Biosafety in Biomedical Laboratories

June 2, 2009

Marshall University

Summer Students/Recent Hires

Please complete the sign in sheet

Major Safety Concerns

Safe handling + containment of infectious agents Recombinant DNA (rDNA), D. Primerano June 2 Management of Biohazard Waste (N. Douglas) Bloodborne Pathogen Exposure (N. Douglas) Chemical Safety (Nathan Douglas), June 3rd Use of Animals in Research(Dr. Billy Howard June 4) Radioisotope Use (Dr. Will McCumbee, June 8) Human Subject Protection (ORI, Mr. Bruce Day)

Key Biosafety Personnel

Chemical & Biological Safety Officer Mr. Nathan Douglas

Marshall University Safety & Health

Sorrell Maintenance Building, 215

(304) 696-3461

(304) 208-7385 cell

douglas2@marshall.edu

Institutional Biosafety Committee, ChairDr. Donald Primerano, Room 336F BBSC

304-696-7338

primeran@marshall.edu

Everything we cover will be posted to the IBC website

http://musom.marshall.edu/biosafety/

Main Principle of Biosafety: Containment

Purpose of Containment: to reduce or eliminate exposure of lab workers and others to hazardous biological agents Bacteria, viruses, fungi, parasites Recombinant DNA Potentially dangerous cell lines

General Principles of Biosafety

Three elements of containment: Personal protective equipment Facility design Lab practice and techniques

Extent of containment depends on level of risk of exposure nature of agent

Examples of Safety Equipment

Biological Safety Cabinets (Laminar Flow Hood) Protect you and cells you are working with!!!

Centrifuge Safety Canisters Gloves, lab coats, gowns, face shield, goggles,

shoe covers, respirators

Biological Safety Cabinets (BSC)

Class I and II BSCs provide effective containment system for safe handling of moderate/high risk organisms (BSL2 and BSL3 agents)

Class II BSCs also protect the research material itself through high efficiency particulate filtration (HEPA) of the air flow down across the work surface (vertical laminar flow which forces outside air into the plenum)

BBSC has shared facilities with Class II Type A2 BSC

Biological Safety Cabinet II type 2

Facility Design and Construction(Secondary Barriers)

Separation of lab work areas from areas of public access

Availability of decontamination stations (autoclaves and hand-washing facilities)

Separate ventilation systems Controlled access labs

HOMEWORK ASSIGNMENT

On the way back to your lab today take note of the location of Showers Eye wash stationsFire extinguishersEmergency exit routes

Biosafety Levels (BSLs)

A biosafety level is the level of biocontainment precautions required to isolate dangerous biological agents in an enclosed facility

Each level represents a combination of lab practices, techniques, safety equipment lab facilities

designed to minimize release and exposure.

Biosafety Level 1 (BSL 1)

appropriate for work with organisms known to not cause disease in healthy adult humans.

“Avirulent organisms” Minimal hazard to lab personnel and the

environment Examples:

E coli, Bacillus spp, Exempt categories of rDNA work

Biosafety Level 2 BSL 2: indigenous moderate risk agents

Mild or treatable disease in humans Not spread by aerosol. Primary Hazard: Skin break, mucous

membrane exposure or ingestion.

Examples: Hepatitis A,B+C, HIV, some Salmonellae Human derived blood and blood products Cell culture work and some rDNA

Biosafety Level 3 (BSL 3)

work with indigenous or exotic agents with potential for respiratory transmission or lethal consequences.

Examples: M. tuberculosis, B. anthracis SARS and West Nile viruses Primary and Secondary barriers to protect

personnel in contiguous areas

Biosafety Level 4 (BSL4) BSL 4: lethal exotic agents especially where

there is no vaccine or therapy. BSL 4 facility is separate facility or HVAC

isolated zone Examples: Ebola, Marburg and Lassa Fever

viruses NO BSL3 or BSL4 work at MU.

Which Biosafety Level Should You

Be Using? (more homework) Ask your mentor first!! Read Sections I-III of Biosafety in Microbiological

and Biomedical Laboratories Go to ABSA Website for a listing of infectious

agents and corresponding BSL Read the NIH rDNA guidelines

American Biological Safety Association (ABSA)

If don’t which risk group your agent falls into:Call or email Don Primerano ORConsult the ABSA website:http://www.absa.org/resriskgroup.html

Biosafety Levels

Safe Practices

Biosafety Level 1 Practices: “Standard Microbiological Practices”

Access to lab is limited or restricted when experiments are in progress

Workers wash hands after handling viable organisms, after removing gloves and before leaving the lab

Eating, drinking, smoking, handling contact lens, brushing teeth, storing food is absolutely forbidden.

Biosafety Level 1

Mouth pipetting is prohibited- YOU MUST USE MECHANICAL PIPETTING DEVICES

Safe handling of sharps (disposal) Procedures minimize splashing and aerosols Work surfaces are decontaminated with disinfectant

on a daily basis All cultures, stocks and other infectious materials

are decontaminated by an approved method (usually autoclaving)

Biohazard signs are posted at lab entrance Insect and rodent control program in place

Biosafety Level 2

Similar to BSL-1 and suitable for work with agents of moderate hazard.

