Biosafety Laboratories: Containment vs Exclusion - Planning ...

Biosafety Laboratories: 

Containment vs Exclusion 

Planning & Design Considerations 

Janet Baum, AIA 

Washington University in St. Louis

Containment Laboratories 

• Maintain under negative pressure to ALL 

surroundings 

• Treat effluent air 

• Biosafety Labs 

• High Toxicity Labs

Exclusion Laboratories 

• Maintain under positive pressure to ALL 

surroundings 

• Treat supply air 

• Clean Room Laboratories 

• Trace Analysis Laboratories

Neutral-Pressure Laboratories 

• NO hazardous or toxic materials handled or 

stored 

• NO volatile or malodorous materials handled 

or stored 

• NO animals housed

Containment Laboratories 

BIOSAFETY LABORATORY 

• Is a containment laboratory 

• U.S.A. Biosafety Levels are from 1 to 4 

• U.S.A. CDC / NIH designate hazard level of 

individual organisms on a scale of 1 to 4 

• U.S.A. Dept. of Agriculture designates hazard 

level of organisms on a scale of 1 to 3

U.S.A. Standards and Guidelines 

Biological Safety 

Centers for Disease Control with National Institutes of Health 

Biosafety in Microbiological and Biomedical Laboratories 

U.S. Department of Agriculture 

Agricultural Research Service (ARS) 

Facility Design Standards 

Animal & Plant Health Inspection Service (APHIS) 

Quarantine Facility Guidelines for Microorganisms 

Quarantine Facility Guidelines for Weeds 

Quarantine Facility Guidelines for the Receipt and Containment 

Non-indigenous Arthropod Herbivores, Parasitoids, Predators 

American Committee of Medical Entomology 

Arthropod Containment Guidelines



National Institutes of Health 

Guidelines for Recombinant DNA Research 

Laboratory Safety Supplement to the Guidelines 

Occupational Safety and Health Administration 

Bloodborne Pathogens 

Tuberculosis Standard 

National Cancer Institute with NIH 

Guidelines for Oncogenic Viruses

International Standards & Guidelines 


• World Health Organization 

Laboratory Safety Guidelines 

• CEN (Comité Européen de Normalisation) 

CWA #15793 document, February, 2008, 

Laboratory Biorisk Management Standard 

• International Union Against Tuberculosis and Lung Disease 

1998 The Public Health Service National Tuberculosis 

Reference Laboratory Minimum Requirements, 

Role and Operation in a Low Income Country


Impacting Biosafety Laboratories 

AAALAC – Guide for Care and Use of Laboratory Animals 

Local Building Code 

National Building Code 

NFPA Standards 

Fire Protection Code 

Mechanical Code 

Plumbing Code 

Electrical Code 

Energy Code 

Nuclear Regulatory Commission 

OSHA 

Americans with Disabilities Act of 1990

U.S.A. Regulations 

U.S. Federal Laws 

Public Law 107- 56: 

U.S.A. Patriot Act of 2001 (Uniting and Strengthening America by 

Providing Appropriate Tools Required to Intercept and Obstruct Terrorism) 

Prohibits restricted persons from shipping, possessing, receiving select agents 

Public Law 107- 188: 

U.S.A. Public Health Security and Bioterrorism Preparedness 

and Response Act of 2002 

U.S.A. Agricultural Bioterrorism Protection Act of 2002 

Requires institutions to notify DHHS or USDA of possession of specific 

pathogens or toxins 

42 CFR Part 73 / Part 1003: 

U.S.A. Select Agent Act of 2002 (Possession, Use and Transfer of Select 

Agents and Toxins) 

(Department of Health and Human Services)


Select Agents 

U.S.A. Public Health Security and Bioterrorism Preparedness 

and Response Act of 2002 

Requires DHHS to regulate the possession, use, and transfer of biological 

agents or toxins that could pose a severe threat to public health and safety. 

DHHS require entities to register with the U.S. Centers for Disease Control 

U.S.A. Agricultural Bioterrorism Protection Act of 2002 

Requires USDA to regulate the possession, use, and transfer of biological 

agents or toxins that could pose a severe threat to animal or plant health 

or to animal or plant products. USDA require entities to register with APHIS. 

These Acts require establishment of a national database of registered entities 

Set criminal penalties for failing to comply with requirements of these Acts. 

CDC and APHIS coordinate regulatory activities for agents that would be 

Registered by both Agencies. The F.B.I. has been delegated authority for 

conducting security risk assessments.


