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Bioaerosol Measurement in Animal
             Environments




          Continuing Professional Development


     Lingjuan Wang-Li & Otto D. Simmons III

Department of Biological & Agricultural Engineering
          North Carolina State University
                                                      1
Bioaerosol Measurement in Animal
          Environments



       Part I: Classroom Lecture



     Part II: Bioaerosol Sampling:
   Demonstration & Hands-on Practice



                                       2
Part I: Classroom Lecture




                            3
Overview:


   • Bioaerosol fundamentals

   • Bioaerosol in animal environments

   • Bioaerosol sampling

   • Bioaerosol sampler selection

   • Biological analyses



                                         4
Bioaerosol Fundamentals




                          5
Bioaerosol Fundamentals:
                          Definitions

• Aerosol: a suspension of solid/liquid particles in a gas
    Includes both the particles and the suspending gas, e.g. air

• Bioaerosol: an aerosol of biological origin, or
    Particles of biological origin suspended in the air

• Particulate matter (PM): the generic term for a broad
  class of chemically and physically diverse substances that exist
  as discrete particle in liquid droplets or solids forms in the air
  (EPA’s definition)
    PM2.5/PM10 : criteria pollutant - NAAQS                           6
Bioaerosol Fundamentals:

          Airborne Microbes & Aerosols

  • Airborne transmission is possible for essentially
    all classes of microbes: viruses, bacteria, fungi,
    and protozoans
  • Any respiratory pathogen able to survive
    aerosolization and air transport is considered a
    potential cause of airborne disease




                                                         7
Bioaerosol Fundamentals:

               Bioaerosol Classification

   • Viruses, parasites
   • Living organisms
         bacteria
         fungi
   • Parts of products of organisms
         fungal spores
         pollen
         endotoxin
         allergens from dogs, cats and insects
                                                  8
Bioaerosol Fundamentals:
Particle size and natural background concentration of bioaerosols:




Source: Hinds, W.C. Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles, 2 nd edition.

• Bioaerosol particles often occur as agglomerates, as clusters of
  organisms in droplets or attached to other airborne debris
• Bioaerosols can be subdivided into two groups:
    viable: living organisms
    nonviable: dead organism, pollen, animal dander, etc.                                                           9
Bioaerosol Fundamentals:
                                     Bacteria
  Bacteria are single-celled organisms with size from 0.3 ~ 10 µ m
                                           • spherical or rod shaped
                                           • occur as clusters or chains
                                           • pathogens – cause human disease
                                           •ambient – colonize water or soil
                                           and released as aerosols when the
                                           water or soil is disturbed
                                           •indoor – colonize accumulations of
                                           moisture in ventilation systems and
                                           become aerosolized by air currents
Source for the photo:                      or vibration
http://student.nu.ac.th/u46410908/lesson
2.htm
                                                                            10
Bioaerosol Fundamentals:
Bacteria: two groups based upon
    the ability of the cell wall to
    retain crystal violet dye
• Gram-positive: retain the dye;
    lack the outer membrane
    Most pathogenic bacteria –
     Gram-positive
• Gram-negative: cannot retain
  the dye
      Escherichia coli, Salmonella

Endotoxins: a structural component in bacteria released when bacteria are
    lysed
•   Chemically stable and heat resistant                                    11
Bioaerosol Fundamentals:

                                        Fungi
   Fungi: a unique group of organism – 70,000 have been identified




Yeast cell: single celled organisms
                                                                 Mold - hyphae

                                      Fungal hyphae
                          • Most fungi disperse by releasing spores into the air
                          • Fungal spores often occur as individual particles
                                                    Source for the photos:
Fungal spores: 0.5 – 30 µ m
                                          http://www.microbe.org/microbes/fungi1.asp   12
Bioaerosol Fundamentals:

                                    Viruses
 Viruses are intracellular parasites that can reproduce only inside a host cell



                                a cluster of influenza viruses




                             viruses that cause tobacco mosaic
                             disease in tobacco plants

   • naked viruses: from 0.02 – 0.3 µ m
   • airborne viruses – part of droplet nuclei or attached to other particles
   • transmitted by direct contact, or by inhalation of aerosolized viruses
   • aerosolization by coughing, sneezing or talking
   • can survive for weeks on fabric or carpets

              Source for the photos: http://www.ucmp.berkeley.edu/alllife/virus.html   13
Bioaerosol Fundamentals:

                                       Pollen
                                             Pollen grains: 10 – 100 µ m with most
                                                      between 25 - 50 µ m
                                             • near spherical particles
                                             • transmission of genetic material
                                             • anemophilous (wind-pollinated)
                                                      plants – produce abundant
                                                      bioaerosol pollen – wind-
                                             borne pollen
                                             • insect-pollinated plants – produce
                                                      sticky pollen that is not
                                             readily aerosolized
Pollen from a variety of common plants:      • causes allergic diseases of the upper
sunflower, morning glory, hollyhock, etc.             airways (hay fever)

          Photo source: http://en.wikipedia.org/wiki/Image:Misc_pollen.jpg             14
Bioaerosol Fundamentals:

          Microbial Viability & Infectivity


      • Viability (survival): ability to replicate


      • Infectivity: ability to cause infection




                                                     15
Bioaerosol Fundamentals:

      Aerosol Factors Influencing Airborne Infection
• Particle size: <5 um "droplet nuclei" from coughing & sneezing
     Deposition site depends on particle size and hygroscopicity
     Chemical composition of the aerosol particle
• Relative humidity (RH): dessication (loss of moisture)
• Temperature: generally greater inactivation at higher temp.
• Sunlight: UV inactivation of microbes
• Factors influencing air movement: winds, currents,
  mechanical air handlers, etc.
• Seasonal factors: precipitation, air currents, pollen sources, etc.
• Air pollution:
     chemicals inactivating airborne microbes (OAF= Open Air Factor)
     enhancing their ability to cause infection in a host
                                                                        16
Bioaerosol Fundamentals:

      Some Common Airborne Infectious Diseases
       Virus           Disease              Bacteria            Disease
     Adenovirus       Respiratory     Bordetella pertussis     Whooping
                       infection                                cough
    Herpesvirus      Chicken pox         Yersinia pestis       Pneumonic
                                                                 plague
      Poxvirus        Small pox      Staphylococcus aureus       Wound
                                                                infection
     Togavirus         Rubella          Mycobacterium              TB
                                         tuberculosis
   Orthomyxovirus      Influenza     Legionella pneumophila   Legionellosis
   Paramyxovirus    Measles, mumps     Bacillus anthracis       Anthrax
    Rhabdovirus       Newcastle         Coxiella burnetii       Q fever

                                                                              17
Bioaerosol Fundamentals:
            Diseases Caused by Bioaerosols:
           Hypersensitivity or Allergic Diseases
  Result from exposure to antigens (of indoor bioaerosols)
    that stimulate an allergic response by the body's
    immune system.

