Biology 9700/33 — October/November 2010

Background Concept

A serial dilution is a stepwise dilution of a stock solution in which each new concentration is a fixed fraction of the previous one. It is the standard practical way of producing a range of known concentrations quickly and accurately, because each step uses the same volumes and arithmetic. A dilution by half gives concentrations ½, ¼, ⅛ … of the original; a dilution by ten gives 1/10, 1/100, 1/1000 …; equal-interval dilutions give constant steps such as 0.3, 0.2, 0.1, 0.0.

For this investigation the stock copper sulfate solution provided is 0.3%. The candidate has to decide on a sensible range of concentrations to test, including 0% (distilled water) as a control, and show exactly how each 10 cm³ of test solution will be made up from the stock and from distilled water.

Understanding the Question

The question asks the candidate to design the concentration range for the independent variable and to present it as a table showing the three pieces of information requested: the final concentration, the volume of CuSO₄ solution used, and the volume of distilled water used. The mark scheme requires at least four (volumes/concentrations), the highest concentration must lie between 0.15 and 0.3 (i.e. the stock may be used directly or diluted to 0.15), and three consecutive concentrations must have intervals that are equal, halved or ten-fold. The lowest should normally be 0% (a water control).

Approach

The simplest way to satisfy all three marking points is a serial dilution by half starting from the 0.3% stock. This gives five concentrations, includes 0.3% as the highest, and the consecutive intervals (0.15 → 0.075 → 0.0375) are exactly halved. Volumes are calculated from $C_1V_1 = C_2V_2$: for a 10 cm³ volume of 0.15% from a 0.3% stock, $V_1 = (0.15 \times 10) / 0.3 = 5.0$ cm³. The same logic gives 5.0 cm³ of each preceding dilution for the next step.

Step-by-Step Reasoning

1. Identify the stock concentration and target final volume: 0.3% stock, 10 cm³ of each test concentration.
2. Use the highest concentration directly: 10.0 cm³ of 0.3% stock + 0.0 cm³ water gives 10 cm³ of 0.30%.
3. For 0.15%: take 5.0 cm³ of the 0.3% stock and add 5.0 cm³ of water.
4. For 0.075%: take 5.0 cm³ of the 0.15% solution (just made) and add 5.0 cm³ of water.
5. For 0.0375%: take 5.0 cm³ of the 0.075% solution and add 5.0 cm³ of water.
6. For 0.00% (control): 0.0 cm³ stock + 10.0 cm³ water.
7. Tabulate concentrations down the first column with the matching volumes of stock and water in the second and third columns.

Key Takeaways

• A serial dilution by half from the 0.3% stock is the cleanest design for this practical.
• A 0% (water) control is essential; it shows what happens to the tissue in the absence of CuSO₄.
• Always label the source of each diluted solution so it is clear where each volume is taken from.

Common Mistakes

• Forgetting to include 0% (a water control).
• Using unequal intervals (e.g. 0.30, 0.20, 0.10, 0.05) which fail the mark-scheme condition that the intervals be the same, halved or ten-fold.
• Volumes that do not add to 10 cm³ in total (e.g. listing 5 cm³ stock + 4 cm³ water).
• Reading the mark scheme's "0.3 to 0.15" as requiring the highest to be diluted; the stock may be used directly at 0.3%.

Things to Be Careful About

• The volumes in the body of the table are the volumes pipetted for that particular concentration, not the cumulative amount. The 0.15% row is 5 cm³ of 0.3% stock + 5 cm³ water; the 0.075% row is 5 cm³ of the freshly made 0.15% + 5 cm³ water, and so on.
• Mix each solution thoroughly before taking the next aliquot, otherwise the concentrations will be inaccurate.
• Use a separate, clean pipette (or wash and rinse the same one) between concentrations to avoid carry-over of stock.

Background Concept

In any fair test, only the independent variable is allowed to change between samples. Every other variable that could affect the dependent variable must be held constant (standardised) so that any difference in the results can be attributed to the independent variable. These standardised variables are sometimes called controlled variables.

