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Aerobiology
Microbiology of the Atmosphere
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Aerobiology
The interdisciplinary science that deals with
the movement and dispersal of bioaerosols
The movement of bioaerosols is generallypassive and is greatly influenced by the
environment
The survival of viable bioaerosols is also
dependent on the environmental conditions
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Bioaerosols
Biological agents carried in the air as large
molecules, volatile compounds, single
particles, or clusters of particles that are living
or were released from a living organism
Particles sizes - 0.5m to 100 m
Capable of eliciting diseases that may be
infectious, allergic, or toxigenic with theconditions being acute or chronic
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Bioaerosols in Our Environment
Outdoor Sources
Fungal Spores
Pollen Bacteria
Indoor Sources
Viruses
Bacteria Fungal Spores
Dust mites
Cockroaches Animal Dander -
especially cats
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Outdoor Aerobiology
Atmosphere
Transport
Fungal Spores Pollen
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Spores, Pollen and Gravity
In still airspores (and pollen) fall to the
ground at a rate (based on Stokes law)
that is proportional to the square of itsradius
i.e. the bigger the spore the faster it will fall
Aerodynamics also influenced by
non-spherical shape or irregular shape
ornamentation and aggregation
these increase drag and delay deposition
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Stokes Law
V =2 s r
9
gr2
V = terminal velocity in cm/s
s= density of sphere
r = density of air
g = acceleration due to gravity
= viscosity of air )1.8 x 10-4g/cm/sec
r = radius of sphere
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0.5 - 14.0
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Settling rates of particles
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Airborne Transport
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Effects of
environmental
factorsImpact
Deposition
Dispersal
Take-off
Source
The Aerobiological Pathway
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Airborne Transport
Perfectly still air seldom occurs
Prevailing air currents delay deposition by
gravity
Air flow is complex allowing bioaerosols to be
transported over short ranges to global
distances
Transport occurs in the turbulent layer of the
atmosphere
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Turbulent Layer of Atmosphere
Air movement shifting and unpredictable
Depends on wind speed, direction,temperature, and local eddies caused byroughness of the terrain
Fungal spores are a normal component of
the turbulent layer possibly up to 200,000spores/m3of air; however, pollen levelsnormally two orders of magnitude lower
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Long distance transport
Well documented in palynology literature as
well as in aerobiology literature
Typically these are reports of one time
incursions of exotic pollen types or preseason
pollen
Exoticpollen in sediments in Canada
Preseason birch pollen in Scandinavian countriesfrom southern areas in Europe
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Well studied examples of long
distance transport
Wheat rust sporesStarting in the 1920s the
movement of Pucciniagraminisspores has
been demonstrated
Tobacco blue moldSince late 1980s, themovement of Peronospora tabaccina spores
has been followed
Mountain cedar pollenMy lab has been
following the LDT since 1980
African dust and associated microorganisms
have been studied over past decade
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Juniperus asheipollen
Juniperus ashei (mountain cedar) has a limiteddistribution Arbuckle Mountains of Oklahoma
Edwards Plateau of Texas
Scattered areas of Ozarks
Highly allergenic (cedar fever in Texas) Tulsa allergist reported 15% patient sensitivity
Mid-winter pollination (Dec. and Jan.) Tulsa pollen season Feb through early Nov
Evidence of pollen in Tulsa atmosphere Cedar pollen detected since 1980
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Juniperus asheigrows amongst the dissected
slopes of Cretaceous limestone of the Edwards
Plateau. The species co-occurs withJ. virginiana
to the east and J. pinchotii otherJuniperus spp. to
the west.
