Archive for year: 2023

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3 Concepts and Tools for Studying Microorganisms

We think we have life down; we think we understand all the conditions of its existence; and then along comes an upstart bacterium, live or fossil- ized, to tweak our theories or teach us something new. —Jennifer Ackerman in Chance in the House of Fate (2001)

The oceans of the world are a teeming but invisible forest of micro- organisms and viruses. For example, one liter of seawater contains more than 25,000 different bacterial species.

A substantial portion of these marine microbes represent the phy- toplankton (phyto = “plant”; plankto = “wandering”), which are floating communities of cyanobacteria and eukaryotic algae. Besides forming the foundation for the marine food web, the phytoplankton account for 50% of the photosynthesis on earth and, in so doing, supply about half the oxygen gas we and other organisms breathe.

While sampling ocean water, scientists from MIT’s Woods Hole Oceanographic Institution discovered that many of their samples were full of a marine cyanobacterium, which they eventually named Prochlorococcus. Inhabiting tropical and subtropical oceans, a typical sample often contained more than 200,000 (2 × 105) cells in one drop of seawater.

Studies with Prochlorococcus suggest the organism is responsible for almost 50% of the photosynthesis in the open oceans ( FIGURE 3.1 ). This makes Prochlorococcus the smallest and most abundant marine photosyn- thetic organism yet discovered.

Chapter Preview and Key Concepts

3.1 The Bacteria/Eukaryote Paradigm 1. Bacterial cells undergo biological processes

as complex as in eukaryotes. 2. There are organizational patterns common to

all living organisms. 3. Bacteria and eukaryotes have distinct

subcellular compartments. 3.2 Classifying Microorganisms

4. Organisms historically were grouped by shared characteristics.

5. The three-domain system shows the taxonomic relationships between living organisms. MICROINQUIRY 3: The Evolution of Eukaryotic Cells

6. The binomial system identifies each organism by a universally accepted scientific name.

7. Species can be organized into higher, more inclusive groups.

8. Identification and classification of microorganisms may use different methods.

3.3 Microscopy 9. Metric system units are the standard for

measurement. 10. Light microscopy uses visible light to

magnify and resolve specimens. 11. Specimens stained with a dye are contrasted

against the microscope field. 12. Different optical configurations provide

detailed views of cells. 13. Electron microscopy uses a beam of electrons

to magnify and resolve specimens.

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CHAPTER 3 Concepts and Tools for Studying Microorganisms 65

isms influence our lives and life on this planet. Microbial ecologists study how the phytoplank- ton communities help in the natural recycling and use of chemical elements such as nitrogen. Evolutionary microbiologists look at these micro- organisms to learn more about their taxonomic relationships, while microscopists, biochemists, and geneticists study how Prochlorococcus cells compensate for a changing environment of sun- light and nutrients.

This chapter focuses on many of the aspects described above. We examine how microbes maintain a stable internal state and how they can exist in “multicellular”, complex communities. Throughout the chapter we are concerned with the relationships between microorganisms and the many attributes they share. Then, we explore the methods used to name and catalog microorgan- isms. Finally, we discuss the tools and techniques used to observe the microbial world.

The success of Prochlorococcus is due, in part, to the presence of different ecotypes inhabiting different ocean depths. For example, the high sun- light ecotype occurs in the surface waters while the low-light type is found below 50 meters. This latter ecotype compensates for the decreased light by increasing the amount of cellular chlorophyll that can capture the available light.

In terms of nitrogen sources, the high-light ecotype only uses ammonium ions (NH4+) (see MicroFocus 2.5). At increasing depth, NH4+ is less abundant so the low-light ecotype compensates by using a wider variety of nitrogen sources.

These and other attributes of Prochlorococcus illustrate how microbes survive through change. They are of global importance to the function- ing of the biosphere and, directly and indirectly, affect our lives on Earth.

Once again, we encounter an interdisciplinary group of scientists studying how microorgan-

FIGURE 3.1 Photosynthesis in the World’s Oceans. This global satellite image (false color) shows the distribution of photosynthetic organisms on the planet. In the aquatic environments, red colors indicate high levels of chlorophyll and productivity, yellow and green are moderate levels, and blue and purple areas are the “marine deserts.” »» How do the landmasses where photosynthesis is most productive (green) compare in size to photosynthesis in the oceans?

Ecotypes: Subgroups of a species that have special charac- teristics to survive in their ecological surroundings.

Biosphere: That part of the earth— including the air, soil, and water—where life occurs.

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66 CHAPTER 3 Concepts and Tools for Studying Microorganisms

3.1 The Bacteria/Eukaryote Paradigm

In the news media or even in scientific magazines and textbooks, bacterial and archaeal species often are described as “simple organisms” compared to the “complex organisms” representing multicellu- lar plant and animal species. This view represents a mistaken perception. Despite their microscopic size, bacterial and archaeal organisms exhibit every complex feature, or emerging property, common to all living organisms. These include:

• DNA as the hereditary material control- ling structure and function.

• Complex biochemical patterns of growth and energy conversions.

• Complex responses to stimuli. • Reproduction to produce offspring. • Adaptation from one generation to the

next.

Focusing on the Bacteria, what is the evidence for complexity?

Bacterial Complexity: Homeostasis and Biofilm Development KEY CONCEPT 1. Bacterial cells undergo biological processes as com-

plex as in eukaryotes.

Historically, when one looks at bacterial cells even with an electron microscope, often there is little to see ( FIGURE 3.2A ). “Cell structure,” representing the cell’s physical appearance or its components and the “pattern of organization,” referring to the configuration of those structures and their rela- tionships to one another, do give the impression of simpler cells.

But what has been overlooked is the “cellular process,” the activities all cells carry out for the continued survival of the cell (and organism). At this level, the complexity is just as intricate as in any eukaryotic cell. So, in reality, bacteria cells carry out many of the same cellular processes as eukaryotes—only without the need for an elabo- rate, visible structural organization.

Homeostasis. All organisms continually bat- tle their external environment, where factors such as temperature, sunlight, or toxic chemicals can have serious consequences. Organisms strive to maintain a stable internal state by making appro- priate metabolic or structural adjustments. This ability to adjust yet maintain a relatively steady

internal state is called homeostasis (homeo = “sim- ilar”; stasis = “state”). Two examples illustrate the concept ( FIGURE 3.2B ).

The low-light Prochlorococcus ecotype mentioned in the chapter introduction lives at depths of below 50 meters. At these depths, transmitted sunlight decreases and any one nitrogen source is less accessible. The ecotype compensates for the light reduction and nitro- gen limitation by (1) increasing the amount of cellular chlorophyll to capture light and (2) using a wider variety of available nitrogen sources. These adjustments maintain a steady internal state.

For our second example, suppose a patient is given an antibiotic to combat a bacterial infection. In response, the infecting bacterium compensates for the change by breaking the structure of the antibiotic. The adjustment, antibiotic resistance, maintains homeostasis in the bacterial cell.

In both these examples, the internal environ- ment is maintained despite a changing environ- ment. Such, often complex, homeostatic controls are critical to all microbes, including bacterial species.

Biofilm Development. One of the emerging properties of life is that cells must cooperate with one another. This is certainly true in animals and plants, but it is true of most bacterial organisms as well.

The early studies of disease causation done by Pasteur and Koch (see Chapter 1) certainly required pure cultures to associate a specific dis- ease with one specific microbe. However, today it is necessary to abolish the impression that bac- teria are self-contained, independent organisms. In nature few species live such a pure and solitary life. In fact, it has been estimated that up to 99% of bacterial species live in communal associations called biofilms; that is, in a “multicellular state” where survival requires chemical communication and cooperation between cells.

As a biofilm forms, the cells become embed- ded in a matrix of excreted polymeric substances produced by the bacterial cells ( FIGURE 3.2C .) These sticky substances are composed of charged and neutral polysaccharides that hold the bio- film together and cement it to nonliving or living surfaces, such as metals, plastics, soil particles,

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3.1 The Bacteria/Eukaryote Paradigm 67

(A)

Stage 1: Initial Attachment. Formation begins with the reversible attachment of free- floating bacteria to a surface.

1

Stage 3: Maturation I. The first colonists facilitate the arrival of other cells by providing more diverse adhesion sites and beginning to build the polysaccharide matrix that holds the biofilm together. As nutrients accumulate, the cells start to divide.

Stage 4: Maturation II. A fully mature biofilm is now established and may only change in shape and size.The matrix acts as a protective coating for the cells and is a barrier to chemicals, antibiot- ics, and other potentially toxic substances.

2 3 4111

Stage 2: Irreversible At- tachment. Many pioneer cells anchor themselves irre- versibly using cell adhesion structures as they secrete sticky, extracellular polysaccharides.

Dispersion. Important to the biofilm lifecycle, single di- viding cells (dark cells on the figure) will be periodically dispersed from the biofilm. The new pioneer cells can then colonize new surfaces.

(C)

MICROORGANISM

Microorganism

Compensation fails Compensation succeeds

Microorganism dies Microorganism lives

external change affects

loss of homeostasis

homeostasis maintained

attempts to compensate

(B)

FIGURE 3.2 Simpler, Unicellular Organisms? (A) This false-color electron microscope image of Staphylococcus aureus gives the impression of simplicity in structure. (Bar = 0.5µm) (B) A concept map illustrating how bacterial organisms, like all microorganisms, have to compensate for environmental changes. Survival depends on such homeostatic abilities. (C) The formation of a biofilm is an example of intercellular cooperation in the development of a multicellular structure. »» Using the concept map in (B), explain how Prochlorococcus compensates for low-light conditions in its environment. (C) Modified from David G. Davies, Binghamton University, Binghamton NY.

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68 CHAPTER 3 Concepts and Tools for Studying Microorganisms

oping but persistent infection. As mentioned, the polysaccharide matrix acts as a protective coating for the embedded cells and impedes penetration by antibiotics and other antimicrobial substances. As a result, the infection can be extremely hard to eradicate.

On the other hand, biofilms can be useful. For example, sewage treatment plants use biofilms to remove contaminants from water (Chapter 26). As mentioned in Chapter 1, bioremediation uses microorganisms to remove or clean up chemically- contaminated environments, such as oil spills or toxic waste sites. Such biofilms have been used at sites contaminated with toxic organics, such as “polycyclic aromatic hydrocarbons” that can lead to cancer. Perchlorate (ClO4–) is a soluble anion that is a component in rocket fuels, fire- works, explosives, and airbag manufacture. It is toxic to humans and is highly persistent in drinking water, especially in the western United States. Natural subterranean biofilms are being genetically modified so the cells contain the genes needed to degrade perchlorate from groundwater. In both these cases, a concentrated community of microorganisms—a biofilm—can have positive effects on the environment. CONCEPT AND REASONING CHECKS 3.1 Support the statement “Bacterial cells represent

complex organisms.”

medical indwelling devices, or human tissue. The mature, fully functioning biofilm is like a living tissue with a primitive circulatory system made of water channels to bring in nutrients and eliminate wastes. A biofilm is a complex, metabolically coop- erative community made up of peacefully coexist- ing species.

It is during this colonization that the cells are able to “speak to each other” and cooperate through chemical communication. This process, called quorum sensing, involves the ability of bacteria to sense their numbers, and then to com- municate and coordinate behavior, including gene expression, via signaling molecules. Thus, biofilms are characterized by structural heterogeneity, genetic diversity, and complex community inter- actions. The cells within the community are pro- foundly different in behavior and function from those of their independent, free-living cousins. MICROFOCUS 3.1 describes a few examples.

Biofilms can also be associated with infec- tions. Development of a fatal lung infection (cystic fibrosis pneumonia), middle ear infections (otitis media), and tooth decay (dental caries) are but a few examples ( FIGURE 3.3A ). Biofilms also can develop on improperly cleaned medical devices, such as artificial joints, mechanical heart valves, and catheters ( FIGURE 3.3B ), such that when implanted into the body, the result is a slow devel-

FIGURE 3.3 Biofilms in Disease. (A) A false-color electron microscope image of a tooth surface showing the plaque biofilm (pur- ple) containing bacteria cells. The red cells are red blood cells. (Bar � 60 µm.) (B) An electron microscope image of Staphylococcus aureus contamination on a catheter. The fibrous-looking substance is part of the biofilm. (Bar � 3 µm.) »» What is the best way to minimize such biofilms on the teeth?

(A) (B)

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3.1 The Bacteria/Eukaryote Paradigm 69

3.1: Environmental Microbiology The Power of Quorum Sensing

As the chapter opener stated, the microbial world is truly immense and we are continually surprised by what we find. Take quorum sensing for example. The discovery that bacterial cells can communicate with each other changed our general perception of bacterial species as single, simple organisms inhabiting our world. Here are two examples.

Vibrio fischeri Vibrio fischeri is a light-emitting, marine bacterial organism found at very low concentrations around the world. At these low concentrations, the cells do not emit any light (see figure). However, juvenile Hawaiian bobtail squids selectively draw up the free-living V. fischeri and the bacterial cells take up resi- dence in what will be the squids’ functional adult light organ called the photophore. The bacterial cells are maintained in this organ for the entire life of the squid. Why take up these bacterial cells?

The bobtail squid is a nocturnal species that hunts and feeds in shallow marine waters. On moonlit nights, the light casts a moving shadow of the squid on the sandy bottom. Such movements can attract squid predators. The V. fischeri cells confined to the photophore grow to high concentrations (about 1011 cells/ml). Sensing their high numbers, the V. fischeri cells start chemically “chatting” with one another and produce a signaling molecule that triggers the synthesis of the bacterial enzyme luciferase. This enzyme oxidizes bacterial luciferin to oxyluciferin and energy. Now here is the quorum sensing finale: The energy is given off as cold light (bioluminescence)—the squid’s photophore shines. The squid modulates the light to match that of the moonlight and directs the bacterial glow toward the bottom of the shallow waters, eliminating the bottom shadows and camouflaging itself from any predators.

Myxobacteria One of the first organisms in which quorum sensing was observed was in the myxobacteria, a bacterial group that predominantly lives in the soil. Individual myxobacterial cells are always evaluating both their own nutritional status and that of their community. The myxobacterial cells can move actively by gliding and, on sensing food (bacterial, yeast, or algal cells), typically travel in “swarms” (also known as “wolf packs”) that are kept together by intercellular molecular signals. This form of quorum sensing coordinates feeding behavior and provides a high concentration of extracellular enzymes from the “multicellular” swarm needed to digest the prey. Like a lone wolf, a single cell could not effectively carry out this behavior.

Under nutrient starvation, a different behavior occurs—the cells aggregate into fruiting bodies that facilitate species survival. During this developmental program, approximately 100,000 cells coordinately construct the macroscopic fruiting body. In Myxococcus xanthus, the myxobacterial cells first respond by triggering a quorum-sensing A-signal that helps them assess starvation and induce the first stage of aggregation. Later, the morphogenetic C-signal helps to coordinate fruit body development, as many myxobacterial cells die in forming the stalk while the remaining viable cells differentiate into environmen- tally resistant and metabolically quiescent myxospores.

Photographs of Vibrio fischeri growing in a culture plate (left) and triggered to bioluminesce (right).

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70 CHAPTER 3 Concepts and Tools for Studying Microorganisms

have a single, circular DNA molecule without an enclosing membrane ( FIGURE 3.4 .) Eukaryotic cells, however, have multiple, linear chromosomes enclosed by the membrane envelope of the cell nucleus.

Compartmentation. All organisms have an organizational pattern separating the internal compartments from the surrounding environ- ment but allowing for the exchange of solutes and wastes. The pattern for compartmentation is represented by the cell. All cells are surrounded by a cell membrane (known as the plasma mem- brane in eukaryotes), where the phospholipids form the impermeable boundary to solutes while membrane proteins are the gates through which the exchange of solutes and wastes occurs, and across which chemical signals are communicated. We have more to say about membranes in the next chapter.

Metabolic Organization. The process of metabolism is a consequence of compartmenta- tion. By being enclosed by a membrane, all cells

Bacteria and Eukaryotes: The Similarities in Organizational Patterns KEY CONCEPT 2. There are organizational patterns common to all living

organisms.

In the 1830s, Matthias Schleiden and Theodor Schwann developed part of the cell theory by demonstrating all plants and animals are com- posed of one or more cells, making the cell the fundamental unit of life. (Note: about 20 years later, Rudolph Virchow added that all cells arise from pre-existing cells.) Although the concept of a microorganism was just in its infancy at the time, the theory suggests that there are certain organi- zational patterns common to all organisms.

Genetic Organization. All organisms have a similar genetic organization whereby the heredi- tary material is communicated or expressed (Chapter 9). The organizational pattern for the hereditary material is in the form of one or more chromosomes. Structurally, most bacterial cells

Cytoplasm

Ribosome

Cell membrane

Cell wall

DNA (chromosome)(a)

Ribosomes attached to endoplasmic reticulum

DNA (chromosomes)

Nuclear envelope Lysosome

Cytoplasm

Plasma membrane

Cytoskeleton

Free ribosomes

Cilia

Flagellum

Mitochondrion

Smooth endoplasmic reticulum

Rough endoplasmic reticulum

Centrosome

Golgi apparatus

FIGURE 3.4 A Comparison of Prokaryotic and Eukaryotic Cells. (A) A stylized bacterial cell as an example of a prokaryotic cell. Relatively few visual compartments are present. (B) A protozoan cell as a typical eukaryotic cell. Note the variety of cellular subcompartments, many of which are discussed in the text. Universal structures are indicated in red. »» List the ways you could microscopically distinguishing a eukaryotic microbial cell from a bacterial cell.

(A) (B)

Metabolism: All the chemical reactions occurring in an organism or cell.

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3.1 The Bacteria/Eukaryote Paradigm 71

port. Lysosomes, somewhat circular, membrane- enclosed sacs containing digestive (hydrolytic) enzymes, are derived from the Golgi apparatus and, in protozoal cells, break down captured food materials.

Bacteria lack an endomembrane system, yet they are capable of manufacturing and modifying proteins and lipids just as their eukaryotic rela- tives do. However, many bacte rial cells contain so-called microcompartments surrounded by a protein shell ( FIGURE 3.5 .) These microcom- partments represent a type of organelle since the shell proteins can control transport similar to membrane-enclosed organelles.

Energy Metabolism. Cells and organisms carry out one or two types of energy transfor- mations. Through a process called cellular res- piration, all cells convert chemical energy into cellular energy for cellular work. In eukary- otic microbes, this occurs in the cytosol and in membrane-enclosed organelles called mito- chondria (sing., mitochondrion). Bacterial (and archaeal) cells lack mitochondria; they use the cytosol and cell membrane to complete the energy converting process.

have an internal environment in which chemical reactions occur. This space, called the cytoplasm, represents everything surrounded by the mem- brane and, in eukaryotic cells, exterior to the cell nucleus. If the cell structures are removed from the cytoplasm, what remains is the cytosol, which consists of water, salts, ions, and organic com- pounds as described in Chapter 2.

Protein Synthesis. All organisms must make proteins, which we learned in Chapter 2 are the workhorses of cells and organisms. The structure common in all cells is the ribosome, an RNA- protein machine that cranks out proteins based on the genetic instructions it receives from the DNA (Chapter 8). Although the pattern for pro- tein synthesis is identical, structurally bacterial ribosomes are smaller than their counterparts in eukaryotic cells. CONCEPT AND REASONING CHECKS 3.2 The cell theory states that the cell is the fundamental

unit of life. Summarize those processes all cells have that contribute to this fundamental unit.

Bacteria and Eukaryotes: The Structural Distinctions KEY CONCEPT 3. Bacteria and eukaryotes have distinct subcellular com-

partments.

In the cytoplasm, eukaryotic microbes have a variety of structurally discrete, often membrane- enclosed, subcellular compartments called organelles to carry out specialized functions (Figure 3.4). Bacterial cells also have subcellular compartments—they just are not readily visible or membrane enclosed.

Protein/Lipid Transport. Eukaryotic microbes have a series of membrane-enclosed organelles that compose the cell’s endomembrane system, which is designed to transport protein and lipid cargo through and out of the cell. This system includes the endoplasmic reticulum (ER), which consists of flat membranes to which ribosomes are attached (rough ER) and tube-like membranes without ribosomes (smooth ER). These portions of the ER are involved in protein and lipid synthesis and transport, respectively.

The Golgi apparatus is a group of indepen- dent stacks of flattened membranes and vesicles where the proteins and lipids coming from the ER are processed, sorted, and packaged for trans-

Vesicles: Membrane-enclosed spheres involved with secretion and storage.

FIGURE 3.5 Microcompartments. Purified bacterial microcompartments from Salmonella enterica are composed of a complex protein shell that encases metabolic enzymes. (Bar � 100 nm.) »» How do these bacterial microcompart- ments differ structurally from a eukaryotic organelle?

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72 CHAPTER 3 Concepts and Tools for Studying Microorganisms

the flagella are structurally different and without a cell membrane covering. The pattern of motility also is different, providing a rotational propeller- like force for movement (Chapter 4).

Some protozoa also have other membrane- enveloped appendages called cilia (sing., cilium) that are shorter and more numerous than flagella. In some motile protozoa, they wave in synchrony and propel the cell forward. No bacterial cells have cilia.

Water Balance. The aqueous environment in which many microorganisms live presents a situ- ation where the process of diffusion occurs, spe- cifically the movement of water, called osmosis, into the cell. Continuing unabated, the cell would eventually swell and burst (cell lysis) because the cell or plasma membrane does not provide the integrity to prevent lysis.

Most bacterial and some eukaryotic cells (fungi, algae) contain a cell wall exterior to the cell or plasma membrane. Although the structure and organization of the wall differs between groups (see Chapter 2), all cell walls provide support for the cells, give them shape, and help them resist the pressure exerted by the internal water pressure.

A summary of the bacteria and eukaryote pro- cesses and structures is presented in TABLE 3.1 .

CONCEPT AND REASONING CHECKS 3.3 Explain how variation in cell structure between bac-

teria and eukaryotes can be compatible with a simi- larity in cellular processes between these organisms.

A second energy transformation, photosyn- thesis, involves the conversion of light energy into chemical energy. In algal protists, photosyn- thesis occurs in membrane-bound chloroplasts. Some bacteria, such as the cyanobacteria we have mentioned, also carry out almost identical energy transformations. Again, the cell membrane or elab- orations of the membrane represent the chemical workbench for the process.

Cell Structure and Transport. The eukary- otic cytoskeleton is organized into an intercon- nected system of cytoplasmic fibers, threads, and interwoven molecules that give structure to the cell and assist in the transport of materials throughout the cell. The main components of the cytoskeleton are microtubules that originate from the centro- some and microfilaments, each assembled from different protein subunits. Bacterial cells to date have no similar physical cytoskeleton, although proteins related to those that construct micro- tubules and microfilaments aid in determining the shape in some bacterial cells as we will see in Chapter 4.

Cell Motility. Many microbial organisms live in watery or damp environments and use the process of cell motility to move from one place to another. Many algae and protozoa have long, thin protein projections called flagella (sing., fla- gellum) that, covered by the plasma membrane, extend from the cell. By beating back and forth, the flagella provide a mechanical force for motility. Many bacterial cells also exhibit motility; however,

Diffusion: The movement of a sub- stance from where it is in a higher concentration to where it is in a lower concentration.

TABLE

3.1 Comparison of Bacterial and Eukaryotic Cell Structure Cell Structure or Compartment

Process Bacterial Eukaryotic

Genetic organization Circular DNA chromosome Linear DNA chromosomes Compartmentation Cell membrane Plasma membrane Metabolic organization Cytoplasm Cytoplasm Protein synthesis Ribosomes Ribosomes Protein/lipid transport Cytoplasm Endomembrane system Energy metabolism Cytoplasm and cell membrane Mitochondria and chloroplasts Cell structure and transport Proteins in cytoplasm Protein filaments in cytoplasm Cell motility Bacterial flagella Eukaryotic flagella or cilia Water balance Cell wall Cell wall

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3.2 Classifying Microorganisms 73

3.2 Classifying Microorganisms

If you open any catalog, items are separated by types, styles, or functions. For example, in a fash- ion catalog, watches are separated from shoes and, within the shoes, men’s, women’s, and children’s styles are separated from one another. Even the brands of shoes or their use (e.g., dress, casual, athletic) may be separated.

With such an immense diversity of organisms on planet Earth, the human drive to catalog these organisms has not been very different from cata- loging watches and shoes; both have been based on shared characteristics. In this section, we shall explore the principles on which microorganisms are classified and cataloged.

Classification Attempts to Catalog Organisms

KEY CONCEPT 4. Organisms historically were grouped by shared

characteristics.

In the 18th century, Carolus Linnaeus, a Swedish scientist, began identifying living organisms according to similarities in form (resemblances) and placing organisms in one of two “kingdoms”— Vegetalia and Animalia ( FIGURE 3.6 ). This system was well accepted until the mid-1860s when a German naturalist, philosopher, and physician, Ernst Haeckel, identified a fundamental problem in the two-kingdom system. The unicellular (micro- scopic) organisms being identified by Haeckel, Pasteur, Koch, and their associates did not con- form to the two-kingdom system of multicellular organisms. Haeckel constructed a third kingdom, the Protista, in which all the known unicellular organisms were placed. The bacterial organisms, which he called “moneres,” were near the bottom of the tree, closest to the root of the tree.

With improvements in the design of light microscopes, more observations were made of bacterial and protist organisms. In 1937, a French biologist, Edouard Chatton, proposed that there was a fundamental dichotomy among the Protista. He saw bacteria as having distinctive properties (not articulated in his writings) in “the prokaryotic nature of their cells” and should be separated from all other protists “which have eukaryotic cells.” With the development of the electron microscope

in the 1950s, it became apparent that some pro- tists had a membrane-enclosed nucleus and were identified, along with the plants and animals, as being eukaryotes while other protists (the bacte- ria) lacked this structure and were considered to be prokaryotes (see Chapter 1). Thus, in 1956, Herbert Copland suggested bacteria be placed in a fourth kingdom, the Monera.

But there was still one more problem with the kingdom Protista. Robert H. Whittaker, a botanist at the University of California, saw the fungi as yet another kingdom of organisms. The fungi are the only eukaryotic group that must externally digest their food prior to absorption and, as such, live in the food source. For this and other rea- sons, Whittaker in 1959 refined the four-kingdom system into five kingdoms, identifying the king- dom Fungi as a separate, multicellular, eukaryotic kingdom distinguished by an absorptive mode of nutrition (Chapter 17).

The five kingdom system rested safely for about 15 years. In the late 1970s, Carl Woese, an evolu- tionary biologist at the University of Illinois, began a molecular analysis of living organisms based on comparisons of nucleotide sequences of genes cod- ing for the small subunit ribosomal RNA (rRNA) found in all organisms. These analyses revealed yet another dichotomy, this time among the prokary- otes. By 1990, it was clear that the kingdom Monera contained two fundamentally unrelated groups, what Woese initially called the Bacteria and Archaebacteria. These two groups were as different from each other as they were different from the eukaryotes. CONCEPT AND REASONING CHECKS 3.4 What four events changed the cataloging of micro-

organisms.

Kingdoms and Domains: Trying to Make Sense of Taxonomic Relationships KEY CONCEPT 5. The three-domain system shows the taxonomic rela-

tionships between living organisms.

What many of these scientists are or were doing is systematics; that is, studying the diversity of life and its evolutionary relationships. Systematic biologists—systematists for short— identify, describe, name, and classify organisms

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74 CHAPTER 3 Concepts and Tools for Studying Microorganisms

the new information fits into the known classifi- cation schemes—or how the schemes need to be modified to fit the new information. This is no clearer than the most recent taxonomic revolution that, as the opening quote states, has come along to “tweak our theories or teach us something new.”

(taxonomy), and organize their observations within a framework that shows taxonomic relationships.

Often it is difficult to make sense of taxonomic relationships because new information that is more detailed keeps being discovered about organisms. This then motivates taxonomists to figure out how

FIGURE 3.6 A Concept Map Illustrating the Development of Classification for Living Organisms. Over some 140 years, new observations and techniques have been used to reclassify and reorganize living organisms. »» Of the plants, algae, fungi, bacteria, protozoa, and animals, which are in each of the three domains? Modified from Schaechter, Ingraham, and Neidhardt. Microbe. ASM Press, 2006, Washington, D.C.

Living organisms

AnimaliaVegetalia1735

Approximate date

Plants

ProtistaPlantae1866

Bacteria

Prokaryota1937

Monera1959

Bacteria1990 Archaea Eukarya

Eukaryota

Fungi Algae Protozoa

Algae Fungi

FungiProtista Animalia Plantae

separated into kingdoms

consisting of

grouped into kingdom

separated into domains

combined into domain

forming the kingdom

separating into kingdoms

containing

Protozoa Animals

consisting of

remaining as kingdom

Animalia

remaining as kingdom

based on structure and metabolism to form the

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3.2 Classifying Microorganisms 75

among species. Because, like all hypotheses, they are revised as scientists gather new data, trees change as our knowledge of diversity increases.

In Woese’s three-domain system, one branch of the phylogenetic tree includes the former archaebacteria and is called the domain Archaea ( FIGURE 3.7 ). The second encompasses all the remaining true bacteria and is called the domain Bacteria. The third domain, the Eukarya, includes the four remaining kingdoms (Protista, Plantae, Fungi, and Animalia).

In 1996, Craig Venter and his coworkers deci- phered the DNA base sequence of the archaean Methanococcus jannaschii and showed that almost two thirds of its genes are different from those of the Bacteria. They also found that proteins repli- cating the DNA and involved in RNA synthesis have no counterpart in the Bacteria. The three- domain system now is on firm ground.

MICROINQUIRY 3 examines a scenario for the evolution of the eukaryotic cell.

CONCEPT AND REASONING CHECKS 3.5 It has been said that Woese “lifted a whole sub-

merged continent out of the ocean.” What is the “submerged continent” and why is the term “lifted” used?

Carl Woese, along with George Fox and coworkers at the University of Illinois, Urbana- Champaign, proposed a new classification scheme with a new most inclusive taxon, the domain. The new scheme initially came from work that com- pared the DNA nucleotide base sequences for the RNA in ribosomes, those protein manufacturing machines needed by all cells. Woese and Fox’s results were especially relevant when comparing those sequences from a group of bacterial organ- isms formerly called the archaebacteria (archae = “ancient”). Many of these bacterial forms are known for their ability to live under extremely harsh envi- ronments. Woese discovered that the nucleotide sequences in these archaebacteria were different from those in other bacterial species and in eukary- otes. After finding other differences, including cell wall composition, membrane lipids, and sensitivity to certain antibiotics, the evidence pointed to there being three taxonomic lines to the “tree of life”.

One goal of systematics, and the main one of interest here, is to reconstruct the phylogeny (phylo = “tribe”; geny = “production”), the evolu- tionary history of a species or group of species. Systematists illustrate phylogenies with phyloge- netic trees, which identify inferred relationships

FIGURE 3.7 The Three-Domain System Forms the “Tree of Life”. Fundamental differences in genetic endowments are the basis for the three domains of all organisms on Earth. Some 3.5 billion years ago, a universal ancestor arose from which all modern day organisms descended. »» What cellular characteristic was the major factor stimulating the development of the three-domain system?

Prokaryotes Eukaryotes

BACTERIA (>70 major phyla)

Gram-positive bacteria

Proteobacteria

Cyanobacteria

ARCHAEA (2 major phyla)

Euryarchaeota

Crenarchaeota

MULTICELLULAR ORGANISMS

EUKARYA (>30 major phyla)

Diplomonads

Mitochondrion degenerates

Parabasalids

Kinetoplastids

Ciliates

Plants

Fungi

Animals

Slime molds

Amoebas

Endosy mbiosis

UNIVERSAL ANCESTOR

Taxon (pl., taxa): Subdivisions used to classify organisms.

