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PART 1 |Acellular and Procaryotic Microbes

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LEARNING OBJECTIVES AFTER STUDYING THIS CHAPTER, YOU SHOULD BE ABLE TO: Describe the characteristics used to classify viruses (e.g., DNA vs. RNA) List five specific properties of viruses that distinguish them from bacteria List at least three important viral diseases of humans Discuss differences between viroids and virions, and the diseases they cause List various ways in which bacteria can be classified State the three purposes of fixation Define the terms diplococci, streptococci, staphylococci, tetrad, octad, coccobacilli, diplobacilli, streptobacilli, and pleomorphism Define the terms obligate aerobe, microaerophile, facul- tative anaerobe, aerotolerant anaerobe, obligate anaer- obe, and capnophile State key differences among rickettsias, chlamydias, and mycoplasmas Identify several important bacterial diseases of humans State several ways in which archaea differ from bacteria INTRODUCTION Imagine the excitement that Anton van Leeuwenhoek experienced as he gazed through his tiny glass lenses and became the first person to see live microbes. In the years that have followed his eloquently written late 17th to early 18th century accounts of the bacteria and protozoa that he observed, tens of thousands of mi- crobes have been discovered, described, and classified. In this chapter and the next, you will be introduced to the diversity of form and function that exists in the microbial world. As you will recall, microbiology is the study of microbes, which are too small to be seen by the naked eye. Microbes can be divided into those that are truly cellular (bacteria, archaea, algae, protozoa, and fungi) and those that are acellular (viruses, viroids, and prions). The cellular microorganisms can be subdivided into those that are procaryotic (bacteria and archaea) and those that are eucaryotic (algae, protozoa, and fungi). For a variety of reasons, acellular microorganisms are not considered by most scientists to be living organisms. Thus, rather than using the term microorganisms to 40 MICROBIAL DIVERSITY PART 1 | Acellular and Procaryotic Microbes 4 CHAPTER OUTLINE INTRODUCTION ACELLULAR MICROBES Viruses Origin of Viruses Bacteriophages Animal Viruses Latent Virus Infections Antiviral Agents Oncogenic Viruses Human Immunodeficiency Virus Mimivirus Plant Viruses Viroids and Prions THE DOMAIN BACTERIA Characteristics Cell Morphology Staining Procedures Motility Colony Morphology Atmospheric Requirements Nutritional Requirements Biochemical and Metabolic Activities Pathogenicity Genetic Composition Unique Bacteria Rickettsias, Chlamydias, and Closely Related Bacteria Mycoplasmas Especially Large and Especially Small Bacteria Photosynthetic Bacteria THE DOMAIN ARCHAEA
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• Describe the characteristics used to classify viruses (e.g.,DNA vs. RNA)

• List five specific properties of viruses that distinguishthem from bacteria

• List at least three important viral diseases of humans• Discuss differences between viroids and virions, and the

diseases they cause• List various ways in which bacteria can be classified• State the three purposes of fixation• Define the terms diplococci, streptococci, staphylococci,

tetrad, octad, coccobacilli, diplobacilli, streptobacilli,and pleomorphism

• Define the terms obligate aerobe, microaerophile, facul-tative anaerobe, aerotolerant anaerobe, obligate anaer-obe, and capnophile

• State key differences among rickettsias, chlamydias, andmycoplasmas

• Identify several important bacterial diseases of humans• State several ways in which archaea differ from bacteria


Imagine the excitement that Anton van Leeuwenhoekexperienced as he gazed through his tiny glass lensesand became the first person to see live microbes. In theyears that have followed his eloquently written late17th to early 18th century accounts of the bacteria andprotozoa that he observed, tens of thousands of mi-crobes have been discovered, described, and classified.In this chapter and the next, you will be introduced tothe diversity of form and function that exists in themicrobial world.

As you will recall, microbiology is the study ofmicrobes, which are too small to be seen by the nakedeye. Microbes can be divided into those that are trulycellular (bacteria, archaea, algae, protozoa, and fungi)and those that are acellular (viruses, viroids, and prions).The cellular microorganisms can be subdivided intothose that are procaryotic (bacteria and archaea) andthose that are eucaryotic (algae, protozoa, and fungi).For a variety of reasons, acellular microorganisms arenot considered by most scientists to be living organisms.Thus, rather than using the term microorganisms to


MICROBIAL DIVERSITYPART 1 | Acellular and Procaryotic Microbes4



Origin of VirusesBacteriophagesAnimal VirusesLatent Virus InfectionsAntiviral AgentsOncogenic VirusesHuman Immunodeficiency Virus

MimivirusPlant Viruses

Viroids and PrionsTHE DOMAIN BACTERIACharacteristics

Cell MorphologyStaining ProceduresMotilityColony MorphologyAtmospheric RequirementsNutritional RequirementsBiochemical and Metabolic Activities

PathogenicityGenetic Composition

Unique BacteriaRickettsias, Chlamydias, and Closely

Related BacteriaMycoplasmasEspecially Large and Especially

Small BacteriaPhotosynthetic BacteriaTHE DOMAIN ARCHAEA

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describe them, viruses, viroids, and prions are morecorrectly referred to as acellular microbes or infectiousparticles.


VirusesComplete virus particles, calledvirions, are very small and sim-ple in structure. Most virusesrange in size from 10 to 300 nmin diameter, although some—like Ebola virus—can be up to 1 �m in length. The small-est virus is about the size of the large hemoglobin mole-cule of a red blood cell. Scientists were unable to seeviruses until electron microscopes were invented in the1930s. The first photographs of viruses were obtained in1940. A negative staining procedure, developed in 1959,revolutionized the study of viruses, making it possible toobserve unstained viruses against an electron-dense, darkbackground.

No type of organism is safefrom viral infections; virusesinfect humans, animals, plants,fungi, protozoa, algae, and bac-terial cells (Table 4-1). Manyhuman diseases are caused by viruses (refer back to Table1-1). Many of the viruses that infect humans are shown inFigure 4-1. Some viruses—called oncogenic viruses or

oncoviruses—cause specific types of cancer, includinghuman cancers such as lymphomas, carcinomas, andsome types of leukemia.

Viruses are said to have five specific properties thatdistinguish them from living cells:

• The vast majority of viruses possess either DNA orRNA, unlike living cells, which possess both.

• They are unable to replicate (multiply) on their own;their replication is directed by the viral nucleic acidonce it has been introduced into a host cell.

• Unlike cells, they do not divide by binary fission, mito-sis, or meiosis.

• They lack the genes and enzymes necessary for energyproduction.

• They depend on the ribo-somes, enzymes, and meta-bolites (“building blocks”) ofthe host cell for protein andnucleic acid production.

A typical virion consists ofa genome of either DNA orRNA, surrounded by a capsid(protein coat), which is com-posed of many small proteinunits called capsomeres.Together, the nucleic acid andthe capsid are referred to asthe nucleocapsid (Fig. 4-2).Some viruses (called en-veloped viruses) have an outer envelope composed oflipids and polysaccharides (Fig. 4-3). Bacterial virusesmay also have a tail, sheath, and tail fibers. There are noribosomes for protein synthesis or sites of energy pro-duction; hence, the virus must invade and take over afunctioning cell to produce new virions.

Viruses are classified by the following characteris-tics: (a) type of genetic material (either DNA or RNA),(b) shape of the capsid, (c) number of capsomeres,(d) size of the capsid, (e) presence or absence of an en-velope, (f) type of host that it infects, (g) type of diseaseit produces, (h) target cell, and (i) immunologic or anti-genic properties.

There are four categories of viruses, based on thetype of genome they possess. The genome of mostviruses is either double-stranded DNA or single-stranded RNA, but a few viruses possess single-strandedDNA or double-stranded RNA. Viral genomes are usu-ally circular molecules, but some are linear (having twoends). Capsids of viruses have various shapes and sym-metry. They may be polyhedral (many sided), helical(coiled tubes), bullet shaped, spherical, or a complexcombination of these shapes. Polyhedral capsids have20 sides or facets; geometrically, they are referred to asicosahedrons. Each facet consists of several capsomeres;thus, the size of the virus is determined by the size ofeach facet and the number of capsomeres in each.

Viruses are extremelysmall. They areobserved usingelectron microscopes.

Viruses are not alive. To replicate, virusesmust invade live hostcells.

Except in very rarecases, a particular viruscontains either DNA orRNA—not both.

The simplest of humanviruses consists ofnothing more thannucleic acid surroundedby a protein coat (thecapsid). The capsid plusthe enclosed nucleicacid are referred to asthe nucleocapsid.


Animal VirusesVaccinia DNA Complex 200 � 300Mumps RNA Helical 150–250Herpes simplex DNA Polyhedral 100–150Influenza RNA Helical 80–120Retroviruses RNA Helical 100–120Adenoviruses DNA Polyhedral 60–90Retroviruses RNA Polyhedral 60–80Papovaviruses DNA Polyhedral 40–60Polioviruses RNA Polyhedral 28

Plant VirusesTurnip yellow RNA Polyhedral 28

mosaicWound tumor RNA Polyhedral 55–60Alfalfa mosaic RNA Polyhedral 18 � 36–40Tobacco mosaic RNA Helical 18 � 300

BacteriophagesT2 DNA Complex 65 � 210L DNA Complex 54 � 194Fx-174 DNA Complex 25

Relative Sizes and Shapes of Some Viruses


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Frequently, the envelope around the capsid makes thevirus appear spherical or irregular in shape in electronmicrographs. The envelope is acquired by certain ani-mal viruses as they escape from the nucleus or cyto-plasm of the host cell by budding (Figs. 4-4 and 4-5). Inother words, the envelope is derived from either thehost cell’s nuclear membrane or cell membrane.Apparently, viruses are then able to alter these mem-branes by adding protein fibers, spikes, and knobs thatenable the virus to recognize the next host cell to be in-

vaded. A list of some viruses, their characteristics, anddiseases they cause is presented in Table 4-2. Sizes ofsome viruses are depicted in Figure 4-6.

Origin of VirusesWhere did viruses come from? Two main theories havebeen proposed to explain the origin of viruses. One theorystates that viruses existed before cells, but this seems un-likely in view of the fact that viruses require cells for theirreplication. The other theory states that cells came first and

42 SECTION II ■ Introduction to Microbes and Cellular Biology

FIGURE 4-1. Some of the viruses that infect humans. Note that some viruses contain RNA, whereasothers contain DNA, and that the nucleic acid that they possess may either be single or double stranded.Within the host cell, single-stranded positive sense RNA functions as messenger RNA (mRNA), whereassingle-stranded negative sense RNA serves as a template for the production of mRNA. Some of the virusespossess an envelope, whereas others do not. (From Engleberg NC et al. Schaechter’s Mechanisms ofMicrobial Diseases, 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2007.)


