MGY277H1 Study Guide - Final Guide: Mordant, Peptidoglycan, Biome
8 Jun 2018
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MGY277 Final Exam Notes
Unit 1 – Perspectives
Unit 2 – The Bacteria
• Define the terms “magnification”, “resolution”, “contrast”, and “refraction” and apply them when looking at an image from
a microscope.
- Magnification: the increase in the apparent size of the object compared to the size of the actual object
- Refraction: light rays change direction due to change in medium (refractive index – measure of speed of light as it passes
through a medium)
- Resolution: minimum distance at which two points can be distinguished as individuals
- Contrast: the ability to see objects against the background
• Recite the steps of a Gram stain and why the stain can differentiate between different types of bacteria.
1. Crystal violet (primary stain) – bacteria stain purple
2. Iodine (mordant) – bacteria remain purple, cells less soluble now
3. Alcohol (decolourizer) – Gram-positive remain purple, gram-negative become colourless
4. Safranin (counterstain) – gram-positive cells remain purple, gram-negative cells appear pink
• Recall the different types of staining and how they are usefully applied.
- Gram stain: differentiate negative from positive gram cells
- Acid-fast stain: detect small group of organisms that don’t readily take up stain, i.e. Mycobacterium genus
- Capsule stain: capsules stain poorly, negative stain used, or India ink for suspension of carbon particles
- Endospore stain: Bacillus, Clostridium form dormant endospore, resist gram stain; use heat to facilitate uptake of
primary dye – often malachite green – by endospore, counterstain Safranin used to visualize other cells
- Flagella stain where flagella is for prokaryotic motility
• Know what the basic components of the Gram-positive and Gram negative cell envelope are
- Gram-positive: thick peptidoglycan, teichoic acid stick out above peptidoglycan, no outer membrane – hence no porin, no
LPS, sensitive to lysozyme
- Gram-negative: thin peptidoglycan, no teichoic acid, outer membrane – w/ porin, LPS, not sensitive to lysozyme
• Describe the outer membrane of the Gram-negative bacteria
- Outer membrane is unique lipid bilayer embedded with protein; porins and lipoprotein within outer membrane with lipoprotein
attaching to peptidoglycan
• Describe the structure of peptidoglycan: cell wall made of peptidoglycan, subunits N-acetylmuramic acid (NAM) & N-
acetylglucosamine (NAG) form glycan chains; glycan chains are linked by tetrapeptide chains (4 AA strings)
- NAG found in mammals, NAG is a derivative of NAM, unique to bacteria
- Wall peptides are short and have unique “D” AA’s (D found in bacteria, L in proteins)
• Describe the basics of how bacteria obtain nutrients
- Trans-membrane highly specific proteins (transport systems) allows for movement of nutrients, small molecules, waste &
other compounds
- Facilitated diffusion: passive transport, not useful in low-nutrient environments & rarely used by prokaryotes
- Active transport: movement against [gradient], requires energy – Proton motive force & ATP (ABC transporter), commonly
used by bacteria
- Group translocation: transported molecule chemically altered upon entry via phosphorylation (i.e. glucose, where energy used
during transport is regained when sugar is broken down)
• Describe how bacteria sense their environment
- Membrane-spanning sensor kinase (SK), transfers a phosphate from ATP to response regulator (RR) – usually found as homo-
dimer – RR is transcription factor which turns genes on or off in response
- RR controls response output – RR usually DNA binding protein changing affinity for DNA once phosphorylated
o I.e. Salmonella sense acid to recognize if within host, or sense O2 to regulate genes needed for anaerobic metabolism
• Describe a “typical” bacteria genome
- E. Coli strain CFT073 genome has about 5 million BPs, about 5000 genes
o Other E. coli strains have slight differences in genes they contain, due to frequent gene exchange amongst bacteria
- Contains anywhere from <500 to >8000 genes but only subset expressed at any given time
- Bacteria don’t have histones, introns or exons
• Describe bacterial gene regulation (i.e. how genes are turned on and off)
- Mechanism to control transcription: DNA-binding proteins & alternative sigma factors
- Alternative sigma factors: Standard sigma factor is component of RNA polymerase that recognizes specific promoters for
genes expressed during routine growth conditions
- Alternative sigma factors can replace the standard factor & recognize different sets of promoters to control expression of
specific groups of genes (i.e. heat shock)
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MGY277 Final Exam Notes
Unit 3 – The Virus
• Describe the physical and genomic features of viruses.
