EPA/600/R-18/326 | September 2018
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
oEPA
Reaerosolization of Bacillus
anthracis Spores by Low-
Technology Remediation Methods
when Utilized in a Contaminated
Indoor Environment
Office of Research and Development
Homeland Security Research Program
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*>B>A
EPA/600/R-18/326
Reaerosolization of Bacillus anthracis Spores by
Low-Technology Remediation Methods when
Utilized in a Contaminated Indoor Environment
Assessment and Evaluation Report
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded and managed the research
described herein under contract EP-D-10-070 to Alion Science and Technology and Interagency
Agreement DW-21-92421801-0 with Edgewood Chemical Biological Center. This document has
been subjected to the Agency's review and has been approved for publication. Note that approval
does not signify that the contents necessarily reflect the views of the Agency. Any mention of
trade names, products, or services does not imply an endorsement by the U.S. Government or
EPA. The EPA does not endorse any commercial products, services, or enterprises.
The contractor role did not include establishing Agency policy.
Questions concerning this document, or its application should be addressed to:
John Archer, MS, CIH
U.S. Environmental Protection Agency
109 T.W. Alexander Drive
Mail Code: E343-06
Research Triangle Park, NC 27709
archer, i ohn@epa. gov
919-541-1151
li
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Acknowledgments
The authors would like to acknowledge the support of Marshall Gray (retired) as the U.S.
Environmental Protection Agency (USEPA) National Homeland Security Research Center
(NHSRC) Principal Investigator. The authors would also like to acknowledge work performed
by the following individuals and organizations.
Report Authorship
John Archer, USEPA NHSRC
Katrina McConkey, Booz Allen Hamilton
Peer Reviewers
Worth Calfee, USEPA NHSRC
Leroy Mickelsen, USEPA OLEM/CBRN CMAD
Research Conducted by Alion Science and Technology and RTI International
Laurie Brixey, Alion Science and Technology
Adam Hook, Alion Science and Technology
Howard Walls, RTI International
Jerome Gilberry, RTI International
Robert Yaga, RTI International
Jean Kim, RTI International
J. Randall Newsome, RTI International
Research Conducted by U.S. Army Edgewood Chemical Biological Center Research and
Technology Directorate
Vipin K. Rastogi
Lisa Smith
Jana Kesavan
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Table of Contents
DISCLAIMER II
ACKNOWLEDGMENTS Ill
TABLE OF CONTENTS IV
LIST OF TABLES IV
LIST OF FIGURES V
ACRONYMS AND ABBREVIATIONS VI
EXECUTIVE SUMMARY VIII
1 INTRODUCTION 1
2 LITERATURE REVIEW 2
2.1 Summary of Findings 2
3 LABORATORY EXPERIMENTS 3
3.1 Bench-scale Surface Wiping Experiments 4
3.1.1 Experimental System 5
3.1.2 Spore Preparation 6
3.1.3 Coupon Preparation and Inoculation 6
3.1.4 Measurement of Spore Removal and Reaerosolization 7
3.1.5 Results 8
3.1.6 Discussion 11
3.2 Evaluation of Household Vacuums 14
3.2.1 Experimental System 15
3.2.2 Spore Preparation 16
3.2.3 Coupon Preparation and Inoculation 16
3.2.4 Measurement of Spore Removal and Reaerosolization 18
3.2.5 Results 20
3.2.6 Discussion 26
4 QUALITY ASSURANCE/QUALITY CONTROL 27
4.1 Literature Review 27
4.2 Evaluations 32
4.2.1 Bench-Scale Wiping Experiment 32
4.2.2 Evaluation of Household Vacuums 35
5 SUMMARY AND CONCLUSIONS 38
6 REFERENCES 40
APPENDICES 42
Appendix A - Procedure for Fabrication of 14" X14", 28" X28", and 42" X 42" Material Coupons A-l
Appendix B - Aerosol Deposition of Spores Onto Material Coupon Surfaces Using the Aerosol Deposition Apparatus -
High Dosing B-l
List of Tables
Table 3-1. EPA Laboratory Studies Conducted Based on Information Gaps Related to Surface Wiping and Vacuuming.. .4
Table 3-2. Test Matrix for Wiping Experiments 5
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Table 3-3. MDI Mass and Viable Spore Delivery and Surface Concentration for Each Day 8
Table 3-4. Reaerosolization Results (CFU/cm2) for Surface Wiping Experiments 10
Table 3-5. Reaerosolization of viable spores (CFU/cm2 of surface wiped) from tests surfaces 11
Table 3-6. Evaluation of Household Vacuums Test Matrix 15
Table 3-7. Quantity of Samples from Flooring Materials per Test 19
Table 3-8. Filter Identification Number and Locations Per Test 20
Table 4-1. Critical Measurement Acceptance Criteria 32
Table 4-2. Quality Control Checks 34
Table 4-3. Quality Objectives for Test Measurements 36
Table 4-4. Specifications and Acceptance Criteria for Consumables 37
List of Figures
Figure 3-1. Schematic of the AerosolTest Box 6
Figure 3-2. Placement of SS Test Coupons Usedto Estimate Surface Concentration 8
Figure 3-3. Illustration of the Average Concentration Gradient of Viable Spores (CFU/cm2) Observed Over the Area
Wiped on the Coupon 9
Figure 3-4. Average Surface Wipe Recoveries Compared to Average MDI Control Recoveries (Stainless Steel Coupon). 11
Figure 3-5. Average Reaerosolization of Viable Spores (CFU/cm2 Wiped) for Each of the Wipes by Surface Type 12
Figure 3-6. Vacuums Used in this Study from Leftto Right, Hoover WindTunnel, Olympus Canister, and Hoover Linx.,14
Figure 3-7. ECBC AerosolTest Chamber 16
Figure 3-8. A 4x4 Panel of Wool-Blend Carpet 17
Figure 3-9. Two-Fluid Pneumatic Sonic Nozzle usedto Aerosolize Spores for Coupon Inoculation 18
Figure 3-10. Spore Removal from Floor Surfaces by Vacuum Cleaner Type 21
Figure 3-11. Spore Removal from Two Floor Surfaces with and Without the Beater BarOn 22
Figure 3-12. Spore Reaerosolization during Vacuuming at Floor Level Corners of Panels 23
Figure 3-13. Spore Reaerosolization during Vacuuming at 1-ft Distance from the Floor 24
Figure 3-14. Spore Reaerosolization during Vacuuming atThree Heights 25
Figure 3-15. Spore Reaerosolization during Bag or Canister Emptying 26
Figure 4-1. Flowchart for Information Source Evaluation 31
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Acronyms and Abbreviations
|iL
ADA
AFM
AGI
B. anthracis
Btk
BSC
°C
CBRN
cm
CMAD
cm2
CFU
CV
DI
DQI
ECBC
ft
ft2
g
HEPA
hr
in
L
MDI
mg
min
ml
mm
NHSRC
NIST
OLEM
OSB
HSRP
pAB
PBST
PM
PMA
PPE
QA
QAPP
QC
RH
Micrometer(s)
Microliter(s)
Aerosol deposition apparatus
Atomic force microscopy
All-glass impinger
Bacillus anthracis
Bacillus thuringiensis var. kurstaki
Biological safety cabinet
Degree(s) Celsius
Chemical, biological, radiological, and nuclear
Centimeter(s)
Consequence Management Advisory Division
Square centimeter(s)
Colony forming unit(s)
Coefficient of variation
Deionized
Data quality indicator
Edgewood Chemical Biological Center
Foot/feet
Square foot/feet
Gram(s)
High efficiency particulate air
Hour(s)
Inch(s)
Liter(s)
Metered-dose inhaler
Milligram(s)
Minute(s)
Milliliter(s)
Millimeter(s)
National Homeland Security Research Center
National Institute of Standards and Technology
Office of Land and Emergency Management
Oriented strand board
Homeland Security Research Program
pH-adjusted bleach
Phosphate buffered saline with Tween®20
Particulate matter
Preventative Maintenance Agreement
Personal protective equipment
Quality assurance
Quality Assurance Project Plan
Quality control
Relative humidity
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SOP Standard operating procedure
SS Stainless steel
TSA Tryptic soy agar
U.S. United States
USEPA U.S. Environmental Protection Agency
vii
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Executive Summary
In the event of a release of a biological agent in an urban area, a wide range of surfaces may be
contaminated, including indoor surfaces in homes and businesses. It will be important to develop
options for effective cleaning of these indoor surfaces that may have low levels of biological
contamination. The U.S. Environmental Protection Agency (US EPA) conducted this research to
identify and evaluate low-technology (low-tech) remediation methods that could be used to
reduce surface contamination while minimizing the reaerosolization of contaminants during
routine in-home cleaning of contaminated surfaces.
The research began with a thorough literature review of previous work conducted on
reaerosolization of spores/particles during residential cleaning activities. The review focused on
identifying information that could assist residents, business owners, and hired contractors in
selecting effective remediation approaches that minimize the reaerosolization and migration of
Bacillus anthracis (B. anthracis) spores. Remediation approaches surveyed included vacuuming
(with and without a beater bar), sweeping, dry wiping, and wet wiping of surfaces. Findings from
the literature review identified information gaps on reaerosolization potential due to surface
wiping and vacuuming. Following the literature review, exploratory experimental tests were
conducted by EPA to fill some of the information gaps and evaluate the reaerosolization
potential of common household activities that could be deployed as low-tech remediation
methods for indoor B. anthracis contamination. Two separate studies were conducted: 1) the
evaluation of reaerosolization caused by surface wiping, and 2) the evaluation of spore removal
and reaerosolization caused by vacuuming.
The surface wiping study compared the potential reaerosolization of spores from various surfaces
(glass, wood laminate, and linoleum) by several types of wiping methods (dry paper towels,
premoistened disinfecting towelettes [Lysol® wet wipes], and electrostatic-based Swiffer®
cleaning wipes). Bacillus thuringiensis var. kurstaki (Btk) spores were used as a surrogate for B.
anthracis. The spore reaerosolization potential for each wipe-based remediation approach was
evaluated by determining the fraction of total contaminants resuspended while using each wipe
method. Results from all test conditions were compared and the results were used to inform
whether use of a particular wiping method leads to reaerosolization of spores and if so, which
residential cleaning activities should be avoided to minimize potential exposure.
The main findings from the surface wiping study include the following:
• The data set indicates that surface type has an effect, with laminate wood flooring
showing the highest reaerosolization for the three types tested.
• The data set indicates that wiping with a dry paper towel resulted in the highest
reaerosolization while an electrostatic Swiffer® dry wipe and Lysol® pre-moistened wet
wipe indicated similar yet lower likelihoods for reaerosolization.
• Additional studies are needed to evaluate the statistical significance of these findings.
The vacuum study included a series of tests using three commercially available vacuum cleaners
of different types (upright, canister type, and cordless stick type) equipped with high efficiency
particulate air filters to determine the spore removal and reaerosolization from common
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residential surfaces. Experiments were conducted on three different floor types that are common
in homes including three carpet types (nylon, wool blend and polyester), linoleum, and slate
laminate tile flooring. Btk spores were used as a surrogate for B. anthracis and were deposited as
an aerosol powder on test coupons (4x4-foot. panels of each of the five flooring materials).
Additionally, tests were conducted to evaluate the impact of vacuum beater bar usage on spore
removal from nylon carpet and laminate flooring coupons. To estimate the quantity of spores that
were not collected by the vacuum cleaner, 3x1-inch, core samples (cut out and left in place on
the panel before inoculation) were collected after the panel had been vacuumed. Spore removal
was determined by comparing the spore recoveries per square foot before vacuuming (from
reference coupons) to the recoveries per square foot after vacuuming (from core samples). It is
important to note that this calculation considers the amount of spores (based on colony forming
units [CFU]) removed from the surface but does not incorporate spores captured by the vacuum
itself.
The findings from the vacuum study include the following:
• The results show spore removal of approximately 70-90% of CFUs from all five surfaces.
The results for the slate laminate using the Olympus Canister vacuum was the only
deviation (showing significantly lower spore removal).
• The tests conducted with the beater bar indicated a lower percentage of spore removal
than without the beater bar. It is not clear if these lower removal results are due to lower
recovery by the vacuums or displacement of spores due to the beater bar operations.
Therefore, additional studies to quantify the fate and migration of contaminated surfaces
are recommended.
• Reaerosolization from the mechanical motion of the vacuuming itself as well as during
bag/canister emptying may pose an inhalation hazard.
Based on the findings from the literature review and laboratory experiments, the report includes
some general preferred options for low-tech remediation methods used for surface
decontamination and limiting the reaerosolization of spores by cleaning activities for the indoor
environments. Future research needs are also discussed.
