Basic Biosafety: Safety in Experiments Environmental Health and Safety October 2009 Environmental Health and Safety EHS EHS has been charged with ensuring the safety of this campus. We work to: Ensure that biological research is conducted in a safe fashion Ensure that biological research meets regulatory requirements Support the Institutional Biological Safety Committee (IBSC) Biosafety means Practicing Safe Science Ask Questions BEFORE you start Plan safety into your experiment Biosafety manuals and UA manual – each lab should have these and provide training on their contents Good lab practices Hazard Communication – you must notify anyone who enters your area of risks Reduce risks to acceptable levels Concern for others and the environment – we have an obligation to protect the public and the environment Biohazard Symbol Universal Symbol Communicates potential exposure Typically red or orange Symbol should be defaced when hazard is no longer present Use sparingly, explicitly Use for cultures of pathogens Human blood, tissue Equipment used with above Storage areas of above Cages of infected animals Door into laboratory Risk Assessment Risk assessments on lab procedures should be done before initiating them. They should answer the following: What are the hazards? What might happen? How likely is it to happen? How serious are the consequences if it happens? What are the possible exposures? How can I mitigate exposure? What is the WORST that can happen? Remember! Familiarity affects your perception of risk! Tools for Risk Assessment Risk Groups Biosafety Levels Guidance Documents Risk Groups Based on transmissibility, invasiveness, virulence and lethality of the specific pathogen RG1 – not associated with disease RG2 – associated with disease that is rarely serious or for which there is treatment RG3 – serious or lethal human disease with treatments RG4 – serious or lethal human disease with no treatment options Biosafety Levels Correlates to Risk Groups More commonly used on campus Describes containment practices, equipment, and facility design features recommended for safe handling of these organisms Biosafety Level 1 Class 1 agents are not associated with disease in healthy adult humans However, increased precautions may be appropriate when using these agents for transfection since foreign genes can be delivered even though disease is not caused Some examples include: E. coli nonpathogenic laboratory strains Saccharomyces cerevisiae Agrobacterium tumefaciens Baculovirus Duck hepatitis B virus Biosafety Level 1 “Good Microbiological Techniques” No eating, drinking, applying makeup, etc. No mouth pipetting Safety glasses worn Lab coats stay in lab Wash you hands Safe handling of sharps Decontaminate cultures and waste Laboratory access limited when work is in progress Biosafety Level 2 Class 2 agents are associated with human disease which is rarely serious and for which preventative or therapeutic interventions are often available. Some examples would include: E. coli, pathogenic strains Adenovirus Herpes simplex virus Chicken pox Moloney murine retrovirus, amphotropic Biosafety Level 2 BSL 1 practices in effect Biohazard or restricted access sign on door Door closed – negative air pressure Limit/restrict access to laboratory Minimize aerosols Biosafety cabinet for aerosol control PPE required: gloves, lab coats, respirators in some cases High degree of precaution with sharps Decontaminate surfaces and equipment Biosafety Level 3 Class 3 agents are associated with serious or lethal human diseases for which preventative or therapeutic interventions may be available (high individual risk but low community risk) Francisella tularensis Human immunodeficiency virus Histoplasma capsulatum Mycobacterium tuberculosis Aerosol is a common route of transmission, and increases the risk potential for these agents Biosafety Level 4 Class 4 agents are 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) Ebola virus Herpes B virus (Cercopithecine) Lassa fever virus Relationships Risk BSL Level Examples Practices Facilities, Group Equipment 1 Basic Basic Teaching, Good Microbiological None required; open BSL 1 Research Techniques (GMT) benchwork, directional airflow 2 Basic Primary health services, Level 1 plus protective Open bench plus BSL 2 diagnostic, teaching, clothing, biohazard Biological Safety public health signage Cabinet (BSC) for aerosols 3 Containment Special diagnostic, Level 2 plus special BSC and/or other BSL 3 research clothing, controlled primary containment access devices for all activities 4 Maximum Dangerous pathogen unit Level 3 plus airlock Class III BSC or Containment entry, shower exit, positive pressure BSL 3 special waste disposal suits, double door autoclave, filtered air Recombinant DNA As an institution receiving research funds from the National Institutes of Health, we are subject to the NIH Guidelines for Research Involving Recombinant DNA Molecules All recombinant DNA experiments (rDNA) at the University of Alabama must be reviewed by the Institutional Biological Safety Committee All recombinant DNA work must be approved before work begins Protocols must be reviewed periodically Applicable Sections of the NIH Guidelines III-D, IBSC review prior to initiation BSL2 or higher microbes as vectors Introduction of rDNA into BSL 2 or higher microbes Large scale rDNA production (>10L) III-E, IBSC notification Most BSL1 research Many plant experiments Many transgenic rodent experiments Exempt from NIH Guidelines Section III-F A common misconception is that low risk research is exempt from the Guidelines; in fact, exemptions are limited and specific. S. cerevisiae and some E. coli K-12 hosts Viral genome (<1/2) in tissue culture Sequencing and PCR DNA propagated solely in the species Gene transfer between species