CDC List of Select Agents 

Viruses Bacteria Fungi & Rickettsae Toxins 

• Hendra Virus -Equine 

Morbillivirus 

• Herpesvirus Simiae (B) 

• HPAI Influenza 

• 1918 Influenza 

• Arboviruse & 

Related Zoonotic 

Virus 

• Venezuelan Equine 

Encephalitis 

• Eastern Equine 

Encephalitis 

• Rift Valley Fever 

• Bacillus Anthracis 

• Burcella Abortus 

• Burkholderia Mallei 

• Burkholderia Pseudomallei 

• Neurotoxin Clostridium 

(Botulinum) 

• Francisella Tularensis 

• Coccidioides Immitis 

• Coxiella Burnetii 

• Rickettsia Prowazekii 

• Rickettsia Rickettsii 

• Abrin 

• Aflatoxins 

• Botulinum 

• Clostridium 

Perfringens Epsilon 

• Conotoxin 

• Diacetoxyscirpenol 

• Ricin 

• Saxitoxin 

• Sigatoxin 

• Staphylococcal 

Enterotoxin 

• Tetrodotoxin 

• T-2 Mycotoxin


CDC List of Bacterial Agents


CDC List of Viral Agents


CDC List of Other Agents

Biosafety Level 1 Laboratory (BSL-1) 

• Basic lab 

• Open bench operations 

• Standard lab practices 

• Operable windows have insect screens 

• Biosafety Cabinets (BSCs) not required 

• Biosafety cabinets should be used in introductory 

microbiology courses to teach students how to use 

BSCs.


in addition to all requirements for BSL-1 

• Basic lab 

• Limited access into lab 

• Biohazard warning signs 

• Lab coats and protective gloves used 

• Decontamination facility needed in building 

• Biosafety Cabinets are required when 

operations may produce aerosols 

• Negative pressure is achievable

Lab Planning Issues – BSL-2 

Agents 

Practices 

Safety Equipment 

(Primary Barriers) 

Facilities 

(Secondary Barriers) 

Suitable for 

work 

involving 

agents of 

moderate 

potential 

hazard to 

personnel 

and the 

environment 

BSL-1 plus: 

• Controlled 

access 

• Hand washing 

• Keep lab 

clothing within 

lab, do not wear 

outside lab 

• Baseline serum 

levels of staff 

• Needles / sharps 

precautions 

• Class II BSC’s or 

other physical 

containment 

devices used for 

all open 

manipulations of 

agents 

• PPE: protective 

gear as needed 

• Eyewash available 

• Sink for hand 

wash 

• Autoclave 

available 

BSL-1 plus: 

• Physical 

separation, & 

locked, selfclosing 

doors from 

access corridors 

• Sturdy, easily 

cleaned furniture 

and work surfaces 

• Exhausted air not 

recirculated 

• Negative airflow 

into laboratory 

• Insect screens on 

windows

Goals of a Containment Facility 

• Protect personnel in the laboratory 

• Provide productive scientific environment 

Liquid decon pass-thru 

– Protect research, samples, and products 

• Protect support staff who operate and maintain 

containment and ventilation systems/ controls 

• Protect environment 

– Surrounding personnel and functions 

– Outside environment, adjacent communities 

UV decon pass-thru pass-thru autoclave HEPA filter bank

Definition of a BSL-3 Facility 

Suitable for work with infectious agents which may cause 

serious or potentially lethal disease as a result of exposure by 

the inhalation route 

• Exposure potential to pathogens spread by aerosol 

• Infection serious, possibly lethal 

Canada National Biosafety Laboratory


in addition to all requirements for BSL-1 and BSL-2 

• Designed specifically for biological 

containment 

• Special protective clothing worn 

• All work with biological materials is in 

biosafety cabinets (BSCs) 

• Access is through two doors separated by an 

airlock that may be used for clothing change.

