Testing for biological molecules

• Starch — add iodine in potassium iodide. Orange-brown → blue-black.
• Reducing sugars — add Benedict’s reagent and heat in a water bath. Blue → green → yellow → orange → brick-red precipitate. The colour is semi-quantitative: the more reducing sugar, the further along the sequence it goes.
• Non-reducing sugar (e.g. sucrose) — a negative Benedict’s test; then hydrolyse with dilute HCl and heat, neutralise with sodium hydrogencarbonate, and repeat Benedict’s. A brick-red result now means a non-reducing sugar was present.
• Protein — add Biuret reagent (sodium hydroxide then copper(II) sulfate). Blue → lilac/purple. No heating.
• Glucose specifically — a test strip using glucose oxidase: unlike Benedict’s, it does not respond to other reducing sugars.

To make a test quantitative, use a colorimeter: filter the Benedict’s solution, measure the absorbance, and read the concentration off a calibration curve made from solutions of known concentration.

Which reagent?

Tap a result, then tap the test that produced it.

Chromatography and R_f values

Chromatography separates a mixture (here, amino acids) because the components have different solubilities in the solvent and different affinities for the paper.

• Draw the origin line in pencil (ink would run) and keep it above the solvent — if the solvent touches the origin, the sample simply dissolves away.
• Apply a small, concentrated spot; let it dry and reapply, several times, to get a strong result without a large diffuse spot.
• Run in a sealed container, so the atmosphere is saturated and the solvent does not evaporate off the paper.
• Mark the solvent front immediately, and develop the colourless amino acids with ninhydrin.

Identify each amino acid by comparing its Rf with a table of known values for that solvent — Rf is only reproducible if the solvent, paper and temperature are the same.

Your turn — R_f value

The eyepiece graticule and stage micrometer

An eyepiece graticule is a scale in the eyepiece — but its divisions are arbitrary: they mean a different real length at every magnification. To turn them into micrometres you must calibrate with a stage micrometer, a slide with a scale of known length (typically 1 mm divided into 100 divisions of 10 µm).

1. Focus on the stage micrometer at the chosen objective.
2. Line up the two scales and count how many eyepiece divisions correspond to a known length on the stage micrometer.
3. Divide that known length by the number of eyepiece divisions: this gives the length of one eyepiece division.
4. Recalibrate for every objective — the eyepiece divisions do not change, but what they represent does.

Then measure the specimen in eyepiece divisions and multiply.

Your turn — measuring a cell

Calibration check

Serial dilutions and the colorimeter

A serial dilution makes a series of concentrations from one stock, each a fixed fraction of the last. For a 1 in 10 dilution, take 1 cm³ of solution and add 9 cm³ of distilled water, mix, then take 1 cm³ of that for the next tube. Each step multiplies the concentration by 0.1.

A colorimeter measures how much light of a chosen wavelength a solution absorbs.

• Select the filter of the complementary colour to the solution (a red solution absorbs most strongly in blue/green light).
• Zero the colorimeter with a blank (distilled water, or the reagent alone) — this is the calibration step.
• Plot a calibration curve of absorbance against known concentration, then read your unknown off the line.

It is used to follow a starch–amylase reaction (absorbance falls as the blue-black colour disappears) and to quantify membrane permeability in beetroot (absorbance of the leaked pigment rises as the membrane is disrupted).

Your turn — serial dilution

Measuring water potential and incipient plasmolysis

Average water potential of plant tissue (e.g. potato): cut cylinders of equal size, record the initial mass or length, leave one in each of a range of sucrose concentrations, and re-measure. Plot percentage change in mass against concentration. Where the line crosses zero change, there was no net osmosis — so the water potential of the tissue equals the water potential of that solution.

Average solute potential at incipient plasmolysis: place strips of epidermis in a range of sucrose concentrations and count the proportion of cells plasmolysed. At incipient plasmolysis, 50 % of cells are just beginning to plasmolyse; the protoplast is no longer pushing on the wall, so ψ_p = 0 and therefore ψ = ψ_s — the cell’s solute potential equals that of the external solution.

The root tip squash

1. Cut the tip (about 5 mm) of a growing root — this is the meristem, where mitosis is happening.
2. Warm in hydrochloric acid to hydrolyse the middle lamella, so the cells separate and can be squashed into a single layer.
3. Stain with acetic orcein (or toluidine blue), which binds to the chromosomes and makes them visible.
4. Squash gently under a coverslip so light passes through a single layer of cells.

Your turn — mitotic index

Accuracy, precision, error and reliability

• Accuracy — how close a measurement is to the true value.
• Precision — how closely repeated measurements agree with each other. You can be precisely wrong: a badly calibrated balance gives precise but inaccurate results.
• Reliability — comes from repeats, and from calculating a mean (discarding anomalies first).
• Validity — comes from controlling the other variables, so that the effect you measure really is caused by your independent variable.

This is why you should use as large a measurement as is practical: measuring 25.0 cm³ with a ±0.5 cm³ cylinder gives a 2 % error, but measuring 5.0 cm³ with the same cylinder gives a 10 % error. A random error is reduced by repeating; a systematic error (e.g. a balance that reads 0.2 g high) is not — you must calibrate the instrument.

Your turn — percentage error

Match the term to its meaning

Tap a meaning on the left, then the correct term.

Designing it properly

The big ideas to know

You have covered the AS practical skills. Press Finish to see your score.