Development of Sampling Protocols and Strategies for Solid Waste Sampling During Biological Incidents


EPA/600/R-22/117 | September 2022 |
www.epa.gov/research

United States
Environmental Protection
Agency

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Office of Research and Development

Homeland Security arid Materials Management Division

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Development of Sampling
Protocols and Strategies for
Solid Waste Sampling During
Biological Incidents

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EPA/600/R-22/117
September 2022

Development of Sampling Protocols and
Strategies for Solid Waste Sampling During

Biological Incidents

EPA Contract Number: EP-C-16-014
Task Order 68HERC1 9F0090

Kent Hofacre, Scott Nelson, and Ryan James
Battelle Memorial Institute
Columbus, Ohio 43201

Paul M. Lemieux and Michael Worth Calfee
U.S. Environmental Protection Agency

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Disclaimer

This study was funded through the Analysis for Coastal Operational Resiliency (AnCOR) Project by the
U.S. Department of Homeland Security Science and Technology Directorate under interagency
agreement IA 70RSAT18KPM000084 (RW-070-95937001). This report was prepared by Battelle
Memorial Institute under EPA Contract Number EP-C-16-014; Task Order 68HERC19F0090. This
document has been reviewed in accordance with EPA policy and approved for publication. Any mention
of trade names, manufacturers, or products does not imply an endorsement by the United States
Government or the EPA. EPA and its employees do not endorse any commercial products, services, or
enterprises.

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

Dr. Paul Lemieux

U.S. Environmental Protection Agency

109 T.W. Alexander Drive

Mail Code: E343-06

Research Triangle Park, NC 27711

919-541-0962

lemieux.paul@epa.gov

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Foreword

The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the Nation's
land, air, and water resources. Under a mandate of national environmental laws, the Agency strives to
formulate and implement actions leading to a compatible balance between human activities and the
ability of natural systems to support and nurture life. To meet this mandate, EPA's research program is
providing data and technical support for solving environmental problems today and building a science
knowledge base necessary to manage our ecological resources wisely, understand how pollutants affect
our health, and prevent or reduce environmental risks in the future.

The Center for Environmental Solutions and Emergency Response (CESER) within the Office of
Research and Development (ORD) conducts applied, stakeholder-driven research and provides
responsive technical support to help solve the Nation's environmental challenges. The Center's research
focuses on innovative approaches to address environmental challenges associated with the built
environment. We develop technologies and decision-support tools to help safeguard public water
systems and groundwater, guide sustainable materials management, remediate sites from traditional
contamination sources and emerging environmental stressors, and address potential threats from
terrorism and natural disasters. CESER collaborates with both public and private sector partners to foster
technologies that improve the effectiveness and reduce the cost of compliance, while anticipating
emerging problems. The Center provides technical support to EPA regions and programs, states, tribal
nations, and federal partners, and serve as the interagency liaison for EPA in homeland security research
and technology. In addition, the Center is a leader in providing scientific solutions to protect human
health and the environment.

This report focuses on the evaluation of spore recovery using three different methods applied to
candidate solid waste streams likely to be generated during the decontamination and restoration of a
biological incident at a United States Coast Guard (USCG) station. Candidate solid waste streams were:
personal protective equipment, line (rope), and boat seat covers. The spore recovery and analytical
methods used here were adapted using existing EPA spore recovery and analysis methods but applied to
the solid waste materials so that no new methods needed to be developed. Results from this study can be
used to better plan and execute the recovery of a USCG base, or any large outdoor urban area, following
a biological contamination incident.

Gregory Sayles, Director, Center for Environmental Solutions and Emergency Response

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Table of Contents

Page

Disclaimer	ii

Foreword	iii

Acronyms and Abbreviations	ix

Acknowledgments	x

Executive Summary	xi

1.0 INTRODUCTION	1

1.1	Objective	2

1.2	Scope	2

2.0 MATERIALS AND METHODS	3

2.1	Solid Waste Material Selection	3

2.2	Solid Waste Material Description	5

2.2.1	Personal Protective Equipment - Nitrile Gloves	6

2.2.2	Personal Protective Equipment - Tyvek or Tychem Sleeve	6

2.2.3	Double-Braided Nylon Rope	7

2.2.4	Marine Fabric - Faux Leather Boat Seats	7

2.3	Solid Waste Material Preparation	8

2.3.1	Solid Waste Sample Swatch Preparation	8

2.3.2	Spore Stock	9

2.3.3	BaS Application (Spiking)	9

2.4	Spore Recovery Methods from Solid Waste Materials	10

2.4.1	50-mL Conical Tube Spore Recovery Method	11

2.4.2	400-mL Stomacher Bag Spore Recovery Method	12

2.4.3	1-L Bottle Spore Recovery Method	12

2.5	Test Matrix	13

2.6	Overall Method Implementation	14

2.7	Microbiological Methods	15

2.7.1	BaS Culture Method	16

2.7.2	RV-PCR Methods	17

2.8	Data Reduction and Analysis	20

2.8.1	Percent Recovery of Presumptive BaS Colonies	20

2.8.2	RV-PCR Method	21

2.8.3	Presentation of Results	21

3.0 RESULTS AND DISCUSSION	22

3.1	50-mL Conical Tube Spore Recovery Method Analyses Results	22

3.1.1	50-mL Conical Tube Spore Recovery Sample Culture Analyses	22

3.1.2	50-mL Conical Tube Spore Recovery BaS Colony Confirmation	25

3.1.3	50-mL Conical Tube Spore Recovery Sample RV-PCR Analyses	25

3.2	400-mL Stomacher Bag Spore Recovery Method Analyses Results	27

3.2.1	400-mL Stomacher Bag Spore Recovery Sample Culture Analyses	27

3.2.2	400-mL Stomacher Bag Spore Recovery BaS Colony Confirmation	30

3.2.3	400-mL Stomacher Bag Spore Recovery Sample RV-PCR Analyses	30

3.3	1-L Bottle Spore Recovery Method Analyses Results	33

3.3.1	1-L Bottle Spore Recovery Sample Culture Analyses	33

3.3.2	1-L Bottle Spore Recovery BaS Colony Confirmation	37

3.3.3	1-L Bottle Spore Recovery Sample RV-PCR Analysis	38

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3.4	Spore Recovery Method Comparison	42

3.5	Method Observations	43

4.0 QUALITY ASSURANCE/QUALITY CONTROL	44

4.1	Equipment Calibration	44

4.2	QC Results	44

4.3	Operational Parameters	44

4.4	Audits	44

4.4.1	Performance Evaluation Audit	44

4.4.2	Technical Systems Audit	45

4.4.3	Data Quality Audit	45

4.5	QA/QC Reporting	45

4.6	Data Review	45

5.0 CONCLUSIONS	46

6.0 REFERENCES	48

Development of Sampling Protocols and Strategies for Solid Waste Sampling During
Biological Incidents: Appendices A-J	49

V

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Table of Figures

Page

Figure 1. Representative image of PPE worn during a decontamination operation	4

Figure 2. Photograph of mooring line (rope) common at USCG stations	5

Figure 3. Photograph of a typical boat seat on a small or medium response boat	5

Figure 4. Photograph of nitrile gloves used as a solid waste stream test material	6

Figure 5. Photograph of (A) Tychem and (B) Tyvek sleeve used as a solid waste stream

test material	7

Figure 6. Photograph of double-braided nylon rope (A) 1/4-inch and (B) 1-inch diameter

used as a solid waste stream test material	7

Figure 7. Photograph of faux leather boat seat (marine fabric) used as a solid waste

stream test material	8

Figure 8. Photographs of cutting of bulk solid waste material (A) Tychem sleeve, (B)

double-braided nylon rope, and (C) boat seat covers to obtain sample swatches

for analysis	9

Figure 9. Spiking of nitrile gloves with suspension of BaS (A) spiking palm of nitrile glove,
(B) spiked nitrile glove with dried BaS droplets donned, and (C) doffing of spiked

nitrile glove	10

Figure 10. Spiking of swatches of (A) rope, (B) boat seat cover (marine fabric), and (C)

Tyvek sleeve	10

Figure 11. Solid waste material swatches (A) boat seat (marine fabric) and (B) 1/4-inch

double-braided nylon rope in a 50-mL conical tube with 25 ml_ of buffer	11

Figure 12. Solid waste material swatches (A) boat seat (marine fabric), (B) nitrile glove,
and (C) 1/4-inch double-braided nylon rope in a 400-mL Stomacher bag conical

tube with 90 ml_ of PBSTE	12

Figure 13. Solid waste material (A) Tyvek sleeve, (B) Tychem sleeve, (C) nitrile glove, and
(D) 1-inch double-braided nylon rope in a 1-L Nalgene bottle containing 500 ml_

PBST	13

Figure 14. Process flow chart depicting key process steps in chronological order	15

Figure 15. (A) Manifold containing 16 filter vials, (B) capping tray, and (C) capped filter

vials containing BHIB	18

Figure 16. Recovery of presumptive BaS spores from 1/4-inch diameter double-braided

nylon rope using the 50-mL conical tube spore recovery method	23

Figure 17. Recovery of presumptive BaS spores from boat seat covers (marine fabric)

using the 50-mL conical tube spore recovery method	24

Figure 18. RV-PCR detection of BaS spores from 1/4-inch diameter double-braided nylon

rope using the 50-mL conical tube spore recovery method	26

Figure 19. RV-PCR detection of BaS spores from boat seat cover (marine fabric) using the

50-mL conical tube spore recovery method	27

Figure 20. Recovery of presumptive BaS spores from 1/4-inch diameter double-braided

nylon rope using the 400-mL Stomacher bag spore recovery method	28

Figure 21. Recovery of presumptive BaS spores from nitrile glove using the 400-mL

Stomacher bag spore recovery method	29

Figure 22. Recovery of presumptive BaS spores from boat seat cover (marine fabric) using

the 400-mL Stomacher bag spore recovery method	29

Figure 23. RV-PCR detection of BaS spores from 1/4-inch diameter double-braided nylon

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rope using the 400-mL Stomacher bag spore recovery method	32

Figure 24. RV-PCR detection of BaS spores from nitrile gloves using the 400-mL

Stomacher bag spore recovery method	32

Figure 25. RV-PCR detection of BaS spores from boat seat cover (marine fabric) using the

400-mL Stomacher bag spore recovery method	33

Figure 26. Recovery of presumptive BaS spores from Tyvek sleeve using the 1-L bottle

spore recovery method	35

Figure 27. Recovery of presumptive BaS spores from Tychem sleeve using the 1-L bottle

spore recovery method	35

Figure 28. Recovery of presumptive BaS spores from nitrile glove using the 1-L bottle

spore recovery method	36

Figure 29. Recovery of presumptive BaS spores from 1-inch diameter double-braided

nylon rope using the 1-L bottle spore recovery method	36

Figure 30. RV-PCR response to BaS spores from Tyvek sleeve using the 1-L bottle spore

recovery method	40

Figure 31. RV-PCR response to BaS spores from Tychem sleeve using the 1-L bottle

spore recovery method	40

Figure 32. RV-PCR response to BaS spores from nitrile glove using the 1-L bottle spore

recovery method	41

Figure 33. RV-PCR response to BaS spores from 1-inch double-braided nylon rope using

the 1-L bottle spore recovery method	41

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Table of Tables

Page

Table 1. Test Matrix for Solid Waste Materials and Extraction Vessel	4

Table 2. Test Matrix for Solid Waste Materials and Spore Recovery Method	14

Table 3. Presumptive BaS Spore Recovery from Solid Waste Materials Using the 50-mL

Conical Tube Spore Recovery Method and SBA Media Culture Analysis Method .... 23
Table 4. Summary of the Accuracy of Identification of Presumptive BaS Colonies by PCR
Confirmation from Solid Waste Materials of Double-Braided Nylon Rope and Boat

Seat Cover	25

Table 5. BaS Spores Detected from Solid Waste Materials Using the 50-mL Conical Tube

Spore Recovery Method and RV-PCR Analysis Method	26

Table 6. Presumptive BaS Spore Recovery from Solid Waste Materials Using the 400-mL

Stomacher Bag Spore Recovery Method and SBA Media Culture Analysis Method. 28
Table 7. Summary of the Accuracy of Identification of Presumptive BaS Colonies by PCR
Confirmation from Solid Waste Materials of Double-Braided Nylon Rope, Nitrile

Gloves, and Boat Seat Cover	30

Table 8. BaS Spores Detected from Solid Waste Materials Using the 400-mL Stomacher

Bag Spore Recovery Method and RV-PCR Analysis Method	31

Table 9. Presumptive BaS Spore Recovery from Solid Waste Materials Using the 1-L

Bottle Spore Recovery Method and SBA Media Culture Analysis	34

Table 10. Summary of the Accuracy of Identification of Presumptive BaS Colonies by PCR
Confirmation from Solid Waste Materials of Tychem Sleeve, Tyvek Sleeve, Nitrile

Gloves, and 1-Inch diameter Double-Braided Nylon Rope	38

Table 11. BaS Spores Detected from Solid Waste Materials Using the 1-L Bottle Spore

Recovery Method and RV-PCR Analysis Method	39

Table 12. Analytical Method Comparison Displaying Culture Presumptive Percent

Recovery at the 300 CFU BaS Spore Load Level	42

Table 13. Performance Evaluation Audits	45

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Acronyms and Abbreviations

AnCOR

Analysis for Coastal Operational Resiliency

B. anthracis

Bacillus anthracis

BaS

Bacillus anthracis Sterne

BHIB

Brain Heart Infusion Broth

CESER

Center for Environmental Solutions and Emergency Response (EPA)

CFU

colony forming unit(s)

Ct

cycle threshold

ACt

change in (or Delta) cycle threshold

dH20

distilled water

EPA

United States Environmental Protection Agency

HSMMD

Homeland Security and Material Management Division (EPA)

HSPD

Homeland Security Presidential Directive

HSRP

Homeland Security Research Program (EPA)

IT

interagency team

jiL

microliter(s)

(im

micrometer(s)

ModG

modified G medium

NTC

no template control

ORD

Office of Research and Development (EPA)

PBS

phosphate buffered saline

PBST

phosphate buffered saline with 0.05% Tween 20

PBSTE

phosphate buffered saline with 0.05% Tween 20 and 30% ethanol

PCR

polymerase chain reaction

PE

performance evaluation

Pg

picogram

PMP

paramagnetic particle

PPE

personal protective equipment

QA

quality assurance

QAPP

quality assurance project plan

QC

quality control

QMP

quality management plan

qPCR

quantitative PCR

rcf

relative centrifugal force

rpm

revolution(s) per minute

RV-PCR

rapid viability PCR

SBA

Trypticase Soy Agar with 5% Sheep's Blood

SOP

standard operating procedure

STREAMS

Scientific, Testing, Research, and Modeling, Support

TSA

technical systems audit

USCG

United States Coast Guard

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Acknowledgments

This document was developed by the EPA's Homeland Security Research Program (HSRP) within
EPA's Office of Research and Development (ORD). Drs. Paul Lemieux and Worth Calfee were the
project leads. Contributions of the following individuals and organizations to the development of this
document are acknowledged.

United States Environmental Protection Agency

Dr. Sara Taft, Center for Environmental Solutions and Emergency Response

Dr. Shannon Serre, Office of Land and Emergency Management

Mr. Leroy Mickelsen, Office of Land and Emergency Management

Ms. Erin Silvestri, Center for Environmental Solutions and Emergency Response

Mr. John Archer, Center for Environmental Solutions and Emergency Response

Ms. Katrina McConkey, Booz-Allen Hamilton

Mr. Stuart Willison, Center for Environmental Solutions and Emergency Response
Mr. Neil Norrell, Region 2

Ms. Emily Snyder, Center for Environmental Solutions and Emergency Response
Ms. Kim Kirkland, Office of Land and Emergency Management
Mr. Jason Musante, Region 9

United States Coast Guard

Mr. Edward J. Primeau
Mr. Emile Benard
LT. Omar Borges
CDR. Benjamin Perman
LCDR. Clifton Graham

Battelle Memorial Institute

Mr. Ken Connelly
Mr. Dave Albertson
Ms. Hiba Shamma
Mr. Anthony Smith
Ms. Lindsay Catlin
Mr. Nate Poland
Mr. Zachary Willenberg

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Executive Summary

The United States Environmental Protection Agency (EPA) is charged by Congress with protecting the
Nation's land, air, and water resources. EPA is designated as a coordinating agency, under the National
Response Framework, to prepare for, respond to, and recover from a threat to public health, welfare, or
the environment caused by hazardous materials incidents. Hazardous materials include chemical,
biological, and radiological substances, whether accidentally or intentionally released. The anthrax
incidents in 2001 highlighted the need to improve the United States Government's response to terrorist
attacks. Congress passed the Public Health Security and Bioterrorism Preparedness and Response Act
(Bioterrorism Act) in 2002, and the Office of the President issued a series of Homeland Security
Presidential Directives (HSPDs) to specify the responsibilities of federal agencies. The EPA's role and
responsibilities related to homeland security are protecting human health and the environment from
bioterrorism.

EPA and United States Coast Guard (USCG) have formed an interagency team (IT) to support research
under the Analysis for Coastal Operational Resiliency (AnCOR) program for which this study is a part.

This study evaluated spore recovery and analytical methods for the detection of Bacillus anthracis
Sterne (BaS) spores that could be applicable to biologically contaminated solid waste samples for
various types of solid waste streams. Three spore recovery methods were based on established EPA
methods utilizing 50-milliliter (mL) conical tubes, 400-mL Stomacher bags, or 1-liter (L) bottles,
depending on the material type and size. Five (5) distinct, solid waste materials were selected in
coordination with EPA to use as representative, potential solid waste streams to assess in this study. The
solid waste streams comprised mooring lines (rope), personal protective equipment (PPE) items (i.e.,
nitrile gloves, Tyvek and Tychem sleeves), and boat seats representative of marine fabric materials.
Analytical methods consisted of culture and rapid viability-polymerase chain reaction (RV-PCR)
analysis.

As few as six BaS spores applied to common solid waste materials (Tychem sleeve, Tyvek sleeve,
mooring line (rope), nitrile gloves, and marine fabric found on boat seats) can be recovered using the
applied recovery method (50-mL conical tube, Stomacher, or 1-L bottle) and detected using culture and
RV-PCR analytical methods. Overall, there were only three sample swatches that were negative for both
culture and RV-PCR analytical methods: these three samples were Tychem sleeves spiked with six BaS
spores and recovered using the 1-L bottle spore recovery method.

Solid waste materials used in this study were new materials that had not been used in the field or been
through a decontamination procedure that might be implemented in the field. Therefore, these materials
had relatively low levels of background microorganisms present to interfere with detection or potential
assay inhibitors that might be introduced during decontamination. It is recommended to evaluate these
methods using solid waste materials that have been worn in the field or processed using a field
decontamination procedure.

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1.0 INTRODUCTION

Following a bioterrorism attack, materials contaminated with biological agent pose significant health
threats. The EPA's Homeland Security and Material Management Division (HSMMD) within the Center
for Environmental Solutions and Emergency Response (CESER) conducts research to develop methods
and technologies able to rapidly and cost-effectively remediate areas affected by a bioterrorism attack.
The research that CESER performs addressing these topics falls under EPA's Homeland Security
Research Program (HSRP). Waste management is an integral part of the remediation process and must
be included as a specific function during response planning. Sampling and analysis of waste generated
during remediation needs to be performed to meet applicable waste acceptance criteria as determined by
state regulatory decision-makers and receiving facilities, to determine whether the waste requires
treatment and, if so, has been adequately treated to allow for transportation as conventional solid waste.
Currently, there is no federal regulatory framework for management of biocontaminated waste, so each
state or military installation will have specific requirements specified by their individual regulations.
Regardless of whether the regulations specify sampling requirements to meet applicable waste
acceptance criteria, response personnel will need waste sampling strategies and methods to meet any
regulations and instill confidence for the waste treatment/disposal facilities that the remediation wastes
can be safely accepted.

Methods for spore recovery of biological contaminants in solid waste were evaluated for their
application to different solid waste streams that would be expected to be present from recovery after a
biological contamination incident at a United States Coast Guard (USCG) station. The performance of
the methods could, in part, depend on the solid waste streams being sampled and analyzed. USCG bases
and ports are preparing for waste management needs during possible future contamination incident(s).
By nature of their mission and location, they might have unique solid waste streams that could affect
sampling and analysis methods or provide an opportunity to test sampling and analytical methods
selected in previous EPA studies (US EPA, 2013; Serre and Oudejans, 2017).

This study evaluated spore recovery methods that could be applicable to biologically contaminated solid
waste samples for various types of solid waste streams. Multiple surface types were prepared to
represent the solid waste streams from USCG facilities. These surface types were selected by the
research team based on likely materials that would be managed as waste either before or after
decontamination. Bacillus anthracis Sterne (BaS) spores were applied to select solid waste stream
materials and were recovered using select, established EPA spore recovery (extraction) methods that are
applied to other solid materials. The recovered spores were analyzed using established EPA methods of
culture and rapid viability-polymerase chain reaction (RV-PCR) to determine sample collection
efficiency or confirm presence of BaS spores as biological contaminants. This study characterized
effectiveness of procedures for waste sample processing strategies and methods for wastes generated
during a wide-area outdoor incident typical of USCG facilities.

The USCG bases and ports have unique material that, if biologically contaminated, would need to be
sampled before disposal. EPA and USCG have formed an interagency team (IT) to support research
under the Analysis for Coastal Operational Resiliency (AnCOR) program, for which this study is a part,
to address sampling of solid materials for Bacillus spore contamination before being managed as waste.
This study will help the USCG recover rapidly following a biological contamination incident and return
assets to duty. The outcome of the study described in this report will provide data and information that
can be used to inform asset contamination status necessary to make decisions regarding proper waste
management impacting a USCG base. Ultimately, it is desired that these findings will facilitate recovery
following a large-scale biological incident.

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1.1 Objective

The objective of this study was to gather and generate data useful for EPA, USCG decision-makers, and
waste management personnel regarding Bacillus sampling method and analytical method performance
when applied to representative solid waste streams associated with USCG stations. Methods to sample
(recover) spores from USCG-relevant solid waste streams contaminated with BaS spores in the
laboratory were assessed, along with established EPA analytical methods.

The findings can be used to better plan and execute the recovery of a USCG base, or any large outdoor
urban area, following a biological contamination incident. Specifically, the study evaluated the
performance and limitations of traditional and innovative sampling and analysis methods, as well as
sample collection methods that leverage existing USCG maintenance procedures, when applied to
USCG assets and bases.

1.2 Scope

The work described in this report evaluated spiking of selected solid waste streams with Bacillus spores,
sampling the Bacillus spores from those materials, and analyzing the extract using established EPA
analytical methods. The solid waste streams were representative of those that would be present at USCG
bases or ports and thus potentially encountered in a wide-area contamination incident involving a USCG
base. The items would not be decontaminated for salvage, but rather decontaminated prior to disposal at
landfills, hopefully as conventional solid waste. Those representative solid waste streams were selected
in coordination with the EPA and were: nylon rope (line), personal protective equipment (PPE) items
(i.e., gloves, Tyvek, and Tyvek sleeves that would be used during decontamination of the affected site),
and boat seats representative of marine fabric materials. The solid waste materials were inoculated with
BaS at four target levels (quantities) of spores and allowed to dry before the spore recovery method was
applied. The selected waste materials were subjected to different sample processing (spore recovery)
methods to allow comparison of performance across methods. Different spore recovery methods were
used for solid waste materials so that more than one option would be available to implement in an actual
USCG base recovery operation. Spore recovery methods used were based on established EPA spore
recovery methods applied to other solid materials, such as filtration media. This research did not seek to
develop new spore recovery methods, but rather apply existing methods. Those methods varied in waste
material type, scale, and quantity sampled. The spore recovery methods employed either a 50-milliliter
(mL) conical tube, 400-mL Stomacher bag, or a 1-liter (L) bottle, depending on the material type and
size. All relied on adding an extraction fluid to the container after the selected waste material was added
and physical agitation per established EPA procedures. Recovered spores were identified and quantified
using established EPA analytical methods for culture and molecular (RV-PCR) analyses.

