Chapter 7: Decontamination and Sterilization

Definitions

Decontamination is a process or treatment that renders a device, instrument, or work surface safe to handle. A decontamination procedure can range from sterilization by autoclave or ethylene oxide to simple cleaning with soap and water. Sterilization, disinfection, and antisepsis are all forms of decontamination.

Sterilization is the use of a physical or chemical procedure to destroy all microbial life, including highly resistant bacterial endospores.

Disinfection eliminates virtually all pathogenic, non-spore-forming microorganisms but not necessarily all microbial forms on inanimate objects (work surfaces, equipment, etc.). Effectiveness is influenced by the kindstypes and numbersextent of organisms, the amount of organic matter, and the surface type (i.e. porous or smooth) of the object to be disinfected and chemical exposure time, temperature, and concentration.

Antisepsis is the application of a liquid antimicrobial chemical to skin or living tissue to inhibit or destroy microorganisms. It includes using germicidal solutions for swabbing an injection site on a person or animal and for handwashing. Although some chemicals may be utilized as either a disinfectant or an antiseptic, adequacy for one application does not guarantee adequacy for another. Manufacturers’ recommendations for appropriate use of germicides should always be followed.

General Procedures

Decontamination of cultures and objects contaminated by biological agents is routinely performed in microbiological laboratories. Decontamination is a vital component of microbiological safety practice and serves to protect laboratory personnel (as well as others) from infection and the release of infectious organisms to the outside environment (primarily through person-to-person transmission). Decontamination of media, work surfaces, and equipment is also necessary to prevent contamination of cultured organisms.

• Infectious wastes such as contaminated liquid and solid will be handled, treated and disposed of according to biological waste policies and procedures. Liquid wastes such as bacterial or viral culture media from BSL2 labs will be treated with appropriate disinfectant prior to sink disposal. Solid wastes from the BSL2 laboratories will be segregated and placed in biohazard containers lined with biohazardous waste bags and disposed of as biological wastes. This waste is sealed by the laboratory and shipped off-site for sterilization (see Waste Chart posted in the laboratory for more information).
• All wastes from the BSL3 laboratories will be inactivated before disposal from the laboratory (see Chapter 9)
• A disinfectant should be chosen that is appropriate for the organism in use.
• All liquid biological cultures should be deactivated with appropriate disinfectant.
• All solid biological waste should be disposed of in the biohazard waste containers.
• Waste created in BSL3 laboratories is required to be autoclaved prior to removal from the laboratory (see Chapter 9).

Methods of Decontamination

The three main categories of physical and chemical decontamination are heat, liquid disinfection, and vapors and gases.

Heat: Wet heat is the most dependable method of sterilization. Autoclaving (saturated steam under pressure of approximately 15 psi to achieve a chamber temperature of at least 250° F for a prescribed time) is the best method of rapidly achieving destruction of all forms of microbial life.

• In addition to proper temperature and time, prevention of entrapped air is critical to achieving sterility because of air’s poor heat transfer properties.
• Material to be sterilized must come into contact with steam and heat. Indicators of proper autoclave operation (e.g., autoclave tape or autoclave-sensitive labels) must be used with each load to visually indicate successful processing.
• Use of autoclave tape alone is not an adequate monitor of the sterilization’s success.
• The Massachusetts Department of Public Health Medical Waste Management Act has specific quality-control requirements for autoclaves used for sterilization of medical waste. Appendix D: Autoclave Quality Assurance Program describes the procedures for such tests.

Liquid disinfection: A liquid disinfectant (e.g., 1:10 solution of household bleach yielding a final hypochlorite concentration of 0.5%) is used to wipe or soak potentially contaminated materials for a period of time to kill all pathogenic agents present. Each disinfectant requires varying amounts of contact time.

Gas and vapor: Potentially contaminated articles are exposed to a sterilizing gas (e.g., ethylene oxide, or ETO) or vapors from a chemical (e.g., formaldehyde, vaporized hydrogen peroxide). Because of the hazardous nature of the gases and vapors used, this requires specially designed equipment and facilities.

Autoclaving

Autoclaving uses saturated steam under pressure (approximately 15 psi) to achieve a temperature in the autoclave of at least 121° C (25° F). Autoclaving can be used to destroy vegetative bacteria, bacterial spores, and viruses. When decontaminating biohazardous waste, it is recommended that the temperature in the waste reach a minimum of 115° C for a minimum of 20 minutes. The total processing time required to meet these conditions depends on several loading factors (see below); however, it is recommended that a minimum autoclave cycle of one hour be used when decontaminating waste.Please note that waste that has been designated by the IBC for autoclave treatment must be treated by autoclaving prior to disposal in biohazardous waste boxes and shipped off as regulated biohazardous wastes. The autoclaving process makes them safer for handling and transport, it does not change the disposal endpoint.

When using an autoclave, the following guidelines should be taken into consideration:

• Biohazardous materials should not be left inside the autoclaves overnight in anticipation of autoclaving the next day.
• Autoclaves should not be operated by untrained personnel.
• 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.
• Always use the appropriate PPE when operating the autoclave including lab coat, disposable gloves, heat resistant gloves and face protection.
• Dry hypochlorite, or any other strong oxidizing material, must not be autoclaved with organic materials such as paper, cloth, or oil:
  

  WARNING: OXIDIZER + ORGANIC MATERIAL + HEAT = POSSIBLE EXPLOSION

Three factors in combination determine the effectiveness of autoclaving:

Temperature: An autoclave uses steam under a pressure of approximately 15 psi to achieve a chamber temperature of at least 121° C. Although the autoclave chamber may reach 121° C, this does not necessarily mean that the interior of the load will reach this temperature.

