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skills expected; (b) include test items that assess the material

to be learned at the appropriate level; (c) enable instructors

to accurately judge students’ mastery of the material— what

they know, what they don’t know, and to what degree— based

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mastery allow instructors to focus on areas where students

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The Test Bank authors listed, in terms of importance, the

concepts from each chapter that they believe are the most

important for students to learn. The authors then created a

concept map for each chapter that shows the relationships

among these ideas. Once the concept maps are created,

authors develop three types of questions designed to test stu-

dents’ knowledge of a par tic u lar concept. By asking students

questions that vary in both type and level of diffi culty, instruc-

tors can gather different types of evidence, which will allow

them to more effectively assess how well students understand

specifi c concepts.

Three Question Types:

1. Factual questions (ask What?)— Test declarative knowl-

edge, including textbook defi nitions and relationships

between two or more pieces of information.

2. Applied questions (ask How?)— Pose problems in a con-

text different from the one in which it was learned,

requiring students to draw from their declarative and/or

procedural understanding of important concepts.

3. Conceptual questions (ask Why?)— Ask students to draw

from their prior experience and use critical thinking

ASSESSMENT GUIDELINES BASED ON THE SCIENCE OF LEARNING

When was the last time you were pleased with the consis-

tency and quality of the assessment supplements that come

with introductory texts? If you are like most professors, you

probably fi nd that these assessment packages do not always

meet your needs. To address this issue, W. W. Norton has col-

laborated with Valerie Shute (Florida State University) and

Diego Zapata- Rivera (Educational Testing Ser vices) to

develop a methodology for delivering high- quality, valid, and

reliable assessment supplements through our Test Banks and

extensive suite of support materials.

WHY A NEW APPROACH?

In evaluating the Test Banks that accompany introductory

texts, we found four substantive problem areas associated with

the items: (a) item types are misclassifi ed; (b) there is a preva-

lence of low- level (factual) questions that may misrepresent

the goals of the course; (c) topics are unevenly distributed—

where trivial topics are tested via multiple items while impor-

tant concepts are not tested at all; and (d) links to the topic are

often at a very general vs. specifi c level, thus preventing diag-

nostic use of the item information.

STUDENT COMPETENCIES AND EVIDENCE- CENTERED DESIGN

In December 2007, we conducted a focus group with the

brightest minds in educational testing to create a new model

for assessment. A good assessment tool needs to: (a) defi ne

what students need to know and the level of knowledge and

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cally correct language that suits the diffi culty level of the

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oping the questions, every effort has been made to eliminate

bias (e.g., race, gender, cultural, ethnic, regional, handicap,

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of general knowledge or experience. This ensures accessi-

bility and validity.

A FINAL NOTE

We hope that the ideas and methods described have pro-

duced some new ways of thinking about assessment. W. W.

Norton has a strong commitment to supporting instructors

with high- quality material that is based on the latest research

on the science of learning.

skills to take part in qualitative reasoning about the real

world.

Three Difficulty Levels:

1. Easy questions— Require a basic understanding of the

concepts, defi nitions, and examples.

2. Medium questions— Direct students to use critical think-

ing skills, to demonstrate an understanding of core con-

cepts in de pen dent of specifi c textbook examples, and to

connect concepts across chapters.

3. Hard questions— Ask students to synthesize textbook

concepts with their own experience, making analytical

inferences about biological topics and more.

Each question mea sures and explicitly links to a specifi c com-

petency, and is written with clear, concise, and grammati-

260 | Test Bank


CHAPTER 1: Microbial Life: Origin and Discovery

CONCEPT MAP

I. Section 1.1: From Germ to Genome: What Is a Microbe?

A. Microbes are organisms that require a microscope to be seen i. Prokaryotes—cells lacking a nucleus such as bacteria and archaea

ii. Eukaryotes—cells with nucleus such as algae, fungi, and protists B. Microbial genomes (cell’s genetic information) can be sequenced

i. Can indicate degree of relatedness between species ii. Comparative genomics is the comparison of genomes of all species

II. Section 1.2: Microbes Shape Human History

A. Microbial disease devastates human populations i. Tuberculosis, leprosy, bubonic plague, smallpox, AIDS

ii. Florence Nightingale—developed science of medical statistics to show impact of infectious disease on humans

B. Microscopes reveal the microbial world i. Robert Hooke—built compound microscope; first to publish illustrations

of microbes ii. Antoni van Leeuwenhoek—first to view single-celled organisms

C. Spontaneous Generation—theory that living organisms arise spontaneously i. Lazzaro Spallanzani

a. attempted to disprove theory using sealed flask of meat broth ii. Louis Pasteur

a. Discovered that fermentation is caused by yeast b. Disproved spontaneous generation using unsealed flasks

with bent neck iii. John Tyndall

a. Showed that repeated cycles of heat are necessary to destroy endospores (a heat-resistant form of bacteria)

III. Section 1.3: Medical Microbiology

A. Germ theory of disease—theory that many diseases are caused by microbes B. Robert Koch

i. Developed pure culture techniques to study a single species a. Used media which was solidified with agar and placed in petri dishes

ii. Developed postulates for proving that a specific organism causes a disease C. Immunization prevents disease

i. Edward Jenner developed first vaccination using cowpox virus to protect against smallpox

ii. Louis Pasteur developed immunization technique using attenuated pathogens such as rabies virus

iii. Ignaz Semmelweis and Joseph Lister discovered that use of antiseptics prevent infections

iv. Alexander Fleming discovered first antibiotic, penicillin. Howard Florey and Ernst Chain purified penicillin for commercial use.

D. Discovery of viruses

261

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262 | Chapter 1

IV. Section 1.4: Microbial Ecology A. Sergei Winogradsky

i. First to study microbes in their natural environment ii. Discovered lithotrophs, which use inorganic minerals as food source

iii. Developed enrichment culture techniques for growing specific types of organisms

iv. Winogradsky column is an enclosed ecosystem with regions of enrichment for microbes of diverse metabolism

B. Microbes are responsible for geochemical cycling of many minerals

V. Section 1.5: The Microbial Family Tree A. Microbes are a challenge to classify

i. Difficult to visualize distinguishing features ii. Do not fit classic definition of species

B. Microbes include prokaryotes and eukaryotes C. Lynn Margulis proposed that eukaryotes evolved through endosymbiosis—one cell

internalizes another that grows within it D. Carl Woese discovered new domain of prokaryotes, archaea, using 16s rRNA

sequencing

VI. Section 1.6: Cell Biology and the DNA Revolution A. Electron microscope

i. Invented by Ernst Ruska using electromagnets to focus rays of electrons ii. Can magnify up to a millionfold to reveal cellular structures

