. For the replicate samples 

processed by the IMS/ATP method in 2007, the ARPD varied 

from <1 to 145 percent. The average ARPD was 43 percent, 

which is high compared to what Bushon and others (2009a) 

found in replicate samples collected from a river (24 percent).

Data Analysis and Statistics 

Scatterplots and correlation analyses were used as exploratory tools to examine the data. Correlation analyses were 

used to assess the strength of the relation between two variables. Pearson’s r was calculated to measure linear correlation 

1

 Not shown in figure 1. Cleveland Hopkins International Airport is about 

11 mi southwest of Cleveland Burke Lakefront Airport.

For samples collected in 2007, only the direct-addition 

variation was used for sample analyses. The filtration and elution variation of the method was no longer used (see “Sample 

Processing Versus Direct Addition of Sample” section of this 

report). The volume of sample analyzed was 25 mL. Further, PBS was no longer used to buffer the sample; instead, 

ImmTech diluent (ImmTech, Inc., New Windsor, Md.) was 

used to bring the volume up to 40 mL in a 50-mL centrifuge 

tube. The ImmTech diluent is designed to reduce nonspecific 

signal from samples with a complex matrix.

After the initial processing steps (if done), 100 μL of 

antibody-coated magnetic beads were added to the 50-mL 

centrifuge tube containing the sample or eluate. In 2006, the 

processing laboratory coated the beads with antibodies by 

means of passive adsorption according to directions in TechNote 204 by Bangs Laboratories (1999). The antibodies used 

in 2006 were goat anti-E. coli antibodies (BioDesign International, Saco, Maine). In 2007, the beads were coated with the 

antibodies by a commercial supplier (ImmTech, Inc.); a rabbit 

anti-E. coli antibody was used because the goat anti-E. coli

antibodies were no longer available. 

Samples containing antibody-coated beads were rotated 

at 18 rpm for 30 minutes on a sample mixer (Invitrogen, Carlsbad, Calif.). This step permitted the antibodies to adhere to 

the E. coli cells. After mixing, the 50-mL centrifuge tube was 

placed against a magnet (MPC-50 magnetic particle concentrator; Invitrogen) that pulled the bacteria-antibody-bead complex out of solution. The tube and magnet were rocked gently 

for 2 minutes before decanting. A washing step was done to 

decrease contamination from nontarget bacteria. During 2006, 

the washing step was done with PBS; but in 2007, the PBS 

was replaced with ImmTech diluent. After the washing step, 

the beads and associated target bacteria were transferred to a 

1.5-mL tube using PBS and were placed against another magnet (MPS-S magnetic particle concentrator; Invitrogen). The 

tube and magnet were rocked gently for 2 minutes.

The remaining steps were identical for both years and 

followed the published method (Bushon and others, 2009a). 

Briefly, the bead complex was washed with a somatic-cell 

releasing agent (SRA) (New Horizons Diagnostics, Columbia, 

Md.) to rupture any nonbacterial cells. Another wash step was 

performed with PBS, and then the bead complex was exposed 

to a bacterial-cell releasing agent (BRA) (New Horizons Diagnostics) that ruptured bacterial cells, releasing ATP into solution. By using the MPS-S magnet, the solution containing ATP 

was isolated from the beads and exposed to luciferin-luciferase 

(LL) (New Horizons Diagnostics). The LL reacted with ATP to 

produce light. A microluminometer (New Horizons Diagnostics) was used to measure luminescence in relative light units 

(RLU). Results were reported as RLU/100 mL. Formulations 

of the reagents involved in the final steps of analysis (SRA, 

BRA, and LL) were changed by the manufacturer after the 

sampling season of 2006. 
6 Comparison of the IMS/ATP Rapid Method and the Mod. mTEC Membrane-Filtration Method for Enumeration of E. coli

between culture-based method results for the daily average of 

two individual samples and the associated composite sample. 

Kendall’s tau-b (τ) was calculated to measure the strength of 

the monotonic relationship between two variables. Kendall’s 

tau-b is a nonparametric procedure that is based on ranks 

and is resistant to outliers (Helsel and Hirsch, 2002). This 

procedure was used for comparisons of results of the culturebased method (E. coli concentrations, in CFU/100 mL of 

sample) and results of the IMS/ATP method (luminescence, in 

RLU/100 mL of sample) and environmental or water-quality 

factors. In addition, Kendall-Theil robust lines were included 

on plots to assist with making comparisons among beaches 

and years of data. The Kendall-Theil robust line regression is a 

nonparametric method that was chosen for this study because 

of the number of outliers in the datasets. The slope of the 

Kendall-Theil robust line is the median of all slopes between 

any two data points in the dataset; the intercept of this line 

is calculated by placing the line through the median x and 

y values (Granato, 2006). 

