MCB3020 Textbook - Chapter 8: Control of Microorganisms

8 Control of Microorganisms in the Environment Keeping Infection at Bay I magine going to the hospital for neurosurgery, as thousands of people do every day. Now imagine finding out that as a consequence of this surgery you were infected with an agent that has a 100% mortality rate. Surely this co...

8 Control of Microorganisms in the Environment Keeping Infection at Bay I magine going to the hospital for neurosurgery, as thousands of people do every day. Now imagine finding out that as a consequence of this surgery you were infected with an agent that has a 100% mortality rate. Surely this couldn’t happen in the twenty-first century, right? Sadly, the answer is yes it has happened, but we must stress it is exceedingly rare. Since the days of Joseph Lister, disinfection in health care facilities has been the focus of intense scrutiny. However, in the 1980s, microbiologists were challenged when it became clear that a previously unknown infectious agent, the prion, was not susceptible to any of the disinfection methods in practice at that time. Composed entirely of protein, prions resist decontamination efforts that involve heat, radiation, and chemicals that attack cellular components. Prions are composed only of protein (section 6.7) The need to protect against prion contamination was made frighteningly clear when it was determined that infected individuals exhibit high levels of prions in brain and neural tissue and suffer neurological symptoms that always result in death. In cases of iatrogenic (Greek iatros, healer; genic, producing) prion disease, it was discovered that prioncontaining corneas were transplanted or contaminated neurosurgical instruments were used. This has given rise to the use of disposable products that are later incinerated to over 1,000°C, decontamination of durable goods by ozone exposure, and tighter screening of donated corneas. In addition, U.S. blood banks screen donors who resided in Britain for at least 3 months between 1980 and 1996. During those years, the domestic beef supply was tainted with spinal cord tissue from practices in slaughterhouses, and meat from “mad cows” entered the food supply. Because low levels of prions can be detected in the blood of infected individuals, as a cautionary practice, those meeting these criteria are rejected for blood donation in several countries. Prion proteins transmit disease (section 38.6) Having introduced the breadth of microbial forms in chapters 3 through 6 and how they grow in chapter 7, we turn now to how to control microbial growth. This chapter addresses the means to prevent infection; chapter 9 addresses the means to treat an infection. ©Erproductions Ltd/Blend Images LLC Microorganism control continues to be a hot topic as microorganisms evolve to resist current strategies. Control efforts have a substantial role in public health to prevent disease as well as in therapeutic use to treat disease. Thus this chapter focuses on the control of microorganisms by physical, chemical, and biological agents (such as engineered bacteria). In general, any chemical, physical, or biological product that controls microorganisms is referred to as an antimicrobial agent. Chemotherapeutic agents that are used inside the body to treat human disease are discussed in chapter 9. Readiness Check: Based on what you have learned previously, you should be able to: ✓ Identify the structures and their functions of bacteria, protists, fungi, viruses, and prions, including their replication processes and energy requirements (sections 3.3–3.9, 5.1–5.7, 6.1–6.7, 7.1–7.7) 8.1 Microbial Growth and Replication: Targets for Control After reading this section, you should be able to: a. Compare and contrast actions of disinfection, antisepsis, chemotherapy, and sterilization b. Distinguish between cidal (killing) and static (inhibitory) agents The principles of microbial control are rooted in microbial nutrition, growth, and development, which are discussed in chapter 7. If you can starve or poison microbes, or inhibit or prevent growth or replication, you can control microorganisms. Of course, it is not as simple as this sounds, and a complex vocabulary is applied to describe differences in population density, degree of killing, and even what is used to remove microorganisms. Terminology is especially important when the control of microorganisms is discussed because words such as disinfectant and antiseptic often are used loosely, yet they have different meanings. The situation is even more confusing because a particular treatment can either inhibit growth, inactivate replication, or kill, depending on the conditions. The types of control agents and their uses are 170 wil11886_ch08_170-186.indd 170 23/10/18 9:21 am 8.1 Microbial Growth and Replication: Targets for Control 171 Microbial Control Methods Physical agents Heat Chemical agents Radiation Ionizing Nonionizing X ray, cathode, gamma Sterilization Dry Biological agents Mechanical removal methods Filtration UV Disinfection Predator Virus Toxin Antisepsis Antisepsis Sterilization Air Liquids Sterilization Sterilization Moist Incineration Dry oven Sterilization Sterilization Steam under pressure Boiling water, hot water, pasteurization Sterilization Disinfection Gases Sterilization Liquids Disinfection (Animate) Chemotherapy Antisepsis (Inanimate) Disinfection Sterilization Figure 8.1 Microbial Control Methods. MICRO INQUIRY Which types of agents can be used for sterilization? Which can be used for antisepsis? What is the difference? outlined in figure 8.1. Note that not all agents are chemical. Rather, antimicrobial agents can be physical, chemical, mechanical, and biological. To simplify the terminology, we use the term biocide to mean all antimicrobial agents that can be used to control microorganisms. In general, to control microorganisms a biocide must be evaluated to determine the specific parameters under which it will be effective. The range of microbial cell types presents a broad target for growth control. The outer layer of a microbe presents a permeability barrier that must be overcome for a treatment to be effective. Many bacterial cells are readily destroyed, with Gram-negatives somewhat less susceptible as a consequence of their outer membrane. Exterior surfaces on Mycobacterium spp., bacterial endospores, and protozoan cysts render these cell types especially recalcitrant. Microbial control methods are typically applied to populations of microbes, where they will affect individuals differently. It is critical to consider the diversity of microbes in a given situation when evaluating control options. Sterilization (Latin sterilis, unable to produce offspring), in our context, is the process by which all living cells, spores, and acellular entities (e.g., viruses, viroids, and prions) are either destroyed or removed from an object or habitat. A sterile object is totally free of viable microorganisms, spores, and other infectious agents. When sterilization wil11886_ch08_170-186.indd 171 is achieved by a chemical agent, the chemical is called a sterilant. In contrast, disinfection is the killing, inhibition, or removal of microorganisms that may cause disease. Disinfection therefore results in the substantial reduction of the total microbial population and the destruction of potential pathogens. Disinfectants are agents, usually chemical, used to carry out disinfection