微生物固体培养基凝固剂研究进展*

明胶是最早使用的固体培养基凝固剂,已逐渐被琼脂所代替。琼脂由于形成凝胶后透明度高、保水性好、无毒、不被微生物液化等优点,逐渐成为最常用的凝胶剂。后来,又发现无机硅胶、瓜尔胶、卡拉胶在某些情况下可用作凝固剂。近年来,兴起一种基于微生物快速检测的快速测试片,其所用凝固剂发展到黄原胶、刺槐豆角、聚丙烯酸系等。但新型凝固剂在使用过程中仍存在许多弊端,因此,从吸水和保水的机理出发对其研究和改性是一项重要的任务。     

琼脂固体培养基培养蜜环菌菌索总DNA的提取

野外采集的蜜环菌[Armillaria mellea(Vahl.ex Fr.)Quel]在提取DNA前需要分离获得纯化的菌丝体。常规液体培养获得菌丝团的方法感杂率较高,采集固体培养基表面cellophane膜上形成的菌丝则难以获得足量的DNA提取材料。蜜环菌细胞内含有大量多醣类物质,也使得蜜环菌高质量DNA的提取存在一定的困难。本研究通过改进试验,提供一个直接从琼脂固体培养基培养的蜜环菌菌索中提取高质量DNA的方法。其中样品的预先冻融处理方法可以促使蜜环菌菌索与琼脂分离;而在裂解提取缓冲液裂解材料细胞后加入1.25 mol/L KAc溶液,则有利于除去蜜环菌细胞内的多醣类物质以及残留的少量琼脂。通过琼脂糖电泳、紫外分光光度计对DNA浓度及OD值的测定、ISSR引物的PCR扩增以及酶切产物的PCR扩增等方法的检测,结果均表明该方法提取的DNA质量较好,符合进一步进行分子生物学研究的要求。     

植物组织培养中琼脂浓度和pH对培养基凝固程度的影响

植物组织培养中多数植物pH要求5.0～6.2,琼脂浓度一般选用7～8mg·L-1,最高可达16 mg·L-1,较低的5 mg·L-1,而我们观察到培养基凝固程度受pH影响较大,适当的降低琼脂浓度不仅可以满足外植体的正常支撑需求,且培养效果更佳.本文研究pH和琼脂浓度对培养基凝固程度的影响,以确定组织培养技术中合理的琼脂浓度.     

标准化微生物培养基研究进展

标准化微生物培养基研究进展吴清平,陈素云,阙绍辉,丘万英（广东省微生物研究所,广州５１００７０）７０年代以来,微生物学技术方法已从传统技术方法的改进、完善和应用范围的扩展,逐步在微量、简易监测系统的兴起、发展和应用的基础上,发展到微生物的自动化快速诊...     

培养基琼脂用量计算的商榷

植物组织培养中,大多数采用固体培养基。固体培养基是以琼脂(又称洋菜)为支持物的。目前一些资料中一般报告用培养基琼脂的百分含量来表示培养基的硬度。笔者认为,这种提法不太科学,而应该用胨力强度(又称凝胶强度)来表示琼脂培养基     

分枝杆菌全合成琼脂培养基的研究

本文报道了一种适合常见致病分枝杆菌生长的全合成固体琼脂培养基体系。它为实验室研究分枝杆菌的营养生理、生化特性、遗传变异、药理和耐药机制提供了一种新工具,也为研制更加满意的分枝杆菌培养基创造了条件。试验过的大多数非典型分枝杆菌,在全合成琼脂培养基上比在罗氏培养基上生长快,生长量少于或等于罗氏培养基。标准牛型株的细胞群体中,仅有少量细胞能在全合成琼脂培养基上生长。鸟型．胞内和偶发等分枝杆菌标准株在全合成琼脂培养基上产生两种不同的菌落。     

分枝杆菌简化琼脂培养基的研究

报道三种组分简单、制备方便的简化琼脂培养基.试验结果表明:7个标准菌株在简化琼脂培养基309A和309C上的生长速度和数量均相似或优于改良罗氏培养基和92号土豆汤琼脂培养基.简化琼脂培养基309C和309A可用于结核杆菌分离和药敏试验.     

