14 Antibiotics Summary

Y13 Core Practical
Antibiotics
Context
Antibiotics are either bactericidal or bacteriostatic. Different bacteria are affected by different antibiotics. The effect
of the antibiotic will depend on its potency, its volume and its concentration. Nutrient agar plates are used to
investigate the effect of different antibiotics on bacterial growth. The antibiotic dissolves in the water of the agar
and diffuses into and through the agar from the antibiotic discs.
Variables
Independent Type of antibiotic At least five antibiotics – e.g. chloramphenicol,
penicillin, fuxocillin, amoxicillin, fusidic acid
Dependent Diameter of inhibition zone Measured using a ruler, taking an average of three
around antibiotic disc measurements
Control Type of bacterium in the Use only Staphylococcus albus

agar plate
Concentration of antibiotic If prepared from dry antibiotic powder, add same
mass of antibiotic to set volume of water each time
e.g. 2g of antibiotic to 10cm3 of water. Cannot be
controlled if using mast rings.
Volume of antibiotic added 0.1cm3 to each antibiotic disc. Cannot be
controlled if using mast rings.
Temperature Incubator set at 30ºC. Below 37ºC avoids growth

of pathogens
Incubation time 48 hours timed using a clock
Humidity and oxygen Sealing plates in the same way to allow same
content access of water and oxygen to the plate and
placing in the same incubator
Size of antibiotic molecules Larger molecules will diffuse more slowly than
smaller ones. This cannot be controlled.
Preliminary procedures
 Identify a species of bacterium which grows successfully on the agar and responds to antibiotics
 Find the optimum temperature at which the bacterium grows
 Find the time taken for bacteria to grow
Validity
Precision
 Same procedure each time
 Compare results in different petri dishes for similarities and anomalies
 Place discs in similar positions in the petri dish to avoid inhibition zones extending to the edges of the dish
where they can’t be measured.
Accuracy
 Use a ruler with an appropriate scale to measure diameter of inhibition zone
Reliability
 Three replicates of each petri dish
 Repeat measurement of each diameter e.g. 3 measurements
Safety
 Disinfect the bench before the practical to remove any potentially harmful bacterial spores that could grow
on the agar.
 Disinfect the bench after the practical to remove any spores of Staphylococcus albus.
 Place a lighted Bunsen burner on the bench to carry any bacterial spores upwards towards the ceiling in a
convection current.
 When transferring the bacterium open the agar plate of a maximum of 45º to prevent he exit of bacterial
spores
 Seal the plat with cellotape in a cross-shape to prevent the lid being removed AND to allow oxygen to enter,
and as such, prevent the growth of potentially harmful pathogenic anaerobic bacteria
 Incubate at 30ºC to reduce the multiplication rate of Staphylococcus, whose optimum temperature is 37ºC.
 Flame forceps and inoculating loops after contact with the bacterium
 Autoclave all equipment after use.
Limitations
 Some inhibition zones may extend to the edge of the dish and therefore be un measurable in certain
diameters.
 Some bacteria may become resistant and regrow within the inhibition zone therefore obscuring the original
zone from measurement
 Different antibiotics are different sizes and therefore will diffuse at different rates. Larger molecules may
take longer to show effects
 Concentration and volume of antibiotics cannot be controlled if using a mast ring
Method
1. Prepare an agar plate with molten agar gel and allow to cool.
2. Turn on a Bunsen burner on a medium flame to create a convection current to waft bacterial spores towards
the ceiling and away from faces.
3. Flame an inoculating loop.
4. Use the inoculating loop to spread a bacterial lawn of Staphylococcus albus on the surface of the agar plate
5. Sterilise forceps by flaming them.
6. Pick up an antibiotic mast ring with the forceps , open the lid of the seeded agar plate to 45º and place the
mast ring in the centre of the agar. Tape the edges to ensure contact with the gel.
7. Tape the dish securely with two pieces of adhesive tape in a cross-shape. Do not seal it completely – this
allows oxygen to enter the petri dish and prevents growth of other anaerobic pathogenic bacteria.
8. Incubate the agar plate upside down for 48 hours at 30ºC (i.e. NOT body temperature).
9. Wash your hands with anti-bacterial soap.
10. Disinfect benches and autoclave equipment.
11. After 48 hours take five repeat measurements of the diameter of each inhibition zone.
12. Calculate the mean for each antibiotic and compare them.

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Y13 Core Practical

Control Type of bacterium in the Use only Staphylococcus albus

Temperature Incubator set at 30ºC. Below 37ºC avoids growth

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