United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
vvEPA
Research and Development
EPA-600/S1-81-069 Feb. 1982
Project Summary
APR r: .•
HA'R PKJlECuON
Development and
Evaluation of an Ambient
Viable Microbial Air Sampler
Kerby F. Fannin and Stanley C. Vana
The purpose of this project was to
enhance existing capabilities for
quantitative detection of viable micro-
organisms in the air. The specific
objectives were: (1) to review avail-
able methodologies for ambient viable
microbial air sampling; (2) to design an
ambient viable microbial air sampler;
(3) based on a new or an existing
design, to fabricate an air sampler; and
(4) to evaluate the suitability of this
sampler for detecting aerosols con-
taining bacteria and viruses. In addition
this study was intended to assess the
applicability of various sampling
methods to studies of specific envi-
ronmental microbial aerosol problems.
To fulfill a need for a standard
microbial large volume aerosol sampler,
an air sampler design based on the
principle of staged impaction was
proposed. A major limitation of this
sampler was that the microbial aerosol
collecting substrate, consisting of a
continuously wetted surface, required
a considerable amount of development
and optimization. Thus, to fulfill near-
term needs for ambient viable micro-
bial air sampling, the existing sampling
concept of cyclone scrubbing was
selected.
Studies were performed to evaluate
the suitability of cyclone scrubber
samplers and a continuously wetted
substrate for detection of several
types of microorganisms. The samplers
were evaluated in a dynamic aerosol
chamber using all-glass impingers as
reference samplers. Comparison of a
stainless steel and a glass cyclone
scrubber sampler for detecting Bacil-
lus subtilis var. niger spore aerosols of
about 1.1 - 3.3 fjm count median
diameter showed no significant dif-
ferences in their relative collection
efficiencies. Consequently, based
upon considerations such as ease of
construction, present usage, and
potential availability, the glass cyclone
scrubber was selected for further
evaluation. This sampler showed
geometric mean relative collection
efficiencies for B. subtilis var. niger
spore aerosols of 52% and 68%,
depending upon the composition of
the disseminating fluid. In studies
using different organisms, in similar
sized aerosols, this relative efficiency
was 46% for Serratia marcescens,
76% for f2 coliphage, and 92% for
poliovirus type 1. During the process
of aerosolization and collection, the
greatest viability losses in both the
reference and cyclone scrubber sam-
plers were observed with poliovirus,
followed by fa coliphage, and S.
marcescens.
Based on the studies, it was recom-
mended that the methods used for the
detection of low concentrations of
ambient viable microbial aerosols be
standardized, that a selected standard
sampler be evaluated under a wide
range of conditions for optimization of
critical parameters, and that relative
microbial aerosol evaluations be
performed with a reference sampler
having a sensitivity similar to that of
the test sampler.
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This Project Summary was developed
by EPA's Health Effects Research
Laboratory, Cincinnati, Ohio, to an-
nounce key findings of the research
project that is fully documented in a
separate report of the same title /see
Project Report ordering information at
back).
Introduction
Project Objective
The overall purpose of this project
was to enhance existing capabilities for
quantitative detection of viable micro-
organisms in the ambient air. The
specific objectives were: (1) to review
available methods for ambient viable
microbial air sampling; (2) to design an
ambient viable microbial air sampler, (3)
to fabricate an air sampler, based on a
new or an existing design; and (4) to
evaluate the suitability of this sampler
for detecting aerosols containing bac-
teria and viruses. In addition, this study
was intended to assess the applicability
of various sampling methods to specific
environmental microbial aerosol problems.
Rationale for Study
The extent of environmental exposure
of human populations to infectious
microbiological aerosols and the effects
of such exposure on incidence of
infectious disease have not been
conclusively determined. Such deter-
minations can be made, provided that
reliable and sensitive methods are
available to detect such aerosols or
indices of exposure. Detection of indices
of human exposure requires that
members of a population serve as
sentinels until an observable effect is
demonstrated. Such human exposure
can, however, be mitigated if contami-
nation by infectious aerosols is detected
early and reduced. The detection of
infectious aerosols with both precision
and accuracy is dependent upon the
availability of adequate sampling and
assay methodologies.
