biosafety level 2 worker training
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ONLINE SELF-STUDY
Biosafety Level 2 Worker Training
Basis for Biosafety Level 22
The principles of biological safety at UNC-CH draw on multiple sources:
At the FEDERAL level, from the
– OSHA Bloodborne Pathogens
Standard
– NIH Guidelines for rDNA
– CDC/NIH published Biosafety in Microbiological and Biomedical Research Laboratories
At the STATE level
– Mostly define Regulated Medical Waste
And at the LOCAL/INSTITUTIONAL level
– IBC (Institutional Biosafety Committee)
– Laboratory and Chemical Safety Committee
3
Principles of Biosafety at UNC-CH
What is a Biological Safety Level ?
In the U.S., biosafety levels are defined in two documents
generated at the federal level:
1. Biosafety in Microbiological and Biomedical Research
Laboratories from the Centers for Disease Control and
Prevention (CDC) and the National Institutes of Health
(NIH).
2. The NIH Guidelines for Research Involving Recombinant
DNA Molecules.
These documents designate four biosafety levels that
provide containment criteria from low hazard to high
hazard.
BSL4
BSL3
BSL2
BSL1
High Hazard
Low
Hazard
“Biosafety Level 2 practices, equipment, and facility
design and construction are applicable to clinical,
diagnostic, teaching, and other laboratories. . .”
--BMBL 5th edition
4
Elements of a Biological Safety Level
(1) characteristics of the microorganisms,
(2) planned manipulations in the research,
(3) primary protective barriers from infection,
(4) facilities (secondary barriers) where the
work will occur.
All biosafety levels build upon
“standard microbiological practices.”
Elements of Biosafety
(1) Microorganism
characteristics
(2) Manipulations
(3) Primary Containment
& Protection
(4) Facilities (secondary)
A Biological Safety Level is a level of containment
appropriate for the:
5
Standard Microbiological Practices
Daily practice of these activities is critical in every laboratory safety program:
• Wash hands after handling biologicals, taking off gloves and before leaving the lab.
• Decontaminate work surfaces daily and after spills.
• No eating, drinking, smoking, or applying cosmetics in the lab.
• Always use mechanical pipetting devices (never mouth pipette).
• If you wear contact lenses, consider wearing goggles or a face shield while working.
• Avoid using hypodermic needles. Refer to the Sharps Handling and Disposal section
of the Biological Safety Manual.
• Use procedures that minimize the formation of aerosols.
• Use personal protective equipment (e.g., lab gowns, coats, and gloves).
• Place all solid biological waste in orange autoclavable bags for disposal. Liquids
must be disinfected before sink disposal.
6
Elements of Biosafety Biosafety Level 1
at UNC-CH
Biosafety Level 2
at UNC-CH
(1) Microorganism characteristics Not known to consistently cause diseases in healthy
adults.
Associated with human disease which is rarely
serious and for which preventive or therapeutic
interventions are often available.
(2) Manipulations Standard Microbiological Practices BSL-1 practices plus documentation:
Restricted access; Biohazard warning signs;
Biosafety manual defining
“Sharps” precautions, Biowaste practices,
Medical surveillance, &
Spill Clean-up.
(3) Primary Containment & Protection Gloves, lab coat, and eye protection are used. BSL-1 protection plus:
Physical containment for splashes/
aerosolization; Biosafety Cabinets: aka “tissue
culture hoods”
(4) Facilities (secondary) Handwashing sink, safety shower/eyewash,
autoclave
Same as BSL-1
Why Are We Concerned with BSL-1?
BSL-2 incorporates all aspects of BSL-1.
BSL-1 is designed for work with microbes that are not known to cause disease in
healthy human adults. As such, standard laboratory practices are acceptable including
use of gloves, lab coats and eye protection. Lab facilities at BSL-1 at UNC include a
handwashing sink, safety shower/eyewash, and an autoclave to sterilize biohazard
waste.
7
Knowledge Review
1. Why is BSL-1 a concern for workers at BSL-2?
a. BSL-1 Standard Microbiological Practices are used at BSL-2.
b. The CDC & NIH definitions for BSL-2 build from BSL-1.
c. All of the above.
2. Identify the federal documents that define BSL-2 (select all that apply):
a. Biosafety in Microbiological and Biomedical Research Laboratories
b. The Biological Safety Manual
c. The NIH Guidelines for Research Involving Recombinant DNA Molecules.
d. The OSHA Laboratory Safety Standard
8
Risk Assessment9
Risk Assessment
A proper risk assessment
must consider each element
in the chain.
10
A thorough risk assessment determines the proper biosafety level.
Start with the organism you are working with. Your first question
should be: is the microorganism I’m working with a pathogen of
sufficient virulence? If you are unsure, contact EHS for guidance.
Common microbes worked with at
BSL-1 include E.coli strains used
for cloning, the yeast S. cerevisiae
and other exempt organisms under
the NIH Guidelines.
Sources to Determine Risk Group
Agent summary statements in the CDC/NIH document
Biosafety in Microbiological and Biomedical
research Laboratories.
Appendix B Classification of Human Etiologic Agents
on the Basis of Hazard from the NIH Guidelines.
The OSHA Bloodborne Pathogen Standard
interpretation letter about risk for all human cell
lines (must be used at BSL-2).
The American Biological Safety Association’s (ABSA)
database of Risk Group Classification for Infectious
Agents.
The Public Health Agency of Canada’s MSDS for
Infectious Substances.
Here are resources that are helpful in determining risk group:
11
Risk Assessment for BSL-2
A proper risk assessment
must consider each element
in the chain.
12
Elements of Biosafety
(1) Microorganism
characteristics
(2) Manipulations
(3) Primary Containment
& Protection
(4) Facilities (secondary)
Your risk assessment is a guide for the
selection of appropriate biosafety levels
and microbiological practices, safety
equipment, and facility safeguards.
Your risk assessment will be used to alert
others to the hazards of working at BSL-2
and to the need for developing
proficiency in the use of safe practices
and containment equipment. Successful
control of hazards in the laboratory also
protects persons not directly associated
with the laboratory, such as other
occupants in the building, infrequent
visitors (e.g. maintenance), and the public.
