Biosafety Level 2 for Department of Laboratory Animal Medicine
Transcript of Biosafety Level 2 for Department of Laboratory Animal Medicine
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
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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.
BSL 4
BSL 3
BSL 2
BSL 1
High Hazard
Low
Hazard
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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:
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Standard Microbiological PracticesDaily practice of these activities is critical in every laboratory safety program:
• Wash or sanitize hands after handling biologically contaminated materials, taking off gloves, and before leaving the animal area.
• Decontaminate work surfaces daily and after spills.
• No eating, drinking, smoking, or applying cosmetics in animal areas.
• 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 or clear autoclavable bags for disposal. Liquids must be disinfected before sink disposal.
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Elements of Biosafety Biosafety Level 1at UNC-CH
Biosafety Level 2at 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 plusdocumentation:Restricted access; Biohazard warning signs; Biosafety manual defining“Sharps” precautions,
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. BSL 1 animal facilities at UNC include a
hand-washing sink, safety shower/eyewash, and an autoclave to sterilize biohazard
waste.
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Knowledge Review1. 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
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Risk Assessment
A proper risk assessment
must consider each element
in the chain.
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A thorough risk assessment conducted by the DLAM Vet is a guide for the
selection of appropriate biosafety levels and microbiological practices, safety
equipment, and facility safeguards.
Your first question –should be: will the microorganism that I may come in
contact with cause disease? If you are unsure, contact EHS for guidance.
The risk assessment will be used to alert others to the hazards of working at
the BSL-2 level and the need for developing proficiency in the use of safe
practices and containment equipment.
Successful control of hazards in DLAM animal facilities also protects
persons not directly associated with the Division of Laboratory Animal
Medicine, such as other occupants in the building, infrequent visitors (e.g.
maintenance), and the public.
Sources to Determine Biosafety Level
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 biosafety level:
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Elements Biosafety Level 1at UNC-CH
Biosafety Level 2at 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, Biowastepractices, 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)
Hand-washing sink, safety shower/eyewash, autoclave
Same as BSL 1
With elevated risk, comes elevated containmentFollow the red arrow ( ) to compare the higher level
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Knowledge Review
3. When beginning a new project at BSL 2, what is the first thing to consider after a risk assessment is conducted?
a. What type of biosafety cabinet should be purchased and where will it be located?
b. Will the microorganisms that I may come in contact with cause disease?
c. What manipulations will produce aerosols?
d. Will human pathogens or work be done with concentrated stocks?
e. What is the largest volume of infectious material that could spill?
4. Which is not true of both BSL 1 and BSL 2 labs at UNC?
a. Standard Microbiological Practices are followed.
b. Biohazard waste is collected and autoclaved according to the University Biohazard Waste Disposal Policy.
c. Work is conducted with microbes that are not known to consistently cause disease in healthy adults.
d. A hand-washing sink, eyewash & safety shower should be readily available.5. Which of the following material(s) must be handled at the BSL 2 level?
a. Well characterized human cell lines.
b. Any risk group 2 agent identified by the NIH Guidelines .
c. Human serum samples.
d. All of the above.
e. B & C only.
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Who Implements the Biological Safety Level at UNC?
Principal Investigator/DLAM Vet/
DLAM Supervisor
EHSTrained DLAM
Workers
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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, DLAM Vet,
EHS, properly trained lab workers and
DLAM staff.
There may be instances where agent or procedural characteristics create
unique hazards such as the potential for aerosolization of pathogens (TB lab
McGavran) cultured in concentrations higher than found in nature. In such
instances, Principal Investigators, DLAM Vet, Lab Supervisors, and/or EHS will
seek enhanced biosafety requirements during the risk assessment. These
requirements are then carried out by DLAM workers.
Responsibilities of theDLAM Supervisor at BSL 2
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The DLAM Supervisor assumes the day-to-day
responsibility for ensuring workers’ safety and proper
training to follow BSL 2.
Specific to this responsibility is:
1. Communicating biological/chemical/radiation risks to DLAM employees.
2. Ensuring that DLAM employees know to wash hands and doff PPE after working in
BSL 2 designated DLAM areas.
