Microflora of Soil as Viewed by Transmission Electron Microscopy1

Bae, H. C.; Cota-Robles, E. H.; Casida, L. E.

doi:10.1128/aem.23.3.637-648.1972

Cited by 99 publications

(29 citation statements)

References 15 publications

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“…trifolii are small enough to pass through 0.4plm-pore-size filters (2). This observation supports various reports stating that there is a high proportion of bacterial cells in soil that are atypically small relative to those grown on laboratory media (3,4,6,8,13,26) and that only a small fraction of this small-cell population can be cultured (7).…”

supporting

confidence: 91%

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Bottomley

Dughri

1989

Appl Environ Microbiol

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Bacterial cells small enough to pass through 0.4-,um-pore-size filters made up 5 to 9% of the indigenous bacterial population in 0to 20-cm-depth samples of Abiqua silty clay loam. Within the same soil samples, cells of a similar dimension were stained with fluorescent antibodies specific to each of four antigenically distinct indigenous serogroups of Rhizobium leguminosarum bv. trifolii and made up 22 to 34% of the soil population of the four serogroups. Despite the extensive contribution of small cells to these soil populations, no evidence of their being capable of either growth or nodulation was obtained. The density of soil bacteria which could be cultured ranged between 0.5 and 8.5% of the >0.4-p.m direct count regardless of media, season of sampling, or soil depth. In the same soil samples, the viable nodulating populations of biovar trifolii determined by the plant infection soil dilution technique ranged between 1 and 10% of the >0.4-p.m direct-immunofluorescence count of biovar trifolii. The <0.4-p.m cell populations of both total soil bacteria and biovar trifolii changed abruptly between the 10to 15-cm and 15to 20-cm soil depth increments, increasing from 5 to 20% and from 20 to 50%, respectively, of their direct-count totals. The increase in density of the small-cell population corresponded to a significant increase in soil bulk density (1.07 to 1.21 g cm-3). The percent contribution of the <0.4-p.m direct count to individual serogroup totals increased with soil depth by approximately 2-fold (39 to 87%) for serogroups 17 and 21 and by 12-fold (6 to 75%) for serogroups 6 and 36.

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supporting

confidence: 91%

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Bottomley

Dughri

1989

Appl Environ Microbiol

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“…As reported previously, thin-tical. This not only increases the ease of thinsectioning requires that all gritty soil debris be sectioning but also improves cell recognition in removed from the sample (1,2). In those studies, replicas of frozen-etched preparations.…”

Section: Discussionmentioning

confidence: 99%

“…The resident microorganisms in soil can be released from the soil debris and concentrated so that they can be viewed by transmission electron microscopy as thin sections (1,2) or replicas of frozen-etched preparations (3). For thin-sectioning (1,2),most ofthe gritty soil debriswas removed by an exhaustive centrifugal washing procedure, but the freeze-etching technique (3) used only a 'Received July 4,1974. 'This research was authorized for publication as paper No.…”

Section: Introductionmentioning

confidence: 99%

Simplified procedures for releasing and concentrating microorganisms from soil for transmission electron microscopy viewing as thin-sectioned and frozen-etched preparations

Balkwill¹,

Labeda²,

Casida³

1975

Can. J. Microbiol.

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A simplified procedure is presented for releasing and concentrating indigenous microbial cells from soil for viewing by transmission electron microscopy as thin sections or replicas of frozen-etched preparations. This procedure is compared with two others reported earlier, and their relative merits are discussed as concerns the choice of procedure for the cellular information desired from the soil. Freeze-etching showed that the cell types and size distributions for cells which have been released and concentrated from soil are in general agreement with those for cells in a crude soil slurry in which no attempt to release and concentrate cells was made. Microcolonies were present both in the crude slurry and in the discard soil debris centrifugation pellets from the cell release and concentration procedures. In contrast to the historic assumptions, these microcolonies, as well as some individual cells embedded in soil debris could not be broken up and (or) dislodged so that they would be washed from the soil. The relative numbers of these cells remaining with the soil debris, however, could not be quantitated in the present study.

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“…The majority of soil bacteria are known to be less than 0.5 m in diameter (6,7,8,17,27), and it has been suggested that this population of small bacteria consists mainly of gram-positive organisms (32). These bacteria do not grow well under laboratory conditions (7), and their growth rate, as determined by the thymidine technique, may be lower than the growth rate of larger bacteria (4).…”

mentioning

confidence: 99%

Counting and Size Classification of Active Soil Bacteria by Fluorescence In Situ Hybridization with an rRNA Oligonucleotide Probe

Christensen

Hansen

Sørensen

1999

Appl Environ Microbiol

141

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A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled oligonucleotide probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA probe, EUB338, for the domain Bacteria was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 μm). A nonsense probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the probes and for binding by the rhodamine part of the probe, control experiments in which excess unlabelled probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 × 108 active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 × 108 active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 μm), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4′,6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic probes and for automatic counting of soil bacteria.

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Microflora of Soil as Viewed by Transmission Electron Microscopy1

Cited by 99 publications

References 15 publications

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Simplified procedures for releasing and concentrating microorganisms from soil for transmission electron microscopy viewing as thin-sectioned and frozen-etched preparations

Counting and Size Classification of Active Soil Bacteria by Fluorescence In Situ Hybridization with an rRNA Oligonucleotide Probe

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