Controlling Microbial Growth in the Environment

Controlling Microbial Growthin the Environment How Clean Is Too Clean? ­ ­ ­ ­ overreacts—causing allergies. The hypothesis has also been applied to a wide range of other disorders that may involve the immune system, such as multiple sclerosis, type 1 diabetes, and inflammatory bowel diseases. An idea as sweeping as the hygiene hypothesis is almost impossible to experimentally demonstrate. Decreased rates of infectious diseases and better hygiene are not the only changes that our modern world has undergone. Many of today’s children live in an environment that differs in other ways from the one their ancestors grew up in. For example, their diet tends to include more processed foods, and they may have greater exposure to chemicals through a variety of personal and household products. Lifestyle is also different; today’s children typically spend more time indoors than their great-grandparents did. These and other factors may also play a role. Your group is writing a guide for first-time parents. 1. What will you advise a young couple concerning standards of cleanliness in their home? 2. Should their child sleep with the family pet? The control of microbes in health care facilities, in laboratories, and at home is a significant and practical aspect of microbiology. In this chapter, we study the terminology and principles of microbial control, the factors affecting the efficacy of microbial control, and various chemical and physical means to control microorganisms and viruses. (Chapter 10 considers the use of antimicrobial drugs in patients to assist the body’s defenses against pathogens.) Basic Principles of Microbial Control Scientists, health care professionals, researchers, and government workers should use precise terminology when referring to microbial control in the environment. In the following sections M09_BAUM7206_05_SE_C09.indd 261 Go to the study area to solve the problem. ­ ­ ­ ­ ­ ­ Epidemiologists report that cases of childhood asthma, hay fever, and other kinds of allergies have increased over the last several decades. A number of hypotheses have been proposed to explain these observations. In 1989, British epidemiologist David Strachan suggested that the increase in immune disorders has arisen because children are less frequently exposed to childhood illnesses. This decreased exposure is due to a number of factors. Today children have fewer “opportunities” to be exposed to communicable diseases. Moreover, improvements in sanitation and advances in medicine have significantly reduced the rate of numerous infectious diseases in the developed world. Strachan’s so-called “hygiene hypothesis” has been expanded to consider children’s decreased contact not only with disease-causing microbes but also with everyday microbes in homes and schools. The idea is that fewer childhood illnesses and higher degrees of personal hygiene and cleanliness have so reduced children’s exposure to microbes that when the body does encounter common microbes, the immune system we consider the terminology of microbial control, the concept of microbial death rates, and the action of antimicrobial agents. Terminology of Microbial Control LE A R N I N G O UTC O M E S 9.1 Contrast sterilization, disinfection, and antisepsis, and describe their practical uses. 9.2 Contrast the terms degerming, sanitization, and pasteurization. 9.3 Compare the effects of ­static versus ­cidal control agents on microbial growth. It is important for microbiologists, health care workers, and others to use correct terminology for describing microbial control. 2/28/17 3:26 PM r 9    Controlling Microbial Growth in the Environment Greek a, meaning “not,” and sepsis, meaning “decay.” Latin dis, meaning “reversal,” and inficere, meaning “to corrupt.” 3From Greek anti, meaning “against,” and sepsis, meaning “putrefaction.” 4From Latin sanitas, meaning “healthy.” arena—private versus public—in which the activity takes place. Pasteurization 5 is the use of heat to kill pathogens and reduce the number of spoilage microorganisms in food and beverages. Milk, fruit juices, wine, and beer are commonly pasteurized. So far, we have seen that there are two major types of microbial control—sterilization, which is the elimination of all microbes, and antisepsis or disinfection, which each denote the destruction of vegetative (nonspore) cells and many viruses. Modifications of disinfection include degerming, sanitization, and pasteurization. Some scientists and clinicians apply these terms only to pathogenic microorganisms. Additionally, scientists and health care professionals use the suffixes ­stasis or ­static6 to indicate that a chemical or physical agent inhibits microbial metabolism and growth but does not necessarily kill microbes. Thus, refrigeration is bacteriostatic for most bacterial species; it inhibits their growth, but they can resume metabolism when the optimal temperature is restored. By contrast, words ending in ­cide or ­cidal7 refer to agents that destroy or permanently inactivate a particular type of microbe; virucides inactivate viruses, bactericides kill bacteria, and fungicides kill fungal hyphae, spores, and yeasts. Germicides are chemical agents that destroy pathogenic microorganisms in general. TABLE 9.1 summarizes the terminology used to describe the control of microbial growth. Microbial Death Rates LE A R N I N G O UTC O M E 9.4 Define microbial death rate, and describe its significance in microbial control. Scientists define microbial death as the permanent loss of reproductive ability under ideal environmental conditions. One technique for evaluating the efficacy of an antimicrobial agent is to calculate the microbial death rate, which is usually found to be constant over time for any particular microorganism under a particular set of conditions (FIGURE 9.1). Suppose, for example, that a scientist treats a broth containing 1 billion (109) microbes with an agent that kills 90% of them in 1 minute. The most susceptible cells die first, leaving 100 million (108) hardier cells after the first minute. After another minute of treatment, another 90% die, leaving 10 million (107) cells that have even greater resistance to and require longer exposure to the agent before they die. Notice that in this case, each full minute decreases the number of living cells 10-fold. The broth will be sterile when all the cells are dead. When these results are plotted on a semilogarithmic graph—in which the y-axis is logarithmic and the x-axis is arithmetic—the plot of microbial death rate is a straight line; that is, the microbial death rate is constant. ­ ­ ­ ­ Although many of these terms are familiar to the general public, they are often misused. In its strictest sense, sterilization refers to the removal or destruction of all microbes, including viruses and bacterial endospores, in or on an object. (The term does not apply to infectious proteins called prions, because standard sterilizing techniques do not destroy them.) In practical terms, sterilization indicates only the eradication of harmful microorganisms and viruses; some innocuous microbes may still be present and viable in an environment that is considered sterile. For instance, commercial sterilization of canned food does not kill all hyperthermophilic microbes; because they do not cause disease and cannot grow and spoil food at ambient temperatures, they are of no practical concern. Likewise, some hyperthermophiles may survive sterilization by laboratory methods (discussed shortly), but they are of no practical concern to technicians because they cannot grow or reproduce under normal laboratory conditions. The term aseptic1 (ā-sep’tik) describes an environment or procedure that is free of contamination by pathogens. For example, vegetables and fruit juices are available in aseptic packaging, and surgeons and laboratory technicians use aseptic techniques to avoid contaminating a surgical field or laboratory equipment. Disinfection 2 refers to the use of physical or chemical agents known as disinfectants, including ultraviolet light, heat, alcohol, and bleach, to inhibit or destroy microorganisms, especially pathogens. Unlike sterilization, disinfection does not guarantee that all pathogens are eliminated; indeed, disinfectants alone cannot inhibit endospores or some viruses. Further, the term disinfection is used only in reference to treatment of inanimate objects. When a chemical is used on skin or other tissue, the process is called antisepsis3 (an-tē-sep’sis), and the chemical is called an antiseptic. Antiseptics and disinfectants often have the same components, but disinfectants are more concentrated or can be left on a surface for longer periods of time. Of course, some disinfectants, such as steam or concentrated bleach, are not suitable for use as antiseptics. Degerming is the removal of microbes from a surface by scrubbing, such as when you wash your hands or a nurse prepares an area of skin for an injection. Though chemicals such as soap or alcohol are commonly used during degerming, the action of thoroughly scrubbing the surface may be more important than the chemical in removing microbes. Sanitization4 is the process of disinfecting places and utensils used by the public to reduce the number of pathogenic microbes to meet accepted public health standards. For example, steam, high-pressure hot water, and scrubbing are used to sanitize restaurant utensils and dishes, and chemicals are used to sanitize public toilets. Thus, the difference between disinfect­ ing dishes at home and sanitizing dishes in a restaurant is the ­ te a       Ch p 1From 2From M09_BAUM7206_05_SE_C09.indd 262 5Named for Louis Pasteur, inventor of the process. meaning “to stand”—that is, to remain relatively unchanged. 7 From Latin cidium, meaning “a slaying.” 6Greek, 11/23/16 3:02 AM TABLE 9.1       P Basic rinciples of Microbial Control Terminology of Microbial Control Definition Examples Comments Antisepsis eduction in the number of microorganisms and viruses, particularly potential pathogens, on living tissue Use of iodine or alcohol to prepare skin for an injection Antiseptics are frequently disinfectants whose strength has been reduced to make them safe for living tissues. Aseptic efers to an environment or procedure free of pathogenic contaminants reparation of surgical field; handwashing; flame sterilization of laboratory equipment Scientists, laboratory technicians, and health care workers routinely follow standardized aseptic techniques. -cide Suffixes indicating destruction of a type of microbe Bactericide; fungicide; germicide; virucide Germicides include ethylene oxide, propylene oxide, and aldehydes. Degerming emoval of microbes by mechanical means andwashing; alcohol swabbing at site of injection Chemicals play a secondary role to the mechanical removal of microbes. Disinfection Destruction of most microorganisms and viruses on nonliving tissue Use of phenolics, alcohols, aldehydes, or soaps on equipment or surfaces The term is used primarily in relation to pathogens. Pasteurization Use of heat to destroy pathogens and reduce the number of spoilage microorganisms in foods and beverages Sanitization emoval of pathogens from objects to meet public health standards Washing tableware in scalding water in restaurants Standards of sanitization vary among governmental jurisdictions. -stasis Suffixes indicating inhibition but not complete destruction of a type of microbe Bacteriostatic; fungistatic; virustatic Germistatic agents include some chemicals, refrigeration, and freezing. Destruction of all microorganisms and viruses in or on an object reparation of microbiological culture media and canned food Typically achieved by steam under pressure, by incineration, or by ethylene oxide gas. -static Sterilization 109 Number of living microbes 108 107 106 90% die Constant percentage of the extant population is killed each minute 1 min 105 eat treatment is brief to minimize alteration of taste and nutrients; microbes remain and eventually cause spoilage. H P asteurized milk and fruit juices P R P R -cidal H R R Term those that disrupt the integrity of cells by adversely altering their cell walls or cytoplasmic membranes, and those that interrupt cellular metabolism and reproduction by interfering with the structures of proteins and nucleic acids. Alteration of Cell Walls and Membranes Action of Antimicrobial Agents A cell wall maintains cellular integrity by counteracting the effects of osmosis when the cell is in a hypotonic solution. If the wall is disrupted by physical or chemical agents, it no longer prevents the cell from bursting as water moves into the cell by osmosis (see Chapter 3, Figure 3.21c). Beneath a cell wall, the cytoplasmic membrane essentially acts as a bag that contains the cytoplasm and controls the passage of chemicals into and out of the cell. Extensive damage to a membrane’s proteins or phospholipids by any physical or chemical agent allows the cellular contents to leak out—damage that, if not immediately repaired, causes death. In enveloped viruses, the envelope is a membrane composed of proteins and phospholipids that is responsible for the attachment of the virus to its target cell. Damage to the envelope by physical or chemical agents prevents viral replication. The lack of an envelope in nonenveloped viruses accounts for their greater tolerance of harsh environmental conditions, including antimicrobial agents. LE A R N I N G O U TC O M E Damage to Proteins and Nucleic Acids 104 90% die 103 ­ 1 min 102 101 100 1 2 3 4 5 6 Time (min) 7 8 9 10 R ▲ FIGURE 9.1 A plot of microbial death rate. Microbicidal agents do not simultaneously kill all cells. ather, they kill a constant percentage of cells over time—in this case, 90% per minute. On this semilogarithmic graph, a straight line indicates a constant death rate. How many minutes are required for sterilization in this case? Figure 9.1 For this microbe under these conditions, this microbicidal agent requires 9 minutes to achieve sterilization. 