海水营养盐测定中水样的保存技术

水样的保存和前处理技术直接关系到分析结果的精确度。针对海水氮、磷营养元素测定中影响水样保存期的容器和保存剂的选择进行了讨论 ,通过实验研究探索其行之有效又方便实用的保存方法以提高实验数据的精确度。     

水样储存期间无机磷酸盐含量的变化

水样的储存直接影响到测定结果的可靠性。水样储存主要包括储存容器的选择、对储存环境的要求以及容器和水样的预处理。关于磷酸盐样品的储存,已有较多的报道。一般认为,水样储存于聚乙烯瓶中,PO_4-P会很快消失、如果快速深度冷冻,则能使样品稳定相当一段时间。有的报道认为,在水样中加入防腐剂氯仿可以减少PO_4-P变化速率;但有的意见认为加氯仿等     

不同保存方法对厚壳贻贝样品蛋白提取效果的比较

为了解厚壳贻贝(Mytilus coruscus)稚贝样品在冷冻保存过程中的蛋白变化情况,作者采用直接冷冻法、添加海水冷冻法、蛋白裂解液冷冻法等分别对保存1周至6个月的厚壳贻贝样品蛋白提取的不同效果进行了比较。SDS-PAGE胶结果显示,3种冷冻保存方法均出现4个有规律条带(a、b、c、d),其相对分子量依次约为55、46、38和26kDa。SDS-PAGE蛋白条带灰度值的检测结果表明,1个月内蛋白裂解液法保存的样品最佳;直接冷冻法和添加海水冷冻法的研究发现保存过程中的蛋白均出现不同程度的降解现象,因而可能不适宜用于该种稚贝样品的保存。     

三门湾夏季浮游植物现存量和初级生产力

该海区的生物、化学、水文等项目进行调查。用HQM-1型有机玻璃采水器采集水样,大面观测站仅采集表层(0～1m)水样,用于测定叶绿素a质量浓度和光合作用速率。在N2和N11两个连续观测站,每隔3h采集表层和5m层的水样,用于分析叶绿素a质量浓度及营养盐浓度。同时,用浅 型浮游生物网由近底层向表层垂直拖网采集浮游植物样品,所采样品被装入容积为600cm3的塑料瓶中,并加入20cm3的福尔马林溶液固定保存,供对浮游植物细胞计数和种属鉴定之用。营养盐样品用经酸预清洗过的孔径为0.45μm的醋酸纤维膜过滤,滤液用饱和HgCl2溶液固定后,低温避光保存。图…     

用于单-藻细胞PCR扩增反应的样品保存方法比较

用室内培养的塔玛亚历山大藻（Alezandrium tamarense ATHK藻株）作为实验对象,分别选择了5％福尔马林固定后常温保存、95％乙醇固定后常温保存、-20℃冷冻保存、鲁哥氏液（Lugol’s solution）固定后常温保存等方法,在5,15,30,60d后对保存的样品进行单细胞PCR反应,并与新鲜样品进行比较。实验结果表明,用95％乙醇保存、-20℃冷冻保存、鲁哥氏液保存的样品在60d后仍可扩增出目标条带,与新鲜样品没有差别,但是样品中的藻细胞形态有一些改变;而以5％福尔马林固定保存的样品只能在5d后扩增出目标条带,但藻细胞形态比较完好。因此,对用于单细胞PCR实验的微藻样品,短期内（不多于5d）可以采用福尔马林保存,而需要长期保存的样品,应当采用乙醇或鲁哥氏液固定,或者采用-20℃冷冻保存的方法。     

用于单一藻细胞PCR扩增反应的样品保存方法比较

用室内培养的塔玛亚历山大藻(Alexandrium tamarense ATHK藻株)作为实验对象,分别选择了5%福尔马林固定后常温保存、95%乙醇固定后常温保存、-20℃冷冻保存、鲁哥氏液(Lugols solution)固定后常温保存等方法,在5,15,30,60 d后对保存的样品进行单细胞PCR反应,并与新鲜样品进行比较。实验结果表明,用95%乙醇保存、-20℃冷冻保存、鲁哥氏液保存的样品在60 d后仍可扩增出目标条带,与新鲜样品没有差别,但是样品中的藻细胞形态有一些改变;而以5%福尔马林固定保存的样品只能在5 d后扩增出目标条带,但藻细胞形态比较完好。因此,对用于单细胞PCR实验的微藻样品,短期内(不多于5 d)可以采用福尔马林保存,而需要长期保存的样品,应当采用乙醇或鲁哥氏液固定,或者采用-20℃冷冻保存的方法。     

