Microscopy and Microorganisms Overview

Microscopy

• Compound Microscope Parts and Functions:
  • Ocular lens: Magnifies the image from the objective lens.
  • Objective lens: Gathers light, forms initial magnified image.
  • Stage: Platform for the specimen slide.
  • Condenser: Focuses light on the specimen.
  • Iris diaphragm: Controls light amount to the condenser.
  • Coarse adjustment knob: Initial image focus.
  • Fine adjustment knob: Precise image focus.
  • Light source: Illuminates the specimen.
• Key Microscopy Terms:
  • Resolving power: Ability to distinguish close objects.
  • Working distance: Distance between objective lens and specimen.
  • Total magnification: Ocular x Objective lens magnification.
  • Parfocalization: Microscope stays in focus when changing objectives.
  • Resolution limit: Limited by light wavelength; shorter wavelengths provide better resolution.

Microorganisms

• Microbial Groups and Characteristics:
  • Protozoa: Single-celled eukaryotes, often motile, diverse shapes/habitats.
  • Algae: Photosynthetic eukaryotes, unicellular or multicellular, aquatic.
  • Cyanobacteria: Photosynthetic prokaryotes, often blue-green, nitrogen fixers.
• Aseptic Technique: Prevents unwanted microbe contamination, essential for pure cultures and preventing pathogen spread.
• Bacteria: Prokaryotic, lack nucleus/membrane-bound organelles; diverse metabolism (respiration, fermentation, photosynthesis). Classified by shape (cocci, bacilli, spirilla).
• Fungi: Eukaryotic, unicellular (yeasts) or multicellular (molds), heterotrophic (absorb organic matter). Important in decomposition/nutrient cycling.
• Yeast vs. Mold:
  • Yeast: Unicellular, bud reproduction, used in fermentation.
  • Mold: Multicellular, filamentous (hyphae), spore reproduction.

Microbiological Techniques

• Smear Preparation:

  • Liquid media: Spread thin drop of culture on slide.
  • Solid media: Mix culture with water drop, spread on slide.

• Heat-Fixing: Kills microbes, attaches cells to slide to prevent washout.

• Staining Types:

  • Simple staining: Single dye to visualize cell morphology.
  • Differential staining: Multiple dyes for distinction based on cell structures/chemicals.

• Dye Types:

  • Basic dyes: Positively charged, bind to negative cellular components.
  • Acidic dyes: Negatively charged, repel negative cells, stain background.

• Capsule Staining:

  • Capsule: Gelatinous layer around some bacteria; protection, adhesion.
  • Difficulty: Non-ionic, does not readily bind to dyes.

• Gram Staining:

  • Procedure: Crystal violet (primary), iodine (mordant), alcohol (decolorizer), safranin (counterstain).
  • Reagent Functions:
    • Crystal violet: Stains all cells purple.
    • Iodine: Forms crystal violet-iodine complex.
    • Alcohol: Removes complex from Gram-negative, not Gram-positive cells.
    • Safranin: Stains Gram-negative pink.
  • Gram-positive vs. Gram-negative:
    • Gram-positive: Purple, thick peptidoglycan layer.
    • Gram-negative: Pink, thin peptidoglycan layer.

• Endospore Staining (Schaffer-Fulton):

  • Endospores: Dormant, resistant structures produced by some bacteria.
  • Genera: Bacillus and Clostridium produce endospores.
  • Challenge: Impermeable to most dyes.
  • Stains: Malachite green (primary, heated), safranin (counterstain).

• Acid-Fast Staining:

  • Significance: Identifies Mycobacterium (tuberculosis).
  • Acid-fast organisms: Waxy cell walls, resistant to acid-alcohol decolorization.
  • Stains: Carbol fuchsin (primary, heated), acid-alcohol, methylene blue (counterstain).

• Motility Determination:

  • Hanging drop: Suspended culture over slide in a drop.
  • Tube method: Observe growth spread from inoculation line in semi-solid agar.

• Culture Media:

  • Definition: Nutrient substances for microbial growth.
  • Agar: Solidifying agent in media.

