Biosafety Levels Biological Safety Cabinets and Biosafety Laboratory Construction Dr Ravi Kant Agrawal, MVSc, PhD Senior Scientist (Veterinary Microbiology) Food Microbiology Laboratory Division of Livestock Products Technology ICAR-Indian Veterinary Research Institute Izatnagar 243122 (UP) India Biosafety Level 1 (BSL-1 and ABSL-1) Suitable for work involving well-characterized agents not known to consistently cause disease in immuno-competent adult humans. Prophylactic treatment available. Minimal potential hazard to laboratory personnel and the environment. Laboratories are not necessarily separated from the general traffic patterns in the building. Work is typically conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is not required. Animals in open cage system or open environment (outdoors) Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist with training in microbiology or a related science. BSL-1 Practices & Procedures Only standard practices required at this level: Good laboratory practices Frequent hand washing, after removing gloves & before leaving lab Door that can be kept closed when working Limits on access to the lab space when working No smoking, eating, drinking, storage of food in laboratory Open bench-top work allowed Biosafety cabinet not required (unless creating aerosols) Care to minimize splashes and actions that may create aerosols (tiny droplets) Daily De-contamination (Decontamination of work surfaces after every use after any spills) Red bag waste Decontamination of laboratory wastes Use of Mechanical pipetting only (no mouth pipetting) “Sharps" precautions, including special containers for disposing of needles and other sharp objects Maintenance of insect/ rodent control program Use of personal protective equipment (lab coats, latex gloves, eye protection or face shields) Open bench top sink for hand washing Biosafety Level 1 Biosafety Level 1 Risk Group 1 Agents E. coli K-12, Bacillus subtilis, Adenoasociated viruses 1-4, T4 bacteriophages, Saccharomyces cerevisiae, Rhizopus stolonifer, Candida albicans, Pseudomonas, Infectious canine hepatitis Transgenic Plants Plasmids Fungi Mold Yeast BSL-1 Containment Overview RG-1 Agents Not known to cause disease in healthy/immunocompetent adult humans Practices Standard microbiological practices Primary Barrier (Safety equipment) Minimal requirements Secondary Barrier (Facilities) Open bench top work Biosafety Level 2 Builds upon BSL-1 BSL-2 is suitable for work involving agents that pose moderate hazards to personnel and the environment. Agents associated with human disease Treatment for disease available (Antibiotics/Vaccines) Laboratory personnel have specific training in handling pathogenic agents Personnel are supervised by scientists competent in handling infectious agents and associated procedures Access to the laboratory is restricted when work is being conducted All procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets (BSCs) or other physical containment equipment. Biosafety manual with definitions of needed waste decontamination or medical surveillance policies Biosafety Level 2 (BSL-2) In working with BSl-2 agents, th eprimary hazards to personnel are – Ingestion of infectious materials Direct contact or exposure Accidental Needle sticks or per-cutaneous exposure by Scratch, Puncture Potential infection through exposure to Eyes, Mouth, nose, open cut (Mucus membrane) BSL-2 agents do not cause lethal infections, are not transmissible via airborne route (do not cause infection if tiny droplets become airborne and are inhaled, which might occur if the material were spattered) Extreme care should be taken with contaminated needles and sharp lab instruments when they are contaminated with agents. BSL-2 Practices &Procedures Include BSL-1 plus Limited access to lab when work in progress Daily de-contamination Mechanical pipetting Lab coat, safety glasses and gloves required Red bag & sharps containers required Biohazard warning sign posted at entrance to lab with contact information: MANDATORY Label all equipment (incubators, freezers, etc.) TC room – negative air flow Documented training Special Entry Procedures: Baseline serology or prevaccination/Immunizations may be required Risk Group 2 Agents S. aureus, Bordetella pertussis, Corynebacterium diphthriae, Other E coli, Nisseria gonorrhoea, Streptococcus pyogenes, Vibrio cholerae, Klesiella spp., Proteus, Serratia marcescens, Salmonella, L. monocytogenes Rabies, Hepatitis A, B, C Cryptococcus neoformans Most parasitic agents Human or Primate Cells Herpes Simplex Virus Replication Incompetent Attenuated Human Immunodeficiency Virus Patient specimens BSL-2 Containment Overview RG-2 Agents Practices BSL-1 plus limited access. Primary Barrier (Safety equipment and PPE) Associated with mild to moderate disease in humans biosafety cabinets or other approved containment devices Personal protective equipment: lab coats, gloves, face protection as needed Protective clothing removed when