Elements Biosafety Level 1
at UNC-CH
Biosafety Level 2
at UNC-CH
(1) Microorganism
characteristics
Not known to consistently
cause diseases in healthy
adults.
Associated with human disease which is
rarely serious and for which preventive
or therapeutic interventions are often
available.
(2) Manipulations Standard
Microbiological Practices
BSL-1 practices plus documentation:
Restricted access; Biohazard warning
signs; Biosafety manual defining
“Sharps” precautions, Biowaste practices,
Medical surveillance, &
Spill Clean-up.
(3) Primary
Containment &
Protection
Gloves, Lab coat, eye
protection
BSL-1 protection plus:
Physical containment for splashes/
aerosolization; Biosafety Cabinets:
(tissue culture hoods)
(4) Facilities
(secondary)
Handwashing sink, safety
shower/eyewash,
autoclave
Same as BSL-1
With elevated risk, comes elevated containmentFollow the red arrow ( ) to compare the higher level
13
Knowledge Review
1. When you begin a new project at BSL-2, what is the first thing you should consider in your risk assessment?
a. What type of biosafety cabinet should be purchased and where will it be located?
b. What is the risk group of the material that will be used?
c. What manipulations will produce aerosols?
d. Will I concentrate human pathogens or work with concentrated stocks?
e. What is the largest volume of infectious material that could spill?
2. If you increase the concentration or the volume of experiments with risk group 2 agents, you should
a. Do the experiment in a biosafety cabinet
b. Review the original risk assessment to determine safety enhancements
c. Do the experiment at night so nobody knows
d. Warn others in the lab of your plan
Which is not true of both BSL-1 and BSL-2 at UNC?
Standard Microbiological Practices are followed.
Biohazard waste is collected and autoclaved according to the University Biohazard Waste Disposal Policy.
Work is conducted with microbes that are not known to consistently cause disease in healthy adults.
A handwashing sink, eyewash & safety shower should be readily available.
Which of the following material(s) must be handled at BSL-2?
Well characterized human cell lines.
Any risk group 2 agent identified by the NIH Guidelines .
Human serum samples.
All of the above.
B & C only.
14
Biosafety Level 2
at UNC15
Who Implements the Biological Safety Level at UNC?
Principal Investigator
EHSTrained Lab
Workers
16
Biosafety Level 2 is always
designated before actual work with
the potentially infectious agent
begins. Because of the elevated risk
status, implementation of BSL-2
requires communication between the
Principal Investigator, EHS, and
properly trained lab workers.
There may be instances where agent or procedural characteristics create
unique hazards such as the potential for aerosolization of pathogens
cultured in concentrations higher than found in nature. In such instances,
Principal Investigators, lab supervisors, and/or EHS will seek enhanced
biosafety requirements during the risk assessment. These requirements
are then carried out by lab workers.
Responsibilities of the Principal Investigator at BSL-2
Principal Investigator
EHSTrained Lab
Workers
17
The Principal Investigator assumes the day-to-day
responsibility for ensuring workers’ safety and proper
training to follow BSL-2.
Specific to this responsibility is:
1. Completing the Laboratory Safety Plan Biohazards Registration (Schedule F) to register
biohazards used at BSL-2 on UNC’s campus. The Biohazards Registration form establishes
criteria for each entry such as vaccinations, personal protective equipment, handling practices
including disinfection procedures, and medical surveillance as applicable. It also prompts labs to
complete the required BSL-2 Checklist.
2. Adopting policies/procedures from the UNC Biological Safety Manual and ensuring workers have
access to this manual at all times.
3. Ensuring that BSL-2 lab workers demonstrate proficiency in standard and special microbiological
practices before working at BSL-2.
4. Ensuring that all lab workers complete their online review of the Lab Safety Plan annually after
the Lab Safety Plan is updated with EHS.
5. Restricting access to the BSL-2 work as appropriate.
“Restricted Access”
Restricted access is important at BSL-2. This means:
Immunocompromised individuals are advised of the increased risk and an individual risk assessment can be conducted for these individuals
Entry requirements are communicated to workers entering the lab (such as immunizations, if appropriate);
Doors are kept closed during experiments and;
An EHS-approved sign indicating the presence
of biohazards is to be posted at all access
areas of the BSL-2 lab. Signs are available on
the EHS website.
18
19
No sandals or open-toed
shoes in the BSL-2 (or any)
laboratory.
Eliminate accidental routes of entry
Workers not wearing closed-toed shoes at BSL-2 is evidence of a
lack of training in standard microbiological practices.
Appropriate footwear
BSL-2 Procedures
are Documented by the PI at UNC
Federal Requirements
(CDC/NIH, BMBL 5th ed.):
“Incorporated into
Laboratory Safety Plan”
“Personnel are advised of
special hazards”
“Personnel are required to
read and follow Biological
Safety Manual”
Compliance at
UNC:
Update Biological Hazards
Form (Schedule F) & PI
completes the online BSL-2
Checklist.
Minimum: Orientation &
annual in-house training
UNC Biological Safety
Manual is adopted, PI
completes the online BSL-2
Checklist.20
BSL-2 Checklist
21
Completing the BSL-2 checklist ensures
you are meeting the latest
requirements from the federal
(2007 changes to the BMBL--5th
edition) , state (waste and OSHA
requirements), and local
(including university requirements)
levels for BSL-2.
A completed BSL-2
Checklist is a one-time
requirement for every
space registered at BSL-2
on the Laboratory Safety
Plan. It must be completed
online.
Responsibilities of EHS at BSL-2
Principal Investigator
EHS
Trained Lab Workers
22
UNC devotes resources to laboratory and biological
safety. The department of Environment, Health &
Safety supports BSL-2 by:
Developing the University Biological Safety Manual;
Review and approval of BSL-2 Checklist and Biohazards Registration Form
(Schedule F)
Providing safety training;
Providing consultation and advice;
Inspecting laboratories for compliance, and;
Establishing and enforcing safety policies instituted by the Laboratory and
Chemical Safety Committee and the Institutional Biosafety Committee.
