37623 CH03 069 101 - Jones & Bartlett · PDF file70 CHAPTER 3 Concepts and Tools for ... Within this structure, a cell-secreted slime forms ... multicellular structure? FIGURE 3.3

69

3Concepts and Tools for StudyingMicroorganisms

The oceans of the world are a teeming but invisible forest ofmicroorganisms and viruses. As we discovered in Chapter 1, thesemicroscopic agents are not only found around the world but, as StevenGiovannoni had pointed out for SAR11, being in such numbersimplies they must be doing something important.

A substantial portion of the marine microbes represent the phyto-plankton (phyto = “plant”; plankto = “wandering”), which are floatingcommunities of prokaryotic cyanobacteria and eukaryotic algae.Besides forming the foundation for the marine food web, the phyto-plankton account for 50 percent of the photosynthesis on earth and, inso doing, supply about half the oxygen gas we and other organismsbreathe.

While sampling ocean water, scientists from MIT’s Woods HoleOceanographic Institution discovered that many of their samples werefull of a marine cyanobacterium, which they eventually namedProchlorococcus. Inhabiting tropical and subtropical oceans, a typicalsample often contained more than 200,000 (2 × 105) cells in one dropof seawater.

Studies with Prochlorococcus suggest the organism is responsiblefor more than 50 percent of the photosynthesis among the phyto-plankton ( ). This makes Prochlorococcus the smallest andmost abundant marine photosynthetic organism yet discovered.

FIGURE 3.1

It’s as if [he] lifted a whole submerged continent out of the ocean.—A prominent biologist speaking of Carl Woese, whose work led to thethree-domain concept for living organisms

3.1 The Prokaryotic/Eukaryotic Paradigm• Prokaryotes undergo biological processes as

complex as in eukaryotes.• There are organizational patterns common to all

prokaryotes and eukaryotes.• Prokaryotes and eukaryotes have distinct

subcellular compartments.MicroInquiry 3: The Evolution of EukaryoticCells

3.2 Cataloging Microorganisms• Organisms historically were grouped by shared

characteristics.• The binomial system identifies each organism by

a universally-accepted scientific name.• Species can be organized into higher, more

inclusive groups.• Historically, most organisms were assigned to one

of several kingdoms.• The three domain system uses nucleotide

sequence analysis to catalog organisms.• Taxonomy now includes many criteria to

distinguish one prokaryote from another.3.3 Microscopy

• Metric system units are the standard formeasurement.

• Light microscopy uses visible light to magnify andresolve specimens.

• Specimens stained with a dye are contrastedagainst the microscope field.

• Different optical configurations provide detailedviews of cells.

• Electron microscopy uses a beam of electrons tomagnify and resolve specimens.

Chapter Preview and Key Concepts

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70 CHAPTER 3 Concepts and Tools for Studying Microorganisms

The success of Prochlorococcus is due, inpart, to the presence of different ecotypesinhabiting different ocean depths. For exam-ple, the high sunlight ecotype occurs in thetop 100 meters while the low-light type isfound between 100 and 200 meters. This latterecotype compensates for the decreased light byincreasing the amount of cellular chlorophyllthat can capture the available light.

In terms of nitrogen sources, the high-lightecotype only uses ammonium ions (NH4

+) (seeMicroFocus 2.5). At increasing depth, NH4

+ isless abundant so the low-light ecotype compen-sates by using a wider variety of nitrogensources.

These and other attributes of Prochlorococ-cus illustrate how microbes survive throughchange. They are of global importance to thefunctioning of the biosphere as well as affect-ing our lives on Earth.

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

isms influence our lives and life on this planet.Microbial ecologists study how the phytoplank-ton communities help in the natural recyclingand use of chemical elements such as nitrogen.Evolutionary microbiologists look at thesemicroorganisms to learn more about their taxo-nomic relationships, while microscopists, bio-chemists, and geneticists study how Prochloro-coccus cells compensate for a changingenvironment of sunlight and nutrients.

This chapter focuses on many of theaspects described above. We examine howprokaryotes maintain a stable internal stateand how they can exist in multicellular, com-plex communities. Throughout the chapterwe are concerned with the relationshipsbetween prokaryotic and eukaryotic organ-isms and the many attributes they share.Then, we explore the methods used to nameand catalog microorganisms. Finally, we dis-cuss the tools and techniques used to observemicroorganisms.

Ecotype:A subgroup of specieshaving specialcharacteristics tosurvive in its ecologicalsurroundings.

Biosphere:That part of theearth—including theair, soil, and water—where life occurs.

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

FIGURE 3.1

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3.1 The Prokaryotic/Eukaryotic Paradigm 71

In Chapter 1 we introduced the concept ofprokaryotes and eukaryotes. In the news mediaor even in scientific magazines and textbooks,bacterial and archaeal cells—the prokaryotes—often are described as “simple organisms” com-pared to the “complex organisms” representingmulticellular plants and animals. This view rep-resents a mistaken perception. Despite theirmicroscopic size, the prokaryotes exhibit everycomplex feature, or emerging property, com-mon to all living organisms. These include:

• DNA as the hereditary material andcomplex gene control.

• Complex biochemical patterns ofgrowth and energy conversions.

• Complex responses to stimuli.• Reproduction.• Adaptation.• Complex organization.

Why then have the prokaryotes inheritedthe misnomer of “simple organisms?” Toanswer that question, we need to examine thesimilarities between prokaryotes and eukary-otes. They share many universal processeseven though in some cases the structures tocarry out these processes may be different.Having done this, we can then better distin-guish the significant differences that setprokaryotes apart. In the next chapter, welook more closely at the differences betweenBacteria and Archaea.

Prokaryote/Eukaryote SimilaritiesKEY CONCEPT

• Prokaryotes undergo biological processes as complexas in eukaryotes.

All organisms continually battle their externalenvironment, where factors such as tempera-ture, sunlight, or toxic chemicals can have seri-ous consequences. Organisms strive to main-tain a stable internal state by makingappropriate metabolic or structural adjust-ments. This ability to adjust yet maintain a rela-tively steady internal state is called homeostasis(homeo = “similar”; stasis = “state”). Two exam-ples illustrate the concept.

The low-light Prochlorococcus ecotype men-tioned in the chapter introduction lives atdepths of 80 to 200 meters. At these varyingdepths, transmitted sunlight decreases and anyone nitrogen source is less accessible. The eco-type compensates for the light reduction andnitrogen limitation by (1) increasing the amountof cellular chlorophyll to capture light and (2)using a wider variety of available nitrogensources. These adjustments maintain a relativelystable internal state.

Suppose a patient is given an antibiotic tocombat a bacterial infection ( ). Inresponse, the infecting bacterium may com-pensate for the change by pumping the drugout of the cell. The adjustment maintainshomeostasis in the bacterial cell.

In both these examples, the internal envi-ronment is maintained despite a changingenvironment. Such, often complex, homeosta-tic controls are as critical to prokaryotes asthey are to eukaryotes (MicroFocus 3.1).

FIGURE 3.2

3.1 The Prokaryotic/Eukaryotic Paradigm

Chlorophyll:The major pigmentused to convertsunlight into sugar.

MICROORGANISM

Microorganism

Compensation fails Compensation succeeds

Microorganism dies (antibiotic sensitive)

Microorganism lives (antibiotic resistant)

external change affects

loss of homeostasis

homeostasis maintained

attempts to compensate

Homeostasis. A concept map illustrat-ing the ability of a bacterial pathogen to compensate forthe presence of an antibiotic. Survival is dependent onits homeostatic abilities.Q: Redraw the concept map for the low-light response byProchlorococcus.

FIGURE 3.2

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CONCEPT AND REASONING CHECKS

3.1 How does homeostasis relate to the listed emerg-ing properties of living organisms?

Another misunderstanding about prokaryotesis that they represent “unicellular” organisms.And why not? When they are studied, oftenthey appear in the microscope as single cells.The early studies of disease causation done byKoch (see Chapter 1) certainly required purecultures to associate a specific disease withone specific microbe. However, today it is nec-essary to abolish the impression that bacteriaare self-contained, independent organisms. Innature few species live such a pure and soli-tary life. In fact, it has been estimated that asmany as 99 percent of prokaryotic species livein communal associations called biofilms; thatis, in a “multicellular state” where survivalrequires chemical communication and cooper-ation between cells.

One example of prokaryotic multicellular-ity within a species involves a group of soil-dwelling bacteria called the myxobacteria,which exhibit the most complex behavioramong the known prokaryotes. The cells rep-resent a social community dependent on cell-to-cell interactions and communicationthroughout growth and development.

Myxobacterial cells can obtain nutrientsby decomposing dead plant or animal matter.More often, when live prey (e.g., bacterialcells, yeasts, or algae) are “sensed” nearby, cellcommunication within the flat colonies trig-gers (1) a swarming or so-called “wolf pack”behavior characterized by predatory feedingand (2) an increasing secretion of hydrolyticenzymes by the wolf pack to digest the prey.Like a lone wolf, a single cell could not carryout this behavior.

At the other extreme, under starvationconditions and high cell densities, myxobacte-rial cells chemically communicate to form amound of 100,000 cells and cooperate to builda three-dimensional structure called a fruitingbody ( ). Within this structure, acell-secreted slime forms the stalk on top ofwhich the remaining viable cells develop intoresting structures called myxospores.

FIGURE 3.3

Here then is one example where the termunicellular does not apply. MicroFocus 3.2further examines the concept of prokaryoticmulticellularity.

The final misconception to be addressedconcerns the reference to prokaryotes as “sim-ple cells” or “simpler organisms” than theireukaryotic counterparts. Historically, whenone looks at bacterial cells even with an elec-tron microscope, often there is little to see( ). “Cell structure,” representingthe cell’s physical appearance or its compo-nents and the “pattern of organization,” refer-ring to the configuration of those structuresand their relationships to one another, doesgive the impression of simpler cells.

