Difficulty: Introductory | Prerequisites: Basic biology (cell theory, organic chemistry fundamentals)
This first block of General Microbiology covers the foundations you will build on for the rest of the course. You start with what microbiology actually is and why it matters, then move into the physical architecture of microbial cells (walls, membranes, inclusions), how those cells harvest and use energy (metabolism, fermentation, respiration), how genetic information flows from DNA to protein, and finally how microbial populations grow and how we control that growth. If you are coming in cold, think of this section as learning what a microbe is made of, how it eats, how it reads its own instructions, and how it multiplies. Everything from viruses to immunity later in the course assumes you have this material down.
Microorganisms are the most abundant and metabolically diverse life forms on Earth, and studying them requires understanding their unique cell structures, their flexible metabolic strategies for generating ATP, and the molecular machinery that lets them grow and divide. This block covers the physical cell, the chemistry of energy, the central dogma as it works in prokaryotes, and the kinetics and control of microbial growth.
Microbiology
The study of organisms too small to be seen clearly with the unaided eye, including bacteria, archaea, fungi, protists, and viruses. In simple terms, it is the science of life at the microscopic scale.
Prokaryote
A unicellular organism lacking a membrane-bound nucleus. DNA sits in a nucleoid region rather than inside a nuclear envelope. Think of it as the "open-plan office" version of a cell: no walls around the genetic material.
Eukaryote
A cell or organism with a membrane-bound nucleus and organelles (mitochondria, ER, Golgi). In simple terms, the compartmentalised cell plan found in animals, plants, fungi, and protists.
Peptidoglycan (murein)
A polymer of sugars (NAG and NAM) cross-linked by short peptides that forms the rigid mesh of bacterial cell walls. Think of it as the chainmail that stops a bacterial cell from bursting under osmotic pressure.
Gram-positive
Bacteria with a thick peptidoglycan layer and no outer membrane. They retain crystal violet stain during the Gram stain procedure, appearing purple under the microscope.
Gram-negative
Bacteria with a thin peptidoglycan layer sandwiched between an inner membrane and an outer membrane containing lipopolysaccharide (LPS). They lose the crystal violet and pick up the safranin counterstain, appearing pink.
Lipopolysaccharide (LPS)
A large molecule in the outer membrane of Gram-negative bacteria composed of lipid A, core polysaccharide, and O-antigen. Lipid A is the endotoxin component that triggers strong immune responses. In simple terms, it is the "alarm molecule" that makes Gram-negative infections particularly inflammatory.
Cytoplasmic membrane
A phospholipid bilayer (bacteria) or monolayer of ether-linked lipids (archaea) that surrounds the cytoplasm. It is selectively permeable and houses transport proteins, respiratory chain components, and sensory apparatus.
Transport (active vs. passive)
Passive transport moves solutes down a concentration gradient without energy input (simple diffusion, facilitated diffusion). Active transport moves solutes against a gradient and requires energy, either from ATP hydrolysis (ABC transporters) or from the proton motive force (symporters, antiporters). The phosphotransferase system (PTS) in bacteria is a specialised group translocation mechanism that chemically modifies the substrate as it enters.
Inclusion bodies
Intracellular granules of stored nutrients or structural material, such as poly-beta-hydroxybutyrate (PHB) for carbon/energy storage, polyphosphate granules, sulfur globules, or gas vesicles for buoyancy. Think of them as a microbe's pantry or flotation devices.
Metabolism
The sum of all chemical reactions in a cell, divided into catabolism (breaking molecules down, releasing energy) and anabolism (building molecules up, consuming energy).
Oxidation-reduction (redox) reaction
A reaction in which electrons transfer from a donor (which is oxidised) to an acceptor (which is reduced). In simple terms, metabolism is largely a controlled relay of electrons from food molecules to terminal electron acceptors.
Free energy (delta G)
The thermodynamic quantity that predicts whether a reaction is spontaneous. A negative delta G means the reaction releases energy and can do work.
