&EPA
                             EPA/600/R-12/577 | December 2012 | www.epa.gov/ord
   United States
   Environmental Protection
   Agency
       Protocol for Detection of
       Bacillus anthracis in
       Environmental Samples
       During the Remediation Phase
       of an Anthrax Event
      Office of Research and Development
      National Homeland Security Research Center

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Detection of Bacillus anthracis in Environmental Samples
                        f/EPA
         United States Environmental Protection Agency
     National Homeland Security Research Center (NHSRC)
          Threat and Consequence Assessment Division
               26 West Martin Luther King Dr.
                    Cincinnati, OH 45268

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Detection of Bacillus anthracis in Environmental Samples
                                  Table of Contents

Disclaimer	iii
Foreword	iv
Trademarked Products	vii
Acknowledgements	viii
Introduction	1
1.0    Scope and Application	4
2.0    Summary of Methods	4
3.0    Interferences and Contamination	5
4.0    Safety	5
   4.1    Laboratory Hazards	5
   4.2    Recommended Precautions	5
5.0    Supplies and Equipment	7
   5.1  General Laboratory Supplies	7
   5.2  Supplies for RV-PCR Analysis	8
   5.3    Supplies for Real-time PCR Analysis	8
   5.4    Supplies for Culture	9
   5.5    Equipment	9
6.0    Reagents and Standards	10
7.0    Calibration and Standardization	14
8.0    Quality Control (QC)	15
9.0    Site Characterization Procedures	17
   9.1    Sample Processing for Laboratories With RV-PCR Capability	17
   9.2    DNA Extraction and Purification	21
   9.3    Real-time PCR Analyses	22
10.0   Post Decontamination Procedures for Rapid Viability-Polymerase Chain Reaction (RV-PCR)
       Analyses	26
   10.1   RV-PCR	26
   10.2   RV-PCR Sample Processing:  Spore Recovery	29
   10.3   RV-PCR Sample Processing:  Buffer Washes and Broth Culture	33
   10.4   Manual DNA Extraction and Purification	35
   10.5   Real-time PCR Analysis of To and T9 DNA Extracts	38
11.0   Post Decontamination Procedures for Culture Analyses	39
   11.1   Sample Processing and Plating for Sponge-Sticks and Wipes	40

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Detection of Bacillus anthracis in Environmental Samples
   11.2   Sample Processing and Plating for Swabs	42
   11.3   Sample Processing and Plating for Air Filters	44
   11.4   Sample Processing and Plating for Vacuum Socks and Filters	46
   11.5   Sample Processing and Plating for Water Samples	50
   11.6   Confirmation of B. anthracis Colonies by Real-time PCR Analysis	52
12.0   Data Analysis and Calculations	53
   12.1   Real-time PCR During the Site Characterization Phase	53
   12.2   RV-PCR	54
   12.3   Culture	54
13.0   Method Performance	56
14.0   Pollution Prevention	56
15.0   Waste Management	56
16.0   References	56
Appendix A
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Detection of Bacillus anthracis in Environmental Samples
                                      Disclaimer

This document has been reviewed in accordance with U.S. Environmental Protection Agency (EPA)
policy and approved for publication. Note that approval does not signify that the contents necessarily
reflect the views of the Agency.  Mention of trade names, products, or services does not convey EPA
approval, endorsement, or recommendation.

Questions concerning this document or its application should be addressed to:

SanjivR. Shah, Ph.D.
National Homeland Security Research Center (NHSRC)
U.S. Environmental Protection Agency
1300 Pennsylvania Avenue, NW
USEPA-8801RR
Washington, DC 20460
(202) 564-9522
shah.sanj iv@epa.gov

If you have difficulty accessing this PDF document, please contact Kathy Nickel
(Nickel.Kathy@epa.gov) or Amelia McCall (McCall.Amelia@epa.gov) for assistance.
                                             /'/'/'                                 December 2012

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Detection of Bacillus anthracis in Environmental Samples
                                        Foreword

Following the 2001 terrorist attacks and the anthrax bioterrorism event, several Presidential Directives
broadened the U.S. Environmental Protection Agency's (EPA's) mission to include key aspects of
homeland security. EPA was directed to protect human health and the environment from harmful effects
of chemical, biological and radiological contamination. To effectively respond to such events, EPA
established the Environmental Response Laboratory Network (ERLN) and Water Laboratory Alliance
(WLA), the laboratory network responsible for analyses of environmental and water samples.  In the
event of the release of a bioterrorism agent such as Bacillus anthracis (B. anthracis), hundreds to
thousands of environmental samples will need to be processed and analyzed in a timely manner to support
decontamination planning efforts. Analytical results, in addition to technical expertise, will be provided
to those responsible for making clearance decisions. To address these critical needs, EPA's National
Homeland Security Research Center (NHSRC), in collaboration with the Centers for Disease Control and
Prevention (CDC) and Lawrence Livermore National Laboratory (LLNL), has developed this protocol for
detection of B.  anthracis in environmental samples, including drinking water.

The "Protocol for Detection of Bacillus anthracis in Environmental Samples During the Remediation
Phase of an Anthrax Event" includes response-phase-appropriate sample processing and analytical
procedures for determining the presence or absence of B. anthracis spores in a cost-effective and time-
efficient manner. This protocol includes sample processing procedures appropriate for the real-time
polymerase chain reaction (PCR) analytical technique used during  the site characterization phase. Since
determination of whether viable B. anthracis spores are present in the samples will be  required during the
post decontamination phase of the response, sample processing methods appropriate for traditional
microbiological culture and EPA's Rapid Viability (RV)-PCR analytical procedures are also included.

Although Laboratory Response Network (LRN) laboratories will provide analytical support to EPA by
analyzing environmental samples, they will be using LRN-specific protocols as opposed to this protocol.
NHSRC has made this protocol available to ERLN and WLA labs for the analysis of samples to assist in
preparing for and recovering from disasters involving contamination from B.  anthracis spores. This
milestone specifically represents a strong and continuous commitment of NHSRC's research in support of
the ERLN and WLA. It also exhibits EPA's commitment to fulfill its homeland security mission and its
overall mission to protect human health and the environment.
Jonathan Herrmann, Director
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
                                                                                 December 2012

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Detection of Bacillus anthracis in Environmental Samples
                                    List of Tables

Table 1. Sample Processing Negative Controls	16
Table 2. Example EPA-2 PCR Assay Master Mix Preparation for 70 Reactions	24
Table 3. Example EPA-1 and BC3 PCR Assay Master Mix Preparation for 70 Reactions	25
                                   List of Figures

Figure 1. PCR Amplification	23
Figure 2. Example PCR Amplification Curves for the Initial T0 Aliquot and the Endpoint Aliquot	27
Figure 3. Flow Chart for RV-PCR Sample Analysis	28
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Detection of Bacillus anthracis in Environmental Samples
                                       Acronyms

ABI          Applied Biosystems®
BD           Becton, Dickinson and Company
BHI          Brain heart infusion
BMBL        Biosafety in Microbiological and Biomedical Laboratories
BSC          Biological safety cabinet
BSL          Biological safety level
CBR          Chemical, biological, radiological
CDC          Centers for Disease Control and Prevention
CFR          Code of Federal Regulations
CT            Cycle threshold
DI            Deionized
DNA          Deoxyribonucleic acid
DQO          Data quality objectives
EDTA        Ethylenediaminetetraacetic acid
EIC          External inhibition control
EPA          United States Environmental Protection Agency
ERLN        Environmental Response Laboratory Network
FEM          Forum  on Environmental Measurement
ICLN         Integrated Consortium of Laboratory Networks
IEC          International Electrotechnical Commission
ISO          International Organization for Standardization
LLNL        Lawrence Livermore National Laboratory
LRN          Laboratory Response Network
MPN          Most probable number
MSDS        Material safety data sheets
NHSRC       National Homeland Security Research Center
NIST          National Institute of Standards and Technology
NTC          No template controls
OSHA        Occupational Safety and Health Administration
PAPR        Powered air purifying respirator
PBST         Phosphate buffered saline with Tween® 20
PC            Positive control
PCR          Polymerase chain reaction
PES          Polyethersulfone
PMP          Paramagnetic particle
PNC          [Sample] processing negative control (Blank)
PPE          Personal protective equipment
psi            Pounds per square  inch
PT            Proficiency testing
QA           Quality assurance
QC           Quality control
RNA          Ribonucleic acid
rpm          Revolutions per minute
RV-PCR      Rapid Viability-polymerase chain reaction
SBA          Sheep blood agar
SOP          Standard operating procedure
TO            Time zero (no incubation)
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Detection of Bacillus anthracis in Environmental Samples
T9
TE
TNTC
UV
WLA
Nine hour incubation
Tris(hydroxymethyl)aminomethane-HCL-EDTA
Too numerous to count
Ultraviolet
Water Laboratory Alliance
                           Trademarked Products
Trademark
Acrovent™
BD Clay Adams™ Nutator Mixer
Biopur® Safelock®
Applied Bio Systems®
Autocup™
BBL™
Black Hole Quencher®
Costar®
Cole Farmer®
Dispatch®
Dynamag™
Fluoropore™
GN-6 Metricel®
Invitrogen®
Jiffy-Jack®
Kendall™
Life Technologies™
MagneSil® Blood Genomic
Masterflex®
MaxQ™
MicroFunnel™
Microcon®
Nalgene®
Stomacher®
TaqMan®
Trypticase™ soy agar
Tween®
Vacushield™
Versalon™
Whatman™
Holder
Pall Corporation
BD Diagnostics
Eppendorf
Life Technologies™
Whatman™ Ltd.
BD Diagnostics
Biosearch Technologies, Inc.
Corning
Cole Farmer®
Clorox Company
Life Technologies™
EMD Millipore
Pall Corporation
Life Technologies™
Cole Farmer®
Covidien, Inc.
Life Technologies™
Promega
Cole Farmer®
Thermo Scientific Inc
Pall Corporation
EMD Millipore
Nalge Nunc Corporation
Seward
Life Technologies™
BD Diagnostics
Sigma-Aldrich
Pall Corporation
Covidien, Inc.
Whatman™ Ltd.
Location
Ann Arbor, MI
Sparks, MD
United States
Carlsbad, CA
Maidstone, United Kingdom
Sparks, MD
Novato, CA
Tewksbury, MA
Vernon Hills, IL
United States
Carlsbad, CA
Billerica, MA
Ann Arbor, MI
Carlsbad, CA
Vernon Hills, IL
Mansfield, MA
Carlsbad, CA
Madison, WI
Vernon Hills, IL
Lenexa, KS
Ann Arbor, MI
Billerica, MA
Rochester, NY
United Kingdom
Carlsbad, CA
Sparks, MD
St. Louis, MO
Ann Arbor, MI
Mansfield, MA
Piscataway, NJ
                                                                       December 2012

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Detection of Bacillus anthracis in Environmental Samples
                                Acknowledgements

This protocol was prepared under the leadership and direction of Sanjiv Shah of the National Homeland
Security Research Center within the U.S. Environmental Protection Agency's Office of Research and
Development.

The contributions of the following organizations and persons to this protocol are gratefully
acknowledged:

Centers for Disease Control and Prevention (CDC) - Laboratory Response Network (LRN)
•  Richard Kellogg and Stephen Morse for their support and cooperation in the preparation and review of
   this protocol
•  Laura Jevitt for providing the LRN protocols that were adapted for inclusion in the protocol

Lawrence Livermore National Laboratory (LLNL)
•  Staci Kane for her technical contribution and critical review of the protocol

U. S. Environmental Protection Agency (EPA) National Homeland Security Research Center
(NHSRC)
•  Erin Silvestri, Tonya Nichols and Hiba Ernst for the overall support and review of the protocol
•  Eletha Brady-Roberts and Ramona Sherman for the QA review of the protocol

EPA Office of Emergency Management (OEM)
•  Marissa Mullins for the overall support and review of the protocol

Computer Sciences Corporation (CSC)
•  Yildiz Chambers and Emily King for support in development and review of the protocol
•  Rashmi Ghei for review of the protocol

Technical Reviewers
•  Laura Rose and Vincent Hill (CDC)
•  Sonia Letant (LLNL)
•  James Anthony (Department of Homeland Security)
•  Douglas Anders (Federal Bureau of Investigation)
•  Stephanie Harris (EPA Region 10)
•  Jafirul Hasan (EPA Office of Pesticides Program Microbiology Laboratory)
•  Frank Schaefer, Gene Rice, Worth Calfee and Vincent Gallardo (EPA NHSRC)
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Detection of Bacillus anthracis in Environmental Samples
                                       Introduction

The series of 2001 terrorist attacks and the anthrax bioterrorism incidents that resulted in human
casualties, and public and private facility closures, prompted enhanced and expanded national safeguards.
Multiple Presidential Directives have designated the U.S. Environmental Protection Agency (EPA) as the
primary federal agency responsible for the protection and decontamination of indoor/outdoor structures
and water infrastructure vulnerable to chemical, biological and radiological (CBR) terrorist attacks.
EPA's mission, to protect human health and the environment, was thereby expanded to address critical
homeland security related needs.

The National Homeland Security Research Center (NHSRC) within the Office of Research and
Development is EPA's hub for providing expertise on CBR agents and for conducting research to meet
EPA's homeland security mission needs.  A focus of NHSRC's research is to support the EPA's
Environmental Response Laboratory Network (ERLN), a nationwide network of federal, state, local and
commercial environmental laboratories which includes the Water Laboratory Alliance (WLA). Along
with the Centers for Disease Control and Prevention's (CDC's) Laboratory Response Network (LRN), the
ERLN can be activated in response to a large-scale environmental disaster to provide analytical
capability, increased capacity and produce quality data in a systematic and  coordinated manner.
Preparedness against potential indoor or outdoor wide-area anthrax attacks is currently the highest priority
for the ERLN. Based on the realities of response activities after the 2001 anthrax event and continued
preparedness efforts since then, it is anticipated that in the event of an intentional (bioterrorist attack) or
accidental release of Bacillus anthracis (B. anthracis) spores, hundreds to thousands of diverse
environmental sample (e.g., aerosols, particulates and drinking water) will need to be rapidly processed
and analyzed in order to assess the extent of contamination and support the planning of decontamination
efforts.  A similar number of samples will also need to be analyzed to determine the efficacy of
decontamination activities during the remediation phase of the response. During an anthrax event, EPA's
decision makers will need timely results from rapid sample analyses for planning the decontamination
efforts.  To address these critical needs, NHSRC, in collaboration with CDC and Lawrence Livermore
National Laboratory (LLNL), has generated this protocol for detection of B. anthracis in environmental
samples.

To complement an effective sample collection strategy during an anthrax event, a systematic approach for
timely and cost-effective sample analyses is critical.  Such a systematic approach also helps in effectively
managing and increasing the analytical laboratory capacity. This protocol attempts to present such an
approach for the detection of B. anthracis spores in various environmental samples (e.g., aerosol,
particulate [surface swabs, wipes, vacuum socks and filters, and Sponge-Sticks], drinking water). During
the remediation phase, prior to decontamination activities, EPA has to quickly determine the extent of
contamination in an affected building, facility, area or water system. Thus, for sample analyses during the
site characterization phase, detecting presence or absence of the deoxyribonucleic acid  (DNA) of B.
anthracis by real-time polymerase chain reaction (PCR) is usually the most appropriate, and both time-
and cost-effective technique.  Since determination of whether viable B. anthracis spores are present in the
samples will be required during the post decontamination phase of the response, either traditional
microbiological culture or EPA's Rapid Viability-PCR (RV-PCR) analytical procedures must be used.
Accordingly, the protocol presented here includes response-phase-appropriate sample processing and
analytical procedures.  It should be noted that LRN laboratories will support EPA environmental sample
analyses during remediation using LRN-specific protocols.
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Detection of Bacillus anthracis in Environmental Samples
Sample processing procedures are provided for real-time PCR analyses conducted during the site
characterization phase.  In addition, sample processing procedures appropriate for RV-PCR and culture
methods (for the post decontamination phase) are included. For drinking water samples, large volume
samples may need to be analyzed to detect low concentrations of B. anthracis spores or vegetative cells.
Therefore, the protocol also includes an ultrafiltration-based concentration procedure.  For the post
decontamination phase culture analyses, selected isolated colonies will be analyzed using real-time PCR
to confirm the identity of B. anthracis as opposed to traditional biochemical and serological testing.

Many sample processing and analysis procedures in this document have been derived from LRN
protocols. However, these procedures have been modified, as necessary, to address  EPA's homeland
security mission needs during the remediation phase of an anthrax event.  Therefore, these modified
procedures or this protocol itself must not be designated, referred to, or misconstrued as LRN procedures
or protocol.

Since this protocol was developed to include the analyses of diverse environmental samples, it
emphasizes appropriate sample processing as well as the DNA extraction and purification steps to
significantly remove any growth- and/or PCR-inhibitory materials present in the samples. This protocol
will be revised as better sample processing procedures and real-time PCR assays become available.

It should be noted that as of the publication date of this protocol has not been validated.  During any
B. anthracis related  emergency situations, EPA's use of non-validated methods in the absence of
validated methods must adhere to the EPA's Forum on Environmental Measurement (FEM) policy
directive on method validation:

According to Agency Policy Directive FEM-2010-01, Ensuring the Validity of Agency Methods
Validation and Peer Review Guidelines: Methods of Analysis Developed for Emergency Response
Situations:


       It is EPA's policy that all methods of analysis (e.g., chemical, radiochemical,
       microbiological) must be validated and peer reviewed prior to issuance as Agency
       methods.  There are emergency response situations that require methods to be developed
       and utilized, which may or may not have previously been validated or peer reviewed
       prior to use. This policy directive addresses those situations in which a method must be
       developed, validated and/or peer reviewed expeditiously for utilization in an emergency
       response situation. Also, in such emergency response situations only, an analytical
       method may be employed that has been validated by another established laboratory
       network (e.g., the Center for Disease Control and Prevention's Laboratory Response
       Network, the U.S. Department of Agriculture/Food and Drug Administration's Food
       Emergency  Response Network). In those instances, the responsible federal  agency will
       indicate that the level of validation and/or peer review that their analytical method
       underwent is consistent with the Integrated Consortium of Laboratory Networks (ICLN)
                                                                    5
       Guidelines for Comparison of Validation Levels between Networks.  The responsible
       federal agency may also refer to the Validation Guidelines for Laboratories Performing
                                            6
       Forensic Analysis of Chemical Terrorism  in order for the receiving federal  agency to
       determine if the analytical method meets the intended purpose.
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Detection of Bacillus anthracis in Environmental Samples
        Any EPA regional or program office that proposes to utilize a method in an emergency
        response situation is responsible for establishing and documenting to what level and by
        what process the method has been validated and/or peer reviewed in accordance with this
        policy. A regional or program office may determine the level of validation and/or peer
        review that is necessary to provide the objective evidence that a method is suitable for its
        intended purpose; however, the office must document the validation and/or peer review
        information supporting use of the method. All documentation should be preserved in
        accordance with the Agency's records management policy.
        5
         U.S. Department of Homeland Security, Integrated Consortium of Laboratory Networks (ICLN),
        ICLN Guidelines for Comparison of Validation Levels between Networks, Original Version,
        http ://www. icln. org/docs/sop.pdf.
        6
         Federal Bureau of Investigation (FBI), Scientific Working Group on Forensic Analysis of
        Chemical Terrorism (SWGFACT), Validation Guidelines for Laboratories Performing Forensic
        Analysis of Chemical Terrorism, Forensic Science Communications, Volume 7, Number 2, April
        2005.

The above policy is available at:

http://www.epa.gov/fem/pdfs/FEM  Policv2010-
01  Emergency Response Methods Final July 2010.pdf

Also, EPA recognizes that having analytical data of known and documented quality is critical in making
proper decisions during all phases of a response to an anthrax event and strives to establish data quality
objectives (DQOs) for each response activity. : These DQOs are based upon needs for both quality and
response time.  EPA's ERLN, which is tasked with providing laboratory support following homeland
security-related events, also has established data reporting procedures.  Requirements for receiving,
tracking, storing, preparing, analyzing and reporting data are specified in the Environmental Response
Laboratory Network Laboratory Requirements Document at:
http://epa.gov/erln/techsupport.html; project-specific requirements also are included in individual
Analytical Service Requests.

1 Information regarding EPA's DQO process, considerations and planning is available at:
http://www.epa.gov/QUALITY/dqos.html
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Detection of Bacillus anthracis in Environmental Samples
     Protocol for Detection of Bacillus anthracis in Environmental
     Samples During the Remediation Phase of an Anthrax Event

                                   December 2012

1.0    Scope and Application

The purpose of this protocol is to detect Bacillus anthracis (B. anthracis) spores in environmental
samples using real-time polymerase chain reaction (PCR) during the site characterization phase and to
detect viable B. anthracis spores using either Rapid Viability-PCR (RV-PCR) or culture followed by
isolate confirmation by real-time PCR during the post decontamination phase of the response. The real-
time PCR assays included in this protocol have been only partially characterized for specificity, however,
the use of these assays is currently recommended.  These assays will be replaced with fully characterized
and validated assays upon availability. This protocol is intended for the analyses of swabs, wipes,
Sponge-Sticks, vacuum socks and filters, air filters, drinking water and decontamination waste water for
B. anthracis spores.


2.0    Summary of Methods

2.1    Site Characterization Phase - Sample Analysis for Detection (Real-time PCR): After samples
       have been appropriately processed, the deoxyribonucleic acid (DNA) is extracted and purified.
       DNA extracts are analyzed by real-time PCR using the Applied Biosystems® (ABI) 7500 Fast
       Real-Time PCR System thermocycler.  Direct DNA-based analysis of samples allows for high
       throughput and rapid results.  Unless advised otherwise, real-time PCR should be performed
       using only the most sensitive assay, EPA-2 (Section 6.15).

2.2    Post Decontamination Phase - Sample  Analyses for Viability: After samples have been
       appropriately processed, they are cultured by either inoculating into nutrient rich broth (RV-PCR
       procedures) or plating on sheep blood agar (SBA), culture procedures, to allow for germination of
       viable spores.

       2.2.1   RV-PCR Procedure
              The RV-PCR is a combination of rapid broth culture and real-time PCR. Culturing the
              samples allows the germination of viable B. anthracis spores recovered from processed
              samples. Real-time PCR, via change in cycle-threshold (CT) value, offers a rapid
              determination of the viability and identity of B. anthracis bacteria that grow from
              germinated spores in broth culture. Samples (air filter, wipe, Sponge-Stick, swab,
              vacuum sock or filter, or drinking water) are processed in multiple spore extraction and
              wash steps.
              Recovered spores are incubated in brain heart infusion (BHI) broth for optimum growth
              of B. anthracis. After vortexing, an aliquot is withdrawn for baseline analysis. This is
              the Time Zero (T0) aliquot and is stored at 4°C. Then the broth culture remaining in the
              filter cup is incubated at 36°C for 9 hours on a rotary shaker incubator. After the broth
              culture has incubated for 9 hours, another aliquot is withdrawn. This is the T9 aliquot.
              Both the TO and T9 aliquots are processed to extract and purify total DNA. The T0 and T9
              DNA extracts, in triplicate, are then analyzed in real-time PCR assay(s) to detect
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Detection of Bacillus anthracis in Environmental Samples
               B. anthracis DNA.  The CT values for both the T0 and T9 DNA extracts are recorded and
               compared. Unless advised otherwise, real-time PCR should be performed using only the
               most sensitive assay, EPA-2 (Section 6.15).
       2.2.2   Culture Procedure
               The culture option includes plating serial dilutions of the sample on a non-selective agar
               SB A followed by rapid confirmation of typical isolated colonies using real-time PCR.
               Unless advised otherwise, real-time PCR should be performed using only the most
               sensitive assay, EPA-2 (Section 6.15).
3.0   Interferences and Contamination

3.1    Low recoveries of B. anthracis spores may be caused by the presence of high numbers of
       competing or inhibitory organisms, background debris, or toxic substances (e.g., metals or
       organic compounds).
3.2    Metals and organic compounds may also inhibit PCR reactions.
3.3    Problems related to sample processing, such as clogging of filters and inefficient extraction, may
       also result in low spore recoveries.
4.0   Safety

       Note:   This protocol should not be misconstrued as a laboratory standard operating procedure
               (SOP) that addresses all aspects of safety; the laboratory should adhere to their
               established safety guidelines.
4.1    Laboratory Hazards
       Direct contact of skin or mucous membranes with infectious materials, accidental parenteral
       inoculation, ingestion, and exposure to aerosols and infectious droplets has resulted in B.
       anthracis infection. Due to the infectious nature of this organism, all samples should be handled
       using biosafety requirements as dictated by Biosafety in Microbiological and Biomedical
       Laboratories [BMBL], 5th Edition, CDC 2009. (Reference 16.1) and/or organizational health and
       safety plans.