Enveloped Nonenveloped

Single stranded Positive sense

Single stranded Positive sense

Single stranded Negative sense











Oncoviruses Rhabdoviruses







Double stranded


Nonenveloped Enveloped

Double stranded Linear

Single stranded Linear

Double stranded Linear

Double stranded Circular

Double stranded Circular



Parvoviruses Adenoviruses Papillomaviruses



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that viruses represent ancientderivatives of degenerate cells orcell fragments. The question ofwhether viruses are alive de-pends on one’s definition of lifeand, thus, is not an easy questionto answer. However, most sci-entists agree that viruses lackmost of the basic featuresof cells; thus, they consider viruses to be nonliving entities.

BacteriophagesThe viruses that infect bacteria are known as bacterio-phages (or simply, phages). Like all viruses, they are obli-gate intracellular pathogens, in that they must enter abacterial cell to replicate. There are three categories ofbacteriophages, based on their shape:

• Icosahedron bacteriophages: an almost spherical shape,with 20 triangular facets; the smallest icosahedronphages are about 25 nm in diameter.

• Filamentous bacteriophages: long tubes formed bycapsid proteins assembled into a helical structure; theycan be up to about 900 nm long.

• Complex bacteriophages: icosahedral heads attached tohelical tails; may also possess base plates and tail fibers.

In addition to shape, bacteriophages can be catego-rized by the type of nucleic acid that they possess; thereare single-stranded DNA phages, double-stranded DNAphages, single-stranded RNA phages, and double-stranded RNA phages. From this point, only DNAphages will be discussed.

Bacteriophages can be categorized by the events thatoccur after invasion of the bacterial cell: some are viru-lent phages, whereas others are temperate phages.Phages in either category do not actually enter the bacte-rial cell—rather, they inject their nucleic acid into thecell. It is what happens next that distinguishes virulentphages from temperate phages.

Virulent bacteriophages al-ways cause what is known as thelytic cycle, which ends with thedestruction (lysis) of the bacte-rial cell. For most phages, thewhole process (from attachmentto lysis) takes less than 1 hour.The steps in the lytic cycle are shown in Table 4-3.

FIGURE 4-2. Viral nucleocap-sids. (A) Nucleocapsid of a heli-cal virus. (B) Nucleocapsid of anicosahedral virus. (From HarveyRA et al. Lippincott’s IllustratedReviews: Microbiology, 2nd ed.Philadelphia: Lippincott Williams& Wilkins, 2007.)

FIGURE 4-3. Enveloped viruses.(A) Enveloped helical virus.(B) Enveloped icosahedral virus.(From Harvey RA et al. Lippincott’sIllustrated Reviews: Microbiology,2nd ed. Philadelphia: LippincottWilliams & Wilkins, 2007.)

Because they are notcomposed of cells,viruses are notconsidered to be livingorganisms. They arereferred to as acellularmicrobes or infectiousparticles.

Once it enters a hostcell, a virulentbacteriophage alwaysinitiates the lytic cycle,which results in thedestruction of the cell.

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The first step in the lyticcycle is attachment (adsorp-tion) of the phage to the sur-face of the bacterial cell. Thephage can only attach to bac-terial cells that possess the ap-propriate receptor—a proteinor polysaccharide molecule onthe surface of the cell that is recognized by a moleculeon the surface of the phage. Most bacteriophages arespecies- and strain-specific, meaning that they only in-fect a particular species or strain of bacteria. Those thatinfect Escherichia coli are called coliphages. Some bacte-riophages can attach to more than one species ofbacterium. Figure 4-7 shows numerous bacteriophagesattached to the surface of a Vibrio cholerae cell.

The second step in the lytic cycle is called penetration.In this step, the phage injects its DNA into the bacterialcell, acting much like a hypodermic needle (Fig. 4-8).From this point on, the phage DNA “dictates” what oc-curs within the bacterial cell. This is sometimes de-scribed as the phage DNA taking over the host cell’s“machinery.”

The third step in the lytic cycle is called biosynthesis. Itis during this step that the phage genes are expressed, re-sulting in the production (biosynthesis) of viral pieces. Itis also during this step that the host cell’s enzymes (e.g.,DNA polymerase and RNA polymerase), nucleotides,amino acids, and ribosomes are used to make viral DNAand viral proteins. In the fourth step of the lytic cycle,called assembly, the viral pieces are assembled to producecomplete viral particles (virions). It is during this stepthat viral DNA is packaged up into capsids.

The final step in the lytic cycle, called release, iswhen the host cell bursts open and all of the new virions

44 SECTION II ■ Introduction to Microbes and Cellular Biology

FIGURE 4-4. Virus particle becoming envelopedin the process of budding from a host cell.(From Harvey RA et al. Lippincott’s Illustrated Reviews:Microbiology, 2nd ed. Philadelphia: Lippincott Williams& Wilkins, 2007.)












FIGURE 4-5. Herpesviruses acquiring their en-velopes as they leave a host cell’s nucleus by bud-ding. (1–3) Viruses within the nucleus. (4) Virus in theprocess of leaving the nucleus by budding. (5, 6) Virusesthat have already acquired their envelopes. (From VolkWA, et al. Essentials of Medical Microbiology, 5th ed.Philadelphia: Lippincott-Raven, 1996.)

Bacteriophages canonly attach to bacteriathat possess surfacemolecules (receptors)that can be recognizedby molecules on thephage surface.

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CHAPTER 4 ■ Microbial Diversity 45

(about 50–1,000) escape from the cell. Thus, the lyticcycle ends with lysis of the host cell. Lysis is caused byan enzyme that is coded for by a phage gene. At the ap-propriate time—after assembly—the appropriate viralgene is expressed, the enzyme is produced, and thebacterial cell wall is destroyed. With certain bacterio-phages, a phage gene codes for an enzyme that inter-feres with cell wall synthesis, leading to weakness and,finally, collapse of the cell wall. The lytic cycle is sum-marized in Figure 4-9.

The other category of bacteriophages—temperatephages (also known as lysogenic phages)—do not immedi-ately initiate the lytic cycle, but rather, their DNAremains integrated into the bacterial cell chromosome,generation after generation. Temperate bacteriophagesare discussed further in Chapter 7.

Bacteriophages are in-volved in two of the fourmajor ways in which bacteriaacquire new genetic informa-tion. These processes—calledlysogenic conversion andtransduction—are discussed inChapter 7.

Because bacteriophages de-stroy bacteria, there has beenmuch speculation and experi-mentation through the yearsregarding their use to destroy bacterial pathogens andtreat bacterial infections. The earliest research of thisnature was conducted in the 1930s, but ended when an-tibiotics were discovered in the 1940s. However, since


Poxviruses Large, brick shape with Variola Smallpoxenvelope, dsDNA Vaccinia Cowpox

Polyoma-papilloma dsDNA, polyhedral Papillomavirus WartsPolyomavirus Some tumors, some cancer

Herpesvirus Polyhedral with envelope, Herpes simplex I Cold sores or fever blistersdsDNA Herpes simplex II Genital herpes

Herpes zoster ShinglesVaricella Chickenpox

Adenovirus dsDNA, icosahedral, with Respiratory infections, envelope pneumonia, conjunctivitis,

some tumors

Picornaviruses ssRNA, tiny icosahedral, Rhinovirus Colds(the name means with envelope Poliovirus Poliomyelitissmall RNA viruses) Hepatitis types A and B Hepatitis

Coxsackievirus Respiratory infections,meningitis

Reoviruses dsRNA, icosahedral Enterovirus Intestinal infectionswith envelope

Myxoviruses RNA, helical with envelope Orthomyxoviruses Influenzatypes A and B

Myxovirus parotidis MumpsParamyxovirus Measles (rubeola)Rhabdovirus Rabies

Arbovirus Arthropodborne RNA, cubic Mosquitoborne type B Yellow feverMosquitoborne types A and B Encephalitis (many types)Tickborne, coronavirus Colorado tick fever

Retrovirus dsRNA, helical with envelope RNA tumor virus TumorsHTLV virus LeukemiaHIV AIDS

ds, double-stranded; ss, single-stranded.

Selected Important Groups of Viruses and Viral DiseasesTABLE 4-2

Unlike virulentbacteriophages,temperatebacteriophages do notimmediately initiatethe lytic cycle. TheirDNA can remainintegrated into thehost cell’s chromosomefor generation aftergeneration.

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46 SECTION II ■ Introduction to Microbes and Cellular Biology

Escherichia coli (one-half)

FIGURE 4-6. Comparative sizes ofvirions, their nucleic acids, andbacteria. (From Davis BD, et al.Microbiology, 4th ed. Philadelphia:JB Lippincott, 1990.)


1 Attachment (adsorption) The phage attaches to a protein or polysaccharide molecule (receptor) on the surface of the bacterial cell

2 Penetration The phage injects its DNA into the bacterial cell; the capsid remains on theouter surface of the cell

3 Biosynthesis Phage genes are expressed, resulting in the production of phage pieces or parts (i.e., phage DNA and phage proteins)

4 Assembly The phage pieces or parts are assembled to create complete phages

5 Release The complete phages escape from the bacterial cell by lysis of the cell

Steps in the Multiplication of Bacteriophages (Lytic Cycle)TABLE 4-3

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FIGURE 4-7. A partially lysed cell of a Vibriocholerae bacterium, with many attached virions ofphage CP-T1. (Courtesy of R.W. Taylor and J.E. Ogg,Colorado State University, Fort Collins, CO.)




Protein coat

Cell wall



End plate

Tail fiber



FIGURE 4-8. Bacteriophages.(A) The bacteriophage T4 is anassembly of protein components.The head is a protein membranewith 20 facets, filled with DNA. Itis attached to a tail consisting ofa hollow core surrounded by asheath and based on a spiked endplate to which six fibers are at-tached. (B) The sheath contracts,driving the core through the cellwall, and viral DNA enters the cell.

the emergence of multidrug-resistant bacteria (“super-bugs”), research into the use of bacteriophages to treatbacterial diseases has been renewed. Additionally, bacte-riophage enzymes that destroy cell walls or prevent theirsynthesis are currently being studied for use as therapeu-tic agents. It is possible that, in the future, certain bacte-rial diseases will be treated using orally administered orinjected pathogen-specific bacteriophages or bacterio-phage enzymes.

Animal VirusesViruses that infect humans and animals are collectively re-ferred to as animal viruses. Some animal viruses are DNAviruses; others are RNA viruses. Animal viruses may consistsolely of nucleic acid surrounded by a protein coat (capsid),or they may be more complex. For example, they may beenveloped or they may contain enzymes that play a role inviral multiplication within host cells. The steps in the mul-tiplication of animal viruses are shown in Table 4-4.

The first step in the multi-plication of animal viruses isattachment (or adsorption) ofthe virus to the cell. Like bac-teriophages, animal virusescan only attach to cells bearing

the appropriate protein or polysaccharide receptors ontheir surface. Did you ever wonder why certain virusescause infections in dogs, but not in humans, or viceversa? Did you ever wonder why certain viruses causerespiratory infections, whereas others cause gastroin-testinal infections? It all boils down to receptors.Viruses can only attach to and invade cells that bear areceptor that they can recognize.

