What is a biological safety cabinet (BSC)?
Biological safety cabinets are primary devices intended to contain and minimize exposure when working with biohazardous materials. They are often, but incorrectly referred to as laminar flow hoods, tissue culture hoods, and biological fume hoods. Biological safety cabinets are designed to protect laboratory personnel against exposure during experimental procedures (personnel protection), as well as protect experimental materials from contamination (product protection). They are recommended for manipulations of infectious agents that are likely to create aerosols (i.e., aspirating with a syringe, removing caps from tubes after centrifugation, vortexing of open tubes, sonication, etc.). Additionally, they are also used when manipulating human blood and body fluids, generating high concentrations or large volumes of infectious agents, and when working with and maintaining sterile cell and tissue cultures.
IMPORTANT: Although BSCs are extremely effective and are the single most important containment device, they are only one part of a comprehensive biosafety program and are not a substitute for careful work practices and good aseptic technique. Since aerosols can escape, precautions must be taken to minimize their production and ensure protection from possible exposure to infectious material.
Biological safety cabinets utilize vertical laminar airflow (i.e., uniform air velocity in one direction along parallel flow lines) to achieve a barrier of protection against airborne contaminants, such as microorganisms. The laminar airflow reduces turbulence in the work area, and minimizes the potential for cross-contamination. HEPA (High Efficiency Particulate Air) filters, 
creating a nearly sterile work environment remove airborne particles going into the work area and out to the environment. The filters have an efficiency of 99.97% with particles that are 0.3um, and higher efficiencies (99.99%) with particles above and below that size. HEPA filters are made of borosilicate fibers, pleated to increase the overall surface area, divided by separators, and glued into a frame. Careless handling of the filters or cabinet (during movement) can damage the filter medium at a joint or cause a tear that will compromise the filter integrity and render the BSC unusable.
Biological safety cabinets must be routinely inspected for proper airflow and filter integrity to ensure that they are providing protection to the worker and the environment. Certification must be performed when a new BSC is first installed (damage or misadjustments can occur during shipment) and annually thereafter by a qualified technician according to the National Sanitation Foundation Standard No. 49, "Class II (Laminar Flow) Biohazard Cabinetry." Your BSC should have a label indicating the date it was last tested. In addition, BSCs must be inspected and certified when they are moved to a new location, or when the HEPA filters are changed. Please call EH&S (5-8200) to obtain the names and telephone numbers of licensed contractors.
Work practices and procedures:
The proper use of biological safety cabinets (BSC) can complement good microbiological practices and result in effective containment and control of biohazardous and infectious agents. General guidelines include:
Locating the BSC "deep" in the laboratory away from air currents produced by ventilation inlets, opening/closing of the laboratory door(s), and away from areas of heavy traffic. If possible, close laboratory doors and limit entry, egress and walking traffic. Air currents and movements create turbulence that disrupt the protective envelope of the cabinet. Additionally, other nearby laboratory equipment such as centrifuges, vacuum pumps, etc. can affect the performance of the BSC. Cabinets should not be located directly opposite of each other or opposite a chemical fume hood, as laminar airflow will be hindered.
Observing the magnehelic gauge and noting its relative position each time that the BSC is operated. The magnehelic gauge measures the pressure drop across the HEPA filters and thus indicates filter load and integrity. A significant increase or decrease in the pressure over a short period of time may indicate clogging or leaking of the filter.
Planning and preparing for your work in the cabinet by having a checklist of materials needed and placing those materials in the BSC before commencing work. This reduces the number of arm movements across the air barrier of the cabinet, thereby preserving the protective envelope and containment properties. Slow movement of arms in and out of the cabinet will reduce the risk of potential contamination.
Operational Procedures:
- Ready the work area. Operate cabinet blowers for five min before beginning work to allow the cabinet to purge or remove particulates from the cabinet.