4.2    Recommended Precautions
       4.2.1   A biological safety level (BSL)-3 laboratory is required for handling and manipulating
               samples and cultures presumptive for B. anthracis. B. anthracis analyses should be
               conducted using BSL-3 practices, containment and facilities (BMBL, 5th Edition, CDC
               2009. [Reference 16.1]). Additional biosafety and select agent information, as well as
               statutory requirements for possession, use and transfer of select agents, can be found in
               the Code of Federal Regulations (42 CFRpart 73).
       4.2.2   All drinking water concentration activities should be performed within a BSL-3 facility
               using BSL-3 practices. If a biological safety cabinet (BSC) is not used, due to space
               limitations, approved respiratory equipment should be used while concentrating samples
               within the BSL-3 suite.  Caution should be used throughout the process (e.g., switching
               carboys, manipulation of the apparatus, adding wash solutions) to minimize spills/leaks.
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Detection of Bacillus anthracis in Environmental Samples
               Analysts should be properly trained prior to concentrating samples using the protocol
               provided in Appendix A.
       4.2.3   BSL-3 Practices

               The laboratory supervisor must ensure that laboratory personnel demonstrate proficiency
               in standard and special microbiological practices before working with BSL-3 agents. All
               procedures involving manipulation of infectious material must be conducted within a
               BSC (preferably Class II or Class III) or other physical containment device. Protective
               clothing (e.g., laboratory coats, gloves and respirator) should be worn while processing
               and analyzing samples.  Personal protective equipment (PPE) should never be worn
               outside the laboratory.

       4.2.4   Disposable materials are recommended for sample manipulations.
       4.2.5   The analyst must know and observe normal safety procedures required in a microbiology
               laboratory while preparing, using and disposing of media, cultures, reagents and
               materials.  Analysts must be familiar with the operation of sterilization equipment.
       4.2.6   Personal Protective Equipment (PPE)

           •   Laboratory personnel processing and conducting analyses of samples for B. anthracis
               place themselves at risk for exposure. However, the use of appropriate PPE can reduce
               the exposure risk. Laboratory personnel should familiarize themselves with the specific
               guidance for levels of protection and protective gear developed by the U.S. Department
               of Labor, Occupational Safety and Health Administration (OSHA), as provided in
               Appendix B of 29 CFR 1910.120
               (http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p
                id=9767). In addition to OSHA guidance, the Centers for Disease Control and
               Prevention (CDC) has developed recommendations for PPE based on BSL (Reference
               16.1, http://www.cdc.gov/biosafety/publications/bmbl5/index.htm).

           •   Goggles or a face shield should be used for protection against splashes, spills and sprays.
               Protective coats, gowns, smocks or uniforms designated for laboratory use must be worn
               while working with samples and potentially contaminated materials.  It may also be
               necessary to use a powered air purifying respirator (PAPR) to reduce the risk of
               inhalation. After use, protective clothing should be placed in sealed bags for appropriate
               decontamination, disposal or laundering.  Disposable gloves should be worn to protect
               hands from contact with potentially contaminated samples.  Wearing two pairs of gloves
               may be appropriate, but should not compromise needed dexterity.
       Note:   Gloves should be removed appropriately to avoid contaminating hands and surfaces
               between processing of each sample to prevent cross-contamination and disposed of
               whenever they become visibly contaminated or the integrity of the gloves is
               compromised. After all work with potentially infectious materials is completed, gloves
               should be removed and hands should be washed with soap and water.
       4.2.7   Depending on each laboratory's biosafety requirements, analysts may be required to be
               vaccinated prior to working with samples that could contain B. anthracis.
       4.2.8   This protocol does not address all safety issues associated with its use. Please refer to
               BMBL, 5th Edition, CDC 2009. (Reference 16.1) for additional safety information. A
               reference file of Material Safety Data Sheets (MSDS) should be available to all personnel
               involved in analyses.
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Detection of Bacillus anthracis in Environmental Samples
5.0   Supplies and Equipment
       Note:  Refer to Appendix A for supplies and equipment for large volume drinking water
              sample processing.
5.1    General Laboratory Supplies
       5.1.1  Gloves (e.g, latex, vinyl, or nitrile)
       5.1.2  Sterile gloves (e.g., latex, vinyl, or nitrile)
       5.1.3  Bleach wipes (Dispatch® Cat. No. 69150 or equivalent)
       5.1.4  Wipes
       5.1.5  Ziplock bags (large -20" x 28", medium -12" x 16", small -7" x 8")
       5.1.6  Sharps waste container
       5.1.7  Absorbent pad
       5.1.8  Medium and large biohazard bag(s) and rubber band(s)
       5.1.9  Sterile scalpels
       5.1.10 Sterile stainless steel scissors
       5.1.11 Squeeze bottle with 70% isopropyl alcohol
       5.1.12 Squeeze bottle with deionized (DI) water
       5.1.13 Autoclave tape
       5.1.14 Autoclave bags, aluminum foil, or kraft paper
       5.1.15 Large photo-tray or similar tray for transport of racks
       5.1.16 Laboratory marker
       5.1.17 Timer
       5.1.18 Disposable aerosol barrier pipet tips: 1000 \\L, 200 \\L, 10 \\L (Rainin Cat. No.
              SR-L1000F, SR-L200F, GP-10F or equivalent)
       5.1.19 1.5 mL Eppendorf Snap-Cap Microcentrifuge Biopur® Safe-Lock® tubes (Fisher
              Scientific Cat. No. 05-402-24B or equivalent)
       5.1.20 50 mL conical tubes (Fisher Scientific Cat. No. 06-443-18 or equivalent)
       5.1.21 15 mL conical tubes (Fisher Scientific Cat. No. 339650 or equivalent)
       5.1.22 250 mL and 1 L filter systems, polyethersulfone (PES), 0.2 \\m (Fisher Scientific Cat.
              No. 09-741-04, 09-741-03 or equivalent)
       5.1.23 0.1 urn Ultrafree-MC, VV filter unit (Millipore Cat. No. UFC30VVOO or equivalent)
       5.1.24 Tubes, sterile 2 mL DNase, RNase-free, gasketed, screw caps (National Scientific Cat.
              No. BC20NA-PS or equivalent)
       5.1.25 Glass Petri dishes, 100 * 15 mm
       5.1.26 Glass beads, acid washed, 106 \\m and finer (Sigma Cat. No. G4649 or equivalent)
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Detection of Bacillus anthracis in Environmental Samples
       5.1.27  Glass beads, acid washed, 425 - 600 (im and finer (Sigma Cat. No. G8772 or equivalent)
       5.1.28  PCR 8 cap strips (VWR Cat. No. 83009-684 or equivalent)
       5.1.29  Microcon® Centrifugal Filter Devices, Micron YM-100, blue Microcon® filters,
               (Amicon/Millipore Cat. No. 42413)
       5.1.30  Wide mouth screw cap containers (Fisher Scientific Cat. No. 4-375-459 or equivalent)
       5.1.31  Gauze wipes, 2" x 2" 50% rayon/50% polyester (Kendall™ Versalon™ Cat. No. 8042 or
               equivalent)
       5.1.32  Air filters, 37 mm, Fluoropore™ (Millipore Cat. No. FSLW04700 or equivalent)
       5.1.33  Swabs, macrofoam (VWR Cat. No. 10812-016 or equivalent)
       5.1.34  Vacuum socks (Midwest Filtration Co. Cat. No. FAB-07-03-006PS or equivalent)
       5.1.35  Vacuum filters (3M Forensic, Precision Data Products Cat. No. FF-1 with 4" diameter
               filter or equivalent)
       5.1.36  Sponge-Stick sampling tools (3M Inc. Cat. No. SSL100 or equivalent).
5.2   Supplies for RV-PCR Analysis
       5.2.1   30 mL screw cap tubes (E&K Scientific Cat. No. EK-T324S or equivalent)
       5.2.2   Disposable nylon forceps (VWR Cat. No. 12576-933 or equivalent)
       5.2.3   Monofilament polyester mesh disc (McMaster Carr Cat. No. 93185T17 or equivalent) or
               2" x 2" cut squares from mesh sheets (McMaster Carr Cat. No. 9218T13 or equivalent)
       5.2.4   Whatman™ Autocup™, filter cups (VWR Cat. No. 1602-0465 or equivalent)
       5.2.5   Polyethylene caps, blue with pull-tabs (McMaster Carr Cat. No. 94075K56 or
               equivalent), for vortexing and incubation steps
       5.2.6   50 mL Tube Cap (E&K Scientific, Cat. No. T3251-C or equivalent)
       5.2.7   Polyethylene quick turn tube fittings (Ark-Plas Products Cat. No. 51525K365 or
               equivalent)
       5.2.8   50 mL conical tubes, skirted, sterile (VWR Cat. No. 82050-322 or equivalent)
       5.2.9   Disposable serological pipets: 25 mL, 10 mL, 5 mL
       5.2.10  Single 50 mL conical tube  holder (Bel-Art Cat. No. 187950001 or equivalent)
       5.2.11  Screw cap tubes, 2 mL (VWR Cat. No. 89004-298 or equivalent)
       5.2.12  96-well tube rack(s) for 2 mL tubes (8 x 12 layout) (Bel-Art Cat. No. 188450031 or
               equivalent)
       5.2.13  2 mL Eppendorf tubes (Fisher Scientific Cat. No. 05-402-24C or equivalent)
       5.2.14  96-well 2 mL tube rack (8  x 12 format) (Bel-Art Cat. No. 188450031)
5.3   Supplies for Real-time PCR Analysis
       5.3.1   96-well PCR plates (ABI Cat. No. 4346906 or equivalent)
       5.3.2   96-well plate holders,  Costar®, black (VWR Cat. No. 29442-922 or equivalent)
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Detection of Bacillus anthracis in Environmental Samples
       5.3.3   Edge seals for 96-well PCR plates (Adhesive Plate Sealers, Edge Bio Cat. No. 48461 or
               equivalent)
       5.3.4   Foil seals for 96-well PCR plates (Polar Seal Foil Sealing Tape, E& K Scientific Cat. No.
               T592100 or equivalent) - for longer storage of the plates
       5.3.5   Optical seals (ABI Cat. No. 4311971 or equivalent)
5.4   Supplies for Culture
       5.4.1   Petri dishes, sterile, disposable, 100 x 15 mm
       5.4.2   Inoculating loops and needles, sterile, disposable
       5.4.3   Disposable cell spreaders (such as L-shaped, Fisher Scientific Cat. No. 03-392-150 or
               equivalent)
       5.4.4   MicroFunnel™ Filter Funnels, 0.45 (im pore-size (VWR Cat. No. 55095-060 or
               equivalent)
       5.4.5   Racks for 15  mL and 50 mL centrifuge tubes
       5.4.6   Sterile, plastic, screw cap 50 mL centrifuge tubes (Becton, Dickinson and company [BD]
               Cat. No. 352070 or equivalent)
       5.4.7   Sterile, plastic, screw cap 15 mL centrifuge tubes (BD Cat. No. 352097 or equivalent)
       5.4.8   Pipet tips with aerosol filter for 1000 (iL and 100 (iL (Rainin Cat. No. SR-L1000F and
               GP-100F or equivalent)
       5.4.9   Biotransport carrier (Nalgene® , Thermo Scientific Cat. No. 15-251-2 or equivalent)
5.5   Equipment
       5.5.1   Biological Safety Cabinet (BSC) - Class II or Class III
       5.5.2   PCR preparation hood (optional)
       5.5.3   Shaker incubator for RV-PCR (Thermo Scientific, MaxQ™ 4000 Cat No. SHKE4000 or
               equivalent) and Universal 18" x  18" shaker platform (Thermo Scientific, MaxQ™ Cat.
               No. 30110)
       5.5.4   Balance, analytical, with Class S reference weights, capable of weighing 20 g ± 0.001 g
       5.5.5   Applied Biosystems® (ABI) 7500 Fast Real-Time PCR System (Life Technologies™)
       5.5.6   Refrigerated centrifuge with PCR plate adapter and corresponding safety cups
               (Eppendorf Cat. No. 5804R, 5810R or equivalent) or PCR plate spinner (placed in BSC)
               (VWR, Cat. No. 89184-608 or equivalent)
       5.5.7   Refrigerated micro-centrifuge for Eppendorf tubes with aerosol-tight rotor (Eppendorf
               Cat No. 5415R or equivalent)
       5.5.8   Filter cup manifold-top and bottom for RV-PCR (DV Manufacturing. Cat. No. DVM-
               OSH-24 and DVM-BCP-24)
       5.5.9   Allen wrench for manifold for RV-PCR (9/64 Hex key)
       5.5.10  Capping tray for RV-PCR (DV Manufacturing Cat. No. DVM-LB-24) and screws to
               attach to shaker platform (DV Manufacturing)
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Detection of Bacillus anthracis in Environmental Samples
       5.5.11  30 mL tube rack for RV-PCR (DV Manufacturing Cat. No. DVM-24 VC)
       5.5.12  Vacuum pump with gauge (Cole Farmer® Model EW-07061-40 or equivalent) or vacuum
               source capable of < 10 pounds per square inch (psi)
       5.5.13  Vacuum pump filters for pump (Acrovent™ Cat. No. 4249 or equivalent)
       5.5.14  Vacuum trap accessories
       5.5.15  Platform vortexer for RV-PCR (VWR Cat. No. 588 16-1 15 or equivalent) with velcro
               straps
       5.5.16  Single-tube vortexer (Fisher Scientific Cat. No. 02-215-365 or equivalent)
       5.5.17  Heating block for RV-PCR (VWR Cat. No. 12621-096 or equivalent) and 2 mL tube
               blocks (VWR Cat. No. 12985-048 or equivalent) or water bath set at 95°C
       5.5.18  Single-channel micropipettors (1000 jiL, 200 nL,100 (iL, 20 (iL, 10
       5.5.19  Serological pipet aid
       5.5.20  Dynamag™ magnetic racks for RV-PCR (Invitrogen® Cat. No. 123 -2 ID or equivalent)
       5.5.21  Incubator(s), microbiological type, maintained at 37.0°C
       5.5.22  Autoclave or steam sterilizer, capable of achieving 121°C (15 psi) for 30 minutes
       5.5.23  Manifold incubator rack to hold up to 4 manifold/capping trays (DV Manufacturing Cat.
               No.  1701 190)  and/or peg kit for securing individual manifold/capping trays to the
               shaking incubator platform (Shaker Kit, DV Manufacturing Cat. No.  1701 189) for
               RV-PCR
       5.5.24  Cold block for 2 mL tubes (Eppendorf Cat. No. 3880 001.018 or equivalent)
       5.5.25  Bead-beater (BioSpec Products, Inc. Cat. No. 693 [8 place] or 607  [16 place] or
               equivalent)
       5.5.26  Tube racks, 80 place (VWR Cat. No. 30128-282 or equivalent)
       5.5.27  40 kHz Sonicator bath (Branson Ultrasonic Cleaner Model 1510, Process Equipment and
               Supply, Inc. Cat. No. 952-1 16 or equivalent)
       5.5.28  Stomacher® 400 Circulator (Seward Cat. No. 0400/00 1/AJ or equivalent) with closure
               bags (Cat. No. BA6141/CLR or equivalent) and rack (Cat. No. BA6091 [1 place] and
               BA6096 [10 place] or equivalent)
6.0  Reagents and Standards
6.1    Reagent-grade chemicals must be used in all tests.  Unless otherwise indicated, reagents shall
       conform to the specifications of the Committee on Analytical Reagents of the American
       Chemical Society (Reference 16.2).  For suggestions regarding the testing of reagents not listed
       by the American Chemical Society, see 'AnalaR' Standards for Laboratory Chemicals, BDH
       Ltd., Poole, Dorset, U.K. (Reference 16.3); and United States Pharmacopeia and National
       Formulary 24, United States Pharmacopeial Convention, Md. (Reference 16.4).
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Detection of Bacillus anthracis in Environmental Samples
6.2    10X Wash Buffer for RV-PCR (250 mM KH2PO4, pH 7.4)
       6.2.1   Composition:
              KH2PO4                          34 g
              Reagent-grade water               ~1 L
       6.2.2   Dissolve KH2PO4in 500 mL of reagent-grade water. Addition of NaOH is required if
              reagent-grade water has a low pH (pH ~5).  Add 1 N NaOH to bring to pH 7.4 (> 100 mL
              of 1 N NaOH). Bring volume to 1 L with reagent-grade water. Filter sterilize using 250
              mL, 0.22 (im PES filtering system.  Store solution at 4°C until time of use for a
              maximum of 90 days.
6.3    IX Wash Buffer for RV-PCR (Low Salt Buffer)
       6.3.1   Composition:
              1 OX wash buffer                lOOmL
              Reagent-grade water             900 mL
       6.3.2   Mix with magnetic stirrer: when mixed, measure pH.  Final pH should be 7.4. Filter
              sterilize using a 1 L, 0.22 (im PES filtering system with disposable bottle.  Store solution
              at 4°C until time of use for a maximum of 90 days.
6.4    Tween® 80 (Fisher Cat. No. T164 or equivalent)
6.5    Extraction Buffer with Tween® 80 for RV-PCR
       6.5.1   Composition:
               1 OX wash buffer                  1 mL
              200 proof ethanol               300 mL
              Tween® 80                     0.5 mL
              Reagent-grade water           698.5 mL
       6.5.2   Add 10X wash buffer, ethanol and Tween® 80 to 500 mL of reagent-grade water and mix
              well.  Bring volume to 1 L with reagent-grade water; mix well. Filter sterilize using a 1
              L, 0.22 (im PES filtering system with disposable bottle. Store solution at 4°C until time
              of use for a maximum of 90 days.
6.6    Extraction Buffer without Tween® 80 for RV-PCR
       6.6.1   Composition:
              10X wash buffer                  1 mL
              200 proof ethanol                300 mL
              Reagent-grade water             699 mL
       6.6.2   Add 10X wash buffer and ethanol to 500 mL of reagent-grade water mix well. Bring
              volume to 1 L with reagent-grade water; mix well. Filter sterilize using a 1 L, 0.22  (im
              PES filtering system with disposable bottle.  Store solution at 4°C until time of use for a
              maximum of 90 days.
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Detection of Bacillus anthracis in Environmental Samples
6.7    High Salt Wash Buffer for RV-PCR
       6.7.1 Composition:
              KH2PO4                       28.2 g
              Reagent-grade water                1 L
       6.7.2   Dissolve KH2PO4 in 500 mL of reagent-grade water. Addition of NaOH is required if
              reagent-grade water has low pH (pH ~5). Add 1 M NaOH to bring to pH 6.0 (> 100 mL
              of 1 M NaOH). Bring volume to 1 L with reagent-grade water.  Filter sterilize using a 1
              L, 0.22 (im PES filtering system with disposable bottle. Store solution at 4°C until time
              of use for a maximum of 90 days.
6.8   70% Ethanol for RV-PCR - Aseptically mix 70 mL of ethanol (100%) with 3 0 mL of sterile PCR-
      grade water.  Dispense into 2-3, sterile 50 mL conical tubes and store at 4°C, for a maximum of
      one week.  Unopened tubes may be stored for up to one month at 4°C.
6.9   PCR-grade water, sterile (Teknova Cat. No. W3350 or equivalent)
6.10  Phosphate buffered saline Tween® 20 (PBST) buffer for RV-PCR (Teknova Cat. No. P0201 or
      equivalent)
6.11  PBST buffer with 0.005% Anti-Foam reagent (Section 6.14) for Ultrafiltration secondary water
      sample processing; prepare aseptically mix well by vortexing before use.
6.12  0.1 M Sodium Phosphate/10  mM EDTA buffer/0.01% Tween-20, pH = 7.4 (Teknova Cat. No.
      S2216 or equivalent)
6.13  TE buffer (IX Tris-HCl-EDTA [Ethylenediaminetetraacetic acid]) buffer, pH 8.0 (Fisher
      Scientific Cat. No. BP2473-500 or equivalent)
6.14  Promega reagents for DNA extraction and purification procedure for RV-PCR:
      • MagneSil® Blood Genomic, Max Yield System, Kit (Promega Cat. No. MD1360; VWR Cat.
        No. PAMD1360)
      • Salt Wash (VWR Cat. No. PAMD1401 or equivalent)
      • Magnesil Paramagnetic Particles (PMPs) (VWR Cat. No. PAMD 1441 or equivalent)
      • Lysis Buffer (VWR, Cat. No. PAMD 1392 or equivalent)
      • Elution Buffer (VWR Cat. No. PAMD 1421 or equivalent)
      • Alcohol Wash, Blood (VWR Cat. No. PAMD 1411 or equivalent)
      • Anti-Foam Reagent (VWR Cat. No. PAMD 1431 or equivalent)
6.15  TaqMan® Fast Advanced PCR Master Mix (Life Technologies™ Cat. No. 4444557)
6.16  Primers and probe for EPA-2 PCR assay targeting the capB gene on the pXO2 plasmid (Francy et
       al, 2009 [Reference 16.5])
      • Forward Primer (BA-EPA-2F) - 5' -TGCGCGAATGATATATTGGTTT-3'
      • Reverse Primer (BA-EPA-2R) - 5' -GCTCACCGATATTAGGACCTTCTTTA-3'
      • Probe (BA-EPA-2Pr) - 5' -6FAM-TGACGAGGAGCAACCGATTAAGCGC-BHQ1 -3'
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Detection of Bacillus anthracis in Environmental Samples
6.17  Optional Real-time PCR Assays
      EPA-1 targeting thepagA gene on pXOl plasmid (Francy et al, 2009 [Reference 16.5])

      • Forward Primer (BA-EPA-1F) - 5' -GCGGATAGCGGCGGTTA-3'

      • Reverse Primer (BA-EPA-1R) - 5' -TCGGTTCGTTAAATCCAAATGC-3'

      • Probe (BA-EPA-IPr) -
        5' -6FAM-ACGACTAAACCGGATATGACATTAAAAGAAGCCCTTAA-BHQ1 -3'
      BC3 targeting a hypothetical gene on the chromosome of B. anthracis

      • Forward Primer (BA-BC3-F) - 5'-TTTCGATGATTTGCAATGCC-3'

      • Reverse Primer (BA-BC3-R) - 5'-TCCAAGTTACAGTGTCGGCATATT-3'

      • Probe (BA-BC3-Pr) - 5'-6FAM-ACATCAAGTCATGGCGTGACTACCCAGACTT-BHQ1 -3'
6.18   Trypticase™ Soy Agar with 5% Sheep Blood (SBA)

       6.18.1 The use of commercially prepared media plates is recommended (VWR Cat. No. 90001-
             276 or 90001-282 or equivalent), however dehydrated media (BBL™ Cat. No. 227300 or
             equivalent), with the addition of sheep blood (Oxoid Cat. No. SR0051 or equivalent),
             may be used.  If commercially prepared media are not available, prepare media using
             procedures in Sections 6.17.2 - 6.17.4.
       6.17.2 Composition:
             Tryptone H                         15 g
             Soytone                               5 g
             Sodium chloride                        5 g
             Agar                               12 g
             Sheep blood                        50 mL
             Reagent-grade water               -900 mL
       6.18.3 Add reagents except sheep blood to 850 mL  of reagent-grade water and mix thoroughly
             using a stir bar and hot plate. Boil for 1 minute with rapid stir bar agitation to dissolve
             completely. Adjust pH to 7.3 ± 0.2  with 1.0  N HC1 or 1.0 N NaOH and bring to 950 mL
             with reagent-grade water. Autoclave at 121°C (15 psi) for 15 minutes. Do not overheat.
             Cool to 45°C - 50°C in a water bath.
       6.18.4 Prepare plates by aseptically adding 50 mL of sterile sheep blood (5.0% final
             concentration) to the cooled media and mix well.  Aseptically pour 12-15 mL into each
             15 x 100 mm sterile Petri dish. Store at 4°C for a maximum of two weeks.
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Detection of Bacillus anthracis in Environmental Samples
6.19   Brain Heart Infusion Broth for RV-PCR (BHI broth, Fisher Scientific Cat. No. DF0037-15-0)
       6.19.1  Composition:
               Calf brains, infusion from 200 g         7.7g
               Beef heart, infusion from 250 g          9.8g
               Proteose peptone                      lO.Og
               Sodium chloride                       5.0 g
               Disodium phosphate (Na2HPO4)         2.5 g
               Dextrose                              2.0 g
               Reagent-grade water                    l.OL
       6.19.2  Add reagents to 1 L of reagent-grade water, mix thoroughly and heat to dissolve
               completely. Autoclave at  121°C (15 psi) for 15 minutes.  Final pH should be 7.4 ± 0.2.
               Store at 4°C for a maximum of three months in screw cap containers.
6.20   10% Bleach-pH amended (prepared daily)
       6.20.1  Prepare bleach solution by adding 1 part bleach, 1 part acetic acid and 8 parts reagent-
               grade water as described below.
       6.20.2  Add 2 parts water to 1 part bleach, then add 5% acetic acid (1 part) and remaining  water
               (6 parts). Measure pH and add bleach (to increase pH) or acetic acid (to decrease pH) as
               needed to obtain a final pH between 6 and 7.  A pH meter should be used to measure pH
               as opposed to  pH strips or kit.  When mixed, place a lid on the mixture to reduce chlorine
               escape and reduce worker exposure.

7.0   Calibration and Standardization
7.1    Check temperatures in incubators twice daily with a minimum of four hours between each reading
       to ensure operation within stated limits. Record the temperature in a log book.
7.2    Check temperature in refrigerators/freezers at least once daily to ensure operation is within stated
       limits of the method. Record daily measurements in a refrigerator/freezer log book.
7.3    Check thermometers including those on instrumentation (e.g., digital display) at least annually
       against a National Institute of Standards and Technology (NIST) certified thermometer or one that
       meets the requirements of NIST Monograph SP 250-23.  Check columns for breaks.
7.4    Calibrate pH meter prior to each use with two of three standards (e.g., pH 4.0, 7.0 or 10.0) closest
       to the range being tested.
7.5    Micropipettors should be calibrated at least annually and tested for accuracy on a weekly basis.
7.6    Follow manufacturer instructions for calibration of real-time PCR instruments.
7.7    Re-certify BSCs annually. Re-certification must be performed by a qualified technician.
7.8    Autoclave maintenance should be conducted at least annually. Autoclave temperature and total
       sterilization cycle time  should be checked on a quarterly basis. Record the data in a log book.
       Spore strips or spore ampules should be used monthly as bioindicators to confirm sterilization.
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Detection of Bacillus anthracis in Environmental Samples
7.9    Refrigerated centrifuges should be checked to confirm temperature and revolutions per minute
       (rpm) on a quarterly basis. Record the data in a log book.
8.0   Quality Control (QC)

8.1    Each laboratory that uses this protocol is required to operate a formal quality assurance (QA)
       program that addresses and documents instrument and equipment maintenance and performance,
       reagent quality and performance, analyst training and certification, and records storage and
       retrieval. International Organization for Standardization (ISO)/International Electrotechnical
       Commission (IEC) 17025 (International Standard: General requirements for the competence of
       testing and calibration laboratories, Section Edition 2005-05-15) provides a quality framework that
       could be used to develop a formal QA program.
8.2    Sample integrity — Samples should be checked for integrity (e.g., improperly packaged,
       temperature exceedance, leaking). Samples may be rejected if the integrity has been
       compromised.  Alternately, if sample integrity has been compromised it may be analyzed and the
       data qualified and marked accordingly (e.g., sample exceeded temperature during transport - data
       is flagged and marked as exceeding temperature), so that a decision can made regarding whether
       the data should be considered valid/invalid.
8.3    Analyst qualifications — Only those analysts that have been trained and have demonstrated
       proficiency with these analytical techniques should perform this procedure.
8.4    Proficiency testing (PT) — The laboratory should have analysts analyze test samples annually at a
       minimum to ensure they are maintaining proficiency. In addition, analysts should analyze PT
       samples to demonstrate proficiency prior to analyzing field samples. For laboratories not routinely
       using this protocol, analysts should analyze PT samples biannually. In the event of a PT failure
       the laboratory should identify and resolve any issues and then request and analyze additional PT
       samples.  Field samples should not be analyzed until the laboratory  passes the PT.
8.5    Media sterility check — The laboratory should test media sterility by incubating a single unit (tube
       or Petri dish) from each batch of medium (BHI broth or SB A) at 37°C ± 2°C for 24 ± 2 hours and
       observe for growth. Absence of growth indicates media sterility.  On an ongoing basis, the
       laboratory should perform a media sterility check every day that samples are analyzed.
8.6    PCR: Positive control (PC) — The laboratory should analyze a PC in triplicate reactions with each
       PCR run. Prepare the PC at a concentration of 50 pg of purified B. anthracis total DNA per 5 (iL
       of PCR-grade water. All PCs should result in a CT < 40 and replicates should be within ±  1 CT of
       each other.

8.7    Culture: Positive control (PC) — The laboratory should analyze PCs (known quantity of viable
       spores) to ensure that all media and reagents are performing properly.  PCs should be analyzed
       whenever a new batch of media or reagents is used.  On an ongoing basis, the laboratory should
       run a PC every day that samples are analyzed.
8.8    External inhibition control (EIC) 50 pg genomic DNA from B. anthracis Ames — The laboratory
       should analyze an EIC for site characterization phase sample DNA extracts to determine if the
       matrix is causing inhibition potentially resulting in false  negative  results. Prepare the EIC at a
       concentration of 50 pg of purified B. anthracis DNA per 1  \\L of PCR-grade water. Using a 10 \\L
       pipettor, carefully add 1 \\L of the DNA to the EIC wells on a PCR  plate and then add 5 \\L of
       sample DNA extract to each well and mix thoroughly. The PCR results from the PC and EICs
                                               15                                 December 2012

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Detection of Bacillus anthracis in Environmental Samples
       (both containing 50 pg of B. anthracis DNA) are then compared. Lower or similar CT values for
       the EIC indicate there is no inhibition. A higher CT value for the EIC (>3 CT values) is indicative
       of matrix inhibition.

Note:  To minimize cross contamination the EICs should not be placed next to the field samples when
       setting up the PCR plate.
8.9
8.10
8.11
8.12
No template controls (NTC) — The laboratory should analyze NTCs (5 (iL of PCR-grade water is
added to the NTC wells on a PCR plate in place of the DNA or the sample DNA extract) to ensure
that reagents are not contaminated.  On an ongoing basis, the laboratory should analyze NTCs in
triplicate PCR reactions with each PCR run.  The negative controls (NTCs) must not exhibit
fluorescence above the background level (i.e., no CT value). If CT values are obtained as a result
of a possible contamination or cross-contamination, prepare fresh PCR Master Mix and repeat the
analysis.
Field blank — The laboratory should request that the sampling team provide a field blank with
each batch of samples. A field blank is defined as either a sample collection tool (e.g., wipe,
swab) or sterile  reagent-grade water that is taken out to the field, opened and exposed to the
environment, but not used to collect a sample, and then placed in a bag and sealed and transported
to the laboratory along with the field samples. The field blank is treated as a sample in all
respects, including exposure to sampling location conditions, storage, preservation and all
analytical procedures.  Field blanks  are used to assess any contamination contributed from
sampling location conditions, transport, handling and storage. The laboratory should process and
analyze this control along with each batch of environmental samples.  The field blanks should not
exhibit fluorescence (i.e., no CT value).

Sample processing negative control  (PNC) or method blank — The laboratory should process and
analyze a PNC in the same manner as a sample to verify the sterility of equipment, materials and
supplies. Absence of growth indicates lack of contamination from the target organism. Please
refer to Table 1 for appropriate PNC.
Table 1. Sample Processing Negative  Controls
Matrix
Wipes
Swabs
Vacuum socks/filters
Air filters
Sponge-Sticks
Drinking water and decontamination
waste water
PNC
Clean (unused) wipe
Clean (unused) swab
Clean (unused) vacuum sock/filter
Clean (unused) air filter
Clean (unused) Sponge-Stick
100 mL of sterile reagent-grade water
For RV-PCR based analysis, the T0 and T9 extracts are analyzed (in triplicate). PCR positive and
negative controls must be analyzed using the same preparation of the PCR Master Mix and must
be run on the same 96-well plate as the T0 and T9 extracts.
                                               16
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Detection of Bacillus anthracis in Environmental Samples
9.0   Site Characterization Procedures

       For sample analyses during the site characterization phase of the response to an anthrax event,
       only real-time PCR based analytical procedures will be used. Accordingly, this section includes
       appropriate sample processing and real-time PCR procedures.
       Acceptable sample types: Gauze wipes (2" x 2" 50% rayon/50% polyester), air filters (37 mm),
       swabs (macrofoam), vacuum socks (large dust collection sample bags, 9 1/8" x 4", mean pore size
       6.7 (im), vacuum filters (4" diameter filter), Sponge-Stick sampling tools, drinking water and
       decontamination waste water.
       Note: For laboratories with RV-PCR capability please follow the sample processing procedures
             for spore recovery and concentration as outlined below (Section 9.1). For all other
             laboratories, either the same procedures (Section 9.1) could be followed or those
             described for culture analyses as outlined in Sections 11.1 -11.5 could be used.

9.1    Sample Processing for Laboratories With RV-PCR Capability
       Note: Sterile gloves should be used and changed between samples and as indicated below.
       Prepare monofilament polyester mesh (Section 5.2.3) supports by cutting 2" x 2" squares using
       sterile scissors and place squares into a clean ziplock bag. Since the supports are not sterilized
       prior to use, ensure that the working surface has been disinfected and sterile gloves are worn
       during the process.
       Fill sample tube rack with 30 mL or 50 mL screw cap conical tubes as appropriate. All sample
       types (except water samples) may be placed behind a mesh support in the tube to prevent
       interference from pipetting activities and to improve efficiency of spore extraction during
       vortexing. Using two pairs of sterile forceps, coil the mesh support and then grasp both ends of
       the coil with one pair of forceps.  Place the support into the tube by holding the sample to the side
       of the tube with one pair of sterile forceps and placing the coiled mesh support on top with the
       other set of forceps.
       9.1.1    Wipe and Air Filter Samples
               Place mesh support over wipe or air filter samples in 30 mL tube by holding the wipe or
               air filter to the side of the tube with sterile forceps and placing the coiled mesh support on
               top (Section 9.1). Ensure the sample and mesh are in the bottom half of the tube
               (avoiding the conical portion). Change gloves in between each sample. The support
               keeps the wipe or air filter from interfering with pipetting activities and also improves
               efficiency of spore extraction during vortexing. Proceed to Section 9.1.7.
       9.1.2    Vacuum Samples (Socks and Filters)
               For vacuum socks, using sterile scissors, cut the top blue portion off and discard prior to
               folding the sock from the top opening down.  Make four 1/2" folds in order to reduce the
               sock size to about 1" x 2". Cut the sock five times above the 30 mL tube using scissors,
               making sure to cut through all the folds. Place cut, folded sock in bottom half of tube
               (avoiding conical portion) and using two pairs of sterile forceps, place the coiled mesh
               support on top of sock (Section 9.1). Bleach the BSC working surface and don a fresh
               pair of gloves in between samples.  Proceed to Section 9.1.7.
                                               17                                 December 2012

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Detection of Bacillus anthracis in Environmental Samples
      Note:    The sample processing procedure provided is for up to 1 tablespoon (~1 g of test dust)
               of debris per sock sample. If socks received contain higher levels of debris, follow
               procedure in Section 11.4.1.
               For vacuum filters, ensure that the exposed filter surface (with debris) is facing up and
               carefully cut through the evidence tape with a sterile scalpel in order to remove the top of
               the cartridge.  Using a pair of sterile forceps, transfer large pieces of debris into the
               appropriate 50 mL tube, then fold filter in half with dirty, exposed filter side in, and then
               fold in half again in order to fit it into the 50 mL tube. Place folded filter in bottom half
               of tube (avoiding conical portion) and using two pairs of sterile forceps place the coiled
               mesh support on top of filter (Section 9.1).  Proceed to Section 9.1.7.

      9.1.3    Sponge-Stick Sampling Tools

          •    If the Sponge-Sticks/wipes are not in  Stomacher® bags, aseptically transfer each sample
               to a Stomacher® bag using sterile forceps. Change forceps between samples.

          •    Add 90 mL of phosphate buffered saline with Tween® 20 (PBST) to each bag.  Set
               Stomacher® (Section 5.5.28) to 260 rpm.

          •    Place a bag containing  a sample into the Stomacher® (Section 5.5.28) so the Sponge-
               Stick/wipe rests evenly between the homogenizer paddles and stomach each sample for 1
               minute.

          •    Open the door of the Stomacher® (Section 5.5.28) and remove the bag.  Grab the wipe
               from the outside of the bag with hands.  With the bag closed, move the  Sponge-
               Stick/wipe to the top of the bag while using hands to squeeze excess liquid from the
               Sponge-Stick/wipe.

          •    Open the bag, remove and discard the Sponge-Stick/wipe using sterile forceps.

          •    Repeat steps described above for each sample.

          •    Allow bags to sit for 10 minutes to allow elution suspension foam to settle.

          •    Gently mix the elution  suspension in the Stomacher® bag up and down  3 times with a
               sterile 50 mL pipet.  Remove half of the suspension volume (-45 mL) and place it in a 50
               mL screw capped centrifuge tube. Place the remaining suspension (~45 mL) into a
               second 50 mLtube.

          •    Record suspension volumes on tubes and data sheet.

          •    Process elution suspension for each sample as described above.

          •    Place 50 mL tubes into sealing centrifuge buckets and decontaminate centrifuge buckets
               before removing them from the BSC.

          •    Centrifuge tubes at 3500 x g, with the brake off, for 15 minutes in a swinging bucket
               rotor.

      Note:    A higher x g is preferred as long as the speed is within the tube specifications.

          •    Remove the supernatant from each tube with a 50 mL pipet and discard leaving
               approximately 3 mL in each tube. The pellet may be  easily disturbed and not visible, so
               keep the pipet tip away from the tube  bottom. Use a sterile 50 mL pipet for each sample.

          •    Set the vortexer (Section 5.5.16) to  high intensity. Set the sonicator water bath to high.
                                               18                                  December 2012

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Detection of Bacillus anthracis in Environmental Samples
          •    Vortex the tubes for 30 seconds and transfer the tubes to the sonicator bath and sonicate
               for 30 seconds. Repeat the vortex and sonication cycles twice.

          •    Remove suspension from one tube with a sterile 5 mL pipet and combine it with the
               suspension in the other tube from the same sample. Measure final volume  of suspension
               with 5 mL pipet and record the result on the tube and data sheet.

          •    Repeat vortexing and sonication steps for each sample.

          •    Proceed to Section 9.1.19.

      9.1.4    Swabs
               Place swab into the 30 mL tube and cut handle with sterile scissors if necessary to fit into
               the tube.  Using sterile forceps, place the coiled mesh support over the swab (Section
               9.1). Proceed to Section 9.1.7.

      9.1.5    Water Samples (Large Volume [10 L -100 L], Drinking Water)
               Please see Appendix A for primary (Section 2.0) and secondary (Section 3.0)
               concentration of large volume (10 L - 100 L) water samples. For water samples < 10 L,
               please refer to Appendix A, Section 3.0, secondary concentration.

          •    Add 15 mL of PBST buffer to the 50 mL conical tube with membrane (Appendix A,
               Section 3.5).

          •    Set vortexer (Section 5.5.16) to high intensity.

          •    Vortex membrane in 10 second bursts for 2 minutes to dislodge spores.

          •    Remove membrane and centrifuge at 3500 x g5 with the brake off, for 30 minutes at 4°C.
      Note:    A higher x g is preferred as  long as the speed is within the tube specifications.

          •    Remove 12 mL of the supernatant without disturbing/dislodging the pellet; resuspend the
               pellet by vortexing in the remaining volume.

          •    Use a 0.5 mL aliquot for DNA extraction using bead-beating, as described  in Section 9.2.

      9.1.6    Water Samples (Small Volume [< 50 mL], Surface or Drinking Water)

          •    Transfer 30 mL of water sample into a 50 mL screw cap conical tube.

          •    Add 10 mL of PBST buffer (Section 6.10) and mix by vortexing for 30 seconds.

          •    Centrifuge at 3500 x g5 with  the brake off, for 30 minutes at 4°C.

      Note:    A higher x g is preferred as  long as the speed is within the tube specifications.

          •    Remove 37 mL of the supernatant without disturbing/dislodging the pellet. The volume
               of supernatant remaining should not be below the conical portion of the tube. Resuspend
               the pellet by vortexing in the remaining volume.

          •    Use a 0.5 mL aliquot for DNA extraction using bead-beating, as described  in Section 9.2.
      9.1.7    Add 20 mL (5 mL for swabs) of cold (4°C) extraction buffer with Tween®  80 to
               environmental samples (Sections 9.1.1, 9.1.2, 9.1.4) placed in 30 mL tubes in tube rack.
               Use a new serological pipet to transfer buffer from a sterile, 250 mL screw capped bottle
               to each tube (keep bottle cap loosely over opening between transfers). Uncap one tube  at
                                              19                                  December 2012

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Detection of Bacillus anthracis in Environmental Samples
               a time, add 20 mL extraction buffer with Tween® 80, close tube, and place it back in tube
               rack.  Repeat for each sample tube.  Check that all caps are on tubes securely. Label
               tubes, as appropriate, and document location in rack.
       9.1.8    Place tube rack in plastic bag, seal, bleach bag and double bag, prior to transferring to
               platform vortexer (outside BSC).
       9.1.9    Vortex samples for 20 minutes on platform vortexer (Section 5.5.15), with speed set to 7.
       9.1.10   After vortexing, transfer sample tube rack to BSC.  Remove tube rack from plastic bag.
       9.1.11   Vortex one sample tube on single-tube vortexer (Section 5.5.16), in the BSC, for 3-5
               seconds. For samples containing large amounts of debris, let sample sit for 30 seconds to
               allow large particles to settle prior to aliquoting.
       9.1.12   Uncap tube.  Using a 25 mL serological pipet, transfer as much liquid volume as possible
               (while avoiding any particles) to a fresh appropriately labeled 50 mL conical tube and
               place in tube rack. Dispose pipets in waste container.  Cap sample tube and place tube
               back in rack.  Change gloves.
       9.1.13   Repeat Sections 9.1.11 -9.1.12 for each sample tube.
       9.1.14   Perform second spore extraction. Uncap one sample tube at a time.
       9.1.15   Add 14 mL (5 mL for swabs) of cold (4°C) extraction buffer without Tween® to each
               sample tube, one at a time, with a new 25 mL serological pipet and a fresh pair of gloves
               for each sample.  Keep buffer bottle loosely covered between transfers. Recap sample
               tube after buffer addition.
       9.1.16   After adding extraction buffer to all tubes, check that all caps are on securely. Place rack
               in plastic bag, seal and bleach bag.  Transfer bagged tube rack to platform vortexer
               (Section 5.5.15) outside BSC.
       9.1.17   Vortex rack for 10 minutes, with speed set to 7.
       9.1.18   Repeat Sections 9.1.11-9.1.12.
       9.1.19   Centrifuge the 50 mL conical tube containing the combined suspension at 3500  x g5 with
               the brake off, for 30 minutes at 4°C.
       Note:   A higher x g is preferred as long as the speed is within the tube specifications.
       9.1.20   Leaving approximately 3 mL in the tube, carefully discard the supernatant using a
               serological pipet without disturbing/dislodging the pellet. Ensure that the volume of
               liquid remaining  is not below the conical portion of the tube. Resuspend  the pellet by
               vortexing.
       9.1.21   Add 25 mL of Phosphate/EDTA/Tween buffer (Section 6.11) and mix the suspension by
               vortexing.
       9.1.22   Centrifuge the suspension at 3500 x g5 with the brake off, for 30 minutes  at 4°C.
       Note:   A higher x g is preferred as long as the speed is within the tube specifications.
       9.1.23   Carefully discard 22 mL of supernatant without disturbing/dislodging the pellet.
               Resuspend the pellet by vortexing in the remaining volume.
       9.1.24   Use a 0.5 mL aliquot for DNA extraction using bead-beating, as described in Section 9.2.
                                               20                                  December 2012

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Detection of Bacillus anthracis in Environmental Samples
9.2    DNA Extraction and Purification
       9.2.1    Using the 8 cap strips, transfer one level capful (~50 mg) of the 106 (im glass beads and
               one level capful (-50 mg) of the 425 - 600 (im glass beads (using a clean strip of caps
               between bead sizes) into each gasketed, capped bead-beating tube.
       9.2.2    In the BSC, pipet 0.5 mL of the suspension (Section 9.1.23) into pre-labeled, gasketed,
               capped bead-beating 2 mL tube containing glass beads. Replace cap on tube securely.
               Wipe outside of tube with bleach wipe.
       9.2.3    Insert tube in tube holder of the bead-beater set at 4800 rpm for 3 minutes (180 seconds).
               Instrument settings: Speed = 48; Time = 18. Press start. Bead-beating disrupts the cells
               and spores to release the DNA.
       9.2.4    Remove tube from bead-beater (tube will be warm), place in a cold block for 2 minutes
               (or until cool to touch). If tube leaks during bead-beating, wipe tube and bead-beater
               thoroughly with bleach wipe.
       9.2.5    Supernatant separation and transfer

          •    Set up tubes; label one, 1.5 mL microcentrifuge tube, one yellow-top filter unit, two blue
               Microcon® filter inserts and five blue Microcon® collection tubes with sample  ID.

          •    In BSC, centrifuge the screw capped tube containing sample at 7000 rpm for 1 minute in
               a microcentrifuge to pellet beads and particulate matter.

          •    Using a micropipettor, carefully transfer supernatant to yellow top filter collection tube
               (try to avoid beads and particulate matter at bottom of tube).

          •    Centrifuge at 7000 rpm for 4 minutes.

          •    Open tube; remove the yellow top filter with sterile disposable forceps, gripping  only on
               the sides. Transfer filtrate to blue Microcon® filter insert. Do not transfer any particulate
               matter that may be evident at bottom of tube. Replace filter and cap tube.

          •    Centrifuge at 7000 rpm for 1 minute.

          •    Remove the blue Microcon® filter insert with disposable forceps (gripping only the sides)
               and transfer to a new collection tube.
       9.2.6    First wash

          •    Add 400 nL of IX TE buffer to the filter.

          •    Centrifuge at 7000 rpm for 2 minutes.

          •    Carefully remove retentate from the top of the  blue Microcon® filter insert, avoiding any
               particulate matter visible on filter surface (tilt the tube for better viewing) and transfer
               liquid into a new blue Microcon® filter insert and collection tube.
       9.2.7    Second wash

          •    Add 400 jiL IX TE buffer to the  filter.

          •    Centrifuge at 7000 rpm for 2 minutes.

          •    Transfer blue Microcon® filter insert with disposable forceps (gripping only the sides) to
               a new collection tube.
                                               21                                   December 2012

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Detection of Bacillus anthracis in Environmental Samples
       9.2.8    Third wash

          •    Add 400 jiL IX TE buffer to the filter.

          •    Centrifuge at 7000 rpm for 2 minutes.

          •    Transfer blue Microcon® filter insert with disposable forceps (gripping only the sides) to
               a new collection tube.

       9.2.9    Fourth wash

          •    Add 400 (iL PCR-grade water to the filter.

          •    Centrifuge at 7000 rpm for 1 minute.

          •    Check fluid level in blue Microcon® filter insert. If above 200 uL, pulse spin for about
               10 seconds (or less) until about 100 \\L of fluid is retained on top of white base.
       9.2.10   Sample retrieval from the filter unit

          •    Use a micropipettor to carefully remove all of the retentate from the blue Microcon® filter
               insert and transfer into clean, labeled 1.5 mL microcentrifuge tube.  Avoid any particulate
               matter.

          •    If there is less than 100 (iL of extract, transfer extract back to the same blue Microcon®
               filter insert and add 100 \\L PCR-grade water and pulse spin to obtain about 100 \\L on
               filter. Transfer retentate into clean, labeled 1.5 mL microcentrifuge tube.

          •    Using clean gloves, place tubes containing filter extracts in DNA loading station/hood in
               preparation for PCR analyses (Section 9.3).

