Microbiology - Unit I

Lectures 1, 2, 3, 4, 5

What is a microbe?

A microscopic organism (often unicellular) with a genome that directs its structure and function

What size range do microbes typically fall into?

From millimeters to 0.2 micrometers; often only visible with electron microscopes.

What group of microbes are extremophiles that rarely infect humans?

Archaea

How have microbes shaped human history?

By making food (e.g., bread, wine), aiding mining (lithotrophs), and causing epidemics.

Who built the first compound microscope and coined the term "cell"?

Robert Hooke

Who first observed single-celled microbes?

Antonie van Leeuwenhoek

What experiment disproved spontaneous generation?

Louis Pasteur’s swan-neck flask experiment.

What is the Germ Theory of Disease?

The idea that many diseases are caused by microbes

What are Koch’s Postulates used for?

To link a specific microbe to a specific disease.

Who pioneered vaccination using cowpox to protect against smallpox?

Edward Jenner

Who developed vaccines from attenuated strains (e.g., rabies)?

Louis Pasteur

What is the principle of immunization?

Stimulating immunity with an attenuated or inactive pathogen

Who promoted handwashing with chlorine to reduce mortality in 1847?

Ignaz Semmelweis

Who developed antiseptic surgery using carbolic acid?

Joseph Lister

Who discovered penicillin?

Alexander Fleming

What did Ivanovsky discover about tobacco mosaic disease?

It passed through filters that block microbes, suggesting viruses

What role do microbes play in ecology?

Cycling essential minerals and nutrients (e.g., nitrogen, oxygen)

What is geochemical cycling?

The global interconversion of minerals by living organisms

What did Carl Woese contribute to microbiology?

He proposed the three domains of life based on 16S rRNA

What is the endosymbiotic theory?

Eukaryotic organelles (mitochondria, chloroplasts) evolved from engulfed prokaryotes

What two instruments transformed cell biology?

The electron microscope and the ultracentrifuge

Who discovered bacterial transformation?

Frederick Griffith (1928).

Who demonstrated that DNA is the transforming principle?

Avery, MacLeod, and McCarty (1944).

Who used x-ray crystallography to reveal DNA’s structure?

Rosalind Franklin

Who determined the structure of DNA through complementary base pairing?

Watson and Crick.

What is PCR and what enzyme makes it possible?

Polymerase chain reaction; uses Taq polymerase (heat-stable)

What are the three domains of life?

Bacteria, Archaea, and Eukarya

Whose swan-neck flask experiment disproved spontaneous generation?

Louis Pasteur.

Who developed Postulates to link microbes with disease?

Robert Koch

Who pioneered vaccination with cowpox against smallpox?

Edward Jenner

Who promoted handwashing with chlorine to reduce mortality in hospitals?

Ignaz Semmelweis.

Who discovered penicillin by observing mold killing bacterial colonies?

Alexander Fleming but mostly Florey and Chain

How did Pasteur’s swan-neck flask experiment directly challenge earlier beliefs in spontaneous generation?

It showed that microbes did not appear in sterilized broth unless exposed to outside air, disproving spontaneous generation.

Why are Koch’s Postulates still important today in microbiology and medicine?

They provide a framework for proving a microbe causes a specific disease, forming the basis of infectious disease research.

How did Semmelweis’ and Lister’s work together advance public health?

Semmelweis reduced infections with handwashing, and Lister extended the idea to surgery with antiseptics—both laid the foundation for sterile medical practice.

Why was Griffith’s discovery of transformation so groundbreaking even before DNA was understood as the genetic material?

It showed that “something” (later proven to be DNA) could transfer heritable traits between bacteria.

How did Franklin’s crystallography data contribute to Watson and Crick’s model of DNA?

Her images provided critical evidence of the helical structure and dimensions of DNA, guiding the double helix model.

What is resolution in microscopy?

The smallest distance by which two objects can be separated and still be distinguished.

What is the resolution limit of the human retina?

About 150 micrometers (1/7 mm).

