Definitive Microbiology Study Notes

Fundamentals of Microbiology and Historical Milestones

• Definition and Scale of Microorganisms:

  • Microbes are defined as microscopic living organisms (often colloquially termed germs) found universally across the planet.

  • Most single microbial cells are smaller than $1\,mm$ across, ranging in size from $10^{-7}\,m$ ($100\,nm$) to $10^{-3}\,m$ ($1\,mm$).

  • Major taxonomic groups studied in microbiology include:

  • Prokaryotes: Bacteria and Archaea.

  • Eukaryotes: Algae, Protists, and Fungi.

  • Non-cellular entities: Viruses.

  • Key exceptions to the general microscopic rule:

  • Thiomargarita namibiensis: A giant marine sulfur bacterium that grows up to $100\,\mu m$ to $750\,\mu m$ ($0.75\,mm$), making it visible to the naked eye.

  • Caulerpa taxifolia: A single-celled, multinucleated green alga ("killer alga") that can grow up to several meters long.

  • Multicellular microbial structures: Biofilms and complex fruiting bodies.

• Historical Discovery of Microscopic Life:

  • Light microscopy was invented in the 1600s, undergoing continuous optical improvements.

  • Robert Hooke (mid-1600s) constructed compound light microscopes and published Micrographia, observing small eukaryotic structures such as fungal sporangia.

  • Antonie van Leeuwenhoek (1676) designed high-quality single-lens simple microscopes and became the first to observe single-celled bacteria (which he termed "animalcules").

• Cell Theory and the Spontaneous Generation Controversy:

  • Cell Theory establishes that all living organisms are composed of cells, and that microbes arise exclusively from pre-existing microbes (no spontaneous generation).

  • Francesco Redi (1688) demonstrated that maggots on meat arise from fly eggs rather than spontaneous generation.

  • Lazzaro Spallanzani demonstrated that boiled broth in sealed flasks remained sterile unless exposed to air.

  • Louis Pasteur (1861) definitively disproved spontaneous generation using swan-necked flasks. Boiled nutrient broth remained sterile for over a year because dust and airborne microbes were trapped in the curved neck; tipping the flask allowed broth to contact trapped microbes, causing rapid growth.

• Prebiotic Chemistry and the Origins of Life:

  • Early Earth lacked atmospheric oxygen ($O_2$); anaerobic microbes preceded all plant and animal life.

  • Stanley Miller (1953) conducted electrical discharge experiments simulating early Earth conditions, demonstrating the abiotic synthesis of organic amino acids.

  • Joan Oró (working at the University of Houston) demonstrated the abiotic synthesis of adenine and other essential biochemicals under reaction conditions mimicking comets.

• The Germ Theory of Disease:

  • Formulated by Louis Pasteur, the Germ Theory posits that specific diseases are caused by the transmission and growth of microscopic pathogens within a host.

  • A scientific theory is an overarching explanatory framework supported by extensive testing, not an unproven guess.

• Koch's Postulates:

  • Developed by Robert Koch to establish a rigorous causal relationship between a specific pathogenic microbe and a specific disease:

  1. The microbe is present in all cases of the disease and absent from healthy individuals.

  2. The microbe is isolated from the diseased host and grown in pure culture.

  3. When the isolated microbe is inoculated into a healthy, susceptible host, the identical disease occurs.

  4. The same strain of microbe is re-isolated from the newly diseased experimental host.

• Immunization and Medical Interventions:

  • Early variolation techniques in Asian and African nations involved transferring smallpox lesion material to healthy individuals to induce immunity.

  • Lady Mary Wortley Montagu observed variolation in Turkey and introduced the practice to England.

  • Edward Jenner (1796) developed the first vaccine by inoculating patients with cowpox matter obtained from a milkmaid's hand, conferring immunity against smallpox.

  • Dr. Kizzmekia Corbett led the NIH-Moderna research team in developing mRNA vaccines against COVID-19, introducing lipid-encapsulated mRNA to prompt host cells to express the SARS-CoV-2 spike protein antigen.

  • Antiseptics: Joseph Lister (1865) pioneered antiseptic surgical techniques using carbolic acid (phenol) to prevent bacterial infection of wounds.

  • Antibiotics: Alexander Fleming (1929) discovered penicillin produced by Penicillium mold contaminating a bacterial plate. Penicillin was purified for clinical use between 1929 and 1941.

• Microbial Ecology and Biogeochemical Cycling:

  • Most environmental microbes cannot be cultured on standard laboratory media.

  • Sergei Winogradsky developed the Winogradsky column, a stratified mud ecosystem where oxygen and sulfur gradients establish microenvironments for distinct metabolic guilds (e.g., cyanobacteria, iron oxidizers, purple and green sulfur/nonsulfur bacteria, sulfate reducers).

• Microbes drive key biogeochemical cycles:

  • Nitrogen Cycle: Soil bacteria and plant symbionts fix atmospheric $N_2$ into ammonium ($NH_4^+$); nitrifying microbes convert $NH_3$ to nitrite ($NO_2^-$) and nitrate ($NO_3^-$).

  • Carbon Cycle: Photosynthetic microbes fix $CO_2$ into organic compounds; heterotrophs return $CO_2$ via respiration.

  • Sulfur and Phosphorus Cycles.

