Biological Safety Cabinets (BSCs), or biosafety cabinets, are primary containment devices utilized in laboratories for the handling of biohazardous agents. They are routinely used for a wide variety of applications, such as human and animal tissue culture, bacterial and viral work, transfection or infection of cells with recombinant DNA (rDNA), clinical sample manipulation, and animal care.

 

The Role of Biological Safety Cabinets in Research

BSCs are designed to protect laboratory personnel and the environment from exposure to biohazards through the combined use of laminar flow and High Efficiency Particulate Air (HEPA) filters. For this reason, they are considered Clean Air Devices. HEPA filters trap the Most Penetrating Particle Sizes (MPPS) of 0.3 μm with an efficiency of at least 99.97%. Larger and smaller particles are captured with greater efficiency. Thus, bacteria, spores, and viruses are removed from the air by HEPA filters.

HEPA filters are very fragile - Avoid storing items on top of the BSC and bumping the filters or exposing them to dust or liquids.

When used correctly, BSCs shield workers from the inhalation of infectious particles/microorganisms and prevent their spread into the laboratory. In addition, most BSCs (all classes and types except for Class I BSCs) protect sensitive research material from external contamination.

Remember, the main purpose of a BSC is to protect you and the enviornment from exposure to biohazards while working with infectiours agents. Class II and III BSC's will protect your research materials from airborne contaminants with the aid of HEPA supply filters.

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How to Work Safely in a Biological Safety Cabinet

Biosafety cabinets afford the best protection and are more effective when maintained and used properly. To accomplish this goal, it is necessary that:

  • The BSC be certified upon installation, after it is moved, after repairs and annually thereafter.
  • Researchers learn how to work in and take care of a biological safety cabinet.

To extend the life of your BSC and ensure a safe working environment, follow these tips:

  • Minimize the storage of materials in and around the BSC. Avoid storing items on top of the cabinet since it could damage the HEPA filters.
  • Never operate the blower with the sash closed - an excessive air inflow could rip the HEPA filters.
  • Turn the BSC off after use - do not leave it running overnight.
  • If an UV light is present, use only as needed - never have the UV on when somebody is in the room.
  • Clean any spill immediately to prevent staining of the surfaces.

To operate a BSC safely and effectively, follow these instructions

Prior to use:

  1. Wash your hands with soap and water and put on the appropriate PPE (at a minimum gloves and a buttoned down or disposable gown).
  2. If available, turn UV light on for 15 minutes and switch it off afterwards.
  3. Raise the sash to the proper height.
  4. Turn on the blower and light.
  5. Wipe down the surface, walls, grills, and viewscreen of the BSC with an appropriate disinfectant.
  6. Load the cabinet with the necessary materials and reagents - Make sure that you disinfect the outer surface of medium bottles and pipettes before putting them in the BSC and, if possible, place a container with disinfectant and/or sharps containers to discard serological/pasteur pipettes and tips.
  7. Let the air purge for 10 minutes.

While working in the BSC:

  • Do not block the front and rear air intake grills.
  • Do not disrupt the protective airflow pattern - Avoid rapidly moving your arms in and out of the cabinet, people walking rapidly behind you, and open laboratory doors, which may reduce the effectiveness of the BSC.
  • Establish working areas and always work from the clean to the dirty area.
  • Clean spills as soon as they occur and replace broken/dirty gloves.

After Use:

  • Disinfect the materials that have been inside the cabinet before taking them out and wipe down the BSC.
  • Purge the air inside the BSC for a few minutes.
  • Turn off the blower and light and close the sash.
  • If the BSC is equipped with a UV light, turn it on for 15 minutes if nobody is present and abandon the room. Post a sign on the door notifying others that the UV light is on. Return later to turn the UV light off. Do not expose yourself unnecessarily to the UV light, since this can result in skin burns and eye lesions.
  • Remove PPE and wash hands.

