Legionella Testing / Biosafety Training

NSF 49 - 2016 Annex E

NSF 49 - 2016 Annex E


NSF 49 - 2016 Annex E "Biosafety cabinet selection, installation, [use], lifespan and decommissioning" is a free, informative section of NSF/ANSI 49 - 2016 "Biosafety Cabinetry: Design, Construction, Performance, and Field Certification" published by NSF International - an independent, not for-profit, non-governmental organization, dedicated to being the leading global provider of public health and safety-based risk management solutions while serving the interests of all stakeholders.

Annex E is available online: Portions are detailed below:

All BSCs should be placed in a laboratory at a location that provides a minimum of:
— 6 inches (150 mm) from adjacent walls or columns.
— 6 inches (150 mm) between two BSCs.
— 6 inches (150 mm) space between both sides of the cabinet and 6 inches (150 mm) behind the BSC to allow for service operations.
— 40 inches (1020 mm) of open space in front of the BSC
— 60 inches (1520 mm) from opposing walls, bench tops and areas of occasional traffic.
— 20 inches (510 mm) between BSC and bench tops along a perpendicular wall.
— 100 inches (2540 mm) between two BSCs facing each other.
— 60 inches (1520 mm) from behind a doorway.
— 40 inches (1020 mm) from an adjacent doorway swing side.
— 6 inches (150 mm) from an adjacent doorway hinge side.

BSC techniques
— have proper BSC training including proper aseptic technique before use.
— decontaminate BSC work surface before use.
— ensure that sash is at the proper height depending on BSC model.
— do not use an open flame.
— do not use high pressure gases.
— do not use volatile chemicals without a risk assessment by qualified chemical safety personnel system (Refer to Section E.3.1.3).
— do not place large objects on the BSC work surface.
— do not place fan-cooled devices in the BSC work area.
— minimize room activity
— activity in the room should be held to a minimum. Unnecessary activity may create disruptive air currents as well as interfere with BSC operation. A person walking past the front of a BSC may cause draft velocities up to 175 fpm (89 m/s), which can disrupt the air inside the BSC.
— utilize unidirectional air flow
— keep two important facts in mind: (1) Air supplied to the work area through a supply filter from the top, is contaminant free; and (2) Airborne contamination generated in the work area is controlled by flow of airstreams in a top-to-bottom direction.
— a solid object placed in the sterile air stream will disrupt the air flow and consequently, the capability to control lateral movement of airborne particulates. A cone of turbulence will extend below the object and uniform flow of the air stream will not be regained until for approximately equal to three to six times the size of the object. Within this cone of disturbed air, particles may be carried laterally by multidirectional eddy currents.
— transfer of viable materials and manipulations which may generate aerosols should not be performed above sterile materials.
— employ Aseptic Technique
— do not assume that the BSC will prevent contamination when performing procedures. BSCs will control airborne contamination of viable agents (i.e., microorganisms), but the BSC will not eliminate contact transmission of contamination. Contamination control procedures such as aseptic technique
is required to obtain maximum benefit from the BSC.
— open bottles, tubes or flasks should be kept as parallel as possible to the downflow air to minimize capture of particulates. This precaution is merely good aseptic technique.
— equipment in direct contact with agents must remain in the cabinet until bagged or until surface decontaminated. Trays of discard pipettes must be covered before removal from the BSC.
— when there is a spill or splatter of agent in the work area, all surfaces and items in the BSC should be surface-decontaminated before materials are removed.
— using natural gas to flame flask or tube necks is not recommended. A gas burner flame in BSC also contributes significantly to the heat build-up inside the BSC and also disrupts the sterile supply air stream. If a procedure demands use of a flame, a Bunsen burner with an on-demand ignition is recommended. Do not use a constant flame gas burner. Bunsen burners must be placed near the rear of the work space where air turbulence will have a minimal effect.
— a plastic-backed absorbent paper should be placed on the work surface during mixing procedures and replaced whenever significant spills occur and when the work is completed.
— vials should be opened with a tool specifically designed for that purpose. Vials should be vented with a filter needle to eliminate internal pressure or vacuum.
— before opening ampules, make sure that no liquid remains in the tip of the ampule. A sterile gauze pad should be wrapped around the neck of the ampule before it is opened.
— a collection vessel should be available on the work surface for discard of solutions.

