KM

Introduction to Microbiology: Prokaryotes, Cell Structure, Taxonomy, and Growth

Domains, cell types, and basic taxonomy

  • Three domains of life: Bacteria, Archaea, and Eukarya.
    • Bacteria and Archaea are prokaryotic (no nucleus).
    • Eukarya are eukaryotic (nucleus-containing).
  • Historical framing mentioned in lecture: kingdoms under domains include Monera as a kingdom (with Archaea and Bacteria as two conserved domains) and four kingdoms under Eukarya (Plant, Fungi, Animalia, and Protista/Protozoa).
  • Binomial nomenclature (Genus species):
    • Genus: capitalized first letter, italicized when printed, e.g., Staphylococcus.
    • Species: lowercase, italicized, e.g., aureus.
    • Full names are emphasized for learning; abbreviation like S. aureus is discouraged in early study here.
  • Common examples invoked: "Staphylococcus aureus", "Staphylococcus epidermidis", and others like Escherichia coli strains.
  • Quick reminder on formatting in notes: use whole genus and species in full (no abbreviations) to reinforce recognition.

Bacterial surface features and their roles

  • Glycocalyx (external glycocalyx): a sugary/protein coating around the cell
    • Two main forms:
    • Capsule: tighter, thicker layer; helps in adherence and immune evasion (slows phagocytosis).
    • Slime layer: looser, helps with dehydration protection.
    • Also involved in biofilm formation and immune evasion; capsules can hinder macrophage uptake.
  • Capsule/slime relevance in disease and environment: common in biofilm form; capsules tie into immune protection and persistence.
  • Glycocalyx details: composed of sugars and amino acids; bacteria also produce a pellicle on surfaces (e.g., dental enamel) mediated by glycoproteins.

Attachment structures and modes of movement

  • Two major groups of appendages:
    • Motility structures (flagella): for movement (chemotaxis-guided).
    • Attachment structures (fimbriae/pili): for adherence and genetic exchange.
  • Flagella basics:
    • Attachment and movement depend on membrane structure. If bacteria have an outer membrane, there are two rings anchoring the basal body; if there is only a cell wall, there is a single ring arrangement.
    • The hook/sheath acts to position the flagellum so it can move without damaging the membrane; the filament is the long, repeating protein structure that propels the cell.
  • Fimbriae: short, hair-like structures used primarily for attachment (colonization).
  • Pili (sex pili): specialized for genetic exchange via conjugation; a pilus forms a tube for DNA transfer.
  • Attachments and biofilm relevance: attachment triggers extracellular matrix production, leading to biofilms (see below).

Biofilms, attachment, and dental plaque as an example

  • Biofilm concept: bacteria settle on a surface, synthesize extracellular matrix, and reach a quorum (high cell density) to coordinate gene expression and community behavior.
  • Quorum sensing: communication via signaling molecules that regulate community-wide gene expression.
  • Plaque as a classic biofilm example on teeth:
    • Initial conditioning film (pellicle) forms from salivary components and adheres to enamel within seconds.
    • Early colonizers (e.g., Streptococcus species) adhere and establish a base layer.
    • Later, anaerobes like Fusobacterium and spirochetes join, building a mature, multi-species biofilm.
    • Biofilms on medical devices (catheters, pacemakers) are a major concern due to persistent attachment.

