Bacterial Growth and Culture Techniques

Understanding how bacteria grow, divide, and respond to their environment is fundamental to microbiology. This lesson covers the bacterial growth curve, methods of culturing bacteria, and the use of culture media, as required by the Edexcel A-Level Biology (9BI0) specification.

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Binary Fission

Bacteria reproduce asexually by binary fission — a single cell divides into two identical daughter cells.

Steps of Binary Fission

1. The circular DNA molecule replicates — each copy attaches to the cell membrane at different points.
2. The cell elongates and the two DNA copies move apart.
3. The cell membrane invaginates (grows inward) at the centre of the cell.
4. A new cell wall forms across the centre, creating a septum.
5. The cell divides into two identical daughter cells, each with a copy of the chromosome.

Generation Time

The generation time (doubling time) varies between species and depends on conditions:

Organism	Approximate generation time
Escherichia coli (optimal conditions)	~20 minutes
Mycobacterium tuberculosis	~15–20 hours
Staphylococcus aureus	~30 minutes

Under ideal conditions, a single E. coli bacterium could theoretically produce over 4 × 10²¹ cells in just 24 hours. In practice, this does not occur because nutrients become depleted and waste products accumulate.

Exam Tip: Be prepared to calculate population sizes using the formula: N = N₀ × 2ⁿ, where N₀ is the initial population, n is the number of generations, and N is the final population.

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The Bacterial Growth Curve

When bacteria are inoculated into a fresh nutrient medium in a closed system (batch culture), the population follows a characteristic growth curve with four phases:

The Four Phases

Phase	Description	Explanation
1. Lag phase	Little or no increase in cell number	Bacteria are adapting to the new environment; synthesising enzymes and other molecules needed for growth. No cell division yet.
2. Log (exponential) phase	Rapid, exponential increase in cell number	Nutrients are abundant; conditions are optimal. The population doubles at a constant rate (generation time is constant).
3. Stationary phase	Population remains approximately constant	The rate of cell division equals the rate of cell death. Nutrients are depleted; toxic waste products accumulate; space is limited.
4. Decline (death) phase	Population decreases	The rate of cell death exceeds the rate of division. Nutrient exhaustion and waste toxicity dominate.

Plotting the Growth Curve

The growth curve is typically plotted as:

• Log₁₀ (number of cells) on the y-axis (to accommodate the exponential range)
• Time on the x-axis

Using a logarithmic scale linearises the exponential phase, making it easier to determine the generation time from the gradient.

Exam Tip: In the lag phase, although cell number does not increase, the cells are metabolically active — they are producing enzymes and growing in size. Do not state that "nothing is happening" in the lag phase.

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Culture Media

Bacteria are grown on or in culture media — substances that provide the nutrients required for growth.

Types of Culture Media

Type	Description	Example
Nutrient broth	Liquid medium containing peptone, beef extract, NaCl, and water	Used for growing bacteria in liquid suspension (e.g. for population counts)
Nutrient agar	Nutrient broth solidified with agar (a polysaccharide from seaweed)	Used for growing bacteria as colonies on plates
Selective media	Contains specific agents that inhibit the growth of certain organisms while allowing others	MacConkey agar (selects for Gram-negative bacteria)
Differential media	Contains indicators that differentiate between organisms based on metabolic activity	MacConkey agar (turns pink if bacteria ferment lactose)
Minimal media	Contains only the minimum nutrients for growth (a carbon source, mineral salts, water)	Used for genetic and metabolic studies

Agar Plates

• Agar melts at ~85°C and solidifies at ~40°C. It is poured into sterile Petri dishes and allowed to set.
• Bacteria are inoculated onto the agar surface using a streak plate technique or spread plate technique.
• Plates are incubated upside down to prevent condensation dripping onto the colonies.
• In schools, plates are incubated at 25°C (not 37°C) to reduce the risk of growing human pathogens.

Exam Tip: Always state that in school laboratories, agar plates are incubated at a maximum of 25°C. This reduces the likelihood of culturing pathogenic organisms that thrive at human body temperature (37°C).

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Methods of Measuring Bacterial Growth

1. Total Cell Count (Haemocytometer)

• A small sample is placed on a haemocytometer slide with a grid of known dimensions.
• Cells are counted under a microscope.
• Limitation: Counts both living and dead cells; does not distinguish viable cells.

2. Viable Cell Count (Dilution Plating)

• The sample is serially diluted and known volumes are plated onto agar.
• After incubation, colonies are counted (each colony originates from a single viable cell).
• The number of colony-forming units per cm³ (CFU/cm³) is calculated.
• Advantage: Only counts living cells.