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76 CHAPTER 3 Concepts and Tools for Studying Microorganisms

INQUIRY 3 The Evolution of Eukaryotic Cells

Biologists and geologists have speculated for decades about the chemical evolution that led to the origins of the first prokaryotic cells on Earth (see Micro- Focus 2.1 and 2.6). Whatever the ori- gin, the first ancestral prokaryotes arose about 3.8 billion years ago and remained the sole inhabitants for some 1.5 billion years.

Scientists also have proposed various scenarios to account for the origins of the first eukaryotic cells. The oldest known fossils thought to be eukaryotic are about 2 billion years old.

A key concern here is figuring out how different membrane compartments arose to evolve into what are found in the eukaryotic cells today. Debate on this long intractable problem continues, so here we present some of the ideas that have fueled such discussions.

At some point around 2 billion years ago, the increasing number of metabolic reactions occurring in presumably larger prokaryotes started to interfere with one another. As cells increased in size, the increasing volume of cell cytoplasm outpaced the ability of the cell surface (membrane) to be an effective “work- bench” for servicing the metabolic needs of the whole cell. Complexity would neces- sitate more extensive workbench surface through compartmentation.

The Endomembrane System May Have Evolved through Invagination Similar to today’s bacterial and archaeal cells, the cell membrane of an ancestral prokaryote may have had specialized regions involved in protein synthesis, lipid synthesis, and nutrient hydroly- sis. If the invagination of these regions occurred, the result could have been the internalization of these processes as independent internal membrane systems. For example, the membranes of the endo- plasmic reticulum may have originated by multiple invagination events of the cell membrane (Figure A1).

Biologists have suggested that the elaboration of the evolving ER surrounded the nuclear region and DNA, creating the nuclear envelope. Surrounded and protected by a double membrane, greater genetic complexity could occur as the primitive eukaryotic cell continued to evolve in size and function. Other inter- nalized membranes could give rise to the Golgi apparatus.

Chloroplasts and Mitochondria Arose from a Symbiotic Union of Engulfed Bacteria Mitochondria and chloroplasts are not part of the extensive endomembrane sys- tem. Therefore, these energy-converting organelles probably originated in a differ- ent way.

The structure of modern-day chloro- plasts and mitochondria is very similar to a bacterial cell. In fact, mitochondria, chloroplasts, and bacteria share a large number of similarities (see Table). In addition, there are bacte rial cells alive today that carry out cellular respira- tion similarly to mitochondria and other bacterial cells (the cyanobacteria) that can carry out photosynthesis similarly to chloroplasts.

These similar functional pat- terns, along with other chemical and molecular similarities, suggested to Lynn Margulis at the University of Massachusetts, Amherst, that present- day chloroplasts and mitochondria represent modern representatives of what were once, many eons ago, free- living prokaryotes. Margulis, therefore, proposed the endosymbiont model for the origin of mitochondria and chlo- roplasts. The hypothesis suggests, in part, that mitochondria evolved from a prokaryote that carried out cellular respiration and which was “swallowed” (engulfed) by a primitive eukaryotic cell. The bacterial partner then lived within (endo) the eukaryotic cell in a

mutually beneficial association (symbio- sis) (Figure A2).

Likewise, a photosynthetic prokary- ote, perhaps a primitive cyanobacte- rium, was engulfed and evolved into the chloroplasts present in plants and algae today (Figure A3). The theory also would explain why both organelles have two membranes. One was the cell membrane of the engulfed bacterial cell and the other was the plasma membrane resulting from the engulfment process. By engulfing these prokaryotes and not destroying them, the evolving eukary- otic cell gained energy-conversion abili- ties, while the symbiotic bacterial cells gained a protected home.

If the first ancestral prokaryote appeared about 3.5 billion years ago and the first single-celled eukaryote about 2 billion years ago, then it took some 1.5 billion years of evolution for the events described above to occur (see Figure 8.2). With the appearance of the first eukaryotic cells, a variety of single-celled forms evolved, many of which were the very ancient ancestors of the single-celled eukaryotic organ- isms that exist today.

Obviously, laboratory studies can only hypothesize at mechanisms to explain how cells evolved and can only suggest—not prove—what might have happened bil- lions of years ago. The description here is a very simplistic view of how the first eukaryotic cells might have evolved. Short of inventing a time machine, we may never know the exact details for the origin of eukaryotic cells and organelles.

Discussion Point Determine which endosymbiotic event must have come first: the engulfment of the bac- terial progenitor of the chloroplast or the engulfment of the bacterial progenitor of the mitochondrion.

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3.2 Classifying Microorganisms 77

FIGURE A Possible Origins of Eukaryotic Cell Compartments. (A1) Invagination of the cell membrane from an ancient prokaryotic cell may have led to the development of the cell nucleus as well as to the membranes of the endomembrane system, including the endoplasmic reticulum. (A2) The mitochondrion may have resulted from the uptake and survival of a bacterial cell that carried out cellular resipration. (A3) A similar process, involving a bacterial cell that carried out photo- synthesis, could have accounted for the origin of the chloroplast.

(A1) Ancient eukaryotic cell (A2) Early respiratory eukaryotic cell

(A3) Early photosynthetic eukaryotic cell

DNA

Membrane- bound ribosomes

Ancient prokaryotic cell

Respiratory bacterium

Photosynthetic bacterium

Nucleus

Nuclear membrane

Endoplasmic reticulum

Mitochondrion

Chloroplast

TABLE

Similarities between Mitochondria, Chloroplasts, Bacteria, and Microbial Eukaryotes

Characteristic Mitochondria Chloroplasts Bacteria Microbial Eukaryotes

Average size 1–5 µm 1–5 µm 1–5 µm 10–20 µm Nuclear envelope present No No No Yes DNA molecule shape Circular Circular Circular Linear Ribosomes Yes; bacterial-like Yes; bacterial-like Yes Yes; eukaryotic-like Protein synthesis Make some of Make some of Make all of Make all of their proteins their proteins their proteins their proteins Reproduction Binary fission Binary fission Binary fission Mitosis and cytokinesis

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78 CHAPTER 3 Concepts and Tools for Studying Microorganisms

the genus name using only its initial genus letter or some accepted substitution, together with the full specific epithet; that is, E. coli or H. sapiens. A cau- tionary note: often in magazines and newspapers, proper nomenclature is not followed, so our gut bacterium would be written as Escherichia coli. CONCEPT AND REASONING CHECKS 3.6 Which one of the following is a correctly written sci-

entific name for the bacterium that causes anthrax? (a) bacillus Anthracis; (b) Bacillus Anthracis; or (c) Bacillus anthracis.

Classification Uses a Hierarchical System KEY CONCEPT 7. Species can be organized into higher, more inclusive

groups.

Linnaeus’ cataloging of plants and animals used shared and common characteristics. Such similar organisms that could interbreed were related as a species, which formed the least inclusive level of the hierarchical system. Part of Linnaeus’ innova- tion was the grouping of species into higher taxa

Nomenclature Gives Scientific Names to Organisms KEY CONCEPT 6. The binomial system identifies each organism by a

universally accepted scientific name.

Another goal of systematics is the naming of spe- cies and their placement in a classification. In his Systema Naturae, Linnaeus popularized a two- word (binomial) scheme of nomenclature, the two words usually derived from Latin or Greek stems. Each organism’s name consists of the genus to which the organism belongs and a specific epi- thet, a descriptor that further describes the genus name. Together these two words make up the spe- cies name. For example, the common bacterium Escherichia coli resides in the gut of all humans (Homo sapiens) (MICROFOCUS 3.2).

Notice in these examples that when a species name is written, only the first letter of the genus name is capitalized, while the specific epithet is not. In addition, both words are printed in italics or underlined. After the first time a species name has been spelled out, biologists usually abbreviate

3.2: Tools Naming Names

As you read this book, you have and will come across many scientific names for microbes, where a spe- cies name is a combination of the genus and specific epithet. Not only are many of these names tongue twisting to pronounce (many are listed with their pronunciation inside the front and back covers), but how in the world did the organisms get those names? Here are a few examples.

Genera Named after Individuals Escherichia coli: named after Theodore Escherich who isolated the bacterial cells from infant feces in

1885. Being in feces, it commonly is found in the colon. Neisseria gonorrhoeae: named after Albert Neisser who discovered the bacterial organism in 1879. As

the specific epithet points out, the disease it causes is gonorrhea.

Genera Named for a Microbe’s Shape Vibrio cholerae: vibrio means “comma-shaped,” which describes the shape of the bacterial cells that

cause cholera. Staphylococcus epidermidis: staphylo means “cluster” and coccus means “spheres.” So, these bacterial

cells form clusters of spheres that are found on the skin surface (epidermis).

Genera Named after an Attribute of the Microbe Saccharomyces cerevisiae: in 1837, Theodor Schwann observed yeast cells and called them

Saccharomyces (saccharo = “sugar”; myce = “fungus”) because the yeast converted grape juice (sugar) into alcohol; cerevisiae (from cerevisia = “beer”) refers to the use of yeast since ancient times to make beer.

Myxococcus xanthus: myxo means “slime,” so these are slime-producing spheres that grow as yellow (xantho = “yellow”) colonies on agar.

Thiomargarita namibiensis: see MicroFocus 3.5.

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3.2 Classifying Microorganisms 79

CONCEPT AND REASONING CHECKS 3.7 How would you describe an order in the taxonomic

classification?

Many Methods Are Available to Identify and Classify Microorganisms KEY CONCEPT 8. Identification and classification of microorganisms

may use different methods.

There are several traditional and more modern criteria that microbiologists can use to identify and classify microorganisms. For example, a medical identification usually uses physical, staining, and biochemical methods (metabolic tests). In fact, Bergey’s Manual of Determinative Bacteriology, now in its ninth edition, is the primary source for mak- ing routine medical identifications of bacterial pathogens. On the other hand, many emerging biotechnologies (Chapter 9) depend on a thorough knowledge of a microorganism’s biochemistry, molecular biology, and phylogenetic relatedness. More molecular methods will be required here.

Let’s briefly review some of the more deter- minative methods and a few molecular methods for classification.

Physical Characteristics. These include dif- ferential staining reactions to help determine the organism’s shape (morphology), and the size and arrangement of cells. Other characteristics can include oxygen, pH, and growth temperature requirements. Spore-forming ability and motility are additional determinants. Unfortunately, there

that also were based on shared, but more inclusive, similarities.

Today several similar species are grouped together into a genus (pl., genera). A collection of similar genera makes up a family and families with similar characteristics make up an order. Different orders may be placed together in a class and classes are assembled together into a phylum (pl., phyla). All phyla would be placed together in a kingdom and/or domain, the most inclusive level of classification. TABLE 3.2 outlines the taxonomic hierarchy for three organisms.

