Observing Microorganisms Through a Microscope

Advanced Light Microscopy Techniques

Modern microscopy provides several methods for visualizing microorganisms, each with unique advantages for resolution, contrast, and three-dimensional imaging.

• Confocal Microscopy: Illuminates one plane at a time and rejects out-of-focus light, producing clear two-dimensional slices. Computer software can combine these slices to construct a three-dimensional image.

• Two-Photon Microscopy: Uses two long-wavelength red photons to excite dyes, allowing imaging of living cells up to one millimeter deep and tracking activity in real time.

• Super-Resolution Microscopy: Employs two laser beams and computer scanning to localize fluorescence down to about one nanometer, surpassing the diffraction limit of light.

Non-Light-Based Microscopy

• Scanning Acoustic Microscopy (SAM): Measures sound waves reflected from a specimen, useful for studying cells attached to surfaces (e.g., cancer cells, arterial plaque, bacterial biofilms). Resolution is about one micrometer.

• Electron Microscopy: Uses electrons instead of visible light, allowing much higher resolution due to the shorter wavelength of electrons. Images are originally black and white; color may be added digitally.

  • Transmission Electron Microscopy (TEM): Electrons pass through ultrathin sections of the specimen. Useful for internal structures. Magnification: 10,000x to 10,000,000x; resolution limit: about 0.2 nanometers.

  • Scanning Electron Microscopy (SEM): Scans the surface with a focused electron beam. Provides three-dimensional images of surfaces. Magnification: 1,000x to 500,000x; resolution limit: about 0.5 nanometers.

• Scanned Probe Microscopy: Uses a fine probe to examine surfaces at atomic or molecular resolution without modifying the specimen.

  • Scanning Tunneling Microscopy (STM): Measures tunneling current as a sharp probe scans the surface. Can resolve features as small as individual atoms and image molecules like DNA.

  • Atomic Force Microscopy (AFM): Uses a metal-and-diamond probe to scan the surface, producing three-dimensional images with near-atomic detail.

Specimen Preparation and Staining

Staining increases contrast and reveals specific structures in microorganisms.

• Smear: A thin film of material containing microorganisms spread on a slide.

• Fixation: Attaches microorganisms to the slide, kills them, and preserves their structure. Methods include heat fixation (passing through a flame) and chemical fixation (using methanol).

• Staining: Uses dyes to color microorganisms. Dyes contain positive and negative ions; the colored ion is the chromophore.

  • Basic Dyes: Chromophore is a positively charged cation (e.g., crystal violet, methylene blue, safranin). Attracted to negatively charged bacterial cells.

  • Acidic Dyes: Chromophore is a negatively charged anion (e.g., eosin, acid fuchsin, nigrosin). Repelled by cells, staining the background (negative staining).

• Simple Stain: Uses one basic dye to color the entire microorganism, making shape and arrangement visible. A mordant may be used to enhance staining.

Differential Staining Techniques

• Gram Stain: Classifies bacteria as Gram-positive or Gram-negative based on cell wall structure.

  • Gram-positive: Thick peptidoglycan wall; retains purple dye.

  • Gram-negative: Thin peptidoglycan layer plus outer membrane; loses purple dye during decolorization, appears pink/red after counterstaining.

  • Steps:

    1. Apply crystal violet (primary stain) – both cell types purple.

    2. Add iodine (mordant) – forms dye complex.

    3. Wash with alcohol – Gram-positive remains purple; Gram-negative becomes colorless.

    4. Apply safranin (counterstain) – Gram-positive remains purple; Gram-negative becomes pink/red.

• Acid-Fast Stain: Identifies bacteria with waxy cell walls (e.g., Mycobacterium, Nocardia).

  • Primary stain: Carbolfuchsin (both cell types red).

  • Decolorize with acid-alcohol: Acid-fast cells remain red; non-acid-fast become colorless.

  • Counterstain with methylene blue: Acid-fast cells remain red; non-acid-fast become blue.

  • Example: Mycobacterium tuberculosis appears as red cells against a blue background.

• Capsule Stain: Capsules do not accept most dyes. Negative staining with India ink or nigrosin darkens the background; a simple stain colors the cell, leaving the capsule as a clear halo.

