December 2011
Environmental Technology
Verification Report
Mycometer -test Rapid Fungi Detection and
Bactiquant®-test Rapid Bacteria Detection
Technologies
Prepared by
Bafteiie
/HI.' Business of Innovation
Under a cooperative agreement with
CrTr\ U.S. Environmental Protection Agency
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December 2011
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
Mycometer®-test Rapid Fungi Detection and
Bactiquant®-test Rapid Bacteria Detection
Technologies
by
Mary Schrock, Carol Riffle, Amy Dindal, Battelle
John McKernan and Julius Enriquez, U.S. EPA
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Notice
The U.S. Environmental Protection Agency, through its Office of Research and Development,
funded and managed, or partially funded and collaborated in, the research described herein. It
has been subjected to the Agency's peer and administrative review. Any opinions expressed in
this report are those of the author(s) and do not necessarily reflect the views of the Agency,
therefore, no official endorsement should be inferred. Any mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
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Foreword
The EPA is charged by Congress with protecting the nation's air, water, and land resources.
Under a mandate of national environmental laws, the Agency strives to formulate and implement
actions leading to a compatible balance between human activities and the ability of natural
systems to support and nurture life. To meet this mandate, the EPA's Office of Research and
Development provides data and science support that can be used to solve environmental
problems and to build the scientific knowledge base needed to manage our ecological resources
wisely, to understand how pollutants affect our health, and to prevent or reduce environmental
risks.
The Environmental Technology Verification (ETV) Program has been established by the EPA to
verify the performance characteristics of innovative environmental technology across all media
and to report this objective information to permitters, buyers, and users of the technology, thus
substantially accelerating the entrance of new environmental technologies into the marketplace.
Verification organizations oversee and report verification activities based on testing and quality
assurance protocols developed with input from major stakeholders and customer groups
associated with the technology area. ETV consists of six environmental technology centers.
Information about each of these centers can be found on the Internet at http://www.epa.gov/etv/.
Effective verifications of monitoring technologies are needed to assess environmental quality
and to supply cost and performance data to select the most appropriate technology for that
assessment. Under a cooperative agreement, Battelle has received EPA funding to plan,
coordinate, and conduct such verification tests for "Advanced Monitoring Systems for Air,
Water, and Soil" and report the results to the community at large. Information concerning this
specific environmental technology area can be found on the Internet at
http ://www. epa.gov/etv/centers/centerl .html.
in
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Acknowledgments
The authors wish to thank Dr. Timothy Dean of the U.S. EPA Air Pollution Prevention and
Control Division; Dr. Connie Schreppel, Director of Water Quality of the Mohawk Valley Water
Authority; and Dr. Nancy Clark Burton, Industrial Hygiene Team Leader for the Centers for
Disease Control and Prevention/National Institute of Occupational Safety and Health, for their
review of the Quality Assurance Project Plan and this verification report. Quality assurance
oversight was provided by Michelle Henderson and Laurel Staley, U.S. EPA, and Rosanna Buhl,
Battelle.
IV
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Contents
Page
Foreword iii
Acknowledgments iv
List of Abbreviations viii
Chapter 1 Background 9
Chapter 2 Technology Description 10
Chapters Test Design and Procedures 12
3.1 Introduction 12
3.2 Test Overview 12
3.3 Experimental Design 13
3.3.1 Mycometer -test for Fungi 13
3.3.2 Bactiquant -test for Bacteria 17
3.3.3 Data Completeness and Operational Performance Parameters 20
Chapter 4 Quality Assurance/Quality Control 21
4.1 Quality Control Samples 21
4.2 Audits 21
4.2.1 Technical Systems Audits 21
4.2.2 Data Quality Audit 22
4.3 Deviations 23
Chapters Statistical Methods 26
5.1 Linearity 26
5.2 Repeatability 26
5.3 Inter-Assay Reproducibility 26
5.4 Data Completeness 27
Chapter 6 Test Results 28
6.1 Characterization of Columbus, Ohio Tap Water Used for Testing 28
6.2 Mycometer®-test for Fungi 28
6.2.1 Linearity 28
6.2.2 Repeatability 32
6.2.3 Inter-Assay Reproducibility 33
6.2.4 Data Completeness 33
6.2.5 Operational Factors 34
6.3 Bacti quant®-test for Bacteria 35
6.3.1 Linearity 35
6.3.2 Repeatability and Inter-Assay Reproducibility 39
6.3.3 Data Completeness 40
6.3.4 Operational Factors 40
Chapter 7 Performance Summary 42
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7.1 Results for Mycometer®-test 42
7.2 Results for Bactiquant®-test 44
Chapter 8 References 47
Appendix Data from Tap Water Analyses 49
Figures
Figure 2-1. Mycometer®-test and Bactiquant®-test Fluorometer Kit Components 10
Figure 2-2. Principle of Fluorogenic Detection of Fungal or Bacterial Related Enzymatic
Activity in an Environmental Sample 11
Figure 3-1. ARCA Chamber 15
Figure 3-2. Sampling Cartridges Inside the ARCA Chamber with Tubing Leading to Pumps
Outside the ARCA Chamber 15
Figure 3-3. Bactiquant®-test Water Sample Filtration System 18
Figure 6-1. Plot of Mycometer®-test fluorescence response vs. A.flavus spore counts 30
Figure 6-2. Plot of Mycometer®-test fluorescence response vs. C. herbarum spore counts 32
Figure 6-3. Plot of Bactiquant®-test BQ Values vs. Lake Water Indigenous Bacteria
Concentration in CFU/mL -Responses less than 20,000 fu 37
Figure 6-4. Plot of Bactiquant®-test BQ Values vs. P. aeruginosa Concentration in CFU/mL.. 39
Tables
Table 3-1. Solutions Used to Generate Mycometer®-test Linearity Data 14
Table 3-2. Repeatability and Inter-assay Reproducibility Test Scheme for Mycometer®-test.... 16
Table 3-3. Solutions Used to Generate Bactiquant -test Linearity Data 19
Table 3-4. Repeatability and Inter-assay Reproducibility Test Scheme for Bactiquant®-test 20
Table 6-1. Mycometer®-test Linearity Data for Aspergillus flavus ATCC 58870 29
Table 6-2. Summary of Replicate Measurements for A. flavus ATCC 58870 Mycometer®-test
Linearity Data 30
Table 6-3. Mycometer®-test Linearity Data for Cladosporium herbarum ATCC 58927 31
Table 6-4. Summary of Replicate Measurements for C. herbarum ATCC 58927 Mycometer -test
Linearity Data 32
Table 6-5. Mycometer®-test Repeatability: Air Samples Containing A. flavus 33
Table 6-6. Mycometer®-test Inter-Assay Reproducibility: Air Samples Containing A. flavus .... 33
Table 6-7. MYCOMETER™ Analysis Equipment Kit 35
Table 6-8. Bactiquant®-test Linearity Data for Lake Water Indigenous Bacteria 36
Table 6-9. Summary of Replicate Measurements for Lake Water Indigenous Bacteria
Bactiquant®-test Linearity Data 37
Table 6-10. Bactiquant -test Linearity Data for Pseudomonas aeruginosa ATCC 27853 38
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Table 6-11. Summary of Replicate Measurements for P. aemginosa ATCC 27853 Linearity Data
- Adjusted Fluorescence 39
Table 6-12. Bactiquant -test Repeatability and Inter-Assay Reproducibility: 40
Indigenous Bacteria from Lake Water 40
Table 6-13. Bactiquant®-test Repeatability and Inter-Assay Reproducibility: 40
P. aeruginosa ATCC 27853 40
Table 7-1. Linearity Results for Mycometer®-test Adjusted Fluorescence vs. Total Spores Tested
42
Table 7-2. Mycometer®-test Repeatability: Air Containing A. Flavus 43
Table 7-3. Mycometer®-test Inter-Assay Reproducibility: Air Containing A Flavus 43
Table 7-4. Bactiquant®-test Linearity: BQ Value vs. Concentration 45
Table 7-5. Bactiquant®-test Repeatability and Inter-Assay Reproducibility 45
Table Al-1. Water Quality Parameters for Characterizing Columbus, Ohio Tap Water Used for
Testing 50
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List of Abbreviations
ADQ audit of data quality
AMS Advanced Monitoring Systems
ARCA aerosol research and component assessment
ASTM American Society for Testing and Materials
ATCC American Type Culture Collection
BQ Bacti quant value
CPU colony forming unit
cm centimeter(s)
COA certificate of analysis
°C degrees Celsius
EPA U.S. Environmental Protection Agency
ETV Environmental Technology Verification
fu fluorescence units
HPC heterotrophic plate counts
in inch(es)
LPM liters per minute
uL microliter(s)
mL milliliter(s)
min minutes
NRMRL National Risk Management Research Laboratory
NTU nephelometric turbidity unit
QA quality assurance
QAPP Quality Assurance Proj ect Plan
QC quality control
QMP Quality Management Plan
R2 coefficient of determination
RPD relative percent difference
RSD relative standard deviation
rtPCR real-time polymerase chain reaction
SM Standard Methods
TSA technical systems audit
UV ultraviolet
Vlll
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative 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.
ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
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 or 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 EPA's National Risk Management Research Laboratory (NRMRL) and its verification
organization partner, Battelle, operate the Advanced Monitoring Systems (AMS) Center under
ETV. The AMS Center recently evaluated the performance of Mycometer®-test rapid fungi
detection and Bactiquant®-test rapid bacteria detection technologies that are commercially
available from Mycometer A/S in Europe and Mycometer, Inc. in North America. These
technologies are based on fluorogenic detection of enzyme activities found predominantly in
fungal biomass for the Mycometer®-test and bacterial biomass for the Bactiquant®-test.
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Chapter 2
Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of
environmental monitoring technologies for air, water, and soil. This report provides results for
the verification testing of the Mycometer®-test rapid fungi detection and BactiQuant®-test rapid
bacteria detection technologies developed and patented by Mycometer A/S based in
Copenhagen, Denmark and available for distribution in the U.S. through Mycometer, Inc. The
following is a description of the Mycometer®-test and Bactiquant®-test, based on information
provided by the vendor.
Both the Mycometer®-test rapid fungi detection and
Bactiquant -test rapid bacteria detection technologies
(Figure 2-1) are based on fluorogenic detection of
enzyme activities found predominantly in a
taxonomic group of organisms. For the Mycometer -
test (correlating to fungal biomass) and the
Bactiquant®-test (correlating to bacterial biomass), a
sample (e.g., filter or swab) is contacted with a test
solution containing a synthetic enzyme substrate. The
enzyme present in the fungal cells or bacterial cells
hydrolyzes the synthetic enzyme substrate as shown
in Figure 2-2. When the synthetic substrate molecule
is cleaved into two molecules by the enzyme, one of
the molecules (as indicated by the yellow asterisk in
Figure 2-1. MycometerR-test and
Bactiquant®-test Fluorometer Kit
Components
Figure 2-2) can be made to fluoresce upon excitation with ultraviolet (UV) light at a wavelength
of 365 nanometers. The amount of fluorescence is measured using a handheld fluorometer after
processing for a reaction time based on the ambient temperature. This fluorescence correlates to
the fungal or bacterial biomass. The same fluorometer may be used to measure fluorescence for
both the Mycometer®-test and Bactiquant®-test. Fluorescence measurements can be captured
electronically and downloaded to a computer, or can be transcribed by hand. Sample preparation
and analysis can be performed on site in less than one hour.
