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Bioaerosol Sampling. John Scott Meschke 4225 Roosevelt Way NE, suite 2338 jmeschke@u.washington.edu 206-221-5470. Bioaerosols.

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bioaerosol sampling

Bioaerosol Sampling

John Scott Meschke

4225 Roosevelt Way NE, suite 2338

jmeschke@u.washington.edu

206-221-5470

bioaerosols
Bioaerosols
  • A collection of aerosolized biological particles (e.g. microbes, by-products of living organisms) capable of eliciting diseases that may be infectious, allergic, or toxigenic with the conditions being acute or chronic
  • Size range 0.02–100 micrometers (typically 2-10 microns size range of most concern)
  • Composition of the particles varies with source and environmental conditions
  • Particles can contain varying amounts of water
  • Some are colloidal particles of soil, vegetation, other material
  • Viruses, bacteria and fungi (spores and hyphae) in aerosols due to small size
  • Many protozoa too large to remain airborne
examples agents of respiratory infections
Examples: Agents of Respiratory Infections

Viruses: influenza, measles (rubeola), chickenpox (herpes varicella‑zoster) and rhinoviruses (colds); Hantavirus (from a rodent; mouse)

Bacteria: Legionella spp., tuberculosis and other mycobacteria (Mycobacterium spp.), anthrax (Bacillus anthracis), and brucellosis (Brucella spp.).

Fungi: diseases: histoplasmosis, cryptococcosis, blastomycosis, coccidiodomycosis, and aspergillosis

Protozoans: Pneumocystis carinii pneumonia; prevalent in immunodeficient hosts such as AIDS patients.

Acanthamoeba encephalitis; primary amebic meningoencephalitis (PAM)

reservoirs and amplifiers of airborne microbes
Reservoirs and Amplifiers of Airborne Microbes

Wide range, overall

Depends on the microbe

    • humans,
    • animal,
    • soil
    • dust
    • water
    • air

Amplifiers:

  • Places where microorganisms multiply or proliferate.
  • Most reservoirs are potential amplifiers.
airborne microbes and their reservoirs
Airborne Microbes and their Reservoirs

Viruses:

  • Mostly humans but some animals
  • Some rodent viruses are significant: ex: Lassa Fever Virus and Hantavirus.

Bacteria:

  • Humans (TB & staphylococci),
  • other animals (brucella and anthrax),
  • water (Legionella)
  • soil (clostridia).

Fungi:

  • soil and birds (Cryptococcus and Histoplasma)
  • dead plant material
  • wet surfaces (wood and other building materials)
  • indoor air (mycotic air pollution)
  • stagnant water for the opportunistic fungi (e.g., Aspergillus sp.).
disseminators
Disseminators
  • Devices causing microbes to enter airborne state or be aerosolized; often the reservoir or amplifier.
  • Any device able to produce droplets and aerosols:
    • Humans and other animals: coughs and sneezes, esp.
    • Mechanical ventilation systems
    • Nebulizers and vaporizers
    • Toilets (by flushing)
    • Showers, whirlpools baths, Jacuzzi, etc.
    • Wet or moist, colonized surfaces (wet walls and other structures in buildings)
    • Environments that are dry and from which small particles can become airborne by scouring or other mechanisms:
      • Vacuuming or walking on carpets and rugs
      • Excavation of contaminated soil
      • Demolition of buildings
bioaerosol samplers
Bioaerosol Samplers
  • Numerous sampler types
  • Some adapted from dust or particle samplers
  • Some designed specifically for microbes
  • Few specifically for non-microbial bioaerosols (e.g. endotoxin), but generally thought samplers used for microbe collection are adaptable
bioaerosol samplers10
Bioaerosol Samplers
  • Gravitational samplers (e.g. settle plates)
    • No special equipment only coated microscope slide, agar plates, etc.
    • Passive (non-volumetric), relies on collection of particles by gravity settling
    • Oversamples for larger particles
    • Poor for collection in turbulent air; affected by turbulent deposition or shadowing
inertial bioaerosol samplers
Inertial Bioaerosol Samplers
  • Allow collection of particles by size selective sampling
  • Includes impactors, sieves, stacked sieves
  • Relies on particle tendency to deviate from air flow streamlines due to inertia
  • Particles deposited to solid or semi-solid surface
spore traps
Spore Traps
  • E.g. Hirst, Burkhard, Air-o-cell, Allergenco
  • Initially designed for fungal spore and pollen
  • Sample at 10-20 Liters/minute
  • Particles impacted on to coated glass slide or adhesive tape
  • Advantages: non-selective, direct analysis after collection
  • Disadvantages: may mask problem species, does not assess viability
impactors
Impactors
  • Similar to spore trap, but collection on slide or agar plates
  • Several designs tend to undersample smaller particles; particle bounce can also be an issue
  • Used at air flows of 10-30 Liters/minute
  • Types:
    • Single Stage or Multistage (e.g. Anderson)
    • Rotary arm samplers (e.g. Rotorod, Mesosystems BT550)
    • Slit to agar samplers
    • Sieve Samplers and Stacked Sieves (e.g. SAS)
impingers
Impingers
  • Air drawn through liquid (e.g. water, broth, mineral oil), particles removed by impingement
  • Allows dilution
  • Problems with pass through, particle bounce, bubbling, evaporation of liquid loss of viability
  • Inlet efficiency decreased for particles above 10 microns
  • Sampling rate 0.1-15 liters/minute (12.5 for AGI 30)
  • Types:
    • AGI
    • Biosampler
    • Shipe
    • Multistage
cyclones or centrifugal samplers
Cyclones or Centrifugal Samplers
  • Creation of vortex creating sufficient inertia to trigger deposition of particles onto collection surface; recovered in liquid (cyclone) or semisolid medium (centrifugal)
  • Allows dilution; high air sampling rates (up to 75-1000 LPM for cyclones, 40-100 LPM for centrifugal samplers); small pressure drop
  • Oversamples larger particles (can be used as trap); poor collection below 5 micron
  • Can be used in series or paired with other samplers to overcome sampling bias (e.g. Innovatek)
large volume aerosol samplers
Large Volume Aerosol Samplers
  • Biocapture BT 550 (Mesosystems)
    • Rotary arm impactor, liquid collection
    • 150L/min (~15 min)
  • Bioguardian (Innovatek)
    • Wet-walled multi cyclone, w/centrifugal impactor for removal of large particles
    • 100-1000L/min (1 min-12 hours)
  • Spincon (Sceptor)
    • Centrifugal wet concentrator, w/cyclonic preseparation
    • 450L/min (5 min-6 hours)
non inertial samplers
Non-Inertial Samplers
  • E.g. Filtration, Electrostatic Precipitation, thermal precipitators, and Condensation traps
  • Do not rely on inertia of particles for operation, thus less reliant on particle size (less particle size bias)
filtration
Filtration
  • Simple equipment requirements
  • Adaptable to personal sampling
  • Less particle size bias (allows large and small particle collection; dependent on inlet size/shape)
  • Continuous sampling over extended period
  • Wide variety of sampling rates
  • However, problems with desiccation leading to reduced viability and difficulties with particle recovery efficiencies
filter media
Filter Media
  • Fiborous- mesh of material whose fibers are randomly oriented (creating nominal pore size); depth filter entrainment
    • Glass fiber (works for proteinaceous bioaerosols)
  • Membrane- a gel with interconnected pores of uniform size (absolute pore size); depth filter entrainment
    • Cellulose esters (commonly used for water and other liquids for microbe concentration), PVC, PTFE, nylon, gelatin
  • Flat disc or etched membranes- defined holes or pores (absolute pore size); surface collection
    • Silver, aluminum oxide, polycarbonate (most commonly filter media for collection of microbes from air)
electrostatic precipitators
Electrostatic Precipitators
  • Particles removed from air stream by electrical rather than inertial forces
  • Low pressure drop; low power; capable of large volume sampling and high rates
  • Draws air across high voltage field or corona discharge inducing charge; surface collection
  • Can be effective for very small particles, as well as larger ones
  • Problem with ozone production; loss of viability
  • Examples-
    • LVAS
    • LEAP
thermal precipitation and condensation traps
Thermal Precipitation and Condensation Traps
  • Thermal precipitation
    • Not commonly used
    • Based on Thermophoretic motion
    • Air passed between two plates (one heated and one cooled); particles collected on cooler plate
  • Condensation trap
    • Relies on manipulation of relative humidity
    • Bioaerosol used as condensation nuclei
    • Particles collected by settling
recovery from air
Recovery from Air
  • Factors that will affect the recovery of microbes from air samples:
    • Sampling Rate
    • Environmental Factors may reduce sampling efficiency (e.g. Swirling winds)
    • Sampling Time
    • Organism Type and Distribution
    • Particle Size and Distribution
    • Target of detection method to be utilized
    • Sampler Choice
      • Collection efficiency
      • Recovery efficiency
      • Particle Size Bias
recovery from air30
Recovery from Air
  • Factors that will affect the recovery of microbes from air samples:
    • Sampling Rate and Sampling Time (sampled volume)
    • Concentration factor
    • Environmental Factors may reduce sampling efficiency (e.g. Swirling winds)
    • Organism Type and Distribution (need for replication)
    • Target of detection method to be utilized
    • Sampler Choice
      • Collection efficiency (d50)
      • Retention efficiency
      • Recovery efficiency
      • Particle Size Bias
      • Loss of viability
    • Sampler Calibration
sample line losses
Sample Line Losses
  • To minimize make as short as possible, minimize angles
separation and purification methods
Separation and Purification Methods
  • Purification, separation and secondary concentration of target microbes in primary sample or sample concentrate
    • Separate target microbes from other particles and from solutes
    • Reduce sample size (further concentrate)
separation purification methods
Separation/Purification Methods
  • Variety of physical, chemical and immunochemical methods:
    • Sedimentation and flotation (primarily parasites)
    • Precipitation (viruses)
    • Filtration (all classes)
    • Immunomagnetic separation or IMS (all classes)
    • Flow cytometry (bacteria and parasites); an analysis, too
secondary concentration and purification
Secondary Concentration and Purification
  • PEG (polyethylene glycol)
  • Organic Flocculation
  • IMS (Immunomagnetic separation)
  • Ligand capture
  • BEaDs (Biodetection Enabling Device)
  • Capillary Electrophoresis
  • Microfluidics
  • Nucleic Acid Extraction
  • Spin Column Chromatography
  • Floatation
  • Sedimentation
  • Enrichment
chemical precipitation methods
Chemical Precipitation Methods
  • Viruses: precipitate with polyethylene glycol or aluminum hydroxide
    • resuspend PEG precipitate in aqueous buffer
    • dissolve aluminum floc in dilute acid solution
    • both have been used as second-step concentration and purification methods
  • Parasites: precipitate with calcium carbonate
    • dissolve precipitate in dilute sulfamic acid
other recovery and concentration methods
Other Recovery and Concentration Methods
  • Minerals, such as iron oxide and talc; used to adsorb viruses
  • Synthetic resins: ion exchange and adsorbent
  • Other granular media: glass beads and sand

