Chapter 03 Lecture Notes: Nester's Microbiology — Prokaryotic vs Eukaryotic Cells, Gram Stain, Cell Structures, and Microscopy

Prokaryotic vs Eukaryotic Cells

Two fundamental cell types revealed by cell study: prokaryotic and eukaryotic
Bacteria and archaea are composed of prokaryotic cells
Animals, plants, protozoa, fungi, and algae are composed of eukaryotic cells
Relevance to health: cell-type differences create targets for antibacterial drugs by focusing on bacterial-specific components
Implication: selectivity in antimicrobial therapy relies on exploiting bacterial cell features absent or different in human cells

History of the Gram Stain

Hans Christian Gram (1853–1938) developed staining methods to differentiate bacteria
One staining method yielded unequal dye retention by bacteria, revealing two distinct groups
Basis for modern Gram stain that identifies two major groups by cell wall structure/chemistry
Gram-positive vs Gram-negative cell wall distinction hinges on dye retention after decolorization
Practical outcome: Gram status influences antibiotic susceptibility and stain-based identification

Prokaryotic Cell Structures (Overview)

Prokaryotic cell envelope components: cytoplasmic membrane, cell wall, capsule (if present)
Cytoplasm
Nucleoid: location of chromosome
Locomotor appendages (if present)
Key takeaway: prokaryotes lack a nucleus; their simpler organization contrasts with eukaryotic cells, but they possess diverse structures aiding survival and pathogenicity

The Cytoplasmic Membrane

Defines the boundary of the cell; a phospholipid bilayer embedded with proteins
Hydrophobic tails face inward; hydrophilic heads face outward
Proteins serve many functions: selective gates, environmental sensors, enzymes
Fluid mosaic model: proteins drift within the lipid bilayer
Similar cytoplasmic membrane structure in Bacteria and Archaea but with distinct phospholipid chemistries
Archaea lipids are not fatty acids and connect differently to glycerol
Relevance: membrane components are targets for antibiotics; permeability influences drug entry

Permeability and Transport Across the Cytoplasmic Membrane

Cytoplasmic membrane is selectively permeable
Small uncharged molecules (O2, CO2, N2) and water pass freely; aquaporins facilitate water transport
Larger or charged molecules require transport systems
Simple diffusion: high to low concentration until equilibrium; rate depends on concentration gradient
Osmosis: diffusion of water across a selectively permeable membrane; water moves from hypotonic (high water/low solute) to hypertonic (low water/high solute); isotonic solutions show no net water flow
Osmotic environment of prokaryotes is typically dilute (hypotonic) relative to cytoplasm; cell wall helps prevent lysis
Energy-transforming membrane: ETC embedded in cytoplasmic membrane creates a proton motive force used to synthesize ATP and drive transport/motility
Transport systems (transporters/permeases/carriers) span membranes and are highly specific
Efflux pumps expel wastes and antimicrobials, contributing to drug resistance
Facilitated diffusion: passive transport down gradient; not efficient in low-nutrient environments
Active transport: requires energy; can be proton-motive-force driven or ATP-powered (ABC transporters)
Group translocation: common in bacteria; chemically modifies substrates (e.g., phosphorylation) during entry; often used for glucose uptake
Protein secretion: secretion requires signal sequences guiding polypeptides; transport across cytoplasmic membrane to exterior

The Bacterial Cell Wall and Peptidoglycan

Cell wall provides structural strength to prevent bursting; distinguishes Gram-positive from Gram-negative
Peptidoglycan (murein) is a layered polymer
Subunit composition: N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG)
Peptide chains attach to NAM; tetrapeptide linkages connect glycan chains
Direct cross-linking in Gram-negative cells; peptide interbridges in Gram-positive cells
Gram-positive cell wall: relatively thick peptidoglycan; teichoic acids project above the layer; periplasmic-like space lies below peptidoglycan
Gram-negative cell wall: thin peptidoglycan layer; outer membrane present