Gram staining: purpose, protocol, and structural interpretation

  • Purpose: Differentiates bacteria into Gram-positive and Gram-negative based on cell envelope differences.
  • Historical figure: Hans Christian Gram.
  • Visual outcome:
    • Gram-positive cells: purple (crystal violet retained).
    • Gram-negative cells: red/pink (decolorized and counterstained with safranin).
  • Cell envelope differences driving Gram results:
    • Gram-positive: thick peptidoglycan (PG) layer, ~80 nm tall, with thick cross-linked NAM-NAG polymers; lacks an outer membrane.
    • Gram-negative: thin PG layer (~11 nm) and outer membrane containing lipopolysaccharide (LPS); periplasmic space between inner and outer membranes; porins present in outer membrane.
  • Peptidoglycan; NAM/NAG polymers and cross-linking:
    • Repeating units: N-acetylmuramic acid (NAM) alternating with N-acetylglucosamine (NAG).
    • Strength from cross-links via a tetrapeptide to form a sturdy lattice; NAM is cross-linked by peptide bridges (e.g., L-alanine, D-glutamic acid, meso-diaminopimelic acid or lysine, D-alanine).
    • A short glycine bridge can connect peptide chains; the cross-linking is the major determinant of wall strength.
  • Gram-positive envelope components:
    • Thick PG layer with teichoic and lipoteichoic acids that help anchor the wall.
    • These features attract cations (e.g., Mg2+, Ca2+) and contribute to wall charge and integrity.
  • Gram-negative envelope features:
    • Outer membrane with LPS, lipoproteins anchoring to the PG layer.
    • Periplasmic space contains PG and various enzymes; porins enable diffusion of small molecules.
  • Gram stain steps (live protocol overview):
    1) Primary dye: crystal violet (purple) binds to cell walls.
    2) Mordant: iodine (forms a complex with crystal violet, locking dye in for Gram-positive walls).
    3) Decolorization: alcohol (ethanol, ~95%) dehydrates walls; thick layered Gram-positives retain dye; Gram-negatives become colorless due to dye removal.
    4) Counterstain: safranin (red/pink) stains decolorized Gram-negatives.
  • Practical note: identifying Gram result early helps narrow identification and guides antibiotic therapy.
  • Special cases:
    • Mycobacteria have a waxy, mycolic-acid-rich outer layer that resists standard Gram staining; require acid-fast staining.
    • Archaea generally lack peptidoglycan; cell wall composition varies and differs from bacterial PG.
    • Mycoplasmas lack a cell wall entirely and rely on a membrane rich in sterols for rigidity.

Cell envelope comparison: Gram-positive vs Gram-negative versus special cases

  • Gram-positive:
    • Very thick PG layer (~80 nm).
    • No outer membrane; cytoplasmic membrane present.
    • Teichoic/l lipoteichoic acids contribute to charge and rigidity.
  • Gram-negative:
    • Very thin PG layer (~11 nm).
    • Outer membrane with LPS; periplasmic space present; outer membrane adds barrier to many antibiotics.
    • Porins provide selective permeability.
  • Mycobacteria:
    • Waxy, lipid-rich outer layer (mycolic acids) that hinders typical Gram staining; require acid-fast stain.
  • Archaea:
    • No universal PG; walls may be made of protein or polysaccharides; adapted to extreme environments.
  • Mycoplasmas:
    • No cell wall; membranes with sterol-like molecules; highly flexible.

The bacterial cell interior: nucleus, chromosomes, ribosomes, and plasmids

  • Nucleoid: bacterial chromosome is a circular, double-stranded DNA molecule without a nuclear membrane; exists in a defined region.
  • Introns: generally absent in bacterial genomes (no introns in bacterial genes).
  • Plasmids: extrachromosomal DNA elements that replicate independently and can transfer between cells; often carry antibiotic resistance genes or metabolic traits.
  • Ribosomes: the bacterial ribosome is 70S (50S + 30S subunits).
    • 70S = 50S + 30S
    • Eukaryotic ribosomes are typically 80S (40S + 60S) and differ in composition, which is a key drug target.
  • Translation and ribosome assembly occur in the cytoplasm and are separate from the nucleus in eukaryotes; in bacteria, ribosomes function in the cytoplasm and are targets for many antibiotics.
  • Plasmid utilities in biotechnology:
    • Example discussed: inserting a gene (e.g., human insulin gene) under a promoter to drive expression; antibiotic resistance markers used for selection in lab strains.
  • Transduction and conjugation basics (mentioned conceptually):
    • Conjugation involves a pilus and transfer of plasmid DNA between bacteria; not sexual reproduction but genetic exchange.

Cytoplasm organization, inclusions, and cytoskeleton

  • Inclusions (storage granules):
    • Stores nutrients (e.g., sulfur, iron, glycogen, polyphosphate).
    • Some cyanobacteria have gas vesicles to aid buoyancy for photosynthesis.
  • Cytoskeleton in bacteria: provides shape, aids in intracellular transport, and supports cellular architecture (not all bacteria have a pronounced cytoskeleton).
  • The cell envelope largely dictates shape; cytoskeletons support complex morphologies in some bacteria (rods, spirals, pleomorphic forms).