9.5 Describe how antimicrobial agents act against cell walls, cytoplasmic membranes, proteins, and nucleic acids. There are many types of chemical and physical microbial controls, but their modes of action fall into two basic categories: M09_BAUM7206_05_SE_C09.indd 263 Proteins regulate cellular metabolism, function as enzymes in most metabolic reactions, and form structural components in membranes and cytoplasm. A protein’s function depends on its exact three-dimensional shape, which is maintained by hydrogen and disulfide bonds between amino acids. When these 11/23/16 3:02 AM te a       Ch p r 9    Controlling Microbial Growth in the Environment bonds are broken by extreme heat or certain chemicals, the protein’s shape changes (see Figure 5.8). Such denatured proteins cease to function, bringing about cellular death. Chemicals, radiation, and heat can also alter and even destroy nucleic acids. Given that the genes of a cell or virus are composed of nucleic acids, disruption of these molecules can produce fatal mutations. Additionally, that portion of a ribosome that actually catalyzes the synthesis of proteins is a ribozyme—that is, an enzymatic RNA molecule. For this reason, physical or chemical agents that interfere with nucleic acids also stop protein synthesis. Scientists and health care workers have at their disposal many chemical and physical agents to control microbial growth and activity. In the next section, we consider the factors and conditions that should be considered in choosing a particular control method, as well as some ways to evaluate a method’s effectiveness. TELL ME WHY Why does milk eventually go “bad” despite being pasteurized? The Selection of Microbial Control Methods Ideally, agents used for the control of microbes should be inexpensive, fast acting, and stable during storage. Further, a perfect agent would control the growth and reproduction of every type of microbe while being harmless to humans, animals, and objects. Unfortunately, such ideal products and procedures do not exist—every agent has limitations and disadvantages. In the next section, we consider the factors that affect the efficacy of antimicrobial methods. fragile objects, such as artificial heart valves and plastic utensils. Moreover, when performing medical procedures, medical personnel must choose a method and level of microbial control based on the site of the procedure because the site greatly affects the potential for subsequent infection. For example, the use of medical instruments that penetrate the outer defenses of the body, such as needles and scalpels, carries a greater potential for infection, so they must be sterilized; however, disinfection may be adequate for items that contact only the surface of a mucous membrane or the skin. In the latter case, sterilization is usually required only if the patient is immunocompromised. Relative Susceptibility of Microorganisms Though microbial death rate is usually constant for a particular agent acting against a single microbe, death rates do vary— sometimes dramatically—among microorganisms and viruses. Microbes fall along a continuum from most susceptible to most resistant to antimicrobial agents. For example, enveloped viruses, such as HIV, are more susceptible to antimicrobial agents and heat than are nonenveloped viruses, such as poliovirus, because viral envelopes are more easily disrupted than the protein coats of nonenveloped viruses. The relative susceptibility of microbes to antimicrobial agents is illustrated in (FIGURE 9.2). Often, scientists and medical personnel select a method to kill the hardiest microorganisms present, assuming that such a treatment will kill more fragile microbes as well. The most resistant microbes include the following: • Bacterial endospores. The endospores of Bacillus (ba-sil’ŭs) and Clostridium (klos-trid’ē-ŭm) are the most resilient forms of life. They can survive environmental extremes of temperature, acidity, and dryness and can withstand many Most resistant Prions Factors Affecting the Efficacy of Antimicrobial Methods Bacterial endospores Cysts of Cryptosporidium (protozoan) Mycobacteria LE A R N I N G O U TC O M E S Cysts of other protozoa 9.6 List factors to consider in selecting a microbial control Small nonenveloped viruses method. 9.7 Identify the three most resistant groups of microbes, and explain why they are resistant to many antimicrobial agents. 9.8 Discuss environmental conditions that can influence the effectiveness of antimicrobial agents. Active-stage protozoa (trophozoites) Fungal spores Most Gram-negative bacteria Vegetative fungi Large nonenveloped viruses In each situation, microbiologists, laboratory personnel, and medical staff must consider at least three factors: the nature of the sites to be treated, the degree of susceptibility of the microbes involved, and the environmental conditions that pertain. In many cases, the choice of an antimicrobial method depends on the nature of the site to be treated. For example, harsh chemicals and extreme heat cannot be used on humans, animals, and M09_BAUM7206_05_SE_C09.indd 264 Enveloped viruses Most susceptible ▲ FIGURE 9.2 Relative susceptibilities of microbes to antimicrobial agents. Why are nonenveloped viruses more resistant than enveloped viruses? Figure 9.2 A protein coat is less fragile than a phospholipid membrane (envelope). Site to Be Treated Most Gram-positive bacteria 11/23/16 3:02 AM       The Selection of Microbial Control Methods Number of living microbes 105 ­ chemical disinfectants. For example, endospores have survived more than 20 years in 70% alcohol, and scientists have recovered viable endospores that were embalmed with Egyptian mummies thousands of years ago. • Species of mycobacteria. The cell walls of members of the genus, Mycobacterium (mī-kō-bak-tēr’ē-ŭm) such as Mycobacterium tuberculosis (too-ber-kyū-lō’sis), contain a large amount of a waxy lipid. The wax allows these bacteria to survive drying and protects them from most water-based chemicals; therefore, medical personnel must use strong disinfectants or heat to treat whatever comes into contact with tuberculosis patients, including utensils, equipment, and patients’ rooms. • Cysts of protozoa. A protozoan cyst’s wall prevents entry of most disinfectants, protects against drying, and shields against radiation and heat. ­ Prions, which are infectious proteins that cause degenerative diseases of the brain, are more resistant than any cell or virus; it takes heating to 482°C for 4 hours to denature them. The effectiveness of germicides can be classified as high, intermediate, or low, depending on their proficiency in inactivating or destroying microorganisms on medical instruments that cannot be sterilized with heat. High­level germicides kill all pathogens, including bacterial endospores. Health care professionals use them to sterilize invasive instruments such as catheters, implants, and parts of heart-lung machines. Intermediate­level germicides kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria, but not bacterial endospores. They are used to disinfect instruments that come in contact with mucous membranes but are noninvasive, such as respiratory 104 103 205C 455C 102 101 100 2 4 6 Time (min) 8 10 ▲ FIGURE 9.3 Effect of temperature on the efficacy of an antimicrobial chemical. This semilogarithmic graph shows that the microbial death rate is higher at higher temperatures; to kill the same number of microbes, this disinfectant required only 2 minutes at 45°C but 7 minutes at 20°C. equipment and endoscopes. Low­level germicides eliminate vegetative bacteria, fungi, protozoa, and some viruses; they are used to disinfect items that contact only the skin of patients, such as furniture and electrodes. Environmental Conditions Temperature and pH affect microbial death rates and the efficacy of antimicrobial methods. Warm disinfectants, for example, generally work better than cool ones because chemicals react faster at higher temperatures (FIGURE 9.3). Acidic conditions R EME GING DISEASE CASE STUDY Acanthamoeba Keratitis M09_BAUM7206_05_SE_C09.indd 265 This eukaryotic microbe commonly lives in water, including rivers, hot springs, and lakes. When trapped under a contact lens, the amoeba can penetrate the eye to cause keratitis. Very occasionally, Acanthamoeba may also enter the body through the nasal mucous membrane or through a cut in the skin. It has become an emerging menace in our modern society because it can live in hot tubs, pools, showerheads, and sink taps. The physician prescribed a solution of antiseptic agent that Tim had to drop into his eyes every 30 minutes, day and night, for three weeks. The treatment was painful and time consuming, but at least Tim retained his sight without needing a corneal transplant. e also learned to remove his contact lenses at the lake! H ­ Tim liked the lake; in fact, his girlfriend suggested he was more fish than man. They spent all of their free time swimming, waterskiing, diving, and sunbathing, until Tim met a single-celled amoeba, Acanthamoeba. Tim noticed something was wrong when his left eye began to hurt, turned red, and became so sensitive to light that he could not stand to be outside. Two days later, the pain was excruciating, like nothing Tim had ever experienced. It felt as if someone were pounding pieces of broken glass into the front of his eye and at the same time quickly inserting a thousand tiny needles into the back of the eye. With his eye swollen shut and tears flowing down his face, Tim sought medical aid. The doctor diagnosed Acanthamoeba keratitis, which is inflammation of the covering of the eye (the cornea) caused by the amoeba. 1. Why are there many more cases of amebic keratitis than there were 100 years ago? 11/23/16 3:02 AM te a       Ch p r 9    Controlling Microbial Growth in the Environment enhance the antimicrobial effect of heat. Some chemical disinfectants, such as household chlorine bleach, are more effective at low pH. Organic materials, such as fat, feces, vomit, blood, and the intercellular matrix of biofilms, interfere with the penetration of heat, chemicals, and some forms of radiation, and in some cases these materials inactivate chemical disinfectants. For this reason, it is important to clean objects before sterilization or disinfection so that antimicrobial agents can thoroughly contact all the object’s surfaces. Biosafety Levels LE A R N I N G O U TC O M E 9.9 Describe four levels of biosafety, and give examples of microbes handled at each level. The Centers for Disease Control and Prevention (CDC) has established guidelines for four levels of safety in microbiological laboratories dealing with pathogens. Each level raises personnel and environmental safety by specifying increasingly strict laboratory techniques, use of safety equipment, and design of facilities. Biosafety Level 1 (BSL­1) is suitable for handling microbes, such as Escherichia coli (esh-ĕ-rik’ē-ă kō’lī), not known to cause disease in healthy humans. Precautions in BSL-1 are minimal and include handwashing with antibacterial soap and disinfecting surfaces. BSL­2 facilities are similar to those of BSL-1 but are designed for handling moderately hazardous agents, such as hepatitis and influenza viruses and methicillin-resistant Staphylococcus aureus (staf’i-lō-kok’ŭs o’rē-ŭs) (MRSA). Access to BSL-2 labs is limited when work is being conducted, extreme precautions are taken with contaminated sharp objects, and procedures that might produce aerosols are conducted within safety cabinets (see Figure 9.11). BSL­3 is stricter, requiring that all manipulations be done within safety cabinets containing high­efficiency particulate air (HEPA) filters. BSL-3 also specifies special design features for the laboratory. These include entry through double sets of doors and lower air pressure in the laboratory, such that an open door only allows air to move into, not out of, the room. Air leaving the room is HEPA-filtered before being discharged. BSL-3 is designed for experimentation on microbes such as bacteria of tuberculosis and anthrax and viruses of yellow fever and Rocky Mountain spotted fever. The most secure laboratories are BSL­4 facilities, designated for working with dangerous or exotic microbes that cause severe or fatal diseases in humans, such as Ebola, smallpox, and Lassa fever viruses. BSL-4 labs are either separate buildings or completely isolated from all other areas of their buildings. Entry and exit are strictly controlled through electronically sealed airlocks with multiple showers, a vacuum room, an ultraviolet light room, and other safety precautions designed to destroy all traces of the biohazard. All air and water entering and leaving the facility are filtered to prevent M09_BAUM7206_05_SE_C09.indd 266 ▲ FIGURE 9.4 A BSL-4 worker carrying Ebola virus cultures. accidental release. Personnel wear “space suits” supplied with air hoses (FIGURE 9.4). Suits and the laboratory itself are pressurized such that microbes are swept away from workers. TELL ME WHY Why are BSL-4 suits pressurized? Why not just wear tough regular suits? Now that we have studied the terminology and general principles of microbial control and biosafety levels, we turn our attention to the actual physical and chemical agents available to scientists, medical personnel, and the general public to control microbial growth. Physical Methods of Microbial Control LE A R N I N G O UTC O M E 9.10 Describe five types of physical methods of microbial control. Physical methods of microbial control include exposure of the microbes to extremes of heat and cold, desiccation, filtration, osmotic pressure, and radiation. Heat-Related Methods LE A R N I N G O UTC O M E S 9.11 Discuss the advantages and disadvantages of using moist heat in an autoclave and dry heat in an oven for sterilization. 