Fluorescence microscopy for studying the viability of micro-organisms in natural whey starters

AIMS: The aim of this work was to study the viability and cultivability of microbial populations of different natural whey starters and to evaluate their resistance to thermal treatments (such as exposure to high or low temperatures). METHODS AND RESULTS: Twenty-three natural whey starters for Grana Padano cheese were investigated and subsequently pH measurement, plate count agar using Man-Rogasa-Sharpe (MRS) pH 5.4 agar and whey agar medium (WAM) were performed using these samples. LIVE/DEAD BacLight bacterial viability kit was used. Total count and viability of all the 23 samples were high and similar to each other (CV 20%). However, the cultivable population was lower in terms of cfu ml(-1) and number of cells per millilitre than the viable fraction and highly variable, although its count value was higher in WAM than in MRS pH 5.4. The heating (60 degrees C for 5 min and 54 degrees C for 1 h) and freezing (-20 and -80 degrees C) treatments affected the cultivability and viability of the microbial population. CONCLUSIONS: This study demonstrated the effectiveness of LIVE/DEAD BacLight bacterial viability kit, which has already been used to evaluate bacterial populations, in investigating microbial viability in a complex ecosystem such as a natural whey starter. Significance and Impact of the Study: The aim of this study was to quantify the presence of damaged nonviable bacterial cells in natural whey starters. The Thoma Glass is a useful method to obtain fluorescence microscopy counts to evaluate the technological performance of natural whey starters.     

Viable Cell Detection by the Combined Use of Fluorescent Glucose and Fluorescent Glycine

The combined use of a fluorescent glucose (2NBDG) and a fluorescent glycine (NBD-Gly) was tried for the detection of viable cells of significant foodborne pathogenic strains in addition to several Escherichia coli strains and coliforms. Thirty-five out of 41 strains showed marked uptake of 2NBDG but 6 strains were not able to take in 2NBDG. Five out of these 6 strains showed NBD-Gly uptake.     

Microtubules and morphogenesis in ordinary epidermal cells ofVigna sinensis leaves

Summary Undifferentiated ordinary epidermal cells (ECs) ofVigna sinensis leaves possess straight anticlinal walls and cortical microtubules (Mts) scattered along them. At an early stage of EC differentiation cortical Mts adjacent to the above walls form bundles normal to the leaf plane, loosely interconnected through the cortical cytoplasm of the internal periclinal wall. At the upper ends of the Mt bundles, Mts fan out towards the external periclinal wall and form radial arrays. Mt bundles and radial arrays exhibit strict alternate disposition between neighbouring ECs. An identical reticulum of cellulose microfibril (CM) bundles is deposited outside the Mt bundles. Local wall pads rise at the junctions of anticlinal walls with the external periclinal one, where the CM bundles terminate. They display radial CMs fanning towards the external periclinal wall. The CM bundles and radial CM systems prevent local cell bulging, but allow it in the intervening wall areas. In particular, the radial CM systems dictate the pattern of EC waviness by favouring local tangential expansion of external periclinal wall. As a result, ECs obtain an undulate appearance. Constrictions in one EC correspond with protrusions of adjacent ECs. ECs affected by colchicine entirely lose their Mts and do not develop wavy walls, an observation substantiating the role of cortical Mts in EC morphogenesis.Abbreviations CM cellulose microfibril - DTT dithiothreitol - EC epidermal cell - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride     

The determination of cellular viability of hybridoma cells in microtitre plates: A colorimetric assay based on neutral red

A colorimetric assay utilising Neutral Red (C.I. 50040), a nuclear stain, was developed to determine the cellular viability of hybridoma cells in microtitre plates. A linear correlation (r=0.99) was found to exist between the uptake of Neutral Red by viable cells and the viable cell count determined by Trypan blue exclusion test. The linearity stretched over the range of cell concentrations normal in batch cultures (2–30×10⁴/0.2 ml) with as little as ±6% intra-plate well-to-well variation and ±10.2% inter-assay variation.Microscopical examinations of viable hybridoma cells stained with Neutral Red showed that it was located in the nucleus. The possble bifunctional activity of the Neutral Red assay as a test for cellular viability and estimating the DNA content of hybridoma cells is discussed along with its application in a drug screening programme.     

Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate

Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.     

Models for the morphogenesis of the molluscan shell

We give a review of current theories of morphogenesis of both the general coiling and the ornamentation of molluscan shells. These two aspects of shell growth are closely connected, as ornamentation is primarily due to local perturbations of the general apertural growth field controlling coiling. Also, a new, generalized, free-form apertural growth map model is presented in this paper, illustrating some aspects of the regulation of logarithmic spiral growth. This model is used to simulate the formation of megastriae in ammonoids. We emphasize the importance of damaged specimens and how they regenerated, as illustrated with examples from ammonoids. The phenomenon of ornamental compensation can be explained by a mechanism involving a pre-pattern in the mantle. However, simple reaction-diffusion models for ornamental pattern formation should be regarded only as useful abstractions.     

