SEPA
EPA/600/R-17/374 September 2017
www.epa.gov/homeland-securitv-research
United States
Environmental Protection
Agency
Sample Collection Information
Document for Pathogens
Companion to Selected Analytical Methods for
Environmental Remediation and Recovery (SAM)
2017
Office of Research and Development
Homeland Security Research Program
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EPA/600/R-17/374
September 2017
Sample Collection Information
Document for Pathogens
Companion to Selected Analytical Methods for
Environmental Remediation and Recovery
(SAM) 2017
by
Sandip Chattopadhyay, Ph.D.
Threat and Consequence Assessment Division
National Homeland Security Research Center
Cincinnati, OH 45268
U.S. Environmental Protection Agency
Office of Research and Development
Homeland Security Research Program
Cincinnati, OH 45268
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Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research and
Development funded and managed the research described herein. It 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.
Questions concerning this document or its application should be addressed to:
Sandip Chattopadhyay, Ph.D.
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
26 W. Martin Luther King Drive, MS NG16
Cincinnati, OH 45268
Phone: 513-569-7549
Fax: 513-487-2555
E-mail: chattopadhyay.sandip@epa.gov
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Table of Contents
DISCLAIMER II
LIST OF TABLES IV
LIST OF ATTACHMENTS IV
ACRONYMS AND ABBREVIATIONS V
ACKNOWLEDGEMENTS VII
1.0 BACKGROUND 1
2.0 SCOPE AND APPLICATION 1
2.1 Sample Collection Informa tion Tables 3
2.2 Document Development. 3
2.3 LIMITATIONS 4
3.0 HEALTH AND SAFETY CONSIDERATIONS 5
3.1 Health and Safety Plans 5
3.2 Personal Protective Equipment 5
3.3 Training 5
4.0 PREPARATION FOR SAMPLE COLLECTION 6
4.1 Field Sampling Equipment and Supplies 6
4.2 Field Da ta Document a tion 6
4.3 Field Screening 7
4.4 Quality Assurance/Quality Control 7
5.0 SAMPLE HANDLING 9
6.0 SAMPLE ACCEPTANCE 10
7.0 DEFINITIONS 11
8.0 LABORATORY SUPPORT 11
8.1 Defining Analytical Support Requirements: Capabilities and Capacity 11
8.2 ESTABLISHING ANALYTICAL SUPPORT NETWORKS 12
8.3 COORDINATING WITH ANALYTICAL SUPPORT NETWORKS 13
8.4 Laboratory Networks and Associations 13
9.0 TOOLS AND DATABASES 14
10.0 ADDITIONAL RESOURCES 16
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List of Tables
Table 1. Pathogens and Media Addressed in this Sample Collection Information Document.... 2
Table 2. Key Laboratory Networks and Associations 13
Table 3. Representative Tools and Databases 15
List of Attachments
Attachment A: Sample Collection Information for the Environmental Media (Soil, Surface,
Liquid, and Aerosol)
Attachment B-1: Sample Collection Information for Pathogens (Bacteria, Viruses, Protozoa,
and Helminths) in Solids (Soil, Powder)
Attachment B-2: Sample Collection Information for Pathogens (Bacteria, Viruses, Protozoa,
and Helminths) in Surfaces (Swab, Wipe, Dust Socks)
Attachment B-3: Sample Collection Information for Pathogens (Bacteria, Viruses, Protozoa,
and Helminths) in Liquids (Water and Wastewater)
Attachment B-4: Sample Collection Information for Pathogens (Bacteria, Viruses, Protozoa,
and Helminths) in Aerosols
Attachment C: Holding Time, Packaging Requirements, and Shipping Label of Sample
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Acronyms and Abbreviations
AAVLD
American Association of Veterinary Laboratory Diagnosticians
AOAC
Association of Official Analytical Chemists
APHIS
Animal and Plant Health Inspection Service
APHL
Association of Public Health Laboratories
ASM
American Society for Microbiology
BSL
biosafety level
CBR
chemical, biological, and radiological
CDC
Centers for Disease Control and Prevention
CFR
Code of Federal Regulations
Ch.
Chapter
COC
chain-of-custody
dso
cut-off sizes correspond to 50% particle collection efficiency mark
DGR
Dangerous Goods Regulations
DHS
Department of Homeland Security (U.S.)
DOL
Department of Labor (U.S.)
DOT
Department of Transportation (U.S.)
DQO
data quality objectives
DWRPTB
Drinking Water Utility Response Protocol Toolbox
EMAC
Emergency Management Assistance Compact
EOC
EPA Emergency Operations Center
EPA
U.S. Environmental Protection Agency
ERLN
EPA's Environmental Response Laboratory Network
FBI
Federal Bureau of Investigation
FDA
Food and Drug Administration
FERN
Food Emergency Response Network
g
gram(s)
GPS
Global Positioning System
HASP
health and safety plan
HCV
Hepatitis C Virus
HEV
Hepatitis E virus
HFV
Hemorrhagic Fever Viruses
HSRP
Homeland Security Research Program
IATA
International Air Transportation Association
ICLN
Integrated Consortium of Laboratory Networks
ISO
International Organization for Standardization
L
liter
Lab Compendium
Compendium of Environmental Testing Laboratories
LRN
Laboratory Response Network
MCE
mixed cellulose ester
mL
Milliliter
MS/MSD
Matrix Spike/Matrix Spike Duplicates
NAHLN
National Animal Health Laboratory Network
NEMI
National Environmental Methods Index
NHSRC
National Homeland Security Research Center
NIFA
National Institute of Food and Agriculture
NIOSH
National Institute for Occupational Safety and Health
NIST
National Institute of Standards and Technology
°C
degrees Celsius
OSHA
Occupational Safety and Health Administration
PPE
personal protective equipment
psi
Pound-force per square inch
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PTFE
Polytetrafluoroethylene (Teflon®)
QA
quality assurance
QC
quality control
SAM
Selected Analytical Methods for Environmental Restoration Following
Homeland Security Events
SCID
sample collection information document
spp.
species
UN
United Nations
URL
uniform resource locator
USAMRIID
United States Army Medical Research Institute of Infectious Diseases
USGS
United States Geological Survey
VCSB
voluntary consensus standards body
WCIT
Water Contaminant Information Tool
WLA
Water Laboratory Alliance
WWRPTB
Wastewater Utility Response Protocol Toolbox
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Acknowledgements
The following individuals and organization have been acknowledged for their contributions
towards the development and/or review of this document.
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research Center (NHSRC)
Sandip Chattopadhyay, Ph.D. (Principal Investigator)
Sarah Taft, Ph.D.
Sanjiv Shah, Ph.D.
Eric Rhodes, Ph.D.
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Risk Management Research Laboratory
Ralph Ludwig, Ph.D.
Chris Marks, Ph.D.
Office of Land and Emergency Management
Terry Smith
Francisco J. Cruz
Marti Sinclair (Alion Science and Technology) is acknowledged for technical editing; and quality
assurance reviewer Eletha Brady-Roberts (ORD/NHSRC) is acknowledged for contributions to
this report.
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1.0 Background
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research
Program (HSRP) has worked with experts from across EPA and other federal agencies
since 2003 to develop a compendium of analytical methods to be used when
responding to national homeland security related incidents. These sample collection
methods are to be used by laboratories designated by EPA to perform the analyses of
environmental samples following incidents resulting in the intentional or unintentional
release of contaminants. Analytical methods have been selected for chemicals,
radiochemicals, pathogens, and biotoxins for the types of environmental sample
matrices that are anticipated in such incidents. The results of these efforts have been
published in several revisions of EPA's Selected Analytical Methods for Environmental
Remediation and Recovery - 2017. The HSRP periodically reviews and updates the
Selected Analytical Methods document to address the needs of homeland security,
reflect improvements in analytical methods and new technologies, and incorporate
changes in target pathogens.
During development of the Selected Analytical Methods document, EPA recognized the
need for a companion document to provide information regarding collection of samples
for analysis by the listed methods. This Sample Collection Information Document
(SCID) is intended to address this need, in part, by providing complementary
information on sample collection, containers, preservation, size, and packaging, and by
providing additional information sources to support the collection of samples to be
analyzed for the selected pathogens, using the methods listed in Selected Analytical
Methods for Environmental Remediation and Recovery - 2017 (herein referred to as
"the Selected Analytical Methods document"). As with the Selected Analytical Methods
document, HSRP plans to update the information in this document periodically, to
reflect changes to the list of pathogens and/or methods.
The information contained in this document is intended to support and be used
with the methods listed in Selected Analytical Methods for Environmental
Remediation and Recovery -2017 for analysis of selected pathogens. The
information will be reviewed and updated periodically, along with the Selected
Analytical Methods document, to reflect advances in technologies, results of
method evaluation and validation studies, and additional pathogens or matrices.
2.0 Scope and Application
This document provides general information for use by EPA and other users when
collecting samples for pathogen analysis during environmental remediation following an
intentional or unintentional release. The document is intended to be used with the
Selected Analytical Methods document, and to provide information needed for collection
of samples to be analyzed using the specific selected methods. Where possible, the
information provided was obtained from the sample collection requirements and
guidelines included in the Selected Analytical Methods for Environmental Remediation
and Recovery - 2017 analytical methods. Where this information was not available,
additional sources were used (see Section 10.0 and additional resources).
A pathogen or infectious agent is a biological agent that causes disease or illness to its
host. This document includes following pathogens: bacteria, viruses, protozoa, and
helminths in a variety of environmental media (Table 1).
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Table 1. Pathogens and Media Addressed in this Sample Collection Information
Document
Media
Pathogen<>^^^^
(Size*)
Soil Surface Liquid Aerosol
• Bacillus anthracis • Legionella pneumophila
Bacteria
g
• Brucella spp. • Leptospira spp.
• Burkholderia mallei • Listeria monocytogenes
• Burkholderia pseudomallei • Non-typhoidal Salmonella
• Campylobacter jejuni • Salmonella Typhi
• Chlamydophila psittaci • Shigella spp.
• Coxiella burnetii • Staphylococcus aureus
(0.2 - 5 pm)
• Escherichia coli • Vibrio cholerae 01 and 0139
• Francisella tularensis • Yersinia pestis
• Adenoviruses: enteric and non-enteric
Viruses
> "V
• Astroviruses
• Caliciviruses: Norovirus and Sapovirus
UStel
• Coronaviruses: SARS-associated human coronavirus
• Hepatitis E virus (HEV)
(0.02-0.2 pm)
• Influenza H5N1 virus
• Picornaviruses: Enteroviruses and Hepatitis A virus (HAV)
• Reoviruses: Rotavirus (Group A)
Protozoa
$
• Cryptosporidium spp.
• Entamoeba histolytica
Tx
• Giardia spp.
• Naegleria fowleri
(4 - 20 pm)
• Toxoplasma gondii
Helminths
¦Q,
• Baylisascaris procyonis
(40-100 pm)
* Sizes shown in the diagrams are not to scale.
The information in this document is intended to be used during site assessment,
remediation, and clearance activities following an intentional or unintentional release of a
contaminant; it assumes that samples will be collected by personnel trained in the
collection of environmental samples containing the target pathogens, and trained in
dealing with the corresponding health and safety concerns. Information is included
regarding containers, collection volume or weight, sample preservation, sample holding
times, and the packaging of samples representing the various matrices and pathogens
of concern.
Certain information in this report may need to be modified to address site- or
event-specific data needs; for example, additional sample volume may be
needed for quality control (QC) or in cases when a low concentration of pathogen
is suspected. Sample collection plans should be in place and consulted for
specific sample collection requirements prior to initiation of sample collection
activities. Site- or event-specific sample collection plans include information
regarding laboratory capacity, the extent of contamination, target pathogens, data
quality objectives (DQOs), sample locations, the number and type of samples
needed, and other details.
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2.1 Sample Collection Information Tables
This document contains the following tables listing information for collection of
samples that will be analyzed for measurement of the selected pathogens.
• Attachment A: Sample collection information for pathogens in various
environmental media (soil, surface, liquid, and aerosols). It should be noted
that the surfaces include porous and non-porous surfaces; aerosols include
natural aerosols and bioaerosols; solids include soils, granular and powder
forms of debris and/or natural materials; and liquids include drinking water,
surface water, and wastewater present at the pathogen impacted area.
• Attachment B: Sample collection information for pathogens.
• Attachment C: Holding times, packaging requirements, and shipping label
requirements for samples.
Each table provides the sample size that should be collected to support sample
analysis, the preservatives and/or temperature needed to maintain sample
integrity prior to analysis, the maximum amount of time that should elapse
between sample collection and the initiation of analytical procedures (e.g.,
sample analysis, digestion, inoculation), the appropriate type of container, the
sample label and packaging procedures needed for sample shipment, and the
source(s) used to provide the information. Unless otherwise specified, the
following sample storage protocol may be followed:
• Ensure samples maintain integrity, and are not contaminated, lost, damaged.
• Samples requiring thermal preservation at other than < 6°C shall be stored at
± 2° C of stated temperature.
• Samples are to be kept separate from reagents, standards, and other
interfering items in refrigerators.
2.2 Document Development
EPA developed a hierarchy of references to prioritize the documents and
resources that were used to identify the information that is included in this
document. The first sources consulted were the methods listed in Selected
Analytical Methods for Environmental Remediation and Recovery -2017. If
those methods included sample collection information, the information was
evaluated and, if appropriate, included in the sample collection information
tables. The second sources consulted were EPA procedures for collection of
samples that address the specific pathogen/matrix pair. If there were no EPA
procedures available, other federal agency or voluntary consensus standards
body (VCSB) methods were consulted. If no procedures were identified for
collection of a particular pathogen/matrix combination, methods for that pathogen
in other matrices were considered, followed by procedures described and
supported by data in peer-reviewed research literature, such as journal articles.
The following agencies, organizations, and publications are representative
examples:
• EPA - United States Environmental Protection Agency
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• AOAC - AOAC International (formerly Association of Official Analytical
Chemists)
• CDC - Centers for Disease Control and Prevention
• CFR - Code of Federal Regulations
• U.S. DHS - United States Department of Homeland Security
• U.S. DOL - United States Department of Labor
• U.S. DOT - United States Department of Transportation
• U.S. FDA - United States Food and Drug Administration
• USGS - United States Geological Survey
• IATA - International Air Transport Association
• ISO - International Organization for Standardization
• LRN - Laboratory Response Network
• NEMI - National Environmental Methods Index
• NIOSH - National Institute for Occupational Safety and Health
• OSHA - Occupational Safety and Health Administration
• Rice et al. 2017. Standard Methods for the Examination of Water and
Wastewater 23rd edition. Washington, DC: American Public Health
Association
• Journals: Analyst, Applied and Environmental Microbiology, Current
Protocols in Microbiology, FEMS Microbiology Letters, Journal of Virological
Methods, Public Health Reports, and others.
2.3 Limitations
This document provides summary information only regarding collection of
samples to be analyzed for selected target pathogens. This document includes
the information based on the sampling protocols and analytical methods that
were available at the time of publication. The document is expected to be
updated with the advance of technologies. For example, research is needed to
determine appropriate preservation and holding times for many of the biological
agents. In addition, many of the pathogens listed in this document have only
recently become an environmental concern, and EPA is actively pursuing
development and validation of appropriate sample collection procedures.
Sample collection plans must be consulted for site- or event-specific
requirements, including quality control (QC) and reporting. The information
sources cited in this document also should be consulted for additional details
regarding sample collection, including QC requirements, and sample handling,
packaging, shipping, and safety procedures. Samplers should check with the
incident commanders for special instructions regarding evidentiary matters prior
to sample collection.
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3.0 Health and Safety Considerations
This document assumes that a site- or event-specific health and safety plan (HASP) is in
place that includes the safety concerns and requirements for the specific types of
hazards that should be considered during a sample collection event. At a minimum, all
sampling team members should be trained in Occupational Safety and Health
Administration (OSHA) requirements for hazardous waste operations and emergency
response (29 CFR 1910.120 or 29 CFR 1926.65) and should have current medical
screening.
3.1 Health and Safety Plans
Health and safety plans (HASPs) will vary depending on the site, nature and
extent of contamination, the sampling phase (site assessment, remediation, or
final status determination), and the responsible organization. The purpose of
these plans is to ensure maximum protection to workers, the environment, and
surrounding communities, in a way that is consistent with requirements needed
to perform operational activities.
Sample collection and decontamination procedures should address
personnel monitoring and decontamination during ingress and egress.
3.2 Personal Protective Equipment
Each site or event also will dictate the level of personal protective equipment
(PPE) that will be required. Specific guidance for selection of PPE is provided in
29 CFR 1910.120, Appendix B. Factors that should be considered during
selection include: contaminant identification, routes of exposure (i.e., inhalation,
skin absorption, ingestion, and injection), performance of equipment in protecting
against exposure, activity duration, and the stress that will be induced by work
requirements.
3.3 Training
Sample collectors must be trained in collection and handling of samples
suspected of containing the contaminants of concern, must be up to date
regarding medical screening requirements, and must be approved for site entry.
Additionally, sample collectors must be trained in the following:
• Ability to select and work with the appropriate level of PPE
Decontamination procedures
Prevention of sample cross-contamination.
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4.0 Preparation for Sample Collection
During the early stages of an event, upon initial contact by the by the analytical services
requester or other responsible party, coordination and communications with the primary
responding laboratory may be performed to gather critical information pertaining to the
nature of the samples to be collected, the number of samples required, prioritization of
samples, and to alert member laboratories.
It is highly recommended that sampling kits be used during sample collection, and that
these kits be properly equipped, maintained, and organized before deployment of
sample collection personnel. Sample collectors should consult with project managers
and the sample collection plan to determine what equipment and materials should be
assembled. Sample kits should contain all sample containers, materials, supplies, and
forms needed to perform sample collection, decontamination, documentation, and field
packaging activities.
4.1 Field Sampling Equipment and Supplies
Before starting field sampling activities, all necessary equipment and supplies
should be identified and available. The following is a preliminary list of
equipment that needs to be specified and available:
Sampling devices (e.g., air filters, soil samplers, water samplers, air filter
samplers)
Sample preservation equipment (e.g., acids, dechlorinating reagents)
Sample volumetric measuring devices and/or weighing devices
Sample containers and packaging equipment
• PPE
Record keeping devices (e.g., logs, chain-of-custody [COC] forms, writing
instruments)
Site maps, Global Positioning System (GPS) recorders, etc.
Sample location markers
Pre-labeled and pre-weighed sampling containers
Shipping containers, shipping forms, and shipping labels.
4.2 Field Data Documentation
All data collected in the field should be adequately documented. Documented
information should include (for example):
Names of field sampling personnel
Sample collection plan
Sample location(s)
Sampling depth
Physical and meteorological conditions
Date and time of sampling
Sample medium
Expected radionuclides (if applicable)
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Sample identification number
Sample size (weight, volume), sample duration (air filters), air volume
Sample handling precautions.
4.3 Field Screening
Field screening procedures are typically qualitative or semi-quantitative in nature
and are performed using special screening equipment or techniques, such as
probes or portable hand-held instruments and meters. Some field screening is
performed using field testing methods, and special kits that are designed for use
in a field environment. Because the quality control and analytical sophistication
of field screening is not as controlled as it is for laboratory testing, a
representative set of split or duplicate samples should be submitted to a
laboratory for comparison with the field results.
4.4 Quality Assurance/Quality Control
Sampling personnel should employ quality assurance/quality control (QA/QC)
program requirements when collecting samples to include information on the
collection of equipment blanks, field blanks, and field replicates, when available
and as appropriate for the intended analyses. Field QA/QC requirements should
be specified in sampling or site plans, and analytical support laboratories should
be included in the discussion as analytical QA/QC requirements should greatly
impact field sampling. The purpose of such QA/QC protocol is to ensure that (1)
the laboratory receives samples that accurately represent the conditions existing
at the sample site, (2) appropriate method-specific controls are provided to the
analytical laboratory, and (3) the results of the analyses are traceable to the
specific sample location or event. The following QC procedures should be
included, as appropriate:
Decontamination of Sampling Equipment: The field sampling plan should
address the extent of decontamination and specify the procedures to prevent
sample contamination that could be introduced from contaminated collection
equipment. Sampling may be performed using separate laboratory-cleaned
equipment for each sample location.
Sample Container Cleanliness Requirements: The field sampling plan
should also address the extent and type of sample container cleaning, to
prevent sample contamination from containers. Pre-cleaned containers
meeting EPA method-specific cleanliness protocols are available from many
suppliers. If pre-cleaned containers are used, the serial number and QA
batch number of each container should be recorded in the field log
book/notes or field form. If sample containers are re-used, they should be
decontaminated, and field blank samples should be submitted to the
laboratory to verify container cleanliness.
Field Duplicates and Split Samples: Field duplicates are two separate
samples taken from the same source and are used to determine data
repeatability based on field conditions. Field duplicate samples are assigned
different sample numbers, specified in the field log book/notes or on the field
form, distinguished from the regular field samples on the COC form, and
often submitted blind to the laboratory to provide objectivity. The
comparability of the results provides information on the repeatability of the
field extraction and analytical procedures. Split samples are two or more
representative portions taken from one sample and submitted to different
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laboratories for identical analyses to obtain information on inter-laboratory
repeatability.
Equipment Decontamination Blank: These samples provide information on
the levels of cross-contamination resulting from field or laboratory sample
preparation actions. The equipment blank is reagent water that is free of the
pathogen of interest, transported to the site, opened in the field, and poured
over or through the sample collection device, collected in a sample container,
and returned to the laboratory and analyzed. Equipment blanks are collected
for each type of equipment used in sampling during the day. Equipment
blanks are assigned sample numbers and are not distinguished from regular
field samples on the COC form. To decontaminate, sampling equipment
(e.g., scoops, spoons, bowls, etc.) will first be cleaned with a laboratory-grade
detergent such as Alconox® using plastic brushes to remove soil and surface
matter, and then rinsed with water to remove the remaining soapy material.
The equipment will then be allowed to air dry. If the equipment is not to be
used immediately, it will be wrapped with aluminum foil and stored in a clean,
dry place. Verification of the effectiveness of the decontamination procedure
will be acquired through equipment rinsate samples. Drill stems, rods,
augers, tools, split spoons, sample barrels, and associated equipment will be
cleaned prior to initial sampling and between sampling. Cleaning and
decontamination of all equipment will occur at a designated area on the site.
Equipment that is steam cleaned will be placed on racks or sawhorses at
least two feet above the floor of the decontamination pad. After cleaning, all
surfaces will be thoroughly rinsed. Cleaned equipment will be allowed to air
dry.
Field Blanks: Field blanks check the cleanliness of sample containers, for
environmental contamination, for the purity of reagents, or for the purity of
solvents used in the field. A sample container is filled with laboratory grade
reagent water in the field, preserved, and submitted for analysis for the same
parameters as the regular field sample.
Trip Blank: A trip blank is a container of laboratory reagent water that is
shipped, unopened, to and from the field, with empty and full sample
containers. Its purpose is to identify contaminants introduced into samples
during transit to and from the laboratory. At no time after their preparation
are the sample containers opened before they reach the laboratory.
Matrix Spike/Matrix Spike Duplicates (MS/MSD): Some analytical methods
require that the laboratory spike a portion or duplicate portions of the sample
matrix with a predetermined quantity of analytes prior to sample extraction
and analysis. A spiked sample is processed and analyzed in the same
manner as the sample. The results of the spike compared with the non-spike
sample indicate the ability of the test procedures to repeat recovery of the
analyte from the matrix and also provides a measure of the performance of
the analytical method. Additional containers may be specified to provide
enough material for this procedure. The sample containers are assigned the
same sample number as the regular field sample and are designated
MS/MSD on the COC form.
Equipment Maintenance and Calibration: All sampling equipment should
be maintained on a regular basis, consistent with the documented criteria of
the laboratory and normally accepted codes of practice/standards, which are
well within the limits normally established and recommended for the care of
the particular piece of equipment. Frequent checks on the reliability of
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equipment and the calibration checks on all relevant equipment must be
performed. Equipment calibration and maintenance records should be kept
for all equipment, thus allowing the repair status of each piece of apparatus to
be monitored. This reduces the likelihood that malfunctioning equipment will
be used for sampling (thereby leading to poor bioassay data), and allows any
problems with equipment to be more quickly diagnosed and corrected.
5.0 Sample Handling
A key aspect of biological research revolves around the gathering and collection of
samples and their preservation for examination and analysis at a future date. Since time
elapses between when a sample is collected and when it is analyzed, and biological
samples often degrade over time, it is imperative to have a process of storage (short and
long term) that is efficient and preserves sample integrity over time. Good storage
practices of biological materials are essential component of any sampling activity.
Biological samples often degrade over time when stored at room temperature, but some
samples may also lose integrity at low temperatures if subjected to multiple freeze-thaw
cycles. Many bio-specimens can be safely stored at a range between -20°C and 5°C,
known as cold storage. Enzymes and antibodies can lose much of their functional
activity if they are repeatedly frozen and thawed, so these samples are often refrigerated
at around 2°C. Biological specimen storage in a range of 15°C to 27°C is known as
room temperature storage. The best storage temperature for a given biological sample
or reagent often varies depending on the type of biological material, the solution it is
suspended in, the sample's intended use, and how long the material will be stored.
Many variables go into making ideal storage temperature decisions for biological
materials. For reagents and biological assays, it is often best to follow
manufacturer/bioassay laboratory recommendations for both short-term and long-term
storage temperatures. When storing samples, it is important to consider the sample's
molecular structure (Holland et al., 2003; Budowle et al., 2006; NRC, 2014; Shabihkhani
et al., 2015), the preservatives or solutions it is suspended with, and the degree of
biological integrity required for analytical or research goals.
Samples that require low temperature preservation shall be considered acceptable if the
arrival temperature of a representative sample container meets the method or mandated
temperature requirement.
• Samples that are delivered to the laboratory on the same day they are collected may
not meet the temperature or method requirements, if the time frame between
collection and delivery is too short for the cooling process to complete. In these
cases, the samples shall be considered acceptable if the samples were received
nestled in ice with evidence that the cooling process has begun and the temperature
of the sample(s) (or representative sample) is recorded upon receipt and is less than
the temperature recorded at the time of sampling.
• Low temperature preservation along with temperature monitoring might not be
required in the field if the laboratory receives the sample and either begins the
analysis or refrigerates the sample within fifteen (15) minutes of collection.
Microbiological samples from known chlorinated sources, unknown sources where
chlorine usage is suspected and all potable water supplies (including source water) shall
be checked for absence of chlorine residual in the laboratory unless all of the following
conditions are met:
• the laboratory can show that the received sample containers are from their laboratory
or have been appropriately chlorine tested and documented;
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• sufficient sodium thiosulfate was in each container before sample collection to
neutralize at minimum 5 mg/L of chlorine for drinking water and 15 mg/L of chlorine
for wastewater samples;
• one container from each batch of laboratory prepared containers or lot of purchased
ready-to-use containers is checked to ensure efficacy of the sodium thiosulfate to 5
mg/L chlorine or 15 mg/L chlorine as appropriate and the check is documented;
• chlorine residual is checked in the field and actual concentration is documented with
sample submission.
6.0 Sample Acceptance
Acceptance or rejection of samples may be based on individual samples (i.e., a
laboratory can accept or reject samples at any time during the lifetime of the event). A
laboratory's participation in a specific incident is at the discretion of the individual
laboratory's management and may require consultation with higher level management in
the parent organization before the laboratory agrees to provide analytical support. If
samples are collected, shipped, and/or preserved in a manner that may affect sample
integrity, the notification should be communicated as soon as possible. Consideration of
possible impacts on data quality should be weighed against the monitoring objectives
(e.g., the need to obtain rapid preliminary identification of the pathogen) before making a
decision to accept or reject samples. Any results generated from analysis of samples
with shipping or preservation issues should be appropriately qualified. Although sample
acceptance (or rejection) is ultimately the laboratory director's or higher level
management's prerogative, laboratories must consider the following before accepting
samples:
• Sample integrity (i.e., condition)
• Sample packaging and preservation
• Sample volume
• Chain of custody provided
• Minimum documentation provided
• Potential sample hazards
• Field/safety screening results
• Law enforcement involvement or requirements
• Special instructions, if any
• Availability of additional, identical samples (splits)
Sample must be rejected if:
• Hold time is exceeded
• Improper preservation is noted
• Sample is in the wrong container
• Absence of chain of custody.