For this experiment the independent variable is the concentration of CuSO₄, and the dependent variable is the colour of the solution (or, equivalently, the colour of the tissue) after a set time. Anything else that could change how much dye leaves the cells must be kept the same across all five test-tubes.

Understanding the Question

The question asks for a single variable that must be controlled when preparing the plant-tissue samples. The mark scheme accepts length, size, surface area, dimensions, volume (and also methylene blue as the staining variable).

Approach

Think about what is being done differently to each piece of tissue: each piece is placed in a different concentration of CuSO₄. Everything else about the pieces themselves should therefore be identical. The most obvious property is the size of the piece: a larger piece has more cells, more dye, and a larger surface area for CuSO₄ to act on, which would confound the effect of concentration.

Step-by-Step Reasoning

1. The independent variable is CuSO₄ concentration — this changes between tubes.
2. The dependent variable is the colour observed after 5 minutes — this will be measured.
3. Anything else that could change the colour must be controlled. A bigger piece of tissue contains more methylene blue and exposes more membrane to the CuSO₄, so it would look different even at the same concentration.
4. Therefore the size (length, surface area, volume) of each piece must be the same.

Key Takeaways

• A controlled variable is anything that could affect the result and is not the independent or dependent variable.
• In this practical the size of the tissue piece is the most important controlled variable because the dye it contains is what is being observed.

Common Mistakes

• Stating the independent variable (concentration of CuSO₄) by mistake.
• Stating the dependent variable (colour of solution) by mistake.
• Giving a vague answer such as "amount of plant tissue" without specifying what is being held constant about it (length, size, surface area, volume).

Things to Be Careful About

• The mark scheme allows either length, size, surface area, dimensions, or volume — any one of these is sufficient.
• "Methylene blue" is also accepted because the amount/concentration of stain is another variable that could affect how much colour is released.

Background Concept

Once a controlled variable has been identified, the practical description must explain how it will actually be kept constant. For tissue samples, the size is usually standardised by cutting to a measured length with a ruler and scalpel, or by using a cork borer to give the same cross-sectional area and length. Skin contact must be avoided when the tissue is stained because methylene blue is a strong dye and is harmful; the standard technique is to use blunt forceps, and to rinse off surface dye so that colour released into the solution later comes from inside the cells and not from dye smeared on the outside.

Understanding the Question

The question follows on from (ii) and asks for two things: (a) how the controlled variable identified in (ii) is actually controlled, and (b) how the samples of plant tissue are prepared ready to be put into the test-tubes. The mark scheme awards one mark for the control (measure, cut, rinse, "the same", e.g. a length of $3\ \text{cm}$ or less) and a second mark for the preparation (use of scalpel/knife or ruler; rinsing; or using methylene blue-stained water).

Approach

Describe the cutting and rinsing procedure in a logical order: measure → cut → handle → rinse. The two marks are best earned by giving a specific length (or use of cork borer) for the control and a separate point about handling or rinsing for the preparation.

Step-by-Step Reasoning

1. Measure: lay a ruler next to the tissue and mark the same length on each piece (e.g. $3.0\ \text{cm}$).
2. Cut: use a scalpel or sharp knife to cut each piece to that length; for cylindrical tissue a cork borer gives identical cross-sectional area and length simultaneously.
3. Handle: use blunt forceps at all times to pick up and transfer the tissue; this protects the experimenter from methylene blue and prevents crushing the cells.
4. Rinse: dip each piece briefly in distilled water (or rinse under a wash bottle) to remove any methylene blue clinging to the outer surface, so the colour that later appears in the solution comes from dye released by damaged membranes rather than from dye that was never inside the cells.

Key Takeaways

• "Same length, cut with ruler and scalpel" is the simplest way to standardise the size of tissue samples.
• Forceps are used both for safety (methylene blue) and to avoid damaging the tissue.
• Rinsing removes surface dye so the results reflect dye leaving the cells, not dye washed off the outside.

