Comprehensive Study Notes: Bacterial and Archaeal Growth (Chapter 7)
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Chapter 7 focus: Bacterial and Archaeal Growth.
Dramatic intro cues:
"DON'T ANYBODY TOUCH THAT CHIP YET!" followed by countdown (1…2…3…4…).
A flash of light that causes… SYPHILIS, TREPONEMA PALLIDUM!!
Light humor: scientist jokes about naming microbial species as Harry Potter spells for diseases; reflects a playful approach to taxonomy and pathogenicity.
Takeaway for study: establishes context that microbes vary widely in growth and form, and naming conventions are part of microbiology language.
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Learning objectives overview:
Describe the general process of asexual reproduction (binary fission) in bacteria.
Describe how chromosome partitioning and cytokinesis occur in bacteria.
Define cardinal temperatures: minimum, optimum, and maximum growth temperatures, and how growth/survival shifts impact organisms as temperatures change.
Define growth-temperature-related categories: thermophilic, psychrophilic, psychrotolerant, mesophilic, halophilic, acidophilic, alkalophilic, etc.
Identify the four phases of bacterial batch culture growth and describe cellular activities in each phase.
Explain how extreme temperatures, pH, or salt concentration affect growth (e.g., membrane stability, enzyme activity, proton motive force).
Explain how oxygen availability influences growth of aerobes, obligate anaerobes, aerotolerant anaerobes, and facultative anaerobes.
Summarize what a chemostat is and its用途/uses.
Given starting culture concentration and number of generations, calculate the final concentration.
Predict microbial growth behavior under varying conditions (temperature, nutrients, aeration, etc.).
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Binary fission as the typical replication mode for many Bacteria and Archaea:
Growth refers to an increase in cell number, not just cell size.
In binary fission, the cell elongates and increases in volume.
The chromosome replicates and moves toward opposite poles.
A septum forms to separate the cytoplasm into two daughter cells.
In cells that form arrangements, the septum may remain attached; this occurs almost never in Gram-negative bacteria due to the outer membrane (OM).
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The bacterial cell cycle is well studied in model organisms (e.g., E. coli, B. subtilis, C. crescentus).
Three phases of the cell cycle:
1) Elongation: increase in cell size (similar to G1 phase in eukaryotes).
2) Chromosome replication and partitioning: involves S-phase-like replication and mitosis-like partitioning, occurring concurrently.
3) Cytokinesis: occurs prior to complete replication/partitioning; septum development mediates division.
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Chromosome partitioning in C. crescentus (well studied):
After replisome formation and bidirectional initiation of DNA replication, chromosomes are partitioned.
parS sites near oriC bind ParB; one parS stays near the stalk, the other moves to the opposite pole.
ParA forms an increasing gradient from the stalk-associated pole toward the opposite side.
The parS site is handed from one ParA to the next like a baton to drive partitioning.
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Cytokinesis and septation (definition and sequence):
Cytokinesis: formation of two daughter cells after division.
Septation: formation of a cross-wall (septum) between the two daughter cells.
Steps:
Selection of the septum formation site.
Assembly of the Z-ring (a polymer of FtsZ).
Formation of the divisome (protein complex involved in peptidoglycan synthesis at midcell).
Constriction of the cell and septum formation.
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FtsZ and cell division architecture:
FtsZ polymers form the bacterial cytoskeleton and are distributed around the plasma membrane when not dividing.
The Min system accumulates near the cell poles when division is imminent and prevents FtsZ polymerization at the poles.
Consequently, FtsZ polymerizes at midcell where Min is absent.
Nucleoid occlusion: SlmA coats the chromosome to prevent premature FtsZ polymerization before DNA partitioning.
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Divisome formation details:
FtsZ assembles at midcell and anchors to the membrane via ZipA and FtsA.
The Z-ring exhibits treadmilling: it grows by removing FtsZ from one end and adding to the other.
