Microbiology Exam 2 Study Guide PDF

Chapter 7: Microbial Growth and Counting − Counting microbes: direct counts, viable counts and turbidity measurements (techniques associated with these counts) Direct Count: Petroff Houser Counting Chamber: Specially constructed microscope slide with a chamber of defined...

Chapter 7: Microbial Growth and Counting − Counting microbes: direct counts, viable counts and turbidity measurements (techniques associated with these counts) Direct Count: Petroff Houser Counting Chamber: Specially constructed microscope slide with a chamber of defined depth and a grid marking off squares of defined area; used for determining cell concentration via microscope Drawbacks: Accounts for all bacteria (dead or alive), labor intensive Viable Counts: 1. Spread Plate Technique 2. Pour Plate Technique (serial dilutions (less than 300 and greater than 10 bacteria) Allows calculation and count of # of viable Colonies Turbidity: Turbidometry: Degree of cloudiness relative to population size Turbidostat + Chemostat: Both continuous cultures, grown in open systems, provided regulated amounts of media to maintain culture in log phase Turbidostat: Photocell regulates flow rate of media through vessel to maintain high levels of dilution and turbidity (cloudiness) Chemostat: Old medium removal = new addition of medium to maintain microbes in log phase for continuous growth What is a CFU, how do we calculate? - Colony Forming Unit: Number of viable microbial colonies within a sample - Calculation: Spread Plate and Pour Plate Techniques to Count # Colonies - Equation: (Bacteria Count x Dilution Factor/(Culture Plate Volume) The growth curve: what it is, what it looks like, all 4 phases and what happens during each 1. Lag Phase 2. Log Phase 3. Stationary Phase 4. Death Phase VBNC and programmed cell death Two hypotheses for death phase of growth curve: Viable but non-culturable: Cells living, but cannot produce in initial media environment (low nutrients, high waste), until move to more suitable environment Apoptosis: Some of microbe populus programmed to die  Balanced growth vs. Unbalanced Balanced: Cell components formed at the same rate (faster metabolism, cell division + proliferation) Unbalanced: Cell components formed at different rates from each other (sudden changes in media) Important points about doubling time and how we find it Time required for population to double in size (generation time) Calculation: Take two points in which the population doubles (log phase) and compare the times via subtracting them Varies depending on bacterial species and environment - Bacteria double in population faster than eukaryotic cells (binary fission) How an open system differs from a closed system Open System: System in which there is a continuous exchange of nutrients/materials Closed System: System in which there is no exchange of materials/nutrients - Ex: Media (No turbidostat, no chemostat) Compare and contrast chemostat and turbidostat: Turbidostat + Chemostat: Both continuous cultures, grown in open systems, provided regulated amounts of media to maintain culture in log phase Turbidostat: Photocell regulates flow rate of media through vessel to maintain high levels of dilution and turbidity (cloudiness) Chemostat: Old medium removal = new addition of medium to maintain microbes in log phase for continuous growth Detection of bacteria without cultivation: 1. PCR Amplification (Rapid amplification and creation of genes) 2. FISH (Complementary DNA is fluoresced and tracks target sequence) Chapter 2: Bacterial Structure and Morphology: 1. Bacterial Morphology Shapes: Cocci, Bacillus, Spirilla, Vibrio - Pleomorphic: Varying shape morphologies (Ex: genus Mycoplasma – lack a cell wall) Multicellular Organization: - Hyphae: Irregular branching filaments (Ex: fungi, bacteria) - Mycelia: 3D network of hyphae (tufts) - Trichomes: unbranched, smooth chains (Ex: cyanobacteria) - A. Size Variations - Bacteria: Typically, smaller than Eukaryal cells (less than 5 µm in diameter) - Mainly between 0.5 and 5 µm - Ultra small bacteria (approximately 0.2 µm in diameter) - Small eukaryotes greater than 5 µm 