流式细胞术在细菌快速检测中的应用

流式细胞仪(Flow cytometer)是集应用流体学、光学、电子学、生物学、免疫学等多门学科和技术于一体的新型高科技仪器。它的核心技术是流式细胞术(Flow cytometry,FCM),该技术是利用流式细胞仪,使单个细胞或其他微小生物粒子处于快速直线流动状态,且逐个通过光束,从而对单个细胞或微粒进行多参数(数量、大小、核酸含量、细胞活性、特定菌群或物种等)定量分析和分选的检测技术,具有快速、灵敏、精确以及便于操作等突出优点。本文简要介绍流式细胞仪的原理,并论述流式细胞技术在实验室研究、工业生产、临床诊断、环境评估等领域的细菌快速检测应用。     

FISH-FCM方法检测酵母-细菌二元体系中微生物数量

以废水生物处理系统和生物发酵系统出现的酵母-细菌二元体系作为研究对象,探讨了二元体系生物样品的最佳超声分散条件以及同时检测酵母和细菌数量时荧光原位杂交（FISH）-流式细胞术（FCM）技术的测量精度。染色-FCM检测表明：同时适用于混合酵母样品（酵母假菌丝）和混合细菌样品（活性污泥絮体）的最佳超声分散条件为100W、60～90s,但过强超声条件（120s）对酵母Candida tropicalis纯培养物（或细菌Escherichia coli纯培养物）的超声粉碎效果完全不同于混合酵母样品（或混合细菌样品）。在此基础上,以C．tropicalis和E．coli分别作为酵母和细菌的模式微生物,采用双探针杂交的FISH—FCM技术检测了背景微生物（C．tropicalis或Ecoll）浓度为10^7个／ml时二元体系中目标微生物（10^2～10^7个／ml）的数量。流式细胞仪能够明显区分噪音和二元体系中的酵母和细菌,因而一次进样时能够同时分析两种微生物数量,并且目标微生物浓度可以精确到10^4个／ml,此时微生物含量仅为0．1％,其中细菌浓度甚至可以精确到10^3个／ml（相应含量仅为0．01％）。然而,当二元体系中目标微生物浓度过低时（〈10^4个酵母／ml或〈10^4个细菌／ml）,背景微生物的存在会严重影响FISH—FCM技术的测量准确性。     

生物质炭配施有机物料对贫瘠红壤酶活性和微生物碳源代谢功能的影响

为进一步促进红壤固碳培肥,于2017和2018年通过田间试验研究了两种有机物料(玉米秸秆和羊粪)单施以及与生物质炭配施对贫瘠红壤养分含量、碳转化相关酶活性和微生物底物利用速率的影响。试验设置6个处理,即不施有机物料(对照)、玉米秸秆、羊粪、单施生物质炭、玉米秸秆与生物质炭配施、羊粪与生物质炭配施。结果表明:与对照相比,有机物料施用显著增加了土壤pH值、有机碳、全氮、有效磷和速效钾含量;与单施秸秆和羊粪相比,生物质炭与秸秆或羊粪配施显著增加了土壤有机碳、速效钾和碱解氮含量,但两者无交互效应。与对照相比,有机物料施用显著提高了β-葡萄糖苷酶(BG)、纤维二糖水解酶(CB)、β-木聚糖苷酶(XYL)和过氧化物酶(PERO)活性;与单施秸秆相比,生物质炭与秸秆配施处理酚氧化酶、过氧化物酶活性分别显著降低了28.6%、22.2%;与单施羊粪相比,生物质炭与羊粪配施处理α-葡萄糖苷酶(AG)、BG、XYL和PERO活性分别显著降低了46.1%、50.9%、41.6%和31.3%。与对照相比,有机物料施用显著提高了土壤基础呼吸和微生物对碳水化合物的利用速率,而生物质炭配施处理对碳水化合物、羧酸类底物的利用速率存在显著抑制作用。微生物碳源利用速率与BG和PERO活性呈显著正相关。因此,有机物料与生物质炭配施更有利于提高土壤养分含量,降低有机碳分解酶和微生物碳源代谢活性,从而促进红壤固碳培肥,有利于贫瘠红壤的地力提升。     

