纳帕海高原湿地浮游病毒丰度及其与可溶性有机碳关系

【背景】浮游病毒在有机碳循环中具有重要作用。【目的】研究纳帕海高原湿地不同季节水样及土样中的浮游病毒和细菌丰度,并分析不同季节浮游病毒丰度与可溶性有机碳的关系。【方法】利用荧光显微镜技术检测不同季节水样中浮游病毒和细菌丰度,利用流式细胞仪技术检测不同季节土壤样品中病毒颗粒和细菌丰度。【结果】雨季所有样品浮游细菌和浮游病毒丰度分别为3.38×10~6/mL和4.38×10~7/mL,旱季所有样品的浮游细菌和浮游病毒丰度分别为8.85×10~5/mL和9.66×10~6/mL,浮游细菌和浮游病毒丰度年平均值分别为2.13×10~6/mL和2.67×10~7/mL。不同季节浮游细菌和浮游病毒的丰度有显著性差异,雨季明显高于旱季(P0.01)。雨季细菌碳产量为8.01μg C/(L·h),旱季为10.30μg C/(L·h)。雨季浮游病毒裂解细菌贡献的可溶性有机碳(Dissolved organic carbon,DOC)占总DOC库的32.38%-76.38%,而旱季为8.23%-47.87%。【结论】浮游病毒在纳帕海高原湿地有机碳循环中具有重要作用。     

纳帕海高原湿地浮游病毒丰度及与环境因子相关性研究

目的 利用EFM技术对纳帕海湿地不同季节7个水样样品中的浮游病毒丰度开展了调查,并对影响浮游病毒丰度的环境因子进行了相关性分析。方法 元素检测,叶绿素a检测,荧光显微镜检测浮游细菌及浮游病毒的丰度。结果 从生态分布上来看旱季和雨季浮游病毒丰度的平均值分别为3.63×106/mL和3.71×107/mL,浮游病毒丰度季节性变化不显著。从对影响浮游病毒丰度的环境因子相关性分析来看,旱季浮游病毒与Chl-a具有弱负相关性（r=0.49,P<0.05）;而雨季浮游病毒丰度与Chl-a具有显著正相关性（r=0.37,P<0.05）,表明雨季纳帕海湿地浮游病毒丰度受Chl-a含量的影响较大。旱季浮游病毒丰度与温度显著负相关,虽然在雨季浮游病毒丰度与温度也呈负相关,但并不显著;在雨季,水体pH变化较明显,而浮游病毒丰度与水体pH呈显著负相关,表明湿地的来水主要在雨季,而离子组成变化大。虽然在雨季和旱季,从多点采样的平均值来看浮游病毒丰度并无明显差异,但从一些局部的采样点来看,雨季的浮游病毒丰度显著高于旱季,说明在纳帕海区域浮游病毒的分布并不平均,因采样点环境的不同会产生明显的区别。结论 对纳帕海高原湿地浮游病毒生态分布的调查,是对该地域生态学研究的补充,使得这一独特地理环境的微生物研究更加系统化。     

浙江近海海域浮游病毒和异养细菌的时空分布

为了解浙江近海海域浮游病毒和异养细菌的时空生态分布, 于2014 年11 月(秋)、2015 年1 月(冬)、2015 年5 月(春)和2015 年7 月(夏)连续4 个季节采集了浙江近海海域表层海水样品, 采用流式细胞仪技术对样品浮游病毒和异养细菌丰度进行了检测, 对其时空分布特征及与环境因子的相关性做了分析。从水平分布来看, 在4 个季节中浮游病毒、异养细菌丰度均为宁波、沈家门、岱山等沿岸海域站位的丰度低, 远陆海域东极和枸杞站位的丰度高。从季节变化来看, 浮游病毒、异养细菌丰度的季节分布特征同为夏>春>秋>冬, 相关性分析结果表明, 春、夏、秋、冬4 个季节, 浮游病毒丰度与异养细菌丰度均为显著正相关。浮游病毒丰度在春、秋、冬季节均与病毒/细菌比值(VBR)显著正相关; 夏、秋季节均与盐度显著正相关; 春、夏季节均与总磷显著负相关; 春季分别于与溶解氧、pH、化学耗氧量(COD)显著正相关。异养细菌在春、秋、冬季节均与VBR 显著正相关; 春、夏季节与溶解氧显著正相关, 冬季与溶解氧显著负相关; 春、夏季节与总磷显著负相关; 秋、冬季节均与温度、盐度显著正相关; 春、冬季节均与COD 显著正相关。     

