Biosafety Training PowerPoint - Environmental Health and Safety ...

University Biosafety Officer 

Research and Biological Safety Office 

Environmental Health and Safety Department 

Phone: 206.221.7770 

Email: rbso@u.washington.edu

To ensure potentially biohazardous material is 

handled safely from start to finish 

1) Minimize Risk to lab Personnel and the Environment 

• By 1978 Pike/Sulkin identified 4,079 laboratory-associated infections (LAI), 168 

deaths occurring between 1930-1978. 

• Potential release to the environment - wild plants/animals, agriculture, livestock 

2) Regulatory Requirements 

• UW Administrative Policy Statement 12.3 

• UW Institutional Biosafety Committee 

• Washington State Bloodborne Pathogen Regulations 

• Biosafety in Microbiological and Biomedical Laboratories 

• NIH recombinant DNA guidelines 

9/17/2012 UW EH&S - Biosafety General Training

Research projects involving biological hazards and 

recombinant DNA must be reviewed by the Institutional 

Biosafety Committee (IBC) 


1) Faculty, Staff, Public Members 

2) Monthly meetings 

3) Review / approve use of biohazardous material in 

Research 

• Pathogenic agents (bacteria, rickettsia, fungi, viruses, protozoa, 

parasites, prions, and select agents) 

• Recombinant DNA molecules, organisms, vectors (e.g., plasmids, 

viral vectors), and viruses containing recombinant DNA molecules 

• Human and non-human primate tissue, body fluid, and cell culture 

(primary or continuous) 

• Plants, animals or derived waste which contain or may contain 

pathogenic hazards (including xenotransplantation tissue) 


• Applies to all those working with human blood, tissue, 

body fluid with visible blood, and cells. Also those working 

with HIV/HBV/HCV or other blood borne pathogens. 

• Requires initial and annual training for the duration of 

work: http://www.ehs.washington.edu/psotrain/corsdesc.shtm 

• A lab specific Exposure Control Plan must be in place: 

http://www.ehs.washington.edu/forms/rbs/researchlaboratorysupplementalform.d 

oc 

• Must be offered Hepatitis B vaccination within 10 days of 

starting work 


Introduced in 1984 

Now in its 5 th Edition (2007) 


• General risk assessment 

information 

• Description of Biosafety 

Levels 1-4 

(facility, equipment, practices) 

• Agent Specific information 

• Additional information 

regarding biosafety 

cabinets, decontamination, 

transportation, biosecurity, 

toxins, training, and more.... 


•Under the current NIH Guidelines, these are molecules 

constructed outside of living cells by joining natural or 

synthetic DNA segments to DNA molecules that can replicate 

in a living cell, or molecules that result from their replication. 

•Includes: viral vectors, E. coli/yeast cultures used for cloning, 

plasmid DNA, naked DNA from PCR or DNA sequencing) 


Must be followed for all recombinant DNA research at the 

University of Washington regardless of funding source 

• Identifies types of experiments covered and the level of 

oversight required (Section III) 

• Roles and Responsibilities for NIH, UW, PI, IBC, BSO 

(Section IV) 

• Classification of biological agents on the basis of hazard 

(virus, bacteria, parasites, fungus - Appendix B) 

• Description of Biosafety Levels 1-4 (Appendix G,P,Q) 


Section of NIH 

Guidelines 

Section III-A 

Section III-B 

Section III-C 

Level of Review 

Experiments that Require Institutional Biosafety 

Committee Approval, RAC review and NIH Director 

Approval Before Initiation 

Experiments that Require NIH/OBA and IBC Approval 

before Initiation 

Experiments that Require IBC and Institutional Review 

Board (IRB) Approvals and RAC Review Before Research 

Participant Enrollment 

Example of projects covered under the 

section 

Deliberate transfer of drug resistance to a 

microorganism that is not known to acquire it 

naturally 

Cloning of toxin molecules with LD less tan 

100 ng/kg 

Human gene therapy 

Section III-D Experiments that IBC Approval Before Initiation Use in pathogenic microorganisms, animals, 

plants, gene transfer in Risk Group 2 or 3 

microorganisms 

Section III-E 

Experiments that Require IBC Notice Simultaneous with 

Initiation 

Generation of transgenic rodents requiring 

BSL-1 containment 

Section III-F Exempt Experiments Recombinant DNA that is not in organisms or 

viruses, DNA/RNA that falls under the 

Exempt Experiments Under Section III-F

Submission to the IBC 

1) Determine level of review required 

2) Submit the Biological Use Authorization (BUA) 