The options contained in this report could be combined with data collected from upcoming
experiments and ultimately used in the development of a risk-based analysis of the effectiveness
and exposure risks of low-tech remediation methods.
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1 Introduction
The U.S. Environmental Protection Agency (US EPA) is the agency responsible for
environmental cleanup after the release of chemical, biological, radiological, and nuclear
(CBRN) contaminants. USEPA's Homeland Security Research Program (HSRP) conducts
research to investigate ways to mitigate and decontaminate contaminated areas. For wide-area
biological incidents, such as the intentional release of the biological agent, Bacillus anthracis (B.
anthracis), residential areas may be impacted by biological contamination. Information on low-
technology (low-tech) remediation methods may be useful to local Public Health officials if self-
help remediation guidance is preferred in areas with likely low-level contamination, or residents
whose homes may not be within the contaminated zone but are concerned about undetected
contamination. Low-tech remediation methods are based upon normal cleaning activities
residents undertake to maintain a clean and sanitary home. In the event of a release of biological
agent in an urban area, a wide range of surfaces may be contaminated including indoor surfaces
in homes and businesses. Because spores or other biological agents can be reaerosolized from
surfaces via mechanical agitation, it is necessary to provide information to the public on the
potential risks of low-tech remediation methods, such as vacuuming, sweeping, and wiping of
indoor surfaces.
To assess the effectiveness and risks of various cleaning activities, experimental studies were
conducted to evaluate surface wiping and vacuuming of indoor surfaces under controlled
conditions. The objectives of these research studies were as follows:
1. Perform a thorough literature review of previous work conducted on reaerosolization of
spores/particles during residential cleaning activities.
2. Fill information gaps from the literature on reaerosolization of spores during residential
cleaning activities through exploratory laboratory experiments focusing on vacuuming
and wiping.
3. Utilize results from the literature review and laboratory experiments in the development
of future guidance on the risks of employing low-tech remediation methods following the
release of a biological agent.
This research effort began with a literature review on the generation and redistribution of
aerosols in the respirable size range as a result of implementing residential cleaning activities.
The review focused on identifying information that could be applicable to residents, business
owners, and hired contractors to reduce the reaerosolization and migration of B. anthracis spores
as a result of the aforementioned cleaning activities as well as identifying any information gaps
that exist.
Following the literature review, limited experimental testing was conducted to fill some of the
information gaps that were identified. These laboratory studies evaluated the reaerosolization
potential of common household activities that could be deployed as low-tech remediation
methods for B. anthracis contamination, specifically surface wiping and vacuuming.
Following the literature review and laboratory experiments, some observations were made of
preferred methods to minimize reaerosolization of spores when cleaning indoor environments
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that may be lightly contaminated due to infiltration from outdoors. Additionally, findings from
the literature review and laboratory experiments could be combined with data collected from
additional experiments to develop a qualitative risk-based assessment of the reaerosolization
potential of B. anthracis spores from low-tech remediation methods.
2 Literature Review
During the past 60 years, numerous experiments have identified the potential for biological
agents to reaerosolize following an initial release with deposition on surfaces. Because only
limited quantitative information has been obtained and the experiments were focused primarily
focused on outdoor environments, it is difficult to predict the reaerosolization hazard that might
occur during routine in-home cleaning outside a heavily contaminated area following the
intentional release of a biological agent. EPA conducted a literature review to identify previous
work conducted on reaerosolization of spores/particles during residential cleaning activities and
any information gaps that exist. The search focused on identifying information that could reduce
the reaerosolization and migration of B. anthracis spores as a result of vacuuming (with and
without a beater bar), sweeping, dry wiping, and wet wiping of surfaces.
2.1 Summary of Findings
Research on the reaerosolization of particles due to vacuuming, walking, and sweeping
constitutes most of the work conducted thus far on indoor reaerosolization. A major study on
secondary aerosolization of B. anthracis spores in a contaminated U.S. senate office showed
dangerous levels of reaerosolization during normal office activities (Weis, 2002). This study
showed the necessity of research examining reaerosolization in indoor environments.
Unfortunately, most studies on reaerosolization and transport of particles in indoor environments
have focused primarily on allergens and dust, only some of which is in the size range (mean
length and diameter of 1.5 |im and 0.8 |im, respectively) of B. anthracis spores (Carrera et al.,
2007) Even when discussing particles of the same size, there are questions about applicability of
these data to B. anthracis reaerosolization because of the differences in density and surface
chemistry between the particles.
Reaerosolization results are reported in a variety of ways, making direct comparison between
studies difficult (Qian et al., 2014). Even papers reporting similar experiments show a strikingly
varied set of results, likely due in large part to the variability in experimental conditions. These
two issues make it nearly impossible to draw direct scientifically-robust conclusions, though
general statements can still be made. Two review articles, Sehmel (1980) and Qian et al. (2014),
highlight this variability. These articles discuss the results of studies of reaerosolization by
mechanical motion in controlled indoor settings. Qian et al. recalculate results to represent
findings in different ways to make direct comparisons between studies when possible. It is
evident from these reviews that any amount of motion indoors causes reaerosolization. However,
the reaerosolization factors reported for different walking experiments and sweeping varied by
six orders of magnitude (Sehmel, 1980), with a sweeping experiment showing the highest
reaerosolization factor (Mitchell and Eutsler, 1967). Even though none of the experiments
reviewed focused directly on particle sizes comparable to B. anthracis spores, it is evident that
sweeping with a broom should be avoided in a possible contamination area as any spore removed
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from the surface could immediately aerosolize. This observation is supported by findings by
Lehtonen et al. (1993), who showed that sweeping increased indoor fungal spore concentrations
tenfold above the background level. Qian et al. reported that reaerosolization fractions for
particles of the size of B. anthracis spores vary between 10"5 and 10"2 for walking experiments,
and that walking and vacuuming have similar emission rates, though the variability within each
study was approximately two orders of magnitude.
A paper by Thatcher and Layton (1994) discussed aerosol concentrations and particle size
distributions measured indoors during a variety of activities. The authors showed that vigorous
vacuuming increased the concentration of aerosols in the 1- to 5-[j,m size range, increasing by a
factor of 2.5 times compared to just walking. In this case they did not report the type of vacuum
used, nor was there any reference to the surfaces involved or the types of particles aerosolized.
Studies on the use of high efficiency particulate air (HEP A) filtered vacuums versus traditional
vacuums have also shown variable results, though HEPA filtration does show some
improvement over non-HEPA filtration. A study by Knibbs et al. (2012) of 21 different types of
vacuum cleaners showed fine particulate matter (PM2.5) emissions from all vacuums, and there
was variation from one vacuum to another. The HEPA-filtered vacuums did emit fewer particles
in general. The major source of dust emission appeared to be due to the mechanical motion of the
vacuuming process (i.e., the movement of the vacuum over the surface). This finding is
supported by work by Van Strien et al. (2004) and Gore et al. (2006). Van Strien et al. showed
that central vacuum systems that filter to the outside do not significantly reduce airborne
allergens as compared to conventional vacuum cleaners. Gore et al. also observed mite allergen
exposure during both HEPA vacuuming and non-HEPA vacuuming. Gore et al. also showed
reaerosolization of dust mites during canister and bag removal and disposal of contents.
Considering these results, vacuuming should be used with caution. Vacuuming studies assessing
the beater bar usage have not been conducted and would be of significant value. Also, with the
variation demonstrated from vacuum to vacuum, it should be recognized that on an individual
basis, that variability in manufacturing quality could lead to significantly different results for
comparable models of vacuums.
No experimental studies could be found reporting the potential for surface wiping to contribute
to reaerosolization. Tang et al. (2004) mention the preference of cleaning with a wet wipe over
cleaning with a dry wipe, though no supporting research could be identified, and no
reaerosolization data were recorded. In the occupational health and safety field, wet wiping is
generally preferred to dry wiping to minimize the potential for reaerosolization. This type of
exposure control preference can be found when dealing with cleanup of many types of dry
materials.
3 Laboratory Experiments
Two laboratory studies (described in Table 3-1) were conducted to evaluate the reaerosolization
potential of common household activities that could be deployed as low-tech remediation
methods for B. anthracis contamination. These studies focused on the findings from the literature
review, specifically the information gaps on surface wiping and vacuuming.
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Table 3-1. EPA Laboratory Studies Conducted Based on Information Gaps Related to Surface
Wiping and Vacuuming.
Year
Information Gap
Focus of Laboratory Study
2015
No supporting investigations conducted on
reaerosolization potential due to surface
wiping activity.
Limited bench-scale study to investigate
reaerosolization from wiping of surfaces
with different types of wipes.
2016
Variability in reaerosolization exists
between vacuum cleaner models as well as
HEPA-filtered vacuum cleaners versus
traditional vacuum cleaners.
Studies assessing beater bar usage have not
been conducted.
Limited chamber study to determine spore
removal and reaerosolization using three
different vacuum cleaners. A portion of
the tests were conducted with and without
the beater bar.
The experimental approach and test results for both studies are reported in the following
sections. The experiments were not meant to be comparative, but rather to collect information
that can be consolidated with future experiments and findings to develop a qualitative risk-based
assessment of the reaerosolization potential of B. anthracis spores from low-tech remediation
methods. The two experiments were conducted independently and varied in several aspects such
as objectives, surrogate spore preparation, data analyses, and reporting of results.
The non-pathogenic test surrogate used in both laboratory studies was Bacillus thuringiensis var.
kurstaki (Btk). Btk is commonly used as a surrogate for B. anthracis for aerosol-based
experiments because of its similarity in physical properties (USEPA, 2012). Btk is a gram-
positive bacterium commonly found in soil. The endotoxin protein produced during sporulation
is commonly used as a pesticide. The average hydrated spore size is 0.8 micrometers (|im) by 1.4
|im, which is very similar to the average hydrated spore size for B. anthracis (Carrera et al.,
2007). The Btk spore preparations used in the both studies varied and are described in Sections
3.1.2 and 3.2.2. respectively. Bar-coded Btk was used in the surface wiping experiments. Bar-
coded Btk is a genetically modified strain developed by Edgewood Chemical Biological Center
(ECBC). The genetic modification alters the DNA sequence so that polymerase chain reaction
analysis clearly distinguishes these spores from the common naturally occurring strain.
3.1 Bench-scale Surface Wiping Experiments
The purpose of these experiments was to fill information gaps related to reaerosolization of
spores due to surface wiping. EPA conducted a limited bench-scale study in 2015 to investigate
reaerosolization from wiping of surfaces with different types of wipes. Btk spores were used as a
surrogate for B. anthracis and were dry-deposited on test surfaces. Experiments were conducted
on three different surface types that are common in homes (glass, wood laminate, and linoleum)
and with three different types of cleaning wipes (dry paper towels, premoistened disinfecting
towelettes [Lysol® wet wipes], and electrostatic-based Swiffer® cleaning wipes). The
experimental matrix is shown in Table 3-2.
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Table 3-2. Test Matrix for Wiping Experiments
Wipe Type
Glass
Wood
Linoleum
Laminate
Dry paper towel
3
3
3
Electrostatic Swiffer®
3
3
3
Lysol® wet wipe
3
3
3
Three main questions were addressed in this limited bench-scale study.
1. Does wiping surfaces contaminated with Bacillus spores cause reaerosolization of the
spores?
2. Does the surface type have an impact on Bacillus spore reaerosolization from wiping
activities?
3.1.1 Experimental System
All testing was conducted inside a humidity- and temperature-controlled environmental chamber.
A small aerosol sampling box (Figure 3-1) was fabricated for this project. The box was
constructed primarily of stainless steel and was designed for laminar flow. The box includes an
upstream HEPA-filtered section large enough to accommodate a 14-in. by 14-in. square test
surface (coupon) laid flat. The upstream section allowed placement of a rotating shaft and wiping
arm directly over the sample coupon. The box was designed so that reaerosolized particles would
be carried downstream by the air being pulled through two high-velocity all-glass impingers
(AGIs). Each AGI contained 20 milliliters (mL) of phosphate-buffered saline with 0.05 %
TWEEN®20 which served as the collection fluid in the impingers. The entire air volume was
pulled through the AGIs for complete sampling. The tunnel flow rate was approximately 25 liters
per minute (L/min), as defined by the AGI flow rate for maximum collection efficiency of 12.5
L/min each. The environmental conditions inside the chamber were kept constant at 22 degrees
Celsius (°C) and 35% relative humidity (RH) for every experiment, and these environmental
conditions were monitored by the chamber temperature and humidity controls. The
environmental chamber was kept at a slightly negative pressure relative to the laboratory to
prevent accidental contamination of the laboratory. After each experiment, the AGIs were
autoclaved and the chamber was sprayed with 10% bleach that was allowed to dry and
crystallize. Then, the chamber was wiped with deionized (DI) water and then isopropyl alcohol.
