应用竞争PCR方法检测亚硝化细菌的研究

选用竞争PCR技术建立了亚硝化细菌的快速定量检测方法.实验以实验室分离、鉴定的一株亚硝化细菌提取的DNA为模板,设计特异性引物,制备特异性内标模板,进行竞争PCR实验;同时采用传统的平板计数法和吖啶橙荧光显微镜计数法两种方法进行细菌计数,与竞争PCR结果比较分析,建立了样品细菌数量与内标模板量的线性方程,并将其应用于海水养殖环境中亚硝化细菌的检测中.该方法具有简化检测过程、缩短细菌检测时间、无须昂贵仪器设备等优点,并克服有些环境微生物难以培养的困难,为将来有益菌在应用中实际存活和繁殖情况的监测提供了有力的技术支撑.     

绿色荧光蛋白标记的嗜水气单胞菌在越冬水体的饥饿存活及复苏

将绿色荧光蛋白标记的嗜水气单胞菌（Ah4332^GFP）置模拟越冬水体（8-10℃）内,进行饥饿存活试验,用三种计数方法检查水体中的细菌数量变化,在41d后平板计数法（PC）降至0。而细菌总数法（DC）和活菌直接计数法（DVC）结果相似,只是DVC计数结果低于细菌总数1-2个数量级,显示细菌已经变成活的非可培养（Viable but nonculturable,VBNC)状态,复苏试验表明,升高温度、添加鱼血清或新鲜培养的Ah4332^GFP细菌上清及通过兔肠管结扎,均使VBNC状态的Ah4332^GFP得到复苏,荧光显微镜和电镜观察处于可培养的VBNC状态的Ah4332^GFP,后者的细菌细胞比前者体积明显缩小,形态由杆状变成了球形,但细胞膜和细胞壁是完整的,不是细菌L型。     

MTT法检测细菌细胞数的主要影响因素分析

MTT法是生物活细胞计数的灵敏、快速和便捷方法。报告对MTT法应用于细菌细胞数定量分析时的分析前培养时间、MTT反应时间、MTT剂量和检测波长等主要影响因素的分析结果,同时报告MTT反应产物溶解检测与直接检测,以及MTT法与平板菌落计数法定量检测的结果比较。结果如下：Formazan生成量与细菌活细胞数呈正相关,对数生长期变化最明显;MTT为0.25 mg/mL,反应2 h时吸光值与菌落数具有良好的线性关系（r=0.999 8）;Formazan的最大吸收波长为570～580 nm;MTT反应产物溶解检测和直接检测结果经统计学分析无显著性差异（P〉0.05）;细菌细胞数在107～109 cfu/mL范围内,MTT法与菌落计数法定量结果呈现良好的线性关系（r≥0.991 7）。     

"活的非可培养状态"细菌荧光观察方法

目的：建立一套稳定、简便、快捷,可靠的细菌活的非可培养状态（VBNC）荧光显微镜观察方法。利用市售的黑色钢笔墨水着染微孔滤膜,然后采用手动压片方式,将经LIVE/DEAD BacLightTM Bacterial Viability Kit（13152）染色的细菌样本固定在滤膜上,用落射荧光显微镜直接观察。结果：该法所得的荧光图像背景无光亮且黑暗,菌体荧光、形态、排列等均清晰可见,而且呈现绿色荧光的活菌与呈现红色荧光的死菌较易分辨。与现行技术相比,还能缩短染色时间近12～16h。结论：建立的VBNC细菌荧光观察方法能有效地克服现有技术存在的适用范围狭窄、取材困难、观察结果不准确、操作时间长等缺点,可为细菌VBNC研究提供值得借鉴的方法。     

