BIOSAFETY MANAGEMENT.ppt
- 1. BIOSAFETY Dr. Esther Shoba R Assistant Professor Kristu Jayanti College
- 2. Introduction Bio-related research activities may involve manipulation of microbial, animal or plant cells. The risks associated with these activities arise from the samples and /or the procedural requirements. Adherence to standard microbiological techniques and using facilities suitable to the risk level of the pathogen helps to protect the researcher from laboratory-acquired infections.
- 3. Bio hazards Hazards related to bio research can be classified into two categories. • hazards related with the pathogen or human/animal cells being used in research. • related with the procedures and practices followed in the lab.
- 4. Pathogenic risks Cell cultures • Researchers who handle or manipulate human or animal cells are at risk of possible exposure to potentially infectious pathogens that may be present in those cells/ tissues. • The human cell lines may contain blood borne pathogens, which can be transmitted due to improper handling.
- 5. Routes of entry for pathogen The probable routes of entry are •Inhalation of infectious aerosols. •Contact of the agent with the skin, eyes or mucous membrane. •Inoculation by contaminated sharps. •Bites from infected animals or contact with their body fluids. •Ingestion of infectious agent through mouth pipetting or contaminated hands.
- 6. Aerosols Aerosols generated during research activities remain undetected and can spread easily and remain suspended in the laboratory atmosphere for a long time. They possess a serious hazard to the person performing the task and also to others who are exposed to the air from the laboratory.
- 7. Aerosols Aerosols can be generated during the following activities •Pipetting •Blending •Centrifugation •Use of sonicators and vortex mixers These respirable size particles when inhaled are retained in the lungs and can cause infection to the person.
- 8. Pathogenic risks The risk from the pathogen handled depends on the following factors. •Capability to cause infection in the host and the severity of the same. •Preventive measures and treatment available. •Route of entry •Infective dose level •Stability in the environment •The range of cells/strains that can act as a host. Based on the above factors the microorganisms are classified into four risk groups.
- 9. Classification of pathogenic microorganisms Risk group I A pathogen that is unlikely to cause any disease in humans or animals. All bacterial, fungal and parasitic agents not included in higher groups.
- 10. Classification of pathogenic microorganisms Risk group II A pathogen that can cause disease in humans or animals but is unlikely to be a serious hazard. Effective treatment and preventive measures are available and the risk of spread of infection is limited. • Bacterial- Vibrio cholerae • Fungal- Aspergillus fumigatus, Actinomycetes • Parasitic- P.falciparum, Plasmodium thcilera • Viral and Rickettssial -Vole rickettsia, Mumps virus
- 11. Classification of pathogenic microorganisms Risk group III A pathogen that can cause serious human or animal disease , but does not ordinarily spread from one infected person to another. Effective treatment and preventive measures are available. •Bacterial - Clostridium botulium, Francisella tularensis •Fungal - Coccidioides immitis,Histoplasma capsulatum •Parasitic- Schisistosoma mansomi •Viral and Rickettssial - Foot-and- Mouth disease virus
- 12. Classification of pathogenic microorganisms Risk group IV A pathogen that usually causes serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly. Effective treatment and preventive measures are not usually available. •Korean hemorrhagic fever •Omsk hemorrhagic fever and •Central European Encephalitis viruses
- 13. Containment The term containment is used to describe the safe work practices in handling infectious agents to reduce exposure to laboratory personnel and others. Types of containment •Biological containment •Physical containment
- 14. Biological containment (BC) Any combination of vector and host which is to provide biological containment must be chosen or constructed to limit the infectivity of vector to specific hosts and control the host-vector survival in the environment.
- 15. Physical Containment (PC) Physical containment helps to confine the pathogenic organisms being handled and prevent exposure to personnel. Physical containment is achieved by Primary containment • Laboratory practices • Containment equipment • Special laboratory design Secondary containment Primary containment offers protection to personnel and immediate laboratory environment whereas secondary containment offers protection to the environment outside the laboratory.