_®
According to the vendor, the Mycometer -test for fungi is designed to measure both viable and
non-viable spores, hyphae and fungal particles such as hyphal fragments in air, on surfaces, or in
bulk materials to give a representation of the contamination in the environment. Although the
Mycometer®-test cannot distinguish between fungal genera or viable/non-viable fungi, it
provides a semi-quantitative measure of the total fungal biomass present. Air samples can be
collected with traditional air sampling pumps onto filter media. Typically 300 liters of air are
collected by sampling 20 liters per minute (LPM) for 15 minutes, or 15 LPM for 20 minutes.
Surface samples are collected by swabbing a nine square centimeter area and bulk material
samples are weighed. Enzyme substrate is added to the filter, swab, or bulk material and fungal
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enzyme reacts with the substrate to release a fluorescent product. The amount of fungi in the
sample is estimated by measuring the fluorescence produced.
In solution, the fluorophore absorbs UV light and
Enzyme re-emits visible light that can be measured with a
substrate with fluorophore fluorometer
attached
Microbial enzyme
hydrolyzes substrate
releasing fluorophore in
solution
Figure 2-2. Principle of Fluorogenic Detection of Fungal or Bacterial Related Enzymatic
Activity in an Environmental Sample
According to the vendor, the Bactiquant®-test is designed to provide a rapid method to estimate
total bacteria in water samples. With the Bactiquant -test, bacteria are concentrated from water
samples by passing the sample (typically 250 milliliters (mL)) through a membrane filter.
Enzyme substrate is added to the filter unit and left to react over a period of time based on
temperature. Bacterial enzyme reacts with the substrate, releasing a fluorescent product. The
amount of bacteria in the water sample correlates to the amount of the fluorescent product
released into the solution during the reaction period. 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 applied
to surface and air samples.
The fluorometer used for both Mycometer®-test and Bactiquant®-test is provided in a hard-cover
carrying case. The carrying case has dimensions of 45 centimeters (cm) wide x 15 cm deep x
32 cm high (17.5 inches (in) wide X6 in deep x 12.5 in high) and weighs approximately 7.2
kilograms (16 pounds). The fluorometer is provided with the components shown in Figure 2-1.
The Mycometer®-test and Bactiquant®-test reagents are sold separately in lots of 5-20 tests
depending on the type and includes the sampling media (swab, filter or other), enzyme substrate,
developer and calibration standard. Stationary vacuum manifolds for filtering up to five water
samples simultaneously or portable manifolds for up to two samples simultaneously are sold
separately through Mycometer, Inc. The recommended air sampling pumps (Gast 3-30 LPM IAQ
Pump w/Tubing & Rotameter) are commercially available. For both the Mycometer -test and
Bactiquant®-test, the filter material and the type and material of the air sampling filter cartridge
are critical for both sampling and the enzyme reaction that takes place directly on the filter.
Therefore, it is important 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.
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Chapter 3
Test Design and Procedures
3.1 Introduction
The ETV AMS Center Water and Air Stakeholder Committees identified the use of rapid fungal
and bacterial detection technologies as an area of interest for technology verification. Rapid
technologies (results available same day of testing) to detect fungi and 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 or water systems, and for
monitoring progress during cleanup or remediation processes. Microbial contamination has the
potential to cause health problems. Fungi are known to produce allergens, irritants, and
potentially toxic substances1 resulting in asthma, respiratory infections and a variety of allergic
reactions2, and bacteria ingested through the water supply can cause illnesses.3
Traditional methods of analysis for bacteria in drinking water include plate count and
microscopy for total counts (e.g., heterotrophic plate count or direct total microbial counts) and
specialized analysis for indicator organisms such as Escherichia coll (E. coll). Plating methods
are time consuming and can take up to seven days for results using traditional methods such as
heterotrophic plate counting. Microscopic techniques such as direct microbial counts using
epifluorescence are faster, but do not estimate microbial biomass or viability, and require an
experienced analyst for differentiation of cells from other water constituents. Detection of fungi
in air is tenuous using methods such as spore trap air sampling and analysis for identifying fungi
present, quantifying spores, and assessing background debris (such as pollen). This technique is
subject to variation due to concentrations of airborne particles (spores, hyphae, and debris) and
analyst-to-analyst variability associated with microscopic techniques.
Technologies such as real-time polymerase chain reaction (rtPCR) can provide same day results,
typically within a few hours or overnight and have increased accuracy and sensitivity. However,
cost and time are the trade-offs to be considered with this type of technology. Screening
technologies to monitor changes in water or air quality that are fast and affordable would help to
control microbial outbreaks, expedite remediation efforts, and protect public health.
It should be noted that U.S. ETV verification does not represent an approval of methods for
regulatory compliance.
3.2 Test Overview
This verification test was conducted according to procedures specified in the Quality Assurance
Project Plan for Verification of Mycometer®-test Rapid Fungi Detection and Bactiquant®-test
Rapid Bacteria Detection Technologies4 (QAPP), and adhered to the quality system defined in
the ETV AMS Center Quality Management Plan (QMP).5 As indicated in the QAPP, the testing
conducted satisfied EPA QA Category III requirements. The QAPP and this verification report
were reviewed by:
• Dr. Timothy Dean, U.S. EPA Air Pollution Prevention and Control Division
• Dr. Connie Schreppel, Director of Water Quality of the Mohawk Valley Water Authority
• Dr. Nancy Clark Burton, Industrial Hygiene Team Leader for the Centers for Disease
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Control and Prevention/National Institute of Occupational Safety and Health
In addition, the QAPP in general was reviewed with the broader AMS Center Stakeholder
Committee as a presentation during regular stakeholder teleconferences, including the September
11, 2008 and January 22, 2009 meetings. Input from the water and air committees was also
solicited during email updates of AMS Center activities in June 2010.
The Mycometer®-test and Bactiquant®-test technologies were verified for repeatability and inter-
assay reproducibility by detecting fungi in air samples and bacteria in water samples,
respectively. Linearity was assessed for both technologies using dilutions of stock cultures in tap
water. The linearity test for fungi was a modification of test procedures in place for air and
surface samples. In addition, sustainable operational factors are reported such as ease of use,
required reagents, analysis time, laboratory space, and utilities required.
3.3 Experimental Design
3.3.1 Mycometer®-test for Fungi
®
3.3.1.1 Mycometer -test Linearity. Mycometer -test linearity was demonstrated using two
fungal cultures from American Type Culture Collection (ATCC), Cladosporium herbarum
ATCC 58927 and Aspergillus flavus ATCC 58870. These two fungal cultures were chosen
based on their presence in indoor fungal isolates as reported in the literature.6'7'8'9 The specific
strains (species designation and ATCC number) were selected based on their being isolated from
air samples as indicated by ATCC. The ATCC cultures were confirmed based on a Certificate of
Analysis (COA) provided by ATCC. A dilution series for each of the fungal cultures (based on
the spore counts) was performed using dechlorinated tap water. Tap water used for dilution was
collected and dechlorinated with sodium thiosulfate as detailed in the QAPP Section B2.2.4 The
tap water was characterized for pH, free chlorine, and total chlorine. It was also characterized by
Pace Analytical (Columbus, OH) for turbidity, total organic carbon, specific conductivity,
alkalinity, hardness, and dissolved oxygen. These characterization results were not used in
evaluating the technologies, but were included in the appendix for informational purposes since
tap water characteristics can vary from location to location.
The dilution series originally targeted a test range of approximately 500 to 50,000 spores/mL of
enzyme substrate based on vendor communication that these concentrations should be expected
to generate fluorescence in the range typically encountered by a user. However, during the
training session with the vendor, it became apparent that these concentrations did not provide
sufficient fluorescence response. As a result, a deviation (Deviation Number 2) was prepared to
change the target test concentration to approximately 2.4 x 105to 4.8 x 106 spores/mL of enzyme
substrate. Test solutions were prepared by diluting a stock solution with dechlorinated tap water
to target a range of 5.0 x 106 to 1.0 x 108 spores/mL. The neat stock solution, along with three
dilutions of the stock solution (1:5, 1:10, and 1:20) were used for linearity testing. Preliminary
testing conducted during training confirmed that the stock solution generated sufficient
fluorescence and that the dilutions were likely to be detectable. The actual stock solution
concentration in spores/mL for each fungal culture was evaluated using a hemocytometer
following procedures in American Society for Testing and Materials (ASTM) D4300-01 Annex10
and was based on five replicate analyses for each fungal stock. Table 3-1 shows the actual
concentrations used in testing. The stock solution and each of the three dilution concentrations
were sub-sampled in five separate iterations. Each iteration involved processing one sample per
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concentration level plus a blank. All samples were processed identically by adding 100
microliters (uL) of test sample (dechlorinated tap water for the blank) to 2 mL of enzyme
substrate and incubating the solution for approximately 32 minutes based on temperature. The
contents of the Mycometer®-test kit developer vial were then added to the test sample/enzyme
substrate mix and the fluorescence was measured.
.®
Table 3-1. Solutions Used to Generate Mycometer -test Linearity Data
Actual
Concentration of
Stock Dilutions*
(spores/mL)
Volume (ml)
of Stock
Dilution added
to 2 ml
Enzyme
Substrate to
create Test
Solution
Total
Spores
Tested
Final
volume
of Test
Solution
(ml)
Actual Test
Solution
Concentration
(spores/mL of
Enzyme
Substrate)
Number of
Aliquots
Processed
Equivalent
Concentration
in Air**
(spores/m3)
Aspergillus flavus ATCC 58870
NEAT: 6.2x10'
1:5: 1.2x10'
1:10: 6.2x10°
1:20: 3.1 x10°
0.10
0.10
0.10
0.10
6.2x10°
1.2x10°
6.2x10°
3.1 x10D
2.1
2.1
2.1
2.1
3.0x10°
5.9x10°
3.0x10°
1.5x10°
5
5
5
5
2.0x10'
3.9x10°
2.0x10°
9.8x10°
Cladosporium herbarum ATCC 58927
NEAT: 9.6x10'
1:5: 1.9x10'
1:10: 9.6x10°
1:20: 4.8x10°
0.10
0.10
0.10
0.10
9.6x10°
1.9x10°
9.6x10°
4.8x10°
2.1
2.1
2.1
2.1
4.6x10°
9.1 x10°
4.6x10°
2.3x10°
5
5
5
5
3.0x10'
6.1 x10°
3.0x10°
1.5x10°
*NEAT solution concentration measured from hemocytometer counts. Dilutions calculated by dividing the NEAT
solution concentration by the dilution factor.
"""Calculated as the air concentration necessary to generate a test solution concentration the same as the actual test
solution when a 300 L (0.30 m3) air sample is collected and processed with 2 mL enzyme substrate (e.g. actual test
solution concentration in spores/mL of enzyme substrate * 2.0 mL enzyme substrate/0.30 m3).
