Current practices and knowledge about sterilization of pharmaceuticals, medical devices, and other related products flow from thousands of years of discovery. Steam sterilization began during the renaissance when Denis Pavan invented the “digester” or pressure cooker.1
It was found that heating water in a sealed vessel heated the air, causing it to expand. The pressure generated from this closed system caused the boiling point of water to increase from 100°C to 121°C at 15 psi, allowing food to cook more quickly. Later embodiments of this invention, which include microprocessor controls of temperaturepressure ranges, air removal, and pharmaceutical clean steam, have led to the present day commercial moist heat steam sterilizer.
In 1683, microbes became visible to the naked eye for the first time as Antonie van Leeuwenhoek improved on a primitive microscope that proved the existence of microorganisms. From 1860 to 1864, the French biologist Louis Pasteur convincingly demonstrated the germ theory of disease, namely that disease was caused by microorganisms and not by spontaneous generation. Pasteur demonstrated that processing by heat (ie, pasteurization) of wine and beer eliminated unwanted microbes. He recognized the importance of moist and dry heat as sterilization techniques. Charles Chamberlain, working in Pasteur’s laboratory, improved on the digester design and, in 1879, invented the autoclave sterilization process wherein time and sterilization temperature was controlled and monitored. By the late 1800s, governments began to enact legislation to protect the quality of food. The US canning industry adopted a very conservative heat treatment, known as the “12D” process that reduces the probability of survival of heat-resistant Clostridium botulinum
spores to one in a billion by causing a theoretical reduction of the initial population by 12 logarithms.2
This is the historical thread that links today’s 10-6
level (SAL) approach used for the heat processing of medical devices, instruments, and pharmaceuticals to food microbiology safety (see chapter 1
In the 21st century, heat processing is a critical component throughout a broad spectrum of industries. Moist heat steam sterilization is perhaps the most well-known and most practiced form of sterilization because an “autoclave” can essentially be found in every university, hospital, research center, dental office, tattoo shop, testing laboratory, and health care manufacturing facility still to this day. Dry heat processes are also used for a variety of applications. In the pages that follow, we begin with the modes used to explain the lethal effect of heat on microorganisms followed by the theoretical foundation and practical applications of processing by moist steam heat and dry heat. Both processes have many favorable attributes that benefit many applications such as in product manufacturing of pharmaceuticals and medical devices, equipment sterilization, and waste disposal.
MODELS USED TO DESCRIBE THE LETHAL EFFECT OF HEAT ON MICROORGANISMS
The theoretical framework for heat sterilization comes from research on the effects of time and temperature on bacterial spores such as from Geobacillus
species (see chapters 7
). Sterilization by steam or dry heat are lethal processes that destroy microbes by heat. Specifically, lethality is attributed to the transfer of steam or dry heat energy to its surroundings. This energy transfer acts directly on any contaminating microorganism’s structure, replication, or metabolic process that is necessary to provide viability for survival.
The basis of the models used to explain lethality comes from the work of many investigators. They sometimes encountered results that were complex and difficult to extrapolate into a model. It was observed that exposure to a heat process was a lethal event and that the rate of death varied because the sensitivity of bacteria to heat was variable. Many factors were identified to explain the variation. For example, one was that two populations of the same species may be present in a sample and these two populations may have markedly different susceptibility to heat processing. One population may be much more resistant to the lethal process than the other. It is well understood that deviation from the linear logarithmic nature of the survival curve is usually due to two basic factors. The first is the presence of a hump or “lag” in the initial portion of the survival curve of a heat-resistant population of spores, due to Anand’s “heat stimulation” or what is understood as “heat activation.”3
The second, the tailing of the final portion, is doubtless due to more than one thermoresistant variant in the population. Because of this, when the rate of death observed as a function of heat was graphed, nonlinear curves were often observed. In addition, many other factors are now known that significantly impact the rate at which a population of microorganisms are killed by heat processes. These factors are summarized in Table 28.1
. Determining heat resistance for a specific microorganism can be a difficult task because many environmental and physiologic factors play a role.
