RP2: Required practical activity 2: Bacterial growth

Independent variable: type (or concentration) of antiseptic or antibiotic being tested.

Dependent variable: diameter of the zone of inhibition around each disc (mm). A larger zone indicates a more effective treatment.

Control variables:

same strain of bacteria

same agar type and volume

same incubation temperature (25 °C) and time (48 hours)

same filter paper disc size

same volume of antiseptic/antibiotic applied to each disc

Spray the bench with disinfectant and wipe clean with paper towels. Wash hands with antibacterial hand wash.

Using a wax pencil, mark the base (not the lid) of the agar plate: divide it into three equal sections, number them 1–3 around the edge, place a dot at the centre of each section, and add your initials, the date and the name of the bacterium.

Soak three filter paper discs in three different antiseptics (or use antibiotic discs). One disc may be soaked in distilled water as a negative control.

Carefully lift the lid of the agar plate at an angle — do not open it fully. Use forceps to place each disc flat onto its labelled dot. Replace the lid immediately.

Secure the lid with two or three small pieces of clear tape. Do not seal all the way around — this would create anaerobic conditions that prevent normal bacterial growth and encourage dangerous anaerobic bacteria.

Invert the plate and incubate upside down at 25 °C for 48 hours (upside down prevents condensation dripping onto the colonies).

Without opening the plate, measure the diameter of the clear zone (zone of inhibition) around each disc using a ruler. Measure twice at 90° to each other and calculate the mean diameter. The area of each zone can then be calculated using πr².

Bacteria are potentially harmful — use aseptic technique throughout (keep the lid closed as much as possible; never open a sealed plate after incubation).

Do not fully seal plates — anaerobic conditions encourage dangerous bacteria; use only 2–3 small pieces of tape.

Do not incubate at 37 °C — this is human body temperature and encourages growth of human pathogens; school labs must use 25 °C.

Bench disinfection — disinfect work surfaces with 1% VirKon solution before and after the practical; wear eye protection when using VirKon.

Hand hygiene — wash hands thoroughly with antibacterial hand wash before and after the practical.

Disposal — all equipment that has contacted bacteria must be sterilised (autoclaved) or soaked in 1% VirKon disinfectant before disposal; never place live cultures in the normal waste bin.