1. Biological Safety Cabinets and Disinfection Sean Fitzgerald Harvard Biosafety

2. Biological Safety Cabinets HEPA filtration Classes and uses Certification Decontamination

3. HEPA Filter High Efficiency Air Particulate (HEPA) Filter –Particles at 0.3 microns are captured with an efficiency of 99.97% –Particles larger than 0.3 microns and particles smaller than 0.3 microns are captured with a greater efficiency than 99.97%

4. HEPA Filter HEPA filters use three mechanisms to trap particles 1.Interception – Particles being carried in the airflow around the filter fibers adhere to the filter. 2.Impaction – Large particles cannot adjust to sudden changes in airflow around the filter and essentially run into the filter fiber and become embedded. 3.Diffusion (Brownian motion) – Molecules move in a random, zig-zag pattern because they collide with surrounding molecules, which slows down a particle’s path through the HEPA filter and increases the probability that the particle will be captured by either interception or impaction.

5. Biological Safety Cabinets Provides three basic types of protection: –Personnel protection from harmful agents inside the cabinet. –Product protection to avoid contamination of the work, experiment, or process. –Environmental protection from contaminants contained within the cabinet. NOTE: Chemical Fume Hoods are used for hazardous chemicals NOT biohazardous materials

6. DOs and DON’Ts DOs Move arms in and out slowly, perpendicular to the face opening Clean up spills immediately Work in the center of the work area (at least 4 inches from front grille) Separate clean from dirty Adjust chair height so that your face is above the front sash opening and the bottom of the glass screen is even with your underarms DON’Ts Use highly flammable chemicals – BSC fans not spark proof Go in and out rapidly Tape the biohazard bag to the outside Overload cabinet Block front or rear grilles Work with more than two people in the same BSC Store Materials within-major source of contamination

7. Classes of Biological Safety Cabinets Class I Class II, A1 Class II, B1 Class II, B2 Class II, A2 Class III

8. Class I Biological Safety Cabinets This type of cabinet provides personnel and environmental protection, but no protection for research materials. The air exhausted from the BSC is HEPA filtered, but unfiltered room is drawn into the work surface. Use has declined with the advent of the Class II cabinets, though they may still be in use for equipment that may generate an aerosol. These cabinets are traditionally hard-ducted into the building exhaust.

9. Laminar Flow Unidirectional air movement at a fixed velocity along parallel lines to reduce turbulence.

10. Biological Safety Cabinet Air Flow

11. Class II Biological Safety Cabinets Laminar flow is utilized along with HEPA filtration to allow for consistent removal of airborne contaminants. Airflow is drawn into the front grille of the BSC and then this air is HEPA filtered prior to being recirculated back into the cabinet providing a down flow over the work area of clean air. This flow design has allowed for protection of personnel, the environment, and the research materials.

12. Class II Biological Safety Cabinets

13. CLASSIFICATIONS AND TYPES Class/ Type Face Velocity Airflow Pattern Volatile Chemicals II, A175 70% recirculated to the cabinet work area through HEPA; 30% balance can be exhausted through HEPA back into the room or to the outside through a thimble unit NO II, A2100 Same as II, A1, but plenums are under negative pressure to room; exhaust air can be thimble-ducted to the outside through a HEPA filter NO II, B1100 30% recirculated to the cabinet work area through HEPA. Exhaust cabinet air must pass through a dedicated duct to the outside through a HEPA filter NO II, B2100 No recirculation; total exhaust to the outside through hard-duct and a HEPA filter YES [small amounts]

14. Biological Safety Cabinet Certification BSCs are tested and certified on-site: –At the time of installation –At least annually thereafter –At any time the BSC is moved If BSC not certified within the designated time period then you should not use it Common Certification Failures –Clogged or damage HEPA filter –Motor not operating properly –Cracked sash

15. Biological Safety Cabinet Certification Changes to Biological Safety Cabinet field certification. –As of April 15, 2016, NSF field certifiers will no longer certify either direct-connected Type A cabinets or canopy connected Type A cabinets without air flow alarms installed.

