1. Biocontainment Practices Inside the Animal Lab
Guglielmo Vismara

2. Topics Biological Risk Primary and Secondary Barriers
Biosafety Cabinets and Decon
Layouts

3. Biocontainment Microorganisms, Animals manipulation, Allergens…
Biological risk: risk associated to biological agents
Microorganisms, Animals manipulation, Allergens…
Invisible risk, it could be underestimated
It must be assessed by experienced and responsible people (biosafety officer), for evaluation of the optimal working conditions
Risk = probability of accident x consequence of accident
The risk is always present. It can be minimized but never eliminated at all
Pathogens associated with living animals increase the level of biological risk

4. (Animal) Biological Safety Levels
ABSL-1
Defined organisms
Not known to cause disease in healthy adults
Basic
Facility
ABSL-2
Moderate-risk agents present
Disease of varying severity
ABSL-3
Indigenous or exotic agents, aerosol transmission
Serious and potentially lethal infection
Containment
Facility
ABSL-4
Dangerous or exotic high risk agents,
Aerosol transmission
Serious and potentially lethal infection

5. The importance to know what pathogen is in use
Micobacterium Tuberculosis: level 3 (CDC-NIH)
Avian Influenzae H5N1: level 2/3 (CDC), level 3 (NIH)
Human adherent cancer cells: generally level 2
- Herpes, Papilloma, Citomegalovirus: level 2

6. Risk Assesment and Management
Evaluation of the risk
Multiple Factors
Managment of the risk
Risk assessment
Risk management
Validation and surveillance
Corrective actions

7. Risk Assesment – examples of factors
Evaluation of exposed personnel: who is exposed to animals, who enters the vivarium, who do the service, who clean the rooms.
P.I. – Animal care takers – Technicians, …..
Exposition to the biological agent: skin contact, eyes contact, inhalation, ingestion, inoculation.
Splashes – Aerosols – Puntures – Animal bites and scratches,…
Evaluation of the vivarium rooms layout and all the available equipment
(barriers)

8. Assesment/Management - examples
Example: contaminated ferret urine spilled on the floor
Is the microorganism in the urine?
Can the urine be infective?
What is the volume of contaminant?
Choice of the absorbent material
What waste container to be used
What personal protective equipment to wear
What tools to use to remove the waste
How to decontaminate the floor surface

9. Managing the Biological Risk
To involve all the aspects in the lab activities
Equipment choice, and best use
Organization of the room layouts
Organization of logistic in the room
Evaluate the people and material flow in the vivarium
Write and “publish” the SOPs
Train of people
All this process is DYNAMIC and subjected to revisions
Biological
Agent
Host
Environment
Risk

10. Enclosure for the Biological Agents
Primary barrier:
first protective barrier from biohazard to the operators
to protect people and environment close to the source of contamination
physical shield
Biosafety cabinet, animal ventilated/sealed cage, tube, centrifuge…
Every equipment must be associated with SOPs to be an effective barrier

11. When working with an infected animal, it is the cage
Primary barriers
When working with an infected animal, it is the cage

12. Features of a Good Primary Barrier
Tight Sealed, no air leakage
Filtration by HEPA filters – high efficiency, biological filters
Autoclavable
Compatible with disinfectants and chemicals
Compatible with the size of a Biosafety Cabinet
Ventilation controlled by pressure
Air proof – Water proof

13. Personal Protective Equipments
- If animals are housed in open cages, PPEs are the primary barrier
- In case of sealed primary enclosures, PPEs can be downgraded, anyway always represent an important protection
PPEs:
Gloves
Coats/Gowns
Respirators
Goggles
Face shields
Shoe covers

14. Secondary barriers Depend on the transmission risk of agents used.
The design, engineering and construction of the facility provides protection for the laboratory workers’ & provides a barrier to protect persons outside the laboratory.

15. ABSL3 vivarium: what’s “HOT”???
HEPA
AUTOCLAVE
Cages on Rack
Classic Changing station
MICROISOLATORS?
OPEN CAGES?
IVCs’?

16. BSL3 vivarium: what’s “HOT”???
HEPA
AUTOCLAVE
MICROISOLATORS?
OPEN CAGES?
IVCs’?
Area fully contaminated
PPE
Risk assessment
Cages on Rack
Laminar Flow

17. BSL3 vivarium: what’s “HOT”???
HEPA
AUTOCLAVE
Biocontainment at cage level
Laminar Flow

18. BSL3 vivarium: what’s “HOT”???
HEPA
AUTOCLAVE
Contamination at enclosures level
PPE
But different risk assessment

19. Some peculiarities of a Biocontainment Lab
Equipment
Changing station = Biosafety Cabinet (Class II, Biohazard…)
Animal cage = sealed containment cage
PPE = very well defined, often respirators
Rooms
Very restricted access, locked doors
Negative pressure, constant monitoring
Decontaminate all is taken out
Anteroom, gradients of pressure, showers
Strong Procedures

20. Biosafety Cabinets
Very important equipment for procedures/animal cage change/biological agents manipulation
Mandatory for BSL and ABSL > 1
Certified equipment, requires training and ability to use
Laminar Flow, protects product, people and environment under specific certifications

21. Biosafety Cabinets Class II Cabinet – EN12469, NSF49
Frontal aperture, the operator can manipulate the product inside the cabinet
Frontal air barrier to protect the operator
Laminar flow from the top to protect the product
HEPA filtered exhausted air to protect the environment
Partial recirculation of air (60-70%)

22. Decontamination Different words around this topic
Sanitization: reduction of the microorganisms up to a level considered safe for public health
Disinfection: chemical or physical process to eliminate a biological agent
Sterilization: eliminates-inactivates all the biological agents
In a Biocontainment lab/facility the decon equipment
is on the barrier

23. Decontamination What is the microorganism (virus, bacteria, spore)
Need effective decontamination
What is the media: surface/liquid waste/room
Right concentration and volume
Is the material resistant or not to the treatment
Chemical or physical treatment
Huge choice of chemicals available on the market
Don’t be disoriented!

24. Decontamination by gas
Cold sterilization: useful for what cannot be treated by heat or liquid
Rooms: vaporization and ventilation, routine vivarium shutdown
Equipment: bulky, not removable from the room, or sensitive (electrics)
Performed in decon-locks: flexible system
Peroxide – Formaldehide – Clorine Dioxide
MUST KNOW:
NOT EFFECTIVE BELOW THE SURFACE!

25. Some BSL3 Layouts Similar to BSL-2 personal protective equipment
Primary barriers:
Similar to BSL-2 personal protective equipment
Respiratory equipment if risk of infection through inhalation
Secondary barriers:
Autoclave in the barrier
Corridors separated from direct access to lab
Access through self-closing double doors, locked
Air handling systems to ensure negative air flow (air flows into the lab)
BSC II or BSC III

26. BSL 3
Some BSL3 Layouts

28. Guidelines
BMBL 5th Edition (CDC/NIH)

29. Guidelines

30. Thanks for your attention!