11/23/16 3:02 AM       P hysical Methods of Microbial Control techniques. 9.13 Explain the importance of pasteurization, and describe three different pasteurization methods. ­ Heat is one of the older and more common means of microbial control. High temperatures denature proteins, interfere with the integrity of cytoplasmic membranes and cell walls, and disrupt the function and structure of nucleic acids. Heat can be used for sterilization, in which case all cells and viruses are deactivated, or for commercial preparation of canned goods. In so-called commercial sterilization, hyperthermophilic prokaryotes remain viable but are harmless because they cannot grow at the normal (room) temperatures in which canned foods are stored. Though microorganisms vary in their susceptibility to heat, it can be an important agent of microbial control. As a result, scientists have developed concepts and terminology to convey these differences in susceptibility. Thermal death point is the lowest temperature that kills all cells in a broth in 10 minutes, and thermal death time is the time it takes to completely sterilize a particular volume of liquid at a set temperature. As we have discussed, cell death occurs logarithmically. When measuring the effectiveness of heat sterilization, researchers calculate the decimal reduction time (D), which is the time required to destroy 90% of the microbes in a sample (FIGURE 9.5). This concept is especially useful to food processors because they must heat foods to eliminate the endospores of anaerobic Clostridium botulinum (bo-tū-lī’ num), which could germinate and produce life-threatening botulism toxin inside sealed cans. The standard in food processing is to apply heat such that a population of 1012 C. botulinum endospores is reduced to 100 (i.e., 1) endospore (a 12-fold reduction), which leaves only a very small chance that any particular can of food Number of living microbes 105 104 103 90% 102 D 90% 101 100 2 4 6 8 10 12 14 Time (min) D 16 18 20 22 24 ▲ FIGURE 9.5 Decimal reduction time (D) as a measure of microbial death rate. D is defined as the time it takes to kill 90% of a microbial population. Note that D is a constant that is independent of the initial density of the population. What is the decimal reduction time of this heat treatment against this organism? What is the thermal death time? Figure 9.5 D = 5 minutes; thermal death time = 22.5 minutes. M09_BAUM7206_05_SE_C09.indd 267 contains a single endospore. Researchers have calculated that the D value for C. botulinum endospores at 121°C is 0.204 minute, so it takes 2.5 minutes (0.204 * 12) to reduce 1012 endospores to 1 endospore. ­ 9.12 Explain the use of Bacillus endospores in sterilization Moist Heat Moist heat (which is commonly used to disinfect, sanitize, sterilize, and pasteurize) kills cells by denaturing proteins and destroying cytoplasmic membranes. Moist heat is more effective in microbial control than dry heat because water is a better conductor of heat than air. An example from your kitchen readily demonstrates this: you can safely stick your hand into an oven at 350°F for a few moments, but putting a hand into boiling water at the lower temperature of 212°F would burn you severely. The first method we consider for controlling microbes using moist heat is boiling. Boiling Boiling kills the vegetative cells of bacteria and fungi, the trophozoites of protozoa, and most viruses within 10 minutes at sea level. Contrary to popular belief, water at a rapid boil is no hotter than water at a slow boil; boiling water at normal atmospheric pressure cannot exceed boiling temperature (100°C at sea level), because escaping steam carries excess heat away. It is impossible to boil something more quickly simply by applying more heat; the added heat is carried away by the escaping steam. Boiling time is the critical factor. Further, it is important to realize that at higher elevations, water boils at lower temperatures because atmospheric pressure is lower; thus, a longer boiling time is required in Denver than in Los Angeles to get the same antimicrobial effect. Bacterial endospores, protozoan cysts, and some viruses (such as hepatitis viruses) can survive boiling at sea level for many minutes or even hours. In fact, because bacterial endospores can withstand boiling for more than 20 hours, boiling is not recommended when true sterilization is required. Boiling is effective for sanitizing restaurant tableware or disinfecting baby bottles. Autoclaving Practically speaking, true sterilization using heat requires higher temperatures than that of boiling water. To achieve the required temperature, pressure is applied to boiling water to prevent the escape of heat in steam. The reason that applying pressure succeeds in achieving sterilization is that the temperature at which water boils (and steam is formed) increases as pressure increases (FIGURE 9.6). Scientists and medical personnel routinely use a piece of equipment called an autoclave to sterilize chemicals and objects that can tolerate moist heat. Alternative techniques (discussed shortly) must be used for items that are damaged by heat or water, such as some plastics and vitamins. An autoclave consists of a pressure chamber, pipes to introduce and evacuate steam, valves to remove air and control pressure, and pressure and temperature gauges to monitor the procedure (FIGURE 9.7). As steam enters an autoclave chamber, it forces air out, raises the temperature of the contents, and increases the pressure until a set temperature and pressure are reached. 11/23/16 3:02 AM E       Chapter 9    Controlling Microbial Growth in the nvironment 25 20 15 10 5 100 110 120 Temperature (5C) 130 140 ▲ FIGURE 9.6 The relationship between temperature and (a) pressure. Note that higher temperatures—and consequently, greater antimi- crobial action—are associated with higher pressures. Ultra-high-temperature pasteurization of milk requires a temperature of 134°C; what pressure must be applied to the milk to achieve this temperature? Pressure gauge Manual exhaust to atmosphere Figure 9.6 29 psi. Exhaust valve Steam Air Steam jacket Door Material to be sterilized Trap Steam supply Thermometer (b) A ▲ FIGURE 9.7 An autoclave. (a) autoclave. (b) functions. photo of a laboratory schematic of an autoclave, showing how it Video utor In a variation on this technique, the endospores are on a strip in one compartment of a vial that also includes, in a separate compartment, a growth medium containing a pH color indicator. After autoclaving, a barrier between the two compartments is broken, putting the endospores into contact with the medium (FIGURE 9.8). In this case, the absence of a color change after incubation indicates sterility. ­ Pasteurization Louis Pasteur developed a method of heating beer and wine just enough to destroy the microorganisms that cause spoilage without ruining the taste. Today, pasteurization is also used to kill pathogens in milk, ice cream, yogurt, and fruit juices. Brucella melitensis (broo-sel’lă me-li-ten’sis), Mycobacterium bovis (bō’vis), and Escherichia coli, the causative agents of undulant fever, bovine tuberculosis, and one kind of diarrhea, respectively, are controlled in this manner. Pasteurization is not sterilization. Thermoduric and thermophilic—heat-tolerant and heat-loving—prokaryotes survive pasteurization, but they do not cause spoilage over the relatively short times during which properly refrigerated and ­ Scientists have determined that a temperature of 121°C, which requires the addition of 15 pounds per square inch (psi)8 of pressure above that of normal air pressure (see Figure 9.6), destroys all microbes generally encountered in a laboratory or medical setting in a small volume in about 15 minutes. Sterilizing large volumes of liquids or solids slows the process because they require more time for heat to penetrate. Thus, it requires more time to sterilize 1 liter of fluid in a flask than the same volume of fluid distributed into smaller tubes. Autoclaving solid substances, such as meat, also requires extra time because it takes longer for heat to penetrate to their centers. Sterilization in an autoclave requires that steam be able to contact all liquids and surfaces that might be contaminated with microbes; therefore, solid objects must be wrapped in porous cloth or paper, not sealed in plastic or aluminum foil, both of which are impermeable to steam. Containers of liquids must be sealed loosely enough to allow steam to circulate freely, and all air must be forced out by steam. Since steam is lighter than air, it cannot force air from the bottom of an empty vessel; therefore, empty containers must be tipped so that air can flow out of them. Scientists use several means to ensure that an autoclave has sterilized its contents. A common one is a chemical that changes color when the proper combination of temperature and time has been reached. Often such a color indicator is impressed in a pattern on tape or paper so that the word sterile or a pattern or design appears. Another technique uses plastic beads that melt when proper conditions are met. A biological indicator of sterility uses endospores of bacteria such as Bacillus impregnated into tape. After autoclaving, the tape is aseptically inoculated into sterile broth. If no bacterial growth appears, the original material is considered sterile. Valve for steam to chamber Safety valve T 0 A Pressure above normal (psi) 30 8The Standard International (SI) equivalent of 1 psi is 6.9 * 103 pascals. M09_BAUM7206_05_SE_C09.indd 268 06/03/2018 07:03       P hysical Methods of Microbial Control ◀ FIGURE 9.8 Sterility indicators. A commercial Cap that allows steam to penetrate Flexible plastic vial Crushable glass ampule Red medium means spores were killed; autoclaved objects are sterile. ­ Ultra-High-Temperature Sterilization The dairy industry and other food processors can also use ultra­high­temperature sterilization, which involves flash heating milk or other liquids to rid them of all living microbes. The process involves passing the liquid through superheated steam at about 140°C for 1 to 3 seconds and then cooling it rapidly. Treated liquids can be stored indefinitely at room temperature without microbial spoilage, though chemical degradation after months of storage results in flavor changes. Small packages of dairy creamer served in restaurants are often sterilized by the ultra-high-temperature method. TABLE 9.2 summarizes the dairy industry’s use of moist heat for controlling microbes in milk. TABLE 9.2 Moist Heat Treatments of Milk H Process istorical (batch) pasteurization Flash pasteurization Treatment 63°C for 30 minutes 72°C for 15 seconds Ultra-high-temperature pasteurization 135°C for 1 second Ultra-high-temperature sterilization 140°C for 1–3 seconds Dry Heat Some substances, such as powders and oils, cannot be sterilized by boiling or with steam. Others, such as some metal objects, can be damaged by repeated exposure to steam. For such materials, sterilization can be achieved by the use of dry heat, as occurs in an oven. Hot air is an effective sterilizing agent because it denatures proteins and fosters the oxidation of metabolic and structural chemicals; however, in order to sterilize, dry heat requires higher temperatures for longer times than moist heat because dry heat penetrates more slowly. For instance, whereas an autoclave needs about 15 minutes to sterilize an object at 121°C, an oven at the same temperature requires at least 16 hours to achieve sterility. Scientists typically use higher temperatures—171°C for 1 hour or 160°C for 2 hours—to sterilize objects in an oven, but objects made of rubber, paper, and many types of plastic oxidize rapidly (combust) under these conditions. Complete incineration is the ultimate means of sterilization. As part of standard aseptic technique in microbiological laboratories, inoculating loops are sterilized by heating them in the flame of a Bunsen burner or with an electric heating coil until they glow red (about 1500°C). Health care workers incinerate contaminated dressings, bags, and paper cups, and field epidemiologists incinerate the carcasses of animals that have diseases such as anthrax or bovine spongiform encephalopathy (mad cow disease). ­ After autoclaving, flexible vial is squeezed to break ampule and release medium onto spore strip. pasteurized foods are stored before consumption. In addition, such prokaryotes are generally not pathogenic. The combination of time and temperature required for effective pasteurization varies with the product. Because milk is the most familiar pasteurized product, we consider the pasteurization of milk in some detail. Historically, milk was pasteurized by the batch method for 30 minutes at 63°C, but most milk processors today use a high-temperature, shorttime