Estimation of the disruption in freeze-pressed Saccharomyces cerevisiae by an electronic particle counter

An electronic particle counter has been used to estimate the disintegration by freeze-pressing baker's yeast. A counter threshold level which just yielded the maximum count for intact cells was selected. The conductivity of the suspending medium was chosen such that maximum counts were obtained. Under these conditions, the electronic counts agreed well with the visual counts. At a certain threshold level the maximum count was obtained at a lower resistivity (higher conductivity) in the suspending solution with the freeze-pressed suspension than with untreated cells, indicating that damage to the permeability barrier may occur without disruntion of the cell envelope. Fresh baker's yeast cells do not behave as nonconducting particles. This has to be taken into account when volume determinations with electronic particle counters are performed.     

Determination of Mammalian Cell Counts,Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls^1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques⁶.Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.     

On the Correlation of the Volume of a Prolate-Spheroidal Cell, Tetrahymena as Determined by Electronic Particle Counters and by Morphometry

ABSTRACT. The cell volume provided by electronic particle counters may be incorrect. As a particle, or cell, passes the counting device, its volume is calculated as a sphere. The electronically derived, mean cell volume (electronic MCV) of a population of Tetrahymena (prolate spheroid) is smaller than the volume (morphometric MCV) calculated from measured cell length and width. This discrepancy was studied using a Coulter Multisizer particle counter and cell morphometry. The electronic MCV averaged 0.70 of the morphometric MCV (1.00) but changed from 0.72 (fast growth) to 0.63 and 0.76 (slow or no growth) for cells having a mean length/width of 2.05, 2.33, and 1.61, respectively. The measured diameter of latex particles (used for calibration) was identical to that stated, but the diameter of the electronic MCV was larger than the width of the cells which related to wehther the length/width of the cells was above, or below, 2.00. Hence, electron particle counters register primarily the width of a prolate-spheroidal cell, oriented with its long axis in the direction of flow, and uses this value as diameter for the calculated sphere, whereas for more spherical cells, tumbling without any orientation, a mean of the axes is used. Factors for correction of the electronic MCV of Tetrahymena are provided.     

Fixation artifacts of phytoplankton in zooplankton grazing experiments

The influence of various fixatives on size distribution and cell concentration was investigated using two types of electronic particle counters. Both in autotrophic and heterotrophic flagellates drastic losses of cells and changes of size were found in all preservatives used. Also preserved natural seston samples were not representative of the original fresh sample, and they continued changing and varying in the course of time. In diatoms also changes in size were noticed due to the fixative, however, doubt arises whether they were real or only measured differently by the particle counter. Even filtered sea water could not be preserved without obvious changes in particle distribution. Apart from diatoms, commonly used fixatives must be regarded worthless to preserve most phytoplankton and protozooplankton species in grazing experiments with zooplankton.     

Tunable pores for measuring concentrations of synthetic and biological nanoparticle dispersions

Scanning ion occlusion sensing (SIOS), a technique that uses a tunable pore to detect the passage of individual nano-scale objects, is applied here for the rapid, accurate and direct measurement of synthetic and biological nanoparticle concentrations. SIOS is able to characterize smaller particles than other direct count techniques such as flow cytometry or Coulter counters, and the direct count avoids approximations such as those necessary for turbidity measurements. Measurements in a model system of 210-710 nm diameter polystyrene particles demonstrate that the event frequency scales linearly with applied pressure and concentration, and that measured concentrations are independent of particle type and size. Both an external-calibration and a calibration-free measurement method are demonstrated. SIOS is then applied to measure concentrations of Baculovirus occlusion bodies, with a diameter of ~1 μm, and the marine photosynthetic cyanobacterium Prochlorococcus, with a diameter of ~600 nm. The determined concentrations agree well with results from counting with microscopy (a 17% difference between the mean concentrations) and flow cytometry (6% difference between the mean concentrations), respectively.     

