双层平板法检测嗜盐古菌铁载体

采用双层平板法应用于嗜盐古菌铁载体的原位检测.双层平板的上层为不添加铁离子的嗜盐古菌培养基,嗜盐古菌可在其上生长,在缺铁胁迫下可向外界分泌铁载体;下层为含有CAS检测液用于铁载体检测的琼脂.当上层平板生长的嗜盐古菌分泌的铁载体透过培养基渗透到下层检测琼脂后,即可在下层检测平板上产生明显的特征性的铁载体螯合晕圈,表明双层平板法在嗜盐古菌的铁载体检测中确实可行,且较原有的嗜盐古菌铁载体检测方法简便、直接.     

CAS平板覆盖法检测氢氧化细菌铁载体

【目的】用CAS平板覆盖法检测氢氧化细菌铁载体,解决通用CAS琼脂平板法中十六烷基三甲基溴化铵对真菌和某些细菌的生长抑制问题。【方法】将改良的CAS检测培养基覆盖在长满菌落的无铁培养基上,生长抑制问题因微生物未与十六烷基三甲基溴化铵直接接触而解决。【结果】3株氢氧化细菌SDW-5、SDW-9和AaP-13均能产生单菌落,加入CAS检测培养基1 h后,菌落周围产生明显的铁载体晕圈。【结论】本方法成功解决了生长抑制问题,可以作为检测微生物铁载体的通用方法。     

假单胞菌荧光与非荧光铁载体对铁离子的应答差异

假单胞菌既能产荧光铁载体也能产非荧光铁载体.通过对假单胞菌在不同铁离子浓度下,在通用CAS(Chrome azroul S)检测平板、改进的蔗糖-天冬氨酸(SA)平板(MSA)上以及通用液体CAS培养基和MSA培养基内的铁载体产生情况的比较,发现在通用CAS的液体培养基上产生的主要为非荧光铁载体(pyochelin),而在改进的MSA培养基上产生的主要为荧光铁载体(pyoverdine);在铁离子的应答方面,pyoverdine较pyochelin灵敏,较低的铁离子浓度即可抑制荧光铁载体的产生,但是不能抑制非荧光铁载体.     

高灵敏假单胞菌铁载体的平板检测方法*

CAS蓝色检测平板是一种筛选、检测各类细菌铁载体的常用方法,而蔗糖-天冬酰氨培养基被用于假单胞菌产铁载体规律的研究。用天冬氨酸替代天冬酰氨,将CAS蓝色检测液与蔗糖-天冬氨酸培养基(MSA培养基)相结合,得到一种改进的MSA-CAS检测平板。通过对假单胞菌属7个种8个株进行荧光与非荧光铁载体检测方面的比较研究,结果表明MSA-CAS检测平板假单胞菌铁载体的检测灵敏度比通用CAS检测平板高,而且在检测荧光铁载体方面具有荧光背景低、荧光铁载体晕圈明显和晕圈与背景的对比度大的优点。     

异源表达脂肪酶的重组酵母筛选培养基的改良

在脂肪酶产生菌的筛选中,最常用的方法是三丁酸甘油酯琼脂平板透明圈法和橄榄油琼脂平板变色圈法。而传统的三丁酸甘油酯平板缺乏有效营养成分,重组酵母无法生长;橄榄油平板灵敏度较低,低脂肪酶活力的重组酵母不能产生变色圈,这给产脂肪酶基因工程菌的鉴定带来了困难。本研究在三丁酸甘油酯平板的基础上,添加适合酵母生长的营养成分,获得了新的改良培养基;重组酵母在该培养基上生长良好、水解圈清晰,检测脂肪酶灵敏度高。     

高灵敏假单胞菌铁载体的平板检测方法

CAS蓝色检测平板是一种筛选、检测各类细菌铁载体的常用方法,而蔗糖-天冬酰氨培养基被用于假单胞菌产铁载体规律的研究。用天冬氨酸替代天冬酰氨,将CAS蓝色检测液与蔗糖-天冬氨酸培养基（MSA培养基）相结合,得到一种改进的MSA-CAS检测平板。通过对假单胞菌属7个种8个株进行荧光与非荧光铁载体检测方面的比较研究,结果表明MSA-CAS检测平板假单胞菌铁载体的检测灵敏度比通用CAS检测平板高,而且在检测荧光铁载体方面具有荧光背景低、荧光铁载体晕圈明显和晕圈与背景的对比度大的优点。     

噬菌蛭弧菌的分离及初步鉴定

采用双层平板法,以滤膜过滤的方法来收集噬菌蛭弧菌,以嗜水气单胞菌(Aeromonas hudrophila)、荧光假单胞菌(Pseudomonas fluorescent)和绿脓杆菌(Pseudomonas aeruginosa)为宿主菌,进行噬菌蛭弧菌的分离研究;并在此基础上,通过接触酶检测和寄生性确认对噬菌蛭弧菌(Bdellovibrio bacteriovorus)进行了初步的鉴定.结果表明未使用滤膜过滤,采用自来水琼脂双层平板法分离噬菌蛭弧菌的效果较好;并经过接触酶和寄生性检测初步鉴定此BD-SPOI菌株为噬菌蛭弧菌.     

拟康氏木霉菌YIM PH30002铁载体活性化学成分北大核心CSCD

通过双层琼脂平板法对拟康氏木霉菌YIM PH30002在三种培养基条件下产铁载体能进行了半定量分析检测,该菌株在PDB培养基中,产铁载体成分的量最多。利用多种色谱技术从菌株YIM PH30002的PDB发酵液的乙酸乙酯提取物中共获得了5个聚酮类代谢产物,经波谱数据分析与文献比对,化合物分别鉴定为：konginginin A（1）、konginginin B（2）、konginginin D（3）、konginginin F（4）、konginginin M（5）。化合物1~4表现有铁载体活性,其中化合物2的铁载体活性相对较强,MTC值约为300μg/m L。但化合物均无显著抗菌生物活性。     

拟康氏木霉菌YIM PH30002铁载体活性化学成分

通过双层琼脂平板法对拟康氏木霉菌YIM PH30002在三种培养基条件下产铁载体能进行了半定量分析检测,该菌株在PDB培养基中,产铁载体成分的量最多。利用多种色谱技术从菌株YIM PH30002的PDB发酵液的乙酸乙酯提取物中共获得了5个聚酮类代谢产物,经波谱数据分析与文献比对,化合物分别鉴定为:konginginin A(1)、konginginin B(2)、konginginin D(3)、konginginin F(4)、konginginin M(5)。化合物1~4表现有铁载体活性,其中化合物2的铁载体活性相对较强,MTC值约为300μg/m L。但化合物均无显著抗菌生物活性。     

高产铁载体荧光假单胞菌Pseudomonas fluorescens sp-f的筛选鉴定及其铁载体特性研究

采用改进的CAS检测平板从东湖中筛选得到了一株高产铁载体细菌sp-f,并用CAS检测液定量检测其分泌铁载体量,发现其As/Ar仅0.09(OD680),Su(Siderophore Unit)为90%,达到产铁载体菌最高级。用BIOLOG检测板,结合细菌生理生化反应、形态观察和16S rDNA序列比对分析等分类鉴定方法,确定sp-f为一株荧光假单胞菌。P.fluorescenssp-f生长过程中胞外铁载体的量在对数生长前期累积达到最高后有所减少,至稳定期时菌液中铁载体量达到稳定。在已知铁载体特异吸收峰波长下,用反向高效液相色谱检测无铁环境和高铁环境下培养液上清,比较发现sp-f上清含有3种含儿茶酚胺类基团铁载体,其中包括荧光和非荧光性的脓菌素,200μmol/L Fe2 可完全抑制荧光性质脓菌素的分泌,但非荧光脓菌素的分泌不受抑制,并且对非脓菌素的儿茶酚胺类铁载体的合成分泌反而具有一定的诱导作用。     

Method for the Isolation of Proteolytic Marine Bacteria

A two-layer plate consisting of a lower indicator layer of milk-agar and an upper nutrient agar layer was developed for isolating proteolytic marine bacteria.     

