Methodological issues in the quantification of subgingival microorganisms using the checkerboard technique

• Reproducibility and reliability of the checkerboard (CKB) method were evaluated.
• Reproducibility of the CKB method to quantify bacteria in a clinical sample is high.
• Reproducibility of the CKB method varies between different subgingival bacteria.
• Storage time of DNA samples for CKB was marginal compared to probe quality.
• High quality DNA probes and standards have a strong impact on the reproducibility.

The reproducibility and reliability of quantitative microbiological assessments using the DNA–DNA hybridization “checkerboard method” (CKB) were assessed. The data originated from 180 chronic periodontitis patients, who were enrolled in a clinical trial and sampled at baseline, and 3 and 12 m post-therapy. The samples were divided into two portions allowing evaluation of reproducibility. In total, 531 samples were analyzed in a first run, using standard bacterial preparations of cells and 513 samples were accessible for analysis in the second, using standards based on purified DNA from the species. The microbial probe panel consisted of periodontitis marker bacteria as well as non-oral microorganisms. Three different ways of quantifying and presenting data; the visual scoring method, VSM, the standard curve method, SCM, and the percent method, PM, were compared. The second set of analyses based on the use of standard preparations of pure DNA was shown to be more consistent than the first set using standards based on cells, while the effect of storage time per se up to 2.5 y seemed to be marginal. The best reproducibility was found for Tannerella forsythia, irrespective of quantification technique (Spearman's rho = 0.587, Pearson's r ≥ 0.540). The percent method (PM) based on percent of High Standard (106 cells) was more reliable than SCM based on a linear calibration of the High Standard and a Low Standard (105 cells). It was concluded that the reproducibility of the CBK method varied between different bacteria. High quality and pure specific DNA whole genomic probes and standards may have a stronger impact on the precision of the data than storage time and conditions.