Microbial Growth PDF

In the Clinic As a nurse in a plastic surgery clinic, you...

In the Clinic As a nurse in a plastic surgery clinic, you instruct patients on postsurgical care of their sutures. You tell patients to wash hands before removing bandages, to wash gently around the surgical site with soap and water, and to swab the wound with hydrogen peroxide. One day a patient calls, alarmed that the hydrogen peroxide caused her wound to bubble. What would you tell the patient? Hint: Read about catalase on page 155. NOTE: Answers to In the Clinic questions are found online at MasteringMicrobiology 6W Microbial Growth hen we talk about microbial growth, we are really referring to the number of cells, not the size of the cells. Microbes that are “growing” are increasing in number, accumulating into colonies (groups of cells large enough to be seen without a microscope) of hundreds of thousands of cells or populations of billions of cells. Although individual cells approximately double in size during their lifetime, this change is not very significant compared with the size increases observed during the lifetime of plants and animals. Many bacteria survive and grow slowly in nutrient-poor environments by forming biofilms. The Serratia marcescens bacteria in the photo may form biofilms on urinary catheters or on contact lenses. Biofilms are frequently sources of health care associated infections such as the one described in the Clinical Case. Microbial populations can become incredibly large in a very short time. By understanding the conditions necessary for microbial growth, we can determine how to control the growth of microbes that cause diseases and food spoilage. We can also learn how to encourage the growth of helpful microbes and those we wish to study. In this chapter we will examine the physical and chemical requirements for microbial growth, the various kinds of culture media, bacterial cell division, the phases of microbial growth, and the methods of measuring microbial growth. Serratia marcescens on a cracker. This gram-negative rod produces the pigment prodigiosin, causing bright red colonies when the bacteria grow at room temperature. 149 M06_TORT9150_12_CH06_pp149-175.indd 149 10/10/14 10:25 AM  150 Part one Fundamentals of Microbiology The Requirements for Growth Clinical Case: Glowing in the Dark Learning Objectives Reginald MacGruder, an investigator at the Centers for Disease 6-1 Classify microbes into five groups on the basis of preferred Control and Prevention (CDC) in Atlanta, Georgia, has a temperature range. mystery on his hands. Earlier this year, he was involved in the 6-2 Identify how and why the pH of culture media is controlled. recall of an intravenous heparin solution that was blamed 6-3 Explain the importance of osmotic pressure to microbial growth. for causing Pseudomonas fluorescens bloodstream infections 6-4 Name a use for each of the four elements (carbon, nitrogen, sulfur, in patients in four different states. It seemed that everything and phosphorus) needed in large amounts for microbial growth. was under control, but now, three months after the recall, 19 patients in two other states develop the same P. fluorescens 6-5 Explain how microbes are classified on the basis of oxygen bloodstream infections. It makes no sense to Dr. MacGruder; requirements. how could this infection be popping up again so soon after 6-6 Identify ways in which aerobes avoid damage by toxic forms the recall? Could another heparin batch be tainted? of oxygen. What is P. fluorescens? Read on to find out. The requirements for micro- bial growth can be divided ASM: The survival and growth of any into two main categories: microorganism in a given environment depends on its metabolic characteristics. 150 ▲ 162 170 172 physical and chemical. Physi- cal aspects include tempera- ture, pH, and osmotic pressure. Chemical requirements include sources of carbon, nitrogen, sulfur, phosphorus, oxygen, trace Most bacteria grow only within a limited range of temperatures, elements, and organic growth factors. and their maximum and minimum growth temperatures are only about 30°C apart. They grow poorly at the high and low Physical Requirements temperature extremes within their range. Each bacterial species grows at particular minimum, opti- Temperature mum, and maximum temperatures. The minimum growth Most microorganisms grow well at the temperatures that temperature is the lowest temperature at which the species humans favor. However, certain bacteria are capable of growing will grow. The optimum growth temperature is the tempera- at extremes of temperature that would certainly hinder the sur- ture at which the species grows best. The maximum growth vival of almost all eukaryotic organisms. temperature is the highest temperature at which growth is Microorganisms are classified into three primary groups on possible. By graphing the growth response over a temperature the basis of their preferred range of temperature: psychrophiles range, we can see that the optimum growth temperature is usu- (cold-loving microbes), mesophiles (moderate-temperature– ally near the top of the range; above that temperature the rate of loving microbes), and thermophiles (heat-loving microbes). growth drops off rapidly (Figure 6.1). This happens presumably Figure 6.1 Typical growth rates of different types of microorganisms in response to Thermophiles temperature. The peak of the curve represents optimum growth (fastest reproduction). Notice Hyperthermophiles that the reproductive rate drops off very quickly Mesophiles at temperatures only a little above the optimum. At either extreme of the temperature range, the Psychrotrophs reproductive rate is much lower than the rate at the Psychrophiles optimum temperature. Q  hy is it difficult to define psychrophile, W mesophile, and thermophile? Rate of growth –10 0 10 20 30 40 50 60 70 80 90 100 110 Temperature (°C) M06_TORT9150_12_CH06_pp149-175.indd 150 10/10/14 10:25 AM  Chapter 6  Microbial Growth 151 °F °C the term psychrotrophs, which food microbiologists favor, for this group of spoilage microorganisms. 130 Refrigeration is the most common method of preserving 260 120 household food supplies. It is based on the principle that microbial 240 110 reproductive rates decrease at low temperatures. Although microbes 220 100 Temperatures in this range destroy most usually survive even subfreezing temperatures (they might microbes, although lower temperatures 200 90 take more time. become entirely dormant), they gradually decline in number. 