Design an experiment to confirm that the decolorization of methylene blue in skim milk is catalyzed by a heat-labile enzyme (dehydrogenase).

Here is the hypothesis and procedure for an experiment to confirm the findings of the reductase (dehydrogenase) test:

a. Hypothesis:
The decolorization of methylene blue in skim milk is catalyzed by a heat-labile enzyme (dehydrogenase), meaning its activity will be significantly reduced or abolished at high temperatures.

b. Procedure:

Aim: To confirm that the decolorization of methylene blue in skim milk is due to the activity of a heat-labile enzyme (dehydrogenase).

Materials:

• Fresh skim milk (non-sterilized)
• Methylene blue solution (0.005%)
• Three clean test tubes
• Water bath set at 37°C
• Boiling water bath (100°C)
• Ice bath (0°C)
• Stopwatch
• Measuring cylinder or pipette (for milk)
• Dropper (for methylene blue)

Method:

1. Preparation:

   • Label three clean test tubes as A, B, and C.
   • Add 5 ml of fresh skim milk to each test tube.
   • Add 5 drops of methylene blue solution to each test tube.
   • Gently mix the contents of each test tube.

2. Experimental Setup:

   • Test Tube A (Boiled Control): Place test tube A in the boiling water bath (100°C) for 5 minutes to denature any enzymes present. After boiling, cool the test tube to 37°C (e.g., by placing it in the 37°C water bath).
   • Test Tube B (Optimal Temperature): Place test tube B directly into the water bath set at 37°C.
   • Test Tube C (Low Temperature): Place test tube C into the ice bath (0°C).

3. Observation:

   • Once all test tubes have reached their target temperatures, start the stopwatch simultaneously for all three.
   • Observe each test tube and record the time taken for the methylene blue to completely decolorize (turn colorless).
   • If no decolorization occurs within 60 minutes, record "no decolorization" or "decolorization time > 60 minutes".

4. Data Collection:

   • Record the treatment/temperature and the corresponding decolorization time for each test tube in a table.

Expected Results and Confirmation:

• Test Tube A (Boiled): The methylene blue is expected to remain blue or decolorize very slowly, indicating that the enzyme was denatured by boiling.
• Test Tube B (37°C): The methylene blue is expected to decolorize relatively quickly, as 37°C is often an optimal temperature for many biological