and normally used only on inanimate objects. A disinfectant does not necessarily sterilize an object because viable spores and a few microorganisms may remain. In sanitization, the microbial population is reduced to levels that are considered safe by public health standards. The inanimate object is usually cleaned as well as partially disinfected. For example, sanitizers are used to clean eating utensils in restaurants. Viroids and satellites: nucleic acidbased subviral agents (section 6.6); Prions are composed only of protein (section 6.7) It also is frequently necessary to control microorganisms on or in living tissue. Antisepsis (Greek anti, against; sepsis, putrefaction) is the destruction or inhibition of microorganisms on living tissue; it is the prevention of infection or sepsis. Antiseptics are chemical agents applied to tissue to prevent infection by killing or inhibiting pathogen growth; they also reduce the total microbial population. Because they must not cause too much harm to the host, antiseptics are generally not as toxic as disinfectants. The exposure of 23/10/18 9:21 am 172 CHAPTER 8 | Control of Microorganisms in the Environment Log (microbial population) Antisepsis Sanitization Disinfection Sterilization 0 Time Figure 8.2 Impact of Biocide Exposure. Three exponential plots of survivors versus time of biocide exposure, indicating the potential kinetics of biocide action. Note how the general terms referencing microbial control are reflected by decreasing numbers of microbes. For example, sterilization refers to the absence of viable organisms regardless of biocide kinetics. microorganisms to increasing biocide concentrations decreases the number of viable organisms. Figure 8.2 shows three possible population reduction curves resulting from three different biocides. The shape of the curve reflects conditions that influence biocide effectiveness. Note that in each case, the eventual decline in viable microorganisms can occur as a staged interval of viability from antisepsis to sterilization. Chemotherapy is the use of chemical agents to kill or inhibit the growth of microorganisms within host tissue and is the topic of chapter 9. The type of antimicrobial agent can be discerned from its suffix. Substances that kill organisms often have the suffix -cide (Latin cida, to kill); a cidal agent kills microbes but not necessarily endospores. A disinfectant or antiseptic can be particularly effective against a specific group, in which case it may be called Table 8.1 a bactericide, fungicide, or viricide. Other chemicals do not kill but rather prevent growth. If these agents are removed, growth will resume. Their names end in -static (Greek statikos, causing to stand or stopping)—for example, bacteriostatic and fungistatic. It is worth noting here that resistance to antimicrobial biocides has been increasing nearly as rapidly as resistance to antibiotics. Similar to antibiotic resistance mechanisms (discussed in chapter 9), biocide resistance mechanisms include the induction of efflux pumps, modified membrane permeability, and target modification. In fact, a number of studies have linked the acquisition of biocide resistance mechanisms to resistance to antibiotics. In many cases they are shared on plasmids and upregulated in response to ubiquitously low environmental biocide concentrations. It is clear that microorganisms adapt well to new situations, resulting in the sharing of successful resistance strategies. Comprehension Check 1. Define the following terms: sterilization, sterilant, disinfection, disinfectant, sanitization, antisepsis, antiseptic, chemotherapy, biocide. 2. What is the difference between bactericidal and bacteriostatic? To which category do you think most household cleaners belong? Why? 8.2 The Pattern of Microbial Death Mirrors the Pattern of Microbial Growth After reading this section, you should be able to: a. Calculate the decimal reduction time (D value) b. Correlate antisepsis, sanitization, disinfection, and sterilization with agent effectiveness A microbial population is not killed instantly when exposed to a lethal agent. Population death is generally exponential; that is, the population is reduced by the same fraction at constant intervals (table 8.1). If the logarithm of the population number A Theoretical Microbial Heat-Killing Experiment 1 Assume that the initial sample contains 106 vegetative microorganisms per milliliter and that 90% of the organisms are killed during each minute of exposure. The temperature is 121°C. wil11886_ch08_170-186.indd 172 23/10/18 9:21 am 8.3 Mechanical Removal Methods Rely on Barriers 173 6 5 10000 Log10 number of survivors 4 1000 2 Log10 D value 3 D value 1 100 10 Z value 1 0 –1 0 1 2 3 4 5 6 100 7 110 115 120 125 Temperature (°C) Minutes of exposure (a) 105 (b) Figure 8.3 The Pattern of Microbial Death. (a) An exponential plot of the survivors versus the minutes of exposure to heating at 121°C. In this example, the D value is 1 minute. The data are from table 8.1. (b) An exponential plot of D values versus temperature. The temperature change at a given D value that reduces the population by one log unit is known as the Z value. In this example, the Z value is 5°C. MICRO INQUIRY Examine graph (a). How long would it take to kill one-half of the original population of microorganisms? Keep in mind that the y axis is exponential. remaining is plotted against the time of exposure of the microorganism to the agent, a straight-line plot will result (figure 8.3). When the population has been greatly reduced, the rate of killing may slow due to the survival of a more resistant strain of the microorganism. One measure of an agent’s killing efficiency is the decimal reduction time (D) or D value. The decimal reduction time is the time required to kill 90% of the microorganisms or endospores in a sample under specified conditions. For example, in a semilogarithmic plot of the population remaining versus the time of heating, the D value is the time required for the line to drop by one log cycle or 10-fold (figure 8.3a). It is also possible to determine the temperature change at a given D value that decreases the microbial population by one log cycle (90%). This temperature change is referred to as the Z value and is predicted from a semilogarithmic plot of D values versus temperature (figure 8.3b). To study the effectiveness of a lethal agent, one must be able to decide when microorganisms are dead, which may present some challenges. A microbial cell is often defined as dead if it does not grow when inoculated into culture medium that would normally support its growth. In like manner, an inactive virus cannot infect a suitable host. This definition has flaws, however. wil11886_ch08_170-186.indd 173 It has been demonstrated that when bacteria are exposed to certain conditions, they can remain alive but are temporarily unable to reproduce (see figure 7.34). In conventional tests to demonstrate killing by an antimicrobial agent, these viable but nonculturable bacteria would be thought to be dead. This is a serious problem because the bacteria may regain their ability to reproduce and cause infection after a period of recovery. Most microbes live in growth-arrested states (section 7.6) 8.3 Mechanical Removal Methods Rely on Barriers After reading this section, you should be able to: a. Explain the mechanism by which filtration removes microorganisms b. Propose filtration methods