Investigating anti-microbial action
This protocol can be used to investigate the effects of a range of substances that may have anti-microbial action. You can adapt it to see the effects of bactericides (that kill bacteria), bacteriostatic substances (halt microbial growth, such as, some bactericides at low dilutions). The method could be used to compare the efficacy of a range of antimicrobials in personal hygiene products (toothpastes, mouthwashes, deodorants), disinfectants for domestic use, or in extracts from plant materials such as garlic, herbs, spices and essential oils.
This practical is based on ideas for an open-ended investigation
Investigating the effects of antimicrobials published in Practical Microbiology for Secondary Schools © Society for General Microbiology.
Students could make their own pour plates (see Standard technique Making a pour plate), or you could demonstrate how to make one and provide plates freshly prepared by technical staff.
There is scope for developing or assessing a range of experimental and investigative skills (including students' ability to make a risk assessment) if this is set up as an open-ended student-led investigation. Students could develop their own hypotheses to test, based on ideas introduced by the teacher and researched further on-line.
Students could compare:
- different brands of similar products
- the effects of extracts from different plant materials
- the effect of changing concentration on the effectiveness of anti-microbials
- the effect of anti-microbials on different microbes.
Apparatus and Chemicals
For the class – set up by technician/ teacher:
Microbial broth culture – such as Bacillus subtilis, Escherichia coli or Micrococcus luteus
Sterile Petri dishes, 1 or 2 per group
Sterile forceps, 1 or 2 per group
Disinfectant solution, 1 discard beaker per group
For each group of students:
Nutrient agar – in a sterile McCartney/ Universal bottle, 1
Materials with anti-microbial action, according to the students’ hypotheses
Paper discs, Whatman antibiotic assay paper discs, or new filter/ chromatography paper cut with a hole punch then sterilised by autoclaving, 4-8
Health & Safety and Technical notes
Carry out a full risk assessment before planning any work in microbiology (see Note 1 for more details).
Some strains of the bacteria listed above have been associated with health hazards. Use microbes from the ‘safe micro-organisms’ list provided by SGM (Appendix 2, page 31 in Basic Practical Microbiology) or CLEAPSS (Table 15.2 in Section 15 of the Laboratory Handbook). These microbes present the minimum risk given good practice. Recognised educational suppliers should provide safe strains. The E. coli K12 strain is not thought to have any harmful effects, and is preferable because of its more rapid and consistent growth.
Ensure that no members of the group are debilitated or taking immuno-specific medication, as this may increase their risk of infection by the bacteria used.
When using any commercial products, refer to manufacturers’ guidelines, avoid contact with eyes, and limit skin contact.
Take particular care that ethanol used to sterilise instruments or make plant extracts is kept away from lit Bunsen burners. See CLEAPSS Hazcard: ethanol is HIGHLY FLAMMABLE and IDA is HARMFUL.
2 Making plant extracts: Crush 3 g of plant material with 10 cm3 of ethanol (IDA). The CLEAPSS Hazcard describes this as HIGHLY FLAMMABLE (flash point 13 ºC) and HARMFUL (because of presence of methanol). Shake from time to time for 10 minutes.
3 Preparing Whatman antibiotic assay paper discs: Soak the paper discs in the extract or solution to be tested. Remove them with sterile forceps. Allow them to dry on an open, sterile Petri dish, next to a lit Bunsen burner to create an updraught to limit aerial contamination. If the extract contains ethanol, make sure it has evaporated completely before using. Consider the value of using a control disc soaked in ethanol (and dried as above).
Students will need to observe basic hygiene rules and follow aseptic techniques. They need to be aware of what to do with spills and how to dispose of cultures and contaminated equipment.