2. S/V Ratio Increased surface-to-volume ratio increases nutrient uptake efficiency Decreased surface-to-volume ratio decreases nutrient uptake efficiency 3. Cell Structure: A. Cytoplasm - Nucleoid: Convoluted mass of circular DNA (+ cations, supercoiling, nucleoid proteins) in bacteria (contain RNA and protein/lacks membrane) - Plasmids: Variable, encode non-chromosomal genes for certain functions (Ex: Episome – can exist independent from genome and self-replicate) (Plasmids: Can be inherited stably or lost from cell division) - Ribosomes: Protein synthesis (70S – Bacteria/Archaea) (80S – Eukarya) Bacteria: 16S rRNA = 30S small subunit, 23S and 5S = 50S large subunit Archaea: Contain 5.8S = large subunit - Inclusion bodies: Granule within bacteria which store aggregated cellular and viral material (sulfur, nitrogen, carbon, phosphorus) 1. PHB Granules – Store carbon 2. Sulfur Globules – Store elemental sulfur for energy 3. Gas Vesicles – Store gas for buoyancy 4. Carboxysomes: Carbon fixation (photosynthesis) 5. Magnetosomes: Membrane enclosed structure containing magnetite (movement/motility via assistance from flagella)  B. Cytoskeleton FtzS: Forms Z-ring which assists in cell division (evolutionarily related to tubulin) MamK: Assembles into filaments which organize magnetosomes (like actin) MreB: Forms helical bands on the inside of the plasma membrane to provide shape of the cell (like actin/nearly universal in non-spherical bacteria) CreS: Maintains the curved shape of crescent shaped bacteria (vibrio) via filaments (like intermediate filaments) ParM: Responsible for moving copies of plasmids to opposite ends of the cell ParR: Recognizes plasmid and connects to ParM filaments C. Plasma Membrane - Selectively permeable bilayer mainly composed of phospholipids regulating material entry and exit, environmental sensing/signaling, and the capture and storage of energy - Features: Hopanoids – Homologs of sterols (cholesterol) which stabilize the plasma membrane (not common in bacteria) Aquaporins – Facilitate the movement of water, (osmosis: diffusion of water from high [C] to low [C]) - Fluid Mosaic Model: Biological model of the cell membrane Phospholipids: Laterally moving lipids consisting of hydrophilic polar head (phosphate groups) and hydrophobic non-polar tails Membrane proteins: Integral: Amphipathic, exist as microdomains, carbohydrates often attached, can move laterally along the membrane Peripheral: Loosely connected to the membrane, make up 30% of membrane proteins 4. Material Movement: Sec System: 1. Proteins identified for exit contain signal peptide (hydrophobic amino acids connected to the N-terminus of the protein) 2. SecB: Inhibits protein folding via binding itself and delivers to SecA 3. SecA: Associates with SecYEG (membrane protein) and with ATP hydrolysis, facilitates the movement of the protein through the membrane protein 4. Signal peptidase: Cuts off signal peptide signal from protein and proteins returns to functional conformation Gram (-) Secretion Systems: Various pathways for protein export. Nutrition Transport: - Porin: Channel proteins which facilitate the movement of essential nutrients of Gram (-) (located in outer membrane) - Gram (-) bacteria usually resistant to large antibiotics (ex: vancomycin) due to the mass size of it, which porins do not allow in (600 daltons max) TonB-Dependent Receptors: Bind scarce nutrients with high affinity and deliver through periplasm via active transport ExbB & ExbD: Complex connected to TonB and allow conformational change of TonB via active transport and the PMF Protein Transport: Type III Secretion – Protein complex in gram (-) that serve as injection agents to deliver virulence factors, such as toxins directly into the target cell by forming a tunnel that passes through bacterial membranes and through the host cell membrane Cell Wall: - Consists of peptidoglycan which resists damage from osmotic pressure, mechanical forces and shearing (non-permeable) Peptidoglycan synthesis: 1. NAM synthesized in the cytoplasm binded to UDP, containing a short peptide chain (amino acids) 2. NAM links to bacterophol via two phosphates 