Process for rapid and ultrasensitive detection and counting of microorganisms by fluorescence

A method for detecting and counting separated fluorescent microorganisms arrayed in two dimensions on a solid support and separated by a distance greater than the diameter of an incident laser beam, comprising the steps of:

• collecting microorganisms from a specimen on said solid support, such as a filter membrane;
• labelling said microorganisms with a dye selected from the group consisting of vital dyes, viability markers, fluorescent substances carried by antibodies or nucleic probes, or generated by an enzyme linked probe system, which, when excited, produce an emission fluorescent at a wavelength .lambda..sub.1 ;
• scanning said solid support with said incident laser beam, forming a laser spot having said diameter on said solid support, said laser spot diameter being greater than the microorganism to be detected and wherein said laser spot diameter is between 4 and 14 .mu.m, and wherein a distance between two adjacent scanning lines is such that the entirety of the support is scanned at least twice, by partial overlapping of adjacent scanning paths;
• and simultaneously:
• detecting the resultant fluorescent light at least at one wavelength, by selecting as samples only detected signals exceeding a given threshold, wherein a set of adjacent samples on a scan-line represents a feature;
• establishing a set of correlated-features by line-to-line correlation of individual features, by comparing features on each pair of adjacent lines in time-synchrony, counting the number of lines over which said set of correlated-features occur, each of set of correlated-features forming an event, and eliminating any single uncorrelated-feature;
• comparing said correlated-features on each pair of adjacent lines in time synchrony, at least at two different wavelengths .lambda..sub.1 and .lambda..sub.2 for selecting the correlated-features having an emission intensity ratio at said two wavelengths lower than a predetermined number, said predetermined number being determined by a spectral nature of the dye labelling and said predetermined number being specified that if the emission ratio at said wavelength generated by any correlated samples is greater than a predefined value, the complete event is eliminated;
• determining if, for retained events, the events energy profile in three dimensions is within predetermined Gaussian shape criteria predetermined by the Gaussian energy profile of the laser beam and rejecting events not within said predetermined Gaussian shape criteria.