Lab 1: Experimental Design and Diffusion Concepts

Lab 1: Experimental Design, Lab Fundamentals, and Diffusion

• Hypothesis: A prediction about the outcome of an experiment.
• Dependent Variable: Outcome being measured, influenced by the independent variable.
• Independent Variable: Variable manipulated by the researcher to observe its effect on the dependent variable.
• Random Errors: Unpredictable variations in measurements.
• Systematic Errors: Consistent, repeatable errors caused by flaws in equipment or methods.
• Metric Conversions: 1 milliliter (mL) = 1000 microliters (μL); 1 liter (L) = 1000 milliliters (mL).
• Micropipette Use: Set desired volume, use appropriate tip, avoid air bubbles, dispense smoothly.
• Volume Inaccuracy Factors: User error, wrong tip, bubbles in the tip.
• Rate of Diffusion: Influenced by temperature, molecule size, and concentration gradient.
  • Higher temperature → Increased diffusion.
  • Larger molecule size → Decreased diffusion.
  • A steeper concentration gradient (larger difference in concentration between areas) increases the rate of diffusion.

Lab 2: Microscopy

• Microscope Parts: Eyepiece, objective lenses, stage, coarse/fine focus knobs.
• Microscope Types: Light microscopes use light; electron microscopes use electrons.
• Total Magnification: Magnification of eyepiece x magnification of objective lens.
• Field of View: Area visible through the microscope; decreases as magnification increases.
• Magnification vs. Resolution: Magnification enlarges, resolution clarify.
• Cell Staining: Enhances contrast to view transparent structures.

Lab 3: Biological Molecules

• Monomers vs. Polymers: Monomers are single units; polymers are chains of monomers.
• Categories of Biological Molecules: Carbohydrates, proteins, lipids, nucleic acids.
• Dehydration Reaction: Joins monomers by removing water.
• Amino/Carboxyl Groups: Structural components of amino acids.
• Protein Structures:
  • Primary: Amino acid sequence.
  • Secondary: Alpha helices or beta sheets.
  • Tertiary: 3D folding.
  • Quaternary: Multiple polypeptides.
• Triglycerides vs. Monoglycerides: Three vs. one fatty acid chain.
• DNA Structure: Double helix; bases pair as A-T and G-C.
• Purines/Pyrimidines: Purines = Adenine, Guanine; Pyrimidines = Thymine, Cytosine.
• Indicators:
  • Benedict's Test (for reducing sugars):
    • Detects the presence of reducing sugars, like glucose and fructose.
    • A sample is mixed with Benedict's reagent and heated.
    • Positive Result: The solution changes color from blue to green, yellow, orange, or brick-red, depending on the amount of sugar.
    • Negative Result: The solution remains blue.
  • Biuret Test (for proteins):
    • Identifies the presence of peptide bonds, which are indicative of proteins.
    • Biuret reagent (a blue solution) is added to the sample.
    • Positive Result: The solution turns violet or black when proteins are present.
    • Negative Result: The solution remains blue.
  • Iodine Test (for starch) (carbohydrate):
    • Detects the presence of starch (a polysaccharide).
    • Iodine solution (brownish-yellow) is added to the sample.
    • Positive Result: The solution turns a dark blue or black color if starch is present.
    • Negative Result: The solution remains yellowish-brown.
  • Sudan IV Test (for lipids):
    • Detects lipids (fats and oils).
    • Sudan IV, a fat-soluble dye, is added to the sample.
    • Positive Result: Lipids absorb the dye and appear as red-stained oil droplets.
    • Negative Result: No red-staining, and the solution remains uncolored or uniformly pink.
  • Ninhydrin Test (for amino acids):
    • Detects free amino acids, which are the building blocks of proteins.

Lab 4: Enzyme Kinetics

• Spectrophotometer: Measures absorbance to determine concentration.
• Absorbance vs. Concentration: Higher absorbance = higher concentration.
• Enzyme: A biological catalyst that speeds up chemical reactions in cells without being consumed in the process.
• Substrate: The specific molecule that an enzyme acts upon in a reaction.
• Enzyme-Substrate Complex: A temporary molecule formed when the enzyme binds to its substrate at the enzyme's active site.
• Active Site: The region on the enzyme where the substrate binds.
• Activation Energy: The minimum amount of energy required to initiate a chemical reaction.
• How Enzymes Work: Enzymes facilitate reactions by lowering the activation energy.
• Factors Affecting the Rate of a Reaction:
  • Substrate Concentration: Increasing substrate concentration increases the reaction rate until the enzyme becomes saturated.
  • Enzyme Concentration: More enzyme molecules increase the rate of the reaction, provided there is enough substrate available.
  • Temperature: Reaction rates typically increase with temperature, but high temperatures can denature the enzyme (change its structure), reducing activity.
  • pH: Each enzyme has an optimal pH at which it works best. Deviation from this pH can alter the enzyme’s shape and function.
• Enzyme Kinetics:
  • Vmax: Maximum reaction rate.
  • Km: Substrate concentration at half Vmax.
  • pH and Temperature: Affect enzyme activity by altering shape or stability.
• Michaelis-Menten Kinetics: Describes the rate of enzyme-catalyzed reactions.
  • Vmax: The maximum velocity or rate of the reaction when the enzyme is fully saturated with substrate.
  • Km (Michaelis constant): The substrate concentration at which the reaction rate is half of Vmax. It represents the enzyme’s affinity for the substrate.

Lab 5: Chemotaxis and Bioconvection

• Chemotaxis: Movement of organisms (e.g., Tetrahymena) toward or away from chemicals.
• Cellobiase: An enzyme that catalyzes the breakdown of cellobiose, a disaccharide, into two glucose molecules.
• Detection System: Uses a substrate analog (synthetic molecule resembling cellobiose) that releases a colored product upon enzymatic cleavage.
• How Chemotaxis Detection Works:
  1. Cellobiase reacts with the substrate analog, breaking it down.
  2. A colored product is released, which can be measured using a spectrophotometer to quantify enzyme activity.

Dive into the fundamentals of experimental design and diffusion in this Lab 1 quiz. Explore key concepts such as variables, types of errors, and the principles of micropipette use. Perfect for students aiming to reinforce their understanding of core lab techniques.