B. Ultracentrifuge i. Invented by Theodor Svedberg

ii. Uses centrifugal force to separate and determine sizes of cellular components C. Discovery of structure and function of DNA led to many scientific advances

i. DNA sequencing ii. Polymerase chain reaction

iii. Recombinant DNA D. Microbial discoveries enable production of new pharmaceuticals and industrial

products


CHAPTER 1: Microbial Life: Origin and Discovery

MULTIPLE CHOICE

1. The eukaryotic predators of the microscopic world are:

a. viruses b. bacteria c. algae d. cyanobacteria e. protists

ANS: E DIF: Easy REF: Introduction OBJ: Factual TOP: Introduction

2. A human body contains __________ times as many microbes as it does human cells.

a. 2 b. 5 c. 10 d. 50 e. 100

ANS: C DIF: Easy REF: Introduction OBJ: Factual TOP: Introduction

3. What are the primary producers of major food webs?

a. plants b. microbes c. animals d. fungi e. viruses

ANS: B DIF: Easy REF: Introduction OBJ: Factual TOP: Introduction

4. What type of gas do cyanobacteria produce for planet Earth?

a. oxygen b. hydrogen c. nitrogen d. carbon dioxide e. water vapor

ANS: A DIF: Easy REF: Introduction OBJ: Factual TOP: Introduction

5. Bacteria that produce enzymes used in the polymerase chain reaction were isolated from:

a. lake water b. ocean c. soil d. intestines e. hot springs


263


264 | Chapter 1 6. What percentage of microbes in our biosphere can be cultured in the laboratory?

a. less than 1% b. approximately 10% c. approximately 25% d. approximately 50% e. more than 90%

ANS: A DIF: Medium REF: Introduction OBJ: Factual TOP: Introduction

7. Which of these is currently the number one cause of human mortality?

a. cardiovascular disease b. cancer c. accidents d. microbial disease e. strokes

ANS: D DIF: Easy REF: Introduction OBJ: Applied TOP: Introduction

8. The presence of __________ on Mars today would increase the chance that microbial life exists there.

a. liquid water b. oxygen c. nitrogen gas d. ammonia e. DNA

ANS: A DIF: Easy REF: Introduction OBJ: Applied TOP: Introduction

9. Which of these groups are considered to be microbes but NOT considered to be cells?

a. viruses b. bacteria c. archaea d. protists e. prions

ANS: A DIF: Easy REF: 1.1 OBJ: Applied TOP: I.A

10. The first genomes to be sequenced were those of:

a. humans b. bacteria c. viruses d. prions e. fungi

ANS: C DIF: Easy REF: 1.1 OBJ: Factual TOP: I.B.i


Microbial Life: Origin and Discovery | 265 11. Which century is known as the golden age of microbiology?

a. the seventeenth b. the eighteenth c. the nineteenth d. the twentieth e. the twenty-first

ANS: C DIF: Easy REF: 1.2 OBJ: Factual TOP: II

12. All of the following have been found in mummies and tomb art EXCEPT:

a. tuberculosis b. polio c. leprosy d. smallpox e. prions

ANS: E DIF: Medium REF: 1.2 OBJ: Applied TOP: II

13. How did European invaders to North America kill much of the native population?

a. tuberculosis b. leprosy c. smallpox d. HIV e. bubonic plague

ANS: C DIF: Medium REF: 1.2 OBJ: Applied TOP: II.A.i

14. Who developed the concept of medical statistics?

a. Francis Crick b. Florence Nightingale c. Edward Jenner d. Louis Pasteur e. Alexander Fleming

ANS: B DIF: Easy REF: 1.2 OBJ: Factual TOP: II.A.ii

15. Which technique was developed to distinguish bacteria from human cells?

a. Gram stain b. electron microscopy c. X-ray diffraction d. DNA sequencing e. PCR

ANS: A DIF: Medium REF: 1.2 OBJ: Factual TOP: I.B


266 | Chapter 1 16. The first person to visualize individual microbes was:

a. Antoni van Leeuwenhoek b. Robert Hooke c. Louis Pasteur d. Lady Montagu e. Edward Jenner

ANS: A DIF: Easy REF: 1.2 OBJ: Factual TOP: II.B.ii

17. Suppose Pasteur’s swan-necked flasks containing boiled broth became cloudy 24 hours after boiling.

Which choice could best explain the turbidity or cloudiness in the broth without supporting spontaneous generation? a. Endospores in the broth survived boiling and grew after the broth cooled. b. Contaminating organisms in the broth killed by boiling became alive again after the broth

cooled. c. Chemicals in the broth came together to form living organisms. d. The broth allowed light to pass through it with less interference after boiling. e. Solid material in the broth dissolved during boiling.

ANS: A DIF: Difficult REF: 1.2 OBJ: Applied TOP: II.C.ii.b

18. How is most sterilization performed for the controlled study of microbes?

a. boiling b. pasteurization c. filter sterilization d. autoclaving e. irradiation

ANS: D DIF: Medium REF: 1.2 OBJ: Applied TOP: II.C.iii

19. The use of agar as the gelling agent in solid media was suggested by:

a. Robert Koch b. Ignaz Semmelweis c. Angelina Hesse d. Louis Pasteur e. Richard Petri

ANS: C DIF: Easy REF: 1.3 OBJ: Factual TOP: III.B.i.a

20. Robert Koch’s greatest accomplishment in the field of medical bacteriology was with:

a. Escherichia coli b. Bacillus subtilis c. Mycobacterium tuberculosis d. rabies e. smallpox

ANS: C DIF: Medium REF: 1.3 OBJ: Applied TOP: III.B.ii


Microbial Life: Origin and Discovery | 267 21. You have isolated a bacterium that you believe to be the causative agent of a new disease in frogs.

How would you test the third of Koch’s postulates? a. Determine the shape of the bacterial cells. b. Inject the bacteria into a healthy frog. c. Isolate the bacterium from a sick frog. d. Show that the bacterium is NOT present in healthy frogs. e. Grow a pure culture of the bacterium outside the frog.

ANS: B DIF: Difficult REF: 1.3 OBJ: Applied TOP: III.B.ii

22. It took the advent of the PCR to detect the presence of the causative agent for which disease?

a. anthrax b. tuberculosis c. AIDS d. rabies e. smallpox

ANS: C DIF: Difficult REF: 1.3 OBJ: Applied TOP: III.B.ii

23. The word “vaccination” is derived from the Latin word vacca, which means:

a. inject b. smallpox c. immunize d. cow e. pustule

ANS: D DIF: Easy REF: 1.3 OBJ: Factual TOP: III.C.i

24. What is the basis for the modern smallpox vaccine?

a. chickenpox virus b. cowpox virus c. rabies virus d. smallpox virus e. anthrax

ANS: B DIF: Easy REF: 1.3 OBJ: Factual TOP: III.C.i

25. Penicillin was first used to save the lives of many people during which war?

a. Civil War b. Korean War c. Vietnam War d. World War I e. World War II

ANS: E DIF: Easy REF: 1.3 OBJ: Factual TOP: III.C.iv


268 | Chapter 1 26. Which of the following can safely be ingested to fight bacterial infections?

a. antiseptics b. disinfectants c. phenol d. chlorine e. antibiotics

ANS: E DIF: Easy REF: 1.3 OBJ: Applied TOP: III.C.iv

27. All of the following are true about penicillin EXCEPT:

a. It was discovered by Alexander Fleming. b. It was an accidental discovery. c. It is produced by a bacterium. d. It was the first antibiotic used by humans. e. It was purified by Florey and Chain.

