Biological Safety Cabinets (BSCs)

Class II, Type A1

• Internal fan draws room air through the front grille to maintain average inflow velocity of at least 75 lfm at the face opening of the cabinet.
• Supply air flows through a HEPA filter and provides particulate-free air to the work surface.
• Downward air splits as it approaches the work surface and the internal fan draws air to the front grille and the rear grille.
• Approximately 30% of air passes through the exhaust HEPA filter and 70% is recirculated through the supply HEPA back onto the work surface of the cabinet.

Class II, Type A2

• Differs from Class II, Type A1 in the inflow velocity; Class II, Type A2 cabinets have an inflow velocity of 100 lfm.
• Small quantities of volatile toxic chemicals may be used in a Class II, Type A2 cabinet ONLY if it exhausts to the outside via a canopy or thimble connection with an exhaust alarm.

Source: CDC Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition

Class II, Type B1

The Class II, Type B1 cabinet is suitable for work with biohazardous materials that also requires the use of small quantities of toxic volatile chemicals, including organic solvents or carcinogens.

• Supply blower draws room air and a portion of the cabinets recirculated air through the front grille and through the supply HEPA filter.
• Particulate-free air flows upward through a plenum on each side then downward to the work area.
• Room air is drawn through the face opening at an inflow velocity of 100 lfm.
• As with Type A cabinets, the downward airflow splits just above the work surface.
• Approximately 70% of the downflow air exits through the rear grille, passes through the exhaust HEPA filter, and is discharged from the building.
  • Because the air flowing to the rear grille is discharged into the building exhaust, work with toxic volatile chemical vapors or gases should be conducted towards the rear of the cabinet.
• The remaining 30% of downflow air is drawn through the front grille, passes through the supply HEPA filter, and is recirculated.

Source: CDC Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition

Class II, Type B2

The Class II, Type B2 cabinet provides both biological and (small quantity) chemical containment. 

• Supply blower draws room or outside air in at the top of the cabinet, passes it through a supply HEPA filter and down over the work surface.
• The building exhaust system pulls air through the front and rear grilles. This captures the supply air plus room air needed to create an inflow velocity of 100 lfm.
• All air entering this cabinet is passed through a exhaust HEPA filter and exhausted via the building exhaust system.
• A Class II, Type B2 cabinet will exhaust as much as 1,200 cubic feet per minute of conditioned room air.
• Expensive to operate and maintain.

Source: CDC Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition

• Biosafety cabinets will be certified at least annually by NSF-certified technicians and according to NSF 49 standards. 
• All BSCs and LFBs (laminar flow benches) at the University of Kentucky shall be placed in the University of Kentucky Department of Biological Safety inventory database. 
• Any BSCs or LFBs not certified will be reported to the Department Chair, Executive Vice-President of Research, and Provost as a serious lab violation. 
• It is the responsibility of the Principal Investigator (PI) to participate in the required annual certification process. 
• Prior to certification the user will be responsible for decontaminating the interior surfaces of the cabinet using an approved method and disinfectant specific to the agent. 
• Copies of annual certification reports will be maintained by the Principal Investigator or Laboratory Manager.

As of 10/01/2022, the following vendors are authorized to certify, repair, or gas decontaminate BSCs or LFBs on UK campus:

SafetyPlus, LLC. (877) 821-9822

Precision Air Technology (919) 812-0340

All users should be trained prior to utilizing a BSC. 

Online BSC training is available in the SciShield Course Directory - https://uky.scishield.com/raft/training/courses 

In-person BSC training and demonstration is available upon request. Email biosafety@uky.edu for more information.

• Conduct all procedures involving the manipulation of potentially infectious materials, including aerosol-producing activities, when using aerosol-producing equipment, or when using high concentrations/large volumes of organisms, inside a biological safety cabinet.
• Storage of excessive materials or equipment inside a BSC can disrupt airflow, resulting in turbulence, cross-contamination, or breach of containment. Therefore, only materials and equipment necessary for immediate work will be placed in the BSC. Additionally, storing items on top of the BSC can interrupt exhaust airflow and can result in damage to the BSC. Place large items close to the sidewalls, rather than at the back of the cabinet where they will interfere with airflow. Blocking the intake grills in the front and rear of the cabinet will interfere with proper functioning of the cabinet, which can cause a loss of containment of infectious organisms.
• Prior to each use examine the cabinet to ensure it is clean and in good repair. 
  • Verify that the cabinet blower is on and functioning properly based upon observance of Magnehelic gauges and/or digital readouts. 
  • Ensure that UV lights are disengaged prior to commencing work in the BSC.
• Wipe interior of BSC (work surface, walls & interior surface of window) and items placed in the cabinet with an efficacious and approved disinfectant. 
• Ensure the cabinet drain valve is closed prior to starting work, to contain spills.
• Place items necessary for the procedure (including a receptacle for waste) in BSC prior to initiating work. This will minimize the number of arm-movement disruptions to the air barrier, which can compromise the containment of the BSC. 
• Arrange the materials within the cabinet in a manner to ensure good aseptic technique; e.g., all workflows from one side of the cabinet (clean) to the other side (dirty). Contaminated items never cross over uncontaminated/clean supplies. 

