UR Requirements for Class II Biosafety Cabinets
The Basics:
A biosafety cabinet (BSC) is not chemical fume hood. Fume hoods are designed to remove chemical fumes and aerosols away from the work area. Biological safety cabinets are designed to protect personnel and the environment external to the cabinet while providing a suitable workspace for tissue culture or microbiological procedures. Frequently they are referred to as vertical laminar flow hoods, tissue culture hoods, or bio hoods. The use of Class II biosafety cabinets is required for controlling manipulations of infectious agents likely to generate aerosols.
Biosafety cabinets use vertical laminar airflow and HEPA filters (High Efficiency Particulate Air filter) to provide protection.
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Laminar airflow is uniform air velocity in one direction along parallel flow lines to achieve a barrier of protection against contaminants entering and exiting the cabinet. The laminar airflow reduces the turbulence in the work area and minimizes the potential for cross-contamination within cabinet’s the workspace.
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HEPA filters remove particulate contamination through filtration, but do not trap or capture volatile chemicals or gases.
These filters have an efficiency of 99.97% for particles that are 0.3um in size and higher filtering efficiencies for particles that are smaller or larger than 0.3um. 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 the cabinet during movement can damage the filter medium causing failure in the cabinet’s protective feature.
Class II biosafety cabinets are classified into two types, the A cabinet and the B cabinet. This classification is based on airflow velocities and patterns, and exhaust systems.
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Class II type A cabinets are divided into sub categories, A1 and A2, and are suitable for microbiological work without volatile or toxic chemicals. The air from an A cabinet is directly exhausted into the laboratory. Type A1 cabinets are not appropriate for hazardous biologicals because the biologically contaminated plenum is under positive pressure and may leak into the laboratory. The biologically contaminated plenums of Type A2 cabinets are either under negative pressure or are surrounded by plenums under negative pressure thereby preventing leaks of contaminated air into the lab.
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Class II type B cabinets are divided into sub categories, B1 and B2, and are ducted to the house exhaust system. The B cabinets are divided into sub-categories based on the amount of air that is recirculated within the cabinet. B1 cabinets have approximately 30% recirculated supply air and B2 cabinets have no recirculated supply air (100% exhaust). Type B cabinets also have a higher face velocity, which allows for work including volatile chemicals or radionuclides.
Biological safety cabinets must be certified periodically to ensure proper airflow and filter integrity. Certification must be performed upon installation at least annually thereafter by a qualified technician. Additionally, biosafety cabinets must be certified when they are moved to a new location, when HEPA filters are replaced, or when the cabinet is serviced. Certification parameters are dictated by the
National Sanitation Foundation Standard No. 49 “Class II (Laminar Flow) Biohazard Cabinetry”. A label is placed on the cabinet by the certifier, which indicates the last date of certification. Please call the Biosafety Officer (X5-3014) for the names of approved contractors.
Although biosafety cabinets are extremely effective and are the single most important biological 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. Aerosols or particulates containing infectious agents can escape from the biosafety cabinet, so steps must be taken to minimize their production and ensure protection from possible exposure to infectious materials.

Work Practices:
The proper use of a biosafety cabinet complements good microbiological techniques and results in effective containment and control of infectious agents. As with any other pieces of laboratory equipment, personnel must be trained in its proper use. General guidelines for the safe and effective use of biosafety cabinets are below.
1.
Locate your biosafety cabinet deep in the laboratory away from doors, traffic patterns, fans, and ventilation air inlet (supply) diffusers.
2.
Avoid locating other laboratory equipment such as fume hoods, centrifuges, and vacuum pumps near biosafety cabinets. The air currents / turbulence associated with these types of equipment may adversely affect the biosafety cabinet’s performance.
3.
Avoid activities that may disrupt the inward flow of air through the sash opening. These activities include:
A.
Repeated insertion and withdrawal of the worker’s arms into and out of the cabinet.
B.
Opening and closing the doors to the laboratory.
C.
Brisk walking past the cabinet.
4.
Use the magnehelic gauge to track the performance of the HEPA filters over time. The magnehelic 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 the filter is clogging or leaking.
Magnehelic gauge
5.
Plan and prepare for your work in the cabinet by having a checklist of materials needed and by placing those materials in the cabinet before beginning your work. This reduces the number of times you break the protective air current and thereby reduces the amount of contamination entering the cabinet. Slow movement of arms in and out of the cabinet will also reduce the risk of contamination entering and exiting the cabinet.

