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Health & Safety Manual - Biological Safety

2.3 Engineering Controls

2.3.1 Biological Safety Cabinets (BSCs)
2.3.1.1 Class I BSC
2.3.1.2 Class II, type A1 BSC
2.3.1.3 Class III BSC
2.3.1.4 Clean benches
2.3.1.5 Procedures for Effective Use of BSCs
2.3.2 Vacuum Line HEPA Filters
2.3.3 Sharps Containers and Safe Needle Devices
2.3.3.1 Sharps Containers
2.3.3.2 Safe Needle Devices
2.3.4 Centrifuge Safety

Engineering controls are devices and equipment that isolate and contain a hazard. The best engineering controls function with a minimum of user input and may, to a degree, compensate for human error.

2.3.1 Biological Safety Cabinets (BSCs)
Biosafety cabinets are the primary engineering control for the minimization of exposure to potentially infectious materials. BSCs combine directional air flow and high efficiency particulate air (HEPA) filters to protect researchers and the environment from aerosolized microorganisms. Air enters the cabinet through the face (where the investigator sits), preventing contaminants generated at the work surface from entering the laboratory. Air discharged from the cabinet first passes through a HEPA filter, removing 99.97% of particles with an aerodynamic diameter of 0.3 microns; smaller or larger particles are removed with greater efficiency. Most BSCs also protect materials used within them from contamination. All open manipulation of organisms requiring BSL-3 containment and activities with a BSL-2 organism having potential for splashes or aerosol generation, must be performed in a BSC or similar type of containment device.

2.3.1.1 Class I BSC - Room air enters at face (A), circulates within the work space, and exits through the HEPA filter (C) after passing through rear plenum (D). Class I cabinets closely resemble the earliest manufactured BSCs; they are infrequently used for modern research activities, because while protecting the investigator and the immediate environment, they do not protect research materials from environmental contamination.

2.3.1.2 Class II, type A1 BSC - Room air (A) is drawn through the supply grille at the front of the work surface and passed through fan (F), entering the rear plenum, (D). Portions of airstream pass through exhaust filter (C) or supply filter (E). Only HEPA-filtered air contacts the work area, providing protection from environmental contamination of research materials. Class II, type A1 BSCs are the most common type of BSC used in Columbia research facilities

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BSC

Class I BSC

Class II, type A1 BSC

Please note, Class I and Class II, cabinets exhaust filtered air back into the laboratory. Because HEPA filters do not capture gases or vapors, volatile, toxic chemicals must not be used in these BSCs. Limited quantities of these materials may be used in Class II, type B cabinets, which discharge into building exhaust systems.

2.3.1.3 Class III BSC – Often referred to as 'glove boxes,' these are totally enclosed, gas-tight cabinets designed for work with the highest risk pathogens. Exhaust from Class III cabinets is filtered before being discharged through dedicated ventilation systems.

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2.3.1.4 Clean benches - Some laboratories have Clean Air Benches, devices that may be confused with BSCs because of their physical similarities. Clean Air Benches draw air through a filter and direct a filtered airstream, (and any contaminants, if present) from the inside of the work space into the laboratory-a pattern just the opposite of a BSC. They are designed for handling sterile materials or when a dust-free environment is needed. They are not safety devices and should never be used for handling infectious materials.

2.3.1.5 Procedures for Effective Use of BSCs 
Appropriate user protection and contamination prevention provided by a BSC is directly related to the activities of the operator.  Below are the steps to ensure that the BSC functions effectively.

  • Cabinets must be certified under the following conditions:
    • Annually
    • Following relocation (including within-room). BSC on castors may be moved carefully without subsequent recertification.
    • Following HEPA filter change
    • Following service that may have affected containment ability. 
    • Semi-annual certification is recommended when cabinets are used for work with airborne-transmitted organisms or other high risk agents, e.g. M. tuberculosis. 
    • If the airflow, indicated by magnehelic gauges fall out of an established range.
  • A list of qualified vendors can be found on EH&S’s web site: http://www.ehs.columbia.edu/bsccert.html.
  • To maintain proper directional airflow, do not block the front air intake or the rear exhaust grille and minimize the amount of material kept inside the cabinet.
  • Heat from a Bunsen burner may damage HEPA filters and disrupt the protective airflow pattern.  The use of disposable inoculating supplies combined with the sterile atmosphere of the BSC, should eliminate the need for heat decontamination throughout the procedure.  When heat sterilization is necessary, use a microincinerator - a small oven that eliminates the need for an open flame.  Microincinerators are available from lab supply vendors, e.g. VWR.
  • Work 4-6 inches from the front of the cabinet, over the tray and not over the grille; avoid rapid arm movements that can disrupt airflow.
  • In order to minimize arm movement in and out of the cabinet, place all needed materials in BSC at the start of procedures, arranging them to so that 'dirty' items do not pass over 'clean' ones.  Clean cultures (left) can be inoculated (center); contaminated pipettes can be discarded in the shallow pan and other contaminated materials
  • Allow cabinet fan to run 5 minutes prior to and at the completion of work; wipe interior with 70% ethanol before and after work.
  • Locate BSCs in low-traffic areas away from air supply grilles and doorways; drafts may disrupt protective air flow.
  • Many BSCs are equipped with UV lights, but routine disinfection of work surfaces is more critical in ensuring a contaminant-free work area, and relying heavily upon the disinfection activity of the UV light is not recommended.  Turn off UV lights when the cabinet is in use. UV lights should be wiped with an alcohol-moistened cloth weekly; a dust covered bulb is ineffective.  Bulbs must be disposed using EH&S’s hazardous waste program:http://www.ehs.columbia.edu/HazardousWaste.html.
  • Close the room door when working in a BSC, particularly if it is close to a laboratory door.
  • Most BSCs have a removable work surface tray and front grille, and the space beneath it requires regular cleaning to avoid contamination problems. A schedule for regular removal of the work surface tray and disinfection of the space beneath with 10% bleach followed by 70% ethanol is recommended. The drain valve under the work surface can facilitate cleaning.

