Volume 10, Number 4, 2005 - American Biological Safety Association

Volume 10, Number 4, 2005

213
214
216
258
Journal of the American Biological Safety Association
Volume 10, Number 4, 2005
Letter to the Editors
President’s Page
Articles
Comparison of the Canadian Industrial Security Manual and the United States National
Industrial Security Program Operating Manual
Andrew Hammond .......................................................................................................................216
The Infectious Dose of Francisella tularensis (Tularemia)
Rachael M. Jones, Mark Nicas, Alan Hubbard, Matthew D. Sylvester, and Arthur Reingold .....227
High-Dose Ultraviolet C Light Inactivates Spores of Bacillus Atrophaeus and Bacillus Anthracis
Sterne on Nonreflective Surfaces
Marie U. Owens, David R. Deal, Michael O. Shoemaker, Gregory B. Knudson,
Janet E. Meszaros, and Jeffery L. Deal.....................................................................................240
Autoclave Testing in a University Setting
Richard N. Le, Amy L. Hicks, and Janice Dodge..........................................................................248
Operating a BSL-4 Laboratory in a University Setting
Tradeline Publications ..................................................................................................................253
Special Features
Use of Multiple SOP Styles to Increase Personnel Compliance and Safety
Within a BSL-2/BSL-3 Animal Facility
Andrea Mitchell, Jeri Ellis, and Tim Ruddy..................................................................................258
Book Review—Revenge of the Microbes by Abigail A. Salyers and Dixie D. Whitt
Reviewed by George A. Pankey.....................................................................................................265
Book Review—Biodefense: Principles and Pathogens Edited by Michael S. Bronze
and Ronald A. Greenfield
Reviewed by Michael P. Owen......................................................................................................266
(continued on page 210)

(continued from page 209)
273
210
Ask the Experts—HEPA Filtered Supply Air for BSL-3 Laboratories?
John H. Keene...............................................................................................................................268
Biosafety Tips—Lymphocytic Choriomeningitis Virus—A Hazard in Rodent Animal Colonies
Karen B. Byers ...............................................................................................................................270
ABSA News
2005 ABSA Conference Photos.................................................................................................273
2005 ABSA Service Award Recipients......................................................................................274
New ABSA Members for 2006 ...................................................................................................277
2005 ABSA Conference Sponsors.............................................................................................279
Calendar of Events .......................................................................................................................280
ABSA Journal Subscription Information ...................................................................................281
ABSA Anthology Books Information and Order Form.............................................................282
ABSA Chapters, Affiliates, and Affiliated Organizations........................................................283
About the Cover
Fransicella tularensis is the causative agent of tularemia. Exposure to F. tularensis has resulted in numerous
laboratory acquired infections, some of which may have been due to aerosol exposures. Read more about
infectious dose modeling on pages 227-239, “The Infectious Dose of Francisella tularensis (Tularemia)” by
Rachael M. Jones, Mark Nicas, Alan Hubbard, Matthew D. Sylvester, and Arthur Reingold.
One natural reservoir for this zoonotic disease is the rabbit. It can be transmitted to humans by handling
infected blood or tissue, or consuming undercooked infected meat. The tick, an arthropod vector, can transmit
the disease through its bite. The symptoms developed depend on the type of exposure route.
Images from the CDC Public Health Image Library are: D. variabilis tick photo, taken by Andrew Brooks
of CDC; Tularemia lesion on the dorsal skin of right hand photo, taken by Dr. Brachman of the CDC; and
F. tularensis colony characteristics when grown on Chocolate, Martin Lewis or Thayer-Martin medium at
48-72 hours, courtesy of Larry Stauffer, Oregon State Public Health Laboratory.

Applied Biosafety: Journal of the American Biological Safety
Association (ISSN 1535-6760) is published quarterly by the American
Biological Safety Association (ABSA). ABSA members receive the
journal as a benefit of membership. An additional annual subscription
for members is $60. Nonmembers and institutions/libraries may
subscribe at the annual rates of $92 and $122 respectively. Single issue
rates are as follows: members $18; nonmembers $28; and institutions/
libraries $35.
Authorization to Copy: No part of this publication may be
reproduced, stored in a retrieval system, or transmitted in any form or
by any means, electronic, electrostatic, magnetic tape, photocopying,
recording, or otherwise, without permission in writing from the
copyright holder.
Change of Address: A change of address notice should be sent at
least 6 weeks in advance to the ABSA National Office to ensure that
all mailings, including the journal and newsletter, will reach you.
ABSA is not responsible for misrouted mail as a result of insufficient
notification of an address change. Undelivered copies resulting from
an insufficient address change notification will not be replaced, but
issues may be purchased at the single issue price as detailed above.
ABSA National Office
American Biological Safety Association
1202 Allanson Road
Mundelein, IL 60060-3808, USA
847-949-1517 / Fax 847-566-4580
E-mail: absa@absa.org
Web Site: www.absa.org
Advertising Rates
Rates 1x 2x 4x
Outside back cover $800 $720 $680
Inside front cover $600 $540 $510
Inside back cover $600 $540 $510
Full page $500 $450 $425
1/2 page $300 $270 $255
1/4 page $200 $180 $170
Color rates: $350 for first color (after black) and $300 each additional color.
15% discount for agencies (orders must be supplied on agency letterhead).
Mechanical Requirements Width Height
Outside back cover (full bleed) 8-1/2” 11”
Inside front cover (full bleed) 8-1/2” 11”
Inside back cover (full bleed) 8-1/2” 11”
Full page 7” 10”
1/2 page - horizontal 7” 4-7/8”
1/2 page - vertical 3-3/8” 10”
1/4 page 3-7/8” 4-7/8”
Trim size—8-1/2” x 11”
Film—133 line screen, right reading, emulsion side down, color separated
Submission Deadlines
February 1 for Spring issue May 1 for Summer issue
August 1 for Fall issue November 1 for Winter issue
The publication of any advertisement by this journal is not an
endorsement of the advertiser or of the products or services advertised.
ABSA is not responsible for any claims made in any advertisement.
ABSA Journal Editorial Board
Co-Editors
Barbara Johnson, Department of Defense, Arlington, VA
Karen B. Byers, Dana Farber Cancer Institute, Boston, MA
Associate Editor
Elizabeth Gilman Duane, Wyeth, Cambridge, MA
Lynn Harding, Biosafety Consultant, Chattanooga, TN
Assistant Editors
Richard Fink, Wyeth BioPharma, Andover, MA
John H. Keene, Biohaztec Associates, Inc., Midlothian, VA
Thomas A. Kost, GlaxoSmithKline, Research Triangle Park, NC
Ed Krisiunas, WNWN International, Burlington, CT
International Editors
Allan Bennett, European Biological Safety Association (EBSA),
United Kingdom
Maureen Best, International Biosafety Working Group (IBWG),
Canada
Otto Doblhoff-Dier, European Biological Safety Association (EBSA),
Austria
Betty Kupskay, ABSA Canada, Canada
Ai Ee Ling, Asia Pacific Biosafety Association (APBA), Singapore
Leila Oda, Associação Nacional de Biossegurança (ANBio), Brazil
Production Editor
Karen D. Savage
Editorial Board
Matthew J. Bankowski, ViroMed (LabCorp), Minnetonka, MN
Franklin R. Champlin, Mississippi State University, Mississippi
State, MS
Mary L. Cipriano, Abbott Laboratories, Abbott Park, IL
Robert P. Ellis, Colorado State University, Fort Collins, CO
Glenn A. Funk, Lawrence Livermore National Laboratory,
Livermore, CA
Raymond W. Hackney, Jr., University of North Carolina, Chapel
Hill, NC
Philip Hagan, Georgetown University, Washington, DC
Robert J. Hawley, Midwest Research Institute, Frederick, MD
Richard Henkel, Centers for Disease Control and Prevention,
Atlanta, GA
Debra L. Hunt, Duke University, Durham, NC
Peter C. Iwen, University of Nebraska Medical Center, Omaha, NE
John H. Keene, Biohaztec Associates, Inc., Midlothian, VA
Paul Michael Kivistik, University of Nevada, Reno, NV
Joseph P. Kozlovac, USDA-ARS, Beltsville, MD
Jens H. Kuhn, Harvard Medical School, Southborough, MA
Margy S. Lambert, University of Wisconsin, Madison, WI
R. Thomas Leonard, University of Virginia, Charlottesville, VA
Paul J. Meechan, Merck Research Laboratories, West Point, PA
Mark Nicas, University of California, Berkeley, CA
Beryl J. Packer, Iowa State University, Ames, IA
Tim Ravita, Constella Health Sciences, Atlanta, GA
Richard Rebar, GlaxoSmithKline R&D, King of Prussia, PA
Jonathan Y. Richmond, Jonathan Richmond & Associates, Inc.,
Southport, NC
Deanna S. Robbins, Department of Veterans Affairs, Baltimore, MD
Richard J. Shaughnessy, University of Tulsa, Tulsa, OK
Allan Showler, USDA-ARS, Weslaco, TX
Cecil R. Smith, Ohio State University, Columbus, OH
Gerard J. Spahn, The Salk Institute, La Jolla, CA
Donald Vesley, University of Minnesota, Minneapolis, MN
Catherine L. Wilhelmsen, United States Army Medical Research
Institute of Infectious Diseases (USAMRIID), Fort Detrick, MD
Linda B. Wolfe, Whitehead Institute for Biomedical Research,
Cambridge, MA
Jeffrey D. Wolt, Iowa State University, Ames, IA
Alan G. Woodard, International Environmental Health Alliance,
Gansevoort, NY

Vision
212
ABSA, the leader in the profession of biological safety.
Mission Statement
The American Biological Safety Association is dedicated to expanding biological safety awareness to
prevent adverse occupational and environmental impact from biohazards.
Goals
• Expand professional and public awareness of biological safety through effective communication.
• Participate in the development of biological safety and biosecurity standards, guidelines, and
regulations.
• Develop ABSA as the recognized resource for professional and scientific expertise in biological
safety and biosecurity.
• Advance biological safety as a scientific discipline through education, research, and professional
development.
ABSA Officers
President
Glenn A. Funk, Lawrence Livermore National Laboratory, Livermore, CA
President-Elect
Robert J. Hawley, Midwest Research Institute, Frederick, MD
Secretary
Rosamond Rutledge-Burns, National Institute Standards & Technology, Gaithersburg, MD
Treasurer
Leslie Delpin, University of Connecticut, Storrs, CT
Past-President
Elizabeth Gilman Duane, Wyeth, Cambridge, MA
Council Members
Robert P. Ellis, Colorado State University, Fort Collins, CO
Joseph P. Kozlovac, USDA Agricultural Research Service, Beltsville, MD
Patricia Olinger, Pharmacia Corporation, Kalamazoo, MI
Chris Thompson, Greenfield, IN
Executive Director
Edward J. Stygar, Jr.

Letter to the Editors
James J. Coogan
Siemens Building Technologies, Buffalo Grove, Illinois
Let me commend Allan Bennett et al. on valuable
piece of research and an illuminating article
entitled “Development of Particle Tracer Techniques
to Measure the Effectiveness of High Containment
Laboratories” in Applied Biosafety (Volume 10, Number
3, 2005). The facts and observations presented
support some common engineering assumptions and
challenge others. Both results are valuable.
Definition of Laboratory Protection Factor and
the test methods described advance our ability to
discuss effective pressurization. The result that containment
correlates more closely to infiltrating air
flow than to pressure difference is intriguing. I expect
it surprises many engineers as much as it does
me.
The explanation offered is that the air flowing
inward through the door catches contaminants and
prevents their escape while the door is open and a
person walks through it. The authors themselves
seem to find it unlikely that such low air velocity
captures contaminants. (The air velocity reported
through the open door is 0.14 m s-1 and lower:
much less than the velocity of the swinging door or
the walking person.)
Perhaps it is appropriate to propose another explanation
for the results. Consider the effect that the
Editorial Note
Letters to the Editors (approximately 400 words)
discuss information published in Applied Biosafety in
the past nine months or discuss topic areas of general
interest in the biosafety profession. Letters can
Applied Biosafety, 10(4) p. 213 © ABSA 2005
infiltrating air flow has on contaminant concentrations
while the door is closed before and after entry
or exit. Just inside the door, contaminated air is continuously
replaced by the clean infiltrating air. This
lowers the concentration near the door. When entry
or exit occurs, certainly some air leaves the room,
but this air is cleaner than it would be with a lower
infiltrating air flow rate, so less contaminant leaves
the room. After the entry or exit is complete and the
door is closed, some quantity of contaminant lingers
outside the room. A portion of it is picked up by the
particle counters, but presumably, some of it is
drawn back into the room by the on-going infiltration.
In short, the supposition is that infiltrating air
flow has a continuous cleaning effect near the door,
and that this on-going effect (rather than several seconds
of infiltration at very low velocity during entry
or exit) increases the Laboratory Protection Factor.
Do the authors have more information that will
help us choose one mechanism or the other to explain
the results? In some cases, the question is
moot. Containment is the issue and the mechanism
may be unimportant, but there are ventilation systems
where the distinction is crucial.
be submitted electronically to Karen D. Savage,
Production Editor, at ksavage@covad.net or by mail
to ABSA National Office, Applied Biosafety, 1202
Allanson Road, Mundelein, IL 60060. Letters published
in part or whole are subject to editing for clarity
and special formatting.
213

I suspect every new organization president wrestles
with questions like “What do I want to achieve
during my presidency?” and “Where do I want to
lead the organization during the next year?” These
are weighty issues that focus on the best interests of
the membership in general and the institution in
particular. They’re made especially challenging by
the outstanding leadership and successes provided
by recent past presidents. I have, appropriately, a
very high standard to uphold.
With 1,500 members representing 30 different
countries and 16 Affiliates and affiliated organizations,
ABSA is no longer a small club of friends. We
are gaining the mass to be noticed, to be listened to,
to be asked for advice and help, and to make a difference
in the realms of science we impact. It would
be easy for me to suggest that we grow the organization
because as we grow larger, we grow stronger and
we have a louder voice. There is truth in this, and
growing ABSA is a worthy goal that we should pursue.
However, at this point, I believe it is also important
to “grow our profession”—to bring more highly
qualified scientists into our ranks, to strengthen our
knowledge base, and to provide additional ABSA
members who will enrich our organization. I believe
there are many scientists and health and safety professionals
who are unaware of the challenges and
rewards of biosafety as a career, or of the need for
highly skilled biosafety professionals in the scientific
community.
To keep our profession attractive to others and
respected within the scientific community, we must
maintain, strengthen, and continue to demonstrate
the high professional standards to which we adhere
every day. Each of us must set the Gold Standard for
professionalism, for technical competency, for cus-
214
President’s Page
Glenn A. Funk
Gualala, California
Applied Biosafety, 10(4) pp. 214-215 © ABSA 2005
tomer service, and for flexibility and adaptability to
meet the varied and often unanticipated needs of
our customers. We must not simply continue to be
the best of the best; we must continually get better at
what we do. As “Neutron Jack” Welch, ex-CEO of
General Electric, used to say, “You can’t just talk the
talk; you’ve also got to walk the walk.”
One of the things we do best in ABSA is share
our knowledge through networking, seminars, training
sessions, and professional courses. ABSA is your
organization, and I urge you to use the opportunities
it offers to improve your skills as a biosafety professional.
Meanwhile, your ABSA Council and I will
continue to make ABSA better for you. The management
consultants who helped us restructure our
business model also helped us identify important
areas for Task Force study; we held off implementing
these during the first year in order to focus on the
business infrastructure. This year we’ll put Task
Forces to work defining the parameters of the biosafety
profession, enhancing the value and benefits
of professional Registration and Certification, and
determining the feasibility and usefulness of an
ABSA Emergency Response capability to provide
assistance during national and international emergencies
that involve biosafety. If you have an interest
in serving on one of these Task Forces, please let me
know (gafunk@absa.org).
One other effort soon to be underway is a renewed
push to catalog the ABSA Historical Collection
and transfer it to safe archival storage. Once
that’s done, we can look at ways to make it accessible
to members through, for example, displays at meetings
and articles in this Journal. Ultimately, I’d like
to see at least parts of the collection available online.
In future columns I’ll discuss other ideas for

moving ABSA forward. As always, I greatly appreciate
your volunteer efforts to help make ABSA what
it is. We wouldn’t be here today if it weren’t for the
active and positive participation of our many talented
members. As President, my door is open to
Corrections and Clarifications
G. A. Funk
you. Please send me e-mails with your ideas and suggestions.
We’ll talk about them, I’ll take the great
ideas to Council, and together we’ll continue to
make ABSA better and stronger.
Special Features, Ask the Experts: “USDA BSL-3 Facility Requirements: What’s the
Concern?” authorship attribution in the Table of Contents is incorrect (Volume 10, Number
3, page 133). It should be: John H. Keene.
EPA Pesticide Program Update from EPA’s Office of Pesticide Programs
10/11/05: EPA Approves New Non-Chemical Control for Corn Rootworm
www.epa.gov/pesticides
After an intensive, multi-year scientific analysis, EPA has approved applications submitted by Mycogen
Seeds (c/o Dow AgroSciences, LLC) and Pioneer Hi-Bred International, Inc. for the use of a new corn
plant-incorporated protectant (PIP) designed to control corn rootworm. Corn rootworm is a widespread
and destructive insect pest responsible for the single largest use of conventional insecticides in the United
States. The new product is the second PIP to offer protection against corn rootworm and is expected to
result in a further reduction of chemical insecticide use by growers.
The new corn plant-incorporated protectant, Event DAS-59122-7 Corn, produces its own insecticide
within the corn plant derived from Bacillus thuringiensis (Bt), a naturally occurring soil bacterium. The Bt
proteins used in this product, called Cry34Ab1 and Cry35Ab1 (Cry 34/35), control corn rootworm.
To reduce the likelihood of corn rootworm developing resistance to Bt, EPA is requiring Mycogen and
Pioneer to ensure that buffer zones within the planted acreage be planted with corn that is not protected
from corn rootworm to serve as a “refuge.” The insect populations in the refuges will help prevent
resistance development when they cross-breed with insects in the Bt fields. This resistance management
strategy was developed as a condition of the registration, and EPA will require routine monitoring and
documentation that these measures are followed.
The reduction in chemical pesticide use will benefit the environment directly and can mean less
chemical exposure to people who apply pesticides to corn. The availability of multiple corn rootwormprotected
corn products will also increase grower choice and price competition, resulting in lower seed
prices for consumers and higher adoption rates. The product provides yet another way to combat corn
rootworm, as well as indirect benefits such as energy savings resulting from reduced chemical insecticide
use. As with similar products, EPA has approved Cry 34/35 for time-limited use, which will be subject to
reevaluation in five years. For more information on EPA’s regulation of biopesticide products, see
www/epa.gov/pesticides/biopesticides/.
215

Abstract
Because of the potential for use as a bioterrorism
agent or bioweapon, many governments have imposed
strict regulations regarding the possession, use, and
transfer of “select” biological agents. Consequently,
much of the information surrounding the possession and
use of these agents is potentially classified, and those
contractors and their employees who require access to
this information must receive Facility (contractor) and
Personnel (employees) clearances. Both Canada and
the United States (U.S.) have produced industrial security
manuals—the Industrial Security Manual (ISM)
(Canadian and International Industrial Security Directorate,
2004) and the National Industrial Security
Program Operating Manual (NISPOM) (Defense
Technical Information Center, 1995)—for use by
cleared government contractors. These documents set
forth the requirements, restrictions, and other safeguards
that are necessary to prevent unauthorized disclosure
of classified information and assets provided to
or produced by private government contractors. This
article compares and contrasts the requirements set
forth in the ISM and the NISPOM. The results of this
comparison present a valuable security management
tool for private organizations that wish to engage in
classified work for the Canadian, U.S., or both governments.
216
Article
Introduction
Applied Biosafety, 10(4) pp. 216-226 © ABSA 2005
Comparison of the Canadian Industrial Security
Manual and the United States National
Industrial Security Program Operating Manual
Andrew Hammond
Constella Health Sciences, Atlanta, Georgia
As a result of the October 2001 anthrax letter
attacks, both the United States and Canada enacted
new laws imposing additional restrictions on certain
hazardous biological agents and toxins. The U.S.
enacted the Public Health Security and Bioterrorism
Preparedness and Response Act of 2002 (Public Law
107-188) (U.S. Government Printing Office, 2002)
and Canada passed the Public Safety Act, 2002
(Parliament of Canada, 2002). Because of the potential
for “select” biological agents and toxins being
used as bioterrorism agents or in a bioweapons program,
both Acts impose strict regulations regarding
their possession, use, and transfer. Consequently,
much of the information surrounding the possession
and use of these agents is potentially classified (or
confidential), and those organizations and their employees
who require access to this information must
receive Facility (organization) and Personnel
(employee) clearances. Both Canada and the U.S.
have produced industrial security manuals—Industrial
Security Manual (ISM) (Canadian and International
Industrial Security Directorate, 2004) and the National
Industrial Security Program Operating Manual
(NISPOM) (Defense Technical Information Center,
1995)—for use by cleared government contractors.
These documents set forth the requirements, restrictions,
and other safeguards that are necessary to prevent
unauthorized disclosure of classified information
(and assets) provided to or produced by private

government contractors and to control the authorized
disclosure of classified information (and assets)
released by the governments to their contractors.
United States—National Industrial
Security Program Operating Manual
Security Classifications
An original classification decision at any level
can be made only by a U.S. Government official
who has been delegated this authority in writing.
Contractors may make derivative classification decisions
based on the guidance provided by the Contract
Security Classification Specification that is issued
with each classified contract. Derivative classification
is the act of classifying a specific item of information
or material on the basis of an original classification
decision already made by an authorized
original classification authority. The source of authority
for derivative classification ordinarily consists
of a previously classified document or a classification
guide issued by an original classification authority.
Top Secret
Top secret information or material is that information
or material whose unauthorized disclosure
could be reasonably expected to cause exceptionally
grave damage to the national security that the original
classification authority is able to identify or describe.
Examples include armed hostilities against the
United States or its allies, disruption of foreign relations
vitally affecting the national security, and the
disclosure of scientific or technological developments
vital to national security.
Secret
Secret information or material is that information
or material whose unauthorized disclosure
could be reasonably expected to cause serious damage
to the national security that the original classification
authority is able to identify or describe. Examples
of serious damage include significant impairment
of a program or policy directly related to the
national security and compromise of significant scientific
or technological developments relating to national
security.
A. Hammond
Confidential
Confidential information or material is that information
or material whose unauthorized disclosure
could be reasonably expected to cause damage to
the national security that the original classification
authority is able to identify or describe. Examples
include documents relating to clearance or assignment
of personnel who will have knowledge of, or
access to, classified information or materials or details
pertaining to features of routes and schedules of
shipments of confidential materials.
Facility Security
Facility Clearances
A facility security clearance (FCL) is an administrative
determination that a facility is eligible for access
to classified information at the same or lower
classification category as the clearance being granted.
Contractors are eligible for custody of classified material,
if they have an FCL and storage capability approved
by the Cognizant Security Agency (CSA).
A procuring activity of the Government or
cleared contractor may request a facility clearance for
a contractor or prospective contractor/
subcontractor when a definite, classified procurement
need has been established. Also, the contractor
must be organized and existing under the laws of any
of the 50 states, the District of Columbia, or Puerto
Rico, and be located in the U.S. and its territorial
areas or possessions.
Meetings
Classified disclosure at a meeting (e.g., conference,
seminar, symposium, exhibit, convention,
training course, or other such gathering) which
serves a government purpose and at which adequate
security measures have been provided in advance
may be conducted by a cleared contractor provided
the meeting is authorized by a Government Agency
that has agreed to assume security jurisdiction. The
Government Agency must approve security arrangements,
announcements, attendees, and the location
of the meeting. (Classified meetings shall be held
only at a Federal Government installation or a
cleared contractor facility where adequate physical
security and procedural controls have been ap-
217

proved.) Contractors wishing to conduct classified
meetings shall submit their requests to the Government
Agency having principal interest in the subject
matter of each meeting.
Personnel Security
Security Officers
The Facility Security Officer (FSO) shall be a
U.S. citizen employee appointed by the contractor
who is cleared as part of the facility clearance. The
FSO will supervise and direct security measures necessary
for implementing the NISPOM and related
Federal requirements for classified information.
The senior management official and the FSO
must always be cleared to the level of the Facility
Clearance (FCL). Other officials, as determined by
the CSA, must be granted a Personnel Clearance
(PCL) or be excluded from classified access.
Personnel Clearances
An industrial personnel security clearance is an
administrative determination that an industrial employee
is eligible for access to classified information.
This determination is based on investigation and
review of available personal data and a finding that
access is clearly consistent with national interests.
An individual may be processed for a personnel
security clearance only when employed by a cleared
contractor in a job requiring access to classified information.
As an exception, a candidate for employment
may be processed for a PCL provided a written
commitment for employment that prescribes a fixed
date for employment within the ensuing 180 days
has been made by the contractor, and the candidate
has accepted the employment offer in writing.
Under rare circumstances, a non-U.S. citizen
may be issued a Limited Access Authorization for
access to classified information. Specific criteria and
limitations are provided in the NISPOM.
Contractors have no authority to grant, deny, or
revoke personnel clearances for their employees.
This authority is reserved by the U.S. Government.
Subcontracting
Before a prime contractor may release, disclose
classified information to a subcontractor, or cause
218
Comparison of the Canadian Manual and the U.S. Manual
classified information to be generated by a subcontractor,
he or she must determine the security requirements
of the subcontract and determine clearance
status of prospective subcontractors. The prime
contractor shall verify the clearance status and safeguarding
capability of the subcontractor from the
CSA. If a prospective subcontractor does not have
the appropriate FCL or safeguarding capability, the
prime contractor shall request the CSA of the subcontractor
to initiate the necessary action.
The prime contractor shall ensure that a Contract
Security Classification Specification is incorporated
in each classified subcontract. The contractor
shall also review the security requirements during
the different stages of the subcontract and provide
the subcontractor with applicable changes in these
security requirements. Upon completion of the subcontract,
the subcontractor may retain classified material
received or generated under the subcontract
for a 2-year period, provided the prime contractor or
GCA does not advise to the contrary.
Education, Training, and Briefings
Contractors shall provide all cleared employees
with security training and briefings commensurate
with their involvement with classified information.
Contractors shall also be responsible for ensuring
that the FSO, and others performing security duties,
complete security training deemed appropriate by
the CSA. (Training, if required, should be completed
within 1 year of appointment to the position
of FSO.) The contractor is responsible for providing
all cleared employees with some form of security
education and training at least annually.
The SF 312 is an agreement between the United
States and an individual who is cleared for access to
classified information. An employee issued an initial
PCL must execute an SF 312 prior to being granted
access to classified information. The employee must
also receive an initial security briefing that includes a
Threat Awareness Briefing, a Defensive Security
Briefing, an overview of the security classification
system, employee reporting obligations and requirements,
and security procedures and duties applicable
to the employee’s job.
Contractors shall debrief cleared employees at
the time of termination (discharge, resignation, or