- Viewed as DNA or RNA within protective coating, not alive outside of a cell (not metabolically active or replicating)
- Obligate intracellular parasites – reproduction reliant on intracellular resources
- Can be eukaryotic or prokaryotic (bacteria infected by bacteriophage – don’t infect humans)
- Difficult to study in lab: can’t be grown in pure culture, require live host, EM (expensive) to see
- Small size: 10 – 10000 x smaller than cells they infect (smallest is 17 nm diameter needs 2 genes to replicate), largest is
mimivirus – 2x larger than mycoplasma bacterium
- Virion: nucleic acid + protein coat (capsid), where nucleocapsid comprised of capsid containing genome
- Viruses have spikes for attachment to receptor sites & aid in entry to host cell
- Naked virus: Nucleoplasmid with capsomere subunits & spikes
- Enveloped virus: nucleoplasmid in matrix protein (immediate role during infection), surrounded by envelope w/spikes (outer
bilayer)
- Icosahedral: 20 flat triangles, efficient design using least energy to assemble
- Helical: spiral staircase like helix arrangement, can be short & rigid or long & filamentous
- Complex: intricate structures, i.e. phage w/ icosahedral head and long helical tail w/spikes & tail fibers
- Genomic features: DNA or RNA; linear or circular; double or single-stranded; always segmented; genome sizes vary, DNA
virus typically larger than RNA – large RNA are not stable
• Understand how viruses are classified and grouped, both formally and informally.
- ICVT 2009 report: > 6000 viruses, 2288 species, 348 genera, 87 families & 6 orders
- Formal classification based on: genome structure (ss/ds, RNA or DNA), hosts infected (bacteria, archea, animal, plant or
insect), viral shape & structure (enveloped or naked, icosahedral/helical, dimensions of capsid), disease symptoms
- Families end in –viridae, some names indicate appearance (coronae – crown like appearance), others named for geographic
area first isolated
- Genus ends in –virus (Enterovirus or Herpesvirus)
- Species name often name of disease, i.e. Poliovirus causes poliomyelitis
- Viruses commonly referred to only by species name, in contrast to virus nomenclature of genus, species
- Informal: Epstein-Barr for herpesvirus (Michael Epstein & Evon Barr discovered herpesvirus)
o Based on shared route of infection: Enteric viruses through fecal-oral route, Respiratory viruses through respiratory
route, Zoonotic viruses animal to human transmission, Arboviruses spread by arthropods such as ticks & mosquitos
▪ Arboviruses can infect widely different species, i.e. West Nile encephalitis, yellow fever, dengue fever
- Viral Taxonomy: Nucleic Acid (ds DNA, ss DNA, ds RNA or ss RNA), ss DNA & ds RNA are only naked, reverse
transcribing viruses all enveloped
o Nucleic Acid > Outer covering > Family > Members (family have viruses w/ similar structural features, infect similar
host & cause similar diseases)
• 5-step infection cycle for enveloped and non-enveloped viruses.
1. Attachment: > 1 spikes binds specific receptor on plasma – specificity of spike to receptors accounts for resistance
2. Penetration & uncoating: Membrane fusion of enveloped virus, receptor-mediated endocytosis of enveloped + naked virus
a. Membrane fusion leads to nucleocapsid entering cytoplasm & envelope remaining with plasma membrane
b. Receptor-mediated endocytosis: enveloped virus enters intact with enveloped virus staying within endosomal
membrane. Naked virus cannot fuse with endosome of host membrane, must damage endosome & release
nucleocapsid into cytoplasm
o Virion must localize to site of replication, may be nucleus or cytoplasm
o Uncoating of NA & disassembly of virus may occur simultaneously upon entry, or upon final destination
3. Synthesis of viral proteins & genome replication: Requires viral gene expression + replication
a. DNA viruses: usually replicate in nucleus, requires host machinery for gene expression & DNA synthesis, encodes
own DNA polymerase (ss – or ss + to ds ± DNA to ss + RNA (mRNA) to protein)
b. RNA viruses: usually replicate in cytoplasm, require RNA polymerase replicase – enzyme catalyzing replication of
mRNA/+ ss RNA from RNA template (ss – RNA or ds ± DNA)
▪ Replicase lacks proofreading ability – accounts for genetic mutations where pre-existing immunity ineffective,
as seen in seasonal influenza (new vaccine needed due to mutations)
▪ Segmented RNA viruses undergo re-assortment to alter host specificity, non-human to human transmission
c. Reverse transcribing viruses: encode reverse transcriptase which makes DNA from RNA; retroviruses have ss + RNA
genome, single ds ± DNA complementary strand synthesized => integrated into host genome (can’t eliminate)
4. Assembly: protein capsid must be formed; genome & enzymes packaged within capsid, occurs in nucleus or organelles, many
assemble near plasma membrane close to site of release
5. Release: enveloped virus released via budding (integrates into host plasma membrane upon budding & becomes coated
w/matrix proteins), naked virus released when host cell dies (apoptosis initiated by virus or causing lysis of cell during release)
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MGY277 Final Exam Notes
• List the difference between acute and persistent infections, and understand how these concepts influence the viral disease
pathogenesis.
- Acute infections: rapid onset of symptoms with short duration, immune system eliminates virus to undetectable level in time
o Influenza is an example of an acute infection
- Persistent infections are prolonged infections which could last years, may remain with host for lifetime
o Chronic: continuous production of low levels of virus particles in absence of disease symptoms, transmitted unknowingly.