For those samples analyzed, all data must be reported with qualifiers. All associated
results must be reported. Result qualification may be required when:
• Samples are improperly preserved
10
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• The wrong container is used
• Holding time is exceeded
• Insufficient sample volume is available to perform analysis
• Known sampling errors are noted
The analytical laboratory may reject or require re-sample as alternative to qualification of
sample results based on the appropriate bioassay qualification criteria established for
the specific condition.
If discrepancies between sample collection records and sample receipts are noted, the
laboratory must consult with the sample collector and other experts to determine if
samples can still be analyzed and reported with qualification, or whether re-sampling is
required.
7.0 Definitions
The following definitions are provided to describe the information listed in the sample
collection tables:
Container - The type of container (e.g., bottle, bag) that must be used to hold the
sample. The container must be sufficient to maintain sample integrity and be
composed of materials that will remain inert when in contact with the sample.
Holding Time - The maximum amount of time allowable from sample collection until
sample analysis, extraction, or inoculation.
Matrix - The principal material of which the sample is composed.
Packaging - Sample container packaging requirements for shipment of the sample
to the laboratory.
Preservation - Conditions and/or chemicals used to maintain the integrity of a
sample (e.g., sodium thiosulfate and refrigeration at temperatures < 10°C but above
freezing for biological samples).
Sample Size - The minimum amount of sample that should be collected to support
analysis of a single sample. Volume and weight requirements depend on the target
pathogen(s), the analytical method that will be used, and the data requirements.
Shipping Label - U.S. DOT shipping label requirements under 49 CFR 172 and
173.
8.0 Laboratory Support
8.1 Defining Analytical Support Requirements: Capabilities and Capacity
The inherent rigidity in a standard operating protocol for biological incident
sampling and processing could be unwieldy and require consultation among the
different entities involved in a response should provide best-practice options. If
the pathogen incident fits a pattern or template for which a sample collection
methodology and/or sampling strategy already has been validated, then the
sampling activities could be well-defined and more focused. However, in most
pathogenic incident cases, the sampling area, location, type of agent, substrates,
and combinations of these variables are almost always novel (Budowle et al.,
2006). A network of laboratories with technical infrastructure (centralized
communication, personnel, standardized reagents and equipment and test
11
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protocols, reporting policies, shipping and transportation guidelines assay
development, and new or renovated facilities to increase levels of biosafety
containment), provides the necessary infrastructure for a tiered capability of
response to an event. Most field sample collectors might not be responsible for
analyzing the samples. Water utilities, if involved, might have unique capabilities
to collect samples and analyze them in their laboratories. For this reason, it is
critical that the role of the microbiology laboratory in incident response be
evaluated based on internal analytical capabilities and response capacity. Some
contaminants (for example, select biological agents) should be analyzed by
qualified laboratories using specialized or restricted analytical methods. It is
important that utilities are familiar with analytical support networks. They are
encouraged to look into the resources offered by EPA's Environmental Response
Laboratory Network (ERLN) and Water Laboratory Alliance (WLA), such as the
WLA response plan, as well as other members of the Integrated Consortium of
Laboratory Networks (ICLN) including the CDC Laboratory Response Network
(LRN). (Table 1 provides descriptions of these laboratory networks.) Internal and
external analytical support networks should be in place and operational prior to
initiating any baseline sampling and analysis activities, and in preparation for an
event.
8.2 Establishing Analytical Support Networks
Establishing a support network of laboratory analytical capabilities and capacity
should ensure that samples can be processed properly and expeditiously. To
assist in locating laboratories capable of providing the necessary support, the
EPA's Compendium of Environmental Testing Laboratories (Laboratory
Compendium) provides users with real-time data related to laboratory contact,
capability and capacity information, and ERLN/WLA Membership status, through
a secure web-based tool. The Laboratory Compendium is available to
emergency response, laboratory and water utility personnel, at the federal, state,
and local levels. Access is secured through an application process at
https://cfext.epa.gov/cetl.
Each EPA region maintains an EPA regional laboratory, which may be able to
analyze samples or to help identify potential analytical support. Access the list of
EPA regional laboratory contacts at http://www2.epa.gov/aboutepa/regional-
science-and-technology-rst-organizations#branches.
LRN laboratories have response teams available 24 hours a day/7 days a
week/365 days a year who may be able to assist with sample collection needs
after routine business hours. Usually the closest LRN laboratory should be the
state's department of health laboratory; also, consider contacting the local public
health laboratory. For more information, CDC can be contacted at (800) CDC-
INFO, (888) 232-6348 (TTY) or www.cdc.gov/info. More information is also
available at: https://emergency.cdc.gov/lrn/biological.asp. Another resource for
state laboratory contact information is maintained by Association of Public Health
Laboratories (APHL) at
https://www.aphl.org/membership/Pages/memberlabs.aspx.
EPA Headquarters might also be able to provide help in identifying support for
analysis and collection of samples. The ERLN/WLA Helpline may be reached at
(703) 461-2400, Monday-Friday from 8:30 AM to 5:00 PM ET, except for federal
holidays. The WLA may also be reached at
https://www.epa.gov/waterlabnetwork. Outside of regular business hours, the
12
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EPA Emergency Operations Center (EOC) Hotline may be reached at (202) 564-
3850.
8.3 Coordinating with Analytical Support Networks
Once appropriate analytical laboratory support has been identified, it is
imperative to establish a chain of communication between and among the entity
affected by a contamination incident and the supporting laboratories. Support
laboratories should be consulted regarding specific sample collection, container,
volume, preservation, holding time, and shipping requirements. In some cases,
support laboratories should train sampling teams in specialized sample collection
procedures. The support laboratory may also provide the affected entity with, or
assist with the preparation of, sampling kits to ensure that the samples are
properly prepared and preserved for the required analyses, particularly for
sampling unknown or tentatively identified contaminants, as appropriate. It is
important to follow specific laboratory requirements since this may impact the
quality of the analytical results. Depending on the method and event,
laboratories should request specific quality control (QC) samples such as field
duplicates, field blanks, trip blanks, and field matrix spikes and may require
specific chain of custody (COC), notification, and shipping procedures.
8.4 Laboratory Networks and Associations
Table 2 provides the key laboratory networks and associations.
Table 2. Key Laboratory Networks and Associations
Laboratory
Networks/
Associations
Description
Additional
Information Source*
Environmental
Response
Laboratory
Network
(ERLN)
EPA's ERLN is a national network of laboratories that
provides analytical capability and enhanced capacity to
meet project-specific data quality objectives on an as-
needed basis. The ERLN integrates capabilities of
existing public sector laboratories with accredited private
sector laboratories to support environmental responses.
httDsV/www.eoa.aov/
emeraencv-
resDonse/environmen
tal-resoonse-
laboratorv-network
CDC
Laboratory
Response
Network (LRN)
In response to the threat of bioterrorism and following a
presidential order, officials at the Centers for Disease
Control and Prevention (CDC), Association of Public
Health Laboratories (APHL), Federal Bureau of
Investigation (FBI), and United States Army Medical
Research Institute of Infectious Diseases (USAMRIID)
established the Laboratory Response Network (LRN) in
1999. This national system is designed to link state and
local public health laboratories with other advanced-
capacity clinical, military, veterinary, agricultural, water,
and food-testing laboratories, including those at the
federal level. The LRN is a critical component of CDC's
public health mission, enhancing U.S. readiness to
detect and respond to bioterrorism incidents.
httDs://emeraencv.cd
c.aov/lrn/
Water
Laboratory
Alliance (WLA)
The WLA provides the Water Sector with an integrated
nationwide network of laboratories with the analytical
capability and capacity to respond to intentional and
unintentional drinking water contamination events
involving chemical, biological, and radiochemical
contaminants. The WLA structure consists of three tiers
of laboratories: sentinel, confirmatory, and reference
httDsV/www.eoa.aov/
waterlabnetwork
13
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Laboratory
Networks/
Associations
Description
Additional
Information Source*
laboratories. Sentinel labs will perform routine
monitoring and surveillance and will rule out or refer
samples to confirmatory labs for further analysis.
Confirmatory labs will perform rapid, high-confidence
presumptive and confirmatory identification of samples
referred by sentinel labs. These labs generally have
facilities with biosafety levels (BSLs) of 2 and 3.
Reference labs will provide definitive characterization of
agents and attribution of the source. These labs will
also have highly specialized containment facilities (BSL
levels of 3 and 4), and highly trained staff. Confirmatory
and reference labs will likely participate in several
laboratory networks including the LRN and the
Environmental Response Laboratory Network (ERLN).
Food
Emergency
Response
Network
(FERN)
FERN integrates the nation's food-testing laboratories at
the local, state, and federal levels into a network that is
able to respond to emergencies involving biological,
chemical, or radiological contamination of food. The
FERN structure is organized to ensure federal and state
interagency participation and cooperation in the
formation, development, and operation of the network.
The FERN plays a number of critical roles related to
food security and food defense, including prevention,
preparedness, response, and recovery. FERN provides
training, proficiency testing, method development and
validation, surveillance, electronic communication, and
laboratory outreach/cooperative agreements.
httD://www.fernlab.ora
/
Association of
Public Health
Laboratories
(APHL)
APHL promotes the role of public health laboratories in
shaping national and global health objectives, and
promotes policies, programs, and technologies which
assure continuous improvement in the quality of
laboratory practice and health outcomes. A membership
must be purchased to access most APHL publications
and services.
htt d ://www. a oh I. o rci/P
aaes/default.asox
National Animal
Health
Laboratory
Network
(NAHLN)
The USDA's NAHLN is a network of laboratories that is
organized and supported to have the capacity to
respond to animal-disease outbreaks nationwide. The
network is a cooperative effort between the USDA
Animal and Plant Health Inspection Service (APHIS),
the National Institute of Food and Agriculture (NIFA),
and the American Association of Veterinary Laboratory
Diagnosticians (AAVLD).
httDs://www.nahln.ora
/ and
httDs://www.aDhis.usd
a.aov/aDhis/ourfocus/
animalhealth/lab-info-
services/nahln
Emergency
Management
Assistance
Compact
(EMAC)
EMAC is a congressionally ratified organization that
provides form and structure to interstate mutual aid.
The EMAC mutual aid agreement and partnership
between member states exist because—from hurricanes
to earthquakes, wildfires to toxic waste spills, and
terrorist attacks to biological and chemical incidents—all
states share a common enemy: the threat of disaster.
httD://www.emacweb.
org/
*Last accessed September 11, 2017.
9.0 Tools and Databases
Table 3 lists the representative tools and databases. Uniform resource locator (URL)
can be accessed for additional information.
14
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Table 3. Representative Tools and Databases
Tool/Database
Name
Description
Additional
Information Source*
Compendium of
Environmental
Testing
Laboratories (Lab
Compendium)
EPA's Lab Compendium is a secure Web-based system
that provides users the ability to access and identify
appropriate laboratories to support specific analytical
needs. The Lab Compendium contains laboratory
records for several hundred public and private sector
environmental testing laboratories.
https://cfext.epa.aov/
cetl/lbloain.cfm?actio
n=None&CFID=3240
4&CFTOKEN=83271
178
Drinking Water
Utility Response
Protocol Toolbox
(DWRPTB) and
Wastewater Utility
Response Protocol
Toolbox
(WWRPTB)
Organized in modular format, this set of toolboxes
assists with emergency response preparedness and is
of value to drinking water and wastewater utilities,
laboratories, emergency responders, state drinking
water programs, technical assistance providers and
public health and law enforcement officials. These
modules provide emergency response planning tools
that are designed to help the water sector to effectively
and appropriately respond to intentional contamination
threats and incidents.
https ://www. e pa. a o v/
waterutilitvresponse/d
rinkina-water-and-
wastewater-utilitv-
response-protocol-
toolbox
Water Contaminant
Information Tool
(WCIT) for Priority
Contaminants
WCIT is a secure on-line database with methods for
more than 800 analytes, including detailed profiles for
over 100 chemical, biological, and radiological (CBR)
contaminants of concern for the water sector. It allows
users to compare and contrast the performance, speed,
and relative cost of analytical methods for response to
all-hazard incidents from CBR type contaminants. This
tool compiles drinking water and wastewater-specific
data in a single location to help plan for and respond to
drinking water contamination incidents. WCIT
functionality and data were shaped and validated by
water utility professionals, scientists, and public health
experts. WCIT also features a search function capable
of scanning searchable fields in the database. Users
must apply to gain access to WCIT.
https ://www. e pa. a o v/
waterdata/water-
contaminant-
information-tool-wcit
WaterlSAC
WaterlSAC is a community of water sector professionals
who share a common purpose: to protect public health
and the environment. WaterlSAC serves as a
clearinghouse for government and private information
that helps subscribers identify risks, prepare for
emergencies and secure the nation's critical water
infrastructure. Users must apply to gain access to
WaterlSAC.
http://www.waterisac.
org/
Sampling Guidance
for Unknown
Contaminants in
Drinking Water
This document provides comprehensive guidance that
integrates recommendations for pathogen, toxin,
chemical, and radiochemical sample collection,
preservation, and transport procedures to support
multiple analytical approaches for the detection and
identification of potential contaminants in drinking water.
The guidance is intended to support sampling for routine
and baseline monitoring to determine background
concentrations of naturally occurring pathogens,
sampling in response to a triggered event, and sampling
in support of remediation or decontamination efforts.
https ://www. e pa. a o v/
sites/production/files/
2017-
02/documents/sampli
na auidance for unk
nown contaminants i
n drinkina water 02
152017 final.pdf
Pathogen
Research
Databases
Los Alamos National Laboratory maintains pathogen
research databases including hepatitis C virus (HCV),
hemorrhagic fever viruses (HFV)/Ebola, and human
immunodeficiency virus.
http://lanl.aov/collabo
ration/pathoaen-
database/index.php
*Last accessed September 11, 2017.
15
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10.0 Additional Resources
American Society for Microbiology (ASM). 2013. Sentinel Level Clinical Laboratory Guidelines
for Suspected Agents of Bioterrorism and Emerging Infectious Diseases: Bacillus anthracis.
Washington DC: American Society for Microbiology.
http://www.swacm.orq/annualmeetinq/2013/handouts/20130904/Qrqanisms%20of%20Bioter
rorism 8.pdf (accessed July 27, 2016).
AOAC International. 1994. Method 993.06: "Staphylococcal Enterotoxins in Selected Foods." In:
Official Methods of Analysis of AOAC International. 16th Edition, 4th Revision, Volume I.
Gaithersburg, MD: AOAC International.
Budowle, B., Schutzer, S. E., Burans, J. P., Beecher, D. J., Cebula, T. A., Chakraborty, R., et al.
2006. Quality Sample Collection, Handling, and Preservation for an Effective Microbial
Forensics Program. Appl. Environ. Microbiol. 72(10):6431-6438.
Camarillo, M.K., Stringfellow, W.T., and Jain, R. 2014. Drinking Water Security for Engineers,
Planners, and Managers: Integrated Water Security Series. Waltham, MA: Elsevier Inc.
Centers for Disease Control and Prevention. "Emergency Preparedness and Response,
Bioterrorism Agents/Diseases." http://emerqencv.cdc.gov/aqent/aqentlist.asp (accessed July
20, 20016).
Duchaine, C., Thorne, P.S., Meriaux, A., Grimard, Y., Whitten, P., and Cormier, Y. 2001.
"Comparison of Endotoxin Exposure Assessment by Bioaerosol Impinger and Filter-
Sampling Methods." Applied and Environmental Microbiology. 67(6): 2775-2780.
Fout, G.S., Martinson, B.C., Moyer, M.W.N., and Dahling, D.R. June 2003. "A Multiplex Reverse
Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater." Applied
and Environmental Microbiology. 69(6): 3158-3164.
Holland, N.T., Smith, M.T., Eskenazi, B., and Bastaki, M. 2003. Biological Sample Collection
and Processing for Molecular Epidemiological Studies. Mutat Res. 543(3):217-34.
Huq, A., Grim, C., Colwell, R., and Nair, G.B. September 2006. "Detection, Isolation, and
Identification of Vibrio cholerae from the Environment." Current Protocols in Microbiology.
6A.5.1-6A.5.3.8.
Hunt, M.E. and Rice, E.W. 2005. Part 9000, "Microbiological Examination." In: Eaton, A.D.,
Clesceri, L.S., Rice, E.W., Greenberg, A.E., and M.A.H. Franson (eds.). Standard Methods
for the Examination of Water and Wastewater, 21st Edition. American Public Health
Association, American Waterworks Association, and Water Environment Federation, pp.
9.1-9.169.
International Air Transport Association. 2009. "Guidance Document - Infectious Substances."
International Civil Aviation Organization Dangerous Goods Panel.
http://www.icao.int/publications/Documents/quidance doc infectious substances.pdf
(accessed July 20, 2016).
Jothikumar, N., Kang, G., and Hill, V.R. 2009. "Broadly Reactive TaqMan® Assay for Real-Time
RT-PCR Detection of Rotavirus in Clinical and Environmental Samples." Journal of
Virological Methods. 155(2): 126-131.
Lawton, L.A., Edwards, C., and Codd, G.A. 1994. "Extraction and High-Performance Liquid
Chromatographic Method for the Determination of Microcystins in Raw and Untreated
Waters." Analyst. 119(7): 1525-1530.
Mangal, C.N. and Maryogo-Robinson, L. 2014. "Leveraging the Laboratory Response Network
Model for the Global Health Security Agenda." Biosecurity and Bioterrorism: Biodefense
Strategy, Practice, and Science. 12(5):274-283.
Metcalf, J.S., Beattie, K.A., Saker, M.L., and Codd, G.A. 2002. "Effects of Organic Solvents on
the High Performance Liquid Chromatographic Analysis of the Cyanobacterial Toxin
Cylindrospermopsin and Its Recovery from Environmental Eutrophic Waters by Solid Phase
Extraction." FEMS Microbiology Letters. 216(2): 159-164.
National Environmental Methods Index (NEMI). EPA, U.S. Geological Survey, National Water
Quality Monitoring Council, https://www.nemi.gov/home/ (accessed July 20, 20016).
16
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NRC. 2014. Science Needs for Microbial Forensics: Developing Initial International Research
Priorities. Board on Life Sciences, Division on Earth and Life Studies, National Research
Council, The National Academies Press, Washington, D.C.
Rees, H.B., Smith, M.A., Spendlove, J.C., Fraser, R.S., Fukushima, T., Barbour, A.G., and
Schoenfeld, F.J. 1977. "Epidemiologic and Laboratory Investigations of Bovine Anthrax in
Two Utah Counties in 1975." Public Health Reports. 92(2): 176-186.
Shabihkhani, M., Lucey, G. M., Wei, B., Mareninov, S., Lou, J. J., Vinters, H. V., Singer, E.J.,
Cloughesy, T.F., and Yong, W. H. 2014. The Procurement, Storage, and Quality Assurance
of Frozen Blood and Tissue Biospecimens in Pathology, Biorepository, and Biobank
Settings. Clinical Biochemistry 47(0):258-266.
U.S. Department of Health and Human Services, Centers for Disease Control and Prevention
and National Institutes of Health. 2007. Biosafetyin Microbiological and Biomedical
Laboratories (BMBL), 5th Edition, http://www.cdc.gov/biosafetv/publications/bmbl5/bmbl.pdf
(accessed July 20, 20016).
U.S. Department of Health and Human Services. October 2012. Federal Register Part III: 42
CFR 73. "Possession, Use, and Transfer of Select Agents and Toxins; Final Rule."
https://www.qpo.gov/fdsvs/pkq/FR-2012-10-05/pdf/2012-24389.pdf (accessed July 20,
2016).
U.S. Department of Labor. 29 CFR 1910.120, "Hazardous Waste Operations and Emergency
Response," 29 CFR 1910.120, Appendix B, "General Description and Discussion of the
Levels of Protection and Protective Gear," and 1910.38 "Emergency Action Plans,"
http://www.osha.gov/pls/oshaweb/owasrch.search form?p doc type=STANDARDS&p toe
level=1&p kevvalue=1910 (accessed July 20, 2016).
U.S. Department of Labor. 29 CFR 1926.65. "Hazardous Waste Operations and Emergency
Response."
http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=STANDARDS&p id=1
0651 (accessed September July 20, 2016).
U.S. Department of Transportation. 49 CFR 172.132. "Class 6, Division 6.1 - Definitions."
https://www.govregs.com/regulations/title49 chapterl part173 subpartD section 173.132
(accessed July 20, 2016).
U.S. Department of Transportation. 49 CFR 172.301. "General Marking Requirements for Non-
Bulk Packagings." https://www.gpo.gov/fdsys/pkg/CFR-2002-title49-vol2/pdf/CFR-2002-
title49-vol2-sec172-302.pdf (accessed July 20, 2016).
U.S. Department of Transportation. 49 CFR 173. "Shippers - General Requirements for
Shipments and Packagings." http://www.ecfr.gov/cgi-bin/text-
idx?tpl=/ecfrbrowse/Title49/49cfr173 main 02.tpl (accessed October 9, 2009).
U.S. Department of Transportation. 49 CFR 173.153. "Exceptions for Division 6.1 (poisonous
materials)." http://www.ecfr.gov/cgi-bin/text-
idx?SID=b5cfaecde208fdd9e351f389baa62305&mc=true&node=se49.2.173 1153&rgn=div
8 (accessed July 20, 2016).
U.S. Department of Transportation. 2016. 49 CFR 173.134. "Class 6, Division 6.2 - Definitions
and Exceptions." http://www.ecfr.gov/cgi-bin/text-
idx?SI D=91621 d8e4a95a61154541448bb0927fa&node=se49.2.173 1134&ron=div8
(accessed July 20, 2016).
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Substances." https://www.gpo.gov/fdsvs/granule/CFR-2013-title49-vol2/CFR-2013-title49-
vo!2-sec173-199 (accessed July 21, 2016).
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Hazardous Materials in Packing Groups I - III."
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e+49%2FSubtitle+B%2FChapter+l%2FSubchapter+C%2FPart+173%2FSubpart+E%2FSect
ion+173.211&granuleld=CFR-2010-title49-vol2-sec173-211&packageld=CFR-2010-title49-
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17
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U.S. Department of Transportation. 49 CFR 173.4. "Small Quantities for Highway and Rail."
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Villena, I., Aubert, D., Gomis, P., Ferte, H., Inglard, J-C., Dinise-Bisiaux, H. Dondon, J-M.,
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Oocyst Detection in Water." Applied and Environmental Microbiology. 70(7): 4035-4039.
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ua=1 (accessed July 20, 2016).
18
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Sample Collection Information Document - Attachment A
Attachment A:
Sample Collection Information for the Environmental Media
(Soil, Surface, Liquid, and Aerosol)
A-1
-------
Sample Collection Information Document - Attachment A
Attachment A: Table of Contents
1. Soil Sampling for Pathogens 3
2. Surface Samples 10
3. Swab Samples 11
4. Wipe Samples 11
5. Vacuum Samples 12
6. Macrofoam Swab Procedure 13
7. Cellulose Sponge Procedure 15
8. Gauze Procedure 17
9. Liquid Sampling for Pathogens 19
10. Sampling of Bioaerosols 25
11. Instrument and System Calibration 28
12. Optimal Sampling Time Determination 30
13. Air Impactor Samples 35
14. Impinger (Wet Method) Air Samples 36
15. Passive Samplers 38
16. References 43
17. Additional Bibliography 44
Attachment A Tables
Table A-1. Soil Sampling for Pathogens 4
Table A-2. Representative Soil Sampling Devices 7
Table A-3. Liquid Sampling for Pathogen 21
Table A-4. Sources and Particle Size Distribution of Bioaerosols 26
Table A-5. Bioaerosol Samplers - Common Devices and Mechanisms Involved 33
Table A-6. Comparison of Commercially Available Representative Aerosol Samplers. 34
Table A-7. Advantages and Challenges of Passive Samplers 38
Table A-8. Key Features of Bioaerosol Sampling 40
Table A-9. Manufacturers of Representative Aerosol Samplers 42
Attachment A Figures
Figure A-1. Schematic diagram of bioaerosol sampling procedure 25
Figure A-2. Typical sampling times for representative bioaerosol samplers 32
Figure A-3. Schematic diagram of passive samplers: (a) Diffusion, (b) Permeation. ... 38
A-2
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Sample Collection Information Document - Attachment A
1. Soil Sampling for Pathogens
This sample collection procedure describes the activities and considerations for the collection of
pathogens from representative soil samples. There are a wide variety of reasons for collecting
samples and various sampling strategies for different situations. Sample containers of the
proper size/composition are shown in Table A-1 and identification/selection of sampling
equipment/device are shown in Table A-2. Use of a device constructed of unsuitable material
might compromise quality by the material leaching into the sample or sorbing materials from the
sample. Even the most well designed, constructed and cleaned sampling device will yield a
non-representative sample if used improperly. Identification of the physical environment is
important in determining the potential distribution of pathogens at a given site. Pathogens can
be deposited and distributed on the surface soil with greatest concentrations in the top few
centimeters. If only a few large samples are taken at depth (e.g., 0 to 30 cm) to meet the soil
volume requirements for testing, the pathogen concentrations in the test samples after
homogenization will be diluted and probably not be representative of site conditions. A better
approach may be to collect and composite many smaller samples at shallower depths (e.g., 0 to
5 cm).
A-3
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Sample Collection Information Document - Attachment A
Table A-1. Soil Sampling for Pathogens
Soil is a natural body comprised of solids (minerals and organic matter), liquid, and gases that occurs on the
land surface, and is characterized by one or both of the following: horizons, or layers, that are distinguishable
from the initial material as a result of additions, losses, transfers, and transformations of energy and matter or
the ability to support rooted plants in a natural environment.
Soil Sampling Strategies: Size, Number and Type of Samples
Soil
Sample
Collection -
Planning/
Preparation
and
Process
Sampling Materials
• Identification and selection of sampling
equipment/device
• Sample containers of the proper size and
composition
• Quality control samples (e.g., field and/or
trip blanks, duplicates, performance
evaluation samples)
• Bound field logbook, writing instruments
(pens, pencils and permanent markers),
camera and extra charged batteries.
• Appropriate paperwork (e.g., chain of
custody, logging and calibration forms)
• Sample labels
• Reagents, preservatives, coolers and a
means to maintain sample temperature at
4°C
• Portable instrumentation and GPS unit
• Decontamination equipment for personnel
and/or equipment
• Absorbent pads
• Plastic bags for containerizing contaminated
items
• Packaging materials for sample shipment
and custody seals, appropriate shipping
containers that meet U.S. DOT/IATA or
appropriate standards
Sampling Process Most pathogenic tests can be conducted with discreet soil samples or
composite samples. The test end points measured are often the same, however, the test
design (e.g., number of replicates, test species per replicate, volume of soil per test) can be
different. Once composited samples (soil cores) are received at the testing facility, they
should be stored immediately and remain undisturbed (to mimic the field conditions) until they
are tested. Composited soil samples should be tested as soon as possible and may not be
frozen as freezing and thawing can disrupt soil structure and could influence the biological
activity.
Sample size: The minimum volume (or mass) of soil required depends on the overall objective, site
conditions, and the tests to be conducted. A few examples of impact of soil and site characteristics are
indicated below.
Bulk Density: Soil with high bulk density (e.g., sandy soil or clay rich subsurface soil) might require a greater
mass of sample compared to low bulk density soil (e.g., peat or organic rich forest soil).
Moisture Content: Moisture content at the time of collection can influence sample quantity as soil mass
requirements in a test method are recommended based on dry weight of the soil. If a site soil is very moist,
more soil should be collected than if the soil at a site is dry.
Impurities: If the site soil contains significant amount of large (>6 cm) stones, industrial debris, or plant roots,
then additional quantity of soil should be collected.
Nature, Extent, and Distribution of Pathogens: Pathogens may be deposited and distributed on the
surface soil with greatest concentrations in the top few centimeters. If a few large samples are taken at depth
(e.g., 0 to 30 cm) to meet the soil volume requirements for testing, after homogenization the pathogen
concentrations in the test samples will be diluted and probably no longer represent the site. A better
approach would be to collect many smaller samples at depths that represent the depth of contamination (e.g.,
0 to 5 cm).