16. BSC Post-work Disinfect materials before removal from BSC working area Seal and remove biohazardous waste Disinfect work surface, rear wall, sides, and inside front window NOTE: If using bleach solution as the disinfectant, this may result in pitting. Therefore, follow in 5 to 10 minutes with 70% ethanol to prevent pitting of the stainless steel, and allow to dry. Leave cabinet running if possible or close sash Routine housekeeping including catch basin Clean daily, weekly, monthly, semi-annually Don’t store materials on top of the BSC

17. Disinfection Sterilization vs. Disinfection Levels of Disinfection Keys to Disinfection Disinfection of Large Spaces

18. Sterilization “Any item, device, or solution is considered to be sterile when it is completely free of all living microorganisms and viruses. The definition is categorical and absolute (i.e., an item is either sterile or it is not). A sterilization procedure is one that kills all microorganisms, including high numbers of bacterial endospores. Sterilization can be accomplished by heat, ethylene oxide gas, hydrogen peroxide gas, plasma, ozone, and radiation (in industry). From an operational standpoint, a sterilization procedure cannot be categorically defined. Rather, the procedure is defined as a process, after which the probability of a microorganism surviving on an item subjected to treatment is less than one in one million (10-6).” BMBL 5 th edition, CDC

19. Disinfection Less lethal than Sterilization. It will eliminate nearly all forms of vegetative microorganism, but not necessarily all microbial life (such as bacterial spores). It is a reduction of the level of "microbial contamination".

20. Descending Order of Resistance to Chemical Disinfection Bacterial Spores Mycobacteria Nonlipid or Small Viruses Fungi Vegetative Bacteria Lipid or Medium-size Viruses

21. Common Disinfectants

22. High-Level Disinfection "This procedure kills vegetative microorganisms and inactivates viruses, but not necessarily high numbers of bacterial spores. Such disinfectants are capable of sterilization when the contact time is relatively long (e.g., 6 to 10 hours). As high-level disinfectants, they are used for relatively short periods of time (e.g., 10 to 30 minutes). These chemical germicides are potent sporicides and, in the United States, are classified by the FDA as sterilant/disinfectants. They are formulated for use on medical devices, but not on environmental surfaces such as laboratory benches or floors." BMBL 5th edition, CDC

23. Intermediate-Level Disinfection "This procedure kills vegetative microorganisms, including Mycobacterium tuberculosis, all fungi, and inactivates most viruses. Chemical germicides used in this procedure often correspond to Environmental Protection Agency (EPA)-approved “hospital disinfectants” that are also “tuberculocidal.” They are used commonly in laboratories for disinfection of laboratory benches and as part of detergent germicides used for housekeeping purposes." BMBL 5th edition, CDC

24. Low-Level Disinfection "This procedure kills most vegetative bacteria except M. tuberculosis, some fungi, and inactivates some viruses. The EPA approves chemical germicides used in this procedure in the US as “hospital disinfectants” or “sanitizers.”" BMBL 5th edition, CDC

25. Keys to Disinfection Concentration –Whether using a chemical or heat, the concentration of the disinfectant of choice is vital. Contact Time –The disinfectant of choice will require a specified amount of time to be in contact with the material being disinfected. Without the proper concentration or contact time, disinfection may be ineffective.

26. Disinfection of Large Spaces Disinfection is sometimes needed on a much larger scale. –Whole lab spaces, specialized areas, etc. This task should only be performed by specialists with the proper training and equipment. Methods used: –Paraformaldehyde Gas –Chlorine Dioxide Gas –Vaporized Hydrogen Peroxide

27. Biological Safety Cabinets and Disinfection Biological Safety Cabinets –Choose the right one for the right task –Make sure certifications are up to date! Disinfection –Proper disinfectant for the proper material –Contact time and Concentration

28. Clicker Questions

29. Is a 4 micron particle efficiently captured by a HEPA filter? A.True B.False

30. What should you NOT do when working in a Biological Safety Cabinet? A.Move arms in and out slowly, perpendicular to the face opening B.Work in the center of the work area (at least 4 inches from front grille) C.Block front or rear grilles D.Separate clean from dirty

31. Open flames are not allowed in a Biological Safety Cabinet because: A.They disrupt the normal airflow patterns designed to protect user and product B.Heat may damage HEPA filters C.They can cause a fire in the BSC D.All of the above

32. Sterilization will eliminate nearly all forms of vegetative microorganism, but not necessarily all microbial life (such as bacterial spores). A.True B.False

33. Without the proper concentration or contact time, disinfection may be ineffective. A.True B.False

34. Questions?