新的酵母细胞自动计数仪

BioScience Technology 05／2005（增刊）10页报道：美国新不伦瑞克科学公司最近推出了一种可以在30秒内自动计数酵母细胞的新的仪器,从而结束了手工计数酵母细胞的历史。与原先可以自动计数哺乳动物细胞的核计数仪相似,这种新的可用于计数酵母细胞的YC-100型核计数仪,包含荧光显微镜、CCD照相机和整合影像分析软件,可快速而准确地对酵母细胞进行计数。它可以根据DNA在数秒钟内进行细胞计数,     

激光共聚焦显微镜在肿瘤免疫治疗检测中的应用

目的:通过激光共聚焦显微镜对肿瘤生物治疗后患者的外周血淋巴细胞进行亚群计数,为生物治疗后外周血淋巴细胞无法分群的肿瘤患者提供新的监测免疫功能状态的方法。方法:收集35例肿瘤生物治疗后患者的外周血标本,通过激光共聚焦显微镜和流式细胞仪两种方法分别对患者外周血淋巴细胞亚群进行分类计数。结果:流式细胞仪和激光共聚焦显微镜同时分类计数的患者外周血细胞标本30例,两种方法在检测CD3、CD3~+/CD4~+、CD3~+/CD8~+、CD3-/CD16~+56~+、CD3-/CD19~+细胞时均无统计学差异(P值0.05);5例流式细胞仪无法将患者外周血淋巴细胞分群的样本,通过激光共聚焦显微镜可以进行分类计数。结论:激光共聚焦显微镜亦可以用于外周血淋巴细胞的分类计数。     

VBNC细菌荧光观察技术问题分析及对策

以SYTO-9和PI两种荧光染料对细菌进行核染,以市售的黑色钢笔墨水替代伊拉克黑,利用手动压片方式对标本进行固定,自建一种"活的非可培养状态"(VBNC)细菌荧光显微镜观察技术。通过总结分析该项技术方法在应用过程中所出现的问题,如存在背景荧光、菌体聚集、荧光图像模糊、滤膜吸附能力差、菌体形态不鲜明、菌体荧光减淡等,并提出相应的解决策略,旨在给予相关研究者以帮助和启迪。     

应用双光子显微镜和流式细胞仪定性及定量确定小鼠巨噬细胞的吞噬功能

建立了流式细胞仪和双光子激光共聚焦荧光显微镜进行定性和定量检测小鼠巨噬细胞吞噬鸡红细胞的方法,并同传统光学显微镜细胞化学染色观察方法相比较,探讨其检测巨噬细胞吞噬效应的优越性。常规方法获取小鼠腹腔和脾脏巨噬细胞,制备巨噬细胞悬液。常规制备鸡红细胞,计数并调整活细胞数,用5-二醋酸羧基荧光素琥珀酸单胞菌酯(5-carboxyfluorescein diacetate succinimidyl ester,CFSE)染色,与巨噬细胞共温育一定时间后,小鼠巨噬细胞特异性荧光抗体F4/80标记巨噬细胞。应用流式细胞仪检测巨噬细胞中CFSE阳性百分率来表示巨噬细胞吞噬率;应用双光子显微镜观察被吞噬的CFSE阳性鸡红细胞动态分布情况。同时,采用传统光学显微镜吉姆萨染色观察巨噬细胞吞噬百分率。结果显示,流式细胞仪结合双光子显微镜检测巨噬细胞吞噬率与传统的显微镜计数法比较,两者有明显的正相关性。双光子显微镜和流式细胞仪可以定性与定量检测巨噬细胞吞噬功能,该方法具有灵敏、快捷、重复性好以及准确率高的特点,是进行免疫学研究的可行方法。     

海洋病毒荧光显微计数法的优化与应用

【目的】建立一种快速、稳定、可靠的海洋病毒计数方法。【方法】海水水样经福尔马林固定后,滤过孔径为0.02μm的Anodisc Al2O3膜。滤膜经SYBR Green I染色后,在相应波长的激发光下进行观察。借助荧光显微镜目镜网格尺,计数视野中的病毒颗粒,换算后获得样品中病毒的浓度。【结果】对具体实验方法进行了优化,可快速、稳定地对海水中的病毒计数。【结论】建立了一种适用于国内实验条件的、可靠的海洋病毒计数方法。     

