PAG 7.1

Question 1

Equipment

Answer 1

• Bunsen burner
• Antibacterial spray
• Antibacterial waste pot
• Paper towels
• Forceps
• Agar plate
• 5 ml culture of Bacillus subtilis
• Tray
• 100 µl pipette
• Glass spreader
• Marker pen
• 3 different antibiotic discs, A , B and C
• Filter paper disc soaked in distilled water (D)
• Ethanol
• Tape
• Graph or squared paper

Question 2

Method Part 1 1.

Answer 2

1. At all times in this investigation, a Bunsen burner should be burning on the bench.
2. Wipe your bench with antibacterial spray and a paper towel.
3. Take a sterile agar plate and a 5 ml culture of Bacillus subtilis and place in your tray.
4. Working near the flame, carefully open the culture and use the pipette to remove 100 µl of culture, flaming the top of the culture bottle when opening and closing.
5. Lift the lid of the agar plate very slightly and pipette the 100 µl of culture into the centre of the plate, close the lid. Discard the pipette tip into the antibacterial waste pot.
6. Next, take the glass spreader, dip it in ethanol and flame it in a blue Bunsen flame, then use it to spread the culture evenly across the surface of the agar plate.
7. Label the agar plate with the date and your name. Tape the plate securely but do not seal it completely.
8. Incubate the plate upside down for at least 24 hours at 20-25C.

Question 3

method Part 2

Answer 3

9. Collect your plate and observe the lawn of bacterial colonies that have grown.
10. On the bottom of the plate, draw a cross and label the 4 sections “A”, “B”, “C” and “D” using the marker pen.
11. Dip the forceps in ethanol and flame them in a blue Bunsen flame.
12. Take a disc of the first antibiotic to be tested in the forceps.
13. Lift the lid of the agar plate very slightly, only enough to be able to slide the forceps in and gently place the disc in the centre of area A.
14. Dip the forceps in ethanol and flame them in a blue Bunsen flame.
15. Repeat for the other two antibiotic discs and place them in areas B and C, remembering to flame the forceps each time.
16. In area D, place a filter paper disc that has been soaked in distilled water, flaming the forceps as before.
17. Tape the plate securely but do not seal it completely. 18. Incubate the plate upside down for three days at 20-25C.

Question 4

method Part 3

Answer 4

19. After three days, look at the plate but do not open it. Draw and annotate a diagram of the agar plate indicating any clear zones that have appeared in the four areas.
20. Measure any clear zones around the antibiotic and water discs (graph or squared paper can be used).
21. Use your drawing and measurements to draw conclusions about the anti-bacterial properties of each

Question 5

1. What was the purpose of the lit Bunsen burner while you were working?

Answer 5

To create an up-draught to limit aerial contamination.

Question 6

2. Why did you only lift the lid slightly from the agar plate when placing the discs on the agar?

Answer 6

To avoid contamination from microbes in the air.

Question 7

3. Why did you not seal the plate completely?

Answer 7

To avoid the growth of anaerobic microbes.

Question 8

4. Why is 20-25C a more suitable incubation temperature than 37C?

Answer 8

To avoid growing microbes likely to inhabit the human body as this is human body temperature.

Question 9

5. What are your conclusions about the anti-bacterial properties of the antibiotics that you tested?

Answer 9

Depends on antibiotics investigated and student’s own results

Question 10

6. What was the purpose of using a filter paper disc soaked in distilled water in area D?

Answer 10

Control