• Microbial Classification and the Three Domains of Life:

  • Microbial taxonomy relies on metabolic traits, Gram-staining, and molecular gene sequencing due to morphological similarities and horizontal gene transfer (HGT).

  • Carl Woese and George Fox (University of Houston) analyzed small-subunit ribosomal RNA ($16S\text{ rRNA}$) sequences to discover that Archaea are distinct from Bacteria, establishing three primary domains of life: Bacteria, Archaea, and Eukarya.

• Endosymbiont Theory:

  • Proposed by Lynn Margulis, stating that eukaryotic organelles evolved via intracellular endosymbiosis:

  • Mitochondria evolved from ancestral respiring proteobacteria.

  • Chloroplasts evolved from ancestral phototrophic cyanobacteria.

• Molecular Genetics and Molecular Biology Techniques:

  • Structural tools: Rosalind Franklin produced Photo 51 via X-ray crystallography; James Watson and Francis Crick elucidated the double-helix structure of DNA.

  • Recombinant DNA technology relies on bacterial enzymes (restriction endonucleases, DNA polymerases for PCR).

  • Escherichia coli possesses the most thoroughly characterized genome.

  • Claire Fraser and TIGR completed the first cellular genome sequence of Haemophilus influenzae ($1,830,137\,bp$) in 1995.

  • Thomas Cech (1982) discovered catalytic RNA molecules (ribozymes), providing evidence for the RNA World hypothesis.

  • Emmanuelle Charpentier and Jennifer Doudna adapted the bacterial adaptive immune system, CRISPR-Cas9, for targeted genome editing.

• Primary Research Fields in Microbiology:

  • Experimental Microbiology: Fundamental questions regarding microbial structure, genetics, and physiology.

  • Medical Microbiology: Mechanisms, diagnosis, and treatment of infectious diseases.

  • Epidemiology: Distribution and tracking of disease in populations.

  • Immunology: Host defense systems against pathogens.

  • Food Microbiology: Fermentation processes and food preservation.

  • Industrial Microbiology: Production of pharmaceuticals, biofuels, and cloned proteins.

  • Environmental Microbiology: Microbial diversity in natural environments.

  • Bioremediation: Degradation of pollutants and human waste.

  • Forensic Microbiology: Analysis of microbial strains for legal investigations.

  • Climate Science: Microbial impacts on greenhouse gases and climate dynamics.

Bacterial Cell Structure and Function

• Subcellular Organization of Bacteria:

  • Bacterial cells consist of a central cytoplasm containing the nucleoid, enclosed by a cell envelope composed of a lipid membrane boundary and a structural peptidoglycan cell wall.

  • Model organism Escherichia coli (Gram-negative gut bacterium) structural dimensions and molecular complexes:

  • Outer Membrane Proteins: Sugar porins ($10\,nm$), Braun lipoprotein ($8\,nm$).

  • Inner Membrane Proteins: Substrate transporters, Sec secretory complex, ATP synthase ($20\,nm$ membrane diameter; $32\,nm$ total height).

  • Periplasmic Proteins: Arabinose-binding protein ($3 \times 3 \times 6\,nm$), Acid resistance chaperone HdeA ($3 \times 3 \times 6\,nm$), Disulfide bond protein DsbA ($3 \times 3 \times 6\,nm$).

  • Cytoplasmic Proteins: Pyruvate kinase ($5 \times 10 \times 10\,nm$), Phosphofructokinase ($4 \times 7 \times 7\,nm$), Proteasome ($12 \times 12 \times 15\,nm$), Chaperone GroEL ($14 \times 18\,nm$).

  • Transcription and Translation Machinery: RNA polymerase ($10 \times 10 \times 16\,nm$), Ribosomes ($21 \times 21 \times 21\,nm$).

  • Nucleoid Components: Double-stranded DNA ($2.4\,nm$ wide; $3.4\,nm$ per $10\,bp$ helical turn), DNA-binding protein HU ($3 \times 3 \times 5\,nm$), DNA-bridging protein H-NS ($3 \times 3 \times 5\,nm$).

• Methodologies for Studying Cell Structure:

  • Cell Fractionation: Breaking open cells via enzymatic or mechanical lysis followed by ultracentrifugation to separate organelles and complexes by size and density.

  • Biochemical Separation: Gel chromatography and gel electrophoresis separate macromolecules based on charge, size, and chemical properties.

  • Atomic Structure Determination: X-ray crystallography requires rigid, purified protein crystals; Nuclear Magnetic Resonance (NMR) spectroscopy reveals dynamic regions in small proteins.

  • Genetic Analysis: Evaluates biological function through phenotype observation (e.g., motility, colony morphology). Fluorescent gene reporter constructs (e.g., GFP fused to ftsZ) localize proteins in living cells.

• Bacterial Membrane Chemistry:

  • Membranes consist of a phospholipid bilayer embedded with proteins and stiffening lipids.

  • Bacterial phospholipids (e.g., phosphatidylglycerol and phosphatidylethanolamine) consist of a glycerol backbone linked via ester bonds to two fatty acid side chains, with a phosphoryl head group bound to terminal groups like ethanolamine or glycerol.

• Bacterial membranes contain hopanoids, pentacyclic non-polar lipids that fit between fatty acid chains to adjust membrane fluidity and stability.