9.3    Real-time PCR Analyses
       As compared to traditional PCR, real-time PCR uses a sequence-specific hybridization probe
       sequence internal to the amplification primers, in addition to two target gene-specific amplification
       primers. The probe is fluorescently labeled at the 5' end with a reporter dye/fluorophore and at the
       3' end with a quencher dye (usually, Black Hole Quenchers).  The emission of light/fluorescence
       by the reporter dye is normally quenched by virtue of its proximity to the quencher dye. At the
       annealing step in a PCR,  along with the amplification primers, depending upon its orientation, the
       probe sequence also hybridizes to its target site on the DNA strand downstream from the binding
       site of one of the primers. During the enzymatic extension step when the probe comes in the way
       of Taq DNA polymerase enzyme, the 5' exonuclease activity of the enzyme hydrolyzes the probe
       sequence by cleaving individual nucleotides from the 5' end.  Cleavage of the probe releases the
       reporter dye from the proximal quencher allowing emission of measurable fluorescence.
       Therefore, this assay is also known as the 5' exonuclease assay as it relies on the 5' to 3'
       exonuclease activity of the Taq DNA polymerase enzyme to hydrolyze the probe.  Thus, the PCR
       amplification of a specific gene sequence can be detected by monitoring the increase in
       fluorescence (Figure 1).
                                               22                                 December 2012

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Detection of Bacillus anthracis in Environmental Samples
                            1) Reaction Mtaui e
                                             Target ckDNA
                                                             Quenched
                                                            TaqMan' Probe
                            3) Anneal Primer; nud Probe
                                                       -

                                           ,,  .1...T  TTT 1 1.
                                                  \
                            4) Extension, Hydroh -sis,
                             and Detection

                                               Polymerase

                                   Figure 1. PCR Amplification.
       As the amplification reaction proceeds, more amplicons become available for probe binding and
       hydrolysis, and consequently, the fluorescence  signal intensity per cycle increases. The increase in
       fluorescence can be detected in real time on PCRthermocyclers. When the fluorescence level
       crosses a set threshold value at a certain cycle number during the PCR, the result indicates the
       presence of the target gene sequence in the DNA in the sample, which in turn indicates the
       presence of a target pathogen in the sample. The PCR can specifically amplify a single copy of
       target gene sequence and generate millions of copies in a matter of minutes.
       The TaqMan® fluorogenic probe hydrolysis-based real-time PCR assays are commonly used in
       biodetection. Using established computer software (e.g., Primer Express) and genome sequence
       databases, bioagent-specific primers and probe nucleotide sequences for these assays are selected
       in such a way that they are present only in a specific location on the unique gene and/or virulence
       factor gene of interest for the detection and identification of a specific pathogen. These primers
       and probe sequences are absent in any other gene of that pathogen or in the genes of any near
       neighbor organisms. The primers generate a PCR product (amplicon) of a definite length/size.
       For a high-confidence identification of pathogens, PCR assays for multiple pathogen-specific
       genes are usually used.  For example, for detection of B. anthracis, real-time PCR assays generally
       target three separate genes.  They include one gene each on pXOl and pXO2 plasmids (usually
       targeting virulence genes) and one gene on the  chromosome/genome.  An algorithm based on the
       positive detection of all three gene targets in a sample indicates the presence of virulent B.
       anthracis spores. However, since this protocol is developed for the  remediation phase of the
       response where the B. anthracis strain has usually been identified and characterized, real-time
                                               23                                  December 2012

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Detection of Bacillus anthracis in Environmental Samples
      PCR using only the EPA-2 assay targeting the capsular antigen gene on the pXO2 plasmid is
      recommended. Although, if needed, all three assays or any combination thereof can be performed
      using the same sample DNA extract. Accordingly, the primers and probe sequences, and PCR
      conditions for the two optional assays, EPA-1 (targeting the protective antigen gene on the pXOl
      plasmid) and BC3 (targeting a hypothetical gene on the B. anthracis chromosome) are also
      included.
      The real-time PCR assays included in this protocol have been only partially characterized for
      specificity, however, the use of these assays is currently recommended. These assays will be
      replaced with fully characterized and validated assays upon availability.
      Note:    This procedure is to be carried out in an area designated for PCR only. A PCR-
              workstation that is equipped with an ultraviolet (UV) light for sterilization must be used
              for PCR Master Mix preparation. Micropipets and corresponding sterile, aerosol-
              resistant pipet tips are  used throughout this procedure for the addition of reagents.
              Aseptic technique must be used throughout and all reagents must be kept at or near
              4°C.
      9.3.1   Decontaminate the PCR workstation by treating all work surfaces with a 10% pH
              amended bleach solution, allowing the bleach to contact the work surface for a minimum
              of 15 minutes prior to rinsing with sterile water. Turn on UV light for 15 minutes.  After
              decontamination, discard gloves and replace with a new clean pair.
      Note:    If gloves become contaminated, they should be disposed of and fresh gloves donned.
              Only open one tube at  a time throughout the process. At no point, should more than
              one tube be open.  Do not allow hands (gloved or otherwise) to pass over an open tube,
              PCR plate, or any reagent container. All used pipet tips, gloves and tubes must be
              discarded in a biohazard autoclave bag.
      9.3.2   Determine the number of reactions that are to be run. Include four replicate reactions
              each (for each assay) for a NTC, PC and three replicates of the PNC (blank) per run.  In
              addition, include three reactions for each sample and two reactions for the EIC for each
              sample.  Prepare a sufficient volume of Master Mix to allow for one extra reaction for
              every ten reactions, so that there is enough Master Mix regardless of pipetting variations.
              For example, if ten samples are to be analyzed a total of 61 reactions would be included
              in the run [4-NTC, 4-PC,  3-PNC, 30-samples and20-EICs].  The amount of PCR Master
              Mix prepared should be sufficient to run 70 reactions.

      9.3.3   Based on the example provided above (i.e., 10 samples) the amount of Master Mix
              required would be  as indicated in Table 2.
              Table 2. Example EPA-2 PCR Assay Master Mix Preparation for 70 Reactions
Reagent
TaqMan® 2X Universal Master Mix
Forward primer, 25 joM
Reverse primer, 25 joM
Probe, 2 uM
PCR-grade water
Total Volume
Volume
(HL)
12.5
0.3
0.3
1
5.9
20
Total
Volume (jiL)
875
21
21
70
413
1400
Final Concentration
(UM)
IX
0.3
0.3
0.08
N/A

                                              24
December 2012

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Detection of Bacillus anthracis in Environmental Samples
         Optional PCR Assays
              If required the Master Mix for the optional assays should be prepared according to Table
              3 based on the example provided in Section 9.3.2.
              Table 3. Example EPA-1 and BC3 PCR Assay Master Mix Preparation for 70 Reactions
      Note:

      9.3.4

      9.3.5
      9.3.6


      Note:

      9.3.7
      9.3.8

      9.3.9


      9.3.10
      9.3.11
      9.3.12
Reagent
TaqMan® 2X Universal Master Mix
Forward primer, 25 uM
Reverse primer, 25 uM
Probe, 2 uM
PCR-grade water
Total Volume
Volume
(HL)
12.5
1
1
1
4.5
20
Total
Volume (jiL)
875
70
70
70
315
1400
Final Concentration
(UM)
IX
1.0
1.0
0.08
N/A

        The PC andNTC controls must be analyzed prior to sample analyses to verify that the
        Master Mix works properly and is free of contamination.
        In a clean PCR-preparation hood, pipet 20 (iL of Master Mix to four wells of the PCR
        plate. Label two wells as NTC and two as PC.
        Add 5 (iL of PCR-grade water into the NTC wells.
        Cover the plate with edge seal and transfer the PCR plate to the BSC. Remove the seal
        and add 5 (iL of the PC (B. anthracis DNA  [10 pg/^L]) to the PC wells.
        This step must be performed in the BSC outside the PCR clean room set-up area.
        Change gloves.
        Seal PCR plate with optical seal, using plate sealer for good contact. Change gloves.
        Centrifuge sealed PCR plate for 1 minute at 2000 rpm and 4°C, using the PCR plate
        safety cups or mini-plate centrifuge in the BSC.
        Open the centrifuge safety cup and transfer PCR plate to the ABI 7500 Fast
        thermocycler.

        The PCR cycling conditions on the ABI 7500 Fast include an initial cycle of 50°C for 2
        minutes, followed by one cycle of 95°C for 10 minutes, followed by 45 cycles of 95°C for
        5 seconds, and 60°C for 20 seconds (use Fast Temperature Ramp rate: 3.5°C/s up and
        3.5°C/s down). Fluorescence is automatically measured at the end of the 60°C annealing-
        extension combined step.
        If the Master Mix test results show "True Positive" assay detection for the PC and "True
        Negative" assay detection for the NTC, then proceed with analyses of samples. If the
        results are not "True" then repeat the PCR Master  Mix preparation and testing protocol
        and reanalyze.
        In a clean PCR-preparation hood, pipet 20 (iL of Master Mix into the required number of
        wells of a new PCR plate (as per the number of samples to be analyzed). An eight
        channel micropipettor can be used to add the Master Mix to the plate. Label two wells as
        NTC and two as PC. Label the rest of the wells such that there are five wells for each
        sample (three wells for actual sample analyses and two wells for EICs for each sample).
9.3.13   Add 5 (iL of PCR-grade water into the NTC wells.
                                              25
                                                                          December 2012

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Detection of Bacillus anthracis in Environmental Samples
      9.3.14  Cover the plate with edge seal and transfer the PCR plate to the BSC. Remove the seal
              and add 5 (iL of the PC (B. anthracis DNA [10 pg/nL]) to the PC wells.
      Note:   This step must be performed in the BSC outside the PCR clean room set-up area.
              Change gloves.
      9.3.15  Add 5 (iL of the PNC extract to the three PNC wells.
      9.3.16  Add 5 (iL of each sample DNA extract to the sample and EIC wells.
      9.3.17  Add 1 jiL of the PC (B. anthracis DNA [50 pg/jiL]) to all the EIC wells.
      Note:   To minimize cross contamination the EICs should not be placed next to the field
              samples when setting up the PCR tray.
      9.3.18  Seal PCR plate with optical seal, using a plate sealer for good contact. Change gloves.
      9.3.19  Centrifuge sealed PCR plate for one minute at 2000 rpm and 4°C, using the PCR plate
              safety cups or mini-plate centrifuge in the BSC.
      9.3.20  Transfer PCR plate to the ABI  7500 Fast thermocycler.
      9.3.21  Run PCR using the thermocycling conditions as  described in  Section 9.3.10.
      9.3.22  After completion of thermocycling, discard sealed PCR plate.
      Note:   PCR plates with amplified product should not be opened in the laboratory.
      9.3.23  Laboratory clean-up procedures
           •  Dispose of all biological materials in autoclave bags (double bagged).
           •  Autoclave all waste materials at the end of the work day.
           •  Decontaminate counters and equipment with fresh bleach (Section 6.19), followed by
              70% isopropyl and a DI water final rinse.
      9.3.24  Refer to section 12.1 for Data Analyses and Calculations.
10.0 Post Decontamination Procedures for Rapid Viability-Polymerase Chain
      Reaction (RV-PCR) Analyses
      Acceptable sample types: Gauze wipes (2" x 2" 50% rayon/50% polyester), air filters (37 mm),
      swabs (macrofoam), vacuum socks (large dust collection sample bags, 9 1/8" x 4", mean pore size
      6.7 (im), vacuum filters (4" diameter filter), Sponge-Stick sampling tools, water and
      decontamination waste water.
      Note:   Neutralization of decontamination agent(s) may be required prior to sample processing
              and analyses.
10.1  RV-PCR
      The RV-PCR method (Figures 2 and 3) is most useful for the analyses of samples collected during
      and after decontamination because determination of the presence or absence of viable B. anthracis
      spores (in the presence of a large number of dead spores) is a key analytical requirement during
      this phase of the response. This method can be more sensitive than the traditional culture-based
      method because almost all the spores recovered from the sample are used for analysis. It is
                                             26                                December 2012

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Detection of Bacillus anthracis in Environmental Samples
       relatively rapid, cost-effective, less labor-intensive, less prone to inhibition by environmental
       matrices, and less prone to interferences from the outgrowth of other bacteria, fungi, other
       microbes and presence of other biological material in the sample. It also provides higher-
       throughput and generates significantly less biohazardous and general laboratory wastes than the
       culture-based method.  Using the current version of the RV-PCR procedure a batch of 16 samples
       can be analyzed in 15 hours.  If the laboratory is running 7/24 approximately 150 samples can be
       analyzed in 48 hours. If additional equipment and personnel are available, the throughput could be
       increased.
                                                           The RV-PCR method is based on
                                                           a shift in PCR Ct value indicating
                                                           an increase in DMA, which is itself
                                                           due to an increase in cell number
                                                           due to organism viability

                                                           The method accurately
                                                           distinguishes live cells from dead
                                                           spores based on Ct0, Ctfinai and
                                                           ACt

                                                           ACt (Ct[T0] - Ct[T9]) > 9
                                                           ACt> 9 fora positive result
                           20        30
                             PCR Cycle
                                              40
       Figure 2. Example PCR Amplification Curves for the Initial T0 Aliquot and the Endpoint (Final)
       Aliquot.
       The RV-PCR method is a combination of culture and real-time PCR, a rapid, highly sensitive and
       specific analytical method to detect and identify bioterrorism agents. Culturing the samples allows
       the germination of viable B. anthracis spores recovered from processed samples. Real-time PCR
       via a change in the CT value (Figure 2) offers a rapid determination of the viability and identity of
       B. anthracis bacteria that grow from germinated spores in broth culture.  Samples (air filter,
       particulate [wipe, Sponge-Stick, swab, vacuum sock or filter], drinking water or decontamination
       waste water) are processed in multiple spore extraction and wash steps.  Recovered spores are
       incubated in BHI broth for optimum growth of B.  anthracis.  After vortexing, an aliquot is
       withdrawn for baseline analysis before incubating the broth culture in the filter cup at 36°C for 9
       hours on a rotary shaker incubator. This is the T0 aliquot and is stored at 4°C for immediate
       processing or at -20°C for an extended period until analysis.  After the broth culture is incubated
       for 9 hours, another aliquot is withdrawn. This is the T9 aliquot. Both the T0 and T9 aliquots are
       then extracted and purified to obtain B. anthracis total DNA. The T0 and T9 DNA extracts are
       then analyzed, in triplicate, using real-time PCR to detect the presence of B. anthracis DNA.
       Figure 3 provides a flowchart for the RV-PCR analyses. The CT values for both the T0 and T9
       DNA extracts are recorded and compared.  A change in CT for the T9 aliquot relative to the CT for
       the TO aliquot is calculated as follows:  ACT (CT [T0] - CT [T9]).  A ACT > 9 (i.e., the endpoint PCR
       CT of < 36 for the T9 DNA extract in a 45-cycle PCR) is set as a cut-off value for a positive
       detection of viable B. anthracis spores in the sample. The ACT > 9 criterion represents an
       approximate three log increase in DNA concentration at T9 relative to T0. The increase in DNA
       concentration at T9 is as a result of the presence of viable spores in the sample that germinated and
       grew during the 9 hours of incubation in growth medium. Depending upon the end user's
       requirement, sample complexity (dirtiness) and the phase of response during an event, a lower  AQ
       criterion of > 6 (a two log difference in DNA concentration) and a corresponding higher endpoint
                                               27
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Detection of Bacillus anthracis in Environmental Samples
      PCR CT of < 39 could be set.  The current protocol provides qualitative (presence or absence)
      results. However, introduction of the Most Probable Number (MPN) technique in the RV-PCR
      protocol (i.e., performing serial dilutions and replicate subsamples) could provide semi-
      quantitative results.
                     Samples in
                    conical tube
                    with support
                        _L
                   Add extraction
                   buffer, vortex
                   Collect spores
                    by vacuum
                     filtration
                      Repeat
                   extraction step
                    and collect
                      spores
                  Wash with high
                    and low salt
                      buffers
                    Add growth
                      medium
                   Vortex, take T0
                   aliquot for PCR
                    Incubate at
                       37°C
                   Vortex, take L
                   aliquot for PCR
                                             Sample Processing and Incubation
Ct[T0J




CtFJJ
ACt >9
 Viable
 spores
                 T0= Zero Hour Incubation      I. = Final Incubation-Hours (Endpoint)

                        Figure 3. Flow Chart for RV-PCR Sample Analysis.
                                            28
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Detection of Bacillus anthracis in Environmental Samples
10.2  RV-PCR Sample Processing: Spore Recovery
      Note:    Gloves should be used and changed between samples and as indicated below.
      10.2.1   Prior to sample processing, prepare the following items:

           •   Fill sample tube rack with 30 mL or 50 mL screw cap conical tubes and label as
               appropriate.

           •   In a BSC, assemble manifold by connecting upper part (with 24 openings as 3 columns of
               8 openings each) to lower part (with port) using six Allen screws.  Place filter cups into
               the manifold in the two outer columns (leaving the center column vacant). Verify that all
               filter cups are completely pushed down in manifold such that the filter cup bottom is
               touching the top surface of the manifold. Place a 50 mL tube cap (Section 5.2.6) on each
               filter cup. Place blue pull-tab caps in a beaker (one for each filter cup with a couple extra)
               and put inside a 37°C incubator (caps are easily to apply when pre-warmed).

           •   Vacuum: Prepare vacuum pump or house vacuum source; connect vacuum source to in-
               line filter and to waste container filled with -150 mL of fresh undiluted bleach resulting
               in a final concentration of approximately 10%,  once filtration is complete (final waste
               volume will be  ~1.5 L).
      Note:    If using external vacuum pump, tape pump exhaust tube to BSC to vent exhaust inside
               BSC.

           •   Capping tray set up: Add bottom caps to capping tray.

           •   For TO and T9 sample aliquots: In the BSC, for each sample 96-well tube rack or control,
               set up the following tubes, each labeled with the sample ID, the time-point (T0 or T9), the
               date, and the operator's initials:
               -   One 2 mL screw cap tube
               -   One 2 ml Eppendorf tube
               -   Two 1.5 mL Eppendorf tubes
               Use a different tube rack (96-well tube rack in 8 x 12 format) for T0 and T9 tubes,
               following the sample layout.

           •   Tape filter cup layout on outside glass window of the BSC.

           •   Special Instructions

               -   All procedures in the laboratory are to follow protocols that maintain a safe and clean
                  environment for the operators and reagents.
               -   All the procedures must be carried out in a BSC.
               -   Before and after sample analysis, decontaminate the BSC, pipet aids, centrifuge and
                  other equipment. Also, decontaminate all working areas suspected to be
                  contaminated.
               -   When operating in a BSC and throughout the laboratory, perform all steps of the
                  process using aseptic techniques. These precautionary techniques are to be used to
                  prevent contamination of equipment and individual reagents.
               -   Wear safety glasses, a lab coat, and gloves throughout the process.
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Detection of Bacillus anthracis in Environmental Samples
               -  If the gloves have somehow become contaminated, the gloves must be disposed of
                  and fresh gloves must be donned.
               -  Open one tube at a time throughout the process.  At no point may more than one tube
                  be opened.  Do not allow hands (gloved or otherwise) to pass over an open tube or
                  container.

               -  All used pipette tips, gloves, and tubes must be discarded in a biohazard autoclave
                  bag in the biosafety cabinet for later disposal after autoclaving.
       10.2.2   Wipe and Air Filter Samples
               Place mesh support over wipe or air filter samples  in 30 mL tube by holding the wipe or
               air filter to the side of the tube with sterile forceps  and placing the coiled mesh support on
               top (Section 9.1). Ensure the sample and mesh are in the bottom half of the tube
               (avoiding the conical portion). Change gloves in between each sample. The support
               keeps the wipe or air filter from interfering with pipetting activities and also improves
               efficiency of spore extraction during vortexing. Proceed to Section 10.2.8.

       10.2.3   Vacuum Samples (Socks and Filters)
               For vacuum socks, using sterile scissors, cut top blue portion off and discard prior to
               folding the sock from the top opening down. Make four, 1/2" folds in order to reduce the
               sock size to about 1" x 2".  Cut the sock five times above the 30 mL tube using scissors,
               making sure to cut through all the folds. Place cut, folded sock in bottom half of tube
               (avoiding conical portion) and using sterile forceps, place mesh support on top of sock
               (Section 9.1). Bleach the BSC working surface and don a fresh pair of gloves in between
               samples.  Proceed to Section 10.2.8.
       Note:    The sampling processing procedure provided is for up to 1 tablespoon (~1 g of test
               dust) of debris per sock sample. If socks received contain higher levels of debris,
              follow procedure in Section 11.4.1.
               For vacuum filters, ensure that the exposed filter surface (with debris) is facing up and
               carefully cut through the evidence tape with a sterile  scalpel  in order to remove the top of
               the cartridge. Using a pair of sterile forceps, transfer large pieces of debris into the
               appropriate 30 mL tube, then fold filter in half with dirty, exposed filter side in, and then
               fold in half again in order to fit it into the 30 mL tube. Place folded filter in bottom half
               of tube (avoiding conical portion) and using two pairs of sterile forceps, place mesh
               support on top of filter (Section 9.1). Proceed to Section 10.2.8.

       10.2.4   Sponge-Stick Sampling  Tools

           •   If the Sponge-Sticks/wipes are not in Stomacher® bags, aseptically transfer each sample
               to a Stomacher® bag using  sterile forceps. Change forceps between samples.

           •   Add 90 mL of PBST to each bag. Set Stomacher® (Section 5.5.28) to 260 rpm.

           •   Place a bag containing a sample into the Stomacher® (Section 5.5.28) so the Sponge-
               Stick/wipe rests evenly between the homogenizer paddles and stomach each sample for 1
               minute.

           •   Open the  door of the Stomacher® (Section 5.5.28)  and remove the bag.  Grab the wipe
               from the outside of the bag with hands. With the bag closed, move the Sponge-
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Detection of Bacillus anthracis in Environmental Samples
               Stick/wipe to the top of the bag while using hands to squeeze excess liquid from the
               Sponge-Stick/wipe.
           •   Open the bag, remove and discard the Sponge-Stick/wipe using sterile forceps.
           •   Repeat steps (Section 10.2.4) described above for each sample.
           •   Allow bags to sit for 10 minutes to allow elution suspension foam to settle.
           •   Process one stomacher bag sample at a time, transfer 20 mL at a time to the
               corresponding filter cup (with vacuum set to 5 - 10 psi), allowing complete filtration
               prior to the subsequent addition, until entire liquid volume has been processed. Use a
               new 25 mL serological pipet for each transfer.
           •   Turn off vacuum pump.
           •   Add 20 mL of cold (4°C) extraction buffer without Tween® 80 to each filter cup, and
               wait for 5 minutes prior to turning on the vacuum to 5  - 10 psi. Complete filtration
               through the filter cups. Washing the filter with  extraction buffer containing 30% ethanol
               may reduce the number of vegetative cells that may compete with B. anthracis spore
               outgrowth.
           •   Proceed to Section 10.3.
       10.2.5   Swabs
               Place swab into the 30 mL tube and cut handle with sterile scissors if necessary to fit into
               the tube. Using sterile forceps, place the mesh support over the swab (Section 9.1).
               Proceed to Section 10.2.8.
       10.2.6   Water Samples (Large volume [10 L - 100 L],  drinking water)
           •   Please see Appendix A for primary (Section 2.0) and secondary (Section 3.0)
               concentration of large volume (10 L - 100 L) water samples.  For water samples < 10 L,
               please refer to Appendix A, Section 3.0, secondary concentration.
           •   After secondary water concentration, using a 25 mL serological pipette add 15 mL of
               PBST buffer to the 50 mL conical tube with membrane filter (Appendix A,  Section 3.5).
               Repeat for each tube.
           •   Set vortexer (Section 5.5.16) to high intensity.
           •   Vortex membrane in 10 second bursts for 2 minutes to dislodge spores, taking care to
               prevent the liquid from entering the tube cap. Repeat for each tube.
           •   Let tubes settle for 2 minutes.
           •   Using a new 25 mL serological pipette, transfer as much liquid as possible to the
               corresponding filter cup while  avoiding any settled particles during pipetting.
           •   Repeat for each sample tube.
           •   Turn on vacuum pump to 5 - 10 psi in order to  collect spores onto filter cups.
           •   Repeat extraction of membrane filter by adding another 15 mL of PBST buffer to the 50
               mL conical tube with membrane and vortex for 2 minutes with 10 second bursts, as
               described above.
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Detection of Bacillus anthracis in Environmental Samples
           •   After 2 minute settling, transfer as much liquid as possible to the corresponding filter cup
               using a 25 mL serological pipet while avoiding any settled particle.
           •   Complete filtration of liquid through filter cups.  Turn off the vacuum pump.

           •   Add 20 mL of cold (4°C) extraction buffer without Tween® 80 to each filter cup, and
               wait for 5 minutes prior to turning on the vacuum to 5 - 10 psi.  Complete filtration
               through the filter cups. Washing the filter with extraction buffer containing 30% ethanol
               may reduce the number of vegetative cells that may compete with B. anthracis spore
               outgrowth.

           •   Proceed to Section 10.3.
       10.2.7   Water Samples (small volume [< 50 mL], surface or drinking water)

           •   Place manifold and filter cups in BSC. Label all filter cups, following the sample tube
               rack layout. Document filter cup and sample tube labels.

           •   Turn on vacuum pump to 5  -  10 psi.

           •   Mix water sample  by vortexing 5-10 seconds, then using a 25 mL serological pipet
               transfer 20 mL of water to the corresponding filter cup.

           •   Dispose of pipet in waste container. Perform second transfer of 20 mL to same filter cup
               using a new serological pipet.
       Note:    Additional 20 mL  transfers may be conducted.

           •   Cap sample tube.  Change gloves.

           •   After performing transfers to the corresponding filter cup for each water sample, check
               that all sample tube caps are secure. Place tube rack in plastic bag, seal and bleach bag.

           •   Store water sample tubes at 4°C as an archive until analyses are completed or until
               directed to discard samples.

           •   Turn off the vacuum pump.

           •   Add 20 mL of cold (4°C) extraction buffer without Tween® 80 to each filter cup, and
               wait for 5 minutes prior to turning on the vacuum to 5 - 10 psi.  Complete filtration
               through the filter cups. Washing the filter with extraction buffer containing 30% ethanol
               may reduce the number of vegetative cells that may compete with B. anthracis spore
               outgrowth.