湖泊浮游细菌与浮游植物的溶原病毒诱导率检测方法优化

溶原性感染作为浮游病毒生态学效应表达方式之一,常以24 h化学诱导得到的溶原性宿主可诱导率来量化。为了获得适合淡水环境的溶原性宿主可诱导率研究方法,本研究采集6个淡水湖泊水样,对每个水样采用直接法、滤膜法、滤膜振荡法和直接振荡法4种诱导培养方法,通过宿主诱导致死率计算6个淡水湖泊浮游细菌和浮游植物溶原诱导率。结果表明:无论是浮游细菌还是浮游植物,直接法获得的诱导结果普遍高于其他方法,表明游离病毒的裂解性感染能够显著影响溶原诱导率的测定结果;直接振荡法获得的6个湖泊溶原诱导率与滤膜法及滤膜振荡法获得结果间均无显著差异,说明实验中振荡培养能有效抑制裂解性病毒对宿主的裂解性感染。本研究通过直接诱导结合振荡培养能够获得与滤膜法一致的诱导效果,但相比于滤膜法,直接振荡法实验过程更加简单,是一种更为高效的测定方法。     

南海北部海域春季浮游细菌和病毒空间分布及其影响因素

应用流式细胞检测技术测定了2014年春季南海北部海域浮游细菌和病毒丰度,研究了其水平和垂直分布特征并对其与环境因子的相关性进行了分析。结果表明,调查海区浮游细菌和病毒丰度分别介于1.28×10~4—9.96×10~5个/m L和4.69×10~5—5.39×10~7个/m L之间,二者丰度随水深的增加基本呈现逐渐下降的趋势,而水平分布趋势不明显。浮游细菌和病毒丰度与温度、p H和溶解氧显著正相关,与水深、盐度、活性磷酸盐、硅酸盐、硝酸盐和总氮则呈显著负相关关系(P0.01),说明该海域细菌和病毒数量受到上述环境因子的共同调控。分析浮游细菌和病毒的相互关系发现,VBR(Virus to bacteria ratio)平均32.23,最小值位于S11站位25m层,最大值则位于S7站位75m层,分别为4.80和264.63,VBR值小于100的站位占到调查站位总数的95.6%。VBR值除与细菌呈显著负相关关系外(P0.01),与其它环境因子相关性不明显(P0.05),说明该海区细菌是病毒的主要寄主,病毒可能主要是以噬菌体的状态存在。     

武汉东湖浮游病毒的丰度及多样性

运用透射电镜及荧光显微技术 ,对富营养化的武汉东湖中浮游病毒丰度及多样性进行了研究。电镜观察和计数结果显示 ,东湖中浮游病毒的丰度达到 10 8个 /mL。荧光显微镜计数结果约是电镜计数结果的 2 72倍 ,差异极显著 (P <0 0 1)。在东湖超富营养区的水样中观察到了多种与各种病毒形态类似的颗粒 ,其中大部分与噬菌体和噬藻体类似 ,具多种形态的尾部和六边形头部。还有一些线状、杆状、子弹形等形态的病毒粒子。研究结果显示东湖水环境中的浮游病毒不仅丰度极高 ,而且种类丰富。提示浮游病毒在东湖水环境和水生态系统中可能扮演重要角色。     

用流式细胞仪检测大黄鱼三倍体

通过对大黄鱼二倍体和三倍体的倍性分析,建立流式细胞仪检测三倍体的方法。大黄鱼受精卵经三倍体诱导处理后,胚胎期进行染色体滴片证实在处理组中有三倍体细胞存在。接着对该组胚胎进行育苗,获得1￣3cm的鱼苗,用流式细胞仪进行检测。以二倍体大黄鱼的肌肉组织或血液细胞DNA含量的峰值道数作为对照,用同样的方法取样处理、上机、测定处理组样本个体细胞的DNA含量的峰值道数。如果处理组个体细胞的DNA含量的峰值道数是二倍体组的1.5±0.1倍,则认为该个体为三倍体。实验结果经冷休克或静水压诱导处理的样本共检测182个,三倍体检出率为12.09%,其中有一组检出率高达55.56%。     

淡水湿地浮游病毒的空间分布

在2006年3～7月间,对湖北省内15个营养水平不同的湿地水体中浮游病毒的分布规律开展了大规模研究.采用荧光显微直接计数法测定了浮游病毒丰度,同时还测量水体透明度、水温、pH、总氮、总磷、COD、叶绿素a浓度及活菌数. 结果显示,浮游病毒丰度不但与活菌数和叶绿素a浓度显著相关(P<0.05),而且也与COD和水温极显著相关(P<0.01),这一结果说明有机物浓度和水温分别是决定淡水湿地中浮游病毒空间和时间分布的重要因素. 进一步的分析还表明在富营养化水体中,浮游病毒与活菌数的相关性(P<0.05)高于与叶绿素a浓度的相关性(P>0.05),说明噬菌体(而不是浮游植物病毒)是富营养化水体中浮游病毒的优势种类.     

