Culture of NB from Serum
NB specimens were cultured from FBS or HS as described before [5], [85]. Approval for the use of human samples in this study was obtained from the Institutional Review Board of Chang Gung Memorial Hospital (Gueishan, Taiwan, Republic of China). Written informed consents were signed by the individuals who provided blood samples. Human blood was obtained from healthy volunteers by venipuncture following sterilization of the skin with alcohol. The blood was withdrawn into sterile Vacutainer tubes containing no anticoagulant (Becton, Dickinson & Company, Sparks, MD, USA). Whole blood was centrifuged at 1,500× g for a period of 15 min at room temperature. The supernatant corresponding to HS was retrieved and placed into another tube. The FBS (Biological Industries, Kibbutz Beit Haemek, Israel; PAA Laboratories, Pashing, Austria) and HS used throughout this study were sterilized by filtration through both 0.2-µm and 0.1-µm membranes (Pall Corp., Ann Arbor, MI, USA) prior to use. To culture NB, both sera were diluted into DMEM (Gibco, Carlsbad, CA, USA) to final concentrations ranging from 0.1% to 10%. Culture was performed in 24-well plates with flat-bottom wells and covering lid (Corning, Inc., Corning, NY, USA) using a final volume of 1 ml per well. Culture was also performed in 75-cm2 flasks with 0.2-µm vented caps (Corning) using a final volume of 20 ml per flask. Untreated DMEM was used as a negative control. The culture plates and flasks were incubated at 37°C for several months in the humidified atmosphere of a cell culture incubator. Nanobacterium sp. strain DSM 5820 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany). Culture of “nanons”, which was initially called “Nanobacterium sp. strain Seralab 901045” [46], [86], was kindly provided by Dr. Didier Raoult (Unité des Rickettsies, Centre National de la Recherche Scientifique UMR 6020, Faculté de Médecine, Marseille, France). Both NB strains DSM 5820 and “nanons” were originally isolated from commercially available FBS used for cell culture purposes [86].
To prepare NB samples for electron microscopy and spectroscopy analyses, the cell culture medium of a flask containing a 1-month-old culture of NB was discarded and the NB sample which consisted of a white precipitate adherent to the flask was scraped using a sterile cell scraper (Corning). The precipitate was resuspended in 1 ml of DMEM and centrifuged at 16,000× g for 15 min at room temperature. The pellet was washed twice with DMEM, HEPES buffer (20 mM HEPES, 1 mM CaCl2, 2 mM Na2HPO4, 0.02% sodium azide, and 0.15 M NaCl, pH 7.4), or double-distilled water using the same centrifugation steps. The NB specimen was resuspended in a small volume of double-distilled water and used for the microscopy and spectroscopy analyses.