Proper Use of a Biosafety Cabinet
Biosafety cabinets are the primary means of containment for working safely with infectious microorganisms. The commonly used type II biosafety cabinets also provide product protection to prevent contamination of cultures. However, adequate training and proper technique are required to ensure the user obtains optimum performance from the biosafety cabinet.
|Air Flow Within a Class II Biosafety Cabinet|
A. Front Opening
C. Exhaust HEPA filter
D. Supply HEPA filter
E. Rear plenum
Proper Use of a Biosafety Cabinet
Materials or equipment placed inside the cabinet may cause disruption to the airflow, resulting in turbulence, possible cross-contamination, and/or breach of containment. Extra supplies (e.g., additional gloves, culture plates or flasks, culture media) should be stored outside the cabinet. Only the materials and equipment required for the immediate work should be placed in the biosafety cabinet.
Cabinet blowers should be operated at least five minutes before beginning work to allow the cabinet to "purge" by removing any particulates in the cabinet. The work surface, the interior walls, and the interior surface of the sash should be wiped with 70% alcohol (EtOH) or other disinfectant, as determined by the investigator, to meet the requirements of the particular activity. Please be aware that bleach is corrosive and any bleach used to decontaminate the surfaces of the biosafety cabinet must be removed by rinsing with water or 70% ethanol to protect the metal surfaces after allowing suficient contact time for surface decontamination.
Personnel should don clean disposable gloves and lab coats when working in the biosafety cabinet. The surfaces of all materials and containers placed into the cabinet should be wiped with 70% alcohol to reduce the introduction of contaminants to the cabinet environment. Users' arms should enter the biosafety cabinet directly and avoid sweeping motions to prevent disruption of the airflow within the cabinet.
Personnel should plan the manipulations they will perform within the biosafety cabinet in advance in order to avoid accidental contamination of cultures. One such approach involves the segregation of sterile cultures from decontaminating and contaminated items. A typical layout for working “from clean to dirty” within a Class II BSC is illustrated below. Clean cultures (left) can be inoculated (center); contaminated pipettes can be discarded in the shallow pan and immersed in 10% bleach to decontaminate while other contaminated materials can be placed in the biohazard bag (right). This arrangement is reversed for left-handed persons.
All containers and equipment should be surface decontaminated and removed from the cabinet when work is completed. At the end of the workday, the final surface decontamination of the cabinet should include a wipe-down of the work surface, the cabinet’s sides and back, and the interior of the glass. Investigators should remove their gloves and gowns and wash their hands as the final step in safe microbiological practices.
Biohazardous waste generated within the biosafety cabinet must be properly contained, treated and disposed. Solid waste (culture dishes, pipets, contaminated gloves, etc.) must be placed in an infectious waste bag bearing the biohazard symbol and autoclaved prior to disposal in one of the infectious waste disposal sites on campus. Liquid waste must be treated with bleach to a final concentration of 10% bleach by volume and allowed 30 minutes of contact time prior to disposal in a lab sink with copious amount of running water. All biohazardous waste must be adequately decontaminated or autoclaved prior to disposal.
Liquid waste is commonly aspirated into vaccuum flasks using a system similar to the one depicted below. The left suction flask (A) is used to collect the contaminated fluids into a suitable decontamination solution; the right flask serves as a fluid overflow collection vessel. A glass splarger in flask B minimizes splatter. An in-line HEPA filter (C) is used to protect the vacuum system (D) from aerosolized microorganisms. Aspirator bottles can be filled with bleach to the first 10% of their volume to facilitate decontamination of collected liquid waste. The in-line HEPA filter in the vacuum tubing provides protection to the central building vacuum system or vacuum pump, as well as to the personnel who service this equipment. HEPA filters should be replaced if wetted or noticeably blocked.
The following link provide examples of recommended supplies for cell and viral culture with ordering information (e.g. in-line HEPA filters, pipet sterilization trays and pipet keepers).
Protection against exposure from fumes generated by radionuclides and toxic chemicals is only provided by Class II, Type B biosafety cabinets, which are ducted directly to the facility exhaust. Other types of biosafety cabinets exhaust air to the laboratory and must not be utilized in conjunction with hazardous volatile materials.
Ultra Violet Lights
According to the CDC: “Ultraviolet (UV) lamps are not recommended in biological safety cabinets nor are they necessary. If installed, UV lamps must be cleaned weekly to remove any dust and dirt that may block the germicidal effectiveness of the ultraviolet light. The lamps should be checked weekly with a UV meter to ensure that the appropriate intensity of UV light is being emitted. UV lamps must be turned off when the room is occupied to protect eyes and skin from UV exposure, which can burn the cornea and cause skin cancer. If the cabinet has a sliding sash, close the sash when operating the UV lamp." Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets
For additional information, please read the following articles:
- Position Paper on the Use of Ultra Violet Lights in Biological Safety Cabinets. Applied Biosafety 11(4): 228-230.
- Use of Ultraviolet Lights in Biological Safety Cabinets: A Contrarian View. Applied Biosafety 11(4): 222-227.
Common Errors to Avoid
1. Do not block the perforated grills at the front and rear of the biosafety cabinet. The biosafety cabinet's air intake is dirupted by blocking the grills. Blocking the grills at the opening of the biosafety cabinet may also allow contaminated room air from entering the cabinet without filtration.
2. Do not store unecessary equipment or supplies in the cabinet. The more cluttered the cabinet, the greater the disruption to it's air flow.
3. Do not use the top of the cabinet for storage. The HEPA filter could be damaged and the airflow disrupted.
4. Never disengage the alarm, as it indicates improper airflow, thereby effecting performance and endangering the researcher or the experiment.
5. Never completely close the window sash with the motor running as it may cause the motor to burn out.
6. U.V. lights can accumulate dust and dirt that will block the germicidal effectiveness of the light. Clean U.V. lamps regularly to remove such build-up.
|Air Flow Within a Class II Biosafety Cabinet||Blocked Grates Interfere with Air Flow.|
Avoid Fires within Biosafety Cabinets
- Fans in biosafety cabinets are not spark proof. Avoid the use of highly volatile flammable chemicals as a fire or explosion may occur.
- Avoid the use of Bunsen burners or other flames which can affect airflow and damage HEPA filters.
Open flames are not required in the near microbe-free environment of a biosafety 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 biosafety cabinet, however, creates turbulence that disrupts the pattern of air supplied to the work surface. When deemed absolutely necessary, touch-plate microburners 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. While an open flame is being used in the cabinet, any alcohol or flammable liquid in its vicinity could cause a fire, so additional care must be taken. Small electric furnaces are available for decontaminating bacteriological loops and needles and are preferable to an open flame inside the BSC. Use of pretreated sterile loops is also a good option.
The Stanford University Open Flames Memo justifies the university's efforts to prohibit open flames in biosafety cabinets.