Spores (endospores): formation, resistance, and relevance

  • Spores are not a reproductive form in bacteria; they are a dormant, highly resistant state formed under adverse conditions.
  • Key producers: Bacillus and Clostridium species (bacilli and rod-shaped).
  • Sporulation sequence (high-level):
    • DNA replication and chromosome segregation occur while the mother cell (sporangium) forms a spore precursor.
    • Spore layers are deposited around the core, yielding a highly resistant endospore.
    • Water is removed to dry the spore; the mother cell eventually lyses, releasing a mature endospore.
    • Spores can survive extreme heat, desiccation, chemicals, and radiation for long periods (historical examples include spores found in ancient contexts).
  • Practical note: heat and pressure are required to kill endospores; autoclaving is the standard practice.
    • Typical autoclave settings: T
      ightarrow 121^ ext{°} ext{C} at around P
      ightarrow 15 ext{ psi} (some settings mention ~20 psi as cited in lecture).
    • For manual context: high temperature and pressure together are needed to inactivate endospores.

Morphology and cellular arrangement of bacteria

  • Morphology terms (single cells):
    • Coccus: spherical.
    • Bacillus (bacilli): rod-shaped.
    • Coccobacillus: short, rounded rod.
    • Vibrio: curved rod (comma-shaped).
    • Spirillum: rigid spiral.
    • Spirochete: flexible, helical with axial filaments (drives motility in a corkscrew fashion).
  • Pleomorphism: some bacteria lack a single fixed shape (e.g., Mycoplasma) and can vary morphologically; biochemical testing is crucial for identification.
  • Arrangements (based on division patterns in cocci or bacilli):
    • Diplococci: two cocci together.
    • Streptococci: chains of three or more cocci.
    • Tetrads: groups of four cocci in a square (or packets of 8-16 if divisions occur in multiple planes).
    • Staphylococci: irregular clusters (multiple planes of division).
    • Palisades: chained, hinge-like alignment (less common; appearance akin to a chain with side-to-side attachment).
  • Genus-level implications: terms like "Staphylococcus" reflect morphology (spherical) and arrangement (irregular clusters); species differentiate by properties and antigens.

Taxonomy, species, strains, and the naming system

  • Taxonomy aims to classify organisms by relatedness using several criteria:
    • Morphology and cell structure.
    • Physiology and biochemistry (what enzymes they have, metabolic capabilities).
    • Serology (antibody-based identification; e.g., antibodies against surface antigens).
    • Genetic methods (e.g., ribosomal RNA sequencing to differentiate domains like Archaea vs Bacteria).
  • Nomenclature and databases:
    • Biologists reference works like the Berkey’s Manual of Systematic Bacteriology (a comprehensive taxonomy reference; very detailed and sometimes considered the “bacteria bible”).
  • Species, strains, and types:
    • Species: e.g., within the genus Staphylococcus, species include S. epidermidis and S. aureus.
    • Strain: within a species, many strains exist (e.g., Escherichia coli strains), e.g., O157:H7 (pathogenic) vs other laboratory strains.
    • Type/Subtype: antigenic types, phage types, or serotypes differentiate strains within a species.
  • Examples of diversity:
    • Escherichia coli has many strains (e.g., E. coli K-12 vs pathogenic O157:H7).
    • Salmonella has thousands of strains (e.g., over 2,500). Different strains can differ in virulence and antigenicity.
  • Concept of serology and antigens:
    • Antigens are surface markers used by the immune system to recognize pathogens; antibodies against specific antigens can differentiate strains.

Metabolism, nutrition, and energy flow in microbes

  • Nutritional types:
    • Autotrophs generate organic substances from inorganic materials (e.g., chemolithotrophs oxidize inorganic compounds; phototrophs use light energy).
    • Heterotrophs obtain organic carbon by consuming other organisms or organic molecules.
  • Metabolic energy sources:
    • Chemoautotrophs and chemoorgano-trophs: derive energy from chemical reactions; chemo organisms may be aerobic or anaerobic.
    • Phototrophs: derive energy from light (photosynthesis).
  • Examples given:
    • Rhizobium (nitrogen-fixing bacteria) symbiotically associates with legumes, converting atmospheric nitrogen to ammonium (NH4+) via nitrogenase.
    • Leghemoglobin in plant roots creates a low-oxygen microenvironment to allow nitrogenase to function (nitrogen fixation is inhibited by oxygen).
    • Carbon fixation pathways: turning CO2 into organic carbon skeletons; nitrogen metabolism ties into amino acid synthesis.
  • Essential nutrients:
    • Essential nutrients cannot be synthesized in adequate amounts and must be supplied (e.g., certain amino acids like lysine in monogastric animals).
    • Macro-nutrients: large amounts (e.g., nitrogen, phosphorus, sulfur).
  • Energy and electron acceptors: aerobic vs anaerobic respiration and fermentation pathways; oxygen can form toxic byproducts (e.g., reactive oxygen species) unless detoxified by enzymes.
  • Oxygen radical detoxification:
    • Enzymes like Superoxide Dismutase (SOD) and Catalase help neutralize reactive oxygen species.