细菌测试片的冷水可凝凝固剂初步研究和应用

微生物测试片具有携带方便、不用配制培养基的优点, 为一种新型的检测工具。测试片的技术关键是冷水可凝胶, 本文通过研究新的冷水可凝凝固剂(HKG)的保水性能、pH、强度、微生物利用及降解和抑菌效果, 再组装成细菌菌落总数测试片, 进行特异性和食品样品效果检验, 并与倾注平板法比对, 评价其作为微生物测试片凝固剂的可行性。结果显示HKG对16种微生物均无抑菌作用, pH为中性, 强度为340.31 g/cm2 ? 48.71 g/cm2; 具有良好的保水性能; HKG在短时期(5 d)内不支持微生物生长, 不被分解利用, 在测试片和倾注培养法中5种测试菌株均可达相同的灵敏度(P = 0.438 > 0.05), 各菌株菌落数(P = 0.442 > 0.05)和食品样品菌落数(P = 0.718 > 0.05)比较在统计学上无显著差异。结果表明, HKG能较好地应用于细菌测试片计数, 并能提高检测效率。     

培养基琼脂浓度及抗生素对3种海洋微藻生长的影响

目的:确定用于分离、纯化及保种所用的固体培养基中优化的琼脂浓度和利于微藻生长并抑菌的抗生素浓度。方法:设计固体培养基中的琼脂浓度以及培养液中抗生素的种类和浓度,定时测定培养体系中三种微藻的生物量及细菌菌落随培养时间的变化。结果:利于固体培养基上三种微藻生长的琼脂浓度均为0.6%~0.8%;牟氏角毛藻用200IU/ml的庆大霉素和200IU/ml的青霉素联合作用,球等鞭金藻8701和盐藻分别用300IU/ml和400IU/ml的青霉素时可保证微藻较好生长。结论:不同微藻生长所需的抗生素浓度不同,抗生素对微藻生长的影响不完全取决于抑菌作用,青霉素促进球等鞭金藻8701生长主要是产生促生长因子,庆大霉素抑制生长可能是产生抑制因子。     

Differential performance of marubakaido apple rootstock shoots grown in culture media containing different agar brands: dynamic rheological analysis

Shoots of the marubakaido apple rootstock grown in culture medium containing BBL agar presented significantly lower multiplication rate (MR) compared to MRs found for shoots grown in medium containing A-7002, A-7921, Select, and Phytagar as gelling agents. In addition, significant hyperhydricity was found for shoots grown in Phytagar and A-7921 agar-containing media. Analysis of elastic (G′) and viscous (G″) modulus showed that for all of the five agar brands used in this study, G′ was always much higher, i.e., typically one order of magnitude higher than G″, which characterizes a strong gel. G′ changed randomly with time for all of the agar brands studied, except for BBL, which presented progressive decline in G′ throughout the culture cycle. Examination of G′, within the same week, showed that Select agar always had the smallest G′, while Phytagar always had the highest G′. Analysis of the loss tangent (tan δ = G″/G′), a better indicator for gel behavior compared to G′ isolated, showed that tan δ for Select and Phytagar were always between tan δ values found for A-7002 and BBL. In addition, analysis of tan δ also indicated that BBL and Select agars showed a significantly weaker gel network, compared to Phytagar, A-7002 and A-7921 agars after the third week of culture. When seen together, these results indicate that shoot performance for the marubakaido rootstock is not related to agar gel strength. In addition, the high hyperhydricity rate found for shoots grown on agars A-7921 and Phytagar could not be related to agar gel strength, as well. Analysis of HPSEC profiles indicated that the best performance, i.e., multiplication rate, of marubakaido shoots in agars A-7002 and A-7921 is likely to be related to their lower polydispersity and/or smaller amount of high molecular weight fractions, compared to BBL, Phytagar, and Select agars.     

`Isubgol' as an alternative gelling agent in plant tissue culture media

`Isubgol', the mucilaginous husk derived from the seeds of Plantago ovata, was successfully used as a gelling agent in tissue culture media for in vitro seed germination, shoot formation and rooting in Syzygium cuminii and anther culture in Datura innoxia. For seed germination, Knop's basal medium supplemented with 1% sucrose was employed, whereas for the development of shoots the epicotyl segments excised from in vitro-developed seedlings were cultured on MS basal medium supplemented with 4% sucrose and 1 mg/l 6-benzyladenine. Shoots that developed from the epicotyl segments were rooted on Knop's medium enriched with 2% sucrose and 1 mg/l indole-3-acetic acid. The anthers of D. innoxia excised at the late uninucleate to early binucleate stages of microspore development were cultured on Nitsch's basal medium containing 2% sucrose. Media were either gelled with 0.9% agar or 3% `Isubgol'. The response on media gelled with `Isubgol' in each of the cases was similar to that on media solidified with agar. Received: 9 October 1996 / Revision received: 22 July 1996 / Accepted: 30 July 1997     

