Detection of Viable but Nonculturable Microbial Cells in Chicken Mince

The relevance of the study and the presence of gaps in the relevant knowledge on the topic. Potential existence of hazardous viable but nonculturable (VBNC) cells of pathogenic microorganisms in foodstuffs that can be formed under the influence of various factors, their detection and determination of conditions for formation of VBNC cells of various contaminant bacteria are relevant for preventing contamination of meats.Methods. In the study, a search was conducted for VBNC cells in chicken mince in real time and during experimental infection of it by Staphylococcus aureus 209P. In order to detect VBNC cells in chicken mince, total number of microbes, number of bacterial colonies (CFU), and the portion of living (dead) cells were determined in 1g of the product using a commercial set of fluorescent dyes. A second study was carried out after 5 h of incubation of tested samples at room temperature.Results and discussion. In samples of minced meat on the 4th day after production, more than 99% of all detected living cells were VBNC. After 5-hour incubation of the sample, the number of CFU/g increased by 22.5 times, but the portion of VBNC cells remained higher than 99% of viable bacteria. During artificial infection of the same batch of mince with S. aureus in broth culture at the stage of logarithmic growth, the amount of VBNC cells for 0 hours was 97.3%. After 5 hours their number increased to 99.99%. Probably, in the introduced culture of Staphylococcus at the stage of active reproduction, formation of VBNC bacteria did not occur, which initially reduced their number in the sample. After 5-h incubation, transition of bacteria to VBNC state was accelerated, possibly due to unfavorable conditions for the cell population (changes in trophic substrate, temperature, pH, etc.).Conclusions. Experimental data confirm presence of VBNC bacteria in chicken products that don’t grow on traditional nutrient media, and showing a false negative result in traditional microbiological expertise. Because of the biohazard of such dormant cells, it is advisable to provide regulated testing of foodstuffs for presence of VBNC cells.

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Conformation of Ribosomes from Escherichia Coli

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051062 ◽

1974 ◽

Vol 32 ◽

pp. 210-211

Author(s):

M. Boublik ◽

W. Hellmann ◽

F. Jenkins

Keyword(s):

Binding Sites ◽

Ribosomal Proteins ◽

Fluorescent Dyes ◽

Protein Biosynthesis ◽

Three Dimensional ◽

Spatial Arrangement ◽

Dimensional Structure ◽

Complete Understanding ◽

And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

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Assessment and determination of lyoprotectant for survival of freeze-dried Lactobacillus rhamnosus

Acta Universitatis Cibiniensis Series E Food Technology ◽

10.1515/aucft-2016-0009 ◽

2016 ◽

Vol 20 (1) ◽

pp. 105-113

Author(s):

Zongcheng Miao ◽

Yang Zhao ◽

Xiaoping Huo

Keyword(s):

Amino Acids ◽

Fractional Factorial Design ◽

Biological Activities ◽

Lactobacillus Rhamnosus ◽

Fractional Factorial ◽

Practical Application ◽

Steepest Ascent ◽

Freeze Dried ◽

Viable Bacteria

Abstract Currently research of lactic acid bacteria focus primarily on the functional probiotics, which are major beneficial biota in the gastrointestinal tract, have been industrial manufactured. Probiotics confer health benefits on the host need adequate amounts. However, the absence of data makes it difficult to ensure the maintenance biological activities and population of probiotic. In this research, a fractional factorial design and steepest ascent experiment were used to analyze the influence of lyoprotectant as carbohydrates, prebiotics and amino acids on the survival of the probiotic Lactobacillus rhamnosus. The results indicated a maximum survival rate and population of viable bacteria of L. rhamnosus to be 55.84 % and 1.60 ×1011 CFU/g after freeze-dried by using a combination of 10 g/100mL Sucrose, 2.5 g/100mL Isomaltooligosaccharide, 12 g/100mL Hydroxyproline. To a large extent, the survival and viability were dependent on the cryoprotectant used and make probiotics more attractive from a practical application in industrial viewpoint.

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Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

10.1101/407684 ◽

2018 ◽

Author(s):

Robert B. Quast ◽

Fataneh Fatemi ◽

Michel Kranendonk ◽

Emmanuel Margeat ◽

Gilles Truan

Keyword(s):

Single Molecule ◽

Conformational Equilibrium ◽

Fluorescent Dyes ◽

Photophysical Properties ◽

Accurate Determination ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

General Strategy

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

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Use of fluorescent dyes in the determination of adherence of human leucocytes to endothelial cells and the effect of fluorochromes on cellular function

Journal of Immunological Methods ◽

10.1016/0022-1759(94)90384-0 ◽

1994 ◽

Vol 172 (1) ◽

pp. 115-124 ◽

Cited By ~ 179

Author(s):

Luc S. De Clerck ◽

Chris H. Bridts ◽

Annemie M. Mertens ◽

Marleen M. Moens ◽

Wim J. Stevens

Keyword(s):

Endothelial Cells ◽

Fluorescent Dyes ◽

Cellular Function

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Pattern Analysis Meets Cell Biology

