Fluorescence assay

The present application discloses a method of identifying and characterising intact pathogens in a sample using a fluorescence imaging system, in particular for characterising bacteria and viruses, and associated systems and kits for implementing the imethod. The method involves incubating a sample with fluorescent probes from at least two of the following categories: a fluorescent probe for binding to a pathogen surface carbohydrate; a nucleic acid stain; and a membrane stain. The sample is then imaged using fluorescence imaging apparatus to detect candidate objects, and the fluorescence characteristics of the candidate objects used to identify whether the object is a pathogen and, if so, the type of pathogen. Particularly preferred implementations employ flow to improve data acquisition, and machine learning classification algorithms to distinguish different pathogen types.