一种简便鉴定细菌、真菌的显微镜

最近,以色列Ben-Gurion大学应用生物科学研究所Erukhimovitch等介绍了一种傅立叶转换红外显微镜[Fourier-transform infrared（FTIR）microscopy]广泛而又敏感地测定细胞的分子转换而用于鉴定细菌、真菌。这种新型显微镜比通常已广泛应用的傅立叶转换红外光谱仪（FTIR spectroscopy）有明显优势,即后者只能测定一个样品的一定局限范围,而前者则可测定更广范围的标本,是更简便、快速且准确可信的鉴定细菌、真菌的新仪器,用于各种人类和动物感染细菌和真菌的鉴别。     

Electronic sizing and counting of bacteria

An electronic apparatus is described that permits rapid determination of the concentration and size distribution of bacteria in electrolyte suspensions by a resistance method. The resulting size-concentration distribution may be displayed on an oscilloscope and recorded with an X–Y plotter and an electric typewriter-tape punch unit. The paper tape is analyzed with a computer program. Comparisons are made between electronic measurements of bacterial cell concentration and size distribution and values obtained by other methods. Effects of heat-killing and disruption of the cell membrane on the electrical counting characteristics of the organisms are discussed.     

最大或然数法在光合细菌计数中的应用及效果研究

1引言光合细菌(photosynthetic bacteria,PSB)是能进行不放氧光合作用的一大类细菌的总称,属水圈微生物,广泛分布于地球生物圈,无论江、河、湖、海,水田、旱地都有存在[3].光合细菌在水体自净、调节微生态平衡、促进动植物生长、增加产量和提高产品质量、防病、固氮等方面具有重要作用[3,22].近年来,光合细菌菌剂在水产养殖、家畜养殖、污水处理及植物生产上的应用日益广泛[3,13,19,27,28,31],相继推出多种产品,包括单菌菌剂、复合菌剂,剂型上有液体菌剂、浓缩菌剂和固体菌剂(粉剂)等[20].为保证产品质量和应用效果,有必要对光合细菌菌剂…     

The viable but non-culturable state of wine micro-organisms during storage

Colony counting and DEFT did not give the same results when wine micro-organisms were enumerated. Both methods were used to monitor the population of acetic acid bacteria (AAB) and lactic acid bacteria (LAB) during wine storage. Results suggest that part of the populations had reached a viable but non-culturable (VBNC) state. These cells were unable to produce colonies but could hydrolyse fluorescent esters and could be counted by DEFT. For AAB, O2 deprivation quickly induced this state. Recovery from this state was very rapid as soon as O2 was available. The response was not so clear for LAB during wine storage. However, a similar state was induced by sulfiting. Moreover, filtration of wine stored in barrels and contaminated by Brettanomyces, AAB and LAB demonstrated that cell size was not homogeneous. Cells which remained in wine after several weeks could pass through a 0.45-microm membrane. However, when they re-entered a growing phase, they were again retained by membrane filtration. During and after the decline phase, wine micro-organisms might survive as smaller cells in a VBNC state.     

DIRECT ENUMERATION OF BACTERIA FROM MACROALGAE BY EPIFLUORESCENCE MICROSCOPY AS APPLIED TO THE FLESHY RED ALGAE KAPPAPHYCUS ALVAREZII AND GRACILARIA SPP. (RHODOPHYTA)1

This report describes a simplified method for direct counting of total bacteria associated with the fleshy red algae Kappaphycus alvarezii (Doty) Doty and Gracilaria spp. A Nuclepore® polycarbonate membrane (0.2–μm pore size) fitted to a vacuum filtration apparatus was used to filter algal tissue homogenate after serial dilution and staining with the fluorochrome 4′,6-diamidino-2-phenylindole. Using epifluorescence microscopy, it is possible to count bacteria without preseparating them from the algae. The technique requires homogenized algal tissue diluted with 0.2-μm-filtered, autoclaved seawater to a level appropriate for counting. Dilution reduces the amount of autofluorescent algal debris, which may interfere with Counting. The membrane filtration method yielded a bacterial count two orders of magnitude higher than that of the conventional agarspread plate technique. This method offers a more accurate approach to counting the total number of bacteria on macroalgae.     

