Replacement of milk fat by rapeseed oil stabilised emulsion in commercial yogurt.

The incorporation of lipid droplets and further characterization of matrices within dairy products may be possible using such adjacent particles as protein complexes/lipids. Among the range of varied emulsions and their functionalities, great attention has recently focused on the fabrication of high internal phase types. Feasibly, stable alternatives structured with health-beneficial lipids like those derived from plants could replace saturated fatty acids. As a fat replacement strategy, the fate of incorporated HIPE would require some adjustments either with storage stability and/or structural feat for the food matrix. Therefore, the replacement of milk fat by rapeseed oil stabilised emulsion in commercial yogurt was investigated. This involved 25%, 50% and 75% rapeseed oil respectively assigned as low (LIPE), medium (MIPE), and high internal phase emulsion (HIPE). Specifically, emulsions were examined by droplet size, encapsulation, pH, zeta potential, phase separation, and rheology. The fat free yogurt supplemented by HIPE were examined by droplet size, zeta potential, pH, color, sensory, texture and microbiological aspects against positive (regular milk fat) and negative (fat free) yogurt controls. Results showed increasing rapeseed oil contents would form smaller droplet-like emulsions. Within the yogurt matrix however, incorporating HIPE would seemingly reduce oil droplet size without much compromise to bacterial viability, sensory, or texture. Overall, this simple method of lipid alternation shows promise in dairy products.

User-friendly analysis of droplet array images.

Water-in-oil droplets allow performing massive experimental parallelization and high-throughput studies, such as single-cell experiments. However, analyzing such vast arrays of droplets usually requires advanced expertise and sophisticated workflow tools, which limits accessibility for a wider user base in the fields of chemistry and biology. Thus, there is a need for more user-friendly tools for droplet analysis. In this article, we deliver a set of analytical pipelines for user-friendly analysis of typical scenarios in droplet experiments. We built pipelines that combine various open-source image-analysis software with a custom-developed data processing tool called "EasyFlow". Our pipelines are applicable to the typical experimental scenarios that users encounter when working with droplets: i) mono- and polydisperse droplets, ii) brightfield and fluorescent images, iii) droplet and object detection, iv) signal profile of droplets and objects (e.g., fluorescence).

Binary RuO2-CuO Electrodes Outperform RuO2 Electrodes in Measuring the pH in Food Samples.

Glass electrodes are the only type of pH-sensitive electrodes currently used in the food industry. While widely used, they have several disadvantages, especially in the areas of brittleness and price. Ruthenium(IV) oxide (RuO2) pH electrodes are a well-known alternative to conventional glass electrodes, providing improved durability and lower price. Nevertheless, partial substitution of RuO2 with cupric oxide (CuO) would further lower the price and reduce the toxicity of the electrode. In this paper, we present the applicability of RuO2-CuO electrodes for pH measurement in food samples. The electrodes were fabricated by screen printing and covered with a protective Nafion membrane. In the experiments with food samples, the RuO2-CuO electrodes outperformed RuO2 electrodes in measuring the pH with an almost twofold higher rate of accurate measurements. The utilization of CuO for the fabrication of pH electrodes allowed the accurate measurement of pH in a larger variety of food samples without compromising the response time.

Physicochemical Properties Predict Retention of Antibiotics in Water-in-Oil Droplets.

Water-in-oil droplet microfluidics promises capacity for high-throughput single-cell antimicrobial susceptibility assays and investigation of drug resistance mechanisms. Every droplet must serve as an isolated environment with a controlled antibiotic concentration in such assays. While technologies for generation, incubation, screening, and sorting droplets mature, predictable retention of active molecules inside droplets remains a major outstanding challenge. Here, we analyzed 36 descriptors of the antibiotic molecules against experimental results on the cross-talk of antibiotics in droplets. We show that partition coefficient and fractional polar surface area are the key physicochemical properties that predict antibiotic retention. We verified the prediction by monitoring growth inhibition by antibiotic-loaded neighboring droplets. Our experiments also demonstrate that transfer of antibiotics between droplets is concentration- and distance-dependent. Our findings immediately apply to designing droplet antibiotic assays and give deeper insight into the retention of small molecules in water-in-oil emulsions.

