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流式细胞术检测单增李斯特菌与酿酒酵母黄生权1，付萌1，唐青涛1，黄韵2，胡双芳2，余以刚2，肖性龙2（1.无限极（中国）有限公司，广东江门 529156）（2.华南理工大学轻工与食品学院，广东广州 510640）摘要：为探讨流式细胞术对单增李斯特菌和酿酒酵母的活菌与热灭活菌的检出效果，本文采用荧光染色试剂SYTO-9和碘化乙锭（PI）对单增李斯特菌和酿酒酵母的活菌与热灭活菌的细胞悬液进行染色，采用流式细胞仪同时测量红色荧光与绿色荧光从而得出细胞悬液中的细菌和酵母的含量。
结果表明经核酸荧光染料染色后，再结合流式细胞术对细菌与酵母菌进行检测，步骤简单、耗时短。
该法不仅简化了测量步骤且分辨率高，对单增李斯特菌和酿酒酵母均具有良好的检出结果，能分辨同一体系中同一菌种的活细胞与热灭活细胞和同一体系中的细菌与酵母活细胞；该法检出限低，将单增李斯特菌稀释后，最低检出限可达1.2×104 cells/mL，将酿酒酵母稀释后，最低检出限可达6×103 cells/mL，因此能大大缩短增菌时间或者避免繁复的增菌步骤。
关键词：流式细胞术；荧光；李斯特菌；酿酒酵母文章篇号：1673-9078(2014)3-195-200Detection of Saccharomyces cerevisiae and Listeria monoeytogenes byFlow CytometryHUANG Sheng-quan1, FU Meng1, TANG Qing-tao1, HUANG Yun2, HU Shuang-fang2, YU Yi-gang2,XIAO Xing-long 2(1.Infinitus (China) Co. Ltd., Jiangmen 529156, China)(2.College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China)Abstract: The viable and heat-treated microorganisms such as Listeria monocytogenes and Saccharomyces cerevisiae were investigated in this study. They were stained by fluorescent staining reagents SYTO-9 and Propidium Iodide (PI) and then the red and green fluorescence signals were detected using flow cytometry to get the concentration of cells in samples. The results showed that flow cytometry was able to detect bacteria and yeast after the nucleic acid fluorescent staining. This method simplified the procedure for L. monocytogenes and S. cerevisiae detection, shortened the time of detection procedures, and also distinguished viable with heat-treated micrograms and viable bacteria with yeast in the same system. This method was capable of detecting as few as 1.2×104 cells/mL L. monocytogenes and 6 × 103 cells/mL S. cerevisiae. This improvement might shorten the time consuming of culture enrichment or simplify the process.Key words:flow cytometry; fluorescent; Listeria; Saccharomyces cerevisiae由于保健品原料中污染的微生物往往含量很低，或在加工过程中受到损伤活性低，给快速检测带来许多困难，由此造成的漏检给保健品生产企业带来重大的经济损失与资源浪费[1]。
ASTM美国材料标准中文版ASTM A488/A488-2007 钢铸件焊接工艺和人员资格评定的标准实施规程( Standard Practice for Steel Castings, Welding, Qualifications of Procedures and Personnel )ASTMA 802/A 802M-1995(R2006重新审批) 视觉检测铸钢表面验收标准规程 (STANDARPDR ACTICEFOR STEEL CASTINGS, SURFACE ACCEPTANCE STANDARDS, VISUAL EXAM)INATIONASTM B108-2006 铝合金永久型铸件标准规范( STANDARD SPECIFICATION FOR ALUMINUM-ALLOY PERMANENT MOLD CAST)INGSASTM B179-2006 铸造用铝合金原锭及熔融锭在各铸造过程的标准技术规范( STANDARD SPECIFICATION FOR ALUMINUM ALLOYS IN INGOT AND MOLTEN FORMS FOR CASTINGS FROM ALL CASTING PROCESS)ESASTM B26/B26M-2005铝合金砂铸件标准规范( STANDARD SPECIFICATION FOR ALUMINUM-ALLOY SAND CASTING)SASTM D256-2006 测定塑料抗悬臂梁摆锤冲击性的标准试验方法( STANDARD TEST METHODS FOR DETERMINING THE IZOD PENDULUM IMPACT RESISTANCE OF PL)ASTICSASTM D2794-1993(R2004) 有机涂层抗快速形变(冲击)作用的标准试验方法( STANDARD TEST METHOFDO RR ESISTANCOE FO RGANI C OATINGTSO T HEE FFECTSO FR APIDD EFORMATIO(INM PACT) )ASTM D3359-2008 胶带试验用测定粘合性的标准试验方法( STANDARD TEST METHODS FOR MEASURING ADHESION BY TAPE T)ESTASTM D3363-2005 铅笔试验法测定涂膜硬度的标准试验方法( STANDARD TEST METHOD FOR FILM HARDNESS BY PENCIL TE)STASTMD 4060-2007 用泰伯尔磨蚀机测定有机涂层耐磨性的标准试验方法 ( STANDARTDE STM ETHODFOR ABRASION RESISTANCE OF ORGANIC COATINGS BY THE TABER A)BRASERASTM D4674-2002A暴露在室内办公室环境下的塑料颜色稳定性加速试验的标准实施规范(STANDARTDE STM ETHOFDO RA CCELERATETDE STINGF ORC OLORS TABILITY OF PLASTICSE XPOSED TO INDOOR OFFICE ENVIRONME)NTSASTM D4752-2003 用溶剂擦试法测定硅酸乙酯( 无机)富锌底漆耐甲乙酮的标准试验方法(STANDARD TEST METHOD FOR MEASURING MEK RESISTANCE OF ETHYL SILICATE (INORGANIC) ZINC-RICH PRIMERS BY SOLVENT R)UBASTMD 4828-1994E1(R2003) 有机覆层实际可洗性的标准试验方法( STANDARTDE STM ETHODFSO R PRACTICAL WASHABILITY OF ORGANIC COAT)INGSASTMD 638-2003 塑料拉伸性能标准测试方法 (STANDARTDE STM ETHOFDO RT ENSILEP ROPERTIESOF PLASTICS)ASTM E1316-2007 无损检测标准术语( STANDARD TERMINOLOGY FOR NONDESTRUCTIVE EXAMINATION)SASTM E1444-2005 磁粉检测标准规程( STANDARD PRACTICE FOR MAGNETIC PARTICLE TE)STING ASTM E155-2005 铝、镁铸件检验用标准参考射线底片( STANDARD REFERENCE RADIOGRAPHS FOR INSPECTION OF ALUMINUM AND MAGNESIUM CAS)TINGSASTME 165-2002 液体渗透剂检查标准测试方法( STANDARTDE STM ETHOFDO RL IQUID PENETRANT EXAMINATIO)NASTM E165-2002 液体渗透检查的标准试验方法王倩译( STANDARD TEST METHOD FOR LIQUID PENETRANT EXAMINAT)IONASTME 192-2004 航天设备蜡模钢铸件的参考放射线照相( STANDARRDE FERENCREA DIOGRAPHOSF INVESTMENT STEEL CASTINGS FOR AEROSPACE APPLIC)ATIONSASTM E242-2001(2005年重新批准) 在某些参数变化时射线图像外观用标准参考射线底片(STANDARD REFERENCE RADIOGRAPHS FOR APPEARANCES OF RADIOGRAPHIC IMAGES AS CER PARAMETERS ARE CHAN)GEDASTM E385-2007 使用14 兆电子伏特的中子活化和直接计数技术测定含氧量的试验方法(STANDARD TEST METHOD FOR OXYGEN CONTENT USING A 14-MEV NEUTRON ACTIVATION AND DIRECT-COUNTING TECHNIQ)UEASTM E426-1998(2007重新审批) 无缝及焊接管产品、沃斯田不锈钢及类似合金的电磁(涡电流)检测操作规程( Standard Practice for Electromagnetic (Eddy-Current) Examination of Seamless and Welded Tubular Products, Austenitic Stainless Steel and Similar Alloys )ASTM E446-98(2004 年重新批准) 用于厚度在2in(51mm)以下钢铸件的标准参考射线底片(STANDARD REFERENCE RADIOGRAPHS FOR STEEL CASTINGS UP TO 2 IN. 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F38-2000版垫片材料的蠕变松弛的标准试验方法ASTM F112-2000版包复垫片密封性能的标准试验方法ASTM F146-2004版垫片材料耐液体标准试验方法ASTM F1311-1990(R2001)版大口径组装式碳钢法兰标准规范ASTM G1-2003版腐蚀试样的制备、清洁处理和评定用标准实施规范ASTM G36-73(R1981) 参考资料标准实用规程：在沸的氯化镁溶液中进行的应力腐蚀裂纹试验ASTM G46-1976(R1986) 参考资料标准实用规程：麻点腐蚀的检验和评定ASTMG 48-2003 版使用三氯化铁溶液做不锈钢及其合金的耐麻点腐蚀和抗裂口腐蚀性试验的标准方法ASTM标准中译本丛书(一) 碳钢、铸铁、不锈钢及合金钢材料标准规范(含18个标准)1.ASTM A105/A105M-2002 版管道部件用碳钢锻件2.ASTM A126-1995(R2001)版阀门、法兰和管道附件用灰铁铸件3.ASTM A181/A181M-2001 版通用管路用碳钢锻件标准规范4.ASTM A193/A193M-2001 版高温用合金钢和不锈钢螺栓材料5.ASTM A194/A194M-2001a版高温、高压或高温高压螺栓用碳钢及合金钢螺母标准规范6.ASTM A216/A216M-2001a版高温用可熔焊碳钢铸件标准规范7.ASTM A217/A217M-2002 版高温承压件用马氏体不锈钢和合金钢铸件标准规范8.ASTM A276-2002a 版不锈钢棒材和型材9.ASTM A278/A278M-2001 版高温不超过650°F(350℃)的承压部件用灰铸铁件10.ASTM A320/A320M-2002 版低温用合金钢栓接材料11.ASTM A350/A350M-2002 版要求冲击韧性试验的管件用碳钢及低合金钢锻件标准规范12.ASTM A351/A351M-2000 版承压件用奥氏体、奥氏体- 铁素体(双相)钢铸件规范13.ASTM A352/A352M-1993(R1998)版低温承压件用铁素体和马氏体钢铸件标准规范14.ASTM A395/A395M-1999 版高温用铁素体球墨铸铁承压铸件15.ASTM A439-1983(R1999) 版奥氏体球墨铸铁件16.ASTM A536-1984(R1999) 版球墨铸铁件17.ASTM A694/A694M-2000 版高温输送用管法兰、管件、阀门及零件用碳钢和合金钢锻件标准规范18.ASTM A965/A965M-2002 版高温高压部件用奥氏体钢锻件ASTM标准中译本丛书(二) 法兰、管件、阀门及部件(含9 个标准)1.ASTM A182/A182M-2002版高温用锻制或轧制合金钢法兰、锻制管件、阀门和部件2.ASTM A961-2002 版管道用钢制法兰、锻制管件、阀门和零件的通用要求标准规范3.ASTMB 462-2002 版高温耐腐蚀用锻制或轧制的UNSN O6030、UNSN O6022、UNSN O6200、UNS NO8020、UNS NO802、4 UNS NO802、6 UNS NO836、7 UNS NO1027、6 UNS N10665、UNS N10675和UNS R20033合金管法兰、锻制管件、阀门和零件标准规范4.ASTM F885-1984(R2002)版公称管径为NPS 1/4～2的青铜截止阀外形尺寸标准规范5.ASTM F992-1986(R2001) 版阀门铭牌标准规范6.ASTM F993-1986(R2001) 版阀门锁紧装置标准规范7.ASTM F1030-1986(R1998) 版阀门操作装置的选择准则8.ASTM F1098-1987(R1998) 版公称管径有NPS2～24 的蝶阀外形尺寸标准规范9.ASTM F1565-2000 版蒸汽用减压阀规范。