Increased mass of reticulo-rumen tissue and contents with advancing age in Svalbard reindeer

Little is known about the effect of age on digestive processes in wild cervids. One potentially important mechanism is that tooth wear alters the occlusal surface topography, causing gradual loss of functionality. Mastication efficiency is crucial to digestion processes among ruminants, as a larger particle size is associated with longer retention times and potentially reduced digestion efficiency. Using data from 49 adult Svalbard reindeer Rangifer tarandus platyrhynchus females, we investigated whether the mass of reticulo-rumen (RR) contents and tissue and the amount of back fat showed age-related changes. Older animals had higher RR content mass than younger individuals. This is consistent with the prediction that altered particle size due to decreased mastication efficiency led to increased rumen retention time either through increased RR capacity or filling. Additional data on RR particle size distribution were available for a subset ( n =30). The correlation between the proportion of small particles (<1.0 mm) and RR contents was much weaker than the correlation between age and RR contents. This suggests that additional factors apart from particle size contribute to the age-related pattern in RR content mass. The mass of RR tissue in older animals was higher than that in younger animals. This possibly reflects an adaptation to the higher organ fill. The amount of back fat decreased with age, suggesting that alterations in digestion-related processes are not sufficient to compensate for reduced mastication efficiency in Svalbard reindeer. Our results present one possible link between foraging, digestive processes and life-history patterns.     

Measurement of the nucleation of atmospheric aerosol particles

The formation of new atmospheric aerosol particles and their subsequent growth have been observed frequently at various locations all over the world. The atmospheric nucleation rate (or formation rate) and growth rate (GR) are key parameters to characterize the phenomenon. Recent progress in measurement techniques enables us to measure atmospheric nucleation at the size (mobility diameter) of 1.5 (±0.4) nm. The detection limit has decreased from 3 to 1 nm within the past 10 years. In this protocol, we describe the procedures for identifying new-particle-formation (NPF) events, and for determining the nucleation, formation and growth rates during such events under atmospheric conditions. We describe the present instrumentation, best practices and other tools used to investigate atmospheric nucleation and NPF at a certain mobility diameter (1.5, 2.0 or 3.0 nm). The key instruments comprise devices capable of measuring the number concentration of the formed nanoparticles and their size, such as a suite of modern condensation particle counters (CPCs) and air ion spectrometers, and devices for characterizing the pre-existing particle number concentration distribution, such as a differential mobility particle sizer (DMPS). We also discuss the reliability of the methods used and requirements for proper measurements and data analysis. The time scale for realizing this procedure is 1 year.     

Optically induced flow cytometry for continuous microparticle counting and sorting

This paper reports a new microfluidic device capable of performing optically induced flow cytometry (OIFC). This enables it to continuously count and to sort microparticles based on optically induced dielectrophoretic (ODEP) forces. Gravity was used to drive the particles instead of using syringe pumps. The particles were then focused inside a sample channel by the ODEP forces and then passed through a detection region. A pair of optical fibers were embedded into fiber channels to count the number of particles and analyze the particle size in real time. Using 20.9 and 9.7 microm polystyrene microparticles, the average light intensity were about 63.67 and 8.80 units, with a coefficient-of-variation (CV) of 7.46 and 25.57%, respectively. This demonstrated that these two particle sizes could be successfully distinguished. After detecting the number and size of the microparticles, an optically induced dynamic switch (ODS) was used to sort microparticles to downstream fluidic outlets. The ODS used ODEP forces generated by different illumination intensities or optical line widths. The ODS was composed of two virtual electrodes which controlled particle movement in two dimensions. The ODS can successfully sort microparticles with different sizes continuously. The development of the OIFC device is a major advancement in the design of microparticle counting and sorting devices. Applications in future biomedical applications for cell counting and manipulation are envisioned.     

Electronic particle counting for evaluating the quality of air in operating theatres: a potential basis for standards?

