临床厌氧菌的分离和鉴定

我们从口腔、胸部、腹部和盆腔等处采集了110份临床感染标本,以自制的输送培养基立即送实验室,在厌氧手套箱(霍尔玛厌氧系统1029型)或Gas-pak罐中,行厌氧菌的分离和培养,其中64份标本检出厌氧菌,阳性率为58％。临床标本中牙周炎标本厌氧菌检出率高达100％,牙髓炎标本为85％,阑尾脓肿和腹膜炎标本为83％,胆道标本为39％,脓胸标本为57％,盆腔标本为33％,早期单纯性阑尾炎和甲状腺囊肿合并感染的标本各5份,都未检出厌氧菌。从64份阳性分离的标本中共分离到厌氧菌370株,经鉴定分别属于11个菌属32个菌种(未定种的有74株),其中类杆菌最多占45.9％(类杆菌属中脆弱类杆菌占37.6％),次为梭杆菌属和消化链球菌属,各占15.1％。革兰氏阳性无芽胞厌氧菌占8％左右,而梭菌属为8.9％,其余是二氧化碳噬纤维菌属(2.9％)、韦荣氏球菌属(1.3％)、链球菌属和纤毛菌属(1.5％)等。在厌氧菌鉴定中,我们使用了微量生化直接酶测定技术和代谢产物的气相色谱分析技术,这些方法在厌氧菌鉴定中比常规方法敏感且有较大的价值。     

鸡骨骼肌卫星细胞的分离培养、鉴定及生物学特性研究

采用I型胶原酶和胰蛋白酶二步消化,经体外培养获得鸡骨骼肌卫星细胞,并通过检测肌卫星细胞特异基因的表达进行鉴定。结果表明：二步消化法适用于鸡骨骼肌卫星细胞的分离和获取,此方法分离得到的鸡骨骼肌卫星细胞表达卫星细胞特异的标志基因desmin和Pax7,并具有良好的增殖和分化能力,为鸡骨骼肌细胞增殖、分化和再生机制的研究提供技术平台。      

Isolation of Anaerobic Bacteria from Human Gingiva and Mouse Cecum by Means of a Simplified Glove Box Procedure

An anaerobic glove box constructed of clear flexible vinyl plastic is described. It is sufficiently inexpensive and simple in operation to be used not only in research but also in a clinical laboratory by technicians without special training. Conventional bacteriological techniques may be used inside the glove box for culturing and transferring anaerobic bacteria. The box may be heated to 37 C and thus serve as an anaerobic incubator as well, permitting inspection of cultures at any time. Media may be prepared and agar plates may be poured on the laboratory bench in the conventional manner. An overlay of trace amounts of palladium black catalyst over plated agar media reduces the medium to an oxidation-reduction (O-R) potential of - 300 mv within 2 days after introduction into the glove box. In spite of its greater simplicity, the system matched or excelled the roll tube method with respect to all parameters tested, including O-R potential obtainable in the media, O(2) concentration in the gas phase, and efficiency in isolating anaerobic bacteria from the mouse cecum. Comparative studies indicate that the conventional anaerobic jar method was inadequate for the isolation of strict anaerobes from human gingival specimens and from the mouse cecum. This was due to the exposure of specimens and media to air during plating on the open laboratory bench. Anaerobic jars were adequate for maintaining the proper conditions for growth of anaerobic bacteria once these had been established in the glove box.     

好氧反硝化菌的分离鉴定及特性研究

从土壤中分离到1株能以硝酸钠为氮源进行好氧反硝化作用的细菌,命名为Rhodococcussp.DN,分离菌株革兰氏染色为阳性,球状或杆状,菌落颜色为橙红色。该细菌能以乙酰胺为惟一碳源和氮源,能进行氨化和硝化作用并产生亚硝酸。部分长度的16S rDNA序列分析表明,所分离的细菌与Rhodococcus ruber的16S rDNA序列具有99%相似性。     

一株石油烃降解菌分子鉴定及特性分析

从受污海水中分离筛选了具有石油烃降解能力的降解菌Bac1020,经PCR扩增得到1 497 bp长16S rDNA序列,通过Blast比对,与主要石油烃降解菌属16S rDNA序列构建系统发生树,鉴定其为不动杆菌(Acinetobactersp.)。降解菌(Acinetobactersp.)在72 h内生长稳定,对石油烃的降解率随时间的延长而不断增加。建立了快速筛选及鉴定石油烃降解菌的方法,应用于海洋石油烃污染的生物降解。     

