Unit 3 Study Guide: Scientific Method and Daphnia Heart Rate Experiment

Key Terminology & Scientific Method Principles

• Independent Variable:

  • Definition: The variable in an experiment that is directly manipulated or changed by the researcher at the outset of the experiment.

  • Conceptualization: An easy way to recall this concept is that Independent begins with "I"—representing "I, the researcher, manipulate this variable."

  • Example: In a study examining the effect of sleep on student performance, the number of hours slept before a test is the independent variable.

• Dependent Variable:

  • Definition: The variable in an experiment that is measured to assess the effect of the experimental conditions; it changes in response to alterations made to the independent variable.

  • Example: In a study examining the effect of sleep on student performance, the student's test grade is the dependent variable.

• Hypothesis:

  • Definition: A tentative answer to a well-framed scientific question, phrased as a statement and grounded in previously supported data or observations. It is not merely an "educated guess."

  • Falsifiability: A valid hypothesis must be falsifiable, meaning it must be structured in a way that allows empirical testing to either support or nullify it.

  • Experimental Outcome: Scientific experiments do not "prove" a hypothesis to be absolute truth; rather, experimental data either support or nullify/reject the hypothesis.

  • Example: Based on prior lab observations, when deionized water ($\text{diH}_2\text{O}$) reaches a temperature at or above $100^\text{o}\text{C}$, it will boil. If $\text{diH}_2\text{O}$ reaches $100^\text{o}\text{C}$ and boils, the hypothesis is supported; if it does not boil, the hypothesis is nullified.

• Steps of the Scientific Method:

  • Scientific inquiry follows a systematic sequence: making observations, formulating specific questions, constructing falsifiable hypotheses, designing and executing controlled experiments, analyzing collected data, and determining whether the hypothesis is supported or nullified.

Physiological Effects of Stimulants and Depressants

• Chemical Classification and Cardiovascular Impact:

  • Stimulants: Chemical substances that increase activity within the central nervous system and cardiovascular system. Caffeine—a common stimulant found in coffee, tea, chocolate, and certain medications—causes an increase in heart rate.

  • Depressants: Chemical substances that decrease or slow down central nervous system and cardiovascular function. Alcohol (ethanol) acts as a nervous system depressant, causing a decrease in heart rate, slowed physiological activity, and drowsiness/sleepiness.

• Model Organism: Daphnia magna:

  • Classification: A small freshwater crustacean.

  • Anatomical Advantage: The external body of Daphnia is encased in a translucent exoskeleton made of the polysaccharide chitin.

  • Experimental Utility: Because the chitin layer is transparent, internal anatomical structures—specifically the compound eye, paired antennae, and rapidly beating heart—can be non-invasively observed and quantified under a light microscope.

Concentration and Dilution Calculations

• Dilution Equation Formula:

  • To prepare a lower concentration solution from a concentrated stock solution, use the dilution formula:     $C_1 V_1 = C_2 V_2$

  • Where:

  • $C_1$ = Concentration of the stock solution

  • $V_1$ = Volume of the stock solution needed

  • $C_2$ = Desired final concentration of the diluted solution

  • $V_2$ = Desired total final volume of the diluted solution

• Solving for Stock Volume ($V_1$):

  • Rearranging the dilution equation to isolate stock volume yields:     $V_1 = \left(\frac{C_2}{C_1}\right) \times V_2$

• Calculating Solvent Volume:

  • To determine the required volume of water (or other solvent) to mix with the stock volume:     $\text{Volume of Solvent} = V_2 - V_1$

• Worked Example 1 (Lab Manual Protocol):

  • Problem: Calculate how to prepare $15\,\text{mL}$ of a $5\%$ solution using a $30\%$ stock solution.

  • Step 1: Identify given values ($C_1 = 30\%$, $C_2 = 5\%$, $V_2 = 15\,\text{mL}$).

  • Step 2: Calculate $V_1$:     $V_1 = \left(\frac{5\%}{30\%}\right) \times 15\,\text{mL}$     $V_1 = 0.167 \times 15\,\text{mL} = 2.5\,\text{mL}$

  • Step 3: Calculate required solvent ($\text{diH}_2\text{O}$) volume:     $\text{Volume of } \text{diH}_2\text{O} = 15\,\text{mL} - 2.5\,\text{mL} = 12.5\,\text{mL}$

  • Result: Combine $2.5\,\text{mL}$ of $30\%$ stock solution with $12.5\,\text{mL}$ of water to yield $15\,\text{mL}$ of $5\%$ solution.

• Worked Example 2 (Video Demonstration):

  • Problem: Calculate how to prepare $20\,\text{mL}$ of a $2\%$ ethanol solution from a $10\%$ ethanol stock solution.

  • Step 1: Identify given values ($C_1 = 10\%$, $C_2 = 2\%$, $V_2 = 20\,\text{mL}$).

  • Step 2: Calculate $V_1$:     $10\% \times V_1 = 2\% \times 20\,\text{mL}$     $V_1 = \frac{2 \times 20\,\text{mL}}{10} = \frac{40\,\text{mL}}{10} = 4\,\text{mL}$

  • Step 3: Calculate required solvent volume:     $\text{Volume of Water} = 20\,\text{mL} - 4\,\text{mL} = 16\,\text{mL}$

  • Result: Mix $4\,\text{mL}$ of $10\%$ stock ethanol with $16\,\text{mL}$ of water to produce $20\,\text{mL}$ of $2\%$ ethanol solution.