Application to the Institutional Biosafety Committee 

(IBC) for review and approval (rbso@u.washington.edu) 

• Required for new projects and renewal every 3 years 

• Must be complete and accurate 

• BUA application reviewed by IBC and BSO 

• Review process may include lab inspection 

• Biological Use Authorization Letter sent to PI if approved 


Sample 

Biological Use 

Authorization 

Letter 

9/17/2012

Prior to Initiation 

• Instruct and train lab staff on: 

• Lab specific biosafety practices 

• spill / exposure procedures 

• shipping requirements 

• Inform lab staff of precautionary 

medical practices that are advised 

(e.g. vaccinations, etc.) 


While research is on-going 

• Supervise to ensure the required safety practices are in use 

• Investigate and report any significant problems, violations, 

accidents, or exposures (rbso@u.washington.edu , OARS ) 

• Remain in communication with the IBC to keep your 

biological use approval current (e.g. change in location, 

new agents). Biological Use Authorization Change Form 

• Ensure no project is modified prior to IBC approval 


• Standard microbiological practices 

• Special practices (e.g. decontamination procedures, 

vaccinations) 

• Safety equipment (e.g. containment devices, PPE) 

• Lab facilities (e.g. benches, chairs, sinks) 


•Well-characterized agents 

•Do not consistently cause disease in 

immunocompetent adult humans 

•Minimal potential hazard to laboratory 

personnel and the environment. 

E. coli-K12, Bacillus subtilis, canine hepatitis 

virus, murine cell lines 


Well-characterized agents 

Moderate hazard to personnel and the environment. 

Preventative and therapeutic interventions are often available 

Measles virus, Human cells, Toxoplasma, Adenovirus, Lentiviral 

vectors (non-HIV pseudotyped) 

9/17/2012

BSL-2 Lab with the following additions: 

• Lab specific Biosafety Manual 

• Lab specific Training 

• Rear opening gowns, inward airflow, limit sharps 

• Access controlled by PI (e.g. locks, keypad) 

• Increased review / oversight by EH&S 

Examples: HIV, Hepatitis C, 

Lentiviral vectors with known 

oncogenic inserts 


Indigenous or exotic agents 

May cause serious or potentially lethal disease through 

inhalation 

Preventative and therapeutic interventions may be available 

BSL-2 plus: 

Additional training 

Lab specific Biosafety Manual 

Protective equipment (inhalation) 

Engineering and design features 

Increased oversight by BSO 

Anthrax, SARS, TB, St. Louis encephalitis virus 


Dangerous and exotic agents 

High individual risk of life-threatening disease 

Aerosol transmission 

Preventative / therapeutic interventions are not usually 

available 

None at the University of Washington 


Marburg Virus, Congo-Crimean hemorrhagic fever, Ebola 


Specific Equipment and Practices 


Access to the lab is limited 

Sink for handwashing when 

leaving lab 

Eating, drinking, smoking, 

handling contact lenses, 

applying cosmetics, storing 

food is prohibited 

 

 

 

Fly screens recommended if 

windows are operable 

Decontaminate at least daily, or 

after spill 

Bench tops are impervious to 

water, resistant to heat /solvents 

/ acids / alkalis 

 

No mouth pipetting 

 

no carpets / rugs 

 

Lab coats recommended 

 

No fabric furniture or chairs 

 

Minimize aerosols 

 

Surfaces accessible for cleaning 


Fabric Chair to be discarded 


Sharps include needles, IV tubing with needles attached, scalpel blades, lancets, glass 

tubes that could be broken during handling and syringes that have been removed from 

their original sterile containers 

Don’t bend, break, recap, or remove needles from disposable syringes 

Develop and implement specific policies for safe handling 

Adopt improved engineering and work practice controls that 

reduce the risk of injuries 

Use a red puncture resistant sharps container for disposal 

Do not handle broken glassware directly by hand. 

