微丝菌(Microthrix parvicella)原位荧光杂交(FISH)定量过程的条件优化

微丝菌(Microthrix parvicella)是世界范围内诱发活性污泥膨胀现象的主要丝状菌之一,它在活性污泥中准确的原位定量解析对污泥膨胀现象及控制策略研究具有非常重要的意义.由于微丝菌自身的特殊生理生化性质(如表面高疏水性及较厚细胞壁)易导致常规荧光原位杂交(FISH)过程中定量结果偏低.本研究针对FISH过程中存在的探针渗透率低、荧光信号偏弱等现象,从活性污泥样品前处理、杂交过程条件等方面对Microthrix parvicella的FISH定量过程进行了优化.结果表明,在前处理使用溶菌酶(浓度为36 000 U·m L~(-1)),探针浓度为4.5 ng·μL~(-1),杂交时间延长至4 h的条件下,Microthrix parvicella的FISH定量结果可从1.12%提高至96.70%,并与定量PCR(q-PCR)结果和EikelboomJenkins法(镜检观察)定量结果更为趋近一致.     

FISH技术定量解析亚硝酸盐氧化菌的条件优化

利用基于亚硝酸盐氧化菌(nitrite-oxidizing bacteria,NOB)16S rRNA序列的荧光探针,优化了针对亚硝酸盐氧化菌的FISH技术实验条件.确定了FISH技术定量检测亚硝酸盐氧化菌的具体方法,并优化了样品预处理的实验条件为热固定2h,4%多聚甲醛固定15min,乙醇脱水5min;杂交过程的实验条件为杂交温度46℃,杂交时间3h,洗脱液中NaCl浓度为60mmol·L-1.运用建立的FISH技术检测了7d内亚硝酸盐氧化菌菌群的丰度变化,充分显示了荧光原位杂交技术(Fluorescence in Situ Hybridization,FISH)的快速定性定量检测优势.     

基于秸秆厌氧干发酵的FISH预处理条件优化

优化了荧光原位杂交技术(FISH)定量测定秸秆厌氧干发酵基质中微生物的样品预处理条件,并在最优条件下做了验证性实验。结果表明:使用无水乙醇作为清洗液,FISH样品的荧光信号强度和丰度比用PBS清洗液(磷酸缓冲盐溶液)时有显著提高;样品预处理过程中加无菌玻璃珠震荡能提高微生物的分散度,从而提高FISH技术对秸秆厌氧发酵基质中微生物定量的准确性;采用离心转速为13 000 r/min时,微生物菌体收集率提高,而且还能保证完好的细胞形态,为后续的FISH杂交奠定基础。     

荧光原位杂交法在活性污泥硝化细菌检测中的应用

采用荧光原位杂交(FISH)法对活性污泥样品中的硝化细菌进行了检测研究。与传统方法相比,FISH法具有快速便捷的特点,可原位检测出活性污泥菌胶团上的硝化细菌的分布情况,结合稀释的方法,可对环境样品中的硝化细菌进行定量分析。此法既可定性,又可对环境中的硝化细菌进行直接计数,为研究硝化细菌类群的空间和数量分布提供有效的检测工具。     

应用FISH法对污水生物处理系统中细菌数量的计数

近年来,FISH(Fluorescence in Situ Hybridization,荧光原位杂交)法作为一种应用分子生物学技术对细胞及细菌等微生物进行定性以及定量分析的研究方法,在国际上得到广泛的应用。本文介绍了应用FISH法对污水处理工艺中细菌数量的计数方法。同时介绍了活性污泥试样制作、FISH法观测试样的制作以及各种试剂的配制、作用以及使用方法,基因探针的选择原则、杂交方法等实际操作方法与过程。     

