DECONTAMINATION, STERILIZATION, DISINFECTION

While Biosafety Level -3 and the higher risk BL - 2 agents must be autoclaved prior to further processing, (See the list of agents required to be autoclaved prior to disposal)  most infectious material can be disposed safely through proper handling and packaging directly for incineration.  The following guidelines are intended to ensure that all biohazardous waste is processed in a safe and timely manner, as well as in accordance with all applicable regulations.

Each generator of biohazardous waste has an obligation to handle and dispose their material in a manner which affords protection from leakage and injury or exposure to anyone handling their waste material.

1. Each individual working with biohazardous material or contaminated items is responsible for their decontamination, disinfection, and appropriate preparation prior to disposal or reuse.
2. All laboratories, in which work with biohazardous materials is carried out, must have labeled, leak-proof, covered containers for temporary holding of infectious materials awaiting disinfection or disposal.
3. When autoclaving:
a. test tape or another suitable indicator must be used on each load placed in the autoclave.  This will aid in determining which items have been sterilized.
b. bagged waste must be placed into a containment pan prior to autoclaving.   The purpose of the containment pan is to prevent release of material in the event that the bag loses its integrity during the cycle.  This prevents waste from building up within drainage pipes, allowing for proper function of the autoclave.
c. only approved autoclavable bags are to be used.  Red bags are reserved for incineration, and are not acceptable.
d. autoclaves may only be operated by trained individuals.  Operators must never attempt to open an autoclave door while the chamber contains any pressure.   Doing so may result in severe burns, forceful release of the autoclave door and injury, as well as damage to the unit.
e. log sheets must be available at each autoclave to record the name of the user, time of run, and amount being autoclaved.
4. All floors, laboratory benches, and other surfaces in areas where biohazardous materials are handled must be disinfected upon completion of operations involving plating, pipetting, centrifugation and similar procedures.
5. Floors should be mopped with disinfectant.  Avoidance of dry sweeping and dusting will reduce the formation of aerosols.  If sweeping is necessary, a push broom and floor sweeping compound should be used.  Waxing and buffing should be done only after mopping.
6. Floor drains must be flooded with water periodically in order to fill traps and prevent the backflow of sewer gases.

Specific Disinfection and Sterilization Methods

    Wet Heat

1. The destruction of all forms of microorganisms is most readily accomplished by wet heat or autoclaving (saturated steam under pressure).
a. Higher pressures give higher internal temperatures.
b. Appropriate biological indicators should be used in containers or between densely packed materials to determine the effectiveness of the decontamination cycle.
2. Other critical factors which ensure the effectiveness of the autoclaving (besides saturated steam and proper temperature) are the removal of air from the chamber and its contents and adequate exposure time as relate to the "soil" load on contaminated items.
a. Heavily "soiled" items, especially if the "soil" is of proteinaceous nature, should not be flash autoclaved because that "soil" may briefly protect the microorganism from the lethal effects of the wet heat.
b. Autoclave times are directly proportional to the volume of materials to be autoclaved.  Twenty minutes at 121°C is adequate for the smallest loads.  When volumes in excess of 500 ml are autoclaved, times must be increased.  Consult the autoclave manufacturers' handbook for your unit.
c. It should also be noted that over or underloading of an autoclave also reduces the efficiency of decontamination.

Suggested Temperatures and Exposure Times from NIH Biohazards Guideline

Laundry 121°C  (250°F) 30 minutes
Trash 121°C  (250°F) 1 hour
Glassware 121°C  (250°F) or
160°C  (320°F)		 1 hour
4 hours (dry heat)
Liquids 121°C  (250°F) 1 hour, (each gallon)
Small Animals 121°C  (250°F) 8 hours

CAUTION:
Never autoclave hazardous chemicals!  Doing so, can create hazardous conditions.  Very few chemicals are considered acceptable to autoclave.

Dry hypochlorites, or any other strong oxidizing material, must not be autoclaved with organic materials such as paper, cloth or oil:

OXIDIZER + ORGANIC MATERIAL + HEAT = MAY PRODUCE AN EXPLOSION.

   Dry Heat

1. The use of dry heat for the disinfection or sterilization of biohazardous materials and contaminated items is less efficient than autoclaving and requires a longer exposure time with higher temperatures.
a. It may be possible to disinfect "soiled" materials by exposing them to 160°C (320°F) for four hours.
b. If items are heat sensitive, a temperature of 120°C (248°F) must be used, and exposure time necessary for disinfection or sterilization is usually greater than 24 hours.
2. The use of biological indicators (Bacillus subtilis spores) is also necessary with dry heat to determine the effectiveness of the sterilization cycle, and to determine the most effective temperature and/or exposure time for sterilization of materials or equipment.

CAUTION:
Dry heat at high temperatures and for long durations should be used to sterilize oils and anhydrous materials such as powders.

University of the Sciences in Philadelphia • 600 South Forty-third Street • Philadelphia, PA 19104-4495 • phone: 215-596-8800 • email: safety@usp.edu