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Lactobacilli have been consistently associated with dental caries for decades; however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit identification of oral Lactobacillus species is possible, despite their taxonomic complexity. Here we describe a combined approach involving both cultivation and genetic methods to ascertain and characterize the diversity and abundance of the Lactobacillus population in the oral cavities of children with severe early childhood caries (S-ECC). Eighty 3- to 6-year-old children (40 S-ECC and 40 caries free) who were seeking dental care at the Pediatric Dental Clinic of Bellevue Hospital in New York City were invited to participate in this study. Clinical data on socio-demographic information and oral health behavior were obtained from the primary caregiver. The data included a detailed dental examination, children’s medical history, and a questionnaire survey. Combined non-stimulated saliva and supra-gingival plaque samples were collected from each child and cultivated on selective media for quantitative measures of lactobacilli levels. The procedure for Lactobacillus species screening will include the random selection of 50 colonies per plate, extraction of DNA from each colony, and genotyping by arbitrarily primed polymerase chain reaction (AP-PCR). Each unique Lactobacillus AP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis. Lactobacillus species will be identified by comparing the 16S rRNA sequences with the Ribosomal Database and the Human Oral Microbiome Database. Meanwhile, the same set of clinical samples will be independently subjected to genomic DNA isolation, 16S rRNA amplification with Lactobacillus genus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of these Lactobacillus species will be compared between children with or without S-ECC. One of the main objectives of this study is to establish a study protocol for the identification and characterization of lactobacilli in the oral cavity. Future caries risk assessments can include lactobacilli counts (quantitative) and the presence/absence of specific cariogenic genetic signatures of a Lactobacillus species (qualitative) associated with S-ECC.