A Science and Plants for Schools – Student Project Starter
Investigating Antimicrobials
This is a project starter, suitable for Advanced Higher biology investigations or A-level
extended projects. Don’t forget to credit this resource in your bibliography by including the
title, the website, the web address and the date you accessed it.
Background information
One often sees claims that a wide range of products, both synthetic and
natural, are antibacterial, antifungal or both. Disc diffusion tests provide a
simple and reliable method of testing antibacterial or anti-yeast properties.
Sterile discs of filter paper can be impregnated with standard quantities of the
substance to be tested. They are then placed on a freshly spread lawn plate
of the test organism. After incubation, the effectiveness of the substance is
determined by measuring the area of inhibition of growth around the disc.
For materials which are not liquid, extracts or infusions can be prepared. The
effects of preparation method of the material could be explored. Emulsions or
creams may need to be mixed with water, another useful point for
consideration.
A number of variables that influence the diffusion of the antimicrobial
substance may affect the size of areas of inhibition of growth. For example:
 depth of the agar
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concentration of agar in the medium
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age of the plates (they may have dried out)
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pH of the medium
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temperature and other relevant conditions of incubation
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size of inoculum
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spread of inoculum
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rate of growth of inoculum
These are useful discussion points in the planning and evaluation of project
work.
An investigation from Science and Plants for Schools, www.saps.org.uk/students
Wells
Another way of applying the test material to a plate is by placing it in wells
made in the agar plate.
Wells can be made in agar plates by using the wide end of a blunted sterile
Pasteur pipette, inserting it and twisting it slightly to remove the plug of agar.
Alternatively, cork borers sterilised with alcohol may be used. A mounted
needle or a pair of forceps, sterilised by flaming in alcohol, may be required to
remove the agar.
The zone of inhibition can be traced on to an acetate grid with felt pen and
measured against squared or graph paper to determine its size.
Antimicrobials incorporated in agar
The above techniques are not suitable for examining the effects of
antimicrobials on moulds. However, the agar plug technique for making wells
can be used to provide a standard size inoculum of a mould which is
inoculated on to agar containing the test substance. Some substances may
not be suitable for incorporation into agar e.g. if they are too oily.
The inoculum should be taken from a non-sporing region of a 3 – 4 day old
mould colony. Note: If the culture is old or producing large numbers of
spores, it should not be opened.
After incubation, the size of the colony can be measured, either by measuring
the diameter or determining the area from a squared acetate grid.
All of the above techniques provide good quantitative results which can be
graphed and analysed.
An investigation from Science and Plants for Schools, www.saps.org.uk/students
Technical information
A student who does a project to investigate antimicrobial activity is
likely to need the following equipment and materials
Equipment
 Broth cultures of bacteria or yeasts
 Sterile nutrient or yeast glucose agar plates
 Sterile spreader
 Sterile Pasteur pipettes
 Forceps
 Alcohol
 Sterile filter discs
 Marker pen
 Antimicrobials e.g. disinfectants, antibacterial handwash or other
products, T-tree oil, garlic, cinnamon
Preparation of Materials
All work involving micro-organisms including preparation of sterile materials
and disposal should be carried out with reference to the following publications
by SSERC:
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Microbiology and Biotechnology: A Code of Practice for Safe Working
with Micro-organisms.
Microbiology and Biotechnology: Microbiology Techniques Cards
Microbiology Techniques CD-ROM
Sterile nutrient agar should be prepared in accordance with manufacturer’s
guidelines.
Note: It should be pointed out to students that the outcome on a plate does
not necessarily reflect efficacy in vivo.
An investigation from Science and Plants for Schools, www.saps.org.uk/students
Starter experiment
The effect of different disinfectants on a range of bacteria
Introduction
Disinfectants are chemicals which inhibit the growth of bacteria. They are
used to cleanse areas where it is important to have a low risk of
contamination e.g. hospitals and domestic kitchens and bathrooms (N.B. they
do not sterilise the area).
Materials
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Broth culture of Micrococcus luteus
2 sterile nutrient agar plates
Sterile Pasteur pipettes
Sterile spreader
Forceps
Alcohol
Discard jar containing appropriate disinfectant
6 sterile filter discs
Marker pen
Disinfectants (4 different types)
Instructions
1. Label the underside of each plate with initials, date, name of organism
and disinfectants as shown in diagram.
A
B
C
D
2. Using a sterile Pasteur pipette, aseptically transfer a pipette-ful of bacterial
culture to the centre of a sterile agar plate.
3. Flame the spreader with alcohol and allow to cool.
4. Lift the lid of the Petri dish, spread the culture across the plate, replace lid,
then flame the spreader in alcohol again.
5. Flame forceps in alcohol. Allow to cool.
An investigation from Science and Plants for Schools, www.saps.org.uk/students
6. Partially lift the lid of the Petri dish and remove a sterile filter disc with the
forceps.
7. Using aseptic technique, dip the filter disc into disinfectant A, hold till it
stops dripping and place carefully above the label ‘A’ on a plate seeded
with M luteus.
8. Repeat steps 2, 3, and 4 on to ‘B’, ‘C’, and ‘D’ for the other disinfectants.
9. Seal the plates diametrically with sellotape.
10. Incubate for 48 hours at 30C.
Results
1. Draw the appearance of the resulting plates.
2. Record in a table the diameter of the zones of inhibition of growth for each
disinfectant.
3. Make a bar chart of the class results.
Conclusion
Which disinfectant is most effective against your organism?
Can you think of any ways to improve this experiment? Think about quantities
of disinfectant, uniformity of the bacterial lawn and other variables.
Possible project titles
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Investigate the effect of disinfectants against gram positive and gram
negative bacteria (e.g. using Micrococcus luteus or Bacillus subtilis as
Gram positives,
E coli as Gram negative).
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Investigate the effects of natural substances against bacteria and yeasts
(e.g. from garlic, ginger, herbs etc – or proprietary natural substances such
as T-tree oil).
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Investigate the effects of proprietary antibacterial preparations such as
antibacterial hand-washes, surface cleaners etc.
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Investigate the most effective concentration of proprietary antibacterial
substances.
An investigation from Science and Plants for Schools, www.saps.org.uk/students