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HALVORSON, H. 0. AND ZIEGLER, N. R. 1933 Application of statistics to problems in bacteriology. III. A consideration of the accuracy of dilution data obtained by using several dilutions.

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A recent review (Carlucci and Pramer, 1959) of present knowledge of factors affecting the survival of bacteria in sea water provided no satisfactory explanation for the rapid decrease in bacterial numbers with distance from land. Numerous suggestions have been made but few were tested experimentally. Since the practice of disposing of wastes in the sea has increased, causing pollution of shellfish areas and bathing beaches, information regarding the survival of bacteria in sea water has assumed public health significance. An evaluation of the contribution of various physical, chemical, and biological factors to the death of bacteria in a marine environment appeared desirable. It was possible that a study of this nature would also indicate why bacteria die less rapidly in autoclaved than in untreated sea water. Although the beneficial effect of heat sterilization on the survival of bacteria in sea water was first described in 1885 (Nicati and Reitsch) and was reported periodically by various investigators located throughout the world (de Giaxa, 1889; Kiribayashi and Aida, 1934;ZoBell, 1936;Krassilnikov, 1938;Ketchum et al., 1949;Vaccaro et al., 1950;Nusbaum and Garver, 1955;and Richou et al., 1955) the identity of the factors responsible for the phenomenon remains to be established.Before the survival of Escherichia coli could be investigated, a method was needed whereby the test organism was recovered quantitatively from sea water. The present paper describes the development, standardization, and evaluation of a suitable procedure. EXPERIMENTAL PROCEDURES AND RESULTSMedium and plating procedure. Various selective media for the isolation and enumeration of coliform bacteria are available commercially. , La Lima, Honduras, C. A.agar (Levine, 1943) and Tergitol 7 agar (Chapman' 1947) favor growth of E. coli and facilitate its identification. The development of E. coli on selective as well as on nonselective agar media was examined. For this purpose approximately 10 ml of agar were added to each of a number of sterile Petri dishes and allowed to solidify. One-tenth ml of appropriate dilutions of 24-hr cultures grown on nutrient-lactose agar was distributed over the surface of the various media. To overcome spreading and coalescing colonies, absorbent discs3 were placed in the Petri dish tops. The plates were incubated for 24 hr at 37 C and colony counts were made. The composition of the media tested and the results obtained are shown in table 1.Escherichia coli developed best on Tergitol 7 agar producing a yellow colony surrounded by a yellow

Although the source and identity of these substances remain obscure, they are believed to be of microbial origin and have been described as antibiotics (Rosenfeld and Zo