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。     

如何防止花粉流失

植物花粉肉眼难以看到,在按扫描电镜常规方法制备样品很容易流失。如果在取材时将花药整体采取一起固定,操作时不碰破花药囊,花粉就不会流失。这样可按扫描电镜常规方法处理样品(双固定时间可延长到12小时以上,固定时需抽真空),待临界点干燥做完之后,再用镊子把花药轻轻夹到贴有双面胶纸的样品座上,捣碎花药囊花粉就会从囊中落出而粘在胶纸上,喷金后即可进行电镜观察。注:实验样品由林志华同志供给,蔡继炯同志拍摄电镜照片.     

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     

冻结速率对血小板冷冻干燥保存的影响

冷冻干燥法是使血小板能够长期保存的一种理想方法。冻结过程对血小板的冻干保存至关重要。采用梯度降温、搁板预冷、液氮冻结等三种冻结方式,研究了冻结速率对血小板冷冻干燥保存恢复率的影响。实验结果表明用搁板预冷的方式冻结并干燥的血小板复水后的恢复率最高,达到(93.0.2)%,此时的冻结速度约为10℃/min。扫描电镜照片显示冻干复水后的血小板保持了完整的细胞结构,但与新鲜血小板相比略呈球形。冻干复水后的血小板对1U/ml凝血酶的最大聚集率接近于新鲜血小板,但聚集速度比新鲜血小板慢。     

不同干燥方法对白芍中6种化学成分的影响与评价

研究不同干燥方法对白芍中单萜苷类和酚酸类化学成分的影响,为白芍适宜干燥方法的确定提供依据;采用HPLC同时测定经不同干燥方法制备的样品中4种单萜苷类、2种酚酸类化学成分的含量;利用SPSS软件进行主成分分析,利用主成分得分进行综合评价。结果表明:不同干燥方法所得白芍均能达到2015年版《中国药典》对白芍含量和水分的要求,采用真空冷冻干燥单萜苷类和酚酸类总量最高,为6.248%,热风60℃干燥次之,为6.141%;经主成分分析,不同干燥方法处理的白芍中单萜苷类和酚酸类成分含量综合评分依次为:真空冷冻干燥热风60℃干燥微波干燥远红外干燥晒干热风40℃干燥热风80℃干燥阴干热风100℃干燥。不同干燥方法对白芍中单萜苷类和酚酸类成分有一定的影响。因此,从各成分含量、成本和实用性方面综合分析,热风60℃干燥为白芍适宜的干燥方法。     

纤维素酶降解人参细胞胞壁产生的激发子诱导人参培养细胞的反应

用纤维素酶降解人参细胞壁制备的激发子刺激人参细胞后,电子显微镜的观察结果显示激发子处理诱导了细胞内淀粉粒的降解,出现了细胞内动员能量物质的应急反应;同时还出现了大量拟脂类圆球体。进一步的研究表明,涉及降解淀粉的淀粉酶活力是升高了,而降解脂类物质的酯酶活力是降低的;同时细胞中可溶性糖和总类脂含量是增加的。这些都说明了人参细胞经激发子处理后能量物质的动员以及脂类代谢途径的调整。另外,适当浓度激发子的处理会诱导人参细胞内人参皂苷含量和苯丙氨酸解氨酶活力的升高,而过氧化物酶和多酚氧化酶活力是下降的。     

一种用于观察纤毛虫表膜下结构的扫描电镜样品制备新方法

该实验摸索出通过扫描电镜观察纤毛虫表膜下三维结构的新方法:用适当浓度的KMnO_4作为固定剂,固定虫体细胞表膜,调整固定液的渗透压使细胞在低渗溶液中胀破、细胞质溶出,表膜剥落下来、内外翻转,经脱水、冷冻干燥、喷金后,在扫描电镜下对爽口虫(Climacostomumsp.)、尾草履虫(Paramecium caudatum)及拟尾柱虫(Paraurostyla weissei)的表膜下结构进行了观察。结果表明:利用此方法能够观察到表膜下层次分明而又清晰的三维立体构象。此方法可为纤毛虫表膜及其它细胞质膜的研究提供可借鉴的样品制备新方法。     