Sharp increase of virus in early stage, followed by disease symptom, after which virus persists asymptomatically
▪ Hepatitis B is an example of a chronic persistent infection
o Latent: viral genome can remain silent between infections, integrated into host genome (provirus) – similar to plasmid
▪ Provirus cannot be eliminated, can be reactivated at later date to carry out productively/lyse cell
▪ Cold sores & herpex simplex viruses I & II are latent persistent infections; HSV latent in CN V)
▪ Viruses such as HIV or Cytomegalovirus undergo latency in immune cells – leukocyte or T cells
- HIV initially acute, after integrating to host becomes persistent
• Understand the concepts required to cultivate viruses in a laboratory
- Virus grown in live animal, embryonated eggs (influenza, newcastle, mumps & avian adenovirus) & cell cultures
- Cell culture where typically animal cells are grown in flasks and bathed in liquid medium – adherent or suspension cells (i.e.
blood cells), pink color of flask caused by pH indicator like phenol red, neutral pH (pink-red) preferred by most cells
- Animal tissue processed to obtain primary culture – most natural state.
1. Protease trypsin used for incubation – digest/separate cells of tissue
2. Tissue placed in flask + growth medium
3. Cells settle in flask and grown in culture
- Drawbacks of cultivating animal viruses: slow growing, divide limited # of times, not all viruses can be grown in this manner
o To work around slow dividing viruses tumour cells can be used
• Describe the effects that virus replication has on cells in culture
- Cytopathic effects: change shape, detachment, lysis, form inclusion body (site of viral replication), fuse into giant multinuclear
cell (syncytium)
o Syncytium caused by HIV, paraximal virus & HSV type I – syncytium allows virus to transmit to nearby cells without
lysing host, allowing virus to enter multiple cells, virus uses syncytium as base
• List and differentiate between the four methods used to quantify viruses in a laboratory
1. Plaque assay – precise method; serially dilute stock of virus then add to monolayer of cells (where white circle indicates cell
death, so low dilution = high cell death). Titer = # plaques x dilution / inoculum volume (mL), units: PFU/mL
2. Quantal assay: ID50 or LD50 – estimate, titer determined at dilution where 50% of inoculated host demonstrates signs of
cytopathic effect. Titer = dilution @ LD50 / inoculum volume (mL) – measures viable viral particles only
3. Direct counts via EM – done if viral count is high, empty capsid not counted & cannot distinguish b/w defective & viable
virus
4. Hemagglutination: relative concentration – assay carried out by mixing serial dilutions of viral suspensions w/standard amount
of RBCs, added to culture dish & diluted as you move right. Titer of virus determined by highest dilution showing
hemagglutination (where hemagglutinated cells don’t sink & non-hemagglutinated RBC sink)
Unit 4 – Eukaryotic Microbes
• Know the difference between an opportunistic and primary pathogen
- Opportunistic pathogen causes a disease in immunocompromised individuals
- Primary pathogen can cause disease in an otherwise healthy individual
• Know what differentiates a eukaryotic cell from a prokaryotic cell
- Eucarya domain include animals, plants, fungi & Protista
- Eukaryotic cells: 5 – 50 µm, nuclear membrane & membrane-bound organelles, no peptidoglycan & found in non-extreme
environments.
- Bacteria (prokaryotic): 0.3 – 2 µm, no nuclear membrane or membrane-bound organelles, peptidoglycan present & found in all
environments
• Be able to list the defining characteristics of fungi
- Eukaryote, cell wall contains chitin (molecule which also forms exoskeleton of insects), cell membranes composed of
ergosterol (animal cell membranes composed of cholesterol) – ergosterol biosynthesis is a major anti-fungal drug target
- Exist in multiple forms: single-celled yeast, multi-celled filaments called hypae or pseudohyphae & spores
o Hyphae are long branching filamentous structure aiding for movement towards nutrient
• Be familiar with some of the most common causative agents of fungal disease and risk factors for acquiring each disease.
- Person develops an allergic or asthmatic reaction to either fungus or spores
- Fungus can grow on or in human body, aka mycosis
- Fungus produces toxin causing illness after ingestion
- Since fungal organisms share same molecular machinery as us (they are eukaryotes) – drug targets problematic
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Unit 2 the bacteria: define the terms magnification , resolution , contrast , and refraction and apply them when looking at an image from a microscope. Magnification: the increase in the apparent size of the object compared to the size of the actual object. Refraction: light rays change direction due to change in medium (refractive index measure of speed of light as it passes through a medium) Resolution: minimum distance at which two points can be distinguished as individuals. Gram stain: differentiate negative from positive gram cells. Acid-fast stain: detect small group of organisms that don"t readily take up stain, i. e. mycobacterium genus. Capsule stain: capsules stain poorly, negative stain used, or india ink for suspension of carbon particles. Endospore stain: bacillus, clostridium form dormant endospore, resist gram stain; use heat to facilitate uptake of primary dye often malachite green by endospore, counterstain safranin used to visualize other cells.