胚胎干细胞试验预测胚胎毒性替代方法进展程树军;秦瑶;喻欢;陈彧【摘要】小鼠胚胎干细胞试验(EST)是通过ECVAM正式验证的胚胎和发育毒性的动物试验替代方法.在新技术的推动下,多项优化的EST方法通过基因转染、利用人来源的干细胞、增加检测通量、组合检测终点、量化检测指标等方式,提高了EST 方法预测胚胎毒性的科学相关性和检测效率.在整合测试策略的原则之下,经过大规模化学物质的前验证和标准化试验,新的EST方法可单独或作为组合试验的一部分可以应用于先导药物的研发和化学品的胚胎毒性预测及机制研究.【期刊名称】《中国比较医学杂志》【年(卷),期】2016(026)001【总页数】5页(P81-85)【关键词】胚胎干细胞试验;胚胎和发育毒性;替代方法;预测【作者】程树军;秦瑶;喻欢;陈彧【作者单位】广东出入境检验检疫技术中心,广州510623;广东省动植物与食品进出口技术措施研究重点实验室,广州510623;广州市华代生物科技有限公司,广州510623;中山大学公共卫生学院,广州510080;广东出入境检验检疫技术中心,广州510623;广州市华代生物科技有限公司,广州510623【正文语种】中文【中图分类】R-332胚胎干细胞是毒性预测的有用工具，2003年，欧洲替代方法验证中心(european centre for validation of alternative methods, ECVAM)发布了通过验证的胚胎干细胞试验方法(embryonic stem cell test，EST)，成为胚胎和发育毒性动物试验的三个替代方法之一[1]。
经典的EST方法周期偏长、通量低，形态观察的检测终点量化程度低。
10多年来，传统的EST方法已取得了重要进展，例如诱导多能干细胞(induced pluripotent stem cells，iPS)技术的革命性突破，解决了测试系统来源困难的问题[2]；基于微孔培养技术与流式标志物分析的高通量测试提高了定量检测毒性终点的效率[3-4]；“组”学技术和信号转导技术的运用优化了检测终点的特异性[5-6]。
眼科常用英语主诉complain [kəmˈplein]视物模糊blurred vision视力减退visual deterioration [dɪˌtɪrɪəˈreɪʃən]视力疲劳asthenopia / eye strain [ˌæsθiˈnəupiə]（Asth [英汉医学名词] 视疲劳。
）视物变形metamorphosis [ˌmetəˈmɔ:fəsis] (metamorphosis变态，变形变质：尤指发育上从一个阶段转向另一阶段的变化，例如从幼虫变为成虫。
meta-denoting a change of position or condition 表示“变化”，“变位”，“改变”，“变换”morphosis [mɔ:ˈfəusis] 形态形成。
)流泪lacrimation [ˌlækriˈmeiʃən] (lacrima [ˈlækrimə] n.泪lacrimat adj.泪的,流泪的,啜泣的,泪腺的lacrimalD.J.[ˈlækrəməl] K.K.[ˈlækrəməl] adj.泪腺的,泪的,满是泪水的)溢泪epiphora [iˈpifərə] (语源late 16th cent. (in sense 2): via Latin from Greek epi 'upon' +pherein 'to bear or carry'. Sense 1 dates from the mid 17th cent)眼痛ophthalmalgia怕光photophobia [ˌfəutəuˈfəubiə](phobia D.J.[ˈfəʊbi:ə];K.K.[ˈfobiə] n.恐惧, 厌恶) 复视diplopia [diˈpləupiə]眼球突出exophthalmos [ˌeksɔfˈθælmɔs] (exophthalmus exo- prefix external; fromoutside表示“外部的"；“从外部ophthalmic D.J.[ɔfˈθælmik] K.K.[ɑfˈθælmɪk,ɑp-] adj.眼的,眼科的n.眼药语源：early 17th cent.: from modern Latinexophthalmus, from Greek exophthalmos 'having prominent eyes', from ex- 'out'+ ophthalmos 'eye')飞蚊症Muscae volitantes [ˌmʌsiː ˌvɒlɪˈtantiːz](Muscae (所有格)苍蝇座muscae［拉］〔单musca〕蝇; volitation D.J.[ˌvɔliˈteiʃən] n.飞翔volitantD.J.[ˈvɔlitənt] adj.能飞的,飞的,活动的)虹视halo vision (halo D.J.[ˈheɪləʊ] K.K.[ˈhelo] n.(图画中圣人头上的)光环,灵光)异物感foreign body sensation眼干dryness of eyes眼磨scratching sensation (scratching 刮痕)烧灼感burning sensation刺痛stabbing pain眼睑eyelid睑缘炎blepharitis 语源mid 19th cent.: from Greek blepharon 'eyelid' + -itis睫毛eyelash睑下垂ptosis [ˈptəusis]睑外翻ectropian [ekˈtrəupjən] (ec- 外的tropia，斜视)裂隙灯slit lamp （slit 切开，撕开；裂缝）发痒itch [itʃ]睑内翻entropion （en- 在。
INSTRUCTIONSPierce® Immunostain Enhancer46644 Pierce Immunostain Enhancer, 20mL, sufficient for 100 large (~3cm2) tissue section slides46645 Pierce Immunostain Enhancer, 2mL, sufficient for 10 large (~3cm2) tissue section slidesStorage: Upon receipt store at 4︒C. Product shipped at ambient temperature.IntroductionThe Thermo Scientific Pierce Immunostain Enhancer reduces the required amount of antibody for various detection applications and alleviates common immunostaining problems such as low signal and sensitivity and suboptimal specificity. The enhancer is supplied ready to use and does not add steps to the workflow because it is used to dilute the primary and secondary antibodies. The Pierce Immunostain Enhancer is compatible with fluorescence and chromogenic detection and routinely increases both signal intensity and detection sensitivity. Signal enhancement is antibody-dependent and typically ranges from 3- to 12-fold. Because of the strong signal enhancement, the Pierce Immunostain Enhancer reduces the amount of antibody required to achieve optimal detection.Important Product Information∙Read all instructions before beginning the procedure. Use any suitable protocol for immunostaining cells or tissues.∙For all steps, use sufficient volumes to completely immerse the specimen (cells or tissue section). Refer to the recommended volumes in the procedure section.∙Keep tissue section(s) or cells at high humidity and do not let them become dry.Additional Materials RequiredNote: Use the Pierce Immunostain Enhancer to dilute the primary and secondary antibodies. The enhancer enables significant antibody dilution. A dilution range of 5- to 20-fold beyond the antibody’s vendor recommendations has been used successfully; however, the dilution factor varies depending on the antibody, tissue and antigen.∙Primary antibody diluted in the Pierce Immunostain Enhancer; empirically determine the optimal antibody dilution for each specific antigen type being tested∙Secondary antibody conjugate diluted in the Pierce Immunostain Enhancer; empirically determine the optimal antibody dilution for each specific antigen type and detection system. Use an antibody labeled with a fluorescent dye (e.g.,Thermo Scientific DyLight Dye-conjugated Antibodies) or with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).∙Wash buffer: Phosphate-buffered saline (PBS), Tris-buffered saline (TBS), or ultrapure water. Include 0.05% Tween®-20 Detergent in the buffer or water.