Columbia cna agar

Both the aerobic and anaerobic blood culture flasks (BACTEC Plus Aerobic/F and Anerobic/F) were incubated for a maximum of 14 days in the BACTECTM FX blood culture machine (BD, Heidelberg, Germany). After cultural growth was indicated, a Gram staining was performed. Furthermore, an agar diffusion test was carried out using a Müller-Hinton agar (bioMérieux, Nürtingen, Germany) and test platelets (bestbion dx, Köln, Germany) for specific antibiotics to provide a provisional resistogram. Cultivation from aerobic culture was performed by plating sample on a Columbia blood agar (bioMérieux, Nürtingen, Germany), on chocolate agar (bioMérieux, Nürtingen, Germany), bile Chryosidine Glycerol Agar (Becton Dickinson, Heidelberg, Germany), Sabauroud glucose agar (Becton Dickinson, Heidelberg, Germany) and on Columbia CNA agar with 5% sheep blood (Becton Dickinson, Heidelberg, Germany). HCG agar (Sifin Diagnostics GmbH, Berlin, Germany) was used to detect anaerobic bacterial. It was incubated in a culture pot “AnaeroJar” (Becton Dickinson, Heidelberg, Germany) under absorption of oxygen using AnaeroGen (Oxoid, Wesel, Germany). The aerobic cultures were incubated at 37 degrees for 24 h. The HCG agar was incubated for 48 h. As soon as bacterial colonies were detectable, the species were identified using MALDI TOF MS (Bruker Daltonics GmbH, Bremen, Germany).

The hemolytic activity of the compounds was assessed using the method described by Bechlars et al. [58 (link)]. Briefly, 2 μL of each peptide solution in DMSO (25 mg/mL), corresponding to 50 μg, was spotted on the surface of a blood agar plate (Columbia CNA Agar, Becton Dickinson Italia S.p.A., Florence, Italy). The plate was then incubated at 35 ± 2 °C for 24 h and visually inspected for the presence of hemolysis zones (see Figure S13, Supplementary Material). The experiment was performed in triplicate. Table 2 reports the average value of the diameter of hemolysis zones measured in each individual experiment. Triton X-100 was used as a positive control (diameter of the hemolysis zone: 13 mm), while DMSO was used as a negative control.

Three vaginal swabs were collected from each woman enrolled in this study. One swab was used for pH measurement using pH-Fix strips (Macherey-Nagel GmbH & Co. KG, Düren, Germany).
One swab was soaked in 1 ml of saline. A certain volume of this sample was cultured on BD Columbia CNA Agar containing 5% Sheep Blood (Becton Dickinson, New Jersey, USA). For G. vaginalis identification, mass-spectrometry (MALDI-TOF MS) analysis (Bruker Daltonik GmbH, Bremen, Germany) was performed. The remaining volume of the sample was used for Gram stain (Nugent’s criteria) for the diagnosis of BV. The last swab was soaked in 1 ml of saline and vortexed for at least one minute. Around 100 μL of the sample was examined under a light microscope (Olympus, Milan, Italy) to evaluate the presence of neutrophils (PMNs) and EC exfoliation. The numbers of PMNs and ECs were counted in four fields at ×400 magnification and expressed as the average number of PMNs or ECs/field, as previously described^{10 (link),19 (link),27 (link)}.
The remaining sample (900 μl) was centrifuged at 1,600 rpm for 10 min, and the cellular fraction was used for flow cytometric analysis or for assessment of EC damage or EC apoptosis.
Due to the limited amount of ECs in our samples, we were unable to perform all analyses for all samples. In each figure, we have reported the number of BV and healthy specimens used.