一种快速、简易的扫描电镜植物样品干燥新方法

扫描电子显微镜是观察植物样品表面超微结构的有效方法,大部分新鲜植物样品需经过干燥处理才可以进行扫描电镜观察。该研究在传统叔丁醇冷冻干燥法的基础上,建立了叔丁醇一步冷冻干燥法,省略了固定、脱水、置换等步骤,简便易行,干燥后的样品形态饱满,最大程度保持了样品原貌。用叔丁醇一步冷冻干燥法干燥的样品可以与CO_2临界点干燥法和常规叔丁醇冷冻干燥法的效果相媲美。通过对不同的样品进行干燥处理,结果证明,该方法具有广泛适用性。     

高水分、富含淀粉植物组织的扫描电镜制备技术优化

应用常规高真空扫描电子显微镜观察生物样品必须经过脱水和干燥处理,但无论采用临界点干燥还是冷冻干燥方法,都存在样品表面不同程度失真的问题。植物高水分、富含淀粉组织样品经处理后,容易出现淀粉流失、细胞壁变形等现象,从而造成扫描图像粗糙,无法获得真实的细胞内部结构。本文通过对CO_2临界点干燥、化学固定样品冷冻干燥和新鲜样品冷冻干燥3种扫描电镜样品制备技术中后期制样进行机械断裂和液氮脆断改进,优化出两种植物高水分、富含淀粉组织的扫描电镜样品制备方法:(1)样品首先进行FAA化学固定,经冷冻干燥后用液氮脆断,对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞结构完整,细胞壁整齐,淀粉粒和蛋白轮廓明确,可用于分析淀粉粒和蛋白颗粒在细胞内的分布。(2)新鲜样品直接进行冷冻干燥,经液氮脆断后对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞壁整齐,淀粉粒轮廓更清晰,并且无蛋白颗粒干扰,用于分析淀粉粒在细胞内的分布更加理想。     

扫描电镜标本制备

扫描电镜主要用以观察物体表面,可观察较大的范围,同时由于它有很大的焦深,因此立体感很强,弥补了透射式电镜对复杂的立体形貌观察上的不足。扫描电镜样品制备方法有多种,下面介绍常用的几种:(一)临界点干燥法本文介绍用液体 CO_2进行临界点干燥的方法。1. 固定:把样品放在缓冲液或生理盐水中洗净后,放在1-3%戊二醛中固定30分钟至     

用准饱和CO_2干操法制备两种肿瘤细胞的扫描电镜试样

采用准饱和CO_2干燥法(QSCD)制备SGC-7901人体胃癌细胞株贴壁细胞和HAC小鼠腹水型肝癌游离细胞的扫描电镜试样,结果显示,细胞形貌与临界点干燥(CPD)对照组无明显差异,但收缩率较后者减小。QSCD法突破了传统的无表面张力干燥条件,操作顺序简单;一次可干燥较多试样;样品室压力不超过70大气压,较CPD法安全。     

一种适合野外使用的被子植物分子标本干燥方式

该研究通过比较不同干燥方式对被子植物分子标本的影响,试图得到一种野外采集过程中可以替代硅胶干燥法的更为便捷的植物分子标本的干燥方法。选取40、80和150℃烘干和吸水纸压制干燥、硅胶干燥法对日本晚樱(Prunus serrulatavar.lannesiana)和山麦冬(Liriope spicata)两种植物的新鲜叶片进行干燥处理,提取各种处理样品的DNA,并将DNA进行电泳检测、分光光度计检测及PCR扩增,以此评价不同的干燥方式对植物基因组DNA的影响。分光光度计检测及总DNA电泳结果显示,经40℃烘干或硅胶干燥处理的样品总DNA浓度及长片段DNA浓度较其他干燥方式高;PCR产物浓度统计学分析显示,40℃烘干处理的样品PCR产物浓度高于其他干燥方式。基于以上结果,建议在野外采集被子植物分子标本时,使用40℃烘干干燥法对分子标本进行干燥处理,避免分子标本快速降解以及硅胶干燥法的污染问题,同时可节省携带、更换大量硅胶材料所耗费的人力。     

