This course covers biosafety concepts including risk assessment, standard microbiological practices, containment levels, and proper use and maintenance of biological safety cabinets to minimize exposure to biological agents in the laboratory. The document discusses determining appropriate biosafety levels based on risk assessment and outlines biosafety levels 1 and 2, including standard practices, safety equipment, and facility requirements for each level.
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Biosafety Essentials for Containment and Risk Assessment
1. MILWAUKEE SCHOOL OF ENGINEERING
CONCEPTS OF BIOSAFETY, BIOCONTAINMENT & RISK
ASSESSMENT
MARIAN DOWNING, RBP, CBSP, SM(NRCM)
2. Objectives of this course
• By the end of this course, you will:
– Know why a risk assessment is
necessary for all research with
biological materials
– Know the basic considerations for
a risk assessment
– Understand standard
microbiological practices for the
lab and use of the biosafety
cabinet
– Have a basic understanding of
how to dispose of waste, clean up
a spill, and what to do in an
accidental exposure situation
4. What is Biosafety?
• Fundamental objective:
– Containment of potentially harmful biological agents
• Purpose of containment:
– Reduce or eliminate exposure of lab workers, other
persons, and outside environment
5. Proliferation of Containment
• BMBL Outlines:
– Standard Microbiological Practices
– Special Microbiological Practices
– Safety Equipment
– Facilities
http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
6. What is a biohazard?
– An agent of biological origin that has the capacity to produce
deleterious effects in humans
• HBV, HCV, HIV, West Nile Virus
(WNV), Malaria, Toxoplasma gondii, Mycobacterium
tuberculosis, Staph aureus
– Infectious clinical specimens
– Infected animals
– Toxins and allergens derived from living organisms
– Materials or equipment that have come in contact with
biohazardous materials
Mycobacterium tuberculosis
7. Risk Groups of Viruses, Bacteria, Fungi and Parasites
Risk Group Agents
1 Not associated with disease in healthy adult humans
Associated with human disease which is rarely serious
2 and for which preventive or therapeutic interventions are
often available
Associated with serious or lethal human disease for which
3 preventive or therapeutic interventions may be available
(high individual risk but low community risk)
Likely to cause serious or lethal human disease for which
4 preventive or therapeutic interventions are not usually
available (high individual risk and high community risk)
8. Biosafety Levels
Risk Group Biosafety Level Agents
1 Basic – BSL1 Not generally pathogenic
2 Basic – BSL2 Community pathogens
3 High Containment – BSL3 Agents spread by aerosol route
4 Maximum containment – Extreme pathogens, no antimicrobials
BSL4 or other drugs
9. Biosafety Levels (BSLs)
• Combinations of lab practices and techniques, safety
equipment, and laboratory facilities
• Specifically appropriate for:
– Operations performed
– Known or suspected routes of infection
– Lab function or activity
• NOTE: The Risk Group of an organism
may not correspond with the
Biosafety Level
– This is determined by the Risk
Assessment
10. How to Determine a Biosafety Level?
• Conditions under which the agent ordinarily can be handled in
the laboratory
• Based on risk assessment
• Established by responsible scientists
• Resources include:
– Published data
– Biological safety officer (BSO)
– Institutional Biosafety Committee (IBC)
11. Risk Assessment
• Consideration of:
– Route of infection
– Infectious dose
– Manipulations to be done
– Volumes handled
– Virulence and pathogenicity
– Antibiotic resistance patterns
– Vaccine and treatment availability
– Other factors
12. Assessing risk
• Helps to assign the biosafety level that reduces to an absolute
minimum the worker’s exposure to agents, their risk of an LAI
(lab associated infection), and potential impact on the
environment.
• Will also assist in QA/QC and product protection.
13. Chain Of Infection Model
• What is the primary risk of working with agents?
– infecting self and others
– release of agent to the environment
• What factors have to be in place to cause an infection?
– presence of an infectious agent
– infectious dose of agent
– virulence of agent
– route of infection
– susceptible host
• All 5 factors must be present for an infection to occur
• Only 1 factor needs to be eliminated to avoid infection!
15. Individual Risk Factors
• Susceptibility to Infection
– Age
• Some infections more severe in children (RSV, West
Nile)
• Some infections more severe in adults (mumps, chicken
pox, measles)
• Immune function usually decreases > middle age
– Sex
• TB = greater infectivity in women
• Listeria monocytogenes greater risk for women
• Mumps infection in men (testicle inflammation)
CDC/James Gathany
16. Susceptibility to Infection
– Genetics
• Inherited immune problems
– “Healthy Adult”
• Factors affecting immune status: CDC/James Gathany
– Chemotherapy, smoking, immune suppressive drugs,
– Lupus, HIV, etc.
