An immunologist is evaluating the antibacterial efficacy of three novel essential oils (tea tree, eucalyptus, and lavender) against a strain of Staphylococcus aureus using the disc-diffusion method. The researcher inoculates nutrient agar plates with the bacterial culture and places paper discs impregnated with each essential oil onto the agar surface.
The plates are then incubated at a constant temperature of 25∘C25^\circ\text{C}25∘C for 48 hours48\text{ hours}48 hours.
Identify two variables, other than incubation temperature and incubation time, that must be controlled to ensure a valid comparison of the essential oils' efficacy.
