An in vitro model of supragingival plaque associated with gingivitis was characterized by traditional culture techniques, comparative 16S rRNA gene sequencing of isolates, and quantitative PCR (QPCR). Actinomyces naeslundii, Prevotella spp., and Porphyromonas gingivalis increased under conditions emulating gingivitis. Gram-negative species and total bacteria were dramatically underestimated by culture compared to the estimates obtained by QPCR.