Airborne particle counting in eight size ranges (0.5- greater than 20 microns), by computerized electronic equipment, was compared with the numbers of bacteria-carrying particles (BCP) assessed by slit sampling in ultra-clean and turbulently ventilated operating theatres. In the ultra-clean theatre the number of particles of 5-7 microns size range correlated with BCP while peaks in the numbers of particles less than 3 microns and greater than 15 microns corresponded with activity. Comparative relationships also occurred in the turbulently ventilated theatre but the use of this equipment in that environment cannot yet replace counts of airborne bacteria. We consider that electronic particle counting in the 0-20 microns size range may be used to judge the performance of a clean air operating theatre distribution system, including efficiency and integrity of the filter/seal systems and the presence or absence of entrainment of bacteria and other particles. The sampling techniques and analysis of particle concentration results described here may be a suitable basis for standards.     

Use of the electronic particle counter to investigate phosphorus on particles in streams

A simple and rapid method is described for the measurement by electronic particle counter of the relative proportions of sediment particles, of diameters 1 to 40 µm, which could enter the stream suspended load. The method is useful to further investigations into nutrients borne upon such material because the amounts of phosphate per unit weight of particles varies through orders of magnitude depending upon the particle size. An example is given to illustrate the application of the method.     

Feasibility of Focused Beam Reflectance Measurement (FBRM) for Analysis of Pharmaceutical Suspensions in Preclinical Development

This study examined the use of focused beam reflectance measurement (FBRM) for qualitative and quantitative analysis of pharmaceutical suspensions with particular application to toxicology supply preparations for use in preclinical studies. Aqueous suspensions of ibuprofen were used as prototype formulations. Initial experiments were conducted to examine the effects of operational conditions including FBRM probe angle, probe location, and mixing (method and rate of mixing) on the FBRM analysis. Once experimental conditions were optimized, the homogeneity and sedimentation-redispersion of particles in the suspensions were assessed. Ibuprofen suspension under continuous agitation was monitored using FBRM for 60 h to study particle size change over time. Another study was performed to determine if particle count rates obtained by FBRM could be correlated to suspension concentration. The location and the angle of the FBRM probe relative to the beaker contents, and the rate and the method of mixing the suspension were found to be sensitive parameters during FBRM analysis. FBRM was able to monitor the process of particle sedimentation in the suspension. The attrition of ibuprofen particles was detectable by FBRM during prolonged stirring with an increase in the number of smaller particles and decrease in the number of larger particles. A strong correlation was observed between particle count rate by FBRM and ibuprofen concentration in the suspension. Also, change in content uniformity in the suspension at different locations of the beaker was represented by FBRM particle count. Overall, FBRM has potential to be a useful tool for qualitative and quantitative analysis of pharmaceutical suspensions.     

Immobilized enzyme reaction stability: Attrition of the support material

One of the main reasons for immobilizing an enzyme is to enable its reuse, or continuous use, in a reactor. Consequently immobilized enzyme stability is an important factor in enzyme reactor design. The performance of the reactor will decrease if during operation the support material disintegrates into smaller particles that pass out of the reactor system. When β-galactosidase is immobilized by covalent attachment to AE-cellulose, the smaller particles have a higher activity. After subjection of the immobilized enxyme to a shear stress the average particle size decreases and the total enzymic activity increases. A loss of small particles from the reactor, although constituting a small weight percent loss of support, will result in a disproportionately large loss in activity. The relevance of these observations to reactor performance is discussed.     

Insulin particle formation in supersaturated aqueous solutions of poly(ethylene glycol)

Protein microspheres are of particular utility in the field of drug delivery. A novel, completely aqueous, process of microsphere fabrication has been devised based on controlled phase separation of protein from water-soluble polymers such as polyethylene glycols. The fabrication process results in the formation of spherical microparticles with narrow particle size distributions. Cooling of preheated human insulin-poly(ethylene glycol)-water solutions results in the facile formation of insulin particles. To map out the supersaturation conditions conducive to particle nucleation and growth, we determined the temperature- and concentration-dependent boundaries of an equilibrium liquid-solid phase separation. The kinetics of formation of microspheres were followed by dynamic and continuous-angle static light scattering techniques. The presence of PEG at a pH that was close to the protein's isoelectric point resulted in rapid nucleation and growth. The time elapsed from the moment of creation of a supersaturated solution and the detection of a solid phase in the system (the induction period, t(ind)) ranged from tens to several hundreds of seconds. The dependence of t(ind) on supersaturation could be described within the framework of classical nucleation theory, with the time needed for the formation of a critical nucleus (size <10 nm) being much longer than the time of the onset of particle growth. The growth was limited by cluster diffusion kinetics. The interfacial energies of the insulin particles were determined to be 3.2-3.4 and 2.2 mJ/m(2) at equilibrium temperatures of 25 and 37 degrees C, respectively. The insulin particles formed as a result of the process were monodisperse and uniformly spherical, in clear distinction to previously reported processes of microcrystalline insulin particle formation.     

