“ is an inexact science, and Biosafety Levels the interacting system of agents and activities and the people performing them are constantly changing.”

Betsy Weirich, MS, SM(NRM), CBSP Every etiologic agent is different. Centers for Disease Control and Prevention Every laboratory is different. Every person is different.

Principles of Biosafety Primary Barriers Protect the POTENTIAL Worker and the Lab Environment BSL-4 HIGHEST Large research Labs – CDC, NIH • Safety equipment

BSL-3 – Personal Protective Equipment (PPE) University research laboratories – Biological Safety Cabinets (BSC) BSL-2 – Mechanical pipetting device Clinical laboratories – Safety centrifuge cups BSL-1 High school laboratories LOWEST – Removable rotors

Secondary Barriers Protect the Biosafety Levels (BSL) 1-4 Worker and the Lab Environment • Facility design • Each level builds on previous levels – Separation of lab from public access • Increasing emphasis on safety – facilities procedures and practices – Handwashing and eyewash • Increasing need for training, facilities preparation and competent – Specialized ventilation systems supervision – Directional airflow • Increasing requirements for PPE and facility containment – Restricted access zones

CDC 1 1 Biosafety Level 1 (BSL-1) Biosafety Level 1 (BSL-1) • Suitable for work involving well – Vaccine strains should not be characterized agents not known to considered avirulent simply consistently cause disease in immunocompetent adult humans and because they are vaccine strains present minimal potential hazard to (RB51) laboratory personnel and the environment (most college training labs) – May cause opportunistic in the young, the elderly and the Lactobacillus sp. immunocompromised individuals

Needles and Sharps Safe Handling, Minimizing, Precautions and Disposal of Sharps • Use sharps containers • Don’t break, bend, re-sheath or reuse for disposal syringes or needles • Never fill sharps • Use retractable or container to the top shielded needles • Use plastic vs. glass • Use shielded scalpels • Don’t touch broken • Collect reusable sharps in labeled, leak proof glass with bare hands container

Minimize Aerosols Aerosols • Procedures that impart energy to a microbial suspension produce aerosols • Ubiquitous in laboratory procedures • Usually undetected • Extremely pervasive, putting all at risk • Likely to be the cause when other causes are ruled out and the person just “worked in the room” where the agent was

CDC 1 2 Procedures That Emit Aerosols Aerosols from • Catalase • Lasers, cell sorters Laboratory Equipment • Inoculating • Inoculating 10 biochemicals or biochemicals or blood (10 /ml culture - 10 min. use)0 blood culture bottles culture bottles 6 • Pipetting • Splashes Blender, open 10 Sonicator with bubbling 106 • Mixing • Opening lyophilized Pipetting, vigorous 106 • Centrifugation cultures Dropping culture 3 X 105 • Grinding • Flaming loops Splash on a centrifuge rotor 105 • Vortexing • Entering or opening Blender, opened after 1 minute 2 X 104 • Pouring vessels at non- ambien t pressures, fermenters, Pipetting, carefully 104 • Loading syringes freezer vials

Minimize Aerosols Minimize Aerosols • Do • Don’t – Discharge liquid down side of container – Use Bunsen burners – Deliver as close as possible – Drop liquids onto hard surfaces to contents – Blow out last drop in pipette – Use capped tubes when mixing or vortexing – Mix by suction + expulsion – Use care with needles – Open centrifuge before it stops (gauze pad with alcohol on septum of blood culture bottle)

Minimize Aerosols Protect Your Eyes and – Use pipette aids with Mucous Membranes Against filters Splashes and Aerosols! – Use horizontal pipette trays • Safety glasses? – Use incinerators • Plastic shields? – Pour liquids carefully – Work over absorbent – Use centrifuge safety cups – Use sealed rotors

CDC 1 3 Training Biosafety Training Should Be… – Biohazards • Done routinely – Risks of different types of • Made realistic exposures • Use proper equipment • Be “Hands on” – Available vaccinations and side • Practiced effects – Post-incident first aid and remediation – Signs and symptoms of infection What I hear, I forget – Emergency response procedures What I see, I remember – Reporting procedures What I do, I understand

Importance of First Aid First Aid Response for Exposures and Injuries Adequacy and timeliness of wound cleansing after an occupational – Pre-defined exposure occurs may be the – Available and accessible most critical determinant in immediately and 24/7 preventing infection – Simple, easy to follow guidance – Widely known about and reviewed often (are kit contents checked regularly?) – Linked to further assessment and reporting – Practiced!!

