scholarly journals PHENOS: a high-throughput and flexible tool for microorganism growth phenotyping on solid media

2017 ◽  
Author(s):  
David B. H. Barton ◽  
Danae Georghiou ◽  
Neelam Dave ◽  
Majed Alghamdi ◽  
Thomas A. Walsh ◽  
...  
Keyword(s):  
High Throughput ◽  
Growth Curves ◽  
Cost Effective ◽  
Flexible Tool ◽  
Hybrid Techniques ◽  
Single Plate ◽  
Solid Media ◽  
Solid Agar ◽  
Colony Cell ◽  
Different Strains

ABSTRACTBACKGROUNDMicrobial arrays, with a large number of different strains on a single plate printed with robotic precision, underpin an increasing number of genetic and genomic approaches. These include Synthetic Genetic Array analysis, high-throughput Quantitative Trait Loci (QTL) analysis and 2-hybrid techniques. Measuring the growth of individual colonies within these arrays is an essential part of many of these techniques but is useful for any work with arrays. Measurement is typically done using intermittent imagery fed into complex image analysis software, which is not especially accurate and is challenging to use effectively. We have developed a simple and fast alternative technique that uses a pinning robot and a commonplace microplate reader to continuously measure the thickness of colonies growing on solid agar, complemented by a technique for normalizing the amount of cells initially printed to each spot of the array in the first place. We have developed software to automate the process of combining multiple sets of readings, subtracting agar absorbance, and visualizing colony thickness changes in a number of informative ways.RESULTSThe “PHENOS” pipeline (PHENotyping On Solid media), optimized for Saccharomyces yeasts, produces highly reproducible growth curves and is particularly sensitive to low-level growth. We have empirically determined a formula to estimate colony cell count from an absorbance measurement, and shown this to be comparable with estimates from measurements in liquid. We have also validated the technique by reproducing the results of an earlier QTL study done with conventional liquid phenotyping, and found PHENOS to be considerably more sensitive.CONCLUSIONS“PHENOS” is a cost effective and reliable high-throughput technique for quantifying growth of yeast arrays, and is likely to be equally very useful for a range of other types of microbial arrays. A detailed guide to the pipeline and software is provided with the installation files at https://github.com/gact/phenos.

scholarly journals PHENOS: a high-throughput and flexible tool for microorganism growth phenotyping on solid media

2018 ◽  
Vol 18 (1) ◽  
Author(s):  
David B. H. Barton ◽  
Danae Georghiou ◽  
Neelam Dave ◽  
Majed Alghamdi ◽  
Thomas A. Walsh ◽  
...  
Keyword(s):  
High Throughput ◽  
Flexible Tool ◽  
Solid Media

TEM processing procedure for a bacillus organism grown on solid media

1990 ◽  
Vol 48 (3) ◽  
pp. 624-625
Author(s):  
Jane Payne ◽  
Philip Coudron
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Fume Hood ◽  
Vacuum Aspiration ◽  
Solid Media ◽  
Transmission Electron ◽  
Solid Agar ◽  
Processing Procedure ◽  
Agar Media ◽  
Rat Bone

This transmission electron microscopy (TEM) procedure was designed to examine a gram positive spore-forming bacillus in colony on various solid agar media with minimal artifact. Cellular morphology and organization of colonies embedded in Poly/Bed 812 resin (P/B) were studied. It is a modification of procedures used for undecalcified rat bone and Stomatococcus mucilaginosus.Cultures were fixed and processed at room temperature (RT) under a fume hood. Solutions were added with a Pasteur pipet and removed by gentle vacuum aspiration. Other equipment used is shown in Figure 3. Cultures were fixed for 17-18 h in 10-20 ml of RT 2% phosphate buffered glutaraldehyde (422 mosm/KgH2O) within 5 m after removal from the incubator. After 3 (30 m) changes in 0.15 M phosphate buffer (PB = 209-213 mosm/KgH2O, pH 7.39-7.41), colony cut-outs (CCO) were made with a scalpel.

scholarly journals Local adaptation mediated niche expansion in correlation with genetic richness

2021 ◽  
Author(s):  
Masaomi Kurokawa ◽  
Issei Nishimura ◽  
Bei-Wen YING
Keyword(s):  
Escherichia Coli ◽  
Local Adaptation ◽  
High Throughput ◽  
Experimental Evolution ◽  
Environmental Gradient ◽  
Growth Curves ◽  
Quantitative Relationship ◽  
Central Issue ◽  
Wild Type ◽  
Niche Expansion

