Post on 08-May-2018
BIOSAFETY TRAINING NH IN-BRE May 22, 2014 Brenda Petrella, PhD Dartmouth College Biosafety Officer
DARTMOUTH COLLEGE Environmental Health & Safety
Objectives • Biosafety overview
• Background • Principles • Containment • Risk Assessment • Bloodborne Pathogens • Best Practices • Laboratory Inspections
• rDNA overview • What is recombinant or synthetic DNA • Safety considerations of rDNA experiments
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What is Biosafety? The principles, practices, procedures, and containment measures designed to prevent the accidental exposure to or release of biological agents and toxins.
What is Biosecurity? The protection, control, and accountability measures implemented to prevent the loss, theft, misuse, diversion or intentional release of biological agents and toxins.
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What is a Biohazard? An agent or material of biological origin that has the capacity to produce deleterious effects in humans, animals, or the environment:
• Viruses, bacteria, fungi, parasites, prions • Blood, body fluids, cells, and tissues from humans or
animals • Biological toxins, allergens, venoms • Recombinant DNA
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Why Biosafety? Laboratory Acquired Infections (LAIs)
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Route Practice Inhalation Aerosol generation – pipetting,
vortexing, centrifuging, sonication Inoculation Needlestick accidents; sharps;
animal or insect bites scratches Mucous Membrane Spills/splashes into eyes, mouth,
nose; aerosols Ingestion Splashes into mouth;
contaminated fingers near or in mouth; eating in lab
Skin Contamination
Spills/splashes without PPE; contaminated surfaces or equipment
Sewell, DL. Clinical Microb. Rev., July 1995, p. 389–405. http://wwwnc.cdc.gov/eid/article/12/1/05-1126_article.htm
Why Biosafety? Laboratory Acquired Infections (LAIs)
Pike, R.M. 1979. Annu. Rev. Mocrobiol. 33:41-66. Harding L.H. and Byers, K.B. 2006. Epidemiology of laboratory-acquired infections. Biological Safety Principles and Practices.
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4,079 LAIs
168 deaths
1935 1978 43 yrs
1,141 LAIs
24 deaths
1979 2005 26 yrs
Update: (1979-2013)
2033 LAIs 37 deaths
and counting
Outbreak of Salmonella Typhimurium LAIs Teaching and Clinical Microbiology Labs
http://www.cdc.gov/salmonella/typhimurium-laboratory/011712/index.html
BSL2 agent 109 LAIs 4 were in children
2010-2011
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Containment Primary goal of biosafety: reduce or eliminate human and environmental exposure to potentially harmful agents
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Primary Containment • Protects lab personnel and the immediate lab environment • Biological Safety Cabinet (BSC) • Good microbiological techniques (PPE, decontamination, etc.) • Safety centrifuge cups
Biological Containment • Inactivating mutations • Reducing virulence or pathogenicity
Secondary Containment • Protects environment external to the lab • Facility design and operations
• lab doors and/or anterooms • specialized air handling systems for contamination control • autoclaves • hand washing, eyewash, shower stations
Clean bench vs Fume hood vs BSC
Biological Safety Cabinet • Protects user, sample, and
environment from biologics • Does not provide chemical
protection
Fume Hood • Protects user from volatile
compounds • Does not protect against
infectious agents
Clean bench Protects sample ONLY
No/low individual risk No/low community risk
Moderate individual risk Low community risk
High individual risk Low community risk
High individual risk High community risk
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Risk Groups
What are Biosafety Levels (BSLs)?