180 80 Some species decline faster than others. Psychrotrophs do not 160 70 grow well at low temperatures, except in comparison with other 140 60 Very slow bacterial growth. organisms; given time, however, they are able to slowly degrade 120 50 food. Such spoilage might take the form of mold mycelium, slime 100 Danger 40 on food surfaces, or off-tastes or off-colors in foods. The tem- Rapid growth of bacteria; some may zone 30 produce toxins. perature inside a properly set refrigerator will greatly slow the 80 20 growth of most spoilage organisms and will entirely prevent the 60 10 Many bacteria survive; some may grow. growth of all but a few pathogenic bacteria. Figure 6.2 illustrates 40 0 Refrigerator temperatures; may allow slow the importance of low temperatures for preventing the growth 20 growth of spoilage bacteria, very few pathogens. –10 of spoilage and disease organisms. When large amounts of food 0 No significant growth below freezing. must be refrigerated, it is important to remember that a large –20 –20 –30 quantity of warm food cools at a relatively slow rate (Figure 6.3). Mesophiles, with an optimum growth temperature of 25–40°C, are the most common type of microbe. Organisms that have adapted to live in the bodies of animals usually have an optimum temperature close to that of their hosts. The ­optimum Figure 6.2 Food preservation temperatures. Low temperatures decrease microbial reproduction rates, which is the basic principle of 80 refrigeration. There are always some exceptions to the temperature responses shown here; for example, certain bacteria grow well at high 70 temperatures that would kill most bacteria, and a few bacteria can actually Darker band shows 15 cm (6") deep approximate temperature grow at temperatures well below freezing. 60 range at which Bacillus Q  hich bacterium would theoretically be more likely to grow W 50 cereus multiplies in rice at refrigerator temperatures: a human intestinal pathogen or a soilborne plant pathogen? 43°C Temperature (°C) 40 5 cm (2") deep because the high temperature has inactivated necessary enzy- 30 matic systems of the cell. The ranges and maximum growth temperatures that define 20 bacteria as psychrophiles, mesophiles, or thermophiles are not 15°C 10 rigidly defined. Psychrophiles, for example, were originally considered simply to be organisms capable of growing at 0°C. 0 However, there seem to be two fairly distinct groups capable of Refrigerator air growth at that temperature. One group, composed of psychro- –10 philes in the strictest sense, can grow at 0°C but has an optimum growth temperature of about 15°C. Most of these organisms are 0 1 2 3 4 5 6 7 8 so sensitive to higher temperatures that they will not even grow Hours in a reasonably warm room (25°C). Found mostly in the oceans’ Figure 6.3 The effect of the amount of food on its cooling depths or in certain polar regions, such organisms seldom cause rate in a refrigerator and its chance of spoilage. Notice that in problems in food preservation. The other group that can grow this example, the pan of rice with a depth of 5 cm (2 in) cooled through at 0°C has higher optimum temperatures, usually 20–30°C the incubation temperature range of the Bacillus cereus in about 1 hour, and cannot grow above about 40°C. Organisms of this type are whereas the pan of rice with a depth of 15 cm (6 in) remained in this temperature range for about 5 hours. much more common than psychrophiles and are the most likely to be encountered in low-temperature food spoilage because Q  Given a shallow pan and a deep pot with the same volume, which they grow fairly well at refrigerator temperatures. We will use would cool faster? Why? M06_TORT9150_12_CH06_pp149-175.indd 151 10/10/14 10:25 AM  152 Part one Fundamentals of Microbiology Figure 6.4 Plasmolysis. Plasma Plasma membrane Cell wall H2O membrane Q   Why is osmotic pressure an important factor in microbial growth? Cytoplasm Cytoplasm NaCl 0.85% NaCl 10% (a) Cell in isotonic solution. Under these (b) Plasmolyzed cell in hypertonic solution. conditions, the solute concentration in the If the concentration of solutes such as NaCl cell is equivalent to a solute concentration is higher in the surrounding medium than in of 0.85% sodium chloride (NaCl). the cell (the environment is hypertonic), water tends to leave the cell. Growth of the cell is inhibited. temperature for many pathogenic bacteria is about 37°C, and few bacteria grow at an acidic pH below about pH 4. This is why a incubators for clinical cultures are usually set at about this tem- number of foods, such as sauerkraut, pickles, and many cheeses, perature. The mesophiles include most of the common spoilage are preserved from spoilage by acids produced by bacterial and disease organisms. ­fermentation. Nonetheless, some bacteria, called acidophiles, Thermophiles are microorganisms capable of growth at are remarkably tolerant of acidity. One type of chemoautotrophic high temperatures. Many of these organisms have an opti- bacteria, which is found in the drainage water from coal mines mum growth temperature of 50–60°C, about the temperature and oxidizes sulfur to form sulfuric acid, can survive at a pH 1. of water from a hot water tap. Such temperatures can also be Molds and yeasts will grow over a greater pH range than bacteria reached in sunlit soil and in thermal waters such as hot springs. will, but the optimum pH of molds and yeasts is generally below Remarkably, many thermophiles cannot grow at temperatures that of bacteria, usually about pH 5 to 6. Alkalinity also inhibits below about 45°C. Endospores formed by thermophilic bacteria microbial growth but is rarely used to preserve foods. are unusually heat resistant and may survive the usual heat treat- When bacteria are cultured in the laboratory, they often ment given canned goods. Although elevated storage tempera- produce acids that eventually interfere with their own growth. tures may cause surviving endospores to germinate and grow, To neutralize the acids and maintain the proper pH, chemical thereby spoiling the food, these thermophilic bacteria are not buffers are included in the growth medium. The peptones and considered a public health problem. Thermophiles are impor- amino acids in some media act as buffers, and many media also tant in organic compost piles (see Figure 27.8 on page 779), in contain phosphate salts. Phosphate salts have the advantage of which the temperature can rise rapidly to 50–60°C. exhibiting their buffering effect in the pH growth range of most Some microbes, members of the Archaea (page 4), have bacteria. They are also nontoxic; in fact, they provide phospho- an optimum growth temperature of 80°C or higher. These rus, an essential nutrient. organisms are called hyperthermophiles, or sometimes extreme thermophiles. Most of these organisms live in hot springs Osmotic Pressure associated with volcanic activity, and sulfur is usually impor- Microorganisms obtain almost all their nutrients in solu- tant in their metabolic activity. The known record for bacterial tion from the surrounding water. Thus, they require water for growth and replication at high temperatures is about 121°C near growth, and their composition is 80–90% water. High osmotic deep-sea hydrothermal vents. See the Applications of Microbi- pressures have the effect of removing necessary water from a ology box “Life in the Extreme.” The immense pressure in the cell. When a microbial cell is in a solution whose concentra- ocean depths prevents water from boiling even at temperatures tion of solutes is higher than in the cell (the environment is well above 100°C. hypertonic to the cell), the cellular water passes out through the plasma membrane to the high solute concentration. (See the pH ­discussion of osmosis in Chapter 4, pages 88–89, and review Recall from Chapter 2 (pages 33–34) that the pH refers to the Figure 4.18 for the three types of solution environments a cell acidity or alkalinity of a solution. Most bacteria grow best in a may encounter.) This osmotic loss of water causes plasmolysis, narrow pH range near neutrality, between