冬虫夏草蛋白图谱及干燥条件对超氧化物歧化酶活性影响

本文采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和双向电泳(2-DE)技术对3个野生冬虫夏草样本和3个培植冬虫夏草样本进行了比较分析。SDS-PAGE结果显示6个样品的蛋白质分子量主要分布在3–66.4k Da之间,Quantity one软件处理凝胶图谱显示蛋白条带在20–23条之间;采用PDQuest软件对双向电泳图谱处理,共检测到670–936个蛋白点,蛋白质主要分布在等电点为p I 4.5–6、分子量为29.0–66.7k Da和14.3–20.1k Da两个区域。尽管在不同样品之间存在一定程度的蛋白质差异,但是并没有发现野生冬虫夏草和培植冬虫夏草之间有显著差异。以超氧化物歧化酶(SOD)活力为评价指标对新鲜冬虫夏草以及冷冻干燥、20℃阴干和60℃烘干冬虫夏草样品进行比较,发现新鲜冬虫夏草SOD活力最高,冷冻干燥对冬虫夏草SOD活力的影响小于20℃阴干干燥,60℃烘干干燥对冬虫夏草SOD活力破坏最大,结果表明干燥条件对冬虫夏草SOD活性极其重要。     

银杏胚乳淀粉体的积累规律和发生特性

以银杏（Ginkgo biloba L.）核用品种‘七星果’、‘马铃’和‘龙眼’不同发育天数的胚乳为材料,采用透射电镜和扫描电镜技术,对其胚乳细胞内淀粉体的积累规律和发生特性进行研究。结果显示：3种银杏胚乳形态差异显著,‘七星果’呈梭形、‘马铃’呈椭圆形、‘龙眼’呈卵圆形;3种银杏胚乳早期均为嫩绿色,后期为黄色;授粉后65~125 d是胚乳体积快速增长时期。淀粉体的积累规律为：在胚乳组织内,淀粉体由糊粉层-外胚乳-内胚乳逐渐积累;在单个胚乳细胞内,淀粉体由细胞壁边缘向内部逐渐充实。银杏淀粉质体起源于类叶绿体质体,淀粉粒最初在类叶绿体质体的内膜上发生。淀粉体通过出芽、缢缩以及出芽和缢缩同时进行的增殖方式产生新淀粉体,成熟淀粉体形态有圆形、椭圆形和不规则形,属于单粒淀粉。研究结果表明银杏淀粉体在胚乳组织内具有由外向内的空间积累规律,淀粉质体起源于类叶绿体质体并通过出芽、缢缩、出芽和缢缩同时存在的方式增殖。     

舞花姜种子的解剖学和组织化学研究

舞花姜种子表面只许多表皮毛,基部具黄白色的种阜状结构。假种皮着生于种阜结构内缘,基部筒状,中上部指状分裂;假种皮细胞长条形．内含许多细小淀粉粒。种皮由外珠被发育而来．可划分为外种皮、中种皮与内种皮。外种皮由具３—５（６）层表皮细胞的复表在构成,最外层的一些表皮细胞向强突起形成表皮毛。中种皮由下皮层、半透明细胞层、中种皮薄壁细胞层与色素层构成。下皮层由一层下皮细胞构成,细胞内充满结合有单字的红褐色色素;半透明细胞含有与脂类结合的淡黄褐色无定形块状物,中种皮薄壁细胞内无色素或任何颗粒状物;色素层为中种皮最内方的一层,细胞体积大、充满与单宁结合的红褐色色素。内种在由一层体积小、壁局部增厚的砖形薄壁细胞构成,其机械保护作用小。种子在珠孔区分化出珠孔领、孔盖及种阜状结构。珠孔领为异形型,孔盖不具石细胞硬层。种阜状结构以其细胞层数增多、壁增厚并本质化的复表皮加强了珠孔区的机械保护作用。合点区内种皮出现缺口．缺口间充满通常呈多角形的合点区色素细胞,其整体轮廓为长条形。外胚乳细胞壁平直,细胞内充满淀粉粒,部分细胞还含有少量的蛋白质与脂类：近合点外胚乳形成一薄区。内胚乳细胞含有蛋白质、脂类与淀粉．其细胞轮廓清楚,椭圆形或不规?     

花菇的冷冻干燥技术研究

实验研究用板层导热法研究了花菇的冻干特性,获得了新鲜花菇的冻干曲线,分析了花菇冻干过程,测定和比较了新鲜花菇和冻干花菇的营养成份。证实试验机的适应性并确定了花菇的冻干工艺,为工业生产提供了理论依据和参考价值。     

Collapse temperature of solutions important for lyopreservation of living cells at ambient temperature