生物活性炭用微生物菌种的选育及评价

目的:强化生物活性炭在净化饮用水中的作用,采用人为补充有益微生物的办法来提高活性炭的净化水效果。方法:利用生物技术从原水中、运行的活性炭中等分离出适用于制备生物活性炭微生物菌种,将选育的菌种与要处理的水进行培养处理,然后根据测定微生物对水的净化效果以及微生物的菌种对环境的适应能力来建立活性炭用微生物菌株的评价方法,并对分离的菌种进行评价、鉴定。结果:一些菌株对饮用水中TOC的处理效率达90%以上。培养条件要求低,最适温度20-28℃,在处理水中即可生长。并建立了菌剂的发酵生产工艺。     

增温和外源碳输入对泥炭地土壤碳氮循环关键微生物功能基因丰度的影响

为探讨温度升高和外源碳输入对泥炭地土壤碳氮循环关键微生物的影响,于2017年7月采集多年冻土区泥炭地表层(0—10 cm和10—20 cm)土壤样品,在10、15℃两个温度下开展为期42d的增温模拟试验,同时设置葡萄糖添加处理,利用荧光定量PCR技术分析泥炭地土壤碳氮循环关键微生物丰度变化,同时分析增温和外源碳输入对泥炭地土壤活性碳组分和无机氮含量的影响。结果表明:温度升高可导致北方泥炭地表层土壤微生物丰度以及群落结构变化,0—10 cm土壤微生物比10—20 cm土壤微生物更加敏感。增温条件下微生物首先快速分解活性有机碳,同时温度升高加快土壤氮周转速率,增加有效氮含量。外源碳输入整体提高了深层土壤微生物丰度,使得10—20 cm土壤细菌、产甲烷菌、甲烷氧化菌、氨氧化细菌以及反硝化细菌丰度显著增加,说明外源碳输入可能会促进10—20 cm土壤甲烷氧化过程、氨氧化过程和反硝化过程。温度和葡萄糖的交互作用对泥炭地表层土壤碳氮循环关键微生物丰度均有显著影响。在增温和外源碳输入条件下,北方泥炭地表层土壤微生物丰度受土壤碳氮活性基质的影响。     

基于流式细胞术快速检测人鼻黏膜组织淋巴细胞亚群的实验研究

目的：基于流式细胞术检测正常人的鼻黏膜组织中的淋巴细胞亚群(CD3+T、CD4+T、CD8+T、NK、CD19+B)的比例,初步探讨鼻局部黏膜免疫功能的意义,为鼻局部黏膜免疫性疾病的研究提供更多的参考。方法：用鼻黏膜刮匙获取21 例正常人的鼻黏膜组织,并抽取其外周血2 mL。采用流式细胞术分别检测其鼻黏膜和外周血中的淋巴细胞亚群(包括CD3+T、CD4+T、CD8+T、NK、CD19+B)分别所占的比例。结果：鼻黏膜和外周血的淋巴细胞亚群存在很大差异,与外周血相比,CD3+T 比例增加（t=15.34,P<0.0001),CD4+T 比例降低(t=5.952,P<0.0001)、CD8+T 比例增加(t=12.44,P<0.0001)、NK 比例降低(t=4.865,P<0.0001)、CD19+B 比例降低(t=15.56,P<0.0001),CD4+T/CD8+T 降低。结论：流式细胞术可以用来检测鼻黏膜的淋巴细胞亚群,鼻黏膜的淋巴细胞亚群和外周血的淋巴细胞亚群存在很大差异,这种差异体现鼻黏膜组织独特的局部黏膜免疫功能,本方法为变应性鼻炎的研究提供了新的研究途径。     

