Bioaerosols in university animal care facilities

Transcription

The University of Toledo
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Theses and Dissertations
2004
Bioaerosols in university animal care facilities
Heather Lorenz
Medical College of Ohio
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Bioaerosols in University Animal Care Facilities
Heather Lorenz
Medical College of Ohio
2004
DEDICATION
I would like to dedicate this work to my husband for his continued love and support; my
parents for always encouraging and believing in me; Dr. Brian Harrington for his love of
microbiology; and my major advisor, Dr. Michael Bisesi, for sharing his knowledge and
guidance.
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ACKNOWLEDGEMENTS
I would like to acknowledge the following for their willingness to contribute and make
this work possible: my major advisor, Dr. Michael Bisesi; committee members Dr. Brian
Harrington and Dr. Sheryl Milz; April Ames and Sara Fuenger for their guidance and
many hours of work they put into the lab; and Dr. Jeffrey Jablonski for giving me the
tools to prepare myself.
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TABLE OF CONTENTS
DEDICATION................................................................................................................... i.
ACKNOWLEDGEMENTS ............................................................................................ ii.
TABLE OF CONTENTS ............................................................................................... iii.
INTRODUCTION............................................................................................................. 1
LITERATURE REVIEW ................................................................................................ 5
METHODS ...................................................................................................................... 23
RESULTS ........................................................................................................................ 33
DISCUSSION .................................................................................................................. 47
CONCLUSIONS ............................................................................................................. 54
REFERENCES................................................................................................................ 56
APPENDICES ................................................................................................................. 66
ABSTRACT................................................................................................................... 100
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INTRODUCTION
Overview
Air quality concerns have been a prominent health issue in the indoor environment.
According to the American Conference of Governmental Industrial Hygienists
(ACGIH’s) Bioaerosols: Assessment and Control Book (Macher, [Ed.] 1999a), sick
building syndrome has been used to describe symptoms that appear to be associated with
building occupation, however, a specific cause for the symptoms cannot be identified.
Another designation defined by the book, building related illness, is a term that is often
used to describe symptoms that appear to be associated with building occupation where a
specific physical, chemical, or biological agent has been identified as the contributing
factor to a diagnosable disease. Biologicals have become the indoor air quality agent of
interest to many researchers, medical, public and occupational health professionals
because of their known association with a variety of adverse health effects (Douwes et
al., 2003). A common route of exposure and the one of most interest is inhalation.
According to Hirst (1995), “A bioaerosol is an aerosol comprising particles of biological
origin or activity which may affect living things through infectivity, allergenicity,
toxicity, pharmacological or other processes.” Health effects cover allergic response,
infectious disease and even toxic responses. Bioaerosols encompass a wide range of
biologic material; however, bacteria and fungi are of great importance in relation to
human health and have been the focus of many indoor air quality studies.
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Bioaerosols originate from both indoor and outdoor reservoirs. One major biological
sources is animals. Therefore, it is reasonable to believe that animal care settings such as
animal laboratories housing mice, rabbits, birds, and other small animals, provide the
conditions to support biological growth and promote the generation of bioaerosols.
Accordingly, this study was designed to determine a profile and characterization of viable
bacteria and fungi during different animal activities throughout the work-shift at two
university animal care facilities. Rooms studied in the animal care facilities included
rooms where the animals were housed, rooms where cage cleaning occurred, office areas,
and outdoors. The identification of specific tasks performed throughout the day that
produce an increased level of exposure, may be used by occupational and public health
professionals to determine adequate control measures.
Statement of Problem
Scientific research has supported the fact that animal settings present a potential
inhalation occupational health concern for animal caretakers. The major potential indoor
air quality contaminants in animal houses include viruses, bacteria, fungi, and other
organic dust (Wathes, 1995). Wathes further pointed out that animals themselves
generate bioaerosols, specifically bacteria and fungi, through secretions, excretions, feed,
and bedding material. Health effects, in relation to both bacteria and fungi, include
allergenic responses, infectious disease, and toxic responses. Although many studies have
been published on bioaerosols in various animal settings in the agricultural industry, there
is a paucity of published data on bioaerosol levels in other animal care settings. This
study was designed to collect air samples to determine bacterial and fungal levels before
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and during cleaning activities throughout the work shift to determine if certain tasks
created an increased risk for employee fungal and bacterial exposure via inhalation.
Purpose and Significance
The purpose of this study was to research and conduct an industrial hygiene air sampling
investigation in two university animal care facilities to determine if there were
statistically significant different levels of fungi and bacteria between outdoors, rooms
housing animals, cage cleaning rooms, and rooms not housing animals during different
activities throughout the work shift. The significance of the study was to provide
assistance to occupational health professionals in order to anticipate, recognize, evaluate,
and control bacterial and fungi concentrations in non-agricultural animal care facilities.
Hypothesis
Hypotheses
The hypotheses tested were:
1a. There is no significant difference in measured mean airborne bacteria levels
before and during cleaning activities throughout the work shift.
1b. There is no significant difference in measured mean airborne fungi levels before
and during cleaning activities throughout the work shift.
2a. There is no significant difference in measured mean airborne bacteria levels
between rooms housing animals, cage cleaning rooms, outdoors, and rooms not
housing animals.
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2b. There is no significant difference in measured mean airborne fungi levels between
rooms housing animals, cage cleaning rooms, outdoors, and rooms not housing
animals.
Objectives
The objectives of this study were to:
Conduct an industrial hygiene air sampling investigation for viable bacteria and
fungi in two university animal care facilities and determine concentrations.
Compare viable bacteria and fungi results, between areas within each location, to
determine if specific area concentrations were statistically significantly higher.
Compare viable bacteria and fungi results, between areas within each location, to
determine if specific activities were statistically significantly higher.
Compare viable bacteria and fungi results to background samples (outdoors and
office areas).
Classify bacteria into gram-positive or negative, rod or cocci, and determine the
genus of fungi.
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LITERATURE REVIEW
Overview
Many studies have been published on bioaerosols in various animal care settings such as
swine houses, dairy farms and cow barns, poultry housing and other farming
environments indicating high concentrations of bacteria, fungi, endotoxin, organic dust,
and ammonia among other species specific air contaminants (Chang et al., 2001; Kullman
et al., 1998; Debey et al., 1995). These indoor contaminants may affect the respiratory
system creating various human health concerns for building occupants (Kaliste et al.,
2002). Although a few studies have investigated bioaerosols in other animal housing
environments, there is a lack of research on bioaerosol levels in non-agricultural animal
care settings.
Conceptual Framework
Association between employee complaints and a causative agent are often difficult to
determine, resulting in the frequent use of a less specific term, sick building syndrome.
Research in the area of bioaerosols is helpful in identifying and narrowing down the
etiology of symptoms often associated with sick building syndrome in order to anticipate,
recognize, evaluate and control the causative agent. Because of the nature of animal care
environments, it is reasonable to anticipate that elevated bioaerosol exposure exists,
presenting a potential occupational heath concern for the animal caretakers. An industrial
hygiene air sampling investigation for bioaerosol concentrations, specifically bacteria and
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fungi, was warrented to determine potential exposure risks for animal caretakers in order
to determine proper control measures.
Sources and Generation of Bioaerosols
Bioaerosols are airborne organic substances or organisms consisting of and/or originating
from microbes, animals, and plants (Douwes et al., 2003). Fungi and bacteria are found in
outdoor air and are either tracked indoors by humans and other animals or forced into the
indoor environment through a Heating Ventilating and Air Conditioning (HVAC) system
or natural ventilation. A major outdoor source that produces both bacteria and fungi is the
agriculture industry including; composting, crop processing and farming (Stetzenbach,
2002). Therefore, microorganisms are always present to some degree in the indoor
environment. However, indoor reservoirs present a higher potential for occupant
exposure.
Some examples of indoor reservoirs include water damaged building material, air
handling systems, wastewater treatment facilities and animals (Stetzenbach, 2002). Water
damaged building material including ceiling tile, carpet, drywall etc. has been a source of
indoor bioaerosol exposure in many buildings. According to published studies, mold
growth found on building material is often accompanied by gram-negative bacteria,
endotoxin, and mycobacteria (Andersson et al., 1997; Dales et al., 1999). Air handling
systems have been known to contain Legionella pneumophila which causes Legionnaires’
disease. Bacteria found in sewage may include gram-negative bacteria that can cause
gastroenteritis (Institute of Inspection, Cleaning and Restoration Certification, 1999).
Indoor reservoirs occur in animal care settings as result of the animals themselves.
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Bioaerosols originate from fur, skin, animal waste, feed, and bedding (Kaliste et al.,
2002). According to Wathes (1995), “In general, bioaerosol concentrations will be higher
in animal houses than in other industrial, residential or ambient settings.”
Microorganisms require a nutrient source and water to support growth. Nutrient sources
are plentiful in the indoor environment. A variety of different sources include building
material and specific to the animal care settings: feed and bedding material. If moisture is
not present, the nutrient source alone cannot support growth. Microorganisms can grow
on water-damaged materials within 24 – 48 h if not completely dried. Determinants
including temperature and relative humidity affect the ability for microorganisms to
proliferate (Shaughnessy et al., 1999).
Fungi
Fungi can be either multicellular, consisting of spores and hyphae (branched filaments)
that grow as a mycelium, or unicellular. Mullins (2001) explains that while some
varieties use water as a mechanism to release spores into the air, others disseminate
spores directly into the air. He also stated that spores that are produced asexually, usually
passively rely on wind for dispersal, while those that reproduce sexually release spores
more forcefully into the air. The type of dispersion can greatly influence concentrations.
Spore concentration and speciation, in outdoor air, varies significantly depending on time
of day, location, season, temperature, and precipitation. A year round study from 1991 –
1993 completed in Kitchener-Waterloo, Ontario, Canada on the comparison of total
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fungal spores in indoor residential houses and outdoor air found a mean outdoor level of
3301 spores/m3 and a mean indoor level of 2254 spores/m3 (Li and Kendrick, 1995).
Fungi (not specified) found in animal facilities range in concentration between 100
cfu/m3 to 1000 cfu/m3 (Stetzenbach, 2002). An unpublished study (Ames, 2003),
conducted during the months of April, May, and June of 2003 in Ohio, found fungal
concentrations outdoors ranging from 0 cfu/m3 to 1653 cfu/m3.
Bacteria
Bacteria are described as single or multi cellular organisms that come in a variety of
shapes including cocci or small spheres, straight rods, spiral rods, and branched
filaments. Some bacteria, notably Bacillus spp. also produce endospores which are thick
walled spores produced within the bacterial cell, and are resistant to adverse conditions
such as drying, heat, and radiation, often becoming airborne when disturbed (Burge and
Otten, 1999a).
Burge and Otten (1999a) discuss bacteria at length describing that bacteria can be
classified as either gram-positive or gram-negative. Bacteria usually originate outdoors
from plants and animals such as animal feces and decaying organic matter. The most
common outdoor varieties include gram-positive rods and cocci including Micrococcus
spp., Bacillus spp., and Pseudomonas spp., a gram-negative bacteria. The most common
indoor varieties include gram-positive rods and cocci including Micrococcus spp. and
Bacillus spp. Typically indoor environments contain higher concentrations and a more
diverse species population of bacteria, originating primarily from humans.
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Bacteria (not specified), found in animal facilities, range in concentration between 1,000
cfu/m3 to 100,000 cfu/m3 (Stetzenbach, 2002). An unpublished study (Ames, 2003),
conducted during the months of April, May, and June of 2003 in Ohio, found bacteria
concentrations outdoors ranging from 14 cfu/m3 to 768 cfu/m3.
Health Effects Associated with Exposure to Bioaerosols
According to ACGIH’s Bioaerosols: Assessment and Control book (Macher, [Ed.],
1999b), health effects resulting from exposure to bioaerosols have been known to elicit
allergenic responses including hypersensitivity and opportunistic diseases, diseases with
known etiology and even toxic responses. Indoor sources of these substances are usually
attributed to fungi or bacteria.
Allergenicity
An allergic response or hypersensitivity results from exposure to a substance that elicits
an immune response referred to as an antigen. When exposed to an antigen the immune
system may produce antibodies in the form of proteins classified as either IgA, IgD, IgE,
IgG, or IgM. Bioaerosols such as fungi and bacteria are common sources of airborne
antigens (Rose, 1999). When bioaerosols such as bacteria and fungi are inhaled, they
deposit in the respiratory system and may cause an allergic reaction among those who are
hypersensitive or allergic. The organisms do not need to be viable to trigger an allergic
reaction.
