四种微藻对氨苄青霉素敏感性的研究

海洋微藻培养中抗生素的大量使用对微藻的生长产生一定的影响。为了研究抗生素在海洋微藻培养过程中对微藻的影响,本文探讨了四种海洋微藻(海链藻、小硅藻、塔胞藻、小球藻)对氨苄青霉素的敏感性,通过培养过程中加入抗生素进行培养并测定其光密度值进行试验。研究表明,氨苄青霉素对海链藻的生长影响较大,对其它的三种微藻影响较小。     

四种微藻对氨苄青霉素敏感性的研究

为了研究抗生素在海洋微藻培养过程中对微藻的影响,实验探讨了4种海洋微藻（海链藻、小硅藻、塔胞藻、小球藻）对氨苄青霉素的敏感性,通过培养过程中加入抗生素进行培养并测定其光密度值进行实验.研究表明,氨苄青霉素对海链藻的生长影响较大,对其它的3种微藻影响较小.     

纤细裸藻对5种抗生素的胁迫响应

裸藻(Euglena)是一种重要的资源微藻,含有丰富的氨基酸、不饱和脂肪酸、维生素和裸藻糖等营养物质,并且裸藻没有细胞壁,其营养成分相对于其他藻类更容易被人体、水产动物吸收,开展裸藻集约化培养,对优化养殖环境具有十分重要的意义。为了研究裸藻的无菌化养殖,实验生态条件下,研究了不同浓度的5种抗生素(遗传霉素、氯霉素、青霉素、土霉素和链霉素)胁迫对纤细裸藻(Euglena gracilis)细胞密度及叶绿素a水平的影响。结果表明,在遗传霉素、土霉素和链霉素胁迫作用下,裸藻细胞密度显著降低(P0.05),低于25μg/m L的遗传霉素即可对裸藻生长与叶绿素a合成产生抑制,200μg/m L的土霉素和25μg/m L、50μg/m L的链霉素对裸藻叶绿素a合成有一定的促进作用;氯霉素和青霉素对裸藻生长有一定的促进效应,200μg/m L的青霉素可以显著提高纤细裸藻的细胞密度和叶绿素a水平(P0.05)。实验结果说明纤细裸藻对遗传霉素、土霉素和链霉素的敏感性较强,而氯霉素和青霉素可以作为纤细裸藻无菌系建立的备选抗生素,研究结果可为纤细裸藻的基因工程选择标记和无菌培养体系的建立提供参考。     

G418和氨苄青霉素对海水小球藻生长及光化学活性的影响

采用不同质量浓度的2种抗生素———G418硫酸盐(简称G418,试验浓度为0、40、80、160、240、320μg/mL)和氨苄青霉素(试验浓度为0、200、400、800、1 200、1 600μg/mL)对海水小球藻(Chlorella sp.)进行无菌化处理,研究抗生素种类及其质量浓度对海水小球藻细胞密度、叶绿素含量及光化学活性的影响,以确定对海水小球藻细胞无害并能抑制伴杂菌生长的抗生素种类及使用浓度。结果显示,不同质量浓度的G418均能显著抑制小球藻的生长,破坏其潜在光合活性(F_v/F_0),降低光合系统II(PSⅡ)的最大量子产量(F_v/F_m),阻碍其相对电子传递速率(ETR),降低光化学淬灭(q P),抑制作用随G418质量浓度的增加而增强;而氨苄青霉素对海水小球藻的影响则呈现低质量浓度促进、高质量浓度抑制的特征,具体表现为低质量浓度氨苄青霉素(≤800μg/mL)能够促进海水小球藻的生长,增强其潜在光合活性(F_v/F_0),提高其光合系统Ⅱ的最大光化学量子产量(F_v/F_m),促进其相对电子传递速率(ETR)并且增强光化学淬灭(q P)的能力,高质量浓度氨苄青霉素(1200μg/mL)对海水小球藻的生长、叶绿素含量以及荧光参数等指标则呈现抑制作用。研究结果表明,G418不适用于海水小球藻的无菌培养,可选择添加质量浓度不高于800μg/mL的氨苄青霉素对海水小球藻进行抑菌处理,建议添加的质量浓度为200μg/mL。     

