Topic 4: Antibacterial properties core practical Flashcards
Define antimicrobial (1 point)
- Something that kills microorganisms or inhibits their growth
Plants produce ________ which are toxic to ______________ and store them in hairs on leaves or in roots to protect plants from __________.
chemicals
microorganisms
diseases
What chemicals that are toxic to microorganisms does mint make?
(2 chemicals)
- Menthol
2. Carvone
What chemical that is toxic to microorganisms does garlic make?
(1 chemical)
- Allicin
What chemicals that are toxic to microorganisms does basil make?
(2 chemicals)
- Linalool
2. Rosmarinic acid
What chemicals that are toxic to microorganisms does lemon make?
(1 type of chemical + example)
- Alkaloid compounds e.g. saponin
How do you prepare the agar plates?
5 steps
- The agar has been boiled to sterilise it – the water bath keeps it molten
- Label plate - divide base into 4 and
label each quarter on side of base
– also add date, 3C and initials - Remove agar from water bath and allow
to cool slightly (not too long or it will set in the bottle) - Add the bacteria to the molten agar,
using aseptic technique, then swirl
to mix - Quickly pour the agar into the
petri dish base, swirl gently,
place on flat surface and
leave to set
How do you prepare the discs of plant extract?
3 steps
- Add the same volume of plant extract to each disc
(garlic, mint, lemon) and control (solvent) - Write G, M, L or S next to each disc
- Allow the discs to DRY
How do you put the discs on the agar plate?
2 steps
- Use sterile forceps to place the discs onto the agar,
taking care not to lift the lid off the plate completely - Space out the discs evenly and don’t put them too
close to the edge of the plate
How do you seal the plates?
2 steps + give reason for each step
- Use two small pieces of tape to the plate to fix the lid to the base
– allows oxygen to enter for aerobic conditions to allow
chosen microorganism to grow - Do not seal completely
– this creates anaerobic conditions which encourages
pathogenic anaerobic microorganisms to grow which
could infect humans
How do you incubate the agar plates?
4 points + reason for 3 of the points
- At 25 C
- optimum temperature for the
bacterial species, to allow bacteria
to grow and multiply;
- optimum temperature for the
- Not at 37 C
- prevents growth of pathogenic
microorganisms, which grow
at 37C, which can infect humans;
- prevents growth of pathogenic
- Upside down
- to stop condensation dripping on
bacteria which may stop them growing
- to stop condensation dripping on
- If many separate plates then all at the …
same temperature - 25C
for the same time - 24 hours
What should the result be if the plant extract has antibacterial properties?
(1 point)
If the plant extract has antibacterial properties, it will
produce a zone of inhibition (clear circle around the disc)
which can be measured.
What causes the clear zone of inhibition? (1 point)
This is due to the plant extract diffusing out of disc and
killing the bacteria or inhibiting bacterial growth, so
bacteria do not grow.
Define zone of inhibition
clear area where bacteria do not grow/no bacteria are present
(Antibacterial properties core practical)
Name the dependent variable
Area of zone of inhibition
(Antibacterial properties core practical)
How is the dependent variable measured? (2 steps)
- Measure diameter of zone of inhibition with a ruler
2. Calculate area of zone of inhibition using pi*r^2
The LARGEST zone of inhibition/diameter, the
more bacteria killed/fewer grow, so the _______
antibacterial properties.
greatest
(Antibacterial properties core practical)
What is the method for this practical? (7 steps + 1 point explaining safety)
- Prepare sterile agar plates using aseptic technique and add bacteria
- Soak microbiology discs of same area with the same volume of different plant extracts (independent variable) + one in solvent (water/oil/ethanol) as a control
- Controlled variables for validity - extracts made with same mass of plant, same volume of solvent
- Place discs on surface of agar using sterile forceps
- Same incubation time -24 hours, at same temperature - 25C
- Measure diameter of zones of inhibition and calculate area for each disc (dependent variable)
- Repeat 5 times for each plant extract to identify and repeat
anomalies, obtain 3 concordant results, calculate mean area to
increase reliability
Safety: pathogenic bacteria on plates could infect humans
– do not incubate at 37C, allow oxygen into plate, use aseptic
technique e.g. flame sterilise necks of bottles
(Antibacterial properties core practical)
Describe the aseptic technique (safety of humans/prevent infection + prevents contamination) for this practical
(9 points)
- boil agar before use to sterilise it
- work near a Bunsen burner to provide a convection current
- sterilise/disinfect work surfaces
- heat sterilise equipment eg. flame necks of bottles in a hot blue Bunsen flame
- limit time containers are open - only partially lift lids
from Petri dishes when pouring agar plates, put lids
back on bottles quickly after use, do not put lids on
the bench - use sterile pipettes - only remove from wrapper when just about to use and do not touch end to be used
- place all used pipettes in Virkon disinfectant and wipe up drops or spills immediately using Virkon
- keep lids on all bottles and test tubes when not in use (lids off for minimum time)
- At the end of the experiment: disinfect bench with Virkon, wash hands, sterilise all equipment and used plates in an autoclave
(Antibacterial properties core practical)
Name the independent variable
Type of plant extract
(Antibacterial properties core practical)
How is the independent variable varied?
