Biology 9700/34 — May/June 2015

Background Concept

A serial dilution is a step-wise dilution in which each new solution is prepared from the previous one. The concentration is reduced by a constant factor at every step. A common factor is 10 (a tenfold serial dilution), in which a small volume of the previous solution is added to a larger volume of diluent to make the next concentration.

The general dilution equation is

where $c_1$ and $V_1$ are the concentration and volume of the stock, and $c_2$ and $V_2$ are the concentration and total volume of the diluted solution. To dilute by 10 the ratio of stock to total must be $1:10$ — for example, $1\ \text{cm}^3$ of stock + $9\ \text{cm}^3$ of diluent = $10\ \text{cm}^3$ of a one-tenth-strength solution.

In this experiment the diluent is $\text{W}$ (distilled water), the total volume per beaker must be $10\ \text{cm}^3$, and $9\ \text{cm}^3$ of each concentration is needed for the investigation. The remaining $1\ \text{cm}^3$ is used to start the next dilution.

Understanding the Question

You are given a partially completed Fig. 1.1 with four beakers. Beaker 1 is already complete: $10\ \text{cm}^3$ of $0.3\%$ $\text{X}$ + $0\ \text{cm}^3$ $\text{W}$, with $9\ \text{cm}^3$ available to use. A curved arrow shows $1\ \text{cm}^3$ of $0.3\%$ $\text{X}$ being transferred to Beaker 2. You must complete the figure by:

• writing the concentration and volume available to use under Beakers 2, 3 and 4;
• adding arrows above Beakers 2, 3 and 4 showing the volume of $\text{X}$ transferred from the previous beaker and the volume of $\text{W}$ added.

Approach

Apply the tenfold dilution rule at each step: $1\ \text{cm}^3$ of previous + $9\ \text{cm}^3$ of $\text{W}$ = $10\ \text{cm}^3$ of a one-tenth solution. Work from Beaker 1 outwards, halving the digits in the concentration each time.

Step-by-Step Reasoning

1. Beaker 1 (given): $10\ \text{cm}^3$ of $0.3\%$ $\text{X}$ + $0\ \text{cm}^3$ $\text{W}$ → $0.3\%$ solution, $9\ \text{cm}^3$ to use.
2. Beaker 2: $1\ \text{cm}^3$ from Beaker 1 + $9\ \text{cm}^3$ $\text{W}$ → $0.03\%$ solution, $9\ \text{cm}^3$ to use.
3. Beaker 3: $1\ \text{cm}^3$ from Beaker 2 + $9\ \text{cm}^3$ $\text{W}$ → $0.003\%$ solution, $9\ \text{cm}^3$ to use.
4. Beaker 4: $1\ \text{cm}^3$ from Beaker 3 + $9\ \text{cm}^3$ $\text{W}$ → $0.0003\%$ solution, $9\ \text{cm}^3$ to use.

The general rule at every step (except the first) is $1\ \text{cm}^3$ of the previous concentration + $9\ \text{cm}^3$ of $\text{W}$ = $10\ \text{cm}^3$ of a solution one-tenth as strong.

Key Takeaways

• A tenfold serial dilution always uses $1$ part stock + $9$ parts diluent to give $10$ parts of a solution one-tenth as concentrated.
• The same volume of diluent ($9\ \text{cm}^3$ of $\text{W}$) is added at every step after the first.
• The volume available to use is always $9\ \text{cm}^3$ — the remaining $1\ \text{cm}^3$ is left behind to start the next dilution.
• Concentrations must be written with a leading zero (e.g. $0.03\%$, not $.03\%$).

Common Mistakes

• Forgetting the leading zero in very small concentrations.
• Adding $\text{X}$ to all four beakers, or $\text{W}$ to all four beakers — only the second, third and fourth beakers receive both $\text{X}$ and $\text{W}$.
• Drawing the transfer arrow from the wrong beaker (it must come from the previous, more concentrated, beaker).
• Labelling only the percentage without including the $\%$ sign, or omitting the volume available to use.

