副溶血性弧菌显色培养基检测效果初步评价

副溶血性弧菌(Vibrio parahaemolyticus)是一种重要的食源性致病菌, 广泛存在于各种海产品中。由于传统培养基和检测方法费时费力, 本研究设计开发了一种新型显色培养基HKC vibrio, 通过应用于人工污染样品和实际样品检验, 以法国科玛嘉弧菌显色平板CHROMagar vibrio和柠檬酸钠-硫代硫酸钠-氯化钠-蔗糖琼脂平板(TCBS)为对照, 对显色培养基HKC vibrio的灵敏性、特异性和检测效果进行了初步评价。结果表明, HKC vibrio的灵敏性与CHROMagar vibrio和TCBS相当, 并具有较好的特异性, HKC vibrio是非常有价值的分离平板, 可大大提高副溶血性弧菌的检测效率。     

副溶血性弧菌显色培养基检测效果初步评价

副溶血性弧菌(Vibrio parahaemolyticus)是一种重要的食源性致病菌,广泛存在于各种海产品中.由于传统培养基和检测方法费时费力,本研究设计开发了一种新型显色培养基HKC vibrio,通过应用于人工污染样品和实际样品检验,以法国科玛嘉弧菌显色平板CHROMagar vibrio和柠檬酸钠-硫代硫酸钠-氯化钠-蔗糖琼脂平板(TCBS)为对照,对显色培养基HKC vibrio的灵敏性、特异性和检测效果进行了初步评价.结果表明,HKC vibrio的灵敏性与CHROMagar vibrio和TCBS相当,并具有较好的特异性,HKC vibrio是非常有价值的分离平板,可大大提高副溶血性弧菌的检测效率.     

大肠杆菌O157显色培养基检测效果的评价

【目的】评价显色培养基对大肠杆菌O157:H7(Escherichia coli O157:H7)的检测效果。【方法】本实验室研制的大肠杆菌O157显色培养基(HKM),与国外梅理埃、科玛嘉及国内厂家的同类产品及传统培养基CT-SMAC作比较,对相关菌株以及污染样品和实际样品进行对比测试。【结果】实验室研制的HKM大肠杆菌O157显色培养基与科玛嘉同类产品在特异性、灵敏度及检测效果方面均无明显差异,均优于梅里埃、国内厂家产品及CT-SMAC。【结论】HKM大肠杆菌O157显色培养基具有高检出率及高特异性的特点,具有较好的应用价值和前景。     

一种副溶血性弧菌显色培养基的应用

副溶血性弧菌(Vibri oparahemolyticus)是一种在海产品中广泛存在的嗜盐性细菌,也是重要的食源性致病菌。传统检验方法检测周期长、特异性低,为提高检测效率、缩短检测周期,开发出一种副溶血性弧菌显色培养基(HKMVPM),与国外同类产品KHVPM和TCBS在灵敏度、特异性及检测效果方面进行了比较和初步评价。结果显示,HKMVPM显色培养基和KHVPM显色培养基均具有较好的选择性和特异性,这3种培养基对天然污染样品的分离率高低顺序为HKMVPMKHVPMTCBS。显色培养基具有高检出率及高特异性的特点,有良好的应用前景。     

阪崎肠杆菌显色培养基的应用研究

阪崎肠杆菌(Enterobacter sakazakii)是新近引起广泛关注的一种危险的条件致病菌, 主要存在于婴幼儿奶粉、婴幼儿补充食品中。由于目前日常使用的传统检验方法存在检测周期长等方面的不足之处, 本实验室研究设计出一种新的显色培养基(HKMCES), 通过与OXOID公司的同类产品(OXCES)比较, 分别应用于质控菌株、污染样品和实际样品的测试, 对这2种显色培养基的灵敏度、特异性、检测效果以及前增菌方法进行了初步评价。结果表明, 合适的增菌方法更有利于样品中阪崎肠杆菌的检出, 本实验室研制的显色培养基和OXOID公司的显色培养基均具有较好的选择性和特异性, 检测效果相当。这种新的显色培养基能使检测周期缩短, 具有较好的应用价值。     