To further examine the relation between environmental 

or water-quality variables and E. coli concentrations, observations were placed into groups of nearly equal size on the basis 

of magnitude of the explanatory variable. Tukey-Kramer multiple-comparison tests on the data were used to compare all 

possible pairs of treatment group means of log10-transformed 

concentrations. This test is powerful even when treatment 

group sizes are unequal (Helsel and Hirsch, 2002). The alpha 

(α) level for all tests of significance in this report was 0.05.

During 2006, for individual beaches, 26 to 40 percent 

of sample results from the IMS/ATP method (direct-addition 

variation) exceeded the maximum measureable luminescence 

of the luminometer. These data were described as rightcensored because, when plotted in a scatterplot, the censored 

values fall on the right side of the graph. Because different 

volumes of sample were analyzed by means of the IMS/ATP 

method, some sample results for luminescences within the 

measureable range exceeded those that were censored. For 

example, if 30 mL of sample A was analyzed and the luminescence was greater than 900,000 RLU, it would be reported 

as greater than 3,000,000 RLU/100 mL. However, if 5 mL 

of sample B was analyzed and the luminescence was measureable at 200,000 RLU, it would be reported as 4,000,000 

RLU/100 mL. Because only one volume was analyzed per 

sample, there is no way to know whether the luminescence 

was higher for sample A or sample B. Therefore, to treat all 

data equally, any measured values that were greater than 

900,000 RLU per 30 mL (highest volume of any sample 

analyzed by means of the IMS/ATP method) were classified as 

censored. The additional censoring increased the percentage of 

censored values to 33 to 55 percent. The censored data were 

all assigned the same value (3,000,000 RLU/100 mL) for use 

in nonparametric correlation analysis. Changes in reagents and 

antibodies and amendments to the procedure for the IMS/ATP 

method between years eliminated the higher range of luminescence so that no sample results were censored during 2007.

Results

Comparison of Composite Sampling to 

Average of Multiple-Point Samples

Bacteria concentrations can vary along a beach. Concentrations along the beach may be affected by the location 

relative to the bacteria source, by breakwaters or groins, or by 

exposure to sunlight. Because of this spatial variability, agencies who monitor beach water quality may choose to stratify 

the beach into several areas and collect a sample from each 

area for analysis. Current practice at Edgewater, Villa Angela, 

and Huntington is to collect two samples at fixed locations at 

each beach and analyze them using the culture-based method 

for determining concentrations of E. coli. The arithmetic mean 

concentration is then computed from the individual concentrations determined for the two samples. The mean concentration is then used to determine whether a beach is posted with 

an advisory based on the water-quality standard set by Ohio 

Environmental Protection Agency. 

For all samples collected during this study, E. coli concentrations in the composited samples were compared to the 

arithmetic mean of the two individual samples collected for 

the same beach and date. Prior to statistical analysis, E. coli 

concentrations were log10 transformed. The mean concentration (determined by averaging concentrations from the two 

point samples) and the composite sample concentrations for 

Edgewater, Villa Angela, and Huntington beaches combined 

were highly correlated (r = 0.976, p <0.0001) (fig. 2). The 

mean E. coli concentrations in the composite samples were not 

significantly different from the E. coli concentrations determined by averaging the concentrations of two point samples, 

as determined by paired t-tests at Edgewater (t = –0.56, 

p = 0.5725), Villa Angela (t = –1.86, p = 0.0641), or Huntington (t = –0.75, p = 0.4567). Combining two point samples 

into a single composite is more economically feasible than 

analyzing individual samples by the culture-based method 

(Bertke, 2007). The difference in cost is even greater for the 

IMS/ATP method, because the IMS/ATP method is more labor 

intensive and supplies are more expensive than the culturebased method. Therefore, it is important to determine whether 

composited-sample results provide similar measures of water 

quality as results from averaging concentrations from two 

point samples.

