Required Practicals

AQA A-Level Biology includes 12 required practicals that you must carry out during your course. Although these are not assessed through coursework, questions about them appear frequently on all three exam papers. You must understand the method, variables, expected results, potential sources of error, and how to improve each practical.

Key Principle: Questions on required practicals can appear on ANY paper — not just the paper that covers the relevant topic. Paper 3 is synoptic and can include questions linking required practicals to content from any part of the specification.

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Required Practical 1: Effect of a Named Variable on the Rate of an Enzyme-Controlled Reaction

Specification link: Topic 3.1 (Biological molecules — Enzymes)

Aim

To investigate the effect of a named variable (e.g., temperature, pH, substrate concentration, or enzyme concentration) on the rate of an enzyme-controlled reaction.

Method — Example: Effect of Temperature on Catalase Activity

1. Set up a water bath at the required temperature (e.g., 20 °C)
2. Add 5 cm³ of hydrogen peroxide solution to a boiling tube and place it in the water bath for 5 minutes to equilibrate
3. Add 1 cm³ of catalase (potato extract) to a separate boiling tube and place in the same water bath for 5 minutes
4. Add the catalase to the hydrogen peroxide and immediately connect to a gas syringe or inverted measuring cylinder over water
5. Record the volume of oxygen gas collected every 30 seconds for 5 minutes
6. Repeat at different temperatures (e.g., 30, 40, 50, 60, 70 °C)
7. Repeat each temperature at least 3 times and calculate a mean

Variables

Variable	Details
Independent	Temperature (or pH / substrate concentration / enzyme concentration)
Dependent	Rate of reaction (measured as volume of O₂ produced per unit time)
Control	Volume and concentration of substrate, volume and concentration of enzyme, same enzyme source, equilibration time

Expected Results

• Rate increases with temperature up to the optimum (typically around 37–40 °C for mammalian enzymes)
• Above the optimum, rate decreases sharply as the enzyme denatures (hydrogen bonds and ionic bonds maintaining tertiary structure are broken)
• At very high temperatures, rate falls to zero

Sources of Error

• Temperature fluctuations in the water bath
• Oxygen escaping before the gas syringe is connected
• Difficulty in determining the exact moment the reaction starts
• Catalase activity may vary between different potato samples

Exam-Style Considerations

• Calculate initial rate from a tangent to the curve at time = 0
• Use Q₁₀ to compare rates at different temperatures
• Explain the difference between denaturation and inhibition

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Required Practical 2: Preparation of Stained Squashes to Observe Mitosis

Specification link: Topic 3.2 (Cells)

Aim

To observe the stages of mitosis in actively dividing cells using a root tip squash.

Method

1. Cut approximately 1 cm from the tip of a growing root (e.g., garlic or onion)
2. Place the root tip in a watch glass with 1 mol/dm³ hydrochloric acid and warm gently at approximately 60 °C for 5 minutes (this dissolves the middle lamella, separating cells)
3. Transfer the root tip to a clean slide and add a few drops of acetic orcein (or toluidine blue) stain
4. Place a coverslip over the root tip and squash firmly by pressing down with the blunt end of a pencil (place filter paper over the coverslip to absorb excess stain and prevent the coverslip from sliding)
5. Observe under a light microscope, starting at low power and increasing to high power
6. Identify cells in interphase, prophase, metaphase, anaphase, and telophase
7. Count the number of cells in each stage and calculate a mitotic index

Mitotic Index

Mitotic index = number of cells undergoing mitosis ÷ total number of cells observed

A high mitotic index suggests a rapidly dividing tissue — this is expected in root tips, bone marrow, and cancerous tumours.

Variables

Variable	Details
Independent	Stage of mitosis (categorical)
Dependent	Number of cells observed in each stage
Control	Same species, same stain, same acid treatment time, same magnification

Expected Results

• Most cells will be in interphase (the cell spends most of its time here)
• Prophase is typically the most commonly observed mitotic stage
• Anaphase is the shortest stage and fewest cells are usually seen here

Sources of Error

• Cells may overlap, making identification difficult
• Staining may be uneven
• Squashing too hard can distort cells; too gently leaves cells in layers
• Subjective identification of stages

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Required Practical 3: Production of a Dilution Series of Glucose to Calibrate a Colorimeter

Specification link: Topic 3.1 (Biological molecules)

Aim

To use serial dilutions and a colorimeter to produce a calibration curve for glucose concentration.

Method

1. Prepare a stock solution of glucose (e.g., 1.0 mol/dm³)
2. Produce a serial dilution series (e.g., 1.0, 0.8, 0.6, 0.4, 0.2, 0.0 mol/dm³) by diluting with distilled water
3. Add equal volumes of Benedict's reagent to each solution
4. Heat all tubes in a water bath at 80 °C for 5 minutes
5. Allow tubes to cool and filter or centrifuge to remove the precipitate
6. Set the colorimeter to the appropriate wavelength (blue/green filter, ~500 nm) and zero it using a blank (water + Benedict's reagent, heated)
7. Measure the absorbance (or % transmission) of each solution
8. Plot a calibration curve — glucose concentration (x-axis) vs absorbance (y-axis)
9. Use the calibration curve to determine the glucose concentration of unknown samples

Variables

Variable	Details
Independent	Glucose concentration
Dependent	Absorbance (or % transmission)
Control	Volume of Benedict's reagent, heating time, temperature, wavelength of light in colorimeter

Expected Results

• Higher glucose concentration → more Cu₂O precipitate formed → more light absorbed → higher absorbance
• A linear (or near-linear) relationship is expected within the working range

Sources of Error

• Incomplete reaction if heating time is insufficient
• Precipitate may not settle evenly, affecting colorimeter readings
• Parallax error when measuring volumes
• Air bubbles in the cuvette

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Required Practical 4: Effect of a Named Variable on the Permeability of Cell-Surface Membranes

Specification link: Topic 3.2 (Cells)

Aim

To investigate the effect of a named variable (e.g., temperature, alcohol concentration, or solvent) on the permeability of cell-surface membranes using beetroot.

