Using Physical Methods to Control Microorganisms Quiz

PDF page 571: C. botulinum) being more heat tolerant. A similar parameter, the thermal death time (TDT), is the length of time

PDF page 571: needed to kill all microorganisms in a sample at a given temperature. These parameters are often used to describe sterilization procedures that use high heat, such as autoclaving. Boiling is one of the oldest methods of moist-heat control of microbes, and it is typically quite effective at killing vegetative cells and some viruses. However, boiling is less effective at killing endospores; some endospores are able to survive up to 20 hours of boiling. Additionally, boiling may be less effective at higher altitudes, where the boiling point of water is lower and the boiling time needed to kill microbes is therefore longer. For these reasons, boiling is not considered a useful sterilization technique in the laboratory or clinical setting. Many different heating protocols can be used for sterilization in the laboratory or clinic, and these protocols can be broken down into two main categories: dry-heat sterilization and moist-heat sterilization. Aseptic technique in the laboratory typically involves some dry-heat sterilization protocols using direct application of high heat, such as sterilizing inoculating loops (Figure 13.6). Incineration at very high temperatures destroys all microorganisms. Dry heat can also be applied for relatively long periods of time (at least 2 hours) at temperatures up to 170 °C by using a dry-heat sterilizer, such as an oven. However, moist-heat sterilization is typically the more effective protocol because it penetrates cells better than dry heat does.

PDF page 571: needed to kill all microorganisms in a sample at a given temperature. These parameters are often used to describe sterilization procedures that use high heat, such as autoclaving. Boiling is one of the oldest methods of moist-heat control of microbes, and it is typically quite effective at killing vegetative cells and some viruses. However, boiling is less effective at killing endospores; some endospores are able to survive up to 20 hours of boiling. Additionally, boiling may be less effective at higher altitudes, where the boiling point of water is lower and the boiling time needed to kill microbes is therefore longer. For these reasons, boiling is not considered a useful sterilization technique in the laboratory or clinical setting. Many different heating protocols can be used for sterilization in the laboratory or clinic, and these protocols can be broken down into two main categories: dry-heat sterilization and moist-heat sterilization. Aseptic technique in the laboratory typically involves some dry-heat sterilization protocols using direct application of high heat, such as sterilizing inoculating loops (Figure 13.6). Incineration at very high temperatures destroys all microorganisms. Dry heat can also be applied for relatively long periods of time (at least 2 hours) at temperatures up to 170 °C by using a dry-heat sterilizer, such as an oven. However, moist-heat sterilization is typically the more effective protocol because it penetrates cells better than dry heat does.

PDF page 571: needed to kill all microorganisms in a sample at a given temperature. These parameters are often used to describe sterilization procedures that use high heat, such as autoclaving. Boiling is one of the oldest methods of moist-heat control of microbes, and it is typically quite effective at killing vegetative cells and some viruses. However, boiling is less effective at killing endospores; some endospores are able to survive up to 20 hours of boiling. Additionally, boiling may be less effective at higher altitudes, where the boiling point of water is lower and the boiling time needed to kill microbes is therefore longer. For these reasons, boiling is not considered a useful sterilization technique in the laboratory or clinical setting. Many different heating protocols can be used for sterilization in the laboratory or clinic, and these protocols can be broken down into two main categories: dry-heat sterilization and moist-heat sterilization. Aseptic technique in the laboratory typically involves some dry-heat sterilization protocols using direct application of high heat, such as sterilizing inoculating loops (Figure 13.6). Incineration at very high temperatures destroys all microorganisms. Dry heat can also be applied for relatively long periods of time (at least 2 hours) at temperatures up to 170 °C by using a dry-heat sterilizer, such as an oven. However, moist-heat sterilization is typically the more effective protocol because it penetrates cells better than dry heat does.