           •   Proceed to Section 10.3.
       10.2.8   Place manifold and filter cups with 50 mL tube caps in BSC.  Label all filter cups,
               following the sample tube rack layout. Document filter cup and sample tube labels.
       10.2.9   Add 20 mL of cold (4°C) extraction buffer with Tween® 80 to samples (use 5 mL for
               swabs) placed in 30 mL conical tubes (50 mL conical tubes for Sponge-Stick samples) in
               tube rack (up to 16 tubes per rack). Use a new serological pipet to transfer buffer from a
               sterile, 250 mL screw capped bottle to each tube  (keep bottle cap loosely over opening
               between transfers). Uncap one tube at a time, add extraction buffer, close tube and place
               it back in tube rack. Check that all caps are on tubes securely. Label tubes as appropriate
               and document location in rack.
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Detection of Bacillus anthracis in Environmental Samples
       10.2.10  Place tube rack in plastic bag, seal, double bag and bleach the bag prior to transferring to
               the platform vortexer located outside the BSC.
       10.2.11  Vortex samples for 20 minutes on platform vortexer (Section 5.5.15), with the speed set
               to 7.
       10.2.12  After vortexing, transfer sample tube rack to the BSC.  Remove tube rack from plastic
               bag and discard the bag.
       10.2.13  Vortex up to 8 sample tubes on a single-tube vortexer (Section 5.5.16) in the BSC, for 3 -
               5 seconds each. Let sit for at least 2 minutes to allow large particles to settle prior to
               aliquoting (for samples containing debris). If required, allow the tubes to settle up to 5
               minutes.
       10.2.14  Uncap tube one at a time.  Using a 25 mL serological pipet (10 mL pipet for swabs)
               carefully transfer 13 mL (5 mL for swabs) to corresponding filter cup by lifting 50 mL
               tube cap slightly (same  position in the tube rack as in filter cup manifold). Dispose of
               pipet in waste container. Cap sample tube and place tube back in rack. Change gloves.
               Turn on vacuum pump at 5 - 10 psi.
       10.2.15  Repeat Section 10.2.14 for each sample tube using a new serological pipet for each
               sample, processing one sample at a time and up to 8 samples as a set, not exceeding 2 sets
               of 8 (16 sample) per manifold (in rows 1 and 3).  Repeat Section 10.2.13 for second set of
               up to 8 sample tubes.
       10.2.16  Complete filtration of liquid through  filter cups.  Change gloves.
       10.2.17  Perform the second spore extraction.  Uncap one sample tube at a time.
       10.2.18  Add 20 mL (5  mL for swabs) of cold (4°C) extraction buffer without Tween® 80 to each
               sample tube, one at a time with a new 25 mL serological pipet for each sample. Keep the
               buffer bottle loosely covered between transfers.  Recap the sample tube after each buffer
               addition.
       10.2.19  After adding extraction buffer to all of the tubes, check that all caps  are tight.  Place rack
               in plastic bag, seal and bleach the bag. Transfer bagged tube rack to platform vortexer
               (Section 5.5.15) located outside the BSC.
       10.2.20  Vortex rack for only 10 minutes, with speed set to 7.
       10.2.21  Repeat sections 10.2.12 - 10.2.16 except transfer 15-16 mL to the corresponding filter
               cup instead of 13 mL, taking care to avoid settled particles during aliquoting. Proceed to
               RV-PCR processing section (Section 10.3) below, with filter cup manifold.
       10.2.22  Check that all caps are on sample tubes securely. Place tube rack in a plastic bag, seal
               and bleach the bag. Store the samples at 4°C as an archive until analyses are completed
               or until directed to discard samples.
10.3   RV-PCR Sample Processing: Buffer Washes and Broth Culture
       10.3.1   Place into a 37°C incubator: Blue filter cup caps, one for each filter cup, in  a ziplock
               bag.
       Note:   Caps are easier to place on filter cups when pre-warmed.
       10.3.2   Place into BSC: 25 mL and 10 mL (for swab samples) serological pipets and cold (4°C)
               high salt wash buffer (pH 6.0) in 250 mL screw cap bottle.
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Detection of Bacillus anthracis in Environmental Samples
       10.3.3   To each filter cup, transfer 20 mL (10 mL for swab samples) of cold (4°C) high salt wash
               buffer (pH 6.0) using a 25 mL serological pipet (10 mL pipet for swabs) and lifting 50
               mL tube cap slightly, keeping the bottle covered between transfers. Use new pipet for
               each filter cup. Complete filtration of liquid through the filter cups. Change gloves.
       10.3.4   Place into the BSC:  10 mL serological pipets and cold (4°C) IX low salt wash buffer, pH
               7.4, in 250 mL screw cap bottle.
       10.3.5   Transfer 20 mL of cold (4°C) IX low salt wash buffer (pH 7.4) to each filter-cup using a
               25 mL serological pipet (by lifting 50 mL tube cap slightly). Keep the bottle covered
               between transfers and use new pipet for each filter cup.
       10.3.6   Complete filtration of liquid through filter cups. Remove 50 mL tube caps and dispose to
               waste. Turn off vacuum pump.  Change gloves.
       10.3.7   Unscrew the manifold top using an Allen wrench. Break the seal on manifold to ensure
               there is no vacuum by inserting a plate sealer between manifold top and bottom.  Using
               gloves hold the sides of the filter cup manifold top and remove it from the bottom
               vacuum manifold and place on top of the capping tray, fitted with bottom caps (Section
               10.2.1).  Press down firmly to ensure caps are securely fastened to filter cup bottom ports.
               Place bleach wipes over the manifold bottom until it can be disinfected. Change gloves.
       10.3.8   Place into the BSC:  5 mL serological pipets, 200 \\L pipettor, 200 \\L  tips, cold (4°C)
               BHI broth aliquoted in 50 mL conical tubes, sharps container and blue filter cup caps
               (pre-warmed in 37°C ± 1°C incubator).
       10.3.9   Pipet 3.5 mL of cold BHI broth into each filter cup using a 5 mL serological pipet. Use a
               new pipet for each filter cup.

       10.3.10  Firmly press blue caps onto filter cups prior to vortexing. Record the  time of the BHI
               broth addition since this represents T0.
       10.3.11  Place the capped filter cup manifold in a plastic bag, seal, double bag  and bleach the bag.
       10.3.12  Vortex the filter cups for 10 minutes on the platform vortexer (Section 5.5.15), setting 7.
       10.3.13  Place 2 mL screw cap tubes for T0 aliquots into the cold block in the BSC.
       10.3.14  After vortexing, transfer filter cup manifold in capping tray to the BSC. Remove bag.
       10.3.15  Uncap one filter cup at a time and open the corresponding 2 mL tube.  Using a 1 mL
               pipettor while gently pipetting up and down 10 or more times to mix the sample (and to
               avoid aerosol generation), transfer 1 mL (T0 aliquot) from each cup to the corresponding
               pre-chilled (cold block) 2 mL tube. Cap the tube and place  it back into the cold block.
       10.3.16  Repeat Section 10.3.15 for each filter cup.
       10.3.17  After transferring the T0 aliquot for all of the samples, place the capped filter cup
               manifold in a plastic bag, seal, double bag and bleach the outer bag.
       10.3.18  Transfer bagged filter cup manifold in capping tray to the shaker incubator. Secure the
               manifold using manifold rack or pins (for single manifold).  Incubate  at 37°C ± 1°C at
               230 rpm, for 9 hours (i.e., 9 hours from the addition of BHI broth to the filter cups).
       10.3.19  Process  1 mL T0 aliquots in 2 mL screw cap tubes using the Manual DNA Extraction and
               Purification Protocol (starting from Section 10.4.9), below.
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Detection of Bacillus anthracis in Environmental Samples
10.4   Manual DNA Extraction and Purification
       Prepare lysis buffer with anti-foam according to manufacturer's instructions in the Magnesil
       Blood Genomic, Max Yield System, Kit. Prepare the alcohol wash solution by adding ethanol and
       isopropyl alcohol according to manufacturer's instructions.  Transfer sufficient volume of buffer
       to sterile, 100 mL reservoir immediately before use. Pre-heat heat block to 80°Cprior to Section
       10.4.8.
       Note:    1 mL T0 and T9 aliquots are processed in the same manner, as described below.
       10.4.1   The TO aliquots can be extracted and purified during incubation of the T9 aliquots.
       10.4.2   After the 9 hour incubation, remove the filter cup manifold from the  shaker incubator.
       10.4.3   Vortex filter cups for 10 minutes on platform vortexer (Section 5.5.15) with speed set to
               7.

       10.4.4   Transfer the filter cup manifold to the BSC, remove and discard bags.
       10.4.5   Set up 2 mL screw cap tubes for T9 aliquots in a 96-well tube rack (8 x 12) and verify
               that 2 mL tube labels match the filter cup layout.  Maintain the tube layout when
               transferring tubes between the magnetic stand and the 96-well tube rack. Do not use 1.5
               mL tubes. Transfer T9 aliquot screw cap tubes to the BSC.
       10.4.6   Uncap one filter cup at a time and open the corresponding 2 mL tube. Using a 1 mL
               pipettor, swirl pipet tip gently in filter cup, while gently pipetting up and down 10 or
               more times to mix sample (and to avoid aerosol generation), transfer 1 mL (T9 aliquot)
               from each cup, and transfer to  corresponding T9 aliquot tube  in the 96-well tube rack; cap
               the tubes.
       10.4.7   Repeat Section 10.4.6 for each filter cup.

       10.4.8   Centrifuge 2 mL  screw cap tubes at 14,000 rpm for 10 minutes (4°C). Remove  800 (iL of
               the supernatant from each tube, using a 1000 (iL pipettor and dispose to waste.  Do not
               disturb the pellet (use a new tip for each sample and discard tips in a sharps container).  If
               processing for DNA extraction immediately, proceed to Section 10.4.11.

       10.4.9   Store T9 aliquots  at -20°C until further processing, if the remaining steps of the protocol
               cannot be conducted immediately.
       Note:    T0 and T9 extractions can be completed separately.
       10.4.10  Thaw T0 and T9 aliquots if they were stored at -20°C.
       10.4.11  Add 800 \\L of lysis buffer using a 1000  \\L pipettor with a new tip for each sample. Cap
               the tubes and mix by vortexing on high (-1800 rpm) in 10 second pulses for a total of 60
               seconds and place in 96-well tube rack at room temperature.  Change gloves in between
               the samples.
       10.4.12  Vortex each screw-cap tube briefly (low speed, 5-10 seconds) and transfer the sample
               volume to a 2 mL Eppendorf tube (ensure the tubes are labeled correctly during transfer).
               Change gloves in between each sample.  Incubate the T0 and T9 lysate tubes at room
               temperature for 5 minutes.
       10.4.13  Vortex the PMPs on high (-1800 rpm) for 30 - 60 seconds, or until they are uniformly
               resuspended.  Keep PMPs in suspension by briefly vortexing (3-5 seconds) before
               adding to each T0 and T9 lysate tube.
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Detection of Bacillus anthracis in Environmental Samples
       10.4.14  Uncap one tube at a time and add 600 (iL of PMPs to each T0 and T9 lysate (containing 1
               mL sample), hereafter referred to as "T0 and T9 tubes" using a new pair of gloves for each
               tube. Mix by briefly vortexing (use a new tip for each sample and discard used tips in a
               sharps container).  Change gloves in between each tube.
       10.4.15  Repeat Section 10.4.14 for all T0 and T9 tubes, vortexing the PMPs suspension (10.4.13)
               between each T0 and T9 tube.
       10.4.16  Vortex each T0 and T9 tube for 5-10 seconds (high), incubate at room temperature for 5
               minutes, briefly vortex, and then place on the magnetic stand with hinged-side of the tube
               facing toward the magnet. After all the tubes are in the stand, invert tubes 180 degrees
               (upside-down) turning away from you, then right side-up, then upside down toward you,
               then right side-up (caps up) position. This step allows all PMPs to contact the magnet.
               Check to see if any beads are in the caps and if so, repeat the tube inversion cycle again.
               Let the tubes sit for 5  - 10 seconds before opening. Maintain the tube layout when
               transferring tubes between the magnetic stand and the 96-well tube rack.
       10.4.17  Uncap each tube, one at a time and withdraw all liquid using a 1000 \\L pipettor with the
               pipet tip placed in the bottom of 2 mL tube, taking care not to disturb the PMPs. Ensure
               that all the liquid is removed. Use a new pipet tip to remove any residual liquid, if
               necessary. If liquid remains in the tube cap, remove by pipetting. Dispose tip and liquid
               in a sharps container.  Recap tube.  Change gloves.
       Note:    Section 10.4.17 can be combined with Section 10.4.18. After withdrawing the liquid in
               Section 10.4.17, add 360 [iL of Lysis buffer using a separate pipettor and new tip.
       10.4.18  Uncap each T0 and T9tube, one at atime, and add 360 \\L of lysis buffer using a 1000  \\L
               pipettor. Use a new tip for each sample and discard tips in a sharps container. Cap and
               vortex on low setting  for 5-10 seconds, and transfer to 96-well tube rack.

       10.4.19  After adding lysis buffer to all of the T0 and T9 tubes, vortex each tube for 5-10 seconds
               (low) and place back on the magnetic stand. After all tubes are in the stand, follow tube
               inversion cycle, as described in Section 10.4.16.

       10.4.20  Remove all the liquid as described in Section 10.4.17, except that a glove change
               between samples is not required. Use a new tip for each T0 and T9 tube (discard used tips
               in a sharps container). Recap the tube.
       Note:    Section 10.4.20 can be combined with Section 10.4.21. After withdrawing the liquid in
               Section 10.4.20, add 360 [iL of Lysis buffer using a separate pipettor and new tip.  If
               the steps are combined, cap the tube after the buffer addition.
       10.4.21  Repeat Sections 10.4.18 - 10.4.20 for all tubes.

       Note:    Section 10.4.21 can be combined with Section 10.4.22. After withdrawing the liquid in
               Section 10.4.17, add 360 [iL of Salt Wash buffer using a separate pipettor and new tip.
               If the steps are combined, cap the tube after the buffer addition.
       10.4.22  1st Salt Wash: Uncap each T0 and T9 tube, one at a time, and add 360 \\L of Salt Wash
               solution (VWR Cat. No.  PAMD1401 or equivalent).  Use a new tip for each T0 and T9
               tube and discard used tips in a sharps container.  Cap and transfer to 96-well tube rack.
       10.4.23  After adding the Salt Wash solution to all of the T0 and T9 tubes, vortex each tube for 5 -
               10 seconds (low) and place on the magnetic stand. After all tubes are in the stand, follow
               tube inversion cycle, as described in Section 10.4.16.
                                               36                                 December 2012

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Detection of Bacillus anthracis in Environmental Samples
       10.4.24  Remove liquid as described in Section 10.4.17, except that a glove change between T0
               and T9 tubes is not required. Use a new tip for each T0 and T9 tube and discard used tips
               in a sharps container. Recap the tube. Repeat for all T0 and T9 tubes.
       Note:    Section 10.4.24 can be combined with Section 10.4.25. After withdrawing the liquid in
               Section 10.4.24, add 360 [iL of Salt Wash buffer using a separate pipettor and new tip.
               If the steps are combined, cap the tube after the buffer addition.
       10.4.25  2nd Salt Wash: Repeat Sections 10.4.22 - 10.4.24 for all T0 and T9 tubes.
       Note:    Section 10.4.25 can be combined with Section 10.4.26. After withdrawing the liquid in
               Section 10.4.25, add 500 [tL of Alcohol Wash buffer using a separate pipettor and new
               tip.  If the steps are combined, cap the tube after the buffer addition.
       10.4.26  1st Alcohol Wash: Uncap each T0 and T9 tube, one at a time, and add 500 \\L of alcohol
               wash solution. Use a new tip for each sample and discard used tips in a sharps container.
               Cap and transfer to 96-well tube rack.
       10.4.27  After adding alcohol wash solution to all of the T0 and T9 tubes, vortex each tube for 5 -
               10 seconds (low speed) and place on the magnetic stand. After all T0 and T9 tubes are in
               the stand, follow the tube inversion cycle, as described in Section 10.4.16.
       10.4.28  Remove liquid as described in Section 10.4.17, except that a glove change between T0
               and T9 tubes is not required. Use a new tip for each T0 and T9 tube and discard used tips
               in a sharps container. Recap the tube.
       Note:    Section 10.4.28 can be combined with Section 10.4.29. After withdrawing the liquid in
               Section 10.4.28, add 500 [iL of Alcohol Wash buffer using a separate pipettor and new
               tip. If the steps are combined, cap the tube after the buffer addition.
       10.4.29  2nd Alcohol Wash: Repeat Sections 10.4.26 - 10.4.28 for all T0 and T9 tubes.
       Note:    Section 10.4.29 can be combined with Section 10.4.30. After withdrawing the liquid in
               Section 10.4.29, add 500 [iL of Alcohol Wash buffer using a separate pipettor and new
               tip. If the steps are combined, cap the tube after the buffer addition.
       10.4.30  3rd Alcohol Wash:  Repeat Sections 10.4.26 - 10.4.28 for all T0 and T9 tubes.
       Note:    Section 10.4.30 can be combined with Section 10.4.31. After withdrawing the liquid in
               Section 10.4.30, add 500 [iL of 70% ethanol wash buffer using a separate pipettor and
               new tip. If the steps are combined, cap the tube after the buffer addition.
       10.4.31  4th Alcohol Wash: Repeat Sections 10.4.26 - 10.4.28 except use 70% ethanol wash
               solution for all tubes. After the liquid is removed, recap the tube and transfer to the 96-
               well tube rack.
       10.4.32  Open all T0 and T9 tubes and air dry for 2 minutes.
       10.4.33  Heat the open T0 and T9 tubes in the heat block at 80°C until the PMPs are  dry (-20
               minutes). Allow all the alcohol solution to evaporate since alcohol may interfere with
               analysis.
       10.4.34  DNA elution: While they are  in the heating block add 300 \\L of elution buffer to each T0
               and T9 tube, and close tube.
       10.4.35  Vortex for 10 seconds and let the tubes sit in the heating block for 80 seconds.
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Detection of Bacillus anthracis in Environmental Samples
       10.4.36  Briefly vortex the tubes (5-10 seconds) taking care to prevent the liquid from entering
               the tube cap and let the tube sit in the heating block for 1 minute.
       10.4.37  Repeat Section 10.4.36 four more times.

       10.4.38  Remove the tubes from the heating block, place them in a 96-tube rack in the BSC, and
               let them sit at room temperature for at least 5 minutes.

       10.4.39  Briefly vortex each tube (5-10 seconds) on low speed and centrifuge at 2000 rpm at 4°C
               for 1 minute. Place tube in 96-well tube rack.
       10.4.40  Briefly vortex each tube and place on the magnetic stand for at least 30 seconds.  Bring
               the cold block to the BSC.
       10.4.41  Collect liquid from each T0 or T9 tube with a micropipettor and transfer to a clean,
               labeled, 1.5 mL tube (-170 (iL) on a cold block (check tube labels to ensure the correct
               order). Use a new tip for each tube and discard tips in a sharps container.  Visually verify
               absence of PMP carryover during final transfer. If magnetic bead carryover occurred,
               place 1.5 mL tube on magnet, collect liquid, and transfer to a clean, labeled, 1.5 mL tube
               (ensure the tubes are labeled correctly during transfer).
       10.4.42  Centrifuge tubes at 14,000 rpm at 4°C for 5 minutes to pellet any particles remaining
               with the eluted DNA; carefully remove  supernatant and transfer to a new 1.5 mL tube
               using a new tip for each tube (ensure the tubes are labeled correctly during transfer).
       10.4.43  Store T0 and T9 DNA extract tubes "referred to as T0 and T9 DNA extracts" at 4°C until
               PCR analysis (use photo-tray to transport 1.5 mL tubes in a rack).
       Note:    If PCR cannot be performed within 24 hours, freeze DNA extracts at -20"C.
       10.4.44  Laboratory Clean-up procedures

            •  Dispose of all biological materials in autoclave bags (double bagged).

            •  Autoclave all waste materials at the end of the work day.

            •  Decontaminate counters and equipment with fresh pH amended bleach (Section 6.19),
               followed by 70% isopropyl and a DI water final rinse.
10.5   Real-time PCR Analysis of T0 and T9 DNA Extracts
       For real-time PCR, follow Sections 9.3 - 9.3.16 with the following exceptions and changes:

            •  No EIC control is required for the samples.

            •  PCR Master Mix for 6 reactions per sample is required to accommodate the T0 and T9
               DNA extracts.

            •  For each batch of samples, PCR Master Mix should be made for 4 PCs, 4 NTCs,  3 PNCs
               and 6 DNA extracts per sample (3 for T0 and 3 for T9 DNA extracts).
       10.5.1   TO DNA extracts: Label  1.5 mL tubes with the sample identifier and "10-fold dilution".
               Add  90 (iL of PCR-grade water to the tubes.
       10.5.2   Mix TO DNA extracts by vortexing (3-5 seconds), spin at 14,000 rpm for 2 minutes, and
               transfer 10 |oL of supernatant to 1.5-mL Eppendorf tubes with 90 |oL of PCR-grade water,
               maintaining the plate layout.
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Detection of Bacillus anthracis in Environmental Samples
      Note:   No centrifugation is required (Section 10.5.2) ifPCR analysis is conducted
              immediately after DNA elution (Section 10.4.42).
      10.5.3  Mix diluted T0 DNA extracts by vortexing (5 seconds at low speed), and transfer 5 joL
              from the tubes to the PCR plate (with PCR Master Mix).
      10.5.4  T9 DNA extracts: Label 1.5 mL tubes with the sample identifier and "10-fold dilution".
              Add 90 (iL of PCR-grade water to the tubes.
      10.5.5  Mix T9 DNA extracts by vortexing (3-5 seconds), spin at 14,000 rpm for 2 minutes, and
              transfer 10 \\L of supernatant to 1.5-mL Eppendorf tubes with 90 \\L of PCR-grade water,
              maintaining the plate layout.
      Note:   No centrifugation is required (Section 10.5.5) if PCR analysis is conducted
              immediately after DNA elution (Section 10.4.42).
      10.5.6  Mix diluted T9 DNA extracts by vortexing (5 seconds), and transfer 5 |oL from the tubes
              to the PCR plate (with PCR Master Mix).
      10.5.7  Seal PCR plate with optical seal, using a plate sealer to ensure good contact. Change
              gloves.
      10.5.8  Centrifuge sealed PCR plate for 1 minute at 2000 rpm and 4°C using the PCR plate
              safety cups or mini-plate centrifuge in the BSC.
      10.5.9  Open the centrifuge safety cup and transfer PCR plate to ABI thermocycler.
      10.5.10 Transfer the PCR plates to the ABI 7500 Fast thermocycler.
      10.5.11 Run PCR using the thermocycler conditions described in Section 9.3.10.
      10.5.12 After the PCR run, discard sealed PCR plate.
      Note:   PCR plates with amplified product should not be opened in the laboratory.
      10.5.13 Follow laboratory cleanup protocol provided in Section 10.4.44.
      10.5.14 Refer to Section 12.2 for Data Analyses and Calculations.
11.0 Post Decontamination Procedures for Culture Analyses
      Acceptable sample types: Gauze wipes (2" x 2", 50% rayon/50% polyester), air filters (37 mm),
      swabs (macrofoam), vacuum socks (large dust collection sample bags, 9 1/8" x 4", mean pore size
      6.7 (im), vacuum filters (4" diameter filter), Sponge-Stick sampling tools, drinking water and
      decontamination waste water.
      Note:   Neutralization of decontamination agent(s) may be required prior to sample processing
              and analyses.
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Detection of Bacillus anthracis in Environmental Samples
11.1  Sample Processing and Plating for Sponge-Sticks and Wipes
      Note:   Sterile gloves should be used and changed between samples and as indicated below.

      11.1.1  Dislodge spores from the sample (Sponge-Sticks and wipes)
      Note:   All subsequent procedures involving manipulation of Sponge-Sticks, wipes, or spore
              suspensions must be carried out in a BSC using appropriate PPE (e.g., gloves, lab
              coat).

           •  If the Sponge-Sticks/wipes are not in Stomacher® bags, aseptically transfer each sample
              to a Stomacher® bag using sterile forceps. Change forceps between samples.

           •  Add 90 mL of phosphate buffered saline with Tween® 20 (PBST) to each bag. Set
              Stomacher® (Section 5.5.28) to 260 rpm.

           •  Place a bag containing a sample into the Stomacher® (Section 5.5.28) so the Sponge-
              Stick/wipe rests evenly between the homogenizer paddles and stomach each sample for 1
              minute.

           •  Open the door of the Stomacher® (Section 5.5.28) and remove the bag. Grab the wipe
              from the outside of the bag with hands. With the bag closed, move the Sponge-
              Stick/wipe to the top of the bag while using hands to squeeze excess liquid from the
              Sponge-Stick/wipe.

           •  Open the bag, remove and discard the Sponge-Stick/wipe using sterile forceps.

           •  Repeat steps (Section 11.1.1) described above for each sample.

           •  Allow bags to sit for 10 minutes to allow elution suspension foam to settle.
      Note:   For RV-PCR analysis, proceed to Section 10.2.4. Do not proceed to Section 11.1.2.
      11.1.2  Concentrate elution suspension

           •  Gently mix the elution suspension in the Stomacher® bag up and down 3 times with a
              sterile 50 mL pipet.  Remove half of the suspension volume (~45 mL) and place it in a 50
              mL screw capped centrifuge tube. Place the remaining suspension (-45 mL) into a
              second 50 mL tube.