Distribution of Juniperus ashei
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Incursion of J. asheipollen into Tulsa
Each winter since 1980 J. asheipollen has
been registered by our Tulsa air samplers
Pollen recorded on 20% to 60% of the days
in Dec and Jan
Concentrations typically low however very
high concentrations have been registered on
several occasions (based on NAB level ofvery high >1500 pollen grains/m3)
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Mountain Cedar Forecasting
Started in Dec 1998
Daily forecasts in Dec and Jan
Forecasts posted on internet athttp://pollen.utulsa.edu
During past 6 season 812 forecasts issued Three sites in TexasAustin, Junction, San
Angelo One site in OklahomaArbuckle Mountains
One site in ArkansasOzark Mountains (5 yrs)
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Forecast Components
Release forecastbased on meteorologicalconditions and phenology of the plant Temperatures above 45o F
R.H. below 50%
Sunshine
No rain in previous 24 hours
Pollen cone maturity
Downwind forecast based on model projections
using: HY-SPLIT model trajectories
Meteorological conditions along the path
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University of Tulsa
Mountain Cedar Pollen Forecast
Date Issued: 15 January 2000
Mountain Cedar location(s): Arbuckle Mountains, OK
Regional weather: Friday/Saturday, January 14/15 - TX/OK: High pressure will control
the weather for the weekend. Moist flow off the Gulf of Mexico is causing some clouds
and showers in deep South TX and some variable cloudiness in south-central TX.
Cloudiness will increase today for other parts of southern TX and there may be somedrizzle tonight. Otherwise, fair weather will prevail, and the chance of rain will diminish
for southern TX as the center of the High moves away to the east. Late-night/early
morning clouds in some areas Saturday night are expected. Warming trend through the
weekend. Highs today 50's and 60's, lows tonight 30's to 50's, highs Saturday 60's and
70's.
Trajectory weather: Mostly sunny today, high in the 60's. Partly cloudy tonight, low
near 40. Dry with a high around 60 on Sunday.
Trajectory confidence: High
OUTLOOK: *** Serious Threat*** Conditions are favorable for pollen release.
Strong release expected. Temperatures and humidities are favorable much of the day.
Airborne pollen will be moving into northeast OK (including the Tulsa area) later
this afternoon.... and across southern MO and southern IL tonight. Residents of
western KY may also be affected. TK
T j t St t( ) ( h b th * ) S lf Okl h
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Trajectory Start(s)(shown by the *on map): Sulfur, Oklahoma
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1998-2004 Tulsa Cedar Profile
During Dec and Jan of the 6 yearscedarpollen present on 233 days
Most days lowlevels
Over 50 days moderate to very highlevels 12 days with highlevels
One day had very highlevel (2,109 pollen/m3)
Trajectories crossed Tulsa >150 times with
the majority of trajectories from the ArbuckleMts.
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London, Ontario
Air sampling data from Jim Anderson from
London, Ontario
Juniperuspollen recorded 22 times in Dec
and Jan from 1998-2004
Maximum was 58 pollen grains/m3
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Continental transport
27 Jan 99, Jim Anderson inLondon, Ontario reportedatmospheric Juniperuspollen - 58 pollen grains/m3
Trajectories show that the
source of this pollen wasTexas population ofJuniperusashei
Our Jan 26 forecastindicated that the pollenhas the potential to travelvery long distances.
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Fungal Bioaerosols
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THE FUNGI
Kingdom Protista
Div Myxomycota
Div Oomycota
Kingdom Eumycota
Div Chytridiomycota
Div Zygomycota
Div Ascomycota
Div Basidiomycota
Asexual Fungi
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FUNGAL SPORES
Majority of spore types adapted for airborne
dispersal
Spores unicellular to multicellular from 1 to
100 m
Main sources in the environment
Leaf surfaces (phylloplane fungi)
Soil
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Spore Release Mechanisms
Passive
Wind - generally the most abundant airborne
spores in the atmosphere - include membersof the Dry Air Spora which peak in afternoon
Rain - rain splash as well as tap and puff
Active
Generally require moisture
Common mechanism for ascospores and
basidiospores
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Wind Dispersed Spores
Frequently related to wind speed and
turbulence
Typically borne on erect conidiophores or
sporangiophores that elevate spores above
the substrate
Dry Air Spora Cladosporium, Alternaria,
Epicoccum, Drechslera, Pithomyces,Curvularia, smut spores
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Dry Air Spora
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Diurnal Rhythm of Cladospor ium
Hourly CladosporiumLevels
Tulsa May 24, 1999
0
5,000
10,000
15,000
20,000
25,000
30,000
12:00
AM
2:00
AM
4:00
AM
6:00
AM
8:00
AM
10:00
AM
12:00
PM
2:00 PM 4:00 PM 6:00 PM 8:00 PM 10:00
PM
S
pores/m3
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Cladospor iumspores peak hourly concentration of
>120,000 spores/m3 during a spore plume
Cladosporium ,
30 September 1998
Tulsa
0
20,000
40,000
60,000
80,000
100,000
120,000
140,000
0:00 2:00 4:00 6:00 8:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00
Spores/m3
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Cladospor ium
Common fungal genusoccurring both indoorsand outdoors
Most abundant outdoorspore type with aworldwide distribution
Normally exists as asaprobe or weak plant
pathogen Spores are known to be
allergenic
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Cladospor iumspp.