真菌的冷冻扫描电镜观察

冷冻扫描电镜法是根据低温生物学原理,生物样品经过超低温处理后进行电镜观察的一种方法。经超低温冷冻固定的生物样品可保持其原有的形态特征和生物活性。样品内部水份在超低温(—150℃)下,即使在真空中升华速度     

扫描电镜研究节肢动物形态的简便方法

<正> 近年来,扫描电镜在研究动物形态时广泛应用。但是,供扫描电镜观察的生物样品的制备比较复杂,需要经过预处理,如临界点干燥,金属喷镀等。一些体表柔软的动物体经过这些处理,受抽真空及热辐射等影响,使样品损伤、变形,或由于镀膜有一定厚度(100—200A),小于此厚度的细节则不能分辨。同时,标本经喷镀后再不能继续保存供其他观察用,故扫描电镜技术的运用受到一定限制。 Howden等(1973)曾探索改进扫描电镜,降低加速电压(1.5—2.5KV),用来观察未喷镀的     

低pH孵放法对马破伤风免疫球蛋白F(ab′)_2病毒灭活效果的验证

目的采用低pH孵放法对马破伤风免疫球蛋白F(ab′)_2样品中辛德毕斯病毒(sindbis virus, SINV)、水疱性口炎病毒(vesicular stomatitis virus, VSV)、脑心肌炎病毒(encephalomyocarditis virus, EMCV)的灭活效果进行验证。方法以SINV、VSV、EMCV作为指示病毒,将3批马破伤风免疫球蛋白F(ab′)_2的pH调至4.1±0.1,每批12瓶(3 mL/瓶),分别加入333μL指示病毒,25℃放置21 d灭活病毒,同时设置中性对照和阳性对照,并于0 d、1 d、3 d、7 d、14 d、21 d取样测定剩余病毒滴度,验证低pH孵放法的病毒灭活效果。结果马破伤风免疫球蛋白F(ab′)_2样品经低pH孵放处理21 d后,SINV、VSV和EMCV等3种指示病毒残余病毒滴度均≤0.5 lgTCID_(50)/0.1 mL,灭活效果分别达到5.50～5.83、5.70～6.00和6.00～6.17 lgTCID_(50)/0.1 mL。结论采用低pH孵放法处理马破伤风免疫球蛋白F(ab′)_2中SINV、VSV、EMCV,病毒滴度下降值均>4 lgTCID_(50)/0.1 mL,灭活效果较好。     

不同干燥方法对灰毡毛忍冬花蕾中活性成分含量的影响

目的:比较不同干燥方法对灰毡毛忍冬花蕾中4种活性成分含量的影响.方法:采用生晒法、烘干法、蒸制生晒法和蒸制烘干法对灰毡毛忍冬花蕾进行干燥处理.参照《中国药典》,利用高效液相色谱(HPLC)-紫外扫描(UV)法测定干燥样品中绿原酸和木犀草苷的含量;利用HPLC-蒸发光散射检测器(ELSD)法测定干燥样品中灰毡毛忍冬皂苷乙和川续断皂苷乙的含量.结果:蒸制烘干法处理的灰毡毛忍冬花蕾中绿原酸含量和皂苷总量最高,分别为3.705％和11.834％,木犀草苷含量为0.103％,均高于《中国药典》对山银花和金银花指标物质含量的规定.结论:不同干燥方法对灰毡毛忍冬花蕾中活性成分的含量有较大影响,以蒸制烘干法最优.     

植物染色体SSG分带方法与带型

植物染色体Giemsa C-带技术,目前广泛使用BSG法(Barium hydroxide-Saline-Giemsa)。但是,此法用Ba(OH)_2进行变性处理,容易发生制片污染,因此我们改用NaHCO_3代替Ba(OH)_2进行变性处理,首先在蚕豆上分带成功,并取名为SSG法(sodium b carbonate-Saiine-     

A Chemical Drying Technique for Plant Materials Applied to the Scanning Electron Microscopy

There are several drying methods for biological materials for the use of scanning electron microscopy. Applying bexamethyl disilazane (HMDS) as a drying treatment is a new method and it's application on drying plant tissue has not been previously reported. The advantage of this method is the treatment only for a few minutes and is also good and stable for very small biological specimens. The method is simple, low cost and time saving and does not need any apparatus. The features of the tissue structure observed are satisfactory.     

