光密度测定法在百日咳杆菌培养菌液浓度测定中的应用

本实验通过抽取百日咳杆菌悬液样品,分别用比浊法和光密度法测量其浓度,并进行各种比较,证明在百日咳杆菌培养中使用分光光度计测量菌悬液光密度值来代替比浊法测定菌悬液浓度的方法是准确可行的,并能建立光密度值与菌悬液浓度的关系,从而提高工作效率、更好的控制培养参数。试验数据表明,利用光密度法测定的百日咳杆菌悬液浓度数据稳定,重复性好,可信度高。证明了光密度法在百日咳杆菌悬液的浓度测定中的可行性,初步建立了光密度值与菌悬液浓度之间的联系。     

枯草芽胞杆菌紫外线诱变实验方案的优化

【目的】优化枯草芽胞杆菌紫外线诱变实验教学方案,以便学生在实验中获得预期结果。【方法】从菌体培养、活菌浓度估测和辐射参数三方面探讨影响实验结果的因素。【结果】采用液体培养法活化菌体有利于制备均匀的菌悬液,采用比浊法估测初始菌悬液浓度可以指导学生将其稀释至102-103 CFU/m L的浓度;涂布接种后平板表面干燥、培养皿盖内无明显水珠附着,是获得单菌落的关键;在紫外辐射过程中采用固定的菌悬液体积和搅拌速度,可以获得规律性的辐射剂量——致死效应曲线。【结论】优化后的方案实验结果易得、重复性好,可供教学和研究实验参考。     

吸光度法快速确定菌悬液浓度及其适用范围

采用麦氏比浊法,将大肠埃希菌的菌悬液浓度调整到约8×108cfu/g,再将其稀释成多个梯度,在600nm的波长下用紫外分光光度计测定吸光度值,同时将各个梯度的菌悬液分别做菌落总数试验,得到不同吸光度值对应的菌落总数,并制作标准曲线,通过测量菌悬液的吸光度值确定菌悬液浓度及其适用范围。试验证明,用此法确定菌悬液浓度时,其菌落总数宜在105~108cfu/g之间,不适用于在105cfu/g以下的菌悬液,所得结果比麦氏比浊法准确。     

利用废水液体发酵生产单细胞蛋白的实验研究

利用工厂废水或动物血制作培养基,以液体发酵方式培养产朊假丝酵母NCTC3576菌及甘饲8501菌.离心分离菌体,测定菌体生物生长量,并观察蔗糖、温度、酸碱度、发酵时间、种子液接种量对生物生长量的影响.生化方法分析NCTC3576菌单细胞蛋白的品质,氨基酸分析仪分析单细胞蛋白的氨基酸组成.结果表明,玉米淀粉厂废水,不必调pH,不用添加其它任何组分,即可直接用于液体发酵产朊假丝酵母NCTC3576菌;pH4.0～7.0均对生长量无明显影响;24h培养已达到最好生长量,再延长时间已不能提高产量;28℃及37℃培养无明显差别,在3%～17%种子液接种量范围内,接种量与生长量有正相关关系.研究表明,玉米淀粉厂废水单一成分即可用于液体发酵产朊假丝酵母NCTC3576菌,原料成本低,且易于产品分离纯化和工业化生产中的连续培养,是单细胞蛋白生产的良好途径.     

脯氨酰内肽酶培养条件的优化及高密度发酵

基因工程菌E.coliBL21/pGEMPEP可以组成型表达重组的点状产气单胞菌脯氨酰内肽酶(PEP),但培养条件极大地影响着酶的产量,为了获得高效表达,首先测定了工程菌表达PEP的稳定性并考察了培养温度、pH、发酵时间、碳源、氮源、无机盐等对产酶的影响,得到了优化的发酵条件,L9(3⁴)正交试验进一步明确了摇床转速、培养温度、pH值、培养时间对产酶量的影响都有高度的统计学意义。在此基础上利用NBSBioFlo3000型5L自控发酵罐进行了高密度、高表达发酵、经20h培养,最终菌体密度达OD₆₀₀60(相当于干菌体225g/L),PEP表达量为28%,每升发酵液中含PEP酶315g。     

