scholarly journals Comparison of Fluorescence Microscopy and Solid-Phase Cytometry Methods for Counting Bacteria in Water

2004 ◽  
Vol 70 (9) ◽  
pp. 5343-5348 ◽  
Author(s):  
John T. Lisle ◽  
Martin A. Hamilton ◽  
Alan R. Willse ◽  
Gordon A. McFeters
Keyword(s):  
Bacterial Abundance ◽  
Solid Phase ◽  
Fluorescent Microscopy ◽  
Industrial Applications ◽  
Bacterial Cells ◽  
Abundance Estimate ◽  
Statistical Confidence ◽  
Quality Of Water ◽  
Microscopic Count ◽  
Direct Counts

ABSTRACT Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 106 cells filter−1. In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 105, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.

scholarly journals A Stringent Analysis of Polyphosphate Dynamics inEscherichia coli

2019 ◽  
Vol 201 (9) ◽  
Author(s):  
Michael Downey
Keyword(s):  
Escherichia Coli ◽  
Infection Control ◽  
Bacterial Species ◽  
Stringent Response ◽  
Industrial Applications ◽  
Bacterial Cells ◽  
Inescherichia Coli ◽  
Content Type ◽  
Polyphosphate Accumulation ◽  
Inorganic Phosphates

ABSTRACTDuring stress, bacterial cells activate a conserved pathway called the stringent response that promotes survival. Polyphosphates are long chains of inorganic phosphates that modulate this response in diverse bacterial species. In this issue, Michael J. Gray provides an important correction to the model of how polyphosphate accumulation is regulated during the stringent response inEscherichia coli(M. J. Gray, J. Bacteriol, 201:e00664-18, 2019,https://doi.org/10.1128/JB.00664-18). With other recent publications, this study provides a revised framework for understanding how bacterial polyphosphate dynamics might be exploited in infection control and industrial applications.

scholarly journals Glassy Carbon: A Promising Material for Micro- and Nanomanufacturing

Materials ◽  
2018 ◽  
Vol 11 (10) ◽  
pp. 1857 ◽  
Author(s):  
Swati Sharma
Keyword(s):  
Glassy Carbon ◽  
Carbon Material ◽  
Large Scale ◽  
Solid Phase ◽  
Three Dimensional ◽  
Industrial Applications ◽  
Degradation Temperature ◽  
Pass Through ◽  
Semi Solid ◽  
Device Research

When certain polymers are heat-treated beyond their degradation temperature in the absence of oxygen, they pass through a semi-solid phase, followed by the loss of heteroatoms and the formation of a solid carbon material composed of a three-dimensional graphenic network, known as glassy (or glass-like) carbon. The thermochemical decomposition of polymers, or generally of any organic material, is defined as pyrolysis. Glassy carbon is used in various large-scale industrial applications and has proven its versatility in miniaturized devices. In this article, micro and nano-scale glassy carbon devices manufactured by (i) pyrolysis of specialized pre-patterned polymers and (ii) direct machining or etching of glassy carbon, with their respective applications, are reviewed. The prospects of the use of glassy carbon in the next-generation devices based on the material’s history and development, distinct features compared to other elemental carbon forms, and some large-scale processes that paved the way to the state-of-the-art, are evaluated. Selected support techniques such as the methods used for surface modification, and major characterization tools are briefly discussed. Barring historical aspects, this review mainly covers the advances in glassy carbon device research from the last five years (2013–2018). The goal is to provide a common platform to carbon material scientists, micro/nanomanufacturing experts, and microsystem engineers to stimulate glassy carbon device research.

