Microbiology Study Guide: Chapters 1 & 2 – The Microbial World and Microbial Cell Structure & Function

Microbiology Intro

What is Microbiology?

Microbiology is the study of cellular and acellular biological particles that are invisible to the unaided eye. Microorganisms play essential roles in human activities and the web of life on Earth.

• Biomass: Microbes comprise a major fraction of Earth's biomass, exceeding that of plants and animals. They are key contributors to macronutrients such as nitrogen and phosphorus.

• Diversity: Most major branches on the tree of life are microbial.

• Time: Microbial fossils date back to 3.8 billion years ago, predating multicellular fossils (600 million years ago). Cyanobacteria oxygenated the planet.

• Habitat Diversity: Microbes inhabit extreme environments (boiling hot springs, Antarctic sea ice, salt ponds, deep sea, human body).

• Asexual Reproduction: Microbes reproduce asexually and acquire genes through various methods.

Cellular and Acellular Microorganisms

• Cellular Microorganisms: Unicellular microbes perform all functions within one cell, unlike multicellular plants and animals.

• Bacterial Population: Group of cells derived from a single parent by successive divisions.

• Microbial Community: Mixture of microbial species in nature.

• Habitat: The environment where a species normally lives.

• Microbial Ecology: Study of microorganisms in their natural environment.

Macromolecules in Living Cells

• Lipids: Form cytoplasmic membranes.

• Nucleic Acids: DNA (genes) in nucleus/nucleoid; RNA in ribosomes.

• Proteins: Throughout cell, membrane wall, flagellum.

• Polysaccharides: Cell wall, storage granules.

Microbial Morphology

• Coccus: Spherical shape.

• Bacillus: Rod-shaped.

• Spirillum: Spiral-shaped.

• Spirochete: Flexible, spiral-shaped.

• Filaments: Thread-like.

• Budding/Appendage Bacteria: Stalk or hypha structures.

The Importance of Being Small

Microorganisms are measured in millimeters (mm), micrometers (μm), and nanometers (nm). Smaller cells have a larger surface area-to-volume ratio, allowing faster membrane transport, metabolism, and cell division. Small cells can inhabit microhabitats.

• Example: ~1000 bacteria fit across a pinhead (1 mm).

Acellular Microbes/Particles

• Viruses: Non-living infectious agents with DNA or RNA, surrounded by a protein coat; replicate in living cells. Some have lipid membranes. There are 10x more viruses than microbial cells.

• Prions: Infectious proteins lacking nucleic acids; cause conformational changes in host proteins.

Characteristics of Living Systems

• Structure: All cells have a cytoplasmic membrane (semi-permeable), cytoplasm, genome (DNA), and ribosomes.

• Metabolism: Cells use DNA to make RNA and protein (replication, transcription, translation). They take up nutrients, transform them, conserve energy, and expel waste. Metabolism includes catabolism (energy generation) and anabolism (biosynthesis).

• Growth: Cell division is growth, not reproduction. Proteins convert nutrients into new cells.

• Evolution: Mutations in DNA lead to new properties, driving evolution via mutation and natural selection.

Properties of Some Microbial Cells

• Differentiation: Formation of specialized cell types.

• Communication: Cells interact via signaling.

• Motility: Movement in response to environment.

• Genetic Exchange: Exchange of genetic material between cells.

Why Study Microbiology?

• Biochemistry: Microbes are essential for understanding biochemistry.

• Disease Agents: Historically, microbes were the leading cause of death; antibiotics have reduced this, but superbugs are emerging.

• Food: Microbes improve food safety and preservation (yeast for alcohol/bread, bacteria for cheese/yogurt).

• Agriculture: Nitrogen fixation (legumes), cellulose digestion (rumen).

• Energy Production: Methane, biodiesel, ethanol from microbial processes.

• Environment: Wastewater treatment, bioremediation (microbes degrade pollutants).

• Biotechnology: PCR uses enzymes from thermophilic bacteria for research, forensics, diagnostics.

Historical Roots of Microbiology

Antonie van Leeuwenhoek

• Invented the microscope; first to observe bacteria and their morphologies.

• Observed motile rods and estimated their size.

Louis Pasteur

• Disproved spontaneous generation.

• Developed aseptic technique (sterilization via heat and pressure, autoclave).

• Founded applied microbiology; discovered yeast require nitrogen to grow.

• Germ Theory of Disease: Microbes cause disease.

• Developed pasteurization (heat kills bacteria in food).

Robert Koch

• Father of clinical microbiology; studied diseases (e.g., Bacillus anthracis causes anthrax).

• Developed pure culture techniques, Petri dish, solid media.

• Koch’s Postulates: Criteria to prove a microbe causes disease.

• Proved Mycobacterium tuberculosis causes TB.

Martinus Beijerinck

• Developed enrichment culture technique to isolate specific microbes.