ANS: C DIF: Difficult REF: 1.3 OBJ: Applied TOP: III.C.iv

28. Which of the following does NOT contain DNA or RNA?

a. prokaryote b. eukaryote c. virus d. viroid e. prion

ANS: E DIF: Medium REF: 1.3 OBJ: Factual TOP: III.D

29. The environment of early Earth may have contained all EXCEPT:

a. ferrous iron b. methane c. ammonia d. oxygen e. hydrogen gas

ANS: D DIF: Easy REF: Special Topic 1.1 OBJ: Applied TOP: Special Topic 1.1

30. The development of the theory of the “RNA world” resulted from the discovery of:

a. archaea b. prions c. bacteria d. ribozymes e. endosymbionts

ANS: D DIF: Medium REF: Special Topic 1.1 OBJ: Applied TOP: Special Topic 1.1


Microbial Life: Origin and Discovery | 269 31. Which microbes may resemble those of the earliest life forms?

a. archaea b. photosynthetic microbes c. viruses d. cyanobacteria e. protists

ANS: A DIF: Medium REF: Special Topic 1.1 OBJ: Applied TOP: Special Topic 1.1

32. Early metabolism may have been catalyzed by:

a. DNA b. RNA c. protein d. amino acids e. carbohydrates

ANS: B DIF: Medium REF: Special Topic 1.1 OBJ: Applied TOP: Special Topic 1.1

33. Which types of compounds have a strong tendency to accept electrons?

a. oxidized b. reduced c. neutral d. protons e. neutrons

ANS: B DIF: Medium REF: Special Topic 1.1 OBJ: Applied TOP: Special Topic 1.1

34. How did Sergei Winogradsky grow lithotrophs?

a. enrichment culture b. aseptic technique c. pure culture d. endosymbiosis e. chain of infection

ANS: A DIF: Easy REF: 1.4 OBJ: Applied TOP: IV.A.iii

35. Organisms which live symbiotically inside a larger organism are known as:

a. organelles b. cyanobacteria c. mitochondria d. endosymbionts e. chloroplasts

ANS: D DIF: Easy REF: 1.4 OBJ: Factual TOP: IV.B


270 | Chapter 1 36. Which group of microorganisms includes many that grow in extreme environments?

a. algae b. bacteria c. protists d. archaea e. fungi

ANS: D DIF: Easy REF: 1.5 OBJ: Applied TOP: V.B

37. The genetic expression machinery of archaea is most similar to:

a. monera b. prokaryotes c. bacteria d. eukaryotes e. mitochondria

ANS: D DIF: Medium REF: 1.5 OBJ: Applied TOP: V.B

38. In the three-domain model, the bacterial ancestor of mitochondria derives from ancient:

a. fungi b. cyanobacteria c. proteobacteria d. archaea e. protists

ANS: C DIF: Medium REF: 1.5 OBJ: Applied TOP: V.C

39. Which of the following organelles are thought to be of prokaryotic origin?

a. chloroplast b. mitochondria c. nucleus d. chloroplast and mitochondria e. chloroplast and nucleus

ANS: A DIF: Medium REF: 1.5 OBJ: Applied TOP: V.C

40. In the three-domain model, the bacterial ancestor of chloroplasts derives from ancient:

a. fungi b. cyanobacteria c. proteobacteria d. archaea e. protists

ANS: B DIF: Medium REF: 1.5 OBJ: Applied TOP: V.C


Microbial Life: Origin and Discovery | 271 41. Carl Woese’s discovery replaced the classification scheme of five kingdoms with a scheme of three:

a. phyla b. domains c. classes d. orders e. genera

ANS: B DIF: Easy REF: 1.5 OBJ: Factual TOP: V.D

42. How are microbes classified today?

a. comparative genomics b. microscopy c. X-ray diffraction d. DNA sequencing e. rRNA sequencing

ANS: E DIF: Medium REF: 1.5 OBJ: Applied TOP: V.D

43. What is used to focus the beam of electrons in an electron microscope?

a. electromagnets b. condenser lens c. light rays d. X-ray diffraction e. glass

ANS: A DIF: Easy REF: 1.6 OBJ: Factual TOP: VI.A

44. The X-ray diffraction studies by which of the following scientists concluded that DNA was a double

helix? a. James Watson b. Rosalind Franklin c. Francis Crick d. Maurice Wilkins e. Kary Mullis

ANS: B DIF: Easy REF: 1.6 OBJ: Factual TOP: VI.C

45. The Asilomar Conference was held to regulate and restrict the field of:

a. recombinant DNA b. comparative genomics c. DNA sequencing d. DNA amplification e. forensic microbiology

ANS: A DIF: Easy REF: 1.6 OBJ: Factual TOP: VI.C


272 | Chapter 1 46. What type of analysis was used to discover the overall structure of the DNA double helix?

a. microscopy b. X-ray diffraction c. Polymerase chain reaction d. DNA sequencing e. recombinant DNA

ANS: B DIF: Medium REF: 1.6 OBJ: Factual TOP: VI.C

47. Which scientist first discovered the process of transformation?

a. Francis Crick b. Robert Koch c. Edward Jenner d. Louis Pasteur e. Frederick Griffith

ANS: E DIF: Difficult REF: 1.6 OBJ: Factual TOP: VI.C

48. Taq polymerase formed the basis of a multibillion-dollar industry of:

a. comparative genomics b. recombinant DNA c. X-ray diffraction d. DNA amplification e. DNA sequencing

ANS: D DIF: Difficult REF: 1.6 OBJ: Applied TOP: VI.C.ii

49. The study of and cause of disease in humans, animals, and plants is called:

a. microbiology b. phylogeny c. genomics d. epidemiology e. forensics

ANS: D DIF: Easy REF: 1.6 OBJ: Factual TOP: VI.D

50. The analysis of microbial strains as evidence in criminal investigations is known as:

a. forensic microbiology b. recombinant DNA c. comparative genomics d. classification e. gene regulation

ANS: A DIF: Easy REF: 1.6 OBJ: Factual TOP: VI.D


Microbial Life: Origin and Discovery | 273 SHORT ANSWER

1. Why did it take so long for humans to determine that microbes cause infectious diseases?

ANS: Microbes are too small to be seen with the naked eye so until microscopes were invented, humans did not know that microbes existed. Even after humans were aware of the presence of microbes, they did not suspect them of causing disease until people such as Joseph Lister and Ignaz Semmelweis performed experiments that showed antiseptics decrease the incidence of infection.

DIF: Difficult REF: Introduction | 1.2 OBJ: Conceptual TOP: II

2. How are prokaryotes and eukaryotes different?

ANS: A prokaryote lacks a nucleus and membrane-bounded organelles, whereas a eukaryote has a nucleus and membrane-bounded organelles.

DIF: Easy REF: 1.1 OBJ: Applied TOP: I.A

3. What is the most recent evidence suggesting that all life on Earth shares a common ancestry?

ANS: Many genomes have now been sequenced and those sequences are available in databases for comparison. This field is referred to as comparative genomics. Comparisons have revealed that there is a set of core genes shared by all organisms.