According to the vendor, data can be transferred from the fluorometer to a computer. Originally,
this was the intended method of data transfer for further data reduction; however, training for the
data transfer software was not included as part of the vendor-provided training to use the
technology. All fluorescence readings were recorded by hand onto data sheets, and transcribed
into spreadsheets provided by Mycometer for further calculation. This change in the data
recording procedure was documented as Deviation Number 7. The Mycometer®-test adjusted
fluorescence values (fluorescence unit reading for the test solution - fluorescence unit reading
for the blank) were plotted against the concentration of spores in each test solution displayed as
the total number of spores tested to generate linearity data.
3.3.1.2. MycometerR -test Repeatability and Inter-Assay Reproducibility. Mycometer®-test
repeatability and inter-assay reproducibility were evaluated by producing controlled air samples
in Battelle's Aerosol Research and Component Assessment (ARCA) chamber (Figure 3-1) and
sampling and analyzing the air using the Mycometer®-test technology. A total of eight air pumps
(GAST Model 1532 Pumps with IAQ option) supplied by the vendor was used. These pumps
were placed outside of the chamber and were connected by tubing to the Mycometer®-test air
sampling cartridges inside the chamber. The eight air sampling cartridges were arranged in close
proximity inside the chamber on two tripod stands holding four Mycometer®-test sampling
cartridges each (Figure 3-2). The flow rate for each pump was adjusted using a calibrated flow
meter (Sierra Instruments, Model 821 or 822, Monterey, CA). Originally, samples were to be
collected using a sampling flow rate of 20 LPM for 15 minutes to provide a total air volume of
300 L. This was based on guidance in the Mycometer 2008 air sampling protocol.11 During
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repeatability and inter-assay reproducibility tests, a flow rate of 20 LPM could not be established
in all eight vendor-supplied pumps. This resulted in a deviation for the sampling flow rate and
collection time (Deviation Number 4). Sampling was conducted using a flow rate of 15 LPM for
20 minutes, providing a total air volume of 300 L following an option in an updated air sampling
protocol provided by Mycometer.12 One fungal stock, A.flavus, was used to determine
repeatability and inter-assay reproducibility. A. flavus was selected based on the fluorescence
response observed during linearity testing and the consistency of linearity data.
Figure 3-1. ARCA Chamber
Figure 3-2. Sampling Cartridges Inside the ARCA Chamber with Tubing Leading to
Pumps Outside the ARCA Chamber
In order to produce an aerosol sample resulting in a fluorescence signal approximately 300
fluorescence units (fu) above the level of a blank, an initial characterization run was performed.
The starting spore stock concentration for generation of the aerosol used in the initial
characterization (1.24 x 107 spore/mL) was estimated based on the fluorescence responses
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observed during the A. flavus linearity test and calculations outlined in the QAPP.4 Analysis of
eight samples generated with this stock solution generated fluorescence ranging from 38 to 47 fu,
below the goal of 300 fu above the blank; therefore, for testing, a spore stock concentration of
6.2 x 107 spores/mL was used and the chamber flow rate was lowered in attempt to increase the
air sample fluorescence readings.
For testing, the 6.2 x 107 spores/mL of A. flavus solution was released into the chamber using a
generation rate of 0.5 mL/minute (min) through a Collison nebulizer aerosol generator and a
chamber velocity of 5,000 L/min for an expected chamber aerosol concentration of 6.2 x 103
spores/L of air. Once the system reached steady state as determined by checking aerodynamic
particle sizer concentration readings, sampling commenced. A total of 300 L of air was pumped
through each of the eight sampling filters simultaneously for an approximate expected sample
concentration of 1.9 x 106 spores/sample. During the inter-assay reproducibility chamber run,
the chamber air flow between the beginning and middle of the run dropped by 18%, falling
below the ± 10% measurement quality objective. The flow was not adjusted to avoid further
fluctuation in air flow and remained steady from the middle to the end of the run. At this lower
flow rate, the A. flavus spores were still uniformly collected on all of the filters. This slight
change to flow rate was considered to have no impact on the test since a specific concentration
on the filters was not targeted.
Repeatability was determined by having one vendor-trained analyst process samples from
cartridges connected to all eight pumps from one chamber test using one fluorometer. For inter-
assay reproducibility, two vendor-trained analysts each processed samples on cartridges
connected to four pumps during one chamber test. Each analyst used a separate fluorometer. The
repeatability and inter-assay reproducibility test scheme is described further in Table 3-2. The
QAPP4 originally stated that one analyst would perform both sampling and analysis from all
eight pumps for repeatability testing and two analysts would each perform sampling and analysis
for the inter-assay reproducibility testing. Because of ARC A chamber access restrictions, a
deviation to the sampling scheme was required (Deviation Number 5). Only one analyst, serving
the role of Analyst 2 in Table 3-2, physically assembled and removed the sample filter for all air
samples collected in the ARCA chamber. However, all sample processing was carried out as
intended and as outlined in Table 3-2. Eight background air samples were collected in the
chamber prior to release of the fungal culture. Each repeatability and inter-assay reproducibility
air sample set (test sample and background) was processed with a blank. Blanks consisted of an
air sampling filter through which no air passed. Blanks were handled, processed, and analyzed
in the same manner as the air samples.
Table 3-2. Repeatability and Inter-assay Reproducibility Test Scheme for Mycometer®-test
Analyst
Analyst 1
Analyst 2
Fluorometer Unit
A
B
Number of Repeatability
Samples
8
None
Number of Inter-assay
Reproducibility Samples
4
4
The chamber schedule used for generation of repeatability and inter-assay reproducibility
samples is listed below:
• Decontaminate the ARCA test chamber (prior to test),
• Set up,
• Characterization run to check test parameters and stock concentration,
• Perform an air wash of the chamber,
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• Collect eight background samples (Mycometer®-test analysis),
• Run one test run with A. flavus to collect eight repeatability samples (Mycometer®-test
analysis),
• Perform an air wash of the chamber,
• Surface decontaminate the tripod stands,
• Collect eight background samples - split between two analysts (Mycometer®-test
analysis),
• Run one test with an aerosolized fungal stock to collect eight inter-assay reproducibility
samples - split between two analysts (Mycometer®-test analysis),
• Perform an air wash of the chamber,
• Remove tubing and surface decontaminate the tripod stands, and
• Decontaminate the chamber (after test day).
3.3.2 Bactiquant*-test for Bacteria
3.3.2.1 Bactiquanf-test Linearity. Bactiquant®-test linearity was determined using two types of
bacterial stocks: a quality control (QC) strain consisting of Pseudomonas aeruginosa ATCC
27853 obtained as a QuantiCult culture from Remel, Inc. and a consortium of indigenous flora
in water from a local lake. Both bacterial stocks were diluted with dechlorinated Columbus,
Ohio tap water to prepare solutions for linearity testing. Tap water was collected and
dechlorinated as described in the QAPP Section B2.24 and as noted above for the Mycometer®-
test. The P. aeruginosa QC strain was prepared following the manufacturer's directions. The
working stock for spiking into tap water was initially planned to be grown on a low nutrient R2A
agar. Prior to testing, the vendor expressed concern that the content of hydrolyzed milk protein
in R2A agar might have an effect on the Bactiquant -test analysis as the vendor had never used
this medium to generate bacteria for testing with their technology. The vendor did have
considerable experience using yeast extract and, therefore, a deviation was prepared to use yeast
extract agar to grow the working stocks of P. aeruginosa (Deviation Number 1).
The original target concentration for the bacterial stocks ranged from approximately 50 to 50,000
colony forming units per milliliter (CFU/mL). However, during the technology training session
with the vendor, it was apparent that these concentrations would not provide sufficient
fluorescence for the P. aeruginosa QC strain using the reaction time and sample volumes agreed
to for verification testing. Therefore, adjustments were made to the concentrations for both the
indigenous flora from lake water (Deviation Number 3) and the P. aeruginosa QC strain
(Deviation Number 6).
Indigenous Bacteria from Lake Water. The lake water was first analyzed neat (no dilution with
dechlorinated tap water) to determine the water fluorescence reading. Following the
Bactiquant®-test processing procedures, 250 mL of lake water were filtered and processed using
the Bactiquant®-test reagents. The fluorescence response of the neat water was 48,384 fu. Based
on this, the four sample concentrations selected for testing were a 1:5, 1:10, 1:20, and 1:100
dilution of the neat lake water using dechlorinated tap water. Each of the four test concentrations
were sub-sampled five times and processed using the Bactiquant®-test reagents. The samples
(250 mL) were filtered as shown in Figure 3-3. Each filter was processed by flushing the filter
with 2.5 mL of enzyme substrate and incubating the filter for 30 minutes at room temperature.
This reaction was terminated by flushing the filter with developer solution provided in the kit.
The fluorescence of the resulting solution was then measured. The actual bacterial concentration
of each solution used for testing was determined using heterotrophic plate counts (SM 921513)
17
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conducted in triplicate. For the 1:100 dilution, the heterotrophic plate counts (HPC) were too
low for reliable count; therefore, the concentration of this solution was determined by taking the
neat water HPC- determined concentration and dividing by the dilution factor of 100. The HPC
results for the lake water solutions used in testing are shown in Table 3-3. Linearity data was
generated by plotting the Bactiquant®-test fluorescence results against the HPC-determined
concentration of bacteria in each testing solution (CFU/mL).
Figure 3-3. Bactiquant -test Water Sample Filtration System
Pseudomonas aeruginosa ATCC 27853. Following the training session, the vendor provided
additional information from the vendor's experiments with a different strain of P. aeruginosa. In
these experiments, solution concentrations of 5.0 x 103 to 5.0 x 106 CFU/mL resulted in
Bactiquant (BQ) values ranging from 50 to 55,000. BQ values are calculated values adjusting
the fu results to the standard reaction time (30 minutes), temperature (23 °C), and sample volume
(250 mL) and are lower than actual fluorescence readings. The BQ value is calculated as:
250 30
BQ = (Fs - Fb} x 0.59 x — x — x 3.3344e(-ao522xT)
Where Fs is the sample fluorescence, Fb is the blank fluorescence, V is the volume of the water
sample in milliliters, R is the reaction time in minutes, and T is the room temperature in degrees
Celsius.
In this equation, 0.59 is a transformation constant that adjusts the results to an earlier Bactiquant
protocol. The exponential function adjusts for the influence of temperature on the reaction rate.
The vendor also noted in separate communication that high fluorescence readings (> 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, to generate
detectable fluorescence that would not exceed 20,000 fu, a P. aeruginosa solution containing
approximately 5.0 x 105 CFU/mL was prepared from a working stock with turbidity equivalent
to a 0.5 McFarland standard (estimated concentration ranging from 1 x 107 to 1 x 108 CFU/mL).
18
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This working stock was diluted by a factor of 100 with dechlorinated tap water to make the
starting solution with a target concentration of approximately 5.0 x 105 CFU/mL.
