冰冻切片技术在海滨锦葵细胞学研究中的应用

为解决普通石蜡切片观察植物样品繁琐与耗时长的问题,利用改进的蔗糖保护—液氮速冻切片法,进行海滨锦葵不同器官细胞学研究。结果表明:(1)适合于海滨锦葵不同器官的最适蔗糖浓度不同,适于含水量较大的营养器官(根、茎和叶)的最适蔗糖浓度比含水量较小的花器官(柱头裂片、花柱、子房和花药)高。(2)利用最适蔗糖浓度的蔗糖保护-液氮速冻切片方法,获得了完整而清晰的海滨锦葵各器官组织细胞结构的切片;海滨锦葵具有典型的双子叶草本植物营养器官的组织细胞结构特征,花柱为闭合型花柱,子房为多室复子房。表明基于蔗糖保护-液氮速冻的冰冻切片技术在植物细胞学研究中具有广阔的应用前景。     

冰冻切片技术在铁皮石斛显微结构上的应用

为了快速高效的观察兰科植物铁皮石斛的显微结构,利用光镜和改进的蔗糖保护--液氮速冻冰冻切片法,观察铁皮石斛根、茎、叶的显微结构。该技术方法是将铁皮石斛器官经过蔗糖磷酸缓冲液保护液处理后抽真空,再经过液氮速冻、包埋、切片、展片观察、染色以及拍照等步骤,制作出铁皮石斛根、茎和叶较完整的显微结构切片。研究结果表明,适合铁皮石斛根的最适条件为:蔗糖质量体积分数为8%、冷凝温度-25℃、切片厚度20μm;茎的最适条件为:蔗糖质量体积分数为16%、冷凝温度-20℃、切片厚度15μm;叶的最适条件为:蔗糖质量体积分数为4%、冷凝温度-20℃、切片厚度10μm。该研究在兰科植物显微结构观察和组织化学研究中将具有广阔的应用前景。     

植物冰冻切片条件的优化及其与石蜡切片在组织化学应用中的比较

冰冻切片是植物组织学研究中一项重要的实验技术,冷冻温度和冷冻时间是决定切片质量的关键因素。通过比较15种冰冻切片条件,得出植物组织直接冰冻切片较适宜的冷箱温度、冷台温度和冷冻时间。同时,通过对5种植物的不同组织进行组织化学染色,比较了新鲜材料直接冰冻切片与常规石蜡切片在不同化学成分鉴定上的异同及各自的适用范围。结果表明,对于多糖、蛋白质和角质,两种切片方法的鉴定结果比较一致,但对于脂肪只能采用冰冻切片技术。研究结果对植物组织学实验和研究方法的改进具有一定的参考价值。     

一种简单快速植物组织冰冻切片方法

比较不同冷冻方法对植物细胞超微结构的影响,结果表明:直接包埋法处理的植物细胞超微结构保存较好,而液氮冷冻处理的植物细胞内膜系统损伤严重.建立了一种直接包埋冷冻和适当回温相结合的方法,不仅可以制作出植物细胞基本结构保存完整的组织切片,而且避免了使用冰冻保护剂的弊端.其操作程序是:样品固定→冰冻与包埋→适当回温→快速切片→展片→染色.此法制作的切片可进行不同的染色和组织细胞化学测定,具有操作简便,易于推广的特点.     

微小组织冰冻快速制片技术

目的摸索提高微小组织冰冻快速制片质量的方法。方法针对小组织冰冻切片中常常出现组织切完、多粒组织切片不全、冰晶、染色不均匀等问题,采用预先制作冷冻平台、快速冷冻等方法来制作优质的冰冻切片。结果组织铺平可使组织切片完整,快速冷冻可减少冰晶形成,使组织结构更清晰,及时固定和加温染色可使细胞核染色更鲜艳,核、质对比明显。结论组织铺平、速冻、固定、染色等都是提高微小组织制片质量的关键。     

