1. Biosafety- working Safely in a BSL-3 Laboratory
Parampal Deol, Ph.D, MBA, Director R&D Microbiology
Anne Hutchins, Ph.D, Associate Staff Scientist

2. Agenda Biosafety: Principles/concepts Areas of Biosafety
Biological Risk Groups
Standards
Types of Biosafety Laboratories
Engineering controls
Best practices
Operational Controls
Administrative controls
BSL 3 laboratory in bMX
Select Agents

3. Biological Safety
Biosafety - the discipline addressing the safe handling & containment of infectious microorganisms & hazardous biological materials
application of knowledge & the use of appropriate techniques & equipment
prevent personal, laboratory & environmental exposure to potentially infectious agents or biohazards
Source: BMBL, 5th Ed.

4. Areas of Biosafety Laboratory Safety
Bloodborne pathogens (BBP) & other infectious agents
Recombinant DNA (rDNA)
Biological waste disposal
Infectious substance shipping
Respiratory Protection (TB)
Bioterrorism & select agents/toxins
Mold & indoor air quality
Occupational safety & health in the use of research animals

5. Hazard Classifications of Microbial Agents
Classification is based on Human Disease
Risk Group 1: Agents not associated with disease
K12 strain of E. coli
Risk Group 2: Agents associated with disease
rarely serious & preventive / therapeutic interventions are often available
Salmonella species, HIV, Listeria monocytogenes
Risk Group 3: Agents associated with serious or lethal disease
preventive/ therapeutic interventions may be available
Mycobacterium tuberculosis, Eastern Equine Encephalitis (EEE) Virus, Bacillus anthracis
Risk Group 4: Agents likely to cause serious or lethal disease
preventive or therapeutic interventions are not usually available
Ebola virus, Lassa virus, Variola virus (smallpox)

6. Laboratory Design and Operational Practices
Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th edition
Centers for Disease Control and Prevention (CDC)
National Institute of Health (NIH)
Federal Select Agent Program (FSAP)
World Health Organization (WHO)
United States Department of Agriculture (USDA)

7. Regulations & Guidelines
Biosafety in Microbiological and Biomedical Laboratories, 5th Edition, CDC/NIH (BMBL)
NIH Guidelines for Research Involving Recombinant DNA Molecules, April 2002 as amended
Occupational Safety and Health Administration (OSHA) Bloodborne Pathogens Standard 29 CFR
Respiratory Protection including Tuberculosis 29 CFR (independent standard repealed)
State regulations concerning biological waste disposal
IATA Dangerous Goods Regulations, DOT 49 CFR , & USPS 39 CFR Part 111
Select Agents and Toxins Regulations (HHS/CDC 42 CFR 73; USDA/APHIS 7 CFR 331 & 9 CFR 121)

8. Biosafety: Where to Start?
Your laboratory wants to start working with an infectious agent and you need to have a lab space or renovate current lab.
How do you decide what changes need to be made to the current space to safely handle the bacteria.
What are the guidelines?

9. Biosafety Measures Illustrated
Administrative Controls
Training
Operations
Maintenance
Risk Assessment
Emergency Response
Plans
Lab Inspections
Biosecurity
Biosafety Manual
SOPs
Medical Surveillance
Respiratory Protection
Inventory
Record Keeping
Microbiological
Practices
Standard
Special
Safety Equipment
(Primary Barriers)
PPE
BSCs
Facility Design
(Secondary Barriers)

10. Principles of Biosafety
Containment
microbiological practices
safety equipment
facility safeguards
protect laboratory workers, the environment & the public from exposure to infectious agents that are handled & stored in the laboratory
Risk assessment
process
enables the appropriate selection of microbiological practices, safety equipment & facility safeguards that can prevent laboratory-associated infection
The backbone of the practice of biosafety is risk assessment
WHO Biosafety Manual, 3rd ed.