Lab Planning Issues – BSL-3 

Agents 

Practices 

Safety Equipment 

(Primary Barriers) 

Facilities 

(Secondary Barriers) 

Indigenous or 

exotic agents 

with potential 

for aerosol 

transmission; 

disease may 

have serious 

or lethal 

consequences 

BSL-2 plus: 

• Controlled 

access 

• Work in certified 

BSC 

• Decontamination 

of all waste 

• Decontaminate 

lab clothing 

before laundering 

• Baseline serum 

levels of staff 

• Class II or III BSC’s 

or other physical 

containment 

devices used for all 

open manipulations 

of agents 

• PPE: protective lab 

clothing, gloves, 

respiratory 

protection as 

needed 

• Autoclave in lab 

suite 

• HEPA filter vacuum 

lines 

BSL-2 plus: 

• Physical separation 

from access 

corridors 

• Locked self-closing, 

double-door access 

• Exhausted air not 

recirculated 

• Negative airflow into 

laboratory 

• Room penetrations 

are sealed 

• Room surfaces are 

water resistant for 

cleaning

Biosafety Level 3 Lab – ABSL-3 

in addition to all requirements for BSL-1, BSL-2, BSL-3 


containment of small research animals 

• Entrance is through a pressure-controlled 

airlock used for clothing change. 

• Exit optional through an airlock used for 

personnel showers and clothing change. 

• Supply and exhaust systems separate and 

independent of other building ventilation. 

• Interlocked controls to maintain pre-set 

pressures

Biosafety Level 3 Lab – BSL-3Ag 



containment of plants and large animals 



• Exit is through an airlock used for personnel 

showers and clothing change. 




pressures




containment. 



• Exit is through an airlock used for personnel 

showers and clothing change. 

