The direct count method for enumerating bacteria in natural environments is widely used. This paper analyzes the sources of variation contributed by the various levels of the method: subsamples, filters, and microscope fields. Based on a nested analysis of variance, we show that most of the variance (less than 80%) is caused by the fields and that the filters contributed nearly all of the remaining variance. The replication at each of the levels determines the total cost and error of a measurement. We compared several sampling schemes, including an optimal strategy which gives the lowest possible variance for a given cost. We recommend that preparing one filter from one subsample is adequate only if the samples are closely spaced in time or distance; otherwise, one filter should be prepared from two or preferably three subsamples. This sampling scheme emphasizes the importance of the highest level of replication. Our analysis shows that the accuracy of the direct count method can be substantially improved (by 20 to 50%) without a large increase in cost when the proper degree of replication at each level is performed.

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Sublethal injury was noted in Escherichia coli after cells were exposed to solar radiation. Injury was detected by differential plate counts between complete and minimal media that were observed with sunlight-exposed cells but not with cells kept in the dark. Since addition of catalase or pyruvate to minimal medium overcame or repaired this injury, the catalase system appeared to be the site of injury.

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One approach to exploring the behavior of microbial cultures during transient conditions of unbalanced growth is to experimentally observe continuous-flow biological reactors which have been subjected to perturbations in the influent flowrate and/or concentration of growth-limiting substrate. Proper interpretation of such experiments requires that appropriate account be taken of reaction stoichiometry, the distribution and abundance of microbial populations within the reactor, and the nonideality of mixing and flow distribution in the reactor. These aspects of proper experimental design are particularly critical when the system of interest involves methanogenic consortia and is not a completely-mixed, suspended-growth reactor.