Number: The number of soil samples to collect depends on the study objectives, the DQOs, the desired level
of certainty, and site-specific considerations such as predicted distribution of pathogens, the heterogeneity of
the soil, test requirements, and the size of the site. The number and location of samples can be determined
using two dimensional sampling patterns (random, transect, two-stage, and grid sampling) orthree-
dimensional sampling (information concerning depth is needed).
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Sample Collection Information Document - Attachment A
Type of Soil Samples - Point, Composite and Bulk: Point samples (or sample increments) are individual
blocks of soil removed from one location by a sampling device. Composite samples are samples comprising
two or more point samples. When point samples from different sampling locations are pooled together, the
pooled sample is a composite sample. Bulk samples are large (e.g., >1L) point samples that consist of
individual blocks of soil removed from one location by a sampling device and often collected to satisfy the
large volume requirements for biological testing.
Surface soil: Bulk soil samples are easily obtained with a shovel or a soil auger. Soil augers can be more
precise than simple shovels because they ensure that samples are taken to exactly the same depth on each
occasion as several soil factors can vary considerably with depth. To minimize pathogen contamination a
sterile spatula can be used to scrape away the outer layer of the core and use the inner part of the core for
analysis. Pathogen cross-contamination can also occur between samples, which can be avoided by cleaning
the auger after each sample is taken. The cleaning procedure involves washing the auger with water, then
rinsing it with 75% ethanol or 10% bleach, and a final rinse with sterile water. Rhizosphere soil volumes are
variable. Soil adhering to the plant roots is considered to be rhizosphere soil. Roots are normally excavated
and shaken gently to remove bulk or non-rhizosphere soil. Surface soil samples usually undergo sieving
through a 2-mm mesh to remove large stones and debris. Prior to separation, air drying may need to be
performed to facilitate sieving. However, care should be taken so that the soil moisture content does not
become too low to reduce microbial populations.
Subsurface soil: Subsurface soil samples generally have lower pathogen contents and microbial
contamination from extraneous sources during sample collection may significantly affect the numbers
counted. Mechanical approaches (such as drill rigs) may be necessary for collecting deep or shallow
subsurface samples. Air rotary drilling can be used for unsaturated systems; however, if the core barrel
overheats, pathogens within the sample may be effectively sterilized rendering the sample unrepresentative
and unusable. To avoid potential contamination from water and surfactants that are normally injected to
control dust and prevent overheating, coring can be performed slowly to avoid the need for these additives.
To limit or prevent contamination from air, all air used in the coring process can be pre-filtered through a
0.3"|jm high-efficiency particulate air (HEPA) filter. Immediately following core collection, the surface layer
from the core can be scraped away with a sterile spatula, and sub-cores can then be taken using a sterile
plastic (e.g. 60-mL) syringe with the end removed. The sample can subsequently be placed in a sterile
plastic bag or sleeve and either analyzed immediately or frozen for future analysis.
Sample Storage: Preservation Method and Maximum Holding Time
• Pathogen analyses should be performed as soon as possible (dependent on the specified holding times
for the pathogen of interest) after collection of a soil to minimize the effects of storage on pathogens.
Once removed from the field, pathogen populations within a sample can and will change regardless of
the method of storage. If immediate testing is not possible, guidance needs to be obtained for storage
and holding times allowed for the specific pathogen of interest.
• Samples should be stored in darkness (to avoid growth of algae) with free access to air (to avoid
development of anaerobic conditions).
• Samples should not be stacked, nor be too large as anaerobic conditions might develop. If samples are
stored, care should be taken to ensure that samples do not dry out and that anaerobic conditions do not
develop at the bottom of the sample.
• Samples must not dry out or become waterlogged during storage.
• Samples that are to be tested for pathogenic DNA/RNA or enzyme activity should be tested immediately.
If this is not possible, samples for DNA and phospholipids fatty acid analyses and dehydrogenase activity
analyses can be stored at -20°C for 1 to 2 years. Samples for RNA analyses can be stored at -80°C for 1
to 2 years after an initial shock-freezing with liquid nitrogen.
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Sample Collection Information Document - Attachment A
Containers for Soil Samples Collected for Pathogen Testing
Container Material of
Construction and Type
Sample Volume
(L)
Advantages
Disadvantages
HDPE bucket
10-20
• Widely available
• Inexpensive
• Rugged
• Suitable for long-term storage
• Can influence organic
co-contaminants
analyses
SS bucket with push-fit
lids
5-20
• Commercially available
• Reasonably priced
• Rugged
• Suitable for VOCs
• Suitable for long-term storage
• Need specialized
equipment to seal
buckets
Polyethylene bag
Up to 60
• Usable as a bucket liner for
samples contaminated with
inorganics
• Not rugged
Teflon bag
Up to 60
• Chemically inert and solvent
resistant to most chemicals
• Can be used as a bucket liner or
as a sample container by itself
• Not rugged
Glass wide-mouthed jars
with
polyethylene/polypropyle
ne caps or HDPE lids
0.125-2
• Widely available
• Inexpensive
• Suitable for long-term storage
• Not rugged
• Can only contain small
sample volumes
Plastic* wide-mouthed
jars with plastic caps
and HDPE lids
0.125-4
• Widely available
• Inexpensive
• Rugged
• Suitable for long-term storage
• Can only contain small
sample volumes
• Not suitable for non-
weathered organics
HDPE, high density polyethylene; SS, stainless steel; VOC, volatile organic compound
*Plastic materials include polypropylene, polystyrene, HDPE, and polystyrene.
A-6
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Sample Collection Information Document - Attachment A
Table A-2. Representative Soil Sampling Devices
Device
Sample
Type
Soil Type
Soil Sample
Area/Volume
Penetration
Depth
Advantage
Disadvantage
References
Shovel.
Scoop,
Spoon,
Trowel,
Spade
U
All soil
types
including
non-
cohesive
sandy or
loose soils
0.5 to 4 L
Surface,
shallow
subsurface
• Collection of large volumes
of soil can be done quickly
and easily
• Collects blocks of soil
• Easy to decontaminate
• Samples can be biased
because of shape and
imprecise volume. Bias can
be minimized by careful
sample collection.
Prevost and
Antoun,
2008
Cutting/
Sampling
Frame
u
Organic
horizon(s),
mineral A
horizon(s)
100 to 900
cm3
Surface
• Efficient way to collect
representative bulk sample
• Can be difficult to remove
all soil within frame
Belanger
and Van
Rees, 2008
Ring Sampler
C orU
Cohesive
soils
0.5 to 20 cm
diameter
Surface
• Easy to use
• Precise core
• Not as useful for
unconsolidated soils or hard
clay
ISO, 2002
Bulb Planter
C or U
Cohesive
soils
1.5 L
Surface (0
to 15 cm)
• Large core - higher volume
• Not useful for hard soils
Dalpe and
Hamel,
2008
Cutting
Cylinder (Soil
Punch)
C or U
Organic, A
horizon
59 to 556 cm2
Surface
• Soil cores are large and can
efficiently collect large
volume
• Can compress soil samples
Belanger
and Van
Rees, 2008
Soil Corer
C or U
Cohesive
soils
2.5 to 10 cm
(dia.)
30 to 60 cm
(height)
0 to 60 cm
• Easy to use
• Precise core
• Easy to clean
• Can use liner or sample
tube
• Compaction when driving
corer into soil
• Cores not truly disturbed
unless linear used
USEPA,
2006
Slide-hammer
Core Sampler
Co or
U
Cohesive
soils
2.5 to 10 cm
(dia.)
30 to 60 cm
(height)
0 to 60 cm
• Easy to use
• Precise core
• Easy to clean
• Can use liner or sample
tube
• Compaction when driving
corer into soil
• Cores not truly disturbed
unless linear used
EC and
SRC, 2007
Auger
U
Cohesive
soils
2.5 to 15 cm
long
0 to 60 cm
• Easy to use
• Can handle various types of
soils
• Less precise sample than
coring device
• Hard to decontaminate
• Modifies soil matrix
• Can introduce artifacts into
soil sample
Mason,
1992
A-7
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Sample Collection Information Document - Attachment A
Split Spoon/
Tube Sampler
C or U
Cohesive
soils and
hard soils
Variable (up
to 10 cm
(dia.) and up
to 2 kg
sample
• Easy to use
• Precise core
• Large cores
• Can use liner
• Deep cores can only be
obtained using drilling rig
Weinfurtner
and Kordel,
2007
Shelby Tube
Sampler
C or U
Cohesive
soils and
hard soils
Variable (up
to 10 cm
(dia.)
0 to 40 cm
or
0 cm to
bedrock
• Easy to use
• Precise core
• Large cores
• Can use liner
• Deep cores can only be
obtained using drilling rig
• Not durable in hard soils
CCME,
1993
Non-
cohesive
Piston
Samplers
C or U
soils, wet
soils, wet
clay, dry
and wet
peat
Variable
Surface
Shallow
subsurface
• Holds moisture and fine
materials in place in sample
• Can be difficult to operate
Mason,
1992
Tubes:5 or 7
Direct Push
Corer
(GeoProbe™)
cm (dia.) and
• Saturated sands and silts
• Must use a drill rig
c
Cohesive
1.2 m long
Surface
can be collected
• Not optimal in wet condition
ASTM,
soils
Size of
probes and
liners vary
Subsurface
• Consolidated samples used
to classify soils
with stony soils or soils with
high clay content
2008
Rotary Auger
with lined or
c
Cohesive
soils and
Variable
Surface to
• Saturated sands and silts
• Must use a drill rig
• Not suitable for stony soils
ASTM,
unlined core
soft
bedrock
can be collected
• Modified soil matrix
2009
barrels
bedrock
• Can introduce artifacts
• Must use a drill rig
Cohesive
• Limited by stony soils
Rotary (solid
u
soils,
frozen
15 cm and
Surface to
• Easy to use
• Faster than hollow stem
• Sample depth
determination can be
ASTM,
stem) Auger
soils, and
soft
bedrock
larger
bedrock
• Provides continuous
lithology information
imprecise due to auger
sample spin up
• Modified soil matrix
• Can introduce artifacts
2009
PIPE and Emergency Equipment
Depending on site and pathogen specific health and safety plan (HASP) to be followed.
C, consolidated; U, unconsolidated
A-8
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Sample Collection Information Document - Attachment A
References for Table A-2
ASTM (American Society for Testing and Materials). 2008. Standard practice for
decontamination of field equipment used at nonradioactive waste sites. D5088-90. American
Society for Testing and Materials, West Conshohocken, PA.
ASTM. 2009. Practice for soil investigation and sampling by auger borings. D1452. American
Society for Testing and Materials, West Conshohocken, PA.
Belanger, N., and Van Rees, K.C.J. 2008. Sampling forest soils. In: Carter, M.R., Gregorich,
E.G., (eds.) Soil sampling and methods of analysis. Sponsored by the Canadian Soil
Science Society. Boca Raton, FL: Taylor and Francis, pp. 15-24.
CCME (Canadian Council of Ministers of the Environment). 1993. Guidance manual on
sampling, analysis and data management for contaminated sites - Volume I. CCME
Subcommittee on Environmental Quality Criteria for Contaminated Sites, the National
Contaminated Sites Remediation Program, Winnipeg, Manitoba.
Dalpe, Y., Hamel, C. 2008. Vesicular-arbuscular mycorrhiza. In: Carter, M.R. (editor) Soil
sampling and methods of analysis. Lewis, Boca Raton, pp 287-302.
Drielak, S.C. 2004. Hot Zone Forensics: Chemical, Biological, and Radiological Evidence
Collection. Charles C. Thomas Publisher, Ltd., Springfield, Illinois.
EC and SRC (Environment Canada and Saskatchewan Research Council). 2007. Validation of
toxicology test methods for assessing petroleum hydrocarbon and brine spills in boreal
forest soils. Prepared for: Environmental Research Advisory Council, Canadian Association
of Petroleum Producers. Prepared by: Biological Methods Division Environment Canada and
Environment and Forestry Division Saskatchewan Research Council.
ISO. 2002. 10381-2. Soil quality Sampling — Part 2: Guidance on sampling techniques.
International Organization for Standardization, Geneva, Switzerland.
Mason, B.J. 1992. Preparation of soil sampling protocols: Sampling techniques and strategies.
U.S. Environmental Protection Agency, Office of Research and Development, Washington,
DC 20460, EPA/600/R-92/128.
Prevost, D., and Antoun, H. 2008. Root nodule bacteria and symbiotic nitrogen fixation. In:
Carter, M.R., Gregorich, E.G. (eds.), Soil sampling and methods of analysis (2nd edition).
Boca Raton, FL: CRC Press, pp 379-397.
U.S. Environmental Protection Agency (USEPA). 2006. Wadeable stream assessment: A
collaborative survey of the Nation's streams. U.S. EPA, Office of Water, Washington, DC.
EPA 841-B-06-002.
Weinfurtner, K., and Kordel, W. 2007. Umweltprobenbank des Bundes. Guidelines for
sampling and sample processing. Soil. Guidelines for sampling, transport, storage and
chemical characterization of environmental and human-organ samples, Umweltbundesamt,
Germany.
Additional Resources
ISO. 2009. Soil quality - Sampling - Part 6: Guidance on the collection, handling and storage of
soil under aerobic conditions for the assessment of microbiological processes, biomass and
diversity in the laboratory. International Organization for Standardization, Geneva,
Switzerland. NFIS010381-6.
ISO 15799, Soil quality - Soil Quality - Guidance on the ecotoxicological characterization of
soils and soil materials. International Organization for Standardization, Geneva, Switzerland.
NSTC. 2009. Planning Guidance for Recovery Following Biological Incidents, Biological
Decontamination Standards Working Group, Subcommittee on Decontamination Standards
and Technology Committee on Homeland and National Security, National Science and
Technology Council.
A-9
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Sample Collection Information Document - Attachment A
U.S. EPA. 2002. Guidance on Choosing a Sampling Design for Environmental Data Collection.
U.S. Environmental Protection Agency, Washington, DC. EPA/240/R-02/005.
U.S. EPA/USGS. 2014. Sample Collection Protocol for Bacterial Pathogens in Surface Soil.
EPA/600/R-14/027.
2. Surface Samples
Surface sampling involves collecting microbial contaminants from a surface using an
appropriate sampling device to determine the presence of pathogens. Swabs, wipes, Sponge-
Sticks (3M, Solar-Cult, or equivalent), and vacuum filter, socks or cassettes are the primary
collection devices for spores on surfaces and are used during all phases (identification,
characterization, decontamination, and clearance) of a response (CDC 2012).
Determining the most appropriate type of surface sample collection method depends on whether
porous or non-porous surfaces are to be sampled. Wipes and swabs should be used on non-
porous surfaces while vacuum socks or filter cassettes should be used on porous surfaces
(Raber, 2006). Examples of non-porous surfaces include: stainless steel, painted wallboard,
glass, floor tile, and wood laminate. Examples of porous surfaces include: ceiling tile, fabrics,
carpet, clothing, rugs, and upholstered furniture.
When collecting samples for pathogen on porous surfaces, use of wipes can be considered,
because some studies have demonstrated higher recovery efficiencies when wipes were used
to sample carpet and upholstery than when vacuum methods were used (Buttner et al. 2004,
Estill et al. 2009, Valentine et al. 2008). Rayon/polyester or cellulose/polyester blends are
superior to cotton wipes (Valentine et al. 2008). Vacuum sampling is also effective for spore
collection from carpet or upholstery and could be used on these surfaces if high concentrations
(> 102 spores/cm2) are expected (Brown et al. 2007).
Certain solutions (wetting agents) can be used to pre-moisten biological collection devices to
enhance their overall performance. Common solutions include sterile water, sterile saline,
neutralizing buffer, sterile phosphate buffer, and peptone buffer. In addition, surfactants (such
as Tween® 80, Tween® 20, or Pluronic®) can be added to these pre-moistening solutions to
improve removal of spores from surfaces. Neutralizing solutions block the continued action of a
disinfectant after sampling. These neutralizing solutions are important during post-
decontamination activities (verification and clearance sampling) to ensure that samples, when
analyzed properly, are not falsely negative due to the presence of residual disinfectant. Among
available neutralizing solutions are:
• Butterfield's buffer with 0.02% Tween 80 (Tween 80 is effective in neutralizing phenolic
compounds and acting as a surfactant)
• Day Engley broth (Becton Dickinson, Sparks, MD) [neutralizes chlorine compounds and
iodine, but may encourage growth during transport]
• Neutralizing Buffer (Becton Dickinson) [contains sodium thiosulfate to neutralize chlorine
compounds and aryl sulfonate complex to neutralize quaternary ammonium compounds]
• Neutralizing Buffer (Hardy Diagnostics) [contains aryl sulfonate complex to neutralize
quaternary ammonium compounds, sodium thiosulfate to neutralize chlorine compounds,
potassium phosphate to maintain the pH, and sodium hydroxide]
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Sample Collection Information Document - Attachment A
• Letheen broth (Becton Dickinson [neutralizes quaternary ammonium compounds, but may
encourage growth during transport]
• Phosphate Buffered Saline, pH 7.2 with 0.02% Tween 80 [Tween 80 is effective at
neutralizing phenolic compounds at appropriate concentrations and acts as a surfactant]
Similar recovery efficiencies (26.8 — 39.0%) have been obtained with wipes pre-moistened with
each of these neutralizing buffers that were processed by the LRN processing procedure. The
choice of neutralizing solution depends on the disinfectant used. During the initial identification
and characterization of a contaminated building, collection devices with a neutralizing solution
are less important.
There are factors that will affect the choice of which wetting solutions to use for pre-moistening
swabs and wipes for sampling. For example, phosphate-containing solutions (e.g., Butterfield's
buffer and phosphate buffered saline [PBS]) may inhibit polymerase chain reaction (PCR)
assays if appropriate DNA extraction and purification is not performed; the use of Dey Engley or
Letheen broth may encourage germination and growth during transport. Sterile saline will not
neutralize the action of a sporicide or chemical. However, neutralization may not be a concern
during characterization sampling (on surfaces that do not already contain sporicides).
Some of the sampling devices can be purchased pre-moistened or they can be pre-moistened
prior to collecting a sample. The Centers for Disease Control and Prevention (CDC)
recommends the use of a neutralizing buffer as the pre-moistening solution in their validated
swab and wipe-sampling and analysis methods (CDC 2012). The CDC developed methods for
processing macrofoam swab and cellulose sponge wipe samples collected on environmental
surfaces. These processing protocols use traditional culture methods and yield semi-
quantitative estimates of the amount of pathogen contamination in a sample. The CDC
collection procedures for the validated swab and wipe method and a non-validated gauze
method are provided on the CDC website at http://www.cdc.gov/niosh/topics/emres/surface-
sampling-bacillus-anthracis.html.
3. Swab Samples
Swabs are appropriate for sampling small [26 square centimeters (cm2)] non-porous surfaces.
Swabs work best for small areas like crevices, corners, supply air diffusers, air return grills, and
hard-to-reach places. The CDC currently recommends using macrofoam swabs for the
collection of Bacillus anthracis spores on smooth, non-porous surfaces (CDC 2012). The
Laboratory Response Network (LRN) laboratories are capable of processing samples collected
in accordance with this sample collection protocol using the prescribed swab type.
4. Wipe Samples
Wipes are appropriate for sampling larger (e.g., 645 cm2 per CDC sampling method) non-
porous surfaces, such as walls, desks, and non-carpeted floors. Wipe sampling can be
performed using either cellulose sponges or gauze. Sponge-Sticks (3M, Solar-Cult, or
equivalent) are sponge wipes with handle and are therefore preferred for surface sampling. The
CDC currently recommends using a cellulose sponge wipe for the collection of B. anthracis
spores on smooth, non-porous surfaces (CDC 2012). The LRN laboratory or laboratories that
will be analyzing the sponge wipe samples should be consulted prior to using this collection
method to determine if that laboratory is capable of processing and analyzing the sample.
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Sample Collection Information Document - Attachment A
5. Vacuum Samples
The primary sample collection method for sampling large porous surfaces (> 600 cm2) for B.
anthracis spores is vacuum sampling using filter socks or cassettes. Collecting samples by
vacuuming is advantageous for covering large, non-porous and porous surfaces such as
carpeting, ceiling tiles, ventilation systems filters, and upholstered furniture. This type of
sampling also works well for capturing bulk powder or dust in hard-to-reach places. Vacuum
sampling is also the best choice if sensitive items such as electronics and personal items are a
concern, since it is less likely to cause damage compared to pre-moistened swabs and wipes.
The laboratories analyzing the vacuum filter socks or cassettes should be consulted prior to
using this collection method to determine if that laboratory is capable and willing to process this
sample type, since at this time there are no LRN-approved processing methods for either
device. Vacuum sampling and analysis methods have been evaluated for their performance to
collect a surrogate spore (B. atrophaeus) contamination from carpet, concrete, upholstery and
HVAC filters (USEPA 2013).
During vacuum sampling, bulk material is trapped by the dry collection media/filter by utilizing a
small, HEPA vacuum cleaner or a small sampling pump to draw air through the filter. A number
of sampling devices can be used to collect samples from porous materials including filter socks,
3M Forensics Vacuum filters, or 37 mm cassettes. The filter sock method utilizes a filter sock
and attachment nozzle that fits onto the inlet nozzle of a HEPA vacuum hose. The 3M
Forensics Vacuum filter is favored by law enforcement groups due to its ease of use in evidence
collection protocols. This filter also attaches to a HEPA vacuum cleaner hose for sampling,
though care should be exercised to regulate the power of the vacuum so the filter integrity is not
compromised during sampling. The last option uses micro-vacuuming techniques to collect a
sample using personal sampling pumps or carbon vane pumps. These pumps utilize a suitable
filter contained in a closed-face, conductive sampling cassette to which a short section of plastic
tubing cut at a 45° angle is added to the inlet. In the EPA comparison (USEPA 2013) the 37mm
vacuum cassettes were found to be more efficient than the vacuum socks at collecting the
spores from multiple surfaces. Filter cassettes were also determined to be safer for samplers
and laboratorians to handle because the filter is sealed within a plastic case, thus reducing
potential for exposures. The EPA methods for collecting vacuum filter sock samples and 37 mm
vacuum cassettes samples (USEPA 2013) are described in Attachment C. Information on
proper packaging and shipping of vacuum socks can be found on the CDC website (CDC 2012).
Vacuum sock samples must be collected using only HEPA filtered vacuum pumps.
Conventional home or industrial vacuum cleaners should not be used for sample collection,
because they can further disperse spores as filtration is not highly efficient.
Three of the CDC surface sampling procedures (macrofoam swab, Cellulose Sponge, and
gauze) for Bacillus anthracis spores from smooth, non-porous surfaces are indicated as
examples in the following sections.
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Sample Collection Information Document - Attachment A
6. Macrofoam Swab Procedure
Swab Materials
1. Gloves, nitrile
2. Ruler, disposable, and masking tape or sample template, disposable, sample area size 4
in2 (26 cm2)
3. Macrofoam swab, sterile, 3/16-inch thick medical-grade polyurethane foam head, 100
pores per inch, thermally bonded to a polypropylene stick (such as the Sterile Foam
Tipped Applicators Scored with Thumb Stop [Puritan, Guilford, Maine; catalog number
25-1607 1PF SC] or equivalent)
4. General neutralizing buffer that will inactivate halogen disinfectants and quaternary
ammonium compounds, 10 milliliter (ml_), sterile (such as the Neutralizing Buffer [Hardy
Diagnostics, Santa Maria, California; catalog number K105] or equivalent)
5. Screw-cap centrifuge tubes, sterile, 15 ml_ (such as 15 ml_ High-Clarity Polypropylene
Conical Centrifuge Tube [Becton Dickinson, Franklin Lakes, New Jersey; catalog
number 352097] or equivalent)
6. Sample labels or permanent marker
7. Re-sealable plastic bag, 1-quart or smaller
8. Re-sealable plastic bag, 1-gallon or larger
Swab Sampling Procedure
1. Wearing a clean pair of gloves over existing gloves, place the disposable template over
the area to be sampled and secure it. If the template cannot be used, measure the
sampling area with a disposable ruler, and delineate the area to be sampled with
masking tape.
2. Remove the sterile swab from its package. Grasp the swab near the top of the handle.
Do not handle below the thumb stop.
3. If the sterile swab is not pre-moistened, moisten the sterile swab by dipping it in the 10
ml_ container of neutralizing buffer solution. Remove any excess liquid by pressing the
swab head on the inside surface of the neutralizing buffer solution container.
Note: Once a sterile swab has been moistened, the remaining neutralizing buffer
solution and container must be discarded.
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Sample Collection Information Document - Attachment A
4. Swab the surface to be sampled using the moistened sterile swab. Use an overlapping
'S' pattern to cover the entire surface with horizontal strokes.
Note: Depending on the design of the swab, a rolling motion can be used when
swabbing the surface to maximize swab contact with the surface.
5. Rotate the swab and swab the same area again using vertical 'S'-strokes.
6. Rotate the swab once more and swab the same area using diagonal 'S'-strokes.
7. Place the head of the swab directly into a sterile screw-capped centrifuge tube. Break off
the head of the swab by bending the handle. The end of the swab handle, touched by
the collector, should not touch the inside of the tube. Securely tighten the screw-cap and
label the tube (e.g., unique sample identifier, sample location, initials of collectors and
date and time sample was collected). Collection tubes and re-sealable bags may be pre-
labeled to assist with sampling efficiency.
8. Place the sample container in a re-sealable 1-quart plastic bag. Securely seal and label
the bag (e.g., sample location, date and time sample was collected, and name of
individual collecting the sample).
Note: Remove excessive air from the re-sealable plastic bags to increase the number of
samples that can be shipped in one container.
9. Dispose of the template, if used.
10. Remove outer gloves and discard. Clean gloves must be worn for each new sample.
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7. Cellulose Sponge Procedure
Cellulose Sponge Materials
1. Gloves, nitrile
2. Ruler, disposable, and masking tape or sample template, disposable, sample area size
100 in2 (645 cm2)
3. Sponge, sterile, pre-moistened with 10 ml_ neutralizing buffer solution, 1.5 by 3 inches
cellulose sponge folded over a handle (such as the 3M™ Sponge-Stick [3M, St. Paul,
Minnesota; catalog number SSL-10NB] or equivalent)51 or sponge, sterile, dry, 1.5 by 3
inches cellulose sponge folded over a handle (such as the 3M™ Sponge-Stick [3M, St.
Paul, Minnesota; catalog number SSL-100] or equivalent) and general neutralizing buffer
that will inactivate halogen disinfectants and quaternary ammonium compounds, sterile,
10 ml_ (such as the Neutralizing Buffer [Hardy Diagnostics, Santa Maria, California;
catalog number K105] or equivalent)
4. Screw-cap specimen container, sterile, individually wrapped 4 ounce (such as General
Purpose Specimen Container [Kendall Healthcare, Mansfield, Massachusetts; catalog
number 8889-207026] or equivalent)
5. Sample labels or permanent marker
6. Re-sealable plastic bag, 1-quart or smaller
7. Re-sealable plastic bag, 1-gallon or larger
Cellulose Sponge Sampling Procedure
1. Wearing a clean pair of gloves over existing gloves, place the disposable template over
the area to be sampled and secure it. If a template cannot be used, measure the
sampling area with a disposable ruler, and delineate the area to be sampled with
masking tape. The surface area sampled should be less than or equal to 100 in2 (645
cm2).
2. Remove the sterile sponge from its package. Grasp the sponge near the top of the
handle. Do not handle below the thumb stop.
3. If the sterile sponge is not pre-moistened, moisten the sponge by pouring the 10 mL
container of neutralizing buffer solution over the dry sponge.
Note: The moistened sponge should not be dripping neutralizing buffer solution.
Note: Any unused neutralizing buffer solution must be discarded.
