Topic 4: Antibacterial properties core practical

Question 1

Define antimicrobial (1 point)

Answer 1

1. Something that kills microorganisms or inhibits their growth

Question 2

Plants produce ________ which are toxic to ______________ and store them in hairs on leaves or in roots to protect plants from __________.

Answer 2

chemicals

microorganisms

diseases

Question 3

What chemicals that are toxic to microorganisms does mint make?
(2 chemicals)

Answer 3

1. Menthol

2. Carvone

Question 4

What chemical that is toxic to microorganisms does garlic make?
(1 chemical)

Answer 4

1. Allicin

Question 5

What chemicals that are toxic to microorganisms does basil make?
(2 chemicals)

Answer 5

1. Linalool

2. Rosmarinic acid

Question 6

What chemicals that are toxic to microorganisms does lemon make?
(1 type of chemical + example)

Answer 6

1. Alkaloid compounds e.g. saponin

Question 7

How do you prepare the agar plates?

5 steps

Answer 7

1. The agar has been boiled to sterilise it – the water bath keeps it molten
2. Label plate - divide base into 4 and
   label each quarter on side of base
   – also add date, 3C and initials
3. Remove agar from water bath and allow
   to cool slightly (not too long or it will set in the bottle)
4. Add the bacteria to the molten agar,
   using aseptic technique, then swirl
   to mix
5. Quickly pour the agar into the
   petri dish base, swirl gently,
   place on flat surface and
   leave to set

Question 8

How do you prepare the discs of plant extract?

3 steps

Answer 8

1. Add the same volume of plant extract to each disc
   (garlic, mint, lemon) and control (solvent)
2. Write G, M, L or S next to each disc
3. Allow the discs to DRY

Question 9

How do you put the discs on the agar plate?

2 steps

Answer 9

1. Use sterile forceps to place the discs onto the agar,
   taking care not to lift the lid off the plate completely
2. Space out the discs evenly and don’t put them too
   close to the edge of the plate

Question 10

How do you seal the plates?

2 steps + give reason for each step

Answer 10

1. 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
2. Do not seal completely
   – this creates anaerobic conditions which encourages
   pathogenic anaerobic microorganisms to grow which
   could infect humans

Question 11

How do you incubate the agar plates?

4 points + reason for 3 of the points

Answer 11

1. At 25 C
   
   • optimum temperature for the
     bacterial species, to allow bacteria
     to grow and multiply;
2. Not at 37 C
   
   • prevents growth of pathogenic
     microorganisms, which grow
     at 37C, which can infect humans;
3. Upside down
   
   • to stop condensation dripping on
     bacteria which may stop them growing
4. If many separate plates then all at the …
   same temperature - 25C
   for the same time - 24 hours

Question 12

What should the result be if the plant extract has antibacterial properties?
(1 point)

Answer 12

If the plant extract has antibacterial properties, it will
produce a zone of inhibition (clear circle around the disc)
which can be measured.

Question 13

What causes the clear zone of inhibition? (1 point)

Answer 13

This is due to the plant extract diffusing out of disc and
killing the bacteria or inhibiting bacterial growth, so
bacteria do not grow.

Question 14

Define zone of inhibition

Answer 14

clear area where bacteria do not grow/no bacteria are present

Question 15

(Antibacterial properties core practical)

Name the dependent variable

Answer 15

Area of zone of inhibition

Question 16

(Antibacterial properties core practical)

How is the dependent variable measured? (2 steps)

Answer 16

1. Measure diameter of zone of inhibition with a ruler

2. Calculate area of zone of inhibition using pi*r^2

Question 17

The LARGEST zone of inhibition/diameter, the
more bacteria killed/fewer grow, so the _______
antibacterial properties.