EFFECT OF PASTEURIZATION ON THE DIRECT MICROSCOPIC COUNT OF EGGS

1971 ◽  
Vol 34 (4) ◽  
pp. 209-211 ◽  
Author(s):  
H. E. Hall ◽  
D. F. Brown ◽  
R. B. Read
Keyword(s):  
Methylene Blue ◽  
Bacterial Count ◽  
Bacterial Cells ◽  
Bacterial Counts ◽  
The North ◽  
Counting Chamber ◽  
Blue Stain ◽  
Microscopic Count ◽  
Whole Egg

When pasteurized whole eggs from breakers were examined by the Direct Microscopic Count (DMC) procedure, the bacterial count frequently appeared to be too low to correlate with the observed state of decomposition. The DMC of whole egg was found to decrease during pasteurization. To determine why, DMC's were done using the North Aniline Oil - Methylene Blue Stain and the Levowitz-Weber modification of the Newman-Lampert stain. Total bacterial counts also were made using the Petroff-Hausser counting chamber. Results indicated that the reduction in count resulted from lysis of some of the bacterial cells in egg rather than to loss of stainability. Crystalline lysozyme at the concentration found in egg and whole egg preparations produced similar reductions in the DMC of bacteria isolated from egg.

scholarly journals The physical boundaries of public goods cooperation between surface-attached bacterial cells

2017 ◽  
Author(s):  
Michael Weigert ◽  
Rolf Kümmerli
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Soft Tissue ◽  
Social Interactions ◽  
Single Cell ◽  
Fluorescent Microscopy ◽  
Social Consequences ◽  
Social Effects ◽  
Bacterial Cells ◽  
Soft Tissue Infections ◽  
Natural Conditions

AbstractBacteria secrete a variety of compounds important for nutrient scavenging, competition mediation and infection establishment. While there is a general consensus that secreted compounds can be shared and therefore have social consequences for the bacterial collective, we know little about the physical limits of such bacterial social interactions. Here, we address this issue by studying the sharing of iron-scavenging siderophores between surface-attached microcolonies of the bacterium Pseudomonas aeruginosa. Using single-cell fluorescent microscopy, we show that siderophores, secreted by producers, quickly reach non-producers within a range of 100 μm, and significantly boost their fitness. Producers in turn respond to variation in sharing efficiency by adjusting their pyoverdine investment levels. These social effects wane with larger cell-to-cell distances and on hard surfaces. Thus, our findings reveal the boundaries of compound sharing, and show that sharing is particularly relevant between nearby yet physically separated bacteria on soft surfaces, matching realistic natural conditions such as those encountered in soft tissue infections.

Solid-phase enzyme-receptor assay (SPERA): a competitive-binding assay for glycopeptide antibiotics of the vancomycin class.

1985 ◽  
Vol 31 (10) ◽  
pp. 1606-1610 ◽  
Author(s):  
A Corti ◽  
C Rurali ◽  
A Borghi ◽  
G Cassani
Keyword(s):  
Human Serum ◽  
Binding Assay ◽  
Solid Phase ◽  
Quantitative Detection ◽  
Synthetic Analog ◽  
Bacterial Cells ◽  
Glycopeptide Antibiotics ◽  
Competitive Binding Assay ◽  
Analytical Recovery ◽  
Receptor Assay

Abstract A solid-phase enzyme-receptor assay (SPERA) has been developed for glycopeptide antibiotics of the vancomycin class such as teicoplanin, vancomycin, ristocetin, avoparcin, actaplanin, A-47934, A-41030, and A-35512-B. The assay exploits the mechanism of most action of these antibiotics, which is based on their interaction with acyl-D-alanyl-D-alanine, a constituent of the walls of most growing bacterial cells. The antibiotics and enzyme-labeled teicoplanin compete for a synthetic analog of the biological receptor, albumin-epsilon-aminocaproyl-D-alanyl-D-alanine. The various antibiotics produced different competition curves, 50% displacement being obtained with antibiotic concentrations ranging from 0.04 to 4 mg/L, vancomycin and actaplanin being the weakest and strongest competitors, respectively. For teicoplanin in human serum the intra-assay CV was 7.2%, the interassay CV was 11.2%, and the analytical recovery 94%. Teicoplanin concentrations obtained by SPERA (chi) correlated well with those obtained by microbiological assay (y): y = 1.03 chi + 0.053 (r = 0.943; n = 60). We conclude that SPERA is a powerful tool for identification and quantitative detection of glycopeptide antibiotics, even in complex media.

scholarly journals A Novel Biocompatible Titanium–Gadolinium Quantum Dot as a Bacterial Detecting Agent with High Antibacterial Activity

