Optimisation and standardisation of a method for detecting and enumeratingbacteriophages infecting Bacteroides fragilis

A method for the detection and enumeration of bacteriophages infecting Bact eroides fragilis has been standardised. The recommended host strain is RYC2 056 (ATCC 700786) because of the relatively high counts (10(4)-10(5) PFU/10 0ml) that it recovers in sewage from very different geographical areas. The addition of 0.25% bile to the culture and assay media and the manipulation of the host strain under strict anaerobic conditions resulted in a signifi cant increase (more than 100%) in the number of phages detected, No other c hanges in the media and culture conditions resulted in changes in the phage counts detected. However, these increases do not justify changing the cult ure conditions and media described, taking into consideration that bile ren ders the media cloudy making it difficult to follow the host growth and tha t most laboratories do not have the: facilities to work under strict anaero bic conditions. Nalidixic acid (100 mug/ml) and kanamycin (100 mug/ml) in t he assay medium significantly reduce the background flora from polluted sam ples without affecting the phage counts. Freezing cultures just before the end of the log-phage growth at ( -70 +/- 10)degreesC with BSA-sucrose as cr yoprotector, storing of 1-2 mi in glass vials at (-70 +/- 10)degreesC and u sing them directly to inoculate fresh broth allows the obtention of culture s ready for phage enumeration in about 2.5 h. All these developments have b een incorporated into a procedure that makes the method for detecting phage s infecting B. fragilis as workable as the standardised methods available f or the detection of coliphages. (C) 2001 Elsevier Science B.V. All rights r eserved.