     • Susceptiblity varies among people.
     • Diseases usually are diagnosed by a physician.
     • Once an individual has developed a hypersensitivity
       disease, a very small amount of the antigen may cause a
       severe reaction.
     • Hypersensitivity diseases account for most of the health
       problems due to indoor bioaerosols                         18
Bioaerosol Fundamentals:




                           19
Bioaerosol Fundamentals:

           Regions of the Respiratory System

 The cellular composition and anatomy of the respiratory
   system influence particle deposition

 • Nasopharynx Region: the head region, including the
   nose, mouth, pharynx, and larynx
 • Tracheobronchial Region: includes the trachea, bronchi,
   and bronchioles
 • Pulmonary (Alveolar) Region: comprised of the alveoli;
   the exchange of oxygen and carbon dioxide through the
   process of respiration occurs in the alveolar region
                                                             20
Bioaerosol Fundamentals:
Regional Deposition in Respiratory Tract vs. Particle Size




                                                         21
Bioaerosol Fundamentals:

          Aerosols & Respiratory Deposition

   Aerosols > 5 microns in diameter are removed in
   the upper respiratory tract, especially the nose.
      • Particles are brought to the pharynx by
        mucociliary activity of the upper respiratory
        epithelial mucosa, where they are expectorated
        or swallowed.


      Swallowed particles containing enteric
      microbes can initiate enteric infections
                                                         22
Bioaerosol Fundamentals:

            Aerosols & Respiratory Deposition
 Particles <5 microns in diameter, esp. 1‑ 3 microns diameter,
 penetrate to the lower respiratory tract
    • Can be deposited in the bronchioles, alveolar ducts and
      alveoli
    • Deposition efficiency in lower respiratory tract is ~50%
      for particles 1‑ 2 microns diameter.
    • Can also be deposited in the lower respiratory tract,
      especially particles <0.25 microns dia.
    • Particles deposited in the lower respiratory tract can be
      phagocytized by respiratory (alveolar) macrophages
       can be destroyed or carried to the ciliary escalator,
        where they are transported upward to the pharynx
                                                                  23
Bioaerosol Fundamentals:
         Hygroscopicity & Aerosol Deposition
              in the Respiratory Tract

   When inhaled, aerosol particles derived from
    aqueous fluids pick up moisture (water) while
    traveling in the respiratory passageways, thereby
    increasing in size.
      Increased size changes deposition site

        H2O       H2O             H2O



                                                        24
Bioaerosol in Animal Environments

  (Important issue, and yet understudied)




                                            25
Bioaerosol in Animal Environments:
       Common Pathogenic Bioaerosols in Poultry & Pig houses




Source: Cox, C.S. and C.M. Wathes. 1995. Bioaerosols Handbook.   26
Bioaerosol in Animal Environments:
       Common infectious disease of farm animals and pathogens
      Host       diseases              Factors implicated in causation
                                       pathogens                         environment
      pigs       Atrophic rhinitis     Bordetella bronchiseptica         Crowding
                                       Pasteurella multocida             Poor ventilation
                 Enzootic              Mycoplasma suipneumoniae          Poor drainage, high
                 pneumonia                                               relative humidity
      cattle     Diarrhea              Rotavirus, E. Coli, etc.          Weaning, hygiene, cold
                 pneumonia
                                       Mycoplasma bovis, dispar          Crowding, poor feeding
                 Shipping fever        P. haemolytica etc.               High relative humidity,
                                                                         stress
                 Environmental         E. Coli, strep. uberis            Contaminated bedding,
                 mastitis                                                stage of lactation
      horses     Obstructive           Mycropolyspora faeni              Dusty feed and bedding,
                 pulmonary disease                                       poor ventilation
                                       Aspergillus fumigatus
Source: Cox, C.S. and C.M. Wathes. 1995. Bioaerosols Handbook.                                     27
Bioaerosol Sampling




                      28
General Considerations:

• Why? – sampling objectives
• What? – measurement variables
• Where? – take representative samples:
            (sampling locations, number of sites)
• When? – frequency of sampling (statistical replicates)
• How? – sampler selection & sampling processes/steps
• Results? – determination of concentration and/or
            emission rate

                                                           29
Sampling Objectives:


  • Verify and quantify the presence of bioaerosols
    (specific species or total bioaerosol?)
  • Identify their sources for control

  • Evaluate the effectiveness of control measures

  • Others (fate and transport …)




                                                      30
Taking Representative Samples:

 Temporal and spatial variations in bioaerosol speciation
   and concentrations:
 • Sampling locations: horizontal (building layout, ventilation
       system…) & vertical (animal or human height)

 • Number of sampling sites: statistical replicates
 • Frequency of sampling: diurnal, seasonal variations
 • Optima sampling duration: concentration dependent



                                                                  31
Sampling Procedures:


   Step 1: Agar or nutrient broth preparation

   Step 2: Sampler flowrate calibration

   Step 3: Sample collection with a viable sampler

   Step 4: Sample transportation

   Step 5: Sample condition

   Step 6: Sample analysis


                                                     32
Result Analysis:

      Sample Concentration Determination

             N
         C=                  [cfu/m3 = colony-forming units /m3]
            Q*t
  • C = bioaerosol concentration in cfu/m3
  • N = total bioaerosol counts on the agar plate (in the
    sample, #)
  • Q = sampling flowrate of the viable sampler
    (m3/min)
  • t = sampling duration (min.)                                   33
Result Analysis:

           Emission Rate Determination

       ER = concentration * ventilation rate

   • ER = emission rate of bioaerosol from animal
        housing in cfu/min
   • Concentration = in-house bioaerosol concentration
        in cfu/m3
   • Ventilation rate = air flowrate of animal housing
        ventilation fans in m3/min
                                                         34
Bioaerosol Sampler
     Selection




                     35
Bioaerosol Samplers:

                    Viable Sampling Systems
Size selective system                            Non-size- selective system
                        Sampled air
                                                              Sampled air
 Size selective sampling head, nozzles

