1. By :
DR RAGYA BHARADWAJ
FIRST YEAR POST GRADUATE STUDENT
DATE 13.03.2012

2. INDEX
 OBJECTIVES
 DEFINITION OF BIOSAFETY CABINETS
 BIOSAFETY LEVELS
 TYPES OF BIOSAFETY CABINETS
 MECHANICS OF BIOSAFETY CABINETS
 SAFE WORK PRACTICES
 BIOHAZARD SPILL CONTROL MANAGEMENT
 CERTIFICATION
 SUMMARY

3.  To know in brief about Biosafety levels
 To know the types of Biological safety
cabinets
 To understand the mechanics of BSC
 To understand the safe work practices
while handling a BSC

4. What are Biological Safety Cabinets
(BSC)?
Containment and protection devices used in
laboratories working with biological agents with a
primary purpose of protecting the laboratory worker
and the environment from viable organisms.
http://www.cdc.gov

5. http://www.cdc.gov/od/ohs/pdffiles/ Module%202%20-%20Biosafety.pdf

6. BIOSAFETY LEVEL 1
 BSL‐1 is suitable for well‐characterized agents not know to cause disease in
immunocompetent adult humans, and present minimal potential hazard to
laboratory personnel and the environment.
 Examples of agents handled at BSL‐1 include: Bacillus subtilis, Eschericia coli.

7. BIOSAFETY LEVEL 2
 BSL‐2 is suitable for work involving agents that pose moderate hazards to
personnel and the environment.
 It differs from BSL‐1 in that:
1) Laboratory personnel have specific training in handling pathogenic agents
2) Access to the laboratory is restricted when work is being conducted
3) All procedures involving infectious aerosols or splashes are conducted in
BSCs or other physical containment equipment.

8. BSL‐2 Viral Agents:
 Adenovirus
 Creutzfeld‐Jacob agent
 Cytomegalovirus
 Eastern equine encephalitis
 Epstein‐Barr virus
 Hepatitis A, B, C, D, E
 Herpes simplex viruses
 HTLV types I and II
 Kuru
 Monkeypox virus
 SIV
 Spongiform encephalopathies
 Vaccinia virus
 HIV
 VSV (lab adapted strains)
BSL‐2 Bacterial/Rickettsial Agents
•Campylobacter fetus, coli, jejuni
•Chlamydia psittaci, trachomatis
•Clostridium botulinum, tetani
•Corynebacterium diphtheriae
•Legionella spp
•Neisseria gonorrhoeae
•Neisseria meningitidis
•Pseudomonas pseudomallei
•Salmonella spp
•Shigella boydii, dysenteriae, flexneri, sonnei
•Treponema pallidum
•Vibrio cholera (including El Tor)
•Vibrio parahemolyticus
•Vibrio vulnificus
•Yersinia pestis
http://www.cdc.gov

9. BSL‐2 Fungal Agents:
•Blastomyces dermatitidis
•Cryptococcus neoformans
•Microsporum spp
•Sporothrix schenkii
•Trichophyton spp
BSL‐2 Parasitic Agents:
•Entamoeba histolytia
•Crytosporidium spp
•Giardia spp
•Naegleria fowleri
•Plasmodium spp
•Strongyloides spp
•Tania solium
•Toxoplasma spp
•Trypanosoma spp
http://www.cdc.gov/od/ohs/biosfty

10. BIOSAFTEY LEVEL 3
• Laboratory personnel must receive specific training in handling pathogenic and
potentially lethal agents supervised by scientists competent in handling
infectious agents and associated procedures.
• All procedures must be conducted within BSCs or other physical containment
devices.

11. BSL‐3 Viral Agents:
•Rift Valley Fever (Zinga)
•VSV exotic strains (Piry)
•Yellow fever (wild type)
BSL‐3 Bacterial/Rickettsial Agents:
•Bacillus anthracis
•Francisella tularensis
•Mycobacterium tuberculosis
•Mycobacterium bovis
•Rickettsia rickettsii
•Yersenia pestis (resistant strains)
BSL‐3 Fungal Agents:
•Coccidioides immitis
•Histoplasma capsulatum

12. Biosafety level 4
 This level deals with dangerous and exotic agents that pose a high
individual risk of aerosol-transmitted laboratory infections (severe to
fatal disease in humans)
• Positive pressure personnel suit, with a segregated air supply,
is mandatory.
• The entrance and exit of a level four biolab contains multiple
showers, a vacuum room, an ultraviolet light room.
• Multiple airlocks are employed and are electronically secured to
prevent both doors opening at the same time.

