Microbial Nutrition and Growth

Microbial Nutrition and Growth The Microbes Ate My Homework ­ ­ ­ ­ Paper in the form of cardboard and shipping containers is one of the lar gest contributors to landfills. What if we could turn this waste into fuel? Paper is mainly composed of cellulose, a branched polymer of glucose. Cellulose makes up the majority of the cell wall material of plants. A number of species of bacteria, including members of the phyla Actinobacteria and Firmicutes, can degrade cellulose by releasing free sugars from the polymer using enzymes called cellulases. Other bacteria can ferment these sugars into fuels such as ethanol or butanol, which are called biofuels when they are derived by metabolic activity. An exciting idea is to genetically engineer a single bacterial species to secrete many different kinds of cellulases and also ferment the released sugars into biofuel. This new organism could be added to cardboard waste dissolved in water and would turn the waste into fuel, solving two problems at once. Adversaries of this idea are opposed to genetically modifying bacteria in this manner. What if such a microbe escaped into the wild? Could it become a new plant pathogen, attacking the very fabric of plants’ cell walls? Imagine you are on a scientific team at the Environmental Protection Agency charged with reviewing an application from a company that wants to use engineered bacteria in cellulosic bioreactors. • • • How can these organisms be used safely? Would you approve this application? What additional information is required to make an informed decision? Metabolism—the set of controlled chemical reactions within cells—is an essential characteristic of all living things. The ultimate outcome of metabolic activity is reproduction, an increase in the number of individual cells or organisms. When speaking of the reproductive activities of microbes in general and of bacteria in particular, microbiologists typically use the term growth, referring to an increase in the size of a population of microbes rather than to an increase in size of an individual. The result of such microbial growth is either a discrete colony, which is an aggregation of cells arising from a single parent cell, or a biofilm, which is a collection of microbes living on a surface in a complex community. Put another way, the reproduction of individual microorganisms results in the growth of a colony or biofilm. Further, common expressions such as “The microorganisms grow in salt-containing media” are widely understood to mean that the organisms metabolize and reproduce rather than that they increase in size. In this chapter, we consider the characteristics of microbial growth from two different but related perspectives: We examine the requirements of microbes in natural settings, including their chemical, physical, and energy requirements, M06_BAUM7206_05_SE_C06.indd 161 Glucose Go to the study area to solve the problem. and we explore how microbiologists try to create similar conditions to grow microorganisms in the laboratory so that they can be transported, identified, and studied. We conclude by examining laboratory analysis of bacterial population dynamics and some techniques for measuring bacterial population growth. Growth Requirements Organisms use a variety of chemicals—called nutrients—to meet their energy needs and to build organic molecules and cellular structures. The most common of these nutrients are compounds containing necessary elements such as carbon, oxygen, nitrogen, and hydrogen. Like all organisms, microbes obtain nutrients from a variety of sources in their environment, and they must bring nutrients into their cells by passive and active transport processes (see Chapter 3). When they acquire their nutrients by living in or on another organism, they may cause disease as they interfere with their hosts’ metabolism and nutrition. 11/23/16 3:00 AM r 6    Microbial Nutrition and Growth Nutrients: Chemical and Energy Requirements LE A R N I N G O U TC O M E S 6.1 Describe the roles of carbon, hydrogen, oxygen, nitrogen, 6.2 6.3 6.4 6.5 trace elements, and vitamins in microbial growth and reproduction. Compare four basic categories of organisms based on their carbon and energy sources. Distinguish among anaerobes, aerobes, aerotolerant anaerobes, facultative anaerobes, and microaerophiles. Explain how oxygen can be fatal to organisms by discussing singlet oxygen, superoxide radical, peroxide anion, and hydroxyl radical; describe how organisms protect themselves from toxic forms of oxygen. Define nitrogen fixation, and explain its importance. We begin our examination of microbial growth requirements by considering three things all cells need for metabolism: a source of carbon, a source of energy, and a source of electrons or hydrogen atoms. Sources of Carbon, Energy, and Electrons Organisms can be categorized into two broad groups based on their source of carbon. Organisms that utilize an inorganic source of carbon (i.e., carbon dioxide) as their sole source of carbon are called autotrophs1 (aw´tō-trōfs), so named because they “feed themselves.” More precisely, autotrophs make organic compounds from CO2 and thus need not acquire carbon in organic compounds from other organisms. In contrast, organisms called heterotrophs2 (het´er-ō-trōfs) catabolize organic molecules (such as proteins, carbohydrates, amino acids, and fatty acids) they acquire from other organisms. Organisms can also be categorized according to whether they use chemicals or light as an energy source for such cellular processes as anabolism, intracellular transport, and motility. Organisms that acquire energy from redox reactions involving inorganic and organic chemicals are called chemotrophs (kēm´ō-trōfs). These reactions are either aerobic respiration, anaerobic respiration, or fermentation, depending on the final electron acceptor (see Chapter 5). Organisms that use light as an energy source are called phototrophs3 (fō´tō-trōfs). Thus, we see that organisms can be categorized on the basis of their carbon and energy sources into one of four basic groups: photoautotrophs, chemoautotrophs, photoheterotrophs, and chemoheterotrophs (FIGURE 6.1). Plants, some protozoa, and algae are photoautotrophs, whereas animals, fungi, and other protozoa are chemoheterotrophs. Bacteria and archaea exhibit greater metabolic diversity, uniquely having members in all four groups. In addition to carbon and energy sources, cells require electrons or hydrogen atoms (which are composed of an electron orbiting a simple nucleus) for redox reactions. Hydrogen is the most common chemical element in cells, and it is so common in organic molecules and water that it is never a limiting nutrient; that is, metabolism is never interrupted by a lack of hydrogen. Hydrogen is essential for hydrogen bonding and in electron transfer. Heterotrophs acquire electrons (typically as part of hydrogen atoms) from the same organic molecules that provide them carbon and are called organotrophs (ōr´gān-ō-trōfs); alternatively, autotrophic organisms acquire electrons or hydrogen ◀ FIGURE 6.1 Four basic groups Carbon source Energy source Light (photo-) Chemical compounds (chemo-) Carbon dioxide (auto-) Photoautotrophs • Plants, algae, and cyanobacteria use H2O as an electron source to reduce CO2, producing O2 as a by-product • Green sulfur bacteria and purple sulfur bacteria use H2S as an electron source; they do not produce O2 Chemoautotrophs • Hydrogen, sulfur, and nitrifying bacteria, some archaea Organic compounds (hetero-) Photoheterotrophs • Green nonsulfur bacteria and purple nonsulfur bacteria, some archaea Chemoheterotrophs • Aerobic respiration: most animals, fungi, and protozoa, and many bacteria • Anaerobic respiration: some animals, protozoa, bacteria, and archaea • Fermentation: some bacteria, yeasts, and archaea 2From 1From Greek auto, meaning “self,” and trophe, meaning “nutrition.” M06_BAUM7206_05_SE_C06.indd 162 3From of organisms based on their carbon and energy sources. Additionally, organo- trophs utilize electrons from organic molecules, and lithotrophs utilize electrons from inorganic molecules. ­ hapte       C Greek hetero, meaning “other,” and trophe, meaning “nutrition.” Greek photos, meaning “light,” and trophe, meaning “nutrition.” 11/23/16 3:00 AM atoms from inorganic molecules (such as H2, NO2 - , H2S, and Fe2+ ) and are called lithotrophs4 (lith´ō-trōfs). other metabolic reactions) contains peroxide anion, another highly reactive oxidant. It is peroxide anion that makes hydrogen peroxide an antimicrobial agent. Aerobes contain catalase or peroxidase enzymes that detoxify peroxide anion. Catalase converts hydrogen peroxide to water and molecular oxygen: Oxygen Requirements Oxygen is essential for obligate aerobes because it serves as the final electron acceptor of electron transport chains, which produce most of the ATP in these organisms. By contrast, oxygen is a deadly poison for obligate anaerobes. How can oxygen be essential for one group of organisms and yet be a fatal toxin for others? The key to understanding this apparent incongruity is understanding that neither atmospheric oxygen (O2) nor covalently bound oxygen in compounds such as carbohydrates and water is poisonous. Rather, the toxic forms of oxygen are those that are highly reactive. They are toxic for the same reason that oxygen is the final electron acceptor for aerobes: They are excellent oxidizing agents, so they steal electrons from other compounds, which in turn steal electrons from still other compounds. The resulting chain of vigorous oxidations causes irreparable damage to cells by oxidizing important compounds, including proteins and lipids. The cellular components most sensitive to oxidative damage are proteins with active sites involved in redox reactions, especially iron-sulfur clusters. There are four toxic forms of oxygen: 2O2 - + 2H + S H2O2 + O2 One reason that anaerobes are susceptible to oxygen is that they lack superoxide dismutase; the oxidizing reactions of superoxide radicals that have been formed in the presence of oxygen cause their death. • Peroxide anion (O2 2- ). Hydrogen peroxide formed during reactions catalyzed by superoxide dismutase (and during M06_BAUM7206_05_SE_C06.indd 163 A simple test for catalase involves adding a sample from a bacterial colony to a drop of hydrogen peroxide. The production of bubbles of oxygen indicates the presence of catalase. Peroxidase breaks down hydrogen peroxide without forming oxygen, using a reducing agent such as the coenzyme NADH: H2O2 + NADH + H + S 2H2O + NAD + Obligate anaerobes either lack both catalase and peroxidase or have only a small amount of them, so they are susceptible to the toxic action of hydrogen peroxide. • Hydroxyl radical (OH·). Hydroxyl radicals result from ionizing radiation and from the incomplete reduction of hydrogen peroxide: H2O2 + e- + H + S H2O + OH # Hydroxyl radicals are the most reactive of the four toxic forms of oxygen, but hydrogen peroxide does not accumulate in aerobic cells (because of the action of catalase and peroxidase); so in aerobic cells, the threat of hydroxyl radical is virtually eliminated. ­ Besides the enzymes superoxide dismutase, catalase, and peroxidase, aerobes use other antioxidants, such as vitamins C and E, to protect themselves from toxic oxygen products. These antioxidants provide electrons that reduce toxic forms of oxygen. Not all organisms are either strict aerobes or anaerobes; many organisms can live in various oxygen concentrations between these two extremes. For example, some aerobic organisms can maintain life via fermentation or anaerobic respiration, though their metabolic efficiency is often reduced in the absence of oxygen. Such organisms are called facultative anaerobes. Escherichia coli (esh-ĕ-rik´ē-ă kō´lī) is an example of a facultatively anaerobic bacterium. Note that although the term facultative aerobe could possibly have the same meaning, for consistency scientists use only the term facultative anaerobe. Aerotolerant anaerobes do not use aerobic metabolism, but they tolerate oxygen by having some of the enzymes that detoxify oxygen’s poisonous forms. The lactobacilli that transform cucumbers into pickles and milk into cheese are aerotolerant. These organisms can be kept in a laboratory without the special conditions required by obligate anaerobes. ­ • Superoxide radical (O2 - ). A few superoxide radicals form during the incomplete reduction of O2 during electron transport in aerobes and during metabolism by anaerobes in the presence of oxygen. Superoxide radicals are so reactive and toxic that aerobic organisms must produce enzymes called superoxide dismutases (dis´myu-tās-es) to detoxify them. These enzymes, which have active sites that contain metal ions—Zn2+ , Mn2+ , Fe2+ , Ni2+ , or Cu2+ , depending on the organism—combine two superoxide radicals and two protons to form hydrogen peroxide (H2O2) and molecular oxygen (O2): 2H2O2 S 2H2O + O2 ­ • Singlet oxygen (1O2). Singlet oxygen is molecular oxygen with electrons that have been boosted to a higher energy state, typically during aerobic metabolism. Singlet oxygen is a very reactive oxidizing agent. Phagocytic cells, such as certain human white blood cells, use it to oxidize pathogens. Singlet oxygen is photochemically produced by the reaction of light with oxygen in the presence of certain light-absorbing pigments, such as chlorophyll. To protect themselves from singlet oxygen, phototrophic microorganisms often contain pigments called carotenoids (ka-rot´enoyds) that prevent toxicity by removing the excess energy of singlet oxygen.       R Growth equirements 4From Greek lithos, meaning “rock,” and trophe, meaning “nutrition.” 11/23/16 3:00 AM Oxygen concentration High Low r 6    Microbial Nutrition and Growth hapte       C Loosefitting cap (a) Obligate aerobes (b) Obligate (c) Facultative (d) Aerotolerant anaerobes anaerobes anaerobes ▲ FIGURE 6.2 Using a liquid thioglycolate growth medium to identify the oxygen requirements of organisms. The surface is exposed to atmospheric oxygen and is aerobic. Oxygen concentration decreases with depth; the bottom of the tube is anaerobic. (a) Obligate aerobes cannot survive below the depth to which oxygen penetrates the medium. (b) Obligate anaerobes cannot tolerate any oxygen. (c) Facultative anaerobes can grow with or without oxygen, but their ability to use aerobic respiration pathways enhances their growth near the surface. (d) Aerotolerant aerobes can grow equally well with or without oxygen; their growth is relatively evenly distributed throughout the medium. Where in such a test tube would the growth zone be for a microaerophilic aerobe? Figure 6.2 Microaerophiles would be found slightly below the surface but neither directly at the surface nor in the depths of the tube. Microaerophiles, such as the ulcer-causing pathogen Helicobacter pylori5 (hel´ĭ-kō-bak´ter pī´lō-rē), require oxygen levels of 2% to 10%. This concentration of oxygen is found in the stomach. The 21% concentration of oxygen in the atmosphere damages microaerophiles, presumably because they have limited ability to detoxify hydrogen peroxide and superoxide radicals. Microbial groups contain members with each of the five types of oxygen requirement. Algae, most fungi and protozoa, and many prokaryotes are obligate aerobes. A few yeasts and numerous prokaryotes are facultative anaerobes. Many prokaryotes and a few protozoa are aerotolerant, microaerophilic, or obligate anaerobes. The oxygen requirement of an organism can be identified by growing it in a medium that contains an oxygen gradient from top to bottom (FIGURE 6.2). Though nitrogen constitutes about 79% of the atmosphere, relatively few organisms can utilize nitrogen gas (N2), because it is a triple-bonded, highly stable gas. A few bacteria, notably many cyanobacteria and Rhizobium (rī-zō´bē-ŭm), reduce nitrogen gas to ammonia (NH3) via a process called nitrogen fixation. Nitrogen fixation is essential for life on Earth because nitrogen fixers provide nitrogen in a form usable by other organisms. (Chapters 11 and 26 discuss nitrogen-fixing prokaryotes as well as nitrifying prokaryotes—those