                                         collecting medium (filter, or, agar
 collecting medium (filter, or, agar          plate, or nutrient broth )
      plate, or nutrient broth )


           Calibrated flow                       Calibrated flow
        monitoring/control unit)              monitoring/control unit)


      Air                                     Air
                                                        pump
  discharged     pump                     discharged                           36
Bioaerosol Samplers:

                    Total Sampling Efficiency
 The overall sampling efficiency of a bioaerosol sampler:
     the inlet sampling efficiency – the same as for non-bioaerosol
    sampling – depends on the size, shape and aerodynamics of         the
    particles being sampled – first stage
    collection/deposition efficiency onto glass slides, a semisolid
    culture medium – second stage
    the biological aspect of sampling efficiency – depends on the
    sampling and removal of biological particles without altering their
    viability or biological activity – biological analysis to identify & quantify
    the biological particle presents – third stage

 None of the presently available samplers for culturable bioaerosols can be
 considered as reference method
     • Glass liquid impingers (AGI, HAM, MIL)
     • Six-stage Andersen impactor (AND)                                            37
Bioaerosol Samplers:

               Principles of Bioaerosol Collection

 Inertial impaction: the inertial of the particle forces its impaction onto a
         solid or semisolid impaction surface – a cultural medium, or an
 adhesive surface – be examined microscopically

                            Single-stage impactors:
                                    the surface air sampler, PBI, SPI
                            Cascade impactors:
                                    two or more impaction stages (the Anderson
                                    cascade impactor)
                            Slit samplers:
                                   the impaction stage consists of one or more
                                   slits instead of one or more circle holes
                            (CAS, NBS, BAS cultural plate samplers)
                                                                                 38
Bioaerosol Samplers:

           Principle of Collection - Inertial Impaction
Impaction ~ a special case of curvilinear motion ~ application in the collection and
                         measurement of aerosol particles




Assumption: particles stick to the surface
of the impaction plate once they hit it
                                                                                   39
Bioaerosol Samplers:

             Principle of Collection - Inertial Impaction
Assumptions: the flow velocity is uniform in the jet; the streamlines are arcs of a circle with centers at A

                          Y


                                                                                τU 2
             X

                                                                    Vr = τa r =
                                                                                 r

                                                               τU 2  2πr  π
                                                    ∆ = Vr t =            = τU
                                                                r  4U  2

                                                                     ∆ πτU π
                                                                 EI = =    = Stk
                                                                     h  2h  2
                                                                                                           40
Bioaerosol Samplers:

        Principle of Collection - Inertial Impaction

 Stk. to characterize inertial impaction:
                                      The characterization dimension :
                         2            • the radius of the nozzle jet = Dj/2 for a
          τV        ρp d p VC c                  circular jet
   Stk =      =
         Dj 2         9ηD j           • the jet half-width = W/2 for a
                                      rectangular jet
                                                              1.00




                                      Collection efficiency
                                                              0.80


                                                              0.60



                  9ηD jStk 50                                 0.40


   d 50 C c =                                                 0.20

                     ρp V                                     0.00
                                                                     0     2       4        6        8     10

                                                                         Aerodynamic equivalent diameter
                                                                                                           41
Aerosol Samplers In General:

      Sampler Fractional Efficiency Curve (FEC)


                                           Ideally, it is desired that all particles
                                           greater than a certain size are collected
                                           and all particles smaller than that size
                                           pass through – Cut-off size



                                           FEC: relates collection efficiency to the
                                                   particle diameters
                                           Cut-point , cut-off size (d50): is the AED
                                                     of the particle with 50%
                                           efficiency
                                           Slope: it the sharpness of the cut
    Aerodynamic equivalent diameter (µm)
                                                                                        42
Bioaerosol Samplers:

                       Principle of Collection –
                        Multi-stage Impaction




                        Cut-points for different stages




                                                          43
Bioaerosol Samplers:

        Principles of Bioaerosol Collection

              Centrifugal inertial impaction: particle
              separation by centrifugal force in a radial
              geometry - the Reuter centrifugal sampler
              (BIO)


              Liquid impingement: the particles are collected
                       by inertial impaction into a liquid, and
              particle diffusion within the bubbles (the AGI-4
              and AGI-30 impingers)


              Tangential impinger: collects particles by
              inertial impaction and centrifugation
              (BioSampler SKC)
                                                                  44
Bioaerosol Samplers:

             Principles of Bioaerosol Collection

Filtration: impaction, interception, diffusion, gravitational
settling,      etc. − particle physical properties, filter pore size,
air flow

         Challenges: inlet – isokinetic sampling ?
filter: dehydration effect – desiccation stress?

Gravitational Settling: the least effective methods of bioaerosol
       collection – particle size, shape and airflow dictate the
deposition of particles

Electrostatic Precipitation: overcome some of physical damage
       caused by impinger, or impactor
                                                                        45
Bioaerosol Samplers:

               Viable Samplers – Inertial Impactors




Anderson single stage Anderson two-stage   Anderson six-stage   Stage with Petri-
  viable impactor      viable impactor      viable impactor           dish



                                                                                    46
Bioaerosol Samplers:

                 Viable Samplers – Impingers




   All glass AGI-30 liquid impinger   Multistage all glass liquid impinger



                                                                             47
Bioaerosol Samplers:

       Viable Samplers – Centrifugal Sampler




    “Aerojet” cyclone sampler   RCS Biotest centrifugal sampler


                                                                  48
Bioaerosol Samplers:




             High impact velocity can result in metabolic and structural
                injuries of the collected microorganisms 1 – 265 m/s

           Selection of sampler: cutoff size and the aerodynamic particle size
                                                                                 49
Bioaerosol Samplers:

            Selection of Sampler
                       SAS samplers – portable one stage
                              multiple-hole impactors
                       Air-O-Cell and Bukard samplers –
                               the slit impactors, on
                       microscope slide or tape
                       The Reuter centrifugal sampler
                       (RCS) – portable – d50 ~ 3.8µ m
                       The AGI-30 and the AGI-40 can only
                             be used with water-based
                             collection fluids
                       The BioSampler can be used with
                               nonevaporative liquids
                       (mineral oil) – permit long
                       sampling time                        50
Bioaerosol Samplers:

              Collection Time
                  Bioaerosol concentration vary greatly with
                           time – sample collection time is
                  essential
                   t1 – t2 – low concentration
                   t3 – t4 – high concentration
                   Sampling time – sufficiently long Average
                          concentration: Ca
                     ts – starting time               V = Qt
                     tf – finish time
                                                      N = CaQt
                     Q – sampling flow rate
                                                      N Ca Q
                   The surface density:          δ=     =    t
                                                      A   A
                    δo optimal surface density                        51
                                                        A - viewing area
Bioaerosol Samplers:

             Optimal Collection Time
      Optimal sampling time for solid surface sampler

                               δo          optimal surface density
                               δ < < δ o Insufficiently loaded samples
                               δ > > δ o overloaded samples

                               Adjusting the sampling period to obtain
                                       optimal surface density

                                                                       δ A
                               Optimal sampling time:             t=
                                                                       Ca Q
                    The optimal sampling time for a given bioaerosol concentration is
                     different for each sampler – sampler’s flow rate and collection
                                              surface area                           52
Bioaerosol Samplers:

                Optimal Collection Time

  Optimal collection time for impingers

   Impinger samples are not sensitive to overloading or under-
     sampling because the liquid sample can be either diluted or
     concentrated depending on the concentration of collected
     bioaerosol particles in the liquid.


   Evaporation of sampling liquid and reaerosolization of already
     collected particles limit the sampling time in most impingers



                                                                     53
Bioaerosol Samplers:

                        Optimal Collection Time




Permissible sampling parameter ranges for less than 10% change in collection efficiency
 with the AGI-4 and AGI-30 impingers when operated at a sampling rate of 12.5 L/min
                                                                                          54
Bioaerosol Analysis




                      55
Intermediate Processing:
• Manipulate samples to be compatible with detection
  methodology
   Take into account liquid or solid surface collection
    techniques
   Ex. - microscopy – sample on solid surface (i.e. filter)

• If samples in liquid media – dilutions to achieve
  countable concentrations




                                                           56
Non-viability Assays:
           Microscopy – Brightfield (Light)
• Limit of resolution => 0.2 µ m
• Time-consuming, tedious, expensive



                                         Salmonella (DIC)




                                        Cryptosporidium (DIC)


                                                            57
Non-viability Assays:
          Microscopy – Electron Microscopy
• Electronically magnified images
• Electrons => short wavelengths
• Magnification up to 2,000,000x

                                     Hepatitis A virus




                                      Influenza virus    58
Non-viability Assays:
          Flow Cytometry – Cell Sorting




                                          59
Non-viability Assays:
                 Molecular Detection
• Polymerase Chain Reaction (PCR)




• Genetic hybridization



                                       60
Viability Assays:
          Bacteria and Fungi – Agar Culture
 • Non-selective: Plate Count Agar, R2A Agar
 • Selective: mFC Agar, EC Agar, Salmonella-Shigella
   Agar, Malt Extract Agar (MEA – fungi)




         R2A Agar            Salmonella-Shigella Agar   61
Viability Assays:

                        Virus – Cell Culture




   uninfected   Late cytophathic effects:   Cell degeneration   Plaque formation
                Enlarged cells
                Nuclear inclusions
                                                                                   62
Further Characterization:
             Biochemical Analyses




                                    63
Further Characterization:

                Antibiotic Resistance




      Microdilution                Disk Diffusion

                                                    64
Further Characterization:
             Molecular Sequencing




                      Sequence Info
                                      Dendrogram
                                                   65
Further Characterization:

      Molecular Characterization: Ribotyping




                                               66
Part II: Bioaerosol Sampling:
Demonstration & Hands-on Practice




                                    67
Bioaerosol Sampling Illustration:

    Illustration of Bioaerosol Sampling Procedure
              (Impactor - culture method)

    Step 1: Agar preparation

    Step 2: Sampler flowrate calibration

    Step 3: Sample collection with a viable sampler

    Step 4: Sample transportation

    Step 5: Sample condition

    Step 6: Sample analysis
                                                      68
Pre-sampling – agar plate preparation:




                                         69
Pre-sampling – agar plate preparation:




                                         70
Pre-sampling – agar plate preparation:




                                         71
Pre-sampling – agar plate preparation:




                                         72
Pre-sampling- sampler flowrate calibration:




                                              73
Pre-sampling- sampler flowrate calibration:




                                              74
Taking Samples in the Field:




                               75
Taking Samples in the Field:




                               76
Taking Samples in the Field:




                               77
Taking Samples in the Field:




                               78
Post-Sampling – agar plate incubation:




                                         79
Post-Sampling – agar plate reading:




                                      80
Post-Sampling – agar plate reading:




                                      81
Post-Sampling – agar plate reading:




                                      82
Concentration Determination:

                        Example #1

• A bioaerosol sampling campaign was conducted at a ambient
  location in vicinity of a egg production farm. AGI-30 viable
  sampler was used to take total bacteria samples. The air flow
  rate of the AGI-30 was controlled at 12.5 l/min and the
  sampling duration was 30 min. After the field sampling, the
  samples were transported to the lab at 4 oC.




                                                                  83
Concentration Determination:

                  Example #1 – Cont.

 • Impinger fluid from each sample was transferred to a sterile
   tube and its volume determined. Impinger fluid samples,
   and/or dilutions in F-tab containing 0.1% Tween 80, were
   plated in duplicate on Trypticase Soy Agar (TSA) for growth
   of bacteria. The TSA plates were incubated at 37º C. Plates
   were checked daily for growth of colonies and moved to 4º C
   when colonies were of appropriate size for identification and
   counting.
                                                                   84
Concentration Determination:

                           Example #1 – Cont.

• Lab results of an Impinger sample:

          Total Bacteria
                                                                         Total
                                                         Impinger       bacteria
                                      Recip. Bacteria/ml fluid vol.        in
Count 1      Count 2       Average   Dilution Imp fluid     (ml)       Impinger
  11           24           17.5        10        ?          16             ?


 • Bacteria/ml Imp fluid = average count * Recip. Dilution

 • Total bacteria in impinger = Bacteria/ml Imp fluid * Impinger fluid vol.

                                                                               85
Concentration Determination:

                  Example #1 – Cont.