微塑料对稻田土壤温室气体排放和微生物群落的影响

微塑料因在土壤环境中广泛存在及其潜在的生态风险而受到越来越多的关注。微塑料的赋存会改变土壤理化性质,并对土壤微生物群落及其驱动的生物地球化学过程产生影响,而相关研究尚处于起步阶段。可生物降解塑料作为传统塑料的替代品,越来越多地应用于农业活动,并释放到土壤中。然而,可生物降解微塑料对土壤微生物特性产生影响的研究鲜有报道。基于此,本试验以我国三江平原水稻田土壤为研究对象,选取了2种常见的微塑料为试验材料,分别为传统型微塑料聚丙烯（Polypropylene, PP）和可降解微塑料聚乳酸（Polylactic acid, PLA）,进行了为期41d的微宇宙培养实验,旨在分析不同浓度与类型的微塑料对土壤可溶性有机碳（Dissolved Organic Carbon, DOC）含量及官能团特征、温室气体排放以及微生物群落结构的差异性影响。结果表明,传统型微塑料PP与可降解微塑料PLA添加均对土壤理化性质与微生物群落产生显著影响。其中,微塑料添加大体上增加了土壤DOC含量,PLA的促进作用较为明显,且增加含量与微塑料添加量呈正相关;PP和PLA均影响土壤DOC分子结构,削弱了土壤团聚化程度并促进了大分子量DOC化合物的生成;微塑料的添加促进土壤CH₄排放,而有效抑制了土壤CO₂排放;微塑料显著改变了土壤细菌和真菌群落的丰富度与多样性。相关分析结果表明,土壤CO₂累计排放量与芳香族化合物结构及疏水性等官能团特征、变形菌门（Proteobacteria）与放线菌门（Actinobacteria）均呈显著正相关关系。以上结果表明,微塑料添加改变了土壤DOC含量及官能团特征与微生物环境,进而影响土壤温室气体排放。本研究为今后微塑料对土壤地球化学和微生物特性的影响研究提供了科学的思路,同时也有助于评估微塑料对土壤生态系统的生态风险。     

油松-辽东栎混交林地表凋落物与氮添加对土壤微生物生物量碳、氮及其活性的影响

2010年9月-2011年10月,在山西省灵空山油松和辽东栎混交林样地采取随机区组设计,研究了地表凋落物和氮添加处理对土壤微生物生物量碳、氮和微生物活性的影响.凋落物处理包括:剔除凋落物(N)、叶凋落物加倍(L)、枝果凋落物加倍(B)和混合凋落物加倍(LB);氮添加量分别为0(N0)、5 g· m-2·a-1(N1)和10 g·m-2·a-1(N2).结果表明:剔除地表凋落物且无氮添加时,油松和辽东栎混交林地的土壤有机碳(SOC)含量显著降低,其他试验处理间对SOC的影响无显著差异.土壤微生物生物量碳(MBC)、氮(MBN)及其活性(MR)的变化范围依次为:262.42 ～ 873.16 mg·kg-1、73.55 ～ 173.85 mg·kg-1和2.38～3.68mg·kg-1·d-1.MBC、MBN和MR两两间呈极显著正相关.氮添加对MBC、MBN和MR均无显著影响;凋落物处理对MR影响显著,表现为混合凋落物加倍处理的MR最高,叶凋落物加倍处理次之,剔除凋落物处理最低,而对MBC和MBN无显著影响.凋落物和氮添加处理在整个试验过程中未表现出交互作用.短期的氮添加处理和森林地表凋落物变化对土壤微生物过程的影响有限.     

油松 辽东栎混交林地表凋落物与氮添加对土壤微生物生物量碳、氮及其活性的影响

2010年9月-2011年10月,在山西省灵空山油松和辽东栎混交林样地采取随机区组设计,研究了地表凋落物和氮添加处理对土壤微生物生物量碳、氮和微生物活性的影响.凋落物处理包括: 剔除凋落物(N)、叶凋落物加倍(L)、枝果凋落物加倍(B)和混合凋落物加倍(LB);氮添加量分别为0(N₀)、5 g·m^-2·a^-1(N₁)和10 g·m^-2·a^-1(N₂).结果表明: 剔除地表凋落物且无氮添加时,油松和辽东栎混交林地的土壤有机碳(SOC)含量显著降低,其他试验处理间对SOC的影响无显著差异.土壤微生物生物量碳(MBC)、氮(MBN)及其活性(MR)的变化范围依次为: 262.42～873.16 mg·kg^-1、73.55～173.85 mg·kg^-1和2.38～3.68mg·kg^-1·d^-1.MBC、MBN和MR两两间呈极显著正相关.氮添加对MBC、MBN和MR均无显著影响;凋落物处理对MR影响显著,表现为混合凋落物加倍处理的MR最高,叶凋落物加倍处理次之,剔除凋落物处理最低,而对MBC和MBN无显著影响.凋落物和氮添加处理在整个试验过程中未表现出交互作用.短期的氮添加处理和森林地表凋落物变化对土壤微生物过程的影响有限.     

