The quantification of cellular metabolic activity via MTT assay has become a widespread practice in eukaryotic cell studies and is progressively extending to bacterial cell investigations. This study pioneers the application of MTT assay to evaluate the metabolic activity of biofilm-forming cells within bacterial biofilms on nanofibrous materials. The biofilm formation of Staphylococcus aureus and Escherichia coli on nanomaterials electrospun from polycaprolactone (PCL), polylactic acid (PLA), and polyamide (PA) was examined. Various parameters of the MTT assay were systematically investigated, including (i) the dissolution time of the formed formazan, (ii) the addition of glucose, and (iii) the optimal wavelength for spectrophotometric determination. Based on interim findings, a refined protocol suitable for application to nanofibrous materials was devised. We recommend 2 h of the dissolution, the application of glucose, and spectrophotometric measurement at 595 nm to obtain reliable data. Comparative analysis with the reference CFU counting protocol revealed similar trends for both tested bacteria and all tested nanomaterials. The proposed MTT protocol emerges as a suitable method for assessing the metabolic activity of bacterial biofilms on PCL, PLA, and PA nanofibrous materials.
- MeSH
- biofilmy * růst a vývoj MeSH
- Escherichia coli * fyziologie MeSH
- glukosa metabolismus MeSH
- nanovlákna * chemie MeSH
- nylony chemie MeSH
- polyestery * chemie MeSH
- spektrofotometrie metody MeSH
- Staphylococcus aureus * fyziologie MeSH
- tetrazoliové soli * metabolismus chemie MeSH
- thiazoly metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Klíčová slova
- mapování barev, translucence,
- MeSH
- design s pomocí počítače MeSH
- lidé MeSH
- preparace zubu * metody přístrojové vybavení MeSH
- spektrofotometrie * metody přístrojové vybavení MeSH
- výsledek terapie MeSH
- zubní cementy MeSH
- zubní fazety MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
During recent years, interest concerning selenium has considerably increased. It is due to the combined behaviour it can have in humans, as either a toxic or an essential element depending on its concentration. For these reasons, its reliable quantification is extremely important. Blood serum is one of the most often analysed biological fluids when focusing on selenium concentration. Many detection methods can be used for the quantification of selenium, electrothermal atomic absorption spectrometry being one of the most suitable ones to perform such analyses. This is due to the possibility of a direct analysis (even though it is a complicated biological matrix) and the use of a small volume of the samples (mostly 20 µl for a single analysis). This article offers an overview of the selenium concentrations found in blood serum for healthy populations in European countries. The data presented here indicate that selenium status is not optimal in most European countries. The estimated mean value is 75 µg L–1 and 65 µg L–1 for adults and children, respectively. These results, combined with growing knowledge of the importance of selenium to overall health, require more systematic studies aimed to a reliable quantification of selenium in biological fluids for large populations correlated with various parameters, in order to subsequently ensure adequate selenium supplementation for those populations where selenium intake is significantly reduced.
- MeSH
- biochemická analýza krve metody MeSH
- epidemiologické monitorování MeSH
- epidemiologické studie MeSH
- lidé MeSH
- selen * analýza krev MeSH
- spektrofotometrie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.
The main objective of the presented research was to prepare an innovative carrier as a filler for detection tubes in the form of double-coated pellets with a very significant color transition during the detection of cholinesterase inhibitors such as nerve agents, organophosphorus or carbamate insecticides in liquids that is observable visually and also spectrophotometrically at 412 nm. The pellet cores were prepared by the extrusion/spheronization method. Consecutively, two different coats were applied on the pellet cores in the coating device using the Wurster column method. To increase the color change intensity, the second semipermeable coat based on Eudragit® RL was applied on top of the first coat, which was formed by butyrylcholinesterase immobilized in hydroxypropyl methylcellulose. Prepared samples differing in thickness of the second coat were evaluated for their quality parameters, enzymatic activity and inhibition. The detection mechanism was based on the standard Ellman's colorimetric reaction. It was observed that the semipermeable coat prevented leaching of the enzyme into the solution and led to an increased intensity of color transition from white - yellow to white - deep yellow/orange, thus enabling a more accurate visual detection. This system allows easy, rapid and safe identification of cholinesterase inhibitors in liquids, especially chemical warfare agents.
Procedures for the extraction-spectrophotometric determination of tris(2-chloroethyl)amine, an alkylating agent known as a drug as well as a chemical warfare agent (nitrogen mustard HN-3), with 7 acid-base indicators of a triphenylmethane lactone type, phthaleins, were developed. Representatives of phthaleins without an oxygen bridge (thymolphthalein, o-cresolphthalein, naphtholphthalein) and with an oxygen bridge (fluorescein, 2',7'-dichlorofluorescein, eosin B and eosin Y) were used. The methods were based on the formation of ion pair complexes. Chloroform was used as a non-polar solvent for an extraction. The conditions to determine were optimized for the optimal pH of the buffer and the concentration of a phthalein as a reagent. The dependence on the reaction time in a water phase and the stoichiometry of extraction products were studied. The detection limits and the limits of the determination of separate procedures and conditional extraction constants were determined. Comparison with the spectrophotometric method of the group determination of alkyl halides and acyl halides using alkaline ethanol-water solution of thymolphthalein, the so-called T-135 agent, was conducted. While studying the selectivity, the possible interference of bis(2-chloroethyl)sulphide and 3 nitrogen mustards in the proposed procedures were verified. Copyright © 2016 John Wiley & Sons, Ltd.