Siderophores of halophilic archaea and their chemical characterization

Nine halophilic archaea viz., Halobacterium salinarum, Halobacterium sp.1, Halobacterium sp.2, Halobaculum sp., Halococcus saccharolyticus, Halorubrum saccharovorum, Haloterrigena turkmenica, Halogeometricum sp. and Natrialba sp. isolated from marine salterns around Bhavnagar coast were screened for siderophore production. Five isolates viz., Halococcus saccharolyticus, Halorubrum saccharovorum, Haloterrigena turkmenica, Halogeometricum sp. and Natrialba sp. produced siderophores as evidenced by positive reaction in FeCl3 test, CAS assay and CAS agar plate test. Determination of chemical nature of siderophores by chemical assays and bioassays identified them as carboxylates. Quantification of siderophores indicated Halorubrum saccharovorum to be the maximum siderophore producer (2.62 RE mg/ml) and Halococcus saccharolyticus to be the least (1.33 RE mg/ml). The present study is the first report on siderophore production in Indian haloarchaeal strains. Mechanism of iron assimilation in four non-siderophore isolates still needs to be investigated further.     

A simple agar plate assay for screening siderophore producer yeasts.

Yeasts produce hydroxamate-type siderophores (iron-binding compounds) in response to Fe-stress conditions. Because these siderophores are important to the biocontrol of postharvest diseases of apple and pears, a method for screening siderophore producer yeast was developed.The screening method was carried out in special Petri dishes with eight or nine wells (25-mm diameter). These wells were filled with siderophore production medium and seeded with yeasts isolated from epiphytic apple microflora. After yeasts grew (24-48 h), holes (2-mm diameter) were made in the agar of each well. Holes were filled with an acid solution of ferric perchlorate. After 10-15 min, reddish halos appeared in the bottom of the plate and their intensities were compared with standards. Standards were prepared in the same special dish with rhodotorulic acid solutions (concentrations between 0.05 and 1 g/l) plus 2% agar. When agar solidified into wells, holes were made and filled with ferric perchlorate solution. Color intensities of reddish halos were proportional to siderophore concentration and the detection limit was 0.1 g/l. It was possible to correlate the production of siderophore in solid medium with the results obtained in liquid medium. The methodology was also a useful tool for making a preliminary assessment of the influence of different factors on the siderophore production.     

Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate

The dynamics of a bacterial population exposed to the minimum inhibitory concentration (MIC) of an antibiotic is an important issue in pharmacological research. Therefore, a novel antibiotic susceptibility test is urgently needed that can both precisely determine the MIC and accurately select antibiotic-resistant strains from clinical bacterial populations. For this purpose, we developed a method based on Fick's laws of diffusion using agar plates containing a linear gradient of antibiotic. The gradient plate contained two layers. The bottom layer consisted of 15 mL agar containing the appropriate concentration of enrofloxacin and allowed to harden in the form of a wedge with the plate slanted such that the entire bottom was just covered. The upper layer consisted of 15 mL plain nutrient agar added with the plate held in the horizontal position. After allowing vertical diffusion of the drug from the bottom agar layer for 12 h, the enrofloxacin concentration was diluted in proportion to the ratio of the agar layer thicknesses. The uniform linear concentration gradient was verified by measuring the enrofloxacin concentration on the agar surface. When heavy bacterial suspensions were spread on the agar surface and incubated for more than 12 h, only resistant cells were able to form colonies beyond the boundary of confluent growth of susceptible cells. In this way, the true MIC of enrofloxacin was determined. The MICs obtained using this linear gradient plate were consistent with those obtained using conventional antibiotic susceptibility tests. Discrete colonies were then spread onto a gradient plate with higher antibiotic concentrations; the boundary line increased significantly, and gene mutations conferring resistance were identified. This new method enables the rapid identification of resistant strains in the bacterial population. Use of the linear gradient plate can easily identify the precise MIC and reveal the dynamic differentiation of bacteria near the MIC. This method allows the study of genetic and physiological characteristics of individual strains, and may be useful for early warning of antibiotic resistance that may occur after use of certain antimicrobial agents, and guide clinical treatment.     

Isolation of a small rod with lytic activity against Vibrio parahaemolyticus from fresh sea water.