etirement); when an employee’s PCL is terminated,
suspended, or revoked, and upon termination of the
FCL.
Visits
The contractor must determine that the visit is
necessary and that the purpose of the visit cannot be
achieved without access to, or disclosure of, classified
information. All classified visits require advance
notification to, and approval of, the organization
being visited. In urgent cases, visit information may
be furnished by telephone provided that it is followed
up in writing. The contractor shall issue a
Visit Authorization Letter (VAL) to the organization
being visited that shall include the following:
• Contractor’s name, address, and telephone number,
assigned CAGE Code, and certification of the
level of the FCL
• Name, date, place of birth, and citizenship of the
employee intending to visit
• Certification of the proposed visitor’s PCL and
any special access authorizations required for the
visit
• Name of person(s) to be visited
• Purpose and sufficient justification for the visit
to allow for a determination of the necessity of the
visit
• Date or period during which the VAL is to be
valid
Contractors shall maintain a record of all visitors
to their facility who have been approved for access
to classified information.
Document Security
General Marketing
All classified material shall be marked on the
face of the document to show the name and address
of the facility responsible for its preparation and the
date of preparation. The highest level of classified
information contained in a document is its overall
marking. The overall marking shall be conspicuously
marked or stamped at the top and bottom on the
outside of the front cover, on the title page, on the
first page, and on the outside of the back cover. Interior
pages of classified documents shall be marked at
A. Hammond
the top and bottom with the highest classification of
the information appearing thereon or marked UN-
CLASSIFIED if all the information on the page is
UNCLASSIFIED. The major components of complex
documents are likely to be used separately.
Therefore, each major component shall be marked
as a separate document. Also, each section, part,
paragraph, or similar portion of a classified document
shall be marked to show the highest level of its
classification, or that the portion is unclassified. Unclassified
subjects and titles shall be selected for classified
documents, if possible. If a classified subject or
title must be used, it shall be marked with the appropriate
symbol—(TS), (S), or (C)—placed immediately
following and to the right of the item.
All classified information shall be marked to
reflect the source of the classification and declassification
instructions. This required information shall
be placed on the cover, first page, title page, or in
another prominent position.
General Storage
Cognizant security officials shall work to meet
appropriate security needs according to the intent of
the NISPOM and at an acceptable cost.
TOP SECRET material shall be stored in a
GSA-approved security container, an approved vault,
or an approved Closed Area. Supplemental protection
is required.
SECRET material shall be stored in the same
manner as TOP SECRET material without supplemental
protection.
CONFIDENTIAL material shall be stored in the
same manner as TOP SECRET or SECRET material
except that no supplemental protection is required.
Reproduction
Contractors shall establish a reproduction control
system to ensure that reproduction of classified
material is held to the minimum consistent with
contractual and operational requirements. Classified
reproduction shall be accomplished by authorized
employees knowledgeable about the procedures for
classified reproduction. The use of technology that
prevents, discourages, or detects the unauthorized
reproduction of classified documents is encouraged.
219

All reproductions of classified material shall be
conspicuously marked with the same classification
markings as the material being reproduced.
Domestic Transmission Standards
(Outside of Facility)
Top Secret
• Written authorization of the Government Contracting
Activity (GCA)
• Sealed, opaque inner and outer covers with the
inner cover being a wrapper or envelope plainly
marked with the assigned classification and addresses
of both sender and addressee
• A receipt that identifies the sender, the addressee,
and the document shall be attached to or
enclosed in the inner cover
• Via:
a. Defense Courier Service (DCS), if authorized
by GCA
b. A designated courier or escort cleared for
access to TOP SECRET information
c. By electrical means over CSA-approved secured
communications security circuits provided
such transmission conforms with the
NISPOM, the telecommunications security
provisions of the contract, or is authorized
by the GCA
Secret
• Sealed, opaque inner and outer covers with the
inner cover being a wrapper or envelope plainly
marked with the assigned classification and addresses
of both sender and addressee
• A receipt that identifies the sender, the addressee,
and the document shall be attached to or
enclosed in the inner cover
• Via:
a. TOP SECRET methods
b. USPS Express or Registered mail
c. A cleared “Commercial Carrier”
d. A cleared commercial messenger service engaged
in the intracity/local area delivery
(same day delivery only) of classified material
e. A commercial delivery company approved by
the CSA
f. Other methods as directed, in writing, by
the GCA
220
Comparison of the Canadian Manual and the U.S. Manual
Confidential
• Packaged by SECRECT material methods except
that a receipt is required only if the sender deems it
necessary
• Via:
a. SECRET methods
b. USPS Certified mail
International Transmission Standards
Top Secret
• Domestic requirements
• Via:
a. Defense Courier Service
b. Department of State Courier System
c. Courier service authorized by GCA
Secret and Confidential
• Domestic requirements
• Via:
a. Registered mail through U.S. Army, Navy,
or Air Force postal facilities
b. Appropriately cleared contractor employee
c. U.S. civil service employee or military person
designated by the GCA
d. U.S. and Canadian registered mail with registered
mail receipt to and from Canada and
via a U.S. or Canadian government activity
e. As authorized by the GCA
Destruction
Contractors shall destroy classified material in
their possession as soon as possible after it has
served the purpose for which it was intended.
Classified material may be destroyed by burning,
shredding, pulping, melting, mutilation, chemical
decomposition, or pulverizing. Pulpers, pulverizers,
or shedders may be used only for the destruction of
paper products. Residue shall be inspected during
each destruction to ensure that classified information
cannot be reconstructed. Crosscut shredders
shall be designed to produce residue particle size not
exceeding 1/32 inch in width by 1/2 inch in length.
Public destruction facilities may be used only
with the approval of the CSA, and classified material
removed from a cleared facility for destruction shall
be destroyed on the same day it is removed.
Destruction shall be performed only by appropri-

ately cleared employees of the contractor. For destruction
of TOP SECRET material, two persons are
required. For destruction of SECRET and CONFI-
DENTIAL material, one person is required.
Destruction records that indicate the date of
destruction, identify the material destroyed, and are
signed by the individuals designated to destroy and
witness the destruction are required for TOP SE-
CRET material.
Information System Security
Information systems (IS) that are used to capture,
create, store, process, or distribute classified
information must be properly managed to protect
against unauthorized disclosure of classified information
and loss of data integrity, and to ensure the
availability of the data and system.
Protection requires a balanced approach including
IS security features to include. but not limited
to, administrative, operational, physical, computer,
communications, and personnel controls. Protective
measures commensurate with the classification of
the information, the threat, and the operational requirements
associated with the environment of the
IS are required.
The requirements outlined in the NISPOM apply
to all information systems processing classified
information. Additional requirements for high-risk
systems and data are covered in the NISPOM Supplement.
The CSA is the Designated Accrediting/
Approving Authority (DAA) responsible for accrediting
information systems used to process classified
information in industry. A formal certification and
accreditation (C&A) occurs after the protection
measures have been implemented and any required
IS protection documentation has been approved.
Certification validates that the protection measures
described in the System Security Plan (SSP) have
been implemented on the system and that the protection
measures are functioning properly. Accreditation
is the approval by the CSA for the system to
process classified information.
A. Hammond
Canada—Industrial Security Manual
Security Classifications
The originator of the information and assets
determines the classification level.
Top Secret
TOP SECRET refers to information and assets
related to the national interest that may qualify for
an exemption or exclusion under the Access to Information
Act or Privacy Act and that the compromise
of which would reasonably be expected to cause
exceptionally grave injury to the national interest.
Secret
SECRET refers to information and assets related
to the national interest that may qualify for an exemption
or exclusion under the Access to Information
Act or Privacy Act and that the compromise of
which would reasonably be expected to cause serious
injury to the national interest.
Confidential
CONFIDENTIAL refers to information and
assets related to the national interest that may qualify
for an exemption or exclusion under the Access
to Information Act or Privacy Act and that the compromise
of which would reasonably be expected to
cause injury to the national interest.
Protected “C”
PROTECTED “C” refers to information and
assets related to other than the national interest that
may qualify for an exemption or exclusion under the
Access to Information Act or Privacy Act that could
reasonably be presumed to cause extremely serious injury,
such as loss of life, if compromised.
Protected “B”
PROTECTED “B” refers to information and
assets related to other than the national interest that
may qualify for an exemption or exclusion under the
Access to Information Act or Privacy Act that could
reasonably be expected to cause serious injury if compromised.
221

Protected “A”
PROTECTED “A” refers to information and
assets related to other than the national interest that
may qualify for an exemption or exclusion under the
Access to Information Act or Privacy Act that could
reasonably be presumed to cause injury if compromised.
Facility Security
Facility Clearances
A Facility Security Clearance is an administrative
determination that an organization is eligible, from a
security viewpoint, for access to CLASSIFIED and
PROTECTED information and assets of the same
or lower classification level as the clearance being
granted.
There are three types of Facility Security Clearances
each of which may be authorized at the classification
level of CONFIDENTIAL, SECRET, or TOP
SECRET:
1. Personnel Assigned (PA). This is the most basic
type of Facility Security Clearance which involves
security screening of the organization’s Key Senior
Officials and employees. There is NO requirement
to evaluate the physical security status of the organization’s
facilities. The organization is not authorized
to possess or store CLASSIFIED information and
assets.
2. Document Safeguarding Capability (D.Sc.). In addition
to the security screening of the organization’s
Key Senior Officials and employees, the physical security
of the organization’s facilities is assessed to
ensure safeguarding requirements are met. The organization
is authorized to possess and store CLAS-
SIFIED information and assets.
3. Production (PROD). This includes all of the elements
of a Document Safeguarding Facility Security
Clearance. In addition, the security of the manufacturing,
repairing, modifying, or otherwise working
on CLASSIFIED components or items is assessed to
ensure government security requirements are met.
A Designated Organization Screening (at the PRO-
TECTED level) is an administrative determination
that an organization is eligible, from a security viewpoint,
for access to PROTECTED information and
assets of the same or lower level as the clearance be-
222
Comparison of the Canadian Manual and the U.S. Manual
ing granted. The three types of Designated Organization
Screening are equivalent to the three types of
Facility Security Clearances except they pertain only
to PROTECTED information and assets. Each of
the three types may be authorized at one of the following
levels: PROTECTED “A,” PROTECTED
“B,” or PROTECTED “C.”
An organization is eligible to obtain an organization
security screening/clearance only if it is sponsored
by an authorized sponsor in support of an existing
or impending contract or bid solicitation
which calls for access to CLASSI-
FIED/PROTECTED information, assets, and/or
certain restricted work sites.
Meetings
(No provisions are established within the Canadian
Industrial Security Manual.)
Personnel Security
Security Officers
All organizations that require a Designated Organization
Screening or a Facility Security Clearance
shall appoint a Company Security Officer. The Company
Security Officer shall be appointed by the
Chief Executive Officer (CEO) or the designated Key
Senior Official (KSO) of the organization. The CSO
must be a Canadian citizen employee, report to a
designated KSO, and be security screened or cleared
to the Reliability Status level or Facility Security
Clearance level of the facility. The appointment of
the Company Security Officer must be approved by
the Canadian and International Industrial Security
Directorate (CIISD).
When a facility-cleared Canadian parent organization
owns one or more cleared subsidiaries in Canada,
a Corporate Company Security Officer (CCSO)
should be appointed to oversee government industrial
security matters for the entire corporation.
Personnel Clearances
Personnel Security Screening must be carried
out according to the highest sensitivity level of information
and assets that will be accessed during the

contracting process and/or required for access to
restricted work sites. Access to PROTECTED information,
assets, and restricted work sites requires that
an individual has Reliability Status, and access to
CLASSIFIED information, assets, and/or restricted
work sites requires a Security Clearance at the appropriate
level of sensitivity.
Only individuals employed or under a contract
to commence employment within 60 days by a private
sector organization on a contract/subcontract
requiring access to CLASSIFIED/PROTECTED
information, assets, and/or certain restricted work
sites may be security screened. Non-Canadian citizens
may be security cleared with access limitations.
The limitations include denying access to CLASSI-
FIED/PROTECTED information and assets which
are not of Canadian origin, do not come from the
country of which the person is a citizen, or are not
releasable to his/her nation of origin.
Contractors have no authority to deny or revoke
Personnel Security Clearances for employees. This
authority is reserved by the Canadian Government.
The contractor may suspend the access of an individual,
while notifying CIISD of the circumstances.
Subcontracting
Contractors shall subcontract work only to companies
holding a current Designated Organization
Screening or a Facility Security Clearance of the type
and at the level appropriate to the work to be performed
under the subcontract. CIISD approval of
the subcontractor must be obtained before award of
the subcontract and the Designated Organization
Screening or Facility Security Clearance for the proposed
subcontractor(s) must be verified by CIISD
before issue of bid solicitation documents. Contractors
shall not assign a subcontract to organizations
located outside of Canada without the prior written
approval of CIISD and the Public Works and Government
Services Canada (PWGSC) contracting authority.
The prime contractor shall ensure the security
safeguarding of work placed with subcontractors.
Education, Training, and Briefings
A major objective of the Company Security
Officer in conducting a Security Education Program
A. Hammond
involves working closely with management, from the
top down, to ensure proper company security. Managers
and supervisors at all levels are responsible not
only for their own personal security measures, but
also for ensuring that proper security procedures are
followed by all employees in the organization. An
initial security briefing, reinforced by an ongoing Security
Education and Awareness Program, is essential
to the maintenance of an effective security program.
Upon receiving a Personnel Security Clearance
an employee acknowledges his or her responsibilities
by reading and signing the Security Screening Certificate
and Briefing Form, TBS/SCT 330-47 Rev.
2002/06. A briefing from the Company Security
Officer, which details the individual’s specific responsibilities
and duties relative to security in the
facility, must be presented. (New employees, even
though not yet security-screened and therefore prohibited
from access to CLASSIFIED information
and assets, should be given a security briefing appropriate
to their duties.)
Visits
A Visit Clearance Request (VCR) (submitted to
CIISD via a Request for Visit form) is required
when a security-cleared individual must visit a government/commercial
organization in Canada or
abroad, for the purpose of having access to CLASSI-
FIED information and assets or where access to the
installation is restricted in the interest of national
security. Visitors must not proceed with CLASSI-
FIED visits without prior visit clearance authorization
from CIISD. The host organization shall deny
access to CLASSIFIED information and assets or
access to certain restricted work sites until the visitors’
Personnel Security Clearance level and their
need-to-know have been verified and confirmed by
the CIISD through official visit protocol.
Submission of a VCR initiates verification by
CIISD that confirms:
• The organization requesting the visit has an Facility
Security Clearance to the required level
• Each of the proposed visitors has a valid Personnel
Security Clearance to the required level
• Foreign disclosure limitations are identified and
strictly observed
Visit Clearance Request is approved when the
223

equesting organization is notified by CIISD. Visitors
must not proceed on CLASSIFIED visits without
prior visit clearance authorization.
Visit Clearance Request (VCR) requires strict
lead-times imposed by the authorities of foreign nations.
Every effort must be made to ensure that leadtimes
are observed, as failure to do so will likely result
in rejection of the RFV.
Organizations shall maintain a record of all individuals
who visit the facility for the purpose of having
access to CLASSIFIED information. This record
shall be separate from the record of unclassified visits.
Document Security
General Marketing
All documents shall be marked on the outside of
both the front and back covers with the highest level
of classification and loose documents shall be
marked on every sheet. Security markings should
include the applicable classification/protection and
the date or event at which declassification or downgrading
is to occur. All covering or transmittal letters
or forms or circulation slips must be marked to show
the highest level of classification or protection of the
attachments.
For TOP SECRET information, mark the classification
in the upper right corner of each document
page and show the total number of pages on each
page of the document.
For SECRET information, mark the classification
in the upper right corner of each document
page.
For CONFIDENTIAL information, mark the
classification in the upper right corner of the face of
the document.
For PROTECTED information, mark the word
“PROTECTED” in the upper right corner of the
face of the document and, where required, with the
letter “A,” “B,” or “C” to indicate the level of protection.
General Storage
PROTECTED B and PROTECTED C information
and assets and all CLASSIFIED information
must be stored in an approved security container.
224
Comparison of the Canadian Manual and the U.S. Manual
PROTECTED A information and assets shall be
stored in a locked container.
CLASSIFIED or PROTECTED information
and assets may be stored on open shelving in a secure
room, only after inspection and approval by
CIISD and only to the level approved by CIISD.
Also, CLASSIFIED and PROTECTED information
and assets shall not be stored in the same container
as negotiable or attractive assets.
Reproduction
Reproduction of CLASSIFIED information
shall be done only with the authorization of the
Company Security Officer or an authorized Alternate
Company Security Officer. Reproductions must
be marked, registered, and accounted for in the
same manner as for the originals. Reproductions of
PROTECTED information must be marked in the
same manner as the originals. TOP SECRET and
PROTECTED C information shall NEVER be reproduced
without written authorization from
CIISD.
Domestic Transmission Standards
(Outside of Facility)
Top Secret
• Documents must be double enveloped (gum
sealed, heavy duty) and sealed with government approved
security tape.
• A self-addressed receipt is enclosed in the inner
envelope or wrapping and the inner envelope or
wrapping is closed with an approved security tape.
• Inner envelope or wrapping must bear the security
marking and the recipient’s address.
• Shipment must be recorded prior to leaving a
Security Zone and the recipient must be notified in
advance of shipment.
• Documents are sent via a securitycleared/reliability-checked
individual employed by
the dispatching/receiving Facility Security Cleared
Canadian organization.
Secret, Confidential, and Protected “C”
• Documents must be double enveloped (gum
sealed, heavy duty) and sealed with government approved
security tape.
• A self-addressed receipt is enclosed in the inner

envelope or wrapping and the inner envelope or
wrapping is closed with an approved security tape.
• Inner envelope or wrapping must bear the security
marking and the recipient’s address.
• Via:
a. Priority courier
b. Registered mail
c. A security-cleared/reliability-checked individual
employed by the dispatching/receiving
Facility Security Cleared Canadian organization
Protected “A” and “B”
• Single, gum-sealed, heavy duty envelope
• Via:
a. First class mail
b. An individual employed with the organization
c. Classified/Protected “C” methods
International Transmission Standards
Top Secret, Secret, Confidential, and Protected “C”
• Double enveloped (gum sealed, heavy duty) and
sealed with government approved security tape
• Via CIISD
Protected “B”
• Single, gum sealed, heavy-duty envelope
• Via CIISD
Protected “A”
• Single, gum sealed, heavy-duty envelope
• Via first class mail, priority courier, or registered
mail
Destruction
Unless otherwise specified, TOP SECRET, and
PROTECTED “C” information and assets must be
returned to CIISD for disposal.
Unless otherwise specified, SECRET, CONFI-
DENTIAL, and PROTECTED “A” and “B” information
and assets of Canadian origin may be destroyed
by the organization with the approval of
CIISD.
CLASSIFIED and PROTECTED information
and assets which have been authorized for destruction
must be disposed of in accordance with the
following:
A. Hammond
• It must be destroyed only by approved destruction
equipment, or at a facility authorized by CIISD.
• Information awaiting destruction or in transit to
destruction must be safeguarded in the manner prescribed
for the most highly CLASSIFIED and PRO-
TECTED information asset involved.
• CLASSIFIED and PROTECTED information/assets
awaiting destruction must be kept separate
from other information/assets awaiting destruction.
• An employee with a proper security clearance or
with Reliability Status, as applicable, must be present
to monitor the destruction of CLASSIFIED and
PROTECTED information, respectively.
• Surplus copies and waste that could reveal
CLASSIFIED and PROTECTED information must
be protected to the appropriate level and should be
promptly destroyed.
Information System Security
The ISM establishes operational standards in
Canadian industry for the safeguarding of Government
information electronically processed, stored, or
transmitted. This also applies to the safeguarding of
technology assets. The administrative, organizational,
physical, and personnel security standards as
documented in the ISM also apply to the information
technology environment.
The Government Security Policy requires that
the degree of safeguarding provided by industry be
commensurate with the level of the information and
assets and the associated threats and risks. The contracting
authority is responsible for ensuring that the
requirements of the Government Security Policy are
met and that the security standards are applied by
the private sector contractor. The security standards
contained in the Government Security Policy, Information
Technology Standards, are the minimum
standards for security in the private sector. Assessments,
advice, and guidance regarding these standards
are available from the Canadian and International
Industrial Services Directorate (CIISD) of
Public Works and Government Services Canada
(PWGSC).
The prime contractor’s Information Technology
Facility(s) must be approved by CIISD prior to processing
government information.

Conclusions
“It’s important to be responsible here and to be particularly
careful after 9/11 that we’re not giving our
enemies information or materials that would make
their job easier.” (Chui, 2003)
John H. Marburger III,
Director, Office of Science & Technology Policy
(and science adviser to President George W. Bush)
To no surprise, the anthrax letter attacks of
2001 led directly to national policy changes since
they specifically targeted both lawmakers and media
personnel at their workplaces. To better protect
their citizens, the United States and Canadian governments
established controls not only over the possession
and use of hazardous biological agents, but
also over the information pertaining to their possession
and use. Legislation is now in place that forbids
the disclosure of information that may identify
which biological agents are possessed, who possesses
that agent(s) and where, and any safeguard and security
measures used to protect unauthorized access to
the agent(s). Because of the genuine threat of bioterrorism,
biodefense research has become a vital and
necessary component of an overall national security
program. The United States alone has committed
billions of dollars towards biodefense research and
development. To protect biodefense information
and assets, organizations working on projects
deemed to be “classified” (for the sake of national
security) must follow precise requirements, restrictions,
and safeguards established by their federal government.
For Canada and the United States, these
requirements are conveyed in the Canadian Industrial
Security Manual (ISM) and the U.S. National Industrial
Security Program Operating Manual (NISPOM). These
manuals provide guidance in implementing a uni-
226
Comparison of the Canadian Manual and the U.S. Manual
form and cost-effective security system, thus allowing
an organization to focus mainly on research rather
than the burden of developing and implementing
security procedures. Without these standards and
consistent security policies and practices the potential
for compromise leading to a serious national
security threat is enormous.
References
Canadian and International Industrial Security Directorate.
(2004). Industrial security manual. Available
at www.ciisd.gc.ca/ism/text/preface-e.asp. Accessed
online 2004.
Chui, G. (2003). Security concerns imperil research:
Restrictions shackle scientists, some say. The Mercury
News, March 3, 2003. Available at www.
mercurynews.com/mld/mercurynews/news/
5303757.htm?1c. Accessed online 2004.
Defense Technical Information Center. (2004). National
industrial security program operating manual
(DoD 5220.22-M). Available at www.dtic.mil/whs/
directives/corres/html/522022m.htm. Accessed
online 2004.
Parliament of Canada. (2004). Public Safety Act,
2002. Available at www.parl.gc.ca/37/3/parlbus/
chambus/house/bills/summaries/c7-e.pdf. Accessed
online 2004.
U.S. Government Printing Office. (2004). Public
Health Security and Bioterrorism Preparedness and Response
Act of 2002. Available at frwebgate.access.gpo.
gov/cgi-bin/getdoc.cgi?dbname=107_cong_public_
laws&docid=f:publ188.107.pdf. Accessed online
2004.