仙客来愈伤组织的超低温保存

为了避免连续继代造成仙客来愈伤组织的变异, 对仙客来愈伤组织进行了超低温冷冻保存研究。以继代后处于对数生长期的愈伤组织为实验材料, 首先在含有不同蔗糖浓度的培养基上预培养不同时间, 转至不同的冰冻保护剂中直接液氮冷冻或-20oC预冷冻2 h, 然后液氮超冷冻保存, 37oC水浴迅速解冻, 并用相应蔗糖浓度的液体培养基洗涤, 以中性红染色测定细胞的存活率, SPSS13.0软件进行统计学分析。结果表明: 预培养基中蔗糖浓度、预培养时间、降温方式、冷冻保护剂等对解冻后材料相对存活率存在不同程度的影响, 筛选出4%蔗糖浓度预培养3 d、9号保护剂、0oC停留30 min后直接冷冻为超低温保存的最佳方案, 通过简单的方法获得了较好的愈伤组织保存效果。     

仙客来愈伤组织的超低温保存

为了避免连续继代造成仙客来愈伤组织的变异, 对仙客来愈伤组织进行了超低温冷冻保存研究。以继代后处于对数生长期的愈伤组织为实验材料, 首先在含有不同蔗糖浓度的培养基上预培养不同时间, 转至不同的冰冻保护剂中直接液氮冷冻或-20oC预冷冻2 h, 然后液氮超冷冻保存, 37oC水浴迅速解冻, 并用相应蔗糖浓度的液体培养基洗涤, 以中性红染色测定细胞的存活率, SPSS13.0软件进行统计学分析。结果表明: 预培养基中蔗糖浓度、预培养时间、降温方式、冷冻保护剂等对解冻后材料相对存活率存在不同程度的影响, 筛选出4%蔗糖浓度预培养3 d、9号保护剂、0oC停留30 min后直接冷冻为超低温保存的最佳方案, 通过简单的方法获得了较好的愈伤组织保存效果。     

两种方法制备的脑组织切片激光共聚焦显微镜成像效果的比较研究

该文比较研究了振动切片法与冰冻切片法制作的脑组织切片的质量及其激光共聚焦显微镜成像的效果。振动切片法通过灌流固定、取小鼠脑、琼脂糖包埋后利用振动切片机连续冠状切片。冰冻切片法通过蔗糖脱水、OCT包埋液氮骤冷后利用冰冻切片机连续冠状切片。制备的脑组织切片进行免疫荧光染色,激光共聚焦显微镜观察成像效果。结果表明,与冰冻切片相比,振动切片法简单快速,且制片质量较高,脑组织切片无冰晶形成,抗原免遭破坏,组织结构较完整,更适用于脑组织样品的激光共聚焦显微镜的成像观察。     

肾移植穿刺标本冰冻切片制作方法及体会

目的介绍一种肾移植穿刺标本冰冻切片的制作方法并分享制片过程中的体会。方法选取2017年武汉大学人民医院手术室提供的肾移植供体穿刺标本10例,分别记录每例冰冻切片的制作方法,切片质量,制片时间。收到标本后,首先在样本托上均匀涂抹一层普通胶水,于冷冻锤下压平成型30s,取出样本托,将标本铺展成一条直线于样品托上之后,在放置好的标本上均匀涂抹一层普通胶水覆盖标本,并用冷冻锤轻轻压平1min,取出进行切片。切片经改良AFA固定液固定,苏木素-伊红(hematoxylin-eosin staining,HE)染色制片后,显微镜下观察。结果切片完整平坦,无褶皱、卷缩、刀痕;染色核质分明,红蓝适度,对比度好;制片时间在13min左右。结论肾移植穿刺标本经两步法包埋再切片,联合改良AFA固定液固定及HE染色,大大缩短了制片时间,提高了制片质量,易于观察,且重复性好,可以推广。     

病理快速冷冻切片染色的质控切片制备

目的探索一种较佳的病理快速冷冻切片染色质控片的制备方法。方法随机抽取60例快速冷冻组织,每例取2块组织,随机分为A、B二组,在-25℃速冻后,A组(标准组)切片放入AAF液(由95%乙醇A、乙酸A、甲醛F按85:5:10的比例配制而成)固定30s后立即进行HE染色,B组切片放入AAF液固定30s后取出室温晾干后放进冷冻切片机内过夜。余下的A组及B组组织进行后续2种不同的操作。C组:得到A组切片后的组织不做任何处理,直接放冷冻切片机机箱内,并将冷冻切片机箱体温度调高至-10℃,第二天上午再次调至-25℃;D组:得到B组切片后的组织于组织上加包埋剂覆盖后放冷冻切片机机箱内,余操作同C组。C、D两组于第二天上午8点再次进行冷冻切片后同B组一同进行HE染色,以十分制评定切片有无裂隙、皱褶或冰晶,染色后组织结构、细胞核着色和核质对比清晰度等6项指标的得分,分别与A组的染色片进行比较。结果 A、B两组切片质量评价指标得分无明显差别,C组切片无裂隙指标得分低于A组,D组无冰晶指标得分低于A组;B、C两组的组织结构清晰、细胞核鲜艳、核质对比清晰指标得分与A组比较无显著差异;D组组织结构清晰、细胞核鲜艳、核质对比清晰指标得分均低于A组。结论新鲜的组织冷冻切片后,切片立即放入AAF液固定30s,随后取出,常温晾干后再次放进冷冻切片机机箱内过夜直至第二天上午放入全自动染色机内进行HE染色,观察染液的染色力,适用于每天快速冷冻切片工作开展前的全自动染色机的染色质控。     