method known as flash pasteurization, in which milk flows through heated tubes that raise its temperature to 72°C for only 15 seconds. This treatment effectively destroys all pathogens. Ultra­high­temperature pasteurization heats the milk to at least 135°C for only 1 second, but some consumers claim that it adversely affects the taste. M09_BAUM7206_05_SE_C09.indd 269 H Incubation Nutrient medium containing pH color indicator Endospore strip H Yellow medium means spores are viable; autoclaved objects are not sterile. endospore-test ampule, which is included among objects to be sterilized. After autoclaving is complete, the ampule is broken, and the medium, which contains a p color indicator, is released onto the endospore strip. If the endospores are still alive, their metabolic wastes lower the p , changing the color of the medium. Refrigeration and Freezing LE A R N I N G O UTC O M E 9.14 Describe the use and importance of refrigeration and freezing in limiting microbial growth. In many situations, particularly in food preparation and storage, the most convenient method of microbial control is either refrigeration (temperatures between 0°C and 7°C) or freezing 11/23/16 3:02 AM te a       Ch p r 9    Controlling Microbial Growth in the Environment metabolism requires liquid water. Drying inhibits the spread of most pathogens, including the bacteria that cause syphilis, gonorrhea, and the more common forms of bacterial pneumonia and diarrhea. However, many molds can grow on dried raisins and apricots, which have as little as 16% water content. Scientists use lyophilization (lī-of′-i-li-zā′shŭn), a technique that combines freezing and drying, to preserve microbes and other cells for many years. In this process, scientists instantly freeze a culture in liquid nitrogen or frozen carbon dioxide (dry ice); then they subject it to a vacuum that removes frozen water through a process called sublimation, in which the water is transformed directly from a solid to a gas. Lyophilization prevents the formation of large, damaging ice crystals. Although not all cells survive, enough are viable to enable the culture to be reconstituted many years later. Filtration LE A R N I N G O UTC O M E ­ ­ (temperatures below 0°C). These processes decrease microbial metabolism, growth, and reproduction because chemical reactions occur more slowly at low temperatures and because liquid water is not available at subzero temperatures. Note, however, that psychrophilic (cold-loving) microbes can still multiply in refrigerated food and spoil its taste and suitability for consumption. Refrigeration halts the growth of most pathogens, which are predominantly mesophiles. Notable exceptions are the bacteria Listeria (lis-tēr’ē-ă), which can reproduce to dangerous levels in refrigerated food, and Yersinia (yer-sin’ē-ă), which can multiply in refrigerated blood products and be passed on to blood recipients. (Chapters 19 and 20 discuss these pathogens in more detail.) Slow freezing, during which ice crystals have time to form and puncture cell membranes, is more effective than quick freezing in inhibiting microbial metabolism, though microorganisms also vary in their susceptibility to freezing. Whereas the cysts of tapeworms perish after several days in frozen meat, many vegetative bacterial cells, bacterial endospores, and viruses can survive subfreezing temperatures for years. In fact, scientists store many bacteria and viruses in low-temperature freezers at -30°C to -80°C and are able to reconstitute the microbes into viable populations by warming them in media containing proper nutrients. Therefore, we must take care in thawing and cooking frozen food because it can still contain pathogenic microbes. Desiccation and Lyophilization LE A R N I N G O U TC O M E 9.15 Compare and contrast desiccation and lyophilization. sterilization. Filtration is the passage of a fluid (either a liquid or a gas) through a sieve designed to trap particles—in this case, cells or viruses—and separate them from the fluid. Researchers often use a vacuum to assist the movement of fluid through the filter (FIGURE 9.10a). Filtration traps microbes larger than the pore size, allowing smaller microbes to pass through. In the late 1800s, filters were able to trap cells, but their pores were too large to trap the pathogens of such diseases as rabies and measles. These pathogens were thus named filterable viruses, which today has been shortened to viruses.9 Now, filters with pores small enough to trap even viruses are available, so filtration can be used to sterilize such heat-sensitive materials as ophthalmic solutions, antibiotics, vaccines, liquid vitamins, enzymes, and culture media. Over the years, filters have been constructed from porcelain, glass, cotton, asbestos, and diatomaceous earth, a substance composed of the innumerable glasslike cell walls of single-celled algae called diatoms. Scientists today typically use thin (only 0.1 mm thick), circular membrane filters manufactured of nitrocellulose or plastic and containing specific pore sizes ranging from 25 mm to less than 0.01 mm in diameter (FIGURE 9.10b). The pores of the latter filters are small enough to trap small viruses and even some large protein molecules. Microbiologists also use filtration to estimate the number of microbes in a fluid by counting the number deposited on the filter after passing a given volume through the filter (see Figure 6.24). TABLE 9.3 lists some pore sizes of membrane filters and the microbes they do not allow through. Health care and laboratory workers routinely use filtration to prevent airborne contamination by microbes. Medical personnel wear surgical masks to prevent exhaled microbes from ­ ­ ­ Desiccation, or drying, has been used for thousands of years to preserve such foods as fruits, peas, beans, grain, nuts, and yeast (FIGURE 9.9). Desiccation inhibits microbial growth because 9.16 Describe the use of filters for disinfection and ▲ FIGURE 9.9 The use of desiccation as a means of preserving apricots in Pakistan. In this time-honored practice, drying inhibits microbial growth by removing the water that microbes need for metabolism. M09_BAUM7206_05_SE_C09.indd 270 9Latin, meaning “poisons.” 11/23/16 3:02 AM       P hysical Methods of Microbial Control ◀ FIGURE 9.10 Filtration equipment used for microbial control. (a) Assembly for sterilization by vacuum filtration. (b) Membrane filters composed of various substances and with pores of various sizes can be used to trap diverse microbes. ere, a bacterium known as vancomycin-resistant Enterococcus (V E) is trapped. H Nonsterile medium R Membrane filter To vacuum pump Sterile medium (a) (b) Outside Membrane Filters Pore Size (Mm) Smallest Microbes That Are Trapped 5 Multicellular algae, animals, and fungi 3 Yeasts and larger unicellular algae P 1.2 1 µm rotozoa and small unicellular algae Blower 0.45 Largest bacteria 0.22 Largest viruses and most bacteria Supply HEPA filter 0.025 Larger viruses and pliable bacteria (mycoplasmas, rickettsias, chlamydias, and some spirochetes) Light 0.01 Safety glass viewscreen Exhaust HEPA filter Smallest viruses contaminating the environment, and cotton plugs are placed in culture vessels to prevent contamination by airborne microbes. Additionally, high-efficiency particulate air (HEPA) filters are crucial parts of biological safety cabinets (FIGURE 9.11). HEPA filters are mounted in the air ducts of some operating rooms, rooms occupied by patients with airborne diseases such as tuberculosis, and rooms of immunocompromised patients, such as burn victims and AIDS patients. ▲ FIGURE 9.11 The roles of high-efficiency particulate air (HEPA) filters in biological safety cabinets. E A filters protect workers from exposure to microbes (by maintaining a barrier of filtered air across the opening of the cabinet). ospitals also use E A filters in air ducts of operating rooms and of the rooms of highly contagious or immunocompromised patients. 9.17 Discuss the use of hypertonic solutions in microbial control. Another ancient method of microbial control is the use of high concentrations of salt or sugar in foods to inhibit microbial M09_BAUM7206_05_SE_C09.indd 271 P H LE A R N I N G O U TC O M E H H Osmotic Pressure High-velocity air barrier P TABLE 9.3 SEM growth by osmotic pressure. Osmosis is the net movement of water across a semipermeable membrane (such as a cytoplasmic membrane) from an area of higher water concentration 11/23/16 3:02 AM te a       Ch p r 9    Controlling Microbial Growth in the Environment ­ ­ to an area of lower water concentration. Cells in a hypertonic solution of salt or sugar lose water, and the cell shrinks (see Figure 3.21b). The removal of water inhibits cellular metabolism because enzymes are fully functional only in aqueous environments. Thus, osmosis preserves honey, jerky, jams, jellies, salted fish, and some types of pickles from most microbial attacks. Fungi have a greater ability than bacteria to tolerate hypertonic environments with little moisture, which explains why jelly in your refrigerator may grow a colony of Penicillium (pen-i-sil’ē-ŭm) mold but is not likely to grow the bacterium Salmonella (sal’mŏ-nel’ă). Radiation LE A R N I N G O U TC O M E 9.18 Differentiate ionizing radiation from nonionizing radia- tion as they relate to microbial control. ­ ­ Another physical method of microbial control is the use of radiation. There are two types of radiation: particulate radiation and electromagnetic radiation. Particulate radiation consists of high-speed subatomic particles, such as protons, that have been freed from their atoms. Electromagnetic radiation can be defined as energy without mass traveling in waves at the speed of light (3 * 105 km/sec). Electromagnetic energy is released from atoms that have undergone internal changes. The wavelength of electromagnetic radiation, defined as the distance between two crests of a wave, ranges from very short gamma rays; to X-rays, ultraviolet light, and visible light; to long infrared rays; and, finally, to very long radio waves (see Figure 4.1). Though they are particles, electrons also have a wave nature, with wavelengths that are even shorter than gamma rays. The shorter the wavelength of an electromagnetic wave, the more energy it carries; therefore, shorter-wavelength radiation is more suitable for microbial control than longer-wavelength radiation, which carries less energy and is less penetrating. Scientists describe all types of radiation as either ionizing or nonionizing according to its effects on the chemicals within cells. Gamma rays, which are emitted by some radioactive elements, such as radioactive cobalt, penetrate much farther than electron beams but require hours to kill microbes. The U.S. Food and Drug Administration (FDA) has approved the use of gamma irradiation for microbial control in meats, spices, and fresh fruits and vegetables (FIGURE 9.12). Irradiation with gamma rays kills not only microbes but also the larvae and eggs of insects; it also kills the cells of fruits and vegetables, preventing both microbial spoilage and overripening. Consumers have been reluctant to accept irradiated food. A number of reasons have been cited, including fear that radiation makes food radioactive and claims that it changes the taste and nutritive value of foods or produces potentially carcinogenic (cancer-causing) chemicals. Supporters of irradiation reply that gamma radiation passes through food and cannot make it radioactive any more than a dental X-ray produces radioactive teeth, and they cite numerous studies that conclude that irradiated foods are tasty, nutritious, and safe. X-rays travel the farthest through matter, but they have less energy than gamma rays and require a prohibitive amount of time to make them practical for microbial control. Nonionizing Radiation Electromagnetic radiation with a wavelength greater than 1 nm does not have enough energy to force electrons out of orbit, so it is nonionizing radiation. However, such radiation does contain enough energy to excite electrons and cause them to make Ionizing Radiation Electron beams, gamma rays, and some X-rays, all of which have wavelengths shorter than 1 nm, are ionizing radiation because when they strike molecules, they have sufficient energy to eject electrons from atoms, creating ions. Such ions disrupt hydrogen bonding, oxidize double covalent bonds, and create highly reactive hydroxyl radicals (see Chapter 6). These ions in turn denature other molecules, particularly DNA, causing fatal mutations and cell death. Electron beams are produced by cathode ray machines. Electron beams are highly energetic and therefore very effective in killing microbes in just a few seconds, but they cannot sterilize thick objects or objects coated with large amounts of organic matter. They are used to sterilize spices, meats, microbiological plastic ware, and dental and medical supplies, such as gloves, syringes, and suturing material. M09_BAUM7206_05_SE_C09.indd 272 Non-irradiated Irradiated ▲ FIGURE 9.12 A demonstration of the increased shelf life of food achieved by ionizing radiation. Both sets of strawberries are the same age and type. The circular radura symbol is used internationally to label irradiated foods. 