13. BIOSAFTEY LEVEL 4 LAB SCHEMATIC

14. • Marburg virus
• Ebola virus
• Lassa fever virus
• Crimean-Congo hemorrhagic fever
• Smallpox

15. What is NOT a BSC!!!
 Chemical Fume Hoods
 Conventional lab fume
hoods should NEVER be
used to contain biological
hazards.

16. This is also NOT a BSC!
 Clean Benches discharge HEPA filtered
air across the work surface toward the
user.
 They provide protection only for
product inside the bench.
 They are commonly used for sterile
assembly or dust free electronic
production.
 Clean benches should NEVER be used
when handling cell culture materials,
drug formulations, or manipulating
potentially infectious materials.
Laboratory Laminar Flow Clean Benches

17. Find a Biological Safety Cabinet!

18. TYPES OF BIOLOGICAL SAFETY
CABINETS
 Class I Biological Safety Cabinets
 Class II Biological Safety Cabinets
 Type A
 Type B ( B1 , B2 , B3 )
 Class III Biological Safety Cabinets
http://www.cdc.gov/od/ohs/biosfty

19. Class I Biological Safety Cabinets
 Class I BSC is a ventilated cabinet with
an inward airflow and HEPA filters at its
outlets.
 It was previously referred to as the CDC
Hood and served a valuable function in its
time to protect personnel and the
environment.
 Because it offers no product protection, it
has been essentially obsolete for the
past several decades.

20. • Room air is drawn in through the front
opening at a minimum velocity of 0.38 m/s.
• The directional flow of air whisks aerosol
particles away from the laboratory worker and
into the exhaust duct.
• The front opening allows the operator’s arms
to reach the work surface inside the cabinet
while he or she observes the work surface
through a glass window.
• The air from the cabinet is exhausted through a
HEPA filter to outside.

21. Class I Biological Safety Cabinet Schematic

22. Class II Biological Safety Cabinets
 As biomedical researchers began to use sterile animal and cell
tissue culture systems, BSC’s utility needed to be expanded.
 Class II BSC’s protect Personnel, Product and
Environment.
 Provides an inward airflow to protect personnel, a downward
flow of HEPA filtered air to the work area to protect the
product, and then exhaust HEPA filtered air to protect the
environment from particulate and aerosol hazards.

23. • Class II differ from Class I BSCs by allowing only HEPA-filtered (sterile)
supply air to flow over the work surface.
• The Class II BSC can be used for working with infectious agents in
Risk Groups 2 and 3.
• Class II BSCs can be used for working with infectious agents in Risk Group 4
when positive pressure suits are used.

24. • Depending on their inlet flow velocity and percent of HEPA
filtered air recirculated ; Class 2 are divided further into A or B.

25. Class II Type A BSC
 These hoods offer personnel protection
through negative pressure airflow into
the cabinet.
 To protect the product, the work area in
the cabinet is continuously bathed with
ultra-clean air provided by the supply
HEPA filter.
 Approximately 70% of the air of each
cycle is recirculated through this
supply HEPA filter.
 The remaining air is discharged from
the hood through the exhaust HEPA
filter, protecting the environment.

26. Class II Type A BSC Schematic
A- Front Opening
B- Sash
C- Exhaust HEPA Filter
D- Supply HEPA Filter
E- Rear Plenum
F- Blower

27. Class II Type B BSC
 It was created for manipulations
of minute quantities of hazardous
chemicals such as carcinogens
when used in research.
 Carcinogens used in cell culture
or microbial systems require both
biological and chemical
containment.

28. Class II Type B 1 BSC Schematic
A- Front Opening
B- Sash
C- Exhaust HEPA Filter
D- Plenum
E- Supply HEPA Filter
F- Blower
G- Filter screen
Connection to building exhaust
System required
.

30. Class II Type B 2 BSC
A total-exhaust cabinet
No air is recirculated within it.
This cabinet provides simultaneous
primary biological and chemical
containment.
The supply blower draws in room air at the
top of the cabinet, passes it through a HEPA
filter and down into the work area of the
cabinet.

31. A. front opening
B. sash
C. exhaust HEPA filter
D. supply HEPA filter
E. negative pressure
exhaust plenum
F. supply blower
G. filter screen
Note:
The cabinet exhaust
needs to be connected
to the building exhaust
system.

32. Class II Type B 3 BSC
All positive pressure contaminated plenums are surrounded by a negative air
pressure plenum.
Thus, leakage in a contaminated plenum will be into the cabinet and not into the
environment.