Responsibilities of Lab Workers at BSL-2
Principal Investigator
EHS
Trained Lab Workers
23
Working at BSL-2 requires strict
adherence to biosafety requirements and
proficiency in performing various
procedures. The remainder of this course
is dedicated to providing a foundation of
BSL-2 practices and regulatory
requirements:
•Communicating Biohazards
•Recombinant DNA
•Spills and Disinfection
•Biohazard Waste Management
•Exposure
•Aerosolizing Procedures
•The Biological Safety Cabinet
•Prudent Practices at BSL-2
Communicating Biohazards at BSL-2
The standardized label with the biohazard symbol
communicates the presence of potentially
biohazardous materials to lab workers, visiting
professors, Facilities Services workers, housekeeping
personnel, vendors, and others who may come into
contact with a BSL-2 laboratory.
At UNC, employees are trained to use and recognize the universal
biohazard symbol and the word “BIOHAZARD.”
24
Post all Equipment at BSL-2
All equipment used in BSL-2 or greater containment must be posted with a biohazard label. For example, hoods, freezers, incubators, and centrifuges, etc. are labeled if they are used with material on your Lab Safety Plan Biohazards Registration form (Schedule F).
Also, transport containers and biohazard waste containers must display a biohazard label on the outermost part.
25
Knowledge Review
1. Who is ultimately held accountable for the policies, practices, training, and documentation of BSL-2 safety in the laboratory?
a. The Principle Investigator
b. EHS
c. The lab workers
d. Both B and C
2. A proper risk assessment for biosafety
a. Is documented in the Lab Safety Plan Biohazards Registration section and updated at least annually.
b. Includes microorganism characteristics, manipulations, containment and protection, and facilities.
c. Is referenced by the PI in alerting all lab workers and others to the hazards of working in this BSL-2 laboratory space.
d. All of the above
e. A & B only
3. The universal biohazard symbol and the word “BIOHAZARD” provide a standardized communication method to inform others of the hazard. At
BSL-2, they must be located where?
a. At the access door to BSL-2 areas.
b. On lab equipment used to manipulate or store material handled at BSL-2.
c. On biohazard waste including contaminated broken glass boxes.
d. On containers used to transport BSL-2 material within or between buildings.
e. All of the above
f. B & C only
26
CDC/NIH Vertebrate Animal Biosafety
Level Criteria27
CDC/NIH Vertebrate Animal Biosafety
Level Criteria
For all Animal Biosafety Levels (1 - 4)
IACUC Approval
Authorized access to facilities
Animal handling training
Medical surveillance program
Written safety manual(s)
Pest control program
CDC/NIH Verterbrate Animal
Biosafety Level Criteria
For all Animal Biosafety Levels (1 - 4)
no eating, drinking, smoking, touching face
no food or drink storage within facilities
minimize aerosols
decontaminate work surfaces, equipment
transport wastes in leak-proof covered containers
handle sharps safely
CDC/NIH Verterbrate Animal
Biosafety Level Criteria
For all Animal Biosafety Levels (1 - 4)
Protective clothing recommended, not worn outside
facility
Facilities separate from general building traffic
External doors self-closing/self-locking
Animal room doors open inward, self-closing
Water resistant, easily cleaned surfaces (horizontal
surfaces kept to a minimum)
CDC/NIH Verterbrate Animal
Biosafety Level Criteria
For all Animal Biosafety Levels (1 - 4)
Sealed, break resistant windows
Floor drain traps filled with water or disinfectant
No recirculation of exhaust air
Rooms have negative pressure gradient to adjacent
hallway
Hand washing sink available in facility
180 F cage wash rinse temperature
Adequate illumination
CDC/NIH Vertebrate Animal Biosafety
Level Criteria
Animal Biosafety Level 2 (ABSL2)
Restrict access to few as possible (advise support staff
of potential hazards)
Biohazard sign posted on animal room entrance
(hazard ID, contact info., & entry requirements)
Immunizations, serum surveillance
Labeled leak-proof carriers, exterior disinfected before
transport (wastes, tissues, etc.)
Safe sharps policies (plasticware, safe sharps devices,
limited use of needles/syringes)
A sign incorporating the universal biohazard symbol must be
posted at the entrance to areas where infectious materials
and/ or animals are housed or are manipulated when infectious
agents are present. The sign must include the animal biosafety
level, general occupational health requirements, personal
protective equipment requirements, the supervisor’s name (or
names of other responsible personnel), telephone number, and
required procedures for entering and exiting the animal areas.
Identification of all infectious agents is necessary when more
than one agent is being used within an animal room.
http://ehs.unc.edu/ehs/forms.shtml
FOR ANIMAL USE Example of appropriate signage for ABSL2 laboratory doorway: PLEASE COMPLETE and
POST IT!
AUTHORIZED PERSONNEL ONLY!
BIOHAZARD
ANIMAL BIOSAFETY LEVEL
Principal Investigator: ________________________
Agent (s): ___________________________________
Bldg: ___________ Room: _____________ (must be space approved by DLAM for
ABSL2)
Special Instructions/ Requirements Prior to Entry or Exit (i.e. personal protective
equipment, vaccination):
EMERGENCY CONTACT/ ADVICE
CONTACT
WORK PHONE
HOME PHONE or PAGER
PRIMARY
SECONDARY
BIOSAFETY OFFICER
2
CDC/NIH Vertebrate Animal
Biosafety Level Criteria
Animal Biosafety Level 2 (ABSL2)
Limited to animals dedicated to work
Exposures, spills reported immediately
Gloves, gowns, uniforms or lab coats worn in rooms
(removed prior to exit)
Face protection (goggles, safety glasses, full face
shields, masks) selected on basis of risk
Biosafety cabinets used to confine aerosol
procedures
CDC/NIH Vertebrate Animal
Biosafety Level Criteria
Animal Biosafety Level 2 (ABSL2)
Filter top cages, cage dumping containment
stations used where appropriate
Autoclave available
hand washing sink in animal room
Recombinant DNA38
Institutional Biosafety Committee (IBC)
Have your rDNA projects been registered with the IBC?