Comparisons were made between the mean and composite concentrations for samples collected at the same beach 

and on the same date to determine whether one concentration 

exceeded the WQS whereas the other did not. Discordance 

occurred in 5.0 percent of the samples collected at all beaches. 

More discordances were observed at Villa Angela (8.3 percent) 

than at Edgewater (2.7 percent) and Huntington (3.6 percent). 

The low percentage of discordances and the strong correlations between the concentrations indicate that composite 

sampling appears to be a suitable alternative to multiplepoint sampling.
Results 7

Figure 2. Scatterplot of Escherichia coli concentrations from composite samples and the arithmetic average of multiple-point samples 

at three Lake Erie beaches, May through September 2006 and May through August 2007. The water-quality standard is the Ohio singlesample bathing-water standard for E. coli of 235 colony-forming units per 100 milliliters. (r, the Pearson’s correlation coefficient for 

results from all beaches combined.)

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

Average concentration of two point samples, in colony-forming units per 100 milliliters

1

10

100

1,000

10,000

100,000

Compo

site

s

ample

con

centration, in

c

olony-forming units per 100 milliliter

s

Villa Angela

Huntington Beach

Edgewater Park

1:1 Line

Water-quality standard

r = 0.976
8 Comparison of the IMS/ATP Rapid Method and the Mod. mTEC Membrane-Filtration Method for Enumeration of E. coli

Table 1. Summary statistics for concentrations of Escherichia coli as determined by the culture-based 

method in samples collected from three northeastern-Ohio beaches, May through September 2006 and 

May through August 2007. The water-quality standard is the Ohio single-sample bathing-water standard of 

235 colony-forming units per 100 milliliters. 

[WQS, water-quality standard; CFU/100 mL, colony-forming units per 100 milliliters]

Year Number of 

samples

Number exceeding WQS

(percent)

Escherichia coli, in CFU/100 mL 

Minimum Median Maximum Average

Edgewater

2006 82 18 (22) 4 90 1,900 180

2007 62 22 (35) 10 110 2,000 280

Villa Angela

2006 82 39 (48) 4 230 4,100 430

2007 62 37 (60) 10 340 24,000 1,800

Huntington

2006 53 12 (23) 7 75 3,100 220

2007 60 10 (17) 5 50 1,800 150

General Patterns in Escherichia coli, Turbidity, 

and Rainfall

Including both years of study at all beaches, concentrations of E. coli ranged from 4 to 24,000 CFU/100 mL 

and included several exceedances of the WQS (table 1). 

At Edgewater, concentration ranges were similar for both 

years of data collection (4 to 1,900 CFU/100 mL in 2006 

and 10 to 2,000 CFU/100 mL in 2007) (table 1). During 

2006 at Villa Angela, the median concentration of E. coli

was near the WQS; but in 2007, the median was above the 

WQS, and approximately 60 percent of the sample concentrations exceeded the WQS. The largest E. coli concentration 

was observed at Villa Angela on July 17, 2007, a second 

consecutive day without rainfall on which a large number of birds were observed at the beach. Yearly ranges of 

concentrations were similar at Huntington: 7 to 3,100 and 

5 to 1,800 CFU/100 mL for 2006 and 2007, respectively.

Throughout the duration of the study at all beaches, 

turbidity ranged from 0.8 NTRU to 260 NTRU (table 2). The 

largest observed turbidity was at Huntington Beach on August 

29, 2006; a large rainfall event occurred the previous day 

(1.3 in.), and wave heights were recorded at 5 ft—the highest 

waves observed during this study. Samples were not collected 

at Edgewater or Villa Angela on this date because of unsafe 

sampling conditions. Turbidity remained elevated at Huntington over the next 2 days (162 and 83 NTRU, respectively). 

With the exception of this event, turbidity rarely exceeded 

50 NTRU (n=8 for samples with greater than 50 NTRU for all 

beaches). Median turbidity values for all beaches and years 

sampled did not exceed 7.9 NTRU.

The largest rainfall amount observed for a 24-hour perior 

prior to sampling was 3.33 in. at Cleveland Hopkins International Airport on August 8, 2007 (table 3). For both years 

during reported periods, rainfall in excess of 0.05 in. occurred 

on approximately 27–29 percent of the days for both airports. 
Results 9

Table 2 Summary statistics for turbidity measurements from samples collected at 

three northeastern-Ohio beaches, May through September 2006 and May through 

August 2007.