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Wipe Holder Rotation Actuator
Wipe Holder Plate
Stainless Steel
Sampling Box Cap
Stainless Steel
Base for coupon
Filtered Air intake
AGI Sampling Manifold
Airflow Direction
Figure 3-1. Schematic of the Aerosol Test Box
3.1.2 Spore Preparation
The bar-coded Btk spores used in this study were developed by ECBC (Emanuel, 2012 and
Buckley, 2012). The Btk preparation was obtained from the U.S. Army Dugway Proving Ground
Life Science Division. The bar-coded Btk cells were cultured by 10-L batch fermentation.
Following sporulation, the spores were concentrated into a wet pellet, washed three times, and
lyophilized. The lyophilized spores were a dry but agglomerated cake rather than a loose dry
powder. Spores were suspended in 100% ethanol, combined with Dymel 134a (DuPont,
Wilmington, DE) propellant, and loaded into metered-dose inhalers (MDIs) by Cirrus
Pharmaceuticals (Research Triangle Park, NC) (Calfee et al., 2014).
3.1.3 Coupon Preparation and Inoculation
Coupons of surface materials (14 x 14-inch) were fabricated according to the procedures found
in Appendix A. Five small (9.7 square centimeter [cm2]) stainless steel (SS) reference coupons
were also cut from thin SS shim stock. All coupons were disinfected using the following
procedure immediately prior to inoculation:
1. Wipe all coupon surfaces with 10% pH-adjusted (acidified) household bleach
solution.
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2. Wait at least 2 min and then wipe all decontaminated coupon surfaces with DI
water.
3. Wipe all decontaminated coupon surfaces with 70% isopropanol solution.
4. Wait 5 min for isopropanol to dry prior to inoculating the coupon.
Material and SS reference coupon inoculation is described fully in Appendix B. Briefly, each
coupon was inoculated inside the test chamber using its own aerosol deposition apparatus (ADA)
pyramid (Lee et al., 2011), which is designed to fit one 14-in. square coupon of any thickness.
The MDI was discharged a single time into each dosing chamber. The spores were allowed to
settle onto the coupon surfaces for a minimum of 18 hours (hr) and the AD As remained on the
coupons until transfer to the sampling box for testing.
3.1.4 Measurement of Spore Removal and Reaerosolization
The critical measurement for this project was the number of spores captured by the impingers
during each test, measured as CFUs. The results of these measurements were used to compare
the reaerosolization of spores due to each type of wipe from each surface.
Each test included one coupon loading level using ADA pyramids, one surface type, and one
type of wipe. The target spore loading was 106 CFU per square foot (ft2) for every test. The three
surface types were glass, linoleum, and laminate flooring. The wiping process was performed by
an electric motor connected to a rotating rectangular pad on which the wipes were mounted. The
motor rotated at a constant rate clockwise and counterclockwise for each test. Each coupon was
wiped with a single type of wipe (dry paper towels, Lysol® wet wipes, and electrostatic-based
Swiffer® cleaning wipes).
The AGIs were connected to the sampling box and the AGI pumps were turned on for 1 min
before wiping began. The sampling pumps remained on for 1 min after the wiping process was
completed and the wiping pads were lifted from the surface. Each AGI contained 20 mL of
phosphate-buffered saline with 0.05 % TWEEN®20 which served as the collection fluid in the
impingers. After the pumps were turned off, the AGIs were disconnected from the box and taken
to the microbiology laboratory for fluid removal and plating.
A reference coupon was also prepared to estimate the surface concentration of Btk spores in the
central area of the coupon where wiping was conducted. Five small (9.7 cm2) sterile, SS test
coupons with a polished surface were placed in a pattern on the coupon in the area where wiping
would take place (Figure 3-2). The ADA was placed on top, secured, and the prescribed
procedure for Btk deposition was conducted. After 24 hr each SS test coupon was aseptically
transferred to a sterile, labeled container. Spores were extracted, and the extract was plated to
estimate the surface concentration of Btk spores in CFU/cm2
7
-------
Figure 3-2. Placement of SS Test Coupons Used to Estimate Surface Concentration.
The area covered is that swept out by the wiper. Letters denote the location key used in collecting
data.
Additionally, a reference sample was taken from the MDI each day before Btk depositions. For
the reference sample, the MDI was discharged (1 actuation) directly into an AGI with impinger
fluid. The collected sample was then processed to determine the total CFU of Btk spores in a
single actuation.
3.1.5 Results
3.1.5.1 Depositions and Surface Concentrations
The capability of the MDI to deliver a repeatable and precise dose of spores is shown in Table 3-
3.
Table 3-3. MDI Mass and Viable Spore Delivery and Surface Concentration for Each Day
Direct deposition to AGI
Measurement of Surface Concentration with S.S. test coupons
Date
Puff mass
0)
Spores
(CFU/act)
Spore sfrriass
(CFU/g act)
Puff mass
(g)
Deposition area concentration (CFU/cm2)
A | B C D | E
Avg Cone
(CFU/cm 2)
3/10/2015
0 0578
2.96E+06
5.12E+07
not measured
3/11/2015
0.0579
1.38E+07
2 38E + 08
0 0558
5 50E+04
3 40E+04
2 85E+05
361E+04
2 99E+04
8.80E+04
3/12/2015
0.0572
2 69E+07
4 70E+08
0 0576
9 04E+04
8 04E+04
3 54E + 05
4 98E+04
3 61E+04
1.22E+05
3/13/2015
0.0576
9.56E+06
166E+08
0 0569
4 91E+04
3 23E+04
313E+05
4 30E+04
3 16E+04
9.37E+04
3/16/2015
0 0572
2 09E+07
3 65E+08
0 0577
3 68E+04
1 86E+04
1 03E+05
1 72E+04
1 07E+05
5.65E+04
3/17/2015
0.0581
9.15E+06
157E+08
0 0581
2 85E+04
1 19E+04
1 94E+05
5 91E+03
4 09E+04
5.62 E+04
3/18/2015
0.0581
1.21E+07
2.08E+08
0.0587
1 14E+04
3.85E+04
7.87E+04
2.96E+04
5.10E+04
4.18E+04
Avg
0.0577
1.36E+07
2.37E+08
0.0575
4.52E+04
3.60E+04
2.21E+05
3.03E+04
4.94E+04
7.64E+04
Stdev
0.0004
7.97E+06
1.40E+08
0.0010
2.70E+04
2.40E+04
1.14E+05
1.64E+04
2.92E+04
3.01 E+04
CV
0.007
0.585
0.591
0.018
0.597
0.668
0.516
0.542
0.590
0.394
Notes:
act = actuation
8
-------
The MDI drop in mass by each actuation (loss of propellant, particles, and spores) was very
consistent at 0.0577 ± 0.0004 gram (g) per actuation, with a coefficient of variation (CV) of
0.007. However, the CFU/actuation or CFU/g of actuation varied considerably. Spores delivered
by the MDI were on the order of 107 CFU/actuation with CV of approximately 0.6. There are
many potential sources for this variability including: how well-mixed the solution in the MDI
was each time an actuation was delivered; the variability of the number of viable spores in the
particles and the size distribution; experimental variability in actuating the MDI into the AG I;
and experimental error in processing, plating, and enumerating the AGI fluid.
The surface concentration exhibited a gradient from the center to the edge of the wiping area.
The surface concentration at each measured point and for the average of all the measurement
points exhibited considerable variability. Figure 3-3 illustrates the average surface concentration
gradient; discrete measurements were taken at the center and the edges, and the contour shown is
an interpolation from the center to the edges. Overall the average surface concentration was 7.6
(± 3.0) >< 104 CFU/cm2. One major source of the variability in the surface concentration
measurements is the variability in the amount delivered by the MDI. Other factors that could
contribute to variability include: differences in electrostatics of the surface that the SS test
coupon was placed on; variability in the AD As and the mating of the MDI to the ADA inlet; and
experimental error in processing, plating, and enumerating the SS coupon samples.
Figure 3-3. Illustration of the Average Concentration Gradient of Viable Spores (CFU/cm2)
Observed Over the Area Wiped on the Coupon
9
-------
3.1.5.2 Wiping Experiments
Table 3-4 summarizes the experiments conducted each day with the results for reaerosolized Btk
spores reported as total collected CFU/cm2 of coupon surface wiped.
Table 3-4. Reaerosolization Results (CFU/cm2) for Surface Wiping Experiments
Day 1: All wipe types, one surface type.
Glass
Wood Laminate
Linoleum
Paper towel
0.059
-
-
Swiffer®
0.034
-
-
Wet wipe
0.0038
-
-
Day 2: One wipe type, all surface types.
Glass
Wood Laminate
Linoleum
Paper towel
3.0
10
0.00
Swiffer®
-
-
-
Wet wipe
-
-
-
Day 3: One wipe type, one surface type.
Glass
Wood Laminate
Linoleum
Paper towel
-
-
-
Swiffer®
-
0.20
-
0.15
0.46
Wet wipe
-
-
-
Day 4: All wipe types, one surface type AND one wipe type, all surface types.
Glass
Wood Laminate
Linoleum
Paper towel
-
-
0.088
Swiffer®
-
-
0.011
Wet wipe
0.43
0.048
0.078
Day 5: All wipe types, one surface type AND one wipe type, all surface types.
Glass
Wood Laminate
Linoleum
Paper towel
1.7
2.5
0.22
Swiffer®
-
-
0.0019
Wet wipe
-
-
0.021
Day 6: One wipe type, all surface types AND one replicate.
Glass
Wood Laminate
Linoleum
Paper towel
-
-
-
Swiffer®
-
-
-
Wet wipe
0.16
0.42
0.021
0.076
Day 7: Random AND one replicate.
Glass
Wood Laminate
Linoleum
Paper towel
-
8.3
-
Swiffer®
0.090
-
0.0038
0.038
Wet wipe
-
-
-
10
-------
The reaerosolization results for all wipe experiments are summarized in Table 3-5. Considerable
variability was observed within each surface and wipe type (i.e., large standard deviation). The
variability could be due to a combination of factors such as: the large variation in surface
concentration of spores; natural variations in the individual wiping materials and surfaces wiped;
and experimental error during the wipe experiments and sample processing.
Table 3-5. Reaerosolization of viable spores (CFU/cm2 of surface wiped) from tests surfaces.
Glass
avg. s.d.
Wood
Laminate
avg. s.d.
Linoleum
avg. s.d.
All Surfaces
avg. s.d.
Paper towel
Swiffer®
Wet wipe
1.58 1.46
0.054 0.031
0.20 0.21
7.04 4.07
0.27 0.17
0.18 0.20
0.10 0.11
0.0057 0.0051
0.04 0.03
2.91 3.83
0.11 0.15
0.14 0.17
All Wipes
0.61 1.04
2.50 3.97
0.05 0.072
3.1.6 Discussion
3.1.6.1 Spore Deposition and Surface Concentration
There was variability in the surface concentration of viable spores in the wipe area (CFU/cm2).
One source of this variability was the inconsistency in the CFU/g of actuation delivered by the
MDI. The prescribed procedure was to deposit on a reference coupon for each day of testing.
This reference coupon should represent the surface concentration for the coupons used in the
wiping experiments that testing day, however there was no correlation between the control
recoveries and the recoveries resulting from surface sampling (Figure 3-4). Because of this the
surface sampling results were not predicted by MDI control sample recoveries. The average
value of 7.6 x io4 CFU/cm2 can be used for all experiments as the approximate concentration of
spores on the surface.
Spore Surface Concentration as a Function of
Viable Spore Concentration per MDI Actuation
1.4E+05
1.2E+05
(n 1.0E+05
E
8.0E+04
£ 6.0E+04
< 4.0E+04
2.0E+04
0.0E+00
0.0E+00 5.0E+06 1.0E+07 1.5E+07 2.0E+07 2.5E+07 3.0E+07
CFU/Actuation
R2 = 0.2553
•
•
•
•
•
Figure 3-4. Average Surface Wipe Recoveries Compared to Average MDI Control Recoveries
(Stainless Steel Coupon)
11
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3.1.6.2 Reaerosolization from Wiping
A wide range of reaerosolization values was observed between the various wipe/surface
combinations and between repeated experiments with the same wipe/surface combination.