that oxidize nitrogenous compounds to acquire electrons for electron transport.) Other Chemical Requirements Together, carbon, hydrogen, oxygen, and nitrogen make up more than 95% of the dry weight of cells; phosphorus, sulfur, calcium, manganese, magnesium, copper, iron, and a few other elements constitute the rest. Phosphorus is a component of phospholipid membranes, DNA, RNA, ATP, and some proteins. Sulfur is a component of sulfur-containing amino acids, which bind to one another via disulfide bonds that are critical to the tertiary structure of proteins, and in vitamins such as thiamine (B1) and biotin. Other elements are called trace elements because they are required in very small (“trace”) amounts. For example, a few atoms of selenium dissolved out of the walls of glass test tubes provide the total requirement for the growth of green algae in a laboratory. Other trace elements are usually found in sufficient quantities dissolved in water. For this reason, tap water can sometimes be used instead of distilled or deionized water to grow microorganisms in the laboratory. Some microorganisms—for example, algae and photosynthetic bacteria—are lithotrophic photoautotrophs; that is, they can synthesize all of their metabolic and structural needs from inorganic nutrients. They have every enzyme and cofactor they need to produce all their cellular components. Most organisms, however, require small amounts of certain organic chemicals that they cannot synthesize in addition to those that provide carbon and energy. These necessary organic chemicals are called growth factors (TABLE 6.1). For example, vitamins are Nitrogen Requirements ­ ­ ­ 5From the semihelical shape of the cell and from Greek pyle, meaning “gate,” in reference to the pylorus, the distal portion of the stomach, which is the gate to the small intestine. M06_BAUM7206_05_SE_C06.indd 164 TABLE 6.1 Some Growth Factors of Microorganisms and Their Functions ­ Another essential element is nitrogen, which is an important element in many organic compounds, including the amine group of amino acids and as part of nucleotide bases. Nitrogen makes up about 14% of the dry weight of microbial cells. Nitrogen is often a growth-limiting nutrient for many organisms; that is, their anabolism ceases because they do not have sufficient nitrogen to build proteins and nucleotides. Organisms acquire nitrogen from organic and inorganic nutrients. For example, most photosynthetic organisms can reduce nitrate (NO3 - ) to ammonium (NH4 + ), which can then be used for biosynthesis. In addition, all cells recycle nitrogen from their amino acids and nucleotides. Growth Factor Function Amino acids Components of proteins Cholesterol Used by mycoplasmas (bacteria) for cell membranes Heme Functional portion of cytochromes in electron transport system NADH Electron carrier Niacin (nicotinic acid, vitamin B 3) Precursor of NAD + and NADP + Para-aminobenzoic acid (PABA) Precursor of folic acid, which is involved in metabolism of one-carbon compounds and nucleic acid synthesis 11/23/16 3:00 AM Growth rate growth factors for some microorganisms. Recall that vitamins constitute all or part of many coenzymes. (Note that vitamins are not growth factors for microorganisms that can manufacture them, such as E. coli.) Growth factors for various microbes include some amino acids, purines, pyrimidines, cholesterol, NADH, and heme.       R Growth equirements Physical Requirements Minimum Maximum LE A R N I N G O U TC O M E 6.6 Explain how extremes of temperature, pH, and osmotic Temperature and hydrostatic pressure limit microbial growth. Optimum (a) In addition to chemical nutrients, organisms have physical requirements for growth, including specific conditions of temperature, pH, osmolarity, and pressure. Temperature 22°C 30°C 37°C (b) ▲ FIGURE 6.3 The effects of temperature on microbial growth. (a) Minimum, optimum, and maximum growth temperatures are ­ revealed by growth rate plotted against temperature. (b) Growth of Escherichia coli on nutrient agar after 18 hours of incubation at three different temperatures. If microorganisms can survive at temperatures lower than their minimum growth temperature, then why is it called “minimum”? Figure 6.3 The minimum growth temperature is defined as the lowest temperature that supports metabolism. Though organisms might survive at lower temperatures, they do not actively metabolize, grow, or reproduce. Temperature plays an important role in microbial life through its effects on the three-dimensional configurations of biological molecules. Recall that to function properly, proteins require a specific three-dimensional shape that is determined in part by temperature-sensitive hydrogen bonds, which are more likely to form at lower temperatures and more likely to break at higher temperatures. Proteins denature and lose their shape and function when hydrogen bonds break. Additionally, lipids, such as those that are components of the membranes of cells and organelles, are temperature sensitive. If the temperature is too low, membranes become rigid and fragile; if the temperature is too high, lipids can become too fluid, and the membrane cannot contain the cell or organelle. Because temperature plays an important role in the three-dimensional structure of many types of biological molecules, different temperatures have different effects on the survival and growth of microbes (FIGURE 6.3). The lowest temperature at which an organism is able to conduct metabolism is called the minimum growth temperature. Note, however, that many microbes, particularly bacteria, survive (although they do not thrive) at temperatures far below their minimum growth temperature despite the fact that cell membranes are less fluid and transport processes are too slow to support metabolic activity. The highest temperature at which an organism continues to metabolize is called its maximum growth tempera­ ture; when the temperature exceeds this value, the organism’s proteins are permanently denatured, and it dies. The temperature at which an organism’s metabolic activities produce the highest growth rate is its optimum growth temperature. Each organism thus survives over a temperature range within which its growth and metabolism are supported. Based on their preferred temperature ranges—the temperatures within which their metabolic activity and growth are best supported—microbes can be categorized into five overlapping groups (FIGURE 6.4). Psychrophiles6 (sī´krō-fīls) grow best at temperatures below about 15°C and can even continue to grow at temperatures below 0°C. Most die at temperatures above 20°C. In nature, psychrophilic algae, fungi, archaea, and bacteria live in snowfields, ice, and cold water (FIGURE 6.5). They do not cause disease in humans because they cannot thrive at body temperature, though some do spoil food in refrigerators. Psychrotolerant organisms (also called psychrotrophs) tolerate but don’t grow best in the cold. They may also infect warmblooded animals, including humans; for example, Listeria mono­ cytogenes is a pathogen that, can grow in refrigerated foods. Mesophiles7 (mez´ō-fīls) are organisms that grow best in temperatures ranging from 20°C to about 40°C, though they can survive at higher and lower temperatures. Because normal body temperature is approximately 37°C, most human pathogens are mesophiles. Thermoduric8 organisms are mesophiles that can survive brief periods at higher temperat ures. Greek mesos, meaning “middle,” and philos, meaning “love.” Greek therme, meaning “hot.” The organisms are so named because of their ability to endure or tolerate heat. 7From 6From Greek psuchros, meaning “cold,” and philos, meaning “love.” M06_BAUM7206_05_SE_C06.indd 165 8From 11/23/16 3:01 AM r 6    Microbial Nutrition and Growth hapte       C Growth rate Thermophiles Mesophiles Hyperthermophiles Psychrotolerants Psychrophiles Geogemma barossii (jē´ō-jem-ă ba-rōs´ē-ē). Geogemma grows and reproduces near submarine hot springs at temperatures between 85°C and 121°C and can survive for at least two hours at 130°C! Thermophiles and hyperthermophiles stabilize their proteins with extra hydrogen and covalent bonds between amino acids. Heat-stable enzymes are useful in industrial, engineering, and research applications. Heat-loving organisms do not cause disease because they “freeze” at body temperature. Beneficial Microbes: A Nuclear Waste-Eating Microbe? highlights an unusual thermophile that can also withstand radiation. pH –10 0 10 20 30 40 50 60 70 Temperature (°C) 80 90 100 110 120 ▲ FIGURE 6.4 Five categories of microbes based on temperature ranges for growth. Categorize the bacterium Thermus aquaticus, which has an optimum growth temperature of 70°C. Figure 6.4 Vibrio marinus is a thermophile. ­ Inadequate heating during pasteurization or canning can result in food spoilage by thermoduric mesophiles. Thermophiles9 (ther´mō-fīls) grow at temperatures above 45°C in habitats such as compost piles and hot springs. Some bacteria and archaea, called hyperthermophiles, grow best at temperatures above 80°C; others live in water heated above its boiling point (100°C)10. The current record holder is an archaeon, pH is a measure of the concentration of hydrogen ions in a solution; that is, it is a measure of the acidity or alkalinity of a substance. A pH below 7.0 is acidic; the lower the pH value, the more acidic a substance is. Alkaline (basic) pH values are higher than 7.0. Organisms are sensitive to changes in acidity because hydrogen ions interfere with hydrogen bonding within proteins and nucleic acids; as a result, organisms have ranges of acidity that they prefer and can tolerate. Most bacteria and protozoa, including most pathogens, are neutrophiles (nū´trō-fīls), because they grow best in a narrow range around a neutral pH—that is, between pH 6.5 and pH 7.5, which is also the pH range of most tissues and organs in the human body. By contrast, other bacteria and many fungi are acidophiles (ā-sīd´ō-phīls), organisms that grow best in acidic habitats. One example of acidophilic microbes is the chemoautotrophic prokaryotes that live in mines and in water that runs through mine tailings (waste rock), habitats that have pHs as low as 0.0. These prokaryotes oxidize sulfur to sulfuric acid, further lowering the pH of their environment. Whereas obli­ gate acidophiles require an acidic environment and die if the pH approaches 7.0, acid­tolerant microbes merely survive in acid without preferring it. Many organisms produce acidic waste products that accumulate in their environment until eventually they inhibit further growth. For example, many cheeses are acidic because of lactic acid produced by fermenting bacteria and fungi. The low pH of these cheeses then acts as a preservative by preventing any further microbial growth. Other acidic foods, such as sauerkraut and dill pickles, are also kept from spoiling because most organisms cannot tolerate the foods’ low pH. The normal acidity of certain regions of the body also inhibits microbial growth and retards many kinds of infection. At one site, the vaginas of adult women, acidity results from the fermentation of carbohydrates by normal resident bacteria. If the growth of these normal residents is disrupted—for instance, by antibiotic therapy—the resulting higher pH may allow yeasts to grow and lead to a yeast infection. Another site, the stomach, is inhospitable to most microbes because of the normal production of stomach acid. However, the acidtolerant bacterium Helicobacter pylori neutralizes stomach acid by secreting bicarbonate and urease, an enzyme that converts urea to alkaline ammonia. The growth of Helicobacter is the cause of most gastric ulcers. Alkaline conditions also inhibit the growth of most microbes, but alkalinophiles live in alkaline soils and water (a) (b) LM 80 µm ▲ FIGURE 6.5 An example of a psychrophile. (a) The alga Chlamydomonas nivalis colors this summertime snowbank on Cuverville Island, Antarctica. (b) Microscopic view of the red-pigmented spores of C. nivalis. Greek therme, meaning “hot,” and philos, meaning “love.” can remain a liquid above 100°C if it has a high salt content or is under pressure, such as occurs in geysers or deep ocean troughs. 9From 10Water M06_BAUM7206_05_SE_C06.indd 166 11/23/16 3:01 AM Osmotic Pressure Osmosis is the diffusion of water across a membrane and is driven by unequal solute concentrations on the two sides of such a membrane. The osmotic pressure of a solution is the pressure exerted on a membrane by a solution containing solutes (dissolved material) that cannot freely cross the membrane. Osmotic pressure is related to the concentration of dissolved molecules and ions in a solution. Solutions with greater concentrations of such solutes are hypertonic relative to those with a lower solute concentration, which are hypotonic. Osmotic pressure can have dire effects on cells. For example, a cell placed in freshwater (a hypotonic solution relative to the cell’s cytoplasm) gains water from its environment and swells to the limit of its cell wall. Cells that lack a cell wall—animal cells and some bacterial, fungal, and protozoan cells—will swell until they burst in hypotonic solutions. By contrast, a cell placed in seawater, which is a solution containing about 3.5% solutes and thus hypertonic to most cells, loses water into the surrounding saltwater. Such a cell can die from crenation, or shriveling of its cytoplasm. Osmotic pressure accounts for the preserving action of salt in jerky and salted fish and of sugar in jellies, preserves, and honey. In those foods, the salt and sugar are solutes that draw water out of any microbial cells that are present, preventing growth and reproduction. Osmotic pressure restricts organisms to certain environments. Some microbes, called obligate halophiles,11 are adapted to growth under high osmotic pressure such as exists in the Great Salt Lake and smaller salt ponds. They may grow in up to 30% salt and will burst if placed in freshwater. Other microbes are facultative halophiles; that is, although they do not require high salt concentrations, they can tolerate them. One potential bacterial pathogen, Staphylococcus aureus (staf´i-lō-kok´ŭs o´rē-ŭs), can tolerate up to 20% salt, which allows it to colonize the surface of the skin—an environment that is too salty for most microbes. S. aureus causes a number of different skin and mucous membrane diseases ranging from pimples, sties, and M06_BAUM7206_05_SE_C06.indd 167 ­ ­ ­ ­ ­ ­ Microorganisms require water; they must be in a moist environment if they are to be metabolically active. Water is needed to dissolve enzymes and nutrients; also, it is an important reactant in many metabolic reactions. Even though most cells die without water, some microorganisms—for example, the bacterium Mycobacterium tuberculosis (mī´kō-bak-tēr´ē-ŭm too-berkyū-lō´sis)—have cell walls that retain water, allowing them to survive for months under dry conditions. Additionally, some microbes have endospores and cysts that cease most metabolic activity in a dry environment and might survive for years; these cells are in essence in a state of suspended animation because they neither grow nor reproduce in their dry condition. We now consider the physical effects of water on microbes by examining two topics: osmotic pressure and hydrostatic pressure. Hydrostatic Pressure Water exerts pressure in proportion to its depth. For every additional 10 m of depth, water pressure increases 1 atmosphere (atm). Therefore, the pressure at 100 m below the surface is 10 atm—10 times greater than at the surface. Obviously, the pressure in deep ocean basins and trenches, which are thousands of meters below the surface, is tremendous. Organisms that live under such extreme pressure are called barophiles12 (bar´ō-fīls). Their membranes and enzymes do not merely tolerate pressure; they also depend on pressure to maintain their threedimensional, functional shapes. Thus, barophiles brought to the surface die quickly because their proteins denature. Obviously, barophiles cannot cause diseases in humans, plants, or animals that do not live at great depths. Associations and Biofilms LE A R N I N G O UTC O M E 6.7 Describe how quorum sensing can lead to formation of a biofilm. Organisms in solitary culture in a laboratory are living very differently from organisms in nature, which live in association with other individuals of their own and different species. The relationships between organisms can be viewed as falling along a continuum stretching from causing harm to providing benefits. Relationships in which a microbe harms or even kills another organism are considered antagonistic relationships. Viruses are especially clear examples of antagonistic microbes; they require cells in which to replicate themselves and almost always kill their cellular hosts. Beneficial relationships take at least two forms: synergistic relationships and symbiotic relationships. In synergistic relation­ ships, the individual members of an association cooperate such that each receives benefits that exceed those that would result if each lived by itself, even though each member could live separately. In symbiotic relationships, organisms live in such close nutritional or physical contact that they become interdependent; the members rarely (if ever) live outside the relationship. (Chapter 14 discusses symbiotic relationships in greater detail, particularly as they relate to the production of disease.) Biofilms are examples of complex, synergistic relationships among numerous microorganisms, often different species, attached to surfaces such as teeth (dental plaque), rocks in streams, shower curtains (“soap scum” is really a biofilm), implanted medical devices (e.g., catheters), and mucous membranes of the digestive system. Biofilms are the primary residence of microorganisms in nature. For example, one study showed that more than 10 billion bacteria per square centimeter form the slippery biofilm on rocks in a streambed. The Centers ­ Physical Effects of Water boils to life-threatening scalded skin syndrome and toxic shock syndrome. (Chapter 19 covers these diseases more fully.) ­ up to pH 11.5. For example, Vibrio cholerae (vib´rē-ō kol´er-ī), the causative agent of cholera, grows best outside the body in water at pH 9.0.       