[NTRU, neophelometric turbidity ratio units]

Year Number of 

samples

Turbidity, in NTRU

Minimum Median Maximum Average

Edgewater

2006 81 1.7 7.9 46 11

2007 61 1.6 4.0 52 7.4

Villa Angela

2006 81 1.3 5.1 61 10

2007 61 0.8 3.2 90 7.4

Huntington

2006 53 1.9 7.6 260 21

2007 60 1.7 7.8 90 14

Table 3. Summary statistics for rainfall at two Cleveland, Ohio, airports on days samples were collected.

[Wet days are days on which rainfall over the previous 24 hours prior to sampling exceeded 0.05 inch.Total rainfall is the 

sum of all observed rainfall in the previous 24 hours prior to sampling. Source information from the National Weather 

Service.]

Dates Number of 

observations

Number of wet days 

(percent)

Maximum rainfall, 

in inches

Total rainfall, 

in inches

Cleveland Hopkins International Airport

2006: 5/31–9/28 82 23 (28) 2.26 10.1

2007: 6/5–8/31 62 18 (29) 3.33 12.1

Cleveland Burke Lakefront Airport

2006: 5/31–9/28 82 24 (29) 1.60 11.3

2007: 6/5–8/31 62 17 (27) 3.18 10.5
10 Comparison of the IMS/ATP Rapid Method and the Mod. mTEC Membrane-Filtration Method for Enumeration of E. coli

Table 4. Summary of Kendall’s tau correlations between log10-transformed Escherichia coli

concentrations in water as measured by the culture-based method and environmental or 

water-quality variables at three northeastern-Ohio beaches in 2006 and 2007. 

[Bold values are significant at α = 0.05. na, data not available]

Year Number of 

samples

Log10-transformed 

turbidity Rainfall Wave height

Number of 

birds

Edgewater

2006 82 0.40 0.18 0.25 0.28

2007 61 .28 .37 .37 –.049

Both years 143 .30 .26 .30 .15

Villa Angela

2006 81 .33 .35 .41 –.024

2007 61 .30 .11 .42 .15

Both years 142 .27 .24 .37 .011

Huntington

2006 53 .32 .31 .40 na

2007 60 .52 .38 .55 na

Both years 113 .44 .35 .47 na

Relations Between Environmental or WaterQuality Factors and E. coli Concentrations

Correlation analysis and scatterplots were done to evaluate the correlations between E. coli concentrations measured 

by the culture-based method and environmental or waterquality factors. Statistically significant relations were found 

between log10-transformed E. coli concentrations and log10-

transformed turbidity, rainfall, and wave height at all three 

beaches during both years of sampling (table 4). The number 

of birds at the beach was significantly correlated with E. coli

concentrations at Edgewater during 2006 and when both years 

were combined, but the relations were not significant at Villa 

Angela. At Huntington, the correlations between E. coli and 

turbidity and wave height were strong and persisted over both 

years of sample collection. 

Relations Between Culture-Based and 

IMS/ATP Methods

Sample Processing Versus Direct Addition 

of Sample

During sample processing in 2006, the filtration and 

elution variation was compared to the direct-addition variation for the IMS/ATP method to determine which variation 

produced results most strongly correlated with those of the 

culture-based method, as measured by Kendall’s tau-b correlation coefficient. The results of the two methods were significantly correlated for each beach. The direct-addition variation 

at Villa Angela and Huntington produced results that were 

more strongly correlated with results from the culture-based 

method (data not shown) than did the filtration and elution 

variation. At Edgewater, in contrast, the relation between the 

filtration and elution variation and the culture-based method 

was slightly stronger than that between the direct addition 

variation and the culture-based method (τ = 0.26 and τ = 0.20, 
Results 11

respectively). On the basis of this statistical analysis and 

increased ease of use of the direct-addition variation compared 

to the filtration and elution variation, the direct-addition variation was chosen for use during the 2007 field season.