Although the variability in the surface concentration was problematic, the large difference in
surface concentration and the amount reaerosolized does provide for some limited analysis. The
average surface concentration was three orders of magnitude higher than even the largest number
of spores reaerosolized by wiping.
Improvements in the experimental processes and a larger sample size are needed to make
accurate determinations of the effects of surface and wipe types. However, examination of the
data did indicate trends as a function of both wipe type and surface type. Figure 3-5 summarizes
the results as total observed reaerosolized viable spores (CFU/cm2 of surface wiped) for each
wipe by surface type.
-a 12
Q)
Q.
5 10
CM
E
u
^ *
LL-
o
Wood Laminate Glas
Linoleum
Lysol Wet Wipe
U 0.25
Q) 0.05
Wood Laminate
Glass
Linoleum
Figure 3-5. Average Reaerosolization of Viable Spores (CFU/cm2 Wiped) for Each of the Wipes
by Surface Type.
Error bars are the standard deviation of the mean. Note: The y-axis scale for Dry Paper Towel is
different than the other two surface wipes due to the magnitude of reaerosolization.
12
-------
Ideally the reaerosolization fraction would be calculated, but the un-normalized results are
presented due to the variability in surface concentration. However, the surface concentration of
viable spores over the area wiped was approximately 7.6 (± 3.0) x io4 CFU/cm2.
For the various wipes tested there was a trend for reaerosolization of
paper towel > wet wipe > Swiffer®.
However, because of the small sample size and wide variability, a Student's t-test does not reveal
a statistical significance at the 95% confidence level. Likewise, for the surfaces the apparent
trend for reaerosolization is
wood laminate > glass > linoleum.
Again, although there is a trend, the small sample size and high variability result no differences
at the 95% confidence level (Student's t test).
The observed trends appear stronger when looking at the individual wipe type and the impact of
surface type. For the dry paper towel and the wet wipe, the apparent reaerosolization trend is
wood laminate > glass > linoleum.
However, there is still not a statistical significance at the 95% confidence level.
The Lysol® wet wipes yielded widely varying results and reaerosolization was observed even in
the presence of moisture. One possible explanation is that the hydrophobic spores do not initially
interact with the aqueous solution of the wet wipe and are repelled, resulting in reaerosolization
when they are disturbed. Further research would be needed to investigate if this is happening.
The least amount of reaerosolization was observed from linoleum. This could be due to the
surface properties of the linoleum providing better adhesion of the spores to the surface.
Investigation of the surface properties and interaction with the spore would be needed to better
understand the results. One method to do this is atomic force microscopy (AFM). A spore could
be attached to the tip of an AFM probe and the adhesion forces between the spore and the
linoleum surface explored.
Although the small sample size and large variability in the results makes it difficult to conclude
definitively the effect of wipe type and surface type on spore reaerosolization, we can make
some general statements about wiping a spore-contaminated surface.
• Reaerosolization of spores on a surface can occur due to various wiping activities
regardless of the wipe type used.
• The amount of spores reaerosolized is very small compared to the total surface
concentration. The highest reaerosolization fraction observed was less than 0.02% of the
surface contamination level.
• Wiping with a dry paper towel resulted in reaerosolization up to an order of magnitude
higher than with an electrostatic Swiffer® or wet wipe.
• Laminate wood flooring resulted in the highest, reaerosolization fraction, while linoleum
appeared to have the lowest.
13
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Based on the literature review and limited wiping study that was conducted, the following
observations were made on preferred methods to limit reaerosolization of spores by cleaning
activities for indoor environments that may be lightly contaminated.
• Sweeping is not preferred, as it provides mechanical disturbance to reaerosolize spores
but provides no potential for trapping the spores (i.e., decontamination of the area).
• Wiping with an electrostatic Swiffer® wipe or a premoistened wipe is preferred over
wiping a surface with a dry paper towel because a dry paper towel has little potential to
contain removed spores.
• Premoistening a surface with a spray bottle prior to wiping has not been tested and cannot
be recommended at this time as the force of the spray has the potential to resuspend
spores. Premoistening a surface with a spray bottle should be investigated in the future.
3.2 Evaluation of Household Vacuums
The purpose of these experiments was to fill information gaps related to reaerosolization of
spores due to vacuuming. This study was conducted by ECBC in 2016 and included a series of
tests using three commercially available vacuum cleaners of different types (upright, canister
type, and cordless stick type) equipped with HEPA filters (Figure 3-6) to determine spore
removal and reaerosolization from common residential surfaces.
Figure 3-6. Vacuums Used in this Study from Left to Right, Hoover Wind Tunnel, Olympus
Canister, and Hoover Linx
Btk spores were used as a surrogate for B. cmthrcicis and were deposited as a dry aerosol onto test
surfaces. Experiments were conducted on three different surface types that are common in homes
including three carpet types (nylon, wool blend and polyester), linoleum, and slate laminate tile
flooring. A limited set of experiments was also conducted to evaluate spore removal when
vacuuming with the beater bar on nylon carpet and laminate flooring coupons. Additionally,
spore reaerosolization was assessed when the vacuum bag or canister was removed and emptied
after vacuuming. The experimental matrix is shown in Table 3-6.
14
-------
Table 3-6. Evaluation of Household Vacuums Test Matrix
(number of tests conducted per surface type and vacuum cleaner type)
Vacuum Cleaner
Carpet
Slate
Laminate
Linoleum
Bag/
Canister
Emptying
Nylon
Wool
Blend
Polyester
Hoover Wind Tunnel
T-series Rewind Plus
UH70120
(Upright and Bagless)
3 (1 with
beater bar on)
2
2
3 (1 with
beater bar on)
2
2
The Classic CI
Olympus Canister
(With Bag)
3 (1 with
beater bar on)
2
2
3 (lwith beater
bar on)
2
2
Hoover Linx Cordless
Stick BH5 0010
(Cordless and Bagless)
3 (1 with
beater bar on)
2
2
3 (1 with
beater bar on)
2
2
The three main questions addressed in this study include:
1. Can household cleaning methods, such as vacuuming, effectively remove spores from
indoor surfaces?
2. Are the spores reaerosolized during vacuuming?
3. Are the spores reaerosolized while changing or emptying a contaminated vacuum bag or
canister.
3.2.1 Experimental System
All testing was done at ECBC in a 64-cubic meter Biological Safety Level 1+ aerosol chamber
(Figure 3-7). Temperature and RH of the chamber were set using a computer and maintained at
24+5 °C and 35+5% RH. Power receptacles inside the chamber were also controlled by this
computer. HEPA filters were installed at the inlet to filter air entering the chamber to achieve
very low background particle concentrations in the chamber. Similarly, HEPA filters were
installed at the exhaust port to filter all particles leaving the chamber.
The aerosol chamber was cleaned between test runs by exhausting the chamber air through the
HEPA filters, and by pumping HEPA-filtered air into the chamber. The maximum amount of
airflow that can be exhausted from the chamber by the exhaust pump is approximately 2 x 104
L/min. There was also a small recirculation system that removes air from the chamber, passed it
through a HEPA filter, and delivered it back to the chamber. This system was useful when the
aerosol concentration in the chamber needed to be reduced incrementally. In addition, the
chamber walls and floor were wiped down with 10% bleach between runs. If air sampling
showed a significant number of spores, the chamber was further decontaminated with vaporous
hydrogen peroxide.
15
-------
Figure 3-7. ECBC Aerosol Test Chamber
3.2.2 Spore Preparation
The fluidized, milled Btk spores used in this project were procured from Dugway Proving
Ground. Spores were prepared by milling the preparation until particles consisted of single
spores and then adding to the fluidizer. Before testing a known mass of powder (20 milligram
[mg]) was suspended in 10 mL volume of 0.01% Tween "-80 and enumerated by serial dilution
and plating on tryptic soy agar (TSA) plates. Three separate enumerations were performed to
determine the number of viable spores per gram of dry powder. All plating was performed in
duplicate for each dilution.
3.2.3 Coupon Preparation and Inoculation
Coupons, procured by ECBC, consisted of 4x4-ft panels of each of the five flooring materials.
The flooring material was secured to plywood (see Figure 3-8). Reference coupons, 2x1
centimeter(cm), were cut from the same material as the panel. The five flooring materials were
nylon carpet, wool blend carpet, polyester carpet, linoleum type flooring material, and slate
laminate tile flooring. The nylon carpet, 24x24-incommercial carpet tiles, was from the Dean
Flooring Company and purchased from Amazon. The wool carpet pieces were from Learning
Carpets. The remaining flooring, mentioned hereafter, was purchased from The Home Depot.
The polyester carpet coupons were TrafficMaster Gunmetal Ribbed 18xl8-in Caipet Tiles,
Model # CP44N4716PK. The linoleum type flooring materials were HDX 10-ft. wide castle
travel-time linoleum universal flooring Model # HXW70CT10X1AC009TR. The laminate
flooring was Hampton Bay Canyon Slate Clay 8-millimeter (mm) thick x 15-5/8-in (wide) x 50-
3/4-in (length) laminate flooring, Model # 195151.
16
-------
Figure 3-8. A 4x4 Panel of Wool-Blend Carpet
(four pieces were joined together using lxl-ft pieces)
Prior to coupon inoculation, all chamber surfaces were cleaned with bleach wipes and the
chamber was air-washed with sterile air for 30 minutes (20 air exchanges), before exiting
through a HEP A filter. All the test flooring panels were irradiated with UV light for 1-2 hr to
reduce or eliminate background vegetative bacterial contamination.
The fluidized Btk spores were aerosolized onto the flooring panel using a two-fluid pneumatic
sonic nozzle (SRI International, Menlo Park, CA). The nozzle was connected to the powder to be
aerosolized and a compressed air source that exited out through a small annular opening at the
top of the nozzle (see Figure 3-9). The low pressure created in the exit region due to the air flow
caused the powder to be pulled through an axial tube at a very low feed rate due to the Bernoulli
Effect. The desired air to powder mass ratio was 80-100:1. Powder was fed into the nozzle
through a 1/4 -in (internal diameter) stainless tube. The nozzle air pressure can be varied, thereby
varying the dispersing airflow rate. The compressed air (800-1300 L/min) was passed through a
3/8-in (outer diameter) by 5/16-in (internal diameter) orifice. Measurements show that at a flow
rate of 1300 L/min, the disperser produced a vacuum of 12.5 cm Hg.
17
-------
Release
point
Fluidized Btk
spores
Compressed air
supply
Figure 3-9. Two-Fluid Pneumatic Sonic Nozzle used to Aerosolize Spores for Coupon Inoculation
3.2.4 Measurement of Spore Removal and Reaerosolization
3.2.4.1 Spore Removal
Each test was conducted using the three vacuum cleaners per the manufacturer's
recommendations. For spore removal tests, all flooring materials (4x4-ft panels) were vacuumed
in a left to right pattern over the full length of the panel, then repeated a second time at a 90°
angle to the first vacuum pattern. Care was taken to ensure that the vacuum cleaner operator or
other personnel did not step onto the flooring panels. Five panels (nylon carpet, wool blend
carpet, polyester carpet, linoleum, and slate laminate tile) were vacuumed with each vacuum
cleaner with one replicate performed. After vacuuming each panel, the exposed bottom surface
and accessible areas of each vacuum cleaner were scrubbed with Hype Wipe8' disinfectant bleach
towelettes (Current Technologies Inc., Crawfordsville, IN) and allowed to remain in contact for
15 min, followed by 70% ethanol wipe down and a 15 min drying period before use on the next
panel.
Additionally, a limited experiment was conducted to evaluate spore removal when vacuuming
with the beater bar on nylon carpet and laminate flooring panels. The above-mentioned
procedures for vacuuming were followed with the beater bar operating (Note: the Hoover Wind
Tunnel was not equipped with a beater bar and was operated in the carpet setting).
Five 2x1-cm reference coupons, cut from the same material as the panel, were placed on top of
the panel before the inoculation of spores and removed the next morning before the panel was
vacuumed. To estimate the quantity of spores that were not collected by the vacuum cleaner,
core samples, 3x1-in size, were taken from the surface panel used for each test, after the panel
had been vacuumed. Before inoculations, the core samples were cut out and left in place. A total
of five core samples were taken from each surface tested, one from each corner and a fifth one
18
-------
from the middle of the panel. The total number of reference coupons and core samples per
experimental run are shown in Table 3-7.
Table 3-7. Quantity of Samples from Flooring Materials per Test
Tests with All Three Vacuum Cleaners (One Replicate)
Nylon Carpet
Wool Blend Carpet
Polyester Carpet
Linoleum
Slate Laminate
Ref.
Core
Ref.
Core
Ref.