– Vaccination Status
• Reduces chance of infection
• Reduces severity of infection (chickenpox)
• Never reduce risk 100%
– Reproductive Risks
• Risks to fetus: Toxoplasma, Listeria, CMV, Rubella, HIV
• Mother may be asymptomatic, but fetal effects are severe
17. Biocontainment Concepts
• Containment Barriers
Primary - BSC’s, personnel protective gear, containment
equipment
Secondary - Room, systems
Tertiary - Containment around systems
• Access Control and Separation
• Redundancy and Reliability
• Decontamination
18. Biosafety Level 1
• Basic lab for working with non-infectious agents
– Not known to cause disease in healthy adults
– May still have hazardous chemicals, radiation, magnets
– Work with established animal tissue culture cell lines, pcr
testing (polymerase chain reaction), E. coli, yeast, etc.
• Low individual risk, low community risk
• May still be able to cause an infection
– Immune suppressed individuals
19. Biosafety Level 2
• Hospital clinical lab or standard R&D Lab
– Work with infectious agents
• Non-aerosol spread (HIV, HCV, Shigella)
• Moderate individual risk, low community risk
• Usually treatments or vaccines available
– Eyewash station
– Waste decontamination available
– BSCs installed as needed
– Inward air flow
– Recirculated to lab areas only
Bacillus anthracis
Photo: CDC
20. Biosafety Levels 1 & 2
BSL1 BSL2
Agents Not known to cause Associated with human disease, but not
disease in life-threatening
Healthy adults
Practices Standard Microbiological BSL1 plus:
Practices •Limited access
•Biohazard signs
•Sharps precautions
•Biosafety manual with waste handling and
medical surveillance
Safety No special equipment Biosafety cabinets or other physical
Equipment required containment for splashes or aerosols
PPE: lab coats, gloves, eye/face protection
Facilities Sink required BSL1 plus:
•Autoclave
20
23. Hierarchy Of Controls
Most effective
Avoidance
Engineering Controls
Work Practices
PPE Least effective
24. Engineering Controls
• Centrifuges with aerosol safety cups
• Substitution, replacing sharps with non-sharp objects
• biosafety cabinets
• Hand and eye washing facilities
• Safe needles/scalpels
• Sharps containers
BD Disposable Scalpel
25. Safe Work Practices
Fill line
• Do not bend or recap needles
• Place sharps in the appropriate container
• Do not eat, drink, smoke, apply cosmetics or handle contact lenses in the work
areas
• Do not store food in the laboratory
• Minimize splashing, spraying, and
generation of droplets
• Do not reach into trash/sharps
containers
• Minimize glassware/sharps hazards
26. Laboratory Work Practices, continued
• Wash hands frequently
• Keep your hands away from your face
– May infections are spread by
mucous membrane contact
• Do not mouth pipet
– Also mucous membrane contact
• Wipe down work area with
disinfectant (daily)
27. Hand Decontamination
• Hand washing is the single most important procedure for
preventing infections
http://www.cdc.gov/handhygiene/training.html#posters
27
28. Routine Hand Hygiene
• Soap and water
– Most of benefit is from
removal of transient
organisms on surface
epidermal layers
– Also removes soil, such as
dirt, blood, feces
– Soap and water, if available,
are better than hand
sanitizers in most cases
30. Other Aerosol Generators
• Vigorous shaking
• Any fluid manipulation
• Pouring
• Opening lyophilized cultures
• Flaming loops/needles (Bunsen burner)
• Removing needle from rubber
diaphragm on vial or sampling port
CDC/Greg Knobloch
31. Aerosols
• Lab equipment
– Produces aerosols more efficiently than
natural methods (coughing, sneezing, etc.)
• Materials encountered
– Higher titered than in natural setting
• Modest aerosol-producing activity could be infectious
– Larger quantities being manipulated
– Agent that is not normally aerosol-transmissible
may be transmitted under these circumstances
• Class I or II BSC for containment
of small equipment
(blenders, homogenizers, etc.)
