Microbiology Exam

4 categories of microorganisms

1. Bacteria

2. Archaea

3. Protists (Eukarya)

4. Fungi (Eukarya)

Koch’s Postulates

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

2. The suspected pathogen must be grown in a pure culture.

3. Cells from a pure culture must cause disease in healthy individuals.

4. The suspected pathogen must be re-isolated and shown to be the same as the original.

Bacteria

• Prokaryotic (no membrane-bound organelles)

• Heterotrophs and autotrophs

• Cell wall with peptidoglycan

• Photosynthetic or non-photosynthetic

• Unicellular or filamentous

Archaea

• Prokaryotic (no membrane-bound organelles)

• Unicellular

• Non-photosynthetic

• Contains extremophiles

• Most have a cell wall (no peptidoglycan)

Magnification

The ratio of the size of an image to the size of the object.

Resolution

The ability to distinguish adjacent objects as distinct and seperate.

Compound light microscope

Ocular and objective lens.

At high magnifications, the resolution of the object becomes compromised. Oil immersion can help increase resolution.

Bright field: can visualize objects by differences in contrast against the background.

Electron microscopy

Improves resolution by using electrons instead of photons of light (electrons have shorter wavelegths).

Cells must be dead.

Requires more sample prep than light microscopy.

Scanning electron microscopy (SEM)

Specimes are covered in a gold or palladium film, and their surface is scanned by electron beam. Internal structures are not visible.

Transmission electron microscopy (TEM)

Allows us to see subcellular structures.

Requires thin sections of cells (sample prep is time consuming).

Electrons pass through the sample.

Types of membrane proteins

Integral proteins: are firmly embedded in the membrane.

Peripheral proteins: are associated with the membrane, but not embedded.

How does the Gram stain work?

1. Stain the heat-fixed sample with crystal violet. This stains all cells purple.

2. Add iodine solution. This gets embedded into the peptidoglycan, forming the crystal-violet iodine complex.

3. Rinse with alcohol. This locks in the crystal violet in Gram + bacteria and washes it out of Gram - bacteria.

4. Counterstain with safranin red. Gram + cells are purple, Gram - cells are pink.

Gram + vs. Gram - cells

Gram + cells have thick peptidoglycan cell wall.

Gram - cells have thin peptidoglycan cell wall and an outer membrane.

Lipopolysaccharide (LPS) function

LPS are found in the outer membrane of Gram negative cells. They anchor the outer membrane to the thin peptidoglycan layer.

Has 3 components: O-specific polysaccharide, a core polysaccharide, and lipid A.

Highly toxic to mammals.

Periplasm

The space between the two membranes in a Gram - cell. Contains hydrolytic enzymes for food digestion and transport proteins.

Defined media

Contains precise and known amounts of pure chemicals.

More difficult to prepare and may support one or a few organisms.

Complex media

Mixtures of compounds of imprecise composition.

Easier to prepare and supports a wide range of organisms.

However, nutritional composition is not precisely known.

Steps of cell division

1. DNA replication

2. Formation of the divisome complex

3. Septum formation

Formation of the divisome complex

FtsZ protein forms a ring around the middle of the cell and stacks together a bunch of monomer units inside the cell wall, then removes them one at a time, making the Z ring contract.

FtsA and ZipA anchor FtsZ to the cytoplasmic membrane.

Phases of the growth cycle in batch culture

1. Lag phase: cells adjust to the growth conditions, there is little change in the number of cells.

2. Exponential phase: phase of rapid growth.

3. Stationary phase: nutrients are depleted and waste products accumulate, no net growth or death of cells.

4. Death phase: nutrients are depleted and waste products accumulate and become toxic, causing cells to die off.

Direct counts for measuring growth

Microscopic: uses a microscope to count the number of cells on a grid.

Flow cytometer: a laser counts the number of cells in a flow of liquid.

Viable plate counts for measuring growth

Count the colonies formed on an agar plate (colony forming units, CFU). The sample must be diluted until the CFU is between 10 and 300.

Optical density

A measure of cell turbidity, which is a measure of cell growth. The more turbid a sample is, the more cells there are. OD is faaster than direct counts or plate counts, but does not accound for dead cells or distinguish between cell size and number.

The Great Plate Count Anomaly

Discrepancy between the large number of cells that are counted under a microscope in environmental samples compared to the small fraction that grow in lab cultures and are counted on plate counts.

Chemoorganotroph

Energy source: chemical reactions

Electron source: organic molecules (sugar, acetate, ethanol, etc)

Chemolithotroph

Energy source: chemical reactions

Electron source: inorganic molecules (H2S, H2, NH3, etc)

Net products of glycolysis

2 pyruvates, 2 ATP, 2 NADH

What process creates ATP during glycolysis?

Substrate-level phosphorylation: a phosphate groupe is transferred off of an organic molecule (like glucose) to ADP, turning it into ATP.

Products of fermentation

No ATP

Ethanol and CO2 or lactate

NAD+

Fermentation

NADH reduces pyruvate to form fermentation products.

Aerobic respiration

Net products of TCA cycle

ATP, NADH, FADH2, and CO2

3 parts of respiration (aerobic or anaerobic)

Pyruvate oxidation

TCA cycle

Electron transport chain

Components of the ATP synthase

F0 subunit is embedded in the membrane. Protons enter via F0.

F1 subunit protrudes from the membrane into the cytoplasm. F1 catalyzes oxidative phosphorylation to ATP.