Method — Example: Effect of Temperature on Membrane Permeability

1. Cut beetroot into cylinders of equal size using a cork borer
2. Rinse the cylinders thoroughly in distilled water to remove pigment released during cutting
3. Place each cylinder into a separate test tube containing 5 cm³ of distilled water
4. Place each test tube in a water bath at a different temperature (e.g., 20, 30, 40, 50, 60, 70, 80 °C) for 30 minutes
5. Remove the beetroot cylinders
6. Use a colorimeter (with a green/blue filter) to measure the absorbance of the surrounding solution — a higher absorbance indicates more betacyanin (red pigment) has leaked out
7. Repeat each temperature at least 3 times and calculate a mean

Variables

Variable	Details
Independent	Temperature
Dependent	Absorbance of the surrounding solution (indicates pigment leakage)
Control	Size of beetroot cylinders, volume of distilled water, time in water bath, same beetroot source

Expected Results

• At lower temperatures (20–40 °C): low absorbance — membrane is intact
• At higher temperatures (50–70 °C): absorbance increases sharply — membrane proteins denature and phospholipid bilayer becomes more fluid and disordered, increasing permeability
• Above 70 °C: maximum leakage — membrane structure is completely disrupted

Sources of Error

• Beetroot cylinders may not be identical in size
• Pigment on the outside of cylinders from cutting (must rinse thoroughly)
• Temperature fluctuations in the water bath
• Time delay between removing beetroot and reading colorimeter

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Required Practical 5: Dissection of Animal or Plant Gas Exchange or Mass Transport System

Specification link: Topic 3.3 (Organisms exchange substances)

Aim

To dissect an organ or system to examine gas exchange or mass transport structures.

Examples

• Fish head — to examine gill structure (lamellae and gill filaments)
• Insect (locust) — to examine tracheal system
• Leaf — to examine stomata, spongy mesophyll, palisade mesophyll
• Heart — to examine chambers, valves, and associated vessels

Method — Example: Dissection of a Fish Head

1. Place the fish head on a dissecting board
2. Use scissors to cut away the operculum (gill cover)
3. Identify the gill arches, gill filaments, and gill rakers
4. Carefully remove one gill arch and place it in water on a white tile
5. Use a hand lens or dissecting microscope to observe the gill filaments and lamellae
6. Note the large surface area created by the many lamellae
7. Prepare a simple drawing with labels

Key Points for Exam Answers

• Gills have a large surface area (many lamellae), thin epithelium (short diffusion distance), and a rich blood supply (maintaining the concentration gradient)
• Counter-current flow maintains a concentration gradient along the entire length of the lamella — this is more efficient than parallel flow
• In insects, the tracheal system delivers air directly to cells via spiracles → tracheae → tracheoles
• In leaves, gas exchange occurs through stomata; guard cells control their opening and closing

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Required Practical 6: Use of Aseptic Technique to Investigate the Effect of Antimicrobials

Specification link: Topic 3.2 (Cells — immune system context) and Topic 3.4 (Genetic diversity)

Aim

To use aseptic technique to investigate the effect of antimicrobial substances (e.g., antibiotics, disinfectants, or plant extracts) on bacterial growth.

Method

1. Sterilise all equipment — autoclave agar plates, sterilise the inoculating loop by passing through a Bunsen flame until red-hot, and allow to cool
2. Inoculate a nutrient agar plate with a bacterial culture using an aseptic technique:
   • Lift the lid of the Petri dish at an angle (do not remove completely)
   • Zigzag the inoculating loop across the agar surface
   • Replace the lid promptly
3. Place antibiotic discs (multidiscs or individual discs) on the inoculated agar using sterile forceps
4. Secure the lid with two strips of tape (do NOT seal completely — this prevents anaerobic conditions that could encourage the growth of harmful pathogens)
5. Incubate upside down at 25 °C for 24–48 hours (25 °C in school labs to prevent growth of human pathogens; clinical labs use 37 °C)
6. Measure the diameter of the clear zone (zone of inhibition) around each disc
7. Calculate the area of the zone of inhibition using A = πr²

Variables

Variable	Details
Independent	Type of antimicrobial (or concentration)
Dependent	Area of the zone of inhibition
Control	Same bacterial species, same volume of bacterial culture, same incubation temperature and time, same agar type

Sources of Error

• Contamination from poor aseptic technique
• Uneven spreading of bacteria
• Discs not pressed firmly onto agar (may not make proper contact)
• Zones may not be perfectly circular — measure at widest and narrowest and take mean

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Required Practical 7: Use of Chromatography to Investigate Pigments from Leaves