The second step in the multiplication of animalviruses is penetration, but, unlike bacteriophages, the en-tire virion usually enters the host cell, sometimes becausethe cell phagocytizes the virus (Figs. 4-10, 4-11, and 4-12). This necessitates a third step that was not requiredfor bacteriophages—uncoating—whereby the viral nu-cleic acid escapes from the capsid.

As with bacteriophages, from this point on, the viralnucleic acid “dictates” what occurs within the host cell.The fourth step is biosynthesis, whereby many viral pieces(viral nucleic acid and viral proteins) are produced. Thisstep can be quite complicated, depending on what type ofvirus infected the cell (i.e., whether it was a single-stranded DNA virus, a double-stranded DNA virus, asingle-stranded RNA virus, or a double-stranded RNAvirus). Some animal viruses do not initiate biosynthesisright away, but rather, remain latent within the host cellfor variable periods. Latent viral infections are discussedin more detail in a subsequent section.

The fifth step—assembly—involves fitting the virus piecestogether to produce completevirions. After the virus parti-cles are assembled, they mustescape from the cell—a sixthstep called release. How theyescape from the cell dependson the type of virus that it is. Some animal viruses escapeby destroying the host cell, leading to cell destructionand some of the symptoms associated with infection withthat particular virus. Other viruses escape the cell by aprocess known as budding. Viruses that escape from thehost cell cytoplasm by budding become surrounded with

Like bacteriophages,animal viruses can onlyattach to and invadecells bearingappropriate surfacereceptors.

Animal viruses escapefrom their host cellseither by lysis of thecell or budding. Virusesthat escape by buddingbecome envelopedviruses.

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diagnostic tool to identify certain viral diseases.Inclusion bodies may be found in the cytoplasm (cyto-plasmic inclusion bodies) or within the nucleus(intranuclear inclusion bodies), depending on the par-ticular disease. In rabies, the cytoplasmic inclusion bod-ies in nerve cells are called Negri bodies. The inclusionbodies of AIDS and the Guarnieri bodies of smallpoxare also cytoplasmic. Herpes and poliomyelitis virusescause intranuclear inclusion bodies. In each case, inclu-sion bodies may represent aggregates or collections ofviruses. Some important human viral diseases includeAIDS, chickenpox, cold sores, the common cold, Ebolavirus infections, genital herpes infections, Germanmeasles, Hantavirus pulmonary syndrome, infectiousmononucleosis, influenza, measles, mumps, po-liomyelitis, rabies, severe acute respiratory syndrome(SARS), and viral encephalitis. In addition, all humanwarts are caused by viruses.

Latent Virus InfectionsHerpes virus infections, such as cold sores (fever blis-ters), are good examples of latent virus infections.Although the infected person is always harboring thevirus in nerve cells, the cold sores come and go. A fever,stress, or excessive sunlight can trigger the viral genesto take over the cells and produce more viruses; in theprocess, cells are destroyed and a cold sore develops.Latent viral infections are usually limited by the defensesystems of the human body—phagocytes and antiviralproteins called interferons that are produced by virus-infected cells (discussed in Chapter 15). Shingles, apainful nerve disease that is also caused by a her-pesvirus, is another example of a latent viral infection.After a chickenpox infection, the virus can remain latentin the human body for many years. Then, when thebody’s immune defenses become weakened by old ageor disease, the latent chickenpox virus resurfaces tocause shingles.

Antiviral AgentsAntibiotics function by inhibit-ing certain metabolic activitieswithin cellular pathogens, andviruses are not cells. However,for certain patients with coldsand influenza, antibiotics maybe prescribed in an attempt toprevent secondary bacterial in-fections that might followthe virus infection. In recentyears, a relatively small numberof chemicals—called antiviral agents—have been devel-oped to interfere with virus-specific enzymes and virusproduction by either disrupting critical phases in viral cy-cles or inhibiting the synthesis of viral DNA, RNA, orproteins. Antiviral agents are discussed further inChapter 9.

48 SECTION II ■ Introduction to Microbes and Cellular Biology

1 Attachment of phage to cell surface receptor





Cell wall of bacterium

2 Injection of phageDNA into cell

3 Synthesis of newphage DNA and phage proteins

4 Assembly of newphage particles

5 Lysis of cell and release ofprogeny phage






FIGURE 4-9. Summary of the lytic process.(From Harvey RA et al. Lippincott’s Illustrated Reviews:Microbiology, 2nd ed. Philadelphia: Lippincott Williams& Wilkins, 2007.)

It is very important forhealthcare professionalsto understand thatantibiotics are noteffective against viralinfections.

pieces of the cell membrane, thus becoming envelopedviruses. If it is an enveloped virus, you know that it es-caped from its host cell by budding.

Remnants or collections of viruses, called inclusionbodies, are often seen in infected cells and are used as a

Drugs used to treatviral infections arecalled antiviral agents.

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FIGURE 4-10. Penetration of a host cell by anonenveloped virus via endocytosis. (From Harvey RAet al. Lippincott’s Illustrated Reviews: Microbiology, 2nded. Philadelphia: Lippincott Williams & Wilkins, 2007.)

FIGURE 4-11. Penetration of a host cell by an en-veloped virus. (From Harvey RA et al. Lippincott’sIllustrated Reviews: Microbiology, 2nd ed. Philadelphia:Lippincott Williams & Wilkins, 2007.)

Oncogenic VirusesViruses that cause cancer arecalled oncogenic viruses or on-coviruses. The first evidence thatviruses cause cancers camefrom experiments with chick-ens. Subsequently, viruses were shown to be the cause ofvarious types of cancers in rodents, frogs, and cats.

Although the causes of many (perhaps most) types ofhuman cancers remain unknown, it is known that somehuman cancers are caused by viruses. Epstein-Barr virus(a type of herpesvirus) causes infectious mononucleosis(not a type of cancer), but also causes three types ofhuman cancers: nasopharyngeal carcinoma, Burkitt lym-phoma, and B-cell lymphoma. Kaposi sarcoma, a type ofcancer common in AIDS patients, is caused by human

Viruses that causecancer are known asoncogenic viruses oroncoviruses.


1 Attachment (adsorption) The virus attaches to a protein or polysaccharide molecule (receptor) on the surface of a host cell

2 Penetration The entire virus enters the host cell, in some cases because it was phagocytizedby the cell

3 Uncoating The viral nucleic acid escapes from the capsid

4 Biosynthesis Viral genes are expressed, resulting in the production of pieces or parts of viruses (i.e., viral DNA and viral proteins)

5 Assembly The viral pieces or parts are assembled to create complete virions

6 Release The complete virions escape from the host cell by lysis or budding

Steps in the Multiplication of Animal VirusesTABLE 4-4

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50 SECTION II ■ Introduction to Microbes and Cellular Biology

herpesvirus 8. Associations between hepatitis B and Cviruses and hepatocellular (liver) carcinoma have been es-tablished. Human papillomaviruses (HPV; wart viruses)can cause different types of cancer, including cancers ofthe cervix and other parts of the genital tract. A retrovirusthat is closely related to human immunodeficiency virus(HIV; the cause of acquired immunodeficiency syndrome[AIDS]), called human T-lymphotrophic virus type 1(HTLV-1), causes a rare type of adult T-cell leukemia.All of the mentioned oncogenic viruses, except HTLV-1,are DNA viruses. HTLV-1 is an RNA virus.

Human Immunodeficiency VirusHuman immunodeficiency vi-rus, the cause of AIDS, is an en-veloped, single-stranded RNAvirusa (Fig. 4-13). It is a mem-ber of a genus of viruses calledlentiviruses, in a family ofviruses called Retroviridae (retroviruses). HIV is able toattach to and invade cells bearing receptors that the virusrecognizes. The most important of these receptors is des-ignated CD4, and cells possessing that receptor are calledCD4� cells. The most important of the CD4� cells is thehelper T cell (discussed in Chapter 16); HIV infectionsdestroy these important cells of the immune system.Macrophages also possess CD4 receptors and can, thus,

FIGURE 4-12. Infection of host cells by Herpes simplex virus. Adsorption (A),penetration (B–D), and uncoating and digestion of the capsid (E–G) of herpes sim-plex on HeLa cells, as deduced from electron micrographs of infected cell sections.Penetration involves local digestion of the viral and cellular membranes (B, C), result-ing in fusion of the two membranes and release of the nucleocapsid into the cytoplas-mic matrix (D). The naked nucleocapsid is intact in E, is partially digested in F, andhas disappeared in G, leaving a core containing DNA and protein. (From Morgan C, etal. J Virol 1968;2:507.)

a The HIV virion contains two single-stranded RNA molecules.








FIGURE 4-13. Human immunodeficiency virus(HIV). HIV is an enveloped virus, containing two identi-cal single-stranded RNA molecules. Each of its 72 surfaceknobs contains a glycoprotein (designated gp120) capableof binding to a CD4 receptor on the surface of certainhost cells (e.g., T-helper cells). The “stalk” that supportsthe knob is a transmembrane glycoprotein (designatedgp41), which may also play a role in attachment to hostcells. Reverse transcriptase is an RNA-dependent DNApolymerase. (From Porth CM. Pathophysiology: Conceptsof Altered Health States, 5th ed. Philadelphia: LippincottWilliams & Wilkins, 1998.)

AIDS is caused by asingle-stranded RNAvirus known as humanimmunodeficiency virus(HIV).

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be invaded by HIV. In addition, HIV is able to invadecertain cells that do not possess CD4 receptors, but dopossess other receptors that HIV is able to recognize.

MimivirusAn extremely large double-stranded DNA virus, calledMimivirus, has been recovered from amebas. The viruswas given the name Mimivirus because it “mimics” bac-teria. It is so large that it can be observed using a standardcompound light microscope. The Mimivirus particle hasa 7 nm thick capsid with a diameter of 750 nm. An arrayof 80- to 125-nm long closely packed fibers project out-ward from the capsid surface (Fig. 4-14). Within thecapsid, its DNA is surrounded by two 4-nm thick lipidmembranes. Its genome is at least 10 times larger thanthat of the large viruses in the smallpox family and largerthan the genome of some of the smallest bacteria. Someof its genes code for functions which were previouslythought to be the exclusive province of cellular organ-isms, such as the translation of proteins and DNA repair

enzymes. Mimivirus contains several genes for sugar,lipid, and amino acid metabolism. And, unlike mostDNA viruses, Mimivirus contains some RNA molecules.A limited number of reports suggest that Mimivirus maybe the cause of some cases of human pneumonia.

Plant VirusesMore than 1,000 different viruses cause plant diseases,including diseases of citrus trees, cocoa trees, rice, barley,tobacco, turnips, cauliflower, potatoes, tomatoes, andmany other fruits, vegetables, trees, and grains. Thesediseases result in huge economic losses, estimated to bein excess of $70 billion per year worldwide. Plant virusesare usually transmitted via insects (e.g., aphids, leaf hop-pers, whiteflies); mites; nematodes (round worms); in-fected seeds, cuttings, and tubers; and contaminated tools(e.g., hoes, clippers, and saws).

