Principles of Biosafety

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Principles of Biosafety

• IBC
• Laboratory Safety
• Education Module

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Introduction

• It is the policy of North Carolina AT State
  University that all research involving
  biohazardous agents be reviewed and approved by a
  Biosafety Committee in accordance with
  requirements set forth in the National Institutes
  of Health "Guidelines for Research Involving
  Recombinant DNA Molecules, Occupational Safety
  Health Association (OSHA) "Occupational Exposures
  to Hazardous Chemicals in Laboratories" (referred
  to as the "Laboratory Standard") and the Center
  for Disease Control (CDC).

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Introduction

• Although the "Laboratory Standard" does not refer
  to biological agents, the policy of this
  University is to apply the same basic
  requirements and responsibilities for training,
  Laboratory Safety Plan, and reporting of
  accidents/exposures, to biological agents
• This policy applies to research that
  is sponsored by the University conducted by
  employees or agents of the University or
  conducted using the University's property,
  facilities, students or non-public
  informationThe University is responsible for
  ensuring that research and teaching activities
  are carried out in a manner that protects
  students, University employees, and the community.

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Introduction

• The Institutional Biosafety Committee has
  developed this
• education module to ensure that faculty, staff,
  and students at
• North Carolina Agricultural and Technical
  State University who
• are working with infectious agents and
  recombinant DNA are
• knowledgeable regarding the guidelines set by
  the National
• Institutes of Health (NIH), the Center for
  Disease Control (CDC),
• and the University for such research.

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Target Audience

• Principal Investigators
• Students
• Research Laboratory Staff
• (Includes research assistants/technicians)

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Module Features

• Pre-Test/Test Out Feature
• Faculty PIs only
• 95 passing rate
• Post-Test
• 80 passing rate
• Certification

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Learning Objectives

• Upon completion of the education modules,
  participants will be able to
• Practice safety in the laboratory environment
• Understand resources available for maintaining
  research compliance in the laboratory environment
• Understand what agents can be studied at NC AT
  and the requirements for working with agents
  based on their assigned biosafety level
• Be aware of the principles of the Patriot Act
  relating to research with infectious agents
  included on the select agents list.
• http//www2.ncsu.edu/ncsu/ehs/www99/left/bioSafe/f
  orms/PATRIOT.pdf

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Target Audience Required Modules

• Principal Investigators
• Research Laboratory Assistants
• Summer Research Associates

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Principles of Biosafety

• The term "containment" is used in describing safe
  methods for managing infectious materials in the
  laboratory environment where they are being
  handled or maintained. The purpose of containment
  is to reduce or eliminate exposure of laboratory
  workers, other persons, and the outside
  environment to potentially hazardous agents.

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Containment

• Primary containment
• The protection of personnel and the immediate
  laboratory environment from exposure to
  infectious agents is provided by both good
  microbiological technique and the use of
  appropriate safety equipment. The use of vaccines
  may provide an increased level of personal
  protection.

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Principles of Biosafety

• Secondary containment
• The protection of the environment external to
  the laboratory from exposure to infectious
  materials is provided by a combination of
  facility design and operational practices.

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Principles of Biosafety

• The three elements of containment include
• laboratory practice and technique
• safety equipment
• and facility design.
• The risk assessment of the work to be done with a
  specific agent will determine the appropriate
  combination of these elements.
• The Principal Investigator is responsible for
  performing the laboratory risk assessment

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Primary Barriers

• Safety Equipment
• Safety equipment includes biological safety
  cabinets (BSCs), enclosed containers, and other
  engineering controls designed to remove or
  minimize exposures to hazardous biological
  materials. The biological safety cabinet (BSC) is
  the principal device used to provide containment
  of infectious splashes or aerosols generated by
  many microbiological procedures.

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Looks Matter

• Biological safety cabinets can only protect the
  worker and the experiment if they have been
  properly selected for the intended containment
  function. Selection is dependent on
• The hazard classification of the agent
• The need for protection of research material or
  personnel
• The extent to which hazardous aerosols are
  involved
• There are three basic types of biological safety
  cabinets. Class I, Class II, and Class III.