Clean-to-Dirty

A typical layout for working from the clean to the dirty side within a Class II BSC. Clean cultures (left) can be inoculated (center); contaminated pipettes can be discarded and other contaminated materials placed in the biohazard bag (right). Reverse this arrangement for left-handed persons.

Source: CDC Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition

• Verify that the sash is set at the appropriate height. The operating height of a BSC is not variable; so follow the manufacturer’s recommendations for the BSC that you are using. 
• Cabinets are designed for a single operator. More than one person working in the BSC at one time (even in a six-foot BSC) produces enough disturbances in the airflow to breach the containment of the BSC. Never lean into a BSC or place head into a BSC.  

BSC Surface Decontamination

The BSC must be emptied and decontaminated at the end of each procedure and/or workday. Every individual is responsible for cleaning the BSC when they have finished working.

• Remove all items from the BSC. The exterior of all potentially contaminated material must be surface decontaminated with an appropriate disinfectant prior to being removed from the unit.
• Place all biohazardous waste in an autoclave bag in the BSC and seal prior to removing.
• Wipe the interior surface of the unit (work surface, sides and back, interior of the glass) with an appropriate disinfectant after all items have been removed from the unit. Ensure adequate contact time. 
• DO NOT rely on UV light as a means of decontamination. 

Avoid the following common mistakes:

• Air currents and drafts can disrupt BSC airflows. Locate BSCs away from doors, vents/diffusers and traffic paths. 
• Do not store equipment or supplies inside the cabinet. 
• Make sure that items necessary for procedures are inside the BSC prior to starting work in the BSC. 
• To prevent disruption of airflow and damage to the HEPA filter, do not store items on the top of the cabinet. 
• Prevent damage to cabinet by keeping all objects (i.e. paper towels, Kim Wipes, work surface diapers, etc.) from being pulled into the back, front, and side grills or slots.
• Never disengage the alarm. The alarm indicates when the cabinet has improper airflow or reduced performance, which may endanger the researcher and/or the experiment. 
• Avoid rapid motions at the front of the unit and minimize movement of arms in and out of cabinet which may disrupt the air curtain. 

Vacuum Line Trap System

Vacuum line trap systems are utilized across campus research laboratories to collect liquid biohazardous waste and prevent suction of infectious and non-infectious materials into vacuum lines (house vacuum line or vacuum pump). Setting up your vacuum line trap system correctly will allow for safe collection and decontamination of liquid biohazardous waste.

Source: CDC Biosafety in Microbiological and Biomedical Laboratories, 6th Edition

The diagram above details the correct setup of two vacuum flasks and an in-line hydrophobic/HEPA filter in series connected by vacuum lines to a port for house vacuum. Alternatively, a vacuum pump may be used if house vacuum is not available.

Liquid waste material is drawn into the catch flask A which is pre-filled with an appropriate disinfectant*. Flask A is connected to the overflow flask B. Note that flask B is empty. Flask B provides overflow protection for flask A. Care must be taken to prevent overflow of flask A. An in-line hydrophobic/HEPA filter C is located between overflow flask B and the vacuum port D. The only item missing from this diagram is a spill tray. Both flasks should be seated in a spill tray to contain any spill should the flasks be knocked over. The spill tray must be non-porous and autoclavable.

*Household bleach is commonly used for disinfection of liquid biohazardous waste. When utilizing household bleach for disinfection of liquid biohazardous waste, the catch flask A should be pre-filled with household bleach such that when the flask is 2/3 full, the final concentration is 10% bleach.  Ensure appropriate contact time (20 minutes for 10% bleach). Once the appropriate concentration and contact time have been met, test the pH of the liquid using a pH meter or pH test strip (available upon request from Research Safety) to ensure it is within acceptable limits for drain disposal (5.5-11.5 SU). Once the mixture has met all requirements (10% bleach, 20 minutes, pH 5.5-11.5), it may be poured carefully and slowly down the laboratory sink drain with copious amounts of water.