The primary risks that determine levels of containment are:
• Risk Group category • Infectivity • Severity of disease • Transmissibility • Nature of the work conducted • Route of exposure
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Biosafety Level 1 (BSL1) Risk Group 1 Agents:
• E.coli K-12 • Transgenic plants • Fungi • Mold • Yeast
Lab Practice • Standard microbiological practices • Open bench work ok unless aerosols generated, then use BSC • Daily decontamination • Required hand-washing • Autoclave waste
Safety Equipment • PPE – gloves, lab coat, eye protection as needed • BSC – if generating aerosols
Facility • Sink available for hand washing • Door on lab with appropriate signage DARTMOUTH COLLEGE
Environmental Health & Safety
Biosafety Level 2 (BSL2)
Builds on BSL1 requirements
Risk Group 2 Agents: • Human or primate cells • Herpes Simplex Virus (HSV) • Replication-incompetent attenuated HIV • Patient specimens • Staphylococcus aureus
Lab Practice • Access to the laboratory is restricted when
work is being conducted • PPE = lab coats, gloves, eye protection,
face shields or masks as needed. • Decontamination after each procedure • Autoclave waste • Document training
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BSL2 cont. Safety Equipment
• Biosafety cabinet (BSC) • Sharps containers • Autoclave waste bags/containers
Facility • Self-closing doors with BSL2 signage • Autoclave • Sink and eyewash station readily
available • Label all equipment in contact with
biohazard
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Biosafety Level 3 (BSL3)
Builds on BSL2 requirements
Risk Group Agents: • Respiratory transmission • Mycobacterium tuberculosis • HIV (wild-type) • Coxiella burnetii
Lab Practice • Medical surveillance, baseline serology • Immunizations available or required • Autoclave all waste at end of day • Avoid use of sharps • No bench top – all work in BSC • PPE – double gloves, PAPR, wrap around
disposable gown
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BSL3 cont. Safety Equipment
• All work with microbes must be performed within an appropriate BSC
Facility • A hands-free sink and eyewash • Exhaust air cannot be recirculated • Sustained directional airflow by drawing air into the lab
from clean areas towards potentially contaminated areas • Entrance to the lab is through two sets of self-closing and
locking doors • Access to the laboratory is restricted and controlled at all
times.
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Biosafety Level 4 (BSL4) Maximum containment facility - builds on BSL3 requirements
Risk Group Agents • high risk of aerosol-transmitted infections • exotic, frequently fatal • examples: Ebola and Marburg viruses
Lab Practice • Change clothing before entering • Shower upon exiting • Decontaminate all materials before exiting
Safety Equipment • All work performed in Class III, wearing a full body,
air-supplied, positive pressure suit.
Facility • separate building or restricted zone • dedicated supply and exhaust air, and vacuum lines
and decontamination systems DARTMOUTH COLLEGE Environmental Health & Safety
Biosafety Risk Assessment 1. Identify the hazard
2. Assess potential risk to personnel and/or environment: • Identify Risk Group • What is the scale of work – volume, concentration • What are the procedures/manipulations – aerosol generating? Use of
sharps? • Determine pathogenicity – is it wild-type or attenuated? What is the
infectious dose? • What is the route of entry? • Are prophylaxis treatments available? • Are personnel trained? • Any risk to immuno-compromised or pregnant personnel?
3. Determine appropriate containment/biosafety level (BSL) DARTMOUTH COLLEGE Environmental Health & Safety
RG1 RG2 RG3 RG4
Question 1 An undergraduate is studying the regulation of circadian rhythm genes using the mold, Neurospora crassa (RG1), as a model system. Methodology will be limited to quantitative PCR and western blotting to assess gene regulation and expression.
BSL1
What PPE should be worn? Can this work be conducted on the bench top?
At what BSL level should this work be conducted?
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Question 2 A principal investigator just received IN-BRE funding to study antibiotic resistance of Pseudomonas aeruginosa (RG2) in cystic fibrosis. He will be using a human lung cell line model system for infectivity studies using various clinical isolates of the bacteria.
BSL2
What PPE should be worn? Can this work be conducted on the bench top?
What types of containment should be considered? At what BSL level should this work be conducted?