两种检测水产品中副溶血性弧菌方法的比较分析

为了比较传统检测方法与快速检测方法的优劣,本实验室采用国标法、显色培养基+API20E法对50组水产品进行副溶血性弧菌的检测,结果两种方法检测结果一致,即样品S006、S043中检出副溶血性弧菌,检出率为4%,其余样品未检出该菌。显色培养基+API20E法在检测时间和操作步骤方面要优于传统检测方法。     

九种单核细胞增生性李斯特菌检测技术效果比较及评价

采用传统生物学方法、显色培养基方法、自制三抗体夹心ELISA试剂盒(TAS-ELISA)、基因组直接PCR(DNA Direct-PCR)、菌裂解直接PCR(Bacterium Direct-PCR)、免疫捕捉PCR(IC-PCR)、生物PCR(Bio-PCR)、SYBR Green染料实时荧光PCR(SYBR Green Real time-PCR)、探针实时荧光PCR(TaqMan Real time-PCR)检测纯菌液及模拟带菌食品中的单核细胞增生性李斯特菌(Listeria monocytogenes,LM)。结果表明:传统生物学培养方法出现假阳性;TAS-ELISA、DNADirect-PCR、Bacterium Direct-PCR、IC-PCR最低检测限分别为106、102、105、104CFU/ml;显色培养基、SYBR Green Real time-PCR灵敏度达102CFU/ml;Bio-PCR、TaqMan Real time-PCR检测灵敏度最高,均为101CFU/ml。显色培养基、TAS-ELISA操作简单,适合大量样品的初检;IC-PCR具有灵敏、特异、快速、经济的优点,特别适合大体积样品中微量病原的检测;Bio-PCR、TaqMan Real time-PCR灵敏度高、特异性好,适合阳性样品的复检以及科研使用。     

海产品中副溶血性弧菌的直接定量法-疏水网格滤膜法

本研究建立了一种疏水网格滤膜法,直接定量检测海产品中的副溶血性弧菌。该方法包括4~5 h复苏步骤以使受损细胞得以修复,然后37℃下在副溶血性弧菌显色培养基上培养过夜后计数。从显色培养基上挑取典型菌落进行确证试验,确证率大于98%。与传统MPN法比较,对经过冷藏、冷冻和热处理的样品进行检测时,新方法检出菌量明显高于MPN法(p0.01)。     

2005-2013年肠道致病菌中志贺菌和沙门菌的分布及检测方法经验总结

目的分析杭州市第三人民医院2005-2013年肠道致病菌中志贺菌和沙门菌的分布并对检测方法进行经验总结。方法采用定位显色培养基和营养琼脂初筛疑似志贺菌和沙门菌,并参考该院历年菌种分离情况进行血清凝集。采用EXCEL统计处理数据,χ2检验比较采用定位显色培养基和营养琼脂平板之前和之后,粪便培养志贺菌和沙门菌的分离率之间差异的显著性。结果采用定位显色培养基和营养琼脂平板初筛疑似菌株后,志贺菌和沙门菌的分离率差异存在统计学意义。结论采用定位显色培养基和营养琼脂初筛疑似菌株,并对本院历年菌种分离情况进行总结,对改善志贺菌和沙门菌的分离率,提升检验科微生物室的工作效率有较大帮助。     

阪崎肠杆菌在不同品牌显色培养基上的对比研究

目的:对比不同生产单位同种选择性培养基的差异,为乳粉的阪崎肠杆菌分离提供参考。方法:采用盲样考核样品,考察A、B、C、D、E五家生产单位生产的不同品牌的阪崎杆菌显色培养基的选择性强弱。结果:不同品牌的显色培养基的准确率分别为100%、66.67%、20.00%和13.33%。结论:各生产单位生产的不同品牌阪崎肠杆菌显色培养基质量参差不齐,建议使用者在进行阪崎肠杆菌检查时尽量选择不同生产单位的培养基进行筛查,避免因培养基的质量差异,出现检验结果误判。     

