Hospital Epidemiology and Infection Control
3rd Edition

Chapter 86
Sterilization and Pasteurization
John H. Keene
Although chemicals have been used empirically for centuries to preserve foods, sterilization methods for medical equipment and devices are a relatively recent development. The recognition and acceptance of germs as a causative agent of disease led, in the mid- to late 1800s, to the realization that removal of germs from surgical instruments and other hospital equipment would protect patients from life-threatening infections. Pasteur’s laboratory experiments led him to discover the dust on his laboratory instruments and that passing them through a flame before use prevented contamination of his experiments (1). The use of heat as a sterilizing agent was not readily accepted, but as the positive results became known, more emphasis was placed on sterilization processes in the medical field.
Sterilization has become a prerequisite for certain procedures and devices, and a whole industry has been developed to provide new, better, and more cost-effective ways of ensuring sterilization of medical equipment and devices. It is important to understand the terminology and technology of sterilization and to recognize the importance of following appropriate procedures to achieve sterilization. In addition, it is important to understand the need for and methodology of validating and documenting sterilization procedures. Failure to implement the appropriate sterilization process leads to contamination of critical instrumentation, infection of patients, and potential loss of life.
In many cases, the destruction of common pathogens is sufficient for a particular process. However, it is important to remember that any microorganism in the wrong place at the right time is a potential pathogen. Because the current patient population is at particular risk for infection due to immune incompetence (patients infected with the human immunodeficiency virus, transplant patients, cancer chemotherapy patients, and elderly patients), sterilization verification becomes even more important.
In addition to their concern for proper sterilization processes and protection of patients, healthcare administrators must also be concerned with the health and safety of their personnel and the potential for environmental contamination. Sterilization processes designed to destroy microorganisms carry with them the potential to harm the personnel who must perform the processes and the potential to result in environmental damage. Appropriate mechanisms for minimizing personnel exposure and environmental release must be developed and incorporated in the operation of the healthcare facility.
Disinfection is a process that results in the destruction of infectious agents on inanimate objects but does not necessarily destroy all bacterial spores. The process may be a result of treatment with chemicals or physical agents. Although the term disinfection is often used synonymously with sterilization, it is not the same, and the two processes should be considered separately. Disinfection is the subject of Chapter 85 and is not further considered here.
Sterilization, on the other hand, is defined as a process that results in the destruction or elimination of all forms of life, including bacterial spores. The term is most often used in the context of destroying microorganisms. Sterilization is an absolute in that a material, when sterile, cannot be contaminated with any form of viable microorganism. However, the term has been used to denote the filter treatment of fluids that removes bacteria, fungi, and spores but not viruses. Therefore, one must understand the limitations of the sterilization process before accepting the product as truly sterile.
The verification of sterility would necessarily depend on the ability of personnel to demonstrate the destruction of all living microorganisms. Such verification would suppose that we have knowledge of all living microorganisms and can demonstrate their existence. Because this is virtually impossible and totally impractical, the efficacy of sterilization processes is most often demonstrated through the use of known highly resistant microorganisms as indicators, and the verification of sterility becomes a matter of probability. The assurance of the completion of the sterilization process is different for differing operations and is measured by the percentage of reduction, or log reduction (D value), in initial counts of biologic indicators that is accomplished by the process.
Pasteurization is historically defined as the heating of materials to temperatures of around 60°C for 30 minutes to destroy pathogens that may be present, although other time/temperature relationships have also been used. The process of pasteurization is also used for the reduction of infectious agents in liquids and has been tried in the processing of various devices, particularly anesthesia equipment and various types of scopes. It should be noted that this is not sterilization and should not be used for devices when there is a critical need for a sterilized product.

Filtration is another mechanism for treatment of air and fluids to reduce microbial contamination. Although filtration is often referred to as a sterilization process, it is possible for viral particles and bacteria to pass through many filters. Properly chosen and controlled, however, this process can be used to ensure that fluids and air are free from bacterial, mold, and particulate contamination.
Several criteria of sterilization as an absolute process should be recognized:
  • Thermal death time: the time required to kill all spores at a specified temperature;
  • D value: the time required to reduce the microbial population by 90% or 1 log;
  • F value: the time in minutes required to kill all the spores in suspension when at a temperature of 121°C or 250°F.
The D and F values can be used to evaluate various methods of sterilization.
The kinetics of inactivation and principles of thermal destruction of microorganisms are beyond the scope of this chapter; interested readers are referred to the excellent treatises of Wickamanayake and Sproul (2) and Pflug and Holcomb (3).
The efficacy of various sterilization processes depends on a number of factors. Each factor that must be considered in choosing a particular process and determining the efficacy of that process is discussed below.
Natural Resistance
Sterilization depends on the inactivation of microbial life processes faster than the microorganism can replace or repair the destroyed cell material. Death curves for microorganisms are generally accepted to be logarithmic, and variations from log death curves are considered to be due to variations in the nature of the microorganisms in question (4). Some antimicrobial agents (chemical disinfectants and antibiotics) react with crucial enzymes or interfere with enzyme systems within the cell. Cells that are not killed by the initial insult often are genetically different from those that are killed and have altered enzymes or enzyme systems that allow for survival. These microorganisms then multiply and become the resistant population. Sterilization processes do not lead to resistant populations because, by definition, all microorganisms are killed by the process.
However, even in regard to sterilization processes, genetics do play a significant role in protection of microorganisms. For example, bacterial spores are significantly more resistant to various sterilization processes than are vegetative cells. The natural resistance of spores is primarily due to the chemical composition of the spore coat and differs for different genera and species.
The requirements for sterilization differ for different microorganisms according to the genetic makeup of the microorganism. Some microorganisms grow well at temperatures normally used for pasteurization and must be treated at higher temperatures. Some microorganisms can rapidly repair radiation damage; thus, the level and duration of radiation treatment must be increased to ensure inactivation. Some microorganisms can find their way through filter materials, and the final use of the filtrate must be considered. This is not to imply that sterilization cannot be accomplished, but only to point out that the process must be carefully determined and scrupulously monitored to ensure the desired result.
