Biological Safety Training Title

EHS staff works 24/7 to ensure safety of this campus

Call 413-545-2682 with any questions or visit http://www.ehs.umass.edu/

Biosafety Office: 413-545-2682 Biological Safety staff works to:

Judy LaDuc, Biosafety Officer: [email protected] • Ensure that biological research is conducted in a safe fashion Myron Lizak, Associate Biological Safety Officer: [email protected] • Ensure that biological research meets regulatory requirements

Biological Agents & Routes of Exposure Biological Agents & Routes of Exposure Risk Groups Risk Group NIH Guidelines Biological agents: Classification Working with biological agents may create risks to: Risk Group 1 Agents not associated with disease in healthy adult • Bacteria, viruses, fungi, rickettsia humans. (ex. Escherichia coli K-12) • Other microorganisms and/or their associated toxins •workers • environment Risk Group 2 Agents associated with human disease that is rarely • community serious and for which preventive or therapeutic Can be transmitted to humans via: interventions are often available. • Ingestion, inhalation, mucous membrane exposure, (ex. E. coli O157:H7) inoculation (e.g. needlestick) Biosafety regulations were developed to mitigate all of these Risk Group 3 Agents associated with serious or lethal human disease for which preventive or therapeutic interventions may be risks available (high individual risk but low community risk). (ex. HIV) Risk Group 4 Agents likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk). (ex. Ebola virus) Biosafety Level Chart Biosafety Levels Biosafety Level BSL-1 BSL-2 BSL-3 BSL-4 Biosafety Level BSL-1 BSL-2 BSL-3 BSL-4 Safety glasses Each biosafety level has its own specific √√ √ √ Sample • E. Coli K-12 • E. Coli O157:H7 • Yellow Fever • Ebola Virus containment controls that are required : Organisms Strain • Staphylococcus Virus • Marburg Gloves • Lactobacillus aureus • West Nile Virus Viruses √√ √ √ acidophilus • HIV • Mycobacterium • Bacillus subtilis • Hepatitis B virus tuberculosis Proper shoes √√ √ √ • Laboratory Practices Buttoned lab coats Facility • Doors for access • Biosafety cabinet • Pass-through • Pass-through √√ • Laboratory Facilities Requirements control for aerosol autoclave with autoclave • Sink for hand procedures Bioseal with Bioseal Tyvek suit washing • Centrifuge safety required required √√ • Safety Equipment • Sanitizable caps • Negative air • Highest surfaces and • Sharps flow security Respiratory frequent containers • HEPA filter protection √√ disinfection • Biosecurity exhaust • Screens on windows • Sharps

NO Engineering Controls Engineering Controls • Cell Phones • Biosafety cabinet used for procedures likely • Ear Buds to generate splashes or aerosols • Mouth Pipetting • Centrifuge must have sealed rotor head or Acceptable Better • Loose Hair centrifuge safety cups • Handling Contact Lenses • Autoclaves are large pressure cookers used to sterilize media and biological • Applying Cosmetics waste. • Shorts/Skirts • Eating/Drinking • Animals (pets) 12 inches • Plants (house plants)

Photo Credits: Power Vision Heating Systems Working with Human, Mammalian Cells and Tissues NIH Guidelines for Research Involving HOW TO REMOVE YOUR GLOVES PROPERLY Recombinant DNA or Synthetic Nucleic Acid Molecules • Always follow Universal/Standard Precautions •Register the research with Institutional Biosafety Committee when working with human blood, body fluids, Pull one glove near your wrist towards your finger tips until the glove folds over tissues, primary cell lines etc. •The principal investigator must make sure that his/her laboratory complies with the NIH Guidelines for such research •Non-compliance can result in UMass losing all NIH funding* • ATCC (American Type Culture Collection) does not Carefully grab the fold and pull towards your finger screen for every possible organism (mycoplasma, •Injuries/ incidents to rDNA or synthetic nucleic acids must be reported to the tips, as you pull, turn the inside of the glove viruses) in the cell lines that it sells Biosafety Officer ASAP outwards •Proper containment of these materials must be maintained. They are not Slide your finger from your glove free hand under • Handle all human cell lines, (including all cancer allowed to enter the food chain, water supply, air stream, etc. the remaining glove. Turn you finger 180 degrees cell lines) at biosafety level 2 and pull the glove outwards and towards your finger •Proper disposal requires destruction of the research material by autoclaving tips • Self inoculation, needle stick, is a potential risk or incineration before the final product is discarded While you do this, the first glove will be encased in with human cancer cell lines. There has been one the second glove. Dispose of the gloves properly •Example: Fish are maintained in sealed tanks and destroyed by autoclaving reported case of a tumor from a needle-stick and… or incineration, they are not allowed to go “down the drain” WASH YOUR HANDS