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2.0 MATERIALS AND METHODS

The materials and methods section of this report includes information on the solid waste material
selection, description, preparation, and spore recovery methods. It also includes specific details on the
test matrix, microbiological methods, and data reduction analyses. The materials that were selected for
the study are intended to reflect some of the high-volume waste materials likely to be generated as waste
from cleanup of USCG assets.

2.1 Solid Waste Material Selection

EPA and Battelle staff toured three USCG stations in August 2019: 1) Chincoteague Station; 2) Curtis
Bay Station/Coast Guard Yard; and 3) Portsmouth Station to identify potential solid waste streams to
consider for the study. These three stations represented a range of sizes, missions, and operations that
were expected to influence the types and quantities of solid waste generated. Those visits resulted in
EPA and Battelle categorizing the waste types into: 1) vessel waste (e.g., seat material, mooring lines,
vessel surfaces); 2) indoor building waste (e.g., barrack furniture, ceiling tiles, carpet, vinyl, electronics);
and 3) outdoor waste (e.g., vegetation, roofing, siding, vehicles, industrial equipment) associated with
items present on the grounds. Following those trips, discussions were held with the EPA project team
about the best way to down-select from the extremely large list of possible solid waste materials to a
number of solid waste material types that would be reasonable for this project. The EPA project team
performed a prioritization survey where mooring lines (rope), decontamination PPE, and vessel seat
material were determined as the highest priorities.

Prior discussion had also included consideration of several types of spore recovery methods (wipes, air,
extractive). Subsequently, the project team recognized the value of evaluating methodologies that could
apply to various sample sizes to provide data that indicate what methods might be options for analysis in
the future and are amenable to field operations. It was decided to use an extractive method only for the
recovery of spores from the solid waste materials, varying in extraction vessel size (and thus extraction
liquid volume) and type. Purposely, the three extraction methods selected were established EPA spore
recovery methods applied to other solid materials such as gravel or filtration media and thus no new
method development was required. Each of the three extraction methods differed by the vessel type and
size, volume of extraction liquid, and liquid agitation method in the vessel.

It is highly likely that waste materials would be bagged at the contamination site and moved to a
separate waste staging location to facilitate recovery operations without waste management activities
affecting the overall critical path to reopening the contaminated areas for reuse. Therefore,
considerations were also made as to the operational practicality of procuring the samples from
previously sealed bags while minimizing potential cross-contamination of the waste staging area and
reducing potential personnel exposures to contaminants present in the waste. The test matrix is shown in
Table 1.

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Table X* Test Matrix for Solid Waste Materials and Extraction Vessel

Extraction Vessel

Solid Waste Type

Waste Item Description

1-L Bottle

Tyvek® coverall sleeve

Tyvek 400

1-L Bottle

Tychem' coverall sleeve

Tychem 6000

50-mL Conical Tube

Line (small)

1/4-inch diameter double-
braided nylon rope

400-mL Stomacher Bag

Line (medium)

1/4-inch diameter double-
braided nylon rope

1-L Bottle

Line (large)

1-inch diameter double-braided
nylon rope

400-mL Stomacher Bag

Glove(s)

Standard nitrile gloves

1-L Bottle

50-mL Conical Tube

Marine fabric

Bycast65 Black Matte Correct-
Grain Faux Leather Marine
Vinyl Fabric

400-mL Stomacher Bag

Representative images of the materi als as they exist on a USCG station are depicted in Figure 1 (PPE),
Figure 2 (line), and Figure 3 (marine fabric/boat seats).

Figure 1. Representative image of PPE worn during a decontamination operation.

Note: Includes gloves and Tyvek or Tychem protective suit material worn and potentially exposed to biological
contamination.

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Figure 3. Photograph of a typical boat seat on a small or medium response boat.

2.2 Solid Waste Material Description

The solid waste streams were representative of those that would be present at most USCG bases or ports
and thus potentially encountered in a wide-area contamination incident involving a USCG base and their
assets. The waste items would likely be decontaminated prior to possible secondary treatment and
subsequent disposal at landfills, hopefully as conventional solid waste, although recycling and reuse
remain as potential waste minimization options. Those representative solid waste streams were selected
in coordination with the EPA. Five (5) distinct, solid materials were selected in coordination with EPA
to use as representative, potential solid waste streams to assess in this study. The solid waste streams
comprised mooring lines (rope), PPE items (i.e., nitrile gloves, Tyvek and Tychem sleeves), and boat
seats representative of marine fabric materials.

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2.2.1 Personal Protective Equipment - Nitrile Gloves

Nitrile gloves (Part No. 19-130-1597D, Fisher Scientific, Hampton, NH) are a commonly worn type of
glove for handling potentially hazardous materials. Other glove materials might be encountered such as
butyl rubber or latex, depending on the application. For the purposes of this study, it was agreed to use
nitrile gloves, recognizing that there could be differences in physical recoveiy of spores from different
material surfaces. A photograph of the nitrile glove is shown in Figure 4.

Figure 4. Photograph of nitrile gloves used as a solid waste stream test material.

2.2.2 Personal Protective Equipment - Tyvek or Tychem Sleeve

Full body protection during decontamination operations typically includes the use of Tyvek or Tychem
protective garments. For purposes of this study, both materials were used. For Tyvek (Part No.
TY500SWH00020000, DuPont, Wilmington, DE), the entire sleeve was used and for Tychem (Part No.
TF145TGYLG000600, DuPont, Wilmington, DE), an equivalent sleeve length (18 inches) was excised
because it was anticipated that the physical recovery of Tyvek or Tychem sleeve material from a solid
waste stream would easily be accessed from collected solid waste, which would allow for a consistent
and reproducible means of collecting a physical sample for analysis that would be readily implemented
in the field. If smaller samples of material are desired, they can be cut from the removed sleeve. A
photograph of a Tychem sleeve and Tyvek sleeve are shown in Figure 5.

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Figure 5. Photograph of (A) Tychem and (B) Tyvek sleeve used as a solid waste stream test material.
2.2.3 Double-Braided Nylon Rope

Review of USCG information pertaining to boats and ships, combined with discussions with USCG
personnel, indicated that double-braided nylon was the predominate material of construction for
mooring lines (rope). It was evident from the tour of USCG stations that a wide range of rope diameters
would and could be present, ranging from 1/4-inch diameter to well over 1-inch diameter. Considering
the organism extraction methods implemented for this study, an upper size limit of about 1-inch
diameter was determined for the study. Rope was a test material for all three recovery methods used in
the study with diameters of 1/4-inch (Part No. 0089, Knot & Rope Supply, Perrysburg, OH) and 1-inch
(Part No. 0094, Knot & Rope Supply, Perrysburg, OH). The sample length was also recovery-method-
dependent. Photographs of varying rope diameters are shown in Figure 6. For rope with diameters
greater than 1 inch as specified in the spore recovery methods evaluated in this study, an alternate
approach (basically a larger extraction vessel and quantity of extraction solution) would need to be
developed.

i s iiS

Figure 6. Photograph of double-braided nylon rope (A) 1/4-inch and (B) 1-inch diameter used as a solid
waste stream test material.

2.2.4 Marine Fabric - Faux Leather Boat Seats

Faux leather boat seats were evaluated using the 50-mL conical tube and Stomacher spore recovery
methods. Swatches of marine fabric (Part No. Bycast65 Black Matte Correct-Grain Faux Leather Marine
Vinyl Fabric, VViViD, St-Laurent, Quebec) were cut from the fabric shown in Figure 7.

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Figure 7. Photograph of faux leather boat seat (marine fabric) used as a solid waste stream test material.
2.3 Solid Waste Material Preparation

All solid waste stream materials purchased were used "as-received" without any cleaning or
decontaminating.

2.3.1 Solid Waste Sample Swatch Preparation

Consideration was given to how the solid waste materials would be obtained in a field operation in the
preparation of sample swatches for use in this study. Tyvek disposable sleeves were used in their
entirety. The Tychem sleeve, however, was obtained by cutting from an entire protective suit of
Tychem, by measuring 18 inches from the wrist cuff. Nitrile gloves were used whole. The mooring line
(nylon rope) was cut to desired length using a hot knife (Part No. 122177, Sailrite, Columbia City, EST).
A hot knife was used to minimize generation of aerosol and droplets during the cutting of the material
from a larger sample. Although the field operators collecting the line sample would be wearing PPE, to
include respiratory protection, cutting rope with a blade or saw would be expected to generate more
droplets and aerosols potentially containing hazardous materials. The use of a hot knife to cut also
reduced the chance of rope fraying during the physical agitation associated with the extraction method,
which was important to minimize extraneous inert debris from the analytical method that could clog
filtration media. Potential drawbacks to using the hot knife are that a solid cutting surface is needed, the
melting rope produces smoke and potential inhalation/exposure to hazardous material, contact with
combustible or flammable material needs to be avoided, and laceration or burns could result from the
hot blade. The boat seat cover material was cut with scissors in a manner anticipated to be used in the
field. Static illustrations of swatches of the various materials being prepared are provided in Figure 8. It
is expected that a hot-knife or scissors would also be an appropriate method for gathering samples from
other large pieces of waste material that were outside the scope of these tests.

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A

g i

C

/









Figure 8. Photographs of cutting of bulk solid waste material (A) Tychem sleeve, (B) double-braided nylon
rope, and (C) boat seat covers to obtain sample swatches for analysis.

2.3.2	Spore Stock

BaS spores were used as the biological test agent for the entire study. This organism is a vaccine strain
produced by Colorado Serum Company and is frequently used as a surrogate to fully virulent B.
cmthracis strains such as Ames. The BaS strain was handled as a Risk Group II agent following the
Biosafety in Microbiological and Biomedical Laboratories guidelines and Battelle biosafety work
practices for such agents. A spore bank was produced using sporulation broth as follows and used as
needed for the duration of the study.

A cell bank of BaS 34F2 prepared previously at Battelle (BEI N R-1400; BEI Resources, Manassas, VA)
was streaked for isolation on Trypticase Soy Agar with 5% Sheep's Blood (SBA, Part No. B21261X,
Fisher Scientific, Hampton, NH), then incubated overnight at 36 ± 2 degrees Celsius (°C). An isolated
colony was then used to inoculate a 50-mL aliquot of nutrient broth and incubated overnight at 36 ± 2°C
with shaking at 200 revolutions per minute (rpm). Modified G (ModG) (500 mL) of sporulation broth
(Appendix A, Table 1) was inoculated with 50 mL of the overnight BaS culture, and then incubated in a
3-L Fernbach flask at 36 ± 2°C with shaking at 200 rpm. The culture was observed via wet mount
microscopy every one to three days for sporulation. Following five days of incubation, the ModG
culture reached > 99% sporulation.

The sporulated culture was centrifuged at 10,000 relative centrifugal force (rcf) for 12 minutes (min) at
4°C in multiple 250-mL bottles. After removing and discarding the supernatant, the resulting pellets
were resuspended to a total volume of approximately 100 mL with sterile distilled water (dLkO),
transferred into a sterile glass vessel, and heat shocked at 60 to 65°C for 1 hour (hr) in a water bath with
gentle agitation. (Note: A control flask with a thermometer was used to ensure the desired temperature
was achieved and maintained during the heat-shock step). The spores were then washed twice by
repeated centrifugations at 10,000 rcf for 12 min at 4°C using 100 mL dFLO per wash. After the final
centrifugation, the spores were resuspended to a total volume of 100 mL in sterile dFkO yielding a titer
of-1.5 x 10lu colony forming units (CFU)/mL. That spore stock was then subsequently diluted as
needed by the project. The spore bank was assigned a unique lot number and stored refrigerated at 2 to
8°C.

2.3.3	BaS Application (Spiking)

On the day of sample processing, BaS spores were vortex-mixed and diluted using phosphate buffered
saline with 0.05% Tween 20 (PBST, Part No. NC0847251, Fisher Scientific, Hampton, NH) and used to
directly spike the sample swatches with a defined quantity of BaS spores (6, 60, 600, or 6,000 CFUs).
Spores were applied to swatches using 20 5-microliter (jiL) droplets within at 4-centimeter (cm) x 4-cm
grid of four rows with five droplets for Tyvek (near wrist cuff), Tychem (near wrist cuff), nitrile glove

9

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(on palm), and marine fabric. For the mooring line, the 20 droplets were distributed evenly across the
length of double-braided nylon rope. Photographs of BaS spore spiking of solid waste materials are
depicted in Figure 9 for the nitrile gloves and Figure 10 for the line, boat seat cover, and Tyvek sleeve.

All spiked materials were allowed to dry at ambient laboratory temperature and humidity, and once
visibly diy, they were processed using their associated sample recovery method. Prior to spore recovery
of nitrile gloves, the spiked glove was donned and doffed (inside-out) prior to placing the glove into the
spore recovery container (Figure 9B and Figure 9C, respectively). The spiking procedure is further
outlined in Appendix B, "Work Instaiction for Spiking with Bacillus anthracis Sterne Spores."

Figure 9. Spiking of nitrile gloves with suspension of BaS (A) spiking palm of nitrile glove, (B) spiked nitrile
glove with dried BaS droplets donned, and (C) doffing of spiked nitrile glove.

Figure 10. Spiking of swatches of (A) rope, (B) boat seat cover (marine fabric), and (C) Tyvek sleeve.
2.4 Spore Recovery Methods from Solid Waste Materials

Three (3) spore recovery methods were selected to recover deposited BaS spores from the solid waste
material: 50-mL conical tube with vortex agitation (small mooring line and marine fabric); 400-mL
Stomacher bags (Part No. 14-285-20, Fisher Scientific, Flampton, NFI) for stomaching (medium
mooring line, gloves, and marine fabric); and 1-L bottles for manual shaking (large mooring line, Tyvek
and Tychem sleeves, and gloves). The spore recovery methods are based on existing EPA methods and
were thus selected for use because development of new methods was not desired within the scope of this
project. The three different methods provided a range of scale (50-mL to 1-L) to be used with a range of
sample sizes. Different solid waste streams will potentially yield different sizes of samples to be
analyzed. The spore recovery methods were summarized as work instaictions for the laboratory staff to

10

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execute (Appendix C). (Note, spore recovery methods do not pertain to collecting the sample of solid
waste from the waste stream, only the spore extraction process.)

2.4.1 50-mL Conical Tube Spore Recovery Method

Spores were recovered by adding 5-cm x 5-cm swatches of marine fabric or 1/4-inch double-braided
nylon rope (5-cm length) into a 50-mL conical tube. The marine fabric swatch was positioned in the
bottom half of the conical tube with the spike side facing away from the tube wall with a 2-inch x 2-inch
mesh support (Part No. 9218T13, McMaster Carr Supply Company, Douglasville, GA) covering the
spiked side to hold the swatch in place. Then an initial buffer volume of 15 mL cold (2 to 8°C)
phosphate buffered saline with 0.05% Tween 20 and 30% ethanol (PBSTE) was added to each tube and
vortex-mixed on a platform vortex for 20 min with speed set to seven. The PBSTE was transferred into a
new 50-mL conical tube for pooling the initial and second buffers used for spore recovery. Ten
milliliters (10 mL) of the second buffer (phosphate buffered saline with 30% ethanol was added to each
swatch and vortex-mixed on a platform vortex for 10 min with speed set to seven. The suspension was
pooled with the initial buffer and vortex-mixed for 10 seconds (sec). This 25-mL pooled volume was
vortex-mixed, allowed ~2 min of settling time, and then split in half for culture-based analysis described
in Section 2.7.1, and RV-PCR analysis as described in Section 2.7.2. Solid waste samples of the marine
fabric and 1/4-inch double-braided nylon rope are shown in Figure 11. This method was adapted from
the wipe and air filter sample spore recovery method described in "U.S. EPA Protocol for Detection of
Bacillus anthracis in Environmental Samples During the Remediation Phase of an Anthrax Incident,
Second Edition" (EPA, 2017).

Figure 11. Solid waste material swatches (A) boat seat (marine fabric) and (B) 1/4-inch double-braided
nylon rope in a 50-mL conical tube with 25 mL of buffer.

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2.4.2 400-mL Stomacher Bag Spore Recovery Method

Spores were recovered from inoculated waste material swatches by adding a 15-cm x 15-cm swatch of
marine fabric, doffed nitrile glove, or 15-cm length of 1/4-inch double-braided nylon rope to a
Stomacher bag containing 90 mL cold (2 to 8°C) PBSTE, then homogenized for 1 min at 260 rpm in a
Stomacher 400 (Seward; Bohemia, NY). Each sample then sat for 10 min to allow foam to settle before
removing the sample swatch. Absorbed liquid was expelled from the swatch into the Stomacher bag and
the swatch was removed. The suspension (-90 mL) was gently mixed by pipetting up and down three
times with a steril e 50-mL pipet, then the suspension was split in half in two 50-mL sterile conical tubes
and centrifuged at 3,500 rcf for 15 min in a swinging bucket rotor at 4°C with the brake off. To
concentrate the sample, -65 mL of supernatant was removed and the remaining -25 mL of supernatant
was used to suspend the pellets. The suspension was split in half and used for culture-based analysis
described in Section 2.7.1, and RV-PCR analysis as described in Section 2.7.2. Solid waste samples of
the 15-cm x 15-cm marine fabric, doffed nitrile glove, and 1/4-inch double-braided nylon rope are
shown in Figure 12. This method was adapted from the sponge-stick spore recovery method described in
"U.S. EPA Protocol for Detection of Bacillus anthracis in Environmental Samples During the
Remediation Phase of an Anthrax Incident, Second Edition" (EPA, 2017).

Figure 12. Solid waste material swatches (A) boat seat (marine fabric), (B) nitrile glove, and (C) 1/4-inch
double-braided nylon rope in a 400-mL Stomacher bag conical tube with 90 mL of PBSTE.

2.4.3 1-L Bottle Spore Recovery Method

Spores were recovered from inoculated waste material swatches by adding a Tyvek sleeve, Tychem
sleeve, one doffed nitrile glove, or 15-cm length of 1-inch double-braided nylon rope to a 1-L bottle
(Part No. 02-925-141, Fisher Scientific, Hampton, NH) containing 500 mL of sterile PBST. The
material was shaken vigorously with one hand on the bottom and the other on the top using an over the
shoulder back-and-forth motion for 2 min. The sample was allowed to settle for 30 sec and then the
recovery solution was either poured into a clean, sterile 500-mL container or directly concentrated onto
a filter funnel membrane. The recovery solution was mixed vigorously by hand for 30 sec, then the
liquid was poured into a 0.45-micrometer (urn) filter funnel (MicroFunnel Filter; Cat. 4804; Pall
Corporation, Washington, NY) to the 100-mL gradation line. If the volume passed through the filter
without becoming clogged, an additional 100-mL aliquot and 50-mL aliquot was added for a total of up
to 500 mL. If a 100-mL or 50-mL aliquot took longer than 10 min to pass through the filter, no further
volume was added. At 30-min post-sample addition, if the sample did not completely pass through, the
remaining volume in the filter unit was carefully removed. The total volume vacuum filtered was
documented. The filter membrane was then removed using sterile forceps and transferred to a 50-mL

-------
conical tube such that it was positioned in the bottom half of the tube with the inlet side of the
membrane facing the center of the tube. Ten milliliters (10 mL) of PBSTE was added and vortex-mixed
at maximum speed on a platform vortex with 10-sec bursts for 2 min to dislodge the spores. The
suspension in tubes was allowed to settle for 2 min, then transferred to a 50-mL conical tube. An
additional 10 mL of PBSTE was added to the 50-mL tube containing the membrane and vortexed as
described for the first 10 mL, then pooled with the first 10 mL for each sample. This 20-mL pooled
volume was vortex-mixed, allowed 30 sec of settling time, and then split in half for culture-based
analysis described in Section 2.7.1, and RV-PCR analysis as described in Section 2.7.2.

Solid waste samples of the Tyvek sleeve, Tychem sleeve, one doffed nitrile glove, and 15-cm length of
1-inch double-braided nylon rope in a 1-L bottle with 500 mL of PBST are shown in Figure 13. This
method was adapted from the established EPA method for sampling rail ballasts (Serre and Oudejans,
2017).

Figure 13. Solid waste material (A) Tyvek sleeve, (B) Tychem sleeve, (C) nitrile glove, and (D) 1-inch
double-braided nylon rope in a 1-L Nalgene bottle containing 500 mL PBST.

2.5 Test Matrix

The extract collected from each of the solid waste samples that were spiked with BaS and extracted as
described in Sections 2.4.1 through Section 2.4.3 were analyzed to quantify and identify recovered BaS
using EPA-developed culture and RV-PCR methods. The completed test matrix is provided in Table 2.
In total, 179 solid waste samples were analyzed (excluding any additional spike controls or laboratory
controls or blanks), comprising 38 samples extracted using the 50-mL conical tube method, 58 samples
analyzed using the 400-mL Stomacher bag method, and 83 samples analyzed using the 1-L bottle
method.

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Table 2. Test Matrix for Solid Waste Materials and Spore Recovery Method

Spore Recovery
Method

Material
(Material Code)

Solid Waste Material
Description

Target Spore Load
(CFU)1,1

Replicates 11)1

Total
Samples

50-mL Conical
Tube

Line - Small
(DBN1/4)

1/4-inch diameter double-
braided nylon rope; 5-cm long

0/6/60/600/6,000

3/3/5/5/3

19

Marine Fabric
(MFAB)

VViViD Bycast 65 Black Matte;
5-cm x 5-cm

0/6/60/600/6,000

3/3/5/5/3

19

400-mL Stomacher

Line - Medium
(DBN1/4)

1/4-inch diameter double-
braided nylon rope; 15-cm long

0/6/60/600/6,000

3/3/5/5/3

19

Glove
(GLV)

Standard nitrile gloves
(size Large)

0/6/60/600/6,000

3/3/5/5/4

20

Marine Fabric
(MFAB)

VViViD Bycast 65 Black Matte;
15-cm x 15-cm

0/6/60/600/6,000

3/3/5/5/3

19

1-L Bottle

Tyvek Sleeve
(TYVEK)

Tyvek 400, Part # TY122SWH

0/6/60/600/6,000

4/5/5/6/3

23

Tychem Coverall
Sleeve
(TYCHEM)

Tychem 6000, Part#
19141750

0/6/60/600/6,000

4/5/5/5/3

22

Glove
(GLV)

Standard nitrile gloves
(size Large)

0/6/60/600/6,000

3/3/5/5/3

19

Line - Large
(DBN1)

1-inch diameter double-
braided nylon rope; 15-cm long

0/6/60/600/6,000

3/3/5/5/3

19

00 Target spike levels were 6, 60, 600, and 6,000 BaS spores per material swatch.

(b) Nominal number of replicates was three (3) replicates for the 6 and 6,000 BaS spore target spike and five (5) replicates for
the 60 and 600 BaS spore spike level. However, some additional replicates were added for selected conditions to fill available
analysis sample sets.