Viroids and PrionsAlthough viruses are extremelysmall nonliving infectiousagents, viroids and prions areeven smaller and less complexinfectious agents. Viroids con-sist of short, naked fragments of single-stranded RNA(about 300–400 nucleotides in length) that can interferewith the metabolism of plant cells and stunt the growthof plants, sometimes killing the plants in the process.They are transmitted between plants in the same man-ner as viruses. Plant diseases thought or known to becaused by viroids include potato spindle tuber (produc-ing small, cracked, spindle-shaped potatoes), citrus exo-cortis (stunting of citrus trees), and diseases of chrysan-themums, coconut palms, and tomatoes. Thus far, noanimal diseases have been discovered that are caused byviroids.

Prions (pronounced “pree-ons”) are small infectious pro-teins that apparently cause fatalneurological diseases in animals,such as scrapie (pronounced“scrape-ee”) in sheep and goats;bovine spongiform encephalopathy (BSE; “mad cow dis-ease”; see “Insight: Microbes in the News: ‘Mad CowDisease’” on the CD-ROM ); and kuru, Creutzfeldt-Jakob(C-J) disease, Gerstmann-Sträussler-Scheinker (GSS) dis-ease, and fatal familial insomnia in humans. Similar dis-eases in mink, mule deer, Western white-tailed deer, elk,





Inner Membranes


FIGURE 4-14. Mimivirus structure. The Mimivirusvirion consists of a double-stranded DNA core, surroundedby two lipid membranes and a protein capsid. Numerousfibrils extend outward from the capsid surface. (Courtesyof Xanthine at http://en.wikipedia.org.)

Prions are infectiousprotein molecules thatcause a variety ofanimal and humandiseases.

Viroids are infectiousRNA molecules thatcause a variety of plantdiseases.

STUDY AID Beware of Similar Sounding TermsA virion is a complete viral particle (i.e., one that hasall its parts, including nucleic acid and a capsid). Aviroid is an infectious RNA molecule.

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and cats may also be caused by prions. The name “scrapie”comes from the observation that infected animals scrapethemselves against fence posts and other objects in an ef-fort to relieve the intense pruritus (itching) associated withthe disease. The disease in deer and elk is called “chronicwasting disease,” in reference to the irreversible weightloss that the animals experience.

Kuru is a disease that was once common among na-tives in Papua, New Guinea, where women and childrenate human brains as part of a traditional burial custom(ritualistic cannibalism). If the brain of the deceased per-son contained prions, then persons who ate that brain de-veloped kuru. Kuru, C-J disease, and GSS disease involveloss of coordination and dementia. Dementia, a generalmental deterioration, is characterized by disorientationand impaired memory, judgment, and intellect. In fatalfamilial insomnia, insomnia and dementia follow diffi-culty sleeping. All these diseases are fatal spongiform en-cephalopathies, in which the brain becomes riddled withholes (spongelike).

Scientists have been investigating the link between“mad cow disease” and a form of C-J disease (calledvariant CJD or vCJD) in humans. As of December2008, 207 cases of vCJD had been diagnosed world-wide, including 164 in the United Kingdom; these casesprobably resulted from eating prion-infected beef. Thecattle may have acquired the disease through ingestionof cattle feed that contained ground-up parts of prion-infected sheep.

The 1997 Nobel Prize for Physiology or Medicine wasawarded to Stanley B. Prusiner, the scientist who coinedthe term prion and studied the role of these proteinaceousinfectious particles in disease. Of all pathogens, prions arebelieved to be the most resistant to disinfectants. Themechanism by which prions cause disease remains a mys-tery, although it is thought that prions convert normalprotein molecules into nonfunctional ones by causing thenormal molecules to change their shape. Many scientistsremain unconvinced that proteins alone can cause disease.


CharacteristicsRecall from Chapter 3 that there are two domains of pro-caryotic organisms: Domain Bacteria and Domain Archaea.The bacteriologist’s most important reference (sometimesreferred to as the bacteriologist’s “bible”) is a five-volumeset of books entitled Bergey’s Manual of SystematicBacteriology (Bergey’s Manual for short), which is currentlybeing rewritten. (An outline of these volumes can befound on CD-ROM Appendix 2: “Phyla and MedicallySignificant Genera Within the Domain Bacteria.”) Whenall five volumes have been completed, they will containdescriptions of more than 5,000 validly named species ofbacteria. Some authorities believe that this number repre-sents only from less than 1% to a few percent of the totalnumber of bacteria that exist in nature.

According to Bergey’s Manual, the Domain Bacteriacontains 23 phyla, 32 classes, 5 subclasses, 77 orders,14 suborders, 182 families, 871 genera, and 5,007 species.Organisms in this domain are broadly divided into threephenotypic categories (i.e., categories based on their phys-ical characteristics): (a) those that are Gram-negative andhave a cell wall, (b) those that are Gram-positive and havea cell wall, and (c) those that lack a cell wall. (The termsGram-positive and Gram-negative are explained in a sub-sequent section of this chapter.) Using computers, micro-biologists have established numerical taxonomy systemsthat not only help to identify bacteria by their physicalcharacteristics, but also can help establish how closely re-lated these organisms are by comparing the compositionof their genetic material and other cellular characteristics.(Note: as previously mentioned, throughout this book, theterm “to identify an organism” means to learn the organ-ism’s species name [i.e., to speciate it].)

Many characteristics ofbacteria are examined to pro-vide data for identification andclassification. These charac-teristics include cell shape andmorphological arrangement,staining reactions, motility,colony morphology, atmos-pheric requirements, nutri-tional requirements, biochem-ical and metabolic activities, specific enzymes that theorganism produces, pathogenicity (the ability to causedisease), and genetic composition.

Cell MorphologyWith the compound light microscope, the size, shape,and morphologic arrangement of various bacteria areeasily observed. Bacteria vary greatly in size, usuallyranging from spheres measuring about 0.2 �m in diame-ter to 10.0-�m–long spiral-shaped bacteria, to evenlonger filamentous bacteria. As previously mentioned,the average coccus is about 1 �m in diameter, and the av-erage bacillus is about 1 �m wide � 3 �m long. Some un-usually large bacteria and unusually small bacteria havealso been discovered (discussed later).

There are three basicshapes of bacteria (Fig. 4-15):(a) round or spherical bacte-ria—the cocci (sing., coccus);(b) rectangular or rod-shapedbacteria—the bacilli (sing.,bacillus); and (c) curved and spi-ral-shaped bacteria (sometimesreferred to as spirilla).

Recall from Chapter 3 thatbacteria divide by binary fis-sion—one cell splits in half tobecome two daughter cells.The time it takes for one cell to

52 SECTION II ■ Introduction to Microbes and Cellular Biology

A bacterium’s Gramreaction (Gram-positiveor Gram-negative),basic cell shape, andmorphologicalarrangement of thecells are very importantclues to the organism’sidentification.

The three general shapes of bacteria areround (cocci), rod-shaped (bacilli), and spiral-shaped.

Bacteria reproduce bybinary fission. The timeit takes for one bacterial cell to splitinto two cells is referred to as thatorganism’s generationtime.

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split into two cells is referred to that organism’s genera-tion time. After binary fission, the daughter cells mayseparate completely from each other or may remain con-nected, forming various morphologic arrangements.

Cocci may be seen singly orin pairs (diplococci), chains(streptococci), clusters (staphylo-cocci), packets of four (tetrads),or packets of eight (octads), de-pending on the particularspecies and the manner inwhich the cells divide (Figs. 4-16 and 4-17). Examples ofmedically important cocci include Enterococcus spp.,Neisseria spp., Staphylococcus spp., and Streptococcus spp.

Bacilli (often referred to as rods) may be short orlong, thick or thin, and pointed or with curved or bluntends. They may occur singly, in pairs (diplobacilli), inchains (streptobacilli), in long filaments, or branched.Some rods are quite short, resembling elongated cocci;they are called coccobacilli. Listeria monocytogenes andHaemophilus influenzae are examples of coccobacilli.Some bacilli stack up next to each other, side by sidein a palisade arrangement, which is characteristic ofCorynebacterium diphtheriae (the cause of diphtheria) andorganisms that resemble it in appearance (called diph-theroids). Examples of medically important bacilli in-clude members of the family Enterobacteriaceae (e.g.,Enterobacter, Escherichia, Klebsiella, Proteus, Salmonella,and Shigella spp.), Pseudomonas aeruginosa, Bacillus spp.,and Clostridium spp.

Curved and spiral-shaped bacilli are placed into athird morphologic grouping. For example, Vibrio spp.,such as V. cholerae (the cause of cholera) and V. para-haemolyticus (a cause of diarrhea), are curved (comma-shaped) bacilli. Curved bacteria usually occur singly,but some species may form pairs. A pair of curved bacilli

Curved and spiral-shaped


Cell Shape

FIGURE 4-15. Categories of bacteria based on the shape of their cells.

STUDY AID Bacterial Names Sometimes Provide a Clue toTheir ShapeIf “coccus” appears in the name of a bacterium, you au-tomatically know the shape of the organism—spherical.Examples include genera such as Enterococcus,Peptococcus, Peptostreptococcus, Staphylococcus, andStreptococcus. However, not all cocci have “coccus” intheir names (e.g., Neisseria spp.). If “bacillus” appears inthe name of a bacterium, you automatically know theshape of the organism—rod-shaped or rectangular.Examples include genera such as Actinobacillus, Bacillus,Lactobacillus, and Streptobacillus. However, not all bacillihave “bacillus” in their names (e.g., E. coli).

Pairs of cocci areknown as diplococci.Chains of cocci areknown as streptococci.Clusters of cocci areknown as staphylococci.

STUDY AID Beware the Word “Bacillus”Whenever you see the word Bacillus, capitalized andunderlined or italicized, it is a particular genus of rod-shaped bacteria. However, if you see the word bacillus,and it is not capitalized, underlined, or italicized, itrefers to any rod-shaped bacterium.

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Arrangement Description Appearance Example Disease

Diplococci Cocci in pairs Neisseria gonorrhoeae Gonorrhea

Streptococci Cocci in chains Streptococcus pyogenes Strep throat

Staphylococci Cocci in clusters Staphylococcus aureus Boils

Tetrad Micrococcus luteus

Rarely pathogenic

A packet of 4 cocci

Octad Sarcina ventriculi

Rarely pathogenic

A packet of 8 cocci

FIGURE 4-16. Morphologicarrangements of cocci and exam-ples of bacteria having thesearrangements.

A B FIGURE 4-17. Morphologic arrangements of cocci. (A) Photomicrograph of Gram-stainedStaphylococcus aureus cells illustrating Gram-positive (blue) cocci in grapelike clusters. Apink-stained white blood cell can also be seen in the lower portion of the photomicrograph.(B) Scanning electron micrograph of Streptococcus mutans illustrating cocci in chains.([A] From Winn WC Jr, et al. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology,6th ed. Philadelphia: Lippincott Williams & Wilkins, 2006. [B] From Volk WA, et al. Essentialsof Medical Microbiology, 5th ed. Philadelphia: Lippincott-Raven, 1996.)

