Required practical 02: Microbiology

Question 1

Describe how you could investigate the effect of antibiotics on bacterial growth using zones of inhibition. (8)

Triple only

Answer 1

1. Spray the bench you are working on with disinfectant then wipe dry with paper towels.
2. On the bottom of the agar plate (not the lid) mark with a wax pencil / permanent marker:
   - (a) 3 segments
   - (b) a dot in the middle of each segment
   - (c) your initials, date, name of bacteria as seen below
3. Wash hands with antibacterial handwash.
4. Place the different antiseptics onto different filter paper discs.
5. Lift the lid of the agar plate at an angle carefully and use forceps to place each filter paper disc onto the dots, noting down the antiseptic applied to each zone.
6. Tape the lid onto the agar plate securely, but loosely enough that oxygen can still reach the
   bacteria.
7. Place the agar plate in the incubator at 25ºC​ for 48 hours.
8. Keeping the lid on, measure the diameter of the clear zones using a ruler and measure again at 90º from your first measurement to take a mean for the diameter.
9. Record the results in a table as seen below. Calculate the area using the formula πr².​

Question 2

Why should you not completely seal the Agar plate?

Triple only

Answer 2

To allow oxygen to enter the agar plate, preventing the growth of harmful anaerobic bacteria.

Question 3

What is the white area surrounding the antiseptic paper disk; what does it determine?

Answer 3

The area of inhibition; the greater the area, the more effective the antibiotic.

Question 4

Why is it necessary to measure the diameter of the zone of inhibition twice?

Triple only

Answer 4

Clear zones are not always uniform - taking more than one measurement allows a mean diameter to be calculated.

Question 5

What equation is used to calculate the area of clear zones?

Triple only

Answer 5

Area = πr²