But what has been overlooked is the “cel-lular process,” the activities all cells carry outfor the continued survival of the cell (andorganism). At this level, the complexity is justas intricate as in any eukaryotic cell. So, inreality, prokaryotes carry out many of thesame cellular processes as eukaryotes—only

FIGURE 3.4

A Myxobacterial Fruiting Body. Thestalk (lower left) is made of slime secreted by the cellsthat eventually form the resting spores in the head. (Bar = 10 μm.)Q: How does the myxobacterial fruiting body represent amulticellular structure?

FIGURE 3.3

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3.1: Being SkepticalAre Cyanobacterial Food Supplements Better for What’s Ailing You?

The food supplement business is always looking for new products to boost human health. Take the phy-toplankton. The chapter introduction highlighted the importance of these microbial communities to themarine food web. Some individuals therefore have perceived this as indicating these microorganismsmust be very nutritious.

Among the items found on today’s health food shelves and the Internet are capsules, pills, and pow-ders consisting of dried cyanobacteria, primarily from the fresh-water species Aphanizomenon flos-aquae(AFA) and the genus Spirulina (see figure). These products are promoted as a treatment or cure for awide variety of human ailments, including asthma, allergies, chronic viral hepatitis, anxiety, depression,fatigue, hypoglycemia, digestive problems, and attention deficit disorder. The microbial products pur-port to help individuals lose weight, improve memory and mental ability, boost immune system func-tion, and restore overall cellular balance.

Are these claims reasonable? What potentially unique nutrients must these cyanobacterial supple-ments have to provide relief from all these ailments? The answer—none that we know. The organismscontain large amounts of chlorophyll, but humans cannot carry out photosynthesis so why eat chloro-phyll? Cyanobacteria do contain small amounts of protein, beta carotene, and a few vitamins and miner-als. However, you would have to eat shovels full of the cyanobacterial powders to get a sufficient levelof these nutrients—there are more sensible and cheaper ways to get these same nutrients in easily con-sumable amounts.

Importantly, having comefrom natural lakes, the productsmay be contaminated with toxicsubstances, such as heavy metalsthat polluted the lake water.

The verdict? Yes, microbes dohave many useful and importantroles in the human body. How-ever, medical organizations agreethere is not enough evidence tosupport their use for treatmentof any medical condition. When-ever a product proclaims such awide-range of curative powers,be skeptical. As the old adagesays, “If it is too good to betrue,” it probably is. A mass of Spirulina cells. (Bar = 5 lm.)

Simpler Organisms? This false-colorphoto of the bacterial species Yersinia pestis, thecausative agent of plague, was taken with an electronmicroscope. (Bar =1 μm.)Q: Why does this view suggest to the observer thatprokaryotes are simple cells?

FIGURE 3.4

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without the need for an elaborate, visible pat-tern of organization.

Let’s now look at the cell structure rela-tionships in terms of organized patterns andprocesses.CONCEPT AND REASONING CHECKS

3.2 Respond to the statement that prokaryotes aresimple, unicellular organisms.

Prokaryotes and Eukaryotes: The Similarities in Organizational PatternsKEY CONCEPT

• There are organizational patterns common to allprokaryotes and eukaryotes.

In the 1830s, Matthias Schleiden and TheodorSchwann developed part of the cell theory bydemonstrating all organisms are composed ofone or more cells, making the cell the funda-mental unit of life. (Note: about 20 years later,Rudolph Virchow added that all cells arisefrom pre-existing cells.) Although the conceptof a microorganism was just in its infancy atthe time, the doctrine suggests that there arecertain organizational patterns common toprokaryotes and eukaryotes.

Genetic organization. All organisms havea similar genetic organization whereby thehereditary material is communicated or

3.2: Environmental MicrobiologyMulticellular Prokaryotes

As microbiologists continue to study prokaryotic organisms, it is becoming quite obvious that thesemicrobes are social creatures. Whether it is studying myxobacteria, mixed bacterial populations in abiofilm, or other communities of microorganisms, there is more to bacterial life than just existing in amulticellular community.

Quorum SensingSeveral bacterial behaviors are dependent on population density. When population numbers reach a criti-cal level, chemical signals are produced that alter gene activity. Such quorum sensing (QS) can controlspore formation as described for the myxobacteria or enhance the ability of a pathogen to cause disease.

Chemical communication can occur between different bacterial species. This QS cross talk allows thecells of one species to detect the presence of other species, which could be a good thing or a badthing. In biofilms, QS is necessary for the development of the complex structures within the biofilm.However, Pseudomonas aeruginosa, a pathogen of the human gut, can sense an immune system responseto infection. Through QS, P. aeruginosa counters immune activation by strengthening or enhancing itsability to cause disease by disrupting the function of epithelial cells in the gut. These chemical signalswould not be produced by solitary, non-communicative cells.

Programmed Cell DeathThe multicellular nature of microbial communities also demonstrates cell suicide; that is, geneticallyprogrammed cell death (PCD). Such a phenomenon (called apoptosis) was discovered in eukaryotesmany years ago, but a death program in bacterial cells is a newly discovered phenomenon that is basedon multicellular behavioral responses.

One example of PCD is the myxobacteria, where up to 80 percent of the cells die in the process offruiting body formation, the remaining 20 percent becoming the myxospores. Also, during biofilm devel-opment, overcrowding can be prevented by cells dying within the biofilm, which hollows out the struc-ture and makes room for new cell reproduction. “Sensed” by the multicellular aggregate, specific toxicproteins trigger this form of PCD.

Perhaps most interesting (and controversial) is research looking at the death of defective or injuredcells resulting from antibiotics. In the face of a chemical attack, cells damaged by the antibioticundergo PCD so they do not become a burden to the remaining cells. Yet other damaged cells in thecommunity apparently turn off the PCD program and become persistor cells—they survive by growing soslowly, they are not susceptible to the action of antibiotics.

Interestingly, many of the multicellular behaviors observed only occur in wild strains in nature. Evi-dently, the “good life” in a culture plate has made such cell-cell behaviors unnecessary—the cells havebecome unicellular “couch bacteria.”

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expressed (Chapter 7). The organizationalpattern for the hereditary material is in theform of one or more chromosomes. Struc-turally, most prokaryotic cells have a single,circular DNA molecule without an enclosingmembrane ( .) Eukaryotic cells,however, have multiple, linear chromosomesenclosed by the membrane envelope of thecell nucleus.

Compartmentation. All prokaryotes andeukaryotes have an organizational pattern sep-arating the internal compartments from thesurrounding environment but allowing for theexchange of solutes and wastes. The patternfor compartmentation is represented by thecell. All cells are surrounded by a cell mem-brane (known as the plasma membrane ineukaryotes), where the phospholipids formthe impermeable boundary to solutes whileproteins regulate the exchange of solutes and

FIGURE 3.5

wastes across the membrane. We have more tosay about membranes in the next chapter.

Metabolic organization. The process ofmetabolism is a consequence of compartmen-tation. By being enclosed by a membrane, allcells have an internal environment in whichchemical reactions occur. This space, calledthe cytoplasm, represents everything sur-rounded by the membrane and, in eukaryoticcells, exterior to the cell nucleus. If the cellstructures are removed from the cytoplasm,what remains is the cytosol, which consists ofwater, salts, ions, and organic compounds asdescribed in Chapter 2.

Protein synthesis. All organisms mustmake proteins, which we learned in Chapter 2are the workhorses of cells and organisms.The structure common to all prokaryotes andeukaryotes is the ribosome, an RNA-proteinmachine that cranks out proteins based on the

Cytoplasm

Ribosome

Cell membrane

Cell wall

DNA (chromosome) (a)

Ribosomes attached to endoplasmic reticulum

DNA (chromosomes)

Nuclear envelope Lysosome

Plasma membrane

Nucleolus

Free ribosomes

Cilia

Flagellum

Basal body

Mitochondrion

Smooth endoplasmic reticulum

Rough endoplasmic reticulum

Centrioles Microtubules

Actin filaments

Golgi apparatus

Nuclear pore

A Comparison of Prokaryotic and Eukaryotic Cells. (A) A stylized bacterial cell as an example of a prokaryotic cell. Relatively fewvisual compartments are present. (B) A protozoan cell as a typical eukaryotic cell. Note the variety of cellular subcompartments, many of which are dis-cussed in the text.Q: List the ways you could microscopically distinguishing a eukaryotic microbial cell from a prokaryotic cell.

FIGURE 3.5

(A) (B)

Metabolism:All the chemicalreactions occurring inan organism or cell.

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genetic instructions it receives from the DNA(Chapter 7). Although the pattern for proteinsynthesis is identical, structurally prokaryoticribosomes are smaller than their counterpartsin eukaryotic cells.CONCEPT AND REASONING CHECKS

3.3 The cell theory states that the cell is the funda-mental unit of life. Summarize those processes allcells have that contribute to this fundamental unit.

Prokaryotes and Eukaryotes: The Structural DistinctionsKEY CONCEPT

• Prokaryotes and eukaryotes have distinct subcellularcompartments.

Eukaryotic microbes have a variety of struc-turally discrete, often membrane-enclosed,subcellular compartments called organelles(Figure 3.5). Prokaryotic cells also have sub-cellular compartments—they just are notreadily visible or membrane enclosed.

Protein/lipid transport. Eukaryoticmicrobes have a series of membrane-enclosedorganelles in the cytosol that compose thecell’s endomembrane system, which isdesigned to transport protein and lipid cargothrough and out of the cell. This systemincludes the endoplasmic reticulum (ER),which consists of flat membranes to whichribosomes are attached (rough ER) and tube-like membranes without ribosomes (smoothER). These portions of the ER are involved inprotein and lipid synthesis and transport,respectively.

The Golgi apparatus is a group of inde-pendent stacks of flattened membranes andvesicles where the proteins and lipids comingfrom the ER are processed, sorted, and pack-aged for transport. Lysosomes, somewhat cir-cular, membrane-enclosed sacs containingdigestive (hydrolytic) enzymes, are derivedfrom the Golgi apparatus and are vital to thekilling of many pathogens by white blood cells.

Prokaryotes lack an endomembrane sys-tem, yet they are capable of manufacturingand modifying proteins and lipids just astheir eukaryotic relatives do. However, manybacterial cells contain so-called microcom-partments surrounded by a protein shell.These microcompartments represent a typeof organelle since the shell proteins can con-

trol transport similar to membrane-enclosedorganelles.