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resembles a bird and is described as having a gull-wingmorphology. Campylobacter spp. (a common cause of di-arrhea) have a gull-wing morphology. Spiral-shapedbacteria are referred to as spirochetes. Different speciesof spirochetes vary in size, length, rigidity, and thenumber and amplitude of their coils. Some are tightlycoiled, such as Treponema pallidum, the cause of syphilis,with a flexible cell wall that enables them to move read-ily through tissues (Fig. 4-18). Its morphology andcharacteristic motility—spinning around its long axis—make T. pallidum easy to recognize in wet preparationsof clinical specimens obtained from patients with pri-mary syphilis. Borrelia spp., the causative agents ofLyme disease and relapsing fever, are examples of lesstightly coiled spirochetes (Fig. 4-19).

Some bacteria may losetheir characteristic shape be-cause adverse growth condi-tions (e.g., the presence ofcertain antibiotics) prevent theproduction of normal cell walls. They are referred to ascell wall–deficient (CWD) bacteria. Some CWD bacteriarevert to their original shape when placed in favorablegrowth conditions, whereas others do not. Bacteria in thegenus Mycoplasma do not have cell walls; thus, when ex-amined microscopically, they appear in various shapes.Bacteria that exist in a variety of shapes are described asbeing pleomorphic; the ability to exist in a variety of shapesis known as pleomorphism. Because they have no cell walls,mycoplasmas are resistant to antibiotics that inhibit cellwall synthesis.

Staining ProceduresAs they exist in nature, most bacteria are colorless,transparent, and difficult to see. Therefore, variousstaining methods have been devised to enable scientiststo examine bacteria. In preparation for staining, thebacteria are smeared onto a glass microscope slide (re-sulting in what is known as a “smear”), air-dried, andthen “fixed.” (Methods for preparing and fixing smearsare further described in CD-ROM Appendix 5:“Clinical Microbiology Laboratory Procedures.”) Thetwo most common methods of fixation are heat fixationand methanol fixation. Heat fixation is usually accom-plished by passing the smear through a Bunsen burnerflame. If not performed properly, excess heat can dis-tort the morphology of the cells. Methanol fixation,which is accomplished by flooding the smear with ab-solute methanol for 30 seconds, is a more satisfactoryfixation technique. In general, fixation serves threepurposes:

1. It kills the organisms.2. It preserves their morphology (shape).3. It anchors the smear to the slide.

Specific stains and staining techniques are used to ob-serve bacterial cell morphology (e.g., size, shape, mor-phologic arrangement, composition of cell wall, capsules,flagella, endospores).

A simple stain is sufficient to determine bacterial shapeand morphologic arrangement (e.g., pairs, chains, clus-ters). For this method, shown in Figure 4-20, a dye (such

FIGURE 4-18. Scanning electron micrograph ofTreponema pallidum, the bacterium that causessyphilis. (Courtesy of Dr. David Cox and the Centers forDisease Control and Prevention.)

FIGURE 4-19. Spiral-shaped Borrelia hermsii(arrows), a cause of relapsing fever, in a stainedblood smear. (From Volk WA, et al. Essentials ofMedical Microbiology, 5th ed. Philadelphia: Lippincott-Raven, 1996.)

A bacterial specieshaving cells ofdifferent shapes is saidto be pleomorphic.

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as methylene blue) is applied to the fixed smear, rinsed,dried, and examined using the oil immersion lens of themicroscope. The procedures used to observe bacterialcapsules, spores, and flagella are collectively referred toas structural staining procedures.

In 1883, Dr. Hans Christian Gram developed astaining technique that bears his name—the Gram stainor Gram staining procedure. The Gram stain has be-come the most important staining procedure in the bac-teriology laboratory, because it differentiates between“Gram-positive” and “Gram-negative” bacteria (theseterms will be explained shortly). The organism’s Gramreaction serves as an extremely important “clue” when

attempting to learn the identity (species) of a particularbacterium. The steps in the Gram staining procedureare described in CD-ROM Appendix 5: “ClinicalMicrobiology Laboratory Procedures” and illustrated inFig. 4-21.

The color of the bacteria at the end of the Gramstaining procedure depends on the chemical compositionof their cell wall (Table 4-5). If the bacteria were not de-colorized during the decolorization step, they will beblue to purple at the conclusion of the Gram stainingprocedure; such bacteria are said to be “Gram-positive.”The thick layer of peptidoglycan in the cell walls ofGram-positive bacteria makes it difficult to remove thecrystal violet–iodine complex during the decolorizationstep. Figures 4-22 through 4-26 depict various Gram-positive bacteria.

If, on the other hand, thecrystal violet was removedfrom the cells during thedecolorization step, and thecells were subsequently stainedby the safranin (a red dye),they will be pink to red at theconclusion of the Gramstaining procedure; such bac-teria are said to be “Gram-negative.” The thin layer of peptidoglycan in the cellwalls of Gram-negative bacteria makes it easier to re-move the crystal violet–iodine complex during decol-orization. In addition, the decolorizer dissolves thelipid in the cell walls of Gram-negative bacteria; thisdestroys the integrity of the cell wall and makes it mucheasier to remove the crystal violet–iodine complex.Figures 4-27 and 4-28 depict various Gram-negativebacteria.

56 SECTION II ■ Introduction to Microbes and Cellular Biology

A. Smear loopful of microbes onto slide

D. Flood slide with stain

E. Rinse with water Blot dry

F. Examine with ×100 objective (oil immersion)

C. Drip methanol onto specimen to fix

B. Air-dry FIGURE 4-20. Simple bacterialstaining technique. (A) With aflamed loop, smear a loopful of bacte-ria suspended in broth or water onto aslide. (B) Allow slide to air-dry.(C) Fix the smear with absolute(100%) methanol. (D) Flood the slidewith the stain. (E) Rinse with waterand blot dry with bibulous paper orpaper towel. (F) Examine the slidewith the �100 microscope objective,using a drop of immersion oil directlyon the smear.

HISTORICAL NOTEThe Origin of the Gram StainWhile working in a laboratory in the morgue ofa Berlin hospital in the 1880s, a Danish physiciannamed Hans Christian Gram developed what was tobecome the most important of all bacterial stainingprocedures. He was developing a staining techniquethat would enable him to see bacteria in the lung tis-sues of patients who had died of pneumonia. The pro-cedure he developed—now called the Gram stain—demonstrated that two general categories of bacteriacause pneumonia: some of them stained blue and someof them stained red. The blue ones came to be knownas Gram-positive bacteria, and the red ones came to beknown as Gram-negative bacteria. It was not until1963 that the mechanism of Gram differentiation wasexplained by M.R.J. Salton.

If a bacterium is blueto purple at the end ofthe Gram stainingprocedure, it is said to be Gram-positive. If, on the other hand,it ends up being pink to red, it is said to beGram-negative.

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Key: = Gram-positive violet. = Gram-negative red. = Colorless.







Heat-fix specimen to slide. Flood slide with crystal violet solution; allow to act for 1 minute.

Rinse the slide, then flood with iodine solution; allow iodine to act for 1 minute. Before acetone decolorization (next step), all organisms appear purple, that is, gram-positive.

Rinse off excess iodine. Decolorize with acetone,approximately 5 seconds (timedepends on density of specimen).

Wash slide immediately in water. After acetone decolorization, those organisms that are gram-negative are no longer visible.

Wash in water, blot, and dry in air.Gram-negative organisms are visualized after application of the counterstain.

Crystal violet solution



Apply safranin counterstain for 30 seconds.

Iodine solution

FIGURE 4-21. Steps inthe Gram staining tech-nique. (From Harvey RA etal. Lippincott’s IllustratedReviews: Microbiology, 2nded. Philadelphia: LippincottWilliams & Wilkins, 2007.)


Color at the end of the Gram staining procedure Blue-to-purple Pink-to-red

Peptidoglycan in cell walls Thick layer Thin layer

Teichoic acids and lipoteichoic acids in cell walls Present Absent

Lipopolysaccharide in cell walls Absent Present

Differences between Gram-Positive and Gram-Negative BacteriaTABLE 4-5

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FIGURE 4-22. Chains of Gram-positive streptococciin a Gram-stained smear from a broth culture. (FromWinn WC Jr, et al. Koneman’s Color Atlas and Textbook ofDiagnostic Microbiology, 6th ed. Philadelphia: LippincottWilliams & Wilkins, 2006.)

FIGURE 4-23. Gram-positive Streptococcus pneumo-niae in a Gram-stained smear of a blood culture.Note the pairs of cocci, known as diplococci (arrows).(From Winn WC Jr, et al. Koneman’s Color Atlas andTextbook of Diagnostic Microbiology, 6th ed.Philadelphia: Lippincott Williams & Wilkins, 2006.)

FIGURE 4-24. Gram-positive bacilli (Clostridiumperfringens) in a Gram-stained smear prepared froma broth culture. Individual bacilli and chains of bacilli(streptobacilli) can be seen. (From Winn WC Jr, et al.Koneman’s Color Atlas and Textbook of DiagnosticMicrobiology, 6th ed. Philadelphia: Lippincott Williams &Wilkins, 2006.)

FIGURE 4-25. Gram-positive bacilli (Clostridiumtetani) in a Gram-stained smear from a broth culture.Terminal spores can be seen on some of the cells (arrows).(From Winn WC Jr, et al. Koneman’s Color Atlas andTextbook of Diagnostic Microbiology, 6th ed. Philadelphia:Lippincott Williams & Wilkins, 2006.)

FIGURE 4-26. Many Gram-positive bacteria can beseen on the surface of a pink-stained epithelial cellin this Gram-stained sputum specimen. Severalsmaller pink-staining polymorphonuclear leukocytes canalso be seen. (From Winn WC Jr, et al. Koneman’s ColorAtlas and Textbook of Diagnostic Microbiology, 6th ed.Philadelphia: Lippincott Williams & Wilkins, 2006.)

FIGURE 4-27. Gram-negative bacilli in a Gram-stained smear prepared from a bacterial colony.Individual bacilli and a few short chains of bacilli can beseen. (From Koneman E, et al. Color Atlas and Textbookof Diagnostic Microbiology, 5th ed. Philadelphia:Lippincott Williams & Wilkins, 1997.)

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Figure 4-29 illustrates the various shapes of bacte-ria that may be observed in a Gram-stained clinicalspecimen. Some strains of bacteria are neither consis-tently blue to purple nor pink to red after Gram stain-ing; they are referred to as Gram-variable bacteria.Examples of Gram-variable bacteria are membersof the genus Mycobacterium, such as M. tuberculosisand M. leprae. Refer to Table 4-6 and Figures 4-22through 4-28 for the staining characteristics of cer-tain pathogens.

