A high-throughput quality control method for assessing the serial dilution performance of dose-response plates with acoustic ejection mass spectrometry.

This study aimed to develop a streamlined method for evaluating the dilution ratio of drug dose-response plates created by automated liquid handlers in the early stages of drug discovery. The quantitative techniques commonly used for this purpose have restrictions due to their limited linear dynamic range and inaccuracies in assessing serial dilution performance. To address this challenge, we describe a method based on acoustic ejection mass spectrometry (AEMS). The method involves using standard compounds and an internal standard to evaluate each dilution point in quality control (QC) plates. The samples are transferred to a chromatography-free tandem mass spectrometry system through an acoustic source, enabling the analysis of one sample per three seconds from a microtiter plate. This approach provides precise, accurate, label-free, and rapid data acquisition to support high-throughput screening efforts.

Effects of microbial diversity loss on the stability of CO2 production and N2O emission in agricultural soils.

The relationship between biodiversity and ecosystem stability is a hot topic in ecology. However, current studies focus mainly on aboveground system with plants, little attention has been paid to belowground system with soils. In this study, we constructed three soil suspensions with varying microbial diversity (100, 10-2, 10-6) by the dilution method and inoculated separately into agricultural Mollisols and Oxisols to examine the stability (indicated by resistance and resilience) of soil CO2 production and N2O emission to copper pollution and heat stress. Results showed that the stability of CO2 production in Mollisols was not influenced by microbial diversity loss, while the resistance and resilience of N2O emission in Mollisols were significantly decreased at the 10-6 diversity. In the Oxisols, the resistance and resilience of N2O emission to copper pollution and heat stress started to decrease even at the 10-2 diversity, and the stability of CO2 production decreased at the 10-6 diversity. These results suggested that both soil types and the identity of soil functions influenced the relationship between microbial diversity and the stability of function. It was concluded that soils with ample nutrients and resistant microbial communities tend to have higher functional stability, and that the fundamental soil functions (e.g., CO2 production) are more resistant and resilient than the specific soil functions (e.g., N2O emission) in response to environmental stress.

Analysis of two sampling treatments of beef for microbiome studies based on metataxonomic.

Microbial enumeration by serial dilution is one of the best resources to estimate cellular density for microbiological analysis. However, for metataxonomic analysis, it is not clear if serially diluted samples may accurately be used for metataxonomic analysis to represent species composition in beef samples. In this study, the effect of sampling preparation of beef samples on the bacterial composition was evaluated by the comparison of dilution and exudate. Based on the obtained results, data obtained from the exudate of the samples were more robust in terms of number of generated reads, but no significant differences in terms of biological diversity were observed (P < .05, Wicoxon Test). Besides, both sample preparation procedures evidenced equivalent results of bacterial composition as well as its relative abundances. In conclusion, the use of exudate allows bacterial enumeration and metataxonomic analysis, which is interesting for the point of view of food microbiologists as cellular loads and microbial composition of culturable and unculturable bacteria could be compared.

Maltodextrin-Coated Peppermint and Caraway Essential Oils Effects on Soil Microbiota.

Essential oils exhibit strong antimicrobial effects that can serve as a substitute for synthetic pesticides. However, many reports mention the use of essential oils in protecting above-ground plant organs and storing raw materials and seeds, but only a few address the effects of treatments on soil microbiota. Regarding this, it is necessary to find a solution that will prevent the rapid degradation of oils in soil and extend the period of their action on the soil microbiota. The solution to this problem can be microencapsulation, where the choice of carrier plays a key role. In our experiment, maltodextrin was studied, often used in the microencapsulation of essential oils. It was examined independently in two doses (M1 and M2, with 50 and 200 g kg-1, respectively) and a combination with two essential oils known for their antimicrobial activity. We hypothesized that the selected microbial communities would react differently to the stress caused by maltodextrin-encapsulated essential oils. The serial dilution method assessed the number of colony-forming units (CFU) of bacteria, fungi, and actinomycetes. As the goal of microencapsulation was to prolong the effect of essential oils, their reaction was observed over a longer period. The soil microbial populations were examined in sandy and loamy soil at 1, 7, 14, and 78 days after encapsulated essential oils were mixed with the soil samples. In both types of soil, a significant increase in bacteria and actinomycetes was observed with maltodextrin in both doses. Encapsulated peppermint and caraway oils had different effects on microbes, both inhibitory and stimulatory. It is also important to note that peppermint with a smaller dose of maltodextrin significantly inhibited the growth of fungi in sandy soil in all measurements, as well as that caraway oil with a higher dose of maltodextrin significantly stimulated the growth of bacteria and actinomycetes in sandy soil. The higher dose of maltodextrin could explain this stimulation. Further research is recommended to test different doses of essential oils and maltodextrin, which would lead to the optimal dose of both wall and core materials.

