Microbiology Exam 1 Giorno

Define the term Microbiology:

Microbiology is the study of microscopic organisms, usually less than 1 millimeter in diameter, that can be unicellular or acellular. This includes bacteria, archaea, fungi, protists, and acellular entities like viruses, viroids, satellites, and prions.

Explain Carl Woese’s contributions in establishing the three-domain system for classifying cellular life:

Carl Woese used small subunit ribosomal RNA (SSU rRNA) sequencing to compare organisms. His work showed that prokaryotes were not one single group, but two distinct domains: Bacteria and Archaea. Along with Eukarya, this led to the modern three-domain system of classification.

bacteria, archaea, fungi, protists-

Cellular

viruses, viroids, satellites, prions-

Acellula

unicellular, prokaryotic, peptidoglycan in cell wall, many shapes (rods, cocci, spirals):

Bacteria

unicellular, prokaryotic, no peptidoglycan (different cell wall/membrane), often in extreme environments (salt, heat, methane):

Archaea

eukaryotic, mostly unicellular, may be algae (photosynthetic) or protozoa (animal-like):

Protists

eukaryotic, unicellular (yeast) or multicellular (molds/mushrooms), cell wall made of chitin:

Fungi

nucleic acid (DNA or RNA) + protein coat, need host to replicate:

Viruses

RNA only, infect plants:

Viroids

nucleic acid + helper virus required:

Satellites

infectious proteins, cause neurodegenerative diseases (mad cow):

Prions

How does it affect humans? (Bacteria)

• Positive: Part of our microbiome — help digest food, make vitamins (e.g., E. coli in the gut makes vitamin K).

• Negative: Can cause diseases like plague, tuberculosis, strep throat.

How does it affect humans? (Archaea)

• Positive: Methanogens produce methane used as biofuel; also play a role in nutrient cycling.

• Negative: None are major human pathogens (but some live in extreme environments that affect industries, like corrosion).

How does it affect humans? (Protists)

• Positive: Algae produce oxygen and form the base of aquatic food chains.

• Negative: Protozoa like Plasmodium cause malaria.

How does it affect humans? (Fungi)

• Positive: Source of antibiotics (penicillin), food production (bread, beer, cheese).

• Negative: Can cause infections (athlete’s foot, candidiasis)

How does it affect humans? (Viruses)

• Negative: Cause human diseases like influenza, COVID-19, rabies.

• Positive: Some are used in gene therapy and biotechnology.

How does it affect humans? (Viroids)

Negative: Infect plants, damaging agriculture (e.g., potato spindle tuber disease).

How does it affect humans? (Satellities)

Negative: Cause diseases in plants and animals, but only when paired with a helper virus.

How does it affect humans? (Prions)

Negative: Cause neurodegenerative diseases like Creutzfeldt–Jakob disease and mad cow disease.

What is the RNA World Hypothesis?

The idea that early life was based on RNA, which could both store genetic information and act as a catalyst before DNA and proteins evolved.

What evidence supports the RNA World Hypothesis?

• Ribozymes discovered (RNA molecules with enzymatic activity).

• RNA plays central roles in modern cells (e.g., rRNA in ribosomes catalyzes peptide bonds).

• Experiments show RNA can self-replicate.

• Liposomes could have enclosed RNA to form protocells.

How would you experimentally place a new microbe on a phylogenetic tree using SSU rRNA sequences?

1. Extract DNA from the microbe.

2. Use PCR to amplify SSU rRNA genes.

3. Sequence the gene.

4. Align sequence with known organisms.

5. Count nucleotide differences.

6. Use computer programs to build a phylogenetic tree.

7. Place the new organism based on closest relatives and evolutionary distance.

How did mitochondria and chloroplasts evolve?

Both evolved by endosymbiosis.

What is the origin of mitochondria?

From aerobic proteobacteria.

What is the origin of chloroplasts?

From photosynthetic cyanobacteria.

What similarities do mitochondria and chloroplasts share?

