Comprehensive Microbiology Study Notes

Chapter 1 — What is Microbiology?

• Historical Context
• Anton van Leeuwenhoek
• Textile merchant turned microscopist after reading Robert Hooke’s Micrographia.
• Built simple single-lens microscopes (≈300× magnification).
• Observed objects one-tenth the size Hooke had seen.
• Robert Hooke
• Constructed compound microscopes (~30×).
• Coined the term “cell” while examining cork.

• Microbes & Their Importance
• Enormously abundant, diverse, and ancient (billions of years before humans).
• Essential for biosphere health; interact with each other and multicellular life (humans included).
• Applications: agriculture, industry, human microbiome studies.
• Some cause disease (e.g., SARS-CoV-2).

• Characteristics of Living Organisms
• Growth, reproduction, genetic blueprint, variation/evolution, response & adaptation, homeostasis.
• Edge cases:
• Bacterial endospores – dormant yet carry full living machinery.
• Viruses – replicate/evolve but are sub-cellular.
• Definition of “life” applied holistically.

• Cellular Organization
• Cell = minimal unit executing all life processes.
• Unicellular vs. multicellular; some single-celled microbes exhibit social/complex behaviors.

• Macromolecular Composition (> 90 % dry weight)
• Polypeptides/Proteins (≈50–55 %) – enzymes, transporters, structure.
• Nucleic acids (DNA, RNA) (≈2–20 %) – information & catalysis.
• Lipids (≈10 %) – membranes.
• Polysaccharides (≈6–7 %) – energy storage/structure.

• Domains of Life
• Traditional split: prokaryotes vs. eukaryotes.
• Carl Woese’s SSU rRNA comparisons → three-domain system: Bacteria, Archaea, Eukarya.
• Archaea & Eukarya share a more recent common ancestor than either with Bacteria.
• Replaces earlier five-kingdom model.
• Genomic architecture:
• Prokaryotes – usually one circular chromosome.
• Eukaryotes – multiple linear chromosomes, typically diploid (2n).

• Information Flow (“Central Dogma”)
• DNA (replication) → mRNA (transcription) → protein (translation).
• tRNAs deliver amino acids; ribosomes read universal genetic code.

• Genetic Variation & Evolution
• Mutation = ultimate source of variation.
• Genomics & bioinformatics reveal evolutionary relationships.
• Horizontal gene transfer common among microbes → mosaic genomes.

• Microbes as Model Systems
• Simple structure, fast growth, low cost.
• Escherichia coli, Saccharomyces cerevisiae central to biochemistry & genetics discoveries.

• Microbes & Disease
• Ancient beliefs: gods, “bad air,” spontaneous generation.
• Louis Pasteur’s swan-neck flask disproved spontaneous generation.
• Robert Koch linked Bacillus anthracis to anthrax → Koch’s postulates.
• Historic plagues: smallpox, influenza, Black Death (Yersinia pestis).

• Control of Infectious Disease
• Joseph Lister – antiseptic surgery.
• Antimicrobials: Salvarsan, penicillin, sulfa drugs.
• Vaccination (Edward Jenner); modern challenge – antibiotic resistance.

• Polymerase Chain Reaction (PCR)
• Invented 1983 (Kary Mullis).
• Reagents: template DNA, dNTPs, primers, thermostable DNA polymerase (e.g., Taq).
• Cycle:
1. Denature (~95 °C)
2. Anneal (primers bind)
3. Extend (polymerase synthesizes)
• Applications: microbial identification via SSU rRNA, pathogen detection, viral load monitoring.

Chapter 2 — What do Bacterial Cells Look Like?

• Morphology
• Coccus (spheres)
• Bacillus (rods)
• Vibrio (curved rods)
• Spirilla (spirals)
• Pleiomorphy – variable shapes in some species.

• Multicellular Arrangements
• Hyphae → mycelia (Actinomycetes).
• Trichomes (cyanobacteria) with polysaccharide sheath, interior channels.

• Size Range
• Typical: 0.5–5 µm.
• Gigantic exceptions: Thiomargarita namibiensis (≈700 µm), Epulopiscium fishelsoni (200–700 µm).