Numerous sampling techniques exist
for detecting microbial aerosols. These
techniques operate on a wide variety of
principles and their use for applications
requiring high sensitivity has not been
standardized. Research in experimental
infectious aerobiology has involved
aerosol generation in static or dynamic
chambers at concentrations detectable
with conventional instrumentation,
usually at relatively low air sampling
rates and short operational time limits.
Determination of low concentrations of
microbial aerosols in the ambient
outdoor environment, however, requires
more sensitive instrumentation. Eval-
uation of the risk of exposure and
potential infection with source-related
infectious microbial aerosols often
requires an extensive sampling pro-
gram. The methods used for such
sampling must be sufficiently sensitive
to detect such aerosols at very low
concentrations. This may necessitate
sampling at relatively high air flow rates
for long time periods. The applicability of
available air samplers to ambient
environmental aerobiological studies is
determined by the inherently designed
capabilities. Ideally, such a sampler
should have a high collection efficiency,
maintain the viability of collected micro-
organisms without permitting growth,
discriminate between respirable and
non-respirable particles and collect the
sample so that it can be easily assayed.
In addition it should be easily sterilized,
highly reliable, simple to operate, and
capable of remote-control operation,
and its cost should be such that it can be
used in routine monitoring programs.
Before undertaking the task of de-
veloping a new sampler, the available
air samplers were examined to deter-
min and select operational characteris-
tics that would be applicable to the final
design. Main emphasis was placed on
air sampling devices used for detection
of viable microbial aerosols. The princi-
pal aerosol collection concepts reviewed
for their application to viable microbial
aerosol sampling included sedimenta-
tion, filtration, impingement, precipita-
tion, centrifugal separation, and impac-
tion.
Experimental Approach
The research effort was performed as
several consecutive task. The direction
of each task was dependent upon the
findings of previous tasks and program
decisions made in consultation with the
U.S. EPA Project Officer. The project
tasks can be summarized as follows:
Review of Literature
A review of literature related to air
samplers having application to the
project objective was performed. The
purpose was to determine the availabil-
ity and suitability of existing instrumen-
tation as samplers of ambient viable
microbial aerosols.
Air Sampler Design
The need fora new air sampler design
was based on the review of existing
concepts of microbial air samplers. '"^
design of the new air sampler was>
based on such criteria as estimated
collection efficiency, sensitivity, reli-
ability, ease of sterilization, viability of
collected organisms, ease of sample
assay, capacity for remote operation,
particle size discrimination, and cost of
construction and operation. The con-
ceptual design was reviewed by several
staff members of organizations desig-
nated by the U.S. EPA Project Officer.
Air Sampler Construction
Samplers based on existing designs
were fabricated. The number of samplers
constructed was sufficient to perform
evaluation studies, and to provide
additional samplers as required by the
U.S. EPA Project Officer.
Air Sampler Evaluation
The sampler selected for further study
was evaluated by comparing its per-
formance in collection of viable and
non-viable aerosols to that of a reference
sampler. The purpose of these compara-
tive studies was to determine the
relative collection efficiency of the two
samplers for the recovery of aerosols
containing uranine dye, bacteria (Bacil-
lus subtilis var. niger spores and
Serratia marcescens), and viruses (fl
coliphage and poliovirus type 1). This
relative aerosol collection efficiency
was expressed as:
CE =
where
(R, x R2)/2
100
CE = relative aerosol collector
efficiency
S = aerosol concentration ai
determined by test sample
2= aerosol concentration a;
determined by paired ref
erence samplers at pre-S
position.
The aerosol slippage through the tes
sampler was determined by comparin<
the chamber aerosol concentration:
detected with reference sampler;
located at the pre- and post-sample
positions. The pre-sampler position wa
upstream from the test sampler whili
the post-sampler position was down
stream. The percent survival of bacteri
and viruses during the aerosohzatio
and sampling process was determine
relative to B. subtilis var. niger spore
aerosolized simultaneously with th
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test organism. The percent survival was
calculated as follows:
PS =
where
100
(2)
B,
PS = percent survival
B = B. subtilis var. niger concentra-
tion in the spray suspension (B0)
or in the aerosol sampler (61)
T = test organism concentration in
the spray suspension (T0) or in the
aerosol sampler TI).
The method of sampler operation,
including the sampling medium used,
air sampling rates, duration of collec-
tion, and sampling fluid flow rates, was
typical of that used in field studies and
did not necessarily include an evalua-
tion of multiple methods of operation.
