Modification of the disk assay method for detection of antibiotics by direct seeding of spores of Bacillus stearothermophilus

By means of direct inoculation of spores of the assay organism, Bacillus stearothermophilus var. B. calidolactis C953, a modification of the IDF standard method for the detection of antibiotics in milk and milk products was investigated. A new sporulation medium, composed of peptone 1%, polypepton 1%, MnSO4·5H2O 0.004%, and agar 2%, was developed. High yield of spores of the assay organism was obtained on this medium. With crude or purified spore suspensions prepared from the agar plate cultures of the organism, the modified method was examined and it gave the same results to those obtained by the original IDF method, provided the spores were inoculated in the range of 2×107 to 5×107 in 1ml of the disk plate agar. Spore suspensions could be prepared readily by usual means in the laboratory, and purification was not necessary. The spore suspensions could be preserved without any deterioration for 12 months at 5°C. It was enable us to shorten the time required for the assay of antibiotics in this modified method, since the time consuming steps of preparation of the seed cultures could be omitted.