17.5 Required Practicals: Organising an Ecosystem

A free GCSE Biology lesson for AQA (8461) from OBA School: required practical: the effect of temperature on the rate of decay of fresh milk, measured by pH change. Watch the video, read the explanation and worked examples, then try the revision questions. You can work through the full lesson, with every video, practice quizzes and printable worksheets with mark schemes, in our free online course.

What you will learn

• Describe how to investigate the effect of temperature on the rate of decay of milk
• Identify the independent, dependent and control variables
• Carry out a risk assessment for the practical
• Calculate means and rates, spot anomalies and draw a suitable graph
• Evaluate the method and suggest improvements

Key words

• Lipase: An enzyme that breaks down lipids (fats) into fatty acids and glycerol.
• Indicator: Phenolphthalein is a pH indicator. It is pink in alkaline solutions (above about pH 8.3) and colourless at lower pH.
• Sodium carbonate: An alkali added to the milk so the indicator starts pink.
• Independent variable: The variable you change: here, temperature.
• Dependent variable: The variable you measure: here, the time for the pink colour to disappear.
• Control variable: A variable kept the same so the test is fair.

The science behind the practical

Milk contains fats (lipids). When milk decays, enzymes from microorganisms break the fats down. In this practical the enzyme lipase stands in for the decomposers:

lipid (fat) → (lipase) fatty acids + glycerol

The fatty acids lower the pH. The milk starts alkaline and pink with phenolphthalein. When enough fatty acid has been made, the pH falls below about 8.3 and the pink colour disappears. The shorter the time, the faster the rate of decay.

Method

• 1. Add 5 drops of phenolphthalein to a test tube. Add 5 cm³ of milk and 7 cm³ of sodium carbonate solution. The mixture turns pink.
• 2. Put about 5 cm³ of lipase solution in a second test tube.
• 3. Stand both tubes in a water bath at the chosen temperature (e.g. 20 °C) for 10 minutes, so they reach that temperature. Check with a thermometer.
• 4. Use a pipette to add 1 cm³ of lipase to the milk tube. Start the stopwatch and stir.
• 5. Stop the stopwatch when the pink colour disappears. Record the time. Use a white tile and a colour standard to judge the end point.
• 6. Repeat at other temperatures, e.g. 10, 20, 30, 40, 50 and 60 °C. Do each temperature three times and calculate a mean.

Option: Instead of an indicator, you can use a pH probe and data logger to record pH every 30 seconds. This gives a pH–time graph and removes the judgement of a colour change.

Variables

• Independent; In this practical Temperature of the water bath (°C)
• Dependent; In this practical Time for the pink colour to disappear (s), used to calculate the rate
• Control; In this practical Volume and type of milk; volume and concentration of lipase; volume of sodium carbonate; number of drops of indicator; same end point (same colour)

Risk assessment

• Phenolphthalein (dissolved in ethanol); Risk Irritant to eyes; flammable; Precaution Wear eye protection; keep away from flames
• Lipase solution; Risk Enzymes can cause allergic reactions; Precaution Avoid skin contact; wash spills off hands; wear eye protection
• Hot water bath; Risk Scalds at high temperatures; Precaution Take care at 50–60 °C; use tongs or a test-tube rack
• Milk / sodium carbonate; Risk Irritant / spills cause slips; Precaution Do not taste anything; wipe up spills

Processing the results

Example results (time in seconds for the pink colour to disappear):

• 10; Test 1 610; Test 2 590; Test 3 626; Mean time / s 608.7; Rate (1000 ÷ t) 1.64
• 20; Test 1 342; Test 2 355; Test 3 337; Mean time / s 344.7; Rate (1000 ÷ t) 2.90
• 30; Test 1 198; Test 2 205; Test 3 213; Mean time / s 205.3; Rate (1000 ÷ t) 4.87
• 40; Test 1 121; Test 2 126; Test 3 119; Mean time / s 122; Rate (1000 ÷ t) 8.20
• 50; Test 1 260; Test 2 247; Test 3 431*; Mean time / s 253.5; Rate (1000 ÷ t) 3.94
• 60; Test 1 –; Test 2 –; Test 3 –; Mean time / s no change; Rate (1000 ÷ t) 0

Worked example: mean and rate at 50 °C

431 s (*) is an anomaly – much larger than the other two. Leave it out.
Mean = (260 + 247) ÷ 2 = 253.5 s.
Rate = 1000 ÷ 253.5 = 3.94 s⁻¹ (3 s.f.). (Using 1000 ÷ t just makes the numbers easier to plot.)

Answer: 253.5 s, rate 3.94 s⁻¹

Conclusion: the rate increases as temperature rises up to about 40 °C, because molecules move faster and collide with the lipase active site more often. Above the optimum the rate falls, and at 60 °C the lipase is denatured – its active site changes shape, so it can no longer bind to fat molecules.

Evaluation: errors and improvements

• Judging when the pink has gone is subjective; Improvement Use a colour standard and white tile, or a pH probe and data logger
• Temperature of the tube changes during the test; Improvement Keep the tube in the water bath; use a thermostatically controlled water bath
• Reaction starts before the tube reaches temperature; Improvement Pre-warm both solutions for 10 minutes before mixing
• Large gaps between temperatures, so optimum unclear; Improvement Repeat with 2 °C steps between 35 °C and 45 °C

Exam tip: Repeating and calculating a mean improves repeatability and helps identify anomalies. It does not make each reading more accurate.

Common mistakes

• Saying the milk goes “acidic” at the end point. The pH only needs to fall below about 8.3 for phenolphthalein to go colourless.
• Mixing the lipase with the milk before both are at the right temperature.
• Including an anomalous result in the mean.
• Confusing time and rate: a longer time means a slower rate.
• Saying the enzyme is “killed” at high temperature. Enzymes are not alive; they are denatured.

Revision questions

Try these, then check your answers below each one.

Question 1. In the milk practical, what does the enzyme lipase do? (Choose from: Breaks down fats into fatty acids and glycerol, Breaks down starch into sugars, Breaks down proteins into amino acids, Makes the milk more alkaline.)

Answer: Breaks down fats into fatty acids and glycerol

Working: Lipase breaks lipids (fats) into fatty acids and glycerol.

Question 2. Why does the pink colour of the phenolphthalein disappear? (Choose from: Fatty acids are produced, so the pH falls, The milk is heated, so the dye breaks down, Sodium carbonate is used up by the lipase, Oxygen is used up by the milk.)

Answer: Fatty acids are produced, so the pH falls

Working: Fatty acids lower the pH below about 8.3, so the indicator turns colourless.

Question 3. True or false? At 70 °C the pink colour would disappear faster than at 40 °C.

Answer: False

Working: False. Lipase is denatured at high temperatures, so the colour change would be very slow or would not happen.

Related lessons

• Previous lesson: 17.4 Rates of Decomposition
• Next lesson: 18.1 The Human Population Explosion
• In this chapter: 17.1 Feeding Relationships
• In this chapter: 17.2 Materials Cycling
• In this chapter: 17.3 The Carbon Cycle

Continue in the full course

This lesson is section 17.5 (ARPBH1705) of our free GCSE Biology course for AQA. In the full course you get every video for this section, a self-marking practice quiz, a printable worksheet with a mark scheme, chapter tests, and your progress saved. Go to the full GCSE Biology course.