Use of Membrane Filter Technique in the Microbiological Control for the Brewing Industry

A physical method was developed involving serial filtration with membrane filters for separating yeast cells from bacteria. Such a method eliminates the need for antibiotics previously required to permit differential counting of such populations. All yeast cells filtered were successfully retained and cultivated on a 1.2-mu membrane filter by use of a synthetic medium. All bacteria filtered avoided entrapment on a 1.2-mu membrane filter and were successfully retained and cultivated on a 0.22-mu membrane filter with the same synthetic medium. Final filtrates from these serial filtrations were free from all yeast cells and bacteria when tested with Fluid Thioglycollate Medium.     

Development of detection system of extraterrestrial microorganisms]

A noble method for the exploration of terrestrial and extraterrestrial soil microorganisms, especially targeted for Mars, has been developed. The method is based on the microscopic observation using fluorescence techniques. Microorganisms could be fluorescent by adsorption, enzymatic cleavage of extrinsic fluorescence chromophores such as acridine orange, ANS and SFDA, and also by intrinsic chromophores. The characteristic points of our fluorescence method are shown below. 1. The present method detected all the culturable cells tested (about 200 species from bacteria to eukaryofic cells). 2. Microorganisms in soil were much brighter than background fluorescence of soil. Cell shapes and location were clearly observed. 3. An esterase substatum SFDA, discriminated vital (reproductive) cells from dead. On the other hand, a membrane probe, ANS, detected both vital and dead cells. 3. Pre-treatment of cells with bleaching reagents improved the detection efficiency. Especially, this pretreatment was effecfive in Fungi with black chromophores. 4. Some anaerobic microorganisms such as methanogenic bacteria with intrinsic chromophores can be detected without stain. 5. Application of the technique to terrestrial soil revealed that more than 100 times larger cell density was obtained compared to the value obtained by the classic plate counting technique. Vertical distribution of microorganism of soil microorganisms from Mt. Shigayama showed that, at surface, cell density was small and maximum was shown below 15 cm from surface. 6. Some pre-biotic cell (cell like aggregates composed of amino acids) could be detected by SFDA or ANS. It can be concluded that the fluorescence technique is one of the most promising method for the exploration of extraterrestrial microorganisms.     

Association of Rhizobium Strains with Roots of Trifolium repens

TWO TECHNIQUES WERE USED TO ASSESS THE BINDING OF RHIZOBIA TO CLOVER ROOTS: indirect counting after radiolabeling the bacteria and direct counting by using phase-contrast microscopy. Microscopic observations revealed a large variability in the number of bacteria associated with individual root hairs. This variability made unbiased counting by microscopy difficult. Systematic examination of all visible root hairs and "blind" counting of coded strains and treatments were adopted to minimize observer bias. The validity of the radiolabeling method was also examined in some detail. The reproducibility of results from this method was satisfactory. However, drawbacks of this method included its lack of sensitivity and its failure to distinguish between bacteria attached to mature root hairs, emerging root hairs, and undifferentiated epidermal cells. The method also failed to distinguish between individual bacteria and any aggregates that may be present. The ability of a number of chosen mutant strains of Rhizobium trifolii and their corresponding parent strains, as well as a number of nonhomologous strains, to bind to clover roots was assessed by using both of these methods. Our results gave no indication of specificity of R. trifolii binding to clover roots. 2-Deoxy-d-glucose did not appear to have a major inhibitory effect on the attachment of rhizobia to the host root, which suggests that lectin cross-bridging is not an obligatory step in the initiation of infection even though it may occur under some conditions. The presence or absence of the symbiotic plasmid was not correlated with bacterial adherence to the host plant root. Since host specificity functions are carried on this plasmid, our results suggest that binding of rhizobia to the legume root is not the basis of host specificity.     