PDF page 572: Autoclaves Autoclaves rely on moist-heat sterilization. They are used to raise temperatures above the boiling point of water to sterilize items such as surgical equipment from vegetative cells, viruses, and especially endospores, which are known to survive boiling temperatures, without damaging the items. Charles Chamberland (1851–1908) designed the modern autoclave in 1879 while working in the laboratory of Louis Pasteur. The autoclave is still considered the most effective method of sterilization (Figure 13.7). Outside laboratory and clinical settings, large industrial autoclaves called retorts allow for moist-heat sterilization on a large scale. In general, the air in the chamber of an autoclave is removed and replaced with increasing amounts of steam trapped within the enclosed chamber, resulting in increased interior pressure and temperatures above the boiling point of water. The two main types of autoclaves differ in the way that air is removed from the chamber. In gravity displacement autoclaves, steam is introduced into the chamber from the top or sides. Air, which is heavier than steam, sinks to the bottom of the chamber, where it is forced out through a vent. Complete displacement of air is difficult, especially in larger loads, so longer cycles may be required for such loads. In prevacuum sterilizers, air is removed completely using a high-speed vacuum before introducing steam into the chamber. Because air is more completely eliminated, the steam can more easily penetrate wrapped items. Many autoclaves are capable of both gravity and prevacuum cycles, using the former for the decontamination of waste and sterilization of media and unwrapped glassware, and the latter for sterilization of packaged instruments.

PDF page 572: Autoclaves Autoclaves rely on moist-heat sterilization. They are used to raise temperatures above the boiling point of water to sterilize items such as surgical equipment from vegetative cells, viruses, and especially endospores, which are known to survive boiling temperatures, without damaging the items. Charles Chamberland (1851–1908) designed the modern autoclave in 1879 while working in the laboratory of Louis Pasteur. The autoclave is still considered the most effective method of sterilization (Figure 13.7). Outside laboratory and clinical settings, large industrial autoclaves called retorts allow for moist-heat sterilization on a large scale. In general, the air in the chamber of an autoclave is removed and replaced with increasing amounts of steam trapped within the enclosed chamber, resulting in increased interior pressure and temperatures above the boiling point of water. The two main types of autoclaves differ in the way that air is removed from the chamber. In gravity displacement autoclaves, steam is introduced into the chamber from the top or sides. Air, which is heavier than steam, sinks to the bottom of the chamber, where it is forced out through a vent. Complete displacement of air is difficult, especially in larger loads, so longer cycles may be required for such loads. In prevacuum sterilizers, air is removed completely using a high-speed vacuum before introducing steam into the chamber. Because air is more completely eliminated, the steam can more easily penetrate wrapped items. Many autoclaves are capable of both gravity and prevacuum cycles, using the former for the decontamination of waste and sterilization of media and unwrapped glassware, and the latter for sterilization of packaged instruments.

PDF page 572: displacing the air in the chamber with increasing amounts of steam, pressure increases, and temperatures exceeding 100 °C can be achieved, allowing for complete sterilization. (b) A researcher programs an autoclave to sterilize a sample. (credit a: modification of work by Courtney Harrington; credit b: modification of work by Lackemeyer MG, Kok-Mercado Fd, Wada J, Bollinger L, Kindrachuk J, Wahl-Jensen V, Kuhn JH, Jahrling PB) Standard operating temperatures for autoclaves are 121 °C or, in some cases, 132 °C, typically at a pressure of 15 to 20 pounds per square inch (psi). The length of exposure depends on the volume and nature of material being sterilized, but it is typically 20 minutes or more, with larger volumes requiring longer exposure times to ensure sufficient heat transfer to the materials being sterilized. The steam must directly contact the liquids or dry materials being sterilized, so containers are left loosely closed and instruments are loosely wrapped in paper or foil. The key to autoclaving is that the temperature must be high enough to kill endospores to achieve complete sterilization. Because sterilization is so important to safe medical and laboratory protocols, quality control is essential. Autoclaves may be equipped with recorders to document the pressures and temperatures achieved during each run. Additionally, internal indicators of various types should be autoclaved along with the materials to be sterilized to ensure that the proper sterilization temperature has been reached (Figure 13.8). One common type of indicator is the use of heat-sensitive autoclave tape, which has white stripes that turn black when the appropriate temperature is achieved

