1. The microorganisms must be found in abundance in all organisms from the diseases but should not be found in health organisms.
2. The microorganisms must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be re-isolated from the inoculated, disease experimental host and identified as being identical to the original causative agent.