Pearson Edexcel International A Level Biology: Culturing Microorganisms and Bacterial Growth (YBI11) – Revision Notes

These notes condense statements 6.1 to 6.4 of Topic 6, including Core Practical 13, from the Pearson Edexcel International Advanced Subsidiary/Advanced Level Biology specification (YBI11), Issue 2. Topic 6 is International A2 (IA2) and there are no tiers. Read the full study guide for explanations and test yourself with the practice questions. The course hub, the printable checklist and the free diagnostics show where this fits.

6.1 Culturing and aseptic technique

• Aims: keep unwanted microbes out of the culture, and keep the culture away from you.
• Medium: carbon source, nitrogen source, mineral ions, sometimes vitamins. Broth is liquid. Agar makes it solid.
• Sterilise media and equipment first, in an autoclave (steam under pressure).
• Aseptic technique: wipe the bench with disinfectant; keep a Bunsen burner lit nearby; heat the loop to red then wait for it to cool; pass bottle necks through the flame; keep lids open briefly; use sterile pipettes; short tape strips on plates; inverted incubation at a modest temperature; never reopen incubated plates; sterilise all waste.
• Streak plate: thin spread gives single cells, each making a colony that is a clone.

Step	Reason
Flame loop, then cool	Kills contaminants; hot loop would kill the sample
Work near flame	Rising air carries microbes away from the open vessel
Short tape strips	Lets oxygen in, so anaerobes are not encouraged
Invert plates	Condensation does not fall on colonies
Modest temperature	Does not favour human pathogens

6.2 Measuring growth

Method	What it measures	Living and dead?	Weakness
Haemocytometer count	Cells per volume	Both, unless stained	Slow; suits large cells such as yeast
Dilution plating	Viable cells as CFU per cm³	Living only	Slow; clumps count as one
Dry mass	Biomass	Both	Destroys the sample; needs large volume
Turbidity	Light scatter (absorbance)	Both	Not proportional at high density

Dilution plating formula

CFU per cm³ = (mean colonies ÷ volume plated in cm³) × dilution factor

Method in steps: serial dilution

1. Put 9 cm³ of sterile diluent in each tube.
2. Transfer 1 cm³ of culture into the first tube and mix, giving 10⁻¹.
3. Transfer 1 cm³ of that into the next tube, giving 10⁻². Use a fresh sterile pipette each time.
4. Plate a known volume of the chosen dilutions in triplicate on agar.
5. Incubate, then count plates with a countable number of colonies, about 30 to 300.

Haemocytometer: one large square holds 1 × 10⁻⁴ cm³ (1 mm × 1 mm × 0.1 mm). Cells per cm³ = (mean per square ÷ 10⁻⁴) × dilution factor. Count cells touching two of a square’s edges and ignore those on the other two. Methylene blue stains dead yeast cells blue.

Turbidity: blank with sterile medium, mix before each reading, build a calibration curve against known cell concentrations, dilute dense samples.

Small reminder. 0.5 cm³ of culture added to 4.5 cm³ of broth gives 1 in 10, the same as 1 cm³ in 9 cm³. Four such steps give 10⁻⁴. If 0.1 cm³ gives a mean of 62 colonies: 62 ÷ 0.1 = 620, then 620 × 10⁴ = 6.2 × 10⁶ CFU cm⁻³.

Small reminder: dry mass. A 20 cm³ sample increases from 0.031 g to 0.109 g. The increase is 0.078 g in 20 cm³. Multiply by 50 to give per dm³: 3.9 g dm⁻³.

Which method when? Use dilution plating if you need to know how many cells are alive. Use turbidity if you want many readings from one culture without opening it often. Use dry mass if the cells clump or are filamentous and cannot be counted. Use a haemocytometer for a rapid count of larger cells.

6.3 The growth curve

Phase	What happens	Why
Lag	Little change in number	Cells adapt, make enzymes, take up nutrients
Exponential	Numbers double at regular intervals; log₁₀ N against time is a straight line	Nutrients plentiful, maximum rate of division
Stationary	Number of viable cells roughly constant	Division rate equals death rate; nutrients low, waste high
Death	Viable cells fall	Death exceeds division; nutrients gone, toxic waste

Turbidity stays high in the death phase because dead cells still scatter light. A fall in viable count therefore shows up in dilution plating before it shows up in a colorimeter.

Formulae (exponential phase only)

Quantity	Expression
Population after n generations	N = N₀ × 2ⁿ
Generations	n = (log₁₀ N − log₁₀ N₀) ÷ 0.301
Growth rate constant	k = n ÷ t (generations per unit time)
Generation time	g = 1 ÷ k
Gradient of log₁₀ N against t	0.301 × k

If a question supplies a different form of the formula, use that one.