氯嘧磺隆降解菌的分离鉴定及其降解特性

利用富集培养技术从长期施用氯嘧磺隆的土壤中分离得到1株能够降解氯嘧磺隆的细菌L-7。通过生理生化特性和16SrRNA序列分析,初步鉴定L-7为寡养单胞菌属(Stenotrophomonas sp.)。并分析了氯嘧磺隆的初始浓度、接种量、温度和pH值对L-7菌株降解氯嘧磺隆效果的影响,确定了最佳降解条件。结果表明,该菌在氯嘧磺隆浓度为100mg/L、接种量为5%、pH4.0、温度30°C条件下,接种5d后对氯嘧磺隆的降解效率达到80%以上。     

麝鼠肠道纤维素分解菌的分离鉴定及其酶学特性分析

本研究旨在从麝鼠(Ondatra zibethicus)肠道中分离出高效分解纤维素的菌株,为开发纤维素分解菌微生物制剂提供菌种资源。本研究利用以羧甲基纤维素钠(CMC-Na)为单一碳源的培养基,从麝鼠盲肠内分离出--株高效分解纤维素的菌株WJ-3,并对该菌株进行形态鉴定、生理生化鉴定和16S.rDNA分子鉴定。对菌株WJ-3所产羧甲基纤维素酶(CMCase)进行酶学特性实验,分析此纤维素酶的最佳反应pH和最佳反应温度,以及此纤维素酶对不同温度和不同酸碱度的耐受性。结果表明,菌株WJ-3属于空气芽孢杆菌(Bacillus aerius),并将其命名为Bacillus aerius WJ-3。菌株WJ-3所产羧甲基纤维素酶在pH 4.0~6.0的范围内反应时,酶活性随pH值升高而增加,其最佳反应pH为6.0,且此纤维素酶在pH4.0~8.0范围内保存30min后均能保持80%以上的相对酶活性:菌株WJ-3所产羧甲基纤维素酶在温度30~50 ℃范围内反应时,随温度上升酶活性逐渐增加,在50 ℃时酶活性最高,之后随温度的升高酶活性逐渐下降,且纤维素酶在此温度范围内保存30 min后均能保持较高的酶活性。综上所述,菌株Bacillus aerius WJ-3所产羧甲基纤维素酶的酶活性较高,并且此纤维素酶的耐酸碱性及热稳定性良好,是具有一定利用价值的菌种资源。     

Identification of Anaerobic Selenate-Respiring Bacteria from Aquatic Sediments

The diversity population of microorganisms with the capability to use selenate as a terminal electron acceptor, reducing it to selenite and elemental selenium by the process known as dissimilatory selenate reduction, is largely unknown. The overall objective of this study was to gain an in-depth understanding of anaerobic biotransformation of selenium in the environment, particularly anaerobic respiration, and to characterize the microorganisms catalyzing this process. Here, we demonstrate the isolation and characterization of four novel anaerobic dissimilatory selenate-respiring bacteria enriched from a variety of sources, including sediments from three different water bodies in Chennai, India, and a tidal estuary in New Jersey. Strains S5 and S7 from India, strain KM from the Meadowlands, NJ, and strain pn1, categorized as a laboratory contaminant, were all phylogenetically distinct, belonging to various phyla in the bacterial domain. The 16S rRNA gene sequence shows that strain S5 constitutes a new genus belonging to Chrysiogenetes, while strain S7 belongs to the Deferribacteres, with greater than 98% 16S rRNA gene similarity to Geovibrio ferrireducens. Strain KM is related to Malonomonas rubra, Pelobacter acidigallici, and Desulfuromusa spp., with 96 to 97% 16S rRNA gene similarity. Strain pn1 is 99% similar to Pseudomonas stutzeri. Strains S5, S7, and KM are obligately anaerobic selenate-respiring microorganisms, while strain pn1 is facultatively anaerobic. Besides respiring selenate, all these strains also respire nitrate.     