酵母乙醇脱氢酶电极的研究

从面包酵母中筛选到一株乙醇脱氢酶活力高、杂酶干扰小的菌株。对该菌株进行扩增培养,在72h收获,用此酵母制成测定乙醇浓度的生物电极,该电极测量的线性范围为3．4×10^－5～2．04×10^－3mol/L,r=0．9996,响应时间小于2min,甲醇、叔丁醇、异成醇的干扰分别为1．2％、2.0％、34％,醋酸和葡萄糖不干扰。测定乙醇标准样品的相对标准偏差为1．6％（n=6）,测定白酒发酵样的相对标准偏差为3．1％（n＝6）。     

营养要素对原油降解菌生长的影响

目的:研究营养要素对原油降解菌生长的影响.方法:通过紫外分光光度计于600nm处测量菌体OD值检测6种碳源、5种氮源、无机离子含量和氯化钠含量对原油降解菌K5生长的影响.通过正交实验确定了原油降解菌K5生长的最佳营养条件.结果:当蔗糖含量0.6%、硫酸铵含量0.2%、氯化钠含量0.6%、微量元素含量0.1%时,原油降解菌生长得最好.结论:确定了原油降解菌K5生长的最佳营养条件,并且发现了培养基中的氮源和碳源对菌体生长量的影响最大以及不同微量元素对菌体生长量有不同程度的促进或抑制作用.     

秦皮素对大肠埃希菌作用机制的初步研究

目的以大肠埃希菌ATCC 25922为供试菌,探讨秦皮素的抑菌活性及其作用机制。方法利用TTC法测定秦皮素对大肠埃希菌ATCC 25922的最低抑菌浓度;通过测定加药前后菌体培养液电导率和大分子的变化及观察扫描电镜和透射电镜电镜结果,分析秦皮素对其细胞膜的影响;通过SDS-PAGE测定秦皮素对供试菌株蛋白含量的影响;采用逐个检出法研究秦皮素对大肠埃希菌ATCC 25922质粒合成的抑制作用。结果秦皮素可抑制大肠埃希菌ATCC 25922的生长,其最低抑菌浓度为40μg/mL。秦皮素作用菌体5 h后,培养液中的电导率比对照组增加1.96%,但DNA和RNA大分子增加的不明显。秦皮素作用大肠埃希菌20 h后,菌体可溶性蛋白总量比对照组降低42%。秦皮素对大肠埃希菌的质粒有消除作用,药物作用48 h后,秦皮素对大肠埃希菌的质粒消除率为60.3%。结论秦皮素可抑制大肠埃希菌的生长,其抑菌作用机制与抑制菌体内蛋白质合成和消除菌体内的质粒有关,但对大肠埃希菌细胞膜的影响不大。     

GL-7-ACA酰化酶的分离纯化及性质研究

CU334是高表达GL-7-ACA酰化酶工程菌,其菌悬液用超声波处理后,经硫酸铵分级沉淀、DEAE-Sephadex A-50离子交换柱层析、DEAE—纤维素DE-52柱层析、Sephadex G-200凝胶过滤及羟基磷灰石吸附柱层析等步骤,得到了凝胶电泳均一的GL-7-ACA酰化酶蛋白,纯化了22倍,得率4.0％,比活力为13.8U/mg。用浓度梯度PAGE测得GL-7-ACA酰化酶的分子量为134kD,用SDS-PAGE测得两个亚基分子量分别为15.5kD和58.4kD。用PI法测得等电点为3.5。GL-7-ACA酰化酶反应最适pH为7.0。反应最适温度为37℃,GL-7-ACA酰化酶对底物GL-7-ACA的K_m值为0.50mmol/L,V_(max)为13.10U·mg~(-1)。Ca²⁺、EDTA和巯基乙醇对该酶有激活作用,Cu²⁺、Fe²⁺和Mg²⁺等有一定程度的抑制作用。产物7-ACA、戊二酸均为GL-7-ACA酰化酶的反竞争性抑制剂,其K_1值分别为16.58mmol·L~(-1)和9.88mmol·L~(-1)。     

