CHAPTER 3a :

CONTROL IN BIOSAFETY

3.1 : Facility Design

3.2 : Containment Equipment – Biological Safety
Cabinets
Biocontainment Facilities
Engineering Controls (Biocontainment)
 Primary barriers (biosafety equipment)
◦ Biosafety cabinets (BSCs), Animal caging (IVCs)
◦ Specialized lab equipment (pipetting devices, waste
containers, safety centrifuge cups, fermenters, etc.)

 Secondary barriers (lab design & facilities)

◦ Building & room construction – the floor plan
◦ HVAC issues – directional airflow, filtration
◦ Waste treatment (liquid, solid, air)
◦ Access controls
◦ People and product flow
“Biocontainment Engineering”

 Biocontainment : preventing the

unintentional release of biological agents
 Engineering : is the discipline, skill, and
profession of acquiring and applying
scientific, economic, social, and practical
knowledge, in order to design and build
structures, machines, devices, systems,
materials and processes.
Biocontainment: Engineering Controls
 Primary barriers – contain the agent at the source
◦ Biological safety cabinet
◦ Animal caging
◦ Specialized lab equipment (centrifuges, fermenters, etc.)

 Secondary barriers – contain the agent within the room or facility

in case an agent escapes from the primary barriers
◦ Building & Room Construction
◦ Heating,Ventilation, Air Conditioning (HVAC)
 Directional airflow
 Exhaust filtration
◦ Other Engineering Controls: Building Room BSC
 Solid waste treatment Tube
 Wastewater treatment
 Containment Barriers
⚫ Containment laboratories are layers of open
and closed systems
⚫ So what is the containment envelope?

Building
Room
BSC
Tube
Biosafety Levels – 1, 2, 3, 4
How much containment?
Types of biocontainment facilities
 Low containment (BSL1, ABSL 1),*A = animal
 Medium containment (BSL 2, ABSL 2)
 High containment (BSL 3, ABSL 3)
 Maximum containment (BSL4, ABSL4, BSL3-
Ag)
 More engineering to achieve the higher
containment
BSL-1

WHO Biosafety
Manual 3rd Ed., 2004
BSL 1
• open bench
• easy-clean surfaces
• closed or screened windows
• hand-wash basin
• impervious, chemical resistant benchtops
• hand wash basin
• doors
BSL-2

WHO Biosafety
Manual 3rd Ed., 2004
BSL 2
BSL 1
+ signage
+ restricted access
+ self-closing lockable doors
+ eye wash and shower station*
+ Biological Safety Cabinet Class II*
+ Autoclave*
+ annual inspection/ audit
BSL-3

WHO Biosafety
Manual 3rd Ed., 2004
BSL 3
• controlled access
• directional air control and filtration
• two sets of interlocking doors
• enhanced PPEs + powered respirators
• Immunization & medical surveillance
• certification
BSL 4
• Full containment
• Full body positive pressure suits
+ air supply
• Isolated building
• Full decontamination facilites
 Examples

BSL-3 Laboratory
BSL-2 Laboratory

BSL-4 Lab & Animal

BSL-3 Animal Facility
BSL-3 AG Lab & Animal
BSL-3 Laboratory – enh.
Directional Airflow
Double Door Entry
Autoclave Available
Pass-through Autoclave
Seamless Floors
Monolithic Ceilings
HEPA Filtered Exhaust
HEPA Filtered Supply
Supply/exhaust interlock
Personnel Shower
Airlock Entry
Pressure Differential
HEPA Plumbing Vents
Effluent Decontamination
Pressure Decay Testing
Chemical Decon Shower
Breathing Air System
Engineering Features for Biosafety Levels
3.2 Containment Equipment -
BioSafety Cabinets
Biosafety and Containment
• Protection of :

➢ Personnel

➢ Environment

➢ Product
 AEROSOL GENERATION:

• Aerosol -- suspension of solid or liquid particles in a gas (air)

• Infectious aerosols are to be avoided at all costs in the laboratory
• Containment and ventilation of aerosol is a major concern
Aerosol Producing Activities
 Sonication, blending, mixing, vortexing
 Almost any liquid manipulation
 Centrifuging
 Pouring
 Pipetting
 Opening containers at non-ambient pressures,
(e.g. fermenters, freezer vial)
 Loading syringes and injections
Airflow control
 By using Air Filters and Air Pumps
 Negative pressure area
◦ Air constantly flowing into room
◦ Particles/ aerosols do not escape
◦ Keeps things IN
 Positive pressure area
◦ Air constantly flowing out of room
◦ Contaminations do not enter
◦ Keeps things OUT
 All air movement filtered thru HEPA filters
High Efficiency Particulate Air Filters
(HEPA)
HEPA Filters
DO NOT
filter
gases and vapors
they should not
be used with
chemicals and
radionuclides

 Constructed of pleated boro-silicate glass fiber paper

 Filters 99.97% of airborne particles
How do HEPA Filters Operate?
i. Inertial Impaction
ii. Interception
iii. Diffusion

◦ Filters 99.97% of particle of size 0.3 um

◦ Particles that arelarger or smaller
are filtered with higher efficiency
Particle Size Ranges

Typical aerosolized bacteria

Droplet nuclei

Fungi

Viruses Bacteria

.01 .10
0.3 1.0 10 50

microns
Filtration Efficiency

Collection Efficiency (%)

Inertial Impaction

99.97 Interception
Diffusion
Overall Efficiency
Most
viruses

Tularemia Anthrax spores

Smallpox

.01 0.1 1.0 10.0

Particle Diameter (microns)