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Passive Discharge during Rain
Vibration and shaking as raindrops hit leaf
May explain increases in Cladosporium
during rainfall
Release of basidiospores by puffballs shows
similar puffing when raindrop strikes mature
fruiting body
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Rain Splash
Generally spores surrounded by mucilage
Mucilage protects from desiccation but also
prevent dispersal by wind
First raindrops dissolve mucilage
Resulting spore suspension dispersed by
subsequent raindrops
Spores commonly thin, colorless, elongate
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Active Discharge - Ascospores
Hygroscopic material within ascus absorbs
moisture
Ascus swells and develops high osmotic
pressure
Pressure causes ascus to burst, explosively
shooting spores into the atmosphere
Need for rainfall will often override diurnalrhythm of release
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Basidiospore discharge
from basidium
Moisture condense around
crystal of manitol
Bullers drop enlarges
Fuses with a film of water
around spore
Shifts center of gravity
Result of discharge
Spores shot only a fractionof a millimeter
Spores freed from gill or
pore and able to fall free
and reach turbulent layer
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Basidiospore Rhythm
Need for moisture
confines spore release
to periods of highhumidity
Typically peak levels in
pre-dawn hours and
low levels in afternoon
May of 1998
0
500
1000
1500
2000
2500
2:00AM
4:00AM
6:00AM
8:00AM
10:00AM
12:
00PM
2:
00PM
4:
00PM
6:
00PM
8:
00PM
10:
00PM
12:00AM
spores/m
3
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Airborne Fungal Spore
Concentrations in Tulsa 2002
0
10,000
20,000
30,000
40,000
50,000
60,000
J F M A M J J A S O N D
Spores/m3
0
10,000
20,000
30,000
40,000
50,000
60,000
J F M A M J J A S O N D
Spores/m3
2002
23 T id tifi d Cl d i A
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23 Taxa identified: Cladospor ium, Ascospores,Basidiospores, and Alternar iaComprised 90% of Total
Basidiospores
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 1 1/1 12/1
spores/m3
Ascospores
0
2000
4000
6000
8000
10000
12000
1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 11/1 12/1
spores/m
3
Cladospor ium
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
1 /1 2 /1 3 /1 4 /1 5 /1 6 /1 7 /1 8 /1 9 /1 10/1 11 /1 12/1
spores/m3
A l t ernari a
0
500
1000
1500
2000
2500
1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 11/1 12/1
spores/m3
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Indoor Aerobiology
Outdoor spores enter readily
Many fungi can also amplify indoors
anytime moisture is available fungi can grow
on many indoor substrates
Penicillium, Aspergillus,andCladosporium
are most common indoors
Many can form mycotoxinsAspergillussppand Stachybotrys
E i t l f t th t i fl
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Environmental factors that influence
indoor fungal contamination
Outdoor concentration and type
Type and rate of ventilation
Activity levels Indoor moisture levels
Modern building materials
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Typical Yearly Spore Levels 1998
0
10,000
20,000
30,000
40,000
50,000
60,000
J F M A M J J A S O N D
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Spore plumesshow the influence of
environmental conditions
Total Spore Concentration and Wind Speed
Hectorville, OK - Sept 21, 1998
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
12:00
a.m.
