Thiomargarita namibia is the smallest bacteria known to man

MICROBIOLOGY

Unit outcomes addressed in this Assignment:

· List important discoveries in microbiology and their importance

· Discuss the classification schema

· Select appropriate microscopic method to study different types of microorganisms

Instructions

· In an essay, describe the various mechanisms utilized within the field of microscopy for studying microbes.

· Be sure to include the appropriate staining techniques.

Requirements

· Your essay should be a minimum of 500 words

· Be sure that your grammar, sentence structure, and word usage is appropriate.

· APA FORMAT

Guidelines

· Identifies light and electron as two main branches of Microscopes

· Identifies functional differences between the two main branches of microscopes

· Identifies the differences in staining techniques

· Provides specific microbial staining examples

· Identifies how microscopy is utilized in identifying unknown

microbial specimen.

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.

64

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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-