You can grow bacteria, and other microorganisms
safely in a school or college
laboratory by using the correct procedures.
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 (nutrient broth solution) 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
use of aseptic techniques
Aseptic techniques are designed to
avoid contamination by unwanted microorganisms which could
affect your results i.e. the growth of pathogens.
Disinfect all work surfaces in the
laboratory - alcohol is very effective, but very inflammable!
All glassware and other equipment must
be sterilised before use.
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.
This can be done in an autoclave which
uses steam at high pressure to kill any
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!
The liquid bacterial cultures should be
kept in special culture vials with lids.
The lids should only be removed
briefly, when transferring the bacteria to the petri dishes,
to stop other microorganisms getting in.
You can briefly flame the neck of a
glass container of bacteria just after its opened and just
before its closed - the hot convecting air moves air out of
the container preventing microbes in the air getting in.
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.
NOTE: An antibiotic kills bacteria
in the body.
Antiseptics kill bacteria
outside the body e.g. on your skin or disinfecting a worktop in the
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/antiseptics 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.
Using this set-up you can test antibiotics,
antiseptics and plant extracts (*) to investigate their
effectiveness in killing or inhibiting the growth of cultured
(* Some plants produce their own antiseptics
as part of their defence systems against pathogens))
The antibiotics/antiseptics (samples A1 to A4
on the diagram) soaked into the circular paper
discs will diffuse out into the agar jelly and may/may not kill the
If the antibiotic/antiseptic works the bacteria are killed,
inhibiting growth, a 'cleared' area will grow around the disc - called an
inhibition zone - see diagram above.
If the bacteria are resistant to the
antibiotic/antiseptic, the colony will continue to grow on the agar
gel around the paper discs.
The bigger the inhibition zone, the more effective
is the antibiotic/antiseptic against the particular strain of bacteria
growing on the agar gel.
If you have an antibiotic/antiseptic resistant bacteria, then
the bacteria will continue to grow around the paper disc.
You can use this experimental procedure to test both
antibiotics and antiseptics.
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/antiseptic 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/antiseptic A2 has weakly antibacterial
action - small inhibition zone.
Antibiotic/antiseptics/ A3 is a 'moderately'
effective in its antibacterial action.
Antibiotic/antiseptic A4 is very effective in
killing this particular strain of bacteria - the largest inhibition
You can quantitatively measure the
effectiveness of the antibiotics/antiseptics by calculating the area of the dead
bacteria - better and more accurate than just a superficial
You accurately measure, as best you can, the diameter of the circular
area with a ruler (e.g. in mm) where no bacteria are growing any
longer - see on the right of the experiment diagram above.
relative effect of antibiotic/antiseptic
of circle = π x r2 e.g. in mm2.
(pi = 3.14, r = diameter/2)
Sample calculation of relative
Suppose in the experiment the diameter of
the inhibition zones was 10 mm for test sample A3 and 20 mm for
Relative effect of A3 = 3.14 x (10/2)2
Relative effect of A4 = 3.14 x (20/2)2
314/78.5 = 4.0: therefore
antibiotic/antiseptic A4 is four times more effective than A3.
You can use the same diameter measurement
and calculation technique to calculate the area of a colony.
of a strain of bacteria.
Variations on the experiment
You can keep the antibiotic/antiseptic 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.