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

This paper reports the development of a protocol allowing hybridizatio n and detection of DNA fixed to nylon colony lifts from up to three sp ecies of bacteria simultaneously. Half ml samples of serial dilutions of pure cooked-meat broth (CMB) cultures of Capnocytophaga ochracea, A ctinobacillus actinomycetemcomitans and Prevotella intermedia were gro wn on trypticase soy blood agar (TSBA) plates for 7 days in an anaerob ic chamber. From the same CMBs a further set of dilutions was complete d that contained all three species. Samples from these dilutions produ ced mixed-growth TSBA plates following anaerobic incubation for 7 days . After incubation, colony counts on pure-growth TSBA plates were enum erated by colony counter. Colony counts of C. ochracea, A. actinomycet emcomitans and P. intermedia on mixed-growth TSBA plates were enumerat ed by nylon colony lift, simultaneous hybridization with non-isotopic whole chromosomal DNA probes and alkaline phosphatase substrates gener ating three colours. The results indicate that the protocol correctly identified and differentiated between the three species on mixed-growt h TSBA plates. The proportions of each species and mean total colony c ount expected by counting pure plates were in agreement with the propo rtions of each species and total colony counts enumerated by DNA probe s on mixed growth plates. The development of simultaneous hybridizatio n and multicolour detection will result in improved data recovery from dental plaque samples, in addition to reducing the cost and labour re quired in current colony-lift protocols, without affecting the specifi city or sensitivity of the probes used.