The first and the fourth test tube exhibit a redish/brown color and the second and third test tubes show a dark blue/black color.
This variation exists due to the fact that amylase was added to the first and fourth test tubes. Amylase acts upon starch and breaks it down to simpler molecules. Therefore, when the IKI solution is added to check for the presence of starch, a negative result is shown. Meanwhile, the starch in the second and third test tubes remained and the IKI test showed a positive result for the presence of starch.