- 16. Primary containment Laboratory practices Consists of adhering to standard microbiological practices and techniques and awareness of potential hazards.
- 17. Primary containment Containment equipment This includes biological safety cabinets and enclosed containers (e.g. safety centrifuge cup).
- 18. Secondary containment Proper design of the facility helps in protecting personnel inside the facility and also prevents the release of pathogenic organisms outside the facility. Facility designs are of three types Basic Laboratory (for Risk Group I and II) Containment Laboratory (for Risk Group III) Maximum Containment Laboratory (for Risk Group IV)
- 19. CONTAINMENT LEVELS Biosafety containment levels have to be designated for a facility depending on the level of risk associated with the biological and chemical agents used and released from it. Following NIH (National Institute of Health, USA) and DBT (Department of Biotechnology, India) guidelines, different facilities for biological research have been classified under three containment levels)
- 20. CONTAINMENT LEVELS - I Viable organisms should be handled in a production system which physically separates the process from the environment; Exhaust gases should be treated to minimize (i.e. to reduce to the lowest practicable level consistent with safety) the release of viable organisms; Sample collection, addition of materials to the system and the transfer of viable organisms to another system should be done in a manner which minimizes release; Bulk quantities of culture fluids should not be removed from the system unless the viable organisms have been inactivated by validated means; Effluent from the production facility should be inactivated by validated means prior to discharge.
- 21. CONTAINMENT LEVELS - II •Viable organisms should be handled in a production system which physically separates the process from the environment; •Exhaust gases should be treated to prevent the release of viable organisms; •Sample collection, addition of materials to a closed system and the transfer of viable organisms to another closed system should be done in a manner which prevents release;
- 22. CONTAINMENT LEVELS – II - CONTD •Culture fluids should not be removed from the closed system unless the viable organisms have been inactivated by validated chemical or physical means; •Seals should be designed to prevent leakage or should be fully enclosed in ventilated housings; •Closed systems should be located in an area controlled according to the requirements; •Effluent from the production facility should be inactivated by validated chemical or physical means prior to discharge.
- 23. CONTAINMENT LEVELS – III •Viable organisms should be handled in a production system which physically separates the process from the environment; •Exhaust gases should be treated to prevent the release of viable organisms; •Sample collection, addition of materials to a closed system and the transfer of viable organisms to another closed system should be done in a manner which prevents release; •Culture fluids should not be removed from the closed system unless the viable organisms have been inactivated by validated chemical or physical means; •Seals should be designed to prevent leakage or should be fully enclosed in ventilated housings;
- 24. CONTAINMENT LEVELS – III - CONTD •Production systems should be located within a purpose built controlled area according to the requirements; •Entry should be restricted in the laboratory area and only persons with appropriate authority should be allowed access to the working area. Effluent from the production facility should be inactivated by validated chemical or physical means prior to discharge. Different containment levels have been assigned for rDNA GILSP (Good industrial large scale practice) micro-organisms. Examples of containment approaches for recombinant organisms are discussed
- 25. CONTAINMENT LEVELS - I
- 26. It consists of a combination of laboratory practices, equipment and facilities suitable to the procedures being performed and hazards of the pathogen. The four biosafety levels corresponds to four risk groups. A lower risk group can be assigned a higher biosafety level, if a biological risk assessment carried out requires so. Biosafety levels
- 27. Biosafety level I Suitable for teaching laboratories and for facilities in which work is done with defined and characterised strains of agents not known to cause any disease. Good microbiological techniques(GMT) to be followed.