• Described filterable infectious agents (viruses).

• Modified defined media for microbial growth.

Microscopy

Light Microscopy

Used to observe intact cells at low magnification. Key concepts include magnification and resolution.

• Resolution: Function of light wavelength and numerical aperture of objective lens.

  • Formula: $\text{Diameter of object resolvable} = 0.5 \times \text{wavelength} / \text{numerical aperture}$

  • Shorter wavelengths (blue light) yield better resolution.

• Magnification: Calculated by multiplying objective lens (10x, 40x, 100x) by ocular lens (10x).

• Staining: Increases contrast but kills cells. Gram stain differentiates Gram-positive (purple) and Gram-negative (pink) bacteria.

• Phase Contrast Microscopy: Amplifies differences in light phase between cell and surroundings.

• Dark Field Microscopy: Illuminates from the side; background appears dark, improving resolution.

• Fluorescence Microscopy: Objects emit light of one wavelength when illuminated by another.

• 3D Microscopy: DIC (polarized light), Confocal Scanning Laser Microscopy (high contrast, 3D images).

Electron Microscopy (EM)

• Transmission EM: Used to examine internal cell structures; 1000x better resolution than light microscopy.

• Scanning EM: Views surface structures by detecting scattered electrons.

Theory of Spontaneous Generation

• Belief that life arises from non-living materials (favored by Aristotle).

• Redi disproved with maggot experiments; Spallanzani boiled seeds in sealed flasks to show sterilization.

Terms Related to Cultivation

• Medium: Nutrient solution for growing microbes (solid or liquid).

• Sterilization: Killing all microbes in/on objects.

• Pure Culture: Culture containing a single kind of microbe.

• Turbid: Cloudy liquid medium indicating microbial growth.

Structure of a Prokaryotic Cell

Macromolecule Locations

• Proteins: Throughout cell, membrane, wall, flagellum.

• Lipids: Cytoplasmic membrane.

• Polysaccharides: Cell wall, storage granules.

• Nucleic Acids: DNA (nucleus/nucleoid), RNA (ribosomes).

Cell Membrane Charge and Strength

• Net Negative Charge: Due to phosphate head groups on membrane surface.

• Strength: Sterols (eukaryotes) and hopanoids (bacteria) strengthen membranes. Archaea lack hopanoids.

Archaeal Membranes

• Contain ether bonds between glycerol and hydrophobic side chains.

• Lack fatty acids; side chains are isoprene units.

• Major lipids: 20-C phytanyl and 40-C biphytanyl (monolayers).

Membrane Proteins

• Integral: Embedded in membrane.

• Transmembrane: Span entire membrane.

• Peripheral: Surface-associated.

• Higher protein content increases membrane strength.

Functions of Cytoplasmic Membranes

• Permeability Barrier: Controls entry/exit of substances.

• Protein Functions: Enzymatic and transport roles.

• Energy Conservation: Site of proton motive force generation and dissipation.

Transport Systems

• Simple Transport: Membrane-spanning protein; driven by proton motive force (PMF). Examples: Lac permease (symporter), sodium-proton antiporter, potassium uniporter (does not require energy).

• Group Translocation: Series of proteins; transports sugars (glucose, mannose, fructose); energy from phosphoenolpyruvate (PEP).

• ABC Systems: Three components; transport sugars, amino acids, inorganic nutrients; use ATP.

• All require energy (PMF, high-energy phosphate bonds, or ATP).

• Secretion: Translocases transport proteins across membranes.

Cell Envelopes of Bacteria

Gram-Positive vs. Gram-Negative

• Gram-Positive: Two layers (thick peptidoglycan, membrane); contains teichoic acid.

• Gram-Negative: Four layers (thin peptidoglycan in periplasm, membrane, periplasm, outer membrane with lipopolysaccharide [LPS]).

Gram-Negative Cell Envelope Structure

Layer	Components
Outer Membrane	Lipopolysaccharide (LPS), proteins (porins)
Periplasm	Peptidoglycan (thin), enzymes
Inner Membrane	Lipids, proteins

• LPS Structure: O-specific polysaccharide, core polysaccharide, lipid A (endotoxin), fatty acids from disaccharide (glucosamine phosphate), KDO, heptoses, N-acetylglucosamine.

• Lipid A: Toxic to humans; causes gastrointestinal symptoms.

Peptidoglycan

• Polymer of sugars: N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG), linked by peptide side chains.

• Crosslinking: Amino group of DAP and carboxyl group of D-Ala (Gram-negative).

• Gram-Positive: Thick (20-35 nm, >15 layers), contains teichoic acid (polyalcohols with phosphate, sugars, D-amino acids).

• Gram-Negative: Thin (2-7 nm, 1-3 layers).

Lysozyme and Antibiotics

• Lysozyme targets β(1,4) linkage between NAM and NAG.

