# An Invisible World

## What Our Ancestors Knew

### Learning Objectives

By the end of this section, you will be able to:
1. Describe how our ancestors improved food with the use of invisible microbes
2. Describe how the causes of sickness and disease were explained in ancient times, prior to the invention of the microscope
3. Describe key historical events associated with the birth of microbiology
Most people today, even those who know very little about microbiology, are familiar with the concept of microbes, or “germs,” and their role in human health. Schoolchildren learn about bacteria, viruses, and other microorganisms, and many even view specimens under a microscope. But a few hundred years ago, before the invention of the microscope, the existence of many types of microbes was impossible to prove. By definition, microorganisms, or microbes, are very small organisms; many types of microbes are too small to see without a microscope, although some parasites and fungi are visible to the naked eye.
Humans have been living with—and using—microorganisms for much longer than they have been able to see them. Historical evidence suggests that humans have had some notion of microbial life since prehistoric times and have used that knowledge to develop foods as well as prevent and treat disease. In this section, we will explore some of the historical applications of microbiology as well as the early beginnings of microbiology as a science.

### Fermented Foods and Beverages

People across the world have enjoyed fermented foods and beverages like beer, wine, bread, yogurt, cheese, and pickled vegetables for all of recorded history. Discoveries from several archeological sites suggest that even prehistoric people took advantage of fermentation to preserve and enhance the taste of food. Archaeologists studying pottery jars from a Neolithic village in China found that people were making a fermented beverage from rice, honey, and fruit as early as 7000 BC.P.E. McGovern et al. “Fermented Beverages of Pre- and Proto-Historic China.”
Production of these foods and beverages requires microbial fermentation, a process that uses bacteria, mold, or yeast to convert sugars (carbohydrates) to alcohol, gases, and organic acids (). While it is likely that people first learned about fermentation by accident—perhaps by drinking old milk that had curdled or old grape juice that had fermented—they later learned to harness the power of fermentation to make products like bread, cheese, and wine.

### The Iceman Treateth

Prehistoric humans had a very limited understanding of the causes of disease, and various cultures developed different beliefs and explanations. While many believed that illness was punishment for angering the gods or was simply the result of fate, archaeological evidence suggests that prehistoric people attempted to treat illnesses and infections. One example of this is Ötzi the Iceman, a 5300-year-old mummy found frozen in the ice of the Ötzal Alps on the Austrian-Italian border in 1991. Because Ötzi was so well preserved by the ice, researchers discovered that he was infected with the eggs of the parasite Trichuris trichiura, which may have caused him to have abdominal pain and anemia. Researchers also found evidence of Borrelia burgdorferi, a bacterium that causes Lyme disease.A. Keller et al. “New Insights into the Tyrolean Iceman's Origin and Phenotype as Inferred by Whole-Genome Sequencing.”  Some researchers think Ötzi may have been trying to treat his infections with the woody fruit of the Fomitopsis betulinus fungus, which was discovered tied to his belongings.L. Capasso. “5300 Years Ago, the Ice Man Used Natural Laxatives and Antibiotics.”  This fungus has both laxative and antibiotic properties. Ötzi was also covered in tattoos that were made by cutting incisions into his skin, filling them with herbs, and then burning the herbs.L. Capasso, L. “5300 Years Ago, the Ice Man Used Natural Laxatives and Antibiotics.”  There is speculation that this may have been another attempt to treat his health ailments.

### Early Notions of Disease, Contagion, and Containment

Several ancient civilizations appear to have had some understanding that disease could be transmitted by things they could not see. This is especially evident in historical attempts to contain the spread of disease. For example, the Bible refers to the practice of quarantining people with leprosy and other diseases, suggesting that people understood that diseases could be communicable. Ironically, while leprosy is communicable, it is also a disease that progresses slowly. This means that people were likely quarantined after they had already spread the disease to others.
The ancient Greeks attributed disease to bad air, mal’aria, which they called “miasmatic odors.” They developed hygiene practices that built on this idea. The Romans also believed in the miasma hypothesis and created a complex sanitation infrastructure to deal with sewage. In Rome, they built aqueducts, which brought fresh water into the city, and a giant sewer, the Cloaca Maxima, which carried waste away and into the river Tiber (). Some researchers believe that this infrastructure helped protect the Romans from epidemics of waterborne illnesses.
Even before the invention of the microscope, some doctors, philosophers, and scientists made great strides in understanding the invisible forces—what we now know as microbes—that can cause infection, disease, and death.
The Greek physician Hippocrates (460–370 BC) is considered the “father of Western medicine” (). Unlike many of his ancestors and contemporaries, he dismissed the idea that disease was caused by supernatural forces. Instead, he posited that diseases had natural causes from within patients or their environments. Hippocrates and his heirs are believed to have written the Hippocratic Corpus, a collection of texts that make up some of the oldest surviving medical books.G. Pappas et al. “Insights Into Infectious Disease in the Era of Hippocrates.”  Hippocrates is also often credited as the author of the Hippocratic Oath, taken by new physicians to pledge their dedication to diagnosing and treating patients without causing harm.
While Hippocrates is considered the father of Western medicine, the Greek philosopher and historian Thucydides (460–395 BC) is considered the father of scientific history because he advocated for evidence-based analysis of cause-and-effect reasoning (). Among his most important contributions are his observations regarding the Athenian plague that killed one-third of the population of Athens between 430 and 410 BC. Having survived the epidemic himself, Thucydides made the important observation that survivors did not get re-infected with the disease, even when taking care of actively sick people.Thucydides.  This observation shows an early understanding of the concept of immunity.
Marcus Terentius Varro (116–27 BC) was a prolific Roman writer who was one of the first people to propose the concept that things we cannot see (what we now call microorganisms) can cause disease (). In Res Rusticae (On Farming), published in 36 BC, he said that “precautions must also be taken in neighborhood swamps. . . because certain minute creatures [animalia minuta] grow there which cannot be seen by the eye, which float in the air and enter the body through the mouth and nose and there cause serious diseases.”Plinio Prioreschi.

### The Birth of Microbiology

While the ancients may have suspected the existence of invisible “minute creatures,” it wasn’t until the invention of the microscope that their existence was definitively confirmed. While it is unclear who exactly invented the microscope, a Dutch cloth merchant named Antonie van Leeuwenhoek (1632–1723) was the first to develop a lens powerful enough to view microbes. In 1675, using a simple but powerful microscope, Leeuwenhoek was able to observe single-celled organisms, which he described as “animalcules” or “wee little beasties,” swimming in a drop of rain water. From his drawings of these little organisms, we now know he was looking at bacteria and protists. (We will explore Leeuwenhoek’s contributions to microscopy further in How We See the Invisible World.)
Nearly 200 years after van Leeuwenhoek got his first glimpse of microbes, the “Golden Age of Microbiology” spawned a host of new discoveries between 1857 and 1914. Two famous microbiologists, Louis Pasteur and Robert Koch, were especially active in advancing our understanding of the unseen world of microbes (). Pasteur, a French chemist, showed that individual microbial strains had unique properties and demonstrated that fermentation is caused by microorganisms. He also invented pasteurization, a process used to kill microorganisms responsible for spoilage, and developed vaccines for the treatment of diseases, including rabies, in animals and humans. Koch, a German physician, was the first to demonstrate the connection between a single, isolated microbe and a known human disease. For example, he discovered the bacteria that cause anthrax (Bacillus anthracis), cholera (Vibrio cholera), and tuberculosis (Mycobacterium tuberculosis).S.M. Blevins and M.S. Bronze. “Robert Koch and the ‘Golden Age’ of Bacteriology.”  We will discuss these famous microbiologists, and others, in later chapters.
As microbiology has developed, it has allowed the broader discipline of biology to grow and flourish in previously unimagined ways. Much of what we know about human cells comes from our understanding of microbes, and many of the tools we use today to study cells and their genetics derive from work with microbes.

### Key Concepts and Summary

1. Microorganisms (or microbes) are living organisms that are generally too small to be seen without a microscope.
2. Throughout history, humans have used microbes to make fermented foods such as beer, bread, cheese, and wine.
3. Long before the invention of the microscope, some people theorized that infection and disease were spread by living things that were too small to be seen. They also correctly intuited certain principles regarding the spread of disease and immunity.
4. Antonie van Leeuwenhoek, using a microscope, was the first to actually describe observations of bacteria, in 1675.
5. During the Golden Age of Microbiology (1857–1914), microbiologists, including Louis Pasteur and Robert Koch, discovered many new connections between the fields of microbiology and medicine.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# An Invisible World

## A Systematic Approach

### Learning Objectives

By the end of this section, you will be able to:
1. Describe how microorganisms are classified and distinguished as unique species
2. Compare historical and current systems of taxonomy used to classify microorganisms
Once microbes became visible to humans with the help of microscopes, scientists began to realize their enormous diversity. Microorganisms vary in all sorts of ways, including their size, their appearance, and their rates of reproduction. To study this incredibly diverse new array of organisms, researchers needed a way to systematically organize them.

