New Method For Culture Research Articles

Feasibility of attached cultivation for polysaccharides production by Porphyridium cruentum.

Porphyridium cruentum is one of the most valued microalgae species able to produce both pigments and exopolysaccharides. Conventional liquid suspended cultivation in ponds and photobioreactors show its disadvantages in lower cultivation efficiency and higher stirring power consumption due to the high viscosity of the medium by the accumulation of polysaccharides. In this work, a new method of culture (called attached cultivation) based on the growth of microalgae using a supporting surface was successfully applied to the cultivation of P. cruentum and the effect of the main influential parameters on its growth rate and polysaccharides production has been investigated. Higher values of these factors resulted in a faster growth rate and, in particular, optimum values of 6.98gm-2 for initial biomass density, 100µmolm-2s-1 for light intensity, continuous illumination, 2.0% for CO2 concentration, and 0.1vv-1min-1 for aeration rate produced the best polysaccharide production of 42% dry weight. The nutrition profile of P. cruentum obtained in attached and suspended cultivations was similar. Overall these results demonstrate that the attached cultivation is a promising technique which greatly improves the growth rate of P. cruentum as well as its production of polysaccharides and, therefore, it is worth enhancing to be exploited for commercial application.

Culture and adenoviral infection of sinoatrial node myocytes from adult mice.

Pacemaker myocytes in the sinoatrial node of the heart initiate each heartbeat by firing spontaneous action potentials. However, the molecular processes that underlie pacemaking are incompletely understood, in part because of our limited ability to manipulate protein expression within the native cellular context of sinoatrial node myocytes (SAMs). Here we describe a new method for the culture of fully differentiated SAMs from adult mice, and we demonstrate that robust expression of introduced proteins can be achieved within 24-48 h in vitro via adenoviral gene transfer. Comparison of morphological and electrophysiological characteristics of 48 h-cultured versus acutely isolated SAMs revealed only minor changes in vitro. Specifically, we found that cells tended to flatten in culture but retained an overall normal morphology, with no significant changes in cellular dimensions or membrane capacitance. Cultured cells beat spontaneously and, in patch-clamp recordings, the spontaneous action potential firing rate did not differ between cultured and acutely isolated cells, despite modest changes in a subset of action potential waveform parameters. The biophysical properties of two membrane currents that are critical for pacemaker activity in SAMs, the "funny current" (If) and voltage-gated Ca(2+) currents (ICa), were also indistinguishable between cultured and acutely isolated cells. This new method for culture and adenoviral infection of fully-differentiated SAMs from the adult mouse heart expands the range of experimental techniques that can be applied to study the molecular physiology of cardiac pacemaking because it will enable studies in which protein expression levels can be modified or genetically encoded reporter molecules expressed within SAMs.

Development and characterization of an endometrial tissue culture system

A culture system has been developed for pieces of whole endometrium, comprising many different cell types. The system maintains the integrity of all compartments important for tissue function and allows functional studies of the human endometrium. Endometrial biopsy strips were collected from fertile women. The strips were cut into small pieces and cultured for 8 h on dishes pre-coated with Matrigel. The oxygen concentration was kept low (5%), since it was shown to be beneficial for the culture in preliminary studies. Scanning electron microscopy and immunohistochemical detection of proliferating cell nuclear antigen (PCNA), oestrogen receptor alpha (ERα), progesterone receptor (PR) A + B and B and TUNEL were used to characterize the endometrium. During culture, the luminal and glandular epithelial cells began to cover the surface injured when cutting the sample, suggesting a healing process. The luminal epithelial surface appeared normal after culture. There was an increased sensitivity to culture after the implantation window. This new method for culture of endometrial strips in vitro has proven successful and may be used for functional studies of the human endometrium.

New method for culture of zona-included or zona-free embryos: The Well of the Well (WOW) system

Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 × 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 μl medium, 200 μl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 μl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P < 0.05 for all). The developmental rate was independent of the number of WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production. Mol Reprod Dev 55:256–264, 2000. © 2000 Wiley-Liss, Inc.

New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.

Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 x 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 microl medium, 200 microl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 microl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P < 0.05 for all). The developmental rate was independent of the number of WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production.

