Differences In Cell Concentrations Research Articles

The viability of human cells irradiated with 470-nm light at various radiant energies in vitro.

Blue light is known to be antimicrobial, but its effect on normal cutaneous and subcutaneous cells remains unclear. Therefore, we studied the effect of 470-nm light on the viability of adult and neonatal human dermal fibroblasts, Jurkat T-cells, and THP-1 monocytes in vitro. Each culture was irradiated with 0, 3, 55, or 110 J/cm2 of 470-nm light and subjected to trypan blue assay to ascertain viability. Further, MTT, neutral red, and fluorescence assays of fibroblasts were performed, and cell morphology visualized using bright field and fluorescence microscopy. At each dose and in each of the four celllines, there was no significant difference in cell concentration between irradiated and non-irradiated cultures, even though irradiation with 55 J/cm2 or 110 J/cm2 slightly decreased cell count. Light microscopy showed progressive morphological changes in the fibroblasts as energy fluence increased from 55 to 110 J/cm2. Irradiation at 3 J/cm2 produced a slight but non-significant increase in the viability of Jurkat T-cells and THP-1 monocytes. In contrast, at 110 J/cm2 radiant exposure, irradiation slightly decreased the viability of all four cells. While 3 J/cm2 appears stimulatory, our finding that 110 J/cm2 produces a slight decrease in viability and engenders morphological changes in fibroblasts, suggesting that such high doses should be avoided in blue light treatments.

Insights into Heap Bioleaching at the Agglomerate-Scale

Previous agglomerate-scale heap bioleaching studies have outlined the variations in cell numbers of the liquid and attached phases during colonisation of sterilised ore by a pure culture. In this study, a mixed mesophilic culture was used in agglomerate-scale columns containing non-sterilised low-grade copper ore. Over a six - month period, columns were harvested at various intervals to provide snapshots of the metal distribution and the quantity, location, and ecological variations of mineral-oxidizing microbes within the ore bed. The initial colonisation period in this experiment was dissimilar to previous work, as the indigenous community was retained within the ore-bed throughout acid agglomeration. The overall colonisation phase lasted for approximately 1,000 hours until cell concentrations stabilised. In each column, less than 0.05% of the total cells were found in the leachate, 15-20% in the interstitial phase and the remaining ~80% were attached to the mineral surface. Once cell numbers had stabilised, interstitial cell concentrations were approximately 2,000× greater than those in the leachate. This difference persisted for the duration of the experiment. Copper concentrations in the two liquid phases generally decreased over time, but were on average 50× higher in the interstitial phase. Iron concentrations were more stable, but again were 30× higher in the interstitial phase. This demonstrates that that the difference in cell concentration between the leachate and interstitial phases cannot be explained through diffusion gradients within the system as it is much greater than those observed for the dissolved metals. It also shows that the specific environmental conditions of the interstitial and attached cells are very different to those inferred through analysis of leachates alone.

Estimating Cell Concentration in Three-Dimensional Engineered Tissues Using High Frequency Quantitative Ultrasound

Histology and biochemical assays are standard techniques for estimating cell concentration in engineered tissues. However, these techniques are destructive and cannot be used for longitudinal monitoring of engineered tissues during fabrication processes. The goal of this study was to develop high-frequency quantitative ultrasound techniques to nondestructively estimate cell concentration in three-dimensional (3-D) engineered tissue constructs. High-frequency ultrasound backscatter measurements were obtained from cell-embedded, 3-D agarose hydrogels. Two broadband single-element transducers (center frequencies of 30 and 38MHz) were employed over the frequency range of 13-47MHz. Agarose gels with cell concentrations ranging from 1×10(4) to 1×10(6)cellsmL(-1) were investigated. The integrated backscatter coefficient (IBC), a quantitative ultrasound spectral parameter, was calculated and used to estimate cell concentration. Accuracy and precision of this technique were analyzed by calculating the percent error and coefficient of variation of cell concentration estimates. The IBC increased linearly with increasing cell concentration. Axial and lateral dimensions of regions of interest that resulted in errors of less than 20% were determined. Images of cell concentration estimates were employed to visualize quantitatively regional differences in cell concentrations. This ultrasound technique provides the capability to rapidly quantify cell concentration within 3-D tissue constructs noninvasively and nondestructively.