• Total containment Class III biosafety cabinets 

or 

• Partial containment Class II biosafety 

cabinets used by personnel clothed in 

positive-pressure, full-body, suits with 

separate and protected air supply






pressures 

• HEPA filtered supply air 

• Dual HEPA filtered exhaust air, in sequence 

• In-place filter testing facilities

Cost Premium-Containment Facilities

Cost Premium-Containment Facilities

Cost Premium-Containment Facilities 

Diagram of BSL-4 Containment Concept

Containment Planning – BSL-3 and 4 

Primary/Secondary 

Barriers 

Camera 

Bubble 

Air 

Supply 

Diffuser 

Breathing 

Air 

Biosafety cabinets used 

Gas tight room tested 

and certified 

Air Locks with air 

pressure doors 

Electrical 

Outlets 

Floor/Sink 

Drains 

Window 

Frame 

Waste Management 

All waste rendered harmless 

autoclave waste from lab 

decontaminate effluent waste 

HEPA filter air


• Design of containment barriers 

• Quality research = containment 

• Ventilation requirements 

Sealed pipe penetrations 

BSL-3 lab in Canada


Containment Barriers 

• Primary barrier 

– Biological safety cabinet 

– Personal protective gear 

• Secondary barrier 

– Room enclosure 

– Engineering systems 

• Tertiary barrier 

– Building around lab 

– Containment around 

systems 

BSL-3 lab in Canada

Concentrate/ Minimize – BSL-3 and 4 

• Limit number of individuals and processes that 

must work under BSL-3 and BSL-4 conditions 

• Provide appropriate service support outside 

containment zone 

• Provide adequate functional support inside 

containment zone 

• Estimate capital and life cycle costs for operating 

facility 

Capital cost includes design, construction, commissioning, 

and equipment fit-out of the laboratory 

Life cycle cost includes utility expense for ventilation and 

electric power, filter testing / replacement, inspections, 

insurance / liability

Plan for Flexibility 

• Regulatory and research changes 

• New pathogens 

• New equipment

Planning Concepts – BSL-3 and 4 

• Hazard zoning 

• Egress in direction of lower hazard 

• Zone from clean to dirty 

• Separate hazards that may interact 

• Isolate highest hazard processes & agents 

Enter 

Exit 

Prep Lab 

Air Flow 

Hazard 

Contaminated 

Hot Zone 

Lowest Hazard 

Highest Hazard


• One-way passage 

• Enter BSL-3 lab from one passage, exit from 

another 

• Sequential work flow, if possible 

• Reduce intersections in circulation pathways 

Process 

Path 

ENTRY 

Process 

#1 

Process 

#2 

Process 

#3 

EXIT 

Process 

#5 

Process 

#4


• Suites 

• Cluster support 

functions around 

basic work zone to 

reduce distances 

between functional 

zones 

• Good for repetitive 

functions and 

processes 

Decon 

Entry 

Vestibule 

Specimen 

Drop-off 

Equipment 

BSL-3 

Solutions/ 

Media Prep 

Culture 

Labs

Planning Concepts – BSL-3 

Graphic Program 

Blocking Diagram 

AL 

BSL-2 

AL 

AL 

Micro 

Test 

Decon 

BSL-3 Cell 

Culture 

BSL-3 

Cell 

Culture 

Degown 

BSL-2 

Degown / 

Shower 

Gown 

AL 

AL 

BSL-3 

Gown 

Decon


Flow Diagram 

AL 

Micro 

Test 

Decon 

BSL-3 Cell 

Culture 

BSL-2 

Degown 

AL 

BSL-3 

Gown 

AL 

Personnel Supplies 

Infectious Agent / Waste Material 

Animals


• Locate biosafety cabinets and fume hoods 

out of high traffic zones 

• Develop understanding on how to deal with 

emergencies within containment zone 

• Understand how to manage emergencies 

from outside zone 

• Investigate how containment zone can 

expand in future, if needed


Primary Entry 

and Exit 

Emergency Exit 

Only 

Phones 

Phones 

BSC BSC BSC 

EQ 

FH 

Bench 

EQ 

EQ 

EQ 

AL 

Air 

Locks 

BSC 

Degown 

& Gown 

Micro 

Test 

AC 

AC 

Shower 

Air 

Locks 

Decon 

EQ 

Bench 

Cell 

Culture 

EQ 

BSC 

BSC 

BSC BSC 

BSL-2 

BSL-3


Expansion Options 


Only 

Primary Entry 

and Exit 


Only 

Phones 

Phones 

Phones 

BSC 

Cell 

Culture 

Decon 

AC 

AC 

Micro 

Test 

BSC 

EQ 

AL 

BSC 

Micro 

Test 

AC 

AC 

Decon 

Cell 

Culture 

BSC 

Bench 

or 

Equip 

BSC 

BSC 

BSC 

BSC 

EQ 

Shower 

AL 

Air 

Lock 

Degown 

& Gown 

EQ 

Bench 

FH EQ 

EQ 

Air 

Lock 

Degown 

& Gown 

Shower 

AL 

EQ 

Bench 

or 

Equip 

EQ 

BSC 

BSC BSC 

BSL-3 

BSL-2 

BSL-3


Expansion Options 

Primary Entry 

and Exit 


Only 

Phones 

Phones 

BSC BSC BSC 

Bench 

EQ 

AL 

Air 

Lock 

BSC 

Micro 

Test 

AC 

AC 

Shower 

Decon 

Bench 

Equip 

Or BSC 

Equip 

Decon 

BSC BSC 

EQ EQ 

EQ BSC 

Cell Culture 

EQ BSC 

EQ 

FH 

EQ 

EQ 

Degown 

& Gown 

AL 

EQ 

EQ 

BSC 

EQ 

EQ 

BSC 

BSL-2 

BSL-3

Design Concepts – BSL-3 and 4 

• Understand how laboratory can be cleaned 

– Shape of each space is simple (minimize corners) 

– Finishes are seamless, smooth and coved 

– Seal ALL penetrations through interior surfaces, 

walls, floors, and ceilings 

– Conduct Integrated Pest Management Program 

– Equipment is located so floor can be cleaned; 

provide clearances above, below and beside them 

enough for mop and head of shop-vacuum machine 

– Provide cleaning tools and supplies within lab for 

daily as well as emergency cleaning procedures


• Understand how laboratory and equipment 

can be maintained 

– Provide tools for simple repairs 

– Locate laboratory HVAC controls exterior to 

laboratory so maintenance staff do not have to 

enter containment zone to do tests and repairs 

– Locate HVAC dampers and actuators exterior to 

laboratory so maintenance staff do not enter 

– Clearances above, below and beside adequate 

for access to controls motors, fans, filters.


