El objetivo del estudio fue evaluar el efecto de dietas con alta concentración de Saccharomyces cerevisiae sobre la proliferación de hemocitos en camarones Cryphiops caementarius machos. Los camarones (5.3 ± 1.2 cm de longitud total y 7.4 ± 2.9 g de peso

El objetivo del estudio fue evaluar el efecto de dietas con alta concentración de Saccharomyces cerevisiae sobre la proliferación de hemocitos en camarones Cryphiops caementarius machos. Los camarones (5.3 ± 1.2 cm de longitud total y 7.4 ± 2.9 g de peso total) se colectaron del río Pativilca (Perú). Cada camarón se mantuvo en un recipiente instalado dentro del acuario (seis camarones por acuario). Se empleó una dieta control (3% de levadura) y tres dietas experimentales (6, 9 y 12% de levadura) con dos repeticiones por tratamiento durante 28 días de cultivo. El número total de hemocitos fue mayor (p<0.05) con 6% de levadura (134.75 x 105 cél/ml), así mismo el número de granulocitos (31.44 x 105 cél/ml) y semigranulocitos (102.44 x 105 cél/ml). El número de hialinocitos disminuyó en todos los tratamientos y se mantuvo entre 0.31 y 1.56 x 105 cél/ml. El número de hemocitos atípicos se mantuvieron bajos en todos los tratamientos y sin diferencias con el basal (0.31 x 105 cél/ml). La dieta con 6% de levadura incrementó (p<0.05) el número total de hemocitos y los hemocitos granulocitos y semigranulocitos en los camarones machos C. caementarius. En cambio, las dietas con 9 y 12% de levadura afectaron (p<0.05) la proliferación de hemocitos totales y diferenciales.The aim of this study was to evaluate the effect of diets with high concentration of Saccharomyces cerevisiae on the proliferation of hemocytes in male shrimp Cryphiops caementarius. The shrimp (5.3 ± 1.2 cm of total length and 7.4 ± 2.9 g of total weight) were collected from the Pativilca River (Peru). Each shrimp was kept in a container installed inside the aquarium (six shrimp per aquarium). A control diet (3% yeast) and three experimental diets (6, 9 and 12% yeast) were used with two repetitions per treatment during 28 days of culture. The total number of hemocytes was higher (p<0.05) with 6% yeast (134.75 x 105 cells/ml), as well as the number of granulocytes (31.44 x 105 cells/ml) and semigranulocytes (102.44 x 105 cells/ml). The number of hyalinocytes decreased in all treatments and remained between 0.31 and 1.56 x 105 cells/ml. The number of atypical hemocytes remained low in all treatments and without differences with the baseline (0.31 x 105 cells/ml). The diet with 6% yeast increased (p<0.05) the total number of hemocytes and the granulocyte and semigranulocyte hemocytes in C. caementarius male shrimps. In contrast, diets with 9 and 12% of yeast affected (p<0.05) the proliferation of total and differential hemocytes

Variations in virulence of three (3) Escherichia coli serotypes confirmed in experimental mammary gland infections

An experiment was conducted to confirm the pathogenicity of three (3) serotypes of Escherichia coli (E. coli 037, 02a and 109) in mammary glands of experimental cows (cow 105, 107 and 102 respectively). Pathogenicity ofthe E. coli which is a measure of virulence was observed to vary in the cows. Following inoculation bacterial number peak at 160,000 CFU/ml, 7,6000,000 CFU/ml and 3,600 CFU/ml respectively. Also milk somatic cell count (SCC) were observed to peak at 15,000 x 103 cell/ml, 58,700 x 103 cell/ml and 360 x 103 cells/ml respectively. The time taken for maximum bacterial number and somatic cell count to reach varied. There was leukemia with relative neutropenia in all cases. Typical responses included fever, painful inflammation of glands and gradual weakness of the experimental cows. Time to peak rectal temperature, also varies. The control quarter of teat of each cow infused with 1.0ml of saline showed little or no response. Milk SCC never exceeded 100,000 cells/ml in the control quarter. Systemic effects were little and cows appeared normal externally. E. Coli serotypes varied in virulence with the degree of variation highlydetermined by the organism tried

