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Binary Fission and Culturing Microorganisms

Bacteria reproduce by binary fission, can be cultured in nutrient broth or on agar and are investigated using aseptic technique, antibiotic inhibition zones and area and population calculations. AQA GCSE Biology Higher, specification references 4.1.1.6, 2.2, 2.4, 8.2.2 and 2.1.
  • GCSE
  • AQA
  • Biology
  • Higher
  • Spec 4.1.1.6 · 2.2 · 2.4 · 8.2.2 · 2.1
  • 8 min read
  • 10 revision boxes

Introduction

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1 min read

Growing and killing bacteria

Bacteria are prokaryotic cells, and they reproduce by a type of simple cell division called binary fission. Given plenty of nutrients and a suitable temperature, some bacteria can divide as often as once every 20 minutes, so numbers build up fast.
In the lab you can grow (culture) bacteria in a nutrient broth solution or as colonies on an agar plate. Cultures let you investigate how effective antibiotics, antiseptics and disinfectants are at killing them.
Two calculations come with this topic: working out how many bacteria a population contains after a given time, and working out the cross-sectional area of a colony or a clear area using πr2\pi r^2.

Learning

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"\"4 stages\""

Binary fission step by step

Prokaryotic cells such as bacteria reproduce by a type of simple cell division called binary fission. The cell splits into two, but there is a bit more going on than that.
Flow chart of binary fission: circular DNA and plasmids replicate, the cell grows and the DNA moves to opposite poles, the cytoplasm constricts and new cell walls form, then two daughter cells separate, each with one copy of the circular DNA and a variable number of plasmids.
Bacteria only divide this quickly if the conditions are right: a warm temperature and plenty of nutrients. Some bacteria, such as E. coli, can divide in as little as 20 minutes. If conditions turn unfavourable, the cells stop dividing and eventually begin to die.
  1. The circular DNA and the plasmid(s) replicate, so there are two copies of each ready to be shared out.
  2. The cell gets bigger and the two circular DNA strands move to opposite 'poles' (ends) of the cell.
  3. The cytoplasm begins to divide and new cell walls begin to form between the two halves.
  4. The cytoplasm finishes dividing and two daughter cells are produced. Each one gets a single copy of the circular DNA, but the number of plasmids each cell ends up with is variable.
Follow the four stages in order: replicate the DNA and plasmids, grow and move DNA to opposite poles, begin cytoplasm division and form new cell walls, then produce two daughter cells.
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2 min read

Culture media and making an agar plate

To grow (culture) microorganisms in the lab you need a culture medium: something that supplies the proteins, carbohydrates, minerals and vitamins they need. This can be a liquid nutrient broth solution, or a solid jelly called agar.
To make an agar plate, hot agar jelly is poured into shallow round plastic dishes called Petri dishes and left to cool and set. Once it has set, microorganisms are transferred onto the surface with a wire inoculating loop, which is sterilised first. To get an even covering instead, a sterile dropping pipette and a spreader are used.
A Petri dish holding a layer of set agar jelly, with a wire inoculating loop held above the agar surface ready to transfer microorganisms.
The microorganisms then multiply. Depending on how they were transferred, you either see separate visible colonies dotted on the surface of the jelly, or an even layer of bacteria covering it.
In industrial conditions, cultures are incubated at higher temperatures than in a school lab, so the microorganisms grow a lot faster.
5 precautions

Aseptic technique and why each step matters

If unwanted microorganisms get into your culture, they grow alongside the bacteria you are testing and spoil your results. Worse, some of them could be pathogens (microorganisms that cause disease). An uncontaminated culture means any effect you measure is due to the treatment you are investigating alone.
Aseptic technique is the set of precautions that keeps a culture uncontaminated.
An inoculating loop being sterilised in a Bunsen flame, and a Petri dish with its lid lightly taped on being stored upside down.

  • Sterilise the Petri dishes and culture medium

    Heat them to a high temperature before use. This kills any unwanted microorganisms already lurking on them.

  • Sterilise the inoculating loop

    Pass the wire loop through a hot flame before you transfer the microorganisms, so unwanted microorganisms on the loop are killed.

  • Tape the lid on lightly

    Secure the lid with adhesive tape to stop microorganisms in the air getting into the dish. Tape it lightly rather than sealing it all the way round.

  • Store the dish upside down

    This stops drops of condensation falling onto the agar surface.