ATP (adenosine triphosphate)
The universal energy currency of cells. Energy is released when the terminal phosphate bond is hydrolysed. In simple terms, it is the rechargeable battery that powers nearly every cellular process.
Fermentation
An anaerobic catabolic process in which an organic compound serves as both the electron donor and the terminal electron acceptor. Substrate-level phosphorylation generates a small yield of ATP. Common end products include ethanol, lactic acid, and mixed acids.
Aerobic respiration
A catabolic process using oxygen as the terminal electron acceptor. Electrons pass through an electron transport chain embedded in the membrane, generating a proton motive force that drives ATP synthesis via ATP synthase (oxidative phosphorylation). Yields far more ATP than fermentation.
Proton motive force (PMF)
The electrochemical gradient of protons across the cytoplasmic membrane, composed of a chemical gradient (delta pH) and an electrical potential (delta psi). It is the energy intermediate that couples electron transport to ATP synthesis. Think of it as a dam of protons: when they flow back through ATP synthase, that flow does work.
Electron transport chain (ETC)
A series of membrane-associated electron carriers (flavoproteins, iron-sulfur proteins, quinones, cytochromes) that pass electrons in stepwise redox reactions from a donor to a terminal acceptor, pumping protons outward at each step.
Substrate-level phosphorylation
Direct transfer of a phosphate group from a high-energy organic substrate to ADP, forming ATP. This happens in glycolysis and the TCA cycle, not at the membrane.
Oxidative phosphorylation
ATP synthesis driven by the proton motive force via ATP synthase, coupled to the electron transport chain. This is the high-yield ATP pathway.
Central dogma
The flow of genetic information: DNA is replicated, transcribed into mRNA, and translated into protein. In prokaryotes, transcription and translation are coupled (they happen simultaneously in the cytoplasm).
Replication
Copying of DNA by DNA polymerase. In bacteria, replication is bidirectional from a single origin of replication (oriC) and semiconservative (each daughter molecule has one old strand and one new strand).
Transcription
Synthesis of RNA from a DNA template by RNA polymerase. In bacteria, sigma factors direct RNA polymerase to specific promoter sequences.
Translation
Synthesis of a polypeptide at the ribosome using mRNA as a template and tRNAs to deliver amino acids. Prokaryotic ribosomes are 70S (composed of 30S and 50S subunits), distinct from eukaryotic 80S ribosomes, which is why certain antibiotics can target bacterial translation without harming human cells.
Codon
A three-nucleotide sequence in mRNA that specifies a particular amino acid (or a stop signal). The genetic code is nearly universal.
Operon
A cluster of co-transcribed genes under the control of a single promoter, common in prokaryotes. In simple terms, it is a way for a bacterium to switch a whole metabolic pathway on or off with one regulatory event.
Generation time (doubling time)
The time required for a microbial population to double in number. It varies enormously: roughly 20 minutes for fast-growing E. coli under ideal conditions, hours or days for slower organisms.
Growth curve (batch culture)
The four phases a closed population passes through: lag phase (adaptation, no net increase), exponential (log) phase (constant maximal growth rate), stationary phase (growth rate equals death rate, nutrients deplete), and death phase (viable count declines).
Binary fission
The primary mode of cell division in prokaryotes. The chromosome replicates, the cell elongates, a septum forms at midcell (coordinated by the FtsZ protein ring), and two daughter cells separate.
Chemostat
A continuous culture device that maintains a microbial population in exponential growth by continuously adding fresh medium and removing culture at the same rate (dilution rate). Useful for studying growth at a steady state.
Sterilisation
The complete destruction or removal of all viable organisms, including endospores. Achieved by autoclaving (121 °C, 15 psi, 15 min), filtration, or certain chemical treatments.
Pasteurisation
Heat treatment sufficient to kill pathogens and reduce spoilage organisms without sterilising. Named after Louis Pasteur.
Antimicrobial agent
A chemical that kills (bactericidal/cidal) or inhibits growth of (bacteriostatic/static) microorganisms. Includes antibiotics, disinfectants, and antiseptics.