### The Science of Taxonomy

Taxonomy is the classification, description, identification, and naming of living organisms. Classification is the practice of organizing organisms into different groups based on their shared characteristics. The most famous early taxonomist was a Swedish botanist, zoologist, and physician named Carolus Linnaeus (1701–1778). In 1735, Linnaeus published Systema Naturae, an 11-page booklet in which he proposed the Linnaean taxonomy, a system of categorizing and naming organisms using a standard format so scientists could discuss organisms using consistent terminology. He continued to revise and add to the book, which grew into multiple volumes ().
In his taxonomy, Linnaeus divided the natural world into three kingdoms: animal, plant, and mineral (the mineral kingdom was later abandoned). Within the animal and plant kingdoms, he grouped organisms using a hierarchy of increasingly specific levels and sublevels based on their similarities. The names of the levels in Linnaeus’s original taxonomy were kingdom, class, order, family, genus (plural: genera), and species. Species was, and continues to be, the most specific and basic taxonomic unit.

### Evolving Trees of Life (Phylogenies)

With advances in technology, other scientists gradually made refinements to the Linnaean system and eventually created new systems for classifying organisms. In the 1800s, there was a growing interest in developing taxonomies that took into account the evolutionary relationships, or phylogenies, of all different species of organisms on earth. One way to depict these relationships is via a diagram called a phylogenetic tree (or tree of life). In these diagrams, groups of organisms are arranged by how closely related they are thought to be. In early phylogenetic trees, the relatedness of organisms was inferred by their visible similarities, such as the presence or absence of hair or the number of limbs. Now, the analysis is more complicated. Today, phylogenic analyses include genetic, biochemical, and embryological comparisons, as will be discussed later in this chapter.
Linnaeus’s tree of life contained just two main branches for all living things: the animal and plant kingdoms. In 1866, Ernst Haeckel, a German biologist, philosopher, and physician, proposed another kingdom, Protista, for unicellular organisms (). He later proposed a fourth kingdom, Monera, for unicellular organisms whose cells lack nuclei, like bacteria.
Nearly 100 years later, in 1969, American ecologist Robert Whittaker (1920–1980) proposed adding another kingdom—Fungi—in his tree of life. Whittaker’s tree also contained a level of categorization above the kingdom level—the empire or superkingdom level—to distinguish between organisms that have membrane-bound nuclei in their cells (eukaryotes) and those that do not (prokaryotes). Empire Prokaryota contained just the Kingdom Monera. The Empire Eukaryota contained the other four kingdoms: Fungi, Protista, Plantae, and Animalia. Whittaker’s five-kingdom tree was considered the standard phylogeny for many years.
 shows how the tree of life has changed over time. Note that viruses are not found in any of these trees. That is because they are not made up of cells and thus it is difficult to determine where they would fit into a tree of life.

### The Role of Genetics in Modern Taxonomy

Haeckel’s and Whittaker’s trees presented hypotheses about the phylogeny of different organisms based on readily observable characteristics. But the advent of molecular genetics in the late 20th century revealed other ways to organize phylogenetic trees. Genetic methods allow for a standardized way to compare all living organisms without relying on observable characteristics that can often be subjective. Modern taxonomy relies heavily on comparing the nucleic acids (deoxyribonucleic acid [DNA] or ribonucleic acid [RNA]) or proteins from different organisms. The more similar the nucleic acids and proteins are between two organisms, the more closely related they are considered to be.
In the 1970s, American microbiologist Carl Woese discovered what appeared to be a “living record” of the evolution of organisms. He and his collaborator George Fox created a genetics-based tree of life based on similarities and differences they observed in the gene sequences coding for small subunit ribosomal RNA (rRNA) of different organisms. In the process, they discovered that a certain type of bacteria, called archaebacteria (now known simply as archaea), were significantly different from other bacteria and eukaryotes in terms of their small subunit rRNA gene sequences. To accommodate this difference, they created a tree with three Domains above the level of Kingdom: Archaea, Bacteria, and Eukarya (). Analysis of small subunit rRNA gene sequences suggests archaea, bacteria, and eukaryotes all evolved from a common ancestral cell type. The tree is skewed to show a closer evolutionary relationship between Archaea and Eukarya than they have to Bacteria.
Scientists continue to use analysis of RNA, DNA, and proteins to determine how organisms are related. One interesting, and complicating, discovery is that of horizontal gene transfer—when a gene of one species is absorbed into another organism’s genome. Horizontal gene transfer is especially common in microorganisms and can make it difficult to determine how organisms are evolutionarily related. Consequently, some scientists now think in terms of “webs of life” rather than “trees of life.”

### Naming Microbes

In developing his taxonomy, Linnaeus used a system of binomial nomenclature, a two-word naming system for identifying organisms by genus and specific epithet. For example, modern humans are in the genus Homo and have the specific epithet name sapiens, so their scientific name in binomial nomenclature is Homo sapiens. In binomial nomenclature, the genus part of the name is always capitalized; it is followed by the specific epithet name, which is not capitalized. Both names are italicized. When referring to the species of humans, the binomial nomenclature would be Homo sapiens.
Taxonomic names in the 18th through 20th centuries were typically derived from Latin, since that was the common language used by scientists when taxonomic systems were first created. Today, newly discovered organisms can be given names derived from Latin, Greek, or English. Sometimes these names reflect some distinctive trait of the organism; in other cases, microorganisms are named after the scientists who discovered them. The archaeon Haloquadratum walsbyi is an example of both of these naming schemes. The genus, Haloquadratum, describes the microorganism’s saltwater habitat (halo is derived from the Greek word for “salt”) as well as the arrangement of its square cells, which are arranged in square clusters of four cells (quadratum is Latin for “foursquare”). The species, walsbyi, is named after Anthony Edward Walsby, the microbiologist who discovered Haloquadratum walsbyi in 1980. While it might seem easier to give an organism a common descriptive name—like a red-headed woodpecker—we can imagine how that could become problematic. What happens when another species of woodpecker with red head coloring is discovered? The systematic nomenclature scientists use eliminates this potential problem by assigning each organism a single, unique two-word name that is recognized by scientists all over the world.
In this text, we will typically abbreviate an organism’s genus and species after its first mention. The abbreviated form is simply the first initial of the genus, followed by a period and the full name of the species. For example, the bacterium Escherichia coli is shortened to E. coli in its abbreviated form. You will encounter this same convention in other scientific texts as well.

### Bergey’s Manuals

Whether in a tree or a web, microbes can be difficult to identify and classify. Without easily observable macroscopic features like feathers, feet, or fur, scientists must capture, grow, and devise ways to study their biochemical properties to differentiate and classify microbes. Despite these hurdles, a group of microbiologists created and updated a set of manuals for identifying and classifying microorganisms. First published in 1923 and since updated many times,  of Determinative Bacteriology and Bergey’s Manual of Systematic Bacteriology are the standard references for identifying and classifying different prokaryotes. (Appendix D of this textbook is partly based on Bergey’s manuals; it shows how the organisms that appear in this textbook are classified.) Because so many bacteria look identical, methods based on nonvisual characteristics must be used to identify them. For example, biochemical tests can be used to identify chemicals unique to certain species. Likewise, serological tests can be used to identify specific antibodies that will react against the proteins found in certain species. Ultimately, DNA and rRNA sequencing can be used both for identifying a particular bacterial species and for classifying newly discovered species.
1. Carolus Linnaeus developed a taxonomic system for categorizing organisms into related groups.
2. Binomial nomenclature assigns organisms Latinized scientific names with a genus and species designation.
3. A phylogenetic tree is a way of showing how different organisms are thought to be related to one another from an evolutionary standpoint.
4. The first phylogenetic tree contained kingdoms for plants and animals; Ernst Haeckel proposed adding kingdom for protists.
5. Robert Whittaker’s tree contained five kingdoms: Animalia, Plantae, Protista, Fungi, and Monera.
6. Carl Woese used small subunit ribosomal RNA to create a phylogenetic tree that groups organisms into three domains based on their genetic similarity.
7. Bergey’s manuals of determinative and systemic bacteriology are the standard references for identifying and classifying bacteria, respectively.
8. Bacteria can be identified through biochemical tests, DNA/RNA analysis, and serological testing methods.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# An Invisible World

## Types of Microorganisms

### Learning Objectives

By the end of this section, you will be able to:
1. List the various types of microorganisms and describe their defining characteristics
2. Give examples of different types of cellular and viral microorganisms and infectious agents
3. Describe the similarities and differences between archaea and bacteria
4. Provide an overview of the field of microbiology
Most microbes are unicellular and small enough that they require artificial magnification to be seen. However, there are some unicellular microbes that are visible to the naked eye, and some multicellular organisms that are microscopic. An object must measure about 100 micrometers (µm) to be visible without a microscope, but most microorganisms are many times smaller than that. For some perspective, consider that a typical animal cell measures roughly 10 µm across but is still microscopic. Bacterial cells are typically about 1 µm, and viruses can be 10 times smaller than bacteria (). See  for units of length used in microbiology.
Microorganisms differ from each other not only in size, but also in structure, habitat, metabolism, and many other characteristics. While we typically think of microorganisms as being unicellular, there are also many multicellular organisms that are too small to be seen without a microscope. Some microbes, such as viruses, are even acellular (not composed of cells).
Microorganisms are found in each of the three domains of life: Archaea, Bacteria, and Eukarya. Microbes within the domains Bacteria and Archaea are all prokaryotes (their cells lack a nucleus), whereas microbes in the domain Eukarya are eukaryotes (their cells have a nucleus). Some microorganisms, such as viruses, do not fall within any of the three domains of life. In this section, we will briefly introduce each of the broad groups of microbes. Later chapters will go into greater depth about the diverse species within each group.