pH 6.5 and 7.5. Very or shrinkage of the cell’s cytoplasm (Figure 6.4). M06_TORT9150_12_CH06_pp149-175.indd 152 10/10/14 10:25 AM  153 Applications of Microbiology Life in the Extreme Until humans explored the deep-ocean floor, create an environment that supports higher These enzymes can be used in automatic scientists believed that only a few forms of life life forms. Hydrothermal vents in the seafloor thermalcyclers to repeat the heating and could survive in that high-pressure, completely supply the H2S and CO2. cooling cycles, allowing many copies of DNA dark, oxygen-poor environment. Then, in 1977, to be made easily and quickly. New Products from Alvin, the deep-sea submersible, carried two Hydrothermal Vents scientists 2600 meters below the surface at Terrestrial fungi and bacteria have had the Galápagos Rift (about 350 km northeast a major impact on the development of of the Galápagos Islands). There, amid the biotechnology. Hydrothermal vents are vast expanse of barren basalt rocks, the the next frontier in the hunt for new scientists found unexpectedly rich oases of life. products. In 2010 a peptide produced Superheated water from beneath the seafloor by Thermovibrio ammonificans was was rising through fractures in the Earth’s crust shown to induce apoptosis (cell death) called vents. They discovered that mats of and thus potential anticancer activity. bacteria were growing along the sides of the Researchers are growing Pyrococcus vents, where temperatures exceeded 100°C furiosus because it produces alternative (see the figure). fuels, hydrogen gas and butanol. DNA Ecosystem of the Hydrothermal Vents polymerases (enzymes that synthesize Life at the surface of the world’s oceans DNA) isolated from two archaea living Biofilm depends on photosynthetic organisms, such near deep-sea vents are being used in as plants and algae, which harness the sun’s the polymerase chain reaction (PCR), energy to fix carbon dioxide (CO2) to make a technique for making many copies Tubeworm carbohydrates. At the deep-ocean floor, of DNA. In PCR, single-stranded DNA where no light penetrates, photosynthesis is made by heating a chromosome is not possible. The scientists found that the fragment to 98°C and cooling it so 1m primary producers at the ocean floor are that DNA polymerase can copy each A white microbial biofilm is visible on this chemoautotrophic bacteria. Using chemical strand. DNA polymerases from Thermococcus deep-sea hydrothermal vent. Giant tubeworms energy from hydrogen sulfide (H2S) as a source litoralis, called VentR, and from Pyrococcus, are visible. Water is being emitted through the ocean floor at temperatures above 100°C. of energy to fix CO2, the chemoautotrophs called Deep VentR, are not denatured at 98°C. The growth of the cell is inhibited as the plasma membrane organisms. A few species of facultative halophiles can tolerate pulls away from the cell wall. Thus, the addition of salts (or even 15% salt. other solutes) to a solution, and the resulting increase in osmotic Most microorganisms, however, must be grown in a medium pressure, can be used to preserve foods. Salted fish, honey, and that is nearly all water. For example, the concentration of agar sweetened condensed milk are preserved largely by this mecha- (a complex polysaccharide isolated from marine algae) used nism; the high salt or sugar concentrations draw water out of any to solidify microbial growth media is usually about 1.5%. If microbial cells that are present and thus prevent their growth. markedly higher concentrations are used, the increased osmotic These effects of osmotic pressure are roughly related to the pressure can inhibit the growth of some bacteria. number of dissolved molecules and ions in a volume of solution. If the osmotic pressure is unusually low (the environment is Some organisms, called extreme halophiles, have adapted hypotonic)—such as in distilled water, for example—water tends so well to high salt concentrations that they actually require to enter the cell rather than leave it. Some microbes that have a them for growth. In this case, they may be termed obligate relatively weak cell wall may be lysed by such treatment. halophiles. Organisms from such saline waters as the Dead Sea Check Your Understanding often require nearly 30% salt, and the inoculating loop (a device for handling bacteria in the laboratory) used to transfer them ✓ Why are hyperthermophiles that grow at temperatures above 100°C seemingly limited to oceanic depths? 6-1 must first be dipped into a saturated salt solution. More com- mon are facultative halophiles, which do not require high salt ✓ Other than controlling acidity, what is an advantage of using phosphate salts as buffers in growth media? 6-2 concentrations but are able to grow at salt concentrations up ✓ Why might primitive civilizations have used food preservation to 2%, a concentration that inhibits the growth of many other techniques that rely on osmotic pressure? 6-3 M06_TORT9150_12_CH06_pp149-175.indd 153 10/10/14 10:25 AM  154 Part one Fundamentals of Microbiology Chemical Requirements Trace Elements Microbes require very small amounts of other mineral ele- Carbon ments, such as iron, copper, molybdenum, and zinc; these are Besides water, one of the most important requirements for referred to as trace elements. Most are essential for the func- microbial growth is carbon. Carbon is the structural backbone tions of certain enzymes, usually as cofactors. Although these of living matter; it is needed for all the organic compounds that elements are sometimes added to a laboratory medium, they are make up a living cell. Half the dry weight of a typical bacte- usually assumed to be naturally present in tap water and other rial cell is carbon. Chemoheterotrophs get most of their carbon components of media. Even most distilled waters contain ade- from the source of their energy—organic materials such as pro- quate amounts, but tap water is sometimes specified to ensure teins, carbohydrates, and lipids. Chemoautotrophs and photo- that these trace minerals will be present in culture media. autotrophs derive their carbon from carbon dioxide. Oxygen Nitrogen, Sulfur, and Phosphorus We are accustomed to thinking of molecular oxygen (O2) as In addition to carbon, microorganisms need other elements a necessity of life, but it is actually in a sense a poisonous gas. to synthesize cellular material. For example, protein synthe- Very little molecular oxygen existed in the atmosphere during sis requires considerable amounts of nitrogen as well as some most of Earth’s history—in fact, it is possible that life could not sulfur. The syntheses of DNA and RNA also require nitro- have arisen had oxygen been present. However, many current gen and some phosphorus, as does the synthesis of ATP, the forms of life have metabolic systems that require oxygen for molecule so important for the storage and transfer of chemical aerobic respiration. Hydrogen atoms that have been stripped energy within the cell. Nitrogen makes up about 14% of the dry from organic compounds combine with oxygen to form water, weight of a bacterial cell, and sulfur and phosphorus together as shown in Figure 5.14 (page 125). This process yields a great constitute about another 4%. deal of energy while neutralizing a potentially toxic gas—a very