Biosafety Cabinets

Biosafety cabinets can be used with any biological agent including bacteria, viruses, viral vectors, fungi, parasites, human/animal tissue and cell lines, and prions. They should not be used with:

• Large amounts of volatile or toxic chemicals
• Concentrated flammable chemicals
• Volatile radionuclides
• Open flames (for more information see the Policies section below)

A biosafety cabinet provides three layers of protection:

1. Personnel — Air curtain and HEPA filters protect users from biohazardous aerosols generated inside the chamber
2. Sample Protection — Recirculating and unidirectional HEPA filtered air protect samples from contamination from unsterile lab air
3. Lab/Environmental protection — HEPA filtered exhaust from top of cabinet protects the lab environment from contamination by biohazardous aerosols generated inside the chamber

HEPA filter (High-Efficiency Particulate Air or High-Efficiency Particulate Arresting filters) are fibrous filters that remove particles from air passing through them. HEPA filters consist of a metal or wood frame holding a long, folded strip of cellulose or borosilicate fiber. The edges are sealed with Epoxy or polyurethane.

To be designated as HEPA, the filter must remove 99.97% of all particles at a 0.3 um size. This particle size is the Most Penetrating Particle (MPP) size.

• Fibrous material is used to separate biological material from air passing through the filter
• Particles are “trapped” by the fibers and removed from the air as it flows through the filter
• Multiple, folded sheets of fibrous material drastically increase the surface area of the filter
• Increased surface area drastically increases the efficiency of filtration

HEPA filters remove biological aerosols through several mechanisms:

• Fast-moving particles are filtered through direct impact with fibers
• Large particles are removed by straining effect when particles become trapped between two fibers
• Smaller particles are removed by interception
• Very small particles move by Brownian motion and are removed by diffusion when they come in contact with fibers
• Negatively charged particles (such as some viral particles) are removed by electrostatic attraction to the slightly positive charge of the fibers

1. Nonsterile room air is drawn into the front of the cabinet and mixes with contaminated air from the chamber
2. Contaminated air:
   • pushed below the work surface
   • then drawn up through the plenum
   • then pushed through the HEPA filters by the cabinet blower motor
3. Around 30% of the air passes through the exhaust HEPA filter at the top of the cabinet and recirculates into the room or is removed by the canopy exhaust
4. Around 70%  of the air passes through the supply HEPA filter, enters the cabinet from above, and flows back down on work surfaces under unidirectional flow

• Class II Type A2 cabinets are designed to function independently from the building or room exhaust (non-ducted BSC)
• Biological material passes through the exhaust HEPA filter of the cabinet and is removed
• Exhaust air can safely recirculate back into lab if handling biological material at BL1 or BL2 containment
• Volatile or toxic chemicals and volatile radionucleotides require a canopy connected cabinet
• Most work at BL2+ containment requires a canopy connected cabinet as an extra precaution; for some BL2+ research, the PI can request an exemption from the CAB/ESCRO which will be reviewed on a case-by-case basis

Class II Type A2 cabinets can be connected to the building exhaust through the addition of a canopy or thimble connection:

• This leaves a small air gap between the exhaust of the cabinet and the connection to the building exhaust which avoids the airflow reversal problems of hard ducted cabinets describe below
• A canopy can be used with minute amounts of volatile or toxic chemicals or volatile radionuclides
• The HEPA filters will not remove chemicals but these particles will be exhausted through the building exhaust
• If building exhaust fails, the canopy will allow the exhaust to flow back into the room rather than pressurizing and blowing non-HEPA filtered air back into the operators’ face
• NSF standard 49 also calls for the addition of a canopy airflow alarm which warns operators that building exhaust is no longer sufficient to remove the exhaust air from the canopy (see below)
  

NSF standard 49 requires that canopy connected Class II Type A2 biosafety cabinets have an airflow alarm. Airflow alarms monitor the airflow passing through the canopy and measures whether it is sufficient to capture the exhaust air exiting the cabinet:

• When airflow is disrupted (typically because an exhaust fan has failed or lost capacity), the alarm will alert the operator they have lost containment of exhaust and the cabinet is now recirculating back into the lab
• This does not pose a safety risk for experiments only handling biological agents since the exhaust air has already passed through the HEPA filters
• For experiments using minute amounts of volatile toxic chemicals or volatile radionuclides, this could cause an exposure risk depending on the nature and concentration of the material

Alarms can be integrated into the cabinet or installed as a separate piece of hardware.