长江口及邻近海域夏、冬季浮游病毒丰度分布

应用荧光显微计数法,对2006年夏季和2007年冬季长江口浮游病毒丰度(virus direct count,VDC)进行了检测.结果表明:夏季该海域VDC在2.22×106～9.97×107个·ml-1,高值分布在近海B区(122.5°-123.5°E)的表层海域;冬季VDC在1.99×106～2.66×107个·ml-1,高值分布在近岸A区(120.5°-122.5°E)海域,且由近岸向外海逐渐降低.夏季VDC与浮游细菌生物量、叶绿素含量关系密切,与营养盐相关性不显著(P＞0.05);冬季VDC与浮游细菌、营养盐含量关系密切,与叶绿素a含量相关性不显著(P＞0.05).夏季VDC显著高于冬季(P＜0.01),且两季的分布特征存在不同,此种差异主要与浮游细菌、浮游植物等病毒寄主的分布有关.冬季的营养盐含量也是影响其浮游病毒分布的重要因素.     

太湖浮游细菌与春末浮游藻类群落结构演替的相关分析

为研究浮游细菌与浮游藻类群落演替的相关性,2005年4月至6月在太湖5个观测点采集浮游细菌及浮游藻类样本。分别采用聚合酶链式反应-变性梯度凝胶电泳（PCR—DGGE）和显微观察的方法分析浮游细菌及浮游藻类群落组成。结果表明,春末夏初,浮游细菌与藻类均呈现较高的多样性,浮游细菌DGGE图谱具有43种不同条带,浮游藻类的常见种有29种。浮游细菌群落聚类分析显示,丝藻（Ulothrix sp．）和微囊藻（Microcystis spp．）占优势时,浮游细菌群落基因组成存在明显差异。以藻类种群Shannon—Wiener多样性指数（Hp）,浮游藻类总细胞数（N）以及Microcystis spp．（M）百分含量为变量,典型对应分析（CCA）结果显示浮游细菌与浮游藻类群落结构变化的相关系数为30．9％,表明春末夏初太湖浮游细菌与浮游藻类群落演替具有较高的相关性。     

Fundamentals of flow cytometry

Flow cytomelers arc instruments that arc used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytomcters are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/ particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytomety is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.     

流式细胞术在细菌快速检测中的应用

流式细胞仪(Flow cytometer)是集应用流体学、光学、电子学、生物学、免疫学等多门学科和技术于一体的新型高科技仪器。它的核心技术是流式细胞术(Flow cytometry,FCM),该技术是利用流式细胞仪,使单个细胞或其他微小生物粒子处于快速直线流动状态,且逐个通过光束,从而对单个细胞或微粒进行多参数(数量、大小、核酸含量、细胞活性、特定菌群或物种等)定量分析和分选的检测技术,具有快速、灵敏、精确以及便于操作等突出优点。本文简要介绍流式细胞仪的原理,并论述流式细胞技术在实验室研究、工业生产、临床诊断、环境评估等领域的细菌快速检测应用。     

Multiparametric flow cytometry allows rapid assessment and comparison of lactic acid bacteria viability after freezing and during frozen storage

Freezing is widely used for the long-term preservation of lactic acid bacteria, but often affects their viability and technological properties. Different methods are currently employed to determine bacterial cryotolerance, but they all require several hours or days before achieving results. The aim of this study was to establish the advantages of multiparametric flow cytometry by using two specific fluorescent probes to provide rapid assessment of the viability of four strains of Lactobacillus delbrueckii after freezing and during frozen storage. The relevance of carboxyfluorescein diacetate and propidium iodide to quantify bacterial viability was proven. When bacterial suspensions were simultaneously stained with these two fluorescent probes, three major subpopulations were identified: viable, dead and injured cells. The cryotolerance of four L. delbrueckii strains was evaluated by quantifying the relative percentages of each subpopulation before and after freezing, and throughout one month of storage at -80 degrees C. Results displayed significant differences in the resistance to freezing and frozen storage of the four strains when they were submitted to the same freezing and storage procedures. Whereas resistant strains displayed less than 10% of dead cells after one month of storage, one sensitive strain exhibited more than 50% of dead cells, together with 14% of stressed cells after freezing. Finally, this study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of lactic acid bacteria, and was well correlated with plate count results. Moreover, it made it possible to differentiate strains according to their susceptibility to freezing and frozen storage.     