Microbial Load and Extraneous Organic Materials
The final outcome of the sterilization process depends on the numbers of microorganisms initially present in the material to be sterilized, and the values that define the process all depend on the initial number of microorganisms. Therefore, the higher the number of microorganisms in or on the materials to be treated, the longer or more concentrated the treatment must be to achieve sterilization.
In addition to the effect of bioburden on the final outcome of the sterilization process, extraneous organic materials also contribute to the efficacy of the process. As with microorganisms, excess organic material increases the duration of, and changes the requirements for, the sterilization process. Organic material serves to protect the microorganisms from the effects of the specific process and may cause process failure.
Sterilization processes have been developed to ensure successful sterilization. Because, in a practical sense, sterilization is a statistical phenomenon, we must assume that a given process will result in complete kill of any microorganisms present, and our assumption must be based on the monitoring data obtained from that process.
As in disinfection processes, a number of factors, aside from the natural resistance of spores and other microorganisms, affect the efficacy of any sterilization process. These factors include time, temperature, relative humidity (RH), pH, and standardization of loads.
All sterilization processes require time for completion. The time required primarily depends on the process (e.g., wet heat, dry heat, gas, radiation). In addition, the time depends on the presence or absence of organic material and bioburden. The time required for adequate processing is determined by the use of indicator microorganisms that are known to be particularly resistant to the process being used. The sterilization process is defined in terms of time required to kill all spores present or to reduce the

number of microorganisms present by 90%. Because microbial death curves tend to be exponential, extrapolations can ensure that appropriate times are used to allow for destruction of the microorganisms that might be present.
Microorganisms generally have an optimal growth temperature above which they do not grow well or they die. Therefore, increasing the temperature of a sterilization process above the optimal growth temperature for the microorganism in question would increase the efficacy of a process.
Relative Humidity
The role of RH has been studied with regard to both heat sterilization processes and chemical (gas) processes. RH is defined as the ratio of the actual water vapor pressure in a system to the saturated water vapor pressure of the system at the same temperature. This term describes the water conditions in the atmosphere and as such inherently describes the water condition of the microbial cell or spore. Water activity is the relative water availability in a cell or spore and depends on the RH. There is an inverse relationship of cell resistance to water activity. It appears that, in most instances, the more water available to the vegetative cells or spores, the faster the heat inactivation process. Any sterilization process should account for the RH (5).
As with disinfectant activity, the pH of the suspending medium appears to play a role in the sensitivity of microorganisms and spores to heat inactivation. A number of studies have demonstrated that a lowered pH may result in decreased resistance to heat treatment for bacterial species and spores, but the opposite is true for yeasts (3). It is postulated that pH changes alter the degree of dissociation of materials in solution, resulting in a shift of the oxidation reduction potential, thus affecting the survival of microorganisms.
Load Standardization
For all contained sterilization processes, it is important that loads be standardized to ensure a uniform process. Loads can vary in a number of ways, including the number of packs, the volume of the packs, the size of the packs, and the contents of the packs. Failure to standardize loads (i.e., instrument packs, linens, routine loads) adds another variable to the process. Theoretically, if a sterilization container/process [e.g., autoclave, ethylene oxide (ETO) unit] is tested and validated with a given load, any change in that load could result in a failure of the process. However, as a practical matter, the parameters of sterilization are chosen to ensure overkill, and failure of the process is most often due to actual equipment failure or to failure of personnel to monitor or to adequately follow the instructions for performing the process.
Since the beginning of recorded time, heat in one form or another has been used to cleanse and purify. In the medical field, hot air ovens, which require extended process times to be effective, have been used to sterilize materials and equipment that must be kept dry. On the other hand, moist heat (steam sterilization) processes have been found to be a more rapid and effective method of sterilization for those materials with which they are compatible.
Steam Sterilization
Steam sterilization is the most common of all the sterilization procedures used in the healthcare facility, because steam under pressure has been found to effectively destroy even the most resistant bacterial spores during a brief exposure. Steam sterilization is universally used except where heat and moisture damage may occur to the material being sterilized.
Various types of steam sterilization equipment (autoclaves) have been developed and used with success over the years. It has been demonstrated that moisture is a necessary part of the steam sterilization process, because without moisture the process reverts to a dry heat process and requires longer exposure times. The major design features of steam sterilization equipment involve the mechanisms for removal of air from the load, thus ensuring complete mixing of the steam and elimination of cold spots in the autoclave. These mechanisms include gravity displacement, mass flow dilution, pressure pulsing, high vacuum, and pressure pulsing with gravity displacement. All these methods have been developed to help remove air from the system and from the materials to be sterilized to optimize efficiency and efficacy. Each method has its own deficiencies because of the physics and thermodynamics of steam, air, and water mixtures. The pressure pulsing gravity displacement system has been found to be most useful for general use, because it reduces the thermal lag on heating of the load to the desired exposure temperature.
Factors that can affect the efficacy of the steam sterilizer include the air tightness of the sterilizer, atmospheric pressure, quality of steam, and characteristics of the load. In autoclaves that use the vacuum process, air from outside the vessel may be brought in through leaks in the system. This may result in a failure of the system because of uneven heating and spot dry conditions. Autoclaves operated at or below atmospheric pressure are inherently subject to air leaks, and continued vigilance with regard to maintenance of equipment is necessary to minimize potential problems. Joslyn (4) has described a new mechanism whereby sterilization is performed using a pulse method in which the steam pulses are performed at pressures above atmospheric pressure. This process should eliminate problems associated with air leaks because it is performed completely under positive pressure conditions.
The quality of the steam introduced into the sterilizer is also important in ensuring appropriate operation of the device. The quality of steam is defined by the weight of dry steam in a mixture of dry saturated steam and water in the system. Ideally, 100% saturated steam

is required for proper operation of steam sterilizing equipment. Most equipment is designed with a steam separator and baffle that removes the water from the steam and directs the pure saturated steam to the chamber at the required velocity.
An appropriately designed autoclave operates efficiently regardless of the quality of the steam delivered to the equipment except when the separator or baffle malfunctions. Decreased steam quality (i.e., increased water content) may result in saturation of the materials such as dressings, wrappings, or linens. Excessive moisture then reduces the diffusion of steam throughout the load and, specifically, throughout the moisture laden packs. This may result in trapped air in the pack and increased time requirements for sterilization. In addition, grossly wet materials do not dry easily when the sterilization cycle is completed, and wet packs easily become contaminated.