to selectively remove one type of microbe from a mixed population Filtration is an excellent way to reduce the microbial population in solutions of heat-sensitive material and can be used to sterilize liquids and gases (including air). Rather than directly 23/10/18 9:21 am 174 CHAPTER 8 | Control of Microorganisms in the Environment Liquid Filter Pore Filter Vacuum pump suction Sterilized fluid (a) 1/4" stepped hose connector Vent Durapore filters Filter support destroying contaminating microorganisms, the filter simply acts as a barrier to retain them. There are two types of filters. Depth filters consist of fibrous or granular materials that have been bonded into a thick layer filled with narrow, twisting channels. The solution containing microorganisms is sucked through this layer under vacuum, and microbial cells are removed by entrapment and by adsorption to the surface of the filter material. Membrane filters have replaced depth filters for many purposes. These filters are porous membranes, a little over 0.1 mm thick, made of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride, or other synthetic materials. Although a wide variety of pore sizes are available, membranes with pores about 0.2 μm in diameter are used to remove most vegetative cells, but not viruses, from liquids. The membranes are held in special holders (figure 8.4), and their use is often preceded by depth filters to remove larger particles that might clog the membrane filter. The liquid is forced through the filter and collected in previously sterilized containers. Membrane filters remove microorganisms by screening them out much as a sieve separates large sand particles from small ones (figure 8.5). These filters are used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils, antibiotics, and other heat-sensitive solutions. Air also can be filtered to remove microorganisms. Two common examples are N95 disposable masks used in hospitals and labs, and high-efficiency particulate air (HEPA) filters that let air move freely but restrict microorganisms. N95 masks exclude 95% of particles that are larger than 0.3 μm. HEPA filters (a type of depth filter made from fiberglass) remove 99.97% of particles 0.3 μm or larger by both physical retention and electrostatic interactions. HEPA filters sterilize air by removing viruses that are 0.1 μm and Filling bell Bell cap Cross section Millipak-40 filter unit (b) Figure 8.4 Membrane Filter Sterilization. The liquid to be sterilized is pumped through a membrane filter and into a sterile container. (a) Schematic representation of a membrane filtration setup that uses a vacuum pump to force liquid through the filter. The inset shows a cross section of the filter and its pores, which are too small for microbes to pass through. (b) Cross section of a membrane filtration unit. Several membranes are used to increase its capacity. MICRO INQUIRY How might one verify that filtration removed all microorganisms? Figure 8.5 Membrane Filter. Enterococcus faecalis resting on a polycarbonate membrane filter with 0.4-μm pores. ©Callista Images Cultura/Newscom wil11886_ch08_170-186.indd 174 23/10/18 9:21 am 8.3 Mechanical Removal Methods Rely on Barriers 175 MICROBIAL DIVERSITY & ECOLOGY 8.1 The Cleanest Place on Earth? Many environments must be as devoid of microbes as possible, for example, operating rooms or pharmaceutical manufacturing facilities. Microbial contamination in these places can result in severe infection or fatality. Spacecraft assembly sites, too, must be free of microbes, but the consequences of contamination could affect an entire planet. The National Air and Space Administration (NASA) is planning two missions to look for signs of past life or current habitable conditions elsewhere in the solar system. Mars 2020 and the Europa Clipper mission to Jupiter’s moon take the search for extraterrestrial life directly to those locations. Two concerns face astrobiologists: first, avoiding falsepositive signals for life by detecting stowaway microbes; and second, avoiding contamination of extraterrestrial environ- (a) ments. Although complete spacecraft sterility is the ideal goal, it is currently unattainable, as sensitive instruments cannot withstand heat treatment. Extremophiles and spores are the primary concern because they have the greatest probability for survival in space. The major contamination source, the engineers who assemble the spacecraft, work in special suits to confine their natural microbial shedding. These clean rooms operate with HEPA-filtered air and surfaces are regularly treated with ultraviolet light. Surprisingly, the lack of dust and organic matter in NASA clean rooms has created an extreme environment that enriches for the presence of certain microbes. Tersicoccus phoenicis has been isolated from only two places on Earth: spacecraft assembly clean rooms on two continents. (b) NASA Clean Room Conditions. (a) Staff in NASA clean rooms dress in protective garb when working on spacecraft to minimize contamination. (b) Tersicoccus phoenicis, whose only known natural environment is clean rooms where spacecraft are assembled. (a) Source: NASA/Leif Heimbold; (b) Source: NASA/JPL-Caltech smaller. HEPA-filtered air is critical in clean room environments, as discussed in Microbial Diversity & Ecology 8.1. Laminar flow biological safety cabinets or hoods force air through HEPA filters, then project a vertical curtain of sterile air across the cabinet opening. This protects a worker from microorganisms being handled within the cabinet and prevents contamination of the room (figure 8.6). Dangerous agents such as Mycobacterium tuberculosis, pathogenic fungi, or tumor viruses must be confined to biological safety cabinets. Comprehension Check 1. What are depth filters and membrane filters, and how are they used to sterilize liquids? 2. Describe the operation of a biological safety cabinet. 176 CHAPTER 8 | Control of Microorganisms in the Environment 8.4 Physical Control Methods Alter Microorganisms to Make Them Nonviable After reading this section, you should be able to: a. Describe the application of heat and radiation to control microorganisms b. Explain the mechanisms by which heat and radiation kill microbes c. Design novel antimicrobial control applications using heat and radiation Heat and other physical agents are normally used to control microbial growth and sterilize objects, as can be seen from the operation of the autoclave. The most frequently employed physical agents are heat and radiation. (a) Heat HEPA-filtered air Room air Contaminated air Most microorganisms require specific temperatures for normal growth and replication; temperatures that exceed those damage structures and alter chemical reactions. Moist and dry heat readily destroy viruses, bacteria, and fungi in this way (table 8.2). Moist heat destroys cells and viruses by degrading nucleic acids, denaturing proteins, and disrupting cell membranes. Exposure to boiling water for 10 minutes is sufficient to destroy vegetative cells and eukaryotic spores. Unfortunately, the temperature of boiling water (100°C at sea level) is not sufficient to destroy bacterial endospores, which may survive hours of boiling. Therefore boiling can be used for disinfecting drinking water and objects not harmed by water, but boiling does not sterilize. To destroy bacterial endospores, moist heat sterilization must be carried out at temperatures above 100°C, and this requires the use of saturated steam under pressure. Steam sterilization is carried out with an autoclave (figure 8.7), a device somewhat like a fancy pressure cooker. The development of the autoclave by Charles Chamberland in 1884 tremendously stimulated the growth of microbiology as a science. Steam is released Table 8.2 Approximate Conditions for Moist Heat Inactivation Side view (b) Figure 8.6 A Biological Safety Cabinet. (a) A technician pipetting potentially hazardous material in a safety cabinet. (b) A schematic diagram showing the airflow pattern within a class II safety cabinet. (a) ©miR156/Alamy Stock Photo wil11886_ch08_170-186.indd 176 1 Conditions for mesophilic bacteria. 