4. Wipe the surface to be sampled using the moistened sterile sponge by laying the widest
part of the sponge on the surface, leaving the leading edge slightly lifted. Apply gentle
but firm pressure and use an overlapping 'S' pattern to cover the entire surface with
a Additional sponges with limited recovery efficiency data available include the Versalon Non-Woven Ail-Purpose Gauze Sponge (Kendall
Healthcare, Mansfield, Massachusetts; catalog number 8042), Bacti-Sponge (Hardy Diagnostics, Santa Maria, California; catalog number SK711),
Cellulose Sponge with DE Broth (Solar Biological, Ogdensburg, New York; catalog number BS-10BPB-1), and Sponge-Wipe (Micronova, Torrance,
California; catalog number SWU-99 [cut into 2 by 2 inches).
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horizontal strokes.
5. Turn the sponge over and wipe the same area again using vertical 'S'-strokes.
6. Use the edges of the sponge (narrow sides) to wipe the same area using diagonal 'S'-
strokes.
8. Place the head of the sponge directly into a sterile specimen container. Break off the
head of the sponge by bending the handle. The end of the sponge handle, touched by
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the collector, should not touch the inside of the specimen container. Securely seal and
label the container (e.g., unique sample identifier, sample location, initials of collector
and date and time sample was collected).
9. Place the sample container in a re-sealable 1-quart plastic bag. Securely seal and label
the bag (e.g., sample location, date and time sample was collected, and name of
individual collecting the sample). Specimen containers and re-sealable bags may be pre-
labeled to assist with sampling efficiency.
Note: Remove excessive air from the re-sealable plastic bags to increase the
number of samples that can be shipped in one container.
10. Dispose of the template, if used.
11. Remove outer gloves and discard. Clean gloves should be worn for each new sample.
8. Gauze Procedure
Gauze Materials
Note: This sampling and analytical method has not been validated by CDC. A standard
sampling procedure is provided in the event that the macrofoam swab or cellulose sponge
methods cannot be utilized.
1. Gloves, nitrile
2. Gloves, sterile, nitrile
3. Ruler, disposable, and masking tape or sample template, disposable, sample area
between 144 in2 (929 cm2)
4. Gauze, sterile, non-cotton, polyester blend sponge or rayon/polyester blend, 2 inches x
2 inches (such as the Versalon Ail-Purpose Sponge [Kendall Healthcare, Mansfield,
Massachusetts; catalog number 8042; includes two gauze squares/packet] or
equivalent)
5. General neutralizing buffer that will inactivate halogen disinfectants and quaternary
ammonium compounds solution, 10 ml_, sterile (such as the Neutralizing Buffer [Hardy
Diagnostics, Santa Maria, California; catalog number K105] or equivalent)
6. Pipette, 5 ml_, sterile, individually wrapped (such as the Greenwood Products' Sterile
5ml_ Standard Transfer Pipette [Greenwood Products, Inc., Middlesex, New Jersey;
catalog number GS137038] or equivalent)
7. Screw-cap specimen container, 4-ounce, sterile, individually wrapped (such as General
Purpose Specimen Container [Kendall Healthcare, Mansfield, Massachusetts; catalog
number 8889-207026] or equivalent)
8. Sample labels or permanent marker
9. Re-sealable plastic bag, 1-quart or smaller
10. Re-sealable plastic bag, 1-gallon or larger
Gauze Sampling Procedure
1. Wearing a pair of gloves over existing gloves, place the disposable template over the
area to be sampled and secure it. If the template cannot be used, measure the sampling
area (144 in2) with a disposable ruler, and delineate the area to be sampled with
masking tape.
2. Partially peel open the sterile gauze package carefully exposing the gauze.
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Sample Collection Information Document - Attachment A
Note: The sterile gauze should riot be touched without sterile gloves.
3. Measure 5 mL of neutralizing buffer solution from the 10 mL container using a
disposable pipette and apply to sterile gauze in its original packaging. Remove outer
gloves.
Note: The moistened gauze should not be dripping neutralizing buffer solution.
Note: Any unused neutralizing buffer solution and the pipette must be discarded.
4. Don a pair of sterile gloves.
Note: Sterile gloves are required when sampling with gauze because of the direct
contact with the sampling media.
5. Remove one of the sterile gauze (if two per package) and dispose of or retain the other
gauze as a field blank (see section 4.1).
6. Completely unfold the remaining moistened sterile gauze, and then fold in half.
7. Wipe the surface to be sampled using the moistened sterile gauze, fingertips should be
held together and apply gentle but firm pressure. Use an overlapping S' pattern to cover
the entire surface with horizontal strokes.
8. Fold the exposed side of the gauze in and wipe the same area again using vertical'S'-
strokes.
9. Fold the exposed side of the gauze in once more and wipe the same area using
diagonal 'S'-strokes.
10. Fold the gauze, exposed side in, and place it into a sterile screw-cap specimen
container.
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11. Securely tighten the screw-cap and label the container (e.g., unique sample identifier,
sample location, initials of the collectors and date and time sample was collected).
12. Place the sample container into a re-sealable 1-quart plastic bag. Securely seal and
label the bag (e.g., sample location, date and time sample was collected, and name of
individual collecting the sample). Specimen containers and re-sealable bags may be pre-
labeled to assist with sampling efficiency.
Note: Remove excessive air from the re-sealable plastic bags to increase the
number of samples that can be shipped in one container.
13. Dispose of the template, if used.
14. Remove outer gloves and discard. Clean sterile gloves should be worn for each new
sample.
9. Liquid Sampling for Pathogens
Liquids are often easier to collect but obtaining representative samples may still be difficult.
Density, solubility, temperature, and other factors/properties can cause changes in the
composition of a liquid in both time and space. Sampling must be responsive to these dynamics
to ensure collection of representative samples. The objective prior to sample collection must
always be clear. Indoor (e.g., small fish tank in an office to large storage tank or indoor pool in
multistoried building) or outdoor settings may include a variety of liquids: surface water,
wastewater, and containerized liquids. Liquid sampling in a flowing indoor conduit/channel
should proceed from downstream locations to upstream locations so that disturbances related to
sampling do not affect sampling quality. The opening of the sampling device or container
should face upstream. If water and solid samples need to be collected during the same
sampling event, they must be co-located, and the aqueous samples should be collected first.
When possible, sumps and monitoring manholes at which sampling is required should be
suctioned to remove any accumulated silt or floating layer, then allowed to refill before sampling
begins. It is essential to prevent accidental intake of such material into a sampler when
intending to assess qualities of bulk liquids. When taking a grab sample, the entire mouth of the
container should be submerged below the surface of the liquid. A wide mouth bottle with an
opening of at least two inches can be used for this type of sampling.
For shallow waters, samples may be collected by directly filling the sample bottle. For deeper
water layers, below about 0.5 m, these methods may not work, so dedicated water samplers
can be used. They are lowered in an open condition on a rope or steel cable and remotely
triggered to close. A third option is the use of pumps (e.g., peristaltic pumps offer the option of
collecting larger amounts of water). For example, a biological agent grab sample can be
obtained in the following manner:
• Take a bacteriological sample container and remove the covering and closure (protect from
contamination).
• Grasp the container at the base with one hand and plunge the container (opening down) into
the water to avoid introducing surface scum.
• Do not rinse the container.
• Position the mouth of the container into the current away from the hand of the collector and
away from the sampler location.
• The sampling depth could be 15 to 30 cm (6 to 12 inches) below the water surface under
certain conditions. If the water is static, an artificial current can be created by moving the
container horizontally in the direction it is pointed and away from the sampler.
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Sample Collection Information Document - Attachment A
• Tip the container slightly upward to allow air to exit and the container to fill.
• After removal of the container from the water, pour out a small portion of the sample to allow
an air space of 2 to 3 cm (1 inch) above the sample for proper mixing of the sample before
analysis.
• Tightly close and label the container.
When collecting a sample at a depth greater than an arm's reach use a Kemmerer or weighted
container sampler. The devices are lowered into the water in the open position, and a water
sample is collected in the device. A drop messenger closes the sampler. Appropriate
sterilization and cleaning protocols should be followed. Sample collection frequency for
pathogens should be appropriate for the investigation objectives.
Table A-3 provides representative liquid samplers for a variety of environmental settings, the
procedures, advantages and disadvantages. Appropriate sampling methods and sampling
devices should be determined based on the site specific conditions. Appropriate care should be
taken to avoid limitations such as (a) spot water sampling that reflect residue composition only
at the moment of sampling and may fail to detect episodic contamination; (b) quality control
issues when, for example, large volumes of water must be collected and extracted for
quantifying and assessing biological pathogens. An ideal sampling device for water should be
one that is:
• Made of materials that are inert to or non-interfering with the pathogen detection method
• Able to deliver sample without causing biological, chemical or physical alteration
• Compatible with the bioassay sensitivity
• Easily operated under the indoor settings
• Easily disassembled for cleaning and maintenance
• Easily transported to indoor locations
• Reliable and durable to use and able to withstand potentially hostile environments
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Table A-3. Liquid Sampling for Pathogen
Designation
Typical Setting
Salient Features/Procedure
Advantage
Disadvantage
Dipper/Pond
sampler/
Swing
sampler
Water/wastewater
from aquarium,
pits, or other
reservoirs
• Assemble the pond sampler to be performed by
making sure that the sampling container and
fixtures are secured to the pole.
• Slowly submerge the container with minimal
surface disturbance. Retrieve the sampler from
the surface water with minimal disturbance.
• Remove the cap from the sample bottle and
slightly tilt the mouth of the bottle below the
dipper/device edge.
• Empty the sampler slowly, allowing the stream
to flow gently down the inside of the bottle with
minimal entry turbulence.
• Repeat above three steps until sufficient sample
volume is acquired. Dismantle the sampler, if
applicable and store in plastic bags for
subsequent decontamination.
• Relatively inexpensive to
fabricate
• Can sample depths or
distances up to 3.5m
• Difficult to obtain
representative samples in
stratified liquids
• Difficult to decontaminate
when handling viscous
liquids
Weighted
Bottle
Sampler
Tanks, wells,
sumps, or other
reservoirs
• Sampler consists of a bottle, usually glass or
plastic, a weight sinker, and a bottle stopper.
• Assemble the weighted bottle sampler. Lower
the sampling device to the predetermined depth.
• When the sampler is at the required depth, pull
out the bottle stopper with a sharp jerk of the
sampler line and allow the bottle to fill
completely (usually evidenced by the cessation
of air bubbles)
• Retrieve sampler. Transfer sample into
laboratory cleaned sample bottles, if applicable.
Follow procedures for preservation and
transport.
• Sampler remains unopened
until at sampling depth
• Laboratory supplied bottle
may not fit into sampler,
thus requiring additional
equipment.
• Some mixing of sample
may occur when retrieving
the sampler from depth.
Open Tube
Thief
Sampler
Versatile, e.g.
may be used to
sample water
from sump areas
in homeowner
basements
• A hollow glass or rigid plastic tube, which is
anywhere from four to five feet in length. It
generally has an inside diameter of %-inch or 1/4-
inch.
• Inexpensive
• Simplicity of operation
• Small puddle of liquid can
be collected, which other
samplers may not
• Disposable
• Sample leakage
• Small sample volume
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Sample Collection Information Document - Attachment A
Syringe
Collects
representative
small volume
liquid samples in
puddles
• Use the syringe to draw the sample from the top
of the container or puddle by pulling the plunger.
Syringe plunger may become difficult to push
while handling slurry due to clogging. Once you
encounter moderate resistance, do not push
harder and you may have to start again.
Syringes should be kept in clean containers or
original packaging until ready for use to prevent
contamination (e.g., keep both wrapped in
original package or in new/clean plastic baggies
until actually collecting and/or filtering the
sample). Under certain indoor conditions,
accessory equipment may be necessary for
operation of syringe sampler is a hand pump
and a length of tubing to supply
negative/positive pressure to the syringe to
actuate the piston.
• Samples does not come in
contact with atmospheric
gas and is subjected to a
negative pressure, thus
neither aeration nor
degassing of the sample
occurs
• Syringes are or can be
made inert or nearly inert
materials
• Syringe can be utilized as
sample container, thus
removing the possibility of
cross-contamination
• Inexpensive, highly portable
and simple to operate
• Inefficient to collect large
volume of samples
• Limited to water with a low
suspended solids content
• Leakage may occur
around the plunger when
syringes are used to
sample high levels of
suspended solids.
Kemmerer
Depth
Sampler/
Van Dorn
sampler/
Niskin bottle
Liquid samples in
storage tank, tank
trailer, vacuum
tanks, or other
situations where
collection depth
prevents use of
other sampling
devices
• Sampling device consists of an open tube with
two sealing end pieces. Niskin sampler has the
same design as the Van Dorn sampler except
that it can be cast in a series on a single line for
simultaneous sampling at multiple depths with
the use of auxiliary messengers.
• Set the sampling device so that the sealing end
pieces are pulled away from the sampling tube,
allowing the substance to pass through the tube.
• Lower the pre-set sampling device to the
predetermined depth.
• When the sample is at the required depth, send
down the messenger, closing the sampling
device.
• Retrieve sampler. Transfer sample into
laboratory cleaned sample bottles (if applicable)
and follow procedures for preservation and
transport
• Able to sample at discrete
depths
• Able to sample great depths
• Open sampling tube is
exposed while traveling
down to sampling depth
• Transfer of sample into
sample bottle may be
difficult
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Sample Collection Information Document - Attachment A
Bailer
Well, deep sump
pit
• Bailer should be cleaned and handled with
surgical gloves to prevent cross contamination.
Surgical gloves must be changed between each
sample location.
• Lower bailer slowly until it contacts the water
surface. Allow bailer to sink and fill with a
minimum of disturbance to the sample. Slowly
raise the bailer to the surface. Avoid contact of
the bailer line to the well casing and/or ground.
Tip the bailer to allow a slow discharge from the
top gently down the side of the sample bottle to
minimize turbulence.
• Repeat above steps until a sufficient sample
volume is acquired.
• Place used bailer in bag for return to lab for
decontamination and dispose of polyethylene
line.
• No external power source
required
• Economical enough that a
separate laboratory cleaned
bailer may be utilized for
each sampling to eliminate
cross contamination
• PTFE
(polytetrafluoroethylene) or
stainless steel construction
available
• Simple to use, lightweight,
portable
• Limited volume of sample
collected
• Unable to collect discrete
samples from a depth
below the water surface
• Leakage due to wear,
dimension distortion and
silt buildup may aerate
succeeding sample and
may gather unwanted
material.
• Aeration and turbidity may
bias the result.
Suction-lift
mechanisms
Well, deep sump
pit, large storage
tank
• Low volume pump that, by applying vacuum,
causes water to be drawn upward through a
suction line. Two types of suction-lift pumps are
generally available for shallow water sampling:
centrifugal pumps and peristaltic pumps.
• Flow rate of suction-lift
pumps is easily controlled
• Highly portable and readily
available.
• A drop in pressure due to
negative pressure
(suction) causes
degassing of the sample
• Where the sample comes
in contact with pump
rotating parts or tubing,
the choice of appropriate
material for impeller or
flexible pump tubing may
be restrictive.
Liquid Grab
Sampler
Collect liquid and
slurry samples
from surface
impoundments,
pool or
containers.
• Grab samples can be obtained at discrete
depths. The sample bottle might be attached to
the end of a 6-ft. long handle. The control valve
is operated from the top of the handle once the
sampler is at the desired depth. The general
procedure would be:
• Assemble the sampler. Operate the sampler
several times to ensure proper adjustment,
tightness of the cap, etc. Submerge sampler into
liquid to be sampled. When the desired depth is
reached, pull valve finger ring to open control
valve and allow sample to enter container.
• Retrieving sampler by closing valve. Transfer
sample into laboratory cleaned sample bottles
and follow procedures for preservation and
transport.
• Allows discrete samples to
be taken at depth
• Depth of sampling is
limited by length of pole
• Hard to decontaminate
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10. Sampling of Bioaerosols
The term biological aerosol particle is defined as a solid airborne particle derived from biological
organisms, including microorganisms and fragments of biological materials such as plant debris
and animal dander (IGAP, 1992). The term primary biological aerosol is more or less equivalent
to the term bioaerosol (Reponen et al., 1995; Hinds, 1999). The term bioaerosol is used in a
broad sense to include any particle with biological activity/toxicity (Hirst, 1995). This document
uses the term bioaerosol to include airborne particles (dead or alive), large molecules or volatile
compounds that are or were derived from living organisms, including micro-organisms and
fragments of all varieties of living materials (viruses [0.02 to 0.3|jm], bacterial cells [0.5 to
30|jm], fungal spores [0.5 to 30|jm], pollen [10 to 100|jm], and protozoa [>10|jm]). Physical
characterization of bioaerosols is the concentration of pathogens that can be cultured, which is
expressed as the number of colony forming units per unit volume of air (cfu/m3). A schematic
diagram of bioaerosol sampling procedure is shown in Figure A-1, and examples of sources of
bioaerosols are shown in Table A-4.
Figure A-1. Schematic diagram of bioaerosol sampling procedure.
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Sample Collection Information Document - Attachment A
Table A-4. Sources and Particle Size Distribution of Bioaerosols
Typical Bioaerosol
Source/Activity
Particle Size Distribution12
Reference
Surgical/dental procedure
Up to 50 jjm
Jewett et al. 1992; Szymariska,
2007
Hospital air
<2 (jm (22%), 2 to 6 jjm (30%), >5
|jm (48%)
Greene et al., 1962
Mechanical ventilators, bed
making, resuspension on
dust or skin squamae
0.3 jjm to >5 jjm
Tang et al., 2006; Roberts et al.,
2006
Cooling tower
<5 jjm up to > 100 jjm (bimodal
peaks at <5 jjm and 20-40 jjm)
Rothman et al., 1975
Wastewater irrigation
1.0 to 5.9 jjm
Bausum et al., 1982
Grain harvesting, food
processing, animal farming
activities
0.9 to 18.9 jjm (0.5 to >5 jjm)
Lee at al., 2006; Olsen et al., 2009
Mail sorting and opening
0.3 jjm to >5 jjm; 19.6-fold increase
in particles >5 jjm
Brandl et al., 2005
Mist machine
Between 40 and 70 jjm
Barrabeig et al., 2010
Whirlpools
<1 and 15 jjm depending on
turbulence
Baron et al., 1986
Breathing
<0.8 to 2 jjm
Morawska et al., 2009
Speaking
16 to 125 jjm
Chao et al., 2009; Xie et al. 2009
Shouting
0.5 to 10 jjm (mean = 1.0 jjm)
Lai et al. 2011
Sneezing
7 to 125 jjm
Duguid et al. 1945; Jennison et al.,
1942
Showering
Hot water 5.2 to 7.5 jjm
Cold water 2.5 to 3.1 jjm
Zhou et al. 2007; Chattopadhyay et
al. 2017
1: aerodynamic diameter
2: distribution should be considered with caution as often tests used samplers with cut off limits <15 |jm and therefore
were preferentially selective for particles smaller than this size.
References for Table A-4
Baron, P.A., and Willeke, K. 1986. Respirable droplets from whirlpools: measurements of size
distribution and estimation of disease potential. Environ Res. 39:8-18.
Barrabeig, I., Rovira, A., Garcia, M., Oliva, J.M., Vilamala, A., Ferrer, M.D., Sabria, M.,
Dormnguez, A. 2010. Outbreak of Legionnaires' disease associated with a supermarket
mist machine. Epidemiol Infect. 138:1823-8.
Bausum, H.T., Schaub, S.A., Kenyon, K.F., and Small, M.J. 1982. Comparison of coliphage
and bacterial aerosols at a wastewater spray irrigation site. Appl Environ Microbiol. 43:28-
38.
Brandl, H., Bachofen, R., and Bischoff, M. 2005. Generation of bioaerosols during manual mail
unpacking and sorting. J Appl Microbiol. 99:1099-107.
Chao, C.Y.H., Wan, M.P., Morawska, L., Johnson, G.R., Ritovski, Z.D., Hargreaves, M., et al.
2009. Characterization of expiration air jets and droplet size distributions immediately at the
mouth opening. Aerosol Sci. 40:122-33.
Chattopadhyay, S., Perkins, S.D., Shaw, M., and Nichols, T.L. 2017. Evaluation of Exposure of
Brevundimonas diminuta and Pseudomonas aeruginosa during Showering. Journal of
Aerosol Science. 114:77-93. https://doi.orq/10.1016/i.iaerosci.2017.08.008.
Duguid, J.P. 1945. The numbers and the sites of origin of the droplets expelled during
expiratory activities. Edinb Med J. 52:385-401.
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Sample Collection Information Document - Attachment A
Greene, V.W., Vesley, D., Bond, R.G., and Michaelsen, G.S. 1962. Microbiological
contamination of hospital air. I. Quantitative studies. Appl Microbiol. 10:561-6.
Jennison, M.W. 1942. Atomizing of mouth and nose secretions into the air as revealed by high-
speed photography. Aerobiol. 17:106-28.
Jewett, D.L., Heinsohn, P., Bennett, C., Rosen, A., and Neuilly, C. 1992. Blood-containing
aerosols generated by surgical techniques: a possible infectious hazard. Am Ind Hyg Assoc
J. 53:228-31.
Lai, K.M., Bottomley, C., and McNerney, R. 2011. Propagation of respiratory aerosols by the
vuvuzela. PLoS One. 6:e20086.
Lee, S-A., Adhikari, A., Grinshpun, S.A., McKay, R., Shukla, R., and Reponen, T. 2006.
Personal exposure to airborne dust and microorganisms in agricultural environments. J
Occup Environ Hyg. 3:118-30.
Morawska, L., Johnson, G.R., Ristovski, Z.D., Hargreaves, M., Mengersen, K., Corbett, S., et al.
2009. Size distribution and sites of origin of droplets expelled from the human respiratory
tract during expiratory activities. Aero Sci. 40:256-69.
Olsen, K.N., Lund, M., Skov, J., Christensen, L.S., and Hoorfar, J. 2009. Detection of
Campylobacter bacteria in air samples for continuous real-time monitoring of Campylobacter
colonization in broiler flocks. Appl Environ Microbiol. 75:2074-8.
Roberts, K., Hathway, A., Fletcher, L.A., Beggs, C.B., Elliott, M.W., and Sleigh, P.A. 2006.
Bioaerosol production on a respiratory ward. Indoor Built Environ. 15:35-40.
Rothman, T., and Ledbetter, J.O. 1975. Droplet size of cooling tower fog. Environ Lett. 10:191-
203.
Szymanska J. 2007. Dental bioaerosol as an occupational hazard in a dentist's workplace. Ann
Agric Environ Med. 14:203-7.
Tang, J.W., Li, Y., Eames, I., Chan, P.K.S., and Ridgway, G.L. 2006. Factors involved in the
aerosol transmission of infection and control of ventilation in healthcare premises. J Hosp
Infect. 2006;64:100-14.
Xie, X., Li, Y., Sun, H., and Liu, L. 2009. Exhaled droplets due to talking and coughing. J R
Soc Interface. 6(Suppl 6):S703-14.
Zhou, Y., Benson, J.M., Irvin, C., Irshad, H., and Cheng, Y-S. 2007. Particle size distribution
and inhalation dose of shower water under selected operating conditions. Inhal Toxicol.
19:333-42.
Bioaerosol samplers are designed for sampling biological aerosols under various conditions
such as short sampling cycles, long sampling cycles, high temperature, and low temperature.
Knowledge and use of efficient air samplers enhance the ability to protect users, first
responders, and the general public from airborne agents. Sampling devices and detection
systems need to be tested and their performance efficiencies determined so that they can be
appropriately matched for various challenges. Each air sampler has multiple components such
as an inlet, transmission tubes, a pre-separator skimmer to reject large particles, aerosol
concentrating stages, and a collector such as an impactor. The performance of an aerosol
sampler, or the sampling efficiency, is the overall end-to-end ratio of the amount of aerosol
contained in the sample produced by the sampler to the amount of aerosol contained in the
volume of ambient air sampled by the system's inlet. In a well-designed, well-fabricated, well-
assembled system, it is the product of the performance efficiencies of the sampler's individual
components, variously: aspiration, transmission, collection, retention, and recovery efficiencies.
The aspiration efficiency of a sampler's inlet describes the efficiency with which particles are
extracted from the air and transmitted through the sampler inlet and is dependent on particle
aerodynamic size and wind speed. Transmission efficiency describes the efficiency with which
particles are transported from the intake of a component to its collector, and the collection
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efficiency describes the efficiency with which particles are captured by the collector. Retention
efficiency indicates how efficiently particles are retained by the sampler during a long sampling
time, e.g., either in an impinger or in a wetted cyclone that stores the collected particles in the
active collection fluid throughout the collection time. Particles in the collection fluid can escape
into the air (reaerosolization) and be ejected with the exhaust. The collected particles are
recovered for assay, and the efficiency with which they are recovered is indicated by the
recovery efficiency. These efficiencies described above can depend on particle size, density,
charge, composition, and biological factors. Organisms have two additional issues: survival
fraction and culturable fraction. Survival of an organism can be measured by flow cytometry
using different dyes that reveal viable versus non-viable organisms, and by other life function
measures such as ATP. The culturability is determined by plating. These are reported as
fractions rather than efficiencies because they are characteristics of the aerosol in the sample
not the amount of aerosol in the sample.
The key factors affecting aerosol characteristics during sampling include:
• Aspiration efficiency and deposition in the sampling inlet
• Deposition during transport
• Extremes or inhomogeneity in the ambient aerosol concentration
• Agglomeration of particles during transport
• Evaporation and/or condensation of aerosol material during transport
• Retainment of deposited aerosol back into the sample flow
• High local deposition causing flow restriction or plugging
Desirable sampling conditions are:
• Constant free stream flow rate during sampling
• Stable aerosol condition during sampling
• Sufficiently low sampling gas velocity so that the sampled particles can accommodate
themselves to the sampling gas flow within a distance comparable to the inlet diameter
(inertial condition)
• Sufficiently high sampling gas velocity so that the sampled particles do not settle
appreciably (gravitational settling condition)
• Application of larger inlet diameters (of the order of a centimeter) as they are less
susceptible to deposition caused by free-stream turbulence
11. Instrument and System Calibration
Instrument and system calibration are essential for successful measurement of bioaerosol
properties in a sampling environment. Calibration can be conducted via direct measurement or
using primary standards, e.g. latex spheres size calibration; currently no concentration
standards are available; gravimetric techniques are applicable for larger particles only. Reliable
and accurate calibration requires:
• A proper selection of a desired test aerosol
• A complete understanding of the principles and procedures of operation
• A thorough investigation of the relevant parameters
• A sufficient knowledge of the capabilities and limitations of the instrument
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Before setting up of a sampling system, it should be determined whether standard procedures
for this type of sampling are available. There are prescribed standard sampling procedures for
certain types of measurements, such as:
• ASTM E2720 - 16: Standard Practice for Evaluation of Effectiveness of Decontamination
Procedures for Air-Permeable Materials when Challenged with Biological Aerosols
Containing Human Pathogenic Viruses.
• NIST Technical Note 1737: Challenges in Microbial Sampling in the Indoor Environment.
National Institute of Standards and Technology.
• NIOSH Manual of Analytical Methods: Bioaerosol Sampling (Indoor Air); Sampling and
Characterization of Bioaerosols.
The sampling methods and sampling devices available today are shown in Table A-5 with the
mechanisms involved, ability, availability, advantages and disadvantages. The selection criteria
of sampling devices for pathogens are dependent on the needs of post-sampling analysis
method, the fate and transport of and exposure to the bioaerosols through size resolved
measurements, and conditions dictated by the indoor environment. Generally, the desired
properties exist in the variety of aerosol samplers, but rarely in a single sampler. There is lack
of standard protocols for aerosol sampling and sample preparation. Without standard protocols
that contain information on efficiencies associated with sample collection and sample
preparation, quantitative bioaerosol data may lack both accuracy and precision. Standards are
necessary to provide consistency in investigations in order to compare data sets. Challenges
with bioaerosol sampling technology include the need for compact and portable sampling
devices, and the significant contamination issues association with high volume liquid impingers.
Regarding the application of molecular techniques, many of the sampling techniques provide
sufficient material for PCR-based analysis, but significant limitations still occur in concentrating
the samples into small volumes, and collecting sufficient samples for non-PCR based analyses.
Table A-6 provides a comparison of commercially available representative aerosol samplers.
