Impact of sample storage on detection of periodontal bacteria

Background/aims: Information on the impact of sample storage prior to analysis by DNA methods is limited. The aim of this study was to investigate the effect of subgingival sample storage on bacterial detection and enumeration. Material and methods: Subgingival plaque samples were studied by a) checkerboard DNA-DNA hybridization by immediate processing, b) storage at + 4degreesC for 6 weeks, c) storage at - 20degreesC for 6 months or d) storage at - 20degreesC for 12 months. Results: No differences in total DNA were found between protocol 1 and 2, or between protocol 3 and 4. Protocol 1 yielded 2.4 times more total bacterial DNA than did protocol 3 (P < 0.001). Actinobacillus actinomycetemcomitans and Campylobacter gracilis were detected in 21.1% of the immediately processed samples but only in 6.6% of the samples after 12 months of storage. Similar changes were noticed for Treponema denticola, which was detected in 22.3% and 9.2%, respectively. Streptococci spp., Fusobacterium nucleatum and Tannerella forsythia did not seem to be affected by storage. In contrast, the level of Campylobacter rectus detection frequency changed from 2.6% if processed immediately to 15.8% if samples were stored for 12 months. Conclusions: In longitudinal clinical studies including microbiological samples and processed with DNA-DNA hybridization methods, samples should be stored for the same period of time before processing to avoid loss of microbiological information.