2.6 Overall Method Implementation

The procedures used to spike/recover/analyze the solid waste materials (double-braided nylon rope,
Tyvek sleeve, Tychem sleeve, marine fabric, and nitrile gloves) are shown as they occur in
chronological order, as depicted graphically in the process flow diagram in Figure 14. The depicted
process flow chart describes the workflow followed for this project. Calendar time could be reduced for
both culture and RV-PCR methods. For culture, colonies from agar plates and Brain Heart Infusion
Broth (BHIB) enrichment culture could be PCR screened following overnight incubation for
confirmation of target. A note in the EPA protocol for a culture method allows for faster analysis of
samples by combining and concentrating the remaining spore suspension and remainder of all dilutions
from plating onto a MicroFunnel membrane filter and plate, instead of BHIB enrichment (EPA, 2017).
Additional analysis of BHIB enrichment of the swatch or filter by streaking turbid BHIB onto solid
media would require an extra day of incubation followed by PCR; however, there is not an equivalent
analysis for RV-PCR. RV-PCR only analyzes the whole spore recovery suspension. For RV-PCR, if the
enrichment time is reduced from 16 hrs (overnight) to 9 hrs, the DNA extraction could be performed by
a third-shift staff or automated method and PCR analysis could potentially be completed within 24 hrs.

14

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Spore Spike
and Spore
Recovery

Extracted
Swatch or

filter

Blood Agar,
Incubate
Overnight

BHIB

Enrichment

BHIB

Incubate
Overnight

BHIB,
Incubate 2
Days

DNA
Extraction

ft and t,}

Colony PCR

PGR
Analysis

Day 1
(Monday)

Day 2
(Tuesday)

Streak for
Isolation If
Colony PCR
Negative

Day 3

(Wednesday)

Select Presumptive BaS
Colonies and Prepare
BHIB Broth for PCR

Day 4
(Thursday)

Day 5

(Wednesday of
Following Week)

Figure 14. Process flow chart depicting key process steps in chronological order.

The methods implemented, in the form of work instructions followed by the analytical staff, are
provided in Appendices B through J. These work instructions also complement those microbiological
methods described in Section 2.7, and emphasize glove-changing schedules that were implemented to
minimize cross-contamination. Work instructions were reviewed, as needed, with the EPA project team
to ensure consistency with published methods.

The above method was used to analyze a batch of 16 samples, with one batch conducted per week. For
each weekly batch, non-spiked samples and spiked samples were processed. Samples were spiked with a
target spore load (6, 60, 600, or 6,000 CFU) per "Work Instruction for Spiking with Bacillus anthracis
Sterne Spores" in Appendix B. The collected samples were recovered following the "Work Instruction
for Bacillus anthracis Sterne Spore Recovery," as described in Appendix C. The recovered volume was
then split between the culture method and RV-PCR. The culture aliquot was plated onto SBA media and
incubated overnight as described in the "Work Instruction for Culture of Bacillus anthracis Sterne
Spores Recovered from Swatches" in Appendix D. The RV-PCR aliquot was processed as described in
the "Work Instruction for RV-PCR Processing of Bacillus anthracis" in Appendix E. The To RV-PCR
aliquot was stored frozen while the recovered spores enriched overnight, then the Tfinal aliquot was
removed, and the DNA was extracted from both To and Tfmai aliquots per "Work Instruction for Manual
DNA Extraction and Purification from Bacillus Species " in Appendix F. The extracted DNA was then
analyzed using the assay described in Section 2.7.2.4 and per "Work Instruction for Real-time PCR
Analysis of Bacillus anthracis" in Appendix G. Real-time PCR was also used to confirm or refute
presumptive BaS spores selected from the culture analysis per "Work Instruction for Selecting
Presumptive Bacillus anthracis Sterne Colonies for PCR Confirmation" in Appendix H. Selected
samples for which the culture was a non-detect were further analyzed using an enrichment procedure per
"Work Instruction for BHIB Enrichment for Culture" in Appendix I.

2.7 Microbiological Methods

Spores recovered from samples were processed using analytical methods (both a culture and RV-PCR
analytical method) described in the "U.S. EPA Protocol for Detection of Bacillus anthracis in
Environmental Samples During the Remediation Phase of an Anthrax Incident, Second Edition"
(EPA, 2017), with modification to spore recovery to allow for sample splitting. The following are

-------
sections that summarize specific procedures and steps applied to conduct the study.

2.7.1 BaS Culture Method

Culture-based microbiological analysis was performed on each sample by filtering the recovered extract
through MicroFunnel filters (Part No. 4804 Pall Corporation, Washington, NY) then placing the filters
onto SBA or spread-plating 0.1 mL of the recovered extract onto SBA. A modification to the "U.S. EPA
Protocol for Detection of Bacillus anthracis in Environmental Samples During the Remediation Phase
of an Anthrax Incident, Second Edition" (EPA, 2017) used in the current study was that sample analysis
proceeded directly to filter-plate of undiluted samples, rather than inclusion of spread plate analysis of
10"1 and 10"2 dilutions, since spore levels were expected to be low. Work instructions for the culture
method are detailed in Appendix D.

For MicroFunnel filter analysis, initially, each filter was pre-wetted with 5 mL of PBST, then 10 mL of
PBST was added to each MicroFunnel filter to suspend aliquots, milliliter volumes of the spore recovery
were applied, and vacuum filtered. The walls of each MicroFunnel filter were rinsed with 10 mL of
PBST and filtered through the MicroFunnel filter, then the filter membrane was removed and placed
onto SBA media.

Colonies with a typical BaS morphology following overnight incubation at 36 ± 2°C were counted to
determine percent spore recovery. Typical BaS morphology on SBA is 2 to 5 millimeters in diameter,
flat or slightly convex with edges that are irregular, are gamma hemolytic, and have a ground-glass
appearance.

Two different microbiologists enumerated colonies over the course of the project, all of whom were
trained by the lead microbiologist on the project to most consistently identify presumptive BaS based on
colony morphology. The lead microbiologist periodically reviewed the enumeration results to help
ensure consistency and integrity, which is an important consideration and factor in the application of the
method because the culture analysis was subjective to the assessment of colony morphology. Colonies
identified during culture analysis are reported as presumptive BaS.

A small subset of presumptive BaS colonies were screened using real-time PCR assay targeting the
chromosome and pXOl of BaS. A portion of a single colony was suspended in 100 |iL of PCR-grade
water, heated for 5 min at 95 ± 2°C, and centrifuged at 18,400 rcf for 2 min and the supernatant was
analyzed in triplicate. An average cycle threshold (Ct) value of < 40 was recorded as a positive result.
The work instruction for colony PCR is located in Appendix H.

For BHIB enrichment, the spores that remained on the sample or filter (for 1-L bottle samples) were
enriched following spore recovery within the 50-mL conical tube by adding 25 mL of BHIB, then
incubated at 36 ± 2°C for 24 to 48 hrs. Note, sample swatches of marine fabric (15-cm x 15-cm) that
were processed using the Stomacher method were not BHIB enriched because 25 mL BHIB would not
adequately cover a swatch of this size and a container larger than a 50-mL tube would need to be
consumed for this process. If BaS colony morphology was not observed on SBA plates from culture
analysis (spread plate or MicroFunnel Filters), turbid BHIB culture was then streaked onto three SBA
plates for isolation and incubated overnight at 36 ± 2°C. Colonies with BaS morphology that were
isolated on these streak plates were screened using a real-time PCR assay targeting the chromosome and
pXOl of BaS. If colonies with BaS morphology were not isolated on streak plates, an aliquot of the
BHIB culture suspension (50 |iL) was pelleted by centrifugation at 12,000 rcf for 2 min, supernatant was
discarded, and the pellet was suspended in 100 |iL of PCR-grade water. The suspended pellet was lysed

-------
at 95 ± 2°C for 5 min, then screened using the duplex real-time PCR assay. The work instruction for
BHIB enrichment is located in Appendix I.

2.7.2 RV-PCR Methods

The extracts collected from each of the solid waste samples that were spiked with BaS and extracted
were analyzed to quantify and identify recovered BaS using EPA-developed culture and RV-PCR
methods. This section discusses these methods in more detail.

2.7.2.1 Further Sample Processing for RV-PCR

Following filtration of half of the recovered spore suspension (12.5 mL for samples processed using
Stomacher and 50-mL tubes, 10 mL for samples processed using 1-L bottles) through the Whatman
Autovial filter vials (with polyvinylidene difluoride membrane; Part No. AV125NPUAQU, Whatman,
Marlborough, MA) set in a manifold, two buffer washes were performed according to "U.S. EPA
Protocol for Detection of Bacillus anthracis in Environmental Samples During the Remediation Phase
of an Anthrax Incident, Second Edition" (EPA, 2017). The first wash was 12.5 mL of cold (4°C) high
salt buffer (10X PBS) followed by 12.5 mL of cold (4°C) low salt wash buffer (IX PBS). The top
portion of the manifold was then removed and placed into a capping tray with pre-filled luer plugs
(Cat No. LPC14-PP0; Ark-Plas Products, Flippin, AR, or equivalent) to seal the filter vials. Five (5) mL
of cold (4°C) BHIB was then added to each filter vial, and the vials were capped and vortex-mixed for
10 min on a setting of seven. Images of the manifold and capping tray are depicted in Figure 15.
Following the vortex step, the broth was mixed by pipetting up and down -10 times and a 1-mL aliquot
was transferred to a screw cap tube and stored at -20°C as the time zero (To) aliquot. The capped filter
vials were then incubated overnight (-16 hrs, Tf) in an incubator shaker set to 36 ± 2°C at 230 rpm.

-------
* i * J Vj J 4

1 ^ jjp

' - ' J ~ W -

^ jB

A 4 ,

4 W «r «n

VI

A





B







^ 8 V f <5 4

CMr ^ W

i[ y 1y





s * ' *. *

(i -tf : v

c



Figure 15. (A) Manifold containing 16 filter vials, (B) capping tray, and (C) capped filter vials containing
BH1B.

Following overnight incubation (~16 hrs), filter vials were mixed on a platform vortex for 10 min with
speed set to seven. Note the "U.S. EPA Protocol for Detection of Bacillus anthracis in Environmental
Samples During Remediation Phase of an Anthrax Incident" specifies 9 hrs or longer for complex and
post-decontamination samples; however, the 16-hr incubation allowed for a standard work schedule to
be maintained rather than require an overnight shift that would have been required by a 9-hr incubation
(EPA, 2017). The culture suspension was mixed by pipetting up and down -10 times, and a 1-mL
aliquot was transferred to screw cap tubes and labeled as the final time ffjj aliquot (Appendix E).

2.7.2.2 DNA Extraction arid Purification

Prior to extraction of deoxyribonucleic acid (DNA), the lysis buffer with anti-foam reagent and the
alcohol wash was added according to the manufacturer's instructions in the Magnesil Blood Genomic,
Max Yield System Kit (Part No. MD1360, Promega, Madison, WI) and a heat block was pre-heated to
80°C. All screw-capped, 1-mL aliquots were thawed and centrifuged at 18,400 rcf for 10 min (4°C), and
800 |iL of the supernatant from each tube was removed and discarded. To extract the DNA, 800 lj. L of
lysis buffer was added to each tube and the samples were mixed by vortexing on high (-1,800 rpm) in
10-sec pulses for a total of 60 sec. Each tube was then vortex-mixed for 10 sec at low speed directly
before the lysate was transferred to a 2-mL labeled Eppendorf tube. The lysate tube was then incubated
at room temperature for 5 min. Uniformly resuspended paramagnetic particles (PMPs) (600 liL) were
added to each lysate tube and the samples were mixed by vortexing. After vortexing each To and Tfinai
tube for 10 sec (high, -1,800 rpm), the samples were incubated at room temperature for 5 min.

18

-------
The samples were then placed on the magnetic stand with the hinged-side of the tube facing toward the
magnet after briefly resuspending the particles by vortexing. The magnetic rack was then inverted to
ensure all PMPs were contacting the magnet. After 10 sec, the tubes were opened, and the liquid
removed without disturbing the PMPs. Lysis buffer (360 |iL) was then added to each To and Tfmaitube
and vortexed for 10 sec. The tubes were then placed on the magnetic stand and inverted again. The
supernatant was then removed and 360 |iL of salt wash solution was added to each tube. The tubes were
capped and vortexed for 10 sec, placed on the magnetic stand, and inverted. The supernatant was
removed without disturbing the PMP pellet. The pelleted PMPs were washed a second time with 360 |iL
of salt wash solution.

After removal of the second salt wash supernatant, 500 |iL of alcohol wash was added to each tube. The
tubes were vortexed for 10 sec, placed on the magnetic stand, and inverted. The supernatant was then
removed, and two more alcohol washes were conducted for a total of three 500-|iL alcohol washes. A
fourth alcohol wash was then conducted using 500 |iL of 70% ethanol. After the supernatant from the
70% ethanol wash was removed, all tubes were opened and allowed to air dry for 2 min. The open tubes
were then heated at 80°C in a heat block inside a biosafety cabinet until the PMPs were dry (-20 min).
DNA was then eluted from the PMPs by the addition of 200 |iL of elution buffer to each To and Tfmai
tube. The tubes were then closed, vortexed for 10 sec, and incubated in the heat block for 80 sec. The
tubes were then vortexed another 10 sec and incubated in the heating block for 1 min. The vortexing and
heating for 1 min was repeated four more times for a total of five times. The tubes were then removed
from the heating block and incubated at room temperature for at least 5 min. Each tube was briefly
vortexed and then centrifuged at 376 rcf at 4°C for 1 min. The tubes were then vortexed and placed on
the magnetic stand for at least 30 sec. The elute was collected (-80 to 90 |iL) and transferred to clean,
labeled, 1.5-mL tubes on a cold block. The tubes were centrifuged at 18,400 rcf at 4°C for 5 min to
pellet any particles remaining with the eluted DNA. The supernatant was carefully removed and
transferred to a new 1,5-mL tube using a new tip for each tube. The To and Tfmai DNA extracts were
stored at 4°C until RV-PCR analysis or at -20°C if RV-PCR could not be performed within 24 hrs. The
work instruction for DNA purification is Appendix F.

2.7.2.3 BaS DNA Preparation

Genomic DNA of BaS was extracted for use as a positive control for RV-PCR based analysis. The BaS
vegetative cell culture that DNA was extracted from originated from the spore stock used for spike/
recovery tests. The Wizard Genomic DNA Kit (Promega, Madison, WI) was used following an internal
Battelle method specific for extracting B. anthracis. The resulting DNA was quantified by Quant-iT
PicoGreen dsDNA Assay Kit (Part No. PI 1496, Invitrogen, Waltham, MA). The purified DNA was
assigned a unique lot number, dispensed as multiple aliquots, stored frozen at < -20°C, and used as
needed as the positive control for PCR analysis.

2.7.2.4 Real-Time PCR Assay

The real-time PCR assay used for detection of BaS was a duplex TaqMan real-time PCR assay that
utilized FAM and VIC reporter dyes for detection of two BaS DNA sequence targets simultaneously in a
single reaction. (Note: FAM and VIC are Applied Biosystems trademark fluorescent reporter dyes on 5'
end of PCR probe that emit at -517 nm and -551 nm, respectively.) The two assays target sequences on
the B. anthracis chromosome and pXOl plasmid and were previously described as singleplex real-time
PCR assays (Letant et al., 2011) and "U.S. EPA Protocol for Detection of Bacillus anthracis in
Environmental Samples During Remediation Phase of an Anthrax Incident" (EPA, 2017). The duplex
PCR assay Master Mix was prepared using the conditions provided in Table 2 of Appendix A. Each
sample DNA extract was assayed in triplicate reactions. Controls consisted of four positive control wells

-------
containing 50 picogram (pg) of DNA extracted from BaS 34F2 (NR-1400, BEI Resources) and four no
template controls (NTCs) were also included with each assay. Applied Biosystems 7500 Fast Real-Time
PCR Instrument was used for PCR assay development and testing. Thermocycler conditions with a fast
ramp rate were:

Stage 1: 1 cycle at 50°C for 2 min
Stage 2: 1 cycle at 95°C for 2 min

Stage 3: 45 cycles at 95°C for 3 sec followed by 60°C for 30 sec
The work instruction for real-time PCR is Appendix G.

2.8 Data Reduction and Analysis

This section discusses the data reduction of the results including the percent recovery calculations, the
RV-PCR results, and the presentation of the results.

2.8.1 Percent Recovery of Presumptive BaS Colonies

The percent recovery (Erecovery) of BaS spores from each spiked sample swatch was calculated by
dividing the number of presumptive BaS CFUs recovered (Nrecover) from the sample by the actual
number of BaS spores spiked (Nspike), as determined by stock suspension titer for each test, then
multiplied by 100. Nrecover is a product of the presumptive BaS spore concentration (Crecover) (CFU/mL)
and the total volume of extract used to recover the spores (Vextract) (mL). Mathematically, the percent
recovery is expressed as follows in Equation 1:

Erecaverym = f * 100% (1)

Further, the number of presumptive BaS spores present in the volume of recovered suspension plated
onto spread plates or via MicroFunnel filter membrane was divided by the suspension volume analyzed,
to yield a presumptive BaS spore concentration (Crecover) in CFU/mL. The recovered suspension volume
(Vextract) was used to determine BaS CFUs recovered from the sample. The percent recovery was
calculated for all volumes plated. The reported percent recovery was determined using the below rules:

1) Report the percent recovery from the aliquot that has between 20 to 80 CFU on MicroFunnel
Filter membranes.

2) Report the higher-volume aliquot percent recovery if the CFU counted from both aliquots is less
than 20.

3) Report the higher-volume aliquot percent recovery if the CFU counted from both aliquots is
between 20 to 80.

4) Report the lower-volume aliquot percent recovery if the background microbial flora on the high-
volume aliquot produces numerous colonies or a lawn of growth, thus complicating the
identification of BaS colonies.

5) Report the percent recovery from the spread plate that has between 25 and 250 CFU. Note, since
spike levels were at or near the method detection limit for samples processed in this study, 10"1
and 10"2 dilutions were not spread-plated as described in "U.S. EPA Protocol for Detection of
Bacillus anthracis in Environmental Samples During the Remediation Phase of an Anthrax

-------
Incident, Second Edition" (EPA, 2017).

The number of CFUs were reported as presumptive BaS colonies. PCR analysis of presumptive colonies
is required to positively confirm the presence of BaS. To perform this task, a portion of the presumptive
colony was collected into 100 |iL of PCR-grade water in microcentrifuge tubes. The colony suspension
was then heated for 5 min on a heat block at 95°C. The lysate was cooled and then centrifuged at
14,000 rpm (18,188 rcf) for 2 min and the supernatant was analyzed using the real-time PCR assays
targeting the chromosome and pXOl plasmid of BaS.

2.8.2 RV-PCR Method

Ct values for the To and Tfmai timepoints as well as the delta Ct value (ACt) were reported. The ACt is
generated by subtracting the average Ct (from triplicate reactions) generated by the Tfinal aliquot from the
average Ct (triplicate reactions) value generated by the To aliquot. A positive ACt value indicates that
viable BaS spores were detected in the sample if all the below acceptance criteria were met:

The ACt must be greater than or equal to 9 for both chromosome and pXOl BaS gene targets
(Equation 2):

ACt = Ct (To) - Ct (Tfi„ai) > 9 (2)

Additional criteria exist for the positive confirmation of a sample if analyzing samples obtained from an
actual incident, but for this study the above criterion was used.

2.8.3 Presentation of Results

The method employed to recover BaS spores was based on current EPA methods, as described in
Section 2.7. In the instance of an actual biological release, the entire extract would be analyzed either
using a culture method or an RV-PCR method. In the study performed and reported here, however, the
sample extract was split as described in Sections 2.7.1 and 2.7.2, so that approximately half of the
sample extract was used for culture analysis and the other half for RV-PCR analysis. Consequently,
neither the culture nor the RV-PCR method processed the total quantity of spores available in the extract
for analysis. Rather, each split sample extract had a maximum of nominally half the actual spiked spore
quantity available for their respective analyses. Therefore, in the presentation of results in tables and
figures, unless explicitly noted otherwise, column headers or axis labels denote the nominal maximum
number of recovered spores available in the sample for its respective analysis, which was half of the
target spore load.

-------
3.0 RESULTS AND DISCUSSION

Since recovered spore suspensions from each collected sample were split for the two analysis methods,
culture and RV-PCR, summary result tables indicate one-half the nominal target spore load and one-half
the determined spore load that was applied to the surfaces sampled, assuming 100% recovery efficiency.
This convention of presenting the results was considered the most accurate and consistent representation
and allowed for the most unambiguous discussion and interpretation of results across all the surface
types and analytical methods, recognizing that the surfaces were originally sprayed with target quantities
of BaS double the quantity that is represented in the tables and plots.

Note that the determined number of spores available for analysis represents the maximum number of
spores available and is half of the quantity of spores estimated to have been applied based on the
quantity and concentration of BaS in the suspension used to spike the solid waste material (see
Section 2.3.3). The number assumes 100% recovery from the surface and no physical losses associated
with processing of samples, and is not an absolute indication of the analytical method's limit of
identification; rather, it is a measure of the method's end-to-end performance to identify BaS.

3.1 50-mL Conical Tube Spore Recovery Method Analyses Results

This section includes information on the 50-mL conical tube spore recovery sample culture results, the
BaS colony confirmation results, and the sample RV-PCR analysis results.

3.1.1 50-mL Conical Tube Spore Recovery Sample Culture Analyses

A summary of the average and standard deviation of the measured recovery efficiencies of presumptive
BaS spores recovered from solid waste materials using the 50-mL conical tube extraction method and as
determined by culture analysis using the SB A plates is presented in Table 3. The nominal quantity
represented one-half the target spore load applied to the materials based on the BaS spore concentration
in the spiking suspension and quantity spiked. The recovery of presumptive BaS colonies recovered are
plotted in Figure 16 and Figure 17 for the 1/4-inch diameter, 5-cm-long double-braided nylon rope and
the boat seat cover (marine fabric), respectively.

More than half of all applied BaS spores were recovered from the two spiked waste stream materials
using the 50-mL conical tube method. The recoveries that appeared to exceed 100% were attributed to
the artifact that too few spores were available to accurately enumerate, thus not yielding a statistically
sound quantity. The scatter in the data was attributed to variability in the various processing and
measuring steps, to include recovery of spores from the surface, any dilutions for analysis, and in the
culture analytical method. Typically, the scatter decreased at the highest loading of 3,000 CFU relative
to the lower-level spore loadings.

22

-------
Table 3. Presumptive BaS Spore Recovery from Solid Waste Materials Using the 50-mL Conical Tube
Spore Recovery Method and SBA Media Culture Analysis Method

Waste Material

Spores Available for Analysis
(CFU)

Replicates

Spore Recovery (CFU)

(X ± cr)'°>

Spore
Recovery
Efficiency

(%)
(X ± <7)'d>

Nominal'3'

Determined'13'

Double-Braided Nylon
Rope (1/4-inch diameter,
5-cm long)

0

0

3

0

N/A

3

3.6 ± 0.2

3

4.2 ±2.4

110 ± 62

30

37 ± 0.0

5

25 ±21

68 ± 56

300

370 ± 0.0

5

340±150

93 ±41

3,000

3,700 ± 130

3

2,700 ± 450

74 ± 12

Boat Seat Cover
(Marine Fabric)

0

0

3

0

N/A

3

3.5 ± 0.0

3

5.2 ± 3.9

150 ±110

30

37 ± 0.0

5

37 ±6.9

100 ± 19

300

370 ± 0.0

5

350 ±4.3

95 ± 12

3,000

3,600 ± 58

3

2,600 ± 380

71 ± 12

(a) Nominally one-half of the target BaS spore load on the swatch and assuming 100% recovery of spores.
ft1 Based on the spiking suspension titer measured each test trial. 100% recovery efficiency, and one-half of extract used for
RV-PCR analysis.

?C I Presumptive BaS colonies based on morphology and one-half of spore recovery used for culture analysis.

® Calculated using the actual BaS spore load on each swatch and total presumptive BaS spores recovered from each sample.

Double Braided Nylon 1/4" (50 mL)

140


O
u
OJ

en

\P

2 120
o
a.
to

c 100

u.

0)

4-»

CO

d

80 -

60 -

40 -

20 -

30	300	3,000

Nominal Spores Available for Analysis (CFU)

Figure 16. Recovery of presumptive BaS spores from 1/4-inch diameter double-braided nylon rope using
the 50-mL conical tube spore recovery method.