39
Institutions that receive support from the National Institutes of Health (NIH) for
recombinant DNA (rDNA) research are required to establish and register an
Institutional Biosafety Committee (IBC) with the NIH Office of Biotechnology Activities
(OBA) in compliance with the NIH Guidelines.
The IBC reviews protocols when there could be a safety concern with rDNA
research.
This process is separate from the Lab Safety Plan Biohazards Registration
form (Schedule F).
Major Actions under NIH Guidelines
Experiments that compromise the control of disease agents in
medicine through deliberate transfer of a drug resistance trait Require IBC, RAC, NIH Director Review and Approval prior to the
initiation of work
Containment determined by NIH/OBA
Example: Deliberate Cloning of Toxin Molecules Lethal to
Vertebrates at an LD50 of Less Than 100 Nanograms/Kg of
Body Weight (e.g., Botulinum Toxin)
Requires: NIH/OBA, IBC Review and Approval prior to the initiation of
work
40
Exempt Experiments
Some recombinant DNA work is exempt from the NIH
Guidelines (Section III-E). All such research must
be conducted at BSL-1. This group includes (but is
not limited to) experiments that:
use as host vector systems E. coli K 12,
Saccharomyces cerevisiae, Saccharomyces
uvarum, or Bacillus subtilis, and their plasmids;
use rDNA molecules containing less than one-
half of any eukaryotic genome that are
propagated and maintained in cells in tissue
culture.
41
For more information about rDNA work that is exempt, visit the EHS
website at http://ehs.unc.edu/ih/biological/dna.
Experiments Requiring Prior Approval
The following are examples of rDNA experiments that require prior approval from either the NIH,
Recombinant DNA Advisory Committee (RAC), Food and Drug Administration, and/or the IBC.
These experiments are to be described on the appropriate registration form, and sent to
Environment, Health and Safety.
Gene transfer experiments in humans;
Genes for toxins lethal to vertebrates;
Release of genetically engineered organisms to the environment;
Those using human or animal pathogens (at risk group 2 and higher) as host-vector systems,
including adenovirus vectors and murine retroviruses that infect human cells;
Cloning DNA from human or animal pathogens (biosafety level 2 and higher) into a non-
pathogen host vector system;
Cultures of more than 10 liters; and,
Experiments involving whole plants or animals, including creation of transgenic organisms.
42
An in-depth training required for Principal Investigators that are
responsible for rDNA research at UNC-CH is located at
http://ehs.unc.edu/biological/dna/
Knowledge Review
1. What do these items have in common: NIH OBA; IBC; rDNA
a. They offer authoritative resources for determining risk group
b. They are defined in the BSL-2 Checklist
c. They are defined in the NIH Guidelines
d. They are effectively contained by a HEPA filter that is certified annually
2. Responsibilities of the Principal Investigator at BSL-2 include?
a. Updating the Lab Safety Plan Biohazards Registration annually and providing proper PPE to employees.
b. Registering non-exempt rDNA experiments with the IBC.
c. Ensuring workers have access to and follow the Lab Safety Plan and Biological Safety Manual at all times.
d. All of the above.
e. A & C only.
43
Spills and Disinfection44
SPILLS
Every lab member working at BSL-2 must follow the proper protocol in the
event of a biohazardous spill.
There are three standard spill protocols listed on the Biohazards
Registration Form (Schedule F) of your Laboratory Safety Plan:
1. Small spill in a biological safety cabinet
2. Large spill in a biological safety cabinet
3. Spill outside the biological safety cabinet (in the open lab)
These should be posted in your lab when the Lab Safety Plan is completed.
45
Protocol for Small Spill in a
Biological Safety Cabinet 46
1. Contain spill with absorbent paper.
2. Dampen paper with disinfectant. Allow to stand for 20
minutes.
3. If sharps/glass are present, use mechanical means to
collect the waste (eg. forceps, cardboard flaps).
4. Remove gloves after area is decontaminated.
5. Wash hands.
Protocol for Large Spill in a Biological
Safety Cabinet 47
Large Spill Inside BSC:
♦ Cover spill with paper towel, pour bleach around area, remove arms from cabinet, bring shield down
and allow air to flow for 10 min.
♦ Discard any contaminated PPE into appropriate containers and put on fresh PPE.
♦ After 10 min, lift shield, collect spill with paper towels and discard soiled towels into double autoclaved
bags.
♦ Wipe down with 10% bleach, the inside, sides and top, followed by thoroughly wiping down the same
areas with 70% Ethanol.
♦ Flood top tray, drain pans and catch basin below work surfaces with disinfectant and allow to stand for
20 minutes.
♦ Lift out tray and removable exhaust grille work. Wipe off top and bottom (underside) surfaces with
disinfectant sponge or cloth. Replace in position.
♦ Remove all PPE and discard in an autoclave bag. Wash hands.
♦ This procedure does not decontaminate the interior parts of the cabinet such as the filters, blowers
and air ducts. If the entire cabinet needs to be decontaminated with formaldehyde gas, contact EHS
(962-5507).
Protocol for Spill Outside the Biological Safety Cabinet
(in the open lab)48
1. Remove contaminated outer gloves.
2. Notify others in lab of spill.
3. Decontaminate all personnel, clothing and exit laboratory
4. Notify PI and EHS.
5. Allow aerosols to settle for 30 minutes.
6. Re-enter wearing PPE (including respiratory protection).
7. Carefully dilute spill from outside edges using appropriate disinfectant.
8. Allow 20 minutes contact time.
9. Clean-up with paper towels.
10. Decontaminate clean-up materials.
11. Wash hands thoroughly.
Remember your sharps precautions! Contaminated broken glassware is cleaned
up by mechanical means (e.g. tongs, forceps, pieces of cardboard).