 • Concentration calculation:

       N           ???cfu
   C=      =                        = ???cfu / m 3
      Q * t 0.0125m 3 / min* 30 min


  N = total bioaerosol counts = ??? cfu

  Q = sampling flowrate = 12.5 l/min * 0.001 m3/l = 0.0125 m3/min

  t = sampling duration = 30 min.
                                                                86
Concentration Determination:

                          Example #2

• If a one-stage Anderson viable sampler with a no-selective R2A
  agar plate was used to take total bacteria samples in a
  residence home. The air flow rate of the sampler was
  controlled at 28.3 l/min and the sampling duration was 10 min.
  After the field sampling, the samples were transported to the
  lab at 4 oC. The agar plates were incubated for growth of
  colonies. It was observed that colonies counts of a plate was 82.
  What was the concentration of the total bacterial in the air of
  this home?                                                          87
Concentration Determination:

                  Example #2 – Cont.

 • Concentration calculation:

       N           ???cfu
   C=      =                        = ???cfu / m 3
      Q * t 0.0283m 3 / min* 10 min


  N = total bioaerosol counts = ??? cfu

  Q = sampling flowrate = 28.3 l/min * 0.001 m3/l = 0.0283 m3/min

  t = sampling duration = 10 min.
                                                                88
References:

  •Baron, P.A. and K. Willeke. 2001. Aerosol Measurement:
Principles, Techniques, and Applications, 2nd edition. John
Wiley & Sons, New York.

  •Hinds, W.C. 1999. Aerosol Technology: Properties, Behavior,
      and Measurement of Airborne Particles, 2nd edition. John
      Wiley & Sons, New York.

  •Cox, C.S. and C.M. Wathes. 1995. Bioaerosols Handbook.
Lewis Publishers. Washington D.C.




                                                                 89
Acknowledge:


• Supported by a National Research Initiative grant
 from the National Institute of Food and Agriculture,
 Air Quality Program (No. 2007-55112-17856)




                                                        90
Hands-on Practice: sampler flow
      calibration/check




                                  91

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Bioaerosol Measurement in Animal Environments