Investigations of microbial inactivation in plant soil (or earth) have demonstrated many of these variables. Dry heat destruction characteristics of the microflora associated with soil particles suggest the following:
In summary, bacterial spores are known to survive for long periods in harsh environmental conditions. This can be influenced by the spore structure itself, the presence of external protective factors, and water retention. These factors can allow some species of bacterial spores to survive even harsh sterilization processes.43
In experiments evaluating the effect of heat stress on a microbial population, aliquots of a suspension of microorganisms are subjected to heat for several different periods and then the number of survivors for each heating time determined. In addition, the number of microorganisms in the unheated control is determined as the starting point. To communicate these results visually (the change in the number of surviving microorganisms with increased heat stress), a survivor curve graph is prepared in which the number of survivors is plotted as a function of the length of heating time at temperature T. There are several ways to plot the data. When plotting microbial survivor data on a graph with an arithmetic scale on both the x- and the y-axes, the data form an exponential decay-type curve. This is a nondistorted, visual perspective on the effect of a constant stress on a microbial population.
At longer heating times, the number of surviving organisms approaches zero, but because the y-axis scale is arithmetic 1, it does not identify with precision survival numbers smaller than approximately 2000; therefore, this graph does not tell us much about the area of low levels of survival. Learning from this, it came to be known that the lethal effect of heat is more clearly demonstrated in graphs when the number of microorganisms is expressed as log10
. The number of organisms that survive (NT
) exposure to the heat process is plotted on the y-axis, and the time of
exposure to the heat process is plotted on the arithmetic x-axis. Semilogarithmic graphs are optimal for this purpose because the magnitude of the initial target population (N0
) is very large (eg, 1 million colony-forming units [CFUs]). Using the semilogarithmic curve, it is possible to see with accuracy when there are only a small number of surviving organisms (chapters 7
TABLE 28.1 Factors affecting the heat resistance of microorganisms
Destruction rate of microbial cells is a function of water content, which is determined by the relative humidity of the atmosphere surrounding the cells or the water activity of the environment in which the cells are suspended. Spores are more resistant to heat because they have a very low water content. Their water activity is much lower than vegetative bacteria. There is a free exchange of water between the spore and its environment, and the water activity is expected to change in relation to the suspending medium or the atmospheric environment.9 The presence of humidity can raise the water activity of the spore making it susceptible to inactivation processes.
Vegetative microbial cells show differing degrees of susceptibility to adverse influences at various stages of the growth cycle.10,11 Young cells are more susceptible to heat destruction than older and more mature cells.12,13 Increased resistance of thermoduric streptococci to heat destruction was observed during the early logarithmic phase,14 and greater heat resistance is exhibited during the stationary phase than during exponential growth.15,16
Spores produced at higher temperatures were more resistant than spores produced at lower temperatures.17,18,19,20,21,22,23,24,25,26 Spores of thermophilic organisms are inherently more resistant than those of mesophilic or psychrophilic species. The spores of a given species grown at a maximum temperature are in general more resistant than those grown at optimum or minimum temperature.27
Organisms, to a limited extent, have an adaptive response to protect themselves from the lethal effect of a heat process. Enhancement of resistance to lethal heating may result from prior sublethal heating of dormant spores, which reportedly can sometimes induce an increase in dormancy. Sporulation in the presence of cadmium, known to induce stress shock proteins, resulted in spores with increased resistance to heat. A mutant lacking the ability to produce heat shock proteins was more temperature sensitive.28 The induction of increased heat resistance by sublethal heating has also been observed in vegetative cells. Heat resistance is increased not only by prior sublethal heating but also during the process of heating the cells to lethal temperature, with the extent of increase depending on the slowness of the rise in temperature.29
Extensive studies of the effect of nutrient conditions and other factors on the resistance of spores of a strain of Bacillus atrophaeus have been performed.19 Nutrient conditions can either increase or decrease the resistance compared with a standard nutrient condition. Different brands of peptone resulted in a change in resistance, although resistance appeared to be independent of concentration with any one peptone. Various digest media resulted in spores of low resistance, except casein digest, which enhanced resistance. Spores of high resistance were obtained using media prepared from vegetable extracts as well as isoelectric gelatin. The addition of either phosphate or magnesium to the standard peptone medium increased resistance. The addition of available carbohydrates, organic acids, or amino acids in some cases increased resistance. The increases in resistance obtained with varying nutrient conditions were reflected only in the specially produced spores; transfer to a standard medium restored the original resistance.