• Cell wall prevents osmotic lysis; lysozyme digestion leads to lysis or protoplast formation.

• Beta-Lactam Antibiotics (e.g., Penicillin): Bind and inactivate transpeptidases, preventing cross-linking in peptidoglycan, leading to cell lysis.

Phylogenetic Tree of Bacteria

• Gram-Positive Phyla: Actinobacteria (Actinomycetota), Firmicutes (Bacillota).

• Some bacteria do not stain reliably: Mycoplasmas (wall-less), Mycobacteria (acid-fast), obligate intracellular and spiral bacteria (too small or structurally distinct).

Stages of Gram Stain

Step	Procedure	Result
1	Crystal violet (1 min)	All cells purple
2	Iodine (3 min)	All cells remain purple
3	Alcohol (20 sec)	Gram-positive: purple; Gram-negative: colorless
4	Safranin (1-2 min)	Gram-positive: purple; Gram-negative: pink/red

• Alcohol dehydrates peptidoglycan in Gram-positive cells, trapping dye.

• Gram-negative cells require counterstain to be visible.

Prokaryotic Life Without Cell Walls

• Mycoplasmas (Tenericutes): Pathogenic, wall-less bacteria; live in osmotically protected habitats; contain sterols for membrane rigidity.

• Thermoplasm (Archaea): Tough cytoplasmic membranes (monolayers).

Archaeal Cell Walls & Surface Layers

• Peptidoglycan is exclusive to bacteria; absent in Archaea.

• Pseudomurein: β(1,3) linkages (lysozyme-insensitive), different cross-link sugars, no D-amino acids.

• S-layer: Paracrystalline protein/glycoprotein layer; always outermost; provides structural strength, protects from osmotic lysis, confers shape, allows cell surface interactions.

Other Bacterial Extracellular Layers

• Capsule: Dense polysaccharide/protein layer; virulence factor; gives halo appearance.

• Slime Layer: Unorganized, loosely attached; often called extracellular polysaccharide (EPS); promotes attachment and protects cells (e.g., in biofilms).

Extracellular Filaments

• Fimbriae: Thin, short protein structures; multiple copies; involved in attachment.

• Pilus: Thin, longer protein structure; usually single copy; used for cell-cell attachment, DNA transfer (conjugation), twitching motility.

• Functions: Attachment, conjugation, twitching motility.

Bacterial Locomotion

• Allows exploration, colonization, escape from predators/toxins, access to nutrients.

• Flagella Arrangements:

  • Atrichous: No flagella

  • Monotrichous: One flagellum

  • Amphitrichous: Flagella at both ends

  • Lophotrichous: Cluster at one end

  • Peritrichous: Flagella all around

• Flagella Structure: Composed of flagellin protein, helically wound, hollow core, attached via hook to membrane complex.

• Mechanism: Proton motive force drives motor proteins (Mot) to spin flagella.

• Flagellar Synthesis: Requires ~50 genes; assembly starts at base, proceeds to rings, hook, flagellin; cap directs polymerization.

Prokaryotic Cell Inclusions

Movement-Related Inclusions

• Gas Vesicles: Increase buoyancy; allow phototrophs and sea ice bacteria to access light/nutrients.

• Magnetosomes: Contain magnetite (Fe3O4); orient cells in magnetic fields; common in microaerophilic bacteria.

• Surface Motility: Twitching (Type IV pili, ATP-driven), gliding (PMF-driven, smooth movement).

• Taxes: Movement toward/away from stimuli (chemotaxis, phototaxis, magnetotaxis).

Motility Mechanisms

• Capillary Tube Assay: Used to study swimming behavior.

• Tumble and Run: Random movement via sensor proteins.

• Peritrichous Flagella: Bundled (CCW rotation) for run, tumble (CW rotation) for reorientation.

• Polar Flagella: Reversible (CCW forward, CW backward; e.g., Pseudomonas aeruginosa), unidirectional (CW forward, stop/reorient; e.g., Rhodobacter sphaeroides).

Bacterial Inclusions for Storage

• Phosphate: Stored as polyphosphate granules; used for nucleic acid and phospholipid synthesis.

• Carbon: Stored as poly-β-hydroxybutyrate (PHB) or glycogen.

• Elemental Sulfur: Stored for use when H2S is depleted; oxidized for energy or carbon fixation.

• Storage granules are insoluble, reducing osmotic stress and aiding metabolism.

Bacterial Endospores

• Endospores are differentiated cells highly resistant to heat, drying, and nutrient depletion.

• Enable survival during unfavorable conditions; can persist for millions of years.

• Formed only by Firmicutes (Bacillota).

• Notorious Spore-Formers: Bacillus anthracis (anthrax), Clostridium tetani (tetanus).

• Endospores differ from vegetative cells in microscopic appearance, calcium content, and presence of dipicolinic acid.