           •  Record suspension volumes on tubes and data sheet.

           •  Repeat steps above (Section 11.1.2) for each  sample.

           •  Place 50 mL tubes into sealing centrifuge buckets and decontaminate centrifuge buckets
              before removing them from the BSC.

           •  Centrifuge tubes at 3500 x g, with the brake off, for 15 minutes in a swinging bucket
              rotor.

      Note:   A higher x g is preferred as long as the speed is within the tube specifications.

           •  Remove the supernatant from each tube with a 50 mL pipet and discard leaving
              approximately 3 mL in each tube. The pellet may be easily disturbed and not visible, so
              keep the pipet tip away from the tube bottom. Use a sterile 50 mL pipet for each sample.

           •  Set the vortexer (Section 5.5.16) to high intensity.  Set the sonicator water bath to high.
                                              40                                 December 2012

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Detection of Bacillus anthracis in Environmental Samples
           •   Vortex the tubes for 30 seconds and transfer the tubes to the sonicator bath and sonicate
               for 30 seconds.  Repeat the vortex and sonication cycles twice.

           •   Remove suspension from one tube with a sterile 5 mL pipet and combine it with the
               suspension in the other tube from the same sample.  Measure final volume  of suspension
               with 5 mL pipet and record the result on the tube and data sheet.

           •   Repeat vortexing and sonication steps for each sample.

      Note:    For real-time PCR analyses (for the site characterization phase) proceed to Sections
               9.2 (DNA Extraction and Purification)  and 9.3 (Real-time PCR analyses). For culture
               analyses proceed to 11.1.3,  below.
       11.1.3   Serially dilute the spore elution suspension in PBST

           •   Vortex the elution suspension on high for 30 seconds.
               a. Transfer 1 mL of the suspension from the 50 mL tube to a 15 mL containing 9 mL of
                 PBST.  Recap the tube and vortex it on high for 30 seconds.  This is the 10"1
                 suspension.
               b. Open the cap of the 10"1 suspension and transfer 1 mL of this suspension into a new 15
                 mL tube containing 9 mL of PBST. Recap the tube and vortex on high for 30 seconds.
                 This is the 10"2 suspension.

               c. The above results in 3 spore suspensions: the initial wipe elution suspension
                 (undiluted) and 2 serial dilutions  of the suspension in PBST (10"1 and 10"2).

           •   Repeat steps (a) through (b) for each sample.
       11.1.4   Culture spore suspensions on SBA
      Note:    Plating of 100 [iL results in an additional 1:10 dilution of each of the suspensions.
               Each of the following will be conducted in triplicate, resulting in the evaluation of 9
               spread plates per sample:
               a. After vortexing tubes, pipet 0.1 mL of undiluted suspension onto surface of pre-dried
                 SBA plate (labeled 10'1).
               b. After vortexing tubes, pipet 0.1 mL of 10"1 suspension onto surface of pre-dried SBA
                 plate (labeled 10"2).
               c. After vortexing tubes, pipet 0.1 mL of 10"2 suspension onto surface of pre-dried SBA
                 plate (labeled 10'3).

           •   After pipetting the 3 spread plates for each dilution, use a  sterile L-Shaped spreader to
               distribute the inoculum over the surface of the medium by rotating the dish by hand or on
               a turntable. Please ensure that inoculum is evenly distributed over the entire surface of
               the plate. Repeat for the next 2 replicate plates.

           •   Allow inoculum to absorb into the medium completely.
       11.1.5   Invert the plates and incubate them at 37°C ± 1°C for a maximum of 3  days. Plates
               should be examined within 18-24 hours after starting the incubation and at 72 hours of
               incubation. Count the number of colonies and record results. B.  anthracis produces flat
               or slightly convex, 2-5  mm colonies, with edges that are slightly irregular and have a
               "ground glass" appearance.  Comma-shaped projections may arise from the colony edge.
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Detection of Bacillus anthracis in Environmental Samples
              B. anthracis is not (3-Hemolytic. However, weak hemolysis may be seen under areas of
              confluent growth in aging cultures and should not be confused with (3-hemolysis.
              a. If the number of colonies is < 300/plate, record actual number.
              b. If the number of colonies is > 300/plate, record as "too numerous to count" (TNTC).
              c. If no target colonies are observed, record as "None detected" and proceed to
                 evaluation of growth on MicroFunnel™ plates (11.1.7).
              A minimum of 3 typical colonies should be confirmed using real-time PCR (Section
              11.6).

       11.1.6  Capture spores on MicroFunnel™ filter membranes and culture on SBA

           •  Place 3, 0.45 (im (pore-size) MicroFunnel™ filter funnels (Section 5.4.4) on the vacuum
              manifold and moisten membrane with 5 mL PBST.  All filtering should be done with a
              vacuum pressure < 20 mm Hg.

           •  With the vacuum valve closed (and vacuum pressure released), place 10 mL of PBST
              into each filter cup. Add 1.0 mL of the undiluted elution suspension each to two
              MicroFunnel™ cups and to the third cup add the remainder.

           •  Open the vacuum valve and filter the suspension. Close the valve  and release the vacuum
              pressure. Rinse the walls of each MicroFunnel™ cup with 10 mL  of PBST and filter.
              Open the valve and complete filtration.

           •  Squeeze the walls of the MicroFunnel™ cup gently and separate the walls from the base
              holding the filter. Discard cup. Remove each membrane with sterile forceps and place it
              grid-side up on a SBA plate.  Make sure that the filter is in contact with the  surface of the
              agar. If an air pocket occurs under the filter, use the sterile forceps to lift the edge of the
              filter to release the air pocket.

           •  Record the exact volume of the undiluted elution suspension filtered (1.0 mL) on each
              plate.

           •  Repeat steps (Section 11.1.6) described above for each sample.
       11.1.7  Invert and incubate SBA plates with membranes at 37°C ± 1°C for a maximum of 3 days.
              Plates should be examined within 18-24 hours after starting the incubation and at 72
              hours of incubation.  Count the number of colonies and record results.  Confirm 1-3
              colonies using real-time PCR (Section 11.6).
11.2   Sample Processing and Plating for Swabs

       11.2.1  Dislodge spores from swabs
       Note:   All subsequent procedures involving manipulation of swabs  or spore suspensions must
              be carried out in a BSC using appropriate PPE (e.g., gloves, lab coat).

           •  If the swabs are not in screw cap centrifuge tubes, transfer each swab to sterile, plastic 15
              mL screw cap  centrifuge tube using sterile forceps.

           •  If necessary, cut the handle of the swab to fit into the tube using sterile scissors. Use
              sterile forceps  and scissors for each sample.
       Note:   Flaming scissors with an alcohol lamp will not be sufficient to sterilize scissors in
              between samples due to the presence of Bacillus spores in the samples.
                                              42                                 December 2012

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Detection of Bacillus anthracis in Environmental Samples
           •   Add 5 mL of PB ST to each tube.

           •   Set vortexer (Section 5.5.16) to high intensity.

           •   Vortex swab in 10 second bursts for 2 minutes to dislodge spores from swab.

           •   Using sterile forceps, remove the swab from the 15 mL centrifuge tube.  Use the forceps
               to press the tip of the swab against the inside of the tube to remove extra liquid from the
               foam tip before discarding the swab.

           •   Repeat vortexing steps for each swab sample.
      Note:    For real-time PCR analyses (for the site characterization phase) proceed to Section
               9.1.19. For culture analyses proceed to 11.2.2, below.
      11.2.2   Serially dilute the spore elution suspension in PBST

           •   Vortex the elution suspension on high for 30 seconds.
               a.  Transfer 1 mL of the suspension from the 50 mL tube to a 15 mL tube containing 9
                  mL of PBST. Recap the tube and vortex it on high for 30 seconds. This is the 10"1
                  suspension.
               b.  Open the cap of the 10"1 suspension and transfer 1 mL of this suspension into a new 15
                  mL tube containing 9 mL of PBST.  Recap the tube and vortex on high for 30 seconds.
                  This is the 10"2 suspension.

               c.  The above results in 3 spore suspensions: the initial wipe elution suspension
                  (undiluted) and 2 serial dilutions of the suspension in  PBST (lO^and  10"2).

           •   Repeat steps (a) through (b) for each sample.

      11.2.3   Culture spore suspensions on SBA
      Note:    Plating of 100 [iL results in an additional 1:10 dilution  of each of the suspensions.
               Each of the following will be conducted in triplicate, resulting in the evaluation of 9
               spread plates per sample:
               a.  After vortexing tubes, pipet 0.1 mL  of undiluted suspension onto surface of pre-dried
                  SBA plate (10"1 mL of the original elution suspension).

               b.  After vortexing tubes, pipet 0.1 mL  of 10"1 suspension onto surface of pre-dried SBA
                  plate (10~2 mL of the original elution suspension).
               c.  After vortexing tubes, pipet 0.1 mL  of 10"2 suspension onto surface of pre-dried SBA
                  plate (10~3 mL of the original elution suspension).

           •   After pipetting the 3 spread plates for each dilution, use a sterile L-shaped spreader to
               distribute inoculum over the surface of the medium by rotating the dish by hand or on a
               turntable.  Please ensure that inoculum is evenly distributed over entire surface of the
               plate.  Repeat for the next 2  dilutions.

           •   Allow inoculum to absorb into the medium completely.
      11.2.4   Invert the plates and incubate them at 37°C ± 1°C for a maximum of 3 days. Plates
               should be examined within 18 - 24 hours after starting the incubation and at 72 hours of
               incubation. Count the number of colonies and record results. B. anthracis produces flat
               or slightly convex, 2-5 mm colonies, with edges that are slightly irregular and have a
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Detection of Bacillus anthracis in Environmental Samples
               "ground glass" appearance. Comma-shaped projections may arise from the colony edge.
               B. anthracis is not (3-Hemolytic. However, weak hemolysis may be seen under areas of
               confluent growth in aging cultures and should not be confused with (3-hemolysis.
               a.  If the number of colonies is < 300/plate, record actual number.
               b. If the number of colonies is > 300/plate, record as "too numerous to count" (TNTC).
               c. If no target colonies are observed, record as "None detected" and proceed to
                 evaluation of growth on MicroFunnel™ plates (11.2.6).

               A minimum of 3 typical colonies should be confirmed using real-time PCR (Section
               11.6).

       11.2.5   Capture spores on MicroFunnel™ filter membranes and culture on SBA

           •   Place 3, 0.45 (im (pore-size) MicroFunnel™ filter funnels (Section 5.4.4) on the vacuum
               manifold and moisten membrane with 5 mL PBST.  All filtering should be done with a
               vacuum pressure < 20 mm Hg.

           •   With the vacuum valve closed (and vacuum pressure released), place 10 mL of PBST
               into each filter cup. Add 1.0 mL of the undiluted elution suspension each to two
               MicroFunnel™ cups and to the third cup add the remainder.

           •   Open the vacuum valve and filter the suspension. Close the valve  and release the vacuum
               pressure. Rinse the walls of each MicroFunnel™ cup with 10 mL  of PBST and filter.
               Open the valve and complete filtration.

           •   Squeeze the walls of the MicroFunnel™ cup gently and separate the walls from the base
               holding the filter. Discard cup. Remove each membrane with sterile forceps and place it
               grid-side up on a SBA plate. Make sure that the filter is in contact with the  surface of the
               agar. If an air pocket occurs under the filter, use the sterile forceps to lift the edge of the
               filter to release the air pocket.

           •   Record the exact volume of the undiluted elution suspension filtered (1.0 mL) on each
               plate.

           •   Repeat steps (Section 11.2.5) described above for each sample.
       11.2.6   Invert  and incubate  SBA plates with membranes at 37°C ± 1°C for a maximum of 3 days.
               Plates  should be examined within 18 - 24 hours after starting the incubation and at 72
               hours of incubation.  Count the number of colonies and record results.  Confirm 1-3
               colonies using real-time PCR (Section 11.6).
11.3   Sample Processing and Plating for Air Filters
       11.3.1   Dislodge spores from air filter

       Note:    All subsequent procedures involving manipulation of filters or spore suspensions must
               be carried out in a BSC using appropriate PPE (e.g., gloves, lab coat).

           •   Add 5  mL of sterile PBST into a leak-proof, 50 mL conical tube containing an air filter,
               and close tube.

           •   Set vortexer (Section 5.5.16) to high intensity.

           •   Vortex membrane in 10 second bursts for 2 minutes to  dislodge spores.
                                              44                                 December 2012

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Detection of Bacillus anthracis in Environmental Samples
           •   Using sterile forceps remove the air filter from the conical tube and discard.

           •   Repeat the vortexing steps for each air filter.

      Note:    For real-time PCR analyses (for the site characterization phase) proceed to Sections
               9.2 (DNA Extraction and Purification) and 9.3 (Real-time PCR analyses).  For culture
               analyses proceed to 11.3.2, below.
      11.3.2   Serially dilute the suspension in PBST

           •   Vortex the elution suspension on high for 30 seconds.
               a.  Transfer 1 mL of the suspension from the 50 mL tube to a 15 mL tube containing 9
                  mL of PBST. Recap the tube and vortex it on high for 30 seconds. This is the 10"1
                  suspension.
               b.  Open the cap of the 10"1 suspension and transfer 1 mL of this suspension into a new 15
                  mL tube containing 9 mL of PBST. Recap the tube and vortex on high for 30 seconds.
                  This is the 10"2 suspension.
               c.  Open cap of the 10"2 suspension and transfer 1 mL of this suspension into a new 15
                  mL tube containing 9 mL of PBST. Recap the PBST tube and vortex on high for 30
                  seconds. This is the 10"3  suspension.

               d.  The above results in 4 spore suspensions: the initial sock elution suspension
                  (undiluted) and three serial dilutions of the suspension in PBST (10"1, 10"2 and 10"3).

           •   Repeat steps (a) through  (c)  for each sample.
      11.3.3   Culture spore suspensions on SBA
      Note:    Plating of 100 [iL results in an additional 1:10 dilution of each of the suspensions.
               Each of the following will be conducted in triplicate, resulting in the evaluation of 12
               spread plates per sample:

               a.  After vortexing tubes, pipet 0.1 mL of undiluted suspension onto surface of pre-dried
                  SBA plate (10"1 mL of the original elution suspension).
               b.  After vortexing tubes, pipet 0.1 mL of 10"1 suspension onto surface of pre-dried SBA
                  plate (10~2 mL of the original elution suspension).

               c.  After vortexing tubes, pipet 0.1 mL of 10"2 suspension onto surface of pre-dried SBA
                  plate (10~3 mL of the original elution suspension).
               d.  After vortexing tubes, pipet 0.1 mL of 10"3 suspension onto surface of pre-dried SBA
                  plate (10~4 mL of the original elution suspension).

           •   After pipetting the 3 spread plates for each dilution, use a sterile L-shaped spreader to
               distribute inoculum over the surface of the medium by rotating the dish by hand or on a
               turntable.  Please ensure that inoculum is evenly distributed over entire surface of the
               plate.  Repeat for next 3 dilutions.

           •   Allow inoculum to absorb into the medium completely.
      11.3.4   Invert the plates and incubate them at 37°C ± 1°C for a maximum of 3 days.  Plates
               should be examined within 18-24 hours after starting the incubation and at 72 hours of
               incubation. Count the number of colonies and record results. B. anthracis produces flat
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Detection of Bacillus anthracis in Environmental Samples
              or slightly convex, 2-5 mm colonies, with edges that are slightly irregular and have a
              "ground glass" appearance.  Comma-shaped projections may arise from the colony edge.
              B. anthracis is not (3-Hemolytic. However, weak hemolysis may be seen under areas of
              confluent growth in aging cultures and should not be confused with (3-hemolysis.
              a. If the number of colonies is < 300/plate, record actual number.

              b. If the number of colonies is > 300/plate, record as "too numerous to count" (TNTC).
              c. If no target colonies are observed, record as "None  detected" and proceed to
                 evaluation of growth on MicroFunnel™ plates (11.3.6).

                 A minimum of 3 typical colonies should be confirmed using real-time PCR (Section
                 11.6).

       11.3.5  Capture spores on MicroFunnel™ filter membranes and culture on SBA

           •  Place 3, 0.45 (im (pore-size) MicroFunnel™ filter funnels (Section 5.4.4) on the vacuum
              manifold and moisten membrane with 5 mL PBST.  All filtering should be done with a
              vacuum pressure < 20 mm Hg.

           •  With the vacuum valve closed (and vacuum pressure released), place 10 mL of PBST
              into each filter cup. Add 1.0 mL of the undiluted elution suspension each to two
              MicroFunnel™ cups and to the third cup add the remainder.

           •  Open the vacuum valve and  filter the suspension. Close the valve and release the vacuum
              pressure. Rinse the walls of each MicroFunnel™ cup with 10 mL of PBST and filter.
              Open the valve and complete filtration.

           •  Squeeze the walls of the MicroFunnel™ cup gently and separate the walls from the base
              holding the filter.  Discard cup. Remove each membrane with sterile forceps and place it
              grid-side up on a SBA plate.  Make sure that the filter is in contact with the surface of the
              agar. If an air pocket occurs under the filter, use the sterile forceps to lift the edge of the
              filter to release the air pocket.

           •  Record the exact volume of the undiluted elution suspension filtered (1.0 mL) on each
              plate.

           •  Repeat steps (Section 11.3.5) described above for each sample.
       11.3.6  Invert and incubate SBA plates with membranes at 37°C ± 1°C for a maximum of 3 days.
              Plates should be examined within 18-24 hours after starting the incubation and at 72
              hours of incubation. Count the number of colonies and record results. Confirm 1-3
              colonies using real-time PCR (Section 11.6).
11.4   Sample Processing and Plating for Vacuum Socks and Filters

       11.4.1  Dislodge spores from the vacuum socks and concentrate elution suspension
       Note:   All subsequent procedures involving manipulation of vacuum socks or spore
              suspensions must be  carried out in a BSC using appropriate PPE (e.g., gloves, lab
              coat).

           •  Place 50 mL of PBST into sterile, leak-proof, wide-mouth screw cap plastic container.

           •  Remove the sock from the bag by holding onto the upper blue plastic material. Wet the
              sock by dipping the lower 1" of the vacuum sock into the PBST in the container.
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Detection of Bacillus anthracis in Environmental Samples
           •   While holding the sock over the container, cut lower edge of sock as close to the lower
               edge seam as possible with disposable sterile scissors.  Use sterile scissors for each
               sample.
      Note:    Flaming scissors with an alcohol lamp will not be sufficient to sterilize scissors in
               between samples due to the presence of Bacillus spores in the samples.

           •   Submerge the sock in the PBST so that the liquid is allowed to enter the opening and wet
               the contents inside.

           •   When the liquid appears to have wet the sock beyond about 1" from the bottom, cut a 1"
               vertical slit up the center from the bottom of the sock.  Then cut horizontally from side to
               side, about  1" from the bottom, allowing the two pieces to fall into the container with
               PBST.

           •   Submerge the lower edge of the sock again to allow wetting of the contents inside. Again
               cut a 1" vertical slit up the center and horizontally from side to side to allow two
               additional sections to fall into the container with PBST.

           •   Continue to submerge and cut the sock until all of the white filter part of the sock is in
               pieces in the jar.

           •   Discard the upper blue portion of the vacuum sock and change gloves.

           •   Tightly close the container, seal with parafilm, and place on a platform shaker/rotator
               with lock bars. Agitate samples at 300 rpm for 30 minutes.
      Note:    If shaker/rotator is outside of the BSC, the containers should be enclosed in plastic
               bags and a sealed biotransport carrier.

           •   Remove the biotransport carrier from the shaker and place it in the BSC. Allow settling
               of samples for 1 minute, then transfer 30 mL of supernatant from each sample into
               corresponding 50 mL sterile, screw cap, conical tubes.

           •   Discard the settled material.

           •   Place the conical tubes into sealing centrifuge buckets within the BSC. Transport to
               centrifuge and place them in a swinging bucket rotor.

           •   Centrifuge the supernatant at 3500 x g, with the brake off, for 15 minutes.
      Note:    A higher x g is preferred as long as the speed is within the tube specifications.

           •   After centrifugation, move the sealed centrifuge buckets back to  the BSC.

           •   Carefully pipet off 25 mL of the supernatant and resuspend the pellet in the remaining 5
               mL by vortexing the 5 mL sample for 1 minute with 10 second bursts.

      11.4.2   Dislodge spores from the vacuum filters and concentrate the  elution suspension

           •   Place 50 mL of PBST into a sterile, leak-proof, wide-mouth screw cap plastic container.

           •   Ensure that the exposed filter surface (with debris) is facing up and carefully cut through
               the evidence tape with a sterile scalpel in order to remove the top of the cartridge.
               Using a pair of sterile forceps, transfer large pieces of debris into the appropriate screw
               cap container.
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Detection of Bacillus anthracis in Environmental Samples
           •   Fold the filter in half with dirty, exposed filter side in, and then fold in half again in order
               to fit it into the screw cap plastic container.

           •   Place folded filter in screw cap plastic container.

           •   Submerge the filter in the PBST so that the liquid is allowed to wet the entire filter.

           •   Tightly close the container, seal with parafilm. Place on a platform shaker/rotator with
               lock bars.  Agitate samples at 300 rpm for 30 minutes.
      Note:    If the shaker/rotator is outside of the BSC, the containers should be enclosed in plastic
               bags and a sealed biotransport carrier.

           •   Remove biotransport carrier from the shaker and place in the BSC. Allow settling of
               samples for 1 minute, then transfer 30 mL of supernatant from each sample into
               corresponding 50 mL sterile, screw cap, conical tubes.

           •   Discard the settled material.

           •   Place conical tubes into sealing centrifuge buckets within the BSC.  Transport them to
               centrifuge and place on swinging bucket rotor.

           •   Centrifuge the supernatant at 3500 x g, with the brake off, for  15 minutes.

      Note:    A higher x g is preferred as long as the speed is within the tube specifications.

           •   After centrifugation, move the sealed centrifuge buckets back to the BSC.

           •   Carefully pipet off 25 mL of the supernatant and resuspend the pellet in the remaining 5
               mL by  vortexing the 5 mL sample for 1 minute with 10 second bursts.
      Note:    For real-time PCR analyses (for the site characterization phase) proceed to Sections
               9.2 (DNA Extraction and Purification) and 9.3 (Real-time PCR analyses). For culture
               analyses proceed to 11.4.3, below.
      11.4.3   Serially dilute the spore elution suspension  in PBST

           •   Vortex the elution suspension on high for 30 seconds.
               a.  Transfer 1 mL of the suspension from the 50 mL tube to a 15 mL tube containing 9
                  mL  of PBST. Recap the tube and vortex it on high for 30 seconds. This is the 10"1
                  suspension.
               b.  Open the cap of the 10"1 suspension and transfer 1 mL of this suspension into a new 15
                  mL tube containing 9 mL of PBST.  Recap the tube and vortex on high for 30 seconds.
                  This is the 10"2 suspension.
               c.  Open cap of the 10"2 suspension and transfer 1 mL of this suspension into a new 15
                  mL tube containing 9 mL of PBST.  Recap the PBST tube and vortex on high for 30
                  seconds.  This is the 10"3 suspension.

               d.  Resulting in 4 spore suspensions: the initial sock elution suspension (undiluted) and
                  three serial dilutions of the suspension in PBST (10"1, 10"2 and  10"3).

           •   Repeat steps (a) through  (c) for each sample.
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Detection of Bacillus anthracis in Environmental Samples
       11.4.4   Culture spore suspensions on SBA
      Note:    Plating of 100 [iL results in an additional 1:10 dilution of each of the suspensions.
               Each of the following will be conducted in triplicate, resulting in the evaluation of 12
               spread plates per sample:
               a. After vortexing tubes, pipet 0.1 mL of undiluted suspension onto surface of pre-dried
                 SBA plate (1CT1 mL of the original elution suspension).

               b. After vortexing tubes, pipet 0.1 mL of 10"1 suspension onto surface of pre-dried SBA
                 plate (10~2 mL of the original elution suspension).
               c. After vortexing tubes, pipet 0.1 mL of 10"2 suspension onto surface of pre-dried SBA
                 plate (10~3 mL of the original elution suspension).

               d. After vortexing tubes, pipet 0.1 mL of 10"3 suspension onto surface of pre-dried SBA
                 plate (10~4 mL of the original elution suspension).

           •   After pipetting the 3 spread plates for each dilution,  use a sterile L-shaped spreader to
               distribute inoculum over the surface of the medium by rotating the dish by hand or on a
               turntable. Please ensure that inoculum is evenly distributed over entire surface of the
               plate. Repeat for next 3 dilutions.