23/10/18 9:21 am 8.4 Physical Control Methods Alter Microorganisms to Make Them Nonviable 177 (a) Pressure regulator Safety valve Steam from jacket to chamber Door gasket Steam jacket Steam supply (b) Figure 8.7 The Autoclave. (a) A modern, automatically controlled autoclave or sterilizer. (b) Longitudinal cross section of a typical autoclave showing some of its parts and the pathway of steam. (a) ©BSIP SA/Alamy Stock Photo into the autoclave chamber (figure 8.7b). The air initially present in the chamber is forced out until the chamber is filled with saturated steam and the outlets are closed. Hot, saturated steam continues to enter until the chamber reaches the desired temperature and pressure, usually 121°C and 15 pounds per square inch (psi) of pressure. Autoclaving must be carried out properly so that the saturated steam can destroy all vegetative cells and endospores within the processed materials. If all air has not been flushed out of the chamber, it will not reach 121°C, even though it may reach a pressure of 15 psi. The chamber should not be packed too tightly because the wil11886_ch08_170-186.indd 177 steam needs to circulate freely and contact everything in the autoclave. Bacterial endospores will be killed only if they are kept at 121°C for 10 to 12 minutes. When a large volume of liquid must be sterilized, an extended sterilization time is needed because it takes longer for the center of the liquid to reach 121°C; 5 liters of liquid may require about 70 minutes. In view of these potential difficulties, a biological indicator is often included in the autoclave. This indicator commonly consists of a culture tube containing a sterile ampule of medium and a paper strip covered with endospores of Geobacillus stearothermophilus. After autoclaving, the ampule is aseptically broken and the culture incubated. If the test bacterium does not grow, the sterilization run has been successful. Sometimes indicator tape or paper that changes color upon heating is autoclaved with a batch of material. These indicate heating has occurred and are convenient but are not as reliable as techniques that directly demonstrate the inactivation of bacterial endospores. Many heat-sensitive substances, such as milk, are treated with controlled heating at temperatures well below boiling, a process known as pasteurization in honor of its developer, Louis Pasteur (1822–1895). In the 1860s the French wine industry was plagued by wine spoilage, which made wine storage and shipping difficult. Pasteur examined spoiled wine under the microscope and detected microorganisms that looked like the bacteria responsible for lactic acid and acetic acid fermentations; these bacteria do not form endospores. He discovered that a brief heating at 55° to 60°C would destroy these microorganisms and preserve wine for long periods. In 1886 German chemists V. H. Soxhlet and F. Soxhlet adapted the technique for preserving milk. Milk pasteurization was introduced in the United States in 1889. Milk, beer, and many other beverages are now pasteurized. Pasteurization does not sterilize, but it does kill pathogens and drastically slows spoilage by reducing the level of nonpathogenic spoilage microorganisms (see table 41.2). Some materials cannot withstand the high temperature of the autoclave, and endospore contamination precludes the use of other methods to sterilize them. For these materials, a process of intermittent sterilization, also known as tyndallization (for John Tyndall [1820–1893], the British physicist who used the technique to destroy heat-resistant microorganisms in dust) is used. The process also uses steam (30–60 minutes) to destroy vegetative bacteria. However, steam exposure is repeated for a total of three times with 23- to 24-hour incubations between exposures. The incubations permit remaining endospores to germinate into heat-sensitive vegetative cells that are destroyed upon subsequent steam exposures. Many objects are best sterilized by dry heat. For instance, inoculating loops, which are used routinely in the laboratory, can be sterilized in a small, bench-top incinerator (figure 8.8). Other items are sterilized in an oven at 160° to 170°C for 2 to 3 hours. Microbial death results from the oxidation of cell constituents and protein denaturation. Dry air heat is less effective than moist heat. The endospores of Clostridium botulinum, the cause of botulism, are killed in 5 minutes at 121°C by moist heat but only after 2 hours at 160°C by dry heat. However, dry heat has some advantages. It does not 23/10/18 9:21 am 178 CHAPTER 8 | Control of Microorganisms in the Environment Incinerator Figure 8.8 Dry Heat Incineration. Bench-top incinerators are routinely used to sterilize inoculating loops used in microbiology laboratories. ©McGraw-Hill Education/James Redfearn, photographer corrode glassware and metal instruments as moist heat does, and it can be used to sterilize powders, oils, and similar items. Despite these advantages, dry heat sterilization is slow and not suitable for heat-sensitive materials such as plastic and rubber items. beta radiation (accelerated electrons from high-voltage electricity) are used in the cold sterilization of antibiotics, hormones, sutures, and plastic disposable supplies such as syringes. Gamma radiation and electron beams have also been used to sterilize and “pasteurize” meat and other foods (figure 8.9). Irradiation can eliminate the threat of such pathogens as E. coli O157:H7, which causes a life-threatening intestinal disease; Staphylococcus aureus, which causes skin and blood infections, and readily colonizes medical devices used on patients; and Campylobacter jejuni, which contaminates poultry, causing intestinal disease when undercooked meat is eaten. Both the U.S. Food and Drug Administration and the World Health Organization have approved irradiated food and declared it safe for human consumption. Currently irradiation is used to treat meat, fruits, vegetables, and spices. Human diseases caused by bacteria (chapter 39); Various methods are used to control food spoilage (section 41.2) Comprehension Check 1. Describe how an autoclave works. What conditions are required for sterilization by moist heat? What three things must one do when operating an autoclave to help ensure success? 2. In the past, spoiled milk was responsible for a significant proportion of infant deaths. Why is untreated milk easily spoiled? 3. List the advantages and disadvantages of ultraviolet light and ionizing radiation as sterilizing agents. Provide a few examples of how each is used for this purpose. 