Tris not only controls the pH in microalgal cultures,but also feeds bacteria

Tris (Tris(hydroxymethyl)amino methane), a compound often used as a buffer in microalgal culture media, sustains active bacterial growth in non-axenic microalgal cultures when sodium phosphate is present. The low pH levels caused by bacterial growth and probably the depletion of phosphorus in the medium caused the collapse ofPhaeodactylum tricornutum cultures resulting in a reduction of microalgal growth from 32 x 10⁶ to 1.1 x 10⁶ cells ml^–1. This emphasizes the need for care when interpreting the results of non-axenic microalgae cultures in which Tris or other organic buffer is added.     

基于双重芯片式数字PCR快速鉴定KPC型碳青霉烯耐药肺炎克雷伯菌的方法

【背景】由于碳青霉烯类药物的泛用和滥用,致使肺炎克雷伯菌碳青霉烯耐药株与日俱增,产碳青霉烯酶是肺炎克雷伯菌对碳青霉烯类药物耐药的主要原因。目前对肺炎克雷伯菌碳青霉烯耐药株的检测方法存在费时费力、特异性差、灵敏度低等问题。【目的】建立一种能同时检测肺炎克雷伯菌和碳青霉烯酶基因bla_KPC的双重芯片式数字PCR方法。【方法】依据肺炎克雷伯菌的特有基因yhaI和碳青霉烯耐药基因bla_KPC保守序列设计特异性引物和探针,确定双重芯片式数字PCR同时对yhaI和bla_KPC两个基因核酸浓度绝对定量的检测范围、检出限和最佳实验体系,并进行方法特异性、灵敏度、重复性分析及临床菌株的检测。【结果】双重芯片式数字PCR检测灵敏度比双重实时荧光定量PCR提高了约1.5个数量级,在两基因同时检出的情况下,最低检出限分别为3.74 copies/μL (yhaI基因)和1.93 copies/μL (bla_KPC基因);优化后的双重芯片式数字PCR对参考菌株检测特异性的结果与双重实时荧光定量PCR结果一致;利用优化后的双重芯片式数字PCR方法共检测58株临床菌株,其中肺炎克雷伯菌43株,属肺炎克雷伯菌且含有bla_KPC基因的菌株13株,这与质谱及耐药谱检测结果一致。【结论】利用双重芯片式数字PCR技术建立了产KPC型碳青霉烯酶肺炎克雷伯菌的绝对定量检测方法。该方法特异性强、灵敏度高、准确度好,可用于检测具有碳青霉烯酶基因bla_KPC的肺炎克雷伯菌的核酸检测和定量分析,也为产其他类型碳青霉烯酶的病原菌检测提供了新的技术参考。     

Heterogeneic modulation of malignant behavior in human glioma cells in defined and serum-containing media

Summary Malignant features in three glioma cell lines were studied in four defined media of various complexity. The cell lines D37MG, D54MG, and GaMG were able to grow in monolayer culture in all media examined, and as multicellular tumor spheroids in the two most nutrient-rich media. In the defined media, none of the cell lines were able to migrate in a migration assay on poly-D-lysine-coated plastic surfaces. Flow cytometric analysis of the GaMG cell line demonstrated no medium-dependent selection of subclones of glioma cells in spheroids cultured for 30 d. Morphological diversity of spheroids varied according to the supplementation of the media. The capacity of glioma cells to invade cellular rat brain aggregates was intact in the media examined. However, glioma migration was severely inhibited by the lack of specific serum components. This study demonstrates that glioma growth and invasion was heterogenously preserved in the defined media used. Depending on the assay to be used in the study of glioma cell behavior, the degree of medium supplementation has to be considered.     