Energy metabolism. Cells and organismscarry out one or two types of energy transfor-mations. Through a process called cellular res-piration, all cells convert chemical energy intocellular energy for cellular work. In eukaryoticmicrobes, this occurs in the cytosol and inmembrane-enclosed organelles called mito-chondria (sing., mitochondrion). Bacterialand archaeal cells lack mitochondria; they usethe cytosol and cell membrane to completethe energy converting process.

A second energy transformation, photo-synthesis, involves the conversion of lightenergy into chemical energy. In algal protists,photosynthesis occurs in membrane-boundchloroplasts. Some prokaryotes, such as thecyanobacteria we have mentioned, also carryout almost identical energy transformations.Again, the cell membrane or elaborations ofthe membrane represent the chemical work-bench for the process.

Cell structure and transport. The eukary-otic cytoskeleton is organized into an inter-connected system of fibers, threads, and inter-woven molecules that give structure to the celland assist in the transport of materialsthroughout the cell. The main components ofthe cytoskeleton are microtubules and actinfilaments, each assembled from different pro-tein subunits. Prokaryotes to date have nosimilar physical cytoskeleton, although pro-teins related to those that construct micro-tubules and actin filaments aid in determiningthe shape in some bacterial cells as we will seein Chapter 4.

Cell motility. Many microbial organismslive in watery or damp environments and usethe process of cell motility to move from oneplace to another. Many algae and protozoahave long, thin protein projections called fla-gella (sing., flagellum) that, covered by theplasma membrane, extend from the cell. Bybeating back and forth, the flagella provide amechanical force for motility. Many prokary-otic cells also exhibit motility; however, theflagella are structurally different and without acell membrane covering. The pattern of motil-ity also is different, providing a rotational propeller-like force for motility (Chapter 4).

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3.2 Cataloging Microorganisms 77

Some protozoa also have other membrane-enveloped appendages called cilia (sing., cil-ium) that are shorter and more numerousthan flagella. In some motile protozoa, theywave in synchrony and propel the cell for-ward. No prokaryotes have cilia.

Water balance. The aqueous environmentin which many microorganisms live presents asituation where the process of diffusionoccurs, specifically the movement of water,called osmosis, into the cell. Continuingunabated, the cell would eventually swell andburst (cell lysis) because the cell or plasmamembrane does not provide the integrity toprevent lysis.

Many prokaryotic and eukaryotic cells con-tain a cell wall exterior to the cell or plasmamembrane. Although the structure and organi-zation of the wall differs between groups, allcell walls provide support for the cells, givethem shape, and help them resist the pressureexerted by the internal water pressure.

A summary of the prokaryote and eukary-ote processes and structures is presented in

. MicroInquiry 3 examines a scenariofor the evolution of the eukaryotic cell.CONCEPT AND REASONING CHECKS

3.4 Explain how variation in cell structure betweenprokaryotes and eukaryotes can be compatible witha similarity in cellular processes between prokary-otes and eukaryotes.

TABLE 3.1

Diffusion:The movement of asubstance from whereit is in a higherconcentration to whereit is in a lowerconcentration.

TABLE

3.1 A Comparison of Prokaryotes and Eukaryotes

Microbial Cell Structure or Compartment

Process Prokaryotic Eukaryotic

Genetic organization Circular DNA chromosome Linear DNA chromosomesCompartmentation Cell membrane Plasma membraneMetabolic organization Cytoplasm CytoplasmProtein synthesis Ribosomes RibosomesProtein/lipid transport Cytoplasm Endomembrane systemEnergy metabolism Cytoplasm and cell membrane Mitochondria and chloroplastsCell structure and transport Proteins in cytoplasm Protein filaments in cytoplasmCell motility Prokaryotic flagella Eukaryotic flagella or ciliaWater regulation Cell wall Cell wall

If you open any catalog, items are separatedby types, styles, or functions. For example, ina fashion catalog, watches are separated fromshoes and, within the shoes, men’s, women’s,and children’s styles are separated from oneanother. Even the brands of shoes or their use(e.g., dress, casual, athletic) may be separated.

The human drive to catalog objectsextends to the sciences, especially biologywhere historically the process was not muchdifferent from cataloging watches and shoes; itwas based on shared characteristics. In thissection, we shall explore the principles onwhich microorganisms are cataloged.

Classification Attempts to Catalog Organisms

KEY CONCEPT

• Organisms historically were grouped by shared characteristics.

The science of classification, called taxonomy,involves the systematized arrangement or cat-aloging of related organisms into logical cate-gories. Taxonomy is essential for understand-ing the relationships among living organisms,discovering unifying concepts among organ-isms, and providing a universal “language” forcommunication among biologists.

3.2 Cataloging Microorganisms

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INQUIRY

Biologists and geologists have speculatedfor decades about the chemical evolutionthat led to the origins of the firstprokaryotic cells on Earth (see Micro-Focus 2.1 and 2.6). Whatever the ori-gin, the first ancestral prokaryotes aroseabout 3.8 billion years ago.

Scientists also have proposed variousscenarios to account for the origins of thefirst eukaryotic cells. A key concern hereis figuring out how different membranecompartments arose to evolve into whatare found in the eukaryotic cells today.Debate on this long intractable problemcontinues, so here we present some of theideas that have fueled such discussions.

At some point around 2 billion yearsago, the increasing number of metabolicreactions occurring in some prokaryotesstarted to interfere with one another.Complexity would necessitate more exten-sive compartmentation.

The Endomembrane System May HaveEvolved through InvaginationSimilar to today’s prokaryotic cells, thecell membrane of an ancestral prokaryotemay have had specialized regions involvedin protein synthesis, lipid synthesis, andnutrient hydrolysis. If the invagination ofthese regions occurred, the result couldhave been the internalization of theseprocesses as independent internal mem-brane systems. For example, the mem-branes of the endoplasmic reticulum mayhave originated by multiple invaginationevents of the plasma membrane (see Figure A1).

Biologists have suggested that theelaboration of the evolving ER created the

nuclear envelope. Surrounded and pro-tected by a double membrane, greatergenetic complexity could occur as theprimitive eukaryotic cell continued toevolve in size and function.

Chloroplasts and Mitochondria Arosefrom a Symbiotic Union of EngulfedProkaryotesMitochondria and chloroplasts are notpart of the extensive endomembrane sys-tem. Therefore, these energy-convertingorganelles probably originated in a differ-ent way.

The structure of modern-day chloro-plasts and mitochondria is very similar tothe description of a bacterial cell. In fact,mitochondria, chloroplasts, and bacteriashare a large number of similarities (see Table). In addition, there are bacter-ial cells alive today that carry out cellular respiration similarly to mitochon-dria and other bacterial cells that cancarry out photosynthesis similarly tochloroplasts.

These similar functional patterns,along with other chemical and molecu-lar similarities, suggested to Lynn Mar-gulis that present-day chloroplasts andmitochondria represent modern repre-sentatives of what were once, manyeons ago, free-living prokaryotes. Mar-gulis, therefore, proposed theendosymbiotic theory for the originof mitochondria and chloroplasts. Thehypothesis suggests, in part, that mito-chondria evolved from a prokaryote thatcarried out cellular respiration andwhich was “swallowed” (engulfed) by aprimitive eukaryotic cell. The bacterial

partner then lived within (endo) theeukaryotic cell in a mutually beneficialassociation (symbiosis) (see Figure A2).

Likewise, a photosynthetic prokary-ote, perhaps a primitive cyanobac-terium, was engulfed and evolved intothe chloroplasts present in plants andalgae today (see Figure A3). The theoryalso would explain why both organelleshave two membranes. One was the cellmembrane of the engulfed bacterial celland the other was the plasma membraneresulting from the engulfment process.By engulfing these prokaryotes and notdestroying them, the evolving eukary-otic cell gained energy-conversion abili-ties, while the symbiotic bacterial cellsgained a protected home.

Obviously, laboratory studies can onlyguess at mechanisms to explain how cellsevolved and can only suggest—notprove—what might have happened bil-lions of years ago. The description here isa very simplistic view of how the firsteukaryotic cells might have evolved. Shortof inventing a time machine, we maynever know the exact details for the ori-gin of eukaryotic cells and organelles.

Discussion PointDetermine which endosymbiotic eventmust have come first: the engulfmentof the bacterial progenitor of thechloroplast or the engulfment of thebacterial progenitor of themitochondrion.

3The Evolution of Eukaryotic Cells

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

(A3) Early photosynthetic eukaryotic cell

DNA

Membrane-boundribosomes

Ancient prokaryotic cell

Respiratory bacterium

Photosyntheticbacterium

Nucleus

Nuclearmembrane

Endoplasmicreticulum

Mitochondrion

Chloroplast

Possible Origins of Eukaryotic Cell Compartments. (A1) Invagination of the cell membrane from an ancient prokaryotic cell mayhave lead 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 respiration. (A3) A similarprocess, involving a bacterial cell that carried out photosynthesis, could have accounted for the origin of the chloroplast.

FIGURE A

TABLE

Similarities between Mitochondria, Chloroplasts, Prokaryotes, and Microbial Eukaryotes

Characteristic Mitochondria Chloroplasts Prokaryotes Microbial Eukaryotes

Average size 1–5 μm 1–5 μm 1–5 μm 10–20 μmNuclear envelope present No No No YesDNA molecule shape Circular Circular Circular LinearRibosomes Yes; bacterial-like Yes; bacterial-like Yes Yes; eukaryotic-likeProtein synthesis Make some of Make some of Make all of Make all of

their proteins their proteins their proteins their proteinsReproduction Binary fission Binary fission Binary fission Mitosis and cytokinesis

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The Swedish botanist Carl von Linné, bet-ter known to history as Carolus Linnaeus, laiddown the foundation of modern taxonomy.Until his time, naturalists referred to organ-isms using long cumbersome phrases ordescriptive terms, which often varied by coun-try. In his Systema Naturae, published in sev-eral editions between 1735 and 1759, Linnaeusestablished a uniform system for namingorganisms based on shared characteristics. As aresult, he named about 7,700 species of plantsand 4,400 species of animals. In reflecting thescant knowledge of microorganisms (animal-cules) at the time and his general disinterest,Linnaeus grouped them separately under theheading of Vermes (vermis = “worm”) in thecategory Chaos (chaos = “an abyss”).

