Abstract
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.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ashby R. E., Rhodes-Roberts M. E. The use of analysis of variance to examine the variations between samples of marine bacterial populations. J Appl Bacteriol. 1976 Dec;41(3):439–451. doi: 10.1111/j.1365-2672.1976.tb00657.x. [DOI] [PubMed] [Google Scholar]
- Fuhrman J. A., Azam F. Bacterioplankton secondary production estimates for coastal waters of british columbia, antarctica, and california. Appl Environ Microbiol. 1980 Jun;39(6):1085–1095. doi: 10.1128/aem.39.6.1085-1095.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hobbie J. E., Daley R. J., Jasper S. Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl Environ Microbiol. 1977 May;33(5):1225–1228. doi: 10.1128/aem.33.5.1225-1228.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones J. G., Simon B. M. An investigation of errors in direct counts of aquatic bacteria by epifluorescence microscopy, with reference to a new method for dyeing membrane filters. J Appl Bacteriol. 1975 Dec;39(3):317–329. doi: 10.1111/j.1365-2672.1975.tb00578.x. [DOI] [PubMed] [Google Scholar]
- Kaper J. B., Mills A. L., Colwell R. R. Evaluation of the accuracy and precision of enumerating aerobic heterotrophs in water samples by the spread plate method. Appl Environ Microbiol. 1978 Apr;35(4):756–761. doi: 10.1128/aem.35.4.756-761.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- King J. D., White D. C. Muramic acid as a measure of microbial biomass in estuarine and marine samples. Appl Environ Microbiol. 1977 Apr;33(4):777–783. doi: 10.1128/aem.33.4.777-783.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kirchman D., Mitchell R. Contribution of particle-bound bacteria to total microheterotrophic activity in five ponds and two marshes. Appl Environ Microbiol. 1982 Jan;43(1):200–209. doi: 10.1128/aem.43.1.200-209.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meyer-Reil L. A. Autoradiography and epifluorescence microscopy combined for the determination of number and spectrum of actively metabolizing bacteria in natural water. Appl Environ Microbiol. 1978 Sep;36(3):506–512. doi: 10.1128/aem.36.3.506-512.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Newell S. Y., Christian R. R. Frequency of dividing cells as an estimator of bacterial productivity. Appl Environ Microbiol. 1981 Jul;42(1):23–31. doi: 10.1128/aem.42.1.23-31.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Palmer F. E., Methot R. D., Staley J. T. Patchiness in the distribution of planktonic heterotrophic bacteria in lakes. Appl Environ Microbiol. 1976 Jun;31(6):1003–1005. doi: 10.1128/aem.31.6.1003-1005.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watson S. W., Novitsky T. J., Quinby H. L., Valois F. W. Determination of bacterial number and biomass in the marine environment. Appl Environ Microbiol. 1977 Apr;33(4):940–946. doi: 10.1128/aem.33.4.940-946.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]