3. Adopting policies/procedures from the UNC Biological Safety Manual and ensuring
workers have access to this manual at all times.
4. Ensuring that DLAM employees working at BSL 2 are aware of decontamination
procedures.
5. Restricting access to DLAM BSL 2 areas as appropriate.
Principal Investigator/DLAM Vet/
DLAM Supervisor
EHSTrained DLAM
Workers
“Restricted Access”
Restricted access is important at DLAM 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 animal areas (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 animal facility.
Signs are available on the EHS website.17
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No sandals or open-toed
shoes in the DLAM BSL 2
(or any) animal area.
Eliminate accidental routes of entry
Workers not wearing closed-toed shoes at the BSL 2 level is
evidence of a lack of training in standard microbiological practices.
Appropriate footwear
Procedures are Documented by the PI/DLAM Vet/DLAM Supervisor
Compliance at UNC includes:
• BSL 2 Checklist
• Use of biohazards in animals
• Use of radiation in animals
• Use of chemicals in animals
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BSL 2 Checklist
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EHS will complete the BSL 2 checklist while conducting an inspection. Action items listed on the BSL 2 checklist will be sent to the DLAM Operations Director and must be resolved before the following inspection.
Use of Biohazardous Materials in Laboratory Animals
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PI must have EHS
approval before using
biohazardous
materials in laboratory
animals.
This form is completed
online by the PI
through the ACAP
webpage
(https://cfx2.research.u
nc.edu/acap/) and
posted on the cubicle
door.
Use of Hazardous Chemical Materials in Laboratory Animals
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PI must have EHS
approval before using
hazardous chemical
materials in laboratory
animals.
This form is completed
online by the PI through
the ACAP webpage
(https://cfx2.research.unc.
edu/acap/) and posted on
the cubicle door.
Responsibilities of EHS at BSL 2
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UNC devotes resources to laboratory and biological
safety. The department of Environment, Health &
Safety supports BSL 2 by:
Developing the University Biological Safety Manual;
Providing safety training;
Providing consultation and advice;
Inspecting DLAM facilities for compliance;
Establishing and enforcing safety policies instituted by the Laboratory and
Chemical Safety Committee, IACUC and the Institutional Biosafety Committee.
Principal Investigator/DLAM
Vet/ DLAM Supervisor
EHSTrained DLAM
Workers
Responsibilities of DLAM Workers at BSL 2
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Working at BSL 2 requires strict
adherence to biosafety requirements. The
remainder of this course is dedicated to
providing a foundation of BSL 2 practices
and regulatory requirements:
•Communicating Biohazards
•Spills and Disinfection
•Biohazard Waste Management
•Exposure
•Aerosolizing Procedures
•The Biological Safety Cabinet
•Prudent Practices at BSL 2
Principal Investigator/DLAM
Vet/ DLAM Supervisor
EHSTrained DLAM
Workers
Communicating Biohazards at BSL 2
The standardized label with the biohazard symbol
communicates the presence of potentially biohazardous
materials to DLAM workers, PIs, visiting professors, facilities
services workers, housekeeping personnel, vendors, and others
who may enter a BSL 2 animal area.
At UNC, employees are trained to use and recognize the universal
biohazard symbol and the word “BIOHAZARD.”
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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, incubators and centrifuges, etc. are labeled if they are used with biohazardous materials.Also, transport containers and biohazard waste containers must display a biohazard label on the outermost part.
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Knowledge Review
6. Who is ultimately held accountable for ensuring that DLAM employees work safely and are properly trained in BSL 2 procedures?
a. The Principle Investigator
b. EHS
c. The lab workers
d. The DLAM Supervisor
7. A proper risk assessment for biosafety
a. Is documented in the Use of Biohazardous Materials in Laboratory Animals form.
b. Includes microorganism characteristics, manipulations, containment and protection, and facilities.
c. Is communicated by the DLAM supervisor by alerting all animal facility workers and others to the hazards of working in BSL2 animal areas.
d. All of the above
e. A & B only
8. 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
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SPILLSEvery DLAM member working at BSL 2 must follow the proper protocol in
the event of a biohazardous spill.