Alarm States

The following states refer to one common style of alarm: the Rooster alarm from Degree Controls Inc.  Other styles of alarm may have slight variations.

Ready/Normal state

• Alarm functioning properly
• Building exhaust appropriate to capture canopy exhaust
• Generally indicated by a GREEN indicator light for the Rooster; the Green LED flashes every 2 seconds
• If airflow drops below threshold for more than 5 seconds, alarm will go into alarm state

Alarm State

• Indicates low building exhaust airflow
• Red light flashes quickly and an audio alarm sounds
• Audio alarm can be silenced by pressing the “Reset” or “Mute” button; the red light will continue to flash
• Alarm will automatically return to normal operation state when proper airflow is re-established (this is a default setting for the Rooster alarms but may vary with other alarms depending on alarm style and settings)

Error State

Some alarms such as the Rooster alarm have an error state represented by a yellow caution light. This state indicates the alarm has received a fault (sometimes caused by a power failure):

• Reset button will light up yellow and an audible alarm will sound
• Unplug alarm power, wait 10 seconds, restore power plug
• Alarm will restart and automatically return to normal state
• If monitor goes into alarm state again, the building exhaust is problematic and you should repeat the previous steps

Operator Response

Response action will vary depending on the type of material being used:

• For biological material only (no toxic chemical or volatile radionuclide material) –
  • Silence audible alarm by pressing “Reset” or “Mute” button
  • Finish your experiment
• With minute volatile chemical or radionuclide material, or BL2+ containment work:
  • Stop experiment and close sash
  • Silence audible alarm by pressing “Reset” or “Mute” button

In both cases:

• Alert your EHS rep and place a warning sign on the cabinet
• Inform the EHS Coordinator (may vary depending on the department, lab, or center)
• Contact MIT Facilities and request they check the building exhaust
• If building exhaust is sufficient, contact your BSC certification vendor to inspect the alarm

Per a 2016 update to NSF/ANSI standard 49, Class II Type A2 cabinets may no longer be connected directly to the building exhaust (hard or direct ducted) due to the following safety reasons:

• If the building exhaust fails, the internal motors in the biosafety cabinet will continue to operate causing a pressurization of the ductwork
• Pressurization is not sufficient to push the exhaust air through the ductwork to the roof exhaust vent
• Airflow will reverse and blow non-HEPA filtered air back out the sash and into the operators face leading to potential exposure

For these reasons, use the following guidelines:

• Recertifiers can no longer certify direct or hard ducted cabinets
• All newly installed biosafety cabinets must be non-ducted or canopy connected to comply with updated NSF 49 standards
• Existing hard ducts were modified through a campus-wide facilities project
• If you still have a hard ducted Class II Type A2 biosafety cabinet, contact your biosafety officer for guidance

Note: Other types of biosafety cabinets (Class II Type B1/B2 and certain Type C1 cabinets) are still required to be hard-ducted.  These cabinets handle larger amounts of volatile toxic chemicals or radionuclides, and this style of cabinet has interlocks that turn off the blower-motor if the building/room exhaust fan fails.  The cabinet also goes into alarm, and the users are instructed to shut the sash and contact Facilities.

• Turn on blower and light; allow cabinet to run for 2-3 minutes prior to use to purge stagnant air inside BSC
• Ensure window sash is at proper operating height (typically 8 or 10 inches according to manufacturer instructions)
• Monitor alarms, pressure gauge, or flow indicators for any major fluctuations; a piece of tissue or Kimwipe held at the sash opening is a quick test to ensure the cabinet has proper airflow (tissue should be pulled inward)
• Avoid bringing exposed skin into the chamber – gloves should be tucked underneath the cuff of your lab coat or your lab coat should be tucked beneath the cuff of your glove (depending on your preference)
• Spray appropriate disinfectant on paper towel and wipe cabinet surfaces from back to front (clean to dirty); a tool such as a swifter handle can be used for hard to reach spaces – Do NOT place your head inside the cabinet
• Wipe all materials with disinfectant (typically 70% ethanol) before placing inside the chamber to ensure a sterile environment is maintained
• Ensure the back and front grates are clear
  • Equipment near back grates should be at least 1 inch away from the grates
  • Do not place anything on the front grates (such as lab notebooks or protocols)
• Before use, check the certification sticker to ensure the cabinet has been certified within the past year; if certification has expired, do not use the cabinet and alert your lab EHS representative to schedule recertification

Bring all material into the chamber prior to beginning experiment and perform experiments at least 4-6 inches beyond the front grill to ensure best unidirectional airflow and containment.