• Both contain their own DNA and ribosomes.

• Both replicate by binary fission.

• Both have double membranes.

• Both are genetically similar to bacteria.

What are the key differences between mitochondria and chloroplasts?

• Mitochondria are in almost all eukaryotes, used for ATP production.

• Chloroplasts are only in plants and algae, used for photosynthesis.

What were Robert Hooke’s contributions to microbiology?

Published Micrographia (1665), the first book of microscopic observations; coined the term "cell."

What were Antonie van Leeuwenhoek’s contributions to microbiology?

: First to observe and describe microorganisms (“animalcules”) with simple microscopes (1674–1676).

What was Louis Pasteur’s contribution to microbiology?

Disproved spontaneous generation with swan-neck flask experiments; developed pasteurization; demonstrated microbes cause fermentation and disease; developed vaccines for rabies and anthrax.

What was Joseph Lister’s contribution to microbiology?

Developed antiseptic surgery techniques, reducing infections by sterilizing instruments and using disinfectants.

What was Robert Koch’s contribution to microbiology?

Proved that specific microbes cause specific diseases; developed Koch’s postulates; discovered anthrax and tuberculosis causation.

What was Martinus Beijerinck’s contribution to microbiology?

Isolated nitrogen-fixing root nodule bacteria; showed viruses (tobacco mosaic virus) are infectious agents smaller than bacteria.

What was Sergei Winogradsky’s contribution to microbiology?

Studied soil microbes; discovered chemolithotrophy and the cycling of sulfur and nitrogen; developed enrichment culture techniques.

What was Emil von Behring’s contribution to microbiology?

Discovered diphtheria antitoxin (serum therapy), advancing immunology.

What was Shibasaburo Kitasato’s contribution to microbiology?

Worked with von Behring on diphtheria antitoxin; also studied tetanus.

What was Elie Metchnikoff’s contribution to microbiology?

Discovered phagocytosis; showed white blood cells engulf and destroy pathogens, laying the foundation of immunology.

Outline an experiment to decide if a microbe causes a disease (Koch’s Postulates).

1. The microbe must be present in every case of the disease but absent from healthy organisms.

2. Isolate and grow the microbe in pure culture.

3. Inoculate a healthy host; disease must result.

4. Re-isolate the same microbe from the experimentally infected host.

What difficulties arise when applying Koch’s postulates to human-specific diseases?

• Some microbes cannot be cultured outside a host (e.g., viruses, obligate parasites).

• Lack of appropriate animal models for human diseases.

• Ethical issues prevent experimental infection of humans.

• Genetic variability of microbes (mutations may alter virulence).

Why do microbiologists say microbiology is experiencing a “second golden age”?

Because advances in molecular and genomic methods allow scientists to study microbes at the DNA and systems level, leading to rapid discoveries in microbial diversity, host-pathogen interactions, and biotechnology — rivaling the first golden age of microbiology.

Construct a concept map/table that illustrates the diverse nature of microbiology and how it has improved human conditions.

• Medical microbiology → Identifies pathogens, develops treatments, prevents disease.

• Public health/epidemiology → Controls disease spread in populations, monitors outbreaks.

• Immunology → Vaccines, antibodies, autoimmune disease research.

• Microbial ecology → Studies nutrient cycling, climate impact, human microbiome, bioremediation.

• Agricultural microbiology → Soil fertility, nitrogen-fixation, crop yield, pest control, livestock health.

• Food microbiology → Improves food production (yogurt, cheese, beer), prevents spoilage, ensures safety.

• Industrial microbiology → Produces antibiotics, vaccines, enzymes, vitamins, biofuels.

• Microbial physiology/genetics/molecular biology → Understand metabolism, stress responses, gene regulation; drives biotechnology.

How did the methods used to classify microbes change, particularly in the last half of the 20th century? What was the result?

Earlier classification was based on morphology & metabolism. With electron microscopy and molecular sequencing (especially SSU rRNA by Carl Woese), microbes were reclassified.