Divisome is coordinated by anchoring proteins and comprises roughly 30 proteins that catalyze peptidoglycan synthesis across midcell.
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Uiowa Microbiology Faculty Study Divisome Proteins:
Research by Weiss and Ellermeier on divisome architecture in Clostridioides difficile (C. diff).
Identify novel divisome proteins by tagging candidate genes with RFP to detect localization.
Use reverse genetics to observe phenotypes after deleting suspected divisome genes.
Potential antibiotic targets: divisome components in C. diff may be exploited to minimize impacts on the microbiome.
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Peptidoglycan synthesis is essential for septum formation:
NAG-NAM-pentapeptide building blocks are synthesized in the cytoplasm.
Building blocks are attached to bactoprenol and flipped across the membrane by MurJ flippase.
In the periplasm, peptidoglycan glytransferases cleave existing strands to insert new subunits.
Transpeptidases (penicillin-binding proteins, PBPs) form crosslinks.
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How peptidoglycan synthesis determines cell shape:
Cocci: limited elongation; synthesis mainly at the central septum.
Bacilli: elongasome-driven elongation via MreB (cytoskeletal protein forming bands for lateral growth).
Vibrio: crescentin slows synthesis on one side, inducing curvature.
MreB forms structures similar to FtsZ in guiding PG synthesis in elongated cells; crescentin contributes to curvature in certain shapes.
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The Archaeal Cell Cycle is Unique:
Archaea (e.g., Sulfolobus spp.) show a different timing of replication, partitioning, and cytokinesis, not all occurring simultaneously as in bacteria.
SegA is analogous to ParA; SegB functions like ParB, but with distinct structure (convergent evolution).
Z-ring in archaea is involved in synthesizing the S-layer rather than a typical bacterial cell wall.
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Microbial growth in batch culture basics:
Growth is typically evaluated in pure liquid cultures in closed vessels (batch culture) with no new resource input.
Growth is plotted as a log (Y-axis) of cell number versus time (X-axis) for better visualization of doubling.
Five phases in batch culture: lag, exponential, stationary, death, long-term stationary.
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Lag phase details:
Cells prepare for division (ribosome and ATP synthesis).
Acclimation to new conditions.
Initiation of replication begins during lag.
Exponential (log) phase:
Maximum replication rate; population becomes uniform.
Higher nutrient availability supports higher density and growth rate; growth eventually saturates due to resource limits.
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Stationary and death phases:
Stationary: replication and death occur at similar rates due to limited nutrients and oxygen; toxic wastes accumulate.
Death: cells die faster than they replicate due to resource depletion and waste accumulation.
Long-term stationary phase: dying cells release materials that fuel persisting cells; waves of replication and death occur; harsh conditions can drive evolution.
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Using generation number to determine total cells:
Generation time is the time required for the population to double.
Let N0 be the initial population; Nt the population at time t; n is the number of generations in time t.
For binary fission, the relationship is:
This formulation highlights exponential growth in discrete generations.
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Generation times of commonly studied microbes (examples):
Bacteria:
Escherichia coli — Incubation temp: 40°C; Generation time: 0.35 h
Staphylococcus aureus — 37°C; 0.47 h
Mycobacterium tuberculosis — 37°C; ≈12 h
Treponema pallidum — 37°C; 33 h
Archaea:
Pyrococcus abyssi — 90°C; 0.67 h
Sul furisphaera tokodaii — 75°C; 6 h
Nitrososphaera viennensis — 37°C; 45 h
Eukaryotes:
Euglena gracilis — 25°C; 22 h
Ceratium tripos — 20°C; 48–72 h
Saccharomyces cerevisiae — 30°C; 2 h
Monilinia fructicola — 25°C; 30 h
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Continuous culture is used in biotechnology to avoid stationary/death phases:
Continuous culture: an open system where fresh resources are added and waste products and cells are removed at the same rate.
Chemostat: a continuous culture device with a known volume added and spent media removed at a constant rate; an essential nutrient is limited to maintain a low growth rate.
