Preparing a Bacterial Culture Using Aseptic Technique

In order to carry out experiments on bacteria, you need to be able to grow them. This is called preparing a bacterial culture.

On this page, you will learn how to prepare a bacterial culture on an agar gel plate.

A bacterial culture on an agar gel plate.

When preparing a bacterial culture, it is very important to prevent contamination

Contamination is when microorganisms from the surroundings get into your culture, or when microorganisms from your culture escape into the surroundings.

It is important to prevent contamination for the following reasons:

• If microorganisms from the surroundings get into your culture, they could disrupt the experiment (e.g. by killing the bacteria you are trying to culture).
• Microorganisms from the surroundings could be pathogens (microorganisms that cause disease in humans). If they get into your culture they could grow and multiply, increasing the chance of someone getting infected.
• If the bacteria from your culture escape they could cause harm - especially if they are pathogens.

An agar plate which has been contaminated with various microorganisms other than the one the scientist was trying to culture. This has ruined the experiment. Image: Garnhami via Wikimedia Commons (CC BY-SA 4.0 - https://creativecommons.org/licenses/by-sa/4.0/deed.en)

Aseptic technique is used to minimise the chances of contamination

When preparing a bacterial culture, there are steps that you must take to minimise the chances of contamination. Collectively, these steps are called aseptic technique.

Different aspects of aseptic technique will be explained throughout the rest of this page. Then, they will all be summarised at the end of the page.

Equipment for preparing a bacterial culture

To prepare a bacterial culture on an agar gel plate, you will need the following:

• A Bunsen burner.

A Bunsen burner. Image: Eunice Laurent via Wikimedia Commons (CC BY-SA 4.0 - https://creativecommons.org/licenses/by-sa/4.0/deed.en)

• An inoculation loop (a tool used to transfer the bacteria).

An inoculation loop. This one is metal, but sometimes they are made out of plastic. Image: Jeffrey M. Vinocur via Wikimedia Commons (GNU Free Documentation License - https://commons.wikimedia.org/wiki/Commons:GNU_Free_Documentation_License,_version_1.2)

• A bottle of nutrient broth solution with bacteria growing in it.

A nutrient broth solution with bacteria growing in it. Image by Nothingserious on Wikimedia Commons. (CC BY-SA 4.0 - https://creativecommons.org/licenses/by-sa/4.0/deed.en)

• An agar gel plate (to grow the bacteria on). This must be sterilised before you use it.

Agar gel plates. Image: Y tambe via Wikimedia Commons (GNU Free Documentation License - https://commons.wikimedia.org/wiki/Commons:GNU_Free_Documentation_License,_version_1.2)

• Tape (for taping the lid onto the plate).

Tape.

• An incubator.

An incubator.

How to prepare a bacterial culture using aseptic technique

The method is as follow:

1. Light the Bunsen burner. All of the following steps should be done close to the lit Bunsen burner, since it creates an up-draft which prevents microorganism in the air from settling on the equipment.

Light the Bunsen burner.

2. If you are using a metal inoculation loop, pass the end of it through the flame in order to sterilise it. If you are using a plastic inoculation loop, it will have already been sterilised inside its packaging. Take it out of the packaging and hold it in your hand - do not put it in the flame.

If using a metal inoculation loop, pass it through the flame.

3. Remove the lid from from the bottle of nutrient broth solution that has the bacteria in it. Pass the neck of the open bottle through the Bunsen flame to sterilise it.

Remove the lid from the bottle and pass the neck of the bottle through the flame.

4. Place the end of the inoculation loop into the nutrient broth solution. Bacteria will transfer onto the inoculation loop.

Place the end of the inoculation loop into the nutrient broth solution to collect bacteria.

5. Remove the inoculation loop from the bottle. Pass the neck of the bottle through the flame again and then put the lid back on the bottle.

Remove inoculation loop, flame bottle neck again, and put lid back on.

6. Partially lift the lid of the agar gel plate and use the inoculation loop to spread bacteria over the surface of the agar. Then remove the inoculation loop and close the lid.

Use the inoculation loop to spread bacteria over the surface of the agar.

7. If the inoculation loop is metal, pass it through the flame again to sterilise it. If it is plastic, dispose of it in the appropriate contaminated waste bin.

If using a metal inoculation loop, pass it through the flame again.

8. Tape the lid onto the agar gel plate. Then turn the plate upside down so that the agar is at the top and the lid is on the bottom. This will prevent condensation from the lid, which could contain microorganisms, from dripping onto the surface of the agar.

Tape the lid onto the plate and turn it upside down.

9. Place the agar gel plate into the incubator with the temperature set to 25°C. Higher temperature helps the bacteria to grow, however the temperature must be kept below 37°C (human body temperature) since many pathogens of humans grow well at this temperature. Therefore, 25°C is used as a safe temperature which still allow the desired bacteria to grow.

Place the agar gel plate in an incubator at 25°C.

Summary of aseptic technique

The procedure described above includes the following steps which are all part of aseptic technique:

Flashcards

Flashcards help you memorise information quickly. Copy each question onto its own flashcard and then write the answer on the other side. Testing yourself on these regularly will enable you to learn much more quickly than just reading and making notes.