- 28. Biosafety Level II Applicable to facilities in which work is done with indigenous moderate-risk agents present in the community and associated with human disease of varying severity. BSL II is appropriate when work is done with any human- derived blood, body fluids, tissues, or primary human cell lines, in which presence of an infectious agent may be unknown BSL II requires •Following GMT •Use of personal protective equipment •Use of BSC •Use of autoclaves
- 29. Biosafety level III Applicable to facilities in which work is done with indigenous or exotic agents where the potential for infection by aerosols is real and the disease may have serious or lethal consequences. BSL III requires in addition to that of BSL II requirements •Special clothing •Directional airflow •Controlled access •Double door entry/Anteroom •Supervision
- 30. Biosafety level IV Applicable to work with dangerous and exotic agents which pose a high individual risk of life-threatening disease. BSL IV requires in addition to BSL III requirements •Positive pressure personnel suits •Strictly limited access •Double ended autoclave •Class III BSC •Airlock with shower •Supervision
- 31. BIOSAFETY GUIDE LINES - RDNA Biosafety guidelines are designed and applied to research involving recombinant DNA (rDNA) techniques. Handling, production, storing and transportation of genetically modified organism (GMOs) involve different biosafety issues under different category. Biosafety practices deal with the application of standard safety principles handling hazardous material/agents to minimize potential harmful effect on human health and environment.
- 32. BIOSAFETY MANAGEMENT - RDNA Biosafety regulatory principles and protocols regulates the potential risk and allow access to the benefits of rDNA technology. RISK ASSESSMENT RISK MANAGEMENT Are the components of biosafety
- 33. BIOSAFETY MANAGEMENT - RDNA The foundation of any safety program is the use of control measures appropriate for the risk posed by the activities and the agents in use. The process of analyzing and determining the risk associated with recombinant DNA work is called as Risk analysis. The principle behind biosafety regulations is to minimize the risk to human health and safety, and the conservation of environment including safe handling of hazardous material. Risk analysis consists of three components: risk assessment, risk management and risk communication.
- 34. BIOSAFETY MANAGEMENT - RDNA Risk Assessment: Estimation and determination of risk associated with the handling and production of a recombinant DNA molecule. Risk Management: The process of analyzing possible prevention measures to minimize the risk and designing policies accordingly including implementation of them. Risk Communication: The exchange of information and opinions on risk management between academic parties, industry, consumers and policy makers.
- 35. RISK ASSESSMENT - RDNA The biosafety level is determined based on the risk associated with the work. The principle investigator is responsible for implementing the necessary safety requirements in his/her laboratory. Risk assessment process accounts the following criteria to determine biosafety level: i.Pathogenicity – The ability of an organism to cause disease in human system. ii.Virulence – The severity of the disease (lethal/non lethal, availability of cure etc) in a healthy adult. iii.Proliferation – the subsequent multiplication, genetic reconstruction, growth, transport, modification and die-off of these micro-organisms in the environment, including possible transfer of genetic material to other micro- organisms.
- 36. RISK ASSESSMENT - RDNA iii.Transmission route – The possible route of transmission (mucous membrane, inhalation etc) to establish the disease in human or other organism. iv.Infectious dose (ID) – The amount of infectious agent required to cause disease in healthy human. vi.Antibiotic/disinfectant resistance – The resistance acquired by the infectious agent to available antibiotic/disinfectant.
- 37. RISK MANAGEMENT - RDNA Risk management in biosafety issues is related to the target site where the practice is conducting (laboratory, industry, agriculture field etc.). Recommendations: General i.Harmonization of approaches to rDNA techniques can be facilitated by exchanging principles or guidelines for national regulations; developments in risk analysis; and practical experience in risk management. Therefore, information should be shared as freely as possible. ii.There is no scientific basis for specific legislation for the implementation of rDNA techniques and applications.
- 38. RISK MANAGEMENT - RDNA Member countries should examine their existing oversight and review mechanisms to ensure that adequate review and control may be applied while avoiding any undue burdens that may hamper technological developments in this field. iii. Any approach to implement guidelines should not impede future developments in rDNA techniques. International harmonization should recognize this need.
- 39. RISK MANAGEMENT - RDNA iv. To facilitate data exchange and minimize trade barriers between countries, further developments such as testing methods, equipment design and knowledge of microbial taxonomy should be considered at both national and international levels. Due account should be taken of ongoing work on standards within international organizations. v. Special efforts should be made to improve public understanding of the various aspects of rDNA techniques.