• Nutritional Categories:

  • Autotrophs: Synthesize organic compounds from inorganic sources.
    • Photoautotrophs: Light energy for synthesis.
    • Chemoautotrophs: Chemical energy for synthesis.
  • Heterotrophs: Require preformed organic compounds.
    • Chemoheterotrophs: Obtain energy by breaking down organic molecules.
    • Photoheterotrophs: Light energy, but organic carbon.

• Growth Factors: Organic compounds needed for growth; organism cannot synthesize.

• Culture Media Types:

  • Synthetic: Known, defined components.
  • Non-synthetic: Complex, undefined components (extracts, digests).

• Selective and Differential Media:

  • Selective: Inhibits certain microbes, allows growth of others.
  • Differential: Differentiates microbes based on their appearance on the media.

• Autoclaving Conditions: 121°C (249°F) at 15 psi for 15-20 minutes.

• Isolation of Pure Cultures: Streak plating, spread plating, pour plating to separate individual colonies.

• Quantitative Plating: Known volume diluted sample, spread on plate; count colonies (30-300 colony count).

• Microbial Growth Factors:

  • Optimum temperature: Fastest growth rate.
  • Maximum temperature: Highest growth.
  • Minimum temperature: Lowest growth.
  • Mesophiles: Moderate temps (20-45°C).
  • Psychrophiles: Low temps (below 20°C).
  • Thermophiles: High temps (above 45°C).
  • Thermal death time (TDT): Time to kill all cells at a temp.
  • Thermal death point (TDP): Lowest temp killing all cells in given time.

• Tonicity:

  • Hypotonic: Lower solute, water enters.
  • Hypertonic: Higher solute, water exits.
  • Isotonic: Same solute, no net water movement.
  • Plasmolysis: Shrinkage due to H2O loss in hypertonic solution.

• Osmophiles: High osmotic pressure growth.

• Halophiles: High salt concentrations growth

• Effects of Tonicity:

  • Hypertonic solution: Cells shrink (plasmolysis).
  • Hypotonic solution: Cells swell and potentially burst.

• Antimicrobial Agents

  • Antiseptics: Inhibit/kill on living tissues.
  • Disinfectants: Inhibit/kill on inanimate surfaces.
  • Antibiotics: Substances from microbes/synthetic, inhibit/kill other microbes.
  • Zone of inhibition: Clear area around disk where growth is inhibited.

• Kirby-Bauer method: Standardized method to test antibacterial sensitivity (zone of inhibition).

• Metabolic Processes & Enzymes:

  • Fermentation: Sugar energy release without oxygen.
  • Metabolism: All chemical reactions.
  • Endoenzymes: Enzymes function inside the cell.
  • Exoenzymes: Enzymes secreted outside the cell.
  • Oxidation: Electron loss.
  • Enzymes: Biological catalysts.

• Biochemical Tests:

  • Durham tubes: Detect gas production during fermentation.
  • Mixed acid/Butanediol fermentations: Detect acidic/butanediol production.
  • Catalase production: Breakdown of hydrogen peroxide (protection).
  • Oxidase production: Tests respiratory pathways, cytochrome oxidase presence.
  • Starch hydrolysis: Ability to break down starch.
  • Casein hydrolysis: Ability to break down casein.
  • Fat hydrolysis: Ability to break down fats.
  • Tryptophan hydrolysis: Ability to break down tryptophan (indole).
  • Urea hydrolysis: Presence of enzyme urease breaking down urea.
  • Hydrogen sulfide production.
  • Citrate utilization.
  • IMViC: Four tests used to differentiate Enterobacteriaceae family (Indole, Methyl Red, Voges-Proskauer, Citrate)

• Significance of Tests:

  • Oxidase test: Differentiates respiratory pathways.
  • IMViC tests: Important for identifying Enterobacteriaceae family, identifying pathogens.

Quiz & Answer Key

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Essay Questions

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Glossary

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Explore the fundamental components and functions of compound microscopes as well as key microscopy terms. Additionally, delve into the characteristics of different microbial groups such as protozoa. This quiz will enhance your understanding of microscopy techniques and microbial biology.