• Understand overall health & safety issues 

– Perform periodic risk assessments 

– Personnel Medical Surveillance Program 

– Ergonomics 

– Visibility to all parts of lab 

– Communications – internal and external 

– Alarm systems – internal and external 

– Safety equipment located in critical areas 

– Back-up systems for PPE, critical supplies

Design Concepts – TB Testing Labs 

• In developing countries 3 types of labs 

supported by W.H.O. and other NGOs 

– Peripheral Laboratories in rural regions, villages 

for initial TB screening from sputum samples 

– Intermediate Laboratories in towns and cities 

for initial TB screening and confirmation of 

samples brought from Peripheral Laboratories 

– Central Laboratories capitol of nation or region 

for initial TB screening, confirmation of samples 

brought from Intermediate Laboratories, and 

testing for MDR and XDR TB

Design Concepts – WHO Level 3 

PERIPHERAL LABORATORY 1




PERIPHERAL LABORATORY 1 

STORAGE 

CABINETS 

LOG-IN 

TABLE 

WET 

BENCH 

HAND 

WASH 

SINK 

REF 

MICROSCOPE & 

WORK TABLES






PERIPHERAL LABORATORY 2 

STORAGE 

CABINETS 

LAB 

ENTRY 

LOG-IN 

TABLE 

HAND 

WASH 

SINK 

WORK TABLE & 

STORAGE CABINET 

REF 

WET 

BENCH 

MICROSCOPE & 

WORK TABLES & 

STORAGE CABINET




INTERMEDIATE LABORATORY 1


INTERMEDIATE LABORATORY 1 

PPE ANTEROOM 

SPUTUM RECEIVING 

& LOG-IN



PPE ANTEROOM 

SPUTUM RECEIVING 

& LOG-IN 

LAB 

ENTRY 

LOG-IN 

TABLE 

PASS-THRU 

BOX 

MAIN 

ENTRY 

STORAGE 

CABINET 

PASS-THRU 

BOX 

HAND 

WASH 

SINK



CULTURE LAB 

INCUBATION 

LAB



BSC 

CULTURE LAB 

CENTRI 

-FUGE 

LAB 

ENTRY 

REF 

WET 

BENCH 

HAND 

WASH 

SINK 

LAB 

ENTRY 

INCUBATION 

LAB 

STORAGE 

SHELVES 

PASS- 

THRU 

BOX & 

TABLE 

MICROSCOPE 

TABLES 

STORAGE 

SHELVES



STERILIZATION 

LAB 

MEDIA & REAGENT 

PREP LAB



MEDIA & REAGENT 

PREP LAB 

PASS-THRU 

AUTOCLAVE 

LAB 

ENTRY 

STORAGE 

CABINET 

STERILIZATION 

LAB 

HAND 

WASH 

SINK 

PASS-THRU 

AUTOCLAVE 

STORAGE 

CABINET 

REF 

PASS- 

THRU 

BOX & 

TABLE 

WET 

BENCH 

WET 

BENCH



Mechanical Ventilation Zone 

51 SM or 550 SF



Natural Ventilation Zone 

24 SM or 258.0 SF 

Prevailing 

Winds 

Mechanical Ventilation Zone 

51 SM or 550 SF


CENTRAL LABORATORY 1 

For COLD CLIMATES



For WARM CLIMATES



MDR TESTING 

LAB 

CULTURE LAB


CULTURE LAB 


MDR TESTING 

LAB 

BSC 

MICROSCOPE 

TABLES 

BSC 

BSC 

WET 

BENCH 

WET 

BENCH 

HAND 

WASH 

SINK 

PASS- 

THRU 

BOX & 

TABLE 

HAND 

WASH 

SINK 

MICROSCOPE 

TABLES


CENTRAL LABORATORY 2


WAITING ROOM 


RECEIVING 

ROOM


WAITING ROOM 


PASS- 

THRU 

BOX 

LAB 

ENTRY 

HAND 

WASH 

SINK 

HAND 

WASH 

SINK 

LAB 

ENTRY 

PASS- 

THRU 

BOX & 

TABLE 

RECEIVING 

LAB 

DESKS or 

TABLES 

STORAGE 

CABINETS


MDR TESTING LAB 

CENTRAL LABORATORY 2



MDR TESTING LAB 

LAB 

ENTRY 

HAND 

WASH 

SINK 

LAB 

SINK 

LAB 

TABLES 

PASS- 

THRU 

BOX 

BSC 

WET 

BENCH 

BSC 

MICROSCOPE 

TABLE


PCR AMPLIFICATION 

LAB 


STEP 2 

DNA SEQUENCING 

LAB 

DNA EXTRACTION 

LAB 

STEP 1 

STEP 3


DNA EXTRACTION 

LAB 

PASS- 

THRU 

BOX 

PCR AMPLIFICATION 

LAB 

HAND 

WASH 

SINK 

COMPUTER 

TABLE 

HAND 

WASH 

SINK 

BSC 

WET 

BENCH 


LAB 

ENTRY 

EQUIPMENT 

BENCH 

2 PASS-THRU 

TRANSFER 

BOXES 

EQUIPMENT 

TABLES 

WET 

BENCH 

DNA SEQUENCING 

LAB 

PASS- 

THRU 

BOX 

REF 

LAB 

ENTRY 

HAND 

WASH 

SINK

Biosafety Laboratories: 

Containment vs Exclusion 

Planning & Design Considerations 

Janet Baum, AIA 

Washington University in St. Louis

Biosafety Laboratories: Containment vs Exclusion - Planning ...

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