The comparative studies were performed
in a dynamic aerosol chamber system
using aerosol concentrations of dye or
microorganisms that were detectable
with both the test and reference
samplers. The aerosol particle size, >1
//m count median diameter (CMD), was
similar to that which might be observed
in field studies of source-related ambient
viable microbial aerosols.
Continuously Wetted Substrate
Evaluation
A continuously wetted substrate was
evaluated in a test stand device to
determine its potential suitability for
use in a viable microbial aerosol
impacting-type sampler. The substrate
was evaluated to determine:
The recovery of B. subtilis var. niger
spores after collection in nutrient
broth and transferring the membrane
surface to nonselective nutrient agar;
Its applicability for recovery of total
coliform (Enterobacter aerogenes),
fecal coliform (Eschenchia coli], and
fecal streptococcus (Streptococcus
fecalis) by plating onto selective
media; and
The performance of the substrate-
containing device compared to that of
a slit sampler for recovery of B.
subtilis and S. marcescens.
Conclusions
1. There is a need for standardization
of methods for detecting low-level
ambient viable microbial aerosols.
2. An air sampler based on the
principle of staged impaction, with
aerosol classification into respirable
and non-respirable size ranges,
may be superior to existing samplers.
3. A major limitation of this sampler
design, however, is that the sub-
strate for final microbial aerosol
collection required further devel-
opment and optimization prior to
use in such a sampler.
4. A continuously wetted surface
developed as a potential collecting
substrate is suitable for collecting
bacterial aerosols. Additional studies
are needed to determine operating
conditions optimal for survival of
collected organisms on such a
surface.
5. Until the collection substrate is
optimized and the sampler design
concept developed, an existing
sampler, such as a cyclone scrub-
ber, should be used for viable
microbial aerosol studies where
low concentrations are expected.
6. The existing sampling concept that
demonstrates the greatest overall
superiority, in terms of the criteria
used for sampler evaluation, is that
of cyclone scrubbing.
7. Comparison of a glass and a
stainless steel cyclone scrubber
showed that there was no signifi-
cant difference in their relative
efficiencies for collecting B. subtilis
var. niger spore aerosols.
8. Further studies with the glass
cyclone scrubber showed that the
geometric mean relative collection
efficiency for 1 to 3.5 fjrr\ CMD
aerosols containing B. subtilis var.
niger spores was 52% and 68%,
depending upon the aerosol com-
position. The efficiency for S.
marcescens was 46%, for f2 coli-
phage 76%, and for poliovirus type
1 92%.
9. S. marcescens, ^2 coliphage, and
poliovirus type 1 showed viability
losses during the aerosolization-
sampling process. The survival of
poliovirus was less than or equal to
1 % of that observed for the coliphage
in samples collected with all-glass
impinger reference samplers and
with a glass cyclone scrubber.
10. Because of the great disparity
between the air volumes sampled
with the all-glass impinger and the
cyclone scrubber test samplers,
substantial fluctuations in relative
aerosol collection efficiencies can
be observed.
Recommendations
1. Methods used for detecting low-
level ambient viable microbial aero-
sols should be standardized. These
methods should employ a sampler
that is specifically designed for this
application, and that is reliable,
robust, and can be effectively steri-
lized by conventional methods. Until
such a sampler is developed, an
existing device such as a cyclone
scrubber sampler, should be used.
2. The standard sampler should be
evaluated under varied and controlled
conditions to optimize collection
efficiency by selecting appropriate
collecting media for the organisms
being studied and by controlling
critical parameters such asfluidflow
and air sampling rates.
3. These evaluations should employ a
viable microbial aerosol reference
sampler that operates at an air
sampling rate similar to that of the
device under evaluation
Kerby F Fann/n is with IIT Research Institute, Chicago, IL 60616, and Stanley C
Vana is with the Institute of Gas Technology, Chicago, IL 60616
Walter Jakubowski is the EPA Project Officer (see below)
The complete report, entitled "Development and Evaluation of an Ambient
Viable Microbial Air Sampler," (Order No PB 82-113 689, Cost $11 00,
subject to change) will be available only from
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone 703-487-4650
The EPA Project Officer can be contacted at
Health Effects Research Laboratory
U S. Environmental Protection Agency
Cincinnati, OH 45268
U S GOVERNMENT PRINTING OFFICE, 1982 - 559-017/7452
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