There are three standard spill protocols that should be posted in the animal area:
1. Small spill in a biological safety cabinet2. Large spill in a biological safety cabinet3. Spill outside the biological safety cabinet (in the open animal
area)
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Protocol for Small Spill in a Biological Safety Cabinet
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.
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Protocol for Large Spill in a Biological Safety Cabinet
Large Spill Inside BSC:
♦ Cover spill with paper towel, pour bleach or other disinfectant 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 grill 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).
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Protocol for Spill Outside the Biological Safety Cabinet
(in the open animal areas)
1. Remove contaminated outer gloves.
2. Notify others of the spill.
3. Personnel should follow decontamination procedures and exit the animal room.
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 Resistant33
An appropriate disinfectant is determined during the initial risk
assessment. Each worker needs to ensure they allow for the proper
contact time for the selected disinfectant.
Decontaminate Work Surfaces
Work surfaces must be
decontaminated:
•After work,
•After spills,
•At the end of each day,
•Prior to the repair or
surplus of equipment
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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+
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Facility Design and Use
The DLAM animal area 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 for the PI listed on their Lab Safety Plan.
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Cage Washing
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Final rinse temperature for
animal cages should reach at
least 180o F. This should be
documented in the cage
wash notebook.
Biohazard Waste
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Any DLAM employee handling untreated biohazard waste must
complete the Laboratory Worker Bloodborne Pathogen training
course annually, if applicable.
Any DLAM worker working in a BSL 2 animal area must take the
DLAM BSL 2 training.
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UNC Biohazard Waste Disposal Policy
The UNC Biohazard Waste Disposal Policy is based on the North Carolina
Medical Waste Rules and the OSHA Bloodborne Pathogens Standard.
The complete policy is available at the EHS website at
http://ehs.unc.edu/biological/policy/
The single page flow-diagram of the Biohazard Waste Disposal
Policy is available here.
NO YES
BIOHAZARD WASTE DISPOSAL CHART (UNC Research Laboratories)
LIQUIDS RESEARCH LAB/
CLINIC PIPETTING
SOLIDS SHARPS
Razor blades, scalpels, lancets, syringes
with/without needles, slide covers, specimen
tubes, inoculating loops, stirring devices,
broken glass
Puncture resistant,
outer container
bearing the
biohazard symbol
(“x” with indicator
tape) lined with
orange autoclave bag
(“x” over biohazard
symbol before lining).
PIPETTES PIPETTE TIPS
Small
bench top
biohazard
bag with
indicator
tape “x”.
Collect in red plastic
sharps containers
bearing biohazard
symbol and “x” with
indicator tape.
Autoclave with test indicator.
Dispose with
pipettes or
solid waste
Culture dishes & flasks,
Petri dishes, solid waste
cultures/stocks from the
production of biological,
gloves, gowns, masks, shoe
covers, and any other solid
materials potentially
contaminated with
biohazardous material
Biohazard waste collection
container (red, hard-walled,
closeable) lined with an orange
autoclave bag bearing “x” over
biohazard symbol.
Human blood, animal
blood, human tissue
culture, body fluids,
liquid growth media
Was liquid waste used for propagating
microbes/viral vectors/toxins?
Chemical
disinfection
with
appropriate
contact time,
dispose of
down sanitary
sewer
Autoclaved
and
disposed of
down
sanitary
sewer
Apply to NC
Medical
Waste
Division for
approval of
chemical
disinfection
if necessary
Disposal for removal by housekeeping.
For special circumstances, please
contact EHS @ 962-5507
Revised 8/2011
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Disposal of Biohazard Waste: SHARPS
When the container is no more than 2/3 full, place autoclave indicator tape over the biohazard symbol in an “X” pattern as shown.
Contaminated sharps must be placed in red, plastic sharps containers labeled with the biohazard symbol as shown.
Disposing of sharps in the proper container helps keep others safe.
Remember, any contaminated object that can penetrate the skin,
including needles, scalpels and glass objects requires extra attention.
Needles & Sharps Precautions
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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.
DON’T Break, bend,
re-sheath, or reuse syringes or needles
DOUse approved sharps containers ;
Only fill sharps containers to 2/3; Close the container when it is not in use.