Gelling agents for tissue culture of the seaweed Hizikia fusiformis

Callus and blade formation of the seaweed Hizikia fusiformis depended on the gelling agents used under axenic culture conditions. Excised cylindrical pieces (5 mm) of the hold fast were cultured on seven different gelling agents in seawater with added Provasoli's enrichment (PESI), at 40 μmol m⁻² s⁻¹ light intensity, 18 −C for 1 month. The highest percent of callus formation (47%), from holdfast pieces, was produced on solid medium composed of 2.0% high gel strength agar. No callus was formed in liquid medium. Blades, from holdfast pieces, were formed in PESI liquid medium at the rate of 45%, while the high level of axenic blade formation (30%) on solid support was observed on 0.5% high gel strength agar. Callus and blade were identified with the original strain, at the DNA level, using random amplified polymorphic DNAs. This revised version was published online in August 2006 with corrections to the Cover Date.     

耐碳青霉烯类肠杆菌科细菌体外抗菌活性分析

目的 了解临床分离的59株碳青霉烯酶类抗生素耐药的肠杆菌科细菌（CRE）体外药敏情况。方法 采用法国生物梅里埃公司VITEK-2 Compact进行细菌鉴定及药敏,MALDI-TOF MS确认。琼脂稀释法测定临床常用抗生素的最低抑菌浓度。结果 分离的菌株以肺炎克雷伯菌为主。59株CRE除对亚胺培南和美罗培南耐药率100.0%外,对哌拉西林/他巴唑坦和阿莫西林/克拉维酸同样完全耐药。头孢他啶、头孢噻肟、氨曲南耐药率分别为91.5%、98.5%和94.8%。阿米卡星有较好的抗菌活性,耐药率为44.8%。替加环素耐药率最低,为22.0%。结论 CRE耐药率较高,头孢类、青霉素类及其复合制剂单独用药不适合作为本地区CRE菌株抗菌药物,需联合用药。替加环素目前可作为CRE理想用药,阿米卡星也可作为次选。     

Factors influencing in vitro shoot regeneration from leaf segments of Chrysanthemum

The objective of this research was to develop an efficient protocol for shoot regeneration from leaf segments of the Chrysanthemum cv. Vivid Scarlet by examining the effects of plant growth regulators, dark incubation period, gelling agents, and silver nitrate. The highest number of shoots per explant (12.3) was regenerated from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with a combination of 1 mg L⁻¹ of 6-benzyladenine (BA) and 2 mg L⁻¹ of α-naphthaleneacetic acid (NAA) under light conditions without any initial dark period. Gelrite was the most effective gelling agent for shoot regeneration among those tested, whereas the presence of silver nitrate distinctly inhibited shoot regeneration. Superior plant growth and rooting was observed on a hormone-free MS medium solidified with Gelrite. Flow cytometry analysis revealed no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions. The established protocol was applicable to shoot regeneration for four out of six cultivars tested. This research will facilitate the genetic transformation and micropropagation of Chrysanthemum cultivars.     

Endogenous cytokinins in shoots of Aloe polyphylla cultured in vitro in relation to hyperhydricity, exogenous cytokinins and gelling agents

The process of hyperhydricity in tissue cultured plants of Aloe polyphylla is affected by both applied cytokinins (CKs) and the type of gelling agent used to solidify the medium. Shoots were grown on media with agar or gelrite and supplemented with different concentrations of N⁶-benzyladenine (BA) or zeatin (0, 5 and 15 μM). Endogenous CKs were measured in in vitro regenerants after an 8-weeks cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. On media with agar a reduction in hyperhydricity occurred, while the gelrite treatment produced both normal and hyperhydric shoots (HS). The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of HS was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene.     

New insights in agar biorefinery with arylsulphatase activities

Agar is a major gelling agent used both in food and pharmaceutical applications. Traditional purification of agar is generally performed by sequential time consuming chemical and/or physical steps, leading to both poor recovery yields and low productivities. As a consequence, only 30% of the amount of agar produced is actually available under purified form to feed the world market.The current limiting factor for purification is the presence of sulphated compounds such as sulphated-agaropectin, which strongly affect the technological properties of the agar gel such as gel strength, melting and fusion temperatures and electroendosmosis.In this context, this communication aims at discussing about the development of a biorefining agar purification approach which allows overcoming the current limitations associated with traditional purification methods. More specifically, this article focuses on the potential role of arylsulphatases in agar purification processes to reduce the number of purification steps and to improve recovery yields.This review first presents the global gelling agents market before focusing on agar characteristics and production processes. Then, after a brief reminder of the sulphur metabolism, the roles, classes and properties of the different arylsulphatases are described to draw perspectives on their integration in current or new agar production processes.