Separation of Non‐filamentous Micro‐organisms from Soil by Density Gradient Centrifugation in Percoll

Abstract

Soil suspensions were homogenized, and desorbed non‐filamentous micro‐organisms were concentrated in a minimum volume of buffer by low speed centrifugation. The cells were separated from inanimate material by flotation at the interface between the buffer and a silica sol/polyvinyl pyrrolidone density gradient medium (Percoll). Cell suspensions were removed from the interface and fractionated according to density by high speed centrifugation on discriminating density gradients in Percoll. Preliminary experiments indicated that most non‐filamentous soil micro‐organisms had densities in the range 1.081–1.123 g%sol;ml while Rhizobium isolated from crushed root nodules on Percoll was split into two bands of densities 1.081–1.110 and 1.041–1.073 g/ml. The lighter cells were the more pleomorphic. The efficiency of extraction of cells from soil was governed by the extent of their desorption from inanimate particles. As rigorous desorption procedures damage cells, extraction efficiencies were low; 10–20% of cells counted microscopically in soil were recovered from density gradients. Electron microscopy of soil micro‐organisms isolated by this method showed an unusual range of surface ornamentations on cell‐like structures of bacterial dimensions.

The bacterial population of a blanket peat

“…The values for the biomass are only approximate estimates, since they are based on measurements of stained preparations of laboratory cultures and an assumed density of 1.1. The latter value is, however, close to the actual density range of soil micro-organisms measured by Martin & MacDonald (1981). In view of the complex wet spongy structure of the peat soil, expressing the results on a counts/m2 basis rather than counts/g dry peat gives a better impression of the ecological significance of each group.…”

Kinetically Limited Differential Centrifugation as an Inexpensive and Readily Available Alternative to Centrifugal Elutriation

“…(e.g., 1.1 g/mL for Pseudomonas (27); 1.13 g/mL for Streptococcus (28), and 1.135 g/mL for Bacillus (29)). They are thus likely to have sedimentation velocities comparable to E. coli in both blood culture broth and Histopaque-1083, and consequently, the operating window should be similar as well.…”

An ion‐exchange based extraction method for the detection of salmonellas in soil

“…A soil with a high clay content was selected for the Chelex work because it was regarded as a more severe test of the method than sandy soil, from which bacteria are extracted more readily. The extraction method of Martin and MacDonald (1981) involved homogenization of soil in buffer, followed by low and high speed centrifugation steps and the separation of cells from soil contaminants by density gradient centrifugation in Percoll. They reported low extraction efficiencies of 1&20%.…”

“…Some food industry wastes which can contain salmonellas are also applied to agricultural land. Survival of salmonellas in soil depends on many factors, including temperature, moisture, soil microflora, sunlight, soil type and pH (Wray 1975;Ellis and McCalla 1976;Chandler and Craven 1980;Papaconstantinou et al 1981 ;Reddy et al 1981 ;Chao et al 1987;Davies and Evison 1991).…”