Which observation by Alexander Fleming provided the crucial clue that led to the discovery of penicillin?

A mold colony on a bacterial culture plate was surrounded by a clear zone where bacteria failed to grow

A heated culture had fewer bacteria after incubation, indicating heat-labile substances kill microbes

Patient sera from people recovering from infection always killed bacteria in vitro, implying antibodies were antibiotics

All bacterial isolates from hospital patients were found to be naturally resistant to mold-derived compounds