Rutala et al. (6) showed that the type of container in which bags of waste were treated in a gravity displacement steam sterilizer had a significant effect on the sterilization time. These workers found that stainless steel containers allowed for optimal heat transfer and decreased the time required to sterilize the waste.
Although steam sterilization is the most common sterilization process used in the healthcare facility and personnel are most familiar with the process, the maintenance and operation of the equipment must still be closely monitored. The process is extremely complex and can be affected by a number of variables. Personnel responsible for steam sterilization in a healthcare facility should be familiar with all requirements for proper operation of the process. An excellent reference on the subject is found in the Association for the Advancement of Medical Instrumentation’s recommended practice (7). Failure to understand the steam sterilization process, the equipment operation, or the validation process could lead to sterilization failure and contamination of critical medical supplies.
Flash Sterilization
The process of flash sterilization is often used for treatment of items that have become contaminated in the operating suite and will be needed again in a short time. As mentioned in the section on steam sterilization, sterilization requires removal of air and replacement of that air with saturated steam. Materials to be flash sterilized may inherently trap air in the system (e.g., porous linen, lumens of instruments), and varying conditions are necessary with these types of materials to ensure appropriate treatment. Flash sterilization for nonporous items is accomplished by heating to 270°F (132°C) for 3 minutes, or 10 minutes for porous materials, in a gravity displacement steam sterilizer. The actual sterilization cycle is the time required to heat up, treat, and cool down the sterilizer and therefore can take as long as 5 to 7 minutes for the 3-minute cycle and 12 to 18 minutes for the 10-minute cycle (8).
This process undoubtedly results in the destruction of most vegetative cells and viruses provided they are not protected by excess organic matter and the bioburden is low. Experimental evidence in the laboratory also indicates that the times and temperatures are sufficient to inactivate spores of Bacillus stearothermophilus. However, spore testing with commercial self-contained biologic indicators is often misleading (9). In addition, when this process is used, there is rarely time, before the use of the sterilized item, to allow for incubation of biologic indicators. Because of difficulties in verifying the validation process, Garner and Favero (10) have recommended against the use of flash sterilization for implantable items.
The cleanliness of the instruments to be sterilized, the condition of the autoclave, failure to document loads, and autoclave parameters all may affect the outcome of the process. It is important to recognize the shortcomings of flash sterilization and to use this process sparingly if protection of patients from wound contamination is to be ensured.
Dry Heat Sterilization
Dry heat (hot air ovens) has been used for many years as a method for sterilizing glassware, instruments, and other critical supplies that, for various reasons, could not be sterilized by steam sterilization procedures. Although wet heat sterilization is defined as sterilization at an RH of 1% or 100%, the parameters of dry heat treatment are not so easily determined. Dry heat sterilization takes place at an RH of between 0% and greater than 99%. The conditions for effective dry heat sterilization depend on the amount of water in the materials to be sterilized and in the environment of the dry heat sterilizer. At any given temperature, the lower the RH, the longer the time required for sterilization in a dry heat process. An understanding of this phenomenon explains the conflicting requirements established by various regulatory agencies in different countries with regard to the parameters of dry heat sterilization. Generally, in the United States, the requirement for dry heat treatment of containers for pharmaceutical products is 170°C for 2 hours. The American process includes a significant protection factor if one assumes that the British Pharmacopeia requirement of 150°C for 1 hour is also efficacious (11).
Dry heat sterilization advantages include low corrosiveness and deep penetration. However, the heating process is slow, and long sterilizing times are required. Materials may also be damaged by exposure to high temperatures for long periods.
Although a number of testing procedures have been developed to demonstrate the dry heat inactivation of microbial cells and spores, none of these is readily acceptable as a routine mechanism for deciding the exact time and temperature to be used for the process in the hospital. A description of these tests is beyond the scope of this chapter. It should be sufficient to recognize that the process recommended in the U.S. Pharmacopeia includes an appropriate protection factor and therefore should be a safe procedure when necessary.
Since before the time of Hippocrates and the proclamation of the belief that infections were caused by miasmas and bad vapors, humans have sought a means of combating infectious diseases through the use of gaseous agents. The use of incense and frankincense was associated with concepts of purification of the air. Spices were placed in foods in the hope that their strong odors would prevent spoilage. In more recent times, the aerosolization of carbolic acid in operating rooms by Joseph

Lister, the use of sulfur dioxide and chlorine for terminal disinfection of a sick room, and the introduction of the use of formaldehyde for the same purpose did much to stimulate research on methods and mechanisms of gas sterilization. During the early part of this century, it was discovered that the terminal gas sterilization of sick rooms with formaldehyde was not as important as previously thought, and the emphasis on gas sterilization in the medical field declined.
With the advent of modern medical science and its plastics, electronics, disposables, and other heat-labile components, a new interest has developed in gaseous sterilization procedures for the medical field. Several gaseous agents have been used successfully to sterilize medical devices, instruments, and equipment. However, these agents can be toxic to people as well as the microorganism they are designed to destroy, and caution is needed to ensure appropriate protection of personnel and patients from exposure to many of these gaseous sterilants.
Ethylene Oxide
Phillips and Kaye (12), in a series of reports, reviewed the early literature concerning the use of ETO as a bactericidal agent. They proposed an alkylation reaction as the mechanism of action of this material and established the basic conditions under which ETO was most effective as a sterilizing agent. This was the beginning of a new era in the field of medical device sterilization. The development of ETO sterilization methods and procedures, pioneered by Phillips and Kaye and continued by numerous other investigators, has led to the widespread use of disposable equipment and supplies in the hospital industry. This trend has done much to decrease the possibility of cross-contamination and has aided in the battle against hospital-acquired infections. It has, however, also led to the discovery of the toxic effects of ETO, and the safety procedures for the use of this material must be carefully considered to avoid personnel, patient, and environmental exposure.
ETO is a colorless gas that is highly reactive with many different types of chemicals. ETO gas is highly flammable and explosive. However, mixture of the gas with carbon dioxide (13) or other gaseous carriers (fluorocarbons) (14) significantly reduces the fire hazards associated with the pure substance and allows its use, in special vessels, for sterilization. Mixtures of ETO in fluorocarbons appear to be more advantageous for use in hospitals, but information on the potential environmental hazards of these compounds has limited their use (15, 16, 17).