• Cytoplasmic Components
• Nucleoid: single circular chromosome + proteins/RNA; DNA compacted via cations, DNA-binding proteins, topoisomerases.
• Ribosomes; inclusion bodies (PHB, sulfur globules); gas vesicles; magnetosomes (guided by MamK).

• Cytoskeleton Proteins
• FtsZ (tubulin-like) – forms Z-ring for septation.
• MreB (actin-like) – shape maintenance.
• ParM (actin-like) – plasmid segregation.

• Cell Envelope
• Plasma Membrane
• Phospholipid bilayer (fluid mosaic).
• No sterols; some use hopanoids.
• Functions: transport (ABC, symport, antiport), energy capture (ETC → proton motive force → ATP synthase), sensing, secretion (general secretory pathway).
• Peptidoglycan Cell Wall
• Backbone: \text{NAG}–\text{NAM} repeats (β-1,4 linkages).
• Cross-linked peptides (species-specific bridges, D-amino acids).
• Degradation:
• Lysozyme (cuts β-1,4 glycosidic bond).
• Lysostaphin (cuts pentaglycine bridge in S. aureus).
• β-lactam antibiotics inhibit transpeptidases; β-lactamases confer resistance.
• Gram Stain
• Procedure: crystal violet → iodine → alcohol → safranin.
• Gram-positive – thick peptidoglycan + teichoic/lipoteichoic acids, stain purple; some form endospores.
• Gram-negative – thin peptidoglycan + outer membrane with LPS; stain pink.
• Periplasm resides between membranes.
• LPS = Lipid A (endotoxin) + core + O-side chain.
• Porins, TonB transport, Type III secretion system (injectisome).

• Surface Structures
• Flagella – helical propellers powered by proton motive force; assembly at tip.
• Pili/Fimbriae – attachment; sex pilus for conjugation.
• Stalks with holdfasts (e.g., Caulobacter).
• Capsules/Slime layers – polysaccharide shields; resist phagocytosis, desiccation.
• S-layers – crystalline protein lattices against phages/immune attack.
• Biofilms – surface-attached microbial communities.

• Classification & Phyla
• Taxonomic hierarchy: Domain → Species (type strain).
• Modern classification uses DNA (SSU rRNA).
• Major phyla: Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus-Thermus, Actinobacteria, Bacteroidetes.

Chapter 3 — What do Eukaryal Cells Look Like?

• Defining Features
• Membrane-bound nucleus; transcription separated from translation.
• Generally larger; contain membrane-bound organelles; complex cytoskeleton.

• Key Organelles
• Nucleus (DNA replication/transcription).
• Rough ER (translation, protein folding).
• Golgi (modification/sorting).
• Mitochondria (respiration; own DNA).
• Chloroplasts (photosynthesis; own DNA).
• Vacuoles, lysosomes, peroxisomes, hydrogenosomes.

• Secretory Pathway
• ER → vesicles → Golgi → destination; signal sequences guide trafficking; extensive post-translational modification.

• Plasma Membrane
• Phospholipid bilayer with sterols (cholesterol); lipid rafts (signaling, viral budding).
• Homeostasis via facilitated diffusion, active transport.
• Flagella/cilia are membrane-sheathed.

• Cell Wall (when present)
• Fungi: chitin.
• Algae: cellulose.
• Absent in protozoa (except cyst stages).

• Cytoskeleton
• Microtubules (α/β-tubulin), microfilaments (actin), intermediate filaments.
• Eukaryal flagella/cilia: 9 + 2 microtubule arrangement, dynein motors (wave-like motion).
• Pathogens (Shigella, Listeria) hijack actin for motility.

• Microscopy Tools
• Immunofluorescence (antibody staining of fixed cells).
• GFP fusions (live-cell protein localization).

• Diversity & Supergroups
• Six proposed supergroups: Archaeplastida, Excavata, Rhizaria, Amoebozoa, Opisthokonta, Chromalveolata.
• Model microbes: Saccharomyces cerevisiae, Giardia duodenalis, Dictyostelium discoideum, Chlamydomonas.