∙Blocking buffer: Pierce Immunostain Enhancer is compatible with most commonly used blocking buffers and has been used successfully with Thermo Scientific Blocker BSA (Product No. 37520 in TBS or 37525 in PBS), SuperBlock Blocking Buffer (Product No. 37535 in TBS or 37515 in PBS) and normal serum.∙Optional reagents depending on the detection method: Ethanol and xylene for processing paraffin-embedded tissue sections;endogenous enzyme suppressor (e.g., Thermo Scientific Peroxidase Suppressor, Product No. 35000 or 2mM levamisol for alkaline phosphatase); enzyme substrate for color development (e.g., Thermo Scientific Metal Enhanced DAB Substrate Kit, Product No. 34065 for peroxidase or 1-Step NBT/BCIP plus Suppressor, Product No. 34070 for alkaline phosphatase);counterstain (nuclear or cytoplasmic); mounting agent; Pap pen; humidifying box; glass coverslip; and microscope.Example Procedure for Staining Formalin-fixed Paraffin-embedded Tissue Sections1.Perform all necessary steps preceding incubation with primary antibody.2.Wash the tissue section three times for two minutes with wash buffer.3.Incubate the tissue section with 100µL of blocking buffer.e a Pap pen to delineate the specimen.5.Apply the primary antibody diluted with Pierce Immunostain Enhancer (see Table 1) to the tissue section. Incubate for1 hour at room temperature or overnight at 4°C.Table 1. Suggested volumes to use for different tissue section sizes.Large Tissue Section (~ 3cm2)Small Tissue Section (~ 1cm2)Primary Antibody Solution diluted in Pierce Immunostain Enhancer 100µL 50µLSecondary Antibody Solution diluted in Pierce Immunostain Enhancer 100µL 50µLNote: Volumes are valid only when the Pap pen is used to circle the tissue.6.Wash the tissue section three times for 5 minutes with wash buffer.7.Add the secondary antibody conjugate diluted with Pierce Immunostain Enhancer (see Table 1) to the tissue section.Incubate for 45 minutes at room temperature.8.Wash the tissue section three times for 5 minutes each with wash buffer.9.For immunodetection with a fluorescent dye-labeled secondary antibody proceed to Step 11.10.For immunodetection with enzyme-labeled secondary antibody or conjugate, use the appropriate substrate (e.g., MetalEnhanced DAB Substrate Kit or 1-Step NBT/BCIP plus Suppressor) according to the manufacturer’s instructions. The time required for color development varies greatly depending on the experimental system. The reaction must be monitored under a microscope and stopped at the appropriate time.11.Optional: Incubate the tissue section in an appropriate counterstain according to the manufacturer’s instructions.12.Mount the specimen using the appropriate method and image on a microscope.Example Procedure for Staining Cells Cultured in a 96-well PlateA.Solution Preparation (96-well plate)Note: The following solutions are required in addition to the solutions listed in the Additional Materials Required Section. Adjust volumes accordingly when using more than one plate.Fixation Solution (4% formaldehyde) In a ventilated fume hood, add 3mL of 16% formaldehyde to 9mL of PBS. Warm solution to 37︒C. Make this solution immediately before use.Permeabilization Buffer PBS or TBS containing 0.1% Triton X-100. For example, add 500µL of Thermo ScientificTriton® X-100 Surfact-Amps Detergent Solution (10%) to 49.5mL PBS or TBS. Store buffer at4︒C for up to 7 days.B.Staining ProcedureNote: See Table 2 for suggested solution volumes for different cell culture systems.1.Plate cells in 96-well plate. Culture cells as desired and remove media before fixation.2.Add 100μL of warmed Fixation Solution to each well. Incubate plate in a ventilated fume hood at room temperature for15 minutes.3.Aspirate Fixation Solution and wash plate three times with 100µL/well of wash buffer.4.Aspirate wash buffer, add 100μL/well of Permeabilization Buffer and incubate for 15 minutes at room temperature.5.Aspirate Permeabilization Buffer, add 100μL/well of blocking buffer and incubate at room temperature for 15 minutes.6.Aspirate blocking buffer and add 50μL/well of the primary antibody diluted with the Pierce Immunostain Enhancer.7.Incubate plate for 1 hour at room temperature or overnight at 4°C.8.Aspirate Primary Antibody Solution and wash plate three times with 100μL/well of wash buffer.9.Aspirate wash buffer and add 50μL/well of secondary antibody diluted with the Pierce Immunostain Enhancer.10.Incubate plate for 45 minutes at room temperature. If using a fluorescent dye-labeled antibody, protect plate from lightduring incubation.11.Aspirate the secondary antibody solution and wash plate three times with 100μL/well of wash buffer. For detection offluorescent-labeled secondary antibody proceed to Step 13.12.Detect the enzyme-labeled secondary antibody or conjugate using the appropriate substrate (e.g., Metal Enhanced DABSubstrate Kit or 1-Step NBT/BCIP plus Suppressor), according to the manufacturer’s instructions. The time required for color development varies depending on the experimental system. The reaction must be monitored under a microscope and stopped at the appropriate time.13.Add 50μL/well of a counterstaining solution as needed. Incubate plate for 5 minutes at room temperature. If using afluorescent dye-labeled antibody, protect plate from light during incubation.14.Aspirate counterstaining solution and wash plate three times with 100μL/well of wash buffer.e the appropriate system to image cells (e.g., fluorescence or light microscope, Thermo Scientific ArrayScan HCSReader).Table 2. Suggested volumes to use for different cell culture systems.96-Well Plates (μL/well) Small Coverslip(μL)8-Well Chamber Slide(μL/well)Permeabilization Buffer 100 100 400Blocking buffer 100 100 400Primary antibody diluted inPierce Immunostain Enhancer50 50 100Secondary antibody diluted inPierce Immunostain Enhancer50 50 100TroubleshootingProblem PossibleCause Solution Primary antibody