Effects of Acetic Acid on the Regrowth of Heterotrophic Bacteria in the Drinking Water Distribution System

Summary Three laboratory-scale water pipe systems were set up to study the effects of adding two levels of acetic acid (10 and 50 μg acetate eq-C l⁻¹) on the bacterial regrowth in water pipes. The results of the water pipe test showed that nearly all carbon in the acetic acid could be readily utilized by bacteria and resulted in an increase in biomass concentration. The maximum heterotrophic plate counts in biofilm were equal to 3.5 × 10⁴, 8.9 × 10⁵ and 2.9 × 10⁷ c.f.u. cm⁻² while the maximum heterotrophic plate counts of free bacteria were equal to 1.2 × 10³, 5.0 × 10³ and 6.8 × 10⁴ c.f.u. ml⁻¹ for the blank and with addition of 10 and 50 μg acetate eq-C l⁻¹. These results showed that addition of acetic acid to drinking water has a positive effect on the assimilable organic carbon content of drinking water and bacterial regrowth in the distribution system. This effect is enhanced with addition of high-level acetic acid. Batch tests were also conducted using water samples collected from a Taiwanese drinking water distribution system. The bacterial regrowth potentials of the blank were equal to 4.3 × 10³, 1.5 × 10⁴, 4.9 × 10⁴ and 7.5 × 10⁴ c.f.u. ml⁻¹ for water samples collected from treatment plant effluent, commercial area, mixed area, and residential area, respectively. These results showed that the biological stability of drinking water is the highest in treatment plant effluent, followed by distributed water of the commercial area, distributed water of the mixed area, and then the distributed water of residential area.     

Assessing the effect of different ultrasonic frequencies on bacterial viability using flow cytometry

Aims: This research investigated the effect of sonication at frequencies of 20, 40 and 580 kHz and approximately the same acoustic intensity on the viability and declumping of two micro‐organisms (Escherichia coli and Klebsiella pneumonia). Methods and Results: Two analytical methods were employed; viable plate counts (CFU ml^?1) and flow cytometry to identify and quantify both live/viable and dead bacteria in the bulk liquid. Flow cytometry results for E. coli and Kl. pneumonia indicated a high sensitivity to 20 and 40 kHz frequency with a continuous decrease in the viable cells and an increase in dead cells during experiments. In contrast, results using the higher frequency of 580 kHz indicate predominantly deagglomeration of bacterial clumps rather than cell membrane disruption (Joyce et al. 2003). Results indicate a good correlation between flow cytometry and viable plate count methodology. Conclusions: Sonication has two different effects on bacteria (i) inactivation and (ii) declumping; however, the scale of these effects is dependent on intensity and frequency. Flow cytometry provides a method to distinguish between and quantify the effects through the observation of two subpopulations: (i) live/viable and (ii) dead bacterial cells. Significance and Impact of the study: Treatment using power ultrasound has been shown to have a significant impact on microbial activity. This is the first time a study has compared the influence of a range of different frequencies, but at similar power settings on the survival of bacteria in phosphate buffer saline (PBS). This work is of importance for applications where ultrasound has been considered for use in industry as a means of disinfection including the treatment and pretreatment of water and also for the sterilization of liquid foods.     