- 40. RISK MANAGEMENT - RDNA v.For rDNA applications in industry, agriculture and the environment, it will be important for member countries to watch the development of these techniques. vi.For certain industrial applications and for environmental and agricultural applications of rDNA organisms, some countries may wish to have a notification scheme. vii.Recognizing the need for innovation, it is important to consider appropriate means to protect intellectual property and confidentiality interests while assuring safety.
- 41. BIOSAFETY LEVELS - RDNA All the facilities handling microorganisms and materials containing recombinant DNA molecules have risk assessment program. Depending on the risk possessed by the samples, four biosafety levels have been assigned to rDNA research facilities. Each BSL facility has requirement of unique design features and safety equipments
- 42. BIOSAFETY LEVEL I (BSL –I) - RDNA •Agents: Characterized strains of microorganisms known to cause no disease in healthy adults. eg. E. coli, S. cerevesiae, B. subtilis etc. •Recombinant DNA based research activities involving non- pathogenic micro-organisms for expression of genes using plasmid vectors or low risk viral vectors. •Work practice: Standard aseptic microbiological techniques. •Safety equipment requirement: Lab coats and eye protection recommended. •Facilities: Bench top, sink etc.
- 43. BIOSAFETY LEVEL II (BSL - II) - RDNA •Agents: Handling of micro-organisms which possess moderate hazard to personal and environment. •rDNA based research activities in micro-organisms using non-viral or viral vectors. •Work practice: Standard BSL-I practices with addition of limited access, biohazard sign, defined procedure for disposal of “Regulated Medical Waste”, proper training to lab personal and medical surveillance. •Safety equipment: Class-II biological safety cabinet, lab coats, gloves, eye/face protection, physical containment equipment to reduce infectious aerosol exposure or splashes. •Facility: BSL-I facility with addition of autoclave, decontamination facility and proper airflow.
- 44. BIOSAFETY LEVEL III (BSL -III) - RDNA •Agents: Handling of micro-organisms which are designated as hazardous or potentially lethal agents to personal and environment. •Laboratory personnel must have specific training in handling infectious micro-organisms and should be supervised by scientist competent in handling infectious agents. •Work practices: BSL-2 practices, with the addition of: controlled access, on-site decontamination of all waste and lab clothing and medical surveillance.
- 45. BIOSAFETY LEVEL III (BSL -III) - RDNA •Safety equipment: Class-III biological safety cabinet, lab coats, gloves, eye/face protection, respiratory protection, physical containment equipment to reduce infectious aerosol exposure or splashes. •Facility: BSL-III facility has specific criteria to meet. Lab should have double door entry with physical separation of working area from the access corridors, directional airflow in lab, and no recirculation of exhaust air in the lab, sufficient decontamination facility, in lab autoclave etc.
- 46. BIOSAFETY LEVEL IV (BSL -IV) - RDNA •Agents: Hazardous and potentially lethal organisms that posses high individual risk of laboratory transmitted disease for which there is no vaccine or treatment, or a related agent with unknown risk of transmission. •Laboratory personnel must have specialized training in handling BSL-IV agents and should be supervised by scientist competent in handling infectious agents.
- 47. BIOSAFETY LEVEL IV (BSL -IV) - RDNA •Safety equipment: Class-IV biological safety cabinet, lab coats, gloves, eye/face protection, respiratory protection, physical and containment equipment to reduce infectious aerosol exposure or splashes. •Facility: BSL-IV facility requires specialized design to minimize the exposure to risk and only the authorized entry should be permitted in laboratory area in BSL-IV labs.
- 48. Good microbiological techniques (GMT) • Specimen containers must be correctly labelled for easy identification. • Use secondary containers (autoclavable) while transporting specimens to contain spill. • Specimen containers received from external agencies must be opened in the biosafety cabinet. • Use mechanical pipettes.
- 49. Good microbiological techniques • Open flame must not be used in BSC as it can distort the air flow pattern and damage the filters. •Always use disposable gloves. Do not touch mouth, eyes and face with contaminated hands.
