1. The microorganism must be present in diseased organisms and absent in healthy organisms
2. The microorganisms must be isolated from the diseased organism and grown in pure culture
3. The cultured microorganism must produce the same disease upon inoculation into a healthy organism
4. The microorganism must be re-isolated and re-cultured form the inoculated organism, and upon culturing, it should be identical to the original isolated microorganism