A COST Action on microbial responses to low pH: Developing links and sharing resources across the academic-industrial divide.

We present work of our COST Action on "Understanding and exploiting the impacts of low pH on micro-organisms". First, we summarise a workshop held at the European Federation of Biotechnology meeting on Microbial Stress Responses (online in 2020) on "Industrial applications of low pH stress on microbial bio-based production", as an example of an initiative fostering links between pure and applied research. We report the outcomes of a small survey on the challenging topic of developing links between researchers working in academia and industry that show that, while people in different sectors strongly support such links, barriers remain that obstruct this process. We present the thoughts of an expert panel held as part of the workshop above, where people with experience of collaboration between academia and industry shared ideas on how to develop and maintain links. Access to relevant information is essential for research in all sectors, and because of this we have developed, as part of our COST Action goals, two resources for the free use of all researchers with interests in any aspects of microbial responses to low pH. These are (1) a comprehensive database of references in the literature on different aspects of acid stress responses in different bacterial and fungal species, and (2) a database of research expertise across our network. We invite the community of researchers working in this field to take advantage of these resources to identify relevant literature and opportunities for establishing collaborations.

Droplet-based methods for tackling antimicrobial resistance.

Application of droplet-based methods enables (i) faster detection, (ii) increased sensitivity, (iii) characterization of the level of heterogeneity in response to antibiotics by bacterial populations, and (iv) expanded screening of the effectiveness of antibiotic combinations. Hereby, we discuss the key steps and parameters of droplet-based experiments to investigate antimicrobial resistance. We also review recent findings accomplished with these methods and highlight their advantages and capacity to yield new insights into the problem of antimicrobial resistance.

Investigation of Different Free Image Analysis Software for High-Throughput Droplet Detection.

Droplet microfluidics has revealed innovative strategies in biology and chemistry. This advancement has delivered novel quantification methods, such as droplet digital polymerase chain reaction (ddPCR) and an antibiotic heteroresistance analysis tool. For droplet analysis, researchers often use image-based detection techniques. Unfortunately, the analysis of images may require specific tools or programming skills to produce the expected results. In order to address the issue, we explore the potential use of standalone freely available software to perform image-based droplet detection. We select the four most popular software and classify them into rule-based and machine learning-based types after assessing the software's modules. We test and evaluate the software's (i) ability to detect droplets, (ii) accuracy and precision, and (iii) overall components and supporting material. In our experimental setting, we find that the rule-based type of software is better suited for image-based droplet detection. The rule-based type of software also has a simpler workflow or pipeline, especially aimed for non-experienced users. In our case, CellProfiler (CP) offers the most user-friendly experience for both single image and batch processing analyses.

Can 3D Printing Bring Droplet Microfluidics to Every Lab?-A Systematic Review.

In recent years, additive manufacturing has steadily gained attention in both research and industry. Applications range from prototyping to small-scale production, with 3D printing offering reduced logistics overheads, better design flexibility and ease of use compared with traditional fabrication methods. In addition, printer and material costs have also decreased rapidly. These advantages make 3D printing attractive for application in microfluidic chip fabrication. However, 3D printing microfluidics is still a new area. Is the technology mature enough to print complex microchannel geometries, such as droplet microfluidics? Can 3D-printed droplet microfluidic chips be used in biological or chemical applications? Is 3D printing mature enough to be used in every research lab? These are the questions we will seek answers to in our systematic review. We will analyze (1) the key performance metrics of 3D-printed droplet microfluidics and (2) existing biological or chemical application areas. In addition, we evaluate (3) the potential of large-scale application of 3D printing microfluidics. Finally, (4) we discuss how 3D printing and digital design automation could trivialize microfluidic chip fabrication in the long term. Based on our analysis, we can conclude that today, 3D printers could already be used in every research lab. Printing droplet microfluidics is also a possibility, albeit with some challenges discussed in this review.

Techniques Used for Analyzing Microplastics, Antimicrobial Resistance and Microbial Community Composition: A Mini-Review.