Compartmental models with restricted movement

A restriction is imposed on the number of particles that can possibly move at any time from a compartment, so that any other particles present in the compartment must wait until such particles have moved out. The equations for such a system are formulated and the solution is given for a single compartment system; increased variability of the compartmental particle count is one effect of this restriction.     

Untersuchungen zur Separation aufgeschlossener Hefesuspensionen

By the means of the electronic particle counting, characteristics of industrial contrifuges are found out in order to characterize the performance in the case of the separation of cell debris of mechanically disrupted yeast cells. The knowledge about the particle size distribution complements the usual determination of the dry matter content in the supernatant. Thus an optimization of the separation of tiny particles is facilitated.     

No distinct difference in the excretion of large particles of varying size in a wild ruminant,the banteng (<Emphasis Type="Italic">Bos javanicus</Emphasis>)

The forestomach of ruminants and camelids does not only allow a differential excretion of fluids and small particles but also a differential excretion of small and large particles. The question whether larger particles of different size classes are also retained for different time periods, or whether simply a particle-size threshold exists above which all particles of a size higher than this threshold are retained in an undifferentiated manner, has not been addressed so far. We determined the mean retention time (MRT) of different-sized large particles (10 and 20 mm) in three banteng (Bos javanicus) on two forage only diets, grass and grass hay. We used cerium-mordanted fibre (10 mm) and lanthanum-mordanted fibre (20 mm) as particle markers, mixed in the food. Average total tract MRT for large and very large particles at the grass diet was 58 and 56 h and at the grass hay diet 66 and 64 h, respectively. Very large particles moved slightly faster than large particles through the gut of the banteng. Three interpretations are possible: Very large particles are resubmitted to rumination sooner than large particles; ingestive mastication of the particle markers could have reduced the difference in the size of the particle markers; alternatively, particle retention may be governed by a threshold, above which all particles of a size higher than this threshold are retained in an undifferentiated manner. In order to test these possibilities, experiments with fistulated animals would have to be performed.     

Electronic and utermöhl comparative techniques in quantitative analysis of freshwater particulates

The Coulter Counter Model B and the Millipore Particle Measurement Computer System were compared with the Utermöhl technique in counting and sizing test objects and suspended freshwater particulate matter. Replicate samples of lake water and pulp mill effluent were analyzed with the electronic instruments for an estimate of counting error % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaaeWaaeaada% WcaaqaaiGacofaciGGfbGaaiOlaiaacshacaGGUaGaaiymaiaaccda% caGGWaaabaGabiiEayaaraaaaaGaayjkaiaawMcaaaaa!3EB7!\[\left( {\frac{{\operatorname{S} \operatorname{E} .t.100}}{{\bar x}}} \right)\] Coulter Counter error for determinations of number, volume, and diameter of homogeneous particles was less than io%, and for natural populations of freshwater particles, less than 2o%. Millipore errors of number, area, and horizontal chord were roughly twice these values. Precision was least at low concentration of particles. In practical applications, pulp mill effluent analyses were characterized by close correlation between electronic methods of counting, but lake water particle determinations failed to relate. Microscope counts and sizes of freshwater particles were generally unrelated to electronic measurements. A definite electronic instrument specificity for homogeneous particles was apparent. Wide variations in determinations of characteristics by electronic and manual methods existed. The greatest problem in the. estimation of freshwater biomass is the determination of the unknown detrital fraction.     

