BIOL 3200 - Exam 1

Microbiology Exam 1

Microbes

a living organism that requires a microscope to be seen

What are the exceptions to Microbes?

1. Supersize microbial cells

2. Microbial communities

3. Viruses

Robert Hooke

1. built the first compound microscope

2. coined the term “cell” after observing a cork

3. observed mold

4. published Micrographia, which was the first manuscript that illustrated objects under the microscope

Anton Van Leeuwenhoek

1. built single-lens magnifiers, complete with a sample holder and focus adjustment

2. first to observe single-celled microbes and called them “small animals"

3. a cloth draper

Francesco Redi

1. showed that maggots in decaying meat were offspring of flies

John Needham

Lazzaro Spallanzani

1. showed that a sealed flask of meat broth sterilied by boiling failed to grow microbes

Louis Pasteur

1. devised “swan-neck” flasks

2. showed that after boiling, the contents remain free of microbial growth, despite access to air

3. disproved spontaneous generation

4. proposed Biogenesis

5. developed the first vaccines based on attenuated (weakened) strains

   • Fowl cholera aka chicken cholera = bacterial disease

   • Rabies = viral disease

Mr. Onesimus and Lady Montagu

1. immunization through innocuation, which was the process of immunizing someone against a disease by introducing infective materials or microorganisms into the body

2. Onesimus = African slave, introduced into the American Colonies

3. Montagu = introduced to Europe from Turkey

Edward Jenner

1. deliberately infected patients with matter from cowpox lesions

2. practice of cowpox innoculation was called vaccination

Florence Nightingale

1. first to recognize disease in warfare

2. showed the deaths of soldiers due to various causes, she devised the “polar area chart”

Robert Koch

1. german physician and founder of the scientific method of microbiology

2. applied methods to numerous lethal diseases around the world - known as the Koch’s Postulates

   1. Microbe is always present in diseased host - not always true

      • absent in healthy - immune system (could appear helthy, but not actually health)

   2. Microbe is grown in pure culture - not always true

      • no other microbes present - majority of microbes do not grow in a media

   3. Introduce pure microbe into healthy host - not always true

      • individual becomes sick - immune system (could not be affected)

   4. same microbe re-isolated from now-sick individual - usually true

Ignaz Semmelweis

1. ordered doctors to wash their hands with chlorine, an antiseptic agent

   • mortality rates of woman in childbirth fell

Joseph Lister

1. developed carbolic acid to treat wounds and clean surgical instruments

   • from this, aseptic surgery was developed - environments completed microbe-free

Alexander Fleming

1. rediscovered that Penicillium mold generated a substance that kills bacteria

   • Later, Howard Florey and Ernest Chain purified penicillin (first commerical antibiotic to save human lives)

Sergei Winogradsky

1. Russian scientist who was among the first to study microbes in natural habitats

2. Discovered lithotrophs (feed on inorganic minerals)

3. Developed enrichment cultures

4. Built the Winogradsky column

   • a wetland model ecosystem containing regions of enrichment for microbes of diverse metabolism

Carolus Linnaeus

called the microbial world “chaos”

Lynn Margulis

1. proposed mitochondria and chloroplasts, evolved by endosymbiosis from prokaryotic cells engulfed by pre-eukaryotes

   • proposed the endosymbiosis theory - species evolved from more than one ancestry

     • mitochondrial and chloroplast DNA

       • has double membrane

       • has own DNA very similar to Bacterial DNA

     • mitochondrial and chloroplast ribosomes

       • ribosomes usually on rough ER for human cells, here they are much closer related to Bacterial ribosomes

     • size and morphology

Carl Woese

1. studying recently discovered prokaryotes that

   • live in hot springs

   • produce methane

2. analysis of the DNA that codes for SS rRNA revealed that these prokaryotes were a distinct form of life

   • he and George Fox, called them archaea (based on rDNA sequence, archae are similar to eukarya)

Rosalind Franklin

used x-ray crystallography to determine that DNA is a double helix

What difficulties did early taxonomists encounter when attempting to classify microbes?