• Archaeal Membrane Adaptations:

  • Archaea feature ether linkages ($C-O-C$) connecting glycerol to hydrophobic tails, offering greater chemical stability against hydrolysis.

  • Hydrophobic tails consist of branched terpenoid units (isoprenoids) rather than linear fatty acids.

  • Some hyperthermophilic archaea form diglycerol tetraether lipid monolayers (condensed diether chains) and incorporate cyclized cyclopentane rings to prevent membrane melting at extreme temperatures.

• Energetics and Proton-Motive Force:

  • Electron transport chains pump protons ($H^+$) out of the cell across the inner membrane, generating an electrochemical gradient known as the Proton-Motive Force (PMF).

  • Driven by PMF, protons flow back into the cytoplasm through ATP Synthase ($F_0F_1$ complex), powering the phosphorylation of ADP to ATP.

  • ATP Synthase Subunit Composition:

  • $F_0$ trans-membrane proton channel: Subunits $a$, $b$, and $c$ ring.

  • $F_1$ cytoplasmic catalytic core: Subunits $\alpha$, $\beta$, $\gamma$, and $d$ assembly.

• Peptidoglycan Cell Wall Architecture:

  • The cell wall (sacculus) is a single, mesh-like macromolecule surrounding the cell.

  • Composed of repeating disaccharides: $N\text{-acetylglucosamine}$ (NAG) and $N\text{-acetylmuramic acid}$ (NAM).

  • Glycan chains are cross-linked by short peptide chains attached to NAM consisting of alternating D- and L-amino acids: L-Alanine, D-Glutamic acid, m-Diaminopimelic acid (m-DAP), and D-Alanine.

  • Cross-bridge formation occurs between the free amino group of m-DAP on one peptide and the carboxyl group of D-Ala on an adjacent peptide, releasing the terminal D-Alanine residue.

• Cell Wall Biosynthesis and Antibiotic Targets:

  • Transpeptidases that catalyze cross-linking are termed Penicillin-Binding Proteins (PBPs, e.g., PBP1A, PBP2).

  • The peptidoglycan synthesis complex in rod-shaped cells coordinates PBPs with MreB, RodA, RodZ, MreC, and MreD.

• Penicillin acts as a structural analog of D-Ala-D-Ala, competitively inhibiting transpeptidases (PBPs).

• Vancomycin binds directly to the D-Ala-D-Ala peptide terminus, sterically preventing cross-bridge formation.

• Inhibition leads to cell wall weakening, causing osmotic lysis in growing bacteria.

  • Comparative Envelope Structures:

• Gram-Positive Envelope:

  • Thick cell wall consisting of $3$ to $20$ layers of peptidoglycan.

  • Threaded with negatively charged teichoic acids and lipoteichoic acids.

  • Narrow periplasmic space.

  • Plasma membrane.

  • Optional outer S-layer (crystalline protein array) and polysaccharide capsule.

• Gram-Negative Envelope:

  • Thin cell wall consisting of $1$ or $2$ peptidoglycan layers.

  • Extended periplasm bounded by inner membrane and outer membrane.

  • Outer membrane outer leaflet contains Lipopolysaccharide (LPS), acting as an endotoxin.

  • Outer membrane contains trimeric porins (e.g., OmpF) and is anchored to peptidoglycan via Braun lipoproteins.

• Mycobacterial Envelope (Mycobacterium tuberculosis):

  • Cell wall core contains peptidoglycan linked to galactans, attached to complex branched arabinans.

  • Arabinans are esterified to long-chain mycolic acids, forming an outer mycomembrane embedded with porins (e.g., OmpA Tb) and fibronectin-binding proteins (Fbp).

  • Ethambutol specifically blocks arabinan synthesis.

  • Outer thick capsule composed of glycolipids and sugar mycolates.

• Bacterial Cytoskeletal Elements:

  • FtsZ: Tubulin homolog; polymerizes into a dynamic Z-ring at the cell midpoint, required for septation and division.

  • MreB: Actin homolog; forms helical filaments beneath the inner membrane in rod-shaped bacteria (E. coli), organizing cell wall synthesis.

  • CreS (Crescentin): Intermediate filament homolog; localizes along the inner concave curve of Caulobacter crescentus, inducing crescent morphology.

• Nucleoid Organization and Gene Expression:

  • Contains a single, circular double-stranded DNA chromosome ($\approx 4 \times 10^6\,bp$) organized into supercoiled loops radiating from a central core attached to the envelope.

  • Lacks a nuclear membrane; transcription by RNA polymerase and translation by ribosomes are coupled in the cytoplasm.

• Antibiotic Targets in Expression:

  • Rifampin: Inhibits bacterial RNA polymerase.

  • Tetracycline: Binds the $30S$ ribosomal subunit, blocking aminoacyl-tRNA entry.

  • Erythromycin: Binds the $50S$ ribosomal subunit, inhibiting peptide chain elongation.

  • Specialized Internal Structures:

• Thylakoids: Concentric folded intracellular membranes in cyanobacteria (Prochlorococcus) housing photosystems and chlorophyll.

• Carboxysomes: Protein-shelled microcompartments filled with Rubisco for $CO_2$ fixation.

• Phycobilisomes: Light-harvesting protein complexes attached to thylakoid membranes.