Things to Be Careful About

• Concentrations must be in the correct sequence: $0.3\% \rightarrow 0.03\% \rightarrow 0.003\% \rightarrow 0.0003\%$. Each is exactly one-tenth of the previous.
• The total volume in every beaker is $10\ \text{cm}^3$; the $9\ \text{cm}^3$ available to use is what remains after the $1\ \text{cm}^3$ is removed for the next dilution.
• The same syringe must be used for $\text{X}$ throughout (the question states "syringe labelled X should be used for solution X only") so that the $1\ \text{cm}^3$ transfer is accurate.

Background Concept

A results table is the standard way to record experimental data. A well-drawn table should have:

• A clear heading for every column, including the quantity and its unit.
• The independent variable (the one you deliberately change) in the first column.
• The dependent variable (the one you measure) in the next column.
• Replicate readings wherever possible, to give a measure of repeatability.
• Values written to a consistent number of decimal places, and a sensible range of the independent variable including a control.

The experiment tests how different concentrations of solution $\text{X}$ affect the activity of catalase. Catalase hydrolyses hydrogen peroxide to oxygen and water; the rate of reaction is estimated by counting the bubbles of $\text{O}_2$ released in 2 minutes. If $\text{X}$ is an inhibitor, more concentrated $\text{X}$ should give fewer bubbles.

Understanding the Question

The procedure runs five test-tubes: one with $\text{P} + \text{W}$ (the 0% control) and one each for the four concentrations of $\text{X}$ made in (b)(i). You must draw a results table and record the number of bubbles counted in 2 minutes for each tube. At least one concentration must be repeated.

Approach

Draw a two-column table. The first column is the independent variable (concentration of $\text{X}$, including the W control). The second column is the dependent variable (number of bubbles in 2 minutes). Add a row for one repeat. Record your actual counts; the marks are for the table structure and trend, not for any particular number.

Step-by-Step Reasoning

1. The independent variable has five values: $0\%$ (W), $0.0003\%$, $0.003\%$, $0.03\%$ and $0.3\%$.
2. The dependent variable is the number of bubbles counted in 2 minutes — a count has no units, but the time interval must be stated in the heading.
3. A repeat must be performed for at least one concentration (the procedure tells you to "Consider how you will obtain results which are as accurate as possible"). Repeating the highest concentration ($0.3\%$) is a good choice because it is the most likely to be erratic.
4. Expected trend: because $\text{X}$ inhibits catalase (see (b)(iv)), increasing the concentration of $\text{X}$ should decrease the number of bubbles. The $0\%$ (W) control should give the most bubbles, and the $0.3\%$ tube should give the fewest.
5. Representative counts that match this trend are given in the answer table above. The student's own numbers will differ but the trend should be the same.

Key Takeaways

• A results table must have a heading with units for every column, and must include a control (here, 0% $\text{X}$).
• Repeats are essential for assessing repeatability and identifying anomalous readings.
• The trend in the data is the biological signal — here, bubble count falls as [X] rises, indicating inhibition.

Common Mistakes

• Forgetting the units in the column heading (e.g. writing "concentration" without "%" or "bubbles" without "in 2 minutes").
• Omitting the W (0%) control row.
• Recording the same value for every concentration (a sign that the bubbles were not actually counted, or that a step was missed).
• Showing a reverse trend (more bubbles at higher [X]) — this contradicts the biology in (b)(iv) and is unlikely to be correct.
• Forgetting to include a repeat.

Things to Be Careful About

• Use whole numbers for bubble counts (you cannot count a fraction of a bubble).
• Keep the same number of decimal places down a column.
• Record the actual reading, not what you "expected" to see — examiners can spot invented data when it does not match the trend.

Background Concept

The rate of an enzyme-catalysed reaction can be measured in several ways. Here the volume of oxygen released is measured, so the rate has units of volume per unit time — for example, $\text{cm}^3\ \text{s}^{-1}$ or $\text{cm}^3\ \text{min}^{-1}$. The simplest way to find a rate is to measure how long it takes to collect a fixed volume of gas, and then divide that volume by the time:

In this experiment the volume is fixed at $2\ \text{cm}^3$ (read off the syringe of Fig. 1.2 as the water is displaced downwards), and the time is the one the student measures with a stop-clock.

