Reverse plaque formation method for titration of non-cytopathogenic bovine viral diarrhea-mucosal disease virus

The reverse plaque formation (RPF) method with a semi-micro plate was applied to the titration of a non-cytopathogenic (non-CP) strain of bovine viral diarrhea-mucosal disease (BVD-MD) virus. All the five non-CP strains used in this experiment formed reverse plaques (RP) on bovine testicle cell culture under methyl cellulose overlay. The RPF was inhibited by the pretreatment of a non-CP virus strain with immune rabbit serum to a reference strain. The specificity of the RPF method was demonstrated by the linear test and Poisson distribution test. Comparative titration of commercial BVD-MD vaccines was carried out by the semi-micro RPF method and the tube method based on the exaltation on Newcastle disease virus. The virus titer obtained by the former was slightly higher than that obtained by the latter. The former was proved to be a method of high sensitivity for determining non-CP virus.     

快速提取类球红细菌中辅酶Q10的方法研究

目的：建立一种从类球红细菌中快速分离纯化辅酶Q10的方法。方法：对影响超声提取辅酶Q10的各因素,包括提取试剂、超声频率、循环次数及工作时间的最佳条件进行正交试验,比较超声破碎法与碱醇皂化法提取辅酶Q10的差异。结果：在超声提取中,提取试剂和循环次数对辅酶Q10提取效果具有显著性影响;在超声频率0.5s、丙酮提取3min、循环3次的条件下提取的辅酶Q10的含量比碱醇皂化法提高了近6倍。结论：超声破碎法是一种简单、迅速、高效的提取辅酶Q10方法。     

A method for characterizing ultrasonically-derived follicular data in heifers

A method was developed for ultrasonically characterizing follicular waves in heifers without the necessity of maintaining day-to-day identities of individual follicles (nonidentity method). Results were compared to a method in which the identities of individual follicles were maintained from day to day (identity method). Data collected daily during 5 estrous cycles were processed by each method, independently, by different operators. The nonidentity method involved grouping and then profiling follicles in order of decreasing diameters without regard to day-to-day identities. The profiling scheme distinguished between follicles of the left versus the right ovary. The dominant and subordinate follicles were readily distinguishable in the nonidentity profiles. When successive dominant follicles developed in the opposite ovary, the follicles were profiled directly. When two successive dominant follicles developed on the same ovary, size information was obscured for a few days where the profiles for each follicle crossed, but continuity of the profiles on each side of the area of ambiguity was maintained. The nonidentity method seemed equivalent to the identity method in determining characteristics of the dominant follicle (e.g., maximal diameter, growth rate, regression rate). Day of emergence of a wave and day of divergence in diameters between dominant and subordinate follicles were readily determined by inspection of the nonidentity profiles. A greater number of subordinate follicles per wave was detected by the nonidentity method due to the inability to individually identify all detected follicles by the identity method. Regression of follicles from a previous wave into the subordinate follicles of a succeeding wave was apparent by either method. The nonidentity method seemed suitable for most needs, was less tedious, and required less skill than the identity method.     

Improvement of the micromethod for the limulus lysate test.

Frauch's micro-slide method was improved to facilitate the endpoint-determination of the Limulus test. Two precise observations, by inverted phase contrast microscopy and with a staining procedure, were newly performed as additions to the slide test. The staining procedure was proposed as an improved method for the Limulus test since it is simple and convenient. In the staining method, bromophenol blue (BPB) solution was used as the staining solution. A negative (-), a strong positive (++) and a weak positive reaction (+) were characterized by a "ring" formation, a "cloud-like" spread of gel and a "spot" in the "cloud" respectively. Since the distinction between (-) and (+) reactions was obvious in the proposed method, determination of the endpoint was easier than in the ordinary tube and Frauch's method. The sensitivity of the present method was equal to or higher than that of other methods. Inverted phase contrast microscopy was utilized to confirm the findings obtained by the staining method. The volume of the lysate used in this method was as little as 1/10 of that used in the tube method.     