As was mentioned above, ETO mixtures have been used in sterilization processes. With the concern over fluorocarbons, processes have been developed using vacuum vessels in which a series of evacuations and backfills with nitrogen are used to ensure that the oxygen level remaining is insufficient to support combustion when pure ETO is added. The equipment used for the sterilization process is complex and has been developed to ensure appropriate mixing and control of the factors required (e.g., temperature, RH, ETO concentration) to achieve sterilization (18).
Formaldehyde has been shown, under appropriate conditions of temperature and humidity, to be both sporicidal and bactericidal. Although the use of formaldehyde in the decontamination of sick rooms in hospitals was considered helpful in the early part of this century, further study of the practice indicated that such a drastic method was not necessary to ensure cleanliness of rooms occupied by contagious persons. However, formaldehyde is still used as a fumigant for rooms and buildings in which massive contamination has occurred, such as mold growth in water-damaged buildings and in the terminal decontamination of high-containment biologic laboratories. It is the sterilizing gas of choice for decontamination of biologic safety cabinets and high-efficiency particulate air (HEPA) filter units.
Formaldehyde is generated by heating either paraformaldehyde or formalin to release the gaseous formaldehyde, and the activity of the formaldehyde depends on its condensation on contaminated surfaces. A procedure for microbiologic decontamination using paraformaldehyde has been published by the National Sanitation Foundation International (19).
Formaldehyde has also been demonstrated to be toxic to humans and has been classified as a potential carcinogen. The Occupational Safety and Health Administration (OSHA) has thus developed a standard regarding potential personnel exposure (20). Anyone using this material would be wise to review current federal and state regulations that might apply to both personnel exposure and environmental release.
Low-Temperature Steam Formaldehyde Process
Although pure formaldehyde has not been found to be particularly useful for medical device sterilization in the United States, European investigators have demonstrated that a combination of low-temperature steam and formaldehyde (LTSF) can be used. This process was first described by Alder et al. (21) in England. It was initially designed for the processing of cystoscopes and similar devices. Since that time, equipment has been developed that provides the necessary controlled conditions for sterilization of a wide variety of medical devices. Kanemitsu et al. (22) evaluated an LTSF sterilizer and concluded that this methodology was particularly useful because of its excellent efficacy, short handling time, and safety. However, these authors warned that the size of the load in the sterilizer affected its efficacy and that small loads were preferable to larger ones for processing.
The process involves the injection of dry formaldehyde gas into the treatment vessel followed by injection of steam to ensure an internal temperature of about 73°C and a holding time of 2 hours. The process is completed, and the residual formaldehyde is removed by further steam flushes and an introduction of sterile filtered air (23). As with ETO sterilization, the potential for residual formaldehyde on the surface of sterilized devices is a major concern. Nystrom (24) reported that studies in Sweden have shown that residual formaldehyde can consistently be kept under 5 mg/cm3. Nystrom also stated that the occupational exposure resulting from the operation of this process is well below the threshold limit value of 0.6 mg/m3 mandated by Swedish occupational health regulations.
Proponents of the process point to the facts that treated equipment needs less aeration than ETO does and that the in

creased temperature of operation increases the probability of sterilization success. In the U.S., however, this process has not been well accepted, probably because a reliable commercial process has not been validated, and there is considerable concern regarding the toxic and allergenic nature of formaldehyde.
Alternatives to Ethylene Oxide Sterilization
Concern over the hazards associated with the use of ETO have prompted many investigators to evaluate alternative methods for sterilizing heat-labile devices and instruments, including vapor phase hydrogen peroxide (VPHP) and various gas plasma technologies. Although not yet in widespread use, these technologies are beginning to be evaluated by healthcare facilities as safer more environmentally friendly alternatives to ETO processes.
Vapor Phase Hydrogen Peroxide
Liquid hydrogen peroxide (H2O2) has long been known for its ability to sterilize and its relative safety. Graham and Rickloff (25) reported on the development of a process using gaseous H2O2 at low concentrations and ambient temperatures to sterilize equipment and devices. It appears that sterilization can be achieved with this material with relatively short contact times. One of the major concerns of using relatively powerful oxidants for sterilizing medical devices has been the potential for damage to the devices. The short contact times required for the vapor phase H2O2 process appear to allow for reduced potential damage to devices because of possible oxidation.
Vapor phase H2O2 technology seems to have considerable potential in its use to replace ETO for the sterilization of heat-labile materials. Johnson et al. (26) and Klapes and Vesley (27) reported that VPHP generators have shown sporicidal activity and that, in their studies, the process shows promise as an effective and safe alternative method of sterilization. However, much work is still to be done with regard to such factors as compatibility studies and efficacy. In addition, the penetrability of H2O2 vapor through cellulosics is limited by absorption, which further limits the type of packaging available for this process. Nonetheless, VPHP sterilization is a promising alternative to more toxic and potentially environmentally hazardous methods of sterilization.
Plasma Gas Sterilization
Other alternatives to ETO sterilization have been developed and are currently available for use in healthcare facilities for the processing of heat-sensitive devices. These low-temperature plasma technologies include the Plaslyte (AbTox, Mundelein, IL) system that uses gaseous peracetic acid and the Sterrad (Advanced Sterilization Products, Irvine, CA) system that uses low-temperature H2O2 gas plasma (LT-HPGP). The ion plasma sterilization processes operate at relatively low temperatures by exposing peracetic acid or H2O2 to either strong electric or magnetic fields. Such exposure results in the formation of an ion plasma that contains reactive radicals that are known to be reactive with almost all molecules essential for metabolism and reproduction of living cells (e.g., DNA, RNA, proteins, etc.).
These technologies have stimulated interest in healthcare facility personnel, because they have short turnaround times compared with ETO sterilizers and are both more environmentally friendly and safer to use. Rutala and Weber (28) summarized the disadvantages of these methodologies. The authors stated that the use of the peracetic acid plasma method was limited to stainless steel surgical instruments (excluding lumen devices and hinged instruments). In addition, no liquids or materials that might be harmed by vacuum could be treated. The LT-HPGP process was limited by U.S. Food and Drug Administration (FDA) restrictions on treatment, by this method, of endoscopes and other medical devices with lumina longer than 12 inches or having a lumen diameter less than one-quarter inch (6 mm). Cellulose, linens, and liquids also cannot be processed in this device. Finally, the LT-HPGP process requires special packaging of devices and a special tray for processing.