R Growth equirements 11From 12From Greek halos, meaning “salt,” and philos, meaning “love.” Greek baros, meaning “weight,” and philos, meaning “love.” 11/23/16 3:01 AM r 6    Microbial Nutrition and Growth hapte       C R BENEFICIAL MIC OBES A Nuclear Waste-Eating Microbe? for Disease Control and Prevention estimates that biofilms cause up to 70% of bacterial diseases in industrialized countries, including kidney infections, tooth and gum decay, infections that occur with cystic fibrosis, and healthcare-associated infections, such as might occur with implantation of medical devices. Cells within biofilms communicate and coordinate with one another, acting similar to a tissue in a multicellular organism. 1 Free-swimming microbes are vulnerable to environmental stresses. R have discovered organisms that can use an energy source other than light or chemicals. Since radiation is available in space, perhaps future astronauts will be able to grow fungi as a continual food supply for long space voyages. The bacterium Kineococcus radiotolerans is notable among radiation-tolerant extremophiles because it can also break down herbicides, chlorinated compounds, and other toxic substances. esearchers at the U.S. Department of Energy would like to shape K. radiotolerans into a biological tool that can clean up environments contaminated with radioactive wastes. Using microbes to break down toxic chemicals in the environment, a process known as bioremediation, is often cheaper, quicker, and more effective than conventional methods. K. radiotolerans could potentially slash the cost of nuclear cleanup. ­ ­ 10 µm ­ Gamma rays emitted by radioactive decay are usually deadly. However, some fungi and bacteria can survive Kineococcus being bombarded with radiation at levels higher than radiotolerans. thousands of times the level that would kill a person. Currently, researchers are studying these organisms in the hope that we can use their biochemistry to develop ways to protect our cells from radiation and use the microbes to clean up nuclear wastes. Microbes that can survive in extremely hostile environments are called extremophiles. Scientists have discovered amazing species of fungi in the genera Wangiella and Cladosporium inhabiting the walls of the damaged nuclear reactor in Chernobyl, Ukraine, which is considered the site of the worst nuclear accident in history. The fungi absorb radiation with the help of a pigment called melanin, the same pigment that colors human skin and hair. Incredibly, the fungi harness radioactive energy so as to grow faster—the first time we LM Biofilm development can involve at least six steps (FIGURE 6.6). Free-living cells 1 settle on a surface and attach 2 . They develop a gooey, extracellular polymeric substance (EPS) or matrix, often composed of DNA, proteins, and polysaccharides 3 . This slimy matrix adheres cells to one another, sticks the cells to their substrate, forms microenvironments within the biofilm, sequesters nutrients, forms water channels between groups of cells, and may protect individuals in a Water flow Chemical structure of one type of quorum-sensing molecule O Bacteria O Escaping microbes O N H Water channel O Matrix 2 Some microbes land on a surface, such as a tooth, and attach. 3 The cells begin producing an extracellular matrix and secrete quorum-sensing molecules. 4 Quorum sensing triggers cells to change their biochemistry and shape. 5 New cells arrive, possibly including new species, and water channels form in the biofilm. 6 Some microbes escape from the biofilm to resume a free-living existence and, perhaps, to form a new biofilm on another surface. ▲ FIGURE 6.6 Biofilm development. Quorum sensing allows microbes to change their behavior in the presence of other microbes to form microbial ­ communities called biofilms. M06_BAUM7206_05_SE_C06.indd 168 11/23/16 3:01 AM       Culturing Microorganisms Low cell density below threshold High cell density above threshold different roles in different areas of a biofilm, much as cells and tissues of multicellular organisms have different functions in different parts of the body. Some individual cells or groups of cells in streamers may leave the biofilm 6 . Given that many microorganisms become more harmful when they are part of a biofilm, scientists are seeking ways to prevent biofilms from forming in the first place. Researchers have learned that drugs that block microbial cell receptors, thereby disrupting communication among cells, can inhibit biofilm formation and thus prevent disease. Such receptorblocking drugs have successfully blocked biofilm formation and prevented disease in mice; they are being considered for use in humans. Scientists are investigating another potential new tool against biofilm infections in the lungs—enzymes that degrade the EPS material to enhance penetration of antimicrobial drugs. Another possible approach to preventing biofilm formation involves artificially amplifying quorum sensing while bacteria are still relatively few in number. Some pathogens hide within capsules and in blood clots, and only after they have multiplied significantly and formed a biofilm do they emerge and become “visible” to the immune system. With amplified quorum sensing, bacteria might “reveal” themselves sooner to the immune system, which may then eliminate them before they can cause disease. Dental plaque is a common biofilm that can lead to dental caries—cavities. Plaque formation usually begins with colonization of the teeth by Streptococcus mutans (strep-tō-kok´ŭs mū´tanz). This bacterium breaks down carbohydrates, particularly the disaccharide sucrose (table sugar), to provide itself with nutrition and a glycocalyx. One of its enzymes catabolizes sucrose into its component monosaccharides—glucose and fructose—which the cells use as energy sources. A second enzyme polymerizes glucose into long, insoluble polysaccharide strands called glucan molecules, which form a sticky matrix around the bacteria. Glucan adheres S. mutans to the tooth, provides a home for other species of oral bacteria, and traps food particles. A biofilm has formed. Bacteria in the biofilm digest nutrients and release acid, which is held against the teeth by the biofilm’s matrix. The acid gradually eats away the minerals that compose the tooth, resulting in dental caries and eventually total loss of the teeth. The Clinical Case Study: Cavities Gone Wild deals with an especially severe case of a biofilm running amok in a small boy’s mouth. ­ ­ biofilm from environmental stresses, including ultraviolet radiation, antimicrobial drugs, and changes in pH, temperature, and humidity. Biofilms form as a result of a process called quorum sensing, in which microorganisms respond to the density of nearby microorganisms (FIGURE 6.7). The microbes secrete quorum­sensing molecules that act to communicate number and types of cells among members of the biofilm 4 . Many cells possess receptors for these signal molecules. When the density of microorganisms increases, the concentration of quorum-sensing molecules also increases such that more and more receptors bind them. Once the binding exceeds a certain threshold amount, just like the quorum required for Congress to vote, the cells activate previously suppressed genes. The microbes develop new characteristics, such as the production of enzymes, changes in cell shape, the formation of mating types, and the ability to form and maintain biofilms. Some researchers estimate quorum sensing may regulate 10% of the genes in a cell. An EPS not only attaches a biofilm and positions the cells but also may allow members of the biofilm to concentrate and conserve digestive enzymes, directing them against the underlying structure rather than having them diffuse away in the surrounding medium. New microbes can arrive, and the synergistic relationships allowed in a biofilm continue to organize the biofilm community, so that individual members display metabolic and structural traits different from those expressed by the same cells living individually 5 . Members assume TELL ME WHY Why should cardiac nurses and respiratory therapists care about biofilms? Quorum signal Signal receptor ▲ FIGURE 6.7 Quorum sensing. To measure their population density, bacteria synthesize and secrete a small signaling molecule. A protein receptor can detect this signal. At low density, the signal diffuses away from cells. At high density, the signal reaches the receptor. M06_BAUM7206_05_SE_C06.indd 169 Culturing Microorganisms One of Koch’s postulates for demonstrating that a certain agent causes a specific disease requires that microorganisms be isolated and cultivated (Chapter 1). Medical laboratory personnel must also grow pathogens as a step in the diagnosis of 11/23/16 3:01 AM r 6    Microbial Nutrition and Growth hapte CLINICAL CASE STUDY Cavities Gone Wild Five-year-old Daniel appears to be shy. He always looks at the floor, has no friends, never plays with the other children, and will rarely speak to adults. When he does speak, it is difficult to understand his broken enunciation. His skinny frame and the dark circles under his eyes make him appear malnourished. Daniel cries frequently and misses many days of school. A speech specialist at school finds that only two of Daniel’s teeth are healthy; all the others have rotted away to the gum line. The little guy is in constant pain, and it hurts to chew. A doctor later determines that many diseases. To cultivate or culture microorganisms, a sample called an inoculum (plural: inocula) is introduced into a collection of nutrients called a medium. Microorganisms that grow from an inoculum are also called a culture; thus, culture can refer to the act of cultivating microorganisms or to the microorganisms that are cultivated. Cultures can be grown in liquid media called broths or on the surface of solid media. Cultures that are visible on the surface of solid media are called colonies. Bacterial and fungal colonies often have distinctive characteristics—including color, size, shape, elevation, texture, and appearance of the colony’s margin (edge)—that taken together help identify the microbial species that formed the colony (FIGURE 6.8). Microbiologists obtain inocula from a variety of sources. Environmental specimens are taken from such sources as ponds, streams, soil, and air. Clinical specimens are taken from patients and handled in ways that facilitate the examination of microorganisms or testing for their presence. Another source of inocula is a culture originally grown from an environmental or clinical specimen and maintained in storage in a laboratory. Next, we briefly examine clinical sampling. Clinical Sampling LE A R N I N G O U TC O M E 6.8 Describe methods for collecting clinical specimens from the skin and from the respiratory, reproductive, and urinary tracts. Diagnosing and treating disease often depend on isolating and correctly identifying pathogens. Health care professionals must properly obtain samples from their patients and must then transport them quickly and correctly to a microbiology M06_BAUM7206_05_SE_C06.indd 170 bacteria from the cavities in his mouth have entered his bloodstream and infected his heart, causing an irregular heartbeat and poor blood circulation. 1. How does knowledge of biofilms help explain the bulk of Daniel’s problems? 2. What can Daniel, his parents, and health care professionals do to cure his diseases? 3. What is the scientific name of the nutrient that Daniel’s parents should eliminate from his diet to help prevent a repeat of this condition? 4. A recent study has shown that brushing does more than clean plaque from the teeth; it also disrupts associations between oral bacteria. How does this simple act help prevent the formation of biofilms? laboratory for culture and identification. They must take care to prevent contaminating samples with microorganisms from the environment or other regions of the patient’s body, and they must prevent infecting themselves with pathogens while sampling. In this regard, the Centers for Disease Control and Prevention has established a set of guidelines, called standard precautions, to protect health care professionals from contamination by pathogens. In clinical microbiology, a clinical specimen is a sample of human material, such as feces, saliva, cerebrospinal fluid, or blood, that is examined or tested for the presence of microorganisms. As summarized in (TABLE 6.2), health care professionals collect clinical specimens using a variety of techniques and equipment. Specimens must be properly labeled and promptly transported to a microbiological laboratory to avoid death of the pathogens and to minimize the growth of microbes normally found at the collection site. Clinical specimens are often transported in special transport media that are chemically formulated to maintain the relative abundance of different microbial species or to maintain an anaerobic environment. ­       C Obtaining Pure Cultures LE A R N I N G O UTC O M E 6.9 Describe the two most common methods by which microorganisms can be isolated for culture. Clinical specimens are collected in order to identify a suspected pathogen, but they also contain normal microbiota, which are microorganisms associated with a certain area of the body without causing diseases (see Table 14.2 on p. 414). As a result, the suspected pathogen in a specimen must be isolated from the normal microbiota in culture. Scientists use several techniques 11/23/16 3:01 AM       Culturing Microorganisms Shape Circular Rhizoid Irregular Filamentous Spindle TABLE 6.2 Clinical Specimens and the Methods Used to Collect Them Type or Location of Specimen Margin Elevation Size Entire Flat Undulate Lobate Curled Filiform Small Moderate Texture Smooth or rough Appearance Glistening (shiny) or dull Large Pigmentation Nonpigmented (e.g., cream, tan, white) Pigmented (e.g., purple, red, yellow) Optical property Skin, accessible membrane (including eye, outer ear, nose, throat, vagina, cervix, urethra), or open wounds Sterile swab brushed across the