Microbiology encompasses the study of bacteria, archaea, eukarya (fungi, algae, protozoa), and viruses
Microorganisms drive global biogeochemical cycles (carbon, nitrogen, sulfur) and are essential for life on Earth
Historical milestones to know:
Antonie van Leeuwenhoek: first to observe microorganisms with a microscope (1670s)
Louis Pasteur: disproved spontaneous generation, developed pasteurisation, pioneered germ theory
Robert Koch: Koch's postulates (four criteria for establishing a microorganism as the cause of a disease)
Martinus Beijerinck and Sergei Winogradsky: founders of environmental microbiology and the enrichment culture technique
Koch's postulates:
The suspected pathogen must be present in every case of the disease and absent from healthy individuals
The pathogen must be isolated and grown in pure culture
The cultured pathogen must cause the disease when introduced into a healthy host
The same pathogen must be re-isolated from the experimentally infected host
The three domains of life: Bacteria, Archaea, Eukarya. Determined by comparative rRNA sequencing (Carl Woese)
Cell morphology: cocci (spheres), bacilli (rods), spirilla (spirals), vibrios (curved rods). Shape is maintained largely by the peptidoglycan sacculus and cytoskeletal elements (MreB, FtsZ)
Cell wall differences are clinically important:
Gram-positive: thick peptidoglycan (up to 90% of wall), teichoic acids and lipoteichoic acids embedded in the wall
Gram-negative: thin peptidoglycan, outer membrane with LPS, periplasmic space between inner and outer membranes
Archaea: lack peptidoglycan entirely; instead use pseudopeptidoglycan (pseudomurein), S-layers, or other polymers
Outer membrane of Gram-negatives: contains porins (channels for small hydrophilic molecules) and LPS. The lipid A portion of LPS is the endotoxin
Surface structures:
Capsules and slime layers (collectively, glycocalyx): polysaccharide or polypeptide coatings that protect against phagocytosis, desiccation, and help with biofilm formation
Fimbriae and pili: thin protein appendages; fimbriae for adhesion, type IV pili for twitching motility and DNA uptake
Flagella: rotating protein filaments for motility; driven by the proton motive force. Bacterial flagella rotate; archaeal flagella (archaella) are structurally different and assembled differently
Endospores (e.g., Bacillus, Clostridium): highly resistant dormant structures formed under nutrient stress. Contain dipicolinic acid and small acid-soluble proteins (SASPs) that protect DNA. Relevant to food safety and clinical sterilisation
Bacterial membranes: phospholipid bilayer with ester-linked fatty acids. Hopanoids serve a stabilising role similar to cholesterol in eukaryotic membranes
Archaeal membranes: ether-linked isoprenoid lipids, sometimes forming a monolayer (tetraether lipids) that is exceptionally stable at extreme temperatures and pH
Transport mechanisms:
Simple diffusion: small nonpolar molecules (O₂, CO₂) cross freely
Facilitated diffusion: carrier proteins or channels, no energy required, down the gradient
Active transport: energy-dependent; ABC transporters (ATP-driven), symporters and antiporters (PMF-driven)
Group translocation (PTS): substrate is phosphorylated during transport, so the intracellular form differs from the extracellular form
Surface-area-to-volume ratio: smaller cells have a higher ratio, allowing faster nutrient exchange per unit volume. This is a major reason prokaryotes can grow so much faster than larger cells
Energetics basics:
Catabolic reactions release free energy (exergonic, negative delta G)
Anabolic reactions require free energy input (endergonic, positive delta G)
Coupled reactions: an exergonic reaction drives an endergonic one, often through ATP as an intermediary
Glycolysis (Embden-Meyerhof pathway): glucose (6C) is split into two pyruvate (3C), yielding 2 ATP (net) and 2 NADH per glucose
TCA cycle (Krebs cycle): acetyl-CoA is oxidised to CO₂, generating NADH, FADH₂, and 1 GTP per turn. Runs twice per glucose
Electron transport and oxidative phosphorylation:
NADH and FADH₂ donate electrons to the ETC