    BSC1    bsc3

bcs2

Most BSCs have a removable work surface tray and front grille, and the space beneath it requires regular cleaning to avoid contamination problems.

2.3.2 Vacuum Line HEPA Filters
Vacuum lines require periodic maintenance by University personnel and it is vital to ensure that exposures to research materials are prevented. All vacuum lines, both inside BSCs and on bench tops must be protected with a HEPA filter and a disinfectant-filled collection flask. See the following diagram for setup instructions.

The left suction flask (A) is used to collect contaminated fluids into a suitable decontamination solution; the right flask (B), serves as a fluid overflow collection vessel. An in-line HEPA filter (C) is used to protect the vacuum system (D) from aerosolized microorganisms. Use flask(s) large enough to collect a day's worth of aspirate, nothing larger. Compression of the Tygon tubing may indicate that the filter requires replacement. Keep flasks in the BSC, not on the floor, to avoid accidental breakage or spilling. If flasks must be kept on the floor, use secondary containment such as a plastic bucket. Filters may be obtained through VWR, Fisher, and other lab supply companies. Empty flasks daily, providing fresh disinfectant each day, to reduce the likelihood of contamination problems.  

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HEAPA filter

The left suction flask (A) is used to collect contaminated fluids into a suitable decontamination solution; the right flask (B),  serves as a fluid overflow collection vessel. An in-line HEPA filter (C) is used to protect the vacuum system (D) from aerosolized microorganisms.  Use flask(s) large enough to collect a day’s worth of aspirate, nothing larger.  Keep flasks in the BSC, not on the floor, to avoid accidental breakage or spilling.  Filters may be obtained through VWR, Fisher, and other lab supply companies.  Empty flasks daily.

2.3.3 Sharps Containers and Safe Needle Devices

2.3.3.1 Sharps Containers
Needles, razor and scalpel blades, Pasteur pipettes, serological pipettes, micropipette tips and similar items must be discarded as Regulated Medical Waste, including organisms and materials containing recombinant DNA in puncture-resistant sharps containers. Many 'sticks' and cuts are caused by improperly disposed sharp items or sharps that were left 'lying around'; keep a sharps container as close as possible to where these items are used, if possible within arm's reach.

Glass items (pipettes, test tubes) should be substituted whenever possible by plastic ones. The use of needles, blades and other sharp objects should be limited to those situations where no other alternative exists.

2.3.3.2 Safe Needle Devices
These include 'needleless systems' and sharps incorporating automatic protection features. They allow for the elimination of exposure to or automatic shielding of needles during use, minimizing the risk of 'sticks' and cuts. While mostly applicable to clinical settings, they must be incorporated whenever there is the risk of exposure to materials containing recombinant DNA, human blood, body fluids, cells, unfixed tissue or any other material covered by OSHA's Bloodborne Pathogens Standard.

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needles

2.3.4 Centrifuge Safety
Centrifuge accidents may release large volumes of infectious, aerosolized material.

  • Keep accurate rotor use logs; decommission rotors as per manufacturers’ recommendations.
  • Inspect rotors, particularly the chambers, for corrosion and pitting.
  • Use “safety cups” or covers (gasketted containers into which tubes are placed during centrifugation). If a tube breaks, the material will be contained. An example of a centrifuge safety cup is illustrated on the next page. These safety devices can be obtained from the manufacturer. Check with personnel in your laboratory to confirm that they have already been obtained for your lab’s centrifuge(s).
  • If a safety cup is unavailable, be sure the rotor cover or chamber lid is tightly closed - never use an uncovered rotor.
  • For infectious materials or materials containing recombinant DNA, fill tubes and load/unload rotors or safety cups inside a BSC.

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If a tube breaks during centrifugation:

  • Allow aerosols to settle for 15 minutes before opening the chamber.
  • Don personal protective equipment as described in Section 2.8, Spill Procedures (below).
  • Use a squeeze bottle to carefully apply disinfectant solution to contaminated surface, taking care to minimize splashing.
  • Allow 20 minutes contact time, remove buckets and rotors to nearest BSC, aspirate residual disinfectant, and wipe down surfaces with clean water.
  • Place debris in sharps containers or red bags.
  • Follow manufacturers’ instruction for selection of disinfectants for use on rotors and buckets. These items are usually corrosion-sensitive.
safety Container

Centrifuge safety container with threaded lid.

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