In prokaryotes, an organism may belong to a rank below the species level to indicate a spe- cial characteristic exists within a subgroup of the species. Such ranks have practical usefulness in helping to identify an organism. For example, two biotypes of the cholera bacterium, Vibrio cholerae, are known: Vibrio cholerae classic and Vibrio chol- erae El Tor. Other designations of ranks include subspecies, serotype, strain, morphotype, and variety.

David Hendricks Bergey devised one of the first systems of classification for the bacterial species in 1923. Today, the proper taxonomic classification for the Bacteria and Archaea can be found in the second edition of Bergey’s Manual of Systematic Bacteriology. The first two volumes of this 5-volume compendium have been published. The tremendous changes that have taken place in taxonomy can be seen by the addition of more than 2,200 new species and 390 new genera to the first volume of the new second edition.

TABLE

3.2 Taxonomic Classification of Humans, Brewer’s Yeast, and a Common Bacterium

Humans Brewer’s Yeast Escherichia coli

Domain Eukarya Eukarya Bacteria Kingdom Animalia Fungi Phylum Chordata Ascomycota Proteobacteria Class Mammalia Saccharomycotina Gammaproteobacteria Order Primates Saccharomycetales Enterobacteriales Family Hominidae Saccharomycetaceae Enterobacteriaceae Genus Homo Saccharomyces Escherichia Species H. sapiens S. cerevisiae E.coli

Biotypes: Populations or groups of individuals having the same genetic constitution (genotype).

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80 CHAPTER 3 Concepts and Tools for Studying Microorganisms

such collected antibodies, called antisera, are commercially available for many medically important pathogens. For example, mixing a Salmonella antiserum with Salmonella cells will cause the cells to clump together or agglutinate. If a foodborne illness occurs, the antiserum may be useful in identifying if Salmonella is the pathogen. More information about serological testing will be presented in Chapter 22.

Nucleic Acid Analysis. In 1984, the editors of Bergey’s Manual of Systematic Bacteriology noted that there is no “official” classification of bacte- rial species and that the closest approximation to an official classification is the one most widely accepted by the community of microbiologists. The editors stated that a comprehensive classifica- tion might one day be possible. Today, the fields of molecular genetics and genomics have advanced the analysis and sequencing of nucleic acids. This has given rise to a new era of molecular taxonomy.

Molecular taxonomy is based on the univer- sal presence of ribosomes in all living organisms. In particular, it is the RNAs in the ribosome, called ribosomal RNA (rRNA), which are of most interest and the primary basis of Woese’s construction of the three-domain system. Many scientists today believe the genes for rRNA are the most accurate measure for precise bacterial classification in all taxonomic classes. Other techniques, including the polymerase chain reaction and nucleic acid hybridization, will be mentioned in later chapters.

The vast number of tests and analyses avail- able for bacterial cells can make it difficult to know which are relevant for pathogen identification pur- poses. One widely used technique in many disci- plines is the dichotomous key. There are various forms of dichotomous keys, but one very useful construction is a flow chart where a series of posi- tive or negative test procedures are listed down the page. Based on the dichotomous nature of the test (always a positive or negative result), the flow chart immediately leads to the next test result. The result is the identification of a specific organism. A simplified example is shown in MICROFOCUS 3.4.

CONCEPT AND REASONING CHECKS 3.8 Why are so many tests often needed to identify a

specific bacterial species?

are many bacterial and archaeal organisms that have the same physical characteristics, so other distinguishing features are needed.

Biochemical Tests. As microbiologists better understood bacterial physiology, they discovered there were certain metabolic properties that were present only in certain groups.

Today, a large number of biochemical tests exist and often a specific test can be used to elimi- nate certain groups from the identification process. Among the more common tests are: fermentation of carbohydrates, the use of a specific substrate, and the production of specific products or waste products. But, as with the physical characteristics, often several biochemical tests are needed to dif- ferentiate between species.

These identification tests are important clini- cally, as they can be part of the arsenal available to the clinical lab that is trying to identify a patho- gen. Many of these tests use rapid identification methods (MICROFOCUS 3.3) or automated systems ( FIGURE 3.8 ).

Serological Tests. Microorganisms are antigenic, meaning they are capable of trigger- ing the production of antibodies. Solutions of

FIGURE 3.8 A Biolog MicroPlate®. The BIOLOG system is capable of identifying bacteria by assessing the bac- terium’s ability to use any of 95 different substrates in a 96-well microtiter plate. The use of any substrate results in a reduction of the dye in each well, resulting in purple color development. The intensity of the purple coloration indicates the degree of substrate usage and is read by a computer-linked automated microtiter reader. The first well (upper left) is a negative control with no substrate. »» Of the methods described on this page, which is/are most likely to be used in this more automated system? Explain.

Antibodies: Proteins produced by the immune system in response to a specific chemical con- figuration (antigen).

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3.2 Classifying Microorganisms 81

3.3: Tools Rapid Identification of Enteric Bacteria

In recent years, a number of miniaturized systems have been made available to microbiologists for the rapid identification of enteric bacteria. One such system is the Enterotube II, a self-contained, sterile, compartmentalized plastic tube containing 12 different media and an enclosed inoculating wire. This system permits the inoculation of all media and the performance of 15 standard biochemical tests using a single bacterial colony. The media in the tube indicate by color change whether the organism can carry out the metabolic reaction. After 24 hours of incubation, the positive tests are circled and all the circled numbers in each boxed section are added to yield a 5-digit ID for the organism being tested. This 5-digit number is looked up in a reference book or computer software to determine the identity of the bacterium.

(A) An uninoculated tube.

(B) An inoculated tube incubated for 24 hours.

As seen from the reference, 24160 is Escherichia coli.

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82 CHAPTER 3 Concepts and Tools for Studying Microorganisms

3.4: Tools Dichotomous Key Flow Chart

A medical version of a taxonomic key (in the form of a dichotomous flow chart) can be used to identify very similar bacterial species based on physical and biochemical characteristics.

In this simplified scenario, an unknown bacterium has been cultured and several tests run. The test results are shown in the box at the top. Using the test results and the flow chart, identify the bacterial species that has been cultured.

UNKNOWN BACTERIUM

Negative Positive

Negative Positive

Gram staining

ability to ferment lactose

Negative Positive

Negative Positive

Citrobacter intermedius

Escherichia coli

indole production

use of citrate as sole carbon source

Negative Positive

Citrobacter freundii

Enterobacter aerogenes

methyl red reaction

Microbiology Test Results

• Gram stain: gram-negative rods

Biochemical tests: • Citrate test: negative • Lactose fermentation: positive • Indole test: positive • Methyl red test: positive

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3.3 Microscopy 83

3.3 Microscopy

The ability to see small objects all started with the microscopes used by Robert Hooke and Antony van Leeuwenhoek. By now, you should be aware that microorganisms usually are very small. Before we examine the instruments used to “see” these tiny creatures, we need to be familiar with the units of measurement.

Many Microbial Agents Are In the Micrometer Size Range KEY CONCEPT 9. Metric system units are the standard for measurement.

One physical characteristic used to study micro- organisms and viruses is their size. Because they are so small, a convenient system of measurement is used that is the scientific standard around the

world. The measurement system is the metric sys- tem, where the standard unit of length is the meter and is a little longer than a yard (see Appendix A). To mea sure micro organisms, we need to use units that are a fraction of a meter. In microbiology, the common unit for measuring length is the microm- eter (µm), which is equivalent to a millionth (10–6) of a meter. To appreciate how small a micrometer is, consider this: Comparing a micrometer to an inch is like comparing a housefly to New York City’s Empire State Building, 1,472 feet high.

Microbial agents range in size from the rel- atively large, almost visible protozoa (100 µm) down to the incredibly tiny viruses (0.02 µm) ( FIGURE 3.9 ). Most bacterial and archaeal cells are about 1 µm to 5 µm in length, although notable exceptions have been discovered recently

0.1nm 1nm 10nm 100nm 10µm 100µm 1mm 1cm 0.1m 1m 10m

Electron microscope

Light microscope

Unaided eye

Molecules

Viruses Unicellular algae

FungiProtozoa Bacterial and

archaeal organisms Multicellular organisms

Atoms Colonial algae

Carbon atom

Glucose molecule

DNA double helix

Poliovirus

HIV

Rickettsiae

Rod-shaped and spherical-shaped

bacteria

Protozoan

Alga

Mold

Yeast

Fluke

Red blood cells

Tapeworm

1µm

Cyanobacterium

FIGURE 3.9 Size Comparisons Among Various Atoms, Molecules, and Microorganisms (not drawn to scale). Although tapeworms and flukes usually are macroscopic, the diseases these parasites cause are studied by microbiologists. »» Which domain on average has the smallest organisms and which has the largest?

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84 CHAPTER 3 Concepts and Tools for Studying Microorganisms

closely spaced objects to be clearly distinguished. For example, a car seen in the distance at night may appear to have a single headlight because at that distance the unaided eye lacks resolving power. However, using binoculars, the two head- lights can be seen clearly as the resolving power of the eye increases.

When switching from the low-power (10×) or high-power (40×) lens to the oil-immersion lens (100×), one quickly finds that the image has become fuzzy. The object lacks resolution, and the resolving power of the lens system appears to be poor. The poor resolution results from the refraction of light.

Both low-power and high-power objec- tives are wide enough to capture sufficient light for viewing. The oil-immersion objective, on the other hand, is so narrow that most light bends away and would miss the objective lens FIGURE 3.10C . The index of refraction (or refrac-

tive index) is a measure of the light-bending abil- ity of a medium. Immersion oil has an index of refraction of 1.5, which is almost identical to the index of refraction of glass. Therefore, by immersing the 100× lens in oil, the light does not bend away from the lens as it passes from the glass slide and the specimen.

The oil thus provides a homogeneous pathway for light from the slide to the objective, and the resolution of the object increases. With the oil- immersion lens, the highest resolution possible with the light microscope is attained, which is near 0.2 µm (200 nm) (MICROFOCUS 3.6).

CONCEPT AND REASONING CHECKS 3.10 What are the two most important properties of the

light microscope?

Staining Techniques Provide Contrast KEY CONCEPT 11. Specimens stained with a dye are contrasted against

the microscope field.

Microbiologists commonly stain bacterial cells before viewing them because the cytoplasm lacks color, making it hard to see the cells on the bright background of the microscope field. Several stain- ing techniques have been developed to provide contrast for bright-field microscopy.

(MICROFOCUS 3.5). Because most viruses are a frac- tion of one micrometer, their size is expressed in nanometers. A nanometer (nm) is equivalent to a billionth (10–9) of a meter; that is, 1/1,000 of a µm. Using nanometers, the size of the poliovi- rus, among the smaller viruses, mea sures 20 nm (0.02 µm) in diameter. CONCEPT AND REASONING CHECKS 3.9 If a bacterial cell is 0.75 µm in length, what is its

length in nanometers?