Influence of external factors on secondary embryogenesis and germination in somatic embryos from leaves of Quercus suber

Somatic embryogenesis was obtained in cultures of leaves from young seedlings of Quercus suber L. A two-stage process, in which benzyladenine and naphthaleneacetic acid were added first at high and then at low concentrations, was required to initiate the process. Somatic embryos arose when the explants were subsequently placed on medium lacking plant growth regulators. The embryogenic lines remained productive, by means of secondary embryogenesis, on medium without growth regulators. However, this repetitive induction was influenced by the macronutrient composition of the culture medium. Both low total nitrogen content and high reduced nitrogen concentration decreased the percentage of somatic embryos that showed secondary embryogenesis. Our results suggest that alternate culture on medium that increases embryo proliferation and a low salt medium prohibiting embryo formation will partially synchronize embryo development. Chilling slightly reduced secondary embryogenesis but gave a modest increase in germination. Maturation under light followed by storage at 4 °C for at least 30 days gave the best results in switching embryos from an embryogenic pathway to a germinative one. Under these conditions 15% of embryos showed coordinated root and shoot growth and 35% formed either shoots or mostly roots. These percentages were higher than those of embryos matured in darkness. This result indicates that a specific treatment is required after maturation and before chilling to activate the switch from secondary embryo formation to germination.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA indolebutyric acid - MS Murashige & Skoog (1962) medium - SH Schenk & Hildebrandt (1972) medium - G Gamborg (1966, PRL-4-C) medium (macronutrients in mg l^–1: NaH₂PO₄·H₂O, 90; Na₂HPO₄, 30; KCl, 300; (NH₄)₂SO₄, 200; MgSO₄·7H₂O, 250; KNO₃, 1000, CaCl₂·2H₂O, 150) - PGR plant growth regulator     

Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media

The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (a(w) = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol-containing enzymes were employed, higher reaction rates and less hydrolysis were observed. Immobilization of chymotrypsin resulted in high activity and operational stability, while the nonimmobilized enzyme was stable only in the presence of sorbitol. The activity of all preparations diminished after washing them with pyridine to remove sorbitol. Furthermore, severe stability problems occurred in the preparations lacking sorbitol. Sorbitol treatment, even after removal of the sorbitol itself, improved the activity of nonimmobilized chymotrypsin relative to the washed control. On the other hand, washing to remove sorbitol had a negative effect on the activity of both coimmobilized lipase and coimmobilized chymotrypsin. Addition of a substrate analogue, N-acetyl-L-phenylalanine, to chymotrypsin yielded a preparation that exhibited higher activity than both the control and its sorbitol-containing counterpart. Differential scanning calorimetry measurements revealed that the chymotrypsin-sorbitol complex was stable against thermal denaturation, undergoing transition at a high temperature (89 degrees C). The transition temperatures of the substrate-containing chymotrypsin and of the control were identical (72 degrees C). (c) 1995 John Wiley & Sons, Inc.     

The effects of domestic compost upon the germination and emergence of barley and six arable weeds

In a bioassay, leachate from composted household waste was found to decrease both cress germination and the germination of barley and six arable weeds, especially in the light. The effect of compost depth upon the emergence of these species was examined in a glasshouse experiment. Compost had no significant effect on barley emergence. However, weed emergence, particularly of those with small seeds, was reduced greatly by the physical effect of compost and, to a lesser extent, by its chemical effect. The emergence of the large-seeded species was relatively unaffected by increasing depths of compost, whereas that of the smaller-seeded weeds steadily decreased, being almost completely prevented by a 3 cm layer. The use of compost to control arable weeds in the field is discussed.     

Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

Roche Susceptibility Test (RST) medium,a defined formulation for susceptibility testing: I. Nutrient interaction and optimization studies

Roche Susceptibility Test (RST) Medium represents the most completely optimized and convenient fully defined medium described. It requires no post-autoclaving supplementation with vitamins, supports good growth of all common aerobic and anaerobic pathogens and may be used as a broth or agar gel on which the swarming of Proteus spp. is virtually eliminated. The broth as a superior buffering capacity to most complex media and an osmolality and pH close to those of human serum. RST is highly satisfactory for the susceptibility testing of commonly used antibiotics and meets the requirements of the National Committe for Clinical Laboratory Standards of the U.S.A. in almost every respect.     

Cultured hepatoma cells for the study of enzyme regulation: Induction of ornithine decarboxylase by insulin and asparagine

Summary The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combintion, of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10⁻⁵ M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present. This work was supported in part by Grants CA-07175, CA-22484, and CA-17334 from the National Cancer Institute. D. P. G. is a Predoctoral Fellow at the Food Research Institute, supported by a fellowship from the Monsanto Fund and by NIH Grant R01-AI 15693 to Prof. Michael W. Pariza, Food Research Institute, University of Wisconsin, Madison.