Understanding the Question

You are asked to (a) record the time for $2\ \text{cm}^3$ of oxygen to be collected, and (b) use that time to calculate the rate of activity of catalase in $\text{cm}^3\ \text{s}^{-1}$. The question warns that you may lose marks if you do not show your working or use appropriate units.

Approach

Time the displacement carefully with a stop-clock. Read the time to the nearest whole second. Then substitute into $\text{rate} = V / t$ with $V = 2\ \text{cm}^3$, and quote the answer with units and a sensible number of significant figures.

Step-by-Step Reasoning

1. Start the stop-clock as soon as the end of the delivery tube is placed under the syringe (step 18 of the procedure).
2. Watch the meniscus of water in the syringe and stop the clock the moment $2\ \text{cm}^3$ of water has been displaced.
3. Record the time in whole seconds (e.g. 20 s).
4. Calculate the rate using the formula above. With $t = 20\ \text{s}$: $$\text{rate} = \frac{2\ \text{cm}^3}{20\ \text{s}} = 0.1\ \text{cm}^3\ \text{s}^{-1}$$
5. Quote the answer to the same number of significant figures as the time (1 sig fig if $t$ is given to the nearest 10 s, 2 sig figs if $t$ is given to the nearest second).
6. Always include the unit $\text{cm}^3\ \text{s}^{-1}$ — a bare number with no unit scores 0.

The student's actual time will depend on the activity of their catalase extract; the calculation method is what earns the marks, not the particular value.

Key Takeaways

• Rate of gas production = volume of gas ÷ time, with units of volume per unit time.
• Always show your working and quote the answer with its unit.
• Match the number of significant figures in the rate to the precision of the time measurement.

Common Mistakes

• Writing the time as a decimal (e.g. 20.5 s) — the procedure uses a stop-clock reading in whole seconds.
• Inverting the calculation (writing $t / 2$ instead of $2 / t$).
• Omitting the unit $\text{cm}^3\ \text{s}^{-1}$, or writing it as "$\text{cm}^3 / \text{s}$" without the exponent form (the negative-exponent form $\text{cm}^3\ \text{s}^{-1}$ is the SI convention).
• Quoting too many significant figures (e.g. $0.13333$ from $t = 15\ \text{s}$) — round to match the precision of the time.

Things to Be Careful About

• The question marks showing working as part of the mark scheme — write the equation, substitute the values, and then the final answer.
• The volume in the formula is $2\ \text{cm}^3$ (the fixed volume in the procedure), not the total volume of hydrogen peroxide added.

Background Concept

Enzyme inhibition is any process that reduces the rate of an enzyme-catalysed reaction. There are two broad classes:

• Competitive inhibition — the inhibitor is similar in shape to the substrate and binds to the active site, blocking substrate entry. Competitive inhibition can be overcome by increasing the substrate concentration.
• Non-competitive inhibition — the inhibitor binds to a site other than the active site (an allosteric site), changing the shape of the active site so the substrate can no longer bind effectively. Non-competitive inhibition cannot be overcome by adding more substrate.

In both cases the result is the same: fewer enzyme–substrate (E–S) complexes form per unit time, and the rate of reaction falls.

The data from (b)(ii) show that as the concentration of $\text{X}$ increases, the number of oxygen bubbles released in 2 minutes decreases. This is the classic signature of an inhibitor.

Understanding the Question

You are asked to use your knowledge of enzymes to suggest how $\text{X}$ might be changing the activity of catalase. The mark scheme rewards two ideas: that $\text{X}$ inhibits activity, and that it does so by interfering with substrate binding.

Approach

Link the observed trend (fewer bubbles at higher [X]) to a mechanism. The most direct mechanism is that molecules of $\text{X}$ occupy the active site of catalase, so hydrogen peroxide cannot bind and fewer E–S complexes form.