Common Mistakes

• Picking up the tissue with fingers (stains skin, also crushes cells and may alter results).
• Stating "cut to the same size" without saying how this is done (ruler, scalpel, cork borer, micrometer).
• Forgetting to rinse, which leaves surface dye that will colour every test-tube regardless of CuSO₄ concentration.

Things to be Careful About

• The mark scheme specifically looks for "ruler" or "scalpel/knife" and an example length (e.g. $3\ \text{cm}$ or less) — give both.
• "Cork borer" is also accepted and is a good way to guarantee identical cross-sectional area.
• The pieces should not be so long that they do not fit fully submerged in the $10\ \text{cm}^3$ of solution in the test-tube.

Background Concept

Qualitative observations are recorded in a table rather than as free text, so that the pattern across the independent variable can be read at a glance. Cambridge Paper 3 has firm conventions for any observation table:

• All cells must be ruled.
• The independent variable is the first column (or top row) and must include a clear heading with units in the heading only — never in the body of the table.
• The dependent variable(s) is the next column (or row), again with a clear heading.
• A key is supplied if shorthand symbols are used.
• Extra columns for method, time, length, etc. are not credited and should be omitted.

Understanding the Question

The question requires a table that records what the candidate observes in each of the test-tubes over the 5 minutes before mixing and again after the tube is inverted. The mark scheme awards one mark for a table with all cells drawn and a percentage-concentration heading; one mark for a colour/observation heading; one mark for separate observations before and after mixing; one mark for showing a difference in colour between the lowest and highest concentration; and one mark for recording five or more concentrations (or for including the water control and any replicate).

Approach

Plan the columns first: (1) concentration of CuSO₄, (2) observation before mixing, (3) observation after mixing. List the five concentrations down column 1. Fill columns 2 and 3 with the colour that is actually seen in each tube, and write a brief key if shorthand is used. The colour should be more intense (more blue) at higher CuSO₄ concentrations because more dye leaks out when the membrane is more damaged.

Step-by-Step Reasoning

1. Draw a ruled table with three columns.
2. Head the first column "Percentage concentration of CuSO₄" (no unit in the body; the % is part of the heading).
3. Head the second column "Colour of solution before mixing" and the third "Colour of solution after mixing".
4. Enter the five concentrations: 0.00, 0.0375, 0.075, 0.15, 0.30.
5. Record the colour observed in each tube before mixing and again after mixing. Higher CuSO₄ damages the membrane more, so more methylene blue leaks out and the solution turns a deeper blue.
6. Add a key if shorthand such as "+, ++, +++" is used; otherwise write descriptive words in the cells.
7. Check that the trend across the table is clear: the colour intensifies from colourless (0.00%) to dark blue (0.30%).

Key Takeaways

• Always put the independent variable first and the dependent variable(s) next; units belong in the heading only.
• For "observe for 5 minutes and then mix" questions, the table must show observations before AND after mixing.
• A water (0%) control is a row in its own right and is what makes the trend interpretable.

Common Mistakes

• Putting % or other units in the body of the table (e.g. writing 0.30% instead of just 0.30).
• Adding extra columns for "method", "time", "length of tissue" — the mark scheme rejects these.
• Forgetting the "after mixing" column.
• Not showing a difference between the 0% and 0.30% tubes, or reversing the trend (writing the wrong colours).
• Omitting the water control or a replicate.

Things to be Careful About

• The exact colour description will depend on the candidate's actual observation; the table above shows the expected pattern (deeper blue at higher CuSO₄) but the candidate's wording may differ.
• The heading for the first column should be "Percentage concentration of CuSO₄" (or close equivalent) and the body should be plain numbers.
• A key is only required if shorthand symbols are used; descriptive words do not need a key.