A number of studies have demonstrated the efficacy of the LT-HPGP against viruses and parasites in the laboratory (29, 30). The efficacy of both processes in the treatment of medical devices was evaluated by comparison with the ETO 12/88 process by Alfa et al. (31). These authors concluded that the margin of safety for the methods tested was less than that of the 12/88 method and were concerned that even the 12/88 method failed to kill microorganisms in narrow lumen devices when salt or serum was present. They emphasized the need for scrupulous cleaning of the lumen of medical devices before treatment to ensure sterilization. Such research emphasizes the need for strict adherence to cleaning protocols before treatment of devices and provides valuable insight into a number of problems that can be associated with any alternative sterilization techniques. Bar et al. (32), concerned about reports of mycobacterial contamination of bronchoscopes, studied the use of LT-HPGP for the sterilization of these devices. Their results indicated that bronchoscopes washed and disinfected by conventional “washer/disinfector” as well as “intensive washing” (washing followed by glutaraldehyde treatment) still showed the presence of mycobacterium DNA, by nucleic acid amplification technique. Those scopes sterilized by the LT-HPGP were all negative by this test methodology. The authors concluded that LT-HPGP sterilization would be recommended if the nucleic acid amplification technique was to be used for the diagnostic procedure to verify sterility of the treated bronchoscopes.
Feldman and Hui (33) studied the compatibility of LT-HPGP sterilization with various medical devices and materials. The authors reported that in their studies of over 600 individual resterilizable devices from more than 125 manufacturers, approximately 95% of the devices could be safely sterilized by this process. They listed various materials that could be considered for LT-HPGP processing, including stainless steel (300 series), aluminum (600 series), titanium, glass, silica ceramic, and a number of plastics and elastomers. They also studied numerous adhesives and provided a listing of the adhesives that proved to be most compatible with the process.
Although these alternative methods have been developed in response to the patient, occupational, and environmental safety hazards associated with the use of ETO sterilizers, they may not themselves be without potential hazard. A recent report of several cases of corneal endothelial decompensation resulting from sur

gery with instruments sterilized in the peracetic acid plasma system has raised questions about the possible interaction of the sterilizing agents with the brass-containing parts of the instruments, resulting in release of metal compounds that can cause corneal decompensation. Studies are currently underway to verify the connection between the sterilization process and the injuries (34). Ikarashi et al. (35) also reported on the cytotoxicity of various medical materials exposed to a VPHP sterilization process, thus emphasizing the requirement for further investigation regarding the need for aeration to remove cytotoxic residuals from materials treated by the alternative techniques.
Although ionizing radiation is not commonly used in the hospital for the sterilization of equipment and medical devices, it is an important process in the manufacture and packaging of devices used in the healthcare facility. Many of the devices that are supplied sterile to the hospital, such as plastic hypodermic syringes and catheters, are formulated to be sterilized by gamma radiation and may be damaged or may not properly function when sterilized in any other manner. These items are considered to be single-use items and are not to be resterilized once they have been opened and contaminated, unless the manufacturer guarantees the safety of the device after resterilization (see Chapter 87). Radiation causes little or no damage to the materials treated and leaves no residual radioactivity. Radiation of drugs, pharmaceuticals, and tissues for transplantation has also been successful.
Although there are a number of proposals for explaining the radiation inactivation of microorganisms, the effect of radiation appears to be a result of damage to DNA. Resistance to radiation treatment appears to depend on the microorganism’s ability to repair the DNA damage (36, 37). As with other sterilization processes, it has been generally accepted that bacterial spores are the most resistant microorganisms, and that demonstration of the killing of spores is an appropriate demonstration of the efficacy of the radiation sterilization process. It appears that although bacterial spores are the most resistant and gram-negative rods appear to be the least resistant to radiation damage, a number of inherently radiation-resistant microorganisms do exist and could be present in or on items to be sterilized. Members of the genus Deinococcus appear to be extremely resistant to radiation (38). In addition, other microorganisms (specific Moraxella, Arthrobacter, Acinetobacter, and Pseudomonas species) have been shown to exhibit enhanced resistance to radiation damage.
Procedures for ensuring the sterility of irradiated products have been proposed by the Association for the Advancement of Medical Instrumentation (39). These procedures are based on the known bioburden of the product, dose of irradiation, and good manufacturing procedures as required by the FDA.
Although filtration is an important process in the preparation of a variety of liquid products used in the healthcare facility, in general it cannot be considered a mechanism for sterilization. Strict interpretation of the term sterilization implies killing or removal of all forms of life. Filters in use for the sterilization of such items as intravenous additives, drugs, and vaccines are, for the most part, bacterial filters. They do not, nor are they designed to, remove viruses. On the other hand, the materials that are treated by filtration are not expected to have live virus in them. Still, the fact that this process is designed for removal of bacteria must be considered.
Processing of fluids in the healthcare setting is discussed by Eudailey (40). Procedures for ensuring the quality of filtered materials, with particular reference to hospital pharmacy prepared intravenous fluids and hyperalimentation fluids, are presented in a number of articles on the subject (41, 42, 43, 44).
The type of filter to be used for a particular operation depends on the operation and the requirements for the final product. Different types of filters are used for specific processes. The filter media range from deep filters of various materials (e.g., fiberglass, cotton, resins, porcelain, diatomaceous earth) to membrane filters of cellulose and other polymers. Depth filters have the advantage of being able to handle large amounts of contaminants throughout their thickness and can often retain particles smaller than their normal size rating because of adsorption of the particles on the filter. These filters, however, have some disadvantages. They tend to allow media migration in that the filter media may be released and travel through the filter and in fact may contaminate the product. There may also be a release of microorganisms as material passes through the filter during long process times. The filters may also retain significant amounts of fluid product, which can be a problem if the product is particularly valuable. Membrane filters, on the other hand, do not suffer from the problems of media migration or potential release of filtered microorganisms. They are efficient, and there is no retention of fluid product. The major disadvantage of the membrane filter is the fact that it tends to get clogged by excess dirt in the system.