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BSC Class 1

• Designed as a partial containment cabinet
  offering adequate personnel protection but no
  product or experiment protection. Cross
  contamination may result from contaminated air
  flowing over the work area.
• NIH recommends the use of this type cabinet for
  low to moderate risk infectious biological gents
  (CDC Class 1,2).
• For adequate personnel protection, the front
  opening of the cabinet, through which the worker
  operates, should be approximately 8 inches.
• Air velocity through this opening must be a
  minimum of 75 linear feet per minute (lfpm).

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BSC Class II

• Class II cabinets are for procedures that
  generate aerosols, such as sonicating and
  blending, with low-risk oncogenic viruses, and
  for all procedures with moderate-risk oncogenic
  viruses and Class 3 etiologic agents
• A laminar flow safety cabinet developed to
  protect the worker, the research material and
  environment. The essential components of Class II
  cabinets are
• High efficiency particulate air (HEPA)
  filtered-recirculated airflow
• Front open access
• Downward clean air flow
• Work access opening air flow
• HEPA filtration of the exhaust air
• .
• There are two types of Class II cabinets, Type A
  and Type B.

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BSC Class II/Type A

• Type A cabinets recirculates approximately 70 of
  the total cabinet air.
• Do not use Type A cabinets with flammable
  solvents, toxic agents or radioactive material.

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BSC Class II/Type B

• Type B cabinets exhaust 70 of the air flowing
  through the work area, thereby allowing a wider
  range of chemicals that can be used.
• Type B cabinets are not recommended for use with
  explosive vapors.

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Keep It In Check

• The certification procedure should include
• Halogen leak test to insure the positive pressure
  air flow plenums are gas tight
• Measurement of air inflow velocity
• Measurement of the airflow within the cabinet to
  assure it is uniform and unidirectional
• A leak test of the HEPA filter to verify proper
  installation and that it is leak-free

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PPEs

• Safety equipment also may include items for
  personal protection, such as gloves, coats,
  gowns, shoe covers, boots, respirators, face
  shields, safety glasses, or goggles.
• Personal protective equipment is often used in
  combination with biological safety cabinets and
  other devices that contain the agents, animals,
  or materials being handled.
• In some situations in which it is impractical to
  work in biological safety cabinets, personal
  protective equipment may form the primary barrier
  between personnel and the infectious materials.

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Short Quiz
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Biosafety Levels
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Biosafety Level 1

• Suitable for experiments, involving agents
  of no known or of minimal potential hazard to
  laboratory personnel and the environment. The
  laboratory is not separated from the general
  traffic patterns of the building. Work is
  generally conducted on open bench tops. Special
  containment equipment is not required or
  generally used. Laboratory personnel have
  specific training in the procedures conducted in
  the laboratory, and are supervised by a scientist
  with general training in microbiology or a
  related science.

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Biosafety Level 1

• Suitable for experiments involving
• Microorganisms classified as Risk Group I. These
  organisms present minimal or no biohazard risk to
  healthy workers under ordinary handling
  conditions.
• Recombinant DNA activity that requires BL1
  containment including animal studies that involve
  the construction of transgenic animals. (If you
  wish to review the Guidelines developed by NIH
  for RDNA click here.
• Non-recombinant cell or tissue culture studies
  that do not involve infectious plant or animal
  virus.
• Management of animal populations that are free of
  zoonotic organisms or are not part of a study
  which involves organisms or chemical substances
  which may require higher containment
  considerations.
• Microorganisms in this class include Escherichia
  coli K12, Saccharomyces cerevisiae, Bacillus
  subtilis, Neurospora crassa and non primate cells.

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Biosafety Level 1

• Standard Microbiological Practices

• Access to the laboratory is limited or restricted
  at the discretion of the laboratory director when
  experiments or work with cultures and specimens
  are in progress.
• 2. Persons wash their hands after they handle
  viable materials, after removing gloves, and
  before leaving the laboratory.
• 3. Eating, drinking, smoking, handling contact
  lenses, applying cosmetics, and storing food for
  human use are not permitted in the work areas.
  Persons who wear contact lenses in laboratories
  should also wear goggles or a face shield. Food
  is stored outside the work area in cabinets or
  refrigerators designated and used for this
  purpose only.

