Synchronization of Escherichia coli by zonal centrifugation.

A zonal centrifugation technique that can select the smallest newborn cells in an exponentially growing culture of Escherichia coli B/r is described.     

Loss of cell constituents from hepatocytes on centrifugation.

In studies of the metabolism of isolated hepatocytes, it is often necessary to measure the concentrations of cell constituents both in cells and medium. When hepatocytes are separated in the special tubes of Hems, Lund & Krebs (1975) (Biochem. J. 150, 47--50), they lose much glucose, urea and Na+, whereas there is no loss of K+, glutamate, aspartate and adenine nucleotides. Cell water is also lost, as measured by the distribution of 3H2O. This loss is mainly due to an exchange of cell water with the aqueous solution in the stems of the tubes through which the cells pass on centrifugation. In general, substances are lost only when the intracellular concentration is equal to, or lower than, the extracellular concentration. Probably solutes are lost because they travel with the water unidirectionally out of the cell. A loss of solute does not occur when the cells are centrifuged in conical tubes with a layer of silicone oil between the cell suspension and the deproteinizing layer. The reasons for the loss occurring in the special separation tubes are discussed.     

Numerical solutions of the Lamm equation. III. Velocity centrifugation

We have generated solutions to the Lamm equation to examine the effects of concentration dependence on velocity experiments. Two forms of c dependence are considered: s/s₀ = 1 – kc and s/s₀ = (1 + kc)^?1. Features of these solutions are discussed. The magnitude of the errors resulting from the usual procedure of measuring the rate of movement of schlieren maxima or of the position at which the concentration is one half the plateau value have been examined. These errors are usually negligible after sufficient centrifugation time. The errors in using the half-plateau concentration are less than those using the movement of the peak. We have also examined a technique due to Fujita for determining D from boundary spreading when s/s₀ = (1+kc)^?1. This method is satisfactory when s/s₀ is actually of this form, or under certain limitations when s/s₀ = (1 + kc)^?1. Creeth has shown that under certain conditions the concentration gradient, curve remains virtually unchanged in shape after separating from the meniscus. When this occurs it is possible to estimate s/D from the data. The condition for such a steady state is that kc₀ be sufficiently large. Numerical confirmation of this method is presented in the final section.     

Effect of centrifugation on amniotic fluid phospholipid composition.

The first step in the determination of phospholipid in amniotic fluid is generally the removal of cells and debris from the fluid by centrifugation. Low-speed centrifugation of the supernatant is reported to have the same phospholipidic profile and L/S, PG/S and PI/S ratios similar to those of the uncentrifuged amniotic fluid sample. With high-speed centrifugation almost all the pulmonary surfactant seems to be recovered in pellet with the same characteristics as the uncentrifuged amniotic fluid.     

Centrifugal elutriation (counterstreaming centrifugation) of cells.

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.     

Water balance in rats exposed to chronic centrifugation.

Changes in gravitational load have been shown to alter renal function, which could potentially affect water balance. Therefore, the present study was conducted to determine the effects of chronic centrifugation on water balance. Eight Sprague-Dawley rats were centrifuged (12 days at 2 G), and eight rats were used as a control group. Water balance over the course of the study was determined by quantifying the percentage (%) of total body water [TBW; (TBW/body mass)] and water flux (water consumption - urine volume). TBW was estimated, by means of deuterium oxide dilution, before the study and after 3 days of centrifugation and by means of desiccation after 12 days of centrifugation. %TBW did not change in the centrifuged rats from initial levels or relative to controls over the course of the study. Differences between the sum of water consumption and sum of urine volume for the 12-day period were the same in both groups. Although an initial period of negative water balance was observed, the lack of a change in %TBW among the three measurement periods or in water flux over the 12 days of centrifugation suggests that water balance is not negatively affected as a result of centrifugation at 2 G.     

Modulation by centrifugation of cell susceptibility to chlamydial infection.

Enhancement of chlamydial infection of cell monolayers by centrifugation was shown to depend on induced cell surface changes. Evidence for this came from analysis of two forms of organism attachment which take place during centrifugation. In 'productive binding', organisms attached to cells and then entered and infected them. In 'unproductive binding', organisms became attached to cells but were not ingested. These organisms could be stripped from the cells by treatment with trypsin and could then infect fresh monolayers. Measurement of attachment kinetics during centrifugation showed that cells passed through three different susceptibility states. Only productive binding occurred in the first 20 min; cells then entered a refractory state during which no attachment took place At about 45 min, attachment recommenced but this allowed only unproductive binding. Induced movement of cell surface structures may enhance infection by promoting specific or non-specific interactions. Failure of ingestion may result from insufficient cell 'receptors' for circumferential binding of the whole chlamydial surface so that engulfment cannot take place.     

Purification of filamentous plant viruses by thin layer centrifugation.

The construction of a modified thin layer ultracentrifuge rotor is described. This rotor was used in the purification of five filamentous plant viruses, viz. TMV, SCMV, PVX, SGV and YMC. The purification and concentration of these viruses in their monomeric forms is hazardous when conventional "tube" rotors are used since they invariably result in dissociation and aggregation of the virus particles. Using the thin layer rotor these infective agents may be concentrated in volumes of fluid equal to approximately 1% of the starting suspension and not as pellets obtained after ultracentrifugation is conventional "tube" rotors. Electron microscopy revealed that the virus particles concentrated by thin layer centrifugation were not aggregated and that only few fragments of the virus filaments were present in the final preparations.     

Analysis by isopycnic centrifugation of isolated nucleoids of Escherichia coli.

The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 +/- 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 +/- 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.     

Purification of Synechococcus lividus by equilibrium centrifugation and its synchronization by differential centrifugation

A culture of the thermophilic, unicellular, blue-green alga Synechococcus lividus was freed from two thermophilic bacteria by use of equilibrium (isopycnic) centrifugation in Ficoll density gradients. After removal of the bacteria, the alga would grow only on agar plates in a high carbon dioxide atmosphere. Intermittent illumination, equilibrium centrifugation, and differential centrifugation were tested as techniques for obtaining synchronized cultures of the alga. Daughter cells selected by differential centrifugation in Ficoll density gradients yielded the best synchrony, and a cross-wall index of 85% was observed during the period of cell division.