known to exchange DNA by physiological means Does not present risk, as determined by the NIH Director NIH Guidelines - Compliance “As a condition for NIH funding of rDNA research, institutions shall ensure that such research conducted at or sponsored by the institution, irrespective of the source of funding, shall comply with the NIH guidelines.” UA receives funding from NIH, so all research here must comply with the NIH Guidelines. Noncompliance may result in suspension, limitation, or termination of NIH funds at the institution. Some Available References for Risk Assessment and Lab Safety Laboratory Biosafety Manual, 3rd edition World Health Organizatio Geneva 2004 Pathogen Characteristics We must think of the characteristics of a pathogen in order to determine how best to contain it. Ecotropic pathogens are less risk than amphotropic pathogens because amphotropic can infect humans. Some pathogens are carried by vectors (e.g., mosquitoes and malaria). Survivability in the environment varies greatly among pathogens, from minutes to years. Virulence can also vary greatly, even among different strains of the same microbe. Some pathogens produce toxins, many do not. Whether a pathogen stays local in the host or goes systemic (spreads to other organs) greatly affects virulence and host survival. Routes of Transmission Fecal - oral Vector – e.g., mosquito Mucosal (splash) Cuts, scratches, bites Aerosol, inhalation ** **Aerosol transmission tends to be the most hazardous and hardest to contain. We will give these some additional consideration Some Common Activities That Generate Aerosols Vortexing Pipetting Sonication Electroporation Popping tube caps Flame sterilizing tools Flow cytometry Centrifugation Infected animals Examples of Infectious Doses It is the low infectious doses that make some diseases so dangerous to human life. M. Tuberculosis 10 cells Salmonella typhi 100,000 cells Listeria < 1,000 cells Cryptosporidium <10 cells Rotavirus 10-100 i.u. Vibrio cholera 1,000,000 cells Host Susceptibility Age Immune Competence Medication Nutritional Status Pregnancy Metabolic Disorders Malignancy Some Other Hazards Bloodborne Pathogens – for those working with human blood or tissue Mixed Hazards – for those who use chemical and biological materials combined Physical Sharps Hazard – for needles and broken glass Bloodborne Pathogens Universal Precautions – handle human blood, body fluids and tissues as if they harbor pathogens like HIV, Hep B, etc Use BSL 2 containment: Protect against cuts, needle stick injuries Use a BSC for aerosol control Hepatitis B vaccine recommended Wear gloves, lab coat, safety glasses, etc Decontaminate spills with approved disinfectant Mixed Hazards: Chemical and Biological – Some Lab Practices Door closed: negative air pressure Limit/Restrict access to lab Use of signs/labels Minimize aerosol production Containment: fume hoods for chemicals PPE required: gloves, lab coats, safety glasses, respirators in some cases High degree of caution with sharps Decontaminate surfaces and equipment What are considered Sharps? Hypodermic needles and syringes, IV needles and tubing, blades, etc., are regulated as medical waste Glassware exposed to an infectious agent must be managed as a sharp until it has been autoclaved. Sharps containers must be red in color and display the International Biohazard Symbol, say Medical Waste, or be labeled as Infectious Waste Sharps containers should be puncture proof. Sharps may not be disposed of in red bags. Some Protective Equipment Respiratory Protection Ventilation Devices Fume Hoods Laminar Flow Hoods Biological Safety Cabinets Respiratory Protection Surgical or dust mask for large particles Chemical masks for vapors, acids Filtering facepiece, air purifying respirator for microbes Must be FIT TESTED by EHS for respirator use! Ventilation Devices Fume Hood Laminar Flow Hoods Biological Safety Cabinet Fume Hoods Fume hoods protect workers from chemical vapors. Handle hazardous chemicals in fume hoods whenever possible. Laminar Flow Hoods These hoods are only appropriate for very few purposes and they are often misused. Don’t work with any hazardous in a laminar flow hood. They protect the working surface only, NOT the worker! Biological Safety Cabinets (BSC) BSCs work to protect workers and material from microbes. Avoid working with volatile chemicals in a BSC. Proper Use of a BSC Turn it on! Maintain constant air curtain – minimize movements in/out, traffic No Bunsen Burners Avoid clutter, keep grille clean Disinfect working surface and interior Certify performance annually (EHS does this!) Avoid extended use of UV lamps – use them with care. Disposal of Potentially Hazardous Waste Chemical Disinfection Autoclave Disinfection Aim for total destruction of target organism Autoclave sterilization Bleach - effective against microbial agents of diseases at a concentration of 0.1 percent Detergents Alcohol (70%) Factors Affecting Bleach Efficacy Amount of organic matter Exposure to light Age of bleach solution Concentration We recommend that you make a fresh solution of bleach at least monthly and store it in a opaque container that has a good seal. Date it so you know when to toss it. It is common for bleach to 50% or more of its efficacy when in storage for just one month. Bleach itself is a hazard – it burns the skin and it is corrosive. Do not autoclave a solution that has been treated with bleach. It is hard on the autoclave and can cause chlorine gas to be released. Can I autoclave something then put it into regular trash? The short answer is YES – on some things: So what can I autoclave and dispose? Medical Waste Class 1 Agents Class 2 Agents that can’t be aerosolized to class 3 Autoclavable Waste ADEM regulations have specific prohibitions on the regular trash disposal of