Use plastic labware when possible 



BSL-1 plus 

additional: 

1) Administrative controls 

2) Safety equipment 

3) Safe practices 


• General Biosafety training (e.g. this class) 

• Lab specific hands-on training by supervisor or PI 


Equipment 

centrifuge, freezer, incubator 

Biohazard waste bags / containers 

Transporters / Shippers 


- Restrict access when work is 

in-process 

-Name of the infectious agent 

-Special entry requirements 

(minimum of lab coat 

when work is in-process) 

-Name, phone # of PI or 

other responsible person 

9/17/2012 UW EH&S - Biosafety General Training 

link to Biohazard sign

9/17/2012 UW EH&S - Biosafety General Training 

Must be available in the lab 

Project Review Process 

Biohazard Control Procedures 

Occupational Health Program 

Bloodborne Pathogen Program

• Personal Protective Equipment (PPE) 

• Biological Safety Cabinets 

• Centrifuge Safety Cups / Sealed rotors 

• Autoclaves 


Personal Protective Equipment (PPE) 

Lab Coat 

Gloves 

Additional PPE to be 

determined by risk 

assessment 


- Make sure PPE covers your street cloths - 


Aerosol generating activities at BSL-2 must be 

performed in a BSC or other containment device 


Biological Safety 

Cabinet (BSC) 

- Personnel Protection 

- Product Protection 

- Environmental Protection 


Biological Safety Cabinets 

High Efficiency Particulate Air (HEPA) filters 


Protect the front air curtain, keep items off the grill 

Store equipment outside of the BSC 

Decontaminate in / Decontaminate out 


Biological Safety Cabinets 


All BSCs on campus must be annually certified 

Test integrity of HEPA filters 

Test airflow rates 

For more information see the EH&S website: 

http://www.ehs.washington.edu/fsobiocab/index.shtm 

(Note: EH&S Paperwork is required for purchasing new or relocating a BSC) 


Personnel protection, but no product or environmental protection 


Laminar Flow Cabinet 

Product Protection only. No Personnel or environmental protection 


Centrifuge Safety Cups 

Required when centrifuging biological agents at BSL-2 outside of a 

biosafety cabinet 


Load /unload inside the BSC 

Insure rotor is properly balanced 

Know the speed ratings for rotor and tubes 

If unattended, post a warning sign with contact 

information 

Have an emergency plan in place, many lab associated 

infections have been linked to centrifuge use 


Use of safety cups or sealed 

rotors are not required if 

small enough to be placed in 

the BSC 


All biohazardous waste (including recombinant DNA 

section III-A thru III-F) must be treated prior to disposal 

• Solid waste – autoclave or equivalent 

• Liquid waste – bleach or equivalent 


• All users must adhere to proper operating and 

monitoring procedures. 

9/17/2012 http://www.ehs.washington.edu/rbsbiosafe/autoclave.shtm 

UW EH&S - Biosafety General Training

Autoclave Tape 

Use on every item placed in the autoclave. 

Indicates if temp. has been reached, but not duration 

untreated 

autoclaved 


Chemical Integrators 

Place a Thermalog-S 

integrator in center of each 

autoclave load. 

Attach a string to 

integrator for retrieval at 

end of cycle. 

Biological Indicators 

Perform test at least 

monthly. 

Bacillus stearothermophilus 

(See UW Biosafety Manual Section IV 

pg 47 for procedure) 


• Operators must ensure minimum temperature of 250°F / 

121°C for at least ½ hour. ( > time/temp. may be required) 

• Autoclave thermometer calibration at least annually 

Structural Testing 

• Must be inspected by a qualified pressure vessel inspector 

at least every two years 


Maintain and post a user log 

•Date 

•Time 

•Temperature 

•Pressure 

•Type of waste 

•Type of container(s) 

•Closure on container(s) 

•Pattern of loading 

•Water content 

•Maximum load quantity 

Complete the EH&S 

Quality Control Checklist 

http://www.ehs.washington.edu/forms/rbs/qcchecklist.doc 


Before loading and unloading the 

autoclave: 

• Inspect biohazardous bags and 

containers for protruding 

sharps or lab glass 

• Items with protruding sharps or 

lab glass must be placed in a 

secondary, leak-proof, 

container. 