同步脱氮除磷好氧颗粒污泥形成与反应机制的研究

处理实际生活污水添加不同碳源的A(丙酸钠+乙酸钠)、B(葡萄糖)2个单级SBR中的好氧颗粒污泥(aerobic granularsludge,AGS)均能在常温(18～27℃)和低温(9～13℃)条件下稳定维持同步脱氮除磷的去除效果,利用荧光原位杂交技术(fluorescence in situ hybridization,FISH)、多重荧光染色技术以及扫描电镜(scanning electron microscope,SEM)技术对2个反应器中好氧颗粒污泥的菌群、细菌凋亡和胞外多聚物(extracellular polymeric substances,EPS)的分布以及颗粒污泥的微观形态等进行了研究.FISH结果表明,2个反应器的AGS中,氨氧化菌均位于AGS的最外层,约占总菌的12%,聚磷菌则均位于颗粒的内层,约占总菌的40%,2种颗粒对氮、磷的去除机制没有明显区别,受碳源影响较小.硝化是限速步骤,好氧条件下颗粒内部反硝化除磷的存在加速了吸磷的进行.细菌凋亡荧光染色结果表明,A、B中AGS的活细菌多位于外层,而死细菌均匀分布.EPS多重荧光染色结果表明,2种AGS中的蛋白质和脂类均分布均匀,不受碳源的影响,但蛋白质数量较多,脂类较少;而多糖(α吡喃葡萄糖、α甘露糖和β-D-吡喃葡萄糖)在不同反应器的AGS中呈现出不同的分布规律,表明其分布及含量与外加碳源有着密切的关系,多糖与AGS的形成、稳定维持具有直接的联系.SEM显示B中球菌较多,而A中以杆菌为主,该结果表明不同的碳源会对好氧颗粒污泥的菌种产生影响,并且与最终的去除效果相关,以丙酸钠+乙酸钠为外加碳源更容易维持稳定的同步脱氮除磷去除效果.     

改良剂调控下水稻镉累积和土壤溶解性有机质光谱特征的响应

研究土壤中溶解性有机质(DOM)的化学组成和特征差异对理解改良剂对Cd的稳定机制具有重要意义.土壤改良剂被广泛应用于受污染农田以缓解重金属在农作物中的累积,但改良剂调控下土壤DOM的光谱特征却鲜见报道.采集南方典型Cd污染水稻土为研究对象,通过投加三大类改良剂(有机类、无机类和石灰类,共11种)进行水稻种植盆栽试验,研究了不同改良剂对土壤DOM的影响.利用紫外-可见光谱、三维荧光光谱和平行因子分析法(PARAFAC)对比分析了不同改良剂调控下根际土壤DOM的光谱特征.结果表明,石灰类改良剂提高土壤pH,促进土壤固相中有机质的溶解,从而显著提高了土壤DOM的质量分数.与对照相比,有机类改良剂提高了土壤DOM的相对分子量和新生自生源贡献率,无机类改良剂提高了土壤DOM的芳香性和疏水性组分,石灰类改良剂提高了土壤DOM的显色组分和腐殖化程度.通过PARAFAC分析和OpenFluor数据库验证,解析出了4个荧光组分C1(255/465)、 C2(325/400)、 C3(275/390)和C4(240/460),均为类腐殖质.两种光谱相互印证,表明土壤DOM来源以陆源输入的类腐殖质为主.相关...     

低温域湿地植物根际硝化强度及氨氧化微生物研究

以低温域(0~15 ℃)下黄菖蒲(Iris pseudacorus)、菖蒲(Acorus calamus)和香蒲(Typha orientalis)3种湿地植物为研究对象,分别取其根际土壤测定硝化强度,并采用FISH(荧光原位杂交)技术,考察植物根际AOB(氨氧化细菌)、AOA(氨氧化古菌)的数量变化规律. 结果表明:低温条件下,香蒲根际土壤的硝化强度最高,平均值为1.40 mg/(kg·h),黄菖蒲和菖蒲的平均值均为0.96 mg/(kg·h). 湿地植物根际土壤中的细菌数量(数量级为1010)远高于古菌(数量级为108),其中AOB为优势菌种,3种湿地植物的AOA数量分别约占总古菌数量的46.0%、47.9%和49.7%. 3种湿地植物根际AOB的数量(以湿土计,下同)排序为香蒲(2.57×109 g-1)>黄菖蒲(1.23×109 g-1)≈菖蒲(1.14×109 g-1),AOA的数量(以湿土计)排序为黄菖蒲(2.78×108 g-1)>香蒲(2.57×108 g-1)>菖蒲(1.15×108 g-1). 微生物分布特性和硝化作用效果均表明,不同植物根际氨氧化过程的主要作用微生物具有一定差异,AOA和AOB对于湿地土壤氮转化均具有不可忽视的作用,并与植物本体、土壤硝化过程微环境之间有一定的耦合关系.      