• Storage Granules: Intracellular inclusions storing glycogen (energy), poly-\beta-hydroxybutyrate (PHB, lipid energy store), polyphosphate (essential inorganic phosphate), or elemental sulfur (oxidation byproduct).

  • External Surface Structures and Motility:

• Attachment Structures: Fimbriae and pili (composed of polymerized pilin subunits); stalks (membrane-bound cytoplasmic extensions terminating in a holdfast).

• Flagellar Motor Architecture:

  • Helical filament composed of flagellin (FliC subunits).

  • Rotary motor embedded in envelope: Filament connects to hook, anchored by L ring (outer membrane), P ring (peptidoglycan), MS ring (FliF in inner membrane), and C ring (cytoplasmic face).

  • MotA and MotB form proton channels across the inner membrane; proton flux drives rotation of the rotor protein FliG.

• Chemotaxis Mechanism:

  • Counterclockwise (CCW) rotation bundles flagella together, propelling the cell in a smooth forward "Run".

  • Clockwise (CW) rotation causes flagella to unbundle, causing the cell to "Tumble" and reorient.

  • Attractants (sugars, amino acids) binding to chemoreceptor arrays suppress tumbling, lengthening runs toward higher chemical concentrations (biased random walk).

Microbial Physiology and Environmental Adaptations

• Metabolic Classifications:

  • Energy Source:

  • Phototrophs: Harness light energy.

  • Chemotrophs: Extract energy from chemical electron donors.

  • Electron Source:

  • Lithotrophs: Utilize inorganic electron donors (e.g., $H_2$, $Fe^{2+}$, $H_2S$).

  • Organotrophs: Utilize organic electron donors (e.g., glucose).

  • Carbon Source:

  • Autotrophs: Fix inorganic carbon ($CO_2$) into organic sugars.

  • Heterotrophs / Organotrophs: Assimilate pre-formed organic carbon compounds.

  • Mixotrophs: Combine organic assimilation with phototrophy or lithotrophy (e.g., Chloroflexus aggregans).

• Nutritional Requirements:

  • Macronutrients (large quantities):

  • Structural elements: Carbon ($C$), Hydrogen ($H$), Nitrogen ($N$), Oxygen ($O$), Phosphorus ($P$), Sulfur ($S$).

  • Essential cations: Magnesium ($Mg^{2+}$), Iron ($Fe^{2+}$), Potassium ($K^+$), Calcium ($Ca^{2+}$).

  • Micronutrients (trace amounts as cofactors): Cobalt ($Co^{2+}$), Copper ($Cu^+$), Manganese ($Mn^{2+}$), Molybdenum ($Mo^{2+}$), Nickel ($Ni^{2+}$), Zinc ($Zn^{2+}$).

• Nutrient Transport Mechanisms:

  • Passive Diffusion: Small uncharged molecules ($O_2$, $CO_2$) cross the lipid bilayer down concentration gradients.

  • Facilitated Diffusion: Channel proteins pass solutes down concentration gradients without energy expenditure (e.g., GlpF glycerol channel in E. coli).

• Active Transport (against concentration gradient):

  • Coupled Transport: Energy released by one substrate moving down its gradient powers transport of another substrate against its gradient.

    • Symport: Both substrates move in the same direction (e.g., LacY permease importing protons with lactose).

    • Antiport: Substrates move in opposite directions (e.g., $H^+/Na^+$ antiporter).

- ABC Transporters (ATP-Binding Cassette):
  1. Solute passes through an outer membrane porin and binds a periplasmic binding protein.
  2. Complex binds the membrane transporter, triggering conformational change.
  3. ATP hydrolysis by cytoplasmic nucleotide-binding domains opens the channel, releasing solute into the cytoplasm.

- Group Translocation / Phosphotransferase System (PTS):
  - Chemically modifies the substrate during transport.
  - High-energy phosphate from Phosphoenolpyruvate (PEP) is transferred along cascade proteins (Enzyme I, HPr) to Enzyme II complex (IIA, IIB, IIC).
  - Substrates are phosphorylated as they cross the membrane (e.g., Glucose converted to Glucose-6-phosphate; Mannitol converted to Mannitol-6-phosphate).

• Environmental Limits on Growth (Table 5.1):

• Extremophile Adaptations:

  • Cold Adaptations (Psychrophiles, e.g., Flavobacterium in South Pole ice at $1\,^\circ\text{C}$): Enzymes flexible at low temperatures; membranes enriched in unsaturated fatty acids.

  • Thermal Adaptations (Thermophiles, e.g., Thermus aquaticus, Methanocaldococcus jannaschii): Thermostable enzymes packed with hydrophobic cores; DNA protected by reverse gyrase; membranes enriched in saturated fatty acids or ether monolayers.

  • Heat Shock Response: Exposure to thermal limits activates chaperones to prevent protein denaturation and refold misfolded proteins.

  • Pressure Adaptations (Barophiles, e.g., Shewanella living in Mariana Trench at $11,035\,m$ depth under $1085\text{ atm}$ pressure; ocean average depth $3,730\,m$ at $365\text{ atm}$ versus Mount Everest $8,848\,m$ at $0.296\text{ atm}$): High-pressure adapted membranes contain elevated polyunsaturated fatty acids; specially structured ribosome complexes.