Article
The Infectious Dose of Francisella
tularensis (Tularemia)
Abstract
Quantitatively estimating an individual’s risk of
infection by an airborne pathogen requires knowledge
of the expected dose and the pathogen’s infectious
dose. Based on our review of the published literature on
tularemia, we conclude that the infectious dose of Francisella
tularensis varies among individuals, but that a
substantial proportion of the population can be infected
by a single bacillus. We also conclude that infection can
be initiated by inhaling bacilli carried on respirable particles
(diameters less than 10 µm) or nonrespirable particles
(diameters between 10 µm and 100 µm). Regression
analyses based on two-parameter Weibull and lognormal
models of human inhalation dose-infection data
aggregated across three studies indicate that approximately
30% of individuals who inhale a single F. tularensis
bacillus will develop tularemia. Further, when
the organism is carried on particles with diameters on
the order of 1 µm, it is estimated that the deposition of
a single bacillus produces infection in 40% to 50% of
individuals; thus, when F. tularensis is carried on respirable
particles, the estimated ID50 via inhalation is close
to one deposited bacillus. These results are consistent
with separate analyses using nonparametric methods
and with experimental animal models in which infection
is observed after injection of a single bacillus. The risk
of person-to-person transmission of tularemia is generally
considered negligible, perhaps due to a low concentration
of F. tularensis in respiratory fluids. However,
viable F. tularensis bacilli are present in human respiratory
fluids, and can be carried in inspirable particles
(diameters less than 100 µm) which are emitted during
coughs and sneezes.
Introduction
Applied Biosafety, 10(4) pp. 227-239 © ABSA 2005
Rachael M. Jones, Mark Nicas, Alan Hubbard, Matthew D. Sylvester, and Arthur Reingold
University of California—Berkeley, Berkeley, California
Francisella tularensis (formerly termed Pasteurella
tularensis and Bacterium tularensis) is a bacterium that
causes a spectrum of clinical illnesses termed
“tularemia.” F. tularensis is a candidate agent for
bioterrorism because it can be weaponized readily
and is considered to have a low airborne infectious
dose (Dennis et al., 2001; Franz et al., 1999). The
World Health Organization (1970) has estimated
that aerosol dispersal of 50 kg of F. tularensis over a
metropolitan area with approximately 5 million inhabitants
would result in 250,000 incapacitating
casualties, including 19,000 deaths. Infection by the
respiratory route has been demonstrated in Macaca
mulatta using both respirable particles (diameters less
than 10 m) and inspirable, but nonrespirable, particles
(diameters between 10 m and 100 m) (Day &
Berendt, 1972). Naturally occurring respiratory infection
has been documented in Scandinavian farm
workers exposed when handling hay contaminated
by voles and their waste products (Dahlstrand,
Ringertz & Zetterberg, 1971; Syjala, Kujala, Myllyla,
& Sandstrom, 1996). Historically, laboratory personnel
have become infected by bacterium-containing
aerosols generated during normal laboratory procedures
and accidents (Ledingham & Fraser,
1923/1924; Overholt et al., 1961; Van Metre, Jr. &
Kadull, 1959). Perhaps due to the low infectious
dose by inhalation, implementation of careful handling
procedures for F. tularensis in clinical laboratories
has not entirely eliminated the potential for infection
(Shapiro & Schwartz, 2002).
Secondary (person-to-person) transmission of F.

tularensis is generally considered improbable. For
example, the Centers for Disease Control and Prevention
(2004) states: “Tularemia is not known to be
spread from person-to-person.” Similarly, the Working
Group on Civilian Biodefense (Dennis et al.,
2001) concludes: “Isolation is not recommended for
tularemia patients given the lack of human-tohuman
transmission.” In 1951, however, Fillmore
reported the case of a nurse’s aide who developed
symptoms of tularemia 3 to 4 weeks after attending a
patient with pleuropulmonary tularemia; the nurse’s
aide had no contact with domestic or wild animals.
In determining procedures to control airborne
infection, it is useful to quantitatively estimate infection
risk, which depends on the pathogen’s infectious
dose, the concentration of the pathogen in air,
and the duration of exposure. Variability in host
susceptibility is captured, in part, by interindividual
variability in a deterministic infectious dose. Given
an appreciation of the potential intensity of airborne
exposure to F. tularensis and of the pathogen’s infectious
inhalation dose, one can evaluate existing biosafety
protocols in laboratory and clinical settings. In
this paper, we argue that the infectious dose of F.
tularensis for a substantial portion of the population
is on the order of one bacillus. We also argue that
person-to-person transmission of tularemia is theoretically
possible given low infectious dose values
overall, and the presence of the bacillus in the sputum
of some infected patients. However, the risk of
secondary airborne infection may typically be low
due to low pathogen concentrations in respiratory
fluid and small aerosol volumes emitted in coughs
commonly described as “nonproductive.”
Background on Tularemia
F. tularensis is a Gram-negative coccobacillus,
with diameter ranging from 0.2 to 0.7 m (Evans,
1985). The organism is rickettsial in that it cannot
replicate outside a host cell, and it is pathogenic after
being phagocytized by macrophages (Sjostedt,
Tarnvik, & Sandstrom, 1996). Although originally
described in ground squirrels in Tulare County,
California, in 1911 by McCoy, the organism was
renamed for Edward Francis who described the
clinical and epidemiologic features of the disease
228
The Infectious Dose of Francisella tularensis (Tularemia)
(Francis, 1927; Francis 1983). F. tularensis is found
globally in mammals and arthropod vectors, and two
strains produce infection in humans. Type A is associated
with illness in North America; Type B is less
virulent and is associated with illness in Europe
(Reinjes et al., 2002). Type A has been traditionally
considered for use as a biological weapon (Conlan et
al., 2003), and the Schu strain of Type A, isolated
from a finger ulcer (Bell, Owen, & Larson, 1955),
was frequently used in experimental work until the
late 1960s. At that time, investigators began using
the live vaccine strain (LVS) of F. tularensis for experimental
work because it is less virulent in humans yet
is virulent in mice (Conlan et al., 2003).
Note that the terms “infectivity” and “virulence”
are distinct. Infectivity signifies the ability of a pathogen
to penetrate into host tissue and multiply. Infectivity
can be quantified by the metric of infectious
dose, that is, the number of viable organisms
(colony-forming units) that must penetrate into host
tissue to initiate an infection, where infection is assessed
by serology and clinical indicators. However,
in nonhuman mammalian studies, the infectivity of
F. tularensis is typically reported as the number of
organisms that are lethal to 50% of exposed animals,
termed the LD50. Virulence refers to the intensity of
the disease produced by pathogen infection in a
given host species (Black, 2002); the term is also
used to compare the intensity of disease produced by
a given pathogen in different host species. For F.
tularensis, there is evidence that virulence is influenced
by the dose of organisms received. For example,
among Macaca mulatta receiving Schu S-4 strain
organisms carried on 2.1 m diameter particles by
inhalation, five inhaled bacilli infected 6/6 hosts but
caused death in only 1/6 hosts, whereas higher inhaled
doses caused death in a progressively greater
proportion of animals (McCrumb, 1961).
Infection with F. tularensis can occur through
ingestion, dermal contact, and inhalation of the organism,
and produces an array of clinical features
(Centers for Disease Control and Prevention, 2003;
Dennis et al., 2001). Ingestion of F. tularensis typically
produces oropharnygeal tularemia (pharyngitis
and cervical adenitis) (Reintjes et al., 2002). Dermal
contact, arthropod bites, and intracutaneous inoculation
often produce an ulcerated lesion at the site

of contact and/or swelling of the regional lymph
nodes, although some individuals exposed through
these routes can present with fever and other signs
indicative of systemic infection (Dennis et al., 2001;
Evans, 1985; Saslaw et al., 1961), including pulmonary
involvement (bronchopneumonia and hilar
adenopathy) (Miller & Bates, 1969). Inhalation of F.
tularensis also produces systemic disease in humans
and may produce pneumonia (McCrumb, 1961;
Overholt et al., 1961), oval lesions in the lungs
(Overholt & Tigertt, 1960), and/or bronchial
changes (Syrjala et al., 1986). Although it has been
demonstrated experimentally that humans can develop
tularemia through inhalation of F. tularensis
(McCrumb, 1961; Sawyer et al., 1966), rapid disease
onset and delay in examination make it difficult to
determine in some cases if pulmonary involvement
precedes or follows systemic infection. Cough frequently
occurs in patients with and without objective
pulmonary involvement (Dennis et al., 2001;
Saslaw et al., 1961). Although McCrumb (1961) reports
that patients exhibited a lack of sputum production
and nonproductive cough, case reports indicate
that some patients exhibit increased mucous
and sputum production, and productive cough
(Cluxton, Jr., Cliffton & Worley, 1948; Syrjala et al.,
1986).
If untreated, pulmonary tularemia resulting from
the type A strain has a case fatality proportion of
40% to 60% (McCrumb, 1961). Mortality due
to all clinical manifestations of tularemia has been
approximately 5%, although in the United States,
treatment with streptomycin and gentamicin has
reduced the overall case fatality proportion to below
2% (Dennis et al., 2001). The type B strain found in
Europe is rarely fatal (Dennis et al., 2001).
Experimental Airborne Infection
Study Descriptions
Airborne transmission of F. tularensis has been
demonstrated using investigator-generated aerosol
and respiratory aerosol emitted by infected animals.
Work with investigator-generated aerosols commenced
at Fort Detrick, Maryland in the mid-1940s,
when infection of mice by “clouds” of F. tularensis
was assessed (Rosebury, 1947). Animals were ex-
R. M. Jones, et al.
posed in a stainless steel chamber to aerosol generated
by a Chicago atomizer from cultures suspended
in water; the reported mass median diameter of the
aerosol particles was less than 1 m. The inhalation
dose was estimated based on the animal’s breathing
rate (related to weight), duration of exposure, and
the viable F. tularensis aerosol concentration as measured
by impinger sampling of chamber air. The reported
inhalation doses assumed that 100% of inhaled
particles were retained in the respiratory tract.
For mice, the doses ranged from 14 to 4,500 bacilli.
Only one of thirty (1/30) mice receiving a dose of 14
organisms died, while all 59 mice exposed to doses
of 330 or more organisms died. All animals that did
not die within 16 days subsequent to exposure were
autopsied and found by gross evaluation and spleen
culture to be negative for F. tularensis. It was estimated
that 70 organisms would produce mortality in
50% of exposed mice, with 95% confidence limits of
2 to 2,063 organisms. The wide confidence interval
was attributed to the large variability in recovery of
aerosolized organisms. Rosebury (1947) also reports
the results of Henderson, which were conveyed in a
personal communication. Working at Porton Down
in the United Kingdom, Henderson found that the
50% lethal dose in mice exposed via inhalation was
12 organisms.
Hood (1961) exposed guinea pigs to aged F. tularensis
(Schu D strain) aerosols generated with a
Henderson apparatus and stored in a rotating
stainless steel drum. Of the aerosols generated in the
Henderson apparatus, approximately 98% of the
dried particles had diameters less than 1 m, and
90% of the droplets emerging from the spray apparatus
had diameters less than 10 m (Henderson,
1952). The inhaled dose was estimated based on the
animal’s breathing rate (related to weight), exposure
duration, the viable F. tularensis aerosol concentration
as measured by impinger sampling near the
guinea pigs, and an estimated inhaled particle retention
factor of 0.55. When bacterial suspensions aged
6 to 30 days were aerosolized and held in the drum
for 3 seconds before the animals were exposed, the
LD50 was 1 to 4 organisms. The investigators found
that there was no significant loss of infectivity when
aerosols were aged for 20 minutes in the drum, but
significant infectivity was lost when aerosols were
229

aged for 20 hours. It is unclear, however, what portion
of the decreased infectivity was due to organism
die-off versus deposition on the drum walls.
A similar study was undertaken by Sawyer et al.
(1966), who exposed Macaca mulatta and human
volunteers to aerosols of the F. tularensis Schu-S4
strain, generated with a two-fluid nozzle and stored
in a spherical static chamber. The concentration of
aerosol particles was measured by a total collector,
and an impinger was used to determine the number
of particles with diameters of 5 m or less; it was
estimated that 65% of viable organisms were contained
in particles with diameters of 5 m or less.
Caged monkeys were placed in the test chamber for
3 or 10 minutes; human subjects were exposed
through a facemask for ten 1-liter breaths in 60 seconds.
The inhaled dose was defined as the product
of the duration of exposure, respiratory minute volume,
and the concentration of viable organisms in
particles with diameters 5 m or less; the fraction of
organisms retained was not considered. It was found
that an inhaled dose of 80 to 180 viable organisms
from an aerosol aged for 60 minutes infected three
of four (3/4) humans and seven of eight (7/8) monkeys.
In another series of experiments, it was found
that among human subjects who inhaled 150 viable
organisms, two of four (2/4) became infected when
the aerosol was aged 30 minutes, and three of four
(3/4) became infected when the aerosol was aged 60
minutes. Only results for aerosols aged 60 minutes
or less are reported here because aerosols aged for
120 and 180 minutes showed substantially decreased
infectivity for humans and M. mulatta.
One limitation of the Sawyer et al. study was
that the dose estimate was based on particles with
diameters less than 5 m. Day and Brendt (1972)
noted that all particles with diameters greater than
5 m contained F. tularensis bacilli, and that while
these particles may not penetrate to the pulmonary
region of M. mulatta, infection could result from
deposition in the upper respiratory tract. This observation
suggests that the inhaled doses were larger
than reported by Sawyer et al. On the other hand,
Sawyer et al. did not adjust the inhaled dose for the
fraction of organisms that deposited in the respiratory
tract, which signifies that the retained respirable
dose was less than the dose reported.
230
The Infectious Dose of Francisella tularensis (Tularemia)
Low dose infectivity of F. tularensis aerosols in
vaccinated and nonvaccinated men has been reported
by two investigators (Table 1). Saslaw et al.
(1961) exposed men via a facemask to an aerosol
with an average particle diameter of 0.7 m. The
subjects were instructed to inhale through their nose
and exhale through their mouths. Sixteen of the
twenty (16/20) nonvaccinated men became infected
after inhaling doses of 10 to 52 F. tularensis bacilli.
The four men who did not develop infection had
inhaled 10 to 45 organisms. The lowest doses producing
infection among nonvaccinated and vaccinated
men were 10 and 13 bacilli, respectively. We
note that for particles with an aerodynamic diameter
of 0.7 m, the approximate deposition fraction in
the human pulmonary region is 0.2 (Hinds, 1999).
Therefore, 10 to 52 inhaled F. tularensis bacilli represent
approximately 2 to 10 deposited bacilli.
McCrumb (1961) reported that among nonvaccinated
individuals exposed to 20, 200, and 2,000 F.
tularensis bacilli, all individuals (4/4, 4/4, and 2/2,
in the respective dose groups) became infected;
among 12 vaccinated individuals exposed to 20 organisms,
four (33%) were infected. Unfortunately,
McCrumb provided little information on the methodology
used in these studies and did not account
for the particle deposition fraction.
Statistical Analysis of Aggregate Data
To examine the infectious dose statistically, we
began by pooling the inhalation dose-infection results
obtained by McCrumb (1961) and Saslaw et al.
(1961) for nonvaccinated individuals, and by Sawyer
et al. (1966) for aerosols aged 60 minutes or less
(Table 1). For this analysis, none of the inhaled
doses were adjusted for the deposition fraction of
the challenge particles in the respiratory tract, which
is to say that the true deposited doses were less than
the reported inhaled doses. We alternatively fit a
discrete nonparametric maximum likelihood cumulative
probability distribution and two continuous
cumulative probability distributions (lognormal and
Weibull) to the data to estimate the probability of
remaining uninfected after being exposed to a fixed
dose. These fitted probability distributions describe
the survival distribution; the complement of the survival
distribution is the cumulative infection distri-

ution (i.e., the proportion of individuals infected
with increasing dose).
The survival data structure can be considered
“current status.” For each dose, one knows whether
the dose given was either less than a subject’s minimum
infectious dose (i.e., the subject was not infected)
or greater than the minimum infectious dose
(i.e., the subject was infected). The nonparametric
maximum likelihood estimate of the survival function
for current status data has been referred to as
the pooled adjacent violator (PAV); the analysis involves
a nonparametric, monotonically decreasing
regression on the proportion of subjects left unin-
R. M. Jones, et al.
Table 1
Infection dose-response data in human subjects used to determine low dose infectivity.
Estimated Bacilli Dose
Response
#Infected/#Exposed
Reference
10 1/2 Saslaw, et al., 1961
12 0/1 Saslaw, et al., 1961
13 1/1 Saslaw, et al., 1961
14 1/1 Saslaw, et al., 1961
15 1/1 Saslaw, et al., 1961
16 1/1 Saslaw, et al., 1961
18 1/1 Saslaw, et al., 1961
20 5/6
Saslaw, et al., 1961;
McCrumb, 1961
23 2/2 Saslaw, et al., 1961
25 1/1 Saslaw, et al., 1961
30 1/1 Saslaw, et al., 1961
45 0/1 Saslaw, et al., 1961
46 2/2 Saslaw, et al., 1961
48 1/1 Saslaw, et al., 1961
50 1/1 Saslaw, et al., 1961
52 1/1 Saslaw, et al., 1961
150 5/8 Sawyer, et al., 1966
200 4/4 McCrumb, 1961
350 2/4 Sawyer, et al., 1966
750 4/4 Sawyer, et al., 1966
2,000 2/2 McCrumb, 1961
fected at each dose (Kalbfleisch & Prentice, 2002).
Inference can be derived from nonparametric bootstrapping.
Because the data are sparse and thus the nonparametric
estimates are highly variable, we also explored
parametric regressions by fitting both a twoparameter
Weibull model and a two-parameter lognormal
model to the data. The Weibull survival
function has the form:
Eq. 1 S(d) = exp(— [d] )
where S(d) is the probability of not being infected at
dose d, and where > 0 and > 0 are scale and
231

shape parameters, respectively. The lognormal survival
function has the form:
Eq. 2
where GM and GSD denote the geometric mean
(median value) and geometric standard deviation,
respectively, and (z) denotes the cumulative standard
normal distribution evaluated for the argument
z. For improved readability, all fits are displayed
graphically as the probability of infection, which is
one minus the probability of survival.
We note that the infectious dose models
explored in this analysis are “deterministic” or
232
The Infectious Dose of Francisella tularensis (Tularemia)
“threshold” models. It is assumed that if the host
receives a threshold number of organisms or more,
infection is certain to occur, whereas if the host receives
fewer than the threshold number of organisms,
infection is certain not to occur. Variability in
host susceptibility is reflected by interindividual variability
in the threshold dose value, and the ID50 is
that deposited dose which will infect 50% of the
population with certainty. In contrast, a purely
“probabilistic” model assumes that a single organism
can successfully infect the host with probability
psuccess, such that the probability of infection is
1 —(1 — psuccess) D , where D is the deposited number
of organisms. If the value of psuccess is the same across
individual hosts, the ID50 is that deposited dose
which produces a 50% likelihood of infection in all
Figure 1
Estimated cumulative infection probability of humans experimentally exposed to increasing inhaled doses of
F. tularensis based on the Table 1 data, via a nonparametric maximum likelihood analysis (black line with
dashed lines representing the 95% CI), a lognormal parametric regression, and a Weibull parametric
regression. The Table 1 data points are indicated by the diamonds.

hosts. A probabilistic model can also account for
variability in the value of psuccess between individual
hosts.
Figure 1 displays the data listed in Table 1, the
best fit curves for the lognormal and Weibull model,
and the PAV fit along with the 95% pointwise confidence
interval for the PAV fit. Over the range of
data (inhaled doses of 10 and greater), the lognormal,
Weibull, and PAV fits are consistent (the
lognormal and Weibull fits are within the 95% confidence
interval for the PAV fit). The lognormal and
Weibull models allow extrapolation to inhaled doses
below 10 bacilli. The lognormal model parameter
estimates (GM = 3.71, GSD = 6.01) indicate that
infection risk given inhalation of one bacillus is
23%, while the Weibull model parameter estimates
( = 0.191, = 0.412) indicate that infection risk
given inhalation of one bacillus is 40%. Overall, the
lognormal and Weibull models suggest that 20% to
40% of the population would be infected due to inhaling
a single F. tularensis bacillus.
The PAV fit indicates that inhaling 10 bacilli
would infect approximately 30% of the population;
unfortunately, this nonparametric model does not
permit estimating the percent infected below the
inhaled doses for which data are available. However,
it does not make sense that 10 bacilli is the minimum
inhalation infectious dose. If inhaling 10 organisms
produces infection with 30% probability, it
is reasonable that inhaling 9 organisms should produce
infection with some probability less than 30%
but greater than zero, unless infection is a multihit
process requiring a minimum of 10 organisms. In
regard to the latter idea, we note that the infectivity
of just one F. tularensis organism is supported by animal
data. In mice, guinea pigs, and rabbits, the intracutaneous
and intraperitoneal injection of a single
F. tularensis bacterium is capable of producing
lethal disease (Bell, Owen, & Larson, 1955; Downs
et al., 1947; Emel’yanova, 1961; Rosebury, 1947).
The distributions depicted in Figure 1 are somewhat
confused by aggregating the results from aerosol
exposure studies involving different or unknown
particle size distributions. McCrumb (1961) did not
specify the particle size distribution or describe the
aerosol generating equipment, while Sawyer et al.
(1966) considered the dose to be only those organ-
R. M. Jones, et al.
isms present in particles of diameter 5 m or less.
Because particle size is inversely related to infectivity
(Day & Berendt, 1972), we applied the same statistical
analyses to the data of Saslaw et al. (1961) for
which the aerosol size distribution was described as
homogeneous with an average particle diameter of
0.7 m. We also decreased Saslaw’s reported inhaled
doses to account for a deposition fraction of 0.3 in
the respiratory tract (Hinds, 1999). Figure 2 plots the
Saslaw et al. (1961) data from Table 1, and the cumulative
infection probability as a function of the
deposited dose based on fitted lognormal and
Weibull models; the 95% confidence intervals for
the cumulative probability functions are also shown.
The lognormal model parameter estimates are GM =
1.48 and GSD = 5.75, and the Weibull model parameter
estimates are = 0.427 and = 0.458.
Given the deposition of one bacillus, the lognormal
model parameter estimates indicate that infection
risk is 41%, while the Weibull model parameter estimates
indicate that infection risk is 49%. Overall,
the parametric models suggest that 40% to 50% of
the population would be infected by the deposition
of a single F. tularensis bacillus. Figures 3 and 4 show
that the 95% confidence intervals for the lognormal
and Weibull cumulative probability functions, respectively,
fall within the 95% confidence interval of
the PAV cumulative probability function. Therefore,
the lognormal and Weibull probability estimates are
consistent with the PAV estimates in the dose range
of three bacilli or more.
Person-to-Person Transmission
Based in part on the observation that surgeons
did not become ill after incising or excising suppurating
lymph nodes from tularemia patients, Francis
concluded in 1927 that tularemia is not transmitted
from person-to-person. This conclusion has been
reiterated frequently in the tularemia literature.
However, it is inconsistent with the report by Fillmore
(1951) of tularemia in a health care worker
who had treated a tularemia patient, and with
reports of droplet spray transmission resulting
from experimental animals “sneezing” at persons
(Aagaard, 1944; Francis, 1983; Ledingham & Fraser,
1923/1924). Further, airborne transmission between
233

mice was demonstrated experimentally by Owen and
Bucker (1956), who found that ten of twenty-eight
(10/28) healthy mice strapped into tubes in near
nose-to-nose contact with moribund mice succumbed
to infection. For secondary airborne transmission
to occur, viable organisms must be emitted
from the infected animal and remain viable until
reaching target tissues in the susceptible host. In this
segment of the paper, we evaluate the feasibility that
these events can occur and cause person-to-person
transmission of tularemia.
Viable F. tularensis bacilli are present in the human
body, including respiratory fluids. Emel’yanova
(1961) compared strains of F. tularensis isolated from
234
The Infectious Dose of Francisella tularensis (Tularemia)
Figure 2
Estimated cumulative infection probability of humans experimentally exposed to increasing inhaled doses of
F. tularensis, based on the Saslaw et al. (1961) data adjusted for particle deposition, via a lognormal parametric
regression and a Weibull parametric regression, including 95% confidence intervals. The Saslaw, et al. (1961)
data points are indicated by the diamonds.
the abscesses of two tularemia patients to strains isolated
from well water which had caused a tularemia
outbreak; all strains had the same biological properties
and were lethal to mice, rats, guinea pigs, and
rabbits in the same doses and at the same intervals.
In addition, there are several reports that bacilli
from throat swabs and/or sputum of tularemia patients
with and without pulmonary involvement
were fatal to experimental animals (Johnson, 1944;
Larson, 1945). In a more extensive evaluation, gastric,
pharyngeal, and sputum specimens were collected
from patients with laboratory-acquired tularemia
during the first 3 weeks of their illness; these
specimens were inoculated into guinea pigs or cul-

tured on glucose-cysteine blood agar plates. A total
of 14/16 sputum specimens, 18/32 pharyngeal
specimens, and 22/31gastric specimens either killed
the guinea pigs or produced cultures of F. tularensis
(Overholt et al., 1961). Unfortunately, concentration
data for viable F. tularensis bacilli in respiratory
fluids have not been reported.
The presence of F. tularensis in respiratory fluids
implies that the organism will be present in the aerosol
of respiratory fluids emitted during coughs and
sneezes. The numbers and size distribution of respiratory
aerosol particles have been summarized by
Nicas, et al. (2005). Aerosolized F. tularensis remain
viable in air for a sufficient time to be inhaled by
health care workers or family members attending a
R. M. Jones, et al.
Figure 3
Estimated cumulative infection probability of humans experimentally exposed to increasing inhaled doses of
F. tularensis, based on the Saslaw et al. (1961) data adjusted for particle deposition, comparing the lognormal
parametric regression to the nonparametric PAV regression, including 95% confidence intervals.
tularemia patient. As described previously, Hood
(1961) observed that F. tularensis aerosols retained a
diminished, but still lethal, infectivity in guinea pigs
when aged 20 hours. In humans, Sawyer et al. (1966)
found that the inhaled dose of viable cells required
to produce infection increased when the aerosol
aged more than 120 minutes, but the severity of
clinical illness and the incubation period did not
vary with the age of the aerosol. Ambient temperature
and relative humidity influence the viability of
airborne F. tularensis, although the relationships are
not linear. Peak recovery of viable airborne F. tularensis
was observed between -7 o C and 3 o C (Ehrlich &
Miller, 1973), and when disseminated from a wet
state, survival of the organism in air was greatest at
235

