1. Biosafety in Microbiological and Biomedical Laboratories
S. Wongratanacheewin, Ph.D.
Melioidosis Research Center, Faculty of Medicine, KKU.

2. September 11, 2001

3. ไทยรัฐ 20 กันยายน 2546
ไทยรัฐ 19 กันยายน 2546

4. What is bioterrorism
The US president's 2006 budget requests more than $4 billion for biodefense activities at the Department of Health and Human Services (HHS).
Within budget, approximately $1.7 billion will fund medical research and product development at the National Institute of Allergy and Infectious Diseases (NIAID), including
Vaccines to immunize the public against diseases caused by bioterrorism agents
Diagnostic tests to help first responders and other medical personnel rapidly detect exposure and provide treatment
Therapies to help patients exposed to bioterrorism agents regain their health

5. Biosafety Personal protection Work place practices Administrative
Engineering
All play an equal role in protecting workers from occupational exposures in laboratories

6. Biosafety Personal protection Work place practices
PPE, respirotors, gloves, booties, eye and ear protections
Work place practices
Entry and exit practices, decontamination, emergency procedure, handle sharp.

7. Biosafety Administrative Engineering Training Risk management
Immunization
Security, supervision
Engineering
System, Hepa filter, Accessible

8. CDC Bioterrorism Agents
Category A
Category B
Category C

9. Category A Diseases/Agents
can be easily disseminated or transmitted from person to person;
result in high mortality rates and have the potential for major public health impact;
might cause public panic and social disruption; and
require special action for public health preparedness.

10. Category B Diseases/Agents
Second highest priority agents include those that
are moderately easy to disseminate;
result in moderate morbidity rates and low mortality rates; and
require specific enhancements of CDC's diagnostic capacity and enhanced disease surveillance.

11. Category C Diseases/Agents
Third highest priority agents include emerging pathogens that could be engineered for mass dissemination in the future because of
availability;
ease of production and dissemination; and
potential for high morbidity and mortality rates and major health impact.

16. The US National Institute of
Allergy and Infectious Diseases (NIAID)

17. The US National Institute of
Allergy and Infectious Diseases (NIAID)

18. Helen Quill & Maria Giovanni, Nature Immunology, 2004

19. Microbiological laboratories
Microbiological laboratories are special, often unique work environments that may pose identifiable infectious disease risks to persons in or near them.

20. Containment
Primary containment, the protection of personnel and the immediate laboratory environment from exposure to infectious agents, is provided by both good microbiological technique and the use of appropriate safety equipment.
Secondary containment, the protection of the environment external to the laboratory from exposure to infectious materials,

21. Laboratory Practice and Technique
Develop or adopt a biosafety or operations manual that identifies the hazards that will or may be encountered, and that specifies practices and procedures designed to minimize or eliminate exposures to these hazards.

22. Principles of Biosafety
Laboratory personnel
Safety practices, and techniques must be supplemented
Appropriate facility design and engineering features
Safety equipment, and management practices.

23. Safety Equipment (Primary Barriers)
Biological safety cabinets (BSCs)
Enclosed containers,
Engineering controls designed to remove or minimize exposures to hazardous biological materials.

24. The biological safety cabinet (BSC)
Device used to provide containment of infectious splashes or aerosols.
Three types of biological safety cabinets (Class I, II, III) used in microbiological laboratories.
Open-fronted Class I and Class II biological safety cabinets are primary barriers which offer significant levels of protection to laboratory personnel.
Safety centrifuge cup, an enclosed container designed to prevent aerosols.

28. Personal Protection gloves, coats, gowns, shoe covers, boots,
respirators,
face shields,
safety glasses, or goggles.

29. Facility Design and Construction (Secondary Barriers)
Depend on the risk of transmission of specific agents.
Separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and handwashing facilities.
If an infectious aerosol is present, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment.

32. Biological Safety Biological Hazards Divided into 4 biosafety levels
Levels define the lab requirements, protective equipment, and work practices
4 animal biosafety levels also

33. Biosafety Level 1 Definition
Work with agents not known to cause disease in healthy adults
Minimal potential hazard to personnel and environment

34. Biosafety Level 1 Requirements Wash hands before leaving lab
No eating, drinking, etc. in lab
Follow safe sharps procedures
Decontaminate waste and work surfaces

35. Biosafety Level 1 Examples Escherichia coli K12 Klebsiella oxytoca
Lactobacillus
Saccharomyces

37. Biosafety Level 2 Definition
Work with agents of moderate potential hazard to personnel and the environment. Agents associated with human disease which is rarely serious and for which preventative or therapeutic interventions are often available.