Organisms use nitrogen primarily to form the amino group neat solution, all in all. of the amino acids of proteins. Many bacteria meet this require- Microbes that use molecular oxygen (aerobes) extract more ment by decomposing protein-containing material and rein- energy from nutrients than microbes that do not use oxygen corporating the amino acids into newly synthesized proteins (anaerobes). Organisms that require oxygen to live are called and other nitrogen-containing compounds. Other bacteria obligate aerobes (Table 6.1a). use nitrogen from ammonium ions (NH41), which are already Obligate aerobes are at a disadvantage because oxygen is in the reduced form and are usually found in organic cellular poorly soluble in the water of their environment. Therefore, material. Still other bacteria are able to derive nitrogen from many of the aerobic bacteria have developed, or retained, the nitrates (compounds that dissociate to give the nitrate ion, ability to continue growing in the absence of oxygen. Such NO32, in solution). organisms are called facultative anaerobes (Table 6.1b). In Some important bacteria, including many of the photosyn- other words, facultative anaerobes can use oxygen when it thesizing cyanobacteria (page 137), use gaseous nitrogen (N2) is present but are able to continue growth by using fermen- directly from the atmosphere. This process is called nitrogen tation or anaerobic respiration when oxygen is not available. fixation. Some organisms that can use this method are free-liv- However, their efficiency in producing energy decreases in the ing, mostly in the soil, but others live cooperatively in symbio- absence of oxygen. An example of facultative anaerobes is the sis with the roots of legumes such as clover, soybeans, alfalfa, familiar Escherichia coli that are found in the human intes- beans, and peas. The nitrogen fixed in the symbiosis is used by tinal tract. Many yeasts are also facultative anaerobes. Many both the plant and the bacterium (see Chapter 27). microbes are able to substitute other electron acceptors, such Sulfur is used to synthesize sulfur-containing amino acids as nitrate ions, for oxygen, which is something humans are and vitamins such as thiamine and biotin. Important natural unable to do. (See the discussion of anaerobic respiration in sources of sulfur include the sulfate ion (SO422), hydrogen sul- Chapter 5, page 126). fide, and the sulfur-containing amino acids. Obligate anaerobes (Table 6.1c) are bacteria that are unable Phosphorus is essential for the synthesis of nucleic acids and to use molecular oxygen for energy-yielding reactions. In fact, the phospholipids of cell membranes. Among other places, it is most are harmed by it. The genus Clostridium (klos-TRID- also found in the energy bonds of ATP. A source of phospho- ē-um), which contains the species that cause tetanus and rus is the phosphate ion (PO432). Potassium, magnesium, and botulism, is the most familiar example. These bacteria do use calcium are also elements that microorganisms require, often as oxygen atoms present in cellular materials; the atoms are usually cofactors for enzymes (see Chapter 5, pages 113–114). obtained from water. M06_TORT9150_12_CH06_pp149-175.indd 154 10/10/14 10:25 AM  Chapter 6  Microbial Growth 155 Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria a. Obligate b. Facultative c. Obligate d. Aerotolerant Aerobes Anaerobes Anaerobes Anaerobes e. Microaerophiles Effect of Oxygen on Only aerobic growth; Both aerobic and Only anaerobic Only anaerobic Only aerobic growth; Growth oxygen required. anaerobic growth; growth; growth growth; but growth oxygen required in low greater growth in ceases in presence continues in concentration. presence of oxygen. of oxygen. presence of oxygen. Bacterial Growth in Tube of Solid Growth Medium Explanation of Growth Growth occurs Growth is best Growth occurs only Growth occurs Growth occurs Patterns only where high where most oxygen where there is no evenly; oxygen has only where a low concentrations of is present, but oxygen. no effect. concentration of oxygen have diffused occurs throughout oxygen has diffused into the medium. tube. into medium. Explanation of Presence of enzymes Presence of enzymes Lacks enzymes to Presence of one Produce lethal Oxygen’s Effects catalase and superoxide catalase and SOD neutralize harmful enzyme, SOD, allows amounts of toxic dismutase (SOD) allows allows toxic forms forms of oxygen; harmful forms of forms of oxygen if toxic forms of oxygen to of oxygen to be cannot tolerate oxygen to be partially exposed to normal be neutralized; can use neutralized; can use oxygen. neutralized; tolerates atmospheric oxygen. oxygen. oxygen. oxygen. Understanding how organisms can be harmed by oxygen 3. The hydrogen peroxide produced in this reaction contains requires a brief discussion of the toxic forms of oxygen: the peroxide anion O22− and is also toxic. It is the active principle in the antimicrobial agents hydrogen peroxide 1. Singlet oxygen (1O22) is normal molecular oxygen (O2) that and benzoyl peroxide. (See Chapter 7, page 194). Because has been boosted into a higher-energy state and is extremely the hydrogen peroxide produced during normal aerobic reactive. respiration is toxic, microbes have developed enzymes to 2. Superoxide radicals (O22), or superoxide anions, are neutralize it. The most familiar of these is catalase, which formed in small amounts during the normal respiration converts it into water and oxygen: of organisms that use oxygen as a final electron acceptor, forming water. In the presence of oxygen, obligate 2 H2O2 ¡ 2H2O + O2 anaerobes also appear to form some superoxide radicals, Catalase is easily detected by its action on hydrogen which are so toxic to cellular components that all organisms peroxide. When a drop of hydrogen peroxide is added attempting to grow in atmospheric oxygen must produce to a colony of bacterial cells producing catalase, oxygen an enzyme, superoxide dismutase (SOD), to neutralize bubbles are released. Anyone who has put hydrogen them. Their toxicity is caused by their great instability, peroxide on a wound will recognize that cells in human which leads them to steal an electron from a neighboring tissue also contain catalase. The other enzyme that breaks molecule, which in turn becomes a radical and steals down hydrogen peroxide is peroxidase, which differs from an electron, and so on. Aerobic bacteria, facultative catalase in that its reaction does not produce oxygen: anaerobes growing aerobically, and aerotolerant anaerobes (discussed shortly) produce SOD, with which they convert H2O2 + 2 H+ ¡ 2 H2O the superoxide radical into molecular oxygen (O2) and hydrogen peroxide (H2O2): Another important form of reactive oxygen is ozone (O3) (discussed on page 194). O2 - + O2 - + 2 H + ¡ H2O2 + O2 4. The hydroxyl radical (OH·) is another intermediate form of oxygen and probably the most reactive. It is formed in M06_TORT9150_12_CH06_pp149-175.indd 155 10/10/14 10:25 AM  156 Part one Fundamentals of Microbiology the cellular cytoplasm by ionizing radiation. Most aerobic Check Your Understanding respiration produces traces of hydroxyl radicals, but they ✓ If bacterial cells were given a sulfur source containing radioactive are transient. sulfur (35S) in their culture media, in what molecules would the 35 S be found in the cells? 