Sampling of subgingival microbiota for dark-field microscopy.

The aim of the present investigation was to study the validity of 2 different methods of sampling the subgingival microbiota. The composition of the flora was estimated using the dark-field microscope. Subgingival samples were obtained using either a curette or a Hamilton microsyringe for a subgingival washing. The method of determination of the composition of the subgingival microorganisms was found to be very reproducible when the same investigator performed the counting. Samples obtained by the washing technique had a slight but significantly higher proportion of coccoid cells when compared to samples obtained by curette. The calculation of the reproducibility of the washing technique as assessed in 6 immediately repeated samples from the same sites yielded a coefficient of variation of 40% for spirochetes and motile rods. When 9 repeated samples from each of 28 sites (14 with a curette, 14 with washing) were studied over a period of 32 days, no systemic alterations were observed in the % of spirochetes and motile rods. The variation within each individual site was high. For curette samples and washing samples, the coefficients of variation for spirochetes and motile rods were 85% and 63%, respectively. The present investigation clearly demonstrated that care should be taken in the interpretation of single samples of subgingival microbiota. A more relevant picture of the actual situation is achieved when trends of repeated samples over time are studied.