Bioaerosol sampling aims to take a sample that is physically and biologically representative of
the indoor environment. Air will often contain microorganisms such as viruses, bacteria, spores,
and other microorganisms. Airborne spores can remain viable for much longer periods, even at
low relative humidity and high or low temperature extremes. A proper sampling process
includes determining location and number of sampling locations, selecting an appropriate
sampler or sampling system, and determining sampling duration and frequency. A bioaerosol
sampling plan should begin by determining the purpose of sampling. Sampling objectives may
include verification and quantification of pathogen present, identification of sources that could
lead to control and mitigation, and subsequent monitoring to ensure the effectiveness of control
measures implemented. Sampling parameters that may be considered include type of sample,
duration of samples, potential interferences and expected co-contaminant concentrations in the
indoor environment. The sampling media should be specifically identified, e.g., pore size and
type of filter, concentration and amount of liquid media required, and specific type and amount
of solid sorbent. The sampling pump used to collect the sample must also be compatible with
the sampling needs and the media used. The pump should be capable of maintaining the
desired flow rate over the time period needed using the sampling media specified. Certain
pumps may not be able to handle the large pressure drop due to media, fine mesh (smaller than
40 mesh) solid sorbent tubes, small pore size filters or when attempting to take a short-term
sample on a sorbent tube of a higher than normal pressure drop at a flow rate of 1 L/min or
greater. Factors that can influence collection of pathogens in indoor environments include
relative humidity, temperature, oxygen, indoor pollutants, sampling flow rate/face velocity,
concentration (breakthrough capacity/breakthrough volume), and indoor atmospheric stability
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(degree to which the atmosphere can dampen vertical and horizontal motion) - stable
atmospheric conditions result in low dispersion, and unstable atmospheric conditions (for
example, hot conditions) result in higher dispersion. The flow rate recommended for a specific
device/method can be used for the desired sampling period considering total sample volume,
sampling time, and limit of quantitation. Some of these variables will be fixed by sampling
needs, e.g., sampling time or by the measurement method of choice (limit of quantitation or
maximum sampling volumes).
Bioaerosol concentrations generally have considerable temporal and spatial variation because
pathogen sources may not generate aerosols continuously. The time and space dependent
characteristics in bioaerosol concentrations have a significant effect on determining the optimal
sampling duration and location. The overall performance of an aerosol sampler can be
determined by two factors: physical factors (inlet sampling efficiency and collection efficiency)
and biological factors (preserving biological characteristics of pathogens during sampling and
accurate analysis for identification and quantification). There can be challenges that may be
addressed when determining an appropriate sampling protocol: (a) level of concentrations of
pathogen as high levels may overload some samplers, which may lead to shortened sampling
time or use of a diluter system; (b) comprehensive quantitative and qualitative analysis may
require the use of multiple sampling and analysis methods; and (c) practical constraints (such
as spatial restrictions, proximity to the source, proximity to the ventilation systems, and other
logistical considerations). The number and location of sampling points may be selected
according to the variability, or sensitivity, of the sampling and analytical methods being utilized,
the variability of contaminant concentration over time at the site, and the level of precision
required. The number of locations and placement of samplers can be determined by
considering the nature of the response, indoor location (with respect to other conflicting
background sources), size of the concerned area, and the number, size, and relative proximity
of separate on-site emission sources. The duration of sampling activities should be considered
when choosing the location and number of samples to be collected. Air quality dispersion
models may be used to place samplers in areas of maximum predicted concentrations.
Sampling duration and flow rate dictate the volume of air collected, and to a major degree, the
detection limit. The analytical method selected will provide a reference to flow rate and volume.
Flow rates are limited to the capacity of the pumps being employed and the contact time
required by the collection media. The duration or period of air sampling is commonly divided
into two categories: (a) samples collected over a brief time period are referred to as
instantaneous or grab samples and are usually collected in less than five minutes and (b)
average or integrated samples are collected over a significantly longer period of time.
Integrated samples provide an average concentration over the entire sampling period. The
typical optimal sampling times for representative commercially available bioaerosol samplers
are illustrated in Figure A-2. Case studies on bioaerosol sampling frequency, layout, and
estimates of collectable biological particle are performed by various researchers (LaForce,
1990; Fennelly et al. 2004; Hwang et al., 2011)
Once the pathogen sample has been collected, it must be conditioned and transported to a
laboratory for further analysis. Appropriate care should be taken so the physical and biological
properties of the sample are preserved (i.e., refrigeration, observing sample holding times).
12. Optimal Sampling Time Determination
The concentrations of bioaerosols can vary with time. Sufficiently long collection times or
multiple samples with short collection times may be required during periods of changing
concentration so that collected sample(s) may properly represent the average environmental
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concentration over some time period. During a sampling process within a sampling period t
(start time = ts and final time = tf), the number of particles per unit area varies with bioaerosol
particle concentration in the sampled air. This results in a change in surface density (5) of the
sample, which equates to the number of particles on the surface per viewing area (A), i.e.,
microbial colonies on a petri dish. The surface density of a bioaerosol sample is determined by
the following equation:
g_N_cxQxt Equation (1)
A A
Where 5 is the surface density of a bioaerosol sample in cfu/m2, A is the viewing area (i.e., petri
dish) in m2; C is the average concentration of bioaerosols in cfu/m3, N is the number of viable
bioaerosol particles collected on the impaction substrate, in cfu, Q is the flow rate of the
sampling system in m3/min, and t is the sampling time in minutes.
In general, post analyses of bioaerosol samples include viewing, counting, and identifying the
particles within the sample. This can occur following collection by viewing the collected particles
under a microscope, or it may occur after an incubation period, which allows the colonies to
grow to sufficient size so they can be counted without magnification. An accurate quantification
of bioaerosols in a sample may only be obtained if the surface density of organisms is optimal,
50. If the sample surface density is very low, 5 « 50 sampling and counting errors may be high.
As a result, the calculated concentration may not be accurate and may misrepresent the true
concentration in the original air sampled. On the other hand, if the sample surface density is
very high, 5 » 50, the particles may be located in close proximity to each other, whereby the
collected organisms may grow together or may inhibit each other's growth such that accurate
counting and identification may not be possible. As shown in equation 1, the surface density of
a bioaerosol sample collected on a substrate is linearly related to sampling time. To avoid
insufficiently-loaded samples (5« 50) and overloaded samples (5» 50), the sampling time
should be adjusted accordingly. The optimal sampling time for a given bioaerosol concentration
depends upon sampler flow rate and collection surface area as demonstrated by the following
equation:
to = ^ x 6° Equation (2)
The calculated optimal sampling times for representative commercially available bioaerosol
samplers are illustrated in Figure A-2. Impinger samples are not sensitive to under- or
overloading during sampling because the liquid sample can be diluted or concentrated following
sample collection, depending on the concentration of collected bioaerosol particles in the liquid.
However, evaporation of sampling liquid and reaerosolization of prior-collected particles limit the
sampling time for most impingers.
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10s
1 10 1Q3 iff* 10s Casella Sampler
Concentration of btoaerosol
particles in air (m 3)
Figure A-2. Typical sampling times for representative bioaerosol samplers. (Modified after Baron,
P. A., arid K. Willeke. 2001. Aerosol Measurement: Principles, Techniques, and Applications, 2nd ed. John
Wiley & Sons.)
In general, impactors can be used for cut-off sizes (d50) in the range from 0.1 to 50 pm (cut-size
of the impactor stage corresponds to the 50% particle collection efficiency mark), flow rates from
a few cm3/min to 1000s of m3/min. and sampling times from minutes to hours. Scanning
mobility particle sizer measures the particle size distribution in the range of 5 to 1000 nm,
measurement cycle time 60 to 500 s, and concentration range 20 to 1x107 particles/cm ' The
aerodynamic particle sizer measures particle aerodynamic diameter in real time (1 s to 18 hrs)
within the size range 0.5 to 30 |jm.
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Table A-5. B
ioaerosol Samplers - Common Devices and Mechanisms Involved (Chattopadhyay et al., 2017*)
Device
Mechanism
Typical Model/
Materials
Sampling
Rate
Sampling
Approach
Viability
Advantage
Disadvantage
Cascade
Impactor
Sampling air
stream
makes a
sharp bend
and particles
are stripped
based on
their
aerodynamic
diameter.
Anderson,
MOUDI, BGI, or
equivalent
10-28
L/min
(typical)
>500
L/min
(high
volume)
Provides the
best size
distribution
information. 1
and 12
stages for
aerosols with
aerodynamic
diameters
from 10 nm to
>18 |jm.
Only at 28
L/min collection
rates and
requires direct
sampling onto
agar plates.
• Widely used to define
particle size distributions
• Models available to
perform culturing
• High cost, especially for high
volume
• Inefficiencies due to particle
bounce
• Not sensitive as total sampled
mass is divided among
multiple stages.
Sampling air
passes
through a
small opening
and captured
into a liquid
medium.
Liquid
Impingement
SKC swirl,
Omni, or
equivalent
14 L/min
for glass
impingers
>100
L/min
(high
volume)
Efficiency
drops in low
volume glass
impingers
below
aerodynamic
diameters of
1 |jm.
Impingers are
flexible since
pathogens are
impinged into
liquid media or
buffer and can
be used for
culturing or
molecular
analysis.
• Sample is collected into
liquid and does not
require extraction from
solid
> Low cost of low flow glass
impingers
• Impacts on pathogen viability
due to evaporation of fluid and
collection efficiency are
concerns if an extended
sample collection is desired
• Effective decontamination the
equipment is a concern.
Filtration
Aerosols are
captured on
filters by
impaction or
diffusional
forces.
Anderson, SKC
IMPACT, or
equivalent
Ranges
from 4 to
1000
L/min
Typical for
<10 |jm and
<2.5 |jm size
fractions.
High
diffusional
forces, filters
are efficient
at sampling
sizes down to
the 20 nm
Not
recommended
for viability due
to high
stresses from
impaction and
desiccation
• Available for high
sampling rates
• Common and robust form
of high volume sampling
and low cost
> No possibility for viable
determination
> Limited ability of particle size
distributions
: The evaluations are based on tests performed using selected bioaerosol samplers and selected vegetative bacteria and spores.
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able A-6. Comparison of Commercially Available Representative Aerosol Samplers
Bioaerosol
Able to
Able to
Provide
Bioaerosol
Concentration
Data
Able to
Provide
Reference
Sampler
Name
Effectively
Sample
Spores
Bioaerosol
Size
Distribution
Data
Remarks
AG 1-30
Yes
Yes
No
AGI-30 has been used as a standard bioaerosol sampler for several decades
and its use has been widely published.
SKC
BioSampler®
Yes
Yes
No
SKC sampler is similar in size and operation to the AGI-30.
Gelatin Filter
Yes
Yes
No
The use of gelatin filters for sampling spore-forming bacterial bioaerosols is
well-documented. These filters (in a 47-mm format) can be used for
sampling spores because of their excellent total efficiency and ease of use.
WWC
Yes
Yes
No
The use of somewhat-unique high-volume cyclones is supported in the
literature, though there are no well-documented, commercially available high-
volume cyclones. It has the potential to provide much better detection limits
than the low-volume impingers and filters (approximately two orders-of-
magnitude better detection limit due to its high sampling rate).
ACI has been used as a standard bioaerosol sampler for several decades
and its use has been widely published. The information can be used to
provide both bioaerosol concentration and size distribution information.
ACI
Yes
Yes
Yes
Since particles are impacted directly into the agar, this sampler provides data
about the number of bioaerosol particles, rather than the total number. The
size distribution information should be expressed in terms of the number size
distribution, rather than a mass-weighted distribution.
BCI
Yes
Yes
Yes
BCI provides good data on the effective mass-weighted size distribution of
bioaerosols, and thus these data complement the ACI data well.
MLI
Yes
Yes
Yes
There are publications that cite the use of the MLI for sampling bioaerosols.
It has good potential for providing mass-weighted size distribution
information.
ELPI®
TBD
Yes
Yes
Limited publications available regarding the use of the ELPI for
characterization. It has potential for providing both real-time and culturable
mass-weighted size distribution information.
Note: Mention of trade names, products, or services does not convey official Agency approval, endorsement, or recommendation. The models,
trade names are indicated as examples.
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13. Air Impactor Samples
Below is procedure for collecting air impactor samples with petri dishes specific to the contaminant
being sampled.
Materials and Equipment
• Calibrated high-flow sampling pump (28.3 liters/minute [LPM])
• Rotameter (air flow meter) or dry cell calibrator
• Calibration adapter for impactors
• Sterile single or six stage impactor
• Sterile Petri dish and agent-specific agar for each stage
• Flexible Tygon™ tubing
• Sterile non-powdered sampling gloves
• Sealable plastic bags
• Parafilm M® wax strips
• Sample labels and wax pencil
• Documentation materials, digital camera, indelible ink pen, and logbook
• Custody seals and tags
• Chain-of-custody forms and shipping paperwork
Procedure
1. For each sample collected, ensure that a new pair of sterile gloves is worn.
2. Set the pump flow rate to 28.3 LPM or as specified in the analytical method, and turn it on.
3. To calibrate the impactor, aseptically remove the lids from the calibration set of Petri
dish(es) and keep lids in a clean sealable plastic bag. For the single stage impactor, place
each one calibration Petri dish on the stage and reassemble the impactor. For the 6 stage
impactor, place one of the calibration Petri dishes on each of the impactor stages and
reassemble the stages in the correct numerical order. Attach the calibration adapter to the
top of the impactor. Attach flexible Tygon™ tubing from the impactor calibration adapter to
the calibrator or rotameter inlet. Attach the second piece of tubing from the outlet of the
impactor to the inlet of the sample pump. Turn on the calibrator and record the initial flow
rate in the logbook.
4. Calibration of the sampling train can be performed outside the hot zone such as in the
sample preparation area. If using a rotameter for calibration, then it should be calibrated
with a primary standard such as the dry cell calibrator. Rotameters are considered
secondary standards.
5. After calibration, remove the calibration Petri dishes from each stage of the impactor and
cover with a lid. These can be reused for calibration several times until they begin to dry
out and not more than one day.
6. In preparation to sample, aseptically remove lids from the sample Petri dish(es) and keep in
a clean sealable plastic bag. For the single stage impactor, place one Petri dish on the
stage and reassemble the impactor. For the 6 stage impactor, place on of the 6 Petri
dishes on each impactor stage and reassemble the impactor ensuring that the stages are in
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the correct numerical order. Connect the Tygon™ from the outlet of the impactor to the
inlet of the pump.
7. Place the impactor and pump in desired sample location and photo document and map the
location.
8. Start the pump and record the time sampling began and the time the sampling is
completed. Sampling times should be between 10 to 15 minutes. At completion of sample
time, don sterile gloves and aseptically remove the petri dish(es), cover with lids and seal
each dish with Parafilm M® to secure, label each dish with the wax pencil including the
stage number and place into sterile zippered sample bag upside down (agar oriented up).
9. Double bag each sample.
10. Decontaminate outer bag prior to leaving hot zone. This is usually done at the entrance of
the personnel decontamination line.
11. For post sampling calibration, aseptically remove lids from each of the pre-calibration
sample Petri dishes and place on the impactor stages. Attach the tubing to the calibrator
and the pump as in the initial calibration.
12. Turn on pump and record the post sampling flow rate in the log book. Pre- and post-
calibration flow rates are very important in determining final contaminate concentration.
13. Pre and post sampling train calibration can be done either inside or outside the hot zone.
For calibration outside the hot zone the sampling equipment must be protected from
contamination or easily decontaminated. Otherwise, pre and post sampling train calibration
should be done in the hot zone.
14. Package samples for transport.
15. Fill out chain-of-custody form, and make a copy.
16. Refrigerate samples or package with ice, ensuring agar does not freeze.
17. Secure samples in shipping container with chain-of-custody and attach custody seals.
18. Fill out shipping manifest or contact courier.
19. Prior to use to collect another sample, the impactor must be autoclaved.
14. Impinger (Wet Method) Air Samples
Below is procedure for collecting air samples with an impinger using a wet method.
Materials and Equipment
• High Flow Sampling Pump
• Dry cell calibrator and stand
• Two sterile impinger, pump attachment, and sterile impinger fluid
• Teflon or Parafilm M® tape
• Flexible Tygon tubing
• Sterile sample container bottle
• Sterile non-powdered sample gloves
• Documentation materials, digital camera and logbook
• Custody seals, sealable plastic bags, and tags
• Sample labels, documentation forms, permanent marker(s)
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• Chain-of-custody forms and shipping paperwork
Procedure
1. Aseptically fill an impinger with appropriate sterile fluid and attach to pump. This should be
done outside the hot zone in a clean area.
2. Set up the sampling train by attaching Tygon™ tubing to outlet of impinger and the other
end to inlet of the sample pump.
3. In a clean area, calibrate the sample train by attaching another piece of Tygon ™ tubing to
the outlet of the impinger and the other end to a rotameter or dry cell calibrator. Adjust
pump to the desired flow rate of 12.5 LPM. If using a rotameter for calibration, then it
should be calibrated with a primary standard such as the dry cell calibrator before using.
Rotameters are considered secondary standards.
4. After pre-sampling calibration, remove impinger, place caps or Parafilm M® over both the
inlet and outlet of the impinger and set aside to use to check the flow rate after the sample
is collected.
5. Don a new pair of sterile gloves and attach a second sterile impinger, filled with appropriate
sterile fluid, to the sampling train.
6. Place sampling train in desired sample location and turn on pump.
7. Photo document sample location, draw map and record sample start time in the log book.
8. After sampling time has elapsed, turn off pump, don sterile gloves and aseptically remove
the impinger.
9. Ascetically transfer impinger fluid to sample container bottle can be done either inside or
outside the hot zone. If done outside the hot zone, place a cap or Parafilm M® over the
inlet and outlet of the impinger. It is important to keep impingers upright to prevent loss of
fluid due to leaking or spillage. Fluid transfer done outside the hot zone must be done in an
appropriate fume hood. If impinger fluid will be transferred to sample container bottle in the
hot zone, don sterile gloves and aseptically remove the impinger, transfer fluid to labeled,
sterile sample container and seal the lid with Teflon or Parafilm M® tape.
10. Double bag the sample.
11. For post sampling train calibration, don sterile gloves and attach a fluid filled calibration
impinger to the sample train as described in Step 4. Turn on pump and record flow rate.
Record flow rate in log book.
12. Pre and post sampling train calibration can be done either inside or outside the hot zone.
For calibration outside the hot zone the sampling equipment must have be protected from
contamination or easily decontaminated. Otherwise, pre and post sampling train calibration
should be done in the hot zone.
13. Decontaminate sample bag before leaving hot zone. This is usually done at the entrance of
the personnel decontamination line.
14. Package samples for shipment including ice, if needed.
15. Complete chain-of-custody form and place in sample shipment container.
16. Secure shipment container and complete shipping manifest.
17. Prior to another use, the impinger used to collect the sample must be autoclaved.
Note: For each sample collected, ensure that a new pair of sterile gloves is worn.
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15. Passive Samplers
Selecting pathogen samplers and sampling methods depends on the site-specific questions that
need to be addressed. Since samples for active pathogen sampling methods, described in
previous sections, are collected from single points in time, the data are representative "snapshots"
of the pathogens. Thus, multiple sampling might be used to describe how pathogen conditions
vary over time. Passive pathogenic sampling devices are incubated within the sampled
environment for weeks (typically 15 - 90 days) and depend on the formation and collection of
biofilms that grow on surfaces or within a solid matrix. The passive samplers provide a more time-
integrated sample of pathogens from the sampled environment. In active monitoring a pathogenic
air sampler physically draws a known volume of air through or over a particle collection device
which can be a liquid or a solid culture media or a nitrocellulose membrane and the quantity of
pathogens present is measured (for example in CFU/rrr of air). Passive monitoring uses settle
plates, which are standard Petri dishes containing culture media, that are exposed to the air for a
given time in order to collect biological particles, which settle out and are then incubated. Results
are expressed in CFU/plate/time or in CFU/m2/hour. Passive sampling provides a valid risk
assessment as it measures the harmful part of the airborne population that falls onto a critical
surface (French et al. 1980; Matysik et al. 2009; Napoli et al. 2012; Mills et al. 2014). Table A-9
provides advantages and challenges of commonly used passive samplers.
Table A-T, Advantages and Challenges of Passive Samplers
Advantages
Challenges
• Sampling devices are relatively easy to
deploy and recover.
• Sample collection over an extended period
of time might be desirable at certain
conditions compared to single, grab-
sample collection of pathogen.
• Passive sampling devices can concentrate
contaminants.
• Sampling devices require several days of placement in
the sampled environment and require two mobilizations
to the site to install and then retrieve the sampling
devices.
• The solid matrix of most passive microbial sampling
devices is a surrogate; thus, differences may exist
between pathogens colonizing the sampling device and
native material.
Even though the implementation might vary between different types of passive samplers, nearly all
share certain common characteristics, the most important of which is the presence of a barrier
between the sampled medium and the collecting medium. The barrier defines the rate at which
analytes are collected at a given concentration, which is crucial for quantitative analysis. An
effective sampler should eliminate or minimize the effects of external factors (such as the velocity
of the sampled medium at the face of the sampler, humidity, and temperature) on the sampling
rate. In practice, the barrier usually falls into one of two categories: (1) diffusion or (2) permeation
Schematic diagrams of the two types of samplers are given in Figure A-3. The sampling process is
similar for both categories of samplers.
I • »c°. •
L • • • *
Molecular Diffusion
Figure A-3. Schematic diagram of passive samplers: (a) Diffusion, (b) Permeation.
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For example, the Rutgers Electrostatic Passive Sampler is comprised of a permanently polarized
ferroelectric polymer film, which electrostatically attracts bioaerosol and other particles from the air
onto its surface. Airborne bioaerosol particles are particularly well suited to this electrostatic
collection method because they carry a relatively high electrical charge. Captured particles are
easily washed from the film and assayed. The advantages of this passive sampler are its small
size, customizable shape, ease of use, and the fact that it does not inactivate sampled microbes.
This device does not require a device to pull air through the sample, does not require external
power, and can be placed anywhere for any length of time. It can be easily applied anywhere in
indoor and outdoor environments providing representative data on ambient levels of bioaerosols
and also other particulate matter. The sampler can be used in any area and for personal
applications, where it can be worn by clipping it onto a shirt collar for applications such as
widespread airport bioterrorism monitoring.
Commercially available membranes (such as Zetapor®, gauze, nylon, low-density polyethylene, or
polyvinylidene difluoride) are also used as passive samplers to improve the detection of various
types of pathogens including viruses in water and wastewater systems. These passive samplers
are valuable tools for microbiome analysis with new-generation sequencing. The sorption of
pathogens on membranes is influenced by several parameters including characteristics of the
pathogens (i.e., isoelectric point, pH, particle size), membrane properties (i.e., electric charge,
hydrophobicity) and aqueous solution characteristics (pH, ionic strength). Field applications of
these passive samplers has revealed that short-term exposure allows for qualitative detection, and
long-term exposure gives an integrated concentration over a period of time.
Most traditional methods for the sampling and analysis of bioaerosols are offline and involve the
collection of the investigated particles on solid deposition substrates (membrane or fiber filters,
inertial impaction plates, thermal or electrostatic precipitation plates) or in a liquid (wetted wall
cyclone, impinger, or washing bottle) and intermediate steps of sample storage, transport, and
preparation before analysis. These methods are prone to artifacts caused by evaporation of
particle components, sorption of additional gas phase components, and reaction/alteration during
sample collection, storage, transport, and preparation. The potential for measurement artifacts for
bioaerosols can be minimized or at least quantify the effects outlined above by using elaborate
sampling techniques combining parallel or consecutive trains of denuders, filters, and adsorbent
cartridges. Substantial progress has been made in the development of aerosol mass
spectrometers for real-time measurements of size-selected particles. As the methods of
vaporization, ionization, calibration, and data analysis are improved, these instruments promise
reliable quantitative analyses by allowing differentiation between surface and bulk composition. A
particularly interesting application of aerosol mass spectrometry with high relevance is the
identification of biological particles and pathogens (bacteria, viruses, spores, etc.). Alternative
concepts for online monitoring of bioaerosols are based on aerodynamic sizing and fluorescence
spectroscopy, whereas most other applicable techniques are offline and highly labor intensive
(cultivation, staining, fluorescence and electron microscopy, enzyme and immunoassays, DNA
analysis, etc.). The key features of bioaerosol sampling are shown in Table A-7. Table A-8 lists
manufacturers of representative aerosol samplers.
A-39
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Sample Collection Information Document - Attachment A
Table A-8. Key Features of Bioaerosol Sampling
Passive Sampling1
Settle plates
oConsider using the 1/1/1 scheme (for 1 h, 1 m from the floor, at least 1 m
away from walls or any obstacle - standard index of microbial air
contamination) with 90mm plates
Surface sampling
oConsider using membranes (e.g., nitrocellulose) as an alternative to
contact plates on curved surfaces
o Surface and aerial contamination may have different sources
Active Sampling'1'
Impactors
o Collection on to agar plates
o Collection efficiency highly dependent on particle size (should be sieve-
like in performance)
o Ideal as a particle size classifier
oLoss of bioefficiency: shear forces, desiccation, particle bounce, and
deposition build-up
Virtual impactors
o Collection into liquid, thus minimizing risk of desiccation
o Collection efficiency dependent on particle size
o Useful as particle concentrators
Slit impactors
o Collection on to agar plates
oLoss of bioefficiency: shear forces, desiccation, particle bounce, and
deposition build-up
o Records variation in bioaerosol concentration over a specified time-period
Impingers
o Collection into liquid, thus minimizing risk of desiccation
oLoss of bioefficiency: shear forces, re-aerosolization, evaporation,
adherence to device walls
o Collection efficiency dependent on particle size
Cyclones (wetted)
o Collection into liquid, thus minimizing risk of desiccation
oLoss of bioefficiency: shear forces, liquid carryover, evaporation,
adherence to device walls
o May be used as pre-classifiers for particle size
o Collection efficiency dependent on particle size
oVary considerably in size and airflow rate
Filters
o Small, portable personal samplers
o Loss of bioefficiency: desiccation
oCollection efficiency dependent on particle size (sample head, foam, or
cyclone being used as pre-selectors)
Laboratory Testing
Calibrate the flow rate of the active sampler
o Ensures the maximum collection efficiency
o Influences the size of particles collected
Determine the bioefficiency of the sampler against the target pathogen
oTest in air conditions expected in the field (relative humidity and
temperature)
o Spike sampler with known concentration of the target pathogen
oEach type of pathogen has a unique response to conditions experienced
o Surrogate viruses may be used in place of pathogens; however, response
may differ from target pathogen
o Check that bio-efficiency is maintained throughout planned sampling time
Determine errors in numeration when sampling from a known, repeatable
concentration of the target pathogen
A-40
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Sample Collection Information Document - Attachment A
Ensure that the sampler exhaust is not a source of pathogen contamination
to the environment
Test the storage, enumeration, and identification procedure
Field Testing
Position of the inlet sampler
o Avoid strong airflows around the inlet of the sampler
o If using an inlet nozzle, position horizontally
o Ensure that the sample position is beyond the range of droplet fallout from
a source
Aerial microbial concentration
o Expect non-uniformed concentration in the area studied (expect
associated sampling errors)
o Consider taking samples at various locations in the area studied
oBe aware of airflow patterns due to HVAC and natural ventilation
oNote air quality: relative humidity, temperature, and particle dust
o There may be seasonal variation in concentration of the pathogen
Active samplers: quantification of pathogens
o Expressed as enumeration per cubic meters of air
o Need to know the collection time and flow rate of the sampler.
(1) Results from passive and active samplers should not be assumed comparable.
A-41
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Sample Collection Information Document - Attachment A
Table A-9. Manufacturers of Representative Aerosol Samplers
Impingement Samplers
All Glass lmpinger-30 and -4
(AG 1-30 & AG 1-4)
Ace Glass Incorporated
P.O. Box 688
1430 Northwest Blvd.
Vineland, NJ 08360
(609) 692-3333
Multi-Stage Liquid Impinger
(May)
Burkard Manufacturing Co. Ltd.