6-4 These toxic forms of oxygen are an essential component of ✓ How would one determine whether a microbe is a strict one of the body’s most important defenses against pathogens, anaerobe? 6-5 phagocytosis (see page 450 and Figure 16.7). In the phagolyso- ✓ Oxygen is so pervasive in the environment that it would be very some of the phagocytic cell, ingested pathogens are killed by difficult for a microbe to always avoid physical contact with it. exposure to singlet oxygen, superoxide radicals, peroxide anions What, therefore, is the most obvious way for a microbe to avoid of hydrogen peroxide, and hydroxyl radicals and other oxidative damage? 6-6 compounds. Obligate anaerobes usually produce neither superoxide dis- mutase nor catalase. Because aerobic conditions probably lead Biofilms See how biofilms affect a patient’s health @. to an accumulation of superoxide radicals in their cytoplasm, obligate anaerobes are extremely sensitive to oxygen. Learning Objective Aerotolerant anaerobes (Table 6.1d) cannot use oxygen for 6-7 Describe the formation of biofilms and their potential for growth, but they tolerate it fairly well. On the surface of a solid causing infection. medium, they will grow without the use of special techniques (discussed later) required for obligate anaerobes. Many of the In nature, microorganisms seldom live in the isolated single-spe- aerotolerant bacteria characteristically ferment carbohydrates cies colonies that we see on laboratory plates. They more typically to lactic acid. As lactic acid accumulates, it inhibits the growth live in communities called biofilms, which are a thin, slimy layer of aerobic competitors and establishes a favorable ecological encasing bacteria that adheres to a surface. This fact was not well niche for lactic acid producers. Common examples of lactic appreciated until the development of confocal microscopy (see acid–producing aerotolerant anaerobes are the lactobacilli used page 58) made the three-dimensional structure of biofilms more in the production of many acidic fermented foods, such as pick- visible. A biofilm also can be considered a hydrogel, which is a les and cheese. In the laboratory, they are handled and grown complex polymer containing many times its dry weight in water. much like any other bacteria, but they make no use of the oxy- Cell-to-cell chemical communication, or quorum sensing, allows gen in the air. These bacteria can tolerate oxygen because they bacteria to coordinate their activity and group together into com- possess SOD or an equivalent system that neutralizes the toxic munities that provide benefits not unlike those of multicellular forms of oxygen previously discussed. organisms (see the box in Chapter 3, page 54). Therefore, biofilms A few bacteria are microaerophiles (Table 6.1e). They are are not just bacterial slime layers but biological systems; the bacte- aerobic; they do require oxygen. However, they grow only in ria are organized into a coordinated, functional community. Bio- oxygen concentrations lower than those in air. In a test tube of films are usually attached to a surface, such as a rock in a pond, a solid nutrient medium, they grow only at a depth where small human tooth (plaque; see Figure 25.3 on page 710), or a mucous amounts of oxygen have diffused into the medium; they do not membrane. This community might be of a single species or of a grow near the oxygen-rich surface or below the narrow zone of diverse group of microorganisms. Biofilms also might take other, adequate oxygen. This limited tolerance is probably due to their more varied forms. In fast-flowing streams, the biofilm might sensitivity to superoxide radicals and peroxides, which they be in the form of filamentous streamers. Within a biofilm com- produce in lethal concentrations under oxygen-rich conditions. munity, the bacteria are able to share nutrients and are sheltered from harmful factors in the environment, such as desiccation, Organic Growth Factors antibiotics, and the body’s immune system. The close proxim- Essential organic compounds an organism is unable to synthe- ity of microorganisms within size are known as organic growth factors; they must be directly a biofilm might also have the ASM: Most bacteria in nature live advantage of facilitating the in biofilm communities. obtained from the environment. One group of organic growth factors for humans is vitamins. Most vitamins function as coen- transfer of genetic information zymes, the organic cofactors required by certain enzymes in by, for example, conjugation. order to function. Many bacteria can synthesize all their own A biofilm usually begins to form when a free-swimming vitamins and do not depend on outside sources. However, some (planktonic) bacterium attaches to a surface. (See Figure 1.8 bacteria lack the enzymes needed for the synthesis of certain on page 15.) If these bacteria grew in a uniformly thick mono- vitamins, and for them those vitamins are organic growth fac- layer, they would become overcrowded, nutrients would not be tors. Other organic growth factors required by some bacteria available in lower depths, and toxic wastes could accumulate. are amino acids, purines, and pyrimidines. Microorganisms in biofilm communities sometimes avoid these problems by forming pillar-like structures (Figure 6.5) with M06_TORT9150_12_CH06_pp149-175.indd 156 10/10/14 10:25 AM  Chapter 6  Microbial Growth 157 channels between them, through which water can carry incom- Clumps of bacteria Migrating ing nutrients and outgoing wastes. This constitutes a primitive adhering to surface clump of circulatory system. Individual microbes and clumps of slime bacteria occasionally leave the established biofilm and move to a new location where the biofilm becomes extended. Such a biofilm is generally composed of a surface layer about 10 mm thick, with pillars that extend up to 200 mm above it. The microorganisms in biofilms can work cooperatively to carry out complex tasks. For example, the digestive systems of ruminant animals, such as cattle, require many different microbial species to break down cellulose. The microbes in a ruminant’s digestive system are located mostly within biofilm Surface Water currents communities. Biofilms are also essential elements in the proper functioning of sewage treatment systems, which we will discuss Water currents move, as shown by the blue arrow, among 10 μm pillars of slime formed by the growth of bacteria attached to in Chapter 27. They can also, however, be a problem in pipes solid surfaces. This allows efficient access to nutrients and removal of and tubing, where their accumulations impede circulation. bacterial waste products. Individual slime-forming bacteria or bacteria in clumps of slime detach and move to new locations. Biofilms are an important factor in human health. For exam- ple, microbes in biofilms are probably 1000 times more resistant Figure 6.5 Biofilms. to microbicides. Experts at the Centers for Disease Control and Prevention (CDC) estimate that 70% of human bacterial infec- Q  hy is the prevention of biofilms important in a health care W environment? tions involve biofilms. Most health care associated infections are probably related to biofilms on medical catheters (see Figure 1.8 on page 15 and Figure 21.3 on