Culturing microorganisms

How often do bacteria multiply?

Once every 20mins if enough nutrients are available & the temperature is suitable.

How are the effects of disinfectants and antibiotics on microorganisms investigated?

Using uncontaminated cultures of bacteria grown in the lab.

What are bacteria and some other microorganisms grown (cultured) in?

In a ‘culture medium’ that contains the carbohydrates, minerals, proteins and vitamins they need to grow. The culture medium used can be a nutrient broth solution or a solid agar jelly.

State the 2 culture mediums in which bacteria can be grown:

Nutrient broth solution or colonies on a solid agar gel plate.

What nutrients make up a nutrient broth solution?

All nutrients required for bacteria to grow, including nitrogen for protein synthesis, carbohydrates for energy & other minerals.

What do bacteria require to multiply quickly?

An adequate supply of nutrients (carbohydrates, proteins, minerals and vitamins) and an appropriate temperature (which varies depending on species being grown).

What temperatures promote faster growth?

Higher temperatures.

What is the maximum allowed temperature for growth in school labs?

25°C

Why are cultures of microorganisms in school labs not kept above 25°C?

Because above this temperature, more harmful pathogens are likely to grow.

Describe how to make an agar plate:

• Hot agar jelly is poured into shallow round plastic dishes called Petri dishes

• When the jelly’s cooled and set, inoculating loops can be used to transfer microorganisms to the culture medium

• The microorganisms then multiply

Why is it vital that cultures of microorganisms grown in labs are uncontaminated?

Because the presence of unwanted or competing microorganisms affect the growth of the cultures as well as the the validity of any study performed on them. Also can potentially result in the growth of pathogens.

Describe how to prepare uncontaminated cultures:

1. Whenever working aseptically, all work should be carried out in front of a lit bunsen burner with a yellow flame - the flame creates a convection current above the bench preventing contamination of any microorganisms in the air

2. Pour hot agar jelly into a sterilised petri dish and leave to cool and set

3. Pass an inoculating loop through the hot flame before using it to transfer bacteria to the agar plate

4. Open the petri dish as little as possible, at the side facing the bunsen burner

5. Secure the lid of the petri dish with tape at intervals around the dish & store upside down

6. Do not incubate the cultures at above 25°C in a school lab

Why must petri dishes and culture media be sterilised before use?

To kill any bacteria or microorganisms already present that could contaminate the experiment.

Why is the petri dish and medium heated to a high temperature and the inoculating loop passed through a hot flame?

To kill any potential microorganisms that could contaminate the experiment.

Why is the petri dish stored upside down?

To prevent drops of condensation falling onto the agar surface & contaminating it.

Why is the lid of the petri dish only lifted a little?

To reduce the risk of contamination by other microorganisms. It is not to prevent air from entering as air is required by the bacteria grown in school labs - more harmful bacteria tends to be anaerobic.

Why should you write the information on the bottom of the agar plate?

To ensure that the label stays with the sample even if the lid is swapped or lost and it helps avoid confusion between samples.

What is the zone of inhibition?

An area where no bacterial growth has occurred.

What equation is used to calculate the area of a zone of inhibition?

πr²

REQUIRED PRACTICAL: Investigating the effect of Antibiotics on Bacterial growth

1. Use inoculating loops to spread bacterium onto agar plate. Place paper discs soaked in different types (or concentrations) of antibiotics on the agar plate that has an even covering of bacteria, leaving space between discs

2. Antibiotic-resistant bacteria will continue to grow on the agar, while non-resistant strains will die. A clear area will be left where the bacteria have died - called the inhibition zone

3. For the control variable, use a paper disc that has not been soaked in an antibiotic, instead soak it in sterile water, to ensure that any difference between the growth around the control disc and around one of the antibiotic discs is due to the antibiotic alone.

4. Leave the plate for 48 hours at 25°C. The more effective the antibiotic is, the larger the inhibition zone.

Describe the technique you would use to place the discs containing each antiseptic/antibiotic onto the plate.

Use sterile forceps to carefully pick up each disc and gently lift up the lid of the agar plate just enough to place the disc flat on the surface of the agar. Press down slightly and then quickly replace the lid to prevent contamination.