11/23/16 3:02 AM       P hysical Methods of Microbial Control transcribe or replicate its genetic material. If dimers remain uncorrected, an affected cell may die. The effectiveness of UV irradiation is tempered by the fact that UV light does not penetrate well. UV light is therefore suitable primarily for disinfecting air, transparent fluids, and the surfaces of objects, such as barber’s shears and operating tables. Some cities use UV irradiation in sewage treatment. By passing wastewater past banks of UV lights, they reduce the number of bacteria without using chlorine, which might damage the environment. TABLE 9.4 summarizes the physical methods of microbial control discussed in the previous pages. ­ ­ new covalent bonds, which can affect the three-dimensional structure of proteins and nucleic acids. Ultraviolet (UV) light, visible light, infrared radiation, and radio waves are nonionizing radiation. Of these, only UV light has sufficient energy to be a practical antimicrobial agent. Visible light and microwaves (radio waves of extremely short wavelength) have little value in microbial control, though microwaves heat food, inhibiting microbial growth and reproduction if the food gets hot enough. The more energetic microwaves produced by some commercial microwave ovens can kill fungal spores, preventing treated food from molding. UV light with a wavelength of 260 nm is specifically absorbed by adjacent pyrimidine nucleotide bases in DNA, causing them to form covalent bonds with each other rather than forming hydrogen bonds with bases in the complementary DNA strand (see Figure 7.25). Such pyrimidine dimers distort the shape of DNA, making it impossible for the cell to accurately TABLE 9.4 TELL ME WHY Why are Bacillus endospores used as sterility indicators? (See Figure 9.8.) Physical Methods of Microbial Control Method Conditions Action Representative Use(s) Boiling 10 min at 100°C (at sea level) Denatures proteins and destroys membranes Disinfection of baby bottles and sanitization of restaurant cookware and tableware Autoclaving (pressure cooking) 15 min at 121°C Denatures proteins and destroys membranes Autoclave: sterilization of medical and laboratory supplies that can tolerate heat and moisture; pressure cooker: sterilization of canned food 30 minutes at 63°C; 15 sec at 72°C Denatures proteins and destroys membranes Destruction of all pathogens and most spoilage microbes in dairy products, fruit juices, beer, and wine 1–3 sec at 140°C Denatures proteins and destroys membranes Sterilization of dairy products 2 h at 160°C or 1 h at 171°C Denatures proteins, destroys membranes, oxidizes metabolic compounds Sterilization of water-sensitive materials, such as powders, oils, and metals Incineration 1 sec at approximately 1500°C Oxidizes everything completely Sterilization of inoculating loops, flammable contaminated medical waste, and diseased carcasses Refrigeration 0–7°C Inhibits metabolism Freezing Below 0°C Inhibits metabolism Desiccation (drying) Varies with amount of water to be removed Inhibits metabolism Lyophilization (freeze-drying) - 196°C for a few minutes while drying Inhibits metabolism Long-term storage of bacterial cultures Filtration Filter retains microbes hysically separates microbes from air and liquids Sterilization of air and of heat-sensitive ophthalmic and enzymatic solutions, vaccines, and antibiotics Osmotic Pressure Exposure to hypertonic solutions Inhibits metabolism Ionizing Radiation (electron beams, gamma rays, X-rays) Seconds to hours of exposure (depending on wavelength of radiation) Destroys DNA Sterilization of medical and laboratory equipment and preservation of food Nonionizing Radiation (ultraviolet light) Irradiation with 260-nm-wavelength radiation Formation of thymine dimers inhibits DNA transcription and replication Disinfection and sterilization of surfaces and of transparent fluids and gases P Moist Heat asteurization Ultra-high-temperature sterilization M09_BAUM7206_05_SE_C09.indd 273 P reservation of food P Long-term preservation of foods, drugs, and cultures P ot air P H Dry Heat reservation of food reservation of food 11/23/16 3:02 AM te a       Ch p r 9    Controlling Microbial Growth in the Environment Chemical Methods of Microbial Control OH OH OH Cl CH3 Cl O Cl LE A R N I N G O U TC O M E 9.19 Compare and contrast nine major types of antimicrobial ­ (b) A phenolic (c) A bisphenolic ▲ FIGURE 9.13 Phenol and phenolics. (a) henol, a naturally occurring molecule that is also called carbolic acid. (b) henolics, which are compounds synthesized from phenol, have greater antimicrobial efficacy with fewer side effects. (c) Bisphenols are paired, covalently linked phenolics. are commonly used in health care settings, laboratories, and households. Negative aspects of phenolics include their disagreeable odor and possible side effects; for example, phenolics irritate the skin of some individuals. Alcohols LE A R N I N G O UTC O M E 9.21 Discuss the action of alcohols as antimicrobial agents, and explain why solutions of 70% to 90% alcohol are more effective than pure alcohols. Alcohols are bactericidal, fungicidal, and virucidal against enveloped viruses; however, they are not effective against fungal spores or bacterial endospores. Alcohols are considered intermediate-level disinfectants. Commonly used alcohols include rubbing alcohol (isopropanol) and drinking alcohol (ethanol): ­ Phenol and Phenolics (a) Phenol Triclosan P Although physical agents are sometimes used for disinfection, antisepsis, and preservation, more often chemical agents are used for these purposes. As we have seen, chemical agents act to adversely affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA. As with physical agents, the effect of a chemical agent varies with temperature, length of exposure, and the amount of contaminating organic matter in the environment. The effect also varies with pH, concentration, and freshness of the chemical. Chemical agents tend to destroy or inhibit the growth of enveloped viruses and the vegetative cells of bacteria, fungi, and protozoa more than fungal spores, protozoan cysts, or bacterial endospores. The latter are particularly resistant to chemical agents. In the following sections, we discuss nine major categories of antimicrobial chemicals used as antiseptics and disinfectants: phenols, alcohols, halogens, oxidizing agents, surfac­ tants, heavy metals, aldehydes, gaseous agents, and enzymes. Some chemical agents combine one or more of these. Additionally, researchers and food processors sometimes use antimicrobial drugs—substances normally used to treat diseases—as disinfectants. (Chapter 10 discusses antimicrobial drugs in more detail.) Orthocresol P chemicals, and discuss the positive and negative aspects of each. LE A R N I N G O U TC O M E 9.20 Distinguish between phenol and the types of phenolics, OH and discuss their action as antimicrobial agents. ­ M09_BAUM7206_05_SE_C09.indd 274 CH CH3 Isopropanol CH3 CH2OH Ethanol Isopropanol is slightly superior to ethanol as a disinfectant and antiseptic. Tinctures (tingk’chūrs) of antimicrobial chemicals, which are solutions of the chemicals in alcohol, are often more effective than the same chemicals dissolved in water. Alcohols denature proteins and disrupt cytoplasmic membranes. Surprisingly, pure alcohol is not an effective antimicrobial agent because the denaturation of proteins requires water; therefore, solutions of 70% to 90% alcohol are typically used to control microbes. Alcohols evaporate rapidly, which is advantageous in that they leave no residue but disadvantageous in that they may not contact microbes long enough to be effective. Alcohol-based antiseptics are more effective than soap in removing bacteria from hands but not effective against some viruses, such as diarrhea-causing noroviruses. Swabbing the skin with alcohol prior to an injection removes more microbes by physical action (degerming) than by chemical action. ­ ­ In 1867, Dr. Joseph Lister (1827–1912) began using phenol (also known as carbolic acid) (FIGURE 9.13a) to reduce infection during surgery. Phenolics are compounds derived from phenol molecules that have been chemically modified by the addition of organic functional groups or reactive atoms (FIGURE 9.13b). For instance, chlorinated phenolics contain one or more atoms of chlorine and have enhanced antimicrobial action and a less annoying odor than phenol. Natural oils, such as pine and clove oils, are also phenolics and can be used as antiseptics. Bisphenolics are composed of two covalently linked phenolics. An example is triclosan, which is incorporated into numerous consumer products, including garbage bags, diapers, and cutting boards (FIGURE 9.13c). Phenol and phenolics denature proteins and disrupt cell membranes in a wide variety of pathogens. They are effective even in the presence of contaminating organic material, such as vomit, pus, saliva, and feces, and they remain active on surfaces for a prolonged time. For these reasons, phenolics CH3 11/23/16 3:02 AM       Chemical Methods of Microbial Control Halogens LE A R N I N G O U TC O M E 9.22 Discuss the types and uses of halogen-containing anti- microbial agents. ▲ FIGURE 9.14 Degerming in preparation for surgery on a hand. Betadine, an iodophor, is the antiseptic used here. bleach, and calcium hypochlorite (Ca(OCl)2). The dairy industry and restaurants use these compounds to disinfect utensils, and the medical field uses them to disinfect hemodialysis systems. Household bleach diluted by adding two drops to a liter of water can be used in an emergency to make water safer to drink, but it does not kill all protozoan cysts, bacterial endospores, or viruses. Chlorine dioxide (ClO2) is a gas that can be used to disinfect large spaces; for example, officials used it to sterilize federal buildings contaminated with anthrax spores following the 2001 bioterrorism attack. Chloramines—chemical combinations of chlorine and ammonia—are used in wound dressings, as skin antiseptics, and in some municipal water supplies. ­ Halogens are the four very reactive, nonmetallic chemical elements: iodine, chlorine, bromine, and fluorine. Halogens are intermediate-level antimicrobial chemicals that are effective against vegetative bacterial and fungal cells, fungal spores, some bacterial endospores and protozoan cysts, and many viruses. Halogens are used both alone and combined with other elements in organic and inorganic compounds. Halogens exert their antimicrobial effect by unfolding and thereby denaturing essential proteins, including enzymes. Iodine is a well-known antiseptic. In the past, backpackers and campers disinfected water with iodine tablets, but experience has shown that some protozoan cysts can survive iodine treatment unless the iodine concentration is so great that the water is undrinkable. Medically, iodine is used either as a tincture or as an iodo­ phor, which is an iodine-containing organic compound that slowly releases iodine. Iodophors have the advantage of being long lasting and nonirritating to the skin. Betadine is an example of an iodophor used in medical institutions to prepare skin for surgery (FIGURE 9.14) and injections and to treat burns. Municipalities commonly use chlorine in its elemental form (Cl2) to disinfect drinking water, swimming pools, and wastewater from sewage treatment plants. Compounds containing chlorine are also effective disinfectants. Examples include sodium hypochlorite (NaOCl), which is household chlorine R BENEFICIAL MIC OBES Hard to Swallow? R M09_BAUM7206_05_SE_C09.indd 275 P In 2006, the U.S. Food and Drug Administration (FDA) approved the nonmedical use of a phage that specifically kills Listeria monocytogenes, which is frequently a bacterial contaminant of cheese and lunch meat. Listeria kills about 20% of people who get sick from ingesting it. The approved anti-Listeria phage is available in a solution that food processors, delicatessen owners, and consumers can spray on food to reduce the number of Listeria cells. Some people find the idea hard to swallow—deliberately contaminating food and equipment with viruses sounds like poor hygiene. roponents of using phages point out that an individual consumes millions of phages daily in water and food without ill effect. Indeed, phages in restaurants are more common than mustard and mayonnaise. Medical professionals in the former Soviet Union, particularly the country of Georgia, have used phages to successfully treat disease for over six decades without deleterious side effects. ­ ­ P 90 nm ­ Controlling bacteria in the environment is becoming more difficult because they are Bacteriophages. developing resistance to common disinfectants and antiseptics. So, scientists are turning to natural parasites of bacteria—bacteriophages, also simply called phages—to control bacterial contamination. Bacteriophages, which literally means “bacteria eaters,” is the term for viruses that specifically attack particular strains of bacteria. hages are like “smart bombs”; unlike disinfectants and most antimicrobial drugs, phages attack specific bacterial strains and leave neighboring bacteria unharmed. A phage injects its genetic material into a bacterial cell, causing the bacterial cell to produce hundreds of new phages that burst out of the bacterium and kill it. esearchers are developing phage solutions to control bacteria in medical settings, in food, and in patients. SEM 11/23/16 3:02 AM te a       Ch p r 9    Controlling Microbial Growth in the Environment Chloramines are less effective antimicrobial agents than other forms of chlorine, but they release chlorine slowly and are thus longer lasting. Bromine is an effective disinfectant in hot tubs because it evaporates more slowly than chlorine at high temperatures. Bromine is also used as an alternative to chlorine in the disinfection of swimming pools, cooling