• Osmotic Regulation and Halophiles:

  • Hypotonic environments: Mechanosensitive membrane channels open to dump intracellular solutes, preventing cell explosion.

  • Hypertonic environments: Cells synthesize or import compatible osmolytes to elevate internal osmolarity.

  • Halophiles (e.g., Halobacterium in hypersaline seas and soda lakes like Lake Magadi in Kenya, colored pink by Spirulina cyanobacteria): Maintain elevated internal $KCl$ concentrations to match external $2 - 4\text{ M NaCl}$.

  • Internal pH Homeostasis:

• Neutralophiles (E. coli, Bacillus subtilis) preserve internal pH around $7.8$ despite environmental shifts.

• Weak organic acids act as food preservatives because unprotonated molecules cross membranes and release protons inside cells, collapsing internal pH homeostasis.

• Acidophiles (Acidithiobacillus ferrooxidans, Acidiphilium acidophilum): Transport $K^+$ ions into cytoplasm to generate an inside-positive membrane potential, countering proton influx. Under extreme acid stress (pH 2), amino acid decarboxylases drain protons:

$\text{RCOO}^- + H^+ \rightarrow \text{RH} + CO_2$

• Alkaliphiles (Bacillus pseudofirmus OF4): Rely on $Na^+/H^+$ antiporters to import environmental protons while expelling $Na^+$. An energetic sodium motive force drives motility via flagellar motors and powers solute symport.

• Oxygen Toxicity and Reactive Oxygen Species (ROS):

  • Respiration uses $O_2$ as a terminal electron acceptor.

  • Autoxidation of electron transport carriers (e.g., FAD) transfers electrons to $O_2$, forming toxic Reactive Oxygen Species (ROS).

  • ROS Pathways:

  1. Superoxide radical anion ($\cdot O_2^-$) generated by single-electron transfer to $O_2$.

  2. Superoxide dismutase converts superoxide into Hydrogen Peroxide ($H_2O_2$):

$2\cdot O_2^- + 2H^+ \rightarrow H_2O_2 + O_2$

3. Hydroxyl radical ($\cdot OH$) generated via the Fenton reaction involving ferrous iron:

$Fe^{2+} + H_2O_2 \rightarrow Fe^{3+} + \cdot OH + OH^-$

• Protective Enzymes:

  • Catalase converts hydrogen peroxide to water and oxygen:

$2H_2O_2 \rightarrow 2H_2O + O_2$

- Peroxidase converts hydrogen peroxide to water using reduced NAD:

$2H_2O_2 + NADH + H^+ \rightarrow 2H_2O + NAD^+$

• Categorization of Organisms by Oxygen Sensitivity (Table 5.2):

  • Aerobic Microbes: Azotobacter spp. (soil nitrogen fixer), Neisseria spp. (meningitis, gonorrhea), Pseudomonas fluorescens (degrades TNT and aromatic hydrocarbons), Rhizobium spp. (plant root symbiont).

  • Facultative Microbes: Bacillus anthracis (anthrax), Escherichia coli (gut biota), Saccharomyces cerevisiae (baking yeast), Staphylococcus spp. (skin boils), Vibrio cholerae (cholera).

  • Microaerophilic Microbes: Campylobacter spp. (gastroenteritis), Helicobacter pylori (gastric ulcers), Lactobacillus spp. (yogurt fermentation), Treponema pallidum (syphilis).

  • Anaerobic Microbes: Actinomyces spp. (soil antibiotic producer), Azoarcus tolulyticus (degrades toluene), Bacteroides spp. (gut biota), Clostridium spp. (tetanus, botulism), Desulfovibrio spp. (sulfate reducer).

• Oxygen Growth Zones in Culture Tubes:

  • Top Aerobic Zone: High oxygen concentration ($300\,\mu M\text{ } O_2$).

  • Middle Microaerophilic Zone: Low oxygen concentration ($50\,\mu M\text{ } O_2$).

  • Bottom Anaerobic Zone: No oxygen ($5\,\mu M\text{ } O_2$).

• Anaerobic Growth Systems:

  • GasPak Jars: Sealed jars containing a palladium catalyst that drives:

H_2 + \frac{1}{2}O_2 \rightarrow H_2O

  GasPak envelopes generate $H_2$ and $CO_2$ to evacuate oxygen.

• Anaerobic Glove Boxes: Sealed glove boxes with air locks continuously purged with nitrogen/carbon dioxide mixtures.

Bacterial Growth Dynamics, Culturing, and Development

• Culturing Formulations and Media Classification:

  • Complex / Rich Media: Undefined nutrient mixtures supporting broad microbial growth (e.g., Lysogeny Broth / LB containing Bacto tryptone $10\,g/L$, Bacto yeast extract $5\,g/L$, and NaCl $10\,g/L$ at pH 7).

  • Defined / Synthetic Media: Precise, known chemical quantities (e.g., M9 medium containing glucose $2.0\,g/L$, $Na_2HPO_4\,42\,mM$, $KH_2PO_4\,22\,mM$, $NH_4Cl\,19\,mM$, $NaCl\,9\,mM$, $MgSO_4\,2.0\,mM$, $CaCl_2\,0.1\,mM$ at pH 7; or Sulfur oxidizer medium for Acidithiobacillus thiooxidans at pH 3).