Precision of the daily pollen count. Identifying sources of variation using variance component models

Summary During the pollen season, a daily account of airborne pollen is reported by radio and newpapers in Denmark as «Today's pollen count». The Aerobiological Group under the Danish Asthma and Allergy association is responsible for the daily identification and counting of pollen. In 1984 the Group set up a trial to investigate the reproducibility of the pollen count.Three trained pollen counters independently examined the same specimens from two Burkard pollen traps. The traps were located on the roof of the Meterological Institute in Copenhagen 15 meters above ground level in the northern outskirts of central Copenhagen. This is the usual sampling site. Specimens from each trap from 20 days during the grass pollen season were selected. Four species groups were identified in sufficient numbers for statistical analysis. These were, in order of occurrence, spruce/pine (Picea/Pinus), grass (Poaceae), nettle (Urtica) and mugwort (Artemisia).Identification of sources of variation and assessment of their relative importance were carried out using variance components models. A detailed account of the method is given.Qualitatively equivalent conclusions were reached for each group: i) The expected seasonal variation was identified; ii) The counters contributed significantly to the variation of pollen counts; iii) The traps did not contribute to the variance, but a day-trap interaction was identified. This interaction was interpreted. as a problem of instrumental variation over time of traps of variation in meteorological conditions; iv) The total variance was larger than originally expected. The relative uncertainty was greater than 50 per cent.The variance of the daily pollen count cannot be reduced much by reducing counter variation or day-trap interaction. The variation among pollen counters is small, and the day-trap interaction is difficult to fully comprehend. The basic measurement error can be reduced by examining a larger area of each specimen. Although this implies a greater cost, it is probably the most effective method of reducing the uncertainty of «Today's pollen count».     

New chamber for flow cytometric analysis over an extended range of stream velocity and application to cell adhesion measurements.

When analyzed in a flow cytometer, particles are suddenly accelerated to high velocities (1-10 m.s-1) over very short distances. This feature is essential to obtain high analysis rates and low coincidence levels, but translates into very strong velocity gradients (greater than 10(5) s-1): particles experience strong hydrodynamic stresses that elongate them and tend to dissociate weakly associated complexes. In order to analyze fragile conjugates formed by heterotypic adhesion between two cell types, a flow cytometer was modified to make hydrodynamic stress not only much weaker but also adjustable. A new and easily adaptable flow cell was designed for the instruments of the FACS series; it provided satisfactory hydrodynamic conditions on a wide continuous range of flow rates. Accompanying electronic adaptations permitted standard analysis between 0.01 and 10 m.s-1. At 0.01 m.s-1, the velocity gradient roughly amounts to 50 s-1. Conjugates formed by the adhesion between human B and resting T lymphocytes, disrupted in conventional flow cytometers, could be detected and precisely quantified provided analysis velocity was kept below 0.1 m.s-1. We conclude that low velocity flow cytometry makes possible the quantification of weak intercellular adhesion phenomena, and is potentially useful for the future development of new biomechanical techniques and other applications.     

Investigation of how agitation during precipitation,and subsequent processing affects the particle size distribution and separation of α-lactalbumin enriched whey protein precipitates

In this work, α-lactalbumin (α-la) rich precipitate particles are formed and aged in a batch stirred-tank from a whey protein concentrate (WPC) dispersion. Precipitation of the proteins occurs during a period of acid-addition followed by an ageing period. This study investigates how stirred-tank impeller agitation and subsequent processing, by means of passing precipitate suspensions through a capillary tube or a partially open ball-valve, affect particle size and composition. Precipitate particles are largely unaffected when subjected to laminar capillary tube flow. However, as flow becomes transitional and thereafter turbulent, particle breakage increases, especially for precipitates formed and aged under mild agitation conditions. Precipitates passed through the ball-valve experience even greater particle size reduction as a sharp geometrical transition results in highly turbulent flow. Moreover, particles formed and aged under low shear conditions, though initially larger, are in fact weaker and fragment to a greater extent during turbulent processing through the ball-valve. This has process design implications for separation processes where particle size is important, as shear history can influence particle toughness. Substantial size reduction of particles can best be mitigated by identifying regions of high turbulence or sudden changes in flow geometry, and by redesigning these regions so as to reduce these effects.