Culturing Microorganisms

• When culturing microorganisms, bacteria grown on agar ‘plates’ will form visible colonies on the surface of the jelly, or will spread out to give an even covering of bacteria.
• 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 (wire loops) can be used to transfer microorganisms to the culture medium. Alternatively, a sterile dropping pipette and spreader can be used to get an even covering of bacteria. The microorganisms then multiply.
• In the lab at school, cultures of microorganisms are not kept above 25 degrees C, because harmful pathogens are more likely to grow above this temperature. In industrial conditions, cultures are incubated at higher temperatures so that they can grow a lot faster.
• You can test/investigate the effect/action of antibiotics/antiseptics on cultures of bacteria and their growth. The first step to do this would be to place paper discs soaked in different types (or different concentrations) of antibiotics on an agar plate that has an even covering of bacteria. Leave some space between the discs.
• When you’re investigating the effect of antibiotics on bacterial growth, once you’ve soaked the discs in the antibiotics, it should diffuse (soak) into the agar jelly. Antibiotic-resistant bacteria (i.e. bacteria that aren’t affected by the antibiotic) will continue to grow on the agar around the paper discs, but non-resistant strains will die. A clear area will be left where the bacteria have died - this is called an inhibition zone.
• When testing the action of antibiotics on cultures of bacteria, make sure you use a control. This is a paper disc that has not been soaked in antibiotic. Instead, soak it in sterile water. You can then be sure that any difference between the growth of the bacteria around the control disc and around one of the antibiotic discs is due to the effect of the antibiotic alone.
• When testing the action of antiseptics on bacterial growth, you want to avoid contamination. To avoid this:

  1. The Petri dishes and culture medium must be sterilised before use (e.g. by heating to a high temperature), to kill any unwanted microorganisms that may be lurking on them.
  2. If an inoculating loop is used to transfer the bacteria to the culture medium, it should be sterilised first by passing it through a hot flame.
  3. After transferring the bacteria, the lid of the Petri dish should be lightly taped on - to stop microorganisms from the air getting in.
• To avoid contamination when investigating the effect of antibiotics on cultures of bacteria, the Petri dish should be stored upside down - to stop drops of condensation falling onto the agar surface.
• You can compare the effectiveness of different antibiotics (or antiseptics) on bacteria by looking at the relative sizes of the inhibition zones. The larger the inhibition zone around disc, the more effective the antibiotic is against the bacteria. You can do this by eye if there are large differences in size. But to get more accurate results it’s a good idea to calculate the area of the inhibition zones using their diameter (the distance across).
• To calculate the area of an inhibition zone you need to use the equation: area = pi x r^2.
• The equation used to calculate the area of an inhibition zone (area = pi x r^2) can also be used to calculate the area of a bacterial colony. You just need to measure the diameter of the colony you are interested in first.