Emergency Procedures BSL-1 • Facility Design (secondary barriers) • Written SOP’s and training for: Requirements: – Building exhaust failure – Laboratories have doors – Sink for – BSC failure – Work surfaces easily cleaned – Spills – Sturdy furniture – Bench tops are impervious to water – Loss of power – Windows fitted with flyscreens – Evacuation – Insect and rodent control program – Location: not separated – Medical injuries or exposure – Structure: normal construction – Ventilation: none

CDC 1 4 Biosafety Level 2 (BSL-2)

• Suitable for work Biosafety Level 2 involving agents that pose moderate potential to personnel and the environment (most clinical labs)

BSL-2 BSL-2

• Differs from BSL-1 in that – Access to the laboratory is restricted when work is being conducted – Laboratory personnel have specific training in handling pathogenic – All procedures in which infectious agents and are supervised by aerosols or splashes may be created scientists competent in handling are conducted in BSC’s or other infectious agents and associated physical containment equipment procedures

Biosafety Level 2 Biosafety Level 2: • Supervision Special Practices – Supervisor is a competent • Use biosafety cabinets (Class II) for scientist with increased work with infectious agents involving responsibilities • Limits access if – Aerosols immunocompromised – Large volumes • Restricts access to immunized if necessary – High concentrations of organisms • Lab Personnel – Small Gram negative diplococci – Aware of potential hazards from spinal fluid or blood – Proficient in practices/techniques

CDC 1 5 Biosafety Level 2: Personal Protective Special Practices Equipment

– Small Gram negative or Gram • Act as a barrier to protect skin, variable rods, slow growth on BA, mucous membrane or respiratory no growth on Mac tract from exposure • Prevent spread of contamination • Protect the worker from splash and splatter • Protect product from contamination

BSL-2 PPE Personal Protective • Lab coat-long sleeved and buttoned Equipment

• Eye and face protection • Is appropriate PPE available? • Gloves • Separate waste stream for used PPE? • Is PPE being worn outside the laboratory? • Are personnel trained?

Personal Protective Personal Protective Equipment Equipment Gloves • Check integrity before use • Is appropriate PPE available? • Do not wash or reuse • Separate waste stream for used • or chemicals PPE? enhance permeation • Is PPE being worn outside the • Change often - Integrity laboratory? decreases with use • Do not touch “clean” surfaces • Are personnel trained? Does not eliminate the hazard!

CDC 1 6 When Do You Wear Gloves in When Do You Wear Gloves in the General Micro Lab? the General Micro Lab?

• “Gloves should be worn at the • “Gloves must be worn to protect specimen receiving and set-up areas, hands from exposure to hazardous and in TB/ labs, and when materials” (BMBL 5th edition) hands may contact potentially – Based on a lab-specific risk infectious material, contaminated assessment, the Laboratory Director surfaces or equipment.” (CLSI M29- or supervisor determines laboratory A3) hazards and when to wear gloves

BSL-2 Biosafety Level 2 • Facility Design (secondary barriers) Facility Design Requirements (BSL-1 Facilities PLUS) – Self-closing and lockable doors • (Secondary Barriers) (restricted access) – Automatic hand washing sinks – Air flow into lab without re- (preferably located near exit door) circulation to non-lab areas (for new construction) – Autoclave in or near lab (not – Easily cleanable-no rugs or cloth required) covered chairs – Eye wash – Location: separated from public areas

Biosafety Level 2 Facility Design

– Vacuum system protection Biosafety Level-3 – BSC’s installed so that fluctuations in the air supply or exhaust do not interfere with proper operation

CDC 1 7 Biosafety Level 3 (BSL-3) Biosafety Level 3 • Applicable to clinical, • Differs from BSL-2 in that diagnostic, teaching, – Personnel have specific training to research, or production handle particular facilities where work is – Supervised by scientists experienced with these agents performed with – All manipulations of infectious indigenous or exotic Mycobacterium tuberculosis material carried out in BSCs agents that may cause – Laboratory has special engineering serious or lethal disease and design features through inhalation route – Supervisors evaluate effectiveness exposure of training

What are BSL-3 Practices? What are BSL-3 Practices?