Approved Disinfectants
Characteristics of microorganisms affect their resistance to
disinfection:
Prions
Bacterial spores
Mycobacteria
Nonlipid viruses
Fungi
Bacteria
Lipid Viruses
Most Resistant
Least Resistant
49
An appropriate disinfectant is determined during the initial risk assessment
and listed on the Biohazards Registration form (Schedule F) of the Lab
Safety Plan. Each worker needs to ensure they allow for the proper
contact time for the selected disinfectant.
A list of chemical disinfection methods that are recognized by the National
Institutes of Health, the CDC, or the American Biological Safety
Association can be found on the next slide.
Work surfaces must be
decontaminated
•After work,
•After spills,
•At the end of each day and,
•Prior to the repair or surplus
of equipment
To locate proprietary disinfectants, search for the product name at
http://ppis.ceris.purdue.edu/ or refer to the EPA registered disinfectants
website at http://www.epa.gov/oppad001/chemregindex.htm
Decontaminate Work Surfaces50
51
Paraform-
aldehyde
(gas)
Quaternary
Ammonium
Cmpds.
Phenolic
Cmpds.
Chlorine
Cmpds.
Iodophor
Cmpds.
Alcohol
(ethyl or
isopropyl)
Formaldehyde Glutaral-dehyde
USE PARAMETERS
Conc. of active
ingredient
0.3 g/ft3 0.1-2% 0.2-3% 0.01-5% 0.47% 70-85% 4-8% 2%
Temp. (oC) >23
Relative
humidity (%)
>60
Contact time (min.) 60-180 10-30 10-30 10-30 10-30 10-30 10-30 10-600
EFFECTIVE
AGAINST
Vegetative
Bacteria+ + + + + + + +
Bacterial Spores + + + +
Lipo Viruses + + + + + + + +
Hydrophilic viruses + + + + + + +
Tubercle bacilli + + + + + +
HIV + + + + + + + +
HBV + + + + + + +
APPLICATIONS
Contaminated liquid discard + +
Contaminated glassware + + + + + +
Contaminated
instruments+ + +
Equipment total
decontamination+
Facility Design and Use52
The laboratory should be designed so
that it can be easily cleaned and
decontaminated.
Benchtops must be impervious to water
and resistant to heat, organic solvents,
acids, alkalis, and other chemicals.
Chairs used at BSL-2 must be covered
with a non-porous material that can be
easily cleaned and decontaminated with
your approved disinfectant.
Disinfecting Liquid Microbiological Waste
Liquid waste such as human blood, animal blood, human tissue culture, body fluids, or growth media that has not been used for propagating microbes/vectors/toxins must not be poured down the sanitary sewer without prior steam sterilization or chemical disinfection.
For disposal of liquid microbiological waste that was used for propagating microbes/viral vectors/toxins, NC Medical Waste Rules do not allow chemical disinfection followed by disposal to the sanitary sewer unless approval has been obtained from the NC Division of Waste Management. To see what approvals have been granted at UNC, click here.
The liquid chemical disinfection procedure used prior to disposal down the sanitary sewer must be detailed on the Biohazard Registration form of the Laboratory Safety Plan.
53
Knowledge Review
All of the following are approved disinfecting agents by the NIH, the CDC, or the American Biological
Safety Association, except,
UV irradiation
Liquid chlorine compounds such as bleach
Gaseous paraformaldehyde
Compounds of Quaternary Ammonium
54
Biohazard Waste Management55
Biohazard Waste56
Any lab worker handling untreated biohazard waste must be:
1. registered as a BSL-2 worker on their Lab Worker Registration Form and
2. also complete the Laboratory Worker Bloodborne Pathogens training
course annually, if applicable.
UNC Biohazard Waste Disposal
Policy57
The UNC Biohazard Waste Disposal Policy is based on the North
Carolina Medical Waste Rules, the OSHA Bloodborne Pathogens
Standard, requirements from the County Landfill, and
recommendations from University safety committees.
The complete policy is available at the EHS website at
http://ehs.unc.edu/biological/infectious-waste/
The single page flow-diagram of the Biohazard Waste Disposal
Policy is available here.
58
Disposal of Biohazard Waste: SHARPS
Because percutaneous exposure (through the skin) is a primary route of
transmission at BSL-2, extreme caution should be taken with contaminated
needles or sharp instruments.
Needles/contaminated sharps:
• Must never be bent, recapped or removed unless there is no alternative
• May only be recapped using a mechanical device or one handed technique
• Must never be sheared or broken
Needles & Sharps Precautions59
Contaminated sharps must be
placed in hard walled plastic
containers labeled with the
biohazard symbol as shown.
When the container is no more
than 2/3 full, place autoclave
indicator tape over the biohazard
symbol in an “X” pattern as
shown.
Containers must be placed in
biohazard bag prior to
autoclaving.
Glassware should be limited in BSL-2 areas. Substitute a
plastic alternative whenever possible.
Large biologically contaminated broken glass items must
be autoclaved separately in a hard-walled container
(such as a cardboard box) lined with a biohazard bag.
Mark the outside of the box with a biohazard label. Prior
to treatment, be sure to mark an “X” over the biohazard
symbol with autoclave sterilization tape. The autoclaved
glass waste may then be disposed of in a larger
cardboard box lined with a plastic bag, clearly marked
with the "GLASS AND SHARPS" label.
Always wear gloves and use tongs or a brush and dust
pan to collect broken glassware.
Broken Glassware60
61
Testing Your Autoclave
BSL-2 labs are required under the N.C. Medical Waste Rules to test their autoclave under
conditions of full waste loading with a biological indicator.
Autoclave sterilization tape (required in the shape of an “x” over
the biohazard symbol) is not a biological indicator.
If autoclaves used to treat waste are shared (among departments,
floors, etc.), it may be advisable to designate a rotation so
that each Principal Investigator documents testing on the
same log. Duplicate testing of the same autoclave is not
required by multiple PI’s each week.
Visit the EHS website for more information at
http://ehs.unc.edu/biological/autoclave/
For labs generating biohazard waste at BSL-2 or above, a weekly autoclave testing log must
be completed every week and available at all times. Testing is not required if waste was not
autoclaved that week; however, this must be indicated in the log. Autoclave testing logs must
be kept for 3 years.
