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.

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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