The Gram-Negative Cell Wall (Detailed)

Outer membrane contains lipopolysaccharide (LPS), a key immune system signal (endotoxin)
LPS comprises Lipid A (endotoxin component) and O antigen (serotype identifier)
LPS can trigger strong immune responses; widespread LPS can be lethal in high amounts
Outer membrane acts as a barrier to many molecules, including some antibiotics; porins permit selective passage of small molecules
Periplasmic space lies between inner cytoplasmic membrane and outer membrane; contains gel-like matrix and binding proteins
ABC transport systems and periplasmic binding proteins function in import/export within this space
Implication: Gram-negative bacteria often more resistant to antibiotics due to outer membrane barrier and efflux systems

Antibacterial Substances Targeting Peptidoglycan

Interference with peptidoglycan compromises cell wall integrity, leading to lysis
Penicillin inhibits cross-linking of glycan chains; more effective against Gram-positive bacteria; outer membrane of Gram-negative can block access; derivatives improve entry
Lysozyme cleaves glycan chain bonds; more effective against Gram-positive bacteria
Overall, disruption of peptidoglycan is a classic antibiotic strategy

Bacteria That Lack Cell Walls

Some bacteria naturally lack a cell wall (e.g., Mycoplasma species)
Penicillin and lysozyme are ineffective against these organisms
Survival without a cell wall is aided by sterols in the cytoplasmic membrane, providing structural integrity

Archaea: Cell Walls

Archaeal cell walls vary; many have S-layers made of protein or glycoprotein subunits
No peptidoglycan; some have pseudopeptidoglycan
Adaptations reflect a wide range of environments, including extreme conditions

Capsules and Slime Layers

External, gelatinous layers outside the cell wall
Capsule: distinct, well-defined; slime layer: diffuse
Most capsules are composed of glycocalyx; some are polypeptides
Capsule presence often enables adherence and biofilm formation; can aid in immune evasion
Example: dental plaque as a biofilm

Flagella and Motility

Flagella provide motility; rotate like propellers
Tempered by disease relevance in some pathogens (e.g., Helicobacter pylori)
Flagellar arrangement patterns: peritrichous (around the surface) and polar (at one end)
Structure: basal body anchors to cell wall and membrane; hook; filament made of flagellin
Archaella (archaeal flagella) are biochemically distinct; rely on ATP rather than proton motive force

Chemotaxis and Other Taxis

Chemotaxis: movement toward attractants (e.g., nutrients) or away from repellents (toxins)
Movement comprises runs (straight) and tumbles (direction changes) via flagellar rotation
Other taxis: Aerotaxis (O2), Magnetotaxis (Earth's field), Thermotaxis (temperature), Phototaxis (light)

Pili, Fimbriae, and DNA Transfer

Pili (pilus, singular): thin, shorter than flagella; enable attachment to surfaces
Common pili facilitate adhesion; some pili enable twitching/gliding motility
Sex pili join bacteria for DNA transfer (conjugation)

Internal Components of Prokaryotic Cells

Nucleoid: gel-like region housing a single circular chromosome; tightly packed with binding proteins and supercoiling
Plasmids: extra-chromosomal DNA; non-essential; can spread antibiotic resistance between bacteria
Ribosomes: protein synthesis; prokaryotic 70S ribosomes (composed of 30S and 50S subunits); eukaryotic ribosomes are 80S
Medical relevance: many antibiotics target 70S ribosomes, sparing 80S ribosomes in humans
Cytoskeleton: interior protein framework; supports shape and division; bacterial homologs exist
Storage granules: intracellular polymers for carbon/energy storage (e.g., glycogen, PHB)
Metachromatic granules stain red with methylene blue
Protein-based compartments: specialized microstructures that compartmentalize reactions
- Gas vesicles: adjust buoyancy in aquatic bacteria; allow gas passage; reduce density
- Bacterial microcompartments (BMCs): contain enzymes for specific metabolic pathways; confine to avoid side reactions
- Encapsulins: encapsulate specific proteins (e.g., iron-binding proteins)