• Exercise 1 Protocol: Preparing Simulated Ethanol Dilutions:

  • Goal: Prepare $500\,\mu\text{L}$ of a $2\%$ simulated ethanol solution and $500\,\mu\text{L}$ of a $6\%$ simulated ethanol solution using a $10\%$ simulated ethanol stock (water mixed with dye).

  • Dilution Calculation for $2\%$ Solution:     $V_1 = \left(\frac{2\%}{10\%}\right) \times 500\,\mu\text{L} = 100\,\mu\text{L}\text{ stock}$     $\text{Volume of } \text{diH}_2\text{O} = 500\,\mu\text{L} - 100\,\mu\text{L} = 400\,\mu\text{L}$

  • Dilution Calculation for $6\%$ Solution:     $V_1 = \left(\frac{6\%}{10\%}\right) \times 500\,\mu\text{L} = 300\,\mu\text{L}\text{ stock}$     $\text{Volume of } \text{diH}_2\text{O} = 500\,\mu\text{L} - 300\,\mu\text{L} = 200\,\mu\text{L}$

  • Laboratory Action Steps:

  • Label two microfuge tubes as E2 (for the $2\%$ solution) and E6 (for the $6\%$ solution).

  • Use appropriate micropipettes to measure and transfer stock solution and $\text{diH}_2\text{O}$ into each tube.

  • Cap and invert both microfuge tubes thoroughly to ensure complete mixing.

  • Present prepared tubes and completed calculations to the laboratory instructor for verification.

Laboratory Protocol: Microscopy and Daphnia Preparation

• Microscope Operation and Focus Steps:

  • Step 1: Ensure the microscope stage is lowered to its lowest position and the $4\times$ objective lens is locked in place.

  • Step 2: Position the prepared slide onto the stage, securing it with stage clips.

  • Step 3: Center the Daphnia directly over the light path and adjust light intensity for comfortable viewing.

  • Step 4: Raise the stage to its maximum height using the coarse focus adjustment knob.

  • Step 5: Look through the eyepieces and slowly lower the stage using the coarse focus knob until the organism comes into view.

  • Step 6: Refine image clarity using the fine focus adjustment knob.

  • Step 7 (Contrast Enhancement): If the heart or body structures are difficult to distinguish due to high light intensity, adjust the condenser iris diaphragm to reduce light passage and increase image contrast.

  • Step 8: View the organism under $4\times$ or $10\times$ total objective magnification as needed to facilitate accurate heart rate counting.

• Anatomical Identification:

  • Eye: The compound eye is the most prominent, dark circular structure located in the head region.

  • Antennae: Paired, branched appendages protruding from the head used primarily for swimming locomotion and environmental sensing.

  • Heart: A transparent, rapidly beating structure located in the upper dorsal portion of the body, positioned directly behind the digestive tract.

Experimental Protocol: Heart Rate Determination & Chemical Testing

• Baseline Calculations:     $\text{Average Beats per } 15\,\text{sec} = \frac{\text{Trial 1} + \text{Trial 2} + \text{Trial 3}}{3}$     $\text{Baseline Heart Rate (Beats/Min)} = (\text{Average Beats per } 15\,\text{sec}) \times 4$

• Exercise 3: Testing Effects of Caffeine or Ethanol:

  • Experimental Goal: Determine the concentration of assigned chemical (caffeine or ethanol) required to induce a $20\%$ change ($\pm 20\%$) relative to the baseline heart rate.

  • Chemical Ranges Available:

  • Caffeine Concentrations: $0.2\%$ to $1.0\%$

  • Ethanol Concentrations: $1.0\%$ to $5.0\%$

  • Target Heart Rate Calculations:     $\text{Heart Rate Change Amount (Beats/Min)} = \text{Baseline Heart Rate} \times 0.20$     $\text{Target Heart Rate (Stimulant / Caffeine)} = \text{Baseline Heart Rate} + (\text{Baseline Heart Rate} \times 0.20)$     $\text{Target Heart Rate (Depressant / Ethanol)} = \text{Baseline Heart Rate} - (\text{Baseline Heart Rate} \times 0.20)$

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Data Presentation, Graphing, and Discussion Guidelines

• Data Table Format (Table 2 Guidelines):

  • Organize data in a table containing five distinct columns:

  1. Experiment / Chemical Treatment

  2. Chemical Concentration ($\%$

  3. Beats per $15\,\text{sec}$ (3 individual trials)

  4. Average Beats per $15\,\text{sec}$

  5. Calculated Beats per Minute (BPM)

  • Table rows must include:

  • Control — Untreated Baseline

  • Chemical Test Concentration 1

  • Chemical Test Concentration 2

  • Chemical Test Concentration 3

• Graphing Protocols:

  • Type of Plot: XY Scatter Plot.

  • Independent Variable (X-axis): Solution Concentration ($\%$).

  • Dependent Variable (Y-axis): Average Heart Rate per Minute (Beats/Min).

  • Labeling: Include descriptive axis labels with appropriate units and a clear, descriptive overall title.

  • Interpolation: If no tested concentration produced an exact $20\%$ heart rate change, use the scatter plot trendline to estimate the precise concentration required.