### Prokaryotic Microorganisms

Bacteria are found in nearly every habitat on earth, including within and on humans. Most bacteria are harmless or helpful, but some are pathogens, causing disease in humans and other animals. Bacteria are prokaryotic because their genetic material (DNA) is not housed within a true nucleus. Most bacteria have cell walls that contain peptidoglycan.
Bacteria are often described in terms of their general shape. Common shapes include spherical (coccus), rod-shaped (bacillus), or curved (spirillum, spirochete, or vibrio).  shows examples of these shapes.
They have a wide range of metabolic capabilities and can grow in a variety of environments, using different combinations of nutrients. Some bacteria are photosynthetic, such as oxygenic cyanobacteria and anoxygenic green sulfur and green nonsulfur bacteria; these bacteria use energy derived from sunlight, and fix carbon dioxide for growth. Other types of bacteria are nonphotosynthetic, obtaining their energy from organic or inorganic compounds in their environment.
Archaea are also unicellular prokaryotic organisms. Archaea and bacteria have different evolutionary histories, as well as significant differences in genetics, metabolic pathways, and the composition of their cell walls and membranes. Unlike most bacteria, archaeal cell walls do not contain peptidoglycan, but their cell walls are often composed of a similar substance called pseudopeptidoglycan. Like bacteria, archaea are found in nearly every habitat on earth, even extreme environments that are very cold, very hot, very basic, or very acidic (). Some archaea live in the human body, but none have been shown to be human pathogens.

### Eukaryotic Microorganisms

The domain Eukarya contains all eukaryotes, including uni- or multicellular eukaryotes such as protists, fungi, plants, and animals. The major defining characteristic of eukaryotes is that their cells contain a nucleus.

### Protists

Protists are an informal grouping of eukaryotes that are not plants, animals, or fungi. Some algae are protists and others are bacteria; all protozoa are examples of protists.
Algae (singular: alga) are  mostly made up of protists that can be either unicellular or multicellular and vary widely in size, appearance, and habitat (). Algal protists are surrounded by cell walls made of cellulose, a type of carbohydrate. Algae are photosynthetic organisms that extract energy from the sun and release oxygen and carbohydrates into their environment. Cyanobacteria, a type of bacteria, is also considered an algae, but these organisms are bacterial prokaryotes and therefore have a peptidoglycan-based cell wall, unlike the cellulose-based cell wall of the algal protists. Because other organisms can use the waste products of all algae for energy, algae are important parts of many ecosystems. Many consumer products contain ingredients derived from algae, such as carrageenan or alginic acid, which are found in some brands of ice cream, salad dressing, beverages, lipstick, and toothpaste. A derivative of algae also plays a prominent role in the microbiology laboratory. Agar, a gel derived from algae, can be mixed with various nutrients and used to grow microorganisms in a Petri dish. Algae are also being developed as a possible source for biofuels.
Protozoa (singular: protozoan) are protists that make up the backbone of many food webs by providing nutrients for other organisms. Protozoa are very diverse. Some protozoa move with help from hair-like structures called cilia or whip-like structures called flagella. Others extend part of their cell membrane and cytoplasm to propel themselves forward. These cytoplasmic extensions are called pseudopods (“false feet”). Some protozoa are photosynthetic; others feed on organic material. Some are free-living, whereas others are parasitic, only able to survive by extracting nutrients from a host organism. Most protozoa are harmless, but some are pathogens that can cause disease in animals or humans ().

### Fungi

Fungi (singular: fungus) are also eukaryotes. Some multicellular fungi, such as mushrooms, resemble plants, but they are actually quite different. Fungi are not photosynthetic, and their cell walls are usually made out of chitin rather than cellulose.
Unicellular fungi—yeasts—are included within the study of microbiology. There are more than 1000 known species. Yeasts are found in many different environments, from the deep sea to the human navel. Some yeasts have beneficial uses, such as causing bread to rise and beverages to ferment; but yeasts can also cause food to spoil. Some even cause diseases, such as vaginal yeast infections and oral thrush ().
Other fungi of interest to microbiologists are multicellular organisms called molds. Molds are made up of long filaments that form visible colonies (). Molds are found in many different environments, from soil to rotting food to dank bathroom corners. Molds play a critical role in the decomposition of dead plants and animals. Some molds can cause allergies, and others produce disease-causing metabolites called mycotoxins. Molds have been used to make pharmaceuticals, including penicillin, which is one of the most commonly prescribed antibiotics, and cyclosporine, used to prevent organ rejection following a transplant.

### Helminths

Multicellular parasitic worms called helminths are not technically microorganisms, as most are large enough to see without a microscope. However, these worms fall within the field of microbiology because diseases caused by helminths involve microscopic eggs and larvae. One example of a helminth is the guinea worm, or Dracunculus medinensis, which causes dizziness, vomiting, diarrhea, and painful ulcers on the legs and feet when the worm works its way out of the skin (). Infection typically occurs after a person drinks water containing water fleas infected by guinea-worm larvae. In the mid-1980s, there were an estimated 3.5 million cases of guinea-worm disease, but the disease has been largely eradicated. In 2014, there were only 126 cases reported, thanks to the coordinated efforts of the World Health Organization (WHO) and other groups committed to improvements in drinking water sanitation.C. Greenaway “Dracunculiasis (Guinea Worm Disease).” World Health Organization. “Dracunculiasis (Guinea-Worm Disease).”

### Viruses

Viruses are acellular microorganisms, which means they are not composed of cells. Essentially, a virus consists of proteins and genetic material—either DNA or RNA, but never both—that are inert outside of a host organism. However, by incorporating themselves into a host cell, viruses are able to co-opt the host’s cellular mechanisms to multiply and infect other hosts.
Viruses can infect all types of cells, from human cells to the cells of other microorganisms. In humans, viruses are responsible for numerous diseases, from the common cold to deadly Ebola (). However, many viruses do not cause disease.

### Microbiology as a Field of Study

Microbiology is a broad term that encompasses the study of all different types of microorganisms. But in practice, microbiologists tend to specialize in one of several subfields. For example, bacteriology is the study of bacteria; mycology is the study of fungi; protozoology is the study of protozoa; parasitology is the study of helminths and other parasites; and virology is the study of viruses (). Immunology, the study of the immune system, is often included in the study of microbiology because host–pathogen interactions are central to our understanding of infectious disease processes. Microbiologists can also specialize in certain areas of microbiology, such as clinical microbiology, environmental microbiology, applied microbiology, or food microbiology.
In this textbook, we are primarily concerned with clinical applications of microbiology, but since the various subfields of microbiology are highly interrelated, we will often discuss applications that are not strictly clinical.
1. Microorganisms are very diverse and are found in all three domains of life: Archaea, Bacteria, and Eukarya.
2. Archaea and bacteria are classified as prokaryotes because they lack a cellular nucleus. Archaea differ from bacteria in evolutionary history, genetics, metabolic pathways, and cell wall and membrane composition.
3. Archaea inhabit nearly every environment on earth, but no archaea have been identified as human pathogens.
4. Eukaryotes studied in microbiology include algae, protozoa, fungi, and helminths.
5. Algae are plant-like organisms that can be either unicellular or multicellular, and derive energy via photosynthesis.
6. Protozoa are unicellular organisms with complex cell structures; most are motile.
7. Microscopic fungi include molds and yeasts.
8. Helminths are multicellular parasitic worms. They are included in the field of microbiology because their eggs and larvae are often microscopic.
9. Viruses are acellular microorganisms that require a host to reproduce.
10. The field of microbiology is extremely broad. Microbiologists typically specialize in one of many subfields, but all health professionals need a solid foundation in clinical microbiology.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# How We See the Invisible World

## Introduction

When we look at a rainbow, its colors span the full spectrum of light that the human eye can detect and differentiate. Each hue represents a different frequency of visible light, processed by our eyes and brains and rendered as red, orange, yellow, green, or one of the many other familiar colors that have always been a part of the human experience. But only recently have humans developed an understanding of the properties of light that allow us to see images in color.
Over the past several centuries, we have learned to manipulate light to peer into previously invisible worldsâ€”those too small or too far away to be seen by the naked eye. Through a microscope, we can examine microbial cells, using various techniques to manipulate color, size, and contrast in ways that help us identify species and diagnose disease.
 illustrates how we can apply the properties of light to visualize and magnify images; but these stunning micrographs are just two examples of the numerous types of images we are now able to produce with different microscopic technologies. This chapter explores how various types of microscopes manipulate light in order to provide a window into the world of microorganisms. By understanding how various kinds of microscopes work, we can produce highly detailed images of microbes that can be useful for both research and clinical applications.