Antimicrobial resistance (AMR) is a global health threat. Antibiotics, heavy metals, and microplastics are environmental pollutants that together potentially have a positive synergetic effect on the development, persistence, transport, and ecology of antibiotic resistant bacteria in the environment. To evaluate this, a wide array of experimental methods would be needed to quantify the occurrence of antibiotics, heavy metals, and microplastics as well as associated microbial communities in the natural environment. In this mini-review, we outline the current technologies used to characterize microplastics based ecosystems termed "plastisphere" and their AMR promoting elements (antibiotics, heavy metals, and microbial inhabitants) and highlight emerging technologies that could be useful for systems-level investigations of AMR in the plastisphere.

Front-Face Fluorimeter for the Determination of Cutting Time of Cheese Curd.

The yield of product (cheese) during the cheese-making process depends on the cutting time of the cheese curd. However, the determination of optimal cutting time on an industrial scale is difficult as current standard methods are destructive or analyse only small volumes and not the entire milk to be curdled into cheese. This paper presents a novel front-face fluorimeter (FFF) that is designed to be immersed into a milk batch to enable the determination of the cutting time of cheese curd without the destruction of the sample. The FFF sensor signal corresponds to physical changes in milk during cheese formation and has high predictive power (r > 0.85) and good accuracy (RSE = 30%, considering daily variation between milk samples). The performance of the presented fluorimeter was on par with standard rheological and Berridge methods.

Optical Detection Methods for High-Throughput Fluorescent Droplet Microflow Cytometry.

High-throughput microflow cytometry has become a focal point of research in recent years. In particular, droplet microflow cytometry (DMFC) enables the analysis of cells reacting to different stimuli in chemical isolation due to each droplet acting as an isolated microreactor. Furthermore, at high flow rates, the droplets allow massive parallelization, further increasing the throughput of droplets. However, this novel methodology poses unique challenges related to commonly used fluorometry and fluorescent microscopy techniques. We review the optical sensor technology and light sources applicable to DMFC, as well as analyze the challenges and advantages of each option, primarily focusing on electronics. An analysis of low-cost and/or sufficiently compact systems that can be incorporated into portable devices is also presented.

Understanding How Microorganisms Respond to Acid pH Is Central to Their Control and Successful Exploitation.

Microbes from the three domains of life, Bacteria, Archaea, and Eukarya, share the need to sense and respond to changes in the external and internal concentrations of protons. When the proton concentration is high, acidic conditions prevail and cells must respond appropriately to ensure that macromolecules and metabolic processes are sufficiently protected to sustain life. While, we have learned much in recent decades about the mechanisms that microbes use to cope with acid, including the unique challenges presented by organic acids, there is still much to be gained from developing a deeper understanding of the effects and responses to acid in microbes. In this perspective article, we survey the key molecular mechanisms known to be important for microbial survival during acid stress and discuss how this knowledge might be relevant to microbe-based applications and processes that are consequential for humans. We discuss the research approaches that have been taken to investigate the problem and highlight promising new avenues. We discuss the influence of acid on pathogens during the course of infections and highlight the potential of using organic acids in treatments for some types of infection. We explore the influence of acid stress on photosynthetic microbes, and on biotechnological and industrial processes, including those needed to produce organic acids. We highlight the importance of understanding acid stress in controlling spoilage and pathogenic microbes in the food chain. Finally, we invite colleagues with an interest in microbial responses to low pH to participate in the EU-funded COST Action network called EuroMicropH and contribute to a comprehensive database of literature on this topic that we are making publicly available.

Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population.

Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations.

A dual colour FISH method for routine validation of sexed Bos taurus semen.

BACKGROUND: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. RESULTS: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). CONCLUSIONS: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.

Recent developments of microfluidics as a tool for biotechnology and microbiology.

Academic microfluidics has decisively shifted in recent years from the research on phenomenology and proof-of-concept fluidic functionalities to the developments oriented at applications with biology, medicine and biotechnology in prime focus. Significant efforts are made to demonstrate that microfluidics can be used in unspecialized laboratories to perform previously mundane tasks faster and easier, or to venture into new research areas that were unavailable or unattractive when only classical means of microbiology or biotechnology were employed. Here we review a variety of biological experiments recently performed in microfluidic assays. We categorize the microfluidic systems by the key role they play in the biological experiments as: (i) controlled reaction chambers, (ii) high-throughput arrays, or (iii) micro-positioning systems. We also discuss the outlook for further development and applications of microfluidics in biological sciences.