towers, and other water containers. Fluorine in the form of fluoride is antibacterial in drinking water and toothpastes and can help reduce the incidence of dental caries (cavities). Fluorine works in part by disrupting metabolism in the biofilm of dental plaque. gives air its “fresh smell” after a thunderstorm. Some Canadian and European municipalities treat their drinking water with ozone rather than chlorine. Ozone is a more effective antimicrobial agent than chlorine, but it is more expensive, and it is more difficult to maintain an effective concentration of ozone in water. Peracetic acid is an extremely effective sporicide that can be used to sterilize surfaces. Food processors and medical personnel use peracetic acid to sterilize equipment because it is not adversely affected by organic contaminants, and it leaves no toxic residue. Oxidizing Agents LE A R N I N G O UTC O M E Surfactants 9.24 Define surfactants, and describe their antimicrobial action. LE A R N I N G O U TC O M E Surfactants are “surface active” chemicals. One of the ways surfactants act is to reduce the surface tension of solvents such as water by decreasing the attraction among molecules. One result of this reduction in surface tension is that the solvent becomes more effective at dissolving solute molecules. Two common surfactants involved in microbial control are soaps and detergents. One end of a soap molecule is hydrophobic because it is composed of fatty acids, and the other end is hydrophilic and negatively charged. When soap is used to wash skin, for instance, the hydrophobic ends of soap molecules are effective at breaking oily deposits into tiny droplets, and the hydrophilic ends attract water molecules; the result is that the tiny droplets of oily material—and any bacteria they harbor— are more easily dissolved in and washed away by water. Thus, soaps by themselves are good degerming agents though poor antimicrobial agents; when household soaps are antiseptic, it is largely because they contain antimicrobial chemicals. Synthetic detergents are positively charged organic surfactants that are more soluble in water than soaps. The most popular detergents for microbial control are quaternary ammonium compounds (quats), which are composed of an ammonium cation (NH4+ ) in which the hydrogen atoms are replaced by other functional groups or hydrocarbon chains (FIGURE 9.15). Quats are not only antimicrobial but also 9.23 Describe the use and action of oxidizing agents in ­ microbial control. ­ ­ ­ ­ Peroxides, ozone, and peracetic acid kill microbes by oxidizing their enzymes, thereby preventing metabolism. Oxidizing agents are high-level disinfectants and antiseptics that work by releasing oxygen radicals, which are particularly effective against anaerobic microorganisms. Health care workers use oxidizing agents to kill anaerobes in deep puncture wounds. Hydrogen peroxide (H2O2) is a common household chemical that can disinfect and even sterilize the surfaces of inanimate objects such as contact lenses, but it is often mistakenly used to treat open wounds. Hydrogen peroxide does not make a good antiseptic for open wounds because catalase—an enzyme released from damaged human cells—quickly neutralizes hydrogen peroxide by breaking it down into water and oxygen gas, which can be seen as escaping bubbles. Though aerobes and facultative anaerobes on inanimate surfaces also contain catalase, the volume of peroxide used as a disinfectant overwhelms the enzyme, making hydrogen peroxide a useful disinfectant. Food processors use hot hydrogen peroxide to sterilize packages such as juice boxes. Ozone (O3) is a reactive form of oxygen that is generated when molecular oxygen (O2) is subjected to electrical discharge. Ozone H H N+ ◀ FIGURE 9.15 Quaternary ammonium compounds (quats). Quats are surfactants in which the hydrogen atoms of an ammonium ion (a) are replaced by other functional groups (b). H H (a) Ammonium ion CH3 CH2 N+ CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 Hydrophobic tail Benzalkonium CH2 CH2 CH2 CH2 CH3 CH2 CH2 N+ CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH2 Cetylpyridinium (b) Quaternary ammonium ions (quats) M09_BAUM7206_05_SE_C09.indd 276 11/23/16 3:02 AM       Chemical Methods of Microbial Control At one time, many states required that the eyes of newborns be treated with 1% silver nitrate (AgNO3) to prevent blindness caused by Neisseria gonorrhoeae (nī-se’rē-ă go-nor-rē’ī), which can enter babies’ eyes while they pass through an infected birth canal. Today, hospitals have replaced silver nitrate with less irritating antimicrobial ointments that are also effective against other pathogens. Silver still plays an antimicrobial role in some surgical dressings, burn creams, and catheters. For over 70 years, drug companies used thimerosal, a mercury-containing compound, to preserve vaccines. In 1999, the U.S. Public Health Service recommended that alternatives be used because mercury is a metabolic poison, though the very small amount of mercury in vaccines is considered safe. Today, the recommended children’s vaccines are thimerosal free, except for influenza (flu) vaccines distributed in multidose vials. A few adult vaccines also contain thimerosal, including the whole-cell pertussis vaccine and some vaccines against tetanus, flu, and meningococcal meningitis. Copper, which interferes with chlorophyll, is used to control algal growth in reservoirs, fish tanks, swimming pools, and water storage tanks. In the absence of organic contaminants, copper is an effective algicide in concentrations as low as 1 ppm (part per million). Copper, zinc, or mercury is used to control mildew in some paint. ­ ­ ­ ­ colorless, tasteless, and harmless to humans (except at high concentrations), making them ideal for many industrial and medical applications. If your mouthwash foams, it probably contains a quaternary ammonium compound. Examples of quats are benzalkonium chloride (Zephiran) and cetylpyridinium (used in Cepacol mouthwash). Quats function by disrupting cellular membranes so that affected cells lose essential internal ions, such as potassium ions (K+ ). Quats are bactericidal (particularly against Gram-positive bacteria), fungicidal, and virucidal against enveloped viruses, but they are not effective against nonenveloped viruses, mycobacteria, or endospores. The action of quaternary ammonium compounds is retarded by organic contaminants, and they are deactivated by soaps. Some pathogens, such as Pseudomonas aeruginosa (soo-dō-mō’nas ā-roo-ji-nō’să), actually thrive in quats; therefore, quats are classified as lowlevel disinfectants. Heavy Metals LE A R N I N G O U TC O M E 9.25 Define heavy­metal ions, give several examples, and describe their use in microbial control. Heavy-metal ions are ions of relatively high density metals, such as arsenic, zinc, mercury, silver, and copper, that are toxic at low concentrations. Heavy-metal ions are antimicrobial because they combine with sulfur atoms in molecules of cysteine, an amino acid. Such bonding denatures proteins, inhibiting or eliminating their function. Heavy-metal ions are low-level bacteriostatic and fungistatic agents, and with few exceptions their use has been superseded by more effective antimicrobial agents. FIGURE 9.16 illustrates the effectiveness of heavy metals in inhibiting bacterial reproduction on a Petri plate. Aldehydes LE A R N I N G O UTC O M E 9.26 Compare and contrast formaldehyde and glutaraldehyde as antimicrobial agents. Aldehydes are compounds containing terminal —CHO groups (see Table 2.3 on p. 40). Glutaraldehyde, which is a liquid, and formaldehyde, which is a gas, are highly reactive chemicals with the following structural formulas: O CH CH2 CH2 CH2 Glutaraldehyde O O CH HCH Formaldehyde ­ Aldehydes function in microbial control by cross-linking organic functional groups, including amino, hydroxyl, sulfhydryl, and carboxyl (see Table 2.3), thereby denaturing proteins and inactivating nucleic acids. Hospital personnel and scientists can use 2% solutions of glutaraldehyde to kill bacteria, viruses, and fungi; a 10-minute treatment effectively disinfects most objects. When the time of exposure is increased to 10 hours, glutaraldehyde sterilizes. Glutaraldehyde is less irritating and more effective than for maldehyde, but it is more expensive. Morticians and health care workers use formaldehyde dissolved in water to make a 37% solution called formalin. They use formalin for embalming and to disinfect hospital rooms, instruments, and machines. Formaldehyde must be handled with care because it irritates mucous membranes and is carcinogenic (cancer causing). ▲ FIGURE 9.16 The effect of heavy-metal ions on bacterial growth. Zones of inhibition can form because ions of heavy metals, such as dental amalgam used in fillings and shown in the center of the plate, inhibit bacterial reproduction through their effects on protein function. M09_BAUM7206_05_SE_C09.indd 277 11/23/16 3:03 AM te a       Ch p r 9    Controlling Microbial Growth in the Environment Gaseous Agents LE A R N I N G O U TC O M E 9.27 Describe the advantages and disadvantages of gaseous agents of microbial control. Many items, such as heart-lung machine components, sutures, plastic laboratory ware, mattresses, pillows, artificial heart valves, catheters, electronic equipment, and dried or powdered foods, cannot be sterilized easily with heat or water-soluble chemicals, nor is irradiation always practical for large or bulky items. However, they can be sterilized within a closed chamber containing highly reactive microbicidal and sporicidal gases such as ethylene oxide, propylene oxide, and beta­propiolactone: O CH2 O CH2 Ethylene oxide CH3 CH CH2 Propylene oxide CH2 CH2 O C O Beta-propiolactone These gases rapidly penetrate paper and plastic wraps and diffuse into every crack. Over time (usually 4–18 hours), they denature proteins and DNA by cross-linking organic functional groups, thereby killing everything they contact without harming inanimate objects. Ethylene oxide is frequently used as a gaseous sterilizing agent in hospitals and dental offices, and NASA uses the gas to sterilize spacecraft designed to land on other worlds lest they accidentally export earthly microbes. Large hospitals often use ethylene oxide chambers, which are similar in appearance to autoclaves, to sterilize instruments and equipment sensitive to heat. Despite their advantages, gaseous agents are far from perfect: They are often highly explosive, and they are extremely poisonous, so workers must extensively flush sterilized objects with air to remove every trace of the gas (which adds to the time required to use them). Finally, gaseous agents, especially beta-propiolactone, are potentially carcinogenic. Enzymes LE A R N I N G O U TC O M E 9.28 Describe the use of an enzyme to remove most bac- teria from food and to remove prions from medical instruments. ­ Many organisms produce chemicals that inhibit or destroy a variety of fungi, bacteria, or viruses. Among these are antimicrobial enzymes, which are enzymes that act against microorganisms. For example, human tears contain the enzyme lysozyme, which is a protein that digests the peptidoglycan cell walls of bacteria, causing the bacteria to rupture because of osmotic pressure and thus protecting the eye from most bacterial infections. Scientists, food processors, and medical personnel are researching ways to use natural and chemically modified M09_BAUM7206_05_SE_C09.indd 278 antimicrobial enzymes to control microbes in the environment, inhibit microbial decay of foods and beverages, and reduce the number and kinds of microbes on medical equipment. For example, food processors use lysozyme to reduce the number of bacteria in cheese, and some vintners use lysozyme instead of poisonous sulfur dioxide (SO2) to remove bacteria that would spoil wine. One exciting development is the use of an enzyme to eliminate the prion that causes variant Creutzfeldt-Jakob disease (vCJD), also called mad cow disease. The brain, spinal cord, placenta, eye, liver, kidney, pituitary gland, spleen, lung, and lymph nodes, as well as cerebrospinal fluid, can harbor prions. Medical instruments contaminated by these highly infectious and deadly proteins may remain infectious even after normal autoclaving; boiling; exposure to formaldehyde, glutaraldehyde, or ethylene oxide; or 24 hours of dry heat at 160°C. Until recently, harsh methods, such as autoclaving in sodium hydroxide for 30 minutes or complete incineration, were required to eliminate prions. In 2006, the European Union approved the use of the enzyme Prionzyme to safely and completely remove prions on medical instruments. Prionzyme is the first certified, noncaustic chemical to target prions. Antimicrobial Drugs LE A R N I N G O UTC O M E 9.29 Describe three general types of antimicrobial drugs and their use in environmental control of microorganisms. Antimicrobial drugs include antibiotics, semisynthetics, and synthetics. Specifically, antibiotics are antimicrobial chemicals produced naturally by microorganisms. When scientists chemically modify an antibiotic, the agent is called a semisynthetic. Scientists have also developed wholly synthetic antimicrobial drugs. The main difference between these antimicrobials and the chemical agents we have discussed in this chapter is that antimicrobial drugs are typically used for treatment of disease and not for environmental control of microbes. Nevertheless, some antimicrobial drugs are used for control outside the body. For example, nisin and natamycin are