Turbidostat: similar to a chemostat but maintains a target turbidity (cell density) rather than limiting a specific nutrient.
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Growth rate is impacted by environmental factors – Nutrients:
Macronutrients: required in large amounts; major elements C, O, N, H, P, S constitute ~96% of the dry weight of a bacterial cell; form macromolecules (proteins, lipids, polysaccharides, LPS, nucleic acids).
Micronutrients: required in smaller amounts; K, Na, Ca, Mg, Cl, Fe contribute ~3.7% of dry weight and often function as coenzymes.
Trace metals and growth factors also play crucial roles.
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Osmotic concentration and osmotic stress:
Osmotic conditions affect growth by altering osmotic pressure across membranes.
Hypotonic environments: some microbes tolerate or require lower external solute; hypertonic environments: high external solute concentrations.
Halophiles require high NaCl for growth.
Osmotolerant/halotolerant organisms grow across wide ranges of water activity.
Xerotolerant organisms withstand high solute concentrations.
Microbes often synthesize compatible solutes to balance internal osmotic pressure.
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pH and growth:
Neutrophiles grow best near neutral pH.
Acidophiles prefer low pH; alkalophiles prefer high pH.
Most bacteria and protists thrive at neutral pH; fungi and some photosynthetic protists favor slightly acidic environments.
Individual microbes tend to have narrow pH ranges, but communities can span broad pH spectra.
Buffers help maintain near-neutral cytoplasmic pH even in highly acidic environments (e.g., sauerkraut, pickles, cheeses resist spoilage due to acidity).
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Temperature and growth:
Enzyme activity constrains the temperature range a microbe can tolerate.
Below optimum: reduced catalytic activity; above optimum: protein denaturation.
Every organism has a minimum, optimum (cardinal), and maximum growth temperature; typical growth temperature range is less than 40°C for a single microbe.
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Temperature classifications and adaptations:
Psychrotolerant: grows 0–35°C; Psychrophiles: 0–20°C; enzymes with more α-helices, more polar residues, fewer weak bonds; more unsaturated fatty acids in membranes.
Thermophiles: 45–85°C; Hyperthermophiles: 85–100°C; adaptations include stronger ionic bonding, highly hydrophobic interiors, solute production for protein stabilization, long/saturated membrane lipids; enzymes like Taq polymerase used in biotechnology.
Most human pathogens and microbiome members are mesophiles: roughly 20–45°C.
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Oxygen and growth (aerobic respiration vs ROS):
Oxygen as a terminal electron acceptor enables efficient ATP production but can produce reactive oxygen species (ROS).
Organisms living with oxygen must have ROS-detoxifying enzymes: catalase, peroxidase, superoxide dismutase, and/or superoxide reductase.
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Oxygen-utilization categories:
Obligate aerobes: require full O2 tension (~21%);
Microaerophiles: require O2 at levels lower than air due to respiration limits or sensitivity;
Facultative anaerobes: can grow with or without O2, generally grow better with O2;
Anaerobes: cannot respire O2;
Aerotolerant anaerobes: tolerate O2 but cannot respire;
Obligate anaerobes: inhibited or killed by O2 (found in bacteria/archaea; some fungi and protozoa as exceptions).
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Learning objectives related to biofilms (summary):
List stages of biofilm formation and maturation.
Explain the great plate count anomaly / viable but non-culturable state (VBNC).
Decide which method of analyzing microbial cell density is appropriate for a given situation.
Explain when to use selective, differential, or enrichment media; predict the nature of a medium (selective/differential/enriched) based on ingredients and observed growth.
Determine if a given medium is complex or defined.
Determine if a medium is broth or semi-solid based on ingredients.
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Biofilms and microbial lifestyle:
Planktonic growth: free-floating in liquids.
Sessile growth: microbes attached to surfaces; most microbes favor sessile lifestyle.
