xvEPA
   www.epa.gov
TECH
BRIEF
                            Rapid Fungi and Bacteria Detection  Technologies
Air and water pollution from microbes such as fungi and
bacteria can cause significant health concerns. Certain
species of fungi can produce allergens, irritants,  and
potentially toxic substances resulting in asthma,  respiratory
infections, and a variety of allergic reactions in humans.
Some bacterial species contaminate water supplies causing
gastrointestinal illnesses, liver damage or other negative
health effects.  Rapid and cost-effective detection
technologies are needed to identify these microorganisms to
document, control and prevent microbial outbreaks.  In
2011, the U.S. EPA Environmental Technology  Verification
(ETV) Program's Advanced Monitoring Systems (AMS)
Center, operated by Battelle under a cooperative agreement
with EPA, evaluated the performance of two Mycometer,
Inc. detection technologies, one for fungi in air samples and
one for bacteria in water samples. The technologies provide
semi-quantitative results of bacterial or fungal biomass.
Technology descriptions are provided in Table 1.

Technology Description and Verification Testing
Bacteria Testing:
Battelle used indigenous lake water bacteria and a reference
strain of Pseudomonas aeruginosa from the American Type
Culture Collection (ATCC 27853) to compare the technology
against the reference method of heterotrophic plate counts
(HPC) in triplicate according to Standard Method (SM) 9215.
For each type of bacteria, four different concentrations were
sub-sampled five times.  For each sub-sample, 250 mL of
bacterial solution was filtered and processed using the
technology kit.  Linearity testing determined test kit accuracy
by plotting the fluorescence readings for each sample against
the HPC bacterial concentration. Repeatability and inter-
assay reproducibility testing were also conducted using both
indigenous  lake water  bacteria and the reference strain of .P.
aeruginosa. For both tests, two analysts each processed four
water samples containing the bacteria using the technology
kit and separate fluorometers.
                            Fungi Testing:
                            For linearity testing, Battelle used two different fungal cultures:
                            Cladosporium herbarum ATCC 58927 andAspergillusflavus
                            ATCC 58870. Fungal stocks were diluted with dechlorinated
                            tap water to form five replicates of four different concentrations
                            of testing solution. Linearity was determined by plotting the
                            fluorescence readings for each sample against the actual fungal
                            concentration, measured by a hemocytometer following proce-
                            dures in American Society for Testing and Materials (ASTM)
                            D43 00-01. Repeatability and inter-assay reproducibility were
                            evaluated by producing controlled air samples containing one
                            fungal stock, A. flavus, and sampling and analyzing the air with
                            the technology kit. For repeatability, one analyst processed
                            eight simultaneously-collected air samples using a fluorometer.
                            For inter-assay reproducibility, two analysts processed eight
                            simultaneously-collected samples (four each).  Each analyst
                            used a separate fluorometer.
                              Environmental and Regulatory Background of Bacteria
                                             and Fungi at a Glance

                             EPA has several current regulatory frameworks that aim to
                             monitor and control bacterial and fungal outbreaks. The
                             Federal Insecticide, Fungicide, and Rodenticide Act
                             (FIFRA) is EPA's regulatory framework that allows EPA
                             to monitor the sale and use of fungicides and pesticides.
                             The Safe Drinking Water Act (SDWA) sets national health
                             standards for drinking water to protect against naturally-
                             occurring and man-made contaminants, Long Term 2
                             Enhanced  Surface Water Treatment Rule (LT2) aims to
                             reduce illnesses related to  Cryptosporidium and other
                             microbial contaminants and the Total Coliform Rule (TCR)
                             sets maximum contaminant limits (MCLs) for total
                             coliforms and E. coll in drinking water supplies.
                   Contact Information
         ETV Advanced Monitoring Systems Center

             John McKernan, EPA Project Officer
                   Phone:(513)569-7415
               Email: mckernan.john@epa.gov
                   Amy Dindal, Battelle
                   Phone:(561)422-0113
                 Email: dindala@battelle.org
                                                         Fluorometer kit components of a verified detection technology.
 The ETV Program operates largely as a public-private partnership through competitive cooperative agreements with non-profit research institutes.
The program provides objective quality-assured data on the performance of commercial-ready technologies.
Verification does not imply product approval or effectiveness. EPA does not endorse the purchase or sale of any
products and services mentioned in this document.

