Taurocholic acid sodium salt hydrate

Bile stock solution (12.5%) was prepared by dissolving crude porcine bile extract (Sigma, B8631) in RPMI or YPD. Insoluble components were removed by centrifugation at 12000 ×g, the supernatant filter sterilized and stored at 4°C in the dark. Sodium taurodeoxycholate hydrate (Sigma, T0875), taurocholic acid sodium salt hydrate (Sigma, T4409), sodium glycocholate hydrate (Sigma, G7132), and unconjugated bile salts (UBS) (Sigma 48305, 50% cholic acid sodium salt and 50% deoxycholic acid sodium salt) were dissolved at 100 mg/ml in either YPD or RPMI, filtered and used as stock solutions.

A DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) was used as the spin column reference method. DNA of S. aureus and E. coli was extracted following the manufacturer’s instructions for Gram-positive and -negative bacteria, respectively. The only modification for S. aureus was the inclusion of bead beating as the pellet was resuspended in 400 µL PBS and transferred into a Soil Grinding SK38 Precellys Lysing Tube (Bertin, Montigny-le-Bretonneux, France). The suspension was mixed in Precellys 24 Tissue Homogenizer (Bertin, Montigny-le-Bretonneux, France) at 6500 rpm for one minute. A total of 200 µL of the cell suspension was treated as described in the manufacturer’s basic protocol. For C. parvum, 100 µL of 1.2% taurocholic acid sodium salt hydrate (Sigma Aldrich, St. Louis, MO, USA) was added to the cell suspension followed by incubation at 37 °C for 120 min. The suspension was pelleted (13,000× g, 5 min) and resuspended in 80 µL PBS, 100 µL ATL Buffer (Qiagen, Hilden, Germany) and 20 µL PK. The procedure was continued as the recommendation by the manufacturer’s protocol for Gram-negative bacteria. This was considered a “spin column” reference method.