一株中度嗜盐菌的分离鉴定及特性研究

目的：从盘锦红海滩泥样中分离中度嗜盐菌,对其进行鉴定及特性研究。方法：CZ培养基分离纯化,通过形态学、生理生化实验、16SrDNA序列比对分析进行鉴定,吸光光度法测定生长特性。结果：分离得到一株中度嗜盐菌CNY0802,该菌革兰氏阳性反应,菌体杆状,宽度0.7μm～0.9μm,长度2μm～3μm,产芽孢,接触酶、酯酶、明胶液化和硝酸盐还原反应阳性,氧化酶、淀粉酶、MR和VP反应均为阴性。生长的盐度范围0.5%～25%（NaCl,W/V）,最适盐度5%;温度范围10℃～45℃,最适温度30℃;pH范围4.0～12.0,最适pH8.0～10.0;不同的阴阳离子对CNY0802生长影响显著。经16S rDNA序列比对及系统发育树分析,与Halobacillus salinus亲缘关系最近。结论：该菌的分离鉴定对我国辽宁沿海地区极端环境微生物资源的开发研究有一定意义。     

一株轻度嗜盐反硝化细菌的分离鉴定和反硝化特性初探

从处理高盐度废水的成熟活性污泥中分离筛选得到1株轻度嗜盐反硝化细菌GYL, 通过对该菌株的形态观察、生理生化实验以及16S rDNA序列分析, 确定该菌株为盐单胞菌(Halomonas sp.)。该菌株能在盐度为10%的培养液中生长, 最适盐度为2%~7%, 最适pH为7.5~8.5, 最佳碳源为蔗糖, 在25°C~30°C的温度范围内脱氮效率达到80%以上。对该菌株的异养硝化能力进行了测定, 其对氨氮的去除率可达98.3%, 说明该菌株可实现同步硝化反硝化, 即该菌可以独立完成生物脱氮的全部过程。     

北京油鸡肺间充质干细胞的分离培养及鉴定

以9-14日龄北京油鸡肺脏组织为材料,Ⅳ型胶原酶消化,percoll分离,获得肺间充质干细胞.进行免疫组化和RT-PCR鉴定,并比较3种培养体系对北京油鸡肺间充质干细胞增殖的影响.RT-PCR结果显示,细胞免疫组化签定结果CD44、CD29呈阳性,CD34呈阴性.证实所培养的细胞为北京油鸡肺间充质干细胞;比较不同扩增培养体系,结果表明,培养体系DMEM/F 12+10％FBS+2.5 ng/mL bFGF较有利于北京油鸡肺间充质干细胞的增殖.该试验成功地分离并鉴定了北京油鸡骨骼肌卫星细胞,建立了适于北京油鸡肺间充质干细胞体外扩增的培养体系.     

低营养浓度培养基分离培养生物膜中的细菌及其鉴定

室内潮湿环境固体表面很容易形成细菌生物膜,这些生物膜生长在不易清除或不被人注意的固体表面,研究这一类生物膜中的细菌类群可以了解其中是否滋生着对人体健康有威胁性的致病菌或条件致病菌。选择某公共浴室内塑料表面生长的生物膜作为分离培养对象,同时使用实验室最常用的LB培养基及其10倍稀释培养基(LB/10)作为分离培养基。结果表明在LB培养基上生长的细菌类群相对单一,并且有单一类群占优势或抑制其他类群生长的现象;而在LB/10培养基上生长的细菌具有更丰富的多样性,并且各类菌的数量分布较均匀,没有出现LB培养基中一种细菌占绝对生长优势的现象。对LB/10上生长的8个菌株的16SrDNA片段序列进行测定和系统发育分析,发现与所分离的细菌系统发育相近的包括多株人体临床分离的病原菌以及未培养细菌(Uncultured bacterium)。     

鸡大肠杆菌病病原的分离鉴定和部分生物学特性研究

无菌采取某鸡场送检的疑似大肠杆菌病的病、死鸡的心血、肝、脾、肾,进行病原菌的分离、生化鉴定、动物实验和药敏试验。生化鉴定结果显示引起该鸡场鸡只发病的病原为大肠杆菌,并且该菌对小白鼠和鸡有较强的致病力。药物敏感性试验结果显示,该菌对先锋V、头孢呋肟、头孢噻肟敏感,对庆大霉素和氧氟沙星不敏感,而链霉素、新诺明、红霉素、青霉素、氨苄西林、四环素对它完全没有抑制效果。结果表明:该鸡场出现了耐药的致病性大肠杆菌,在以后的肉鸡生产中应慎用抗生素,在使用药物治疗大肠杆菌病时,应根据药敏试验结果,选择敏感药物,且应注意交替用药,按疗程投药。     

一株甲烷氧化菌的分离鉴定与特性

甲烷氧化菌是一类能以甲烷作为唯一碳源和能源进行同化和异化代谢的微生物。从若尔盖高原不同地点采集的样品中筛选得到一株名为XN1的甲烷氧化菌,根据此菌株的形态与16SrRNA序列同源性分析,证实该菌株属于Methylomonas属。对该菌株的培养条件进行研究的结果表明,以甲烷与甲醇共同作为碳源,硝酸钾和氯化铵共同为氮源时菌生长最好,最适生长温度为25℃,最适生长pH为6.5,培养基中CuSO4·5H2O和FeSO4·7H2O的浓度以0.03mg/L和0.4mg/L为宜。     