Non-linear transformation of enzyme-linked immunosorbent assay (ELISA) measurements allows usage of linear models for data analysis.

BACKGROUND: In research questions such as in resistance breeding against the Beet necrotic yellow vein virus it is of interest to compare the virus concentrations of samples from different groups. The enzyme-linked immunosorbent assay (ELISA) counts as the standard tool to measure virus concentrations. Simple methods for data analysis such as analysis of variance (ANOVA), however, are impaired due to non-normality of the resulting optical density (OD) values as well as unequal variances in different groups. METHODS: To understand the relationship between the OD values from an ELISA test and the virus concentration per sample, we used a large serial dilution and modelled its non-linear form using a five parameter logistic regression model. Furthermore, we examined if the quality of the model can be increased if one or several of the model parameters are defined beforehand. Subsequently, we used the inverse of the best model to estimate the virus concentration for every measured OD value. RESULTS: We show that the transformed data are essentially normally distributed but provide unequal variances per group. Thus, we propose a generalised least squares model which allows for unequal variances of the groups to analyse the transformed data. CONCLUSIONS: ANOVA requires normally distributed data as well as equal variances. Both requirements are not met with raw OD values from an ELISA test. A transformation with an inverse logistic function, however, gives the possibility to use linear models for data analysis of virus concentrations. We conclude that this method can be applied in every trial where virus concentrations of samples from different groups are to be compared via OD values from an ELISA test. To encourage researchers to use this method in their studies, we provide an R script for data transformation as well as the data from our trial.

Pipette Olympics: An Engaging Exercise for Undergraduate Laboratory Training.

Pipetting is an important technique used in almost every molecular neuroscience method including but not limited to, PCR, reverse transcription, immunohistochemistry, chromatin immunoprecipitation, and cell culture. The COVID-19 pandemic has robbed the undergraduate population of time to practice in person laboratory techniques. In response, we have devised a standardized, quick, and fun way to instruct students on the fundamentals of pipetting, serial dilutions, and basic statistical analysis. Here, we offer a standardized protocol for instructors to use to teach undergraduates valuable skills while providing friendly competition. We also offer an example of an undergraduate performing the steps of this protocol with example results and the results from three separate undergrads' first two attempts. This exercise provides laboratories with a method to reintroduce undergraduates to lab basics while standardizing the training thereby saving time lost to the pandemic.

Cultivation of Important Methanotrophs From Indian Rice Fields.

Methanotrophs are aerobic to micro-aerophilic bacteria, which oxidize and utilize methane, the second most important greenhouse gas. The community structure of the methanotrophs in rice fields worldwide has been studied mainly using culture-independent methods. Very few studies have focused on culturing methanotrophs from rice fields. We developed a unique method for the cultivation of methanotrophs from rice field samples. Here, we used a modified dilute nitrate mineral salts (dNMS) medium, with two cycles of dilution till extinction series cultivation with prolonged incubation time, and used agarose in the solid medium. The cultivation approach resulted in the isolation of methanotrophs from seven genera from the three major groups: Type Ia (Methylomonas, Methylomicrobium, and Methylocucumis), Type Ib (Methylocaldum and Methylomagnum), and Type II (Methylocystis and Methylosinus). Growth was obtained till 10-6-10-8 dilutions in the first dilution series, indicating the culturing of dominant methanotrophs. Our study was supported by 16S rRNA gene-based next-generation sequencing (NGS) of three of the rice samples. Our analyses and comparison with the global scenario suggested that the cultured members represented the major detected taxa. Strain RS1, representing a putative novel species of Methylomicrobium, was cultured; and the draft genome sequence was obtained. Genome analysis indicated that RS1 represented a new putative Methylomicrobium species. Methylomicrobium has been detected globally in rice fields as a dominant genus, although no Methylomicrobium strains have been isolated from rice fields worldwide. Ours is one of the first extensive studies on cultured methanotrophs from Indian rice fields focusing on the tropical region, and a unique method was developed. A total of 29 strains were obtained, which could be used as models for studying methane mitigation from rice fields and for environmental and biotechnological applications.