Time: A minimum autoclave cycle time of 20 minutes at a chamber temperature of 121° C (time does not begin as soon as the autoclave cycle is initiated) is commonly recommended for sterilization of clean items.However, the total processing time required to achieve decontamination depends on several loading factors, including the load container (heat transfer properties); the amount of water added to the load; and the weight of the load. For increased loads, an increased cycle time will be required to ensure effective decontamination.

Contact: Steam saturation is essential for maximum heat transfer. Steam must contact all areas of the load. Autoclave bags and other containers should be left partially open (or otherwise permit entry of steam) to ensure adequate contact. Studies have shown that adding water to the interior of the bag improves the time-temperature profile of the autoclave cycle, thereby increasing the autoclave’s sterilization efficiency.

Dry Heat

Dry heat method requires a higher temperature and longer contact time. It is less effective than moist heat (autoclaving). Nevertheless, dry heat is preferable to moist heat for decontamination of anhydrous materials and closed containers because the moisture component of the steam used in an autoclave will not effectively penetrate anhydrous materials and closed containers.

The highest dry heat equivalent temperature that these materials will reach in an autoclave is 121° C. The highest temperature that material will reach in a dry heat oven will be the actual temperature inside the oven. A temperature of 160°-180° C for three to four hours is recommended for decontamination of waste using a dry heat oven.

Chemical Disinfection

Disinfection is the decontamination of work surfaces, equipment, biological safety cabinets, and other inanimate objects using antimicrobial agents. Several chemical agents are used as disinfectants. Laboratory workers should remember that there are hazards associated with all of these chemical disinfectants.

• Inhalation and skin contact should be minimized, and contact with eyes avoided.
• Appropriate gloves and safety eyewear should always be worn when handling these chemicals.

Pertinent information for some of the common chemical disinfectants is summarized in table format at the end of this chapter.

Summary of Chemical Disinfectants

Alcohol (ethyl, isopropyl)

Use Parameters: conc.: 70-85%; contact time: 10-30 min.

Effective Against:

• Vegetative cells: very positive response
• Lipophilic viruses: very positive response
• Tubercle bacilli: very positive response
• Hydrophilic viruses: less positive response

Important Characteristics: Eye irritant, toxic, flammable, inactivated by organic matter

Potential Application: Surfaces: work and equipment

Chlorine Compounds

Use Parameters: conc.: 0.05-0.5% (commercial bleach 0.5%); contact time: 10-30 min.

Effective Against:

• Vegetative cells: very positive response
• Lipophilic viruses: very positive response
• Tubercle bacilli: very positive response
• Hydrophilic viruses: very positive response
• Bacterial spores: less positive response

Important Characteristics: May leave residue; corrosive; skin, eye and respiratory irritant; inactivated by organic matter; make up at least weekly

Potential Application: Spills, equipment surfaces, instruments, glassware, water baths

Quaternary Ammonium Compounds

Use Parameters: conc.: 0.1-2%; contact time: 10-30 min.

Effective Against:

• Vegetative cells: very positive response
• Lipophilic viruses: very positive response

Important Characteristics: Toxic, inactivated by organic matter

Potential Application: Surfaces (work and equipment), BSCs, floor maintenance, glassware, instruments

Phenolic Compounds

Use Parameters: conc.: 0.2-3%; contact time: 10-30 min.

Effective Against:

• Vegetative cells: very positive response
• Lipophilic viruses: very positive response
• Tubercle bacilli: very positive response
• Hydrophilic viruses: less positive response

Important Characteristics: Leaves residue; corrosive; skin, eye and respiratory irritant; toxic; inactivated by organic matter

Potential Application: Surfaces (work and equipment), BSCs, floors, spills, glassware, instruments, water baths

Iodophor Compounds

Use Parameters: conc.: 0.47%; contact time: 10-30 min.

Effective Against:

• Vegetative cells: very positive response
• Lipophilic viruses: very positive response
• Tubercle bacilli: very positive response
• Hydrophilic viruses: less positive response

Important Characteristics: Leaves residue; corrosive; skin and eye irritant; toxic; inactivated by organic matter

Potential Application: Surfaces (work and equipment), BSCs, glassware, water baths

Formaldehyde* (Formalin)

Use Parameters: conc.: 4-8%; contact time: 10-30 min.

Effective Against:

• Vegetative cells: very positive response
• Lipophilic viruses: very positive response
• Tubercle bacilli: very positive response
• Hydrophilic viruses: very positive response
• Bacterial spores: less positive response

Important Characteristics: Leaves residue; skin, eye and respiratory irritant; toxic (carcinogen)

Potential Application: Less effective than other disinfectants but can be used for equipment surfaces, glassware, instruments

*Note: Due to its irritating characteristics and status as a carcinogen, formaldehyde should not be used without good local exhaust ventilation.

Glutaraldehyde

Use Parameters: conc.: 2%; contact time: 10-60 min.

Effective Against:

• Vegetative cells: very positive response
• Lipophilic viruses: very positive response
• Tubercle bacilli: very positive response
• Hydrophilic viruses: very positive response
• Bacterial spores: very positive response

Important Characteristics: Leaves residue; skin, eye and respiratory irritant; toxic

Potential Application: Equipment surfaces, glassware, instruments

From Laboratory Safety: Principles and Practices, second edition, Diane O. Fleming, John H. Richardson, Jerry J. Tulis, and Donald Vesley, eds., American Society for Microbiology, Washington, D. C.