Key Concepts Recap

• Choose a microscope based on the size and detail required.

• Magnification is not the same as resolution.

• Stains create contrast and can differentiate cell types or structures.

Functional Anatomy of Prokaryotic and Eukaryotic Cells

Prokaryotic vs. Eukaryotic Cells

Cells are classified based on the presence or absence of a membrane-enclosed nucleus and internal organization.

• Prokaryotic Cells: No membrane-enclosed nucleus; DNA in a nucleoid region. Generally smaller (1–10 μm), simpler internal structure, lack most organelles. Divide by binary fission.

• Eukaryotic Cells: Membrane-enclosed nucleus; DNA in several linear chromosomes. Larger (10–100 μm), complex internal structure with many organelles. Divide by mitosis.

Bacterial Shapes and Arrangements

• Shapes:

  • Cocci: Spherical

  • Bacilli: Rod-shaped

  • Spiral Forms: Vibrio (comma-shaped), Spirillum (rigid spiral), Spirochete (flexible spiral with axial filament)

• Arrangements:

  • Diplococci: Pairs

  • Streptococci: Chains

  • Tetrads: Groups of four

  • Sarcinae: Cubes of eight

  • Staphylococci: Irregular clusters

• Monomorphic: Single, consistent shape

• Pleomorphic: Variable shape

Basic Structure of a Bacterial Cell

• Plasma Membrane: Inner boundary; controls movement of substances in and out.

• Cytoplasm: Fluid region where chemical reactions occur.

• Ribosomes: Sites of protein synthesis.

• Nucleoid: Contains main circular chromosome (not membrane-bound).

• Plasmids: Small, circular DNA molecules carrying extra genetic information (optional).

• Cell Wall: Provides shape and prevents osmotic lysis; composed mainly of peptidoglycan.

• Glycocalyx: General term for substances surrounding the cell wall; can be a capsule (organized, firm) or slime layer (unorganized, loose).

Functions of the Glycocalyx

• Prevents desiccation (drying out)

• Helps bacteria adhere to surfaces (e.g., Streptococcus mutans to teeth)

• Contributes to virulence by evading phagocytosis (e.g., Streptococcus pneumoniae)

Appendages

• Flagella: Motility structures made of flagellin; consist of filament, hook, and basal body. Arrangement varies:

  • Atrichous: No flagella

  • Peritrichous: Flagella all over

  • Monotrichous: Single flagellum at one pole

  • Lophotrichous: Tuft at one end

  • Amphitrichous: Flagella at both ends

• Axial Filaments: Internal flagella in spirochetes, producing a twisting motion.

• Fimbriae: Numerous, short, hairlike; used for attachment.

• Pili: Longer, fewer; involved in motility (twitching/gliding) and DNA transfer (conjugation).

• Archaella: Motility structures in archaea, made of archaellins (glycoproteins).

Bacterial Cell Wall Structure

• Peptidoglycan: Main structural component; mesh of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) sugars cross-linked by peptides.

• Gram-Positive Cell Wall: Thick peptidoglycan layer, teichoic acids (wall and lipoteichoic), highly susceptible to penicillin, disrupted by lysozyme, often produce exotoxins.

• Gram-Negative Cell Wall: Thin peptidoglycan in periplasm, outer membrane with lipopolysaccharide (LPS), porins, and lipoproteins. LPS contains Lipid A (endotoxin), core polysaccharide, and O polysaccharide (antigenic).

Summary of Prokaryotic Cell Structure

• Prokaryotes are small, organized cells with essential structures for survival.

• Cell wall structure determines Gram reaction and influences susceptibility to antibiotics and immune defenses.

• Appendages and surface structures contribute to motility, attachment, and virulence.

Microbial Metabolism

Overview of Metabolism

Metabolism is the sum of all chemical reactions in a living organism, including both energy-releasing and energy-consuming processes.

• Catabolism: Breakdown of complex molecules into simpler ones, releasing energy (exergonic). Example: Hydrolysis of macromolecules.

• Anabolism: Synthesis of complex molecules from simpler ones, requiring energy (endergonic). Example: Protein synthesis.

