Biosafety Equipment

Biosafety Cabinets

Biosafety Cabinet (BSC) - is an enclosed, ventilated laboratory workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens requiring a defined biosafety level (BSL). The primary purpose of a BSC is to serve as a means to protect the laboratory worker and the surrrounding environment from pathogens.  All air is HEPA-filtered as it exits the BSC, removing harmful bacteria and viruses. Many (but not all) types of BSCs are also designed to maintain the sterility of the materials being worked with.

Note: Chemical fume hoods are not designed nor intended to be used as BSCs.  Chemical fume hoods fail to provide the environmental protection that HEPA filtration in a BSC would provide.  Likewise, a laminar flow clean bench can not be used as a BSC.  A laminar flow clean bench blows unfiltered exhaust air towards the user and is not safe for work with pathogenic agents.

For a thorough description of BSCs, including selection, installation, and use please see Appendix A of the Biosafety in Microbiological and Biomedical Laboratories Guidelines (6th ed.)

If work is being performed in a BSL-2 laboratory, all work in which infectious aerosols may be produced must be conducted in a BSC or other type of physical containment.  If work is being performed in a BSL-3 laboratory, all work must be performed in a BSC or other type of physical containment.

Work Practices and Procedures

1. Cabinet blowers should be operated at least 3-5 minutes before beginning work to allow the BSC to "purge" particulates.
2. Turn off BSC Ultraviolet (UV) light (if present) before beginning work.
3. Appropriate personal protective equipment must be worn. Lab coats must be buttoned. Gloves should be pulled over the knitted wrists of the lab coat, not worn inside the coat.
4. Place only the necessary materials in the BSC before beginning work.
5. Extra supplies (gloves, plates, media, etc.) should be stored outside BSC: material placed inside BSC may cause disruption to the airflow.
6. Move arms in and out of BSC slowly, perpendicular to the face opening to reduce disruption of air curtain. Manipulation of materials should be delayed momentarily to allow air circulation to stabilize.
7. Do not block front grille with papers or materials or rest arms on front grille. This allows room air to flow directly into the work area rather than being drawn through the front grille. Work with arms raised slightly.
8. All operations should be performed at least 4 inches from the front grille on the work surface.
9. Interior walls, interior surface of the window, and the surfaces of all materials to be placed in the BSC should be wiped with 70% ethanol or other appropriate disinfectant before and after each use. If bleach is used, make sure that it is followed by sterile water or 70% ethanol.  Bleach will eventually ruin the stainless steel surfaces of the BSC.
10. Plastic backed absorbent toweling can be placed on the work surface (but not on the front grille) to aid in cleanup and spill containment.
11. Active work should flow from the clean to contaminated area across the work surface.
12. To minimize frequent in/out arm movement and maintain air barrier, do not tape autoclavable biohazard collections bags to the outside of the BSC and upright pipette collection containers should not be used in the BSC nor placed on the floor outside the BSC (use horizontal discard trays containing an appropriate chemical disinfectant within the BSC).
13. Potentially contaminated materials should not be brought out of the BSC until they have been surface decontaminated.
14. Use techniques to reduce splatter and aerosol generation: Open bottles or tubes should not be held in a vertical position, hold the lid above open sterile surfaces to minimize direct impact of downward air, open flames create turbulence which disrupts the pattern of air supplied to the work surface and should not be used. If necessary, touch plate microburners which provide a flame on demand or electric furnaces are available. All flames must be off before disinfectants are used.
15. Aspirator bottles or suction flasks should be connected to an overflow collection plastic flask containing an appropriate disinfectant, and to an in-line HEPA filter.
16. If spilled liquid enters through the front or rear grilles, close the drain valves and pour decontaminating solution into the drain pans. After 20-30 minutes, collect the spilled liquid and disinfectant with paper towels.