Endospores

Dormant, highly resistant cells produced by Bacillus and Clostridium genera
Can remain viable for extremely long periods (potentially >100 years)
Resistant to heat, desiccation, chemicals, UV, and boiling water
Germinate to become vegetative cells under favorable conditions
Sporulation: triggered by nutrient limitation; involves asymmetric cell division and spore formation
Endospore contains a core with DNA-protective proteins and calcium dipicolinate; enveloped by cortex and spore coat
Not a means of reproduction, but a survival strategy

Eukaryotic Cell Structure and Functions (Overview)

Eukaryotic cells are highly variable (e.g., protozoa, animal, plant, fungal, algal cells)
Protozoa can be cell-wall-free; animal cells lack cell walls; fungal cell walls contain chitin; plant cell walls primarily cellulose
Eukaryotic cells have membrane-bound organelles and a nucleus containing DNA
Tissue organization: similar cells form tissues; tissues form organs

Membranes and Transport in Eukaryotic Cells

Cytoplasmic membrane resembles prokaryotic membranes but with differences in composition and receptors
Outer layer proteins: receptors bind ligands; signal transduction is key for communication
Sterols increase membrane strength (cholesterol in animals, ergosterol in fungi)
Lipid rafts help detect and respond to signals; many viruses exploit raft domains for entry/exit
Electrochemical gradients maintained by sodium or proton pumps
Transport across membranes includes aquaporins, channels, and carriers (facilitated diffusion and active transport)

Endocytosis and Exocytosis

Endocytosis: cell takes in material by invaginations of the membrane
- Pinocytosis: uptake of extracellular fluid; forms endosome; fuses with lysosome
- Receptor-mediated endocytosis: specific ligands bind surface receptors before uptake
- Phagocytosis: engulfment of larger particles via pseudopods; forms phagosome that fuses with lysosome
Exocytosis: vesicles fuse with the plasma membrane to release contents

Secretion and Protein Sorting in Eukaryotes

Proteins destined for secretion have signal sequences guiding trafficking
Ribosomes on rough endoplasmic reticulum synthesize proteins that enter the ER lumen
ER-Golgi pathway sorts and modifies proteins (e.g., carbohydrate/phosphate additions) and directs them to final destinations via vesicles
Other organelles have specific targeting tags for delivery

Protein Structures Within Eukaryotic Cells

Ribosomes: eukaryotic 80S ribosomes composed of 60S and 40S subunits; prokaryotic ribosomes are 70S
Antibacterial agents typically do not affect 80S ribosomes, reducing host toxicity
Cytoskeleton: actin filaments, microtubules, and intermediate filaments
- Actin filaments (microfilaments) enable movement; polymerize/depolymerize dynamically
- Microtubules (tubulin) form mitotic spindles, cilia, and flagella; provide tracks for organelle movement
- Intermediate filaments provide mechanical support

Actin Hijacking by Pathogens (Perspective 3.1)

Some pathogens manipulate host actin polymerization to facilitate invasion or spread
Illustrates pathogen-host interactions and cellular susceptibility to manipulation

Flagella and Cilia in Eukaryotes

Eukaryotic flagella and cilia are structurally distinct from prokaryotic flagella
Covered by extensions of the cytoplasmic membrane
Composed of microtubules arranged in a 9+2 pattern
Flagella propel cells; cilia beat in synchrony to move cells or move external material

Membrane-Bound Organelles in Eukaryotic Cells

Nucleus: contains genetic material; double-mated phospholipid bilayer; nuclear pores regulate traffic; nucleolus synthesizes rRNA
Mitochondria: generate ATP; double membranes; inner membrane forms cristae; matrix contains DNA and 70S ribosomes
Chloroplasts (plants/algae): sites of photosynthesis; ATP generation for carbon fixation; contain DNA and 70S ribosomes; thylakoids within stroma; multiple membranes
Endosymbiotic theory: mitochondria and chloroplasts originated as bacteria living inside ancestral eukaryotic cells
- Evidence includes: bacterial-type DNA, 70S ribosomes, double membranes, binary fission-like replication, and related gene sequences

