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PubMed1976

[The fecal flora of man. IV. Communication: Comparison of the newly developed method with the old conventional method for the analysis of intestinal flora (author's transl)].

The results of 42 fecal flora analysis of healthy adult men were compared by using a newly developed method and an old conventional method. Statistical estimate indicate, that with the new method the total counts and the counts of bifidobacteria, catenabacteria (eubacteria and anaerobic lactobacilli), peptostreptococci, and clostridia (except Cl. perfringens) as well as the occurences of peptostreptococci and spirillaceae were significant higher than with the use of the old method. On the other hand the counts and occurences of bifidobacteria, veillonellae, Cl. perfringens, Megasphaerae and aerobes were similar with both method. With the new method the total counts of feces of healthy adult men nubmered 1-2 X 10(11) per g wet weight, and fastidious anaerobes, such as bacteroides, catenabacteria and peptostreptococci, were always occurred as predominant flora. The results suggest that the conventional anaerobic jar method is inadequate for the isolation of fastidiuos anaerobes and indicate that the use of modified medium 10 in combination with strict anaerobic technique is necessary for the analysis of fecal flora.

Adult↗
PubMed1975

[Comparison of conventional methods for the determination of albumin, transferrin and ceruloplasmin in serum with immunological reference methods (author's transl)].

Radial immunodiffusion, which is a presently accepted reference method, was compared with the mechanized immunoprecipitation. Each of these specific methods was compared with two normal clinical-chemical methods for the determination of albumin and transferrin, and an enzymic method for the determination of ceruloplasmin. The correlation coefficients, the mean slope for regression, and the percentage variation of the average values from those of the average values of the reference methods are presented. In the determination of albumin and ceruloplasmin, radial immunodiffusion and mechanized immunoprecipitation give the same results. Surprisingly, these two specific methods give different results in the determination of transferrin. In as far as there is no doubt concerning the accuracy of the mechanized immunoprecipitation, this method is recommended, since it also has the advantage of being less demanding in cost and time.

Autoanalysis↗
PubMed1990

Methods for estimating release rates during high frequency quantal secretion and for testing such methods.

The rate of spontaneous quantal release must be estimated in some investigations of synaptic transmission, even when frequencies are so high that individual quanta cannot be distinguished. An obvious method is to measure the time integrals of the summed MEPPs and then dividing this value by the integral of an average MEPP. The method was tested by recording miniature end-plate potentials (MEPPs) at frog neuromuscular junctions, counting the number of MEPPS, and then adding together records from the same junction to simulate high frequencies. The estimates from the integral method agreed well with the actual counts. The method can readily be used with a microcomputer and does not require stationary data. Methods based on fluctuation analysis were also used to estimate quantal frequencies, but they did not always give good estimates. This was not a thorough test of the fluctuation method, but an example of testing with MEPP data. The integral method might be reasonably reliable, but there are further potential complications, like changes in MEPP size and short-circuiting of the end-plate membrane, which may make it difficult to obtain reliable measurements of high frequency quantal secretion without voltage clamping and protocols that permit measurements of individual MEPCs during the course of the frequency measurements.

Animals↗
PubMed2026

An enhanced multisegment RT-PCR method for influenza A virus sequencing: Improved performance and reduced preparation time over traditional methods.

Influenza A viruses (IAVs) remain a major global health threat, affecting both human and animal populations. Whole-genome sequencing is essential for monitoring viral evolution, zoonotic transmission, and emerging variants. However, conventional RT-PCR methods often result in incomplete gene coverage, amplification biases, and reduced sequencing accuracy, particularly in clinical samples. We developed a robust In-house method for IAV full-genome sequencing using the Oxford Nanopore Technologies (ONT) long-read sequencing platform. This method integrates an in-house multisegment Reverse Transcription PCR (RT-PCR) method with a streamlined 2-pool primer design targeting all eight IAV gene segments. RNA extracted from clinical and stock virus samples was reverse-transcribed and amplified using Superscript IV-based chemistry, followed by magnetic bead purification to ensure high-quality amplicons. Sequencing libraries were prepared with the Native Barcoding Kit 24 (SQK-NBD114.24) and sequenced on R10.4.1 flow cells on the MinION MK1C device. Data analysis using the Iterative Refinement Meta-Assembler (IRMA) confirmed improved read depth, uniform coverage, and complete genome recovery. Compared to conventional methods, our In-House Multisegment 2-Pool (IH-MS2P) RT-PCR method generated higher numbers of matched read counts, minimized chimeric artifacts, and delivered superior genome coverage across human, swine, and avian isolates. This optimized RT-PCR method provides a high-performance, time-efficient, and portable solution for influenza genomics, demonstrating robust applicability even with clinical samples of low RNA yield.