high relative humidity (Cox & Goldberg, 1972).
Cough is frequently associated with tularemia,
and increased sputum production has been observed
in some patients. Coughing emits many particles
that quickly attain diameters less than 10 m; these
particles can penetrate to and deposit in the alveolar
region. Coughing also emits many particles that
quickly attain diameters in the 10 m to 100 m
range; these particles can be inspired and deposit in
the upper respiratory tract (Nicas, et al., 2004). Experimental
evidence indicates that F. tularensis remains
virulent in the human host and retains viability
in air for prolonged periods of time. Coupled
with an infectious dose of one bacillus, these conditions
indicate that airborne person-to-person trans-
236
The Infectious Dose of Francisella tularensis (Tularemia)
Figure 4
Estimated cumulative infection probability of humans experimentally exposed to increasing inhaled doses of
F. tularensis, based on the Saslaw et al. (1961) data adjusted for particle deposition, comparing the Weibull
parametric regression to the nonparametric PAV regression, including 95% confidence intervals.
mission of tularemia is possible. The overall lack of
reported person-to-person transmission may be due
to a low concentration of F. tularensis in respiratory
fluids, such that the airborne concentration of the
pathogen is usually low and the infection risk by inhalation
is small.
Conclusion
Overall, we judge there is adequate evidence that
infection can be produced by the respiratory tract
deposition of a single F. tularensis bacillus. Human
experimental work concerning inhalation infection
by F. tularensis is limited to inhaled doses of 10 organisms
or greater. However, regression parameter

estimates for the two-parameter lognormal and
Weibull models indicate that the deposition of a
single bacillus carried on a respirable particle will
produce infection in 40% to 50% of individuals.
This prediction is consistent with the observation
that a single F. tularensis bacillus can initiate infection
in experimental animals exposed via injection
(Bell, Owen & Larson, 1955; Downs et al., 1947;
Emel’yanova, 1961; Rosebury, 1947).
Our review of the published literature indicates
that airborne tularemia transmission in the laboratory
has been frequent, and that person-to-person
tularemia transmission is possible. In particular, Fillmore
(1951) reported the case of a nurse’s aide who
developed symptoms of tularemia 3 to 4 weeks after
attending a case of pleuropulmonry tularemia, and
virulent bacilli have been recovered from respiratory
fluids of tularemia patients (Johnson, 1944; Larson,
1945; Overholt et al., 1961). Coughing, which is
associated with tularemia, emits many particles of
respiratory fluid that quickly attain diameters less
than 100 m; these particles can be inspired and
deposit in the alveolar region or upper respiratory
tract depending on particle diameter.
The risk of person-to-person airborne infection
is a multivariable function involving the pathogen
concentration in respiratory fluid, the expiratory
event rate, the size and volume distribution of particles
emitted per respiratory event, the receptor’s
breathing rate and exposure duration, and the receptor’s
location in the room relative to the source case
(Nicas, et al., 2005). Given a specified infectious
dose distribution for F. tularensis, and the concentration
of bacilli in respiratory fluids, it is possible to
estimate tularemia infection risk due to bacilli in
emitted respiratory aerosol. Similarly, the risk of airborne
infection in the laboratory involves the pathogen
concentration in the materials being handled,
the size and volume distribution of the aerosolized
particles, the aforementioned receptor parameters,
and the nature of the pathogen’s infectious dose. A
quantitative estimate of infection risk, even if uncertain,
serves to inform biosafety officers in their decision
making about infection control procedures.
R. M. Jones, et al.
Acknowledgements
This work was supported by a collaborative
agreement with the Association of Schools of Public
Health, S2148-22/23. The opinions expressed are
solely those of the authors and do not necessarily
reflect the views of the funding agency.
References
Aagaard, G. N. (1944). Involvement of the heart in
tularemia. Minnesota Medicine, 27, 115-117.
Bell, J. F., Owen, C. R., & Larson, C. L. (1955).
Virulence of Bacterium tularense I: A study of the
virulence of Bacterium tularense in mice, guinea pigs,
and rabbits. Journal of Infectious Diseases, 97, 162-176.
Black, J. (2002). Microbiology: Principles and explorations.
New York: John Wiley and Sons, Inc.
Centers for Disease Control and Prevention. (2003).
Key facts about tularemia. Retrieved August 11,
2004 from www.cdc.gov/agent/tularemia/facts.asp.
Cluxton, Jr., H. E., Cliffton, E. E., & Worley, J. A.
(1948). Primary tularemia of the lungs masquerading
as other forms of lung pathology. The Journal of the
Arkansas Medical Society, 44, 266-275.
Conlan, J. W., Chen, W., Shen, H., Webb, A., &
KuoLee, R. (2003). Experimental tularemia in mice
challenged by aerosol or intradermally with virulent
strains of Francisella tularensis: Bacteriologic and
histopathologic studies. Microbial Pathogensis, 34,
239-248.
Cox, C. S., & Goldberg, L. J. (1972). Aerosol survival
of Pasteruella tularensis and influence of relative
humidity. Applied Microbiology, 23, 1-3.
Dahlstrand, S., Ringertz, O., & Zetterberg, B.
(1971). Airborne tularemia in Sweden. Scandinavian
Journal of Infectious Diseases, 3, 7-16.
237

Day, W. C., & Berendt, R. F. (1972). Experimental
tularemia in Macaca mulatta: Relationship of aerosol
particle size to infectivity of airborne Pasteurella tularensis.
Infection and Immunity, 5, 77-82.
Dennis, D. T., Inglesby, T. V., Henderson, D. A.,
Bartlett, J. G., Ascher, M. S., Eitzen, E., Fine, A.D.,
Friedlander, A. M., Hauer, J., Layton, M., Lillibridge,
S. R., McDade, J. E., Osterholm, M. T.,
O’Toole, T. O., Parker, G., Perl, T. M., Russell, P.
K., & Tonat, K. (2001). Tularemia as a biological
weapon: Medical and public health management.
JAMA, 285, 2763-2773.
Downs, C. M., Coriell, L. L., Pinchot, G. B., Maumenee,
E., Klauber, A., Chapman, S. S., & Owen,
B. (1947). Studies on tularemia I. The comparative
susceptibility of various laboratory animals. Journal of
Immunology, 56, 217-228.
Ehrlich, R., & Miller, S. (1973). Survival of airborne
Pasteurella tularensis at different atmospheric temperatures.
Applied Microbiology, 25, 369-372.
Emel’yanova, O. S. (1961). The virulence of strains
of Pasteurella tularensis isolated from man. Journal of
Microbiology, Epidemiology and Immunobiology, 32, 853-
858.
Evans, M. E. (1985). Francisella tularensis. Infection
Control, 6, 381-383.
Fillmore, A. J. (1951). Tularemia with report of six
cases. Arizona Medicine, 8, 27-33.
Francis, E. (1927). Tularemia. The Atlantic Medical
Journal, 30, 337-344.
Francis, E. (1983). Landmark article April 25, 1925:
Tularemia by Edward Francis. JAMA, 250, 3216-
3224.
Franz, D. R., Jahrling, P. B., Friedlander, A. M.,
McClain, D. J., Hoover, D. L., Byrne, W. R., Pavlin,
J. A., Christopher, G. W., & Eitzen, E. M. (1999).
Clinical recognition and management of patients
238
The Infectious Dose of Francisella tularensis (Tularemia)
exposed to biological warfare agents. In J. Lederberg
(Ed.), Biological weapons: Limiting the threat (pp. 38-
80). Cambridge, MA: MIT Press.
Henderson, D. W. (1952). An apparatus for the
study of airborne infection. Journal of Hygiene, 50, 53-
68.
Hinds, W. C. (1999). Aerosol technology (2nd ed.).
New York: John Wiley and Sons, Inc.
Hood, A. M. (1961). Infectivity of Pasteurella tularensis
clouds. Journal of Hygiene, 54, 497-504.
Johnson, H. N. (1944). Isolation of Bacterium tularense
from the sputum of an atypical case of human
tularemia. Journal of Laboratory and Clinical Medicine,
29, 903-905.
Kalbfleisch, J. D., & Prentice, R. L. (2002). The statistical
analysis of failure time data (2nd ed.). New Jersey:
Wiley-Interscience.
Larson, C. L. (1945). Isolation of Pasteurella tularensis
from sputum: A report of successful isolations from
3 cases without respiratory symptoms. Public Health
Reports, 60, 1049-1053.
Ledingham, J. C. G., & Fraser, F. R. (1923/1924).
Tularemia in man from laboratory infection. The
Quarterly Journal of Medicine, 17, 365-382.
McCrumb, F. R. (1961). Aerosol infection of man
with Pasteurella tularensis. Bacteriological Reviews, 25,
262-267.
Miller, R. P., & Bates, J. H. (1969). Pleuropulmonary
tularemia: A review of 29 patients. American Review
of Respiratory Disease, 99, 31-41.
Nicas, M., Nazaroff, W. W., & Hubbard, A. (2005).
Toward understanding the risk of secondary airborne
infection: Emission of respirable pathogens.
Journal of Occupational and Environmental Hygiene,
2,143-154.

Overholt, E. L., & Tigertt, W. D. (1960). Roentgenographic
manifestations of pulmonary tularemia.
Radiology, 74, 758-765.
Overholt, E. L., Tigertt, W. D., Kadull, P. J., Ward,
M. K., Charkes, N. D., Rene, R. M., Salzman, T. E.,
& Stephens, M. (1961). An analysis of forty-two
cases of laboratory acquired tularemia. American Journal
of Medicine, 30, 785-806.
Owen, C. R., & Buker, E. O. (1956). Factors involved
in the transmission of Pasteurella tularensis
from inoculated animals to healthy cage mates. Journal
of Infectious Diseases, 99, 227-233.
Reintjes, R., Dedushaj, I., Gjini, A., Jorgensen, T.
R., Cotter, B., Lieftucht, A., D’Ancona, F., Dennis,
D. T., Kosoy, M. A., Mulliqi-Osmani, G., Grunow,
R., Kalaveshi, A., Gashi, L., & Humolli, I. (2002).
Tularemia outbreak investigation in Kosovo: Case
control and environmental studies. Emerging Infectious
Diseases, 8, 69-73.
Rosebury, T. (1947). Experimental airborne infection.
Baltimore, MD: The Williams and Wilkins Company.
Saslaw, S., Eigelsbach, H. T., Prior, J. A., Wilson, H.
E., & Carhart, S. (1961). Tularemia vaccine study II.
Respiratory challenge. Archives of Internal Medicine,
107, 702-714.
Safety Library Reference
R. M. Jones, et al.
The National Academies Press provides, free of charge, Prudent
Practices in the Laboratory: Handling and Disposal of Chemicals, 1995,
in a searchable, printable format at:
http://www.nap.edu/openbook/0309052297/html/index.html
Sawyer, W. D., Jemski, J. V., Hogge, Jr., A. L., Eigelsbach,
H. T., Wolfe, E. K., Dangerfield, H. G.,
Gochenour, Jr., W. S., & Crozier, D. (1966). Effect
of aerosol age on the infectivity of airborne Pasteurella
tularensis for Macaca mulatta and man. Journal
of Bacteriology, 91, 2180-2184.
Shapiro, D. S., & Schwartz, D. R. (2002). Exposure
of laboratory workers to Francisella tularensis despite a
bioterrorism procedure. Journal of Clinical Microbiology,
40, 2278-2281.
Sjostedt, A., Tarnvik, A., & Sandstrom, G. (1996).
Francisella tularensis: Host-parasite interaction. FEMS
Immunology and Medical Microbiology, 13, 181-184.
Syrjala, H., Kujala, P., Myllyla, V., & Salminen, A.
(1985). Airborne transmission of tularemia in farmers.
Scandinavian Journal of Infectious Diseases, 17, 371-
375.
Syrjala, H., Sutinen, S., Jokinen, K., Nieminen, P.,
Touuponen, T., & Salminen, A. (1986). Bronchial
changes in airborne tularemia. The Journal of Laryngology
and Otology, 100, 1169-1176.
Van Metre, Jr., T. E., & Kadull, P. J. (1959). Laboratory-acquired
tularemia in vaccinated individuals: A
report of 65 cases. Annals of Internal Medicine, 50,
621-632.
World Health Organization. (1970). Health aspects of
chemical and biological weapons. Geneva: World
Health Organization.
239

240
Article
Introduction
Ultraviolet light (UV) is frequently used in limited
clinical settings to reduce the risk of nosocomial
infections. The World Health Organization Global
Solar UV Index (UVI) divides UV into A (315-400
nm), B (280-315 nm), and C (100-280 nm) bands.
Light in the C band (UV-C) is listed in the UVI as
the most harmful to living organisms because of its
propensity to damage DNA and RNA. It is also the
least relevant in the natural setting since it is completely
filtered by the atmosphere and does not
reach Earth’s surface in levels measurable with commercially
available equipment.
Often small surface areas or airflow in high-risk
areas are treated with UV-C to decrease infectious
microorganism populations. Other means of decontamination
are sometimes employed when a large
area has been contaminated. Gaseous disinfection
with ethylene oxide, chlorine dioxide, or formaldehyde
is costly, hazardous to workers and the environment,
and requires prolonged evacuation of the
treatment area (Rehork et al., 1990). Liquid disinfectants
must be manually applied and removed and
may damage exposed materials such as electrical devices.
Ionizing radiation will kill in adequate doses
but is hazardous to workers, difficult to contain, and
not practical for general working space disinfection
(Rehork et al., 1990).
Applied Biosafety, 10(4) pp. 240-247 © ABSA 2005
High-Dose Ultraviolet C Light Inactivates
Spores of Bacillus Atrophaeus and Bacillus
Anthracis Sterne on Nonreflective Surfaces
Marie U. Owens 1, David R. Deal 2, Michael O. Shoemaker 3, Gregory B. Knudson 3,
Janet E. Meszaros 4, and Jeffery L. Deal 2
1College of Charleston, Charleston, South Carolina; 2UVAS-LLC, Charleston, South Carolina; 3Armed Forces
Radiobiology Research Institute, Bethesda, Maryland; and 4Steris Corporation, Mentor, Ohio
The possibility of using UV-C (254 nanometer
range) to decontaminate or sterilize work areas and
to avoid the problems listed above led to the development
by two of the authors (JLD, DRD) of the
Ultraviolet Area Sterilizer (UVAS). The device is
unique in that it generates intense levels of UV-C
and then utilizes measured UV-C intensities reflected
from the walls, ceilings, floors, or other
treated areas to calculate the operation time to deliver
the programmed lethal dose for infectious microorganisms.
UV-C has been found to be highly
effective against a wide spectrum of microorganisms
(Banrud & Moan, 1999; Druce et al., 1995; Inamoto
et al., 1979; Knudson, 1985). The development of a
method to deliver a lethal and predictable UV-C
dose can greatly increase the potential uses for UV-C
in decontamination. Since the biological activity of
UV-C is not limited to microorganisms, the UVAS
has multiple safety features including remote controls,
motion sensors, and audible voice warnings.
These safety features were active during the course of
this study.
The ability of the UVAS device to deliver lethal
doses of UV-C to bacterial spores on nonreflective
surfaces was evaluated by comparing the susceptibilities
of Bacillus atrophaeus (formerly named B. subtilis
var. niger, and B. globigii) and Bacillus anthracis Sterne
spores to incremental UV-C doses. Additionally, the
susceptibility of Bacillus atrophaeus spores in the pres-

ence of silica-containing powder to simulate a bioterrorism
attack weapon was tested to see if this modification
altered the efficacy of UV-C deactivation.
This investigation has shown that spore viability
of both Bacillus atrophaeus as well as B. anthracis
Sterne was significantly and reproducibly reduced by
3-5 logs under extreme contamination levels following
dosimetric UV-C exposure. Complete kill can be
achieved when the spore contamination level is
lower. These findings are consistent with those of
Nicholson and Galeano (2003) and Knudson
(1986). Spores of Bacillus atrophaeus in 1%-2% silica
were likewise susceptible to killing by UV-C. However,
the presence of gross particulate matter such as
visible powder containing extremely high concentrations
of spores significantly inhibits spore susceptibility
to UV-C inactivation.
Materials and Methods
Bacterial Spore Suspensions/Preparations
B. atrophaeus 93-PBA-1 spore mixture (provided
by the Armed Forces Radiobiology Research Institute
(AFRRI), Bethesda, MD) contained 1%-2% silica
by weight and had an initial concentration of 2.5
x 10 11 Colony Forming Units (CFU)/g. This spore
mix was diluted in 50% ethanol to achieve concentrations
of 10 9 , 10 5 , 10 4 , and 10 3 CFU/ml. The dry,
free-flowing silica spore mixture closely simulates a
weapons-grade product used by bioterrorists. B. anthracis
Sterne spores were produced at AFRRI using
an inoculum from a live-spore veterinary vaccine
(Colorado Serum Company, Denver, CO) as previously
described (Elliott et al., 2002). The B. anthracis
Sterne spores were washed twice in deionized sterile
water and examined by phase-contrast microscopy to
confirm that the refractile spore suspension was free
of vegetative cells. The B. atrophaeus spores ATCC
9372 (Steris Corp., Mentor, OH) were at initial concentrations
of 3.0 x 10 9 CFU/g and 2.5 x 10 10
CFU/ml, respectively. Both of these spore products
were suspended in 50% ethanol and used at a concentration
of 10 9 , 10 5 , and 10 4 CFU/ml.
Test Surfaces
Aluminum plates, which measured 1276 cm 2 ,
were painted with high heat-stable, flat-black enamel
(7778-822, Rust-Oleum, Vernon Hills, IL). These
M. U. Owens, et al.
test surfaces were autoclaved prior to being evenly
spread with suspensions of B. atrophaeus ATCC
9372 and B. anthracis Sterne spores in ethanol. The
dark surface was intended to minimize reflectance
and, therefore, measured primarily the effect of direct
UV-C exposure. For the test using the dry, freeflowing
B. atrophaeus (93-PBA-1) spore powder, sterile
90 mm Petri plates were used.
Spore Distribution
Two milliliters of the liquid spore suspensions in
50% ethanol were uniformly distributed on the
metal test and control surfaces using sterile, plastic,
cell spreaders. An additional 5 ml of 50% ethanol
was used to facilitate the even distribution over the
entire surface area. Test surfaces were air dried for a
minimum of 2 hours before UV-C exposure.
Dry B. atrophaeus (93-PBA-1) spore powder was
spread in the base of sterile 90 mm Petri plates by
adding a uniform dry measure to 16 dishes. Each
dish was gently rotated and tilted by hand to achieve
a relatively uniform distribution of the powder. Excess
spore powder was removed into a beaker by inverting
and tapping the plates. To reduce shadowing
effects during UV-C exposure, the powder was wiped
from the sides and corners of the dishes with sterile
cotton swabs. The mass of spores remaining in each
plate was determined by weighing each plate before
and after addition of spores. Each plate received 20
to 50 mg of spore powder.
Exposure
The UVAS device employs 14 medium-pressure
mercury bulbs (product #TUV 115W HHO, Philips
Corporation, Sommerset, NJ) with a combined
power output of approximately 3,000 microwatts/cm
2 at 1 meter. The device was used to expose
test surfaces in a room measuring 25 x 35 ft. For the
flat-black metal surfaces, cumulative UV-C doses
were measured using two National Institute of Standards
and Technology-calibrated dosimeters
(PMA2100, Solar Light Company, Philadelphia, PA)
placed on either side of the test surfaces and were
reported as the average between both devices. For
the Petri plates containing dry spore powder, a single
PMA2100 dosimeter was placed in the center of the
plate array as shown in Figure 1.

Spore Recovery and Culture Media
Two methods were used to recover spores from
the metal test surfaces following each UV-C exposure.
Rodac 45-mm contact culture plates containing
trypticase soy agar with lecithin and polysorbate 80
(TSALP, PML Microbiologicals, Mississauga, ON,
Canada) were used to recover viable spores from
28.3 cm 2 sample areas of the test surfaces by direct
contact with the metal plates. Swab Dilution Samplers
(MT0010025, Millipore Corp., Billerica, MA)
were used for recovery of 16-cm 2 sample areas of the
test surfaces. For each test surface, a minimum of
three contact plates and one swab is used to obtain
spore recovery at each targeted dose. The recovered
spores adhering to the swabs were eluted in the Millipore
buffer, subsequently diluted, and plated in
triplicate as 150-l aliquots onto 90 mm trypticase
242
Recovered B. atrophaeus spores
(CFU/ 1276 cm 2 )
0
250
High-Dose Ultraviolet C Light Inactivates Spores
Figure 1
Recovery of B. atrophaeus spores containing ~1-2% silica after exposure to UV-C
10 10
10 9
10 8
10 7
10 6
10 5
10 4
10 3
10 2
10 1
10 0
500
750
1000
1250
1500
UV-C Dose (milliJoules/cm 2 )
Incremental doses of UV-C were administered to a total surface inoculum of 6.2 x 10 8 CFU/1276 cm 2 .
Following each dose, spores were recovered using Millipore swabs and Rodac contact plates. The average
number of colonies were reported and used in calculations to determine the total viable spore count over the
entire test surface. The curve represents the best-fit of a single exponential decay equation to the data.
1750
2000
soy agar plates (TSA, BD Diagnostic Systems, Sparks,
MD). Plates from both recovery methods were incubated
at 35 o C for 15 hours before colonies were
counted. Average plate counts were used to calculate
the number of viable spores remaining on the metal
plates and were plotted as a function of UV-C dose
to generate inactivation curves (Figures 1-3).
Spores in dry powder that were dispensed within
the Petri plates were recovered by washing the Petri
plates three times with sterile water (5 ml final volume)
followed by recovering any material adhering
to the bottom of the plate with a sterile rubber cell
spreader. The suspensions were vortexed, diluted,
inoculated as 150 l aliquots onto TSA plates in triplicate,
and incubated at 35 o C for 15 hours before
colonies were counted. Average plate counts were

used to calculate the number of viable spores remaining
in each exposure plate and plotted as a
function of UV-C dose (Figure 4).
Calculations
Total Population per Aluminum Plate Surface Area:
Swab Dilution Samplers:
= Mean Plate Count x Dilution
Factor Vol. Plated (0.150 ml) x
Swab Dilution Sampler Vol. (18
ml) Aluminum Plate Surface Area
Swabbed (16 cm 2 ) x Aluminum
Plate Total Surface Area (1276 cm 2 )
Rodac Contact Plates:
= Mean Plate Count Aluminum
Plate Surface Area Contacted (28.3
cm 2 ) x Aluminum Plate Total Surface
Area (1276 cm 2 )
M. U. Owens, et al.
Photo 1
UVAS prototype in test room with sensor and test plates
containing a thin layer of dry B. atrophaeus spore powder
Results
Inactivation of B. atrophaeus (93-PBA-1)
Spores with Silica
At extremely high spore-inoculum levels, contact
plates could not provide population data from the
control, unexposed plates due to the high number of
colonies, but the swabbing method gave a viable
spore count of 6.2 x10 8 CFU existed over the entire
plate surface. The swab method was also the only
procedure that could be used for recovery studies at
lower UV-C doses of 100-600 milliJoules/cm 2 for
the same reason. Results from the swab method indicated
a 3-log reduction could be obtained at these
UV-C exposure levels. At higher UV-C doses between
600-1,800 milliJoules/cm 2 , a 3-4-log reduction
was demonstrated with the swab recovery method
and a 5-log reduction was obtained using the contact
plates (Figure 1).
For the test plates spread with less concentrated
spore suspensions (theoretically 10 5 , 10 4 , and 10 3
243

CFU per test surface) recoveries were conducted using
the contact plates alone after UV-C exposures of
1,000 to 2,000 milliJoules/cm 2 . All plates indicated
no survivors. Recoveries from unexposed control
plates were 1 log lower than anticipated; therefore,
at least a 4-log reduction can be ensured.
Control surfaces, which were not exposed to
UV-C, did not show any reduction in colony counts
during the course of the experiment, eliminating the
possibility that recoveries varied with time.
Inactivation of B. atrophaeus
(ATCC 9372) Spores
Swab recoveries used for determining viable
spore counts from the test plates spread with highly
concentrated spore suspensions indicated a spore
survival count of 10 8 CFU per test surface. This recovery
method demonstrated a 2-log reduction at 50
244
High-Dose Ultraviolet C Light Inactivates Spores
Figure 2
Recovery of B. atrophaeus (ATCC9372) spores from black-plate surfaces after exposure to UV-C.
Recovered B. atrophaeus spores
(CFU/ 1276 cm 2 )
10 10
10 9
10 8
10 7
10 6
10 5
10 4
10 3
10 2
10 1
10 0
0
500
1000
1500
2000
2500
3000
3500
UV-C Dose (milliJoules/cm 2 )
Recovery of B. atrophaeus spores using the Millipore swab sampling recovery method. Curves represent best-fit
of a single exponential decay equation to data.
4000
4500
5000
milliJoules/cm 2 and a 3–4-log reduction at 100 to
4,647 milliJoules/cm 2 UV-C. The variability was attributed
to the use of data from plates in which usable
counts were less than 30 CFU per plate. Contact
plates, which were used only at the doses from
2,000 to 4,000 milliJoules/cm 2 UV-C, indicated that
a 5-log reduction could be obtained in comparison
to the unexposed spore population determined by
swabbing (Figure 2).
The control, contact plate viability counts from
the less-concentrated test plates, theoretically 10 4 and
10 5 CFU, indicated a 2-log lower population than
expected. The number of survivors was quite often
zero for all the UV-C doses evaluated from 50 to
2,000 milliJoules/cm 2 except in some instances low
colony counts were obtained, which may have been
environmental contamination.