Avoid disrupting the air curtain:

• Use slow, controlled movements
• If you must bring things into and out of the chamber, moving using an inward and outward motion
• Avoid moving your hands from side to side
• Avoid traffic while working in the cabinet – anyone walking by will disrupt the air curtain

Waste should be kept inside the cabinet and only removed at the end of the experiment – this avoids frequent disruption of the air curtain.

Work “clean” to “dirty”:

• “Clean” (sterile) media and glassware is stored on one side of the cabinet
• Manipulation is done in the center of the cabinet to prevent cross-contamination
• As material becomes “dirty” (contaminated), it is moved to the opposite side of the cabinet and collected as biohazardous waste

If necessary, use an appropriately set up vacuum aspiration line:

• Place a hydrophobic* or HEPA filter before the vacuum line; filters are directional so make sure the filter faces the overflow flask
• Add an appropriate volume of disinfectant to the primary flask to disinfect the final volume of liquid – 1:10 (v/v) dilution of household bleach; bleach should be refreshed weekly to ensure disinfecting effectiveness
• If the flasks are on the floor, place them in a secondary container such as a plastic bin to contain any spills if the flasks are knocked over
• Place a biohazard sticker on the primary collection flask or the secondary container

*Hydrophobic filters are available through VWR – item #55095-006, 28137-858, or 28137-737

Do not use an open flame in a biosafety cabinet:

• Chamber is a sterile environment and does not require a heat source for sterility
• Disposable or autoclavable loops/spreaders are available to replace flame sterilization of metal loops or metal/glass plate spreaders
• Heat from an open flame will disrupt unidirectional air currents in the chamber and can lead to cross-contamination of samples
  • Baker recently provided testing data that shows potential sample cross-contamination from using a heat source inside a BSC
  • For more information, visit Baker BSC Mythbusters: https://bakerco.com/communication/bsc-mythbusters/
• Heat can damage supply HEPA filters
• CAB policy prohibits use of open flames outside of special circumstances (which require prior CAB review and approval)
• Other heat sources in a BSC (such as ceramic incinerators) require prior CAB review and approval
• Please see the Policies section below for more details

• When you are finished, leave the BSC blower running for 2-3 minutes to purge all the chamber air
• Wipe down materials with appropriate chemical disinfectant and remove everything from cabinet
• Wipe down cabinet surfaces with appropriate chemical disinfectant working from clean to dirty areas
• Turn off cabinet, close sash, remove personal protective equipment, and wash hands

Notes about UV lights:

• UV lights not a dependable disinfection method – chemical disinfection and proper use of the cabinets are sufficient to maintain sterility
• Are no longer recommended by American Biological Safety Association (ABSA International, 2000), NSF (2004), or Centers for Disease Control (CDC, 2009)
• Have no performance verification standards for testing effectiveness of disinfection
• Bulbs have a limited shelf life
• Research found labs were not maintaining bulbs (replace every 6 months and weekly wiping to remove particles)
• Newer cabinets are no longer constructed with UV lights as a default option
• Please see UV light section for additional information for labs who choose to use UV lights

The National Institutes of Health (NIH) requires biosafety cabinets to be certified on an annual basis. Labs that conduct research in a BSC that has not been properly certified are in violation of the NIH guidelines and could have their grant funding impacted. NSF/ANSI standard 49 discusses the certification standards required for proper certification of biosafety cabinets:

• Labs are required to ensure their cabinets are certified on an annual basis
• Labs call an accredited certification company to perform the certification
• Each individual PI is responsible for ensuring their cabinets are properly certified, though some DLC’s may schedule certification for their research labs
• Annual certification generally costs around $125-200

