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General Biosafety Training Module Biosafety Compliance Program Office of the Vice Chancellor for Research and Graduate Education Eleanor Low Research Compliance Officer [email protected] 956-8420 285-6367 (emergencies) Stephen Case Biosafety Specialist [email protected] 956-8009 BIOSAFETY COMPLIANCE PROGRAM [email protected] http://manoa.hawaii.edu/ovcrge/research/biosafety_program/index.html Learning Objectives: • Describe the differences between the FOUR Biosafety Laboratory Levels • Explain what types of risks are involved when working in a lab • Understand how to minimize the potential risks when working in a lab through RISK ASSESSMENT! • Describe incident response procedures to a biological spill • Describe emergency response procedures Acronyms and Abbreviations commonly used in Biosafety • BSL - Biosafety Level, criteria defined in Biosafety in Microbiological and Biomedical Laboratories, 5th edition. (BMBL) • BSC - Biosafety Cabinet • LAI - Laboratory Acquired Infection • N95 - Filter respirator mask used to protect an individual from airborne particles. Filters at least 95% of airborne particles. Not resistant to oil. Not all N95’s are created equal. • PPE - Personal Protective Equipment Standard Terminology • Biohazard - A biological substance which has or may have the ability to pose a threat to the health of a living organism (human, animal or the environment). Examples include microorganisms, toxins or allergens • Decontamination - The process of removing, neutralizing or destroying a biohazardous agent. • Risk Group Classification - Risk group classifications are used in the research lab as part of a biosafety Risk Assessment to determine the proper containment, PPE used… o http://www.absa.org/riskgroups/index.html Known as the "BMBL" . The standard established guideline for best safe biosafety practices Established to promote safe science practices for research involving the use of or creation of recombinant material. Institutional Biosafety Committee’s comply with the NIH Guidelines and use the BMBL as the reference standard 2 3 Biological Safety Levels? 1 BMBL 5th Ed. 4 • Biosafety Levels were developed to provide increasing levels of protection to Personnel, Materials and the Environment based on an agents risk level. A combination of procedures, engineering controls, work practices, and policies are utilized to develop these different biosafety levels. Low Risk High Risk BSL-1 BSL-2 BSL-3 BSL-4 Basic Biological RISK Groups Risk Group 1 Agents are not associated with disease in healthy adult humans. Risk Group 2 Agents are associated with human disease which is rarely serious and for which preventative or therapeutic interventions are often available. Risk Group 3 Risk Group 4 Agents are associated with serious or lethal human disease for which preventative or therapeutic interventions may be available. Agents are likely to cause serious or lethal human disease for which preventative or therapeutic interventions are usually not available. Basic Biosafety Levels Risk Group BSL Level Basic 1 BSL 1 Basic 2 BSL 2 Containment 3 4 Examples BSL 4 Facilities, Equipment Basic Teaching, Research Good Microbiological Techniques (GMT) None required; open benchwork, directional airflow Primary health services, diagnostic, teaching, public health Level 1 plus protective clothing, biohazard signage Open bench plus Biological Safety Cabinet (BSC) for aerosols Special diagnostic, research Level 2 plus special clothing, controlled access BSC and/or other primary containment devices for all activities Dangerous pathogen unit Level 3 plus airlock entry, shower exit, special waste disposal Class III BSC or positive pressure suits, double door autoclave, filtered air BSL 3 Maximum Containment Practices • No eating, drinking, chewing gum, smoking, handling contact lenses or applying cosmetics • No mouth pipetting. • No open toe shoes in the lab − No sandals − Sandals with socks are still not acceptable!! • Perform procedures carefully to minimize generation of aerosols • Sharps Handling Policy Do Not recap, re-use, bend or break Needles Properly contain and