NO 

NO 

YES 

•Can be stored up to 14 days prior to decontamination 

•Designate a space avoid contaminating lab supplies 


• Chemical Decontamination 

• Pipetting 

• Transportation 

• Spills 

• Exposures 


Use an appropriate chemical decontaminant 

70% Ethanol alone does not meet BBP regulations 

Points to Consider 

Concentration 

Contact time 

Stability 

Toxicity 

Corrosiveness 

Flammability 

Human Source Material – BBP 

Must be EPA registered against 

Hepatitis B and HIV, or TB 

Example: Chlorine Bleach 

1:10 spills 

1:100 surface decontamination 


• Use pipette filters to prevent contamination of equipment 

• Do not forcefully pipette up and down or blow out final volume 

• Pipette down the side of wells, dishes, flasks 

• Collect pipettes or micropipette tips in the BSC for disposal as 

contaminated biohazardous “lab glass” waste 

• Recommended discard containers: 

Pipet keepers 

Bench top keepers 


Materials must be placed in a durable, leakproof container for 

transport from the lab. 


Use a durable / leakproof secondary container 

1) 

2) 

Ziplock Bags 

3) Label with biohazard sign 

with contact information on 

container 

Coolers 

Decontaminate the outer container prior to leaving the lab 


Highly regulated (IATA, 49 CFR, State Regulations) 

Specific shipping containers required 

Additional packaging, absorbent materials, labels, paperwork 

Shippers must attend EH&S Class: Shipping and Transporting hazardous 

9/17/2012 UW EH&S - Biosafety General Materials Training

Have a plan for your specific lab 

Include: 

Chemical decontaminants 

Absorbent material 

Additional PPE 

Tongs / Forceps 

Biohazard waste bags 

http://www.ehs.washington.edu/rbsbiosafe/spillbiokit.shtm 


Life threatening – Dial 911 

Wash area immediately and thoroughly (15 minutes) 

Contact Supervisor / Manager 

Seek Care: 

UW Employee Health Clinic 685-1026 

Harborview Employee Health 744-3081 

After Hours – Go to the nearest ER (e.g. UWMC, Harborview) 

Report using the On-line Accident Reporting System (OARS) 

http://www.ehs.washington.edu/ohsoars/index.shtm#accidents 


BSL – 1 ABSL- 1 

BSL - 2 ABSL- 2 

BSL - 3 ABSL- 3 

BSL- 4 ABSL- 4 

(None at UW) 


• Animal sharps (teeth, fangs, claws, beaks, 

etc.) 

• Size and weight of the animal 

• Zoonotic diseases( e.g. Coxiella burnetii) 

• Will an administered infectious agent 

potentially be shed from the animal 


Hazardous material information for 

animal husbandry staff 

• Project contact information 

• Hazardous material identification 

• Additional personal protection / procedures required 

• Exposure procedures 


• Vaccination recommendations 

• Respirator usage/fit testing 

• Animal Allergies 

• Animal Use medical Screening 

form: AUMS 


• By who: University Biosafety Officer 

• When: New projects, renewal, significant changes 

• How do I Prepare: Request a BSL-1 or BSL-2 checklist 

• What to expect: Assess current facility and practices 

Discuss research needs, compliance options 

Provide additional training if necessary 

• Follow-up E-mail summary to PI / lab manager 

2 nd inspection may be required 


• Centrifuge safety cups not in use 

• Lab coats not in use or designated for lab 

• Updated Biosafety Manual needed (2003) 

• Improper biohazardous waste disposal process 

• Fabric upholstered chairs present in the lab 

• No food in the lab 

• No Biohazard door sign 


Food in lab 

Coffee made on lab 

equipment 

Lab coats stored in hallway – next to food 


• Current lab specific risk assessment 

• Use your safety equipment & PPE (know benefits & limitations) 

• Stay safety conscious 

• Avoid drift of practices (Most convenient is usually not the safest) 

• Plan ahead /stay well organized 

• Address additional issues that may increase susceptibility 

Immuno-compromised, skin rashes, cuts, pregnancy 

• Contact an EH&S Biosafety Officer or Occupational Health Nurse 

with questions (206) 221 7770 


University Biosafety Officer 

Research and Biological Safety Office 

Environmental Health and Safety Department 

Phone: 206.221.7770 

Email: rbso@u.washington.edu

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