Published: 2021-05-24
Autoclave technology has improved over the years to the point where it is now possible to safely and effectively use it. It is possible to achieve a high degree of sterility with this process and maintain the desired level of quality control.
This sterilization process is still necessary in the medical world to ensure that all items are sterilized and stored correctly.
What is Autoclave Sterilization?
In healthcare and industrial applications, autoclaves are typically known as steam sterilizers. In an autoclave, items placed in a pressure vessel are heated up to kill harmful bacteria, viruses, fungi, and spores. It is then heated to the appropriate sterilization temperature and held at that temperature for a predetermined amount of time. As a result, the moisture in the steam affects bacteria and spores by efficiently transferring heat to the items.
The medical profession uses the term "autoclave" in describing Steam Sterilizers. For successful autoclaving, specific parameters and requirements are set. Also, a suitable biological indicator is added to the sterilization process.
Requirements for Autoclaving
Autoclaving is following the primary application of four parameters for successful sterilization. Exposing each piece of equipment to steam under the required temperature and pressure for the specified time. Thus, the principle of making the steam, pressure, temperature and time work together must be conducted systematically. These four parameters are manipulatable to different cycle set-ups to sterilize diverse types of loads.
Pressure and Steam
One of the conditions in autoclaving is the quality of saturated steam. Steam sterilization is achieved with dry saturated steam and entrained water with a dryness fraction of ≥97%. Steam experiences the most significant heat transfer as it approaches boundary conditions.
Condensation is not possible when it is dry or gaseous, so its effectiveness is compromised. High pressure allows microorganisms to be killed more quickly by high temperatures. The efficiency of the analyzing process depends on the accuracy of the equipment's temperature.
Temperature
The temperature is crucial to microbicidal activity. Temperatures from 121°C (250°F) to 132°C (270°F) usually are employed in steam sterilization. The temperatures must be maintained for a minimum period to kill off unwanted microbes.
Time
Different types of items require different lengths of time for sterilization. Metal, rubber, plastic, and lumen items are among the available types. The required time can also be affected by how the package is packaged and the type of sterilizer used. It is essential to maintain the required time when sterilizing any equipment to keep it practical.
Time and Temperature in Autoclaving
When designing an autoclave for the first time or upgrading an existing one to improve efficiency, you will need to consider temperature and time requirements. These are the parameters that control the rate and depth of sterilization. The use of autoclaves for sterile processing needs these conditions to be met.
With the four parameters being set, the minimum sterilizing conditions in gravity autoclaving are 121° C (250°F) for the TDT (Thermal Death Time) of 15 minutes using saturated steam with the pressure of at least 15 psi. On the other hand, 132°C (270°F) for 4 minutes is the minimum sterilizing condition in a pre-vacuum sterilizer. Increasing the cycle time is not required but recommended depending upon the make-up and amount of the load. It is to ensure that you will eliminate all unwanted microbes present.
As mentioned earlier, the required time and temperature can significantly be affected by how the packaged is wrapped and what sterilizer is being used. Below is a chart showing the different time and temperature requirements during autoclaving and other processes:
You should understand temperature and time requirements during the sterilization process. It is to help you understand the importance of using the right equipment. Knowing about these things first can save you from purchasing equipment that is either unsuitable or not working correctly.
Biological Indicators (BIs) in Autoclave Validation
As previously stated, biological indicators substantiate the instruments used to verify the practical function of autoclaving. Additionally, biological indicators are viable microorganisms. These microorganisms are resistant to sterilization processes, enabling monitoring whether the required sterilization conditions are met when eradicating the undesirable microbes present.
The Use of Endospores
Because they can withstand the high-level sterilization process, bacterial spores become the primary microorganisms used in BIs. The following are some of the toughest microorganisms used as indicators:
- Geobacillus stearothermophilus (GS) spores. The spores are used in the sterilization process, which involves steam and vaporized hydrogen peroxide.
- Bacillus atrophaeus (BA). It is used in sterilization processes that include dry heat and ethylene oxide (EO).
The biological indicator's positive result shows that the sterilizer can effectively kill many highly resistant bacterial spores. Users can be confident in the sterilization process by using this indicator.
Effectivity Validation
The use of biological indicators allows qualifying, routing and load monitoring of the autoclave process. They help indicate if the conditions during the steam cycle were satisfactory for producing a particular level of microbial inactivation.
The Association for the Advancement of Medical Instrumentation (AAMI) considers the use of biological indicators to be "the cornerstone for the sterilization process's quality assurance program." The BIs' proof-positive method of confirming the elimination of microbial contaminants is far superior to monitoring with chemical and mechanical indicators. As a result, BI is more reliable than other available indicators.
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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 kinds and numbers of organisms, the amount of organic matter, and 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.
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 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: Biohazardous and Medical Waste Disposal)
- 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 BSL-3 laboratories is required to be autoclaved prior to removal from the laboratory (see Chapter 9: Biohazardous and Medical Waste Disposal).
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). Because of the hazardous nature of the gases and vapors used, this requires specially designed equipment and facilities.
Autoclaving uses saturated steam under pressure (approximately 15 psi) to achieve a temperature in the autoclave of at least 121° C (250° 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 only EHS-approved autoclaves can be used to decontaminate biological waste, and that all decontaminated waste will still be packaged and shipped off-site for destruction as regulated biological wastes. Autoclaving of these materials simply makes them safer for handling and transport, it does not affect the disposal endpoint.
When using an autoclave, the following guidelines should be taken into consideration:
- Biohazardous materials should not be placed in 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.
- 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:
TemperatureAn 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.
TimeA 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.
ContactSteam 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.
Requiring higher temperature and longer contact time, dry heat 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.
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.
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 CompoundsUse 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 CompoundsUse 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
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 CompoundsUse 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.
GlutaraldehydeUse 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.