Quantitative Study of Growth of Some Dairy Psychrophiles

Summary and Conclusions: Samples of pasteurized skim milk were steamed for 25 minutes at approximately 100° C. and inoculated with pure cultures of Pseudomonas fluorescens, A and B, at different levels, Pseudomonas fragi, Brevibacterium lipolyticum. The fifth culture was the normal flora of commercially pasteurized skim milk treated as a pure culture. All samples were stored at 4° C. Bacterial growth was determined using agar plates incubated at 25° C. for 3 days. The generation time was calculated for all cultures which ranged from 5.55 hours for Pseudomonas fragi to 11.24 hours for Brevibacterium lipolyticum. The generation time was found the same (7.22 hours) for Pseudomonas fluorescens cultures A and B regardless of differences in inoculum size for the two cultures. In the pure cultures, bacterial count after 7 days of storage was the highest for Pseudomonas fragi, 3.0 x 107, and the lowest for Brevibacterium lipolyticum, 1.9 x 105 cells per ml. However, the mixed flora culture obtained a slightly lower count, 1.7 x 105 cells per ml. The lag phases for the pure cultures studied ranged from 1 day for Pseudomonas fragi to 3 days for both cultures of Pseudomonas fiuorescens. The mixed flora culture showed still a longer lag phase of 4.2 days. Of all cultures studied, Pseudomonas fragi was found to be the most actively growing culture. It had the shortest generation time, the highest counts after 7 days of storage, and the shortest lag phase

Seasonal and spatial variation of bacterial production and abundance in the northern Levantine Sea

Spatial and temporal heterogeneity in bacterial production and abundance in relation to ambient bio-physicochemical parameters has been investigated in the Levantine Sea. Five stations with different trophic states in an area extending from highly eutrophic Mersin bay to the mesotrophic Rhodes gyre area including the oligotrophic offshore waters were sampled four times. Integrated bacterial production varied between 6.1 and 90.3 µg C m-2 d-1 with higher rates occurring during September 2012 in offshore waters. Bacterial abundance ranged between 0.18 and 7.3 x 105 cells ml-1 within the euphotic zone and was generally higher up to 100 meters throughout the study period. In offshore waters, bacterial production (0.401 to 0.050 µg C m-3 d-1), abundance (4.5 to 1.6 x 105 cells ml-1) and depth of the productive layer decreased from 150 to 75 meters westward along the transect. Although the highest abundance was observed in July 2012 in offshore waters, the highest activity was measured in September 2012. These results indicated that the temperature played a key role in regulating bacterial abundance and production in the area. High chlorophyll concentrations in March did not correspond to high bacterial abundance and production at the same time. Increase in dissolved organic carbon content following spring phytoplankton bloom and the increase in temperature in the mean time might have enhanced the bacterial activity towards summer

Citotoxicity evaluation of three dental adhesives on vero cells in vitro

To evaluate, in vitro, the potential cytotoxicity of three different dental adhesives systems (Adper Single Bond 2 -SB, Silorane System Adhesive Bond -SSAB and Single Bond Universal -SBU) on cultivated Vero cells after different contact times. The cells were cultured in a concentration of 2 x 105 cells/mL for 24h and grown to sub-confluent monolayers. VERO cells were exposed to 25µl of conditioned extracts obtained from 24h, 48h and 72h immersion of adhesive samples in culture medium (DMEM), immediately after polymerization. Fresh DMEM was used as negative control. Cell metabolism was evaluated by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyl-tetrazolium bromide). The data were analyzed statistically by ANOVA, considering a significance of 5%. The values of cell viability ranged from 94.2% at 72h (SBU) to 109.6% at 48h (SB). The mean percentage of viability after exposure to the extracts of SB, SSAB and SBU were 103.2%, 100.63% and 97.43%, respectively. There was no statistically significant difference (p= 0.342) between the experimental and negative control groups. At all exposure times, all adhesives tested in this study presented no cytotoxicity to Vero cells in vitro

Expression of Tumor Assosiated and Epithelial-mesenchymal Transition Markers in 2d and 3d Cell Cultures of Mcf-7