           •   Allow inoculum to absorb into the medium completely.
       11.4.5   Invert the plates and incubate at 37°C ± 1°C for a maximum of 3 days. Plates should be
               examined within 18-24 hours after starting the incubation and at 72 hours of incubation.
               Count the number of colonies and record results. B. anthracis produces flat or slightly
               convex, 2-5 mm colonies, with edges that are slightly irregular and have a "ground
               glass" appearance.  Comma-shaped projections may arise from the colony edge. B.
               anthracis is not |3-Hemolytic. However, weak hemolysis may be seen under areas of
               confluent growth in aging  cultures and should not be confused with |3-hemolysis.
               a. If the number of colonies is < 300/plate, record actual number.
               b. If the number of colonies is > 300/plate, record as "too numerous to count" (TNTC).
               c. If no target colonies are observed, record as "None detected" and proceed to
                 evaluation of growth on MicroFunnel™ plates (11.4.7).
               A minimum of 3 typical colonies should be confirmed using real-time PCR (Section
               11.6).

       11.4.6   Capture spores on MicroFunnel™ filter membranes and culture on SBA

           •   Place 3, 0.45 (im (pore-size) MicroFunnel™ filter funnels (Section 5.4.4) on the vacuum
               manifold and moisten membrane with 5 mL PBST.  All filtering should be done with a
               vacuum pressure < 20 mm Hg.

           •   With the vacuum valve closed (and vacuum pressure released), place 10 mL of PBST
               into each filter cup.  Add 1.0 mL of the undiluted elution suspension each to two
               MicroFunnel™ cups and to the third cup  add the remainder.

           •   Open the vacuum valve and filter the suspension. Close the valve and release the vacuum
               pressure.  Rinse the walls of each MicroFunnel™ cup with 10 mL of PBST and filter.
               Open the valve and complete filtration.
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Detection of Bacillus anthracis in Environmental Samples
           •   Squeeze the walls of the MicroFunnel™ cup gently and separate the walls from the base
               holding the filter. Discard cup. Remove each membrane with sterile forceps and place it
               grid-side up on a SB A plate. Make sure that the filter is in contact with the surface of the
               agar. If an air pocket occurs under the filter, use the sterile forceps to lift the edge of the
               filter to release the air pocket.

           •   Record the exact volume of the undiluted elution suspension filtered (1.0 mL) on each
               plate.

           •   Repeat steps (Section 11.4.6) described above for each sample.
       11.4.7   Invert and incubate SBA plates with membranes at 37°C ± 1°C for a maximum of 3 days.
               Plates should be examined within 18 - 24 hours after starting the incubation and at 72
               hours of incubation. Count the number of colonies and record.  Confirm 1-3 colonies
               using real-time PCR (Section 11.6).

11.5   Sample Processing and Plating for Water Samples
       Note:    All water samples prior to 11.5.1 have been concentrated according to Appendix A
               Sections 2.0 and 3.0 (large volume) or Section 3.0 (small volume).
       11.5.1   Dislodge and elute spores from the MicroFunnel™ membrane (from secondary water
               concentration, Appendix A, Section 3.0)

       Note:    All subsequent procedures involving manipulation of membranes or spore suspensions
               must be carried out in a BSC using appropriate PPE (e.g., gloves, lab coat).

           •   Add 5 mL of sterile PBST into  a screw cap, 50 mL conical tube containing a membrane
               filter, and close tube.

           •   Set vortexer (Section 5.5.16) to high intensity.

           •   Vortex the membrane in 10 second bursts for 2 minutes to dislodge spores.

           •   Using sterile forceps remove membrane from conical tube and discard.

           •   Repeat the vortexing steps for each membrane.
       Note:    For real-time PCR analyses (for the site characterization phase) proceed to Sections
               9.2 (DNA Extraction and Purification) and 9.3 (Real-time PCR analyses). For culture
               analyses proceed to 11.5.2, below.
       11.5.2   Serially dilute the suspension in PBST

           •   Vortex the elution suspension on high for 30 seconds.
               a. Transfer  1 mL of the suspension from the 50 mL tube to a 15 mL tube containing 9
                 mL of PBST. Recap the tube and vortex it on high for 30 seconds.  This is the 10"1
                 suspension.
               b. Open the cap of the  10"1 suspension and transfer 1 mL of this suspension into a new  15
                 mL tube  containing 9 mL of PBST. Recap the tube and vortex on high for 30 seconds.
                 This is the 10"2 suspension.
               c. Open cap of the 10"2 suspension and transfer 1 mL of this suspension into a new 15
                 mL tube  containing 9 mL of PBST. Recap the PBST tube and vortex on high for 30
                 seconds.  This is the 10"3 suspension.
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Detection of Bacillus anthracis in Environmental Samples
               d.  The above results in 4 spore suspensions: the initial sock elution suspension
                  (straight/neat undiluted) and 3 serial dilutions of the suspension in PBST (1CT1, 10~2
                  and 10'3).

           •   Repeat steps (a) through (c) for each sample.
       11.5.3   Culture spore suspensions on SB A
       Note:    Plating of 100 [iL results in an additional 1:10 dilution of each of the suspensions.
               Each of the following will be conducted in triplicate, resulting in the evaluation of 12
               spread plates per sample:
               a.  After vortexing tubes, pipet 0.1 mL of undiluted suspension onto surface of pre-dried
                  SB A plate (1CT1 mL of the original elution suspension).
               b.  After vortexing tubes, pipet 0.1 mL of 10"1 suspension onto surface of pre-dried SBA
                  plate (10~2 mL of the original elution suspension).
               c.  After vortexing tubes, pipet 0.1 mL of 10"2 suspension onto surface of pre-dried SBA
                  plate (10~3 mL of the original elution suspension).

               d.  After vortexing tubes, pipet 0.1 mL of 10"3 suspension onto surface of pre-dried SBA
                  plate (10~4 mL of the original elution suspension).

           •   After pipetting the 3 spread plates for each dilution, use a sterile L-shaped spreader to
               distribute inoculum over the surface of the  medium by rotating the dish by hand or on a
               turntable. Please ensure that inoculum is evenly distributed over entire surface of the
               plate.  Repeat for next 3 dilutions.

           •   Allow inoculum to absorb into the medium completely.
       11.5.4   Invert plates and incubate at 37°C ± 1°C for a maximum of 3 days. Plates should be
               examined within  18-24 hours after starting the incubation and at 72 hours of incubation.
               Count the number of colonies and record results. B. anthracis produces flat or slightly
               convex, 2-5 mm colonies, with edges that are slightly irregular and have a "ground
               glass" appearance. Comma-shaped projections may arise from the colony edge.
               B. anthracis is not (3-Hemolytic.  However, weak hemolysis may be seen under areas of
               confluent growth in aging cultures and should not be confused with (3-hemolysis.
               a.  If the number of colonies is < 300/plate, record actual number.
               b.  If the number of colonies is > 300/plate, record as "too numerous to count" (TNTC).
               c.  If no target colonies are observed, record as  "None detected" and proceed to
                  evaluation of growth on MicroFunnel™ plates (11.5.6).

               A minimum of 3 typical colonies should be confirmed using real-time PCR (Section
               11.6).

       11.5.5   Capture spores on MicroFunnel™ filter membranes and culture on SBA

           •   Place 3, 0.45 (im (pore-size) MicroFunnel™ filter funnels (Section 5.4.4) on the vacuum
               manifold and moisten membrane with 5 mL PBST. All filtering should be done with a
               vacuum pressure  < 20 mm Hg.
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Detection of Bacillus anthracis in Environmental Samples
           •   With the vacuum valve closed (and vacuum pressure released), place 10 mL of PBST
               into each filter cup. Add 1.0 mL of the undiluted elution suspension each to two
               MicroFunnel™ cups and to the third cup add the remainder.

           •   Open the vacuum valve and filter the suspension.  Close the valve and release the vacuum
               pressure.  Rinse the walls of each MicroFunnel™ cup with 10 mL of PBST and filter.
               Open the valve and complete filtration.

           •   Squeeze the walls of the MicroFunnel™ cup gently and separate the walls from the base
               holding the filter. Discard cup.  Remove each membrane with sterile forceps and place it
               grid-side up on a SB A plate. Make sure that the filter is in contact with the surface of the
               agar. If an air pocket occurs under the filter, use the sterile forceps to lift the edge of the
               filter to release the air pocket.

           •   Record the exact volume of the undiluted elution suspension filtered (1.0 mL) on each
               plate.

           •   Repeat steps (Section 11.5.5) described above for each sample.
       11.5.6   Invert and incubate SBA plates with membranes at 37°C ± 1°C for a maximum of 3 days.
               Plates should be examined within 18-24 hours after starting the incubation and at 72
               hours of incubation. Count the number of colonies and record results. Confirm 1-3
               colonies using real-time PCR (Section 11.6).

11.6   Confirmation of B. anthracis Colonies by Real-time PCR Analysis
       11.6.1   Pipet 100 (iL of PCR-grade water into a 1.5 mL Eppendorf microcentrifuge tube (Section
               5.2.13).

       11.6.2   Use a disposable 1 \\L inoculating loop or pre-wetted swab to remove bacterial growth
               from a typical B. anthracis colony grown on SBA.

       Note:    In some cases, it may be difficult to remove the bacterial growth with a loop. If this
               happens, use a sterile applicator swab.  Be sure topre-wet the swab with PCR-grade
               water before removing the bacterial growth.
       11.6.3   Insert the loop or swab into the tube containing the PCR-grade water and immerse the in
               the liquid.
       11.6.4   Gently spin the loop or swab in the liquid to remove and resuspend the bacterial growth
               in the water.  Press the tip of the swab against the tube to remove the liquid from the tip
               prior to discarding.
       11.6.5   Lysate preparation

           •   Cap the microcentrifuge tubes containing the bacterial suspension and briefly vortex.

           •   Place the capped tubes in a floating rack if using the water bath. Otherwise place the
               capped tube in the heat block at 95°C - 98°C.

           •   Heat the sample  for 5 minutes.

           •   Remove the tubes from the water bath or heat block and place it directly in a cold block.
               Chill for a minimum of 2 minutes.

           •   Place the microcentrifuge tubes in the refrigerated microcentrifuge.  Centrifuge at 12,000
               rpm for 2 minutes.
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Detection of Bacillus anthracis in Environmental Samples
       11.6.6   Filtration of lysate using 0.1 um centrifugal filter unit (Section 5.1.23)

           •   Remove top cap from the filtrate collection tube.

           •   Hold the filter unit vertical with the filter cup opening facing up.  Pipet the supernatant
               from each microcentrifuge tube into the corresponding filterate collection tube. Avoid
               removing any visible pellet material.

           •   Replace top cap onto the filtrate collection tube.  Balance the tubes prior to placing the
               filter unit into the microcentrifuge fixed-angle rotor.

           •   Centrifuge the filter units for 2 minutes at 8000 rpm. Ensure all supernatant is collected
               in the filtrate cups.
       Note:    If the supernatant has not passed completely through the filter, centrifuge for an
               additional 2 minutes. Repeat as necessary until all the supernatant has passed through
               thefilter.

           •   Remove top cap and discard the filtrate cup using sterile disposable forceps.  Replace top
               cap.

           •   The liquid in the filtrate collection tube is the lysate for real-time PCR analysis.

           •   Wipe the outside of the tube containing lysate with bleach.

           •   It is safe to remove lysates from the BSL-3 after  filtration and  disinfecting the outside of
               the tube.

           •   Lysates must be stored in a cold block while preparing for the  PCR analysis.

           •   If PCR analysis will not be completed the same day the lysates are prepared,  aliquot and
               freeze them at -20°C.
       Note:    DNA extracted by this procedure should not be  stored for more than one week.
       11.6.7   Use 5 \\L of the lysate as the DNA template to run the PCR analysis in triplicate.
       11.6.8   For real-time PCR, follow Sections 9.3 - 9.3.23 with the following exceptions and
               changes:

           •   No PNC and EIC controls are required for the samples.
           •   For each batch of sample colonies, PCR Master Mix should be made for 4 PCs, 4 NTCs
               and 3 replicates for DNA extracts per colony.
       11.6.9   Refer to Sections 12.1 and 12.3 for Data Analyses and Calculations.
12.0  Data Analysis and Calculations

12.1   Real-time PCR During the Site Characterization Phase
       Calculate the average CT from the replicate reactions for each sample, PC and the EIC, where
       applicable. The average CT < 40 for the sample indicates a positive result suggesting the presence
       of B. anthracis spores in the sample. A minimum of two out of three replicates must show CT < 40
       for a sample result to be considered positive.  If only one out of three PCR replicates for any
       sample gives CT < 40, the PCR analysis of the DNA extract for that sample must be repeated.
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Detection of Bacillus anthracis in Environmental Samples
       If the EIC for a sample results in a CT value (> 3) compared to the CT value for the positive
       control, there may be matrix inhibition.  If the corresponding sample is negative (CT > 40) for B.
       anthracis, the sample should be diluted  1:10 and the PCR assay should be repeated for that sample
       along with the EIC with diluted sample extract. Negative controls (NTCs) should not yield any
       measurable CT values; if CT values are obtained check for potential for cross-contamination and
       repeat analysis. In addition, field blank samples should not yield any measurable CT values.  If CT
       values are obtained as a result of a possible contamination or cross-contamination, depending upon
       the CT value, a careful interpretation of the CT values for the sample DNA extracts must be done
       for the final result or the PCR analyses must be repeated.

12.2   RV-PCR
       Calculate an average CT from the replicate reactions for T0 and T9 DNA extracts of each sample.
       Subtract the average CT of the T9 DNA extract from the average CT of the T0 DNA extract. If
       there is no CT for the T0 DNA extract (i.e., the T0 is non-detect), use 45 (total number of PCR
       cycles used) as the CT.  The change (decrease) in the average CT value from T0 to T9 (ACT) > 9
       indicates a positive result suggesting the presence of viable B. anthracis spores in the sample. If
       an incubation time longer than 9 hours was used for the RV-PCR, instead of T9, appropriate Tx
       (incubation time)  should be used. However, (ACT) > 9 algorithm should still be used for a positive
       result. Depending upon the end user's requirement, sample complexity (dirtiness) and the phase of
       response during an event, a lower ACT criterion of > 6 (a two log difference in DNA
       concentration) and a corresponding higher endpoint PCR CT of < 39 could be set. A minimum of
       two out of three T0 PCR replicates must result in CT values < 44 (in a 45-cycle PCR) to calculate
       the average CT. A minimum of two out of three T9 PCR replicates (or Tx for other incubation
       time) must result in CT values < 36 to calculate the  average CT for a sample result to be considered
       positive. Negative controls (NTCs) should not yield any measurable CT values above the
       background level.  If CT values are obtained as a result of a possible contamination or cross-
       contamination, prepare fresh PCR Master Mix and  repeat analysis. In addition, field blank
       samples  should not yield any measurable CT values. If CT values are observed as a result of a
       possible  contamination or cross-contamination, a careful interpretation of the CT values for the
       sample DNA extracts and field blanks must be done to determine if the data is considered valid or
       if the PCR analyses must be repeated.

12.3   Culture
       12.3.1   Serial dilution plating
               Count the number of typical colonies on replicate culture plates and calculate the average
               number of colonies per plate. Apply the following (a - c) when counting the colonies
               and report results based on the  number of confirmed colonies.
               Media sterility checks should not exhibit growth. Growth should also not be present on
               SBA plates from field blank samples.  If growth  is observed on plates, colony
               morphology should be evaluated to determine if contamination is due to the target
               organism and potential source of contamination.  Samples should be reanalyzed if QC
               plates are contaminated with B. anthracis.
               a. If the number of colonies is < 250/plate, record actual number.
               b. If the number of colonies is > 250/plate, record as "TNTC".
               c. If no target colonies are observed, record as "None detected".
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Detection of Bacillus anthracis in Environmental Samples
               To determine the number of spores per sample divide the total number of B. anthracis
               colonies by the dilution factor plated, and multiply by the conversion factor for 1 mL, and
               the total suspension volume. For example if 201 colonies were observed on the 10"1
               dilution plate, and the total suspension volume was 5 mL, the number of colonies per
               sample would be 100,500.
                                    [(201/0.1) x 10] x 5 = 100,500 spores per sample

       12.3.2   MicroFunnel™ filter plating
               Count the number of typical colonies on each  filter and record. Apply the following (a -
               c) when counting the colonies and report results based on the number of confirmed
               colonies.
               Media sterility checks should not exhibit growth. Growth should also not be present on
               SBA plates from field blank samples.  If growth is observed on filters, colony
               morphology should be evaluated to determine if contamination is due to the target
               organism and potential source of contamination. Samples should be reanalyzed if QC
               plates are contaminated with B. anthracis.
               a. If the number of colonies is < 80/plate, record actual number.
               b. If the number of colonies is > 80/plate, record as "TNTC".
               c. If no target colonies are observed, record as "None detected".
               To determine the number of spores per 5 mL sample using the 1 mL aliquot plates,
               multiply the total number of B. anthracis colonies on each plate by 5 (total suspension
               volume). For example if 60 colonies were observed on one of the 1.0 mL  plates, and the
               total suspension volume was 5 mL, the number of colonies per sample would be 300.
                                        60 x 5 = 300 spores per sample
               To determine the number of spores per 5 mL using the third plate (remainder of the
               volume, [1.7 - 2.7 mL) multiply the total number of B. anthracis colonies by the ratio of
               the total suspension volume (5 mL) to the actual volume plated. For example if 60
               colonies were observed on the plate, the volume plated was 2.7 mL and the total
               suspension volume was 5 mL, the number of colonies per sample would be 111.
                                      60 x (5/2.7) = 111 spores per sample

       12.3.3   Confirmation of Colonies by Real-time PCR
               Presence of B. anthracis typical colonies on the culture plate indicates the  presence of
               viable B. anthracis spores or vegetative bacteria in the sample. A minimum of three
               typical colonies should be confirmed using real-time PCR.  The CT < 40 for the sample
               indicates a positive result suggesting the presence of B. anthracis in the respective
               sample. Report the results based on the number of confirmed colonies.  Negative
               controls (NTCs) should not yield any measurable CT values above the background level.
               If CT values are obtained as  a result of a possible contamination or cross-contamination,
               prepare fresh PCR Master Mix and repeat the  analysis.
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Detection of Bacillus anthracis in Environmental Samples
13.0  Method Performance

       To be completed upon protocol verification and/or validation.


14.0 Pollution Prevention

14.1  The solutions and reagents used in this method pose little threat to the environment when recycled
      and managed properly.

14.2  Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
      the volume of expired materials to be discarded.


15.0 Waste Management

15.1  It is the laboratory's responsibility to comply with all federal, state, and local regulations
      governing waste management, especially the biohazard and hazardous waste rules and land
      disposal restrictions.  Following these regulations protects the air, water and land by minimizing
      and controlling all releases from fume hoods and bench operations. Compliance with all sewage
      discharge permits and regulations is also required.
15.2  Samples, reference materials and equipment known or suspected to be contaminated with or to
      contain viable B. anthracis must be decontaminated prior to disposal.

15.3  Large volume water filtrates should be decontaminated using bleach (10% final concentration) for
      a minimum of 30 minutes prior to disposing to the sanitary sewer (e.g., pouring down the drain).

15.4  For further information on waste management, consult The Waste Management Manual for
      Laboratory Personnel (Reference 16.6) and Less Is Better: Laboratory Chemical Management for
      Waste Reduction (Reference 16.7),  both authored by the American Chemical Society.


16.0 References

16.1  U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and
      National Institutes of Health.  2009. Biosafety in Microbiological and Biomedical Laboratories
      (BMBL), 5th Edition, http://www.cdc.gov/biosafety/publications/bmbl5/index.htm

16.2  American Chemical Society (ACS). 2005. Reagent Chemicals: Specification and Procedures,
      Oxford University Press (USA), New York.
16.3  British Drug Houses, Ltd. 1957. AnalaR Standards for Laboratory Chemicals. 5th Edition.
      BDH Ltd., Poole, Dorset, U.K.
16.4  United States Pharmacopeia.  2005. United States Pharmacopeia and National Formulary 24.
      United States Pharmacopeial Convention, Md.

16.5  Francy, D.S., Bushon, R.N., Brady, A.M., Bertke, E.E., Kephart, C.M., Likirdopulos, C.A.,
      Mailot, B.E., Schaefer, F.W. Ill and Lindquist, H.D.A. 2009. "Performance of Traditional and
      Molecular Methods for Detecting Biological Agents in Drinking Water." U.S. Department of the
      Interior/U.S. Geological Survey. Scientific Investigations Report 2009-5097.


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Detection of Bacillus anthracis in Environmental Samples
16.6  American Chemical Society (ACS) 1990.  The Waste Management Manual for Laboratory
      Personnel.  American Chemical Society Department of Government Relations and Science
      Policy, Washington, DC.
16.7  American Chemical Society (ACS). 2002. Less Is Better: Laboratory Chemical Management for
      Waste Reduction. American Chemical Society Taskforce on RCRA (Resource Conservation and
      Recovery Act of 1976), Washington, DC.
      http://portal.acs.org/portal/fileFetch/CAVPCP 012290/pdf/WPCP 012290.pdf
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Detection of Bacillus anthracis in Environmental Samples
                                     Appendix A
  Ultrafiltration (UF) for the Detection of Bioterrorism Threat (BT)
                       Agents in Potable Water Samples
Note:  This protocol should not be misconstrued as a laboratory standard operating procedure (SOP)
       that addresses all aspects of safety; the laboratory should adhere to their established safety
       guidelines.
       At a MINIMUM these procedures should be performed in a Biological Safety Level (BSL)-3
       facility using BSL-3 practices. It is recommended that all sample manipulations be performed
       within a Class II (or higher) biological safety cabinet (BSC).