surface; care should be taken not to contact neighboring tissues Blood Needle aspiration from vein; anticoagulants are included in the specimen transfer tube Cerebrospinal fluid Needle aspiration from subarachnoid space of spinal column Stomach Intubation, which involves inserting a tube into the stomach, often via a nostril Urine In aseptic collection, a catheter is inserted into the bladder through the urethra; in the “clean catch” method, initial urination washes the urethra, and the specimen is midstream urine Lungs Collection of sputum either dislodged by coughing or acquired via a catheter Diseased tissue Surgical removal (biopsy) Raised Convex Pulvinate Umbonate Crateriform Punctiform Opaque, translucent, transparent Collection Method (a) to isolate organisms in pure cultures, that is, cultures composed of cells arising from a single progenitor. The word axenic13 (ā-zen´ik) is also used to refer to a pure culture. The progenitor from which a particular pure culture is derived may be either a single cell or a group of related cells; therefore, the progenitor is termed a colony-forming unit (CFU). In all microbiological procedures, care must be taken to reduce the chance of contamination, which occurs, for example, when instruments or air currents carry foreign microbes into culture vessels. All media, vessels, and instruments must be sterile—that is, free of any microbial contaminants. Sterilization and aseptic techniques are designed to limit contamination (discussed in Chapter 9). Now we examine two common isolation techniques: streak plates and pour plates. We consider another method, serial dilution, in a later section. (b) LM 20 mm ▲ FIGURE 6.8 Characteristics of bacterial colonies. (a) Shape, margin, elevation (side view), size, texture, appearance, pigmentation (color), and optical properties are described by a variety of terms. (b) Serratia marcescens growing on an agar surface. These colonies are circular, entire, convex, large, smooth, shiny, red, and opaque. Streak Plates The most commonly used isolation technique in microbiological laboratories is the streak-plate method. In this technique, a sterile inoculating loop (or sometimes a needle) is used to spread an inoculum across the surface of a solid medium in Petri14 dishes, which are clear, flat culture dishes with loosefitting lids. The loop is used to lightly streak a set pattern that gradually dilutes the sample to a point that CFUs are isolated from one another (FIGU RE 6.9a) . After an appropriate period of time called incubation, colonies develop from each isolate (FIGURE 6.9b) . The various types of organisms ­ Colony 13From Greek a, meaning “no,” and xenos, meaning “stranger.” for Richard Petri, Robert Koch’s assistant, who invented them in 1887. 14Named M06_BAUM7206_05_SE_C06.indd 171 11/23/16 3:01 AM hapte       C r 6    Microbial Nutrition and Growth 4 3 1 2 (a) (b) ▲ FIGURE 6.9 The streak-plate method of isolation. (a) An inoculum is spread across the surface of an agar plate in a sequential pattern of streaks (as indicated by the numbers and arrows). The loop is sterilized between streaks. In streaks 2, 3, and 4, bacteria are picked up from a previous streak; thus the number of cells is diluted each time. (b) A streak plate showing colonies of Escherichia coli on blood agar. present are distinguished from one another by differences in colonial characteristics (Figure 6.8). Samples from each variety can then be inoculated in new media to establish axenic cultures. Pour Plates ­ In the pour-plate technique, CFUs are separated from one another using a series of dilutions. There are various ways to perform pour-plate isolations. In one method, an initial 1-milliliter sample is mixed into 9 ml of sterile medium in a test tube. After mixing, a new sample from this mixture is then used to inoculate a second tube of liquid medium. The process is repeated to establish a series of dilutions (FIGURE 6.10a). Samples from the more diluted media are mixed in Petri dishes with sterile, warm medium containing agar—a gelling agent derived from the cell walls of red algae. After the agar cools and solidifies, the filled dishes are called Petri plates. Isolated colonies—colonies that are separate and distinct from all others—form in the plates from CFUs that have been separated via the dilution series (FIGURE 6.10b). One difference between this method and the streak-plate technique is that colonies form both at and below the surface of the medium. As before, pure cultures can be established from distinct colonies. Isolation techniques work well only if a relatively large number of CFUs of the organism of interest are present in the initial sample and if the medium supports the growth of that microbe. As discussed later, special media and enriching techniques can be used to increase the likelihood of success. Other Isolation Techniques Streak plates and pour plates are used primarily to establish pure cultures of bacteria, but they can also be used for some fungi, particularly yeasts. Protozoa and motile unicellular algae are not usually cultured on solid media because they do M06_BAUM7206_05_SE_C06.indd 172 not remain in one location to form colonies. Instead, they are isolated through a series of dilutions but remain in broth culture media. In cases of fairly large microorganisms, such as the protists Euglena (yū-glēn´ă) and Amoeba (am-ē´bă), hollow tubes with small diameters called micropipettes can be used to pick up single cells that are then used to establish axenic cultures. Culture Media LE A R N I N G O UTC O M E S 6.10 Describe six types of general culture media available for bacterial culture. 6.11 Describe enrichment culture as a means of enhancing the growth of less abundant microbes. Cult uring microorganisms can be an exacting science. Although some microbes, such as E. coli, are not particular about their nutritional needs and can be grown in a variety of media, some other bacteria, such as Neisseria gonorrhoeae (nī-se´rē-ă go-nor-rē´ī) and Haemophilus influenzae (hē-mof´i-lŭs in-flu-en´zī), and many archaea require specific nutrients, including specific growth factors. The majority of microorganisms have never been successfully grown in any culture medium in part because scientists have concentrated their efforts on culturing commercially important species and pathogens. However, some pathogens, such as the syphilis bacterium Treponema pallidum (trep-ō-nē´mă pal´li-dŭm), have never been cultured in any laboratory medium despite over a century of effort. A variety of media are available for microbiological cultures, and more are developed each year, to support the needs of food, water, industrial, and clinical microbiologists. Most media are available from commercial sources and come in powdered forms that require only the addition of water to 11/23/16 3:01 AM       Culturing Microorganisms Sequential inoculations Initial sample 1 ml 1 ml 9-ml broth 9-ml broth 1 ml 9-ml broth 1 ml to each Petri dish, add 9-ml warm agar, swirl gently to mix Colonies (b) Fewer colonies (a) ▲ FIGURE 6.10 The pour-plate method of isolation. (a) After an initial sample is diluted through a series of transfers, the final dilutions are mixed with warm agar in Petri plates. Individual CFUs form colonies in and on the agar. (b) A portion of a plate showing the results of isolation. make broths. A common medium, for example, is nutrient broth, which contains powdered beef extract and peptones (short chains of amino acids produced by enzymatic digestion of protein) dissolved in water. For some purposes broths are adequate, but if solid media are needed, dissolving about 1.5% agar into hot broth, pouring the liquid mixture into an appropriate vessel, and allowing it to cool provides a solid surface to support colonial growth. Media made solid by the addition of agar to a broth have the word agar in their names; thus, nutrient agar is nutrient broth to which 1.5% agar has been added. Agar, a complex polysaccharide derived from the cell walls of certain red algae, is a useful compound in microbiology for several reasons: into test tubes that are then placed at an angle and left to cool until the agar solidifies, the result is slant tubes, or slants (FIGURE 6.11). The slanted surface provides a larger surface area for aerobic microbial growth while the butt of the tube remains almost anaerobic. Next we examine six types of general culture media: defined media, complex media, selective media (including enrichment culture), differential media, anaerobic media, and transport media. It is important to note that these types of media are not mutually exclusive categories; that is, in some cases a given medium can belong to more than one category. • Most microbes cannot digest agar; therefore, agar media remain solid even when bacteria and fungi are growing on them. • Powdered agar dissolves in water at 100°C, a temperature at which most nutrients remain undamaged. • Agar solidifies at temperatures below 40°C, so temperaturesensitive, sterile nutrients such as vitamins and blood can be added without detriment to cooling agar before it solidifies. Further, cooling liquid agar can be poured over most bacterial cells without harming them. The latter technique plays a role in the pour-plate isolation technique. • Solid agar does not melt below 100°C; thus, it can even be used to culture some hyperthermophiles. Still-warm liquid agar media can be poured into Petri dishes to make Petri plates. When warm agar media are poured M06_BAUM7206_05_SE_C06.indd 173 Slant Butt ▲ FIGURE 6.11 Slant tubes containing solid media. In this case, citrate agar is the medium. 11/23/16 3:01 AM hapte       C r 6    Microbial Nutrition and Growth Defined Media If microorganisms are to grow and multiply in culture, the medium must provide essential nutrients (including an appropriate energy source for chemotrophs), water, an appropriate oxygen level, and the required physical conditions (such as the correct pH and suitable osmotic pressure and temperature). A defined medium (also called a synthetic medium) is one in which the exact chemical composition is known. (TABLE 6.3) gives a recipe for one example. Relatively simple defined media containing inorganic salts and a source of CO2 (such as sodium bicarbonate) are available for autotrophs, particularly cyanobacteria and algae. Defined media for chemoheterotrophs can contain necessary organic molecules, such as glucose, amino acids, or vitamins, which supply carbon and energy or are vital growth factors. Organisms that require a relatively large number of growth factors are termed fastidious. Such organisms may be used as living assays for the presence of growth factors. For example, a scientist wanting to know whether a particular environment contains vitamin B12 could inoculate a sample from the environment with Euglena granulata (gran-yū-lă´tă), an organism that requires the vitamin. If the microbe grows, the vitamin is present in the sample and therefore in the environment. The amount of growth provides an estimate of the amount of vitamin present, scant growth indicating a small amount. Complex Media ­ For most clinical cultures, defined media are unnecessarily troublesome to prepare. Most chemoheterotrophs, including pathogens, are routinely grown on complex media that contain nutrients released by the partial chemical breakdown of yeast, beef, soy, or proteins, such as casein from milk. The exact chemical composition of a complex medium is unknown because partial degradation releases many different chemicals in a variety of concentrations each time it takes place. Complex media have advantages over defined media. Because a complex medium contains a variety of nutrients, including growth factors, it can support a wider variety of different microorganisms. Complex media are also used to culture organisms whose exact nutritional needs are unknown. Nutrient broth, Trypticase soy agar, and MacConkey agar are some common complex media. Blood is often added to complex media to provide additional growth factors, such as NADH and heme. Such a fortified medium is said to be enriched and can support the growth of many fastidious microorganisms. Selective Media Selective media typically contain substances that either favor the growth of particular microorganisms or inhibit the growth of unwanted ones. Eosin, methylene blue, and crystal violet dyes as well as bile salts are included in media to inhibit the growth of Gram-positive bacteria without adversely affecting most Gram-negatives. A high concentration of NaCl (table salt) in a medium selects for halophiles and for salt-tolerant bacteria, such as the pathogen Staphylococcus aureus. Sabouraud dextrose agar has a slightly low pH, which inhibits the growth of bacteria and thereby is selective for fungi (FIGURE 6.12). A medium can also become a selective medium when a single crucial nutrient is left out of it. For example, leaving glucose out of Trypticase soy agar makes the resulting medium selective for organisms that can meet all their carbon requirements by catabolizing amino acids. Enrichment Culture Bacteria that are present in small numbers may be overlooked on a streak plate or overwhelmed by fastergrowing, more abundant strains. This is especially true of organisms in soil and fecal samples that contain a wide variety of microbial species. To isolate potentially important microbes that might otherwise be overlooked, microbiologists enhance the growth of less abundant organisms by a variety of techniques. In the late 1800s, the Dutch microbiologist Martinus Beijerinck (1851–1931) introduced the most common of these methods, called simply enrichment culture. Enrichment cultures use a selective medium and are designed to increase very small numbers of a chosen microbe to observable levels. For example, suppose a microbiologist specializing in environmental cleanup wanted to isolate an organism capable of digesting Bacterial colonies Fungal colonies TABLE 6.3 Ingredients of a Representative Defined (Synthetic) Medium for Culturing E. coli Glucose Na2HPO4 1.0 g 16.4 g KH2PO4 1.5 g (NH4)3PO4 2.0 g MgSO4 # 7H2O CaCl 2 FeSO4 # 7H2O Distilled or deionized water M06_BAUM7206_05_SE_C06.indd 174 0.2 g 0.01 g 0.005 g Enough to bring volume to 1 L pH 7.3 pH 5.6 ▲ FIGURE 6.12 An example of the use of a selective medium. After the medium is inoculated with a diluted soil sample, acidic pH in Sabouraud dextrose agar (right) makes the medium selective for fungi by inhibiting the growth of bacteria. At left, for comparison, is a nutrient agar plate inoculated with an identical sample. 