Investigating Antiseptics & Antibiotics

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 $0\text{ mm}^2$ .
The Petri dish lid is then secured with two small pieces of adhesive tape. It must NOT be sealed all the way around, because an airtight seal prevents oxygen from entering and promotes the growth of dangerous anaerobic bacteria.
The dish is stored upside down to prevent condensation from dripping onto the agar surface and disrupting the colonies.
Finally, the plates are incubated for typically 48 hours at a maximum temperature of 25°C. They must NOT be incubated at higher temperatures (like 37°C) to strictly reduce the risk of cultivating harmful human pathogens.

Calculating Cross-Sectional Areas

To determine which antibiotic or antiseptic is most effective, you must measure the cross-sectional area of the clear zones where bacteria have been killed.
The most effective substance will produce the largest area.
Measurements are taken from one edge of the clear zone to the other, passing directly through the centre of the paper disc to find the diameter.
Because bacterial growth can be uneven, you should take two measurements at 90° angles to calculate a mean diameter.
The area is then calculated using the formula for the area of a circle:

\text{Area} = \pi r^2

Worked Example: Area Calculation

Calculate the cross-sectional area of a non-circular zone of inhibition. Diameter 1 is $18\text{ mm}$ and Diameter 2 is $20\text{ mm}$ . Use $3.14$ for $\pi$ .

Step 1: Calculate the mean diameter.

Add the two diameters together and divide by 2.
$\text{Mean diameter} = (18 + 20) / 2 = 19\text{ mm}$

Step 2: Calculate the radius ( $r$ ).

Divide the mean diameter by 2.
$r = 19 / 2 = 9.5\text{ mm}$

Step 3: Substitute the radius into the area formula.

$\text{Area} = \pi \times r^2$
$\text{Area} = 3.14 \times 9.5^2$
$\text{Area} = 3.14 \times 90.25$

Step 4: State the final answer with units.

$\text{Area} = 283.385\text{ mm}^2$

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Exam Tips

Common Mistake
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.
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.
3
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.
4
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.

Key Terms(9)

Zone of inhibition: The clear area around an antibiotic or antiseptic disc where bacterial growth has been prevented or bacteria have been killed.
Culture medium: A substance (either liquid nutrient broth or solid agar jelly) that provides the carbohydrates, minerals, proteins, and vitamins needed for microorganisms to grow.
Agar gel: A solid nutrient-rich medium used for the cultivation of bacteria and other microorganisms.
Colony: A visible cluster of identical bacteria grown on a solid medium, all originating from a single parent cell.
Aseptic technique: A set of laboratory procedures and practices performed under carefully controlled conditions to prevent the contamination of cultures with unwanted microorganisms from the environment.
Autoclave: A device that uses high-pressure steam at high temperatures to sterilise equipment and agar before use.
Inoculating loop: A tool made of wire used to transfer and smear a small sample of a microorganism culture onto agar.
Cross-sectional area: The area of the two-dimensional circular shape formed by the inhibition zone on the agar surface.
Mean diameter: The average of two or more diameter measurements taken at different angles (usually 90°) to account for uneven, non-circular bacterial growth.

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Key Terms(9)

Zone of inhibition: The clear area around an antibiotic or antiseptic disc where bacterial growth has been prevented or bacteria have been killed.
Culture medium: A substance (either liquid nutrient broth or solid agar jelly) that provides the carbohydrates, minerals, proteins, and vitamins needed for microorganisms to grow.
Agar gel: A solid nutrient-rich medium used for the cultivation of bacteria and other microorganisms.
Colony: A visible cluster of identical bacteria grown on a solid medium, all originating from a single parent cell.
Aseptic technique: A set of laboratory procedures and practices performed under carefully controlled conditions to prevent the contamination of cultures with unwanted microorganisms from the environment.
Autoclave: A device that uses high-pressure steam at high temperatures to sterilise equipment and agar before use.
Inoculating loop: A tool made of wire used to transfer and smear a small sample of a microorganism culture onto agar.
Cross-sectional area: The area of the two-dimensional circular shape formed by the inhibition zone on the agar surface.
Mean diameter: The average of two or more diameter measurements taken at different angles (usually 90°) to account for uneven, non-circular bacterial growth.