溶藻菌A2对4种微藻的溶藻效果分析

将溶藻菌菌株A2以初始浓度107CFU·m L-1接入到锥状斯氏藻(Scrippsiella trochoidea)、条纹小环藻(Cyclotella striata)、蛋白核小球藻(Chlorella pyrenoidosa)和四尾栅藻(Scenedesmus quadricauda)的纯培养与混合培养藻液中,研究A2对不同微藻的溶藻专一性,并通过16S r DNA序列分析对A2菌株进行菌株鉴定。结果表明,经16S r DNA鉴定,菌株A2属于海杆菌属(Marinobacter sp.);在微藻纯培养与混合培养环境下,A2皆可有效溶解甲藻类的锥状斯氏藻,对硅藻类的条纹小环藻具有一定的溶解效果,对绿藻类的四尾栅藻生长无影响,可以促进绿藻类蛋白核小球藻的生长。在多种微藻共存环境下,A2菌组锥状斯氏藻死亡率可高达98.29%和95.37%,蛋白核小球藻的浓度较对照组提高237.6%。结果表明,菌株A2具有良好的溶藻专一性,可作为研发养殖池塘甲藻调控剂的备选菌株。     

温度和起始密度比对舟形藻和小球藻生长和竞争的影响

为利用种间竞争进行有益微藻共培养和构建池塘优良藻相,文章探究了不同温度(10、15、20、25、30、35℃)和起始密度比[小皮舟形藻(Navicula pelliculosa)∶小球藻(Chlorella vulgaris)分别为1∶10、1∶1、1∶0.1]对2种藻类生长竞争的影响。结果显示,单种培养中,10～15℃小皮舟形藻的细胞密度呈先升高后降低的趋势,20～30℃呈逐渐升高的趋势,最大值为0.50×106个·mL-1,35℃时停止生长,适宜生长温度为25～30℃;10～15℃小球藻生长缓慢甚至停止,20～35℃细胞生长迅速,最大值为14.15×106个·m L-1,适宜生长温度为35℃。混合培养中小皮舟形藻生长速率均高于单种培养,且随小球藻接种比例增加逐渐升高,在适宜温度下,混合培养的细胞峰值显著高于单种培养;混合培养中小球藻的接种密度越小生长速率则越大,1∶0.1组显著高于单种培养组,1∶10组则显著低于单种培养组。小球藻对小皮舟形藻的竞争抑制作用较小,混合培养中,小球藻对小皮舟形藻的竞争抑制参数(α)随温度升高和小球藻密度增加而增大,小皮舟形藻对小球藻的竞争抑制参数(β)随温度和小皮舟形藻比重增加而增大。2种微藻能够稳定共存。     

菹草对小球藻和卵囊藻的形态化感效应研究

研究生态修复过程中沉水植物对藻类的影响,为水生态修复提供理论依据。选用常见沉水植物菹草(Potamogeton crispus)研磨液为培养液,对常见绿藻普通小球藻(Chlorella vulgaris)和卵囊藻(Oocystis sp.)开展室内模拟试验培养,通过绿藻生长和形态特征的变化,了解新鲜菹草是否对普通小球藻和卵囊藻存在一定的化感效应。研究结果表明,菹草对普通小球藻和卵囊藻的生长均未呈现显著抑制效应;普通小球藻对菹草具有形态响应,一次性添加的菹草研磨液在试验初期显著促进普通小球藻群体的形成,随着时间的推移,群体普通小球藻逐渐裂开转化为小群体或者是单细胞;试验结果还表明,菹草对卵囊藻形态无显著影响。     

铜绿微囊藻和小球藻室内竞争实验研究

对铜绿微囊藻和小球藻进行藻类竞争实验,并通过模拟计算,分别得到铜绿微囊藻和小球藻纯培养和竞争条件下的生长模式,并计算了2种藻之间的相互竞争抑制强度。纯培养时铜绿微囊藻和小球藻的增长速率r分别为0.451/d和0.419/d,最大环境容量K分别为1414×104个/mL和812×104个/mL;混合培养时铜绿微囊藻对小球藻的种间竞争强度为1.44,小球藻对铜绿微囊藻的种间竞争强度为0.9。     

两种微藻在无菌和带菌状态下生长特点比较研究

对无菌和带菌的波吉卵囊藻(Oocystis borge)和小球藻(Chlorella sp.)生长特点进行比较研究,并且研究光照和盐度对无菌和带菌的波吉卵囊藻和小球藻生长的影响.结果表明:与带菌藻相比,无菌藻更加稳定,不易老化. 光照和盐度对无菌和带菌的波吉卵囊藻和小球藻的生长均有显著影响(P<0.05),但在相同条件下,无菌和带菌藻的增殖率差异不显著(P>0.05).这说明可以直接利用无菌化微藻代替自然带菌藻进行微藻的生理特征以及微藻与其它生物之间关系的研究.     