In this study, the collapse temperature was determined using the freeze‐drying microscopy (FDM) method for a variety of cell culture medium‐based solutions (with 0.05–0.8 M trehalose) that are important for long‐term stabilization of living cells in the dry state at ambient temperature (lyopreservation) by freeze‐drying. Being consistent with what has been reported in the literature, the collapse temperature of binary water‐trehalose solutions was found to be similar to the glass transition temperature (T′_g ～ ?30°C) of the maximally freeze‐concentrated trehalose solution (～80 wt% trehalose) during the freezing step of freeze‐drying, regardless of the initial concentration of trehalose. However, the effect of the initial trehalose concentration on the collapse temperature of the cell culture medium‐based trehalose solutions was identified to be much more significant, particularly when the trehalose concentration is less than 0.2 M (the collapse temperature can be as low as ?65°C). We also determined that cell density from 1 to 10 million cells/mL and ice seeding at high subzero temperatures (?4 and ?7°C) have negligible impact on the solution collapse temperature. However, ice seeding does significantly affect the ice crystal morphology formed during the freezing step and therefore the drying rate. Finally, bulking agents (mannitol) could significantly affect the collapse temperature only when trehalose concentration is low (<0.2 M). However, improving the collapse temperature by using a high concentration of trehalose might be preferred to the addition of bulking agents in the solutions for freeze‐drying of living cells. We further confirmed the applicability of the collapse temperature measured with small‐scale (2 µL) samples using the FDM system to freeze‐drying of large‐scale (1 mL) samples using scanning electron microscopy (SEM) data. Taken together, the results reported in this study should provide useful guidance to the development of optimal freeze‐drying protocols for lyopreservation of living cells at ambient temperature for easy maintenance and convenient wide distribution to end users, which is important to the eventual success of modern cell‐based medicine. Biotechnol. Bioeng. 2010;106: 247–259. © 2010 Wiley Periodicals, Inc.     

FTIR spectroscopy for the detection and evaluation of live attenuated viruses in freeze dried vaccine formulations

This article examines the applicability of Fourier Transform Infrared (FTIR) spectroscopy to detect the applied virus medium volume (i.e., during sample filling), to evaluate the virus state and to distinguish between different vaccine doses in a freeze dried live, attenuated vaccine formulation. Therefore, different formulations were freeze dried after preparing them with different virus medium volumes (i.e., 30, 100, and 400 µl) or after applying different pre‐freeze‐drying sample treatments (resulting in different virus states); i.e., (i) as done for the commercial formulation; (ii) samples without virus medium (placebo); (iii) samples with virus medium but free from antigen; (iv) concentrated samples obtained via a centrifugal filter device; and (v) samples stressed by 96h exposure to room temperature; or by using different doses (placebo, 25‐dose vials, 50‐dose‐vials and 125‐dose vials). Each freeze‐dried product was measured directly after freeze‐drying with FTIR spectroscopy. The collected spectra were analyzed using principal component analysis (PCA) and evaluated at three spectral regions, which might provide information on the coated proteins of freeze dried live, attenuated viruses: (i) 1700–1600 cm^?1 (amide I band), 1600–1500 cm^?1 (amide II band) and 1200–1350 cm^?1 (amide III band). The latter spectral band does not overlap with water signals and is hence not influenced by residual moisture in the samples. It was proven that FTIR could distinguish between the freeze‐dried samples prepared using different virus medium volumes, containing different doses and using different pre‐freeze‐drying sample treatments in the amide III region. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1107–1118, 2015     

Rheological properties of suspensions containing cross-linked starch nanoparticles prepared by spray and vacuum freeze drying methods

The rheological behavior of suspensions containing vacuum freeze dried and spray dried starch nanoparticles was investigated to explore the effect of these two drying methods in producing starch nanoparticles which were synthesized using high pressure homogenization and mini-emulsion cross-linking technique. Suspensions containing 10% (w/w) spray dried and vacuum freeze dried nanoparticles were prepared. The continuous shear viscosity tests, temperature sweep tests, the frequency sweep and creep-recovery tests were carried out, respectively. The suspensions containing vacuum freeze dried nanoparticles showed higher apparent viscosity within shear rate range (0.1-100s(-1)) and temperature range (25-90°C). The suspensions containing vacuum freeze dried nanoparticles were found to have more shear thinning and less thixotropic behavior compared to those containing spray dried nanoparticles. In addition, the suspensions containing vacuum freeze dried particles had stronger elastic structure. However, the suspensions containing spray dried nanoparticles had more stiffness and greater tendency to recover from the deformation.     

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.     