Post treatment of groundwater denitrification fluidized bed reactor effluents to achieve drinking water quality

Post treatment of effluents from heterotrophic groundwater denitrification fluidized bed reactors (FBR) designed to achieve drinking water quality has been investigated. The denitrification process adds to the dissolved organic compounds, biomass and bacteria in the effluent. They are also lacking dissolved oxygen. Effluents from the process were treated in combined post treatment processes based on either a trickling filter and sedimentation unit (‘TF combination’) or contact flocculation (‘CF combination’). Both processes were followed by sand filtration, granular activated carbon (GAC) and chlorination. Results regarding total suspended solids (TSS) and turbidity removal showed an advantage to the ‘CF combination’, and the target turbidity (NTU <1) was always achieved when the alum dose was 10 or 20 mg l⁻¹. Backwash of the sand filter and GAC column was required after 27 h of operation (average value). An average total reduction in dissolved organic carbon (DOC) of 40% was observed with a final DOC of 3.5–4 mg L⁻¹. Most of the removal of the DOC occurred in the sand filter (28%), while the GAC contribution was smaller (18%). No regrowth potential was observed using the Werner method when a pure culture of Pseudomonas fluorescens P17 was used as inoculum in samples of chlorinated effluent (post chlorination). When a mixed culture of indigenous bacteria was used as inoculum, a high regrowth potential was observed. Installing an additional chlorination unit before the sand filter column (pre and post chlorination) resulted in effluent with no regrowth potential for both Pseudomonas fluorescens P17 and indigenous bacteria. Received 17 October 1997/ Accepted in revised form 29 May 1998     

土壤有机碳分组方法及其在农田生态系统研究中的应用

农田土壤有机碳成分复杂,活性有机碳对管理措施具有敏感性,而惰性有机碳具有固碳作用.碳分组技术主要包括物理技术、化学技术和生物学技术.物理分组的依据是密度、粒径大小和空间分布,可分离出有机碳的活性组分和惰性组分.化学分组基于土壤有机碳在各种提取剂中的溶解性、水解性和化学反应性从而分离出各种组分:溶解性有机碳是生物可代谢有机碳,包括有机酸、酚类和糖类等;酸水解方法可将有机碳分成活性和惰性成分;利用KMnO4模拟酶氧化可分离出活性碳和非活性碳.利用生物技术可测定出微生物生物量碳和潜在可矿化碳.在不同农田管理措施下,有机碳组分的化学组成和库容会发生不同变化,对土壤有机碳沉积速率产生不同影响.为了探明土壤有机碳组分与碳沉积之间的定性或定量关系,今后应该加强对各种分组方法的标准化研究,探索不同分组方法的整合应用,针对不同农田管理措施,总结出适合的有机碳分组方法或联合分组方法.     

Utilization of oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater by Flavobacterium johnsoniae

Aims: To obtain a bacterial strain that can be used to quantify biodegradable polysaccharides at concentrations of a few micrograms per litre in freshwater. Methods and Results: Flavobacterium johnsoniae strain A3 was isolated from tap water supplemented with laminarin, pectin or amylopectin at 100 μg C l^?1 and river Rhine water. The organism utilized 14 of 23 oligo‐ and polysaccharides, and 1 of 9 monosaccharides, but none of the sugar acids, sugar alcohols, carboxylic acids or aromatic acids tested at 10 μg C l^?1. Amino acids promoted growth of strain A3, but not in coculture with assimilable organic carbon (AOC) test strain Pseudomonas fluorescens P17, which utilized these compounds more rapidly than strain A3. Compounds released by strain P17 and AOC test strain Spirillum sp. NOX grown on acetate promoted the growth of strain A3 at N_max values of ≥ 2 × 10⁵ CFU ml^?1 of strain P17 and ≥ 5 × 10⁵ CFU ml^?1 of strain NOX. Significant growth of strain A3 was observed in surface water and in tap water in the presence of strain P17 (N_max P17 < 2 × 10⁵ CFU ml^?1). Conclusions: Strain A3 utilizes oligo‐ and polysaccharides at microgram‐per‐litre levels. In surface water and in tap water, the organism was able to utilize compounds that were not utilized by strain P17. These compounds may include oligo‐ and/or polysaccharides. Significance and Impact of the Study: Phytoplanktonic and bacterial polysaccharides can constitute an important biodegradable fraction of natural organic matter in water and may promote growth of heterotrophic bacteria during water treatment and drinking water distribution. Strain A3 can be used to quantify a group of compounds that includes oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater.     

