BSL 2/DLAM Training

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Basis for Biosafety Level 2

Part I

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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

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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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Biosafety Level 2at UNC

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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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Spills and Disinfection

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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.

Part II

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Biohazard Waste Management

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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

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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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The Biological Safety Cabinet

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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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Prudent Practices at BSL 2

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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.

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Questions??

If you have any questions, please contact

Environment, Health and Safety at 962-5507

during normal university office hours. \