Oxygen requirements, pH, and environmental adaptation

  • Oxygen use and microbial niches:
    • Aerobic organisms require oxygen to survive and proliferate.
    • Obligate anaerobes cannot tolerate oxygen and will be harmed/killed by it.
    • Facultative anaerobes prefer oxygen but can grow without it, albeit less efficiently.
    • Microaerophiles require only small amounts of oxygen.
    • Aerotolerant organisms do not use oxygen but can survive in its presence.
  • Testing oxygen tolerance with thioglycolate broth (historical method):
    • Thioglycolate broth creates a gradient of oxygen; after autoclaving, air-tight sealing results in an oxygen gradient: oxygen-rich at the top, anoxic at the bottom.
    • Observing where growth occurs helps determine oxygen requirements (obligate aerobes at the top, obligate anaerobes at the bottom, facultative and microaerophiles across the gradient).
  • Temperature categories and growth preferences:
    • Psychrophiles: optima around 0–15°C (cold environments).
    • Mesophiles: optima around 20–45°C (most human pathogens fall here; typical lab temps ~25–37°C).
    • Thermophiles: optima ~45–80°C.
    • Hyperthermophiles/extremophiles: optima above ~80°C, some up to ~130°C.
    • The Goldilocks concept: minimum, optimum, maximum temperatures define viability and growth rate.
  • pH adaptation:
    • Acidophiles: thrive at low pH.
    • Neutrophiles: prefer neutral pH (~7).
    • Alkaliphiles: prefer high pH (>8).
  • Important environmental factors affecting microbial enzymes and survival:
    • Temperature, oxygen, pH, osmotic pressure, and barometric pressure all influence enzyme stability and cellular processes.
  • Nutritional and environmental interplay:
    • The lecture emphasizes that real-world habitats (e.g., stomach vs. mouth vs. duodenum) host different microbial communities adapted to local pH and oxygen conditions.

Growth and reproduction: binary fission and growth curves

  • Binary fission: primary bacterial reproductive mode (asexual, no sex)
    • Growth process overview:
    • Cell elongates; chromosome and plasmid (if present) replicate.
    • A septum forms; FtsZ ring constricts to divide the cell into two daughter cells.
    • Depending on species, daughter cells may separate or remain attached (e.g., diplococcus in pairs).
    • Result: two genetically identical daughter cells (clones).
  • Doubling time and growth rate:
    • Doubling time varies broadly; shortest reported: about 5–10 minutes; longest can be days to weeks depending on species and conditions.
  • Growth curve phases (typical batch culture):
    • Lag phase: little apparent growth as cells adapt to new environment and synthesize needed enzymes; metabolic activity is high but biomass does not increase much yet.
    • Exponential (log) phase: rapid, geometric growth; population doubles regularly; most active growth occurs here.
    • Stationary phase: nutrients become limited and waste products accumulate; growth rate slows and equals death rate; population size plateaus.
    • Death phase: depletion of nutrients and accumulation of toxins cause a decline in viable cells.
  • Practical note for experiments:
    • For biochemical tests and identification, sampling is often done during the late lag/early exponential window (roughly 18–24 hours after inoculation) to capture characteristic growth without the confounding changes seen in stationary/death phases.

Nitrogen fixation, legume symbiosis, and energy flow in soil-plant microbiomes

  • Rhizobium and legume symbiosis:
    • Rhizobium bacteria fix atmospheric nitrogen (N2) to ammonia (NH3) via nitrogenase in root nodules of legumes (e.g., alfalfa, soybeans, peanuts).
    • Leghemoglobin, produced by the plant, binds O2 to create a low-oxygen microenvironment that protects nitrogenase while allowing respiration for ATP production.
    • This symbiosis supplies ammonia to the plant, enabling growth in nitrogen-poor soils; the plant provides carbohydrates to the bacteria.
  • Ecological significance:
    • This relationship is a key example of mutualism that supports plant growth and soil fertility.