• Replication Strategies
• Asexual: mitosis (diploid → two identical diploid).
• Sexual: meiosis (diploid → four diverse haploid).
• Life-cycle variations (e.g., yeast, slime molds).

• Endosymbiotic Theory
• Mitochondria from α-proteobacteria; chloroplasts from cyanobacteria.
• Evidence: size, double membrane, bacterial-type DNA & ribosomes, independent fission.
• Primary vs. secondary endosymbiosis; hydrogenosome origin; nucleus origin debated (possible archaeal ancestor).

• Environmental Interactions
• Pathogenic eukaryotes: Plasmodium, Trypanosoma, Leishmania, Entamoeba, Giardia, etc.
• Treatment difficult due to host-like cellular machinery.
• Beneficial roles: primary production (algae), cellulose degradation (e.g., Trichonympha in termites), extremophiles.

Chapter 4 — What do Archaeal Cells Look Like?

• Discovery & Evolution
• Woese & Fox (1977) – SSU rRNA sequencing revealed Archaea distinct from Bacteria.
• Possible closer ancestry with Eukarya; two-domain hypothesis (Eukarya from Asgard archaea).
• Habitats: extremophiles and common environments.

• Cytoplasmic Features
• Circular chromosome; some possess histones (H3/H4-like) → DNA condensation.
• Actin/MreB-like cytoskeletal elements.

• Plasma Membrane Distinctions
• Isoprenoid chains attached to glycerol-1-phosphate.
• Ether linkages (vs. ester).
• Some form monolayers (biphytanyl) for thermal stability.
• Ignicoccus: inner & outer membrane, energy generation at outer membrane.
• Archaeosomes (liposomes of archaeal lipids) = stable vaccine platforms.

• Cell Wall
• Often pseudomurein (NAG-NAT, β-1,3 linkages).
• Unaffected by penicillin/lysozyme.

• Surface Structures
• S-layer; cannulae networks (Pyrodictium); archaeal flagella (thinner, glycosylated, basal assembly).

• Superphyla & Representative Groups
• Euryarchaeota
• Methanogens (CH₄ producers; e.g., Methanobrevibacter smithii in human gut; syntrophy with Bacteroides).
• Halophiles (high-salt; Halobacterium salinarum with bacteriorhodopsin phototrophy; adaptations: high intracellular K⁺, high GC DNA, acidic proteins).
• Thermo/acidophiles (e.g., Picrophilus).
• TACK
• Crenarchaeota – thermophiles/hyperthermophiles (> 55/80 °C), acidophiles, barophiles; adaptations: tetraether monolayers, protein salt bridges, thermosome chaperone, reverse DNA gyrase; industrial enzymes (Pfu polymerase).
• Thaumarchaeota – ammonia-oxidizers; some psychrophiles.
• Korarchaeota – uncultured; genomic mosaics.
• DPANN
• Nanoarchaeota – Nanoarchaeum equitans (490 kb genome) obligate symbiont of Ignicoccus.
• Asgard
• Lokiarchaeota, etc.; genomes with eukaryotic signature proteins; Prometheoarchaeum syntrophicum cultivated 2020.

Chapter 5 — What is a Virus?

• Definition
• Obligate intracellular parasites; genome (RNA or DNA) + protein capsid → nucleocapsid; some with lipid envelope.
• Depend on host for translation and (often) transcription/replication.

• Historical Milestones
• Ramses V mummy (smallpox).
• Yellow fever (Walter Reed, 1901).
• Virology advanced with cell culture techniques.

• Structure
• Size: 10–100 nm typical; giant viruses (APMV 400 nm, Pandoravirus 2.47 Mbp).
• Genome variations: ss/ds, RNA/DNA, linear/circular, segmented.
• Capsid symmetries: helical (TMV), icosahedral (rhinovirus), complex (bacteriophage, poxvirus).
• Envelopes: acquired from host; contain viral spikes – aid entry; fragile in environment; absent in “naked” viruses.

• Replication Cycle

  1. Attachment – specific receptor binding.

  2. Entry/Uncoating
    • Endocytosis (non-enveloped), fusion (enveloped), direct injection (phages), wound entry (plants).

