Microbiology required practical

Aim: To investigate how different antiseptics or antibiotics affect the growth of bacteria by measuring the clear zones they produce on an agar plate.

Variables

• Independent: The type (or concentration) of antiseptic or antibiotic.
• Dependent: The area of the clear zone (zone of inhibition) around each disc.
• Control: Type and volume of bacterial culture spread on the plate; Size of the paper discs and volume of liquid soaked into each; Depth and type of agar; Incubation temperature and time

Equipment

• Sterile agar plate (Petri dish with nutrient agar)
• Broth culture of a safe bacterium
• Sterile pipette or spreader
• Filter paper discs
• Antiseptic solutions and sterile water
• Sterile forceps
• Bunsen burner and heat-proof mat
• Adhesive tape, marker pen, incubator set to 25 °C
• Ruler

Method

1. Wipe the bench with disinfectant and work close to a lit Bunsen burner so rising air carries microbes away.
2. Label the base of the agar plate with your name, date and the positions of the discs.
3. Pass the neck of the culture bottle through the flame, then use a sterile pipette to transfer bacteria onto the agar, lifting the lid only slightly.
4. Spread the bacteria evenly over the whole surface with a sterile spreader to make a lawn.
5. Use sterile forceps to dip each paper disc into a different antiseptic and place it on the agar, spaced well apart.
6. Add one disc soaked in sterile water as a control.
7. Secure the lid with two short strips of tape, not sealed all the way round, and store the plate upside down.
8. Incubate at 25 °C for about 48 hours.
9. Measure the diameter of the clear zone around each disc without opening the dish.

Safety

• Use aseptic technique throughout and never open the plates after incubation.
• Incubate at no more than 25 °C in school so that harmful human pathogens are unlikely to grow.
• Do not seal the lid completely, as a lack of oxygen could encourage dangerous anaerobic bacteria.
• Wash hands afterwards and hand plates to a technician for autoclaving before disposal.

Results and calculations

Record the diameter of each clear zone, halve it to find the radius, and calculate the area using area = πr². Present the mean area for each antiseptic in a table and bar chart; the largest area shows the most effective antiseptic.

Common mistakes and improvements

• Contaminating the plate with microbes from the air or skin; flame equipment, lift the lid only a little and work near the Bunsen.
• Discs placed too close together so clear zones overlap; space them evenly around the plate.
• Irregular clear zones giving unreliable diameters; measure across two directions at right angles and find the mean.
• Uneven spreading of bacteria; spread over the entire surface so growth is a uniform lawn.

Exam tips

• Explain each aseptic step: flaming the loop or bottle neck kills microbes, the lid is taped to stop entry of airborne microbes, and the plate is upside down so condensation does not drip onto the agar.
• Practise calculating the area of a clear zone with πr², remembering to halve the diameter first, and give units such as mm².
• State why a control disc with sterile water is used: to show that the clear zone is caused by the antiseptic and not the disc itself.
• Be ready to calculate bacterial numbers after division using number = starting number × 2 to the power of the number of divisions, for the related culturing calculations.