- Disinfect the work area. Wipe the work surface, interior walls and surface of the window with a suitable disinfectant such as 70% ethanol, an iodophor, or quaternary ammonium compound.
- Assemble material. Introduce only those items that are required to perform the procedures and arrange in a logical order. Each item should be wiped with disinfectant prior to placing it into the cabinet to reduce the introduction of contaminants, such as mold spores. The flow of work should proceed across the work surface from clean to contaminated areas. Similarly, pipette discard trays containing disinfectant, biohazard bags, sharps containers, etc. should be placed to one side inside the BSC. This limited motion in and out of the cabinet preserves the protective envelope, and prevents the release of infectious materials outside of the BSC.
- Don protective clothing. Laboratory coats or solid front gowns should be worn over street clothing and long-cuffed latex or other appropriate (e.g., nitrile, vinyl) gloves should be worn for hand protection. The cuffs of the gloves should be pulled up and over the cuffs of the coat sleeves.
- Perform procedures. Slowly move arms when working and when moving items in and out of the cabinet. Avoid rapid movements during procedures. After placing arms/hands inside the BSC manipulations should be delayed to permit the cabinet to stabilize and allow the flow of air to remove surface contaminants from your arms/hands.
- Do not block the front grille with papers, equipment, etc. as this may cause air to enter the work space area instead of being drawn through the front grille and to the HEPA filter. Arms should be raised slightly, and operations should be performed on the work surface at least 4 in from the front grille. The middle third area is ideal. Likewise, no operations or equipment should block the rear exhaust grille. Any equipment generating aerosols, such as a microcentrifuge, vortex or blender, should be placed near the rear of the cabinet. A disinfectant-soaked towel can be placed on the work surface to contain any spills or splatters that may occur.
- Open flames inside the cabinet create turbulence that can disrupt the pattern of air and compromise the safety of the operator and affect product protection (i.e., cause contamination). Flames can also damage the interior of the cabinet as well as the HEPA filters. If a burner is necessary to sterilize tools such as a loop or needle, consider the use of a touch plate burner that provides a flame on demand, and place it to the rear of the cabinet. Alternatively, electric furnaces or disposable, sterile tools can be used.
- If culture media or other fluids need to be aspirated, suction or aspirator flasks should be connected to an overflow collection flask containing disinfectant (the aspirated materials can then be discarded as noninfectious waste). The flasks should then be coupled to an inline HEPA or equivalent filter designed to protect the vacuum system.
- When work is completed all items within the cabinet should be wiped down with disinfectant and removed from the cabinet. Do not use the interior of the BSC as a storage area since stray organisms may become "trapped" and contaminate future experiments. The interior surfaces of the cabinet should also be cleaned with a suitable disinfectant. Let the blowers operate for five minutes, with no activity inside the cabinet, to purge the cabinet of contaminants.
- Investigators should remove their gowns and gloves and thoroughly wash their hands before exiting the laboratory.
Use of ultraviolet lights in the biosafety cabinet:
Ultraviolet lights are a common accessory of many BSCs. These lamps are regarded as biocidal devices, "protecting" the operator from exposure to infectious agents and experimental materials from contamination. However, the actual effectiveness of UV light in providing this "sterile" environment has been questioned. Additionally, there are potential occupational hazards that carry significant risks (e.g., serious eye and skin injury) associated with the use and misuse of these lamps. Ultraviolet lamps must be periodically tested to ensure that the energy output is adequate to kill microorganisms. The radiation output should be at least 40microwatts/ cm2 at 254 nm when measured with a UV flux meter placed in the center of the work surface. The output performance of the lamps is adversely affected by dust accumulating on the surface of the lamps (UV light is unable to penetrate through dust or other materials), and microorganisms adhering to floating dust particles or other fixed objects are also protected and unaffected by UV illumination. Ultraviolet exposure damaging to the eyes and skin exists well after the output of the lamps has dropped below the biocidal level. The effective life spans of the lamps are relatively short and the bulbs are expensive to replace. As a result, Environmental Health & Safety dissuades operators from using UV lights to maintain a clean working environment. A significantly more effective and recommended strategy to reduce or eliminate contamination utilizes well-practiced microbiological procedures, good aseptic techniques, operational procedures outlined in this document, and thorough decontamination procedures before and after BSC use.