The influence of pH on heat resistance of microorganisms depends on different factors, such as the strain investigated, suspending medium, water activity, and test temperature. Thus, because of the strong influence of the type of substrate on the effect of pH on heat resistance, resistance calculations based on one set of experimental conditions (pH, substrate, and temperature, among others) are not necessarily applicable to other experimental conditions.30,31
Physical environmental conditions
“Enclosure effect” on heat resistance, when Geobacillus stearothermophilus spores were dried on filter paper discs and placed in nonhermetic paper or foil envelopes, which in turn were heated in saturated steam, survival times were shorter than when no envelope was used.32,33,34 Speculated that the envelope effect may occur because the spores are at an elevated temperature in an envelope at ambient temperature before they become saturated with water unlike “naked” exposed spores.35 This is considered the result of a difference in the level of stabilization of critical molecules in the spore.
or the z-value model is the temperature coefficient model that is used worldwide in designing and monitoring heat sterilization processes. It is also derived from experimental data on the effect of heat on the survival of microorganisms and is portrayed graphically. Bigelow observed that when the logarithm of the destruction time was plotted on the y-axis against temperature on an arithmetic scale on the x-axis, the resultant shape of the survivor curve over the range of temperatures studied was a straight line. The time at a given temperature that causes a 1 log reduction (ie, 90%) in the specific target microbial population is referred to as D-value. The graph of log D versus temperature is called the thermal resistance (TR) curve and from it, one learns the change in temperature that causes a 10-fold change in the D, known as the z-value. Thus, D- and z-values are measures of the destruction of microorganisms caused by the lethality of a heat process. These were recommended for the analysis of microbial survival data generated from a heat process.45
Thus, since that time, survivor curves are typically plotted on semilogarithmic scales.
Today, commercially available biological indicators (BIs) are widely used as biological reference standards for sterilization processes and are manufactured according to US Food and Drug Administration, International Organization for Standardization 11138, or other current good manufacturing practices. The BIs are challenge devices that contain biological spores (as the most resistant microorganisms to heat inactivation processes) at a known count and resistance (see chapter 65
). The BIs are often used to develop, validate, and monitor the effectiveness of the heat process. They can also verify the process parameters relied on for the parametric release of sterile products. As a challenge device, they are manufactured to contain higher numbers of resistant bacterial spores than the organisms expected to be found in manufacturing facilities or on surfaces. A certificate of quality is provided with each shipment certifying the population per BI (N0
) and its resistance to the heat process (D-value). Thus, BIs can be used to biologically verify and/or validate the lethality of heat processes.
Lethality is also referred to as an F-value in moist heat steam (F0
) and dry heat processes (FD or H
). The F0
-value is the equivalent exposure time in a moist heat steam process related to the temperature of 121°C and to z = 10 (see chapter 2
). A standard moist heat steam process is defined as 121°C for 15 minutes, whereas a standard dry heat sterilization process is defined at a minimum of 160°C for at least 120 minutes.47
Accordingly, commercial cycles used by the pharmaceutical and medical device industries are designed to be very conservative such that after the heat process, there is a very high probability that the product sterilized is sterile.