长江口内外表层沉积物中营养元素的分布特征研究

通过对长江口内外9个表层沉积物中不同形态碳、氮、磷含量的测定,得到了长江口内外表层沉积物中营养元素的分布特征,探讨了影响其分布的主要因素。长江口内外表层沉积物中的营养元素在河流段的分布波动较大,在口门附近海域分布较平缓。在三种营养元素中,IC、ON和IP分别为C、N、P的优势形态。在影响表层沉积物中营养元素分布的因素中,沉积物的粒度和上覆水体的盐度在其中起着重要的作用,同时还受到近岸排污和海洋生物生产的影响。     

海水中总氮和总磷的同时测定

早期总氮和总磷的过硫酸钾法氧化分析分别是在碱性和酸性介质中进行［１,２］,样品分别保存,然后分别氧化测定,工作较繁琐。１９７７年Ｋｏｒｏｌｅｆｆ用过硫酸钾氧化法同时氧化水体中的总氮和总磷,使样品的保存和氧化分析工作简化。１９８１年Ｖａｌｄｅｒｒａｍａ对该方法测定天然海水中的总氮和总磷做了进一步研究,总氮和总磷同时氧化后,水样中的硝酸盐和磷酸盐用手工分别进行分析。Ｗａｌｓｈ１９８９年和Ｋａｒｌ等１９９３年曾采用ＵＶ光氧化法对海水中总溶解氮和总溶解磷进行同时氧化后,用自动分析对氧化后的无机氮和磷进行…     

水样中氮磷营养盐的短期保存技术研究

用8种保存方法对淡水和海水水样中四个氮磷营养盐（NH3-N、NO3—N、NO3-N、PO4—P）参数及总氮的稳定性进行了7d保存效果的比较研究。结果表明,淡水与海水氮磷营养盐的最佳保存方法与效果各不相同,但淡水和海水的总氮值在各种方法保存下均十分恒定。综合考虑有效性及可操作性后提出了5d内的短期保存技术：（1）海水水样用5‰氯仿4℃条件下保存,供NH3-N、NO2—N和N03-N三个营养盐的测定,用5‰甲醛4℃条件下保存供PO4-P测定;（2）淡水用5‰氯仿4℃条件下保存,供NH3-N测定,5‰甲醛4℃条件保存供NO2-N、NO3-N和PO4-P测定。     

海水中总溶解糖分析水样的保存方法

海水总溶解糖的数据是海洋初级生产力测定中的一项重要参数。该组份已有多种化学测定方法,但尚缺乏船用自动快速分析方法,待分析水样往往须要送回实验室测定。到目前为止,只有个别零星的报道涉及此问题,例如Walsh曾指出采样后3小时内水样的总溶解糖浓度基本不变,Handa在印度洋和大西洋调查中采用深冷法保存过滤水样,而Strickland和Parsons以及格拉斯霍夫则分别推荐以氯仿和氯化汞作为葡萄糖标准溶液的保护剂,     

Effects of preservatives on stable isotope analyses of four marine species

The aim of the present study is to quantify the effect of formalin–ethanol preservation on the carbon and nitrogen stable isotope signatures of four taxonomical groups of marine species (Himanthalia elongata, Anemonia sulcata, Mytilus galloprovincialis and Patella vulgata). To examine temporal changes in the effects of preservation and to determine if preservation induced predictable shifts in δ¹³C and δ¹⁵N signatures, repeated analyses were carried out after 6, 12 and 24 months of preservation. Data from our study showed highly variable effects of the formalin–ethanol preservation on carbon and nitrogen isotope signatures between species. The use of a general correction factor was not possible, or else it should be species-specific. Differences in nitrogen isotopic values between preserved and unpreserved samples were minor compared to the assumed enrichment between trophic levels. The combined use of data from preserved and unpreserved samples could lead to biases in the estimation of the trophic level of organisms. Changes that preservatives caused in carbon values were variable between species and not always small enough to be ignored. So the use of data from preserved samples could change the interpretation of the mixing models used to determine the importance of multiple sources of carbon. In order to elucidate the effects that preservatives have in other species, further studies will be necessary.     