A single 250 mL sample of this starting solution was filtered and processed. If the fluorescence
reading for this starting solution was between 1,000 and 20,000 fu, testing was to proceed using
the starting solution and dilutions of the starting solution with dechlorinated tap water by factors
of 1:2, 1:5, and 1:10 (filtering 250 mL of each dilution). The actual fluorescence reading of the
starting solution was 12,784 fu and so the starting solution, 1:2, 1:5 and 1:10 dilutions were used
for testing. The actual bacterial concentration of each solution used for testing was determined
using HPC conducted in triplicate. The HPC results for the P. aeruginosa solutions used in
testing are shown in Table 3-3. The concentration of the P. aeurignosa starting solution was
approximately two logs lower than the target concentration. The working stock and all dilutions
prepared in dechlorinated tap water may have had reduced viability or stressed the organisms as
a result of the change in osmotic pressure when the culture was introduced into the water
resulting in lower counts. Linearity data was generated by plotting the Bactiquant®-test
fluorescence results against the HPC-determined testing solution concentrations (CFU/mL).
3.3.2.2. Bactiquant®-test Repeatability and Inter-Assay Reproducibility. Inter-assay
reproducibility and repeatability were determined by having two vendor-trained analysts each
perform sampling and analysis of four sub-samples taken from one concentration of tap water
spiked with indigenous flora (3.7 x 102 CFU/mL) and four sub-samples from one concentration
of tap water spiked with P. aeruginosa (4.7 x 103 CFU/mL). Each analyst used a separate
fluorometer. This testing scheme is further described in Table 3-4. Repeatability evaluated
variability of the performance of the technology by each analyst and inter-assay reproducibility
evaluated variability of the performance of the technology between analysts and fluorometers.
Table 3-3. Solutions Used to Generate Bactiquant®-test Linearity Data
Actual Concentration of
Bacteria in Testing Solution*
(CFU/mL)
Volume (ml) of
Testing Solution
Filtered
Total CFU
Tested
Number of
Aliquots
Processed
Indigenous Bacteria from Lake Water
NEAT: 3.7x10"
1:5: 6.0x10J
1:10: 3.0x10J
1:20: 1.3x10J
1:100: 3.7x10^
250
250
250
250
1.5x10°
7.5x10°
3.3x10°
9.3x10"
5
5
5
5
Pseudomonas aeruginosa ATCC 27853
Starting solution: 8.0 x 10J
1:2: 4.7x10J
1:5: 2.1 x10J
1:10: 8.7x10^
250
250
250
250
2.0x10°
1 .2 x 1 0D
5.3x10°
2.2x10°
5
5
5
5
*Each testing solution concentration was determined from heterotrophic plate count measurements conducted in
triplicate. Plates with counts outside of the 30-300 target were estimated.
19
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Table 3-4. Repeatability and Inter-assay Reproducibility Test Scheme for BactiquantR-test
Analyst
Analyst 1
Analyst 2
Fluorometer
Unit
A
B
Number of
Repeatability* Samples-
Indigenous Flora
4
4
Number of
Repeatability* Samples-
P. aeruginosa
4
4
*Repeatability sample results were also used to generate inter-assay reproducibility data.
3.3.3 Data Completeness and Operational Performance Parameters.
For both technologies, data completeness was determined from a review of the valid data (i.e.,
data that met all measurement quality objectives [MQO]) collected during the verification testing
period against the expected amount of total data to be generated. Operational performance
parameters such as maintenance requirements, ease of use, sustainability factors, and portability
were determined from observations by the Battelle testing staff.
20
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Chapter 4
Quality Assurance/Quality Control
QA/QC procedures were performed according to the QAPP for this verification test4 and the QMP
for the AMS Center5. QA/QC procedures and results are described in the following subchapters.
During testing, there were eight deviations from the QAPP. Deviations are described in Section
3.3.2.1 (Deviation 1), 3.3.1 (Deviation 2), 3.3.2.1 (Deviation 3), 3.3.1.2 (Deviations 4 and 5),
3.3.2.1 (Deviation 6), 3.3.1.1 (Deviation 7) and 6.1 (Deviation 8) and discussed in Section 4.4.
These deviations were judged by the Battelle Verification Test Coordinator to not result in any
adverse impacts on the quality of the data generated. The deviations were reviewed by the EPA
ETV AMS Center Project Officer and EPA ETV AMS Center Quality Manager.
4.1 Quality Control Samples
A blank was processed with every sample set. For the Mycometer®-test linearity testing, the
blank consisted of 100 uL of dechlorinated tap water processed with the kit reagents and
procedures as a sample. For Mycometer®-test repeatability and inter-assay reproducibility, the
blank consisted of an air sampling filter through which no air passed, processed with the kit
reagents and procedures as a sample. For the Bactiquant®-test, the blank was prepared using the
blank reagents provided in the Bactiquant®-test kit by adding 0.35 mL of enzyme substrate to a
cuvette containing the developer, and processing it as a sample. All blanks had fluorescence
readings below the measurement quality objective specified for this verification test of 300 fu.
For the HPC tests used to determine bacterial concentrations of test solutions, a positive media
control, negative media control, and diluent blank controls were prepared each day that test
solutions were plated. All positive media controls exhibited growth, and the negative media
controls and diluent blanks exhibited no growth each day plating was conducted.
4.2 Audits
Two types of audits were performed during the verification test; a technical systems audit (TS A)
of the verification test procedures, and an audit of data quality (ADQ). Audit procedures for the
TSAs and ADQs are described further below.
4.2.1 Technical Systems Audits
The Battelle AMS Center Quality Manager or designee performed two TSAs throughout testing.
The first TSA for Mycometer®-test was conducted in two phases on May 19 and June 9-10, 2011
at Battelle's microbiology laboratory in Columbus, OH. The EPA AMS Center Project Officer
21
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participated in the audit on May 19. The second ISA for Bactiquant®-test was conducted on June
2, 2011. The TSAs consisted of interviews with Battelle personnel, observations of test sample
preparation and observation of sample analysis during testing at Battelle. The purpose of these
audits was to verify that:
• Sample preparation procedures were performed by Battelle according to the QAPP
requirements;
• Reference methods for analyzing test samples conformed to the QAPP and reference
method requirements;
• Technology testing was performed according to the QAPP and vendor instructions;
• Test documentation provided a complete and traceable record of sample preparation and
analysis; and
• Equipment used in the test was calibrated and monitored according to QAPP requirements
and standard laboratory procedures.
Zero (0) Findings, two (2) Observations, and one (1) Comment were identified during the first
TSA. Zero (0) Findings, three (3) Observations, and zero (0) Comments were identified during
the second TSA. It was determined by Battelle that none of these had an adverse impact on the
test results and all Observations and Comments have received a satisfactory response.
In response to these audit reports, the following actions were taken:
• Deviation 5 was prepared to correctly describe the collection of samples generated in the
ARC A chamber;
• Deviation 7 was prepared to describe that data was recorded by hand, not electronically;
• Deviation 8 was prepared to describe the tap water fluorescence measurements made;
• Data records were updated where clarification was needed to facilitate understanding of
procedures.
Two separate TSA reports were prepared and distributed to EPA.
4.2.2 Data Quality Audit
Records generated in the verification test received a one-over-one review before these records
were used to calculate, evaluate, or report verification results. Data were reviewed by a Battelle
technical staff member involved in the verification test. The person performing the review added
his/her initials and the date to a hard copy of the record being reviewed.
In addition, ADQs were conducted for Day 1 Mycometer test results on May 19; for Day 1
Bactiquant test results on July 7; and for Days 3 and 4 test results and the final report on August
4 - 11, 2011. During the audits, laboratory data generated at Battelle using the Mycometer®-test
and Bactiquant®-test were reviewed and verified for completeness, accuracy and traceability. The
EPA quality system utilizes a "graded approach" for establishing the appropriate level of QA/QC
for various types of research activities based on the intended use of the data and the visibility of
the research effort dictate the required level of quality. The verification of rapid fungi and
bacteria detection technologies was determined by the EPA AMS Center Project Officer to be a
Category III test. "Category III" establishes the QA/QC requirements for projects involving
applied research or technology evaluations. In addition to preparation of the QAPP, Category III
projects require a technical systems audit and maintenance of project data for 20 years. The ETV
program further requires an audit of data quality for each project. Accordingly, at least 10% of
22
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the results for each of the testing scenarios were verified versus the raw data, and 100% of the QC
sample results were verified. The data were traced from the initial acquisition, through reduction
and statistical analysis, to final reporting to ensure the integrity of the reported results. Data
verification included re-calculation of intermediary and final test results from the raw data files.
In all, four (4) Findings and seven (7) Observations were identified during the ADQs. The
findings involved sample custody, missing test data, missing documentation, and failed quality
control (QC) objectives. Battelle believes that none of these had an adverse impact on the test
results and all have received a satisfactory response.
In response to these audit reports, the following actions were conducted:
• Testing forms were updated to prompt for documentation of calibration readings;
• The excel spreadsheet for Bactiquant®-test repeatability was corrected for a cell marked as
text that was not being included in calculations;
• Data records were updated where clarification was needed to facilitate understanding of
procedures; and
• The report was revised to discuss the impact of the failed QC.
Three separate ADQ reports were prepared and distributed to EPA.
4.3 Deviations
Eight deviations were documented during testing:
Deviation 1 (4-15-11): The QAPP stated that the working stock of P. aeruginosa would be grown
on the low nutrient medium, R2A agar. However, the vendor expressed concern after QAPP
approval that the content of hydrolyzed milk protein in R2A agar might have an effect on the
Bactiquant®-test analysis and recommended that yeast extract agar be used to grow the working
stocks of P. aeruginosa. Yeast extract agar was used to grow the P. aeruginosa working stocks.
Impact: None; the yeast extract agar was a suitable growth medium that eliminated the vendor's
concern with R2A agar.
Deviation 2 (5-18-2011): The QAPP stated that fungal linearity testing would target a test
concentration range of 500-50,000 spores/mL of enzyme substrate; however, during the
technology training session with the vendor, it became apparent that these concentrations did not
provide sufficient fluorescence response. The target test solution concentration range was
changed to approximately 240,000 to 4,800,000 spores/mL of enzyme substrate based on range
finding experiments conducted during the training session. Impact: None, the revised
concentration range provided test solutions that had a response with the vendor's technology in a
range that will be useful to the user.
Deviation 3 (5-27-2011): The QAPP stated that bacterial linearity testing would target using
stock solutions of 50-50,000 CFU/mL; however, during the technology training session with the
vendor using the P. aeruginosa strain, it became apparent that these concentrations did not
provide sufficient fluorescence response. For the indigenous bacteria in lake water, in order to
determine solution concentrations that would provide sufficient fluorescence response, the lake
water was processed neat to obtain a base fluorescence measurement (48,384 fu). Based on this
result, the four sample concentrations selected for testing were a 1:5, 1:10, 1:20, and 1:100
dilution of the neat lake water. The actual bacteria concentrations of each solution were
23
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determined using HPC. Impact: None, the revised approach to obtaining concentrations of
indigenous bacteria that would provide a fluorescence response were obtained in a manner that
more closely simulated collection and analysis of a real-world water sample, as opposed to
targeting a specific bacteria count that might not have provided sufficient fluorescence.