诺卡氏菌形放线菌β甘露聚糖酶的纯化和性质

产β１ ,４Ｄ甘露聚糖酶的诺卡氏菌形放线菌（ Ｎｏｃａｒｄｉｏｆｏｒｍ ａｃｔｉｎｏｍｙｃｅｔｅｓ） 菌株ＮＡ３５４０ ,发酵培养７２ｈ ,发酵液离心去菌体,上清经硫酸铵沉淀,９５ ％ 乙醇沉淀,ＣＭＳｅｐｈａｄｅｘ Ａ５０ 柱层析、羟基磷灰石柱层析、ＤＥＡＥ纤维素离子交换及Ｓｅｐｈａｄｅｘ Ｇ１００ 分子凝胶过滤柱等步骤,β甘露聚糖酶的比活提高了１３７ 倍,获得凝胶电泳均一的蛋白样品。经ＳＤＳＰＡＧＥ 和凝胶过滤法分别测定β甘露聚糖酶分子量为４１ｋＤ和４０ｋＤ,证明该酶为单聚体;用等电聚焦电泳测得其等电点为４－８ ;经氨基酸组成分析,发现蛋白中有较高含量的Ｇｌｙ、Ａｓｐ、Ａｌａ 及Ｇｌｕ 残基。该酶的最适反应温度为７５ ℃,在不超过６０ ℃时酶活较稳定;酶反应的最适ｐＨ 为８－０ ,ｐＨ 稳定范围为６－５～１１－０ 。重金属离子Ｈｇ^２＋ 、Ｃｕ^２＋ 、Ｐｂ^２＋ 、Ｆｅ^3＋ 、Ｃｏ^２＋ 、Ｚｎ^２＋ 能强烈抑制该酶活性,而Ｍｎ^２＋ 、Ｆｅ^２＋ 、Ａｇ^＋ 对该酶有部分的抑制作用,低浓度的Ｎａ^＋ 、Ｋ^＋ 、Ｌｉ^＋ 对该酶基本没有影响。     

HPLC法测定病毒性疫苗中EDTA二钠残余量

实验中用高效液相色谱法测定病毒性疫苗中乙二胺四乙酸二钠残余量。EDTA二钠与FeCl3反应生成的络合物NaFeEDTA在257nm波长处有明显吸收,以C18柱将它与其余组分分开后,用外标法建立五级校正曲线可测定制品中EDTA-2Na含量。该方法准确,快速,可用于检测病毒性疫苗中残余乙二胺四乙酸二钠。     

Simultaneous Determination of EDTA, Sorbic Acid, and Diclofenac Sodium in Pharmaceutical Preparations Using High-Performance Liquid Chromatography

A simple high-performance liquid chromatographic method for simultaneous determination of ethylenediaminetetraacetic acid (EDTA), sorbic acid, and diclofenac sodium was developed and validated. Separation was achieved on a C₁₈ column (10 cm × 4.6 mm) using gradient elution. The mobile phase consisted of acetonitrile–ammonium dihydrogen phosphate buffer solution (0.01 M, pH = 2.5, containing 0.8% tetra-n-butyl ammonium hydroxide). The detector wavelength was set at 254 nm. Under these conditions, separation of three compounds was achieved in less than 10 min. The effect of two metal salts and metal concentration on peak area of EDTA was investigated. The pH effect on retention of EDTA and sorbic acid was studied. The method showed linearity for EDTA, sorbic acid, and diclofenac in the ranges of 2.5–100.0, 5.0–200.0, and 20.0–120.0 μg/mL, respectively. The within- and between-day relative standard deviations ranged from 0.52 to 1.94%, 0.50 to 1.34%, and 0.78 to 1.67% for EDTA, sorbic acid, and diclofenac, respectively. The recovery of EDTA, sorbic acid, and diclofenac from pharmaceutical preparation ranged from 96.0–102.0%, 99.7–101.5%, to 97.0–102.5%, respectively. To the best of our knowledge, this is the first report about simultaneous determination of EDTA, sorbic acid, and diclofenac.KEY WORDS: diclofenac sodium, EDTA, high-performance liquid chromatography, pharmaceutical preparations, sorbic acid     