dispose • Limited/Restricted access when work is in progress • Decontaminate work surfaces before and after use with an effective disinfectant. • Decontaminate waste from all Infectious and potentially infectious materials. • Biohazard signage posted when infectious agents are present or in use • Training. PI/lab supervisor must ensure laboratory personnel demonstrate proficiency (written documentation) prior to working in lab; − Standard and special microbiological − Lab specific training • Always remove gloves before touching clean surfaces; phone, computers etc.. • Always wash hands with warm water and soap for 10-15 seconds after: − Handling any viable material − Removing gloves − Before leaving lab • Maintain a Pest Management program MSU Lab Wars Again remember one of the most effective means of preventing disease spread is proper hand washing!! http://www.cdc.gov/Features/HandWashing/ BIOSAFETY CONTAINMENT Basic Elements Each element plays a role in protecting the individual researcher, the environment, and the material from contamination. – Practices - Standard Microbiological Procedures, SOP’s – Personal Protective Equipment (PPE) - primary barrier – Facility Design - secondary barriers – Containment Equipment - secondary barriers (examples include BSC, HEPA filtered centrifuges) Clearly written Standard Operating Procedures are a resource for working safely in the lab. Steps in writing SOPs for the laboratory: • Identify each task that will be performed • Develop procedures based on risk assessment • Incorporate safety measures (PPE, Equipment, Personal Behavior, etc) • Procedures for − Mitigating aerosol generation − Safe sharps use and handling − Decontamination − Equipment certification PPE- Protect the skin, body, mucous membranes, respiratory system and clothing from potential biohazards when working in the lab. • Gloves- protect hands, act as a barrier. Must be removed and disposed of when contaminated. Never re-use contaminated gloves! • Lab coats/solid-front gowns- protect street clothing and skin • Eye Protection- protect against splashes • Additional PPE that may be required: − − − − Sleeve covers, Shoe Covers, booties Full face protection (Shields) Respiratory protection, such as N95, if needed/required The Facility acts as a secondary barrier to minimize potential contamination and prevent spread of disease. Biosafety Level 1-4 Facilities • Signage - Door sign - Restricted Access • Hand washing sinks • Eye wash • Autoclave • Biosafety Cabinet • Communication outside the lab (phone) Biosafety Level 3-4 • Anteroom • Negative pressure gradient • Airflow monitor • Set Air changes per hour (10-15) • Sealed penetrations, covered flooring • Facility alarms/interlocks • Biological Safety Cabinets (BSC); Class I, II, III − BSC’s are NOT fume hoods. Fume hoods remove chemical fumes and aerosols away from the working area. BSC’s provide a clean work area for personnel and environmental protection while working with a biological agent. • • • • Safety centrifuges; HEPA filtered, sealed Sealed sonicators, blenders, homogenizers Sealed tubes, transport carriers Safe sharps, needle boxes, medical waste bags, tongs, forceps, etc. Dedicated Metal Sharps Safety Needle Container CDC Routes of Exposure Discovery Channel, Coughing Robot 2010 AEROSOL generated from a simple cough! Route Inhalation Microbiological practices/accident Procedures that produce aerosols: Centrifugation Mixing, sonication, vortexing, blending Spills and splashes Pouring/decanting culture fluids Manipulation of inoculation loop Inoculation Needle stick Lacerations from sharp objects (e.g. blades, broken glass) Ingestion Splashes to the mouth Placing contaminated articles/fingers in mouth Consumption of food in the laboratory Mouth pipetting Contamination of skin and mucous membranes Splashes Contact with contaminated fomites From: Sewell, DL. 2006, Clinical Micro Newsletter 28:1-6 • Aerosols are a serious hazard because they are common in lab procedures and usually go undetected. • Aerosols are the probable cause of many LAIs, particularly in cases where there was no known accident. • Lab procedures which could potentially produce aerosols: • − Homogenization, sonication, blending, mixing, grinding, shaking, vortexing, spills, animals excreting agent, etc. − Opening vials; snap cap tubes, vials that have developed pressure − Pipetting − Animal inoculations − Use of a Flow Cytometer • Pipetting: − Cotton plugged pipettes − Gentle mixing and expelling. − NO MOUTH PIPETTING!! − Mechanical pipetting devices • Centrifugation: − Use O-ring screw caps, recommended speeds, plastic tubes, open lid slowly, decontaminate regularly • Mixing (sonication, homogenizers, etc) operations: − Operate equipment in BSC, use O-ring caps, open tubes in BSC, decontaminate after use • Sharps/needles: − Use in BSC, fill syringe slowly (avoid bubbles), extract needle carefully from insertion points − Never re-cap or un-cap a used needle!! Discard used needles immediately MSU lab wars In the Lab What Are Sharps? ANY OBJECT that can puncture or cut the skin and cause injury….. – – – – – – – Needles Any broken glass Microscope Slides Cover Slips Petri Dish Pipet tips Broken plastic ware …..and ANY OBJECT from the lab that can puncture a plastic trash bag should also be considered a SHARP!! • Sharps? Needles, Scalpels or any object that can puncture or cut skin and cause injury including microscope slides and cover slips • Major Sharps Injuries: − Skin puncture from needle OUCH ! CDC − Aerosols − Spills caused by leaking syringe/needle − Poor work practices; tapping on syringe or recapping needle − Inappropriate disposal such as overflowing sharps container(s) OUCH ! CDC • • • • • Adequate training prior to use of sharps. Use needle-locking syringes When working with animals use restraints when possible Immediate collection/disposal of sharps, needles Do not recap or bend the needle!! CDC • Location of sharps container should be within easy reach and proper height • Do not allow sharp containers to overflow • Eliminate/minimize use/ of Sharps. Use safe sharp devices whenever possible • Avoid − glass Pasteur pipettes − sharps use with infectious agents or pathogens Proper Disposal • Contaminated Sharps: − For Biological/Infectious contaminated; decontaminate first (autoclave) − Radioisotope contaminated- Contact Radiation Safety − Chemical contaminated- Contact Chemical Safety • Contact EHSO for disposal − Use appropriate Sharps Container to contain sharps. Do not overfill. Follow guidelines on container • Non Contaminated Sharps: − Use appropriate Sharps Container to dispose of used sharps. Do not overfill. Follow guidelines on container (usually the capacity is about 50 to 75% full) − Follow appropriate guidelines for disposal General Practices • All contaminated wastes should be separated into sharps, liquid, solid biological wastes. • Waste should be placed in autoclave bags inside a durable container (secondary container) that is foot operated to minimize contamination of the lid area. (Non foot operated containers must have a lid/cover) • Do not overfill the bags • Autoclave when ¾ full Lid/Cover Foot Pedal Operation MSU Lab Wars 1. STERILIZATION • The destruction of ALL forms of microbial life. Kills all microorganisms including spores. • Method: Steam sterilization through the use of an autoclave • Applications: Regulated waste and critical instruments or media 2. DISINFECTION • Eliminates NEARLY ALL pathogenic microorganisms, but not all forms (e.g. bacterial spores). Less destructive than sterilization • Method: Use of chemical liquid germicides known as disinfectants • Applications: Biological spills, general decontamination of lab surfaces and equipment Place in a sturdy secondary container – plastic or metal. Add small volume of water to the bag (10ml-250ml). Loosely close bag. Never tightly seal containers or bags. Record in autoclave log book. Monitor the temperature, pressure and time during the autoclave cycle. • Routinely run Biological Indicators (BI’s) (monthly, quarterly). • • • • • – May use indicator strips (i.e. Class 5 CI) to the load. (opp.) Use Caution when removing autoclaved material. Do not open door quickly. • Maintenance. Autoclaves should have a regular maintenance check performed by certified technician. • Effectiveness of disinfection is