In our previous reports, we showed in detail the process of identification and coliform measurement in milk samples [14 (link)]. We plated each sample on a
5% sheep blood agar plate (Nissui Plate Sheep Blood Agar, Nissui Pharmaceutical Co., Tokyo, Japan) and cultured the samples aerobically at 37°C for 24 hr. We identified the coliforms based
on the shape and other characteristics of the colonies that grew on each plate. In addition, we measured the coliform bacterial count by spreading a 1-ml aliquot of each milk sample on 3M™
Petrifilm™ Coliform Count Plates (3M, Minneapolis, MN, USA), which is known to be suitable for identifying mastitis pathogens [3 (link), 11 (link), 13 (link)], then incubated the plates at 37°C for 24 hr, followed by colony counting. We used the counts to calculate the
colony-forming units (CFU)/ml. In addition, we considered culture-positive milk to be milk in which coliforms were detected by this method. Furthermore, to confirm that the bacteria on the
plates were coliforms, colonies on blood agar plates were applied to the direct PCR amplification kit (RR180A, Takara, Kusatsu, Japan). We sequenced all PCR products at the authorized
inspection agency. Obtained sequences were blasted with the GenBank database (http://www.ncbi.nlm.nih.gov/Blast ; 16S ribosomal RNA gene sequences [Bacteria and Archaea]) for species or genus assignment.
5% sheep blood agar plate (Nissui Plate Sheep Blood Agar, Nissui Pharmaceutical Co., Tokyo, Japan) and cultured the samples aerobically at 37°C for 24 hr. We identified the coliforms based
on the shape and other characteristics of the colonies that grew on each plate. In addition, we measured the coliform bacterial count by spreading a 1-ml aliquot of each milk sample on 3M™
Petrifilm™ Coliform Count Plates (3M, Minneapolis, MN, USA), which is known to be suitable for identifying mastitis pathogens [3 (link), 11 (link), 13 (link)], then incubated the plates at 37°C for 24 hr, followed by colony counting. We used the counts to calculate the
colony-forming units (CFU)/ml. In addition, we considered culture-positive milk to be milk in which coliforms were detected by this method. Furthermore, to confirm that the bacteria on the
plates were coliforms, colonies on blood agar plates were applied to the direct PCR amplification kit (RR180A, Takara, Kusatsu, Japan). We sequenced all PCR products at the authorized
inspection agency. Obtained sequences were blasted with the GenBank database (