11. Biological Containment
Laboratory Practice & Technique
Adherence to standard microbiological practices & techniques
Awareness of hazards & training
Biosafety or laboratory Standard Operating Procedures (SOPs)
Safety Equipment (Primary Barriers)
Biological safety cabinets (BSC)
Safety centrifuge cups/sealed rotor heads
Personal protective equipment (PPE)
lab coats, gowns, gloves, safety glasses, face shields, shoe covers, respirators, Tyvek suit
Facility Design & Construction (Secondary Barriers)
Restricted Access from public
Autoclave
Directional airflow
HEPA filter units
Airlocks as lab entrances

12. Biological Containment
Biological Safety Cabinets (BSC) Class I, Class II & Class III

13. Biological Safety Level - 1 (BSL-1)
Standard microbiological practices:
Hand washing sink
No eating, drinking, smoking, applying cosmetics, handling contact lenses, or storing food
No mouth pipetting
Safe handling of sharps
Decontaminate work surfaces after completion of work, & any spill or splash; any waste generated
Biohazard symbol when infectious agents present
Wear lab coats or gowns, protective eye wear, gloves
Windows to exterior have screens
WHO Biosafety Manual 3rd Ed., 2004

14. Biological Safety Level – 2 (BSL-2)
All of BSL-1 practices plus:
Access is restricted to trained personnel
Lab doors should be self-closing & have locks
BSC used when potential for aerosol production
Sealed centrifuge cups/rotors
Eyewash station must be readily available
Method for decontaminating laboratory waste should be available in the facility
WHO Biosafety Manual 3rd Ed., 2004

15. Biological Safety Level – 3 (BSL-3)
All of BSL-2 practices plus:
Lab is separated from unrestricted traffic flow
Access to lab is restricted to entry through a series of 2 self-closing doors (ante-room)
All manipulations must be conducted in a BSC, other containment, or when wearing proper PPE
Protective clothing with a solid front (tie-back or wrap-around gowns, scrubs, coveralls) is worn
Eye, face, & respiratory protection may be worn
Hands-free sink near exit door
WHO Biosafety Manual 3rd Ed., 2004

16. Biological Safety Level – 4 (BSL-4)
All of BSL-3 practices plus:
Generally a separate facility; dedicated mechanical, electrical, plumbing, HVAC
Agents handled in Class III BSC or with one-piece positive pressure personnel suit
Shower out practices
Lab clothing decontaminated prior to laundering
Liquid effluent decontaminated prior to release to sewer
Redundant supply fans recommended, redundant exhaust fans required, interlocked supply & exhaust
CDC workers in BSL-4

17. Biological Safety Level - 3 Laboratory Design
Containment, ability to clean
Full containment
Controlled access
Surfaces design for cleaning
Sustained directional (negative) airflow
Visual monitoring device at entry, gown in and prior to entrance in lab space
Utilities
Lab air supply and exhaust fans with HEPA
HEPA filters certified and replaced as needed
Utilities (exhaust) are separated
Decontamination/Spill Management
Waste disposal autoclave within lab
Equipment (surface)
Spill cleaning

18. BSL-3 in at bioMerieux in Durham
Emergency Response
Natural Disasters
Tornado warnings
Transport
Import/export of cultures
Operational SOPs/GMP
All records electronic, if possible
Medical Surveillance
Sustainability and permits
Annual certification

19. Select Agent Regulations
What are the Select Agents?
Biological agents /toxins pose a severe threat to human health
65 agents and toxins
Reconstructed 1918 Influenza virus
Ricin toxin
Ebola virus *
Bacillus anthracis*
Francisella tularensis*
* denotes Tier 1
Established to keep the agents/toxins out of possession of individuals who may misuse them
USA Patriot Act
Public Health Security and Bioterrorism Preparedness and Response Act of 2002
The Select Agent Regulations (7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73)
2001 Anthrax Letter
22 illnesses
5 deaths
Cost $23 million

20. Who Can Possess Select Agents?
Individuals
FBI security clearance
Entities
Facility Design Requirements
Biosafety Plan
Security Plan
Physical, control of select agents, inventory, and information system
Incident Response Plan
Natural Disaster events
Failures- spills, equipment failures, unsecured containment, loss of power
Need to Train and SA requires annual drills

21. Tier 1 Select Agents
Greatest risk of deliberate misuse for mass casualties or devastating effect to the economy, critical infrastructure, or public confidence
Requires additional measures such as:
personnel suitability assessment
occupational health program
visitor procedures
response time of security forces within 15 minutes
incident response procedures
annual training on reporting suspicious behavior
> 3 physical security barriers
intrusion detection system (IDS) with backup during power failure

22. Non-Tier 1 (Clinical laboratories)
Can still test for and identify a Tier 1 agent
Can not ‘possess’ a Tier 1 agent
Have 7 days to transfer or destroy a Tier 1 agent
State Public Health Laboratories
Laboratory Response Network (LRN)
LRN Network (