荧光原位杂交法检测反应器中聚磷菌实验条件优化及应用

文章简要介绍了荧光原位杂交（FISH）技术的基本原理和操作步骤。通过正交试验筛检荧光原位杂交（FISH）技术在检测反应器中聚磷菌时的最佳及适合的实验条件。结果显示,样品预处理较优的条件为：样品在固定前应先经过1×PBS清洗两次,37℃热固定3h,乙醇脱水3min;FISH技术检测反应器中聚磷菌的最佳实验条件为：杂交温度46℃,杂交时间2h,清洗缓冲液中NaCl浓度70 mmol/L。FISH技术检测反应器样品中聚磷菌与传统检测方法相比具有快捷、简便、淮确的优点,在研究环境微生物方面有较好的应用前景。     

辽河水体中溶解有机质的光谱初步研究

文章分析了辽河流域水体中溶解有机质的吸收光谱,荧光特性及其主要来源。对比紫外可见吸收和散射2种模型,结果表明DOM对紫外可见光既有吸收也有散射。运用平行因子分析法(parallel factor analysis,PARAFAC),对三维荧光激发-发射矩阵(EEM)进行分析,发现3种荧光团,包括1种类蛋白荧光团和2种类腐殖质荧光团。荧光指数(FIX)为1.60～2.10,初步认为此时段辽河流域DOM的来源为多重来源,生物源比重较大。大多数SUVA_(254)在1.22～2.82L/(mg·m)之间,同文献报导的来自于土壤或者高等植物的DOM相比,值相对较小,DOM的芳构化程度较低,进一步表明DOM来源以生物源为主。     

Micelle dyeing with low liquor ratio for reactive dyes using dialkyl maleic acid ester surfactants

Micelle dyeing with low liquor ratio is novel fabric dyeing method for cleaner production. Preparation and interaction of dye-surfactant micelles using dibutyl maleic acid ester sodium sulfate surfactant were investigated by UV-Vis spectra. Dyeing properties of cellulose fabrics using micelle dyeing with low liquor ratio were discussed by color yields (K/S), color differences (ΔE), exhaustion (E), fixation (F) and reactivity (R) of three reactive dyes. The results show that reactive dye molecules could be solved in surfactant micelles and form stable dye-surfactant micelles. Micelles formed by dibutyl maleic acid ester sodium sulfate had good anti-agglomeration effect on dye solutions. Liquor ratio of micelle dyeing for cellulose fabrics could be reduced from 1:15 to 1:5. Leveling properties of three reactive dyes using micelle dyeing were good. The exhaustion, fixation and reactivity of all three dyes using micelle dyeing were similar with the conventional dyeing technology. Wet rubbing, washing and perspiration fastnesses of dyed samples using micelle dyeing were also good. Micelle dyeing with low liquor ratio can save a large amount of water and energy.     

原料材质、产品类型及生产工序对印染废水产生特性的影响研究

选取嘉兴市82家印染企业,针对棉、毛、化纤3种典型原材料,研究了织物、纱线、纤维产品在前处理、染色和印花等工段的废水产生量和水质,基于产污强度算法估算了各印染工序的废水产污强度.结果表明：棉、毛、化纤织物印染废水总产生强度分别为90.21、206.02、109.66 L·kg^-1织物,漂洗为废水主要产生工序.印染各工段化学需氧量（COD）产生强度普遍较高,特别是棉、化纤织物前处理工段COD产生强度高达136.98和131.67 g·kg^-1织物.印染部分工序氮、磷产污强度高,如棉、毛、化纤织物的洗网废水总氮（TN）产生强度为4.98、1.25、2.21 g·kg^-1织物,棉纤维、毛织物和毛纱线的染色工序总磷（TP）产生强度为0.28、0.18、0.18 g·kg^-1产品,还有棉、化纤的洗网废水TP产生强度为0.30、0.15 g·kg^-1产品.利用产污强度及治理设施削减系数估算得出的印染企业各废水污染物排放量,与污染物在线监测数据相比较,相对误差低于30%;将本研究所得各产污强度与第二次全国污染源普查产污系数比较,除印花工段外,两者相对偏差均低于20%.本研究获得的印染行业各产污节点特征和产污强度,可为印染行业的环境精细化管理和产污节点管控提供数据支撑.     

