Methodological issues in the quantification of subgingival microorganisms using the checkerboard technique G. Dahlen a, ⁎, H.R. Preus b , V. Baelum c a Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, PO Box 450, SE 40530 Gothenburg, Sweden b Department of Periodontology, Institute of Clinical Odontology, Faculty of Dentistry, University of Oslo, PO Box 1109 Blindern, 0317 Oslo, Norway c Department of Dentistry, Health, Aarhus University, Vennelyst Boulevard 9, 8000 C Aarhus, Denmark abstract article info Article history: Received 8 October 2014 Received in revised form 12 January 2015 Accepted 14 January 2015 Available online 17 January 2015 Keywords: Checkerboard DNA hybridization Periodontitis Subgingival plaque 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 sec- ond 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 mar- ginal. 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 (10 6 cells) was more reliable than SCM based on a linear calibration of the High Standard and a Low Standard (10 5 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. © 2015 Elsevier B.V. All rights reserved. 1. Introduction While it is generally agreed that periodontitis is an infectious dis- ease, initiated and sustained by bacteria (Socransky and Haffajee, 1994), the traditional focus on a few key putative pathogenic microbes with direct damaging effects on the host has gradually diminished. In- stead, there has been a growing interest in the ecology of the subgingival microflora (Socransky et al., 1998; Kolenbrander, 2000; Jenkinson and Lamont, 2005; Haffajee et al., 2008; López et al., 2011a; Duran-Pinedo et al., 2011), and its dynamic interplay with the host (Jenkinson and Lamont, 2005; Jiao et al., 2014; Nibali et al., 2014). This development has been promoted in part by a growing understanding of periodontitis as a disease reflecting complex interaction between a multitude of indigenous microorganisms (Jenkinson and Lamont, 2005) and interactions between the host and symbionts or pathobionts (Jiao et al., 2014). This development has been spurred, in part, by the de- velopment of high-throughput microbiological techniques that allow the rapid quantification of a wide range of microbes across a multitude of samples, such as the “checkerboard” DNA–DNA hybridization tech- nique (Socransky et al., 1994). The DNA–DNA checkerboard technique (Socransky et al., 1994) is a two-step procedure including DNA–DNA hybridization followed by a second step detecting the probes with a fluorescent antibody. The chemiluminescent/fluorescent signals elicited may either be visually scored (Haffajee et al., 1997; Socransky et al., 1998) by reference to the signals generated by standard samples of the test species (typically 10 5 and 10 6 cells (Socransky et al., 2004)); or they may be quantified by means of a charge-coupled device (CCD) camera and appropriate soft- ware for signal quantification. As such, the DNA–DNA checkerboard technique is subject to the same kinds of errors that affect immunoblots in general (Schilling et al., 2005). So far, the assessment of microbial communities in periodontal health and disease has commonly made use of color-coding of individ- ual species belonging to different microbial complexes such as the red (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and orange (e.g. Prevotella intermedia, Campylobacter rectus, Fusobacterium nucleatum) complexes (Socransky et al., 1998; Socransky and Haffajee, 2002; Teles et al., 2013) identified by means of principal components and correspondence analyses (Socransky et al., 1998; Haffajee et al., 2008); or of graphic visualization of bacterial Journal of Microbiological Methods 110 (2015) 68–77 ⁎ Corresponding author. E-mail address: dahlen@odontologi.gu.se (G. Dahlen). http://dx.doi.org/10.1016/j.mimet.2015.01.010 0167-7012/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth