Culturing Microorganisms

Bacteria reproduce by binary fission — a single cell copies its genetic material and splits into two identical daughter cells. This is a form of asexual reproduction, so every cell in the colony is a clone of the original.

Given enough nutrients and a suitable temperature, bacteria can divide as often as once every 20 minutes. This short division time is why a tiny starting population becomes enormous within hours.

The number of cells doubles each division, producing exponential growth:

Divisions	Time (at 20 min each)	Population (from 1 cell)
0	0 min	1
1	20 min	2
2	40 min	4
3	60 min	8
4	80 min	16

Binary fission: one bacterial cell divides into two identical daughter cells. Each daughter can then divide again, so the population grows by repeated doubling.

This topic (culturing microorganisms, including Required practical 2) is in the Biology specification only, not in Combined Science Trilogy.

To find how many bacteria are present after a given time, work in two steps.

Step 1 — find the number of divisions:

$\text{number of divisions}=\frac{\text{total time}}{\text{mean division time}}$

Step 2 — apply the doubling:

$\text{final number}=\text{starting number}\times 2^{(\text{number of divisions})}$

Worked example — start with 100 bacteria, dividing every 20 minutes, for 5 hours:

• Total time = $5\times 60=300$ minutes
• Number of divisions = $300÷20=15$
• Each division doubles the population, so multiply by $2^{15}$
• $2^{15}=32768$
• Final number = $100\times 32768=3276800$ bacteria

(Higher Tier only — express large populations in standard form: $3276800=3.2768\times 10^6$ bacteria.)

A common trap is to divide instead of multiply, or to use the time in hours. Always convert the time and the division time into the same unit first.

To study bacteria, they must be grown on a controlled food supply. There are two standard methods.

• Nutrient broth — a sterile liquid containing nutrients. Bacteria spread throughout it, and growth is measured by how cloudy (turbid) the liquid becomes.
• Agar gel plate — a Petri dish set with nutrient agar. Bacteria fixed in place grow into visible colonies, each one a clump of millions of cells descended from a single bacterium.

Agar plates are preferred for the Required practical because separate colonies and clear zones can be seen and measured.

Investigating the action of disinfectants and antibiotics requires an uncontaminated culture — one containing only the species being studied. If unwanted microbes from the air, skin, or apparatus get in, they compete for nutrients and ruin the results. This is why aseptic technique matters.

Colony: a visible mass of identical bacteria on an agar plate, all descended from one original cell by binary fission.

Aseptic technique is the set of precautions that keeps a culture free from unwanted microorganisms. Each step prevents contamination from a specific source.

Step	Reason
Sterilise Petri dishes and culture media before use	Kills any microbes already present, so only the chosen species grows
Pass the inoculating loop through a flame	Sterilises the loop so it does not introduce other microbes to the media
Work near a Bunsen flame	The rising hot air keeps airborne microbes away from the open plate
Secure the lid with adhesive tape	Stops airborne contaminants getting in, while still allowing some air
Store the plate upside down	Stops condensation dripping from the lid onto the colonies
Incubate at 25 °C in school labs	Limits growth of pathogens that thrive at human body temperature (37 °C)

Exam tip: the tape should hold the lid but should not seal it completely — a fully airtight dish could allow harmful anaerobic bacteria to grow.

In school laboratories, cultures are generally incubated at 25 °C rather than 37 °C, reducing the risk of culturing microbes that are dangerous to humans.

Required practical 2 investigates the effect of antiseptics, antibiotics, or plant extracts on bacterial growth.

The method:

1. Spread bacteria evenly over an agar plate to make a "lawn".
2. Soak paper discs in different antibiotics or antiseptics (plus one control disc soaked in sterile water).
3. Place the discs on the lawn, tape the lid, and incubate at 25 °C.
4. After incubation, measure the clear ring around each disc where bacteria could not grow.

The clear ring is the zone of inhibition. A larger zone means the substance is more effective at killing or stopping the bacteria.

The control disc (sterile water) should show no zone. This confirms that any clear zone is caused by the test substance and not the disc itself.

The zone of inhibition is circular, so its cross-sectional area is found with:

$A=\pi r^2$

where $r$ is the radius of the clear zone. Measurements are usually taken as a diameter, so halve it first.

Worked example — a clear zone has a measured diameter of 16 mm:

• Radius $r=16÷2=8$ mm
• $A=\pi r^2=\pi \times 8^2=\pi \times 64$
• $A=201.06\dots \approx 201.1$ mm² (to 1 decimal place)

Repeats improve reliability, so a result is reported as a mean of several discs.

Worked example — mean zone diameter for three repeat discs of the same antibiotic: 18 mm, 20 mm and 16 mm:

$\bar{x}=\frac{18+20+16}{3}=\frac{54}{3}=18\text{ mm}$

Exam tip: check whether the question gives a diameter or a radius. Using the diameter as the radius is the single most common way to lose marks on this calculation.

1. Dividing instead of doubling

The population multiplies by $2^n$, where $n$ is the number of divisions — it does not grow by adding a fixed amount. With 15 divisions the factor is $2^{15}=32768$, not $15\times 2$.

2. Mixing up time units

If division time is in minutes, the total time must also be in minutes. For 5 hours that is $5\times 60=300$ minutes before dividing by 20.

3. Using the diameter as the radius in $\pi r^2$

A 16 mm zone has radius 8 mm. Substituting 16 gives roughly four times the correct area.

4. Sealing the Petri dish lid completely

Tape secures the lid against airborne contaminants but must not make it airtight — full sealing can encourage harmful anaerobic bacteria to grow.

5. Forgetting the control disc

Without a sterile-water control, you cannot show that a clear zone is due to the antibiotic rather than the paper disc, and the conclusion is weakened.

6. Leaving Higher Tier answers out of standard form

On Higher Tier, a population such as $3276800$ should be written as $3.2768\times 10^6$ when the question asks for standard form.