  • Incubate at 25 °C, no higher

    In a school lab, cultures are not kept above 25 °C because harmful pathogens are more likely to grow above this temperature. Industrial cultures are incubated hotter.
"\"6 steps\""

Testing antibiotics and antiseptics on bacteria

Before you start, write a hypothesis about which treatment you expect to be most effective.
  1. Spread an even covering of bacteria over an agar plate, using aseptic technique throughout.
  2. Soak small paper discs in different antibiotics, or in different concentrations of the same one, and place them on the plate with space left between them. The antibiotic diffuses out into the agar jelly.
  3. Add a control disc soaked in sterile water instead of antibiotic, so you can be sure any difference in bacterial growth is due to the antibiotic alone and not the paper itself.
  4. Leave the plate for 48 hours at 25 °C.
  5. Look for inhibition zones (clear zones): areas around a disc where the bacteria have not been able to grow. The larger the zone, the more effective that antibiotic is against those bacteria. Antibiotic-resistant bacteria are not affected, so they carry on growing on the agar right up to the discs.
    An agar plate covered with bacteria, with paper discs on the surface. Clear circular zones with no bacterial growth surround the antibiotic discs, while the control disc has bacteria growing right up to it.
  6. Measure the zones carefully so your comparison is accurate, and follow the health and safety precautions for handling bacterial cultures.
Compare the sizes of the inhibition zones to judge the effectiveness of the antibiotics or antiseptics.
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"4 steps"

Calculating the area of an inhibition zone or colony

Working out each clear zone's cross-sectional area gives a more accurate comparison than judging by eye. The bigger the area, the more effective the treatment is against the bacteria.
The same method works for bacterial colonies.
  1. Measure the diameter of the zone (the distance straight across it) with a ruler, reading through the bottom of the dish. Do not open the Petri dish.
  2. Halve the diameter to get the radius, r. It is easy to slot the diameter straight into the equation, but the equation needs the radius.
  3. Put r into area = πr2\pi r^2, the equation for the area of a circle, using the π\pi button on your calculator or the value 3.14.
  4. Give the answer in square units, either mm2\text{mm}^2 or cm2\text{cm}^2, matching the units you measured in.
diameter ÷2\div 2 = radius rr, then area = πr2\pi r^2
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"3 steps"

Working out bacterial numbers from mean division time

The mean division time is the average time it takes for one bacterial cell to divide into two. If you know it, you can work out how many divisions have happened in a given time, and from that how many cells there are.
Each division doubles the population, so the number of cells goes up by a power of 2 every time. That means the totals get very large very quickly, even over a few hours. On Higher tier, write very large answers in standard form.
  1. Put the total time and the mean division time into the same units, usually minutes, so the numbers can be compared. E. coli, for example, has a division time of about 20 minutes.
  2. Divide the total time by the mean division time. This gives you the number of divisions that have taken place.
  3. Raise 2 to the power of that number of divisions: number of cells = 2n2^n, where nn is the number of divisions. Convert the result to standard form if it is a very large number.
Number of divisions = total time ÷ mean division time.
Number of cells = 2number of divisions2^{\text{number of divisions}}.
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Examples

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How many cells after 2 hours 40 minutes?

An E. coli cell has a mean division time of 20 minutes. How many cells will a single E. coli cell produce in 2 hours and 40 minutes?
3 marks

Comparing two antibiotics using zone areas

Two antibiotics, A and B, were tested on a plate covered evenly with bacteria. The inhibition zone around antibiotic A has a diameter of 14 mm and the zone around antibiotic B has a diameter of 20 mm.
Use the areas of the inhibition zones to compare the effectiveness of the two antibiotics.
Agar plate with two clear inhibition zones around paper discs, zone A measuring 14 mm across and zone B measuring 20 mm across.
4 marks

Practice: antiseptic concentrations on an agar plate

Helena investigated four antiseptic concentrations (A to D) on bacteria.
Agar plate covered in bacteria with five paper discs: discs A, B, C and D each have a clear zone with no bacterial growth, C's being the largest and D's the smallest, while the control disc has no clear zone.
a) Suggest which concentration of antiseptic was the strongest. (1 mark)
b) Explain how to calculate the area of Concentration C's inhibition zone. (2 marks)
c) Suggest what Helena used as a control in this investigation. (1 mark)

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Common questions

Why must the control disc contain sterile water?
It shows whether any difference in bacterial growth is caused by the antibiotic rather than the paper disc. The control disc should not contain antibiotic.
Why is a Petri dish stored upside down?
Storing it upside down stops drops of condensation falling onto the agar surface. This helps prevent the culture from being disturbed or contaminated.
Why are school bacterial cultures kept at 25 °C or below?
Harmful pathogens are more likely to grow at higher temperatures. Industrial cultures can be incubated at higher temperatures, but school cultures are kept at 25 °C or below for safety.