Evaluation of 2006 and 2007 Data

Because of optimization steps and changes in antibodies and reagents between years, the IMS/ATP method results 

could not be combined across years. However, several observations are worth noting by comparing the datasets between 

years for the direct-addition variation. The most notable difference between years was the change in magnitude of luminescence observed. During 2006, IMS/ATP results often exceeded 

the upper limit of the luminometer; however, no samples collected during 2007 required upper-limit censoring. At Edgewater (fig. 3) and Villa Angela (fig. 4), the slopes of the KendallTheil robust lines were similar for both years (censored data 

are not shown on the plots); however, the y-intercepts were 

different. At Huntington, the slope for 2006 is noticeably 

steeper and the y-intercept higher than for 2007 (fig. 5). 

Correlations between the culture-based and IMS/ATP 

methods were different between 2006 and 2007 (table 5). At 

Edgewater, the correlation coefficient was significant in 2007 

but was not significant in 2006. At Villa Angela and Huntington, these relations were stronger in 2006 than in 2007. 

The reason for this difference may be the result of changes in 

reagents and the antibody used. In addition, differences in correlations between the two methods could be influenced by the 

censoring of the IMS/ATP method results for some samples 

collected during 2006.

Although the majority of the correlation coefficients were 

significant, there was noticeable scatter around the KendallTheil roubust line for all plots (figures 3, 4, and 5). This scatter 

may be the result of differences in the what is being measured 

by the two different methods, that is, culturable versus viable 

cells. The culture-based method measures cells that can be cultured on a specific agar plate; whereas, the IMS/ATP method 

measures culturable cells, as well as viable but not culturable 

cells that contain ATP.

Figure 3. Relation between the culture-based Escherichia coli concentrations and IMS/ATP luminescence at 

Edgewater, Cleveland, Ohio, during May through September 2006 and June through August 2007. The solid line 

is the Kendall-Theil robust line. The water-quality standard is the Ohio single-sample bathing-water standard for 

E. coli of 235 colony-forming units per 100 milliliters.

103 104 105 106 107 108

Luminescence, in relative light units per 100 milliliters

100

101

102

103

104

Escherichia

c

oli, i

n colony-forming units per 100 milliliters

2006 (censored data not shown)

2007

Water-quality standard

log10(y) = 0.53 10(x) - 1.5

log10(y) = 0.42 log10(x) + 0.22

x log

x
12 Comparison of the IMS/ATP Rapid Method and the Mod. mTEC Membrane-Filtration Method for Enumeration of E. coli

Figure 4. Relation between the culture-based Escherichia coli concentrations and IMS/ATP luminescence at 

Villa Angela, Cleveland, Ohio, during May through September 2006 and June through August 2007. The solid line is the 

Kendall-Theil robust line. The water-quality standard is the Ohio single-sample bathing-water standard for E. coli of 

235 colony-forming units per 100 milliliters.

102 103 104 105 106 107 108

Luminescence, in relative light units in 100 milliliters

100

101

102

103

104

105

Escherichia

coli, in colony-forming units per 100 milliliters

2006 (censored data not shown)

2007

Water-quality standard

log10(y) = 0.32 log10(x) + 1.2

log10(y) = 0.43 10 x log (x) - 0.38

x
Results 13

Figure 5. Relation between the culture-based Escherichia coli concentrations and IMS/ATP luminescence at 

Huntington Reservation, Bay Village, Ohio, during May through September 2006 and June through August 2007. 

The solid line is the Kendall-Theil robust line. The water-quality standard is the Ohio single-sample bathing-water 

standard for E. coli of 235 colony-forming units per 100 milliliters

102 103 104 105 106 107 108

Luminescence, in relative light units in 100 milliliters

100

101

102

103

104

Es

che

richia c

oli, in c

olony-forming units per 100 milliliter

s

2006 (censored data not shown)

2007

Water-quality standard

log10(y) = 0.24 log10(x) + 0.54

log10(y) = 0.71 log10 x (x) - 2.7

x
14 Comparison of the IMS/ATP Rapid Method and the Mod. mTEC Membrane-Filtration Method for Enumeration of E. coli

Table 5. Summary of Kendall’s tau correlations between results for the culture-based method 

and direct-addition variation of the IMS/ATP method at three northeastern-Ohio beaches. 

[Bold values are significant at α = 0.05.]

Year Number of 

samples

Number of censored values

(percent)

Kendall’s tau 

correlation 

coefficient

p value

Edgewater

2006 73 40(55) 0.12 0.1699

2007 61 0 (0) .24 .0076

Villa Angela

2006 72 24 (33) .36 <.0001

2007 61 0 (0) .16 .0646

Huntington

2006 46 19 (41) .40 .0002

2007 57 0 (0) .20 .0312
Results 15

Table 6. Summary of Kendall’s tau correlations between results 

for the culture-based method and direct-addition variation of the 

IMS/ATP method at three northeastern-Ohio beaches, by month 

sampled in 2007. 