PDF page 573: during a successful autoclave run. This type of indicator is relatively inexpensive and can be used during every run. However, autoclave tape provides no indication of length of exposure, so it cannot be used as an indicator of sterility. Another type of indicator, a biological indicator spore test, uses either a strip of paper or a liquid suspension of the endospores of Geobacillus stearothermophilus to determine whether the endospores are killed by the process. The endospores of the obligate thermophilic bacterium G. stearothermophilus are the gold standard used for this purpose because of their extreme heat resistance. Biological spore indicators can also be used to test the effectiveness of other sterilization protocols, including ethylene oxide, dry heat, formaldehyde, gamma radiation, and hydrogen peroxide plasma sterilization using either G. stearothermophilus, Bacillus atrophaeus, B. subtilis, or B. pumilus spores. In the case of validating autoclave function, the endospores are incubated after autoclaving to ensure no viable endospores remain. Bacterial growth subsequent to endospore germination can be monitored by biological indicator spore tests that detect acid metabolites or fluorescence produced by enzymes derived from viable G. stearothermophilus. A third type of autoclave indicator is the Diack tube, a glass ampule containing a temperature-sensitive pellet that melts at the proper sterilization temperature. Spore strips or Diack tubes are used periodically to ensure the autoclave is functioning properly.

PDF page 573: Pasteurization Although complete sterilization is ideal for many medical applications, it is not always practical for other applications and may also alter the quality of the product. Boiling and autoclaving are not ideal ways to control microbial growth in many foods because these methods may ruin the consistency and other organoleptic (sensory) qualities of the food. Pasteurization is a form of microbial control for food that uses heat but does not render the food sterile. Traditional pasteurization kills pathogens and reduces the number of spoilage-causing microbes while maintaining food quality. The process of pasteurization was first developed by Louis Pasteur in the 1860s as a method for preventing the spoilage of beer and wine. Today, pasteurization is most commonly used to kill heat-sensitive pathogens in milk and other food products (e.g., apple juice and honey) (Figure 13.9). However, because pasteurized food products are not sterile, they will eventually spoil.

PDF page 574: The methods used for milk pasteurization balance the temperature and the length of time of treatment. One method, high-temperature short-time (HTST) pasteurization, exposes milk to a temperature of 72 °C for 15 seconds, which lowers bacterial numbers while preserving the quality of the milk. An alternative is ultra-high-temperature (UHT) pasteurization, in which the milk is exposed to a temperature of 138 °C for 2 or more seconds. UHT pasteurized milk can be stored for a long time in sealed containers without being refrigerated; however, the very high temperatures alter the proteins in the milk, causing slight changes in the taste and smell. Still, this method of pasteurization is advantageous in regions where access to refrigeration is limited.

PDF page 574: The methods used for milk pasteurization balance the temperature and the length of time of treatment. One method, high-temperature short-time (HTST) pasteurization, exposes milk to a temperature of 72 °C for 15 seconds, which lowers bacterial numbers while preserving the quality of the milk. An alternative is ultra-high-temperature (UHT) pasteurization, in which the milk is exposed to a temperature of 138 °C for 2 or more seconds. UHT pasteurized milk can be stored for a long time in sealed containers without being refrigerated; however, the very high temperatures alter the proteins in the milk, causing slight changes in the taste and smell. Still, this method of pasteurization is advantageous in regions where access to refrigeration is limited.

PDF page 574: Just as high temperatures are effective for controlling microbial growth, exposing microbes to low temperatures can also be an easy and effective method of microbial control, with the exception of psychrophiles, which prefer cold temperatures (see Temperature and Microbial Growth). Refrigerators used in home kitchens or in the laboratory maintain temperatures between 0 °C and 7 °C. This temperature range inhibits microbial metabolism, slowing the growth of microorganisms significantly and helping preserve refrigerated products such as foods or medical supplies. Certain types of laboratory cultures can be preserved by refrigeration for later use. Freezing below −2 °C may stop microbial growth and even kill susceptible organisms. According to the US Department of Agriculture (USDA), the only safe ways that frozen foods can be thawed are in the refrigerator, immersed in cold water changed every 30 minutes, or in the microwave, keeping the food at temperatures not

PDF page 575: conducive for bacterial growth. In addition, halted bacterial growth can restart in thawed foods, so thawed foods should be treated like fresh perishables. Bacterial cultures and medical specimens requiring long-term storage or transport are often frozen at ultra-low temperatures of −70 °C or lower. These ultra-low temperatures can be achieved by storing specimens on dry ice in an ultra-low freezer or in special liquid nitrogen tanks, which maintain temperatures lower than −196 °C (Figure 13.10).