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
¢ concentration of agar in the medium
¢ age of the plates (they may have dried out)
¢ pH of the medium
¢ temperature and other relevant conditions of incubation
¢ size of inoculum
¢ spread of inoculum
¢ rate of growth of inoculum
These are useful discussion points in the planning and evaluation of project work.
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.
A student who does a project to investigate antimicrobial activity is likely to need the following equipment and materials
¢ Broth cultures of bacteria or yeasts
¢ Sterile nutrient or yeast glucose agar plates
¢ Sterile spreader
¢ Sterile Pasteur pipettes
¢ 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:
¢ 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 manufacturers guidelines.
Note: It should be pointed out to students that the outcome on a plate does not necessarily reflect efficacy in vivo.
The effect of different disinfectants on a range of bacteria
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).
¢ Broth culture of Micrococcus luteus
¢ 2 sterile nutrient agar plates
¢ Sterile Pasteur pipettes
¢ Sterile spreader
¢ Discard jar containing appropriate disinfectant
¢ 6 sterile filter discs
¢ Marker pen
¢ Disinfectants (4 different types)
1.Label the underside of each plate with initials, date, name of organism and disinfectants as shown in diagram.
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.
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.
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.
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
¢ 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).
¢ 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).
¢ Investigate the effects of proprietary antibacterial preparations such as antibacterial hand-washes, surface cleaners etc.
¢ Investigate the most effective concentration of proprietary antibacterial substances.