A new spin on cytocentrifugation.

The patented Cytopro system design makes significant advances in the convenience and reliability of cytocentrifugation. The chamber and pad are designed to facilitate prewetting and to better control fluid flow rates. The sealed rotor is durable, convenient, and reliable with significant patented features impacting sample quality and convenience of operation.

Influence of fluid absorption time on cell recovery in cytocentrifugation.

OBJECTIVE: To determine the absorption time required to give complete cell recovery in cytocentrifugation. STUDY DESIGN: A simplified model for cell recovery was outlined that relates cell recovery to the relative magnitude of cell sedimentation velocity (SV) and fluid velocity (FV) during cytocentrifugation. The recovery of blood leukocytes was measured at various relative values of the SV/FV ratio and plotted to provide an indication of the effect of the ratio on recovery and to identify the conditions necessary to yield complete cell recovery. RESULTS: As SV/FV approaches zero, cell recovery also approaches zero. As the SV/FV ratio increases, recovery increases toward a maximum of 100%. The fluid absorption time is a key factor in the SV/FV ratio, which also includes centrifugal force and sample volume. A preliminary evaluation of commercial equipment yielded a wide range of absorption characteristics. In addition, fluid flow can be slowed and the SV/FV ratio increased by adding macromolecules--e.g., bovine serum albumin--to the sample fluid. CONCLUSION: Cytocentrifugation has the capability of nearly complete cell recovery if conditions are selected that yield a high SV/FV ratio. Such recovery usually requires slowing the fluid flow rate beyond what is normally obtained in most commercial equipment.

Effect of sample volume on cell recovery in cytocentrifugation.

The purpose of this study was to improve cell recovery and distribution in cytocentrifugation by optimizing sample volume. The experimental design used equal cell numbers in different diluent volumes from 25 to 500 microL. As the diluent volume increased, the fractional recovery of cells also increased, from 10% to a virtual 100%, even though the number of cells per sample remained constant. Optimal cell recovery was achieved with sample volumes between 200 and 500 microL. The importance of using a 200-microL volume with samples of high cellularity as well as those of low cellularity, such as cerebrospinal fluid, is discussed.

Enumeration of total bacteria in raw and pasteurized milk by reflectance colorimetry: collaborative study.

Seven out of 9 laboratories completed a collaborative study comparing a reflectance colorimetric (RC) bioactivity monitor (Omnispec 4000) method to the standard plate count (SPC) method for estimation of total bacteria in raw and homogenized pasteurized milk. Each laboratory analyzed 12 different samples by the SPC method and 24 samples (12 blind duplicates) by the RC method. For the RC method RSDr was 1.7%, and RSDR was 4.5%. RSDR for the SPC method was 20.8%. The method was adopted first action by AOAC INTERNATIONAL.

Rapid transfer of oxygens from inorganic phosphate to glutamine catalyzed by Escherichia coli glutamine synthetase.

Measurements are reported on certain isotopic fluxes during the net conversion of glutamine, ADP and Pi to glutamate, NH3, and ATP by Escherichia coli glutamine synthetase (adenylylated form, Mn2+ activated) in presence of a hexokinase/glucose trap to remove the ATP formed during the reaction. The results show that the transfer of oxygens from Pi to glutamine is the most rapid of the measured isotopic interchanges, over five oxygens from Pi being transferred to glutamine for each glutamate formed by net reaction. Under similar conditions, the oxygen transfer from Pi to glutamate, was stimulated somewhat by an increase in the glutamate concentration but inhibited by an increase in the ammonia concentration. The enzyme from brain or peas did not show the rapid transfer of 18O from Pi to glutamine shown by the E. coli enzyme. Deductions are also made from the data about the availability of the oxygens of gamma-carboxyl of bound glutamate for reaction. The most logical explanation of the results with the E. coli enzyme is that the gamma-carboxyl group of bound glutamate has sufficient rotational freedom so that under conditions of rapid substrate interconversion either carboxylate oxygen can participate in the reaction. The results with the pea enzyme are consistent with hindered rotation of the gamma-care additional findings make likely a relative order of certain catalytic steps for the E. coli enzyme as follows: ATP release less than NH3 release less than glutamate release less than substrate interconversion less than glutamine release and Pi release and glutamate release less than ADP release.

Probes of initial phosphorylation events in ATP synthesis by chloroplasts.

Rapid mixing and quenching techniques have been used with chloroplasts activated by an acid-base transition or by light to assess the nature and characteristics of the substances initially labeled by inorganic [32P]phosphate during ATP synthesis. With light-activated chloroplast fragments, but not with acid-base-activated preparations, an initial rapid labeling of a small amount of ADP is observed. With the acid-base activated preparations a slower continued labeling of ADP occurs that is uncoupler-sensitive, that does not proceed via [gamma-32]ATP of the medium and for which medium ADP furnishes the AMP moiety. The results point to ADP as the initial acceptor of phosphate for ATP synthesis, with a slow side reaction in which bound ATP phosphorylates bound AMP to give a bound ADP. The phosphorylation of bound ADP by medium [32P]phosphate in the absence of added ADP is confirmed, but the reaction is too slow to serve as an intermediate in photophosphorylation. The appearance of label from [32P]phosphate in ATP in the acid-base transition at 25 degrees shows a lag of only about 3 to 7 ms, consistent with the absence of any phosphorylated intermediate. The lag is followed by a linear rate of [gamma-32]ATP formation that is about as fast as that observed in steady photophosphorylation, consistent with a proton gradient serving for transmission of energy from electron transfer reactions to the ATP-synthesizing complex.

Coupling of "high-energy" phosphate bonds to energy transductions.

Recent results suggest consideration of a new concept for oxidative phosphorylation in which a prime function of energy is to bring about release of ATP formed at the catalytic site by reversal of hydrolysis. Data with submitochondrial particles include properties of an uncoupler insensitive Pi=HOH exchange, a rapid reversible formation of bound ATP in presence of uncouplers, and predictable patterns of 32-Pi incorporation into ATP in rapid mixing experiments. ADP is confirmed as the primary Pi acceptor in mitochondrial ATP synthesis, but with chloroplasts ADP is also rapidly labeled. Other findings with pyrophosphatase and with transport ATPase harmonize with the new concept. Measurements of the reversal of ATP cleavage and binding by myosin suggest that oxygen exchanges result from reversible cleavage of ATP to ADP and Pi at the catalytic site and that the principal free energy change in ATP cleavage occurs in ATP binding. Reversal of conformational changes accompanying ATP binding and cleavage is proposed to drive the actin filament in contraction. Thus energy transductions linked to ATP in both mitochondria and muscle may occur primarily through protein conformational change.