B. anthracis Sterne Spores
Millipore swab recoveries from nonirradiated
test surfaces indicated 3.6 x 10 8 CFU. Swab recoveries
from UV-C doses of 150 and 1,000 milli-
Joules/cm 2 resulted in a three and four log reduction
respectively. Contact plates used for recoveries
on the test surface exposed to UV-C doses of 2,000
to 4,000 milliJoules/cm 2 demonstrated a 4 log reduction
(Figure 3).
Test surfaces spread with the diluted spore inoculum
had 10 4 CFU per test surface and showed
total kill when exposed to 50 to 1,000 milli-
Joules/cm 2 .
Inactivation of Dry Spore Powder
The last evaluation used dry, free-flowing B. atrophaeus
spore powder in open Petri plates, with spore
counts of 10 8 to 10 9 CFU per plates. A 1-log reduc-
M. U. Owens, et al.
Figure 3
Recovery of B. anthracis Sterne spores from black plate surfaces after exposure to UV-C.
Recovered B. anthracis spores
(CFU/ 1276 cm 2 )
10 10
10 9
10 8
10 7
10 6
10 5
10 4
10 3
10 2
10 1
10 0
0
500
1000
1500
2000
2500
3000
tion was obtained from duplicated plates after exposures
of 10,000 or 16,000 milliJoules/cm 2 (Figure 4).
Discussion
3500
UV-C Dose (milliJoules/cm 2 )
Recovery of B. anthracis Sterne spores using the Millipore swab sampling recovery method. Curves represent
best-fit of a single exponential decay equation to data.
4000
4500
5000
This investigation has demonstrated that UV-C
generated by the UVAS in the absence of visible particulate
matter can be delivered at lethal doses on
nonreflective, non-porous surfaces for partial spore
reduction even when the contamination levels are
extremely high and for total spore reduction in the
presence of less-concentrated spore populations. A 3-
5-log reduction can be assured following UV-C exposure
to contamination levels simulating those used
in bioterrorist weapons—10 8 to10 9 CFU/1276 cm 2 or
10 5 to 10 6 CFU/cm 2 —using doses of one hundred to
several thousand milliJoules/cm 2 (Figures 1, 2, and
3). In situations where the bioburden levels are more
245

epresentative of the contamination in areas such as
operating/emergency rooms (10 2 CFU/cm 2 ), UV-C
is capable of completely deactivating the entire population
at lower doses, most likely less than 100 milli-
Joules/cm 2 . Test surfaces contaminated with high
numbers of spores that are subsequently spread
across the surface area as may occur during precleaning
were readily decontaminated or sterilized with
adequate doses of UV-C.
Spores at contamination levels of 10 5 to 10 6
CFU/cm 2 and applied in powder dense enough to
be visible to the naked eye indicated a 1-log reduction
after UV-C doses of 1,000 to 16,000 milli-
Joules/cm 2 (Figure 4). These findings emphasize the
need for precleaning contaminated surfaces soiled
with gross material. Use of a precleaning step, such
246
Recovered B. atrophaeus spores
(CFU/ 90 mm Petri plate)
10 10
10 9
10 8
10 7
10 6
10 5
10 4
10 3
10 2
10 1
10 0
0
2000
High-Dose Ultraviolet C Light Inactivates Spores
Figure 4
Recovery of a visible layer of dry B. atrophaeus spores containing
~1% silica on Petri plates after exposure to UV-C.
4000
6000
The curve represents the best-fit of a single exponential decay equation to the data.
8000
10000
12000
14000
UV-C Dose (milliJoules/cm 2 )
16000
18000
20000
as HEPA-vacuuming or damp wiping, for heavily
contaminated surfaces in the presence of visible soil,
followed by UV-C exposure, should effectively decontaminate
the area or surface. This is substantiated
by the data in which total kill was demonstrated
from surfaces contaminated with less-concentrated
spore suspensions in the absence of visible powder.
The presence of 1%-2% silica does not impede
the germicidal effect of UV-C since the lethality was
similar to that observed in the absence of silica. This
finding demonstrates that the efficacy of UV-C is
not altered in the presence of low concentrations of
particulate matter or soil that could be present after
precleaning.
Prior studies in animal laboratory settings using
UV light showed a significant reduction of bacterial

loads, and the addition of a chemical disinfectant
followed by UV-C treatment was “particularly successful,
reducing bacterial loads to extremely low levels”
(Dix et al., 1992). In this study where the organisms
were spread on the test surface without deactivation
or removal by any cleaning agent, a significant
spore reduction was observed after UV-C irradiation.
A recent study by Nicholson and Galeano (2003)
found “the data indicate that standard UV treatments
that are effective against B. subtilis spores are
likely also sufficient to inactivate B. anthracis spores,
and spores of standard B. subtilis strains could reliably
be used as a biodosimetry model for the UV inactivation
of B. anthracis spores” Our investigations
have confirmed this finding.
Previous experience with the UVAS device suggested
smooth materials that reflected UV-C may be
more readily decontaminated than rough, nonreflective
materials. Data obtained through this investigation
will be useful in planning surface decontamination
for many environmental applications, since determining
the required decontamination doses
should be based upon information obtained using
the least reflective test surfaces, thus avoiding underexposure.
From these experimental findings one may reasonably
conclude that the UVAS device, or other
UV-C generating devices, could decontaminate areas
in which surface contamination was 10 2 CFU/cm 2
and could be used to decontaminate extremely concentrated
surfaces as long as a precleaning step was
instituted.
Author’s Note
The B. anthracis Sterne spores were washed twice
in deionized sterile water and examined by phasecontrast
microscopy to confirm that the refractile
spore suspension was free of vegetative cells.
References
Banrud, H., & Moan, J. (1999). [Use of short wave
ultraviolet radiation for disinfection in operating
rooms]. Tidsskr Nor Laegeforen, 119, 2670-2673.
M. U. Owens, et al.
Dix, J., Nolan, G., Schlick, H., & Elliot, H. B.
(1992). The efficiency of ultraviolet irradiation and
chemical disinfection as a means of reducing bacterial
contamination of animal rooms. Fifth FELASA
Symposium: Welfare and Science.
Druce, J. D., Jardine, D., Locarnini, S. A., & Birch,
C. J. (1995). Susceptibility of HIV to inactivation by
disinfectants and ultraviolet light. Journal of Hospital
Infection, 30, 167-180.
Elliott, T. B., Brook, I., Harding, R. A., Bouhaouala,
S. S., Shoemaker, M. O., Knudson, G. B. (2002).
Antimicrobial therapy for Bacillus anthracis-induced
polymicrobial infection in 60 Co gamma-irradiated
mice. Antimicrobial Agents and Chemotherapy, 46,
3463-3471.
Inamoto, H., Ino, Y., Jinnouchi, M., Sata, K., Wada,
T., Inamoto, N., & Osawa, A. (1979). Dialyzing
room disinfection with ultra-violet irradiation. Journal
of Dialysis, 3, 191-205.
Knudson, G. B. (1985). Photoreactivation of UVirradiated
Legionella pneumophila and other Legionella
species. Applied Environmental Microbiology, 49, 975-
980.
Knudson, G. B. (1986). Photoreactivation of ultraviolet-irradiated
plasmid-bearing and plasmid-free
strains of Bacillus anthracis. Applied. Environmental
Microbiology, 52, 444-449.
Nicholson, W. L., & Galeano, B. (2003). UV resistance
of Bacillus anthracis spores revisited: Validation
of Bacillus subtilis spores as UV surrogates for
spores of B. anthracis Sterne. Appied Environmental
Microbiology, 69, 1327-1330.
Rehork, B., Martiny, H., Weist, K., & Ruden, H.
(1990). [Performing surface and room disinfection in
the hospital]. Offentl Gesundheitswes, 52, 36-45.
247

Abstract
248
Article
Potentially infectious wastes, if not properly
treated, could expose both humans and the environment
to untreated microbes and toxins, and create a potential
for illness. Therefore, all pathogenic materials used in
research must be destroyed by heat or chemical treatment
prior to disposal as biomedical waste. Additionally,
bacterial waste from nonpathogenic bacteria carrying
antibiotic resistance must be treated before placement
in the waste stream to avoid transmission of
the antibiotic-resistant trait. In biomedical laboratories,
glassware, beakers, test tubes, and other contaminated
research material are usually treated by autoclaving before
placing them into the waste stream or recycling for
continued use in the laboratory.
Introduction
Florida Administrative Code (F.A.C.) Chapter
64E-16, under the Biomedical Waste Code, requires
an infectious waste treatment facility to maintain
records of temperature and dwell time when wastes
are rendered noninfectious by gas or steam
sterilization. The F.A.C. further requires biological
culture testing to assure proper sterilization of all
autoclaved materials. Since this University is not a
treatment facility but a generator facility, the
University is not required to follow these records
and testing requirements. However, in the best
interest of the University’s environmental and
research goals, the Department of Environmental
Health & Safety (EH&S) is voluntarily complying
with the sterilization aspect of the F.A.C.
As a service, EH&S conducts annual sterilization
testing for all departmental autoclave steam cycles
Applied Biosafety, 10(4) pp. 248-252 © ABSA 2005
Autoclave Testing in a University Setting
Richard N. Le, Amy L. Hicks, and Janice Dodge
Florida State University, Tallahassee, Florida
used for biological waste and materials decontamination.
This testing is to ensure that the autoclave cycles
used are sterilizing all microbial waste.
Background on Steam
Sterilization Testing
Researchers who work with potentially infectious
materials are at a higher risk of exposure, especially
when exposed to untreated infectious waste.
Infections may be transmitted through several different
routes, including direct contact with untreated
infectious waste, indirect contact with contaminated
instruments or environmental surfaces, or inhalation
of airborne contaminants. Infection via any of these
routes requires that all five of the following conditions
be present to form “the chain of infection”:
1. A pathogen with sufficient infectivity and numbers
to cause infection
2. A reservoir or source that allows the pathogen to
survive and multiply
3. A mode of transmission from the source to the
host
4. A portal through which the pathogen may enter
the host
5. A susceptible host (CDC, 2003)
Effective control measures are intended to break
one or more of these “links” in the chain, thereby
reducing the risk of, or completely preventing, exposure
to potentially infectious materials. A key component
in this link-breaking process is the proper use
of steam sterilizers (autoclaves), which serve as an
essential step toward eliminating the “pathogen
links” in the chain of infection (DePaola, 2003;
Quality America, Inc., 2001).
The Occupational Safety and Health Association

(OSHA) relies on guidelines published by the Centers
for Disease Control and Prevention (CDC) as a
widely recognized and accepted standard to be followed
by employers in carrying out their responsibilities
under the Occupational Safety and Health Act.
The CDC and OSHA recommend the use of Biological
Indicators (BI) for monitoring steam sterilization
cycles in autoclaves. The CDC states [for medical
autoclaves]...“proper functioning of sterilization
cycles should be verified by the periodic use (at
least weekly) of biological indicators (i.e., spore
tests). Heat-sensitive chemical indicators (e.g., those
that change color after exposure to heat) alone
do not ensure the adequacy of sterilization cycle…”
(CDC, 2004).
In addition, F.A.C. 64E-16.007 states that a
treatment facility that disposes of biomedical waste
must utilize steam sterilization, incineration, or an
alternative process approved by the Department of
Health prior to disposal. Treatment shall occur
within 30 days of collection from the generator. Additionally,
steam treatment facility units should be
evaluated for effectiveness with spores of Geobacillus
stearothermophiluss at least once every 7 days for permitted
treatment facilities, or once every 40 hours of operation
for generators that treat their own biomedical
waste.
Sterilizer manufacturers also recognize the importance
of routine testing of sterilizers and autoclaves.
They recommend that a biological spore test
indicator be used weekly in a representative load for
sterilization assurance (Ritter; Tilton et al., 2004).
Background on PROSPORE2
Biological Indicators
PROSPORE2 (HealthLink) is a self-contained
biological indicator for validating and monitoring
steam sterilization of solids. It consists of a paper
disc carrier containing Geobacillus stearothermophilus
(ATCC 7953) spores. The disc is enclosed in a plastic
tube along with a glass vial containing growth media
for the bacterial spores. Bromocresol purple has
been added as a pH indicator to detect spore growth.
The outgrowth of spores decreases pH, causing a
color change from purple to yellow following a 24hour
incubation period.
R. N. Le, et al.
A PROSPORE2 biological indicator is placed
inside the autoclave and a specific cycle is selected.
When the cycle is complete, the PROSPORE2 indicator
is sealed by firmly depressing the cap. The glass
ampoule of media is crushed inoculating the Geobacillus
stearothermophilus disc. Then the indicator is
incubated at 55 o -60 o C for a 24-hour time period
along with an untreated PROSPORE2 indicator that
serves as a control to ensure spore viability.
Once the minimum incubation time of 24 hours
has been achieved, both the control and autoclaved
PROSPORE2 indicator are examined. If the autoclaved
PROSPORE2 indicator retains its purple
color, then the sterilization cycle is adequate and the
result is recorded as passing. A failed sterilization
cycle is indicated by turbidity or a change in color
toward yellow, indicating spore growth due to a
change in pH. The control indicator should exhibit
turbidity and/or a color change to yellow after incubation
to ensure the viability of the biological indicators.
If the control indicator does not show signs of
growth, the test is considered invalid and all the results
are unacceptable and not considered.
Evaluation of Autoclaves
PROSPORE2 biological indicators were used to
test the steam sterilization cycles of several departmental
autoclaves. A total of three runs were conducted
for each autoclave. For each autoclave, a test
trial run using a PROSPORE2 biological indicator
was run to see if the autoclaves were capable of sterilization
without any load. The biological indicator
in the test trial was placed in a horizontal position,
as recommended by the manufacturer, in various
locations within the autoclave chamber. This was
done to verify the effectiveness of steam sterilization
in different areas within the autoclave.
Following the test trial run, two additional runs
were performed in which PROSPORE2 biological
indicators were placed in a representative load or
challenge load to be autoclaved. The challenge load
test consisted of placing a PROSPORE2 biological
indicator in the center of an autoclave bag containing
empty plastic pipette tip boxes. This bag was left
partially open to allow for steam penetration and
sterilization.

For the results, a “PASS” indicated that the
PROSPORE2 biological indicator retained its purple
color signifying an adequate sterilization cycle. A
“FAIL” resulted in the PROSPORE2 biological indicator
having turbidity or a color change toward
yellow for that sterilization cycle. Untreated control
PROSPORE2 biological indicators were incubated
with each autoclaved ran PROSPORE2 biological
indicator.
The following results were compiled over a 3month
testing period for several departmental autoclaves.
These results are for the challenge load testing
only. Challenges were performed using the temperatures
preset by the research groups using the
autoclaves.
Test Findings
Each test trial run resulted in a passing test for
every autoclave. Based on this result, it was concluded
that the autoclave was functioning properly.
250
Autoclave Testing in a University Setting
An adequate amount of steam and heat were produced
to sterilize any unobstructed contents such as
glassware. However, problems occurred with the
challenge load testing. Almost every challenge load
test failed to sterilize the biological indicator. Therefore,
the testing procedure was modified in an effort
to sterilize the enclosed biological indicator. The
challenge load volume was reduced by approximately
half its original volume and the bag’s opening was
increased. In addition, approximately 250 mL of
water was added directly to the autoclave bag to generate
additional steam within the bag to achieve sterilization.
Biological indicators were also given to two separate
departments to autoclave with their normal biohazardous
waste load. One was autoclaved for 15
minutes at 121°C gravity cycle and the other load
was autoclaved at 121°C for 35 minutes on a liquid
setting. Since the biological indicators were placed in
actual bags of biohazardous waste, these tests were
true representatives of what laboratories were gener-
Autoclave
Table 1
Cycle Pass Fail
Amsco 3021-S-A
(Amsco)
G15/D30
L60
Amsco 3041
All settings (Liquid and
(Amsco)
Gravity)
Consolidated
Addition of water to bag
G60/D15
(Consolidated) G30/D15
Primus Autoclaves-A
(Primus)
G20/D15 (Room 511)
G15/D1 (Room 312)
Amsco 3021-S-B All liquid cycles
Amsco Scientific
(Amsco)
Amsco 3021-S-C
(Amsco)
G30/D30 (132°C)
L30
L15
G15/D30 (132°C)
L30
L15
Primus Autoclave(B) G15
Steris, Stage 1
(Amsco)
G30/D15
G30/D15 (addition of water)
(“L”=liquid cycle, “G”=gravity cycle; and “D”=Dry time. Unless otherwise
indicated, the standard 121°C and 15 psi were used at each autoclave)

ating. Unfortunately, both of these tests failed.
Based on the information in the preceding table,
it was evident that the results varied greatly with
each autoclave due to the type of autoclave and differences
in cycle times and temperatures. However,
it can be shown that the shorter-timed cycles failed
consistently for each challenge load tested. The exceptions
to this were the results generated from the
Amsco 3041 autoclave and the Amsco Scientific
autoclave. For the Amsco 3041 autoclave, the 250
mL of water added to the autoclave bag helped with
the sterilization of the materials, resulting in a passing
test for the challenge load. The Amsco Scientific
autoclave was able to sterilize the PROSPORE2 biological
indicator using a 30-minute cycle because of
the higher temperature setting. However, the bags
used for this autoclave were not compatible with the
higher temperature, resulting in complete deterioration
of the bags after autoclaving.
Additional Findings
It was observed that not all autoclaves had clear
instructions available detailing proper usage and that
no user log was present to record the users, cycle
times, settings, and autoclave contents. In addition,
it was unknown if the temperature sensors on each
autoclave were properly calibrated due to inadequate
record keeping concerning general maintenance on
several autoclaves. However, each autoclave chamber
temperature was verified during the runs using a
high-low thermometer that was placed in the autoclave
with each challenge load run. There was good
consistency between the high-low thermometer temperature
readings and the temperature indicated on
the autoclaves.
In addition, no records were kept to indicate
when general or safety maintenance was last conducted
on several autoclaves. Maintenance should be
done yearly or as recommended by the autoclave
manufacturer.
Recommendations
In keeping with the CDC and OSHA recommendations
and using F.A.C. 64E-16.007 as a guide-
R. N. Le, et al.
line, EH&S will use biological indicators for validating
the steam sterilization cycles on an annual basis.
EH&S recommends that the following adjustments
to policies and practices be implemented in order to
better achieve sterilization and ensure proper maintenance
for each autoclave:
1. Readjustment of Cycle Times
The standard 121°C, 15-psi, and 15-minute
dwell time are adequate for sterilization of clean
items or smaller loads, but were found to be inadequate
for large bulky loads of biohazardous waste.
For this type of waste, it is recommended that the
sterilization time be increased to a minimum of 60
minutes, while still maintaining the standard temperature
and pressure. This setting has proven effective
for sterilizing larger loads. The addition of water
is not recommended because results were not consistent
enough for this to be considered an effective
option. It is also recommended not to leave the autoclave
bag open for actual autoclaving of materials
even though this procedure was used in the test procedure.
The bag should be secured loosely with a
rubberband or closed loosely with a small opening
(at least 1 inch in diameter). This will minimize an
individual’s risk of becoming exposed to hot materials
within the bag. It is suggested that a “kill cycle”
be set on each autoclave intended specifically for
sterilization of contaminated dry waste loads.
The Amsco Scientific autoclave did yield a passing
result due to a higher temperature setting. However,
the autoclave bags used could not withstand
the higher temperature and the bags deteriorated
during the procedure. This is a safety concern because
the contents within the bag could spill onto
the user exposing him or her to the extremely hot
contents. To use this autoclave at the higher temperature
settings, it is important to use an autoclave
bag that is compatible with the 132°C temperature.
In addition, the autoclaves should be retested using
the new autoclave bags.
2. Proper Autoclave Use
The autoclave bag should never be over-packed
or sealed too tightly. It is also important to make
sure that the contents of the autoclave bag are kept
at a minimum. An over-packed bag will insulate mi-
251

crobes at the center of the bag compromising effective
sterilization for the core contents (Churchill,
2003).
It is the responsibility of each department to ensure
that all users understand and know how to use
the autoclave correctly. EH&S recommends that
proper personal protective equipment be worn when
unloading materials from the autoclave. Any exposed
skin should be covered when reaching into
the autoclave after operation to prevent burns. Heatresistant,
elbow-length gloves and other personal
protective equipment are necessary to prevent burns
from occurring when removing hot items. In addition,
a shallow pan or container should be used
when autoclaving bags of biohazardous waste to prevent
potential spills of this material onto the user.
3. Record Keeping
The use of an autoclave logbook is recommended
for each autoclave. Prior to autoclaving any
items, users must fill in all information requested in
the autoclave logbook. The logbook should be located
adjacent to the autoclave and be maintained
by the department. Information that should be part
of the logbook includes: user’s name, cycle time, cycle
setting, materials being autoclaved, contact number,
time in, time out, and verification results.
4. Safety Maintenance
General maintenance should be conducted on
an annual basis or as recommended by the manufacturer.
Specifically, the safety valve should be checked
and replaced as required. This information should
be posted on the autoclave or maintained in a maintenance
logbook for easy reference. In addition, a
quick check of the autoclave should be conducted
prior to each use to ensure that all parts are properly
functioning (e.g., door closes and is sealed properly,
the rack is in place, correct settings are being used,
the interior is clean, etc.). Autoclaves should be
cleaned after every use and the work area should be
disinfected as needed. Appropriate cleaning protocols
should be obtained from each manufacturer.
252
Autoclave Testing in a University Setting
5. Training
Each autoclave user should be trained on the
proper use of the autoclave. The cycle settings
should be posted next to the autoclave to inform
each user of the types of settings available, such as
gravity and liquid cycles, the temperatures for each
cycle, and run times. A written sterilization procedure
should be kept near each autoclave and a standard
operating procedure should be developed. It
should include the appropriate sterilization times for
liquid and dry waste goods, identification of standard
treatment containers and proper load placement
procedures, personal protection equipment
required for removing materials from the autoclave,
instructions for loading and unloading the autoclave,
and instructions on cleaning and maintaining
the autoclave.
References
Centers for Disease Control and Prevention. (2003).
Guidelines for infection control in dental healthcare
settings-2003. MMWR, December 19/52(RR17), 1-
98.
DePaola, L. G. (2003). Infection control and the
prevention of disease transmission in the dental office:
Back to the basics. The Infection Control FO-
RUM, 1(2).
HealthLink. Prospore and Prosure Technical Bulletin 1.
Frequency of Use for Biological Indicators.
Quality America, Inc. (2001). Break the chain of infection.
OSHA Watch, January/February, 3(1).
Ritter©—Installation and Operation Manual M9/
M11 UltraClave TM Steam Sterilizer. Sterilizer Manufacture
Monitoring Recommendations.
Tilton, G., & Kauffman, M. (2004). Sterilization: A
review of the basics. STERIS Corporation white papers,
document number M2712EN. www.steris.
com/resources/resources_wp.cfm#. June.