Influenza A virus↗
PubMed1978

Comparison of the conventional lecture method and the self-instruction method for teaching clinical pathology.

Two separate courses in clinical pathology were presented to two groups of second-year medical students. The first (large) group was taught by the conventional lecture method; the other (small) group, by a self-instructional method for roughly half of the course, followed by the conventional lecture method for the second half of the course. The effectivenesses of the two methods were compared, based on performances on the final written examination. It was found that the self-instructional method was as effective as the conventional method in teaching clinical pathology, at least in teaching basic principles and interpretive clinical chemistry, as judged by the performances of the two groups. Surprisingly, the two groups differed significantly in performances on the portion of the course taught by similar methods.

Education, Medical, Undergraduate↗
PubMed1976

A comparative study of the Bessey method and Hausamen method for serum alkaline phosphatase determination.

The methods of Bessey et al. and Hausamen et al. for serum alkaline phosphatase determination were compared. Determinations were carried out using sera from healthy blood donors and sera from patients suffering from different hepatobiliary diseases. A high reproducibility of values obtained using both methods was noted. The sensitivity of the method of Hausamen et al. is about four times greater than that of the method of Bessey et al., even though the former method employed a lower reaction temperature. A high degree of correlation exists between the two methods and the data reported indicate that both methods are completely valid.

Adult↗
PubMed1991

Collagen-gel-embedded three-dimensional culture of human thyroid epithelial cells: comparison between the floating sandwich method and the dispersed embedding method.

Human thyroid epithelial cells were isolated from surgically resected human thyroid gland with collagenase and cultured for one week under EGF-supplemented conditions to allow them to proliferate. Then the cells were transferred to the following three-dimensional culture systems. One was a culture of isolated cells between floating double layers of collagen gel, designated the "floating sandwich method." The other was a culture of isolated cells mixed with collagen gel, designated the "dispersed embedding method." Many folliclelike structures with lumina of appreciable size were obtained by the former method. The cells cultured by the floating sandwich method exhibited a distinct polarity shown by the presence of numerous microvilli at the apical surface and close contact with collagen gels at the basal surface. On the other hand, only a few folliclelike structures were obtained by the dispersed embedding method, in which the folliclelike structures were small in size and the cells showed less distinct polarity than those observed in the floating sandwich method. Thus, the floating sandwich method appears to be suitable for studying the process and mechanism of in vitro organization of follicular structures by human thyroid epithelial cells.

Cell Division↗
PubMed1990

Particle design of tolbutamide by the spherical crystallization technique. III. Micromeritic properties and dissolution rate of tolbutamide spherical agglomerates prepared by the quasi-emulsion solvent diffusion method and the solvent change method.

With the objective of modifying the micromeritic properties of tolbutamide (i.e., to manufacture a highly functional powder form), particle design was attempted using a quasi-emulsion solvent diffusion (QESD) method, and the micromeritic properties and dissolution rate of the obtained spherical agglomerates were evaluated by comparison with agglomerates prepared by the solvent change (SC) method. For the production of tolbutamide agglomerates by the QESD method, a necessary condition was the addition of a sucrose fatty acid ester to the system as an emulsifying agent. The particle diameter of the agglomerates obtained by the QESD method depended on the size of the initially formed quasi-emulsion droplets, which in turn depended on the viscosity of the solution. In addition, the agglomerates were nearly perfectly spherical in shape. In the QESD method, the quasi-emulsion droplets crystallized instantaneously from the droplet surface inward. The resultant agglomerates were dense, had great mechanical strength and showed excellent flowability due to their perfect spherical shape. On the other hand, the agglomerates produced by the SC method were conglomerates of primary crystals, and fine, needle-like crystals formed on their surface. As a result, these agglomerates had a large specific surface area, and they therefore showed greater solubility than the agglomerates prepared by the QESD method.

Chemistry, Pharmaceutical↗
PubMed1992

[Development of a new method for calibration of pneumotachograph according to flow rate--evaluation of a computer-controlled-system for assessing respiratory gas exchange using this method].