Autoclave Waste Treatment
Validation Procedure at UNC
1. Safety First: Ensure BBP training and Hep B vaccination are completed.
2. Minimum protective apparel for operating the autoclave is a lab coat, safety glasses, gloves, and closed-
toed shoes.
3. Ensure biohazard waste is disposed of in an orange autoclavable bag.
4. Place autoclave tape “x” over biohazard symbol.
5. Always leave the bag open in the autoclave (to allow steam penetration).
6. Always use a leak-proof secondary container in the autoclave.
7. Secure the indicator to the end of a string, paper, or serological pipette to aid retrieval.
8. Carefully place the indicator in the densest portion of the biohazard waste load.
9. Run the autoclave cycle as usual & log the cycle parameters and indicator lot number.
10. Carefully remove the indicator from the waste, and incubate with a control indicator.
11. Document the results on the log.
12. A positive result on the indicator requires the autoclave to be posted for
“NO USE” and EHS contacted and/or repairs initiated.
Waste does not need to be held until the indicator grows out. But once the result is positive, the autoclave may
not be used and must be reported.
62
63
Autoclave Sterilization Indicator Tape
Every container of biohazard waste must bear autoclave sterilization
indicator tape in the shape of an “x” directly over its biohazard symbol.
This is a requirement of the County Landfill. After treatment in the
autoclave, seal the bags with more tape and place in a lined
Rubbermaid Brute container (provided by your department). Bags are
required to be orange by 2012 (phase out your red bags).
64
POLICY CHANGE:
Beginning June 1, 2008, all UNC laboratories were
required to collect biohazard waste in outer containers
that are red in color.
This policy also placed a maximum limit on the size of biohazard waste containers at 15-gallons (57-L).
A review of biohazard waste containers available through Fisher Scientific is available here. However, EHS has located economical alternatives to help ease the cost associated with replacing waste containers to meet the new standard (available here).
Please note that this policy change does not impact the requirements for sharps collection containers (i.e. use of metal cans) or small containers located on the benchtop.
Knowledge Review
Which item is a prerequisite for a new lab worker handling materials (including just waste) in the BSL-2 lab:
Register as a BSL-2 worker on the online Lab Worker Registration form.
Complete all required vaccinations or serum samples.
Complete all required training courses (including Bloodborne Pathogen training for laboratory workers,
if appropriate).
Review the Laboratory Safety Plan to be familiar with all hazards in the laboratory.
All of the above.
Regarding BSL-2 liquid waste used in propagating microbes/viral vectors/toxins, which of the following is not accurate:
It is regulated by NC Division of Waste Management.
It can be autoclaved prior to disposing down the sanitary sewer.
EHS maintains a list of methods approved by NC Division of Waste Management on its website.
Chemical treatment (bleach, etc.) approvals are not necessary prior to disposal down the sanitary sewer as part of the
initial Biohazards Registration on the Lab Safety Plan.
Which statement is not true regarding autoclave testing of biohazard waste at UNC:
Biological indicator testing is required by state statute.
If a lab autoclaves biohazard waste less than once a week (e.g. every other week), bioindicator testing is necessary with
each load.
Autoclave Sterilization indicator tape is a biological indicator.
BSL-2 labs are required to document bioindicator testing.
65
Exposure66
67
Exposure Reporting
Following any exposure event during work hours, employees must be seen by the University Employee Occupational Health Clinic (UEOHC) for treatment and documentation of exposure.
Any exposure event involving a needlestick injury or exposure to blood after work hours or on the weekend, should be reported to HealthLink (966-9119) for instructions in the event that further treatment is necessary.
Immediately report the incident to your Principal Investigator and/or call the University Employee Occupational Health (UEOHC) at 919-966-9119.
Emergency Contact Numbers
UEOHC Clinic: 919-966-9119
Locations of University Employee Occupational Health Clinic,
Campus Health Services, and UNC Healthcare Emergency Room
68
University
Employee
Occupational
Health Clinic
Student
Health
Emergency
Room
Immediate Precautions: Through the Skin
69
Needlesticks
Cuts from contaminated objects
Splashes to unprotected areas with broken skin
Animal bites or scratches.
In the event of exposure, follow the proper procedure:
1. Remove contaminated gloves.
2. Wash the wound with soap and water for 5 minutes and apply sterile gauze or a bandage, if necessary.
3. Decontaminate and remove protective lab clothing and proceed immediately to UEOHC. If the injury requires immediate medical attention, go to the Emergency Room.
At BSL-2, percutaneous (through the skin) exposure is a recognized primary route of
transmission. Every BSL-2 laboratory requires a designated handwash sink with soap
and disposable paper towels in the event that the following occur:
70
Immediate Precautions: Mucous Membrane
In the event of mucous membrane exposure:
1. Rinse tissue surface with copious amounts of water.
2. Eyes should be irrigated for at least 15 minutes using the emergency
eyewash station.
3. Decontaminate and remove protective lab clothing and proceed
immediately to UEOHC.
•Within 75 feet or 10 seconds
•Without obstruction
•Able to provide single motion
activation and hands free use•Able to provide dual eye irrigation
Every BSL-2 laboratory on UNC campus must meet the ANSI Eyewash
Standard Z358.1-2004. This means the emergency eyewash must be:
Knowledge Review
Which of the following is not discussed in this training regarding exposure procedure?
The location of handwashing sinks and eyewashes to support proper practices.
The importance of discussing your research organisms with your personal physician.
The number to call in the event of an exposure (966-9119).
The location of the University Employee Occupational Health Clinic.
71
Aerosolizing Procedures72
Are you working with concentrated stocks?
Will your procedures concentrate the risk group 2 pathogen (e.g. high-
speed centrifugation of the liquid supernatant from cells)?
Does your procedure generate aerosols?
Sulkin and Pike (1951), estimated approximately 65% of laboratory acquired
infections are caused by aerosols of pathogenic microorganisms.