Mucin-lectin interactions assessed by flow cytometry

The O-glycosylated domains of mucins and mucin-type glycoproteins contain 50-80% of carbohydrate and possess expanded conformations. Herein, we describe a flow cytometry (FCM) method for determining the carbohydrate-binding specificities of lectins to mucin. Biotinylated mucin was immobilized on streptavidin-coated beads, and the binding specificities of the major mucin sugar chains, as determined by GC-MS and MALDI-ToF, were monitored using fluorescein-labeled lectins. The specificities of lectins toward specific biotinylated glycans were determined as controls. The advantage of flexibility, multiparametric data acquisition, speed, sensitivity, and high-throughput capability makes flow cytometry a valuable tool to study diverse interactions between glycans and proteins.     

Evaluation of fresh and frozen-thawed fowl semen by flow cytometry

The aim of this study was to assess the spermatozoal viability, acrosome integrity, mitochondrial activity, and DNA status in the frozen-thawed fowl semen with the use of flow cytometry. The experiment was carried out on 10 sexually adult roosters of meat type line Flex. The semen was collected three times a week by dorso-abdominal massage method, then pooled and subjected to cryopreservation using “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. For cytometric analysis the fresh and frozen-thawed semen was extended with EK diluent to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI). Acrosomal damages were evaluated using phycoerythrin-conjugated lectin PNA from Arachis hypogaea. The percentage of live spermatozoa with functional mitochondria was estimated using Rhodamine 123 (R123) and PI. The spermatozoal DNA integrity was measured by sperm chromatin structure assay (SCSA). The freezing-thawing process decreased the viability, mitochondrial activity in the chicken sperm and increased the percentage of dead cells with ruptured and intact acrosomes, and also the percentage of spermatozoa with fragmented DNA. In conclusion, the present study indicates that fluorescent staining and flow cytometry may be useful for assessment of the changes of fowl semen quality caused by cryopreservation process. This technique allows precise examination of spermatozoa functional characteristic in a very short time.     

Cytokeratin analysis of breast and leukemia tumor cell lines by flow cytometry

Using four human tumor cell lines, MCF-7 and T47-D from breast tumors, MOLT-4 and K-562 from leukemia, flow cytometric DNA analysis of pure and mixed cell population was performed using monoclonal antibodies to cytokeratin to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratins. Surprisingly, on pure or mixed K-562 cells, we found positive labeling with KL1, CK8, and CK18 antibodies (results confirmed by immunocytology). This preliminary study has allowed a DNA analysis on epithelial cells of human breast tumors.     

Real-time flow cytometry analysis of permeability transition in isolated mitochondria

Mitochondrial membrane permeabilization (MMP) is a key event in necrotic and (intrinsic) apoptotic processes. MMP is controlled by a few major rate-limiting events, one of which is opening of the permeability transition pore (PTP). Here we develop a flow cytometry (FC)-based approach to screen and study inducers and blockers of MMP in isolated mitochondria. Fixed-time and real-time FC permits to co-evaluate and order modifications of mitochondrial size, structure and inner membrane (IM) electrochemical potential (DeltaPsi(m)) during MMP. Calcium, a major PTP opener, and alamethicin, a PTP-independent MMP inducer, trigger significant mitochondrial forward scatter (FSC) increase and side scatter (SSC) decrease, correlating with spectrophotometrically detected swelling. FC-based fluorescence detection of the DeltaPsi(m)-sensitive cationic lipophilic dye JC-1 permits to detect DeltaPsi(m) variations induced by PTP openers or specific inducers of inner MMP such as carbonylcyanide m-chlorophenylhydrazone (mClCCP). These simple, highly sensitive and quantitative FC-based methods will be pertinent to evaluate compounds for their ability to control MMP.     

The characterization and isolation of plant heterokaryons by flow cytometry

Summary Plant heterokaryons were identified and isolated from a population of protoplasts following fusion. Endogenous chlorophyll autofluorescence and exogenously supplied fluorochromes were utilized to differentiate between parental and heterokaryon populations. Flow cytometric analysis detected heterokaryons based upon the simultaneous presence of both chlorophyll and an exogenously supplied fluorochrome within one cell. These parameters were utilized to sort large numbers of heterokaryons from the fusion mixture using modified flow instrumentation. Modifications to the instrumentation which allowed this sorting are discussed.