API NH (BioM��rieux SA) for Eikenella spp., Haemophilus spp., Neisseria spp. and Actinobacillus spp. (Gram-negative cocci and rods; facultative, oxidase-positive). API C AUX (BioM��rieux SA) for yeast (Candida spp.). Bacterial detection (Polymerase chain reaction – PCR 16S rDNA) Reference bacteria strains used in this selleck chemicals llc study were acquired from the American Type Culture Collection (ATCC) and are listed as follows: Enterococcus faecalis (ATCC 4034), Filifactor alocis (ATCC 35896), Fusobacterium nucleatum (ATCC 25586), Gemella morbillorum (ATCC 27824), Parvimonas micra (ATCC 33270), Porphyromonas endodontalis (ATCC 35406), Porphyromonas gingivalis (ATCC 33277), Prevotella intermedia (ATCC 25611), Prevotella nigrescens (ATCC 33536), Prevotella tannerae (ATCC 51259), Tannerella forsythia (ATCC 43037), Treponema denticola (ATCC 35405), and Treponema socranskii (ATCC 35536).

DNA extraction Microbial DNA from all stages of endodontic retreatment samples (S1, S2, and S3) and control sample, as well as from ATCC bacteria, were extracted and purified by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The DNA concentration (absorbance at 260 nm) was determined with a spectrophotometer (Nanodrop 2000; Thermo Scientific, Wilmington, DE, USA). PCR assay The PCR reaction was performed in a thermocycler (My-Cycler; Bio-Rad, Hercules, CA, USA) with total volume of 25 ��L containing 2.5 ��L of 10X Taq buffer (1x) (Invitrogen, Eugene, OR, USA), 0.5 ��L of dNTP mix (25 ��mol/L of each deoxyribonucleoside triphosphate �C dATP, dCTP, dGTP, and dTTP) (Invitrogen), 1.

25 ��L of 25 ��mol/L MgCl2, 0.25 ��L of forward and reversal universal primers (0.2 ��mol/L) (Invitrogen), 1.5 ��L of sample DNA (1 ��g/50 ��L), 1.5 ��L of Taq DNA polymerase (1 U) (Invitrogen), and 17.25 ��L of nuclease-free water. The primer sequences and PCR cycling parameters were previously optimized[16] and are listed in Table 1. Table 1 PCR primer pairs and cycling parameters used for detection of bacteria species in post-treatment apical periodontitis Statistical analysis Effectiveness of each treatment step in rendering root canals free of cultivable bacteria was recorded as percentage of cases yielding negative cultures. In this regard, the one-tailed Fisher exact test was used to compare S2 and S3 samples.

The percent reduction in the number of CFUs after each treatment step was calculated based on quantitative data obtained from samples S1, S2, and S3. Quantitative data were statistically analyzed for Drug_discovery differences by using the Mann-Whitney U test comparing pairs of groups. Significance level was always set at 5% (P < 0.05). RESULTS All disinfection control samples yielded no growth, confirming effective disinfection of the tooth surfaces by culture and PCR technique (16s rDNA). Colony-forming unit The bacterial counting at each stage of retreatment is shown in Table 2. High bacterial counting was found in all S1 samples.