Short-term culturing of teleost crystalline lenses combined with high-resolution optical measurements

Culturing whole lenses is a frequently used method for studying regulatory events on the lens in controlled environments. The evaluation methods used often fall under two categories, molecular or optical. The main benefit from optical measurements is that they directly detect changes in the lens’ main function, i.e. refracting light. However, these measurements often have rather low resolution or yield results open for subjective interpretation. Here we present a short-term crystalline lens culturing technique combined with a high-resolution optical measuring method. There are two main advantages of using teleost lenses compared to mammalian lenses. Teleost tissue generally has a higher tolerance than mammalian tissue with regard to temperature and nutrient fluctuations. Teleost lenses are structurally more robust and can be excised from the eye without disturbing form or function. The technique is developed for short-term culturing (3 h), however, the lenses appear viable for at least 24 h and longer culturing may be possible. The technique is resistant to small variations in osmolarity and yields quantitative datasets for further analyses and statistical treatment.     

A Thick-Walled Organism Isolated from the Cockroach Gut by Using a Spent Medium Technique

By using a conditioning technique whereby complex media are inoculated several times with bacteria from the hindgut of the cockroach Eublaberus posticus, a succession of bacterial types occurred. An obligately anaerobic, pleomorphic, thick-walled, gram-positive organism is described which was isolated by this culturing technique.     

未培养微生物的研究与微生物分子生态学的发展*

近年来现代分子技术和基因组学逐渐渗透到有关生命科学的整个领域,也为微生物生态学提供了新的研究方法和机遇。16S rRNA基因序列分析、DNA-DNA杂交、核酸指纹图谱以及宏基因组学等分子技术检查自然环境中的微生物,可以克服传统纯培养技术的不足,是一条探知未培养微生物、寻找新基因及其产物的新途径,开启了我们认识微生物多样性和获得新资源的大门。     

Chromosome preparations of human blood lymphocytes—Evaluation of techniques

Several parameters of human lymphocyte culturing techniques and metaphase chromosome preparation procedures were studied and quantitatively evaluated in regard to their influence on the results of Q and G banding procedures. The culturing conditions were studied using³H thymidine incorporation as a parameter. A whole blood culturing technique using Ham's F12 medium was found to give optimal and consistent results. Colcemid concentration proved to be of no influence on chromosome contraction or on the number of metaphases obtained over the concentration range investigated. Prolonged exposure to colcemid was found to cause a decrease in the mean chromosome length but the absolute number of metaphases with a low degree of chromosomal contraction hardly decreased. Different spreading techniques were quantitatively analysed and factors important for the spreading of chromosomes were evaluated. Based on the results obtained, an optimal procedure is described which over a period of one year has given consistent results.     

微生物生态学研究方法进展

微生物培养及显微技术作为鉴定微生物种群的手段有很大的局限性,因为环境中大多数微生物处于“存活但不能培养”的状态。因此．不依赖于微生物培养的生物化学以及分子生物学方法正被广泛地用于微生物生态学研究。主要介绍了荧光技术。基于PCR的分析技术和PLFA等技术在表征微生物多样性研究中的某些进展。     

Comparison of two methods for callus culture and plant regeneration in wheat (Triticum aestivum)

Callus growth and development involve a complex relationship between the explants used to initiate callus, the constituents of the medium and the environmental conditions during culturing. Use of high molecular weight osmotica such as polyethylene glycol (PEG-4000) results in non-solidification of agar medium used for culturing and selection. Thus, a new filter paper bridge technique was compared with the existing agar medium for callus initiation, multiplication, and plant regeneration of wheat. The yield of both total and embryogenic callus was doubled and significantly higher number of regenerants was obtained on filter paper bridges compared to agar medium.     

Surface immobilization of plant cells

A novel technique has been developed to immobilize plant cells. The cells are deposited on a surface of manmade fibrous material that provides for strong binding of the plant tissue biomass growing in the submerged culture. The immobilized plant cells remain fully viable. Relatively uniform biomass loadings of up to 20 mg d.w. plant cells/cm(2) support material have been attained. All plant cells from the inoculum suspension became attached within the first 24-48 h depending on the support matrix configuration and hydraulic culture conditions. The advantages and scale-up potential of this technique are discussed and compared to other culturing modes.     

Isolation of L-Forms in a Clinical Microbiology Laboratory

Previous studies have demonstrated that L-forms of bacteria may play a role in persistent, chronic, or recurrent urinary-tract infections. A 2-year program was initiated to determine the feasibility of culturing for L-forms on a routine basis, and to determine the effectiveness of such a program. In relation to the total number of specimens, few L-forms were actually isolated. In comparison with the amount of equipment and technician time required, the return was negligible; only 0.5% of all urine specimens were positive for L-forms. An increase to only 1.2% was noted when culturing for L-forms was limited to patients with a diagnosis of bacteriuria or pyelonephritis. It is recommended that this technique be reserved for those patients with a long history of recurrent urinary-tract infections, after other attempts to cure the patient have met with failure.     