# How We See the Invisible World

## The Properties of Light

### Learning Objectives

By the end of this section, you will be able to:
1. Identify and define the characteristics of electromagnetic radiation (EMR) used in microscopy
2. Explain how lenses are used in microscopy to manipulate visible and ultraviolet (UV) light
Visible light consists of electromagnetic waves that behave like other waves. Hence, many of the properties of light that are relevant to microscopy can be understood in terms of light’s behavior as a wave. An important property of light waves is the wavelength, or the distance between one peak of a wave and the next peak. The height of each peak (or depth of each trough) is called the amplitude. In contrast, the frequency of the wave is the rate of vibration of the wave, or the number of wavelengths within a specified time period ().

### Interactions of Light

Light waves interact with materials by being reflected, absorbed, or transmitted. Reflection occurs when a wave bounces off of a material. For example, a red piece of cloth may reflect red light to our eyes while absorbing other colors of light. Absorbance occurs when a material captures the energy of a light wave. In the case of glow-in-the-dark plastics, the energy from light can be absorbed and then later re-emitted as another form of phosphorescence. Transmission occurs when a wave travels through a material, like light through glass (the process of transmission is called transmittance). When a material allows a large proportion of light to be transmitted, it may do so because it is thinner, or more transparent (having more transparency and less opacity).  illustrates the difference between transparency and opacity.
Light waves can also interact with each other by interference, creating complex patterns of motion. Dropping two pebbles into a puddle causes the waves on the puddle’s surface to interact, creating complex interference patterns. Light waves can interact in the same way.
In addition to interfering with each other, light waves can also interact with small objects or openings by bending or scattering. This is called diffraction. Diffraction is larger when the object is smaller relative to the wavelength of the light (the distance between two consecutive peaks of a light wave). Often, when waves diffract in different directions around an obstacle or opening, they will interfere with each other.

### Lenses and Refraction

In the context of microscopy, refraction is perhaps the most important behavior exhibited by light waves. Refraction occurs when light waves change direction as they enter a new medium (). Different transparent materials transmit light at different speeds; thus, light can change speed when passing from one material to another. This change in speed usually also causes a change in direction (refraction), with the degree of change dependent on the angle of the incoming light.
The extent to which a material slows transmission speed relative to empty space is called the refractive index of that material. Large differences between the refractive indices of two materials will result in a large amount of refraction when light passes from one material to the other. For example, light moves much more slowly through water than through air, so light entering water from air can change direction greatly. We say that the water has a higher refractive index than air ().
When light crosses a boundary into a material with a higher refractive index, its direction turns to be closer to perpendicular to the boundary (i.e., more toward a normal to that boundary; see ). This is the principle behind lenses. We can think of a lens as an object with a curved boundary (or a collection of prisms) that collects all of the light that strikes it and refracts it so that it all meets at a single point called the image point (focus). A convex lens can be used to magnify because it can focus at closer range than the human eye, producing a larger image. Concave lenses and mirrors can also be used in microscopes to redirect the light path.  shows the focal point (the image point when light entering the lens is parallel) and the focal length (the distance to the focal point) for convex and concave lenses.
The human eye contains a lens that enables us to see images. This lens focuses the light reflecting off of objects in front of the eye onto the surface of the retina, which is like a screen in the back of the eye. Artificial lenses placed in front of the eye (contact lenses, glasses, or microscopic lenses) focus light before it is focused (again) by the lens of the eye, manipulating the image that ends up on the retina (e.g., by making it appear larger).
Images are commonly manipulated by controlling the distances between the object, the lens, and the screen, as well as the curvature of the lens. For example, for a given amount of curvature, when an object is closer to the lens, the focal points are farther from the lens. As a result, it is often necessary to manipulate these distances to create a focused image on a screen. Similarly, more curvature creates image points closer to the lens and a larger image when the image is in focus. This property is often described in terms of the focal distance, or distance to the focal point.

### Electromagnetic Spectrum and Color

Visible light is just one form of electromagnetic radiation (EMR), a type of energy that is all around us. Other forms of EMR include microwaves, X-rays, and radio waves, among others. The different types of EMR fall on the electromagnetic spectrum, which is defined in terms of wavelength and frequency. The spectrum of visible light occupies a relatively small range of frequencies between infrared and ultraviolet light ().
Whereas wavelength represents the distance between adjacent peaks of a light wave, frequency, in a simplified definition, represents the rate of oscillation. Waves with higher frequencies have shorter wavelengths and, therefore, have more oscillations per unit time than lower-frequency waves. Higher-frequency waves also contain more energy than lower-frequency waves. This energy is delivered as elementary particles called photons. Higher-frequency waves deliver more energetic photons than lower-frequency waves.
Photons with different energies interact differently with the retina. In the spectrum of visible light, each color corresponds to a particular frequency and wavelength ().The lowest frequency of visible light appears as the color red, whereas the highest appears as the color violet. When the retina receives visible light of many different frequencies, we perceive this as white light. However, white light can be separated into its component colors using refraction. If we pass white light through a prism, different colors will be refracted in different directions, creating a rainbow-like spectrum on a screen behind the prism. This separation of colors is called dispersion, and it occurs because, for a given material, the refractive index is different for different frequencies of light.
Certain materials can refract nonvisible forms of EMR and, in effect, transform them into visible light. Certain fluorescent dyes, for instance, absorb ultraviolet or blue light and then use the energy to emit photons of a different color, giving off light rather than simply vibrating. This occurs because the energy absorption causes electrons to jump to higher energy states, after which they then almost immediately fall back down to their ground states, emitting specific amounts of energy as photons. Not all of the energy is emitted in a given photon, so the emitted photons will be of lower energy and, thus, of lower frequency than the absorbed ones. Thus, a dye such as Texas red may be excited by blue light, but emit red light; or a dye such as fluorescein isothiocyanate (FITC) may absorb (invisible) high-energy ultraviolet light and emit green light (). In some materials, the photons may be emitted following a delay after absorption; in this case, the process is called phosphorescence. Glow-in-the-dark plastic works by using phosphorescent material.

### Magnification, Resolution, and Contrast

Microscopes magnify images and use the properties of light to create useful images of small objects. Magnification is defined as the ability of a lens to enlarge the image of an object when compared to the real object. For example, a magnification of 10⨯ means that the image appears 10 times the size of the object as viewed with the naked eye.
Greater magnification typically improves our ability to see details of small objects, but magnification alone is not sufficient to make the most useful images. It is often useful to enhance the resolution of objects: the ability to tell that two separate points or objects are separate. A low-resolution image appears fuzzy, whereas a high-resolution image appears sharp. Two factors affect resolution. The first is wavelength. Shorter wavelengths are able to resolve smaller objects; thus, an electron microscope has a much higher resolution than a light microscope, since it uses an electron beam with a very short wavelength, as opposed to the long-wavelength visible light used by a light microscope. The second factor that affects resolution is numerical aperture, which is a measure of a lens’s ability to gather light. The higher the numerical aperture, the better the resolution.
Even when a microscope has high resolution, it can be difficult to distinguish small structures in many specimens because microorganisms are relatively transparent. It is often necessary to increase contrast to detect different structures in a specimen. Various types of microscopes use different features of light or electrons to increase contrast—visible differences between the parts of a specimen (see Instruments of Microscopy). Additionally, dyes that bind to some structures but not others can be used to improve the contrast between images of relatively transparent objects (see Staining Microscopic Specimens).