Formation of Fine Oil-in-Water Emulsions by the Gel Emulsification Method with Saccharide and Protein

An emulsification method using a gel-like phase of a saccharide and protein mixture has been developed. In the method, which is called a gel emulsification method, an oil is added to the highly concentrated saccharide solution containing protein to form a clear gel-like phase, which followed by dilution with water to form a fine oil-in-water emulsion. This emulsion was investigated as to its emulsifying activity and emulsion stability as compared with that obtained by high-shear equipment, which was called a homomixer method. The emulsifying activity of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method.

The emulsions prepared by both methods were highly stable in terms of the stability against coalescence. On the other hand, the stability against creaming of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method.

The surface hydrophobicity of the protein and the unfreezable water content in the highly concentrated saccharide solution containing protein were not correlated to the emulsifying properties of the emulsions prepared by the gel emulsification method, which appeared to be dependent on the viscosity of the highly concentrated saccharide solution containing protein.     

T细胞亚群CD4测定方法的研究

为比较外周血T淋巴细胞亚群CD4不同测定方法的差别,以流式细胞术为定量手段,测定了猴外周血中三种不同方法处理后CD4的表达.结果表明：先标后溶法——先用异硫氰荧光素标记的单克隆抗体（FITC-CD4 McAb）标记后,再加入红细胞溶解液溶掉红细胞的处理方法,结果基本等同于传统的淋巴细胞分离法,但样本用量仅为传统方法的1/5,且操作简单.激光共焦显微术的形态学研究也证实：先标后溶法与淋巴细胞分离法相似,其细胞膜表面荧光标记清晰,优于先溶后标法.     

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: comparative study of preparations extracted by the phenol-water and the phenol-chloroform-petroleum ether methods

An R-form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN-111 (O3-:K1-) by the phenol-chloroform-petroleum ether method was compared with that extracted by the phenol-water method in the ability to form a hexagonal assembly. The LPS which was extracted by the phenol-water method and dialyzed against tap water to remove phenol showed ribbon-like structures, and it formed a hexagonal lattice structure with a lattice constant of 14.5 +/- 0.3 nm when it was precipitated by addition of two volumes of 10 mM MgCl2-ethanol. The LPS which was extracted by the phenol-chloroform-petroleum ether method and lyophilized consisted of ribbon-like structures and their fragments and it often formed small pieces of a hexagonal lattice, although the LPS before lyophilization did not form such a lattice. When the LPS extracted by the phenol-chloroform-petroleum ether method was precipitated by addition of two volumes of 10 mM MgCl2-ethanol, it formed essentially the same hexagonal lattice structure as that formed by the LPS extracted by the phenol-water method. From these results it is concluded that the ability of the LPS to form a hexagonal lattice structure does not depend upon the method of its extraction from bacterial cells.     

Determination of cortisol in human saliva using liquid chromatography-electrospray tandem mass spectrometry

The aim of this work was to develop a method for determination of cortisol in saliva by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Saliva was sampled on Salivette tubes. These were centrifuged, deuterium-labeled cortisol was added as internal standard and the proteins precipitated by acetonitrile. The supernatant was evaporated, dissolved in methanol acidified with acetic acid and analyzed by LC-MS-MS. The with-in run precision, tested by pooling saliva samples from volunteers and then analyzing these in a single run, was found to be 7% at 0.7 microgram l(-1). The between-run precision was tested by analysis of the same samples at different days and found to be 11% at 2.5 microgram l(-1). The limit of quantification was 0.5 microgram l(-1). The method was applied for analysis of saliva samples from three volunteers during their last week before vacation and the first and second week on vacation. In addition, the method was compared to analysis by an immunological method. The values from the immunological method were 2.7 times higher than the LC-MS-MS results.     

A quantitative method to evaluate neutralizer toxicity against Acanthamoeba castellanii.