concentration wastoo lowIncrease primary antibody concentrationInappropriate antibody was used Find an antibody validated for the specific applicationAntigen poorly accessible in the sample Optimize the dilution of the primary antibody or use antigen retrieval methods to unmask the epitopeExcessive blocking Optimize the blocking time as the length of blocking cangreatly affect staining or try a different blocking agentPrimary and secondary antibodies were not compatible Use secondary antibody conjugate that was raised against the species in which the primary antibody was raisedAntigen retrieval was inappropriate Empirically determine optimal unmasking conditions –excessive unmasking can cause loss of antigenicity Antigenicity was lost Optimize the method for fixation or antigen retrieval Weak or no signalSecondary antibody concentration was too low Optimize secondary antibody concentration and consult instructions for the detection system being usedContinued on next pageContinued from previous pageProblem PossibleCause SolutionBlocking of nonspecific sites or washing was inadequate Empirically determine the optimal blocking buffer and optimize washing stepsThe primary antibody concentration was too high Empirically determine the optimal primary antibody concentration (using antibody at a lower concentration with longer incubation time favors antigen/antibody reaction and typically results in lower background)Endogenous enzymes were active Use endogenous enzyme inhibitors (e.g., 2mM levamisolfor alkaline phosphatase or 0.3% v/v H2O2 forperoxidase)Phosphate-based wash buffer was used for alkaline phosphatase detection Use Tris-buffered saline for washing when detecting alkaline phosphataseHigh backgroundThe secondary antibody conjugate concentration is too high Reduce the concentration of the secondary antibody conjugateA high concentration of primary antibody combined with the antibody buffer and the enhancer caused precipitation Further dilute antibody to reduce speckling; alternatively, filter the primary antibody through 0.2µm filter suitable for small volumes (e.g., Nalgene® 4mm syringe filter, Product No. 171-0020)HRP-conjugated secondary antibody contains aggregates Briefly centrifuge HRP conjugate immediately before use and remove aliquot from top of vialAppearance of intensespecklingPowder from gloves contaminatedthe sampleWear only powder-free glovesRelated Thermo Scientific ProductsVisit our website for a complete list of immunostaining products including secondary antibodies labeled with DyLight Fluorescent Dyes and enzymes, blockers, buffers and stains.37520 Blocker BSA Blocking Buffer in TBS (10X), 125mL37525 Blocker BSA Blocking Buffer in PBS (10X), 200mL31872 Normal Goat Serum36000 Pierce Peroxidase IHC Detection Kit62249 Hoechst 33342 Solution, 5mL62248 DAPI Solution, 1mLTriton® is a registered trademark of Rohm & Haas.Tween® is a registered trademark of ICI Americas.This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than the original purchaser of the Product (“Buyer”).No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular purpose, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or refund for the non-conforming Product(s).There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii) use of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products.Current product instructions are available at /pierce. For a faxed copy, call 800-874-3723 or contact your local distributor.© 2010 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA.。
Journal of China Pharmaceutical University2021，52（1）：100-103学报抗炎三肽KdPT对小鼠眼干燥症的治疗作用王华，许元生，宋燕*（广州领晟医疗科技有限公司，广州510663）摘要为探讨抗炎三肽KdPT治疗眼干燥症的疗效，用0.2%苯扎氯铵溶液处理8周龄雄性BALB/c小鼠，建立眼干燥症模型；造模4周后，将小鼠随机分为模型对照组，阳性对照组和KdPT的低、中、高剂量组。
每组分别给予生理盐水、人工泪液和1、10、100μg/mL KdPT。
治疗3、5、7、10、14d后，观察眼表面形态，进行荧光素钠染色评分；治疗14d后用酚红棉线测定泪液分泌量；治疗14d后处死动物取出右眼角膜进行组织病理学分析。
实验结果显示，治疗3、5、7、10、14d后，各组小鼠的状态和体重无明显异常。
治疗14d后，KdPT可促进泪液分泌，修复受损角膜上皮，对小鼠眼干燥症有显著治疗作用。
KdPT有可能被开发为一种新型的眼干燥症治疗药物。
关键词眼干燥症；三肽；KdPT；荧光素钠染色；泪液分泌中图分类号R965.1文献标志码A文章编号1000-5048（2021）01-0100-04doi：10.11665/j.issn.1000-5048.20210114引用本文王华，许元生，宋燕.抗炎三肽KdPT对小鼠眼干燥症的治疗作用［J］.中国药科大学学报，2021，52（1）：100–103.Cite this article as：WANG Hua，XU Yuansheng，SONG Yan.Therapeutic effect of anti-inflammatory tripeptide KdPT on ophthalmoxerosis in mice［J］.J China Pharm Univ，2021，52（1）：100–103.Therapeutic effect of anti-inflammatory tripeptide KdPT on ophthalmoxero⁃sis in miceWANG Hua,XU Yuansheng,SONG Yan*Guangzhou Link Health Pharma Co.,Ltd.,Guangzhou510663,ChinaAbstract This study was designed to investigate the therapeutic effect of anti-inflammatory tripeptide KdPT on ophthalmoxerosis.Male BALB/c mice,8-week old,were treated with0.2%benzalkonium chloride solution to establish the ophthalmoxerosis model.Four weeks after modeling,the mice were randomly divided into control group,positive group and the low,medium,high dose groups of KdPT.Each group was given normal saline,artifi⁃cial tears and1,10,100μg/mL KdPT,respectively.After3,5,7,10and14days of treatment,the morphology of the eye surface was observed,and the fluorescein sodium staining score was performed.The amount of tear secre⁃tion was measured by phenol red cotton thread and the right corneas were taken out for histopathological analysis after14days of treatment.Data showed that there was no significant abnormality in general state and the weight of mice in each group at each time point of treatment.After14days of treatment,KdPT can promote the secre⁃tion of tear,repair the damaged corneal epithelium,and showed a significant therapeutic effect on ophthalmoxero⁃sis in mice.Based on the data,it is possible for KdPT to be developed as a novel drug for ophthalmoxerosis.Key words ophthalmoxerosis;tripeptide;KdPT;sodium fluorescein staining;tear secretion眼干燥症是由于泪液的量或质或流体动力学异常引起的泪膜不稳定和（或）眼表损害，从而导致眼不适症状及视功能障碍的一类疾病。
