You can grow bacteria, and other microorganisms in a school/college
laboratory. You can then test the cultures of the bacteria for the
effectiveness of various antibiotics, antiseptics and disinfectants in
inhibiting and killing a particular bacterial growth.
The setup - equipment and materials (diagram ==>)
The experiments are conducted in glass petri dishes - shallow round
plastic/glass containers over which a tight fitting lid can be fitted.
The bacteria are grown ('cultured') in a culture medium such as agar
jelly (gel) which contains the necessary food for the
microorganism-bacteria to grow. The agar jelly contains carbohydrates,
minerals, proteins and vitamins.
The culture medium can be a nutrient broth solution or the semi-solid
Hot fluid agar jelly is poured into the Petri dish
and left to cool and set to a firm gel-like state.
The selected microorganism must then be
transferred onto the surface of the culture medium.
You can use a dropping pipette and spreader to get
an even coating of bacteria across the surface of the agar jelly.
When a particular bacteria is spread over the surface of the agar gel
(e.g. with an inoculating loop) you will see colonies growing
that will eventually spread over the whole surface, hopefully giving an
even coating of the selected bacteria as it multiplies.
Safety notes and ensuring uncontaminated
cultures prior to testing 'antibacterial agents'
Cultures of microorganisms should not be kept
above 25oC because there is less chance of harmful pathogens
(microorganisms that cause disease) growing at the cooler temperatures.
In research laboratories in universities and
industry, cultures can be safely incubated at higher temperatures to
grow them faster - time is money!
If the culture is contaminated with unwanted
microorganisms, these will affect your results and some of them maybe
Precautions to be taken
The Petri dishes and culture medium - agar
gel, must be all sterilised before conducting the experiment by
heating to a high temperature e.g. ~100oC ???
The higher temperature should kill any
The metal inoculating loop is sterilised
by placing it in a roaring blue bunsen flame until it glows red
- no microorganism will survive this heat treatment!
When the Petri dish is ready with the agar
gel added and set, a lightly taped lid should be placed on it to
stop any microorganisms in air getting in.
The Petri dishes should be stored upside
down to prevent drops of condensation falling on the agar jelly.
Preparation of the test samples and conducting
the experimental investigation
You can use petri dishes of agar jelly plus a
single selected bacteria to test the effectiveness of various
antibiotics, antiseptics and disinfectants in inhibiting and killing a
particular the selected bacterial growth.
You soak small circular paper discs (all the same
size) with different types of antibiotics and place them on the surface
so they are spread out across a evenly bacteria coated
surface of the agar gel.
The bacteria must be evenly spread out to make
it a fair test, and the antibiotic test discs spread out to allow
for the formation of inhibition zones - where the antibiotic is
effective in killing the bacteria (see diagram below, with a
fictitious bacteria strain and four fictitious antibiotics).
The petri dish and contents are left for e.g.
48 hours at ~25oC after which it is ready to be examined
and the results analysed.
The antibiotics soaked into the circular paper
discs will diffuse out into the agar jelly and may/may not kill the
If the antibiotic works the bacteria are killed,
inhibiting growth, a 'cleared' area will grow around the disc - an
inhibition zone - see diagram above.
The bigger the inhibition zone, the more effective
is the antibiotic against the particular strain of bacteria.
If you have an antibiotic resistant bacteria, then
the bacteria will continue to grow around the paper disc.
Analysing the results
On the diagram
C is just a paper disc soaked in
sterile water to act as a control ...
... it should have no effect on bacterial
... neither should it introduce any other
... this is all about a fair test to show that
any inhibition is due to the antiseptic
... and any lack of inhibition is due the
antibacterial properties of the bacteria being investigated.
Antibiotic A1 is an ineffective
antibiotic with respect to the particular bacteria under
investigation - this bacterial strain is antibiotic-resistant with
respect to A1 only.
Antibiotic A2 has weakly antibacterial
action - small inhibition zone.
Antibiotic A3 is a 'moderately'
effective in its antibacterial action.
Antibiotic A4 is very effective in
killing this particular strain of bacteria - the largest inhibition
You can quantitatively measure the
effectiveness of the antibiotics by calculating the area of the dead
bacteria - better and more accurate than just a superficial
You measure the diameter of the circular
area with a ruler (e.g. in mm) where no bacteria are growing any
relative effect of antibiotic = area
of circle =
π x r2 e.g. in mm2.
(pi = 3.14, r = diameter/2)
Variations on the experiment
You can keep the antibiotic constant and vary
You can keep the antibiotic constant and coat
the agar surface with 'strips' of different strains of bacteria.
Alternatively, you can mix an antibiotic with the agar gel and then
treat the surface with various strains of bacteria. You can then
measure the area of growth to test the effectiveness of the
antibiotic in killing that particular bacterium.