Environmental Health and Safety

SDSU Biosafety Manual

San Diego State University
BIOHAZARD CONTROL PROGRAM

Part XIV:
Whole Building Safety

As laboratories are obviously just a portion of a whole building, the PI and laboratory staff should familiarize themselves with aspects of the building which are not necessarily restricted to their own laboratories, but which can impact their work or their neighbors' work. Included among such building issues are the following:

A. ACCESS

B. VENTILATION (including air supply, exhaust and distribution)

C. CENTRIFUGES

D. EMERGENCY EQUIPMENT

A. ACCESS

Corridors, stairways and exits are important to all persons in the building. Of special interest in the biohazard laboratory is control of access to the laboratory; doors must be kept closed while all BSL 2 or greater work is in progress.

B. VENTILATION

Some laboratory workers refer to BSCs as "hoods." It is important to know the difference between a BSC and a chemical fumehood. BSCs are designed to protect the individual and the environment from biological agents and to protect the research materials from contamination. Chemical fumehoods, however, are designed solely to protect the individual from exposure to chemicals and noxious gases. Since chemical fumehoods are not equipped with HEPA filters, they must not be used for work with biohazardous materials.

C. CENTRIFUGES

Hazards associated with centrifuging include mechanical failure (e.g., rotor failure, tube or bucket failure) and the creation of aerosols. To minimize the risk of mechanical failure, centrifuges must be maintained and used according to the manufacturer's instructions. Users should be properly trained and operating instructions that include safety precautions and regular maintenance should be prominently posted on the unit.

Aerosols are created by practices such as filling centrifuge tubes, removing plugs or caps from tubes after centrifugation, removing supernatant, resuspending sedimented pellets and by the very process of centrifugation. The greatest aerosol hazard is created if a tube breaks during centrifugation. To minimize the generation of aerosols when centrifuging biohazardous material, the following procedures should be followed:

• Use sealed tubes and safety buckets that seal with O-rings. Before use, inspect tubes, O-rings and buckets for cracks, chips, erosions, bits of broken glass, etc. Do not use aluminum foil to cap centrifuge tubes; it may detach or rupture during centrifugation.
  
  
• Fill and open centrifuge tubes, rotors and accessories in a BSC. Avoid overfilling of centrifuge tubes so that closures do not become wet. After tubes are filled and sealed, wipe them down with disinfectant.
  
  
• Add disinfectant to the space between the tube and the bucket to disinfect material in the event of breakage during centrifugation.
  
  
• Always balance buckets, tubes and rotors properly before centrifugation.
  
  
• If the centrifuged specimen contains biohazardous material, open the centrifuge tubes inside a BSC with the tube pointed away from you.
  
  
• Do not decant or pour off supernatant. Use a vacuum system with appropriate in-line reservoirs and filters.
  
  
• Work in a BSC when resuspending sedimented material. Use a swirling rotary motion rather than shaking. If shaking is necessary, wait a few minutes to permit the aerosol to settle before opening the tube.
  
  
• Small low-speed centrifuges may be placed in a BSC during use to reduce the aerosol escape.
  
  
• High-speed centrifuges pose additional hazards. Precautions should be taken to filter the exhaust air from centrifuge vacuum lines. Manufacturers' recommendations must be meticulously followed to avoid metal fatigue, distortion and corrosion. For high speed spins (.3500g) use only glass corex tubes, not test tubes whether Pyrex or not.
  
  
• Avoid the use of celluloid (cellulose nitrate) tubes with biohazardous materials. Celluloid centrifuge tubes are highly flammable and prone to shrinkage with age. They distort on boiling and can be highly explosive in an autoclave. If celluloid tubes must be used, an appropriate chemical disinfectant must be used to decontaminate them.
  
  

D. EQUIPMENT THAT CREATES AEROSOLS

The use of blenders, homogenizers, ultrasonic disrupters, grinders and lyophilizers can result in considerable aerosol production. This equipment should be used in a BSC when working with biohazardous materials.

Safety blenders are designed to prevent leakage from the bottom of the blender jar, provide a cooling jacket to avoid biological inactivation and to withstand sterilization by autoclaving. If blender rotors are not leakproof, they should be tested with sterile saline or dye solution prior to use with biohazardous materials. The use of glass blender jars is discouraged because of the potential for breakage. If they must be used, glass jars should be covered with a polypropylene jar to prevent spraying of glass and contents in the event the blender jar breaks. A towel moistened with disinfectant should be placed over the top of the blender during use. Before opening the blender jar, allow the unit to rest for at least one minute to allow the aerosol to settle and then open in a BSC. The device should be decontaminated promptly after use.

Lyophilizers and ampoules. Depending on lyophilizer design, aerosol production may occur when material is loaded or removed from the lyophilizer unit. If possible, sample material should be loaded in a BSC. The vacuum pump exhaust should be filtered to remove any hazardous agents or, alternatively, the pump can be vented into a BSC. After lyophilization is complete, all surfaces of the unit should be disinfected. If the lyophilizer is equipped with a removable chamber, it should be closed off and moved to a BSC for unloading and decontamination. Handling of cultures should be minimized and vapor traps should be used wherever possible.

Opening ampoules containing liquid or lyophilized culture material should be performed in a BSC to control the aerosol produced. Gloves must be worn. To open, nick the neck of the ampoule with a file. Wrap it in a disinfectant soaked towel. Hold the ampoule upright and snap it open at the nick. Reconstitute the contents of the ampoule by slowly adding liquid to avoid aerosolization of the dried material. Mix the contents without bubbling and withdraw it into a fresh container. Discard the towel and ampoule top and bottom as biohazardous material waste in a sharps container since broken glass ampoules used to store biohazardous material in liquid nitrogen have exploded causing eye injuries. The use of polypropylene tubes eliminates this hazard. These tubes are available dust-free and pre-sterilized and are fitted with polyethylene caps with silicone washers. Heat-sealable polypropylene tubes are also available.