STUDY AID A Method of Remembering a Particular Bacterium’sGram ReactionA former student used this method to remember theGram reaction of a particular bacterium. In her note-book, she drew two large circles. She lightly shaded inone circle, using a blue colored pencil. The other cir-cle was lightly shaded red. Within the blue circle, shewrote the names of bacteria studied in the course thatwere Gram-positive. Within the red circle, she wrotethe names of bacteria that were Gram-negative. Shethen studied the two circles. Later, whenever she en-countered the name of a particular bacterium, shewould remember which circle it was in. If it was in theblue circle, then the bacterium was Gram-positive.If it was in the red circle, the bacterium was Gram-negative. Clever!

FIGURE 4-28. Loosely coiled Gram-negative spiro-chetes. Borrelia burgdorferi is the etiologic agent(cause) of Lyme disease. (From Winn WC Jr, et al.Koneman’s Color Atlas and Textbook of DiagnosticMicrobiology, 6th ed. Philadelphia: Lippincott Williams& Wilkins, 2006.)

FIGURE 4-29. Various forms of bacteria that mightbe observed in Gram-stained smears. Shown here aresingle cocci, diplococci, tetrads, octads, streptococci,staphylococci, single bacilli, diplobacilli, streptobacilli,branching bacilli, loosely coiled spirochetes, and tightlycoiled spirochetes. (See text for explanation of terms.)

Mycobacterium species aremore often identified using astaining procedure called theacid-fast stain. In this proce-dure, carbol fuchsin (a brightred dye) is first driven into thebacterial cell using heat (usu-ally by flooding the smear with carbol fuchsin, and thenholding a Bunsen burner flame under the slide untilsteaming of the carbol fuchsin occurs). The heat is nec-essary because the cell walls of mycobacteria containwaxes, which prevent the stain from penetrating the cells.The heat softens the waxes, enabling the stain to pene-trate. A decolorizing agent (a mixture of acid and alcohol)is then used in an attempt to remove the red color fromthe cells. Because mycobacteria are not decolorized bythe acid–alcohol mixture (again owing to the waxes intheir cell walls), they are said to be acid-fast. Most otherbacteria are decolorized by the acid–alcohol treatment;they are said to be non–acid-fast. The acid-fast stain is es-pecially useful in the tuberculosis laboratory (“TB lab”)where the acid-fast mycobacteria are readily seen as redbacilli (referred to as acid-fast bacilli or AFB) against ablue or green background in a sputum specimen from atuberculosis patient. Figures 4-30 and 4-31 depict the ap-pearance of mycobacteria after the acid-fast staining pro-cedure. The acid-fast staining procedure was developedin 1882 by Paul Ehrlich—a German chemist.

The Gram and acid-fast staining procedures are re-ferred to as differential staining procedures because theyenable microbiologists to differentiate one group of bac-teria from another (i.e., Gram-positive bacteria fromGram-negative bacteria, and acid-fast bacteria fromnon–acid-fast bacteria). Table 4-7 summarizes the vari-ous types of bacterial staining procedures.

The acid-fast stain is ofvalue in the diagnosisof tuberculosis. Acid-fast bacteria are red atthe end of the acid-faststaining procedure.

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Gram-positive Cocci in clusters Staphylococcus aureus Wound infections, boils, pneumonia,septicemia, food poisoning

Cocci in chains Streptococcus pyogenes Strep throat, scarlet fever, necrotizingfasciitis, septicemia

Diplococci Streptococcus pneumoniae Pneumonia, meningitis, ear and sinus infections

Bacillus Corynebacterium diphtheriae DiphtheriaSpore-forming bacillus Bacillus anthracis Anthrax

Clostridium botulinum BotulismClostridium perfringens Wound infections, gas gangrene, food

poisoningClostridium tetani tetanus

Gram-negative Diplococci Neisseria gonorrhoeae gonorrheaNeisseria meningitidis Meningitis, respiratory infections

Bacillus Bordetella pertussis Whooping cough (pertussis)Brucella abortus BrucellosisChlamydia trachomatis Genital infections, trachomaEscherichia coli Urinary tract infections, septicemiaFrancisella tularensis TularemiaHaemophilus ducreyi ChancroidHaemophilus influenzae Meningitis; respiratory, ear and sinus

infectionsKlebsiella pneumoniae Urinary tract and respiratory infectionsProteus vulgaris Urinary tract infectionsPseudomonas aeruginosa Respiratory, urinary, and wound

infectionsRickettsia rickettsii Rocky Mountain spotted feverSalmonella typhi Typhoid feverSalmonella spp. GastroenteritisShigella spp. GastroenteritisYersinia pestis Plague

Curved bacillus Vibrio cholerae CholeraSpirochete Treponema pallidum Syphillis

Acid-fast, Branching bacilli Mycobacterium leprae Leprosy (Hansen disease)Gram-variable Mycobacterium tuberculosis Tuberculosis

Characteristics of Some Important Pathogenic BacteriaTABLE 4-6

FIGURE 4-30. Many red acid-fast mycobacteria canbe seen in this acid-fast stained liver biopsy specimen. (From Winn WC Jr, et al. Koneman’s ColorAtlas and Textbook of Diagnostic Microbiology, 6th ed.Philadelphia: Lippincott Williams & Wilkins, 2006.)

FIGURE 4-31. Many red acid-fast bacilli(Mycobacterium tuberculosis) can be seen in thisacid-fast stained concentrate from a digestedsputum specimen. (From Koneman, E, et al. Color Atlasand Textbook of Diagnostic Microbiology, 5th ed.Philadelphia: Lippincott Williams & Wilkins, 1997.)

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MotilityIf a bacterium is able to “swim,” it is said to be motile.Bacteria unable to swim are said to be nonmotile. Bacterialmotility is most often associated with the presence of fla-gella or axial filaments, although some bacteria exhibit atype of gliding motility on secreted slime. Bacteria neverpossess cilia. Most spiral-shaped bacteria and about onehalf of the bacilli are motile by means of flagella, but cocciare generally nonmotile. A flagella stain can be used todemonstrate the presence, number, and location of flagellaon bacterial cells. Various terms (e.g., monotrichous, am-phitrichous, lophotrichous, peritrichous) are used to de-scribe the number and location of flagella on bacterial cells(see Chapter 3).

Motility can be demonstrated by stabbing the bacte-ria into a tube of semisolid agar or by using the hanging-drop technique. Growth (multiplication) of bacteria insemisolid agar produces turbidity (cloudiness).Nonmotile organisms will grow only along the stab line(thus, turbidity will be seen only along the stab line), butmotile organisms will spread away from the stab line(thus, producing turbidity throughout the medium; seeFig. 4-32). In the hanging-drop method (Fig. 4-33), adrop of a bacterial suspension is placed onto a glass cov-erslip. The coverslip is then inverted over a depressionslide. When the preparation is examined microscopically,motile bacteria within the “hanging drop” will be seendarting around in every direction.


Simple staining procedure Staining with methylene blue Merely to stain the cells so that their size, shape,and morphologic arrangement can be determined

Structural staining procedures Capsule stains To determine whether the organism is encapsulatedFlagella stains To determine whether the organism possesses flagella

and, if so, their number and location on the cellEndospore stains To determine whether the organism is a spore-former

and, if so, to determine whether the spores areterminal or subterminal spores

Differential staining procedures Gram stain To differentiate between Gram-positive and Gram-negative bacteria

Acid-fast stain To differentiate between acid-fast and non–acid-fastbacteria

Types of Bacterial Staining ProceduresTABLE 4-7

A. B. C. D.


FIGURE 4-32. Semisolid agarmethod for determining motility.(A) Uninoculated tube of semisolidagar. (B) Same tube being inocu-lated by stabbing the inoculatingwire into the medium. (C) Patternof growth of a nonmotile organism,after incubation. (D) Pattern ofgrowth of a motile organism, afterincubation.

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Colony MorphologyA single bacterial cell that landson the surface of a solid culturemedium cannot be seen, butafter it divides over and overagain, it produces a mound orpile of bacteria, known as a bac-terial colony (Fig. 4-34). A colony contains millions of or-ganisms. The colony morphology (appearance of thecolonies) of bacteria varies from one species to another.Colony morphology includes the size, color, overall shape,elevation, and the appearance of the edge or margin of thecolony. As is true for cell morphology and staining charac-teristics, colony features serve as important “clues” in theidentification of bacteria. Size of colonies is determined bythe organism’s rate of growth (generation time), and is animportant characteristic of a particular bacterial species.Colony morphology also includes the results of enzymatic

activity on various types of culture media, such as thoseshown in Figures 8-3 through 8-5 in Chapter 8.

Atmospheric RequirementsIn the microbiology laboratory, it is useful to classify bac-teria on the basis of their relationship to oxygen (O2) andcarbon dioxide (CO2). With respect to oxygen, a bacterialisolate can be classified into one of five major groups: ob-ligate aerobes, microaerophilic aerobes (microaerophiles),facultative anaerobes, aerotolerant anaerobes, and obli-gate anaerobes (Fig. 4-35). In a liquid medium such asthioglycollate broth, the region of the medium in whichthe organism grows depends on the oxygen needs of thatparticular species.

To grow and multiply, obli-gate aerobes require an atmos-phere containing molecularoxygen in concentrations com-parable to that found in roomair (i.e., 20%–21% O2).Mycobacteria and certain fungiare examples of microorganismsthat are obligate aerobes.Microaerophiles (microaerophilicaerobes) also require oxygen for

62 SECTION II ■ Introduction to Microbes and Cellular Biology

A. B.


Petroleum jelly

FIGURE 4-33. Hanging-drop preparation for study of living bacteria.(A) Depression slide. (B) Depression slide with coverglass over the depressionarea. (C) Side view of hanging-drop preparation showing the drop of liquid culturemedium hanging from the center of the coverglass above the depression.

Obligate aerobes andmicroaerophiles requireoxygen. Obligateaerobes require anatmosphere containingabout 20% to 21%oxygen, whereasmicroaerophiles requirereduced oxygenconcentrations (usuallyaround 5% oxygen).

8 hr.

12 hr.

No. ofcells




4 hr.

0 hr.




Visible colony

Singlebacterial cell

FIGURE 4-34. Formation of a bacterial colony onsolid growth medium. In this illustration, the genera-tion time is assumed to be 30 minutes. (From Harvey RA,et al. Lippincott’s Illustrated Reviews: Microbiology, 2nded. Philadelphia: Lippincott Williams & Wilkins, 2007.)








Percent oxygen (O2)More Less

FIGURE 4-35. Categories of bacteria based on theirrelationship to oxygen.

A mound or pile ofbacteria on a solidculture medium isknown as a bacterialcolony.

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multiplication, but in concentrations lower than thatfound in room air. Neisseria gonorrhoeae (the causativeagent of gonorrhea) and Campylobacter spp. (which aremajor causes of bacterial diarrhea) are examples of mi-croaerophilic bacteria that prefer an atmosphere contain-ing about 5% oxygen.

