Abstract
A simple method for direct and accurate enumeration of bacteriophage is still lacking. In many instances it is desirable to determine the bacteriophage unit directly. For this purpose 2 general methods are now in use, (1) the counting of plaques formed when bacteriophage and susceptible bacteria are spread on the surface of agar plates, and (2) the determination of the highest dilution in broth for a given bacteriophage to cause complete lysis of susceptible bacteria. The best agreement by the dilution method of titration has been calculated by Clark 1 to be 60%. The difficulty of spreading evenly on an agar surface and the adsorption of a variable amount of bacteriophage by the spreader are the disadvantages of the streak method. If bacteriophage and susceptible bacteria are mixed thoroughly in meat infusion agar and then poured into plates, we find that the technique is not only simplified but its accuracy is also increased. It seems worthwhile, therefore, to test the practicability of the pour plate method and the optimal conditions for the demonstration of bacteriophage plaques under various factors of growth.
A dysentery Shiga bacteriophage isolated from a single plaque was used. Pour plates were made by mixing 1 cc. of diluted bacteriophage, 0.5 cc. of an 18-hour agar slant growth of susceptible bacteria in concentration of 1:50 in saline and 15 cc. of 2% meat infusion agar (pH 7.6). Upon incubation at 37°C. for 24 hours. 2 kinds of plaques appear: (a) surface plaque, which is large, clear, and extending through the depth of the medium, and, (b) deep plaque, which is small, less clearly outlined, and situated below the surface.
In agreement with Bronfenbrenner and Korb 2 we found the size of the individual plaque to be bigger as the concentration of agar is decreased (Fig. 1).
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