23

-------
Marine Fabric {50 mL)

160

i" 140 -I

120

0)
>
o

u
-------
3.1.2 50-mL Conical Tube Spore Recovery BaS Colony Confirmation

Presumptive BaS colonies were identified for all sample replicates spiked with BaS spores and were
confirmed positive by colony PCR. All zero-spike samples were negative for the culture analytical
method. Results from PCR confirmatory testing are shown in Table 4.

Table 4. Summary of the Accuracy of Identification of Presumptive BaS Colonies by PCR Confirmation
from Solid Waste Materials of Double-Braided Nylon Rope and Boat Seat Cover

Waste Material

Nominal Spore
Load(CFU)

Culture
Replicates
Presumptive
Positive

Colonies from
Initial Culture
Plates PCR
Screened
(# PCR +)lbl

Colonies from
BHIB Streak
Plates PCR
Screened
(# PCR+)1:1

BHIB PCR
Screened
(# PCR +)ldl

Double-Braided Nylon
Rope (1/4-inch diameter,
5-cm long)

1 (0)

0(0)

3(0)

5(3)

0(0)

35 (5)

1 (0)

2(0)

300

50 (5)

0(0)

3,000

11 (3)

0(0)

Boat Seat Cover (Marine
Fabric)

1 (0)

0(0)

2(0)

10(3)

0(0)

50 (5)

0(0)

300

50 (5)

0(0)

3,000

12(3)

0(0)

(a) Presumptive BaS was present on initial culture plates.

(b) Number of colonies PCR screened from initial plating, with number of PCR positive replicates in parentheses.

(d) Number of samples with PCR screening of BHIB enrichment culture, with number of PCR positive replicates in
parentheses.

3.1.3 50-mL Conical Tube Spore Recovery Sample RV-PCR Analyses

A summary of the average and sample standard deviation of the RV-PCR ACt values for the detection of
BaS spores recovered from solid waste materials using the 50-mL conical tube extraction method are
presented in Table 5. The nominal quantity represented one-half the target spore load applied to the
materials based on the BaS spore concentration in the spiking suspension and quantity spiked. The
RV-PCR ACt results are plotted in Figure 18 for the 1/4-inch diameter, 5-cm-long double-braided nylon
rope and in Figure 19 for the boat seat (marine fabric). All samples spiked with BaS spores were
RV-PCR positive (RV-PCR ACt value > 9), indicating that viable BaS spores were recovered, and all
zero-spike sample replicates were RV-PCR negative (RV-PCR ACt value < 9). The plots all depict an
area shaded in red that is the region of a negative detection result and an area of green that is a positive
detection result, delineated by the BaS barcode target ACt value > 9 to be a positive result.

These results indicated that as few as six BaS spores applied to the target solid waste stream sample
could be recovered in the spore suspension and detected to correctly determine the presence or absence
of BaS.

-------
Table 5. BaS Spores Detected from Solid Waste Materials Using the 50-mL Conical Tube Spore Recovery
Method and RV-PCR Analysis Method

Waste Material

Spores Available for
Analysis (CFU)

Replicates

ACt (X ± ct)

RV-PCR
Replicates
Positive(c)

Nominal'3'

Determined'151

Chromosomal
Gene Target

pXOl Gene
Target

Double-Braided
Nylon Rope
(1/4-inch
diameter, 5~cm
long)

0.0 ± 0.0

0.6 ± 1.1

3.6 ± 0.2

14.8 ±2.4

14.4 ±2.9

37 ± 0.0

18.8 ±2.7

18.4 ±2.7

300

370 ± 0.0

23.4 ± 1.3

23.1 ± 1.3

3,000

3,700 ± 130

27.3 ±2.5

26.8 ±2.0

Boat Seat Cover
(Marine Fabric)

0.6 ± 1.1

0.5 ± 0.8

3.5 ± 0.0

20.6 ±4.8

19.8 ±4.5

37 ± 0.0

24.6 ± 3.3

23.9 ± 3.2

300

370 ± 0.0

27.5 ± 1.0

26.5 ± 0.9

3,000

3,600 ± 58

27.7 ± 0.4

26.9 ± 0.3

"" Nominally one-half of the target BaS spore load on the swatch and assuming 100% recovery of spores.
0» presumptive BaS colonies based on morphology and one-half of spore recovery used for RV-PCR analysis.
Number of replicates with a RV-PCR ACt value > 9 for both chromosomal and pXOl gene targets.

30 -

W in

< 20
-------
35

4 20

Nominal B. a. Sterne Spore Available for Analysis (CFU)

Figure 19. RV-PCR detection of BaS spores from boat seat cover (marine fabric) using the 50-mL conical
tube spore recovery method.

3.2 400-mL Stomacher Bag Spore Recovery Method Analyses Results

This section includes information on the 400-mL Stomacher bag spore recovery sample culture results,
the BaS colony confirmation results, and the sample RV-PCR analysis results.

3.2.1400-mL Stomacher Bag Spore Recovery Sample Culture Analyses

A summary of the average and standard deviation of the measured recovery efficiencies of presumptive
BaS spores recovered from solid waste materials using the 400-mL Stomacher bag extraction method
and as determined by culture analysis using the SBA plates is presented in Table 6. The nominal
quantity represented one-half the target spore load applied to the materials based on the BaS spore
concentration in the spiking suspension and quantity spiked. The recovery of presumptive BaS colonies
recovered are plotted in Figure 20, Figure 21, and Figure 22 for the solid waste materials of 1/4-inch,
15-cm-long double-braided nylon rope, nitrile glove, and boat seat cover (marine fabric), respectively.

More than half of all applied BaS spores were recovered from the spiked waste stream materials of
nitrile gloves and boat seat cover using the 400-mL Stomacher bag method. Spore recovery from the
1/4-inch, 15-cm-long double-braided nylon rope material was slightly lower at 12-54%. A clear reason
for the lower recovery for the rope was not identified.

-------
Table 6. Presumptive BaS Spore Recovery from Solid Waste Materials Using the 400-mL Stomacher Bag
Spore Recovery Method and SBA Media Culture Analysis Method

Waste Material ID

Spores Available for Analysis
(CFU)

Replicates

Spore Recovery (CFU)
(X ± CT)'°>

Spore
Recovery
Efficiency

(%)
(X ± <7)'d>

Nominal'3'

Determined'13'

Double-Braided Nylon
Rope (1/4-inch diameter,
15-cm long)

2.4 ±4.2

N/A

3.8 ± 0.2

0.5 ± 0.8

12 ± 20

32 ± 6.3

16 ±7.3

54 ± 30

300

320 ± 63

130 ±23

41 ± 14

3,000

3,300 ± 460

1,600 ± 750

46 ± 18

Nitrile Glove

0.0 ± 0.0

N/A

3.6 ± 0.0

6.8 ± 5.0

190 ±140

3.4 ± 0.0

29 ± 10

85 ± 30

300

340 ± 0.0

180 ± 80

54 ±24

3,000

3,400 + 0.0

2,000 ± 210

58 ± 6.0

Boat Seat Cover (Marine
Fabric)

0.0 ± 0.0

N/A

3.8 ± 0.2

4.0 ± 1.5

110 ± 47

32 ± 6.3

27 ± 9.9

85 ± 36

300

320 ± 63

230 ± 48

71 ± 12

3,000

3,300 ± 460

1,700 ± 880

50 ± 22

w Nominally one-half of the target BaS spore load on the swatch and assuming 100% recovery of spores.
ft1 Based on the spiking suspension titer measured each test trial, 100% recovery efficiency, and one-half of extract used for
RV-PCR analysis.

?CI Presumptive BaS colonies based on morphology, and one-half of spore recovery used for culture analysis.

;d) Calculated using the actual BaS spore load on each swatch and total presumptive BaS spores recovered from each sample.

Double Braided Nylon 1/4" (Stomacher)

100

U
CD
CC

r 90 q

CO
0)

E 70 }

<3J
¦*->

60 -

40 -

30 -

> 20 :

30 300 3,000

Nominal Spores Available for Analysis (CFU)

Figure 20. Recovery of presumptive BaS spores from 1/4-inch diameter double-braided nylon rope using
the 400-mL Stomacher bag spore recovery method.

-------
Nitrile Glove (Stomacher)

200

SO*

0s
to

180

160

i—

140

120

100

O
u

<11

30 300 3,000

Nominal Spores Available for Analysis (CFU)

Figure 21. Recovery of presumptive BaS spores from nitrile glove using the 400-mL Stomacher bag spore
recovery method.

Marine Fabric (Stomacher)

120

S ioo

CL
CO

O
u
0)

40 -

20 ¦

30 300 3,000

Nominal Spores Available for Analysis (CFU)

Figure 22. Recovery of presumptive BaS spores from boat seat cover (marine fabric) using the 400-mL
Stomacher bag spore recovery method.

-------
3.2.2 400-mL Stomacher Bag Spore Recovery BaS Colony Confirmation

Presumptive BaS colonies were identified for all sample replicates spiked with BaS spores except for a
3 CFU nominal BaS spore load 1/4-inch diameter, 15-cm-long double-braided nylon rope sample; this
sample was the only spiked sample not confirmed PCR positive using the culture method. All other
sample replicates spiked with BaS spores were confirmed positive by PCR. All zero-spike sample
replicates were negative for the culture analytical method, with only one 1/4-inch diameter double-
braided nylon rope sample with presumptive BaS morphology present; all five of the presumptive BaS
colonies were screened by colony PCR and were negative. Results from PCR confirmatory testing are
shown in Table 7.

Table 7. Summary of the Accuracy of Identification of Presumptive BaS Colonies by PCR Confirmation
from Solid Waste Materials of Double-Braided Nylon Rope, Nitrile Gloves, and Boat Seat Cover

Waste Material

Nominal Spore
Load (CFU)

Culture
Replicates
Presumptive
Positive

Colonies from
Initial Culture
Plates PCR
Screened
(# PCR +)lbl

Colonies from
BHIB Streak
Plates PCR
Screened
(# PCR +)Kl

BHIB PCR
Screened
(# PCR +)ldl

Double-Braided Nylon

Rope (1/4-inch
diameter, 15-cm long)

5(0)

0(0)

1 (1)

0(0)

2(1)

42 (5)

0(0)

300

50 (5)

0(0)

3,000

11 (3)

0(0)

Nitrile Glove

0(0)

2(0)

13(3)

0(0)

50 (5)

0(0)

300

50 (5)

0(0)

3,000

40 (4)

0(0)

Boat Seat Cover
(Marine Fabric)

0(0)

8(3)

0(0)

50 (5)

0(0)

300

50 (5)

0(0)

3,000

12(3)

0(0)

(a) Presumptive BaS was present on initial culture plates.

(b) Number of colonies PCR screened from initial plating, with number of PCR positive replicates in parentheses.

(d) Number of samples with PCR screening of BHIB enrichment culture, with number of PCR positive replicates in
parentheses.

3.2.3 400-mL Stomacher Bag Spore Recovery Sample RV-PCR Analyses

A summary of the average and sample standard deviation of the RV-PCR ACt values for the detection of
BaS spores recovered from solid waste materials using the 400-mL Stomacher bag extraction method is
presented in Table 8. The nominal quantity represented one-half the target spore load applied to the
materials based on the BaS spore concentration in the spiking suspension and quantity spiked. The
RV-PCR ACt results are plotted in Figure 23 for the 1/4-inch diameter, 15-cm-long double-braided
nylon rope, in Figure 24 for the nitrile glove, and in Figure 25 for the boat seat cover (marine fabric). All
sample replicates spiked with BaS spores were RV-PCR positive (RV-PCR ACt value > 9), indicating

-------
that viable BaS spores were recovered, and zero-spike sample replicates were RV-PCR negative
(RV-PCR ACt value < 9). The plots all depict an area shaded in red that is the region of a negative
detection result and an area of green that is a positive detection result, delineated by the BaS barcode
target ACt value > 9 to be a positive result.

Table 8. BaS Spores Detected from Solid Waste Materials Using the 400-mL Stomacher Bag Spore
Recovery Method and RV-PCR Analysis Method

Waste Material

Spores Available for
Analysis (CFU)

Replicates

ACt (X ± ct)

RV-PCR
Replicates
Positivelcl

Nominal1"

Determined"51

Chromosomal
Gene Target

pXOl Gene
Target

Double-Braided
Nylon Rope
(1/4-inch
diameter, 5-cm
long)

5.9 ± 1.6

5.8 ± 1.7

3.8 ± 0.2

18.4 ± 1.9

18.2 ±2.0

32 ±6.3

18.4 ± 2.0

18.0 ± 1.9

300

320 ± 63

21.9 ± 1.7

21.7 ± 1.8

3,000

3,300 ±460

25.0 ± 0.7

24.7 ± 1.0

Nitrile Glove

2.5 ±4.3

3.7 ± 3.9

3.6 ± 0.0

26.6 ± 2.2

26.0 ±2.3

3.4 ± 0.0

25.1 ± 3.1

24.8 ± 3.2

300

340 ± 0.0

24.8 ± 3.4

24.7 ± 3.5

3,000

3,400 ± 0.0

26.7 ± 1.5

25.9 ± 1.4

Boat Seat
Cover (Marine
Fabric)

3.8 ± 3.6

5.5 ±2.1

3.8 ± 0.2

21.8 ± 6.2

21.2 ±6.2

32 ±6.3

26.2 ± 1.5

25.9 ± 1.1

300

320 ± 63

25.8 ± 1.3

25.3 ± 0.9

3,000

3,300 ± 460

27.9 ± 0.4

27.1 ± 0.6

(a) Nominally one-half of the target BaS spore load on the swatch and assuming 100% recovery of spores.

(b) Presumptive BaS colonies based on morphology and one-half of spore recovery used for RV-PCR analysis.

-------
35

Double Braided Nylon 1/4" (Stomacher)

i i Chromosome i i pXOl

• Threshold

< 20

QJ
00

QJ 1 c

3 15

'On

Nominal B. a. Sterne Spore Available for Analysis (CFU)

Figure 23. RV-PCR detection of BaS spores from 1/4-inch diameter double-braided nylon rope using the
400-ni L Stomacher bag spore recovery method.

Nitrile Glove (Stomacher)

4 20

-------
35

4 20

Nominal B. a. Sterne Spore Available for Analysis (CFU)

Figure 25. RV-PCR detection of BaS spores from boat seat cover (marine fabric) using the 400-m L
Stomacher bag spore recovery method.

3.3 1-L Bottle Spore Recovery Method Analyses Results

This section includes information on the 1-L bottle spore recovery sample culture results, the BaS
colony confirmation results, and the sample RV-PCR analysis results.

3.3.11-L Bottle Spore Recovery Sample Culture Analyses

A summary of the average and standard deviation of the measured recovery efficiencies of presumptive
BaS spores recovered from solid waste materials spiked with BaS then extracted using the 1-L bottle
method and as determined by EPA culture analysis method using the SBA plates is presented in
Table 9. The nominal quantity represented one-half the target spore load applied to the materials based
on the BaS spore concentration in the spiking suspension and quantity spiked. The recovery of
presumptive BaS colonies recovered are plotted in Figure 26, Figure 27, Figure 28, and Figure 29.

BaS spores were recovered from Tychem (36 to 63%), Tyvek (49-63%), and nitrile gloves (47 to 56%)
at a similar level when comparing 30-3,000 CFU nominal spore load levels. The percentage of BaS
spores recovered from double-braided nylon rope (1-inch diameter) was lower (23 to 47%) for the 30 to
3,000 CFU nominal spore load levels, perhaps due to the spore spiking solution being absorbed into the
inner layers of the double-braided nylon rope.

-------
Table 9. Presumptive BaS Spore Recovery from Solid Waste Materials Using the 1-L Bottle Spore
Recovery Method and SBA Media Culture Analysis

Waste Material

Spores Available for Analysis
(CFU)

Replicates

Spore Recovery
(CFU)
(X ± CT)l:l

Spore
Recovery
Efficiency (%)
(X ± CT)ldl

Nominal1"

Determined"51

Tychem Sleeve

0.6 ± 1.3

N/A

4.2 ± 0.2

3.3 ±4.2

77 ± 98

33 ± 0.3

21 ± 2.2

63 ±65

300

390 ± 52

140 ± 26

36 ± 10

3,000

3,600 ± 290

1,700 ± 190

47 ± 2.7

Tyvek Sleeve

0± 0

N/A

4.2 ± 0.2

2.1 ± 1.7

49 ±41

37 ± 4.1

23 ±2.8

63 ± 11

300

410 ± 14

220 ± 52

54 ± 11

3,000

3,900 ± 120

1,600 ± 470

41 ± 13

Nitrile Gloves

0.0 ± 0.0

N/A

3.9 ± 0.0

2.1 ± 0.7

55 ± 19

39 ± 0.8

18 ± 6.5

47 ± 17

300

390 ± 8.2

220 ± 55

56 ± 15

3,000

3,900 ± 120

1,800 ± 730

48 ±20

Double-Braided Nylon
Rope (1-inch diameter)

0.0 ± 0.0

N/A

3.9 ± 0.0

0.5 ± 0.8

12 ±20

35 ± 3.0

16 ± 16

47 ±48

300

350 ± 30

97 ± 30

30 ± 10

3,000

3,600 ± 290

820 ±410

23 ± 12

(a) Nominally one-half of the target BaS spore load on the swatch and assuming 100% recovery of spores.

(b) Based on the spiking suspension titer measured each test trial, 100% recovery efficiency, and one-half of extract used for
RV-PCR analysis.

(d) Calculated using the actual BaS spore load on each swatch and total presumptive BaS spores recovered from each sample.

-------
100

r 90

80 -

E 70 -

a;
+•»
co

§ 50

<~)
CD
fa
Q-

40 -

30 -

> 20 :

Tyvek Sleeve (1 L Extractive)

Figure 26.
method.

30 300 3,000

Nominal Spores Available for Analysis (CFU)

Recovery of presumptive BaS spores from Tyvek sleeve using the 1-L bottle spore recovery

180

~160H

CD
O

£ 140

CD
C

® 120

OJ
CO

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>

100 -

S. 80 H
E

i—

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a>
>
o
u

(U
Q£

40 -

Tychem Sleeve (1 L Extractive)

30 300 3,000

Nominal Spores Available for Analysis (CFU)

Figure 27. Recovery of presumptive BaS spores from Tychem sleeve using the 1-L bottle spore recovery
method.

-------
100

O)
>
o
u
CD

40 -

30 -

20 :

10 -

Nitrile Glove (1 L Extractive)

30 300 3,000

Nominal Spores Available for Analysis (CFU)

Figure 28.
method.

Recovery of presumptive BaS spores from nitrile glove using the 1-L bottle spore recovery

100

r 90 q

CL 80

g 50

70 -

60 -

E
d

wo
0)

la.

Q-

v*—
O

0)
>
o

u
0

40 -

30 -

Double Braided Nylon 1" (1 L Extractive)

30 300 3,000

Nominal Spores Available for Analysis (CFU)

Figure 29. Recovery of presumptive BaS spores from 1-inch diameter double-braided nylon rope using the
1-L bottle spore recovery method.

-------
3.3.2 1-L Bottle Spore Recovery BaS Colony Confirmation

Presumptive BaS colonies were identified for all spiked samples except for two Tychem sleeves (3 CFU
nominal BaS spore load), a Tyvek sleeve (3 CFU nominal BaS spore load), and two 1-inch diameter
double-braided nylon ropes (3 CFU nominal BaS spore load) samples. Only four (4) spiked samples
were not confirmed PCR positive: three replicates of Tychem sleeve (3 CFU nominal BaS spore load),
and one Tyvek sleeve (3 CFU nominal BaS spore load). All other sample replicates that were spiked
with BaS spores were confirmed positive by PCR.

Presumptive BaS colonies were identified on one Tychem sleeve zero-spike sample that had two
presumptive BaS colonies, although they were negative for chromosome and pXOl signature when
screened using colony PCR. All other zero-spike sample replicates had zero presumptive BaS colonies
and PCR analysis on the BHIB enrichment culture was negative for all zero-spike samples. A
background colony from a zero-spike 1-inch diameter double-braided nylon rope sample that was PCR
screened as a negative control resulted in average Ct values of 35.5 and 36.2 for chromosome and pXOl
targets, respectively. Repeat PCR analysis of this background colony resulted in average Ct value of
41.0 for both the chromosome and pXOl targets. Additionally, PCR analysis on the BHIB enrichment
culture was negative for this sample with a Ct value of 45 for both the chromosome and pXOl targets.
The cause of this low-level amplification in the colony screen might have been cross-contamination or
non-specific amplification. For comparison, colony PCR screening of presumptive BaS colonies
recovered from 1-inch diameter double-braided nylon rope samples that were spiked with BaS spores
resulted in average Ct values of 17.4 ± 1.3 and 17.7 ± 1.3 for chromosome and pXOl targets,
respectively. Results from PCR confirmatory testing are shown in Table 10.

-------
Table 10. Summary of the Accuracy of Identification of Presumptive BaS Colonies by PCR Confirmation
from Solid Waste Materials of Tychem Sleeve, Tyvek Sleeve, Nitrile Gloves, and 1-Inch diameter Double-
Braided Nylon Rope

Waste Material

Nominal Spore
Load(CFU)

Culture
Replicates
Presumptive
Positive1,1

Colonies from
Initial Culture
Plates PCR
Screened
(# PCR+)1151

Colonies from
BHIB Streak
Plates PCR
Screened
(# PCR+)1:1

BHIB PCR
Screened
(# PCR +)ldl

Tychem Sleeve

2(0)

0(0)

4(0)

11 (2)

0(0)

3(0)

30 (5)

0(0)

300

50 (5)

0(0)

3,000

30 (3)

0(0)

Tyvek Sleeve

0(0)

3(0)

10 (4)

0(0)

1 (0)

50 (5)

0(0)

300

60 (5)

7(0)

1 (1)

3,000

30 (3)

0(0)

Nitrile Gloves

0(0)

3(0)

5(3)

0(0)

48 (0)

0(0)

300

50 (5)

0(0)

3,000

30 (3)

0(0)

1-inch diameter Double-
Braided Nylon Rope

1 Background (1)

0(0)

1 (0)

1 (1)

0(0)

2(2)

31 (5)

0(0)

300

50 (5)

0(0)

3,000

30 (3)

0(0)

(a) Presumptive BaS was present on initial culture plates.

(b) Number of colonies PCR screened from initial plating, with number of PCR positive replicates in parentheses.

(d) Number of samples with PCR screening of BHIB enrichment culture, with number of PCR positive replicates in
parentheses.

3.3.3 1-L Bottle Spore Recovery Sample RV-PCR Analysis

A summary of the average and sample standard deviation of the RV-PCR ACt values for the detection of
BaS spores recovered from solid waste samples recovered using a 1-L bottle is presented in Table 11.
The nominal quantity represented one-half the target spore load applied to the materials based on the
BaS spore concentration in the spiking suspension and quantity spiked. The RV-PCR ACt results are
plotted in Figure 30, Figure 31, Figure 32, and Figure 33. The plots all depict an area shaded in red that
is the region of a negative detection result and an area of green that is a positive detection result,
delineated by the BaS barcode target ACt value > 9 to be a positive result.

Nine (9) samples spiked with BaS spores were negative for RV-PCR, all at the 3 CFU nominal BaS
spore load: five (5) Tychem sleeves, two (2) Tyvek sleeves, one (1) nitrile glove, and one (1) 1-inch
diameter double-braided nylon rope. All zero-spike samples were negative using the RV-PCR analytical
method.

-------
These results indicated that as few as six BaS spores applied to the target solid waste stream sample
could be recovered in the spore suspension and detected to correctly determine the presence or absence
of BaS.