Ferrowax microvalves for fully automated serial dilution on centrifugal microfluidic platforms.

We herein describe a centrifugal microfluidic system to accomplish a fully automated serial dilution. The liquid flow on the disc was regulated by utilizing ferrowax microvalves systematically integrated into the channels within specially designed metering structures. By opening the differently positioned microvalves through irradiation of IR laser to allow metering, the same amount of diluent was serially eluted to the dilution chamber from the same diluent chamber. After dilution, the diluted samples were automatically delivered to the respective final product chambers by appropriately opening or closing the microvalves in the connecting channels, followed by rotating the disc. Based on this unique design principle, six consecutive two-fold and 10-fold dilutions were successfully achieved, yielding excellent accuracy in a wide dynamic range up to six orders of magnitude. Very importantly, the overall serial dilution process, including the diluent addition, mixing, and product transfer steps, was completed very rapidly within 5 min, due to the minimized procedures enabled by the automated actuation of the ferrowax microvalves at the rationally designed positions. We expect our centrifugal microfluidic system would serve as a powerful elemental tool to realize fully automated diagnostic microsystems involving the serial dilution process.

An Integrated Centrifugal Degassed PDMS-Based Microfluidic Device for Serial Dilution.

We propose an integrated serial dilution generator utilizing centrifugal force with a degassed polydimethylsiloxane (PDMS) microfluidic device. Using gas-soluble PDMS as a centrifugal microfluidic device material, the sample can be dragged in any arbitrary direction using vacuum-driven force, as opposed to in a single direction, without adding further actuation components. The vacuum-driven force allows the device to avoid the formation of air bubbles and exhibit high tolerance in the surface condition. The device was then used for sample metering and sample transferring. In addition, centrifugal force was used for sample loading and sample mixing. In this study, a series of ten-fold serial dilutions ranging from 100 to 10-4 with about 8 µL in each chamber was achieved, while the serial dilution ratio and chamber volume could easily be altered by changing the geometrical designs of the device. As a proof of concept of our hybrid approach with the centrifugal and vacuum-driven forces, ten-fold serial dilutions of a cDNA (complementary DNA) sample were prepared using the device. Then, the diluted samples were collected by fine needles and subject to a quantitative polymerase chain reaction (qPCR), and the results were found to be in good agreement with those for samples prepared by manual pipetting.

Isolation and identification of milk oligosaccharide-degrading bacteria from the intestinal contents of suckling rats.

We report the isolation of bacteria capable of degrading milk oligosaccharides from suckling infant rats. The bacteria were successfully isolated via a selective enrichment method, in which the serially diluted intestinal contents of infant rats were individually incubated in an enrichment medium containing 3'-sialyllactose (3'-SL), followed by the isolation of candidate strains from streaked agar plates and selection of 3'-SL-degrading strains using thin-layer chromatography. Subsequent genomic and phenotypic analyses identified all strains as Enterococcus gallinarum. The strains were capable of degrading both 3'-SL and 6'-SL, which was not observed with the type strain of E. gallinarum used as a reference. Furthermore, a time-course study combining high-performance anion-exchange chromatography with pulsed amperometric detection revealed that the representative strain AH4 degraded 3'-SL completely to yield an equimolar amount of lactose and an approximately one-fourth equimolar amount of sialic acid after 24 hr of anaerobic incubation. These findings point to a possibility that the enterococci degrade rat milk oligosaccharides to "cross-feed" their degradants to other members of concomitant bacteria in the gut of the infant rat.

Light microscopy and scanning electron microscopy of bacterial colonies isolated from ras malai samples from different markets of Lahore, Pakistan.