ULPA and SULPA
Ultra Low Penetration Air (ULPA)
 Theoretically can remove 99.999% of particles of 0.12
microns.
◦ HEPA removes 99.97% of particles 0.3 microns in size
 Some manufactures are offering ULPA filters in their BSCs
◦ More commonly used for clean room applications
◦ Used for Nanotechnology enclosures

Super Ultra Low Penetration Air (SULPA)

 Theoretically can remove 99.9999% of particles of 0.12
microns.
Common Ventilated Engineering Controls
Chemical Fume Hood Laminar Flow Clean Air Center Biological Safety Cabinet

• Closes completely: • Usually no sash or sash • Fixed sash opening (8 in.)

either horizontally or is fixed (alarmed)
vertically • Positive pressure – air • Sash moves up but does
• Negative pressure blowing into face or not close completely
breathing zone • Negative pressure
Chemical Fume Hoods
◦ Remove chemical fumes and vapors to outside
 May have charcoal filters (radioactive materials)
◦ Not for use with highly infectious materials or
environmentally dangerous organisms
 Exhaust air not HEPA filtered
 Not easily decontaminated
 No product protection
Chemical Fume Hoods
 When working with chemical hazards
 Personnel protection only
 Exhausts air to the outside
 Does not typically provide environmental protection

100
ft/
min
Clean Benches
 “Laminar Flow cabinet”
 Creates airflow generated
through a HEPA filter
 Discharge air goes directly into
the room
 Air flows toward the worker
 Used when the product is not
hazardous but must be kept
sterile
 Preparation of non-hazardous
materials
 Provides product protection only
Biological Safety Cabinets
Contain biological hazards
 Inward airflow provides personnel protection
 Filtered exhaust air provides environmental protection
 Supply air filter provides product protection

Classes of BSC
• Class I
– protects worker and environment
• Class II
– worker, product, environmental
protection
• Class III
– totally enclosed, ventilated, air-tight
– suitable for work at BSL3 and BSL4
– absolute containment
Class I BSC
Where no product protection needed:
• Opening suspicious mail
• Running a centrifuge
Class I BSC
 Exhausted into room or hard
ducted
 Typical uses of Class I BSCs
include:
◦ Housing centrifuges
◦ Housing fermenters
◦ Cage dumping (animal capability)
◦ Aerating cultures
 Class II BSC
 “Curtain” of air at the opening
 Laminar flow of filtered air
within the BSC
 Filtration of exhausted air
Airflow in a Class II BSC
Types of Class II BSC
Class II Recirculated Exhausted
Exhaust to
Type Air Air
A1 70% 30% Room

A2 70% 30% Outside

B1 30% 70% Outside

B2 0% 100% Outside

Type B2 BSCs may be used for work with volatile toxic chemicals
and radionuclides required as an adjunct to microbiological studies.
 Class III BSC
 Gas-tight and under negative pressure
 Minimum air intake velocity through a glove port: 100 fpm
 Exhaust air is double HEPA-filtered or HEPA-filtered and
incinerated before being exhausted to the outdoors
 Supply air is HEPA filtered
 NO recirculation of air

 Operation in BSC performed

through rubber gloves
 Must be connected to a
double door autoclave and/or
chemical dunk tanks to
sterilize or disinfect all exiting
materials
Comparing Levels of Protection
Primary Barrier Personnel Product Environment

Fume Hood x

Clean Bench x

BSC Class I x x

BSC Class II x x x

BSC Class III x x x

BSC Selection
Conduct a Risk Assessment!
Select BSC type based on
 Organisms to be handled
 Manipulations required of the laboratorian
 Equipment needed
 Use of toxic chemicals and radionuclides
Locating a BSC in the Lab
 Airflow at the front of the BSC can be compromised
when it’s velocity is not greater than other sources of
airflow
 Minimize this effect by locate the BSC away from:
◦ Doors and windows
◦ Personnel traffic
◦ Supply air ducts
◦ Other lab equipment
Before use, check :
• Certification
• certified by who
• date certified
• validity period
• certification ensures
that the BSC is
functioning within
specification
Placement of Materials
 Work from clean to dirty across the work
surface
 Materials should be as far back from the front as
possible
 Place a paper towel or disposable pad where
biological materials will be manipulated
While working in a BSC
 Keep doors closed
 Refrain from constantly moving arms in and out
of a BSC
 IF necessary, move arms in and out slowly and
deliberately to the front opening
 Immediately clean-up spills
 Minimize aerosols
Working in a BSC
✓ Use proper PPE
✓ Do not block grilles
✓ Do not dispose of
waste materials
outside of the BSC
✓ Do not use open
flames
Working in a BSC

✓ Do not overload materials

✓ Do not rest arms across
the front grill
✓ Don’t store materials on
top of BSC
Critical Containment Components
• Room Integrity
• Service Penetrations and seals, floors, walls, ceiling
• Inward Directional Airflow
• Door Interlocks
• Autoclaves
• Effluent Decontamination System
• Pass-boxes, Fumigation chambers
• Biological Safety Cabinets
• HEPA filters and housings
• Ductwork Integrity
• Control Systems (prevent positive pressurization)
• Alarms (containment systems)
• Communication devices
• Security devices
• access control, alarms, cctv
 Impact of Containment Level
BSL2 BSL3 BSL3+ BSL3++ BSL-4

HEPA Floor
Lab Floor
Waste Floor

Decon

Lab Area

Entry

Gross SM 100 110 140 450 450

Net to Gross 68% 68% 41% 27% 27%

Cost/GSM $ $$ $$$ $$$$$ $$$$$$$$

 Impact of Containment Level
BSL2 BSL3 BSL3+ BSL3++ BSL-4

HEPA Floor
Lab Floor
Waste Floor

PRODUCTIVITY

OPERATING COSTS