- MeSH
- alkylační látky analýza izolace a purifikace MeSH
- chemické bojové látky analýza izolace a purifikace MeSH
- fenolftaleiny chemie MeSH
- koncentrace vodíkových iontů MeSH
- limita detekce MeSH
- pufry MeSH
- sloučeniny dusíkatého yperitu analýza izolace a purifikace MeSH
- spektrofotometrie metody MeSH
- voda analýza MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
This article reviews the Trinder chromogenic reaction and describes its discovery, further development, current state and use in practice. It has been known for nearly half a century as a universal and analytically specific chromogenic method that is abundantly used in clinical biochemistry tests. The article provides information on the benefits and shortcomings of the Trinder reaction in comparison to alternative methods. It also reviews substances interfering in this reaction. This may be particularly helpful for a biochemical analyst or a physician in determination the cause of discrepant laboratory results, and thus prevent an application of improper patient treatment.
- Klíčová slova
- Trinderova reakce,
- MeSH
- cholesterol analýza MeSH
- glukosa analýza MeSH
- klinické laboratorní techniky * dějiny metody MeSH
- kreatinin analýza MeSH
- kyselina močová analýza MeSH
- spektrofotometrie * metody MeSH
- triglyceridy analýza MeSH
- Publikační typ
- práce podpořená grantem MeSH
Spectrophotometric analysis of human pigmentation characteristics plays an important role in medicine and cosmetology. However, to correctly interpret the results, one must know the normal range of variability of pigmentation and erythema in the population. This is of utmost importance for quantitative assessment of melanocytic nevi, hemangiomas, and allergic reactions. The objective of the present work was to determine the physiological range of variability of skin and hair pigmentation and of erythema in Polish prepubertal children. The study encompassed Polish children aged 7 to 10 years without any abnormalities in skin or hair pigmentation. A total of 802 children were examined. Constitutive skin and hair pigmentation and skin erythema were evaluated using a dermaspectrometer. The LMS method (Cole 1990, Pan, Cole 2004) was used for the construction of percentile charts presenting the distribution of skin melanin index (SMI), hair melanin index (HMI), and erythema index (EI) values in the various age and sex groups. The presented normal ranges of variability of SMI, HMI, and EI are the first Polish reference standards for these indices. The manner in which the data are presented makes it possible to graphically assess these characteristics (percentile charts) as well as to conduct their normalization using L, M, and S parameters. The normalized values may also be converted to percentiles. Standards may be used in clinical practice, e.g., for evaluating pigmentation abnormalities and skin reactions to inflammations, irritations, and allergies. Furthermore, they may be applied in designing dermatological and pharmacological research.
- MeSH
- barva vlasů MeSH
- biologická variabilita populace MeSH
- dítě MeSH
- erytém * epidemiologie MeSH
- lidé MeSH
- melaniny * analýza MeSH
- pigmentace kůže MeSH
- spektrofotometrie metody MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Geografické názvy
- Polsko MeSH
Úvod: Spektrofotometrie mozkomíšního moku (SPFM) je vyšetření používané k diagnostice subarachnoidálního krvácení, zejména v případech, kdy nativní CT hlavy krvácení neprokazuje, avšak nadále přetrvávají klinické příznaky. Vzorek mozkomíšního moku by měl být doručen do laboratoře a analyzován co nejrychleji, nejdéle však do jedné hodiny od odběru. V případech, kdy vzorek není zpracován nejdéle do jedné hodiny od odběru, by měl být supernatant skladován ve tmě při 4°C. Cílem studie bylo zjistit vliv časové prodlevy zpracování mozkomíšního moku na výsledek spektrofotometrického vyšetření. Metodika: Do studie bylo zařazeno 48 vzorků doručených do laboratoře do 30 minut od provedení lumbální punkce. Současně s provedením základního biochemického a cytologického vyšetření, zahrnujícího stanovení počtu erytrocytů, byly vytvořeny dva alikvoty (A a B). Alikvot „A“ byl ihned po doručení vzorku zcentrifugován a bylo provedeno SPFM vyšetření dle britských doporučení se stanovením net oxyhemoglobin absorbance (NOA) a net bilirubin absorbance (NBA). Alikvot „B“ byl ponechán při pokojové teplotě na denním světle 60 minut, poté centrifugován a následně bylo provedeno SPFM vyšetření jako v případě alikvotu „A“. Pro statistické porovnání hodnot NOA a NBA ve skupině „A“ a ve skupině „B“ byla užita párová verze Wilcoxonova