Microorganisms eluted from the day 1 samples were spread on Luria–Bertani agar plates and incubated at 25 °C for 5 days to enrich the culturable strains as much as possible. Loopfuls of inoculum from the plates were then grown in shake flasks in 100 mL of MSM with 400 mg L−1 dichlorvos as the sole carbon source. A suitable control was set using the same medium containing 400 mg L−1 of dichlorvos without inoculum. After cultivation for 2 days, 1 mL of the supernatant was collected and analysed for its dichlorvos concentration by HPLC. The isolates of dichlorvos-degrading bacteria are available upon request from the

corresponding author. Total DNA of the microbial pellets from the controls and treated samples of experiment 1 and pure cultures of the

isolates were selleck chemicals selleckchem extracted with the method of Zhou et al. (1996). The PCR universal primer pair PRBA338f and PRUN518r (Øvreås et al., 1997) was used to amplify the bacterial V3 region of the 16S rRNA genes, and PRBA338f with a GC clamp was used for DGGE (Muyzer et al., 1993). The 16S rRNA genes of the pure isolates were amplified by PCR using the universal primers 27F and 1492R (Lillo et al., 2006). All the PCR products were confirmed by agarose gel electrophoresis and staining with ethidium bromide. DGGE was performed with the Dcode™ universal mutation detection system (Bio-Rad). The PCR products (40 μL) were loaded onto a 10% w/v acrylamide gel (acrylamide/bis solution, 37.5 : 1; Amresco) containing a linear chemical gradient ranging from 30% to 60% denaturant (where the 100% denaturing solution contained 7 M urea and 40% v/v formamide). The electrophoresis was run for 16 h at 60 °C at 100 V in 1 × TAE buffer. Aprepitant The gel images

were transformed into digital data using quantity one from Bio-Rad. The DNA isolated in experiment 1 from the control and treated samples on days 0 and 1 were used as the templates to amplify the complete 16S rRNA genes with primers 27F and 1492R, as described above. The products were purified and cloned into the pGEM-T Easy vector (Promega) and then transformed into competent Escherichia coli DH5α cells. The inserts were PCR amplified from randomly selected colonies using the T7 forward and SP6 reverse primers. The resulting library of PCR products was sequenced (Beijing Aoke Biotechnology Co. Ltd) based on different restriction fragment length polymorphism genetic profiles using the RsaI restriction enzyme. The DNA bands of interest were excised from the DGGE gel. The gel slices were placed into microcentrifuge tubes and the DNA was eluted at 37 °C for 2 h in sterile distilled water by allowing it to passively diffuse into the water. The eluted DNA was further amplified using the primers PRBA338f and PRUN518r, as described above.