1.2.2(f)-(g), 1.2.2(i) - Qualitative testing, separation and microbiology
In this lesson you are learning three laboratory technique families that OCR expects you to use safely and explain precisely: qualitative tests for biological molecules, separation methods, and aseptic microbiology. The biology is not just the name of a test; it is the link between a method step, a reliable observation, and a justified conclusion. The focus is practical skill and risk management, so each technique is taught as evidence-gathering rather than as a list of instructions to memorise.
Techniques As Evidence
A practical technique is useful only if it turns a biological question into evidence. In this lesson the evidence is usually a visible change: a colour change, a separated pattern of compounds, or microbial growth that can be observed without opening the container.
Qualitative test
A qualitative test shows whether a substance or condition is present. It usually gives a visible positive or negative result rather than an exact numerical concentration.
For a qualitative result to be worth trusting, the method needs controls. A positive control contains the substance you expect to detect, so it checks that the reagent and method can work. A negative control lacks the substance you are testing for, so it checks that the method is not giving a false positive.
For example, if you use Benedict's reagent to test an unknown food extract for reducing sugar, a glucose solution would be a positive control and distilled water would be a negative control. If the glucose tube stays blue, the test has failed. If the water tube changes colour, something has contaminated the test or the reagent.
Risk management is part of the technique, not an optional extra. Heating in a water bath, wearing eye protection, working near a Bunsen burner for aseptic work, handling solvents carefully, and observing cultures without opening them all protect the student and improve the validity of the evidence.
When a question asks why a practical step is used, link the step to a result quality or safety reason. "To make it fair" is usually too vague; "to reduce contamination", "to prevent solvent touching the sample spot", or "to reduce spitting when heated" is much stronger.
Qualitative Reagent Tests
Qualitative reagents identify biological molecules by producing characteristic visible results. The sample should usually be ground or finely broken up, mixed with water, and tested as a solution or fine suspension. This gives the reagent better contact with the biological molecules.
The core tests you should be able to carry out and interpret are:
| Molecule tested for | Reagent or method | Key method point | Positive result |
|---|---|---|---|
| Reducing sugar | Benedict's reagent | Heat with the sample in a water bath | Blue changes to green, yellow, orange or brick-red precipitate |
| Non-reducing sugar | Acid hydrolysis, neutralisation, then Benedict's reagent | First test should be negative; hydrolyse, neutralise, then heat with Benedict's reagent | Benedict's reagent gives a coloured precipitate after hydrolysis |
| Starch | Iodine in potassium iodide solution | Add to solution, suspension, or directly to a solid sample | Orange-brown changes to blue-black |
| Protein | Biuret test | Add alkali and copper sulfate; allow time for colour to develop | Blue changes to lilac, mauve or purple |
| Lipid | Ethanol emulsion test | Shake with ethanol, then add the ethanol extract to water | White cloudy emulsion |
Benedict's test must be heated safely. A water bath gives controlled heating and reduces the chance of the contents spitting out of the tube. Direct heating in a flame is poor practice because the mixture can bump violently.
The non-reducing sugar test is a good example of a method decision. Some sugars do not reduce Benedict's reagent directly. Acid hydrolysis breaks them into reducing sugars, but the acid must then be neutralised before the Benedict's test is repeated. Otherwise, the conditions of the reagent test are changed.
Interpreting A Food-Test Sequence
A student tests a food extract with Benedict's reagent. The sample remains blue after heating. The student then heats a fresh sample with dilute acid, neutralises it, adds Benedict's reagent, and heats it again. This time the mixture forms an orange precipitate.
The first result suggests no reducing sugar was detected in the original extract. The second result suggests a non-reducing sugar was present, because hydrolysis produced reducing sugars that then reacted with Benedict's reagent.
A common error is to describe every positive Benedict's result as "red". Green, yellow and orange precipitates can also be positive; they usually indicate a lower amount of reducing sugar than a brick-red/brown precipitate.
Separating Biological Compounds
Some biological samples contain several compounds mixed together. Separation techniques make the mixture visible as separate spots or bands, so the sample can be compared with known substances.
Chromatography
Chromatography separates compounds because they move differently between a mobile phase and a stationary phase.
In paper chromatography, the stationary phase is the chromatography paper and the mobile phase is the solvent that moves up the paper. In thin-layer chromatography, the stationary phase is a thin layer of solid material on a plate, and the mobile phase is again the solvent.
A good chromatography method depends on careful setup:
- Draw a pencil baseline near the bottom of the paper or plate.
- Place a small concentrated spot of sample on the baseline.
- Let the spot dry; if more sample is needed, add another small spot on top and let it dry again.
- Put the paper or plate in a tank with the solvent level below the baseline.
- Allow the solvent to rise.
- Remove the paper or plate before the solvent reaches the top, then mark the solvent front in pencil.
- Compare the separated spots with known samples or standards.
The solvent must start below the sample spot. If the sample is submerged, it dissolves into the solvent reservoir instead of being carried up the paper or plate. The spot should be small and dry, because a large wet spot smears and produces poor separation.