Core
Ref.
Core
Ref.
Core
Coupon
Sample
Coupon
Sample
Coupon
Sample
Coupon
Sample
Coupon
Sample
30
30
30
30
30
30
30
30
30
30
Tests with All Three Vacuum Cleaners - with Beater Bar Operating
Nylon Carpet
Slate Laminate
Ref. Coupon
Core Sample
Ref. Coupon
Core Sample
15
15
15
15
The core samples and reference coupons underwent the same extraction and enumeration
process. The samples were placed in a 50 mL conical tube containing 20 mL 0.01% Tween®-80,
as extraction solution. The samples were sonicated for 10 min and vortexed for 2 min. A 0.1-mL
aliquot of each was plated on TSA plates. The spread-plates were incubated at 37 °C for 16-24
hr. The CFU on plates were counted after overnight incubation using QCount™ (Advanced
Instruments Inc., Norwood, MA). The total spore count from spread plates was estimated as
follows:
CFU x 10 (volume factor) x 10 (dilution factor) (Test [core] samples)
CFU x 10 (volume factor) x (1/dilution plated) (Reference samples)
Spore load removal from the five surfaces was estimated by comparing the spore number per
square foot before vacuuming (from reference coupons) to the number per square foot after
vacuuming (from core samples).
3.2.4.2 Spore Reaerosolization
Spore reaerosolization was assessed by capturing aerosolized spores on a glass fiber filter
connected to a vacuum pump. A 47-mm glass fiber filter (type A/E, Pall Corporation, Port
Washington, NY) was placed in a 47-mm BGI filter holder (Mesa Labs, Butler, NJ) and
connected via a flow rate controller to a vacuum pump. Several of these filter holder systems
were used to collect aerosolized spores. Three filter holder systems were placed at 2-, 4-, and 6-ft
height in the aerosol chamber. Eight filter holder systems were placed at each corner of the 4x4-
ft panel, four at 1-2 inches from the floor and four at 1 ft above the floor. The filters were placed
in the holders after the overnight spore settling period but before vacuuming the panels and were
collected after vacuuming of all five panels/material types. The pumps operated with the same
filter for a total time of 75 min at a flow rate of 15 L/min. The pumps were turned off and did not
sample during panel replacement and vacuum decontamination.
19
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Additionally, two filter holder systems were placed 1-ft above the floor in the area close to where
the vacuum bag or canister was removed and emptied after vacuuming. The bag from the
Olympus canister was inverted over a biohazard bag to empty the contents. The canisters from
the Hoover Wind Tunnel and Hoover Linx were emptied by releasing the bottom lid over a
biohazard bag. The vacuum pumps ran for 10 minutes at a flow rate of 15 L/min during bag and
canister emptying. Table 3-8 lists filter locations for each experimental run.
Table 3-8. Filter Identification Number and Locations Per Test
6 ft
Height
4 ft Height
2 ft Height
Surface Corners
1-2 in Height
1 ft Height
above floor
Corners
Vacuum
Emptying
Filter 1
Filter 2
Filter 3
Filters 4-7
Filters 8-11
Filters 12-13
The collected filters were placed in a 50-mL tube containing 0.01% Tween®-80 and processed in
the same manner as the reference coupons and core samples. The spore numbers (CFU) were
quantified by dilution plating.
3.2.5 Results
3.2.5.1 Depositions and Surface Concentrations
In preliminary baseline experiments, 1 gram of powder was aerosolized and reference coupons
(2x1 cm) were placed across a 4x4-ft panel to determine spore loading density per coupon. The
spore deposition averaged 7-9 logs CFU/ft2for all material types/panels.
3.2.5.2 Spore Removal During Vacuuming
Spore removal data for three vacuum cleaners from five surfaces are summarized in Figure 3-10.
The data show spore removal, as a percent of initial spore load (CFU) inoculated. Spore percent
removal was calculated as a ratio of spore number recovered from reference coupons vs. core
samples for each material type. This calculation considers the number of spores removed from
the surface but does not incorporate spores captured by the vacuum itself.
20
-------
Spore Removal During Vacuuming per Material Type
100
¦ Nylon Carpet ¦ Polyester Carpet ¦ Wool Blend Carpet ¦ Linoleum ¦ Slate Laminate
Hoover Wind Tunnel Hoover Linx Olympus Canister
Vacuum Cleaner Type
Figure 3-10. Spore Removal from Floor Surfaces by Vacuum Cleaner Type
The results per material type show spore removal of approximately 70-90% from all five
surfaces. The results for the slate laminate using the Olympus Canister vacuum was the only
deviation (showing significantly lower spore removal). No other significant differences were
observed for the three vacuum cleaner types.
The results for the beater bar tests are summarized in Figure 3-11. Spore removal with the beater
bar on ranged from approximately 40-88%. This percentage is lower than removal without the
beater bar, which was approximately 70-95%. No replicate tests were conducted for the beater
bar tests, so no error bars are included to show the variability in the data.
21
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Spore Removal During Vacuuming With and Without Beater Bar On
(Two Material Types)
¦ Nylon Carpet ¦ Slate Laminate
With Beater Bar Without Beater Bar With Beater Bar Without Beater Bar With Beater Bar Without Beater Bar
Hoover Wind Tunnel Hoover Linx Olympus Canister
Vacuum Cleaner Type
Figure 3-11. Spore Removal from Two Floor Surfaces with and Without the Beater Bar On
3.2.5.3 Spore Reaerosolization during Vacuuming
Reaerosolization of all five flooring materials was assessed simultaneously. These samples
demonstrate the magnitude of resuspension potential within the test chamber, by each vacuum
type. The pumps operated with the same filter for a total time of 75 min at a flow rate of 15-
L/min for each experimental run. The results for the four air samples collected from the corners
of the panel (1-2 inches from the floor) are summarized in Figure 3-12 and show a consistent 5-
6 log CFU captured per air sample for each vacuum type.
22
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Reaerosolization During Vacuuming Captured at the Four Corners of Panels
(1-2 Inches from the Floor)
¦ Corner 1 ¦ Corner 2 ¦ Corner 3 ¦ Corner 4
Hoover Wind Tunnel Hoover Linx Olympus Canister
Vacuum Cleaner Type
Figure 3-12. Spore Reaerosolization during Vacuuming at Floor Level Corners of Panels
Figure 3-13 summarizes the data from the four air samples collected from the corners of the
panel (1 ft from floor). The number of spores aerosolized at 1 ft from the floor were comparable
to those captured very close to floor.
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Reaerosolization During Vacuuming Captured at 1-ft Height at the Four
Corners of Panels
7.00
¦ 1-ft Corner 1 ¦ 1-ft Corner 2 ¦ 1-ft Corner 3 ¦ 1-ft Corner 4
Hoover Wind Tunnel Hoover Linx Olympus Canister
Vacuum Cleaner Type
Figure 3-13. Spore Reaerosolization during Vacuuming at 1-ft Distance from the Floor
Figure 3-14 summarizes the spore capture by the filters placed at three heights, 2, 4, and 6 ft, to
assess the vertical extent of spore reaerosolization and the ability of spores to reach breathing
zone height. The results span 3-6 log CFU captured per air sample at three heights with the
Hoover Wind Tunnel and the Olympus Canister showing slightly less reaerosolization than the
Hoover Linx. There does not appear to be a significant difference in spore concentration per
vacuum at the three heights above the floor, with the exception of the 2 ft height for the Hoover
Wind Tunnel.
24
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Reaerosolization During Vacuuming Captured at Three Heights
7.00
¦ eft ¦ 4 ft ¦ 2 ft
Hoover Wind Tunnel Hoover Linx Olympus Canister
Vacuum Cleaner Type
Figure 3-14. Spore Reaerosolization during Vacuuming at Three Heights
3.2.5.4 Spore Reaerosolization during Bag and Canister Emptying
The vacuum pumps operated for 10 minutes at a flow rate of 15-L/min during each experimental
run. Assessment of spore reaerosolization during bag or canister emptying was conducted at the
end of each experimental run by placement of filter holder systems at two locations at 1 ft from
the floor in the area close to where the vacuum bag/canister was removed and emptied. The
results are summarized in Figure 3-15 and show 4-5 log CFU per air sample were aerosolized
during bag emptying. A consistent level of spores was aerosolized for all three vacuum cleaner
types while emptying the bag or canister.
25
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6.00
Reaerosolization During Emptying of Vacuum Bag or Canister
i Location 1 ¦ Location 2
u
DO
o
5.00
4.00
3.00
2.00
1.00
0.00
Hoover Wind Tunnel
Hoover Linx
Olympus Canister
Vacuum Cleaner Type
Figure 3-15. Spore Reaerosolization during Bag or Canister Emptying
3.2.6 Discussion
The tests previously described serve as a basis for investigating the capture and reaerosolization
of spores by several types of commonly used household vacuums. This information is important
in determining which types of cleaning activities are preferred. For spore removal tests
conducted with three vacuum types, the results per material type (nylon carpet, wool blend
carpet, polyester carpet, linoleum, and slate laminate tile) show spore removal of approximately
70-90% from all five surfaces. The results for the slate laminate using the Olympus Canister
vacuum was the only deviation (showing significantly lower spore removal). However, this spore
removal does not account for displacement and reaerosolization of spores and cannot be reported
accurately as removal by the vacuuming process alone as the vacuum canisters/bags were not
analyzed for spore capture.
When evaluating low-tech remediation methods for removing low level biological contamination
from indoor surfaces, the focus should be on the ability of the cleaning technique (i.e.,
vacuuming) to both remove the contaminant and prevent it from being reaerosolized and
presenting an inhalation hazard to the resident or business occupant. Reaerosolization was also
assessed during the vacuum spore removal tests using stationary filter samplers at various
26
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heights above the floor surface. The results for the four air samples collected from the corners of
the panel (1-2 inches from the floor) were comparable to those captured from the samples placed
1 ft from the floor. Spore capture by the filters at these heights was 5-6 log CFU. This spore
capture demonstrates the potential for the vacuuming process to reaerosolize spores off the
flooring surface by mechanical action. The results for the filters placed at three heights (2, 4, and
6 ft) span 3-6 log CFU with the Hoover Wind Tunnel and Olympus Canister showing slightly
less reaerosolization than the Hoover Linx. Additionally, an assessment of spore reaerosolization
during bag or canister emptying was conducted at the end of each experimental run by placing
filter holder systems at two locations at 1-ft from the floor in the area close to where the vacuum
bag/canister was removed and emptied. These results show that approximately 4-5 log CFU per
air sample were aerosolized during bag emptying for the three vacuum cleaner types. These data
confirm a significant amount of spore reaerosolization during vacuuming and bag/canister
emptying. This factor is important in considering whether to use household vacuums with
canisters or bags to remove biological contamination. Significant reaerosolization was observed
during vacuuming regardless of the vacuum type with thousands to millions of CFUs detected in
the air near the breathing zone height (6 ft), implying that the collection efficiency of household
vacuums is poor for spores deposited on flooring surfaces. Since all flooring types were assessed
simultaneously, it cannot be determined if reaerosolization varies by flooring type.
A limited follow-up experiment was conducted to assess spore removal only (not
reaerosolization) when vacuuming with the beater bar on nylon carpet and laminate flooring
coupons. All tests up to this point had been conducted without the vacuum beater bar. Results
from the beater bar tests indicate spore removal from the surface ranging between 40-85%. It is
not clear if these lower percent removal results are due to less recovery by the vacuums or
displacement of spores due to the beater bar operations. Therefore, additional studies to quantify
the fate and migration of contaminated surfaces are recommended.
Reaerosolization from the mechanical motion of the vacuuming itself as well as during
bag/canister emptying may pose an inhalation hazard to persons conducted the vacuuming.
Future experiments should incorporate a mass balance approach for calculation of spore recovery
by the vacuums, reaerosolization, and displacement and re-deposition onto vertical and/or
horizontal surfaces.
Information gaps in understanding how vacuum cleaners can be used for low-tech remediation
still exist. Future experiments designed to evaluate removal, reaerosolization, and migration of
spores resulting from vacuuming with high-end commercial vacuums, robotic floor cleaners, wet
vacuums, and beater bar functions may help fill some of these information gaps.
4 Quality Assurance/Quality Control
4.1 Literature Review
The literature search was conducted through a methodical iterative process by which results of a
previous search and review of linking citations informed the next search etc. The search was
conducted primarily through internet searches and through the EPA's library resources. Internet
search engines included but were not necessarily limited to Web of Science, Google Scholar, and
27
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Science Direct. The sources of data were primarily nationally or internationally recognized
scientific publications that went through a peer review process. The standard of these nationally
or internationally recognized scientific publications was primary research and review articles in
peer reviewed journals. Other peer reviewed sources include dissertations/theses,
government/industry reports, and scientific manuals.