4. What is the correlation between radiation “energy” and the mechanisms of sterilization? Radiation Ultraviolet (UV) radiation around 260 nm (see figure 7.20) is quite lethal. It causes thymine-thymine dimerization of DNA, preventing replication and transcription (see figure 16.5). However, UV radiation does not penetrate glass, dirt films, water, Radiation room and other substances effectively. Because of this disadvantage, Chamber with radiation shield UV radiation is used as a sterilizing agent only in a few situaConveyor system with pallets tions. UV lamps are sometimes placed on lab ceilings or in bioof sterilized materials logical safety cabinets to sterilize the air and any exposed surfaces. Because UV radiation burns the skin and damages eyes, the UV lamps are off when the areas are in use. Commercial UV units are available for water treatment. Microbes are destroyed when a thin layer of water is passed under the lamps. Purification and sanitary analysis ensure safe drinking water (section 43.1) Ionizing radiation is an excellent sterilizing agent that penetrates deep into objects. Ionizing radiation has sufficient energy to dislodge electrons from atoms or moleRadioactive cules, producing chemically reactive free radisource cals. The free radicals react with nearby matter to weaken or destroy it. Ionizing radiation destroys bacterial endospores and all microbial cells; however, it is not always effective against viruses. Figure 8.9 Sterilization with Ionizing Radiation. An irradiation facility that uses radioactive Gamma radiation (from a cobalt 60 source) and cobalt 60 as a gamma radiation source to sterilize fruits, vegetables, meats, fish, and spices. wil11886_ch08_170-186.indd 178 23/10/18 9:21 am 8.5 Microorganisms Are Controlled with Chemical Agents 179 8.5 Microorganisms Are Controlled with Chemical Agents After reading this section, you should be able to: a. Describe the use of and mechanism of action for phenolics, alcohols, halogens, heavy metals, quaternary ammonium compounds, aldehydes, and oxides to control microorganisms b. Design novel antimicrobial control applications using phenolics, alcohols, halogens, heavy metals, quaternary ammonium compounds, aldehydes, and oxides Chemicals can be employed for sterilization, disinfection, and antisepsis. The proper use of chemical agents is essential for personal safety. Chemicals also are employed to prevent microbial growth in food, and certain chemicals are used to treat infectious disease. The use of chemical agents for chemotherapy in humans is covered in chapter 9 and their use in food is discussed in chapter 41. Here we discuss chemicals used outside the body. Many different chemicals have been specifically formulated as disinfectants, each with its own advantages and disadvantages. Ideally the biocide is effective against a wide variety of infectious agents (bacteria, bacterial endospores, fungi, viruses, and prions) at low concentrations and in the presence of organic matter. Although the chemical must be toxic for infectious agents, it should not be toxic to people or corrosive for common materials. In practice, this balance between effectiveness and low toxicity is hard to achieve. Some chemicals are used despite their low effectiveness because they are relatively nontoxic. The ideal disinfectant should be stable upon storage, odorless or with a pleasant odor, and soluble in water and lipids for penetration into microorganisms; have a low surface tension so that it can enter cracks in surfaces; and be relatively inexpensive. Other chemical biocides are used as antiseptics. Recall that antiseptics are less toxic to humans than disinfectants and as such may be less effective at killing all the microorganisms that disinfectants can kill. In general, antiseptics should reduce the number of pathogens on human tissue to prevent infection. Examples of antiseptics include hand sanitizers, silver threads woven into clothing, and dilute iodine solutions that can be sprayed onto wounds. One potentially serious problem is the overuse of antiseptics. For instance, resistance to the antibacterial agent triclosan (found in products such as deodorants, mouthwashes, soaps, cutting boards, and baby toys) has become a problem. Because of its overuse, triclosan was banned from sale in the United States in 2017, but it persists in many products in consumers’ homes. There are several mechanisms of drug resistance (section 9.8) The properties and uses of common disinfectants and antiseptics are surveyed next. Many of the characteristics of disinfectants and antiseptics are summarized in table 8.3. Structures of some common agents are shown in figure 8.10. wil11886_ch08_170-186.indd 179 Phenolics Phenol was the first widely used antiseptic and disinfectant. In 1867 Joseph Lister employed it to reduce the risk of infection during surgery. Today phenol and phenol derivatives (phenolics) are used as disinfectants in laboratories and hospitals. The commercial disinfectant Lysol is a mixture of phenolics. Phenolics denature proteins and disrupt cell membranes. They have some important advantages as disinfectants: Phenolics are tuberculocidal, effective in the presence of organic material, and remain active on surfaces long after application. However, they have a disagreeable odor and can cause skin irritation. Alcohols Alcohols are among the most widely used disinfectants, antiseptics, and sanitizers. They are bactericidal and fungicidal but not sporicidal; some enveloped viruses are also destroyed. The two most popular alcohol germicides are ethanol and isopropanol, usually used in about 60 to 80% concentration. They act by denaturing proteins and possibly by dissolving membrane lipids. A 10- to 15-minute soaking is sufficient to disinfect small instruments, while rubbing hands with specially formulated alcohol products sanitizes them by killing many pathogens. Halogens The halogens iodine and chlorine are important antimicrobial agents. Iodine is used as a skin antiseptic and kills by oxidizing cell constituents and iodinating proteins. At higher concentrations, it may even kill some endospores. Iodine often is applied as tincture of iodine, 2% or more iodine in a water-ethanol solution of potassium iodide. Although it is an effective antiseptic, the skin may be damaged, a stain remains, and iodine allergies can result. Iodine can be complexed with an organic carrier to form an iodophor. Iodophors are water soluble, stable, and nonstaining, and release iodine slowly to minimize skin irritation. They are used in hospitals for cleansing preoperative skin and in hospitals and laboratories for disinfecting. Some popular brands are Wescodyne for skin and laboratory disinfection, and Betadine for wounds. Chlorine is the usual disinfectant for municipal water supplies and swimming pools, and is also employed in the dairy and food industries. It may be applied as chlorine gas (Cl2), sodium hypochlorite (bleach, NaOCl), or calcium hypochlorite [Ca(OCl)2], all of which yield hypochlorous acid (HOCl): Cl2 + H2O → HCl + HOCl NaOCl + H2O → NaOH + HOCl Ca(OCl)2 + 2H2O → Ca(OH)2 + 2HOCl The result is oxidation of cellular materials and destruction of vegetative bacteria and fungi. Death of almost all microorganisms usually occurs within 30 minutes. Two important eukaryotic pathogens are not killed by chlorine, Cryptosporidium and Giardia. Both are transmitted via water. Food and water are vehicles for fungal and protozoal diseases (section 40.5) Chlorine is also an excellent disinfectant for individual use because it is effective, inexpensive, and easy to employ. Small quantities of drinking water can be disinfected with halazone 23/10/18 9:21 am 180 CHAPTER 8 Table 8.3 | Control of Microorganisms in the Environment Activity Level of Selected Biocides 1 High-level disinfectants destroy vegetative bacterial cells including M. tuberculosis, bacterial endospores, fungi, and viruses. Intermediate-level disinfectants destroy all of these except endospores. Low-level agents kill bacterial vegetative cells except for M. tuberculosis, fungi, and medium-sized lipid-containing viruses (but not bacterial endospores or small, nonlipid viruses). 