Determination of multi-dimensional location, species and genomic copy number by comparative fluorescence imaging of three stained microbial cultures including a lower genomic copy number, slow growth culture

Abstract

The present invention provides a microbial genomic analysis tool, named Spatial Analytical Microbial Imaging (SAMI), which provides the spatiotemporal and comparative intracellular ploidy, indicating the relative growth rate of the cells in situ. Firstly, pure cultures of two microbial species were pre-evaluated for their validity using SAMI. Secondly, the same pure cultures were split aliquot to pure culture and mixed culture, and grown concurrently to reduce error. Another set of pure culture can also be grown as a standard to evaluate inferential comparative genomic copy number if necessary. Thirdly, the genomes of individual cells in the pure culture and the mixed culture are stained with membrane permeable fluorescent DNA markers and analyzed by confocal laser scan microscopy and an image software. The average fluorescent intensity (AFI) and the total genomic fluorescent biding area (GFA) of slow growth pure cultures indicate the population AFI and GFA using inferential statistics. They are used as standards in comparison with the results of the mixed culture to specify the genera, 3D locus and the inferential comparative genomic copy number or a different category of each cell. Fourth, the final results are presented in 3D.