There are two major elements to Linnaeus’classification system: the use of binomialnomenclature and a hierarchical organizationof species.

Nomenclature Gives Scientific Namesto OrganismsKEY CONCEPT

• The binomial system identifies each organism by auniversally-accepted scientific name.

In his Systema Naturae, Linnaeus popularizeda two word (binomial) scheme of nomencla-ture, the two words usually derived from Latinor Greek stems. Each organism’s name con-sists of the genus to which the organismbelongs and a specific epithet, a descriptorthat further describes the genus name.Together these two words make up thespecies name. For example, the common bac-terium Escherichia coli resides in the gut of allhumans (Homo sapiens) (MicroFocus 3.3).

Notice in these examples that when aspecies name is written, only the first letter ofthe genus name is capitalized, while the spe-cific epithet is not. In addition, both words areprinted in italics or underlined. After the first

3.3: ToolsNaming Names

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

Genera Named after IndividualsEscherichia 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 ShapeVibrio 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 MicrobeSaccharomyces cerevisiae: in 1837, Theodor Schwann observed yeast cells and called them Saccha-

romyces (saccharo = “sugar;” myce = “fungus”) because the yeast converted grape juice (sugar) intoalcohol; cerevisiae (from cervisia = “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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time a species name has been spelled out,biologists usually abbreviate the genus nameusing only its initial genus letter or someaccepted substitution, together with the fullspecific epithet; that is, E. coli or H. sapiens. Acautionary note: often in magazines and news-papers, proper nomenclature is not followed,so our gut bacterium would be written asEscherichia coli.CONCEPT AND REASONING CHECKS

3.5 Which one of the following is a correctly writtenscientific name for the bacterium that causesanthrax? (a) bacillus Anthracis; (b) BacillusAnthracis; or (c) Bacillus anthracis.

Classification Uses a Hierarchical SystemKEY CONCEPT

• Species can be organized into higher, more inclusivegroups.

Linnaeus’ cataloging of plants and animalsused shared and common characteristics. Suchsimilar organisms that could interbreed wererelated as a species, which formed the leastinclusive level of the hierarchical system. Partof Linnaeus’ innovation was the grouping ofspecies into higher taxa that also were basedon shared, but more inclusive, similarities.

Today several similar species are groupedtogether into a genus (pl. genera). A collectionof similar genera makes up a family and fami-lies with similar characteristics make up anorder. Different orders may be placed togetherin a class and classes are assembled togetherinto a phylum (pl. phyla) or division (in bac-

teriology and botany). All phyla or divisionswould be placed together in a kingdom and/ordomain, the most inclusive level of classifica-tion. outlines the taxonomic hierar-chy for three organisms.

In prokaryotes, an organism may belong toa rank below the species level to indicate a spe-cial characteristic exists within a subgroup ofthe species. Such ranks have practical useful-ness in helping to identify an organism. Forexample, two biotypes of the cholera bac-terium, Vibrio cholerae, are known: Vibriocholerae classic and Vibrio cholerae El Tor.Other designations of ranks include subspecies,serotype, strain, morphotype, and variety.CONCEPT AND REASONING CHECKS

3.6 How would you describe an order in the taxonomicclassification?

Kingdoms and Domains: Trying to Make Sense of TaxonomicRelationshipsKEY CONCEPT

• Historically, most organisms were assigned to one ofseveral kingdoms.

As more microorganisms were described afterLinnaeus’ time, some were considered plants(bacterial, algal, and fungal organisms) whilethe protozoa were categorized as animals.

In 1866, the German naturalist Ernst H.Haeckel disturbed the tidiness of Linnaeus’plant and animal kingdoms. How couldmushrooms be plants when they do not carryout photosynthesis? Haeckel therefore coinedthe term “protist” for a microorganism, and

TABLE 3.2

Taxa (sing. Taxon):Subdivisions used toclassify organisms.

Biotype: A populationor group of individualshaving the samegenetic constitution(genotype).

TABLE

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

Human Being Brewer’s Yeast Escherichia coli

Domain Eukarya Eukarya BacteriaKingdom Animalia FungiPhylum (Division) Chordata Ascomycota ProteobacteriaClass Mammalia Saccharomycotina Gamma-ProteobacteriaOrder Primates Saccharomycetales EnterobacterialesFamily Hominidae Saccharomycetaceae EnterobacteriaceaeGenus Homo Saccharomyces EscherichiaSpecies H. sapiens S. cerevisiae E.coli

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he placed all bacterial, protozoal, algal, andfungal species in this third kingdom.Although not everyone liked his system, itdid provoke discussions about the nature ofmicroorganisms.

During the twentieth century, advances incell biology and interest in evolutionary biol-ogy led scientists to question the two- orthree-kingdom classification schemes. In1969, Robert H. Whittaker of Cornell Univer-sity proposed a system that quickly gainedwide acceptance in the scientific community.Further expanded in succeeding years byLynn Margulis of the University of Massachu-setts, the five kingdom system of organismswas established ( ).

In the five-kingdom system, bacterialorganisms were so different from other organ-isms they must be in their own kingdom. Pro-tista were limited to the protozoa and the uni-cellular algae. Fungi included non-green,non-photosynthetic eukaryotic organisms

FIGURE 3.6

that, along with other characteristics, had cellwalls that were chemically different fromthose in bacterial, algal, and plant cells.CONCEPT AND REASONING CHECKS

3.7 Could an organism be assigned to a kingdom basedon one characteristic? Two characteristics? Howmany characteristics?

The Three-Domain System Places the Prokaryotes in Separate LineagesKEY CONCEPT

• The three domain system uses nucleotide sequenceanalysis to catalog organisms.

Often it is difficult to make sense of taxo-nomic relationships because new informationthat is more detailed keeps being discoveredabout organisms. This then motivates taxono-mists to figure out how the new informationfits into the known classification schemes—orhow the schemes need to be modified to fitthe new information. This is no clearer thanthe most recent taxonomic revolution that, asthe opening quote states, “It’s as if [he] lifted awhole submerged continent out of the ocean.”

“He” is Carl Woese, who along with hiscoworkers at the University of Illinois proposeda new classification scheme with a new mostinclusive taxon, the domain. The new schemeprimarily came from work that compared thenucleotide base sequences of the RNA in ribo-somes, those protein manufacturing machinesneeded by all cells. Woese’s results in the late1970s were especially relevant when comparingthose sequences from a group of bacterial organ-isms formerly called the archaebacteria (archae =“ancient”). Many of these bacterial forms areknown for their ability to live under extremelyharsh environments. Woese discovered that thenucleotide sequences in these archaebacteriawere different from those in other bacterialspecies and in eukaryotes. After finding otherdifferences, including cell wall composition,membrane lipids, and sensitivity to certainantibiotics, the evidence pointed to there beingthree taxonomic lines to the tree of life.

In Woese’s three domain system, onedomain includes the former archaebacteria andis called the domain Archaea ( ).The second encompasses all the remainingtrue bacteria and is called the domain Bacteria.The third domain, the Eukarya, includes the

FIGURE 3.7

InsectsVertebrates

Unicellular algae

Cyanobacteria BacteriaProkaryoticorganisms

Eukaryotic,unicellularorganisms

Eukaryotic, "multicellular"organisms

FlatwormsRoundworms

Molluscs

ANIMALIA PLANTAEFUNGI

MossesFlowering

plants

Redalgae

Mushrooms

Molds

Yeasts

Greenalgae

PROTISTA

MONERA

Protozoa

UNIVERSAL ANCESTOR

Five-Kingdom System. This system implies an evolutionary lineage,beginning with the Monera and extending to the Protista. In this scheme, certain Pro-tista were believed to be ancestors of the Plantae, Fungi, and Animalia.Q: How many of the kingdoms consist of microorganisms?

FIGURE 3.6

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four remaining kingdoms (i.e., Protista, Plan-tae, Fungi, and Animalia).

Scientists initially were reluctant to acceptthe three-domain system of classification, andmany deemed it a threat to the tenet that allliving things are either prokaryotes or eukary-otes. Then, in 1996, Craig Venter and hiscoworkers deciphered the DNA base sequenceof the archaean Methanococcus jannaschii andshowed that almost two thirds of its genes aredifferent from those of the Bacteria. They alsofound that proteins replicating the DNA andinvolved in RNA synthesis have no counter-part in the Bacteria. It appears that the three-domain system now is on firm ground.CONCEPT AND REASONING CHECKS

3.8 What is the “whole submerged continent” thatWoese lifted out of the ocean and why is the term“lifted” used?

Distinguishing between ProkaryotesKEY CONCEPT

• Taxonomy now includes many criteria to distinguishone prokaryote from another.

We shall consider the taxonomy of mostgroups of microorganisms in their respectivechapters. Prokaryotic taxonomy (referring tothe Bacteria and Archaea), however, merits

special attention because these organismsoccupy an important position in microbiologyand have had a complex system of taxonomy.David Hendricks Bergey devised one of thefirst systems of classification for the prokary-otes in 1923. His book, Bergey’s Manual ofDeterminative Bacteriology, was updated andgreatly expanded in the decades that followed.Now in its 9th edition, Bergey’s Manual isavailable for use as a reference guide to identi-fying unknown bacteria that previously havebeen isolated in pure culture.

The five-volume Bergey’s Manual of Sys-tematic Bacteriology is intended as a classifica-tion guide and the official listing for all recog-nized Bacteria and Archaea. Since the firstedition in 1984, bacterial taxonomy has gonethrough tremendous changes. For example, inVolume 1 of the second edition (2001), morethan 2,200 new species and 390 new generahave been added.

There are several traditional and moremodern criteria that microbiologists can useto identify (and classify) prokaryotes. Let’sbriefly review these methods.

