Microscopes & Cultures - Bacterial Cultures and Aseptic Techniques (GCSE Biology)

Bacterial Cultures and Aseptic Techniques

Bacterial Growth

Bacteria (and all prokaryotic cells) replicate themselves in a process called binary fission

1. Plasmids and DNA are copied. First, all the genetic material in the cells are duplicated. This includes the plasmids and circular DNA.
2. DNA strands move to opposite poles of the cells. Next the cells grow in size and the circular DNA move to the two ends (poles) of the cells.
3. The cytoplasm of each cells splits. The cytoplasm of each cell starts to be split in two and new cells wall start being formed.
4. Daughter cells are formed. Two new cells called daughter cells are formed and have one copy of the circular DNA. The two new cells formed will each have one copy of circular DNA and are called daughter cells.
5. For some bacteria, this process occurs every 20 minutes. In the right environment, E. coli are able to duplicate themselves in just 20 minutes

Culturing Bacteria

Culturing is defined as growing microorganisms/ tissues in a controlled medium.

Bacteria can multiply by cell division (binary fission). They can divide as often as once every 20 minutes providing they have sufficient nutrients and are kept at a suitable temperature.

Growing a Bacterial Culture

Bacteria can be cultured and investigated for its properties. However, it must be grown in a culture medium which contains nutrients like vitamins, proteins, minerals and carbohydrates to help the cells grow.

The culture medium can either be a a broth solution or solid agar jelly plate on which bacterial colonies form.

A colony is defined as a cluster of identical cells grown on the surface or within a solid medium derived from a single parent cell.

Preparing a Bacterial Culture Using Aseptic Techniques

It is important when preparing a culture you make sure not to contaminate anything to avoid growing harmful pathogens in the process. This can be done by using aseptic techniques which is a process used to prevent contamination from pathogens.

The following steps outline how you can culture bacteria following aseptic techniques:

1. You should try to work next to a bunsen burner. This will help create an aseptic environment and kill any surrounding microorganisms. You can also keep some alcohol handy to sterilise any equipment.
2. Prepare your agar plate. In a round, clear petri dish, pour hot agar jelly and leave this to set. (In schools, this will usually be done for you already). The petri dish and agar jelly must be heated to a high temperature. This can be done in an autoclave – a machine that uses steam at a high temperature to kill any microorganisms present.
3. Use sterilised inoculating loops to transfer some bacteria to the culture medium (agar jelly plate). The inoculating loop can be sterilised by passing it through a hot flame. Alternatively, a sterile dropping pipette can also be used.
4. Use the inoculating loop to carefully spread the bacteria around the surface of the agar jelly plate. This will give an even spread of the bacterial cells.
5. Close the petri dish lid and lightly tape the sides. This avoids any microorganisms from entering the petri dish.
6. Place the petri dish upside down. This prevents condensation droplets falling on to the agar jelly surface.
7. Leave the plate at around 25°C to allow the culture to grow. In schools, bacteria are not grown at temperatures above 25°C as harmful pathogens are more likely to grow above this temperature.
8. After leaving the petri dish for some time, you will notice bacterial colonies appearing on the surface of the agar jelly.

Investigating the Effects of Antibiotics on Bacterial Growth

The effect of different antibiotics/ aseptics on bacterial growth can be tested using a similar method as described above.

1. Follow steps 1-4 as above to prepare your agar jelly plate with your bacteria spread evenly on its surface.
2. Soak paper discs in different types of antibiotics and place them on top of the agar jelly which has been covered with bacteria. Make sure you know which paper disc represents which antibiotic so you don’t get confused.
3. Use a control paper disc. This shouldn’t be soaked in any antibiotic but instead should be soaked in sterile water. This is to ensure that you know any difference in colony growth is due to the antibiotic alone and not anything else e.g. the paper itself.
4. Cover and tape the petri dish.
5. At the back of the petri dish, label which paper disc represents which antibiotic/ control.
6. Leave the petri dish for 48 hours at no more than 25°C.
7. Interpret your results:
   • You will find your petri dish looks something like this:

• A – it is the control, it has no antibiotics so the bacteria continue to grow around it.
• B – is the strongest antibiotic as it has the largest Zone of Inhibition. A Zone of Inhibition is the clear area on the agar jelly plate where there are no bacteria present as they have been killed by the antibiotic.
• C – is the weakest antibiotic as it has the smallest Zone of Inhibition. The antibiotic has only managed to kill a few non-antibiotic resistant bacteria but other resistant bacteria have survived and continued to grow.
• D – this is the second strongest antibiotic as it has the second largest Zone of Inhibition.

FAQs