Practical Investigations

Food tests (qualitative)

Food type	Reagent	Positive result
Starch	Iodine solution	Orange to blue-black
Reducing sugar	Benedict's solution + heat	Blue to brick-red precipitate
Protein	Biuret reagent	Blue to purple/lilac
Fat	Brown paper / Sudan III	Translucent spot / red-stained droplets

In every food test the control is the same test carried out on distilled water. Distilled water must give a negative result, which shows any colour change came from the food sample and not from the reagent itself.

Method points examiners look for: crush or dissolve the food in water first so the reagent can reach it; add the same volume of reagent to test and control; heat the Benedict's test in a water bath rather than over a flame; and describe the change as a colour from and to (for example "blue to brick-red"), not just the final colour.

Habitat study

A named habitat is studied by random sampling. Quadrats are thrown using random number coordinates so the sample is unbiased, and a pitfall trap, beating tray or sweep net is used for animals. Percentage frequency and percentage cover are calculated, organisms are identified with a key, and abiotic factors (pH, light, temperature) are measured.

Why it is done this way: sampling is used because counting every organism is impossible; randomness removes personal bias; and several quadrats are used because a single quadrat may land on an unusual patch. Frequency tells you how widespread a species is, while cover tells you how much space it occupies, so the two together describe the community far better than either alone.

Microscopy

To prepare a slide of onion epidermis: peel a thin layer with forceps, lay it flat on a slide in a drop of water, stain with iodine, and lower a cover slip at an angle with a mounted needle to avoid air bubbles. View on the lowest magnification first, then move up.

For cheek cells: scrape gently inside the cheek with a sterile swab, smear onto a slide, add a drop of dilute methylene blue, add a cover slip, and view on low power. Always record the magnification with your labelled drawing.

Common exam points: the section must be thin so light can pass through it; stain increases contrast so structures such as the nucleus and cell wall stand out; air bubbles are round with thick dark edges and are easily mistaken for cells; and total magnification = eyepiece lens x objective lens.

Factors affecting enzyme activity

Celery (a source of catalase) is added to hydrogen peroxide and the height of foam produced is measured. Changing temperature while keeping pH and concentration fixed shows an optimum near 30°C: most foam at the optimum, far less above or below it because the enzyme works slowly when cold and is denatured when too hot. Repeating the procedure with buffers at different pH values gives an optimum pH in the same way.

Explaining the shape of the graph: below the optimum the molecules move slowly, so there are fewer successful collisions between the enzyme and hydrogen peroxide. Above the optimum the bonds holding the enzyme's shape break, the active site no longer fits the substrate, and the change is permanent - which is why the curve falls away much more steeply on the hot side than it rose on the cold side.

Fair test controls: same mass of celery, same volume and concentration of hydrogen peroxide, same pH buffer, same time allowed, and readings repeated three times and averaged.

Heat denaturation of catalase

Boiled celery is compared with unheated celery at the same temperature (25°C) and pH. Result: the unheated enzyme produces foam, the heated enzyme produces none, because heat has permanently changed the shape of the active site.

Enzyme immobilisation

Yeast is mixed with sodium alginate and dropped from a syringe into calcium chloride solution, forming beads that trap the cells. Free yeast and immobilised yeast are each placed in a separating funnel with sucrose. Glucose test strips show glucose in both funnels, but the liquid from the immobilised funnel is clear rather than cloudy, which is why industry prefers immobilised enzymes: the product is not contaminated and the enzyme can be reused.

The beads work because calcium ions cross-link the alginate into a jelly that traps the yeast but still lets small molecules such as sucrose and glucose diffuse in and out. The enzyme inside is also more stable at higher temperatures and across a wider pH range than free enzyme.

Osmosis using visking tubing

One length of visking tubing is filled with sucrose solution and a second (the control) with distilled water. Both are dried, weighed and suspended in beakers of distilled water. After 20 minutes the sucrose tube has gained mass and become turgid because water moved in by osmosis; the control is unchanged.

Visking tubing models a selectively permeable membrane: water molecules pass through the tiny pores but the much larger sucrose molecules cannot. Water therefore moves from the beaker, where water concentration is higher, into the tubing, where it is lower. Drying the tubing before weighing matters, because surface droplets would add mass and give a false result.

Rate of photosynthesis and light intensity

Elodea is placed cut-end upwards in pondwater in a water bath. The lamp is set at a measured distance, the plant is left five minutes to adjust, and the bubbles of oxygen released per minute are counted three times and averaged. Moving the lamp further away lowers light intensity and the bubble rate falls. The water bath keeps temperature constant.

Light intensity falls off sharply with distance, so doubling the distance reduces intensity by far more than half. At very high intensity the graph levels off because another factor, usually carbon dioxide supply or temperature, has become the limiting factor. Sodium hydrogencarbonate is often added to the water so carbon dioxide does not run short.

Alcohol production by yeast

Two flasks of 10% glucose solution are set up; one receives 5 g of yeast, the other is the control with no yeast. Fermentation locks let carbon dioxide out but keep air (and contaminants) out. After incubating overnight at 30°C the filtrates are tested with potassium iodide and sodium hypochlorite and warmed gently: a yellow precipitate of iodoform forms in the yeast flask only.