  3. Gene expression & protein synthesis.

  4. Genome replication.

  5. Assembly & exit (budding for enveloped, lysis for naked/phages).

• Cultivation
• Phages: bacterial liquid cultures → clearing; agar overlays → plaques.
• Animal viruses: mammalian cell lines (e.g., HeLa); observe cytopathic effects (CPE).

• Purification
• Filtration (0.2–0.45 µm) removes cells; ultrafiltration for virus removal.
• Ultracentrifugation: differential & density gradients (sucrose).

• Quantification (Titer)
• Direct counts (EM, fluorescence, flow cytometry).
• Hemagglutination assay.
• Plaque assay – counts infectious units; often 1/100–1/1000 of total particles.
• Endpoint (LD₅₀, TCID₅₀).
• RT-PCR for viral load.

• Classification
• ICTV hierarchy (Order → Species).
• Baltimore scheme: seven groups based on pathway to mRNA recognizable by host ribosomes.
• Naming: site, physical traits, host, disease; WHO discourages geographic/personal names.

• Origins Hypotheses
• Coevolution, progressive (escape), regressive (reductive).
• Large NCLDVs may blur lines between viruses & cells; nucleus origin speculation.

• Virus-Like Pathogens
• Viroids – naked RNA (< 400 nt), highly base-paired, plant diseases; lack protein coat.

Chapter 7 — How do Microorganisms Grow?

• Growth Determinants
• Nutritional, chemical, physical factors + genetic traits.
• Prototrophs vs. auxotrophs; nutrient concentration initially proportional to growth then saturates.
• Phenotype Microarrays (tetrazolium dye) test utilization/tolerance.

• Oxygen Relationships
• Obligate aerobes, obligate anaerobes, aerotolerant, facultative, microaerophiles.
• Reactive oxygen species defenses: carotenoids, SOD, superoxide reductase, glutathione.
• Anaerobic culture: evacuated containers, anaerobic jars (H₂ + palladium catalyst removes O₂).

• pH Preferences
• Acidophiles (0.5-5.5), neutrophiles (5.5-8.5), alkaliphiles (8.5-13.5).

• Osmotic Pressure & Water Activity
• Hypotonic vs. hypertonic stress; cell wall critical.
• Halophiles accumulate solutes (K⁺).
• Water activity aw: most bacteria require aw > 0.9; fungi tolerate lower.

• Temperature Classes
• Psychrophiles (< 15 °C), mesophiles (15-45 °C), thermophiles (> 55 °C), hyperthermophiles (> 80 °C).

• Media & Cultivation
• Broth vs. agar; agar melts high T, solidifies ~40 °C.
• Complex vs. defined media.
• Selective (inhibit non-targets), differential (colony appearance), enrichment (increase rare microbes).

• Pure Culture Isolation
• Streak, spread, pour plate methods produce individual colonies.

• Unculturable Microbes
• Culture-independent: SSU rRNA sequencing, FISH, metagenomics, SSMS consortia cultivation.

• Measuring Growth
• Direct counts (microscope, flow cytometry).
• Viable counts – CFU via serial dilution & plating (10–300 colonies ideal).
• Turbidimetry – spectrophotometer OD₆₀₀ (10⁷-10⁹ cells/mL linear).

• Batch Culture Growth Curve
• Lag → exponential → stationary → death.
• Exponential math: Nt = N0 \times 2^n; n = generations; g = \frac{t}{n}; k = \frac{n}{t}.

• Continuous Culture (Chemostat)
• Constant influx/efflux keeps cells in exponential phase.
• Dilution rate controls growth rate; nutrient concentration controls cell density; risk of washout if D > \mu_{max}.

• Eliminating/Inhibiting Microbes
• Filtration – 0.2-0.45 µm (cells) or ultrafiltration (viruses).
• Heat
• Boiling, pasteurization, autoclave (121 °C, 15 psi).
• Cold – freezing with cryoprotectants stores cultures.
• Radiation
• UV (surface; thymine dimers).
• Ionizing (gamma/X-ray; deep penetration).
• (Additional chemical methods mentioned but details beyond provided excerpt.)