### Key Concepts and Summary

1. Light waves interacting with materials may be reflected, absorbed, or transmitted, depending on the properties of the material.
2. Light waves can interact with each other (interference) or be distorted by interactions with small objects or openings (diffraction).
3. Refraction occurs when light waves change speed and direction as they pass from one medium to another. Differences in the refraction indices of two materials determine the magnitude of directional changes when light passes from one to the other.
4. A lens is a medium with a curved surface that refracts and focuses light to produce an image.
5. Visible light is part of the electromagnetic spectrum; light waves of different frequencies and wavelengths are distinguished as colors by the human eye.
6. A prism can separate the colors of white light (dispersion) because different frequencies of light have different refractive indices for a given material.
7. Fluorescent dyes and phosphorescent materials can effectively transform nonvisible electromagnetic radiation into visible light.
8. The power of a microscope can be described in terms of its magnification and resolution.
9. Resolution can be increased by shortening wavelength, increasing the numerical aperture of the lens, or using stains that enhance contrast.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# How We See the Invisible World

## Peering Into the Invisible World

### Learning Objectives

By the end of this section, you will be able to:
1. Describe historical developments and individual contributions that led to the invention and development of the microscope
2. Compare and contrast the features of simple and compound microscopes
Some of the fundamental characteristics and functions of microscopes can be understood in the context of the history of their use. Italian scholar Girolamo Fracastoro is regarded as the first person to formally postulate that disease was spread by tiny invisible seminaria, or “seeds of the contagion.” In his book  (1546), he proposed that these seeds could attach themselves to certain objects (which he called fomes [cloth]) that supported their transfer from person to person. However, since the technology for seeing such tiny objects did not yet exist, the existence of the seminaria remained hypothetical for a little over a century—an invisible world waiting to be revealed.

### Early Microscopes

Antonie van Leeuwenhoek, sometimes hailed as “the Father of Microbiology,” is typically credited as the first person to have created microscopes powerful enough to view microbes (). Born in the city of Delft in the Dutch Republic, van Leeuwenhoek began his career selling fabrics. However, he later became interested in lens making (perhaps to look at threads) and his innovative techniques produced microscopes that allowed him to observe microorganisms as no one had before. In 1674, he described his observations of single-celled organisms, whose existence was previously unknown, in a series of letters to the Royal Society of London. His report was initially met with skepticism, but his claims were soon verified and he became something of a celebrity in the scientific community.
While van Leeuwenhoek is credited with the discovery of microorganisms, others before him had contributed to the development of the microscope. These included eyeglass makers in the Netherlands in the late 1500s, as well as the Italian astronomer Galileo Galilei, who used a compound microscope to examine insect parts (). Whereas van Leeuwenhoek used a simple microscope, in which light is passed through just one lens, Galileo’s compound microscope was more sophisticated, passing light through two sets of lenses.
Van Leeuwenhoek’s contemporary, the Englishman Robert Hooke (1635–1703), also made important contributions to microscopy, publishing in his book  (1665) many observations using compound microscopes. Viewing a thin sample of cork through his microscope, he was the first to observe the structures that we now know as cells (). Hooke described these structures as resembling “Honey-comb,” and as “small Boxes or Bladders of Air,” noting that each “Cavern, Bubble, or Cell” is distinct from the others (in Latin, “cell” literally means “small room”). They likely appeared to Hooke to be filled with air because the cork cells were dead, with only the rigid cell walls providing the structure.

### Key Concepts and Summary

1. Antonie van Leeuwenhoek is credited with the first observation of microbes, including protists and bacteria, with simple microscopes that he made.
2. Robert Hooke was the first to describe what we now call cells.
3. Simple microscopes have a single lens, while compound microscopes have multiple lenses.

### Multiple Choice

### Fill in the Blank

### Short Answer


# How We See the Invisible World

## Instruments of Microscopy

### Learning Objectives

By the end of this section, you will be able to:
1. Identify and describe the parts of a brightfield microscope
2. Calculate total magnification for a compound microscope
3. Describe the distinguishing features and typical uses for various types of light microscopes, electron microscopes, and scanning probe microscopes
The early pioneers of microscopy opened a window into the invisible world of microorganisms. But microscopy continued to advance in the centuries that followed. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy, which uses an ultraviolet light source, and electron microscopy, which uses short-wavelength electron beams. These advances led to major improvements in magnification, resolution, and contrast. By comparison, the relatively rudimentary microscopes of van Leeuwenhoek and his contemporaries were far less powerful than even the most basic microscopes in use today. In this section, we will survey the broad range of modern microscopic technology and common applications for each type of microscope.

### Light Microscopy

Many types of microscopes fall under the category of light microscopes, which use light to visualize images. Examples of light microscopes include brightfield microscopes, darkfield microscopes, phase-contrast microscopes, differential interference contrast microscopes, fluorescence microscopes, confocal scanning laser microscopes, and two-photon microscopes. These various types of light microscopes can be used to complement each other in diagnostics and research.

### Brightfield Microscopes

The brightfield microscope, perhaps the most commonly used type of microscope, is a compound microscope with two or more lenses that produce a dark image on a bright background. Some brightfield microscopes are monocular (having a single eyepiece), though most newer brightfield microscopes are binocular (having two eyepieces), like the one shown in ; in either case, each eyepiece contains a lens called an ocular lens. The ocular lenses typically magnify images 10 times (10⨯). At the other end of the body tube are a set of objective lenses on a rotating nosepiece. The magnification of these objective lenses typically ranges from 4⨯ to 100⨯, with the magnification for each lens designated on the metal casing of the lens. The ocular and objective lenses work together to create a magnified image. The total magnification is the product of the ocular magnification times the objective magnification:
For example, if a 40⨯ objective lens is selected and the ocular lens is 10⨯, the total magnification would be
The item being viewed is called a specimen. The specimen is placed on a glass slide, which is then clipped into place on the stage (a platform) of the microscope. Once the slide is secured, the specimen on the slide is positioned over the light using the x-y mechanical stage knobs. These knobs move the slide on the surface of the stage, but do not raise or lower the stage. Once the specimen is centered over the light, the stage position can be raised or lowered to focus the image. The coarse focusing knob is used for large-scale movements with 4⨯ and 10⨯ objective lenses; the fine focusing knob is used for small-scale movements, especially with 40⨯ or 100⨯ objective lenses.
When images are magnified, they become dimmer because there is less light per unit area of image. Highly magnified images produced by microscopes, therefore, require intense lighting. In a brightfield microscope, this light is provided by an illuminator, which is typically a high-intensity bulb below the stage. Light from the illuminator passes up through condenser lens (located below the stage), which focuses all of the light rays on the specimen to maximize illumination. The position of the condenser can be optimized using the attached condenser focus knob; once the optimal distance is established, the condenser should not be moved to adjust the brightness. If less-than-maximal light levels are needed, the amount of light striking the specimen can be easily adjusted by opening or closing a diaphragm between the condenser and the specimen. In some cases, brightness can also be adjusted using the rheostat, a dimmer switch that controls the intensity of the illuminator.
A brightfield microscope creates an image by directing light from the illuminator at the specimen; this light is differentially transmitted, absorbed, reflected, or refracted by different structures. Different colors can behave differently as they interact with chromophores (pigments that absorb and reflect particular wavelengths of light) in parts of the specimen. Often, chromophores are artificially added to the specimen using stains, which serve to increase contrast and resolution. In general, structures in the specimen will appear darker, to various extents, than the bright background, creating maximally sharp images at magnifications up to about 1000⨯. Further magnification would create a larger image, but without increased resolution. This allows us to see objects as small as bacteria, which are visible at about 400⨯ or so, but not smaller objects such as viruses.
At very high magnifications, resolution may be compromised when light passes through the small amount of air between the specimen and the lens. This is due to the large difference between the refractive indices of air and glass; the air scatters the light rays before they can be focused by the lens. To solve this problem, a drop of oil can be used to fill the space between the specimen and an oil immersion lens, a special lens designed to be used with immersion oils. Since the oil has a refractive index very similar to that of glass, it increases the maximum angle at which light leaving the specimen can strike the lens. This increases the light collected and, thus, the resolution of the image (). A variety of oils can be used for different types of light.

### Darkfield Microscopy

A darkfield microscope is a brightfield microscope that has a small but significant modification to the condenser. A small, opaque disk (about 1 cm in diameter) is placed between the illuminator and the condenser lens. This opaque light stop, as the disk is called, blocks most of the light from the illuminator as it passes through the condenser on its way to the objective lens, producing a hollow cone of light that is focused on the specimen. The only light that reaches the objective is light that has been refracted or reflected by structures in the specimen. The resulting image typically shows bright objects on a dark background ().
Darkfield microscopy can often create high-contrast, high-resolution images of specimens without the use of stains, which is particularly useful for viewing live specimens that might be killed or otherwise compromised by the stains. For example, thin spirochetes like , the causative agent of syphilis, can be best viewed using a darkfield microscope ().

### Phase-Contrast Microscopes

Phase-contrast microscopes use refraction and interference caused by structures in a specimen to create high-contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. To create altered wavelength paths, an annular stop is used in the condenser. The annular stop produces a hollow cone of light that is focused on the specimen before reaching the objective lens. The objective contains a phase plate containing a phase ring. As a result, light traveling directly from the illuminator passes through the phase ring while light refracted or reflected by the specimen passes through the plate. This causes waves traveling through the ring to be about one-half of a wavelength out of phase with those passing through the plate. Because waves have peaks and troughs, they can add together (if in phase together) or cancel each other out (if out of phase). When the wavelengths are out of phase, wave troughs will cancel out wave peaks, which is called destructive interference. Structures that refract light then appear dark against a bright background of only unrefracted light. More generally, structures that differ in features such as refractive index will differ in levels of darkness ().
Because it increases contrast without requiring stains, phase-contrast microscopy is often used to observe live specimens. Certain structures, such as organelles in eukaryotic cells and endospores in prokaryotic cells, are especially well visualized with phase-contrast microscopy ().