11/23/16 3:01 AM       Culturing Microorganisms ­ ­ Differential Media Differential media are formulated such that either the presence of visible changes in the medium or differences in the appearance of colonies help microbiologists differentiate among the kinds of bacteria growing on the medium. Such media take advantage of the fact that different bacteria utilize the ingredients of any given medium in different ways. One example of the use of a differential medium involves the differences in organisms’ utilization of red blood cells in blood agar (FIGURE 6.13). Streptococcus pneumoniae (nū-mō´nē-ī) partially digests (lyses) red blood cells, producing around its colonies a greenish brown discoloration denoted alpha­hemolysis. By contrast, Streptococcus pyogenes (pī-oj´en-ēz) completely digests red blood cells, producing around its colonies clear zones termed beta­hemolysis. Enterococcus faecalis (en´ter-ō-kok´ŭ fē-kă´lis) does not digest red blood cells, so the agar appears little changed, a reaction called gamma­hemolysis even though no lysis occurs. In some differential media, such as carbohydrate utilization broth tubes, a pH-sensitive dye changes color when bacteria metabolizing sugars produce acid waste products (FIGURE 6.14). Some common differential complex media are described in (TABLE 6.4 on p. 176). Many media are both selective and differential; that is, they enhance the growth of certain species that can then be distinguished from other species by variations in appearance. For example, bile salts and crystal violet in MacConkey agar inhibit the growth of Gram-positive bacteria and also differentiate between lactose-fermenting and non-lactose-fermenting Gram-negative bacteria (FIGURE 6.15). ­ crude oil on an oil-soaked beach. Even though a sample of the beach sand might contain a few such organisms, it would also likely contain many millions of unwanted common bacteria. To isolate oil-utilizing microbes, the scientist would inoculate a sample of the sand into a tube of selective medium containing oil as the sole carbon source and then incubate it. Then a small amount of the culture would be transferred into a new tube of the same medium to be incubated again. After a series of such enrichment transfers, any remaining bacteria will be oil-utilizing organisms. Different species could be isolated by either streak-plate or pour-plate methods. Cold enrichment is another technique used to enrich a culture with cold-tolerant species, such as Vibrio cholerae, the bacterium that causes cholera. Stool specimens or water samples suspected of containing the bacterium are incubated in a refrigerator. Cold enrichment works because Vibrio cells are much less sensitive to cold than are more common fecal bacteria, such as E. coli; therefore, Vibrio continues to grow in the cold while the other species are inhibited. The result of cold enrichment is a culture with a greater percentage of Vibrio cells than the original sample; the Vibrio cells can then be isolated by other methods. Anaerobic Media Obligate anaerobes require special culture conditions to protect their cells from free oxygen. Anaerobes can be introduced with a straight inoculating wire into the anoxic (oxygen-free) depths of solid media to form a stab culture, but special media called Alpha-hemolysis Beta-hemolysis Durham tube (inverted tube to trap gas) No hemolysis (gamma-hemolysis) No fermentation Acid fermentation with gas ­ ▲ FIGURE 6.14 The use of carbohydrate utilization tubes as differential media. Each tube M06_BAUM7206_05_SE_C06.indd 175 ideo ducing a clear zone termed beta-hemolysis. Streptococcus pneumoniae (middle) partially uses red blood cells, producing a discoloration termed alpha-hemolysis. Enterococcus faecalis (right) does not use red blood cells; the lack of any change in the medium around colonies is termed gamma-hemolysis, even though no red cells are hemolyzed. contains a single kind of simple carbohydrate (a sugar) as a carbon source and the dye phenol red as a pH indicator. Alcaligenes faecalis V in the tube on the left did not ferment this carbohydrate; because T r no acid was produced, the medium did not turn yellow. Escherichia coli in the tube on the right fermented the sugar, producing acid and lowering the pH enough to cause the phenol red to turn yellow. This bacterium also produced gas, which is visible as a small bubble in the Durham tube. uto ▲ FIGURE 6.13 The use of blood agar as a differential medium. Streptococcus pyogenes (left) completely uses red blood cells, pro- 11/23/16 3:01 AM r 6    Microbial Nutrition and Growth ­ TABLE 6.4 Representative Differential Complex Media Medium and Ingredients Use and Interpretation of Results MacConkey Medium Peptone (20.0 g) For the culture and differentiation of enteric bacteria based on the ability to ferment lactose Agar (12.0 g) Lactose (10.0 g) Lactose-fermenters produce red-to-pink colonies; non-lactosefermenters form colorless or transparent colonies Bile salts (5.0 g) NaCl (5.0 g) Neutral red (0.075 g) Crystal violet (0.001 g) Water to bring volume to 1 L Blood Agar Agar (15.0 g) Pancreatic digest of casein (15.0 g) Papaic digest of soybean meal (5.0 g) NaCl (5.0 g) Water to bring volume to 950.0 ml Sterile blood (50.0 ml, added to medium after autoclaving and cooling) For culture of fastidious microorganisms and differentiation of hemolytic microorganisms Partial digestion of blood: alphahemolysis; complete digestion of blood: beta-hemolysis; no digestion of blood: gamma-hemolysis reducing media provide better anaerobic culturing conditions. These media contain compounds, such as sodium thioglycolate, that chemically combine with free oxygen and remove it from the medium. Heat is used to drive absorbed oxygen from thioglycolate immediately before such a medium is inoculated. The use of Petri plates presents special problems for the culture of anaerobes because each dish has a loose-fitting lid that allows the entry of air. For the culture of anaerobes, inoculated Petri plates are placed in sealable containers containing reducing chemicals (FIGURE 6.16). Of course, the airtight lids of anaerobic culture vessels must be sealed so that oxygen cannot enter. Only anaerobes that can tolerate brief exposure to oxygen can be cultured by this method because inoculation and Escherichia coli Escherichia coli transfer occur outside the anaerobic environment. Laboratories that routinely study strict anaerobes have large anaerobic glove boxes, which are transparent, airtight chambers with special airtight rubber gloves, chemicals that remove oxygen, and air locks. These chambers allow scientists to manipulate equipment and anaerobic cultures in an oxygen-free environment. Transport Media Health care personnel use special transport media to carry clinical specimens of feces, urine, saliva, sputum, blood, and other bodily fluids in such a way as to ensure that people are not infected and that the specimens are not contaminated. Speed in transporting clinical specimens to the laboratory is extremely important because pathogens often do not long survive outside the body. Specimens are transported in buffered media designed to maintain the ratio and life of different microbes. Anaerobic specimens may occasionally be transported for less than an hour inside a syringe, but longer times require the use of anaerobic transport media. Special Culture Techniques LE A R N I N G O UTC O M E 6.12 Discuss the use of animal and cell culture and low- ­ hapte       C oxygen culture. Not all organisms can be grown under the culture conditions we have discussed. Scientists have developed other techniques to culture many of these organisms. Animal and Cell Culture Microbiologists have developed animal and cell culture techniques for growing some microbes for which artificial media are inadequate. For example, the causative agents of leprosy and syphilis are grown in animals because all attempts to grow these bacteria using standard culture techniques have been unsuccessful. Mycobacterium leprae (lep´rī) is cultured in Escherichia coli ◀ FIGURE 6.15 The use of MacConkey agar as a selective and differential medium. (a) Whereas both the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli grow on nutrient agar, MacConkey agar (b) selects for Gramnegative bacteria and inhibits Gram-positive bacteria. (c) MacConkey agar also differentiates Gram-negative bacteria based on their ability to ferment lactose. The colonies of the lactose-fermenting E. coli are easily distinguished from those of the non-lactose-fermenting Salmonella enterica serotype Choleraesuis. Staphylococcus aureus (a) Nutrient agar M06_BAUM7206_05_SE_C06.indd 176 Staphylococcus aureus (no growth) Salmonella enterica serotype Choleraesuis (b) MacConkey agar (c) MacConkey agar 11/23/16 3:01 AM       Growth of Microbial Populations anaerobes. The use of packets of chemicals that remove most of the oxygen from the jar has replaced candles in modern microbiology labs. Clamp Airtight lid 2H2 + O2 Chamber H2 CO2 Envelope containing chemicals to release CO2 and H2 O2 Palladium pellets to catalyze reaction removing O2 Methylene blue (anaerobic indicator) LE A R N I N G O UTC O M E 6.13 Contrast refrigeration, deep-freezing, and lyophilization as methods for preserving cultures of microbes. To store living cells, a scientist slows the cells’ metabolism to prevent the excessive accumulation of waste products and the exhaustion of all nutrients in a medium. Refrigeration is often the best technique for storing bacterial cultures for short periods of time. Deep-freezing and lyophilization are used for long-term storage of bacterial cultures. Deep-freezing involves freezing the cells at temperatures from -50°C to -95°C. Deep-frozen cultures can be restored years later by thawing them and placing a sample in an appropriate medium. Lyophilization (lī-of´i-li-zā´shŭn; freeze-drying) involves removing water from a frozen culture using an intense vacuum. Under these conditions, ice sublimates (directly becomes a gas) and is removed from cells without permanently damaging cellular structures and chemicals. Lyophilized cultures can last for decades and are revived by adding lyophilized cells to liquid culture media. ­ ▲ FIGURE 6.16 An anaerobic culture system. The system utilizes chemicals to create an anaerobic environment inside a sealable, airtight jar. Methylene blue, which turns colorless in the absence of oxygen, indicates when the environment within the jar is anaerobic. ­ ­ Petri plates 2H2O Preserving Cultures TELL ME WHY Why do clinical laboratory scientists keep many different kinds of culture media on hand? ­ armadillos, whose internal conditions (including a relatively low body temperature) provide the conditions this microbe prefers. Rabbits meet the culture needs for Treponema pallidum, the bacterium that causes syphilis. Because viruses and small bacteria called rickettsias and chlamydias are obligate intracellular parasites—that is, they grow and reproduce only within living cells—bird eggs and cultures of living cells are used to culture these microbes. M06_BAUM7206_05_SE_C06.indd 177 6.14 Describe binary fission as a means of reproduction. Most unicellular microorganisms reproduce by binary fission, a process in which a cell grows to twice its normal size and divides in half to produce two daughter cells of equal size. (Chapters 11 and 12 discuss other reproductive strategies of prokaryotes and eukaryotes.) Binary fission generally involves four steps, as illustrated in (FIGURE 6.17 ), for a prokaryotic cell. ▶ ANIMATIONS: Bacterial Growth: Overview 1 2 As we have discussed, many types of organisms prefer oxygen conditions that are intermediate between strictly aerobic and anaerobic environments. Carbon dioxide incubators, machines that electronically monitor and control CO2 levels, provide atmospheres that mimic the environments of the intestinal tract, the respiratory tract, and other body tissues and thus are useful for culturing these kinds of organisms. Smaller and much less expensive alternatives to CO2 incubators are candle jars. In these simple but effective devices, culture plates are sealed in a jar along with a lit candle; the flame consumes much of the O2, replacing it with CO2. The candle eventually extinguishes itself, creating an environment that is ideal for many aerotolerant anaerobes, microaerophiles, and capnophiles, which are organisms such as Neisseria gon­ orrhoeae that grow best with a relatively high concentration of carbon dioxide (3–10%) in addition to low oxygen levels. Remaining oxygen in the jar prevents the growth of strict LE A R N I N G O UTC O M E Low-Oxygen Culture Growth of Microbial Populations The cell replicates its chromosome (DNA molecule). The duplicated chromosomes are attached to the cytoplasmic membrane. (In eukaryotic cells, chromosomes are attached to microtubules.) The cell elongates, and growth between attachment sites pushes the chromosomes apart. (Eukaryotic cells segregate their chromosomes by the process of mitosis, described in Chapter 12.) 11/23/16 3:01 AM 3 Cell wall 4 The cell forms a new cytoplasmic membrane and wall (septum) across the midline. When the septum is completed, the daughter cells may remain attached, as shown in Figure 6.17, or they may separate completely. When the cells remain attached, further binary fission in parallel planes produces a chain. Clusters form when attached cells continue to divide in different planes (as shown in Figure 6.17). The process repeats. ▶ ANIMATIONS: Binary Fission ­ Cytoplasmic membrane Chromosome r 6    Microbial Nutrition and Growth hapte       C ­ ­ 1 3 4 ­ Replicated chromosome 5 30 minutes Septum Completed septum 2 Here we consider the growth of populations by binary fission, using bacterial cultures as examples. We begin with a brief discussion of the mathematics of population growth. Generation Time LE A R N I N G O UTC O M E 5 6.15 Explain what is meant by the generation time of bacteria. 60 minutes 120 minutes (a) ­ 90 minutes The time required for a bacterial cell to grow and divide is its generation time. Viewed another way, generation time is also the time required for a population of cells to double in number. Generation times vary among populations and are dependent on chemical and physical conditions. Under optimal conditions, some bacteria (such as E. coli and S. aureus) have a generation time of 20 minutes or less. For this reason, food contaminated with only a few of these organisms can cause food poisoning if not properly refrigerated and cooked. Most bacteria have a generation time of 1 to 3 hours, though some slow-growing species such as Mycobacterium leprae require more than 10 days before they double. Mathematical Considerations in Population Growth Septum LE A R N I N G O UTC O M E 6.16 Describe logarithmic growth. (b) TEM 0.5 µm ▲ FIGURE 6.17 Binary fission. (a) The events in binary fission. All the cells may divide in parallel planes and remain attached to form a chain, or they may divide in different planes to form a cluster (as shown at 120 minutes). (b) Transmission electron micrograph of Bacillus licheniformis undergoing binary fission. M06_BAUM7206_05_SE_C06.indd 178 With binary fission, any given cell divides to form two cells; then each of these new cells divides in two to make four, and then four become eight, and so on. This type of growth, called logarithmic growth or exponential growth, produces very different results from simple addition, known as arithmetic growth. We can compare these two types of growth by considering what would happen over time to two hypothetical populations, as shown in (FIGURE 6.18). In this case, we assume that a hypothetical population of a pink species increases by adding one new cell every 20 minutes, whereas the cells of a purple species divide by binary fission every 20 minutes. After the initial 20 minutes, each population, which started with a single cell, would have two cells; after 40 minutes, the pink species would have three cells, whereas the purple species would have 11/23/16 3:01 AM       Growth of Microbial Populations 70 60 60 40 20 10 10 1 2 (a) growth. 30 20 0 Time (hours) 6.17 Draw and label a bacterial growth curve. 6.18 Describe what occurs at each phase of a population’s 1 2 Time (hours) (b) ▲ FIGURE 6.18 A comparison of arithmetic and logarithmic growth. Given two hypothetical initial populations consisting of a single cell each, (a) after 2 hours an arithmetically growing species will have 7 cells, whereas (b) a logarithmically growing species will have 64 cells. 