The target effects on the expression of epithelial-mesenchymal transition regulation molecules are promising for cancer therapy, including breast cancer. 3D cell culture is a model for studying epithelial-mesenchymal transition in vitro and may become a test system for anticancer therapy.Aim of research. The aim of this research was to evaluate and compare the expression of tumor associated and epithelial-mesenchymal transition markers in tumor cells of breast adenocarcinoma (MCF-7 cell line) in 2D and 3D cell culture.Methods. For realization of the aim MCF-7 cell line (breast adenocarcinoma) was chosen as an experimental model in vitro. The monolayer cell culture was cultured in standard conditions (37 0C, 5 % CO2, humidity 95 %). The initial density of inoculated cells was 2 x 104 cells/cm2. The cells were incubated for two days before their use in the experiment. For the initial generation of spheroids the monolayer cell culture was removed off the substrate after the four days of incubation, using 0,25 % Trypsin-EDTA, and placed in nutrient medium with 5 % carboxymethyl cellulose (Bio-Rad, USA) at concentration of 5 x 105 cells/ml. Then the plates were incubated on an orbital shaker (Orbital shaker, PSU-10i, Biosan, Latvia) at 50 rpm for 3–5 hours. Half of culture medium was replenished every 3 days. A spheroid culture was maintained for 14 days. Detection of markers (ER, p53, EpCAM, vim, AE1/AE3, panCK, EGFR) in 2D and 3D cell culture was performed using immunohistochemistry method with primary monoclonal antibodies. Histological samples of cells were photographed to compare the morphological characteristics and the expression of proteins in monolayer and spheroid cultureResults. The results demonstrated that the percentage of tumor marker positive cells (ER+, EGFR+, EpCAM+, panCK+, AE1/AE3+) in monolayer culture is 1.25–2 times than more in spheroid culture. In contrast, tumor spheroids consist of fewer cells with the expression of epithelial markers such as EpCAM and AE1/AE3, but they contain a large number of cells that expressed mesenchymal marker vimentin by 5 % and p53 by 10 %. This may indicate that the cells acquire a mesenchymal phenotype. However, tumor cells of monolayer cell culture were not expressed vimentin.Conclusions. Our results demonstrated the differences of expression of tumor associated and epithelial-mesenchymal transition markers in 2D and 3D breast cancer cell cultures. Thus, the percentage of epithelial markers (Cytokeratines and epithelial cell adhesion molecule) in tumor spheroids is less than in cells of monolayer however spheroids cells begin expressing a mesenchymal marker – vimentin. In 3D cell culture only the outer cell layers expressed tumor associated proteins unlike 2D cell culture in which all of cells showed equally expression. Reduced of manifestation of tumor associated markers in 3D cell culture may indicate an increase of stem properties. These data showed that 3D cell culture more than 2D cell culture characterized processes of epithelial-mesenchymal transition

Effect of glycosaminoglycans on growth factor-stimulated trophoblast invasion

Objectives: To determine the effect of glycosaminoglycans and a series of growth factors on the viability and invasion of the extravillous trophoblast cell line SGHPL4. Methods: Cells were cultured in Hams F10 media supplemented with fetal bovine serum and L-glutamine. For viability studies cells were seeded into 96-well culture plates (104 cells/well), maintained in serum free medium for 24h and then incubated with glycosaminoglycans (heparin, heparin sulphate and hyaluronic acid; each 100ng/ml) ± growth factors (VEGF, FGFand HB-EGF). Cell viability was measured in cells using the MTS assay. Cellular invasion was assessed using the FluoroBlok invasion assay. Cells were serum-starved for 24 h, incubated with the fluorescent dye DiIC12(3) (10mg/ml) for 1 hour prior to seeding onto an artificial extracellular matrix coated 8 mm FluoroBlok porous membrane inserts (2.5 x 105 cells per insert). Growth factors ± GAGs were added to the cell suspension and the inserts were lowered into a 96-well plate containing 10% fetal calf serum. Plates were incubated at 37°C for 24h. Invasion was determined by measurement of fluorescence of invaded cells using a fluorescent plate reader (Ex549/Em565 nm). Results: Cell numbers were significantly increased following incubation with VEGF, FGF and HB-EGF. Cell number was also increased after incubation with each of the glycosaminoglycans tested. The largest increase was observed following incubation with heparin sulphate. Cell numbers were further increased when the GFs were incubated with HS and heparin, but not with hyaluronic acid. Invasion was increased following incubation with VEGF, HBEGF and HGF. Heparan sulphate and heparin increased invasiveness in a dose-dependent manner. In contrast, hyaluronic acid had no significant effect. Conclusion: This study demonstrates a role for glycosaminoglycans in key features of trophoblast function