Woodcock Hill Industrial Estate
Rickmansworth, Hertfordshire
WD3 1PJ
England
0923-773134
Impaction Samplers
Andersen 6-Stage, 2-Stage,
and 1-Stage
Graseby Andersen
500 Technology Court
Smyrna, GA 30082-5211
(404) 319-9999
(800) 241-6898
S/4S, and Compact SAS
Spiral Biotech, Inc.
7830 Old Georgetown Road
Bethesda, MD 20814
(301)657-1620
Allergenco MK-2
Allergenco/Blewstone Press
P.O. Box 8571
Wainwright Station
San Antonio, TX 78208
(210) 822-4116
Casella Slit Sampler
BGI Incorporated
58 Guinan Street
Waltham, MA 02154
(617) 891-9380
Reuter Centrifugal Sampler
BIOTEST Diagnostics Corp.
66 Ford Road, Suite 131
Denville, NJ 07834
(201)625-1300
(800) 522-0090
Mattson-Garvin Slit-to-Agar
Barramundi Corporation
P.O. Drawer 4259
Homosassa Springs, FL 32647
(904) 628-0200
Aeroallergen Rotorod®
Sampling Technologies, Inc.
26338 Esperanza Drive
Los Altos, CA 94022
(415) 941-1232
Volumetric Spore Traps
(Indoor/Outdoor,
1- & 7-day; Personal)
Burkard Manufacturing Co. Ltd.
Woodcock Hill Industrial Estate
Rickmansworth, Hertfordshire
WD3 1 PJ
England
0923-773134
SKC Biosampler®
SKC, Inc.
863 Valley View Rd.
Eighty Four, PA 15330
(724) 941-9701
Biocapture™, BioBadge™
MesoSystems Technology, Inc.
1021 N. Kellogg Street
Kennewick, WA 99336
(509) 737-8383
General Air Sampling
Equipment Vendors
Industrial Hygiene News
Buyer's Guide
Circulation Department
8650 Babcock Blvd.
Pittsburgh, PA 15237
(412) 364-5366
(800) 245-3182
American Chemical Society
Environmental Buyer's Guide
1155 16th Street, NW
Washington, DC 20036
(202) 872-4600
Dycor Technologies Ltd.
1851 94 St NW, Edmonton, AB
T6N 1E6, Canada
(780) 486-0091
EMD Chemicals, Inc.
480 S Democrat Rd.
Gibbstown, NJ 08027
(856) 224-0094
Filtration Samplers
Samplers and Supplies
Costar Nuclepore™
One Alewife Center
Cambridge, MA 02140
(617) 868-6200
(800) 492-1110
Gelman Sciences Inc.
600 South Wagner Road
Ann Arbor, Ml 48106
(313) 665-0651
Millipore Corporation
80 Ashby Road
Bedford, MA 01730
(617) 275-9200
(800) 225-1380
Sandia Met-One Sampler
Sandia National Laboratories
1515 Eubank Blvd. SE
Albuquerque, NM 87123 BioGuardian®
(505) 845-0011 InnovaTek
350 Hills Street, #104
Richland, WA 99352
(509) 375-1093
A-42
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Sample Collection Information Document - Attachment A
16. References
Brown G.S., Betty, R.G. Brockmann, J.E., Lucero, D.A., Souza, C.A., Walsh, K.S., Boucher, R.M.,
Tezak, M., Wilson, M.C., Rudolph, T., Lindquist, H.D.A. and Martinez, K.F. 2007. Evaluation
of Rayon Swab Surface Sample Collection Method for Bacillus Spores from Nonporous
Surfaces. Journal of Appl. Microbiology 103(4): 1074-80.
Buttner, M.B., Cruz, P., Stetzenbach, L., Klima-Comba, A., Stevens, V., and P. Emanuel. 2004.
Evaluation of the Biological Sampling Kit (BiSKit) for Large-Area Surface Sampling. Appl.
Environ. Microbiol. 12:7040-7045.
CDC. 2012. Surface Sampling Procedures for Bacillus anthracis Spores from Smooth, Non-
Porous Surfaces. Centers for Disease Control and Prevention. Cincinnati, OH.
http://www.cdc.gov/niosh/topics/emres/surface-sampling-bacillus-anthracis.html. Accessed on
September 18, 2017.
Chattopadhyay, S., Perkins, S.D., Shaw, M., and Nichols, T.L. 2017. Evaluation of Exposure of
Brevundimonas diminuta and Pseudomonas aeruginosa during Showering. Journal of
Aerosol Science. 114:77-93. https://doi.Org/10.1016/i.iaerosci.2017.08.008
Estill, C.F., Baron, P.A., Hein, M.J., Larsen, L.D., Rose, L., Schaeferlll, F.W., Noble-Wang, J.,
Hodges, L., Lindquist, H.D., Deye, G.J., and Arduino, M.J. 2009. Recovery Efficiency and
Limit of Detection of Aerosolized Bacillus anthracis Sterne from Environmental Surface
Samples. Appl. Environ. Microbiol. 75(13): 4297-4306.
Fennelly, K.P., Davidow, A.L., Miller, S.L., Connell, N. and J.J. Ellner. 2004. Airborne Infection
with Bacillus anthracis—from Mills to Mail. Emerging Infectious Diseases 10(6):996-1001.
French, M.L.V., Eitzen, H.E., Ritter, M.A., and D.S. Leland. (1980). Environmental control of
microbial contamination in the operating room. In: Wound Healing and Wound Infection. Hunt
T.K. (Editor). New York: Appleton-Century Crofis. pp. 254-261.
Hinds, W.C. 1999. Aerosol Technology. New York, Wiley.
Hirst, J.M. 1995. Introduction, Retrospect and Prospect. In: Bioaerosol Handbook (eds S. Cox
and C. M. Wathes). CRCPress, Boca Raton, FL, 5-14.
Hwang G.M., DiCarlo, A.A., Lin, G.C. 2011. An Analysis on the Detection of Biological
Contaminants aboard Aircraft. PLoS ONE 6(1): e14520. doi:10.1371/journal.pone.0014520
IGAP 1992. The International Global Aerosol Program. Deepak Publishing, Hampton, VA.
LaForce, F.M. 1990. Biological Contaminants in Indoor Environments: Gram Positive Bacteria
with Particular Emphasis on Bacillus anthracis. Biological Contaminants in Indoor
Environments.
Matysik, S., Herbarth, O., and A. Mueller. (2009) Determination of microbial volatile organic
compounds (MVOCs) by passive sampling onto charcoal sorbents. Chemosphere 76:114-119.
Mills, G.A., Gravell, A., Vrana, B., Harman, C., Budzinski, H., Mazzella, N., and T. Ocelka. (2014).
Measurement of environmental pollutants using passive sampling devices - an updated
commentary on the current state of the art. Proceedings for the 6th International Passive
Sampling Workshop and Symposium (IPSW2013), Bordeaux, France.
Napoli, C, Marcotrigiano, V., and M.T. Montagna. (2012). Air sampling procedures to evaluate
microbial contamination: a comparison between active and passive methods in operating
theatres. BMC Public Health. 12:594.
Raber, E. 2006. Summary Document: Restoration Plan for Major Airports after a Bioterrorist
Attack. Lawrence Livermore National Laboratory and Sandia National Laboratories. UCRL-TR-
227254.
Reponen, T., Willeke, K., Grinshpun, S. and Nevalainen, A. 1995. Biological particle sampling. In:
Bioaerosol Handbook (eds C. S. Cox, and C. M. Wathes). CRC-Press, Boca Raton, FL, 751-
778.
USEPA. 2013. Evaluation of Vacuum-based Sampling Devices for Collection of Bacillus Spores
from Environmental Surfaces. EPA 600/R-13/137.
A-43
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Sample Collection Information Document - Attachment A
Valentine, N. B., Butcher, M.G., et al. 2008. Evaluation of sampling tools for environmental
sampling of bacterial endospores from porous and nonporous surfaces. J Appl Microbiol
105(4):1107-1113.
17. Additional Bibliography
ASTM STP 1071, Philip R. Morey, James C. Feeley, Sr., James A. Otten (editors). American
Society for Testing and Materials, Philadelphia.
Baron, P. A., and K. Willeke. 2001. Aerosol Measurement: Principles, Techniques, and
Applications, 2nd ed. John Wiley & Sons.
National Academy of Sciences. 2005. Sensor Systems for Biological Agent Attacks: Protecting
Buildings and Military Bases. Washington, DC.
Vincent, J.H. 2007. Aerosol sampling: science, standards, instrumentation and applications. John
Wiley & Sons.
Wang, C-H., Chen, B.T., Han, B-C., Liu, A.C., Hung P., Chen C.Y, and H.J. Chao. (2015). Field
Evaluation of Personal Sampling Methods for Multiple Bioaerosols. PLoS ONE 10(3):
e0120308. doi:10.1371/journal.pone.0120308.
A-44
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Sample Collection Information Document - Attachim
Attachment B-1:
Sample Collection Information
for Pathogens (Bacteria, Viruses, Protozoa, and Helminths)
in Solids (Soil, Powder)
B1-1
-------
Sample Collection Information Document - Attachim
This page left intentionally blank
B1-2
-------
Sample Collection Information Document-Attachment B-1
Attachment B-1: Sample Collection Information for Pathogens in Solid Samples
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Additional Source'4'
Solid Bacteria
Bacillus anthracis
[Anthrax]
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer. Care
should be taken to avoid freezing the
samples.
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
U.S. EPA/USGS,
2014; Mott et al.,
2017; Olm et al.,
2017
Brucella spp.
[Brucellosis]
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Scholz et al., 2008;
USAMRIID, 2016
Burkholderia mallei
[Glanders]'3'
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Velasco et al., 1998;
Prakash et al., 2014;
U.S. EPA/USGS,
2014; USAMRIID,
2016
Burkholderia
pseudomallei
[Melioidosis]'3'
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Velasco et al., 1998;
Prakash et al., 2014;
EPA/USGS, 2014;
USAMRIID, 2016
Campylobacter jejuni
[Campylobacteriosis]'3'
Sterile, leak-proof
container
Keep on ice (secure double-bagged
ice).
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Rivoal et al., 2005;
Carrillo et al., 2017;
Hiett, 2017
Chlamydophila psittaci
[Psittacosis]'3'
Sterile, leak-proof
container
Keep on ice (secure double-bagged
ice).
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Hulin et al., 2016;
Koskela, 2017
Coxiella burnetii
[Q-fever]'3'
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Fitzpatrick et al.,
2010; Bruin et al.,
2013; Duncan et al.,
2013; Hong et al.,
2013
Escherichia coli
0157:H7'3'
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Gagliardi and Karns,
2000; Jiang et al.,
2002; Park et al.,
2015
B1-3
-------
Sample Collection Information Document-Attachment B-1
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Additional Source'4'
Francisella tularensis
[Tularemia]'3'
Sterile, leak-proof
container
Room temperature if held for 1 hour
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Barns et al., 2005;
Petersen et al., 2009;
Berrada and Telford,
2010; Baird et al.,
2012
Legionella
pneumophila
[Legionellosis - a)
Pontiac fever; and b)
Legionnaires' disease]
Sterile, leak-proof
container. Water
and swab samples
must be packed
into a container
that protects the
samples from
exposure to light
and temperature
fluctuation.
Do not pack any samples with chilled
or frozen ice packs or chiller packs.
Samples must reach the laboratory
within 24 hours of collection.
100 g (gravimetric)
Fill >120mL mL (volumetric)
Steele et al., 1990;
Yang, 2004; Kuroki et
al., 2007;
Environmental
Microbiology
Laboratory, 2014
Leptospira spp.
(L. interrogans
serovars: L.
icteroheamorrhagiae,
L. autralis, L. balum, L.
bataviae, L. sejro, L.
pomona)
[Leptospirosis]
Small, tightly
sealed sterile
bottle or plastic
bag. A small
amount of sterile
deionized water
may be added to
prevent drying.
A small amount of sterile deionized
water should be present in container
to prevent drying. Room temperature
within 72 hours of collection; if longer,
keep on ice packs (or secure double-
bagged ice).
10 - 20 g (gravimetric)
Benacer et al., 2013;
Saito et al., 2013
Listeria
monocytogenes
[Listeriosis]'3'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice). If sample is already
frozen, do not thaw until analysis.
At least 100 g (gravimetric)
Beuchat and Ryu,
1997;
Locatelli et al., 2013;
U.S. FDA, 2016
Non-typhoidal
Salmonella
[Salmonellosis]'3'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Hutchison et al.,
2004; Boes et al.,
2005; Courty et al.,
2008
Salmonella Typhi
[Typhoid fever]'3'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Hutchison et al.,
2004; Boes et al.,
2005; Courty et al.,
2008
B1-4
-------
Sample Collection Information Document-Attachment B-1
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Additional Source'4'
Shigella spp.
[Shigellosis]'3'
Sterile plastic
bags or glass or
plastic bottles
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Alvarez et al., 1995;
U.S. EPA/USGS,
2014; Stanley et al.,
2015; Steiner-Asiedu
et al., 2016
Staphylococcus
aureus(3)
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice) if longer.
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Rusin et al., 2003;
Chaudhary et al.,
2013; Mohammed
and Sheikh, 2010
Vibrio cholerae 01 and
0139 [Cholera]'3'
Sterile, leak-proof
container
Store at room temperature. Do not
ship on ice.
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Santamaria and
Toranzos, 2003; Huq
et al., 2012; Djaouda
et al., 2013; Menezes
et al., 2014
Yersinia pestis
[Plague]''
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Pohanka and Skladal,
2009; U.S.
EPA/USGS, 2014;
U.S. EPA, 2016
Solid Viruses
Adenoviruses:
Enteric and non-
enteric
(A-F)'3)
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Horswell et al., 2010;
Rigotto et al., 2010;
Ahmed et al., 2015;
ASTM, 2016
Astroviruses'3'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Fill 50 mL sample tube to at
least 40 mL mark (volumetric)
Rodriguez et al.,
2009; ASTM, 2016;
Amoah et al., 2017
Caliciviruses:
Norovirus'3'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Jones et al., 2007; La
Rosa et al., 2010;
Bibby and Peccia,
2013; Boehm et al.,
2016
Caliciviruses:
Sapovirus'3'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Jones et al., 2007; La
Rosa et al., 2010;
Bibby and Peccia,
2013; Boehm et al.,
2016
B1-5
-------
Sample Collection Information Document-Attachment B-1
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Additional Source'4'
Coronaviruses: SARS-
associated human
coronavirus(3)
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Derbyshire and
Brown, 1978; De
Paoli, 2005;
Staggemeier et al.,
2015
Hepatitis E virus
(HEV)(3)
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Sobsey et al., 1986;
Rigotto et al., 2010;
Parashar et al., 2011
Influenza H5N1 viruslJ'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Vong et al., 2008;
Gutierrez and Buchy,
2012; Horm et al.,
2012
Picornaviruses:
Enteroviruses'3'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Spilki et al., 2013;
Faleye et al., 2016
Picornaviruses:
Hepatitis A virus
(HAV)(3)
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Rodriguez-Lazaro et
al., 2012; Xagoraraki
et al., 2014;
Adefisoye et al., 2016
Reoviruses:
Rotavirus (Group A)
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
50 - 100 g (gravimetric)
Horswell et al., 2010;
Spilki et al., 2013;
Trubl et al., 2016
Solid Protozoa
Cryptosporidium spp.
[Cryptosporidiosis]
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice); do not freeze.
50 - 100 g (gravimetric)
Prystajecky et al.,
2014; Bonilla et al.,
2015
Entamoeba
histolytica(3)
Sterile, sealed,
leak-proof
container
Keep on ice packs (or secure double-
bagged ice); do not freeze.
50 - 100 g (gravimetric)
Branco et al., 2012;
Calegar et al., 2016
Giardia spp.
[Giardiasis]'3'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice); do not freeze.
50 - 100 g (gravimetric)
Covert et al., 1999;
Olson et al., 1999;
Guy et al., 2003
Naegieria fowieri
[Naegleriasis - primary
amoebic
meningoencephalitis
(PAM)/ amebic
encephalitis]
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice); do not freeze.
100 g (gravimetric)
250 mL-10 L (volumetric)
Mullet al., 2013;
Moussa et al., 2013;
Mahittikorn et al.,
2015; Morgan et al.,
2016
B1-6
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Sample Collection Information Document-Attachment B-1
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Additional Source'4'
Toxoplasma gondii
[Toxoplasmosis]'3'
Sterile, sealed,
leak-proof
container
Keep on ice packs (or secure double-
bagged ice); do not freeze.
50- 100 g
Afonso et al., 2008;
Sroka and
Szymanska, 2012;
Krueger et al., 2014
Solid Helminths
Baylisascaris
procyonis
[Raccoon roundworm
infection]
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice). Store at 2 - 5°C at
laboratory; do not freeze samples.
300 - 600 g (gravimetric)
Gavin et al., 2005;
Gatcombe et al.,
2010; Collender et al.,
2015; Amoah et al.,
2017
Footnotes:
(1) Any sample collected for cultivation-based analysis must not be allowed to freeze.
(2) The sample sizes listed are based on the amount needed for analysis of a single sample. If requested by the laboratory, additional sample(s)
must be collected for laboratory quality control analyses (e.g., duplicates, matrix spikes). It is also recommended that additional sample(s) be
collected in case of the need for reanalysis due to sample spillage or unforeseen analytical difficulties.
(3) Currently, no information is available for this analyte in this sample type. Until such time that analyte-specific information is available, collection
procedures described for a similar analyte/sample type are considered to be appropriate.
(4) References for these sources are provided at the end of this attachment.
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U.S. EPA. 2016. Protocol for Detection of Yersinia pestis in Environmental Samples During the
Remediation Phase of a Plague Incident. Cincinnati, OH, U.S. Environmental Protection
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Influenza A virus (H5N1) outbreaks, Cambodia, 2006. Emerging Infectious Diseases,
14(8): 1303-1305.
Xagoraraki, I., Yin, Z. and Svambayev, Z. 2014. Fate of viruses in water systems. Journal of
Environmental Engineering, 140(7):04014020.
Yang, C.S. 2004. Sampling and controlling Legionella bacteria in domestic water systems.
Cherry Hill, NJ: P & K Microbiology Services.
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Attachment B-2:
Sample Collection Information for
Pathogens (Bacteria, Viruses, Protozoa, and Helminths) in Surfaces
(Swab, Wipe, Dust Socks)
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Sample Collection Information Document - Attachment B-2
Attachment B-2: Sample Collection Information for Pathogens in Surfaces
[Swab, Wipe, Dust Socks)
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Surfaces (Swab, Wipe, Dust Socks) Bacteria
Bacillus anthracis
[Anthrax]
Sterile, leak-proof
container
Room temperature if held for 1 hour
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
ASTM, 2010; Brown
et al., 2007a; Brown
et al., 2007b; Hodges
et al., 2010; Rose et
al., 2011; CDC, 2012;
Piepel et al., 2015;
Hutchison et al., 2015
Brucella spp.
[Brucellosis]
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
USAMRIID, 2016;
Arizona Department
of Health Services,
2017; Ohio
Department of Health,
2013
Burkholderia mallei
[Glanders]'4'
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
USAMRIID, 2016;
Arizona Department
of Health Services,
2017; Downey et al.,
2012
Burkholderia
pseudomallei
[Melioidosis]'4'
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
USAMRIID, 2016;
Arizona Department
of Health Services,
2017; Downey et al.,
2012; Hong-Geller et
al., 2010
Campylobacter jejuni
[Campylobacteriosis]'4'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Vidal et al., 2016;
Arizona Department
of Health Services,
2017; Standard
Methods, 2006;
Standard Methods,
2007
B2-3
-------
Sample Collection Information Document-Attachment B-2
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Chlamydophila psittaci
[Psittacosis]'4'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
U.S. EPA, 2015;
Hulin et al., 2016;
NRC, 2014; Madico
et al., 2000
Coxiella burnetii
[Q-fever](4)
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
USAMRIID, 2016;
Arizona Department
of Health Services,
2017; Kersch et al.,
2010
Escherichia coii
0157:H7(4)
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Ismail et al., 2013;
Downey et al., 2012;
Arizona Department
of Health Services,
2017
Franciseiia tuiarensis
[Tularemia]'4'
Sterile, leak-proof
container
Room temperature if held for 1 hour
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
USAMRIID, 2016;
Arizona Department
of Health Services,
2017; U.S. EPA/CDC,
2012; U.S. Army Test
and Evaluation
Command, 2016;
Rastogi et al., 2008
Legionella
pneumophila
[Legionellosis - a)
Pontiac fever; and b)
Legionnaires' disease]
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Arizona Department
of Health Services,
2017; OSHA, 2016
B2-4
-------
Sample Collection Information Document-Attachment B-2
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Leptospira spp.
(L. interrogans
serovars: L.
icteroheamorrhagiae,
L. autralis, L. balum, L.
bataviae, L. sejro, L.
pomona)
[Leptospirosis]
Sterile, leak-proof
container. A small
amount of sterile
deionized water
may be added to
prevent drying.
Ambient temperature within 72 hours
of collection; keep on ice (e.g., secure
double-bagged ice) if longer.
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
U.S. EPA, 2008; U.S.
EPA, 1978; Firth et
al., 2014; Burroughs
et al., 2007; Riediger
et al., 2016
Listeria
monocytogenes
[Listeriosis]'4'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice). If frozen, do not thaw
until analysis.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Lahou and
Uyttendaele, 2014;
Gomez, et al., 2012;
Zhu et al., 2012;
Downey et al., 2012;
Lim et al., 2005;
Non-typhoidal
Salmonella
[Salmonellosis]'4'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Williams et al., 2015;
Tu et al., 2015; Rose
et al., 2004
Salmonella Typhi
[Typhoid fever]'4'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Weir, 2016; U.S.
EPA, 2010; Zewde et
al., 2009; Rusin et al.,
2002
Shigella spp.
[Shigellosis]'4'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Lim et al., 2005;
Sehulster and Chinn,
2003; Rusin et al.,
2002; Page et al.,
2014
Staphylococcus
(4)
aureus('
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Lutz et al., 2013;
Landers et al., 2010
Vibrio cholerae 01 and
0139 [Cholera]'4'
Sterile, leak-proof
container
Store at room temperature. Do not
ship in ice.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Ley et al., 2012; Lim
et al., 2005; Page et
al., 2014; U.S. EPA,
1978
B2-5
-------
Sample Collection Information Document-Attachment B-2
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size(2)
Source'3'
Yersinia pestis
[Plague](4)
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Silvestri et al., 2016;
AFQTP, 2015; Gilbert
et al., 2014; Da Silva
et al., 2012; Dauphin
et al., 2010; Petrovick
et al., 2007
Surfaces (Swab, Wipe, Dust Socks) Viruses
Adenoviruses:
Enteric and non-
enteric
(A-F)(4)
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Williams et al., 2001;
ASTM 2016;
Xagoraraki et al.,
2014; Tuladhar et al.,
2012
Astroviruses14'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Williams et al., 2001;
U.S. EPA, 2015;
ASTM 2016; Scherer
et al., 2009; Tuladhar
et al., 2012
Caliciviruses:
Norovirus(4)
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Williams et al., 2006;
U.S. EPA, 2015;
Kimmitt and Redway
2016; Tuladhar et al.,
2012
Caliciviruses:
Sapovirus(4)
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Williams et al., 2006;
U.S. EPA, 2015;
Kimmitt and Redway,
2016; Tuladhar et al.,
2012
Coronaviruses: SARS-
associated human
coronavirus(4)
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Weir, 2016; Julian et
al., 2011; Casanova
et al., 2010
Hepatitis E virus
(HEV)(4)
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Givens et al., 2016;
Julian et al., 2011
Influenza H5N1 virus14'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Ip et al., 2012;
Indriani et al., 2010
B2-6
-------
Sample Collection Information Document-Attachment B-2
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Picornaviruses:
Enteroviruses'4'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Ronnqvist 2014;
Tuladhar et al., 2012;
Sanderson et al.,
2010
Picornaviruses:
Hepatitis A virus
(HAV)(4)
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Ronnqvist, 2014;
Tuladhar et al., 2012
Reoviruses:
Rotavirus (Group A)
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Savage and Jones,
2003
Surfaces (Swab, Wipe, Dust Socks) Protozoa
Cryptosporidium spp.
[Cryptosporidiosis]
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Edmonds et al., 2009;
McDermott, 2004;
Carlsen et al., 2001
Entamoeba
histolytica(4)
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Miller et al., 2010
Giardia spp.
[Giardiasis]'4'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Rhodes et al., 2012;
Palomar Health, 2014
Naegieria fowieri
[Naegleriasis - primary
amoebic
meningoencephalitis
(PAM)/ amebic
encephalitis]
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
NIST,2012; Khan,
2008
Toxoplasma gondii
[Toxoplasmosis]'4'
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice).
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Hoorfar, 2011;
NHANES, 2006;
Dumetre. and Darde,
2003
B2-7
-------
Sample Collection Information Document-Attachment B-2
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size(2)
Source'3'
Surfaces (Swab, Wipe, Dust Socks) Helminths
Baylisascaris
procyonis
[Raccoon roundworm
infection]
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice). Samples may be stored
at 2°C-5°C in the laboratory.
Care should be taken to avoid
freezing the samples.
At least 2 sterile, synthetic, and
moistened wipes, swabs, or
dust socks
Ogdee et al., 2016;
Hernandez et al.,
2013; Sorvillo et al.,
2002; Gavin et al.,
2005
Footnotes:
(1) Any sample collected for cultivation-based analysis must not be allowed to freeze.
(2) The sample sizes listed are based on the amount needed for analysis of a single sample. If requested by the laboratory, additional sample(s) must be
collected for laboratory quality control analyses (e.g., duplicates, matrix spikes). It is also recommended that additional sample(s) be collected in case of the
need for reanalysis due to sample spillage or unforeseen analytical difficulties.
(3) Additional resources. References for these sources are provided at the end of this attachment.
(4) Currently, no information is available for this analyte in this sample type. Until such time that analyte-specific information is available, collection procedures
described for a similar analyte/sample type are considered to be appropriate.
Notes:
• Sample transport containers are packed outside the contaminated area. Samples must be packed in a manner that protects the integrity of the sample
containers and provides temperature conditions required for sample preservation. Primary receptacles should be leak-proof with a volumetric capacity of not
more than 500 mL (liquid) or 4 kilograms (solid). If several individual primary containers are placed in a single secondary packaging, they must be individually
wrapped or separated so as to prevent contact between them. Secondary packaging should be leak-proof and surrounded by shock- and water-absorbent
packing materials or ice (if required for preservation) and shipped in a cooler to ensure sample temperatures do not exceed preservation requirements. Ice
should be placed in separate plastic bags or cold packs should be used to avoid leakage, and the bags placed around, among, and on top of the secondary
sample containers. Further guidance can be obtained from 49 CFR 173.199
-------
Sample Collection Information Document - Attachim
References
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Force, Headquarters U.S. Air Force, Washington, DC. AFQTP 4B051-10. http://static.e-
publishing.af.mil/production/1/af sg/publication/qtp4b051-10/qtp4b051-10.pdf.
Arizona Department of Health Services. 2017. Guide to Laboratory Services: Microbiology.
Arizona Department of Health Services, Bureau of State Laboratory Services, Phoenix,
Arizona, http://www.azdhs.gov/documents/preparedness/state-laboratory/public-health-
microbiology/lab-guide.pdf.
ASTM. 2010. ASTM E2458-10, Standard practices for bulk sample collection and swab
sample collection of visible powders suspected of being biological agents from nonporous
surfaces. ASTM International, West Conshohocken, Pennsylvania.
ASTM. 2016. ASTM E2721-16, Standard practice for evaluation of effectiveness of
decontamination procedures for surfaces when challenged with droplets containing
human pathogenic viruses. ASTM International, West Conshohocken, Pennsylvania.
Brown, G.S., Betty, R.G., Brockmann, J.E., Lucero, D.A., Souza, C.A., Walsh, K.S., Rudolph,
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from nonporous surfaces. Applied and Environmental Microbiology, 73(3):706-710.
Brown, G.S., Betty, R.G., Brockmann, J.E., Lucero, D.A., Souza, C.A., Walsh, K.S., Rudolph,
T. 2007b. Evaluation of rayon swab surface sample collection method for Bacillus spores
from nonporous surfaces. Journal of Applied Microbiology, 103(4): 1074-80.