In summary, the international experimental approach to validate the lethality of a sterilization heat process is appropriate for heat-stable materials. It is conservatively based on experiments with typical starting populations of millions of heat-resistant spores. The results are plotted semilogarithmically as described earlier, and the resultant survivor curve will typically be a straight line. Its slope is used to extrapolate the exposure time needed to achieve a theoretical reduction greater than the starting spore population log N0
, for example, a 12 log reduction of N0
. The attainment of a theoretical log reduction greater than what can be empirically measured (because there are no surviving organisms) is known as an SAL. For heat-sensitive materials, the SAL can be obtained by substituting the highly resistant spores with the relevant bioburden typically found on the surfaces being sterilized as discussed later. For further analysis of the mathematical theories behind thermal survivor curves, see chapter 10
and previous editions of this book.48
Moist Heat Steam Sterilization
Sterilization by steam is perhaps the most common and frequently used sterilization modality. This is carried out using sterilization equipment designs that operate at high levels of pressure and temperature. Steam is an ideal form of sterilization except for materials that are compromised by high heat, high pressures, and moisture. Sterilization exposures inside the typical steam sterilization temperature range from 110°C to 135°C and are optimal to achieve microbial lethality with a high degree of confidence. Due to the mass heat transfer generated as the steam condenses to liquid, steam sterilization is more effective than most other sterilization processes at destroying resistant bacterial spores in relatively short periods of exposure. In addition, steam sterilization is also considered one of the most economically friendly sterilization modalities. The main principle of a steam sterilizer design is the vaporization of water by raising its temperature in a closed vessel, producing pressurized saturated steam. The exact control of this process will depend on the specific cycle parameter design to be able to accommodate the load being sterilized and to attain the minimum requirements for sterilization (in these cases, temperature over time). Pure saturated steam is recommended to be made from highly purified water to provide the optimal physical (eg, lack of appreciable levels of air or other noncondensable gases) and chemical (noncontaminated) characteristics of steam.
Pure steam (clean steam or high-purity steam) is steam condensate that complies with a defined water specification (eg, water for injection [WFI], as defined by
pharmaceutical monographs or in associated standards such as EN 285:2015 for steam sterilizers50
) (Table 28.2
TABLE 28.2 Recommended levels of contaminants in condensate from steama
Acceptable Levels in Steam Condensate
Other heavy metals (except iron, cadmium, lead)
Conductivity (at 20°C)
Colorless, no sediment
aReprinted with permission from British Standards Institution.50
To have optimal pure steam for sterilization, it is important to ensure that saturated steam (water vapor in a state of equilibrium between its liquid and gas phases) is free from noncondensable gases and superheat. Both factors have the potential to adversely affect sterilization lethality, especially for loads containing porous/hard goods. Noncondensable gases such as air, nitrogen, and carbon dioxide (CO2) can change steam from a pure state to a mixture of steam and gas, thereby limiting the effectiveness of steam to reliably condense onto desired surfaces. “Super-heated” steam occurs when the liquid/gas equilibrium no longer exists, and steam is in excess relative to water, therefore taking on drier and less efficacious state because it is less likely to efficiently condense on desired surfaces. It is important to monitor that the cycle is free from these effects because it is pure, saturated steam that is most effective at achieving sterilization.
Saturated steam has many properties that make it an excellent heat source, particularly at temperatures of 100°C (212°F) and higher (Table 28.3
). When discussing the use of steam as a sterilant, it is important to note the differences between water and steam because it relates to pressure and temperature relationships. According to Boyle law and the Gay-Lussac pressure temperature law, for a fixed mass of gas, the product of pressure and volume is constant, and the temperature is directly proportional to pressure:
Pressure × Volume = Constant
Pressure ∝ Temperature
TABLE 28.3 Advantageous properties of saturated steam as a sterilant
Rapid, even heating through latent heat transfer
Improved product quality and productivity
Pressure can control temperature.
Temperature can be quickly and precisely established.