DIF: Difficult REF: 1.1 OBJ: Conceptual TOP: I.B.i

4. Identify at least three women and their discoveries that have contributed significantly to the field of

microbiology.

ANS: Answers may vary. Several examples are listed in Table 1.2 in the textbook. They include Lady Montagu’s rendition of a smallpox vaccine, Florence Nightingale’s biomedical statistics, Barbara McClintock and transposons, and Rosalind Franklin and X-ray crystallography studies of DNA.

DIF: Difficult REF: 1.2 OBJ: Applied TOP: II

5. Describe the effects of three microbial diseases that have significantly affected human populations

throughout history.

ANS: Answers may vary. Some examples include bubonic plague, which killed one-third of Europe’s population in the fourteenth century; tuberculosis, which was common in the nineteenth century; AIDS, which affects many people today; and smallpox, which killed a large number of native North Americans.

DIF: Medium REF: 1.2 OBJ: Applied TOP: II.A


274 | Chapter 1 6. Antoni van Leeuwenhoek worked as a cloth draper, inspecting the quality of cloth. How did this lead

to his interest in microscopy?

ANS: Briefly, his work introduced him to magnifying lenses. He began the hobby of grinding lenses, ultimately making a microscope that enabled him to observe single-celled microbes.

DIF: Medium REF: 1.2 OBJ: Conceptual TOP: II.B.ii

7. What was the major complaint about Lazzaro Spallanzani’s experiment to disprove spontaneous

generation, and how did Louis Pasteur’s swan-neck flasks overcome this?

ANS: Spallanzini’s flasks were plugged so as not to let organisms accidentally enter the boiled medium. Opponents argued that no growth was observed simply due to the lack of oxygen. Pasteur’s swan-neck flasks did not allow organisms to enter the flask, but did allow oxygen to enter. Growth was still not observed.

DIF: Medium REF: 1.2 OBJ: Applied TOP: II.C

8. Describe the discoveries of Louis Pasteur while working with the French beer and wine manufacturers.

ANS: Previously, it was believed that the conversion of grapes and grain to wine and beer was a spontaneous chemical process. He discovered that this fermentation was caused by living yeast which did not require oxygen for growth. He also discovered that when the grapes or grain is contaminated with bacteria instead of yeast, acetic acid is produced instead of alcohol.

DIF: Medium REF: 1.2 OBJ: Applied TOP: II.C.ii

9. How would you use Robert Koch’s postulates to prove that a specific organism causes a new disease

in mice?

ANS: See Figure 1.18 in the textbook. 1. The suspected organism is found in all diseased mice, but absent from healthy mice. 2. The suspected organism is isolated from the diseased mice and grown in pure culture. 3. When the suspected organism is introduced into a healthy mouse, the same disease occurs. 4. The same strain of microbe is obtained from the newly diseased mouse.

DIF: Medium REF: 1.3 OBJ: Applied TOP: III.B.ii

10. Robert Koch’s postulates have not been used to prove HIV as the causative agent of AIDS. Why not?

ANS: Answers may vary, but a major reason is that humans cannot be injected with HIV to see if they develop AIDS!

DIF: Difficult REF: 1.3 OBJ: Conceptual TOP: III.B.ii


Microbial Life: Origin and Discovery | 275 11. Define attenuation and describe some mechanisms used to attenuate pathogens.

ANS: Attenuation results in a weakened organism that will not produce full-blown disease, but will generate immunity. Answers for mechanisms may vary. See discussion in textbook Section 1.3 entitled “Immunization Prevents Disease.”

DIF: Medium REF: 1.3 OBJ: Applied TOP: III.C.i

12. What is the significance of the work of Ignaz Semmelweis and Joseph Lister?

ANS: They showed that use of antiseptics on doctor’s hands and medical instruments drastically reduced the mortality rate of hospital patients. They made these observations before Robert Koch’s germ theory of disease.

DIF: Medium REF: 1.3 OBJ: Conceptual TOP: III.C.iii

13. Wilderness protection worldwide has been fueled by the need to find novel strains of antibiotic-

producing bacteria and fungi.

ANS: Many new and powerful antibiotics have been discovered during the second half of the twentieth century. Most of these were produced by obscure strains of bacteria and fungi from dwindling ecosystems. It is important to preserve wilderness worldwide because there are likely many undiscovered antibiotic-producing organisms.

DIF: Difficult REF: 1.3 OBJ: Conceptual TOP: III.C.iv

14. Explain why the organisms that were studied by Sergei Winogradsky could not be grown on Robert

Koch’s plate media containing agar or gelatin.

ANS: The organisms studied by Winogradsky were lithotrophs, which feed solely on inorganic substances. Koch’s plate media contained organic nutrient sources.

DIF: Difficult REF: 1.4 OBJ: Conceptual TOP: IV.A.ii

15. Define the term “endosymbiont” and give an example of an endosymbiotic relationship found in

nature.

ANS: An endosymbiont is an organism living symbiotically inside a larger organism. Examples may vary, but include the following: Rhizobium in a leguminous plant, bioluminescent bacteria in the light organs of fish and squid, photosynthetic algae and coral.

DIF: Medium REF: 1.4 OBJ: Applied TOP: IV.B


276 | Chapter 1 16. Is it true that only culturable bacteria contribute to ecology and pathology? Explain your answer.

ANS: No, this is not a true statement. It is estimated that barely 0.1% of microbial species can be cultured. The work of Winogradsky and later microbial ecologists showed that bacteria are necessary for geochemical cycling. Many of these organisms can’t be grown in pure culture on laboratory media but can be grown in enrichment culture such as a Winogradsky column.

DIF: Difficult REF: 1.4 OBJ: Conceptual TOP: IV.B

17. Give two reasons why microbes have been difficult to classify.

ANS: First, even with the use of light microscopes, only the basic shape of microbes can be determined and many microbes have similar shapes even though they are very different in other ways. Second, microbes do not fit the classic definition of a species, which is a group of organisms that interbreed. Microbes typically reproduce asexually. When they do exchange genes, they may do so with distantly related species.

DIF: Medium REF: 1.5 OBJ: Conceptual TOP: V.A

18. Briefly explain the endosymbiosis theory and the evidence that supports it.

ANS: The endosymbiosis theory proposes that mitochondria and chloroplasts evolved from bacteria that were engulfed by pre-eukaryotic cells and that over time these endosymbiotic prokaryotic cells lost the ability to survive outside of the host cell but were maintained as organelles. Evidence supporting the endosymbiosis theory includes the fact that mitochondria and chloroplasts possess circular DNA with similarity to modern bacteria.

DIF: Difficult REF: 1.5 OBJ: Applied TOP: V.C

19. Briefly describe how the ultracentrifuge is used to determine the sizes of cellular macromolecules.

ANS: The ultracentrifuge uses centrifugal forces to separate cell components. Svedberg calculated that the particle sizes could be determined based on the rate of sedimentation of the particles in an ultracentrifuge.