5000 1015 4,096 4000 3000 2000 ­  ­ four cells. At this point, there is little difference in the growth of the two populations, but after 2 hours, the arithmetically growing pink species would have only seven cells, whereas the logarithmically growing purple species would have 64 cells. Clearly, logarithmic growth can increase a population’s size dramatically—after only 7 hours, the purple species will have over 2 million cells! The number of cells arising from a single cell by binary fission is calculated as 2n, where n is the number of generations; in other words, multiply 2 times itself n number of times. To calculate the total number of cells in a population, we multiply the original number of cells by 2n. If, for example, we had begun with three purple cells instead of one, then after 2 hours there would be 192 cells (3 * 26 = 3 * 64 = 192). A visible culture of bacteria may consist of trillions of cells, so microbiologists use scientific notation to deal with the huge numbers involved. One advantage of scientific notation is that large numbers are expressed as powers of 10, making them easier to read and write. For example, consider our culture of the purple species. After 10 hours (30 generations), there would be 1,073,741,824 (230) cells. This large number can be rounded off and expressed more succinctly in scientific notation as 1.07 * 109. After 30 more generations, scientific notation would be the only practical way to express the huge number of cells in the culture, which would be 1.15 * 1018(260). Such a number, A graph that plots the numbers of organisms in a growing population over time is known as a growth curve. When drawn using an arithmetic scale on the y-axis, a plot of exponential growth presents two problems (FIGURE 6.19a): it is difficult or impossible to distinguish numbers in early generations from the baseline, and as the population grows it becomes impossible to accommodate the graph on a single page. The solution to these problems is to replace the arithmetic scale on the y-axis with a logarithmic (log) scale (FIGURE 6.19b). Such a log scale, in which each division is 10 times larger than the preceding one, can accommodate small numbers at the lower end of the graph and very large numbers at the upper end. This kind of graph is semilogarithmic because only one axis uses a log scale. When bacteria are inoculated into a closed vessel of liquid medium, the population’s growth curve has four ­ Number of cells Number of cells 30 LE A R N I N G O UTC O M E S Species growing logarithmically 50 40 0 Phases of Microbial Population Growth Number of cells Species growing arithmetically 50 if written out (the digits 115 followed by 16 zeros), would be impractically large. Number of cells (log scale) 70 M06_BAUM7206_05_SE_C06.indd 179 1000 8 0 512 64 5 10 105 104 103 102 101 0 Time (hours) (a) 1010 5 10 Time (hours) (b) ▲ FIGURE 6.19 Two growth curves of logarithmic ­ growth. The generation time for this E. coli population is 20 minutes. (a) An arithmetic graph. Using an arithmetic scale for the y-axis makes it difficult to ascertain actual numbers of cells near the beginning and impossible to plot points after only a short time. (b) A semilogarithmic graph. Using a logarithmic scale for the y-axis solves both of these problems. Note that a plot of logarithmic population growth using a logarithmic scale produces a straight line. 11/23/16 3:01 AM r 6    Microbial Nutrition and Growth hapte       C Log Phase Number of live cells (log) Stationary phase Death (decline) phase Log (exponential) phase Time ▲ FIGURE 6.20 A typical microbial growth curve. The curve ­ shows the four phases of population growth. Why do cells trail behind their optimum reproductive potential during the lag phase? ­ ­ ­ Lag phase Eventually, the bacteria synthesize the necessary chemicals for conducting metabolism in their new environment and then enter a phase of rapid chromosome replication, growth, and reproduction. This is the log phase, so named because the population increases logarithmically, and the reproductive rate reaches a constant as DNA and protein syntheses are maximized. Researchers are interested in the log phase for many reasons. Populations in log phase are more susceptible to antimicrobial drugs that interfere with metabolism, such as erythromycin, and to drugs that interfere with the formation of cell structures, such as the inhibition of cell wall synthesis by penicillin. Populations in log phase are preferred for Gram staining because most cells’ walls are intact—an important characteristic for correct staining. Further, because the metabolic rate of individual cells is at a maximum during log phase, this phase may be preferred for industrial and laboratory purposes. Figure 6.20 During lag phase, cells are synthesizing the metabolic machinery and chemicals required for optimal reproduction. Stationary Phase Lag Phase Death Phase ­ ­ During a lag phase, the cells are adjusting to their new environment; most cells do not reproduce immediately but instead actively synthesize enzymes to utilize novel nutrients in the medium. For example, bacteria inoculated from a medium containing glucose as a carbon source into a medium containing lactose must synthesize two new types of proteins: membrane proteins to transport lactose into the cell and the enzyme lactase to catabolize lactose. The lag phase can last less than an hour or can last for days, depending on the species and the chemical and physical conditions of the medium. If bacterial growth continued at the exponential rate of the log phase, bacteria would soon overwhelm the Earth. This does not occur because as nutrients are depleted and wastes accumulate, the rate of reproduction decreases. Eventually, the number of dying cells equals the number of cells being produced, and the size of the population remains constant—hence the name stationary phase. During this phase, the metabolic rate of surviving cells declines. Cells in this phase are stress tolerant because they are adapted for challenging conditions, such as heat, high salt concentrations, and the presence of antibiotics. ­ distinct phases: the lag, log, stationary, and death phases (FIGURE 6.20). ▶ ANIMATIONS: Bacterial Growth Curve CLINICAL CASE STUDY If nutrients are not added and wastes are not removed, a population reaches a point at which cells die at a faster rate than Boils in the Locker Room convex, and golden. The bacterium is Gram-positive, mesophilic, and facultatively halophilic and can grow with or without oxygen. The cells are spherical and remain attached in clusters. 1. What color are the Gram-stained cells? ­ ­R For several weeks, faculty, students, and staff at ayburn 1. Kineococcus radiotolerans. High School have been dealing with an epidemic of unsightly, painful boils and skin infections. Over 40 athletes have been too sore to play sports, and 13 students have been hospitalized. Nurses take clinical samples of the drainage of the infections from hospitalized students, and medical laboratory scientists culture the samples. Isolated bacterial colonies are circular, M06_BAUM7206_05_SE_C06.indd 180 2. What does the term "facultatively halophilic" mean? 3. What is the scientific description of the bacterium’s oxygen requirement? 4. If the bacterium divides every 30 minutes under laboratory conditions, how many cells would there be in a colony after 24 hours? 11/23/16 3:01 AM       Growth of Microbial Populations they are produced. Such a culture has entered the death phase (or decline phase). Bear in mind that during the death phase, some cells remain alive and continue metabolizing and reproducing, but the number of dying cells exceeds the number of new cells produced so that eventually the population decreases to a fraction of its previous abundance. In some cases, all the cells die, whereas in others, a few survivors may remain indefinitely. The latter case is especially true for cultures of bacteria that can develop resting structures called endospores (see Chapter 3). Chemostats make possible the study of microbial population growth at steady but low nutrient levels, such as might be found in biofilms. Such studies are essential to understand interactions of microbial species in nature and can reveal aspects of microbial interactions that are not apparent in a closed system. Scientists also use chemostats to maintain log phase population growth for experimental inquiries into aspects of microbial metabolism, such as enzyme activities. Food and industrial microbiologists use chemostats to maintain constant production of useful microbial products. Continuous Culture in a Chemostat Measuring Microbial Reproduction LE A R N I N G O U TC O M E LE A R N I N G O UTC O M E 6.20 Contrast direct and indirect methods of measuring bacte- 6.19 Explain how a chemostat can maintain a microbial cul- rial reproduction. ture in a continuous phase. ­ Researchers and industrialists can continuously maintain a particular phase of microbial population growth by using a special culture device called a chemostat (FIGURE 6.21). A chemostat is an open system; that is, fresh medium is continuously supplied while an equal amount of old medium (containing microbes) is removed. As nutrients enter the culture vessel of a chemostat, the cells can metabolize, grow, and reproduce—but only to the extent that a limiting nutrient (e.g., a particular amino acid) is available. By controlling the amount of limiting nutrient entering the culture vessel, a chemostat maintains a culture in a particular phase, typically log phase. This is impossible in a closed system because as nutrients are depleted and wastes accumulate, the culture enters the stationary phase and then the death phase. Fresh medium with a limiting amount of a nutrient Flow-rate regulator Sterile air or other gas Direct Methods Not Requiring Incubation It is possible to directly count cells without having to incubate cultures. Here we consider two such direct methods. Microscopic Counts Microbiologists can count microorganisms directly through a microscope rather than inoculating them onto the surface of a solid medium. In this method, particularly suitable for stained prokaryotes and relatively large eukaryotes, a sample is placed on a cell counter (also called a Petroff­Hausser counting chamber), which is a special glass slide composed of an etched grid positioned beneath a glass cover slip (FIGURE 6.22). A cell counter is designed to precisely hold the cover slip 0.02 mm above the grid; therefore, the volume of a suspension of microbes in water over a 1 mm2 portion of the grid is 1 mm * 1 mm * 0.02 mm = 0.02 mm3. Each 1 mm2 grid contains 25 large squares, so a microbiologist can count the Overflow tube ­ Culture ­ ­ Culture vessel We have discussed the concepts of population growth and have seen that large numbers result from logarithmic growth, but we have not discussed practical methods of determining the size of a microbial population. Because of each cell’s small size and incredible rate of reproduction, it is not possible to actually count every one in a population. For one thing, they grow so rapidly that their number changes during the count. Therefore, laboratory personnel must estimate the number of cells in a population by counting the number in a small, representative sample and then multiplying to estimate the number in the whole specimen. For example, if there are 25 cells in a microliter (ml) sample of urine, then there are approximately 25 million cells in a liter of urine. Estimating the number of microorganisms in a sample is useful for determining such things as the severity of urinary tract infections, the effectiveness of pasteurization and other methods of food preservation, the degree of fecal contamination of water supplies, and the usefulness of particular disinfectants and antibiotics. Microbiologists use either direct or indirect methods to estimate the number of cells. We begin with direct methods of measuring bacterial reproduction. ▲ FIGURE 6.21 Schematic of chemostat. A microbial culture is continuously maintained in a predetermined phase, typically log phase, by controlling the amount and type of fresh medium added while removing an equal amount of old culture. M06_BAUM7206_05_SE_C06.indd 181 11/23/16 3:01 AM r 6    Microbial Nutrition and Growth hapte       C Cover slip Bacterial suspension Pipette number of bacteria in several of the large squares and then calculate the mean number of bacteria per square. The number of bacteria per milliliter (cm3) can be calculated as follows: Location of grid mean number of bacteria per square * 25 squares = number of bacteria per 0.02 mm3 no. of bacteria per 0.02 mm3 * 50 = no. of bacteria per mm3 no. of bacteria per mm3 * 1000 = no. of bacteria per cm3 (ml) Overflow troughs (a) Place under oil immersion Bacterial suspension 1 mm 1 mm (b) This means that one needs only to multiply the mean number of bacteria per square by 1,250,000 (25 * 50 * 1000) to calculate the number of bacteria per milliliter of bacterial suspension. Direct microscopic counts are advantageous when there are more than 10 million cells per milliliter or when a speedy estimate of population size is required. However, direct counts can be problematic because it is often difficult to differentiate between living and dead cells and nearly impossible to count rapidly moving microbes. Electronic Counters A Coulter15 counter is a device that directly counts cells as they interrupt an electrical current flowing across a narrow tube held in front of an electronic detector. This device is useful for counting the larger cells of yeasts, unicellular algae, and protozoa; it is less useful for bacterial counts because of debris in the media and the presence of filaments and clumps of cells. Flow cytometry is a variation of counting with a Coulter counter. A cytometer uses a light-sensitive detector to record changes in light transmission through the tube as cells pass. Scientists use this technique to distinguish among cells that have been differentially stained with fluorescent dyes or tagged with fluorescent antibodies. They can count bacteria in a solution and even count host cells that contain fluorescently stained intracellular parasites. We have considered direct methods not requiring incubation. Now we consider direct methods with incubation. Direct Methods Requiring Incubation ­ ­ microbial numbers. (a) The counter is a glass slide with an etched grid that is exactly 0.02 mm lower than the bottom of the cover slip. A bacterial suspension placed next to the cover slip through a pipette moves under the cover slip and over the grid by capillary action. (b) View of a 1 mm2 portion of the grid through the microscope. Each square millimeter of the grid has 25 large squares, each of which is divided into 16 small squares. (c) Enlarged view of one large square containing 15 cells. The number of bacteria in several large squares is counted and averaged. The calculations involved in estimating the number of bacteria per milliliter (cm3) of suspension are described in the text. Serial Dilution and Viable Plate Counts What if the number of cells in even a very small sample is still too great to count? If, for example, a 1-ml sample of milk containing 20,000 bacterial cells per milliliter were plated on a Petri plate, there would be too many colonies to count. In such cases, microbiologists make a serial dilution, which is the stepwise dilution of a liquid culture in which the dilution factor at each step is constant. The scientists plate a set amount of each dilution onto an agar surface and count the number of colonies resulting on a plate from each dilution. They count the colonies on plates with 15Named M06_BAUM7206_05_SE_C06.indd 182 ­ ▲ FIGURE 6.22 The use of a cell counter for estimating ­ (c) Among the many direct techniques are techniques requiring incubation—viable plate counts following dilution, membrane filtration, and the most probable number method. for Wallace Coulter, American inventor. 