2:00
a.m.
4:00
a.m.
6:00
a.m.
8:00
a.m.
10:00
a.m.
12:00
p.m.
2:00
p.m.
4:00
p.m.
6:00
p.m.
8:00
p.m.
10:00
p.m.
Time
Tota
lSporeConcentration
s
(spores/m^3)
0
1
2
3
4
5
6
7
WindSpeed(m/s)
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Water Leak
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Humidity
Indoor relative humidity Below 30% R.H. no mold growth
Above 70% optimal for mold
Usually mold growth can occur above 50% Humid air condenses on cool surfaces
Cold windows in winter - molding and sills become wetand suitable for fungal growth
Cold floors in winter
Cooling coils in AC units in summer Humid air allows hygroscopic materials to absorb
water
Hi h h idit i h h
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High humidity in home where
subslab ducts failed
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Moisture Problems
Become worse in past 20 - 30 yrs
increased use of washing machines, dishwashers
vaporizers and humidifiers actively spray droplets
into the air (often contaminated)
tighter buildings for energy conservation trap
moisture
Anytime moisture available fungi will grow
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Cladosporium one of the most common
indoor fungi growing on a diffuser
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Penic i l l ium
One of the mostcommon soil fungi innatural environment
Over 250 species
Well known allergen
Some species producemycotoxins
Some species produceantibiotics
Produce VOCs
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Penic i l l ium in culture
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Aspergi l lus
Also common soil fungi
Cause rot of stored grain
Over 150 species
Well known allergens
Several species form
mycotoxins
Some species can grow at
high temperatures
Several species causeinfections in lung, sinuses,
and hypersensitivity
pneumonitis
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Aspergi l lus niger
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Asperg i llus fum igatus
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Penic i ll ium and Asperg i llus
Small spores passively
aerosolized when spore
clusters disturbed
Spores extremely buoyant,remain airborne for
extended time
PenicilliumandAspergillus
spores look alikedistinguished in culture
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Stachybotrys chartarum
Soil fungus in nature
Commonly found indoors on
wet materials containing
cellulose, such as
wallboard, jute, wicker,straw baskets, and paper
materials
Spores in slimy mass
Thought to be allergenicalthough little is known
May produce potent
mycotoxins
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Indoor air sample
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Air SamplingTechniques
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Principal Collection Methods
Gravity
Impaction
Impingement
Filtration
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Gravity
Simplest but least accurate method ofcollecting airborne biological samples
Coated microscope slide or open petri dish
containing agar exposed to atmosphere Non quantitative for atmospheric
concentrations
Affected by particle size and shape and airmovement
Biased deposition of large particles
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IMPACTIONSAMPLERS
Separate particles from the air stream by
using inertia of particles
Forces deposition onto a solid or agar
surface
Most commonly used method
Instruments available for culturable sampling
and total spore sampling
Impaction Samplers
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Impaction SamplersTotal Spore Samplers
Outdoor Samplers
Hirst Spore TrapBurkard and others
Rotorod Sampler
Samplair MK-3 Sampler
Indoor Samplers
Burkard Continuous Recording Air Sampler
Samplair MK-3 Sampler
Burkard Personal Volumetric Sampler
Air-O-Cell Sampler
Cyclex-D Sampler
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Burkard Spore Trap
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Advantages of Burkard Spore Trap
High efficiency down to less than 5 m
Allows for accuracy for small fungal spores such
as basidiospores and small ascospores
Time discrimination Permits analysis for diurnal rhythms
Permanent slides for future reference
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Burkard 7-day sampler head
Standard is the 7-day sampling head
Sampler drum mounted on 7-day clock
Drum moves by orifice at 2 mm per hr
Melenex tape mounted on drum and greased(Lubriseal, High Vacuum Grease, other)
Air is brought in at 10 l/min and impacts on greased
Melenex tape
Drum changed each week
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Seven Day Sampling Head
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Processing the 7-day drum
Melenex tape removed from drum
Tape cut into seven 24 hour segments each 48 mmlong
Segments mounted on microscope slides in 10%gelvatol (polyvinyl alcohol) and dried
Glycerin-jelly mounting medium added and a 50 mmcover slip
Mounting medium contains pollen stain - either basicfuchsin or phenosafarin
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Melenex tape on cutting board
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24 hour sampling head
Outdoor Air Sample from Burkard Spore Trap
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Outdoor Air Sample from Burkard Spore Trap
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Rotorod Samplers
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Rotorod Samplers
Models most often used have retracting rodsfor intermittent operation (10% sampling timetypical)
Head rotates at 2400 rpm Leading edge of rod coated with grease
Pollen and spores impacted on greasedsurface
Efficient for pollen and spores >10 m
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Rotorod Sampler
Rods spin at 2400 rpm. Particles (spores and pollen) are impacted on
leading face of the rod which is greased. Efficiency decreases dramatically
below 10 m. Intermittent head spins 10% of time.