Anaerobes can be defined as organisms that do not re-quire oxygen for life and reproduction. However, theyvary in their sensitivity to oxygen. The terms obligateanaerobe, aerotolerant anaerobe, and facultative anaer-obe are used to describe the organism’s relationship tomolecular oxygen. An obligate anaerobe is an anaerobe thatcan only grow in an anaerobic environment (i.e., an envi-ronment containing no oxygen) (see “Insight: Life in theAbsence of Oxygen” on the CD-ROM ).

An aerotolerant anaerobe doesnot require oxygen, grows bet-ter in the absence of oxygen, butcan survive in atmospheres con-taining molecular oxygen (suchas air and a CO2 incubator).The concentration of oxygenthat an aerotolerant anaerobe can tolerate varies from onespecies to another. Facultative anaerobes are capable ofsurviving in either the presence or absence of oxygen; any-where from 0% O2 to 20% to 21% O2. Many of thebacteria routinely isolated from clinical specimens arefacultative anaerobes (e.g., members of the familyEnterobacteriaceae, most streptococci, most staphylococci).

Room air contains less than1% CO2. Some bacteria, re-ferred to as capnophiles(capnophilic organisms), growbetter in the laboratory in thepresence of increased concen-trations of CO2. Some anaer-obes (e.g., Bacteroides and Fusobacterium species) arecapnophiles, as are some aerobes (e.g., certain Neisseria,Campylobacter, and Haemophilus species). In the clinicalmicrobiology laboratory, CO2 incubators are routinelycalibrated to contain between 5% and 10% CO2.

Nutritional RequirementsAll bacteria need some form of the elements carbon, hy-drogen, oxygen, sulfur, phosphorus, and nitrogen forgrowth. Special elements, such as potassium, calcium,iron, manganese, magnesium, cobalt, copper, zinc, anduranium, are required by some bacteria. Certain mi-crobes have specific vitamin requirements and some needorganic substances secreted by other living microorgan-isms during their growth. Organisms with especiallydemanding nutritional requirements are said to be fastid-ious; think of them as being “fussy.” Special enrichedmedia must be used to grow fastidious organisms in thelaboratory. The nutritional needs of a particular organ-ism are usually characteristic for that species of bacteriaand sometimes serve as important clues when attempting

to identify the organism. Nutritional requirements arediscussed further in Chapters 7 and 8.

Biochemical and Metabolic ActivitiesAs bacteria grow, they produce many waste products andsecretions, some of which are enzymes that enable themto invade their host and cause disease. The pathogenicstrains of many bacteria, such as staphylococci and strep-tococci, can be tentatively identified by the enzymes theysecrete. Also, in particular environments, some bacteriaare characterized by the production of certain gases, suchas carbon dioxide, hydrogen sulfide, oxygen, or methane.To aid in the identification of certain types of bacteria inthe laboratory, they are inoculated into various substrates(e.g., carbohydrates and amino acids) to determinewhether they possess the enzymes necessary to breakdown those substrates. Learning whether a particular or-ganism is able to break down a certain substrate serves asa clue to the identity of that organism. Different types ofculture media are also used in the laboratory to learn in-formation about an organism’s metabolic activities (to bediscussed in Chapter 8).

PathogenicityThe characteristics that enable bacteria to cause diseaseare discussed in Chapter 14. Many pathogens are able tocause disease because they possess capsules, pili, or endo-toxins (biochemical components of the cell walls of Gram-negative bacteria), or because they secrete exotoxins andexoenzymes that damage cells and tissues. Frequently,pathogenicity (the ability to cause disease) is tested by in-jecting the organism into mice or cell cultures. Somecommon pathogenic bacteria are listed in Table 4-6.

Genetic CompositionMost modern laboratories are moving toward the identi-fication of bacteria using some type of test procedure thatanalyzes the organism’s deoxyribonucleic acid (DNA) orribonucleic acid (RNA). These test procedures are col-lectively referred to as molecular diagnostic procedures.The composition of the genetic material (DNA) of an or-ganism is unique to each species. DNA probes make itpossible to identify an isolate without relying on pheno-typic characteristics. A DNA probe is a single-strandedDNA sequence that can be used to identify an organismby hybridizing with a unique complimentary sequenceon the DNA or rRNA of that organism. Also, throughthe use of 16S rRNA sequencing (see Chapter 3), aresearcher can determine the degree of relatednessbetween two different bacteria.

Unique BacteriaRickettsias, chlamydias, and mycoplasmas are bacteria,but they do not possess all the attributes of typical bacte-rial cells. Thus, they are often referred to as “unique” or“rudimentary” bacteria. Because they are so small and dif-ficult to isolate, they were formerly thought to be viruses.

Obligate anaerobes,aerotolerant anaerobes,and facultativeanaerobes can thrive inan atmosphere devoidof oxygen.

For optimum growth inthe laboratory,capnophiles require anatmosphere containing5% to 10% carbondioxide.

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Rickettsias, Chlamydias, and CloselyRelated BacteriaRickettsias and chlamydias are bacteria with a Gram-negative–type cell wall. They are obligate intracellularpathogens that cause diseases in humans and other ani-mals. As the name implies, an obligate intracellularpathogen is a pathogen that must live within a host cell.To grow such organisms in the laboratory, they must beinoculated into embryonated chicken eggs, laboratoryanimals, or cell cultures. They will not grow on artificial(synthetic) culture media.

The genus Rickettsia was named for Howard T.Ricketts, a U.S. pathologist; these organisms have noconnection to the disease called rickets, which is the re-sult of vitamin D deficiency. Because they appear to haveleaky cell membranes, most rickettsias must live insideanother cell to retain all necessary cellular substances(Fig. 4-36). All diseases caused by Rickettsia species arearthropod-borne, meaning that they are transmitted byarthropod vectors (carriers); see Table 4-8.

Arthropods such as lice, fleas, and ticks transmit therickettsias from one host to another by their bites orwaste products. Diseases caused by Rickettsia spp. includetyphus and typhuslike diseases (e.g., Rocky Mountainspotted fever). All these diseases involve production of arash. Medically important bacteria that are closely re-lated to rickettsias include Coxiella burnetii, Bartonellaquintana (formerly Rochalimaea quintana), Ehrlichia spp.,and Anaplasma spp. C. burnetii (the cause of Q fever) is

transmitted primarily by aerosols, but can be transmittedto animals by ticks. B. quintana is associated with trenchfever (a louseborne disease), cat scratch disease, bac-teremia, and endocarditis. Ehrlichia and Anaplasma spp.cause human tickborne diseases such as human mono-cytic ehrlichiosis (HME) and human granulocytic

64 SECTION II ■ Introduction to Microbes and Cellular Biology

FIGURE 4-36. Rickettsia prowazekii (arrows), thecause of epidemic louseborne typhus, in experimen-tally infected tick tissue. (From Volk WA, et al.Essentials of Medical Microbiology, 5th ed. Philadelphia:Lippincott-Raven, 1996.)


Rickettsia R. akari Rickettsialpox (a miteborne disease)R. prowazekii Epidemic typhus (a louseborne disease)R. rickettsii Rocky Mountain spotted fever (a tickborne disease)R. typhi Endemic or murine typhus (a fleaborne disease)

Ehrlichia spp. E. chaffeensis Human monocytic ehrlichiosis

Anaplasma spp. Anaplasma phagocytophilum Human granulocytic ehrlichiosis

Chlamydia (and Chlamydia– Chlamydophila pneumoniae Pneumonialike bacteria)

Chlamydophila psittaci Psittacosis (a respiratory disease; a zoonosis; sometimes calledChlamydia trachomatis “parrot fever”)

Different serotypes cause different diseases, including trachoma(an eye disease) inclusion conjunctivitis (an eye disease),nongonococcal urethritis (NGU; a sexually transmitted disease), lymphogranuloma venereum (LGV; a sexually transmitted disease)

Mycoplasma M. pneumoniae Atypical pneumoniaM. genitalium Nongonococcal urethritis (NGU)

Orientia O. tsutsugamushi Scrub typhus (a miteborne disease)

Ureaplasma U. urealyticum Nongonococcal urethritis (NGU)

Human Diseases Caused by Unique BacteriaTABLE 4-8

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ehrlichiosis (HGE). Ehrlichia and Anaplasma spp. are in-traleukocytic pathogens, meaning that they live withincertain types of white blood cells.

The term “chlamydias”refers to Chlamydia spp. andclosely related organisms(such as Chlamydophila spp.).Chlamydias are referred to as“energy parasites.” Althoughthey can produce adenosinetriphosphate (ATP) molecules, they preferentially useATP molecules produced by their host cells. ATPmolecules are the major energy-storing or energy-carrying molecules of cells (see Chapter 7). Chlamydiasare obligate intracellular pathogens that are transferredby inhalation of aerosols or by direct contact betweenhosts—not by arthropods. Medically important chlamy-dias include Chlamydia trachomatis, Chlamydophila pneu-moniae, and Chlamydophila psittaci. Different serotypesof C. trachomatis cause different diseases, includingtrachoma (the leading cause of blindness in the world),inclusion conjunctivitis (another type of eye disease),and nongonococcal urethritis (NGU; a term givento urethritis that is not caused by Neisseria gonorrhoeae).C. pneumoniae causes a type of pneumonia, and C.psittaci causes a respiratory disease called psittacosis.Chlamydial diseases are listed in Table 4-8.

MycoplasmasMycoplasmas are the smallestof the cellular microbes (Fig.4-37). Because they lack cellwalls, they assume manyshapes, from coccoid to fila-mentous; thus, they appear pleomorphic when exam-ined microscopically. Sometimes they are confused withcell wall–deficient (CWD) forms of bacteria, describedearlier; however, even in the most favorable growthmedia, mycoplasmas are not able to produce cell walls,

which is not true for CWD. Mycoplasmas were for-merly called pleuropneumonia-like organisms (PPLO),first isolated from cattle with lung infections. They maybe free-living or parasitic and are pathogenic to manyanimals and some plants. In humans, pathogenic my-coplasmas cause primary atypical pneumonia and geni-tourinary infections; some species can grow intracellu-larly. Because they have no cell wall, they are resistantto treatment with penicillin and other antibiotics thatwork by inhibiting cell wall synthesis. Mycoplasmas canbe cultured on artificial media in the laboratory, wherethey produce tiny colonies (called “fried egg colonies”)that resemble sunny-side-up fried eggs in appearance.The absence of a cell wall prevents mycoplasmas fromstaining with the Gram stain procedure. Diseasescaused by mycoplasmas and a closely related organism(Ureaplasma urealyticum) are shown in Table 4-8.

Especially Large and Especially Small BacteriaThe size of a typical coccus (e.g., a Staphylococcus aureuscell) is 1 �m in diameter. A typical bacillus (e.g., an E. colicell) is about 1.0 �m wide � 3.0 �m long, although somebacilli are long thin filaments—up to about 12 �m inlength or even longer—but still only about 1 �m wide.Thus, most bacteria are microscopic, requiring the use ofa microscope to be seen.