personnel leave laboratory area Secondary Barrier (Facilities) BSL-1 plus the availability decontamination (autoclave). of a mechanism for Biosafety Level 2 Biosafety Level 2 Biosafety Level 3: Working in High Containment Is applicable to clinical, diagnostic, teaching, research, or production facilities where work is performed with indigenous or exotic agents that may cause serious or potentially lethal disease through inhalation route exposure. Primary hazards: needle sticks, ingestion, exposure to infectious aerosols Treatment may or may not exist Laboratory personnel must receive specific training in handling pathogenic and potentially lethal agents Must be supervised by scientists competent in handling infectious agents and associated procedures. Biosafety Level 2 plus all procedures involving the manipulation of infectious materials must be conducted within BSCs, or other physical containment devices Personnel wear additional appropriate personal protective equipment including respiratory protection as determined by risk assessment A BSL-3 laboratory has special engineering and design features. Directional air flow BSL-3 Practices & Procedures Standard practices include BSL-2 plus: Strictly controlled access to the lab; Specific training for lab personnel in handling potentially lethal agents; Decontaminating all waste; Changing contaminated protective lab clothing, Decontaminating lab clothing before laundering; Daily decontamination upon completion of experiment and after spill Autoclave required and waste is disposed at the end of day Required foot activated hand washing sink and controls No sharps unless absolutely necessary Aerosol minimization procedures required Wrap around disposable clothing is required. Specialized equipment may be required depending upon procedures BSL-3 Practices & Procedures (contd…) Biohazard Signs and labels posted Air flow from low hazard to high hazard-“Pressure Mapping” Bench top work not permitted Documented training and personnel competency certification (for BSL-3 procedures) Baseline serology Spills – report immediately and treat accordingly Vaccinations/ post exposure protocols SOP’s Biosafety Manual Biosafety Officer Risk Group 3 Agents SARS Rift valley fever Human Immunodeficiency Virus Yellow fever virus VEE virus Hanta virus Prions M. tuberculosis, M. bovis Coxiella burnetii Franciella tulerensis B. abortus Bacillus anthracis, Pasteurella multocida Yersinia pestis Coccidiodes immitis Plasmodium Trypanosoma No parasitic agents BSL-3 Containment Overview RG-3 Agents Associated with serious or potentially lethal disease in humans Practices BSL-2 plus controlled access. Primary Barrier (Safety equipment) Biological Safety Cabinet and personal protective equipment required similar to BSL-2. Respiratory equipment if risk of infection through inhalation Secondary barrier (Facilities): All BSL-2 barriers with Access through self-closing double doors Corridors separated from direct access to lab Single-pass negative directional airflow- Air handling systems to ensure negative air flow (air flows into the lab) Air pumped into lab not re-circulated in building BSL-3 Biosafety Level 3 Biosafety Level 3 Biosafety Level 4 Required for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, aerosol transmission or related agent with unknown risk of transmission. Agents with a close or identical antigenic relationship to agents requiring BSL-4 containment must be handled at this level until sufficient data are obtained either to confirm continued work at this level, or re-designate the level. Dangerous/exotic agents Life threatening disease No known treatment available Aerosol transmission/mucous membrane exposure/accidental prick Agents of unknown risk of transmission or health affects Laboratory staff must have specific and thorough training in handling extremely hazardous infectious agents. Laboratory staff must understand the primary and secondary containment functions of standard and special practices, containment equipment, and laboratory design characteristics. All laboratory staff and supervisors must be competent in handling agents and procedures requiring BSL-4 containment. Access to the laboratory is controlled by the laboratory supervisor in accordance with institutional policies Biosafety Level-4: Working in High Containment Maximum containment facilities Builds on BSL-3/ ABSL-3 practices Standard practices include BSL-3 plus: strictly controlled access to the laboratory; changing clothing before entering and exiting lab (showering upon exiting recommended-Chemical decontamination showers) decontaminating all material exiting facility (Liquid effluent collection / decontamination) Personnel must receive specialized training in handling extremely dangerous infectious agents, containment equipment and functions Immunocompromised persons are never allowed to enter the lab Two types of laboratory providing absolute separation of the worker from the infectious agents Pressurized Containment