The current study investigates the total bacterial contamination in various packed and unpacked ras malai samples of 14 different localities of Lahore, Pakistan. The bacterial colonies such as Bacillus sp. and Gamella sp. were isolated from ras malai samples and grown on agar-broth media under sterile environmental conditions. Serial dilution technique was used to compose the replicates to get a viable count of bacteria in the samples. Results indicated that in case of packed ras malai samples, maximum bacterial count was observed in Sample 1 (422 × 10-2 to 402 × 10-6 ) and minimum bacterial count was in Sample 4 (21 × 10-2 to 9.3 × 10-6 ). For unpacked ras malai samples, maximum bacterial count was in Sample 3 (200.3 × 10-2 to 181.3 × 10-6 ) and minimum bacterial count was observed in Sample 1 (110 × 10-2 to 90.4 × 10-6 ). It was concluded that the marketed samples contain more bacterial count as compared to the standard sterilization values. Such products could possibly become the cause of many health problems in children.

Synthesis, Characterization and Antimicrobial Evaluation of Some N-Substituted Benzimidazole Derivatives.

BACKGROUND: Due to the appearance of communicable microbial diseases and the toxicity related with presently used several antimicrobials such as ß-lactam antibiotics, tetracyclines, quinolones, macrolides, glycopeptides (vancomycin) etc, demand for new antimicrobial agents has become a great concern in new technologies to improve efficacy and safety. METHODS: In search of new antimicrobial agents with higher potency, some N-substituted benzimidazole derivatives (4, 5a-5h & 6) were obtained by chloroacetylation of benzimidazole followed by reaction with different amines, which were characterized by spectroscopic methods. All the target compounds were evaluated for their antibacterial and antifungal activity against microorganisms using two-fold serial dilution method. RESULTS: Among the compounds evaluated, compounds 4 and 5d exhibited potent activity against Bacillus thuringiensis and Candida albicans while showed moderate activity against Escherischia coli when compared to amoxicillin and fluconazole. Compound 5a showed significant activity against tested microorganisms. CONCLUSION: From the current study, it may be concluded that synthesized compounds are fulfilling in terms of their structural distinctiveness and marked biological properties. These compounds might be encouraged to initiate a new class of antimicrobial agents.

Measurements of Endpoint Titers Based on the Fluorescence Intensity Trend in Anti-Nuclear Antibody Testing.

BACKGROUND: Automated systems for antinuclear antibody (ANA) testing provide endpoint titers that are predicted based on the fluorescence intensity (FI) value at a screening dilution (single-well titration [SWT]) showing frequent titration errors (more than plus or minus 1 dilution). METHODS: Line slope titration (LST) was based on the trend of FI values on dilutions. Three dilutions per specimen were prepared considering a patient's previous titer or FI at the screening dilution. On the XY plot, with the reciprocal of dilution as the X-axis and FI value as the Y-axis, a fitted line was drawn to obtain the endpoint titers. RESULTS: The titration error rate (no. of errors/total no.) of LST using a regression line was lower than that of SWT (31/710 [4.4%] and 152/674 [22.6%], respectively; P < .000000001), with serial dilution as a reference. When comparing a regression line using 3 dilution points with a line using 2 dilution points, the error rate of the former was not significantly different from that of the latter (31/710 [4.4%] and 31/746 [4.2%], respectively; P = .842). CONCLUSIONS: This LST method is useful as an accurate, cost-effective, and rapid approach to measure endpoint titers in routine ANA testing.

Nonspecific probe binding and automatic gating in flow cytometry and fluorescence activated cell sorting (FACS).

Flow cytometry is extensively used in cell biology to differentiate cells of interest (mutants) from control cells (wild-types). For mutant cells characterized by expression of a distinct membrane surface structure, fluorescent marker probes can be designed to bind specifically to these structures while the cells are in suspension, resulting in a sufficiently high fluorescence intensity measurement by the cytometer to identify a mutant cell. However, cell membranes may have relatively weak, nonspecific binding affinity to the probes, resulting in false positive results. Furthermore, the same effect would be present on mutant cells, allowing both specific and nonspecific binding to a single cell. We derive and analyze a kinetic model of fluorescent probe binding dynamics by tracking populations of mutant and wild-type cells with differing numbers of probes bound specifically and nonspecifically. By assuming the suspension is in chemical equilibrium prior to cytometry, we use a two-species Langmuir adsorption model to analyze the confounding effects of non-specific binding on the assay. Furthermore, we analytically derive an expectation maximization method to infer an appropriate estimate of the total number of mutant cells as an alternative to existing, heuristic methods. Lastly, using our model, we propose a new method to infer physical and experimental parameters from existing protocols. Our results provide improved ways to quantitatively analyze flow cytometry data.