4.1.1.1 Source Selection
An assessment of each information source (article, report, website, etc.) was conducted as
depicted in Figure 4-1 using the following guidelines and questions:
• Applicability: The extent to which the information is relevant for the intended use.
- How useful or applicable is the scientific theory applied in the study to the
intended use of the analysis?
- How relevant are the study's purpose, design, outcome measures, and results to
the intended use of the analysis?
• Soundness: The extent to which the scientific and technical procedures, measures,
methods, or models employed to generate the information are reasonable for and
consistent with the intended application.
- Is the purpose of the study reasonable and consistent with its design?
- Is the study based on sound scientific principles?
- To what extent are the procedures, measures, methods, or models employed to
develop the information reasonable and consistent with sound scientific theory or
accepted approaches?
- How do the study's design and results compare with existing scientific theory and
practice?
- Are the assumptions, governing equations, and mathematical descriptions
employed scientifically and technically justified?
- How internally consistent are the study's conclusions with the data and results
presented?
• Clarity and Completeness: The degree of clarity and completeness with which the data,
assumptions, methods, quality assurance, and analyses employed to generate the
information are documented.
- To what extent does the documentation clearly and completely describe the
underlying scientific theory and the analytical methods used?
- To what extent have key assumptions, parameter values, measures, and
limitations been described and characterized?
- To what extent are the results clearly and completely documented as a basis for
comparing them to results from other similar tests?
28
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- If novel or alternative theories or approaches are used, how clearly are they
explained and the differences from accepted theories or approaches highlighted?
- Is the complete data set accessible, including metadata, data-dictionaries, and
embedded definitions (e.g., codes for missing values, data quality flags)?
- To what extent are the descriptions of the study design clear, complete, and
sufficient to enable the study or survey to be reproduced?
- Has the sponsoring organization(s) for the study/information product and the
author(s) affiliation(s) been documented?
- To what extent are the procedures for quality assurance and quality control of the
data documented and accessible?
• Uncertainty and Variability: The extent to which variability and uncertainty (quantitative
and qualitative) related to results, procedures, measures, methods, or models are
evaluated and characterized.
- To what extent have appropriate statistical techniques been employed to evaluate
variability and uncertainty?
- To what extent have the sensitive parameters of models been identified and
characterized?
- To what extent do the uncertainty and variability impact the conclusions that can
be inferred from the data and the utility of the study?
- What are the potential sources and effects of error and bias in the study design?
- Did the study identify potential uncertainties such as those due to inherent
variability in environmental and exposure-related parameters or possible
measurement errors?
• Evaluation and Review: The extent of independent verification, validation, and peer
review of the information or of the procedures, measures, methods, or models.
- To what extent has there been independent verification or validation of the study
method and results?
- What were the conclusions of these independent efforts, and are they consistent?
- To what extent has independent peer review of the study method and results been
conducted, and how were the conclusions of this review considered?
- Has the procedure, method or model been used in similar peer reviewed studies?
- Are the results consistent with other relevant studies?
- In the case of model-based information, to what extent has independent evaluation
and testing of the model code been performed and documented?
All information sources that passed the "cite or cite with explanation" evaluation shown in
Figure 4-1 were subjected to further assessment. The following factors were also considered:
• Focus: The extent to which the work addresses the area of inquiry under consideration.
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- Is the work germane to the issue?
- Does it contribute to the understanding of the issue?
• Verity: The extent to which data are consistent with accepted knowledge in the field or, if
not, the new or varying data are explained within the work.
- Do the data fit within the context of the literature?
- Is the information intellectually honest and authentic?
• Integrity: The degree to which data are structurally sound and present a cohesive story.
- Is the design or research rationale logical and appropriate?
- Is the information clear, concise, and well presented?
• Rigor: The extent to which the work is important, meaningful, and non-trivial relative to
the field.
- Does the work exhibit significant depth of intellect rather than superficial or
simplistic reasoning?
The Literature Assessment Factor Rating form was completed for all cited sources to indicate the
degree to which the acceptance criteria have been met. These forms as well as references
available as electronic copies are stored in an EPA SharePoint directory.
30
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Is it applicable7
Yes
No
T
Is it peer-reviewed?
H
Yes
No
T
Address topics not in peer-
reviewed literature?
Provide useful background
information?
Support conclusions found in
peer reviewed literature7
Yes to one
or more.
No to all.
- Do not cite.
Is it sound?
Is it clear?
Is it complete''
Does it document uncertainty
and variability?
Yes to all.
No to one or
more.
I
Cite.
Cite with
explanation.
Figure 4-1. Flowchart for Information Source Evaluation
4.1.1.2 Data Quality Objectives
Our objective was to cite literature that conforms in full to all five criteria in section 3.1.
However, from previous search efforts, we learned that the preponderance of literature on some
topics does not fully conform to all aspects of the outlined criteria, specifically, in the case of
non-peer reviewed sources. Non-peer reviewed references addressing topics not found in the
peer reviewed literature, providing useful background information, or corroborating conclusions
in the peer reviewed literature, were cited with clear explanation. A clear explanation was also
offered for references that did not fully conform to one of the other criteria. However,
applicability was deemed the most important criterion for inclusion as data that have no
applicability did not need to be tested further for quality.
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4.1.1.3 Quality Assurance/Quality Control Checks
Twenty percent of all citations were quality-checked against the source for correctness and
completeness with zero tolerance for errors. A minimum of twenty percent of data used to
generate human activity forces was checked against the source citation.
4.2 Evaluations
4.2.1 Bench-Scale Wiping Experiment
4.2.1.1 Quantitative Acceptance Criteria
The critical measurements for this project included the enumeration of spores recovered from
the surface of the coupons and AGIs. Table 4-1 lists the quantitative acceptance criteria for
critical measurements. Failure to provide a measurement method or device that met these goals
resulted in a rejection of results derived from the critical measurement.
Table 4-1. Critical Measurement Acceptance Criteria
Critical
Measurement
Measurement
Device
Accuracy
Precision
Detection
Limit
Completeness
Plated Volume
Pipette
±2%
± 1%
N/A
100%
CFU/plate
Hand counting
± 10% (between
2 counters)
±5%
1 CFU
100%
Substantial effort was expended to ensure that samples and measured parameters were
representative of the media and conditions being measured. All data were calculated and reported
in units that are consistent with similar measurements from other organizations to allow for
comparability of data among organizations. Data quality indicators (DQIs) for precision and
accuracy are based on prior knowledge of the measurement system employed and method
verification studies, which include the use of replicate samples and duplicate analyses.
Definitions of DQIs are given below.
Completeness: a measure of the amount of verified data obtained from a measurement
system compared to the amount of data that were expected to be obtained under normal
conditions. Completeness was assessed by reviewing laboratory data logs and laboratory
logbooks to ensure that all data were verified within established indicators.
Accuracy: the degree of agreement of measurements (or an average of measurements) with
an accepted reference or true value. Accuracy is a measure of the bias or systematic error in a
system.
Precision: a measure of mutual agreement among individual measurements of the same
property, usually under prescribed similar conditions. Precision is best expressed in terms of the
standard deviation. Various measurements of precision exist depending on the prescribed similar
conditions.
Representativeness: the degree to which data accurately and precisely represent the
characteristics of a population, process, or environmental condition, or parameter variations at a
32
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sampling point. Representativeness was assessed by the collection of appropriate numbers as
outlined in the Quality Assurance Project Plan (QAPP).
Comparability: the confidence with which one data set can be compared to another.
Comparability of experimental and numerical data will be ensured by using standard comparison
and reporting methods. All data were presented in specified and documented units.
Comparability was ensured by using approved standard operating procedures (SOPs) for all
instrumentation.
4.2.1.2 Procedures to Assess QA Objectives
Uniformity of the test materials is a critical attribute to assure reliable test results. Uniformity
was maintained by obtaining a large enough quantity of material that multiple coupons were
constructed with presumably uniform characteristics. Samples were stored away from other
samples that could contaminate them.
Supplies and consumables were acquired from reputable sources and were National Institute of
Standards and Technology (NIST) traceable when possible. Supplies and consumables were
examined for evidence of tampering or damage upon receipt and prior to use. Supplies and
consumables showing evidence of tampering or damage were not be used. Project personnel will
check supplies and consumables prior to use to verify that they meet specified task quality
objectives and do not exceed expiration dates.
Quantitative standards do not exist for biological agents. Quantitative determinations of
organisms in this investigation do not involve the use of analytical measurement devices. Rather,
CFU was enumerated manually and recorded. Critical QC checks are shown in Table 4-2. The
acceptance criteria were set at the most stringent level that can be routinely achieved.
RTFs microbiology laboratory included positive controls and procedural blanks along with the
test samples in the experiments so that well-controlled quantitative values were obtained. SOPs
using qualified, trained, and experienced personnel were used to ensure data collection
consistency. The confirmation procedure, controls, blanks, and method validation efforts were
the basis of support for biological investigation results.
Potential confounding organisms were excluded or controlled by sterilization of test coupons,
ADA components, and materials used to analyze samples and by use of aseptic technique,
procedural blanks, and a pure initial culture. Aseptic technique was used to ensure that the
culture remained pure. Procedural blanks (negative control) were run in parallel with the
contaminated materials to identify any confounding organisms.
Specific procedures were put in place to prevent cross-contamination. Adequate cleaning of all
common materials and equipment was critical in preventing cross-contamination. Proper
procedures for handling, cleaning, and decontamination of materials and equipment are
described in the laboratories miscellaneous operating procedures.
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Table 4-2. Quality Control Checks
Sample Type
Purpose
Acceptance
Criteria
Corrective Actions
Frequency
Procedural
blank
(negative
control)
Determine extent
of sample box and
wiping mechanism
contamination
No biological
particles detected
If detected, discuss
potential impact on
results with EPA;
repeat test if necessary
after identifying and
removing source of
contamination
1 per test day
Laboratory
materials
Verify sterility of
sterilized test
coupons, ADA
components,
PBST, and
materials used to
analyze viable
spore count
No detectable
spores
Determine source of
contamination and
remove
1-3 per
material
per test
Blank tryptic
soy agar
sterility control
(plate
incubated, but
not inoculated)
Controls for
sterility of plates
No observed
growth following
incubation
All plates are incubated
prior to use, so any
contaminated
ones will be discarded
Each plate
Stainless steel
reference
coupons
Used to determine
extent of
inoculation on
coupon
1E6 CFU ±0.5 log
Outside target range:
discuss potential impact
on results with EPA;
correct loading
procedure for next test
and repeat depending
on decided impact
2 per
deposition
Used to determine
drift in the MDI
CFU recovered
from two reference
coupons must be
within ±0.5 log of
each other
Reject results and
repeat test
Biological
samples
Controls for
outliers in colony
growth
CFU counts
between 30 and
300 AND each
CFU count within
50% of the other
two replicates
Dilute or filter plate if
CFU outside criteria
Each sample
Notes:
PBST = Phosphate buffered saline with Tween®20
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4.2.2 Evaluation of Household Vacuums
4.2.2.1 Quality Control Requirements
In the measurement of spore removal, experimental error or variability can be introduced by
inaccurate measurement of volume of spore suspensions being applied and non-consistent
vacuuming between experimental runs. The data quality objectives for test measurements,
provided in Table 4-3, limited the error introduced into the evaluation.
4.2.2.2 Instrument/Equipment Testing, Inspection, and Maintenance
The equipment used during the evaluation was maintained and operated according to the quality
requirements and documentation of the evaluation facility. Equipment included biological safety
cabinets (BSC), pipettes, incubators, and orbital shakers. There were no critical experimental
parameters of the BSC and orbital shaker equipment that required calibration. Pipettes are
calibrated every six months. The laboratory staff checked the temperature of the incubator daily
and the results were entered into a registered facilities data collection form. The incubators are
calibrated semi-annually on a schedule maintained by the ECBC.
Prior to use and following the frequency specified in Table 4-3, all calibrated equipment was
checked by the user to verify the equipment was within calibration.