Perhaps the largest of all bacteria—large enough tobe seen with the unaided human eye—is Thiomargaritanamibiensis, a colorless, marine, sulfide-oxidizing bac-terium. Single spherical cells of T. namibiensis are 100 to300 �m, but may be as large as 750 �m (0.75 mm). Interms of size, comparing a T. namibiensis cell to an E. colicell would be like comparing a blue whale to a newlyborn mouse. Other marine sulfide-oxidizing bacteria inthe genera Beggiatoa and Thioploca are also especially

STUDY AID “Strains” versus “Serotypes”Within a given species, there are usually differentstrains. For example, there are many different strainsof E. coli. If the E. coli that has been isolated fromPatient X is producing an enzyme that is not beingproduced by the E. coli from Patient Z, the two E. coliisolates are considered to be different strains. Or, ifone isolate of E. coli is resistant to ampicillin (an an-tibiotic), and the other E. coli isolate is susceptible toampicillin, then these isolates are considered to be dif-ferent strains of E. coli. Also, there are usually differentserotypes (sometimes called serovars) within a givenspecies. Serotypes of an organism differ from eachother as a result of differences in their surface mole-cules (surface antigens). Sometimes, as is true for C.trachomatis and E. coli, different serotypes of a givenspecies cause different diseases.

Because they do notpossess cell walls,Mycoplasma spp. arepleomorphic.

Rickettsias andchlamydias are examplesof obligate intracellularorganisms—organismsthat can only existwithin host cells.

FIGURE 4-37. Scanning electron micrograph ofMycoplasma pneumoniae. (From Strohl WA, et al.Lippincott’s Illustrated Reviews: Microbiology.Philadelphia: Lippincott Williams & Wilkins, 2001.)

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large, having diameters from 10 �m to more than100 �m. Although Beggiatoa and Thioploca form fila-ments, Thiomargarita cells do not.

Another enormous bacterium, named Epulopisciumfishelsonii, has been isolated from the intestines of the reefsurgeonfish; this bacillus is about 80 �m wide � 600 �m(0.6 mm) long. Epulopiscium cells are about five timeslonger than eucaryotic Paramecium cells.b The volume ofan Epulopiscium cell is about a million times greater thanthe volume of a typical bacterial cell. Spore-forming bac-teria called metabacteria, found in the intestines of her-bivorous rodents, are closely related to Epulopiscium, butthey reach lengths of only 20 to 30 �m. Although shorterthan Epulopiscium, metabacteria are much longer thanmost bacteria.

At the other end of the spectrum, there are especiallytiny bacteria called nanobacteria. Their sizes are expressedin nanometers because these bacteria are less than 1 �min diameter; hence the name, nanobacteria. In some cases,they are as small as 20 nm in diameter. Nanobacteria havebeen found in soil, minerals, ocean water, human andanimal blood, human dental calculus (plaque), arterialplaque, and even rocks (meteorites) of extraterrestrialorigin. The existence of nanobacteria is controversial,however. Some scientists believe that these tiny structureswere formed by geological, rather than biological,processes. They feel that nanobacteria are smaller thanthe minimum possible size for a living cell.

Photosynthetic BacteriaPhotosynthetic bacteria includepurple bacteria, green bacteria,and cyanobacteria (erroneouslyreferred to in the past as blue-green algae). Although all threegroups use light as an energysource, they do not all carry outphotosynthesis in the same way.For example, purple and green bacteria (which, in somecases, are not actually those colors) do not produceoxygen, whereas cyanobacteria do. Photosynthesis thatproduces oxygen is called oxygenic photosynthesis, whereasphotosynthesis that does not produce oxygen is calledanoxygenic photosynthesis.

In photosynthetic eucaryotes (algae and plants), pho-tosynthesis takes place in plastids, which were discussedin Chapter 3. In cyanobacteria, photosynthesis takesplace on intracellular membranes known as thylakoids.Thylakoids are attached to the cell membrane at variouspoints and are thought to represent invaginations of thecell membrane. Attached to the thylakoids, in orderlyrows, are numerous phycobilisomes—complex proteinpigment aggregates where light harvesting occurs.

Many scientists believe that cyanobacteria were thefirst organisms capable of carrying out oxygenic photo-synthesis and, thus, played a major part in the oxygena-tion of the atmosphere. Fossil records reveal thatcyanobacteria were already in existence 3.3 to 3.5 billionyears ago. Photosynthesis is discussed further in Chapter7. Cyanobacteria vary widely in shape; some are cocci,some are bacilli, and others form long filaments.

When appropriate conditions exist, cyanobacteria inpond or lake water will overgrow, creating a waterbloom—a “pond scum” that resembles a thick layer ofbluish green (turquoise) oil paint. The conditions includea mild or no wind, a balmy water temperature (15°–30°C),a water pH of 6 to 9, and an abundance of the nutrients ni-trogen and phosphorous in the water. Many cyanobacteriaare able to convert nitrogen gas (N2) from the air into am-monium ions (NH4

�) in the soil or water; this process isknown as nitrogen fixation (Chapter 10).

Some cyanobacteria pro-duce toxins (poisons), suchas neurotoxins (which affectthe central nervous system),hepatotoxins (which affect theliver), and cytotoxins (whichaffect other types of cells).These cyanotoxins can cause disease and even death inwildlife species and humans that consume contaminatedwater. Additional information about these toxins can befound in the CD-ROM Appendix 1, entitled “MicrobialIntoxications.”


Procaryotic organisms thus far described in this chapterare all members of the Domain Bacteria. Procaryotic or-ganisms in the Domain Archaea were discovered in 1977.Although they were once referred to as archaebacteria (orarchaeobacteria), most scientists now feel that there aresufficient differences between archaea and bacteria to stopreferring to archaea as bacteria. Archae means “ancient,”and the name archaea was originally assigned when it wasthought that these procaryotes evolved earlier than bacte-ria. Now, there is considerable debate as to whether bac-teria or archaea came first. Genetically, even though theyare procaryotes, archaea are more closely related to eu-caryotes than they are to bacteria; some possess genesotherwise found only in eucaryotes. Many scientistsbelieve that bacteria and archaea diverged from a

66 SECTION II ■ Introduction to Microbes and Cellular Biology

STUDY AID Beware of Similar Sounding NamesDo not confuse Mycoplasma with Mycobacterium. Eachis a genus of bacteria. The unique thing aboutMycoplasma spp. is that they lack cell walls. The uniquething about Mycobacterium spp. is that they are acid-fast.

b Paramecium is a genus of freshwater protozoa.

Photosynthetic bacteriaare bacteria capable ofconverting light energyinto chemical energy.Cyanobacteria areexamples ofphotosynthetic bacteria.

Some cyanobacteriaproduce toxins (calledcyanotoxins) that cancause disease and evendeath in animals andhumans.

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common ancestor relatively soon after life began on thisplanet. Later, the eucaryotes split off from the archaea.

According to Bergey’s Manual of Systematic Bacteriology,the Domain Archaea contains 2 phyla, 8 classes, 12 orders,21 families, 69 genera, and 217 species. Archaea varywidely in shape; some are cocci, some are bacilli, and oth-ers form long filaments. Many, but not all, archaea are “ex-tremophiles,” in the sense that they live in extreme envi-ronments, such as extremely acidic, alkaline, hot, cold, orsalty environments, or environments where there is ex-tremely high pressure (Table 4-9).

Some live at the bottom ofthe ocean in and near thermalvents, where, in addition toheat and salinity, there is ex-treme pressure. Other archaea,called methanogens, producemethane, which is a flammablegas. Although virtually all ar-chaea possess cell walls, their cell walls contain no pepti-doglycan. In contrast, all bacterial cell walls contain pep-tidoglycan. The 16S rRNA sequences of archaea arequite different from the 16S rRNA sequences of bacteria.The 16S rRNA sequence data suggest that archaea aremore closely related to eucaryotes than they are to bacte-ria. You will recall from Chapter 3 that differences inrRNA structure form the basis of the Three-DomainSystem of Classification.


After studying this chapter, answer the following multiple-choice questions.

1. Which one of the following steps occurs during themultiplication of animal viruses, but not during themultiplication of bacteriophages?a. assemblyb. biosynthesisc. penetrationd. uncoating

2. Which one of the following diseases or groups ofdiseases is not caused by prions?a. certain plant diseasesb. chronic wasting disease of deer and elkc. Creutzfeldt-Jacob disease of humansd. “mad cow disease”

3. Most procaryotic cells reproduce by:a. binary fission.b. budding.c. gamete production.d. spore formation.

4. The group of bacteria that lack rigid cell walls andtake on irregular shapes is:a. chlamydias.b. mycobacteria.c. mycoplasmas.d. rickettsias.

5. At the end of the Gram staining procedure, Gram-positive bacteria will be:a. blue to purple.b. green.c. orange.d. pink to red.

6. Which one of the following statements about rick-ettsias is false?a. Diseases caused by rickettsias are arthropod-borne.b. Rickets is caused by a Rickettsia species.c. Rickettsia species cause typhus and typhuslike

diseases.d. Rickettsias have leaky membranes.

7. Which one of the following statements aboutChlamydia and Chlamydophila spp. is false?a. They are obligate intracellular pathogens.b. They are considered to be “energy parasites.”c. The diseases they cause are all arthropod-borne.d. They are considered to be Gram-negative bacteria.

8. Which one of the following statements aboutcyanobacteria is false?a. Although cyanobacteria are photosynthetic, they do

not produce oxygen as a result of photosynthesis.b. At one time, cyanobacteria were called blue-

green algae.c. Some cyanobacteria are capable of nitrogen

fixation.d. Some cyanobacteria are important medically

because they produce toxins.

Type of Extreme Name Given To TheseEnvironment Types of Extremophiles

Extremely acidic Acidophiles

Extremely alkaline Alkaliphiles

Extremely hot Thermophiles

Extremely cold Psychrophiles

Extremely salty Halophiles

Extremely high pressure Piezophiles (formerlybarophiles)

Examples of ExtremophilesTABLE 4-9

Many archaea areextremophiles, meaningthat they live inextreme environments;e.g., environments thatare extremely hot, dry,or salty.

ON THE CD-ROM• Terms Introduced in This Chapter• Review of Key Points• Insight

• Microbes in the News: “Mad Cow Disease”• Life in the Absence of Oxygen

• Increase Your Knowledge• Critical Thinking• Additional Self-Assessment Exercises

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9. Which one of the following statements aboutarchaea is false?a. Archaea are more closely related to eucaryotes

than they are to bacteria.b. Both archaea and bacteria are procaryotic

organisms.c. Some archaea live in extremely hot environments.d. The cell walls of archaea contain a thicker layer

of peptidoglycan than the cell walls of bacteria.

10. An organism that does not require oxygen, growsbetter in the absence of oxygen, but can survive inatmospheres containing some molecular oxygen isknown as a(n):a. aerotolerant anaerobe.b. capnophile.c. facultative anaerobe.d. microaerophile.

68 SECTION II ■ Introduction to Microbes and Cellular Biology

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