Suite Suit Laboratory Cabinet Laboratory BSL-3 + Class III Biosafety Cabinet BSL-4: High Bhopal, IVRI Safety Animal Disease Laboratory, Risk Group 4 agents Ebola Hemmorrhagic Fever Virus Marburg Virus Lassa Fever Virus Machupo virus Crimean congo Haemorrhagic viruses, Bolivian and Argentine Haemorrhagic fever viruses Some encephalitis viruses Herpesvirus simiae No bacterial agents No fungal agents No parasitic agents BSL-4 Containment Overview RG-4 Agents Practices BSL-3 plus controlled access Primary Barrier (Safety equipment) Associated with high risk of life-threatening disease in humans and/or animals Biological Safety Cabinet Full-body air-supplied, positive pressure personnel suit Secondary Barrier (Facilities)- BSL-3 plus dedicated air and exhaust, decontamination procedures for exit, separate building a recommended absence of windows (or sealed and resistant to breakage) Biosafety Level 4 CDC/NIH Guidelines Biosafety Levels (www.cdc.gov) BSL-1 BSL-3 Lower Risk BSL-2 Higher Risk Animal Biosafety Level-4: Working in High Containment Biosafety Level Summary Risk Groups and Biosafety Levels Risk Group 1 Biosafety Level Laboratory Type Laboratory Practices Safety Equipment Basic – Biosafety Level 1 Basic teaching, research GMT None; open bench work Basic – Biosafety Level 2 Primary health services; diagnostic services, research GMT plus protective clothing, biohazards sign Open bench plus BSC for aerosols 2 World Health Organization National Institutes of Health Risk Groups and Biosafety Levels Risk Group 3 4 Biosafety Level Laboratory Type Laboratory Practices Safety Equipment ContainmentBiosafety Level 3 Special diagnostic services, research Level 2 + special clothing, access control, directed airflow BSC and/or other primary devices for all activities Maximum Containment – Biosafety Level 4 Dangerous pathogen units Level 3 + airlock entry, shower exit, special waste disposal Class III BSC, or positive pressure suites with class II BSCs, double ended autoclave World Health Organization National Institutes of Health Biocontainment The principle of holding or being capable of holding or including within a fixed limit or area Preventing the unintentional release of biological agents through a combination of laboratory practices, containment equipment (primary barrier) and laboratory facility design (secondary barrier) Primary Barrier Primary barriers contain the agent at the source Equipment/Engineering Control Biological safety cabinet, fume hood, glove box, animal housing, centrifuge, fermenter Secondary Barrier Secondary barrier is the structure surrounding the primary barrier Facility/Engineering Control Rooms, building Types of Facilities Basic laboratory Containment laboratory Primary Barriers - Equipment Personnel Protection Product Protection Any aerosol generated within the cabinet is contained and kept away from the researcher Air within the work space of the cabinet has been filtered so that it is virtually free of airborne particles and organisms; thus protecting the work from outside contamination Environmental Protection Aerosols generated within the unit are removed from the air before the air is discharged Ventilation Equipment Classes and Types Chemical Fume Hood Offer only personnel protection Do not offer protection to the product or the environment, as there is no filtration of intake and exhaust air (Sometimes air cleaning treatment is added to the exhaust). Always exhaust air to the outside Do draw contaminants in the laboratory air directly over the product being worked on Used for work with chemical hazards 100 fpm face velocity Any Comments? Fumehood - keep hood clean, sash should be closed when hood is not in use, equipment should be 9” from slash Clean Bench / Laminar Flow Hoods Provide product protection only Product protection is provided by creating a unidirectional airflow generated through a HEPA filter Discharge air goes directly into workroom Applications Any application where the product is not hazardous but must be kept contaminant free Preparation of non-hazardous intravenous mixtures and media Particulate free assembly of sterile equipment and electronic devices Eliminate Clean Bench in containment laboratory Biological Safety Cabinets • Biological Safety Cabinets (BSC): primary means of containment developed for working safely with infectious microorganisms Designed to contain biological hazards Supply air HEPA filter for product protection (except Class I) Inward airflow for personnel protection HEPA filtered exhaust air for environmental protection Separated into Classes and Types – Class I – Class II • Type A1, A2 • Type B1, B2 – Class III Microbiological studies, cell cultures, pharmaceutical research etc. Class I Cabinet Provides personnel and environmental protection No product protection In fact, the inward flow of air can contribute to contamination of samples An exhaust blower to pull the air through - usually to the outdoors BSCs of this class are either ducted (connected