Aerosols in the Laboratory73
What are Aerosols?74
l Aerosols are solid or liquid particles
suspended in the air (1 to 100 μm)
The fate of the particles is determined by
their size:
l Larger particles settle more rapidly
becoming a risk for surface contact.
l Smaller particles can remain airborne for
a long period of time, dehydrating to
become “droplet nuclei” and spread wide
distances.
l Smaller particulates (1 to 10 μm) are also
more easily inhaled.
Laboratory doors are to be
closed during procedures at
BSL-2.
Do I Ever Generate Aerosols?75
Here are some common laboratory
procedures that may produce
aerosols:
• animal or human necropsy
• blowing out pipettes
• breakage of culture containers
• cage cleaning and changing animal bedding
• carelessly removing protective gloves
• dropping culture containers
• harvesting infected material
• intranasal inoculation of animals
• flaming inoculating needles, slides or loops
• freeze-drying specimens
• inserting a hot loop into a culture
• opening lyophilized cultures, culture plates,
ampoules, tubes and bottles
• pipetting
• pouring liquids
• removing stoppers
• stirring liquids
• streaking inoculum
In addition, there are many devices
that, if used incorrectly, may create
aerosols, including:
• blenders and vortexers
• bottles and flasks
• cell sorters
• centrifuges
• french press
• homogenizers
• needles and syringes
• pipettes
• pressurized vessels
• rubber stoppers
• shakers
• sonicators
• vacuum and aspirating equipment
Steps to prevent the generation of aerosols in centrifuges and shakers:
1. Routinely inspect the device to ensure that gaskets are properly in place to prevent leakage.
2. Do not overfill your tubes and flasks.
3. Wipe the outside of the tubes and flasks with an appropriate disinfectant after they are filled and sealed.
4. Centrifuge inside a biological safety cabinet. If a biological safety cabinet is not available, internal aerosol containment devices (e.g., sealed canisters, safety cups or buckets with covers, heat sealed tubes or sealed rotors) should be used.
5. After you remove tubes or flasks, open them in a biological safety cabinet. If a biological safety cabinet is unavailable, a minimum of 10 minutes settling time should be allowed before opening.
76
Prevent Aerosols with Centrifuges and Shakers
Knowledge Review
Which of the following would be the most reliable
at containing aerosols from escaping to the lab
while centrifuging?
A centrifuge in the lab with sealed safety cups that are
removed and opened in a biosafety cabinet.
A centrifuge in the lab with a gasket mounted on the
lid.
A centrifuge located in a biosafety cabinet.
A centrifuge in the lab with a sealed rotor.
77
The Biological Safety Cabinet78
Proper Use of Biological Safety Cabinets Contains Aerosols
79
We use biosafety cabinets (tissue culture
hoods) at BSL-1 to maintain an environment
of sterility that is not achieved on the open
bench.
At BSL-2, the biosafety cabinet is relied
upon to protect the worker and the
environment from procedures that may
generate an aerosol.
How Does a Biosafety Cabinet Offer
Protection?
Air from the room never directly contacts the work surface. Instead, air is drawn in at the face opening (A) and immediately drawn through the front grille and under the work surface. The air is then blown through the rear air plenum (B) to the top of the cabinet where it is divided into two chambers. Thirty percent of the air is exhausted out of the cabinet (C) through a high efficiency particulate air (HEPA) filter into the laboratory room. The remaining (seventy percent) of the air is directed through another HEPA filter down onto the work surface (D) in a laminar flow directional air pattern. Most biosafety cabinets at UNC operate in this manner.
A
D
B
C
80
Airflow diagram of Class II
type A-2 biosafety cabinet
non-ducted, stand alone
The biosafety cabinet High
Efficiency Particulate Air (HEPA)
filter is 99.97% efficient at the
most penetrating particle size
~0.3 um.
The filter frame is often
constructed of wood and
gasketed so that air is unable to
escape. Aluminum separates the
long filter sheets.
Biosafety Cabinet HEPA Filter81
EHS requires that biosafety cabinets
be certified annually. Certification
verifies HEPA filter efficiency and
calibration of the airflow that provides
the protective inward air flow at the
face of the cabinet.
If your biosafety cabinet is due for
certification, contact EHS at 962-
5507.
Annual Certification82
Collect Pipettes and Tips Inside the Hood
Preplanning is important to
minimize contamination.
Ensure that your biosafety
cabinet is not overloaded; avoid
having materials in the cabinet
that are not required for your
procedure. This will give you
space to collect your discards
inside the cabinet.
Every movement in and out of the cabinet disrupts the
delicate air barrier. Plan to minimize contamination by
providing a small biohazard waste bag inside the
cabinet during setup. Upon removal, disinfect the
outside of the biohazard waste bag and place it in your
red biowaste bin for autoclaving.
83
Don’t Overload the Biosafety Cabinet
84
Overloading the biosafety cabinet
(BSC) disrupts the laminar air flow
that is integral to providing a
sterile work environment inside
the cabinet. BSC’s are not
engineered to provide storage.
It limits space for the collection of
contaminated discards causing
others to disrupt the inward air
flow when disposing of waste.
Placing items on the grate at the front of the cabinet allows non-HEPA filtered
air to enter the work surface inside the cabinet. This could result in
contaminated cultures or the escape of concentrated pathogens.
The use of open flame burners in the biosafety cabinet is not acceptable at UNC. If
you notice a gas line attached at your cabinet, contact EHS for removal.
For more information, refer to the Policy on the use of Flammable Gases in
Biological Safety Cabinets
at http://http://ehs.unc.edu/manuals/biological/9-i/
Flame Burners in the Biosafety
Cabinet85
Alternatives for researchers that need to disinfect instruments within the BSC include:
•The Electric Bunsen Burner (see example)
•Bact-Cinerator (see example)
•The Fireboy Safety Bunsen Burner by Integra (see example)
Alternatives that avoid the need to disinfect instruments within a BSC include:
•pre-sterilized inoculating loops and needles;
•a glass bead sterilizer;
•pre-autoclaved forceps, scalpels, etc. in covered autoclavable plastic containers or the special sleeves
supplied for this use by various companies. These can be taken into the BSC and used individually, then
placed in an autoclavable discard tray located in the BSC for used/contaminated utensils; or
•a Bunsen burner outside the BSC (> 2 feet away from the BSC) for some applications. However, using a
flame in this manner would cause the researcher to reach in and out of the BSC, disrupting the air curtain
at the front of the cabinet. Flaming the necks of bottles is not necessary due to the protective airflow in
the BSC.