Your risk assessment should take into account your students’ behaviour and all plates should be stopped by treatment with methanal if students cannot be trusted to leave the plates closed, and not to remove them from the laboratory.
a Discuss antimicrobial action of different substances with the students.
b Explain or demonstrate how to make a pour plate. (See Standard technique Making a pour plate.)
c Explain or demonstrate how to make a plant extract. (Note 2)
d Explain or demonstrate how to use Whatman antibiotic assay paper discs to assess microbial action. (Note 3)
e Allow students time to prepare a hypothesis to test, devise a detailed method and write a risk assessment.
f Each group will need to prepare a pour plate seeded with bacteria, or have one provided. See Standard techniques.
g Make up the solutions to test. You can make a plant extract as described in note 2. It might be interesting to test each substance at different dilutions. There are notes in Standard techniques to help students make serial dilutions.
h Prepare 4 paper discs per pour plate as described in note 3.
i When the agar has set, turn the dish upside down. Divide the base into four sections by drawing a cross with the marker pen. Label the sections A, B, C, D.
j Using sterile forceps, dip a paper disc into the plant extracts. Start with distilled water then move through the plant extracts, working from most dilute to most concentrated.
k Using sterile forceps, place a dried paper disc in each section; record the treatment of each disc. Flame forceps (or place in disinfectant) if they contact the surface of the agar and have been contaminated.
l Label the agar plate with your name and date. Tape the lid but do not seal. Incubate inverted for 2-3 days at 20-25 °C.
m Observe the plates without opening them.
n Make any measurements that will help you to compare the anti-microbial properties of the different substances. A piece of squared paper under the agar plate might be helpful here.
Antiseptics are used to disinfect living tissue – both prophylactically to prevent infection and therapeutically to treat infection. Any given antiseptic is usually more effective against some microbes than others. Its activity may be affected by factors such as dilution, temperature, pH, and the presence of detergent or organic matter.
Many types of toothpaste contain low concentrations of anti-microbials, and mouthwashes claim plaque-killing potential.
Billing and Sherman’s 1998 work (see web links below) explored the patterns of use of spicy cooking ingredients in hot countries and cooler countries. They examined the pattern of use of 43 ingredients and tested their antibacterial properties. The ten spices with the most potent antibacterial effects were garlic, onion, allspice, oregano, thyme, cinnamon, tarragon, cumin, cloves and lemon grass. Many spices with relatively weak antibacterial effects become much more potent when combined; examples are in chilli powder (typically a mixture of red pepper, onion, paprika, garlic, cumin and oregano) and five-spice powder (pepper, cinnamon, anise, fennel and cloves). Lemon and lime juice, while weak inhibitors themselves, also have synergistic effects.
Essential oils are also reputed to have antibacterial or antifungal properties particularly tea-tree, oil of cloves, mint and lavender. If your students investigate the antimicrobial properties of essential oils, ensure that none are ingested – many are toxic in concentrated form.
Human secretions such as sweat and tears also contain lysozyme which has antibacterial properties. Refer to CLEAPSS guidelines (Laboratory handbook section 14.4) for working with body fluids if your students investigate the properties of sweat or tears.
A technique for serial dilution is given in Standard techniques which could be used to make dilutions of a range of branded anti-microbials for testing.
Students could consider the consequences for human health of this array of anti-microbial substances now available and widely used.
Health & Safety checked, May 2009
Students will devise their own details for this procedure. Any record sheet will include headings as appropriate to your specific teaching/ learning objectives for this work, and relevant to your examining specification. The teacher should check the students’ procedures before allowing them to proceed.
Society for General Microbiology – source of Basic Practical Microbiology, an excellent manual of laboratory techniques and Practical Microbiology for Secondary Schools, a selection of tried and tested practicals using microorganisms. These include Effects of antiseptics on microbes.
MiSAC (Microbiology in Schools Advisory Committee) is supported by the Society for General Microbiology (see above) and their websites include more safety information and a link to ask for advice by email.
(Websites accessed, October 2011)
Billing, Jennifer and Sherman, Paul W 'Antimicrobial functions of spices: why some like it hot'. Quarterly Review of Biology, March 1998. An interesting reference to research into the effects of herbs and spices used in cooking across a range of human cultures.