rapid, versatile microbiological testing with mycometer

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

3000

4000

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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

)

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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?

Thank you for the opportunity to present

to you today!

Lisa Rogers

813.434.6998

lrogers@mycometer.com