超声强化下多相催化臭氧体系处理印染尾水

随着我国对水环境质量要求逐步提高,对于纺织印染废水处理提出了更为严格的排放要求.臭氧(O₃)工艺已常用于纺织印染废水深度处理,但存在臭氧利用率低、氧化不彻底等问题.该研究通过自制陶粒催化剂,构建多相催化臭氧氧化体系,以期改善接触条件,提高处理效率,同时在此基础上引入超声波进一步强化体系处理效率.采用扫描电子显微镜(SEM)、X射线衍射(XRD)表征了陶粒催化剂,考察了催化剂投加量、超声波频率对印染尾水的COD_Cr去除率,并通过三维荧光光谱、GC/MS等分析了反应前后尾水中有机物的变化特征.结果表明:①自制陶粒催化剂表面较为粗糙,晶面尺寸在36~50 nm之间,投入后可提高臭氧(O₃)体系对印染尾水的COD_Cr去除率(提高了10%~15%),具有较好的催化效率.②引入超声波可进一步提升体系的氧化效率,当超声频率为200 kHz时,出水ρ(COD_Cr)可达到GB 3838—2002《地表水环境质量标准》Ⅴ类要求.③自制陶粒催化剂与超声波的引入主要促进了芳香性蛋白和溶解性微生物代谢产物的氧化分解,且有效地破坏了长链烷烃、环状烷烃、复杂苯系物等有机物,从而实现印染尾水中ρ(COD_Cr)的进一步降解.研究显示,自制陶粒催化剂协同超声波可提高O₃体系对印染尾水的矿化效率且绿色环保,可为我国水环境敏感区域内纺织印染企业或园区废水深度处理工艺的选择提供参考依据.      

江苏省印染污泥现状分析

对江苏省四大纺织基地进行调研,掌握了17家典型的印染企业污水处理量、污泥脱水及处置方式.同时,取样分析了印染污泥中重金属镉(Cd)、铅(Pb)、总铬(Cr)、铜(Cu)、锌(Zn)和镍(Ni)及含水率、低位热值、灰分和氯离子含量的变化情况.研究表明:采用板框压滤脱水的印染污泥平均含水率可达66.42%,便于焚烧处置;将近1/2的污泥低位热值低于污泥助燃焚烧的限值,需对污泥处理工艺和脱水方式加以改进;污泥处理过程中含氯药剂的加入使得Cl-含量波动较大,焚烧时需加强对二英的监测;17家印染企业污泥中重金属平均含量均低于江苏省城市污泥重金属平均含量,但Zn的含量却远高于评价标准.本文可为江苏省印染污泥焚烧及污染防治工作提供技术支持.     

Fully automated FISH examination of amniotic fluid cells

Objective Fluorescence in situ hybridization (FISH) analysis has become a valuable adjunct in cytogenetics, providing a rapid screen for common chromosome abnormalities that is particularly helpful in prenatal diagnosis. FISH analysis using standard microscopy is expensive and labor intensive, requiring both a high skill level and subjective signal interpretation. A reliable fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. Methods The efficacy of an automated system was compared to standard manual FISH analysis. Two sets of slides were generated from each of 152 amniotic fluid samples. Following hybridization with a standard panel of five chromosome FISH probes, one set of slides was evaluated using manual microscopy. The other set was evaluated using an automated microscopy system. Results A diagnostic outcome was obtained for all 152 samples using manual microscopy and for 146 of 152 (96%) samples using automated microscopy. Three cases of aneuploidy were detected. For those samples for which a diagnostic outcome was determined by both manual and automated microscopy, 100% concordance was observed. All FISH analysis results were confirmed by karyotype. Conclusion These data suggest that an automated microscopy system is capable of providing accurate and rapid enumeration of FISH signals in amniocytes. Copyright © 2007 John Wiley & Sons, Ltd.     

降解PVA共生菌的分离及PVA降解性能

对降解PVA的共生菌B1、B2在PVA降解过程中的作用及其B1+B2混合菌的PVA降解性能进行研究 .结果表明 ,B1+B2混合菌在PVA降解过程中 ,B1为B2提供生长因子 ,B2产生PVA降解酶 ,是降解者 .在摇瓶试验中 ,B1+B2混合菌在PVA浓度为 0.1、0.5、1g/L时 ,PVA降解效率均达到80%以上 .用生产废水进行试验 ,当温度为 30℃ ,处理 12h后 ,COD_Cr去除率达到70%左右 .     

Evaluation of X,Y, 18, and 13/21 alpha satellite DNA probes for interphase cytogenetic analysis of uncultured amniocytes by fluorescence in situ hybridization

The major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. This prospective study evaluated the use of four commercially available centromeric DNA probes (DXZ1, DYZ1, D18Z1, and D13Z1/D21Z1) for direct analysis of uncultured amniocytes. One hundred and sixteen amniotic fluid samples were analysed by FISH and standard cytogenetics. This evaluation demonstrated that FISH with, X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory. In contrast, the 13/21 alpha satellite DNA probe hybridizing both chromosomes 13 and 21 was unreliable for prenatal diagnosis in uncultured amniocytes.     