High heat transfer coefficient
Smaller required heat transfer surface area, enabling reduced initial equipment outlay
Originates from water
Safe, clean, and low cost
In water’s liquid form, hydrogen bonding pulls water molecules together. Because it exists below its boiling point and therefore steam is absent, this form of water is referred to as unsaturated water (water in its most recognizable state) and has a relatively compact, dense, and stable structure. Saturated water, on the other hand, occurs at a given temperature-pressure combination where both liquid water and water vapor (ie, steam) are present in equilibrium. Thus, water exists in different phases based on the temperature and pressure of the environment it is present in. The temperature at which water boils changes with ambient pressure, such that a low pressure corresponds to a low boiling point temperature and a high pressure corresponds to an increased boiling point temperature. When a saturation condition is reached, the liquid phase and the vapor phase are in equilibrium with one another, that is, both phases exist simultaneously. If a small amount of energy is added to saturated liquid, it turns into vapor at constant temperature. Likewise, if a small amount of energy is removed from saturated vapor, it will condense to liquid at constant temperature. The transference of heat energy to the microbe from the change of phase from steam to water is the major lethal event that causes sterilization.
For most medical devices and pharmaceutical applications, the most relevant conditions for sterilization are shown in Figure 28.1
. In the optimal region of the temperature-pressure curve, steam and liquid water exist in equilibrium with each other. It is a biphasic mixture of water in its gas and liquid phases that are in thermal equilibrium. Saturated steam can exist at a variety of temperature and pressure combinations. As indicated by the curved line, the optimal conditions for steam sterilization exist at the temperature and pressure where steam is in equilibrium with water. As the process begins to drift to the left (liquid phase) and away from the curved line, steam is considered too “wet,” and as it drifts to the right and away from the curved line (gas), the steam is considered too “dry.” With respect to a moist heat steam
sterilizer, temperature and pressure are carefully controlled and monitored to introduce saturated steam to achieve lethality.
FIGURE 28.1 Phase diagram of water at various temperature and pressure conditions.
When the saturated steam molecules condense from steam to liquid, thermal energy is released and transferred to the materials as well as any organisms present in the load causing the exposed materials to be sterilized. As indicated in Figure 28.1
, saturated steam can occur over a wide range of temperatures and pressures where steam (gas) and water (liquid) can coexist. It occurs when the rate of water vaporization is equal to the rate of condensation. In many applications, the parameters of temperature and pressure used for saturated moist steam heat sterilization are in the range of 115°C to 134°C and 1.1 bar (15.9 psia) to 1.89 bar (27.4 psia), respectively. Where the liquid-vapor boundary terminates is the critical temperature (Tc
) and critical pressure (pc
) (ie, critical point) at which the liquid and gas phases of water are indistinguishable from each other. In water, the critical point occurs at around 647 K (374°C; 705°F) and 22 MPa (220.6 bar). If steam is heated above the saturation point, it becomes superheated steam. Although some degree of superheat can be tolerated, higher degrees result in a loss of sterilizing efficiency.51
Superheated steam can arise from several sources. For one, the sterilization chamber jacket heat can be heated above the sterilizing temperature produces heating of materials with high emissivity characteristics by radiant heat transfer to the surface of materials in the sterilizing chamber. As steam is admitted to the sterilizing environment and passes through the material, it picks up heat from both the chamber walls and the material, resulting in superheat in a surface layer of the material. In the presence of water, the superheated steam loses its additional heat energy when it contacts the water or any surface at a temperature below the superheated steam temperature. Savage52
found that as the deviation from the saturation phase boundary increased, the rate of spore destruction decreased phenomenally. These data indicate that at temperatures above 132°C, superheat was not efficacious; however, this is an area that requires more investigation using appropriately designed test equipment. Rates of reaction are so fast at temperatures above 121°C that the accuracy of test results must be considered questionable for any of the test methods reported to date. In general, based on the bacteriologic results obtained at lower temperatures, superheat below 5°C (approximately 85% RH) should not be objectionable.
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