describe and carry out investigations using redox indicators, including DCPIP and methylene blue, to determine the effects of temperature and substrate concentration on the rate of respiration of yeast

Respiration – Investigating the Effects of Temperature and Substrate Concentration

Learning Objective

Describe and carry out investigations using redox indicators (DCPIP and methylene blue) to determine how temperature and substrate concentration affect the rate of respiration in yeast.

Background Theory

Cellular respiration is a series of redox reactions in which organic substrates are oxidised to produce energy (ATP) and carbon dioxide. The overall equation for aerobic respiration of glucose is:

\$C6H{12}O6 + 6\,O2 \rightarrow 6\,CO2 + 6\,H2O + \text{energy}\$

Yeast ( Saccharomyces cerevisiae ) is a convenient model organism because it ferments sugars rapidly and produces measurable amounts of CO₂.

Redox Indicators

• DCPIP (2,6‑dichlorophenol‑indophenol) – blue when oxidised, colourless when reduced. It accepts electrons from the electron transport chain, allowing visual monitoring of respiration rate.
• Methylene blue – blue in the oxidised form, colourless when reduced to leucomethylene blue (LMB). It is reduced by NADH produced during glycolysis, providing a second, complementary assay.

Principles of the Assays

Both indicators are reduced by the electron flow generated during respiration. The rate at which the blue colour disappears is proportional to the rate of electron transfer, and therefore to the rate of respiration.

DCPIP Reduction

\$\text{DCPIP}{(ox)} + 2e^- + 2H^+ \rightarrow \text{DCPIP}{(red)}\$

Methylene Blue Reduction

\$\text{MB}{(ox)} + 2e^- + 2H^+ \rightarrow \text{LMB}{(red)}\$

Experimental Design

Two separate series of experiments are performed, one using DCPIP and the other using methylene blue. Each series investigates:

1. The effect of temperature (e.g., 15 °C, 25 °C, 35 °C, 45 °C).
2. The effect of substrate (glucose) concentration (e.g., 0 %, 2 %, 4 %, 6 % w/v).

Variables

Materials

• Active dry yeast (S. cerevisiae)
• Glucose solution (stock 10 % w/v)
• DCPIP solution (0.2 mM)
• Methylene blue solution (0.1 mM)
• Distilled water
• Water baths set at required temperatures
• Spectrophotometer (or colourimeter) – wavelength 600 nm for DCPIP, 660 nm for methylene blue
• Cuvettes, pipettes, test tubes, timer

Procedure (example for DCPIP)

1. Label four test tubes for each temperature point.
2. Add 5 mL distilled water to each tube.
3. Add glucose to achieve the desired concentration (0 %, 2 %, 4 %, 6 %).
4. Add 0.5 g wet yeast to each tube and mix gently.
5. Add 1 mL of 0.2 mM DCPIP solution.
6. Place the tubes in the pre‑set water bath for 2 min to equilibrate.
7. Immediately record the initial absorbance (A₀) at 600 nm.
8. Take absorbance readings every 30 s for 5 min (A₁, A₂ … A₁₀).
9. Plot ΔA = A₀ – Aₙ against time; the slope gives the rate of DCPIP reduction.

Data Recording Template

Data Analysis

For each set of conditions calculate the initial rate of colour loss (slope of the linear portion of the ΔA vs. time graph). Convert absorbance change to moles of indicator reduced using Beer‑Lambert law:

\$\Delta c = \frac{\Delta A}{\varepsilon \, l}\$

where \$\varepsilon\$ is the molar extinction coefficient (DCPIP \$\approx 21\,000\ \text{L mol}^{-1}\text{cm}^{-1}\$ at 600 nm) and \$l\$ is the cuvette path length (usually 1 cm).

Plot the initial rates against temperature (Arrhenius plot) and against glucose concentration (Michaelis‑Menten style). Discuss:

• Optimal temperature for yeast respiration and why rates fall at extreme temperatures (enzyme denaturation).
• Effect of substrate saturation – identify \$K_m\$‑like behaviour.
• Comparison of results obtained with DCPIP and methylene blue (different points in the electron transport chain).

Safety and Waste Disposal

• Wear lab coat, gloves and safety glasses.
• DCPIP is a mild irritant; avoid skin contact.
• Methylene blue can stain skin and clothing.
• Dispose of yeast suspensions in the sink with plenty of water; discard indicator solutions according to school chemical waste guidelines.

Possible Extensions

• Investigate the effect of pH on respiration rate using the same indicators.
• Replace glucose with other substrates (e.g., sucrose, maltose) to explore substrate specificity.
• Use a sealed fermentation tube to directly measure CO₂ volume as a complementary method.