THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
&EPA
• «tal Pntectiaa Agacy
Batreiie
'Ike Business of Innovation
ETV Verification Statement
TECHNOLOGY TYPE: RAPID BACTERIA DETECTION
APPLICATION: ANALYSIS OF BACTERIA IN WATER
TECHNOLOGY NAME: Bactiquant®-test
COMPANY: Mycometer A/S Mycometer, Inc.
ADDRESS:
PHONE:
WEB SITE:
E-MAIL:
Lersoe Parkalle 40
2100 Copenhagen
Denmark
+45 3916 1072
www.mycometer.com
info@mycometer.dk
5002 MacDill Avenue
Tampa, FL
33611
813-434-6998
www.mycometer.com
lrogers@mycometer.com
The U.S. Environmental Protection Agency (EPA) has established the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative or improved environmental
technologies through performance verification and dissemination of information. The goal of the ETV
Program is to further environmental protection by accelerating the acceptance and use of improved and cost-
effective technologies. ETV seeks to achieve this goal by providing high-quality, peer-reviewed data on
technology performance to those involved in the design, distribution, financing, permitting, purchase, and
use of environmental technologies. Information and ETV documents are available at www.epa.gov/etv.
ETV works in partnership with recognized standards and testing organizations, with stakeholder groups
(consisting of buyers, vendor organizations, and permitters), and with individual technology developers. The
program evaluates the performance of innovative technologies by developing test plans that are responsive to
the needs of stakeholders, conducting field and laboratory tests (as appropriate), collecting and analyzing
data, and preparing peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality
assurance (QA) protocols to ensure that data of known and adequate quality are generated and that the results
are defensible.
The Advanced Monitoring Systems (AMS) Center, one of six verification centers under ETV, is operated by
Battelle in cooperation with EPA's National Risk Management Research Laboratory. The AMS Center
evaluated the performance of a rapid bacteria detection technology. This verification statement provides a
summary of the test results for the Bactiquant®-test developed by Mycometer A/S and distributed in the
United States by Mycometer, Inc.
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VERIFICATION TEST DESCRIPTION
Rapid technologies (results available same day of testing) to detect bacteria from matrices such as surfaces,
bulk material, air, or water are of interest to improve the efficiency of delineating and documenting microbial
contamination in buildings and water systems, and for monitoring progress during cleanup and remediation
processes. Traditional methods of analysis can take up to seven days for results. Technologies providing
same day or near "real-time" results indicating changes in water quality would help to control microbial
outbreaks, expedite remediation efforts, and protect public health.
The verification test of the Bactiquant®-test technology was conducted from June 2 through June 27, 2011 at
Battelle in Columbus, Ohio. Technology operation, sample handling, and analyses were performed
according to the vendor's instructions.
For this verification, the Bactiquant®-test technology was verified for repeatability and inter-assay
reproducibility by detecting bacteria in water samples. Linearity was assessed using dilutions of stock
cultures in dechlorinated tap water. In addition, sustainable operational factors such as ease of use, required
reagents, analysis time, laboratory space, and utilities required were reported.
QA oversight of verification testing was provided by Battelle and EPA. Battelle and EPA QA staff
conducted technical systems audits of the testing and Battelle QA staff conducted a data quality audit of at
least 10% of the test data. This verification statement, the full report on which it is based, and the quality
assurance project plan for this verification test are available at www.epa.gov/etv/centers/center 1 .html.
TECHNOLOGY DESCRIPTION
According to the vendor, Bactiquant®-test is designed to provide a rapid method to estimate total bacteria in
water samples. The Bactiquant®-test rapid bacteria detection technology is based on fluorogenic detection of
enzyme activities found predominantly in a taxonomic group of organisms. Water samples (typically 250
mL) are passed through a membrane filter. A synthetic enzyme substrate is added to the filter and left to
react over a period of time based on temperature. The enzyme present in the bacterial cells hydrolyzes the
synthetic enzyme substrate. When the synthetic substrate molecule is cleaved into two molecules by the
enzyme, one of the molecules can be made to fluoresce upon excitation with ultraviolet (UV) light (365
nanometers). The amount of fluorescence is measured using a handheld fluorometer. This fluorescence
semi-quantitatively correlates to a measure of the bacterial biomass. Fluorescence measurements can be
captured electronically and may be downloaded to a computer or can be transcribed by hand. The sample
preparation and analyses can be performed on site in less than one hour.
According to the vendor, this technology is designed for application to a range of liquid samples including:
potable water, processed water, CIP (cleaning in place), wastewater, and recreational water. Bactiquant®-test
can also be used for surface and air samples. For Bactiquant®-test, the filter material is critical for both
sampling and the enzyme reaction that takes place directly on the filter. It is important, therefore, to use the
filters provided by the vendor. The vendor provides a proficiency certification training program that is
included with the fluorometer kit (on a flash drive) and is mandatory for use of their technology to document
understanding and proper training.