Electrophoresis separates biological compounds in a different way.
Electrophoresis
Electrophoresis separates charged molecules using an electric field, often through a gel.
In a simple gel electrophoresis setup, samples are loaded into wells in a gel, the gel is covered with buffer, and an electric current is applied. Charged molecules move through the gel. DNA has a net negative charge, so DNA fragments move towards the positive electrode. Smaller DNA fragments usually move further through the gel in the same time because they pass more easily through the gel matrix.
Chromatography and electrophoresis are often confused because both produce a pattern of separated substances. The cause of separation is different:
| Method | Main separation idea | Practical risk or quality point |
|---|---|---|
| Paper/TLC chromatography | Different solubilities and attractions to mobile and stationary phases | Organic solvents may be hazardous; handle plates/paper carefully and avoid smearing |
| Electrophoresis | Charged molecules move through a gel in an electric field | Electrical safety, hot agarose when gels are poured, and safe visualisation dyes matter |
Aseptic Microbiology
Microorganisms are present in the air, on surfaces and on skin. If unwanted microorganisms enter a culture, the results become invalid and the culture may become unsafe. Aseptic technique means working in a way that prevents contamination by unwanted microorganisms.
Aseptic technique
Aseptic technique is the use of procedures that prevent unwanted microorganisms contaminating cultures, apparatus, media or the worker.
An agar plate contains nutrient agar set in a Petri dish. Microorganisms can grow as colonies on the agar surface, so agar plates are useful when growth needs to be seen as separate colonies or inhibition zones. A broth is a liquid nutrient medium. It is useful when microorganisms need to grow throughout a liquid culture, but spills are a greater risk.
Key aseptic steps have clear reasons:
| Aseptic step | Reason |
|---|---|
| Sterilise inoculating loops before and after use | Kills microorganisms already on the loop and those picked up from the culture |
| Let a hot loop cool before touching microorganisms | Prevents killing the microorganisms being transferred |
| Open Petri dish lids for the shortest possible time | Reduces entry of airborne microorganisms |
| Work near a Bunsen burner when appropriate | Convection currents help carry airborne microorganisms away from the open culture |
| Secure lids with tape but do not seal plates fully | Prevents lids falling off while avoiding anaerobic conditions |
| Incubate school plates at low or room temperature | Reduces risk of culturing microorganisms harmful to humans |
| Observe colonies without opening plates | Prevents release or spread of microorganisms |
| Dispose of cultures by sterilising them after use | Kills microorganisms before disposal |
Broth cultures need the same contamination control, plus extra spill control. Working in a tray with a greater capacity than the broth volume helps contain an accidental spill. A student should use sterile pipettes or loops and avoid touching sterile tips or lids to non-sterile surfaces.
Explaining Aseptic Technique
A student opens an agar plate fully for 30 seconds while adding bacteria. This increases the chance that airborne microorganisms land on the agar. A better method is to lift the lid just enough to inoculate the agar, keep the opening time short, and work near a Bunsen burner where appropriate.
The aim is not to make the culture "more sterile" once bacteria have been added. After inoculation, the culture is meant to contain the chosen microorganism. Aseptic technique prevents unwanted microorganisms being added.
Choosing And Evaluating Methods
The strongest practical answers choose a technique for a reason. Start from the evidence you need, then select the method that can produce it.
If the question asks whether a biological molecule is present in a sample, use a qualitative reagent test. If it asks whether a mixture contains several compounds, use a separation method. If it asks about growing or transferring microorganisms, use aseptic technique with agar or broth.
Choosing A Technique
A student has a green leaf extract and wants to show that it contains more than one pigment. A qualitative reagent test would not show the different pigments separately. A paper or thin-layer chromatography method is more suitable because the pigments can move different distances and appear as separate spots or bands.
Evaluation often focuses on validity and safety:
| Method family | Validity or safety improvement | Why it matters |
|---|---|---|
| Qualitative testing | Use positive and negative controls | Shows whether the reagent and method are working correctly |
| Qualitative testing | Use a water bath for Benedict's test | Gives safer, controlled heating |
| Chromatography | Use a small dry spot and keep solvent below baseline | Reduces smearing and prevents loss of sample into solvent |
| Chromatography/electrophoresis | Use known standards or marker samples | Allows comparison with known compounds or fragment sizes |
| Aseptic microbiology | Minimise opening time of plates and sterilise equipment | Reduces contamination |
| Aseptic microbiology | Incubate safely and observe unopened cultures | Reduces risk from harmful microorganisms |
Be careful with conclusions from qualitative techniques. A positive result shows that the target substance or selected microorganism is detected under the conditions of the test. A negative result may mean the substance is absent, but it could also mean the concentration is too low, the reagent has failed, or the method was not carried out correctly. This is why controls and careful records matter.
Choosing And Evaluating Methods Continued
Bring the three technique families back to the same practical logic whenever you revise them: the result is only useful when the method has protected the sample, the student, and the validity of the observation.
For this practical-skills lesson, learn each technique as a chain: purpose, apparatus or reagent, controlled method step, observable result, and a justified conclusion.