As a central issue in evolution and ecology, the quantitative relationship among the genome, adaptation and the niche was investigated. Local adaptation of five Escherichia coli strains carrying either the wild-type genome or reduced genomes was achieved by experimental evolution. A high-throughput fitness assay of the ancestor and evolved populations across an environmental gradient of eight niches resulted in a total of 80 fitness curves generated from 2,220 growth curves. Further analyses showed that the increases in both local adaptiveness and niche broadness were negatively correlated with genetic richness. Local adaptation caused common niche expansion, whereas niche expansion for generality or speciality was decided by genetic richness. The order of the mutations accumulated stepwise was correlated with the magnitude of the fitness increase attributed to mutation accumulation. Pre-adaptation probably participated in coordination among genetic richness, local adaptation and niche expansion.

scholarly journals HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene

2022 ◽  
Vol 8 ◽  
Author(s):  
Ephraim Fass ◽  
Gal Zizelski Valenci ◽  
Mor Rubinstein ◽  
Paul J. Freidlin ◽  
Shira Rosencwaig ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Cost Effective ◽  
Developed Countries ◽  
Spike Protein ◽  
Medical Community ◽  
Effective Vaccine ◽  
Gene Variants ◽  
Spike Gene ◽  
Field Samples

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

scholarly journals Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification

2021 ◽  
Vol 12 ◽  
Author(s):  
Lara Thieme ◽  
Anita Hartung ◽  
Kristina Tramm ◽  
Julia Graf ◽  
Riccardo Spott ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Growth Curves ◽  
Antimicrobial Effect ◽  
Growth Time ◽  
Colony Forming Unit ◽  
Resazurin Assay ◽  
Unspecific Binding ◽  
Biofilm Quantification ◽  
Doubling Times

Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirectly quantifies CFU/mL numbers by evaluating regrowth curves of detached biofilms. For validation, the effect of dalbavancin, rifampicin and gentamicin against mature biofilms of Staphylococcus aureus and Enterococcus faecium was measured by accessing different features of the viability status of the cell, i.e., the cultivability (conventional agar plating), growth behavior (SGT) and metabolic activity (resazurin assay). SGT correlated well with the resazurin assay for all tested antibiotics, but only for gentamicin and rifampicin with conventional agar plating. Dalbavancin treatment-derived growth curves showed a compared to untreated controls significantly slower increase with reduced cell doubling times and reduced metabolic rate, but no change in CFU numbers was observed by conventional agar plating. Here, unspecific binding of dalbavancin to the biofilm interfered with the SGT methodology since the renewed release of dalbavancin during detachment of the biofilms led to an unintended antimicrobial effect. The application of the SGT method for anti-biofilm testing is therefore not suited for antibiotics which stick to the biofilm and/or to the bacterial cell wall. Importantly, the same applies for the well-established resazurin method for anti-biofilm testing. However, for antibiotics which do not bind to the biofilm as seen for gentamicin and rifampicin, the SGT method presents a much less labor-intensive method suited for high-throughput screening of anti-biofilm compounds.

Cost effective microsatellite isolation and genotyping by high throughput sequencing

2019 ◽  
Vol 47 (2) ◽  
pp. 190 ◽  
Author(s):  
Henrik Krehenwinkel ◽  
Susanne Meese ◽  
Christoph Mayer ◽  
Jasmin Ruch ◽  
Jutta Schneider ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Microsatellite Isolation

scholarly journals Review: Cost-Effective Unmanned Aerial Vehicle (UAV) Platform for Field Plant Breeding Application

2020 ◽  
Vol 12 (6) ◽  
pp. 998 ◽  
Author(s):  
GyuJin Jang ◽  
Jaeyoung Kim ◽  
Ju-Kyung Yu ◽  
Hak-Jin Kim ◽  
Yoonha Kim ◽  
...  
Keyword(s):  
Plant Breeding ◽  
Unmanned Aerial Vehicle ◽  
High Throughput ◽  
New Technologies ◽  
Cost Effective ◽  
New Wave ◽  
Breeding Programs ◽  
Modern Agriculture ◽  
Aerial Vehicle ◽  
High Throughput Phenotyping

Utilization of remote sensing is a new wave of modern agriculture that accelerates plant breeding and research, and the performance of farming practices and farm management. High-throughput phenotyping is a key advanced agricultural technology and has been rapidly adopted in plant research. However, technology adoption is not easy due to cost limitations in academia. This article reviews various commercial unmanned aerial vehicle (UAV) platforms as a high-throughput phenotyping technology for plant breeding. It compares known commercial UAV platforms that are cost-effective and manageable in field settings and demonstrates a general workflow for high-throughput phenotyping, including data analysis. The authors expect this article to create opportunities for academics to access new technologies and utilize the information for their research and breeding programs in more workable ways.

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