A fast cell loading and high-throughput microfluidic system for long-term cell culture in zero-flow environments

We present a simple technique for cell loading, culturing, and phenotypic study in a multi-chamber microfluidic device made of polydimethylsiloxane (PDMS). This technique is based on the use of degassing induced aspiration of PDMS which allows loading cells into micro-cavities within 1 min. A large number of triangle cavities are patterned aside main flow channels with narrow connections so that cells can be loaded by aspirating into each cavity. In our device, high throughput and long-term monitoring can be done with minimum shear force of the flow. As a demonstration, we show a controlled loading at single cell level and the phenotypic variation of gene expression of the yeast strain w303 as a function of copper ion concentration of the medium.     

A method of processing first-trimester chorionic villous biopsies for cytogenetic analysis

Chorionic villous biopsy is emerging as a technique for obtaining fetal cells for prenatal diagnosis in the first trimester of pregnancy. Chromosome analysis has been performed on small villous biopsies using either direct harvests of uncultured cells or after culturing villous tissue. Here, we describe a method where both techniques can be used simultaneously; from a single villous biopsy, GTG-banded chromosomes of improved morphology are obtained from direct preparations and from cultured villous cells.     

Culturing chick embryos—a simplification of New's method

A simplified version of New's method for culturing early chick embryos is described. The technique allows continuous observation of the critical first three days of development. The conditions for setting up successful cultures are not at all stringent and the method could become a very useful teaching aid.     

Primary Culture of Dissociated Cells of the Rat Retina under Conditions of Long-Lasting Culturing: Properties of Ganglion Cells

We developed a technique for long-lasting culturing of dissociated cells of the rat retina. Electrophysiological characteristics of cultured retinal ganglion cells were examined using a patch-clamp technique in the whole-cell configuration (current-clamp mode). Morphological and electrophysiological characteristics of cultured retinal ganglion cells corresponded to those obtained under conditions of an acute experiment on the cells of similar-age animals; this is indicative of the possibility of using cultured retinal cells in electrophysiological and pharmacological studies.     

A novel method of encapsulating and cultivating adherent mammalian cells within collagen microcarriers

A novel method of preparing collagen microcarriers was developed and used to entrap adherent cells for cell culturing. This new technique involved seeding of cells in micro gel beads comprised of collagen fibrils dispersed in alginate. The gel beads were washed with phosphate buffered saline (PBS) to remove alginate and the resulting microspheres, about 300-500 microm in diameter, contained evenly distributed collagen fibrils which provided a 3D biomimetic environment for cell growth. The applicability of this microencapsulating system was demonstrated by its ability to support the growth of C2C12 myoblast cells. When seeded and cultured within the 3D collagen microcarriers, the population of C2C12 cells entrapped within the microcarriers increased by 1.5 folds in 7 days after inoculation. This encapsulation technique is potentially useful for culturing cells and especially useful for adherent cells that require a 3D fibrillar collagen environment.     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35?°C for a few days and by then incubating them in the dark at 25?°C. Pre-culturing anthers at 35?°C for 4?days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15?°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.     

Axenizing and Culturing Endomigratory Plant-Parasitic Nematodes using Pluronic F127, Including its Effects,on Population Dynamics of Pratylenchus penetrans

A non-chemical technique for surface sterilizing plant-parasitic nematodes for aseptic cultures is described. The method is most applicable to nematodes with active migratory infective stages and requires only a few starting specimens. Rate of achieving a primary aseptic culture with the technique ranged from 60%-100% depending on the conditions of the specimens collected for culturing. Aseptic cultures of species of Meloidogyne, Rotylenchuluz, Pratylenchus, and Radopholus initiated with the method remained contamination-free after 12 months of maintenance in tomato root explant or alfalfa callus cultures. Further studies of Pluronic F127, a polyol gel medium employed in the technique to confine the spread of contaminating bacteria or fungi associated with the nematodes, showed that the polyol gel was a suitable support medium for culturing corn root explant, alfalfa callus tissues, and consequently Pratylenchus species including P. agilis, P. brachyurus, P. scribneri, and P. penetrans. During the course of 10 months, P. penetrans reared in polyol-base medium followed a standard biological growth curve, multiplied to a higher population density, maintained a similar female-to-male ratio, and possessed a similar tendency to reside inside or outside host tissues as did P. penetrans reared in agar-base medium. The percentages of P. penetrans juveniles in the sub-populations residing outside or inside the host tissues reared in polyol-base medium also were similar to and fluctuated temporally in like manner as those reared in agar-base medium. Members of these sub-populations from the polyol- or agar-base were equally infective and reproductive after 9 months of culturing.     

