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Investigating Bacterial Growth

Every time you use an antibacterial wipe on a kitchen counter, you are relying on chemicals to halt bacterial reproduction.
In the laboratory, we can test how effective different antiseptics and antibiotics are by measuring the zone of inhibition they create.
To do this, microorganisms are grown in a culture medium, which provides the carbohydrates, minerals, proteins, and vitamins they need to survive.
This medium is usually a solid agar gel poured into a Petri dish, where bacteria multiply to form a visible colony.

Aseptic Technique Methodology

Aseptic technique is a set of strict procedures used to prevent the contamination of cultures by unwanted environmental microorganisms.
First, the work surface must be disinfected with a spray (like Virkon) before and after the practical to prevent environmental contamination.
Next, all equipment (including the Petri dish and agar) must be sterilised using an autoclave, which uses high-pressure steam at 121°C to kill existing microbes.
All work must be carried out next to a lit Bunsen burner, as the blue flame creates a convection current that draws airborne microbes upwards and away from the open Petri dish.
To transfer the bacteria, an inoculating loop is heated in the Bunsen flame until it is red hot for sterilisation, and then allowed to cool briefly.
Finally, the lid of the Petri dish is lifted at an angle to smear the bacteria onto the agar. The lid must NOT be removed completely, as this allows microbes from the air to fall onto the gel.

Applying Discs and Incubation

Once the bacteria are smeared, filter paper discs soaked in different antibiotics or antiseptics are placed onto the bacterial lawn.
A control disc soaked in sterile water must also be added. This ensures any bacterial inhibition observed is strictly due to the chemical substance, and should yield an area of .

Students often try to measure the radius directly from the centre of the disc. It is far more accurate to measure the full diameter across the clear zone and then divide by 2.

In 'Describe' questions about aseptic technique, examiners specifically look for three core actions: flaming the inoculating loop, lifting the Petri dish lid at an angle, and disinfecting the work bench.

Always remember to mention the control disc soaked in sterile water; this proves that the chemical substance, and not the paper disc itself, is responsible for inhibiting the bacterial growth.

If an exam question asks you to compare the effectiveness of two antibiotics, calculate the area of both zones of inhibition and state that the most effective substance produces the largest area.

The clear area around an antibiotic or antiseptic disc where bacterial growth has been prevented or bacteria have been killed.

A substance (either liquid nutrient broth or solid agar jelly) that provides the carbohydrates, minerals, proteins, and vitamins needed for microorganisms to grow.

A solid nutrient-rich medium used for the cultivation of bacteria and other microorganisms.

A visible cluster of identical bacteria grown on a solid medium, all originating from a single parent cell.

A set of laboratory procedures and practices performed under carefully controlled conditions to prevent the contamination of cultures with unwanted microorganisms from the environment.

A device that uses high-pressure steam at high temperatures to sterilise equipment and agar before use.

A tool made of wire used to transfer and smear a small sample of a microorganism culture onto agar.

The area of the two-dimensional circular shape formed by the inhibition zone on the agar surface.

The average of two or more diameter measurements taken at different angles (usually 90°) to account for uneven, non-circular bacterial growth.