page 582). In fact, biofilms form on almost all indwelling medical devices, including mechanical Culture Media heart valves. Biofilms, which also can include those formed by Learning Objectives fungi such as Candida, are encountered in many disease con- 6-8 Distinguish chemically defined and complex media. ditions, such as infections related to the use of contact lenses, 6-9 Justify the use of each of the following: anaerobic dental caries (see page 709), and infections by pseudomonad techniques, living host cells, candle jars, selective and bacteria (see page 296). differential media, enrichment medium. One approach to preventing biofilm formation is to incor- 6-10 Differentiate biosafety levels 1, 2, 3, and 4. porate antimicrobials into surfaces on which biofilms might form (see page 54). Because the chemical signals that allow A nutrient material prepared for the growth of microorganisms quorum sensing are essential to biofilm formation, research in a laboratory is called a culture medium. Some bacteria can is under way to determine the makeup of these chemical sig- grow well on just about any culture medium; others require nals and perhaps block them. Another approach involves the special media, and still others cannot grow on any nonliving discovery that lactoferrin (see page 461), which is abundant in medium yet developed. Microbes that are introduced into a many human secretions, can inhibit biofilm formation. Lac- culture medium to initiate growth are called an inoculum. The toferrin binds iron, especially among the pseudomonads that microbes that grow and multiply in or on a culture medium are are responsible for cystic fibrosis biofilms, the cause of the referred to as a culture. pathology of this hereditary disease. The lack of iron inhibits Suppose we want to grow a culture of a certain microorgan- the surface motility essential for the aggregation of the bacte- ism, perhaps the microbes from a particular clinical specimen. ria into biofilms. What criteria must the culture medium meet? First, it must con- Most laboratory methods in microbiology today use organ- tain the right nutrients for the specific microorganism we want isms being cultured in their planktonic mode. However, to grow. It should also contain sufficient moisture, a properly microbiologists now predict that there will be an increas- adjusted pH, and a suitable level of oxygen, perhaps none at all. ing focus on how microorganisms actually live in relation to The medium must initially be sterile—that is, it must initially one another and that this will be considered in industrial and contain no living microorganisms—so that the culture will medical research. contain only the microbes (and their offspring) we add to the medium. Finally, the growing culture should be incubated at the Check Your Understanding proper temperature. ✓ Identify a way in which pathogens find it advantageous to form A wide variety of media are available for the growth of biofilms. 6-7 ­microorganisms in the laboratory. Most of these media, which M06_TORT9150_12_CH06_pp149-175.indd 157 13/10/14 5:43 PM  158 Part one Fundamentals of Microbiology f­actors that serve as a source of carbon and energy. For exam- A Chemically Defined Medium for ple, as shown in Table 6.2, glucose is included in the medium for Growing a Typical Chemoheterotroph, Table 6.2 Such as Escherichia coli growing the chemoheterotroph E. coli. As Table 6.3 shows, many organic growth factors must be Constituent Amount provided in the chemically defined medium used to cultivate Glucose 5.0 g a species of Leuconostoc. Organisms that require many growth factors are described as fastidious. Organisms of this type, Ammonium phosphate, monobasic (NH4H2PO4) 1.0 g such as Lactobacillus (page 310), are sometimes used in tests Sodium chloride (NaCl) 5.0 g that determine the concentration of a particular vitamin in a substance. To perform such a microbiological assay, a growth Magnesium sulfate (MgSO4 # 7H2O) 0.2 g medium is prepared that contains all the growth requirements Potassium phosphate, dibasic (K2HPO4) 1.0 g of the bacterium except the vitamin being assayed. Then the Water 1 liter medium, test substance, and bacterium are combined, and the growth of bacteria is measured. This bacterial growth, which is reflected by the amount of lactic acid produced, will be pro- are available from commercial sources, have premixed compo- portional to the amount of vitamin in the test substance. The nents and require only the addition of water and then sterilization. more lactic acid, the more the Lactobacillus cells have been able Media are constantly being developed or revised for use in the iso- to grow, so the more vitamin is present. lation and identification of bacteria that are of interest to research- ers in such fields as food, water, and clinical microbiology. When it is desirable to grow bacteria on a solid medium, a Defined Culture Medium for solidifying agent such as agar is added to the medium. A com- Table 6.3 Leuconostoc mesenteroides plex polysaccharide derived from a marine alga, agar has long Carbon and Energy been used as a thickener in foods such as jellies and ice cream. Agar has some very important properties that make it valu- Glucose, 25 g able to microbiology, and no satisfactory substitute has yet been Salts found. Few microbes can degrade agar, so it remains solid. Also, NH4Cl, 3.0 g agar liquefies at about 100°C (the boiling point of water) and at sea level remains liquid until the temperature drops to about K2HPO4*, 0.6 g 40°C. For laboratory use, agar is held in water baths at about KH2PO4*, 0.6 g 50°C. At this temperature, it does not injure most bacteria when it is poured over them (as shown in Figure 6.17a, page 168). MgSO4, 0.1 g Once the agar has solidified, it can be incubated at temperatures Amino Acids, 100–200 mg each approaching 100°C before it again liquefies; this property is par- ticularly useful when thermophilic bacteria are being grown. Alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, Agar media are usually contained in test tubes or Petri proline, serine, threonine, tryptophan, tyrosine, valine dishes. The test tubes are called slants when their contents are Purines and Pyrimidines, 10 mg of each allowed to solidify with the tube held at an angle so that a large surface area for growth is available. When the agar solidifies in Adenine, guanine, uracil, xanthine a vertical tube, it is called a deep. Petri dishes, named for their Vitamins, 0.01–1 mg each inventor, are shallow dishes with a lid that nests over the bottom to prevent contamination; when filled, they are called Petri (or Biotin, folate, nicotinic acid, pyridoxal, pyridoxamine, pyridoxine, riboflavin, thiamine, pantothenate, p-aminobenzoic acid culture) plates. Trace Elements, 2–10 mg each Chemically Defined Media Fe, Co, Mn, Zn, Cu, Ni, Mo To support microbial growth, a medium must provide an energy Buffer, pH 7 source, as well as sources of carbon, nitrogen, sulfur, phospho- rus, and any organic growth factors the organism is unable to Sodium acetate, 25 g synthesize. A chemically defined medium is one whose exact Distilled Water, 1000 ml chemical composition is known. For a chemoheterotroph, the chemically defined medium must contain organic growth *Also serves as buffer. M06_TORT9150_12_CH06_pp149-175.indd 