### Differential Interference Contrast Microscopes

Differential interference contrast (DIC) microscopes (also known as Nomarski optics) are similar to phase-contrast microscopes in that they use interference patterns to enhance contrast between different features of a specimen. In a DIC microscope, two beams of light are created in which the direction of wave movement (polarization) differs. Once the beams pass through either the specimen or specimen-free space, they are recombined and effects of the specimens cause differences in the interference patterns generated by the combining of the beams. This results in high-contrast images of living organisms with a three-dimensional appearance. These microscopes are especially useful in distinguishing structures within live, unstained specimens. ()

### Fluorescence Microscopes

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which are capable of absorbing energy from a light source and then emitting this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) as well as fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI) and acridine orange.
The microscope transmits an excitation light, generally a form of EMR with a short wavelength, such as ultraviolet or blue light, toward the specimen; the chromophores absorb the excitation light and emit visible light with longer wavelengths. The excitation light is then filtered out (in part because ultraviolet light is harmful to the eyes) so that only visible light passes through the ocular lens. This produces an image of the specimen in bright colors against a dark background.
Fluorescence microscopes are especially useful in clinical microbiology. They can be used to identify pathogens, to find particular species within an environment, or to find the locations of particular molecules and structures within a cell. Approaches have also been developed to distinguish living from dead cells using fluorescence microscopy based upon whether they take up particular fluorochromes. Sometimes, multiple fluorochromes are used on the same specimen to show different structures or features.
One of the most important applications of fluorescence microscopy is a technique called immunofluorescence, which is used to identify certain disease-causing microbes by observing whether antibodies bind to them. (Antibodies are protein molecules produced by the immune system that attach to specific pathogens to kill or inhibit them.) There are two approaches to this technique: direct immunofluorescence assay (DFA) and indirect immunofluorescence assay (IFA). In DFA, specific antibodies (e.g., those that the target the rabies virus) are stained with a fluorochrome. If the specimen contains the targeted pathogen, one can observe the antibodies binding to the pathogen under the fluorescent microscope. This is called a primary antibody stain because the stained antibodies attach directly to the pathogen.
In IFA, secondary antibodies are stained with a fluorochrome rather than primary antibodies. Secondary antibodies do not attach directly to the pathogen, but they do bind to primary antibodies. When the unstained primary antibodies bind to the pathogen, the fluorescent secondary antibodies can be observed binding to the primary antibodies. Thus, the secondary antibodies are attached indirectly to the pathogen. Since multiple secondary antibodies can often attach to a primary antibody, IFA increases the number of fluorescent antibodies attached to the specimen, making it easier visualize features in the specimen ().

### Confocal Microscopes

Whereas other forms of light microscopy create an image that is maximally focused at a single distance from the observer (the depth, or z-plane), a confocal microscope uses a laser to scan multiple z-planes successively. This produces numerous two-dimensional, high-resolution images at various depths, which can be constructed into a three-dimensional image by a computer. As with fluorescence microscopes, fluorescent stains are generally used to increase contrast and resolution. Image clarity is further enhanced by a narrow aperture that eliminates any light that is not from the z-plane. Confocal microscopes are thus very useful for examining thick specimens such as biofilms, which can be examined alive and unfixed ().

### Two-Photon Microscopes

While the original fluorescent and confocal microscopes allowed better visualization of unique features in specimens, there were still problems that prevented optimum visualization. The effective sensitivity of fluorescence microscopy when viewing thick specimens was generally limited by out-of-focus flare, which resulted in poor resolution. This limitation was greatly reduced in the confocal microscope through the use of a confocal pinhole to reject out-of-focus background fluorescence with thin (<1 μm), unblurred optical sections. However, even the confocal microscopes lacked the resolution needed for viewing thick tissue samples. These problems were resolved with the development of the two-photon microscope, which uses a scanning technique, fluorochromes, and long-wavelength light (such as infrared) to visualize specimens. The low energy associated with the long-wavelength light means that two photons must strike a location at the same time to excite the fluorochrome. The low energy of the excitation light is less damaging to cells, and the long wavelength of the excitation light more easily penetrates deep into thick specimens. This makes the two-photon microscope useful for examining living cells within intact tissues—brain slices, embryos, whole organs, and even entire animals.
Currently, use of two-photon microscopes is limited to advanced clinical and research laboratories because of the high costs of the instruments. A single two-photon microscope typically costs between $300,000 and $500,000, and the lasers used to excite the dyes used on specimens are also very expensive. However, as technology improves, two-photon microscopes may become more readily available in clinical settings.

### Electron Microscopy

The maximum theoretical resolution of images created by light microscopes is ultimately limited by the wavelengths of visible light. Most light microscopes can only magnify 1000⨯, and a few can magnify up to 1500⨯, but this does not begin to approach the magnifying power of an electron microscope (EM), which uses short-wavelength electron beams rather than light to increase magnification and resolution.
Electrons, like electromagnetic radiation, can behave as waves, but with wavelengths of 0.005 nm, they can produce much better resolution than visible light. An EM can produce a sharp image that is magnified up to 100,000⨯. Thus, EMs can resolve subcellular structures as well as some molecular structures (e.g., single strands of DNA); however, electron microscopy cannot be used on living material because of the methods needed to prepare the specimens.
There are two basic types of EM: the transmission electron microscope (TEM) and the scanning electron microscope (SEM) (). The TEM is somewhat analogous to the brightfield light microscope in terms of the way it functions. However, it uses an electron beam from above the specimen that is focused using a magnetic lens (rather than a glass lens) and projected through the specimen onto a detector. Electrons pass through the specimen, and then the detector captures the image ().
For electrons to pass through the specimen in a TEM, the specimen must be extremely thin (20–100 nm thick). The image is produced because of varying opacity in various parts of the specimen. This opacity can be enhanced by staining the specimen with materials such as heavy metals, which are electron dense. TEM requires that the beam and specimen be in a vacuum and that the specimen be very thin and dehydrated. The specific steps needed to prepare a specimen for observation under an EM are discussed in detail in the next section.
SEMs form images of surfaces of specimens, usually from
electrons that are knocked off of specimens by a beam of electrons. This can create highly detailed images with a three-dimensional appearance that are displayed on a monitor (). Typically, specimens are dried and prepared with fixatives that reduce artifacts, such as shriveling, that can be produced by drying, before being sputter-coated with a thin layer of metal such as gold. Whereas transmission electron microscopy requires very thin sections and allows one to see internal structures such as organelles and the interior of membranes, scanning electron microscopy can be used to view the surfaces of larger objects (such as a pollen grain) as well as the surfaces of very small samples (). Some EMs can magnify an image up to 2,000,000⨯.“JEM-ARM200F Transmission Electron Microscope,”

### Scanning Probe Microscopy

A scanning probe microscope does not use light or electrons, but rather very sharp probes that are passed over the surface of the specimen and interact with it directly. This produces information that can be assembled into images with magnifications up to 100,000,000⨯. Such large magnifications can be used to observe individual atoms on surfaces. To date, these techniques have been used primarily for research rather than for diagnostics.
There are two types of scanning probe microscope: the scanning tunneling microscope (STM) and the atomic force microscope (AFM). An STM uses a probe that is passed just above the specimen as a constant voltage bias creates the potential for an electric current between the probe and the specimen. This current occurs via quantum tunneling of electrons between the probe and the specimen, and the intensity of the current is dependent upon the distance between the probe and the specimen. The probe is moved horizontally above the surface and the intensity of the current is measured. Scanning tunneling microscopy can effectively map the structure of surfaces at a resolution at which individual atoms can be detected.
Similar to an STM, AFMs have a thin probe that is passed just above the specimen. However, rather than measuring variations in the current at a constant height above the specimen, an AFM establishes a constant current and measures variations in the height of the probe tip as it passes over the specimen. As the probe tip is passed over the specimen, forces between the atoms (van der Waals forces, capillary forces, chemical bonding, electrostatic forces, and others) cause it to move up and down. Deflection of the probe tip is determined and measured using Hooke’s law of elasticity, and this information is used to construct images of the surface of the specimen with resolution at the atomic level ().
, , and  summarize the microscopy techniques for light microscopes, electron microscopes, and scanning probe microscopes, respectively.