Background Concept

The cell-surface membrane is a phospholipid bilayer with embedded proteins (the fluid-mosaic model). One of its key properties is selective permeability: it allows some molecules through (e.g. water, small non-polar molecules) but prevents the loss of larger or charged molecules. Many heavy-metal ions, including Cu²⁺, disrupt this structure: they denature membrane proteins and/or interfere with the phospholipid packing, so the membrane becomes leaky. A "leaky" membrane no longer retains the contents of the cytoplasm and vacuole, and any dye that was held inside the cells is free to diffuse out down its concentration gradient.

Understanding the Question

The question asks the candidate to suggest, in the context of what they have just observed, how CuSO₄ affects the cell membrane. The mark scheme accepts any of: damage/destruction of the membrane, damage to phospholipids or to membrane proteins, increased permeability, decreased selective permeability, or denaturation of membrane proteins.

Approach

State the effect (damage / increased permeability) and link it to the observation (dye leaks out). A single concise sentence that contains both ideas scores the mark.

Step-by-Step Reasoning

1. The plant tissue was stained with methylene blue and then placed in CuSO₄ solution.
2. In water (0% CuSO₄) the tissue stays blue and the solution stays colourless — the membrane is intact and retains the dye.
3. In higher CuSO₄ concentrations the solution turns progressively more blue — dye is leaving the cells.
4. The only plausible explanation is that CuSO₄ is damaging the cell-surface membrane so it can no longer retain the dye; the membrane has become more permeable (or has lost its selective permeability).

Key Takeaways

• A "leaky" membrane lets the dye out; a "selectively permeable" membrane keeps it in.
• Heavy-metal ions such as Cu²⁺ disrupt the phospholipid bilayer and/or denature membrane proteins.
• The conclusion must be phrased in terms of membrane structure or permeability, not in vague terms such as "kills the cells".

Common Mistakes

• "CuSO₄ kills the cells" — too vague; the question asks specifically about the membrane.
• "CuSO₄ removes the dye" — does not explain the mechanism.
• "CuSO₄ destroys the cell wall" — irrelevant; the cell wall is fully permeable and does not retain the dye.

Things to be Careful About

• The mark scheme accepts any of: damages/destroys the membrane, damages phospholipids, damages/denatures proteins, makes the membrane more permeable, or decreases selective permeability.
• The link to dye leakage is the biological evidence that supports the conclusion, but the mark is for the membrane statement, not for the leakage statement.

Background Concept

A "source of error" in a practical is anything that could cause the measured result to differ from the true value, and that is not already covered by the independent variable. Sources of error can be in the way a measurement is taken (e.g. judging a colour by eye), in the way a sample is prepared (e.g. not exactly the same size), or in the way a procedure is carried out (e.g. not all samples starting at the same time). They must be specific to this investigation and not apply equally to every test-tube (in which case they cannot explain any difference between the tubes).

Understanding the Question

The question asks for three significant sources of error in this investigation. The mark scheme lists several accepted answers, with a maximum of three marks, and explicitly rejects generic errors such as temperature, pH, evaporation, or "human error", and any error that would affect every test-tube equally.

Approach

Walk through the procedure step by step and ask, at each step, "what could vary that would change the result but is not the independent variable?" Cluster the answers into three clear points. Avoid vague phrases such as "human error" or "not accurate" — every mark requires a specific cause.

Step-by-Step Reasoning

1. Observation step — the colour of the solution is judged by eye. Eyes are not equally sensitive to small differences in intensity, and there is no reference standard; this is a genuine source of error. The mark scheme credits "colour/colour change/observations — difficult judging / seeing".
2. Preparation step — the pieces of plant tissue are cut to a similar length, but a small difference in length, cross-sectional area, or surface area is hard to avoid. Larger pieces contain more dye and expose more membrane, so the colour released differs even at the same CuSO₄ concentration. The mark scheme credits "lengths / size / surface areas / volumes — not same".
3. Procedure step — the pieces are added to the five test-tubes one after the other, so the first piece has been in CuSO₄ for slightly longer than the last by the time the clock is started (and certainly by the 5-minute observation). The mark scheme credits "potato / samples (into test-tubes) — time not same / delayed time / not at same time".
4. Other credible sources include: the staining/washing step may not have been identical for every piece, so some pieces contain more dye than others; the tissue may not have been fully submerged; mixing may have made the colours look more uniform than they really were.