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According to Frew (2004), there is no doubt IgE antibodies may be produced in response
to exposure to fungi. Fungi such as Pencicillium spp. and Aspergillus spp. and bacteria
such as Strepromyces spp., Thermoactinomyces spp. and Saccharopolyspora spp. are
common allergens (Institute of Inspection, Cleaning and Restoration Certification, 1999).
Day and Ellis (2001) reported that species such as Alternaria spp., Cladosporiu spp.,
Aspergillus spp., Candidia albicans, Penicillium spp., Stachybotrys spp., Basidiomycetes,
Zygomycetes, and Ascomycetes are the most commonly implicated in allergic disease.
Aspergillus fumigatus, unlike most fungi, not only causes an allergic reaction triggering
IgE response and production of IgG antibodies, but can even grow in the respiratory
system causing allergic bronchopulmonary aspergillosis (Frew, 2004).
The cell walls of microorganisms also have been implicated in asthmatic diseases. B-1, 3D-glucan
is a component of fungi, bacteria, and plants that has been associated with
various respiratory symptoms including inflammatory responses (Wan et al., 1999).
Douwes et al. (2003) pointed out allergic responses to bioaerosols including allergic
asthma, allergic rhinitis, and hypersensitivity pneumonitis (also referred to as extrinsic
allergic alveolititis and farmers lung). Asthma has been associated as a symptom of sick
building syndrome which in many cases is due to specificity of etiology, and is
eventually redefined as a building related illness attributable to bioaerosol exposure
generated from humidifiers, contaminated water sprays, and moisture intrusion. Other
allergic responses including rhinitis and sinusitis induce cold like symptoms.
Hypersensitivity pneumonitis (HP) results in symptoms ranging from fatigue and
shortness of breath to recurrent pneumonia and lung fibrosis diagnosable by patient
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history and clinical signs or symptoms (Macher, [Ed.], 1999b). Hypersensitivity
pneumonitis has specifically been associated with many fungi including Penicillium spp.
and Aspergillus spp. found in water damaged building materials (Douwes et al., 2003)
and thermophilic bacteria including Saccharopolyspora rectivirgula found in
contaminated hay (Duchaine et al., 1999).
Infectivity
Non-allergic respiratory diseases including irritant induced asthma, non-allergic
rhinitis/mucous membrane irritations, chronic bronchitis, chronic airflow obstruction, and
organic dust toxic syndrome, that do not elicit an immunoglobulin response, also have
been associated with bioaerosol exposure including both fungi, bacteria and their
components (Douwes et al., 2003).
Bacteria are a major cause of communicable disease often referred to as infectious
disease. Although transmission of disease occurs through all routes of exposure,
inhalation is of primary concern for bioaerosols. Infectious diseases with known etiology
such as tuberculosis in health care workers, Q-fever in veterinarians, and tularaemia in
forest workers have all been caused by bioaerosols (Douwes et al., 2003). Other bacterial
diseases include Legionnaires’ Disease, Pontiac Fever, and Humidifier Fever (Douwes et
al., 2003). Microorganisms found in soil and compost also have been known to cause
aspergillosis, histoplasmosis, blastomycosis, and coccidioidomycosis (Institute of
Inspection, Cleaning and Restoration Certification, 1999; Center for Disease Control and
Prevention, 1999, 2003; Lenhart et al., 1997). Fungi can also cause fungal infections of
the skin, nails, hair and mucous membranes (Burge and Otten, 1999b).
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Toxicity
According to Douwes et al. (2003) “Mycotoxins or fungal toxins are low molecular
weight biomolecules produced by fungi that are toxic to both animals and humans.” A
toxic response due to mycotoxin exposure has been termed mycotoxicosis (Hossain et al.,
2004). Exposure to certain fungi that contain mycotoxins, such as aflatoxin from
Aspergillus flavus, have been established as a human carcinogen (Hossain et al.). Other
major mcycotoxins include; Ochratoxin from Aspergillus ochraceous, Fumonisin from
Fusarium spp., Trichothecene from Fusarium spp. and S chartarum and Zearalenone
from Fusarium species (Hossain et al., 2004). Toxic responses associated with
mycotoxins can be acute, chronic, mutagenic and teratogenic, possibly resulting in
hepatitis, liver cancer, childhood cirrhosis, immunosuppresion, nephrotoxicity, tumors,
hepatotoxicity, embryotoxicity, gastrointestinal disorders, skin irritation, hemorrhage,
convulsion, gynecomastia in boys, and precocious puberty (Hossain et al, 2004).
Much of the media attention has focused on Stachybotrys chartarum also known as the
infamous “black mold.” As mentioned above, the mycotoxin associated with
Stachybotrys chartarum is trichothecene. According to Hossain et al. (2004),
“Respiratory symptoms range from benign, such as congestion and cough from rhinitis,
to reactive airways disease, to more serious syndromes, including alveolitis,
bronchietasis, and pulmonary fibrosis.”
One of the first studies that broke the “black mold” news headlines originated out of
Cleveland, Ohio. In the early 1990’s a group of infants began experiencing respiratory
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ailments specifically termed acute idiopathic pulmonary hemorrhage (AIPH) (Center for
Disease Control and Prevention, 1994). A correlation was found between AIPH and
water damaged environments with subsequent mold growth including Stachybotrys
chartarum which has been linked to hemorrhagic disorders in animal studies (Center for
Disease Control and Prevention, 1997; Montana et al., 1997). However, the correlation
has been debated and the Center for Disease Control and Prevention has since deemed the
studies unsubstantiated, concluding that further research was needed (Center for Disease
Control and Prevention, 2000). Kuhn et al. (2003) also concurred with the Center for
Disease Control and Prevention, after review of relevant literature, that while certain
toxic responses can be associated with a certain fungus, the relationship between
Stachybotrys spp. and human disease is inconclusive. The Center for Disease Control and
Prevention recently published their plan for conducting research and investigating cases
of AIPH (Center for Disease Control and Prevention, 2004).
Gram-negative bacteria also produce toxic responses including respiratory inflammation
and airway restriction while creating conditions for allergic and infectious disease
(Institute of Inspection, Cleaning and Restoration Certification, 1999). Endotoxins exist
in gram-negative bacteria, making up part of the cell wall and are recognized as a
contributing factor of occupational lung disease (Institute of Inspection, Cleaning and
Restoration Certification). According to Burge and Otten (1999a), while low exposures to
endotoxins assist in the development of the immune system, high exposures cause
irritation and other conditions. They can be found in agriculture, general house dust,
sewage, water sprays containing gram-negative bacteria, compost and many other
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environments and play an important role in the cause of many non-allergic lung diseases
(Douwes et al., 2003).
Sampling Selection
Apparatus
According to Macher (1997a), “To select an appropriate bioaerosol sampler, users must
clearly outline (1) their reasons for collecting samples, (2) what biological agent they
wish to measure, (3) how the samples will be assayed, (4) the estimated or previously
measured bioaerosol concentration at the test site, (5) the aerodynamic diameters of the
particles to be collected, and (6) the velocity of the sampling air stream.”
The best way to characterize bioaerosols, when the make up of the air is unknown, is
through culturing on media (Crook and Sherwood-Higham, 1997). The sampling method
used most frequently for the determination of airborne bacteria and fungi levels and types
has been viable air sampling techniques in which air is collected on a culture medium,
incubated, counted, and identified (Macher, 1997b). Although limitations exist, including
un-representation of total cells, cells that are incapable of growth on the media selected,
and non-culturable viable cells, valuable information including airborne levels and types
of both bacteria and fungi, can be obtained from bioaerosol sampling if performed
properly (Crook and Sherwood-Higham, 1997). A convenient sampling method often
used employs the principle of inertial impaction onto an agar-based medium.
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According to Wathes (1995), the Anderson six-stage impactor is probably the most
common bioaerosol sampler used in animal houses. This multi staged impactor is used to
collect microorganisms in specific particle size ranges with each stage collecting a
smaller particle size aiding in the determination of particle deposition in the lungs. The
single stage sampler is used to collect total particles and is also frequently used. The
Anderson sampler is an aluminum device consisting of an inlet cone, jet classification
stage, and a base plate that holds a petri dish. The impactor stage contains 400 precisiondrilled holes and when assembled is held together by three spring clamps and sealed with
two O-ring gaskets. Air is drawn into the impactor using an ultra high flow pump at a
flow rate of 28.3 liters per min. As air passes through the impactor, multiple jets of air
direct airborne particles onto the agar collection plate where they are retained for
analysis. After suitable incubation, microorganisms collected may grow into visible
colonies.
Media
Although collection techniques for bacteria and fungi are the same, sampling assay
methods must be specifically oriented to the biological material being collected and the
environment in which it resides (Crook and Sherwood-Higham, 1997). Routinely, a
general-purpose medium for bacteria, such as soybean-casein digest agar also known as
tryptic soy agar (TSA), is used in indoor air quality investigations (Burge and Otten,
1999a). Other media, such as MacConkey agar, are specifically designed to detect gramnegative bacteria while suppressing gram-positive bacteria (Burge and Otten, 1999b).
Using both TSA with 5% sheep’s blood and MacConkey agar provides a more accurate
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profile and concentration of airborne bacteria. A general purpose medium used
commonly for fungi is potato dextrose agar (PDA) (Buttner et al., 2002).
Areas and Time
When characterizing bacterial and fungal bioaerosols in animal care facilities, sampling
should take place during normal business hours and specifically during times of
anticipated animal activities. Sampling right after the source is disturbed (cleaning and
feeding) will help determine the number and type of bioaerosols contributing to the entire
load from that source (Burge and Otten, 1999a). According to ACGIH’s Bioaerosols:
Assessment and Control (Macher, [Ed.], 1999), recommended sampling areas include; an
anticipated high-exposure area, an anticipated low exposure area, and outdoors near air
intakes for the building or near windows or doors in naturally ventilated buildings.
According to Folmsbee et al. (2000), sampling run times produce varied results as a result
of stress on the microorganism, impacting total efficiency. Expected levels also vary
greatly, making it difficult to determine proper sampling times to alleviate overloading
and underloading sampling media (Folmsbee et al., 2000). Although there are no
standardized run times for bioaerosol sampling, research has shown that long sampling
times may kill or remove the intended contaminant while short sampling times may not
collect a sample that is representative of the area (Folmsbee et al., 2000). Therefore, each
sampling site must be evaluated for potential load to determine the most suitable
sampling run time.
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Laboratory Analysis
Laboratory analysis using culture based methods consists of incubation, counting and
identification. Both counting and identification are of extreme importance to characterize
levels of bioaerosols and to determine if the types of bacteria and fungi are typical or
unexpected. After the sampling media are cultured, the number of colonies are counted
and identified microscopically; a concentration can be determined by dividing the
number of colony forming units by the volume of air pulled through the sampler. Units
are expressed in colony forming units per meter cubed (cfu/m3).
Occupational Exposure Limits
To date, there has not been a consensus standard developed for bioaerosols. This is due to
the fact that occupational exposure limits are based on a dose-response relationship and
dose-response research in this area is not sufficient enough to develop such a threshold.
Although there are no standardized occupational exposure limits for bioaerosols,
including bacteria and fungi, guidelines for data interpretation do exist. The American
Conference of Governmental Industrial Hygienists does not support any numerical
occupational exposure limit but strongly recommends data gathering and professional
judgment to interpret information and control and remediate the situation in order to
protect employee health and safety (Macher, [Ed.] 1999a). When sampling and
interpreting bioaerosol results, the main objective is to characterize the type and
concentration found in order to determine possible contributing sources so the sources
can be remediated and controlled.
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Bacteria
The dominance of a single species of bacteria may indicate an indoor source while a
variety may indicate the source likely has an outdoor origin. Some bacteria, if found
indoors, are likely to indicate an indoor source because they are rarely found in outdoor
air. The presence of gram-negative bacteria alone indicates poor indoor air quality and
often indicates the presence of contaminated water (Macher, [Ed.], 1999a). Another
indication of an indoor source of bacteria occurs when the number of bacteria of human
origin is relatively high indicating the need for more ventilation. Gram-positive bacteria
are commonly found indoors and outdoors and most bacteria that occur naturally do not
present a health risk under normal circumstances; however, the risk of infection does
increase when bacteria are found in high concentration indoors.
Fungi
The presence of visible fungi indoors is unacceptable. When an indoor source is found,
the source of the problem leading to the environmental conditions required to support
such growth should be identified, corrected, and the visible growth remediated. Air
sampling may provide information leading to the detection of the source but is not
indicative of human health effects. Indoor and outdoor comparisons can provide
information to determine if an indoor source exists.
