A third swab was obtained in a similar manner and placed into Amies transport medium (Nuova Aptaca, Canelli, Italy) for anaerobic culture. Grading of Gram-stained vaginal smears The Gram stained vaginal smears were scored by two independent assessors (GC and RV) according to the criteria previously described by Verhelst et al . Briefly, Gram-stained vaginal smears were categorized as grade I (normal) when only Lactobacillus
cell types were present, as grade II (intermediate) when both Lactobacillus and bacterial vaginosis-associated cell types were present, as grade III (bacterial vaginosis) when bacterial vaginosis-associated cell types were abundant in the absence of lactobacilli, as grade IV when only gram-positive cocci were observed, and as grade I-like when irregularly shaped or curved
selleck compound gram-positive rods were predominant . For the purpose of this study, grade I or Lactobacillus-dominated vaginal microflora is designated as ‘normal vaginal microflora’ and all other grades as ‘abnormal vaginal microflora’. Culture and identification of cultured isolates by tDNA-PCR Trichostatin A The swab on Amies transport medium was streaked onto Schaedler agar enriched with 5% sheep blood, vitamin K, haemin and EPZ004777 price sodium pyruvate (Becton Dickinson, Franklin Lakes, NJ) and incubated anaerobically at 37°C upon arrival at the microbiology laboratory. After 4 days of incubation, all the isolates with different colony morphology were selected for identification. DNA was extracted by simple alkaline lysis: one colony was suspended in 20 μl of 0.25% sodium dodecyl sulfate-0.05 N NaOH, heated at 95°C for 15 min and diluted Amrubicin with 180 μl of distilled water. tDNA-PCR and capillary electrophoresis were carried out as described previously [36, 37]. The species to which each isolate belonged was determined
by comparing the tDNA-PCR fingerprint obtained from each isolate with a library of tDNA-PCR fingerprints obtained from reference strains, using an in-house software program . The library of tDNA-PCR fingerprints is available at our website and the software can be obtained upon request . DNA extraction of vaginal swab samples For DNA extraction from the dry vaginal swabs, the QIAamp DNA mini kit (Qiagen, Hilden, Germany) was used according to the manufacturer’s recommendations, with minor modifications. The dry swab specimen from each patient was swirled for 15 s in 400 μl of lysis buffer (20 mM Tris-HCl, pH 8.0; 2 mM EDTA; 1.2% Triton). Fifty units of mutanolysin (25 U/μl) (Sigma, Bornem, Belgium) were added and the samples were incubated for 30 min at 37°C. After the addition of 20 μl Proteinase K (20 mg/ml) and 200 μl AL buffer (Qiagen), samples were incubated for 30 min at 56°C. Next, 200 μl of ethanol was added and DNA was purified by adding the lysate to the Qiagen columns as described by the manufacturer.