Sterilization and Disinfection

Sterilization is a process that kills all forms of microbial life, including bacterial spores. Disinfection is a process that destroys pathogenic organisms, but not necessarily all microorganisms or spores. Sterilization and disinfection may be accomplished by physical or chemical methods.

Methods of Sterilization

The physical methods of sterilization include:

• Incineration

• Moist heat

• Dry heat

• Filtration

• Ionizing (gamma) radiation

Incineration is the most common method of treating infectious waste. Hazardous material is literally burned to ashes at temperatures of 870° to 980°C. Incineration is the safest method to ensure that no infective materials remain in samples or containers when disposed. Prions, infective proteins, are not eliminated using conventional methods. Therefore incineration is recommended. Toxic air emissions and the presence of heavy metals in ash have limited the use of incineration in most large U.S. cities.

Moist heat (steam under pressure) is used to sterilize biohazardous trash and heat-stable objects; an autoclave is used for this purpose. An autoclave is essentially a large pressure cooker. Moist heat in the form of saturated steam under 1 atmosphere (15 psi [pounds per square inch]) of pressure causes the irreversible denaturation of enzymes and structural proteins. The most commonly used steam sterilizer in the microbiology laboratory is the gravity displacement type (Figure 4-1). Steam enters at the top of the sterilizing chamber; because steam is lighter than air, it displaces the air in the chamber and forces it out the bottom through the drain vent. The two common sterilization temperatures are 121°C (250°F) and 132°C (270°F). Items such as media, liquids, and instruments are usually autoclaved for 15 minutes at 121°C. Infectious medical waste, on the other hand, is often sterilized at 132°C for 30 to 60 minutes to allow penetration of the steam throughout the waste and the displacement of air trapped inside the autoclave bag. Moist heat is the fastest and simplest physical method of sterilization.

Dry heat requires longer exposure times (1.5 to 3 hours) and higher temperatures than moist heat (160° to 180°C). Dry heat ovens are used to sterilize items such as glassware, oil, petrolatum, or powders. Filtration is the method of choice for antibiotic solutions, toxic chemicals, radioisotopes, vaccines, and carbohydrates, which are all heat sensitive. Filtration of liquids is accomplished by pulling the solution through a cellulose acetate or cellulose nitrate membrane with a vacuum. Filtration of air is accomplished using high-efficiency particulate air (HEPA) filters designed to remove organisms larger than 0.3 µm from isolation rooms, operating rooms, and biologic safety cabinets (BSCs). The ionizing radiation used in microwaves and radiograph machines is composed of short wavelength and high-energy gamma rays. Ionizing radiation is used for sterilizing disposables such as plastic syringes, catheters, or gloves before use. The most common chemical sterilant is ethylene oxide (EtO), which is used in gaseous form for sterilizing heat-sensitive objects. Formaldehyde vapor and vapor-phase hydrogen peroxide (an oxidizing agent) have been used to sterilize HEPA filters in BSCs. Glutaraldehyde, which is sporicidal (kills spores) in 3 to 10 hours, is used for medical equipment such as bronchoscopes, because it does not corrode lenses, metal, or rubber. Peracetic acid, effective in the presence of organic material, has also been used for the surface sterilization of surgical instruments. The use of glutaraldehyde or peracetic acid is called cold sterilization.

Chemical Safety

In 1987, the U.S. Occupational Safety and Health Administration (OSHA) published the Hazard Communication Standard, which provides for certain institutional educational practices to ensure that all laboratory personnel have a thorough working knowledge of the hazards of the chemicals with which they work. This standard has also been called the “employee right to know.” It mandates that all hazardous chemicals in the workplace be identified and clearly marked with a National Fire Protection Association (NFPA) label stating the health risks, such as carcinogen (cause of cancer), mutagen (cause of mutations in deoxyribonucleic acid [DNA] or ribonucleic acid [RNA]), or teratogen (cause of birth defects), and the hazard class, for example, corrosive (harmful to mucous membranes, skin, eyes, or tissues), poison, flammable, or oxidizing (Figure 4-2).

