Chapter 4: Required Practical 6

How do you test the effects of antibiotics using agar plates

The bacteria you will use are likely to have been grown in a liquid broth (a mixture of distilled water, bacterial culture and nutrients)

Using a sterile pipette to transfer the bacteria from the broth to an agar plate (a Petri dish containing agar jelly). Spread the bacteria over the plate using a

Use sterile forceps to place paper discs soaked with different antibiotics spaced apart on the plate.sterile plastic spreader

Make sure you add a negative control disc soaked only in sterile water

Lightly tape a lid on, invert, and incubate the plate at about 25C for 48hrs. This allows the bacteria to grow.

How do you know how well an antibiotic works?

Anywhere the bacteria can't grow can be seen as a clear patch in the lawn of bacteria

Called the inhibition zone

The inhibition zone tells you how well an antibiotic works. The larger the zone, the more the bacteria were inhibited from growing

Why are aseptic techniques used to prevent contamination

Aseptic techniques are used to prevent contamination of cultures by unwanted microorganisms. This is important because contamination can affect the growth of the microorganisms that you're working with

What aseptic techniques are used when flooding an agar plate with bactera

Sterilisation of equipment by autoclaving (Autoclave is a big machine which sterilises equipment using pressure)

Rub down surfaces with ethanol in order to disinfect work benches

Use a sterilised pipette/syringe to transfer the bacterial culture. This allows the same/accurate volume to be transferred so allows for comparisons to be made

Contaminated utensils should be placed in a beaker of disinfectant

Pre-sterilised plastic instruments are used once, then discarded

Why is it important to flame the neck of the bottle

Pass the neck of the bottle containing the growth medium through the flame on the bunsen burner, remove the bacterial sample and use, re-pass the neck of the bottle through the flame on the bunsen burner

This is done in order to warm the air around the neck of the bottle and so prevents contamination by bacteria/microbes in the air

Other aespetic techniques

Work near a bunsen flame. Hot air rises, so any microbes in the air should be drawn away from your culture

Minimise the time spent with lid off the agar plate, to reduce the chance of airborne microorganisms contaminating the culture

You should also take steps to protect yourself e.g. wash your hands thoroughly before and after handling cultures

What to scientists use a ring composed of antibiotics for?

A ring composed of antibiotics can be placed on to the agar plates to see what concentration of antibiotic is effective

What do scientists calculate?

Scientists calculated the LC50 value: this is the concentration needed to kill 50% of bacteria.

What is the advantage of calculating the LC50 value?

Save time as don't have to wait for all bacteria to die

If a higher concentration is used on agar plate, then the zone inhibition of other disinfectants would overlap (can't see effect of specific disinfectant)

Will probably have different number of initial bacteria so it allows for comparison

What are the variables to control the type of ring soaked within the antibiotic solution/the agar jelly

Using the same size ring

Length of time ring is placed in solution

Type of material of ring