rapid, versatile microbiological testing with mycometer
TRANSCRIPT
Rapid, Versatile Microbiological Testing with Mycometer
An introduction to the Mycometer technology
EPA Verified!
USEPA Environmental Verification Program
www.epa.gov/etv
What we do
At Mycometer we develop rapid, reproducible and robust microbiological tests for consultants and the industry.
The tests are based on detection and quantification of key enzyme activities using highly sensitive fluorescence technology
Who we are• Danish company established in
1999 , based on research at the University of Copenhagen.
• Headquarters in Copenhagen manufacturing, research and development, and marketing eastern hemisphere
Notable
First field technology developed specifically for the building assessment and included
reproducible interpretation criteria
Intro to Mycometer Technology
Naturally occurring enzymes
Sensitive fluorescence technology
Simple Principle
Why use enzymes
• Culture independent
• Amplifying signal
• High specificity
Why use fluorimetry
• Simple chemistry
• No permeabilization of cells necessary
• Rapid, robust and sensitive methods
Mycometer Principle
sample with microbial presence
Enzyme substrate Fluorescent compound cleaved
+
Multi- purpose
• Mold enyzme
or
• Bacteria enzyme
Mutiple sample matrixes
• Surface
• Bulk
• Slurry
• Water
• Air
One instrument
Technical benefits
• High reproducibility – no extraction procedure
• High sensitivity: Allows measurement of early colonization (growth-germination)
• Quantitative - signal correlates to biomass from non-visual state to end point.
Contingencies
• Proficiency training is mandatory
• Does not identify fungi genera
Mycometer Fungi Test
Total fungal biomass is measured by the determination of the activity of the enzyme
β-n-acetylhexosaminidaseOr, more simply, NAHA
Mycometer Principle
sample with fungal material
Enzyme substrate Fluorescent compound cleaved
+
Mycometer Principle
The more fungi present, the more substrate is broken apart, releasing the Fluorophore
The amount of fluorescense measured is directly proportional to the amount of
fungal biomass present
SPORES GERMINATION
HYPHA
KONIDIOFOR
AIR-HYPHA
BELOW THE
SURFACE
Detection
NAHA enzyme is common to all Filamentous Fungi & some of the yeasts
Organism Organism Organism
Alternaria tenuissima IBT. Humicola sp. AGMf19 Agaricus bisporus
Absidia sp. Mortirella sp.AGM 17 Aspergillus nidulans
Acremonium inflatum AAS – Mk001 Mucor hiemalis AGM 27 Aspergillus niger
Artrobotrys sp. Mucor racemosus AAS 310 Aspergillus oryzae
Ascobolus crenulatus AAS Ophiostoma ulmi AGM 20 Aspergillus tamarii
Aspergillus flavus AGMf19 Paecilomyces farinosus AGM 5 Beauveria bassiana
Aspergillus fumigatus IBT 18111 Penicillium commune IBT 13713
Bipolaris sorokiniana
Aspergillus niger AGM802 Penicillium chrysogenumWisconsin 54-1255
Botrytis cinerea
Aspergillus ochraceus Penicillium solitum Coccidioides immitis
Aspergillus oryzae AGM 11. Penicillium soppii AGM 15 Gliocladium virens
Aspergillus versicolor IBT 13738 Phoma sp. IBT 8996 Metarrhizium anisophiaeAureobasidium pullulans QM 3092 Rhizomucor pussilus AAS 300 Mucor rouxii
Beauveria sp AGMf35. Rhizopus oryzae AGM804 Nomuraea rileyi
Botrytis cinerea CBS 121.39 Schizophyllum commune AGM 25
Penicillium chrysogenum
Candida albicans 898 A Scopulariopsis sp. Penicillium
Cladosporium herbarum AGM 8 Sordaria fimicola AAS 17 oxalicum
Eurotium repens AAS 36 Stachybotrys chatarum IBT 9694
Scizophyllum commune
Fusarium graminearum A 3/5 Trametes versicolor AGM 24 Sclerotinia fructigena
Fusarium graminearum C -106 Trichoderma polysporum AGM f31
Talaromyces emersonii
Fusarium solani AGMf15 Trichoderma viride IBT 9150 Trichoderma harzianum
Geotrichum candidum AGM 3. Ulocladium sp. IBT 9297 Verticillium lecanii
IBT – Technical University of DenmarkQM – The University of Massachusetts, Amherst, USAAGM – Department of GenerelMicrobiology, University of Copenhagen, DenmarkAAS – Department of Mycology and Phycology, University of Copenhagen, DenmarkCBS – Centraalbureau voorSchimmelcultures, Baarn, The Netherlands
Scientific Documentation
0
1000
2000
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4000
5000
En
zym
e act
ivit
y (
arb
. u
nit
s)E
rgo
ster
ol
(ng)
Days since inoculation
0 20 40 60 80 100 120 140 160 180
0
500
1000
1500
2000
2500
A
B
Reeslev et al. (2003) Applied and Environmental
Microbiology, july 3996-3998, VOl 69(7).