抗生素对共培养的萱藻丝状体和膨胀色球藻生长的影响

为抑制萱藻丝状体扩增过程中出现的膨胀色球藻的生长,采用实验生态学方法,研究了链霉素、氨苄青霉素、卡那霉素、庆大霉素和氯霉素等5种常用抗生素对共培养条件下的萱藻丝状体和膨胀色球藻生长的影响。结果显示,(1)链霉素浓度为100μg/mL时可以显著抑制膨胀色球藻生长,而不影响萱藻丝状体的生长;(2)氨苄青霉素和庆大霉素对膨胀色球藻产生明显抑制作用的浓度均为100μg/mL,对萱藻丝状体产生抑制作用的浓度分别为100和500μg/mL;(3)10μg/mL卡那霉素可强烈抑制膨胀色球藻生长,而萱藻丝状体没有出现被抑制的现象;(4)氯霉素对膨胀色球藻和萱藻丝状体均有较强的抑制作用,10μg/mL可以对膨胀色球藻的生长产生显著抑制,浓度达到50μg/mL后,萱藻丝状体生长受到抑制。研究表明,链霉素(100μg/mL)、卡那霉素(100μg/mL)和氯霉素(10μg/mL)可以用于降低萱藻种质保存和扩增过程中萱藻丝状体被膨胀色球藻污染的风险。     

抗生素处理对坛紫菜叶状体生长和藻际微生物的影响

设计了氨苄青霉素、硫酸庆大霉素和卡那霉素3种抗生素的不同组合处理坛紫菜叶状体,旨在筛选出坛紫菜叶状体藻际微生物的最优去除方法;同时,探究紫菜叶状体生长与微生物群落变化之间的关系。结果显示,低浓度(10μg/mL)的硫酸庆大霉素或高浓度(800μg/mL)的卡那霉素处理,对坛紫菜叶状体生长均具一定的抑制作用,而不同浓度的氨苄青霉素处理均促进了叶状体生长。500μg/mL的氨苄青霉素、500μg/mL的硫酸庆大霉素与300μg/mL的卡那霉素3种抗生素混合处理叶状体24 h,不会抑制叶状体的生长;同时,对藻际微生物有较强的抑制性。不同的坛紫菜品系,不同的叶状体日龄和培养密度,以及在不同的海水盐度和不同浓度的营养盐中培养,抗生素处理组圆盘体的生长均比对照组快。通过比较抗生素处理组与对照组分离的菌株后发现,抗生素处理组有两株菌落形态和菌株形态与对照组显著不同的特有菌株。研究表明,在坛紫菜叶状体的生长过程中,某些对叶状体生长起阻碍作用的细菌如果大量繁殖,会抑制对叶状体生长有益的细菌繁殖,当用抗生素抑制了阻碍生长的细菌,就会使益生菌成为优势菌,促进叶状体的生长,益生菌对上述抗生素的处理不太敏感,但由于培养条件的改变,抗生素对阻碍生长的细菌的抑制作用会减弱,使得它们大量繁殖,从而抑制叶状体的生长。     