determined by: • Nature of microorganisms (presence of spores?) • Number/Amount of microorganisms • Amount of organic matter present (soil, feces, blood?) • Temperature • Time allowed for disinfection activity • Concentration of Disinfectant. (Higher concentration is not necessarily more effective!!) • Type of Disinfectant used • • • • • • • • Prions Protozoan cysts Bacterial spores Mycobacteria Non-lipid or small viruses Fungal spores, fungi Vegetative bacteria Lipid or medium-size viruses High level of resistance to germicidal chemicals Low level of resistance to germicidal chemicals BMBL, 5th Edition Practical Requirements Use Contact Time (minutes) Vegetative Dilution Bacteria * Lipid Broad envelope Spectrum( all viruses microbes) Inactivates Lipid Non lipid envelope Viruses viruses Disinfectants Category LIQUID Quaternary Ammonium Compounds Chlorine Compounds Alcohol, Ethyl 0.1%-2.0% 10 Not Effective + + 500 ppm 10 30 + + + 70%-85% 10 Not Effective + + Variable results dependent on virus Alcohol, Isopropyl 70%-85% 10 Not Effective + + Variable results dependent on virus Bacterial Spores + Adapted from Yale University RISK ASSESSMENT? Dean Potter RISK ASSESSMENT A process used to identify the hazardous characteristics of a known infectious or potentially infectious agent or material, the activities that can result in a person’s exposure to an agent, the likelihood that such exposure will cause a LAI, and the probable consequences of such an infection. Definition from CDC WHY do a Risk Assessment? • To better understand how a Lab Acquired Infection may occur. • To assist in determining safe procedures to use to prevent or reduce potential exposure and infection. • A risk assessment is the backbone to SAFE LAB PRACTICES In Lab Setting: • Microorganisms are grown, cultured, purified and used at concentrations that are at much higher levels than in a natural setting. • Microorganisms are treated and used differently than in the natural setting; inoculation studies, purifications, cell culture. Immunocomprimised Individuals. • Exposure can be lethal (Opportunistic Infection) An exposure to a BSl-1 organism may be relatively safe for a normal healthy individual. − Staphylococcus aureus, Pseudomonas aeruginosa, JC polyomavirus, Cytomegalovirus, Cryptococcus neofomans Determine how you will minimize the Identified Risks: • Review the SOP/Procedures and work practices. − Are they adequate to reduce/prevent potential hazards or accidents? • What types of PPE should you use? − Will gloves and lab coat be sufficient? • Training − Are you staff adequately trained to perform the research activity? • Facilities Design (Engineering Controls). − Are you working in and at the correct Biosafety Lab level for the microorganism and research activity? • Equipment. − Is the BSC certified? − Are centrifuges, autoclaves functioning properly? • Signs and Symptoms of Disease vary depending on the microorganism, the persons age, if they are Immunocompromised. • As a lab worker, you should be aware of the symptoms of disease, the transmission route and hazards associated with working with infectious agents References: • ABSA (American Biological Safety Association) • ASM (American Society of Microbiology) • Microbiology, Sherris (Text books) • CDC • Web Review risk assessment 5 Evaluate staff proficiencies 1 Identify agent hazards and perform initial risk assessment RISK ASSESSMENT is always ongoing In the working lab 4 3 Determine Required PPE, Equipment 2 Identify laboratory procedure hazards Review risk assessment 5 Evaluate staff proficiencies 1 Identify agent hazards and perform initial risk assessment Consider all Potential Risks; Actual and Perceived 4 3 Determine Required PPE, Equipment 2 Identify laboratory procedure hazards 1997. A student working in a microbiology was tasked with the cleaning and decontaminating lab ware used in the lab for cultures of Shigella dysenteriae. The student was suppose to perform this step in a dedicated sink for contaminated materials. However the sink was dirty and the student went to the dedicated hand washing sink to clean the materials 12 hours later, Six co-workers became ill and were