SIMULTANEOUS HYBRIDIZATION AND SUBSEQUENT COLOR DETECTION OF SUBGINGIVAL BACTERIAL-DNA ON COLONY LIFTS

This paper reports the development of a protocol allowing hybridization and detection of DNA fixed to nylon colony lifts from up to three species of bacteria simultaneously. Half ml samples of serial dilutions of pure cooked-meat broth (CMB) cultures of Capnocytophaga ochracea, Actinobacillus actinomycetemcomitans and Prevotella intermedia were grown on trypticase soy blood agar (TSBA) plates for 7 days in an anaerobic chamber. From the same CMBs a further set of dilutions was completed that contained all three species. Samples from these dilutions produced mixed-growth TSBA plates following anaerobic incubation for 7 days. After incubation, colony counts on pure-growth TSBA plates were enumerated by colony counter. Colony counts of C. ochracea, A. actinomycetemcomitans and P. intermedia on mixed-growth TSBA plates were enumerated by nylon colony lift, simultaneous hybridization with non-isotopic whole chromosomal DNA probes and alkaline phosphatase substrates generating three colours. The results indicate that the protocol correctly identified and differentiated between the three species on mixed-growth TSBA plates. The proportions of each species and mean total colony count expected by counting pure plates were in agreement with the proportions of each species and total colony counts enumerated by DNA probes on mixed growth plates. The development of simultaneous hybridization and multicolour detection will result in improved data recovery from dental plaque samples, in addition to reducing the cost and labour required in current colony-lift protocols, without affecting the specificity or sensitivity of the probes used.