11/23/16 3:01 AM       Growth of Microbial Populations 25 to 250 colonies and multiply the number on each plate by the reciprocal of the dilution used to inoculate that plate to estimate the number of bacteria per milliliter of the original culture. This method is called a viable plate count (FIGURE 6.23). When a plate has fewer than 25 colonies, it is not used to estimate the number of bacteria in the original sample because the chance of underestimating the population increases when the number of colonies is small. Recall that the number of colonies on a plate indicates the number of CFUs that were inoculated onto the plate. This number differs from the actual number of cells when the CFUs are composed of more than one cell. In cases where a colonyforming unit consists of more than one cell, a viable plate count underestimates the number of cells present in the sample. The accuracy of a viable plate count also depends on the homogeneity of the dilutions, the ability of the bacteria to grow on the medium used, the number of cell deaths, and the growth phase of the sample population. Thoroughly mixing each dilution, inoculating multiple plates per dilution, and using log-phase cultures minimize errors. through a membrane filter with pores small enough to trap the cells. The membrane is then transferred onto a solid medium, and the colonies present after incubation are counted. In this case, the number of colonies is equal to the number of CFUs in the original large sample. ­ ­ Most Probable Number The most probable number (MPN) method is a statistical estimation technique based on the fact that the more bacteria are in a sample, the more dilutions are required to reduce their number to zero. Let’s consider an example of the use of the MPN method to estimate the number of fecal bacteria contaminating a stream. A researcher inoculates a set of test tubes of a broth medium with a sample of stream water. The more tubes that are used, the more accurate is the MPN method; even so, accuracy must be balanced against the time and cost involved in inoculating and incubating numerous tubes. Typically, a set of five tubes is inoculated. The researcher also inoculates a set of five tubes with a 1:10 dilution and another set of five tubes with a 1:100 dilution of stream water. Thus, there are 15 test tubes—the first set of five tubes inoculated with undiluted sample, the second set with a 1:10 dilution, and the third set with a 1:100 dilution (FIGURE 6.25). After incubation for 48 hours, the researcher counts the number of test tubes in each set that show growth. This generates three numbers in this example—growth occurs in four of the undiluted broth tubes, two of the 1:10 tubes, and in only Membrane Filtration Viable plate counts allow scientists to estimate the number of microorganisms when the population is very large, but if the population density is very small—as is the case, for example, for fecal bacteria in a stream or lake— microbes are more accurately counted by membrane filtration (FIGURE 6.24). In this method, a large sample (perhaps as large as several liters) is poured (or drawn under a vacuum) 1 ml of original culture 9 ml of broth + 1 ml of original culture 1 ml 1 ml 1:10 dilution (10–1) 1:100 dilution (10–2) 1 ml 1:1000 dilution (10–3) 1 ml 1:10,000 dilution (10 –4 ) (a) 0.1 ml of each transferred to a plate 0.1 ml 0.1 ml 0.1 ml ◀ FIGURE 6.23 A serial dilution and viable plate count for estimating microbial population size. (a) Serial dilutions. A series of 10-fold 0.1 ml (b) 1:100,000 dilution (10–5) dilutions is made. (b) Plating. A 0.1-ml sample from each dilution is poured onto a plate and spread with a sterile rod. Alternatively, 0.1 ml of each dilution can be mixed with melted agar medium and poured into plates. (c) Counting. Plates are examined after incubation. Some plates may contain so many colonies that they are too numerous to count (TNTC). The number of colonies is multiplied by 10 (because 0.1 ml was plated instead of 1 ml) and then by the reciprocal of the dilution to estimate the concentration of bacteria in original culture—in this case, 65 colonies * 10 * 1000 = 650,000 bacteria/ml. Incubation period Too numerous to count (TNTC) TNTC 65 colonies 6 colonies 0 colonies (c) M06_BAUM7206_05_SE_C06.indd 183 11/23/16 3:01 AM r 6    Microbial Nutrition and Growth hapte       C 1 ml Membrane transferred to culture medium Sample to be filtered Undiluted Membrane filter retains cells 1 ml 1:10 1:100 Inoculate 1 ml into each of 5 tubes Phenol red, pH color indicator, added To vacuum Incubate Incubation Colonies Results 4 tubes positive 2 tubes positive 1 tube positive ▲ FIGURE 6.25 The most probable number (MPN) method for estimating microbial numbers. Typically, sets of five test tubes are (a) used for each of three dilutions. After incubation, the number of tubes showing growth in each set is used to enter an MPN table (see Table 6.5), which provides an estimate of the number of cells per 100 ml of liquid. If the results were 5, 3, 1, what would be the most probable number of microorganisms in the original broth? Figure 6.25 The MPN is 110/100 ml. 2 µm (c) ▲ FIGURE 6.24 The use of membrane filtration to estimate microbial population size. (a) After all the bacteria in a given volume of sample are trapped on a membrane filter, the filter is transferred onto an appropriate medium and incubated. The microbial population is estimated by multiplying the number of colonies counted by the volume of sample filtered. (b) Bacteria trapped on the surface of a membrane filter. (c) Colonies growing on a solid medium after being transferred from a membrane filter. Scientists use a superimposed grid to help them count the colonies. If the colonies in (c) resulted from filtering 2.5 liters of stream water, what is the minimum number of bacteria per liter in the stream? Figure 6.24 83 colonies 2.5 L = 33.2 colonies/L. one of the 1:100 tubes (4, 2, 1). The numbers are compared to the numbers in an MPN table (see TABLE 6.5). How statisticians develop MPN tables is beyond the scope of our discussion, but they accurately estimate the number of cells in a solution. In M06_BAUM7206_05_SE_C06.indd 184 Indirect Methods It is not always necessary to count microorganisms to estimate population size or density. Industrial and research microbiologists use indirect methods that measure such variables as turbidity, metabolic activity, and dry weight instead of counting microorganisms, colonies, or MPN tubes. Scientists can also estimate population size and diversity by analyzing the unique sequences of DNA present in a sample. ­ SEM ­ (b) this case, the MPN table estimates that there were 26 bacteria per 100 ml of stream water. The most probable number method is useful for counting microorganisms that do not grow on solid media, when bacterial counts are required routinely, and when samples of wastewater, drinking water, and food samples contain too few organisms to use a viable plate count. The MPN method is also used to count algal cells because algae seldom form distinct colonies on solid media. Turbidity As bacteria reproduce in a broth culture, the broth often becomes turbid (cloudy) (FIGURE 6.26a). Generally, the greater the bacterial population, the more turbid a broth will be. An indirect method for estimating the growth of a microbial population involves measuring changes in turbidity using a device called a spectrophotometer (FIGURE 6.26b). Researchers most often use this method. 11/23/16 3:01 AM       Growth of Microbial Populations TABLE 6.5 Most Probable Number Table (Partial) Number Out of Five Tubes Giving Positive Results in Three Dilutions Most Probable Number of Bacteria per 100 ml 4 0 0 13 4 0 1 17 4 1 0 17 4 1 1 21 4 1 2 26 4 2 0 22 4 2 1 26 4 3 0 27 4 3 1 33 4 4 0 34 5 0 0 23 5 0 1 30 5 0 2 40 5 1 0 30 5 1 1 50 5 1 2 60 5 2 0 50 5 2 1 70 5 2 2 90 5 3 0 80 5 3 1 110 5 3 2 140 5 3 3 170 5 4 0 130 5 4 1 170 5 4 2 220 5 4 3 280 5 4 4 350 (b) (a) Direct light Spectroph Uninoculated tube Light source otometer Light-sensitive detector Spectroph Light source Inoculated broth culture (c) Scattered light that does not reach reflector otometer ▲ FIGURE 6.26 Turbidity and the use of spectrophotometry ­ M06_BAUM7206_05_SE_C06.indd 185 ­ ­  ­ is useful only if the concentration of cells exceeds 1 million per milliliter; densities below this value generally do not produce turbidity. Further, the technique is accurate only if the cells are suspended uniformly in the medium. If they form either a pellicle (a film of cells at the surface) or a sediment (an accumulation of cells at the bottom), their number will be underestimated. Further, spectrophotometry does not distinguish between living and dead cells. ­ ­ A spectrophotometer measures the amount of light transmitted through a culture under standardized conditions (FIGURE 6.26c). The greater the concentration of bacteria within a broth, the more light will be absorbed and scattered, and the less light will pass through and strike a light-sensitive detector. Generally, transmission is inversely proportional to the population size; that is, the larger the population grows, the less light will reach the detector. Scales on the gauge of a spectrophotometer report percent­ age of transmission and absorbance. These are two ways of looking at the same things; for example, 25% transmission is the same thing as 75% absorbance. Direct counts must be calibrated with transmission and absorbance readings to provide estimates of population size. Once these values are determined, spectrophotometry provides estimates of population size more quickly than any direct method. The benefits of measuring turbidity to estimate population growth include ease of use and speed. However, the technique in indirect measurement of population size. (a) Turbidity (right), an increased optical density or cloudiness of a solution. (b) A spectrophotometer. (c) The principle of spectrophotometry. After a light beam is passed through an uninoculated sample of the culture medium, the scale is set at 100% transmission. In an inoculated sample, the microbial cells absorb and scatter light, reducing the amount reaching the detector. The percentage of light transmitted is inversely proportional to population density. Metabolic Activity Under standard temperature conditions, the rate at which a population of cells utilizes nutrients and produces wastes depends on their number. Once they establish 11/23/16 3:01 AM r 6    Microbial Nutrition and Growth Just a Sore Throat and Cough? ­ At the clinic, Dr. Wyatt gets a medical history, listens to Kalinda’s lungs, and uses a pulse oximeter to measure her pulse and the level of oxygen in her blood. He tells her that he hears a crackling sound in her lungs and that her oxygen levels are slightly lower than normal. Dr. Wyatt orders a chest X-ray exam and blood work, and he obtains a sputum (mixture of saliva and mucus) sample from Kalinda by having her cough deeply. As they view the chest image, Dr. Wyatt points out an area of cloudiness in Kalinda’s right lung and explains that this indicates inflammation in the lung, most likely due to an infection. This also explains the crackling in her lungs, the pain in her chest, her difficulty in breathing, and the lower oxygen saturation. He adds that, based on her medical history, he thinks her sore throat and cough have progressed to pneumonia. Although they need to wait for the blood and sputum test results to come back from the lab in order to make a definitive diagnosis, Dr. Wyatt believes that it’s best to start Kalinda on antibiotics right away. He also prescribes a steroid inhaler to help open up her airways and make breathing easier. M06_BAUM7206_05_SE_C06.indd 186 TELL ME WHY Some students transfer some “gunk” from a 2-week-old bacterial culture into new media. Why shouldn’t they be surprised when this “death-phase” sample grows? Shortly after her visit, Dr. Wyatt phones Kalinda to follow up with her lab results. Molecular analysis (using polymerase chain reaction, or PC ) of the sputum test identified Mycoplasma pneumoniae, a bacterium commonly associated with pneumonia. He tells her to continue taking the antibiotics and to schedule a follow-up appointment. After just a few days on the antibiotics, Kalinda feels a lot better. Her cough tapers off and eventually disappears, and by the time of her follow-up appointment, her right lung is totally clear, and her lung sounds and oxygen levels have returned to normal. 1. Sputum samples are typically plated on blood agar, chocolate agar (blood agar containing lysed red blood cells), and MacConkey agar. Explain why each of these media may be helpful in identifying pathogens in lower respiratory infections. ­ Molecular Methods The majority of bacteria and archaea have not been grown in the laboratory, and representatives of most species are too few in number to study by direct observation. How do scientists estimate the number of such uncultured microbes? Scientists can isolate unique DNA sequences representing uncultured prokaryotic species using genetic techniques such R Dry Weight The abundance of some microorganisms, particularly filamentous microorganisms, is difficult to measure by direct methods. Instead, these organisms are filtered from their culture medium, dried, and weighed. The dry weight method is suitable for broth cultures, but growth cannot be followed over time because the organisms are killed during the process. as polymerase chain reaction (PCR) and hybridization of DNA that codes for ribosomal RNA. For example, one study estimated that a single gram of garden soil contains more than 100 billion bacteria and archaea, representing more than 10 million different species. In the clinical environment, molecular methods have three major advantages. First, PCR and hybridization probes are highly specific, much more than selective media. Second, they are faster because no culture is required. For example, probe tests for Chlamydia can give a result in 2 hours. Third, molecular methods are based on DNA and thus are generic, avoiding the necessity of using a different media type for each microbe. (Chapter 8 discusses molecular methods in more detail.) ­ the metabolic rate of a microorganism, scientists can indirectly estimate the number of cells in a culture by measuring changes in such things as nutrient utilization, waste production, or pH. Scientists studying environmental water samples often use this method. ­ hapte       C 2. The M. pneumoniae in Kalinda’s sputum was identified using PCR, not the culture plates. What are some possible reasons that M. pneumoniae was not found on the plates? Check your answers to Micro in the Clinic Follow-Up questions in the MasteringMicrobiology Study Area. 11/23/16 3:01 AM       Chapter Summary CHAPTER SUMMARY ­ 5. The four toxic forms of oxygen are singlet oxygen (1O2), which is neutralized by pigments called carotenoids; superoxide radicals (O2 -), which are detoxified by superoxide dismutase; peroxide anion (O2 2 - ), which is detoxified by catalase or peroxidase; and hydroxyl radicals (OH #), the most reactive of the toxic forms of oxygen. 6. Microbes are described in terms of their oxygen requirements and limitations as strict aerobes, which require oxygen; as strict anaerobes, which cannot tolerate oxygen; as facultative anaerobes, which can live with or without oxygen; as aerotolerant anaerobes, which prefer anaerobic conditions but can tolerate exposure to low levels of oxygen; or as microaerophiles, which require low levels of oxygen. 7. Nitrogen, acquired from organic or inorganic sources, is an essential element for microorganisms. Some bacteria can reduce nitrogen gas into a more usable form via a process called nitrogen fixation. 8. In addition to the main elements found in microbes, very small amounts of trace elements are required. Vitamins are among the growth factors, which are organic chemicals required in small amounts for metabolism. 9. Though microbes survive within the limits imposed by a minimum growth temperature and a maximum growth temperature, an organism’s metabolic activities produce the highest growth rate at the optimum growth temperature. 10. Microbes are described in terms of their temperature requirements as (from coldest to warmest) psychrophiles, mesophiles, thermophiles, or hyperthermophiles. Psychrotolerant microbes do not normally live in the cold but can tolerate cold temperatures such as in refrigeration. M06_BAUM7206_05_SE_C06.indd 187 ­ 14. Quorum sensing is the process by which bacteria respond to changes in microbial density by utilizing signal and receptor molecules. Biofilms, which are communities of cells attached to surfaces, use quorum sensing. Culturing Microorganisms (pp. 169–177) 1. Microbiologists culture microorganisms by transferring an inoculum from a clinical or environmental specimen into a medium such as broth or solid media. The microorganisms grow into a culture. On solid surfaces, cultures are seen as colonies. 2. A clinical specimen is a sample of human material. Standard precautions are the guidelines to protect health care professionals from infection. 3. Pure cultures (axenic cultures) contain cells of only one species and are derived from a colony-forming unit (CFU) composed of a single cell or group of related cells. To obtain pure cultures, sterile equipment and use of aseptic techniques are critical. 4. The streak-plate method allows CFUs to be isolated by streaking. The pour-plate technique isolates CFUs via a series of dilutions. 5. Petri dishes that are filled with solid media are called Petri plates. Slant tubes (slants) are test tubes containing agar media that solidified while the tube was resting at an angle. 