used to reduce the growth of bacteria and fungi, respectively, in cheese. (Chapter 10 discusses in more detail the nature and use of antimicrobial drugs to treat infectious diseases.) TABLE 9.5 summarizes the chemical methods of microbial control discussed in this chapter. Methods for Evaluating Disinfectants and Antiseptics LE A R N I N G O UTC O M E 9.30 Compare and contrast four methods used to measure the effectiveness of disinfectants and antiseptics. With few exceptions, higher concentrations and fresher solutions of a disinfectant are more effective than more dilute, 11/23/16 3:03 AM       Chemical Methods of Microbial Control TABLE 9.5 Chemical Methods of Microbial Control Action(s) Level of Activity Some Uses Phenol (carbolic acid) Denatures proteins and disrupts cell membranes Intermediate to low Original surgical antiseptic; now replaced by less odorous and injurious phenolics Phenolics (chemically altered phenol; bisphenols are composed of a pair of linked phenolics) Denature proteins and disrupt cell membranes Intermediate to low Disinfectants and antiseptics Alcohols Denature proteins and disrupt cell membranes Intermediate Disinfectants, antiseptics, and as a solvent in tinctures Halogens (iodine, chlorine, bromine, and fluorine) Denature proteins Intermediate Disinfectants, antiseptics, and water purification Oxidizing Agents (peroxides, ozone, and peracetic acid) Denature proteins by oxidation Surfactants (soaps and detergents) Decrease surface tension of water and disrupt cell membranes Low Soaps: degerming; detergents: antiseptic Heavy Metals (arsenic, zinc, mercury, silver, copper, etc.) Denature proteins Low Fungistats in paints; silver nitrate cream: surgical dressings, burn creams, and catheters; copper: algicide in water reservoirs, swimming pools, and aquariums Aldehydes (glutaraldehyde and formaldehyde) Denature proteins igh Disinfectant and embalming fluid Gaseous Agents (ethylene oxide, propylene oxide, and beta-propiolactone) Denature proteins igh Sterilization of heat- and water-sensitive objects Enzymes Denature proteins igh against target substrate Antimicrobials (antibiotics, semisynthetics, and synthetics) Act against cell walls, cell membranes, protein synthesis, and DNA transcription and replication Intermediate to low Disinfectants, antiseptics for deep wounds, water purification, and sterilization of food processing and medical equipment R H H H igh emoval of prions on medical instruments Disinfectants and treatment of infectious diseases effective against Salmonella. Measurement of an agent’s phenol coefficient has been replaced by newer methods because scientists have developed disinfectants and antiseptics much more effective than phenol. ­ older solutions. We have also seen that longer exposure times ensure the deaths of more microorganisms. However, anyone using disinfectants must consider whether higher concentrations and longer exposures may damage an object or injure animals or people. Scientists have developed several methods to measure the efficacy of antimicrobial agents. These include the phenol coef­ ficient, the use­dilution test, the Kelsey­Sykes capacity test, and the in­use test. H Method Phenol Coefficient ­ Joseph Lister introduced the widespread use of phenol as an antiseptic during surgery. Since then, researchers have evaluated the efficacy of various disinfectants and antiseptics by calculating a ratio that compares a given agent’s ability to control microbes to that of phenol under standardized conditions. This ratio is referred to as the phenol coefficient. A phenol coefficient greater than 1.0 indicates that an agent is more effective than phenol, and the larger the ratio, the greater the effectiveness. For example, chloramine, a mixture of chlorine and ammonia, has a phenol coefficient of 133.0 when used against the bacterium Staphylococcus aureus and a phenol coefficient of 100.0 when used against Salmonella enterica (en-ter’i-kă). This indicates that chloramine is at least 133 times more effective than phenol against Staphylococcus but only 100 times more M09_BAUM7206_05_SE_C09.indd 279 Use-Dilution Test Another method for measuring the efficacy of disinfectants and antiseptics against specific microbes is the use-dilution test. In this test, a researcher dips several metal cylinders into broth cultures of bacteria and briefly dries them at 37°C. The bacteria used in the standard test are Pseudomonas aeruginosa, Salmonella enterica serotype Choleraesuis (kol-er-a-su’is), and Staphylococcus aureus. The researcher then immerses each contaminated cylinder into a different dilution of the disinfectants being evaluated. After 10 minutes, each cylinder is removed, rinsed with water to remove excess chemical, and placed into a fresh tube of sterile medium for 48 hours of incubation. The most effective agent is the one that entirely prevents microbial growth at the highest dilution. The use-dilution test is the current standard test in the United States, though it was developed several decades ago before the appearance of many of today’s more troubling pathogens, including hepatitis C virus, HIV, and antibiotic-resistant bacteria and protozoa. Moreover, the disinfectants in use at the time the test was developed were far less powerful than many 11/23/16 3:03 AM r 9    Controlling Microbial Growth in the Environment Kelsey-Sykes Capacity Test The Kelsey-Sykes capacity test is the standard alternative assessment approved by the European Union to determine the capacity of a given chemical to inhibit bacterial growth. In this test, researchers add a suspension of a bacterium such as Psuedomonas aeruginosa or Staphylococcus aureus to a suitable concentration of the chemical being tested. Then at predetermined times, they move samples of the mixture into growth medium containing a disinfectant deactivator. After incubation for 48 hours, turbidity in the medium indicates that bacteria survived treatment. Lack of turbidity, indicating lack of bacterial reproduction, reveals the minimum time required for the disinfectant to be effective. In-Use Test Though phenol coefficient, use-dilution, and Kelsey-Sykes capacity tests can be beneficial for initial screening of disinfectants, they can also be misleading. These types of evaluation are measures of effectiveness under controlled conditions against one or, at most, a few species of microbes at a time, but disinfectants are generally used in various environments against a diverse population of organisms that are often associated with one another in complex biofilms affording mutual protection. A more realistic (though more time-consuming) method for determining the efficacy of a chemical is called an in-use test. In this procedure, swabs are taken from actual objects, Development of Resistant Microbes Many scientists are concerned that Americans have become overly preoccupied with antisepsis and disinfection, as evidenced by the proliferation of products containing antiseptic and disinfecting chemicals. For example, you can now buy hand soap, shampoo, toothpaste, hand lotion, foot pads for shoes, deodorants, and bath sponges that contain antiseptics, as well as kitty litter, cutting boards, scrubbing pads, garbage bags, children’s toys, and laundry detergents that contain disinfectants. There is little evidence that the extensive use of such products adds to human or animal health, but it does promote the development of strains of microbes resistant to antimicrobial chemicals: While susceptible cells die, resistant cells remain to proliferate. Scientists have already isolated strains of pathogenic bacteria, including M. tuberculosis, P. aeruginosa, E. coli, and S. aureus, that are less susceptible to common disinfectants and antiseptics. TELL ME WHY Many chemical disinfectants and antiseptics act by denaturing proteins. Why does denaturation kill cells? her stool sample and that she suspects the raw cheese was the cause of the infection. ublic health officials contact Johnson Dairy and learn that a number of customers have reported getting sick within the last week after drinking milk or eating their cheese. The health department requests that Johnson Dairy cease selling their products until the source of the contamination can be determined. ­P An Uninvited Dinner Guest? such as operating room equipment, both before and after the application of a disinfectant or an antiseptic. The swabs are then inoculated into appropriate growth media that, after incubation, are examined for microbial growth. The in-use test allows a more accurate determination of the proper strength and application procedure of a given disinfection agent for each specific situation. ­ used today. Some government agencies have expressed concern that the test is neither accurate, reliable, nor relevant; therefore, AOAC International—an association of chemists, microbiologists, food science personnel, and others—is developing a new standard procedure for use in the United States. ­ te a       Ch p ­ ­ ­ R ather than feeling fine the next morning, Marcella feels far worse. She also notices blood in her diarrhea; this scares her, so she makes an appointment to see her doctor as soon as possible. Dr. Walker examines Marcella and collects a stool (feces) sample. She tells Marcella that, based on her symptoms, it’s likely that Marcella has food poisoning. They will know for certain after the lab analysis of the stool, but for now the doctor prescribes azithromycin, fluids, and rest. Dr. Walker asks Marcella to try to remember all the food she consumed in the last few days and where she got the food; it’s possible that Marcella and Sarah aren’t the only ones affected, and public health authorities may need to investigate. A few days later, Marcella is feeling much better. Dr. Walker calls and tells Marcella that Campylobacter jejuni was identified in M09_BAUM7206_05_SE_C09.indd 280 1. Why do you think that Dr. Walker suspected the milk as the source of infection? 2. Imagine that you are working for Johnson Dairy. What kinds of practices (both during and after production) would help limit the chance of contamination in raw milk? 11/23/16 3:03 AM       Chapter Summary CHAPTER SUMMARY ­ 3. Antisepsis is the inhibition/killing of microorganisms (particularly pathogens) on skin or tissue by the use of a chemical antiseptic, whereas disinfection refers to the use of agents (called disinfectants) to inhibit microbes on inanimate objects. 4. Degerming refers to the removal of microbes from a surface by scrubbing. 5. Sanitization is the reduction of a prescribed number of pathogens from surfaces and utensils in public settings. 6. Pasteurization is a process using heat to kill pathogens and control microbes that cause spoilage of food and beverages. 7. The suffixes -stasis and -static indicate that an antimicrobial agent inhibits microbes, whereas the suffixes -cide and -cidal indicate that the agent kills or permanently inactivates a particular type of microbe. 6. When used as a microbial control method, filtration is the passage of air or a liquid through a material that traps and removes microbes. Some membrane filters have pores small enough to trap the smallest viruses. HEPA (high-efficiency particulate air) filters remove microbes and particles from air. 7. The high osmotic pressure exerted by hypertonic solutions of salt or sugar can preserve foods such as jerky and jams by removing from microbes the water that they need to carry out their metabolic functions. 8. Radiation includes high-speed subatomic particles and even more energetic electromagnetic waves released from atoms. Ionizing radiation (wavelengths shorter than 1 nm) produces ions that denature important molecules and kill cells. Nonionizing radiation (wavelengths longer than 1 nm) is less effective in microbial control, although UV light causes pyrimidine dimers, which can kill affected cells. ­ 8. Microbial death is the permanent loss of reproductive capacity. Microbial death rate measures the efficacy of an antimicrobial agent. 5. Antimicrobial methods involving drying are desiccation, used to preserve food, and lyophilization (freeze-drying), used for the long-term preservation of cells or microbes. ­ 2. An aseptic environment or procedure is free of contamination by pathogens. 4. Under certain circumstances, microbes can be controlled using ultra-high-temperature sterilization, dry-heat sterilization, incineration, refrigeration, or freezing. ­ 1. Sterilization is the eradication of microorganisms and viruses; the term is not usually applied to the destruction of prions. ultra-high-temperature pasteurization. The methods differ in their combinations of temperature and time of exposure. ­ Basic Principles of Microbial Control (pp. 261–264) 9. Antimicrobial agents destroy microbes either by altering their cell walls and membranes or by interrupting their metabolism and reproduction via interference with proteins and nucleic acids. The Selection of Microbial Control Methods (pp. 264–266) 1. Factors affecting the efficacy of antimicrobial methods include the site to be treated, the relative susceptibility of microorganisms, and environmental conditions. 2. The CDC has established four biosafety levels (BSL) for microbiological laboratories. BSL-1 is minimal; BSL-4 requires special suits, rooms, and other precautions. Physical Methods of Microbial Control (pp. 266–273) 1. Thermal death point is the lowest temperature that kills all cells in a broth in 10 minutes, whereas thermal death time is the time it takes to completely sterilize a particular volume of liquid at a set temperature. Decimal reduction time (D) is the time required to destroy 90% of the microbes in a sample. Chemical Methods of Microbial Control (pp. 274–280) 1. Phenolics, which are chemically modified phenol molecules, are intermediate- to low-level disinfectants that denature proteins and disrupt cell membranes in a wide variety of pathogens. 