Autoclaved pleuropneumonia like organism medium

Serovar 8 of U. urealyticum (Uu8) and serovar 3 of U. parvum (Up3) were acquired from the American Tissue Culture Collection (ATCC; Uu8: ATCC 27618, Up3: ATCC 27815). Isolates were propagated in a liquid in-house medium (“broth”) containing 82% autoclaved pleuropneumonia-like organism medium (Becton, Dickinson & Company, Franklin Lakes, NJ, USA), 10% heat-inactivated horse serum (v/v), 1% urea (w/v), and 0.002% phenol red (w/v) (all: Sigma-Aldrich, St. Louis, CA, USA). The medium was passed through a 0.2 µm filter membrane (Sartorius, Goettingen, Germany) and adjusted to pH 6.5. An endotoxin level of <0.06 EU/mL was verified (ToxinSensor™ Endotoxin Detection System, GenScript, Piscataway, NJ, USA).
As described previously [26 (link)], 10-fold serial dilutions of ureaplasma cultures were incubated to obtain 1 × 10⁹–1 × 10¹⁰ color-changing units (CCU)/mL of viable organisms. The corresponding ureaplasma DNA amounted to 5 × 10⁷–6 × 10⁸ copies/mL (Institute of Medical Microbiology and Hospital Hygiene, Duesseldorf, Germany). Bacterial viability was confirmed by simultaneous culture on selective agar plates (Mycoplasma ureaplasma agar, medco Diagnostika GmbH, Ottobrunn, Germany).

U. urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3) were acquired from the American Tissue Culture Collection (ATCC; Uu8: ATCC 27618, Up3: ATCC 27815). As described previously [31 (link)], ureaplasma isolates were cultured in a liquid in-house medium (“broth”) containing 82% autoclaved pleuropneumonia-like organism medium (Becton, Dickinson & Company, Franklin Lakes, NJ, USA), 10% heat-inactivated horse serum, 1% urea, and 0.002% phenol red (all: Sigma-Aldrich, St. Louis, CA, USA). The medium was adjusted to pH 6.5 after passage through a 0.2-μm filter membrane (Sartorius, Goettingen, Germany). Using the ToxinSensor™ Endotoxin Detection System (GenScript, Piscataway, NJ, USA), we could verify an endotoxin level < 0.06 EU/ml broth. Serial 10-fold dilutions of the ureaplasma cultures were incubated for 18–20 h to obtain titers of 1 × 10⁹–1 × 10¹⁰ color-changing units (CCU)/ml of viable organisms.

U. urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3) were attained from the American Tissue Culture Collection (ATCC; Uu8 ATCC 27618, Up3 ATCC 27815). ureaplasma isolates were cultured in a liquid in-house medium (referred to as “broth”) containing 82% autoclaved pleuropneumonia-like organism medium (Becton, Dickinson & Company, Franklin Lakes, NJ, USA), 10% heat-inactivated horse serum (v/v), 1% urea (w/v), and 0.002% phenol red (w/v) (all from Sigma-Aldrich, St. Louis, CA, USA). After passage through a 0.2-μm filter membrane (Sartorius, Goettingen, Germany), the medium was adjusted to pH 6.5. The ToxinSensor™ Endotoxin Detection System (GenScript, Piscataway, NJ, USA) verified an endotoxin level < 0.06 EU/ml broth. As described previously [17 (link)], serial tenfold dilutions of the ureaplasma cultures were incubated for 18–20 h to obtain titers of 1 × 10⁹–1 × 10¹⁰ color-changing units (CCU)/ml of viable bacteria. Corresponding amounts of ureaplasma DNA were verified and amounted to 5 × 10⁷–6 × 10⁸ copy numbers/ml (Institute of Medical Microbiology and Hospital Hygiene, Duesseldorf, Germany). Simultaneous culture on selective agar plates (medco Diagnostika GmbH, Ottobrunn, Germany) confirmed bacterial viability.