What is detection in microscopy?

The ability to determine the presence of an object, not necessarily to resolve it.

What does magnification mean in microscopy?

An increase in the apparent size of an image to resolve smaller separations between objects.

Typical size range of eukaryotic microbes (protozoa, algae, fungi)?

10–100 micrometers.

Typical size range of prokaryotic microbes (bacteria, archaea)?

1.4–10 micrometers

What pore size is used to filter out most bacteria from a liquid suspension?

0.22 micrometers.

What are the three common bacterial shapes?

Bacilli (rods), Cocci (spheres), Spirilla/Spirochetes (spirals).

Wavelength of visible light?

400–750 nanometers.

Conditions required for electromagnetic radiation to resolve an object?

Contrast between object and medium, wavelength smaller than the object, and magnification.

What happens when light is absorbed by an object?

The photon’s energy is acquired, often increasing heat.

What is reflection?

The wavefront bounces off the surface of an object.

What is refraction?

The bending of light as it enters a substance that slows its speed (basis of magnification).

What is scattering?

a phenomenon where light deviates from its path due to interaction with the sample, and it can be either a detrimental effect that degrades image clarity, contrast, and resolution, or a useful principle for label-free imaging technique

What limits our ability to see detail: magnification or resolution?

Resolution

What is empty magnification?

Magnification without increased detail (like pixelation).

Bright-field microscopy generates what kind of image?

A dark object over a light background.

How can resolution be increased in bright-field microscopy?

Use shorter-wavelength light or immersion oil at high magnification.

What is the total magnification formula in a compound microscope?

Ocular lens magnification × objective lens magnification

Example: With a 10x ocular and 30x objective, what is the total magnification?

300x

What is a wet mount?

A simple specimen prep: microbes in a drop of water on a slide with a coverslip.

Advantages of a wet mount?

Observes cells in natural state, allows motility.

Disadvantages of a wet mount?

Low contrast (no dye), sample may dry quickly.

What does fixation do?

Kills cells and adheres them to slide, but may distort shape.

What does staining do?

Adds color for contrast and visualization of structures.

What is a simple stain? Example?

Adds dark color to cells only (e.g., methylene blue).

What is a differential stain? Example?

Stains one kind of cell/structure but not another (e.g., Gram stain).

Gram-positive bacteria retain crystal violet because?

Thick peptidoglycan cell wall.

Gram-negative bacteria appear what color after Gram stain?

Pink/red (thin peptidoglycan, safranin counterstain).

What does an acid-fast stain detect?

Mycobacterium (stains mycolic acids with carbolfuchsin).

What does a spore stain detect?

Spores of Bacillus and Clostridium (malachite green).

What does a negative stain detect?

Capsules (background stains, capsule is clear halo).

What is required for fluorescence microscopy?

A fluorescent stain or gene (e.g., GFP, DAPI).

What happens in fluorescence microscopy?

Specimen absorbs light at excitation wavelength and emits at longer emission wavelength

What is a fluorophore?

A fluorescent chemical compound used for labeling.

What are three ways fluorophores label cells?

1. Chemical affinity (e.g., DAPI binds DNA)

2. labeled antibodies (immunofluorescence)

3. DNA hybridization (probe bound to fluorophore).

What does confocal laser scanning microscopy (CLSM) provide?

3D reconstructed images (Z-stacks, optical sectioning).

How do electrons behave in electron microscopy?

Like light waves but with much smaller wavelength, allowing nanometer-scale resolution.

How must samples be prepared for electron microscopy?

Coated with heavy metals (e.g., gold) and placed in vacuum.

Transmission electron microscopy (TEM) reveals what?

Internal structures (electrons pass through specimen).

Scanning electron microscopy (SEM) reveals what?

External surface features (electrons scan specimen surface).

What does X-ray crystallography require?

Crystallized samples.

What can X-ray crystallography reveal?

Atomic structure of molecules like proteins, viral capsids, DNA.