Personal Protective Equipment (PPE) Secondary Containment Personal Protective Equipment (PPE) Gloves • No latex or powder gloves Respirator Always transport biological specimens in secondary containment • Change frequently • UMass has a Respiratory Protection Program • Do not reuse; Discard used gloves immediately (if contaminated put • All respirator users on campus (N-95, half-face, full-face, in biohazard waste) powered respirators) will be enrolled in this program • Do not touch door handles with gloves; Never wear outside lab • Use of respirators on UMass campus is not recommended without a fit test • Report any allergies immediately to supervisor and EH&S • Always wash hands after removing the gloves • Visit website https://ehs.umass.edu/respiratory-protection-program or call 413-545-2682 What’s Wrong With This Picture? Biological Safety Cabinet OSHA Bloodborne Pathogen Standard 29 CFR 1910.1030

• Human Blood • Human Blood Components • Human Tissues • Human Cell Lines

Image by Succeed Management Solutions, LLC

OSHA 1910.1030 Lab Acquired Infections (LAIs) Accidental Exposure Procedure Bloodborne Pathogens Standard • The causative incident for most LAI’s is unknown…..ONLY 20% are Needle sticks, Cuts, Animal bite/scratch or Splash to eye/mouth from a recognized incident • Federal standard to protect workers that may be CALL 911 if a serious accident (UMPD: 413-545-3111) • Aerosols most likely route of exposure exposed to human materials (blood, serum, 1: Stop work immediately and wash injured or exposed area ASAP with tissues) that may have HIV, Hepatitis B and soap and water for at least 15 minutes • Always be careful when manipulating Hepatitis C biological materials 2: Do not ignore any exposure; Always seek medical treatment at University Health Services (UHS) or an Emergency room for post exposure • Free vaccinations to Hepatitis B for employees evaluation/treatment within 2 hours of incident that are at risk of exposure Use of Common lab procedures  Vigorous shaking • Free medical evaluations for incidents of  Centrifuge exposure  Vortex  Pouring, Spraying

• Implementation of engineering and work practice  Blender  Opening lyophilized cultures controls  Homogenizer  Flaming loops/needles • Proper labelling of risks  Sonicator  Changing animal bedding Accidental Exposure Procedure Medical Surveillance Principles of Sterilization and Disinfection Needle sticks, Cuts, Animal bite/scratch or Splash to eye/mouth A Sterilization procedure is one that kills all microorganisms, . If immunosuppressed (taking steroids, pregnant, including high numbers of bacterial spores. Sterilization can be 3: All incidents/accidents must be reported to Environmental Health and Safety (EH&S) (413-545-2682) and lab supervisor; NIH may need to be no spleen, etc.), you are more at risk accomplished by autoclaving. notified by EH&S Best This is to decontaminate a space: No Sterilizing pathogens left alive & zero contamination. Complete and submit the Laboratory Incident Report Form: http://www.ehs.umass.edu/lab-incidents-and-lab-incident-report-form . If ill, seek medical attention and inform Doctor Better This is cleaning for Health: Removing what you work with (animals, microbial agents Disinfecting some germs, creating a healthier space. 4: Complete and submit the Notice of Injury (NOI) form to the UMass Amherst Human Resources (HR) within 48 hours of the incident: etc.) Good This is for appearance: Dirt, grime, and Cleaning debris removal. http://www.umass.edu/humres/notice-injury-form Disinfection is generally a less lethal process than sterilization. It eliminates nearly all recognized pathogenic microorganisms but not necessarily all microbial forms (e.g., bacterial spores) on inanimate objects.

Reference: Biosafety in Microbiological and Biomedical Laboratories

Selection and Use of Disinfectants Comparison Chart For Commonly Used Small Scale Biological Spill Clean up Procedure Disinfectants in Labs Call EH&S for assistance with larger spills 413-545-2682 Disinfection is the process that eliminates disease causing micro-organisms on inanimate objects, with the exception of bacterial spores Alcohols • Contact time of 10 min 1.Allow aerosols/dust to settle

70% Ethanol (v/v %) • Challenging due to evaporation 2.Use appropriate PPE Hardest

to kill Bacterial spores (Bacillus anthracis, Clostridium tetani etc.) 10% Bleach • Not effective for blood spills 3.Never pick up broken glass or sharps with bare hands; use tongs, forceps etc.