手工和仪器两种血培养结果差异性分析

目的对手工法双相血培养瓶和BACTEC9120全自动血培养仪的阳性率作回顾性分析。方法将血液标本同时接种双相血培养基和BACTEC9120全自动血培养仪配套血瓶中,将阳性结果移种血平板,如为阴性再移种巧克力平板。结果370例血培养,双相血培养瓶阳性25例,阳性率为6．76％（25／370）,树脂需氧（儿童）瓶BACTEC9120报警显示阳性59例,阳性率为15．9％（59／370）,阳性标本移种到血平板及巧克力平板阳性54例,阳性率为14．6％（54／370）,假阳性5例,假阳性率为1．4％（5／370）,共有29例树脂需氧（儿童）瓶阳性,而双相血培养瓶为阴性,P〈0．001。结论BACTEC9120全自动血培养仪提高阳性率,缩短阳性的报告时间优于传统的双相血培养基。     

平菇汤培养基的研究及应用

研究以平菇汤为基础成分,代替动物组织提取物,制备平菇液体培养基,平菇血琼脂培养基。平菇血琼脂培养基对１４０份痰及中段尿标本阳性菌的检出率为３４．３％,传统血琼脂培养基对阳性菌的检出率为３２．１％。平菇液体培养基和普通营养肉汤培养基同时对８０份分泌物标本培养,其检出率分别为３８．８％和３６．３％（ｐ＞０．０５）。平菇汤培养基制备简便,成本低廉,有较好的实用价值。     

药用植物红根草种质资源的离体保存

以红根草试管苗为材料,研究了不同培养基(MS、1/2MS、1/4MS)、蔗糖浓度和植物生长抑制剂(CCC、PP333、ABA、MH)在红根草试管苗保存中的作用。结果表明:培养基1/2MS对红根草保存最好,保存270d后存活率最高。培养基中不添加蔗糖较添加一定浓度蔗糖时植株的形态和色泽差,但保存时间更长,转继代后能正常恢复生长。添加生长抑制剂能减缓生长速度,延长保存时间,最佳浓度分别为:CCC1.2～1.6mg/L;PP3331.6mg/L;ABA0.5～4.0mg/L;MH0.5mg/L。其中,ABA0.5～4.0mg/L对植株的生长最好,CCC浓度为1.2mg/L和1.6mg/L时,保存时间长,360d时,存活率达90%。     

Detection of salmonellas in animal feeds by electrical conductance

A comparison was made between standard culture methods and electrical conductance using a Malthus AT Microbiological Analyser for the examination of animal feeds for salmonella. Conductance testing with a selenite cystine/trimethylamine-N-oxide/dulcitol medium resulted in the detection of salmonellas in 49 of 55 known positive animal feeds, 13 of 19 spiked feed samples and 36 of 47 salmonella cultures. Testing with a lysine decarboxylase/glucose medium gave significantly better results (P less than 0.05) than with selenite cystine medium but five lysine decarboxylase negative strains of salmonella were undetected. When both media were used in parallel all salmonella positive samples were detected. No difference was found between preenrichment in buffered peptone water containing trimethylamine/mannitol and that containing lysine/glucose. Positive detection criteria for selenite medium of conductance peak at greater than or equal to 500 microsiemens (microS) with a rate of change of greater than or equal to 60 microS/h or 400-499 microS with a rate of change of greater than or equal to 40 microS/h and for lysine medium with a peak of greater than or equal to 100 microS have been established. The method offers savings in media and operating costs over conventional standard culture methods, provides results within 48 h and is recommended for statutory feed monitoring purposes.     

虫草培养基添加剂对Wistar大鼠生长发育和繁殖性能的影响

目的探讨虫草培养基添加剂对Wistar大鼠生长发育和繁殖性能的影响。方法将虫草培养基添加在正常大鼠饲料中进行大鼠喂养试验,观察Wistar大鼠的生长发育和交配以及产仔情况进行统计分析。取大鼠卵巢和睾丸组织进行病理组织学观察。结果虫草培养基添加剂饲喂大鼠后,大鼠增重缓慢,雄性大鼠的饲料利用率有所增加,大鼠交配成功率和窝产仔数基本与对照组没有差异,大鼠卵巢和睾丸组织未受到器质性显微损伤。结论虫草培养基添加剂对大鼠生长发育和繁殖性能无特殊影响。     