Article
The Viral Immunology Center at Georgia State
University (GSU) is recognized as the national resource
facility for research related to the early diagnosis
and effective treatment of dangerous viral diseases,
most notably B virus (Cercopithecine herpesvirus
1), herpesviridae. Funded by the National Institutes
of Health, the Center operates the National B
Virus Resource Laboratory aimed at learning more
about the deadly alpha herpes virus and preventing
its transmission to laboratory workers.
The GSU Center, located in downtown Atlanta,
was the first BSL-4 facility in the country operating in
a university setting. Under the direction of Julia Hilliard,
the goal of the laboratory’s research is not only
to diagnose viral diseases, but also to study the agents
and use their infrastructure to design vaccines.
The B Virus laboratory had been located at the
Southwest Foundation for Biomedical Research in
San Antonio, Texas, and operations were proceeding
well in the BSL-4 environment. However, space constraints
limited the size of the staff to 10 people and
restricted the amount of work that could be done.
Research officials at Georgia State University
called upon Hilliard to move her facility to downtown
Atlanta in 1997, noting the University’s proximity
to Emory University, the Centers for Disease
Control, and the Medical College of Georgia. The
retrofitted lab at the University offers three times the
amount of space that Hilliard was using at the Texas
facility.
“The thrill of working in a rich milieu and having
a new facility was enough to engage my interest
Applied Biosafety, 10(4) pp. 253-257 © ABSA 2005
Operating a BSL-4 Laboratory in a University
Setting: Georgia State University Lab
Studies Deadly Alpha Herpes Virus
Tradeline Publications
Orinda, California
in moving the laboratory,” recalls Hilliard. “We now
know what it’s like to build a BSL-4 laboratory in
a highly concentrated downtown area. We were
told this would not be a big problem because
the residents were already aware that maximumcontainment
laboratories were operating in the city.”
Maximum-containment BSL-3 and -4 suites are
necessary due to the nature of the alpha herpes virus,
or B Virus, which is transmitted by the Macaca
species of monkeys often used in biomedical research.
Although there is a low frequency of infection
among people, the virus is considered a severe
occupational hazard for laboratory workers as a result
of its lethal consequences if not diagnosed
quickly after infection.
“The virus causes death in 80 percent of the infected
individuals (after it literally attacks the cervical
spinal cord),” says Hilliard. “There have been five
fatalities in the last 12 years at four different institutions
in four states. The survivors are our first introduction
to what happens when you put a pathogen
like B virus into the human population. The reason
we work with this agent is because we know that
rapid identification means we can successfully treat
the individuals.”
The cost of moving to Atlanta was approximately
$200,000, which included expenses for shipping the
laboratory equipment, moving staff, and flying back
and forth between the new lab and the Texas lab to
verify that the performance standards of the new
facility matched those of the well established lab.
The cost to renovate the space to accommodate the
253

Center was approximately $1 million, not including
the daily maintenance costs of the hot lab. Both
laboratories had to be operated concurrently in order
to preserve the integrity of the testing until Hilliard’s
team determined that the new Center could
function efficiently on its own.
The architectural firm of Lord, Aeck and Sargent
designed the first phase of the Center, while
construction of the BSL-4 lab was supervised by the
architectural firm of HOK in cooperation with The
Baker Co., which customized the 32-linear-foot
glovebox cabinet.
Designing a BSL-4 Laboratory
The Viral Immunology Center, housed in the
University’s six-floor, 500,000-sf natural sciences
building, features a departmental animal facility in
the basement and BSL-3 and -4 laboratories on the
third floor, including a specialized BSL-4 for small
animal models of disease. The Center, which occupies
about 5,000 sf and employs 30 people, also includes
two support BSL-2 laboratories, a biochemistry
lab, robotics facility, offices, a fermentation lab,
and shared departmental resources, including electron
and confocal microscopy areas, a sequencing
core, and a FACS-analysis core for cell sorting.
The activities that take place in the Natural Science
Center are of particular interest when designing
a BSL-4 laboratory in a university setting. This
building typically houses the departments of biology,
chemistry, biochemistry, physics, and anatomy in
addition to the BSL-4 lab. Research activities that
are not part of the viral laboratories must also be
taken into consideration when deciding where to
locate the BSL-4 suites. Other activities to consider
include teaching sessions and seminars.
Providing access to the laboratories in a manner
that protects visitors is critical to the efficient operation
of the facility. Serious consideration is given to
the type of barriers that are erected between common
access hallways and restricted areas to ensure
that lay visitors and research supporters can view the
work taking place without being exposed to potential
risks. Access is provided via keypad entry for students,
faculty from other disciplines, visiting scientists,
security and maintenance personnel, and inves-
254
Operating a BSL-4 Laboratory in a University Setting
tigators. The BSL-3 and -4 laboratories have a magnetic
card access so that entry and exit into the facilities
can be tracked by a computer. With the additional
restrictions imposed by the new Homeland
Security Program, all individuals who work within
the BSL-3 and -4 labs must first have federal security
clearance after submission of extensive documentation
to the FBI. The facilities must also meet Clinical
Laboratory Improvement Amendments and Select
Agent inspection requirements to carry out the
research and diagnostic missions of the laboratory.
Key Components of a Maximum-
Containment Facility
Each day Hilliard’s facility receives up to 20
boxes from all over the world from individuals who
may have been exposed to the secretions of a Macaca
monkey. Minimizing the transport of materials going
to high-containment areas is critical. When the samples
arrive, they are triaged to determine what tests
must be done so the results can be sent to the concerned
individuals as quickly as possible.
The packages, transported through an isolated
hallway that is sectioned off from other areas of the
facility, are opened in the Level 2 unpackaging
room, attached to the BSL-3 and -4 suites. Each of
these areas can be accessed only by computercontrolled
keypad entry. Any materials that leave the
BSL-4 facility undergo an intensive search for evidence
of any live virus, using virus plaque assays to
verify that all virus has been inactivated. If the assay
is negative, the chain of custody for removal begins.
Items that leave the BSL-4 laboratory must go
through two sets of autoclaves or two sets of crosslinked
glutaraldehyde dunk baths. Dunk baths enable
chemical sterilization of the outside wall of submersed
containers. This allows a live virus to be removed
from the cabinet for ultra-low freezing and
storage within the BSL-4 and passage of inactivated
materials to the Level 2 labs. Live virus never leave
the suite.
The disposal system consists of two redundant
150-gallon tanks that are released into a boilerdecontamination
unit. A chilling mechanism cools
the water, which is released into the public system

once it is steam disinfected and verified as inactivated.
A drain is situated under the tanks to contain
leakage, should that occur.
The specialized HVAC unit dedicated to the
independent air system of the BSL-3 and -4 suites is
located on the roof of the Natural Sciences Center,
while the decontamination facility is positioned under
the BSL-4 laboratory. Computers are used to
analyze temperatures, airflow, and the rooftop exhaust
systems, which are actively engaged. In case of
power failures, a natural gas backup system is in
place to maintain air purification, negative pressure,
and equipment power.
It is critical to be able to monitor the temperature
and air supply for each of the high-containment
suites by computer on site or from remote locations
by the extensively trained and certified engineer required
to maintain the facility. The outer hallways
are kept at negative pressure assessed by magnahelics,
and with each step further into the biocontainment
areas the negative pressure gradient increases
according to the specifications outlined in the
CDC’s Biosafety in Microbiological and Biomedical
Laboratories.
“The goal of the BSL-3 rooms is to isolate viruses
from small samples. In order to process the samples,
pull out the virus, and identify unusual agents, we
have to have incubators that are fed five percent carbon
dioxide and 95 percent air,” explains Hilliard.
“We can’t bring tanks in and out of these rooms, so
we have a separate alcove which houses the gas tanks
feeding the incubators in the BSL-3 and BSL-4 labs.”
It is challenging to keep the BSL-3 laboratories
stocked and tidy at the same time, keeping in mind
that many of the items that come into the labs are
not going to leave until they are thoroughly melted
down by repeated autoclaving. Disabled equipment
cannot be removed from the laboratory for maintenance
or repair. Tools for minor repairs are decontaminated
with gas sterilization before they are removed
from the maximum-containment suites.
Keeping the Scientists Safe
Necessary precautions are taken to protect the
researchers while they are working in the laboratories.
Scientists usually work in a cabinet or a suit lab,
Tradeline Publications
which relies on the standard biocontainment gear,
in the BSL-4 facility. Working in a cabinet lab is extremely
challenging, but considered safer than a suit
lab because the individual uses large glove ports and
is separated from the pathogen by thick stainless
steel. The cabinet lab at the Center is 32-feet long
and can accommodate three or four researchers at a
time. A nearby animal section houses mice, rats, and
rabbits, and a screen built into the cabinet allows
investigators to visualize materials analyzed by an
inverted, phase-contrast microscope.
Working in the cabinet lab can sometimes lead
to fatigue because scientists must do all of their work
using arm-length gloves to maneuver materials
within the stainless steel cabinet while working in a
line with other researchers. Working in the suit lab
allows scientists to use the traditional lab layout and
to move around more freely. However, breathing the
positive-pressure air in the suit can also cause fatigue.
Barrier protection masks are worn at all times in
the BSL-3 and -4 labs. Only 10 people on Hilliard’s
staff are trained to work in the BSL-3 lab, while only
five of these can enter the BSL-4 facility.
“The masks are basically a reminder for us not to
put our double-gloved hands up to our faces. If we
relied on them as filtration or personal protection
devices, we would go to something different,” says
Hilliard. “All agents are handled in a Class 2 cabinet
in the BSL-3, so there is no time at which an agent is
uncapped in the room surrounding the cabinet.”
Important Considerations
Careful consideration must be given to the installation
of all systems and how they will interact.
The HVAC system at GSU had been repaired at an
annual cost of $12,000 every year since Hilliard’s lab
moved into the facility. A new HVAC system specifically
engineered after analysis of previous failures
was installed at a cost of $250,000 and 2003 marked
its first year of operation.
“This is an existing building that we modified. It
wasn’t meant to be under constant negative pressure
and that has caused substantial stress on the walls
and ceiling,” says Hilliard. “Careful consideration of
the systems that are linked is very important. We are
255

ight in the middle of downtown on a rooftop so
there are many issues to address.”
Cabinet labs should be designed so they are ergonomically
comfortable for researchers of varying
physical stature. The cabinet labs at the Center were
designed largely with the input of the former associate
director, who is more than 6-feet tall, creating a
challenge for other lab staff to do their work.
Smaller gloves, extenders, and a few other modifications
enable the other investigators to work relatively
comfortably.
It is also crucial to properly position the laboratories
in a manner that is most conducive to the type
of work being done. For example, a West Nile BSL-3
is located adjacent to the robotics facilities that service
the sera and viral culture laboratories.
The BSL-2 labs have standard virology for lower
containment agents like HSV-1 and HSV-2. The
sum total of the work performed in these labs is to
take core resources of the BSL-3 and -4 labs, combine
them with the research missions, maintain a
high-containment robotics lab for high-throughput
virus identification, as well as serologic screening,
perform microarray analysis, and be flexible enough
to look at new agents as they come to the scientists’
attention.
“All of our work would be for naught if we
didn’t have the supportive BSL-2 labs,” says Hilliard.
The robotics unit can perform multiple tests and
process thousands of sera daily. The viral culture
robot can plate samples and screen them at multiple
times during the day for the presence of a virus. This
allows mass processing of thousands of diagnostic
samples. If a disturbance in cell culture is noted, a
pager is sounded and an investigator is called to the
room for further analyses and to notify the institution
submitting the sample for analysis.
“In the BSL-4 laboratory, the goal of our work
is the production of large amounts of virus and containing
the supply very securely,” says Hilliard. “We
can then look at drug sensitivities and how the virus
256
Operating a BSL-4 Laboratory in a University Setting
behaves in cells in order to understand the pathogen
and how to control it.”
Biography
Julia Hilliard is director of the Viral Immunology
Center at Georgia State University in the Department
of Biology. She is also the Georgia Research
Alliance Eminent Scholar in Molecular Biotechnology
and Director of the National B Virus
Resource Center for global diagnostic resources. She
has worked with the BSL-4, newly classified Select
Agent, B Virus (Cercopithecine herpesvirus 1) for 23
years, providing diagnostic resources to the biomedical
community for the last 16 years.
This report is based on a presentation given by
Julia Hilliard at Tradeline’s International Conference
on Biocontainment Facilities in May 2003.
For more information, please contact: Julia Hilliard,
Director, Viral Immunology Center, Georgia
State University, Box 4118, Atlanta, GA 30302-
4118, 404-651-0811, jhilliard@gsu.edu.
Reprinted with Permission © December 2003 from
TradelineInc.com, a registered product of Tradeline Inc.,
a provider of leading-edge resources to facilities planning
and management through conferences, publications, and
the Internet community. Visit www.TradelineInc.com for
more information.
Resources
Biosafety in Microbiological and Biomedical
Laboratories:
www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm
Clinical Laboratory Improvement Amendments:
www.cms.hhs.gov/clia/
Select Agents:
www.cdc.gov/od/sap/docs/salist.pdf
Viral Immunology Center:
www.gsu.edu/~wwwvir/index.html

Figure 1
The entry area for the maximum-containment
labs includes a pass-through shower. Scientists
prepare for their work by donning either a
suit lab or working in the cabinet lab.
(Photo courtesy of Julia Hilliard.)
Tradeline Publications
Figure 2
Materials that leave the BSL-4 lab go through
two sets of autoclaves or two sets of cross-linked
glutaraldehyde dunk baths. A decontamination
facility is situated under the lab. The disposal
system consists of two redundant 150-gallon
tanks released into a boiler-decontamination unit.
(Photo courtesy of Julia Hilliard.)
Figure 3
When pathogen samples arrive, the packages are transported through an isolated hallway
that is cordoned off from other areas of the facility. They are opened in a Level 2 unpackaging
room, which is attached to the BSL-3 and -4 labs. (Photo courtesy of Julia Hilliard.)
257

Abstract
258
Special Feature
Use of Multiple SOP Styles to Increase
Personnel Compliance and Safety Within
a BSL-2/BSL-3 Animal Facility
Andrea Mitchell, Jeri Ellis, and Tim Ruddy
University of Arizona Animal Care, Tucson, Arizona
Biohazard training and compliance of husbandry
and research staff are often complicated at the University
of Arizona by the sporadic nature of the work
within the BSL-2/BSL-3 suite. Projects may last for a
month and then not be repeated for a significant period
of time. Failure to adhere to approved safety measures
and techniques for specific rooms/projects over time
increased the need for clear standard operating procedures
(SOPs) that were easily accessed and utilized.
SOPs available only in the employee lounge are of little
use when a question arises while changing cages within
a BSL-2 animal room. To this end, new SOP formats
and display locations were developed and instituted to
increase awareness and comprehension of techniques
used within the BSL-2/BSL-3 suite. Changes included
the placement of plain text as well as picture-enhanced
SOPs in common areas such as the employee lounge
and entrance to the biohazard suite, and within the animal
rooms. The introduction of SOPs containing color
photographs has been instrumental in increasing compliance
of in-room procedures by both native English
speakers and those who do not speak English as a first
language. Overall, the use of multiple styles of SOPs
has been a huge success. This process has helped to
reduce confusion and increase compliance by both the
husbandry and research staffs working within the University
Animal Care facilities.
Introduction
In 2002, a review of University Animal Care
(UAC) husbandry and research staff procedures
within the biohazard suite at the Central Animal
Facility identified several areas of concern. Even
though SOPs formalized in the late 1990s were GLPcompliant,
most husbandry technicians did not have
a good understanding of the SOPs or biosafety basics
and were using techniques and procedures that
could potentially cause the spread of contamination
to personnel and/or the environment. Less than
optimal processes were immediately halted and all
husbandry staff were retrained based on existing versions
of SOPs. Fortunately, the agents in use were
primarily ABSL-2, and appropriate personal protective
equipment (PPE) was being worn when the animals
were handled. Research staff used appropriate
technique when working with their animals, but had
difficulty packaging contaminated cages appropriately
for autoclaving out of the suite, often necessitating
the unpacking and repacking of cages.
Data Gathering
Staff Interviews
UAC husbandry and research technicians were
surveyed to establish the cause of the breakdown in
technique and noncompliance with UAC SOPs.
Numerous barriers were identified:

Complex or Unique Procedures. Husbandry
technicians frequently used practices appropriate for
sterile animal rooms instead of the required practices
needed for biohazard rooms. As the bulk of
their workload involved handling “clean” animals,
they found it counter-intuitive to handle the “dirty”
animals differently. The mindset that the animals
must be protected from the technician and the environment
was ingrained and using techniques to protect
personnel, not animals, was reported as “feeling
wrong.”
Individual Variations Among Studies. Procedural
differences based on the agent used confused
the husbandry technicians. Often the techniques
used with one study were applied to the next, without
regard to the differences in agent or route of
infection.
Training. Husbandry technicians were often
trained by the last person to work in the animal
room, with minimal emphasis on SOPs. Incorrect
techniques were passed from one technician to the
next, creating the “we’ve always done it this way”
mindset. A heavy reliance was placed on expecting
the technician to memorize and retain verbal information
rather than to consult written documentation.
Sporadic Nature of the Studies. The sporadic
nature of the biohazard studies hindered the process
of knowledge “ownership” by the husbandry technicians.
Often a study would last several weeks and not
be repeated for 6 months to a year. It was found that
husbandry technicians were trying to rely on memory
rather than written documentation to refresh
their memories about required processes.
Imprecise and Tedious SOPs. A review of existing
husbandry SOPs showed that there were process
inaccuracies, as well as contradictory statements
among SOPs. Husbandry technicians complained
that reading SOPs was tedious and time-consuming.
The inaccuracies caused confusion, often resulting
in the technician abandoning the SOP and relying
on memory to complete a task.
Location of SOPs (distance to animal room).
Husbandry technicians reported distance from the
animal room to the employee lounge, where the
GLP-compliant SOPs were kept, as an issue in the
use of SOPs. They related that if a question arose
A. Mitchell, et al.
while in the animal room, they did not want to
waste time by removing their PPE, leaving the room,
and searching out the appropriate SOP. Instead,
they would rely on memory and check the SOP after
their animal room work had been completed.
Language. Some husbandry and research staff
identified difficulty comprehending written instructions
as one barrier to bagging contaminated caging
appropriately. The individuals experiencing the most
difficulty were those for whom English was not their
primary language.
Communication. Instructions from research
staff as to PPE and husbandry procedures used for
their agent at times varied from the information included
in the Institutional Animal Care and Use
Committee (IACUC)-approved protocol.
Institutional Input
Next, the IACUC-approved protocols were reviewed
for agents and species, PPE and equipment
needed, and procedures involved in the study. This
information was then compared to existing GLPcompliant
UAC husbandry SOPs and the differences
between the two were discussed with the University
Biosafety Officer to facilitate changes needed
in the SOPs to increase compliance with known industry
standards for the agents used.
Materials and Methods
SOPs
Based on the data gathered from the IACUC
protocols and interviews with researchers, UAC husbandry
staff, research staff, and the University Institutional
Biosafety Officer, it was apparent that the
UAC biohazard SOPs needed revamping. The information
presented in the SOPs, as well as the location
and format of the SOPs, needed to be addressed.
It was found that new husbandry staff
needed the in-depth presentation of the GLPcompliant
SOPs during initial training and for occasional
review, whereas experienced staff had no need
to read hundreds of pages of SOPs every time they
were assigned to a biohazard animal room for the
week.
To this end, all biohazard SOPs were reviewed
and revised. Careful attention was paid to ensuring
259

260
Use of Multiple SOP Styles to Increase Personnel Compliance and Safety
that interconnecting SOPs “spoke” to each other
with accuracy. Two non-GLP-compliant SOP formats
were developed to distill the information presented
in the GLP-compliant SOPs to an abbreviated
format for use by experienced, trained husbandry
staff members. SOP placement was also revised:
Employee Lounge SOP Book. University Animal
Care maintains master electronic copies of GLPcompliant
standard operating procedures which are
available for review on a computer located in the
employee lounge. Pictures were added to those SOPs
where a photo could illustrate a complex issue, increase
comprehension, and reduce the wordiness
inherent in complex SOPs. To increase accessibility
specifically to the biohazard SOPs, photocopies were
organized into a three-ring binder and placed in the
employee lounge.
Entrance to the Biohazard Suite SOP Binder.
The biohazard suite is on a different floor from the
employee lounge. Because of this, another three-ring
binder containing photocopies of the GLPcompliant
husbandry biohazard SOPs was placed in
the entry airlock to the biohazard suite. SOPs were
placed in plastic sleeves with closure flaps to keep
the pages clean and facilitate updating the binder
without spending excessive dollars to laminate the
pages. This location allows the husbandry technicians
to review procedures prior to entering the biohazard
suite.
Animal Room. Several changes were implemented
at the animal room level:
• A biohazard questionnaire was designed and
implemented to address several of the issues identified
during the data-gathering phase. The questionnaire
(shown in abbreviated format in Figure 1) is
completed by the PI before a new study begins (new
agent or change in procedure). The Animal Facility
Supervisor compares the information presented on
the questionnaire with the listed IACUC protocol to
determine if there are any differences in the PPE
and procedures listed on the documents. Discrepancies
are addressed prior to the start of the study. The
Animal Facility Supervisor also searches the Web to
gain third-party insight into the hazards associated
with the agent. The multi-page questionnaire is converted
into a single page SOP (Figure 2) for posting
on the outside of the animal room door for the
duration of the study. This SOP provides several
key pieces of information that the husbandry technician
needs prior to entering the animal room: agent
used, species used, contaminated caging/dead animal/trash-processing
procedures, contact name, and
phone number. This SOP varies in format and
length from the GLP-compliant SOPs in an attempt
to increase comprehension and compliance by using
a pared down, bullet-point presentation of information.
The purpose of this SOP is to quickly answer
the question “What do I need to do in this room?”
leaving the “How do I do this task?” to the more extensive
GLP-compliant SOP format. As an example,
Figure 2 instructs the husbandry technician to autoclave
the soiled cages. This translates roughly into 15
individual GLP-compliant SOPs and 25+ pages of
material for the naïve husbandry technician to read.
The experienced husbandry technician needs to
know only “Do I autoclave the dirty caging?” The
naïve technician needs to know how to put on the
PPE, change the cages, bag the cages, operate the
autoclave, etc.
• A photo-enhanced SOP (shown in abbreviated
format in Figure 3) was designed to increase compliance
by research and husbandry staff when bagging
dirty caging for autoclaving. Including pictures with
text has increased procedural compliance while decreasing
the amount of caging that must be reprocessed.
The laminated SOP is posted on each biosafety
cabinet within the animal facility. This SOP
also varies in format from the GLP-compliant SOPs
in an attempt to increase comprehension and compliance
by using bright, colorful, digital pictures to
illuminate a process that is vital to ensure sterilization
of contaminated caging. Autoclave package size
limitations and autoclave operational idiosyncrasies
increase the need for compliance to departmental
SOPs.
• Three-ring binders containing the GLPcompliant
SOPs pertaining specifically to that room
were placed in the BSL-3 anterooms. These rooms
vary radically in equipment, procedures, and personnel
risk from the BSL-2 rooms and each other. The
additional reference material has been a key component
in training new personnel. Once again, SOPs
were placed in plastic sleeves with closure flaps to
facilitate updating the information.

Training to the SOPs
After the GLP-compliant SOPs were revised, the
additional SOP designs formalized, and both styles
put in place, departmental training packets were updated
to reflect the changes in procedures and processes.
Husbandry staff members were retrained and a
“one size fits all” mindset was discouraged. Emphasis
was placed on recognizing the differences in procedures
in their daily routine. Husbandry staff members
are now formally trained using all styles of biohazard
SOPs and are fully aware of where to find the
information they need to work safely in the animal
rooms.
Conclusion
Compliance with standard operating procedures
is a crucial step in ensuring the safety of husbandry
A. Mitchell, et al.
and research technicians working in areas housing
animals exposed to biohazards. It is vital to identify
barriers to comprehension of SOPs by the technicians.
Physical location, language, and accuracy are
all important factors to consider when reviewing
SOPs. Is the SOP in an area where the technician
can review it when actually performing the task? If
not, is it possible to place the information where it
will be readily accessible? Is it important that all of
the information is presented in its entirety or can
the SOP be abbreviated and the format changed to
present a more readily comprehended and consumed
product? Will using pictures reduce confusion
and wordiness? Do all interrelated SOPs speak
to one another accurately? By answering these questions,
University Animal Care was able to produce
stronger SOPs and a safer working environment for
its technicians and research staff.
IMPORTANT .................IMPORTANT......................IMPORTANT...............IMPORTANT
Name
A change of address notice should be sent at least 6 weeks in advance to the ABSA National Office to
ensure that all mailings, including the journal, will reach you. ABSA is not responsible for misrouted mail as
a result of insufficient notification of an address change. Undelivered copies resulting from an insufficient
address change notification will not be replaced, but single issues may be purchased at the single issue price.
Old Address
Old Phone Number
NEW ADDRESS
CHANGE OF ADDRESS FORM
CITY STATE ZIP +4
NEW PHONE NUMBER
E-MAIL ADDRESS
Effective Date
261

FIGURE 1
UAC Biohazard Information Questionnaire
This worksheet has been prepared to aid in the establishment of investigator specific Standard Operating Procedures
(SOPs) for biohazard studies. Completing this worksheet will better enable UAC to care for your biohazardous animals
and assist you in meeting your research goals.
This form must be filled out and submitted to the UAC Facility Supervisor prior to initiation of animal
experiments.
P.I. Office Phone # Research Tech:
Lab Phone # Weekend/Holiday Contact Name and Phone #:
Species used: Protocol #:
Agent used: Please check all that apply and list agent (s). (Note: This form must be filled out in addition to the
Authorization Form for Radioactive Material.)
Carcinogenic agent(s):
Radioactive agent(s):
Human Infectious Agent(s):
Animal Infectious Agent(s):
Study size and duration:
How many cages of animals do you expect to have in use at a time?
How long will the animals be in the room?
If using a radioactive compound, can the animals be housed in a general animal room?
Husbandry Tasks. Please check one:
UAC staff will do all husbandry tasks for the room. This includes changing the cages, removing dead animals,
servicing the cages by adding food and water as needed and changing flooded cages.
Investigator will perform all husbandry tasks. (Note-This includes packaging the used caging/bedding/trash for
autoclaving.)
Investigator and UAC staff will share husbandry tasks. Please explain:
Diets: Please check one
All mice will be fed Teklad NIH-31 diet
All rats will be fed Teklad 4% Mouse/Rat diet
Other, Please explain:
Protective Equipment Needed:
All personnel entering this room need to wear the following: Please check all that apply:
Bonnet N95 Respirator Eye protection Yellow gown Jump suit Shoe covers Gloves
Other:
All personnel handling the animals in this room need to use the following when opening the cages:
Biosafety Cabinet Glove Box Chemical Fume Hood Other:
Check all that apply:
All animal cages and bedding must be autoclaved after use in the animal room.
All animal bedding must be disposed of in yellow radioactive bags and the label filled out.
All trash must be autoclaved prior to disposal from the animal room.
Trash may be disposed in the red barrels without autoclaving.
All animal carcasses must be autoclaved prior to disposal.
All animal carcasses must be disposed of in yellow radioactive bags and the label filled out.
Animal carcasses may be disposed of in the red barrels without autoclaving.
Other:

