B5: Culturing Microorganisms
Binary fission, aseptic technique, zones of inhibition, and bacterial population calculations
Key Definitions
Binary fission — The type of cell division used by prokaryotes (bacteria) where one cell divides into two genetically identical cells.
Agar plate — A Petri dish containing agar jelly (a growth medium) on which microorganisms can be cultured.
Aseptic technique — A set of procedures used to prevent contamination of cultures by unwanted microorganisms.
Zone of inhibition — The clear area around an antimicrobial disc on an agar plate where bacteria cannot grow; indicates the effectiveness of the antimicrobial substance.
Mean division time — The average time taken for one bacterial cell to divide into two by binary fission.
Agar plate — A Petri dish containing agar jelly (a growth medium) on which microorganisms can be cultured.
Aseptic technique — A set of procedures used to prevent contamination of cultures by unwanted microorganisms.
Zone of inhibition — The clear area around an antimicrobial disc on an agar plate where bacteria cannot grow; indicates the effectiveness of the antimicrobial substance.
Mean division time — The average time taken for one bacterial cell to divide into two by binary fission.
Binary Fission
Bacteria reproduce by binary fission — a form of asexual reproduction:
1. The circular DNA and plasmids replicate.
2. The cell elongates and the two copies of DNA move to opposite ends.
3. The cytoplasm divides and a new cell wall forms, producing two genetically identical daughter cells.
Binary fission can happen very rapidly — some bacteria divide every 20 minutes under ideal conditions (warmth, moisture, nutrients, suitable pH). This is why bacterial infections can develop so quickly.
1. The circular DNA and plasmids replicate.
2. The cell elongates and the two copies of DNA move to opposite ends.
3. The cytoplasm divides and a new cell wall forms, producing two genetically identical daughter cells.
Binary fission can happen very rapidly — some bacteria divide every 20 minutes under ideal conditions (warmth, moisture, nutrients, suitable pH). This is why bacterial infections can develop so quickly.
Culturing Bacteria on Agar Plates
Growth medium: Agar jelly is used because it provides the nutrients (carbohydrates, minerals, proteins) that bacteria need to grow. It is poured into a Petri dish and allowed to set.
Inoculation: Bacteria are transferred onto the agar plate using a sterile loop or swab.
Incubation: The Petri dish is placed upside down (to prevent condensation dripping onto the agar) and incubated at a suitable temperature. In school labs, the maximum temperature used is 25°C to prevent the growth of harmful pathogens. Industrial labs may use higher temperatures (e.g. 37°C for human pathogens).
Visible growth: Each bacterial cell on the plate divides repeatedly to form a colony — a visible cluster of millions of identical bacteria, all descended from one original cell.
Inoculation: Bacteria are transferred onto the agar plate using a sterile loop or swab.
Incubation: The Petri dish is placed upside down (to prevent condensation dripping onto the agar) and incubated at a suitable temperature. In school labs, the maximum temperature used is 25°C to prevent the growth of harmful pathogens. Industrial labs may use higher temperatures (e.g. 37°C for human pathogens).
Visible growth: Each bacterial cell on the plate divides repeatedly to form a colony — a visible cluster of millions of identical bacteria, all descended from one original cell.
Exam tip: Petri dishes are incubated upside down to prevent condensation from the lid dripping onto the agar surface, which would spread colonies and make results unreliable. School labs use 25°C max; industrial labs can use higher temperatures.
Aseptic Technique
Aseptic technique prevents contamination by unwanted microorganisms from the air, skin, or surfaces:
— Sterilise the inoculating loop by heating it to red hot in a Bunsen burner flame, then let it cool before use.
— Pass the neck of the bottle of bacterial culture through the Bunsen burner flame before and after pouring (to kill airborne contaminants near the opening).
— Lift the lid of the Petri dish at an angle (like a hinge) — never remove it completely — to minimise exposure to air.
— Tape the lid shut after inoculation and label the base (not the lid).
— Work near a Bunsen burner — the upward convection current draws air away from the work area, reducing contamination.
— Wash hands before and after handling cultures.
— Disinfect surfaces before and after the practical.
— Sterilise the inoculating loop by heating it to red hot in a Bunsen burner flame, then let it cool before use.
