Rapid 48-Hour Viable and Culturable
Fungi Analysis for Indoor
Environment Samples
Harry Neill, CIH 1Source Safety and Health, Inc
Exton, PA
Wei Tang, Ph.D., QLab
Cherry Hill, NJ
AIHce PO 113, June 5, 2007
Why Rapid 48-Hour Viable Count?
2
Why Rapid 48-Hour Viable Count?
It’s much quicker than 7 to 8 days!
3
Existing Technology:
Bacterial Direct Viable Count
Bacterial cells are stained with chemicals
Viable cells are stained differently from nonviable cells.
Difference: DNA, metabolic activity, cell
membrane integrity, etc.
Observed different colors under fluorescence
microscope
Viable count does not always correlate with
culturable count.
4
Traditional Culture Method
Extract fungal cells from samples.
Perform series of 10-fold dilutions.
Inoculate suspensions from 3 – 4
different dilutions onto culturing
media.
Incubate for 7 to 8 days.
Observed colonies.
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Fungal Viable Count
Germination of spores was chosen to
be the method because of its good
correlation with culturable results.
Spores -> germinate -> keep growing
into colonies
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Rapid Viable Count
Extract fungal cells from samples.
Perform series of 10-fold dilutions.
Inoculate suspensions from 3 – 4 different
dilutions onto culturing media.
Incubate for 24 to 48 hours.
Stain the cells.
Observed germinated spores or microcolonies under microscope.
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Fungal Analysis Comparisons
Method
Advantage
Disadvantage
Direct Microscopic
Examination
Total fungi detected
Quick TAT (1 day)
Poor identification
Viable Method
(by Germination)
Viable fungi detected
Quick TAT (2 days)
Viable fungi only
Poor identification
Culture Method
Good identification
Culturable fungi only
Slow TAT (7 days)
PCR
Good identification
Quick TAT (1 day)
Low detection limit
Selected species only
High cost
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MoldSense Rapid Viable Count
Epicoccum
Nigrospora
Alternaia
Pithomyces
Bipolaris
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MoldSense Rapid Viable Count
Cladosporium & alike
Asp/Pen & alike
10
MoldSense Rapid Viable Count
Ulocaldium
Yeasts
Stachybotrys
11
Sample Types and Devices for
Rapid Viable Count
A. Air - QTrap-Viable, QTrap, impinger
Omni 3000, etc.
B. Surface - swabs
C. Bulk - bags, tubes
Dust - filter cassettes
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A. Air Sample
QTrap-Viable (Sterile MCE Filter
Cassette)
QTrap (clean PC Filter Cassette)
Bio-sampler (SKC)
Omni 3000 (Sceptor)
13
B. Surface Sampling - Swab
Transporter Swab
handle too long
sample retention on sponge
RediSwab, neutralizing buffer, BioTrace/3M
(recommended)
14
C. Bulk Sampling
Plastic bag – dry bulk
Centrifuge tube – wet bulk
Dust
15
Rapid Viable Count vs. Culturable Count
MoldSense ViaCount-24 (MCFU/in2)
100,000,000
10,000,000
1,000,000
100,000
10,000
1,000
100
0
00
0,
00
0,
10
00
,0
00
,0
10
0
00
0,
00
1,
0
00
0,
10
00
,0
10
0
00
1,
0
10
Culture Plate Count (CFU/in2)
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Application of
Rapid Viable Fungal Count
(a) When viable fungi is the concern
(b) Quick TAT is essential
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Applications
(a) Hospitals - Aspergillosis investigation
(incubated at 37ºC)
(b) Food industry
(c) Pharmaceutical Manufacture
(d) Indoor Environmental Quality (IEQ)
18
Why Using Rapid Viable Count for
IEQ?
(a) Total fungi spore counts results alone are not
reliable.
(b) Filter cassette: 100% collection efficiency, high
capacity, etc.
(c) Reduces interference of non –viable
Basidiospores and Ascospores on
identification of Aspergillus and Penicillium
spores
(d) Combined with culturable analysis to get a
rapid viable count at 48 hours and complete
identifications after 7 days
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Limitations of 48 hour Viable Analysis
(a)
(b)
(c)
(d)
(e)
Interferences of debris in bulk and surface samples
Desiccation of airborne spores if high volumes are
collected on filter membranes
Inhibition from biocides
Slow growing species of fungi
Limited identification
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Contact information:
Harry Neill, CIH
Vice President
1 Source
Safety and Health, Inc.
Exton, PA
610-524-5525
Wei Tang, Ph.D.
QLab, 5 Allison Drive
Cherry Hill, NJ 08003
856-489-0011(office)
856-229-5789 (cell)
[email protected]