Microscopy and Microanalysis ◽

10.1017/s1431927600015877 ◽

1999 ◽

Vol 5 (S2) ◽

pp. 510-511

Author(s):

Robert F. Murphy ◽

Michael V. Boland

Keyword(s):

Cell Biology ◽

Fluorescent Dyes ◽

Protein A ◽

Chinese Hamster ◽

Subcellular Location ◽

Computer Applications ◽

Double Labeling ◽

A Cell ◽

Widespread Availability

The widespread availability of automated fluorescence microscope systems has led to an explosion in the acquisition of digital images by biologists. This has created a need for computer applications that automate the analysis of these images and an opportunity to develop new approaches to classical problems. An example is the determination of the subcellular location of a protein from immunofluorescence images (or, more recently, images of GFP fluorescence). Current practice is to compare such images to mental images that a cell biologist has developed over time, and to reach a tentative conclusion about the structure (i.e., organelle) that a protein is found in. Since this determination is subjective, it often must be followed up by double labeling with a marker protein from the suspected structure.As an initial exploration of the feasibility of automating the determination of subcellular location, we developed a system that is able to classify the localization patterns characteristic of five cellular molecules (proteins and DNA) in Chinese Hamster Ovary (CHO) cells. Images were acquired on an epifluorescence microscope after the cells had been fixed, permeabilized, and labeled with appropriate fluorescent reagents (usually antibodies conjugated to fluorescent dyes). The labels used were directed against a Golgi protein, a lysosomal protein, a nuclear protein, a cytoskeletal protein, and DNA.

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Evaluation of a High-Throughput Fluorescence Assay Method for hERG Potassium Channel Inhibition

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104272045 ◽

2005 ◽

Vol 10 (4) ◽

pp. 339-347 ◽

Cited By ~ 46

Author(s):

Arnulf Dorn ◽

Francis Hermann ◽

Andreas Ebneth ◽

Hendrick Bothmann ◽

Gerhard Trube ◽

...

Keyword(s):

Drug Development ◽

Potassium Channel ◽

Fluorescent Dyes ◽

False Negative ◽

Qt Prolongation ◽

Fluorescence Assay ◽

Assay Method ◽

Cardiac Side Effects ◽

Channel Inhibition ◽

Wide Range

The number of projects in drug development that fail in late phases because of cardiac side effects such as QT prolongation can impede drug discovery and development of projects. The molecular target responsible for QT prolongation by a wide range of pharmaceutical agents is the myocardial hERG potassium channel. It is therefore desirable to screen for compound interactions with the hERG channel at an early stage of drug development. Here, the authors report a cell-based fluorescence assay using membrane potential-sensitive fluorescent dyes and stably transfected hERG channels from CHO cells. The assay allows semiautomated screening of compounds for hERG activity on 384-well plates and is sufficiently rapid for testing a large number of compounds. The assay is robust as indicated by a Z′ factor larger than 0.6. The throughput is in the range of 10,000 data points per day, which is significantly higher than any other method presently available for hERG. The data obtained with the fluorescence assay were in qualitative agreement with those from patch-clamp electrophysiological analysis. There were no false-positive hits, and the rate of false-negative compounds is currently 12% but might be further reduced by testing compounds at higher concentration. Quantitative differences between fluorescence and electrophysiological methods may be due to the use- or voltage-dependentactivity of the antagonists.

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Laboratory Diagnostics of Primary Hyperaldosteronism and its Peculiarities (Literature Review)

Journal of Medicine and Life ◽

10.25122/jml-2019-0073 ◽

2019 ◽

Vol 12 (3) ◽

pp. 215-220 ◽

Cited By ~ 1

Author(s):

O Shidlovskyi Viktor ◽

◽

V Shidlovskyi Oleander ◽

Mikhail Sheremet ◽

V Zhulkevych Igor ◽

...

Keyword(s):

Magnetic Resonance ◽

Adrenal Glands ◽

False Negative ◽

Photon Emission ◽

Diagnostic Value ◽

Primary Hyperaldosteronism ◽

Laboratory Diagnostics ◽

Resonance Spectroscopy ◽

Morphological Studies

The final stage of the diagnostic of primary hyperaldosteronism is to identify the causes of excessive secretion of aldosterone and determination of its variants. Based on the analysis of literature data, the diagnostic value, sensitivity and specificity of the methods of radiation diagnostics for primary hyperaldosteronism were assessed: ultrasound, computed tomography, magnetic resonance imaging, photon emission tomography, magnetic resonance spectroscopy, scintigraphy with iodine radiopharmaceuticals. The causes of false-positive and false-negative evaluations of changes in adrenal glands in the application of these diagnostics have been analyzed. There are many genetic and morphological studies when searching the literature data on the principles and methods of distinguishing the nosological forms of primary hyperaldosteronism based on the results of the aldosterone level estimation in the separated blood from the central veins of both adrenal glands or segmental veins of one gland with subsequent determination of the concentration gradient. It was noted that topical diagnostics and, especially, the determination of nosological forms of primary hyperaldosteronism are complex and expensive, but their results allow choosing an appropriate treatment approach for each particular case.

