This document discusses biosafety and guidelines for working with biological and biomedical materials in laboratories. It outlines various levels of biosafety from BSL-1 to BSL-4 based on the hazards posed by different pathogens. Personal protective equipment, engineering controls, facility design, safe work practices and waste disposal procedures are described for minimizing exposure risks. Emergency response procedures for spills or accidents are also addressed. The document provides an overview of biosafety principles and regulations for safely handling infectious agents in research and clinical settings.

1. Biosafety in Microbiological and
Biomedical Laboratories
S. Wongratanacheewin, Ph.D.
Melioidosis Research Center, Faculty of
Medicine, KKU.
E-mail: sura_wng@kku.ac.th

2. September 11, 2001

3. ไทยรัฐ 19 กันยายน 2546
ไทยรัฐ 20 กันยายน
2546

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

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

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

7. CDC Bioterrorism Agents
 Category A
 Category B
 Category C

8. 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.

9. 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.

10. 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.

15. 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,

16. 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.

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

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

19. 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.

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

24. 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.

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

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

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

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

32. 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.

33. 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

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

35. 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.

36. 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

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

40. Biosafety Level 3
 Examples
 HIV
 Mycobacterium tuberculosis

41. BSL 3 cabinet

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

43. Class II A Class II B1

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

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

47. 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.

48. 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

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

50. 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

51. 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

52. 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

53. 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

54. 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
 Leave the lab
 Call Officers

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

57. 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

58. 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

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

60. Emergencies
 Safety equipment
 in labs
 Showers
 Eye washes

61. 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

62. (building name & room number, list subrooms, if any)
Hazards Within:
List hazards such as
Flammables, Acids,
Bases, General
Chemicals, high pressure
compressed gases,
Biohazards, Radioactive
materials, etc
Primary Contact: (name & office phone)
Second Contact: (name & office phone)
Building Monitor/Safety: (name & office phone)
Department Head: (name & office phone)
Fire/Police/Ambulance: 911
Envir. Health & Safety
(or RSO, if needed): 646-3327

70. 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.