[Bold values are significant at α = 0.05.]

Month Number of 

samples

Kendall’s tau 

correlation 

coefficient

p value

Edgewater

June 19 0.38 0.0228

July 21 .20 .1939

August 21 .22 .1735

Villa Angela

June 19 .41 .0143

July 21 .076 .6290

August 21 <.00001 1.0000

Huntington

June 18 .48 .0050

July 18 .066 .7047

August 21 –.029 .8561

Effects of Water-Quality and 

Environmental Factors

Because of previously described changes in IMS/ATP 

rapid method, resulting in two distinct datasets, the discussion 

of data analysis that follows will focus on samples analyzed 

with the most up-to-date IMS/ATP protocol (2007). The data 

were divided into groups as a function of environmental factors, water-quality factors, and sampling dates. Groups were 

formed by dividing the dataset into subsets of nearly equal 

size on the basis of the magnitude of the factor. Correlations 

between the IMS/ATP and culture-based methods were determined individually for these groups. 

At all beaches, correlations between the two methods 

were strongest and significant during the month of June 

(table 6) but were not significant during July or August. 

Although ranges in the concentration of E. coli were similar 

during these months, other factors may have affected these 

relations during July and August. For example, there was a 

significant increase in the number of birds at Edgewater during 

July compared to June (p = 0.0058, fig. 6) and at Villa Angela 

during August as compared to June (p = 0.0144, fig. 7). The 

numbers of birds were not recorded at Huntington. Birds, 

therefore, may have been a more dominant fecal source late 

in the summer than early in the summer. Perhaps the relations between culture and IMS/ATP methods were affected by 

fecal source. 

Further, there were more wet days sampled during July 

and August than in June (table 7). Total rainfall for days 

sampled in August was larger than in either June or July. The 

increased number of wet days sampled during July and August 

and the total rainfall during August may have also resulted in 

different fecal sources between the early and late summer. 
16 Comparison of the IMS/ATP Rapid Method and the Mod. mTEC Membrane-Filtration Method for Enumeration of E. coli

Figure 6. Number of birds at Edgewater Beach at time of sampling as a function of month sampled. (Results of 

Tukey-Kramer multiple comparison test are presented as letters, and groups with a letter in common do not differ 

significantly at α = 0.05.)

June July August

0

100

200

300

400

500

600

700

800

900

Number of bird

s

A B A,B

(18) (21) (20)

(18) Number of samples

2

Outliers are defined as values outside

of the whisker span

Outlier

2

Upper whisker

1

75th percentile

Median

25th percentile

Lower whisker

1

Explanation

1

The span of the whiskers is the largest

and smallest values within 1.5 times the 

interquartile range
Results 17

Figure 7. Number of birds at Villa Angela Beach at time of sampling as a function of month sampled. (Results 

of Tukey-Kramer multiple comparison test are presented as letters, and groups with a letter in common do not 

differ significantly at α = 0.05.

June July August

0

100

200

300

400

Number

of bird

s

A A,B B

(18) (20) (21)

(18) Number of samples

2

Outliers are defined as values outside

of the whisker span

Outlier

2

Upper whisker

1

75th percentile

Median

25th percentile

Lower whisker

1

Explanation

1

The span of the whiskers is the largest

and smallest values within 1.5 times the 

interquartile range
18 Comparison of the IMS/ATP Rapid Method and the Mod. mTEC Membrane-Filtration Method for Enumeration of E. coli

Future Steps

The IMS/ATP method is a promising tool that has the 

potential to assist beach managers in determining recreational 

water quality within an hour of the start of sample processing. However, this method is still experimental, and further 

optimization is required before this method can be used for 

regulatory purposes. Ultimately, incorporation of the IMS/ATP 

method in an epidemiological study is needed so that method 

results can be directly correlated to human illness rates. Until 

this is done, further studies are needed to establish the linkage 

between the IMS/ATP method and culture-based method so 

that the IMS/ATP results can be used in recreational water settings that use water-quality standards based on culture-based 

methods. 