diagnosed with Shigella dysenteriae. Other lab workers were identified as asymptomatic (exposed but at lower doses) The procedure generated splash that contaminated the hand washing sink and was spread via the handles as they were turned on and off. Lessons Learned: • All procedures involving a biohazardous material must be completed by trained personnel including the decontamination process. • All surfaces, equipment and areas that may have come in contact with an infectious material must be decontaminated. • Dedicated hand wash area must be respected • Biohazard control procedures should be established and implemented. Risk Assessment: • Training on: − safe and proper handling of the organism including proper decontamination − Know the signs and symptoms of Shigella infection • More experienced staff should do the work • Dedicated room with restricted access • Hands free sinks should have been installed. The most frequent types of lab accidents: • Inhalation (aerosols) • Needle Sticks/Cuts • Splashes/Spills • Animal Bites/Scratches • From mouth pipetting • Direct contact; broken skin cuts CDC Accidents occur when in a hurry, when tired, not paying attention. Review all potential risks before performing the task. Think before you Act LAI’S can occur from any of the above and unknown routes of transmission BSL2 – LAI’s in the News • October 2009 Boston University. Graduate Student became infected with Neisseria meningitides while working in a lab. • 2004 - 3 BU researchers become ill after working with what was believed to be an avirulent strain of Francisella tularensis. Lab sample later found to be contaminated with virulent wild type. • 1987- Researcher becomes infected with HIV when working on HIV. Found “hole in glove” • Most common LAI species include Brucella, Mycobacterium tuberculosis, Shigella, Salmonella. Reports are voluntary and often are unreported because of fear of reproach from reporting. Emergency Response All Lab personnel should Be able to identify the locations of the: • Eye Wash Station • Designated Hand Wash Station • Phone • Spill Kit Find the: • Emergency Contact Information • Procedures on reporting an accident Provide care to injured person first. Always treat injured. Call 911, physician, Campus Security (66911) depending on severity of injury. Percutaneous Injury. Wash affected area with disinfectant/soap and warm water. Apply dressing and seek medical attention. Notify your PI, Biosafety Compliance Program. Splashes to Face. Flush affected area in eyewash for 15 minutes. Notify your PI, Biosafety Compliance Program. Aerosol Exposure. Hold your breath, leave room where aerosol was generated immediately. Carefully remove your PPE (turn inside out) Wash hands with warm water and soap. Post a sign “Do Not Enter; Spill” on door. Lab should be evacuated for a minimum of 30 minutes. • Notify PI, Research Compliance Officer/Biosafety. • Until PI/ Safety Officers gives permission, no one should enter lab. LAB ACCIDENTSINJURY and REPORTING Less than 10 percent of Lab accidents are reported in the Nation. Fewer than 20% of those accidents have a known cause of transmission. (Aerosol is the plausible cause in most cases) By reporting you provide assistance to those who have been injured and provide invaluable help to prevent future similar incidents NBBTP For all exposure and possible exposure incidents: Notify Principal Investigator, lab manager or supervisor to initiate Incident report within 24 hours of accident • PI/supervisor must report the accident to IBC (rDNA & Infectious agents), IACUC (animals), Human Resources, Risk Management • PI notification to Research Compliance Officer within 24 hours 956-8420, 285-6367 • Large spills (> 100-200 ml or when unable to contain) • All BSL-3 spills • All Select Agent spills • rDNA that results in an environmental exposure or injury • Incidents resulting in personal injury • Cover spill with absorbent material. • Saturate with appropriate disinfectant. Starting from the outside and working toward the center of spill. • Allow disinfectant to soak (minimum 30 minutes). • With gloved hands; pick up all wastes and place slowly and carefully into biowaste