A small rod, capable of formine crater-like plaques on lawns of Vibrio parahaemolyticus, was isolated from a marine environment. The isolate was a gram-negative straight rod with round ends and was small in size, equal to that of halophilic Bdellovibrio strain 5501. The isolate appeared to have close taxonomic relationships to Cytophaga, since this bacterium moved slowly in a gliding manner on a solid agar surface, hydrolyzed agar and starch, contained yellow pigment and was halophilic. The isolate was able to grow not only under host-dependent but also under host-independent conditions when low nutrient media were used for cultivation, and its bacteriolytic mode was different from that of Bdellovibrio, an endoparasite. The isolate was halophilic and required Mg++ and Ca++ in addition to 3% saline for growth. The isolate showed a broad host rnage when tested for plaque-forming activity on gram-negative bacteria but not on the gram-positive bacteria tested so far.     

Application of the complex of DNA with the congo red anionic diazo dye for detection of nuclease-producing colonies of marine bacteria

This study was aimed at the development of a method for detection of colonies of nuclease-secreting marine bacteria. The BAL nuclease-producing marine bacterium Pseudoalteromonas espejiana BAL-31 was used as the test object. A new method was developed involving the congo red (CR) anionic dye. The P. espejiana culture was plated on nutrient agar with CR and denatured DNA. In such media. CR was found to form complexes with DNA. After two days of incubation at 30 degrees C, halos were found around the P. espejiana colonies. No halos appeared when DNA was not introduced, when BAL nuclease was inactivated, or when the medium was inoculated with Escherichia coli. It was concluded that the halos around the colonies indicated nuclease excretion. The halos were shown to result from the coagulation of CR released after digestion of the CR-DNA complex by the nuclease. This method for detection of nuclease-producing colonies can probably be used for all marine bacteria and possibly for halophilic bacteria as well.     

Characterization of the dominant halophilic archaea in a bacterial bloom in the dead sea

Abstract A mass bloom of halophilic archaea developed in the Dead Sea in the summer of 1992, with peak densities of more than 3 × 10⁷ cells/ml, imparting a red coloration to the water. Microscopical examination showed a numerical dominance of pleomorphic, flat cells. Attempts to identify the dominant type of halophilic archaea by means of growth experiments, both on agar plates and by dilution in liquid media, were unsuccessful, as viable counts obtained were two or more orders of magnitude lower than the total microscopic counts. Analysis of the polar lipids in the Dead Sea biomass during the bloom showed one major glycolipid to be present in the extracts, corresponding with the sulfated diglycosyl diether lipid (S-DGD-1) characteristic of the genus Haloferax . No indications were found for the presence of significant amounts of other glycolipids that indicate the presence of large numbers of Dead Sea archaea such as Halobacterium sodomense or Haloarcula marismortui , or Halobacterium species such as H. halobium, H. salinarium and H. saccharovorum . Thus, the numerically dominant organisms in the bloom is probably a difficult to culture, not yet isolated, representative of the genus Haloferax .     

岱山盐场可培养嗜盐菌的多样性及其产酶活性筛选

从岱山盐场采集样品,利用选择性培养基分离培养嗜盐菌,对盐田环境中可培养嗜盐菌的多样性及产酶活性进行研究.共分离得到181株嗜盐菌菌株,通过真细菌和古生菌两对通用引物扩增其16S rRNA 基因,并采用限制性内切酶Hinf I进行ARDRA(amplified rDNA restriction analysis)多态性分析,共分为21个不同的操作分类单元(operation taxonomy units, OTUs),其中嗜盐细菌有12个OTUs,嗜盐古菌有9个OTUs.选取具有不同酶切图谱的代表菌株进行克隆测序,BLAST 比对及系统发育分析将嗜盐细菌归于7个属,其中嗜盐单胞菌属(Halomonas)的菌株数占优势,是嗜盐细菌总数的46.8%;嗜盐古菌归于4个属,盐盒菌属(Haloarcula)的菌株数占优势,是嗜盐古菌总数的49.1%.对分离菌株的产酶活性进行检测表明,岱山盐田环境蕴含丰富的产淀粉酶、蛋白酶和脂肪酶等生物活性酶的嗜盐菌, 其中盐盒菌属产酶菌株数最丰富.研究结果表明,岱山盐田环境中具有较为丰富的嗜盐菌多样性,是筛选产酶菌株的重要资源库.