Physical characteristics. These includestaining reactions to help determine the organ-ism’s shape (morphology), and the size and

Prokaryotes Eukaryotes

BACTERIA

Green nonsulfur bacteria

Gram-positive bacteria

Proteobacteria

Cyanobacteria

Flavobacteria

Thermotogales

ARCHAEA

Extremehalophiles

Methanosarcina

Methanobacterium

Methanococcus Thermococcus

Thermoproteus

Pyrodictium

UNIVERSAL ANCESTOR

MULTICELLULAR ORGANISMS

EUKARYA

Diplomonads Microsporidia

Parabasalids

Trypanosomes

Ciliates

Plants

Fungi

Animals Slime molds

Entamoeba

The Three-Domain System. Fundamental differences in genetic endowments are the basis for the three domains of all organisms onEarth. The line length between any two groups is proportional to their genetic differences.Q: What cellular characteristic was the major factor stimulating the development of the three-domain system?

FIGURE 3.7

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arrangement of cells. Other characteristics caninclude oxygen, pH, and growth temperaturerequirements. Spore-forming ability and motil-ity are additional determinants. Unfortunately,there are many prokaryotes that have the samephysical characteristics, so other distinguish-ing features are needed.

Biochemical tests. As microbiologistsbetter understood bacterial physiology, theydiscovered there were certain metabolic prop-erties that were present only in certain groups.

Today, a large number of biochemical testsexist and often a specific test can be used toeliminate certain groups of prokaryotes fromthe identification process. Among the morecommon tests are: fermentation of carbohy-drates, the utilization of a specific substrate,and the production of specific products orwaste products. But, as with the physical char-acteristics, often several biochemical tests areneeded to differentiate between species.

These identification tests are importantclinically, as they can be part of the arsenalavailable to the clinical lab that is trying toidentify a pathogen. Many of these tests can berapidly identified using modern proceduresand automated systems ( ).

Serological tests. Microorganisms are anti-genic, meaning they are capable of triggeringthe production of antibodies. Solutions of suchcollected antibodies, called antisera, are com-mercially available for many medically-impor-tant pathogens. For example, mixing a Salmo-nella antiserum with Salmonella cells will causethe cells to clump together or agglutinate. If afoodborne illness occurs, the antiserum may beuseful in identifying if Salmonella is thepathogen. More information about serologicaltesting will be presented in Chapter 21.

Nucleic acid analysis. In 1984, the edi-tors of Bergey’s Manual noted that there is no“official” classification of bacterial species andthat the closest approximation to an officialclassification is the one most widely acceptedby the community of microbiologists. The edi-tors stated that a comprehensive classificationmight one day be possible. Today, the fields ofmolecular genetics and genomics haveadvanced the analysis and sequencing ofnucleic acids. This has given rise to a new eraof molecular taxonomy.

FIGURE 3.8

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 mostinterest and the primary basis of Woese’s con-struction of the three-domain system. Many sci-entists today believe the rRNA molecule is theultimate “molecular chronometer” that allows forprecise bacterial classification in all taxonomicclasses. Other techniques, including the poly-merase chain reaction and nucleic acid hybridiza-tion, will be mentioned in later chapters.

The vast number of tests and analysesavailable for bacterial and archaeal cells canmake it difficult to know which are relevanttaxonomically for classification purposes ormedically for pathogen identification pur-poses. One widely used technique in many dis-ciplines is the dichotomous key. There are var-ious forms of dichotomous keys, but one veryuseful construction is a flow chart where aseries of positive or negative test proceduresare listed down the page. Based on thedichotomous nature of the test (always a posi-tive or negative result), the flow chart immedi-ately leads to the next test result. The result isthe identification of a specific organism. A sim-plified example is shown in MicroFocus 3.4.CONCEPT AND REASONING CHECKS

3.9 Why are so many tests often needed to identify aspecific prokaryotic species?

Substrate:The reactants on whichan enzyme acts toproduce one or moreproducts.

A Biolog MicroPlate®. New microbio-logical system that tests bacterial respiration simultane-ously with 95 different substrates and then identifiesspecies and types within species by their metabolic fin-gerprint. The 96th well is a negative control with no sub-strate.Q: Of the methods described on the this page, which is/aremost likely to be used in this more automated system?Explain.

FIGURE 3.8

Antibodies:Proteins produced bythe immune system inresponse to a specificchemical configuration(antigen).

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3.4: ToolsDichotomous Key Flow Chart

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

In this simplified scenario, an unknown bacterium has been cultured and several tests run. The testresults are shown in the box at the bottom. Using the test results and the flow chart, identify the bac-terial 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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The ability to see small objects all started withthe microscopes used by Robert Hooke andAnton van Leeuwenhoek. By now, you shouldbe aware that microorganisms usually are verysmall. Before we examine the instrumentsused to “see” these tiny creatures, we need tobe familiar with the units of measurement.

Most Microbial Agents Are In the Micrometer Size RangeKEY CONCEPT

• Metric system units are the standard for measurement.

One physical characteristic used to studymicroorganisms and viruses is their size.Because they are so small, a convenient systemof measurement is used that is the scientificstandard around the world. The measurementsystem is the metric system, where the stan-dard unit of length is the meter and is a littlelonger than a yard (see Appendix A). To meas-ure microorganisms, we need to use units thatare a fraction of a meter. In microbiology, thecommon unit for measuring length is themicrometer (μm), which is equivalent to amillionth (10–6) of a meter. To appreciate howsmall a micrometer is, consider this: Compar-ing a micrometer to an inch is like comparinga housefly to New York City’s Empire StateBuilding, 1,472 feet high.

Microbial agents range in size from the rela-tively large, almost visible protozoa (100 μm)down to the incredibly tiny viruses (0.02 μm)( ). Most bacterial and archael cellsare about 1 μm to 5 μm in length, althoughnotable exceptions have been discovered recently(MicroFocus 3.5). Because most viruses are afraction of one micrometer, their size is expressedin nanometers. A nanometer (nm) is equivalentto a billionth (10–9) of a meter; that is, 1/1,000 ofa μm. Using nanometers, the size of thepoliovirus, among the smaller viruses, measures20 nm (0.02 μm) in diameter.CONCEPT AND REASONING CHECKS

3.10 If a bacterial cell is 0.75 μm in length, what is itslength in nanometers?

FIGURE 3.9

Light Microscopy Is Used to ObserveMost MicroorganismsKEY CONCEPT

• Light microscopy uses visible light to magnify andresolve specimens.

The basic microscope system used in the micro-biology laboratory is the light microscope, inwhich visible light passes directly through thelenses and specimen ( ). Such anoptical configuration is called bright-fieldmicroscopy. Visible light is projected through acondenser lens, which focuses the light into asharp cone ( ). The light then passesthrough the opening in the stage. When hittingthe glass slide, the light is reflected or refracted asit passes through the specimen. Next, light pass-ing through the specimen enters the objectivelens to form a magnified intermediate imageinverted from that of the specimen. This interme-diate image becomes the object magnified by theocular lens (eyepiece) and seen by the observer.Because this microscope has several lenses, it alsois called a compound microscope.

A light microscope usually has at leastthree objective lenses: the low-power, high-power, and oil-immersion lenses. In general,these lenses magnify an object 10, 40, and 100 times, respectively. (Magnification is rep-resented by the multiplication sign, ×.) Theocular lens then magnifies the intermediateimage produced by the objective lens by 10×.Therefore, the total magnification achieved is100×, 400×, and 1,000×, respectively.

For an object to be seen distinctly, the lenssystem must have good resolving power; thatis, it must transmit light without variation andallow closely spaced objects to be clearly dis-tinguished. For example, a car seen in the dis-tance at night may appear to have a singleheadlight because at that distance the unaidedeye lacks resolving power. However, usingbinoculars, the two headlights can be seenclearly as the resolving power of the eyeincreases.

When switching from the low-power(10×) or high-power (40×) lens to the oil-

FIGURE 3.10B

FIGURE 3.10A

3.3 Microscopy

Total magnification:The magnification ofthe ocular multipliedby the magnification ofthe objective lensbeing used.

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3.3 Microscopy 87

immersion lens (100×), one quickly findsthat the image has become fuzzy. The objectlacks resolution, and the resolving power ofthe lens system appears to be poor. The poorresolution results from the refraction oflight.

Both low-power and high-power objec-tives are wide enough to capture sufficientlight for viewing. The oil-immersion objective,on the other hand, is so narrow that mostlight bends away and would miss the objective

lens . The index of refraction (orrefractive index) is a measure of the light-bending ability of a medium. Immersion oilhas an index of refraction of 1.5, which isalmost identical to the index of refraction ofglass. Therefore, by immersing the 100× lensin oil, the light does not bend away from thelens as it passes from the glass slide and thespecimen.

The oil thus provides a homogeneous path-way for light from the slide to the objective, and

FIGURE 3.10C

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

Electron microscope

Light microscope

Unaided eye

Molecules

VirusesUnicellular algae

FungiProtozoaBacterial and

archaeal organismsMulticellularorganisms

AtomsColonial algae

Carbonatom

Glucosemolecule

DNA double helix

Poliovirus

HIV

Rickettsia

Rod-shaped andspherical-shaped

bacteria

Protozoa

Alga

Mold

Yeast

Fluke

Red blood cells

Tapeworm

1µm

Cyanobacterium

Size Comparison among Various Atoms, Molecules, and Microorganisms (not drawn to scale). Although tapeworms and flukes usu-ally are macroscopic, the diseases these parasites cause are studied by microbiologists.Q: Which domain on average has the smallest organisms and which has the largest?

FIGURE 3.9

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the resolution of the object increases. With theoil-immersion lens, the highest resolution possi-ble with the light microscope is attained, whichis near 0.2 μm (200 nm) (MicroFocus 3.6).CONCEPT AND REASONING CHECKS

3.11 What are the two most important properties of thelight microscope?

Staining Techniques Provide ContrastKEY CONCEPT

• Specimens stained with a dye are contrasted againstthe microscope field.

Microbiologists commonly stain bacterial cellsbefore viewing them because the cytoplasm ofbacterial cells lacks color, making it hard tosee the cells on a bright background. Severalstaining techniques have been developed toprovide contrast for bright-field microscopy.