FIGURE 2
UAC Biohazard SOP (Completed Sample)
P.I. John Jones, DVM, PhD P.I. E-mail: jjones@u.arizona.edu Office Phone # 555-1234
Research Technician: Jane Smith E-mail: jsmith@u.arizona.edu Fax # 555-2345
Lab Phone # 555-3456 Weekend/Holiday Contact Name and Phone #: Jane 555-9023
Species used: Mouse Protocol #: 04-555
Agent used: Human and Animal Infectious Agent: Cryptosporidium parvum
Study size and duration: Approximately 10-12 cages will be in the room for 7-10 days.
Husbandry Tasks.
UAC staff will do all husbandry tasks for the room. This includes changing the cages,
removing dead animals, servicing the cages by adding food and water as needed and
changing flooded cages.
Diets: All mice will be fed Teklad NIH-31 non-irradiated mouse chow.
Protective Equipment Needed:
All personnel entering this room need to wear the following: Please check all that apply:
Bonnet N95 Respirator Yellow gown Impervious shoe covers x 2 Gloves x 2
All personnel handling the animals in this room need to use the following when opening the cages: Biosafety
cabinet
Check all that apply:
All animal cages, bedding and trash must be autoclaved after use in the animal room.
Animal carcasses may be disposed of in the red barrels without autoclaving.
Personnel must ungown in the ante room.
The PI will be responsible for terminating all moribund animals.
Notification of Death: Death slips will be filled out and mailed as usual. Investigators will be notified
immediately upon finding dead animals in order for them to harvest tissues or necropsy.
P.I. UAC Supv.
Signature Date Signature Date

264
FIGURE 3
Bagging animal caging using a biosaf ety cabinet with a dump station:
1
4
5
Join a Committee
Use of Multiple SOP Styles to Increase Personnel Compliance and Safety
1. Gown up before entering the ani mal room.
See door SOP for specific PPE worn.
2. Line the dump station barrel with a red, or
orange bi ohazard bag – see door SOP.
3
3. T urn the biosafety cabinet on.
4. Clean cabinet with Clidox. Spray directl y on all surfaces except
“ceiling” and wi pe down with paper towels. Follow the same
process with alcohol.
5. Place clean cages, water bottles, etc. i n the biosafety cabinet if
changing cages.
Have you ever considered joining a committee? When you choose to serve on a volunteer committee, you
open up a world of possibilities for networking, professional growth, and career opportunities while serving
your profession. Volunteer member groups are the backbone of the association because they:
• Serve as a forum for exchange of information
• Advance the science in all specialties of biosafety
• Develop guidelines and standards
• Provide education and training
• Link ABSA to many other institutions
You should explore committees in areas of the profession where you are active or have an interest. There
is a great variety; you can be sure to find one of interest to you. Please review the list of committees and
identify those areas in which you would like to participate or contact the chair of the committee (http://
www.absa.org/abocommittees.html) that interests you to find out more information about the committee’s
goals. You are also invited to attend the committee’s meeting during our national conference or at any other
time (all committee meetings are open).
2

Special Feature
Book Review
Reviewed by George A. Pankey
Ochsner Clinic Foundation, New Orleans, Louisiana
Revenge of the Microbes
By Abigail A. Salyers and Dixie D. Whitt
Washington, DC: ASM Press for Microbiology
186 pp, $ 29.95
ISBN: 1-55581-298-8
This book, for “the general public about antibiotics
and resistance to them,” is the authors’ first
attempt at such a publication. They are both wellknown
scientists and microbiology educators who
have written textbooks on the subject.
As would be expected, some of the material in
Revenge of the Microbes is a little too advanced for the
“general public”; however, for students of medicine,
and members of ABSA, this is an excellent little
book that makes a strong attempt to orient the
reader to the problems of antimicrobial resistance
and the potential etiologies. It is particularly refreshing
that potential contributors to the problem are
not lambasted. Discussions on the use and abuse of
antibiotics by physicians, veterinarians, farmers, and
industry are presented in a balanced fashion. The
dentists somehow escaped discussion, although they
are big antimicrobial users.
I especially enjoyed the historical coverage and
the issues to ponder that occur at the end of each of
the 11 chapters. Following is my evaluation of each
chapter, in parentheses, after each title:
1. Magic Bullets, Miracle Drugs (Excellent)
2. A Brief Look at the History of Antibiotics (Excellent)
3. Bacteria Reveal Their Adaptability, Threatening the
Brief Reign of Antibiotics (Simple and accurate)
4. Antibiotic-Resistant Bacteria in the News (The re-
porters get their due.)
5. Antibiotics that Inhibit Bacterial Cell Wall Synthesis
(Clearly detailed)
6. Antibiotics that Inhibit the Synthesis of Bacterial Proteins
(Good, but eperezolid has not “hit the market”
yet)
7. Fluoroquinolones, Sulfa Drugs, and Antituberculosis
Drugs (Treatment specifics of antibiotic choices
and side effects are not data-based and material
ventures too far from the principles under discussion.)
8. Bacterial Promiscuity: How Bacterial Sex Contributes
to Development of Resistance (Beautifully written)
9. The Looming Crisis in Antibiotic Availability (A
must read)
10. Antiseptics and Disinfectants (A practical and simple
summary)
11. Antiviral, Antifungal, and Antiprotozoal Compounds
(Shows that bacteria are not the only antimicrobial-resistant
microorganisms)
The authors’ folksy writing style makes reading
the book very pleasant. It reminded me of many of
the authors in the United Kingdom whose writing
style keeps one interested and awake.
Revenge of the Microbes is appropriately illustrated
and has an adequate suggested reading list. I
did not find the “structures of antimicrobial agents
mentioned in the text” in Appendix I or Appendix
II regarding “how clinical laboratories measure resistance”
to be necessary or appropriate for the intended
audience. However, this book should be required
reading for all medical, veterinary, nursing,
and pharmacy students as well as those interested in
biosafety.
265

266
Special Feature
Book Review
Reviewed by Michael P. Owen
U.S. Food and Drug Administration Pacific Regional Laboratory Northwest, Bothell, Washington
Biodefense: Principles and Pathogens
Edited by Michael S. Bronze
and Ronald A. Greenfield
Wymondham, United Kingdom: Horizon Bioscience
838 pp., $335, hardcover
ISBN: 1-9094933-12-2
Disclaimer: The opinions and conclusions expressed
in this article are solely the views of the reviewer
and do not necessarily reflect those of the
U.S. Food and Drug Administration.
The 2001 anthrax attacks on the East Coast of
the United States, the 1984 release of Salmonella bacteria
in Oregon, the multiple attempts by the Aum
Shinrikyo cult in Japan to use biological weapons,
and the regularly-reported threats of Al-Qa’ida and
other terrorist movements attempting to use biological
agents have demonstrated that the public health
sector needs an increased sense of urgency to adequately
prepare for bioterrorism events.
One core component of biodefense is continuing
education. Physicians, scientists, government
officials, and other groups involved in public health
need to be constantly aware of current developments
in disease pathology, treatments, detection, biological
safety, and biosecurity to provide an effective response
to bioterrorism attacks. Michael S. Bronze
and Ronald A. Greenfield, University of Oklahoma
Health Sciences Center in Oklahoma City, Oklahoma,
have edited a book about biodefense that assists
with this challenge. One of the notable aspects
of this text is that its information is current, including
many references published in 2005.
Biodefense: Principles and Pathogens’ 23 chapters
are detailed review articles and the book is actually
two different texts in the same volume. The first
seven chapters are a general discussion on biodefense,
including a history of biological weapons, public
health preparedness, public policy and legal issues
surrounding terrorism in the U.S., hospital preparedness
and infection control, surveillance and detection
methods, and psychosocial issues.
The remaining 16 chapters are detailed presentations
about biological agents and biological toxins.
Most of these chapters have sections on the agent’s
history, microbiology, epidemiology, pathogenesis,
veterinary manifestations, clinical manifestations in
humans, diagnosis, treatment, prognosis, prevention,
and research issues. Substances from all three
CDC Bioterrorism Agent Categories A, B, C are
presented. Chapters about food safety, water safety,
agroterrorism, and emerging infectious diseases such
as multi-drug resistant tuberculosis and severe acute
respiratory syndrome are also included, along with a
timely discussion on highly pathogenic avian influenza.
Biodefense: Principles and Pathogens is almost entirely
text. Although there are few images and tables,
those that are included summarize many of the
book’s important topics. The limited graphics is not
necessarily a disadvantage because Biodefense: Principles
and Pathogens is basically one large review article.
As a result of this format, the book contains more
information than tightly-packed chromatin in an
eukaryotic cell. If the reader wants to see more
graphics, the excellent citations and lists of references
should minimize any effort to locate original
publications containing the desired graphics.
The first section presents many excellent points
on preparedness. For example, the text compares
nations such as the United Kingdom and Israel that

have national bioterrorism response plans in place to
the United States which has 50 different plans, one
for each state. Additionally, when discussing the
Strategic National Stockpile of pharmaceuticals and
vaccines, the text cautions that most communities
will be on their own for at least 72 hours until these
supplies can be distributed. It is a sad irony that a
few months after this book was published, the U.S.
was challenged by these issues during the strikes of
Hurricane Katrina and Hurricane Rita. As a result of
those two storms, adjusting the responsibilities of
federal, state, and local agencies when responding to
natural disasters and terrorist attacks and planning
for the degree of self-sufficiency in disaster response
each must assume will likely be often discussed topics
over the next several years.
The chapter on surveillance and detection is well
written and lists methods that are both state-of-theart
and in development. A longer discussion about
the CDC Laboratory Response Network (LRN)
would have been helpful; however, this subject is
addressed in detail on the CDC web site
and in Biological Weapons Defense:
Principles and Mechanisms for Infectious Diseases
Counter-Bioterrorism by Lindler et al (Humana Press,
December 2004).
The chapters on the agents are comprehensive.
Because of their depth and detail, physicians and
scientists will probably find them more useful than
the general public. As in the first part of the book,
the references continue to be very current and easily
retrievable. The clinical information is written at a
level of detail similar to the PDR Guide to Biological
and Chemical Warfare Response: Diagnosis, Treatment,
Prevention by Sifton et al (Thomson PDR, February
2002) and is oriented toward physicians and other
healthcare personnel. The microbiology sections are
in-depth summaries of current knowledge. The research
issues presented at the end of each agent’s
chapter list several new treatment strategies in development.
The first half of the book could have been ex-
M. P. Owen
panded. These chapters offer only an overview of
bioterrorism preparedness; however, they do provide
a solid launching pad for further investigation into
this topic, and the long lists of references make finding
information on this subject easier.
The food safety, water safety, and agroterrorism
chapters would have been more useful if they were
longer, too. However, this observation is not actually
a weakness of the book. As mentioned above, the
text is meant to be a review of biodefense. The relatively
small size of these sections is most likely
the result of most efforts in biodefense and infectious
diseases being focused on higher-level threats.
Longer reviews will likely appear when research efforts
in these areas are increased.
The main disadvantage of Biodefense: Principles
and Pathogens is the cost, which is higher than many
biodefense and biology books available today. Including
an electronic version of the book on a CD-
ROM, similar to Biological Weapons Defense: Principles
and Mechanisms for Infectious Diseases Counter-
Bioterrorism, would have been a helpful addition. Another
option would have been access to a Web site
that has book updates similar to Molecular Cloning: A
Laboratory Manual by Sambrook and Russell (Cold
Spring Harbor Laboratory Press, January 2001).
Again, the lists of references at the end of each chapter
are quite valuable, saving readers a great deal of
time on literature searches.
Biodefense: Principles and Pathogens is a good
primer on current issues in biodefense. Although it
will be most beneficial to physicians and researchers,
the text will also be helpful to biological safety professionals,
especially when performing risk assessments.
It will be a nice addition to any biodefense,
infectious disease, or biological safety library.
Acknowledgement
I would like to thank Karen B. Byers for providing
the opportunity to review the text and submit a
review for this journal.
267

Do you have a biosafety question and you’re not
sure who to ask? Send your questions to the “Ask
the Experts” column and I’ll get them answered for
you. Drawing from my own experience or that of
other experts in the field, we’ll try to compile a thorough
and comprehensive answer to your question.
Please e-mail your questions to jkeene@biohaztec.
com or to Co-Editor Barbara Johnson at barbara_johnson@verizon.net
or Co-Editor Karen B.
Byers at karen_byers@dfci.harvard.edu.
HEPA-Filtered Supply Air for
BSL-3 Laboratories?
Question
I have observed that NIH Design and Policy
Guidelines (http://orf.od.nih.gov/policy/index.htm)
do not appear to require HEPA filtration of air supply
at the BSL-3 level. Isn’t there a possibility that
microorganisms could be drawn into the intake air
and potentially affect the outcome of the science
being conducted in a BSL-3 lab (or any other lab)? If
so, to satisfy the requirement for “flexibility,” wouldn’t
it be more prudent to suggest that designers provide
Air Handling Units (AHUs) with the capacity of
adding HEPA filters?
If I’m not mistaken, the source of the contamination
in the air supply during the Legionnaire’s
incident turned out to be carried on airborne dust
from a construction site in close proximity to the air
intakes of the hotel. Are the 30% prefilters and the
95% filters that the guidelines require for supply air
adequate to prevent a similar incident?
268
Special Feature
Ask the Experts
John H. Keene
Biohaztec Associates, Midlothian, Virginia
Response
The question of HEPA filtration of supply air
for containment laboratories is being asked more
frequently as the construction of more containment
laboratories is being considered. The intake air in
most facilities is filtered by a prefilter and a 95% filter
(not HEPA). This level of filtration is sufficient
for both the general building and the containment
laboratory. HEPA filtering of the air delivered to the
BSL-3 laboratory is not necessary to protect the work
since all open manipulations of infectious materials
are performed in a biosafety cabinet or other physical
containment devices. Moreover, these are leaky
labs and air is drawn through the door from areas
outside of the laboratory anyway.
HEPA filtration of the air in these containment
labs was considered because of concern about potential
“backflow” of air from the lab to the supply system.
This is not a valid concern for several reasons:
1. The lab is not generally contaminated since the
work is contained.
2. In modern buildings control systems should ensure
that the lab would not go positive in case of an
exhaust fan failure.
3. If for some reason the controls failed and the lab
did go positive, the air would take the route of least
resistance—the spaces around the doors and not the
supply system which has a built-in resistance in the
building filter system, and which, if it is still running,
would be pushing air into the room so it
would have to escape through another route.
4. For any possible release, a highly unlikely sequence
of events would have to occur, i.e., a spill in
the laboratory at the same time that the exhaust

fans, primary and backup, failed as well as the supply
fan failing at the same time.
In addition, installing HEPA filters because of a
concern about contamination of the supply duct
work in a containment lab should be reviewed carefully.
HEPA filtration of clean rooms is performed
to reduce the particulate count in these rooms, not
necessarily to remove infectious particles. The filters
are placed so that the supply air blows against the
filter and against the seal of the filter to the housing,
thus ensuring, when properly installed, the continued
seal of the filter. If one assumes that there is a
possibility of contamination of the ducts (not a realistic
assumption given the above discussion), one
would want to install the HEPA filter so that it
would seal in the direction of the potentially contaminated
air flow, which would be toward the supply
air duct. However, with the supply air blowing in
the opposite direction the majority of the time, it
would be difficult, if not impossible, to ensure that
Fact Sheets on Terrorist Attacks
J. H. Keene
the filter seal was not disrupted by the push of air
against it. Secondly, if it is assumed that the laboratory
air is contaminated when it passes back through
the filter, then the filter must be capable of being
decontaminated and tested—a daunting task in itself.
Consequently, the HEPA filtration of supply air in a
BSL-3 laboratory is neither necessary nor desirable.
Regarding the Legionella question: The outbreak
resulted from outside air contamination, but not
necessarily from construction dust. In fact, the outbreak
resulted from Legionella contamination of the
cooling tower water that was being aerosolized.
While contamination of the intake air with Legionella
or any other infectious agent might affect the
personnel in a containment facility, as it has in many
instances, it would not affect the work since good
microbiological practice assumes that air in the laboratory
is never sterile. In summary, the work in BSL-
3 labs is not “clean room” work and does not have
to be HEPA filtered to keep out potential pathogens.
The National Academies is preparing, in cooperation with the Department of Homeland Security, fact
sheets on four types of terrorist attacks. Drawing on our many reviewed publications, the expertise of our
members, and the knowledge of other esteemed authorities, the fact sheets will provide reliable, objective
information. Go to: www.nae.edu/nae/pubundcom.nsf/weblinks/CGOZ-642P3W?OpenDocument.
They are being designed primarily for reporters as part of the project News and Terrorism:
Communicating in a Crisis, though they will be helpful to anyone looking for a clear explanation of the
fundamentals of science, engineering, and health related to such attacks.
These fact sheets are a product of the National Research Council Division on Earth and Life Studies.
Biological Attack (pdf file, 277 KB)—Where do biological agents originate? What’s the difference between
“infectious” and “contagious”? How long after exposure will symptoms appear?
Chemical Attack (pdf file, 72 KB)—What are the different origins of toxic chemicals that could be used? How
do chemical toxicities vary? What are the practical steps to take if there’s a chemical release?
Radiological Attack (pdf file, 68 KB)—What are radiological dispersal devices, a.k.a. “dirty bombs”? How are
they different from nuclear bombs? What are their physical and psychological health effects?
Nuclear Attack (pdf file, 192 KB) NEW!—What is radioactive fallout, and how is it dangerous? What are the
short-term and long-term effects of radiation exposure? What is the likely size of a nuclear explosion from an
attack by terrorists?
269

Biosafety Tips brings you practical approaches to
biosafety or “news you can use.” If you are looking
for a useful and sensible solution to a biocontainment
problem or perhaps a reference to help convince
a skeptical researcher of the need for caution,
this is the place to look. In this column I will share
some biosafety insights for managing a variety of
workplace situations. I welcome feedback or suggestions
for future topics. Please e-mail any comments
or suggestions to karen_byers@dfci.harvard.edu or
to Co-Editor Barbara Johnson at barbara_johnson@
verizon.net.
Lymphocytic Choriomeningitis Virus—
A Hazard in Rodent Animal Colonies
In April 2005, a woman died of a stroke and
after a thorough work-up, her organs were donated
to four recipients. In May, state authorities notified
the CDC that three of the transplant recipients had
died, and the fourth was ill. Extensive testing revealed
that lymphocytic choriomeningitis virus
(LCMV) was present in the transplanted organs. Review
of the organ donor’s history indicated that she
had a new pet hamster, and the hamster was positive
for LCMV infection. Although the donor had a subclinical
(asymptomatic) infection, the immunosuppressed
organ recipients developed fatal or serious
infections from the infected organs (CDC, 2005).
To prevent disease transmission from pet rodents,
CDC has posted advice at: www.cdc.gov/ncidod/
dvrd/spb/mnpages/dispages/lcmv/owners.htm.
Wild, or house, mice are a far more common
source of LCMV. LCMV is a rodent-borne Arenavirus,
and about 5% of adults living in urban populations
have antibodies indicating previous exposure
270
Special Feature
Biosafety Tips
Karen B. Byers
Dana Farber Cancer Institute, Boston, Massachusetts
to LCMV (CDC, 2005). Subclinical LCMV infection
in a healthy adult may go unnoticed, but the
virus may also produce a flu-like illness, or aseptic
meningitis. LCMV is not spread from person to person,
except that it may spread vertically from a pregnant
woman to a developing fetus. Exposures during
the first or second trimester are a serious risk to the
fetus (CDC, 2005). To protect the general public
from the risk of contracting LCMV from wild rodents,
the CDC has posted practical guidelines on
the Web and provided detailed information in the
publication Interim Guidance for Minimizing Risk for
Human Lymphocytic Choriomeningitis Virus Infection
Associated with Rodents (CDC, 2005).
Case Study 1
In the occupational setting, animal facilities are
carefully designed to prevent entry by wild mice and
colonies are routinely screened for LCMV. In addition
to the zoonotic risk, infection with LCMV can
affect a wide range of research results (National Research
Council, 1991). The following case study describes
the chain of events that lead to an LCMV
outbreak at a research institute in 1989 (Dykewicz et
al., 1992).
• In 1964, a cancer research institute developed a
proprietary cancer cell line. This institute routinely
injected the cell line into rodents to induce tumors
in order to study metastasis.
• In 1988, the institute began replacing the hamster
animal model with nude mice.
• A lapse in the routine serological monitoring of
rodent colony health occurred between August 1988
and March 1989. When the monitoring resumed,
the oldest sentinel hamsters were positive for LCMV.

• In May 1989, an animal care worker who had
never worked with hamsters was hospitalized with
aseptic meningitis. With the information that the
sentinel animals had LCMV antibodies, the institute’s
management requested testing to “rule out”
LCMV. The hospitalized staff member was diagnosed
with LCMV.
• How could this infection have occurred? The
proprietary cell line was tested, and it was positive
for LCMV. Other staff members began reporting a
range of complaints, so the CDC was alerted and an
investigation followed.
Results of CDC Investigation
Eighty-two out of 90 staff members consented
to serological monitoring for LCMV antibodies.
Seven were positive, indicating previous exposure to
LCMV, and one was a “probable” previous infection.
The control group included 145 local blood
donors with only one sample positive for LCMV
(Dykewicz et al., 1992). Frozen aliquots of the proprietary
cell line were thawed and it was discovered
that aliquots as far back as 1975 were seropositive
for LCMV. In addition, 13 out of 70 other cell lines
passaged in animals were also positive.
So why did the outbreak occur in 1988, if the
cells had been infected for a long time? A review of
all the factors the eight seropositive staff members
had in common revealed no association with needlesticks
or sharps injuries, bites or scratches, rural residence,
pets, or noncompliance with the use of personal
protective equipment. The facility had a policy
of requiring gloves and a surgical mask. Only one
fact stood out—the eight seropositive staff members
had more contact with nude mice (median, 10 hours
per week) compared to seronegative staff (median, 1
hour per week) (Dykewicz et al.,1992).
Nude mice are hairless, lack a thymus, and have
an impaired immune system. They can become persistently
infected with LCMV and excrete it in high
titer; this characteristic is aptly called “viruria.”
(Dykewicz et al., 1992). LCMV infection of nude
mice does not produce symptoms in the mice and
cannot be detected by direct serological monitoring
since nude mice do not produce an antibody response.
Apparently, the 80-fold increase in the use
of nude mice resulted in a build-up of the viral reser-
K. B. Byers
voir in this facility and led to seven infections with
two hospitalizations and one “probable” infection.
The article goes on to describe the institute’s
effort to contact former staff members and the many
recipients of various cell lines to inform them of the
LCMV infection. If the facility administration had
not requested testing of the staff member hospitalized
with aseptic meningitis, this outbreak might not
have been recognized. Even if an animal colony is
considered pathogen-free, the use of cell lines passaged
in animals in another facility puts the colony,
and the staff, at risk.
Case Study 2
An example similar to the one above was presented
at the 2004 ABSA conference (Braun, 2004).
In 1998, an animal technician developed a persistent
high fever of unknown origin and “worrisome”
symptoms. Several months later, three sentinel animals
in the room where the technician worked were
positive for LCMV infection. All of the other technicians,
veterinarians, and investigators tested negative,
but the animal technician who was ill tested
positive. The source was a human cell line obtained
from another institution that had been passaged in
nude mice for 10 to 12 years without reports of contamination.
Fortunately, that outbreak was limited
to a single case.
The research community must understand the
importance of compliance with a cell line screening
policy, since once a study has been approved, it is
very difficult to monitor compliance. When incorporated
into training programs, case studies such as the
ones described above help researchers understand
that noncompliance results in risks to themselves,
the animal care staff, and their research results.
Additional LCMV Resources
Today, animal care staff and animal researchers
receive training on zoonotic risks, including LCMV.
For example, the University of California-Irvine
web site has the following statement under
“rodent health” at: www.rgs.uci.edu/ular/policies/
rodentcolonyhealth.htm.
271

The overwhelming significant feature of
LCMV is its zoonotic potential. It can be
predicted that T cell-deficient mice will amplify
LCMV infection. The polytropic nature
of LCMV and its wide host range allow
this virus to readily infect transplantable
tumors and cell lines, which can serve as a
source of contamination for mouse colonies.
• Organotropism—kidney, salivary gland,
lymphohematopoietic cells
• A zoonosis
• Interference with research—immunology,
oncology, physiology
Well-established procedures for using immunocompetent
sentinel animals are described at
the University of Washington web site at:
h tt p://depts.washington .edu/compmed/
rodenthealth/#sentinels.
Roughly analogous to the “canary in the
coal mine,” sentinel rodents monitor the
pathogen status of the investigator’s rodents.
Every rack in a room is monitored. As you
are facing a rack, the cage containing the
two sentinels usually resides in the lower
right-hand position. Every time an investigator’s
rodent cage is changed (usually weekly),
about a tablespoon of soiled bedding from
that cage is transferred to the sentinel cage.
In this way, sentinels are exposed to whatever
pathogens may be present in the urine,
feces, fur, saliva, dander, etc. from 100% of
the cages on the rack. Because of this, investigators
must not handle or move sentinels
or sentinel cages.
PCR testing for many zoonotic pathogens is also
commercially available; this allows direct testing of
immunocompromised strains. Charles River Laboratory
has posted an explanation of test methods
available at: www.criver.com/research_models_and_
services/research_animal_diagnostics/LAD_DS_
272
Biosafety Tips
MolecularW.pdf and www.criver.com/research_
m o d e l s _ a n d _ s e r v i c e s / r e s e a r c h _ a n i m a l _
diagnostics/LAD_DS_InVivoBiosafetyW.pdf.
References
Braun, A. (2004). Case report: Transmission of lymphocytic
choriomeningitis virus from laboratory
mice to an animal technician. 47th Annual Biological
Safety Conference, San Antonio, Texas.
Centers for Disease Control Special and Prevention
Pathogens Branch. (2005). Information for pet owners:
Reducing the risk of becoming infected with LCMV from
pet rodents. Available at: www.cdc.gov/ncidod/dvrd/
spb/mnpages/dispages/lcmv/owners.htm. Accessed
online 2005.
Centers for Disease Control and Prevention. (2005).
Lymphocytic choriomeningitis virus infection in organ
transplant recipients. Morbidity and Mortality
Weekly Report, 54(21), 537-539.
Centers for Disease Control and Prevention. (2005).
Interim guidance for minimizing risk for human
lymphocytic choriomeningitis virus infection associated
with rodents. Morbidity and Mortality Weekly Report,
54(30), 747-749.
Dykewicz, C. A., Dato, V. M., Fisher-Hock, S. P.,
Howarth, M. V., Perez-Oronoz, G. I., Ostroff, S. M.,
Gary, H., Shonberger, L. B., & McCormick, J. B.
(1992). Lymphocytic choriomeningitis outbreak associated
with nude mice in a research institute. Journal
of the American Medical Association, 267, 1349-1353.
National Research Council. (1991). A companion
guide to infectious diseases of mice and rats. Washington,
DC: National Academy Press.

