A microassay for tetrazolium-reductase activity of polymorphonuclear leukocytes--comparison with a test-tube technique

A micromethod for quantitative INT test is described to assay the activity of human peripheral blood PMNL. PMNL are obtained from a pellet after the separation of mononuclear cells. The test is performed in microtiter plates with 96 wells. The estimation of the activity is based on the colour change following the INT reduction to formazane. As a stimulating agent starch (rice species--Amylum oryzae) or zymosan is used. Absorbance alternations are measured using a microplate reader (e.g. MR 580, Dynatech). Conditions necessary for standard results (i.e. PMNL concentration, type and concentration of stimulating agent, incubation time, sedimentation time for PMNL isolation) were verified experimentally. Blood samples of 60 blood donors were simultaneously tested by the micromethod and the test-tube method. The statistical analysis (F-test of homogeneity of variances, t-test with paired observations, correlation coefficient of tightness of dependence) proved that the results of the micromethod were comparable to the test-tube assay. The advantages of the micromethod are as follows: low blood consumption, possibility to perform more parallel examinations of the same sample, and to test more samples at a time, standard and highly reproducible results, saving of reagents, reduction of labour time, possibility of some further automation.     

Evaluation of rapid methods for differentiation of Bifidobacterium species

R oy , D. & W ard , P. 1990. Evaluation of rapid methods for differentiation of Bifidobacterium species. Journal of Applied Bacteriology 69 , 739–749.
The fermentation patterns of 20 Bifidobacterium strains were determined by two methods: a carbohydrate utilization test, based on the production of acid from a given range of sugars in modified MRS broth (micromethod), and a gas chromato-graphic (GC) analysis of fermented modified MRS broth. The micromethod provides a qualitative fermentation pattern whereas the GC method gives a quantitative evaluation of carbohydrate utilization. The micromethod and GC analysis can be applied for the differentiation of Bifidobacterium strains used in fermented milks.     

An improved micromethod for infectivity assays and neutralization tests of dengue viruses

An improved micromethod for infectivity assays and neutralization (N) tests of dengue (DEN) type 1-4 viruses was developed, using 96-well plates and the PAP (peroxidase-antiperoxidase) staining technique. The foci formed on BHK-21 cell monolayers in wells of the plate were readily countable under an ordinary stereomicroscope. This micromethod has the advantages over the micromethod of the Lab-Tek 8 chamber slide system of lower cost, requirement for smaller volumes of test sera and applicability to larger number of serum specimens for N tests of DEN viruses.     

Micromethod for Serogrouping Escherichia coli

A micromethod for serogrouping strains of Escherichia coli is described. The method is accurate, rapid, and economical in terms of time and reagents utilized to perform the test.     

Micromethod System for Identification of Anaerobic Bacteria

A micromethod multitest system prepared by Analytab Products, Inc. and conventional tests employed at the Center for Disease Control for identification of anaerobes were compared. All procedures were conducted in an anaerobic glove box. A total of 104 cultures, including 18 reference strains and 86 diagnostic cultures, were examined. Ninety-one percent of the total tests performed with the two systems were in agreement. Greater than 90% agreement between the two systems was obtained with 12 of the 17 differential tests compared. The tests for nitrate reduction and H(2)S production gave the poorest agreement, 77.8 and 80.8%, respectively. Only 66% of the 86 diagnostic cultures could be presumptively identified with the micromethod system supplemented only with microscopy and colonial characteristics. However, when appropriate supplementary tests and gas-liquid chromatography were used with the micromethod system, 85% of the 86 strains could be identified. When Ehrlich reagent, instead of Kovac reagent, was used with the micromethod to test for indole, the agreement in identification was raised to 93%.     

肥达氏反应教学实验新方法

采用20孔U型孔血凝反应板微量法代替传统的肥达氏反应实验方法,结果可见凝集现象清晰,易于判断,解决了试管法易摇动而影响结果观察、需大量试剂和试管等不足。20孔血凝板微量反应法可作为肥达氏反应教学实验的一种新方法。     

Visual micromethod for assay of fungal growth

A visual micromethod for measuring antifungal effects on germination and growth is described. The antifungal agent griseofulvin and the fungus Trichophyton mentagrophytes were used as materials to compare the micromethod with a standard assay based on dry mycelial weight. The micromethod was more sensitive than the weight method in detecting the minimum inhibitory concentration of griseofulvin (0.18 and 35 microgram/ml, respectively). At higher concentrations of griseofulvin (22.5 microgram/ml), the micromethod measured minimal fungal growth that was undetectable on a weight basis. The micromethod showed that griseofulvin does not change the number of spores forming germ tubes. Progressively severe alterations in fungal morphology occured as the concentration of griseofulvin was increased from 0.09 to 22.5 microgram/ml.     

Microadaption of Hemadsorption Inhibition for Neutralization Tests with Pig Hemagglutinating Encephalomyelitis Virus

A microneutralization test is described, in which secondary pig thyroid cells are used and end points are determined by hemadsorption with hamster erythrocytes. The method is evaluated and compared with a macromethod, in which the fluorescent-antibody staining technique is applied for reading titers. The micromethod was found to be useful because of its sensitivity and rapidity.     

Microtiter Plate Agglutination Test for Brucella canis Antibodies

A micro-agglutination test for the antibodies to Brucella canis produced similar results to those obtained with the standard tube agglutination method in human and canine sera. The micromethod does provide an economical means of screening sera for the presence of antibodies.     