1 point
Use mint, garlic and lemon - all made up in ethanol (solvent)
(Antibacterial properties core practical)
Name the negative control? + Give 1 point of why use a negative control
No plant extract/chemical – use the solvent which was used to make the extract
For comparison. This shows that any difference between the discs is due to the plant extract treatment given to those discs and not the paper disc or solvent
(Antibacterial properties core practical)
Why are controlled variables needed? (1 point)
For validity
(Antibacterial properties core practical)
Name the controlled organism variables (3 variables)
- Same volume of bacteria added to agar - graduated pipette
- Same species of bacteria - use E.coli
- Same mass of plant in same volume of solvent - mass balance
(Antibacterial properties core practical)
Name the controlled environmental variable (1 variable)
- Same incubation temperature - 25 degrees Celsius - incubator
(Antibacterial properties core practical)
Name the controlled procedural variables
3 variables
- Same surface area of paper discs - use hole punch
- Same incubation time - 24 hours
- Same volume of plant extract on each disc - use micropipette
(Antibacterial properties core practical)
State the risk of this experiment
How can it be minimised? (3 ways)
Risk - infection of humans with pathogenic bacteria
Minimised by:
- using aseptic techniques
- incubating at 25 degrees Celsius and not 37
- allowing oxygen into plates
(Antibacterial properties core practical)
Prepare sterile agar plates (using aseptic technique) - spread E.coli uniformly over the surface of set agar or mix E.coli with molten agar, pour into plate and allow to set
What is the purpose of this step? (2 points)
- Agar is a source of glucose for energy/amino acids for growth, for the bacteria
- Uniform spreading of bacteria throughout agar or across the agar surface
(Antibacterial properties core practical)
Use aseptic technique
What is the purpose of this step? (3 points)
- Used to prevent contamination of pate with microbes from other sources e.g. air
- e.g. use sterile equipment, such as a sterilised graduated pipette, flame sterilise necks of bottles
- For safety - to prevent microbial contamination of laboratory workers
(Antibacterial properties core practical)
Soak microbiology discs with same area with the same volume of plant extract and allow to dry
What is the purpose of this step? (1 point)
- To allow the plant extract to impregnate the paper disc
(Antibacterial properties core practical)
Soak one disc in suitable solvent (water/oil - same as used to make extracts) and allow to dry
What is the purpose of this step? (3 points)
- As a negative control
- To allow a comparison with other discs
- To show that any difference between the discs is due to the treatment given to those discs and not the paper disc
(Antibacterial properties core practical)
Secure the plate lid with two small pieces of sticky tape
What is the purpose of this step? (2 points)
- reduces contamination of culture by other (potentially pathogenic) species of microorganism, which may compete with selected with selected bacteria for glucose
- entry of oxygen allows aerobic conditions and prevents anaerobic conditions which tend to encourage the growth of pathogenic anaerobic bacteria.
(Antibacterial properties core practical)
Incubate for 24 hours at 25 degrees Celsius and not 37 degrees Celsius.
What is the purpose of this step? (3 points)
- 25 degrees C allows bacteria to grow and multiply
- 37 degrees C is human body temperature which encourages rapid growth of pathogenic bacteria that are harmful to humans
- High temperatures could denature bacterial enzymes and kill bacteria
(Antibacterial properties core practical)
Measure diameter of zones of inhibition and calculate area for each disc/plant extract
What is the purpose of this step? (3 points)
- The extract which gives the largest zone of inhibition area has the greatest antimicrobial properties
- Provides quantitative data
- Allows comparison of zones of inhibition and therefore plant extracts
(Antibacterial properties core practical)
Carry out 5 repeats for each plant extract
What is the purpose of this step? (3 points)
- So anomalous results can be identified, discarded and repeated, to obtain 3 concordant results
- So a mean can be calculated
- To improve reliability
