False results associated with darkground microscopy of subgingival plaque

Abstract

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.

Associations between subgingival plaque bacterial morphotypes and clinical indices?

“…After recording of clinical indices, samples of subgingival plaque from the apical border on the non-extracted teeth were pooled from the labial/buccal and palatal aspects for each selected approximal surface (100 subjects, 1st group), (see Omar & Newman 1986). Subgingival plaque samples from extracted teeth were obtained immediately after extraction; samples were removed from the most apical border on each approximal surface selected on extracted teeth (44 teeth, 22 subjects, 2nd group).…”

“…Although several workers , Lindhe et al 1980, Listgarten & Levin 1981, Armitage et al 1982, Singletary et al 1982, Addy et al 1983, Savitt & Socransky 1984, Tanner et al 1984 have indicated associations between counts of subgingival spirochaetes as well as other motile forms and severity or activity of chronic inflammatory periodontal disease (CIPD), other studies (Evian et al 1982, Claffey et al 1985, Jenkins et al 1986, MacPhee & Muir 1986, Reddy et al 1986, Harley et al 1987, Mtiller & Flores de Jacoby 1987, MacFarlane et al 1988 were not able to demonstrate such relationships. Since differences between investigator approaches as well as problems in methodology have played a role in the differing findings, an attempt was made to standardise subgingival plaque sample preparation and darkground microscopy (DGM) (Omar & Newman 1986). The present study investigated whether differences in other aspects of methodology might influence any association between clinical indices and subgingival plaque morphotype counts as assessed by DGM.…”

A Method for the Identification of Streptococcus mutans in Gingival Margin Plaque by Immunofluorescence

“…Plaque was dispersed by vortexing with RTF and 0.2 mm glass beads for I min, followed by repeated ( x 12) aspi rations of this suspension with a syringe and 25G needle [Omar and Newman, 1986). Films were IF-stained with a 1:200 dilution of the anti-S. mutans antiserum.…”

“…There is considerable evidence implicating Strepto coccus mutans in the aetiology of dental caries [Challacombe, 1986;Marsh, 1986;Newman, 1986]. This oral commensal, facultatively anaerobic bacterium is capable of exploiting the sites most susceptible to ca ries (fissures and approximal surfaces).…”

Microscopic spirochete counts in untreated subjects with and without periodontal tissue destruction

“…However this is not a problem for the estimation of the number of spirochetes which were easily visible amongst the other stained bacteria. The problems in sampling, preparation and microscopic examination of specimens of subgingival plaque have been discussed by others (Omar & Newman 1986, Mac-Farlane et al, 1986) and multiple sampling at a time interval has been advocated.…”