DIF: Medium REF: 1.6 OBJ: Applied TOP: VI.B.ii

20. What were the contributions of Rosalind Franklin toward discovering the structure of DNA and why

wasn’t she one of the recipients of the Nobel Prize for this discovery?

ANS: She was an X-ray crystallographer who studied the structure of DNA. Her X-ray micrographs showed for the first time that DNA was a double helix. A colleague showed her micrographs to James Watson who was also studying the structure of DNA. Watson and Francis Crick published their model of the structure of DNA in the journal Nature and denied that they had used Franklin’s micrographs.

DIF: Medium REF: 1.6 OBJ: Conceptual TOP: I.C


CHAPTER 2: Observing the Microbial Cell

CONCEPT MAP

I. Section 2.1: Observing Microbes

A. Resolution of objects i. Resolution—smallest distance by which two objects can be separated

and still be distinguished B. Resolution differs from detection

i. Detection—the ability to determine the presence of an object. a. The eye can detect the presence of mold but can’t resolve individual

cells ii. Magnification—increase in the apparent size of an object

C. Microbial size and shape i. Prokaryotes are generally between 0.4–10 μm

ii. Three common prokaryotic shapes a. Bacilli—rod shaped b. Cocci—spherical shaped c. Spiral—corkscrew shaped

D. Microscopy for different size scales i. Different types of microscopy are used to view structures of different sizes

II. Section 2.2: Optics and Properties of Light

A. Light carries information i. Electromagnetic radiation interacts with objects and acquires information

that can be used to detect the objects ii. Conditions necessary for electromagnetic radiation to resolve and object

a. Contrast between object and surroundings b. Wavelength smaller than object c. Detector with enough resolution for that wavelength

B. Light interacts with objects i. Particles of light called photons interact with objects in many ways

a. Absorption—light energy is acquired by object b. Reflection—wave front bounces off of object at angle equal to its

incident angle c. Refraction—bending of light when it enters a substance that slows

its speed d. Scattering—wave front interacts with object of smaller dimension

than the wavelength C. Refraction enables magnification

i. Magnification requires refraction of light through medium of high refractive index

D. Magnification and resolution i. Empty magnification—magnification without increase in resolution

277


278 | Chapter 2

III. Section 2.3: Bright-Field Microscopy A. Increasing resolution

i. Resolution depends on a. Wavelength of light b. Contrast c. Lens quality and magnifying power d. Position of the focal plane

B. The compound microscope i. Uses a system of lenses to achieve magnification and resolution

a. Condenser lens—concentrates light from light source to slide b. Objective lenses—closest to specimen; typically magnify

10×, 40×, or 100× c. Ocular lens—closest to eye; typically magnifies 10×

ii. Steps for observing specimen a. Position specimen in middle of field of view b. Optimize light—need more at higher powers c. Focus objective lens

C. Is the object in focus? i. Object is in focal plane of lens when edges of object appear sharp and distinct

D. Fixing and staining improve resolution and contrast i. Wet mount—water and specimen on slide with cover slip

a. Can observe living cells in natural state b. Most cells are transparent so show little contrast

ii. Fixation—cells are treated with alcohol or heat to make them adhere to slide iii. Stains—adhere to bacteria to increase contrast

E. Different kinds of stains i. Simple stain—uses one stain to color all cells

ii. Differential stain—distinguishes between different types of bacteria by using different stains

a. Gram stain—bacteria are classified as Gram positive or Gram negative based on whether they retain crystal violet stain

b. Acid-fast stain—carbolfuchsin is used to stain Mycobacterium species c. Spore stain—malachite green and heat are used to stain endospores d. Negative stain—stains background but not cells; used to view

capsules e. Antibody stain—antibody proteins linked to fluorophores bind

specific components of cells

IV. Section 2.4: Dark-Field, Phase-Contrast, and Interference Microscopy A. Dark-field microscopy

i. Uses scattered light so cells appear bright on dark background ii. Can be used to view very small microbes and motility

iii. Disadvantage—particulates will scatter light; can be difficult to distinguish from microbes from particulates

B. Phase-contrast microscopy i. Enhances differences in refractive index so live cells can be observed without

staining C. Interference microscopy

i. Superimposes interference bands on an image, accenting small differences in refractive index


Observing the Microbial Cell | 279

V. Section 2.5: Fluorescence Microscopy A. Fluorescence requires excitation and emission at different wavelengths B. Fluorophores can label specific parts of cells by

i. Chemical affinity ii. Labeled antibodies

iii. Gene fusion iv. DNA hybridization

C. Laser confocal microscopy i. Fluorescence is used along with laser optics to produce 3D images

VI. Section 2.6: Electron Microscopy

A. The electron microscope focuses beams of electrons and achieves resolution 1000× resolution of light microscopy

i. Transmission electron microscopy a. Electrons are transmitted through specimen

ii. Scanning electron microscopy a. Electrons scan surface of specimen and are reflected to produce 3D

image B. Electron microscopy requires specialized sample preparation

i. Specimen can be embedded in polymer and cut into thins sections using microtome then coated with heavy metal

ii. Specimen can be sprayed onto copper grid then coated with heavy metal iii. Specimen can be flash frozen for cryo-EM

C. Microscopy results require careful interpretation i. Artifact—microscopic structure that is interpreted incorrectly

D. Emerging methods of microscopy i. Cryo-EM

a. Samples are flash frozen in water solution; very high resolution ii. Atomic force microscopy

a. Measure van der Waals forces between atoms on cell surface and a sharp tip; very high resolution

VII. Section 2.7: Visualizing Molecules

A. X-ray diffraction i. Uses X-ray interference patterns to generate computational model

of crystallized macromolecules ii. Cryocrystallography—uses frozen crystals to determine structures

of macromolecular complexes


CHAPTER 2: Observing the Microbial Cell

MULTIPLE CHOICE

1. Who compared “small animals” from his teeth before and after drinking coffee?

a. Hooke b. Fleming c. Gram d. Jenner e. van Leeuwenhoek


2. One __________ is one-thousandth of a millimeter.

a. micrometer b. nanometer c. meter d. centi e. kilo

ANS: A DIF: Medium REF: 2.1 OBJ: Factual TOP: I.A

3. In humans, resolution is achieved by focusing an image on a retina, packed with light-absorbing:

a. rods b. cones c. nerves d. photoreceptor cells e. lenses

ANS: D DIF: Medium REF: 2.1 OBJ: Factual TOP: I.A

4. Having fewer photoreceptors per surface area means higher:

a. resolution b. magnification c. refraction d. reflection e. wavelength

ANS: A DIF: Difficult REF: 2.1 OBJ: Applied TOP: I.A

5. Resolution is the smallest distance by which two objects can be __________ and still be __________.

a. magnified; seen b. separated; distinguished c. magnified; separated d. distinguished; separated e. magnified; distinguished