Table 11. BaS Spores Detected from Solid Waste Materials Using the 1-L Bottle Spore Recovery
Method and RV-PCR Analysis Method

Waste Material

Spores Available for Analysis
(CFU)

Replicates

ACt (X

±CT)

RV-PCR
Replicates
Positive'1^

Nominal1"

Determined"31

Chromosomal
Gene Target

pXOl Gene
Target

1.0 ± 2.0

CO
CO
+l

4.2 ± 0.2

5.0 ±4.6

5.9 ± 3.8

Tychem Sleeve

33 ± 0.3

19.4 ± 5.8

19.2 ± 5.9

300

390 ± 52

20.9 ±2.6

20.5 ± 2.6

3,000

3,600 ± 290

25.6 ± 1.4

25.3 ± 1.1

0.5 ± 1.0

0.4 ± 0.8

4.2 ± 0.2

13.0 ± 8.6

13.1 ± 7.9

Tyvek Sleeve

37 ±4.1

21.8 ± 6.0

21.4 ± 6.0

300

410 ± 14

22.0 ± 3.0

21.5 ± 3.1

3,000

3,900 ± 120

25.6 ± 1.8

24.9 ± 1.6

0.0 ± 0.0

3.9 ± 0.0

14.9 ± 11.3

14.5 ± 11.5

Nitrile Gloves

39 ± 0.8

20.0 ± 5.2

19.4 ± 5.5

300

390 ± 8.2

21.8 ± 7.2

21.2 ± 7.0

3,000

3,900 ± 120

26.7 ±2.2

26.2 ±2.1

0.0 ± 0.0

2.1 ± 2.5

1-inch diameter

3.9 ± 0.0

10.1 ± 7.7

11.3 ± 4.8

Double-Braided

35 ± 3.0

16.2 ± 3.0

15.8 ± 3.3

Nylon Rope

300

350 ± 30

22.4 ±2.8

22.1 ± 2.8

3,000

3,600 ± 290

24.2 ± 1.2

24.0 ± 1.2

(a) Nominally one-half of the target BaS spore load on the swatch and assuming 100% recovery of spores.

(b) Presumptive BaS colonies based on morphology and one-half of spore recovery used for RV-PCR analysis.

-------
35

Tyvek Sleeve {1 L Extractive)

i i Chromosome i i pXOl

• Threshold

< 20

(Die

3 15

Nominal B. a. Sterne Spore Available for Analysis (CFU)

'°o
uo

Figure 30. RV-PCR response to BaS spores from Tyvek sleeve using the 1-L bottle spore recovery method.

TyChem Sleeve (1 L Extractive)

30 ¦

I Chromosome

IpXOl

¦ Threshold

^ in

< 20
m

0) 1 c

I 15

10 •

I—I—I

Nominal B. a. Sterne Spore Available for Analysis (CFU)

Figure 31. RV-PCR response to BaS spores from Tychem sleeve using the 1-L bottle spore recovery
method.

-------
35

< 20

QJ
00

QJ 1 c

3 15

Nitrile Glove (1 L Extractive)

i i Chromosome i i pXOl

• Threshold

'On

Nominal B. a. Sterne Spore Available for Analysis (CFU)

Figure 32. RV-PCR response to BaS spores from nitrile glove using the 1-L bottle spore recovery method.

35
30
25

< 20

01
60
ru

3 15

Double Braided Nylon 1" (1 L Extractive)

i i Chromosome

UpXOl

• Threshold

Nominal B. a. Sterne Spore Available for Analysis (CFU)

Figure 33. RV-PCR response to BaS spores from 1-inch double-braided nylon rope using the 1-L bottle
spore recovery method.

-------
3.4 Spore Recovery Method Comparison

The spore recovery methods evaluated in this study were successful at recovering and detecting nominal
BaS spore loads of 3, 30, 300, and 3,000 CFU. Of the 150 sample swatches spiked with BaS spores, 145
(97%) were confirmed positive by PCR using the culture analytical method and 141 (94%) were positive
by RV-PCR analytical method. Only one zero-spike sample swatch was positive using the culture
analytical method - a background colony that was screened using colony PCR resulted in Ct values of
35.5 and 36.2 for chromosome and pXOl targets, respectively. Repeat PCR analysis of this background
colony resulted in average Ct values of 41.0 and 41.0. Additionally, PCR analysis on the BHIB
enrichment culture was negative for this sample with Ct values of 45. The cause of this low-level
amplification in the colony screen might have been cross-contamination or non-specific amplification.
For comparison, colony PCR screening of presumptive BaS colonies recovered from 1-inch diameter
double-braided nylon rope samples spiked with BaS spores resulted in average Ct values of 17.4 ±1.3
and 17.7 ±1.3 for chromosome and pXOl targets, respectively. All zero-spike sample swatches were
negative using the RV-PCR analytical method.

The only five samples spiked with BaS spores that were negative using the culture analytical method
were three Tychem sleeves with 3 CFU nominal BaS spore load (1-L recovery method), one Tyvek
sleeve with 3 CFU nominal BaS spore load (1-L recovery method), and one 1/4-inch double-braided
nylon rope with 3 CFU nominal BaS spore load (Stomacher method).

Only nine samples spiked with BaS spores were negative using the RV-PCR analytical method, and all
were at the 3 CFU nominal BaS spore load and extracted using the 1-L recovery method. They were
from Tychem (5), Tyvek (2), nitrile glove (1), and 1-inch double-braided nylon rope (1).

The solid waste materials are ranked by average percent recovery using the 300 CFU BaS nominal spore
load, since it was the highest spore load with five replicates analyzed for each material/spore recovery
method and were considered most accurate for quantitation. The highest percent recovery of
presumptive BaS spores was from the 50-mL conical recovery method at 94.9 and 93.5% (Table 12). In
addition to having the highest percent recovery, all sample replicates processed using the 50-mL conical
recovery method spiked with BaS spores were positive using both the culture and RV-PCR analytical
methods. High percent recovery and detection of BaS spores using the 50-mL conical tube recovery
method might have been due to the smaller swatch size and a lower volume of spore recovery buffers
used in the method. The recovery efficiencies would also likely depend on the surface properties of the
materials and agitation method. Assessing those factors was beyond the scope of this project.

Table 12. Analytical Method Comparison Displaying Culture Presumptive Percent Recovery at the
300 CFU BaS Spore Load Level

Material

Spore Recovery
Method

Nominal Spore
Load

Average % Recovery

Standard Deviation

Marine Fabric

50-mL Conical Tube

300

94.9

11.7

Double-Braided Nylon

50-mL Conical Tube

300

93.5

40.7

Marine Fabric

400-mL Stomacher Bag

300

70.5

12.1

Nitrile Glove

1-L Bottle

300

55.9

14.5

Tyvek Sleeve

1-L Bottle

300

54.0

11.4

Nitrile Glove

400-mL Stomacher Bag

300

53.9

23.6

Double-Braided Nylon

400-mL Stomacher Bag

300

41.4

13.7

Tychem Sleeve

1-L Bottle

300

35.9

10.4

Double-Braided Nylon

1-L

300

30.3

10.4

-------
3.5 Method Observations

A hot-knife should be utilized for excising sections of double-braided nylon rope when collecting
samples from the field. Cutting sections of double-braided nylon rope using a knife or other cutting tool
resulted in the rope unraveling. The hot knife successfully fused the ends of the rope so that intact
samples could be sent to the laboratory for processing. The hot-knife also reduced the likelihood of
producing particulates during sample collection that could clog filtration media within the laboratory or
be a source of potential cross-contamination in the waste staging area where the samples were acquired.
Potential drawbacks for using the hot knife were that a solid cutting surface was needed, the melting
rope produced smoke and potential inhalation/exposure to hazardous material, contact with combustible
or flammable material needed to be avoided, and lacerations or burns could result from the hot blade. It
was also possible that the hot knife could thermally degrade spores local to the cutting zone. However,
that would likely be on the order of the width of the cutting blade (~1 mm) and thus relatively small
compared to the 50 to 150-mm-long sample of rope unexposed to the hot cutting edge.

-------
4.0 QUALITY ASSURANCE/QUALITY
CONTROL

Quality assurance (QA)/quality control (QC) procedures were performed in accordance with the
Scientific, Testing, Research, and Modeling, Support (STREAMS III) Program Quality Management
Plan (QMP). The QA/QC procedures and results are summarized below.

4.1 Equipment Calibration

All equipment (e.g., pipettes, incubators, water baths, refrigerators/freezers) used at the time of the
evaluation were verified as being certified, calibrated, or validated.

4.2 QC Results

QC efforts conducted during testing included positive and negative controls for both spread plate
samples and quantitative PCR (qPCR). In addition, quantification of the BaS spike suspensions was
performed to verify either CFU/mL titer or target spike concentrations.

Positive and negative control results were within the target requirements for the qPCR. Applied
Biosystems 7500 Fast system performance was assessed according to internal standard operating
procedures (SOPs) and maintained at regular intervals—monthly (optical and background calibration),
every six months (dye calibration), and annually (RNase P calibration). For culture, the positive control
spore stock maintained a single morphological appearance consistent with BaS throughout the study, as
determined at the beginning of each trial. Media and reagents used for culture analysis were screened
(negative controls) and had no growth, showing that reagents used were not the source of contamination.

4.3 Operational Parameters

Micropipettes, thermometers, and timers used were calibrated against a traceable standard at regular
intervals (every six months or annually) and used only within an acceptable calibration interval
established by internal SOPs.

4.4 Audits

This section includes information on the performance evaluation, technical systems, and data quality
audits and overall QA/QC reporting and data review.

4.4.1 Performance Evaluation Audit

Performance evaluation audits were conducted to assess the quality of the results obtained during these
experiments. Table 13 summarizes the audits that were performed; equipment was within an acceptable
tolerance range.

-------
Table 13. Performance Evaluation Audits

Measurement

Audit

Allowable

Actual

Procedure

Tolerance

Volume of liquid from
micropipettes

Gravimetric evaluation

± 10%

Passed calibration as
found/as returned

Time

Compared to independent
clock

± 2 sec/hr

Passed calibration as
found/as returned

Temperature

Compared to independent
calibrated thermometer

±2°C

Passed calibration as
found/as returned

4.4.2 Technical Systems Audit

Observations and findings from the technical system audit (TSA) were documented and submitted to the
laboratory technical lead for response. The TSA was conducted during the week of July 26, 2021 to
ensure that tests were being conducted in accordance with the appropriate Quality Assurance Project
Plan (QAPP) and QMP. As part of the audit, test procedures were compared to those specified in the
QAPP and work instructions, and data acquisition and handling procedures were reviewed. All
procedures were performed according to documentation and no findings were noted.

4.4.3 Data Quality Audit

At least 10% of data acquired during the evaluation were audited. Data were reviewed from November
16 through November 18, 2021. A QA auditor traced the data from the initial acquisition, biologic plate
counts, PCR ACt calculation, data reduction and statistical analysis, to final reporting to ensure the
integrity of the reported results. All calculations performed on the data undergoing the audit were
verified. No issues were noted with the data collection and reporting process and all calculations were
performed accordingly.

4.5 QA/QC Reporting

Each assessment and audit was documented in accordance with the QAPP and QMP. For these tests, no
findings were noted during the TSA or in the data quality audit, and no follow-up corrective action was
necessary. QA/QC procedures were performed in accordance with the QAPP.

4.6 Data Review

Records and data generated in the evaluation received a QC/technical review before they were utilized
in calculating or evaluating results and prior to incorporation in this report.

-------
5.0 CONCLUSIONS

BaS spores were successfully recovered from candidate USCG solid waste materials using existing EPA
methods previously established for spore recovery from other materials such as filtration media or
gravel. As few as six BaS spores applied to common solid waste materials (Tychem sleeve, Tyvek
sleeve, mooring line (rope), nitrile gloves, and marine fabric found on boat seats) can be recovered using
the applied recovery method (50-mL conical tube, Stomacher, or 1-L bottle) and detected using culture
and RV-PCR analytical methods. Overall, there were only three sample swatches that were negative for
both culture and RV-PCR analytical methods. These three samples were Tychem sleeves spiked with six
BaS spores and recovered using the 1-L bottle spore recovery method.

Of the 31 sample swatches spiked with 6 CFU BaS spores and recovered using three recovery methods,
26 samples (84%) were confirmed positive by PCR using the culture analytical method and 22 samples
(71%) were RV-PCR positive. All sample swatches spiked with a target BaS spore load of 60 CFU (45
samples), 600 CFU (46 samples), and 6,000 CFU (28 samples) that were recovered using the three
recovery methods were confirmed positive for both culture and RV-PCR methods.

The 50-mL conical tube spore recovery method (used on a 5-cm x 5-cm swatch of marine fabric and
1/4-inch double-braided nylon rope cut to 5-cm length) had the highest percent recovery of spores when
comparing 300 CFU nominal spore load across all waste streams/recovery method combinations tested,
and all spiked sample replicates were detected using both culture and RV-PCR analytical methods.
Thirteen (13) of the 14 sample replicates that were spiked, but not detected with the culture and/or RV-
PCR analytical method(s), were processed using the 1-L bottle spore recovery method. Possible
explanations for why recovery and detection might have been better for the 50-mL conical tube and
Stomacher recovery methods compared to the 1-L bottle are that the buffer volumes are lower, and they
rely on mechanical agitation (vortex or paddle homogenization), whereas the 1-L bottle approach is
manual, which might lead to more variability in recovery. Recovery differences might also be due to
differences in the materials and their physical interaction with the applied spores. The sample swatch
sizes were also larger when implementing the 1-L bottle spore recovery approach, perhaps leading to
BaS spores being retained within the swatch. Full nitrile gloves were the only waste stream extracted
using two spore recovery methods (Stomacher and 1-L bottle) with the same size sample, and percent
recoveries were similar between the two methods, 54 ± 24 percent and 56 ± 15 percent, respectively.
However, one nitrile glove spiked with the 3 CFU nominal spore load was RV-PCR negative when
extracted using the 1-L bottle spore recovery method and all spiked nitrile gloves were positive for both
analytical methods when extracted with the Stomacher. Further testing could be performed to compare
spore recovery methods on the same size waste material. For example, a Tyvek sleeve could be
extracted using the Stomacher spore recovery method for comparison to the 1-L bottle spore recovery.

Solid waste materials used in this study were new materials that had not been used in the field or been
through a decontamination procedure that could be implemented in the field. Therefore, these materials
had relatively low levels of background microorganisms present to interfere with detection or potential
assay inhibitors that might be introduced during decontamination. It is recommended to evaluate these
methods using solid waste materials that have been worn in the field or processed using a field
decontamination procedure. In support of an upcoming wide area decontamination field exercise
sponsored by EPA, the procedure to cut solid waste swatches of the bulk material/item was summarized
in a field guide document for each of the four solid materials assessed in this project. Those field guide
documents are provided in Appendix J.

-------
Overall, these sampling procedures appear to be adequate for a wide variety of potential waste materials
that might be generated as the result of cleanup of USCG assets contaminated by a persistent biological
threat. These procedures will be tested in the field during the AnCOR wide-area demonstration.

-------
6.0 REFERENCES

EPA Protocol for Detection of Bacillus anthracis in Environmental Samples During the Remediation
Phase of an Anthrax Incident, 2nd Edition. U.S. Environmental Protection Agency, Washington,
DC, 2017.

Letant, S.E., Murphy, G.A., Alfaro, T.M., Avila, J.R., Kane, S.R., Raber, E., Bunt, T.M., and Shah, S.R.
Rapid viability PCR method for detection of live, virulent Bacillus anthracis in environmental
samples. ApplEnviron Microbiol, 2011. 77(18): p. 6570-8.

Serre, S. and L. Oudejans. Underground Transport Restoration (UTR) Operational Technology
Demonstration (OTD). U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-
17/272, 2017.

U.S. EPA Bio-Response Operational Testing and Evaluation (BOTE) Project: Phase 1: Decontamination
Assessment; EPA/600-R-13-168; 2013.

-------
Development of Sampling Protocols and
Strategies for Solid Waste Sampling During
Biological Incidents: Appendices A-J

-------
LIST OF APPENDICES

Page

APPENDIX A: FORMULATIONS OF RECIPES USED IN BIOLOGICAL TEST METHODS A-l

APPENDIX B: WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE
SPORES B-l

APPENDIX C: WORK INSTRUCTION BACILLUS ANTHRACIS STERNE SPORE
RECOVERY C-l

APPENDIX D: WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS STERNE
SPORES RECOVERED FROM SWATCHES D-l

APPENDIX E:WORK INSTRUCTION FOR RV-PCR PROCESSING OF BACILLUS ANTHRACIS „..E-1

APPENDIX F: WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS SPECIES F-l

APPENDIX G: WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS OF BACILLUS
ANTHRACIS G-l

APPENDIX H: WORK INSTRUCTION FOR SELECTING PRESUMPTIVE BACILLUS ANTHRACIS
STERNE COLONIES FOR PCR CONFIRMATION H-l

APPENDIX I: WORK INSTRUCTION FOR BHIB ENRICHMENT FOR CULTURE 1-1

APPENDIX J: FIELD GUIDES FOR COLLECTING SOLID WASTE SAMPLES J-l

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APPENDIX A: FORMULATIONS OF
RECIPES USED IN BIOLOGICAL TEST
METHODS

-------
Spore Production

Table 1. Components of Modified G Sporulation Medium

Ingredient

A mount/1 ,

Yeast Extract

2.0 g

(NH0:S<>4

2.0 g

CaCi: • 2II2O

0.03 g

CuS04 • 5II2O

0.005 g

I'eSOt • " 11 ()

0.0005 g

MgSO.. • 71i2C)

0.2 g

M11SO1 • 11:0*

0.06 g

XnSOi • 71 l;i()

0.005 g

K2HPO4

0.5 g

dlliO

1000 111L

*MnS()4 • II2O substituted for M11SO1 •

4IhO. 11' M11SO4 • 4IhO is used, add 0.05 g.

Table 2. Real-Time PCR Assay Conditions

Component Volume for one reaction (jiL)

TaqMan Fast Advanced Master Mix 12.5
(Applied Biosystems, Cat. 4444556)

Platinum Taq Polymerase 0.1
(Invitrogen, Cat. 10-966-034)

Btk T1B2 Forward Primer (25 (.iM) 1

Btk T1B2 Reverse Primer (25 (.iM) 1

Btk T1B2 Probe (2 jiM) 1

PCR Grade Water 4.4

Template 5

Total volume 25

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APPENDIX B: WORK INSTRUCTION FOR
SPIKING WITH BACILLUS ANTHRACIS
STERNE SPORES

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WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES
I. PURPOSE/SCOPE

To spike test articles (swatches) with B. anthracis Sterne spores for spore recovery testing.

II. MATERIALS/EQUIPMENT

Materials

Item

Manufacturer

Lot Number

Exp. Date

Storage
Temp.

B. a. Sterne stock (2.2 x
10s CFU/mL)

In house

05282020

5/2025

2-8 °C

PBST

Teknova

R.T.

Blood Agar

2-8 °C

1.5- or 2-mL tubes

Eppendorf

N/A

R.T.

Sterile forceps

N/A

R.T.

Swatch Material and Extraction Container

Material

Lot Number

Material

Extraction

Specification

Code

Containers

Swatch Size

Dupont Tyvek 400,

EV39707865

1 sleeve ~45 cm long (~18 inches)

TY500SWH000200

TYVEK

1 L bottle

Dupont Tychem

CMX1008340

1 sleeve cut ~45 cm long (~18

6000, Part #

inches) or equivalent area Tychem

19141750

TYCHEM

1 L bottle

suit acceptable to use (18" x 16")

1/4" diameter

50-mL

5 cm (~2 inches) of 1/4"

double-braided

N/A

conical

nylon, Knot & Rope

400-mL

15 cm (~6 inches) of 1/4"

Supply, Part # 0089

DBN1/4

stomacher

1" diameter

15 cm (~6 inches) of 1"

double-braided

N/A

nylon, Knot & Rope

Supply, Part # 0094

DBN1

1-L bottle

Nitrile glove, Large,

2006LG, Batch

400-mL

1 glove (size L) doffed

Fisherbrand 19-

20061104LG,

stomacher

130-1597D

exp. 2025-06-

1 glove (size L) doffed

GLV

1-L bottle

Marine Fabric,

50-mL

5 cm x 5 cm (~2 inch x ~2 inch)

VViViD Bycast 65

conical

Black Matte

MFAB

400-mL
stomacher

15 cm x 15 cm (~6 inch x ~6 inch)

Other Supplies and Equipment

• Micropipette filter tips

• Biohazard bags

Performed by:

Solid Waste WI-SPIKE

Date:

Page 1 of 5

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WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES

Equipment

Item

Manufacturer

Serial Number

Thermometer/Rees
#

Calibration
Due

Biosafety
Cabinet (BSC)

The Baker Company

N/A

Micropipette
Type:L1000

Rainin

N/A

Micropipette
Type:L200

Rainin

N/A

Micropipette
Type:L10 or
L20

Rainin

N/A

Refrigerator

Fisher

Incubator

N/A = Not Applicable

III. PROCEDURE

A. Decontaminate the BSC with DNA Erase, bleach, and isopropanol prior to use.

B. Name swatches

1. Label each sample with ID.

i. AAA-BBB-CCC-DDD

1. AAA = Sample #

2. BBB = Material

3. CCC = Extractive Method

4. DDD = Spore Spike Level

ii. Electronically populate below table with sample names to be prepared on each
day from the Sample Log.

Sample
#

Material

Extractive Method

Spore Spike
level (CFU)

Replicate

Sample ID

Performed by:

Solid Waste WI-SPIKE

Date:

Page 2 of 5

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WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES

Sample
#

Material

Extractive Method

Spore Spike
level (CFU)

Replicate

Sample ID

C. Spike test swatches

1. Prepare spiking stocks

i. Fill in information from stock tube.

Organism

Lot

Concentration

B. a. Sterne

05282020

2.2 X 10scfu/mL

ii. Target stock concentrations:

Concentration

Total spores per 100 ^L

6.0 X 104cfu/mL

6,000

6.0 X 103 cfu/mL

600

6.0 X 102 cfu/mL

6.0 X101 cfu/mL

iii. Prepare dilutions of stock in sterile PBST. Vortex stock on high for 30 seconds
prior to preparing dilutions.

Show calculations:

X 10s cfu/mL)*(X)=(6.0 X 107cfu/mL)(l ml.) 273 jiL of stock into 727 PBST

X 107 cfu/mL)*(X)=(6.0 X 10scfu/mL)(l ml.) 100 of Dilution 1 into 900 PBST

X 10s cfu/mL)*(X)=(6.0 X 105cfu/mL)(1.5 mL) 150 nLof Dilution 2 into 1350 PBST

X 10s cfu/mL)*(X)=(6.0 X 104cfu/mL)(1.5 mL) 150 of Dilution 3 into 1350 PBST

X 104 cfu/mL)*(X)=(6.0 X 103 cfu/mL)(1.5 mL) ^ 150 ^L of Dilution 4 into 1350 ^L PBST

X 103 cfu/mL)*(X)=(6.0 X 102cfu/mL)(1.5 mL) 150 nLof Dilution 5 into 1350 ^L PBST

X 102 cfu/mL)*(X)=(6.0X lO^fu/mLHl.S mL) 150 nLof Dilution 6 into 1350 ^L PBST

Spike Tyvek 400, Tychem 6000, Nitrile glove, and/or Marine Fabric materials as
described below.

i. Lay bench paper onto BSC surface.

Date:

Page 3 of 5
Solid Waste WI-SPIKE

Dilution 1: (2.2
Dilution 2: (6.0
Dilution 3: (6.0
Dilution 4: (6.0
Dilution 5: (6.0
Dilution 6: (6.0
Dilution 7: (6.0
2.