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Table 4-3. Quality Objectives for Test Measurements
Test Measurement
Specifications
Instrument Specification
Parameter Measured
Unit
Allowable Test
Measurement Tolerance
Instrument
Instrument
Calibration/C ertification
Instrument Calibration
Frequency
Expected Instrument
Tolerance
Corrective Action if
Expected Instrument
Tolerance Unattained
Volume
(i.e.,
spike or
dilution
volume)
HL
± 10 %
Micro-
pipette
Verified as
calibrated at time of
use by supplier-
recalibrated by
gravimetric
evaluation of pipette
performance by
supplier
Every 6
months
± 10 %
Replace with
calibrated and
sufficiently
accurate
micropipette
Weight
g
±0.1
Balance
Calibrated monthly
and annually
serviced under a
PMA
Every 12
months
±0.ig
Replace with
calibrated and
sufficiently
accurate balance
RHin
chamber
%
± 20 %
full scale
Hygrometer
NIST traceable
certification and/or
checked against
NIST traceable
hygrometer
Once per
quarter
± 0.5 %
from 25
% to 95
% over
the range
of 5 °C
to 55 °C
Replace with
calibrated and
sufficiently
accurate
hygrometer
Tempera
ture
EC
± 2 °C
Thermo-
meter
Checked against
NIST traceable
thermometer
Once
prior to
testing
±0.5 °C
at 25 °C
Replace with
calibrated and
sufficiently
accurate
thermometer
Time
hr
2
seconds/
hr
Timer
Check against NIST
traceable standard
Once per
quarter
2
seconds/
hr
Replace with
calibrated and
sufficiently
accurate timer
Colony
N/A
100%
colonies
must be
counted
QCount™
Calibrated once a
year
Once per
month
1-2 small
colonies
Manually count
missed colonies
Notes:
|iL = Microliter
NIST = National Institute of Standards and Technology
PMA = Preventative Maintenance Agreement
36
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The facility has SOPs for the calibration of all instruments. A list of all instruments requiring
calibration is maintained in a database and calibrations are scheduled by designated staff. All
instruments used at the time of evaluation were verified as being certified, calibrated, or
validated. Calibration of instruments was done at the frequency shown in Table 4-3.
4.2.2.3 Inspection/Acceptance of Supplies and Consumables
Supplies and consumables were acquired from reputable sources. The source and purity of
reagent grade chemicals and standards were documented. Supplies and consumables were
examined for evidence of tampering or damage upon receipt and prior to use. The expiration
dates were noted and recorded. Table 4-4 details the specifications and acceptance criteria for
common consumables used in this evaluation.
Table 4-4. Specifications and Acceptance Criteria for Consumables
Description
pH
CFU/Spread Plate
Corrective Action if
Acceptance Criteria are
not attained
Water, sterile-
filtered, culture
tested
5.0 to 7.0
0 CFU
Seek different vendor
Tween®-80
6.0 to 8.0
0 CFU
Seek different vendor
Tryptic Soy Agar
NA
0 CFU
Seek different vendor
Solutions were prepared following ECBC protocols and were documented in reagent preparation
forms. These forms include preparation instructions; suppliers, catalog numbers, lot numbers,
and expiration dates for components; calculated and actual amounts used; and specific equipment
used with calibration information. A lot number and expiration date are assigned to each reagent
per ECBC requirements. All documents were initialed and dated. Supplies and consumables
showing evidence of tampering or damage were not used. Coupons with anomalies on the test
surface were rejected from use.
4.2.2.4 Data Management
Data acquisition during the evaluation included proper recording of the procedures used in the
testing to assure consistency in the evaluation and adherence to the QAPP; documentation of
sampling/testing conditions; and recording of analytical results and evaluation conditions.
Data acquisition was carried out either electronically by the data logger (e.g., temperature, RH,
and time) or manually by ECBC test personnel. Manually-acquired data were recorded
immediately in a consistent format throughout the evaluation. All written records were in ink,
and any corrections to recorded data were made with a single line through the original entry. The
correction was then entered, initialed, and dated by the person making the correction. Any non-
obvious correction included a reason for the correction. Strict confidentiality of evaluation data
will be maintained.
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Whether collected manually or electronically, relevant data were entered into an electronic
spreadsheet set up to organize the data in a clear and consistent manner. The accuracy of entering
manually recorded data into the spreadsheets was checked at the time the data were entered, and
a portion of the data were checked by the ECBC QA Manager as part of the audit of data quality.
5 Summary and Conclusions
A thorough literature review of previous work conducted on reaerosolization of spores/particles
during residential cleaning activities was conducted. Research on the reaerosolization of particles
due to vacuuming, walking, and sweeping constitutes most of the work done on indoor
reaerosolization. Unfortunately, most studies on reaerosolization and transport of particles in
indoor environments have focused primarily on allergens and dust, only some of which is in the
size range of B. anthracis spores. Even when discussing particles of the same size, there is
question about applicability of these data to B. anthracis reaerosolization because of the
differences in density and surface chemistry between the particles. However, findings from the
literature review did indicate gaps on surface wiping and vacuuming and warranted limited
laboratory studies to evaluate the reaerosolization potential of common household activities that
could be deployed as low-tech remediation methods for B. anthracis contamination, specifically
surface wiping and vacuuming.
In the surface wiping study, reaerosolization of Btk spores was measured during wiping for all
surface types and wipe types used in this study. The findings from this study indicate that it is
likely that surface adhesion and texture properties influence reaerosolization. Roughness
asperities in the spore and surface material determine the contact area for adhesion. Electrostatic
and Van der Waals forces play a role in adhesion and can deform the asperities of the materials,
thereby increasing contact area. The limited data set from this study indicates that surface type
has an effect, with laminate wood flooring showing the highest reaerosolization for the three
types tested (wood laminate, smooth glass, and textured linoleum). However, the small sample
size (three for each wipe and surface type) and broad variability in surface depositions and
reaerosolization results did not result in statistical significance at the 95% confidence level.
Further studies are needed to demonstrate statistical significance between surface types.
The wiping material probably influences reaerosolization as well. The limited data set from this
study indicated that wiping with a dry paper towel resulted in the highest reaerosolization while
an electrostatic Swiffer® dry wipe and Lysol® pre-moistened wet wipe indicated similar yet
lower likelihoods for reaerosolization. However, due to the small sample size and variability in
results, there was no statistical significance at the 95% confidence level. Further studies are
warranted to investigate the quantitative differences in reaerosolization among wiping materials.
The study to determine spore removal and reaerosolization using three different vacuum cleaners
also highlighted several important observations. The additional assessment of spore
reaerosolization during bag or canister emptying was particularly important in identifying
cleaning activities that may lead to reaerosolization.
The results per material type show spore removal of approximately 10-90% from all five
surfaces. The results for the slate laminate using the Olympus Canister vacuum was the only
deviation (showing significantly lower spore removal). The overall findings from the
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reaerosolization study indicate that reaerosolization from the mechanical motion of the
vacuuming itself as well as during bag/canister emptying may pose an inhalation hazard to
persons conducting the vacuuming. Significant levels of spores were detected at breathing zone
height. Future experiments should incorporate a mass balance approach for calculation of spore
recovery by the vacuum cleaners, reaerosolization, and displacement and re-deposition onto
vertical and/or horizontal surfaces to allow for a better understanding of the spore migration
when vacuuming and steps that can be taken to reduce contamination and inhalation exposure
risks. Additionally, the limited experiment conducted with the beater bar indicated a lower
percentage of spore removal than removal without the beater bar. It is not clear if this lower
removal is due to less recovery by the vacuums or displacement of spores due to the beater bar
operations.
When combining results from the literature review and laboratory experiments, some general
observations can be made for low-tech remediation methods used to remove biological
contamination from indoor surfaces. Sweeping and dry wiping are not preferred as significant
reaerosolization can occur. Electrostatic wipes such as the Swiffer® appear to be at least as
effective as wet wipes in limiting reaerosolization. Further reaerosolization data from wiping
methods are needed to expand on this finding. In addition, vacuuming was shown to reaerosolize
spores, and this reaerosolization may pose an inhalation hazard as spores reached breathing zone
height during this study. Large variability in reaerosolization has been shown among vacuum
cleaner models. It is not clear if the same level of variability exists between vacuum cleaners of
the same model. Future experiments designed to evaluate removal, reaerosolization, and
migration of spores resulting from vacuuming with high-end commercial vacuums, robotic floor
cleaners, wet vacuums, and beater bar functions may help provide information on how vacuum
cleaners can be used for low-tech remediation.
The findings from this literature review and laboratory experiments could be combined with data
collected from additional experiments to develop a qualitative risk-based assessment of the
reaerosolization potential of B. anthracis spores from low-tech remediation methods.
39
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6 References
Buckley, P., et al. 2012. Genetic Barcodes for Improved Environmental Tracking of an
Anthrax Simulant. Applied and Environmental Microbiology 78(23): 8272-8280.
Calfee, M.W., et al. 2014. Evaluation of sampling methods for Bacillus spore-contaminated
HVAC filters. Journal of Microbiological Methods 96:1-5.
Carrera, M., et al. 2007. Difference between the spore sizes of Bacillus anthracis and other
Bacillus species. Journal of Applied Microbiology 102(2):303-312.
Emanuel, PA., et al. 2012. Detection and Tracking of a Novel Genetically Tagged Biological
Simulant in the Environment. Applied and Environmental Microbiology 78(23): 8281-8288.
Gore, R.B., et al. 2006. High-efficiency vacuum cleaners increase personal mite allergen
exposure, but only slightly. Allergy 61(1): 119-123.
Knibbs, L.D., He, C., Duchaine, C., and Morawska, L. 2012. Vacuum cleaner emissions as a
source of indoor exposure to airborne particles and bacteria. Environmental Science &
Technology 46(l):534-542.
Lee, S.D., S.P. Ryan, and E.G. Snyder. 2011. Development of an Aerosol Surface Inoculation
Method for Bacillus Spores. Applied and Environmental Microbiology. 77(5): 1638-1645.
Lehtonen, M., Reponen, T., and Nevalainen, A. 1993. Everyday activities and variation of fungal
spore concentration in indoor air. InternationalBiodeterioration & Biodegradation 31(1):25—39.
Mitchell, R.N. and Eutsler, B.C. 1967. A study of beryllium surface contamination and
reaerosolization. Surface Contamination Symposium Proceedings. Gatlinburg, TN; Pergamon
Press, NY. 349-352.
Qian, J., Peccia, J., and Ferro, A.R. 2014. Walking induced particle reaerosolization in indoor
environments. Atmospheric Environment 89:464-481.
Rastogi, V.K., Smith, L.S., Wallace, L. and Kesavan, J. 2016. Evaluation of carpet steam and
heat cleaners as biological sampling devices. Technical Report ECBC-TR-1357.
Sehmel, GA. 1980. Particle reaerosolization: a review. Environment International 4:107-127.
Tang, K.M., et al. 2004. Evaluation of vacuum and wet-wipe methods for removal of World
Trade Center dust from indoor environments. Journal of the Air & Waste Management
Association 54(10): 1293-1298.
Thatcher, T.L. and Layton, D.W. 1994. Deposition, reaerosolization, and penetration of particles
within a residence. Atmospheric Environment 29(13): 1487-1497.
40
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USEPA (Environmental Protection Agency). On the Use of Bacillus thuringiensis as a Surrogate
for Bacillus anthracis in Aerosol Research. U.S. Environmental Protection Agency, Washington,
DC, EPA/600/R-12/596. 2012.
Van Strien, R.T., et al. 2004. Do central vacuum cleaners produce less indoor airborne dust or
airborne cat allergen during and after vacuuming, compared with regular vacuum cleaners?
Indoor Air 14(3): 174-177.
Weis, C.P., et al. Secondary aerosolization of viable Bacillus anthracis spores in a contaminated
US Senate Office. Journal of the American Medical Association 288(22):2853-2858.
41
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Appendices
42
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Appendix A - Procedure for Fabrication of 14" X 14", 28" X 28", and 42" X
42" Material Coupons
This document describes the procedure for construction of 14" x 14", 28" x 28", and 42" x 42"
material coupons. The purpose of this of this document is to ensure consistent manufacture of
these coupons. This document is split into four sections, each section dedicated to one material
type. Sections 1.0 to 4.0 present the methods for fabricating coupons from carpet, drywall,
laminate, and glazed ceramic tile, respectively.
1.0 CARPET
1.1 Equipment
• Beaulieu Solutions Laredo Sagebrush Loop Carpet from Home Depot, SKU 409921
• 7/16" - Trubord Oriented Strand Board (OSB) from Home Depot, SKU 386081
• Roberts Carpet Adhesive from Home Depot, SKU 763258
• V2" staples
• Safety Glasses
• Cut-Resistant Gloves
• Staple Gun
• Safety Razor Utility Knife
• Table saw
• Tape measure
• Straight edge
1.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Cut a 13.5" x 13.5", 27.5" x 27.5", or 41.5" x 41.5" square of OSB using a table saw.