Why can’t light microscopes visualize proteins?

Visible light wavelength is too large compared to proteins (~3 nm).

Which lens property is most important for magnification?

Refraction

If using a 10x ocular and 45x objective lens, what is the total magnification?

450x

What three fundamental traits do most bacteria share?

(1) Thick, complex outer envelope; (2) Compact genome; (3) Tightly coordinated cell functions.

What are the two main domains of prokaryotes? How do they differ from eukaryotes?

• Prokaryotes = Bacteria and Archaea.

• They lack a membrane-bound nucleus and are usually <1 µm

• Eukaryotes have a nucleus and are typically 10-100 µm

Why might small cell size benefit prokaryotes?

Smaller cells have a larger surface-area-to-volume ratio, which speeds up nutrient uptake and waste elimination. This enables faster growth and division, which is advantageous in changing environments.

How was the bacterial cell originally envisioned, and how do we see it now?

Originally seen as a bag of “soup” full of floating enzymes; now known to be an ordered, flexible structure where parts fit together.

How might cell organization affect drug targeting?

• If cellular components are spatially organized, some may be shielded or sequestered, making them harder for drugs to reach.

• Knowing the spatial organization helps design antibiotics that get to the right target.

Which structures are unique to bacteria vs also found in eukaryotes?

• Unique: peptidoglycan cell wall, nucleoid instead of nucleus, hopanoids, pili, magnetosomes.

• Also found in eukaryotes: cytoplasm, membranes, flagella-like structures (though structurally different).

How might temperature adaptation differ in archaea with unique lipids?

• Archaeal membranes have ether-linked isoprenoid chains, which are more stable at high temperatures and extremes of pH than bacterial ester-linked lipids.

• This allows archaea to survive in harsher environments.

How does antibiotic overuse drive resistance evolution?

• Overuse exerts strong selection pressure.

• Bacteria with resistance genes survive and reproduce, spreading those genes.

• Horizontal gene transfer accelerates the spread of resistance.

How might the outer membrane affect antibiotic entry?

• Gram-negative outer membranes act as an extra barrier.

• LPS and porins restrict or slow the entry of many antibiotics, making Gram-negative bacteria more inherently resistant to certain drugs.

Why might different polysaccharides evolve in different lineages?

• Different environments and evolutionary histories led to different structural needs.

• For example, chitin in fungi resists enzymatic degradation in soil; cellulose in algae provides rigidity but is lighter for aquatic conditions.

Why is coupling advantageous in fast-growing bacteria?

• It speeds up protein production because mRNA doesn’t have to be transported out of a nucleus first.

• This rapid response helps bacteria adapt quickly to environmental changes

How might overlapping rounds of replication benefit bacteria?

• They can start a new round of DNA replication before the previous one ends.

• This allows extremely rapid cell division (shorter generation times) when nutrients are abundant.

How might pili relate to pathogenicity?

• Pili enable bacteria to

  • adhere to host tissues

  • form biofilms

  • exchange virulence plasmids.

• All these increase their ability to colonize and cause disease.

Why do antibiotics often target 70S ribosomes but not 80S?

Bacterial (70S) ribosomes have structural differences from eukaryotic (80S) ones.

Antibiotics can exploit these differences to inhibit bacterial protein synthesis without harming host cells.

How might cytoskeleton differences affect motility compared to bacteria?

• Bacteria move mainly via rotary flagella.

• Eukaryotes use actin and microtubules for complex movements like crawling or undulating cilia/flagella.

• The cytoskeleton allows more varied, controlled movement and intracellular transport.

How might combining structural and genetic approaches give a fuller picture?

• Structure shows where parts are; genetics shows what they do.

• Together, you can see both form and function

  • for example, deleting a gene and observing how a structure changes or disappears.

Why is it important to distinguish homologous from analogous structures in microbiology?

• Homologous structures share evolutionary origin

• Analogous ones evolved independently

• Distinguishing them helps avoid incorrect assumptions about function, ancestry, or drug targets.