– All procedures involving the • When a procedure cannot be manipulation of infectious materials performed within a BSC, a MUST be conducted within a BSC or combination of PPE and other other physical containment device containment devices, such as • No work with open vessels is centrifuge safety cups or sealed conducted on the bench rotors, must be used • Restricted access to the laboratory

What are BSL-3 Practices? When Do You Use BSL-3 • Additional PPE (solid-front gown, Practices In A BSL-2 Lab? gloves and eye protection as a • When working with agents that are minimum) are worn in the lab normally handled under BSL-3 – Respiratory protection may be conditions, and a BSL-3 laboratory is indicated not available • Lab personnel must demonstrate proficiency prior to BSL-3 work • When determined by the laboratory • Potentially infectious waste materials director based on their risk should be decontaminated before assessment removal from the facility

CDC 1 8 When Do You Use BSL-3 Biosafety Level 3: Practices In A BSL-2 Lab? Special Practices • When specific high-risk pathogenic • Doors are kept closed organisms are suspected (such as • Persons at increased risk of infection are not Brucella spp., Coccidioides, allowed in lab Blastomyces dermatitidis, Francisella • Only those advised of tularensis, Histoplasma capsulatum, hazards and meet Mtb, etc) requirements can enter • Entry requirements and contacts posted on door

Biosafety Level 3: Biosafety Level 3: Special Practices Special Practices • Use -containing • All cultures, stocks and other equipment regulated wastes are decontaminated before disposal by an approved • NO work in open vessels is decontamination method, such as conducted on the bench! autoclaving • Load/unload – Preferably within centrifuge rotors in the laboratory BSC

Biosafety Level 3: Biosafety Level 3: Special Practices Special Practices • Additional Personal Protective – Other PPE based on risk Equipment assessment – Minimum of solid-front wrap • Coveralls around gowns (preferably with • Booties, head covers tight cuffs), gloves, eye and face • Double gloves protection • • Disposable sleeves – Other PPE based on risk assessment • Scrubs Personnel must be trained!

CDC 1 9 Biosafety Level 3 Biosafety Level 3 Personal Protective Equipment Personal Protective Equipment

• Respiratory protection may be – REDUCE exposure, do NOT indicated eliminate exposure-risk is never – Based on risk assessment zero – Personnel must be fit tested and – Surgical masks are NOT trained (OSHA 29 CFR 1910.134) respirators! – Respirators must be maintained – Facial hair interferes with N95 seal

BSL-3 BSL-3 – Enclosures for aerosol generating • Laboratory Facilities (secondary equipment barriers) – Double door entry (self-closing) • BSL-1 and BSL-2 Facilities PLUS: – Hands-free handwashing sinks – Separate building or isolated zone – Restricted access (locked doors, – Directional inward airflow (verified) biometrics) – Laboratory air is single-pass – Vacuum lines protected – Room penetrations and seams are – Pass-thru (or other sealed means of decon)

Biosafety Level 4 (BSL-4) Laboratory Biosafety Required for work with dangerous and Level Criteria exotic agents that pose a high individual risk of life-threatening disease, aerosol transmission, or related agent with unknown risk of transmission. • Cabinet laboratory

• Suit laboratory See supplemental handout in notebook

CDC 1 10 CLSI Resources CLSI Resources • M29-A3 • GP5-A2 – Protection of Laboratory Workers – Clinical Laboratory Waste From Occupationally Acquired Management-Second Edition -Third Edition • NCCLS Vol. 22 No. 4 • GP17-A2 – Clinical -Second – Implementing a Needlestick and Edition Sharps Injury Prevention Program in the Clinical Laboratory • GP18-A2 – Laboratory Design-Second Edition

Material Safety Data Sheet (MSDS) SafetyFirstPosters.com

http://www.phac-aspc.gc.ca/msds-ftss/index.html#menu?

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