A standard methodology for quantitatively evaluating neutralizer toxicity against Acanthamoeba castellanii does not exist. The objective of this study was to provide a quantitative method for evaluating neutralizer toxicity against A. castellanii. Two methods were evaluated. A quantitative microtiter method for enumerating A. castellanii was evaluated by a 50% lethal dose endpoint method. The microtiter method was compared with the hemacytometer count method. A method for determining the toxicity of neutralizers for antimicrobial agents to A. castellanii was also evaluated. The toxicity to A. castellanii of Dey-Engley neutralizing broth was compared with Page's saline. The microtiter viable cell counts were lower than predicted by the hemacytometer counts. However, the microtiter method gives more reliable counts of viable cells. Dey-Engley neutralizing medium was not toxic to A. castellanii. The method presented gives consistent, reliable results and is simple compared with previous methods.     

Estimation of cardiac output by the CO2 rebreathing method during tethered swimming

The reproducibility of cardiac output (Q) estimated by the CO2 rebreathing method during tethered swimming was studied in five highly trained college swimmers. The reproducibility of the CO2 rebreathing method for estimations of Q during tethered swimming was similar to the reproducibility reported for the CO2 rebreathing method, direct Fick method, or dye-dilution method during either cycling or treadmill walking. All duplicate estimates of Q by the CO2 rebreathing method were within 15% of one another. A comparison was made between the Q's estimated by the CO2 rebreathing method during tethered swimming and previously published data on Q determined by the dye-dilution method during free swimming in a flune. At any given oxygen uptake, Q obtained by the CO2 rebreathing method during tethered swimming was not significantly different from the Q obtained by the dye-dilution method during flume swimming. Estimates of Q by the CO2 rebreathing method made during high intensities of tethered swimming were reproducible and appear to be valid.     

运用微生物法测定保健食品中的维生素B6

介绍运用微生物法测定保健食品中维生素B6的含量。GB／T5009．154—2003将微生物法规定为测定食品中维生素B6的国家标准方法Ⅲ。其原理为卡尔斯伯酵母菌需在有维生素B6存在的条件下才能生长,在一定条件下维生素B6的量与其生长量成正比关系。用分光光度仪在550nm波长下测定该菌的生长．与标准曲线相比较,从而得出该样品中维生素B6的含量。通过对国标方法作较详细的注解,并对有些地方作适当的修改,以期对需要开展此项工作的实验室及其人员会有较大的帮助。     

大肠杆菌O157:H7核酸探针检测方法的建立

目的：应用核酸探针方法快速检测大肠杆菌O157：H7。方法：通过使用吖啶酯标记的特异DNA探针方法检测大肠杆菌O157：H7,对此种方法的特异性、敏感性、准确性进行研究,比较该方法与传统国标法的检测结果。结果：核酸探针方法检测大肠杆菌O157：H7特异性以及敏感性强,检出大肠杆菌O157：H7菌液浓度最低限约为106cfu/ml,检测大肠杆菌O157：H7的结果与国标法相一致;对O157：H7鉴定时间仅需30min,简便快捷。结论：核酸探针方法可用于大肠杆菌O157：H7的快速检测。     

抗MRSA-PBP2a鸡卵黄抗体的制备及乳胶凝集检测法的建立

制备抗耐甲氧西林金黄色葡萄球菌青霉素结合蛋白2a( MRSA- PBP2a)抗原的鸡卵黄免疫球蛋白(IgY),建立检测MRSA的乳胶凝集方法.采用体外诱导的方法制备PBP2a蛋白,胸部肌肉多点注射方式免疫6只海蓝蛋鸡,水稀释法提取IgY,BCA法测定蛋白含量,Western blotting进行特异性分析,用提取的IgY抗体致敏聚苯乙烯乳胶,建立检测PBP2a的乳胶凝集方法.成功诱导并制备获得纯化的PBP2a蛋白,首次免疫后1月每枚鸡蛋提纯后可获得约48 mg IgY抗体,Western blotting结果显示IgY抗体能有效识别纯化的PBP2a蛋白;成功建立检测PBP2a的乳胶凝集法,敏感性达1 mg/L.抗MRSA- PBP2a鸡卵黄抗体具有较高的敏感性和特异性,基于其建立的乳胶凝集检测方法具有较好的灵敏性.     