Answer 17

greatest

Question 18

(Antibacterial properties core practical)

What is the method for this practical? (7 steps + 1 point explaining safety)

Answer 18

1. Prepare sterile agar plates using aseptic technique and add bacteria
2. 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
3. Controlled variables for validity - extracts made with same mass of plant, same volume of solvent
4. Place discs on surface of agar using sterile forceps
5. Same incubation time -24 hours, at same temperature - 25C
6. Measure diameter of zones of inhibition and calculate area for each disc (dependent variable)
7. 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 37C, allow oxygen into plate, use aseptic
technique e.g. flame sterilise necks of bottles

Question 19

(Antibacterial properties core practical)

Describe the aseptic technique (safety of humans/prevent infection + prevents contamination) for this practical
(9 points)

Answer 19

1. boil agar before use to sterilise it
2. work near a Bunsen burner to provide a convection current
3. sterilise/disinfect work surfaces
4. heat sterilise equipment eg. flame necks of bottles in a hot blue Bunsen flame
5. 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
6. use sterile pipettes - only remove from wrapper when just about to use and do not touch end to be used
7. place all used pipettes in Virkon disinfectant and wipe up drops or spills immediately using Virkon
8. keep lids on all bottles and test tubes when not in use (lids off for minimum time)
9. At the end of the experiment: disinfect bench with Virkon, wash hands, sterilise all equipment and used plates in an autoclave

Question 20

(Antibacterial properties core practical)

Name the independent variable

Answer 20

Type of plant extract

Question 21

(Antibacterial properties core practical)

How is the independent variable varied?
1 point

Answer 21

Use mint, garlic and lemon - all made up in ethanol (solvent)

Question 22

(Antibacterial properties core practical)

Name the negative control? + Give 1 point of why use a negative control

Answer 22

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

Question 23

(Antibacterial properties core practical)

Why are controlled variables needed? (1 point)

Answer 23

For validity

Question 24

(Antibacterial properties core practical)

Name the controlled organism variables (3 variables)

Answer 24

1. Same volume of bacteria added to agar - graduated pipette
2. Same species of bacteria - use E.coli
3. Same mass of plant in same volume of solvent - mass balance

Question 25

(Antibacterial properties core practical)

Name the controlled environmental variable (1 variable)

Answer 25

1. Same incubation temperature - 25 degrees Celsius - incubator

Question 26

(Antibacterial properties core practical)

Name the controlled procedural variables
3 variables

Answer 26

1. Same surface area of paper discs - use hole punch
2. Same incubation time - 24 hours
3. Same volume of plant extract on each disc - use micropipette

Question 27

(Antibacterial properties core practical)

State the risk of this experiment

How can it be minimised? (3 ways)

Answer 27

Risk - infection of humans with pathogenic bacteria

Minimised by:

• using aseptic techniques
• incubating at 25 degrees Celsius and not 37
• allowing oxygen into plates

Question 28

(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)

Answer 28

1. Agar is a source of glucose for energy/amino acids for growth, for the bacteria
2. Uniform spreading of bacteria throughout agar or across the agar surface

Question 29

(Antibacterial properties core practical)

Use aseptic technique

What is the purpose of this step? (3 points)

Answer 29

1. Used to prevent contamination of pate with microbes from other sources e.g. air
2. e.g. use sterile equipment, such as a sterilised graduated pipette, flame sterilise necks of bottles
3. For safety - to prevent microbial contamination of laboratory workers

Question 30

(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)

Answer 30

1. To allow the plant extract to impregnate the paper disc

Question 31

(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)

Answer 31

1. As a negative control
2. To allow a comparison with other discs
3. To show that any difference between the discs is due to the treatment given to those discs and not the paper disc

Question 32

(Antibacterial properties core practical)

Secure the plate lid with two small pieces of sticky tape

What is the purpose of this step? (2 points)

Answer 32

1. reduces contamination of culture by other (potentially pathogenic) species of microorganism, which may compete with selected with selected bacteria for glucose
2. entry of oxygen allows aerobic conditions and prevents anaerobic conditions which tend to encourage the growth of pathogenic anaerobic bacteria.

Question 33

(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)

Answer 33

1. 25 degrees C allows bacteria to grow and multiply
2. 37 degrees C is human body temperature which encourages rapid growth of pathogenic bacteria that are harmful to humans
3. High temperatures could denature bacterial enzymes and kill bacteria

Question 34

(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)

Answer 34

1. The extract which gives the largest zone of inhibition area has the greatest antimicrobial properties
2. Provides quantitative data
3. Allows comparison of zones of inhibition and therefore plant extracts

Question 35

(Antibacterial properties core practical)

Carry out 5 repeats for each plant extract

What is the purpose of this step? (3 points)

Answer 35

1. So anomalous results can be identified, discarded and repeated, to obtain 3 concordant results
2. So a mean can be calculated
3. To improve reliability