- 50. Good microbiological techniques • Food and drink must not be stored or consumed in the laboratory. • Glassware must be replaced with plasticware wherever possible.
- 51. Good microbiological techniques • Sharps(e.g., needle sticks, glass) must be avoided wherever possible as it can transmit blood borne pathogens in case of injury.
- 52. Good microbiological techniques • Use engineered sharp-safety devices when syringes and needles are necessary. • Needles must not be recapped, to prevent needle stick injury. • Puncture-proof containers fitted with covers must be used for disposing sharps.
- 53. Good microbiological techniques • Tubes and specimen containers must always be securely capped (screw-capped if possible) for centrifugation. • Refer to manufacturer’s instructions before operating equipments. • Work area must be decontaminated with a suitable disinfectant at the end of the work. • Hands must be thoroughly washed before leaving the lab.
- 54. Personal protective equipment • Personal protective equipment act as a barrier to minimize the risk of exposure to aerosols, splashes and other injuries. •Personal protective equipment must be selected on the basis of the risks involved in the task performed. • Lab coat, safety glasses and toe covered footwear is a minimum requirement while working in the lab. • Face shield must be used if there is any risk of splashing of infectious materials.
- 55. Personal protective equipment • Gloves must be worn for all procedures that may involve direct contact with blood, infectious materials, or infected animals. • Gloves must be removed aseptically and autoclaved with other laboratory wastes before disposal. • If re-usable gloves are used, on removal they must be cleaned and disinfected before re-use. • Lab coats and other personal protective equipment used must not be used outside the laboratory.
- 56. Biosafety cabinets(BSC) Biological safety cabinets provide containment of infectious aerosols generated during the laboratory procedures. Three types of BSCs are used in microbiological laboratories. These are Class I Class II Class III
- 57. Biosafety Cabinets Class I BSC Offers protection to laboratory personnel and to the laboratory environment . It doesn’t protect the samples from external contamination. Class II BSC Provides protection to the samples in the cabinet from external contamination in addition to personnel and laboratory environment protection. Class III BSC Provides the maximum attainable level of protection to personnel and the environment.
- 58. The following factors reduce the efficiency of the BSC • Poor location • Room air currents • Decreased airflow • Leakage in HEPA filters • Working with raised sashes • Overcrowding the work surface • Improper user methodology
- 59. Emergency measures In case of exposure to bio samples • Remove the contaminated clothing. • Wash the skin thoroughly with soap and water. • In case of eye contact flush the eyes with water. • Report the exposure to the Lab in charge. • Get medical attention immediately.
- 60. Decontamination • Decontamination renders an item (work bench, equipment, etc.) safe to handle by reducing the number of organisms to below the threshold infectious dose level such that transmission is unlikely to occur. • Decontamination requirements will depend on the experimental work and the nature of the infectious agent handled. • Decontamination is usually accomplished by steam sterilization or autoclaving. • Sterilization and disinfection are different forms of decontamination.
- 61. Decontamination Sterilisation •Sterilisation makes an item free from all living microorganisms and viruses. • The process of sterilization can be accomplished by applying heat.
- 62. Decontamination Disinfection •Is not as effective as sterilization, as some organisms such as bacterial endospores may survive. •A disinfectant is a chemical or mixture of chemicals used to kill microorganisms, but not spores. They are usually applied to inanimate surfaces or objects.
- 63. Decontamination Disinfectants •Sodium hypochlorite and formaldehyde are the disinfectants recommended for general laboratory use. •For special purposes phenolic compounds, alcohols, iodine etc., can be used effectively.
- 64. Biohazard waste disposal Biohazard waste generated in laboratories must be segregated into the following: •Non-contaminated general waste •“Sharps”-needles, glass pieces, etc •Contaminated material for autoclaving and recycling •Contaminated material for incineration
- 65. • Biohazard waste for autoclaving must be collected in red plastic bags and those for incineration in yellow non chlorinated plastic bags. • Biohazard waste of human and animal origin must be incinerated. Biohazard waste disposal