to the building exhaust system) or un-ducted (re-circulating filtered exhaust back into the laboratory Inward airflow is maintained at 75 fpm velocity Applications – Housing centrifuges, fermenters – Cage dumping in an animal lab – Aerating cultures that potentially generate aerosols Class I BSCs Provides personnel and environment protection only. No product protection. Suitable for low to moderate risk (biosafety level 1, 2, and 3) HEPA filter protects environment by filtering air before it is exhausted Class II Cabinets Provides personnel, product, and environmental protection Widely used in clinical, hospital, life science, research and pharmaceutical laboratories. Have 3 main features: Downward HEPA filtered unidirectional/laminar airflow for product protection Open front with careful maintained inward airflow for personnel protection HEPA filtered exhaust air to the room or to a facility exhaust system for environmental protection Class II BSCs Type A1 (Previously A): Face Velocity of >0.38 m/s or 75 lfpm The filtered makeup air is divided equally over the work surface at about two to six inches above the work surface. Exhaust is drawn at the bottom of the cabinet where it rises to the top. At the top of the cabinet, 70% of the air re-circulates through the supply HEPA filter, the other 30% of air exhausted through the exhaust HEPA filter into the room. This is due to the relative sizes of the two filters, and dampers typically allow the adjustment of this ratio. This type is not safe for work with hazardous chemicals except when ducted, usually with a "thimble" or canopy hood to avoid disturbing internal air flow. Class II A1/A2 BSC Class II BSCs Type A2 (A/B3): Face Velocity of >0.51m/s or >100lfpm HEPA filtered exhaust air may be re-circulated into the room or released outside 70% of air is re-circulated, 30% of air filtered through HEPA and exhausted into the room. A negative air pressure plenum surrounds all contaminated plenums that are under positive pressure. In other respects, the specifications are identical to those of a Type A1 cabinet Class II BSCs Type B1 Face Velocity of >0.51m/s or >100lfpm These cabinets must be hardducted to an exhaust system rather than exhausted through a thimble connection 30% of air is re-circulated to work area through HEPA supply filter and exhausts 70% of circulated air through HEPA exhaust filter Since exhaust air is drawn from the rear grille, Type B1 offers more protection to the personnel if vapor source is at rear of work area. Suitable for work with low levels of volatile toxic chemicals and trace amounts of radionuclides. Class II BSCs Type B2 Face Velocity of >0.51m/s or >100lfpm 0% air re-circulated, 100% exhausted from cabinet These cabinets must be hard-ducted to an exhaust system rather than exhausted through a thimble connection Widely used in toxicology labs and similar labs where clean air is essential. Additionally, there is the risk that contaminated air would flow into the laboratory if the exhaust system for a Type B1 or B2 cabinet were to fail. To mitigate this risk, cabinets of these types generally monitor the exhaust flow, shutting off the supply blower and sounding an alarm if the exhaust flow is insufficient. Any Comments? BSC - remove unnecessary objects, keep grill at front of cabinet unobstructed Class III BSC Used to work with microbiological agents assigned to biosafety level 4 Provides maximum protection to personnel and environment Generally only installed in maximum containment laboratories The enclosure is gas-tight, and all materials enter and leave through a chemical DUNK TANK OR PASS THROUGH BOX ClassIII BSC may be connected to a double-door autoclave. Heavy duty rubber Gloves attached to the front prevent direct contact with hazardous materials (Class III cabinets are sometimes called glove box). Supply air is HEPA filtered and exhaust air is double HEPA filtered. Air flow is maintained by a dedicated exhaust system exterior to the cabinet, which keeps cabinet interior under negative pressure (ABOUT 124.5PA). These custom-built cabinets often attach into a line, and the lab equipment installed inside is usually custom-built as well Applications for Cabinet: Working with high risk agents Working with highly infectious or hazardous experimental materials Working with emerging diseases Working with diseases that are near eradication Weighing and diluting chemical carcinogens Class III Comparison of BSCs Personnel Product Environment Chemical Fumehood x ---- ---(X) ---- x ---- Class I Biosafety Cabinet x ---- x Class II Biosafety Cabinet x x x Class III Biosafety Cabinet x x x Isolators x x x Laminar Flowhood Types of Biosafety Cabinets NSF/ANSI Standard 49 – 2002 Type Face velocity (lfpm) Airflow Pattern Radionuclides/ Toxic Chemicals Biosafety Product Level(s) Protection Class I 75 In at front; rear and top through HEPA filter No 2, 3 No 75 70% recirculated through HEPA; 30% Exhaust through HEPA No 2, 3 Yes 100 30% recirculated through HEPA; 