• Routine maintenance of BSCs will be performed as recommended by the cabinet manufacturer. 
• All maintenance and repairs will be performed by UK approved BSC certification and maintenance contractors. 
• The PI is responsible for any service or repairs that are needed. 
• A risk assessment will be completed prior to maintenance or repairs to determine the extent of decontamination required prior to commencement of work. The Biological Safety Officer will determine the type of decontamination necessary. Typically surface decontamination is adequate, however, there are instances such as extensive repair inside the cabinet envelope or HEPA filter replacement that will require gas decontamination.  
• Recertification of the BSC may be required based upon the type of maintenance or repair work that was completed. The Biosafety Officer will determine the need for recertification. 
• Copies of maintenance or repair reports will be maintained by the Principal Investigator or Laboratory Manager.

Only approved vendors may be contacted to certify, repair or decontaminate biological safety cabinets or laminar flow benches on University of Kentucky campus. 

As of 10/1/2022 the following vendors are authorized to provide service: 

SafetyPlus, LLC. 877-821-9822 

Precision Air Technology 919-812-0340

• Notify UK Biosafety (biosafety@uky.edu or 859.257.1073) at least one-month in advance when you plan to have BSCs or LFBs moved, placed in storage, transferred to a new owner, or surplused. 
• PI is responsible for ensuring proper decontamination of the BSC or LFB.
• AT A MINIMUM, all BSCs or LFBs that will be moved, placed in storage, or transferred to a new owner must undergo a thorough surface decontamination with an appropriate disinfectant. Depending on the biohazardous materials used in the BSC and other factors, gas decontamination may be necessary. UK Biosafety will determine the need for gas decontamination on a case-by-case basis.*

*Gas decontamination should be scheduled as early as possible to ensure vendor will be available prior to move date. Only UK approved vendors are allowed to certify, repair, or decontaminate BSCs or other laminar flow benches on UK campus. 

• All BSCs or LFBs must be tagged by UK Biosafety prior to move. Email biosafety@uky.edu with questions.

Biological safety cabinets (BSCs) are designed to protect personnel, their products, and their environment. Most BSCs at the University of Kentucky are recirculating. The use of natural gas or other flammable gases within a BSC presents several potential safety hazards: 

• Use of natural gas, or other flammable gases, presents a potential fire and/or explosion hazard. Most BSCs at the University of Kentucky are recirculating cabinets, which may allow for flammable gases to quickly accumulate. If a gas leak occurs (i.e. a valve is left on or a tube leaks) inside a recirculating BSC, over time the gas would become more and more concentrated and could reach explosive levels. Since the gas is within a BSC, the user may not be able to detect the leak and, upon ignition, could explode.
• The high efficiency particular air (HEPA) filters—responsible for providing the sterile environment in the cabinet—can act as a mass of combustible material during an uncontrolled fire inside the cabinet. The heat generated by a Bunsen burner can also damage the HEPA filter and/or the filter’s adhesive. This could result in leaks in the filter, adverse flow patterns in the cabinet, and ultimately potential user exposure. 
• The heat generated by an open flame compromises the carefully controlled airflow pattern responsible for providing containment of potentially biohazardous materials. Normal airflow in the cabinet is direction from the top down across the working surface. The addition of a Bunsen burner will produce turbulent airflow due to the heated air rising countercurrent to the normal downward flow. This may result in spread of contamination within the cabinet and potential user exposure.

OPEN FLAMES ARE STRICTLY PROHIBITED IN BSCs!

If you must use open flames within a BSC, first contact UK Biosafety (biosafety@uky.edu or 859.257.1073). 

Always use the smallest quantity of flammable liquid possible. Place flammable liquids in a metal or glass container, and proceed with extreme caution.

If using an alternative flame/heat source, small bottled gas or gas cylinders should be used as the fuel supply for the burner to limit the supply of fuel. Plumbed gas provides an inexhaustible source of fuel.