Floral-induced and constitutive defense against florivory: a comparison of chemical traits in 12 herb species

Little is known about how plants protect flowers—their reproductive organs—against florivory. Additionally, the induced floral defense system has been examined in only a few species. We tested the inducibility of putative floral defenses and investigated the relationship between natural florivory and the floral defenses of 12 naturally growing plant species. The relationships between florivory and four chemical traits (nitrogen, phosphorus, total phenolics, and condensed tannins) were investigated in 12 plant species. We also studied whether flowers induce changes in chemical defenses in response to artificial damage in 10 plant species. A higher concentration of floral nitrogen was associated with a decreasing frequency of florivore attacks. Among the four traits of the 10 plant species studied, no trait changed in response to the artificial damage. We suggest that induced defense systems may not be advantageous for flowers, although it is also possible that these species simply do not use induced defense in any of their plant parts.     

Preparation of smears of isolated hepatocytes and the glycogen preservation in them after cryopreservation of the liver tissue

A method is proposed for preparation of smears of isolated hepatocytes from liver samples frozen in liquid nitrogen or dry ice. The thawing of liver samples and the preparation of smears of isolated hepatocytes were produced by a successive maintenance of the samples in a mixture of 0.067 M phosphate buffer and 5% sucrose (pH 8.0) at 20 degrees C, followed by placing in 0.067 M phosphate buffer (pH 7.4) at the same temperature. Then hepatocytes were fixed by methanol. The cytofluorometric analysis has shown that isolation of cells from the frozen and thawing liver tissue using the proposed method does not influence the level of glycogen preservation in the hepatocytes. The proposed method may be used in the clinical practice.     

Isolation of genomic DNA from medicinal plants without liquid nitrogen

Genomic DNA was extracted from eight medicinal plants using the present DNA extraction protocols (CTAB extraction method) with some modifications. Leaves were fixed in different fixing solutions containing absolute alcohol (99.99%), chloroform and EDTA, but without liquid nitrogen. DNA quality and quantity obtained were comparable to those isolated with liquid nitrogen, as the lambda260/lambda280 ratio with liquid nitrogen was in range 1.3-1.7 and with other fixing solutions it was 1.1-1.5. Absolute alcohol showed best results as fixing solution. Good quality of DNA was isolated without using liquid nitrogen from different medicinal plant species. DNA isolated by this method was suitable for various molecular biology applications.     

Cryopreservation studies of Campylobacter

Seven strains of Campylobacter fetus ss. fetus, one of Campylobacter fetus ss. venerealis, and one of Campylobacter jejuni were preserved using a variety of cryopreservation methods. Organisms were frozen to -150 degrees C in a liquid nitrogen refrigerator, in the freezer compartment of a refrigerator (-20 degrees C), and in a mechanical freezer (-65 degrees C). In the latter two cases, viabilities of the organisms were compared after being frozen in Brucella Albimi broth and 10% glycerol. Viabilities were also examined after Campylobacter species were freeze-dried using rapid or slow cooling, using sucrose or skim milk as cryoprotective agents and in bulb-type vials on a manifold or batch vials. Preservation in liquid nitrogen resulted in no loss in viability after 4 years storage. When Campylobacter species were frozen at -20 degrees C, no cells were recovered after 1 month storage in Brucella Albimi broth or seven months in glycerol. A 6.5 log decrease in viability resulted after organisms were frozen at -65 degrees and subsequently stored at the same temperature for 2 years. In this case, glycerol had no protective advantage over Brucella Albimi broth. Postpreservation viability of organisms cooled slowly was two logs higher than those cooled rapidly prior to freeze-drying. When skim milk or sucrose were employed as cryoprotective agents during freeze-drying, equal viabilities resulted. Equivalent viabilities were also demonstrated when the bulb type or "batch" vials were utilized for freeze-drying. No significant differences were observed between the viabilities of the three species when a given cryopreservation method was employed.     