Investigating Antiseptics & Antibiotics

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 $0\text{ mm}^2$ .
The Petri dish lid is then secured with two small pieces of adhesive tape. It must NOT be sealed all the way around, because an airtight seal prevents oxygen from entering and promotes the growth of dangerous anaerobic bacteria.
The dish is stored upside down to prevent condensation from dripping onto the agar surface and disrupting the colonies.
Finally, the plates are incubated for typically 48 hours at a maximum temperature of 25°C. They must NOT be incubated at higher temperatures (like 37°C) to strictly reduce the risk of cultivating harmful human pathogens.

Calculating Cross-Sectional Areas

To determine which antibiotic or antiseptic is most effective, you must measure the cross-sectional area of the clear zones where bacteria have been killed.
The most effective substance will produce the largest area.
Measurements are taken from one edge of the clear zone to the other, passing directly through the centre of the paper disc to find the diameter.
Because bacterial growth can be uneven, you should take two measurements at 90° angles to calculate a mean diameter.
The area is then calculated using the formula for the area of a circle:

\text{Area} = \pi r^2

Worked Example: Area Calculation

Calculate the cross-sectional area of a non-circular zone of inhibition. Diameter 1 is $18\text{ mm}$ and Diameter 2 is $20\text{ mm}$ . Use $3.14$ for $\pi$ .

Step 1: Calculate the mean diameter.

Add the two diameters together and divide by 2.
$\text{Mean diameter} = (18 + 20) / 2 = 19\text{ mm}$

Step 2: Calculate the radius ( $r$ ).

Divide the mean diameter by 2.
$r = 19 / 2 = 9.5\text{ mm}$

Step 3: Substitute the radius into the area formula.

$\text{Area} = \pi \times r^2$
$\text{Area} = 3.14 \times 9.5^2$
$\text{Area} = 3.14 \times 90.25$

Step 4: State the final answer with units.

$\text{Area} = 283.385\text{ mm}^2$

Sign up to continue reading

Get full access to revision notes, key terms, and exam tips for every subtopic.

Exam Tips

Common Mistake
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.
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.
3
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.
4
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.

Key Terms(9)

Zone of inhibition: The clear area around an antibiotic or antiseptic disc where bacterial growth has been prevented or bacteria have been killed.
Culture medium: A substance (either liquid nutrient broth or solid agar jelly) that provides the carbohydrates, minerals, proteins, and vitamins needed for microorganisms to grow.
Agar gel: A solid nutrient-rich medium used for the cultivation of bacteria and other microorganisms.
Colony: A visible cluster of identical bacteria grown on a solid medium, all originating from a single parent cell.
Aseptic technique: A set of laboratory procedures and practices performed under carefully controlled conditions to prevent the contamination of cultures with unwanted microorganisms from the environment.
Autoclave: A device that uses high-pressure steam at high temperatures to sterilise equipment and agar before use.
Inoculating loop: A tool made of wire used to transfer and smear a small sample of a microorganism culture onto agar.
Cross-sectional area: The area of the two-dimensional circular shape formed by the inhibition zone on the agar surface.
Mean diameter: The average of two or more diameter measurements taken at different angles (usually 90°) to account for uneven, non-circular bacterial growth.

Previous NoteAseptic Techniques & Culturing Next Topic: Cell DivisionChromosomes

Back to Culturing Microorganisms

Put your knowledge into practice — try past paper questions for Biology

Key Terms(9)

Zone of inhibition: The clear area around an antibiotic or antiseptic disc where bacterial growth has been prevented or bacteria have been killed.
Culture medium: A substance (either liquid nutrient broth or solid agar jelly) that provides the carbohydrates, minerals, proteins, and vitamins needed for microorganisms to grow.
Agar gel: A solid nutrient-rich medium used for the cultivation of bacteria and other microorganisms.
Colony: A visible cluster of identical bacteria grown on a solid medium, all originating from a single parent cell.
Aseptic technique: A set of laboratory procedures and practices performed under carefully controlled conditions to prevent the contamination of cultures with unwanted microorganisms from the environment.
Autoclave: A device that uses high-pressure steam at high temperatures to sterilise equipment and agar before use.
Inoculating loop: A tool made of wire used to transfer and smear a small sample of a microorganism culture onto agar.
Cross-sectional area: The area of the two-dimensional circular shape formed by the inhibition zone on the agar surface.
Mean diameter: The average of two or more diameter measurements taken at different angles (usually 90°) to account for uneven, non-circular bacterial growth.