STERILIZATION

Wet or dry heat can be used to kill microorganisms.

Wet Heat

Wet heat is the most dependable procedure for the destruction of all forms of microbial life.  Steam sterilization generally denotes heating in an autoclave employing saturated steam under a pressure of approximately 15 psi to achieve a chamber temperature of at least 121°C (250°F).  The critical factors in insuring the reliability of this sterilization method is: 1) proper temperature and time; and 2) the complete replacement of the air with steam (i.e. no entrapment of air).  Some autoclaves utilize a steam activated exhaust valve that remains open during the replacement of air by live steam until the steam triggers the valve to close.  Others utilize a pre-cycle vacuum to remove air prior to steam introduction.

Physical controls such as pressure gauges and thermometers are widely used but are considered secondary methods of insuring sterilization.  The use of appropriate biological indicators at locations throughout the autoclave is considered the best indicator of sterilization.  The biological indicator most widely used for wet heat sterilization is Bacillus stearothermophilus spores.

Dry Heat

Dry heat is less efficient than wet heat sterilization and requires longer times and/or higher temperatures.  The specific times and temperatures must be determined for each type of material being sterilized.  Generous safety factors are usually added to allow for the variables that can influence the efficiency of this method of sterilization.  The moisture of the sterilization environment as well as the moisture history of organisms prior to heat exposure appear to affect the efficiency of dry heat sterilization.

Higher temperatures and shorter times may be used for heat resistant materials.  The heat transfer properties and the spatial relation or arrangement of articles in the load are critical in insuring effective sterilization.

The advantage of wet heat is a better heat transfer to and into the cell resulting in overall shorter exposure time and lower  temperature. Steam sterilization uses pressurized steam at 121-132° C (250-270° F) for 30 or 40  minutes. This type of heat kills all microbial cells including spores, which are normally heat resistant. In order to accomplish the same effect with dry heat in an oven, the temperature needs to be  increased to 160-170° C (320-338° F) for periods of 2 to 4 hours.

General Procedures

All materials, equipment, apparatus contaminated with or containing potentially hazardous organisms should be steam sterilized before being washed and stored, or discarded.  Autoclaving is the preferred method.  Each individual working with biohazardous materials is responsible for sterilization of materials before disposal.

Biohazardous materials should not be placed in autoclaves overnight in anticipation of autoclaving the next day.

To minimize hazard to firemen or disaster crews, all biohazardous materials must be placed in an appropriately marked refrigerator or incubator, sterilized, or otherwise confined at the close of each work day.

All autoclaves must be certified for operating efficiency by the periodic use of biological indicator controls and records maintained for three years.  Contact the Biosafety Officer for more information and services provided.

Special precautions should be taken to prevent accidental removal of material from an autoclave before it has been sterilized or the simultaneous opening of both doors on a double door autoclave.

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

OXIDIZER + ORGANIC MATERIAL + HEAT =  POSSIBLE EXPLOSION.

All laboratory rooms containing biohazardous materials should designate two separate areas or containers labeled:

BIOHAZARDOUS  TO BE AUTOCLAVED

or

NONINFECTIOUS  TO BE CLEANED

All floors, laboratory benches, and other surfaces in buildings where biohazardous materials are handled should be disinfected as often as deemed necessary by the supervisor.  The surroundings should be disinfected after completion of operations involving plating, pipetting, centrifuging, and similar procedures with biohazardous materials.

It is the responsibility of the supervisor to determine that the disinfectant and the time and method of exposure is effective against the biological agent(s) used in the facility. 

Floor drains should be flooded with water or disinfectant at least once each week in order to fill traps and thus prevent the back flow of sewer gases.

Floor cleaning procedures which minimize the generation of aerosols should be used.  Wet mopping or wet vacuum pick up is recommended.  Water used to mop floors should contain a disinfectant or disinfectantdetergent.  (Dry mopping or dusting should be avoided).  Where wet procedures are not practical, dry vacuum cleaning with a HEPA filter on the exhaust, sweeping compound used with push brooms, or dry dust mop heads treated to suppress aerosolization may be used.

Stock solutions of suitable disinfectants will be maintained in each laboratory for disinfection purposes.  NOTE: working dilutions of certain disinfectants have short shelf lives and must be prepared on a regular basis.

Sterilization Procedures

General criteria for sterilization of typical materials are presented below.  Supervisors are encouraged to review the type of materials being handled and to establish standard conditions for sterilization.  Treatment conditions to achieve sterility will vary in relation to the volume of material treated, volume of the autoclave, the contamination level, the moisture content, and other factors.

Steam Autoclave

• Laundry: 250^oF (121^oC) for a minimum of 30 min.
• Trash: 250^oF (121^oC) for at least 45 minutes per bag.  Size of the autoclave and of the bags greatly effect sterilization time.  Large bags in a small autoclave may require 90 minutes or more.
• Glassware: 250^oF (121^oC) for a minimum of 25 min.
• Liquids: 250^oF (121^oC) for 25 minutes for each gallon.
• Animals & bedding: Steam autoclaving not recommended (sterilization time required would be at least 8 hours).  Incineration in an approved facility is the recommended treatment of these wastes.