Parametric imaging of three-dimensional engineered tissue constructs using high-frequency ultrasound

The goal of this study was to use high-frequency ultrasound to nondestructively characterize three-dimensional engineered tissues. We hypothesized that backscatter spectral parameters, such as the integrated backscatter coefficient (IBC), can be used to quantify differences in cell concentration in engineered tissues. We chose the IBC parameter since it estimates the backscattering efficiency of scatterers per unit volume. In this study, acoustic fields were generated using single-element, focused transducers (center frequencies of 30 and 40 MHz) operating over a frequency range of 13 to 47 MHz. Three-dimensional engineered tissue constructs were fabricated with mouse embryonic fibroblasts homogenously embedded within agarose. Constructs with cell concentrations ranging from 1x104 to 1x106 cells/mL were investigated. The IBC was computed from the backscatter spectra, and parametric images of spatial variations in the IBC were generated. Results showed that the IBC increased linearly with cell concentration. Further, we demonstrated that parametric images detected spatial variations in cell concentration within engineered tissue constructs. Thus, this technique can be used to quantify changes in cell concentration within engineered tissues and may be considered as an alternative to histology. Furthermore, because this technique is nondestructive, it can be employed for repeated monitoring of engineered tissues throughout the duration of fabrication.

Effect of Different Filter Methods on Seawater Quality at a Marine Scallop Hatchery

The effect of using two different filtering methods in the main seawater inlet to a scallop (Pecten maximus) hatchery in Norway was tested. Seawater was filtered through active filter media (AFM) and a protein skimmer, or through a drum filter and a protein skimmer. Seawater quality was characterized and tested on algal growth rate, egg development and larval activity. Tests were performed under winter and spring conditions (March, April and May 2009). Both seawater treatments reduced the dissolved organic carbon concentrations in the inlet seawater. The total bacterial number was stable in both seawater treatments, except for an increase in the drum filter in March. The bacterial community showed seasonal development: Actinobacteria and Alphaproteobacteria dominated in March, while Gammaproteobacteria dominated in April and May. In a cluster analysis, samples from both seawater treatments showed high similarity on similar sampling dates. Vibrio spp. occurred, but was never observed in seawater coming from the skimmer after the drum filter. This sampling point was often clustered as most similar to the incoming seawater. The fraction of scallop eggs that developed into veliger larvae increased from 10% to 50% during the sampling period, and no significant differences were found between the two seawater treatments. The fraction of 8 day old active larvae was lowest in March, in experiments with both undiluted and diluted (1:10, 1:100) seawater from both treatments. No significant difference in activity was found between the treatments, except for undiluted (April) and 100-fold dilution (April and May) from the drum filter, when the larval activity was significantly higher. The effect of both seawater treatments was tested by growing the diatom Chaetoceros muelleri in small volumes for 4-5 days. Daily growth rates (μ) varied between 0.75 and 1.15, and were highest in May. No significant difference in cell concentration was found between the treatments. The results showed that the skimmer attached to the drum filter had the best performance overall in reducing dissolved organic carbon and potentially lethal bacteria. These findings have important implications for hatchery seawater management protocols.

Evolution of fermenting microbiota in tarhana produced under controlled technological conditions

The purpose of this study was to evaluate the evolution of lactic acid bacteria (LAB) and yeasts during the fermentation of tarhana produced with some pasteurised ingredients and carried out at 30 and 40 °C. The chemical parameters were those typical for tarhana production. Coliform bacteria were not detected during fermentation, while LAB and yeasts were in the range 107–108 colony forming units (CFU) g−1. Plate counts showed an optimal development of both fermenting microbial groups and the differences in cell concentrations were not significant (P > 0.05). LAB were isolated during fermentation and grouped on the basis of phenotypic and polymorphic characteristics. LAB isolates were identified by a combined genetic approach consisting of 16S/23S rRNA intergenic spacer region (ITS) and partial 16S rRNA gene sequencing as Pediococcus acidilactici, Lactobacillus plantarum and Lactobacillus brevis. Hence, the pasteurisation of the vegetable ingredients, excluded wheat flour, enhanced the hygienic conditions of tarhana without influencing the normal evolution of LAB. However, the fermentation at 40 °C favoured pediococci, while the production at 30 °C was mainly characterised by lactobacilli. Yeasts, identified by the restriction fragment length polymorphism (RFLP) of the 5.8S ITS rRNA gene, were mainly represented by the species Saccharomyces cerevisiae in both productions.