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What are they? Microorganisms that are transmitted through the bloodstream. • HIV • Hep B • Hep C • Syphilis • Malaria
Where are they found? • Blood or other bodily fluid • Human cell lines, fresh tissues
Routes of exposure • Needle-sticks • Other sharps • Skin scratches • Mucous membrane
Who is at risk? • Lab personnel • Custodial staff
Bloodborne Pathogens
HIV
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Bloodborne Pathogens OSHA Standard (29 CFR 1910.1030)
ü Establish exposure control plan, updated annually ü Implement use of “Universal Precautions” ü Use engineering controls
• sharps disposal containers • self-sheathing needles or other engineered sharps-injury
protection • reduce the need for needles
ü Identify work practice controls • change the way a task is performed • increasing containment • proper labeling
ü Provide appropriate PPE ü Provide training ü Availability of Hepatitis B vaccine and post-
exposure monitoring DARTMOUTH COLLEGE Environmental Health & Safety
Best Practices - Programmatic
• Biosafety Manual • Exposure Control Plan • Training • IBC oversight – NIH Guideline oversight, reporting, (rDNA,
toxins, biohazardous agents) • Standard Operating Procedures (SOPs) • Injury and Accident Reports • Lab Inspections • Occupational Health Program
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Best Practices – Sharps • Bloodborne pathogens
• Use safety-engineered sharps
• Use biohazard sharps containers
• Use plastic pipets instead of glass pasteur pipets for aspiration
Never: • Re-cap needles • Bend or break needles • Re-use needles or syringes • Let sharps container get too full • Put hands into sharps container
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Best Practices - Vacuum Traps
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Trap flask w/ wescodyne HEPA
filter 2° container
Tube from media to top inlet of flask
(part a)
Tube from side of flask to
house vac line (part b)
Direction of vac flow Working end of tube
used to aspirate biological waste
with pipette
Rubber stopper w/pipette
House vac port
Best Practices – Prevention of Aerosols
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Perform manipulations inside BSC • Pipetting • Vortexing • Sonication • Opening of centrifuge tubes • Aspiration rather than decanting
Use safety cups with o-rings for centrifugation
Biosafety Inspection Checklist • PPE, handwashing supplies • Eyewash stations • Sharps eliminated where feasible • Biohazard waste bags (autoclave bags) – no sharps! • Biohazard sharps containers • No recapping of needles • Waste containers not overfilled • Proper disinfection, spill supplies, disposal procedures • Vacuum traps • Equipment (BSC certification, aerosol resistant
centrifuges) • Autoclaving waste - verification
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What is recombinant DNA (rDNA)? According to the NIH Guidelines, recombinant and synthetic nucleic acids are defined as:
• (i) molecules that • a) are constructed by joining nucleic acid molecules • b) that can replicate in a living cell, i.e., recombinant nucleic acids
• (ii) nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, i.e., synthetic nucleic acids
• (iii) molecules that result from the replication of those described in (i) or (ii) above.
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What is recombinant DNA (rDNA)? Recombination = creation of a new DNA molecule from two different DNA molecules
Why does it work? Genetic code is universal!
rDNA
bacterial plasmid carrying human insulin gene
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human insulin gene
bacterial plasmid
Common ways to make rDNA Molecular cloning
• Arts & Crafts: Cut/paste pieces of DNA together; grow up new DNA in bacteria (bugs replicate new DNA for us)
PCR (polymerase chain reaction) • Xerox machine: exponentially duplicate same finite
sequence of DNA
Chemical synthesis (“synthetic DNA”) • Mad scientist • Generation of artificial DNA molecules that do not
exist in nature (no starting sequence) • Able to recombine with naturally occurring nucleic
acid molecules DARTMOUTH COLLEGE Environmental Health & Safety
Common lab uses of rDNA • Overexpression of a gene in cells, animals, or plants
• Knockdown of a gene in cells, animals, or plants
• Copying or mutating a gene
• Sequencing DNA
• Measuring gene expression
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Safety Considerations for rDNA work
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• Increase pathogenicity?
• Change host range? (tropism)
• Inactivate a tumor suppressor gene or activate an oncogene?
• What are the potential downstream effects of release?
• Does it make an organism resistant to treatment or change their growth/development? • GMOs • Antibiotic resistant bacteria • Recombinant bovine growth hormone (rBGH)
Biosafety Summary
Biohazard recognition
Risk Assessment
Biohazard mitigation
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Resources • BMBL: http://www.cdc.gov/biosafety/publications/bmbl5/
• NIH Guidelines:http://osp.od.nih.gov/office-biotechnology-activities/biosafety/nih-guidelines
• ABSA: http://www.absa.org/
• WHO Biosafety Manual: • http://www.who.int/csr/resources/publications/biosafety/
WHO_CDS_CSR_LYO_2004_11/en/
• OSHA Bloodborne Pathogens Standard: https://www.osha.gov/dte/grant_materials/fy09/sh-18796-09/bloodbornepathogens.pdf
• Public Health Agency of Canada – pathogen safety sheets: • http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/pseudomonas-spp-eng.php
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