1.0    Sample Preparation
Note:  Water samples should be concentrated as soon as possible after collection and analyses should
       be initiated immediately, if possible. However, if analyses cannot be accomplished
       immediately, the concentrated sample may be stored at 2°C - 8°Cfor up to 24 hours.
1.1    Laboratory Supplies
       1.1.1   Asahi Kasai Rexeed 25SX Dialyzers (Dial Medical Supply Cat. No. 25SX or equivalent)
       1.1.2   Masterflex® L/S #36 silicon tubing (Cole Farmer® Cat. No. EW-96410-36 or equivalent)
Note:  An alternative to the Masterflex* tubing is #36 BioPharm Silicone tubing (Cole Farmer9 Cat.
       No. EW-96420-36).
       1.1.3   Masterflex® L/S #24 silicon tubing (Cole Farmer® Cat. No. EW-96410-24 or equivalent)
       1.1.4   Masterflex® tubing reducing connectors (Cole Farmer® Cat. No. EW-40610-08 or
              equivalent)
       1.1.5   3-prong extension clamp (Cole Farmer®  Cat. No. EW-08021-36 or equivalent)
       1.1.6   Ring stand (Fisher Cat. No. 14-670C or equivalent)
       1.1.7   Clamp connector/holder (Cole Farmer® Cat. No. EW-08041-20 or equivalent)
       1.1.8   Nalgene® Analytical Filter Unit, 0.45 (im (Fisher Scientific Cat. No. 09-740-2IB or
              equivalent)
       1.1.9   Heavy duty pinchcock, metal clamp (Cole Farmer® Cat. No. EW-08126-0302 or
              equivalent)
       1.1.10  GN-6 Metricel® Membrane Filters, 0.45  (im (VWR Cat. No. 28148-926 or equivalent)
       1.1.11  Forceps, sterile, disposable  (Cole Palmer® Cat. No. U06443-20 or equivalent)
       1.1.12  Hose clamps (Cole Parmer®)
              (a) Large: #10 and #12 white, plastic (Cat. No. EW-06832-10, EW-06832-12 or
                 equivalent)
              (b) Medium size: #6 and #8 white, plastic (Cat. No. EW-06832-06, EW-06832-08 or
                 equivalent) or 7/32" to 5/8" stainless steel (Cat. No. EW-06403-11 or equivalent)
              (c) Small: #4 white, plastic (Cat. No. EW-06832-04 or equivalent) or 7/32" to 5/8"
                 stainless steel (Cat. No. EW-06403-11 or equivalent)
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Detection of Bacillus anthracis in Environmental Samples
        1.1.13  1 L heavy duty polypropylene vacuum bottle (Fisher Scientific Cat. No. 06-443A or
                      equivalent)
        1.1.14  3 port filling/venting closure cap with tubing (Fisher Scientific Cat. No. 02-923-13Y or
                      equivalent)
        1.1.15  Barbed reducing Y connector 1/4 x 3/8 (Cole Farmer® Cat. No. EW-30726-33 or
               equivalent)
        1.1.16  DIN adapters (filter connectors)
               (a) Small connectors for 24 tubing (Molded Products Cat. No. MPC-855NS.250PP or
                  equivalent)
               (b) Large connectors for 36 tubing (Molded Products Cat. No. MPC-855NS.375PP or
                  equivalent)
        1.1.17  Blood Port Storage Cap (screw cap), polypropylene (Molded Products Cat. No. MPC-
               40PP or equivalent)
        1.1.18  Ice bucket and ice
        1.1.19  Flow regulator (Keck) tubing clamps (Cole Farmer® Cat. No. A-06835-07 or equivalent)
        1.1.20  Ziplock bags
        1.1.21  Parafilm
        1.1.22  Bleach Wipes (Dispatch® Cat. No. 69150 or equivalent)
        1.1.23  Bottle, sterile, 100 mL
        1.1.24  Tubes, sterile, 15 mL (Fisher Scientific Cat. No. 339650 or equivalent)
        1.1.25  Bottle, 1-L sterile polypropylene (Thermo Scientific-Nalgene® Cat. No. 2105-0032 or
               equivalent)
        1.1.26  Filter flask, sterile, 500 mL, glass or polypropylene (Fisher Scientific Cat. No. FB-300-
               500; 10-182-5OA; or equivalent).
        1.1.27  10 mL plastic pipets, sterile, T.D. bacteriological (Fisher Scientific Cat. No.  13-678-12E
               or equivalent)
        1.1.28  50 mL plastic pipets, sterile, T.D. bacteriological (Fisher Scientific Cat. No.  13-678-14C
               or equivalent)
        1.1.29  Pliers
        1.1.30  Graduated cylinder (1 L) or graduated beaker (1 L)
        1.1.31  Syringe, 60 mL
        1.1.32  #7 rubber stopper, 500 mL  flask, with hole (Fisher Scientific Cat. No. 14-135L or
               equivalent)
        1.1.33  Collapsible containers, 10 L or 20 L (Cole Farmer® Cat. No. EW-06100-30 or EW-
               06100-40)
        1.1.34  Vacushield™ Vent Device  (HEPA filter) (VWR Cat. No. 55095-006 or equivalent)
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Detection of Bacillus anthracis in Environmental Samples
1.2    Equipment
       1.2.1   Masterflex® Console Drive (Cole Farmer® Cat. No. EW-07554-90 or equivalent)

       1.2.2   Masterflex® EasyLoad II Pump Head (Cole Farmer® Cat. No. SI-77200-52 or equivalent)
       1.2.3   Jiffy-Jack® apparatus positioner (Cole Farmer® Cat. No. A-08057-40 or equivalent)

       1.2.4   BD Clay Adams™ Nutator Mixer (VWR Cat. No. 15172-203 or equivalent)
       1.2.5   Biological safety cabinet (BSC) - Class II or Class III
       1.2.6   Vortex mixer (Fisher Scientific Cat. No. 02-215-365 or equivalent)

1.3    Reagents
       1.3.1   1000X (10%) Sodium Poly-Phosphate (NaPP): Add 10 g of NaPP per 100 mL of sterile
               reagent-grade water in a sterile 100-mL bottle. Cap the bottle and shake vigorously by
               hand for 1 minute to mix the solution. If the water is cold (e.g., from refrigerator), let the
               solution dissolve for 2 hours at room temperature, mixing for Iminute approximately
               every 15 minutes. If water is initially at room temperature, the NaPP should dissolve
               within 30 minutes (mixing vigorously by hand every 15 minutes). If the NaPP is not
               completely dissolved in the water, place the NaPP solution in a water bath at 50°C and
               incubate for 2 hours. Continue incubating until the NaPP is completely in solution. Store
               the 10% NaPP in refrigerator for up to 2 months.
       1.3.2   10% Tween 80-1% Antifoam Y-30 solution: Pipet 0.1 mL Antifoam Y-30 Emulsion
               (Sigma Cat. No. A5758 or equivalent) and 1 mL Tween 80 (Fisher Cat. No. T164 or
               equivalent) into 15 mL conical tube containing 8.9 mL reagent-grade water.  Vortex for
               30 seconds to mix.  Solution can be stored at room temperature for 1 month.
       1.3.3   Elution solution (0.01% Tween® 80. 0.01% NaPP and 0.001% Antifoam Y-30): Add 0.5
               mL of the 10% Tween 80-1% Antifoam Y-30 solution and 0.5 mL of 10% NaPP to 500
               mL of sterile reagent-grade water. Swirl to mix.  Solution can be made up to 24 hours in
               advance and stored in a refrigerator.  Bring to room temperature prior to use.
Note:  The elution solution should be made in or transferred to a sterile 1-L heavy duty polypropylene
       bottle with closed cover.
       1.3.4   0.01% NaPP solution (filter wash) - 1 L: Add 1 mL of 10% NaPP solution to 999 mL of
               reagent-grade water and swirl to mix. Solution can be made up to 24 hours in  advance
               and stored in a refrigerator. Bring to room temperature prior to use.
       1.3.5   1% Bleach solution (for tubing decontamination) - 500 mL: Add 5 mL bleach to 495 mL
               reagent grade or deionized water and swirl to mix. Solution can be stored at room
               temperature for 1 week.
       1.3.6   70% Ethanol solution (Fisher Scientific Cat. No. 04-355-56 or equivalent)
       1.3.7   Sodium thiosulfate solution:  Prepare 10% w/v sodium thiosulfate by adding 100 g
               sodium thiosulfate (Fisher Scientific Cat. No. S446 or equivalent) to 1 L of sterile
               reagent-grade water and mix well. Store at 4°C for up to 1 month.
       1.3.8   Dialyzed Fetal Bovine Serum (FBS) (Fisher Scientific  Cat. No. SH3007303 or
               equivalent)

       1.3.9   Phosphate buffered  saline (PBS) (Fisher Scientific Cat. No. BP3991  or equivalent)
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1.4    Pre-treat the Rexeed ultrafilter with dialyzed 5% FBS in water to block non-specific protein
       binding.
       1.4.1   To make 150 mL of 5% FBS, add 7.5 mL of FBS to 142.5 mL of reagent-grade water.
       1.4.2   Before adding the FBS to the filter, secure the ultrafilter to ring stand with a 3 prong
               clamp. Ensure the bottom port is securely closed with a blood port storage screw cap.
               Place the white port caps that come with the ultrafilter onto both  side ports, leaving them
               loosened to allow air to escape.
       1.4.3   Load the ultrafilter with approximately 120 mL (no more than 150 mL) of 5% FBS by
               injecting it into the top port of the ultrafilter (a 60 mL syringe with no needle works well
               for this application). Attach 3" of #24 tubing to a small filter connector, secure with a #4
               clamp, screw into the filter port, and insert the tip of the syringe into the #24 tubing.
               Pour 60 mL of FBS solution into the syringe and use the plunger to push the solution into
               the ultrafilter. Repeat for remaining volume.

       1.4.4   When the ultrafilter is loaded, close the open end of the ultrafilter with a second blood
               port storage screw cap and ensure side ports are closed with white port caps (they will
               "click" into place).
       1.4.5   Place the ultrafilter on a rocker panel at room temperature; rock for 30 minutes.

Note:  Do not store the filter after pretreating, as this will encourage growth of contaminating
       bacteria which may clog the filter.

1.5    Pre-treat the Water Sample
Note:  Any procedure in which sample containers are opened should be performed inside a BSC.
       1.5.1   If sodium thiosulfate was not added to the sample at the time of collection, add  0.5 mL/L
               of a 10% w/v solution of sodium thiosulfate immediately upon receipt of the sample.
       1.5.2   Pretreatthe water sample withNaPPto reach a final concentration of 0.01%. To achieve
               a 0.01% concentration of NaPP, add 1 mL of 10% NaPP per 1 L  of water  sample.
1.6    Prepare the empty filtrate container(s) by adding a sufficient volume of bleach such that the final
       concentration is at least  1% bleach (e.g., 200 mL bleach in a 20 L container).

1.7    Assembly of the Sample Tubing Set
       1.7.1   Remove both ends (tip and end containing cotton material) of a 10 mL pipet by carefully
               breaking off the ends  while the pipet is within its plastic wrapping (Figure 1, a). Keep the
               plastic sleeve to place the sample pipet in during changing of sample containers.
       1.7.2   Connect the 10 mL pipet to 14" of #24 Masterflex® (MF) tubing  (Figure 1, b) and secure
               with a medium hose clamp (#8 plastic) (Figure 1, #1).
       1.7.3   Connect the #24 tubing to one of the small 1/4" barbs on the Y connector  (Figure 1, c)
               and secure with a small hose clamp (#4 plastic or 7/32" to 5/8" stainless steel) (Figure 1,
               #2).
       1.7.4   Connect 21" of #36 tubing (Figure 1, d) to the end of the large filter connector (Figure 1,
               e) and secure connection with large hose clamp (#10 plastic) (Figure  1, #3).
       1.7.5   Connect the other end of the #36 tubing to the large 3/8" barb on the Y connector (Figure
               1, c) and secure with a large hose clamp (#8 plastic) (Figure  1, #4).
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Detection of Bacillus anthracis in Environmental Samples
        1.7.6   Connect 6" of #24 tubing (Figure 1, f) to the open small 1/4" barb on the Y connector
               (Figure 1, c) and secure with a small hose clamp (#4 plastic or 7/32 to 5/8" stainless steel)
               (Figure 1, #5). Connect the opposite end to the empty port on the 1 L vented cap bottle
               and secure with a stainless steel clamp (vented cap bottle not shown in Figure 1, #6; see
               Figure 7a, #10).
1.8
Figure 1. Sample tubing set assembly.
Assembly of the Retentate Return Tubing Set
1.8.1    Connect 15" of #24 tubing (Figure 2, a) to the small filter connector (Figure 2, b); secure
        connection with a small hose clamp (#4 plastic or 7/32" to 5/8" stainless steel) (Figure 2,
        c).
1.8.2    Attach a flow regulator tubing clamp (Figure 2, d) to the #24 tubing so that the wide end
        is facing the 1 L vented cap bottle (Figure 2, e).
1.8.3    Connect the opposite end of the #24 tubing to the port on the 1 L vented cap bottle that is
        attached to the shorter internal tubing  (Figure 2, f; Figure 7a, #9); secure with a stainless
        steel clamp (Figure 2, g)..
           Figure 2. Retentate return tubing set assembly.
1.9 Assembly of the Filtrate Tubing Set
        1.9.1   Remove both ends (tip and end containing cotton material) of a 10 mL pipet by carefully
               breaking off the ends while the pipet is within its plastic wrapping (Figure 3, a).
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Detection of Bacillus anthracis in Environmental Samples
        1.9.2   Connect the 10 mL pipet to 16" of #36 MF tubing and secure with a medium hose clamp
               (#8 plastic) (Figure 3, b).
           Figure 3. Filtrate tubing set assembly.
1.10    Connection of the Pumping Station
Note:   The pumping station and ultra/liter setup may be assembled inside of a BSC or on the bench
        top (bench top allows ease of assembly, but it is recommended that this protocol not be
       performed on the bench). Additionally, absorbent underpads/diapers may be placed under the
        UF setup and containers to capture any potential leaks or spills that may occur during sample
       processing or disassembly.
        1.10.1  Raise the pump to a height equal to or above the top of the reservoirs by placing the
               pump on a shelf or raising it on a variable height platform such as the Jiffy-Jack®
               apparatus positioner.  Also, secure the filter and the 1 L vented cap bottle in a vertical
               position by using adjustable metal clamps and a ring stand.
        1.10.2  Connect the sample reservoir to the ultrafilter using the sample tubing set. Attach the
               large filter connector on the end of the sample tubing set to the top port of the ultrafilter.
               Feed the #36 tubing through the pump head, and ensure the tubing remains securely
               clamped to the large filter connector.
        1.10.3  Attach the small  filter connector on the end of the retentate return tubing set to the bottom
               port of the ultrafilter.
        1.10.4  Connect the ultrafilter to the filtrate reservoir by connecting the filtrate return tubing set
               to the top side port of the ultrafilter (may secure with #12 plastic clamp, but not required)
               and placing the 10 mL pipet into the filtrate reservoir.  Ensure  the other side port is closed
               with the plastic cap provided with the filter.
        1.10.5  The assembled UF setup should appear as in Figures 7a and 7b.
        1.10.6  Place a 1 L beaker next to the UF setup and another 1 L beaker next to the filtrate setup.
               Use beakers to hold the sample and filtrate tubing pipets when changing sample and
               filtrate containers to prevent potential water droplets from dripping onto the BSC.
        1.10.7  Verify that the MF tubing is threaded through the pump head correctly (Figure 4).
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Detection of Bacillus anthracis in Environmental Samples
        1.10.8  Set the pump to -50% power. If using a digital pump, the flow rate should be set to 1450
               mL/min.
        1.10.9  If the pump has a flow direction toggle switch, confirm the flow direction is set to the
               right (Figure 4).
                                   Figure 4. Photo of the pump head.
1.11   Washing the Ultrafilter
       1.11.1  Wash the 5% FBS from the ultrafilter by placing the sample tubing end (Figure 5, #2)
               into the 1 L bottle containing 0.01% NaPP filter wash prepared in Section 1.3.3 (Figure 5,
               #1).
       1.11.2  Detach the retentate return tubing set (Figure 5, #5) from the 1 L vented cap bottle
               (Figure 5, #7) and place the end in the filtrate reservoir (Figure 5, #6) so that the retentate
               return tubing set and the filtrate tubing are both in the filtrate container.
       1.11.3  Apply the pinchcock to the tubing from port C (clamp the tubing from vented cap bottle
               to the Y connector, getting as close to the Y connector as possible) (Figure 7a, #1).
       1.11.4  Start the pump (Figure 5, #3) and flush the 0.01% NaPP filter wash through the lines and
               the ultrafilter (Figure  5, #4).
Note:  While flushing the NaPP filter wash, be sure to check the tubing and connections for any leaks
       or drips throughout the system.
       1.11.5  When the filter wash is completed, reset the tubing as shown in Figure 7a.
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Detection of Bacillus anthracis in Environmental Samples
           Figure 5. Schematic of Filter Wash
           (1) Sterile water wash, (2) Sample tubing, (3) Pump, (4) Ultrafilter,
           (5) Retentate tubing set, (6) Filtrate reservoir, (7) 1 L vented cap bottle


2.0   Primary Water Sample Concentration

2.1    With the sample container in place and the tubing set per Figure 7a, check to make sure the
       pinchcock is applied to the tubing from port C (Figures 7a and 7c, #1), the vented cap is securely
       tightened to the bottle, and the rubber cap is removed from the vented cap.
Note:  When applying the pinchcock clamp to the tubing, make sure it is clamped as close to the Y
       connector as possible to completely block and/or stop the flow of water through tubing (Figure
       7c, #1).
2.2    Start the pump with the flow switch turned to the right and the pump speed set to the maximum
       (~ 2900 mL/min flow rate).

2.3    Once the 1 L retentate bottle is -2/3 of the way full, quickly close the open port of the vented cap
       with the rubber cap and remove the pinchcock from the tubing (Figures 7a and 7c, #2).  The
       pump will now be drawing water from both the sample container and the 1  L vented cap
       (retentate) bottle. Make sure the water level in the 1 L retentate bottle does not continue to rise.
       If the water level in the 1 L bottle does  rise, remove the rubber cap and apply pinchcock to the
       tubing from port A (Figures 7a and 7c,  #3). Once the water level in the 1 L bottle is 2/3  of the
       way full, close port with rubber cap and remove the pinchcock (Figures 7a and 7c, #2).
2.4    Tighten the flow regulator by rolling the knob to the right until the "K" in the "KT" lettering on
       the front face of the flow regulator is directly in the middle of the adjustment knob (Figure 6).
       The back pressure provided  by the flow regulator should produce a filtrate rate between 1000 -
       1400 mL/min.

Note:  The flow rate does not need to  be measured.
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Detection of Bacillus anthracis in Environmental Samples
                        Figure 6. Position flow regulator rolling clamp over "K" in "KT".
     Y Connector
   Y Connector and pinchcock:
   1. Clamp tubing from port C at
      beginning of filtration. Water
      flows from sample container
      through ultrafilter to fill 1L
      bottle.
   2. Remove pinchcock when the
      volume in the 1L bottle is 2/3
      full. The system then draws
      from both 1L bottle and
      sample container.

   3. Clamp tubing from port A
      once the entire sample is
      drawn from the sample
      container. Sample is now
      being reduced in the 1L bottle
      only.
                  Peristaltic Pump
                        Flow
                                                                                 Hollow Fiber
                                                                                  Ultrafilter
                                                                                           5. Filtrate
                                                                                            tubing
 Sample
container
                          L/S 24 tubing
                            (AandC
                             ports)
                         L/S 36 tubing
                            (B port)
6. Empty container
     (filtrate)
    Figure 7a. Recirculating ultrafiltration assembly.
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   Figure 7b. Photograph of the ultrafiltration system set up inside a BSC.
       Figure 7c. Pinchcock positions.
2.5    When the sample container is empty, apply the pinchcock to clamp the tubing from port A
       (Figures 7a and 7c, #3), loosen the flow regulator and turn off the pump.
Note:  Whenever the pump needs to be stopped, apply thepinchcock to clamp the tubing from port A
       so that the water containing microbes cannot flow back into the sample tank and then quickly
       press the stop button.
2.6    If this is the only sample container, skip to step 2.13.
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Detection of Bacillus anthracis in Environmental Samples
2.7    Remove the sample tubing set from the empty sample container and carefully place it in a sterile
       1 L beaker.  Remove the filtrate pipet in the same manner and place in a second sterile 1 L
       beaker.

Note:  To prevent drips, ensure the pipet is free of any remaining sample while still inside the sample
       container. Labs may consider placing the pipet back into the plastic sleeve prior to placing it
       into the 1L beaker.  Alternatively, if sample container is disposable, the tubing may be cut and
       the pipet can be disposed of inside the sample container.
2.8    Remove the empty sample container and replace with the next sample container to be filtered.
       Also replace the full filtrate container with an empty filtrate container [containing 1% bleach as
       directed in Section 1.3.4].

Note:  Follow Biosafety in Microbiological andBiomedical Laboratories (BMBL) and lab-specific
       safety practices for BSL-3 working conditions while slowly moving containers into and out of
       the BSC.  Let airflow re-establish for a minimum of 15 minutes each time the sash is lifted.
2.9    Place the sample tubing set into the new sample container and the filtrate tubing set into the new,
       empty filtrate container.
2.10   Turn the pump  back on and remove the pinchcock from the tubing from port A (Figures 7a and
       7c, #2) so that the water is once again being drawn from both the container and the 1 L bottle.
2.11   Tighten the flow regulator to the same position described in Section 2.4  and continue filtration.
2.12   Repeat steps 2.7-2.11 for the rest of the containers.
2.13   When the last container has been emptied, apply the pinchcock to clamp the tubing from port A
       (Figures 7a and 7c, #3) so that the sample is only being re-circulated in the 1 L bottle, loosen the
       flow regulator,  and remove the rubber cap from the 1 L bottle.
2.14   When the sample in the 1 L bottle draws down to about 1.5" from the bottom, move  the
       pinchcock to clamp the tubing from port C (Figures 7a and 7c, #1), so that only air is being drawn
       into  the filtration system by the pump.
Note:  Continue flushing until all the retentate is out of the tubing and the filter. The retentate tubing
       may be lifted 2-3 times to help flush the water that has settled in the retentate tubing into the 1
       L bottle.
2.15   Turn off the pump when all of the retentate has been flushed from the tubing and the filter. The
       final volume should be approximately 200 mL - 250 mL.
2.16   Carefully unscrew the steel clamps from the ports on the vented cap bottle and remove the tubing
       connections from the top ports (containing the concentrated sample). Place them onto the ports of
       the second vented cap bottle containing the elution solution and secure the clamps (remove the
       closed cap from the elution solution bottle first).
2.17   Unscrew the vented cap from the first bottle containing the sample. Lift and securely hold the
       cap with one hand (tubing remains inside the  bottle) while using a 25 mL or 50 mL pipet in the
       other hand to measure and transfer the sample retentate into a sterile, 1 L plastic bottle.  Record
       the retentate volume.
2.18   Secure the second vented cap bottle containing the elution solution with the 3-pronged clamp
       from the first vented cap bottle.  Check that the flow regulator is fully opened, the pinchcock is
       clamped to the tubing from port A (Figures 7a and 7c, #3) and the rubber cap is removed from the
       1 L vented cap  bottle. The assembly should now be ready for elution of the ultrafilter.
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Detection of Bacillus anthracis in Environmental Samples
2.19   Turn on the pump, allowing the eluent volume to gradually reduce until the level draws down to
       1.5" from the bottom of the bottle. Move the pinchcock to clamp the tubing from port C (Figure
       7a and 7c, #1) to flush the rest of the eluent from the tubing and filter.  Turn off the pump.
2.20   Unscrew the vented cap assembly from the second bottle containing the concentrated eluent.  Lift
       and securely hold the cap with one hand (tubing remains inside the bottle) while using a 25 mL or
       50 mL pipet in the other hand to measure and transfer the eluent to the 1 L plastic sample bottle
       containing the retentate. The total volume of the final UF concentrate should be 400 mL - 500
       mL.
3.0   Secondary Water Concentration

3.1    Inside of a BSC, assemble the membrane filtration setup shown in Figure 9,  ensuring the HEPA
       filter (Vacushield) is attached with #24 tubing (or alternative vacuum tubing) to the house
       vacuum and the side arm of the back-up flask. Connect the side arm of the main flask to the
       stopper of the back-up flask with vacuum tubing. This tubing length should be long enough such
       that the tubing rests on the surface of the BSC to help stabilize the flasks.  Attach an additional
       piece of vacuum tubing to the side arm of the main flask for connection to the disposable filter
       units.
3.1.1  Use the piece of #24 tubing from the side arm of the main filter flask to attach the quick
       disconnect side arm on the filter unit (Figure 9, #5) and use sterile forceps to place a new filters
       onto the base of each filter unit (Figure 9, #3).
Note:  Disposable filter units (Section 1.1.8) come with cellulose nitrate membrane filters, which must
       be replaced with the mixed ester cellulose filters.  DO NOT remove the cellulose support pad on
       the base of the unit when replacing membrane filters. It is recommended to keep a small
       autoclave bag inside the BSC during processing for safe disposal of filter funnels.
       Figure 9. Membrane filtration setup.
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Detection of Bacillus anthracis in Environmental Samples
3.2    Run a negative control before filtering any sample concentrate. Add 20 mL PBS to the filter cup
       (Figure 9, #2) and turn on the vacuum. Once the PBS has finished filtering, turn off the vacuum
       and remove the cup from the base of the filter unit (Figure 9, #3). Retrieve the membrane filter
       with sterile forceps and place onto a SBA plate. Incubate at 37°C ± 1°C for a maximum of 3
       days.
3.3    Use sterile forceps to place a new 0.45 (im filter onto the support pad on the base of the filter unit.
       Reattach the filter cup to the base.
3.4    Add the retentate slowly to the filter unit (Figure 9, #2) and turn on the vacuum (not all the
       retentate will fit at once). Continue to add retentate to the filter unit taking care to avoid clogging
       the filter.  Once the  sample has finished filtering, rinse the filter cup 3 times with PBS in a squirt
       bottle.  Turn off the vacuum; remove the filter cup from the bottom portion (Figure 9, #3)  of the
       filter unit and set it on a sterile surface.
Note:  If the final retentate is particularly cloudy or has noticeable sediment, it might be necessary to
       split the retentate volume between two or more filter units.
3.5    Using sterile  disposable forceps, grab the edge of the membrane at the filter unit base and  fold it
       toward  the other end.  While holding the 2 edges together, take the forceps and place folded
       membrane into the bottom half of a 50 mL conical tube, avoiding the conical portion. Close tube.
3.6    Repeat  steps  3.1 - 3.4 for the  remaining samples.
Note:  If multiple filtrations were run due to excess sediment, process all of the membrane-filters.
3.7    For polymerase chain reaction (PCR) analysis, proceed to Section 9.1.5 of the protocol.
3.8    For Rapid Viability-PCR (RV-PCR) analysis, proceed to Section 10.2.6 of the protocol.
3.9    For culture analysis, proceed to Section 11.5 of the protocol.
4.0   Limitations

4.1     If the procedure is not performed correctly, it may result in false negative results.
4.2    Water from certain sources may contain higher levels of minerals, organic compounds or other
       substances which may affect the water concentration procedure and subsequent testing
       procedures for the  detection of potential BT agents.
4.3    The presence of chlorine-based disinfectants in the water supply will inhibit or prevent the growth
       of most microorganisms. Sodium thiosulfate should be added as soon as possible to inactivate
       chlorine in the water. Addition of sodium thiosulfate at the time of collection of the water sample
       is recommended.
4.4    Even with the addition of sodium thiosulfate, some organisms may not survive the filtration
       process and may only be detected by real-time PCR.
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Detection of Bacillus anthracis in Environmental Samples
5.0    Acronyms

BMBL    Biosafety in Microbiological and Biomedical Laboratories
BSC      Biological safety cabinet
BSL      Biological safety level
BT       Bioterrorism threat
FBS      Fetal bovine serum
MF       Masterflex®
NaPP     Sodium poly-phosphate
PBS      Phosphate buffered saline
PCR      Polymerase chain reaction
RV-PCR  Rapid viability-polymerase chain reaction
SOP      Standard operating procedure
UF       Ultrafiltration
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     Environmental Protection
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