山核桃林集约经营过程中土壤有机碳和微生物功能多样性的变化

研究集约经营时间为5、10、15、20 a山核桃林的土壤有机碳和微生物功能多样性的演变规律.结果表明: 天然混交林改造为山核桃纯林并经强度经营后,林地土壤有机碳(TOC)、微生物生物量碳(MBC)、水溶性有机碳(WSOC)含量显著下降,但有机碳库的稳定性增强.与天然山核桃 阔叶混交林(0 a)相比,经过5 a的强度经营,TOC、MBC、WSOC含量分别下降28.4%、34.1%和53.3%,20 a后分别下降38.6%、48.9%和64.1%,土壤有机碳组分中的芳香碳、酚基碳和羰基碳比例提高,芳香度提高了23.0%.山核桃的强度经营降低了土壤微生物功能多样性,0、5 a与10、15、20 a土壤微生物活性的平均颜色变化率(AWCD)值差异显著,Shannon指数(H)和均匀度指数(E)在0、5 a与15、20 a间差异显著.林地土壤TOC、WSOC、MBC、AWCD、H和E等6个指标两两之间均显著相关.     

土地利用变化对川西米亚罗林土壤活性碳库的影响

为了揭示土地利用变化对土壤活性有机碳库的影响,在四川省亚高山米亚罗林区,以原始冷杉林(M-Y)和由原始林转化成的45年龄云杉人工林(M-60)、25年龄云杉人工林(M-80)和菜地(M-C)等4种土地利用类型为研究对象,进行了土壤的微生物量碳(MBC)、水溶性有机碳(WDOC)和易氧化有机碳(LOC)的含量和季节变化研究.结果表明,土地利用变化明显影响土壤活性有机碳组分的含量,其中微生物量碳和水溶性有机碳的变化趋势为M-Y>M-60>M-80>M-C,易氧化有机碳的变化趋势则为M-60>M-Y.土地利用变化没有改变活性有机碳各组分的垂直分布,各组分均随着土层深度的增加而降低,季节变化幅度较小,但枯落物层和表层土壤的变化幅度明显高于深层土壤,而各组分的分配比例变化幅度明显小于活性有机碳含量的变化.     

Bio-stabilization of drinking water in supply systems as observed in the tropics

Changes in the bacterial quality of drinking water were examined in the supply system of tropical Dar es Salaam, Tanzania. The water treatment fulfilled WHO standard guidelines. However, the water quality allowed for significant regrowth of bacteria, demonstrating that the WHO guidelines are insufficient to check whether or not the drinking water has an acceptable regrowth potential. An experimental pipe system was examined on site. A growth model was established, based on a zero order bacterial detachment kinetics for the biofilms colonizing the inner surface of the pipes, with a release rate factor R, and a first order growth kinetics in the water flowing in the pipes with a growth rate factor . The rate factors were estimated to be R=1.9×10⁸ m^-2 h^-1 and =2.5 h^-1. It was concluded that growth in the biofilm played a dominant role in the bio-stabilization processes, while the initial microbial quality of the water works water and the growth in the water phase were of minor importance. The bio-stabilization process is understood herein as the reduction in the concentration of nutrients and assimilable organic carbon in the water, and thus as the reduction of the growth potential of the water. Bacterial growth and recontamination were examined during storage at the user site. The results showed that heterotrophic bacteria grew, while faecal coliforms decayed during storage. It was concluded that the users handled the water hygienically.The authors are with the Technical University of Denmark, IMT, CDC-Build, 208 DK-2800 Lyngby, Denmark     

A rapid method for evaluation of cell number and viability by flow cytometry

A simple, rapid and reliable method has been developed for assessing the number and viability of cells, as well as cell size, in suspension culture by the use of flow cytometry. Propidium iodide exclusion is used for viability determination and fluorescent beads serve as an internal standard for cell enumeration. The main advantages of this method are its ability to handle a large number of samples with a high degree of precision and its specificity in detecting viable cells quantitatively in a heterogeneous culture of living and dead cells and debris. The method shows only a fraction of the variation found in the haemacytometer/trypan blue counting method due to its very low operator dependence. CHO - Chinese hamster ovary; FCS - Foetal calf serum; FS - Forward scatter light; MTT - 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NCS - newborn calf serum; PBS - Phosphate buffered saline; PI - Propidium iodide; SS - Side scatter light. This revised version was published online in July 2006 with corrections to the Cover Date.