Comparison of Methods for Isolation of Anaerobic Bacteria from Clinical Specimens

Five different anaerobic culture methods and several different media were compared for their ability to recover anaerobes from clinical specimens. Specimens were obtained from patients with documented infections, avoiding contamination with normal flora, and immediately placed in an anaerobic transporter. Each specimen was cultured by all methods and on all the various media. The comparative data indicate that anaerobic jars (GasPak and evacuation-replacement types) are just as effective in the recovery of clinically significant anaerobes as the more complex roll-tube and chamber methods employing prereduced media. Liquid media were disappointing as a "back-up" system but chopped-meat glucose was superior to two thioglycolate formulations. Growth of all anaerobes was poorer on selective media, but these media were very helpful in the workup of specimens containing mixed growth of anaerobic and facultative organisms. A variety of different anaerobes was isolated, but no very fastidious or extremely oxygen-sensitive organisms were recovered. This suggests that such organisms may not play a significant role in causing clinical infections.     

成年小鼠心肌细胞的分离培养及鉴定

目的探讨成年小鼠心肌细胞分离培养的方法,评价心肌细胞的存活状况。方法应用改良Langendorff方法进行成年小鼠心脏灌流,分离并培养心肌细胞,评价杆状心肌细胞的比例变化。台盼蓝染色判定心肌细胞活力,应用毒胡萝卜素(thapsigargin)治疗心肌细胞24 h后,WST方法测定细胞生存率。结果在每个成年小鼠心脏分离了40~60万的心肌细胞,台盼蓝染色结果68%为存活心肌细胞。心肌细胞培养1 h2、4 h及48 h后,杆状心肌细胞的比例分别为70%、50%及30%。心肌细胞生存率随着毒胡萝卜素浓度的增加逐渐减低,其中1、5及10μmol/L毒胡萝卜素的生存率明显低于对照组(P<0.05,P<0.01)。结论应用改良Langendorff方法及严格控制心脏灌流培养的条件,可以成功地分离培养成年小鼠心肌细胞,有助于进行细胞分子水平的研究。     

犬皮肤成纤维细胞的分离、培养及鉴定

目的探索和建立适用于犬皮肤成纤维细胞的体外分离、培养及鉴定的技术方法。方法采用组织贴块培养法和胰蛋白酶、胶原酶Ⅰ联合消化法对犬皮肤成纤维细胞进行体外培养、传代。并对所培养的细胞进行倒置显微镜观察和苏木素-伊红染色,观察成纤维细胞形态,并对培养细胞行波形蛋白免疫荧光染色。结果倒置相差显微镜下可见长梭形细胞生长,苏木素-伊红染色可见细胞呈漩涡状、平行排列,第5代细胞免疫荧光检测波形蛋白(vimentin)表达阳性。结论建立了高效快速分离和稳定培养成纤维细胞的方法,为诱导犬心房纤维化提供了充足的种子细胞。     

1株蛇足石杉根际铁载体细菌的分离、筛选与鉴定

对蛇足石杉根际铁载体细菌进行了分离、筛选与鉴定,以期筛选到潜在高效促生细菌.采用梯度稀释涂平板法从蛇足石杉根际土中分离细菌,再通过CAS检测平板分析,从中筛选出1株产生铁载体能力较强的菌,并结合16S rDNA基因系统发育分析对可产铁载体的蛇足石杉根际细菌初步鉴定.从蛇足石杉根际中筛选到1株产铁载体细菌JSX 389,经鉴定为Lysinibacillus属菌株.     

油茶根际溶磷菌的分离、鉴定及溶磷能力研究

采用传统的微生物分离培养法,对油茶根际溶磷细菌进行了分离,共筛选得到17株溶磷细菌。利用透明圈法对油茶根际土壤中具有溶磷能力的细菌进行初筛;采用钼锑抗比色法测定发酵液的可溶性磷含量,对解磷菌株进行复筛,得出菌株6-Y-09溶磷活性最强。根据进行菌落形态特征、生理生化特征、16S r DNA序列和系统发育分析等研究,初步鉴定菌株6-Y-09为洋葱伯克霍尔德菌。该菌株在后续微生物菌肥研制中具有较大潜力,为通过生物途径改善油茶磷素供应,促进油茶生长提供了优良的菌株资源。