ABSA News
2005 ABSA Conference Photos
Conference attendees visiting the Exhibit Hall
Betsy Gilman Duane receives Past-
President Award from Glenn Funk
Affiliate Relations Exhibit Booth
Conference attendees at the Scientific Program
273

Award Presentations from the ABSA
Conference, October 2005
Arnold G. Wedum
Distinguished Achievement Award
David G. Stuart, PhD
The Baker Company
Sanford, Maine
The award shall be given to an individual for
outstanding contributions to biological safety through
teaching, research, service or leadership.
David Stuart has had a long and highly successful
career at the Baker Company. He received his
Doctorate in Microbiology, and is a Charter Member
of ABSA. Through his distinguished career he
has served on the original Steering Committee of
ABSA, the Technical Review and Nominating Committees,
and the Journal Editorial Board.
He has mentored numerous colleagues in the
certification profession regarding microbiology, de-
274
ABSA News
2005 ABSA Service Award Recipients
David G. Stuart, PhD, accepting the Arnold G.
Wedum Distinguished Achievement Award
at the 2005 ABSA Conference in Vancouver.
contamination and biocontainment and has been
a constant source of reliable and timely information
to the Controlled Environment Test Association
(CETA), ABSA, and the National Sanitation Foundation
(NSF). He has had a profound influence in
the area of standards writing as a participant in NSF
committees. He is the voice on many panels that
send the clear message that standards be developed
based on scientific fact by ensuring participants provide
research results to support their positions on
standards development. He continues to make contributions
to biosafety on international and national
levels by teaching courses including “Advanced Certification”
in the United States, Russia, and Hong
Kong. He is noted by his colleagues and fellow biosafety
professionals as an individual that leads by
example. He is a dedicated researcher and teacher in
the field of contamination control with an inspirational
work ethic.
Richard C. Knudsen
Memorial Publication Award
Janet E. Meszaros, MS
Steris Corporation
Mentor, Ohio
The award shall be given, when merited, to the
author(s) of an article that reports a significant contribution
in scientific investigation and/or health and safety
in areas of interest to Richard Knudsen during his career.
The award recipient need not be a member of the
American Biological Safety Association.
Research Article: Meszaros, J. E., Antloga, K.,
Justi, C., Plesnicher, C., & McDonnell, G. (2005).
Area Fumigation with Hydrogen Peroxide Vapor.
Applied Biosafety, 10(2), 91-100.
Ms. Meszaros, the senior author of the article
selected for the Richard Knudsen Memorial Publication
Award, earned her BS and MS from Cleveland
State University. She is a Senior Scientist at STERIS

Corp. where she has worked for over a decade, as
well as serving the academic community as a Science
Instructor at Cuyahoga Community College—also
for over a decade.
This original research article was selected for its
contribution of knowledge to the field of biosafety.
Results of this research quantitatively identified the
sterilizing capabilities of hydrogen peroxide vapor
across various classes of resilient organisms that were
applied to numerous common laboratory surfaces
over varied exposure times.
Robert I. Gross Memorial Award
Cassandra Kelly, BS
New York State Department of Health
Albany, New York
Awarded to a student in recognition of academic
achievement in biological safety.
Cassandra Kelly is a Research Scientist and the
Biosafety Level 3 Supervisor in the Biodefense Laboratory
at the Wadsworth Center, New York State
Department of Health (NYSDOH). Cassandra received
a BS in 1997 from Carleton University and is
currently pursuing a PhD in the Biomedical Sciences
Department at the State University of New York,
Albany. Prior to joining the NYSDOH Biodefense
Laboratory in 2002, Ms. Kelly worked at the Canadian
Science Center for Human and Animal Health
conducting research in prion diseases, hepatitis viruses
and emerging blood borne pathogens. Ms.
Kelly is involved in the development of new diagnostics
for select agents as well as training of laboratorians,
first responders and members of the law enforcement
communities in New York State.
Abstract: Effects of bacillus spore decontamination
methods on forensic evidence: an evaluation of
decontamination methods: vaporized hydrogen peroxide,
formaldehyde, and autoclaving.
Many environmental samples submitted to the
New York State Biodefense Laboratory for biothreat
testing are often requested to be transferred, following
select agent testing, to forensic laboratories in order
to proceed with criminal prosecutions. Even after
the presence of potential bioterrorism agents have
been ruled-out, forensic laboratories may be unwilling
to accept samples because they are restricted to working
in biosafety level l or 2 facilities. In order for sam-
2005 ABSA Service Award Recipients
ples to be analyzed safely and expeditiously to the forensic
laboratories, all traces of infectious or toxic biological
material must first be removed or inactivated.
In collaboration with the New York State Police
Forensics Investigation Center, a study has been
conducted to determine which decontamination
method(s) can render a letter/white powder sample
free from infectious biological agents while at the
same time preserving the integrity of forensic evidence.
Several letters seeded with Bacillus spores, representative
inks, drug surrogates, human hair or
DNA, and fingerprints were exposed to three common
decontamination methods: vaporized hydrogen
peroxide, formaldehyde and steam autoclaving. Subsequent
microbiological testing to insure spore inactivation
and concomitant forensic analysis to evaluate the
integrity of trace evidence has determined which decontamination
method will be most useful to safely
release and transfer suspect biothreat samples to forensic
laboratories for additional testing.
John H. Richardson
Special Recognition Award
J. Patrick Condreay, PhD
GlaxoSmithKline
Research Triangle Park, North Carolina
The award shall be given to an individual in
recognition of a specific contribution that has enhanced
the American Biological Safety Association and/or the
profession of biological safety.
Patrick Condreay received his undergraduate
degree in biochemistry from Rice University and his
PhD in microbiology from the University of Texas at
Austin. For over 20 years he has pursued a career in
research, studying the molecular biology of different
microorganisms.
Since first becoming an instructor at an ABSA-
Eagleson Spring Seminar Series in 1998—where he
helped teach the course “Viral Vectors and Biosafety
Considerations”—he has helped develop and taught
13 additional courses on similar topics. His ability to
convey complex scientific topics that pose increasing
challenges to biosafety professionals and his sustained
commitment has made him one of the most
prolific and sought-after teachers promoting biosafety
for ABSA in the last decade. By the close of
the Vancouver conference, he will have increased his
275

number of ABSA or ABSA-related teaching assignments
to 16 classes in 7 years, in topics of extreme
relevance and importance to ABSA members and
biosafety professionals around the globe.
Everett Hanel, Jr. Presidential Award
Robert J. Hawley, PhD
Midwest Research Institute
Frederick, Maryland
The award shall be given to an individual for outstanding
contributions to the American Biological
Safety Association by promoting the field of biological
safety and by fostering the high professional standards
of the Association’s membership.
Bob Hawley is a Senior Advisor, for the Midwest
Research Institute in Frederick, Maryland, and was
previously employed at the U.S. Army Medical Research
Institute of Infectious Diseases at Fort Detrick,
Maryland, as Biosafety Officer, and later as Chief of
the Safety and Radiation Protection Office. He holds
276
2005 ABSA Service Award Recipients
Thanks to the Elizabeth R. Griffin Research Foundation
a PhD in Microbiology from the College of Medicine
and Dentistry of New Jersey; an MS in Microbiology
from the Catholic University of America, and a BS in
Biology from Pennsylvania Military College.
He has authored numerous journal articles and
textbook chapters on biosafety and microbiology,
and further promotes biosafety by teaching preconference
courses, serving on ABSA committees
and participating in IBCs and IACUCs. He is a Registered
Biological Safety Professional serving as the
Chair of the Biological Safety Examination Development
Committee with the National Registry of Microbiology,
and actively encourages biosafety practitioners
to pursue registration and certification as
Biosafety Professionals. He has expressed his commitment
to the biosafety profession in many ways to
include being elected President-Elect of ABSA, President
of ChABSA, Editorial Review Board member
for Applied Biosafety, and as an active promoter of
biosafety in ASM.
The ABSA Council would like to thank the Elizabeth R. Griffin Research Foundation for its continued
support of ABSA activities and the field of biosafety. Through the generous support of the Foundation,
ABSA was fortunate to have Benjamin J. Weigler, DVM, MPH, PhD, as the Elizabeth R. Griffin Research
Foundation Lecturer at the 2005 Conference in Vancouver. Dr. Weigler is the Director of Animal Health
Resources at the Fred Hutchinson Cancer Research Center. His
presentation was titled “A National Survey of Occupationally
Acquired Zoonotic Diseases in Laboratory Animal Workers.” He has
published an article on this research. See Weigler, B. J., Di Giacomo,
R. F., & Alexander, S. (2005, April). A national survey of laboratory
animal workers concerning occupational risks for zoonotic diseases.
[Journal Article]. Comparative Medicine, 55(2), 183-191.
ABSA is also developing an animal biosafety training DVD with
the support of the Foundation. We are grateful to have the
opportunity to work with the Elizabeth R. Griffin Research
Foundation in pursuit of safe science.

ABSA News
New ABSA Members for 2006
Natalie Barnett
Sandia National Laboratories
Albuquerque, NM
Malcolm Barth
Loyola University—Chicago
Chicago, IL
Shelley Belford
Animal Resources Centre, Univ. Health Network
Toronto, Ontario, Canada
Arlisa Benford
University of Houston
Houston, TX
Bill Biebesheimer
Steris Corp.
Rosemount, MN
Asa Bjorndal
Swedish Inst. for Infectious Disease Control
Solna Sweden
Sylvie Blondelle
The Burnham Institute
La Jolla, CA
Mudu Ler Bok San
Kowa Lab Engineering Pte. Ltd.
Singapore, Singapore
Jean-Luc Boudreau
SFBC Anapharm
Ste-Foy, Quebec, Canada
Tiffany A. Brigner
Colorado Department of Agriculture
Denver, CO
Neil Chin
BC Centre for Disease Control
Vancouver, British Columbia, Canada
Benton Daw
East Carolina University
Greenville, NC
Neville Debattista
U of M
Mosta, Malta
Tina M. Ennis
United States Army
APO, AE
Rowelle Enriquez
UCLA Environment Health & Safety
Los Angeles, CA
Jean Francois
University of British Columbia
Vancouver, British Columbia, Canada
Sabrina Frank-DeBose
Constella Group
Snellville, GA
Daniel Frasier
Cornerstone Commissioning, Inc.
Boxford, MA
Claire Fritz
Steris Corporation
Lakewood, CO
Joshua Jenkins
Constella Health Sciences
Stockbridge, GA
277

Stephane Karlen
Swiss Federal Institute of Technology
Lausanne, VD, Switzerland
William R. Lonergan
SAIC-Frederick, Inc.
Frederick, MD
Bruce K. McDowell
Lawrence Livermore National Laboratory
Livermore, CA
Paul Mehta, MD
Constella Group, LLC
Lawrenceville, GA
Janet E. Meszaros
Steris Corp.
Mentor, OH
Thomas Miller
Constella Health Sciences
Atlanta, GA
Matt Mitchell
Steris Corp.
Mentor, OH
Michael Penn
Boston University
Boston, MA
Emily E. Pullins
USDA APHIS BRS
Riverdale, MD
Emily Ranken
MIT
Cambridge, MA
278
New ABSA Members for 2006
Jamie Stalker, MD
Argonne National Laboratory
Argonne, IL
Clarence Stanley, Jr.
Department of Army
Ft. Gordon, GA
Jan Vleck
Western Institutional Review Board
Olympia, WA
Michael Wheatley
City of Hope
Duarte, CA
New Student Members
Donato Aceto
Sandia National Laboratories
Albuquerque, NM
Changhua Chen
University of Iowa Hospitals and Clinics
Iowa City, IA
New Corporate Memberships
UAB-Comite de Bioseguretat
Bellaterra, Barcelona
Pere Ysern Comas
Sebastian Calero Garnica
Josep Santalo Pedro
New Members of Existing Corporations
Mike Brueggerhoff
Siemens Building Technologies
Norcross, GA

ABSA News
2005 ABSA Conference Sponsors
We would like to thank the following sponsors of
the 2005 ABSA Conference in Vancouver.
Featured Sponsor
Elizabeth R. Griffin Research Foundation—
www.ergriffinresearch.org
The Elizabeth R. Griffin Research Foundation is
a 501(c)(3) non-profit foundation dedicated to the
support of professional scientific and educational
organizations that endeavor toward the common
good of humankind. This includes, but is not limited
to, supporting research that aims at the solution
of human health and societal problems and supporting
worker safety training in dealing with nonhuman
primates and other animal subjects. The
Foundation has an expressive interest in research of
the macaque-borne B virus and the prevention of
human exposure to B virus.
Bronze Sponsors
Germfree, Inc.—www.germfree.com
Germfree Laboratories, Inc. was founded in
1962. From its inception, Germfree’s primary objective
has been to manufacture biological safety equipment
for the scientific research and healthcare fields.
Germfree systems have been purchased by more
than 5,000 institutions and companies in the U.S.
and 60 other countries worldwide. Our equipment
has even gone in space.
NuAire, Inc.—www.nuaire.com
NuAire has been universally recognized as a
leader for more than 30 years in providing laboratory
professionals with reliable products such as biological
safety cabinets, CO2 incubators, Laminar
Airflow equipment, animal facility products, and
ultra-low temperature freezers for the most demanding
environments.
The Baker Company—www.bakerco.com
The Baker Company manufactures safety cabinets,
clean benches, and fume hoods. Baker cabinets
are designed for dependability, high performance
and low life cycle costs.
Certek, Inc.—www.certekinc.com
Certek builds modular BL3 laboratories,
safemod/safescan filters for hazardous exhausts, formaldehyde
generators/neutralizers and other contaminations
control equipment.
National Select Agent Registry (NSAR) Sponsored
by CDC and APHIS—www.cri-solutions.com
The National Select Agent Registry (NSAR) is a
system jointly developed by APHIS and CDC to allow
Entities that possess or wish to possess Select
Agents to register or amend their registration
online—eliminating the need to use cumbersome
paper forms. NSAR will be available for use in December
2005.
Siemens—www.sbt.siemens.com
As a leading provider of building controls, fire
safety and security system solutions, Siemens Building
Technologies, Inc., makes buildings comfortable,
safe, productive and less costly to operate. The company
focuses on improving the performance of its
customers’ buildings, so that its customers can focus
on improving their business performance. With U.S.
headquarters in Buffalo Grove, Illinois, Siemens
Building Technologies employs 7,500 people and
provides a full range of services and solutions from
more than 100 locations coast-to-coast. Worldwide,
the company has 29,000 employees and operates in
more than 42 countries.
H.E.P.A. Filter Services Inc./Design Filtration Inc.—
www.hepafilterservices.com
www.designfiltration.com

Calendar of Events
280
ABSA News
October 15-18, 2006
American Biological Safety Association (ABSA) 49th Annual Conference
Marriott Copley Hotel, Boston, Massachusetts
Contact: Phone: 847-949-1517, Fax: 847-566-4580, E-mail: absa@absa.org, Web Site: www.absa.org
October 7-10, 2007
American Biological Safety Association (ABSA) 50th Annual Conference
Opryland Hotel, Nashville, Tennessee
Contact: Phone: 847-949-1517, Fax: 847-566-4580, E-mail: absa@absa.org, Web Site: www.absa.org
October 19-22, 2008
American Biological Safety Association (ABSA) 51st Annual Conference
John Ascuaga’s Nugget, Reno, Nevada
Contact: Phone: 847-949-1517, Fax: 847-566-4580, E-mail: absa@absa.org, Web Site: www.absa.org
ABSA ITEMS FOR SALE
Pin $5.00
Watch $30.00
Men’s Women’s
Total Enclosed: $ Charge my: VISA MasterCard American Express
Card #:
Exp. Date: Signature:
Ship to Name:
Address:
City: State: Zip:
Mail to: ABSA, 1202 Allanson Road, Mundelein, Illinois 60060-3808 or fax your credit card order to
847-566-4580. Please allow two to three weeks for delivery.

Applied Biosafety is the Journal of the American Biological
Safety Association and publishes articles on the research, theory,
and applied practice of biological safety. An expert panel of International
Editors representing ABSA Canada, Associação Nacional de Biossegurança
(ANBio), Asia Pacific Biosafety Association (APBA), European Biological
Safety Association (EBSA), and the International Biosafety Working Group
(IBWG) contribute a global perspective on biosafety and biosecurity. As a
service to the scientific community, online subscriptions are available at cost –
$25/year.
A free preview of Applied Biosafety, Volume 10, Number 1, is at
www.absa.org/resabj.html; for userID, type in “abj273” with password “8f9T74”

ABSA Announces Professional Publications for Purchase
Anthology of Biosafety I:
Perspectives on
Laboratory Design
❑ Member: $41.00
❑ Nonmember: $55.00
Anthology of Biosafety V:
BSL-4 Laboratories
❑ Member: $53.00
❑ Nonmember: $67.00
Anthology of Biosafety II:
Facility Design
Considerations
❑ Member: $42.00
❑ Nonmember: $57.00
Anthology of Biosafety VI:
Arthropod Borne Diseases
❑ Member: $39.00
❑ Nonmember: $52.00
Anthology of Biosafety III:
Application of Principles
❑ Member: $49.00
❑ Nonmember: $64.00
Anthology of Biosafety VII:
Biosafety Level 3
❑ Member: $44.00
❑ Nonmember: $57.00
ADD POSTAGE/HANDLING: U.S. – $3.00 per book; Canada – $5.00 per book; Foreign – $15.00 per book
Name __________________________________________ Member ID# ___________________________________________
Address _____________________________________________________________________________________________________
City/State/Zip ________________________________________________________________________________________________
Total ___________________________ ❑ Check enclosed ❑ Please charge my: ❑ Visa ❑ MasterCard ❑ AmEx
Card # ________________________ Exp. Date ________________________ Signature ____________________________
Mail or fax this order form to:
American Biological Safety Association (ABSA)
1202 Allanson Road, Mundelein, IL 60060-3808
Phone: 847-949-1517 • Fax: 847-566-4580
Anthology of Biosafety IV:
Issues in Public Health
❑ Member: $42.00
❑ Nonmember: $55.00
Anthology of Biosafety VIII:
Evolving Issues in
Containment
❑ Member: $36.00
❑ Nonmember: $49.00
NOTE: Please allow 2-3 weeks for delivery. If using a credit card, please mail or fax the form as we will need a signature on file.
We do not accept purchase orders.

www.absa-canada.org
Please visit our ABSA Chapters, Affiliates, and Affiliated
Organizations for more biosafety information and news.
www.anbio.org.br
www.socalbionet.org
biosafety@comcast.net www.duke.edu/~alder002 www.chabsa.org
www.ebsa.be
EGilman@absa.org
www.mabsa.org www.sebsa.net
Japanese Biological Safety Association (JBSA)
(Note: JBSA affiliate status is in process.)
ksugi@nih.go.jp

Guidelines for Submissions
Authors will be acknowledged of submission receipt by the ABSA National Office. Final decisions regarding publication are
made by the Editorial staff and reviewers. The following are the submission guidelines.
Submission Categories
Articles, Reviews, and Summary Articles—Reviews may focus on the theory, practice, and overarching areas relevant to
biological safety, biosecurity or related areas. Articles must include an abstract not to exceed 250 words summarizing the
main topic of the article. Typically articles do not exceed 20 pages in total length.
Reports—Articles that focus on the results of research are appropriate for this section. Brief reports should include information
on the research design, methods, and results. An abstract not to exceed 250 words must also be included. Articles vary in
length.
Viewpoints—Short articles focusing on personal experiences may be submitted to this section. Articles vary in length.
Book and Video Reviews—Reviews of books and media (videos/CD/other) of interest to biological safety or biosecurity
may be submitted at any time. Reviews typically do not exceed 1-2 pages in length. Books or media which authors wish to
have considered for review may be sent directly to the ABSA National Office.
Commentary/Editorial—Brief comments on submissions published in Applied Biosafety, issues critical to the profession and
practice of biological safety, or letters to the Editor may be submitted to this section. Individuals may be invited by the Editors
to submit a guest editorial article.
Presentations—Articles that recount or summarize information relevant to the field of biological or biosecurity that has
been presented at a conference. Presentation articles vary in length.
Other Requirements
1. Send an electronic submission or one (1) typeset copy with a disk of each submission to: Editor, Applied Biosafety:
Journal of the American Biological Safety Association, c/o ABSA, 1202 Allanson Road, Mundelein,
IL 60060-3808, USA. Submissions that are under consideration by another periodical or publisher or submissions
that have been previously published must be identified as such, and previous citing must be disclosed.
2. Submission guidance:
Format for 8-1/2” x 11” paper using 1” margins, double-spacing, and full-justification.
References, footnotes, table captions, and quotations should be single-spaced, a guide to references can be found at
www.absa.org.
Use Times New Roman, Arial, AvantGarde, Helvetica, or Universal font in 12 point.
Use high resolution laser printing if submission is made in hard copy media.
Primary headings should be flush left, bolded, and have the first letter of all main words capitalized throughout the
submission.
Secondary headings should be flush left, italicized, and have the first letter of all main words capitalized.
3. Submissions should follow ASM guidelines regarding fundamental style and ethics--refer to Applied and Environmental Microbiology
at http://aem.asm.org.
4. The Attention Authors Form may serve as a cover sheet with the full name(s) and degree(s) of the author(s), professional
affiliations, and the return mailing address of the author to whom correspondence can be sent. Authors’ names, positions,
titles, and places of employment should not appear in the body of the paper to facilitate the blind review process.
5. Tables, charts, photographs (at least 3-1/2” x 5”) or diagrams must be computer-generated or professional quality and
submitted as camera ready artwork. Tables, charts, or diagrams should be submitted on a separate page, referenced back
to the text in a vertical (portrait) format including any legend, label, or number associated with them. Refer to each as
Table 1, Table 2, etc., centered above the table. Captions should be single-spaced.
6. It is the author’s responsibility to secure written permission from the original copyright holder to use quotations of over
300 words from one source or use adaptation of tables or figures from copyrighted sources. A copy of the copyright
holder’s written permission must be provided to the Editor immediately upon acceptance of the submission for publication.
The author(s) bear full responsibility for the accuracy of all results, references, quotations, and materials accompanying
their submissions.
7. In the event a diskette it used, it should be prepared on either an IBM or IBM-compatible computer. All submissions
should be formatted using either: Microsoft Word, Microsoft Publisher, or WordPerfect. ASC II files are also acceptable.

Attention Authors
Please complete the following information and include with your submission.
Name
Degrees/Credentials
Affiliation
Address
Phone Numbers Work Home (opt.)
E-mail Address
Type of Submission (check one):
Report Article, Review, Summary Article Viewpoint
Book Review Video Review Presentation Commentary/Editorial
Title of Submission
Checklist
Electronic submission or one (1) hard copy (typeset using 8-1/2” x 11” paper with 1” margins).
Electronic submission or one (1) hard copy original of tables, figures, and/or illustrations.
Submission follows ASM guidelines regarding fundamental style and ethics.
Abstract of approximately 250 words (for Articles, Reviews, and Summary Articles only).
This Attention Authors form.
Author’s Signature Date
Please send the completed form along with your submission to: Editor, Applied Biosafety: Journal of the American
Biological Safety Association, 1202 Allanson Road, Mundelein, IL 60060-3808, USA. Electronic submissions may be
e-mailed to: Production Editor, Karen D. Savage, at ksavage@covad.net. If you have formatting, processing, or general
questions, please contact Ms. Savage at the ABSA National Office Monday through Friday between 9:00 a.m. and
5:00 p.m. Central Time at 847-949-1517.

Register through March 4th 2005
and SAVE $150 US
Learn more about Tradeline Inc
and register today at:
http://www.tradelineinc.com/bio2006
Questions? Call 925.254.1744 x12
Tradeline Inc.
Biocontainment Facilities 2006
Design • Construction • Operation
Biocontainment Facilities 2006 Conference
Design • Construction • Operation
March 27-28, 2006
Renaissance Vinoy Beach and Golf Resort
St. Petersburg, Florida
Here you’ll get the details on:
• Developing efficient BSL-3 solutions for animal facilities
• Sorting out certification, validation, commissioning and design guideline issues
• Getting the design and construction details right
• Benchmarking costs for new construction upgrades and conversions
• Planning for BSL facilities operations and maintenance
• New lab types - Bioaerosol, Bird Flu, Advanced Imaging in BSL environments
Credit: CUH2A/George Ely
Courtesy of Smith Carter Architects and Engineers Inc.