Outdoor levels are usually similar or slightly higher than indoor levels. Therefore, if the
outdoor fungi levels are of similar species and quantity to that of indoor fungi levels, an
indoor source is not likely present. However, in general, if outdoor fungi levels are lower
than indoor fungi levels, an indoor source is likely. Season and weather conditions may
18
suppress or exacerbate outdoors levels, which should be taken into consideration. Some
species, that are indicative of water damage, can also provide an indication that an indoor
source exits regardless of quantity (Macher, [Ed.], 1999a).
Bioaerosol Investigations
Research on bioaerosols in animal housing is not limited to the farming environment.
Although a few studies have investigated bioaerosols in other animals housing
environments, there is a lack of research on bioaerosol levels in animal laboratory
settings. The following summarizes a few investigations in other non-agricultural animal
care settings.
Kaliste et al. (2002) performed an investigation in two conventional animal laboratories
containing 22-24 adult rabbits in individual cages. Both stationary viable airborne fungi
and bacteria samples were collected during 2d at three different times during the day
including 2h before work activities, during work activities, and 2h after work activities.
Total fungi counts, total dust, airborne endotoxins and ammonia also were collected at
the stationary sampling sites. In addition to the area samples, personal dust and endotoxin
samples were collected during work activities.
The results indicated an increased level of mesophilic bacteria during work activities,
returning to pre-work activity levels quickly after completion of the tasks. However, with
a few exceptions, the levels of viable fungi stayed steady throughout the day. Total dust
increased during work activities but was still generally low. Ammonia concentrations did
not increase during work activities and were lower than occupational exposure limits.
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While endotoxins increased during work activities, they decreased back to pre-activity
levels after completion of the tasks producing background levels of 0.5 mg/m3 or lower,
indicating a low risk to human health.
Total bacteria found were generally similar to those levels found in a typical indoor
environment. The source of the fungi and bacteria was found to be the animal bedding,
however, they were still lower than levels traditionally found in other animal facilities. A
follow up study was completed by Kaliste et al. (2004) comparing dust levels of different
types of cleaned and soiled rat and mouse bedding. The study showed that contaminants
such as mesophilic bacteria and fungi, mycobacteria and endotoxins were all present in
the bedding creating the potential for bioaerosol generation when handled and disturbed.
Most of the fungi found in the labs were Cladosporium spp., Penicillium spp., yeasts and
Aspergillus spp. which are common in the indoor and outdoor environment. Therefore,
the study concluded that although the contaminants mentioned above were present they
were not present in harmful concentrations due to proper ventilation, small numbers of
animals, and proper cleaning.
Another study, completed by Thulin et al. (2002) looked specifically at mouse urinary
allergen concentrations during different tasks to determine control efficacy. The study
resulted in findings associating higher exposures during manual cage empting, cage
changing in an unventilated area, and handling of male animals in an unventilated area.
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Bioaerosols in zoos, although research is limited, also have been studied. Fungi
associated with sick building syndrome, such as some species of Penicillium and
Stachybotrys, exist in zoos, indicating the potential for adverse health effects in animals
and zoo employees (Wilson and Straus, 2002). Specifically, bird aviaries have been of
interest to many animal care personnel because of the devastating effects Aspergillus spp.
has on the birds themselves. Deaths of five birds were attributed to Aspergillus spp. in a
forest aviary located in the North Carolina Zoological Park sparking an air sampling
investigation (Dykstra et al., 1997) which found fungal levels up to 1700 cfu/m3.
Although, when compared to outside levels and other areas in the zoo, the levels were
similar. A subsequent study conducted at the North Carolina Zoological Park (Faucette et
al., 1999) compared the mycological content of a penguin housing facility before and
after the birds were introduced. The researchers found that fungal counts, including
Aspergillus spp., were low in the sampled exhibit and that the use of High Efficiency Air
particulate (HEPA) filtration of incoming and re-circulated air in combination with low
air temperatures and the absence of organic material are important in controlling fungal
counts. The City Zoo of Cal, Columbia also was studied for the presence of Cryptococcus
neoformans (Caicedo et al., 1999), an infectious microorganism that causes the fungal
disease cryptococcosis (Lenhart, 1997). The infectious agent was isolated in 2 out of 380
passive samples collected during the study.
The above studies, in addition to the preceding and continuing study of bioaerosols in
agricultural animal care settings, have laid the foundation for future study in other animal
care facilities such as animal laboratories.
21
Control Measures
In order to control bioaerosols in animal care facilities, the sources and activities that
disturb those sources must be identified and evaluated. Following identification and
evaluation, the industrial hygiene paradigm requires hazard control through engineering
controls, administrative controls, and, as a last resort, personal protective equipment.
Summary
Research has shown that elevated levels of bioaerosols are present in animal housing
facilities. Although most of the research completed has focused on agricultural animal
housing, it is reasonable to anticipate that elevated levels of bioaerosols exist in nonagricultural animal care facilities such as animal laboratories. It is likely that the highest
levels will occur during cleaning and other activities that disturb the major sources of
bioaerosols in animal housing including animal waste, feed, and bedding. It is also likely
that bioaerosol levels will increase with the number of animals present.
The literature review also revealed the impact that bioaerosols have on human health,
therefore, making the study of bacteria and fungal levels in non-agricultural animal care
facilities an important area of study. A study of this nature will assist researchers,
medical, occupational health and other environmental disciplines and public health
professional to better identify and evaluate these contaminants in order to control them
and provide a safe and healthy work environment.
22
METHODS
Overview
An industrial hygiene air sampling investigation was conducted at two non-agricultural
animal care facilities in Ohio. Sampling areas within each location included rooms
housing animals, cage cleaning rooms before and during cleaning operations, outdoors,
and rooms not housing animals. Air samples were collected using a single stage sampler
with TSA with 5% sheep’s blood and MacConkey agar for bacteria and PFA for fungi.
Bacteria collected on TSA with 5% sheep’s blood and MacConkey agar were cultured at
35 Co to simulate human body temperature while fungi collected on PFA were cultured at
room temperature. After the agar was given sufficient time to culture, 48 h for bacteria
and 5 d for fungi, the colony forming units were counted and divided by the volume of air
pulled through the sampling apparatus to determine concentration in colony forming units
per meter cubed (cfu/m3) of air. Bacteria were classified as gram-negative or positive, rod
or cocci. Fungi were identified to the genus level. Statistical analysis was used to
determine differences in data sets.
Sampling Sites
Bioaerosol sampling was conducted at two separate university animal care laboratories.
The university animal care laboratories housed animals including rats, mice, rabbits,
birds, and other small animals. Both locations used ground corncob for the rat and mouse
bedding and rodent chow for the feed. The rat and mouse bedding was changed and food
was replenished once a week, in both locations.
23
Source rooms sampled at Location 1, included a mouse room, a rat room and an aviary
containing pigeons. Both the rat and mouse room bedding was changed by removing the
animals and placing them in clean cages with fresh bedding material. The dirty cages
were placed on carts and wheeled to the cage cleaning room where they were dumped
and scraped manually into a garbage can without the use of local exhaust. The dirty cages
then were placed into a cage washing machine for cleaning. The aviary was cleaned once
a week by first scraping the animal waste off of the floor with a long scraping tool into
piles. The material then was removed and the floors were hosed down.
Source rooms sampled at Location 2, included two rat rooms. As in Location 1, the
bedding was changed by removing the animals and placing them in cages with fresh
bedding material. The dirty cages were placed on carts and wheeled to the cage cleaning
room where they were dumped and scraped manually. A portable cage changing
ventilation unit was used during the dumping and scraping. The dirty cages then were
placed in a cage washing machine for cleaning.
Sampling Areas and Times
A pilot study was carried out prior to data collection to ensure sampling times were
adequate. The area with the highest anticipated bioaerosol concentration, the aviary at
Location 1, was used for the pilot study. The aviary, office area and outside samples were
collected for a period of 5 min, two times a day, before and during cleaning activities.
Based on the pilot study results, the actual data collection times ranged from 1-5 min.
Three to four areas were sampled at each location including:
24
•
Outdoors
•
Room not housing animals (Office)
•
Cleaning room (if different than room housing animals)
•
Room housing animals
Indoor samples were collected in the anticipated exposure area, approximately 4 ft from
the ground. Outdoor samples were collected near the air intake of an HVAC system if
possible.
Other parameters measured or recorded during sampling included:
•
Relative humidity and temperature
•
Carbon dioxide
•
Weather conditions
•
Animals (type, number, use)
•
Building layout
•
Feeding (method, frequency, type of feed)
•
Bedding (type, frequency of changing)
•
Cleaning practices (waste removal)
Equipment and Supplies
Sampling was performed using a single stage impactor with TSA with 5% sheep’s blood
and MacConkey agar made by Aerotech Laboratories, Inc. for bacteria and PFA agar
made by Dr. Brian Harrington at the Medical College of Ohio for fungi sampling. A
quality control check of the mediums ability to support adequate and typical growth of
25
enteric gram-negative bacteria, positive controls were performed on both brands of
McConkey agar using Escherichia coli, Salmonella spp. Enterobactor spp. and
Pseudomonas aeruginosa resulting in successful growth. Positive controls for fungi
included using Rhodotorula rubrum, Aspergillus spp., Penicillium spp., Alternaria spp.,
Fusarium spp., and Cladosporium spp. also resulting in successful growth.
Agar was kept refrigerated until 24 h prior to sampling to minimize the potential for presampling growth and unrepresentative sampling results. Sampling equipment included
two Anderson single stage samplers, one Biostage One single stage sampler, three ultra
high flow Gast pumps, calibration cone, tygon tubing, Gilibrator 2 electronic bubble
meter, cooler for transportation to the lab, alcohol pads and hand sanitizer for sanitation
of equipment and field sampler, sampling cart, and TSI Q-Trak for measuring carbon
dioxide, temperature and relative humidity.
Sampling Procedures and Data Collection
Pre-Sampling Calibration
Ultra high flow Gast pumps were pre-calibrated, with representative sampling media in
line, to a flow rate of 28.3 liters per minute using a Gilibrator 2 electronic bubble meter.
Three trial runs were collected and averaged to determine the pre-calibration flow rate.
The sampling train for calibration purposes consisted of the ultra high flow pump,
impactor sampler, and electronic bubble meter connected, respectively, with tygon tubing
and calibration cone. The TSI Q-Track was pre-calibrated for carbon dioxide using
calibration gas regulated to a concentration of 1,000 ppm of carbon dioxide by
26
connecting the instrument to the gas cylinder with tygon tubing. Calibration forms were
developed in a previously unpublished masters thesis (Ames, 2003) and are located in
Appendix A.
Sample Collection
Samples collected at each location included: outdoors, rooms not housing animals, rooms
housing animals and cage cleaning rooms. Bioaerosol samples collected with all three
agars were collected simultaneously. Samples were collected in each room using 10 ft of
tygon tubing and when possible the pumps were left outside the entrance to each area in
order to minimize animal disturbance. Direct reading measurements of relative humidity,
temperature, and carbon dioxide were collected while the bioaerosols samples were
running. All doors and windows in the indoor sampling locations were closed. The
sampling train for bioaerosol sampling purposes consisted of an AC electrically powered
ultra high flow pump and impactor sampler, with a Petri dish containing the desired agar,
connected with tygon tubing. Each sample was collected approximately 4 ft from the
floor, set on a sampling cart. Time between samples was minimized. All data collection
forms were developed in a previously unpublished master’s thesis (Ames, 2003) and are
presented in Appendix A. Sampling location layouts are located in Appendix B. The
following procedure was used when collecting bioaerosol samples:
1. Before each run, the person collecting the sample sanitized his/her hands with
sanitizer and the sampler was wiped with 70% isopropanol, dried, and checked for
blocked holes and damaged O-rings.
27
2. After the sampler was cleaned and holes unblocked (with copper wire), the
sampling medium was loaded into the sampler so the Petri dish rested on top of
the three raised metal pins. The cover was removed and the jet classification stage
was immediately placed over the exposed agar plate followed by the inlet cone.
The unit then was secured by three spring clamps.
3. Each sample was run for a duration of 1-5 min depending on the area. The
rotameter mounted on the ultra high flow pump was checked for approximate
flow rate.
4. After the sample run time was complete, the person collecting the sample
sanitized his/her hands with sanitizer, the Petri dish was removed, covered, taped,
labeled and then placed securely (medium side up) in a cooler for transportation
to the lab.
