RP2: Culturing Microorganisms

Question 1

What is the aim of this experiment ?

Answer 1

• investigate the effect of different antiseptics / antibiotics on bacterial growth using agar plates by measuring zone of inhibition

Question 2

Explain how you would set up the method

Answer 2

1) spray bench with disinfectant spray and wipe with paper towels
2) place Bunsen burner on heatproof mat in middle of working area and light on yellow flame
3) wash hands with antibacterial hand wash
4) mark underneath of nutrient agar plate making sure lid stays in place to avoid contamination
5) divide plate into three equal sections and number 1, 2, 3 around edge
6) place dot in middle of each section
7) around edge, write initials, date and name of bacteria

Question 3

Describe how you could investigate the effect of antibiotics on bacterial growth using zones of inhibition

Answer 3

1) turn bunsen flame to blue
2) remove lid of bottle containing culture of bacteria and flame neck through bunsen flame, quickly twisting bottle from side to side. using disposable pipette collect approx 1ml of bacterial culture
3) quickly flame bottle neck again and replace lid
4) lift lid of agar plate at an angle so that its only fully open on Bunsen burner side
5) pipette bacteria onto agar plate and replace lid
6) place pipette into discard beaker and turn flame back to yellow
7) dip glass spreader into ethanol and tap off excess, then pass through flame
8) allow flame on glass spreader to go out and allow spreader to cool for 20s
9) lift lid of agar plate at an angle so only Bunsen burner side is fully open and spread bacteria round plate using glass spreader
10) lower agar plate lid and place glass spreader into discard beaker
11) place different antiseptics onto three filter paper discs by soaking them in the liquid or spreading cream or paste onto them
12) lift lid of agar plate as before and use forceps to carefully place each disc into one of the dots on agar plate
13) make a note of which antiseptic is in each of the three sections
14) secure lid of agar plate in place using two small pieces of clear tape
15) incubate plate at 25ºC for 48hrs
16) measure diameter of clear zone around each disc by placing ruler across centre of disc. measure again at 90º to first measurement so that mean diameter can be calculated
17) record results in a table

Question 4

Why should you not seal the lid all the way around at the end of the practical?

Answer 4

• because this creates anaerobic conditions, which will prevent the E.coli bacteria from growing and can encourage other dangerous bacteria to grow

Question 5

What are the risks of practical?

Answer 5

• allergic reactions to antibiotics can occur; only handle discs using forceps
• wash hands after handling bacteria

Question 6

Describe the sources of error

Answer 6

• Shape of the clear zone may be irregular and the width is difficult to determine.
• Contamination from other bacteria may have occurred.

Question 7

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

Answer 7

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