Less widely used; less reliable, cumbersome; uncertain elution, desorption, exchange efficiencies

initial recovery and concentration of pathogens
Initial Recovery and Concentration of Pathogens
  • Flotation centrifugation
    • Layer or suspend samples or microbes in medium of density greater than microbe density; centrifuge; microbes float to surface; recover them from top layer
  • Isopycnic or buoyant density gradient centrifugation
    • Layer or suspend samples or microbes in a medium with varying density with depth but having a density = to the microbe at one depth.
    • Microbes migrate to the depth having their density (isopycnic)
    • Recover them from this specific layer

Isopycnic density gradient: microbe density = medium density at one depth

Flotation: microbe density < medium density

immunomagnetic separation
Immunomagnetic Separation

Y

Antibody

Bead

Y

Y

Y

Microbe

virus capture plus rt pcr to detect infectious viruses the scar system
Virus Capture Plus RT-PCR to Detect Infectious Viruses - The sCAR System
  • The cell receptor gene for Coxsackieviruses and Adenoviruses has been cloned and expressed, producing a soluble protein receptor, sCAR
  • Expressed, purified and bound sCAR to solid phases to capture infectious Coxsackieviruses from environmental samples
    • The nucleic acid of the sCAR-captured viruses is RT-PCR amplified for detection and quantitation
slide42

Application of sCAR with Para-Magnetic Beads for Virus Particle Capture and then RT-PCR

sCAR

purification

Covalent coupling

to paramagnetic beads

Culture + media;

:sCAR produced

Blocking

post-coupling

(RT-) PCR

: sCAR

NA extraction

Sample containing viruses

: Virus Particle

: Blocking protein

Amine Terminated Support Magnetic Bead : BioSpheres(Biosource)

Pre-coated to provide available amine groups for covalent coupling

of proteins or other ligands by glutaraldehyde-mediated coupling method