Examination of core samples from the Mount Elbert Gas Hydrate Stratigraphic Test Well, Alaska North Slope: Effects of retrieval and preservation

Collecting and preserving undamaged core samples containing gas hydrates from depth is difficult because of the pressure and temperature changes encountered upon retrieval. Hydrate-bearing core samples were collected at the BPXA-DOE-USGS Mount Elbert Gas Hydrate Stratigraphic Test Well in February 2007. Coring was performed while using a custom oil-based drilling mud, and the cores were retrieved by a wireline. The samples were characterized and subsampled at the surface under ambient winter arctic conditions. Samples thought to be hydrate bearing were preserved either by immersion in liquid nitrogen (LN), or by storage under methane pressure at ambient arctic conditions, and later depressurized and immersed in LN. Eleven core samples from hydrate-bearing zones were scanned using x-ray computed tomography to examine core structure and homogeneity. Features observed include radial fractures, spalling-type fractures, and reduced density near the periphery. These features were induced during sample collection, handling, and preservation. Isotopic analysis of the methane from hydrate in an initially LN-preserved core and a pressure-preserved core indicate that secondary hydrate formation occurred throughout the pressurized core, whereas none occurred in the LN-preserved core, however no hydrate was found near the periphery of the LN-preserved core. To replicate some aspects of the preservation methods, natural and laboratory-made saturated porous media samples were frozen in a variety of ways, with radial fractures observed in some LN-frozen sands, and needle-like ice crystals forming in slowly frozen clay-rich sediments. Suggestions for hydrate-bearing core preservation are presented.     

Effects of long-term sample preservation on flow cytometric analysis of natural populations of pico-and nanophytoplankton

The effects of long-term preservation on flow cytometric parameters of natural oceanic populations of pico-and nanophytoplankton have been examined. Populations collected at oligotrophic subtropical and subarctic locations in the North Pacific were fixed with glutaraldehyde and frozen in liquid nitrogen, according to Vaulot et al. (1989). During six months’ storage, chlorophyll red fluorescence declined in all the groups examined, while forward light scatter was enhanced in Synechococcus and Prochlorococcus, and weakened in nanoeucaryotes. Cell loss was not significant except for Synechococcus. Caution is required when analyzing flow cytometric data of samples stored for more than a month.     

Flow Cytometric Analysis of Coastal Lagoon Bacterioplankton and Picophytoplankton: Fixation and Storage Effects

The effect of different fixation and storage protocols on the flow cytometric (FCM) simultaneous analysis of bacterioplankton and phytoplankton in coastal seawater samples (Mediterranean coastal lagoons) was investigated. FCM measurements (cell number, fluorescence and scatter characteristics) were obtained through DAPI staining. Three fixatives [glutaraldehyde (GA), formaldehyde (FA) and paraformaldehyde (PFA)] and two storage (3 months duration) methods (5 °C and −196 °C) were tested. Two dominant populations were detected in studied samples: bacteria and eukaryotic picophytoplankton. Adding fixatives (2% final concentration) appears necessary to obtain FCM exhaustive counts of all the bacteria and phytoplanktonic cells. This was related to the permeation effect of fixatives which allowed a better DAPI staining of the cells. Maximum fluorescence, i.e. optimal staining of the cells was obtained with FA or PFA, and significant lower fluorescences with GA. Fixed samples stored at 5 °C induced rapid cell loss. Only storage in liquid nitrogen of samples fixed with FA or PFA, allows mid-term (≥4 months) preservation of bacteria or picophytoplankton cell numbers, and limited evolution of DAPI-induced fluorescence and scatter characteristics.     