Lab personnel get specific training in handling pathogens.

Access to the lab is limited when work is being conducted.

Extreme precautions taken with contaminated sharps.

Specific procedures for reducing aerosols or splashes.

Biosafety Levels 3 and 4

Levels of personal protection increases Lab access is restricted Lab design becomes more critical Animal Biosafety Levels: special set of rules

noted in the NIH rDNA guidelines

General Biosafety Advice

Discuss specific safety issues with your mentor and safety committee chairs.

Lab heads should have written safety protocols that are lab/experiment specific.

Read Biosafety in Microbiological and Biomedical Laboratories as needed.

Contact Dr. Primerano at 696-7338 if you have questions or concerns.

General Biosafety Advice

Get a lab coat and safety equipment immediately!

Don’t wear open shoes ever! Keep hair short or tied back Glove allergies: nitrile gloves may help

Transformed or Cancer Cell Lines

Any experiment involving transformed or cancer cell lines falls under Biosafety Level 2 (BL2) practices.

Work with living cells should be done under a biological safety cabinet.

Special precautions are taken to limit aerosols. Discarded media and cells are treated with 10% bleach

before autoclaving (sterilization). Decontaminate spills with bleach and wipe down hood

after working with cells. Protective clothing (lab coat and gloves) should be worn

when working with the cells. Restrict access to the lab when working with cell lines.

rDNA Guidelines

The NIH Rule

Recombinant DNA (rDNA) as defined by the NIH Guidelines

rDNA molecules are either: DNA molecules constructed outside cells

by joining nature or synthetic DNA segments to DNA molecules that can replicate in a living cell, OR

Molecules that result from the replication of those described above

rDNA Regulations

Follow BSL1-4 guidelines. Each lab must have approved protocols for use

of rDNA. Application forms and guidance on the IBC

website BSL1 is appropriate for most rDNA work at

Marshall (but not all). Consult mentor or rDNA guidelines for all cases Each investigator (your mentors) must be

familiar with the guidelines.

Bloodborne Pathogens Standard

OSHA

Bloodborne Pathogen StandardExposure Control Plan

Bloodborne pathogens

Organisms in human blood that cause disease. Includes bacteria and viruses like HbV and HIV

OSHA requires that the university have an Exposure Control Plan (on IBC web page)

Also requires employers to provide Hepatitis B vaccination at no cost to employee

Bloodborne Pathogen Standard

Universal Precautions

Treat all human blood and other body fluids as if they are contaminated with an infectious agent.

Required by the OSHA BBPS

Use Personal Protective Equipment (gloves, face mask, lab coat) when around biohazard materials

Do not take lab coat home, BBSC will launder

Bloodborne Pathogen Standard

No eating, drinking, smoking, applying cosmetics or contact lenses in the labs

No mouth pipetting; use pipetting device

Proper disposal of blood and other biohazard materials

Bleach (1:10 solution of bleach and water)

Autoclave on-site

Off-site shipment for treatment and disposal

Bloodborne Pathogen Standard

If you are working with human blood, you must read and understand the Exposure Control Plan AND meet with Dr. Primerano!

Immediately report exposures to Dr. Donald Primerano 304-696-7338 (w) or 304-697-3815 (h).

Sets in motion the required follow up steps

Infectious Medical Waste Rule

WV Department of Health & Human Resources

Infectious Medical Waste Rule

1. Definition of Infectious Medical Waste

2. Types of Infectious Waste

3. WV DHHR License

4. Infectious Waste Disposal Plan

5. Bloodborne Pathogen Standard Review

Infectious Medical Waste Rule

www.state.wv.us/csr/docs/WPDocs/64-56.wpd

Section 3.9. Infectious Medical Waste is medical waste which is capable of producing an infectious disease.

WV Legislative Rule 64 CSR 56

Infectious Medical Waste Rule

Medical waste shall be considered capable of producing an infectious disease if

1. it has been, or is likely to have been, contaminated by an organism likely to be pathogenic to healthy humans,

2. if such organism is NOT routinely and freely available in the community, AND

3. such organism has a significant probability of being present in sufficient quantities and with sufficient virulence to transmit disease.

Infectious Medical Waste

BBSC Plan: Sterilize all bacterial, fungal, viral and parasitic organisms and cultured cells used in research.

Includes deliberately infected animals

Infected animals go directly to Stericycle

If you have any questions please consult with Don Primerano (696-7338) or Connie Berk (696-7341).