免疫荧光操作流程步骤Fluorescent immunostaining is a valuable technique used in biological research to detect and localize specific proteins of interest within cells and tissues. Immunofluorescence allows researchers to visualize the distribution and expression levels of target proteins, providing valuable insights into cellular function and pathology. The process involves a series of steps that must be carefully followed to ensure accurate and reliable results.免疫荧光染色是生物研究中常用的一种技术，用于检测和定位细胞和组织中感兴趣的特定蛋白质。
免疫荧光使研究人员能够可视化目标蛋白质的分布和表达水平，为细胞功能和病理学提供宝贵的见解。
这个过程涉及一系列必须仔细遵循的步骤，以确保准确和可靠的结果。
To begin the immunofluorescence staining procedure, cells or tissue sections must first be fixed to preserve their structure and antigenicity. This is typically achieved using a fixative such as paraformaldehyde, which cross-links proteins and immobilizes them in place. Proper fixation is essential to prevent cellular components from being lost or damaged during subsequent processing steps.开始免疫荧光染色程序，首先必须固定细胞或组织切片，以保持它们的结构和抗原性。
细菌生物膜荧光染色英文回答：Fluorescent staining of bacterial biofilms is a common technique used to visualize and study the structure and composition of these complex microbial communities. There are several methods available for staining biofilms, each with its own advantages and limitations.One commonly used method for fluorescent staining of biofilms is the use of a fluorescent dye such as SYTO 9 or propidium iodide. These dyes can penetrate the biofilm matrix and stain both live and dead bacteria. SYTO 9 stains live bacteria green, while propidium iodide stains dead bacteria red. This allows researchers to differentiate between viable and non-viable cells within the biofilm.To perform the staining, the biofilm is first fixed with a fixative solution to preserve its structure. The fixed biofilm is then incubated with the fluorescent dyefor a specific period of time. After staining, the biofilm is washed to remove any unbound dye and mounted on a microscope slide for imaging. The stained biofilm can be visualized using a fluorescence microscope, and the images can be analyzed using image analysis software.Another method for fluorescent staining of biofilms is the use of fluorescently labeled antibodies or lectins. Antibodies or lectins specifically bind to certain components of the biofilm, such as specific bacterial species or extracellular matrix proteins. These labeled antibodies or lectins can be incubated with the biofilm, allowing for specific visualization of the targeted components.In addition to fluorescent staining, other techniques such as confocal laser scanning microscopy (CLSM) can be used to obtain three-dimensional images of biofilms. CLSM uses a laser to scan through the biofilm, capturing images at different depths. These images can then be reconstructed to create a three-dimensional representation of the biofilm structure.Overall, fluorescent staining of bacterial biofilms is a powerful tool for studying the organization and composition of these complex microbial communities. It allows researchers to visualize the spatial distribution of bacteria and other components within the biofilm, providing valuable insights into biofilm formation, function, and resistance mechanisms.中文回答：细菌生物膜的荧光染色是一种常用的技术，可用于可视化和研究这些复杂微生物群落的结构和组成。
影响麦芽溶解度的关键制麦参数及参数优化许举飞;康健;谷方红;王德良【摘要】麦芽溶解度是衡量麦芽酿造性能的重要指标,针对影响该指标的关键制麦参数即浸麦度、发芽温度、凋萎温度、干燥温度,以进口大麦为原料,进行了单因素试验,并分别采用荧光染色法和冷场发射扫描电子显微镜观察麦芽胚乳溶解情况来确定麦芽溶解度.并在此基础上采用响应面设计优化制麦工艺.结果表明,各因素对麦芽溶解度影响程度的大小顺序为B(发芽温度)＞D(干燥温度)＞C(凋萎温度)＞A(浸麦度).优化制麦工艺条件为:浸麦度为46％,发芽温度为16℃,凋萎温度为48℃,干燥温度为78℃.优化制麦工艺后,麦芽溶解度从81％提高到87％.%Malt solubility is a significant determination index.The key malting parameters affecting malt solubility include barley steeping degree,germination temperature,searing temperature and drying temperature.Import barley was used as raw material,and single factor experiments were used to study barley malting.Fluorescent staining and ice emission scanning electron microscope were used to to observe malt endosperm dissolving conditions and thus determine malt solubility.Response surface design was used to optimize barley malting process.The experimental results showed that germination temperature was the main factor,and the grade of the factors influencing malt solubility was as follows:B(germination temperature) ＞ D (drying temperature) ＞ C (searing temperature) ＞ A (steeping degree).Optimized barley malting conditions for the best malt solubility were as follows:the barley steeping degree was 46 ％,germination temperature was 16 ℃,searing temperature was 48 ℃,drying temperaturewas 78 ℃.After optimization of ma lting process,malt solubility was increased from 81％ to 87 ％.【期刊名称】《食品与发酵工业》【年(卷),期】2015(041)008【总页数】7页(P146-152)【关键词】麦芽溶解度;关键制麦参数;单因素试验;响应面优化【作者】许举飞;康健;谷方红;王德良【作者单位】新疆大学生命科学与技术学院,新疆乌鲁木齐,830046;新疆大学生命科学与技术学院,新疆乌鲁木齐,830046;中国食品发酵工业研究院,北京,100015;中国食品发酵工业研究院,北京,100015【正文语种】中文麦芽是啤酒的主要原料［1］。
Fluorecent Staining for Study of Extracellular Polymeric Substances in Membrane Biofouling LayersM I N G -Y U A N C H E N ,†D U U -J O N G L E E ,*,†Z .Y A N G ,‡X .F .P E N G ,‡A N D J .Y .L A I §Chemical Engineering Department,National Taiwan University,Taipei,106Taiwan,Department of ThermalEngineering,Tsinghua University,Beijing,100084,China,and Department of Chemical Engineering,R&D Center ofMembrane Technology,Chung Yuan Christian University,Chungli,Taiwan 32023Membrane biofouling by microbial products adversely impacts the feasibility of adopting membrane bioreactors (MBRs)for treating wastewater.The fouling layer structure determines the pressure drop across the fouling layer.Three-dimensional distributions of nucleic acids,proteins,R -D -GLUCOPYRANOSE POLYSACCHARIDES ,AND Β-D -glucopyranose polysaccharides in the fouling layer formed on a mixed cellulose ester membrane were generated utilizing a quadruple staining protocol combined with confocal laser scanning microscopy (CLSM).For the first time,this study constructed a three-dimensional volumetric grids model representing the fouling layer structure on the basis of a series of CLSM images.Quantitative structural information about the fouling layer was extracted from the CLSM images.IntroductionMembrane fouling causes flux decline and increases pressure drop across the membrane,thereby generating operational troubles and even membrane failure (1).Extracellular polymeric substances (EPSs)have been identified as the principal foulants in membrane bioreactor (MBR)processes (2-6).Rojas et al.