2 In autoclave-type equipment at 55° to 60°C. 3 Available iodine. 4 Free chlorine. wil11886_ch08_170-186.indd 180 23/10/18 9:21 am 8.5 Microorganisms Are Controlled with Chemical Agents 181 Phenolics OH OH Cl Phenol Cl Cl HO Cl Cl CH3 CH2 OH Cl Ortho-cresol Hexachlorophene Alcohols CH3 CH CH3 Ethanol Isopropanol Halogenated compound O H2 C C N Cl C H2 C O Halazone Aldehydes O O H H C H O CH2 CH2 C H C CH2 Glutaraldehyde Formaldehyde Aldehydes Quaternary ammonium compounds CH3 Cl– + N C16 H33 CnH2n + 1 N + CH2 Cl – CH3 Cetylpyridinium Benzalkonium Gases CH2 O CH2 Ethylene oxide H H O O Hydrogen peroxide Figure 8.10 Disinfectants and Antiseptics. The structures of some frequently used disinfectants and antiseptics. MICRO INQUIRY Why is it important that all of these compounds are relatively hydrophobic? tablets. Halazone (parasulfone dichloramidobenzoic acid) slowly releases chloride when added to water and disinfects it in about 30 minutes. It is frequently used by campers lacking access to uncontaminated drinking water. Of note is the fact that household bleach (diluted to 10% in water, 10-minute contact time) can be used to disinfect surfaces contaminated by human body fluids and that it is made more effective by the addition of household vinegar. Heavy Metals For many years heavy metal ions such as those of mercury, silver, arsenic, zinc, and copper were used as germicides. These wil11886_ch08_170-186.indd 181 Quaternary Ammonium Compounds Quaternary ammonium compounds are detergents that have broad spectrum antimicrobial activity and are effective disinfectants used for decontamination purposes. Detergents (Latin detergere, to wipe away) are organic cleansing agents that are amphipathic, having both polar hydrophilic and nonpolar hydrophobic components. The hydrophilic portion of a quaternary ammonium compound is a positively charged quaternary nitrogen; thus quaternary ammonium compounds are cationic detergents. Their antimicrobial activity is the result of their ability to disrupt microbial membranes; they may also denature proteins. Cationic detergents such as benzalkonium chloride and cetylpyridinium chloride kill most bacteria but not M. tuberculosis or endospores. They have the advantages of being stable and nontoxic, but they are inactivated by hard water and soap. Cationic detergents are often used as disinfectants for food utensils and small instruments, and as skin antiseptics. OH CH3 CH2 OH have now been superseded by other less toxic and more effective germicides. Silver sulfadiazine is used on burns. Copper sulfate is an effective algicide in lakes and swimming pools. Heavy metals combine with proteins, often with their sulfhydryl groups, and inactivate them. They may also precipitate cell proteins. Both of the commonly used aldehydes, formaldehyde and glutaraldehyde (figure 8.10), are highly reactive molecules that inactivate nucleic acids and proteins, probably by cross-linking and alkylating molecules (figure 8.11). They are sporicidal and can be used as chemical sterilants. Formaldehyde is usually dissolved in water or alcohol before use. A 2% buffered solution of glutaraldehyde is an effective disinfectant. It is less irritating than formaldehyde and is used to disinfect hospital and laboratory equipment. Glutaraldehyde usually disinfects objects within about 10 minutes but may require as long as 12 hours to destroy all endospores. Sterilizing Gases Many heat-sensitive items such as plastic Petri dishes, heart-lung machine components, sutures, and catheters are sterilized with ethylene oxide gas (figure 8.10). Ethylene oxide (EtO) is both microbicidal and sporicidal. It is a strong alkylating agent that kills by reacting with DNA and proteins to block replication and enzymatic activity. It is a particularly effective sterilizing agent because it rapidly penetrates packing materials, even plastic wraps. Sterilization is carried out in an ethylene oxide sterilizer, which resembles an autoclave in appearance. It controls the EtO concentration, temperature, and humidity (figure 8.12). Because pure EtO is explosive, it is usually supplied in a 10 to 20% concentration mixed with either CO2 or dichlorodifluoromethane. The EtO concentration, humidity, and temperature influence the rate of sterilization. A clean object can be sterilized if treated for 5 to 8 hours at 38°C or 3 to 4 hours at 54°C when the relative humidity is maintained at 40 to 50% and the EtO concentration at 700 mg/L. Because it is so toxic to humans, extensive aeration of the sterilized materials is necessary to remove residual EtO. 23/10/18 9:21 am 182 CHAPTER 8 | Control of Microorganisms in the Environment Glutaraldehyde O O Polymerization O O O Polyglutaraldehyde O Cross-linking with microbial protein N N N N G– Amino groups in peptidoglycan G+ Figure 8.11 Effects of Glutaraldehyde. Glutaraldehyde polymerizes and then interacts with amino acids in proteins (left) or in peptidoglycan (right). As a result, the proteins are alkylated and cross-linked to other proteins, which inactivates them. The amino groups in peptidoglycan are also alkylated and cross-linked, which prevents them from participating in other chemical reactions such as those involved in peptidoglycan synthesis. MICRO INQUIRY Why are cross-linking agents such as glutaraldehyde often called “fixatives” or are said to “fix the cells”? after the 2001 anthrax attacks, and to kill molds that contaminated Gulf Coast homes in the aftermath of hurricane Harvey in 2017. ClO2 has a broad killing spectrum, controlling bacteria, endospores, fungi, and protozoa. ClO2 appears to have several mechanisms of action: It reacts readily with amino acids cysteine, tryptophan, and tyrosine, denaturing proteins; with free fatty acids, lysing membranes; and with nucleic acids, inhibiting replication. To date, no resistance to ClO2 has been identified. ClO2 is highly water soluble, especially in cold water, where it does not hydrolyze but remains as a dissolved gas in solution. Thus a number of municipal water treatment facilities use it for water disinfection. Vaporized hydrogen peroxide (VHP) can also be used to decontaminate biological safety cabinets, operating rooms, and other large facilities. VHP is produced from a solution of hydrogen peroxide in water that is passed over a vaporizer to achieve a vapor concentration between 140 and 1,400 parts per million (ppm), depending on the agent to be destroyed. VHP is then introduced as a sterilizing vapor into the enclosure for some time, depending on the size of the enclosure and the materials within. Hydrogen peroxide and its oxy-radical by-products are toxic (75 ppm are dangerous to human health) and kill a wide variety of microorganisms. During the course of the decontamination process, VHP breaks down to water and oxygen, both of which are harmless. Comprehension Check 1. Why are most antimicrobial chemical agents disinfectants rather than sterilants? What general characteristics should one look for in a disinfectant? 