Bacteriophage 80a did not increase in number in cultures containing less than about 1.0 x 104 to 1.5 x 104 CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP, cI was about 3 x 104 CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 x 103 CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 104 CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 104 CFU/ml.

Discriminant analysis of patterns of antibiotic resistance in fecal streptococci was used to differentiate between human and animal sources of fecal pollution in natural waters. A total of 1,435 isolates from 17 samples of cattle, poultry, human, and wild-animal wastes were obtained, and their ability to grow in the presence of four concentrations of five antibiotics (chlortetracycline, halofuginone, oxytetracycline, salinomycin, and streptomycin) was measured. When the resulting antibiotic resistance patterns were analyzed, an average of 74% of the known isolates were correctly classified into one of six possible sources (beef, chicken, dairy, human, turkey, or wild). Ninety-two percent of human isolates were correctly classified. When the isolates were pooled into four possible categories (cattle, human, poultry, and wild), the average rate of correct classification (ARCC) increased to 84%. Human versus animal isolates were correctly classified at an average rate of 95%. Human versus wild isolates had an ARCC of 98%, and cattle versus poultry isolates had an ARCC of 92%. When fecal streptococci that were isolated from surface waters receiving fecal pollution from unknown origins were analyzed, 72% of the isolates from one stream and 68% of the isolates from another were classified as cattle isolates. Because the correct classification rates of these fecal streptococci are much higher than would be expected by chance alone, the use of discriminant analysis appears to hold promise as a method to determine the sources of fecal pollution in natural waters.

The use of antibiotic resistance analysis (ARA) for microbial source tracking requires the generation of a library of isolates collected from known sources in the watershed. The size and composition of the library are critical in determining if it represents the diversity of patterns found in the watershed. This study was performed to determine the size that an ARA library needs to be to be representative of the watersheds for which it will be used and to determine if libraries from different watersheds can be merged to create multiwatershed libraries. Fecal samples from known human, domesticated, and wild animal sources were collected from six Virginia watersheds. From these samples, enterococci were isolated and tested by ARA. Based on cross-validation discriminant analysis, only the largest of the libraries (2,931 isolates) were found to be able to classify nonlibrary isolates as well as library isolates (i.e., were representative). Small libraries tended to have higher average rates of correct classification, but were much less able to correctly classify nonlibrary isolates. A merged multiwatershed library (6,587 isolates) was created and was found to be large enough to be representative of the isolates from the contributing watersheds. When isolates that were collected from the contributing watersheds approximately 1 year later were analyzed with the multiwatershed library, they were classified as well as the isolates in the library, suggesting that the resistance patterns are temporally stable for at least 1 year. The ability to obtain a representative, temporally stable library demonstrates that ARA can be used to identify sources of fecal pollution in natural waters.

Microbial source tracking (MST) uses various approaches to classify fecal-indicator microorganisms to source hosts. Reproducibility, accuracy, and robustness of seven phenotypic and genotypic MST protocols were evaluated by use of Escherichia coli from an eight-host library of known-source isolates and a separate, blinded challenge library. In reproducibility tests, measuring each protocol's ability to reclassify blinded replicates, only one (pulsed-field gel electrophoresis; PFGE) correctly classified all test replicates to host species; three protocols classified 48-62% correctly, and the remaining three classified fewer than 25% correctly. In accuracy tests, measuring each protocol's ability to correctly classify new isolates, ribotyping with EcoRI and PvuII approached 100% correctclassification but only 6% of isolates were classified; four of the other six protocols (antibiotic resistance analysis, PFGE, and two repetitive-element PCR protocols) achieved better than random accuracy rates when 30-100% of challenge isolates were classified. In robustness tests, measuring each protocol's ability to recognize isolates from nonlibrary