A sudden departure from a microbe’s temperature of adaptation is likely to have a detrimental effect on it. As a rule, temperatures that exceed the maximum growth temperature are microbicidal, whereas temperatures below the minimum growth temperature are microbistatic. This essentially means that, for most microbes, the effects of temperatures below minimum can be reversible, whereas the effects of temperatures above maximum are not reversible and most microbes will be destroyed by them.
The two physical states of heat used in microbial control are moist and dry. Moist heat occurs in the form of hot water, boiling water, or steam (vaporized water). In practice, the temperature of moist heat usually ranges from 60°C to 135°C. As we shall see, the temperature of steam can be regulated by adjusting its pressure in a closed container. The expression dry heat denotes air with a low moisture content that has been heated by a flame or electric heating coil. In practice, the temperature of dry heat ranges from 160°C to several thousand degrees Celsius.
Mode of Action and Relative Effectiveness of Heat
Moist heat and dry heat differ in their modes of action as well as in their efficiency. Moist heat operates at lower temperatures and shorter exposure times to achieve the same effectiveness as dry heat (table 1). Although many cellular structures are damaged by moist heat, its most microbicidal effects are the coagulation and denaturation of proteins, which quickly and permanently halt cellular metabolism.
Table1. Comparison of Times and Temperatures to Achieve Sterilization with Moist and Dry Heat
Dry heat with a moderate temperature dehydrates the cell, re moving the water necessary for metabolic reactions, and it alters protein structure. However, the lack of water actually increases the stability of some protein conformations, necessitating the use of higher temperatures when dry heat is employed as a method of microbial control. At very high temperatures, dry heat oxidizes cells, burning them to ashes. This method is the one used in the laboratory when a loop is flamed or in industry when medical waste is incinerated.
Heat Resistance and Thermal Death of Spores and Vegetative Cells
Bacterial endospores exhibit the greatest resistance, and vegetative states of bacteria and fungi are the least resistant to both moist and dry heat. Destruction of spores usually requires temperatures above boiling, although resistance varies widely. The following table compares some times and temperatures required to kill spore- formers using moist versus dry heat:
In general, reliable killing of the most heat-resistant species of spore-formers requires 121°C for 20 minutes in moist heat.
Vegetative cells also vary in their sensitivity to heat, though not to the same extent as spores. Among bacteria, the death conditions for moist heat range from 50°C for 3 minutes (Neisseria gonorrhoeae) to 60°C for 60 minutes (Staphylococcus aureus). It is worth noting that vegetative cells of spore-formers are just as susceptible as vegetative cells of non–spore-formers and that pathogens are neither more nor less susceptible than nonpathogens. Other microbes, including fungi, protozoa, and worms, are rather similar in their sensitivity to heat. Viruses can be surprisingly resistant to heat, with a tolerance range extending from 55°C for 2 to 5 minutes (adenoviruses) to 60°C for 600 minutes (hepatitis A virus). For practical purposes, all non–heat-resistant forms of bacteria, yeasts, molds, protozoa, worms, and viruses are destroyed by exposure to 80°C for 20 minutes.
Practical Concerns in the Use of Heat: Thermal Death Measurements
Adequate sterilization requires that both temperature and length of exposure be considered. As a general rule, higher temperatures allow shorter exposure times, and lower temperatures require longer exposure times. A combination of these two variables constitutes the thermal death time, or TDT, defined as the shortest length of time required to kill all test microbes at a specified temperature. The TDT has been experimentally determined for the microbial species that are common or important contaminants in various heat-treated materials. Another way to compare the susceptibility of microbes to heat is the thermal death point (TDP), defined as the lowest temperature required to kill all microbes in a sample in 10 minutes.
Many perishable substances are processed with moist heat. Some of these products are intended to remain on the shelf at room temperature for several months or even years. The chosen heat treatment must render the product free of agents of spoilage or dis ease. At the same time, the quality of the product and the speed and cost of processing must be considered. For example, in the commercial preparation of canned green beans, one of the cannery’s greatest concerns is to prevent growth of the agent of botulism. From several possible TDTs (that is, combinations of time and temperature) for Clostridium botulinum spores, the cannery must choose one that kills all spores but does not turn the beans to mush. These many considerations produce an optimal TDT for a given processing method. Commercial canneries heat low-acid foods like green beans at 121°C for 30 minutes, a treatment that renders the foods sterile. Because of such strict controls in canneries, cases of botulism from commercially canned foods are rare.
Common Methods of Moist Heat Control
The four ways moist heat is employed to control microbes are (1) steam under pressure, (2) nonpressurized steam, (3) boiling water, and (4) pasteurization.
Sterilization with Steam under Pressure At sea level, nor mal atmospheric pressure is 15 pounds per square inch (psi), or 1 atmosphere. At this pressure, water will boil (change from a liquid to a gas) at 100°C, and the resultant steam will not go above 100°C, regardless of the method of heating. But boiling temperature is not hot enough to reliably kill all targeted microbes. The only way to raise the temperature of steam is to expose it to increased pressure. At higher pressures the temperature at which water boils and the temperature of steam both rise. For example, at a pressure of 20 psi (5 psi above normal), the temperature of steam is 109°C. As the temperature is increased to 10 psi above normal, the steam’s temperature rises to 115°C, and at 15 psi above normal (a total of 2 atmospheres), it will be 121°C. It is not the pressure by itself that is killing microbes but the increased temperature it produces.