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Allergenco Samplair
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Continuous Recording Burkard
24 hour sampling onto a coated microscope slide
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Burkard Personal Sampler
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Air-O-Cell Slide
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Cyclex-d
Andersen (N-6) Single Stage
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( ) g g
Sampler
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Andersen 6-Stage and 2-Stage Impactors
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Biocassette Sampler
Burkard Portable Air Sampler for Agar
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Burkard Portable Air Sampler for Agar
Plates
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Inpingement Samplers
Separate particles from the air stream by
using inertia of particles
Forces deposition into liquid collection
medium (usually a dilute buffer)
Aggregates of cells can be broken apart
Allows for several possible analytic
applications: culture, microscopy,biochemistry, immunoassays, PCR
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Impingers
AGI-30 BioSamplerBurkard
Multi-stage
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Filtration
Separates particles from airstream by
passage through a porous substrate, usually
a membrane filter
Collection depends on filter pore size andflow rate
Loss of viability may occur due to desiccation
Adaptable for a variety of assays
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Sampler Performance
Collection efficiency is the ability to capture
the particles onto the collection medium
Physical characteristics of the inlet and the
airflow rate used to calculate d50 Particle diameter at which 50% of particles are
collected
Because of sharp cut-off it is generally acceptedthat all particles above this size are collected
d50 values for some samplers (CUT SIZE)
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d50values for some samplers (CUT SIZE)
Sampler d50
AGI-30 Impinger 0.30 m
Air-O-Cell Cassette 2.30 m
Andersen Single Stage 0.65 m
BioSampler Impinger 0.30 m
Burkard 7-Day Spore Trap 3.70 m
Burkard Personal Sampler 2.52 m
Buttner MP, Willeke K, Grinshpun SA. Sampling and Analysis of Airborne
Microorganisms. In: Manual of Environmental Microbiology 2nded. ASM Press,
Washington, 2002.
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Analytic Methods
Microscopy
Culture
Biochemistry Molecular Biology
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Microscopy
Identification of total spores (both culturable
and non-culturable) along with pollen and
other particulates
Identification to species level usually notpossible
Identification of morphologically similar
spores not possible
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Culture
Only viable bacteria and fungi
Limited to those taxa able to grow in culture
on medium used
Success based on medium and incubation
time and temperature
Permits speciation when required
Results expressed as CFU/m3
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Biochemistry
Detection of specific compounds
May not be specific for a genus or species
Assay examples:
Ergosterol
B 1,3-glucan
Endotoxins
MycotoxinsStachybotrys toxins and ochratoxin
Various types of assays such as HPLC
I h i
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Immunochemistry
Specific allergen molecules
Not useful for routine air sampling
Typically used for filter samples and impinger
samples but have been used for spore trapsamples
Requires prior development of an antibody
Widely used for dust mite, cockroach, cat,etc.
Few fungal assays available
M l l Bi l
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Molecular Biology
Detection of specific genetic elements
Highly specific and sensitive
Currently restricted to a few organisms