Plumbed gas may be used if the following design criteria and operator procedures are implemented:

• The plumbed natural gas line must have a shutoff valve outside the cabinet. An automatic burner with a foot switch or hand switch to operate the flame must be used. 
• Excess flow check valves or flow limit valves designed to shut off gas flow if a pre-set limit is exceeded should be installed. These devices could prevent the flow of flammable or toxic gases into an area when other conditions have resulted in failure of point-of-use control systems. Use of these valves must be considered early in the design of the piping system.  
• Regardless of gas source, users must be trained to visually inspect the gas petcock or valve and check for the odor of gas before turning on the BSC blowers. The burner should only be used in the rear of the cabinet to minimize the effects of air turbulence. At the end of burner operation, the user must turn the gas petcock or valve off and check for the smell of gas. 
• The BSC should be ducted to an exhaust system with an explosion-proof roof exhaust fan. 
• Butyl rubber hose should be used to connect the burner to the fuel supply. Use of yellow natural rubber or latex tubing is specifically prohibited.

The following are NOT PERMITTED within a BSC:

• Bunsen burners
• Alcohol burners

Open flames are neither required nor recommended in the near microbe-free environment of a biological safety cabinet. On an open bench, flaming the neck of a culture vessel will create an upward air current that prevents microorganisms from falling into the tube or flask. An open flame in a BSC, however, creates turbulence that disrupts the pattern of HEPA-filtered air being supplied to the work surface. When deemed absolutely necessary and approved by the appropriate facility authorities after a thorough risk assessment, touch-plate micro burners equipped with a pilot light to provide a flame on demand may be used. Internal cabinet air disturbance and heat buildup will be minimized. The burner must be turned off when work is completed. Small electric furnaces are available for decontaminating bacteriological loops and needles and are preferable to an open flame inside the BSC. Disposable loops should be used whenever possible.

Appendix A - Primary Containment for Biohazards, CDC Biosafety in Microbiological and Biomedical Laboratories, 6th Edition

Ultraviolet (UV) lamps should not be used as the sole disinfection method in a BSC. If installed, UV lamps should be cleaned regularly to remove any film that may block the output of the lamp. The lamps should be evaluated regularly and checked with a UV meter to ensure that the appropriate intensity of UV light is being emitted. Replace the bulb when the fluence rate is below 40 uW/cm2 . Unshielded UV lamps must be turned off when the room is occupied to protect eyes and skin from UV exposure. If the cabinet has a sliding sash, close the sash when operating the UV lamp. Most new BSCs use sliding sashes that are interlocked when operating the UV lamp to prevent exposure.

CDC Biosafety in Microbiological and Biomedical Laboratories, 6th Edition

Aerosol-producing Activities: May include, without limitation, opening containers with non-ambient pressures; intranasal inoculation of animals; harvesting infected tissues/fluids, or embryonate eggs; transfer operations; necropsy of infected animals; changing animal cage bedding and operating aerosol-producing equipment. 

Aerosol-producing Equipment: May include, without limitation, vortexers, blenders, sonicators, centrifuges, grinders, vigorous shakers, and mixers. However, equipment (e.g. sealed rotors, buckets or centrifuge safety cups) that is designed to contain aerosols does not have to be operated in a BSC, provided that the containers (e.g. rotors or centrifuge safety cups) are opened only in a BSC. 

Biological Safety Cabinet (BSC): Primary containment device which utilizes HEPA filtered directional airflows to contain potentially infectious materials during experimental procedures. The BSC provides protection for the surrounding environment, research personnel and research materials being manipulated. 

Laminar Flow Benches (LFBs): Also referred to as clean benches. Provide an aseptic environment for experimental work by passing HEPA filtered air across the work surface. The Department of Biological Safety actively discourages the purchase and use of LFBs since air is blown across the work surface into the face and torso of the operator. The Institutional Biosafety Committee and the Department of Biological Safety recognize that clean benches do not provide personnel or environmental protection from infectious or potentially infectious agents, allergens, chemicals or radioactive materials. If you are using a clean bench, contact the Department of Biological Safety for a review of your procedures. 

National Sanitation Foundation (NSF): Regulatory agency responsible for development of standards associated with the certification and testing of BSCs. 

Primary Containment: The engineering control which prevents the release of potentially infectious material into the laboratory or outside environment. The use of a primary containment device, such as a BSC, prevents contamination of the room.

NSF/ANSI Standard 49 Class II (laminar flow) biosafety cabinetry - https://d2evkimvhatqav.cloudfront.net/documents/bc_biosafety_cabinetry_testing.pdf 

CDC Biosafety in Microbiological and Biomedical Laboratories, 6th Edition - https://www.cdc.gov/labs/pdf/SF__19_308133-A_BMBL6_00-BOOK-WEB-final-3.pdf