Deviation 4 (6/9/2011): The QAPP stated that air sampling for the Mycometer®-test repeatability
and inter-assay reproducibility tests would be conducted using a sampling flow rate of 20 LPM
for 15 minutes to collect a total air volume of 300 L. This was based on guidance in the
Mycometer protocol "Quantification of mold in air - Protocol for quantification of fungal
propagules in air samples using the Mycometer-test" (2008). For the training session Mycometer
provided an updated protocol, "Mycometer®-air Sampling and analysis," that included the option
of collecting the 300 L volume by either 20 LPM for 15 minutes, or 15 LPM for 20 minutes.
During set up for the repeatability and inter-assay reproducibility tests, a flow rate of 20 LPM
could not be established in all 8 pumps that would be used for testing; therefore, the sampling was
conducted using 15 LPM for 20 minutes for all samples so that all pumps were operating under
identical flow rate and collection time. Impact: None; a 300 L air sample was collected uniformly
with all eight pumps provided for testing.
Deviation 5 (6/9/2011): The QAPP stated that one analyst would perform sampling and analysis
of air samples collected from all eight pumps for the Mycometer®-test repeatability test and two
analysts would each perform sampling and analysis for the Mycometer®-test inter-assay
reproducibility test. Because of limitations on who can be in the ARC A chamber for collection of
samples, only one analyst physically hooked up and removed the sample filter for all air samples
collected in the ARCA for both the repeatability and inter-assay reproducibility tests. The filter
samples were then distributed for sample preparation and analysis as described in QAPP. Impact:
None; the sample processing and analysis were carried out as intended to show repeatability and
inter-assay reproducibility with the Mycometer®-test reagents and fluorometers.
Deviation 6 (6/21/2011): The QAPP stated that bacterial linearity testing would target using stock
solutions of 50-50,000 CFU/mL; however, during the technology training session with the vendor
using the P. aeruginosa strain, it became apparent that these concentrations did not provide
sufficient fluorescence response. Therefore, to attempt to generate detectable fluorescence a P.
aeruginosa solution of approximately 5.0 x 105 CFU/mL was prepared by creating a working
stock with turbidity equivalent to a 0.5 McFarland standard. This working stock was diluted by a
factor of 100 to make a starting solution with a concentration of approximately 5.0 x 105 CFU/mL
"7 o
(based on the turbidity resulting in an estimated concentration ranging from 1x10 to 1x10
CFU/mL). A single 250 mL sample of the starting solution containing approximately 5.0 x 105
CFU/mL was filtered and processed. The fluorescence reading of this starting solution was
12,784 fu and so the starting solution, 1:2, 1:5 and 1:10 dilutions were used for testing. The
actual bacterial concentration of each solution used for testing was determined using UPC.
Impact: None, the revised concentration range provided test solutions that had a response with the
vendor's technology in a range that will be useful to the user.
Deviation 7 (7/7/2011): The QAPP stated that Bactiquant®-test and Mycometer®-test
fluorescence readings, and calculated Mycometer®-test air fungal concentration values would be
recorded electronically by each technology unit and then downloaded to a computer daily.
However, electronic transfer of data was not part of the technology training and so all
fluorescence reading values were recorded by hand onto data sheets and then hand-typed into
24
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Mycometer spreadsheets for further calculation. Impact: Battelle believes that this did not affect
the data generated, only the means in which data was recorded.
Deviation 8 (7/21/2011): The QAPP stated that tap water blanks would be run during sample
analysis to determine the fluorescence associated with the water. This was to be conducted as
part of the tap water characterization and was performed during the days of Mycometer®-test
mold linearity testing and the Bactiquant®-test lake water indigenous bacteria testing, but was
inadvertently omitted from the day of Bactiquant®-test P. aeruginosa testing. Impact: None; a
measure of the tap water fluorescence was not required to generate the verification test data and
was only included to provide information to help characterize the tap water. Tap water was
evaluated at least once with the Mycometer®-test and once with the Bactiquant®-test during
verification testing.
25
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Chapter 5
Statistical Methods
The statistical methods used to evaluate the quantitative performance factors are presented in this
chapter. Qualitative observations were also used to evaluate verification test data.
5.1 Linearity
Linearity with respect to concentration (determined as heterotrophic plate counts for bacteria and
as spore counts for fungi) was assessed by a linear regression analysis of the Mycometer®-test and
Bactiquant®-test fluorescence units using the spore counts or heterotrophic plate counts as
appropriate as the independent variable and the Mycometer®-test and Bactiquant®-test results as
the dependent variable. The results were plotted and linearity expressed in terms of slope,
intercept, and coefficient of determination (R2).
5.2 Repeatability
Repeatability was determined as percent relative standard deviation (%RSD) of the replicate
measurements of fungal and bacterial cultures taken with the Mycometer®-test and Bactiquant®-
test, respectively. Equations 1 and 2 were used to calculate repeatability:
1/2
(1)
S = , „
~" k=\
Where S is the standard deviation, n is the number of replicate samples, A-4 is the technology
fluorescence measurement for the kth sample, and Mis the average technology fluorescence
measurement of the replicate samples.
RSD(%) = =
^ } M
x 100 (2)
5.3 Inter-Assay Reproducibility
Inter-assay reproducibility was evaluated from four measurements of one concentration of each
bacterial or fungal culture by two separate analysts using two separate fluorometer units. The
average and %RSD of each analyst's measurements were calculated. Inter-assay reproducibility
was determined as relative percent difference (RPD) of the average measurements as noted in
Equation 3:
26
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Mi-A/2
Where My is the average of replicate measurements made by the first unit of the technology and
analyst 1 andM2 is the average of replicate measurement made by the second unit of the
technology and analyst 2.
5.4 Data Completeness
Data completeness was assessed based on the overall data return achieved by each Mycometer -
test and Bactiquant®-test analysis during the testing period. For each technology, this calculation
used the total number of valid data points divided by the total number of data points potentially
available from all testing.
27
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Chapter 6
Test Results
As mentioned previously, this verification test included both quantitative and qualitative
evaluations. The quantitative evaluation was conducted to assess the linearity, repeatability, and
inter-assay reproducibility of the Mycometer®-test and Bactiquant®-test. The qualitative
evaluation was performed to document the operational aspects of the Mycometer®-test and
Bactiquant®-test during verification testing. The following sections provide the results of the
quantitative and qualitative evaluations.
6.1 Characterization of Columbus, Ohio Tap Water Used for Testing
The dechlorinated tap water used to prepare Mycometer®-test linearity solutions and Bactiquant®-
test linearity, repeatability, and inter-assay reproducibility solutions was characterized for pH, free
chlorine, and total chlorine. The water was also characterized by Pace Analytical (Columbus,
OH) for turbidity, total organic carbon, specific conductivity, alkalinity, hardness, and dissolved
oxygen. Additionally, fluorescence of the tap water was to be measured as part of the
characterization. Tap water fluorescence was measured with the Mycometer -test on the day of
fungal culture linearity testing and with the Bactiquant®-test on the day of testing linearity,
repeatability, and inter-assay reproducibility with the indigenous bacteria from lake water. A
separate measurement of the tap water used during Bactiquant -test P. aeruginosa testing was
inadvertently omitted and is described in Deviation Number 8. These characterization
measurements were not used in evaluating the technologies, but are included for informational
purposes since tap water can vary from location to location. Results for these characterization
parameters are shown in the appendix.
6.2 Mycometer®-test for Fungi
6.2.1 Linearity
Tables 6-1 and 6-2 summarize the data obtained for linearity testing with A. flavus.
Measurements were made using 3.1 x 105 to 6.2 x 106 total A. flavus spores. Within this range,
replicate measurements of each testing solution had RSDs between 3.2 and 6.7%. Figure 6-1
shows the plot of total A. flavus spore counts as the independent variable and Mycometer®-test
results as the dependent variable. Mycometer®-test results are expressed as adjusted fluorescence
which is the fluorescence reading of the sample minus the fluorescence reading of the blank. The
blank consisted of 100 uL of dechlorinated tap water processed prepared with the same reagents
as the test samples. The relationship between total A. flavus spores in the concentration range
28
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tested and adjusted fluorescence was linear with a slope of 0.0007, a y-intercept of 20.637, and R2
equal to 0.9979.
.®
Table 6-1. Mycometer -test Linearity Data for Aspergittus flavus ATCC 58870
Test
Iteration
1
2
3
4
5
Test
Solution
Blank
NEAT
1:5
1:10
1:20
Blank
NEAT
1:5
1:10
1:20
Blank
NEAT
1:5
1:10
1:20
Blank
NEAT
1:5
1:10
1:20
Blank
NEAT
1:5
1:10
1:20
Test Solution
Concentration *
(spores/mL)
Tap water
6.2 x107
1 .2 x 1 07
6.2 x106
3.1 x106
Tap water
6.2 x107
1 .2 x 1 07
6.2 x106
3.1 x106
Tap water
6.2 x107
1.2x107
6.2 x106
3.1 x106
Tap water
6.2 x107
1 .2 x 1 07
6.2 x106
3.1 x106
Tap water
6.2 x107
1 .2 x 1 07
6.2 x106
3.1 x106
Total Spores
Tested**
N/A
6.2 x106
1 .2 x 1 06
6.2 x105
3.1 x105
N/A
6.2 x106
1 .2 x 1 06
6.2 x105
3.1 x105
N/A
6.2 x106
1.2x106
6.2 x105
3.1 x105
N/A
6.2 x106
1 .2 x 1 06
6.2 x105
3.1 x105
N/A
6.2 x106
1 .2 x 1 06
6.2 x105
3.1 x105
Fluorescence
Reading
(fu)
36
4280
904
506
260
43
4158
944
506
238
43
4413
876
506
254
42
4370
905
536
264
38
4590
940
531
274
Adjusted
Fluorescence***
(fu)
0
4245
868
471
224
0
4115
901
463
196
0
4370
833
463
212
0
4328
863
493
222
0
4552
902
492
235
*NEAT solution concentration measured from hemocytometer counts. Dilutions calculated by dividing the NEAT
solution concentration by the dilution factor.
**Based on adding 0.10 ml_ of test solution to the enzyme substrate.
***Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading and is expressed in
fluorescence units.
Fluorescence readings in the table are rounded to the nearest whole number, adjusted fluorescence calculations were
based on actual measured results.