(7)identified a 10-fold increase in membrane-specific resistance when protein concentrations increased from 30to 100mg L -1.Lee et al.(8)determined that supernatant,at most,accounted for 37%of total resistance during membrane filtration.However,Bouhabila et al.(9)demonstrated that total resistance of filtration by supernatant was 76%.Wisniewski and Grasmick (10)at-tributed roughly 50%of total resistance in filtration to soluble microbial products (SMP)in supernatant.Lee et al.(11)noted that attached cells and SMPs generated a dynamic membrane on the membrane surface during filtration.Pressure drops across a fouling layer and membrane were measured to quantify filtration resistance.The structure of a thin,deposited layer on a filter membrane has been modeled as a homogeneous porous medium with fixed characteristics (12,13).Fluorescently labeled stains were utilized to analyze the cells and EPS distributions in biofilms (14-16).Lee et al.(17)probed the cells,polysaccharides,and proteins in the biofilm using the following three stains:SYBR Green I,fluorescently labeled lectins,and Hoechst 2495.Chen et al.(18)was the first study to profile the distributions of proteins,R -D -glucopyranose polysaccharides,and -D -glucopyranose polysaccharides within a fouling layer on a filter membrane.These authors also applied three stains to the same sample.The structure of a fouling layer determines the pressure drop produced via membrane fouling.Structural information for a fouling layer is essential to understand how a membrane is fouled.The purpose of this work is propose the use of a novel quadruple staining scheme combining with confocal laser scanning microscopy (CLSM)to investigate the distributions of cells (nucleic acids)and EPSs (proteins,R -and -D -glucopyranose polysaccharides)in the fouling layer on a filter membrane.On the basis of CLSM images,the three-dimensional volumetric grid models representing the struc-ture of fouling layer were generated for further analysis.A case study applying dead-end filtration with mixed cellulose and ester membrane demonstrated the feasibility of the use of the proposed scheme.ExperimentalSample.Waste-activated sludge was acquired from a waste-water treatment plant owned by Presidential Enterprise Corp.,Taiwan,which daily treats 250m 3of food-processing wastewater using primary,secondary,and tertiary treatments.The sludge pH was approximately 6.84.The chemical oxygen demand (COD)for the sludge and filtrate (through 0.45-µm membrane)was 16000mg L -1(TCOD)and 86.7mg L -1(SCOD),respectively,as determined by a spectrometer (DR/2000,HACH,United States).The elemental composition of the dried samples,according to an elemental analyzer (Perkin-Elmer 2400CHN),was as follows:C,41.3%;H,6.6%;and N,5.4%.The sludge supernatant obtained after 30-min settling was used as the test sample.Most flocs were removed to focus on the contribution of colloidal fractions in supernatant to fouling layer resistance.Dead-end membrane filtration tests on obtained supernatant samples were conducted in a 30-cm Hg vacuum using a 0.45-µm mixed cellulose and ester membrane (Advantec MFS,Inc.,CA).The membrane and fouling layer were carefully removed from the filtration chamber under fully hydrated conditions for further pro-cessing.Staining.Fluorescein isothiocyanate (FITC)(Molecular Probes,Eugene,OR)was applied to stain the amine-reactive compoundlike proteins and amino sugars.The R -and -D -glucopyranose polysaccharides were stained and probed separately since they have different biodegradability and hence may play different roles in biological systems like MBR.Fluorescently labeled lectin Concanavalin A (Con A,Mo-lecular Probes,Eugene,OR)conjugated with tetramethyl-rhodamine were employed to bind the R -mannopyranosyl and R -glucopyranosyl sugar residues.Calcofluor white (Sigma)was applied to stain the -D -glucopyranose polysac-charides.Since Con A can also bind with proteins and glycoconjugate groups associated with cell walls,SYTO 63(Molecular Probes,Carlsbad,CA),which is a cell-permeative nucleic acid stain,was utilized to differentiate EPS from cells.The SYTO 63(20µM)was first dripped onto the membrane sample and was placed on a shaker table for 30min.Next,0.1M sodium bicarbonate buffer was added to the sample to retain the amine group in a nonprotonated form;then,the FITC solution (10g L -1)was added to the sample for 1h at room temperature.Subsequently,the Con A solution*Corresponding author phone:+886-2-2362-5632;fax:886-2-362-3040;e-mail:djlee@.tw.†National Taiwan University.‡Tsinghua University.