Endosymbiotic Theory: Evidence and Implications

Mitochondria and chloroplasts retain their own DNA sequences similar to bacteria
Ribosomes resemble bacterial 70S ribosomes
Double membranes around these organelles support the endosymbiotic origin
These organelles replicate by binary fission independent of the host cell cycle

Chloroplasts and Photosynthesis

Sites of photosynthesis in plants and algae
Harvest light energy to generate ATP; ATP used to convert CO2 to sugars and starch
Chloroplasts contain DNA, 70S ribosomes, and two membranes
Photopigments reside in thylakoids within the stroma (thylakoids store pigments for light capture)

Endoplasmic Reticulum and Golgi Apparatus

Endoplasmic Reticulum (ER): network of flattened sacs and tubes
- Rough ER: ribosomes synthesize proteins destined for secretion or for organelles
- Smooth ER: lipid synthesis and degradation, calcium storage
Golgi apparatus: site of macromolecule modification (carbohydrate and phosphate additions); sorts and packages molecules for delivery in vesicles

Lysosomes, Peroxisomes, and Other Organelles

Lysosomes contain degradative enzymes; fuse with endosomes/phagosomes to degrade material; autophagy delivers old organelles to lysosomes
Peroxisomes degrade lipids and detoxify chemicals; generate and detoxify reactive oxygen species like hydrogen peroxide and superoxide; protect cells from oxidative damage

Microscopy: Tools for Observing Microorganisms

Light microscope: magnifies up to about 1000\times; common and essential for microbiology
Electron microscope (EM): magnifies up to over 10^5\times; requires vacuum; cannot view living cells
Scanning probe microscopes: atoms-level surface imaging (e.g., AFM)
One major advantage of EM is extremely high resolution; limitation is nonliving specimen due to vacuum and fixation requirements

Principles of Light Microscopy

Light passes through the specimen and through lenses (objective and ocular)
Magnification: product of objective and ocular magnifications (e.g., objective 4x, 10x, 40x, 100x; ocular 10x)
Condenser focuses light on the specimen; does not magnify
Resolution (ability to distinguish two nearby points) depends on lens quality, light wavelength, magnification, and preparation
Maximum resolving power for light microscopy: d\approx 0.2\,\mu\mathrm{m}
Immersion oil (refractive index similar to glass) reduces refraction and improves resolution at high-power objectives (e.g., 100x)
Contrast improves visualization; many microbes are transparent without staining

Microscopy Techniques to Improve Visualization

Dark-field microscopy: bright specimen on dark background; light scattered by specimen enters objective
Phase-contrast: enhances contrast by exploiting differences in refractive indices; cells appear darker/light depending on density
Differential interference contrast (DIC): uses two beams to give a 3D appearance; highlights surface details
Fluorescence microscopy: uses fluorescent dyes or intrinsic fluorescence; molecules emit light at longer wavelengths after UV excitation; often epifluorescent (UV passed through specimen)
Fluorescent dyes and tags: some bind to all cells, others distinguish living vs dead; immunofluorescence uses fluorescently labeled antibodies to tag specific microbes
Scanning laser microscopy (SLM): fluorescently labeled specimens; provides 3D topography views of thick specimens
Confocal microscopy: laser scans successive planes; computer-generated 3D image; like CAT scan for cells
Two-photon microscopy: similar to confocal but gentler on living cells; deeper imaging with less photodamage
Super-resolution microscopy: pushes beyond conventional light limits; resolutions down to ~10 nm

Electron Microscopy (EM)