不同年度收存的褶皱臂尾轮虫休眠卵的孵化及滞育胚胎“激活”效果的研究

比较了 1 985— 1 998年间由同一褶皱臂尾轮虫 (Brachionusplicatilis)种群在实验室内历年产生并收存的休眠卵的孵化效果 ,并初步探讨了胚胎“激活”处理对孵化的影响。结果表明 ,休眠卵的收存年度或时间对其在一定孵育期内的总孵化率 (THR)和日孵化率 (DHR)有极显著影响 (x2 检验 ,p<0 .0 0 1 ) ,但THR和DHR与储存时间间均无显著的相关性 ,而是呈极大波动。同时发现“激活”作用具有促使孵化高峰提前到来、增强孵化同步性的双重功效 ,从而可明显缩短孵育时间 ,提高孵化速率。但“激活”前后多数卵样的总孵化率无显著变化 (p >0 .0 5 )。详细讨论了可能导致孵化率波动的多种因素 ,例如遗传变异、培养历史、储存及孵化的环境条件等。     

纤毛虫单细胞基因组DNA微量提取方法的比较及优化

在纤毛虫的分子生物学研究中,单细胞基因组DNA的微量提取是后续基因分析的关键。本研究以海洋纤毛虫红色伪角毛虫为材料,采用直接取完整虫体、裂解法以及3种改良后的试剂盒微量提取DNA方法,分别提取了新鲜样品与4种不同方法保存样品的基因组DNA,比较了9种组合在PCR产物质量、提取所需时间、成本及便捷性方面的优劣,并进一步比较、讨论和探索了4种样品保存方法和5种DNA微量提取方法在纤毛虫单细胞基因组PCR扩增中的可行性。本工作旨在为后续的纤毛虫分子生物学研究提供可资借鉴的技术路线和方法。     

几种常见保存方法对浮游动物生物量的影响

5%的甲醛,-20°C冰冻和液氮是常见的浮游动物保存方法。本文以中华哲水蚤(Calanus sinicus)和强壮箭虫(Sagitta crassa)为研究对象,以湿重、干重和碳、氮含量为生物量指标,研究了上述几种保存方法对浮游动物生物量的影响。研究结果显示,对于中华哲水蚤,湿重、干重、碳和氮含量,在用甲醛保存2个月后,分别下降为鲜重的81.03%、14.20%、7.02%和1.13%;冰冻保存2个月后,分别下降为鲜重的66.56%、22.34%、10.30%和2.14%;液氮保存2个月后,分别下降为鲜重的91.85%、26.65%、11.25%和2.41%。对于强壮箭虫,湿重、干重、碳和氮含量,在用甲醛保存2个月后,分别变为鲜重的104%、9%、3.49%和0.69%;冰冻保存2个月后,分别下降为鲜重的73.15%、12.39%、4.82%和1.02%;液氮保存2个月后,分别下降为鲜重的70.72%、11.22%、5.23%和1.13%。在保存初期浮游动物的生物量变化较大,后趋于稳定。甲醛保存方法对浮游动物生物量的影响最大,其次是液氮,冰冻的影响最小。中华哲水蚤的生物量受各种保存方法的影响要小于强壮箭虫。     

Natural abundances of 15N as a source indicator for near-shore marine sedimentary and dissolved nitrogen

Because organic matter originating in the euphotic zone of the ocean may have a distinctive nitrogen isotope composition (¹⁵N/¹⁴N), as compared to organic matter originating in terrestrial soils, it may be used to evaluate the relative nitrogen contribution to marine and estuarine sediment. The nitrogen isotope ratios of 42 sediment samples of total nitrogen and 38 dissolved pore-water ammonium samples from Santa Barbara Basin sediment cores were measured. The range of δ¹⁵N values for total nitrogen was $\text{+2.89 – +9.4‰}$ with a mean of $\text{+6.8‰}$ and for pore water ammonium, $\text{+8.2 – +12.4‰}$ with a mean of 10.2‰.The results suggest that the dissolved ammonium in the pore water is produced from bacterial degradation of marine organic matter. The range of δ¹⁵N values for total nitrogen in the sediment is interpreted as resulting from an admixture of nitrogen derived from marine ($\text{+10‰}$) and terrestrial (+2‰ marines. The marine component of this mixture, composed principally of calcium carbonate with smaller amounts of opal and organic matter, contains ～ 1.0% nitrogen. The terrestrial component, which comprises over 80% of the sediment, contains ～ 0.1% organically bound nitrogen and accounts for > 25% of the total nitrogen in Santa Barbara Basin sediment.