70% Ethanol 4.Do not pour disinfectant directly onto the area of spill Mycobacteria (Mycobacterium tuberculosis, Mycobacterium avium etc.) 5.Cover the spill with paper towels or absorbent material Chlorine Non-enveloped viruses (Polio, Rhinoviruses etc.) • Effective for blood spills 6.Pick up as much of the spill as possible and place in biohazard waste 10% Bleach (v/v %) • Contact time of 10 min for surfaces 7.Add appropriate disinfectant and follow manufacturer’s recommendations for Fungi (Candida, Aspergillus, Cryptococcus etc.) contact time • Contact time of 30 min for liquid waste Vegetative Bacteria (Staphylococcus, Salmonella, E.coli etc.) 8.Reapply disinfectant if necessary; wipe down vertical surfaces also • Irritant to skin and mucous membranes Level Disinfectant Level Low Level Disinfectant Level Intermediate Level Disinfectant Level High Enveloped Viruses (Herpes simplex virus, measles virus, Sterilant Easiest 9.Place all clean-up materials in regular waste (second disinfection) to kill mumps virus, influenza virus, rubella virus etc.) • Must make 10% working solution fresh daily Bleach Facts Biological Waste Decontamination of Biological Liquids  Buy 5.25% concentration or higher; if concentration is not listed on Solid the container, don’t use it • Recombinant DNA or synthetic nucleic acid materials (soil, plants,  Check code on bottle for expiration date bacteria, animals, etc.) Biological Liquid Waste can be:  Code cipher: E216145 • Materials containing or contaminated with blood or body fluids; • Cell culture waste Read the last three digits on the right (Julian calendar date) • Other liquid biological waste 145= May 25th • Pipettes and pipette tips; Read the next digit (to left) = year manufactured; 16 = 2016 • Animal waste; specimens 100 mL Concentrated Bleach 900 mL Liquid Waste Ignore other digits/letters  Concentrated bleach is only stabile for one year from date of • Disposable labware (flasks, tubes, agar plates, bottle, vials) manufacture (E216145 = 5/25/2016 which is the date it was made) • Contaminated gloves, other PPE, wipes etc.  Read labels thoroughly Liquid • Cell culture waste • Other liquid biological waste Mixed Biological/Chemical • Call EH&S for information Biohazard Box Waste Contact Time: 30 minutes

Biological Waste Disposal of Biological Waste from BSL-1 and BSL-2 Autoclaving Biological Waste Laboratories (including plants, soil and recombinant DNA) • All Biological waste must be kept in a leak proof End Point: achieve sterilization; kill all living organisms and covered container labeled with a biohazard Biological Waste symbol Mode of Action: Steam Solid Liquid • When the container is not in use, keep the lid Sharps Parameters: closed • Time (minimum) 60 min • Transport full bags in a secondary container; Autoclave Bio-waste box Autoclave Chemical (Preferred Method) (Needs EH&S approval) never place directly on floor as it can lead to a (Preferred Method) Disinfection • Temperature 121oC (250oF) spill/leak Sharps Container • Pressure 15 psi • All biological waste must be deactivated before final disposal (autoclaved, chemically dis- • Do not autoclave chemicals or radioactive materials infected or sent off-site for incineration) Regular Trash Incineration Sanitary Sewer • Do not use as a reaction vessel for experiments Autoclaving Biological Waste Autoclaving Biological Waste What Happened? • Tie bags loosely; Do not tape the bag shut (leave room • Only use clear autoclavable bags for a gloved finger to fit into neck) 1. 2. • Loosen caps for liquid containers or use vented caps • Only use autoclavable secondary containers (Nalgene or Stainless • Do not overload the autoclave Steel) • Place completed label (below) on cooled bag

• Add 50 -100 ml of water into the bag to promote steam production

• Do not overfill the bags; remove the bag from the waste receptacle when it’s approximately 2/3rd full

Photo Credits (top right): http://tea.armadaproject.org/stoyles/11.27.2003.html Photo Credits 1. https://bio.calpoly.edu/content/department-safety-guidelines 2. https://biologyze.wordpress.com/page/5/?pages-list