1) We evaluated the method for calibrating the pneumotachograph according to flow rate by the software on personal computer. Flow volume was detected using the approximated equation (y = 1.294x2 + 0.989x + 35.824, r = 0.976, p less than 0.01) from various mean flow rate and integration of output voltage. The error between syringe volume (21) and the estimated value on the equation was 10 +/- 8 ml (0.48 +/- 0.42%). 2) Using the method described in 1), we developed computer-controlled-system for assessing respiratory gas exchange by mixing chamber method. To evaluate the accuracy of the system as compared with the Douglas Bag method, we measured the gas parameters of the both methods for two male subjects at rest(3 and 5min) and steady state exercise (3 min., 60-240watt). The error between the both methods were within +/- 5% in ventilation(VE), mixed expired O2 (FEO2) and CO2 (FECO2) concentration, and were within +/- 8% in O2 uptake(VO2) and CO2 production (VCO2). There were high positive correlation (r greater than 0.99, p less than 0.01) between the both methods in the all parameters. These results indicate that the system in this study would be practical use, that VE can be measured in only one pneumotachograph, even if the range of flow rate was wide(e.g. from rest to heavy exercise).

Adult↗
PubMedDate not supplied

[Comparative evaluation of 2 methods for the determination of pyruvate dehydrogenase activity in tissues in avitaminosis B 1 induced by various methods].

A method for determination of pyruvate dehydrogenase activity in mitochondria and tissue homogenates was developed. The method was based on a spectrophotometric monitoring of p-nitroaniline acetylation under conditions required to ensure correct stoichiometric course of the reaction. In rat tissues absolute amounts of the enzyme activity, determined by the method, were found to be several-fold lower as compared with the values determined by the conventional ferricyanide method. Within 24 hrs after a single administration of hydroxythiamin into rats (400 mg per 1 kg of body weight) the pyruvate dehydrogenase activity was decreased 1.5-fold in heart (as estimated by the reaction of p-nitroaniline acetylation) and did not alter in liver tissue. While if the determinations were carried out by the method of terricyanide reduction the enzyme activity was decreas 7- and 2-fold, respectively, in heart and liver tissue. The data obtained suggest that hydroxythiamin impaired reactions of electron transport in tissues; on the other hand, the data obtained showed that the method for determination of the pyruvate dehydrogenase activity, based on the acetylation reaction, was more specific than the conventional method which involved measuring of ferricyanide reduction.

Animals↗
PubMed1992

von Willebrand factor antigen: a radial immunodiffusion method evaluated and compared with an ELISA method.

A new commercial kit method for the quantification of von Willebrand factor antigen (vWFAg) by radial immunodiffusion was compared to an established ELISA technique. Major discrepancies were found between the two methods. The radial immunodiffusion method had poorer intra- and inter-assay coefficients of variation than the ELISA method, although there was adequate inter-method agreement when 100 plasma samples from controls and patients were compared. However, there was a great difference in values obtained for vWFAg in the reference sample supplied with the commercial kit and that obtained directly from the National Institute for Biological Standards and Controls. There were also differences in levels of significance when vWFAg was measured by the two techniques in different clinical groups, and standard deviations were larger when the kit method was used. It is suggested that on scientific and economic grounds, the commercial radial immunodiffusion kit does not offer a competitive advantage over the ELISA method.

Autoantigens↗
PubMedDate not supplied

Comparison of two immunochemical methods with thin-layer chromatographic methods for determination of aflatoxins.

Three different methods were compared for the determination of total flatoxins in corn and peanuts naturally contaminated with aflatoxins and in corn, peanuts, cottonseed, peanut butter, and poultry feed spiked with aflatoxins B1, B2, and G1. The 3 methods were an enzyme-linked immunosorbent assay (ELISA) screening test; a monoclonal antibody-affinity column-solid-phase separation method; and the AOAC official thin-layer chromatography (TLC) methods for all except poultry feed, for which Shannon's TLC method for mixed feed was used. The ELISA test is designed to provide only positive results for total aflatoxins at greater than or equal to 20 ng/g or negative results at less than 20 ng/g. The affinity column separation is coupled with either bromination solution fluorometry to estimate total aflatoxins or liquid chromatography (LC) to quantitate individual aflatoxins. Fluorodensitometry was used to determine aflatoxins in commodities analyzed by the TLC methods. The LC and TLC results were in good agreement for all the analyses. The results for the affinity column using bromination solution fluorometry were similar except those for cottonseed, which were about 60% higher. The ELISA screening method correctly identified naturally contaminated corn and peanut positive samples. No false positives were found for controls. The correct response for spiked corn, raw peanuts, peanut butter, and cottonseed at greater than or equal to 20 ng aflatoxins/g was about 90%. The correct response for spiked poultry feed at greater than or equal to 20 ng aflatoxins/g was about 50%.

Aflatoxins↗
PubMed1979

[Critical study of methods of estimation by an enzymatic method of serum triglycerides (author's transl)].