Certification tests the following parameters:

• The HEPA filters are challenged with a particle and the penetration levels are measured to ensure filter integrity
• All the airflow patterns and flow rates are checked and adjusted to make sure they meet manufacturer parameters
• Airflow alarms on canopy connected cabinets are tested and calibrated

The below vendors commonly work on campus:

• Triumvirate Environmental (TEI). Email  mmagnan@triumvirate.com  or Call 888-834-9697.
• Technical Service Systems   Phone: 800-877-7742.
• Air Systems Technology
• AABC Testing & Certification, Contact: Dennis Miller, info@aabc-inc.com, Phone: 844-296-7198
• Life Science Facility Services | DENS Facility Services, Phone: 617-616-5421
• Health and Safety Services Unlimited:  Contact: Kevin Meehan; kmeehan.hssu@comcast.net
• Steris Life Sciences:   Phone: 610-332-3669

Surface decontaminate BSC work surfaces with an appropriate disinfectant prior to using and after each experiment.  Appropriate disinfectants may include:

• An EPA registered product such as quatricide, PREempt, Lysol professional spray, etc.
• 10% household bleach (>0.5% NaOCl final concentration) can be used on a biosafety cabinet but you must always follow with sterile water or 70% ethanol rinse step to prevent corrosion of the stainless steel work surfaces
• 70% ethanol can be used for vegetative or enveloped viral work but it has limited contact time in a BSC due to the high air flow rates (it evaporates quickly)

Items kept in BSC can be source of contamination and should be surface decontaminated between experiments.

Learn more about Decontamination and Disinfection

Every 6-12 months clean the catch basin beneath the work surface:

• Keep BSC running and sash at working level (8-10 inches) to maintain containment
• Do not put your head inside the chamber
• Decontaminate work surface and sides prior to lifting
• Always surface decontaminate the bottom of work surface before removing from the cabinet

Important notes:

• This is generally a two-person job
• Newer cabinets may have tabs to hold work surface elevated during cleaning
• Older cabinets may have obstructions you will need to maneuver around
• Worksurface may need to be removed from the cabinet to access catch basin

Certification companies can also often perform repairs on a BSC. Recertification is required after any repairs.

HEPA Filters

• Can last for 5-10 years or longer depending on usage and lab conditions (i.e. humidity and cleanliness of the lab air)
• Replacement filters require full gaseous decontamination of the BSC before filters can be replaced and the old filters removed
• Most cabinets have 2 filters – a supply and an exhaust (some cabinet models may have a third filter)
• This is generally a 2-day process

Blower Motor

• Blower motors can last for a decade or more
• As they age, they lose capacity and must be replaced
• The cabinet must be gaseously decontaminated before the motor can be replaced
• This is generally a 2-day process

Sensors, Sash, and Controls

• These parts can wear out over time
• They may require gaseous decontamination depending on where the part is located and whether it could have come in contact with contaminated air

Gaseous decontamination is required before the replacement of HEPA filters, repair of blower motors, or disposal of a BSC. There are multiple methods:

Vaporous hydrogen peroxide (VHP) is the preferred method:

• Shorter decontamination time
• Generally an overnight process
• Lower chemical safety risk than formaldehyde gas
• The cabinet is sealed during decontamination, but the lab is generally inaccessible for 8 hours
• Generally costs around $500

Formaldehyde Gas:

• Longer decontamination time (generally 24 hours)
• Higher chemical safety risk due
• Gas must be scrubbed after decontamination
• Cabinet sealed but the lab is inaccessible during decontamination
• Generally costs around $500

UV Lights are only effective for surface decontamination of areas exposed to the UV light. Areas in the shadows of equipment or beneath paper/plastic will not be decontaminated.

UV bulbs have a limited shelf life before they lose effectiveness:

• Average 6-8 month shelf life
• Light will shine blue even after expired
• Only 85% efficiency after 6000 hours of use

Particles can build up on the surface of the bulb:

• Reduces efficiency
• Requires weekly surface decontamination

There are no NSF/ANSI standards for testing and they are not tested during annual certification of the BSC.