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Amelioratory Effect of Nanoconjugated Vancomycin on Spleen during VRSA-Induced Oxidative Stress

Pathology Research International ◽

10.4061/2011/420198 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Subhankari Prasad Chakraborty ◽

Santanu KarMahapatra ◽

Sumanta Kumar Sahu ◽

Panchanan Pramanik ◽

Somenath Roy

Keyword(s):

Oxidative Stress ◽

Reduced Glutathione ◽

Treated Group ◽

Control Group ◽

Myeloperoxidase Activity ◽

Viable Bacteria ◽

Antioxidant Effects ◽

Potential Use

Objective. The aim of the present study was to evaluate the possible antioxidant effects of nanoconjugated vancomycin against VRSA infection on select makers of oxidative damage and antioxidant status in spleen. Methods. A coagulase-positive VRSA strain was used for this study. VRSA infection was developed in Swiss mice by intraperitoneal injection of 5 × 106 CFU/mL bacterial solutions. VRSA-infected mice were treated with nanoconjugated vancomycin at its effective dose for 10 days. After decapitation, blood was used for determination of viable bacteria count and spleen was excised from control and experimental groups, homogenized and used for different biochemical estimations. Results. Nitrate level, myeloperoxidase activity, lipid peroxidation, protein oxidation, oxidized glutathione, and DNA fragmentation level were increased significantly (P<0.05) in spleen of VRSA-infected group as compared to control group, and reduced glutathione level, activity of SOD, CAT, GPx, GR, and GST were decreased significantly (P<0.05); which were increased or decreased significantly (P<0.05) near to normal in nanoconjugated vancomycin-treated group. Conclusion. These findings suggest the potential use and beneficial role of nanoconjugated vancomycin against VRSA-infection-induced oxidative stress and DNA damage in spleen.

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The Basics of Sentinel Lymph Node Biopsy: Anatomical and Pathophysiological Considerations and Clinical Aspects

Journal of Oncology ◽

10.1155/2019/3415630 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Nasuh Utku Dogan ◽

Selen Dogan ◽

Giovanni Favero ◽

Christhardt Köhler ◽

Polat Dursun

Keyword(s):

Lymph Node ◽

Sentinel Lymph Node ◽

Near Infrared ◽

New Technologies ◽

Fluorescent Dyes ◽

False Negative ◽

False Negative Rate ◽

Lymph Node Biopsy ◽

Pathological Examination ◽

Clinical Aspects

Sentinel lymph node (SLN) is the first node to receive the drainage directly from a tumor. Detection and pathological examination of the SLN is an important oncological procedure that minimizes morbidity related to extensive nodal dissection. SLN biopsy was first reported in 1960 but took approximately 40 years to come into general practice following reports of good outcomes in patients with melanoma. After many years of observation and research on its use in various malignancies SLN biopsy has become the standard surgical treatment in patients with malignant melanoma, breast, vulvar, and cervical cancers. Along with the introduction of new technologies, such as the fluorescent dyes indocyanine green (ICG) and near-infrared fluorescence (NIR), and pathologic ultrastaging, SLN detection rate has increased and false-negative rate has decreased. This literature review aimed to present an overview of the basic concepts and clinical aspects of SLN biopsy in the light of the current research.

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A model for determining optimal frequency and fraction of population to be tested, for cost-effective surveillance testing for SARS-CoV2: algorithm development (Preprint)

10.2196/preprints.26747 ◽

2020 ◽

Author(s):

Jonathan Silver

Keyword(s):

Cost Effectiveness ◽

False Negative ◽

False Negative Rate ◽

Cost Effective ◽

Test Frequency ◽

Optimal Test ◽

Current Spread ◽

Graphical Interfaces ◽

Prior Infection

BACKGROUND Because Covid19 can be asymptomatic, surveillance testing with isolation of those who test positive can reduce spread. But what fraction of a population should be tested, how often, and how long should surveillance testing continue as vaccination progresses? OBJECTIVE Develop a model that allows determination of extent and frequency of testing that optimize cost-effectiveness, and time after which surveillance testing ceases to be cost-effective. METHODS Free software from Illumina (Analytica 101) was used to make a model that runs on personal computers. Graphical interfaces demonstrate how the model reaches its conclusions and allow users with little programming experience to choose values for parameters that vary in different circumstances. RESULTS Optimal test frequency and fraction of a population to be tested depend on test cost, test sensitivity, false negative rate, delay (if any) between testing and isolation of those who test positive, and rate of compliance with isolation. Overall cost-effectiveness and optimal duration of testing depend critically on the value placed on saving a life, effective spread rate (R_eff) when surveillance testing is instituted, and fraction of a population already immune due to prior infection or vaccination. CONCLUSIONS For current spread rates (R_eff ca 1.2-1.5) and percent immune (ca 10%), overall cost-effectiveness generally requires value per life saved to be greater than $100,000. Optimal test frequency and fraction tested range from days to months, and ca .1 to 1, respectively, for different types of tests (antigen, pcr) currently on the market.

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