Light Microscopy Is Used to Observe Most Microorganisms KEY CONCEPT 10. Light microscopy uses visible light to magnify and

resolve specimens.

The basic microscope system used in the micro- biology laboratory is the light microscope, in which visible light passes directly through the lenses and specimen ( FIGURE 3.10A ). Such an optical configuration is called bright-field microscopy. Visible light is projected through a condenser lens, which focuses the light into a sharp cone ( FIGURE 3.10B ). The light then passes through the opening in the stage. When hitting the glass slide, the light is reflected or refracted as it passes through the specimen. Next, light passing through the specimen enters the objec- tive lens to form a magnified intermediate image inverted from that of the specimen. This interme- diate image becomes the object magnified by the ocular lens (eyepiece) and seen by the observer. Magnification thus refers to the increase in the apparent size of the specimen being observed. Because this microscope has several lenses, it also is called a compound microscope.

A light microscope usually has at least three objective lenses: the low-power, high-power, and oil-immersion lenses. In general, these lenses mag- nify an object 10, 40, and 100 times, respectively. (Magnification is represented by the multiplication sign, ×.) The ocular lens then magnifies the inter- mediate image produced by the objective lens by 10×. Therefore, the total magnification achieved is 100×, 400×, and 1,000×, respectively.

For an object to be seen distinctly, the lens system must have good resolving power; that is, it must transmit light without variation and allow

Total magnification: The magnification of the ocular multiplied by the magnification of the objec- tive lens being used.

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3.3 Microscopy 85

Ocular

Objectiv e

Stage

Light source

Condenser Oil- immersion lens

Light

Oil

Glass slide

Condenser

Stage

Lost light

WITHOUT OIL

WITH OIL

100X 1.25

160/0.17

Light

Light rays

Eyeball

Ocular lens

Intermediate image

Objective lens

Condenser lens

Light

Bacterium (object)

Image magnified

Object

FIGURE 3.10 The Light Microscope. (A) The light microscope is used in many instructional and clinical laboratories. Note the important features of the microscope that contribute to the visualization of the object. (B) Image formation in the light microscope requires light to pass through the objective lens, forming an intermediate image. This image serves as an object for the ocular lens, which further magnifies the image and forms the final image the eye perceives. (C) When using the oil immersion lens (100�), oil must be placed between and continuous with the slide and objective lens. »» Why must oil be used with the 100� oil-immersion lens?

(A) (C)

(B)

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86 CHAPTER 3 Concepts and Tools for Studying Microorganisms

The negative stain technique works in the opposite manner ( FIGURE 3.11B ). Bacterial cells are mixed on a slide with an acidic (anionic) dye such as nigrosin (a black stain) or India ink (a black drawing ink). The mixture then is pushed across the face of the slide and allowed to air-dry. Because the anionic dye carries a negative charge, it is repelled from the cell wall and cytoplasm. The stain does not enter the cells and the observer sees clear or white cells on a black or gray background. Because this technique avoids chemical reactions and heat fixation, the cells appear less shriveled and less distorted than in a simple stain. They are closer to their natural condition.

The Gram stain technique is an example of a differential staining procedure; that is, it allows the observer to differentiate (separate) bacterial

To perform the simple stain technique, bacterial cells in a droplet of water or broth are smeared on a glass slide and the slide air-dried. Next, the slide is passed briefly through a flame in a process called heat fi xation, which bonds the cells to the slide, kills any organisms still alive, and increases stain absorption. Now the slide is flooded with a basic (cationic) dye such as methy- lene blue ( FIGURE 3.11A ). Because cationic dyes have a positive charge, the dye is attracted to the cytoplasm and cell wall, which primarily have negative charges. By contrasting the blue cells against the bright background, the staining pro- cedure allows the observer to measure cell size and determine cell shape. It also can provide informa- tion about how cells are arranged with respect to one another (Chapter 4).

3.5: Environmental Microbiology Biological Oxymorons

An oxymoron is a pair of words that seem to refer to opposites, such as jumbo shrimp, holy war, old news, and sweet sorrow. One of the characteristics we used for microorganisms is that most are invisible to the naked eye; you need a microscope to see them. Always true? So how about the oxymoron: macro- scopic microorganism?

In 1993, researchers at Indiana University discovered near an Australian reef macroscopic bacterial cells in the gut of surgeonfish. Each cell was so large that a microscope was not needed to see it. The spectacular giant, measuring over 0.6 mm in length (that’s 600 µm compared to 2 µm for Escherichia coli) even dwarfs the protozoan Paramecium.

While on an expedition off the coast of Namibia (western coast of southern Africa) in 1997, Heide Schultz and teammates from the Max Planck Institute for Marine Microbiology in Bremen, Germany, found another bacterial monster in sediment samples from the sea floor. These bacterial cells were spherical being about 0.1 mm to 0.3 mm in diameter—but some as large as 0.75 mm—about the diameter of the period in this sentence (see figure). Their volume is about 3 million times greater than that of E. coli. The cells, shining white with enclosed sulfur granules, were held together in chains by a mucus sheath looking like a string of pearls. Thus, the bacterial species was named Thiomargarita namibiensis (meaning “sulfur pearl of Namibia”). Another closely related strain was discovered in the Gulf of Mexico in 2005.

How does a bacterial cell survive in so large a size? The trick is to keep the cytoplasm as a thin layer plastered against the edge of the cell so mate- rials do not need to travel (diffuse) far to get into or out of the cell. The rest of the cell is a giant “bub- ble,” called a vacuole, in which nitrate and sulfur are stored as potential energy sources. Thus, the actual cytoplasmic layer is microscopic and as close to the surface as possible.

Yes, the vast majority of microorganisms are microscopic, but exceptions have been found in some exotic places.

A phase microscopy image showing a chain of Thiomargarita namibiensis cells. (Bar = 250 µm.)

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3.3 Microscopy 87

3.6: Tools Calculating Resolving Power

The resolving power (RP) of a lens system is important in microscopy because it indicates the size of the smallest object that can be seen clearly. The resolving power varies for each objective lens and is calcu- lated using the following formula:

RP NA

= ×

λ

2

In this formula, the Greek letter λ (lambda) represents the wavelength of light; for white light, it aver- ages about 550 nm. The symbol NA stands for the numerical aperture of the lens and refers to the size of the cone of light that enters the objective lens after passing through the specimen. This number gener- ally is printed on the side of the objective lens (see Figure 3.10C). For an oil-immersion objective with an NA of 1.25, the resolving power may be calculated as follows:

RP = ×

= = 550

2 1 25

550

2 5 220 0 22

nm nm or m

. . . µ

Because the resolving power for this lens system is 220 nm, any object smaller than 220 nm could not be seen as a clear, distinct object. An object larger than 220 nm would be resolved.

cells visually into two groups based on staining differences. The Gram stain technique is named for Christian Gram, the Danish physician who first perfected the technique in 1884.

The first two steps of the technique are straightforward ( FIGURE 3.12A ). Air-dried, heat- fixed smears are (1) stained with crystal violet, rinsed, and then (2) a special Gram’s iodine solu- tion is added. All bacterial cells would appear blue-purple if the procedure was stopped and the sample viewed with the light microscope. Next, the smear is (3) rinsed with a decolorizer, such as 95% alcohol or an alcohol-acetone mixture.

Observed at this point, certain bacterial cells may lose their color and become transparent. These are the gram- negative bacterial cells. Others retain the crystal violet and represent the gram- positive bacterial cells. The last step (4) uses safranin, a red cationic dye, to counterstain the gram-negative organisms; that is, give them a orange-red color. So, at the technique’s conclu- sion, gram-positive cells are blue-purple while gram-negative cells are orange-red ( FIGURE 3.12B ). Similar to simple staining, gram staining also allows the observer to determine size, shape, and arrangement of cells.

Basic dye (+) Bacterial cell Cell stained

(A) Simple stain technique

Dye attracted Dye repelled

Acidic dye (–) Bacterial cell

(B) Negative stain technique

Cell unstained

FIGURE 3.11 Important Staining Reactions in Microbiology. (A) In the simple stain technique, the cells in the smear are stained and contrasted against the light background. (B) With the negative stain technique, the cells are unstained and contrasted against a dark background. »» Explain how the simple and negative staining procedures stain and do not stain cells, respectively.

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88 CHAPTER 3 Concepts and Tools for Studying Microorganisms

when they reproduce, Mycobacterium species often are referred to as “red snappers.” CONCEPT AND REASONING CHECKS 3.11 What would happen if a student omitted the alcohol

wash step when doing the Gram stain procedure?

Knowing whether a bacterial cell is Gram positive or Gram negative is important for micro- biologists and clinical technicians who use the results from the Gram stain technique to classify it in Bergey’s Manual or aid in the identification of an unknown bacterial species (TEXTBOOK CASE 3).

Gram-positive and gram-negative bacte- rial cells also differ in their susceptibility to chemical substances such as antibiotics (gram- positive cells are more susceptible to penicil- lin, gram-negative cells to tetracycline). Also, gram-negative cells have more complex cell walls, as described in Chapter 4, and gram-positive and gram-negative bacterial species can produce dif- ferent types of toxins.

One other differential staining procedure, the acid-fast technique, deserves mention. This tech- nique is used to identify members of the genus Mycobacterium, one species of which causes tuber- culosis. These bacterial organisms are normally difficult to stain with the Gram stain because the cells have very waxy walls that resist the dyes. However, the cell will stain red when treated with carbol-fuchsin (red dye) and heat (or a lipid solu- bilizer) ( FIGURE 3.13 ). The cells then retain their color when washed with a dilute acid-alcohol solu- tion. Other stained genera lose the red color easily during the acid-alcohol wash. The Mycobacterium species, therefore, is called acid resistant or “acid fast.” Because they stain red and break sharply

(A) Gram stain technique

Gram-positive bacterial cell

Gram-negative bacterial cell

Step 1 Crystal violet

Step 2 Iodine

Step 3 Alcohol wash

Step 4 Safranin

Purple Blue-purple Remains blue-purple

Remains blue-purple

Purple Blue-purple Loses stain Orange-red

FIGURE 3.12 Important Staining Reactions in Microbiology. The Gram stain technique is a differential staining procedure. (A) All bacterial cells stain with the crystal violet and iodine, but only gram-negative cells lose the color when alcohol is applied. Subsequently, these bacterial cells stain with the safranin dye. Gram-positive cells remain blue purple. (B) This light micrograph demonstrates the staining results of a Gram stain for differenti- ating between gram-positive and gram-negative cells. (Bar = 10 µm.) »» Besides identifying the Gram reaction, what other characteristics can be deter- mined using the Gram stain procedure?

(B)

Toxins: Chemical substances that are poisonous.

FIGURE 3.13 Mycobacterium tuberculosis. The acid-fast technique is used to identify species of Mycobacterium. The cells retain the red dye after an acid-alcohol wash. (Bar = 10 µm.) »» Why are cells of Mycobacterium resistant to Gram staining?

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3.3 Microscopy 89

Textbook CASE 3

Bacterial Meningitis and a Misleading Gram Stain

1 A woman comes to the hospital emergency room complaining of severe headache, nausea, vom- iting, and pain in her legs. On examination, cerebral spinal fluid (CFS) was observed leaking from a previous central nervous system (CNS) surgical site.

2 The patient indicates that 6 weeks and 8 weeks ago she had undergone CNS surgery after com- plaining of migraine headaches and sinusitis. Both surgeries involved a spinal tap. Analysis of cultures prepared from the CFS indicated no bacterial growth.

3 The patient was taken to surgery where a large amount of CFS was removed from underneath the old incision site. The pinkish, hazy fluid indicated bacterial meningitis, so among the laboratory tests ordered was a Gram stain.

4 The patient was placed on antibiotic therapy, consisting of vancomycin and cefotaxime.

5 Laboratory findings from the gram-stained CFS smear showed a few gram-positive, spherical bac- terial cells that often appeared in pairs. The results suggested a Streptococcus pneumoniae infection.

6 However, upon reexamination of the smear, a few gram-negative spheres were observed.

7 When transferred to a blood agar plate, growth occurred and a prepared smear showed many gram- negative spheres (see figure). Further research in- dicated that several genera of gram-negative bacteria, including Acinetobacter, can appear gram-positive due to under-decolorization during the alcohol wash step.

8 Although complicated by the under-decolorization outcome, the final diagnosis was bacterial meningitis due to Acinetobacter baumanii.

Questions:

(Answers can be found in Appendix D.)

A. From the gram-stained CSF smear, what color were the gram-positive bacterial spheres?

B. After reexamination of the CFS smear, assess the reliability of the gram-stained smear.

C. What reagent is used for the decolorization step in the Gram stain?

Adapted from: Harrington, B. J. and Plenzler, M., 2004. Misleading gram stain findings on a smear from a cerebrospinal fluid specimen. Lab. Med. 35(8): 475–478.

For additional information see http://www.cdc.gov/ncidod/hip/aresist/acin_general.htm.

A gram-stained preparation from the blood agar plate.

Light Microscopy Has Other Optical Configurations KEY CONCEPT 12. Different optical configurations provide detailed views

of cells.

Bright-field microscopy provides little contrast ( FIGURE 3.14A ). However, a light microscope can be outfitted with other optical systems to improve contrast of microorganisms without staining.

Three systems commonly employed are men- tioned here.

Phase-contrast microscopy uses a special condenser and objective lenses. This condenser lens on the light microscope splits a light beam and throws the light rays slightly out of phase. The separated beams of light then pass through and around the specimen, and small differences in the refractive index within the specimen show up as different degrees of brightness and contrast. With

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makes the specimen visible, as the surrounding area appears dark because it lacks background light ( FIGURE 3.14C ). Dark-field microscopy pro- vides good resolution and often illuminates parts of a specimen not seen with bright-field optics. Dark-field microscopy also is the preferred way to study motility of live cells.

Dark-field microscopy helps in the diagnosis of diseases caused by organisms near the limit of resolution of the light microscope. For example, syphilis, caused by the spiral bacterium Treponema pallidum, has a diameter of only about 0.15 µm. Therefore, this bacterial species may be observed in scrapings taken from a lesion of a person who has the disease and observed with dark-field microscopy.

Fluorescence microscopy is a major asset to clinical and research laboratories. The technique has been applied to the identification of many microorganisms and is a mainstay of modern microbial ecology and especially clinical micro- biology.

For fluorescence microscopy, microorganisms are coated with a fluorescent dye, such as fluores- cein, and then illuminated with ultraviolet (UV) light. The energy in UV light excites electrons in fluorescein, and they move to higher energy lev- els. However, the electrons quickly drop back to their original energy levels and give off the excess energy as visible light. The coated microorganisms thus appear to fluoresce; in the case of fluorescein, they glow a greenish yellow. Other dyes produce other colors ( FIGURE 3.15 ).

An important application of fluorescence microscopy is the fluorescent antibody tech- nique used to identify an unknown organism. In one variation of this procedure, fluorescein is chemically attached to antibodies, the protein molecules produced by the body’s immune sys- tem. These “tagged” antibodies are mixed with a sample of the unknown organism. If the antibodies are specific for that organism, they will bind to it and coat the cells with the dye. When subjected to UV light, the organisms will fluoresce. If the organisms fail to fluoresce, the antibodies were not specific to that organism and a different tagged antibody is tried.

More recently, such methods have revolution- ized our understanding of the subcellular organi-

phase-contrast microscopy, microbiologists can see organisms alive and unstained ( FIGURE 3.14B ). The structure of yeasts, molds, and protozoa is typi- cally studied with this optical configuration.

Dark-field microscopy also uses a special condenser lens mounted under the stage. The condenser scatters the light and causes it to hit the specimen from the side. Only light bounc- ing off the specimen and into the objective lens

FIGURE 3.14 Variations in Light Microscopy. The same Paramecium specimen seen with three different opti- cal configurations: (A) bright-field, (B) phase-contrast, and (C) dark-field. (Bar = 25 µm.) »» What advantage is gained by each of the three microscopy techniques?

(A)

(B)

(C)

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3.3 Microscopy 91

in Chapter 1, the early days of electron microscopy produced electron micrographs that showed bac- terial cells indeed were cellular but their structure was different from eukaryotic cells.

The power of electron microscopy is the extraordinarily short wavelength of the beam of electrons. Measured at 0.005 nm (compared to 550 nm for visible light), the short wavelength dramatically increases the resolving power of the system and makes possible the visualization of viruses and detailed cellular structures, often called the ultrastructure of cells. The practical limit of resolution of biological samples with the electron microscope is about 2 nm, which is 100× better than the resolving power of the light micro- scope. The drawback of the electron microscope is that the method needed to prepare a specimen kills the cells or organisms.

Two types of electron microscopes are com- monly in use. The transmission electron micro- scope (TEM) is used to view and record detailed structures within cells ( FIGURE 3.17A ). Ultrathin sections of the prepared specimen must be cut because the electron beam can penetrate matter only a very short distance. After embedding the specimen in a suitable plastic mounting medium or freezing it, scientists cut the specimen into sections with a diamond knife. In this manner, a single bacterial cell can be sliced, like a loaf of bread, into hundreds of thin sections.

Several of the sections are placed on a small grid and stained with heavy metals such as lead and osmium to provide contrast. The microscopist then inserts the grid into the vacuum chamber of the microscope and focuses a 100,000-volt elec- tron beam on one portion of a section at a time. An image forms on the screen below or can be recorded on film. The electron micrograph may be enlarged with enough resolution to achieve a final magnification approaching 2 million ×.

The scanning electron microscope (SEM) was developed in the late 1960s to enable research- ers to see the surfaces of objects in the natural state and without sectioning. The specimen is placed in the vacuum chamber and covered with a thin coat of gold. The electron beam then scans across the specimen and knocks loose showers of electrons that are captured by a detector. An image builds line by line, as in a television receiver. Electrons

FIGURE 3.15 Fluorescence Microscopy. Fluorescence microscopy of sporulating cells of Bacillus subtilis. DNA has been stained with a dye that fluoresces red and a sporulating protein with fluorescein (green). RNA syn- thesis activity is indicated by a dye that fluoresces blue. (Bar = 15 µm.) »» What advantage is gained by using fluo- rescence optics over the other light microscope optical configurations?

zation in bacterial cells. We will see the results in the next chapter. CONCEPT AND REASONING CHECKS 3.12 What optical systems can improve specimen contrast

over bright-field microscopy?

Electron Microscopy Provides Detailed Images of Cells, Cell Parts, and Viruses KEY CONCEPT 13. Electron microscopy uses a beam of electrons to mag-

nify and resolve specimens.

The electron microscope grew out of an engineer- ing design made in 1932 by the German physicist Ernst Ruska (winner of the 1986 Nobel Prize in Physics). Ruska showed that electrons will flow in a sealed tube if a vacuum is maintained to pre- vent electron scattering. Magnets, rather than glass lenses, pinpoint the flow onto an object, where the electrons are absorbed, deflected, or transmit- ted depending on the density of structures within the object ( FIGURE 3.16 ). When projected onto a screen underneath, the electrons form a final image that outlines the structures. As mentioned

Electron micrographs: Images recorded on electron-sensitive film.

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92 CHAPTER 3 Concepts and Tools for Studying Microorganisms

that strike a sloping surface yield fewer electrons, thereby producing a darker contrasting spot and a sense of three dimensions. The resolving power of the conventional SEM is about 10 nm and magnifications with the SEM are limited to about 20,000×. However, the instrument provides vivid

and undistorted views of an organism’s surface details ( FIGURE 3.17B ).

The electron microscope has added immea- surably to our understanding of the structure and function of microorganisms by letting us penetrate their innermost secrets. In the chapters ahead, we

Condenser lens

Specimen

Objective lens

Projector lens Intermediate image

Electron source

Binoculars

Final image on photographic film or screen

FIGURE 3.16 The Electron Microscope. (A) A transmission electron microscope (TEM). (B) A schematic of a TEM. A beam of electrons is emitted from the electron source and electromagnets are used to focus the beam on the specimen. The image is magnified by objective and projector lenses. The final image is projected on a screen, television monitor, or pho- tographic film. »» How does the path of the image for the transmission electron microscope compare with that of the light microscope (Figure 3.10)?

(A) (B)

FIGURE 3.17 Transmission and Scanning Electron Microscopy Compared. The bacterium Pseudomonas aeruginosa (false-color images) as seen with two types of electron microscopy. (A) A view of sectioned cells seen with the transmission electron microscope. (Bar = 1.0 µm.) (B) A view of whole cells seen with the scanning electron microscope. (Bar = 3.0 µm.) »» What types of information can be gathered from each of these electron micrographs?

(A) (B)

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Summary of Key Concepts 93

TABLE

3.3 Comparison of Various Types of Microscopy Type of Special Appearance Magnification Objects Microscopy Feature of Object Range Observed

Light Bright-field Visible light Stained microorganisms 100×–1,000× Arrangement, shape, and illuminates object on clear background size of killed microorganisms (except viruses) Phase-contrast Special condenser throws Unstained micro- 100×–1,000× Internal structures of live, light rays “out of organisms with unstained eukaryotic phase” contrasted structures microorganisms Dark-field Special condenser Unstained micro- 100×–1,000× Live, unstained scatters light organisms on dark microorganisms; motility background of live cells Fluorescence UV light illuminates Fluorescing micro- 100×–1,000× Outline of microorganisms fluorescent-coated organisms on dark coated with fluorescent- objects background tagged antibodies Electron Transmission Short-wavelength Alternating light and 100×–2,000,000× Ultrathin slices of electron beam dark areas contrasting microorganisms and penetrates sections internal cell structures internal components Scanning Short-wavelength Microbial surfaces 10×–20,000× Surfaces and textures of electron beam knocks microorganisms and cell loose electron showers components

SUMMARY OF KEY CONCEPTS

3.1 The Bacteria/Eukaryotic Paradigm 1. All living organisms share the common emergent properties

of life, attempt to maintain a stable internal state called homeostasis, and interact through a multicellular association (a biofilm) involving chemical communication and cooperation between cells.

