Classification: Internal

Core Practical 9: Investigate the antimicrobial properties of plants,

including aseptic techniques for the safe handling of bacteria

Objectives
● To successfully compare the effect of plants on microbial growth
● To understand the safety issues of microbiological work and how to apply good aseptic
techniques

Safety
● Wear eye protection.
● The microorganisms are a potential biological hazard.
● Wash your hands with soap and water before and after the activity.
● Do not open your inoculated plate. Instead, use the alternative plate provided by your teacher.
● Wear gloves when handling mint and garlic.
● Avoid skin contact with the disinfectant.
● Do not decant ethanol near a naked flame and stopper the bottle after use.
● Use aseptic techniques when transferring the bacteria to the Petri dishes.
● Only open the Petri dish slightly when adding the discs with forceps (not fingers) and, once it is
closed with some strips of tape, do not open it again.
● Disinfect the bench before and after working. Leave the disinfectant on the bench for about
10 minutes.
● If the agar plate shows signs of contaminating growth, do not open the plate. A technician will
destroy it by autoclaving.

Procedure Notes on procedure

.Day 1
1. Wash your hands with soap and
water and disinfect your bench area,
leaving it to soak for 10 minutes
before wiping it down.
2. Place a piece of garlic in the mortar
and grind it into a paste with the
pestle. Add 10 cm3 of alcohol to the
garlic paste.
3. Use the forceps to pick up a paper
disc and place it in the mortar to
soak up the garlic and alcohol
solution.
● It is important that students have practised the
required aseptic techniques before the practical
task. Teachers should be familiar with microbiology
techniques. Ensure that good practice (e.g. use of
sterile materials, stoppering of culture flask with
non-absorbent cotton wool, autoclaving of
equipment and culture flask) is carried out.
● This method does not use flaming of equipment on
safety grounds so disinfectant solutions need to be
used instead.
● The temptation for students to embed the dish in
the agar needs to be challenged. A gentle press on
the surface is all that is needed.

4. Use the forceps to place the disc on
the sterile agar plate to dry.
5. Use a marker pen to mark four
quarters on the bottom of the agar
plate that has been seeded with
bacteria; label one garlic, one mint
and two control.
● The paper discs must be dry before placing onto
the seeded agar plate otherwise there will still be
ethanol present which is itself a disinfectant.
● This activity lends itself to planning and evaluation
questionings. There are many aspects that needed
to be controlled to ensure validity e.g mass of plant
material used.

6. Open the lid of the Petri dish
containing the bacteria-seeded agar.
Open it away from yourself and only
open it slightly. Use the forceps to
place the dry disc in the centre of
the quarter labelled garlic.
● Potential for contamination of plates is quite high so
it is useful to have some prepared in advance so
that some data may be collected.
● To increase the accuracy of the practical squared
paper can be used to calculate the area of the zone
of inhibition instead of just the diameter.

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Practical activities have been safety checked but not trialled. Users may need to adapt the risk assessment
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Classification: Internal

7. Close the Petri dish. ● Discuss the need to seal dishes in the criss-cross
8. Clean the pestle and mortar, then pattern as opposed to sealing.
repeat steps 2–7 with the mint. ● The incubation temperature also lends itself to
9. Add a small amount of alcohol to the exploration through questioning.
small beaker. ● Though the method suggests using ready-made
10. Using clean forceps, add two paper agar plates an extension activity could be to allow
discs to the beaker. Remove them students to make their own pour plates. This
and place them on the sterile agar method would need a further risk assessment.
plate dish to dry. ● Extension activities could include creating an
11. Open the lid of the seeded Petri extract using one of the plants suggested and then
dish, making sure you open it away diluting it. Alternatively, the plant extracts could be
from yourself and only open it compared to cleaning products, toothpastes or
slightly. Add one disc to each of the mouth-washes for efficacy.
quarters labelled control.
12. Place one small piece of tape on
each side of the Petri dish lid to hold
it down. Do not tape it all the way
around.
13. Invert the plate and incubate at a
temperature no higher than 30 °C for
24 hours; alternatively, leave at
room temperature for 48 hours.
Day 2
14. Use a ruler to measure the diameter
of the clear zone around each disc.
(It is also possible to measure the
area of the clear zone using squared
paper.) Record your results in a
suitable table.
15. Clear away all the equipment you
have used. Petri dishes should be
returned for sterilisation.
Wash your hands and disinfect
surfaces before leaving the
laboratory.

Answers to questions
1. To allow comparison and to check that the paper discs themselves were not responsible for the
inhibition of growth. The control discs enable us to be sure that the only factor that was different
was the independent variable.
2. It is necessary to incubate for about 2 hours to allow sufficient time for the chemicals in the
extract to diffuse into the agar to inhibit the growth of bacteria.
3. Temperatures above 30 °C are closer to human body temperature, so there is a risk of
incubating human pathogens which could infect you.
4. Aseptic techniques are vital in microbiology to ensure there is no contamination of cultures by
microorganisms from the environment, and that people and the environment are not
contaminated by the microorganisms being handled. Even cultures thought to be low risk should
be treated with caution because:
● bacteria may mutate to form pathogenic strains
● our knowledge of the hazards may be incomplete
● the culture may have become contaminated.

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Practical activities have been safety checked but not trialled. Users may need to adapt the risk assessment
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Classification: Internal

5. Dependent on student results but must include reference to the size of the clear zone (mint)

6. The shape of the clear zone may be irregular, so the diameter may not represent an accurate
measurement. Measuring the area of the clear zone enables a fair comparison to be made
between discs.
7. Size of the molecules in the extract, the rate of diffusion in the agar, concentration and solubility
of the extract

Answers to exam-style questions

1. (a) All numbers should be recorded to the same degree of precision. They should be recorded
to the same number of decimal places so should be 22.50, 22.00, 25.00, 29.50. The data from
the replicates should have been recorded and then the mean values shown.
(b) 397.66 mm2 (πr2 or 1/4πd2)
(c) 39.50-32.00 = 7.5; 7.5/32 x 100 = 23.44% (23%) increase in effectiveness
(d) (i) E.G.: The active ingredients have different molecular sizes and so this affects their
diffusion into the agar gel. Tea tree may have many different ingredients. Some of the
chemicals may remain bound to the paper disc.
(ii) E.G.: because s substance works in a Petri dish does not mean it will work in the same
way on skin; garlic is not poisonous but when ingested may be digested by enzymes and
the active ingredient may not remain intact/active; tea tree oil is poisonous so cannot be
used internally.

© Pearson Education Ltd 2018. Copying permitted for purchasing institution only. This material is not copyright free.
Practical activities have been safety checked but not trialled. Users may need to adapt the risk assessment
information to local circumstances. 3
Classification: Internal

Sample data

Extract used Diameter of zone of inhibition (mm)

mint 22
garlic 17
control 1 0
control 2 0

© Pearson Education Ltd 2018. Copying permitted for purchasing institution only. This material is not copyright free.
Practical activities have been safety checked but not trialled. Users may need to adapt the risk assessment
information to local circumstances. 4