杂交鳢(斑鳢♀×乌鳢♂)内脏类结节病病原菌的分离、鉴定与特性分析

从广东佛山、广州两地养殖场患内脏类结节病杂交鳢(Channa maculata♀×C.argus♂)内脏器官分离到2株细菌,纯化培养后获得2个分离株,编号为WL-1和WL-2,对分离菌株进行了细菌鉴定、致病性分析及药敏实验。应用常规生理生化鉴定和ATB系统细菌自动鉴定仪对分离菌株进行细胞形态学、理化特性分析,初步判定所分离菌为舒伯特气单胞菌。采用16S rRNA基因、DNA促旋酶的B亚单位蛋白(gyrB)基因对分离菌株进行DNA分子鉴定,结果显示,两个菌株间的16S rRNA基因序列相同,gyrB基因序列同源性为99.7%;分离菌株与GenBank上登录的舒伯特气单胞菌16S rRNA基因序列和gyrB基因序列同源性均最高,达99%以上;分离菌株在系统进化树上与舒伯特气单胞菌聚为一族,进一步确认分离株为舒伯特气单胞菌。人工感染健康鱼后出现与自然发病相似的内脏类结节病症状,从发病鱼内脏组织再分离的细菌特性与原感染菌相同。综合理化特性分析、基因鉴定和人工感染实验确认舒伯特气单胞菌是杂交鳢内脏类结节病的致病菌。药敏实验发现分离菌株对头孢唑啉、庆大霉素等14种药物敏感;对青霉素G、苯唑西林2种药物耐受。     

Benefits of live phytoplankton, <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>, as a biocontrol agent against fish pathogen <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

The ability of the probiotic bacterium Sulfitobacter to inhibit the growth of two virulent strains (psh-9019 and ATCC 43306) of the fish pathogenic bacterium Vibrio anguillarum was tested. Probiotic and pathogenic bacteria were inoculated individually or together in three different types of media. Two of the phytoplankton media tested were filtrates of phytoplankton Chlorella vulgaris cultures, either the live phytoplankton (live-CV) or a condensed phytoplankton (condensed-CV). Phytoplankton culture medium, ESM, was used as a control medium without phytoplankton. In ESM, Sulfitobacter decreased the viable cell counts of both V. anguillarum strains by tenfold. In the live-CV filtrate, V. anguillarum was eradicated by Sulfitobacter within one week. Although colony counts of strain ATCC 43306 declined during the two-week co-incubation with Sulfitobacter, its growth was not fully inhibited; however, the counts were tenfold lower than that in control ESM medium. Neither of the pathogenic V. anguillarum strains were inhibited nor eradicated by Sulfitobacter in the condensed-CV filtrate medium. Our study indicates that commercially available condensed phytoplankton can enhance the growth of V. anguillarum. Thus, the addition of live phytoplankton, including the introduction of Roseobacter clade bacteria to fish larvae tanks, leads to better biocontrol of the fish pathogen V. anguillarum.     

Carrier state in furunculosis: secondary infection of trout with different Aeromonas salmonicida strains results in advantage for the primarily harboured one

Abstract. On the basis of previous observations, in vivo experiments were conducted in rainbow trout, Oncorhynchus mykiss (Walbaum), to detect possible competition between strains of Aeromonas salmonicida differing in their pattern of resistance to antimicrobial drugs. Serial infections, or reinfection of naturally-infected trout, were induced by intramuscular injection. Surviving fish were artificially stressed, and all dying fish were examined using selective media to assess the type of latent infection. Secondary infections with virulent strains always resulted in mortality and reisolation of the secondary strain in pure culture. However, when trout having undergone the two successive infections were stressed, the primary strain was always reisolated. Primary strains seem to be able to survive in unknown locations within cells or tissues, regardless of their virulence or growth potency. Although a protective effect conferred by latent infection to secondary challenge could not be clearly proven, such mechanisms resemble what occurs in premunition and could be of epidemiological interest.     

患病草鱼肠道病原菌的分离鉴定及毒力研究

自环洞庭湖区精养鱼池患病草鱼肠道中分离获得4种病原菌,进行16S rRNA基因和gyrB基因序列测定和在线比对,并对草鱼进行回归感染。随后进行4种病原菌的药敏特征、溶血活性试验,并利用PCR方法检测了6种毒力因子的携带情况。试验结果表明,病原菌分别为温和气单胞菌、异常嗜糖气单胞菌、类志贺邻单胞菌和格氏乳球菌;4种病原菌回归感染草鱼,均能使草鱼患病致死,自死亡的草鱼体内均能分离得到相应感染的病原菌;4种病原菌对多种抗生素具有耐药性,且在脱纤维绵羊血血平板上检测到了温和气单胞菌、异常嗜糖气单胞菌AaB007的溶血活性;PCR结果表明,4种病原菌携带多种毒力因子。研究结果可为环洞庭湖区草鱼精养鱼池健康养殖及病害防治提供科学依据。     