• Result: discovery of Archaea as separate from Bacteria → Three-domain system (Bacteria, Archaea, Eukarya)

Explain the “RNA world hypothesis” (Fig. 1.5). What discoveries led them to propose this hypothesis?

The RNA world hypothesis suggests that early life was based on RNA before DNA and proteins became dominant molecules. Scientists proposed this idea because RNA is unique in that it can both store genetic information like DNA and catalyze chemical reactions like proteins. Evidence supporting this includes the discovery of ribozymes, RNA molecules with enzymatic activity, which demonstrated that RNA could potentially catalyze its own replication. The hypothesis was also supported by the idea that an RNA molecule enclosed in a lipid bilayer could represent one of the earliest self-replicating entities on Earth. This dual functionality makes RNA a logical candidate for the first biomolecule of life.

Describe two reasons RNA is thought to be the first self-replicating biomolecule:

• RNA can store genetic information (like DNA).

• RNA can act as a catalyst (ribozymes can catalyze chemical reactions, including self-replication).
  👉 Together, this suggests early life could have depended on RNA before DNA and proteins evolved

Why and how is SSU rRNA used to determine the relatedness between microorganisms?

• SSU rRNA is present in all cellular organisms and performs the same essential function (protein synthesis).

• The gene is highly conserved, but also has variable regions → making it useful for comparing organisms across domains.

• By comparing SSU rRNA sequences, scientists can build phylogenetic trees to show evolutionary relationships (this is what Carl Woese did to establish the three-domain system).

Explain the endosymbiotic hypothesis of the origin of mitochondria, hydrogenosomes, and chloroplasts. List two pieces of evidence that support this hypothesis:

• They originated when early eukaryotic cells engulfed bacterial cells that lived inside them as endosymbionts.

• Evidence:

  1. They have double membranes (consistent with engulfing).

  2. They contain their own circular DNA and 70S ribosomes, similar to bacteria.

  3. They reproduce by a binary fission-like process, not mitosis.

A permanent change in the DNA sequence of an organism; changes are inherited vertically (from parent to offspring):

Mutation

The direct transfer of genetic material between unrelated organisms (e.g., transformation, transduction, conjugation). It allows microbes to rapidly acquire new traits, like antibiotic resistance:

Horizontal Gene Transfer (HGT)

What did Pasteur prove when he showed that a cotton plug that had filtered air would trigger microbial growth when transferred to a sterile medium? What argument was he addressing?

Pasteur proved that microbes are present in the air and can contaminate sterile environments. He was addressing the spontaneous generation argument, showing that microbes do not arise spontaneously but come from existing microorganisms.

What are the three domains of life and which contain microorganisms?

The three domains are Bacteria, Archaea, and Eukarya.

• Microorganisms are found in all three domains:

  • Bacteria → all are microbes.

  • Archaea → all are microbes.

  • Eukarya → microbial members include protists and fungi.

What organism type evolved first?

The first organisms to evolve were microbial prokaryotes (Bacteria and Archaea). They appeared billions of years before multicellular eukaryotes.

What are some of the beneficial products made by microorganisms?

• Antibiotics (e.g., penicillin from fungi).

• Vitamins (e.g., vitamin K from gut bacteria).

• Food & drink (bread, beer, cheese, yogurt).

• Biogeochemical cycling (carbon, nitrogen, sulfur cycles).

• Biotechnology (insulin, enzymes, biofuels).

Explain the evolution of mitochondria and chloroplasts:

• Endosymbiotic hypothesis: Ancestral eukaryotic cells engulfed bacterial cells that became endosymbionts.

• Mitochondria likely came from aerobic bacteria (proteobacteria).

• Chloroplasts came from cyanobacteria.

• Evidence:

  1. Both have double membranes.

  2. Both contain circular DNA and 70S ribosomes.

  3. Both replicate by a process similar to binary fission.

Koch’s postulates: what is its purpose, when is it used, what are the steps, when does it not work?