Nanomaterials ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 778 ◽  
Author(s):  
Vishma Pratap Sur ◽  
Aninda Mazumdar ◽  
Amirmansoor Ashrafi ◽  
Atripan Mukherjee ◽  
Vedran Milosavljevic ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Fluorescent Microscopy ◽  
Oxygen Demand ◽  
Antibacterial Activities ◽  
Dead Cell ◽  
Bacterial Cells ◽  
Growth Curve Analysis ◽  
Cell Content ◽  
Cell Wall Disruption

In this study, the titanium–gadolinium quantum dots (TGQDs) were novel, first of its type to be synthesized, and fully characterized to date. Multiple physical characterization includes scanning electron microscopy (SEM), scanning electrochemical microscope (SCEM), x-ray fluorescence, spectrophotometry, and dynamic light scattering were carried out. The obtained results confirmed appropriate size and shape distributions in addition to processing optical features with high quantum yield. The synthesized TGQD was used as a fluorescent dye for bacterial detection and imaging by fluorescent microscopy and spectrophotometry, where TGQD stained only bacterial cells, but not human cells. The significant antibacterial activities of the TGQDs were found against a highly pathogenic bacterium (Staphylococcus aureus) and its antibiotic resistant strains (vancomycin and methicillin resistant Staphylococcus aureus) using growth curve analysis and determination of minimum inhibitory concentration (MIC) analysis. Live/dead cell imaging assay using phase-contrast microscope was performed for further confirmation of the antibacterial activity. Cell wall disruption and release of cell content was observed to be the prime mode of action with the reduction of cellular oxygen demand (OD).

scholarly journals Impact of Extending Hard-Cheese Ripening: A Multiparameter Characterization of Parmigiano Reggiano Cheese Ripened up to 50 Months

Foods ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 268 ◽  
Author(s):  
Paolo D’Incecco ◽  
Sara Limbo ◽  
John Hogenboom ◽  
Veronica Rosi ◽  
Serena Gobbi ◽  
...  
Keyword(s):  
Solid Phase ◽  
Residual Activity ◽  
Short Chain Fatty Acids ◽  
Bacterial Cells ◽  
Structural Variations ◽  
Ripening Period ◽  
Continuous Release ◽  
Selected Traits ◽  
Parmigiano Reggiano ◽  
Hard Cheese

Extending ripening of hard cheeses well beyond the traditional ripening period is becoming increasingly popular, although little is known about the actual evolution of their characteristics. The present work aimed at investigating selected traits of Parmigiano Reggiano cheese ripened for 12, 18, 24, 30, 40 and 50 months. Two cheeses per each ripening period were sampled. Although moisture constantly decreased and was close to 25% in 50-month cheeses, with a parallel increase in cheese hardness, several biochemical changes occurred involving the activity of both native and microbial enzymes. Capillary electrophoresis demonstrated degradation of αs1- and β-casein, indicating residual activity of both chymosin and plasmin. Similarly, continuous release of free amino acids supported the activity of peptidases deriving from lysed bacterial cells. Volatile flavor compounds, such as short-chain fatty acids and some derived ketones, alcohols and esters, evaluated by gas chromatography with solid-phase micro-extraction, accumulated as well. Cheese microstructure was characterized by free fat trapped in irregularly shaped areas within a protein network, with native fat globules being no longer visible. This study showed for the first time that numerous biochemical and structural variations still occur in a hard cheese at up to 50 months of aging, proving that the ripening extension deserves to be highlighted to the consumer and may justify a premium price.