It should be noted that filtration is also important in the removal of microorganisms and particulates from gases and air. The filtration capacity of such filters primarily depends on impaction, diffusion, and electrostatic charge. Particles traveling in an air stream tend to stay in that stream. Filtration is accomplished when the particles in the air stream have an impact on the surface of the filter fibers. The higher the air velocity, the greater the surface area of the filter, and the smaller the diameter of the fibers, the higher the probability of impaction. Diffusion also plays a part in the filtration process. Low-velocity air flow favors diffusion of the particulates to the filter surface, and very small (low-mass) particles tend to diffuse in the depths of the filter and are intercepted by the filter. HEPA filters have an efficiency of at least 99.97% at 0.3μm. These filters, by design, are more efficient for particle sizes above and below 0.3 μm.
Laminar-flow HEPA filtration units have been suggested for operating rooms, isolation rooms, and laboratories. It should be noted that at the point of release from the filters, the air is sterile, but as with any other sterilization process, the air quickly becomes contaminated from contact with unsterile materials. The use of these units should be tempered with an understanding of their limitations and the potential for recontamination of the air. It should also be noted that the filters used to remove infec

tious agents from the air are considered to be contaminated with those infectious agents. Personnel charged with maintenance, testing, and removal of the filters should be appropriately cautioned with regard to the hazards involved with these procedures.
Pasteurization is a process of inactivation of the vegetative cells of pathogenic bacteria and of viruses by heating at relatively low temperatures. The process has found widespread use in the food industry since its development by Louis Pasteur. The actual time/temperature conditions for pasteurization vary with the type of material being treated and the personnel performing the process. Historically, pasteurization for milk involves heating to approximately 60°C for 30 minutes or to 70°C for 15 to 20 seconds. Anesthesia equipment has been pasteurized by using an exposure to hot water at 75°C for 10 minutes (45). Treatment of plasma fractions at 60°C for 6 hours has been used for inactivation of viruses in the production of blood products (46). All these processes use the principle of heat inactivation of vegetative cells and viruses to ensure appropriate kill times.
The major disadvantages of pasteurization in the treatment of critical materials is the lack of standardization of the equipment and difficulty of validation. Because this is not a sterilization process, extreme care must be taken to ensure that the process is performed so that agents considered to be particularly important are inactivated.
Major research studies on sterilization indicate that there is more to be learned with regard to sterilization processes (2, 3). Research in the laboratory has been directed at the mechanisms of action of various sterilization processes, and the results have been conflicting, because there is so much variation in the conditions of the studies. Although much has been learned, the information gained is not always directly applicable to the real-world process in that microorganisms are not the same and conditions with regard to composition of loads, organic load, and bioburden are constantly changing. Therefore, any validation process must consider the variability inherent in the process and demonstrate overkill if sterilization is to be ensured.
Historically, the spores of bacteria have been thought to be the most resistant microorganisms with regard to heat, radiation, and chemicals. It has been natural to assume that processes that result in inactivation of these spores would provide a significant margin of safety to ensure sterility of the products treated by these processes. Spore suspension testing requires specific laboratory procedures and considerable incubation time. Alternative chemical indicators have been developed and compared with spore tests with good results (47), but spore testing continues to be the standard.
Although indicators are an important part of quality assurance of sterilization processes, the validation of the process and documentation of the actual operating parameters of the process are of paramount importance. It should be noted that spore and chemical indicators testing can only be as good as the placement of the spore suspensions or indicators. Failure to place the indicators in appropriate places in the load leads to false-negative results (i.e., apparent sterility when the items are not really sterilized). All sterilization processes should be thoroughly evaluated before being put into service and at regular intervals. Autoclaves should be mapped with thermocouples to determine potential cold spots. Filter systems should be tested for leakage. Gas sterilization units should be appropriately validated for such factors as gas concentration, temperature, and RH.
The sterility assurance level for a particular sterilization process is not routinely determined in the healthcare facility, because personnel lack expertise in the procedures. Young (48) has discussed cycle times and safety factors for steam and ETO sterilization cycles to be used in hospitals. Validation of healthcare facility sterilization equipment is primarily performed by the manufacturer of the equipment. To ensure appropriate sterilization processes, healthcare facility personnel must ensure that all manufacturer recommendations are met. The daily operation of the sterilizing processes must be documented by personnel performing the process. This documentation should be reviewed for each operation, and any malfunction should be noted and appropriate action taken to ensure that the product either has been properly treated or is returned for reprocessing.
In light of the advent of new medical devices, intricately designed with heat-sensitive parts and narrow lumina, the mechanisms for appropriate sterilization become a matter of concern for patient safety. In a provocative editorial, Rutala and Weber (28) questioned whether or not, because of the development of low-temperature sterilization technologies, there is a need to redefine sterilization. Current FDA requirements stipulate that a sterilizer’s microbicidal performance must be tested under specified simulated use conditions, which include that the test articles must be inoculated with 106 colony-forming units (CFU)/unit of the most resistant test microorganism prepared with inorganic and organic test loads. The inocula must be placed in various locations on the test articles, including those least favorable to penetration and contact with the sterilant (49). Rutala and Weber, however, argue that these requirements may be too restrictive and that the requirements for efficacy should include the demonstration by instrument/device manufacturers that cleaning followed by a sterilization process can inactivate a clinically relevant inoculum of highly resistant microorganisms in the presence of an organic load in the most inaccessible location in the device. They note that the responsibility for defining the efficacy of new sterilization technologies should be met by the FDA, the device manufacturer, or the sterilizer manufacturer.
It seems logical that medical device manufacturers should take the lead in the evaluation of new sterilization processes for their own devices and that they should recommend the safest, most environmentally friendly, and cost-effective technologies available. Healthcare personnel must be aware of the problems associated with new and existing technologies and ensure that whatever process is used, it will be safe and effective.