A simple in vitro culture system for tracheal cartilage development

Semi-circular tracheal cartilage is a critical determinant of maintaining architectural integrity of the respiratory airway. The current effort to understand the morphogenesis of tracheal cartilage is challenged by the lack of appropriate model systems. Here we report an in vitro tracheal cartilage system using embryonic tracheal–lung explants to recapitulate in vivo tracheal cartilage developmental processes. With modifications of a current lung culture protocol, we report a consistent in vitro technique of culturing tracheal cartilage from primitive mouse embryonic foregut for the first time. This tracheal culture system not only induces the formation of tracheal cartilage from the mouse embryonic foregut but also allows for the proper patterning of the developed tracheal cartilage. Furthermore, we show that this culture technique can be applied to culturing other types of cartilage in vertebrae, limbs, and ribs. We believe that this novel application of our in vitro culture system will facilitate the manipulation of cartilage development under various conditions and thus enabling us to advance our current limited knowledge on cartilage biology and development.     

Synaptogenesis in Co-Culture of Neurons of Dorsal Root Ganglia and Spinal Cord

In co-culture of spinal cord and dorsal root ganglion (DRG) neurons, we studied at different terms of culturing postsynaptic currents in DRG neurons evoked by direct electrical stimulation of single spinal neurons using a voltage-clamp technique in the whole-cell configuration. According to the reversal potential and sensitivity to bicuculline, these currents were classified as inhibitory postsynaptic currents (IPSC) carried by Cl^- ions through GABA_A receptors. During neuronal development in dissociated co-culture, the amplitude of evoked IPSC and their time to peak significantly increased. The time to peak of spontaneous IPSC (sIPSC) in DRG neurons remained unchanged, while the frequency of these currents increased with increasing culturing time. It is concluded that under culturing conditions spinal neurons establish inhibitory synaptic contacts with the somata of DRG neurons, and the number of such functional contacts increases in the course of culturing. Our findings show that in dissociated co-culture the process of formation of inhibitory synapses on the axon terminals of primary afferent neurons is akin to that realized in vivo, but with dissimilar topography of distribution of such synapses.     

Evoked inhibitory postsynaptic currents in the dynamics of development of cultured hippocampal neurons of rats

On a low-density culture of the hippocampal neurons of rats, we studied inhibitory transmission through the synaptic connection of a cell pair; a patch-clamp technique in the whole-cell configuration and direct extracellular stimulation of the neurites were used. We found that the mean amplitude of evoked inhibitory postsynaptic currents (eIPSC) and variability of their amplitudes significantly increased within the culturing period. The duration of the current rising phase also increased concurrently with the growth and differentiation of the neurons, but this change was non-monotonic. The coefficient of variation of the current amplitudes, as well as the time constant of current decay (the latter reflects the properties of a postsynaptic unit), showed no clear changes in the course of culturing of the neurons. Our data show that in the course of synaptogenesis the number of unitary inhibitory synaptic contacts between cultured hippocampal neurons increases, while modifications of the transmission mechanism at the level of unitary synaptic contact are less significant.     

大鼠输精管平滑肌细胞的体外培养及形态学特征(英文）

建立大鼠输精管平滑肌细胞的培养方法。取大鼠输精管,剥离外膜和内膜,用组织块法进行体外培养。用抗α-SMA(anti α-smooth muscle actin)免疫组化染色的方法鉴定培养的细胞。结果显示,在倒置显微镜下观察细胞形态多样,表现为长梭形或星形,细胞伸出突起互相接触,彼此融合,部分区域细胞多层重叠,部分区域细胞单层高低起伏,呈"峰-谷"状生长。免疫组化染色鉴定呈阳性反应,用该方法所分离、培养的输精管平滑肌细胞纯度达99%以上。应用组织块法培养大鼠输精管平滑肌细胞,操作简单,结果稳定。     

Mass Production of the Beneficial Nematode Heterorhabditis bacteriophora and Its Bacterial Symbiont Photorhabdus luminescens

Entomoparasitic nematodes (EPNs) are being commercialized as a biocontrol measure for crop insect pests, as they provide advantages over common chemical insecticides. Mass production of these nematodes in liquid media has become a major challenge for commercialization. Producers are not willing to share the trade secrets of mass production and by doing so, have made culturing EPNs extremely difficult to advance existing technologies. Theoretically, mass production in liquid media is an ideal culturing method as it increases cost efficiency and nematode quantity. This paper will review current culturing methodologies and suggest basic culturing parameters for mass production. This review is focused on Heterorhabditis bacteriophora; however, this information can be useful for other nematode species.