Copy of Exer 1_GLP and Biosafety.pdf

Laboratory Biosafety and Good Laboratory Practices Biol 107 Cell and Molecular Biology Intended Learning Outcomes: Execute laboratory safety practices and SOPs when doing laboratory exercises. Enumerate different protocols that have been established at the international and n...

Laboratory Biosafety and Good Laboratory Practices Biol 107 Cell and Molecular Biology Intended Learning Outcomes: Execute laboratory safety practices and SOPs when doing laboratory exercises. Enumerate different protocols that have been established at the international and national levels to ensure biosafety in institutions involved in biotechnology research and development. Introduction GMT (good microbiological techniques) will not suffice to provide safety during early days of the development of rDNA technology June 1976 after Asilomar Conference (Ensuring Safety in Biotechnology) NIH Guidelines for Research Involving Recombinant Nucleic Acid Molecules  UN WHO in 1983 published the Laboratory Biosafety Manual (aka WHO Manual) establishing basic concepts and practices in the safe handling of pathogenic microorganisms encouraged countries to develop national code of practice for implementation Why it is important to adhere to international standards? To protect plant, animal or human life health of its citizen trade in biotech products Risk Categories of Microorganisms NIH guidelines and WHO Manual, recognized 4 categories Based on the risk of infection to: Laboratory workers Community in the event of an escape from the lab How to assign a microorganism to a risk category? Pathogenicity of the organism Host range and mode of transmission of the organism Local availability of effective measures to prevent a disease outbreak Local availability of effective treatment Risk Groups (RG) of Microorganisms: RG 1: unlikely to cause human or animal diseases pose little or no risk to individual and to the community; generally regarded as safe (GRAS) RG 2: pathogenic, but unlikely to pose a serious hazard to lab workers, livestock, community, or environment as effective treatment and preventive measures to limit the spread of infection are available; moderate risk to individual and low risk to the community Risk Groups (RG) of Microorganisms: RG 3: pathogenic and can cause serious human or animal diseases, but are not contagious or have an effective treatment and preventive measures to limit the spread of infection are available; high risk to individual, but low risk to the community RG 4: causes serious diseases in humans and animals and readily transmitted (direct or indirect), effective treatment and preventive measures are unavailable; high risk to individuals and to the community  BIOSAFETY LEVELS (BLs) 4 BLs for handling organisms corresponding to the 4 RGs Relies on: Standard practices of GMT Physical barriers (procedures, equipment, and lab installations based on the estimated biohazard) BLs for specific research work depend on the assessed RG of the organisms handled professional judgement of risk associated with the activity Relation of RGs to BLs, Practices, and Equipment  PHYSICAL CONTAINMENT Strict adherence to good microbial practices all personnel directly or indirectly working with recombinant or synthetic nucleic acids should be trained in GMT 4 Levels of Physical Containment BL1 to BL4 representing facilities in which experiments ranging from low to high potential hazard Standard microbiological practices; Special practices; Containment equipment, and Laboratory facilities BIOLOGICAL CONTAINMENT The growth and dissemination of organisms are naturally limited due to: The infectivity or host specificity of a vector or virus Its spread and survival in the environment Example of biological containment strategies: Survival of the vector in its host outside the lab Transmission of the vector from the propagation host to other nonlaboratory hosts Purpose of Physical and Biological Containment for Reseach to prevent unintentional transmission or release of recombinant or synthetic nucleic acid molecule GOOD LAB PRACTICES (GLP) embodies a set of principles that provide a framework within which laboratory studies are planned, performed, monitored, recorded, reported, and archived (Dolan, 2007) What is the primary purpose of GLP? ensure uniformity, consistency, and reliability of safety tests (nonclinical) for pharmaceuticals, agrochemicals, aroma and color food/feed additives, cosmetics, detergents, novel foods, nutritional supplements for livestock, and other chemicals. GLP is mandatory to evaluate safety or toxicity of products intended to undergo clinical trials References Compliance of GLP requires: Tests conducted by qualified personnel Each study have Study Director The lab study and data are audited by a Quality Assurance Unit All lab activities performed were according to SOPs All test articles and reagents must be ID, characterized, and