2 steps of photosynthesis

1. Photophosphorylation: conversion of light energy to ATP.

2. Carbon fixation: reduction of inorganic CO2 to organic molecules.

Photopigment used by oxygenic phototrophs

Chlorophyll

Photopigment used by anoxygenic phototrophs

Bacteriochlorophyll

Stages of the Calvin Cycle

1. Carbon fixation

2. Reduction

3. Regeneration of RuBP

Purpose of Calvin Cycle

Carbon fixation by green plants, cyanobacteria, algae, etc.

Inputs & outputs of Calvin Cycle

Inputs: 6 CO2, 12 NADH, and 18 ATP

Output: 1 glucose

Function of Rubisco enzyme

Rubisco catalyzes the addition of carbon from CO2 to the ribulose molecule in the first step of the Calvin Cycle.

Nitrification

Conversion of ammonium (NH4+) to nitrite (NO2-) and nitrate (NO3-).

Requires oxygen and is performed by nitrifying bacteria and archaea.

Denitrification

Reduction process that converts nitrates (NO3-) to gaseous nitrogen (N2) in anoxic conditions. NO3- is an important terminal electron acceptor.

Virus

Subcellular particle that can only replicate within a living cell.

Nucleocapsid

Viral genome + capsid

+sense vs. -sense viruses

+sense: RNA acts as mRNA, and can be transcribed directly into amino acids.

-sense: RNA is complementary to mRNA, so must be converted to +sense RNA by RNA polymerase before translation

Endocytosis of an enveloped cell

1. The virus attaches to a receptor on the host cell.

2. Endocytosis is initiated, and the virus is brought into the host cell.

3. The host cell forms an endosome with the virus inside.

4. The nucleocapsid escapes the endosome to the cytoplasm and uncoats to release the viral genome.

Membrane fusion of an enveloped cell

1. Virus attaches to a host cell receptor.

2. A conformational change in the receptor initiates membrane fusion.

3. The viral envelope fuses with the host cell’s plasma membrane

4. The nucleocapsid enters the cytoplasm and uncoats to release the viral genome.

Endocytosis of an enveloped virus

1. Virus attaches to a cell receptor.

2. Endocytosis is initiated.

3. An endosome forms around the virus.

4. The low pH of the endosome initiates the fusion of the viral envelope to the endosome membrane. The nucleocapsid is released to the cytoplasm.

Phage replication

1. Tail fibers of the bacteriophage attach to the host cell receptors.

2. Conformational change in the tail fibers brings the base of the tail in contact with the host cell’s surface.

3. Tail proteins are rearranged to allow the inner core tube proteins to extend down into the host cell wall.

4. Contact with the plasma membrane initiated the transfer of viral DNA through a pore formed in the host’s lipid bilayer.

Egress of enveloped virus

1. Proteins for the viral envelope are inserted into the host cell membrane.

2. Nucleocapsid migrates towards the host cell membrane.

3. Nucleocapsid buds out of the host cell.

Egress of a non-enveloped cell

Lysis of the host cell releases the virus.

Key features of the 16S rRNA gene

Size: large enough to provide information on the species, but small enough to be easily sequenced.

Contains conserved and variable regions.

Conserved vs. variable regions in 16S rRNA gene

Conserved regions: allow comparisons between distinct organisms and track distances in evolutionary relationships.

Variable regions: allow comparisons between closely related organisms due to their higher mutation rates than conserved regions.

Sanger sequencing

As a chain of nucleotides grows, terminator nucleotides (dideoxy nucleotides) ar randomly added, stopping DNA replication. This creates short DNA fragments of different lengths, but all with the same order.

Sequences are run through gel electrophoreses, separating fragments by size, and the results are read to determine the sequence.

Next-generation sequencing

Quickly generates large amounts of sequence data for lower cost. No gel electrophoresis.

Includes 454 pyrosequencing and Illumina sequencing.

454 Pyrosequencing

DNA is attached to a bead, then replicated onto the bead so there are many copies of the DNA on the bead.

Each bead is added to a well.

Nucleotides are added, releasing some phosphate at every addition.

Luciferase enzyme uses phosphate to make ATP, and uses ATP to emit light.

Light signal is detected to determine DNA sequence.

Illumina sequencing

DNA fragments are placed in the wells of a flow cell.

Clusters of DNA are generated on a glass slide, each containing many copies of the same piece of DNA.

Fluerescently labeled removable nucleotides are added, temporarily topping replication. The addition of each base is detected by a camera.

Fluorescent tafs are removed to allow the fragments to keep growing.

Open-reading frames (ORFs)

Sequences of DNA that may encode for a polypeptide (protein). ORFs allow us to know how many genes there are in a genome.

Features of a functional ORF

Start and stop codons.

Ribosome binding site.

Length (long sequence means it likely encodes something).

Similarity to other genes of known function.

Endosymbiosis

Evolutionary process where one organism lives inside another.

Ex: development of the mitochondria from bacteria and of the chloroplast from cyanobacteria.

Root nodule formation

When there is limited nitrogen in the soil, plants release flavonoids.

Flavonoids attract bacteria, which secrete nod factors.

Nod factors induce root curling around the bacterium.

Plant produces infection thread, which allows bacteria to be invaginated into the root hair and spread throughout the plant.

Bacteria differentiate into bacteroids.

Plant grows around bacteroids, forming nodules.

Crown gall formation

Use of T-DNA in genetic engineering of plants