3. Turbidimetry (Spectrophotometry)

• Bacterial growth in liquid culture increases turbidity (cloudiness).
• A spectrophotometer or colorimeter measures the absorbance or % transmission of light through the culture.
• Higher absorbance = more cells.
• Advantage: Quick and non-destructive.
• Limitation: Does not distinguish between living and dead cells.

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Population Growth Calculations

Exponential Growth Formula

N = N₀ × 2ⁿ

Where:

• N = final number of cells
• N₀ = initial number of cells
• n = number of generations

Worked Example

If a culture starts with 1,000 bacteria and the generation time is 30 minutes, how many bacteria are present after 5 hours?

• Number of generations (n) = 5 hours ÷ 0.5 hours = 10
• N = 1,000 × 2¹⁰ = 1,000 × 1,024 = 1,024,000 bacteria

Using Logarithms

To find the number of generations from population data:

n = log₂(N/N₀) = log(N/N₀) / log(2)

Exam Tip: You may be asked to calculate population sizes, generation times, or the number of generations from data. Always show your working and use the correct formula.

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Continuous Culture vs Batch Culture

Feature	Batch culture	Continuous culture
Nutrients	Fixed amount; not replenished	Continuously supplied
Waste removal	Not removed	Continuously removed
Growth curve	Shows all four phases	Maintained in log phase indefinitely
Used for	Laboratory experiments; small-scale production	Industrial fermentation (e.g. antibiotics, insulin production)

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Key Terms

Term	Definition
Binary fission	Asexual reproduction in bacteria; one cell divides into two identical daughter cells
Generation time	The time taken for a bacterial population to double in number
Lag phase	The initial phase of the growth curve where bacteria adapt to new conditions without dividing
Log phase	The phase of exponential growth where the population doubles at a constant rate
Stationary phase	The phase where growth rate equals death rate; population remains stable
Viable cell count	A count of only the living cells in a sample, typically using dilution plating
Turbidimetry	Measurement of bacterial growth by the cloudiness (turbidity) of a liquid culture

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Summary

• Bacteria reproduce by binary fission with a characteristic generation time.
• The bacterial growth curve has four phases: lag, log, stationary, and decline.
• Culture media include nutrient broth, nutrient agar, selective and differential media.
• Growth can be measured by total cell count, viable cell count, or turbidimetry.
• Population calculations use N = N₀ × 2ⁿ.

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A-Level Deep Dive: Bacterial Growth and Culture Techniques

Spec mapping

The Edexcel 9BI0 specification places bacterial population dynamics and laboratory culture within Topic 6: Immunity, Infection and Forensics, with synoptic overlap into Topic 5: On the Wild Side (cellular respiration drives biosynthesis: log-phase cells respire aerobically and ATP synthesis matches the demand of doubling every $\sim 20\,\text{min}$∼20min in E. coli), Topic 1: Lifestyle, Health and Risk (enzyme synthesis as the molecular basis of cell-mass doubling — every protein, lipid and rRNA duplicated each generation), Topic 2: Cells, Viruses and Reproduction (binary fission; prokaryotic chromosome replication initiated at oriC) and Topic 8: Genetics, Populations, Evolution and Ecosystems (bacterial cultures are the workhorse of recombinant-DNA technology — plasmid transformation, blue-white selection and protein expression all assume reliable batch culture). Relevant statements concern: describing the four phases (lag, log/exponential, stationary, death); calculating concentration from serial-dilution viable-count data and from $N = N_0 \times 2^n$N=N0​×2n; distinguishing total counts (haemocytometer) from viable counts (CFU); and applying these techniques to Core Practical 4 (refer to the official Pearson Edexcel 9BI0 specification document for exact wording).

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Worked example with full mark scheme

Question (8 marks):

A student investigates E. coli growth in nutrient broth. After 6 hours of incubation, a $1.0\,\text{cm}^3$1.0cm3 sample is removed and serially diluted in sterile saline by a factor of 10 at each step, through six tubes, to give dilutions of $10^{-1}, 10^{-2}, 10^{-3}, 10^{-4}, 10^{-5}$10−1,10−2,10−3,10−4,10−5 and $10^{-6}$10−6. From the $10^{-6}$10−6 tube, $1.0\,\text{cm}^3$1.0cm3 is spread onto a sterile nutrient-agar plate, incubated at $25°\text{C}$25°C for 48 hours, and the resulting colonies are counted. The plate yields 47 distinct colonies.