  • 1. Bioaerosol Measurement in Animal Environments Continuing Professional Development Lingjuan Wang-Li & Otto D. Simmons III Department of Biological & Agricultural Engineering North Carolina State University 1
  • 2. Bioaerosol Measurement in Animal Environments Part I: Classroom Lecture Part II: Bioaerosol Sampling: Demonstration & Hands-on Practice 2
  • 3. Part I: Classroom Lecture 3
  • 4. Overview: • Bioaerosol fundamentals • Bioaerosol in animal environments • Bioaerosol sampling • Bioaerosol sampler selection • Biological analyses 4
  • 6. Bioaerosol Fundamentals: Definitions • Aerosol: a suspension of solid/liquid particles in a gas  Includes both the particles and the suspending gas, e.g. air • Bioaerosol: an aerosol of biological origin, or  Particles of biological origin suspended in the air • Particulate matter (PM): the generic term for a broad class of chemically and physically diverse substances that exist as discrete particle in liquid droplets or solids forms in the air (EPA’s definition)  PM2.5/PM10 : criteria pollutant - NAAQS 6
  • 7. Bioaerosol Fundamentals: Airborne Microbes & Aerosols • Airborne transmission is possible for essentially all classes of microbes: viruses, bacteria, fungi, and protozoans • Any respiratory pathogen able to survive aerosolization and air transport is considered a potential cause of airborne disease 7
  • 8. Bioaerosol Fundamentals: Bioaerosol Classification • Viruses, parasites • Living organisms  bacteria  fungi • Parts of products of organisms  fungal spores  pollen  endotoxin  allergens from dogs, cats and insects 8
  • 9. Bioaerosol Fundamentals: Particle size and natural background concentration of bioaerosols: Source: Hinds, W.C. Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles, 2 nd edition. • Bioaerosol particles often occur as agglomerates, as clusters of organisms in droplets or attached to other airborne debris • Bioaerosols can be subdivided into two groups:  viable: living organisms  nonviable: dead organism, pollen, animal dander, etc. 9
  • 10. Bioaerosol Fundamentals: Bacteria Bacteria are single-celled organisms with size from 0.3 ~ 10 µ m • spherical or rod shaped • occur as clusters or chains • pathogens – cause human disease •ambient – colonize water or soil and released as aerosols when the water or soil is disturbed •indoor – colonize accumulations of moisture in ventilation systems and become aerosolized by air currents Source for the photo: or vibration http://student.nu.ac.th/u46410908/lesson 2.htm 10
  • 11. Bioaerosol Fundamentals: Bacteria: two groups based upon the ability of the cell wall to retain crystal violet dye • Gram-positive: retain the dye;  lack the outer membrane  Most pathogenic bacteria – Gram-positive • Gram-negative: cannot retain the dye  Escherichia coli, Salmonella Endotoxins: a structural component in bacteria released when bacteria are lysed • Chemically stable and heat resistant 11
  • 12. Bioaerosol Fundamentals: Fungi Fungi: a unique group of organism – 70,000 have been identified Yeast cell: single celled organisms Mold - hyphae Fungal hyphae • Most fungi disperse by releasing spores into the air • Fungal spores often occur as individual particles Source for the photos: Fungal spores: 0.5 – 30 µ m http://www.microbe.org/microbes/fungi1.asp 12
  • 13. Bioaerosol Fundamentals: Viruses Viruses are intracellular parasites that can reproduce only inside a host cell a cluster of influenza viruses viruses that cause tobacco mosaic disease in tobacco plants • naked viruses: from 0.02 – 0.3 µ m • airborne viruses – part of droplet nuclei or attached to other particles • transmitted by direct contact, or by inhalation of aerosolized viruses • aerosolization by coughing, sneezing or talking • can survive for weeks on fabric or carpets Source for the photos: http://www.ucmp.berkeley.edu/alllife/virus.html 13
  • 14. Bioaerosol Fundamentals: Pollen Pollen grains: 10 – 100 µ m with most between 25 - 50 µ m • near spherical particles • transmission of genetic material • anemophilous (wind-pollinated) plants – produce abundant bioaerosol pollen – wind- borne pollen • insect-pollinated plants – produce sticky pollen that is not readily aerosolized Pollen from a variety of common plants: • causes allergic diseases of the upper sunflower, morning glory, hollyhock, etc. airways (hay fever) Photo source: http://en.wikipedia.org/wiki/Image:Misc_pollen.jpg 14
  • 15. Bioaerosol Fundamentals: Microbial Viability & Infectivity • Viability (survival): ability to replicate • Infectivity: ability to cause infection 15
  • 16. Bioaerosol Fundamentals: Aerosol Factors Influencing Airborne Infection • Particle size: <5 um "droplet nuclei" from coughing & sneezing  Deposition site depends on particle size and hygroscopicity  Chemical composition of the aerosol particle • Relative humidity (RH): dessication (loss of moisture) • Temperature: generally greater inactivation at higher temp. • Sunlight: UV inactivation of microbes • Factors influencing air movement: winds, currents, mechanical air handlers, etc. • Seasonal factors: precipitation, air currents, pollen sources, etc. • Air pollution:  chemicals inactivating airborne microbes (OAF= Open Air Factor)  enhancing their ability to cause infection in a host 16
  • 17. Bioaerosol Fundamentals: Some Common Airborne Infectious Diseases Virus Disease Bacteria Disease Adenovirus Respiratory Bordetella pertussis Whooping infection cough Herpesvirus Chicken pox Yersinia pestis Pneumonic plague Poxvirus Small pox Staphylococcus aureus Wound infection Togavirus Rubella Mycobacterium TB tuberculosis Orthomyxovirus Influenza Legionella pneumophila Legionellosis Paramyxovirus Measles, mumps Bacillus anthracis Anthrax Rhabdovirus Newcastle Coxiella burnetii Q fever 17
  • 18. Bioaerosol Fundamentals: Diseases Caused by Bioaerosols: Hypersensitivity or Allergic Diseases Result from exposure to antigens (of indoor bioaerosols) that stimulate an allergic response by the body's immune system. • Susceptiblity varies among people. • Diseases usually are diagnosed by a physician. • Once an individual has developed a hypersensitivity disease, a very small amount of the antigen may cause a severe reaction. • Hypersensitivity diseases account for most of the health problems due to indoor bioaerosols 18
  • 20. Bioaerosol Fundamentals: Regions of the Respiratory System The cellular composition and anatomy of the respiratory system influence particle deposition • Nasopharynx Region: the head region, including the nose, mouth, pharynx, and larynx • Tracheobronchial Region: includes the trachea, bronchi, and bronchioles • Pulmonary (Alveolar) Region: comprised of the alveoli; the exchange of oxygen and carbon dioxide through the process of respiration occurs in the alveolar region 20
  • 21. Bioaerosol Fundamentals: Regional Deposition in Respiratory Tract vs. Particle Size 21
  • 22. Bioaerosol Fundamentals: Aerosols & Respiratory Deposition Aerosols > 5 microns in diameter are removed in the upper respiratory tract, especially the nose. • Particles are brought to the pharynx by mucociliary activity of the upper respiratory epithelial mucosa, where they are expectorated or swallowed. Swallowed particles containing enteric microbes can initiate enteric infections 22
  • 23. Bioaerosol Fundamentals: Aerosols & Respiratory Deposition Particles <5 microns in diameter, esp. 1‑ 3 microns diameter, penetrate to the lower respiratory tract • Can be deposited in the bronchioles, alveolar ducts and alveoli • Deposition efficiency in lower respiratory tract is ~50% for particles 1‑ 2 microns diameter. • Can also be deposited in the lower respiratory tract, especially particles <0.25 microns dia. • Particles deposited in the lower respiratory tract can be phagocytized by respiratory (alveolar) macrophages can be destroyed or carried to the ciliary escalator, where they are transported upward to the pharynx 23