  • MacConkey Agar (Selective and Differential):

  • Selective: Contains bile salts and crystal violet to inhibit Gram-positive organisms, allowing growth of Gram-negative bacteria.

  • Differential: Contains lactose and neutral red pH indicator. Lactose fermenters produce acidic end products, turning colonies pink; non-fermenters form white/uncolored colonies.

• Innovative Culturing Systems:

  • Isolation Chips (iChip): Designed by Kim Lewis and Slava Epstein to culture previously unculturable soil microbes in their native environment, leading to the discovery of Eleftheria terrae (producer of teixobactin).

  • Obligate Intracellular Bacteria: Rickettsia prowazekii (cause of epidemic typhus) cannot grow in cell-free media and must be cultured inside eukaryotic host cells (e.g., chicken embryo fibroblasts).

• Pure Culture Isolation Techniques:

  • Quadrant Dilution Streaking: Successively spreading cells across an agar plate with an inoculated loop, flaming the loop between quadrants to isolate individual single-cell colonies (clones).

  • Serial Dilutions and Spread Plating: Tenfold serial dilutions of a culture plated onto agar yield countable, isolated colonies.

• Cell Quantification Techniques:

  • Direct Microscopic Counting: Using a Petroff-Hausser counting chamber (hemocytometer) with etched grid lines. Live/dead stains (e.g., Syto9 green for live cells, propidium iodide red for dead cells) differentiate viable from non-viable cells.

  • Spectrophotometry: Measures turbidity/optical density ($OD_{600}$) of cell suspensions. Fast and non-destructive, but cannot distinguish live from dead cells; requires cell densities between $10^7$ and $10^{10}\text{ cells/mL}$.

  • Viable Colony Counts: Measures living cells capable of replicating to form visible colonies, expressed as Colony Forming Units (CFU).

  • Fluorescence-Activated Cell Sorting (FACS): Passes single cells through a laser beam, sorting cells electronically based on fluorescence (e.g., GFP expression) and light scatter (size).

• Batch Culture Growth Cycle (4 Phases):

  1. Lag Phase: Cells adapt to new culture conditions, repair damage, and synthesize necessary enzymes and machinery; no immediate change in cell number.

  2. Log / Exponential Phase: Cells divide at a maximal, constant rate, producing an exponential increase in cell numbers (appears as a straight line on a logarithmic scale).

  3. Stationary Phase: Key nutrients are exhausted and toxic wastes accumulate. Growth rate equals death rate; cells shut down growth machinery and turn on stress responses.

  4. Death Phase: Viable cell counts decrease exponentially with a constant half-life.

• Continuous Culture and Chemostats:

  • Chemostats continuously add fresh medium containing a limiting nutrient while removing spent culture fluid and waste at equal flow rates.

  • Maintains bacterial populations in continuous log phase growth at a constant cell density.

• Biofilms and Multicellular Communities:

  • Biofilms are specialized surface-attached cellular communities encased in a self-secreted Exopolysaccharide (EPS) matrix.

  • Stages of Biofilm Development:

  1. Planktonic cells attach to a surface monolayer using flagella.

  2. Microcolonies form as cells lose motility.

  3. Exopolysaccharide (EPS) matrix production is initiated.

  4. Mature biofilm tower structures form, traversed by nutrient channels.

  5. Dissolution and dispersal: Environmental cues trigger matrix degradation, releasing motile planktonic cells.

• Intercellular Communication: Quorum sensing uses autoinducer signals to coordinate collective gene expression.

• Biofilm Surfing Phenomenon: Nonmotile E. coli cells can surf on spreading waves of motile Acinetobacter baylyi colonies (Liyang Xiong et al. 2020).

  • Bacterial Cell Differentiation and Development:

• Endospore Formation (Bacillus, Clostridium):

  • Nutrient starvation induces an asymmetric cell division cascade resulting in a dormant, resistant endospore inside a mother cell.

  • The Seven Stages of Sporulation:

    • Stage I: Asymmetric septum forms near one pole; DNA replicates and extends into an axial filament.

    • Stage II: Septum divides the cell into a forespore and mother cell; DNA is translocated until each compartment receives a chromosome.

    • Stage III: The mother cell engulfs the forespore, enclosing it within a double membrane.

    • Stage IV: The mother cell chromosome disintegrates.

    • Stage V: A thick peptidoglycan cortex layer synthesizes between forespore membranes; coat proteins deposit on the outer membrane.

    • Stage VI: Dipicolinic acid synthesizes, and calcium accumulates within the spore coat.

    • Stage VII: The mother cell lyses, releasing the mature endospore.

• Heterocyst Formation in Cyanobacteria (Anabaena, Nostoc):

  • In response to nitrogen deprivation, every 10th cell in a filament differentiates into a thick-walled heterocyst.

  • Heterocysts shut down Photosystem II to maintain an anaerobic internal environment for nitrogenase ($N_2 \rightarrow NH_4^+$), while adjacent vegetative cells perform photosynthesis and supply carbohydrates.

• Fruiting Bodies (Myxococcus xanthus):

  • Starvation triggers social gliding motility, causing cells to aggregate into multicellular fruiting structures containing stress-resistant myxospores.