Assessment of mold and yeast in some bakery products of Lahore, Pakistan based on LM and SEM.

The present investigation was designed to throw light on the microbial status of bakery products available in bakeries and supermarkets of Lahore. Different bakery samples such as biscuits, pizza, patties were collected from different localities such as Anar Kali, Chauburji, Faisal Town, Iqbal Town, Model Town, Muslim Town were investigated for mold and yeast using serial dilution technique inoculated over malt extract agar and potato dextrose agar under sterilized conditions. Isolated fungi were namely Alternaria alternata, Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus, Curvularia americana, Fusarium solani, Penicillium digitatum, Saccharomyces cerevisiae, and Geotrichum candidum. Results depicted maximum fungal viable count in biscuits, collected from Model Town while minimum count was in the samples of Chauburji. In the case of pizza, the maximum fungal viable count was found in the sample of Muslim Town. In the case of patties, the maximum fungal viable count was found in the sample of Muslim Town while minimum count was found in the sample of Iqbal Town. Prevalence of microorganisms may be due to the use of contaminated raw material, use of polluted waters, human handling, and the use of contaminated containers. Contaminated food intake can lead to measurable signs of liver injury, inflammation, etc. Preventive measures like washing and drying of hands before preparing food, cleaning of food preparation areas, and the use of clean equipment can avoid microbes which cause hazards to human health. RESEARCH HIGHLIGHTS: Bakery products of Lahore, Pakistan is investigated for the first time on the basis of light microscopy (LM) and scanning electron microscopy (SEM) and found very significant to check the quality of bakery products or not.

Comparison of Antibacterial Adhesion When Salivary Pellicle Is Coated on Both Poly(2-hydroxyethyl-methacrylate)- and Polyethylene-glycol-methacrylate-grafted Poly(methyl methacrylate).

Although poly(2-hydroxyethyl methacrylate) (pHEMA) and polyethylene glycol methacrylate (PEGMA) have been demonstrated to inhibit bacterial adhesion, no study has compared antibacterial adhesion when salivary pellicle is coated on polymethyl methacrylate (PMMA) grafted with pHEMA and on PMMA grafted with PEGMA. In this study, PMMA discs were fabricated from a commercial orthodontic acrylic resin system (Ortho-Jet). Attenuated total reflection-Fourier transform infrared spectra taken before and after grafting confirmed that pHEMA and PEGMA were successfully grafted on PMMA. Contact angle measurements revealed PMMA-pHEMA to be the most hydrophilic, followed by PMMA-PEGMA, and then by PMMA. Zeta potential analysis revealed the most negative surface charges on PMMA-PEGMA, followed by PMMA-pHEMA, and then by PMMA. Confocal laser scanning microscopy showed green fluorescence in the background, indicating images that influenced the accuracy of the quantification of live bacteria. Both the optical density value measured at 600 nm and single plate-serial dilution spotting showed that pHEMA was more effective than PEGMA against Escherichia coli and Streptococcus mutans, although the difference was not significant. Therefore, the grafting of pHEMA and PEGMA separately on PMMA is effective against bacterial adhesion, even after the grafted PMMA were coated with salivary pellicle. Surface hydrophilicity, bactericidality, and Coulomb repulsion between the negatively charged bacteria and the grafted surface contributed to the effectiveness.

Hidden Complexity of Yeast Adaptation under Simple Evolutionary Conditions.