Post-Sampling Calibration
At the end of each sampling event, the ultra high flow Gast pump was post-calibrated,
with representative sampling media in line to determine average flow rate. Three trial
runs were collected and averaged to determine the post calibration flow rate. The
sampling train for calibration purposes consisted of the ultra high flow pump, impactor
sampler with a Petri dish containing agar media, and electronic bubble meter connected,
respectively, with tygon tubing and calibration cone. The average of the pre- and postsampling flow rates was used to determine the volume of air by multiplying the average
flow rate by time and then concentration in cfu/m3 by dividing the number of colony
forming units by volume of air. The TSI Q-Track was post- calibrated for carbon dioxide
28
using calibration gas regulated to a concentration of 1,000 ppm of carbon dioxide by
connecting the instrument to the gas cylinder with tygon tubing.
Blanks
A set of field blanks for all types of media, TSA with 5% sheep’s blood, MacConkey,
and PFA, were used as sterility (negative) controls during each sampling event. They
were collected in exactly the same manner as the actual samples, however, without
turning the pump on and running air through the media.
Laboratory Equipment
Laboratory equipment and supplies including reagents for Gram stain (crystal violet,
iodine solution, decolorizer, safranin), water, microscope slides, microscope and
immersion oil for gram staining, were used for gram staining and morphology
identification of bacteria. Clear cellophane tape and lactophenol aniline blue stain were
used for microscopic identification of fungi to the genus level.
Laboratory Analysis
The TSA with 5% sheep’s blood and MacConkey agar for bacteria were incubated at
35Co for 48 h and the PFA agar for fungi was incubated at room temperature for a period
of 5d, after which colonies were examined and counted. Concentrations were determined
by dividing the number of colony forming units by the volume of air in cubic meters. All
laboratory analysis data forms were developed in a previously unpublished master’s
thesis (Ames, 2003) and are presented Appendix A.
29
Bacteria
Gram staining procedures were performed on each bacterial colony to identify the
bacterial growth as gram-negative or gram-positive and rod or cocci. The following
procedures were used when performing the gram staining procedure.
1. The colony was sampled with a sterile loop and a thin smear in saline was made
on a microscope slide and allowed to air dry. After fixation for 1 min with
methanol, it was covered with crystal violet. After 30 sec, the slide was washed
with water for 5 sec.
2. The smear then was covered with iodine, for 30 sec, and then washed with water
for 5 sec.
3. The smear was then treated briefly with the decolorizor and rinsed with water for
5 seconds.
4. The smear was covered with safranin. After the slide remained untouched for 1
min, the slide was washed with water for 5 sec and blotted dry.
5. The slide then was viewed under a microscope at a magnification of 1000 times
for determination of the Gram stain reaction and the morphology of the bacterial
cells.
Fungi
Genus determination was made for each colony using cellophane tape sampling of
colonies for microscopic identification. The following procedures were used when
performing the genus identification.
30
1. Each colony was visually identified by color on the top, bottom, and reverse of
colony, texture, and topography.
2. A piece of clear, sticky cellophane tape was then used to collect a small amount of
the specimen for microscopic identification by lightly touching the surface of the
colony under examination.
3. The tape then was placed on a slide with a small amount of lactophenol aniline
blue stain, covered with a cover slip, and viewed under magnification of 400
times.
4. Each colony was identified to the genus level using reference books (Larone,
2002; Samson et al., 2002; Flannigan et al., 2001).
Data Analysis
The data were analyzed using descriptive statistics and nonparametric statistical tests.
Descriptive statistics including mean, standard deviation, minimum and maximum
concentrations were determined. The Kruskal-Wallis test is non-parametric test used
for multiple comparisons and was used, with a significance value of 0.05, to
determine if a statistically significant difference existed among sampling areas at both
locations. If the Kruskal-Wallis test indicated a significant difference, further
analysis was performed to determine statistically significant differences between
independent comparisons. The Mann-Whitney U test is a non-parametric test used to
determine statistically significant differences between independent comparisons and
was used, with an adjusted significance value, to determine which sampling areas
were statistically significantly different. Because the Mann-Whitney U test does not
account for multiple comparisons an adjusted significance value must be used to
31
reduce the chance of a Type 1 Error. Therefore, the Bonferroni Adjustment was used
with an initial significance value of 0.10 which then was divided by the number of
comparisons to determine the adjusted significance level. The Mann-Whitney U test
also was used to determine if a statistical difference occurred between time periods
before and during cleaning and between a similar area between locations.
32
RESULTS
Overview
Indoor and outdoor air samples were collected in two university animal care settings on 8
separate days on July 19, 20, 22, 26, 27, 29 and August 2 and 16 in 2004. Meteorological
data, the number of animals present in the room during sampling, and other parameters
including carbon dioxide, temperature and relative humidity were collected in addition to
bioaerosols.
The highest mean concentrations of bacteria in the source rooms, by location, were found
in the aviary at Location 1 and in a rat room at Location 2 while the lowest mean
concentrations of bacteria, by location, were found in the mouse room at Location 1 and
the cage cleaning room at Location 2. A majority of bacteria found in all areas at both
locations were gram-positive cocci. However, it was noted that the Aviary at Location 1
did have a mean of 39 gram- negative rods while outdoors had a mean of 1 gram-negative
rod. Growth on the McConkey agar also indicated gram-negative bacteria in the cage
cleaning room and the aviary at Location 1.
The highest mean concentrations of fungi, by location, in the source rooms were found in
the aviary at Location 1 and in cage cleaning room at Location 2 while the lowest
concentrations of fungi were found in the rat rooms at both locations. A majority of fungi
found in all areas at both locations was Cladosporium spp., a species commonly found
outdoors.
33
Meteorological data such as little to no precipitation during sampling, average
temperature ranges from 69.1 oF to 76.6 oF, and average relative humidity ranges from
56.6% to 75.5%. The highest carbon dioxide measurements, by location, were found in
the aviary at Location 1 and in a rat room at Location 2.
Descriptive Analysis
Bioaerosol Concentrations
Comprehensive data sets of bioaerosol concentrations are presented in Appendix C.
Descriptive statistical analysis was performed using total concentrations of bacteria and
fungi.
Growth on the McConkey agar was minimal in all areas at both locations, therefore
statistical analysis was not performed. As mentioned in the materials and methods
sections, a quality control check of the mediums ability to support adequate and typical
growth of enteric gram-negative bacteria, positive controls were performed on both
brands of McConkey agar using Escherichia coli, Salmonella spp. Enterobactor spp. and
Pseudomonas aeruginosa resulting in successful growth.
Field blanks (sterility check plates) for the bacteria sampled did not produce any bacterial
growth after incubation. The highest mean concentrations of bacteria in the source rooms,
by location, were found in the aviary at Location 1 and in a rat room at Location 2 while
the lowest mean concentrations of bacteria, by location, were found in the mouse room at
34
Location 1 and the cage cleaning room at Location 2. The highest quantitative
concentration of bacteria peaked at 26,358 cfu/m3 in the aviary before cleaning
operations at Location 1. However, the sample collected during cleaning on this same day
overloaded the plate and the results were not quantifiable, but qualitatively were much
higher. The employee noted that he did not completely finish cleaning the aviary the
previous week, which may have accounted for the increased level of bioaerosols. The
lowest concentration of the source areas in Location 1 was 358 cfu/m3 found in cage
cleaning room before cleaning. The highest quantitative concentration of bacteria, at
Location 2, peaked at 2,758 cfu/m3 in rat room 87 before the weekly bedding change. The
lowest concentration of the source areas in Location 2 was 135 cfu/m3, found in the cage
cleaning room before cleaning. It was noted during sampling that the employee had
already dumped some bedding material about 10 min prior to sampling in the cage
cleaning room, possibly affecting the results at Location 2. Table I summarizes the
descriptive statistics for the mean concentrations of total bacteria levels in each area by
location.
35
Table I. Total Bacteria Levels at Specific Locations and Areas
12
12
3
2
2
Mean Conc.
(cfu/m3)
87
163
21342
3830
2341
Standard
Deviation
46
134
5653
182
1431
8
3400
4
4
2
2
4
Location
Area
N
1
Outdoors
Office
Aviary
Rat Room
Mouse Room
Cage Cleaning
Room
Outdoors
Office
Rat Room 69
Rat Room 87
Cage Cleaning
Room
2
Minimum
Maximum
14
44
15217
3701
1329
170
521
26358
3958
3353
3344
358
8759
215
163
446
1597
110
63
369
1643
138
74
185
435
377
222
707
2758
435
257
135
744
Field blanks did not contain any fungal growth for all samples collected except for one
sample. The fungal field blank on the second day of sampling at Location 2 had a single
colony of Cladosporium spp. The highest mean concentrations of fungi, by location, in
the source rooms were found in the aviary at Location 1 and in cage cleaning room at
Location 2 while the lowest concentrations of fungi were found in the rat rooms at both
locations. The highest quantitative concentration of fungi, at Location 1, peaked at 14,342
cfu/m3 in the aviary before cleaning operations. However, the sample collected during
cleaning on this same day overloaded the plate and the results were not quantifiable but
qualitatively were much higher. The employee noted that he did not completely finish
cleaning the aviary the previous week, which may have accounted for the increased level
of bioaerosols. The lowest concentration of the source areas in Location 1 was 70 cfu/m3,
found in rat room before the weekly bedding change. In Location 2, the highest
quantitative concentration of fungi in the source areas peaked at 353 cfu/m3 in the cage
cleaning room before cleaning. It was noted during sampling that the employee had
36
already dumped some bedding material about 10 min prior to sampling in the cage
cleaning room, possibly affecting the results. The lowest concentration of the source
areas in Location 2 was 0 cfu/m3 found in rat room 69 before the weekly bedding change.
Table II summarizes the descriptive statistics for the mean concentrations of total bacteria
levels in each area by location.
Table II. Total Fungal Levels at Specific Locations and Areas
12
12
3
2
2
Mean Conc.
(cfu/m3)
734
185
13828
94
106
Standard
Deviation
319
116
665
34
47
8
286
4
4
2
2
4
Location
Area
N
1
Outdoors
Office
Aviary
Rat Room
Mouse Room
Cage Cleaning
Room
Outdoors
Office
Rat Room 69
Rat Room 87
Cage Cleaning
Room
2
Minimum
Maximum
289
29
13077
70
73
1459
402
14342
118
139
167
128
551
1328
129
44
107
328
81
62
74
935
21
0
54
1691
212
88
159
190
143
18
353
Meteorological Data
Data including relative humidity, temperature, and weather conditions at the time of
sampling also were collected using a direct reading indoor air quality instrument called a
Q-Trak. Comprehensive data sets of meteorological data are presented in Appendix D.
Rain was only noted during one occasion, slightly before sampling was completed. Most
samples were collected during sunny conditions with little to no precipitation. Descriptive
statistical analysis was performed using the data collected. The outdoor samples collected
at Location 1 had temperature ranges from 62.5 oF to 79.3 oF and ranges in relative
humidity from 49.5% to 84.8%. The indoor samples ranged in temperatures from 63.6 oF
37
to 76.6 oF with a relative humidity ranging from 50.1% to 87.7%. The outdoor samples
collected at Location 2 had temperature ranges from 57.2 oF to 88.2 oF and ranges in
relative humidity from 43.7% to 84.0%. Indoor samples ranged in temperatures from 70.0
o
F to 79.7 oF with relative humidity ranging from 50.9% to 76.6%. Tables III and IV
summarize the descriptive statistics for the mean of data collected in each area by
location.
Table III. Meteorological Data by Area, Location 1
Area
Outdoors
Office
Aviary
Rat Room
Mice Room
Cage Cleaning
Room
Mean
Temp. (°F)
69.1
72.2
73.7
69.8
72.2
Minimum
Temp. (°F)
62.5
70.3
70.9
68.4
71.4
Maximum
Temp. (°F)
79.3
76.3
76.6
71.2
73.0
Mean RH
(%)
74.8
56.6
75.5
69.0
61.3
Minimum
RH (%)
49.5
50.1
62.6
63.8
61.0
Maximum
RH (%)
84.8
70.3
87.7
74.1
61.6
70.11
63.6
73.6
70.3
60.8
85.7
Table IV. Meteorological Data by Area, Location 2
Area
Outdoors
Office
Rat Room 69
Rat Room 87
Cage Cleaning
Room
Mean
Temp. (°F)
76.6
72.8
74.3
70.2
Minimum
Temp. (°F)
57.2
70.3
73.8
70.0
Maximum
Temp. (°F)
88.2
76.5
74.8
70.4
Mean RH
(%)
60.93
56.8
58.4
60.35
Minimum
RH (%)
43.7
50.9
57.7
60.20
Maximum
RH (%)
84.0
62.8
59.0
60.50
75.4
71.1
79.7
69.1
60.7
76.6
Animal Data
Descriptive statistical analysis was performed on the number of animals counted. Table V
summarizes the descriptive statistics for the mean of data collected in each area by
location. Room 87 containing rats had a large difference in numbers because some rats
were removed between sampling times.