1. Resolution of the light microscope was too low

   • Challenge was overcome via advances in biochemistry and microscope (ex: gram positive and gram negative)

2. Microbial species are hard to define

What type of organisms are included under the kingdom Protista?

1. microbes are neither plant nor animal

   • protista = Monera

2. Later, Monera was divided into two groups

   • Eukaryotic protists (protozoa and algae) = nucleus

   • Prokaryotic bacteria = no true nucleus

What are the five kingdoms proposed by Robert Whittaker?

1. Kingdom of Protists

   • more of a miscullaneous group

2. Kingdom of Monerans

   • more of a miscullaneous group

3. Kingdom of Fungi

4. Kingdom of Plants

5. Kingdom of Animals

What is the endosymbiotic theory?

A theory that states that mitochondria and chloroplasts evolved from free-living prokaryotic cells that were engulfed by early eukaryotic cells and became permanent organelles.

• Evidence: Mitochondrial and Chloroplast DNA, Mitochondrial and Chloroplast Ribosomes, and the size & morphology

How was the ‘Three Domain’ Classification of Life determined?

1. Woese’s discovery grouped the five kingdoms to three equally distinct groups (domains)

   1. Bacteria

   2. Archaea

   3. Eukarya

2. In the three-domain model:

   • Mitochondria derived from proteobacteria (aka pseudomonadota)

   • Chroloplasts derived from cyanobacteria

What arguments support the classification of Archaea as a third domain of life?

1. Archaea DNA sequence

2. Introns are common

3. RNA polymerase: Eukaryotic homologs

4. Transcription factors: Eukaryotic homologs

5. Resistant to ribosome sensitivity to chloramphenicol, kanamycin, and streptomycin

6. Translation initiator: Methionine (except mitochondria and chloroplasts use formylmethionine)

How does a bright-field light microscope work?

1. generates a dark image of an object over a ligher background

2. to increase resolution

   • shorter wavelegnth (400-750 nm)

   • increase contrast - adding color

   • gather as much light - using immerson oil

3. specimen will absorb light to appear darker

What is the purpose for a light microscope?

helps determine:

1. size

2. shape

3. color (gram ±)

4. arrangement

How does a darkfield light microscope work?

1. microbes will be visualized as halos of bright light against a dark background

   • resolves very thin objects

2. light will hit the speciment at an angle and bounce off

What is the purpose of a darkfield light microscope?

1. to view live microorganisms

2. to view very thin organisms

How does a phase-contrast light microscope work?

1. refracted and transmitted light shifted out of phase

   • reveals differences in refractive index (n)

2. changes speed of light

What is the purpose of a phase contrast light microscope?

1. to view internal structures of a living cell

How does a fluorescent light microscope work?

1. speciment aborbs shorter wavelgenth light, and then emits longer wavelength light

   • light is absorbed by a fluorophore and then emits color

What is the purpose of a fluoresence light microscope?

1. to view more details within a structure

What is the difference between excitation and emission wavelength?

1. excitation: absorbs light at a specific wavelength

2. emission: emits light at a longer wavelength

Which has a higher wavelength? Excitation or Emission?

Emission wavelength is HIGHER than excitation wavelength

How does a scanning electron microscope work?

A scanning electron microscope (SEM) works by scanning a focused beam of electrons across the surface of a specimen. The electrons interact with the surface and generate signals that are detected and used to produce a high-resolution, three-dimensional image of external structures

What is the purpose of a scanning electron microscope?

The purpose of a scanning electron microscope (SEM) is to visualize the external surface and morphology of cells or specimens in high detail, producing three-dimensional images of surface structures

How does a transmission electron microscope work?

A transmission electron microscope (TEM) works by passing a beam of electrons through an extremely thin specimen. The electrons that transmit through the sample are detected and used to form a high-resolution image of internal cell structure

What is the purpose of a transmission electron microscope?