§Chung Yuan Christian University.Environ.Sci.Technol.2006,40,6642-664666429ENVIRONMENTAL SCIENCE &TECHNOLOGY /VOL.40,NO.21,200610.1021/es0612955CCC:$33.50©2006American Chemical SocietyPublished on Web 10/05/2006(0.2g L -1)was added to the sample and was incubated for another 30min.Finally,calcofluor white (Sigma,St.Louis,MO)was applied to stain the -linked D -glucopyranose polysaccharides.After each of these four staining stages,the sample was washed twice by phosphate-buffered saline (PBS)to remove excess stain.Some stained membranes were frozen at -20°C and were sectioned into specimens 40-µm thick using a cryomicrotome for imaging from side of fouling layer.CLSM Imaging.Confocal laser scanning microscopy (CLSM)(Leica TCS SP2Confocal Spectral Microscope Imag-ing System,Gmbh,Germany)was performed to investigate the internal structure of the fouling layer.The fluorescence of calcofluor white was detected via excitation at 400nm and from the emission width at 410-480nm (blue).The FITC probe was detected via excitation at 488nm and emission at 500-540nm (green).Excitation at 543nm and emission at 550-600nm were utilized to detect Con A conjugates.The fluorescence of SYTO 63was determined on the basis of excitation at 633nm and emission at 650-700nm (red).Results and DiscussionCLSM Images of Fouling Layer.Figure 1a -d presents the CLSM images scanned at 1µm above the membrane surface,representing the first deposited layer on the membrane surface.In this initial fouling layer,the -D -glucopyranose polysaccharides formed a continuous layer (Figure 1a);proteins were aggregated into clusters (Figure 1b);cells were distributed in dispersed form (Figure 1c);R -D -glucopyranose polysaccharides appeared only in some spots (Figure 1d).Since -D -glucopyranose polysaccharides form a (relatively)continuous layer,the permeability of this substance may contribute significantly to filtration resistance in this initial compact.Figure 2a -d shows the CLSM images scanned at 10µm above the membrane surface,representing a “developed”layer.Like those in the initial layer,the proteins appearedin clusters (Figure 2c)and few R -D -glucopyranose polysac-charides had appeared (Figure 2d).Conversely,both -D -glucopyranose polysaccharides (Figure 2a)and cells (Figure 2b)were largely clustered.How proteins were initially deposited (Figure 1c)clearly impacted the subsequent packing of EPS formed above this layer (Figure 2b).Large channels were observed in this thin layer.Hence,not as noted in Figure 1,most water can flow through these large channels and the permeability of the -D -glucopyranose polysaccha-ride layer likely does not significantly impact filtration resistance.Vertical Distributions of EPS in Fouling Layer.CLSM side-view images suggest that the fouling layer has a thickness of 6.8-17.8mm and has a very nonuniform spatial distribu-tion (images given in Supporting Information).Large chan-nels observed from a top view (Figure 2)were also obvious on the side view,with a mean porosity decreasing toward the membrane surface.Figure 3shows the vertical distributions of the intensities of the fluorescent lights corresponding to protein,nucleic acids (cells),and R -and -polysaccharides probed at specific positions above the membrane (distance )0refers to membrane surface).Figure 3a shows the vertical distributions of light intensities from the “cluster regime”in Figure 2(averaged over 10randomly chosen bright regimes 10×10µm 2in size in Figure 2).On the other hand,Figure 3b shows the vertical distributions of light intensities of the “void regime”in Figure 2(averaged over 10randomly chosen dark regimes 10×10µm 2in size in Figure 2).The fluorescent light collected near membrane surface (distance )0-5µm)is similar in intensity for all of the four foulants in both “cluster”and “void”regimes.This 5-µm thick,initial deposit layer is a mixture of all four foulants,with -polysaccharides presenting as a continuous layer (Figure 1).Above this initial deposit layer,the distribution of EPS became highly stratified.In cluster regimes,the intensities of fluorescent light for proteins,nucleic acids,and R -polysac-FIGURE 1.CLSM images of fouling layer at 1µm above the membrane surface.The scanned regime was 238×238µm 2in size.(a)CLSM image of -D -glucopyranose polysaccharides (calcofluor white);(b)CLSM image of proteins (FITC);(c)CLSM image of nucleic acids (SYTO 63);(d)CLSM image of r -d-glucopyranose polysaccharides (Con A).VOL.40,NO.21,2006/ENVIRONMENTAL SCIENCE &TECHNOLOGY96643charides increased with vertical distance while the corre-sponding distribution for -polysaccharides decreased slightly and then levered off.At the top of the fouling layer,the intensities of proteins were 3.6times to that at the membrane surface,probably becoming one of the dominating foulants for the present system.In the void regimes,the intensities of all of the four foulants declined with vertical distance.The clusters of organic foulants noted in Figure 2were formed on the initial deposit layer.On the basis of the above-mentioned observation,and on the suggestion by a reviewer for this work,very likely the free EPS (not associated with cell membranes)were deposited on the membrane surface first,and then cells with associated surface EPS were deposited on the initial deposit layer to form clusters.Building Organic Fouling Layer ing a series of CLSM images scanned on the fouling layer,the three-dimensional distributions of nucleic acids and EPS can be generated following the methodology proposed by refs 19and 20.(Images combining Figure 2a -d are given in Supporting Information.)The CLSM image could be converted into bileveled image with threshold values determined using Otsu’s scheme (21)(sample images given in Supporting Information).To reduce the resolution of the image and to adjust the physical shape of the voxel to almost a cube,the sliced image resolution was reduced from 512×512to 128×128(image resampling)so that the ratio of all sizes of the voxel was all about unity.The Lanczos filter is used in the resampling.Then,the boundaries in the binary images are classified according to connectivity and whether they lie within the object or its complement.The connectivity of neighboring pixels in all analyses is set to four for edge detection.The marching cubes algorithm for triangulating surfaces was adopted.Finally,the advancing-front algorithm was applied to fill each region defined by the simplified polygonal surface model of pores,with unstruc-tured tetrahedral volumetric grids.The tetrahedral grids were converted to equivalent hexahedral grids to be compactibleFIGURE 2.CLSM images of fouling layer at 10µm above the membrane surface.The scanned regime was 238×238µm 2in size.