38
Table V. Number of Animals by Area, Both Locations
Location
1
2
Area
Aviary
Rat Room
Mouse Room
Rat Room 69
Rat Room 87
Mean
60
94
215
110
144
Minimum
60
88
210
109
70
Maximum
60
100
220
110
217
Other Parameters
Other parameters recorded were temperature, relative humidity and carbon dioxide
concentrations. Carbon dioxide was collected with a Q-Trak, a direct reading indoor air
quality instrument that measures temperature, relative humidity, carbon dioxide and
carbon monoxide. Carbon dioxide can be an indicator of adequate ventilation. If carbon
dioxide levels indoors are no greater than about 700 ppm above outdoor air levels, a
majority of people entering the space will be satisfied with respect to human bioeffluents
(American Society of Heating, Refrigerating and Air-Conditioning Engineers, Inc., 2001)
Comprehensive data sets of carbon dioxide are presented in Appendix D. Descriptive
statistical analysis was performed using the data collected. Carbon dioxide concentrations
ranged from 349 ppm to 621 ppm outdoors and from 332 ppm to 842 ppm inside at
Location 1. Carbon dioxide concentrations ranged from 303 ppm to 438 ppm outdoors
and from 137 ppm to 963 ppm inside at Location 2. Tables VI and VII summarize the
descriptive statistics for the mean of data collected in each area by location.
39
Table VI. Carbon Dioxide (CO2) by Area, Location 1
Area
Outdoors
Office
Aviary
Rat Room
Mice Room
Cage Cleaning
Room
429
521
632
*657
*465
Minimum
CO2
(ppm)
349
332
455
NA
NA
509
399
Mean CO2
(ppm)
Maximum
CO2 (ppm)
621
686
842
NA
NA
597
* Only one measurement was collected.
Table VII. Carbon Dioxide (CO2) by Area, Location 2
Area
Outdoors
Office
Rat Room 69
Rat Room 87
Cage Cleaning
Room
370.1
362
825
488
Minimum
CO2
(ppm)
303
238
687
465
360.8
137
Mean CO2
(ppm)
Maximum
CO2 (ppm)
438
462
963
510
613
* Only one measurement was collected.
Qualitative Identification of Bioaerosols
Bacteria were classified as either gram-positive or negative and morphology was
determined to be rod, cocci, or diphtheroid. Comprehensive data sets of bacteria Gram
stain results are presented in Appendix E. Descriptive statistical analysis was performed
using the data collected. The majority of bacteria found were gram-positive cocci in all
areas at both locations. However, it was noted that the Aviary at Location 1 did have a
mean of 39gram-negative rods while outdoors had a mean of 1 gram-negative rod.
Although growth on the McConkey agar was minimal and statistical analysis was not
performed, it was noted that at Location 1 550 cfu/m3 of gram-negative bacteria was
found in the cage cleaning room while no growth on the McConkey agar was found
outdoors or in the office area. Growth on the McConkey agar for samples collected in the
40
aviary also resulted in a concentration of 105 cfu/m3 compared to that of 7 cfu/m3 found
outdoors on the first day of sampling and concentrations of 34 cfu/m3 before cleaning
activities and 793 cfu/m3 during cleaning activities compared to that of 7 cfu/m3 found
outdoors on the second day of sampling. As mentioned in the literature review, gramnegative bacteria produce toxic responses including respiratory inflammation and airway
restriction while creating conditions for allergic and infectious disease (Institute of
Inspection, Cleaning and Restoration Certification, 1999). Tables VIII and IX summarize
the descriptive statistics for bacteria Gram stain results of the mean of data collected in
each area by location.
Table VIII. Bacteria Gram Stain Results by Area, Location 1
Area
Outside
Office
Aviary
Rat Room
Mouse Room
Cage Cleaning
Room
+ Cocci
5
14
568
279
121
219
Mean Colony Forming Units
- Cocci
+ Rod
- Rod
0
4
1
0
6
1
0
0
39
0
0
0
8
24
0
5
11
Diphtheroid
3
3
0
0
2
1
5
Table IX. Bacteria Gram Stain Results by Area, Location 2
Area
Outside
Office
Rat Room 69
Rat Room 87
Cage Cleaning
Room
+ Cocci
18
13
14
89
11
Mean Colony Forming Units
- Cocci
+ Rod
- Rod
3
7
4
4
6
0
0
11
0
0
5
1
0
10
2
Diphtheroid
0
1
0
0
0
Comprehensive data sets of fungi identified to the genus level are presented in Appendix
E. Genera found included Aspergillu spp., Alternaria spp., Cladosporium spp.,
Eppicoccum spp., Fusarium spp., Rhizopus spp., Trichoderma spp., Rhodotorula spp.,
Scopulariopsis spp., and unidentifiable fungi. The latter group was unidentifiable as no
41
spores or sporing structures were seen under the culture conditions of this study. This is
not an unusual occurrence in any study of bioaerosols (Shelton et al., 2002).
Nonparametric Analysis
Nonparametric analysis of the data was completed using the one-way ANOVA KruskalWallis test to determine statistical differences between areas. The Kruskal-Wallis test was
used with a significance value of 0.05 resulting in the following:
•
A statistically significant difference between the concentrations of total viable
bacteria between sampling areas, excluding the outdoor sample, within Location
1.
•
A statistically significant difference between the concentrations of total viable
fungi between sampling areas, excluding the outdoor sample, within Location 1.
•
No statistical significant difference between the concentrations of total viable
bacteria between sampling areas, excluding the outdoor sample, within Location
2.
•
No statistical significant difference between the concentrations of total viable
fungi between sampling areas, excluding the outdoor sample, within Location 2.
Bacteria
The Kruskal-Wallis test indicated a statistically significant difference between the
concentrations of total viable bacteria at Location 1 with a p value of <0.001. Therefore,
the Mann-Whitney U test was used to compare two independent variables, by area, for
six areas in Location 1 while adjusting for multiple comparisons using the Bonferroni
Adjustment. Comprehensive data sets of comparisons are presented in Appendix G. The
42
results for Location 1 conclude that a statistically significant difference in total bacteria
levels exists between outdoor levels and the aviary, between the office and the aviary,
between outdoor levels and the cage cleaning room, and between the office and the cage
cleaning room. Table X summarizes the total bacteria results of the comparisons between
areas in Location 1.
Table X. Comparison of Mean Total Bacteria Levels by Area, Location 1
Comparison by Area
Calculated p
(Area 1 v. Area 2)
value1
Outdoors v. Office
0.114
Outdoors v. Aviary
0.004
Outdoors v. Rat Room
0.022
Outdoors v. Mouse Room
0.022
Outdoors v. Cage Cleaning Room
<0.001
Office v. Aviary
0.004
Office v. Rat Room
0.022
Office v. Mouse Room
0.022
Office v. Cage Cleaning Room
<0.001
Aviary v. Rat Room
0.200
Aviary v. Mouse Room
0.200
Aviary v. Cage Cleaning Room
0.012
Rat Room v. Mouse Room
0.333
Rat Room v. Cage Cleaning Room
0.711
Mouse Room v. Cage Cleaning Room
1.000
Mann-Whitney U Test comparison of two independent
samples, tested at a significance level of 0.007.
1
Shaded values are statistically significant
Because the Kruskal-Wallis test indicated, with a p value of 0.219 for total viable
bacteria, that there was no statistically significant difference at Location 2 and no other
analysis was needed. Two rat rooms were chosen; a HEPA filtered room and a nonHEPA filtered room. As stated above, there was no significant difference between the
two.
43
The Mann-Whitney U test was used to compare two independent variables by area and
the time period before aggressive cleaning and during aggressive cleaning, by location to
determine if the disturbance of bedding material increased or decreased bioaerosol
concentrations. No significant differences occurred between time periods. Table XI
summarizes the total bacteria results of the comparisons between the time period before
aggressive cleaning and during aggressive cleaning by location.
Table XI. Comparison of Total Bacteria Levels Before Cleaning and During
Cleaning, Both Locations
Location
Comparison Before Cleaning and
Calculated p
During Cleaning
value1
(Time Period 1 v. Time Period 2)
Aviary Before v. Aviary After
1.000
Rats Cage Cleaning Room Before v. Rats
0.333
1
Cage Cleaning Room After
Mouse Cage Cleaning Room Before v.
0.333
Mouse Cage Cleaning Room After
Rats Cage Cleaning Room Before v. Rats
1.000
2
Cage Cleaning Room After
Mann-Whitney U Test comparison of two independent samples, tested at a significance level of 0.05.
location. Areas compared were non-HEPA filtered rat rooms similar in nature. The total
bacteria results of the comparisons between a non-HEPA filtered rat room at Location 1
and a non-HEPA filtered rat room at Location 2 were not statistically significantly
different with a calculated p value of 1.000.
Fungi
The Kruskal-Wallis test indicated a significant difference between the concentrations of
total viable fungi at Location 1 with a p value of 0.015. Therefore, the Mann-Whitney U
44
test was used to compare two independent variables, by area, for six areas in Location 1
while adjusting for multiple comparisons using the Bonferroni Adjustment.
Comprehensive data sets of comparisons are located in Appendixes G. The results for
Location 1 conclude that a significant difference in total fungal levels exists between
outdoor levels and the office levels, between outdoor levels and the aviary, between the
office and the aviary, and between outdoor levels and the cage cleaning room. Table XII
summarizes the total fungi results of the comparisons between areas in Location 1.
Table XII. Comparison of Mean Total Fungus Levels by Area, Location 1
Comparison by Area
Calculated p
(Area 1 v. Area 2)
value1
Outdoors v. Office
<0.001
Outdoors v. Aviary
0.004
0.022
0.022
<0.001
Office v. Aviary
0.004
Office v. Rat Room
0.352
0.440
0.238
Aviary v. Rat Room
0.200
0.200
0.012
0.667
0.044
0.089
Mann-Whitney U Test comparison of two independent
samples, tested at a significance level of 0.007.
1
The Kruskal-Wallis test for Location 2 indicated, with a p value of 0.442 for total viable
fungi, there was no significant difference, therefore no other analysis was needed.
the time period before aggressive cleaning and during aggressive cleaning, by location.
No significant differences occurred between areas. Table XIII summarizes the total fungi
45
results of the comparisons between the time period before aggressive cleaning and during
aggressive cleaning, by location.
Table XIII. Comparison of Total Fungal Levels Before Cleaning and During
Cleaning, Both Locations
Location
Comparison Before Cleaning and
Calculated p
During Cleaning
value1
Aviary A.M. v. Aviary P.M.
1.000
Rats Cage Cleaning Room A.M. v. Rats
1.000
1
Cage Cleaning Room
Mice Cage Cleaning Room A.M. v. Mice
1.000
Cage Cleaning Room P.M.
Rats Cage Cleaning Room A. M. v. Rats
1.000
2
location. Areas compared were non-HEPA filtered rat rooms similar in nature. The total
fungi results of the comparisons between a non-HEPA filtered rat room at Location 1 and
a non-HEPA filtered rat room at Location 2 were not statistically significantly different
with a calculated p value of 1.000.
46
DISCUSSION
Overview
Total bacteria results at Location 1 resulted in statistically significantly higher levels in
the aviary and the cage cleaning room than outdoors and non-source room levels such as
the office. Gram- negative bacteria were also found in both the aviary and cage cleaning
room at Location 1. Total fungi results at Location 1 resulted in statistically significantly
higher levels in the aviary than outdoors and non-source room levels. The resultant higher
concentrations at Location 1 of both bacteria and fungi found in the aviary occur because
the aviary is only cleaned once a week allowing bird feces and dander to accumulate on
the floor. The cage cleaning rooms high bacteria levels are also attributed to the method
of cleaning. The used bedding is dumped manually without the use of local exhaust into
a garbage can.