TEM (Transmission EM): beam of electrons passes through thin specimen sections; darker areas indicate higher density; requires thin-sectioning; cryo-EM and cryo-electron tomography reduce damage and enable 3D visualization
SEM (Scanning EM): electron beam scans surface; produces high-resolution surface images; specimen coated with metal; yields 3D-like appearance
EM advantages: extremely high resolution (≈3\times 10^{-1}\,\text{nm} range for modern systems); useful for ultrastructure
EM limitations: living cells cannot be observed; complex preparation; expensive equipment

Preparing Specimens for Light Microscopy

Wet mount: living organisms in a drop of liquid under a coverslip; useful for observing motility and behavior; easier to observe if cells are not colorless
Smear: drying and fixing the specimen before staining to visualize details

Staining Techniques: Simple, Differential, and Specialized

Simple staining: uses a single basic dye (positive charge) to stain cells; examples: methylene blue, crystal violet
Negative (acidic) staining: uses negatively charged dyes; cells repel dye and appear colorless against a colored background
Differential staining distinguishes groups of bacteria; Gram stain is the most widely used differential stain; separates into Gram-positive and Gram-negative

Gram Stain: Procedure and Considerations

Step 1: Flood smear with primary stain (crystal violet)
Step 2: Apply mordant (iodine) to stabilize dye–cell complex
Step 3: Decolorize briefly with alcohol to remove dye from Gram-negative cells
Step 4: Counterstain with a contrasting dye (e.g., safranin) to color Gram-negative cells
Success depends on the duration of the decolorizing step and the age of the culture
Result: Gram-positive cells retain stain (purple); Gram-negative cells appear pink/red

Other Differential Stains

Acid-fast stain: detects organisms with high mycolic acid content (e.g., Mycobacterium spp.); multi-step process; primary dye retained after acid-alcohol decolorization; counterstain with methylene blue
Capsule stain: visualizes gel-like capsule surrounding some microbes; background is stained (often with India ink) to reveal capsule as clear halo
Endospore stain: visualizes dormant endospores; endospores resist Gram stain; malachite green is applied with heat to facilitate uptake; safranin counterstains remaining cells
Flagella stain: makes thin flagella visible by applying staining agents that adhere to the flagella; distribution aids in identification

Fluorescent Dyes and Immunofluorescence

Dyes may bind to cellular structures in all cells or be activated by cellular processes to distinguish viability
Immunofluorescence uses fluorescently labeled antibodies to tag specific microbial proteins

Summary of Connections and Implications

The Gram stain and cell-wall structure influence antibiotic susceptibility and diagnostic approaches
Prokaryotic features (peptidoglycan, outer membrane, efflux pumps) impact how drugs enter cells and how resistance develops
Endosymbiotic theory explains the origin of mitochondria and chloroplasts, shaping our understanding of eukaryotic cell evolution and organelle genomes
Understanding staining, microscopy, and preparation techniques is essential for accurate identification and study of microorganisms

Notation and Key Terms (Quick Reference)

0.2\,\mu\mathrm{m}: maximum resolving power of light microscope
70S and 80S: ribosome sizes in prokaryotes and eukaryotes
LPS: lipopolysaccharide, outer membrane component; includes Lipid A and O antigen
Endospore: dormant, highly resistant cell form
Endosymbiotic theory: mitochondria and chloroplasts originated from bacteria
9+2: arrangement of microtubules in eukaryotic flagella/cilia
PHB: poly-β-hydroxybutyrate; a storage granule
S-layers: crystalline protein layers on archaeal cell walls
PAMPs and pattern recognition: relevant to immune response to LPS and other bacterial components

References to the Lecture Material

Gram staining, cell-wall architecture, and differential staining principles
Transport and energy systems across cytoplasmic membranes
Archaeal cell envelopes and unique features
Eukaryotic cell organelles, endomembrane system, and endosymbiotic origins
Microscopy techniques: light vs electron vs advanced fluorescence and super-resolution
Conditional statements about antibiotic targets and resistance mechanisms