New methodologies always bring with them new benefits as well as new potential hazards. New sterilization technologies are

no exception. The benefits of new technologies must be reviewed and verified so that decisions can be made regarding the efficacy of medical devices as related to the sterilization process. Nuutinen et al. (50) studied the effect of various sterilization processes on the physical and mechanical properties of self-reinforced bioabsorbable fibers made out of polylactide (PLLA). The intrinsic viscosity, crystallinity, and mechanical properties (modulus of elasticity, yield strength, and ultimate tensile strength) were tested before and immediately after each sterilization treatment, as well as up to 30 weeks in vitro. Compared with unsterilized fibers, the intrinsic viscosity was markedly decreased after radiation sterilization (gamma and electron beam), and the loss in mechanical properties was accelerated during in vitro degradation. Plasma and ethylene oxide (one and two cycles) did not markedly alter the properties of the samples after sterilization or during in vitro degradation. The authors concluded that their data are important for determining the effect of various sterilization processes on the physical and mechanical properties of polylactide-based materials and can be used to predict how fast degradation of the mechanical properties of the self-reinforced PLLA will occur. They can also be used to tailor the degradation kinetics to optimize implant design.
With the advent of new medical devices that are heat sensitive, the search for a safe, effective sterilization methodology that is compatible with the device materials has accelerated. Although the use of various oxidizing agents, coupled with ionization procedures (as in LT-HPGP), or VPHP generators have become more popular and are replacing the more toxic ETO processes, there are potential problems with the integrity of the materials treated by these processes. Hopper et al. (51) postulated that conventional polyethylene liners cross-linked by sterilization with gamma radiation in air had better in vivo wear performance than non–cross-linked liners sterilized with gas plasma. The polyethylene liners that had been sterilized with gamma radiation in air had a significantly lower wear rate than did the gas-plasma–sterilized liners. The authors concluded that in vivo wear of conventional polyethylene liners that had been sterilized with gamma radiation in air was, on average, 50% less than that of non–cross-linked liners sterilized with gas plasma. In a comprehensive study of the safety of plasma-based sterilization, Lerouge et al. (52) used both the Sterrad and Plazlyte processes to evaluate the induction of surface modifications on polymeric medical devices. They observed surface oxidation and wettability changes on all surfaces sterilized by these techniques. The type and severity of the modification varied with the sterilizer and the type of polymer sterilized. It should be noted that these observed changes have not been shown to be particularly detrimental to patients, but further studies need to be performed to ensure the safety of this technology.
Sterilization and disinfection processes for medical devices and equipment have been developed specifically to prevent infections due to contamination of these materials. Obviously, if a material has been sterilized and is kept from being contaminated before use, there is no chance of infection in a patient exposed to it. Failure to appropriately perform or monitor the sterilization process or unvalidated changes in equipment or product, however, may result in an unsterilized product.
Bryce et al. (53) reported on an outbreak of Bacillus cereus in intensive care unit patients on respirators. The infections were traced to ventilator circuitry that had been pasteurized. The infections were due to the presence of a spore-forming microorganism, and the method for treatment of the equipment was not sufficient to kill the spores of the offending microorganism. A similar outbreak involving Flavobacterium meningosepticum was reported by Pokrywka et al. (54). In this outbreak, it was discovered that the pasteurization units were operating at suboptimal temperatures, thus allowing survival of the microorganisms.
Kaczmarek et al. (55) studied disinfection/sterilization practices for endoscopes in healthcare facilities and reported that the disinfection/sterilization procedures are not always optimal and that variation occurred even within hospitals. Pattison et al. (46) reported on an outbreak of hepatitis B associated with transfusion of commercially prepared plasma protein fraction that had undergone pasteurization in the preparation process. Nineteen of 31 patients receiving the plasma fraction had developed illness compatible with hepatitis B. The plasma had been subjected to treatment at 60°C for 10 hours, but the authors suggest that the process was inadequate to destroy the hepatitis B virus.
Although the study of Kaczmarek et al. concentrated on disinfection procedures and a number of outbreaks of nosocomial infection have been traced to inadequately disinfected materials and devices, few cases of nosocomial infection have been traced specifically to failure of sterilization processes. The notable exceptions are the outbreaks of nosocomial sepsis that have been traced to commercial intravenous fluids. Duma et al. (56) reported an outbreak of septicemias specifically related to intravenous infusions in 1971. Goldmann et al. (57) reported a nationwide outbreak of Enterobacter and Erwinia (Enterobacter agglomerans) infections traceable to commercial intravenous fluids that occurred in 1971. Goldmann et al. suggested that appropriate surveillance data were available before the dates of the outbreak and were sufficient to predict that there was a problem and that proper analysis of the data could have prevented the outbreak. In 1981, a second outbreak of nosocomial Enterobacter infections was traced to contaminated commercial intravenous fluids (58). During this outbreak, the contamination was shown to be present in the screw caps of bottles. The contamination was apparently protected from coming in contact with steam during the sterilization cycle by the design of the cap and thus was not subject to appropriate sterilizing conditions. These outbreaks point to the need for constant attention to detail with regard to ensuring the effectiveness of sterilization cycles and to review of surveillance data with particular regard to infections with exotic microorganisms associated with apparently sterile devices and fluids.
Sterilization processes are designed, by definition, to eliminate all forms of life. As a result, these processes are inherently hazardous to those personnel involved with them. It is impera

tive that personnel understand the hazards of the process that they are required to perform. They must be trained in the use of appropriate personal protective equipment and understand and be able to carry out emergency procedures that would minimize personnel exposure to the sterilization process. Table 86.1 shows some of the potential hazards associated with major sterilization processes and includes reference to the OSHA standards that specifically apply. It is obvious that the hazards of sterilization processes involve both potential physical hazards such as heat and radiation and potential exposures to chemically hazardous materials such as the sterilant gases and their carriers. The administrative and supervisory personnel of each facility must recognize the hazards associated with the processes being performed in that facility, must develop appropriate safety procedures to protect the personnel involved, and must ensure that those procedures are being followed.
Ethylene Oxide Safety
A number of reports have demonstrated the dangers of ETO to both patients and personnel. Both human and animal studies suggest that ETO is a potential occupational carcinogen, causing leukemia and other cancers. ETO has also been linked to reproductive damage, including spontaneous abortions, cytogenetic damage, neurologic effects ranging from nausea and dizziness to peripheral paralysis, and tissue irritation (59).