Method in steps: finding k

1. Pick two points in the straight, exponential section.
2. Subtract the logs: log₁₀ N − log₁₀ N₀.
3. Divide by 0.301 to find n.
4. Divide n by the time between the points.
5. Give units, for example generations per hour. For g, invert k and convert to minutes if needed.

Small reminder. 2.5 × 10⁵ cells cm⁻³ rise to 8.0 × 10⁶ in 2 h. The ratio is 32 = 2⁵, so n = 5 and k = 5 ÷ 2 = 2.5 generations per hour, g = 24 minutes.

6.4 Core Practical 13 in brief

• Aim: investigate the rate of growth of a microorganism in a liquid culture.
• Independent variable: one of temperature, glucose concentration, pH or inoculum size.
• Dependent variable: absorbance at set intervals, or viable counts by dilution plating.
• Controlled: broth volume and type, inoculum volume and density, flask, aeration, timing.
• Control: sterile, uninoculated broth.
• Analysis: plot log₁₀ of cell number or a measure of growth against time, find the exponential phase, calculate k or compare gradients, and use repeats and means.
• Safe: risk assessment, approved safe strain, closed vessels, modest temperature, disinfect spills, sterilise waste, wash hands.
• Ethical: do not release cultures; do not grow unknown organisms; dispose responsibly.

Must-know distinctions

• Total count versus viable count. A haemocytometer and turbidity include dead cells. Dilution plating counts only cells able to form colonies.
• Lag versus stationary phase. Both have a flat curve. In lag, cells are adapting and not yet dividing. In stationary, division is matched by death.
• k versus g. k is generations per time, g is time per generation.
• Gradient versus k. The gradient of a log₁₀ graph is 0.301 × k, not k.

Quick self-test

1. What dilution is the fifth tube of a series in which 1 cm³ is added to 9 cm³ each time?
2. A culture of 1.0 × 10³ cells cm⁻³ has a generation time of 20 minutes. How many cells cm⁻³ after 2 hours of exponential growth?
3. A culture grows from 5.0 × 10³ to 1.6 × 10⁵ cells cm⁻³ in 2.5 hours of exponential growth. Calculate k and g.
4. Name the two features of a growth curve that show it is in the exponential phase.
5. Why is the loop allowed to cool after flaming?
6. A 0.2 cm³ sample of a 10⁻⁴ dilution gives 36 colonies. How many CFU cm⁻³ were in the original culture?
7. One large square of a haemocytometer has a mean count of 24 cells in an undiluted sample. How many cells cm⁻³ is this?
8. A log₁₀ graph of cell number against time has a gradient of 0.60 per hour in the exponential phase. Calculate k.
9. Give a reason why dry mass is used in preference to wet mass.
10. Why does absorbance remain high in the death phase?

Answers

1. 10⁻⁵.
2. 2 h = 120 min = 6 generations. 1.0 × 10³ × 2⁶ = 6.4 × 10⁴ cells cm⁻³.
3. n = log₂ (1.6 × 10⁵ ÷ 5.0 × 10³) = log₂ 32 = 5. k = 5 ÷ 2.5 = 2.0 generations per hour. g = 1 ÷ 2.0 = 0.5 h = 30 minutes.
4. Numbers double at regular intervals, and log₁₀ number plotted against time is a straight line.
5. A hot loop would kill the bacteria being transferred.
6. 36 ÷ 0.2 = 180, then 180 × 10⁴ = 1.8 × 10⁶ CFU cm⁻³.
7. 24 ÷ (1 × 10⁻⁴) = 2.4 × 10⁵ cells cm⁻³.
8. k = 0.60 ÷ 0.301 = 2.0 generations per hour (1.99 before rounding).
9. Wet mass includes a variable amount of water, so it is a less reliable measure of the cells.
10. Dead cells still scatter light, so turbidity does not fall as quickly as the viable count.

Where marks are usually lost

• Forgetting the dilution factor or the volume plated in a CFU calculation.
• Counting plates with too few or far too many colonies.
• Writing that stationary phase means “growth stops” rather than division equals death.
• Explaining the lag phase as “bacteria are dead” or “not alive”.
• Using values from outside the exponential phase to calculate k.
• Dividing a log difference by log 2 correctly but then forgetting to divide by time.
• Describing aseptic steps without the reason for each.
• Leaving out a sterile, uninoculated control in a growth investigation.
• Treating turbidity as a count of living cells.

Official syllabus

Pearson Edexcel International Advanced Subsidiary/Advanced Level Biology (YBI11) specification, Issue 2, published by Pearson Education Limited.