• ATP (Adenosine Triphosphate): The cell's energy currency, linking catabolism and anabolism.

ATP Cycle

• Catabolic reactions: ADP + Pi + energy → ATP

• Anabolic reactions: ATP → ADP + Pi + energy

Equation:

Enzymes and Metabolic Pathways

• Enzymes: Biological catalysts that speed up reactions by lowering activation energy. Highly specific for their substrates.

• Active Site: Region where the substrate binds and the reaction occurs.

• Enzyme-Substrate Complex: Temporary association during catalysis.

• Turnover Number: Number of substrate molecules converted per second (typically 1–10,000; some up to 500,000).

• Enzyme Classes:

  • Oxidoreductase: Oxidation-reduction reactions

  • Transferase: Transfer of functional groups

  • Hydrolase: Hydrolysis reactions

  • Lyase: Removal of atoms without hydrolysis

  • Isomerase: Rearrangement within a molecule

  • Ligase: Joining molecules, usually with ATP

• Enzyme Structure:

  • Apoenzyme: Protein portion (inactive alone)

  • Cofactor: Nonprotein helper (inorganic or organic)

  • Coenzyme: Organic cofactor (often derived from vitamins; e.g., NAD+, FAD, CoA)

  • Holoenzyme: Complete, active enzyme (apoenzyme + cofactor)

Factors Affecting Enzyme Activity

• Temperature: Activity increases with temperature up to an optimum, then decreases due to denaturation.

• pH: Each enzyme has an optimum pH; extremes can denature the enzyme.

• Substrate Concentration: Rate increases with concentration until saturation is reached.

• Inhibitors:

  • Competitive: Compete with substrate for active site.

  • Noncompetitive: Bind elsewhere (allosteric site), changing enzyme shape.

• Feedback Inhibition: End-product of a pathway inhibits an early enzyme, preventing overproduction.

Redox Reactions and Energy Transfer

• Oxidation: Loss of electrons

• Reduction: Gain of electrons

• Redox Reaction: Paired oxidation and reduction; electrons lost by one molecule are gained by another.

• OIL RIG: Oxidation Is Loss, Reduction Is Gain

• In biological systems, electrons and protons (hydrogen atoms) are often transferred together (dehydrogenation).

• NAD+ + 2H → NADH + H+

ATP Generation Mechanisms

• Substrate-Level Phosphorylation: Direct transfer of a phosphate group to ADP from a phosphorylated substrate.

• Oxidative Phosphorylation: Electrons pass through an electron transport chain, releasing energy used to generate ATP (chemiosmosis).

• Photophosphorylation: Light energy excites electrons in pigments (e.g., chlorophyll), which pass through carriers to generate ATP.

Carbohydrate Catabolism

• Glycolysis (Embden-Meyerhof Pathway): Series of 10 enzyme-catalyzed reactions converting one glucose to two pyruvic acid, producing ATP and NADH.

• Preparatory Stage: Invests 2 ATP to phosphorylate glucose and split it into two three-carbon molecules (GP and DHAP).

• Payoff Stage: Each three-carbon molecule produces 2 ATP and 1 NADH (total: 4 ATP, 2 NADH, 2 pyruvic acid per glucose).

• Net Gain: 2 ATP (4 produced – 2 used), 2 NADH, 2 pyruvic acid per glucose.

Equation:

Respiration vs. Fermentation

• Cellular Respiration: Includes glycolysis, Krebs cycle, and electron transport chain. Final electron acceptor is external (often oxygen).

• Fermentation: Begins with glycolysis but does not use an external electron acceptor or complete electron transport chain. Pyruvic acid or its derivative accepts electrons from NADH, forming fermentation end-products (e.g., ethanol, lactic acid).

Summary of Microbial Metabolism

• Metabolism includes catabolism (energy release) and anabolism (energy use).

• ATP couples energy transfer between catabolic and anabolic pathways.

• Enzymes control metabolic pathways and are regulated by environmental factors and feedback inhibition.

• Redox reactions and phosphorylation are central to energy capture and storage.

• Glycolysis is the universal starting point for carbohydrate catabolism.