How to Preserve Plant Samples for Carbohydrate Analysis? Test of Suitable Methods Applicable in Remote Areas

Nonstructural carbohydrates (NSC) are frequently studied both by ecologists and plant physiologists because they serve as fundamental carbon and energy sources in plant metabolism. Because of the rapid enzymatic hydrolysis of NSC after harvest, plants are usually placed into liquid nitrogen until subsequent analyses in the laboratory. Nevertheless, when the research is performed in poorly accessible regions such as high mountains, tropical forests or deserts, the use of heavy containers containing vaporizing liquid nitrogen is problematic for logistical reasons. These places are particularly interesting as they harbor plant species with interesting physiological adaptations. In our study we aimed to develop a suitable storage method for plants intended for NSC analyses, which would require minimal equipment. These demands resulted in the idea of using the first step in NSC analyses – extraction in ethanol. Samples were extracted in 50 % and 96 % boiling or non-boiling ethanol and then stored for one month; they were compared with samples immediately frozen in liquid nitrogen. We discovered that for samples containing starch, fructans, soluble sugars and sugar alcohols, the best pretreatment for subsequent storage is extraction in 50 % or 96 % boiling ethanol. For total nonstructural carbohydrates (TNC) assessment, only extracting with 50 % ethanol without boiling gave very good results. Finally, we developed a method that could be used in any remote place without bulky laboratory equipment.     

杂草果实的冰冻切片技术

介绍一种快速、简便的杂草果实解剖技术。以4种木质化程度高且能够漂浮的杂草果实为材料进行冰冻切片, 经过材料固定、前处理、样品冷台固定、冷冻切片、展片、染色和观察并拍照等操作步骤, 可以获得清晰的果实解剖照片, 直观地揭示杂草果实适应漂浮的显微结构。杂草果实冰冻切片方法的优点是不需要对材料进行长时间的软化处理以及繁琐的脱水和包埋等过程。其关键环节是适宜的甘油浓度、冰冻温度和展片步骤。其中, 15%的甘油浓度进行前处理最合适。     

Effects of nutrient contents and defense compounds on herbivory in reproductive organs and leaves of Iris gracilipes

Optimal defense theory (ODT) states that the plant tissue with the highest value to fitness will receive the most protection compared with other plant parts. ODT can be applied to the differences in defenses among floral organs, although most studies have concentrated on the comparison between leaves and flowers. Using Iris gracilipes, we investigated whether ODT is supported when primary and accessory floral organs and leaves are distinguished. We found that anthers and perianths tended to be attacked more severely than ovaries and leaves in the bud and flower stages and that anthers contained the highest nitrogen and phosphorus concentrations. Although ovaries were also found to contain high nitrogen and phosphorus concentrations, they were less severely attacked by herbivores than anthers, perhaps because ovaries contained the highest condensed tannins concentrations among the floral organs except for perianths in the flower stage. Thus, noting that the number of ovules is very much smaller than that of pollen grains, we concluded that ovaries are the most intensively protected, consistent with the prediction of ODT as applied to floral organs. ODT is applicable to the difference in defense allocation among floral organs.     

On the preparation of cryosections for immunocytochemistry

The key preparation steps in the Tokuyasu thawed frozen section technique for immunocytochemistry, namely freezing, sectioning, thawing, and drying, were studied. A spherical tissue culture cell was used as a model system. The frozen hydrated section technique indicated that glutaraldehyde-fixed, 2.1 M sucrose-infused pellets of cells were routinely vitrified by immersion in liquid nitrogen but water was crystallized when lower sucrose concentrations (0.6-1 M) were used. Quantitative mass measurements showed that the fixed cells are freely permeable to sucrose. The frozen hydrated sections were severely compressed but cell profiles regained their circular appearance upon thawing. The average section thickness of our frozen-hydrated sections was 110 nm: this was reduced to 30-50 nm upon thawing, washing, and air-drying. This change was accompanied by severe drying artifacts. By using the methyl cellulose drying technique, this collapse upon air-drying could be significantly reduced, but not completely prevented, giving an average thickness of 70 nm.