False results associated with darkground microscopy of subgingival plaque

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 morphotype 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.