No significant difference was found between areas at Location 2. Location 2 did not have
an area comparable to the aviary, however, did have rooms housing rodents similar in
nature to Location 1. Lower concentrations of both bacteria and fungal were likely
attributable to the method of cleaning. The cage cleaning room is equipped with a
portable local exhaust ventilation system where the used bedding is dumped. A
disinfectant also was used on the shoes to reduce the spread of bacteria before leaving the
cage cleaning area. The cage cleaning room at Location 2 was also visibly much cleaner.
47
Though some limitations exist, the significance of the study was exploratory in nature,
finding a statistically significant elevation of bioaerosols in the aviary and in the cage
cleaning room along with the presence of gram-negative bacteria at one of the two
university animal care facilities studied when compared to outdoor and non–source area
concentrations. The results of this study, including the recommendations for future study,
may be used by occupational health professionals in the future when anticipating,
identifying, evaluating, and controlling occupational hazards.
Concentrations of viable bacteria and fungi were collected, quantified, and analyzed, in
various source areas at two different university animal care facilities. Nonparametric
statistical analysis indicated a significant difference, with a significance value of 0.05,
between areas within Location 1 and indicated no significant difference between areas
within Location 2. Therefore, the Mann-Whitney U test was used to further compare
bioaerosol results between areas independently at Location 1.
The resulting differences in bacteria levels, with associated p values, indicating a
significant difference (tested to an adjusted significance level of 0.007) in the mean
concentration of total bacteria between outdoors and the aviary and cage cleaning room
with the source rooms being higher than outdoor levels are shown in Table X. Significant
differences also were found between the office and the aviary and cage cleaning room
with the source rooms being higher than the office area. The presence of gram-negative
bacteria also was found in both the aviary and cage cleaning room at Location 1.
However, significant differences were not found between either the rat room or the
48
mouse room and outdoor and office levels (background and non-suspect source area,
respectively).
Table XI shows the resulting differences in bacteria levels, with associated p values,
indicating no significant difference (tested to significance value of 0.05) in the mean
concentrations of total bacteria before cleaning and during cleaning operations in any of
the source areas. One comparison was also made between locations using non-HEPA
filtered rat rooms resulting in no significant differences (tested to a significance value of
0.05).
Table XII shows the resulting differences in fungi levels, with associated p values,
indicating a significant difference (tested to an adjusted significance value of 0.007) in
the mean concentration of total fungi between outdoors and the aviary and cage cleaning
room. Significant differences also were found between the office and the aviary and
outdoors. The outdoor levels were found to be statistically significantly higher than office
levels and the aviary was statistically significantly higher than the office. However,
significant differences were not found between either the rat room or the mouse room and
outdoors and office levels (background and non-suspect source area, respectively).
Table XIII shows the resulting differences in fungi levels, with associated p values,
indicating no significant difference (tested to a significance value of 0.05) in the mean
concentrations of total fungi before cleaning and during cleaning operations in any of the
49
source areas. One comparison also was made between locations using non-HEPA filtered
rat rooms resulting in no significant differences (tested to a significance value of 0.05).
Interpretation
The emphasis of the study was exploratory in nature finding a statistically significant
elevation of bacteria in the aviary and in the cage cleaning room at one of the two
university animal care facilities studied when compared to outdoor and non–source area
concentrations. Also, finding statistically higher concentrations of fungi in the aviary at
one of the two university animal care facilities studied when compared to outdoor and
non–source area concentrations. Mean concentrations found in the aviary represented the
greatest exposure. No significant differences were found between areas in the second
location studied. Therefore, the greatest risks of exposure and need for control are in the
aviary and cage cleaning room at Location 1.
The Mann-Whitney U test used to test independent comparisons also indicated that there
were no significant differences between sampling periods before cleaning operations and
during cleaning operations at either location. Because the two locations studied each had
a similar non-HEPA filtered room housing rats, the Mann-Whitney U test was used to
compare the difference. No statistical difference was found between the two locations.
A majority of bacteria identified were gram-positive cocci, in all areas, at both locations.
However, gram-negative bacteria found in both the aviary and cage cleaning room in
Location 1 indicated the potential for adverse health effects. As discussed in the
Literature Review, the indoor environment usually contains higher concentrations and
50
more types of bacteria than outdoors. Gram-positive cocci and gram- positive rods are
commonly found indoors and outdoors; specifically Staphylococcus spp., Micrococcus
spp. and Bacillus spp. Most bacteria that occur naturally do not present a health risk
under normal circumstances; however, the risk of infection does increase when bacteria
are found in high concentration indoors. Bacillus spp. is often associated with HP (Burge
and Otten, 1999a).
The predominant fungus found was Cladosporium spp., in all areas, at both locations. As
discussed in the Literature Review, generally outdoor concentrations of fungi are higher
or equal to indoor concentrations unless an indoor source exists. It is also generally
accepted that indoor species should be similar to outdoor species unless an indoor source
exists. Overall, most species found indoors were also found outdoors. Ten of the 11
fungal genera colonies found were present outdoors at Location 1. The one exception was
Scopulaiopsis spp., present in the office and mouse room, commonly found in the
environment. Results suggest that animal care employees may have a higher risk of
exposure to bioaerosols, which may vary from location to location.
Limitations
Many limitations exist when sampling for bioaerosols. As discussed in the Literature
Review, weather conditions such as rain and snow can decrease bioaerosol concentrations
outdoors and can artificially indicate elevated indoor bioaerosol levels. Weather
conditions were similar with little to no precipitation during all sampling days thereby
reducing variability due to weather conditions. The change in seasons can also affect
51
sampling results. Warmer conditions, including the summer months in Ohio, are often
accompanied by higher levels of outdoor bioaerosol concentrations, which can artificially
indicate low source levels indoors when compared to outdoors. This study was conducted
over a 2 mo span and only represents conditions during July and August in an agricultural
area in Ohio.
As discussed in the Literature Review, sampling time also can be a limiting factor.
Sampling times for viable bioaerosol field collection range from 1 min to 10 min, only
accounting for a snap shot of the entire day. Short sampling times can often under
represent samples and long sampling times can overload the sampling media making it
difficult to quantify. A sample time of 5 min outdoors and indoors was used in the pilot
study in anticipation of high bioaerosol levels. The aviary in Location 1 was chosen as
the likely worse case scenario. Both of the air samples collected in the office area and
outdoors were quantifiable during analysis. However, both the PFA medium chosen for
fungi and the TSA with 5% sheep blood chosen for bacteria were overloaded and
unquantifiable in the aviary. Therefore, a sampling time of 1 min was chosen for the
aviary. Subsequent samples collected in Location 1 in the rooms housing mice and rats,
were collected for a duration of 3 min. However, the sampling time was adjusted to 2 min
for the remaining mouse and rat rooms at both locations due to high counts and possible
overloading. Although these sampling times were adequate for Location 1, they proved to
be too short for Location 2, with little to no growth in some areas, possibly under
representing concentrations.
52
Other limitations include the number of samples collected, total viable bacteria and fungi,
type of media used, and identification to the genus level. Sampling results were only
replicated on two separate days reducing the statistical significance. Total viable samples
do not include dead biological material that can also contribute to adverse health effects,
and the results were limited to those type of bacteria and fungi that grow well on TSA
with 5% sheep blood and PFA, respectively. The fungal samples were only identified to
the genus level and bacteria were only identified by Gram stain results and morphology.
While this information is useful, it does not provide information on specific species found
and their associated health effects.
Recommendations for Future Studies
Recommendations for future study include:
•
Repeat this study over an extended period of time during different times of the
year.
•
Explore the use of different types of sampling media that could capture non viable
fungi.
•
Repeat this study in other university animal care facilities.
•
Use varying sample times dependent on initial results for each area.
53
CONCLUSIONS
The following hypotheses were tested and accepted:
1a. There was no statistically significant difference in measured mean airborne
bacteria levels before and during cleaning activities throughout the work shift at
both Location 1 and Location 2 when tested to a significance level of 0.05.
1b. There was no statistically significant difference in measured mean airborne fungi
levels before and during cleaning activities throughout the work shift at both
Location 1 and Location 2 when tested to a significance level of 0.05.
bacteria levels between rooms housing animals, cage cleaning rooms, outdoors,
and rooms not housing animals at Location 2 when tested to a significance level
of 0.007.
levels between rooms housing animals, cage cleaning rooms, outdoors, and rooms
not housing animals at Location 2 when tested to a significance level of 0.007.
The following hypotheses were tested and rejected:
bacteria levels between rooms housing animals, cage cleaning rooms, outdoors,
and rooms not housing animals at Location 1 when tested to a significance level
of 0.007.
54
levels between rooms housing animals, cage cleaning rooms, outdoors, and rooms
not housing animals at Location 1 when tested to a significance level of 0.007.
55
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65
APPENDICES
66
67
APPENDIX A. FORMS
68
Calibration Data Form
High Flow Air Sampling Pump
Calibration Location: _______________________________________________________________________________________________________________________________
Calibration Conducted By: _______________________________________________________________________________ Calibration Date: _____________________________
Calibration Instruments
(Type/Manufacturer/Model): _________________________________________________________________________________________________________________________
Air Sampling Pump
Air Sampler
Sampling Medium In Line
(Type/Manufacturer): ______________________________________________________________________________________________________________________________
Calibrator Volume: ________________cc Air Temperature: _______________EC Air Pressure: _______________mm Hg Relative Humidity: _______________%
Time of
calibration
Pre-Sampling Calibration Flow Rate (Qpre,L/min)
Q1
Q2
Q3
Qpre-avg
Time of
calibration
Post-Sampling Calibration Flow Rate (Qpost,L/min)
Q1
Q2
Q3
Qpost-avg
Average
Flow Rate
Qavg
% Difference
Pre-Post
Calibration Notes:
69
Field Monitoring Data Form:
Integrated Monitoring for Bacteria and Fungi
Location: ________________________________________________________________________ Contaminant Sampled: _____________________________________________
Monitoring Conducted By: ___________________________________________________________ Date Monitoring Conducted: ________________________________________
Monitoring Instruments (Type/Manufacturer/Model): ______________________________________________________________________________________________________
Collection Medium: ________________________
Area
Field Sample
Identification
No.
Sample
Start-Time
Sample
Stop-Time
Total
Sample Time
(T)
Air Temp,
ºF
RH, %
Number of
Occupants
Comments
(Open windows, etc.)
Field Notes:
70
Laboratory Analysis Data Form:
Analysis of Bioaerosol Samples for Microbial Colonies
Bacterial Analysis Form
Sampling Location: ________________________________________________________ Sampling Area: ___________________________________________________________
Monitoring Conducted By: ___________________________________________________ Date Monitoring Conducted: ________________________________________________
Analysis Conducted By: _____________________________________________________ Date Analysis Conducted: _________________________________________________
Laboratory Instruments (Type/Manufacturer/Model): ______________________________________________________________________________________________________
Collection Medium: ______________________________ Incubation Temperature: _______________________________ Incubation Time: _______________________________
Temperature: _____ºC Relative Humidity: _____% Air Pressure: _____ mm Hg
Field Sample ID
No.
# Colony Forming
Units
(cfu)
Average Flow
Rate
(Q, L/min)
Total Sample
Time
(T, min)
Volume Air
Sampled
3
(Vol, m )
Concentration
3
(cfu/m )
Counts
+ Cocci
- Cocci
+ Rod
- Rod
Yeast
Diptheroid
71
Analysis of Bioaerosol Samples for Microbial Colonies
Fungal Analysis Form
Sampling Location: ________________________________________________________ Sampling Area: ___________________________________________________________
Monitoring Conducted By: ___________________________________________________ Date Monitoring Conducted: ________________________________________________
Analysis Conducted By: _____________________________________________________ Date Analysis Conducted: _________________________________________________
Laboratory Instruments (Type/Manufacturer/Model): ______________________________________________________________________________________________________
Collection Medium: ______________________________ Incubation Temperature: _______________________________ Incubation Time: _______________________________
Field Sample ID
No.
# Colony Forming
Units
(cfu)
Average Flow
Rate
(Q, L/min)
Total Sample
Time
(T, min)
Volume Air
Sampled
3
(Vol, m )
Concentration
3
(cfu/m )
Genus Counts
72
Identification of Bacterial Colonies
Bacterial Analysis Form
Sampling Location: __________________________________________________ Sampling Area: ___________________________________________________________
Monitoring Conducted By: ____________________________________________ Date Monitoring Conducted: ________________________________________________
Analysis Conducted By: ______________________________________________ Date Analysis Conducted: _________________________________________________
Diptheroid
Yeast
- Rod
+ Rod
Color
- Cocci
Identification
+ Cocci
No
Shiny
Hemolytic
Yes
Appear
Dull
Texture
Rough
Crateriform
Umbonate
Convex
Raised
Flat
Lobate
Elevation
Curled
Filiform
Undulate
Entire
Rhizoid
Margin
Filamentous
Irregular
Field
Sample ID
No.