The purpose of a transmission electron microscope (TEM) is to visualize the internal structures of cells at very high resolution by examining thin sections of a specimen

How do you calculate numerical aperture?

NA = n * sin (degrees of angle)

What is the index refreaction (n) for a vaccuum?

1.00

What is the index refreaction (n) for air?

1.000277

What is the index refreaction (n) for water?

1.33

What is the index refreaction (n) for ice?

1.31

What is the index refreaction (n) for glass/oil?

1.5

What is the index refreaction (n) for a diamond?

2.42

What is magnification of a microscope?

the process of enlarging an image so that individual cells and their morphology can be seen more clearly. It can be achieved by lenses that refract light, making objects appear larger than their actual size

What is detection of a microscope?

ability to determine the presence of an object

What is resolution of a microscope?

the smallest distance by which two objects can be seperated and still be distinguished

What is the shape of bacilli bacteria?

rods

What is the shape of a cocci bacteria?

spheres

What spiral form of bacteria has a flexible cell wall?

spirochetes

What spiral form of bacteria has a rigid cell wall?

spirilla

What is a simple stain?

it adds dark color, specifically to cells, but not to the external medium or surrounding tissues

• only 1 color is added; all cells will be that color

What is a differential stain?

it stains one kind of cell one color and the other cell a different color or no color

• ex: gram stain or acid-fast stain

How does immunofluorescence help resolve very thin structures or organisms?

It uses antibodies tagged with fluorescent dyes that bind specifically to target antigens. When exposed to excitation light, the fluorophore emits light at a longer wavelength, producing high contrast that allows thin structures or small organisms to be clearly visualized

What are the structural similarities between Bacteria & Archaea?

Both Bacteria and Archaea are prokaryotic cells that lack a nucleus and membrane-bound organelles. Structurally, both possess a cell membrane composed of phospholipids with embedded proteins, a cytoplasm, and a nucleoid region containing DNA. Many members of both groups also have a cell wall and may contain external structures such as flagella or surface layers

What are the structural differences between Bacteria & Archaea?

• Bacteria have cell membranes made of phospholipids with ester-linked fatty acids and cell walls that typically contain peptidoglycan.

• Archaea have cell membranes with ether-linked, branched isoprenoid lipids and do not contain peptidoglycan in their cell walls; instead, they may have S-layers or other unique wall structures, and their membranes may form a bilayer or a fused monolayer

What are the cell membrane similarities between Bacteria & Archaea?

Both Bacteria and Archaea have a cell membrane composed of phospholipids and proteins that forms a selective barrier around the cell. In both groups, the membrane is involved in transport of molecules, energy production, and maintaining cell integrity

What are the cell membrane differences between Bacteria & Archaea?

• Bacterial cell membranes are composed of phospholipids with ester-linked fatty acids forming a bilayer.

• Archaeal cell membranes contain phospholipids with ether-linked fatty acids, often made of branched isoprenoid chains, and may form a bilayer or a fused monolayer, allowing them to survive extreme environments

How are materials transported across the Cell Membrane?

Materials are transported across the cell membrane through membrane transport proteins or by diffusion. Small or nonpolar molecules move by passive transport down their concentration gradient, while polar or charged molecules require specific transport proteins and may move by active transport using energy (ATP)

What is active transport?

the movement of molecules across the cell membrane against their concentration gradient, which requires energy, usually in the form of ATP, and occurs through specific transport proteins

What is passive transport?

the movement of molecules across the cell membrane along their concentration gradient (from high to low concentration) without the use of energy. This process occurs naturally and does not require ATP

What are the structural similarities between Gram Positive & Gram Negative?

Both Gram-positive and Gram-negative bacteria have a

• cytoplasmic (cell) membrane made of a phospholipid bilayer with embedded proteins

• a cell wall containing peptidoglycan composed of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).