Use of Vacuum Lines

All vacuum lines used to aspirate supernatant, tissue culture media, and other liquids that may contain microorganisms should be protected from contamination by the use of a collection flask and overflow flask. In addition, a hydrophobic vacuum line filter or HEPA filter should be used. This will prevent fluid and aerosol contamination of central vacuum systems or vacuum pumps. Full strength chemical disinfectant should be added to collection flasks and allow the aspirated fluids to complete the dilution (for example: start with 100 ml of undiluted bleach, aspirate 900 ml of fluids, and discard for a 10% final bleach concentration).  Flasks containing aspirated fluids and disinfectants should be changed often and on a regular basis.

Biosafety Cabinet Certifications

1. All BSCs should be certified annually. If Biosafety Level 2 or higher materials are used in the BSC, the cabinet must be certified annually.
2. If the BSC is relocated, it must be re-certified prior to use.

Reference Source

United States. Department of Health and Human Services. Primary Containment for Biohazards:

       Selection, Installation and Use of Biological Safety Cabinets. Washington, D.C: U.S. Government

       Printing Office, 1995.

 

Autoclaves

Autoclaves are used to sterilize equipment, products or culture media prior to use in an experiment.  They can also be used to render items non-infectious prior to disposal. Autoclaves sterilize materials using saturated steam under pressure ("moist heat"). Due to the use of pressure, steam and high temperatures, there is a significant risk for injury.  Individuals that intend to use an autoclave must be properly trained on operational procedures and use appropriate personal protective equipment (PPE). 

General Guidelines

• Autoclaves should be inspected and serviced on a regular basis.
• Individuals using autoclaves must be properly trained and familiar with the autoclave specifications and related SOPs prior to working with them.
• Autoclaves should be tested on a regular basis with a commercial biological indicator (i.e. Geobacilis stearothermophilus ) to ensure that it is functioning properly.
• Tape indicators (autoclave tape) with heat sensitive, chemical indicators should be used in every load. Autoclave tape will only verify that the autoclave has reached normal operating temperatures.  NOTE: It does not indicate proof that organisms were actually killed during an autoclave run.
• High density wastes or materials that insulate agents from heat and steam penetration are not suitable for steam sterilization.  Items covered with dirt or film require additional retention times.
• All autoclaved infectious waste must be placed into red biohazard bags for disposal.
• Upon completion of a cycle wait until the autoclaved has cooled prior to opening the door (Most autoclaves have safety interlocks that prevent the door from opening when the temperature inside is greater than 80°C). Do not stand in front of the autoclave when opening the door, a puff of steam may be ejected immediately after the cycle has been completed.
• Containers with liquids should be allowed to cool for at least 20 minutes before transport to prevent sudden eruption from the containment vessel.

Appropriate Personal Protective Equipment (PPE)

The following PPE should be used during loading and unloading of the autoclave:

• Standard laboratory clothing including long pants and closed-toed shoes
• Eye/face protection
• Heat resistant gloves
• Laboratory coat

When Autoclaving Dry Materials

• Only use approved autoclave bags and ensure that they are not filled beyond 75% of their holding capacity
• Steam resistant autoclaveable bags must be left open (or have holes punched into the top of them to ensure proper steam penetration

When Autoclaving Liquid Materials

• Bottle caps and stoppers must be loosened after placement into the autoclave chamber.  This will allow proper steam penetration to sterilize and prevent the bottles from shattering or bursting from the pressurization and extreme heat during operation.
• Containers should not be filled beyond 25-50% of their capacity to prevent spill and boil over.
• Bottles/flasks/containers can be placed in an autoclave pan with about 5-10 inches of water for even heating, ensure that are no air bubbles under the containers.

Incompatible Materials

Not all materials are autoclaveable as they can generate toxic vapors or create explosive environments. The following materials should not be autoclaved:

• Materials containing solvents, volatile, corrosive or flammable chemicals
• Materials contaminated with chemotherapeutic agents or cytotoxic drugs
• Materials containing bleach
• Carcinogens or mutagens
• Phenol and Trizol
• Polystyrene, polyethylene, low-density polyethylene and high-density polyethylene plastics and polyurethane tubing
• Household glassware
• Radioactive materials