6. A defined medium (also known as synthetic medium) provides exact known amounts of nutrients for the growth of a particular microbe. Complex media contain a variety of growth factors. Selective V media either inhibit the growth of unwanted micror organisms or favor the growth of particular microbes. T Microbiologists use differential media to distinguish among groups of bacteria. Reducing media provide conditions conducive to culturing anaerobes. Transport media are designed to move specimens safely from one location to another while maintaining the relative abundance of organisms and preventing contamination of the specimen or environment. ideo 4. Obligate aerobes require oxygen molecules as the final electron acceptor of their electron transport chains, whereas obligate anaerobes cannot tolerate oxygen and must use an electron acceptor other than oxygen. 13. Barophiles, organisms that normally live under the extreme hydrostatic pressure at great depth below the surface of a body of water, often cannot live at the pressure found at the surface. uto 3. Photoautotrophs use carbon dioxide as a carbon source and light energy to make their own food; chemoautotrophs use carbon dioxide as a carbon source but catabolize organic or inorganic molecules for energy. Photoheterotrophs are photosynthetic organisms that acquire energy from light and acquire nutrients via catabolism of organic compounds; chemoheterotrophs use organic compounds for both energy and carbon. Organotrophs acquire electrons for redox reactions from organic sources, whereas lithotrophs acquire electrons from inorganic sources. ­ 2. Chemical nutrients such as carbon, hydrogen, oxygen, and nitrogen are required for the growth of microbial populations. 12. Osmotic pressure can cause cells to die from either swelling and bursting or from crenation (shriveling). The cell walls of some microorganisms protect them from osmotic shock. Obligate halophiles require high osmotic pressure, whereas facultative halophiles do not require but can tolerate such conditions. ­ 1. A colony, which is a visible population of microorganisms arising from a single cell or colony-forming unit living in one place, grows in size as the number of cells increases. Most microbes live as biofilms, that is, in association with one another on surfaces. 11. Neutrophiles grow best at neutral pH, acidophiles grow best in acidic surroundings, and alkalinophiles live in alkaline habitats. 7. Special culture techniques include the use of animal and cell cultures, low-oxygen cultures, enrichment cultures, and cold enrichment cultures. A capnophile grows best with high CO2 levels in addition to low oxygen levels. ­ Growth Requirements (pp. 161–169) 8. Cultures can be preserved in the short term by refrigeration and in the long term by deep-freezing and lyophilization. 11/23/16 3:01 AM r 6    Microbial Nutrition and Growth 1. A population of microorganisms doubles during its generation time—the time also required for a single cell to grow and divide. 2. Bacteria grow by logarithmic growth, or exponential growth. ▶ ANIMATIONS: Bacterial Growth: Overview; Binary Fission R QUESTIONS FO R 3. A graph that plots the number of organisms growing in a population over time is called a growth curve. When organisms are grown in a broth and the growth curve is plotted on a semilogarithmic scale, the population’s growth curve has four phases. In the lag phase, the organisms are adjusting to their environment. In the log phase, the population is most actively growing. In the stationary phase, new organisms are being produced at the same rate at which they are dying. In the death phase, the organisms are dying more quickly than they can be replaced by new organisms. ▶ ANIMATIONS: Bacterial Growth Curve 4. A chemostat is a continuous culture device that maintains a desired phase of microbial population growth by adding limited amounts and kinds of nutrients while removing an equal amount of old medium. 5. Direct methods for estimating population size that do not require incubation are microscopic counts and electronic counters, including flow cytometry. Direct methods requiring incubation include serial dilution with viable plate counts, membrane filtration, and the most probable number (MPN) method. ­ Growth of Microbial Populations (pp. 177–186) ­ hapte       C 6. Indirect methods include measurements of turbidity, metabolic activity, and dry weight and analysis of numbers and kinds of unique genetic sequences. EVIEW Answers to the Questions for Review (except Short Answer questions) begin on p. A-1. 1. Which of the following can grow in a Petri plate on a laboratory table? a. an anaerobic bacterium c. viruses on an agar surface b. an aerobic bacterium d. all of the above 2. “In the laboratory, a sterile inoculating loop is moved across the agar surface in a culture dish, thinning a sample and isolating individuals.” This statement describes which of the following? a. broth culture c. streak plate b. pour plate d. dilution plate 3. Superoxide dismutase __________________. c. neutralizes singlet oxygen a. causes hydrogen peroxide d. is missing in aerobes to become toxic b. detoxifies superoxide radicals 4. The most reactive of the four toxic forms of oxygen is __________________. a. the hydroxyl radical c. the superoxide radical b. the peroxide anion d. singlet oxygen 5. Microaerophiles that grow best with a high concentration of carbon dioxide in addition to a low level of oxygen are called __________________. a. aerotolerant c. facultative anaerobes b. capnophiles d. fastidious c. die if put under high pressure d. thrive in warm air 9. Which of the following terms best describes an organism that cannot exist in the presence of oxygen? a. obligate aerobe c. obligate anaerobe b. facultative aerobe d. facultative anaerobe ­ Multiple Choice 10. In a defined medium, __________________. a. the exact chemical composition of the medium is known b. agar is available for microbial nutrition c. blood may be included d. organic chemicals are excluded 11. Which of the following is most useful in representing population growth on a graph? a. logarithmic reproduction of the growth curve b. a semilogarithmic graph using a log scale on the y-axis c. an arithmetic graph of the lag phase followed by a logarithmic section for the log, stationary, and death phases d. None of the above would best represent a population growth curve. 12. Which of the following methods is best for counting fecal bacteria from a stream to determine the safety of the water for drinking? a. dry weight c. viable plate counts b. turbidity d. membrane filtration 7. Organisms that preferentially may thrive in icy waters are described as __________________. a. barophiles c. mesophiles b. thermophiles d. psychrophiles 13. A Coulter counter is a(n) __________________. a. statistical estimation using 15 dilution tubes and a table of numbers to estimate the number of bacteria per milliliter b. indirect method of counting microorganisms c. device that directly counts microbes as they pass through a tube in front of an electronic detector d. device that directly counts microbes that are differentially stained with fluorescent dyes 8. Barophiles __________________. a. cannot cause diseases in humans b. live at normal barometric pressure 14. Lyophilization can be described as __________________. a. freeze-drying c. refrigeration b. deep-freezing d. pickling 6. Which of the following is not a growth factor for various microbes? a. cholesterol c. vitamins b. water d. heme M06_BAUM7206_05_SE_C06.indd 188 11/23/16 3:01 AM 2. Describe the type of hemolysis shown by the pathogen Staphylococcus aureus pictured here. ­ 15. Quorum sensing is __________________. a. the ability to respond to changes in population density b. a characteristic allowing secretion of a matrix c. dependent on direct contact among cells d. associated with colonies in broth culture       R Questions for eview Fill in the Blanks 1. All cells require a source of __________________ for redox reactions. 2. A toxic form of oxygen, __________________ oxygen, is molecular oxygen with electrons that have been boosted to a higher energy state. 3. All cells recycle the essential element __________________ from amino acids and nucleotides. 4. __________________ are small organic molecules that are required in minute amounts for metabolism. 5. The lowest temperature at which a microbe continues to metabolize is called its __________________. 6. Cells that shrink in hypertonic solutions such as saltwater are responding to __________________ pressure. 7. Obligate __________________ exist in salt ponds because of their ability to withstand high osmotic pressure. 8. __________________ pigments protect many phototrophic organisms from photochemically produced singlet oxygen. 9. Microbes that reduce N2 to NH3 engage in nitrogen __________________. 10. A student observes a researcher streaking a plate numerous times, flaming the loop between streaks. The researcher is likely using the __________________ method to isolate microorganisms. 11. Chemolithotrophs acquire electrons from (organic/inorganic) __________________ compounds. Short Answer 1. High temperature affects the shape of particular molecules. How does this affect the life of a microbe? 2. Support or refute the following statement: Microbes cannot tolerate the low pH of the human stomach. 3. Explain quorum sensing, and describe how it is related to biofilm formation. 4. Why must media, vessels, and instruments be sterilized before they are used for microbiological procedures? 5. Why is agar used in microbiology? 6. What is the difference between complex media and defined media? VISUALIZE IT! 7. Draw and label the four distinct phases of a bacterial growth curve. Describe what is happening within the culture as it passes through the phases. 1. Label each of these thioglycolate tubes to indicate the oxygen requirements of the microbes growing in them. 8. If there are 47 cells in 1 ml of sewage, how many cells are there in a liter? 9. List three indirect methods of counting microbes. 10. List five direct methods of counting microbes. 11. Explain the differences among photoautotrophs, chemoautotrophs, photoheterotrophs, chemoheterotrophs, organotrophs, and lithotrophs. ­ 12. Contrast the media described in Tables 6.3 and 6.4 on pp. 174 and 176. Why is E. coli medium described as defined, whereas MacConkey medium and blood agar are defined as complex? 13. How does a chemostat maintain a constant population size? M06_BAUM7206_05_SE_C06.indd 189 11/23/16 3:01 AM hapte       C r 6    Microbial Nutrition and Growth 2. Pasteurization is a technique that uses temperatures of about 72°C to neutralize potential pathogens in foods. What effect does this temperature have on the enzymes and cellular metabolism of pathogens? Why does the heat of pasteurization kill some microorganisms yet fail to affect thermophiles? 3. Two cultures of a facultative anaerobe are grown under identical conditions, except that one was exposed to oxygen and the other was completely deprived of oxygen. What differences would you expect to see between the dry weights of the cultures? Why? 4. Some organisms require riboflavin (vitamin B2) to make FAD. For what purpose do they use FAD? 5. A scientist inoculates a bacterium into a complex nutrient slant tube. The bacterium forms only a few colonies on the slanted surface but grows prolifically in the depth of the agar. Describe the oxygen requirements of the bacterium. ­ Not all organisms are easy to classify. For instance, the single-celled eukaryote Euglena granulata typically uses light energy and gets its carbon from carbon dioxide. However, when it is cultured on a suitable medium in the dark, this microbe utilizes energy and carbon solely from organic compounds. What is the nutritional classification of Euglena? ­ 1. A scientist describes an organism as a chemoheterotrophic, aerotolerant, mesophilic, facultatively halophilic coccus. Describe the cell’s metabolic and structural features in plain English. R C ITICAL THINKING 14. Given that Haemophilus ducreyi is a chemoheterotrophic pathogen that requires heme as a growth factor, deduce how this bacterium phosphorylates most of its ADP to form ATP. Defend your answer. 15. Examine the graph in Figure 6.3. Note that the growth rate increases slowly until the optimum is reached and then declines steeply at higher temperatures. In other words, organisms tolerate a wider range of temperatures below their optimal temperature than they do above the optimum. Explain this observation. 7. A scientific article describes a bacterium as an obligate microaerophilic chemoorganoheterotroph. Describe the oxygen and nutritional characteristics of the bacterium in everyday language. 17. Using the terms in Figure 6.8a, describe the shape, margin, pigmentation, and optical properties of two bacterial colonies seen in Figure 6.10b. 8. Microorganisms require phosphorus, sulfur, iron, and magnesium for metabolism. What specifically are these elements used for in microbial metabolism? (Review Chapters 2 and 5.) 18. Why have scientists been unable to axenically culture Treponema pallidum in a laboratory medium? 9. The bacterium Desulforudis audaxviator lives almost two miles underground, deriving energy from sulfate, acquiring electrons from hydrogen, and building organic molecules from inorganic carbon found in surrounding rocks. Describe the nutritional classifications of D. audaxviator. 10. Starting with 10 bacterial cells per milliliter in a sufficient amount of complete culture medium with a 1-hour lag phase and a 30-minute generation time, how many cells will there be in a liter of medium at the end of 2 hours? At the end of 7 hours? ­ 11. Suppose you perform a serial dilution of 0.1-ml sample from a liter of culture medium as illustrated in Figure 6.23. The 10 -3 plate gives 440 colonies, and the 10 -4 plate gives 45 colonies. Calculate the approximate number of bacteria in the original liter. 12. How might the study of biofilms benefit humans? 13. The filamentous bacterium Beggiatoa gets its carbon from carbon dioxide and its electrons and energy from hydrogen sulfide. What is its nutritional classification? M06_BAUM7206_05_SE_C06.indd 190 ­ 6. How can regions within biofilms differ in their chemical content? 16. Over 100 years ago, doctors infected syphilis victims with malaria parasites to induce a high fever. Surprisingly, such treatment often cured the syphilis infection. Explain how this could occur. 19. Examine the ingredients of MacConkey agar as listed in Table 6.4 on p. 176. Does this medium select for Gram-positive or Gram-negative bacteria? Explain your reasoning. 20. The sole carbon source in citrate medium is citric acid (citrate). Why might a laboratory microbiologist use this medium? 21. Using as many of the following terms as apply—selective, differential, broth, solid, defined, and complex—categorize each of the media listed in Table 6.3 on p. 174 and Table 6.4 on p. 176. 22. Beijerinck used the concept of enrichment culture to isolate aerobic and anaerobic nitrogen-fixing bacteria, sulfate-reducing bacteria, and sulfur-oxidizing bacteria. What kind of selective media could he have used for isolating each of these four types of microbes? 23. Viable plate counts are used to estimate population size when the density of microorganisms is high, whereas membrane filtration is used when the density is low. Why is a viable plate count appropriate when the density is high but not when the density is low? 11/23/16 3:01 AM Questions for Review      191 CONCEPT MAPPING Using the following terms, fill in the following concept map that describes culture media. Differential Enriched Fastidious microorganisms Selective and differential Trypticase soy agar Unwanted microorganisms Fermentation broths General purpose Selective Culture media 1 5 3 9 7 such as 2 Nutrient broth support growth of inhibit 4 8 such as such as such as such as MacConkey agar show 6 Sabouraud agar Blood agar Visible differences between microorganisms M06_BAUM7206_05_SE_C06.indd 191 06/03/18 4:43 PM