小球藻psbA基因的克隆与序列分析

psbA基因编码光合系统II反应中心的D1蛋白,是叶绿体基因组中一个重要的光调控基因。对小球藻属4个种的6株小球藻psbA基因进行了PCR扩增和测序,并结合GenBank上已有的2条小球藻psbA基因序列,使用MEGA3.1软件对psbA序列进行了遗传距离值的计算和系统发育树的构建。结果显示8株小球藻psbA基因的遗传距离值为0.012~0.167。除了原始小球藻F-2和蛋白核小球藻820分别与蛋白核小球藻F-5、F-9及与普通小球藻Cvq关系极近之外,其他6株小球藻基本上按照种的划分进行聚类,该结果与用小球藻的ITS和rbcL序列分析的结果一致。     

Recovery of yellow-pigmented bacteria from dead and moribund fish during outbreaks of rainbow trout, Oncorhynchus mykiss (Walbaum), fry syndrome in England

Abstract. During Autumn 1990, the possible presence of microbial pathogens was investigated in two discrete populations of anaemic (rainbow trout fry syndrome; RTFS) rainbow trout fry. Dense pure culture growth of three types of yellow pigmentcd bacteria were recovered from moribund and dead animals on low nutrient media. The cultures were identified as coryneforms. Cytophaga columnaris and Janthinobacterium sp. All taxa induced clinical disease (with similarities to RTFS) in rainbow trout fry.     

凡纳滨对虾养殖体系中群体感应淬灭菌的筛选、安全性评估及发酵条件优化

从凡纳滨对虾(Litopenaeus vannamai)养殖体系中筛选得到具有群体感应淬灭(Quorum quenching, QQ)活性的潜在功能菌,并对菌株进行16S rDNA鉴定、安全性评估及发酵条件优化。采用根癌农杆菌(Agrobacterium tumefaciens) A136液体X-gal法进行活性菌株筛选,发现2株细菌(BDZ5和W1B)的发酵液对信号分子己酰基高丝氨酸内酯(C6-HSL)具有显著的降解作用,但经沸水浴处理后均丧失降解活性。经16S rDNA基因序列分析,菌株BDZ5和W1B分别被鉴定为芽孢杆菌属(Bacillus)和科贝特氏菌属(Cobetia)。安全性评估结果显示,菌株BDZ5和W1B均无溶血性,且对多数抗生素具有敏感性,注射2株活性菌未提高凡纳滨对虾死亡率,且对凡纳滨对虾血清免疫酶活性无显著影响。单因素条件优化结果显示,在起始pH为7.0、盐度为20、0.0005%的MnCl2条件下培养48 h后,菌株BDZ5达到最大生长量;在起始pH为6.0、盐度为40、0.05%的CaCl2条件下培养48 h,菌株W1B达到最大生长量。2株菌的最适信号分子C6-HSL降解条件与其生长条件有所不同,在起始pH为7.5、盐度为30、0.0005%的MnCl2条件下培养48 h,2株活性菌对信号分子C6-HSL的降解率达到最大值。研究表明,菌株BDZ5和W1B可作为益生菌应用于水产养殖,为基于QQ角度防治对虾细菌性病害提供优良菌种。     

Relationships between intracellular bacteria and the bivalve killer dinoflagellate Heterocapsa circularisquama (Dinophyceae)

ABSTRACT: Relationships between the growths of five different clonal strains of Heterocapsa circularisquama and intracellular bacteria were investigated using culture experiments. Although each H. circularisquama strain culture was established from one cell by repeated and careful washings with micropipettes, intracellular and extracellular bacteria in each strain culture were still observed under a epifluorescence microscope using the DAPI (4',6-diamidino-2-phenylindole) staining technique. The extracellular bacteria are derived, presumably, from the inside of algal cells after the death and collapse of algal cells in culture. Using an electron microscope, bacteria were constantly observed in the cytoplasm and food vacuoles of H. circularisquama cells. The growth of five algal strains containing bacteria was compared with that of a bacteria-free strain using culture experiments under combined conditions of five different light intensities and five different strengths of culture medium. Bacteria showed no significant effect on the growth or survival of the algal cells. During the algal exponential growth phase, the intracellular bacterial cell numbers per algal cell decreased, whereas the total bacterial cell density in each algal culture increased. Final cell yield (total number) of the intracellular and extracellular bacteria varied considerably according to the algal strains. These results suggest that the intracellular bacteria of H. circularisquama grow or survive depending on the host alga, and that the alga can grow independently.