UV lights are no longer recommended by:

• American Biological Safety Association (ABSA International, 2000)
• National Sanitation Foundation (NSF International 2004)
• Centers for Disease Control and Prevention (CDC , 2009)

Research showed labs were not replacing and maintaining bulbs regularly which generated a false sense of security regarding sterility.

Newer cabinets are not constructed with UV lights and UV lights must be added as a custom feature (required additional cost).

UV light use can lead to exposure and harm upon skin or eye contact.

• Always use chemical disinfectant before and after BSC use; UV sterilization cannot be used as the primary method of disinfection
• Do not use UV lights while research is being performed in the cabinet; newer cabinets have interlocks that prevent the UV light from activating when the sash is open, but older cabinets may not have this safety feature
• Minimize equipment stored in the BSC to prevent unnecessary exposure; UV light will degrade plastic over time (such as pipettes, waste containers, and vacuum line tubing)
• Use appropriate exposure time:
  • Most agents are inactivated after 10-15 minutes
  • Maximum sterilization time should be limited to 30 minutes – after 30 minutes there is no additional benefit
  • Turn UV light off after sterilization time to conserve bulb life and energy (sustainability)

For additional information, please refer to the following articles:

• American Biological Safety Association (2000). Position Paper on the Use of Ultraviolet Lights in Biological Safety Cabinets
• Burgerner, J. (2006). Position Paper on the Use of Ultraviolet Lights in Biosafety Cabinets. Applied Biosafety 11 (4): 228–230
• Meechan P, Wilson C (2006). Use of Ultraviolet Lights in Biological Safety Cabinets: A Contrarian View. Applied Biosafety 11 (4): 222–227
• Centers for Disease Control and Prevention; The National Institutes of Health. Biosafety in microbiological and biomedical laboratories. 5th ed. Washington, DC. 2009
• NSF International (NSF); American National Standards Institute (ANSI). NSF/ANSI Standard 49-2007. Class II (laminar flow) biosafety cabinetry. Ann Arbor (MI); 2004

Keep cabinet running to contain aerosols and follow the normal biological spill cleanup protocol:

• Keep cabinet running
• Assess situation and ensure you are wearing appropriate PPE
• Gather your biological spill kit and appropriate disinfectant
• Cover spill with paper towels
• Saturate paper towels with disinfectant
• Allow 10 minute contact time
• Pick up paper towels and debris with tongs and dispose as biowaste or biosharps (for any broken glass)
• Surface disinfect to remove any residual contamination and wait 5-10 minutes or until air dry
• Rinse with 70% ethanol or sterile water to remove residual disinfectant (this is required if you use 10% bleach)
• Dispose of all paper towels as biowaste, remove gloves, and wash hands

• Keep cabinet running
• Assess situation and ensure you are wearing appropriate PPE
• Gather your biological spill kit and appropriate disinfectant
• Ensure drain valve is closed
• Pour disinfectant onto surface and through grills
• Allow at least 10 minute contact time
• Use paper towels to soak up residual disinfectant from work surface
• Pick up paper towels and debris with tongs and dispose as biowaste or biosharps (for any broken glass)
• Connect flexible tubing to drain valve
• Drain basin into disinfectant filled container
• Lift work surface
• Decontaminate catch basin (see maintenance section above for details) with appropriate disinfectant and wait 5-10 minutes
• Rinse with 70% ethanol or sterile water to remove residual disinfectant (this is required if you use 10% bleach)
• Dispose of all paper towels as biowaste, remove gloves, and wash hands

Chemical Fume Hoods are used to protect personnel from toxic chemical fumes.  Hazardous chemicals are handled inside the fume hood chamber.  Single pass air draws toxic fumes through building exhaust fans and leaves an air stack at top of the building. Fume hoods can be either constant flow (less energy efficient) or variable flow (more energy efficient as air flow is adjusted depending on whether the hood is in use).

Key Features:

• Lacks internal blower motor so is completely reliant on building exhaust fan to provide airflow
• For use with toxic chemicals
• Provides personnel protection only
• No supply HEPA filters so workspace is a non-sterile environment (no product protection)
• No exhaust HEPA filters (so no environmental protection from biological agents; chemical concentration reduced to acceptable levels through dilution with environmental air)

Clean bench or laminar flow hoods are for use with nonhazardous sterile work (such as PCR or media preparation). They should not be used with hazardous materials (including biological material, hazardous chemicals, or radionuclides).