3. NAG forms a B 1-4 glycosidic linkage with NAM 4. Bacterophol flips NAG-NAM complex to periplasm 5. Disaccharide added to existing peptidoglycan chain which are crosslinked (peptide chain/NAM to NAM) via transpeptidase 6. Bactoprenol is flipped back into the cytoplasm Lysozyme: Degrades peptidoglycan via hydrolyzing NAG and NAM linkage Lysostaphin: Degrades pentaglycine linkage of peptidoglycan of S. aureus B-lactam Antibiotics: Consists of a B-lactam ring which resembles terminal D- Alanine and interferes with synthesis of the bacterial cell wall via fooling crosslinking enzymes into binding, attaching the antibiotic to the enzyme permanently, destroying it  - Only growing cells adding new peptidoglycan are sensitive to antibiotics, where lysozymes aim for both growing and non-growing cells Penicillin: B-lactam antibiotic description Augmentin: Clavulanic acid (strongly inhibits the activity of B-lactamases) combined with B-lactam B-lactamases: Hydrolyze and cleave the C-N bond of C-lactam, deactivating it Peptidoglycan: Polysaccharide-peptide matrix found in bacterial wall cells - Composed of a glycan backbone with two alternating sugars NAG & NAM (beta 1-4 glycosidic bonds), containing penta/tetra amino acid side chains (D or L) Crosslinking: - Note: Fifth amino acid removed when this crosslinking occurs (NAM to NAM) 1. E. coli and many Gram (-) bacteria: Fourth amino acid (D-Ala) attached to NAM attaches to the third amino acid (DAP) of another NAM residue 2. Most Gram (+) bacteria: Vary but tetrapeptides crosslinked via short peptide interbridges (S aureus: Pentaglycine interbridge) B. Gram Stain: Gram (+): Presence of thick peptidoglycan wall (outer membrane) (Purple) consisting of LTA and TA (strong inflammatory) - Teichoic Acid: Polymer of ribitol phosphate or glycerol phosphate attracted to peptidoglycan chains in the cell walls of Gram (+) bacteria - Lipoteichoic Acid: Cell wall molecule of gram (+) bacteria composed of ribose or glycerol polymers anchored to the cytoplasmic membrane by a lipid tail CRIES: Crystal Violet – Stains gram (+) and (-) bacteria with purple Iodine - Stabilizes the crystal violet Alcohol - Decolorizes the crystal violet on (-), while maintaining (+) bacteria crystal violet Safranin - Stains gram (-) bacteria, making them pink, while gram (+) remain violet/purple Gram (-): Presence of thin peptidoglycan wall (periplasm) (pink) consisting of LPS Lipopolysaccharide: Molecule composed of lipid A (strong inflammatory/mainly conserved), core polysaccharide (usually conserved) and the O-side chain (vary) (outer membrane) Flagella and Pili:  Flagella: Motility and movement via rotation of filaments Flagellar System: Like type III secretion system (found in gram + and -), where flagellum subunits enter within the intermembranous basal body, through the hook and into the filament, causing it to grow - Monotrichious (Single flagellum) - Lophotrichous (multiple flagella at one or more ends) - Amphitrichious (one flagellum at each end of the cell) - Peritrichous (evenly spread throughout bacteria) - Polar (flagellum at end of the cell) Chemotaxis: Using chemical signals from the environment to direct movement via chemoreceptors (positive and negative chemotaxis) - Counterclockwise rotation in peritrichous flagella (usually gram (-) and E. coli) Attractants (metabolize) allow forward movement and motion Repellants (deadly to bacterium) cause flagellar motor to switch motion (tumbling) Pilli: For adherence and sticking to surfaces (specifically used for conjugation/horizontal gene transfer) (pilin protein) - Pilli perform ‘twitching’ motility along surfaces (not intrinsic) Sex Pilus: Connects cells during conjugation, the transfer of DNA Fimbria: Another term for Pilli (adherence and surface adhesion) Other Forms of Motion: Gliding Motility: Type of locomotion used by some non-flagellated bacteria to move across a membrane Stalk: Tubular extension of the cell envelope in some Gram (-) increasing cell surface area to volume (held/contained by a holdfast) Twitching Motility: Pilli attached to the and rapidly retract for movement production Capsules and Layers: Capsule: Polysaccharide layer surrounding some bacterial cells that may shield pathogens from host defense systems (defense systems ex: phagocytosis and exocytosis and desiccation (hydrophilic capsules)) Slime Layer: Structure typically consisting of polysaccharides that surrounds cells of some species (less defined than capsule) Biofilm: Microbial community attached to a surface within a matrix of secreted polymers  S Layers: Crystalline like layer of protein bound on surface of many bacterial cells (protective shield of proteins) (Gram (+): Found in peptidoglycan layer/Gram (-): Found in outer membrane − Prevent infection by bacteriophages − Prevention of penetration by predatory bacteria − Prevention of attack by immune system Endospores (formed usually by gram (+)): - Metabolically inert structure increasing bacteria resistant to extreme conditions by compressing Chromosomal DNA with protective proteins - Sudden stressful conditions (lack of imminent nutrients) - Increased resistance to extreme conditions including heat, radiation, chemicals and desiccation Endospore Resistance: 1. Calcium (complexed dipicolinic acid) 2. Small, soluble acid binding DNA Proteins (SASPs) 3. Dehydrated core 4. Spore core and exosporium bring protection Sporulation: Process of endospore formation which commences when growth ceases from a lack of nutrients (up to 10 hours) Germination: Transformation of endospore into vegetative cell complex (multistage process) 1. Enviromental nutrients are detected in the environment 2. Spore swells, absorbing water from the surrounding environment to obtain nutrients (rupture or weakening of wall) 3. Loss of resistance weakens protective spore, making it present in surrounding environment and active 4. Metabolic activity increases and allows further growth of spore Chapter 4: Archaeal Structure Archaeal Morphology: Archaea: Domain in which members are unicellular and lack a membrane-bound nucleus (originally defined as methanogens (strict anaerobes) producing methane as a byproduct of metabolism) Similar To Bacteria’s Morphology: Similar in size (0.5 µm - 5 µm but can vary) and shapes (rods, spheres, spirals, irregular shapes, rectangular shapes), chromosomes of similar size and organization, lack a membrane bound nucleus, differ genetically from bacteria (two distinct evolutionary groups)  DNA Similar to Eukarya: Presence of histones (tetramers wrapped around by 60 base pairs) and homologues of DNA replication enzymes, transcription and translation markers - Mainly live in ‘extreme’ environments, but some live in temperate environments (play a role in biogeochemical nutrient cycling) - Phylogenetic comparisons from rRNA sequences (SSURNA) Unique Structures: - Uniqueness based on the parameters of being an extremophile and having a distinct plasma membrane A. Cytoplasm - Presence of similar molecules as bacteria, DNA/RNA polymerase, ribosomes, gas vesicles B. Cytoskeleton: - Presence of similar protein homologs from bacteria and eukarya (FtsZ, MreB, ParM) - TA0583 resembles eukaryal actin (homolog) - M. thermoautotrophicum and M. kandlieri contain cytoskeletal proteins resembling bacteria C. Plasma Membrane: - Composed of isoprenoids (hydrocarbon molecule in archaeal envelope built from 5C isoprene subunits & G1P) and are linked together via ether linkages - Isoprene polymers (phytanyl: 20-carbon hydrocarbon (bilayer)/biphytanyl: 40- carbon hydrocarbon (monolayer)) D. Cell Wall: - Protection from osmotic pressure changes and mechanical stress - Pseudomurein (not present in all Archaea): Similar to peptidoglycan and found on the cell wall of Archaea - N-acetyltalosaminuronic acid (NAT & NAG) , B 1-3 glycosidic linkages, 1- stereoisomers of amino acids Flagella and S-Layers Archaeal Flagella: Thinner than bacterial archaea (10-14nm vs 20-24 nm) Components:  Flagellin: Comprises the flagellum in bacteria and archaeons (O-linked glycosylation) - Flagellum grows from the base and not the tip in archaea (different from bacterial Type III secretion system) - Signal cascades affect the direct motility of archaeal flagellum (rotational) S-layer: Like that of bacteria (serve as protection from