Key Takeaways

• A valid source of error must be specific to this procedure and must vary between samples (so it could explain differences in result).
• Generic answers ("human error", "not accurate", temperature) do not score and are explicitly rejected.
• Always link the error to the part of the result it could affect.

Common Mistakes

• "Human error" / "not accurate" / "not precise" — too vague; the mark scheme rejects these.
• "Temperature" / "pH" / "evaporation" — these would affect all tubes equally and are explicitly rejected.
• "Wrong concentration of CuSO₄" — this is a mis-calculation, not a source of error in the practical itself.
• Naming only one error and elaborating; the question requires three distinct points.

Things to be Careful About

• The mark scheme allocates separate credit for the cause and the effect — make sure both are present (e.g. "colour is qualitative — difficult to judge").
• The error must be one that would change the comparison between tubes; an error that affects every tube equally is not credited.

Background Concept

A good improvement addresses a named source of error directly and makes the procedure more accurate, more precise, or more reliable. The mark scheme rewards improvements that are specific and achievable with standard laboratory equipment: a colorimeter converts a qualitative colour into a numerical absorbance; a cork borer or vernier callipers guarantees identical sample dimensions; a graduated pipette or burette improves volume accuracy; staggered starts or several stop-clocks fix the timing problem; repeats and a mean improve reliability.

Understanding the Question

The question asks for three improvements. The mark scheme lists many credited improvements with a maximum of three marks. The strongest answers are those that link directly to the three errors identified in (vi).

Approach

Take each of the three errors from (vi) and propose a specific, practical fix. Each improvement must be a single concrete change (with the equipment named), not a vague aspiration such as "be more careful".

Step-by-Step Reasoning

1. Error: qualitative colour judgement → improvement: use a colorimeter (or datalogger with light sensor). The colorimeter measures the absorbance of the solution at a chosen wavelength (e.g. $660\ \text{nm}$ for methylene blue); this gives a numerical value that does not depend on the observer. The mark scheme credits "colorimeter or datalogger with light sensor" and explicitly rejects "calorimeter".
2. Error: tissue pieces not exactly the same size → improvement: use a cork borer / vernier callipers / micrometer so that every piece has the same diameter and length. A ruler with millimetre divisions is also accepted if the candidate already has one. The mark scheme credits "use micrometer / cork borer / vernier callipers / ruler with smaller divisions".
3. Error: time delay when adding samples → improvement: stagger the start or use several stop-clocks / a helper so that all five pieces enter their tubes at effectively the same time. Alternatively, add each piece to its tube individually with a stop-clock so that the 5 minutes is timed precisely per piece. The mark scheme credits "stagger start or do individually or use more stop clocks or use help".
4. Other useful improvements: leave the tissue in methylene blue for longer to give more even staining; use a wider (or narrower) range of concentrations; use a burette or graduated pipette for more accurate volumes; repeat the whole experiment and take a mean.

Key Takeaways

• A good improvement is a single concrete change that uses named equipment.
• Improvements should be matched to the errors in (vi) so the two answers form a coherent critique of the method.
• A colorimeter is the standard way to make a colour-based practical quantitative.

Common Mistakes

• Vague improvements such as "be more careful" or "do it again" — not credited.
• "Use a calorimeter" — a calorimeter measures heat, not colour; the mark scheme rejects this.
• Improvements that do not address a specific error (e.g. "use a better microscope" when the practical does not use a microscope).
• Repeating an error as an improvement (e.g. "cut the pieces to the same length" when this was already done in (iii) and was not the error).

Things to be Careful About

• The improvement must be specific: name the equipment or the action.
• Each improvement earns one mark, so three well-targeted improvements are enough — there is no need to list more.
• "Repeat" or "replicate" is a perfectly valid third improvement if the other two have been used, but it should be phrased as "repeat the experiment and take a mean" rather than simply "do it again".