• Aerial Hyphae and Spores (Streptomyces):

  • Nutrient depletion causes vegetative substrate mycelia to form aerial hyphae that fragment into chains of reproductive spores, accompanied by secondary metabolite/antibiotic production (e.g., actinorhodin).

Physical, Chemical, and Biological Control of Microbes

• Kinetics of Microbial Death:

  • Microbes exposed to lethal agents die exponentially at a logarithmic rate.

  • $D\text{-value}$ (Decimal Reduction Time): The time required at a given temperature or agent concentration to kill $90\%$ ($1\text{ log}_{10}$ reduction) of the microbial population.

  • $2\text{ } D\text{-values}$ reduce the population by $99\%$.

• Physical Control Methods:

  • Heat Treatments:

  • Standard Pasteurization: $63\,^\circ\text{C}$ for $30\text{ minutes}$.

  • Flash Pasteurization (HTST): $72\,^\circ\text{C}$ for $15\text{ seconds}$.

  • Ultra-High Temperature (UHT): $150\,^\circ\text{C}$ for $3\text{ seconds}$ (achieves complete sterility; used for coffee creamers).

  • Autoclaving (Moist Heat under Pressure):

  • Standard parameters: $121\,^\circ\text{C}$ at $15\text{ psi}$ ($2\text{ atm}$ pressure) for $20\text{ minutes}$.

  • Destroys all vegetative cells and resistant endospores (Clostridium botulinum, Bacillus anthracis).

• Cold Preservation: Refrigeration ($4\,^\circ\text{C}$) slows metabolic activity; freezing ($-20\,^\circ\text{C}$ to $-80\,^\circ\text{C}$) halts growth.

• Irradiation: Ultraviolet (UV) light causes thymine dimers (surface disinfection); X-rays and gamma-rays break phosphodiester backbones.

  • Deinococcus radiodurans exhibits extreme radiation resistance via double-stranded DNA repair mechanisms (researched by Lydia Contreras).

• Filtration: Passage through membrane filters ($0.22\,\mu m$ pore size) removes bacteria from heat-labile liquids.

• Laminar Flow Biological Safety Cabinets: Contain HEPA filters to protect researchers and maintain sterile working environments.

  • Chemical Control Agents:

• Disinfectants and Antiseptics: Non-selective chemicals that denature proteins, disrupt membranes, or damage DNA.

  • Disinfectants (for inanimate objects): Chlorine bleach, Betadine (iodine), Phenol, Hexachlorophene, Formaldehyde, Glutaraldehyde, Ethylene oxide gas, Betapropiolactone.

  • Antiseptics (for living tissue): Ethanol, Isopropanol ($70\%$ rubbing alcohol), Cetylpyridinium chloride, Benzalkonium chloride.

• Antibiotics: Agents exhibiting selective toxicity against specific bacterial structures (e.g., cell wall transpeptidases, $30S/50S$ ribosomal subunits) with minimal toxicity toward eukaryotic hosts.

  • Penicillin derivatives block peptidoglycan cross-linking, causing growing cells to lyse.

  • Biological Control Agents:

• Probiotics: Live beneficial microbes administered to displace pathogens from host epithelial tissues.

• Phage Therapy: Application of host-specific bacteriophages to lyse bacterial pathogens without damaging human tissues or host microbiota.

Viral Structure, Classification, and Infection Cycles

• General Properties of Viruses:

  • Non-cellular obligate intracellular parasites that require host machinery for metabolic energy, structural materials, and translation.

  • Total global viral population estimated at $10^{32}$ virions.

  • Genomes consist of single- or double-stranded DNA or RNA, enclosed within a protective protein capsid.

• Detailed Model Viruses (Table 6.1):

• Viral Architecture:

  • Capsid Morphologies:

  • Icosahedral Capsids: $20$ triangular faces with 5-fold, 3-fold, and 2-fold rotational symmetry axes.

  • Filamentous / Helical Capsids: Helical protein tube surrounding the viral genome. Capsid length scales directly with genome length (e.g., Ebola virus; Tobacco Mosaic Virus / TMV with $18\,nm$ width and $2.3\,nm$ helical pitch).

  • Asymmetrical / Irregular Capsids: Large complex viruses (e.g., Vaccinia / Poxvirus) featuring a core envelope, outer membrane, lateral bodies, and surface tubules.

  • Complex Head-Tail Capsids: Combined icosahedral head and helical tail sheath (e.g., T4 and T7 bacteriophages).

  • Envelopes: Lipid bilayers derived from host plasma or organelle membranes during budding, embedded with viral glycoprotein spikes. Bacteriophages lack envelopes.

• Subviral Infectious Agents:

  • Viroids: Unenveloped, infectious circular single-stranded RNA molecules lacking protein capsids or protein-coding genes. Possess catalytic ribozyme activity; infect plants (e.g., Potato spindle tuber viroid).

  • Prions: Proteinaceous infectious particles devoid of nucleic acids. Aberrant, misfolded protein conformations (rich in $\beta$-sheets) induce normal native proteins (rich in $\alpha$-helices) to refold into the pathogenic conformation, causing transmissible spongiform encephalopathies ("mad cow" disease, Creutzfeldt-Jakob disease).