Autoclaving Biological Waste Autoclaving Biological Waste: Cycle Complete Autoclaving Biological Waste: Verification • Chemical Verification: Verifies if autoclave achieved the temperature of Select appropriate cycle for your biological waste (use preprogrammed 121oC (250oF) during the cycle cycles if available) • Wear PPE (heat resistant gloves, buttoned lab coat, covered shoes, safety goggles/ face • Use chemical tape or a chemical integrator every time you run a cycle shield) Remember: Do not use fast exhaust or gravity cycle for autoclaving • Stand behind the autoclave door before opening liquids; liquids will boil over and cause a spill • Allow liquids to cool down for at least 10 min before removing them • Required cycle time for biological waste is 60 min • After autoclaving, place “Autoclave Label” on all • Biological Verification: Verifies if autoclave achieved sterilization autoclaved clear bags of biological waste • Use biological spore vials every month Remember: cycle time will increase depending upon • Before final disposal, place all autoclaved clear number of bags and quantity of biological waste bags (with the autoclave stickers on them) inside a black trash bag

Photo Credits: National Institute of Health and Dartmouth College Complete the Autoclave Log Sheet – Required by Law Documentation: Good Laboratory Practice What Goes In A Sharps Container

• Principles help to define and standardize the planning, performance, • Needles recording, reporting, monitoring and archiving processes within research institutions • ALL Syringes (with or without needles) • Entries must be made in ink (Blue or Black)

• Cross out mistakes using a single line • Lancets

*Rerun the autoclave cycle with a new indicator. If the indicator does not change, • Scalpel blades run spores to validate correct operation. If spores fail, post an “Out of Order” sign on the autoclave; Notify responsible person and/or call autoclave repair service. • Glass Slides and Cover Slips • Razor blades Contact EH&S for alternative waste disposal methods if another autoclave is not • Other devices capable of available. cutting or piercing the skin (e.g. broken glass contaminated with ** If checking “Yes” and performing spore test, document results on this • Glass Pasteur pipettes biological agents) page as well as on the spore testing log at the back of the booklet. Ref: World Health Organization; GLP HANDBOOK, Chapter 2 • Good Laboratory Practice Training

Safety with Sharps Safety with Sharps Containers Sharps Containers Disposal • Visit EH&S website http://www.ehs.umass.edu/ Do Not Shake Container to • Click on “Request It” (see yellow arrow) Make More • Click on “Hazardous Waste Pickup” link on the dropdown list Room!

Photo Credits: The FDA in collaboration with Kwikpoint Photo Credits: The FDA in collaboration with Kwikpoint Sharps Containers Disposal Micropipette Tip Problem Flow Chart for Disposal of Micropipette Tips

• Complete the required fields and submit the form Micropipette Tips

Contaminated with Not Contaminated with Biological Agent(s) Biological Agent(s)

Autoclave Sharps Container Regular Trash (Preferred Method) (heavy duty plastic bag)

Regular Trash (heavy duty plastic bag)

‘Glass Only Box’ only if clean, dry, and NOT Shipping Biological Materials Field Research

CONTAMINATED with biological or chemical hazards Certain biological materials are Have Contingency Plan for: regulated under the Department • Glass slides of Transportation (DOT) and the • Physical hazards extreme heat, extreme • Cover slips International Air Transit Authority cold, noise, weather (IATA) as hazardous materials • Biological hazards vector-borne diseases, • Test tubes and require specific packaging, venomous wildlife and insects, and labeling, and documentation poisonous plants • Glass beakers • Chemical hazards exposure to pesticides • Glass flasks Use eShipGlobal program at: Field Research Considerations http://www.ehs.umass.edu/shippi •Glassvials ng-hazardous-materials- • Vaccination against potential pathogens • Other Glass items instructions (e.g. Tetanus, rabies) • Serological pipettes • Training on when and how to use PPE Contact EHS at 413-545-2682 • Broken or Discarded with any questions Resources American Biological Safety . CDC/NIH Biosafety Manual free PDF download at: Association http://www.cdc.gov/od/OHS/biosfty/b mbl5/BMBL_5th_Edition.pdf

• ABSA was founded in 1984 to promote biosafety as a . UMass Environmental Health and scientific discipline and serve the growing needs of Safety Biological Safety Website: biosafety professionals throughout the world. http://www.ehs.umass.edu/biological- safety • https://absa.org/

. UMass Environmental Health and • Risk Group Database Safety Fact Sheets • Laboratory Acquired Infection (LAI) Database http://www.ehs.umass.edu/fact-sheets