29
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Table 6-2. Summary of Replicate Measurements for A.flavus ATCC 58870 MycometerR-test
Linearity Data
Test
Iteration
1
2
3
4
5
Average
Standard
Deviation
RSD (%)
Adjusted Fluorescence (fu)
6.2 x10b spores
tested
4245
4115
4370
4328
4552
4322
161
3.7
1 .2 x 1 0b spores
tested
868
901
833
863
902
873
29
3.3
6.2 x10D spores
tested
471
463
463
493
492
476
15
3.2
3.1 x10D spores
tested
224
196
212
222
235
218
15
6.7
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
c;nnn
At.r\r\
Annn
01
^ In" 3c;nn -
= ^ 3SUU
Mvcometer®-test Linearity
A -ii a ATffroo-rn y = 0.0007x + 20.637
Aspergillusflavus ATCC 58870 R2 = 0_gg7g
^
^^
^^
.^^
^^
^^
/^
«*
(7^^>0>^^^>
*Q^ '*&?• *y^ *^<^ '^& '^^ '^& '^^
x<^ x<%« x<%« x
-------
.®
Table 6-3. Mycometer -test Linearity Data for Cladosporium herbarum ATCC 58927
Test
Iteration
1
2
3
4
5
Dilution
Blank
NEAT
1:5
1:10
1:20
Blank
NEAT
1:5
1:10
1:20
Blank
NEAT
1:5
1:10
1:20
Blank
NEAT
1:5
1:10
1:20
Blank
NEAT
1:5
1:10
1:20
Test Solution
Concentration *
(spores/mL)
Tap water
9.6 x107
1.9 x107
9.6 x106
4.8 x106
Tap water
9.6 x107
1.9x107
9.6 x106
4.8 x106
Tap water
9.6 x107
1.9 x107
9.6 x106
4.8 x106
Tap water
9.6 x107
1.9x107
9.6 x106
4.8 x106
Tap water
9.6 x107
1.9 x107
9.6 x106
4.8 x106
Total Spores
Tested**
N/A
9.6 x106
1.9 x106
9.6 x105
4.8 x105
N/A
9.6 x106
1.9x106
9.6 x105
4.8 x105
N/A
9.6 x106
1.9 x106
9.6 x105
4.8 x105
N/A
9.6 x106
1.9x106
9.6 x105
4.8 x105
N/A
9.6 x106
1.9 x106
9.6 x105
4.8 x105
Fluorescence
Reading
(fu)
32
3454
598
260
174
38
3490
526
238
165
38
3375
513
228
154
39
3371
523
219
165
40
3442
481
211
154
Adjusted
Fluorescence***
(fu)
0
3422
566
228
142
0
3452
488
201
127
0
3337
476
190
116
0
3332
484
180
126
0
3402
441
171
114
*NEAT solution concentration measured from hemocytometer counts. Dilutions calculated by dividing the NEAT
solution concentration by the dilution factor.
**Based on adding 0.10 ml_ of test solution to the enzyme substrate.
***Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading and is expressed in
fluorescence units.
Fluorescence readings in the table are rounded to the nearest whole number, adjusted fluorescence calculations were
based on actual measured results.
31
-------
Table 6-4. Summary of Replicate Measurements for C. herbarum ATCC 58927
®
Mycometer -test Linearity Data
Test
Iteration
1
2
3
4
5
Average
Standard
Deviation
RSD (%)
Adjusted Fluorescence (fu)
9.6 x10b spores
tested
3422
3452
3337
3332
3402
3389
53
1.6
1.9x10b spores
tested
566
488
476
484
441
491
46
9.3
9.6 x10D spores
tested
228
201
190
180
171
194
22
11.4
4.8 x10D spores
tested
142
127
116
126
114
125
11
8.9
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
Annn
01
tj ^^
01 ±i
(J C
o! = 2500 -
i- Ol
§ g
£ »
« S! 1500 -
3 3
=> 5z innn
<
>
Mycometer®-test Linearity
Cladosporium herbarum ATCC 58927 y = o.OQ04x - 135.25
R2 = 0.9976
^*
^^
^^
.^^
.^^
^^^
^^
\ \ \ \ \ **°>
Total Spores
_®
Figure 6-2. Plot of Mycometer -test fluorescence response vs. C. herbarum spore counts
6.2.2 Repeatability
Table 6-5 shows the results of repeatability testing for eightA.flavus air samples collected in the
ARCA chamber and processed using the Mycometer®-test by one analyst using one fluorometer.
The approximate concentration otA.flavus in the air was 6.2 x 103 spores/L. The RSD for eight
measurements was 8.0%. Eight background air samples before addition oiA.flavus to the air are
also included for reference.
32
-------
®
Table 6-5. Mycometer -test Repeatability: Air Samples Containing A. flavus
Test
Iteration
1
2
3
4
5
Q
7
8
Average
Standard
Deviation
RSD (%)
Adjusted Fluorescence (fu)
A. Flavus
316
313
320
309
343
390
348
334
334
27
8.0
Background
1.9
-0.7
-1.8
2.7
4.0
2.9
4.6
3.1
2.1
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
6.2.3 Inter-Assay Reproducibility
Table 6-6 shows the results of inter-assay reproducibility testing for eight A. flavus air samples
collected in the ARCA chamber. The concentration of A. flavus in the air was approximately 6.2
x 103 spores/L. These eight samples were split into two sets of four for processing using the
Mycometer®-test reagents by two analysts using two different fluorometers. The RSDs for four
samples were 4.7 (Analyst 2) and 8.7% (Analyst 1); the RPD between analysts was 5.3%. Eight
background air samples before the addition of A. flavus to the air were processed in the same way
and are included for reference.
Table 6-6. Mycometer®-test Inter-Assay Reproducibility: Air Samples Containing A. flavus
Test
Iteration
1
2
3
4
Average
Standard
Deviation
RSD (%)
RPD (%)
Adjusted Fluorescence (fu)
A. flavus
Analyst 1
298
320
288
259
291
25
8.7
Analyst 2
314
294
325
297
307
15
4.7
5.3
Background
Analyst 1
-4.6
-7.4
-5.6
-7.4
-6.2
Analyst 2
-1.4
-2.8
2.5
-4.6
-1.6
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
6.2.4 Data Completeness
_®
All of the Mycometer -test data expected to be generated during testing was generated for a
100% data return.
33
-------
6.2.5 Operational Factors
The verification staff found that the Mycometer®-test was easy to use. A Mycometer A/S
representative came to Battelle to train the verification staff in the use of the Mycometer®-test and
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 noted in Chapter 2, the proficiency certification program also focuses on
understanding the principles behind the technology as well as additional applications. The
fluorometer is provided with the components listed in Table 6-7. The verification staff found the
fluorometer and carrying case, described in Chapter 2, to be easy to transport. The fluorometer
operates on four AAA batteries. The fluorometer was found to have push-button operation, a
display that was easy to read, and surfaces that could be wiped clean. The fluorometer required a
calibration check once daily with the black cuvette provided with the fluorometer and a
calibration standard provided in the reagent kit. Both an instruction manual and a quick reference
card were provided for the Mycometer®-test. Verification staff found that the instructions
provided were not always consistent between the manual and the quick reference. For example,
the manual indicated that the blank sample for air testing was to be a blank filter processed
alongside the test filters, while the quick reference guide indicated that the blank was to be an
aliquot of the substrate combined with the developer.
The Mycometer®-test reagents are sold in lots of 10 for air assays and lots of 20 for surface
assays. Each reagent kit included the sampling media (filters for air samples), 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 with the exception of the enzyme
substrate that required re-hydration. Each sample resulted in approximately 5 mL of liquid waste
from the substrate and developer used to process the 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. Once rehydrated, the enzyme substrate could
be stored in a refrigerator for up to one week or at -18 °C for up to 6 months. All components
needed to prepare and analyze a sample were included either in the reagent kit or the fluorometer
kit. Prices for the Mycometer -test reagents and the fluorometer are available from the vendor.
No other laboratory equipment was needed for processing air samples. For air sample collection,
however, a sampling pump must be obtained. The recommended air sampling pumps (Gast 3-30
LPM IAQ Pump w/Tubing & Rotameter) are commercially available.
Verification staff found they were able to collect and analyze eight air samples in one hour given
the availability of enough air sampling pumps to generate eight air samples simultaneously. For
data reduction, a laptop or personal computer is needed. Mycometer provides an Excel
spreadsheet for quantification of mold/fungi in air that converts fluorescence unit values into a
"Mycometer-Air" value and provides suggested interpretation guidelines based on the resulting
value obtained. The Mycometer-Air value calculation converts the fluorescence reading to fu per
volume of air measured in cubic meters. The calculation is (sample fu - blank fu)/volume of air
in cubic meters. This can be used to standardize the results for consistent comparison and
interpretation if there are slight variations in the air volume sampled. Because all sample volumes
used in verification testing were the same, conversion of results to a Mycometer-Air value were
34
-------
not needed for verification testing. In addition, the interpretation guidelines associated with the
Mycometer-Air values were not verified as part of this test.
Table 6-7. MYCOMETER™ Analysis Equipment Kit
Components
1 Field fluorometer
1 Black calibration cuvette
1 Automatic pipette - 100 uL
1 Field carrying case
1 Timer
2 Test racks
1 Calculator
1 Thermometer-ambient air
1 Handbook
1 Certification training flash drive
All batteries
20 assays
(R)
6.3 Bactiquant -test for Bacteria
6.3.1 Linearity
Tables 6-8 and 6-9 summarize the data obtained for Bactiquant®-test linearity testing with
indigenous bacteria from lake water. Measurements were made by filtering 250 mL of solutions
containing from 3.7 x 102 to 6.0 x 103 CFU/mL as measured by HPC and processing with the
Bactiquant®-test reagents. Within this concentration range, replicate measurements of each
testing solution had RSDs between 5.3 and 10.9%. Linearity was evaluated by plotting the lake
water indigenous bacteria concentrations as the independent variable and Bactiquant®-test results
expressed as adjusted fluorescence as the dependent variable. The adjusted fluorescence is the
fluorescence reading of the sample minus the fluorescence reading of the blank. The blank was
prepared using the blank reagents provided in the Bactiquant®-test kit by adding 0.35 mL of
enzyme substrate to a cuvette containing the developer and processing it as a sample. The
relationship between indigenous flora in the concentration range tested and adjusted fluorescence
was linear with a slope of 3.72, a y-intercept of 3502 and R of 0.9147.
Following this set of testing, the vendor provided information that a fluorescence reading greater
than 20,000 fu may generate results that are not linear because the enzyme substrate concentration
will have decreased significantly and the enzyme reaction will have slowed down. Therefore,
linearity was also examined without the 1:5 dilution results since they were consistently greater
than 20,000 fu. The relationship between concentration, using only the 1:10, 1:20, and 1:100
dilutions, and adjusted fluorescence was linear with a slope of 5.54, a y-intercept of 1153 and R2
of 0.9692.
Since the vendor recommends reporting Bactiquant®-test results as BQ values, which standardize
the fu results for reaction time, temperature, and sampling volume, the relationship between
concentration and BQ value was also evaluated. Similar to the linearity results for adjusted
fluorescence, the relationship between concentration and BQ value was linear in the concentration
range tested with a slope of 2.38, a y-intercept of 2243 and R2 of 0.9138 using all data points.
Results using BQ values only from adjusted fluorescence responses less than 20,000 fu plotted
against concentration are shown in Figure 6-3. Using only the 1:10, 1:20, and 1:100 dilutions, the
relationship between concentration and BQ value was linear with a slope of 3.55, a y-intercept of
739 and R2 of 0.9689.