— Pass the neck of the bottle of bacterial culture through the Bunsen burner flame before and after pouring (to kill airborne contaminants near the opening).
— Lift the lid of the Petri dish at an angle (like a hinge) — never remove it completely — to minimise exposure to air.
— Tape the lid shut after inoculation and label the base (not the lid).
— Work near a Bunsen burner — the upward convection current draws air away from the work area, reducing contamination.
— Wash hands before and after handling cultures.
— Disinfect surfaces before and after the practical.
Example 1: Why Aseptic Technique Matters
If aseptic technique is not followed, unwanted microorganisms from the air or skin can contaminate the agar plate. These contaminants grow as colonies that may overlap with or outcompete the intended bacteria, making results unreliable. Contamination could also grow harmful pathogens, especially if the plate is incubated above 25°C.
Zones of Inhibition
When testing the effectiveness of antibiotics or antiseptics:
1. Place paper discs soaked in different antimicrobial substances onto a lawn of bacteria on an agar plate.
2. The antimicrobial diffuses into the agar around the disc.
3. Where the antimicrobial concentration is high enough to kill or inhibit the bacteria, a clear zone appears — the zone of inhibition.
4. The larger the zone of inhibition, the more effective the antimicrobial substance.
5. A control disc (soaked in sterile water) should show no zone of inhibition.
1. Place paper discs soaked in different antimicrobial substances onto a lawn of bacteria on an agar plate.
2. The antimicrobial diffuses into the agar around the disc.
3. Where the antimicrobial concentration is high enough to kill or inhibit the bacteria, a clear zone appears — the zone of inhibition.
4. The larger the zone of inhibition, the more effective the antimicrobial substance.
5. A control disc (soaked in sterile water) should show no zone of inhibition.
Cross-sectional area of zone of inhibition = πr²
where r = radius of the zone (half the diameter)
where r = radius of the zone (half the diameter)
Example 2: Calculating the Area of a Zone of Inhibition
A zone of inhibition has a diameter of 24 mm. Calculate its cross-sectional area.Radius = 24 ÷ 2 = 12 mm
Area = π × 12² = π × 144 = 452 mm² (to 3 significant figures)
Example 3: Comparing Antibiotics Using Zones of Inhibition
Antibiotic A produces a zone with diameter 20 mm. Antibiotic B produces a zone with diameter 30 mm.Area A = π × 10² = 314 mm²
Area B = π × 15² = 707 mm²
Antibiotic B is more effective because it produces a larger zone of inhibition.
Calculating Bacterial Population Growth
Number of bacteria (N) = N₀ × 2n
where N₀ = initial number of bacteria
and n = number of divisions = total time ÷ mean division time
where N₀ = initial number of bacteria
and n = number of divisions = total time ÷ mean division time
How the formula works:
Each division doubles the population (2n = number of new cells from one original cell after n divisions). Multiply by the starting number N₀ to get the total population.
Important: This formula assumes unlimited nutrients, no waste buildup, and no death — it represents the maximum theoretical growth (exponential phase).
Each division doubles the population (2n = number of new cells from one original cell after n divisions). Multiply by the starting number N₀ to get the total population.
Important: This formula assumes unlimited nutrients, no waste buildup, and no death — it represents the maximum theoretical growth (exponential phase).
Example 4: Calculating Population Size
A culture starts with 1 bacterial cell. The mean division time is 30 minutes. How many bacteria will there be after 3 hours?Total time = 3 hours = 180 minutes
Number of divisions (n) = 180 ÷ 30 = 6
N = 1 × 2⁶ = 1 × 64 = 64 bacteria
Example 5: More Complex Population Calculation
A culture starts with 500 bacterial cells. The mean division time is 20 minutes. How many bacteria will there be after 4 hours?Total time = 4 hours = 240 minutes
Number of divisions (n) = 240 ÷ 20 = 12
N = 500 × 2¹² = 500 × 4,096 = 2,048,000 bacteria
Example 6: Working Backwards to Find Division Time
A culture starts with 100 bacteria. After 2 hours there are 6,400 bacteria. What is the mean division time?6,400 = 100 × 2n
2n = 64
2⁶ = 64, so n = 6 divisions
Total time = 2 hours = 120 minutes
Mean division time = 120 ÷ 6 = 20 minutes
Required Practical: Investigating Antimicrobial Action
Aim: To compare the effectiveness of different antiseptics or antibiotics on bacterial growth.