Biofilms: aggregates of microbial communities embedded in exopolymers that protect against harsh conditions, disinfection, and antibiotics.
Microbes secrete polysaccharides, DNA/RNA, and proteins during biofilm development.
Horizontal gene transfer is common within biofilms.
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Biofilm formation and regulation:
Attachment to surfaces initiates biofilm development via pili/fimbriae, flagella, and/or glycocalyx.
Exopolymer production is controlled by quorum sensing, a cell-to-cell communication mechanism using signaling molecules (e.g., N-acylhomoserine lactone, AHL).
Quorum sensing ensures sufficient cell density before robust biofilm formation.
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Growing microbes in the lab: culture media basics
Defined (synthetic) medium: each ingredient is chemically defined with known concentrations.
Complex medium: contains ingredients with nonspecific composition (e.g., extracts, -tones).
Agar media: solidifying agent from algae; when dissolved provides semi-solid media; many bacteria cannot utilize agar as a nutrient source, so it remains largely inert as a support.
Broth media: liquid medium without agar.
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Media types: selective, differential, and enriched
Selective medium: inhibits some microbes while allowing others to grow (e.g., MacConkey agar selects for Gram-negative bacteria and inhibits Gram-positives).
Differential medium: contains indicators (often dyes) that reveal metabolic differences during growth (e.g., blood agar to detect hemolysis).
Enriched medium: supports growth of fastidious organisms by providing additional growth factors, often supplemented with blood.
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Anaerobic culture techniques:
Anaerobic microbes require oxygen-free conditions.
Media can include reducing agents to scavenge residual oxygen.
Methods to create anaerobic conditions:
Anaerobic chambers
Gas packs
Candle jars
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Isolation of pure cultures for diagnostics:
Culturing is used for diagnostics; pure (axenic) culture is required for identification.
A population arising from a single cell yields a pure culture.
Clinical samples are often mixed; plating on selective/differential media helps enrich problematic organisms.
Streak plating is a common method to achieve pure cultures.
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Aseptic technique to minimize contamination:
Aseptic technique aims to prevent contamination from air and non-sterile tools/equipment.
Common practice includes using an inoculating loop and a Bunsen burner to maintain sterile conditions.
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Direct methods for microbial concentration: counting chambers
Cells are counted within a defined grid area.
Can be stained to distinguish dead from living cells (e.g., DAPI stain).
Pros: simple, quick, inexpensive.
Cons: requires a large, evenly dispersed population; cannot distinguish viability without staining.
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Direct methods: viable counting (CFU) method
Cells are spread on a plate or mixed with molten agar and poured.
Plates incubated; colonies counted as CFU (colony forming units).
Requires serial dilution for high concentrations; only viable cells form colonies.
Pros: highly sensitive and accurate; commonly used for water, food, dairy, microbiology.
Cons: time- and resource-intensive; incubation required.
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Direct methods continued: viable counting for low concentrations
For water/food safety, detection limits may require analyzing larger sample volumes.
Large water volumes can be filtered through membranes; membranes placed on agar to detect low concentrations of organisms.
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Limitations of plating and VBNC issue:
Some bacteria persist in the environment in a viable but non-culturable (VBNC) state.
VBNC cells may resuscitate and become vegetative if placed in a host.
The great plate count anomaly: direct microscopic counts often reveal more microbes than viable plate counts; cell numbers can be underestimated by several orders of magnitude.
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Indirect methods for estimating microbial concentration:
Indirect methods rely on a proxy correlated with cell number rather than counting cells directly:
Turbidity measurements: more cells scatter more light, making the culture appear cloudier.
Spectrophotometry: measures light absorption (optical density, OD) of a turbid culture; often denoted as OD at a specific wavelength (e.g., OD_{540}).
A standard curve is typically required to relate optical density to cell concentration.
Pros: quick, easy, non-destructive.
Cons: less reliable at very low or very high densities; can be confounded by dead cells or particulate matter (e.g., milk).