Step-by-Step Reasoning

1. The trend in (b)(ii) — bubble count falls as [X] rises — shows that $\text{X}$ is reducing the activity of catalase. This is the mark-scheme point "inhibits activity".
2. The most common way a small molecule reduces enzyme activity is to bind to the active site in place of the substrate. The substrate (hydrogen peroxide) is then blocked from binding.
3. With fewer successful binding events, fewer enzyme–substrate complexes form per unit time, so less oxygen is produced and fewer bubbles are released.
4. Either wording — "prevents substrate binding to the active site" or "fewer enzyme–substrate complexes are formed" — earns the second mark.

Key Takeaways

• A fall in reaction rate as the concentration of an added substance rises is the signature of an inhibitor.
• Inhibitors typically act by blocking substrate binding at the active site (competitive) or by changing the shape of the active site (non-competitive).
• The result of either mechanism is a reduction in the number of enzyme–substrate complexes formed per unit time.

Common Mistakes

• Stating that $\text{X}$ "stops the reaction" or "destroys the enzyme" — at the concentrations used, $\text{X}$ reduces the rate, it does not abolish the reaction completely.
• Saying $\text{X}$ "uses up the hydrogen peroxide" — $\text{X}$ is a different substance and does not react with the substrate in this way.
• Confusing competitive and non-competitive inhibition without committing to one — either mechanism is acceptable here, but the answer must commit to blocking substrate binding to earn the second mark.

Things to Be Careful About

• The question uses the command word "suggest", so any biologically plausible mechanism is acceptable — but it must be specific to enzymes (e.g. active site, E–S complexes).
• Do not describe the effect of $\text{X}$ on the substrate (hydrogen peroxide) — describe its effect on the enzyme (catalase).

Background Concept

Every measuring technique has limitations. A good practical scientist can name the specific limitations of each method, not just say "human error" or "not accurate". For an experiment measuring gas production, the limitations are usually about:

• the physical nature of the gas bubbles (size, rate of release, tendency to coalesce);
• the transfer of gas from the reaction vessel to the measuring device (leaks from joints, bubbles missing the collection vessel);
• the reading of the measuring device (parallax, end-point judgement).

Understanding the Question

You must identify one significant source of error for each of the two methods used to measure the dependent variable: (1) counting bubbles in 2 minutes, and (2) measuring the volume of oxygen by water displacement in a syringe.

Approach

For each method, think about the chain of events between the reaction happening in the test-tube and the reading being taken. Where could information be lost or distorted? That is your source of error.

Step-by-Step Reasoning

Counting bubbles (method 1)

• The bubbles are produced by an enzyme-catalysed reaction, so they vary in size as the rate changes during the 2 minutes.
• At high reaction rates the bubbles come out so fast that they merge (group together) into a stream, making it hard to count individual bubbles.
• The eye cannot reliably count more than about 5–6 events per second; any rate above this under-counts the true number.
• Credit: different sizes, too fast, or bubbles group together.

Water displacement (method 2)

• The delivery tube is the only route for oxygen to reach the syringe. If the bung is not perfectly tight, gas leaks out around the joint instead of going up the tube.
• Even with a tight bung, some bubbles miss the syringe and float up through the beaker of water to the surface.
• When the syringe fills, the meniscus of water must be read against a scale; if the eye is not level with the meniscus there is a parallax error.
• The water in the beaker may be slightly above room temperature; oxygen is slightly soluble in water, so a small fraction dissolves rather than displacing water.
• Credit: gas escapes from the delivery tube, not all bubbles enter the syringe, or parallax error.

Key Takeaways

• Sources of error must be specific to the apparatus and method, not generic ("human error").
• For gas-collection experiments, the most common errors are: bubble size / coalescence, leakage at joints, bubbles missing the collection vessel, and parallax when reading a scale.

Common Mistakes

• Vague answers such as "human error", "not accurate enough", "the bubbles were hard to count" — these do not name the specific limitation.
• Stating the same error for both methods.
• Describing how to improve the method instead of identifying the error.
• Naming an error that is not really significant (e.g. "the syringe wasn't perfectly clean").