Enumeration of Archaea and Bacteria in seafloor basalt using real-time quantitative PCR and fluorescence microscopy

A SYBR Green real-time quantitative PCR (Q-PCR) assay for the detection and quantification of Bacteria and Archaea present in the glassy rind of seafloor basalts of different ages and water depths is presented. Two sets of domain-specific primers were designed and validated for specific detection and quantification of bacterial and archaeal 16S rRNA genes in DNA extracted from basaltic glass. Total cell numbers were also estimated by fluorescence microscopy analysis of SYBR Gold-stained samples. The results from the two different approaches were concurrent, and Q-PCR results showed that the total number of cells present in basalts was in the range from 6 x 10(5) to 4 x 10(6) cells g(-1) basaltic glass. Further, it was demonstrated that these cells were almost exclusively from the domain Bacteria. When applying the same methods on samples of different ages (22 years-0.1 Ma) and water depths (139-3390 mbsl), no significant differences in cell concentrations or in the relative abundance of Archaea and Bacteria were detected.

The growth rate, biomass production and composition of Chaetoceros sp. grown with different light sources

The diatom Chaetoceros sp. was grown with a continuous photon flow rate of 125 μmol m −2 s −1, provided by Cool White (CW), or by Gro-Lux and Gro-Lux wide spectrum agricultural lamps (GRO and GRO/WS). At the end of exponential and during stationary growth, these gave higher cell numbers and biomass than CW lamps. There were no differences in cell concentration and growth rates between GRO and GRO/WS-grown cultures, but biomass was significantly higher with GRO lamps. In all treatments, protein concentration was higher and lipids were lower at the end of exponential growth than in the stationary phase, but proteins remained consistently higher than other treatments with GRO lamps in both growth phases, possibly because of their high emission of blue light. CW gave the highest lipid and GRO/WS the best carbohydrate production during both phases of growth. This suggests that these consistent differences are caused by different spectral qualities of light, that could be used to obtain different live diets for aquaculture, varying the time of harvest and the type of light sources.

Investigations of cephalosporin C production in an airlift tower loop reactor

Cephalosporin C was produced by Cephalosporium acremonium in a 60 l airlift loop reactor on complex medium (with 30 kg/m3 peanut flour) in fed-batch operation. A final product concentration of 5 kg/m3 and a maximum productivity of 45 g/m3 h were attained. On-line analysis was used to determine ammonia, methionine, phosphate, reducing sugar and cephalosporin C by an autoanalyser, glucose by a flow injection analyser and cephalosporin C, penicillin N, deacetoxycephalosporin C, deacetylce-phalosporin C and methionine by HPLC. The volumetric productivity of the stirred tank reactor was higher than that of the airlift reactor because of differences in cell concentration. Specific productivities in relative to cell mass were similar in the two reactors. The substrate yield coefficient in the airlift reactor was twice that in the stirred tank reactor.

Low density lipoprotein receptor determination in peripheral blood mononuclear cells: Influence of differences in cell concentration

Low density lipoprotein (LDL) receptor determination in peripheral blood mononuclear cells (PBMCs) is influenced by differences in cell concentration. As the cell concentration increases, measured LDL receptor activity decreases. This inter-relationship is caused by a PBMC-induced modification of 125I-LDL. The PBMC-modified 125I-LDL results from shedding of polyanionic cell membrane constituents that subsequently bind to 125I-LDL, and has reduced capacity of binding to the LDL receptors, to the cell membrane independent of the receptors and even to plastic. The cell membrane constituents contain sulphate, have a MW = 200,000-300,000, are heat stable and are rapidly released at 37 degrees C as well as at 4 degrees C. They probably represent a heterogeneous group of proteoglycans, glycoproteins and glycolipids. The higher the cell concentration is, the more polyanionic cell membrane constituents are released, and at high concentrations they may even form aggregates of LDL. We conclude that differences in PBMC concentration interfere with LDL receptor analyses through shedding of different amounts of polyanionic cell membrane constituents into the medium. Thus, standardisation of the experimental procedures with respect to cell number is of great importance in LDL receptor determination in PBMCs.

Cell-released substances interfere with low density lipoprotein receptor determination in fibroblasts.

Low density lipoprotein (LDL) receptor activity in fibroblasts decreases with increasing cell concentration. This was found for radioligand assays at 4 degrees C and 37 degrees C. Thus, it seems that it is the binding of 125I-LDL to the LDL receptor which is the step in the LDL receptor pathway that is influenced by differences in cell concentration. The reduced binding of 125I-LDL with increasing cell concentration seems to result from a modification of 125I-LDL by fibroblasts. Binding of 125I-LDL to cell-free plastic tubes was used to study this modification of 125I-LDL. Such studies indicated that direct cell contact was not required for the modification to occur. The rapid modification of 125I-LDL (within 10 min) took place even at 4 degrees C. These findings suggest that substances released from the cell membrane interact with LDL and decrease its binding to the LDL receptor. The higher the cell concentration is, the more modification of LDL takes place. Fibroblast modified LDL has reduced electrophoretic mobility. We conclude that differences in cell concentration influence LDL receptor activity by interfering with the binding of 125I-LDL to the LDL receptors, rather than affecting the receptor number on the cells' surfaces.