  • 24. Bioaerosol Fundamentals: Hygroscopicity & Aerosol Deposition in the Respiratory Tract When inhaled, aerosol particles derived from aqueous fluids pick up moisture (water) while traveling in the respiratory passageways, thereby increasing in size.  Increased size changes deposition site H2O H2O H2O 24
  • 25. Bioaerosol in Animal Environments (Important issue, and yet understudied) 25
  • 26. Bioaerosol in Animal Environments: Common Pathogenic Bioaerosols in Poultry & Pig houses Source: Cox, C.S. and C.M. Wathes. 1995. Bioaerosols Handbook. 26
  • 27. Bioaerosol in Animal Environments: Common infectious disease of farm animals and pathogens Host diseases Factors implicated in causation pathogens environment pigs Atrophic rhinitis Bordetella bronchiseptica Crowding Pasteurella multocida Poor ventilation Enzootic Mycoplasma suipneumoniae Poor drainage, high pneumonia relative humidity cattle Diarrhea Rotavirus, E. Coli, etc. Weaning, hygiene, cold pneumonia Mycoplasma bovis, dispar Crowding, poor feeding Shipping fever P. haemolytica etc. High relative humidity, stress Environmental E. Coli, strep. uberis Contaminated bedding, mastitis stage of lactation horses Obstructive Mycropolyspora faeni Dusty feed and bedding, pulmonary disease poor ventilation Aspergillus fumigatus Source: Cox, C.S. and C.M. Wathes. 1995. Bioaerosols Handbook. 27
  • 29. General Considerations: • Why? – sampling objectives • What? – measurement variables • Where? – take representative samples: (sampling locations, number of sites) • When? – frequency of sampling (statistical replicates) • How? – sampler selection & sampling processes/steps • Results? – determination of concentration and/or emission rate 29
  • 30. Sampling Objectives: • Verify and quantify the presence of bioaerosols (specific species or total bioaerosol?) • Identify their sources for control • Evaluate the effectiveness of control measures • Others (fate and transport …) 30
  • 31. Taking Representative Samples: Temporal and spatial variations in bioaerosol speciation and concentrations: • Sampling locations: horizontal (building layout, ventilation system…) & vertical (animal or human height) • Number of sampling sites: statistical replicates • Frequency of sampling: diurnal, seasonal variations • Optima sampling duration: concentration dependent 31
  • 32. Sampling Procedures: Step 1: Agar or nutrient broth preparation Step 2: Sampler flowrate calibration Step 3: Sample collection with a viable sampler Step 4: Sample transportation Step 5: Sample condition Step 6: Sample analysis 32
  • 33. Result Analysis: Sample Concentration Determination N C= [cfu/m3 = colony-forming units /m3] Q*t • C = bioaerosol concentration in cfu/m3 • N = total bioaerosol counts on the agar plate (in the sample, #) • Q = sampling flowrate of the viable sampler (m3/min) • t = sampling duration (min.) 33
  • 34. Result Analysis: Emission Rate Determination ER = concentration * ventilation rate • ER = emission rate of bioaerosol from animal housing in cfu/min • Concentration = in-house bioaerosol concentration in cfu/m3 • Ventilation rate = air flowrate of animal housing ventilation fans in m3/min 34
  • 35. Bioaerosol Sampler Selection 35
  • 36. Bioaerosol Samplers: Viable Sampling Systems Size selective system Non-size- selective system Sampled air Sampled air Size selective sampling head, nozzles collecting medium (filter, or, agar collecting medium (filter, or, agar plate, or nutrient broth ) plate, or nutrient broth ) Calibrated flow Calibrated flow monitoring/control unit) monitoring/control unit) Air Air pump discharged pump discharged 36
  • 37. Bioaerosol Samplers: Total Sampling Efficiency The overall sampling efficiency of a bioaerosol sampler:  the inlet sampling efficiency – the same as for non-bioaerosol sampling – depends on the size, shape and aerodynamics of the particles being sampled – first stage collection/deposition efficiency onto glass slides, a semisolid culture medium – second stage the biological aspect of sampling efficiency – depends on the sampling and removal of biological particles without altering their viability or biological activity – biological analysis to identify & quantify the biological particle presents – third stage None of the presently available samplers for culturable bioaerosols can be considered as reference method • Glass liquid impingers (AGI, HAM, MIL) • Six-stage Andersen impactor (AND) 37
  • 38. Bioaerosol Samplers: Principles of Bioaerosol Collection Inertial impaction: the inertial of the particle forces its impaction onto a solid or semisolid impaction surface – a cultural medium, or an adhesive surface – be examined microscopically Single-stage impactors: the surface air sampler, PBI, SPI Cascade impactors: two or more impaction stages (the Anderson cascade impactor) Slit samplers: the impaction stage consists of one or more slits instead of one or more circle holes (CAS, NBS, BAS cultural plate samplers) 38
  • 39. Bioaerosol Samplers: Principle of Collection - Inertial Impaction Impaction ~ a special case of curvilinear motion ~ application in the collection and measurement of aerosol particles Assumption: particles stick to the surface of the impaction plate once they hit it 39
  • 40. Bioaerosol Samplers: Principle of Collection - Inertial Impaction Assumptions: the flow velocity is uniform in the jet; the streamlines are arcs of a circle with centers at A Y τU 2 X Vr = τa r = r τU 2  2πr  π ∆ = Vr t =   = τU r  4U  2 ∆ πτU π EI = = = Stk h 2h 2 40
  • 41. Bioaerosol Samplers: Principle of Collection - Inertial Impaction Stk. to characterize inertial impaction: The characterization dimension : 2 • the radius of the nozzle jet = Dj/2 for a τV ρp d p VC c circular jet Stk = = Dj 2 9ηD j • the jet half-width = W/2 for a rectangular jet 1.00 Collection efficiency 0.80 0.60 9ηD jStk 50 0.40 d 50 C c = 0.20 ρp V 0.00 0 2 4 6 8 10 Aerodynamic equivalent diameter 41
  • 42. Aerosol Samplers In General: Sampler Fractional Efficiency Curve (FEC) Ideally, it is desired that all particles greater than a certain size are collected and all particles smaller than that size pass through – Cut-off size FEC: relates collection efficiency to the particle diameters Cut-point , cut-off size (d50): is the AED of the particle with 50% efficiency Slope: it the sharpness of the cut Aerodynamic equivalent diameter (µm) 42
  • 43. Bioaerosol Samplers: Principle of Collection – Multi-stage Impaction Cut-points for different stages 43
  • 44. Bioaerosol Samplers: Principles of Bioaerosol Collection Centrifugal inertial impaction: particle separation by centrifugal force in a radial geometry - the Reuter centrifugal sampler (BIO) Liquid impingement: the particles are collected by inertial impaction into a liquid, and particle diffusion within the bubbles (the AGI-4 and AGI-30 impingers) Tangential impinger: collects particles by inertial impaction and centrifugation (BioSampler SKC) 44
  • 45. Bioaerosol Samplers: Principles of Bioaerosol Collection Filtration: impaction, interception, diffusion, gravitational settling, etc. − particle physical properties, filter pore size, air flow Challenges: inlet – isokinetic sampling ? filter: dehydration effect – desiccation stress? Gravitational Settling: the least effective methods of bioaerosol collection – particle size, shape and airflow dictate the deposition of particles Electrostatic Precipitation: overcome some of physical damage caused by impinger, or impactor 45
  • 46. Bioaerosol Samplers: Viable Samplers – Inertial Impactors Anderson single stage Anderson two-stage Anderson six-stage Stage with Petri- viable impactor viable impactor viable impactor dish 46