ANS: B DIF: Medium REF: 2.1 OBJ: Applied TOP: I.A.i

280


Observing the Microbial Cell | 281 6. A rod-shaped microbe is referred to as a:

a. bacillus b. coccus c. vibrio d. strepto e. spirochete

ANS: A DIF: Easy REF: 2.1 OBJ: Factual TOP: I.C.ii.a

7. All electromagnetic radiation travels through a vacuum at what speed?

a. 3 × 108 mm/sec b. 3 × 108 cm/sec c. 3 × 108 m/sec d. 3 × 108 ft/sec e. 3 × 108 mph

ANS: C DIF: Difficult REF: 2.2 OBJ: Factual TOP: II.A

8. If an object and its surroundings absorb or reflect radiation equally then the object will be:

a. undetectable b. reflected c. refracted d. radiated e. fluoresced

ANS: A DIF: Medium REF: 2.2 OBJ: Applied TOP: II.A.ii.a

9. Which is the most important property that enables a lens to magnify an image?

a. absorption b. fluorescence c. reflection d. refraction e. scattering

ANS: D DIF: Medium REF: 2.2 OBJ: Applied TOP: II.C

10. What is the key property that enables a lens to magnify an image?

a. reflection b. resolution c. frequency d. refraction e. wavelength

ANS: D DIF: Medium REF: 2.2 OBJ: Applied TOP: II.C


282 | Chapter 2 11. Magnification without resolution is known as __________ magnification.

a. complete b. zero c. maximum d. total e. empty

ANS: E DIF: Easy REF: 2.2 OBJ: Factual TOP: II.D

12. When two waves are out of phase by __________ wavelength, they produce destructive interference,

canceling each other’s amplitude and resulting in contrast in the image. a. one-tenth b. one-eighth c. one-quarter d. one-half e. one

ANS: D DIF: Difficult REF: 2.2 OBJ: Factual TOP: II.D

13. An image is magnified when light passes through a refractive material shaped so as to __________ its

rays. a. absorb b. block c. concentrate d. condense e. spread

ANS: E DIF: Medium REF: 2.2 OBJ: Applied TOP: II.D

14. Increasing the refractive index of the medium between the object and the objective lens increases:

a. refraction b. reflection c. magnification d. resolution e. wavelength

ANS: D DIF: Difficult REF: 2.2 OBJ: Applied TOP: II.D

15. With the 100× lens, the refractive index of light passing through the specimen is maintained by

insertion of __________, with a refractive index comparable to that of glass. a. immersion oil b. water c. air d. bacteria e. stain

ANS: A DIF: Easy REF: 2.3 OBJ: Factual TOP: III.A


Observing the Microbial Cell | 283 16. As lens strength increases, the light cone __________ and the lens must be __________ the object.

a. narrows; nearer to b. narrows; farther from c. widens; nearer to d. widens; farther from e. widens; touch

ANS: C DIF: Medium REF: 2.3 OBJ: Factual TOP: III.A

17. A/An __________ acts to vary the diameter of the light column in a light microscope.

a. condenser b. objective c. ocular d. diaphragm e. lens

ANS: D DIF: Easy REF: 2.3 OBJ: Factual TOP: III.B.i

18. The total __________ of the microscope is obtained by multiplying the magnification of the ocular

lens by that of the objective lens. a. resolution b. magnification c. refraction d. reflection e. wavelength

ANS: B DIF: Easy REF: 2.3 OBJ: Applied TOP: III.B.i

19. Higher-power lenses require more light and thus an open:

a. ocular b. lens c. objective d. condenser e. diaphragm

ANS: E DIF: Easy REF: 2.3 OBJ: Applied TOP: III.B.i

20. If you are using a microscope with a 10× ocular lens and a 100× objective, what is the total

magnification? a. 10-fold b. 100-fold c. 110-fold d. 1,000-fold e. This is not enough information.

ANS: D DIF: Medium REF: 2.3 OBJ: Applied TOP: III.B.i


284 | Chapter 2 21. Which is the counterstain in the Gram stain procedure?

a. crystal violet b. methylene blue c. malachite green d. safranin e. Gram’s iodine

ANS: D DIF: Easy REF: 2.3 OBJ: Factual TOP: III.E.ii.a

22. Which of the following staining processes requires crystal violet?

a. acid-fast stain b. antibody stain c. negative stain d. Gram stain e. spore stain

ANS: D DIF: Easy REF: 2.3 OBJ: Factual TOP: III.E.ii.a

23. Gram’s iodine is the __________ in the Gram staining procedure.

a. primary stain b. counterstain c. decolorizer d. negative stain e. mordant

ANS: E DIF: Easy REF: 2.3 OBJ: Factual TOP: III.E.ii.a

24. Eukaryotes stain:

a. Gram-neutral b. Gram-positive c. Gram-negative d. blue e. no color

ANS: C DIF: Medium REF: 2.3 OBJ: Factual TOP: III.E.ii.a

25. Which of the following is a true statement about Gram staining?

a. The Gram stain differentiates between the three domains. b. In a Gram-negative cell, the crystal violet–iodide complex is retained by multiple layers of

peptidoglycan. c. The outer membrane is disrupted by the decolorizer. d. Human cells appear Gram-positive. e. The mordant is used so that Gram-negative cells may be observed.

ANS: C DIF: Medium REF: 2.3 OBJ: Applied TOP: III.E.ii.a


Observing the Microbial Cell | 285 26. The __________ in the Gram stain process binds to the crystal violet, generating a complex that is

held more tightly within the cell. a. mordant b. safranin c. alcohol d. bacteria e. slide

ANS: A DIF: Medium REF: 2.3 OBJ: Applied TOP: III.E.ii.a

27. Which of the following is a negative stain?

a. acid-fast stain b. endospore stain c. antibody stain d. simple stain e. capsule stain

ANS: E DIF: Easy REF: 2.3 OBJ: Factual TOP: III.E.ii.d

28. X-ray diffraction and phase contrast microscopy both involve:

a. wave interference b. observation of living specimens c. differential stains d. simple stains e. shadowing.

ANS: A DIF: Difficult REF: 2.4 OBJ: Applied TOP: IV.A | IV.B

29. Observations of bacterial flagella during motility are best suited to:

a. bright-field microscopy b. dark-field microscopy c. SEM d. TEM e. NMR

ANS: B DIF: Easy REF: 2.4 OBJ: Applied TOP: IV.A.ii

30. A useful application of dark-field optics is the study of bacterial:

a. motility b. surfaces c. interiors d. shape e. structure

ANS: A DIF: Medium REF: 2.4 OBJ: Applied TOP: IV.A.ii


286 | Chapter 2 31. In which type of microscopy do dust particles interfere the most?

a. bright-field microscopy b. dark-field microscopy c. phase-contrast microscopy d. interference microscopy e. fluorescence microscopy

ANS: B DIF: Easy REF: 2.4 OBJ: Conceptual TOP: IV.A.iii

32. Which form of microscopy is based on the difference in refractive index between cell components and

the surrounding medium? a. bright-field b. dark-field c. phase contrast d. confocal e. fluorescence

ANS: C DIF: Medium REF: 2.4 OBJ: Applied TOP: IV.B

33. The digitally combined images of cryo-EM can achieve resolution comparable to that of:

a. SEM b. TEM c. interference microscopy d. X-ray crystallography e. dark-field microscopy