Laminar Flow Clean Benches
Never use with hazardous or biological materials!
86
The horizontal laminar flow clean bench is not intended
to protect you from aerosols that may be created during
manipulations. They generally draw air in by your feet
and discharge it through HEPA filters across the work
surface to sterilize equipment, tools, media, etc. This
air pattern forces the contaminated air directly into your
breathing zone and the surrounding lab.
This is not a biological safety cabinet.
Protect Vacuum Lines
At UNC, all vacuum lines
must be protected with an
in-line HEPA filter or
aerosol filter of equivalent
or greater efficiency.
Check your media
collection flasks and
suction lines at your
biosafety cabinet often,
and date them according
to manufacturer
standards.
87
Knowledge Review
A biological safety cabinet is not
A cabinet that provides a sterile work environment for manipulations
A cabinet that provides protection of the worker conducting the manipulations
A cabinet that provides protection of the surrounding laboratory and the environment
A cabinet that is a laminar flow clean bench
Which is an example of poor practice in the biosafety cabinet?
Certifying the biosafety cabinet annually.
Use of gas flame burners inside the biosafety cabinet.
Preplanning work and minimizing movement in/out of the biosafety cabinet.
Collecting biohazard waste (discards) inside the biosafety cabinet.
88
Prudent Practices at BSL-289
According to the BMBL, ingestion is a primary route of transmission in the BSL-2
laboratory. This is prevented at UNC by:
•Prohibiting mouth pipetting by
ensuring mechanical pipetting is
used.
•Prohibiting eating, drinking, smoking, and
applying cosmetics, in BSL-2 work areas.
Labs may designate areas for consumption
using approved labels (available here).
•Prohibiting the storage of food and drink in
refrigerators, freezers, shelves, cabinets,
countertops, or benchtops designated for
BSL-2.
Prevent Ingestion90
Acceptable example
Acceptable example
Place specimens in a container
that prevents leaking during:
• Collection
• Handling/Processing
• Storage
• Transport
• Shipping
At BSL-2, transporting specimens down the hall
requires some forethought.
Containers should be gasketed, labeled, and
closable. Use secondary containers (e.g. ziploc
bag w/ a paper towel or other absorbent
material) if the primary container may become
contaminated or punctured. Label the outermost
container with the biohazard symbol.
Use Leak-Proof Transport
Containers91
92
Personal Protective Equipment
Personal protective equipment (PPE) is specialized clothing or
equipment worn by a lab worker for protection against a hazard. Street
clothes are not PPE.
The minimum PPE required for the BSL-2 laboratory is gloves, safety
glasses (or goggles) and lab coats (standard BSL-1 protection).
Elements Biosafety Level 1
at UNC-CH
Biosafety Level 2
at UNC-CH
(3) Primary
Containment &
Protection
Gloves, lab coat, eye
protection
BSL-1 protection (i.e. gloves, lab coat,
eye protection)
plus:
Physical containment for splashes/
aerosolization; Biosafety Cabinets
(tissue culture hoods)
93
Personal Protective Equipment
The minimum PPE required for the BSL-1 and BSL-2 laboratory is gloves, safety glasses (or goggles) and lab coats.
Additional PPE such as surgical masks or
faceshields may be required for procedures
with high probability for splashes, spray,
splatter or droplets.
Work in rooms with infected animals may
require respiratory protection.
94
Gloves
Gloves must be replaced as soon as possible when
contaminated, torn, punctured or compromised.
Disposable gloves cannot be reused.
Utility gloves can be cleaned, disinfected, and reused,
but must be discarded when compromised.
PPE must be placed in an area designated for storage,
washing, decontamination or disposal.
For information about latex allergies at UNC, click here.
95
Personal Protective Equipment
1. PPE is NOT to be worn outside of the
work area.
2. Gloves must be removed prior to
washing hands and leaving the
laboratory.
3. DO NOT wear gloves on elevators
(even if they are “clean”) or use them
to open doors or touch equipment (i.e.
phones, computers) that others will be
handling without gloves.
96
Lab Coats
Employees must not take potentially contaminated lab coats home.
Use these safe and simple measures for handling and washing potentially contaminated lab linens:
1. Handle soiled lab coats as little as possible, using gloves and appropriate protective clothing.
2. Place soiled lab coats in bags that prevent leakage.
3. Lab managers should contact University Auxillary Services at 962-1261 to make arrangements for pick-up and laundering.
Insect and Rodent Control97
Laboratory windows that open to the exterior are not
recommended. However, if a lab does have windows that open
to the exterior, they must be fitted with fly screens.
Animals and plants not associated with the work being performed
are not permitted in the laboratory.
The UNC Chapel Hill campus has an integrated pest management
program. If pests are making their way into the BSL-2 area, contact
your building manager or submit a work to order have the area
evaluated more closely.
Knowledge Review
What are the three primary routes of transmission at BSL-2?
Puncturing skin, ingestion, contact with broken skin or eyes/nose/mouth
Puncturing skin, ingestion, inhaling aerosols
Puncturing skin, inhaling aerosols, contact with broken skin or eyes/nose/mouth
Ingestion, contact with broken skin or eyes/nose/mouth, inhaling aerosols
Lab coats, gloves, eye protection, and closed-toed shoes are examples of
Required PPE at BSL-1
Required PPE at BSL-2
Minimum PPE at BSL-2, additional protection may be determined during the risk assessment.
All of the above
B & C only
98
99
Questions??
If you have any questions, please contact Environment, Health and Safety at 962-5507 during
normal university office hours.
Don’t forget to
take the exam to
get credit for this
course!
To begin the exam, go to
https://itsapps.unc.edu/SelfStudyUnits/?testid=52
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