Detection of the low-germination-rate resting oospores of Pythium myriotylum from soil by PCR

AIMS: To establish a sensitive and specific polymerase chain reaction (PCR)-based method for detecting Pythium myriotylum in soils. METHODS AND RESULTS: Oospores of P. myriotylum were separated from large soil particles by flotation in sucrose solution. The thick-walled oospores were disrupted by vortex with sea sand and its DNA was extracted by the Cetyl trimethyl Ammonium Bromide (CTAB) method. The recovered DNA was verified by PCR amplification of a 150-bp target sequence of P. myriotylum. Samples of 10 g of soil were assayed; thus, the detection limit by PCR-based method was 10 oospores per gram soil. The method was successfully applied for the detection of P. myriotylum in soils collected in March, prior to planting of ginger crops. CONCLUSIONS: A PCR-based method for detecting P. myriotylum from soil was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR method has allowed us to monitor the presence of P. myriotylum in soil prior planting season as a way of reducing or eliminating disease.     

脂质体法和电穿孔法转染哺乳动物细胞研究

用脂质体法和电穿孔法分别转染Cos-7,Vero和Namalwa细胞.发现脂质体法在转染效率和操作方便方面比电穿孔法优越,而电穿孔法对细胞种类的适用性方面似乎比脂质体法广. 结果表明,电穿孔法能转染Cos-7,Namalwa和Vero细胞,而用脂质体法只能转染Cos-7和Vero细胞.     

A method of direct measurement for the enzymatic determination of cholesteryl esters

A direct measurement method for the enzymatic determination of cholesteryl esters (CEs) without measuring total cholesterol (TC) and free cholesterol (FC) is described. In the first step, hydrogen peroxide generated by cholesterol oxidase from FC was decomposed by catalase. In the second step, CE was measured by enzymatic determination using a colorimetric method or a fluorometric method. The measurement sensitivity of the fluorometric method was more than 20 times that of the colorimetric method. Optimal conditions of the assay were determined, and examples of measured CE in human plasma, rat liver, and cultured cells are indicated. The method of directly measuring CE was simple and has exceptional reproducibility compared with the technique of subtracting FC from TC using each measured TC and FC.     

潜生体定植的原位观察技术研究

对小鼠结肠组织原位标本进行潜生体定植的实验研究。染色采用美蓝初染后,高锰酸钾复染的方法。方法简便、有效。     

Use of a new methylene blue derivative for determination of lipid peroxides in foods

To determine lipid peroxides in chloroform-methanol extracts of foods, a simple and sensitive colorimetric method using a new leucomethylene blue derivative was adopted. The amounts obtained by this method coincided well with those by the iodometric method and paralleled those by the thiobarbituric acid method.     

A new method for the separation of the constant and the variable fluorescence of chlorophyll a in in vivo

The constant fluorescence of chlorophyll a in alga Dunaliella tertiolecta was estimated by a method of the least square regression applied to the linearized form of the equation y = axb. The value of the constant fluorescence obtained by this method was compared by the values estimated with simple linear extrapolation. Constant fluorescence, evaluated by the simple linear extrapolation of 10 ms of the initial variable fluorescence, was 50% higher than the value obtained by the method of the least square regression. We demonstrated that the estimation of constant fluorescence by the least square regression is a more correct method and provides a better comparison of results from different laboratories. This method offers a simple way to determine and separate constant fluorescence from variable fluorescence in the total yield of chlorophyll a fluorescence in "in vivo" conditions. Furthermore it facilitates the interpretation of the variable fluorescence phenomena in "in vivo".