158 10/10/14 10:25 AM  Chapter 6  Microbial Growth 159 When the culture must be grown in Petri plates to observe Composition of Nutrient Agar, individual colonies, several methods are available. Laboratories a Complex Medium for the Growth Table 6.4 of Heterotrophic Bacteria that work with relatively few culture plates at a time can use systems that can incubate the microorganisms in sealed boxes Constituent Amount and jars in which the oxygen is chemically removed after the Peptone (partially digested protein) 5.0 g culture plates have been introduced and the container sealed as shown in Figure 6.6. The envelope of chemicals (the active ingre- Beef extract 3.0 g dient is ascorbic acid) is simply opened to expose it to oxygen in Sodium chloride 8.0 g the container’s atmosphere. The atmosphere in such containers usually has less than 5% oxygen, about 18% CO2, and no hydro- Agar 15.0 g gen. In a recently introduced system, each individual Petri plate Water 1 liter (OxyPlate) becomes an anaerobic chamber. The medium in the plate contains an enzyme, oxyrase, which combines oxygen with hydrogen, removing oxygen as water is formed. Laboratories that have a large volume of work with anaer- Complex Media obes often use an anaerobic chamber, such as that shown in Chemically defined media are usually reserved for laboratory Figure 6.7. The chamber is filled with inert gases (typically experimental work or for the growth of autotrophic bacteria. about 85% N2 , 10% H2 , and 5% CO2) and is equipped with air Most heterotrophic bacteria and fungi, such as you would work locks to introduce cultures and materials. with in an introductory lab course, are routinely grown on complex media made up of nutrients including extracts from yeasts, meat, or plants, or digests of proteins from these and Lid with Clamp with other sources. The exact chemical composition varies slightly O-ring gasket clamp screw from batch to batch. Table 6.4 shows one widely used recipe. In complex media, the energy, carbon, nitrogen, and sul- fur requirements of the growing microorganisms are provided Envelope containing primarily by protein. Proteins are large, relatively insoluble inorganic carbonate, molecules that only a minority of microorganisms can utilize activated carbon, ascorbic acid, CO2 directly. Partial digestion by acids or enzymes reduces proteins and water to shorter chains of amino acids called peptones. These small, H2 soluble fragments can be digested by most bacteria. Vitamins and other organic growth factors are provided by meat extracts or yeast extracts. The soluble vitamins and min- Anaerobic indicator (methylene blue) erals from the meats or yeasts are dissolved in the extracting water, which is then evaporated, so these factors are concen- trated. (These extracts also supplement the organic nitrogen and carbon compounds.) Yeast extracts are particularly rich in the B vitamins. If a complex medium is in liquid form, it is Petri plates called nutrient broth. When agar is added, it is called nutrient agar. (This terminology can be confusing; just remember that agar itself is not a nutrient.) Anaerobic Growth Media and Methods Figure 6.6 A jar for cultivating anaerobic bacteria on Petri The cultivation of anaerobic bacteria poses a special problem. plates. When water is mixed with the chemical packet containing Because anaerobes might be killed by exposure to oxygen, special sodium bicarbonate and sodium borohydride, hydrogen and carbon dioxide are generated. Reacting on the surface of a palladium catalyst media called reducing media must be used. These media con- in a screened reaction chamber, which may also be incorporated into tain ingredients, such as sodium thioglycolate, that chemically the chemical packet, the hydrogen and atmospheric oxygen in the jar ­combine with dissolved oxygen and deplete the oxygen in the combine to form water. The oxygen is thus removed. Also in the jar is culture medium. To routinely grow and maintain pure cultures an anaerobic indicator containing methylene blue, which is blue when of obligate anaerobes, microbiologists use reducing media stored oxidized and turns colorless when the oxygen is removed (as shown here). in ordinary, tightly capped test tubes. These media are heated Q  hat is the technical name for bacteria that require a W shortly before use to drive off absorbed oxygen. higher-than-atmospheric-concentration of CO2 for growth? M06_TORT9150_12_CH06_pp149-175.indd 159 10/10/14 10:25 AM  160 Part one Fundamentals of Microbiology Candle jars are still used occasionally, but more often com- mercially available chemical packets are used to generate carbon dioxide atmospheres in containers. When only one or two Petri plates of cultures are to be incubated, clinical laboratory inves- tigators often use small plastic bags with self-contained chemi- cal gas generators that are activated by crushing the packet or Air moistening it with a few milliliters of water. These packets are lock sometimes specially designed to provide precise concentrations of carbon dioxide (usually higher than can be obtained in can- dle jars) and oxygen for culturing organisms such as the micro- aerophilic Campylobacter bacteria (page 302). Some microorganisms, such as the Ebola virus, are so dan- Arm gerous that they can be handled only under extraordinary sys- ports tems of containment called biosafety level 4 (BSL-4). Level 4 labs are popularly known as “the hot zone.” Only a handful of such labs exists in the United States. The lab is a sealed environment within a larger building and has an atmosphere under negative Figure 6.7 An anaerobic chamber. Materials are introduced pressure, so that aerosols containing pathogens will not escape. through the small doors in the air-lock chamber at the left. The operator Both intake and exhaust air is filtered through high-efficiency works through arm ports in airtight sleeves. The airtight sleeves extend particulate air filters (see HEPA filters, page 183); the exhaust into the cabinet when it is in use. This unit also features an internal camera air is filtered twice. All waste materials leaving the lab are ren- and monitor. dered noninfectious. The personnel wear “space suits” that are Q In what way would an anaerobic chamber resemble the Space connected to an air supply (Figure 6.8). Laboratory orbiting in the vacuum of space? Less dangerous organisms are handled at lower levels of biosafety. For example, a basic microbiology teaching labora- tory would be BSL-1. Organisms that present a moderate risk Special Culture Techniques of infection can be handled at BSL-2 levels, that is, on open Many bacteria have never been successfully grown on artificial laboratory benchtops with appropriate gloves, lab coats, or pos- laboratory media. Mycobacterium leprae, the leprosy bacillus, sibly face and eye protection. BSL-3 labs are intended for highly is now usually grown in armadillos, which have a relatively low infectious airborne pathogens such as the tuberculosis agent. body temperature that matches the requirements of the microbe. Biological safety cabinets similar in appearance to the anaero- Another example is the syphilis spirochete, although certain bic chamber shown in Figure 6.7 are used. The laboratory itself nonpathogenic strains of this microbe have been grown on labo- should be