Using supercritical carbon dioxide as solvent to replace water in polyethylene terephthalate (PET) fabric dyeing procedures

Dyeing fabrics in supercritical carbon dioxide (SCD) instead of water can save energy, reduce water use and prevent pollution. The special pilot plant was designed to test dyeing procedures in supercritical carbon dioxide and the analyses of the results indicate major benefits as compared to water based procedures. The dyeing of polyethylene terephthalate (PET) fabric in supercritical carbon dioxide using special pilot plant was investigated. Disperse dye, C.I. (color index) Disperse Blue 79, was used in this study. After dyeing, rinsing in supercritical carbon dioxide, which removes the excess dyes, was also discussed. At the same dyeing conditions, K/S (color yield) of dyed fabric significantly increased with increasing the dye concentration from 1% o.w.f. (on weight of fabric) to 5% o.w.f. Dyeing temperature and pressure had a strong influence on the color yield. When the temperature rose above 110 °C, the increase in color yield was obvious. At 20 MPa, 120–130 °C, dyeing reached equilibrium after 60 min. The excess dye of the dyed PET fabric was small. The suitable condition in supercritical carbon dioxide for removal of excess dye from the dyed fabric was 70 °C, 20 MPa. The PET fabric dyed in supercritical carbon dioxide had good fastness and physical properties.     

Fluorescence in situ hybridization (FISH) for rapid detection of aneuploidy: experience in 911 prenatal cases†

Fluorescence in situ hybridization (FISH) was performed with probes specific for chromosomes 13, 18, 21, X and Y on 911 of 11123 (8.2%) amniotic fluid samples submitted to the present authors' laboratory for cytogenetic analysis over an 8-year period. Altogether 3516 hybridizations were performed with an interpretable FISH result on all chromosomes requested in 884/911 (97%) of cases. An uninformative FISH result occurred in 44 hybridizations among 27 cases (3%). Of a total of 89 karyotypically proven cases with aneuploidy that might have been detected by FISH, the overall detection rate was 84%. An inconclusive or incomplete FISH result occurred in 9/89 (10%) of these proven aneuploid cases. In the remaining 80 informative proven aneuploid cases, correct detection of aneuploidy was accomplished in 75/80 (94%) of samples. A false-negative result occurred in the remaining 5/80 (6%) of such informative cases. Eighteen cases had karyotypically proven abnormalities that could not have been detected by the targeted FISH. Aside from these 18 cases, FISH allowed correct detection of normal disomy in 785/804 (98%) of such cases. An incomplete FISH result occurred in 18 normal disomic cases. There was a single possible ‘false-positive’ FISH result for chromosome 21. Interphase FISH analysis of uncultured amniotic fluid cells has been shown to be a useful laboratory tool for rapid fetal aneuploidy screening during pregnancy. As with all clinical laboratory diagnostic tests, incomplete or inconclusive results (or even interpretive errors) occur in a small percentage of cases. Nevertheless, FISH results accompanied by other data and by appropriate counseling provide clinicians and patients with valuable information for clinical decision-making surrounding family planning and pregnancy management. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal interphase FISH diagnosis of PLP1 duplication associated with Pelizaeus–Merzbacher disease

A submicroscopic genomic duplication in Xq22.2 that contains the entire proteolipid protein 1 gene (PLP1) is responsible for the majority of Pelizaeus–Merzbacher disease (PMD) patients. We previously developed an interphase FISH assay to screen for PLP1 duplications in PMD patients using peripheral blood and lymphoblastoid cell lines. This assay has been utilized as a clinical diagnostic test in our cytogenetics laboratory. To expand usage of the interphase FISH assay to prenatal diagnosis of PLP1 duplications, we examined three PMD families with PLP1 duplications utilizing aminiotic fluid samples. In two families the FISH assay revealed fetuses with PLP1 duplications, whereas the other fetus showed a normal copy number of PLP1. Haplotype analyses, as well as an additional FISH analysis using postnatal blood samples, confirmed the results of the prenatal analyses. Our study demonstrates utility of the interphase FISH assay in the prenatal diagnosis of PLP1 duplications in PMD. Copyright © 2001 John Wiley & Sons, Ltd.