Method:
1. Prepare a sterile agar plate and use aseptic technique.
2. Spread a lawn of bacteria evenly over the agar surface using a sterile spreader.
3. Place paper discs soaked in different concentrations of antiseptic (or different antibiotics) on the agar. Include a control disc soaked in sterile water.
4. Tape the lid, label, and incubate at 25°C for 24–48 hours.
5. Measure the diameter of each zone of inhibition and calculate the area (πr²).
Variables:
— Independent variable: type or concentration of antimicrobial substance.
— Dependent variable: diameter or area of zone of inhibition.
— Control variables: volume of bacteria, volume of antimicrobial on disc, incubation temperature, incubation time, type of bacteria used.
Method:
1. Prepare a sterile agar plate and use aseptic technique.
2. Spread a lawn of bacteria evenly over the agar surface using a sterile spreader.
3. Place paper discs soaked in different concentrations of antiseptic (or different antibiotics) on the agar. Include a control disc soaked in sterile water.
4. Tape the lid, label, and incubate at 25°C for 24–48 hours.
5. Measure the diameter of each zone of inhibition and calculate the area (πr²).
Variables:
— Independent variable: type or concentration of antimicrobial substance.
— Dependent variable: diameter or area of zone of inhibition.
— Control variables: volume of bacteria, volume of antimicrobial on disc, incubation temperature, incubation time, type of bacteria used.
Exam tip: Always include a control disc (soaked in sterile water or the solvent without the active ingredient) to show that any zone of inhibition is caused by the antimicrobial substance and not the disc or solvent itself. If the control shows a zone, the investigation is invalid.
Practice Questions
1. Higher A bacterial culture starts with 2 cells. The mean division time is 25 minutes. Calculate the number of bacteria after 150 minutes.
n = 150 ÷ 25 = 6 divisions. N = 2 × 2⁶ = 2 × 64 = 128 bacteria.
2. Higher A zone of inhibition has a diameter of 28 mm. Calculate its cross-sectional area. Give your answer to 3 significant figures.
Radius = 28 ÷ 2 = 14 mm. Area = π × 14² = π × 196 = 615.75... ≈ 616 mm² (3 s.f.).
3. Higher Describe three aseptic techniques used when culturing bacteria and explain why each is necessary.
1) Sterilise the inoculating loop by heating to red hot — kills any bacteria already on the loop that would contaminate the culture. 2) Lift the Petri dish lid at an angle, not fully off — minimises exposure to airborne microorganisms that could contaminate the plate. 3) Work near a Bunsen burner — the convection current draws air upwards and away from the work surface, reducing the chance of airborne contamination.
4. Higher A culture starts with 200 bacteria. After 3 hours there are 51,200 bacteria. Calculate the mean division time.
51,200 = 200 × 2ⁿ. 2ⁿ = 256. 2⁸ = 256, so n = 8 divisions. Total time = 3 hours = 180 minutes. Mean division time = 180 ÷ 8 = 22.5 minutes.
5. Higher Explain why school laboratories incubate agar plates at a maximum of 25°C, while industrial laboratories may use 37°C.
At 25°C, harmful human pathogens are unlikely to grow because it is below their optimum temperature (which is around 37°C, human body temperature). This reduces the risk of growing dangerous bacteria in a school setting. Industrial labs have stricter safety measures and controlled environments, allowing them to incubate at 37°C to grow human pathogens for research and testing.
🔬 Required Practical
Required Practical: Investigating Antiseptics on Bacterial Growth
Aim: To investigate the effect of different antiseptics on bacterial growth using agar plates.
Method: 1) Prepare a sterile agar plate and use aseptic technique throughout. 2) Use a sterile pipette to spread a lawn of bacteria evenly over the agar surface. 3) Soak paper discs in different antiseptic solutions at the same concentration and place them on the agar. Include a control disc soaked in sterile water. 4) Tape the lid shut, label the base, and incubate upside down at 25°C for 24–48 hours. 5) Measure the diameter of each zone of inhibition and calculate the area using πr².