negatively pressurized and equipped with air filters to ratory media. With few exceptions, the obligate intracellular prevent release of the pathogen from the laboratory. bacteria, such as the rickettsias and the chlamydias, do not grow on artificial media. Like viruses, they can reproduce only in a Selective and Differential Media living host cell. See the discussion of cell culture, page 367. In clinical and public health microbiology, it is frequently Many clinical laboratories have special carbon dioxide incu- necessary to detect the presence of specific microorgan- bators in which to grow aerobic bacteria that require concentra- isms associated with disease or poor sanitation. For this task, tions of CO2 higher or lower than that found in the atmosphere. selective and differential media are used. Selective media are Desired CO2 levels are maintained by electronic controls. High designed to suppress the growth of unwanted bacteria and CO2 levels are also obtained with simple candle jars. Cultures encourage the growth of the desired microbes. For example, are placed in a large sealed jar containing a lighted candle, which bismuth sulfite agar is one medium used to isolate the typhoid consumes oxygen. The candle stops burning when the air in the bacterium, the gram-negative Salmonella typhi (TĪ-fē), from jar has a lowered concentration of oxygen (at about 17% O2, still feces. Bismuth sulfite inhibits gram-positive bacteria and most adequate for the growth of aerobic bacteria). An elevated con- gram-negative intestinal bacteria (other than S. typhi), as well. centration of CO2 (about 3%) is also present. Microbes that grow Sabouraud’s dextrose agar, which has a pH of 5.6, is used to iso- better at high CO2 concentrations are called capnophiles. The late fungi that outgrow most bacteria at this pH. low-oxygen, high-CO2 conditions resemble those found in the Differential media make it easier to distinguish colonies intestinal tract, respiratory tract, and other body tissues where of the desired organism from other colonies growing on the pathogenic bacteria grow. same plate. Similarly, pure cultures of microorganisms have M06_TORT9150_12_CH06_pp149-175.indd 160 10/10/14 10:25 AM  Chapter 6  Microbial Growth 161 Enrichment Culture Because bacteria present in small numbers can be missed, especially if other bacteria are present in much larger num- bers, it is sometimes necessary to use an enrichment culture. This is often the case for soil or fecal samples. The medium (enrichment medium) for an enrichment culture is usually liquid and provides nutrients and environmental conditions that favor the growth of a particular microbe but not others. In this sense, it is also a selective medium, but it is designed to increase very small numbers of the desired type of organism to detectable levels. Suppose we want to isolate from a soil sample a microbe that can grow on phenol and is present in much smaller num- bers than other species. If the soil sample is placed in a liq- uid enrichment medium in which phenol is the only source of carbon and energy, microbes unable to metabolize phenol Figure 6.8 Technicians in a biosafety level 4 (BSL- 4) will not grow. The culture medium is allowed to incubate laboratory. Personnel working in a BSL-4 facility wear a “space suit” that for a few days, and then a small amount of it is transferred is connected to an outside air supply. Air pressure in the suit is higher than into another flask of the same medium. After a series of such the atmosphere, preventing microbes from entering the suit. transfers, the surviving population will consist of bacteria Q If a technician were working with pathogenic prions, how would capable of metabolizing p ­ henol. The bacteria are given time material leaving the lab be rendered noninfectious? (Hint: See to grow in the medium between transfers; this is the enrich- Chapter 7.) ment stage. (See the box in Chapter 28 page 801.) Any nutri- ents in the original inoculum are rapidly diluted out with the successive transfers. When the last dilution is streaked onto a identifiable reactions with differential media in tubes or plates. solid medium of the same composition, only those colonies of Blood agar (which contains red blood cells) is a medium that organisms capable of using phenol should grow. A remarkable microbiologists often use to identify bacterial species that aspect of this particular technique is that phenol is normally destroy red blood cells. These species, such as Streptococ- lethal to most bacteria. cus pyogenes (pĪ-AH-jen-ēz), the bacterium that causes strep throat, show a clear ring around their colonies where they have lysed the surrounding blood cells (Figure 6.9). Sometimes, selective and differential characteristics are Bacterial combined in a single medium. Suppose we want to isolate the colonies common bacterium Staphylococcus aureus, found in the nasal passages. This organism has a tolerance for high concentra- tions of sodium chloride; it can also ferment the carbohy- drate mannitol to form acid. Mannitol salt agar contains 7.5% sodium chloride, which will discourage the growth of com- peting organisms and thus select for (favor the growth of) S. aureus. This salty medium also contains a pH indicator that changes color if the mannitol in the medium is fermented to acid; the mannitol-fermenting colonies of S. aureus are thus Hemolysis differentiated from colonies of bacteria that do not ferment mannitol. Bacteria that grow at the high salt concentration and ferment mannitol to acid can be readily identified by the color 2 mm change (Figure 6.10). These are probably colonies of S. aureus, and their identification can be confirmed by additional tests. Figure 6.9 Blood agar, a differential medium containing red The use of differential media to identify toxin-producing E. coli blood cells. The bacteria have lysed the red blood cells (beta-hemolysis), causing the clear areas around the colonies. is discussed in Chapter 5, page 136. Q Of what value are hemolysins to pathogens? M06_TORT9150_12_CH06_pp149-175.indd 161 10/10/14 4:51 PM  162 Part one Fundamentals of Microbiology Clinical Case Uninoculated P. fluorescens is an aerobic, gram-negative rod that grows best between 25°C and 30°C and grows poorly at the standard hospital microbiology incubation temperatures (35°C to 37°C). The bacteria are so named because they produce a pigment that fluoresces under ultraviolet light. While reviewing the Staphylococcus Staphylococcus facts of the latest outbreak, Dr. MacGruder learns that the epidermidis aureus most recent patients were last exposed to the contaminated heparin 84 to 421 days before onset of their infections. On-site investigations confirmed that the patients’ clinics are no longer using the recalled heparin and had, in fact, returned all unused inventory. Concluding that these patients did not develop infections during the previous outbreak, Figure 6.10 Differential medium. This medium is mannitol salt Dr. MacGruder must look for a new source of infection. The agar, and bacteria capable of fermenting the mannitol in the medium patients all have indwelling venous catheters: tubes that are to acid (Staph ylococcus aureus) cause the medium to change color to inserted into a vein for long-term delivery of concentrated yellow. This differentiates b