Microfluidic screening of antibiotic susceptibility at a single-cell level shows the inoculum effect of cefotaxime on E. coli.

Measurement of antibiotic susceptibility at the level of single cells is important as it reveals the concentration of an antibiotic that leads to drug resistance in bacterial strains. To date, no solution for large-scale studies of antibiotic susceptibility at the single-cell level has been shown. Here, we present a method for production and separation of emulsions consisting of subnanoliter droplets that allows us to identify each emulsion by their spatial position in the train of emulsions without chemical barcoding. The emulsions of droplets are separated by a third immiscible phase, thus forming large compartments-tankers-each filled with an emulsion of droplet reactors. Each tanker in a train can be set under different reaction conditions for hundreds or thousands of replications of the same reaction. The tankers allow for long term incubation - needed to check for growth of bacteria under a screen of conditions. We use microfluidic tankers to analyze susceptibility to cefotaxime in ca. 1900 replications for each concentration of the antibiotic in one experiment. We test cefotaxime susceptibility for different initial concentrations of bacteria, showing the inoculum effect down to the level of single cells for more than a hundred single-cell events per tanker. Lastly, we use tankers to observe the formation of aggregates of bacteria in the presence of cefotaxime in the increasing concentration of the antibiotic. The microfluidic tankers allow for facile studies of the inoculum effect and antibiotic susceptibility, and constitute an attractive, label-free screening method for a variety of other experiments in chemistry and biology.

Droplet microfluidics for microbiology: techniques, applications and challenges.

Droplet microfluidics has rapidly emerged as one of the key technologies opening up new experimental possibilities in microbiology. The ability to generate, manipulate and monitor droplets carrying single cells or small populations of bacteria in a highly parallel and high throughput manner creates new approaches for solving problems in diagnostics and for research on bacterial evolution. This review presents applications of droplet microfluidics in various fields of microbiology: i) detection and identification of pathogens, ii) antibiotic susceptibility testing, iii) studies of microbial physiology and iv) biotechnological selection and improvement of strains. We also list the challenges in the dynamically developing field and new potential uses of droplets in microbiology.

Dodecylresorufin (C12R) Outperforms Resorufin in Microdroplet Bacterial Assays.

This paper proves that dodecylresorufin (C12R) outperforms resorufin (the conventional form of this dye) in droplet microfluidic bacterial assays. Resorufin is a marker dye that is widely used in different fields of microbiology and has increasingly been applied in droplet microfluidic assays and experiments. The main concern associated with resorufin in droplet-based systems is dye leakage into the oil phase and neighboring droplets. The leakage decreases the performance of assays because it causes averaging of the signal between the positive (bacteria-containing) and negative (empty) droplets. Here we show that C12R is a promising alternative to conventional resorufin because it maintains higher sensitivity, specificity, and signal-to-noise ratio over time. These characteristics make C12R a suitable reagent for droplet digital assays and for monitoring of microbial growth in droplets.

Nucleic acid detection technologies and marker molecules in bacterial diagnostics.

There is a growing need for quick and reliable methods for microorganism detection and identification worldwide. Although traditional culture-based technologies are trustworthy and accurate at a relatively low cost, they are also time- and labor-consuming and are limited to culturable bacteria. Those weaknesses have created a necessity for alternative technologies that are capable for faster and more precise bacterial identification from medical, food or environmental samples. The most common current approach is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to the specific nucleic acid profiles that are present. This review aims to give an up-to-date overview of different nucleic acid target sequences and respective analytical technologies.

Label-free, multiplexed detection of bacterial tmRNA using silicon photonic microring resonators.

A label-free biosensing method for the sensitive detection and identification of bacterial transfer-messenger RNA (tmRNA) is presented employing arrays of silicon photonic microring resonators. Species specific tmRNA molecules are targeted by complementary DNA capture probes that are covalently attached to the sensor surface. Specific hybridization is monitored in near real-time by observing the resonance wavelength shift of each individual microring. The sensitivity of the biosensing platform allowed for detection down to 53 fmol of Streptococcus pneumoniae tmRNA, equivalent to approximately 3.16×10(7) CFU of bacteria. The simplicity and scalability of this biosensing approach makes it a promising tool for the rapid identification of different bacteria via tmRNA profiling.