labeled Equipment must be maintained and calibrated STANDARD OPERATING PROCEDURE for GENERAL SAFETY in the LAB Carefully read the activity and systematically plan the workflow before conducting Wear a lab gown while in the lab Clean your lab area & material/equipment with disinfectant before and after use If you do not understand something, ask Any spillage must be immediately addressed STANDARD OPERATING PROCEDURE for BIOSAFETY CABINET Class II Decontaminate the BSC surface and the materials to be placed inside (70% EtOH) Turn on the blower 10 mins before beginning work, turn off UV lamps when the room is occupied Heat sources are strictly prohibited inside the BSCs (will disrupt the laminar flow of air) Keep the work area free of unnecessary equipment or supplies Biosafety Cabinet A partially enclosed workspace that has built in protection for the worker, environment, and the material inside. Outside air is filtered (HEPA High-efficiency particulate absorber) STANDARD OPERATING PROCEDURE for LAMINAR AIR FLOW Close the sash and keep the UV on for 20 mins Turn on the blower 10 mins before beginning work Surface sterilize (70% IPA v/v) all materials to be placed inside Keep the work area free of unnecessary equipment or supplies  Laminar Floodhood aka Tissue Culture Hood enclosed bench designed to prevent contamination of biological samples STANDARD OPERATING PROCEDURE for WASTE DISPOSAL Decontaminate and waste should not be stored for more than 1 week Used sharps should be placed in a safety box (close when 3/4 full) STANDARD OPERATING PROCEDURE for COMPOUND MICROSCOPE Clean the lenses with cotton soaked in xylene (before and after use) Observe with both eyes when using microscope Handle all the parts with care Turn off and cover the microscope when not in use STANDARD OPERATING PROCEDURE for AUTOCLAVE MACHINE Sterilization time and temp are dependent on the composition and nature of items (15 mins at 121C at 15 lbs pressure min) Small items should be bagged or wrapped with label Sterile packs stored on open shelves are safe to use up to 1 month (dry and intact) Autoclave Machine Sterilization refers to the complete killing of all living organisms (wet/dry heat, chemicals, and radiation) STANDARD OPERATING PROCEDURE for PCR MACHINE Protect the outer sleeves from damage or sharp objects Only the power cord of the machine supplied with the workstation should be used to connect PCR Machine aka Thermocycler used to amplify segments of DNA Has a thermal block with holes where tubes can be inserted PERSONAL PROTECTIVE EQUIPMENT To protect against potential chemical exposures It is a supplement and not a substitute for feasible engineering and administrative controls Will vary with the nature of contaminant, route(s) of exposure, and contaminant concentration PERSONAL PROTECTIVE EQUIPMENT RESPIRATORS may be required when engineering controls are not sufficient to reduce the conc of air contaminants below an acceptable level TYPES ranging from filtering face pieces (dust masks) to full face covering, depends on the conc and form of contaminants (dusts, mists, fumes) PERSONAL PROTECTIVE EQUIPMENT EYE and FACE PROTECTION protective eyewear/face shield combination may be needed to prevent projectiles, airborne particles, thermal burns, chemical splashes, vapors, mists, dusts, or radiant energy from damaging the eyes and face PERSONAL PROTECTIVE EQUIPMENT HAND PROTECTION Gloves provide protection for the hands from many types of hazards (chemical absorption) Butyl rubber gloves - chemicals except for alipathic and aromatic hydrocarbons and halogenated solvents Natural (latex) rubber gloves - thin, elastic low barrier protection against acids, alkaline, salts, ketones PERSONAL PROTECTIVE EQUIPMENT Neoprene gloves - similar in texture and appearance to latex gloves but more resistant. Low barrier protection against hydraulic fluids, gasoline, alcohols, organic acids, and alkalis Nitrile gloves - similar in texture and appearance to latex or neoprene gloves. Made of copolymer and provide protection from chlorinated solvents such as trichloroethylene and perchloroethylene PERSONAL PROTECTIVE EQUIPMENT BODY PROTECTION - protective body apparel against potential contact through spills or splashes. In high-risk splash hazards, the body apparel should fully cover the torso, arms, and legs. Low hazard situations - lab coats made of cotton, or flame-retardant material  FIRE EMERGENCY FIRE EXTINGUISHER TYPES: CLASS A- trash, wood, paper involve ordinary combustible materials (quenched by water or multipurpose (ABC) dry chemicals agent) CLASS B - liquid flammable liquid (quenched by dry chemical and CO2) CLASS C- electrical equipment (dry chemicals or CO2)  FIRE EMERGENCY FIRE EXTINGUISHER TYPES: CLASS D- combustible metals like magnesium, titanium, zirconium, and lithium (quenched by sodium carbonate, graphite, bicarbonate, NaCl, and salt based chemicals ) CLASS K fire - Cooking oil