Heat Sterilization - Sterilization Methods

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Chapter: Pharmaceutical Microbiology : Sterilization Procedures And Sterility Assurance

Heat is the most reliable and widely used means of sterilization, affording its antimicrobial activity through destruction of enzymes and other essential cell constituents.


HEAT STERILIZATION

 

Heat is the most reliable and widely used means of sterilization, affording its antimicrobial activity through destruction of enzymes and other essential cell constituents. These lethal events proceed most rapidly in a fully hydrated state, thus requiring a lower heat input (temperature and time) under conditions of high humidity where denaturation and hydrolysis reactions predominate, rather than in the dry state where oxidative changes take place. This method of sterilization is limited to thermostable products, but can be applied to both moisture-sensitive and moisture-resistant items for which dry (160–180 °C) and moist (121–134 °C) heat sterilization procedures are respectively used. Where thermal degradation of a product might possibly occur, it can usually be minimized by selecting the higher temperature range, as the shorter exposure times employed generally result in a lower fractional degradation.

 

Sterilization Processes

 

In any heat sterilization process, the articles to be treated must first be raised to sterilization temperature and this involves a heating-up stage. In the traditional approach, timing for the process (the holding time) then begins. It has been recognized, however, that during both the heating-up and cooling-down stages of a sterilization cycle (Figure 21.4), the product is held at an elevated temperature and these stages may thus contribute to the overall biocidal potential of the process.


 

A method has been devised to convert all the temperature–time combinations occurring during the heating, sterilizing and cooling stages of a moist heat (steam) sterilization cycle to the equivalent time at 121 °C. This involves following the temperature profile of a load, integrating the heat input (as a measure of lethality), and converting it to the equivalent time at the standard temperature of 121 °C. Using this approach, the overall lethality of any process can be deduced and is defined as the F-value; this expresses heat treatment at any temperature as equal to that of a certain number of minutes at 121 °C. In other words, if a moist heat sterilization process has an F-value of x, then it has the same lethal effect on a given organism as heating at 121 °C for x minutes, irrespective of the actual temperature employed or of any fluctuations in the heating process due to heating and cooling stages. The F-value of a process will vary according to the moist heat resistance of the reference organism; when the reference spore is that of B. stearothermophilus with a Z-value of 10 °C, then the F-value is known as the F 0-value.

A relationship between F-and D-values, leading to an assessment of the probable number of survivors in a load following heat treatment, can be established from the following equation: where D is the D-value at 121 °C, and N 0 and N represent, respectively, the initial and final number of viable cells per unit volume.

equation

 

The F-concept has evolved from the food industry and principally relates to the sterilization of articles by moist heat. Because it permits calculation of the extent to which the heating and cooling phases contribute to the overall killing effect of the autoclaving cycle, the F-concept enables a sterilization process to be individually developed for a particular product. This means that adequate sterility assurance can be achieved in autoclaving cycles in which the traditional pharmacopoeial recommendation of 15 minutes at 121 °C is not achieved. The holding time may be reduced below 15 minutes if there is a substantial killing effect during the heating and cooling phases, and an adequate cycle can be achieved even if the ‘target ’ temperature of 121 °C is not reached. Thus, F-values offer both a means by which alternative sterilizing cycles can be compared in terms of their microbial killing efficiency, and a mechanism by which over-processing of marginally thermolabile products can be reduced without compromising sterility assurance. The European Pharmacopoeia emphasizes that when a steam sterilization cycle is designed on the basis of F 0 data, it may be necessary to perform continuous and rigorous microbiological monitoring of the bioburden during routine manufacturing in order consistently to achieve an acceptable sterility assurance level.

 

F 0 values may be calculated either from the area under the curve of a plot of autoclave temperature against time constructed using special chart paper on which the temperature scale is modified to take into account the progressively greater lethality of higher temperatures, or by use of the equation: 


where Δ t is the time interval between temperature measurements, T is the product temperature at time t, and Z is (assumed to be) 10 °C.

 

Thus, if temperatures were being recorded from a thermocouple at 1.00 minute intervals then Δ t 1.00, and a temperature of, for example, 115 °C maintained for 1 minute would give an F 0 value of 1 minute × 10 (115–121)/10, which is equal to 0.25 minutes. In practice, such calculations could easily be performed on the data from several thermocouples within an autoclave using suitable software, and, in a manufacturing situation, these would be part of the batch records. Application of the F-value concept has been largely restricted to steam sterilization processes, although there is a less frequently employed, but direct parallel in dry heat sterilization

 

A)   Moist Heat Sterilization

 

Moist heat has been recognized as an efficient biocidal agent from the early days of bacteriology, when it was principally developed for the sterilization of culture media. It now finds widespread application in the processing of many thermostable products and devices. In the pharmaceutical and medical sphere it is used in the sterilization of dressings, sheets, surgical and diagnostic equipment, containers and closures, and aqueous injections, ophthalmic preparations and irrigation fluids, in addition to the processing of soiled and contaminated items.