Biotech and gene transfer concepts highlighted in class

  • Plasmids as vectors for gene transfer:
    • Plasmids carry genes (e.g., antibiotic resistance) and can be transferred between bacteria via conjugation or introduced into cells by genetic engineering.
    • In laboratory settings, plasmids can be used to express heterologous genes (e.g., human insulin) under control of promoters; selection markers (antibiotic resistance) are used to identify successful transformants.
  • Conjugation versus binary fission:
    • Conjugation is a form of horizontal gene transfer involving a pilus and plasmid exchange; not sexual reproduction in the classical sense.
    • Binary fission is the primary asexual reproduction method in bacteria, producing two identical daughter cells.

Special organisms and exceptions worth noting

  • Mycobacterium (acid-fast bacteria):
    • Waxy outer layer with mycolic acids; requires acid-fast staining due to waxy barrier that resists Gram staining.
  • Mycoplasma:
    • Lack cell walls entirely; membranes contain sterol-like molecules to provide rigidity.
    • Mycoplasma pneumoniae can adhere to lung epithelial cells, illustrating pathogen-host interactions without a rigid cell wall.
  • Archaea:
    • Often inhabit extreme environments; cell wall composition differs from bacterial PG (some have pseudo-PG or protein-based walls).
  • Eukaryotic comparison (context):
    • Prokaryotes use 70S ribosomes (50S + 30S) vs eukaryotes with 80S ribosomes; this difference is a major antibiotic target.
    • Eukaryotes contain nucleus and organelles; the nuclear membrane distinguishes them from bacteria.

Quick reference to common numbers and terms (for quick study)

  • Ribosomes: 70S = 50S + 30S in bacteria; Eukaryotes have 80S ribosomes.
  • Peptidoglycan dimensions: Gram-positive PG ~80 nm thick; Gram-negative PG ~11 nm thick.
  • Growth and timing: doubling time varies; some bacteria double every 5-10 ext{ minutes}; others take hours to days depending on species and conditions.
  • Autoclave basics (sterilization): T
    ightarrow 121^ ext{°}C at about P
    ightarrow 15 ext{ psi} (lecture mentions ~20 psi as an alternate value).
  • pH references in human body contexts: saliva around pH 8; stomach around pH 1.5; duodenum around pH 9.2.
  • Oxygen tolerance categories:
    • Obligate aerobes: require oxygen.
    • Obligate anaerobes: killed by oxygen.
    • Facultative anaerobes: grow with or without oxygen, often better with oxygen.
    • Microaerophiles: require low oxygen.
    • Aerotolerant: tolerate oxygen but do not use it.
  • Temperature categories:
    • Psychrophiles: near 0–15°C.
    • Mesophiles: roughly 20–45°C (humans and many pathogens).
    • Thermophiles: ~45–80°C.
    • Hyperthermophiles/extremophiles: >80°C, some up to ~130°C.
  • Key terms to know by heart:
    • Nucleoid, plasmid, Ŕibosome subunits (50S, 30S), NAM/NAG, teichoic acids, LPS, porins, pilus, fimbriae, capsule, slime layer, biofilm, quorum sensing, endospore, sporulation, conjugation, autotroph, heterotroph, chemolithotroph, phototroph, organotroph, leghemoglobin, nitrogenase, thioglycolate broth.

Practical exam relationships and takeaways

  • The Gram stain remains a foundational diagnostic tool because it rapidly narrows the field into two major groups with distinctly different envelope structures and antibiotic susceptibilities.
  • Knowledge of cell envelope structure (PG thickness, outer membrane, LPS) helps explain antibiotic targets (e.g., beta-lactams destabilize PG cross-links, more effective against Gram-positives).
  • Understanding biofilms is critical for clinical and medical device contexts due to increased resistance and persistence.
  • The growth curve informs timing for sampling and testing in labs; sampling during appropriate phases yields clearer biochemical and phenotypic results.
  • Nitrogen fixation and symbiosis illustrate how microbes directly influence plant nutrition and ecosystem nutrient cycling.
  • Environmental factors (temperature, oxygen, pH) define ecological niches and guide culture conditions in the lab.

Quick glossary reminders

  • Nucleoid: region containing bacterial DNA, no membrane-bound nucleus.
  • Teichoic acids: components of Gram-positive cell walls aiding charge and adhesion.
  • Lipopolysaccharide (LPS): outer membrane component of Gram-negative bacteria; endotoxin in some species.
  • FtsZ: cytoskeletal protein involved in septum formation during binary fission.
  • Endospore: dormant, highly resistant cell form; survives extreme conditions.
  • Quorum sensing: density-dependent regulation of gene expression in biofilms.
  • Leghemoglobin: plant-produced molecule that binds oxygen to enable nitrogenase activity in Rhizobium symbiosis.