What you need to know
- 4.12Core Practical 9: antimicrobialDescribes a substance that kills microorganisms or slows their growth. properties of plants, including aseptic techniqueMethods that stop unwanted microorganisms contaminating a culture and stop cultures escaping, such as flaming loops and using sterile equipment..
- 4.10Conditions required for bacterial growth.
- 4.11Plant substances can have antimicrobial properties.
By the end, you should be able to…
- Describe aseptic technique and why each step is there.
- Control extract concentration, solventA liquid in which other substances (solutes) dissolve. Water is a good solvent for ions and polar molecules. and incubation conditions.
- Interpret zone diameter without over-claiming clinical effectiveness.
Build your understanding
Keep the culture the one you chose
Use only the non-pathogenic speciesA group of organisms that can interbreed to produce fertile offspring. your school provides. aseptic techniqueMethods that stop unwanted microorganisms contaminating a culture and stop cultures escaping, such as flaming loops and using sterile equipment. aims to prevent contamination of the culture, the worker and the room. Work on a cleaned bench, have a lit Bunsen if that is the method, flame the neck of bottles, flame spreaders or forceps between uses, lift lids at an angle, and do not place lids on the bench inner-side down.
Tape plates, incubate inverted at the approved temperature (often about 25 °C, not 37 °C), and do not completely seal the dish. Dispose of all cultures in the designated autoclaveA machine that sterilises equipment and media with steam under pressure, at about 121 °C. bag. Never open an incubated plate to sniff it. Wash hands at the end.
Set up a comparable test
Independent variable: plant species or extract type. Dependent variable: diameter of the zone of inhibitionThe clear area around an antibiotic disc on a bacterial lawn, where bacteria have not grown. after a stated time. Controls: volume of extract per disc, solventA liquid in which other substances (solutes) dissolve. Water is a good solvent for ions and polar molecules., disc size, bacterial lawn density, agar type, incubation temperature and time. Include a solvent-only disc. A known antimicrobialDescribes a substance that kills microorganisms or slows their growth. disc may be used as a comparison if the teacher provides it — it is not a medicine cabinet exercise.
Spread a lawn so colonies merge; otherwise zones are hard to define. Place discs with flamed forceps and do not move them once they have touched the agar. Incubate for the stated time so growth can occur around inactive discs.
Read and evaluate
Measure zone diameter through the centre of the disc, in millimetres, including or excluding the disc consistently and stating which. Read it through the base without opening the lid, and take the mean of two diameters at right angles, because zones are rarely perfect circles. If there is no clear zone beyond the disc, record 0 and say so. Repeat plates. A zero zone means no detectable inhibition on that plate, not that the plant never produces antimicrobials.
Limitations: extracts are mixtures of unknown concentration; diffusionThe net movement of molecules from a region of higher concentration to a region of lower concentration. It is passive. in agar is not the same as distribution in a body; some substances are bacteriostaticDescribes an antibiotic that stops bacteria growing and dividing, without killing them. (stop growth) rather than bactericidalDescribes an antibiotic that kills bacteria. (kill). Contamination invalidates a plate. Do not taste extracts or apply them to skin as a treatment.
| Step | Purpose |
|---|---|
| Flame tools / bottle necks | Kill or reduce microbes on surfaces that will touch the culture |
| Open lids minimally | Reduce the chance of airborne microbes landing on agar |
| Incubate at about 25 °C | Allow the provided species to grow while being less selective for human-body-temperature pathogens |
| Solvent control disc | Separate solvent effects from plant substances |
The plant antimicrobials lab
Spread a lawn of the school’s non-pathogenic bacterium using aseptic technique, add plant-extract discs and a solvent control, incubate, and measure the zones of inhibition. A teaching model: the extracts’ strengths and all results are fictional.
Simulation only. Use only the species your school provides, keep ethanol away from flames, tape lids with two strips, incubate at the approved temperature (about 25 °C), never open an incubated plate, and dispose of everything in the autoclave bag.
Method 8 steps
- Use only the non-pathogenic species your school provides. Disinfect the bench and light a Bunsen on a blue flame if that is the method.
- Label the base of an agar plate with your name, date, species and where each disc will go.
- Flame the neck of the broth bottle, pipette 0.1 cm³ onto the agar while lifting the lid at an angle, and replace the lid.
- Dip the spreader in ethanol (beaker away from the flame), pass it through the flame, let it cool, and spread a confluent lawn.
- Using flamed forceps, place discs soaked in each plant extract, a solvent-only disc and (if provided) a positive-control disc, at least 20 mm apart. Do not move them.
- Tape the lid with two short strips (not sealed all round) and incubate the plate upside down at the approved temperature (about 25 °C) for a stated time, such as 48 h.
- Without opening the plate, measure two perpendicular zone diameters through the base, including the disc, and take the mean. Make at least three plates.
- Put all plates and used equipment in the autoclave bag unopened, disinfect the bench and wash your hands.
Aligned with the order of the specification practical. Simulation only: a teacher sets the real volumes, concentrations and risk controls; this is not a laboratory protocol.
Variables
- Independent
- Plant extract (species of plant).
- Dependent
- Diameter of the zone of inhibition (mm, mean of two perpendicular diameters) after a stated time.
- Controlled
- Volume and concentration of extract per disc
- Solvent, dried before the disc is placed
- Disc size (6 mm)
- Broth volume and lawn density
- Agar type and depth
- Incubation temperature and time
Safety
- Use only the school’s non-pathogenic species. Never open an incubated plate, and never sniff or taste cultures or extracts.