To perform the simple stain technique,bacterial cells in a droplet of water or brothare smeared on a glass slide and the slide air-dried. Next, the slide is passed briefly througha flame in a process called heat fixation,which bonds the cells to the slide, kills anyorganisms still alive, and increases stainabsorption. Now the slide is flooded with abasic (cationic) dye such as methylene blue( ). Because cationic dyes have apositive charge, the dye is attracted to thecytoplasm and cell wall, which primarily havenegative charges. By contrasting the blue cellsagainst the bright background, the stainingprocedure allows the observer to measure cellsize and determine cell shape. It also can pro-vide information about how cells are arrangedwith respect to one another (Chapter 4).

FIGURE 3.11A

3.5: Environmental MicrobiologyBiological Oxymorons

An oxymoron is a pair of words that seem to refer to opposites, such as jumbo shrimp, holy war, oldnews, and sweet sorrow. One of the characteristics we used for microorganisms is that most are invisibleto 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 bacterialcells in the gut of surgeonfish. Each cell was so large that a microscope was not needed to see it. Thespectacular giant, measuring over 0.6 mm in length (that’s 600 μm compared to 2 μm for Escherichiacoli) even dwarfs the protozoan Paramecium.

While on an expedition off the coast of Namibia (western coast of southern Africa), Heide Schultzand teammates from the Max Planck Institute for Marine Microbiology in Bremen, Germany, foundanother bacterial monster in sediment samples from the sea floor (see figure). These bacterial cells werespherical being about 0.1 to 0.3 mm in diameter—but some as large as 0.75 mm—about the diameterof the period in this sentence. Their volume is about 3 million times greater than that of E. coli. Thecells, shining white with enclosed sulfur granules, were held together in chains by a mucus sheath look-ing like a string of pearls. Thus, the bacterialspecies was named Thiomargarita namibiensis(meaning “sulfur pearl of Namibia”).

How does a prokaryotic cell survive in so large asize? The trick is to keep the cytoplasm as a thinlayer plastered against the edge of the cell so mate-rials do not need to travel (diffuse) far to get intoor out of the cell. The rest of the cell is a giant“bubble,” called a vacuole, in which nitrate and sul-fur are stored as potential energy sources. Thus, theactual cytoplasmic layer is microscopic and as closeto the surface as possible.

Yes, the vast majority of microorganisms aremicroscopic, but exceptions have been found insome exotic places.

A phase microscopy micrograph showing a chain ofThiomargarita namibiensis cells. (Bar = 250 μm.)

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3.3 Microscopy 89

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

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 inthe light microscope requires light to pass through the objective lens, forming an intermediate image. This image servesas an object for the ocular lens, which magnifies the image and forms the final image the eye perceives. (C) When usingthe oil immersion lens (100×), oil must be placed between and continuous with the slide and objective lens.Q: Why must oil be used with the 100× oil-immersion lens?

FIGURE 3.10

(A) (C)

(B)

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The negative stain technique works in theopposite manner ( ). Bacterial cellsare mixed on a slide with an acidic (anionic)dye such as nigrosin (a black stain) or Indiaink (a black drawing ink). The mixture then ispushed across the face of the slide and allowedto air-dry. Because the anionic dye carries anegative charge, it is repelled from the cell walland cytoplasm. The stain does not enter thecells and the observer sees clear or white cellson a black or gray background. Because thistechnique avoids chemical reactions and heatfixation, the cells appear less shriveled and lessdistorted than in a simple stain. They are closerto their natural condition.

FIGURE 3.11B

The Gram stain technique is an exampleof a differential staining procedure; that is, itallows the observer to differentiate (separate)bacterial cells visually into two groups basedon staining differences. The Gram stain tech-nique is named for Christian Gram, the Dan-ish physician who first perfected the tech-nique in 1884.

The first two steps of the technique arestraightforward ( ). Air-dried, heat-fixed smears are (1) stained with crystal violet,rinsed, and then (2) a special Gram’s iodinesolution is added. All bacterial cells wouldappear blue-purple if the procedure wasstopped and the sample viewed with the light

FIGURE 3.12A

3.6: ToolsCalculating Resolving Power

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

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 sizeof the cone of light that enters the objective lens after passing through the specimen. This number gen-erally is printed on the side of the objective lens (see Figure 3.10C). For a oil-immersion objective withan NA of 1.25, the resolving power may be calculated as follows:

Because the resolving power for this lens system is 220 nm, any object smaller than 220 nm could notbe seen as a clear, distinct object. An object larger than 220 nm would be resolved.

RP =×

= =550

2 01 25

550

2 5220 0 22

nmnm or m

. .. μ

RPNA

=×λ

2

Basic dye (+) Bacterial (–)cell Cell stained

(A) Simple stain technique

Dye attracted Dye repelled

Acidic dye (–) Bacterial (–)cell

(B) Negative stain technique

Cell unstained

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

FIGURE 3.11

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3.3 Microscopy 91

microscope. Next, the smear is (3) rinsed with adecolorizer, such as 95 percent alcohol or analcohol-acetone mixture. Observed at this point,certain bacterial cells may lose their color andbecome transparent. These are the gram-negative bacterial cells. Others retain the crystalviolet and represent the gram-positive bacterialcells. The last step (4) uses safranin, a redcationic dye, to counterstain the gram-negativeorganisms; that is, give them a orange-red color.So, at the technique’s conclusion, gram-positivecells are blue-purple while gram-negative cellsare orange-red ( ). Similar to thesimple staining, gram staining also allows the

FIGURE 3.12B

observer to determine size, shape, and arrange-ment of cells.

Knowing whether a bacterial cell is gram-positive or gram-negative is important for micro-biologists and clinical technicians who use theresults from the Gram stain technique to classifyit in Bergey’s Manual or aid in the identification ofan unknown bacterial species (Textbook Case 3).

Gram-positive and gram-negative bacterialcells also differ in their susceptibility to chemicalsubstances such as antibiotics (gram-positivecells are more susceptible to penicillin, gram-negative cells to tetracycline). Also, gram-negative cells have more complex cell walls, as

(A) Gram stain technique

Gram-positivebacterial cell

Gram-negativebacterial 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

Important Staining Reactions in Microbiology. The Gram stain technique is a differential staining pro-cedure. (A) All bacterial cells stain with the crystal violet and iodine, but only gram-negative cells lose the color whenalcohol 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 differentiating between gram-positive andgram-negative cells. (Bar = 10 μm.)Q: Besides identifying the Gram reaction, what other characteristics can be determined using the Gram stain procedure?

FIGURE 3.12

(B)

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described in Chapter 4, and gram-positive andgram-negative bacterial species can produce dif-ferent types of toxins.

One other differential staining procedure,the acid-fast technique, deserves mention.This technique is used to identify members ofthe genus Mycobacterium, one species of whichcauses tuberculosis. These bacterial organismsare normally difficult to stain with the Gramstain because the cells have very waxy walls.However, the cell will stain red when treatedwith carbol-fuchsin (red dye) and heat (or a

lipid solubilizer) ( ). The cellsthen retain their color when washed with adilute acid-alcohol solution. Other stainedgenera lose the red color easily during theacid-alcohol wash. The Mycobacterium species,therefore, is called acid resistant or acid fast.Because they stain red and break sharply whenthey reproduce, Mycobacterium species oftenare referred to as “red snappers.”CONCEPT AND REASONING CHECKS

3.12 What would happen if a student omitted the alcoholwash step when doing the Gram stain procedure?

FIGURE 3.13

Textbook CASE 3

Bacterial Meningitis and a Misleading Gram Stain

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

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 ofcultures prepared from the CFS indicated no bacterial growth.

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

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

Laboratory findings from the gram-stained CFS smear showed a few gram-positive, sphericalbacterial cells that often appeared in pairs. The results suggested a Streptococcus pneumoniaeinfection.

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

When transferred to a blood agar plate, growthoccurred and a prepared smear showed many gram-negative spheres (see figure). Further research indi-cated that several genera of gram-negative bacte-ria, including Acinetobacter, can appeargram-positive due to under-decolorization.

Although complicated by the under-decolorizationoutcome, the final diagnosis was bacterial meningi-tis due to Acinetobacter baumanii.

QuestionsA. From the gram-stained CSF smear, what color were the

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 acerebrospinal fluid specimen. Lab. Med. 35(8): 475–478.

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

8

7

6

5

4

3

2

1

Toxins:Chemical substancesthat are poisonous.

A gram-stained preparation from theblood agar plate.

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3.3 Microscopy 93

Light Microscopy Has Other Optical ConfigurationsKEY CONCEPT

• Different optical configurations provide detailedviews of cells.

Bright-field microscopy provides little contrast( ). However, a light microscopecan be outfitted with other optical systems toimprove contrast of prokaryotic and eukary-otic cells without staining. Three systems com-monly employed are mentioned here.

Phase-contrast microscopy uses a specialcondenser and objective lenses. This condenserlens on the light microscope splits a light beamand throws the light rays slightly out of phase.The separated beams of light then pass throughand around the specimen, and small differencesin the refractive index within the specimenshow up as different degrees of brightness andcontrast. With phase-contrast microscopy,microbiologists can see organisms alive andunstained ( ). The structure ofyeasts, molds, and protozoa is studied with thisoptical configuration.

Dark-field microscopy also uses a specialcondenser lens mounted under the stage. Thecondenser scatters the light and causes it tohit the specimen from the side. Only lightbouncing off the specimen and into the objec-tive lens makes the specimen visible, as thesurrounding area appears dark because it lacksbackground light ( ). Dark-fieldFIGURE 3.14C

FIGURE 3.14B

FIGURE 3.14A

Mycobacterium tuberculosis. The acid-fast technique is used to identify species of Mycobac-terium. The cells retain the red dye after an acid-alcoholwash. (Bar = 10 μm.)Q: Why are cells of Mycobacterium resistant to Gramstaining?

FIGURE 3.13

Variations in Light Microscopy. Thesame Paramecium specimen seen with three differentoptical configurations: (A) bright-field, (B) phase-contrast, and (C) dark-field. (Bar = 25 μm.)Q: What advantage is gained by each of the threemicroscopy techniques?

