Light Microscopes

Light Microscopes

• Light microscopes use visible light to view specimens.
• They utilize an ocular lens and an objective lens.
• The final magnification is calculated by multiplying the enlarging power of the ocular and objective lenses (e.g., ×10 times ×4 = ×40).

Types of Light Microscopes

• Bright-field microscope: The background is lighter than the observed specimen; most specimens require fixing and staining.
• Dark-field microscope: Used to view unfixed, unstained specimens such as living organisms; the background is dark, and the specimen is bright.
• Phase-Contrast Microscope: Used to view unfixed, transparent specimens; effective for observing cell division, cell motility, and dynamic states of cell organelles.
• Fluorescence Microscopes: Visualize specimens that contain naturally fluorescent substances or that have been stained with fluorescent stain.
• Confocal Microscopes: Produce sharper images than traditional microscopes; allows visualization of different planes of a specimen; the image can be displayed three-dimensionally with "electronic staining."

Electron Microscopes

• Electron microscopes use electrons as an energy source.
• Transmission Electron Microscope (TEM): Electron beams go through the specimen requiring special preparation, sectioning, and staining, producing two-dimensional images with good internal detail.
• Scanning Electron Microscope (SEM): No sections are needed; it scans the surface of an object, creating a three-dimensional image.

Chemistry of Life

• Matter: Anything that occupies space and has mass (solid, liquid, gas) composed of elements.
• Chemical Compound: Elements in combination.
• Atoms: Smallest particles of an element.
• Atomic Nucleus: Contains protons and neutrons.
• Protons: Positively charged particles.
• Neutrons: Particles without charge.
• Electrons: Negatively charged particles.
• Ions: Electrically charged atoms, molecules, or particles.
  • Cations (+): Due to electron loss.
  • Anions (–): Due to electron gain.
  • Electrolyte: Free ions present in a solvent.

Chemical Reactions

• Synthesis (Dehydration Synthesis): Formation of a larger molecule from smaller ones, removal of H2O; it is an endergonic reaction requiring energy.
• Hydrolysis: Breakdown of large molecules; requires H2O; it is an exergonic reaction that gives off energy.
• Redox (Reduction-Oxidation): Simultaneous electron transfer.
  • Reduction: Gain of electrons.
  • Oxidation: Loss of electrons.

Compounds

• Hydrophilic: Water-soluble (water-loving).
• Hydrophobic: Insoluble in water (water-repelling).
• Inorganic: Does not contain carbon (except CO & CO2); examples include acids, bases, salts (dissolve in water and do not release hydrogen or hydroxyl ions), and water.
• Organic: Contains carbon and hydrogen.

Organic Compounds

• Carbohydrates
• Proteins
• Lipids: Fats and oils
• Nucleic Acids: Pentose sugar, phosphate, nitrogen base.
  • Purines: Adenine (A) and guanine (G).
  • Pyrimidines: Cytosine (C), thymine (T), and uracil (U).
  • DNA: Deoxyribonucleic acid (storage of genetic information).
  • RNA: Ribonucleic acid.
  • ATP: Cell energy source.

Carbohydrates

• Monosaccharides: Glucose, fructose (fruit sugar), galactose.
• Disaccharides: Sucrose (table sugar, sweeteners), maltose, lactose (milk sugar).
• Polysaccharides: Glycogen (storage form of sugar in animals), starch (storage form of sugar in plants)

Solution

• Solution: A mixture of one or more substances called solutes, dispersed in a dissolving medium called a solvent.
• Isotonic Solution: Solute concentration is equal inside and outside the cell.
• Hypertonic Solution: Solute concentration outside the cell is higher than inside the cell.
• Hypotonic Solution: Solute concentration outside the cell is lower than inside the cell.

Body Fluids

• Body fluids make up 60% of total body weight.
  • ⅔ is intracellular fluid (40%).
  • ⅓ is extracellular fluid (20%).
  • ¾ in the interstitial fluid {15%}
  • ¼ in plasma {5%} {intravascular}

Electrolytes

• In ECF: Na+ is the main cation, and chloride is the main anion.
• In ICF: K+ is the main cation, and phosphate is the main anion.

pH Scale

• Measures the alkalinity or acidity of a solution.
• Based on the hydrogen ion concentration.
• The pH scale runs from 0 to 14.
• Pure H2O is neutral with a pH of 7.
• Substances that dissociate in water can form acids, bases, or salts.
  • Acids: pH less than 7 (release hydrogen ions).
  • Bases: pH greater than 7 (release hydroxyl ions).

Microbiological Laboratory Techniques

• A culture can be pure (has one type of organism) or mixed (has two or more species).

Culture Media

• Culture media is classified based on physical state, chemical composition, and functional types.

Physical State

• Liquid Media (broth): Water-based solutions.
• Semisolid Media: Clot at room temperature.
• Solid Media: Solid at room temperature, becomes liquid at the boiling temperature of water.

Chemical Composition

• Chemically defined (synthetic): Contain pure compounds with a chemical content specified by an exact formula.
• Complex Media (nonsynthetic): One or more components is not chemically defined and cannot be represented by an exact chemical formula.

Functional Types of Media

• General Purpose Media: For microbes that do not have special growth requirements; examples include nutrient agar and trypticase soy agar (TSA).
• Enriched Media: Contain complex organic substances, such as blood; examples include blood agar (detects the ability to produce hemolysins), chocolate agar (for respiratory bacteria), and Thayer-Martin agar (for Neisseria).
• Selective Media: Contain one or more agents that inhibit the growth of a certain microbe, favoring or selecting for a certain microbe and allowing it to grow; an example is phenylethyl alcohol agar, which is selective for gram-positive bacteria and inhibits the growth of gram-negative bacteria.
• Differential Media: MacConkey agar (selective & differential): contains neutral red (yellow dye when neutral, red when acidic, by E. coli when lactose fermenter, Salmonella on the other hand is non-lactose fermenter), mannitol salt agar (selective & differential): its 7.5% NaCl inhibits most organisms except staphylococcus.
• Live Media: Certain bacteria require host animals to grow, such as Mycobacterium leprae on mice footpad.

Basic Procedures to Examine & Characterize Microbes (The Five I’s)

• Inoculation: Into a medium.
• Incubation: Incubator temperature is generally between 20°C and 40°C.
• Isolation.
  • Spread Plate: Used to quantify the number of bacteria in a solution; colonies are evenly distributed over the agar surface and are easily counted.
• Inspection: Colonies are observed macroscopically; growth patterns in broths include Pellicle: a thick growth at the top of the tube Sediment at the bottom of the tube Turbid growth throughout the tube.
• Identification: Determination of the type of microbe.

Identification Techniques

• Morphological characteristics.
• Physiological characteristics.
• Biochemical characteristics.