35
-------
Table 6-8. Bactiq
Test
Iteration
1
2
3
4
5
Dilution
Blank
1:5
1:10
1:20
1:100
Blank
1:5
1:10
1:20
1:100
Blank
1:5
1:10
1:20
1:100
Blank
1:5
1:10
1:20
1:100
Blank
1:5
1:10
1:20
1:100
uantR-test Linearity Data for Lake Water Indigenous Bacteria
Test Solution
Concentration *
(CFU/mL)
N/A
6.0 x103
3.0 x103
1.3x103
3.7 x102
N/A
6.0 x103
3.0 x103
1.3x103
3.7 x102
N/A
6.0 x103
3.0 x103
1.3x103
3.7 x102
N/A
6.0 x103
3.0 x103
1.3x103
3.7 x102
N/A
6.0 x103
3.0 x103
1.3x103
3.7 x102
Total CPU
Tested**
N/A
1.5x106
7.5 x105
3.3 x105
9.3 x104
N/A
1.5x106
7.5 x105
3.3 x105
9.3 x104
N/A
1.5x106
7.5 x105
3.3 x105
9.3 x104
N/A
1.5x106
7.5 x105
3.3 x105
9.3 x104
N/A
1.5x106
7.5 x105
3.3 x105
9.3 x104
Fluorescence
Reading
(fu)
109
26240
17676
9768
2546
116
23550
16894
9463
2707
112
20267
18537
8621
2447
113
26693
17981
10492
2151
107
25632
16184
10147
2747
Adjusted***
Fluorescence
(fu)
0
26131
17567
9659
2437
0
23434
16778
9347
2591
0
20155
18425
8509
2335
0
26580
17868
10379
2038
0
25525
16077
10040
2640
BQ Value
N/A
16647
11191
6153
1552
N/A
14852
10633
5924
1642
N/A
12908
11800
5449
1495
N/A
17111
11503
6682
1312
N/A
16518
10404
6497
1708
*Solution concentrations measured from heterotrophic plate counts conducted in triplicate, except for 1:100 dilution.
The 1:100 dilution concentration was based on the neat lake water heterotrophic plate count divided by the dilution
factor of 100.
**Based on filtering 250 ml_ of test solution
***Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading and is expressed in
fluorescence units.
N/A = not applicable
Fluorescence readings in the table are rounded to the nearest whole number, adjusted fluorescence calculations were
based on actual measured results.
36
-------
Table 6-9. Summary of Replicate Measurements for Lake Water Indigenous Bacteria
Bactiquant®-test Linearity Data
Test
Iteration
1
2
3
4
5
Average
Standard
Deviation
RSD (%)
Adjusted Fluorescence (fu)
6.0 x10J CFU/mL
26131
23434
20155
26580
25525
24365
2644
10.9
3.0 x10J CFU/mL
17567
16778
18425
17868
16077
17343
924
5.3
1.3 x10J CFU/mL
9659
9347
8509
10379
10040
9587
717
7.5
3.7x10^ CFU/mL
2437
2591
2335
2038
2640
2408
240
10.0
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
Bactiquant®-test Linearity y = 3 5488x + ?38.8?
R2 = 0.9689
Indigenous Bacteria from Lake Water- without 1:5 dilution
1 Annn
mnnn
01
3 annn
r* finnn
00
Annn
9nnn
<
^^-^
^^^^
^^^^
^^^^
^^^
*
Concentration of Bacteria in Lake Water
(CFU/mL)
,®
Figure 6-3. Plot of Bactiquant -test BQ Values vs. Lake Water Indigenous Bacteria
Concentration in CFU/mL - Responses less than 20,000 fu
Tables 6-10 and 6-11 summarize the data obtained for Bactiquant®-test linearity testing using the
QC strain of P. aeruginosa ATCC 27853. Measurements were made by filtering 250 mL of
solutions containing from 8.7 x 102 to 8.0 x 103 CFU/mL as measured by FLPC and processing
with the Bactiquant -test reagents. Within this range, replicate measurements of each testing
solution had RSDs between 3.2 and 6.3%. In the concentration range tested, the relationship
between concentration and adjusted fluorescence was linear with a slope of 1.45, a y-intercept of-
207 and R2 of 0.9923.
37
-------
Figure 6-4 shows the plot of P. aeruginosa bacteria concentrations as the independent variable
and Bactiquant®-test results expressed as BQ values as the dependent variable. For the
concentration range tested, the relationship between concentration and BQ value was linear with a
slope of 0.95, ay-intercept of-136 andR of 0.9923.
Table 6-10. Bactiquant®-test Linearity Data for Pseudomonas aeruginosa ATCC 27853
Test
Iteration
1
2
3
4
5
Dilution
Blank
starting
solution
1:2
1:5
1:10
Blank
starting
solution
1:2
1:5
1:10
Blank
starting
solution
1:2
1:5
1:10
Blank
starting
solution
1:2
1:5
1:10
Blank
starting
solution
1:2
1:5
1:10
Test Solution
Concentration *
(CFU/mL)
N/A
8.0 x103
4.7 x103
2.1 x103
8.7 x102
N/A
8.0 x103
4.7 x103
2.1 x103
8.7 x102
N/A
8.0 x103
4.7 x103
2.1 x103
8.7 x102
N/A
8.0 x103
4.7 x103
2.1 x103
8.7 x102
N/A
8.0 x103
4.7 x103
2.1 x103
8.7 x102
Total CPU
Tested**
N/A
2.0 x106
1 .2 x 1 06
5.3 x105
2.2 x105
N/A
2.0 x106
1 .2 x 1 06
5.3 x105
2.2 x105
N/A
2.0 x106
1 .2 x 1 06
5.3 x105
2.2 x105
N/A
2.0 x106
1 .2 x 1 06
5.3 x105
2.2 x105
N/A
2.0 x106
1 .2 x 1 06
5.3 x105
2.2 x105
Fluorescence
Reading (fu)
126
11518
6254
2925
1375
109
12440
6182
2818
1355
110
11485
6121
2668
1406
111
11624
6619
2879
1548
118
11732
6374
3063
1493
Adjusted ***
Fluorescence
(fu)
0
11392
6128
2799
1249
0
12332
6074
2710
1247
0
11376
6012
2559
1297
0
11513
6508
2768
1437
0
11614
6256
2945
1375
BQ
Value
N/A
7489
4028
1840
821
N/A
8106
3992
1781
819
N/A
7478
3952
1682
852
N/A
7568
4278
1820
945
N/A
7635
4113
1936
904
*Solution concentrations measured from heterotrophic plate counts conducted in triplicate.
**Based on filtering 250 ml_ of test solution
***Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading and is expressed in
fluorescence units.
N/A = not applicable
Fluorescence readings in the table are rounded to the nearest whole number, adjusted fluorescence calculations were
based on actual measured results.
38
-------
Table 6-11. Summary of Replicate Measurements for P. aemginosa ATCC 27853 Linearity
Data - Adjusted Fluorescence
Test
Iteration
1
2
3
4
5
Average
Standard
Deviation
RSD (%)
Adjusted Fluorescence (fu)
8.0 x10J CPU/mi-
ll 392
12332
11376
11513
11614
11645
396
3.4
4.7 x10J CFU/mL
6128
6074
6012
6508
6256
6196
196
3.2
2.1 x10J CFU/mL
2799
2710
2559
2768
2945
2756
140
5.1
8.7x10^ CFU/mL
1249
1247
1297
1437
1375
1321
83
6.3
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
Bactiquant®-test Linearity y=09542x.13596
Pseudomonas aeruginosa - QC strain Rz _ 0.9923
Qnnn
snnn
?nnn
finnn
01
*^ Annn
00
mnn
<
t
^
^^
^^^
^^-^^^
^^^
^^^
^^^
Concentration of Pseudomonas aeruginosa
(CFU/mL)
Figure 6-4. Plot of BactiquantR-test BQ Values vs. P. aeruginosa Concentration in CFU/mL
6.3.2 Repeatability and Inter-Assay Reproducibility
Tables 6-12 and 6-13 show the results of repeatability and inter-assay reproducibility testing with
indigenous bacteria from lake water and the QC strain of P. aeruginosa, respectively. For both
tests, two analysts each processed four water samples containing the bacteria using the
Bactiquant®-test reagents and separate fluorometers. The RSDs for four samples of indigenous
bacteria from lake water were 2.6% (Analyst 2) and 6.4% (Analyst 1); the RPD between analysts
was 6.0%. For P. aeruginosa, the RSDs for four samples were 1.4% (Analyst 2) and 4.8%
(Analyst 1); the RPD between analysts was 2.9%.
39
-------
,®
Table 6-12. Bactiquant -test Repeatability and Inter-Assay Reproducibility:
Indigenous Bacteria from Lake Water
Test Iteration
1
2
3
4
Average
Standard
Deviation
RSD (%)
RPD (%)
Adjusted Fluorescence (fu)
Indigenous Bacteria from Lake Water
(3.7x102CFU/mL)
Analyst 1
2520
2397
2156
2379
2363
152
6.4
Analyst 2
2284
2218
2247
2149
2225
57
2.6
6.0
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
Table 6-13. BactiquantR-test Repeatability and Inter-Assay Reproducibility:
P. aeruginosa ATCC 27853
Test Iteration
1
2
3
4
Average
Standard
Deviation
RSD (%)
RPD (%)
Adjusted Fluorescence (fu)
P. aeruginosa ATCC 27853
(4.7x103CFU/ml_)
Analyst 1
6830
6618
7370
6736
6888
333
4.8
Analyst 2
6689
6567
6791
6717
6691
93
1.4
2.9
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
6.3.3 Data Completeness
All of the Bactiquant®-test data expected to be generated during testing was generated for a 100%
data return.
6.3.4 Operational Factors
The verification staff found that the Bactiquant®-test was easy to use. The fluorometer used with
Bactiquant®-test is identical to that used with Mycometer®-test and both the fluorometer and
traning provided by the vendor is discussed in Section 6.2.5. 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 measurement with the
black cuvette provided with the fluorometer and a calibration standard provided in the reagent kit.
40
-------
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.
Prices for the Bactiquant®-test reagents and the fluorometer are available from the vendor. No
other laboratory equipment was needed for processing samples; however, for collection of 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 the manual filtration apparatus was used. Verification 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 quantifying bacteria in water that converts fluorescence unit
values into aBQ value as described in Section 3.3.2.1 and provides suggested interpretation
guidelines based on the resulting BQ value obtained. These interpretation guidelines were not
verified as part of this test.
41
-------
Chapter 7
Performance Summary
Rapid technologies (results available same day of testing) to detect fungi and 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 or air quality would help to control microbial outbreaks, expedite
remediation efforts, and protect public health. Therefore, for the purpose of this verification, the
Mycometer®-test and Bactiquant®-test technologies developed by Mycometer A/S were verified
for repeatability and inter-assay reproducibility by detecting fungi in air samples and bacteria in
water samples, respectively. Linearity was assessed for both technologies using dilutions of stock
cultures in tap water. The linearity test for fungi was a modification of test procedures in place for
air and surface samples. In addition, sustainable operational factors such as ease of use, required
reagents, analysis time, laboratory space, and utilities required are reported. The results of the
verification of the Mycometer®-test and Bactiquant®-test technologies are summarized below:
7.1 Results for MycometerR-test
Table 7-1 summarizes the linearity results for Mycometer®-test using two fungal cultures in
water, Aspergillusflavus ATCC 58870 and Cladosporium herbarum ATCC 58927.
