The particle follows the streamline and hits the impaction plate at a distance ∆ from the jet centerline. The impaction efficiency depends on the particle inertia parameter τ and the jet Reynolds number.

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

5. Bioaerosol Fundamentals
5

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

19. Bioaerosol Fundamentals:
19

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

28. Bioaerosol Sampling
28

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

55. Bioaerosol Analysis
55

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

73. Pre-sampling- sampler flowrate calibration:
73

74. Pre-sampling- sampler flowrate calibration:
74

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