FIGURE 3.14

(A)

(B)

(C)

microscopy provides good resolution andoften illuminates parts of a specimen not seenwith bright-field optics. Dark-field microscopyalso is the preferred way to study motility oflive cells.

Dark-field microscopy helps in the diag-nosis of diseases caused by organisms near thelimit of resolution of the light microscope. Forexample, syphilis, caused by the spiral bac-terium Treponema pallidum, has a diameter of

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only about 0.15 μm. Therefore,this bacterialspecies may be observed in scrapings takenfrom a lesion of a person who has the diseaseand observed with dark-field microscopy.

Fluorescence microscopy is a major assetto clinical and research laboratories. Thetechnique has been applied to the identifica-tion of many microorganisms and is a main-stay of modern microbial ecology and espe-cially clinical microbiology. With fluorescencemicroscopy, objects emit a specific color(wavelength) of light after absorbing a shorterwavelength radiation.

Microorganisms are coated with a fluo-rescent dye, such as fluorescein, and thenilluminated with ultraviolet (UV) light. Theenergy in UV light excites electrons in fluo-rescein, and they move to higher energy lev-els. However, the electrons quickly drop backto their original energy levels and give off theexcess energy as visible light. The coatedmicroorganisms thus appear to fluoresce; inthe case of fluorescein, they glow a greenishyellow. Other dyes produce other colors( ).FIGURE 3.15

An important application of fluorescencemicroscopy is the fluorescent antibody tech-nique used to identify an unknown organism.In one variation of this procedure, fluoresceinis chemically attached to antibodies, the pro-tein molecules produced by the body’simmune system. These “tagged” antibodies aremixed with a sample of the unknown organ-ism. If the antibodies are specific for thatorganism, they will bind to it and coat thecells with the dye. When subjected to UVlight, the organisms will fluoresce. If theorganisms fail to fluoresce, the antibodieswere not specific to that organism and a differ-ent tagged antibody is tried.CONCEPT AND REASONING CHECKS

3.13 What optical systems can improve specimen con-trast over bright-field microscopy?

Electron Microscopy Provides DetailedImages of Cells, Cell Parts, and VirusesKEY CONCEPT

• Electron microscopy uses a beam of electrons tomagnify and resolve specimens.

The electron microscope grew out of an engi-neering design made in 1933 by the Germanphysicist Ernst Ruska (winner of the 1986Nobel Prize in Physics). Ruska showed thatelectrons will flow in a sealed tube if a vacuumis maintained to prevent electron scattering.Magnets, rather than glass lenses, pinpoint theflow onto an object, where the electrons areabsorbed, deflected, or transmitted dependingon the density of structures within the object( ). When projected onto a screenunderneath, the electrons form a final imagethat outlines the structures. As mentioned inChapter 1, the early days of electron microscopyproduced electron micrographs that showed bac-terial cells indeed were cellular but their struc-ture was different from eukaryotic cells. This ledto the development of the prokaryotic andeukaryotic groups of organisms.

The power of electron microscopy is theextraordinarily short wavelength of the beam ofelectrons. Measured at 0.005 nm (compared to550 nm for visible light), the short wavelengthdramatically increases the resolving power ofthe system and makes possible the visualizationof viruses and detailed cellular structures, oftencalled the ultrastructure of cells. The practicallimit of resolution of biological samples with

FIGURE 3.16

Fluorescence Microscopy. Fluorescencemicroscopy of sporulating cells of Bacillus subtilis. DNAhas been stained with a dye that fluoresces red and asporulating protein with fluorescein (green). RNA synthe-sis activity is indicated by a dye that fluoresces blue.(Bar = 15 μm.)Q: What advantage is gained by using fluorescence opticsover the other light microscope optical configurations?

FIGURE 3.15

Electron micrographs:Images recorded onelectron-sensitive film.

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3.3 Microscopy 95

the electron microscope is about 2 nm, whichis 100× better than the resolving power of thelight microscope. The drawback of the electronmicroscope is that the method needed to pre-pare a specimen kills the cells or organisms.

Two types of electron microscopes are cur-rently in use. The transmission electronmicroscope (TEM) is used to view and recorddetailed structures within cells ( ).Ultrathin sections of the prepared specimenmust be cut because the electron beam canpenetrate matter only a very short distance.After embedding the specimen in a suitableplastic mounting medium or freezing it, scien-tists cut the specimen into sections with a dia-mond knife. In this manner, a single bacterialcell can be sliced, like a loaf of bread, intohundreds of thin sections.

Several of the sections are placed on asmall grid and stained with heavy metals suchas lead and osmium to provide contrast. Themicroscopist then inserts the grid into the vac-uum chamber of the microscope and focuses a100,000-volt electron beam on one portion ofa section at a time. An image forms on thescreen below or can be recorded on film. The

FIGURE 3.17A

electron micrograph may be enlarged withenough resolution to achieve a final magnifi-cation of over 20 million ×.

The scanning electron microscope (SEM)was developed in the late 1960s to enableresearchers to see the surfaces of objects in thenatural state and without sectioning. The spec-imen is placed in the vacuum chamber andcovered with a thin coat of gold. The electronbeam then scans across the specimen andknocks loose showers of electrons that are cap-tured by a detector. An image builds line byline, as in a television receiver. Electrons thatstrike a sloping surface yield fewer electrons,thereby producing a darker contrasting spotand a sense of three dimensions. The resolvingpower of the conventional SEM is about 7 nmand magnifications with the SEM are limited toabout 50,000×. However, the instrument pro-vides vivid and undistorted views of an organ-ism’s surface details ( ).

The electron microscope has added immea-surably to our understanding of the structureand function of microorganisms by letting uspenetrate their innermost secrets. In the chap-ters ahead, we will encounter many of the

FIGURE 3.17B

Condenser lens

Specimen

Objective lens

Projector lens Intermediate image

Electron source

Binoculars

Final image on photographic plate or screen

The Electron Microscope. (A) A transmission electron microscope (TEM). (B) A schematic of a TEM. Abeam 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, orphotographic film.Q: How does the path of the image for the transmission electron microscope compare with that of the light microscope (Figure 3.10)?

FIGURE 3.16

(A) (B)

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structures displayed by electron microscopy,and we will better appreciate microbial physiol-ogy as it is defined by microbial structures.

The various types of light and electronmicroscopy are compared in .TABLE 3.3

CONCEPT AND REASONING CHECKS

3.14 What type of electron microscope would be used toexamine (a) the surface structures on a Parame-cium cell and (b) the organelles in an algal cell?

Transmission and Scanning Electron Microscopy Compared. The bacterium Pseudomonas aeruginosa asseen 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.)Q: What types of information can be gathered from each of these electron micrographs?

FIGURE 3.17

(A) (B)

TABLE

3.3 Comparison of Various Types of Microscopy

Type of Special Appearance Magnification Objects Microscopy Feature of Object Range Observed

LightBright-field Visible light Stained microorganisms 100× – 1,000× Arrangement, shape, and size

illuminates object on clear background of killed microorganisms (except viruses)

Phase-contrast Special condenser throws Unstained microor- 100× – 1,000× Internal structures of live, light rays “out of ganisms with unstained eukaryotic phase” contrasted structures microorganisms

Dark-field Special condenser Unstained microor- 100× – 1,000× Live, unstained microorganismsscatters light ganisms on dark (e.g., spirochetes)

backgroundFluorescence UV light illuminates Fluorescing microor- 100× – 1,000× Outline of microorganisms

fluorescent-coated ganisms on dark coated with fluorescent-objects background tagged antibodies

ElectronTransmission Short-wavelength Alternating light and 100× – 200,000× Ultrathin slices of

electron beam dark areas contrasting microorganisms and internal penetrates sections internal cell structures components of eukaryotic and

prokaryotic cellsScanning Short-wavelength Microbial surfaces 10× – 50,000× Surfaces and textures of

electron beam knocks microorganisms and cell loose electron showers components

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Learning Objectives 97

3.1 The Prokaryotic/Eukaryotic Paradigm• All living organisms, both prokaryotic and eukaryotic, share

the common emergent properties of life.• All organisms and cells must attempt to maintain a stable

internal state called homeostasis.• Most prokaryotic species interact through a multicellular

association involving chemical communication andcooperation between cells.

• Although prokaryotic and eukaryotic cells may differ in thestructures they contain and the pattern of organization ofthose structures, both groups carry out similar and complexcellular processes.

• Prokaryotic and eukaryotic cells share certain organizationalprocesses, including genetic organization, compartmentation,metabolic organization, and protein synthesis.

• Although prokaryotic and eukaryotic cells carry out manysimilar processes, eukaryotic cells often contain membrane-enclosed compartments (organelles) to accomplish theprocesses.

3.2 Cataloging Microorganisms• Many systems of classification have been devised to catalog

organisms. The work of Linnaeus and Haeckel represents earlyefforts based on shared characteristics.

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

• Organisms are properly classified using a standardizedhierarchical system from species (the least inclusive) todomain (the most inclusive).

• In the 1960s, Whittaker established the five kingdom system.In this classification, bacterial organisms are placed in thekingdom Monera, protozoa and algae in the kingdom Protista,and fungi in the kingdom Fungi.

• Based on several molecular and biochemical differences withinthe kingdom Monera, Woese proposed a three domain system.The Kingdom Monera is separated into two domains, theBacteria and Archaea. The remaining kingdoms (Protista, Fungi,Plantae, and Animalia) are placed in the domain Eukarya.

• Bergey’s Manual is the standard reference to identify and classifybacterial species. Criteria have included traditionalcharacteristics, but modern molecular methods have led to areconstruction of evolutionary events and organism relationships.

3.3 Microscopy• Another criterion of a microorganism is its size, a

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

• The instrument most widely used to observe microorganisms isthe light microscope. Light passes through several lenssystems that magnify and resolve the object being observed.Although magnification is important, resolution is key. Thelight microscope can magnify up to 1,000× and resolveobjects as small as 0.2 μm.

