In the 19th century, there was extensive research on cholera: the disease was generally attributed to miasmatic causes, but this concept was replaced, between about 1850 and 1910, by the scientifically founded germ theory of disease. In 1883, Robert Koch identified the vibrion for the second time, after Filippo Pacini’s discovery in 1854: Koch isolated the comma bacillus in pure culture and explained its mode of transmission, solving an enigma that had lasted for centuries. The aim of this article is to reconstruct the different steps towards the explanation of cholera, paying particular attention to the events occurring in the pivotal year 1854.