微量法鼠疫菌质粒DNA抽提的优化

旨在分析微量法抽提鼠疫菌质粒DNA的效果,探讨其在鼠疫菌分子生物学实验研究中的应用价值.采用微量法分别提取鼠疫菌EV76株,假结核耶尔森菌PstII株及大肠杆菌V517株质粒DNA,琼脂糖凝胶电泳对质粒DNA抽提结果进行分析.结果显示,微量法能在较短时间内获取开环较少的闭合环状鼠疫菌质粒DNA,经琼脂糖凝胶电泳图示其电泳条带清晰、亮度均一.微量法鼠疫菌质粒DNA抽提效率和纯度较好,抽提结果稳定,重复性良好.经微量法抽提的质粒DNA符合多数鼠疫菌分子生物学试验的要求,可广泛应用于鼠疫菌分子生物学试验研究中.     

Micromethod of determining antibodies to nerve tissue galactocerebrosides in the complement fixation reaction

A micromethod has been developed for the determination of serum antibodies to galactocerebroside, a specific antigen of myelin membranes. The method is based on complement fixation test and is used for the analysis of sera from healthy rabbits and those immunized with galactocerebroside.     

A method for detecting the hemagglutinating activity of enteroviruses

Optimal conditions to reveal the hemagglutinating activity in the Coxsackie viruses of group B and hemagglutination test (HAT) performed by the micromethod are studied and determined. The titre of hemagglutinins revealed in HAT is established to depend on pH of the phosphate-buffer solution, concentration of human erythrocytes and their group attribution. The viruses passaged at the suboptimal temperature 33 degrees C are shown to possess the maximal hemagglutinating activity.     

A method for determinations of microgram amounts of chitin in arthropod cuticles

A method is described for determining microgram amounts of chitin in arthropod cuticles. The method, adapted from one described for assaying plant material for fungal content, is based on estimating soluble chitosan derivatives and is not affected by the presence of nonchitinous cuticular components. There is good agreement between the results given by the micromethod and those given by a semimicro method based on material insoluble in hot, dilute alkali. As a qualitative test for chitin the limit for the method is <100 ng.     

Detection of antigen of rickettsia of the tick group in bird droppings by antibody neutralization test]

The author presents methodical materials pointing to a possibility of using a micromethod of the antibody neutralization test for detection in bird pellets of the antigen of rickettsia referred to the Rocky Mountains spotted fever tick group. Specificity of the test, rickettsia antigen and sera was studied. The method is recommended for detection at the territory under study of circulation of the causative agent of tick-borne rickettsiosis by revealing the specific antigen in bird pellets.     

Usefulness of the passive hemagglutination micromethod in the diagnosis of whooping cough

Studies on modification of diagnostic test for pertussis have been continued, they were practically restricted to an application of passive haemagglutination micromethod. Six hundred and twenty eight sera from children suspected to be infected with Bordetella and 38 sera of control children suffering from non-infectious diseases were tested. Despite of the fact that higher titers were obtained with method using red blood cells preserved in Alsevier solution, the passive haemagglutination micromethod may be used in field studies especially during pertussis epidemics what was confirmed by statistical analysis.     

Micro- and macromethod assays for the ecological study of Legionella pneumophila

The survival of a strain of Legionella pneumophila (Lp-1) inoculated in artificial water microcosms was investigated with and without an amoebal host and varying environmental conditions, such as biofilm formation, amount of nutrients and incubation temperature. The results obtained using short (micromethod) and long (macromethod) term methods showed that L. pneumophila Lp-1 dies rapidly at 4 degrees C in the "macromethod" assay. When the same temperature (4 degrees C) was applied to the "micromethod" assay, L. pneumophila Lp-1 survived for three weeks, although it progressively decreased. At an incubation temperature of 30 degrees C, the aquatic environment was more favourable and better survival emerged in the "macromethod"; in contrast, this favourable temperature condition did not improve the survival of L. pneumophila Lp-1 cultured with the "micromethod". The role of the protozoa Acanthamoeba polyphaga proved to be indispensable for legionella survival only when environmental conditions become unfavourable.     

Comparison of "high throughput" micromethods for determination of cytochrome P450 activities with classical methods using HPLC for product identification

Enzyme activities of the CYP enzymes (CYP3A4, CYP2C9 and CYP2A6) were determined using classical substrates (testosterone, diclofenac and coumarin, respectively) as well as with luminogenic or fluorogenic substrates in micromethod arrangement. The luciferin-based luminogenic substrates for CYP3A4 and CYP2C9 as well as coumarin in micromethod for assay of CYP2A6 activity gave results well comparable with the classical methods with determination of reaction products by the HPLC.     

A sensitive and specific method for quantitative estimation of carbamylation in proteins

A simple, rapid, reproducible, and specific micromethod for the estimation of carbamylation of proteins is described. The method is based on permanganate oxidation of radioactive carbamyl derivatives of protein to form urea and on the specific decomposition of urea by urease.     

Ultracentrifugation micromethod for preparation of small experimental animal lipoproteins

Sequential flotation ultracentrifugation is commonly used in the preparation of plasma lipoproteins. However, protocols often require prolonged centrifugation time (48-72 h) and large plasma volumes (2-20 ml), which makes them unsuitable for studies on small laboratory animals. Although analytical techniques such as FPLC have often small sample requirements, further fraction analysis is often limited to the small fraction volume obtained. A sequential ultracentrifugation micromethod is described to obtain rat lipoprotein fractions from 400 microl of plasma in a cumulative centrifugation time of 7.5 h. Fraction volumes were determined and densities were adjusted to those of rat plasma lipoproteins. Polyacrylamide gel electrophoresis and enzymatic measurements of triglycerides, total cholesterol, and phospholipids were used to assess the purity of the lipoprotein fractions. The results were compared with those obtained from a classical sequential ultracentrifugation protocol. The micromethod presented here can be further adapted to other experimental animal species with little modifications.