Bathtub Microbiome Profiling Across Families

Five Japanese families, each consisting of two parents with either 2 or 3 children, were enrolled in this study. Four liters of bathtub water were collected once using sterilized pet bottles within 30 min before (2 families) and after family members (5 families) took a bath, then microbes were concentrated by suction filtration using four pieces of Isopore Membrane filters HTTP04700 (Millipore, Bedford, Massachusetts, USA) with a diameter of 47 mm and a mesh size of 45 µm. One part of the filter, which subsequently was subjected to 16S rRNA gene-based microbiota analysis, was stored at −20 °C until analysis. The remainder of the filter was put onto a TOS propionate agar plate (Eiken Chemical, Tokyo, Japan) supplemented with 50 mg/l mupirocin (Merck KGaA, Darmstadt, Germany), which is a selective medium for bifidobacteria^{18 (link)}, and was then immediately enclosed in plastic bags containing AnaeroPouch (Mitsubishi Gas Chemical, Tokyo, Japan) to create an anaerobic environment so as to retrieve bifidobacterial strains. These plates were anaerobically kept at room temperature and were then transported to the laboratory within 16 hours for cultivation at 37 °C.