S. chartarum growing on
wallpapered gypsum
board
Bio
mass
den
sity
(m
g/c
m2)
0.0
0.5
1.0
1.5
2.0
2.5
Ergost
erol
(ng
)
0
2000
4000
6000
8000
10000
Days since inoculation
En
zym
e a
cti
vit
y (
arb
. u
nit
s)
5000
10000
15000
20000
2 4 6 8
A
B
C
S. Chartarum
growing on V8 agar
Reeslev et al. (2003) Applied
and Environmental Microbiology,
july 3996-3998, VOl 69(7).
Selected Scientific Papers➢The Use Of Fluorogenic Substrates To Measure Fungal Presence And Activity In Soil.
Applied Environmental Microbiology (AEM) Mille r, M., A. Palojärvi, A. Rangger, M. Reeslev, A. Kjøller. 1998.
➢Analytical Instrument Performance Criteria: Application of a Fluorometric Method for the Detection
of Mold in Indoor Environments. Applied Occupational &Environmental Hygiene, Krause, Hamad YY 2003.
➢Quantifying Mold Biomass on Gypsum Board: Comparison of Ergosterol and Beta-N-
Acetylhexosaminidase as Mold Biomass Parameters. AEM. Reeslev M, Miller M, Nielsen KF. 2003.
➢Measuring the efficacy of mold remediation on contaminated ductwork. Proceedings:Indoor Air 2002.
Krause, J.D. and Y.Y.Hammad. 2002.
➢NAGase activity in airborne biomass dust and relationship between NAGase concentrations and
fungal spores. Aerobiologia Vol. 19, p. 97 – 105. A.M., Madsen. 2003.
➢Application of a Fluorometric Method for the Detection of Mold in Indoor Environments. Applied
Occupational and Environmental Hygiene, Krause et al, 2003.
External documentation• Correlation between the biomass of mould and NAHA activity.
Professor Ralph Mitchell, Harvard University 2009.
• NAHA as an indicator of mould problems in buildings (air samples). Professor Ragnar Rylander, Biofact, 2009.
• Correlation between total number of mould spores and NAHA activity in air samples. Ph.D. Anne M. Madsen. Arbejdsmiljø-instituttet, 2003.
• Correlation between NAHA activity and nocturnal asthma. Professor Ragnar Rylander, Biofact, 2011.
• The method is verified by EPA (Environmental Technology Verification program). The study showed that the method has a high reproducibility (2011).
• Enzyme activity in the micro-fragment fraction from air samples (Ph.D. Anne M. Madsen. Arbejdsmiljø-instituttet)
The method’s reproducibility
• The only rapid method for fungi and for bacteria that has been verified through the ETV program (Environmental Technology Verification) of US-EPA. ( 2011)
• ETV found a very high reproducibility with a relative standard deviation of only 5.3% when two people with different equipment analyzed samples taken in the same room.
EPA ETV: Reproducibility
Source Battelle / US EPA -2011
Test Iteration
Adjusted Fluorescence (fu)
8.0 x 103
CFU/mL
4.7 x 103
CFU/mL
2.1 x 103
CFU/mL
8.7 x 102
CFU/mL
1 11392 6128 2799 1249
2 12332 6074 2710 1247
3 11376 6012 2559 1297
4 11513 6508 2768 1437
5 11614 6256 2945 1375
Average 11645 6196 2756 1321
Standard
deviation396 196 140 83
RSD (%) 3.4 3.2 5.1 6.3
Fungi Surface, air or bulk material
sampling
Surface Sample Collection
30
Analysis
Analysis is simple and organized
Results in approximately 30
minutes
31
Calculation sheets
Benefits of Indoor Applications(Compared to other rapid methods)
• Scientifically documented results categories• High reproducibility• Differentiates between bacteria and fungi• Quantifiable, non-destructive• Low false positives/false negatives
Applications
• Initial diagnostic assessment
• Delineation of contamination documentation
• Visual contamination confirmation
• Wood, grout & other building material contamination/remediation assessment
• Rapid remediation clearance testing
Applications
• Pre/Post HVAC cleaning documentation
• Fragile materials assessment
• Expedite disaster response damage assessment
• Healthcare ICRA documentation
• Routine maintenance cleaning confirmation
Interpreting Mycometer Data
CategoryMycometer Value (MM)
A MM 25
C MM > 450
Interpretation
The level of mold is not above normal background level.
The level of mold is high above normal background level indicating mold growth.
B 25 < MM 450The level of mold is above normal background level.
What does a before and after remediation test look like?