• For bacterial cells, staining generally precedes observation.The simple, negative, Gram, acid-fast, and other stainingtechniques can be used to impart contrast and determinestructural or physiological properties.

• Microscopes employing phase-contrast, dark field, orfluorescence optics have specialized uses in microbiology tocontrast cells without staining.

• To increase resolution and achieve extremely highmagnification, the electron microscope employs a beam ofelectrons to magnify and resolve specimens. To observeinternal details (ultrastructure), the transmission electronmicroscope is most often used; to see whole objects orsurfaces, the scanning electron microscope is useful.

SUMMARY OF KEY CONCEPTS

After understanding the textbook reading, you should be capable ofwriting a paragraph that includes the appropriate terms and perti-nent information to answer the objective.

1. Identify the six emerging properties common to prokaryotes andeukaryotes.

2. Assess the importance of homeostasis to cell (organismal) survival.

3. Contrast prokaryotes as unicellular and multicellular organisms.

4. Apply the concepts of cell structure, the pattern of organization,and cellular process to prokaryotes and eukaryotes.

5. Describe the four organizational patterns common to all organisms.

6. Identify the structural distinctions between prokaryotic andeukaryotic cells.

7. Explain how prokaryotic cells carry out cellular processes commonto eukaryotic cells without needing membrane-bound organelles.

8. Distinguish between the processes of invagination and endosym-biosis for the origin of eukaryotic cells.

9. Evaluate Linnaeus’ contributions to modern taxonomy.

10. Write scientific names of organisms using the binomial system.

11. Identify the taxa used to classify organisms from least to mostinclusive taxa.

12. Discuss the assigning of organisms according to the five kingdomsystem of classification.

13. Explain the assignment of organisms to the three domain system ofclassification.

14. Contrast the procedures used to identify and classify bacterial andarchaeal species.

15. Use a dichotomous key.

16. Measure sizes of microbial agents using metric system units.

17. Assess the importance of magnification and resolving power tomicroscopy.

18. Compare and contrast the procedures used to stain bacterial cells.

19. Summarize the Gram stain procedure.

20. Identify the optical configurations that provide contrast with lightmicroscopy.

21. Compare the uses of the transmission and scanning electron microscopes.

LEARNING OBJECTIVES

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98

Answer each of the following questions by selecting the one answerthat best fits the question or statement. Answers to even-numbered questions can be found in Appendix C.

1. ______ describes the ability of organisms to maintain a stableinternal state.A. MetabolismB. HomeostasisC. BiosphereD. EcotypeE. None of the above (A–D) is correct.

2. Which one of the following phrases would not apply to prokaryotes?A. Programmed cell deathB. Multicellular communitiesC. Cell-cell communicationD. Cell cooperationE. Nucleated cells

3. Proteins are made by theA. mitochondria.B. lysosomes.C. Golgi apparatus.D. ribosomes.E. cytoskeleton.

4. Cell walls are necessary forA. nutrient transport regulation.B. DNA compartmentation.C. protein transport.D. energy metabolism.E. water balance.

5. Which one of the following is not found in prokaryotic cells?A. Cell membraneB. RibosomesC. DNAD. MitochondriaE. Cytoplasm

6. The two functions of the endoplasmic reticulum areA. protein and lipid transport.B. cell respiration and photosynthesis.C. osmotic regulation and genetic control.D. cell respiration and protein synthesis.E. sorting and packaging of proteins.

7. Mitochondria differ from chloroplasts in that only mitochondriaA. carry out photosynthesis.B. are membrane-bound.C. are found in the Eukarya.D. convert chemical energy to cellular energy.E. transform sunlight into chemical energy.

8. Who is considered to be the father of modern taxonomy?A. WoeseB. WhittakerC. AristotleD. HaeckelE. Linnaeus

9. Which one of the following is the correct genus name for the bac-terial organism that causes syphilis?A. treponemaB. pallidumC. TreponemaD. pallidumE. T. pallidum

10. Several classes of organisms would be classified into oneA. family.B. genus.C. species.D. order.E. phylum (division).

11. The domain Eukarya includes all the following exceptA. fungi.B. protozoa.C. archaeal cells.D. algae.E. animals.

12. ______ was first used to catalog organisms into one of threedomains.A. PhotosynthesisB. RibosomesC. Ribosomal RNAD. Nuclear DNAE. Mitochondrial DNA

13. Resolving power is the ability of a microscope toA. estimate cell size.B. magnify an image.C. see two close objects as separate.D. keep objects in focus.E. Both B and D are correct.

14. Calculation of total magnification involves which microscope lensor lenses?A. Ocular and condenserB. Objective onlyC. Ocular onlyD. Objective and ocularE. Objective and condenser

15. Before bacterial cells are simple stained and observed with thelight microscope, they must beA. smeared on a slide.B. heat fixed.C. killed.D. air dried.E. All the above (A–D) are correct.

16. The counterstain used in the Gram stain procedure isA. iodine.B. alcohol.C. carbol-fuchsin.D. safranin.E. malachite green.

17. The practical limit of resolution with the transmission electronmicroscope is ______.A. 10 μmB. 1 μmC. 50 nmD. 10 nmE. 2 nm

18. For transmission electron microscopy, contrast is provided byA. heavy metals.B. a coat of gold.C. crystal violet.D. plastic.E. copper ions.

19. If you wanted to study the surface of a bacterial cell, you would useA. 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.E. a light microscope with bright-field optics.

20. If you wanted to study bacterial motility you would most likely useA. 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.E. a light microscope with bright-field optics.

SELF-TEST

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Answers to even-numbered questions can be found in Appendix C.

1. A local newspaper once contained an article about “the famousbacteria E. coli.” How many things can you find wrong in thisphrase? Rewrite the phrase correctly.

2. Microorganisms have been described as the most chemicallydiverse, the most adaptable, and the most ubiquitous organisms onEarth. Although your knowledge of microorganisms still may belimited at this point, try to add to this list of “mosts.”

3. Prokaryotes lack the cytoplasmic organelles commonly found in theeukaryotes. Provide a reason for this structural difference.

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

5. In 1987, in a respected science journal, an author wrote, “Linnaeusgave each life form two Latin names, the first denoting its genusand the second its species.” A few lines later, the author wrote,“Man was given his own genus and species Homo sapiens.” What isconceptually and technically wrong with both statements?

6. A student of general biology observes a microbiology student usingimmersion oil and asks why the oil is used. “To increase the magni-fication of the microscope” is the reply. Do you agree? Why?

7. Every state has an official animal, flower, or tree, but only Oregonhas a bacterial species named in its honor: Methanohalophilus ore-gonese. The specific epithet oregonese is obvious, but can you deci-pher the meaning of the genus name?

QUESTIONS FOR THOUGHT AND DISCUSSION

Applications 99

Answers to even-numbered questions can be found in Appendix C.

1. A student is performing the Gram stain technique on a mixed cul-ture of gram-positive and gram-negative bacterial cells. In reachingfor the counterstain in step 4, he inadvertently takes the methyl-ene blue bottle and proceeds with the technique. What will be thecolors of gram-positive and gram-negative bacteria at the conclu-sion of the technique?

2. Would the best resolution with a light microscope be obtainedusing red light (λ = 680 nm), green light (λ = 520 nm), or bluelight (λ = 500 nm)? Explain your answer.

3. The electron micrograph below shows several bacterial cells. Themicrograph has been magnified 12,000×. At this magnification, thecells are about 25 mm in length. Calculate the actual length of the bacterial cells in micrometers (μm)?

APPLICATIONS

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Match the statement on the left to the term on the right by placing the letter of the term in the available space. Appendix C contains the cor-rect answers to the even-numbered statements.

TermA. Archaea

B. Bergey

C. Binomial

D. Boldface

E. Chloroplast

F. Cyanobacteria

G. Dark-field

H. Eukarya

I. Family

J. Fluorescence

K. Fungi

L. Gram

M. Haeckel

N. Homeostasis

O. Italics

P. Micrometer

Q. Mitochondrion

R. Nanometer

S. Negative

T. Prokaryotes

U. Ribosomes

V. Scanning

W. Simple

X. Taxonomy

Y. Transmission

Z. Woese

Statement1. _____ System of nomenclature used for microorganisms and other

living things.

2. _____ Unit of measurement used for viruses and equal to a bil-lionth of a meter.

3. _____ Major group of organisms whose cells have no nucleus ororganelles in the cytoplasm.

4. _____ Bacterial organisms capable of photosynthesis.

5. _____ Devised the three domain system of classification in whichtwo domains contain prokaryotes.

6. _____ Type of microscope that uses a special condenser to split thelight beam.

7. _____ Type of electron microscope for which cell sectioning is notrequired.

8. _____ The ability of an organism to maintain a stable internalstate.

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

10. _____ The organelle, absent in prokaryotes, which carries out theconversion of chemical energy to cellular energy in eukaryotes.

11. _____ Domain in which many prokaryotes are classified.

12. _____ Staining technique that differentiates bacterial cells intotwo groups.

13. _____ Author of an early system of classification for bacterialorganisms.

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

15. _____ Coined the name protista for microorganisms.

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

17. _____ The convention for writing the binomial name of microor-ganisms.

18. _____ Unit of measurement for bacterial cells and equal to a mil-lionth of a meter.

19. _____ Type of microscopy using UV light to excite a dye-coatedspecimen.

20. _____ Staining technique in which the background is colored andthe cells are clear.

REVIEW

The site features learning, an on-line review area that provides quizzesand other tools to help you study for your class. You can also followuseful links for in-depth information, read more MicroFocus stories, orjust find out the latest microbiology news.

HTTP://MICROBIOLOGY.JBPUB.COM/

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Review—Cell Structure 101

Identify the cell structures indicated in drawings (A) and (B) below. Answers to the even-numbered structures can be found in Appendix C.

REVIEW—CELL STRUCTURE

2

1

3

4

5(A)

10

9

15

7

11

6

8

1213

17

14

16

(A)

(B)

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37623 CH03 069 101 - Jones & Bartlett · PDF file70 CHAPTER 3 Concepts and Tools for ... Within this structure, a cell-secreted slime forms ... multicellular structure? FIGURE 3.3