Detection of living bacterial cells in clay - bentonite 

2021 ◽  
Author(s):  
Veronika Hlavackova ◽  
Katerina Cerna ◽  
Lenka Kejzlarova ◽  
Deepa Bartak ◽  
Rojina Shestra ◽  
...  
Keyword(s):  
Metabolic Activity ◽  
Fluorescent Microscopy ◽  
Extraction Procedure ◽  
Time Frame ◽  
Cementitious Materials ◽  
Gas Production ◽  
Epifluorescence Microscopy ◽  
Nuclear Waste Repository ◽  
Bacterial Cells ◽  
General Applicability

<p>Bentonite is a swelling clay, consisting mainly of montmorillonire,  being planned to be used as a backfill material in the nuclear waste repository. It contains indigenous microbial populations that can negatively influence the long-term safety of the geological repository due to their metabolic activity (canister corrosion, illitization of bentonite, gas production, degradation of cementitious materials). However, reliable detection of microorganisms in clayish material is generally very difficult. Although the compactness of bentonite will undoubtedly limit the microbial activity, in the extremely long-time frame of repository lifetime this condition can fail. It is thus crucial to understand the potential of the naturally present microbial community in bentonite to compromise the safety of repository, if not limited by the compactness. Higher metabolic activity can be mainly expected at the interfaces or in the places with a lower density of bentonite.</p><p>Here we present an optimized cell extraction method enabling direct estimation of bacterial density and viability in bentonite. Indigenous bacterial cells were extracted from bentonite suspensions by an improved step-wise protocol and their viability was detected using live/dead staining and epifluorescence microscopy. We used dispersant (2.5 mM natrium pyrophosphate-based solution or 1% methanol) to partially disintegrate the bentonite and detach the vital and dead microbial cells from its surface. The dispersed material was subsequently stepwise centrifuged over two high-density media (sucrose and Histodenz) to remove most of the heavy bentonite particles while keeping the light bentonite particles and cells in the final extract. We were able to detect and enumerate the cells concentrated at the surface of the light bentonite particles, which served as a sieve to retain all free cells during centrifugation.    </p><p>Different extraction procedures were tested and their efficiency was estimated by comparing live/dead ratios of resulting extracts and was also proved by implementing both NGS and quantitative PCR. The results show that most of the microbial genera present in the original suspension are also present in extracts but as proved by Deseq2 analysis some genera tend to settle down with heavier bentonite particles during the first centrifugation step.</p><p>To conclude, we present a protocol for extraction and detection of metabolically active cells in clayish material – bentonite. The quality of the extraction procedure was estimated both by a combination of fluorescent microscopy and genetic methods. The protocol was successfully tested on different bentonite types showing general applicability of this approach for clay materials.</p>

scholarly journals Oncorhyncin III: a potent antimicrobial peptide derived from the non-histone chromosomal protein H6 of rainbow trout, Oncorhynchus mykiss

2003 ◽  
Vol 373 (2) ◽  
pp. 621-628 ◽  
Author(s):  
Jorge M. O. FERNANDES ◽  
Nathalie SAINT ◽  
Graham D. KEMP ◽  
Valerie J. SMITH
Keyword(s):  
Rainbow Trout ◽  
Antimicrobial Peptide ◽  
Lipid Bilayers ◽  
Solid Phase ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Haemolytic Activity ◽  
Acid Extraction ◽  
Chromosomal Protein ◽  
Bacterial Cells

A 6.7 kDa antimicrobial peptide was isolated from trout skin secretions using acid extraction followed by cation-exchange chromatography, tC18 solid-phase extraction, and C18 reversed-phase HPLC. The molecular mass of this peptide, which is tentatively named oncorhyncin III, is 6671 Da, as determined by matrix-assisted laser-desorption ionization MS. N-terminal amino acid sequencing revealed that the first 13 residues of oncorhyncin III are identical with those of the non-histone chromosomal protein H6 from rainbow trout. Hence these data combined with the MS results indicate that oncorhyncin III is likely to be a cleavage product of the non-histone chromosomal protein H6 (residues 1–66) and that it probably contains two methylated residues or one double methylation. The purified peptide exhibits potent antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations in the submicromolar range. The peptide is sensitive to NaCl, and displays no haemolytic activity towards trout erythrocytes at concentrations below 1 μM. Scanning electron microscopy revealed that oncorhyncin III does not cause direct disruption of bacterial cells. Reconstitution of the peptide in planar lipid bilayers strongly disturbs the membranes, but does not induce the formation of stable ion channels. Taken together, these results support the hypothesis that oncorhyncin III plays a role in mucosal innate host defence.

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