The culturability of bacteria in the bulk soil of an Australian pasture was investigated by using nutrient broth at 1/100 of its normal concentration (dilute nutrient broth [DNB]) as the growth medium. Three-tube most-probable-number serial dilution culture resulted in a mean viable count that was only 1.4% of the mean microscopically determined total cell count. Plate counts with DNB solidified with agar and with gellan gum resulted in viable counts that were 5.2 and 7.5% of the mean microscopically determined total cell count, respectively. Prior homogenization of the soil sample with an ultrasonic probe increased the viable count obtained by using DNB solidified with gellan gum to 14.1% of the mean microscopically determined cell count. A microscopic examination of the cell aggregates that remained after sonication revealed that the potential CFU count was only 70.4% of the total cell count, due to cells occurring as pairs or in clumps of three or more cells. Staining with SYTO 9 plus propidium iodide indicated that 91.3% of the cells in sonicated soil samples were potentially viable. Together, these findings suggest that the maximum achievable CFU count may be as low as 64.3% of the total cell count. Thirty isolates obtained from plate counting experiments performed with DNB as the growth medium were identified by comparative analysis of partial 16S rRNA gene sequences. A large proportion of these isolates represent the first known isolates of globally distributed groups of soil bacteria belonging to novel lineages within the divisions Actinobacteria, Acidobacteria, Proteobacteria, and Verrucomicrobia.It has been established that the genetic diversity of soil bacteria is high (7,34,48) and that soils contain many bacterial species of lineages for which no known cultivated isolates are available (14, 25). Many soil bacteria are referred to as uncultured or even nonculturable. A range of methods have been developed to study these organisms directly in their habitats (2, 10, 35). These methods are extremely useful for studying the ecology of microorganisms as parts of communities, but initial physiological and genetic studies of pure cultures should greatly facilitate such synecological studies. We believe that many of these bacteria are in fact culturable using relatively simple technologies. To test our hypothesis, we used a simple growth medium to investigate the culturability of soil bacteria. We reasoned that if we could increase the culturability of soil bacteria above the level of 5% that often appears to be the upper limit for cultivation studies, we should begin to cultivate bacteria that belong to some of the uncultured groups. MATERIALS AND METHODSSoil and sampling. The soil used in this study was collected from a rotationally grazed pasture dominated by Lolium perenne and Trifolium repens at the Dairy Research Institute, Ellinbank, Victoria, Australia. The soil is a krasnozem clay loam (Gn4.11 [32]; basaltic clay loam; Ferrosol [15]). The management regime, consisting of two cows per ha and 35 kg of...

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Primers were designed and successfully used to screen aromatic hydrocarbon-degrading bacteria for the presence of class II aromatic ring-hydroxylating dioxygenase (RHD) genes and to amplify novel RHD genes from DNA extracted from soil using the polymerase chain reaction. Two previously undiscovered groups of genes encoding putative class II RHDs, designated the S and T clusters, were found in RHD different soil samples. Only one of 70 RHD gene fragments amplified from these soil samples could be assigned to a cluster of previously reported RHD genes. These results suggest that distinct and potentially numerically dominant groups of as-yet unrecognized aromatic hydrocarbon-degrading bacteria exist in soils.

The abundance of genes encoding aromatic ring-hydroxylating dioxygenases (RHDs) in the groundwater at an aromatic hydrocarbon-contaminated landfill near Sydney, Australia, was determined by quantitative DNA-DNA hybridization using class II RHD genes as probes. There were marked differences in hybridization signal intensity against DNA extracted from the groundwater at seven different locations across this heterogeneous site. This was interpreted as indicating variation in RHD gene abundance. Clone libraries of polymerase chain reaction (PCR)-amplified RHD gene fragments were constructed from DNA from each of the groundwater samples. The libraries from the samples with greater RHD gene abundance were dominated by a group of bacterial class II RHD genes, designated the S-cluster, that has yet to be found in cultured isolates. These groundwater samples contained no detectable petroleum hydrocarbons. A second group of class II RHD gene sequences, designated the T-cluster, dominated RHD gene clone libraries prepared from groundwater samples that contained detectable levels of total petroleum and aromatic hydrocarbons but lower RHD gene abundance. The hosts and in situ expression of these novel genes, and the substrates of the enzymes they encode, remain unknown. The scarcity of genes from known aromatic hydrocarbon-degrading bacteria and the numerical dominance of the novel genes suggest that the hosts of these novel genes may play an important role in aromatic hydrocarbon degradation at this site.

About 80 percent of British deep mined coal is obtained from advance longwall faces. This mining method is highly capital intensive. Therefore, it is important to know the geologic structure prior to mining. Small faults can have a large effect on production rates. Channel wave seismology offers a possible method for locating these faults. A coal seam resembles a dispersive acoustic duct, and energy from a seismic source within the seam can be coupled into channel wave modes. Scattered waves from defects within the seam can be recorded and the data analyzed to produce a map of the seam. It has been shown that dispersion does not present a fundamental limitation to the resolution that can be obtained. In addition, broadband numerical holographic mapping procedures in the form of lag sums can be implemented. These techniques have been described previously by Mason et al. (1980a). This paper is divided into two main parts. First, we show how the basic lag‐sum techniques have been extended to produce an adaptive‐lag‐sum (ALS) mapping procedure. Second, we describe a variant to standard common‐depth‐point (CDP) stacking. This variation, called dynamic trace gathering (DTG), stacks only those traces with similar reflection points. Both ALS and DTG are extremely flexible. Targets at large angles to the geophone line may be imaged. Both processes can be used to analyze transmission and reflection seismograms, with or without mode conversion at any target. Dispersive propagation may also be included. The techniques are illustrated using data from underground field trials at three collieries.