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Abstract: The object of this study was to monitor the proportion of vital bacteria (microbial vitality: VF in %) present in subgingival dental plaque following one single subgingival irrigation with saline (S), chlorhexidine (CHX) or povidone iodine (I2), but without any subgingival instrumentation. Its effect on the main composition of the microflora was also assessed. Seventeen patients with adult periodontitis took part in this investigation. In each patient four initially untreated pockets (pocket depth 5–11 mm) associated with bleeding were selected for the standardised pocket irrigation and plaque sampling at baseline (0 h) and after the following 1 h, 24 h, 7 days and 31 days. The subgingival irrigation was only performed once (0 h). One pocket per quadrant was irrigated using 0.9% prereduced S, 0.2% CHX or 0.05% I2 (Iso-Betadine Buccale). The remaining untreated pocket without any irrigation served as an additional control (C). Using an acrylic splint as a guide, paperpoints were inserted into the pocket precisely at the same site to collect subgingival plaque. The bleeding on sampling (BOS) was thereafter noted. The proportions of bacterial morphotypes were examined by darkfield microscopy. VF was evaluated using a vital fluorescence staining. The undisturbed subgingival dental plaque was composed of 86% (median value) vital bacteria. The sampling procedure alone and the saline irrigation led to a decrease in the number of spirochetes but had no influence on the vitality of the flora. Large variations in VF could be observed in the short-term (1 h, 24 h) irrigation effect of CHX and I2. The reduction of VF was still significant after 7 days (VFCHX 30–80%, VFI2 35–80%) but persisted up to 31 days only after I2 irrigation (VFI2 12–90%). The findings indicated that all single subgingival irrigations resulted in a temporary change of the subgingival microflora while povidone iodine produced the longest lasting antimicrobial effect. Any clinical advantage of this situation should be further investigated.