Electrons pass through carriers of increasingly positive reduction potential
Protons are pumped across the membrane, building the PMF
ATP synthase (a rotary molecular motor) uses proton flow back across the membrane to synthesise ATP
Theoretical maximum yield: around 38 ATP per glucose under aerobic conditions (the real yield is lower)
Fermentation pathways:
Used when no external electron acceptor is available (or under facultative conditions)
Organic molecules serve as both electron donor and acceptor
Examples: lactic acid fermentation (homolactic, heterolactic), alcoholic fermentation (ethanol + CO₂), mixed-acid fermentation
Low ATP yield (typically 2 ATP per glucose via substrate-level phosphorylation only)
Anaerobic respiration: uses an alternative terminal electron acceptor instead of O₂, such as nitrate (denitrification), sulfate (sulfate reduction), or Fe³⁺. Less energy yield than aerobic respiration but more than fermentation
Chemolithotrophy: inorganic compounds (H₂, H₂S, NH₃, Fe²⁺) serve as electron donors. Important in biogeochemical cycles. These organisms can be autotrophs, fixing CO₂ via the Calvin cycle
Phototrophy: light energy drives electron flow and proton pumping. Oxygenic photosynthesis (cyanobacteria, using water as electron donor, producing O₂) vs. anoxygenic photosynthesis (purple and green bacteria, using H₂S or organic compounds)
DNA replication in bacteria:
Bidirectional from oriC
DNA polymerase III: main replicative enzyme, synthesises 5' to 3', has proofreading (3' to 5' exonuclease)
Leading strand: continuous synthesis; lagging strand: discontinuous (Okazaki fragments joined by DNA ligase)
Helicase unwinds, primase lays RNA primers, SSB proteins stabilise single strands, topoisomerase relieves supercoiling
Transcription:
RNA polymerase (core enzyme: alpha₂, beta, beta-prime, omega) plus sigma factor for promoter recognition
Sigma-70 is the housekeeping sigma in E. coli; alternative sigma factors direct transcription of specialised gene sets (e.g., sigma-32 for heat shock)
Promoter elements: -10 region (Pribnow box) and -35 region
Termination: intrinsic (rho-independent, via stem-loop/hairpin) or rho-dependent
Translation:
Shine-Dalgarno sequence on mRNA aligns with 16S rRNA of the 30S subunit for initiation
Initiation: 30S + mRNA + fMet-tRNA, then 50S joins to form 70S initiation complex
Elongation: aminoacyl-tRNA enters the A site, peptide bond forms (peptidyl transferase activity of 23S rRNA, a ribozyme), translocation moves the ribosome one codon along
Termination: release factors recognise stop codons
Polyribosomes: multiple ribosomes translate the same mRNA simultaneously
Protein processing:
Chaperones (e.g., GroEL/GroES, DnaK) assist proper folding
Post-translational modifications: limited in bacteria compared to eukaryotes, but include proteolytic cleavage of signal peptides (for secreted proteins) and occasional lipid modification
Protein secretion systems: Sec pathway (general secretory), Tat pathway (twin-arginine translocation, for folded proteins), type I through type VI secretion systems in Gram-negatives
Binary fission overview: chromosome replication, cell elongation, septum formation (FtsZ ring), daughter cell separation
Growth mathematics:
N = N₀ × 2ⁿ, where n = number of generations
Generation time (g) = t / n, where t is elapsed time
Growth rate constant (k) = n / t = 1/g
In log phase, a plot of log₁₀(cell number) vs. time gives a straight line
Batch culture growth curve:
Lag phase: cells adjust enzymes and machinery to the new medium; no increase in cell number
Exponential (log) phase: cells divide at maximum rate for the given conditions
Stationary phase: nutrients deplete, waste products accumulate, growth rate = death rate; cells may produce secondary metabolites, enter stress responses
Death phase: viability drops; some cells may persist (persisters)
Measuring growth:
Direct counts: microscopic count (Petroff-Hausser chamber, includes dead cells), viable plate count (colony-forming units, CFU), membrane filtration
Indirect methods: turbidity (optical density at 600 nm), dry weight, metabolic activity assays
Environmental factors affecting growth:
Temperature: psychrophiles, mesophiles, thermophiles, hyperthermophiles. Optimum, minimum, and maximum growth temperatures define an organism's cardinal temperatures
pH: acidophiles, neutrophiles, alkaliphiles. Most bacteria grow best near pH 7
Osmolarity: halophiles require elevated NaCl; compatible solutes help cells maintain turgor under osmotic stress
Oxygen: obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, aerotolerant anaerobes. Oxygen toxicity involves reactive oxygen species (superoxide, hydrogen peroxide, hydroxyl radical), managed by superoxide dismutase, catalase, and peroxidase
Growth control:
Physical methods: heat (autoclaving, pasteurisation, dry heat), radiation (UV for surfaces, gamma for sterilisation), filtration (0.2 µm membrane filters for heat-sensitive solutions)
Chemical methods: disinfectants (surfaces), antiseptics (living tissue), sterilants (chemical sterilisation). Key agents include alcohols, halogens, phenolics, quaternary ammonium compounds, aldehydes
Measuring effectiveness: minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), decimal reduction time (D value)
Exponential growth equation: N = N₀ × 2ⁿ
Generation time: g = t / n
Growth rate constant: k = 1 / g
Free energy change: ΔG°' = –nF ΔE₀' (relates standard free energy to the difference in reduction potential of the redox couple, where n = electrons transferred, F = Faraday constant)
PMF equation: Δp = Δψ – (2.3RT/F) × ΔpH
Pasteurisation and autoclaving are direct applications of microbial growth control principles; every hospital, food processing plant, and laboratory depends on them daily
Fermentation is the basis of the brewing, baking, dairy, and biofuel industries: when you eat yoghurt or drink beer, you are consuming the metabolic end products of microbial fermentation
The Gram stain is typically the first diagnostic test performed on a clinical specimen; knowing whether an infection is Gram-positive or Gram-negative immediately narrows antibiotic choices
Chemolithotrophs that oxidise ammonia or sulfur compounds are the workhorses of wastewater treatment and bioremediation
Students often think "anaerobic" means "does not use an electron transport chain." Anaerobic respiration does use an ETC; it simply uses a terminal electron acceptor other than oxygen. Fermentation is the pathway with no ETC at all
Students frequently confuse substrate-level phosphorylation with oxidative phosphorylation. Substrate-level phosphorylation is a direct chemical transfer of a phosphate to ADP; oxidative phosphorylation is an indirect, chemiosmotic process driven by the PMF
The Gram stain classifies bacteria by cell wall architecture, not by evolutionary relatedness. Some organisms do not Gram-stain well at all (e.g., Mycoplasma lacks a cell wall entirely)
"Sterilisation" and "disinfection" are not interchangeable. Sterilisation destroys everything, including endospores. Disinfection reduces pathogen load but does not guarantee the elimination of all organisms
⚠️ Be able to compare and contrast Gram-positive and Gram-negative cell wall structure in detail, including the location of peptidoglycan, LPS, teichoic acids, and the periplasmic space
⚠️ Know the difference between fermentation, aerobic respiration, and anaerobic respiration, including ATP yield, electron donors/acceptors, and where each occurs in the cell
⚠️ Understand the PMF: what generates it, what it is composed of (delta psi + delta pH), and what it drives (ATP synthesis, flagellar rotation, transport)
⚠️ Be able to calculate generation time and number of generations from given data
⚠️ Know the four phases of the batch culture growth curve and what is happening physiologically in each
⚠️ Understand Koch's postulates and their limitations (e.g., unculturable organisms, asymptomatic carriers)
True or False: Archaea have peptidoglycan in their cell walls.
Fill in the blank: The terminal electron acceptor in aerobic respiration is ________.
True or False: Fermentation produces more ATP per glucose than aerobic respiration.
Fill in the blank: The Shine-Dalgarno sequence helps position the mRNA on the ________ ribosomal subunit.