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Biosafety Level 1

• Standard Microbiological Practices

4. Mouth pipetting is prohibited mechanical
pipetting devices are used. 5. Policies for the
safe handling of sharps are instituted. 6. All
procedures are performed carefully to minimize
the creation of splashes or aerosols. 7.
Work surfaces are decontaminated at least once a
day and after any spill of viable material.
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Biosafety Level 1

• Standard Microbiological Practices

8. All cultures, stocks, and other regulated
wastes are decontaminated before disposal
by an approved decontamination method such as
autoclaving. Materials to be
decontaminated outside of the immediate
laboratory are to be placed in a durable,
leak proof container and closed for transport
from the laboratory.
Materials to be decontaminated
outside of the immediate laboratory are packaged
in accordance with
applicable local, state, and federal regulations
before removal from the facility. 9.
A biohazard sign can be posted at the entrance to
the laboratory whenever infectious agents
are present. The sign may include the name of the
agent(s) in use and the name and phone
number of the investigator. 10.
An insect and rodent control program is in effect

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Biosafety Level 2

Similar to Biosafety Level 1, is suitable for
work involving agents of moderate potential
hazard to personnel and the environment. It
differs from BSL-1 in that (1) laboratory
personnel have specific training in handling
pathogenic agents and are directed by competent
scientists (2) access to the laboratory is
limited when work is being conducted (3)
extreme precautions are taken with contaminated
sharp items and (4) certain procedures in
which infectious aerosols or splashes may be
created are conducted in biological safety
cabinets or other physical containment
equipment.
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Biosafety Level 2

• Activities include
• Microorganisms of low biohazard potential such as
  those in Risk Group or Biosafety Level 2. (Click
  here for a list of agents)
• Primate cells and/or tissue culture systems.
• Recombinant DNA research requiring BL2 physical
  containment. Oncogenic viral systems research
  classified under low or moderate risk.

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Biosafety Level 2

• Standard Microbiological Practices

Same as Biosafety Level 1 Special
Practices 1. Access to the laboratory is
limited or restricted by the laboratory director
when work with infectious agents is in
progress. In general, persons who are
at increased risk of acquiring
infection, or for whom infection may have serious
consequences, are not
allowed in the laboratory or animal rooms.
For example, persons who are immunocompromised or
immunosuppressed may be at
increased risk of acquiring infections. The
laboratory director has the final
responsibility for assessing each circumstance
and determining who may
enter or work in the laboratory or animal room.
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Biosafety Level 2
Special Practices 2. The
laboratory director establishes policies and
procedures whereby only persons
who have been advised of the potential hazards
and meet specific entry
requirements (e.g., immunization) may enter the
laboratory. 3.
A biohazard sign must be posted on the
entrance to the laboratory
when etiologic agents are in use. Appropriate
information to be posted includes
the agent(s) in use, the biosafety level, the
required immunizations, the
investigator's name and telephone number, any
personal protective equipment
that must be worn in the laboratory,
and any procedures required for exiting
the laboratory.
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Biosafety Level 2
Special Practices 4. Laboratory personnel
receive appropriate immunizations or tests
for the agents handled or potentially present in
the laboratory (e.g., hepatitis B
vaccine or TB skin testing). 5. When
appropriate, considering the agent(s) handled,
baseline serum samples for
laboratory and other at-risk personnel are
collected and stored. Additional
serum specimens may be collected periodically,
depending on the agents handled or the
function of the facility.
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Biosafety Level 2

• Special Practices
• Biosafety procedures are incorporated into
  standard operating
• procedures or in a biosafety manual adopted
  or prepared specifically
• for the laboratory by the laboratory
  director. Personnel are advised of
• special hazards and are required to read
  and follow instructions on
• practices and procedures.