ANS: D DIF: Difficult REF: 2.4 OBJ: Conceptual TOP: IV.D.i

34. When light is absorbed by an object and emitted at a longer wavelength, it is referred to as:

a. fluorescence b. magnification c. reflection d. refraction e. radiation

ANS: A DIF: Easy REF: 2.5 OBJ: Applied TOP: V.A

35. Fluorescence requires excitation and emission at different:

a. refractive indexes b. wavelengths c. contrasts d. refractions e. densities

ANS: B DIF: Easy REF: 2.5 OBJ: Applied TOP: V.A


Observing the Microbial Cell | 287 36. In fluorescence microscopy, incident light is absorbed by the specimen and reemitted at a __________

energy, resulting in a __________. a. lower; longer wavelength b. lower; shorter wavelength c. higher; longer wavelength d. higher; shorter wavelength e. higher; higher contrast

ANS: A DIF: Difficult REF: 2.5 OBJ: Applied TOP: V.A

37. The aromatic groups of the fluorophore DAPI associate exclusively with the:

a. cell wall b. base pairs of DNA c. flagella d. cell membrane e. pili

ANS: B DIF: Medium REF: 2.5 OBJ: Factual TOP: V.B

38. The fluorophore acridine orange specifically binds:

a. cytoplasm b. cell wall c. protein d. RNA e. DNA

ANS: E DIF: Easy REF: 2.5 OBJ: Factual TOP: V.B.i

39. The use of antibodies linked to fluorophores is known as:

a. fluorescence b. immunofluorescence c. X-ray diffraction d. atomic force microscopy e. cryo-EM

ANS: B DIF: Easy REF: 2.5 OBJ: Factual TOP: V.B.ii

40. Which form of microscopy is used with DNA microarrays to observe differences in gene expression?

a. light microscopy b. atomic force microscopy c. SEM d. TEM e. confocal fluorescence microscopy

ANS: E DIF: Difficult REF: 2.5 OBJ: Applied TOP: V.C


288 | Chapter 2 41. Which of the following is true of transmission electron microscopy but NOT scanning electron

microscopy? a. The specimen is usually fixed and embedded. b. The embedded specimen is cut into thin sections with a microtome. c. The specimen is stained with heavy metal. d. The specimen is viewed as three-dimensional. e. The requirement for a vacuum precludes the viewing of live organisms.

ANS: B DIF: Medium REF: 2.6 OBJ: Applied TOP: VI.A | VI.B

42. Transmission electron microscopy commonly has a resolution of __________ times the highest

resolution possible for light microscopy. a. ten b. one hundred c. one thousand d. ten thousand e. one million

ANS: C DIF: Difficult REF: 2.6 OBJ: Applied TOP: VI.A

43. In which one of the following types of microscopy is the specimen shadowed with heavy metal?

a. atomic force microscopy b. SEM c. TEM d. X-ray diffraction e. dark-field

ANS: B DIF: Medium REF: 2.6 OBJ: Factual TOP: VI.A.ii

44. The knife used to cut embedded specimens for observation by TEM is called a:

a. crystallographer b. microtome c. grid d. polymer e. scalpel

ANS: B DIF: Easy REF: 2.6 OBJ: Factual TOP: VI.B.i

45. Fixatives and heavy atom stains used in EM can introduce __________ into an image.

a. colors b. resolution c. refraction d. artifacts e. fluorescence

ANS: D DIF: Easy REF: 2.6 OBJ: Factual TOP: VI.C


Observing the Microbial Cell | 289 46. A microscopic structure that is interpreted incorrectly is a/an:

a. microtome b. crystal c. shadow d. antibody e. artifact

ANS: E DIF: Easy REF: 2.6 OBJ: Factual TOP: VI.C.i

47. In __________, because the frozen sample remains hydrated, the biological molecules retain the same

conformation as in solution. a. cryo-EM b. TEM c. SEM d. fluorescence microscopy e. laser confocal microscopy

ANS: A DIF: Easy REF: 2.6 OBJ: Applied TOP: VI.D.i

48. Atomic force microscopy measures __________ between a probe and an object to map the three-

dimensional topography of a cell. a. hydrogen bonds b. covalent interactions c. van der Waals forces d. pH changes e. magnetic interactions

ANS: C DIF: Easy REF: 2.6 OBJ: Factual TOP: VI.D.ii

49. Which type of microscopy is particularly useful to study the surfaces of live bacteria?

a. atomic force b. SEM c. TEM d. dark-field e. bright-field

ANS: A DIF: Easy REF: 2.6 OBJ: Applied TOP: VI.D.ii

50. Which technique uses interference patterns from crystallized macromolecules to determine structure at

atomic resolution? a. SEM b. TEM c. cryo-EM d. X-ray diffraction analysis e. atomic force microscopy

ANS: D DIF: Medium REF: 2.7 OBJ: Factual TOP: VII.A.i


290 | Chapter 2 SHORT ANSWER

1. What does the phrase “eagle-eyed” mean? Describe why it is scientifically accurate.

ANS: Eagle-eyed means sharp-sighted. Eagles’ eyes can resolve things eight times as small or eight times as far away as humans’ eyes because eagles’ photoreceptors are much more closely packed.

DIF: Medium REF: 2.1 OBJ: Conceptual TOP: I.A

2. Why is it possible to detect microbes but NOT resolve them?

ANS: Detection is the ability to observe the presence of an object, such as when we detect a group of bacteria in a culture tube. Even though we can detect the group, we can’t resolve individual cells without magnification.

DIF: Easy REF: 2.1 OBJ: Applied TOP: I.B.i

3. List and describe three common shapes of bacteria.

ANS: Bacilli (singular, bacillus) are rod-shaped bacteria. Cocci (singular, coccus) are spherical-shaped bacteria. Spirochetes are tightly coiled spirals or corkscrew-shaped bacteria.

DIF: Easy REF: 2.1 OBJ: Factual TOP: I.C.ii

4. Explain when you would use Bacillus versus bacillus.

ANS: Bacillus refers to a genus of organisms. A genus name is always capitalized and italicized. The term bacillus refers to any rod-shaped microbe, which means that not all bacilli belong to the genus Bacillus.

DIF: Easy REF: 2.1 OBJ: Applied TOP: I.C.ii

5. Describe three conditions that are necessary for electromagnetic radiation to resolve and object.

ANS: There must be contrast between the object and its surroundings. The wavelength of the radiation must be equal to or smaller that the size of the object. The detector must have sufficient resolution for the given wavelength.

DIF: Medium REF: 2.2 OBJ: Factual TOP: II.A.ii


Observing the Microbial Cell | 291 6. List and briefly describe four ways that light interacts with objects.

ANS: 1. Absorption. Light energy is absorbed by an object. 2. Reflection. Wave front bounces off of object at angle equal to its incident angle. 3. Refraction. Bending of light when it enters a substance that slows its speed. 4. Scattering wave front interacts with object of smaller dimension than the wavelength.