Summary and Conclusions

Current practices for monitoring concentrations of 

E. coli, a fecal-indicator organism used to assess recreational 

water quality, require a minimum of 18 hours until availability of results. This time lag may result in recreational users 

being exposed to unsafe water; or, alternatively, revenue and 

recreational opportunities may be lost because beaches are 

posted with an advisory when the water is falsely considered 

unsafe for recreation. The need is widely recognized for more 

rapid availability of results so that beach managers can make 

decisions on closures or advisories based on same-day conditions. The U.S. Geological Survey, in cooperation with the 

Ohio Water Development Authority, Northeast Ohio Regional 

Table 7. Summary statistics for rainfall at two Cleveland, Ohio, airports on days samples were 

collected during individual months, 2007. 

[Wet days are days on which rainfall over the previous 24 hours prior to sampling exceeded 0.05 inch. Total rainfall is 

the sum of all observed rainfall in the previous 24 hours prior to sampling during individual months. Source information 

from the National Weather Service.]

Month Number of 

observations

Number of wet days

(percent)

Maximum rainfall, 

in inches

Total rainfall, 

in inches

Cleveland Hopkins International Airport

June 19 3(16) 0.66 1.2

July 21 8 (38) .81 2.4

August 22 7 (32) 3.33 8.6

Cleveland Burke Lakefront Airport

June 19 3 (16) .50 1.0

July 21 7 (33) .36 1.6

August 22 7 (32) 3.18 8.0

Sewer District, Cuyahoga County Board of Health, and 

Cuyahoga County Sanitary Engineer, evaluated a rapid detection method (IMS/ATP) for E. coli that yields results within 

1 hour from the start of sample processing. The IMS/ATP 

method uses magnetic beads coated with antibodies for E. coli 

and an immunomagnetic separation technique to capture 

the E. coli cells in the water sample. Once captured, ATP is 

released from the bacterial cells and measured with a luminometer. Three Lake Erie beaches in northeastern Ohio were 

chosen as the sites for this study because of previous studies 

done at these beaches and the availability of information on 

bacteria contamination sources and (or) levels. 

Beach-water samples were collected during the recreational seasons (May–September) of 2006 and 2007 at 

Edgewater, Villa Angela, and Huntington beaches. In 2006, 

82 samples each were collected at Edgewater and Villa 

Angela, whereas 53 samples were collected at Huntington. 

Approximately 60 samples were collected at each of the three 

beaches in 2007. Each sample was analyzed for E. coli by the 

traditional culture-based method and the IMS/ATP method. 

Environmental data that may affect bacteria concentrations 

on the beaches also were collected, including the number of 

birds at the beach, wave height, sample turbidity, and local 

rainfall amounts. 

Sampling procedures at these beaches involve the collection of two samples from each beach to better represent the 

heterogeneity of bacteria concentrations. The arithmetic mean 

concentration is calculated from the results of the two samples 

and is used to determine whether the water-quality standard 

has been exceeded. In this study, composite samples were 

prepared by adding equal volumes of the two samples. The 

individual samples and the composite sample were analyzed 
Summary and Conclusions 19

by the culture-based method for E. coli. The arithmetic mean 

concentration of the two samples collected at the beach were 

highly correlated with the results of the composite sample 

and had mean concentrations that were not statistically different. A separate study, conducted by the U.S. Geological 

Survey during 2005 at Edgewater, Villa Angela, and Lakeview (Lorain, Ohio) beaches, also found that compositing 

the samples yielded similar measures of recreational water 

quality as the arithmetic average of multiple-point samples at a 

lower cost (Bertke, 2007). Further support of these results was 

found in a previous study at a beach in Racine, Wis., in which 

investigators concluded that analyzing a composited sample 

was a reasonable and cost-effective alternative to results from 

computing the mean of the individual samples (Kinzelman 

and others, 2006). Combining two point samples into a single 

composite is more economically feasible than analyzing individual samples by the culture-based method. The difference 

in cost is even greater for the IMS/ATP method, because the 

IMS/ATP method is more labor intensive and the supplies are 

more expensive than the culture-based method. 