Cultures and Stocks of Microorganisms and Biologicals

Human Blood & Blood Products

All human blood (wet or dried) Products from human blood (plasma) Does not include animal blood

Sharps

Any article that can puncture or cut If sharps have been used in patient care or

treatment OR used to handle or deliver infectious agents, must be placed in a “Biohazard” labeled sharps box or broken glass container

Sharps boxes & containers will be autoclaved Examples: pipette tips, glass Pasteur pipettes,

needles, syringes, scalpel blades, razors, forceps

Pathological Waste

Includes: tissues, organs, body parts, and containers of body fluids

All pathological waste should be packaged by the investigator and picked up by Stericycle

Stericycle provides shipping containers and liners.

Containers and liners are located in BBSC Room 116. Contact Julia Schreiber or Connie Berk if you need packing materials.

Animal Waste

Contaminated animal carcasses, body parts, animal bedding known to have been exposed to infectious agents during research

These items should be taken to the animal facility where Julia or Connie will assist you in preparing for pickup by Stericycle.

Infectious Medical Waste Management Permit

BBSC permit from the WV DHHR is renewed annually

Allows us to generate infectious waste, sterilize on site via autoclave, and dispose waste in regular trash

Allows us to hire a licensed company to remove infectious waste that cannot be autoclaved (e.g. contaminated animal carcasses)

Our company is Stericycle

Infectious Medical Waste Management Plan

OBJECTIVES:

1. Provide a safe environment for faculty, staff, students and visitors

2. Properly manage infectious waste in accordance with WV Legislative Rule 64 CSR 56, Infectious Medical Waste

Infectious Medical Waste Management Plan

Handling of infectious waste

Packaging

Transportation

Sterilization

Storage and disposal

Contingency plan

Spill response

Training

Infectious Waste Packaging

1. Orange biohazard bags must be used for infectious waste, clear and red bags are not acceptable

2. Waste collection containers and bags must be double-bagged

3. Do not fill them more than 2/3 full

4. Loosely gather the top of the bag and place a strip of autoclave tape around it, do not tie the bags closed. Steam must be able to enter the bag.

5. Bags, flasks, and other containers of infectious material must be labeled with primary investigator’s name, room number, phone number, and the contents.

Special Packaging Rules

Labs must have separate containers for contaminated and uncontaminated glassware

Broken glass boxes are acceptable, and must include a biohazard sticker when used for contaminated items

Contaminated pipette tips can easily puncture plastic bags. They must be collected in a sharps box, or in an orange bag, the bag put into a cardboard box, then placed into a biohazard waste collection container

Contaminated Pasteur pipettes must go into a double-bagged, biohazard-labeled contaminated glassware box

Uncontaminated glass must go into a box labeled as “Broken Glass”

Transporting Waste To Autoclave Room 119A

1. All waste must be transported on/in carts with secondary containment trays.

2. Infectious waste may not be sterilized in the satellite autoclaves on the floors.

3. Bags of solid infectious waste must be placed in the large gray bin in Room 119A, accessible 24/7. Lab heads will provide labels and keys to gray bin. Relock the bin, which must be kept locked at all times.

4. Liquid infectious waste must be placed on the metal cart.

5. Waste will be autoclaved, weighed (if solid) and placed into the regular trash. We weigh waste as required by our state permit.

Contingency Plan

In the event that waste cannot be autoclaved at the BBSC, Stericycle will be employed to treat and dispose of the waste.

Waste items sent for treatment and disposal via Stericycle must be packaged and labeled by someone properly trained in DOT Hazardous Materials.

Spill Response

1. Notify Dr. Primerano or Connie Berk as soon as possible.

• Don: 304-696-7338 (w) or 304-697-3815 (h)

• Connie: 304-696-7341 (w) or 304-429-7318

2. If you cannot reach either of them, contact Nathan Douglas (304) 208-7385, 696-3461.

3. Spill kits are located in each satellite autoclave room on every floor, as well as in room 119 and room 121.

Spill Response

Attempt to prevent liquids from running free by laying paper towel or other absorbent material on top.

Do not use spill cleanup kit unless you are comfortable doing so, allow someone trained to clean spill. Make sure you reach someone right away in either case.

Complete a Biohazard Spill Occurrence form, available on IBC web page:

http://musom.marshall.edu/biosafety

Don’t Put Noninfectious Items In Orange Bags

Bubble wrap

Paper towels, either from drying your hands or from wiping down benches with a disinfectant 

Scalpel blade wrappers 

Needle wrappers 

Benchkote or any other bench protector 

Gloves, unless contaminated

Pipettes

Food or drink containers (not allowed in labs)

Packaging materials such as cardboard, Styrofoam “peanuts”, etc.

Paper (copy paper, etc.) Pop cans

All of the above items can be recycled in the blue bins in the middle hallway.

Review

We care about biosafety to prevent the spread of bloodborne pathogens OSHA Standard covers HbV & HIV

Everyone exposed should have completed the 3-shot Hepatitis B vaccine series

Gloves are used to protect us as we work Gloves must be removed before exiting laboratories,

and hands should be washed. If items must be transported, use a cart. If you must carry something, use only 1 gloved hand,

use the other to open doors.

QUESTIONS??