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Classes of Biological Safety Cabinets

There are three classes of BSCs: Class I, II and III. While all three classes afford personnel and environmental protection, only Class II and III cabinets provide product protection thanks to the input of clean air from supply HEPA filters.

Class I BSCs are suitable for work involving low- to moderate-risk agents. Since incoming air is not filtered, Class I BSCs should not be used with research materials (e.g., cell lines) that must be handled under sterile conditions. Class I cabinets are used specifically to enclose equipment (e.g., centrifuges, harvesting equipment or small fermenters) or procedures with potential to generate aerosols (e.g., cage dumping, tissue homogenization or culture aeration). As outgoing air is filtered, research personnel is protected while using a Class I BSC.

The Class II BSC provides an effective partial barrier system for the safe manipulation of low-, moderate-, and high-risk microorganisms (i.e., BSL2 and BSL3 agents). Class II BSCs, which are the most frequently used in research and clinical laboratories, are divided into four types (Types A1, A2, B1 and B2).

The Class III BSC is a totally enclosed, gas-tight ventilated cabinet, and provides the highest level of personnel, environmental, and product protection. Operations within a Class III BSC are conducted through attached rubber gloves.

When purchasing a biological safety cabinet, you should consider the type of work that will be conducted in it. Table 1 provides a brief description of the principal characteristics of all classes and types of BSCs.

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TABLE 1: Characteristics of different classes and types of BSCs
BSC Class/Type Personnel protection Minimum Face Velocity FPM Volatile Toxic Chemicals, Gases or Radionnuculides Allowed?
I Yes 75 No
II / A1 Yes 75 No
II / A2 Yes 100 No, unless thimble connected or hard ducted to the HVAC system
II / B1 Yes 100 Yes: low levels of volatile toxic chemicals & trace radionuculides
II / B2 Yes 100 Yes: volatile toxic chemicals & radionuculides
III Yes 100 Yes

 

Summarizes the type of protection offered by BSCs
BSC Class Personnel protection Environmental Protection Product Protection Biological Risk
I Yes Yes No BSL2-3?
II (A1, A2, B1, B2) Yes Yes Yes BSL2-3?
III (II in suit room with suit) Yes Yes Yes BSL3-4?

Weizmann Institute


Comparison of Biological Safety Cabinets
Cabinets   Applications
Type Face velocity
fpm
Airflow Pattern Radionuclides/
Toxic Chemicals
Biosafety
Level(s)
Product
Protection
Class I:
Open Front
75 In at front; out rear and top through HEPA filter NO 2.3 NO
Class II:
Type A
75 70% recirculated through HEPA; exhaust through HEPA NO 2.3 YES
Type B1 100 30% recirculated through HEPA; exhaust via HEPA and hard-ducted YES
(Low levels volatility)
2.3 YES
Type B2 100 No recirculation: total exhaust via HEPA snd hard-ducted YES 2.3 YES
Type B3 100 Same as IIA, but plan under negative pressure to room, and exhaust air is ducted YES 2.3 YES
Class III NA Supply air inlets and exhaust through 2 HEPA filters YES 3.4 YES

 

Handling biohazardous waste related to BSC operations

To collect liquid biohazardous waste, aspirate liquid into a flask containing bleach (A) connected to a second overflow collection flask (B) and separated from the vacuum system (D) by an in-line filter (C). Biohazardous liquid waste needs to be properly neutralized before disposal.

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Mechanical Operation of a Biological Safety Cabinet

Main Features and Operation Panel

BSCs are equipped with an on/off switch, a blower/fan system, light(s), electrical outlets and, in those cabinets with an alarm, an alarm system bypass. All these functions are controlled through a panel located either on the front or side of the BSC.

The Magnehelic Gauge and Other Indicators

Some BSCs are equipped with a differential pressure gauge, commonly referred to as Magnehelic gauge, that measures the pressure change beween points (pictured below to the lef). If monitored routinely, the Magnehelic gauge may provide a "gross" indication of HEPA filter loading, i.e., the volume of particulate matter the filter has accumulated as the cabinet operates.