3. Cut a 18" x 18", 32" x 32", or 46" x 46" square of carpet using a safety razor utility knife.
4. Place the carpet square with the backing side up on a table. Center the OSB on the carpet
square and use a piece of scrap OSB to mark the corners of the carpet square for trimming
(Figure A-l).
A-l
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12/06/2012
Figure A-1. OSB Centered on the Carpet Square
5. Trim the corners of the carpet (Figure A-2).
12/06/2012
Figure A- 2. Corners of the Carpet Square Trimmed
6. Apply carpet adhesive to the rough side of the OSB and place adhesive side down on the
back of the trimmed carpet square (Figure A-3).
A-2
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Figure A- 3. Carpet Adhesive on OSB
7. Fold the carpet onto the OSB and staple in place (Figure A-4). The front of the finished
carpet coupon will look like Figure A-5.
Figure A- 4. Carpet Folded onto OSB and Stapled in Place
A-3
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Figure A- 5. Front of Finished Carpet Coupon
DRYWALL
2.1 Equipment
• Safety Glasses
• Cut-Resistant Gloves
• Table saw
• W* Gold Bond Drywall from Lowes, SKU 34137
• Sheetrock Brand Drywall Mud from Home Depot, SKU 258717
• Putty Knife
• Joint Tape
• Sanding Block
• KILZ latex primer
• Behr Premium Plus Interior Flat White Latex
• Paint brushes
• Paint Rollers
• Tape measure
2.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Cut a 14" x 14", 28" x 28", or 42" x 42" section of drywall from a drywall sheet using a table
saw.
3. Apply a skim coat of joint compound about 1.5" from each cut edge of the coupon to the cut
edge on the front side using a putty knife.
A-4
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4. Using two-inch joint tape, apply one half of the tape (utilizing the factory fold) to the front
side of the coupon over the joint compound.
5. Apply a second skim coat of joint compound over the tape using a putty knife.
6. Allow to dry.
7. After the compound has dried, apply a skim coat of joint compound to the back side of the
coupon approximately 1" from the cut edge of the coupon to the cut edge using a putty knife.
8. Fold the joint tape over the cut edge to the backside of the coupon (it should extend
approximately 1/2" over the back).
9. Apply a second skim coat of joint compound over the tape using a putty knife.
10. Allow to dry.
11. Smooth out all rough spots in the joint compound using a sanding block.
12. Apply one coat of KILZ latex primer to the front side of the coupon.
13. Allow to dry.
14. Apply one coat of Behr Premium Plus Interior Flat White Latex Paint to the front side of the
coupon.
15. Allow to dry.
16. Seal the back side of the coupon with any non-white latex or enamel paint.
The final coupon front and back will look like Figures A-6 and A-7, respectively.
Figure A- 6. Drywall Coupon Front Figure A- 7. Drywall Coupon Back
A-5
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3.0 LAMINATE
3.1 Equipment
• Pergo Estate Oak Laminate Flooring from Home Depot, SKU 257063
• 7/16" - Trubord OSB from Home Depot, SKU 386081
• Liquid Nails from Home Depot, SKU 119066
3.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Cut a 14" x 14", 28" x 28", or 42" x 42" square of OSB using a table saw.
3. Cut the laminate flooring to the proper length. For each coupon size, one piece of laminate
will have to be ripped lengthwise on the table saw.
4. Apply Liquid Nails to the rough side of the OSB (Figure A-8).
Figure A- 8. Liquid Nails on Rough Side of OSB
5. Snap the pieces of laminate together and place on the OSB ensuring that the laminate is
properly aligned with the edges of the OSB (Figure A-9).
A-6
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Figure A- 9. Laminate Aligned with Edges of OSB
6. Clamp several coupons together (Figure A-10) or stack the coupons in an area where they
can remain undisturbed until the Liquid Nails has dried. The different sizes of fini shed
coupons are shown in Figure A-ll.
Figure A- 10. Several Coupons Clamped Together for Drying
A-7
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Figure A-11. Completed Laminate Coupons
4.0 GLAZED CERAMIC TILE
4.1 Equipment
• 16" x 16" Marazzi Island Sand Glazed Ceramic Tile from Home Depot, SKU UE4L
• James Hardie Hardiebacker 5 ft x 3 ft x 1/4 in Backer Board from Home Depot, SKU
180869
• Simpleset Thin-Set Mortar from Home Depot, SKU 769803
• GE Premium Waterproof Clear Silicone Caulk from Home Depot, SKU 469296
• Circular Saw
• Safety Glasses
• Manual Tile Cutter
• Serrated Trowel
• Tape measure
1.2 14" x 14" Procedure
1. Don safety glasses and cut-resistant gloves.
2. Using a manual tile cutter, cut each 16" x 16" tile down to 14" x 14".
4.3 28" x 28" Procedure
1. Don safety glasses and cut-resistant gloves.
2. Using a manual tile cutter, cut four 16" x 16" tiles down to 14" x 14".
3. Using a circular saw, cut a 28" x 28" piece from the backer board.
4. Apply tile cement to the backer board and spread with a serrated trowel.
A-8
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5. Place the four 14" x 14" tile pieces on the backer board with the factory edge to the inside
and apply pressure to seat the tile to the board.
6. Assure that the tiles are even with the edge of the backer board and set the coupons aside and
allow the cement to dry.
7. Apply a bead of silicone caulk to the crack between the tiles and smooth out with a wet
finger.
8. Set the coupon aside and allow the caulk to cure.
A-9
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Appendix B - Aerosol Deposition of Spores onto Material Coupon Surfaces
Using the Aerosol Deposition Apparatus - High Dosing
This document outlines the procedure for assembly and usage of the Aerosol Deposition
Apparatus (ADA). The purpose of this procedure is to conduct precise and highly repeatable
aerosol deposition of bacterial spores onto material surfaces for detection, sampling, and/or
decontamination studies. The required materials, supplies, and procedure are discussed below.
Materials:
• Aerosol Deposition Apparatus (ADA) (shown in Figure B-l)
• Metered Dose Inhaler (MDI) preloaded with a bacterial spore suspension of known
concentration (i.e., 1 x 109 spores per actuation)
• Vertical MDI Actuator (shown in Figure B-2)
• Material coupon (with dimensions at least that of the ADA)
• ADA-coupon gasket (1 per ADA) (see Figure B-l)
• Clamping devices (i.e., medium-size steel binder clips, C-clamps (8 per ADA))
• Vortex mixer (shown in Figure B-4)
• Aerosol trap (described in Attachment A and shown in Figure B-4)
• Personal Protective Equipment (PPE) (gloves, lab coat, safety goggles)
• pH-adjusted bleach (pAB)
• 0.22 micrometers (|im) pore-size syringe filters (shown in Figure B-l)
• PVC tubing (3/8" outer diameter, 1/4" inner diameter)
• Mass balance (with 0.01-gram accuracy)
• Bench liner
1.0 STERILIZATION OF MATERIALS
Prior to the start of any experiment, all components must be sterilized and stored in a sterile
environment until use. Sterilization is not necessary for binder clips, MDI, vortex, or the aerosol
trap.
AD As can be sterilized by autoclave, vaporous hydrogen peroxide, or by wiping with pAB with
subsequent DI water and ethanol rinse/wipes. The ADA lid should be attached and in the closed
position during the sterilization.
B-l
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ADA with lid in
closed position
2.2um
syringe filter
Gasket
Figure B- 1. ADA Apparatus
The MDI actuator, with attached MDI adaptor, can be wiped with pAB then rinsed with DI
water.
Vertical
Actuator
Figure B- 2. MDI and Vertical Actuator
Sterilization requirements for coupons vary by material. Regardless of the sterilization method,
QC checks (typically by collecting a swab sample) should be administered to ensure the
effectiveness of the sterilization method.
Gasket sterilization may also vary by material. Care should be taken to thoroughly degas gaskets
if sterilized via fumigation.
B-2
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2.0 PROCEDURE
1. Begin by donning PPE (gloves, lab coat, and protective eyewear).
2. Clean the workspace by wiping with pAB, next with DI water, and lastly with a 70-90%
solution of denatured ethanol. Alternatively, new, clean bench liner may be placed on the
work surfaces. Make sure the workspace is clean and free of debris.
3. Discard gloves and replace with fresh pair.
4. Using aseptic techniques (when possible), assemble the coupon/ADA by first placing the
sterilized material coupon onto the clean lab bench or workspace, next place the sterilized
gasket on top of the coupon, and lastly seat the ADA on the coupon + gasket. Orient each
component so that it fits squarely with the previously placed item. Take care not to touch the
inside of the ADA or the coupon surface. Secure these components by attaching medium-size
binder clips, one at each corner, and one at the midpoint of each of the four sides of the
ADA. The binders should firmly secure the coupon to the ADA and apply sufficient pressure
to the gasket to seal the union. If material coupons are too large to use binder clips other
methods may be used to secure the coupon and gasket to the ADA (i.e., larger clamps, weight
added to the ADA, etc.). Lastly, attach 0.2-um syringe filters to each vent tube on all AD As
(4 per ADA). Syringe filters can be attached using PVC tubing (3/8" outer diameter, 1/4"
inner diameter).
5. Determine the weight of the MDI canister using a balance. Record the MDI identification
number and the weight (to the nearest O.Olg) in lab notebook. In addition, keep a record of
the total number of actuations dispensed for each MDI canister.
NOTE: The MDI canister full is approximately 15 g, an empty canister is approximately 9.5
g. To ensure the canister contains adequate spore suspension for dosing, canisters
should be retired from use when their weight falls below 10.5 g.
6. Next, assemble the MDI and actuator by inserting the MDI into the actuator, taking care not
to activate the MDI.
7. Vortex the MDI/actuator assembly for 30 seconds (the MDI canister should be in direct
contact with the vortex mixer).
8. Holding the MDI/actuator assembly upright (Figure B-3), with a swift, firm motion,
dispense three test actuations into the aerosol trap to prime the MDI. It is important to vortex
the assembly 10 seconds before every actuation (the exception being 30 seconds prior to the
initial actuation of the experiment, as prescribed in Step 7).
B-3
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Figure B- 3. MDI Orientation while Dispensing Test Actuations into the Aerosol Trap
9. Vortex the assembly for 10 seconds and then attach to the ADA lid by mating the ADA
adaptor to the hole in the ADA lid. Loosen the lid screws enough to allow the lid to be slid
into the 'open' position. Secure the lid in the open position by tightening the lid screws.
NOTE: The 'open' position is achieved when the hole in the lid aligns with the hole in the
top of the ADA.
10. With a swift, firm motion, dispense the spores by activating the MDI. Hold the MDI in the
activated position for 3 seconds before releasing. Activation is best achieved by grasping the
MDI/actuator with two hands and using a thumb to press the bottom of the MDI canister.
11. Follow the reverse order of the lid opening procedure to close the ADA lid.
12. Determine the weight of the actuator-MDI using a balance and record the weight in lab
notebook.
NOTE: If the dosing actuation is faulty, return to Step 9 and attempt a second actuation on
the current coupon. Do not proceed to the next coupon until a 'successful' actuation
has been delivered. A 'successful' actuation is achieved when the weight of the
B-4
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actuator-MDI assembly has a loss of 0.04 g to 0.07 g. Familiarity and professional
judgment will be needed to determine the success of an actuation.
13. Vortex the assembly for 10 seconds, then proceed to dosing the next coupon (Step 9).
14. Repeat Steps 9 through 13 until all coupons have been dosed.
15. Once all coupons have been dosed, remove the MDI from the actuator and weigh. Record the
final weight and total number of actuation.
16. Allow spores to settle onto the coupon surface for at least 18 hr. Settling time should not
exceed 26 hr.
17. Carefully remove binder clips (or other attachment device), and remove ADA and gasket
from coupon surface, taking care not to disturb the surface of the coupon.
18. Test coupon is now ready for use.
19. Decontaminate the ADA and associated components with the same procedures utilized
during the initial sterilization.
B-5
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Attachment A - Aerosol Trap
Purpose: This device allows test actuations of the MDI to be deployed without contamination of
the surrounding area. Spores are pulled into the trap, contained, and inactivated.
This device consists of a suction source, a trap (containing pAB), and an inlet funnel.
Aerosolized spores are pulled into the funnel and forced into the trap. The spores are collected
and inactivated as the aerosol flows through the pAB solution. The effluent air traveling toward
the suction device is spore-free downstream of the trap. See Figure B-4.
The aerosol trap should be assembled inside a BSC or chemical fume hood.
Aerosol trap
Vortex er
Figure B- 4. Aerosol Trap
B-6
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vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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