VERIFICATION RESULTS
Table 1 summarizes the linearity results for Bactiquant®-test using two types of bacteria in water: indigenous
bacteria from lake water and a QC strain of Pseudomonas aeruginosa ATCC 27853. In Table 1 linearity is
evaluated for Bactiquant BQ values against concentration. The BQ values are fluorescence unit (fu) readings
standardized for reaction time, temperature, and sample volume. During the lake water indigenous bacteria
test, the vendor provided information that fluorescence readings above 20,000 fu may generate results that
are not linear because the enzyme substrate concentration will have decreased significantly, and the enzyme
reaction will slow down. Therefore, the linearity data for indigenous bacteria from lake water were
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Table 1. Linearity Results for Bactiquant®-test: BQ Value vs. Concentration
Test
Organism
Lake Water
Indigenous
Bacteria-
with all test
solutions
Lake Water
Indigenous
Bacteria -
without the
most
concentrated
test solution
P.
aeruginosa
ATCC 27853
Concentration
Range (CFU/mL)
3.7xl02to6.0xl03
3.7xl02to3.0xl03
8.7xl02to8.0xl03
Range of
Average
Adjusted
Fluorescence
Units (fu)
2408 to 24365
2408 to 17343
1321 to 11645
Range of
Average BQ
values
1542 to 15607
1542 to 11106
868 to 7655
Slope
2.38
3.55
0.95
Y-
intercept
2243
739
-136
Coefficient of
Determination
(R2)
0.9138
0.9689
0.9923
examined both with and without the most concentrated test solution that had fluorescence readings
consistently greater than 20,000 fu.
Table 2 summarizes the repeatability and inter-assay reproducibility results for Bactiquant®-test using two
bacterial cultures in water. Two different people analyzed four samples of each bacterial culture, using
different fluorometers.
Table 2. Bactiquant®-test Repeatability and Inter-Assay Reproducibility
Test Iteration
Average
Standard
Deviation
RSD (%)
RPD (%)
Adjusted Fluorescence Units (fu)
Indigenous Bacteria from Lake Water
(3.7xl02CFU/mL)
Analyst 1
2363
152
6.4
Analyst 2
2225
57
2.6
6.0
P. aeruginosa ATCC 27853
(4.7 x 103 CFU/mL)
Analyst 1
6888
333
4.8
Analyst 2
6691
93
1.4
2.9
Operational Factors. The verification staff found that the Bactiquant®-test was easy to use. A Mycometer
A/S representative came to Battelle to train the verification staff in the use of the Bactiquant®-test reagents
and operation of the fluorometer. This training lasted one day and staff felt it was more than sufficient to be
comfortable using the reagent kits and fluorometer without assistance. This on-site training focused on the
technology operating protocols for air and water matrices. While the operational aspects of this training
were similar to the proficiency certification program, the proficiency certification program also focuses on
understanding the principles behind the technology as well as additional applications.
The fluorometer is provided in a hard-cover carrying case. The carrying case has dimensions of 45 cm wide
x 15 cm deep x 32 cm high (17.5 in wide x 6 in deep x 12.5 in high) and weighs approximately 7.2
kilograms (16 pounds). Included with the fluorometer is a black calibration cuvette, a 100 uL automatic
pipette, a timer, two test racks, a calculator, a thermometer, and training materials. The fluorometer operates
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on four AAA batteries and has push-button operation. Testing staff found that the display was easy to read
and surfaces were easy to wipe clean. For the Bactiquant®-test, an instruction manual, a photo manual, and a
quick reference card were provided. Verification staff found that the instructions provided were not always
consistent among all three references and would have been confusing on ocassion had they not had training.
Per the vendor's instruction manual, the fluorometer required a calibration check with each series of
measurements using the black cuvette provided with the fluorometer and a calibration standard provided in
the reagent kit.
The Bactiquant®-test reagents are sold in lots of five for water assays. Each reagent kit included the
sampling filter, enzyme substrate, developer, and calibration standard, all of which were clearly labeled for
identification and storage conditions. Syringes and cuvettes used for processing were also included. All
containers and packaging were easy to open; however, verification staff found there was packaging waste
involved with the different components, particularly if multiple kits were needed to analyze the required
number of samples. All reagents were ready for use. Each sample resulted in approximately 5 mL of liquid
waste from the substrate and developer used to process the sample plus 250 mL of spent sample. Based on
the expiration date stamped on the kits, the shelf life of the kits received for testing was over one year from
receipt date. Several kit components required refrigeration. All components needed to prepare and analyze a
sample were included either in the reagent kit or the fluorometer kit. To process water samples, a vacuum
manifold and pump were needed for filtering the 250 mL samples. Both manual and automated filtration
apparatus are available through the vendor. For verification testing, only manual filtration apparatus was
used. Pricing for the fluorometer, reagent kits and filteration apparatus can be obtained from the provided
vendor contacts. Verification testing staff found they were able to collect and analyze ten water samples in
one hour using a five sample manifold to simultaneously filter five samples.
For data reduction, a laptop or personal computer is needed. Mycometer provides an Excel spreadsheet for
quantification of bacteria in water that converts fluorescence unit values into a 'BQ value' that standardizes
the fluorescence unit readings for reaction time, temperature and sample volume. Mycometer also provides
suggested interpretation guidelines based on the resulting BQ values obtained. These interpretation
guidelines were not verified as part of this test.
Signed by Tracy Stenner 01/06/2012 Signed by Cynthia Sonich-Mullin 02/01/2012
Tracy Stenner Date Cynthia Sonich-Mullin Date
Manager Director
Environmental Solutions Product Line National Risk Management Research Laboratory
Energy, Environment, and Material Sciences Office of Research and Development
Battelle U.S. Environmental Protection Agency
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