Patent ID: 8883441

Claim:
A method for detecting and counting individual intracellularly labeled microorganisms in a food, environment or animal-derived sample, wherein background fluorescence is avoided and wherein detection thresholds are on the order of from 10 to 100 microorganisms per gram of sample, comprising the steps of: a) selectively enriching the microorganism sought in the sample, so as to revive the microorganism being sought while destroying microorganisms that are not being sought, b) inducing or activating at least one enzymatic activity specific to the microorganism being sought, c) immunomagnetically concentrating the microorganism, d) fluorescently labeling the microorganism by adding to the sample containing the microorganisms at least one substrate comprising one part specific to the enzymatic activity to be indicated and one fluorogenic label, wherein the transformation of the substrate takes place inside the microorganism and wherein the fluorescent product resulting from the fluorogenic label is retained in the microorganism thereby avoiding the production of background fluorescence and permitting the counting of each individual microorganism, and e) detecting and counting each fluorescently labeled microorganism by flow cytometry wherein in the method, step a) occurs before step c) and wherein the immunomagnetic concentration of step c) comprises the steps of: i) placing the microorganism sought, present in the sample, in contact with an antibody directed against an antigen specific to the microorganism, the antibody being conjugated with a magnetic bead, wherein the magnetic bead has a diameter that is between 1 and 20 μm, ii) separating the bead-antibody-microorganism complexes from the sample, and iii) separating the microorganism from the rest of the complex.