### Key Concepts and Summary

1. Numerous types of microscopes use various technologies to generate micrographs. Most are useful for a particular type of specimen or application.
2. Light microscopy uses lenses to focus light on a specimen to produce an image. Commonly used light microscopes include brightfield, darkfield, phase-contrast, differential interference contrast, fluorescence, confocal, and two-photon microscopes.
3. Electron microscopy focuses electrons on the specimen using magnets, producing much greater magnification than light microscopy. The transmission electron microscope (TEM) and scanning electron microscope (SEM) are two common forms.
4. Scanning probe microscopy produces images of even greater magnification by measuring feedback from sharp probes that interact with the specimen. Probe microscopes include the scanning tunneling microscope (STM) and the atomic force microscope (AFM).

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# How We See the Invisible World

## Staining Microscopic Specimens

### Learning Objectives

By the end of this section, you will be able to:
1. Differentiate between simple and differential stains
2. Describe the unique features of commonly used stains
3. Explain the procedures and name clinical applications for Gram, endospore, acid-fast, negative capsule, and flagella staining
In their natural state, most of the cells and microorganisms that we observe under the microscope lack color and contrast. This makes it difficult, if not impossible, to detect important cellular structures and their distinguishing characteristics without artificially treating specimens. We have already alluded to certain techniques involving stains and fluorescent dyes, and in this section we will discuss specific techniques for sample preparation in greater detail. Indeed, numerous methods have been developed to identify specific microbes, cellular structures, DNA sequences, or indicators of infection in tissue samples, under the microscope. Here, we will focus on the most clinically relevant techniques.

### Preparing Specimens for Light Microscopy

In clinical settings, light microscopes are the most commonly used microscopes. There are two basic types of preparation used to view specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation is the wet mount, in which the specimen is placed on the slide in a drop of liquid. Some specimens, such as a drop of urine, are already in a liquid form and can be deposited on the slide using a dropper. Solid specimens, such as a skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid used is simply water, but often stains are added to enhance contrast. Once the liquid has been added to the slide, a coverslip is placed on top and the specimen is ready for examination under the microscope.
The second method of preparing specimens for light microscopy is fixation. The “fixing” of a sample refers to the process of attaching cells to a slide. Fixation is often achieved either by heating (heat fixing) or chemically treating the specimen. In addition to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation.
To heat-fix a sample, a thin layer of the specimen is spread on the slide (called a smear), and the slide is then briefly heated over a heat source (). Chemical fixatives are often preferable to heat for tissue specimens. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde can denature proteins, stop biochemical reactions, and stabilize cell structures in tissue samples ().
In addition to fixation, staining is almost always applied to color certain features of a specimen before examining it under a light microscope. Stains, or dyes, contain salts made up of a positive ion and a negative ion. Depending on the type of dye, the positive or the negative ion may be the chromophore (the colored ion); the other, uncolored ion is called the counterion. If the chromophore is the positively charged ion, the stain is classified as a basic dye; if the negative ion is the chromophore, the stain is considered an acidic dye.
Dyes are selected for staining based on the chemical properties of the dye and the specimen being observed, which determine how the dye will interact with the specimen. In most cases, it is preferable to use a positive stain, a dye that will be absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out against the background. However, there are scenarios in which it is advantageous to use a negative stain, which is absorbed by the background but not by the cells or organisms in the specimen. Negative staining produces an outline or silhouette of the organisms against a colorful background ().
Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. Thus, commonly used basic dyes such as basic fuchsin, crystal violet, malachite green, methylene blue, and safranin typically serve as positive stains. On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains. Commonly used acidic dyes include acid fuchsin, eosin, and rose bengal.  provides more detail.
Some staining techniques involve the application of only one dye to the sample; others require more than one dye. In simple staining, a single dye is used to emphasize particular structures in the specimen. A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism. In contrast, differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms in a differentially stained sample may appear to be different colors. Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining.  provides more detail on these differential staining techniques.

### Gram Staining

The Gram stain procedure is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques. The steps of the Gram stain procedure are listed below and illustrated in .
1. First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the cells a purple color.
2. Next, Gram’s iodine, a mordant, is added. A mordant is a substance used to set or stabilize stains or dyes; in this case, Gram’s iodine acts like a trapping agent that complexes with the crystal violet, making the crystal violet–iodine complex clump and stay contained in thick layers of peptidoglycan in the cell walls.
3. Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. Cells that have thick peptidoglycan layers in their cell walls are much less affected by the decolorizing agent; they generally retain the crystal violet dye and remain purple. However, the decolorizing agent more easily washes the dye out of cells with thinner peptidoglycan layers, making them again colorless.
4. Finally, a secondary counterstain, usually safranin, is added. This stains the decolorized cells pink and is less noticeable in the cells that still contain the crystal violet dye.
The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative (). However, there are several important considerations in interpreting the results of a Gram stain. First, older bacterial cells may have damage to their cell walls that causes them to appear gram-negative even if the species is gram-positive. Thus, it is best to use fresh bacterial cultures for Gram staining. Second, errors such as leaving on decolorizer too long can affect the results. In some cases, most cells will appear gram-positive while a few appear gram-negative (as in ). This suggests damage to the individual cells or that decolorizer was left on for too long; the cells should still be classified as gram-positive if they are all the same species rather than a mixed culture.
Besides their differing interactions with dyes and decolorizing agents, the chemical differences between gram-positive and gram-negative cells have other implications with clinical relevance. For example, Gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. Gram-negative bacteria tend to be more resistant to certain antibiotics than gram-positive bacteria. We will discuss this and other applications of Gram staining in more detail in later chapters.

### Acid-Fast Stains

Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool. An acid-fast stain is able to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls, and those that do not. Two different methods for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun technique. Both use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin even after a decolorizing agent (an acid-alcohol solution) is applied. A secondary counterstain, methylene blue, is then applied, which renders non–acid-fast cells blue.
The fundamental difference between the two carbolfuchsin-based methods is whether heat is used during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat. Both techniques are important diagnostic tools because a number of specific diseases are caused by acid-fast bacteria (AFB). If AFB are present in a tissue sample, their red or pink color can be seen clearly against the blue background of the surrounding tissue cells ().

### Capsule Staining

Certain bacteria and yeasts have a protective outer structure called a capsule. Since the presence of a capsule is directly related to a microbe’s virulence (its ability to cause disease), the ability to determine whether cells in a sample have capsules is an important diagnostic tool. Capsules do not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is typically used for capsule staining. The dye stains the background but does not penetrate the capsules, which appear like halos around the borders of the cell. The specimen does not need to be heat-fixed prior to negative staining.
One common negative staining technique for identifying encapsulated yeast and bacteria is to add a few drops of India ink or nigrosin to a specimen. Other capsular stains can also be used to negatively stain encapsulated cells (). Alternatively, positive and negative staining techniques can be combined to visualize capsules: The positive stain colors the body of the cell, and the negative stain colors the background but not the capsule, leaving halo around each cell.

### Endospore Staining

Endospores are structures produced within certain bacterial cells that allow them to survive harsh conditions. Gram staining alone cannot be used to visualize endospores, which appear clear when Gram-stained cells are viewed. Endospore staining uses two stains to differentiate endospores from the rest of the cell. The Schaeffer-Fulton method (the most commonly used endospore-staining technique) uses heat to push the primary stain (malachite green) into the endospore. Washing with water decolorizes the cell, but the endospore retains the green stain. The cell is then counterstained pink with safranin. The resulting image reveals the shape and location of endospores, if they are present. The green endospores will appear either within the pink vegetative cells or as separate from the pink cells altogether. If no endospores are present, then only the pink vegetative cells will be visible ().
Endospore-staining techniques are important for identifying Bacillus and Clostridium, two genera of endospore-producing bacteria that contain clinically significant species. Among others,  (which causes anthrax) has been of particular interest because of concern that its spores could be used as a bioterrorism agent.  is a particularly important species responsible for the typically hospital-acquired infection known as “C. diff.”

### Flagella Staining

Flagella (singular: flagellum) are tail-like cellular structures used for locomotion by some bacteria, archaea, and eukaryotes. Because they are so thin, flagella typically cannot be seen under a light microscope without a specialized flagella staining technique. Flagella staining thickens the flagella by first applying mordant (generally tannic acid, but sometimes potassium alum), which coats the flagella; then the specimen is stained with pararosaniline (most commonly) or basic fuchsin ().
Though flagella staining is uncommon in clinical settings, the technique is commonly used by microbiologists, since the location and number of flagella can be useful in classifying and identifying bacteria in a sample. When using this technique, it is important to handle the specimen with great care; flagella are delicate structures that can easily be damaged or pulled off, compromising attempts to accurately locate and count the number of flagella.

