Design an experiment to test the effect of substrate concentration on enzyme activity using yeast to break down methylene blue.

a) Propose a hypothesis for your experiment:
If the substrate concentration (stale milk) increases, then the rate of enzyme activity (decolorization of methylene blue) will increase until the enzyme active sites become saturated.

b) Procedure:

1. Prepare a series of different substrate concentrations by diluting the stock stale milk with distilled water (e.g., 100%, 75%, 50%, 25%, 0% stale milk).
2. Label five test tubes from 1 to 5.
3. Add $5\,\text{mL}$ of yeast suspension to each test tube using a syringe.
4. Add $1\,\text{mL}$ of methylene blue dye to each test tube.
5. Add $5\,\text{mL}$ of each prepared stale milk concentration to its respective test tube (e.g., $5\,\text{mL}$ of 100% stale milk to tube 1, $5\,\text{mL}$ of 75% stale milk to tube 2, and so on, with $5\,\text{mL}$ of distilled water to tube 5 as a control for 0% stale milk).
6. Shake each mixture gently to ensure even distribution and obtain a uniform blue color.
7. Place all test tubes in a water bath maintained at a constant temperature (e.g., $37\,^{\circ}\text{C}$).
8. Immediately start a stopwatch for each test tube after adding the substrate and mixing.
9. Record the time taken for the methylene blue in each test tube to completely decolorize (turn colorless).
10. Repeat the experiment at least three times for each concentration to ensure reliability and calculate the average decolorization time.

c) Observation and results:

• The time taken for the methylene blue to decolorize will vary depending on the concentration of stale milk.
• As the concentration of stale milk (substrate) increases, the time required for the methylene blue to decolorize will generally decrease, indicating an increase in enzyme activity.
• At very high concentrations of stale milk, the decrease in decolorization time will become less significant, or the time will plateau, suggesting that the enzyme's active sites are saturated with the substrate.
• The control tube (0% stale milk) is expected to show no or very slow decolorization, as there is no substrate for the enzyme to act upon.