BSC start up procedure
— follow manufacturer’s startup procedure. If cabinet alarm condition is present, investigate root cause before continuing.
— inspect air intake grilles for obstructions and foreign materials and remove any obstructions.
Remove all items from the work area.
— adjust view screen to proper height.
— turn on blower and allow five minutes to purge the air.
— wash hands and arms with mild, non-antimicrobial soap for 30 seconds. Put on a rear-fastening, long-sleeved gown with gathered cuffs. Put on a pair (or two pairs) of appropriate long sleeve (11 ½ -12 inch) gloves (nitrile gloves are recommended for biological work). Consider, when appropriate, disposable sleeve protectors and a second or third pair of appropriate gloves. This will minimize the shedding of skin flora into the work area and also protect hands and arms from viable microbial contamination. Before and after work in a BSC, hands and arms should be washed mild, non-antiseptic soap.
— disinfect the interior surfaces of the BSC by wiping down with appropriate disinfectant for an appropriate contact time. 70% alcohol is not considered an appropriate disinfectant because it has no effect on fungal spores.
— place a plastic-backed pad on the work surface without covering the air intake/exhaust grills. This will prevent spills from hitting the stainless steel surface and creating aerosols.
— put all items for the experiment in the BSC and keep clean items segregated from dirty items by 12 inches (300 mm). Organize the material so that dirty "contaminated" items will not be passed over (cross contaminate) clean items.
— exercise care that no items be placed over the front intake grills. Materials should be arranged so that clean materials are separated from dirty (used) virus materials. Passage of contaminated materials over non-inoculated cultures or clean glassware should be avoided to prevent contamination. Transfer of viable materials should be performed as deeply into the cabinet (away from open face) as possible.
— allow air to stabilize for five minutes before starting work. This will rid the area of all "loose" contamination that may have been introduced with the items.
— work from "clean" to "dirty" areas and work at least six inches (150 mm) back from rear of the front air intake grill.
— move arms in and out of the work access opening perpendicular to the front of the BSC in a slow steady motion to minimize disruption of the front air curtain.
— minimize penetration of the work opening air curtain.
— a minimum number of needed items should be placed into the BSC to prevent overloading. Work should be planned to minimize the number of times an operator's hands and arms must enter and leave the air curtain. Ideally, have everything needed for your procedure placed in the BSC before starting, so that nothing needs to pass in or out through the front air curtain until the procedure is completed. Do not raise your hands inside the BSC above the top level of the sash height. If you raise your hands above the sash height, air may flow up your hands to elbows and possibly out of the BSC.
— know your "safe working area". BSC safe working area is the work tray or depressed area. All work should be performed on or above the work tray. The area on or above and within 6 inches (150 mm) from the rear of the front grill is a non-safe working area.
— this is a general operational guideline to control airborne contaminants of low to moderate risk. Procedure protocols defined in terms of the barrier or control concepts unique to BSCs must be developed for maximum safety and protection.
— for preparation of anti-neoplastic drugs, the following procedures summarize the OSHA Technical Manual TED 1-0.15A, Section VI, Chapter 2 "Controlling Occupational Exposure to Hazardous Drugs". This document should be reviewed before preparing anti-neoplastic drugs in a BSC..

Terminal purging & wipedown
— following completion of work, allow the BSC to run for a 5 minute period without personnel activity to purge air in the work area.
— decontamination of the interior surfaces should be repeated after removal of all materials, cultures, apparatus, etc. Check grills and diffuser grids for spilled or splashed materials that may support fungus growth in the work space.
— the interior surfaces of the work space should next be disinfected with an appropriate disinfectant for an appropriate contact time. Use of chlorine bleach in the BSC will damage the BSC stainless steel work surface. Most surface disinfectants require a specific contact time, depending upon the microbiological agents used within the BSC. Consult appropriate disinfectant documents for proper application and suitability against the material used in the BSC.

Use the following procedure to effectively clean or surface disinfect the BSC work zone surfaces.
— raise the sliding sash window to a full-open position.
— silence the audible alarm during the cleaning process.
— wipe all surfaces in parallel strokes from clean to dirty.

Paper catch prefilter
— some BSCs have a paper catch filter installed behind the rear divider panel of the work zone.
This area forms the return air path to the motor/blower. If the airflow is blocked, performance of the
BSC can be compromised. Therefore, the paper catch should be checked and cleaned no less than
weekly or daily if paper products are used for procedures. Removed paper must be properly
discarded as contaminated hazardous waste.

BSC shut down
— turn off blowers and lights. Do not use cabinet as a depository for any lab equipment or materials during periods of non-operation.
— close the sash window.
— if anti-neoplastic agents are prepared in the BSC, it is recommended that the BSC run 24 hours per day. This lessens the possibility that contaminants may escape.