Drug testing and antibioticsEdexcel A-Level Biology A: Revision notes

In the 1700s medicines were usually traditional herbal mixtures given without any systematic testing. In 1785 William Withering studied a herbal remedy for dropsy (fluid retention, often caused by heart failure) known as digitalis soup. He identified foxglove (Digitalis purpurea) as the active ingredient, tested different doses on more than 100 patients and recorded the doses, the effects and the side effects, such as sickness and a very slow pulse. Digitalis is still the source of a heart drug, digoxin.

His approach was an early example of systematic testing, but it had weaknesses: no control group, the patients and the doctor knew what was being given, and patients were not allocated at random, so improvement could have been due to the placebo effect or the doctor's bias.

Before a drug is tried on humans it is tested in the laboratory on cells, tissues, computer models and animals to check for toxicity. Clinical trials then follow three phases:

• Phase 1: a small number of healthy volunteers take the drug at low, then higher, doses to test its safety and side effects and how it is absorbed and removed.
• Phase 2: a small number of patients take the drug to find the effective dose and the effectiveness and side effects.
• Phase 3: a large number of patients, usually compared with a placebo or the best existing treatment, to confirm effectiveness and detect less common side effects.

Only if the results are satisfactory is the drug licensed.

A placebo is an inactive substance that looks, tastes and is given in the same way as the drug. A group given the placebo is the control, so the effect of the drug itself can be separated from the placebo effect.

In a double-blind trial neither the patients nor the researchers who assess them know who is receiving the drug and who is receiving the placebo, so neither the patients' expectations nor the doctors' bias can affect the results. Patients are allocated to groups at random, so that the groups are similar. The code is broken only after the results are collected.

Bacteria in culture need:

• Nutrients: a source of carbon (for example glucose) and nitrogen (for example amino acids), provided in nutrient agar or broth, for respiration and to make proteins and new cells.
• A suitable temperature: enzymes work best at an optimum. Human pathogens grow best at 37 °C, so school laboratories incubate at no more than about 25 °C.
• Oxygen for aerobic bacteria (some bacteria are anaerobic).
• Moisture and a suitable pH.

Bacteria then divide rapidly by binary fission, so the population may double about every 20 minutes under ideal conditions.

Aseptic technique prevents contamination of the culture by other microorganisms and prevents the escape of the bacteria, which could be pathogens.

• Disinfect the bench and wash hands before and after.
• Flame the neck of the culture bottle and the inoculating loop until it is red hot, then let it cool.
• Open the dish lid only slightly and briefly.
• Use sterile equipment, e.g. pipettes and spreaders.
• Tape the lid with two pieces of tape (not all the way around, so air can enter) and invert the dish so condensation does not fall onto the agar.
• Incubate at no more than 25 °C, then autoclave all dishes after use without opening them.

Spread a lawn of bacteria on nutrient agar. Soak paper discs in extracts of plants (for example garlic, mint or ginger) and place them on the lawn with sterile forceps, together with a positive control (antibiotic disc) and a negative control (a disc soaked in sterile water or the solvent). Incubate inverted at 25 °C for 24 to 48 hours.

Where an extract stops the bacteria growing there is a clear zone (zone of inhibition) around the disc. Measure the diameter and calculate the area using πr²; a larger area means a greater antimicrobial effect. Control the size of disc, volume and concentration of extract, the bacterial species, the agar depth and the temperature, and repeat for a mean.

Example: a clear zone of diameter 14 mm has an area of π × 7² = 154 mm².