Normal gauge readings differ from cabinet to cabinet. If your BSC has a Magnehelic gauge, read it every time you use the cabinet to ensure that airflow pressure remains constant. For that purpose, turn on the blower, let it run for 5 minutes, and record the reading. If the differential pressure rises or drops by more than 0.2, a blockage or tear in the HEPA filter(s) may be interfering with the correct functioning of the BSC. Do not use the cabinet and call for service.

Other BSCs (pictured above to the right) are equipped with digital displays of inflow and downflow velocities, and other features showing the capacity of the cabinet to provide a safe working environment. No matter what type of BSC you are using, always pay attention to warning signs and alarms.

Alarms

Many BSCs are equipped with an alarm system that warns users if the airflow in the cabinet gets compromised. The alarm usually goes off when the sash gets raised over the 10-inch mark, rendering user and environmental protection ineffective, or when the filters do not allow air to pass through them anymore. Do not work in the BSC if the alarm sounds continuously, since it indicates a problem with the BSC's performance. Instead, ask a specialized technician to inspect your BSC. For safety reasons, disengaging the alarm to make it stop is not allowed.

UV Lights

UVM Health and Safety has adopted the position of the NSF, NIH, CDC and the American Biological Safety Association (ABSA) in regard to the use of UV lamps within biosafety cabinets; UV lamps are neither recommended nor required in biological safety cabinets.  UVM Health and Safety believes they are not appropriate for disinfection within a BSC and are potentially dangerous when used improperly.  If you have any questions regarding our position on the use of UV light in biosafety cabinets, please contact the UVM Biosafety Officer @ 6-3618.

Outlets

Electrical devices can be used, and vacuum pumps connected, inside the BSC.

Bunsen Burner Use

Warning: Open flames are not allowed inside the BSC. In most instances, flames are not required in the near microbe-free environment provided by a BSC. If absolutely necessary, touch-plate microburners with a pilot light that provide a flame on demand may be used.

Biological Safety Cabinets FAQs

I need to treat my cell culture plates with volatile chemicals. Should I use a Biological Safety Cabinet (BSC) or a Fume Hood (FH) to do my work?

A Fume Hood will not protect your cells from contamination, and therefore, should not be used if you require aseptic conditions for your work (e.g., if you need to pass cells or continue an experiment in which cells must be placed back in the incubator). You could use the Fume Hood for a terminal procedure, such as RNA isolation with Trizol. For regular cell culture work involving small amounts of volatile chemicals, you should use a BSC suitable to that effect. Type xxxx BSCs can be used with limited amounts of chemicals.

What is the difference between a Biological Safety Cabinet and a Clean Bench (CB)? Can I use the clean bench for cell culture?

While all types of biological safety cabinets protect personnel and the environment from exposure to contaminants within the cabinet, clean benches do not. In clean benches, contaminated air is directly blown towards the user and surrounding environment. For this purpose, clean benches should only be used to conduct work that requires sterile conditions (such as preparing sterile solutions) but that does not involve manipulation of biohazards, chemical hazards or radioactivity.

Always use your biological safety cabinet for cell culture work, especially when working with human or primate cell lines, to avoid potential exposure to blood-borne pathogens!

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There are both Clean Benches and Biological Safety Cabinets in my lab. Can I use the Clean Bench for cell culture?

No. The Clean Bench will only protect your cells, but not you, from contamination, since the filtered air is blown directly in the direction of the user. When working with cell cultures, always use a Biological Safety Cabinet suitable to the type of work you are conducting.

My BSC is equipped with a UV light. Do I still need to disinfect the cabinet?

UV lights are not a substitute for chemical disinfection. UV lamps need to be checked periodically to confirm their effectiveness and must be clean and devoid of debris in order to work properly. Chemical disinfection should be always performed, regardless of the absence/presence of a UV light.