Circular
Form
Smooth
73
APPENDIX B. LOCATION LAYOUTS
74
Location 1 Layout
75
Location 2 Layout
76
APEENDIC C. CONCENTRATION OF BIOAEROSOLS
77
Table C-1. Concentration of Bacteria by Area and Date, Location 1
Concentration (cfu/m3)
Area
Time
Outdoors
Before Cleaning
During Cleaning
7/22/04
14
71
7/29/04
48
90
Office
Before Cleaning
During Cleaning
249
249
158
158
Aviary
Before Cleaning
During Cleaning
15217
22452
26358
>13782
Area
Time
Outdoors
Before Cleaning
During Cleaning
7/19/04
44
95
7/26/04
127
133
Office
Before Cleaning
During Cleaning
44
102
521
133
Mouse Room
Before Cleaning
1329
3353
Cage Cleaning
Room
Before Cleaning
During Cleaning
476
1732
1285
6759
Area
Time
Outdoors
Before Cleaning
During Cleaning
7/20/04
170
116
7/27/04
102
34
Office
Before Cleaning
During Cleaning
109
191
61
55
Rat Room
Before Cleaning
3701
3958
Cage Cleaning
Room
Before Cleaning
During Cleaning
1555
7425
358
5612
78
Table C-2. Concentration of Fungi by Area and Date, Location 1
Area
Time
Outdoors
Before Cleaning
During Cleaning
7/22/04
931
1459
7/29/04
674
896
Office
Before Cleaning
During Cleaning
402
155
215
215
Aviary
Before Cleaning
During Cleaning
13077
14064
14342
>14342
Area
Time
Outdoors
Before Cleaning
During Cleaning
7/19/04
907
941
7/26/04
580
646
Office
Before Cleaning
During Cleaning
335
98
66
29
Mouse Room
Before Changing
139
73
Cage Cleaning
Room
Before Cleaning
During Cleaning
209
128
184
551
Area
Time
Outdoors
Before Cleaning
During Cleaning
7/20/04
963
1091
7/27/04
521
500
Office
Before Cleaning
During Cleaning
191
305
134
77
Rat Room
Before Changing
118
70
Cage Cleaning
Room
Before Cleaning
During Cleaning
248
531
299
141
79
Table C-3. Concentration of Bacteria by Area and Date, Location 2
Area
Time
Outdoors
Before Cleaning
During Cleaning
8/2/04
377
138
8/16/04
182
161
Office
Before Cleaning
During Cleaning
189
167
222
74
Rat Room 69
Before Changing
707
185
Rat Room 87
Before Changing
435
2758
Cage Cleaning
Room
Before Cleaning
During Cleaning
744
508
135
353
Table C-4. Concentration of Fungi by Area and Date, Location 2
Area
Time
Outdoors
Before Cleaning
During Cleaning
8/2/04
1208
1477
8/16/04
1691
935
Office
Before Cleaning
During Cleaning
212
162
21
121
Rat Room 69
Before Changing
88
0
Rat Room 87
Before Changing
159
54
Cage Cleaning
Room
Before Cleaning
During Cleaning
353
247
18
143
80
APPENDIX D. METEROLOGICAL CONDITIONS AND
OTHER PARAMETERS
81
Table D-1. Meteorological Conditions, Location 1
7/22/04
Area
Time
Air Temp ºF
Weather
Conditions
Air Temp ºF
RH
%
64.8
83.8
79.3
49.5
Sunny/Windy
80.0
Before Cleaning
During Cleaning
72.7
74.8
65.1
54.0
Indoors
70.3
76.3
55.3
70.3
Indoors
Before Cleaning
During Cleaning
73.8
76.6
82.5
87.7
Indoors
70.9
73.4
69.0
62.6
Indoors
71.2
During Cleaning
Office
Aviary
7/19/04
Area
Time
Outdoors
7/26/04
RH
%
Sunny
64.6
69.0
77.2
70.8
Cloudy
56.8
53.8
Indoors
70.6
72.1
53.8
51.3
Indoors
73.0
61.0
Indoors
71.4
61.6
Indoors
70.7
71.1
69.8
68.0
71.6
60.8
RH%
Before Cleaning
During Cleaning
66.6
70.3
84.4
75.4
Office
Before Cleaning
During Cleaning
71.2
72.3
Mouse
Room
Before
Changing
Cage
Cleaning
Room
Before Cleaning
Weather
Conditions
Indoors
During Cleaning
73.6
85.7
Time
Outdoors
7/27/04
Air Temp ºF
RH
%
Sunny
66.3
62.5
66.6
83.4
Rain/Overcast
56.6
52.2
Indoors
71.1
71.1
60.2
50.1
Indoors
71.2
63.8
Indoors
68.4
74.1
Indoors
71.4
62.8
69.8
70.9
70.4
70.9
Air Temp ºF
RH%
Before Cleaning
During Cleaning
67.1
72.5
78.9
70.6
Office
Before Cleaning
During Cleaning
71.2
72.7
Rat Room
Before
Changing
Before Cleaning
Weather
Conditions
Indoors
During Cleaning
Weather
Conditions
Indoors
7/20/04
Area
Sunny
Air Temp ºF
Air Temp ºF
Cage
Cleaning
Room
Weather
Conditions
No
Data
81.9
Before Cleaning
Outdoors
RH%
7/29/04
63.6
71.8
Weather
Conditions
Indoors
82
Table D-2. Meteorological Conditions, Location 2
8/2/04
Area
Time
Outdoors
Before
Cleaning
During
Cleaning
Office
Before
Cleaning
During
Cleaning
Air Temp ºF
RH%
77.6
62.9
8/16/04
Weather
Conditions
Air Temp ºF
RH
%
57.2
84.0
83.5
43.7
71.1
57.6
70.3
62.8
Sunny
88.2
53.1
73.2
55.7
Sunny
Indoors
76.5
50.9
Weather
Conditions
Indoors
Rat Room 69
Before
Changing
74.8
57.7
Indoors
73.8
59
Indoors
Rat Room 87
Before
Changing
70.0
60.2
Indoors
70.4
60.5
Indoors
Cage
Cleaning
Room
Before
Cleaning
During
Cleaning
73.6
71.7
71.1
60.7
79.7
67.2
Indoors
77.0
76.6
Indoors
83
Table D-3. Other Measured Parameters, Location 1
Area
Time
Before Cleaning
During Cleaning
7/22/04
Carbon Dioxide
397
621
7/29/04
Carbon Dioxide
489
456
Outdoors
Office
Before Cleaning
During Cleaning
686
496
554
332
Aviary
Before Cleaning
During Cleaning
773
455
842
457
Area
Time
Outdoors
Before Cleaning
During Cleaning
7/19/04
Carbon Dioxide
Error
Error
7/26/04
Carbon Dioxide
349
354
Office
Before Cleaning
During Cleaning
Error
Error
475
480
Mouse Room
Before Changing
Error
465
Cage Cleaning
Room
Before Cleaning
During Cleaning
Error
Error
597
399
Area
Time
Outdoors
Before Cleaning
During Cleaning
7/20/04
Carbon Dioxide
Error
Error
7/27/04
Carbon Dioxide
413
355
Office
Before Cleaning
During Cleaning
Error
Error
655
493
Rat Room
Before Changing
Error
657
Cage Cleaning
Room
Before Cleaning
During Cleaning
Error
Error
481
557
84
Table D-4. Other Measured Parameters, Location 2
Area
Time
Before Cleaning
During Cleaning
8/2/04
Carbon Dioxide
438
303
8/16/04
Carbon Dioxide
Error
Error
Outdoors
Office
Before Cleaning
During Cleaning
462
327
419
238
Rat Room 69
Before Changing
687
963
Rat Room 87
Before Changing
465
510
Cage Cleaning
Room
Before Cleaning
During Cleaning
410
613
283
137
85
APPENDIX E. IDENTIFICATION OF BACTERIA
86
Table E-1. Classification of Bacteria, Location 1, 7/22/04
2
10
+ Cocci
1
1
- Cocci
0
0
Counts
+ Rod
1
6
- Rod
0
3
Diphtheroid
0
0
Before Cleaning
During Cleaning
35
35
16
15
0
0
16
19
3
1
0
0
Before Cleaning
During Cleaning
427
630
412
594
0
0
0
1
15
35
0
0
Area
Time
CFUs
Outdoors
Before Cleaning
During Cleaning
Office
Aviary
Area
Time
CFUs
Outdoors
Before Cleaning
During Cleaning
Office
Aviary
7
13
+ Cocci
3
6
- Cocci
0
0
Before Cleaning
During Cleaning
23
23
10
15
0
0
Before Cleaning
During Cleaning
765
>400
698
X
0
0
Counts
+ Rod
3
4
- Rod
0
0
Diphtheroid
1
3
13
4
0
0
0
4
0
0
67
X
0
0
- Rod
0
1
Diphtheroid
0
0
Area
Time
CFUs
Outdoors
Before Cleaning
During Cleaning
Office
Counts
+ Rod
3
3
6
13
+ Cocci
2
9
- Cocci
1
0
Before Cleaning
During Cleaning
6
14
4
8
0
1
0
4
1
1
1
0
Mouse Room
Before Changing
109
46
15
48
0
0
Cage Cleaning
Room
Before Cleaning
During Cleaning
39
142
25
100
3
0
11
42
0
0
0
0
87
Area
Time
CFUs
Outdoors
Before Cleaning
During Cleaning
Office
19
20
+ Cocci
12
12
- Cocci
0
0
Before Cleaning
During Cleaning
78
8
62
6
1
0
Mouse Room
Before Changing
201
196
Cage Cleaning
Room
Before Cleaning
During Cleaning
77
525
69
472
Counts
+ Rod
6
3
- Rod
1
1
Diphtheroid
0
4
9
0
0
1
6
1
1
0
0
4
0
38
4
20
1
0
3
0
- Rod
1
0
Diphtheroid
12
12
Area
Time
CFUs
Outdoors
Before Cleaning
During Cleaning
Office
Counts
+ Rod
12
3
25
17
+ Cocci
0
2
- Cocci
0
0
Before Cleaning
During Cleaning
16
17
2
2
0
0
1
3
0
0
13
12
Rat Room
Before Changing
326
326
0
0
0
0
Cage Cleaning
Room
Before Cleaning
During Cleaning
137
654
108
654
0
0
5
0
0
0
24
0
88
Area
Time
CFUs
Outdoors
Before Cleaning
During Cleaning
Office
15
5
+ Cocci
6
1
- Cocci
1
0
Before Cleaning
During Cleaning
9
8
7
4
0
0
Rat Room
Before Changing
232
232
Cage Cleaning
Room
Before Cleaning
During Cleaning
21
329
12
309
Counts
+ Rod
6
2
- Rod
2
1
Diphtheroid
0
1
1
2
1
2
0
0
0
0
0
0
0
1
3
4
6
1
0
14
- Rod
8
0
Diphtheroid
0
0
Area
Time
CFUs
Outdoors
Before Cleaning
During Cleaning
Office
Counts
+ Rod
20
8
52
19
+ Cocci
24
11
- Cocci
0
0
Before Cleaning
During Cleaning
16
19
13
11
0
0
9
8
1
0
3
0
Rat Room 69
Before Changing
39
23
0
16
0
0
Rat Room 87
Before Changing
24
17
0
6
1
0
Cage Cleaning
Room
Before Cleaning
During Cleaning
41
28
14
24
0
0
24
3
3
0
0
1
89
Area
Time
CFUs
Outdoors
Before Cleaning
During Cleaning
Office
27
24
+ Cocci
13
17
- Cocci
10
0
Before Cleaning
During Cleaning
33
11
18
6
12
2
Rat Room 69
Before Changing
11
5
Rat Room 87
Before Changing
164
Cage Cleaning
Room
Before Cleaning
During Cleaning
8
21
Counts
+ Rod
1
2
- Rod
3
5
Diphtheroid
0
0
3
3
0
0
0
0
0
6
0
0
161
0
3
0
0
6
12
0
0
2
4
0
5
0
0
90
APPENDIX F. IDENTIFICATION OF FUNGI
91
Table F-1. Identification of Fungi, Location 1, 7/22/04
Area
Time
CFU
Aspergillus spp.