Variables: IV: type of antiseptic, DV: diameter or area of zone of inhibition, Control: volume of antiseptic on each disc, concentration of antiseptic, type of bacteria, incubation temperature and time, volume of bacterial lawn
🔢 Maths Skills
Mathematical Skills for this Topic
Cross-sectional area of zones of inhibition: Area = πr², where r = radius = diameter ÷ 2. Always measure the diameter and halve it before squaring.
Bacterial growth calculations: Number of bacteria = N₀ × 2ⁿ, where N₀ is the starting number and n = total time ÷ mean division time. For example, starting with 1 bacterium and a division time of 20 minutes: after 2 hours (120 min), n = 6, so N = 1 × 2⁶ = 64 bacteria.
Example: A zone of inhibition has diameter 18 mm. Area = π × 9² = π × 81 = 254 mm² (3 s.f.). Compare with a zone of diameter 26 mm: Area = π × 13² = 531 mm² — over twice as large, indicating a much more effective antiseptic.
⚠️ Common Misconceptions
Watch Out!
Students often think bacteria always double every 20 minutes. Wrong: Bacteria divide every 20 minutes regardless of conditions. Correct: Bacteria only divide this fast in optimum conditions with unlimited nutrients, suitable temperature, and no waste buildup. Division time depends on temperature, nutrients, and other conditions.
Students often think a bigger zone of inhibition always means a better antiseptic. Wrong: A larger zone of inhibition always indicates a more effective antimicrobial substance. Correct: A larger zone suggests more effective inhibition, but zone size also depends on how well the substance diffuses through agar. A substance that diffuses poorly may be effective but produce a small zone.
✍️ 6-Mark Question
Extended Answer Question
6 marks: Describe how to investigate the effect of different antiseptics on bacterial growth on an agar plate. Explain how to use aseptic technique.
Prepare a sterile agar plate. Using aseptic technique: sterilise the inoculating loop by heating to red hot in a Bunsen burner flame and let it cool; work near the Bunsen burner to create an upward convection current that draws contaminants away; lift the Petri dish lid at an angle like a hinge, never removing it fully, to minimise airborne contamination. Spread a lawn of bacteria evenly over the agar surface using a sterile spreader. Soak paper discs in different antiseptic solutions at equal volumes and concentrations, and place them on the agar surface using sterile forceps. Include a control disc soaked in sterile water to show that any inhibition is due to the antiseptic alone. Tape the lid shut and label the base. Incubate upside down at 25°C for 24–48 hours (not above 25°C in a school lab to prevent growth of harmful pathogens). After incubation, measure the diameter of each zone of inhibition and calculate the area using πr². The antiseptic with the largest zone of inhibition is the most effective.
Mark scheme: 1 mark for preparing the plate, 1 mark for lawn spreading, 1 mark for discs including control, 1 mark for at least two aseptic techniques, 1 mark for incubation conditions, 1 mark for measuring and comparing zones.
📊 AO3: Analyse & Evaluate
Analysis and Evaluation
A student tested three antiseptics (X, Y, Z) and a water control on the same bacterium. Each test was repeated three times. Results (zone diameter in mm):
X: 14, 15, 14 Y: 22, 21, 23 Z: 18, 18, 19 Water: 0, 0, 0
Question 1: Calculate the mean zone of inhibition area for each antiseptic. (X: mean diameter = 14.3 mm, area = π × 7.15² = 161 mm²; Y: mean diameter = 22 mm, area = π × 11² = 380 mm²; Z: mean diameter = 18.3 mm, area = π × 9.15² = 263 mm².)
Question 2: Which antiseptic is most effective? Explain your answer using data. (Y is most effective because it has the largest mean zone area of 380 mm².)
Question 3: Explain why the water control is important. (It shows that the paper disc and solvent do not inhibit bacterial growth; any zone around antiseptic discs is therefore caused by the antiseptic alone, validating the investigation.)
Question 4: Antiseptic Y has a larger zone than Z. Is Y definitely a better antiseptic? Suggest a reason to be cautious. (Y may diffuse through agar more quickly than Z, producing a larger zone even if Z is equally or more effective at killing bacteria. Zone size reflects both effectiveness and diffusion rate.)
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