Koch’s postulates were developed to demonstrate that a specific microorganism is the cause of a specific disease. The purpose of these postulates was to establish a clear link between microbes and disease, forming the foundation of the germ theory of disease. The steps are as follows:

1. The suspected microbe must be present in all cases of the disease but absent from healthy individuals.

2. The microbe must be isolated from the diseased host and grown in pure culture.

3. The pure culture must cause the disease when inoculated into a healthy, susceptible host.

4. The same microbe must be re-isolated from the experimentally infected host.

However, Koch’s postulates have limitations. They do not work for microbes that cannot be cultured in the lab (e.g., certain viruses), for diseases caused by multiple organisms together, or for pathogens that only infect humans (where ethical issues prevent experimental infection). Despite these limitations, they remain a cornerstone of medical microbiology.

Why aren’t viruses, viroids, satellites, and prions included in the three domain system?

The three-domain system classifies all cellular life into Bacteria, Archaea, and Eukarya based on small subunit rRNA sequences. Viruses, viroids, satellites, and prions are excluded because they are acellular and therefore do not share the same characteristics as living cells.

• Viruses consist of nucleic acid (DNA or RNA) surrounded by a protein coat and require a host to replicate.

• Viroids are small, naked RNA molecules that infect plants and lack protein-coding ability.

• Satellites are nucleic acid molecules that require a helper virus to replicate.

• Prions are infectious proteins without nucleic acids, which cause neurodegenerative diseases.

Since these agents lack the basic features of cellular life (such as ribosomes, metabolism, and rRNA genes), they cannot be placed within the three-domain system. Instead, they are studied as unique acellular infectious agents.

What happens to light when it passes from air (low refractive index) into glass (high refractive index)?

It slows down and bends toward the normal.

What happens to light when it passes from glass back into air?

It speeds up and bends away from the normal.

What is the relationship between lens strength and focal length?

• A shorter focal length = stronger lens = higher magnification.

• A longer focal length = weaker lens = lower magnification.

What is refraction?

The bending of light when it passes between substances with different refractive indices.

What is refractive index?

A measure of how much a medium slows the velocity of light compared to air.

What is the focal point?

The spot where parallel light rays converge after passing through a lens.

What is focal length?

The distance from the center of a lens to the focal point.

What is the function of the condenser lens in a light microscope?

The condenser lens focuses light onto the specimen to improve image clarity.

What is the function of the objective lens in a light microscope?

The objective lens magnifies the specimen and forms the primary image.

What is the function of the ocular (eyepiece) lens in a light microscope?

The ocular lens further magnifies the primary image so it can be viewed by the observer.

How does wavelength affect resolution in microscopy?

Shorter wavelengths (e.g., blue light) improve resolution because they can distinguish smaller details.

How does refractive index affect resolution?

A higher refractive index reduces light loss (less bending), improving resolution.

How does numerical aperture (NA) affect resolution?

Higher NA means the lens gathers more light, producing a clearer, higher-resolution image.

What is the relationship between resolution and magnification?

Magnification enlarges the image, but resolution determines clarity; without resolution, magnification is “empty magnification.”

What happens if a microscopist uses an oil immersion objective without oil?

Light rays scatter, lowering numerical aperture → reduced resolution → blurry image.

Why don’t most light microscopes use 30× ocular lenses?

Because magnification would increase without improved resolution → image gets larger but blurrier (empty magnification).

What image does a dark-field microscope produce and what is it used for?

Bright objects on a dark background; useful for live, unstained microbes like spirochetes.

What image does a phase-contrast microscope produce and what is it used for?

Enhanced contrast of internal structures; used to view live, unstained microbial and eukaryotic cells.

What image does a differential interference contrast (DIC) microscope produce and what is it used for?

3D, colorful images; used to view eukaryotic cell details like vacuoles, granules, and endospores.

What image does an epifluorescence microscope produce and what is it used for?