Comparison of Preparation Methods of Bacterial Cell-Mineral Aggregates for SEM Imaging and Analysis Using the Model System of Acidovorax sp. BoFeN1

Innovative microanalytical methods are valuable tools in geomicrobiology. They often require the use of dried samples, demanding a challenging sample preparation. Since geomicrobiological samples typically have a strongly heterogeneous composition, choosing a preparation method is not straightforward. We therefore compared how different drying methods (critical point drying, hexamethyldisilazane drying, air drying and freeze drying) influence the structure of bacterial cell-mineral aggregates. Each method proved suitable for a specific purpose, but none was able to completely preserve the sample structure. Additional information was obtained on surface alterations by sputter coating and on preservation of extracellular polymeric substances during resin embedding.     

Differential mechanisms to induce dehydration tolerance by abscisic acid and sucrose in Spathoglottis plicata (Orchidaceae) protocorms

Abscisic acid (ABA) and sucrose are known to induce dehydration tolerance of in vitro plant cells and tissues. The present study reports the presence of different mechanisms by which sucrose and ABA improve dehydration tolerance of Spathoglottis plicata (orchid) protocorms. Orchid protocorms were generated aseptically from seeds on Murashig and Skoog medium, and then treated for 7 d in medium containing 10 mg L^?1 ABA and/or 10% (w/v) sucrose. Dehydration tolerance of protocorms was determined at ～25 °C under various drying conditions at relative humidity from 7 to 93%. The actual rate of water loss (i.e. drying rate) was determined using the rate constant of tissue water loss during drying according to the first‐order kinetics. Drying rate affected dehydration tolerance. ABA treatment reduced drying rate and increased dehydration tolerance of protocorms at all relative humidity values tested. However, when compared on the basis of actual drying rates, there was no difference in dehydration tolerance between control and ABA‐treated protocorms, suggesting that ABA‐induced tolerance was correlated with the drying rate reduction. Sucrose treatment was more effective than ABA treatment for the induction of dehydration tolerance. Interestingly, sucrose only slightly affected drying rate. ABA treatment significantly enhanced the synthesis of dehydrin, whereas sucrose treatment primarily resulted in sucrose accumulation. Sucrose treatment also affected protein turnover during drying, causing a significant decrease in protein content in protocorms. Slow drying promoted the degradation of high molecular weight proteins and enhanced the synthesis of low molecular weight dehydrin. The data suggest that different physiological mechanisms are probably involved in the induction of dehydration tolerance by ABA and sucrose treatment.     

Xeroprotectants for the stabilization of biomaterials

With the advancement of science and technology, it is crucial to have effective preservation methods for the stable long-term storage of biological material (biomaterials). As an alternative to cryopreservation, various techniques have been developed, which are based on the survival mechanism of anhydrobiotic organisms. In this sense, it has been found that the synthesis of xeroprotectants can effectively stabilize biomaterials in a dry state. The most widely studied xeroprotectant is trehalose, which has excellent properties for the stabilization of certain proteins, bacteria, and biological membranes. There have also been attempts to apply trehalose to the stabilization of eukaryotic cells but without conclusive results. Consequently, a xeroprotectant or method that is useful for the stable drying of a particular biomaterial might not necessarily be suitable for another one. This article provides an overview of recent advances in the use of new techniques to stabilize biomaterials and compare xeroprotectants with other more standard methods.     

植物材料快速石蜡制片方法

真空干燥箱已越来越广泛地应用于现代生物学研究领域。该文利用真空干燥箱温度和负压的可控制性能,将固定、脱水、透明和石蜡渗透等过程在真空干燥箱中进行,建立起一套可行的植物组织快速石蜡制片方法。结果显示,真空干燥箱的应用加速了多种试剂的渗透速率,提高了切片质量,达到了优化实验步骤、节省实验时间和减少室内有毒化学气体污染的目的。     

Dehydration Kinetics of Embryonic Axes from Desiccation-sensitive Seeds:An Assessment of Descriptive Models

The response of desiccation-sensitive plant tissues to dehydration is significantly affected by dehydration conditions, particularly the rate of drying. Consequently it is important to be able to quantify drying rate. The aim of the study was to assess two models that have been proposed to describe drying kinetics, and thus to provide a quantification of non-linear drying rates, of embryonic axes excised from recalcitrant seeds. These models are an exponential drying time course, and a modified inverse relationship, respectively. For the six species investigated here the inverse function was generally found to fit drying data better than the exponential function under both rapid and slow drying conditions, and so is recommended. The rate of drying, under the conditions used here, was determined by axis size and possibly the nature of the axis outer coverings, rather than the water activity difference between the tissue and surrounding air.     