DIF: Medium REF: 2.2 OBJ: Applied TOP: II.B.i

7. Compare and contrast the radiation source, the lenses, and the image-capturing device in light and

TEM.

ANS: The radiation source for light microscopy is a light, whereas for EM it is an electron source or tungsten filament. The lenses in the light microscope are glass, whereas magnets are used in EM. The lenses have similar functions and are arranged in the same order in both types of microscopy. Light microscopy uses a condenser lens, whereas the lens in EM is called the projection lens. The image-capturing device for light is the human eye, or sometimes a camera. The image-capturing device for EM is a fluorescent screen.

DIF: Difficult REF: 2.3 | 2.6 OBJ: Conceptual TOP: III.B | VI.A

8. Describe the lens system of a compound microscope including the location of each lens and the

purpose of each.

ANS: The condenser lens is above the light source and functions to concentrate light rays up through the specimen. The objective lenses are immediately above the specimen and the function is to magnify the specimen. A typical light microscope has objective lenses that magnify 10×, 40×, and 100×. The ocular lens is located in the eyepiece and also magnifies the specimen. A typical ocular lens magnifies 10×.

DIF: Easy REF: 2.3 OBJ: Applied TOP: III.B.i

9. Why are stains used in microscopy? Compare and contrast the stains used in light versus electron

microscopy.

ANS: Stains are used to increase the contrast between an object and its surroundings, so as to make it visible. The stains used in light microscopy usually are charged and interact with different cellular components. Positively charged dyes bind to negatively charged cell surfaces. They also are colored, so they impart color to a cell or its components. The stains used for EM are heavy metals or salts, which increase the density of certain components, again increasing contrast. In EM, the image of the microbe is always black and white.

DIF: Difficult REF: 2.3 | 2.6 OBJ: Conceptual TOP: III.D | VI.B


292 | Chapter 2 10. Briefly explain why or how fixatives and stains used in microscopy may introduce artifacts. How

might this be determined?

ANS: A specimen may be fixed in many different ways depending on the type of microscopy, but two examples are heat and chemical fixation. Heat can denature certain cellular components and chemicals can also alter structural integrity. Most stains are charged and interact with various cellular components. These interactions may also lead to alteration in structural characteristics. Sometimes it can be determined if something is an artifact by comparing the results using different forms of microscopy.

DIF: Difficult REF: 2.3 OBJ: Conceptual TOP: III.D

11. Compare and contrast simple and differential stains. Give examples of each.

ANS: A simple stain is used to increase contrast in order to visualize a specimen. Methylene blue, for example, will stain all cells equally. A differential stain allows one to distinguish between cell types or characteristics. The Gram stain procedure is a differential stain. It can be used to distinguish between Gram-positive and Gram-negative cells.

DIF: Medium REF: 2.3 OBJ: Applied TOP: III.E

12. Briefly state the steps in the Gram stain procedure. What would happen if you accidentally forgot to

use the counterstain?

ANS: The Gram stain procedure involves four steps: 1. the primary stain—crystal violet 2. the mordant—Gram’s iodine 3. the decolorizer—ethanol 4. the counterstain—safranin If you left out the counterstain step it would be difficult to detect any Gram-negative microbes that were present in the specimen.

DIF: Medium REF: 2.3 OBJ: Conceptual TOP: III.E.ii.a

13. Why do some bacteria appear purple after being Gram stained and other appear red?

ANS: Gram-negative cells have a few layers of peptidoglycan cell wall and an outer lipopolysaccharide membrane. Gram-positive organisms have several layers of peptidoglycan and no outer membrane. The multiple layers of peptidoglycan retain the crystal violet–iodine complex so appear purple. Gram-negative cells do not retain the crystal violet because there are few layers of peptidoglycan and the outer membrane is disrupted by the decolorizer.

DIF: Medium REF: 2.3 OBJ: Conceptual TOP: III.E.ii.a


Observing the Microbial Cell | 293 14. Explain how the modified condenser in dark-field microscopy is used to make small microbes visible.

Give one advantage and one disadvantage of dark-field microscopy.

ANS: The condenser contains an opaque disk held by three “spider legs” across an open ring. No light travels directly up through the specimen so the only light that reaches the eye is light that is scattered by objects on the slide. This scattered light allows detection of objects that are too small to be resolved by light rays. Advantages include the ability to detect live organisms without staining, detection of very small organisms that can’t be seen with bright-field microscopy, and ability to visualize motility of microbes. Disadvantages are that shapes of objects can’t be easily resolved and particulates may be mistaken for organisms.

DIF: Medium REF: 2.4 OBJ: Applied TOP: IV.A

15. Explain phase-contrast microscopy and give one advantage and one disadvantage of phase-contrast

microscopy.

ANS: Phase-contrast microscopy exploits differences in refractive index between cell components and transforms them into differences in intensity of transmitted light. Advantages are that live cells can be viewed and the organelles of eukaryotes are visible. A disadvantage is that it is less effective for organisms whose cytoplasm as a low refractive index.

DIF: Medium REF: 2.4 OBJ: Applied TOP: IV.B

16. Define a fluorophore and give three examples of how they can be used to label cells.

ANS: A fluorophore is a fluorescent molecule that can be used to stain a specimen for observation with a fluorescence microscope. Some fluorophores, such as DAPI, have affinity for certain cell chemicals. Antibodies can be labeled with fluorescent dyes and reacted with specific targets in immunofluorescence. Short sequences of DNA attached to a fluorophore can be used to hybridize to and label target DNA.

DIF: Difficult REF: 2.5 OBJ: Applied TOP: V.B

17. How does laser scanning confocal microscopy produce images?

ANS: A laser beam is focused onto the specimen and scanned across it in two planes at right angles to each other. The laser beam excites a fluorophore and both the excitation and emitted light are focused together. This results in images with very high resolution.

DIF: Medium REF: 2.5 OBJ: Applied TOP: V.C


294 | Chapter 2 18. Give a few reasons why living organisms may NOT be observed by TEM or SEM.

ANS: In TEM, the specimens are fixed and embedded into a polymer for sectioning. The specimen is then stained with heavy metal to increase contrast. In SEM, the entire organism is shadowed with heavy metal prior to observation. Most importantly, however, the entire optical column of the EM must be maintained under vacuum, and a living specimen would be quickly destroyed by an electron beam.

DIF: Easy REF: 2.6 OBJ: Conceptual TOP: VI.A | VI.B

19. Why is it that a photographic image from an electron microscope is black and white?

ANS: The original image is produced when the electrons bombard a fluorescent screen. The resultant image is processed by a computer to appear as black and white with intensities in the entire range of grays in between.

DIF: Difficult REF: 2.6 OBJ: Conceptual TOP: VI.A

20. Describe three methods of sample preparation for electron microscopy.

ANS: 1. Samples can be embedded in a polymer and cut into thin sections with a microtome, then coated with a heavy metal. 2. Samples can be sprayed onto a copper grid then treated with a heavy metal. 3. Samples may be flash frozen for cryo-EM.

DIF: Medium REF: 2.6 OBJ: Applied TOP: VI.B


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