In this paper we examine how the bacterial growth is influenced by the availability of copper ions in complex Mueller Hinton growth media. The data shows that the free copper concentration is seven to eight orders of magnitude lower the total copper concentration and that there seems to be a better correlation between the free copper concentration and bacterial growth, than for the total copper concentration and growth. Furthermore, it is shown that the initial pH influences the amount of free copper ions in the media and that this has a direct effect on the ability of bacterial cultures to grow. However, there still remains an effect of pH on bacterial growth which cannot be attributed to the influence of the Cu2+ concentration alone. The study also shows that the sterilization treatment can have some effect on the availability of copper ions in the media over time. Freshly autoclaved and sterile filtered media contain the same level of free copper ions and perform equally well in the biological assay. However, storage in the refrigerator influences the free copper contents in media, which have been autoclaved, but not in media, which were sterile filtered. Therefore, the latter method is to be recommended, when working with bacterial copper testing.