33. A. narrow front opening
B. sash
C. exhaust HEPA filter
D. supply HEPA filter
E. negative pressure
exhaust plenum
F. supply blower
Schematic

35. Class II BSC Modifications
 The front sash can be modified by the manufacturer to
accommodate a microscope
 A rigid plate with arm holes can be added if needed.
 The work surface can be designed to accept a centrifuge,
or other equipment that requires containment
Class II BSCs can be modified to accommodate special tasks:

36. • Long, heavy-duty rubber gloves are
attached in a gas-tight manner to allow
manipulation of the materials isolated
inside(maximizing personal safety)
• Depending on the design of the
cabinet, the supply HEPA filter
provides particulate-free, but
somewhat turbulent, airflow within the
work environment
Class III BSC or Glovebox

37. Class III BSC or Gloveboxes
• Class III cabinets are usually only installed in maximum containment
laboratories with controlled access.
• They require special ventilation systems .

38. Class III BSC Schematic
A- Glove Ports
B- Sash
C- Exhaust HEPA
Filter
D- Supply HEPA
Filter
E- Double Ended
Autoclave or
Pass Through
Box
• The cabinet exhaust needs to be connected to the building exhaust
system.

40. Type of protection BSC selection
Personnel protection, microorganisms in
Risk Group 4
Class III
Personnel protection, microorganisms in
Risk Group 3, suit laboratory
Class II
Product protection Class II, Class III
Volatile radionuclide/chemical
protection, minute amounts
Class IIB1, Class IIA
Volatile radionuclide/chemical
protection
Class I, Class IIB2, Class III
SELECTION OF A BIOLOGICAL SAFETY
CABINET

41. Safe Work Practices for BSC Use
 Do not store equipment or supplies inside the cabinet or the top as
HEPA filter could be damaged and the airflow disrupted.
 Make sure the cabinet is level. If the cabinet base is uneven, airflow can
be affected.
 Never disengage the alarm. It indicates improper airflow and reduced
performance which may endanger the researcher or the experiment.
 Never completely close the window sash with the motor running as this
condition may cause motor burnout.

42. Safe Work Practices for BSC Use
 Cabinets should be placed away from doors, windows, vents or
high traffic areas to reduce air turbulence.
 A clearance of 30–35 cm above the cabinet is required to
provide for accurate air velocity measurement .
 A bunsen burner must be placed at the rear of the work area
where the air turbulence from the flame will have the least
effect on the air stream.
 All equipment which have come in contact with the biological
agent should be decontaminated.

43. • Aerosol-generating equipment (e.g. mixers, centrifuges, etc.) should be
placed towards the rear of the cabinet
• Cabinets should be turned on at least 5 min before beginning work and
after completion of work to allow contaminated air to be removed from
the cabinet environment.
• Ultraviolet lights must be turned off while the room is occupied, to protect
eyes and skin from inadvertent exposure.
• After all items have been removed, wipe the interior surfaces with disinfectant.
Safe Work Practices for BSC Use

44. Safe Work Practices for BSC Use

46. Biohazard Spill Control Inside a BSC
 1. Keep the BSC on.
 2. Put on protective gloves.
 3. Spray & wipe walls, work surfaces, and equipment with
decontamination solution.
 4. Flood tray top, drain pans, and catch basins with
decontamination solution.
 5. Allow to stand for 20 minutes.
 6. Place everything that is contaminated into autoclave pan.
 7. Drain decontamination solution from cabinet base into
AUTOCLAVABLE containers

48. Biological Safety Cabinet Certification
 The cabinet must be certified when first installed and then
annually.
 It must be recertified anytime it is moved even within the same
room.
 Before certification personnel arrive, remove all items from the
cabinet and wipe it down with a disinfectant.
 Any decontaminations, certifications, repairs or adjustments are to
be made by qualified personnel.
 Optional tests for electrical leaks, lighting intensity, ultraviolet light
intensity, noise level and vibration

49. And remember……..
 A laminar flow biological safety
cabinet is a valuable supplement
to good sterile technique, not a
replacement for it.
 If the cabinet is not well
understood and operated
correctly, it will not provide
adequate protection for you or
the environment.

50. BIBLIOGRAPHY
 TOPLEY AND WILSON VOL 2
 MACKIE AND Mc CARTNEY
Practical Medical Microbiology 14th ed
 BAILEY & SCOTT’S
Diagnostic Microbiology 12th ed
 www.cdc.gov

51. THANK YOU