5 cm wide Collins speculum. If introduction of this speculum was judged impossible or if the patient indicated that introduction was too painful, a more slender (2 cm wide) speculum was used. The speculum was only minimally lubricated with a few drops of sterile water. We refrained deliberately from the see more use of anything other than sterile water in order to avoid interference with the vaginal microflora. With the speculum in place, a cotton-tipped swab was rolled around against the mid-portion of one lateral wall to obtain a vaginal smear. The swab was then immediately smeared on a plain glass slide and allowed to dry at room temperature. A second

sterile swab for culture and molecular analysis was rolled around against the same lateral wall of the mid-portion of the vagina and then placed into liquid Amies find more transport medium (Eswab, Nuova Aptaca, Canelli, Italy) and processed at the laboratory within 4 hours. Two commercial (nitrazine) pH strips were used to assess the pH of the neo-vagina: one with pH range from 1 to 10 (with accuracy of 1.0) and another one with pH range from 4 to 7 (with accuracy of 0.1) (Merck, Darmstadt, Germany). These strips were placed against the vaginal wall

until sufficiently moistened and compared with the manufacturer’s standard by a single observer (SW). In one patient insertion of the speculum was impossible due to an almost complete obliteration of the vagina. In this patient all vaginal swabs and pH-strips were taken superficially at the “”introitus”". Chlamydia was determined on a urine sample using a commercial real time PCR assay (Abbott RealTime CT, Abbott Laboratories, Illinois, US). After completion of the study, we were left with some additional questions which we thought might be of Urease influence on the vaginal microflora. Therefore the patients were sent an additional questionnaire in which they were asked about the regular use of vaginal hygiene products (once a month or more) and about

the presence of bad-smelling vaginal discharge (once a month or more). Staining of slides Smears were Gram stained (Mirastainer, Merck-Belgolabo, Overijse, Belgium) and examined under oil immersion at a magnification of 1000 by a single observer (GC). Culture and identification of cultured isolates by tDNA-PCR For the first 30 women, 100 μl of liquid Amies transport medium was streaked onto 5 different agar plates upon arrival at the microbiology laboratory. The culture medium for recovering aerobic bacteria was Tryptic Soy Agar supplemented with 5% sheep blood (Becton Dickinson, Franklin Lakes, NJ). Staphylococci were recovered on Mannitol Salt Agar (Becton Dickinson, Franklin Lakes, NJ). Both media were incubated aerobically at 37°C for 24 h. The culture medium for cultivation of anaerobic bacteria was Columbia based agar with 5% sheep blood (Becton Dickinson, Franklin Lakes, NJ). MRS agar plates (Oxoid, Hampshire, UK) were used for the culture of lactobacilli.