Banthrax Versa Dome
32. Safe Pipeting
• Never blow out last drop in pipette
• Use pipette aids with filters
• Cotton plugged pipettes provide some protection
• Horizontal pipette collection tray w/ disinfectant
– Disinfectant in upright pipet collection vessel
forces aerosols out top of pipette into the air
• Pipet over disinfectant-soaked pad
• Never mix by suction + expulsion
• Discharge liquid down side of container
• Deliver as close as possible to contents
33. Personal Protective Equipment (PPE)
• Safety glasses with side shields
– Wear when entering the work area
• Lab coats
– Leave in the work area
– If contaminated, decontaminate before placing in laundry
bag
– Wear buttoned with sleeves rolled down
• Closed toe shoes in the laboratory – no sandals
34. Gloves
• Check for pinholes before wearing
• Do not touch common items with
gloved hands (e.g., door knobs, phones)
• Consider chemical compatibility (solvents, etc.)
• Don’t reuse disposable gloves
• Waterproof needed for working with human-sourced materials
• Do not use powdered latex gloves
• NOTE: if double gloving, can combine different types (like latex
and nitrile)
• Wash hands after removing
• DO NOT wear outside the lab!
36. Know Your “Hoods”!
Chemical Fume Hood Biological Safety Cabinet Laminar Flow Clean Air Center
• Closes completely: either • Fixed sash opening (8 in.) • HEPA filter visible in
horizontally or vertically (alarmed) rear or top of unit
• Not meant for sitting • Sash moves up but does • Usually no sash or
• Negative pressure not close completely sash is fixed
• May have solvent/chemical • Designed for seated work • Positive pressure – air
storage underneath • Negative pressure blowing into face or
breathing zone
Pictures are courtesy of MSOE
37. Biological Safety Cabinets (BSCs)
• Role:
– To protect the user from the samples
– To protect the environment from the
samples
– To protect the samples from external
elements
• Efficacy
– Dependent on scrupulous work
practices
– Read BSC instructions in
Biosafety Manual
38. Biological Safety Cabinets (Continued)
• There are 3 classes of BSC
– Class I: not often used MSOE Certification Sticker
– Class IIA and B: most often found in laboratories
– Class III: not often used
• Certification:
– Annually
– When moved
– After repairs, filter changes
39. Class II BSC
• Most commonly found BSC in laboratories
• HEPA-filtered vertical laminar air flow
– User and sample protected
• HEPA-filtered exhausted air
– User and environment protected
• There are 2 types and 2 subtypes (A1-2-B1-2)
40. Class II A2 BSCs
• 70% recirculated air through HEPA
filter
• A2 may be used with nonvolatile
toxic chemicals or radionuclides
– minute levels of volatile toxic
chemicals and radionuclides
• If exhausted outside
• A2 has airflow of 100 lfm
Ethanol fire in BSC (AIHA)
41. Class II A2 BSC
Courtesy of MSOE
CDC drawing
The Class II, Type A2 BSC may be connected to the
building exhaust system.
42. Clean Bench
• Not to be confused with Class I BSC
• Inflow air is HEPA filtered
• Exhaust air is not filtered
• Used for Microbiology clean preparation (making agar plates)
and molecular work (PCR)
• Air flow can be vertical or horizontal
Courtesy of MSOE
43. BSC Practices
• Thoroughly clean BSC before/after use
• Do not cover the front grills or block rear vent
• Decontaminate all materials in and out
• Discard containers inside BSC
• Adjust chair height to look down through sash
• Wait 15-20 minutes for HEPA filters
to electrostatically charge before
starting work
44. BSC Work Practices
• Slow arm movements, move in and out in a
straight line, do not sweep.
• Work over absorbent toweling
• Clean spills immediately, wait for BSC to purge
• Open items held at an angle, cap in hand
BMBL, 5th ed.
45. BSC Work Practices
– Work towards the rear of the cabinet
– Equipment in back 1/3 of cabinet
• cease other activities while operating equipment
– Do not rely on UV decontamination
• Bulb must be dusted and tested regularly
– Vacuum trap and filter setup:
BMBL, 5th ed.
In line HEPA filter
Collection vessel Overflow vessel with
disinfectant
47. BSC Practices
• Load only those materials needed inside
cabinet
– No withdrawing hands for discard or
supplies
• Wait 5 minutes after loading BSC before
working
• Do not cross hands while working or sway
side to side
• No Bunsen burners or alcohol burners in
the BSC Corning products
– Use disposable loops and needles
– Bacti-cinerator or micro-burner
48. What Do You Notice About this Picture?
CDC photo: James Gathany
50. Disinfection
• Solutions commonly used
– 2 % bleach
• Prepare fresh daily
– 70% ethanol
• For cleaning only – not considered an
appropriate disinfectant for work with
human specimens
– Cavicide
– BACDOWN Detergent Disinfectant
– Dispatch
• Surfaces
– Bench top
– BSC
– Fume hoods
– Centrifuges
– Equipment
– Incubators
– Floors
51. Disinfectants
• Must follow manufacturer’s label
instructions
• Disinfect bench tops at least daily
• Decontaminate equipment sent for
repair or disposal
– Use Decontamination
Verification Tag
52. Sharps
• Any item capable of puncturing the skin
– needles
– scalpel blades
– razor blades
– glass Pasteur pipettes
– broken contaminated glass
– In Wisconsin – syringes with needles attached
• Eliminate, or substitute safer engineered sharps!
53. Examples of Sharps Alternatives
• Hypodermic needles
• Use safe needles or safe syringes
• Pasteur pipet
• Use plastic transfer pipet
• Scissors
• Use blunt-tipped safety scissors
• Box Cutters
• Use self-retracting utility knife
• Document consideration of
safer devices annually
54. Sharps Precautions
• Do not overfill sharps container
– Seal before waste reaches indicator line
on container
• Keep upright
• Have adjacent to work area
• NOT for chemical disposal
• DO NOT:
– Recap needles before placing in sharps
container
– Remove needles by hand
55. Waste Disposal
• Regular Trash
– Items that are not contaminated
• Paper
• Disposables that have been chemically
decontaminated
– E.g. tissue culture flask, tubes,
plastic vials, paper towels, gloves
• Autoclave
– Microbial cultures
– Tissue culture materials that have not been OR
chemically disinfected
– ALL rDNA materials that have not been
chemically disinfected
– Contaminated gloves, spill cleanup
materials, etc.
• NOTE: ALL RECOMBINANT DNA
CONTAMINATED MATERIALS MUST BE
TREATED AS THOUGH THEY WERE
INFECTIOUS!
57. Laundry Requirements
• Change lab coats regularly
• No sorting in the workplace
• If there are spills on the lab coat:
– Remove
– Spray with disinfectant
– Rinse with water
• Lab coats cannot be taken
home for cleaning
– The lab technician will coordinate
the laundering of lab coats
58. Labeling and Shipping
• Label vials, tubes, etc.
– Biohazard label
• Or, label box or carrier
• Transfer internally in a zip lock bag with absorbent inside.
• Need to ship?
– Only trained employees allowed to package materials for
shipping
• Renewed every 2 years
– Know status of materials to be shipped
• Most MSOE materials will be exempt from shipping
regulations, but must still be packaged appropriately
59. All Biosafety Levels
• Post policies and procedures for entry
• Provide appropriate warning signs
61. Spills
• Clean up and report spills per Biosafety Manual Procedure
• Lab workers/students must be able and equipped to handle spills
of biological material
• Absorb liquid, preclean with detergent (to remove organic
materials), then wipe with an approved disinfectant
• Use disinfectant appropriate for agent
• Use full strength disinfectant on large spills (dilution effect)
• NEVER pick up broken sharps with fingers
Courtesy of MSOE
62. Emergency Procedures
Accidental Exposure in the Lab
• Remove PPE and provide immediate
care to the exposure site:
• Wash wounds and skin with soap and water for 15
minutes
• Flush mucous membranes with water for 15 minutes
• Notify the Laboratory Supervisor or the Lab Technician
• Follow directions of Supervisor, or, if after hours, visit the
Emergency Clinic
63. Thank You!
Be sure to complete the
associated quiz for this training
module.
Editor's Notes
Hepatitis Banc C – RG 2
Decon into cabinet with alcohol or disinfectant (remove environmental contaminants)Decon out with disinfectant, especially if using infectious organisms. This includes vortex mixers, pipets, tip boxes, discard bin, plastic discard bag, etc.
Moldy collection flaskIf there was any disinfectant in the flask, it is not sufficient or fresh (see previous slide)No in line filterNo secondary container (if outside BSC) to prevent breaking and leakageFlask should be netted if glass, or plastic
When laboratorians request a BSC decon because of contamination problems, 99% of the time it is actually due a dirty cabinet, poor technique or incubator problem. It is very difficult to dislodge particles (viruses, bacteria, etc.) once they have lodged in the HEPA filter (Van der Waals forces) and the chances that the HEPA filter is the problem are minimal.
She is actually working too close to the front of the cabinet!Risking infection from her materialsRisking contamination of her materials**Julie would like us to add another thing that is wrong: She generally doesn’t accept a face shield without safety glasses/goggles underneath
Julie’s note: Students should notify the laboratory supervisor or lab technician only. As they will contact EHS (Julie). Students shouldn’t have to contact two sets of people.