Urease Color Test Medium U-9 for the Detection and Identification of “T” Mycoplasmas in Clinical Material

A urease color test fluid medium (U-9) for the detection and identification of T (T-strain) mycoplasmas in clinical material is described which is sensitive and specific for this group of mycoplasmas. The medium was prepared from commercially available components and contained 95% half-strength, tryptic digest broth (pH 5.5), 4% unheated horse serum, 0.05% highest-purity urea, 0.001% sodium phenolsulfonphthalein, and 1,000 units of potassium penicillin G per ml. The final reaction of medium U-9 was pH 6.0. The overall agreement (positive and negative) between urease reactions in U-9 urease color test medium and culture findings in a standard agar primary culture system among 686 clinical specimens was 98.1%. The disagreement consisted of 13 false-positive urease reactions which were recognized visually as false-positive reactions due to other microorganisms. For specimens from the female genitourinary tract, the inclusion of 2.5 mug of amphotericin B (Fungizone) per ml of medium U-9 is recommended for the suppression of growth of Candida species and filamentous fungi.     

Perfusion culture of hybridoma cells for hyperproduction of IgG(2a) monoclonal antibody in a wave bioreactor-perfusion culture system

A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in perfusion culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale.     

酸奶中保加利亚乳杆菌分离条件的优化

为优化从酸奶中分离保加利亚乳杆菌的培养条件,初步建立一种较简单有效的分离方法。以成都市市售的主要酸奶－华西牌瓶装酸牛奶为来源,采用MRS、SL、LS、蕃茄酵母4种经典的乳酸菌选择性培养基,并分别进行有氧及烛缸厌氧分离培养测定其得率。采用X^2检验对不同组的分离得率进行统计学分析。结果表明MRS培养基为酸奶中保加利亚乳杆菌分离首选培养基,分离得率达66．67％;微氧环境优于有氧环境（PO＜0．05）。     

培养山羊痘病毒常用细胞在X-gal环境中的显色差异分析*

目的：为筛选出表达LacZ基因重组山羊痘病毒的适宜培养细胞系。方法：将常用于培养山羊痘病毒(GPV)的乳仓鼠肾细胞(BHK21)、羊肾细胞(SK)和羔羊睾丸细胞(LT)培养至单层,之后单层细胞用含X-gal的培养基继续培养,观察比较各细胞在X-gal环境中呈现的蓝色。提取单层细胞的RNA,进行LacZ基因的RT-PCR,回收PCR产物、连接载体、转化E.coli,挑阳性克隆测序分析。将单层细胞继续培养72 h,检测各细胞产生的β-半乳糖苷酶。结果：随着培养时间的延长,固体培养的BHK21、LT细胞孔中胶颜色变蓝且逐渐加深,显微镜观察可见细胞蓝斑,而SK细胞、空白、无X-gal孔中的胶未变色,镜检无细胞蓝斑;液体培养的单层细胞逐渐脱壁,培养液未见蓝色。RT-PCR产物电泳条带与预期分子量大小相符,测序结果显示目标DNA与LacZ基因序列相似性值达100%,表明3种细胞均有LacZ基因的转录本。β-半乳糖苷酶测定结果显示3种细胞均表达此酶,细胞产酶能力比较为BHK21>LT>SK,尤以SK细胞产β-半乳糖苷酶量极低。结论：显色差异法筛选出的羊肾细胞适宜培养表达LacZ基因的重组山羊痘病毒,结果可为山羊痘病毒新型疫苗研发提供一些参考。     

Design and validation of a pulsatile perfusion bioreactor for 3D high cell density cultures

This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real‐time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48‐h culture period. Cells were uniformly dispersed within the 14.40 mm × 17.46 mm × 6.35 mm chamber. Cells suspended in 6.35‐mm thick gels and cultured in a traditional CO₂ incubator were found to be round and dead. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel. Biotechnol. Bioeng. 2009; 104: 1215–1223. © 2009 Wiley Periodicals, Inc.