卡泊芬净、米卡芬净对8种皮肤癣菌体外抑菌活性的研究

目的评价棘白菌素类抗真菌药物卡泊芬净（caspofungin）、米卡芬净（micafungin）针对常见致病性皮肤癣菌的体外抗菌活性。方法参考CLSI制定的M38-A2方案。测定82株常见皮肤癣菌的最低有效浓度（minimal effective concentrations,MECs）。结果按照MEC90浓度从高到低,米卡芬净对紫色毛癣菌和断发毛癣菌的MEC90是0.25μg/mL;对犬小孢子菌、疣状毛癣菌的MEC90为0.06μg/mL;对红色毛癣菌、须癣毛癣菌、石膏小孢子菌、絮状表皮癣菌的MEC90均在0.03μg/mL。②卡泊芬净对红色毛癣菌、紫色毛癣菌和断发毛癣菌的MEC90为1μg/mL;对须癣毛癣菌、犬小孢子菌、石膏小孢子菌、絮状表皮癣菌和疣状毛癣菌的MEC90为0.5μg/mL。③根据中位数检验,米卡芬净对几种皮肤癣菌的MEC值均低于卡泊芬净的MEC值,统计学比较有显著性差异（P〈0.05）。结论米卡芬净和卡泊芬净对皮肤癣菌有较强的抑菌作用,米卡芬净的MEC值低于卡泊芬净。     

Organization and morphogenesis of the human seminiferous epithelium

Summary The various types of human primary spermatocytes were classified by means of morphological and morphometrical studies. Based on this classification, the topographic arrangement of the spermatocyte populations in the longitudinal course of seminiferous tubules was determined. This analysis revealed human spermatogenesis be to subjected to a complex local plan of organization, which is based upon the geometry of spirals.The centers of gravity of spermatocyte populations of subsequent degrees of differentiation are arranged on he lices that are contracted conically to the lumen of the seminiferous tubule. On these helices the centers of gravity of the populations diverge continuously 173.8°+/-32.4°. Populations of the same degrees of development are arranged on helices with constant diameters. On these helices the centers of gravity of the populations diverge continuously 142.6°+/-14.2°.The present results lead to new aspects of the kinetics and morphogenesis of the seminiferous epithelium, which can be integrated into a comprehensive biological concept.     

Cell traction models for generating pattern and form in morphogenesis

During early development migratory mesenchymal cells navigate to distant sites where they aggregate to form a variety of embryonic organ rudiments. We present here a new model for mesenchymal cell morphogenesis based on the mechanical interaction between motile cells and their extracellular environment. The model is based on two properties of motile cells: (a) they are capable of generating large traction forces which can deform the extracellular matrix through which they move, and (b) the deformations they produce in their environment affect the direction of their movements. We derive field equations which describe the motion of cells in an elastic extracellular matrix and show that these equations can generate a variety of spatial patterns, such as the formations of skin organ primordia, especially feather germs, cartilage condensation patterns which presage bone formation in limb development, and melanocyte density patterns which form animal coat patterns.Support for this work was provided by NSF Grant # MCS-8110557 [GFO]     

A molecular marker for epithelial morphogenesis in the cockroach

Summary A molecular marker has been identified in embryos of the cockroach, Periplaneta americana, that is localized among epithelial cells to those directly involved in morphogenesis. A monoclonal antibody has been developed that selectively binds to epithelial cells undergoing any of three very different morphogenetic movements-invagination, evagination or epiboly. Neighboring cells not involved in these developmental processes are not labeled by the antibody. The antigen is transiently present on the cells for a period just prior to and during the morphogenetic activity. It is localized on the apical surface of the cells. The spatial, temporal and subcellular distributions of antibody binding during development indicate a role for the antigen in epithelial morphogenesis different from that of any previously described molecule.     

Analytical treatment of pattern formation in the Gierer-Meinhardt model of morphogenesis

Summary We first perform a linear stability analysis of the Gierer-Meinhardt model to determine the critical parameters where the homogeneous distribution of activator and inhibitor concentrations becomes unstable. There are two kinds of instabilities, namely, one leading to spatial patterns and another one leading to temporal oscillations. Focussing our attention on spatial pattern formation we solve the corresponding nonlinear equations by means of our previously introduced method of generalized Ginzburg-Landau equations. We explicitly consider the two-dimensional case and find both rolls and hexagon-like structures. The impact of different boundary conditions on the resulting patterns is also discussed. The occurrence of the new patterns has all the features of nonequilibrium phase transitions.     