all items bearing either an international symbol or any wording indicating that the items contain infectious waste, biohazardous waste, or medical waste. In order to dispose of treated medical waste as regular trash the autoclaved bag must not be red or orange nor contain any wording or symbols indicating that it contained medical waste. The state prohibits using an orange/red bag for autoclaving, then placing it into a black trash bag for disposal. How do I handle the collection of material? Many times the materials are biohazardous/medical waste until they are autoclaved – then they can be regular trash. If I can’t trash the red bags, how do I handle these? You should acquire outer secondary containers (ex: trash receptacle) and affix a biohazard symbol to the exterior surface. Use a black autoclave bag inside the secondary container. This allows the material to be clearly identified in the lab and still allows disposal of bagged material in regular trash stream. Autoclave Guidelines Equipment should continuously monitor and record temperature and pressure during the entire length of each cycle. If not so equipped, temp sensitive tape should be affixed to each bag or container. Equivalent tests can be approved by ADEM Effectiveness must be evaluated under a full load at least once every 40 hours of operation In any routine monitoring of autoclave performance, biological indicators or thermocouples should be places at the center of each load. Sterilizers used for waste treatment shall not be used for sterilization of equipment, food, or other related items. Each bag must be exposed to a minimum of: 250 degrees Fahrenheit 15 pounds of pressure At least 30 minutes time AT THESE CONDITIONS Autoclave Recordkeeping A written log or other means of documentation as approved by ADEM shall be maintained for each unit and shall contain the following: Date, time, duration, and operator of each cycle Approximate weight/volume of medical waste treated during each cycle Temperature and pressure maintained during each cycle Method utilized for confirmation of temperature and pressure Dates and results of calibration and maintenance Written log must be maintained for three years Biosecurity Purpose of biosecurity measures is to protect the community, prevent theft, and comply with federal and state regulations CDC guidelines for even BSL 1 labs: “Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures or specimens is in progress.” This means that everyone entering your lab should have approval to be there! Some things you can do to secure your lab: Know the people in your area - question unfamiliar people/activities Maintain inventory of materials and equipment Safeguard hazardous materials – unauthorized persons should not be able to access your inventory Limit access - everyone entering lab has approval to be there Lock lab whenever it is unattended – do not prop lab doors open Summary It is up to you to protect yourself, your co- workers, the community, and the environment! Good lab practices are fundamental Communicate about hazardous materials PLAN PLAN PLAN When in doubt, ASK!!!! How to Reach Us Environmental Health and Safety 15 Research Drive Box 870178 Tuscaloosa, AL 35487 205-348-5905 Marcy Huey firstname.lastname@example.org Last Step! The final step is to take a brief quiz. This will test your knowledge and understanding of this material. Print the quiz, complete it and the information sheet, and send them (email, fax or campus mail) : Marcy Huey Box 870178 Fax: 348-7773 email@example.com We will send you a certificate for training upon grading. You must get a 90% for a passing grade. Quiz – page 1 1. Name “3” things you would want to know about a pathogen that would help you determine an appropriate means of containment: 2. Name “3” things that could make you more susceptible to a pathogen: 3. Name three routes of transmission for an infectious agent: 4. List three references to aid you in Risk Assessment and Lab Safety: 5. Name two common laboratory activities that could generate an aerosol: Quiz – page 2 6. If you handle human blood or body fluids/tissues, what containment level should you use? 7. List the minimum conditions a bag must be exposed to in an autoclave in meet ADEM requirements for Medical Waste treatment: 8. The efficacy of bleach for disinfection depends on: (Mark all that apply) a. Concentration d. The amount of organic material present b. The brand e. The container in which it was stored c. The age of the solution 9. Which of the following can go into regular trash streams? a. Class 4 biological agents c. Class 2 agents that can aerosolize to Class 3 b. Select Agents regulated by the CDC d. Class 2 agents after being autoclaved according to ADEM regs 10. Name three things you can do to protect your lab area: Information Sheet Name: ________________________________________________________ Date:__________________________________________________________ Department: ___________________________________________________ Supervisor: ____________________________________________________ Building/Room: _________________________________________________ Phone: ________________________________________________________ Campus Mailing Address: _________________________________________ Email: ________________________________________________________ Certification Statement: I hereby certify that I have completed the training for Basic Biosafety and that I am submitting my quiz answers for review. Signature: _______________________________________ Print: ___________________________________________ Date: ___________________________________________ Fax this form and quiz to Marcy Huey at 348-7773, email firstname.lastname@example.org or send through campus mail to Box 870178. You should receive your certificate within 2 weeks.