38. Biosafety Level 2 Requirements All requirements for BSL1 plus:
Training to handle organisms
Personal protective equipment (PPE)- lab coats, eyewear, and gloves
Minimize aerosol generation
Biohazard sign posted on door

39. Biosafety Level 2 Examples Pathogenic E. coli Salmonella sp.
Streptococcus sp.
Hepatitis B and C Viruses

40. Biosafety Level 3 Definition
Agents associated with serious or lethal disease for which preventative or therapeutic intervention may be available. High individual risk, low community risk. Often risk is by inhalation route.

41. Biosafety Level 3 Requirements All requirements for BSL2 plus:
Limited lab access
2 doors in series to access lab
Able to wash entire lab
Special exhaust ventilation- not recirculated

44. Biosafety Level 3 Requirements All requirements for BSL2 plus:
High level of training
Personnel receive vaccinations if available
Work in biosafety cabinets

45. Biosafety Level 3
Examples
HIV
Mycobacterium tuberculosis

46. BSL 3 cabinet

50. HEPA Filters
Class I
High Efficiency Particulate Air filter Removes over 99.95% of particles 0.3 mm in diameter or larger

51. Class II A
Class II B1

52. Class II B2
Connection to building exhaust system required.

53. Class II B3
Connection to building exhaust system required.

55. Biosafety Level 4 Definition
Agents likely to cause serious or lethal disease for which preventative or therapeutic intervention is usually NOT available. Dangerous or exotic agents.

56. Biosafety Level 4 Requirements All requirements for BSL3 plus:
Class III biosafety cabinet or positive pressure suits
Shower/change rooms - clothing autoclaved before laundering
Air locks

57. Biosafety Level 4 Examples No Bacteria or Fungi Ebola Virus
Monkey B Virus
Marburg Virus

58. Safe Work Practices
Wash hands after work; before leaving lab; when removing gloves
No eating, drinking, applying cosmetics, handling contact lenses in lab
Maintain labs in clean, orderly fashion
Limit access to lab when work with organisms is in progress
Use good microbiological techniques
No mouth pipetting
When possible use plastic instead of glass

59. Personal Protective Equipment
Contaminated personal protective equipment (PPE) should remain in the work area- do not wear in any "clean" areas
PPE that is contaminated must be discarded as infectious waste or disinfected prior to routine laundering

60. Laminar Flow Equipment
Biosafety cabinets (BSCs)
Contain infectious agents to protect personnel and the environment
Laminar flow clean benches (LFBs)
Non-hazardous work only
Protect work from contamination

61. Laminar Flow Benches DO NOT protect personnel or the environment
Discharges HEPA filtered air across work surface toward user
DO NOT use biohazardous, radioactive, chemical, toxic, mutagenic or carcinogenic agents in a LFB
Should have a pink sticker stating this

63. Biosafety Cabinets 3 Classes
All exhaust is HEPA filtered before leaving cabinet
Class I- do not protect the work from contamination
Air entering cabinet is not filtered

64. Biosafety Cabinets Class II- 4 types- A, B1, B2, B3
Each type recirculates different amount of air
Some are hard-ducted, some exhaust into room
Class III
Totally enclosed, ventilated cabinets
Work through portals with attached gloves

65. Use of Equipment
Minimize airflow disturbances (moving in/out of cabinet, people walking by, opening doors, blocking grilles with equipment)
Wipe down surfaces with alcohol before and after work. Let run 10 minutes to clean it
Minimize use of flames in cabinet
Try not to use chemicals in cabinets- only use in Class I or II hoods that are ducted- contact DOHS
Do not store excess equipment in cabinet

66. Centrifuge Safety
Aerosol generation from broken tubes, opening tubes, decanting supernatant, resuspending materials
Balance load
Use sealed tubes, safety buckets or rotors when possible
When possible fill & open centrifuge tubes or buckets in BSC

67. Centrifuge Accidents If a tube breaks- Turn off centrifuge
Leave the lab
Call Officers
If you find a tube broke when you open the centrifuge-
Close the lid