True or False: During lag phase, cells are dormant and metabolically inactive.
(Answers: 1. False, archaea lack peptidoglycan. 2. Oxygen (O₂). 3. False, fermentation yields far less ATP. 4. 30S. 5. False, cells are metabolically active during lag phase, synthesising enzymes and adjusting to the medium; they simply are not dividing yet.)
Q: A Gram-negative bacterium is treated with lysozyme, which degrades peptidoglycan. What happens to the cell, and why does the outcome differ from a Gram-positive cell treated the same way?
A: In both cases, lysozyme degrades the peptidoglycan layer. A Gram-positive cell, which relies on its thick peptidoglycan for structural integrity, will lyse in a hypotonic environment once the wall is destroyed (forming a protoplast). A Gram-negative cell also loses its peptidoglycan, but it still has an outer membrane; the result is a spheroplast (the outer membrane provides some residual protection, though the cell is still osmotically fragile and will lyse in hypotonic conditions).
Q: Why does anaerobic respiration yield less ATP than aerobic respiration, even though both use an electron transport chain?
A: The amount of energy released (and therefore the number of protons pumped and ATP synthesised) depends on the difference in reduction potential between the electron donor and the terminal electron acceptor. Oxygen has the most positive standard reduction potential of common biological acceptors (+0.82 V). Alternative acceptors like nitrate (+0.43 V) or sulfate (–0.22 V) have less positive potentials, so the overall delta E is smaller, less energy is released, and fewer ATP molecules are produced.
Q: Explain why small cell size is advantageous for prokaryotic growth rate.
A: A smaller cell has a higher surface-area-to-volume ratio, meaning more membrane surface is available per unit of cytoplasm for nutrient uptake and waste export. This allows the cell to exchange materials with its environment more efficiently, supporting a faster metabolic rate and shorter generation time.
Q: A student plates a dilution of a bacterial culture and counts 150 colonies on a plate that received 0.1 mL of a 10⁻⁶ dilution. What is the concentration of viable cells in the original culture?
A: CFU/mL = colonies / (dilution factor × volume plated) = 150 / (10⁻⁶ × 0.1) = 150 / 10⁻⁷ = 1.5 × 10⁹ CFU/mL.
Q: Name two ways the proton motive force is used by bacterial cells besides ATP synthesis.
A: The PMF powers flagellar rotation (motility) and drives active transport of nutrients across the cytoplasmic membrane (e.g., lactose permease, a proton symporter).
Cell wall structure and LPS connect directly to the immunity and pathogenicity material later in the course (Ch. 25–27). Understanding endotoxin here will pay off when studying innate immunity and sepsis
Metabolic diversity (chemolithotrophy, phototrophy) reappears when you study microbial ecosystems and nutrient cycles (Ch. 20–21). The organisms driving the nitrogen and sulfur cycles use the same redox chemistry introduced here
Growth control principles (sterilisation, antimicrobial agents) form the foundation for clinical microbiology and antimicrobial compounds (Ch. 28)
microbiology, prokaryote, eukaryote, peptidoglycan, murein, NAG, NAM, Gram stain, Gram-positive, Gram-negative, LPS, lipopolysaccharide, endotoxin, outer membrane, periplasm, porin, cell membrane, phospholipid bilayer, archaea ether lipids, tetraether, transport, ABC transporter, PTS, group translocation, proton motive force, PMF, chemiosmosis, ATP synthase, oxidative phosphorylation, substrate-level phosphorylation, glycolysis, TCA cycle, Krebs cycle, ETC, electron transport chain, fermentation, aerobic respiration, anaerobic respiration, chemolithotrophy, phototrophy, Calvin cycle, DNA replication, transcription, translation, ribosome 70S, Shine-Dalgarno, sigma factor, operon, binary fission, FtsZ, generation time, doubling time, growth curve, lag phase, log phase, stationary phase, chemostat, sterilisation, autoclave, pasteurisation, MIC, MBC, endospore, Koch's postulates, Brock Biology of Microorganisms Chapter 1 2 3 4 5, MCB 3020