Media Alternatif untuk Pertumbuhan Sel Midgut Spodoptera litura

Cell culture is process when viable cell propagated in vitro in the medium. Midgut cell culture of S. litura is very useful as hosts for SpltMNPV virus that can control S. litura. This study aimed to determine the increase in the number of S. litura midgut cells and long incubation time of S. litura midgut cells in RPMI 1640, DMEM, and mixture of RPMI 1640 medium and DMEM as alternative medium with initial inoculum 4.0 x 105 cell/ml. This research used Completely Randomized Design (CRD) with 4 treatments and 3 repetitions so that there are 12 treatment unit. Data was analyzed using ANOVA (Analysis of Variance). Further tests are LSD (Least Significant Difference). The results showed that the highest increase of the number of S. litura midgut cells was obtained in Grace's medium (control) with 23125 times inoculum, the mixture of RPMI 1640 and DMEM medium was  21950 times inoculum, RPMI 1640 medium was  11725 times, DMEM medium was  10400 times inoculum. Spodoptera litura midgut cells form a monolayer in a 24-hour incubation in Grace's medium, the mixture of RPMI 1640 and DMEM medium, and DMEM, whereas the monolayer formation in RPMI 1640 medium is in a 48-hour incubation. The result showed that the mixture of RPMI 1640 and DMEM medium is an .optimum alternative medium that support the growth of Spodoptera litura midgut cell

Characterization of a unique technique for culturing primary adult human epithelial progenitor/“stem cells”

Abstract Background Primary keratinocytes derived from epidermis, oral mucosa, and urothelium are used in construction of cell based wound healing devices and in regenerative medicine. This study presents in vitro technology that rapidly expands keratinocytes in culture by growing monolayers under large volumes of serum-free, essential fatty acid free, low calcium medium that is replaced every 24 hrs. Methods Primary cell cultures were produced from epidermal skin, oral mucosa and ureter by trypsinization of tissue. Cells were grown using Epilife medium with growth factors under high medium volumes. Once densely confluent, the keratinocyte monolayer produced cells in suspension in the overlying medium that can be harvested every 24 hrs. over a 7–10 day period. The cell suspension (approximately 8 X 105 cells/ml) is poured into a new flask to form another confluent monolayer over 2–4 days. This new culture, in turn produced additional cell suspensions that when serially passed expand the cell strain over 2–3 months, without the use of enzymes to split the cultures. The cell suspension, called epithelial Pop Up Keratinocytes (ePUKs) were analyzed for culture expansion, cell size and glucose utilization, attachment to carrier beads, micro-spheroid formation, induction of keratinocyte differentiation, and characterized by immunohistochemistry. Results The ePUKs expanded greatly in culture, attached to carrier beads, did not form micro-spheroids, used approximately 50% of medium glucose over 24 hrs., contained a greater portion of smaller diameter cells (8–10 microns), reverted to classical appearing cultures when returned to routine feeding schedules (48 hrs. and 15 ml/T-75 flask) and can be differentiated by either adding 1.2 mM medium calcium, or essential fatty acids. The ePUK cells are identified as cycling (Ki67 expressing) basal cells (p63, K14 expressing). Conclusions Using this primary culture technique, large quantities of epithelial cells can be generated without the use of the enzyme trypsin to split the cultures. The cells are small in diameter and have basal cell progenitor/”stem” (P/SC) cell characteristics induced by daily feeding with larger than normal medium volumes. The ePUK epithelial cells have the potential to be used in regenerative medicine and for basic studies of epithelia P/SC phenotype.http://deepblue.lib.umich.edu/bitstream/2027.42/112344/1/12895_2011_Article_127.pd