A note on 'plotless' methods for estimating bacterial cell densities

R oser , D., N edwell , D.B. & G ordon , A. 1984. A note on "plotless" methods for estimating bacterial cell densities. Journal of Applied Bacteriology 56 , 343–347.
'Plotless' techniques for determining population densities have been developed for, and applied to, higher plant populations. They can often be carried out more rapidly than techniques involving total counts of individuals in plots, or quadrants, but such plotless techniques have not been generally applied to the estimation of densities of bacterial cells. Direct microscopical counting of cell numbers in a field of view, an example of a plot-related method, has been traditionally used for micro-bial cell counts. In this study 'plot' and 'plotless' methods on a variety of bacterial samples are compared. Estimates of bacterial cell density were obtained by measuring the distance of cells from a fixed point in a field of view. The values, which were more rapidly obtained, were directly correlated with total cell counts. Although there was some apparent deviation from a perfect 1:1 relationship with total counts, as indicated by a correlation coefficient less than 1.0, there were no significant differences between the replicated counts of bacteria on samples of tissue from the surface of Hypholoma basidiocarps ( P < 0.05). This indicated that the methods of enumeration were comparable. The distance-related estimates could readily be obtained from fields of view with cell densities varying over several orders of magnitude. It was more rapidly applied, particularly at high density, and the method was applicable not only to random cell distributions but also to the non-random distributions encountered when microbial cells aggregated into micro-colonies. The method appears to be particularly well-suited for automated, digitized, direct counting procedures, as well as to estimating bacterial numbers on membrane filters and natural substrates.     

Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present.

Understanding the role of bacteria in microbial food webs is intimately connected to the methods applied in the direct enumeration of bacteria. We have examined over 220 papers describing studies in which fluorochrome staining followed by epifluorescent microscopic direct counts was used to estimate total bacterial abundances. In this review, we summarize patterns in the use of 3,6-bis[dimethylamino]acridinium chloride (acridine orange) and 4',6-diamidino-2-phenylindole (DAPI), the two stains most frequently used in bacterial enumeration. The staining of samples with these fluorochromes, followed by filtration and direct counting of bacterial cells on filter surfaces, has become routine over the past 10 years. We examine trends in features of the standard direct count methods, such as sample preservation and preparation techniques, membrane filter types used, applied stain concentrations, duration of staining, and counting strategies, in relation to the types of samples being examined. The high variability in bacterial counts observed within similar sample types may be partially accounted for by differences in methods. Synthesizing review findings, we include a recommended method for the direct enumeration of bacteria in environmental samples.     

Method for Radiorespirometric Detection of Bacteria in Pure Culture and in Blood

Methods are described for the detection of low numbers of bacteria by monitoring (14)CO(2) evolved from (14)C-labeled substrates. Cell suspensions are filtered with membrane filters, and the filter is then moistened with 0.1 ml of labeled medium in a small, closed apparatus. Evolved (14)CO(2) is collected with Ba(OH)(2)-moistened filter pads and assayed with conventional radioactivity counting equipment. The kinetics of (14)CO(2) evolution are shown for several species of bacteria. Fewer than 100 colony-forming units of most species tested were detected in 2 h or less. Bacteria were inoculated into blood and the mixture was treated to lyse the blood cells. The suspension ws filtered and the filter was placed in a small volume of labeled medium. The evolved (14)CO(2) was trapped and counted. A key development in the methodology was finding that an aqueous solution of Rhyozyme and Triton X-100 produced lysis of blood but was not detrimental to bacteria.