Types of Biological Safety Cabinets:
Three general classes of cabinets are defined; class I, open-front air inflow cabinet; class II, several subtypes of open-front vertical airflow cabinets (very common); and class III, totally enclosed, gas-tight ventilated cabinets with work operations conducted through fixed, attached rubber gloves. Class I and II cabinets (used exclusively on campus) are described below.
Class I: Good protection for the operator, but no product protection, is provided with these cabinets. Air flow, at a minimum inward face velocity of 75 linear feet per minute (lfpm), is directed through the front opening, across the work area and out through the HEPA filter on top. This cabinet is conventionally used with a full width open front, or can be used with an attached armhole front panel with or without attached rubber gloves. Although class I cabinets are simple and economical, and radioisotopes and some toxic chemicals can be used (if exhaust is ducted to the outside), filtered air is not provided over the work area. These cabinets do not protect your materials from contaminants introduced from the environment or the operator.
Class II: Unlike class I cabinets, class II cabinets afford protection for the operator AND the work performed. The capacity to protect materials within the cabinet is provided by the flow of HEPA-filtered air over the work surface. There are four subtypes of Class II cabinets based on the construction, inflow air velocities, and the exhaust systems. These cabinets can be used to manipulate low to moderate risk agents.
Class IIA: Air, at a face velocity of 75 lfpm, is drawn into the front grille of the cabinet away from the work surface. The air is directed through a HEPA filter and downward over the work area. As the air approaches the work surface, the blower draws part of the air through the front grille and the remainder through the rear grille. Approximately 70% of the air is recirculated to the work zone through the supply HEPA filter, and about 30% is exhausted to the room through another HEPA filter. This cabinet is unsuitable for work that involves radioactive materials and toxic chemicals because of the buildup of vapors in the air recirculated within the cabinet and out into the laboratory.
Class IIB1: As with the class IIA cabinet inflow air (face velocity of 100 lfpm) is ultimately forced through a HEPA filter and over the work area, where there is a split of downward flowing air. About 70% of the air is directed through a HEPA filter to the outside (must be hard-ducted, preferably with its own exhaust system), whereas 30% is drawn through the front grille and recirculated. Minute amounts of toxic chemicals and trace amounts of radioisotopes can be used within the hood, although activities should be conducted toward the rear of the cabinet.
Class IIB2: This cabinet is a total exhaust cabinet; no air is circulated within it. A supply blower draws in room air or outside air at the top of cabinet, through a HEPA filter and down into the work area. Additional room air is drawn through the front grilles at a face velocity of 100 lfpm. The air discharged from this cabinet must be 100% exhausted outside through a HEPA filter in a dedicated hard duct. Small quantities of toxic chemicals and radioisotopes can be used within the hood. The exhaust of a large volume of conditioned room air makes this cabinet very expensive to operate. Additionally, the cabinet must be running continuously so as not to interfere with room exhaust. Should building or cabinet exhaust fail, the blower motors should be turned off to prevent a back flow of pressurized air from the cabinet work area into the laboratory.
Class IIB3: This is a combination A/B cabinet with a face velocity of 100 lfpm. This cabinet can be used as a class A cabinet where exhaust air is recirculated in the laboratory. Alternatively, it can be used as a class IIB cabinet and exhaust air vented to the outside with a thimble unit connected to the duct. Biologically contaminated ducts and plenums are under negative pressure to the room and exhaust air. Approximately 70% of the air is exhausted, whereas 30% is recirculated within the cabinet. Minute quantities of toxic chemicals and trace amounts of radioisotopes can be used.