Key Features:

• For use with nonhazardous sterile work (such as PCR or media preparation)
• Supply HEPA filter provides product protection
• No exhaust HEPA filter (so no personnel or environmental protection)

Some laminar flow hoods can be easily mistaken for a biosafety cabinet at a glance. If the instrument blows air into your face, it is not a biosafety cabinet and provides no containment.

Animal transfer or cage changing stations are generally used to reduce allergens when working with animals. They must not be used with animals containing hazardous material including biological material, hazardous chemicals, or radionuclides.

Key Features:

• Supply HEPA filters provide some product protection
• Exhaust HEPA filter provides some room protection
• Does not provide full personnel or environment protection since the sash is open

This is the original style of biosafety cabinet.

Key Features:

• Only provides personnel and environmental protection
• No supply HEPA filter (no product protection); not a sterile chamber
• Largely replaced by Class II biosafety cabinets
• Only used in specialized circumstances where product protection is not needed

This is an older style of biosafety cabinet.

Key Features:

• Similar to Class II Type A2 except that the plenum is under positive pressure due to the motor placement
• Does provide personnel, product, and environmental protection
• Requires plenum seal to be leak tested during annual recertification
• Increased safety risk since a plenum seal leak could lead to non-HEPA filtered and contaminated air escaping into the lab
• Largely replaced by Class II Type A2 cabinets

This BSC is used for biological work with higher concentrations of hazardous chemicals.

Key Features:

• “Partial Exhaust” cabinet in that work done in front part of chamber is recirculated and work done in rear of chamber is completely exhausted
• Hazardous chemical work done in rear of chamber for complete exhaust
• Blower motor interlocked with building exhaust fan
  • If building exhaust fails, blower motor turns off to avoid pressurization
  • Protects operator from exposure to hazardous biological and chemical material
• Airflow patterns are complicated and this BSC requires special installation and recertification
• 3^rd HEPA filters can require more expensive maintenance
• More energy efficient than a Class II Type B2 BSC, but more energy intensive than a Class II Type A2 with a canopy connection
• Only required for specialized situations where higher concentrations of volatile toxic chemicals must be used with biological material under sterile conditions
• Contact your biosafety officer for a risk assessment before purchasing a Class II Type B1 BSC

The BSCs are used for biological work involving higher concentrations of hazardous chemicals.

Key Features:

• Similar to a chemical fume hood with HEPA filters
• “Total Exhaust” cabinet since no air is recirculated (single-pass air)
• Blower motor interlocked with building exhaust fan
  • If building exhaust fails, blower motor turns off to avoid pressurization
  • Protects operator from exposure to hazardous biological and chemical material
• Single pass air is very energy inefficient
• Airflow patterns are complicated and this BSC requires special installation and recertification
• Only required for specialized situations where higher concentrations of volatile toxic chemicals must be used with biological material under sterile conditions
• Contact your biosafety officer for a risk assessment before purchasing a Class II Type B2 BSC

These are a new style of cabinet that can function either as a Class II Type A2 cabinet or a Class II Type B cabinet. Currently only one vendor manufacturers this cabinet. Please visit the Labconco website for more details.

Key Features:

• Allows flexibility since the connection type can be modified to meet changing research needs
• More energy efficient than Class II Type B cabinets
• Has improved safety features for handling hazardous chemicals

Modes of Operation:

• Operate non-ducted for only biological material
• Can be canopy connected for work with biological material and small amounts of chemicals (requires MIT Facilities work to convert into or out of this mode)
• Can be hard ducted for work with biological material and higher concentrations of chemicals (requires MIT Facilities work to convert into or out of this mode)

These BSCs have a gas-tight containment chamber with gas-tight sealed sash.

Key Features:

• User has no direct contact with the samples
• Samples enter chamber through a bypass chamber
• User interacts with samples through thick gloves built into the chamber
• Differs from a chemical glove box used to handle chemicals under inert gas conditions in that Class III BSC’s are under negative pressure (air would flow into the chamber from the room upon a leak) whereas chemical glove boxes are generally under positive pressure (gas would flow out of the chamber into the room upon a leak)