Performed by:

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WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES

ii. Position materials onto bench paper with a flat 4-cm x 4-cm area easily
accessible for spiking.

iii. Prior to spiking, vortex the stock for 30 seconds.

iv. Per material, transfer a 120-piL aliquot of the appropriate Stock tube (Dilution 4
for 6,000 CFU, Dilution 5 for 600 CFU, Dilution 6 for 60 CFU, or Dilution 7 for 6
CFU) into a 1.5-mLtube.

v. Place twenty (20) 5-piL droplets onto each material within a 4-cm x 4-cm grid of
four rows with 5 droplets. The same pipette tip can be used to place all twenty
droplets, dispose of the 120-^L aliquot once each material has been spiked.
Avoid spiking a seam.

vi. Allow droplets to dry, once visibly dry, package into appropriate extraction
container. Note: Nitrile gloves will need to be donned and doffed (inside-out)
following HA 473 prior to placing into extraction containers.

Dry Start Time: Dry End Time:

vii. If samples are not processed immediately, double contain the materials and
store @ 2 - 8 °C.

2-8 °C Start time: Date/Initials:

3. Spike double-braided nylon rope (1/4" or 1" diameter) samples

i. Lay bench paper onto BSC surface.

ii. Position %" rope into petri dishes, position 1" rope onto bench paper for spiking.

iii. Prior to spiking, vortex the stock for 30 seconds.

iv. Per material, transfer a 120-^L aliquot of the appropriate Stock tube (Dilution 4
for 6,000 CFU, Dilution 5 for 600 CFU, Dilution 6 for 60 CFU, or Dilution 7 for 6
CFU) into a 1.5-mLtube.

v. Place twenty 5-piL droplets onto each material evenly distributed across the
length of the rope section. The same pipette tip can be used to place all twenty
droplets, dispose of the 120-piL aliquot once each material has been spiked.

vi. Allow droplets to dry, once visibly dry, package into appropriate extraction
container.

Dry Start Time: Dry End Time:

vii. If samples are not processed immediately, double contain the materials and
store @ 2 - 8 °C.

2-8 °C Start time: Date/Initials:

D. Enumerate stock

i. Spread lOO-piL aliquots of Dilutions 5, 6, and 7 onto Blood Agar in triplicate.

ii. Incubate plates

1. Invert the plates and incubate them at 36°C ± 2°C for 18 - 24 hours. 6.
o. Sterne produces flat or slightly convex, 2-5 mm colonies, with edges

Performed by:

Solid Waste WI-SPIKE

Date:

Page 4 of 5

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WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES

that are slightly irregular and have a "ground glass" appearance and
does not lyse sheep or horse blood.

Incubation start Date/Time: Initials:

Incubation end Date/Time: Initials:

iii. Plate counts

1. Record counts in the below table.

Dilution #

Media Type

Volume/
(Dilution on
Plate)

Plate Counts

Average
Counts

CFU/mL

Plate
1

Plate
2

Plate
3

5 (6.0 X 103 cfu/mL)

Blood Agar

100 |iL/
(10-1)

6 (6.0 X 102cfu/mL)

Blood Agar

100 |iL/
(10-1)

7 (6.0 X 101 cfu/mL)

Blood Agar

100 yU
(10-1)

Reviewed by: Date:

Performed by:

Solid Waste WI-SPIKE

Date:

Page 5 of 5

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APPENDIX C: WORK INSTRUCTION FOR
BACILLUS ANTHRACIS STERNE SPORE
RECOVERY

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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY
I. PURPOSE/SCOPE

To recover B. anthracis Sterne spores from test articles (swatches).

II. MATERIALS/EQUIPMENT

Materials

Item

Manufacturer

Lot Number

Exp.
Date

Storage
Temp.

Initials & Date

(1st) Extraction Buffer,
IX PBS w/0.05%
Tween® 20 + 30%
Ethanol (PBSTE)

Inhouse

2-8 °C

Second Extraction
Buffer (IX PBS with 30%
Ethanol)

IX PBS w/0.05%
Tween20 (PBST)

Teknova

Stomacher Lab Blender
Bags

Seward

N/A

Stomacher Bag Racks

Seward

BA6096

N/A

Conical tubes, 15-mL

N/A

R.T.

Falcon Conical Tube,
50-mL

N/A

R.T.

Sterile screw top flask,
250-mL or 500-mL

N/A

R.T.

2-mL screw cap tubes

N/A

R.T.

Sterile disposable
forceps

Unomedical

N/A

R.T.

N/A = Not Applicable

Equipment

Item

Manufacturer

Serial Number

Thermometer
/Rees #

Calibration
Due

Initials &
Date

Biosafety
Cabinet (BSC)

The Baker
Company

57553

N/A

57544

Micropipette
Type:L1000

Rainin

N/A

Incubator
Shaker

New Brunswick

590644988

Refrigerator

Fisher

C3274822

115

Performed by:

Solid Waste WI-SPORE RECOVERY

Date:

Page 1 of 8

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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY

Item

Manufacturer

Serial Number

Thermometer
/Rees #

Calibration
Due

Initials &
Date

Swinging Bucket
Centrifuge

Beckman Coulter

X59221

N/A

Stomacher

Seward

40142

N/A

N/A = Not Applicable

Samples - Electronically update this table wit

h samples names.

Sample
#

Material

Extractive
Method

Spore Spike
level (CFU)

Replicate

Sample ID

Other Supplies and Equipment

• Forceps

• Biohazard bags

• Bleach

• 5-mL, 25-mL and 100-mL Serological Pipets

• Pipette aid

• Ziplock bags

• 1-L Nalgene bottles (Sterile)

Performed by:

Solid Waste WI-SPORE RECOVERY

Date:

Page 2 of 8

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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY

III. PROCEDURE

A. Spore recovery using Stomacher (15-cm section of V" rope, nitrile glove, and 15-cm x 15-cm marine
fabric)

Note: Process samples from negative control to high inoculation level. Change gloves when working
from an inoculated sample to a sample containing a lower inoculation level, or if contamination of
gloves is suspected. Pre-aliquot reagents from the kit to prevent contamination of reagents between
runs.

1. Prior to sample processing, prepare the following items:

• Fill sample tube rack with 50-mL screw cap conical tubes and label as appropriate, two 50-mL
conical tubes are required per sample.

• Document filter vial and sample tube labels.

• 1,500 mL of Extraction Buffer with Tween® 20 + Ethanol will be needed per set of 16 samples
(90 mL per sample)

2. Add 90 mL cold (4°C) extraction buffer with Tween'1' 20 + Ethanol to each Stomacher bag.

3. Aseptically add sample swatch to a Stomacher bag. Open one bag at a time, close and seal bag prior
to moving to the next sample.

4. Place an unsealed bag containing a sample swatch into the Stomacher so the swatch rests evenly
between the homogenizer paddles and stomach each sample for 1 minute at 260 rpm. Open the
door of the Stomacher and remove the bag. Reseal bag.

5. Stomach all sample swatches, removal of bag from Stomacher begins the settle time. Allow bags to
sit for 10 minutes to allow elution suspension foam to settle.

6. Grab the swatch from the outside of the bag with hands. With the bag closed, move the swatch to
the top of the bag while using hands to expel liquid from the swatch.

7. Open the bag, remove swatch and place into a labeled 50-mL tube using sterile forceps. Store
swatch at 2 - 8 °C until enrichment in BHIB (See Wl #7: BHIB Enrichment for Culture).

8. Follow steps described above for each sample, changing forceps between samples.

9. Gently mix the suspension in the Stomacher bag up and down three times with a sterile 50-mL pipet.
Remove half of the suspension volume (~45-46 mL) and place it in a 50-mL screw cap centrifuge
tube (Aliquot 1). Place the remaining suspension (~45-46 mL) into a second 50-mL tube (Aliquot 2).
Adjust the suspension volumes so that volume is equal in both tubes.

10. Process the suspension for each sample, as described above.

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

Page 3 of 8

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Solid Waste WI-SPORE RECOVERY

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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY

12. Centrifuge tubes at 3,500 x g with the brake off, for 15 minutes in a swinging bucket rotor at 4°C.

13. Each sample has two pelleted aliquots (Aliquot 1 and Aliquot 2). Using a sterile 50-mL pipet, remove
the supernatant from Aliquot 1 and discard it in an autoclavable leak-proof biohazard container. The
pellet might be easily disturbed and not visible, so keep the pipet tip away from the bottom of the
tube. Stop pipetting when meniscus reaches the 5-mL gradation level on the 50-mL Falcon tube,
leaving ~2-3 mL supernatant. Next, using the same pipet, remove 20 mL of supernatant from
Aliquot 2 and add it to Aliquot 1 pellet. Discard the remaining supernatant from Aliquot 2 into an
autoclavable leak-proof biohazard container.

14. Vortex Aliquot 1 (containing ~22 mL of supernatant) for 30 seconds to resuspend the pellet, then
transfer entire volume to Aliquot 2.

15. Vortex Aliquot 2 for 30 seconds to resuspend the pellet. This pooled suspension of ~25 mL will be
used for culture and RV-PCR analytical methods, 12.5 mLfor RV-PCR (See Wl: RV-PCR Processing)
and ~12.5 mL for culture (Wl: Culture of Recovered Spores). Record total volume for each sample in
Table 1. Store aliquot on ice or in refrigerator until processed on same day.

Table 1. Volume of sample recovered from samples swatches.

Sample
Number

Sample ID

Total volume recovered
from sample swatches

Recorded by:

Performed by:

Solid Waste WI-SPORE RECOVERY

Date:

Page 4 of 8

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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY

B. Spore recovery from sample swatches in 50-mL tubes (5-cm section of 1/4" rope, 5-cm x 5-cm of
marine fabric).

Note: Process samples from negative control to high inoculation level. Change gloves when working

from an inoculated sample to a sample containing a lower inoculation level, or if contamination of
gloves is suspected. Pre-aliquot reagents to prevent contamination of reagents between runs.

16. Prior to sample processing, prepare the following items:

• In a BSC, attach the RV-PCR vacuum manifold to the vacuum trap, waste container (with 90 ml
of household bleach [8.25% NaOCI], 5.625 mL per sample), and vacuum source. Attach the filter
vials to the manifold, using outer rows first. Verify that all filter vials are completely pushed
down. Place a red pull tab tapered plug in each filter vial.

• 15-mL and 50-mL conical tube per sample

• 250-mL aliquot of Extraction Buffer with Tween® 20 + Ethanol per set of 16 samples (15 mL per
sample)

• 170-mL aliquot of Extraction Buffer + Ethanol without Tween'1' 20 per set of 16 samples (10 mL
per sample)

17. For marine fabric, using gloved hands, place mesh support over swatches (inoculated side facing
mesh) in 50-mL tubes by holding the swatches to the side of the tube with sterile forceps and
placing the coiled mesh support on top. Ensure the sample and mesh are in the bottom half of the
tube (avoiding the conical portion). Do not touch any other surface with gloved hand that was used
to position mesh over spiked swatch in conical tube. Do not use mesh for 1/4" rope sections.

Change gloves in between each sample.

Note: The support keeps the swatch from interfering with pipetting activities and improves
efficiency of spore extraction during vortexing.

18. First Extraction: Bleach wipe each tube. Add 15 mL cold (4°C) First Extraction Buffer (1 x PBS with
Tween'1' 20 + 30% Ethanol) to samples. Uncap one tube at a time, add extraction buffer, close tube
and Parafilm cap prior to moving to the next sample tube. Bleach wipe each tube.

19. Place tube rack in plastic bag, seal, double bag, and bleach the bag prior to transferring to the
platform vortexer located outside the BSC.

20. Vortex samples for 20 minutes on Platform vortexer with the speed set to 7. Make sure to clamp
tube rack from the top and bottom of the vortexer.

Start time: End Time: Speed:

21. After vortexing, transfer sample tube rack to the BSC. Remove tube rack from plastic bag and
discard the bag.

22. Vortex up to 8 sample tubes on a single-tube vortexer 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 necessary, allow the tubes to settle for up to 5 minutes.

Performed by:

Solid Waste WI-SPORE RECOVERY

Date:

Page 5 of 8

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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY

23. Remove Parafilm, bleach wipe the tube, uncap tubes one at a time. Using a 10-mL serological pipet
carefully transfer 12.5 mLto clean clearly labeled 50-mL conical tube (extract pool). Recap 50-mL
conical tube and move to the next sample.

24. Second Extraction: Add 10 mL of cold (4°C) Second Extraction Buffer (1 x PBS with 30% Ethanol) to
each sample tube, one at a time with a new 10-mL serological pipet for each sample and recapping
each sample tube after buffer addition.

25. After adding extraction buffer to all the tubes, check that all caps are tight and Parafilm each cap.
Place rack in double plastic bags, seal and bleach the outer bag. Transfer double bagged tube rack to
platform vortexer located outside the BSC.

26. Vortex rack for 10 minutes, with speed set to 7.

Start time: End Time: Speed:

27. Move the rack back to the BSC. Discard bags and vortex tubes for 3-5 seconds and allow large
particles to settle for at least 2 minutes.

28. Remove Parafilm, bleach wipe the tube, uncap tubes one at a time. Using a 10-mL serological pipet
carefully transfer ~12.5 mLto corresponding extract pool 50-mL conical tube, but carefully avoid
settled particles during aliquoting. Recap 50-mL conical tube and move to the next sample. Note:
Save swatch and store 2-8 °C until enrichment in BHIB on same day (See Wl: BHIB Enrichment for
Culture).

29. This pooled suspension of ~25 mL will be used for culture and RV-PCR analytical methods, 12.5 mL
for RV-PCR (See Wl: RV-PCR Processing) and ~12.5 mLfor culture (Wl: Culture of Recovered
Spores). Store aliquot on ice or in refrigerator until processed on same day.

C. Spore recovery for 1-L bottle extractive samples (Tyvek400, TyChem6000,1" diameter rope, nitrile
glove)

30. Prior to sample processing, prepare the following items:

• In a BSC, attach vacuum manifold to waste container containing appropriate amount of bleach
for a final concentration of 1% NaOCI after collecting all waste fluids. For extractive methods,
each sample is 500 mL, add 75 mL of household bleach (8.25% NaOCI) per sample. Waste
container may need to be emptied multiple times.

• Document filter vial and sample tube labels.

• 350 mL of Extraction Buffer with Tween® 20 + Ethanol per set of 16 samples (20 mL per sample)

31. Add 20 mL of Extraction Buffer with Tween 20 + 30% Ethanol (PBSTE) to a MicroFunnel unit with
0.45 pirn GN-6 Metricel membrane (Pall ID: 4800 or equivalent), this filtration unit will be referred to
as the filter unit. Apply vacuum, after PBSTE completely passes through membrane, turn off vacuum

Page 6 of 8

Performed by: Date:

Solid Waste WI-SPORE RECOVERY

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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY

and apply the membrane to a TSA plate using sterile forceps. This will serve as a negative control.
Incubate this control at 30 °C overnight and check for sterility.

Start time End time Sterility (Yes/No)

32. Carefully add 500 mL of sterile phosphate buffered saline with 0.05% Tween20 (PBST) to each 1-L
sample bottle. Seal tubes tightly, wipe with bleach, and seal with Parafilm before removing from
BSC.

33. Shake vigorously for 2 minutes by hand. Option to shake outside of hood if sample is placed into
secondary bag with absorbent.

a. Grasp 1-L sample bottle with one hand on bottom of bottle, the other hand around the
bottle near the top. Hold bottle over shoulder and shake vigorously back and forth.

34. (Optional) Pour off eluent into clean, labeled, 500-mL container. Ensure sample labels for each
collection bottle match their respective eluent bottle.

35. Vigorously mix 0.5-L eluate aliquots by hand for 30 seconds. Allow 30 seconds of settle time.

36. Pour mixed eluate into filter unit to the 100-mL gradation line. An inoculating loop or needle can be
used to prevent the sleeve or glove from interfering with liquid transfer.

37. Apply vacuum until entire 100 mL passes through membrane. Once complete, break vacuum
pressure, then close valve. Add additional eluate until full 500-mL recovered volume is filtered onto
one filter membrane per sample.

Note: If filter becomes clogged, less than 500 mL of sample will be processed. Record volume
filtered in the below table. At 30 minutes post-sample addition, if sample has not completely passed
through the filter, the remaining volume in the filter unit will be removed.

Sample
#

Sample ID

Filtration Start
Time

Filtration End
Time

Total Volume
Filtered

Recorded By:

1-TYVK-1L-0-1

2-TYVK-1L-30-1

3-TYVK-1L-30-2

4-TYVK-1L-30-3

5-TYVK-1L-300-1

6-TYVK-1L-300-2

7-TYVK-1L-300-3

8-TYVK-1L-3000-1

9-GLV-1L-0-1

10-GLV-1L-30-1

11-GLV-1L-30-2

12-GLV-1L-30-3

13-GLV-1L-300-1

Performed by:

Solid Waste WI-SPORE RECOVERY

Page 7 of 8

Date:

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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY

Sample
#

Sample ID

Filtration Start
Time

Filtration End
Time

Total Volume
Filtered

Recorded By:

14-GLV-1L-300-2

15-GLV-1L-300-3

16-GLV-1L-3000-1

38. Remove the filter membrane using sterile forceps and transfer to a 50-mL conical tube. Position the
membrane in the bottom half of the conical tube with the inlet side of the membrane facing the
center of the tube. Avoid placing the filter into the conical portion of the tube.

39. Add 10 mL of 1 x PBS with Tween® 20 + 30% Ethanol (PBSTE) to 50-mL conical tubes containing
membrane filters.

40. Vortex at maximum speed on platform vortex in 10 second bursts for 2 minutes to dislodge spores.

41. Let tubes settle for 2 minutes, then transfer volume to a clean 50-mL conical tube.

42. Repeat extraction of each membrane filter by adding another 10 mL of PBSTE to the 50-mL conical
tube with membrane, vortex at max speed on platform vortex in 10 second bursts for 2 minutes,
and let tubes settle for 2 minutes.

43. Transfer volume to the same 50-mL conical tube per sample for a total recovered pooled spore
recovery volume of 20 mL. Observe membrane, and record if particulates were suspended in
solution or remain adhered to filter.

Membrane Observations:

Note: Save swatch. Store at 2 - 8 °C until enrichment in BHIB (See Wl: BHIB Enrichment for Culture).
Full sleeves and gloves may not be BHIB enriched due to large size of sample and media volume
required to cover the sample.

44. Vortex each 20-mL sample, half volume will be used for culture and RV-PCR analytical methods,
10 mL for RV-PCR (See Wl: RV-PCR Processing) and 10 mL for culture (Wl: Culture of Recovered
Spores). Store aliquot on ice or in refrigerator until processed on same day.

IV. Technical Review

Performed by: Date:

Performed by:

Solid Waste WI-SPORE RECOVERY

Date:

Page 8 of 8

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APPENDIX D: WORK INSTRUCTION FOR
CULTURE OF BACILLUS ANTHRACIS
STERNE SPORES RECOVERED FROM
SWATCHES

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WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS STERNE SPORES

RECOVERED FROM SWATCHES

I. PURPOSE/SCOPE

Culture of B. anthracis Sterne spores recovered from swatches.

II. MATERIALS/EQUIPMENT

Materials

Item

Manufacturer

Lot Number

Exp.
Date

Storage
Temp.

Initials & Date

PBS with Tween
(0.05%)

Teknova

2-8 °C

Microfunnel filters

PALL

R.T.

Blood Agar

2-8 °C

N/A = Not Applicable

Equipment

Item

Manufacturer

Serial Number

Thermometer/
Rees#

Calibration
Due

Initials &
Date

Biosafety
Cabinet (BSC)

The Baker Company

N/A

Stationary
Incubator

Precision

9509-003

N/A

Vacuum
manifold

Gelman Sciences

N/A

Micropipette
Type:L200

Rainin

N/A

Micropipette
Type:L1000

Rainin

N/A

N/A = Not Applicable

Other Supplies and Equipment

• Forceps

• Bleach

• 5-mL, 10-mL, and 25-mL Serological Pipettes

• Pipette aid

Performed by:

Solid Waste WI-Culture-4 (vl May 19, 2021)

Date:

Page 1 of 5

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WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS STERNE SPORES

RECOVERED FROM SWATCHES

Electronically update this table wit

h samples names

Sample
#

Material

Extractive Method

Spore Spike
level (CFU)

Replicate

Sample ID

III. PROCEDURE

Note: The following procedure is to be carried out with recovered from spore recovery work
instruction. Process 2 PBST only negative control filter funnels alongside samples, one with first set
and one with last set

A. Culture Method

1. Label filter funnels and Blood Agar plates per sample as indicated below.

Sample #

Sample ID

Volume to plate (0.01 mL, 0.1 mL, 1 mL,
2mL, 4 mL, or 8 mL)

N/A

PBST controls (2)

8 mL

Performed by:

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Date:

Page 2 of 5

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WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS STERNE SPORES

RECOVERED FROM SWATCHES

Sample #

Sample ID

Volume to plate (0.01 mL, 0.1 mL, 1 mL,
2mL, 4 mL, or 8 mL)

2. For samples that are to be spread plated on Blood Agar (0.01 mL, 0.1 mL, or other dilution), spread
0.1 mL of neat sample or appropriate serial dilution in triplicate.

a. For dilutions, serial 1:10 dilutions to be prepared by adding 1 mL of sample (or dilution) into
9 mL of PBST.

3. For samples that are to be concentrated onto a filter membrane, > 1-mL volumes, follow below
steps. Black membranes should be used when possible.

a. Place the filter funnels onto the vacuum manifold in a Class II BSC.

b. Add 5 mL of PBS with 0.05% Tween (PBST) to each filter funnel. Apply vacuum.

c. With the vacuum valve closed and the vacuum pressure released, place 10 mL of PBST into
each filter cup.

d. Vortex each sample. For each sample, add appropriate volume of pooled extract to one
filter funnel. Apply vacuum.

e. Close the vacuum valve and release the vacuum pressure. Rinse the walls of each filter
funnel using 10 mL of PBST. Apply vacuum.

f. With the vacuum valve closed and the vacuum pressure released, remove the membrane
from the filter funnel and place onto TSA. Dispose of filter bases and then change gloves.

B. Plate incubation

1. Incubate plates inverted overnight at 36 ± 2°C.

Incubation start Date/Time: Initials:

Incubation end Date/Time: Initials:

B. a. Sterne produces flat or slightly convex, 2-5 mm colonies, with edges that are slightly
irregular and have a "ground glass" appearance and does not lyse sheep or horse blood.

Performed by:

Solid Waste WI-Culture-4 (vl May 19, 2021)

Date:

Page 3 of 5

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WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS STERNE SPORES

RECOVERED FROM SWATCHES

2. Enter results into enumeration table(s).

Table 1. Filter Membrane Plating

Sample ID

B. anthracis Sterne
colonies

Total colonies (All
morphologies)

CFU/ mL

PBST Negative #1

N/A

PBST Negative #2

N/A

Table 2. Spread Plating

Sample ID

B. anthracis Sterne
colonies

Total colonies (All
morphologies)

0.1 mL

PBST Negative Control

N/A

Performed by:

Solid Waste WI-Culture-4 (vl May 19, 2021)

Date:

Page 4 of 5

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WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS STERNE SPORES

RECOVERED FROM SWATCHES

Sample ID

B. anthracis Sterne
colonies

Total colonies (All
morphologies)

0.1 mL

IV. Technical Review

Reviewed by: Date:

Performed by:

Solid Waste WI-Culture-4 (vl May 19, 2021)

Date:

Page 5 of 5

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APPENDIX E: WORK INSTRUCTION FOR
RV-PCR PROCESSING OF BACILLUS
ANTHRACIS

-------
WORK INSTRUCTION FOR RV-PCR PROCESSING OF BACILLUS ANTHRACIS
I. PURPOSE/SCOPE

Concentrate spore recovery onto filter vials and enrich (RV-PCR).