68. Shipping/ Transporting
Who regulate transportation of biologicals????
Includes infectious materials, diagnostic specimens, dry ice
Program being developed- to include mandatory training

70. Disinfection
All equipment, environmental, and working surfaces must be cleaned and decontaminated after work with infectious materials
Broken glass or sharps shall be cleaned up using mechanical means- NOT by hand
Different levels of cleaning- decontamination, sanitization, disinfection, sterilization

71. Decontamination Use appropriate disinfectant for agents in use
Follow instructions, paying attention to dilution, shelf life, and contact time to assure effective kill
Care must be used to ensure mixing of incompatible materials does not occur

72. Infectious Waste What is infectious waste Procedures for disposal
Solid waste
Liquid waste
Sharps
Autoclaving waste
Containers and pick-ups
Mixed waste

73. Emergencies
Safety equipment
in labs
Showers
Eye washes

74. BSL2 Spills Small spills-
Wipe up with disinfectant-soaked paper towel then clean surface with suitable disinfectant
Large BSL2 Spill in BSC
Keep cabinet running during and 10 minutes after cleanup
Don PPE
Cover spill with disinfectant- let set- wipe up
Clean catch basin under work surface if spill ran there
Remove PPE when done and wash
Leave the lab, close door, post lab off-limits
Decon people if necessary
After 30 minutes, put on PPE and assemble supplies

79. Bacteria Virus Parasite Fungus Agents Lab Animals
B. anthracis, Bordetella pertussis, N. gonorrhoeae, N. meningitidis, B. pseudomallei, L. pneumophila, S. typhi, Y. pestis
Brucella sp, F. tularensis,
Campylobacter spp, C. diphtheriae, E. coli (cytotixin), H. pyroli, L. interrogans, L. monocytogenes, M. leprae, Salmonella spp., Shigella spp., T. pallidum, V. cholera
M. tuberculosis
BSL 2, BSL 3
BSL 2
BSL 3
Virus
Prion, Retrovirus
Rabies virus
Rickettsial agents, LCM, Hepatitis B, C, D
Herpes virus, Hepatitis A, E, influenza, Poliovirus, Poxvirus,
Other zoonosis virus
Parasite
All pathogenic parasites
Fungus
Blastomyces dermatitidis, C.neoformans, Histoplasma capsulatum, Sporothrix schenckii, Miscellaneous Molds
Coccidioides immitis

86. Biosafety Agents Practices Safety Equipment Facilities Level BSL-1
not generally associated with disease
·    Good microbiological practice Hand washing No eating, drinking
Pipeting devices- mouth pipeting is prohibited
BSL-2
These agents are associated with human disease
·    Limited lab access. Most work may be performed on a bench top Biohazard warning. Biosafety manual defining any needed waste decontamination or medical surveillance policies
·    Class I or II Biological Safety Cabinets. Lab coats, gloves, face protection, as needed
·    Open bench-top sink for hand washing is required
Autoclave

87. Agents Practices Safety Equipment Facilities
Level
Agents
Practices
Safety Equipment
Facilities
BSL-3
·    associated with human disease and cause illness by spreading through the air
diseases that may have serious or lethal
BSL-2 practice plus
·    Controlled access
·    Decontamination of all waste
Decontamination of lab clothing before laundering
·    Class I or II Biological Safety Cabinets (BSCs) or other physical containment devices
Protective lab clothing, gloves, respiratory protection as needed
BSL-2 plus
·    Physical separation from access corridors
·    Self-closing, double-door access
·    Exhaust air is not recirculated
·    Negative airflow into laboratory
Design includes back up/redundant systems
BSL-4
·    BSL-3 or have an unknown cause of transmission
Cause diseases that are usually life-threatening
BSL-3 practices plus
·    Clothing change before entering
·    Shower on exit
All material decontaminated on exit from facility
All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive- pressure personnel suit
BSL-3 plus
·    Separate building or isolated zone
·    Dedicated supply and exhaust, vacuum, and decontamination systems
·    Design includes back-up/redundant systems

88. Thanks you Biosafety in the Microbiological and Biomedical Laboratory
More information
Biosafety in the Microbiological and Biomedical Laboratory

94. Acknowledgements T2 (Thailand Tropical Diseases Research Program).
TRF (Thailand Research Fund).
NSTDA
ONSITE, Emory University
Faculty of Science, MU
The Virology Association of Thailand
Faculty of Medicine, KKU.