Fluorescent glowing structures against a dark background; used for detecting specific pathogens or labeled molecules.

What image does a confocal microscope produce and what is it used for?

Sharp 3D images by scanning laser and computer reconstruction; used for biofilms and thick specimens.

What fixation is used for bacteria and archaea?

Heat fixation

What fixation is used for protists?

Chemical fixation

What does simple staining show?

Shape, size, and arrangement of cells.

What does the Gram stain separate bacteria into?

Gram-positive (purple) and Gram-negative (red).

What happens in the first step of Gram staining (crystal violet)?

All cells turn purple

What happens in the iodine step of Gram staining?

Cells remain purple (CV-I complex forms)

What happens when alcohol is added in Gram staining?

• Gram+ stay purple

• Gram– become colorless.

What happens in the safranin step of Gram staining?

• Gram+ stay purple

• Gram– turn red/pink.

What are the two general types of fixation?

• Heat fixation

• chemical fixation.

Which fixation is used for bacteria? For protists?

Heat fixation → bacteria. Chemical fixation → protists.

Why might all cells look red after Gram staining?

Over-decolorization or no iodine used.

Are capsular and flagellar stains differential?

No — they are special stains for specific structures.

Create a concept map, illustration, or table that compares TEMs to light microscopes.

• Light microscope: uses visible light, glass lenses, ~1000–1500× mag, 0.2 μm resolution, live or stained cells.

• TEM: uses electron beam, electromagnets, >100,000× mag, 0.2 nm resolution, very thin slices, specimens dead/fixed.

Decide when it would be best to examine a microbe by TEM, SEM, or cryotomography.

• TEM: best for internal cell details (organelles, structures).

• SEM: best for 3D surface images.

• Cryotomography: best for 3D view of intact cells in near-native state.

Why does an electron microscope have much greater resolution than a light microscope?

Electrons have a much shorter wavelength than visible light → higher resolving power.

Where is the electron gun located relative to the sample? Why must a TEM use high vacuum and very thin sections?

• Electron gun: above the specimen.

• High vacuum: prevents electron scattering by air.

• Thin sections: allow electrons to pass through specimen for clear image.

Difference between SEM & TEM? How to tell from micrograph?

• SEM: scans surface → 3D external view.

• TEM: electrons pass through → detailed internal structures.

• Micrograph clue:

• 3D surface = SEM

• thin, detailed internal slice = TEM

• full intact 3D reconstruction = cryotomography.

Bright field microscope – key facts?

• Illumination: Visible light

• Magnification/Resolution: ~1000–1500× ; 0.2 μm

• Sample state: Alive or dead (usually stained dead)

• Contrast: Stains / light absorption

• Image: 2D, flat, color if stained

Fluorescence microscope – key facts?

• Illumination: UV/blue light excites fluorochromes

• Magnification/Resolution: ~1000–1500× ; 0.2 μm

• Sample state: Dead or alive (with special dyes)

• Contrast: Fluorescent dyes (fluorochromes)

• Image: Bright colors on dark background

Confocal microscope – key facts?

• Illumination: Laser light scans specimen

• Magnification/Resolution: ~1000–1500× ; 0.2 μm

• Sample state: Usually dead (can be live with methods)

• Contrast: Fluorescent dyes + optical sectioning

• Image: Sharp thin “slices,” can compile into 3D, color possible

Transmission electron microscope (TEM) – key facts?

• Illumination: Electron beam passes through thin slice

• Magnification/Resolution: >100,000× ; 0.2 nm

• Sample state: Dead, ultra-thin slices

• Contrast: Heavy metal stains (electron-dense)

• Image: 2D internal structures, black & white

Scanning electron microscope (SEM) – key facts?

• Illumination: Electron beam scans surface

• Magnification/Resolution: ~10,000–50,000× ; 10 nm

• Sample state: Dead, coated in metal

• Contrast: Scattered electrons from surface

• Image: 3D surface details, black & white (often false-colored)

What are the parts of microscope (fig. 2.3)?