### Preparing Specimens for Electron Microscopy

Samples to be analyzed using a TEM must have very thin sections. But cells are too soft to cut thinly, even with diamond knives. To cut cells without damage, the cells must be embedded in plastic resin and then dehydrated through a series of soaks in ethanol solutions (50%, 60%, 70%, and so on). The ethanol replaces the water in the cells, and the resin dissolves in ethanol and enters the cell, where it solidifies. Next, thin sections are cut using a specialized device called an ultramicrotome (). Finally, samples are fixed to fine copper wire or carbon-fiber grids and stained—not with colored dyes, but with substances like uranyl acetate or osmium tetroxide, which contain electron-dense heavy metal atoms.
When samples are prepared for viewing using an SEM, they must also be dehydrated using an ethanol series. However, they must be even drier than is necessary for a TEM. Critical point drying with inert liquid carbon dioxide under pressure is used to displace the water from the specimen. After drying, the specimens are sputter-coated with metal by knocking atoms off of a palladium target, with energetic particles. Sputter-coating prevents specimens from becoming charged by the SEM’s electron beam.

### Preparation and Staining for Other Microscopes

Samples for fluorescence and confocal microscopy are prepared similarly to samples for light microscopy, except that the dyes are fluorochromes. Stains are often diluted in liquid before applying to the slide. Some dyes attach to an antibody to stain specific proteins on specific types of cells (immunofluorescence); others may attach to DNA molecules in a process called fluorescence in situ hybridization (FISH), causing cells to be stained based on whether they have a specific DNA sequence.
Sample preparation for two-photon microscopy is similar to fluorescence microscopy, except for the use of infrared dyes. Specimens for STM need to be on a very clean and atomically smooth surface. They are often mica coated with Au(111). Toluene vapor is a common fixative.

### Key Concepts and Summary

1. Samples must be properly prepared for microscopy. This may involve staining, fixation, and/or cutting thin sections.
2. A variety of staining techniques can be used with light microscopy, including Gram staining, acid-fast staining, capsule staining, endospore staining, and flagella staining.
3. Samples for TEM require very thin sections, whereas samples for SEM require sputter-coating.
4. Preparation for fluorescence microscopy is similar to that for light microscopy, except that fluorochromes are used.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# The Cell

## Introduction

Life takes many forms, from giant redwood trees towering hundreds of feet in the air to the tiniest known microbes, which measure only a few billionths of a meter. Humans have long pondered life’s origins and debated the defining characteristics of life, but our understanding of these concepts has changed radically since the invention of the microscope. In the 17th century, observations of microscopic life led to the development of the cell theory: the idea that the fundamental unit of life is the cell, that all organisms contain at least one cell, and that cells only come from other cells.
Despite sharing certain characteristics, cells may vary significantly. The two main types of cells are prokaryotic cells (lacking a nucleus) and eukaryotic cells (containing a well-organized, membrane-bound nucleus). Each type of cell exhibits remarkable variety in structure, function, and metabolic activity (). This chapter will focus on the historical discoveries that have shaped our current understanding of microbes, including their origins and their role in human disease. We will then explore the distinguishing structures found in prokaryotic and eukaryotic cells.


# The Cell

## Spontaneous Generation

### Learning Objectives

By the end of this section, you will be able to:
1. Explain the theory of spontaneous generation and why people once accepted it as an explanation for the existence of certain types of organisms
2. Explain how certain individuals (van Helmont, Redi, Needham, Spallanzani, and Pasteur) tried to prove or disprove spontaneous generation
Humans have been asking for millennia: Where does new life come from? Religion, philosophy, and science have all wrestled with this question. One of the oldest explanations was the theory of spontaneous generation, which can be traced back to the ancient Greeks and was widely accepted through the Middle Ages.

### The Theory of Spontaneous Generation

The Greek philosopher Aristotle (384–322 BC) was one of the earliest recorded scholars to articulate the theory of spontaneous generation, the notion that life can arise from nonliving matter. Aristotle proposed that life arose from nonliving material if the material contained pneuma (“spirit” or “breath”). As evidence, he noted several instances of the appearance of animals from environments previously devoid of such animals, such as the seemingly sudden appearance of fish in a new puddle of water.K. Zwier. “Aristotle on Spontaneous Generation.” http://www.sju.edu/int/academics/cas/resources/gppc/pdf/Karen%20R.%20Zwier.pdf
This theory persisted into the 17th century, when scientists undertook additional experimentation to support or disprove it. By this time, the proponents of the theory cited how frogs simply seem to appear along the muddy banks of the Nile River in Egypt during the annual flooding. Others observed that mice simply appeared among grain stored in barns with thatched roofs. When the roof leaked and the grain molded, mice appeared. Jan Baptista van Helmont, a 17th century Flemish scientist, proposed that mice could arise from rags and wheat kernels left in an open container for 3 weeks. In reality, such habitats provided ideal food sources and shelter for mouse populations to flourish.
However, one of van Helmont’s contemporaries, Italian physician Francesco Redi (1626–1697), performed an experiment in 1668 that was one of the first to refute the idea that maggots (the larvae of flies) spontaneously generate on meat left out in the open air. He predicted that preventing flies from having direct contact with the meat would also prevent the appearance of maggots. Redi left meat in each of six containers (). Two were open to the air, two were covered with gauze, and two were tightly sealed. His hypothesis was supported when maggots developed in the uncovered jars, but no maggots appeared in either the gauze-covered or the tightly sealed jars. He concluded that maggots could only form when flies were allowed to lay eggs in the meat, and that the maggots were the offspring of flies, not the product of spontaneous generation.
In 1745, John Needham (1713–1781) published a report of his own experiments, in which he briefly boiled broth infused with plant or animal matter, hoping to kill all preexisting microbes.E. Capanna. “Lazzaro Spallanzani: At the Roots of Modern Biology.”  He then sealed the flasks. After a few days, Needham observed that the broth had become cloudy and a single drop contained numerous microscopic creatures. He argued that the new microbes must have arisen spontaneously. In reality, however, he likely did not boil the broth enough to kill all preexisting microbes.
Lazzaro Spallanzani (1729–1799) did not agree with Needham’s conclusions, however, and performed hundreds of carefully executed experiments using heated broth.R. Mancini, M. Nigro, G. Ippolito. “Lazzaro Spallanzani and His Refutation of the Theory of Spontaneous Generation.”  As in Needham’s experiment, broth in sealed jars and unsealed jars was infused with plant and animal matter. Spallanzani’s results contradicted the findings of Needham: Heated but sealed flasks remained clear, without any signs of spontaneous growth, unless the flasks were subsequently opened to the air. This suggested that microbes were introduced into these flasks from the air. In response to Spallanzani’s findings, Needham argued that life originates from a “life force” that was destroyed during Spallanzani’s extended boiling. Any subsequent sealing of the flasks then prevented new life force from entering and causing spontaneous generation ().

### Disproving Spontaneous Generation

The debate over spontaneous generation continued well into the 19th century, with scientists serving as proponents of both sides. To settle the debate, the Paris Academy of Sciences offered a prize for resolution of the problem. Louis Pasteur, a prominent French chemist who had been studying microbial fermentation and the causes of wine spoilage, accepted the challenge. In 1858, Pasteur filtered air through a gun-cotton filter and, upon microscopic examination of the cotton, found it full of microorganisms, suggesting that the exposure of a broth to air was not introducing a “life force” to the broth but rather airborne microorganisms.
Later, Pasteur made a series of flasks with long, twisted necks (“swan-neck” flasks), in which he boiled broth to sterilize it (). His design allowed air inside the flasks to be exchanged with air from the outside, but prevented the introduction of any airborne microorganisms, which would get caught in the twists and bends of the flasks’ necks. If a life force besides the airborne microorganisms were responsible for microbial growth within the sterilized flasks, it would have access to the broth, whereas the microorganisms would not. He correctly predicted that sterilized broth in his swan-neck flasks would remain sterile as long as the swan necks remained intact. However, should the necks be broken, microorganisms would be introduced, contaminating the flasks and allowing microbial growth within the broth.
Pasteur’s set of experiments irrefutably disproved the theory of spontaneous generation and earned him the prestigious Alhumbert Prize from the Paris Academy of Sciences in 1862. In a subsequent lecture in 1864, Pasteur articulated “Omne vivum ex vivo” (“Life only comes from life”). In this lecture, Pasteur recounted his famous swan-neck flask experiment, stating that “…life is a germ and a germ is life. Never will the doctrine of spontaneous generation recover from the mortal blow of this simple experiment.”R. Vallery-Radot.  To Pasteur’s credit, it never has.

### Key Concepts and Summary

1. The theory of spontaneous generation states that life arose from nonliving matter. It was a long-held belief dating back to Aristotle and the ancient Greeks.
2. Experimentation by Francesco Redi in the 17th century presented the first significant evidence refuting spontaneous generation by showing that flies must have access to meat for maggots to develop on the meat. Prominent scientists designed experiments and argued both in support of (John Needham) and against (Lazzaro Spallanzani) spontaneous generation.
3. Louis Pasteur is credited with conclusively disproving the theory of spontaneous generation with his famous swan-neck flask experiment. He subsequently proposed that “life only comes from life.”

### Multiple Choice

### Fill in the Blank

### True/False

### Short Answer

### Critical Thinking