Effect of a Pulsed Electric Field and Osmotic Treatment on Freezing of Potato Tissue

This work discusses the effects of pulsed electric field (PEF) and osmotic pre-treatments on potato tissue structure and on the freezing and freeze-drying behaviour of this tissue. Potato samples (26-mm diameter, 10-mm height) were treated by PEF (400 V/cm) to high level of disintegration (conductivity disintegration index Z was ≈0.95) and were subjected to osmotic treatment in an aqueous solution of NaCl. The samples were either frozen in an air-blast freezer at air temperature of −80 °C and velocity of 2 m/s or freeze-dried at 0 °C and 0.04-mbar pressure. The scanning electron microscope (SEM) images evidenced similarity in structure of the cell walls and area and morphology of starch granules for untreated and PEF-treated potato tissues. However, sequential (PEF + osmotic) pre-treatment of potato tissue resulted in starch granules with rougher surface. The profiles of freezing curves were strongly dependent on pre-treatment. The longest effective freezing time t _f was observed for untreated tissue, and the values of t _f were decreasing in the following sequence: untreated > PEF pre-treated > PEF + osmotically pre-treated. The faster freezing and freeze drying and visually better quality of the dried samples were observed for PEF or sequential PEF + osmotic pre-treatments. The SEM analysis revealed also a noticeable disorder of starch granule surface morphology inside the cells of the freeze-dried potatoes after sequential PEF + osmotic pre-treatment.     

Effects of pericarp alpha-amylase activity on wheat (Triticum aestivum) Hagberg falling number

Pericarp alpha‐amylase activity in wheat has largely been ignored as a potential cause of low Hagberg falling number (HFN) in the UK, because pericarp alpha‐amylase enzymes have been thought too temperature‐sensitive to affect HFN and are usually degraded by harvest. Nevertheless, there are several circumstances where immature grains may be present in harvested grain. Therefore, we tested the hypotheses that pericarp alpha‐amylase enzymes in immature grains could lower HFN, but also that inactivation of the enzymes during drying and storage could ameliorate the effect. The first hypothesis was tested by addition of large amounts of pericarp alpha‐amylase activity from freeze‐dried, defrosted or fresh green grains to high HFN flours of low alpha‐amylase activity, followed by HFN measurement. Enzyme assay and HFN analysis assessed the effect of drying on pericarp alpha‐amylase activity after different storage treatments. Addition of pericarp alpha‐amylase, from all three sources, to high HFN flours caused an exponential decrease in HFN. A drop in HFN from over 470 s to below 250 s resulted from addition of 5–10% by weight of green grains. Between 35–70% of the α‐AMY‐2 activity in immature grains was eliminated by air‐drying at 20°C over 8–10 days, although the residual activity could still lower HFN. Thus, pericarp alpha‐amylase activity may be a cause of low HFN in some UK wheat crops.     

Metabolism of Klebsiella pneumoniae freeze‐dried cultures for the design of BOD bioassays

Aims: The survival rate of freeze‐dried cultures is not enough information for technological applications of micro‐organisms. There could be serious metabolic/structural damage in the survivors, leading to a delay time that can jeopardize the design of a rapid biochemical oxygen demand (BOD) metabolic‐based bioassay. Therefore, we will study the metabolic activity (as ferricyanide reduction activity) and the survival rate (as colony‐forming units, CFU) of different Klebsiella pneumoniae freeze‐dried cultures looking for stable metabolic conditions after 35 days of storage. Method and Results: Here, we tried several simple freeze‐drying processes of Kl. pneumoniae. Electrochemical measurements of ferrocyanide and survival rates obtained with the different freeze‐dried cultures were used to choose the best freeze‐drying process that leads to a rapid metabolic‐based bioassay. Conclusions: The use of milk plus monosodium glutamate was the best choice to obtain a Kl. pneumoniae freeze‐dried culture with metabolic stable conditions after storage at ?20°C without the need of vacuum storage and ready to use after 20 min of rehydration. We also demonstrate that the viability and the metabolic activity are not always directly correlated. Significance and Impact of the Study: This study shows that the use of this Kl. pneumoniae freeze‐dried culture is appropriate for the design of a rapid BOD bioassay.     

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.     

Preservation of Bacteria by Circulating-Gas Freeze Drying

Water-washed Serratia marcescens and Escherichia coli were freeze dried in a circulating-gas system at atmospheric pressure. This convective procedure resulted in a substantially higher survival of organisms than could be obtained by the vacuum method of freeze drying. There was little or no decrease in cell viability during convective drying when the residual moisture content was 15% or higher. Below this level, survival declined with decreasing moisture content. A detailed comparison of the convective and vacuum methods indicated that the advantage gained by freeze drying bacteria in air accrues in the early period of sublimation, at which time cells were found to be sensitive to vacuum drying but insensitive to air drying. An explanation for this difference is proposed, based upon the kinetics of water removal in the two processes. In brief, it is suggested that the convective method permits samples to be dried more uniformly; and regional over-drying, which may be deleterious even if transient, is thus avoided in achieving the optimal level of moisture.