 

Sterilization by moist heat usually involves the use of steam at temperatures in the range 121–134 °C, and while alternative strategies are available for the processing of products unstable at these high temperatures, they rarely offer the same degree of sterility assurance and should be avoided if at all possible. The elevated temperatures generally associated with moist heat sterilization methods can only be achieved by the generation of steam under pressure.

 

By far the most commonly employed standard temperature/time cycles for bottled fluids and porous loads (e.g. surgical dressings) are 121 °C for 15 minutes and 134 °C for 3 minutes, respectively. Not only do high-temperature–short time cycles often result in lower fractional degradation, they also afford the advantage of achieving higher levels of sterility assurance due to greater inactivation factors (Table 21.2). Before the publication of the 1988 British Pharmacopoeia the 115 °C for 30 minute cycle was considered an acceptable alternative to 121 °C for 15 minutes, but it is no longer considered sufficient to give the desired sterility assurance levels for products which may contain significant concentrations of thermophilic spores.

 

i)   Steam as a sterilizing agent

 

To act as an efficient sterilizing agent, steam should be able to provide moisture and heat efficiently to the article to be sterilized. This is most effectively done using saturated steam, which is steam in thermal equilibrium with the water from which it is derived, i.e. steam on the phase boundary (Figure 21.5). Under these circumstances, contact with a cooler surface causes condensation and contraction, drawing in fresh steam and leading to the immediate release of the latent heat, which represents approximately 80% of the total heat energy. In this way, heat and moisture are imparted rapidly to articles being sterilized and dry porous loads are quickly penetrated by the steam.

 


 

Steam for sterilization can either be generated within the sterilizer, as with portable bench or ‘instrument and utensil ’ sterilizers, in which case it is constantly in contact with water and is known as ‘wet ’ steam, or can be supplied under pressure (350–400 kPa) from a separate boiler as ‘dry ’ saturated steam with no entrained water droplets. The killing potential of ‘wet’ steam is the same as that of ‘dry’ saturated steam at the same temperature, but it is more likely to soak a porous load, creating physical difficulties for further steam penetration. Thus, major industrial and hospital sterilizers are usually supplied with ‘dry’ saturated steam and attention is paid to the removal of entrained water droplets within the supply line to prevent introduction of a water ‘fog’ into the sterilizer.

 

If the temperature of ‘dry ’ saturated steam is increased, then, in the absence of entrained moisture, the relative humidity or degree of saturation is reduced and the steam becomes superheated (Figure 21.5). During sterilization this can arise in a number of ways, for example by overheating the steam-jacket, by using too dry a steam supply, by excessive pressure reduction during passage of steam from the boiler to the sterilizer chamber, and by evolution of heat of hydration when steaming overdried cotton fabrics. Superheated steam behaves in the same manner as hot air as condensation and release of latent heat will not occur unless the steam is cooled to the phase boundary temperature. Thus, it proves to be an inefficient sterilizing agent and, although a small degree of transient superheating can be tolerated, a maximum acceptable level of 5 °C superheat is set, i.e. the temperature of the steam is never greater than 5 °C above the phase boundary temperature at that pressure.

 

The relationship between temperature and pressure holds true only in the presence of pure steam; adulteration with air contributes to a partial pressure but not to the temperature of the steam. Thus, in the presence of air the temperature achieved will reflect the contribution made by the steam and will be lower than that normally attributed to the total pressure recorded. Addition of further steam will raise the temperature but residual air surrounding articles may delay heat penetration or, if a large amount of air is present, it may collect at the bottom of the sterilizer, completely altering the temperature profile of the sterilizer chamber. It is for these reasons that efficient air removal is a major aim in the design and operation of a boiler-fed steam sterilizer.

 

ii)  Sterilizer design and operation

 

Steam sterilizers, or autoclaves as they are also known, are stainless steel vessels designed to withstand the steam pressures employed in sterilization. They can be: ‘portable ’ sterilizers, which generally have internal electric heaters to produce steam and are used for small pilot or laboratory-scale sterilization and for the treatment of instruments and utensils; or large-scale sterilizers for routine hospital or industrial use, operating on ‘dry ’ saturated steam from a separate boiler (Figure 21.6). Because of their widespread use within pharmacy this latter type will be considered in greatest detail.