2. Construct a table that compares the chemical nature, mechanism of action, mode of application, common uses and effectiveness, and advantages and disadvantages between phenolics, alcohols, halogens, heavy metals, quaternary ammonium compounds, aldehydes, and ethylene oxide. Air intake Chlorine dioxide (ClO2) gas is also used as a disinfectant. Of note is the fact that the chemistry of chlorine dioxide is very different from that of chlorine gas. ClO2 is typically aerosolized in a humidified environment, 1 mg/liter of air in 60% relative humidity, for at least 4 hours. At this concentration, ClO2 provides a greater than 6 log reduction of endospores and vegetative bacteria. It has been used to sterilize hospital operating and patient rooms. ClO2 fumigation is used in the food industry to sanitize fruits and vegetables of contaminating yeasts and molds. As a disinfecting gas, it is best known for its use in sterilizing the U.S. Senate and Postal facilities wil11886_ch08_170-186.indd 182 Vacuum pump Air filter Chamber Door (a) Figure 8.12 An Ethylene Oxide Sterilizer. (a) An automatic ethylene oxide (EtO) sterilizer. (b) Schematic of an EtO sterilizer. Items to be sterilized are placed in the chamber, and EtO and carbon dioxide are introduced. After the sterilization CO2 cylinder procedure is completed, the EtO and carbon dioxide (b) are pumped out of the chamber and air enters. Gas mixer EtO cylinder (a) ©Anderson Products, www.anpro.com 23/10/18 9:21 am 8.6 Antimicrobial Agents Must Be Evaluated for Effectiveness 183 3. Which disinfectants or antiseptics would be used to treat the following: laboratory bench top, drinking water, patch of skin before surgery, small medical instruments (probes, forceps, etc.)? Explain your choices. 4. How do phenolic agents differ from the other chemical control agents described in this chapter? 5. Which physical or chemical agent would be the best choice for sterilizing the following items: glass pipettes, tryptic soy broth tubes, nutrient agar, antibiotic solution, interior of a biological safety cabinet, wrapped package of plastic Petri plates? Explain your choices. 8.6 Antimicrobial Agents Must Be Evaluated for Effectiveness After reading this section, you should be able to: a. Predict the effects of (1) microbial population size and composition, (2) temperature, (3) exposure time, and (4) local environmental conditions on antimicrobial agent effectiveness b. Describe the processes used to measure microbial killing rates, dilution testing, and in-use testing of antimicrobial agents The assessment of antimicrobial agent effectiveness is a complex process regulated by two different federal agencies. The Environmental Protection Agency regulates disinfectants, whereas agents used on humans and animals are under the control of the Food and Drug Administration. They establish the guidelines under which these agents are used and agent effectiveness is measured. Importantly, there are a number of variables that must also be considered when evaluating antimicrobial agent effectiveness. Destruction of microorganisms and inhibition of microbial growth are not simple matters because the effectiveness of an antimicrobial agent is affected by at least six factors. 1. Population size. Because an equal fraction of a microbial population is killed during each interval, a larger population requires a longer time to die than does a smaller one (table 8.1 and figure 8.3). 2. Population composition. The effectiveness of an agent varies greatly with the nature of the organisms being treated because microorganisms differ markedly in susceptibility. Bacterial endospores are much more resistant to most antimicrobial agents than vegetative forms, and younger cells are usually more readily destroyed than mature organisms. Some species are able to withstand adverse conditions better than others. For instance, M. tuberculosis, which causes tuberculosis, is much more resistant to antimicrobial agents than most other bacteria. 3. Concentration or intensity of an antimicrobial agent. Often, but not always, the more concentrated a chemical agent or intense a physical agent, the more rapidly microorganisms are destroyed. However, agent effectiveness usually is not directly related to concentration or intensity. Over a short range, a small increase in concentration leads to an exponential wil11886_ch08_170-186.indd 183 rise in effectiveness; beyond a certain point, increases may not raise the killing rate much at all. Sometimes an agent is more effective at lower concentrations. For example, 70% ethanol is more bactericidal than 95% ethanol because the activity of ethanol is enhanced by the presence of water. 4. Contact time. The longer a population is exposed to a microbicidal agent, the more organisms are killed (figures 8.2 and 8.3). To achieve sterilization, contact time should be long enough to reduce the probability of survival by at least 6 logs. 5. Temperature. An increase in the temperature at which a chemical acts often enhances its activity. Frequently a lower concentration of disinfectant or sterilizing agent can be used at a higher temperature. 6. Local environment. The population to be controlled is not isolated but surrounded by environmental factors that may either offer protection or aid in its destruction. For example, because heat kills more readily at an acidic pH, acidic foods and beverages such as fruits and tomatoes are easier to pasteurize than more alkaline foods such as milk. A second important environmental factor is organic matter, which can protect microorganisms against physical and chemical disinfecting agents. Biofilms are a good example. The organic matter in a biofilm protects the biofilm’s microorganisms. Furthermore, bacteria in biofilms are altered physiologically, and this makes them less susceptible to many antimicrobial agents. Because of the impact of organic matter, it may be necessary to clean objects, especially medical and dental equipment, before they are disinfected or sterilized. Biofilms are common in nature (section 7.6) The actual testing of antimicrobial agents often begins with an initial screening to see if they are effective and at what concentrations. This may be followed by more realistic in-use testing. The bestknown disinfectant screening test is the phenol coefficient test in which the potency of a disinfectant is compared with that of phenol. A series of dilutions of phenol and the disinfectant being tested are prepared. Standard amounts of Salmonella enterica serovar Typhi and Staphylococcus aureus are added to each dilution; the dilutions are then placed in a 20° or 37°C water bath. At 5-minute intervals, samples are withdrawn from each dilution and used to inoculate growth medium, which is incubated and examined for growth. Growth indicates that the dilution at that particular time of sampling did not kill the bacteria. The highest dilution (i.e., the lowest concentration) that kills the bacteria after a 10-minute exposure but not after 5 minutes is used to calculate the phenol coefficient. The higher the phenol coefficient value, the more effective the disinfectant under these test conditions. A value greater than 1 means that the disinfectant is more effective than phenol. The phenol coefficient test is a useful initial screening procedure, but