The aim of the present study was to evaluate possible associations between subgingival plaque bacterial morphotypes, as assessed by darkground microscopy, and clinical indices of routine adult chronic periodontitis. Clinical indices were plaque index (PlI), gingival index (GI), sulcus bleeding index (SBI), papilla bleeding index (PBI), attachment loss (AL), pocket depth (PD) and probeable pocket depth (PPD). Apical border plaque was sampled in vivo and after extraction to test whether direct or indirect sampling affected any such associations. Similarly, pocket depth and attachment loss were also assessed directly and indirectly on the same teeth, in vivo or after extraction. The influence of the type of index used to record inflammation (GI, SBI, PBI) was also assessed, as were the effects of the numbers of sampled subjects and the method of analysis, which comprised the use of transformed and untransformed data and of parametric and non-parametric tests. Data were collected in relation to the approximal surfaces of 44 teeth extracted from 22 adults (2 teeth each) and from 1 pair of contralateral upper anterior or premolar teeth in each of 100 adults, all which untreated routine chronic periodontitis. Selected subjects had greater than or equal to 4 mm probeable pocket depth and/or attachment loss, and radiographic evidence of bone loss in relation to 1 approximal surface on each of 1 pair of contralateral anterior or premolar teeth, or to 2 teeth scheduled for extraction. Plaque preparation and darkground microscopy were as described previously. Insignificant associations (p greater than 0.05) were demonstrated between supragingival plaque (PlI) and periodontal inflammation (GI, SBI, PBI) or destruction (PPD and AL), as well as between inflammation and attachment level. In contrast, significant moderate associations (r = 0.5-0.77) were demonstrated between each of the 3 morphotype groups; spirochaetes, other motiles and cocci. Spirochaetes showed a significant moderate (r = 0.5) positive association with pocket depth with a 2.43% mean increase of spirochaetes for each 1 mm increase of PPD. Although highly significant associations (r = 0.9) were demonstrable between the 3 inflammation indices (GI, SBI, PBI) themselves, only PBI showed significant positive associations (r = 0.3) with spirochaetes and other motiles. Also, PlI showed significant associations with each of the 4 morphotypes (r = 0.3-0.5). The heterogeneity of spirochaetes and other motiles as well as the multiplicity of possible aetiological microbial agents in plaque may have resulted in underestimated associations between subjects as well as undetectable association within a given mouth using only 4 morphotype groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract. This study considers false results which may arise due to problems in the preparation or examination of specimens for darkground microscopy of subgingival plaque. Subgingival plaque samples obtained with a sterile curette were placed in 0.1–0.3 ml sterile full or 1/4 strength Ringer's solution: 0.85% saline, 1% gelatin in 0.85% saline, formal saline or pyrogen‐free water for injection. Test slides were prepared from the original dispersion, and control slides from the corresponding sterile solution. Optimal dispersion solution, syringe dispersion frequency and the effect on motility of delay in processing samples were tested. Slides were also prepared from dispersions of 11 representative subgingival “periodontopathic” organisms. Problems in sampling included variability in counts between sites with comparable pocket depths, contamination of the sample and reduction of the sample volume after scaling. Problems in dispersion included contamination, uneven distribution of the different morphotypes and destruction of delicate organisms. Problems in slide preparation included slide contamination, limitation in the number of samples that can be assessed by one examiner at a given time without loss of activity of motile cells, and preparation of a cell monolayer. Problems in identification and counting included confusion of Brownian movements with motility, coccoid particles with cocci, spirochetes with campylobacter, flagella with flagella‐like structures, size of cocci, counting of fragmented spirochetes and non‐motile flagellated organisms and motile cells, and also bias in counting. Problems in morpbotype grouping included the observation that many (10 of the 11 representative) periodontitis‐related organisms were in the non‐motile groups and not all cells of the motile species (Campylobacter, Capnocytophaga) showed motility. The results indicate that each stage of subgingival plaque darkground microscopy, sampling, dispersion, slide preparation, counting, morphotype grouping and interpretation may lead to false results if not representative or reproducible. Procedures are suggested for the minimisation of problems in the preparation and examination of subgingival plaque specimens for darkground microscopy.

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Part of the results of a study of subgingival plaque by darkground microscopy was used to investigate morphotype distribution at different levels within the periodontal pocket. Subgingival plaque was sampled at coronal, middle and apical levels from each of 28 approximal surfaces on 28 teeth extracted from 28 adults with untreated moderate to advanced periodontitis. Sample preparation and darkground microscopy were as described previously. The data recorded only the subgingival level at which the maximum count occurred for a given morphotype and subject. It was found that the maximum count of spirochaetes occurred most frequently at the apical level (p less than 0.05) and the maximum count of 'others' at the coronal level (p less than 0.05). Also, coccal counts showed a trend to increase the more apical the sample. Standardised darkground microscopy was able to show that the sampled subgingival level had a significant influence on plaque bacterial morphotype distribution, indicating the importance of accurate sampling of subgingival plaque at the most apical level.