Drying techniques for the visualisation of agarose‐based chromatography media by scanning electron microscopy

The drying of chromatography resins prior to scanning electron microscopy is critical to image resolution and hence understanding of the bead structure at sub‐micron level. Achieving suitable drying conditions is especially important with agarose‐based chromatography resins, as over‐drying may cause artefact formation, bead damage and alterations to ultrastructural properties; and under‐drying does not provide sufficient resolution for visualization under SEM. This paper compares and contrasts the effects of two drying techniques, critical point drying and freeze drying, on the morphology of two agarose based resins (MabSelect?/d _w ≈85 µm and Capto? Adhere/d _w ≈75 µm) and provides a complete method for both. The results show that critical point drying provides better drying and subsequently clearer ultrastructural visualization of both resins under SEM. Under this protocol both the polymer fibers (thickness ≈20 nm) and the pore sizes (diameter ≈100 nm) are clearly visible. Freeze drying is shown to cause bead damage to both resins, but to different extents. MabSelect resin encounters extensive bead fragmentation, whilst Capto Adhere resin undergoes partial bead disintegration, corresponding with the greater extent of agarose crosslinking and strength of this resin. While freeze drying appears to be the less favorable option for ultrastructural visualization of chromatography resin, it should be noted that the extent of fracturing caused by the freeze drying process may provide some insight into the mechanical properties of agarose‐based chromatography media.     

初生休眠解除状态和干燥处理对水曲柳种子萌发的影响

为探究初生休眠解除状态和干燥处理对水曲柳种子萌发的影响，本文以初生休眠的成熟水曲柳种子为材料，研究经不同裸层积（暖温10周+低温8周、暖温12周+低温8周、暖温10周+低温10周、暖温12周+低温10周）和干燥处理（干燥、不干燥）的水曲柳种子在适宜温度和较高温条件下的萌发表现。结果表明，初生休眠解除状态不同的水曲柳种子在不同温度下的萌发表现具有相似的规律，种子的萌发会受到干燥处理的影响。不经干燥处理的种子解除休眠越充分，其萌发能力就越强，但层积处理后的种子若经过干燥处理，则解除休眠越充分（尤其是低温时间越长），种子萌发能力下降越多。水曲柳种子次生休眠（热休眠）的诱导受种子初生休眠解除状态的影响较小，但受干燥处理影响较大。干燥处理会降低水曲柳种子的萌发能力，尤其是较高温条件下的萌发能力，初生休眠解除越充分的种子萌发受干燥处理影响越大。生产中如需对解除休眠的种子干燥处理，选择暖温10周+低温8周的层积方法处理种子效果最好。     

Effect of drying on the biological activities of a red microalgal polysaccharide

The red microalga Porphyridium sp. produces a polysaccharide exhibiting a variety of biological activities with potential for medical and cosmetic uses. For this reason, it is important that the drying process, which is the end point of production, should not destroy the natural characteristics of the material. The objective of this study was to evaluate the effect of drying at temperatures ranging from 40 to 140 degrees C on the bioactivities of the polysaccharide. Drying the polysaccharide at temperatures above 90 degrees C caused a significant decline in its biological activities (antiviral and anti-cell proliferation) and reduced elasticity, viscosity, and intrinsic viscosity relative to lyophilized polysaccharide and to the starting product. The relationship between molecular weight and intrinsic viscosity indicated that the polysaccharide takes a rigid coil conformation, which stiffens as a result of drying. FTIR analysis revealed that drying caused both significant conformational alterations in the polymer chains and changes in the interaction between the polysaccharide and the glycoprotein to which it is noncovalently associated. Differential scanning calorimetry analysis of the water adsorbed on the charged groups of the polysaccharide showed that drying at higher temperatures increased the bound water content due to dissociation of the polymer chains. Thus, it is recommended that the polysaccharide be dried in a two-step process in which free water is removed by convection and bound freezing water is removed by lyphophilization.