The optical density (OD) of each well was measured using a microplate ELISA reader (Emax , USA) at 570nm. Cut-off OD (ODc) is defined as three standard deviations above the mean OD of the negative control. Strains were interpreted as follows:nonbiofilm producers (OD �� ODc);weak biofilm producers (ODc < selleck chemical Seliciclib OD �� 2 �� ODc);moderate biofilm producers (2 �� ODc < OD �� 4 �� ODc);Strong biofilm producers (4 �� ODc < OD).(3) Detection of Exopolysaccharide Production (EPS). Possible EPS-producing isolates were identified via their mucoid or ropy appearance. EPS production was assessed by Indian ink (Art. Material S.A., China) staining of wet mount preparation of each strain after overnight growth on CRA and examined under light microscope [16, 17].

Appearance of a transparent halo zone around the Staphylococcus cells denoted the existence of an organized EPS structure.2.3. Genotypic AnalysisFour biofilm related genes were analyzed by simplex PCR assays to detect the presence of icaA (intercellular adhesion gene A), icaD (intercellular adhesion gene D), bap (encoding biofilm associated protein), and eno (encoding laminin binding protein) in all Staphylococcus isolates. First, crude DNA of the tested strains was extracted using a rapid boiling procedure according to Reischl et al. [18]. Two to 5 loops of Staphylococcus isolates taken from the brain heart infusion agar plate were collected and suspended in 200��L of lysis buffer comprised of 1% Triton X-100, 0.5% Tween 20, 10mM Tris-HCl (pH 8.0), and 1mM EDTA. After boiling for 10min, the suspension was centrifuged for 2min.

to sediment bacterial debris. The supernatant was aspirated, from which 5��L was used directly for PCR amplification. Because of nonavailability of positive controls for the four genes and to exclude any false negative results, the DNA of all isolates was firstly examined by amplification of 16S rRNA gene using Staphylococcus genus specific primers. The names of target genes, nucleotide sequences of primers, references, annealing temperature, and amplicon sizes are shown in Table 1. The amplification cycles were carried out in a PT-100 Thermocycler (MJ Research, USA). With the exception of specific annealing temperatures mentioned in Table 1, the reaction condition was optimized to be 94��C for 4min. as initial denaturation, followed by 40 cycles of 94��C for 60 seconds, annealing for 60 seconds and 72��C for 60 seconds.

A final extension step at 72��C for 10min. was followed. PCR condition was optimized using a total volume of 25��L reaction mixtures which contained 5��L of DNA as template, 25pmol of each primer, and 1X of PCR master mix (Dream Taq Green PCR Master Mix, Batimastat Fermentas Life Science). PCR products were analyzed by electrophoresis in 1.5% agarose gel in 0.5X TBE buffer containing ethidium bromide and visualized under a UV transilluminator.