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Testing DLAM Autoclaves (Daily)
DLAM uses different tests to ensure their autoclaves are
working properly.
Sterilization Monitoring should be used in each
autoclave load. This will let the technician know
whether or not steam has penetrated into the items to be
sterilized.
To accomplish this, DLAM technicians use 2 different
indicators (SteriGage Card & Getinge Steam Monitoring
strips), depending on what is being sterilized.
Testing DLAM Autoclaves (Daily)
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For cages and biohazard bags being
autoclaved, DLAM technicians will use a
SteriGage Card in each load. The Facility
Manager inspects each card for results and
keeps one per day for record keeping. These
cards are saved for 3 months.
For water bottles, a Getinge Steam
Monitoring Strip is used. DLAM visually
checks these strips but, they are not saved.
If any of these tests fail, the Facility
Manager must be immediately informed.
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Testing DLAM Autoclaves (Weekly)The SteriGage Card and Getinge Steam Monitoring Strips indicate whether
or not steam has penetrated, but do not verify whether or not complete
sterilization has occurred.
BSL 2 animal areas are required under the N.C. Medical Waste Rules to test
their autoclave under conditions of full waste loading with a biological
indicator.
Once a week, DLAM techs
use the Getinge Biosign
Biological Indicators to test
complete sterilization.
Weekly Autoclave Biological Testing
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. Carefully place the indicator inside a cage, near the middle of the biohazard waste load.
4. Run the autoclave cycle as usual & log the cycle parameters and indicator lot number.
5. Carefully remove the indicator from the cage, and incubate with a control indicator.
6. Document the results on the log.
7. A positive result on the indicator requires the autoclave to be posted for “NO USE” .
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.
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Documentation
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The results from the Biosign Biological Indicators
are documented in the Autoclave Log Book.
For animal areas generating biohazard waste at
BSL 2 or above, a weekly autoclave testing log
must be completed every week and be available at
all times. Testing is not required if nothing is
autoclaved that week; however, this must be
indicated in the log. Autoclave testing logs must be
kept for 3 years.
Testing DLAM Autoclaves (Weekly)
In addition to the previous
tests, DLAM techs will
also use a Getinge
SMART Pack once a
week to assure proper
vacuum cycle operation.
The Facility Manager keeps
these cards filed for
record keeping for 3
months.
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DLAM Autoclaves
The DLAM Facility Manager or Cage Wash
Supervisor is responsible for training the
staff on how to use each of these tests
mentioned.
Visit the EHS website for more information
at http://ehs.unc.edu/ih/biological/autoclave
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Autoclave Sterilization Indicator Tape
For staff using orange or clear biohazard bags, please be aware 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 landfill. After treatment in the autoclave,
seal the bags with more tape and place in a lined Rubbermaid Brute
container (provided by your department).
Knowledge Review
9. Which statement is not true regarding autoclave testing of biohazard waste at UNC:
a. Biological indicator testing is required by state statute.
b. Minimum protective apparel for operating the autoclave is a lab coat, safety glasses, gloves, and closed-toed shoes
c. Autoclave Sterilization indicator tape is a biological indicator.
d. BSL 2 animal areas are required to document bioindicator testing.
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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 DLAM supervisor and/or call the University Employee Occupational Health (UEOHC) at 6-9119.
Emergency Contact Numbers
UEOHC Clinic: 966-9119
Locations of University Employee Occupational Health Clinic, Campus Health Services, and UNC Healthcare Emergency Room
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University Employee OccupationalHealth Clinic
StudentHealth
EmergencyRoom
Immediate Precautions: Through the Skin
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• Needlesticks• Cuts from contaminated objects• Splashes to breaks/openings in the skin (cut,
eczema, etc)• 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 animal area requires a designated handwash sink with
soap and disposable paper towels in the event that the following occur:
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Immediate Precautions: Mucous Membrane
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.
In the event of mucous membranes
exposure:
•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 animal area on UNC campus must meet the ANSI Eyewash
Standard Z358.1-2004. This means the emergency eyewash must be :
Cage Dumping
• The dumping of animal cages has the potential
to produce aerosols.
• Cages should be dumped in front of the
Biobubble at all times to capture aerosols.
• The waste bin should be within 5 inches of the
pre-filter of the Biobubble.
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Biobubble Maintenance
• The ventilation balloon in the
back of the Biobubble must be
fully inflated at all times when
dumping cages.
• A shrunken balloon indicates
restricted airflow through the
pre-filter.
• The pre-filter must be cleaned
or replaced when the balloon
indicates restricted airflow.
• The Biobubble must be
certified annually.
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What are Aerosols?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.
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Do I Ever Generate Aerosols?
Here are some common laboratory procedures
that may produce aerosols:
• animal or human necropsy
• cage cleaning and changing animal bedding
• carelessly removing protective gloves
• dropping culture containers
• harvesting infected material
• intranasal inoculation of animals
•opening lyophilized cultures, culture plates, ampoules, tubes and
bottles
• pipetting
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Proper Use of Biological Safety Cabinets Contains Aerosols
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The biosafety cabinet is used to protect the worker and the
environment from procedures that may generate an aerosol.
Some models used for animal cage changes may require the
HEPA filter to be changed more frequently due to the dust that
is generated.
Class II BSC: This Biological Safety Cabinet provides
personnel, environmental, and product protection.
There are 4 types of Class II biosafety cabinet.
Type A1, A2, B1 and B2
Type A cabinets are stand alone, re-circulating,
non-ducted cabinets. Chemicals and anesthetic
gases must not be used in re-circulating cabinets.
Gases can build up in the cabinet and cause an
explosion.
Most BSCs in DLAM are Type B2 (total exhaust
cabinets), ducted, non re-circulating.
How Does a Biosafety Cabinet Offer Protection?
Air is drawn in at the face opening (A) and immediately drawn through the front grille and under the work surface. The air is 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. Anesthetic gases must not be used in a non-ducted cabinet.
A
D
B
C
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Airflow diagram of Class II
type A-2 biosafety cabinet
Non-ducted, stand alone
Proper Use of Anesthetic Gases
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This is a B2 ducted total
exhaust cabinet
appropriate for using
anesthetic gases
Annual Certification
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.
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Cage Changing Stations
• The animal cage changing station
provides vertical HEPA filtered
airflow for product protection on
the work surface. The station
provides allergen personnel
protection by using a HEPA filtered
down draft vacuum on the work
surface to assure minimal
exposure to animal hair/dander
and cage bedding material.
• The cage changing station should
not be used as a substitute for a
Class II, Type A2 (Non-ducted)
Biological Safety Cabinet.
• It is not a containment device
for biologicals
Knowledge Review
10. A biological safety cabinet is not
a. A cabinet that provides a sterile work environment for manipulations.
b. A cabinet that provides protection of the worker conducting the manipulations.
c. A cabinet that provides protection of the surrounding laboratory and the environment.
d. A cabinet that is a laminar flow clean bench.
11. Which is an example of poor practice in the biosafety cabinet?
a. Certifying the biosafety cabinet annually.
b. Constantly moving in/out the biosafety cabinet.
c. Disinfecting the biosafety cabinet before and after use, and any spills.
b. Collecting biohazard waste (discards) inside the biosafety cabinet.
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Prevent Ingestion
According to the BMBL, ingestion is a primary route of transmission in the BSL 2
animal areas. 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.
Areas may be designated as
eating/drinking areas using approved
labels (available here).
•Prohibiting the storage of food and drink in
refrigerators, freezers, shelves, cabinets,
countertops, or benchtops designated for
BSL 2.
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Use leak-proof transport containers
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.
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Personal Protective EquipmentPersonal protective equipment (PPE) is specialized clothing or
equipment worn by a DLAM worker for protection against a hazard.
Street clothes are not PPE.
The minimum PPE required for the BSL 2 animal area is gloves, safety
glasses (or goggles) and lab coats (standard BSL 1 protection).
Elements Biosafety Level 1at UNC-CH
Biosafety Level 2at 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)
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Personal Protective Equipment
The minimum PPE required for the BSL 1 and BSL 2 animal area 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.
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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.
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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.
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Insect and Rodent Control
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.