(a)CLSM image of -D -glucopyranose polysaccharides (calcofluor white);(b)CLSM image of proteins (FITC);(c)CLSM image of nucleic acids (SYTO 63);(d)CLSM image of r -polysaccharides (Con A).FIGURE 3.Distributions of EPS in fouling layer.(a)Average intensities of various EPS in “cluster regimes”in Figure 2;(b)average intensities of EPS in “void regimes”in Figure 2.66449ENVIRONMENTAL SCIENCE &TECHNOLOGY /VOL.40,NO.21,2006for the fluid dynamic software.Each tetrahedral grid was converted to four hexahedral grids(Figure4).Over106grids were allocated in the mesh model for describing the interior architecture of the fouling layer in Figure4.Two magnified views of the meshes demonstrated the heterogeneous structure of the fouling layer investigated. This observation contradicts the common assumption of the fouling layer as a homogeneous porous medium of constant property.On the basis of the constructed model in Figure4,the porosity of selected volume of the fouling layer could be calculated by(void space)/(selected volume).The thickness of the fouling layer model in Figure4was divided into10 pieces,each of equal thickness of2µm.Then,the average porosity for each of the10pieces of the fouling layer was reported(Figure5).Near the membrane surface,the porosity of fouling layer is low(<0.28),corresponding to the noted -polysaccharide layer in Figure1.When moving away from the membrane surface,the average porosity increases and reaches about0.5at6µm above membrane surface.This thickness also correlates with the thickness of the initialdeposit layer identified in Figure3.A dense initial deposit layer indicates a higher-pressure drop across this part of the fouling layer than that across the rest of the fouling layer.The box-counting fractal dimension,D P,3,is given by eq 1,when the surface of one fractal object is covered by triangular patches with a characteristic side length,l equi(22), where n(l equi)is the number of triangular patches.Figure6 shows the log-log plot for n(l equi)versus l equi data for the fouling layer,yielding a slope of2.48with r2)0.991.Hence, the interior structure of a fouling layer has a fractal-like characteristic of box-counting fractal dimension of2.48.A fractal-like structure for the organic fouling layer indicated that the most foulants were packed on the membrane surface following similar mechanisms at all scales, with no particular spatial preference.The noted fractal dimension of2.48for the present fouling layer is close to that for a“reaction-limited aggregate”(RLA)and is much higher than that for a“diffusion-limited aggregate”(DLA)or for a “cluster-cluster aggregate”(CCA).The basic assumption of the growth of an RLA is that the approaching particles will be incorporated into the structure if only they hit certain “reactive sites”.Diffusion of particle is hence not a rate-controlling step for the growth of RLA.Certain bounds would be present between the joining particles and the RLA.A common scenario for fouling layer formation is that foulants are carried by filtration flux to the membrane surface andFIGURE4.The tetrahedral volumetric grids of fouling layer.D P,3)-liml equi f0log n(l equi)log l equi(1)FIGURE5.The vertical distribution of porosity in fouling layer.Thefouling layer model in Figure4was divided into10fractions ofequal thickness(2µm).The average porosity averaging over eachof the10fractions over the whole scanned area is reported.VOL.40,NO.21,2006/ENVIRONMENTAL SCIENCE&TECHNOLOGY96645are intersected by the membrane to form a layer.Restated,the flow field is the dominant factor controlling the fouling layer growth.The present results indicate that when a new foulant molecule (or cluster)approaches an existing fouling layer,it may search for certain “sites”to stick on,a process not totally controlled by the diffusion of foulant molecules.In summary,the proposed quadruple staining protocol combined with confocal laser scanning microscopy (CLSM)could be utilized to probe the distributions of EPS in the fouling layer and to construct the three-dimensional volu-metric grids model representing the fouling layer structure.The structural information of the fouling layer could be applied for further study,like to explore the fouling mech-anisms of membrane or to construct a comprehensive fouling layer model.AcknowledgmentsThis work was financially supported by National Science Council,Taiwan,R.O.C.Supporting Information AvailableCLSM side-view images and their combined image.Sample images for original top-view CLSM image and the bileveled image.This material is available free of charge via the Internet at .Literature Cited(1)Ramesh,A.;Lee,D.J.;Wang,M.L.;Hsu,J.P.;Juang,R.S.;Hwang,K.J.;Liu,J.C.;Tseng,S.J.Biofouling in membrane 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thresholding of image of sludgeflocs.Environ.Sci.Technol.2004,38,1161-1169.(22)Baveye,P.;Boast,C.W.;Ogawa,S.;Parlange,J.Y.;Steenhuis,T.Influence of image resolution and thresholding on the apparent mass fractal characteristics of preferential flow patterns in field soilds.Water Resour.Res.1998,34,2783-2796.Received for review May 30,2006.Revised manuscript re-ceived August 24,2006.Accepted August 25,2006.ES0612955FIGURE 6.The number of triangular patches on the constructed surface versus the equivalent voxel size l equi of fouling layer.66469ENVIRONMENTAL SCIENCE &TECHNOLOGY /VOL.40,NO.21,2006。