E. coli concentrations determined by the culturebased method in the composite samples ranged from 4 to 

24,000 CFU/100 mL for both years at all beaches and included 

several water-quality exceedances. At Edgewater, the standard was exceeded in 22 and 35 percent of samples in 2006 

and 2007, respectively. The standard was exceeded in 48 and 

60 percent of samples in 2006 and 2007, respectively, at Villa 

Angela. At Huntington, the percentage of exceedances was 23 

in 2006 and 17 in 2007. The average turbidity measurements 

at all beaches were higher in 2006 than in 2007. The percentage of wet days sampled (sampling days when rainfall over 

the previous 24 hours exceeded 0.05 in.) was similar for both 

years (between 27 and 29 percent of days sampled). E. coli

concentrations measured by the culture-based method were 

significantly correlated to turbidity, rainfall, and wave height 

measurements at all beaches in both years. The number of 

birds was significantly related to E. coli concentrations only 

at Edgewater in 2006 and when 2006 and 2007 data were 

combined. The two strongest relations to E. coli concentrations were at Huntington in 2007; turbidity (r = 0.52) and 

wave height (r = 0.55).

For the IMS/ATP method, two variations of the method 

were tested during the recreational season of 2006. For Villa 

Angela and Huntington, results from the direct-addition variation of the method were more strongly correlated than the 

results of the filtration and elution variation to results from the 

culture-based method. These findings are consistent with an 

earlier study in a recreational river, in which the results from 

the direct-addition variation were found to have a stronger 

correlation with results from the culture-based method than 

the two other variations tested (Bushon and others, 2007). In 

contrast, results for Edgewater from the filtration and elution 

variation were slightly more strongly correlated than those 

from the direct-addition variation to results of the culturebased method. On the basis of these statistical analyses and 

the increased ease of use of the direct-addition variation, 

only the direct-addition variation was used during the 2007 

recreational season. 

Differences between years were noticeable in the relations between the culture-based and IMS/ATP methods (directaddition variation). For Edgewater and Villa Angela, the 

slopes of the Kendall-Theil robust lines were similar for 2006 

and 2007; however, the y-intercepts were quite different. For 

Huntington, both the slopes and y-intercepts were different. 

Correlations of the results of both methods were statistically 

significant for Villa Angela during 2006, for Huntington during 2006 and 2007, and for Edgewater during 2007. Between 

the 2 years of sampling, the manufacturers made changes to 

the antibodies and reagents used for the IMS/ATP method; 

therefore, the data for the 2 years were not combined. Statistically significant relations were also found between the 

methods at a recreational river (Bushon and others, 2009a) 

using the original reagents and antibodies. The updated 

reagents and antibodies used in 2007 were also used in a study 

by Bushon and others (2009b) in which statistically significant 

relations between the two methods were found in untreated 

wastewater samples. 

To further understand the relations between the methods 

in 2007, exploratory data analysis was done by breaking up 

the data into groups based on explanatory variables, including sampling date. At all beaches, relations between the two 

methods were stronger in samples collected earlier in the 

recreational season than in those collected later. At Edgewater 

and Villa Angela, the number of birds was shown to increase 

through the season. Also, there were more wet days sampled 

during July and August compared to June. It is hypothesized 

that the relations between the two methods could be affected 

by fecal source, and the differences in the dominant fecal 

source may explain the change in the relations between the 

two methods. At Huntington, no observed environmental factors could explain the change in relations between the methods 

through the season. The number of birds at time of sampling 

was not recorded at this beach, but counting birds at Huntington is suggested for future studies.

The rapid method shows promise for use in recreational 

water-quality monitoring although the relations between the 

culture-based and IMS/ATP rapid methods were not as strong 

for samples collected in 2007 at Villa Angela and Huntington 

compared to those collected in 2006. Although promising, the 

IMS/ATP method is still undergoing optimization and is not 

ready for use in place of the culture-based method without further refinement. With its lower cost than other currently available rapid methods, IMS/ATP method has an added advantage 

of being cost-effective for smaller beach-monitoring agencies. 

In complex systems, such as beaches where numerous contamination sources affect the area, multiple tools may be required 

to provide beach managers with the best possible information to protect the public. The IMS/ATP rapid method has the 

potential to become a strong tool to assist in this endeavor.
20 Comparison of the IMS/ATP Rapid Method and the Mod. mTEC Membrane-Filtration Method for Enumeration of E. coli

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Brady and others—Comparison of the IMS/ATP Rapid Method and the Modified mTEC Membrane-Filtration Method for Enumeration of E. coli—SIR 2009–5222