Sample
#
Sample
Description
Before
Cleaning
After
Cleaning
MycoMeter
Number
A B C
1 Mortar with mold x 279 x
2 Mortar with mold x 176 x
3 Mortar with mold x 1562 x
4 Mortar with mold x 668 x
5 Mortar with mold x 4 x
6 Mortar with mold x 3 x
7 Mortar with mold x 2 x
8 Mortar with mold x 6 x
9 Mortar with mold x 5 x
10
Remediation in
a hospital
HVAC Evaluation Case Study
• Four story building built in 1950s
• 140,000 m2
• 8 AHU per floor, total 32 AHUs
• 17 AHUs replaced in last 5 years
• Fiberglass lined sheet metal duct
System Type
Range (Mean)
Mean Cat A Cat B Cat C
Old ( 17) 9-130 32 10 (59%) 7(41%) 0
New (15) 3-12,000 1228 9 (60%) 2 (13%) 4 (27%)
HVAC Evaluation Case Study
Surface Characterization- mold vs dirt, algae
Wooden pallet demonstration
Result
MM-test result: 32
MM-test result: 1792
Bactiquant -test
Quantifies total Bacterial presence by determination of a hydrolase enzyme activity.
Total Bacteria can be measured on surfaces, in bulk materials and in water.
Bactiquant -test
The hydrolase enzyme is present in aerobic (both gram negative and gram positive) as well as anaerobic bacteria.
The test does not differntiate bacteria type.
+
Enzyme substrate Fluorescent compound released
Bacteria Sample
Bactiquant Principle
Organism Gram stain Phyllum/ class Athrobacter sp. Negative Actinobacteria / grp. 20
Bacillus cereus Positive Firmicutes / grp. 18
Bacillus subtilis Positive Firmicutes / grp. 18
Bacillus thuringiensis Positive Firmicutes / grp. 18
Clostridium tetani Positive Firmicutes / clostridia
Desulfovibrio desulfouricans Positive Proteobacteria / δ
Enterobacter sp. Negative Proteobacteria / γ
Enterococcous faecalis Positive Firmicutes / bacilli
Eschericia coli Negative Proteobacteria / γ
Escherichia fergusonii Negative Proteobacteria / γ
Flavobacteria bacterium Negative Bacteroidetes / flavobacteria
Flavobacterium johnsoniae Negative Bacteroidetes / flavobacteria
Klebsiella pneumonia Negative Proteobacteria / γ
Lactobacillus acidophilus Positive Firmicutes / bacilli
Lactobacillus reuteri Positive Firmicutes / bacilli
Methanococcus aeolicus Negative Euryarchaeota / methanococci
Micrococcous sodonensis Positive Actinobacteria / actinobacteria
Pseudomonas aeroginosa Negative Proteobacteria / γ
Rhodobacter sphaeroides Negative Proteobacteria / α
Serratia marcescens Positive Proteobacteria / γ
Staphylococcous aureus Positive Firmicutes / bacilli
Sphingomonas Wittichii Negative Proteobacteria / α
Vibrio sp Negative Proteobacteria / γ
Xanthomonas campestris Positive Proteobacteria / γ
Source: Enzyme database Brenda and ExPASy (Expert Protein Analysis System) Proteomic Server.
Specificity
Bacterial biofilm on drain 1206 8
Bacterial hydrolase Fungal NAHA
(FLU) (FLU)
Mold growth on wood 3 473
Mold growth on insulation 35 4670
Correlation between hydrolase activity and endotoxin
(R2 = 0.6469, P<0.0001).
Endotoxin and Hydrolase activity values
in samples from flooded houses, (N=51)
0,1
1
10
100
1000
10000
0,1 1,0 10,0 100,0 1000,0 10000,0
Endotoxin (ng/cm2)
En
zym
e a
cti
vit
y (
AF
U/9
cm
2)
Surface Sample Collection
Categories Characterizing Surfaces
Contaminated surfaces: > 350
Category Enzyme activty (FLU)
Clean surfaces: 0-25
Dirty surfaces: (non-flooded) 26-350
*Based on percentiles between 0.95 and 0.99
Case study: Remediation efficacy
• A flooded house undergoing remediation.
• Tested the efficacy of the cleaning effort with four different cleaning methods.
Remediation efficacy
Room Before After
cleaning cleaning
Living room 468 3
Bedroom 217 0
Living room 344 1
Bedroom 191 9
Treatment: Cleaning with Rodalon
Remediation efficacy
Room Before After
cleaning cleaning
Living room 1032 41
Bedroom 165 50
Living room 718 48
Bedroom 196 54
Treatment: Cleaning with Peroxide tabs
Remediation efficacy
Room Before After
cleaning cleaningLiving room 415 7
Bedroom 189 0
Living room 452 7
Bedroom 178 6
Treatment: Cleaning with Steam
Remediation efficacy
Room Before After
cleaning cleaning
Living room 365 12
Bedroom 178 4
Living room 472 9
Bedroom 194 15
Treatment: Cleaning with Biowash
Evaluating data consistency
Living room Bedroom
365 178
468 217
472 194
344 191
1032 165
415 189
718 196
452 178
Mycometer Summary
• Discriminates between dirt , mold and bacteria
• Only rapid technology verified by the USEPA
• Field portable, no advanced lab facilities required
• Multiple assays with one instrument
• Used for initial assessment, post remediation, routine monitoring
• Broad range of applications
Questions?