Burroughs, E.G, Damer, K.S., Belgrader, P. Raab, B. 2007. Devices for collection and
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Carlsen, T. M., MacQueen, D.H., Krauter, P.W. 2001. Sampling requirements for chemical
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Casanova, L.M., Jeon, S., Rutala, W.A., Weber, D.J., Sobsey, M.D. 2010. Effects of air
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Sample Collection Information Document - Attachim
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B2-11
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Sample Collection Information Document - Attachim
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Hollis, M.A. 2007. Rapid sensors for biological-agent identification. Lincoln Laboratory
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ECBC-TR-595.
Rhodes, E.R., Villegas, L.F., Shaw, N.J., Miller, C. and Villegas, E.N. 2012. A modified EPA
Method 1623 that uses tangential flow hollow-fiber ultrafiltration and heat dissociation
steps to detect waterborne Cryptosporidium and Giardia spp. Journal of Visualized
Experiments, 65:4177.
Riediger, I.N., Hoffmaster, A.R., Casanovas-Massana, A., Biondo, A.W., Ko, A.I. and
Stoddard, R.A. 2016. An Optimized Method for Quantification of Pathogenic Leptospira
in Environmental Water Samples. PLOSONE, 11 (8):e0160523.
Ronnqvist, M. 2014. Noroviruses on surfaces: Detection, transfer, and inactivation.
University of Helsinki, Helsinki, Finland. ISBN: 978-951-51-0128-0.
Rose, L.J., Hodges, L., O'Connell, H. and Noble-Wang, J. 2011. National Validation Study of
a Cellulose Sponge Wipe-Processing Method for Use after Sampling Bacillus anthracis
Spores from Surfaces. Applied and Environmental Microbiology, 77(23):8355-8359.
Rose L, Jensen BJ, Peterson A, Banerjee SN, Arduino MJ. 2004. Swab materials and
Bacillus anthracis spore recovery from nonporous surfaces. Emerging Infectious
Diseases, 10(6): 1023-1029.
Rusin, P., Maxwell, S. and Gerba, C. 2002. Comparative surface-to-hand and fingertip-to-
mouth transfer efficiency of gram-positive bacteria, gram-negative bacteria, and phage.
Journal of Applied Microbiology, 93(4):585-592.
Sanderson, W.T., Hein, M.J., Taylor, L., Curwin, B.D., Kinnes, G.M., Seitz, T.A., Bridges, J.H.
2010. Surface Sampling Methods for Bacillus anthracis Spore Contamination. Emerging
Infectious Diseases. 8(10): 1145—1151.
Savage, C.E. and Jones, R.C. 2003. The survival of avian reoviruses on materials
associated with the poultry house environment. Avian Pathology, 32(4): 417-423.
Scherer, K., Made, D., Ellerbroek, L., Schulenburg, J., Johne, R. and Klein, G. 2009.
Application of a Swab Sampling Method for the Detection of Norovirus and Rotavirus on
Artificially Contaminated Food and Environmental Surfaces. Food and Environmental
Virology, 1:42.
Sehulster, L. and Chinn, R.Y.W. 2003. Guidelines for Environmental Infection Control in
Health-Care Facilities. Recommendations of CDC and the Healthcare Infection Control
Practices Advisory Committee (HICPAC). Morbidity and Mortality Weekly Report,
52(RR10):1-42. Centers for Disease Control and Prevention, Atlanta, GA.
Silvestri, E.E., Yund, C., Taft, S., Bowling, C.Y., Chappie, D., Garrahan, K., Nichols, T.L.
2016. Considerations for estimating microbial environmental data concentrations
collected from a field setting. Journal of Exposure Science and Environmental
Epidemiology, 27:141-151.
Sorvillo, F., Ash, L.R., Berlin, O.G.W., Yatabe, J., Degiorgio, C. and Morse, S.A. 2002.
Baylisascaris procyonis: An Emerging Helminthic Zoonosis. Emerging Infectious
Diseases, 8(4):355-359.
B2-12
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Sample Collection Information Document - Attachim
Standard Methods. 2006. 9060 Samples. Standard Methods for the Examination of Water &
Wastewater. American Public Health Association, American Waterworks Association,
and Water Environment Federation.
Standard Methods. 2007. 9020 Detection of Pathogenic Bacteria, G. Campylobacter.
Standard Methods for the Examination of Water & Wastewater. American Public Health
Association, American Waterworks Association, and Water Environment Federation.
Tu, L.T.P., Hoang, N.V.M., Cuong, N.V., Campbell, J., Bryant, J.E., Hoa, N.T., and Carrique-
Mas, J.J. 2015. High levels of contamination and antimicrobial-resistant non-typhoidal
Salmonella serovars on pig and poultry farms in the Mekong Delta of Vietnam.
Epidemiology & Infection, 143(14):3074-3086.
Tuladhar, E., Hazeleger, W.C., Koopmans, M., Zwietering, M.H., Beumer, R.R. and Duizer, E.
2012. Residual Viral and Bacterial Contamination of Surfaces after Cleaning and
Disinfection. Applied and Environmental Microbiology, 78(21):7769-7775.
USAMRIID. 2016. Specimen Collection and Submission Manual. United States Army
Medical Research Institute of Infectious Diseases, Diagnostic Systems Division, Fort
Detrick, Maryland. Report # TR-16-161.
U.S. Army Test and Evaluation Command. 2016. Test Operations Procedure (TOP) 08-2-
065 developmental testing of liquid and gaseous/vaporous decontamination on bacterial
spores and other biological warfare agents on military-relevant surfaces. Defense
Technical Information Center, Fort Belvoir, VA. DTIC AD No: AD1003462.
U.S. EPA. 1978. Quality Assurance Guidelines for Biological Testing. U.S. Environmental
Protection Agency, Environmental Monitoring and Support Laboratory, Las Vegas, NV.
E P A-600/4-78-043.
U.S. EPA. 2008. Method development and preliminary applications for Leptospira
spirochetes in water samples. U.S. Environmental Protection Agency, Washington, DC.
EPA/600/R-08/017.
U.S. EPA. 2010. Single-laboratory verification of culture-based procedure for detection of
Salmonella Typhi in drinking water and surface water. U.S. Environmental Protection
Agency, Washington, DC. EPA/600/R-10/132.
U.S. EPA/CDC. 2012. Method development for optimum recovery of Yersinia pestis from
transport media and swabs U.S. Environmental Protection Agency, Washington, DC.
EPA/600/R-12/620.
Vidal, A.B., Colles, F.M., Rodgers, J.D., McCarthy, N.D., Davies, R.H., Maiden, M.C.J, and
Clifton-Hadley, F.A. 2016. Genetic Diversity of Campylobacter jejuni and Campylobacter
coli Isolates from Conventional Broiler Flocks and the Impacts of Sampling Strategy and
Laboratory Method. Appl. Environ. Microbiol. 82(8):2347-2355.
Weir, M.H. 2016. Dose-Response Modeling and Use: Challenges and Uncertainties in
Environmental Exposure. In Manual of Environmental Microbiology, 4th ed., 3.5.3-1 -
3.5.3-17. ASM Press.
Williams, J.C., Stone, D., Smith-Arica, J.R., Morris, I.D., Lowenstein, P.R., and Castro, M.G.
2001. Regulated adenovirus-mediated delivery of tyrosine hydroxylase suppresses
growth of estrogen-induced pituitary prolactinomas. Mol Ther. 4:593-602.
Williams, J.V., Wang, C.K., Yang, C.F., Tollefson, S.J., House, F.S., Heck, J.M. etal. 2006.
The role of human metapneumovirus in upper respiratory tract infections in children: a 20-
year experience. J Infect Dis. 193:387-395.
Williams, S., Patel, M., Markey, P., Muller, R., Benedict, S., Ross, I., Krause, V. 2015.
Salmonella in the tropical household environment - Everyday, everywhere. Journal of
Infection, 71(6):642-648.
Xagoraraki, I., Yin, Z. and Svambayev, Z. 2014. Fate of Viruses in Water Systems. Journal
of Environmental Engineering, 140(7)1943-7870.
B2-13
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Sample Collection Information Document - Attachim
Zewde, B.M., Robbins, R., Abley, M.J., House, B., Morgan Morrow, W.E. and Gebreyes, W.A.
2009. Comparison of Swiffer Wipes and Conventional Drag Swab Methods for the
Recovery of Salmonella in Swine Production Systems. Journal of Food Protection,
72(1): 142—146.
Zhu, L., Stewart, D., Reineke, K., Ravishankar, S., Palumbo, S., Cirigliano, M. and Tortorello,
M. 2012. Comparison of Swab Transport Media for Recovery of Listeria monocytogenes
from Environmental Samples. Journal of Food Protection, 75(3):580-584.
B2-14
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Sample Collection Information IDocuim ichiment IB-3
Attachment B-3:
Sample Collection Information for
Pathogens (Bacteria, Viruses, Protozoa, and Helminths) in Liquids
(Water and Wastewater)
B3-1
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Sample Collection Information Document-Attachment IB-3
This page left intentionally blank
B3-2
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Sample Collection Information Document - Attachment B-3
Attachment B-3: Sample Collection Information for Pathogens in Water (Water, Wastewater)
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Liquid (Water, Wastewater) Bacteria
Bacillus anthracis
[Anthrax]
Sterile, leak-proof
container
Room temperature if held for 1 hour or
less; keep on ice (e.g., secure double-
bagged ice) if longer. Care should be
taken to avoid freezing the samples.
200 ml_ (minimum)
Celebi et al., 2016;
Singh et al., 2015;
U.S. EPA, 2012;
Letant et al., 2011;
Perez et al., 2005
Brucella spp.
[Brucellosis]
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
100 ml_ (minimum)
Saraswathy et al.,
2015; Goenka et al.,
2012; Martin et al.,
2012; Corbel 2006
Burkholderia mallei
[Glanders]'4'
Sterile, leak-proof
container
Room temperature if held for 1 hour or
less; keep on ice (e.g., secure double-
bagged ice) if longer.
100 ml_ (minimum)
Prakash et al., 2014;
Thaipadungpanit et
al., 2014;
Vongphayloth et al.,
2012; Baker et al.,
2011; Lever et al.,
2003
Burkholderia
pseudomallei
[Melioidosis]'4'
Sterile, leak-proof
container
Room temperature if held for 1 hour or
less; keep on ice (e.g., secure double-
bagged ice) if longer.
100 ml_ (minimum)
Delgado-Gardea et
al., 2016;
Limmathurotsakul et
al., 2013;
Limmathurotsakul et
al., 2012;
Vongphayloth et al.,
2012
Campylobacter jejuni
[Campylobacteriosis]'4'
Sterile, leak-proof
container
Keep on ice (secure double-bagged
ice).
1 -5 L
Khan et al., 2009;
Pitkanen et al., 2009;
ISO, 2005;
Hanninen et al., 2003
Chlamydia psittaci
(formerly
Chlamydophila psittaci)
[Psittacosis]'4'
Sterile, leak-proof
container
Keep on ice (secure double-bagged
ice).
100 ml_ (minimum)
Hulin et al., 2015;
USDA, 2014b
Coxiella burnetii
[Q-fever]'4'
Sterile, leak-proof
container
Room temperature if held for 1 hour or
less; keep on ice (e.g., secure double-
bagged ice) if longer.
500 ml_ (minimum)
Deshmukh et al.,
2016; Schets et al.,
2013
B3-3
-------
Sample Collection Information Document - Attachment B-3
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Escherichia coli
0157:H7(4)
Sterile, leak-proof
container
Room temperature if held for 1 hour or
less; keep on ice (e.g., secure double-
bagged ice) if longer.
100 mL (minimum)
U.S. EPA, 2010;
Brewster, 2009
Francisella tularensis
[Tularemia]'4'
Sterile, leak-proof
container
Room temperature if held for 1 hour or
less; keep on ice (e.g., secure double-
bagged ice) if longer.
100 mL (minimum)
U.S. EPA, 2015;
Forsman, 1995
Legionella pneumophila
[Legionellosis - a)
Pontiac fever; and b)
Legionnaires' disease]
Sterile, leak-proof
container. Water
and swab samples
must be packed
into a container
that protects the
samples from
exposure to light
and temperature
fluctuation.
Do not pack any samples with chilled
or frozen ice packs or chiller packs. All
samples other than compost material
must reach the laboratory within 24
hours of collection. Compost material
to be reached within three days to the
laboratory. Avoid sampling for at least
72 hours after on-line disinfection or
system decontamination or cleaning.
100 mL (minimum)
ASHRAE, 2015;
AS/NZS, 2011a;
AS/NZS, 2011b;
Flanders et al., 2014
Leptospira spp.
(L. interrogans
serovars: L.
icteroheamorrhagiae, L.
autralis, L. balum, L.
bataviae, L. sejro, L.
pomona)
[Leptospirosis]
Sterile, leak-proof
container
A small amount of sterile deionized
water should be present in container to
prevent drying. Room temperature
within 72 hours of collection; if longer,
keep on ice packs (or secure double-
bagged ice).
100 mL - 1000 mL
Riediger et al., 2016;
Wojcik-Fatla et al.,
2014; Benacer et al.,
2013; U.S. EPA, 2008
Listeria monocytogenes
[Listeriosis]'4'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice). If sample is already
frozen, do not thaw until analysis.
100 mL (minimum)
Gorski et al., 2014;
USDA, 2014;
Taherkhani et al.,
2013
Non-typhoidal
Salmonella
[Salmonellosis]'4'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
1000 mL and above
Cabral, 2010;
Obi et al., 2004
Salmonella Typhi
[Typhoid fever]'4'
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice).
1000 mL. Smaller volumes may
be appropriate for highly
contaminated waters.
McEgan et al., 2012;
Kumar et al., 2006;
Standing Committee
of Analysts, 2006
B3-4
-------
Sample Collection Information Document-Attachment B-3
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Shigella spp.
[Shigellosis]'4'
Sterile plastic bags
or glass or plastic
bottles
Keep on ice packs (or secure double-
bagged ice).
1000 mL. Smaller volumes may
be appropriate for highly
contaminated waters.
Standing Committee
of Analysts, 2006;
Faruque et al., 2003
Staphylococcus
aureusw
Sterile, leak-proof
container
Keep on ice (e.g., secure double-
bagged ice) if longer.
100 mL (minimum)
Piano et al., 2011;
Lechevallier and
Seidler, 1980
Vibrio cholerae 01 and
0139 [Cholera]'4'
Sterile, leak-proof
container
Store at room temperature. Do not
ship on ice.
100 mL (minimum)
Huq et al., 2012;
Schauera et al., 2012;
CDC, 2010
Yersinia pestis
[Plague]^'
Sterile, leak-proof
container
Room temperature if held for 2 hours
or less; keep on ice (e.g., secure
double-bagged ice) if longer.
100 mL (minimum)
Deshmukh et al.,
2016; U.S. EPA, 2015;
Simon et al., 2013
Liquid (Water, Wastewater) Viruses
Adenoviruses:
Enteric and non-enteric
(A-F)'4'
Positively charged
1MDS cartridge
filter
Keep on ice packs (or secure double-
bagged ice).
2 - 20 L (wastewater); 200 - 300
L (surface/recreational water);
1500 - 2000 L (drinking
water/groundwater).
Xagoraraki et al.,
2014; Cashdollar and
Wymer, 2013;
Ikner et al., 2011;
Williams et al., 2001
Astroviruses14'
Positively charged
1MDS cartridge
filter
Keep on ice packs (or secure double-
bagged ice).
2 - 20 L (wastewater); 200 - 300
L (surface/recreational water);
1500 - 2000 L (drinking
water/groundwater)
Filter apparatus should be
allowed to run overnight.
Cashdollar and
Wymer, 2013;
Rodriguez-Lazaro et
al., 2012;
Espinosa et al., 2009;
Williams et al., 2001
Caliciviruses:
Norovirus'4'
Positively charged
1MDS cartridge
filter
Keep on ice packs (or secure double-
bagged ice).
2 - 20 L (wastewater); 200 - 300
L (surface/recreational water);
1500 - 2000 L (drinking
water/groundwater)
Gabrieli et al., 2009;
Karim et al., 2009;
USGS, 2001;
Williams et al., 2001
Caliciviruses:
Sapovirus'4'
Positively charged
1MDS cartridge
filter
Keep on ice packs (or secure double-
bagged ice).
2 - 20 L (wastewater); 200 - 300
L (surface/recreational water);
1500 - 2000 L (drinking
water/groundwater)
Hata et al., 2015;
Williams et al., 2001
B3-5
-------
Sample Collection Information Document - Attachment B-3
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Coronaviruses: SARS-
associated human
coronavirus(4)
Positively charged
1MDS cartridge
filter
Keep on ice packs (or secure double-
bagged ice).
2 - 20 L (wastewater); 200 - 300
L (surface/recreational water);
1500 - 2000 L (drinking
water/groundwater)
AWWA, 2007;
Williams et al., 2001
Hepatitis E virus
(HEV)(4)
Double layer 142
mm diameter
1MDS cartridge
filter
Keep on ice packs (or secure double-
bagged ice).
2 - 20 L (wastewater); 200 - 300
L (surface/recreational water);
1500 - 2000 L (drinking
water/groundwater)
Williams et al., 2001;
Jothikumar et al.,
1993; Rose et al.,
1984
Influenza H5N1 virus14'
Positively charged
1MDS cartridge
filter
Keep on ice packs (or secure double-
bagged ice).
2 - 20 L (wastewater); 200 - 300
L (surface/recreational water);
1500 - 2000 L (drinking
water/groundwater)
Deboosere et al.,
2011; Nazir et al.,
2011; Williams et al.,
2001
Picornaviruses:
Enteroviruses'4'
Positively charged
1MDS cartridge
filter
Keep on ice packs (or secure double-
bagged ice).
2 - 20 L (wastewater); 200 - 300
L (surface/recreational water);
1500 - 2000 L (drinking
water/groundwater)
Filter apparatus should be
allowed to run overnight.
Faleye et al., 2016;
CDC/WHO, 2015;
Spilki et al., 2013;
Williams et al., 2001
Picornaviruses:
Hepatitis A virus
(HAV)(4)
Positively charged
1MDS cartridge
filter
Keep on ice packs (or secure double-
bagged ice).
2 - 20 L (wastewater); 200 - 300
L (surface/recreational water);
1500 - 2000 L (drinking
water/groundwater)
Filter apparatus should be
allowed to run overnight.
Adefisoye et al., 2016;
Xagoraraki et al.,
2014; Rodriguez-
Lazaro et al., 2012;
Fout et al., 2003;
Williams et al., 2001
Reovi ruses:
Rotavirus (Group A)
Positively charged
1MDS cartridge
filter
Keep on ice packs (or secure double-
bagged ice).
2 - 20 L (wastewater); 200 - 300
L (surface/recreational water);
1500 -2000 L (drinking
water/groundwater)
Filter apparatus should be
allowed to run overnight.
Trubl et al., 2016;
Spilki et al., 2013;
USGS/U.S. EPA,
2004; Fout et al.,
2003; Williams et al.,
2001
B3-6
-------
Sample Collection Information Document-Attachment B-3
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size(2)
Source'3'
Liquid (Water, Wastewater) Protozoa
Cryptosporidium spp.
[Cryptosporidiosis]
Sterile, leak-proof
container
or
Filter in sterile
leak-proof
container
Keep on ice (e.g., secure double-
bagged ice); do not freeze
10 L- 15 L
Bonilla et al., 2015;
Prystajecky et al.,
2014; U.S. EPA, 2005
Entamoeba histolytica(4)
Polypropylene
carboys
Keep on ice packs (or secure double-
bagged ice); do not freeze.
10L-50 L
Skotarczak, 2009;
Guy et al., 2003
Giardia spp.
[Giardiasis]'4'
Sterile, leak-proof
container/
Polypropylene
carboys
Keep on ice packs (or secure double-
bagged ice); do not freeze.
100 L - >1000 L through
cartridge filtration
Skotarczak, 2009;
U.S. EPA, 2005; Guy
et al., 2003; McCuin
and Clancy, 2003
Naegieria fowieri
[Naegleriasis - primary
amoebic
meningoencephalitis
(PAM)/ amebic
encephalitis]
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice); do not freeze.
250 mL- 10 L
Morgan et al., 2016;
Mahittikorn et al.,
2015; Moussa et al.,
2013; Mull et al., 2013
Toxoplasma gondii
[T oxoplasmosis](4)
Sterile, sealed,
leak-proof
container/Filter in
sterile leak-proof
container/Polyprop
ylene carboys
Keep on ice packs (or secure double-
bagged ice); do not freeze.
100 L (ten 10 L containers)/4650
L for filter cartridge
Krueger et al., 2014;
Sroka and
Szymariska, 2012;
Villena et al., 2004
Liquid (Water, Wastewater) Helminths
Baylisascaris procyonis
[Raccoon roundworm
infection]
Sterile, leak-proof
container
Keep on ice packs (or secure double-
bagged ice). Store at 2 - 5°C at
laboratory; do not freeze samples.
1 L (minimum)
Graeff-Teixeira et al.,
2016; Gatcombe et
al., 2010
B3-7
-------
Sample Collection Information IDocuim ichiment IB-3
Footnotes:
(1) Any sample collected for cultivation-based analysis must not be allowed to freeze.
(2) The sample sizes listed are based on the amount needed for analysis of a single sample. If requested by the laboratory, additional sample(s) must be
collected for laboratory quality control analyses (e.g., duplicates, matrix spikes). It is also recommended that additional sample(s) be collected in case of the
need for reanalysis due to sample spillage or unforeseen analytical difficulties.
(3) Additional resources. References for these sources are provided at the end of this attachment.
(4) Currently, no information is available for this analyte in this sample type. Until such time that analyte-specific information is available, collection procedures
described for a similar analyte/sample type are considered to be appropriate.
Notes:
• Sample transport containers are packed outside the contaminated area. Samples must be packed in a manner that protects the integrity of the sample
containers and provides temperature conditions required for sample preservation. Primary receptacles should be leak-proof with a volumetric capacity of not
more than 500 mL (liquid) or 4 kilograms (solid). If several individual primary containers are placed in a single secondary packaging, they must be individually
wrapped or separated so as to prevent contact between them. Secondary packaging should be leak-proof and surrounded by shock- and water-absorbent
packing materials or ice (if required for preservation) and shipped in a cooler to ensure sample temperatures do not exceed preservation requirements. Ice
should be placed in separate plastic bags or cold packs should be used to avoid leakage, and the bags placed around, among, and on top of the secondary
sample containers. Further guidance can be obtained from 49 CFR 173.199
-------
Sample Collection Information Document-Attachment IB-3
References
Adefisoye, M.A., Nwodo, U.U., Green, E. and Okoh, A.I. 2016. Quantitative PCR Detection and
Characterisation of Human Adenovirus, Rotavirus and Hepatitis A Virus in Discharged
Effluents of Two Wastewater Treatment Facilities in the Eastern Cape, South Africa. Food
and Environmental Virology, 1-13.
ASHRAE. 2015. Legionellosis: Risk management for building water systems. ANSI/ASHRAE
Standard 188-2015. Atlanta, GA.
AS/NZS. 2011a. Air-handling and water systems of buildings - Microbial control - Design,
installation and commissioning. AS/NZS 3666.1.
AS/NZS. 2011b. Air-handling and water systems of buildings - Microbial control -
Performance-based maintenance of cooling water systems. AS/NZS 3666.3.
AWWA. 2007. Optimizing molecular methods to detect human caliciviruses in environmental
samples. American Water Works Associations Research Foundation, Denver, Colorado.
Baker, A., Pearson.T.. Price,,E.P,, Dale, J., Kejm, P.„ Hornstra, H., Greenhill, A., Padilla, G. and
Warner, J. 2011. Molecular pnylogeny of Burkholderia pseudomalJei from a remote region
of Papua New Guinea. PLoS One, 6(3), p.e18343.
Benacer, D., Woh, P.Y., Zain, S.N.M., Amran, F. and Thong, K.L. 2013. Pathogenic and
Saprophytic Leptospira Species in Water and Soils from Selected Urban Sites in Peninsular
Malaysia. Microbes and Environments, 28(1): 135-140.
Bonilla, J.A., Bonilla, T.D., Abdelzaher, A.M., Scott, T.M., Lukasik, J., Solo-Gabriele, H.M. and.
Palmer, C.J. 2015. Quantification of Protozoa and Viruses from Small Water Volumes.
International Journal of Environmental Research and Public Health, 12:7118-7132.
Brewster, J.D., 2009. Large-Volume Filtration for Recovery and Concentration of Escherichia
Coli 0157: H7 from Ground Beef. Journal of Rapid Methods & Automation in Microbiology
17(2):242-256.
Cabral, J.P.S. 2010. Water Microbiology. Bacterial Pathogens and Water. International Journal
of Environmental Research and Public Health, 7(10):3657-3703.
Cashdollar, J.L. and Wymer, L. 2013. Methods for primary concentration of viruses from water
samples: a review and meta-analysis of recent studies. Journal of Applied Microbiology,
115:1-11.
CDC/WHO. 2015. Enterovirus surveillance guidelines: Guidelines for enterovirus surveillance
in support of the Polio Eradication Initiative. Centers for Disease Control and Prevention and
World Health Organization.
CDC. 2010. Laboratory Methods for the Diagnosis of Vibrio cholera: V. Examination of Food
and Environmental Samples, https://www.cdc.gov/cholera/pdf/laboratorv-methods-for-the-
diaqnosis-of-vibrio-cholerae-chapter-5.pdf.
Celebi, O., Buyuk, F., Pottage, T., Crook, A., Hawkey, S., Cooper, C., Bennett, A., Sahin, M.
and Baillie, L. 2016. The Use of Germinants to Potentiate the Sensitivity of Bacillus
anthracis Spores to Peracetic Acid. Frontiers in Microbiology, 7:18.
Corbel, M.J. 2006. Brucellosis in Humans and Animals. World Health Organization. Report
WHO/CDS/EPR/2006.7.
Deboosere, N., Horm, S.V., Pinon, A., Gachet, J., Coldefy, C., Buchy, P. and Vialette, M. 2011.
Development and validation of a concentration method for the detection of influenza a
viruses from large volumes of surface water. Applied and Environmental Microbiology,
77(11):3802-3808.
Delgado-Gardea, M.C.E., Tamez-Guerra, P., Gomez-Flores, R., Zavala-Dfaz de la Serna, F.J.,
Eroza-de la Vega, G., Nevarez-Moorillon, G.V., Perez-Recoder, M.C., Sanchez-Ramirez, B.,
Gonzalez-Horta, M.D.C. and Infante-Ramirez, R. 2016. Multidrug-resistant bacteria
isolated from surface water in Bassaseachic Falls National Park, Mexico. International
Journal of Environmental Research and Public Health 13(6):597.
B3-9
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Sample Collection Information Document-Attachment IB-3
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Sample Collection Information Document-Attachment IB-3
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Sample Collection Information Document-Attachment IB-3
Morgan, M.J., Halstrom, S., Wylie, J.T., Walsh, T., .Kaksonen, A.H., Sutton, D., Braun, K. and
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Sample Collection Information Document-Attachment IB-3
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B3-13
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Sample Collection Information Document-Attachment IB-3
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Sample Collection Information Document - Attachim
Attachment B-4:
Sample Collection Information for
Pathogens (Bacteria, Viruses, Protozoa, and Helminths) in Aerosols
B4-1
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Sample Collection Information Document - Attachim
This page left intentionally blank
B4-2
-------
Sample Collection Information Document-Attachment B-4
Attachment B-4: Sample Collection Information for Pathogens in Aerosols
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Aerosol Bacteria
Bacillus anthracis
[Anthrax]
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Room temperature if held for 2
hours or less; keep on ice (e.g., ice
packs, secure double-bagged ice) if
longer.
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Teshale et al., 2002;
Estill et al., 2009;
NIST, 2012;
U.S. EPA, 2012;
U.S. EPA, 2013;
Xu et al., 2013;
Clauss, 2015;
Grinshpun et al.,
2016; Haig et al.,
2016
Brucella spp.
[Brucellosis]
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Room temperature if held for 15
minutes or less; keep on ice (e.g.,
ice packs, secure double-bagged
ice) if longer.
