Culturing microorganisms (biology only) — AQA GCSE Biology

Bacteria are cultured in the laboratory to study their growth and behaviour. Two common methods exist. In a nutrient broth solution, bacteria are suspended in a liquid containing nutrients, so they multiply throughout the liquid and the culture becomes turbid; this suits measuring population growth over time. On an agar gel plate, a solid medium containing nutrients is used, and each bacterium divides in place to form a visible colony, a cluster of genetically identical cells. A colony arises from one original cell, so colonies allow separation and counting of viable cells. For example, a serial dilution of a culture spread on an agar plate can produce countable colonies, whereas the same culture in broth gives an overall cloudiness. Both methods supply nutrients, water and suitable conditions for growth.

Investigating disinfectants and antibiotics requires a culture containing only the microorganism being studied. If other microbes are present, results become unreliable because the disinfectant or antibiotic may affect the contaminants as well as the intended species, and contaminant colonies may be mistaken for growth of the test organism. An uncontaminated culture is achieved by sterilising equipment and media, using aseptic technique, and transferring only the chosen microorganism. For example, when testing an antibiotic disc on a lawn of a known bacterium, any stray fungal or bacterial colony would confuse measurement of the inhibition zone. Contamination also risks the safety of the worker. Therefore, valid conclusions about the action of disinfectants and antibiotics depend on starting with a pure culture and keeping it pure throughout the investigation.

Preparing an uncontaminated culture means transferring a known microorganism into sterile medium without introducing others. Work surfaces are cleaned, hands are washed, and equipment is sterilised before use. Nutrient agar plates are poured and set, and the inoculating loop is passed through a flame until red hot, then cooled. The loop is dipped into the culture, and a streak is made across the agar; the lid is replaced and secured with tape, but not sealed completely, to allow gas exchange and reduce the risk of anaerobic conditions. Plates are incubated upside down at a suitable temperature, typically around 25 °C in school laboratories, to reduce condensation and limit the growth of human pathogens. All equipment is sterilised after use. Throughout, the lid is lifted only slightly and the plate is kept closed when not being inoculated.

Culturing microorganisms requires aseptic technique to avoid contamination. Petri dishes and culture media are sterilised before use to kill unwanted microorganisms that would otherwise grow and compete with, or contaminate, the intended culture. Inoculating loops are sterilised by passing them through a flame, which kills microorganisms on the loop so only the intended species is transferred. The lid is secured with adhesive tape to prevent airborne contamination and to stop the culture escaping, and the dish is stored upside down so condensation runs onto the lid rather than onto the culture, preventing colonies from merging. In school laboratories, incubation is generally at 25°C because this reduces the risk of growing harmful human pathogens while still allowing microbial growth.

A colony or a clear area around a colony is roughly circular, so its cross-sectional area is calculated using the formula for the area of a circle, A = πr². First measure the diameter of the colony or clear zone with a ruler, then halve it to find the radius. Substitute the radius into A = πr², using π = 3.14 or the calculator value, and give the area in mm² or cm². For example, a colony of diameter 10 mm has radius 5 mm, so area = π × 5² = 78.5 mm². If comparing clear areas around colonies, calculate each area separately and compare the values to judge which microorganism is more affected by an antibiotic.

Bacteria reproduce by binary fission, so the population can double each mean division time. To find the number after a given time, divide the total time by the mean division time to find the number of divisions, n. The population is then starting number × 2ⁿ. For example, if a bacterium divides every 20 minutes and 2 hours pass, n = 120 ÷ 20 = 6 divisions, so one starting cell becomes 1 × 2⁶ = 64 bacteria. If the starting population is larger, multiply that starting number by 2ⁿ. This assumes ideal conditions with unlimited nutrients and no deaths, so it gives a theoretical maximum.

In culturing work you often calculate numbers of bacteria after growth, or areas of zones of inhibition, and the values can be very large or very small. Standard form writes a number as A × 10ⁿ, where A is at least 1 and less than 10, and n is an integer. For example, 4 500 000 bacteria becomes 4.5 × 10⁶, and 0.000 32 mm becomes 3.2 × 10⁻⁴ mm. To convert, move the decimal point until one non-zero digit remains on its left, then count the moves: left gives a positive index, right gives a negative index. To multiply, multiply the A values and add the indices; to divide, divide the A values and subtract the indices. Always check the final A value is between 1 and 10, and keep units with the answer.

Culture bacteria on a sterile agar plate and place discs soaked in antiseptic or antibiotic on the surface. The substance diffuses into the agar. Where effective, bacteria do not grow, leaving a clear area called a zone of inhibition. After incubation, measure the full diameter of each clear zone in millimetres (including the disc), usually in two perpendicular directions, and calculate a mean. A larger zone indicates greater effectiveness. Control variables include the volume and concentration of each substance, the bacterium, incubation temperature, and disc size. A control disc soaked in sterile water proves the clear zone is caused by the substance. Work aseptically: sterilise equipment, keep lids secured, and do not open plates after incubation.

This practical develops four working scientifically skills. AT 1 is use of appropriate apparatus and techniques, here sterile loops, pipettes, discs and agar plates. AT 3 is recording observations and measurements, so you record zone diameters in millimetres in a suitable table. AT 4 is analysis and interpretation, so you compare zones and decide which substance is most effective. AT 8 is making informed judgements about risks, so you use aseptic technique, keep lids on, and follow safe disposal. These skills are assessed through questions about method, measurements, results and safety rather than by a separate practical mark. For example, you might be asked why the lid is taped and not fully sealed, or how you ensured the comparison was fair.

The zones of inhibition you measure show how well an antibiotic stops bacterial growth. This connects to the topic of antibiotics and painkillers: antibiotics kill bacteria or stop their growth, while painkillers relieve symptoms but do not kill pathogens. Antibiotics are effective only against bacteria, not viruses, which is why viral infections such as colds are not treated with antibiotics. The practical also shows why the wrong antibiotic, or too low a dose, may fail, and why overuse contributes to antibiotic resistance. A clear, large zone around an antibiotic disc indicates that the bacterium is susceptible to that antibiotic. This evidence helps explain why doctors choose particular antibiotics and why completing a prescribed course matters.