Should I leave the UV light on constantly when the BSC is not in use?

The UV light should only be utilized when nobody is in the room. Do not turn the UV on if you have finished your work but are staying in the room, especially if you cannot close the sash. Prolonged exposure to UV light can cause skin cancer and cornea burns. If you leave the UV light on after finishing your work, post a sign outside the room to warn others. If you need to work in the room, switch the UV light off as soon as you enter.

I am always very careful when working in the BSC but my cells have gotten contaminated several times in the past few weeks. I have discarded the medium, decontaminated the incubator, etc. without any luck. What could the problem be?

BSCs are equipped with HEPA filters (except Class I) that supply clean air to the working area. If the supply filter is damaged or leaks, dirty air may be supplied to the BSC, contaminating the area. Schedule a certification with a specialized technician to check the performance of your BSC.

A technician has told me that my BSC has failed the certification. Can I still use it?

If your cabinet has failed the certification, it means that it is not performing as it should. This may compromise the quality of your work (e.g, contaminated cells) or expose you and the environment to aerosols from infectious agents (e.g., exhaust filter damaged). You should not resume work within the BSC until it is repaired and passes the certification. Contact Environmental Safety at esf@uvm.edu if you have any question or concern.

I have never worked in a BSC. Where can I get some training?

BSC training is compulsory for researchers working in laboratories with a minimum biosafety level BSL2.

Biological Safety Cabinet Certification, Decontamination, Repair and Disposal

Certification

Biological safety cabinets must be tested periodically in order to ensure that they are working properly and providing a safe research environment. This process, known as certification, should be performed by a specialized technician. Certifications should be conducted according to the NSF/ANSI Standard 49 for Biosafety Cabinetry of the National Sanitation Foundation.

The NSF Biosafety Cabinetry Program was initiated more than 25 years ago at the request of regulatory organisms including the National Institutes of Health (NIH), Centers for Disease Control (CDC), and the National Cancer Institute (NCI).

As a general rule, a BSC should be certified:

  • Upon initial installation in the laboratory
  • At least once a year thereafter
  • After repairs
  • When moving it to a different laboratory

Depending on the model, age, and condition of the cabinet, the certification process can take somewhere between 2 and 4 hours. Some of the tests conducted include:

  • Testing the inflow (air coming from the room into the BSC) and downflow (air passing through the supply filter into the work area) velocities.
  • Inspecting the HEPA filters for leakages and patching small leakages with silicon if indicated.
  • Testing the airflow smoke pattern to ensure that air is appropriate passing through the HEPA filter and also not escaping from the cabinet.

If the BSC passes the certification, the technician will issue a certification sticker that is valid for one year. If it fails, it should be decontaminated, repaired, and recertified before being used. Remember that a BSC that fails the certification cannot guarantee safe working conditions for you, your research materials, and your lab.

Decontamination, Repair and Disposal

Decontamination of a BSC is necessary when it is:

  • Being opened for repairs (e.g., internal damper adjustment, filter and motor changes)
  • Moving from one laboratory to another
  • Being disposed of

There are several methods to decontaminate BSCs, such as using formaldehyde, Vaporous Hydrogen Peroxide (VHP), or Chlorine Dioxide (CD, ClO2). All these methods relay in strong chemicals to ensure that biological contaminants are inactivated before the BSC is opened or moved, and effectively decontaminate not only the surfaces of the cabinet, but also internal parts. Since chlorine dioxide is less toxic than formaldehyde, and the decontamination can be performed in less time, many technicians are abandoning formaldehyde in favor of ClO2.

For health and safety purposes, only the technician is allowed in the room while the BSC is being decontaminated.

The Environmental Safety Department at UVM assists researchers in scheduling the annual certifications. For decontamination's, repairs, and additional certifications, contact the vendor directly. We encourage you to ask for quotes from several companies since their prices and availability may vary.