Things to Be Careful About

• "Parallax error" is a specific mark-scheme point for the syringe reading, not for bubble counting — make sure each error is matched to the right method.
• One mark is awarded per method, so a single clear statement is enough for each.

Background Concept

A valid fair test requires that only one variable — the independent variable — is changed at a time. Every other variable that could affect the result must be standardised (kept the same in every test). In a temperature investigation, the concentration of $\text{X}$ becomes a control variable, and temperature becomes the new independent variable.

For the temperature itself to be a valid independent variable, it must be:

• Varied across a sensible range that is likely to reveal the effect (too narrow a range misses the optimum; too wide a range is wasteful and may denature the enzyme permanently at high temperatures).
• Measured accurately at each value.
• Held constant during the reaction at each value — a thermostatically-controlled water-bath is the standard piece of apparatus for this.

The dependent variable (rate of catalase activity) is still measured by bubble counting or water displacement, exactly as in (b)(ii)–(iii).

Understanding the Question

You are asked two things:

1. How to standardise the original independent variable (concentration of $\text{X}$) so that it is no longer the variable being changed.
2. How to investigate a new independent variable, temperature, by changing it in a controlled way and measuring its effect on catalase activity.

The mark scheme gives 1 mark for the first part and 2 marks for the second (5 or more temperatures; thermostatically-controlled water-bath).

Approach

• For standardising $\text{X}$: pick one concentration and use it in every test.
• For the temperature investigation: decide on a sensible range of at least 5 temperatures, and use a thermostatically-controlled water-bath to hold each temperature precisely.

Step-by-Step Reasoning

1. Standardising $\text{X}$: the simplest way is to use the same concentration (e.g. the $0.3\%$ solution) in every test-tube. This is exactly what is meant by "control of variables" in CIE marking — keep the variable the same in every test.
2. Range of temperatures: at least 5 evenly spaced values. A typical range for catalase is $0\ ^{\circ}\text{C}$ to $80\ ^{\circ}\text{C}$ in $20\ ^{\circ}\text{C}$ steps, giving 5 temperatures. Anything from 5 to 7 values is fine.
3. Temperature control: a thermostatically-controlled water-bath is required because a beaker of water on a tripod loses heat rapidly and the temperature drifts. The water-bath holds the temperature to within $\pm 1\ ^{\circ}\text{C}$, which is essential for a valid comparison between temperatures.
4. The reaction mixture (catalase extract + $\text{X}$ + hydrogen peroxide) is placed in the water-bath and left to equilibrate to the set temperature before timing is started; otherwise the reading will not be at the intended temperature.

Key Takeaways

• Standardising a variable means keeping it the same in every test — for $\text{X}$, this means using one fixed concentration.
• Investigating a new variable requires (a) a sensible range of at least 5 values, and (b) precise control apparatus (thermostatically-controlled water-bath for temperature).
• Always equilibrate the reaction mixture to the target temperature before starting the reaction, otherwise the temperature during the reaction is not the temperature you set.

Common Mistakes

• "Vary the concentration of $\text{X}$ at each temperature" — this would change two variables at once and make the results impossible to interpret.
• Using only 2 or 3 temperatures — the mark scheme requires at least 5 to reveal a trend (especially to see the optimum and the denaturation drop-off).
• Saying "use a Bunsen burner to heat the water" — a Bunsen burner cannot hold a temperature constant, and an open flame is a hazard near hydrogen peroxide.
• Forgetting to mention equilibration — adding the reactants and then putting the tube in the water-bath means the reaction begins at room temperature, not at the target temperature.

Things to Be Careful About

• The mark scheme credits "thermostatically-controlled water-bath" as a single specific piece of apparatus — a beaker of water on a hot plate, or a Bunsen burner, is not accepted.
• A sensible range of temperatures is implied: values must lie within a range where catalase is active (roughly $0\ ^{\circ}\text{C}$ to $80\ ^{\circ}\text{C}$). Values outside this range are not useful.
• The dependent variable (rate of catalase activity) is still measured by bubble counting or water displacement; do not propose a different measurement method.