Requirement for tumor suppressor Apc in the morphogenesis of anterior and ventral mouse embryo

Tumor suppressor Apc (adenomatous polyposis coli) is implicated in the Wnt signaling pathway that is involved in the early embryonic development and tumorigenesis in vertebrates. While the heterozygous null mutant mice develop intestinal polyps, the homozygous embryos die before gastrulation. To investigate the role of Apc in later embryonic development, we constructed a novel hypomorphic Apc allele whose expression was attenuated by approximately 80%. In the hypomorphic Apc homozygous ES cells, reduction in Apc expression caused beta-catenin accumulation and Wnt signaling activation. The homozygous mutant mouse embryos survived 3 days longer than the null mutant embryos. Interestingly, they showed anterior truncation, partial axis duplication, and defective ventral morphogenesis. To determine the tissues where Apc functions for anterior and ventral morphogenesis, we constructed chimeric embryos whose epiblast was derived predominantly from the Apc hypomorphic homozygous cells but the visceral endoderm was from the wild type. Although these chimeric embryos still showed some anterior defects, their ventral morphogenesis was rescued. In addition, marker studies indicated that the axial mesendoderm was also defective in the homozygous embryos. Our results provide genetic evidence that expression of Apc at the normal level is essential for both anterior and ventral development, in the epiblast derivatives and visceral endoderm.     

Nucleofection as an efficient nonviral transfection method for human monocytic cells

Despite some progress in the field of gene transfer into hard-to-transfect cells, so far an efficient nonviral method for monocytes has not been available. A comparison of plasmid DNA with capped and polyadenylated mRNA for enhanced green fluorescent protein gene delivery into the commonly used monocytic cell lines U937 and THP-1 suggested that limited DNA trafficking may be the underlying cause of poor transfection results. As Nucleofector technology delivers DNA (or mRNA) straight into the nucleus, we obtained nucleofection efficiencies of up to 80% without significant cell toxicity. Moreover, as the DNA quickly reaches the nucleus, nucleofected cells were ready for analysis after only 2–6 h. The technique is suitable not only for monocytes but also for other hard-to-transfect cells.     

Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells

Although green fluorescent protein (GFP) labeling is widely accepted as a tracking method, much remains uncertain regarding the retention of injected GFP-labeled cells implanted in ischemic organs. In this study, we evaluate the effectiveness of GFP for identifying and tracking implanted bone marrow- mesenchymal stem cells (BM-MSCs) and the effect of GFP on the paracrine actions of these cells. MSCs isolated from rat femur marrow were transduced with a recombinant adenovirus carrying GFP. After transplantation of the GFP-labeled BM-MSCs into the infarct zone of rat hearts, the survival, distribution, and migration of the labeled cells were analyzed at 3, 7, 14, and 28 days. To evaluate the effect of GFP on the paracrine actions of BM-MSCs, Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF), b fibroblast growth factor (b FGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases-2 (MMP-2). GFP was successfully expressed by BM-MSCs in vitro. At 14 days after cell transplantation the GFP-positive cells could not be detected via confocal microscopy. By using a GFP antibody, distinct GFP-positive cells could be seen and quantitative analysis showed that the expression volume of GFP was 6.42 ± 0.92 mm³ after 3 days, 1.24 ± 0.76 mm³ after 7 days, 0.33 ± 0.03 mm³ after 14 days, and 0.09 ± 0.05 mm³ after 28 days. GFP labeling did not adversely affect the paracrine actions of BM-MSCs. GFP labeling could be used to track MSC distribution and their fate for at least 28 days after delivery to rat hearts with myocardial infarction, and this stem cell tracking strategy did not adversely affect the paracrine actions of BM-MSCs.     

Force and compliance: rethinking morphogenesis in walled cells

In the turgid cells of plants, protists, fungi, and bacteria, walls resist swelling; they also confer shape on the cell. These two functions are not unrelated: cell physiologists have generally agreed that morphogenesis turns on the deformation of existing wall and the deposition of new wall, while turgor pressure produces the work of expansion. In 1990, I summed up consensus in a phrase: "localized compliance with the global force of turgor pressure." My purpose here is to survey the impact of recent discoveries on the traditional conceptual framework. Topics include the recognition of a cytoskeleton in bacteria; the tide of information and insight about budding in yeast; the role of the Spitzenk?rper in hyphal extension; calcium ions and actin dynamics in shaping a tip; and the interplay of protons, expansins and cellulose fibrils in cells of higher plants.     

Leaving the meristem behind: The genetic and molecular control of leaf patterning and morphogenesis

Leaves, which play an essential role in plant photosynthesis, share common features such as being flat structures, but also show an impressive variability in their sizes and shapes. Following its initiation in the meristems, leaf development is patterned along three polarization axes to establish its basic architecture. This process is further complicated in the case of compound leaves with the formation of new growth axes. Growth and differentiation must be properly coordinated to regulate the size and the flatness of the leaf. This review provides an overview of the genetic and molecular regulatory networks underlying leaf development, with an emphasis on leaf polarity and the comparison of simple and compound leaves.     

高灵敏荧光探针用于肿瘤 Ramos 细胞的快速检测

本文以聚苯乙烯纳米微球为载体,基于适体特异性识别和 DNA 杂交原理,组装了一种 DNA-CdTe 量子点纳米线,制备了具有较高荧光强度的复合型荧光探针,并成功用于 Ramos 细胞的荧光成像。该探针可以用于特异性识别肿瘤细胞,在荧光成像中信号强灵敏度高,为肿瘤细胞的检测提供一种新方法。