Coulter volume

Mack Fulwyler at the Los Alamos Laboratory in New Mexico had decided to investigate a problem well known to everyone looking at red blood cells in Coulter counters. Red cells were known to show a bimodal distribution of their electrical resistance ( Coulter volume ). Anyone looking at erythrocytes under the microscope cannot help but be impressed by the remarkable structural uniformity of these cells Fulwyler wondered if the bimodal Coulter volume distribution represented differences between two classes of these apparently very uniform cells or, alternatively, whether the bimodal profile was simply an artifact based on some quirky aspect of the electronic resistance measurements. The most direct way of testing these two alternatives... 

Coulter volume A cell s Coulter volume is the increased impedance that occurs as the cell displaces electrolyte when it flows through a narrow orifice. This increase in impedance is related to the volume of the cell. 

Volume Volume is a useful and definite characteristic of any particle —but one that is not easily amenable to flow cytometric analysis. Coulter volume does measure volume to a close approximation, but forward scatter does not. 

Fig. 2 Median angle light scatter (DF8) versus Coulter volume histograms for whole blood mixed with various sizes (2.13, 2.40, 3.20, and 4.00pm diameter left to right, top to bottom) of CD3 antibody-conjugated PS latex beads...

Various techniques and equipment are available for the measurement of particle size, shape, and volume. These include for microscopy, sieve analysis, sedimentation methods, photon correlation spectroscopy, and the Coulter counter or other electrical sensing devices. The specific surface area of original drug powders can also be assessed using gas adsorption or gas permeability techniques. It should be noted that most particle size measurements are not truly direct. Because the type of equipment used yields different equivalent spherical diameter, which are based on totally different principles, the particle size obtained from one method may or may not be compared with those obtained from other methods. 

The support and the catalysts were characterised by means of nitrogen adsorption, XPS, TPD and SEM. The nitrogen adsorption isotherms were determined at 77 K in a Coulter Omnisorp 1000 CX equipment, and were analysed by the BET equation (SBet), and by the t-plot for mesopore surface area (Smeso) and micropore and mesopore volume (Vmicr0, Vmeso), using the standard isotherm for carbon materials. The catalyst samples were previously outgassed at 120 °C. 

Procedure of pollen preparation Pollen can be washed off stigmas with an acetone solution as water or other polar solutions often fail to sufficiently break electrostatic bonds holding heterospecific pollen to stigma. However, this means the acetone must be evaporated in an air drying oven (48 h) because a Coulter Counter requires a saline solution of standard volume (usually 10-20 ml) be used to prepare pollen samples. If the solutions are mixed and the volumes are inconsistent, there is a risk that differences in conductivity will create errors. 

Cell titres are determined by diluting of the cell suspension in Isoton and counting an appropriate volume (usually 0.5 ml) with a Coulter counter. Two counts are made per suspension. 

Again, the majority of these parameters are interrelated and highly dependent on the method used to determine them. Red blood cell count (RBC), platelet counts, and mean corpuscular volume (MCV) may be determined using a device such as a Coulter counter to take direct measurements, and the resulting data are usually stable for parametric methods. The hematocrit, however, may actually be a value calculated from the RBC and MCV values and, if so, is dependent on them. If the hematocrit is measured directly, instead of being calculated from the RBC and MCy it may be compared by parametric methods. 

Fig. 4.2.9 Histograms of the size distributions of the particles shown in Fig. 4.2.8. Original and final size distributions are shown by broken and solid lines, respectively. The diameter of an equivalent sphere having the same volume as a nonspherical particle was obtained with a Coulter counter. (From Ref. 9.)...

Other methods that are based on physical volume effects (e.g., the Coulter counter, based on electrical conductivity)... 

Cells were grown in RPMI medium plus 5 % fetal bovine serum in 1 mL total volume as described in Materials and Methods. At 24-hr intervals the cells were counted in a Coulter counter. Control cells 0, cells from cultures containing 1000 lU/mL mouse fibroblast interferon , cells from cultures containing bovine brain gangliosides at a concentration corresponding to 35 p.M sialic acid , cells from cultures containing both interferon (1000 IU/mL) and gangliosides (35 p.M sialic acid). 

CLS13 320 Particle Size Analyzer (Beckman-Coulter Co.), by volume-weight... 

For CS 1.94, several treatments were performed as described above in order to obtain enough volume of hydrolysates for fermentation experiments. Thereafter the hydrolysates were mixed, stored at 4°C and several detoxification methods, including activated charcoal and anion-exchange resin treatments, were applied. Nondetoxified hydrolysate was adjusted to pH 5.5 by adding Ca(OH)2. After 1 h the precipitate was removed by centrifuging (Avanti J-25i Beckman Coulter, Fullerton, CA) at 7500g for 25 min. The detoxification treatments were done as follows. 

Harvest 1 L of induced bacterial culture by centrifugation at 4400 g for 30 min at 4°C (Beckman J2-21 centrifuge, Beckman Coulter, Fullerton, CA). Carefully remove and discard the supernatant. Resuspend bacterial pellet in IX PBS using a 10-mL disposable plastic pipet. Pipet the solution up and down until no visible pellet is left in the suspension. Adjust the final volume to 25 mL with IX PBS. 

FIGURE 6 Particle size distribution of a y-alumina powder measured by an excluded volume method, in this case involving a Coulter counter. 

Cell concentration in suspension can be determined through an optical microscope employing a hemocytometer for manual cell counting, or in a semi-automatic way using an electronic particle counter (such as a Coulter counter), as described in detail by Freshney (2005). Through dye exclusion (such as trypan blue), it is possible to determine viable cell concentration, that is the number of cells in a known sample volume capable of proliferating in favorable culture conditions. 

There are two methods of estimating the number of cells in a suspension. Using a haemocytometer the number of cells in a given volume is counted by direct microscopic examination. Using electronic counters, e.g. the Coulter counter, the cells in a given volume of suspension are drawn through an orifice and registered electronically. 

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