external environment/desiccation, bacteriophages) - Can be composed of proteins, glycoproteins, polysaccharides Cell Surface: - Cannulae: Hollow glycoproteins tubes that connect individual cells to form a complex network (nutrient exchange and connection within periplasm) - Hami: Surface structure used for attachment (biofilm formation) Chapter 5: Viruses Filterable Viruses: - Viruses able to pass through filters small enough to retain the smallest known bacteria - Ex: Bacteriophages (viruses that infect bacteria) Alive vs. Dead: Viruses: Acellular particle including DNA and RNA genome surrounded by a protein coat that can only replicate within host cells - Classified as obligate intracellular parasites (can only replicate within host cells), acellular infections agents Size of Viruses: - Diameter of viral particles is typically between 10 and 100 nanometers - Variola Virus (outlier) could have a diameter of 200nm - Picornaviruses (smaller viruses), Poxviruses (largest viruses) - Genomes vary from 1000+ nucleotides (smaller viruses) to 200000 nucleotides (larger viruses) Viral Capsids: Protein structure surrounding the genome of the virus (useful for genome protection and transfer to host cells) - Protomers (subunits of the capsid/capsomeres) - Can be icosahedral, helical or complex  Icosahedral: Capsid structure forms 20-sided polygon, with each capsomers (pentamers (5 protomers), hexamers (6 protomers)) making up a face of an icosahedron (20 equilateral faces/12 vertices) Helical: Structure of a virus in which capsomeres form a helix and the capsid resembles a hollowed tube - Protomers self-assemble - Shaped like hollow tubes with protein walls and length equates to nucleic acid function Virion: Complete viral capsule outside of the host Complex: Not fitting within the category of icosahedral or helical - Poxvirus: Largest animal virus - Large bacteriophages: Binal symmetry in which the head resembles icosahedral, and the tail is helical Enveloped vs. Naked Viruses: Viral Envelope: Host cell derived membrane that surrounds the capsid of certain viruses - Found mostly in animal viruses and acquired when virus leaves host cell (envelope required for re-entry and hiding it from immune attacks/responses) - Can be degraded in the environment of host - All known animal viruses with helical symmetry have an envelope - Spike embeds itself into the plasma membrane of the cell Naked viruses: Lack the host cell derived lipid envelope - Cannot reenter and invade other host cells - Naked viruses interact with cellular receptor which interacts with amino acid residues within the capsid Viral Surface Proteins: Viral Attachment Proteins: Protein expressed by virus for attachment to the host cell - Virus identification - May have enzymatic activity (enveloped viruses) - May play a role in nucleic acid replication Spikes: Essential for attachment of the virus of the host cell Nucleic Acid Configurations: Viral genome: Can be either DNA or RNA, but never both DNA: Double stranded, but may be single stranded, circular or linear RNA: Single stranded (mainly) or double stranded, segmented into different into separate RNA pieces - Positive-sense RNA: ssRNA ready for immediate translation  - Negative-sense RNA: Must be converted into proper form Life Cycle of Animal Viruses: 1. Attachment: Binding of virus to specific molecules on host cell - Host range: Spectrum of cells virus can invade (Ex: Hep B (Liver), Polio (Primate intestinal and nerve cells) 2. Penetration: Genome enters the host cell - Receptors bind and endocytosis (non-enveloped) or plasma membrane fusion (enveloped) 3. Uncoating: The viral nucleic acid is released from the capsid - Uncoating occurs simultaneously as endocytosis vesicle dissipates - Uncoating occurs after membrane fusion 4. Replication/Synthesis: Viral components are produced - DNA Synthesis: Nucleus - RNA Synthesis: Cytoplasm (+ sense RNA (translation ready)/- sense RNA (conversion to + sense) 5. Assembly: New viral particles are assembled RNA in Cytoplasm DNA in Nucleus 6. Release: Assembled viruses are released by budding (exocytosis) or cell lysis - Budding: Exocytosis – nucleocapsid