2. Alcohols are intermediate-level disinfectants that denature proteins and disrupt cell membranes; they are used either as 70% to 90% aqueous solutions or in a tincture, which is a combination of an alcohol and another antimicrobial chemical. 3. Halogens (iodine, chlorine, bromine, and fluorine) are used as intermediate-level disinfectants and antiseptics to kill microbes by protein denaturation in water or on medical instruments or skin. 4. Oxidizing agents such as hydrogen peroxide, ozone, and peracetic acid are high-level disinfectants and antiseptics that release oxygen radicals, which are toxic to many microbes, especially anaerobes. 3. Pasteurization, a method of heating foods to kill pathoV gens and control spoilage organisms without altering T r the quality of the food, can be achieved by several methods: the historical (batch) method, flash pasteurization, and 6. Heavy-metal ions, such as ions of arsenic, silver, mercury, copper, and zinc, are low-level disinfectants that denature proteins. For most applications they have been superseded by less toxic alternatives. uto M09_BAUM7206_05_SE_C09.indd 281 ­ 5. Surfactants include soaps, which act primarily to break up oils during degerming, and detergents, such as quaternary ammonium compounds (quats), which are low-level disinfectants. ideo 2. An autoclave uses steam heat under pressure to sterilize chemicals and objects that can tolerate moist heat. 11/23/16 3:03 AM te a       Ch p r 9    Controlling Microbial Growth in the Environment 7. Aldehydes are high-level disinfectants that cross-link organic functional groups in proteins and nucleic acids. A 2% solution of glutaraldehyde or a 37% aqueous solution of formaldehyde (called formalin) is used to disinfect or sterilize medical or dental equipment and in embalming fluid. 8. Gaseous agents of microbial control, which include ethylene oxide, propylene oxide, and beta-propiolactone, are high-level disinfecting agents used to sterilize heat-sensitive equipment and large objects. These gases are explosive and potentially carcinogenic. R QUESTIONS FO R 9. Many organisms use antimicrobial enzymes to combat microbes. Humans use them commercially in food preservation and as a noncaustic, nondestructive way to eliminate prions on medical instruments. 10. Antimicrobial drugs, which include antibiotics, semisynthetics, and synthetics, are compounds that are typically used to treat diseases but can also function as intermediate-level disinfectants. 11. Four methods for evaluating the effectiveness of a disinfectant or antiseptic are the phenol coefficient, the use-dilution test, the Kelsey-Sykes capacity test, and the in-use test. The in-use test provides a more accurate determination of efficacy under real-life conditions. EVIEW Answers to the Questions for Review (except Short Answer questions) begin on p. A-1. Multiple Choice 9. The preservation of beef jerky from microbial growth relies on which method of microbial control? a. filtration c. desiccation b. lyophilization d. radiation 2. Which of the following substances or processes kills microorganisms on laboratory surfaces? a. antiseptics c. degermers b. disinfectants d. pasteurization 11. Which of the following substances would most effectively inhibit anaerobes? a. phenol c. ethanol b. silver d. hydrogen peroxide 3. Which of the following terms best describes the disinfecting of cafeteria plates? a. pasteurization c. sterilization b. antisepsis d. sanitization 12. Which of the following adjectives best describes a surgical procedure that is free of microbial contaminants? a. disinfected c. degermed b. sanitized d. aseptic 4. The microbial death rate is used to measure the efficiency of . a. a detergent c. sanitization techniques b. an antiseptic d. all of the above 13. Biosafety Level 3 includes a. double sets of entry doors b. pressurized suits 5. Which of the following statements is true concerning the selection of an antimicrobial agent? a. An ideal antimicrobial agent is stable during storage. b. An ideal antimicrobial agent is fast acting. c. Ideal microbial agents do not exist. d. All of the above are correct. 6. The endospores of which organism can be used as a biological indicator of sterilization? a. Bacillus stearothermophilus c. Mycobacterium tuberculosis b. Salmonella enterica d. Staphylococcus aureus                                10. Which of the following types of radiation is more widely used as an antimicrobial technique? a. electron beams c. radio waves b. visible light waves d. microwaves                                1. In practical terms in everyday use, which of the following statements provides the definition of sterilization? a. Sterilization eliminates organisms and their spores or endospores. b. Sterilization eliminates harmful microorganisms and viruses. c. Sterilization eliminates prions. d. Sterilization eliminates hyperthermophiles. . c. showers in entryways d. all of the above 14. A sample of E. coli has been subjected to heat for a specified time, and 90% of the cells have been destroyed. Which of the following terms best describes this event? a. thermal death point c. decimal reduction time b. thermal death time d. none of the above 15. Which of the following substances is least toxic to humans? a. carbolic acid c. hydrogen peroxide b. glutaraldehyde d. formalin 16. Which of the following chemicals is active against bacterial endospores? a. copper ions c. ethanol b. ethylene oxide d. triclosan 7. A company that manufactures an antimicrobial cleaner for kitchen counters claims that its product is effective when used in a 50% water solution. By what means might scientists best verify this statement? a. disk-diffusion test c. filter paper test b. phenol coefficient d. in-use test 17. Which of the following disinfectants acts against cell membranes? a. phenol c. silver nitrate b. peracetic acid d. glutaraldehyde 8. Which of the following items functions most like an autoclave? a. boiling pan c. microwave oven b. incinerator d. pressure cooker 18. Which of the following disinfectants contains alcohol? a. iodophor c. formalin b. quat d. tincture of bromine M09_BAUM7206_05_SE_C09.indd 282 11/23/16 3:03 AM 20. Which class of surfactant is most soluble in water? a. quaternary ammonium c. soaps compounds d. peracetic acids b. alcohols Short Answer 1. Describe three types of microbes that are extremely resistant to antimicrobial treatment, and explain why they are resistant. 2. Compare and contrast four tests that have been developed to measure the effectiveness of disinfectants. VISUALIZE IT! 1. Calculate the decimal reduction time (D) for the two temperatures in the following graph. 105 104 Number of living microbes 19. Which antimicrobial chemical has been used to sterilize spacecraft? a. phenol c. heavy metal b. alcohol d. ethylene oxide 103 8. Defend the following statement: “Pasteurization is not sterilization.” (b) 100 2 4 6 Time (min) 8 10 2. Indian tradition holds that storing water in brass pitchers prevents disease. Scientists have discovered that there is some truth in the tradition. The researchers collected river water samples and found fecal bacterial counts as high as 1 million bacteria per milliliter. However, they could detect no bacteria in the water after it had been stored for two days in traditional brass pitchers. Bacterial levels in plastic or earthenware containers remained high over the same period. How can brass, which is an alloy of copper mixed with zinc, make water safer to drink? ­ 7. What is the difference between thermal death point and thermal death time? 455C (a) 101 4. Why do warm disinfectant chemicals generally work better than cool ones? 6. Describe five physical methods of microbial control. 205C 102 3. Why is it necessary to use strong disinfectants in areas exposed to tuberculosis patients? 5. Why are Gram-negative bacteria more susceptible to heat than Gram-positive bacteria?       R Questions for eview 9. Compare and contrast desiccation and lyophilization. 10. Compare and contrast the action of alcohols, halogens, and oxidizing agents in controlling microbial growth. 11. Hyperthermophilic prokaryotes may remain viable in canned goods after commercial sterilization. Why is this situation not dangerous to consumers? 12. Why are alcohols more effective in a 70% solution than in a 100% solution? 13. Contrast the structures and actions of soaps and quats. 14. What are some advantages and disadvantages of using ionizing radiation to sterilize food? 15. How can campers effectively treat stream water to remove pathogenic protozoa, bacteria, and viruses? H R C ITICAL T INKING 1. In 2004, a casino paid $28,000 for a grilled cheese sandwich that was purported to have an image of the Virgin Mary on it. The seller had stored the sandwich in a less-than-airtight box for 10 years without decay or the growth of mold. What antimicrobial chemical and physical agents might account for the longevity of the sandwich? M09_BAUM7206_05_SE_C09.indd 283 2. Is desiccation the only antimicrobial effect operating when grapes are dried in the sun to make raisins? Explain. 3. How long would it take to reduce a population of 100 trillion (1014) bacteria to 10 viable cells if the D value of the treatment is 3 minutes? 11/23/16 3:03 AM te a       Ch p r 9    Controlling Microbial Growth in the Environment 4. Some potentially pathogenic bacteria and fungi, including strains of Enterococcus, Staphylococcus, Candida, and Aspergillus, can survive for one to three months on a variety of materials found in hospitals, including scrub suits, lab coats, and plastic aprons and computer keyboards. What can hospital personnel do to reduce the spread of these pathogens? ­ ­ 5. Over 1000 people developed severe diarrhea, and at least four died, from a strain of Salmonella enterica in the fall of 2012. Epidemiologists determined that infection resulted from the consumption of contaminated smoked salmon. Based on this chapter, what methods to control microbial growth are available to fishermen, packers, and stores that might have prevented such an outbreak? What other precautions could consumers have taken? 6. An over-the-counter medicated foot powder contains camphor, eucalyptus oil, lemon oil, and zinc oxide. Only one of the ingredients is a proven antimicrobial. Which one? How does it act against fungi? contains a drop of a chemical with an antimicrobial effect. After 24 hours of incubation, the first tube remains clear, whereas the second tube has become cloudy with bacteria. Design an experiment to determine whether this amount of the antimicrobial chemical is bacteriostatic or bactericidal against E. coli. 11. Would you expect Gram-negative bacteria or Gram-positive bacteria to be more susceptible to antimicrobial chemicals that act against cell walls? Explain your answer, which you should base solely on the nature of the cells’ walls (see Figure 3.15). 12. Where should you place a sterilization indicator within an autoclave? Explain your reasoning. 13. Why is liquid water necessary for microbial metabolism? 14. A virologist needs to remove all bacteria from a solution containing viruses without removing the viruses. What size membrane filter should the scientist use? 8. In what ways might it be argued that the widespread commercial use of antiseptics and disinfectants has hurt rather than helped American health? 15. During the fall 2001 bioterrorist attack in which anthrax endospores were sent through the mail, one news commentator suggested that people should iron all their incoming mail with a regular household iron as a means of destroying endospores. Would you agree that this is a good way to disinfect mail? Explain your answer. Which disinfectant methods would be both more effective and more practical? 9. Explain why quaternary ammonium compounds are not very effective against mycobacteria such as Mycobacterium tuberculosis. 16. What common household antiseptic contains a heavy metal as its active ingredient? 7. Tsunamis and hurricanes severely contaminate water wells and disrupt water supply lines. What immediate steps should people take to lessen the spread of waterborne illnesses such as cholera? 10. A student inoculates Escherichia coli into two test tubes containing the same sterile liquid medium, except that the first tube also M09_BAUM7206_05_SE_C09.indd 284 17. What is the phenol coefficient of phenol when used against Staphylococcus? 11/23/16 3:03 AM       Questions for Review CONCEPT MAPPING Using the following terms, fill in the following concept map that describes moist heat applications to control microorganisms. Sterilization technique Thermoduric microorganisms (2) Ultra-high-temperature pasteurization Flash pasteurization Fungi Most viruses Pasteurization Protozoan trophozoites 140°C, 1–3 sec 63°C, 30 min Autoclave Boiling water Equivalent treatments Ultra-high-temperature sterilization Vegetative bacterial cells Moist heat applications to control microorganisms include does not kill 1005C > 10 min 9 7 1 2 is a 1215C 15 psi > 15 min 15 does not kill 8 is a Sterilization technique 10 16 methods include kills 3 Batch method (classic) 4 11 5 6 12 13 725C 15 sec 1345C 1 sec are 14 M09_BAUM7206_05_SE_C09.indd 285 06/03/18 4:49 PM