Statistical analysis of the direct count method for enumerating bacteria

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.

Spatiotemporal distribution of microbial communities in a coastal, sandy aquifer system (Doñana, SW Spain)

“…Filters were transferred to a labelled microscope slide and kept frozen until samples could be counted (Bölter et al , 2002). Three different filters were prepared for each sampled well per season, and 20 different fields were viewed and counted for each filter (Kirchman et al , 1982) with an Olympus IX50 inverted microscope (Olympus Corporation, Shinjuku-ku, Tokyo, Japan). The results of the counts for bacterial abundance are presented as number of bacteria per millilitre (bacteria mL -1 ) groundwater.…”

Precipitation of dolomite using sulphate‐reducing bacteria from the Coorong Region, South Australia: significance and implications

“…The first lake sediment sample was transferred from storage to the anaerobic cabinet, where a sterile syringe was used to withdraw 5 ml of sediment from the core. The first 1 ml of sediment was wiped off into a sterile Petri dish for injection into a vial containing 9 ml of filter-sterilized 4% formaldehyde, in preparation for acridine orangestained direct counting (Kirchman et al, 1988). Sediment (2AE5 ml) from the syringe was then injected into vial 1 of the dilution series for that sample, being careful to maintain sterility.…”

Taxon‐ and vector‐specific variation in species richness and abundance during the transport stage of biological invasions

“…Bacteria samples were filtered onto 25 mm diameter, 0.2 mm black polycarbonate filters (GE Osmonics Labstore) and stained with acridine orange (final concentration 1.872 3 10 25 g L 21 ; Kirchman et al 1982). Slides were corrected for non-cell staining by counting 5 mL of 0.2 mm filtered distilled water before each sample filtration.…”

“…The slides were observed using an Olympus BH2-RFC epifluorescence microscope, equipped with a 1003 1.30 oil objective (12503 total magnification), a 100 W mercury lamp, and appropriate filter sets (502 nm for excitation, 526 nm for emission). The optimal cell direct counting scheme was followed to optimize the accuracy of the direct count (Kirchman et al 1982).…”