Application of an end point dilution method to bacteriophage assay.

In experiments on the accumulation and elimination of virus particles by Pacific oysters (Crassostrea gigas), an Escherichia coli C2 bacteriophage was used as a virus model. It became necessary to quantitate low concentrations of phage present in samples of seawater, oyster shell liquor, and oyster meats and feces. The agar overlay phage plaque assay is subject to the same limitations with regard to sample size and minimal number of colonies per plate as is the bacterial plate count (G. S. Wilson, J. Bacteriol. 7:405, 1922; M. W. Jennison and G. P. Wadsworth, J. Bacteriol. 39:389, 1940; S. E. Luria, General Virology, John Wiley & Sons, Inc., New York, p. 41, 1953). In addition, when using the plaque assay method with samples such as bJended oyster meat, samples must be diluted 1:10 because the oyster meat particles conceal some of the plaques. Therefore, plaque assays on seawater and shell liquor samples containing fewer than 20 plaque-forming units (PFU)/ml and on oyster meat or feces samples containing fewer than 200 PFU/g were not satisfactory. In efforts to improve phage enumeration in low concentration ranges, a modification of the most probable number (MPN) or end-point dilution method employed in the bacteriological analysis of water (Standard Methods for the Examination of Water and Wastewater, 11th ed., American Public Health Association, Inc., New York, p. 494, 502, 1960) was used. The procedures were as follows: sterile metal-capped tubes (18 by 150 mm) were placed in racks, and 1.5 ml of a 3to 4-hr culture of the phage-sensitive E. coli C2 grown in Trypticase Soy Broth was added to each tube. Tenfold dilutions of the sample were made, and 1.0 ml of each dilution was placed in each of five tubes. The volume of culture placed in the tubes was increased to 3.0 ml when the sample volume was 2.0 ml, as in the case of oyster meats which had been diluted 1: 2 for blending. After incubation at 30 C overnight, the tubes were checked for evidence of phage multiplication. This was done by placing a loopful of the liquid from each tube on designated areas of a Trypticase Soy Agar plate which had been spread with the phage-sensitive E. coli C2 culture. These plates were then incubated overnight and examined for phage activity. Areas showing lysis of the confluent bacterial growth were considered positive. A standard five-tube MPN table was used to convert the results into phage MPN per gram or milliliter (J. H. Hoskins, Public