• Viral Genome Diversity:

  • Simple Genomes: Cauliflower mosaic virus ($8,000\,bp$ dsDNA), Avian leukosis virus ($7,200\,bp$ ssRNA with gag, pol, env genes and LTRs).

  • Giant Genomes: Mimivirus ($1,181,404\,bp$ dsDNA) encoding amino acid transporters, translation factors, and DNA repair enzymes.

• The Baltimore Classification System (Table 6.2):

• Bacteriophage Infection Mechanisms:

  • Receptor Recognition: Phages bind host envelope proteins used for nutrient uptake or motility (e.g., LPS, OmpF, TolC, flagellar motor components MotA, MotB, FliF).

  • Infection Dynamics:

  • Phage T4 attaches via tail fibers; tail sheath contracts to inject double-stranded DNA through the bacterial envelope.

  • Phage T7 virion structural dimensions ($15\,nm$ tail fibers, spanning $30\,nm$ OM-to-IM cell wall distance via an $8\,nm$ channel).

  • Reproductive Pathways:

  • Lytic Cycle: Phage genes express, replicate genome, assemble capsids, and synthesize endolysins to lyse the cell wall, releasing progeny virions.

  • Lysogenic Cycle: Phage DNA integrates into host chromosome as a prophage; replicates alongside host genome. Stress induces prophage excision and transition to the lytic cycle.

- Slow-Release Persistent Cycle (Phage M13): ssDNA enters $\rightarrow$ converts to dsDNA replicative form $

ightarrow$ produces (+)ssRNA progeny extruded through inner and outer membranes without lysing the host cell.

• Lysogenic Conversion (Table 6.3):

  • Prophages supply virulence factors and toxins to bacterial hosts:

  • Phage C1 (Clostridium botulinum) -> Botulinum toxin (c1).

  • Beta phage (Corynebacterium diphtheriae) -> Diphtheria toxin (tox).

  • Lambda (E. coli O157:H7) -> Cell envelope protein (bor).

  • Phage 933 (E. coli O157:H7) -> Shiga toxin (stx).

  • Epsilon 34 (Salmonella enterica) -> LPS synthesis (rfb) & Type III toxin (sopE).

  • TSST-1 (Staphylococcus aureus) -> Toxic shock syndrome toxin (speA).

  • CTX$\phi$ (Vibrio cholerae) -> Cholera toxin (ctxAB).

  • Human Endogenous Retroviruses (HERVs) supply essential functional proteins:

  • HERV-W -> Syncytin-1 (placental fusion).

  • HERV-FRD -> Syncytin-2 (placental fusion).

  • HERV -> INSL4 (insulin-like protein 4).

  • HERV-E -> MID1, Apolipoprotein C1, Endothelin type B receptor.

  • HERV-L -> Beta-1,3-galactosyltransferase.

  • LINE-1 -> ATRN (attractin; modulates inflammation).

• Host Immune Defenses and Viral Counter-Defenses:

  • Genetic Resistance: Mutations in host receptor proteins block viral binding.

  • Restriction-Modification Systems: Host methyltransferases add methyl groups ($Me$) to specific host DNA sequences (e.g., EcoRI site $5'\text{-GAATTC-}3'$). Restriction endonucleases cleave unmethylated foreign viral DNA.

  • CRISPR-Cas Adaptive Immunity:

  • Acquisition: Foreign viral DNA fragments are incorporated as new spacers within the host bacterial CRISPR array.

  • Expression: CRISPR array transcribes into pre-crRNA, processed into small crRNAs.

  • Interference: Cas-crRNA complex binds and cleaves invading viral DNA. Anti-CRISPR phages express Acr proteins to inhibit Cas activity.

• Eukaryotic Viral Life Cycles:

  • Attachment and Entry:

  • Receptor interaction (e.g., SARS-CoV-2 spike protein binding ACE2 receptor).

  • Direct membrane fusion at plasma membrane (e.g., Measles virus).

  • Endocytosis followed by endosomal acidification ($H^+$ pumping induces envelope fusion, e.g., HCV).

  • Endosomal transport and nuclear pore complex docking (e.g., Adenovirus uncoating dsDNA into nucleus).

• Replication Strategies:

  • DNA viruses enter nucleus to use host DNA polymerase, or replicate in cytoplasm using viral polymerases.

  • RNA viruses must encode RNA-dependent RNA Polymerase (RdRp) to transcribe RNA from RNA templates.

• Release Mechanisms: Cell lysis versus budding through host plasma or organelle membranes.

  • Viral Quantification and Culturing:

• One-Step Growth Curve:

  • Eclipse Period: Virions attach and uncoat; intracellular viral component synthesis occurs; free extracellular virions are absent.

  • Rise Period: Intracellular virions assemble and burst release into extracellular medium.

  • Burst Size Calculation:

$\text{Burst Size} = \frac{\text{Extracellular Phages / mL}}{\text{Phage-infected Cells / mL}}$

$\text{Example: } \frac{10^5\text{ phages/mL}}{200\text{ infected cells/mL}} = 500\text{ phages / infected cell}$

• Plaque Assay Protocol: Phage dilutions mixed with host bacteria in molten soft top agar ($0.75\%$) poured over a bottom agar plate ($1.5\%$) at an MOI of $0.1$. Incubation yields clear plaques within the confluent bacterial lawn, each representing a single original viral infection.