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Table 1. Description of Bacteria and Fungi Detection Technologies
Vendor and
Technology
Mycometer, Inc.
Bactiquant®-
test
Mycometer, Inc.
Mycometet®-
test
Type of
Microorganism
Bacteria
Fungi
Technology Descriptions (Provided by Vendors)
The Mycometer, Inc. Bactiquant®-test is a semi-quantitative test kit designed to provide a rapid method to estimate total bacterial
biomass, based on the enzyme activity of viable and some non-viable bacteria, in water. Once samples are passed through a
membrane filter to concentrate the bacteria, an enzyme substrate is then added to the filter. The bacterial enzyme hydrolyzes the
enzyme substrate, cleaving the substrate into two molecules. One of these molecules absorbs UV light at 365 nm and re-emits
visible light that can be measured by a fluorometer. The amount of fluorescence correlates to bacterial concentration. This technol-
ogy can also be applied to surface and air samples.
The Mycometer, Inc. Mycometet®-test is designed to provide a rapid method to estimate total fungal biomass in air, on surfaces, or
in bulk materials. The technology provides semi-quantitative results of viable and non-viable spores, hyphae, hyphael fragments,
and microfragmente. The Mycometet®-test operates under the same principles as the Bactiquan1®-test. The technology measures
the fluorescent product released from the enzyme substrate complex. The amount of fluorescence correlates to the amount of fungi
present in the sample.
 Selected performance results are provided in Table 2. Battelle also evaluated both fungi and bacterial detection technolo-
 gies for data completeness and operational and sustainability factors. The analysis was completed at Battelle's headquar-
 ters in Columbus, Ohio. Additional information is available in the verification reports and statements on ETV's website at
 http ://www. epa. gov/nrmrl/std/etv/vt-ams .html#refdtfb_air or http: //www. epa. gov/nrmrl/std/etv/vt-ams .html#refdtfb_water.

 Potential Outcomes of Verified Bacteria and Fungi Detection  Technologies

 Current bacterial and fungal detection techniques, such as HPC, direct microbial counts using epifluorescence, or real-time poly-
 merase chain reaction, all detect bacteria or fungi but have long analysis times or are expensive. The verified detection technolo-
 gies estimate fungal or bacterial biomass concentrations in a cost-effective manner, achieving on-site results in less than one
 hour. The ability of these technologies to quickly and affordably  screen and monitor water and air quality can help to prevent
 and control microbial outbreaks, expedite remediation efforts, and protect public health.
Table 2. Selected Verification Results for Rapid Fungi and Bacteria Detection Technologies
Microorganism
Fungi
Bacteria
Test Organism
A. flavus
C. herbarum3
Lake water
indigenous bacteria
Lake water
indigenous bacteria3
P. aeruginosa
Linearity Testing
Concentration
Range Tested
(spores/mL or CFU/
mL)
3.1x105 to 6.2x1 06
4.8x1 05 to 9.6x1 06
3.7x1 02 to 6.0x1 03
3.7x1 02 to 3.0x1 03
8.2x1 02 to 8.0x1 03
Range of Adjusted
Fluorescence Units
(fu)
2.2x1 02 to 4.3x1 03
1. 3x1 02 to 3.4x1 03
2.4x1 03 to 2.4x1 04
2.4x1 03 to 1.7x10"
1.3x103to1.2x104
Coefficient of
Determination
(R2)
0.9979
0.9976
0.9138
0.9689
0.9923
Interassay Reproducibility Testing
Concentration
Tested
6.2x1 03
(spore/L of air)
N/A
3.7x1 02CFU/mL
N/A
4.7x1 03CFU/mL
Relative Standard
Deviation (RSD)1.4
6.7%
N/A
4.5%
N/A
3.1%
Relative Percent
Difference (RPD)2
5.3%
N/A
6.0%
N/A
2.9%
1 . RSD was not performed for C. herbarum
2. RPD was not performed for C. herbarum
3. Result from most concentrated test solution is not included
4. Result is the average RSD determined by two analysts using different fluorometers where n=4 for each analyst
5. Parameters are not available due to design of the experiment
References:
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/mold7pdfs/moldguide.pdf

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

World Health Organization, Guidelines for Drinking-water Quality, third edition, 2008. http://www.who.int/water  sanitation health/
    dwq/gdwq3rev/en/index.html
U.S. EPA ETVProgram, http://www.epa.gov/etv
EPA/600/S-12/667
  September 2012

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