Few studies have "quantitatively" probed how adaptive mutations result in increased fitness. Even in simple microbial evolution experiments, with full knowledge of the underlying mutations and specific growth conditions, it is challenging to determine where within a growth-saturation cycle those fitness gains occur. A common implicit assumption is that most benefits derive from an increased exponential growth rate. Here, we instead show that, in batch serial transfer experiments, adaptive mutants' fitness gains can be dominated by benefits that are accrued in one growth cycle, but not realized until the next growth cycle. For thousands of evolved clones (most with only a single mutation), we systematically varied the lengths of fermentation, respiration, and stationary phases to assess how their fitness, as measured by barcode sequencing, depends on these phases of the growth-saturation-dilution cycles. These data revealed that, whereas all adaptive lineages gained similar and modest benefits from fermentation, most of the benefits for the highest fitness mutants came instead from the time spent in respiration. From monoculture and high-resolution pairwise fitness competition experiments for a dozen of these clones, we determined that the benefits "accrued" during respiration are only largely "realized" later as a shorter duration of lag phase in the following growth cycle. These results reveal hidden complexities of the adaptive process even under ostensibly simple evolutionary conditions, in which fitness gains can accrue during time spent in a growth phase with little cell division, and reveal that the memory of those gains can be realized in the subsequent growth cycle.

Simultaneous estimation of detection sensitivity and absolute copy number from digital PCR serial dilution.

Digital polymerase chain reaction (dPCR) is a refinement of the conventional PCR approach to nucleic acid detection and absolute quantification. Digital PCR works by partitioning a sample of DNA or cDNA into many individual, parallel PCR reactions. Current quantification methods rely on the assumption that the PCR reactions are always able to detect single target molecules. When the assumption does not hold, the copy numbers will be severely underestimated. We developed a novel dPCR quantification method which determines whether the single copy assumption is violated or not by simultaneously estimating the assay sensitivity and the copy numbers using serial dilution data sets. The implemented method is available as an R package "digitalPCR".

[Purification Effect of Piggery Wastewater with Chlorella pyrenoidosa by Immobilized Biofilm-Attached Culture].

Piggery wastewater treatment with microalgae is a biological recycling technology. To evaluate the purification effect, this study investigated the treatment of piggery wastewater at different dilution ratios with Chlorella pyrenoidosa by attached cultivation and lipid production of algae cells and explored the tolerance of Chlorella pyrenoidosa to the piggery wastewater, which has high ammonia nitrogen. The piggery wastewater was diluted with purified water 1-, 2-, 5-, and 10-fold in culture media. The removal efficiencies of COD, ammonia nitrogen, total nitrogen, and total phosphorus and the enrichment effect of the heavy metals copper, zinc, and iron were measured. Meanwhile, we investigated the lipid production of Chlorella pyrenoidosa in variously diluted wastewater (1-, 2-, 5-, and 10-fold). It turned out that the purification effects of COD, ammonia nitrogen, total nitrogen, and total phosphorus were best when the piggery wastewater was diluted 5-fold, and the removal efficiencies were 86.8%, 94.1%, 85.2%, and 84.3%, respectively. Correspondingly, the lipid content was as high as 32.7%, and the removal efficiencies of the heavy metals copper, zinc, and iron were 72.9%, 70.0%, and 73.0%, respectively. The biomass productivity was 4.21 g·(m2·d)-1 at the end of the experiment. This research makes an effective connection between microalgae and piggery wastewater, which is difficult to purify deeply, and provides a theoretical basis for achieving algal biofuel production and decreasing the cost of wastewater treatment.

The reactivity of the back revisited. Are there differences in reactivity in different parts of the back?

BACKGROUND: In the contact dermatitis literature, it is regularly stated that the patch test reactivity on various areas of the back differs, which might have a large impact on the reproducibility of patch testing. OBJECTIVES: To investigate the reproducibility of patch testing on the upper back with regard to the left as opposed to the right side, and the medial as opposed to the lateral part of the upper back. The reproducibility over time and with regard to the reactivity pattern was also investigated. METHODS: Thirty-one subjects with contact allergy to the metals gold (n = 19) or nickel (n = 12) were patch tested with serial dilutions, in triplicate applications, on different locations on the upper back. The Friedman test was used for statistical calculations. RESULTS: No significant differences in the reactivity of the back were found. In all gold-allergic patients and 11 of 12 nickel-allergic patients, the allergy could be reproduced with regard to previous patch testing, but the degree of reactivity differed. CONCLUSIONS: When a high level of standardization of the patch test technique with the same test system was used, there were no differences in patch test reactions and sites of application on the upper back.