Air is excluded so the yeast respires anaerobically, converting glucose into ethanol and carbon dioxide. The control flask shows the alcohol came from the yeast and not from the glucose solution or the apparatus.

DNA isolation from onion

Chopped onion is heated in salty washing-up liquid at 60°C for 15 minutes (detergent breaks down membranes, salt makes DNA clump, heat stops enzymes that would digest DNA), cooled quickly on ice, blended for three seconds only so the DNA is not sheared, then filtered. Protease is added to remove protein and ice-cold ethanol is trickled down the side of the tube. White stringy DNA appears at the interface and is spooled out with a glass rod.

Each step has one clear job, and exam questions almost always ask for the reason rather than the step: detergent dissolves the lipid membranes, salt neutralises the negative charge on DNA so the strands stick together, heating and then chilling stops DNase enzymes, brief blending breaks the tissue apart without chopping the long DNA molecules, and cold ethanol makes the DNA come out of solution because DNA is insoluble in alcohol.

Growth of leaf yeast

Ash leaves are attached with Vaseline to the lid of a malt agar plate, one plate using untreated leaves and one using fungicide-treated leaves. Hands, bench, forceps and loop are sterilised, the dishes are taped, labelled, and incubated at 25°C for three days, turned upside down only after the spores have fallen so condensation does not drip onto the agar. Result: pink colonies in the outline of the leaf on the untreated plate, no growth on the treated plate. Plates are soaked in disinfectant for 24 hours before disposal.

Aseptic technique is the part most often examined: sterilise equipment and work surface, flame the loop, keep the lid open for as short a time as possible, seal and label the plate, and never fully open a plate once colonies have grown. The fungicide plate is the control - it shows the colonies came from living yeast carried on the leaf surface, not from contamination of the agar.

Transverse section of a dicot stem

Thin free-hand sections are cut with a razor, floated on water, stained, and mounted. Under the microscope the vascular bundles are arranged in a ring, with xylem on the inside, phloem on the outside and cambium between them.

The section must be very thin so light passes through; floating it on water keeps it flat and unfolded. Working outwards to inwards you should be able to name the epidermis, the cortex, the ring of vascular bundles, and the central pith. The ring arrangement is the quick way to tell a dicot stem from a monocot stem, where the bundles are scattered.

Heart dissection

Place the heart ventral side up. The left side feels firmer than the right. Identify the four chambers and the main vessels, then cut shallowly into the left atrium and ventricle to see the bicuspid valve, and into the right side to see the tricuspid valve with three flaps. The septum separates left from right, and the left ventricle wall is much thicker because it pumps blood to the whole body. Cutting the aorta reveals the half-moon semi-lunar valve and the openings of the coronary arteries just above it. Sterilise all instruments afterwards.

How to tell the sides apart: the left ventricle wall is thick and firm, the right is thin and squashy; the aorta has the thickest wall of the vessels and leads from the left ventricle; the pulmonary artery leads from the right ventricle towards the lungs. Structure links to function throughout - valves prevent backflow, the septum keeps oxygenated and deoxygenated blood separate, and the coronary arteries supply the heart muscle itself.

Effect of exercise on pulse rate

Sit quietly for five minutes, then count pulses per minute three times and average to get the resting rate. Exercise for five minutes, immediately re-count, and compare on a bar chart. The pulse rate rises because muscles need more oxygen and glucose and more carbon dioxide must be removed.

Continue taking readings every minute afterwards to record the recovery time, the time taken to return to the resting rate. A fitter person has a lower resting pulse and a shorter recovery time. Use the same person, the same type and length of exercise, and the same counting method each time.

Effect of IAA on plant tissue

A serial dilution of IAA is prepared: 1 cm³ is moved from bottle to bottle, each already holding 9 cm³ of distilled water, so each is ten times weaker than the last. Five radish seeds are placed on a grid in each matching dish with damp filter paper, the dishes are stood on edge so roots grow down, and incubated at 25°C for 2-3 days. Root and shoot lengths are measured and compared to the water control:

% stimulation or inhibition = (average length - average length of control) ÷ average length of control × 100

Low concentrations of IAA stimulate root growth, while the same concentrations that stimulate shoots will inhibit roots.

Five seeds per dish are used so an average can be taken and one unusual seed does not distort the result. The distilled water dish is the control that the percentages are measured against, and a positive answer means stimulation while a negative answer means inhibition.

Digestive activity during germination

Broad beans are soaked for two days; half are boiled for five minutes to kill them (the control). All seeds are sterilised in disinfectant, rinsed, split, and placed cut face down on starch agar plates, then incubated at 18-20°C for 48 hours. Plates are flooded with iodine for two minutes and drained. Result: a clear halo around the raw seeds where amylase digested the starch, and a uniform blue-black colour around the boiled seeds. This shows germinating seeds release enzymes to break down their food store into glucose for the embryo.

Conditions needed for germination

Cress seeds on cotton wool are set up in four tubes, changing one factor at a time. Only the tube with water, oxygen and a suitable temperature germinates.

Water is needed to activate enzymes and soften the seed coat, oxygen for aerobic respiration to release energy, and a suitable temperature so the enzymes work at a useful rate. Light is not required for germination itself. Each tube differs from the control in one factor only, which is what makes it a fair test.