II. MATERIALS/EQUIPMENT

Materials

Item

Manufacturer

Lot Number

Exp.
Date

Storage
Temp.

Initials & Date

10X PBS

Teknova

2-8 °C

IX PBS (pH 7.4)

Teknova

2-8 °C

BHIB

2-8 °C

Sterile screw top flask,
250-mL or 500-mL

N/A

R.T.

0.45-nm filter vials
(AV125NPUAQU)

Whatman

N/A

R.T.

2-mL screw cap tubes

N/A

R.T.

Equipment

Item

Manufacturer

Serial Number

Thermometer
/Rees #

Calibration
Due

Initials &
Date

Biosafety
Cabinet (BSC)

The Baker
Company

57553

N/A

57544

Micropipette
Type:L1000

Rainin

N/A

Incubator
Shaker

New Brunswick

590644988

Refrigerator

Fisher

C3274822

115

N/A = Not Applicable

Samples - Electronically update this table with samples names.

Sample
#

Sample
Type

Method

Spore Spike

Level

(CFU/mL)

Replicate

Sample ID

Performed by:

Solid Waste WI-RV-PCR

Date:

Page 1 of 5

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WORK INSTRUCTION FOR RV-PCR PROCESSING OF BACILLUS ANTHRACIS

Sample
#

Sample
Type

Method

Spore Spike

Level

(CFU/mL)

Replicate

Sample ID

Other Supplies and Equipment

• Forceps

• Biohazard bags

• Bleach

• 5-mL, 25-mL, and 100-mL Serological Pipets

• Pipette aid

• Ziplock bags

• Vacuum pump set to 5-10 psi
III. PROCEDURE

RV-PCR Processing Method

1. Method Start Time:

2. Prior to sample processing, prepare the following items:

• In a BSC, attach the RV-PCR vacuum manifold to the vacuum trap, waste container (with 90 mL
of household bleach [8.25% NaOCI], 5.625 mL per sample), and vacuum source.

• Attach the filter vials to the manifold, using outer rows first. Verify that all filter vials are
completely pushed down. Place a red pull tab tapered plug in each filter vial.

• Document filter vial and sample tube labels.

• 225-mL aliquot of high salt wash buffer (lOx PBS) in a 250-mL screw capped bottle per set of
16 samples (12.5 mL per sample).

• 225-mL aliquot of low salt wash buffer (lx PBS) in a 250-mL screw capped bottle per set of
16 samples (12.5 mL per sample).

• 100-mL aliquot of BHIB in sterile bottle

3. Place into BSC: a ziplock bag with orange caps (one per filter vial), 10-mL serological pipets and cold
(4°C) 10X PBS in 250-mL screw cap bottle.

Performed by:

Solid Waste WI-RV-PCR

Date:

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WORK INSTRUCTION FOR RV-PCR PROCESSING OF BACILLUS ANTHRACIS

4. Vortex each sample, then allow 30 seconds of settle time to avoid transferring large particulates into
RV-PCR filter vial. Transfer volume of each sample to corresponding labeled RV-PCR filter vial.
Samples with 20-mL volume will need to be added in two steps since the filter vial only holds

~12 mL.

5. Complete filtration of liquid through RV-PCR filter vials. Turn off vacuum pump.

Note 1: At 15 minutes post-sample addition, if sample has not completely passed through the filter
vial, a reduced volume of high salt wash buffer will be added (5 mL) to avoid prolonged filtering
delays - it is desired to add a lower volume of each salt wash buffer (10X and IX) than to omit one
or both entirely.

Note 2: At 1-hour post-sample addition, if sample has not completely passed through the filter vial,
the high salt and low wash steps will be omitted.

Sample
#

Sample ID

Volume
Processed

Sample Addition

Volume of
Wash Buffers

Recorded
by:

(mL)

Start
Time1

End
Time2

10X

Record the time of adding the final sample to filter vial.

2Record end time for samples that have clogging and meet the criteria in Notes 1 and 2 above.

6. Transfer 12.5 mL of cold (4°C) high salt wash buffer (lOx PBS) to each filter-vial using a 10-mL
serological pipet. Change pipet and gloves between each sample.

7. Complete filtration of liquid through the filter vials.

8. Place into the BSC: 10-mL serological pipets and cold (4°C) IX low salt wash buffer in 250-mL screw
cap bottle.

9. Transfer 12.5 mL cold (4°C) low salt wash buffer (lx PBS) to each filter-vial using a 10-mL serological
pipet. Change pipet and gloves between each sample.

Page 3 of 5

Performed by: Date:

Solid Waste WI-RV-PCR

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WORK INSTRUCTION FOR RV-PCR PROCESSING OF BACILLUS ANTHRACIS

10. Complete filtration of liquid through filter vials. Turn off vacuum pump.

11. Using an Allen wrench, unscrew the top of the manifold and break the seal on manifold using a plate
sealer to separate the top of the manifold.

12. Using a tray preloaded with caps, move the top of the manifold with the filters still in place and
firmly press down, capping the bottoms of the filters. Repeat pressing down on each filter vial to
ensure a good seal.

13. Place bleach-soaked wipes onto the manifold to soak up the filtered waste and disinfect for 20
minutes.

14. Place into the BSC: 5-mL serological pipets, 1,000-nL pipet, 1,000-nLtips, cold (2-8°C) BHIB aliquoted
in 50-mL conical tubes, sharps container and orange caps.

15. Pipet 5 ml. of cold BHIB into each filter vial using a 5-mL serological pipet. Use a new pipet for each
filter vial. Dispose of the red cap and place the orange cap firmly into the top of the filter. Change
gloves between each sample.

16. Record the time of the BHIB addition, this represents T0. Bleach wipe the filter vial.

Time of BHIB addition:

17. Place the rack of capped filter vials in a plastic bag, seal, double bag, and bleach the bag.

18. Vortex the filter vials for 10 minutes on the platform vortexer, setting 7.

Start time: End Time: Speed:

19. Place 2-mL screw cap tubes for T0 aliquots onto ice in the BSC.

20. After vortexing, transfer filter vials to the BSC. Remove bag.

21. Uncap one filter vial at a time and open the corresponding 2-mL tube. Using a 1-mL pipette or
serological pipet (if filter deteriorated), gently pipet up and down ~10 to mix. Transfer 1 mLfrom
each vial to the corresponding pre-chilled (on ice) 2-mL screw cap tube for T0. Cap the tube and
place it back onto ice. Wipe the filter vial with a bleach-soaked lab wipe. Change gloves between
each sample.

After transferring the T0 aliquots for all samples, place the filter vial rack in a transfer container,
seal, and bleach the container. Store the T0 aliquot at -20 °C overnight.

To -20 C storage start time: End time: Initial/Date:

22. Transfer the filter vial rack to the shaker incubator. Secure the rack. Incubate at 36 ± 2 °C at 230
rpm, overnight (i.e., 16 hours from the addition of BHIB to the filter vials). These samples are

Performed by:

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Date:

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WORK INSTRUCTION FOR RV-PCR PROCESSING OF BACILLUS ANTHRACIS

referred to as the Tf samples. Following incubation record turbidity observation and volume
remaining in the table below. Incubate leftover BHIB with enriched samples as sterility check of
media (can be incubated stationary).

Start time: End Time: Speed: Temperature:

Sample
Number

Sample ID

Turbid (Yes/No)

Volume

Remaining (mL)

Recorded by:

N/A

BHIB Sterility Check

N/A

23. Proceed to Wl: DNA Purification to process T0 and Tf samples.
IV. Technical Review

Reviewed by: Date:

Performed by:

Solid Waste WI-RV-PCR

Date:

Page 5 of 5

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APPENDIX F: WORK INSTRUCTION FOR
MANUAL DNA EXTRACTION AND
PURIFICATION FROM BACILLUS SPECIES

-------
WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION

FROM BACILLUS SPECIES

I. PURPOSE/SCOPE

Manual DNA extraction and purification Bacillus species from recovered swatches.

II. MATERIALS/EQUIPMENT

Materials

Item

Manufacturer

Lot Number

Exp.
Date

Storage
Temp.

Initials & Date

Lysis Buffer

Promega

PMPs

Promega

Salt Wash Solution

Promega

Alcohol Wash

Promega

70% Ethanol

Inhouse

Elution Buffer

Promega

N/A = Not Applicable

Equipment

Item

Manufacturer

Serial Number

Thermometer/
Rees#

Calibration
Due

Initials &
Date

Biosafety
Cabinet (BSC)

The Baker Company

57544

N/A

Micropipette
Type:L200

Rainin

N/A

Micropipette
Type:L200

Rainin

N/A

Micropipette
Type:L1000

Rainin

N/A

Micropipette
Type:L1000

Rainin

N/A

Ultra-low
Freezer

Woods

X34664

Refrigerator

Thermo Fisher

35840

Centrifuge

Eppendorf

X58983

N/A

Heat block

VWR

949039

N/A

Thermometer

N/A

N/A = Not Applicable

Other Supplies and Equipment

• Micropipette tips

• Biohazard bags

• Bleach

• Prepare tubes

Performed by: Date:

Solid Waste Wl-Manual DNA Extraction and Purification

Page 1 of 5

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WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION

FROM BACILLUS SPECIES

III. PROCEDURE

A. 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. Prepare 70% Ethanol by adding 6 mL of sterile water to
14 mL EtOH. Transfer sufficient volume of buffer to sterile, 100-mL reservoir immediately before use. Pre-
heat heat block to 80°C.

NOTE: Process samples from negative control to high inoculation level. Change gloves when moving
from an inoculated sample to a sample containing a lower inoculation level, or if contamination of
gloves is suspected. Pre-aliquot reagents from the kit to prevent contamination of reagents between
runs.

1. After the overnight (16 h) incubation, remove the filter vial manifold from the shaker incubator. Thaw
To aliquots if they were stored at -20°C.

2. Vortex filter vials for 10 minutes on platform vortexer with speed set to 7.

Start: End: Speed:

3. Transfer the filter vial manifold to the BSC, remove and discard bags.

4. Set up 2-mL screw cap tubes for Tf aliquots in a tube. Do not use 1.5-mL tubes. Transfer Tf aliquot
screw cap tubes to the BSC.

5. Transfer the filter vial rack to the BSC. Uncap one filter vial at a time and transfer 1 mL to
corresponding 2-mL tube after gently pipetting up and down ~10 to mix.

6. Centrifuge 2-mL screw cap tubes (both T0 and Tf) at 14,000 rpm for 10 minutes (4°C).

Start: End: Speed:

7. Remove 800 piL of the supernatant from each tube, using a 1,000-nL pipet and dispose to waste. Do
not disturb the pellet.

8. Add 800 piL of lysis buffer using a 1,000-nL pipet, using a new tip for each sample. Cap the tubes and
mix by vortexing on high (~1,800 rpm) in 10 second pulses for a total of 60 seconds.

9. Vortex each screw-cap tube briefly (low speed, 5-10 seconds) and transfer the entire sample volume
to a 2-mL Eppendorf tube (ensure the tubes are labeled correctly during transfer). Incubate the T0 and
Tf lysate tubes at room temperature for 5 minutes.

10. 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 Tf lysate
tube.

Performed by: Date:

Solid Waste Wl-Manual DNA Extraction and Purification

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WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION

FROM BACILLUS SPECIES

11. Uncap one tube at a time and add 600 piL of PMPs to each T0 and Tf tube (containing a 1-mL sample).

12. Vortex each T0 and Tf tube for 5-10 seconds at high speed. 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.

13. 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, allowing all PMPs to contact the magnet.

14. 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 tube rack.

15. Uncapping one tube at a time, withdraw all liquid using a 1,000-nL pipet, placing the pipet tip in the
bottom of the 2-mL tube. Be sure to remove all liquid without disturbing PMPs. Use a new pipet tip to
remove any residual liquid, if necessary. If liquid remains in the tube cap, remove by pipetting.

16. Uncap each tube one at a time and add 360 piL of lysis buffer using a 1,000-nL pipet. Vortex on low
setting for 5-10 seconds, and transfer to tube rack.

17. 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 Step A. 13.

18. Remove all the liquid as described in Step A. 15. Use a new tip for each T0 and Tf tube.

Wash Steps:

19. Uncap each tube one at a time and add 360 piL of Salt Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
back on magnetic stand. Invert as described in step A.13. Remove all the liquid as described in Step
A. 15. Use a new tip for each T0 and Tf tube. This is 1st Salt Wash.

20. Uncap each tube one at a time and add 360 piL of Salt Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
back on magnetic stand. Invert as described in step A.13. Remove all the liquid as described in Step
A. 15. Use a new tip for each T0 and Tf tube. This is 2nd Salt Wash.

21. Uncap each tube one at a time and add 500 piL of Alcohol Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
back on magnetic stand. Invert as described in step A.13. Remove all the liquid as described in Step
A. 15. Use a new tip for each T0 and Tf tube. This is 1st Alcohol Wash.

22. Uncap each tube one at a time and add 500 piL of Alcohol Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack

Performed by: Date:

Solid Waste Wl-Manual DNA Extraction and Purification

Page 3 of 5

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WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION

FROM BACILLUS SPECIES

back on magnetic stand. Invert as described in step A.13. Remove all the liquid as described in Step
A. 15. Use a new tip for each T0 and Tf tube. This is 2nd Alcohol Wash.

23. Uncap each tube one at a time and add 500 piL of Alcohol Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
back on magnetic stand. Invert as described in step A.13. Remove all the liquid as described in Step
A.15. Use a new tip for each T0 and Tf tube. This is 3rd Alcohol Wash.

24. Uncap each tube one at a time and add 500 piL of 70% Ethanol. Remove tube rack off of magnetic
stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack back on
magnetic stand. Invert as described in step A.13. Remove all the liquid as described in Step A.15. Use a
new tip for each T0 and Tf tube. This is 4th Alcohol Wash.

25. If necessary, use a 200-uL pipet to remove remaining 70% ethanol, being careful to not disturb PMPs.

26. Open all T0 and Tftubes and air dry for 2 minutes.

27. Close tubes and transfer to heat block. Reopen tubes once placed on the heat block at 80°C until the
PMPs are dry (~20 minutes, or until dry). Allow all the alcohol solution to evaporate since alcohol
could interfere with analysis. If residual condensation is present, do not remove, leave it in place.

Start: End: Temperature:

28. DNA elution: While they are in the heating block add 200 piL of elution buffer to each T0 and Tf tube,
and close tube. Vortex for 10 seconds and place back on heating block for 80 seconds.

29. 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. Reduce vortex speed if liquid appears to enter
the tube cap lid.

30. Repeat Step 29 four more times.

31. Remove the tubes from the heating block, place them in a tube rack in the BSC, and incubate at room
temperature for at least 5 minutes.

Start: End:

32. Briefly vortex each tube (5 - 10 seconds) on low speed and centrifuge at 2000 rpm, 4^Cfor 1 minute.

33. Briefly vortex each tube and place on the magnetic stand for at least 30 seconds.

34. Collect liquid from each T0 and Tf tube and transfer ~80-90 uL to a clean, labeled, 1.5-mL tube on ice
(check tube labels to ensure the correct order). Use a new tip for each tube. Visually verify absence of

Performed by: Date:

Solid Waste Wl-Manual DNA Extraction and Purification

Page 4 of 5

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WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION

FROM BACILLUS SPECIES

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.

35. Centrifuge tubes at 14,000 rpm at 4°C for 5 minutes to pellet any particles remaining with the eluted
DNA; carefully remove supernatant from all samples and transfer to a new 1.5-mL tube using a new tip
for each tube.

Start: End:

36. Store T0 and Tf DNA extract tubes at 4°C until PCR analysis. Continue to WI-RV-PCR-STREAMS.

Note: If PCR cannot be performed within 24 hours, freeze DNA extracts at -20°C.

Labeled:

Date/Time:

Storage Temperature:

Storage Location:

IV. Technical Review

Performed by:

Date:

Comments:

Page 5 of 5

Performed by:

Solid Waste Wl-Manual DNA Extraction and Purification

Date:

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APPENDIX G: WORK INSTRUCTION FOR
REAL-TIME PCR ANALYSIS OF BACILLUS
ANTHRACIS

-------
WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS OF BACILLUS ANTHRACIS

I. PURPOSE/SCOPE

Duplex Rapid Viability PCR analysis for B. anthracis nucleic acid detection.

II. MATERIALS/EQUIPMENT

Enter material lot and expiration dates used onto STREAMS Real-Time PCR - FORM A

Materials

Item

Manufacturer

Product Number

TaqMan Fast Advanced
PCR Mix (2x)

Applied Biosystems

4444556

Platinum Taq DNA
Polymerase

Invitrogen

10966-034

Custom Primers and Probes
w/ 6-FAM reporter dye

Applied Biosystems

Custom

PCR Grade Water

Fisher Scientific

BP2484100

Optical Plate Seal

ThermoFisher

4311971

Equipment

Item

Manufacturer

Serial Number

Thermometer/
Rees #

Calibration
Due

Initials &
Date

Biosafety
Cabinet (BSC)

Baker
Thermo Forma

N/A

Micropipette
Type: 10

N/A

Micropipette
Type: 20

N/A

Micropipette
Type: 200

N/A

Micropipette
Type: 1000

N/A

Freezer

Centrifuge

LabNet

K4070898

N/A

7500 Fast

Applied Biosystems

275017115

N/A

N/A = Not Applicable

Page 1 of 6

Solid Waste Wl Real-Time PCR

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WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS OF BACILLUS ANTHRACIS

Other Supplies and Equipment

• Micropipette tips

• 96-well 0.1-mL FAST plates

• Optical caps

• Bleach

• DNA erase

• 70% isopropanol

Attach STREAMS Real-Time PCR - FORM A, Date:

III. PROCEDURE

A. Prepare samples for qPCR

Note: This step must be performed in the BSC outside the PCR clean room set-up area. Prepare afresh

aliquot of PCR-grade water per sample batch to use for 1:10 dilutions and NTCs.

1. To and TfDNA extracts: Label 1.5-mL tubes with the sample identifier and "10-fold dilution." Add
90 piL of PCR-grade water to the tubes.

2. Mix To and 7/ DNA extracts by vortexing (3-5 seconds), spin at 14,000 rpm for 2 minutes, and transfer
10 piL of supernatant to 1.5-mL Eppendorf tubes with 90 piL of PCR-grade water, maintaining the plate
layout.

Note: No centrifugation is required if PCR analysis is conducted immediately after DNA elution.

B. Real-time PCR Analysis of DNA Extracts

1. Decontaminate the PCR workstation by treating all work surfaces with bleach solution, followed by
70% Isopropanol. 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.

2. Determine the number of reactions that are to be run. 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 each batch of samples, PCR Master Mix should be made for 4 PCs, 4 NTCs, and
6 DNA extracts per sample (3 for T0 and 3 for TfDNA extracts). Record sample names and reaction
numbers on STREAMS Real-time PCR - FORMA.

3. In a clean PCR-preparation hood, pipet 20 piL of Master Mix into the wells of the PCR plate. Label four
wells as NTC.

4. Add 5 piL of PCR-grade water into each of the NTC wells.

Solid Waste Wl Real-Time PCR

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WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS OF BACILLUS ANTHRACIS

5. Lightly seal the NTC wells with optical caps, cover all other wells of the plate using optical caps.

6. Vortex each sample briefly, then add 5 piL to each sample well. Lightly seal the sample wells with
optical caps.

7. Vortex the Positive Control (PC), B. anthracis Sterne DNA [10 pg/1 piL or 50 pg/5 piL] and add 5 piL to
each PC well. Tightly seal the wells with an optical plate seal, using optical caps.

Performed by: Date:

C. Within the Post-Amplification Lab, load 96-well plates onto 7500 Fast.

1. Set up 7500 Fast (TaqMan)

a. Open the 7500 Fast Software and select New Experiment

i. Set Experiment Properties:

1. Enter an experiment name

2. Select 7500 Fast (96 wells)

3. Select Quantitation - Standard Curve

4. Select TaqMan Reagents

5. Select Fast (~40 minutes to complete a run)

ii. Plate Step

1. Define the Target and Samples

a. Define a target with designated reporter (6-FAM for Chromosome and
VIC for pXOl) and None as the quencher. Multiple targets can be
selected if more than one target will be run on the plate.

b. Define samples by selecting Add New Sample for all samples, include
NTCs and Standard Curve concentrations as sample names.

2. Assign Targets and Samples

a. Highlight the wells that will be used for this assay, then check the
assign box to assign the target. Check appropriate Task (Unknown,
Standard, or Negative Control).

b. Highlight the sample wells, then check the assign box to assign the
sample.

c. Highlight the standard curve wells, to enter the sample name, then
enter a quantity for each standard under the assign target pane.

d. Select ROX as the passive reference from the Passive Reference drop
down box.

iii. Run Method

1. Under Graphical View, enter 25 piL as the reaction volume.

2. Set thermocycling conditions to match the below settings:

Temperature (°C)

Time

Cvcles

50.0

2:00

Hold

95.0

2:00

Hold

95.0

0:03

60.0

0:30

25 piLTotal Volume

Solid Waste Wl Real-Time PCR

Page 3 of 6

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WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS OF BACILLUS ANTHRACIS

3. Select Save As, assign unique plate file name and save in project folder,
iv. Start Run

1. Centrifuge the plate at 300 x g for 1-2 minutes at room temperature or in
Labnet's MPS-1000 Mini Plate Spinner briefly. Check that the samples are at
the bottom of the wells and no bubbles are at the bottom of the wells.

2. Select Start Run.

3. When run is complete, burn the file to a CD.

4. Remove 96-well plate from the 7500 Fast and dispose.

Performed by: Date:

D. Analysis

1. Open the assay with the most current version of 7500 Fast software

a. Select the Analysis Tab

b. Select Plot Settings:

i. Plot Type: ARn vs Cycle

ii. Graph Type: Log

iii. Plot Color: Well

c. Select Options:

i. Target: Select target that was assigned to wells

ii. Threshold: Uncheck Auto and Auto Baseline

iii. Show: Check Threshold, Baseline Start

d. In Amplification Plot, set the Threshold to 0.1.

e. In View Well Table, view Ct values for all samples. Adjust the baseline manually in the
Amplification Plot so that the Baseline End is two Ct values below the lowest Ct value whole
number, ignoring values to the right of the decimal. For example, if the lowest Ct value is
22.610105, the Baseline End cursor should be set to 20.

f. After moving Baseline End, recheck the Ct values and adjust again if necessary.

2. Save file with the file extension "_Analyzed"

3. Export Results

a. Select Export

b. Check the Results option, one file

c. Enter a unique plate file name with run date and initials

d. Select file type, .xls (Excel)

e. Browse File Location to save in project-specific location

f. Select Start Export, then Close Export Tool

4. Print Report

a. Select Print Report

b. Check the Below Selections and then Print Report:

i. Experiment Summary

ii. Results Summary

iii. Amplification Plot

iv. Standard Curves

v. Results Table (By Well)

c. Under Analysis Setting, Select Multicomponent Plot

i. Highlight all NTC wells, then select Print from the icon on the Multicomponent Plot

Solid Waste Wl Real-Time PCR

Page 4 of 6

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WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS OF BACILLUS ANTHRACIS

ii. Highlight all Standard wells, then select Print from the icon on the Multicomponent
Plot

iii. Highlight all Sample wells, then select Print from the icon on the Multicomponent Plot
d. Annotate Printouts

i. Initial and date every page

ii. Initial, date, and error or otherwise annotate all errors and comments

iii. Indicate which, if any, wells of the standard curve were omitted

iv. Indicate multicomponent results for each well on the Results Table

Performed by: Date:

5. QC Acceptance Criteria

a. Verify the below acceptance criteria are met

• Amplification in PC wells

• NTC wells have no amplification

IV. Data Calculations

Calculate the average Ct value from the replicate reactions for T0 and 7/ DNA extracts of each
sample. Subtract the average Ct value of the 7/ DNA extract from the average Ct value of the T0 DNA
extract to generate delta Ct value (ACt). If there is no Ct value for the T0 DNA extract (i.e., the T0 is
non-detect), use 45 (total number of PCR cycles used) as the Ct value.