• In both groups, peptidoglycan provides rigidity and protection to the cell

What are the structural differences between Gram Positive & Gram Negative?

• Gram-positive bacteria have a thick peptidoglycan layer, lack an outer membrane, and commonly contain teichoic acids in their cell wall.

• Gram-negative bacteria have a thin peptidoglycan layer, an outer membrane containing lipopolysaccharide (LPS), and a periplasmic space between the inner and outer membranes, making their cell envelope more complex

What is being referred to by the number: 1

glycosidic bonds

What is being referred to by the number: 2

D-Alanine (D-Ala)

What is being referred to by the number: 3

• Diaminopimelic acid (DAP) in Gram Negative

• L-Lys in Gram Positive

What is being referred to by the number: 4

N-acetylmuramic acid aka NAM (square)

What is being referred to by the number: 5

N-acetylglucosamine aka NAG (circle)

What is being referred to by the number: 6

Peptide bonds in Gram Negative

Pentaglycine interbridge (linkage) in Gram Positive

What are the peptidoglycan cell wall structure differences and similarities between E. coli and Staphylococcus aureus.

• E. Coli is Gram Negative = peptide bonds between amino acid structures and DAP in the amino acid structure

• Staphylococcus aureus is Gram Positive = pentaglycine interbridge between amino acid structures and L-Lys in the amino acid structure

What is the amino acid structure in the peptitoglycan cell wall?

1. L-Alanine (L-Ala)

2. D-Glutamic Acid (D-Glu)

3. Diaminopimelic Acid (DAP)

4. D-Alanine (D-Ala)

What makes Myobacteria species not typical from gram-positive and gram-negative bacteria?

They contain a very complex cell envelope

• Unusual lipids = mycolic acids

• Unusual sugars = arabinogalactans

• Very hard to stain = stained using Acid fast stain, which requires application of heat

A few prokaryotes lack cell walls. How will they maintain their membrane strength?

They prefer isotonic environments, intracellularlly, where the concentration of solids are very similar to the concentration of solids inside them

What does staph arrangement mean?

Staph (staphylococci) are cells arranged in irregular, grape-like clusters.

What does strep arrangement mean?

Strep (streptococci) are cells arranged in chains.

What does di- arrangement mean?

Di- refers to pairs of bacterial cells.

What does tetrad arrangement mean?

Tetrads are groups of four cells arranged in a square.

What does staphylococci describe?

Spherical bacteria (cocci) arranged in clusters.

What does streptobacilli describe?

Rod-shaped bacteria (bacilli) arranged in chains.

What does diplococci describe?

Spherical bacteria arranged in pairs.

What metric units are used to measure microbes?

Bacteria are measured in micrometers (µm), while viruses are typically measured in nanometers (nm).

What is the approximate minimum size of microbial cells?

About 0.2 µm.

mm (milimeters) ↔ µm (micrometers)

mm → µm → × 1,000
µm → mm → ÷ 1,000

µm (micrometers) ↔ nm (nanometers)

µm → nm → × 1,000

nm → µm → ÷ 1,000

nm (nanometers) ↔ pm (picometers)

nm → pm → × 1,000
pm → nm → ÷ 1,000

Bacteria and archaea are similar when it comes to

size and shape

What is true regarding proteins that are embedded within the cell membrane?

They provide structural support

Which of the following unique characteristics would you expect to find in an archaeon cell membrane living in a hot spring?

Ether linked fatty acids

It is made of a monolayer

What are the two commonalities between Gram negative and Gram positive cell envelope?

Peptidoglycan and Cell Membrane

Which statement is false about the bacterial outer membrane?

It is composed of a phospholipid bilayer

A student made a mistake while performing gram staining. What is the best explanation for a Gram-negative bacterium appearing purple after performing a Gram-stain?

Student failed to decolorize their sample

Which of the following structures are different between Gram positive and Gram negative bacteria?

Presence or absence of outer membrane