Pasteur's work had shown that microbes are associated with diseases, but there was no criterion fixing the difference between association and causation. Finding a microbe in diseased tissue does not prove it caused the disease; it may be there as a consequence.

Robert Koch closed that gap in Berlin with method. His presentation of 24 March 1882 announcing the isolation of the tuberculosis bacillus, Mycobacterium tuberculosis, counts as the first case where the chain of proof was assembled completely. The techniques he used also lasted: obtaining pure colonies on a solid medium — thanks to agar, suggested by Angelina Hesse, the wife of an assistant — special staining methods and photomicrography. A year later he isolated the agent of cholera.

The criterion came to be known as Koch's postulates: the agent must be found in every case of the disease, be isolable in pure culture, produce the same disease when given to a healthy host, and be recoverable from that host. Koch himself knew the conditions could not always be met — some people carry a microbe without falling ill (asymptomatic carriage), and some agents cannot be cultured. Viruses, prions and microbiome research stretched the criterion further, and molecularly updated versions are in use. The core of the principle nevertheless holds: a claim of causation is proved not by observation but by controlled reproduction. Koch received the Nobel Prize in Medicine in 1905; despite vaccines and antibiotics, tuberculosis remains one of the deadliest infectious diseases in the world.