Cell Suspension and Callus Culture from Somatic Tissue of Maize

Cell suspension and callus cultures from somatic tissue of inbred lines of maize (Zea mays L.) were cultured on media that were defined via modification of a Linsmaier and Skoog preparation. Germlings incubated on the primary medium originally employed required long-term incubation for callus induction. Modification of the primary medium with high levels of iron and (ethylene dinitrillo)tetraacetic acid (EDTA), B vitamin amendments and vitamin E, shortened incubation by 75% and nearly doubled the percentage of germlings which produced callus. Callus did not remain viable in subcultures to the secondary medium originally employed, whereas a preparation, developed via modification of the original secondary medium, enabled perpetuation of callus through repeated subculture. Modification with high levels of iron and EDTA, plug B vitamins and vitamin E, with decreased concentrations of five inorganic salts, suppressed aberrant organogenesis and stabilized culture growth as viable callus. Similar modification, with the exception that EDTA was omitted, was employed for the development of a liquid medium. Tonicity of the medium was adjusted with a lowered level of sucrose, with the liquid further modified by addition of acetate. Upon development of this liquid, maize became the sixth monocot species for which somatic cells remain viable through repeated subculture in liquid suspensions.     

一株维氏气单胞菌发酵培养基优化及其制备疫苗免疫效力比较

采用单因素试验、响应面试验法对维氏气单胞菌(Aeromonas veronii)发酵培养基的氮源、碳源、无机盐和磷酸盐成分及用量进行优化组合,确定优化培养基组成：胰蛋白胨10.8 g/L,葡萄糖5.0 g/L,牛肉膏3.0 g/L,磷酸二氢钾2.0 g/L,硫酸镁0.4 g/L,NaCl 5.0 g/L。并与基础培养基的发酵活菌数、制备的灭活疫苗免疫效力进行比较,经过验证试验绘制维氏气单胞菌在优化培养基条件下的7 L发酵罐生长曲线。在优化发酵培养基条件下,维氏气单胞菌活菌数为5.94×10⁹ cfu/mL,比基础培养基增幅43.13%;制备的灭活疫苗相对保护率为77.78%,比基础培养基提高了14.81%。7 L发酵罐发酵培养10 h,活菌数达到最大8.85×10⁹ cfu/mL。通过对发酵培养基的优化,可以获得低成本、优质高效的维氏气单胞菌发酵菌液,为今后维氏气单胞菌灭活疫苗规模化发酵培养提供参考。     

Iron phosphate precipitation in Murashige and Skoog media

Murashige and Skoog revised medium, a standard plant tissue culture medium, precipitated on keeping because iron was ineffectively chelated. Most of the precipitate formed after two days and analysis indicated that it was predominantly iron phosphate. It was found that, after precipitation, the supernatant medium had lost c. 45% of its original iron, 20% zinc and 13% phosphate. The following modifications prevented precipitation: lowering the pH to below 3.2, increasing to 3 the molar ratio of EDTA:Fe or preparing the stock solution of FeEDTA with inadequate heating. Most of these modifications had a deleterious effect on the growth and greening of Ocimum basilicum suspension cultures. However, when an increase in the molar ratio of EDTA:Fe was achieved by reducing the iron concentration to one-third, there was no inhibitory effect on growth or greening of these suspension cultures.     

Reduction of iodate in iodated salt to iodide during cooking with iodine as measured by an improved HPLC/ICP–MS method

Background: Iodate is a strong oxidant, and some animal studies indicate that iodate intake may cause adverse effects. A key focus of the safety assessment of potassium iodate as a salt additive is determining whether iodate is safely reduced to iodide in food. Objective: To study the reduction of iodate in table salt to iodide and molecular iodine during cooking. Materials and Methods: Fifteen food samples cooked with and without iodated salt were prepared in duplicate. The iodine in the cooked food was extracted with deionized water. The iodine species in the extracts were determined by using an improved high-performance liquid chromatography/inductively coupled plasma–mass spectrometry (HPLC/ICP–MS). The cooking temperature and the pH of the food were determined. Results: The conversion rate of iodate in iodated salt to iodide and molecular iodine was 96.4%±14.7% during cooking, with 86.8%±14.5% of the iodate converted to iodide ions and 9.6% ±6.2% converted to molecular iodine to lose. The limit of detection, limit of quantification, relative standard deviation and recovery rate of the method HPLC/ICP–MS were 0.70 μg/L for I⁻ (0.69 μg/L for IO₃⁻), 2.10 μg/L for I⁻ (2.06 μg/L for IO₃⁻), 2.6% and 101.6%±2.6%, respectively. Conclusion: Almost all iodate added to food was converted into iodide and molecular iodine during cooking. The improved HPLC/ICP–MS was reliable in the determination of iodine species in food extracts.     

