Scientists investigated the effectiveness of three different antibiotics (labeled A, B, and C) against a strain of Escherichia coli (E. coli) bacteria.
They placed paper discs containing the antibiotics onto an agar plate inoculated with the bacteria. They also placed a control paper disc, labeled D, containing no antibiotic.
The plate was incubated and the results are shown in the diagram below:

What is the purpose of the control disc D in this investigation?
The diameter of the clear zone of inhibition around antibiotic disc A is 24 mm24\text{ mm}24 mm.
Calculate the area of this zone of inhibition.
Use the equation: Area=πr2\text{Area} = \pi r^2Area=πr2
Take π≈3.142\pi \approx 3.142π≈3.142. Give your answer to 3 significant figures.
A year later, a new outbreak occurred, and antibiotic A was no longer effective against the E. coli bacteria. Suggest why antibiotic A no longer had an effect.