Table 7-1. Linearity Results for Mycometer®-test Adjusted Fluorescence vs. Total Spores
Tested
Test Organism
A. flavus
ATCC 58870
C. herbarum
ATCC 58927
Total Spores
Tested
3.1 x105to6.2x106
4.8x105to9.6x106
Range of
Average
Adjusted
Fluorescence
(fu)
21 8 to 4322
125 to 3389
Slope
0.0007
0.0004
Y-
intercept
20.637
-135.25
Coefficient of
Determination
(R2)
0.9979
0.9976
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
_®
Table 7-2 summarizes the repeatability results for Mycometer -test using eight replicates of one
fungal culture in air, all analyzed by one person.
42
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®
Table 7-2. Mycometer -test Repeatability: Air Containing A. Flavus
Test Iteration
Average
Standard
Deviation
RSD (%)
Adjusted Fluorescence (fu)
A.flavus (6.2 x 103 spores/L)
n=8
334
27
8.0
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
®
Table 7-3 summarizes the inter-assay reproducibility results for Mycometer -test using eight
replicates of one fungal culture in air split into four samples each for analysis by two people with
two different fluorometers.
.®
Table 7-3. Mycometer -test Inter-Assay Reproducibility: Air Containing A. Flavus
Test Iteration
Average
Standard
Deviation
RSD (%)
RPD (%)
Adjusted Fluorescence (fu)
A.flavus (6.2 x 103 spores/L)
n=4
Analyst 1
291
25
8.7
Analyst 2
307
15
4.7
5.3
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
®
Operational Factors. The verification staff found that the Mycometer -test was easy to use. A
Mycometer A/S representative came to Battelle to train the verification staff in the use of the
Mycometer®-test and 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 noted in Chapter 2, 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 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. The fluorometer required a calibration check once daily with the black cuvette provided
with the fluorometer and a calibration standard provided in the reagent kit. Both an instruction
manual and a quick reference card were provided for the Mycometer -test. Verification staff
found that the instructions provided were not always consistent between the manual and the quick
reference. For example, the manual indicated that the blank sample for air testing was to be a
43
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blank filter processed alongside the test filters, while the quick reference guide indicated that the
blank was to be an aliquot of the substrate combined with the developer.
The Mycometer®-test reagents are sold in lots of 10 for air assays and lots of 20 for surface
assays. Each reagent kit included the sampling media (filter for air samples), 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 with the exception of the enzyme
substrate that required re-hydration. Each sample resulted in approximately 5 mL of liquid waste
from the substrate and developer used to process the 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. Once rehydrated, the enzyme substrate could
be stored in a refrigerator for up to one week or at -18 °C for up to 6 months. All components
needed to prepare and analyze a sample were included either in the reagent kit or the fluorometer
kit. No other laboratory equipment was needed for processing air samples. For air sample
collection, however, a sampling pump must be obtained. The recommended air sampling pumps
(Gast 3-30 LPM IAQ Pump w/Tubing & Rotameter) are commercially available. Verification
testing staff found they were able to collect and analyze eight air samples in one hour given the
availability of enough air sampling pumps to generate eight air samples simultaneously.
For data reduction, a laptop or personal computer is needed. Mycometer provides an Excel
spreadsheet for quantification of mold/fugi in air that converts fluorescence unit values into a
"Mycometer-Air" value and provides suggested interpretation guidelines based on the resulting
value obtained. The Mycometer-Air value calculation converts the fluorescence reading to fu per
volume of air measured in cubic meters. This can be used to standardize the results for consistent
comparison and interpretation if there are slight variations in the air volume sampled. Because all
sample volumes used in verification testing were the same, conversion of results to a Mycometer-
Air value were not needed for verification testing. In addition, the interpretation guidelines
associated with the Mycometer-Air values were not verified as part of this test.
7.2 Results for Bactiquant -test
Table 7-4 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 7-4, linearity is evaluated for Bactiquant BQ values (fluorescence unit readings
standardized for reaction time, temperature and sample volume) against concentration. During the lake
water indigenous bacteria test, the vendor provided information that fluorescence readings above
20,000 fu may generate results that are no longer linear because the enzyme substrate
concentration will have decreased significantly and the enzyme reaction will slow down.
Therefore, lake water indigenous bacteria linearity data was examined both with and without the
most concentrated test solution results since they were consistently greater than 20,000 fu.
44
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Table 7-4. BactiquantR-test Linearity: 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)
3.7x102to6.0x103
3.7x102to3.0x103
8.7x102to8.0x103
Range of
Average
Adjusted
Fluorescence
(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
,®
Table 7-5 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 7-5. Bactiquant®-test Repeatability and Inter-Assay Reproducibility
Test Iteration
Average
Standard
Deviation
RSD (%)
RPD (%)
Adjusted Fluorescence (fu)
Indigenous Bacteria from Lake
Water
(3.7 x102 CFU/mL)
Analyst 1
2363
152
6.4
Analyst 2
2225
57
2.6
6.0
P. aeruginosa ATCC 27853
(4.7 x103 CFU/mL)
Analyst 1
6888
333
4.8
Analyst 2
6691
93
1.4
2.9
Adjusted fluorescence = sample fluorescence reading - blank fluorescence reading.
,®
Operational Factors. The verification staff found that the Bactiquant -test was easy to use. The
fluorometer used and training provided by the vendor are the same as that described in Section 7.1
for Mycometer®-test. 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.
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
45
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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. No other laboratory equipment was
needed for processing samples; however, for collection of 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. 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 quantifying 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.
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Chapter 8
References
1. U. S. EPA, A Brief Guide to Mold, Moisture, and Your Home; EPA 402-K-02-0003, U.S.
Environmental Protection Agency, Office of Air and Radiation Indoor Environments
Division, 2002. http://www.epa.gov/mold/pdfs/moldguide.pdf
2. World Health Organization, WHO Guidelines for Indoor Air Quality: Dampness and Mould,
2009. http://www.euro.who.int/_data/assets/pdf_file/0017/43325/E92645.pdf
3. World Health Organization, Guidelines for Drinking-water Quality, third edition, 2008.
http://www.who.int/water sanitation health/dwq/gdwq3 rev/en/index. html
4. Quality Assurance Project Plan for Verification of Mycometer®-test Rapid Fungi Detection
and BactiQuant®-test Rapid Bacteria Detection Technologies, Battelle, Version 1.0, April 5,
2011.
5. Quality Management Plan for the ETV Advanced Monitoring Systems Center, Version 8.
U.S. Environmental Technology Verification Program, Battelle, April 2011.
6. Kuhn, D. M., and M. A. Ghannoum. 2003. Indoor mold, toxigenic fungi, and Stachybotyrus
chartarum: Infectious disease perspective. Clin. Microbiol. Rev., 16(1)144-172.
7. Lee, T., S. A. Grinshpun, D. Martuzevicius, T. Adhikari, C. M. Crawford, and T. Reponen.
2006. Culturability and concentration of indoor and outdoor airborne fungi in six single-
family homes. Atmospheric Science, 40:2902-2910.
8. Shelton, B. G., K. H. Kirkland, W. D. Flanders, and G. K. Morris. 2002. Profiles of airborne
fungi in buildings and outdoor environments in the United States. Appl. Environ. Microbiol.,
68(4)1743-1753.
9. Tsai, F. C., J. M. Macher, and Y. Hung. 2007. Biodiversity and concentrations of airborne
fungi in large US office buildings from the BASE study. Atmospheric Environment, 41:5181-
5191.
10. American Society of Testing and Materials, ASTM D4300 - 01(2008) Standard Test Methods
for Ability of Adhesive Films to Support or Resist the Growth of Fungi
11. Mycometer A/S, Quantification of mold in air - Protocol for quantification of fungal
propagules in air samples using the Mycometer-test, 2008.
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12. Mycometer A/S, Mycometer®-air Sampling and analysis, 2011.
13. American Public Health Association, American Water Works Association, and Water
Environment Federation. 2005. Standard Methods for the Examination of Water and
Wastewater. 21st Editi on.
14. EPA Method 180.1 Turbidity (Nephelometric), Methods for the Determination of Inorganic
Substances in Environmental Samples EPA-600-R-93-100. 1993.
15. Hach Method 8021, Chlorine, Free DOC316.53.01023, February 2008, edition 5,
http://www.hach.com/fmmimghach7/CODE%3ADOC316.53.0102315566%7Cl
16. Hach Method 8167, Chlorine, Total DOC316.53.01027, March 2008, edition 1,
http://www.hach.com/fmmimghach7/CODE%3ADOC316.53.0102715987%7Cl
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Appendix
Data from Tap Water Analyses
49
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Table Al-1. Water Quality Parameters for Characterizing Columbus, Ohio Tap Water
Used for Testing
Analysis
Turbidity*
Total Organic
Carbon*
Specific
Conductivity*
Alkalinity*
Hardness*
Dissolved
Oxygen*
PH
before/after
dechlorination
Free Chlorine
before/after
dechlorination
Total Chlorine
before/after
dechlorination
Heterotrophic
Plate Counts
Fluorescence
Reading
Method/
(reporting
unit)
EPA Method
180.114/(NTU)
SM5310-CIJ/
(mg/L)
SM2510IJ/
u mhos/cm
SM2320-BIJ/
(mg/L)
SM2340-BIJ/
(mg/L)
SM4500-OIJ/
(mg/L)
Battelle SOP
GEN.V-003**/
(pH units)
HACH Method
8021 15/(mg/L)
HACH Method
8 1 67 16 /(mg/L)
SM9215IJ/
(CFU/mL)
Mycometer®-
teston5/19
and
Bactiquant®-
test on 6/2
(fu)
May 19, 2011
Myco mete r®-test
Linearity
<1
2.6
450
50.6
116
9.0
6.66/6.97
1.16/0.15
1.48/0.14
201
38. 82
June 2, 2011
Bactiquant'-test
Linearity, Repeatability,
Inter-Assay
Reproducibility -
Indigenous Lake Water
<1
2.8
340
48.0
111
9.7
7.93/8.01
1.19/0.15
1.52/0.12
201
23.23
June 27, 2011
Bactiquant'-test
Linearity, Repeatability,
Inter-Assay
Reproducibility - QC
Strain P. aeruginosa
<1
2.1
470
50.9
112
9.5
7.46/7.35
1.42/0.17
1.64/0.14
301
N/A
*Analyses provided by Pace Analytical
**Battelle Standard Operating Procedure: GEN. V-003 Standard Operating Procedure for the Use of pH
Meters to Measure pH
1 Estimated value due to low number of counts
2Average of eight measurements made by processing 100 uL of dechlorinated tap water with the same
reagents as the test samples
3 Dechlorinated tap water (250 mL) filtered and processed with the same reagents as the test samples
N/A = not applicable
50
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