SOLID SUBSTRATE FERMENTATION OF CACTUS PULP BY ASPERGILLUS NIGER

A new method of culture is described to study the growth of Aspergillus niger on cactus pulp in the solid-state. After extraction of viscous polyelectrolytes, employed in water treatment, the pulp of Cereus peruvianus was used as a substrate for solid-state fermentation by a wild strain of A. niger . The good conditions for A. niger growth were 60 % moisture, initial pH 3.0, 35 °C, a nitrogen source made of 11 % ammonium and 2 % urea (on a nitrogen basis), 5 % potassium phosphate, and 3.2 g spores/100 g substrate. Related to the dry pulp, maximum fungal growth was observed after 120 hours of fermentation with 14 % of protein production.

Inoculation of Blood Culture Bottles With Ascitic Fluid

The observation that only 42% of the cases of suspected spontaneous bacterial peritonitis at the University of New Mexico, Albuquerque, had positive cultures prompted a change in ascitic fluid culture technique such that a large volume (10 mL in toto) of ascitic fluid was inoculated into blood culture bottles at the bedside. This new method of culture increased the percentage of cases with positive cultures to 91% and decreased the interval between inoculation of the culture and detection of bacterial growth. This is a more sensitive method of culture than the conventional method in detecting spontaneous bacterial peritonitis.

Control of the developmental pathway of tobacco pollen in vitro

We developed a new method for culture of isolated pollen. Using highly homogeneous populations of immature pollen grains of Nicotiana tabacum L. prepared by means of Percoll density gradient centrifugation, we could direct their developmental pathway by regulating certain culture conditions. When the pollen population was cultured in basal medium with glutamine, most pollen grains underwent normal maturation. On the other hand, when first cultured in basal medium without glutamine, most pollen grains did not mature but after transfer to medium with glutamine and sucrose began to divide. This method for inducing pollen cell division was possible only with midbinucleate pollen grains which are characterized by having no central vacuole and no or only a few starch grains. Evidently, some essential changes necessary for the embryogenic response can be induced by glutamine starvation only in pollen grains at a specific stage.

Review of Methods for Studying Maturation of Human Erythroblasts in Vitro: Evaluation of a New Method of Culture of Cell Suspensions in a Clot-Free Medium

Abstract Characteristics of various tissue and cell culture methods for studying maturation of human erythroblasts were reviewed and the basic assumptions required in their use were examined. The relationship of maturation and proliferation of erythroblasts in different types of cultures was considered. Criteria for selection of an in vitro model for studying maturation were established. A new clot-free culture system in roller tubes was found to be superior to those previously described because of convenience, replicability and adaptability to studies of proliferation. The results of challenging the new culture system with various types of cell-plasma relationships indicated that it was effective in distinguishing among erythroblasts and plasmas of iron deficiency anemia, thalassemia, azotemia and erythrocytosis, and that the deductions arrived at were similar to those derived from the use of less convenient in vitro systems in which different parameters of maturation were used. Experience with the new method suggests that it may be useful in the study of humoral factors affecting maturation of human erythroblasts. The chief limitation of the proposed method, as for other in vitro methods, is that only direct effects of humoral agents on erythroblasts can be studied.

A new method of tissue culture for accurate and rapid measurements of the growth

The new method of culture consists in placing the tissue in capillary tubes instead of hanging drop, which was employed heretofore. The glass tube consists of two parts: one part, about 1 mm. in diameter, is used for handling by the operator, the other part, drawn out to about 0.3 mm. in diameter, is filled with the liquid medium, into which is placed the tissue to be cultivated. The thickness of the walls of this second part of the tube is less than 0.1 mm. The length of the whole tube is about 2 inches. 20 such tubes are placed with the thin ends down into a small beaker. A few drops of fluid are poured into the beaker, and this fluid through capillarity fills the tubes. The whole is placed into a test tube and may be sterilized and cooled without handling the individual tubes. A piece of tissue about 0.5 mm. in its widest diameter is placed into the wide part of each tube and then by the aid of a very fine glass rod immersed into the fluid, then with a piece of dry cotton attached to the thin end of the tube a drop of the fluid withdrawn and at the same time the tissue moved deeper in the fluid. The tissue becomes elongated and grows only at both ends and consequently in one direction. It is therefore quite easy to measure the longitudinal increase in size with accuracy of nearly 1/20 of a millimeter. The narrow end of each tube is closed with sealing wax and the thick end by a gas flame. As a result of this manner of sealing the tube the piece of tissue falls out without effort on breaking off of the thin end of the tube.