For a study, it was determined that various culture-dependent and culture-independent methods were used to investigate the human gut microbiota, indicating that most bacteria are still uncharacterized and uncultured. The study aimed to establish whether some gut bacteria remain uncultured because of culture constraints rather than because they fail to reach the conclusion of the gastrointestinal tract. The phenomenon was investigated by examining the microbial viability and culturability of the human gut microbiota in fresh feces from eight healthy persons. The researchers used fluorescence-activated cell sorting (FACS) in conjunction with 16S metagenomics analysis and microbial culturomics for the first time. Through metagenomics and culturomics, a total of 1,020 bacterial OTUs and 495 bacterial isolates were discovered, respectively. Only 735 bacterial OTUs were alive in the FACS metagenomics results, accounting for 42% of identified species and 87% of relative abundance per individual. The bacteria that remained were few, dead, or damaged. The research demonstrated that more understanding of the variety and richness of bacteria in the human gut microbiota required both metagenomics and culturomics approaches. Also, it identified the dark matter of the human gut microbiota. More culture efforts were needed to expand the repertoire of cultured gut bacteria by focusing on low-abundance microorganisms. After the anaerobic sample has been put through several steps, it will be incubated in a vial to improve anaerobic sample conditioning and processing.