70% Exhaust via HEPA and hard ducted No 2, 3 Yes Yes (Low levels/volatility) 2, 3 Yes Class II Type A1 Class II Type A2 Class II Type B1 100 30% recirculated through HEPA; 70% Exhaust via HEPA and hard ducted Class II Type B2 100 No recirculation; total exhaust Yes via HEPA and hard ducted 2, 3 Yes Class III NA Supply air inlets and exhaust through 2 HEPA filters 3, 4 Yes Yes Biological Safety Cabinet Certification First Certification Annually When moved When filter is changed When repaired or modified Note: Certification is paid by the researcher. Other Primary Barriers- Engineering Control Gasketed blenders, homogenizers Cotton plugs, filters for flasks in shakers Filtered pipette tips HEPA and hydrophobic vacuum line filters Plasticware substituted for glassware Gas burners with shield, microincinerator Centrifuges Interlock, solid cover, safety buckets, O-rings Secondary Barrier- Facilities Laboratory Biosafety Level 2 Lockable doors (a must for restricted agents) Sink Bench tops impervious and easily cleaned Biological safety cabinet (if applicable) Eyewash Inward airflow (desirable) BSC Operating Procedures Ready Work Area turn off UV lamp, turn on fluorescent check air grilles for obstructions, switch on blower allow air to purge work space- 5-10 minutes Pre-disinfect spray or swab all interior surfaces with appropriate disinfectant allow to air dry Assemble material introduce only material required to perform procedure place material such that clean and contaminated items do not meet place contaminated material container at right rear ensure view screen is properly located and secured Pre-purge cabinet allow air purge period with no activity inside (leave blower on!) Prepare self - don protective clothing, gloves, mask, etc. as appropriate BSC Operating Procedure Do the procedures DO NOT remove hands from work space until procedures are complete and all critical material is secured, remove gloves into contaminated material container Post-purge cabinet allow air purge period with no activity inside (leave blower on!) Finish personally remove protective clothing, mask, and wash hands Post-disinfect don gloves, remove materials to incubator, to biohazard bag, autoclave as appropriate, spray or swab all interior surfaces with appropriate disinfectant Shutdown cabinet turn off blower and fluorescent lamp, turn on UV lamp Safe Work Practices for BSC Use Do not use the top of the cabinet for storage. The HEPA filter could be damaged and the airflow disrupted. Make sure the cabinet is level. If the cabinet base is uneven, airflow can be affected. Never disengage the alarm. It indicates improper airflow and reduced performance which may endanger the researcher or the experiment. Never completely close the window sash with the motor running as this condition may cause motor burnout. Cabinets should be placed away from doors, windows, vents or high traffic areas to reduce air Safe Work Practices for BSC Use For BSC without fixed exhaust, the cabinet exhaust should have a twelve inch clearance from the ceiling for proper exhaust air flow. Also, allow a twelve inch clearance on both sides of the cabinet for maintenance purposes. Never operate a cabinet while a warning light or alarm is on. The operator should be seated with shoulders level with the bottom of the sash. Perform all work using a limited number of slow movements, as quick movements disrupt the air barrier. Try to minimize entering and exiting your arms from the cabinet, but if you need to, do it directly, straight out and slowly. Keep all materials at least four inches inside the sash opening. To avoid excessive movements in and out of the Safe Work Practices for BSC Use If a bunsen burner must be used, place it at the rear of the work area where the air turbulence from the flame will have the least possible effect on the air stream. Often the use of a flame is redundant in what should be a germ free work space. All equipment which has come in contact with the biological agent should be decontaminated. The cabinet should be allowed to run for at least three minutes with no activity so that the airborne contaminants will be purged from the work area before removing equipment. After all items have been removed, wipe the interior surfaces with disinfectant. Biological Safety Cabinet Certification A cabinet must be certified when first installed and then annually. It must be recertified anytime it is moved even within the same room. Before certification personnel arrive, remove all items from the cabinet and wipe it down with a disinfectant. This will expedite the certification. Any decontaminations, certifications, repairs or adjustments are to be made by qualified personnel. Questions??? Thanks National Sanitation Foundation American National Standards Institute Acknowledgement: All the material/presentations available online on the subject are duly acknowledged. Disclaimer: The author bear no responsibility with regard to the source and authenticity of the content.