Alternaria spp.
Cladosporium
spp.
Eppicoccum spp.
Fusarium spp.
Penicillium spp.
Rhizopus spp.
Trichoderma spp.
Scopulariopsis
spp.
Unknown
Yeast
(Rhodotorula)
Count
Outdoors
Before Cleaning
During Cleaning
132
207
0
0
0
0
102
203
0
0
4
3
8
1
0
0
0
0
0
0
18
0
0
0
Office
Before Cleaning
During Cleaning
57
22
0
0
0
0
18
17
2
0
1
0
4
4
0
1
0
0
0
0
0
0
32
0
Aviary
Before Cleaning
During Cleaning
371
399
0
0
0
0
338
179
1
0
3
0
26
184
0
0
0
0
0
0
3
36
0
0
Blank
NA
0
0
0
0
0
0
0
0
0
0
0
0
Area
Time
CFU
Aspergillus spp.
Alternaria spp.
Cladosporium
spp.
Eppicoccum spp.
Fusarium spp.
Penicillium spp.
Rhizopus spp.
Trichoderma spp.
Scopulariopsis
spp.
Unknown
Yeast
(Rhodotorula)
Count
Outdoors
Before Cleaning
During Cleaning
94
125
0
0
7
0
72
118
0
0
2
1
3
0
0
0
0
0
0
0
0
6
10
0
Office
Before Cleaning
During Cleaning
30
30
0
5
3
4
21
17
0
0
0
0
4
3
0
0
0
0
0
1
2
0
0
0
Aviary
Before Cleaning
During Cleaning
400
>400
0
0
0
0
252
X
0
0
1
0
90
X
0
0
0
0
0
0
57
X
0
0
Blank
NA
0
0
0
0
0
0
0
0
0
0
0
0
92
Area
Time
CFU
Aspergillus spp.
Alternaria spp.
Cladosporium
spp.
Eppicoccum spp.
Fusarium spp.
Penicillium spp.
Rhizopus spp.
Trichoderma spp.
Scopulariopsis
spp.
Unknown
Yeast
(Rhodotorula)
Count
Outdoors
Before Cleaning
During Cleaning
130
135
1
0
0
0
53
113
0
0
0
0
1
2
0
0
1
0
0
0
1
3
73
17
Office
Before Cleaning
During Cleaning
48
14
1
0
0
0
9
10
0
0
0
0
0
1
0
0
0
0
0
0
6
2
32
1
Mouse Room
Before Changing
12
0
0
6
1
0
4
0
0
1
0
0
Cage
Cleaning
Room
Before Cleaning
18
0
0
14
0
0
2
2
0
0
0
0
During Cleaning
11
0
0
7
0
0
2
1
0
0
1
0
Blank
0
0
0
0
0
0
0
0
0
0
0
0
0
Area
Time
CFU
Aspergillus spp.
Alternaria spp.
Cladosporium
spp.
Eppicoccum spp.
Fusarium spp.
Penicillium spp.
Rhizopus spp.
Trichoderma spp.
Scopulariopsis
spp.
Unknown
Yeast
(Rhodotorula)
Count
Outdoors
Before Cleaning
During Cleaning
79
88
0
0
1
1
62
81
0
0
4
1
5
2
0
0
0
0
0
0
7
3
0
0
Office
Before Cleaning
During Cleaning
9
4
0
0
1
1
7
2
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
Mouse Room
Before Changing
10
2
0
2
0
0
5
0
0
0
1
0
Cage
Cleaning
Room
Before Cleaning
4
1
0
2
0
0
1
0
0
0
0
0
During Cleaning
30
0
0
0
0
0
0
1
0
0
29
0
Blank
NA
0
0
0
0
0
0
0
0
0
0
0
0
93
Area
Time
CFU
Aspergillus spp.
Alternaria spp.
Cladosporium
spp.
Eppicoccum spp.
Fusarium spp.
Penicillium spp.
Rhizopus spp.
Trichoderma spp.
Scopulariopsis
spp.
Unknown
Yeast
(Rhodotorula)
Count
Outdoors
Before Cleaning
During Cleaning
136
154
0
0
0
0
90
137
0
0
0
3
1
3
0
1
0
0
0
0
7
1
38
9
Office
Before Cleaning
During Cleaning
27
43
2
0
0
0
0
38
0
0
0
0
2
0
0
1
0
0
0
0
14
4
9
0
Rat Room
Before Changing
10
4
0
3
0
0
3
0
0
0
0
0
Cage
Cleaning
Room
Before Cleaning
21
0
0
18
0
0
2
0
0
0
1
0
During Cleaning
45
0
0
39
0
0
4
1
0
0
0
1
Blank
NA
0
0
0
0
0
0
0
0
0
0
0
0
Area
Time
CFU
Aspergillus spp.
Alternaria spp.
Cladosporium
spp.
Eppicoccum spp.
Fusarium spp.
Penicillium spp.
Rhizopus spp.
Trichoderma spp.
Scopulariopsis
spp.
Unknown
Yeast
(Rhodotorula)
Count
Outdoors
Before Cleaning
During Cleaning
74
71
0
0
0
0
49
27
0
0
3
0
4
2
0
0
0
0
0
0
10
12
8
30
Office
Before Cleaning
During Cleaning
19
11
0
0
0
0
1
3
0
0
0
0
11
1
0
0
0
0
0
0
5
5
2
2
Rat Room
Before Changing
4
0
0
0
0
0
0
0
0
0
4
0
Cage
Cleaning
Room
Before Cleaning
17
0
0
2
0
3
1
0
0
0
10
1
During Cleaning
8
0
0
1
0
0
3
0
0
0
4
0
Blank
NA
0
0
0
0
0
0
0
0
0
0
0
0
94
Area
Time
CFU
Aspergillus spp.
Alternaria spp.
Cladosporium
spp.
Eppicoccum spp.
Fusarium spp.
Penicillium spp.
Rhizopus spp.
Trichoderma spp.
Scopulariopsis
spp.
Unknown
Yeast
(Rhodotorula)
Count
Outdoors
Before Cleaning
During Cleaning
171
209
0
0
0
0
107
148
2
5
0
1
1`
0
0
0
0
0
0
0
61
55
0
0
Office
Before Cleaning
During Cleaning
30
23
2
0
0
0
14
17
0
1
0
0
4
0
0
0
0
0
0
0
10
5
0
0
Rat Room 69
Before Changing
5
1
0
1
0
0
0
0
0
0
3
0
Rat Room 87
Before Changing
9
0
0
2
0
0
1
0
0
0
6
0
Cage Cleaning Room
Blank
Before Cleaning
20
2
0
2
0
0
15
0
0
0
1
0
During Cleaning
14
0
0
0
0
0
11
1
0
0
2
0
NA
0
0
0
0
0
0
0
0
0
0
0
0
Area
Time
CFU
Aspergillus spp.
Alternaria spp.
Cladosporium
spp.
Eppicoccum spp.
Fusarium spp.
Penicillium spp.
Rhizopus spp.
Trichoderma spp.
Scopulariopsis
spp.
Unknown
Yeast
(Rhodotorula)
Count
Outdoors
Before Cleaning
During Cleaning
237
131
0
0
0
0
120
124
0
1
1
0
6
0
0
0
0
0
0
0
0
6
110
0
Office
Before Cleaning
During Cleaning
3
17
0
0
0
0
3
9
0
0
0
0
0
0
0
0
0
0
0
0
0
8
0
0
Rat Room 69
Before Changing
0
0
0
0
0
0
0
0
0
0
0
0
Rat Room 87
Before Changing
3
0
2
1
0
0
0
0
0
0
0
0
Before Cleaning
1
0
0
0
0
0
1
0
0
0
0
0
During Cleaning
8
0
0
0
0
0
8
0
0
0
0
0
NA
1
0
0
0
0
0
0
0
0
0
0
Cage Cleaning Room
Blank
1
95
APPENDIX G. BIOAEROSOL COMPARISONS
96
Table G-1. Comparison of Mean Total Bacteria Levels by Area, Location 1
Comparison by Area
Mean
Mean
Calculated p
(Area 1 v. Area 2)
Area 1
Area 2
value1
Outdoors v. Office
87
163
0.114
Outdoors v. Aviary
87
21342
0.004
87
3830
0.022
87
2341
0.022
87
3400
<0.001
Office v. Aviary
163
21342
0.004
Office v. Rat Room
163
3830
0.022
163
2341
0.022
163
3400
<0.001
Aviary v. Rat Room
21342
3830
0.200
21342
2341
0.200
21342
3400
0.012
3830
2341
0.333
3830
3400
0.711
2341
3400
1.000
Mann-Whitney U Test comparison of two independent samples, tested at a
significance level of 0.007.
1
Table G.2 Comparison of Total Bacteria Levels Before Cleaning and During
Cleaning, Location 1
Comparison Before Cleaning and During
Mean Time
Mean Time
Calculated p
Cleaning
Period 1
Period 2
value1
20788
22452
1.000
Rats Cage Cleaning Room A.M. v. Rats Cage
881
5246
0.333
Cleaning Room P.M.
Mouse Cage Cleaning Room A.M. v. Mouse
957
6519
0.333
1
Table G-3. Comparison of Total Bacteria Levels Before Cleaning and During
Mean Time
Mean Time
Calculated p
Cleaning
Period 1
Period 2
value1
Rats Cage Cleaning Room A. M. v. Rats Cage
440
431
1.000
Cleaning Room P.M.
Mann Whitney U Test comparison of two independent samples, tested at a significance level of 0.05.
1
97
Table G-4. Comparison of Total Bacteria Levels by Area, Both Locations
Comparison by Location
Mean
Mean
Calculated p
(Location 1 v. Location 2)
Location 1
Location 2
value1
Non-HEPA Filtered Rat Room v. Non-HEPA
3830
1597
1.000
Filtered Rat Room
Mann Whitney U Test comparison of two independent samples, tested at a significance level of 0.05.
1
Table G-5. Comparison of Mean Total Fungus Levels by Area, Location 1
Comparison by Area
Mean
Mean
Calculated p
(Area 1 v. Area 2)
Area 1
Area 2
value1
Outdoors v. Office
734
140
<0.001
Outdoors v. Aviary
734
13828
0.004
734
94
0.022
734
106
0.022
734
283
<0.001
Office v. Aviary
140
13828
0.004
Office v. Rat Room
140
94
0.549
140
106
0.791
140
283
0.020
Aviary v. Rat Room
13828
94
0.200
13828
106
0.200
13828
283
0.012
94
106
0.667
94
283
0.044
106
283
0.089
Mann-Whitney U Test comparison of two independent samples, tested at a
significance level of 0.007.
1
Table G-6. Comparison of Total Fungal Levels Before Cleaning and During
Mean Time
Mean Time
Calculated p
Cleaning
Period 1
Period 2
value1
13710
22452
1.000
Rats Cage Cleaning Room A.M. v. Rats Cage
265
330
1.000
Cleaning Room
Mice Cage Cleaning Room A.M. v. Mice Cage
197
340
1.000
Cleaning Room P.M.
1
98
Table G-7. Comparison of Total Fungal Levels Before Cleaning and During
Mean Time
Mean Time
Calculated p
Cleaning
Period 1
Period 2
value1
Rats Cage Cleaning Room A. M. v. Rats Cage
177
195
1.000
Cleaning Room P.M.
1
Table G-8. Comparison of Total Bacteria Levels by Area, Both Location
Comparison by Location
Mean
Mean
Calculated p
(Location 1 v. Location 2)
Location 1
Location 2
value1
Non-HEPA Filtered Rat Room v. Non-HEPA
94
89
1.000
Filtered rat Room
1
99
ABSTRACT
Air sampling was conducted outdoors and indoors in several source areas and non-source
areas at two university animal care facilities. Samples were collected simultaneously,
using single stage samplers, before and during cleaning operations. Concentrations for
both bacteria and fungi were determined. Gram staining and morphology and genus
identification for bacteria and fungi, respectively, also were determined. Bacterial and
fungal levels were statistically significantly different between areas within Location 1 but
were not statistically significantly different between areas within Location 2. Mean
concentrations of bacteria, predominately gram-positive cocci, ranged in source areas
from 435 cfu/m3 to 21,342 cfu/m3. Mean concentrations of fungi ranged in source areas
from 44 cfu/m3 to 13,828 cfu/m3. Eleven genera of fungi were identified, only ten were
found indoors.
100

Bioaerosols in university animal care facilities

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