Such pressure-temperature combinations can be achieved only with a special device that can subject pure steam to pressures greater than 1 atmosphere. Health and commercial industries use an autoclave for this purpose, and a comparable home appliance is the pressure cooker. Autoclaves have a fundamentally similar plan: an enclosed metal chamber with an airtight door on one end and racks to hold materials (figure 1). Its construction includes a network of valves, pressure and temperature gauges, and ducts for regulating and measuring pressure and conducting the steam into the chamber. Sterilization is achieved when the steam condenses against the objects in the chamber and gradually raises their temperature.
Fig1. Sterilization using steam under pressure. (a) A small hospital autoclave for sterilizing surgical instruments. (b) Cutaway section, showing autoclave components and the flow of steam through the autoclave. (a): pittawut/Shutterstock
Experience has shown that the most efficient pressure- temperature combination for achieving sterilization is 15 psi, which yields 121°C. It is possible to use higher pressure to reach higher temperatures (for instance, increasing the pressure to 30 psi raises the temperature to 132°C), but doing so will not significantly reduce the exposure time and can harm the items being sterilized.
The duration of the process is adjusted according to the bulkiness of the items in the load (thick bundles of material or large flasks of liquid) and how full the chamber is. The range of holding times varies from 10 minutes for light loads to 40 minutes for heavy or bulky ones; the average time is 20 minutes.
Nonpressurized Steam Items that would be destroyed by the high temperature of the autoclave, such as seeds for planting, may be subjected to intermittent sterilization, also called tyndallization.1 This technique requires a chamber to hold the materials and a reservoir for boiling water. Items in the chamber are exposed to free-flowing steam for 30 to 60 minutes. This temperature is not sufficient to reliably kill spores, so a single exposure will not suffice. On the assumption that surviving spores will germinate into less resistant vegetative cells, the items are incubated at appropriate temperatures for 23 to 24 hours and then again subjected to steam treatment. This cycle is repeated for 3 days in a row. Because the temperature never gets above 100°C, highly resistant spores that do not germinate could survive even after 3 days of this treatment.
Boiling Water: Disinfection A simple boiling water bath or chamber can quickly decontaminate items in the clinic and home. Because a single processing at 100°C will not kill all resistant cells, this method can be relied on only for disinfection and not for sterilization. Exposing materials to boiling water for 30 minutes will kill most non–spore-forming pathogens, including resistant species such as the tubercle bacillus and staphylococci. Probably the greatest disadvantage with this method is that the items can be easily recontaminated when removed from the water.
Pasteurization: Disinfection of Beverages Fresh beverages such as milk, fruit juices, beer, and wine are easily contaminated during collection and processing. Because microbes have the potential to spoil these foods or cause illness, heat is frequently used to reduce the microbial load and destroy pathogens. Pasteurization is a technique of applying heat to consumable liquids to kill potential agents of infection and spoilage, while at the same time retaining the liquid’s flavor and food value.
A widely used pasteurization technique is the flash method, which exposes the liquid to heat exchangers at 71.6°C for 15 seconds. This first method does not appreciably change flavor and nutrient content, and it is effective against certain resistant pathogens such as Coxiella and Mycobacterium. Although these treatments inactivate most viruses and destroy the vegetative stages of 97% to 99% of bacteria and fungi, they do not kill endospores or thermoduric microbes (mostly nonpathogenic lactobacilli, micrococci, and yeasts). Flash- pasteurized milk is not sterile after regular pasteurization. In fact, it can contain 20,000 microbes per milliliter or more, which explains why even an unopened carton of milk will eventually spoil. Many commercial dairies now produce sterile milk with an increased storage life of several months. This ultrapasteurized milk is processed using ultrahigh temperature (UHT)—134°C for 2 to 5 seconds.
One important aim in pasteurization is to prevent the transmission of milk-borne diseases from infected cows or milk handlers. The primary targets of pasteurization are non–spore-forming pathogens: Salmonella species (a common cause of food infection), Campylobacter jejuni (acute intestinal infection), Listeria monocytogenes (listeriosis), Brucella species (undulant fever), Coxiella burnetii (Q fever), Mycobacterium bovis, M. tuberculosis, and several enteric viruses.
Dry Heat: Hot Air and Incineration
Dry heat is not as versatile or as widely used as moist heat, but it has several important sterilization applications. The temperatures and times employed in dry heat vary according to the particular method, but in general they are greater than with moist heat. Incineration in a flame or electric heating coil is perhaps the most rigorous of all heat treatments. The flame of a Bunsen burner reaches 1,870°C at its hottest point, and furnaces/incinerators operate at temperatures of 800°C to 6,500°C. Direct exposure to such intense heat ignites and reduces microbes and other sub stances to ashes and gas.
Incineration of microbial samples on inoculating loops and needles using a Bunsen burner is a very common practice in the microbiology laboratory. This method is fast and effective, but it is also limited to metals and heat-resistant glass materials. Tabletop infrared incinerators (see table 2), an alternative to Bunsen burners, are safer and prevent splatter of the inoculum.
Table2. Applications Using Heat for Sterilization and Disinfection
The hot-air oven provides another means of dry-heat sterilization. The so-called dry oven is usually electric (occasionally gas) and has coils that radiate heat within an enclosed compartment. Heated, circulated air transfers its heat to the materials in the oven. Depending on the type of oven and the material being decontaminated, a cycle takes 12 minutes to 4 hours to complete and involves temperatures of 150°C to 180°C.
Table 2 presents an illustrated summary of heating methods for microbial control.