Free energy of cell differentiation

The rigid polarity of the coelentrate, hydra, has been examined based on the pattern of cell types found along the body column. The distribution of several cell types was determined by direct counts from histological sections. Treating the individual cell types as separate thermodynamic phases, the distribution of cell types was analyzed using a thermodynamic free energy function. (A steady state free energy function is derived to deal with cell differentiation.) Based on the analysis of the distribution of cell types with the free energy function, it is concluded that cell differentation in hydra is accompanied by mass flow of the components, which are responsible for cell differentiation and/or changes in the free energy of the cell as it is transformed from a precursor to a differentiated cell. The presence of marked differences in cell concentrations at the body extremities is explained as being due to the presence of gradients of growth factors and the continual movement of cells toward the body extremities.

Free energy of cell differentiation

The rigid polarity of the coelenterate, hydra, has been examined based on the pattern of cell types fround along the body cloumn. The distribution of several cell types was determined by direct counts from histological sections. Treating the individual cell types as separate thermodynamic phases, the distribution of cell types was analyzed using a thermodynamic free energy function. (A steady stete free energy function is derived to deal with cell differentiation.) Based on the analysis of the distribution of cell types with the free energy function, it is concluded that cell differentiation in hydra is accompanied by mass flow of the components, which are responsible for cell differentiation and/or changes in the free energy of the cell as it is transformed from a precursor to a differentiated cell. The presence of marked differences in cell concentrations at the body extremities is explained as being due to the presence of gradients of growth factors and the continual movement of cells toward the body extremities.

EFFECT OF CERTAIN VARIATIONS IN DILUENT AND DILUTION PROCEDURE ON SURVIVAL OF PSEUDOMONAS SPECIES GROWN IN VARIOUS MEDIA

Studies on the effect of various diluents on the level of population of pseudomonads grown in skimmilk, nutrient broth, and on agar slants indicate that with skimmilk cultures usually minor increases or decreases in viable counts occurred. With cultures grown in nutrient broth at 25 C, reductions in numbers after holding in phosphate buffered water or distilled water became more prevalent. The viable population of cell suspensions from agar-slant cultures held in either Standard Methods buffer or distilled water decreased rapidly in most instances. The temperature of the diluent did not have a significant effect on the survival of test cultures in various diluents. Studies on the effect of cell concentration on survival of test cultures in various diluents indicate that in general only minor changes in viable count could be attributed to differences in cell concentration. With respect to pH of the diluting fluid, a diluent at pH 7.0 seemed preferable. Experiments with distilled waters from difference sources indicate that the type of water used as diluent can have a great effect on the viable count.

Micromethod for the methyl red test.

SUMMARY: A micromethod for the methyl red (mr) test was worked out for suspensions of bacterial cells. The most important factors in the test are (1) the density of the cell suspension; (2) the ratio of glucose to buffer concentrations. Adaptation of the cells to glucose is essential for the success of the test, and cells from glucose-free media are not satisfactory. At the optimal glucose: buffer ratio Bacterium coli and Klebsiella aerogenes suspensions have different effects on the final pH value attained in the system. Because the mr microtest is dependent on a fine balance between glucose and buffer concentrations, and is so greatly affected by even twofold differences in cell concentration, it is not really suitable for routine work in the characterization of bacteria.

DETERMINATION OF PLASMA VOLUME

The determination of the total volume of the circulating blood or plasma is assuming increasing importance from both the clinical point of view and that of research (Erlanger,¹Rowntree, Brown and Roth,²Stander³). A study of the variation of the blood volume in pregnancy having been projected in this laboratory, it became necessary to investigate the methods for determination of blood volume. This paper deals with the results of the investigation. The extensive literature on the subject discloses two basically different methods in use: the carbon monoxide method of Haldane and Smith⁴and the dye method of Keith, Rowntree and Geraghty.⁵When using the former method, one must consider myohemoglobin (Whipple⁶), the dependence on environmental temperature (Barcroft et al.⁷), the time lag in saturation of the hemoglobin in the splenic spaces (Barcroft and Barcroft⁸) and the local differences in cell concentration (Lamson and Nagayama⁹). An analysis of the data obtained