the phenol coefficient can be misleading if taken as a direct indication of disinfectant potency during actual use. This is because the phenol coefficient is determined under carefully controlled conditions with pure bacterial cultures, whereas disinfectants are normally used on complex populations in the presence of 23/10/18 9:21 am 184 CHAPTER 8 | Control of Microorganisms in the Environment organic matter and with significant variations in environmental factors such as pH, temperature, and presence of salts. To more realistically estimate disinfectant effectiveness, other tests are often used. The rates at which selected bacteria are destroyed with various chemical agents may be experimentally determined and compared. A use dilution test can also be carried out. Stainless steel carriers are contaminated with one of three specific bacterial species under carefully controlled conditions. The carriers are dried briefly, immersed in the test disinfectants for 10 minutes, transferred to culture media, and incubated for 2 days. The disinfectant concentration that kills the bacteria on at least 59 of 60 carriers (a 95% level of confidence) is determined. Disinfectants also can be tested under conditions designed to simulate normal in-use situations. In-use testing techniques allow a more accurate determination of the proper disinfectant concentration for a particular situation. Comprehension Check 1. Briefly explain how the effectiveness of antimicrobial agents varies with population size, population composition, concentration or intensity of the agent, contact time, temperature, and local environmental conditions. 2. How does being in a biofilm affect an organism’s susceptibility to antimicrobial agents? 3. Suppose hospital custodians have been assigned the task of cleaning all showerheads in patient rooms to prevent the spread of infectious disease. What two factors would have the greatest impact on the effectiveness of the disinfectant the custodians use? Explain what that impact would be. 4. Briefly describe the phenol coefficient test. 5. Why might it be necessary to employ procedures such as the use dilution and in-use tests? 8.7 Microorganisms Can Be Controlled by Biological Methods After reading this section, you should be able to: a. Propose predation, competition, and other methods for biological control of microorganisms b. Suggest alternative decontamination and medical therapies using viruses of bacteria, fungi, and protozoa The emerging field of biological control of microorganisms holds great promise. Scientists are learning to exploit natural control processes such as predation of one microorganism on another, viral-mediated lysis, and toxin-mediated killing. While these mechanisms occur in nature, their use by humans is relatively new. Studies evaluating control of the human intestinal pathogens Salmonella spp., Shigella spp., and E. coli by Gramnegative predators such as Bdellovibrio spp. suggest that poultry farms may be sprayed with a predatory bacterium to reduce potential contamination. Another biological control method had its start in the early 1900s at the Pasteur Institute in France. Felix d’Herelle isolated bacteriophage from patients recovering from bacillary dysentery. After numerous tests in vitro, d’Herelle concluded that the bacteriophage participated in the destruction of dysentery-causing bacteria. Bacteriophage therapies were under development when penicillin ushered in the age of antibiotics; they are currently used only in Russia, Poland, and Georgia. However, control of pathogens using bacteriophage is regaining wide support and appears to be effective in the eradication of a number of bacterial species by lysing the pathogenic host. In fact, the U.S. FDA has now approved the use of a bacteriophage spray to eradicate Listeria, Salmonella, and E. coli in foods. Yet to be approved are several designs for treating human infectious diseases by similar methods. This seems intuitive, knowing that the virus lyses its specific bacterial host, yet unnerving when one thinks about swallowing, injecting, or applying a virus (albeit a bacteriophage) to the human body. In essence, microbial control relies on the lytic effect of bacteriophage on cells. The distaste of using bacteriophage as a therapeutic can be overcome by the use of enzybiotics. These are proteins purified from bacteriophage that cause host cell lysis. They are endolysins whose substrate is peptidoglycan, and the exposed Gram-positive cell wall renders these cells especially susceptible. Other potentially useful bacterial proteins are depolymerases that hydrolyze a biofilm matrix and microbial toxins (such as bacteriocins). Viruses and other acellular infectious agents (chapter 6) Class Deltaproteobacteria includes chemoheterotrophic anaerobes and predators (section 22.4); Bacteriocins (section 32.3); Various methods are used to control food spoilage (section 41.2) Comprehension Check 1. How would you explain to a patient that a virus can be used to eliminate a bone infection caused by bacteria that do not respond to antibiotics? 2. Propose the use of specific bacterial, viral, or fungal products that might be used to kill other, more virulent bacteria, viruses, or fungi. Key Concepts 8.1 ! Microbial Growth and Replication: Targets for Control ■ Sterilization is the process by which all living cells, viable spores, viruses, viroids, and prions are either destroyed or removed from an object or habitat. Disinfection is the killing, inhibition, or removal of wil11886_ch08_170-186.indd 184 ■ microorganisms (but not necessarily endospores) that can cause disease. The main goal of disinfection and antisepsis is the removal, inhibition, or killing of pathogenic microbes. Both processes also reduce the total number of microbes. Disinfectants are chemicals used to disinfect 23/10/18 9:21 am Active Learning 185 ■ inanimate objects; antiseptics are used on living tissue. Antimicrobial agents that kill organisms often have the suffix -cide, whereas agents that prevent growth and reproduction have the suffix -static. 8.2 The Pattern of Microbial Death Mirrors the Pattern of Microbial Growth ■ ■ Microbial death is usually exponential (figure 8.3). The decimal reduction time measures an agent’s killing efficiency. It represents the time needed to kill 90% of the microbes under specified conditions. 8.3 Mechanical Removal Methods Rely on Barriers ■ ■ Microorganisms can be efficiently removed by filtration with either depth filters or membrane filters (figure 8.4). Biological safety cabinets with high-efficiency particulate filters sterilize air by filtration (figure 8.6). ■ ■ ■ ■ ■ ■ 8.4 Physical Control Methods Alter Microorganisms to Make Them Nonviable ■ ■ ■ ■ Moist heat kills by degrading nucleic acids, denaturing proteins, and disrupting cell membranes. Although treatment with boiling water for 10 minutes kills vegetative forms, an autoclave must be used to destroy endospores by heating at 121°C and 15 pounds of pressure (figure 8.7). Glassware and other heat-stable items may be sterilized by dry heat at 160° to 170°C for 2 to 3 hours. Radiation of short-wavelength or high-energy ultraviolet and ionizing radiation can be used to sterilize objects (figure 8.9). 8.5 Microorganisms Are Controlled with Chemical Agents ■ ■ ■ Chemical agents usually act as disinfectants or antiseptics because they cannot readily destroy bacterial spores. Disinfectant effectiveness