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Fatah et al., 2007;
NIST, 2012;
Dybwad, 2014
Burkholderia mallei
[Glanders]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Room temperature if held for 15
minutes or less; keep on ice (e.g.,
ice packs, secure double-bagged
ice) if longer.
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Fatah et al., 2007;
Blatny et al., 2008;
Dabisch et al., 2012;
U.S. EPA, 2013;
Grinshpun et al., 2016
Burkholderia
pseudomallei
[Melioidosis]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Room temperature if held for 15
minutes or less; keep on ice (e.g.,
ice packs, secure double-bagged
ice) if longer.
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Fatah et al., 2007;
Dabisch et al., 2012;
U.S. EPA, 2013;
Grinshpun et al., 2016
Campylobacter jejuni
[Campylobacteriosis]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger
Keep on ice (e.g. ice packs, secure
double bagged ice)
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L
Zhao et al., 2011a;
Zhao et al., 2011b;
Dybwad et al., 2014
Chlamydia psittaci
(formerly
Chlamydophila psittaci)
[Psittacosis]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice (e.g. ice packs, secure
double bagged ice)
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Van Droogenbroeck,
et al., 2009;
NIST, 2012;
Dybwad, 2014
Coxiella burnetii
[Q-fever]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Room temperature if held for 15
minutes or less; keep on ice (e.g.,
ice packs, secure double-bagged
ice) if longer.
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
NIST, 2012;
Aarnink et al., 2015;
Nunez et al., 2016
B4-3
-------
Sample Collection Information Document-Attachment B-4
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Escherichia coli
0157:H7(4)
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice (e.g. ice packs, secure
double bagged ice)
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Kesavan et al., 2008;
Riemenschneider et
al., 2010; NIST, 2012;
Xu et al., 2013;
Grinshpun et al., 2016
Francisella tularensis
[Tularemia]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Room temperature if held for 2
hours or less; keep on ice (e.g., ice
packs, secure double-bagged ice) if
longer.
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Burton et al., 2007;
Srikanth et al., 2008;
Dabisch et al., 2012
Legionella
pneumophila
[Legionellosis - a)
Pontiac fever; and b)
Legionnaires' disease]
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep frozen at <-20°C (dry ice or
super cold packs rated for temps
below -70°C)
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
CDC, 2015; AS/NZS,
2011c; Mandal and
Brandl, 2011;
CDC, 2003;
Ishimatsu et al., 2001
Leptospira spp.
(L. interrogans
serovars: L.
icteroheamorrhagiae, L.
autralis, L. balum, L.
bataviae, L. sejro, L.
pomona)
[Leptospirosis]
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice (e.g. ice packs, secure
double bagged ice)
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
VanDyke-
Gonnerman, 2013;
Li et al., 2012
Listeria
monocytogenes
[Listeriosis]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice (e.g. ice packs, secure
double bagged ice). If sample is
already frozen do not thaw until
analysis.
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Kretzer et al., 2008;
Srikanth et al., 2008;
Pillai and Ricke, 2002
Non-typhoidal
Salmonella
[Salmonellosis]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Room temperature if held for 15
minutes or less; keep on ice (e.g.,
ice packs, secure double-bagged
ice) if longer.
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Adell et al., 2014;
Riemenschneider et
al., 2010; Barker
and Jones, 2005
Salmonella Typhi
[Typhoid fever]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice (e.g. ice packs, secure
double bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40- 135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
NIST, 2012;
Woodword et al.,
2004; Pillai and Ricke,
2002
B4-4
-------
Sample Collection Information Document - Attachment B-4
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Shigella spp.
[Shigellosis]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice (e.g. ice packs, secure
double bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Srikanth et al., 2008;
Morey, 2007;
Kalogerakis et al.,
2005
Staphylococcus
(4)
aureus('
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice (e.g. ice packs, secure
double bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Haig et al., 2016;
Chang and Wang,
2015; Tseng et al.,
2014
Vibrio cholerae 01 and
0139 [Cholera]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Store at room temperature. q0 not
ship on ice. Note: unlikely to be
viable - samples should be
collected only for PCR analysis.
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Blatny et al., 2008;
Crook, 1996
Yersinia pestis
[Plague]'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Room temperature if held for 2
hours or less; keep on ice (e.g., ice
packs, secure double-bagged ice) if
longer.
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Dybwad et al., 2014;
Cooper, 2010;
Burton et al., 2007;
Bergman et al., 2005
Aerosol — Viruses
Adenoviruses:
Enteric and non-enteric
(A-F)'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Kienlen, 2015;
Ge et al., 2014;
Cooper, 2010
Astrovi ruses'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
D'Arcy, 2014;
Carducci, 2013;
Uhrbrand et al., 2012
Caliciviruses:
Norovirus'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Ge et al., 2014;
Carducci, 2013;
Grinshpun et al., 2007
Caliciviruses:
Sapovirus'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Ge et al., 2014;
Carducci, 2013;
Grinshpun et al., 2007
Coronaviruses: SARS-
associated human
coronavirus'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Kienlen, 2015;
Xu et al., 2013;
Wu et al., 2013;
Verreault et al., 2008
B4-5
-------
Sample Collection Information Document - Attachment B-4
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Hepatitis E virus
(HEV)(4)
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Aarnink et al., 2015;
Verreault et al., 2008
Influenza H5N1 virus14'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Lednicky et al., 2016;
Fennelly et al., 2015;
Tang et al., 2015;
Cooper 2010
Picornavi ruses:
Enteroviruses'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Kienlen, 2015;
Verreault et al., 2008;
Sattar et al., 1987
Picornaviruses:
Hepatitis A virus
(HAV)'4'
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Kienlen, 2015;
Verreault et al., 2008;
Burton et al., 2007;
Sattar et al., 1987
Reoviruses:
Rotavirus (Group A)
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Fronczek and Yoon,
2015;
Johnson et al., 2013;
Riemenschneider et
al., 2010;
Verreault et al., 2008;
Gerone et al., 1966
Aerosol — Protozoa
Cryptosporidium spp.
[Cryptosporidiosis]
Unlikely to be found.
Entamoeba
histolytica(4)
Unlikely to be found.
Giardia spp.
[Giardiasis]'4'
Unlikely to be found.
Naegieria fowieri
[Naegleriasis - primary
amoebic
meningoencephalitis
(PAM)/ amebic
encephalitis]
Sterile MCE/PTFE
filter'6', gel filter,
impinger, and/or
impactor (agar plate)
Keep on ice packs (or secure
double-bagged ice).
MCE/PTFE filter: 120 - 960 L;
gel filter: 40-135 L;
impinger'5': 750 - 6000 L;
impactor: 84.9 - 849 L
Srikanth et al., 2008;
Fink and Gilman,
2000
B4-6
-------
Sample Collection Information Document - Attachment B-4
Analyte(s) [Disease]
Container
Preservation'1'
Sample Size'2'
Source'3'
Toxoplasma gondii
[Toxoplasmosis]'4'
Unlikely to be found.
Aerosol — Helminths
Baylisascaris procyonis
[Raccoon roundworm
infection]
Unlikely to be found.
B4-7
-------
Sample Collection Information Document - Attachim
Footnotes:
(1) Any sample collected for cultivation-based analysis must not be allowed to freeze.
(2) The sample sizes listed are based on the amount needed for analysis of a single sample. If requested by the laboratory, additional sample(s) must be
collected for laboratory quality control analyses (e.g., duplicates, matrix spikes). It is also recommended that additional sample(s) be collected in case of the
need for reanalysis due to sample spillage or unforeseen analytical difficulties.
(3) Additional resources. References for these sources are supplied at the end of this attachment.
(4) Currently, no information is available for this analyte in this sample type. Until such time that analyte-specific information is available, collection procedures
described for a similar analyte/sample type are considered to be appropriate.
(5) If using impingers that do not replenish the liquid as it is evaporated by the air stream, the maximum recommended sampling volume is 200 L (Applied and
Environmental Microbiology, Duchaine et al., 2001, 67(6): 2775-2780).
(6) Mixed cellulose ester (MCE) and polytetrafluoroethylene (PTFE) filters are available as cassettes.
Notes:
• U.S. DOT and IATA labeling requirements apply to materials that are known to contain, or are suspected of containing, an infectious substance and reflect the
most recent changes, effective October 1, 2006. Further guidance on these changes and lists of substances considered to be either category A (not listed in
this document) or category B can be obtained from the U.S. Department of Transportation, Pipeline and Hazardous Materials Safety Administration (DOT,
PHMSA) (http://www.phmsa.dot.aov/staticfiles/PHMSA/DownloadableFiles/Files/Transportina Infectious Substances brochure.pdf). Definitions and
exceptions for Class 6, Division 6.2 infectious substances are described in 49 CFR 173.134.
• For collection of aqueous samples containing residual chlorine, add a stock solution of filter-sterilized 10% sodium thiosulfate at 0.5 mL/L.
• Sample transport containers are packed outside the contaminated area. Samples must be packed in a manner that protects the integrity of the sample
containers and provides temperature conditions required for sample preservation. Primary receptacles should be leak-proof with a volumetric capacity of not
more than 500 mL (liquid) or 4 kilograms (solid). If several individual primary containers are placed in a single secondary packaging, they must be individually
wrapped or separated so as to prevent contact between them. Secondary packaging should be leak-proof and surrounded by shock- and water-absorbent
packing materials or ice (if required for preservation) and shipped in a cooler to ensure sample temperatures do not exceed preservation requirements. Ice
should be placed in separate plastic bags or cold packs should be used to avoid leakage, and the bags placed around, among, and on top of the secondary
sample containers. Further guidance can be obtained from 49 CFR 173.199
-------
Sample Collection Information Document - Attachim
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Barker, J. and Jones, M.V. 2005. The potential spread of infection caused by aerosol
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Carducci, A. 2013. Sampling strategies for virus detection in foods, food-processing
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Chang, C.W. and Wang, L.J. 2015. Impact of culture media and sampling methods on
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Cooper, C.W. 2010. High volume air sampling for viral aerosols: a comparative approach. Air
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D'Arcy, N. 2014. Exploring the nature and diversity of microorganisms in healthcare and
educational settings. Doctoral Dissertations. Eastman Dental Institute, University College
London, United Kingdom.
Dabisch, P., Bower, K., Dorsey, B. and Wronka, L. 2012. Recovery efficiencies for
Burkholderia thailandensis from various aerosol sampling media. Frontiers in Cellular and
Infection Microbiology, 2:78.
Dybwad, M. 2014. Characterization of airborne bacteria at a subway station: Implications for
testing and evaluation of biological detection, identification, and monitoring systems.
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B4-9
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Sample Collection Information Document - Attachim
Dybwad, M., Skogan, G. and Blatny, J.M. 2014. Comparative Testing and Evaluation of Nine
Different Air Samplers: End-to-End Sampling Efficiencies as Specific Performance
Measurements for Bioaerosol Applications. Aerosol Science and Technology, 48(3): 282-
295.
Estill, C.F., Baron, P.A., Beard, J.K., Hein, M.J., Larsen, L.D., Rose, L., ...Deye, G.J. 2009.
Recovery efficiency and limit of detection of aerosolized Bacillus anthracis Sterne from
environmental surface samples. Applied and Environmental Microbiology, 75(13):4297-
4306.
Fatah, A.A., Arcilesi, Jr., R.D., Chekol, T., Lattin, C.H., Sadik, O.A. and Aluoch, A. 2007. Guide
for the Selection of Biological Agent Detection Equipment for Emergency First Responders.
Guide 101-06. U.S. Department of Homeland Security, Washington, D.C.
Fennelly, K.P., Tribby, M.D., Wu, C.Y., Heil, G.L., Radonovich, L.J., Loeb, J.C. and Lednicky,
J.A. 2015. Collection and measurement of aerosols of viable influenza virus in liquid media
in an Andersen cascade impactor. Virus Adaptation and Treatment, 7:1-9.
Fink, R. and Gilman, E.A. 2000. Biological Contamination of the Building Environment:
Sampling and Analysis. Journal of the American Biological Safety Association, 5(1):19-29.
Fronczek, C.F. and Yoon, J.Y. 2015. Biosensors for Monitoring Airborne Pathogens. Journal
of Laboratory Automation, 20(4):390-410.
Ge, S., Kuehn, T.H., Abin, M., Verma, H., Bekele, A., Mor, S.K.,...Zuo, Z. 2014. Airborne virus
survivability during long-term sampling using a non-viable Andersen cascade impactor in an
environmental chamber. Aerosol Science and Technology, 48(12): 1360-1368.
Gerone, P.J., Couch, R.B., Keefer, G.V., Douglas, R.G., Derrenbacher, E.B. and Knight, V.
1966. Assessment of experimental and natural viral aerosols. Bacteriological Reviews,
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Grinshpun, S.A., Buttner, M.P., Mainelis, G. and Willeke, K. 2016. Sampling for airborne
microorganisms. Manual of Environmental Microbiology, Fourth Edition (pp. 3-2). American
Society of Microbiology.
Grinshpun, S.A., Adhikari, A., Honda, T., Kim, K.Y., Toivola, M., Rao, K.S.R. and Reponen, T.
2007. Control of aerosol contaminants in indoor air: combining the particle concentration
reduction with microbial inactivation. Environmental Science & Technology, 41(2):606-612.
Haig, C.W., Mackay, W.G., Walker, J.T. and Williams, C. 2016. Bioaerosol sampling: sampling
mechanisms, bioefficiency and field studies. Journal of Hospital Infection, 93(3):242-255.
Ishimatsu, S., Miyamoto, H., Hori, H., Tanaka, I. andYoshid, S-l. 2001. Sampling and Detection
of Legionella pneumophila Aerosols Generated from an Industrial Cooling Tower. Annals of
Occupational Hygiene, 45(6):421-427.
Johnson, D.L., Mead, K.R., Lynch, R.A. and Hirst, D.V.L. 2013. Lifting the lid on toilet plume
aerosol: A literature review with suggestions for future research. American Journal of
Infection Control, 41(3):254-258.
Kalogerakis, N., Paschali, D., Lekaditis, V., Pantidou, A., Eleftheriadis, K. and Lazaridis, M.
2005. Indoor air quality—bioaerosol measurements in domestic and office premises.
Journal of Aerosol Science, 36(5):751-761.
Kesavan, J., Bottiger, J.R. and McFarland, A.R. 2008. Bioaerosol concentrator performance:
comparative tests with viable and with solid and liquid nonviable particles. Journal of Applied
Microbiology, 104:285-295.
Kienlen, L.L. 2015. Comparison of Bioaerosol Collection Methods in the Detection of Airborne
Influenza Virus. Masters thesis. The University of Iowa, Iowa City, Iowa.
Kretzer, J.W., Biebl, M. and Miller, S. 2008. Sample preparation-An essential prerequisite for
high-quality bacteria detection. Principles of bacterial detection: Biosensors, recognition
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B4-10
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Sample Collection Information Document - Attachim
Lednicky, J., Pan, M., Loeb, J., Hsieh, H., Eiguren-Fernandez, A., Hering, S., ... Wu, C-Y.
2016. Highly efficient collection of infectious pandemic influenza H1N1 virus (2009) through
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Sample Collection Information Document - Attachim
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B4-12
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Sample Collection Information Document - Attachment C
Attachment C:
Holding Time, Packaging Requirements, and
Shipping Label of Sample
C-1
-------
Sample Collection Information Document - Attachment C
Attachment C: Table of Contents
C.1 Holding time 3
C.2 Packaging Requirements 3
C.3 IATA/DOT Marking and Labeling Requirements 7
C.4 Chain of Custody 8
C.5 Background References 9
Figure C-1. Secondary packaging 6
Table C-1. Transportation Modes and Pathogenic Samples Not Allowed 4
C-2
-------
Sample Collection Information Document - Attachment C
Attachment C: Holding Time, Packaging Requirements, and Shipping Label of Sample
Sample Holding time, packaging requirements, and shipping label of the samples discussed in
this document will follow the following protocol, unless otherwise specified.
C.1 Holding time
Maximum holding time is the time between sample collection and analysis, which is the sum of
the time to transport the sample from the field and storage time at the laboratory. When
samples are to be analyzed for more than one microbiological parameter, due regard must be
given to the appropriate storage conditions. The terms microbial testing can include a wide
range of organisms, some of which may be more or less sensitive to storage times or
temperature. Sample analysis should be prioritized such that the organisms most susceptible to
change are analyzed first. Samples should be shipped to the laboratory without delay so that
analysis can be completed quickly after collection. Samples should be kept in the dark and
measures should be taken to avoid changes in sample moisture content.
Holding Time
Minimize transport and storage time. Analyze or
extract immediately upon receipt at the laboratory.
None of the standards provide published evidence to support the recommended sample
handling guidance and the holding times can appear arbitrary when a single set of instructions
is applied to a large group of organisms. The terms microbial testing or bacteriological
examination can include a wide range of organisms, some of which may be more or less
sensitive to storage times or temperature. A criticism that has been levelled at standards is that
sample holding times were originally established for aqueous media and then blindly applied to
other media (USEPA, 2005).
C.2 Packaging Requirements
This section provides packaging requirements biological materials as needed to safely move the
material from one location to another. Packaging, transportation, and shipping should be in
accordance with:
• U.S. Department of Transportation (DOT) Hazardous Materials Regulations (HMR) for
movement of biological materials in public right-of-ways within the U.S.
• International Air Transport Association (IATA) Dangerous Goods Regulations (DGR) for
shipment of biological materials (e.g., infectious substances) by air.
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Sample Collection Information Document - Attachment C
Table C-1 lists the desired transportation modes that should be considered while shipping
pathogen contaminated samples.
Table C-1. Transportation Modes and Pathogenic Samples Not Allowed
General Transport
Mode
Specific Transport
Mode
Pathogenic Samples That Are Not Allowed
Hand carry between
laboratories
No restrictions on types of biological materials
Hand carry between
buildings
No restrictions on types of biological materials
Personal
Transportation
Personal motor vehicle*
Regulated biological materials are not allowed
except for regulated materials being transported for
research, diagnosis, investigational activities, or
disease treatment or prevention; or that are
biological products. Samples containing "Category
A" infectious substances are not allowed.
Public transportation
Regulated biological materials or other biological
materials that may present a detrimental risk to the
health of humans or other organisms either directly
through infection or indirectly through damage to
the environment are not allowed.
Licensed
Transporter
Common carrier
No restrictions on types of biological materials
unless restricted by the carrier.
* Personal transport in a motor vehicle means transportation in a private or government passenger
vehicle such as a car, van, or pickup truck.
Using the proper packing materials, package, and labels incorrectly can cause the package to
be out of compliance. Proper packaging is the responsibility of the sender. The sender
assumes sole responsibility for compliance with all governmental regulations. Receiving drivers
have the authority and responsibility to refuse any biological substance shipment that does not
meet minimum packaging requirements.
Use well-constructed packaging to cushion the inner containers and enough absorbent material
to absorb the entire contents of the inner packages should they break open during transport.
Inner containers can be glass or plastic with the closure held securely in place (taped closed).
The outer container can be a cardboard box.
Packages may be re-used if they are in good condition and have been disinfected. If packages
are used for items other than infectious substances, all labels and marks for infectious
substances must be removed or completely covered.
C.2.1 Primary Receptacle Requirements
Primary receptacles must be able to be secured with a lid or sealed with a screw top lid or
with tape or Parafilm®. Each of the containers must have the container's content, hazards,
and ownership on or with the container.
• Inner containers:
o Use break-resistant (e.g., plastic) containers, if possible.
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Sample Collection Information Document - Attachment C
o Liquids must be in a leak-proof container. Lids on inner containers must have a
positive means of closure. For example, a screw cap should be used instead of
Parafilm, aluminum foil, or a stopper.
o Container(s) must be disinfected as needed for safety and should be placed in a
Ziploc® bag or an equivalent secondary spill container.
o Information must be placed on or with the container(s) as needed to clearly
communicate the container's contents, hazards, and ownership. Each individual
container must be labeled with enough information to identify its contents. In
addition, the container(s) or secondary bag(s) must also be labeled with the identity
of the material, the name and phone number of the sender, the recipient's name and
phone number if they are different from the sender's, and hazard information. Hazard
information includes a biohazard label if the material is biohazardous, any words
needed to explain the hazard, or words indicating the material is not hazardous.
o Containers for sharps (i.e., sharps container) must be constructed of a rigid material
resistant to punctures and securely closed to prevent leaks or punctures. If several
fragile primary receptacles are placed in a single secondary packaging, they must be
individually wrapped or separated so as to prevent contact between them.
C.2.2 Secondary Packaging Requirements
When placing multiple primary glass receptacles in the same secondary package, each
primary glass receptacle must be wrapped or separated from each other. This will prevent
them from breaking or becoming damaged during transport. The secondary package must
be sealed so that it will not open and spill the contents during transport. See Figure C-1,
below.
• Outer Container Requirements
To prevent a release or leak of the pathogen contaminated substance, place sorbent
material between the primary containers and secondary package. Use enough sorbent
material to absorb the entire contents of the primary containers if they should break. In
addition, the secondary package must fit in the outer package, and it must fit as close as
possible to prevent the secondary package from moving too much during transport.
The outer container should meet the following criteria:
o Must be capable of surviving a drop test at a height of 1.2 meters without leakage
from the primary receptacle. The primary receptacles must remain protected in the
secondary packaging.
o Be adequate in strength
o Have a secure lid (e.g. plastic box, insulated cooler).
o Be rigid so as to retain its original shape and dimensions at all times under all
conditions of transportation.
o Have at least one surface with a minimum dimension of 100-mm X 100-mm (4-
inches x 4-inches).
o Allow the secondary container to fit as closely as possible to prevent excessive
movement during transport, which could damage the primary containers.
NOTE: If there is space between the secondary container and outer container, place padding
between the two containers to prevent the inner container from shifting.
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Sample Collection Information Document - Attachment C
Primary Receptacle
Leakprcofor Sitprcof
7
Secondary Packaging
Leakprcofor Sifproof
(e.g. Seated Plastic Bag)
Sample Material
Sorbent Packing
Material (for Squid)
Cross Section of Closed Package
Rigid Outer Packaging
Cushioning Material ¦
Name and telephone
number of a person
responsible
Figure C-1. Secondary packaging.
Primacy RecefJIacte
Leakp-oof
Secorfla-y Packaging
Leakcroofw Sipnxrf
(e.g. Sea«i Preasac Bag
core' nteninaidfc
packaging)
Sorbent
MaienaJ
C.2.3 Manufacturer's packaging.
When applicable, each regulated biological material must be contained and packaged in the
manufacturer's original container and packaging, or a container and packaging of equal or
greater strength and integrity.
C.2.4 Markings
Markings refer to the information on the outer package and airway bills.
• The marking must be 2 inches * 2 inches (minimum)
• A diamond marking with the appropriate UN number (the four-digit United Nations
number, which identify dangerous goods for transportation purposes)
• The proper shipping name to the marking
• The name, address and phone number of a responsible person must be on the air
waybill or marked on the package.
• If an airway bill is used, the "Nature and Quantity of Goods" box must show the text
"Biological Substance, Category B" and "UN 3373".
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Sample Collection Information Document - Attachment C
C.2.5 Refrigerants
All refrigerants must be placed outside the secondary packaging.
• Gel packs: Use gel-packs in place of wet ice. There are no requirements for marking or
labeling the outer package for use of gel packs. It is difficult to achieve and maintain
lower temperatures using gel packs.
• Dry ice: Class 9 Dangerous Good.
C. 2.6 Packaging Requirements for Dry Ice
Dry ice is a hazardous material and is regulated by both the DOT) and the I ATA. Specific
procedures are required for handling, packaging, and shipping materials refrigerated with
dry ice, if applicable. In addition, refer the IATA/DOT Requirements for Packing Instructions
(PI) 904 and the document ACCEPTANCE CHECKLIST FOR DRY ICE
(https://www.iata.org/whatwedo/cargo/dgr/Documents/acceptance-checklist-dry-ice-en.pdf)
for more information.
• Contact the carrier to ensure proper ventilation will be available for the package and to
determine if the carrier has additional requirements from those specified in the IATA PI
904 regulations.
• Coordinate logistics of the shipment with the recipient. Take into account local holidays
or closings that might delay package receipt.
Refer to package manufacturer's recommendations to determine the correct amount of dry
ice to include in your shipment. The actual time will vary depending on the package used
and the volume and density of the dry ice. In general, however, dry ice will sublimate from a
solid to a gas at a rate of 5-10 pounds (2.27-4.54 kg) per 24 hours when shipped in an
appropriate insulated cooler.
C.3 IATA/DOT Marking and Labeling Requirements
The outermost container must be labeled with a hazard Class 9 Miscellaneous Dangerous Good
label, UN 1845, and net weight of dry ice in kilograms.
J
i
FedEx has no additional restrictions for shipping dry ice. UPS requires the UPS Blue Dry Ice
label in addition to the IATA/DOT requirements for marking and labeling:
DRY ICE
UN 1845
KG NET WT
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Sample Collection Information Document - Attachment C
Shipments of dry ice and other dangerous goods without an approved contract with UPS are
prohibited.
C.4 Chain of Custody
A Chain of Custody (CoC) form documents transfer of sample custody from one individual to
another, from the time the sample is collected until final analytical disposition. Each individual in
possession of the sample must be noted by recording their signature on the form. The CoC
record should include instructions for the laboratory technician as to analytical methods,
potential dangers, and any pertinent handling procedures that should be observed. The CoC
form should be kept separate from the sample (i.e., should not be placed with the sample) in
order to preserve appropriate CoC. The CoC record must include at least the following
information:
• All available information regarding the potential hazards associated with the agent;
Handling procedures associated with the samples;
Sample identification number;
Sample concentration, if known;
Sampling location;
Collection date and time;
Sample matrix;
Names and signatures of the samplers; and
Signatures of all individuals who had custody of the samples.
An unbroken COC must be maintained for all samples from collection through analysis and
archiving. In order to maintain COC, the form must be readily accessible when transferring
samples from one individual to another. Therefore, COC forms should not be placed inside the
primary sample containment. A copy of the record will be kept with the samples until they are
analyzed and returned with the analytical results or will be maintained on site at the laboratory if
samples are archived for later use or collection by law enforcement.
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Sample Collection Information Document - Attachment C
C.5 Background References
DOT. 2014. Federal Register Safety Advisory Notice: Packaging and Handling Ebola Virus
Contaminated Infectious Waste for Transportation to Disposal Sites. 79 FR 64646.
Pipeline and Hazardous Materials Safety Administration, U.S. Department of Transportation.
DOT. 2017. Transporting Infectious Substances. Pipeline and Hazardous Materials Safety
Administration, U.S. Department of Transportation. Retrieved on April 27, 2017 from
https://phmsa.dot.gov/hazmat/transportinq-infectious-substances.
Federal Register. 2005. Possession, Use, and Transfer of Select Agents and Toxins; Final Rule.
42 CFR 72 and 73. Department of Health and Human Services. 70(52): 13294-13325.
IATA. 2017. Dangerous Goods Regulations. 58th edition. The International Air Transport
Association, Miami, Florida.
National Security Council-led Domestic Resilience Group. 2017. Interim - Planning Guidance for
the Handling of Solid Waste Contaminated with a Category A Infectious Substance. Retrieved
on April 27, 2017 from
https://phmsa.dot.gov/staticfiles/PHMSA/DownloadableFiles/Files/lnterim Planning Guidance
for Handling Category A Solid Waste.pdf.
University of Michigan. 2016. Packaging Requirements to Transport Biological Substances &
Hazardous Materials. Retrieved on April 27, 2017 from https://ehs.umich.edu/wp-
content/uploads/sites/37/2017/03/Pack-Reg-BioSub-Manual.pdf.
U.S. Government Publishing Office. 2016. 49 CFR 173.199. Category B infectious substances.
Retrieved on May 1, 2017 from https://www.gpo.gov/fdsvs/pkg/CFR-2006-title49-vol2/pdf/CFR-
2006-title49-vol2-sec173-199. pdf.
USEPA. 2005. Sample Holding Time Reevaluation. Office of Research and Development,
Washington, DC. EPA/600/R-05/124.
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*>EPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development
Homeland Security Research Program
Cincinnati, OH 45268
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