Operational Guidelines for Safe and Effective Biosafety Cabinet Use:
1.
Ready the work area. Operate cabinet blowers for five minutes before beginning work to allow the cabinet to purge or remove particulates from the cabinet.
2.
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. Bleach is highly corrosive and should be avoided. Use of bleach without adequate removal of the bleach will pit and rust the stainless steel.
3.
Assemble materials. Introduce only those items that are required to perform the procedures and arrange them in a logical order. Each item should be wiped with disinfectant prior to placing it into the cabinet to reduce the introduction of contaminants, (i.e. mold spores). The flow of work should proceed across the work surface from clean to contaminated areas. Similarly, pipet discard trays containing disinfectant, biohazard bags, sharps containers, etc. should be placed to one side inside the BSC. Limited motion in and out of the cabinet preserves the protective envelope, and prevents the release of infectious materials outside of the BSC.
4.
Put on protective clothing. Laboratory coats or solid front gowns should be worn over street clothing and long-cuffed gloves should be worn for hand protection. The cuffs of the gloves should be pulled up and over the cuffs of the coat sleeves. Plastic or Tyvek sleeves should be considered if skin is exposed between the cuff and glove.
5.
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.
6.
Do not block the front grille with papers, equipment, etc. as this may cause air to enter the workspace 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 (back 1/3 of cabinet). A disinfectant-soaked towel can be placed on the work surface to contain any spills or splatters that may occur.
7.
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. Only burners such as a touch plate burner that provides a flame on demand are permissible.
Place these heat-generating devices near the rear of the cabinet. Alternatively, electric furnaces or disposable, sterile tools can be used. It is important to note that using flames for sterilization in a biosafety cabinet is unnecessary, potentially dangerous, and ‘old school’ thinking. The combination of proper work practices, disinfection, and the protective features of a biosafety cabinet have been proven to prevent culture contamination.
8.
If culture media or other fluids need to be aspirated, suction or aspirator flasks should be connected to a 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.
9.
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.
10.
Investigators should remove their gowns and gloves and thoroughly wash their hands before exiting the laboratory.

Ultraviolet Lights in the Biosafety Cabinet:
In order for UV light to be an effective biocidal agent, the output must be at least 40microwatts/ cm2 at 254nm. Frequently UV lamps are not maintained adequately to achieve this level of irradiation. Dust accumulating on the surface of the lamp and shadows caused by items left in the biosafety cabinet dramatically decrease the effectiveness of the ultraviolet light as it cannot penetrate dust or other materials. Ultraviolet light also presents risks to personnel by being able to cause serious eye and skin injuries after relatively short exposure times. This risk continues to be present well after the lamp output is no longer sufficient to kill biologicals. As a result
Environmental Health and Safety does not recommend using UV lights in biosafety cabinets.
Biosafety Cabinet Maintenance Requirements:
1.
When do biosafety cabinets have to be certified?
A.
Biosafety cabinets must be certified:
1.
Following new installation and prior to first use,
2.
Following cabinet relocation,
3.
Following HEPA filter replacement, and
4.
Following repair or maintenance on any sealed portion of the cabinet.
B.
Biosafety cabinets must be recertified annually.
C.
An approved contractor must certify biosafety cabinets. Certification of the cabinet must be performed according to the
National Sanitation Foundation Standard Number 49 Class II (Laminar Flow) Biohazard Cabinetry. Contact the Biosafety
Officer (X5-3014) for a list of approved contractors.
2. When do biosafety cabinets require gaseous decontamination?
A. Biosafety cabinets must be decontaminated with paraformaldehyde gas:
1.
Before moving the cabinet to another location,
2.
Before HEPA filter replacement,
3.
Before repair or maintenance in any part of the cabinet that may be contaminated, and
4.
As part of a laboratory decommissioning.
B. Biosafety cabinets must be decontaminated by an approved contractor. Decontamination of the cabinet must be performed according to the National Sanitation Foundation Standard Number 49 Class II (Laminar Flow) Biohazard Cabinetry. Contact the Biosafety Officer (X5-3014) for a list of approved contractors.

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 - Personnel and Environmental Protection Only
The Class I biological safety cabinet is designed to provide personnel and environmental protection only.
 A Class I cabinet does not protect the product from contamination because “dirty” room air constantly enters the cabinet front to flow across the work surface.
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As a partial containment unit, the Class I cabinet is suitable for work involving low to moderate risk agents where there is a need for containment, but not for product protection.
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Unlike conventional fume hoods, the HEPA filter in the Class I cabinet protects the environment by filtering air before it is exhausted.
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Personnel protection is made possible by constant movement of air into the cabinet and away from the user.
Class II - Product, Personnel and Environmental Protection
A Class II cabinet must meet requirements for the protection of product, personnel and the environment.
The Class II biological safety cabinet has three key features:
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A front access opening with carefully maintained inward airflow.
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HEPA-filtered, vertical, unidirectional airflow within the work area of the cabinet.
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HEPA-filtered exhaust air to the room or exhaust to a facility exhaust system.
Vertical, unidirectional airflow and a front access opening are common to Class II cabinets. However, since Class II designs permit different airflow patterns, velocities, HEPA filter position, ventilation rates and exhaust methods, a sub-classification of Type is needed to differentiate Class II designs. Refer to CDC / NIH 5 th edition “Biosafety in Microbiological and
Biomedical Laboratories” Appendix A for additional regarding the Types of biosafety cabinets within the Class II category.

Class III - Total Containment Cabinets
Class III biological safety cabinets are gas-tight, designed for use with high-risk biological agents. Class III cabinets provide the highest level of personnel, product and environmental protection. Contact the Biosafety Officer at X5-3014 if you think you need a
Class III Biosafety Cabinet
CDC/ NIH 3rd Edition Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets
CDC / NIH 5 th edition “Biosafety in Microbiological and Biomedical Laboratories” Appendix A
American Industrial Hygiene Association, Flame / Ethanol Sterilization Fires