The authors describe a critical study of two entirely enzymatic methods of determination of serum triglycerides which differ mainly by the choice of hydrolytic agents: esterase and hydrolase by the Roche methods, lipase and esterase in the case of the Boehringer method. The estimation of the resulting free glycerol requires 3 sequential enzymatic reactions (glycerol kinase, pyruvate kinase, lactate dehydrogenase, ending by the measurement of the consumed reduced NAD. The study of the kinetics of the analysis in two stages is carried out using standard solutions of glycerol and with human sera of variable concentration; the optimal conditions of the reaction time and temperature are drawn up. The trials of repeatability and reproducibility from day to day were carried out together with evaluation of the precision. The method was compared with Beohringer's method (n = 50) using hydrolysis of alcoholic potash. The authors conclude on the use of the methods studied.

Esterases↗
PubMed1977

Combined gel filtration, biuret/copper method compared with an immunochemical method for urinary protein measurement.

We compared an immunochemical method specific for plasma proteins with a chemical method, in which interfering substances are separated by gel filtration and "total" urinary protein is determined by the biuret reaction followed by reaction of protein-bound copper with diethyldithiocarbamate after a second gel filtration to remove nonprotein-bound copper. More than 250 24-h urine samples were analyzed by each method. There was linear agreement and a correlation of 0.96 between the two methods, but urinary protein values determined by the immunochemical method, especially for patients with multiple myeloma, were lower than by the chemical method.

Chromatography, Gel↗
PubMedDate not supplied

Comparative study on two methods for serotyping of Staphylococcus aureus: the Pillet method and the Oeding method. II.-Possible identity between Pillet's antigen 9 and Oeding's antigen h1.

Using two techniques (agglutination and immunoprecipitation), the authors have observed that: -Pillet's strain CK9 and Oeding's strains 670 and 17A are very similar; -antigen 9 is also present in the three strains; the same relation is found in wild strains. Using agglutination and immunoprecipitation, there is a cross reactivity between h1 factor serum, serum 9 and strains CK9, 670 and 17A. A precipitation line involving strain CK9 and serum CK9 is described in the Pillet system. Finally, there is a strong presumption that Oeding's h1 antigen and Pillet's antigen 9 are identical.

Antigens, Bacterial↗
PubMed1991

Verification of the disector method for counting neurons, with comments on the empirical method.

The empirical and disector methods are unbiased sampling methods for determining numbers of neurons. The present study verifies and thus calibrates these methods by determining true numbers of ganglion cells in serial reconstructions and then using each method to estimate the same populations. The empirical method gives accurate counts but is laborious (inefficient). Five separate disector analyses, distinguished by height (h), were done for each ganglion. The findings are: (1) that the estimates are consistently low when h is minimal (reference and look-up sections are adjacent), but (2) the estimates are accurate when h is greater (one to four sections intervene between reference and look-up sections). We ascribe the difficulties with the first disector to "lost" or "invisible" caps. We emphasize that we would not have known of the problem unless we verified our counts. If there is suspicion that difficulties with profile recognition might occur, we recommend that serial sections of an appropriately chosen sample of tissue be prepared and 500-1,000 neurons (or, more generally, particles) be reconstructed. Then the method of choice can be used on the issue of choice to make certain of the necessary accuracy before proceeding with the main study.

Animals↗
PubMed1992

Which method for quantifying "microalbuminuria" in diabetics? Comparison of several immunological methods (immunoturbidimetric assay, immunonephelometric assay, radioimmunoassay and two semiquantitative tests) for measurement of albumin in urine.

We have compared the chemical and clinical characteristics of an immunonephelometric assay (INA), two immunoturbidimetric assays (ITA) and two semi-quantitative methods with those of a solid-phase radioimmunoassay (RIA) for measurement of urinary albumin (UA) concentration in 136 diabetic patients. INA and RIA had similar accuracy, and provided comparable results. However, RIA has slightly greater sensitivity than INA, which is easier and faster. Good agreement was also found between RIA and the two ITA methods, although one of these overestimated RIA values in the low-medium range (5-30 mg/l) of urinary albumin. ITA seems suitable for initial screening of albuminuria in diabetic patients but more sensitive procedures (such as RIA and INA) seem preferable for measurement of UA concentrations in the normal range. The two semi-quantitative methods showed high sensitivity but poor specificity, because of the large number of false positive results. About 50% of diabetic patients "positive" by these methods did not have microalbuminuria. The utility of these methods is questionable, because many samples from diabetic patients need to be reassayed by a more specific and sensitive assay such as the RIA, INA or ITA methods.

Albuminuria↗