OSHA has issued a standard (60) that sets a limit on worker exposure to ETO averaged over an 8-hour day. The standard was amended in 1988 to further reduce the health risk associated with ETO by requiring control of short-term exposures as well.
The key provisions of the ETO standard include a limit on workplace exposure of one part ETO per million parts air (1 ppm) averaged over an 8-hour day, and an excursion limit of 5 ppm averaged over a sampling period of 15 minutes. Employee rotation is prohibited as a means of compliance with the excursion limit.
Where the excursion limit is exceeded, employers must do the following:
  • Use engineering controls and work practices to reduce exposure. These controls and practices may be supplemented by the use of respirators where necessary.
  • Establish and implement a written compliance program to achieve the excursion limit.
  • Establish exposure monitoring and training programs for employees subjected to ETO exposure above the excursion limit.
  • Identify as a regulated area any location in which airborne concentrations of ETO are expected to exceed the excursion limit.
  • Place warning labels on containers capable of releasing ETO to the extent that an employee’s exposure would foreseeably exceed the excursion limit.
Respirators can be used to control exposure only until feasible engineering and work practice controls are being implemented; during maintenance, repair, and other operations for which engineering controls are not feasible; in work situations wherein feasible engineering and work practice controls do not reduce exposures below the permissible exposure limit; and in emergencies.
OSHA has set an action level of 0.5 ppm. If the 8-hour time-weighted airborne concentration of ETO is at or exceeds the action level, employers must begin periodic exposure monitoring and medical surveillance. Employers who demonstrate that worker exposures are below the action level need not comply with most provisions of the standard.
If employers have not monitored worker exposures within the past year, they must do so for each job classification in a work area during each shift; representative sampling is permitted under certain circumstances. The frequency of subsequent monitoring depends on the results of the initial sampling. All monitoring may be observed by workers and their designated representatives.
A comprehensive medical surveillance program must be conducted by or under the supervision of a licensed physician. Workers must receive a medical examination before assignment to an area in which exposure is at or above the prescribed level, annually if they are exposed at this level for 30 days or more during the year, upon request if they develop symptoms suggesting overexposure or want medical advice concerning the effects of ETO exposure on their ability to produce a healthy child, and when they end employment in an area of exposure.
Other requirements include identification of excessive exposure areas, communication of hazard to affected employees, and OSHA record keeping.
Patient Safety
Because of the potential toxicity and resultant hazard to patients, it is important for persons using ETO for sterilization of medical devices to ensure adequate aeration for treated materials. The aeration process reduces ETO residues in and on the devices

to a level that will not cause problems for patients or personnel exposed to the treated materials.
Formaldehyde Safety
Studies indicate that formaldehyde is a potential human carcinogen (20). Airborne concentrations above 0.1 ppm can cause irritation of the eyes, nose, and throat. The severity of irritation increases as concentrations increase; at 100 ppm, exposure to formaldehyde is immediately dangerous to life and health. Dermal contact causes various skin reactions, including sensitization, which might force sensitized persons to find other work.
To protect workers exposed to formaldehyde, the OSHA formaldehyde standard (61) applies to formaldehyde gas, its solutions, paraformaldehyde, and a variety of other materials that serve as sources of the substance. In addition to setting permissible exposure levels and exposure monitoring and training, the standard requires medical surveillance and medical removal of sensitized personnel, record keeping, regulation of potentially hazardous areas, hazard communication, and emergency procedures. Employers are to ensure primary reliance on engineering and work practices to control exposure. Selection and maintenance of appropriate personal protective equipment by employers is also required. If respirators are necessary, compliance with the OSHA respiratory protection standard is required. In addition, training is required at least annually for all employees exposed to formaldehyde concentrations of 0.1 ppm or greater.
The permissible exposure limit for formaldehyde in all workplaces covered by the OSHA Act is 0.75 ppm measured as an 8-hour time-weighted average. The standard includes a 2 ppm short-term exposure limit (STEL) (i.e., maximum exposure allowed during a 15-minute period). The action level is 0.5 ppm measured over 8 hours.
As with the ETO standard, the formaldehyde standard requires that the employer conduct initial monitoring to identify all employees who are exposed to formaldehyde at or above the action level or STEL and to accurately determine the exposure of each employee so identified. If the exposure level is maintained below the STEL and the action level, employers may discontinue exposure monitoring until such time as there is a change that could affect exposure levels. The employer must also monitor employee exposure promptly upon receiving reports of formaldehyde-related signs and symptoms.
A medical removal protection provision is included in the standard for employees suffering significant adverse effects from formaldehyde exposure. This provision requires that such employees are removed to jobs with less exposure until their condition improves, or for a period of 6 months, or until a physician determines that they will not be able to return to any workplace with formaldehyde exposure.
Occupational Safety and Health Administration Hazard Communication
The hazard communication standard (62) requires identification and appropriate labeling of all hazardous chemicals in the workplace. This standard also requires appropriate training and medical monitoring of personnel. In addition to the general requirements of the hazard communication standard, other standards for specific hazardous chemicals also require certain labeling.
The formaldehyde standard specifically delineates requirements for labeling of formaldehyde, including mixtures and solutions composed of 0.1% or greater formaldehyde and for materials capable of releasing formaldehyde in excess of 0.1 ppm. Hazard labeling, including a warning that formaldehyde presents a potential cancer hazard, is required where formaldehyde levels, under reasonably foreseeable conditions of use, could exceed 0.5 ppm. The ETO standard also has provisions for labeling containers that might release substantial quantities of ETO in excess of the excursion limits set by the standard.
Environmental Safety
In addition to the potential for personnel exposure, environmental concerns must be addressed. This is particularly true for the release of agents such as ETO and formaldehyde. The carrier for ETO may also be a potential environmental hazard, because the chlorinated and fluorinated hydrocarbons that have historically been used as a carrier to minimize the explosiveness of the ETO have been banned. These agents can be toxic in the environment and are regulated by either federal or state regulations concerned with toxic releases to air and water. It is important to realize that such environmental regulations are constantly being evaluated and revised by the regulatory sector, and specific references to such regulations in any textbook would undoubtedly be dated. It should be sufficient to warn that administrative and supervisory personnel must evaluate the release of these materials from the facility with regard to specific applicable regulations.
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