 

 

There are two main types of large sterilizers, those designed for use with porous loads (i.e. dressings) and generally operated at a minimum temperature of 134 °C, and those designed as bottled fluid sterilizers employing a minimum temperature of 121 °C. The stages of operation are common to both and can be summarized as air removal and steam admission, heating-up and exposure, and drying or cooling. Many modifications of design exist and in this section only general features will be considered. Fuller treatments of sterilizer design and operation can be found in the relevant Department of Health technical memorandums (DH 1995, DH 1997).

 

General Design Features

 

Steam sterilizers are constructed with either cylindrical or rectangular chambers, with preferred capacities ranging from 400 to 800 L. They can be sealed by either a single door or by doors at both ends (to allow through-passage of processed materials). During sterilization the doors are held closed by a locking mechanism which prevents opening when the chamber is under pressure and until the chamber has cooled to a preset temperature, typically 80 °C.

 

In the larger sterilizers the chamber may be surround ed by a steam jacket which can be used to heat the autoclave chamber and promote a more uniform temperature throughout the load. The same jacket can also be filled with water at the end of the cycle to facilitate cooling and thus reduce the overall cycle time. The chamber floor slopes towards a discharge channel through which air and condensate can be removed. Temperature is monitored within the opening of the discharge channel and by thermocouples in dummy packages; jacket and chamber pressures are followed using pressure gauges. In hospitals and industry, it is common practice to operate sterilizers on an automatic cycle, each stage of operation being controlled by a timer responding to temperature-or pressure-sensing devices.

 

The stages of operation are as follows.

 

           1.     Air removal and steam admission. 


Air can be removed from steam sterilizers either by downward displacement with steam, evacuation or a combination of the two. In the downward displacement sterilizer, the heavier cool air is forced out of the discharge channel by incoming hot steam. This has the benefit of warming the load during air removal, which aids the heating-up process. It finds widest application in the sterilization of bottled fluids where bottle breakage may occur under the combined stresses of evacuation and high temperature. For more air-retentive loads (i.e. dressings), however, this technique of air removal is unsatisfactory and mechanical evacuation of the air is essential before admission of the steam. This can either be to an extremely high level (e.g.2.5 kPa) or can involve a period of pulsed evacuation and steam admission, the latter approach improving air extraction from dressings packs. After evacuation, steam penetration into the load is very rapid and heating-up is almost instantaneous. It is axiomatic that packaging and loading of articles within a sterilizer be so organized as to facilitate air removal.

 

During the sterilization process, small pockets of entrained air may still be released, especially from packages, and this air must be removed. This is achieved in a porous load autoclave with a near-to-steam thermostatic valve incorporated in the discharge channel. The valve operates on the principle of an expandable bellows containing a volatile liquid which vaporizes at the temperature of saturated steam thereby closing the valve, and condenses on the passage of a cooler air-steam mixture, thus reopening the valve and discharging the air. Condensate generated during the sterilization process can also be removed by this device. Small quantities of air will not, however, lower the temperature sufficiently to operate the valve and so a continual slight flow of steam is maintained through a bypass around the device in order to flush away residual air.

 

It is common practice to package sterile fluids, especially intravenous fluids, in flexible plastic containers. During sterilization these can develop a considerable internal pressure in the airspace above the fluid and it is therefore necessary to maintain a proportion of air within the sterilizing chamber to produce sufficient overpressure to prevent these containers from bursting (air ballasting). In sterilizers modified or designed to process this type of product, air removal is therefore unnecessary but special attention must be paid to the prevention of air ‘layering’ within the chamber. This is overcome by the inclusion of a fan or through a continuous spray of hot water within the chamber to mix the air and steam. Air ballasting can also be employed to prevent bottle breakage.

 

2.     Heating-up and exposure. 

When the sterilizer reaches its operating temperature and pressure the sterilization stage begins. The duration of exposure may include a heating-up time in addition to the holding time and this will normally be established using thermocouples in dummy articles.

 

3.      Drying or cooling. 

Dressings packs and other porous loads may become dampened during the sterilization process and must be dried before removal from the chamber. This is achieved by steam exhaust and application of a vacuum, often assisted by heat from the steam-filled jacket if fitted. After drying, atmospheric pressure within the chamber is restored by admission of sterile filtered air.