A pretreatment method for the determination of nitrate in brackish water and seawater based on the hydrazinium reduction technique

The hydrazinium reduction technique has so far been inapplicable to the determination of nitrate in brackish water and seawater due to interference from magnesium ions. We developed a pretreatment method for brackish water and seawater samples for the determination of nitrate based on the hydrazinium reduction technique. Magnesium ions in water samples were converted to a precipitate of a complex with oxine (8-quinolinol) at pH 9.5, and then the precipitate was centrifuged at 3000 rpm for 20 min. The extra oxine in the resulting sample (the supernatant liquid), which inhibits the reduction of nitrate to nitrite, was removed using a Sep-Pak C18 cartridge. Thus we achieved the preparation of a magnesium-free water sample. Using the hydrazinium reduction technique with the proposed pretreatment method, nitrate in brackish water and seawater as well as fresh water was quantitatively determined with high accuracy. Received: July 21, 1999 / Accepted: September 26, 1999     

Levels of Metals in Kidney,Liver, and Muscle Tissue and their Influence on the Fitness for the Consumption of Wild Boar from Western Slovakia

Urine lead level is one of the most employed measures of lead exposure and risk. The urine samples used in this study were obtained from ten healthy male cyclists. Dispersive liquid–liquid microextraction combined with ultraviolet and visible spectrophotometry was utilized for preconcentration, extraction, and determination of lead in urine samples. Optimization of the independent variables was carried out based on chemometric methods in three steps. According to the screening and optimization study, 133 μL of CCl₄ (extracting solvent), 1.34 mL ethanol (dispersing solvent), pH 2.0, 0.00 % of salt, and 0.1 % O,O-diethyl dithiophosphoric (chelating agent) were used as the optimum independent variables for microextraction and determination of lead. Under the optimized conditions, R ² was 0.9991, and linearity range was 0.01–100 μg L^?1. Precision was evaluated in terms of repeatability and intermediate precision, with relative standard deviations being <9.1 and <15.3 %, respectively. The accuracy was estimated using urine samples of cyclists as real samples and it was confirmed. The relative error of ≤5 % was considered significant in the method specificity study. The lead concentration mean for the cyclists was 3.79 μg L^?1 in urine samples. As a result, the proposed method is a robust technique to quantify lead concentrations higher than 11.6 ng L^?1 in urine samples.     

Electron Microscopy and X-ray Analysis of Cr-Containing Precipitates Synthesized by Newly Isolated Actinobacterium,Flexivirga alba ST13T

Chromium(Cr) precipitate synthesized by Cr(VI)-reducing bacterium Flexivirga alba ST13^T was examined using transmission electron microscopy (TEM) and the energy dispersive X-ray (EDX). The strain showed altered-morphology after exposing to Cr(VI) in minimal medium. The resultant precipitate included bacterial pellet and needle-like structure which was similar to the structure made from Cr(OH)₃ precipitate. Cr was observed in bacterial cells using TEM–EDX. Bacteria with high electron density showed the precipitation of Ca in addition to Cr. The isolated strain would be useful to precipitate Cr from Cr(VI)-containing environment.     

Determination of ochratoxin A in fruit juice by high-performance liquid chromatography after vortex-assisted emulsification microextraction based on solidification of floating organic drop

A rapid, simple, and green vortex-assisted emulsification microextraction method based on solidification of floating organic drop was developed for the extraction and determination of ochratoxin A (OTA) with high-performance liquid chromatography. Some factors influencing the extraction efficiency of OTA such as the type and volume of extraction solvent, sample pH, salt concentration, vortex time, and sample volume were optimized. Under optimized conditions, the calibration curve exhibited linearity in the range of 50.0–500 ng L^?1 with a coefficient of determination higher than 0.999. The limit of detection was 15.0 ng L^?1. The inter- and intra-assays relative standard deviations were in a range of 4.7–8.7%. The accuracy of the developed method was investigated through recovery experiments, and it was successfully used for the quantification of OTA in 40 samples of fruit juice.