testu. Za statisticky významnou byla považována hodnota p < 0,05. Jednotlivé vzorky byly dle hodnot NOA a NBA klasifikovány na „positive“, „negative“ a „inconclusive“. Pro statistické zhodnocení byl užit MedCalc software (MedCalc Software, version 16.4.1, MedCalc Software bvba, Ostend, Belgium). Výsledky: Do studie byly zařazeny vzorky s mediánem (min–max) počtu erytrocytů 16/µl (0–119 460). Změny v hodnotách NOA v alikvotech zpracovaných ihned v porovnání s hodnotami ve vzorcích zpracovaných s 60 minutovou časovou prodlevou nedosáhly statisticky významných změn (hodnoty uvedeny jako medián [min–max]): NOA (0 min) = 0,001 (0,001–0,863), NOA (60 min) = 0,001 (0,001–0,898), p=0,67. V případě NBA u alikvotů zpracovaných s 60 minutovým odstupem v porovnání s alikvoty zpracovanými ihned rovněž nedošlo ke statisticky významným změnám: NBA (0 min) = 0,001 (0,001–0,599), NBA (60 min) = 0,001 (0,001–0,601), p=0,12. Při klasifikaci vzorků do skupin „positive“, „negative“ a „inconclusive“ došlo u vzorků zpracovaných s časovou prodlevou 60 minut ke změně v klasifikaci u jednoho vzorku s hraniční hodnotou NBA ze skupiny „positive“ do skupiny „inconclusive“. Závěr: Nebyly zjištěny statisticky významné rozdíly v hodnotách NOA a NBA u vzorků analyzovaných s hodinovou prodlevou v porovnání se vzorky analyzovanými ihned po přijetí do laboratoře. V případech s hraničními hodnotami NOA a NBA je třeba při hodnocení dbát zvýšené opatrnosti zejména v situacích, kdy připadá v úvahu opožděné doručení vzorku do laboratoře o více jak jednu hodinu.
Introduction: Cerebrospinal fluid (CSF) spectrophotometry (SPFM) is a laboratory assessment used in diagnostics of subarachnoid hemorrhage, mainly in cases when it is not proven by computed tomography (CT), but clinical manifestations still persists. Sample of CSF for SPFM examination should be delivered to the laboratory and analyzed as soon as possible, at least within 1 hour. In delay, the superanatant should be stored at 4°C in dark. The aim of the study was to investigate the consequence of delayed processing of CSF samples in SPFM assessment. Methodology: A total of 48 samples delivered to the laboratory until 30 minutes from lumbar puncture. Collection were enrolled in the study. If a sufficient amount of CSF was obtained, the sample was divided into two aliquots (A and B). In the first aliquot (A), routine biochemical and cytological examination, including erythrocyte counting, was performed. SPFM examination was immediately performed on the centrifuged aliquot. Values of net oxyhemoglobin absorbance (NOA) and net bilirubin absorbance (NBA) were calculated according to the UK recommendation. Aliquot “B” was stored at room temperature for 60 minutes in the light, then centrifuged and SPFM determination was performed as in aliquot “A”. A Wilcoxon non-parametric test (paired version) was used for comparisons between groups (group A and B). Results were considered statistically significant at P < 0.05. Upon NOA and NOB, samples were classified as “positive“, “negative“ and “inconclusive“. MedCalc software was used for statistic evaluation. Results: Median erythrocyte count was 16/µL (0–119 460). In 27 (56%) samples, there was a detectable amount of oxyhemoglobin and/or bilirubin. Changes in NOA and NBA levels in samples A in comparison with samples B did not reach statistical significance (P = 0.68 and 0.21, respectively). We found one case classified as “positive” when processed immediately, but an aliquot that was processed after a delay was classified as “inconclusive”. The samples with median (min–max) of erythrocyte counting 16/µl (0–119 460) were used. Changes in values of NOA in aliquots analyzed immediately compare to values of samples analyzed after 60 minutes did were not statistically significant (values of median [min–max]): NOA (0 min) = 0.001 (0.001–0.863), NOA (60 min) = 0.001 (0.001–0.898), p=0.67. In case of NBA aliquots with 60 min delay there also were no statistically significant changes: NBA (0 min) = 0.001 (0.001–0.599), NBA (60 min) = 0.001 (0.001–0.601), p=0.12. In classification of samples to groups „positive“, „negative“ and „inconclusive“ there was, in samples with 60 min delay, the change in classification from group „positive“ to „inconclusive“ in one sample with limit value of NBA. Conclusion: Significant differences in values NOA and NBA were not detected in samples analyzed with 60 min delay compare to those analyzed immediately after collection. In case of limit values of NOA and NBA there is necessary to pay attention in situations of delay in delivery for the sample to laboratory more than 60 minutes.