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Biosafety Level 2
Special Practices 7. The laboratory
director ensures that laboratory and support
personnel receive appropriate training on
the potential hazards associated with
the work involved, the necessary precautions to
prevent exposures, and the exposure
evaluation procedures. Personnel
receive annual updates or additional training as
necessary for procedural or policy
changes.
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Biosafety Level 2
Special Practices-Sharps 8. Needles and
syringes or other sharp instruments should be
restricted in the laboratory for use only when
there is no alternative, such as parenteral
injection, phlebotomy, or aspiration of fluids
from laboratory animals and diaphragm bottles.
Plasticware should be substituted for glassware
whenever possible.
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Biosafety Level 2
Special Practices-Sharps Only
needle-locking syringes or disposable
syringe-needle units (i.e., needle is integral to
the syringe) are used for injection or aspiration
of infectious materials. Used disposable needles
must not be bent, sheared, broken, recapped,
removed from disposable syringes, or otherwise
manipulated by hand before disposal rather, they
must be carefully placed in conveniently located
puncture-resistant containers used for sharps
disposal. Non-disposable sharps must be placed in
a hard-walled container for transport to a
processing area for decontamination, preferably
by autoclaving.
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Biosafety Level 2
Special Practices-Sharps Syringes which
re-sheathe the needle, needleless systems, and
other safety devices are used when
appropriate. Broken glassware must not be
handled directly by hand, but must be removed by
mechanical means such as a brush and dustpan,
tongs, or forceps. Containers of contaminated
needles, sharp equipment, and broken glass are
decontaminated before disposal, according to any
local, state, or federal regulations.
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Biosafety Level 2
Special Practices 9. Cultures, tissues,
specimens of body fluids, or potentially
infectious wastes are placed in a container with
a cover that prevents leakage during collection,
handling, processing, storage, transport, or
shipping.
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Biosafety Level 2
Special Practices 10. Laboratory equipment and
work surfaces should be decontaminated with an
effective disinfectant on a routine basis, after
work with infectious materials is finished, and
especially after overt spills, splashes, or
other contamination by infectious materials.
Contaminated equipment must be decontaminated
according to any local, state, or federal
regulations before it is sent for repair or
maintenance or packaged for transport in
accordance with applicable local, state, or
federal regulations, before removal from the
facility.
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Biosafety Level 2
Special Practices 11. Spills and accidents that
result in overt exposures to infectious
materials are immediately reported to the
laboratory director. Medical evaluation,
surveillance, and treatment are provided as
appropriate and written records are
maintained. 12. Animals not involved in the
work being performed are not permitted in the
lab.
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Biosafety Level 3
Applicable to clinical, diagnostic,
teaching, research, or production facilities in
which work is done with indigenous or exotic
agents which may cause serious or potentially
lethal disease as a result of exposure by the
inhalation route. Laboratory personnel have
specific training in handling pathogenic and
potentially lethal agents, and are supervised by
competent scientists who are experienced in
working with these agents.
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Short Quiz
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Laboratory Practice and Technique
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Knowledge is the Key

• The most important element of containment is
  strict adherence to standard microbiological
  practices and techniques. Persons working with
  infectious agents or potentially infected
  materials must be aware of potential hazards, and
  must be trained and proficient in the practices
  and techniques required to handle such material
  safely. The Principal Investigator in charge of
  the laboratory is responsible for providing or
  arranging the appropriate training of personnel.

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Knowledge is the Key

• The most important element of containment is
  strict adherence to standard microbiological
  practices and techniques. Persons working with
  infectious agents or potentially infected
  materials must be aware of potential hazards, and
  must be trained and proficient in the practices
  and techniques required to handle such material
  safely. The Principal Investigator in charge of
  the laboratory is responsible for providing or
  arranging the appropriate training of personnel.

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Resources Infectious Agents

• CDC HHS Select Agents List
• http//www.cdc.gov/od/sap/docs/salist.pdf
• ABSA Risk Group Classification
• http//www.absa.org/resriskgroup.html

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Resources Chemicals

• Chemical Reactivity Worksheet
• http//www.response.restoration.noaa.gov/chemaids/
  react.html
• Label Maker
• http//beta.ehs.cornell.edu/labels/cgi-bin/label_s
  election.pl

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Resources Animals

• Experiments Involving Transgenic Animals
• http//www.niehs.nih.gov/odhsb/manual/man7b.htm

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Resources Tests and Certifications
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CDC-Safety Survival Skills