binds and pinches membrane, releasing it (cell doesn’t automatically destroy) - Lysis: Non-enveloped viruses release when cell dies and ruptures Cytopathic Effect: Virus induced damage to cells 1. Changes in size and shape 2. Cytoplasmic inclusion bodies 3. Cell lysis 4. Alter DNA 5. Oncogenic cell transformation Persistent Infections vs Transforming Infections: Persistent Infections: Cell harbors the virus and is not immediately lysed - Chronic Latent State: Viruses that can periodically reactivate (weeks to lifetime) Transforming Infections: Cells harboring the virus begin to change, becoming malignant - Viral proteins bind tumor suppressor genes - Carry oncogene into cell and insert it into host genome - Insertion of promoter/enhancer for oncogene Cancer: - Tumor - Growth or lump of tissue - Neoplasia - Abnormal new cell growth and reproduction due to loss of regulation - Anaplasia - Reversion to a more primitive/less differentiated state - Metastasis - Cancerous cells spread throughout the body Viral Cultivation Methods – Inoculation requires active host - Nutrient Agar: Will decrease in turbidity when viral population increase 1. Direct Count - Viral loads viewed under and electron microscope to be visualized using beads (for volume) and viral particles (concentration) - Drawbacks: Cannot differentiate between non-infections and infectious acellular agents and expensive 2. Hemagglutination Assay: - Viruses cause RBCs to clump together (hemagglutination). Through dilution of constant number of RBCs, the hemagglutination titer can be found (concentration of virus) - Drawbacks: Does not accurately reflect the total number of viral particles, placebo effect (hemagglutination occurring without viruses) 3. Plaque Assay: - Can determine the infectious titer via dilutions and nutrient agar for growth of host cells for viruses to invade. The infectious titer is calculated from the number of plaque forming unit (PFU) Noncellular infectious agents: 1. Viroid: Infectious agents of plants composed of naked RNA (closed circular ssRNA) - Require host cell for replication - Resistance to ribonucleases (no RNA degradation) - Utilize DNA dependent RNA polymerases - Do not encode gene products 2. Satellites: Infectious agents of plants composed of an RNA/DNA genome containing a gene that codes for a protein coat - Satellites viruses encode their own capsid proteins with helper virus while satellite RNA/DNA cannot code for their capsid proteins - Require helper viruses for replication (Hep D requires Hep B virus for self-replication) 3. Prions: Infectious agents composed of protein that can replicate within a host cell and cause transmissible spongiform encephalopathies (TSE) -> neurogenerative disease  Virology and Medicine: - Viruses are the biggest cause of acute infections - Major participants in Earth’s ecosystem Treatment: More difficult than other agents Sampling: - Infecting Cell Culture – looking for cytopathic effects - Screen for parts of virus - Screen for antibodies (immune response) Gene Therapy: Treatment of a genetic disorder by introducing a wild-type allele of a mutant gene that codes for an abnormal or missing protein, thereby eliminating the problem Oncolytic viruses: Viruses that target cancerous oncogenic cells and destroy them Nanotechnology Bacteriophages: Viruses that infect bacteria (‘bacteria killers’) - Lytic: Bacteriophages invade bacteria and attempt to self-replicate. Rapid self-replication leads to lysis of the bacteria (bursting), leading to the host cell dying (short term) - Lysogenic: Bacteriophages invade bacteria and introduce themselves to their host’s genome. This integration allows replication of virus within the genome (long term) Other Acellular Partials: - Transposons: Jumping genes within the DNA sequence that can move from one section of the genome to another (can cause mutations (acellular)) - Exosomes: Carry proteins, RNA and molecules which allow for intercellular communication (non-infectious, but acellular) - Defective Interfering Viruses: Viruses which have been mutated, becoming non- infectious viruses and requiring the assistance of infectious viruses