2. Bacterial and eukaryotic cells share certain organizational patterns, including genetic organization, compartmentation, metabolic organization, and protein synthesis.

3. Although bacterial and eukaryotic cells carry out many similar processes, eukaryotic cells often contain membrane-enclosed compartments (organelles) to accomplish the processes.

3.2 Classifying Microorganisms 4. Many systems of classification have been devised to catalog

organisms based on shared characteristics. 5. Based on several molecular and biochemical differences, Woese

proposed a three-domain system where the prokaryotes are

separated into two domains, the Bacteria and Archaea. The kingdoms Protista, Fungi, Plantae, and Animalia are placed in the domain Eukarya.

6. Part of an organism’s binomial name is the genus name; the remaining part is the specific epithet that describes the genus name. Thus, a species name consists of the genus and specific epithet.

7. Organisms are properly classified using a standardized hierarchical system from species (the least inclusive) to domain (the most inclusive).

8. Bergey’s Manual is the standard reference to identify and classify bacterial species. Criteria have included traditional characteristics, but modern molecular methods have led to a reconstruction of evolutionary events and organism relationships.

will encounter many of the structures discovered with electron microscopy, and we will better appreciate microbial physiology as it is defined by microbial structures.

The various types of light and electron micros- copy are compared in TABLE 3.3 .

CONCEPT AND REASONING CHECKS 3.13 What type of electron microscope would be used to

examine (a) the surface structures on a Paramecium cell and (b) the organelles in an algal cell?

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94 CHAPTER 3 Concepts and Tools for Studying Microorganisms

3.3 Microscopy 9. Another criterion of a microorganism is its size, a characteristic

that varies among members of different groups. The micrometer (lm) is used to measure the dimensions of bacterial, protozoal, and fungal cells. The nanometer (nm) is commonly used to express viral sizes.

10. The instrument most widely used to observe microorganisms is the light microscope. Light passes through several lens systems that magnify and resolve the object being observed. Although magnification is important, resolving power is key. The light microscope can magnify up to 1,000× and resolve objects as small as 0.2 µm.

11. For bacterial cells, staining generally precedes observation. The simple, negative, Gram, acid-fast, and other staining

techniques can be used to impart contrast and determine structural or physiological properties.

12. Microscopes employing phase-contrast, dark-field, and fluorescence optics have specialized uses in microbiology to contrast cells without staining.

13. To increase resolving power and achieve extremely high magnification, the electron microscope employs a beam of electrons to magnify and resolve specimens. To observe internal details (ultrastructure), the transmission electron microscope is most often used; to see whole objects or surfaces, the scanning electron microscope is useful.

LEARNING OBJECTIVES After understanding the textbook reading, you should be capable of writing a paragraph that includes the appropriate terms and pertinent information to answer the objective.

1. Assess the importance of homeostasis to cell (organismal) survival and contrast bacteria as unicellular and multicellular organisms.

2. Describe the four organizational patterns common to all organisms. 3. Identify the structural distinctions between bacterial and eukaryotic

cells. 4. Explain how knowledge of shared characteristics changed the classifica-

tion of living organisms from Linnaeus to Woese. 5. Explain the assignment of organisms to the three-domain system of

classification.

6. Write scientific names of organisms using the binomial system. 7. Identify the taxa used to classify organisms from least to most inclu-

sive taxa. 8. Contrast the determinative methods used to identify bacterial species. 9. Identify how microbial agents are measured using metric system units.

10. Assess the importance of magnification and resolving power to microscopy.

11. Summarize the Gram stain procedure. 12. Identify the optical configurations that provide contrast with light

microscopy. 13. Compare the uses of the transmission and scanning electron

microscopes.

STEP A: SELF-TEST Each of the following questions is designed to assess your ability to remember or recall factual or conceptual knowledge related to this chapter. Read each question carefully, then select the one answer that best fits the question or statement. Answers to even-numbered questions can be found in Appendix C.

1. What is the term that describes the ability of organisms to maintain a stable internal state?

A. Metabolism B. Homeostasis C. Biosphere D. Ecotype

2. Which one of the following is NOT an organizational pattern common to all organisms?

A. Genetic organization B. Protein synthesis C. Compartmentation D. Microcompartments

3. Which one of the following is NOT found in bacterial cells? A. Ribosomes B. DNA C. Mitochondria D. Cytoplasm

4. Who is considered to be the father of modern taxonomy? A. Woese B. Whittaker C. Haeckel D. Linnaeus

5. ______ was first used to catalog organisms into one of three domains. A. Photosynthesis B. Ribosomal RNA genes C. Nuclear DNA genes D. Mitochondrial DNA genes

6. Which one of the following is the correct genus name for the bacterial organism that causes syphilis?

A. pallidum B. Treponema C. pallidum D. T. pallidum

7. Several classes of organisms would be classified into one A. order. B. genus. C. phylum. D. family.

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Step B: Review 95

8. An important method used in the rapid identification of a pathogen is ______.

A. rRNA gene sequencing B. polymerase chain reaction C. molecular taxonomy D. biochemical tests

9. Most bacterial cells are measured using what metric system of length? A. Millimeters (mm) B. Micrometers (µm) C. Nanometers (nm) D. Centimeters (cm) 10. Resolving power is the ability of a microscope to A. estimate cell size. B. magnify an image. C. see two close objects as separate. D. keep objects in focus.

11. Before bacterial cells are simple stained and observed with the light microscope, they must be

A. smeared on a slide. B. heat fixed. C. air dried. D. All the above (A–C) are correct. 12. If you wanted to study bacterial motility you would most likely use A. a transmission electron microscope. B. a light microscope with dark-field optics. C. a scanning electron microscope. D. a light microscope with phase-contrast optics. 13. If you wanted to study the surface of a bacterial cell, you would use A. a transmission electron microscope. B. a light microscope with phase-contrast optics. C. a scanning electron microscope. D. a light microscope with dark-field optics.

STEP B: REVIEW Answers to even-numbered questions or statements can be found in Appendix C.

14. Construct a concept map for Living Organisms using the following terms (terms can be used more than once). bacterial cells Golgi apparatus cell membrane lysosomes chloroplasts microcompartments cytoplasm mitochondria cytoskeleton nucleus cytosol RER DNA region ribosomes eukaryotic cells SER flagella

15. Construct a concept map for staining techniques using the following terms only once. acid-fast technique differential stain procedure acidic dye gram negative basic dye gram positive blue-purple cells gram stain technique cell arrangement Mycobacterium cell shape negative stain technique cell size orange-red cells contrast simple stain technique

Match the statement on the left to the term on the right by placing the letter of the term in the available space.

Statement 16. _____ Major group of organisms whose cells have no nucleus or organ-

elles in the cytoplasm.

17. _____ Bacterial organisms capable of photosynthesis.

18. _____ Type of electron microscope for which cell sectioning is not required.

19. _____ These structures carry out protein synthesis in all cells.

20. _____ The organelle, absent in bacteria, that carries out the conversion of chemical energy to cellular energy in eukaryotes.

21. _____ Domain in which fungi and protista are classified.

22. _____ Staining technique that differentiates bacterial cells into two groups.

23. _____ Category into which two or more genera are grouped.

24. _____ The staining technique employing a single cationic dye.

25. _____ Type of microscopy using UV light to excite a dye-coated specimen.

Term A. Bacteria J. Homeostasis B. Chloroplast K. Mitochondrion C. Cyanobacteria L. Negative D. Dark-field M. Phase-contrast E. Eukarya N. Ribosomes F. Family O. Scanning G. Fluorescence P. Simple H. Fungi Q. Taxonomy I. Gram R. Transmission

HTTP://MICROBIOLOGY.JBPUB.COM/9E The site features eLearning, an online review area that provides quizzes and other tools to help you study for your class. You can also follow useful links for in-depth information, read more MicroFocus stories, or just find out the latest microbiology news.

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96 CHAPTER 3 Concepts and Tools for Studying Microorganisms

STEP D: QUESTIONS FOR THOUGHT AND DISCUSSION Answers to even-numbered questions can be found in Appendix C. 30. A local newspaper once contained an article about “the famous bacteria

E. coli.” How many things can you find wrong in this phrase? Rewrite the phrase correctly.

31. Microorganisms have been described as the most chemically diverse, the most adaptable, and the most ubiquitous organisms on Earth. Although your knowledge of microorganisms still may be limited at this point, try to add to this list of “mosts.”

32. Bacteria lack the cytoplasmic organelles commonly found in the eukary- otes. Provide a reason for this structural difference.

33. A new bacteriology laboratory is opening in your community. What is one of the first books that the laboratory director will want to purchase? Why is it important to have this book?

34. In a respected science journal, an author wrote, “Linnaeus gave each life form two Latin names, the first denoting its genus and the second its species.” A few lines later, the author wrote, “Man was given his own genus and species Homo sapiens.” What is conceptually and technically wrong with both statements?

35. A student of general biology observes a microbiology student using immersion oil and asks why the oil is used. “To increase the magnifica- tion of the microscope” is the reply. Do you agree? Why?

36. Every state has an official animal, flower, or tree, but only Oregon has a bacterial species named in its honor: Methanohalophilus oregonese. The specific epithet oregonese is obvious, but can you decipher the meaning of the genus name?

STEP C: APPLICATIONS Answers to even-numbered questions can be found in Appendix C. 26. A student is performing the Gram stain technique on a mixed culture

of gram-positive and gram-negative bacterial cells. In reaching for the counterstain in step 4, he inadvertently takes the methy lene blue bottle and proceeds with the technique. What will be the colors of gram- positive and gram-negative bacteria at the conclusion of the technique?

27. Would the best resolution with a light microscope be obtained using red light (λ = 680 nm), green light (λ = 520 nm), or blue light (λ = 500 nm)? Explain your answer.

28. Identify the cell structures (a–p) indicated in drawings (A) and (B) below.

29. The electron micrograph below shows a group of bacterial cells. The micrograph has been magnified 5,000×. At this magnification, the cells are about 10 mm in length. Calculate the actual length of the bacterial cells in micrometers (µm)?

d

i

b

e

a

c

fg

h

k

j

(A)

p

l

m

n

0(A) (B)

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Inheritance Lab Report – 2

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