 

For bottled fluids the final stage of the sterilization process is cooling, and this needs to be achieved as rapidly as possible to minimize thermal degradation of the product and to reduce processing time. In modem sterilizers, this is achieved by circulating water in the jacket that surrounds the chamber or by spray-cooling with retained condensate delivered to the surface of the load by nozzles fitted into the roof of the sterilizer chamber. This is often accompanied by the introduction of filtered, compressed air to minimize container breakage due to high internal pressures (air ballasting). Containers must not be removed from the sterilizer until the internal pressure has dropped to a safe level, usually indicated by a temperature of less than 80 °C. Occasionally, spray-cooling water may be a source of bacterial contamination and its microbiological quality must be carefully monitored.

 

B) Dry Heat Sterilization

 

The lethal effects of dry heat on microorganisms are due largely to oxidative processes, which are less effective than the hydrolytic damage which results from exposure to steam. Thus, dry heat sterilization usually employs higher temperatures in the range 160–180 °C and requires exposure times of up to 2 hours depending on the temperature employed.

 

Again, bacterial spores are much more resistant than vegetative cells and their recorded resistance varies markedly depending on their degree of dryness. In many early studies on dry heat resistance of spores their water content was not adequately controlled, so conflicting data arose regarding the exposure conditions necessary to achieve effective sterilization. This was partly responsible for variations in recommended exposure temperatures and times in different pharmacopoeias.

 

Dry heat application is generally restricted to glassware and metal surgical instruments (where its good penetrability and non-corrosive nature are of benefit), non-aqueous thermostable liquids and thermostable powders. In practice, the range of materials that are actually subjected to dry heat sterilization is quite limited, and consists largely of items used in hospitals. The major industrial application is in the sterilization of glass bottles which are to be filled aseptically, and here the attraction of the process is that it not only achieves an adequate sterility assurance level, but that it may also destroy bacterial endotoxins (products of Gram-negative bacteria, also known as pyrogens, that cause fever when injected into the body). These are difficult to eliminate by other means. For the purposes of de-pyrogenation of glass, temperatures of approximately 250 °C are used.

 

The F-value concept that was developed for steam sterilization processes has an equivalent in dry heat sterilization although its application has been limited. The FH designation describes the lethality of a dry heat process in terms of the equivalent number of minutes exposure at 170 °C, and in this case a Z-value of 20 °C has been found empirically to be appropriate for calculation purposes; this contrasts with the value of 10 °C which is typically employed to describe moist heat resistance.

 

i)   Sterilizer design

 

Dry heat sterilization is usually carried out in a hot-air oven which comprises an insulated polished stainless steel chamber, with a usual capacity of up to 250 L, surrounded by an outer case containing electric heaters located in positions to prevent cool spots developing inside the chamber. A fan is fitted to the rear of the oven to provide circulating air, thus ensuring more rapid equilibration of temperature. Shelves within the chamber are perforated to allow good airflow. Thermocouples can be used to monitor the temperature of both the oven air and articles contained within. A fixed temperature sensor connected to a chart or digital recorder provides a permanent record of the sterilization cycle. Appropriate door-locking controls should be incorporated to prevent interruption of a sterilization cycle once begun.

 

Recent sterilizer developments have led to the use of dry heat sterilizing tunnels where heat transfer is achieved by infrared irradiation or by forced convection in filtered laminar airflow tunnels. Items to be sterilized are placed on a conveyer belt and pass through a high-temperature zone (250–300 + °C) over a period of several minutes.

 

ii) Sterilizer operation

 

Articles to be sterilized must be wrapped or enclosed in containers of sufficient strength and integrity to provide good post-sterilization protection against contamination. Suitable materials are paper, cardboard tubes or aluminium containers. Container shape and design must be such that heat penetration is encouraged in order to shorten the heating-up stage; this can be achieved by using narrow containers with dull, non-reflecting surfaces. In a hot-air oven, heat is delivered to articles principally by radiation and convection; thus, they must be carefully arranged within the chamber to avoid obscuring centrally placed articles from wall radiation or impending air flow. The temperature variation within the chamber should not exceed ± 5 °C of the recorded temperature. Heating-up times, which may be as long as 4 hours for articles with poor heat-conducting properties, can be reduced by preheating the oven before loading. Following sterilization, the chamber temperature is usually allowed to fall to around 40 °C before removal of sterilized articles; this can be accelerated by the use of forced cooling with filtered air.

 

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