Performed by: Date:

V. Assay Sequences

Below assay sequences from - Letant, S. E., Murphy, G. A., Alfaro, T. M., Avila, J. R., Kane, S. R.,

Raber, E., Bunt, T. M., & Shah, S. R. (2011). Rapid-viability PCR method for detection of live, virulent
Bacillus anthracis in environmental samples. Applied and environmental microbiology, 77(18), 6570-
6578. https://doi.org/10.1128/AEM.0Q623-ll.

Target

Oligo

Sequence

Chromosome

Forward Primer

TTT CG AT G ATTT GCAATGCC

Reverse Primer

T CCAAGTTACAGT GT CG G CAT ATT

Probe

6 F A M - ACAT CA AGT CAT G G CGTG ACT ACCCAG ACTT- M G B N FQ

pXOl

Forward Primer

GCGGATAGCGGCGGTTA

Reverse Primer

TCG GTTCGTT AAATCCAAATG C

Probe

V1C-ACG ACT A A ACCG G AT AT G ACATT AAA AG A AG CCCTT A A-
MGBNFQ

VI. Technical Review

All data will receive technical review and QC review in accordance to QA. 1-005.

Technical Review Initials/Date:

QC Review Initials/Date:

Solid Waste Wl Real-Time PCR

Page 5 of 6

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DNA ASSAY—96 Well Plate Setup for Fast 7500

Project: STREAMS Solid Waste Barcode:

Target: Ba Sterne chromosome and pXOl

1. Calculate the total number of reactions per plate:

Sample wells + 4 NTC wells + 4 positive controls + extras = total rxns/plate (Y)

2. Prepare the Master Mix by combining the following reagents in an appropriate tube according to the following calculation:

Reagent volume (X) x total rxns/plate (Y) = Total Volume of reagent needed

Reagent

Manufacturer

Lot No.

Exp. Date

Total Volume
(ML)

TaqMan Fast Advanced Master Mix
(Cat. 4444556)

Applied
Biosy stems

12.5 |xL

Platinum Taq Polymerase

Invitrogen

0.1 \iL

Chrom. For. primer (25 uM)

1 \iL

Chrom. Rev. primer (25 uM)

1 (iL

Chrom. Probe (2 uM) - 1AM reporter

1 |xL

pXOl For. primer (25 uM)

1 jiL

pXOl Rev. primer (25 uM)

1 jiL

pX() 1 Probe (2 uM) - VIC reporter

1 \iL

PCR grade water

1.4 \iL

Total

20 ul.

Distribute 20 |iL of Master Mix into each reaction well, as indicated in the plate layout, below. Loosely cover all wells containing Master
Mix with caps.

Add 5 (iL of PCR-grade water to each of the NTC Wells. Cap wells tightly.

Add 5 (iL of PNC (Method Blank) to the corresponding wells and secure the caps
Add 5 (iL of Sample to the corresponding wells and secure the caps.

Positive Control

Positive Control lot

prep date

Centrifuge the plate using Labnet's MPS-1000 Mini Plate Spinner at room temperature, and then load the plate onto the 7500 Fast.

A
B

PC
50 PS

NTC

Technicians

Signature

Date

Master Mix, NTC

Samples

Standards

Analyst

Reviewed By: Date:

Real-time PCR - FORM A

Page 6 of 6

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APPENDIX H: WORK INSTRUCTION FOR
SELECTING PRESUMPTIVE BACILLUS
ANTHRACIS STERNE COLONIES FOR PCR
CONFIRMATION

H-l

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WORK INSTRUCTION FOR SELECTING PRESUMPTIVE BACILLUS ANTHRACIS STERNE COLONIES

FOR PCR CONFIRMATION

I. PURPOSE/SCOPE

Select and screen B. anthracis Sterne (BaS) colonies recovered on culture plates using real-time PCR.

II. MATERIALS/EQUIPMENT

Materials

Item

Manufacturer

Lot Number

Exp. Date

Storage
Temp.

PCR-Grade Water

Teknova

R.T.

1-HL loop, 10-nL loop, or
inoculating needles

N/A

R.T.

1.5- or 2-mL tubes

N/A

R.T.

N/A = Not Applicable

Equipment

Item

Manufacturer

Serial Number

Calibration
Due

Biosafety Cabinet
(BSC)

The Baker Company

Heat Block

VWR

N/A

Thermometer

Camera

N/A

N/A = Not Applicable

Other Supplies and Equipment

• Bleach

• 5-mL, 10-mL, and 25-mL Serological Pipettes

III. PROCEDURE

A. Selecting colonies

1. Pipette 100 piL of PCR-grade water into 1.5- or 2-mL tubes.

2. Select colonies. Take pictures of colonies that are selected.

3. Use 1-piL loop, 10 piL loop, or inoculating needle to select the colony.

4. Immerse needle into PCR-grade water and rotate to dislodge cellular material.

5. Colonies from a single sample can be pooled to increase the number of presumptive colonies
screened. Up to 10 colonies can be pooled within a lOO-piL volume of PCR-grade water. Repeat steps
3 and 4 to pool multiple colonies from a single sample and record the number of colonies pooled in
the below table.

Performed by:

Solid Waste Wl Colony Screen

Date:

Page 1 of 2

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WORK INSTRUCTION FOR SELECTING PRESUMPTIVE BACILLUS ANTHRACIS STERNE COLONIES

FOR PCR CONFIRMATION

6. Lyse the colony suspension for 5 minutes on a heat block at 95 ± 2 °C.

Incubation start Date/Time: Initials:

Incubation end Date/Time: Initials:

7. Store lysed suspension at - 20 °C for real-time PCR analysis.

8. Prior to real-time PCR analysis, thaw tubes, centrifuge @ 14,000 rpm for 2 minutes. Use supernatant
for real-time PCR.

Record Sample ID and Morphology for Selected Colonies

Tube
#

Sample ID

Volume
(mL)

Morphology (BaS
or Background)

ff of Colonies
Pooled

PCR Result

IV. Technical Review

Reviewed by: Date:

Performed by:

Solid Waste Wl Colony Screen

Date:

Page 2 of 2

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APPENDIX I: WORK INSTRUCTION FOR
BHIB ENRICHMENT FOR CULTURE

1-1

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WORK INSTRUCTION FOR BHIB ENRICHMENT FOR CULTURE

I. PURPOSE/SCOPE

Enrich extracted swatch in BHIB.

II. MATERIALS/EQUIPMENT

Materials

Item

Manufacturer

Lot Number

Exp.
Date

Storage
Temp.

Initials & Date

PCR-Grade Water

Teknova

R.T.

10-nL loop or
inoculating needles

R.T.

1.5- or 2-mL tubes

R.T.

Blood Agar Plates

2-8 °C

BHIB

R.T.

N/A = Not Applicable

Equipment

Item

Manufacturer

Serial Number

Thermometer/
Rees #

Calibration
Due

Initials & Date

Biosafety
Cabinet (BSC)

The Baker Company

N/A

Incubator

Precision

Thermometer

Traceable

N/A

Heat Block

VWR

Refrigerator

Fisher

C3274822

115

N/A = Not Applicable

Other Supplies and Equipment

• 25-mL Serological Pipettes

III. PROCEDURE

A. Selecting colonies

1. Aliquot 425 mL of BHIB into sterile container. Incubate leftover BHIB with enriched samples as
sterility check of media.

2. Add 25 mL of BHIB to each specimen cup containing the extracted swatch.

3. Incubate cups at 36 °C ± 2 °C for 24-48 hours.

Incubation start Date/Time: Initials:

Incubation end Date/Time: Initials:

BHIB Sterility (Y/N): Initials:

4. Evaluate the BHIB enrichment for samples.

Solid Waste WI-BHIB Enrich

Page 1 of 4

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WORK INSTRUCTION FOR BHIB ENRICHMENT FOR CULTURE

I. If broth is not turbid, record as no growth (NG) and incubate for an additional 24 hours.

II. If broth is turbid, record as positive growth (G+) and proceed to Step 4.

Sample
Number

Sample ID

Growth (G+) or No Growth (NG)

Recorded by:

24 hours

48 hours

5. For samples that have not been confirmed positive by culture membrane plating, streak turbid
samples onto TSA. Cap tightly and mix BHIB with growth for 30 seconds. Remove a loopful of
broth with a lO-piL loop and streak triplicate TSA plates for isolation. Store enriched samples at
2-8 °C.

6. Incubate the isolation plates and BHIB with growth at 36 °C ± 2 °Cfor a maximum of three days.

Incubation start Date/Time: Initials:

Incubation end Date/Time: Initials:

7. Examine plates for B. anthracis Sterne (BaS) colonies.

I. If presumptive BaS colonies are isolated and positive identification has not already
been confirmed by PCR from a representative sample, record the sample in the table
below as a colony selection sample and proceed to PCR confirmation from BHIB streak
plates (Section B).

II. If NO presumptive BaS colonies are isolated and positive identification has not already
been confirmed by PCR from a representative sample, record the sample in the table
below as a BHIB analysis sample and proceed to PCR confirmation of BHIB enriched
samples (Section C).

Solid Waste WI-BHIB Enrich

Page 2 of 4

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WORK INSTRUCTION FOR BHIB ENRICHMENT FOR CULTURE

Sample
#

Sample ID

Colony
Selection or
BHIB Analysis

Number of

Colonies

Screened

PCR Result

Recorded by:

B. Selecting Colonies

1. Pipette 100 piL of PCR-grade water into 1.5- or 2-mL tubes.

2. Select colonies.

3. Use 1-piL loop, 10 piL loop, or inoculating needle to select the colony.

4. Immerse needle into PCR-grade water and rotate to dislodge cellular material.

5. Colonies from a single sample can be pooled to increase the number of presumptive colonies
screened. Up to 10 colonies can be pooled within a lOO-piL volume of PCR-grade water. Repeat
steps 3 and 4 to pool multiple colonies from a single sample and record the number of colonies
pooled in the above table.

6. Proceed to Lysis and Storage (Section D).

C. PCR Confirmation of BHIB Enriched Samples

1. Transfer 50 piL of broth with growth to a microcentrifuge tube.

Page 3 of 4

Solid Waste WI-BHIB Enrich

-------
WORK INSTRUCTION FOR BHIB ENRICHMENT FOR CULTURE

2. Centrifuge at 12,000 x g for 2 minutes.

3. Remove and discard the supernatant in an autoclavable biohazard container. Add 100 piL of
PCR-grade water to the tube containing the bacterial pellet.

4. Resuspend the pellet by flicking the tube.

5. Proceed to Lysis and Storage (Section D).

D. Lysis and Storage

1. Lyse colony screen and BHIB enrichment samples for 5 minutes on a heat block at 95 ± 2 °C.

Incubation start Date/Time: Initials:

Incubation end Date/Time: Initials:

2. Store lysed suspension at - 20 °C for qPCR analysis or refrigerator if processed same day.

3. Prior to qPCR analysis, thaw tubes, centrifuge @ 14,000 rpm for 2 minutes. Use supernatant for
qPCR.

Performed by: Date:

IV. Technical Review

Reviewed by: Date:

Page 4 of 4

Solid Waste WI-BHIB Enrich

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APPENDIX J: FIELD GUIDES FOR
COLLECTING SOLID WASTE SAMPLES

j-i

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EPA Homeland Security Research Program - Solid Waste Sampling
Marine Fabric (Boat Seat) Sample Collection Procedure

1.0 General Sample Handling

• Refer to field site safety plan for appropriate personal protective equipment for sampling.

o This procedure assumes operators will, at a minimum, be wearing a pair of nitrile gloves,
o A second pair of nitrile gloves will be donned over the base pair.

• Collect samples from lowest suspected analyte concentration to highest suspected analyte
concentration to limit potential sample cross-contamination.

o If waste sample collection is collected from both decontaminated and contaminated waste at

the same time, collect from decontaminated waste sources prior to contaminated waste,
o Outer (second) glove change between each sample collection is required to limit cross-

contamination between samples,
o Reusable equipment will be decontaminated between each sample collected.

• Refer to Table 1 for equipment and sample collection supplies. Sample collection containers are pre-
sterilized before use, either by manufacturer, or by autoclaving (1-L with mouth polypropylene
bottles).

Table 1. Equipment and Supplies

Item

Manufacturer

Catalog Number

50-mLConicalTube

Corning

352070

400-mLStomacher Bag

Seward

BA6141/CLR

1-L Wide Mouth Polypropylene Bottle

Nalgene

21050032 or equivalent

Blunt Tip Scissors

Fisher Scientific

12-000-172 or equivalent

Dispatch Disinfectant Towels

Fisher Scientific

50-209-1767 or equivalent

Secondary Containment Bag

Thomas
Scientific

1220N84 for 1-L bottle, 1186T33 for 50-mLand
Stomacher bag or equivalent

Plastic Ruler

Fisher Scientific

12-000-152 or equivalent

2.0 Sample Collection

• Pierce intact boat seat with sharp edge of scissors (Figure 1) and then cut the appropriate swatch size
for each sample collection container (Table 2).

o See sample analysis plan to determine the size and number of samples to collect.

• Place cut boat seat section into the appropriate primary sample collection container and securely
close seal.

• Place the primary sample collection container into a secondary bag and seal.

• Doff outer gloves and don new gloves and place scissors into decontamination container before
collecting the next sample.

o Alternatively, wipe scissors with dispatch towel before collecting the next sample. Scissors
should be dedicated to a single decontamination bag.

• Complete appropriate sample collection documentation and deliver sample to the designated on-site
sampling collection team for subsequent storage, handling, and processing

Table 2. Primary Sample Collection Container for Solid Waste Collection

Sample Collection Container

Waste Item Description

Swatch Size

50-mL Conical Tube

Marine Fabric (Boat Seat)

5 cm x 5 cm

400-mL Stomacher Bag or 1-L
Wide Mouth Bottle

Marine Fabric (Boat Seat)

15 cm x 15 cm

Page 1 of 2

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EPA Homeland Security Research Program - Solid Waste Sampling
Marine Fabric (Boat Seat) Sample Collection Procedure

Pierce intact boat seat

5-cm x 5-cm section
in 50-mL conical tube

15-cm x 15-cm
section in Stomacher
bag

Figure 1. Cutting boat seat with scissors then placing sections into primary sample
collection containers.

Place marine fabric in wide
mouth bottle

Page 2 of 2

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EPA Homeland Security Research Program - Solid Waste Sampling

Glove Collection Procedure

1.0 General Sample Handling

• Refer to field site safety plan for appropriate personal protective equipment for sampling.

o This procedure assumes operators will, at a minimum, be wearing a pair of nitrile gloves,
o A second pair of nitrile gloves will be donned over the base pair.

• Collect samples from lowest suspected analyte concentration to highest suspected analyte
concentration to limit potential sample cross-contamination.

o If waste sample collection is collected from both decontaminated and contaminated waste at

the same time, collect from decontaminated waste sources prior to contaminated waste,
o Outer (second) glove change between each sample collection is required to limit cross-

contamination between samples,
o Reusable equipment will be decontaminated between each sample collected.

Table 1. Equipment and Supplies

Item

Manufacturer

Catalog Number

400-mLStomacher Bag

Seward

BA6141/CLR

1-L Wide Mouth Polypropylene Bottle

Nalgene

21050032 or equivalent

Dispatch Disinfectant Towels

Fisher Scientific

50-209-1767 or equivalent

Secondary Containment Bag

Thomas
Scientific

1220N84 for 1-L bottle, 1186T33 for 50-mL and
Stomacher bag or equivalent

2.0 Sample Collection

• Place glove into the appropriate primary sample collection container (Table 2) and securely close seal
(Figure 1).

o See sample analysis plan to determine the size and number of samples to collect.

• Place the primary sample collection container into a secondary bag and seal.

• Doff outer gloves and don new gloves.

• Complete appropriate sample collection documentation and deliver sample to the designated on-site
sampling collection team for subsequent storage, handling, and processing

Table 2. Primary Sample Collection Container for Solid Waste Collection

Sample Collection Container

Waste Item Description

Size

400-mL Stomacher Bag or 1-L
Wide Mouth Bottle

Nitrile Glove

Various

Page 1 of 2

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EPA Homeland Security Research Program - Solid Waste Sampling

Glove Collection Procedure

Glove in Stomacher bag

Place glove in wide
rnouth bottle

Figure 1. Gloves being placed into primary sample collection containers.

Page 2 of 2

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EPA Homeland Security Research Program - Solid Waste Sampling
Rope/Line Sample Collection Procedure

1.0 General Sample Handling

• Refer to field site safety plan for appropriate personal protective equipment for sampling.

o This procedure assumes operators will, at a minimum, be wearing a pair of nitrile gloves,
o A second pair of nitrile gloves will be donned over the base pair.

• Collect samples from lowest suspected analyte concentration to highest suspected analyte
concentration to limit potential sample cross-contamination.

o If waste sample collection is collecting from both decontaminated and contaminated waste
at the same time, collect from decontaminated waste sources prior to contaminated waste
o Outer (second) glove change between sample collection is required to limit cross-

contamination between samples,
o Reusable equipment will be decontaminated between each sample collected.

Table 1. Equipment and Supplies

Item

Manufacturer

Catalog Number

50-mLConicalTube

Corning

352070

400-mL Stomacher Bag

Seward

BA6141/CLR

1-L Wide Mouth Polypropylene Bottle

Nalgene

21050032 or equivalent

Hot Knife

Sailrite

122177 or equivalent

Cutting Surface or Cutting Foot

Sailrite

121915 or equivalent

Dispatch Disinfectant Towels

Fisher Scientific

50-209-1767 or equivalent

Secondary Containment Bag

Thomas
Scientific

1220N84 for 1-L bottle, 11S6T33 for 50-mLand
Stomacher bag or equivalent

Plastic Ruler

Fisher Scientific

12-000-152 or equivalent

2.0 Hot Knife Setup

• While hot knife is cold, loosen top and bottom locking screws. Insert cutting blade fully and tighten
both screws (Figure 1).

• Attach battery pack until audible click is heard.

• To operate, press safety toggle switch and trigger to heat the blade (allow 5 to 10 sec).

• If cutting blade becomes dirty, clean the cutting blade using wire brush provided by manufacturer.

Figure 1. Hot knife diagram.

Page 1 of 2

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EPA Homeland Security Research Program - Solid Waste Sampling
Rope/Line Sample Collection Procedure

3.0 Sample Collection

• Decontaminate the cutting surface by wiping with Dispatch towel,

o No need to decontaminate the hot cutting element of the hot knife.

• Place double braided nylon rope onto clean cutting surface (Figure 2) and measure to the appropriate
length for each sample collection container (Table 2).

• Alternatively, for 6-inch or longer lengths, fold the rope so that the apex of the loop is the target
cutting point. (See Figure 2)

o See sample analysis plan to determine the size and number of samples to collect.

• Press safety toggle and squeeze hot knife trigger to heat blade (allow 5 to 10 sec).

• Cut rope to length. For thick sections of rope with a diameter greater than the cutting blade, rotate
the rope and cut the outer edge while working toward the inner layers until cut is complete.

• Place cut rope section into the appropriate primary sample collection container and securely close
seal.

• Place the primary sample collection container into a secondary bag and seal.

• Heat hot knife by holding trigger to heat blade for 10 seconds before cutting next section of rope to
decrease likelihood of microbial carryover between samples.

• Doff outer gloves and don new gloves before collecting the next sample.

• If cutting surface is used, wipe with Dispatch towel

• Complete appropriate sample collection documentation and deliver sample to the designated on-site
sampling collection team for subsequent storage, handling, and processing.

Table 2. Primary Sample Collection Container for Solid Waste Collection

Sample Collection Container

Waste Item Description

Length to Cut

50-mL Conical Tube

%-inch diameter double-braided nylon

5 cm (~2 in)

400-mL Stomacher Bag

%-inch diameter double-braided nylon

15 cm (~6 in)

1-L Wide Mouth Bottle

1-inch diameter double-braided nylon

15 cm (~6 in)

Figure 2. Cutting rope using hot knife without (left) and with (right) cutting surface.

Page 2 of 2

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EPA Homeland Security Research Program - Solid Waste Sampling
Tyvek PPE Sample Collection Procedure

1.0 General Sample Handling

• Refer to field site safety plan for appropriate personal protective equipment for sampling.

o This procedure assumes operators will, at a minimum, be wearing a pair of nitrile gloves,
o A second pair of nitrile gloves will be donned over the base pair.

• Collect samples from lowest suspected analyte concentration to highest suspected analyte
concentration to limit potential sample cross-contamination.

o If waste sample collection is collecting from both decontaminated and contaminated waste
at the same time, collect from decontaminated waste sources prior to contaminated waste,
o Outer (second) glove change between sample collection is required to limit cross-

contamination between samples,
o Reusable equipment will be decontaminated between each sample collected.

Table 1. Equipment and Supplies

Item

Manufacturer

Catalog Number

50-mLConicalTube

Corning

352070

400-mLStomacher Bag

Seward

BA6141/CLR

1-L Wide Mouth Polypropylene Bottle

Nalgene

21050032 or equivalent

Blunt Tip Scissors

Fisher Scientific

12-000-172 or equivalent

Dispatch Disinfectant Towels

Fisher Scientific

50-209-1767 or equivalent

Secondary Containment Bag

Thomas
Scientific

1220N84 for 1-L bottle, 1186T33 for 50-mLand
Stomacher bag or equivalent

Plastic Ruler

Fisher Scientific

12-000-152 or equivalent

2.0 Sample Collection

• Decontaminate the cutting surface by wiping with Dispatch towel.

• Place Tyvek onto clean cutting surface (Figure 1) and measure to the appropriate length for each
sample collection container (Table 2).

• Alternatively, for Tyvek sleeves, fold the Tyvek three times at the mid-line so that the material can be
placed into 1-L wide mouth bottle. (See Figure 1)

o See sample analysis plan to determine the size and number of samples to collect.

• Cut Tyvek to length using sterile scissors.

• Place cut Tyvek section into the appropriate primary sample collection container and securely close
seal.

• Place the primary sample collection container into a secondary bag and seal.

• Doff outer gloves and don new gloves and place scissors into decontamination container before
collecting the next sample.

o Alternatively, wipe scissors with Dispatch towel before collecting the next sample. Scissors
should be dedicated to a single decontamination bag.

• Wipe cutting surface with Dispatch towel.

• Complete appropriate sample collection documentation and deliver sample to the designated on-site
sampling collection team for subsequent storage, handling, and processing

Page 1 of 2

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EPA Homeland Security Research Program - Solid Waste Sampling
Tyvek PPE Sample Collection Procedure

Table 2. Primary Sample Collection Container for Solid Waste Collection

Sample Collection Container

Waste Item Description

Length to Cut

50-mL Conical Tube

Tyvek suit or sleeve section

5 cm x 5 cm

400-mL Stomacher Bag

Tyvek suit or sleeve section

15 cm x 15 cm

1-L Wide Mouth Bottle

Tyvek sleeve

45 cm

Fold Tyvek sleeve

5-cm x 5-cm section
in 50-mL conical tube

15-cm x 15-cm
section in Stomacher
bag

Place sleeve in wide
mouth bottle

Figure 1. Placing Tyvek sections into primary sample collection containers.

Page 2 of 2

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oEPA

United States
Environmental Protection
Agency

PRESORTED
STANDARD POSTAGE
& FEES PAID EPA
PERMIT NO. G-35

Office of Research and
Development (8101R)
Washington, DC 20460

Official Business
Penalty for Private Use
$300

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