(a) Calculate the original viable-cell concentration in the broth, in CFU per cm³, showing your working. (4)

(b) State two assumptions that underlie this calculation, and explain why each matters. (4)

Solution with mark scheme:

(a) Step 1 — recognise that 47 colonies on the $10^{-6}$10−6 plate represent the diluted sample. Each colony, by assumption, originated from one viable cell deposited on the plate.

M1 (AO2.1) — identify the dilution factor as $10^{-6}$10−6 and the plated volume as $1.0\,\text{cm}^3$1.0cm3. Common error: candidates use $10^{-5}$10−5 because they miscount tubes, or forget that "diluting by a factor of 10 six times" gives $10^{-6}$10−6.

Step 2 — back-calculate the original concentration. Since the plated $1.0\,\text{cm}^3$1.0cm3 is itself $10^{-6}$10−6 of the original, the colonies on the plate represent the cells that came from $10^{-6}\,\text{cm}^3$10−6cm3 of the original broth.

M1 (AO2.1) — multiply colony count by the reciprocal of the dilution: $47 \times 10^{6} = 4.7 \times 10^{7}$47×106=4.7×107.

A1 (AO1.2) — express the answer with correct units: $4.7 \times 10^{7}\,\text{CFU}\,\text{cm}^{-3}$4.7×107CFUcm−3. Marks lost where candidates forget units, give "$47 \times 10^{6}$47×106" without converting to standard form, or write the mantissa to inappropriate precision (the count of 47 is two significant figures only, so $4.7 \times 10^{7}$4.7×107 — not $4.70 \times 10^{7}$4.70×107).

A1 (AO3.1a) — comment that $30$30–$300$300 colonies per plate is the standard countable range, so $47$47 is statistically acceptable; below 30 the count is unreliable, above 300 colonies merge.

(b) M1 (AO1.1) — assumption 1: each colony originated from a single viable cell. If two cells settled close enough to fuse into one colony, the count under-estimates the true concentration. This is why aseptic spreading distributes cells evenly and dilutions are tuned to keep colonies well-separated.

M1 (AO2.1) — implication: viable counts systematically under-estimate total cell numbers if clumping occurs (e.g. with chain-forming Streptococcus).

M1 (AO1.1) — assumption 2: all viable cells deposited grow into visible colonies under the chosen conditions. Cells that are viable-but-non-culturable (VBNC), injured, or auxotrophic for a missing nutrient will fail to form colonies, again under-estimating viable count.

A1 (AO3.1a) — implication: the chosen medium, temperature ($25°\text{C}$25°C in school labs) and incubation time must be appropriate for the target organism.

Total: 8 marks.

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Specimen question modelled on the Edexcel 9BI0 paper format

Question (6 marks): A liquid culture of E. coli is inoculated and sampled hourly. The plotted growth curve shows that for the first hour cell number remains constant; over the next four hours it rises rapidly along a straight line on a $\log_{10}$log10​ y-axis; for the next two hours the curve plateaus; and beyond that it begins to fall. Explain the underlying biology of each of the four phases, and discuss why $\beta$β-lactam antibiotics are most effective when added during the second phase.

Mark scheme decomposition by AO:

Marking point	AO	Credit-worthy content
1	AO1.1	States that the lag phase represents enzyme induction, repair of cell-wall damage from inoculation, and accumulation of intracellular metabolites — not "warming up".
2	AO1.1	States that the log (exponential) phase shows constant generation time because nutrients are abundant and waste is dilute; appears linear on a log y-axis because $\log N$logN is linear in time when $N$N doubles regularly.
3	AO1.2	States that the stationary phase is not zero growth but rather division rate equal to death rate, with substrate depletion or quorum-sensing inhibition limiting net growth.
4	AO2.1	States that the death phase reflects waste toxicity and substrate exhaustion exceeding any residual division.
5	AO3.1a	Explains that $\beta$β-lactams (e.g. penicillin) inhibit transpeptidase, the enzyme that cross-links peptidoglycan during cell-wall synthesis; therefore they kill only dividing cells which actively remodel their walls.
6	AO3.2a	Concludes by linking phase to therapy: stationary-phase "persisters" survive $\beta$β-lactam exposure because they are not synthesising peptidoglycan — a clinically significant phenomenon underlying chronic infection and antibiotic-tolerance.

Total: 6 marks split AO1 = 2, AO2 = 1, AO3 = 3. Edexcel rewards candidates who explain mechanism (AO2/AO3) rather than merely naming the four phases (AO1).

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Synoptic links