container. • Cover area again with disinfectant then clean up with absorbent material • Remove your gloves and dispose in the biowaste container. • Wash hands with warm water and soap. • Put on new gloves and if necessary new lab coat. • • • • • • • • • Spill Cleanup Protocol/instructions Absorbent material; paper towels or pads Disinfectant -Bleach or other appropriate disinfectant A bottle to make fresh diluted disinfectant Forceps, dust pan, plastic scoop, or other device for handling sharps or broken glass Autoclavable biohazard bags to collect contaminated items Disposable nitrile gloves Durable Utility Gloves Appropriate PPE (lab coat or jumpsuit, safety goggles, shoe covers, face mask, N95**). **Lab personnel requiring N95 respirators must have a medical evaluation from a registered health care professional prior to being fit tested in order to wear a respirator. (N95 fit testing is provided at UH Manoa [email protected] 956-3204 ) Outside of Biosafety Cabinet (BSC) BSL-1 Spills, or Small Scale (<200 mL) BSL-2 Spills Notify Treat Human Injury Treat personal contamination Wear PPE Decontaminate spill Notify others in the immediate area that a spill has occurred. Tend to any injured personnel. Call 911 if necessary Place contaminated clothing in biohazardous bag. Wash skin thoroughly with soap and water. Don lab coat, gloves, face protection. Cover spill absorbent material. Saturate the paper towel with appropriate disinfectant, starting with the edges of the spill and working towards the center. Allow contact time of at least 30 minutes. Clean up After the 30 minute contact time, with gloved hands pick up absorbent material and place in biohazardous bag. Use appropriate equipment (scoop, dustpan, or forceps) to pick up any broken glass. Place in sharps container. Disinfect surrounding area Carefully spray area and surrounding areas with appropriate disinfectant wipe up with absorbent material. Place all waste material in autoclave bag Autoclave contaminated material and PPE. Decontaminate any reusable items with disinfectant. Wash hands and exposed skin areas with soap and warm water Notify PI or supervisor of large spills. If exposure occurred, submit exposure incident report to Supervisor and Biosafety Compliance Proram Decontaminate material and PPE Document and reporting Principal Investigator Responsibilities • Ensure Adequate Training • Equipment Integrity • Proper notification of incidents involving staff, students and visitors of the University of Hawaii • University of Hawaii Approvals − ORS − IACUC, IBC, CHS • Federal and or State permits or licenses • Compliance with all applicable University of Hawaii, State of Hawaii, and Federal policies, guidelines and regulations Principal Investigator Responsibilities (continued ) Develop Biosafety Manual (BMBL 5 th Ed. good reference) Components: − Lab personnel review and acknowledgment (date, name, signature) − General and Organism specific SOP’s /Procedures − Daily Clean Up & General Decontamination − Biological Spill Response Plan − Waste Management Plan − Sharps Injury Prevention Procedures − Emergency response plans for lab • Medical • Fire • Natural disaster − Transportation and Storage of microorganisms Contact Information University of Hawaii • Eleanor Low, Research Compliance Officer, Biosafety (808)956-8420,(808)285-6367 • Stephen Case, Biosafety Specialist (808) 956-8009 • Hubert Olipares, Biosafety Officer (808) 956-3197 • Tim O'Callaghan, Hazardous Material Management Officer (808) 956-3198 • Emma Kennedy, Industrial Hygienist (808) 956-3204 • Irene Sakimoto, Radiation Safety Officer (808) 956-6475 UH HILO • Ken Ikeda Environmental Health & Safety Officer (808)933-3300 Institutional Biosafety Committee (IBC) http://manoa.hawaii.edu/ovcrge/research/ibc/index.html • Eleanor Low @ [email protected] IACUC-Institutional Animal Care and Use Committee IACUC http://www.hawaii.edu/ansc/IACUC/ • Mr. Norman Magno at [email protected] • Ms. Denise Yee at [email protected] Committee on Human Studies http://www.hawaii.edu/irb/index.htm • [email protected] UH Biosafety Compliance Program PROMOTES SAFE LAB PRACTICES Eleanor Low Stephen Case QUESTIONS?