- Ethanol is highly flammable: keep the beaker away from the Bunsen and pass the spreader through the flame only briefly.
- Tape lids with two strips; do not seal all round. Incubate at the approved temperature (often about 25 °C, not 37 °C).
- Dispose of all cultures in the autoclave bag, disinfect the bench and wash your hands.
Follow your school’s risk assessment; these notes are not a substitute for it.
Interpreting
- Mean zone = mean of the plates. For mint, 11, 12 and 13 mm give a mean of 12 mm; solvent control 0 mm (no zone), so mint inhibited this species more than the solvent alone.
- A larger zone means more inhibition here, but zone size also depends on how fast the substance diffuses and its concentration (diameter grows with √ln C), so it is not a direct measure of potency.
- No zone means no detectable inhibition of this species under these conditions, not that the plant has no medical use.
- A zone cannot show whether the extract kills bacteria (bactericidal) or only stops growth (bacteriostatic), and agar is not a human body.
Guided method · step 1 of 13
In the simulator, click the disinfectant spray to wipe down the bench.
Fewer microbes on the work surface means fewer that can reach the agar or your hands.
1 · Your technique
Follow the guided steps: start by disinfecting the bench.
2 · Discs
3 · Plates & incubate
4 · Measure zones
Zone diameter includes the 6 mm disc; no clear zone beyond the disc is recorded as 0. Do one plate by hand first; then auto unlocks. Pick a plate in the rack.
5 · Analyse
Zone size depends on how far the substance diffuses before the lawn is established, and grows only with the square root of ln(concentration ÷ MIC), so a zone twice as wide does not mean an extract twice as strong.
Dispose and evaluate
Plant-extract strengths, zones and contamination chances are fictional teaching values from a simplified diffusion model (Cooper’s critical-time model).
Make your reasoning visible
Name the dependent variable
Zone diameter in mm after T hours at θ °C, mean of n plates.
Do not convert a zone into a tablet
Link the result to 4.11, then stop before claiming a cure.
Avoid this misconception
The claim: No zone means the plant has no medical use of any kind.
The correction: It means no detectable inhibition of that speciesA group of organisms that can interbreed to produce fertile offspring. under those conditions. The plant might affect other microbes, other therapeutic targets, or none.
Check your understanding
Try each question before opening the suggested answer.
Practice question
Retrieval and explanation · suggested answer
Why include a disc soaked only in the solvent used to make the extract?
Show suggested answer
To test whether the solvent itself inhibits the bacteria. Without it you cannot attribute a zone to plant substances.
Practice question
Retrieval and explanation · suggested answer
Give two safety reasons for incubating school plates at about 25 °C rather than 37 °C.
Show suggested answer
First, 37 °C is human body temperature, so it would select for microbes adapted to the body, including pathogens, if any contaminant got onto the plate. Second, any contaminant would grow faster and reach larger numbers at 37 °C in the incubation time, so a contaminated plate would be more hazardous to handle. The school species still grows well at about 25 °C, so the lower approved temperature gives a readable lawn.
Practice question
Retrieval and explanation · suggested answer
Mint extract: zone diameters 11, 13 and 12 mm on three plates (including the 6 mm disc). Solvent control: no zone beyond the disc on any plate, recorded as 0 mm. Calculate the mint mean and state a valid conclusion.
Show suggested answer
Mean = (11 + 13 + 12) ÷ 3 = 12 mm. Under these conditions the mint extract inhibited this bacterium more than the solvent alone. It is not evidence of a safe human treatment.
Take the idea further
Apply the same specification knowledge to an unfamiliar problem. Any extra assumptions are supplied in the question.
Stretch question
A student seals plates completely “to keep them sterile”, incubates at 37 °C “because bacteria like body temperature”, and ranks plants by the darkest extract colour. Identify three faults and the valid alternative for each.
Show a hint
Safety, oxygen and a measured dependent variable.
Explore the reasoning
Complete sealing can favour anaerobes: tape but leave a gas exchange path as instructed. 37 °C increases risk: use the approved lower temperature. Colour is not antimicrobial activity: measure zone diameter against a solvent control.
Recall this lesson
Lesson recall questions
Question 1 of 5: Why do many bacteria need a nitrogen source in the medium?
Conditions for bacterial growth
Practice question
Retrieval and explanation · suggested answer
Why do many bacteria need a nitrogen source in the medium?
Lesson: 4A 6: Plant-based Medicines
Try this from memory first.
Beyond the classroomWhy a new plant extract still has a high barResearch · antimicrobial resistance · Enrichment, not required recall
WHO’s traditional-medicine research agenda exists partly because widely used plant preparations are unevenly tested, while antimicrobialDescribes a substance that kills microorganisms or slows their growth. resistance is increasing the need for genuine new agents. A school zone of inhibitionThe clear area around an antibiotic disc on a bacterial lawn, where bacteria have not grown. is a starting observation in that much larger evidence process.
Back to the spec: 4.12: aseptic, controlled testing is the minimum standard before anyone talks about therapeutic use.
WHO traditional, complementary and integrative medicine research agenda 2025–2034Question
What would a researcher still need after a clear zone on agar?
Explore a possible response
The identity and concentration of the active substance, toxicity data, a reproducible preparation, and later phased clinical evidence if human use is proposed.