  • 47. Bioaerosol Samplers: Viable Samplers – Impingers All glass AGI-30 liquid impinger Multistage all glass liquid impinger 47
  • 48. Bioaerosol Samplers: Viable Samplers – Centrifugal Sampler “Aerojet” cyclone sampler RCS Biotest centrifugal sampler 48
  • 49. Bioaerosol Samplers: High impact velocity can result in metabolic and structural injuries of the collected microorganisms 1 – 265 m/s Selection of sampler: cutoff size and the aerodynamic particle size 49
  • 50. Bioaerosol Samplers: Selection of Sampler SAS samplers – portable one stage multiple-hole impactors Air-O-Cell and Bukard samplers – the slit impactors, on microscope slide or tape The Reuter centrifugal sampler (RCS) – portable – d50 ~ 3.8µ m The AGI-30 and the AGI-40 can only be used with water-based collection fluids The BioSampler can be used with nonevaporative liquids (mineral oil) – permit long sampling time 50
  • 51. Bioaerosol Samplers: Collection Time Bioaerosol concentration vary greatly with time – sample collection time is essential t1 – t2 – low concentration t3 – t4 – high concentration Sampling time – sufficiently long Average concentration: Ca ts – starting time V = Qt tf – finish time N = CaQt Q – sampling flow rate N Ca Q The surface density: δ= = t A A δo optimal surface density 51 A - viewing area
  • 52. Bioaerosol Samplers: Optimal Collection Time Optimal sampling time for solid surface sampler δo optimal surface density δ < < δ o Insufficiently loaded samples δ > > δ o overloaded samples Adjusting the sampling period to obtain optimal surface density δ A Optimal sampling time: t= Ca Q The optimal sampling time for a given bioaerosol concentration is different for each sampler – sampler’s flow rate and collection surface area 52
  • 53. Bioaerosol Samplers: Optimal Collection Time Optimal collection time for impingers Impinger samples are not sensitive to overloading or under- sampling because the liquid sample can be either diluted or concentrated depending on the concentration of collected bioaerosol particles in the liquid. Evaporation of sampling liquid and reaerosolization of already collected particles limit the sampling time in most impingers 53
  • 54. Bioaerosol Samplers: Optimal Collection Time Permissible sampling parameter ranges for less than 10% change in collection efficiency with the AGI-4 and AGI-30 impingers when operated at a sampling rate of 12.5 L/min 54
  • 56. Intermediate Processing: • Manipulate samples to be compatible with detection methodology  Take into account liquid or solid surface collection techniques  Ex. - microscopy – sample on solid surface (i.e. filter) • If samples in liquid media – dilutions to achieve countable concentrations 56
  • 57. Non-viability Assays: Microscopy – Brightfield (Light) • Limit of resolution => 0.2 µ m • Time-consuming, tedious, expensive Salmonella (DIC) Cryptosporidium (DIC) 57
  • 58. Non-viability Assays: Microscopy – Electron Microscopy • Electronically magnified images • Electrons => short wavelengths • Magnification up to 2,000,000x Hepatitis A virus Influenza virus 58
  • 59. Non-viability Assays: Flow Cytometry – Cell Sorting 59
  • 60. Non-viability Assays: Molecular Detection • Polymerase Chain Reaction (PCR) • Genetic hybridization 60
  • 61. Viability Assays: Bacteria and Fungi – Agar Culture • Non-selective: Plate Count Agar, R2A Agar • Selective: mFC Agar, EC Agar, Salmonella-Shigella Agar, Malt Extract Agar (MEA – fungi) R2A Agar Salmonella-Shigella Agar 61
  • 62. Viability Assays: Virus – Cell Culture uninfected Late cytophathic effects: Cell degeneration Plaque formation Enlarged cells Nuclear inclusions 62
  • 63. Further Characterization: Biochemical Analyses 63
  • 64. Further Characterization: Antibiotic Resistance Microdilution Disk Diffusion 64
  • 65. Further Characterization: Molecular Sequencing Sequence Info Dendrogram 65
  • 66. Further Characterization: Molecular Characterization: Ribotyping 66
  • 67. Part II: Bioaerosol Sampling: Demonstration & Hands-on Practice 67
  • 68. Bioaerosol Sampling Illustration: Illustration of Bioaerosol Sampling Procedure (Impactor - culture method) Step 1: Agar preparation Step 2: Sampler flowrate calibration Step 3: Sample collection with a viable sampler Step 4: Sample transportation Step 5: Sample condition Step 6: Sample analysis 68
  • 69. Pre-sampling – agar plate preparation: 69
  • 70. Pre-sampling – agar plate preparation: 70
  • 71. Pre-sampling – agar plate preparation: 71
  • 72. Pre-sampling – agar plate preparation: 72
  • 75. Taking Samples in the Field: 75
  • 76. Taking Samples in the Field: 76
  • 77. Taking Samples in the Field: 77
  • 78. Taking Samples in the Field: 78
  • 79. Post-Sampling – agar plate incubation: 79
  • 80. Post-Sampling – agar plate reading: 80
  • 81. Post-Sampling – agar plate reading: 81
  • 82. Post-Sampling – agar plate reading: 82
  • 83. Concentration Determination: Example #1 • A bioaerosol sampling campaign was conducted at a ambient location in vicinity of a egg production farm. AGI-30 viable sampler was used to take total bacteria samples. The air flow rate of the AGI-30 was controlled at 12.5 l/min and the sampling duration was 30 min. After the field sampling, the samples were transported to the lab at 4 oC. 83
  • 84. Concentration Determination: Example #1 – Cont. • Impinger fluid from each sample was transferred to a sterile tube and its volume determined. Impinger fluid samples, and/or dilutions in F-tab containing 0.1% Tween 80, were plated in duplicate on Trypticase Soy Agar (TSA) for growth of bacteria. The TSA plates were incubated at 37º C. Plates were checked daily for growth of colonies and moved to 4º C when colonies were of appropriate size for identification and counting. 84
  • 85. Concentration Determination: Example #1 – Cont. • Lab results of an Impinger sample: Total Bacteria Total Impinger bacteria Recip. Bacteria/ml fluid vol. in Count 1 Count 2 Average Dilution Imp fluid (ml) Impinger 11 24 17.5 10 ? 16 ? • Bacteria/ml Imp fluid = average count * Recip. Dilution • Total bacteria in impinger = Bacteria/ml Imp fluid * Impinger fluid vol. 85
  • 86. Concentration Determination: Example #1 – Cont. • Concentration calculation: N ???cfu C= = = ???cfu / m 3 Q * t 0.0125m 3 / min* 30 min N = total bioaerosol counts = ??? cfu Q = sampling flowrate = 12.5 l/min * 0.001 m3/l = 0.0125 m3/min t = sampling duration = 30 min. 86
  • 87. Concentration Determination: Example #2 • If a one-stage Anderson viable sampler with a no-selective R2A agar plate was used to take total bacteria samples in a residence home. The air flow rate of the sampler was controlled at 28.3 l/min and the sampling duration was 10 min. After the field sampling, the samples were transported to the lab at 4 oC. The agar plates were incubated for growth of colonies. It was observed that colonies counts of a plate was 82. What was the concentration of the total bacterial in the air of this home? 87
  • 88. Concentration Determination: Example #2 – Cont. • Concentration calculation: N ???cfu C= = = ???cfu / m 3 Q * t 0.0283m 3 / min* 10 min N = total bioaerosol counts = ??? cfu Q = sampling flowrate = 28.3 l/min * 0.001 m3/l = 0.0283 m3/min t = sampling duration = 10 min. 88
  • 89. References: •Baron, P.A. and K. Willeke. 2001. Aerosol Measurement: Principles, Techniques, and Applications, 2nd edition. John Wiley & Sons, New York. •Hinds, W.C. 1999. Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles, 2nd edition. John Wiley & Sons, New York. •Cox, C.S. and C.M. Wathes. 1995. Bioaerosols Handbook. Lewis Publishers. Washington D.C. 89
  • 90. Acknowledge: • Supported by a National Research Initiative grant from the National Institute of Food and Agriculture, Air Quality Program (No. 2007-55112-17856) 90
  • 91. Hands-on Practice: sampler flow calibration/check 91