How do you do the decay required practical?

What does the decay practical investigate?

It tests how temperature affects the rate of decay. When milk goes off, microorganisms break it down and make acids, so its pH falls. In the classroom version, the enzyme lipase does a similar job: it breaks down the fat in milk into fatty acids and glycerol, and the fatty acids lower the pH. An indicator shows the change, and you time it at different temperatures.

This practical is only in separate GCSE Biology. If you take Combined Science (AQA Trilogy), you do not need it. Some schools skip the lipase and leave milk at different temperatures for a few days instead, measuring its pH with a probe. The idea is the same.

What equipment do you need?

The chemistry is simple. Lipase breaks down the fat in milk that has been made alkaline, and an indicator changes colour as the pH falls. The rest of the kit keeps each test at a steady temperature.

• Full-fat milk
• Lipase solution
• Sodium carbonate solution, to make the milk alkaline at the start
• Phenolphthalein indicator
• Test tubes, a rack and labels
• Water baths at a range of temperatures, or beakers of water with a kettle and ice
• A thermometer
• Measuring cylinders or syringes, and a glass rod for stirring
• A stopclock
• A white tile, to see the colour change clearly
• Eye protection

What is the method?

Warm a tube of pink, alkaline milk and a tube of lipase to the same temperature, mix them, and time how long the pink takes to disappear. Then repeat at other temperatures.

• Set a water bath to the first temperature you want to test, for example 20 °C.
• Measure some lipase solution into a test tube labelled "lipase", and stand it in the water bath.
• In a second tube, mix a set volume of full-fat milk with a set volume of sodium carbonate solution and a few drops of phenolphthalein. It should turn pink.
• Stand the milk tube in the water bath too. Wait until both tubes reach the temperature of the water, checking with a thermometer.
• Add a set volume of the warmed lipase to the milk, stir, and start the stopclock.
• Stop the clock as soon as the pink colour has gone, and record the time.
• Repeat at other temperatures, for example 30, 40, 50 and 60 °C, and repeat each one so you can calculate a mean.

What are the variables?

Temperature is what you change, and the time for the colour change is what you measure. The amounts of each liquid must stay the same, because more lipase or less fat would change the time on their own.

How do you work out and present the results?

Find the mean time at each temperature, then work out a rate. A shorter time means faster decay, so rate = 1 ÷ time, with the unit s⁻¹ (per second). Using 1000 ÷ time gives numbers that are easier to plot.

Worked example: at 20 °C, the pink disappears after 400 seconds, so the rate is 1000 ÷ 400 = 2.5. At 30 °C it takes 200 seconds, so the rate is 1000 ÷ 200 = 5. In this example, warming the milk by 10 °C doubled the rate.

Plot rate on the y-axis against temperature on the x-axis. The rate should rise with temperature up to an optimum, then fall at higher temperatures as the lipase is denatured. The same idea explains why milk lasts longer in the fridge: the cold slows down the enzymes of the microorganisms that make it go off.

How do you make it accurate and safe?

The weak spot is deciding exactly when the pink has gone, because the colour fades gradually. Hold the tube against a white tile, compare it with a tube of the same milk and sodium carbonate with no indicator, and have the same person judge every end point. A pH probe gives a number instead of a judgement, which makes it a strong improvement to suggest. Let both tubes reach the water bath temperature before you mix them, or the reaction starts at a different temperature.

Wear eye protection, because sodium carbonate solution can irritate your eyes. Phenolphthalein is usually dissolved in ethanol, which is flammable, so keep it away from flames. Some people react to enzymes such as lipase, so avoid getting it on your skin. Never taste the milk.

What do exam questions ask about it?

Many questions test the chemistry behind the colour change: why sodium carbonate is added (to make the mixture alkaline, so the indicator starts pink) and why the pH falls (lipase makes fatty acids). You may need to explain the shape of the graph, including why the rate drops at high temperatures, or suggest an improvement such as a pH probe. An anomaly might come from a tube that was mixed before it had warmed up. The calculation is a rate from a time, and the 6-mark version asks for the method.

An exam-style question we wrote: "At 35 °C, the indicator lost its pink colour after 125 seconds. Calculate the rate of reaction using rate = 1000 ÷ time." 1000 ÷ 125 = 8.

Common mistakes

• Saying the lipase is "killed" at high temperatures. It is denatured: its active site changes shape.
• Mixing the tubes before they reach the water bath temperature.
• Explaining the fall in pH without the fatty acids. Lipase breaks fat into fatty acids and glycerol, and the fatty acids lower the pH.
• Reading the longest time as the fastest decay. A longer time means a slower rate.