Here is a list of vendors UVM has been working with in the past few years:

Contacts
Vendor Phone Location Website
TSP (UVM) 802-656-3255 Burlington, VT  http://its.uvm.edu/tsp
ATC Associates 802-862-1980 Williston, VT http://www.atcassociates.com
ENV Services 800-292-5255 Hicksville, NY http://www.envservices.com
B&V Testing 800-851-9081 Waltham, MA http://www.bandvtesting.com

 

Use of UV lamps in Biosafety Cabinets

UVM Health and Safety has adopted the position of the NSF, NIH, CDC and the American Biological Safety Association (ABSA) in regard to the use of UV lamps within biosafety cabinets; UV lamps are neither recommended nor required in biological safety cabinets.  UVM Health and Safety believes they are not appropriate for disinfection within a BSC and are potentially dangerous when used improperly. 

If you have any questions regarding our position on the use of UV light in biosafety cabinets, please contact the UVM Biosafety Officer @ 6-5400 or jlabossi@uvm.edu.

Clean Benches

While all types of biological safety cabinets protect personnel and the environment from exposure to contaminants within the cabinet, clean benches do not. In clean benches, contaminated air is directly blown towards the user and surrounding environment. For this purpose, clean benches should only be used to conduct work that requires sterile conditions (such as preparing sterile solutions) but that does not involve manipulation of biohazards, chemical hazards or radioactivity.

In a BSC, the filter(s) are located on the upper part above the work area, which is separated from the user by a sash or viewscreen (left picture). In contrast, the filter can be located above (vertical flow) or in the back (horizontal flow) of a clean bench, and there may or may not be a sash.

Always use your biological safety cabinet for cell culture work, especially when working with human or primate cell lines, to avoid potential exposure to blood-borne pathogens!

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Clean Benches and Fume Hoods are Not Biological Safety Cabinets

Biological safety cabinets must not be mistaken with fume hoods and clean benches. Small amounts of radioactive materials or volatile chemicals can be used in certain types of BSCs (see Classes of Biological Safety Cabinets Table 1 above). However, a BSC is not a substitute for a fume hood and should never be used with large concentrations of hazardous chemicals. Fume hoods are used to protect personnel from exposure to chemical hazards and, in general, are not suitable for work involving biohazards.

Clean benches protect research products from contamination but do not afford protection to the user. Thus, clean benches should not be used when working with potentially infectious materials, chemical hazards or radioactivity.

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Safe Use of Volatile Chemicals and Radioactivity in Biological Safety Cabinets

Work with biohazardous materials involving the use of volatile chemicals and/or radioactivity should exclusively be conducted in a biological safety cabinet that is vented outside the building.

Ducted Class II Type A2, Class II Type B1 and B2 (always hard-ducted), and Class III BSCs may be used with different amounts of chemicals/radioactive materials. Please review the following table for more specific details:

BSC Class / Type Volatile Chemicals Radionuclides
II / A2 If ducted outside building: minute amounts If ducted outside building: trace amounts
II / B1 Low levels Trace amounts
II / B2 Yes Yes
III Yes Yes

 

Biological Safety Cabinets Certification Failure

If your cabinet has failed the certification, it means that it is not performing as it should. This may compromise the quality of your work (e.g, contaminated cells) or expose you and the environment to aerosols from infectious agents (e.g., exhaust filter damaged). You should not resume work within the BSC until it is repaired and passes the certification. Contact Environmental Safety at safety@uvm.edu if you have any question or concern.

Contamination Problems in the Biological Safety Cabinets

Most BSCs (except Class I) are equipped with HEPA filters that supply clean air to the working area. If the supply filter is damaged or leaks, dirty air may be supplied to the BSC, contaminating the area. Schedule a certification with a specialized technician to check the performance of your BSC.

Biological Safety Cabinet Training

BSC training is compulsory for researchers working in laboratories with a minimum biosafety level BSL2. Online training is available. Please, log in with your net ID and password and take the online training "Safe Use of Biosafety Cabinets".

 

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