Each laboratory should have a chemical hygiene plan that includes guidelines on proper labeling of chemical containers, manufacturers’ material safety data sheets (MSDSs), and the written chemical safety training and retraining programs. Hazardous chemicals must be inventoried annually. In addition, laboratories are required to maintain a file of every chemical they use and a corresponding MSDS. The manufacturer provides the MSDS for every hazardous chemical; some manufacturers also provide letters for nonhazardous chemicals, such as saline, so that these can be included with the other MSDSs. The MSDSs include information on the nature of the chemical, the precautions to take if the chemical is spilled, and disposal recommendations. The sections in the typical MSDS include:

Employees should become familiar with the location and organization of MSDS files in the laboratory so that they know where to look in the event of an emergency.

Fume hoods (Figure 4-3) are provided in the laboratory to prevent inhalation of toxic fumes. Fume hoods protect against chemical odor by exhausting air to the outside, but they are not HEPA-filtered to trap pathogenic microorganisms. It is important to remember that a BSC (discussed later in the chapter) is not a fume hood.

Work with toxic or noxious chemicals should always be done wearing nitrile gloves, in a fume hood, or when wearing a fume mask. Spills should be cleaned up using a fume mask, gloves, impervious (impenetrable to moisture) apron, and goggles. Acid and alkaline, flammable, and radioactive spill kits are available to assist in rendering any chemical spills harmless.

Fire Safety

Fire safety is an important component of the laboratory safety program. Each laboratory is required to post fire evacuation plans that are essentially blueprints for finding the nearest exit in case of fire. Fire drills conducted quarterly or annually, depending on local laws, ensure that all personnel know what to do in case of fire. Exit paths should always remain clear of obstructions, and employees should be trained to use fire extinguishers. The local fire department is often an excellent resource for training in the types and use of fire extinguishers.

Type A fire extinguishers are used for trash, wood, and paper; type B extinguishers are used for chemical fires; and type C extinguishers are used for electrical fires. Combination type ABC extinguishers are found in most laboratories so that personnel need not worry about which extinguisher to reach for in case of a fire. However, type C extinguishers, which contain carbon dioxide (CO₂) or another dry chemical to smother flames, are also used, because this type of extinguisher does not damage equipment.

The important actions in case of fire and the order in which to perform tasks can be remembered with the acronym RACE:

Electrical Safety

Electrical cords should be checked regularly for fraying and replaced when necessary. All plugs should be the three-prong, grounded type. All sockets should be checked for electrical grounding and leakage at least annually. No extension cords should be used in the laboratory.

Handling of Compressed Gases

Compressed gas cylinders (CO₂, anaerobic gas mixture) contain pressurized gases and must be properly handled and secured. When leaking cylinders have fallen, tanks have become missiles, resulting in loss of life and destruction of property. Therefore, gas tanks should be properly chained (Figure 4-4, A) and stored in well-ventilated areas. The metal cap, which is removed when the regulator is installed, should always be in place when a gas cylinder is not in use. Cylinders should be transported chained to special dollies (Figure 4-4, B).

Biosafety

Individuals are exposed in various ways to laboratory-acquired infections in microbiology laboratories, such as:

Risks from a microbiology laboratory may extend to adjacent laboratories and to the families of those who work in the microbiology laboratory. For example, Blaser and Feldman¹ noted that 5 of 31 individuals who contracted typhoid fever from proficiency testing specimens did not work in a microbiology laboratory. Two patients were family members of a microbiologist who had worked with S. enterica subsp. Typhi; two were students whose afternoon class was in the laboratory where the organism had been cultured that morning; and one worked in an adjacent chemistry laboratory.

In the clinical microbiology laboratory, shigellosis, salmonellosis, tuberculosis, brucellosis, and hepatitis are frequently acquired laboratory infections. Additional infections have been reported from agents such as Coxiella burnetii, Francisella tularensis, Trichophyton mentagrophytes, and Coccidioides immitis.