scholarly journals Colourless agar for enhanced colour contrast between microbial colonies and solid medium

2015 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Yeast Extract ◽  
Solid Medium ◽  
Low Cost ◽  
Viable Cell ◽  
Automated Image Analysis ◽  
Cell Counting ◽  
Good Growth ◽  
Medium Components ◽  
Screening Experiments ◽  
Colour Contrast

Solid medium enables spatially-resolved simultaneous cultivation of different microbes in a simple and relatively low-cost format useful for preliminary screening of microbial diversity. Lack of colour contrast between colonies and agar, however, hampers colony identification by automated image analysis – thus, presenting a challenge for phenotype screening experiments and viable cell counting. Since microbes secrete pigments of myriad hues, this research sought to develop a colourless agar - which when placed on coloured paper of suitable hue – would enhance the colour contrast between agar and colonies of any colour. Nevertheless, the concept is confounded by formation of coloured compounds between medium components during autoclave sterilisation, which, in this study, was prevented by dissolving glucose and ammonium chloride in two separate solutions containing other medium components. Upon mixing the two solutions after heat sterilisation, a colourless agar was obtained – which remained colourless even after adding sterile yeast extract (max: 1 g/L) for providing essential vitamins to microbes unable to synthesise them. Culture experiments revealed good growth of Escherichia coli DH5α (ATCC 53868), Pseudomonas protegens Pf-5 (ATCC BAA-477), Pseudomonas aeruginosa (ATCC 15442), and Bacillus subtilis (ATCC 8473) with cell yield positively correlated with yeast extract concentration. Additionally, identical viable cell concentration and colonies of similar morphologies were observed on both the colourless and LB agar; thus, suggesting that no inhibitory compounds were formed during agar preparation. Collectively, using commonly used buffer components, salts and nutrients, a colourless agar was prepared by segregating chromogenic compounds during heat sterilisation; thereby, opening up its potential use in enhancing colour contrast between colonies and agar medium for revealing finer details of pure culture colonies, or more accurate automated colony identification and counting in screening and viable cell count assays.

scholarly journals Colourless agar for enhanced colour contrast between microbial colonies and solid medium

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Yeast Extract ◽  
Solid Medium ◽  
Low Cost ◽  
Viable Cell ◽  
Automated Image Analysis ◽  
Cell Counting ◽  
Good Growth ◽  
Medium Components ◽  
Screening Experiments ◽  
Colour Contrast

Solid medium enables spatially resolved simultaneous cultivation of different microbes in a simple and relatively low cost format useful for preliminary screening of microbial diversity. Lack of colour contrast between colonies and agar, however, hampers colony identification by automated image analysis – thus, presenting a challenge for phenotype screening experiments and viable cell counting. Since microbes secrete pigments of myriad hues, this research sought to develop a colourless agar - which when placed on coloured paper of suitable hue – would enhance the colour contrast between agar and colonies of any colour. However, the concept is confounded by formation of coloured compounds between medium components during autoclave sterilization, which, in this study, was prevented by dissolving glucose and ammonium chloride in two separate solutions containing other medium components. Upon mixing the two solutions after heat sterilization, a colourless agar was obtained – which remained colourless even after adding sterile yeast extract (max: 1 g/L) for providing essential vitamins to microbes unable to synthesize them. Culture experiments revealed good growth of Escherichia coli DH5α (ATCC 53868), Pseudomonas protegens Pf-5 (ATCC BAA-477), Pseudomonas aeruginosa (ATCC 15442), and Bacillus subtilis (ATCC 8473) with cell yield positively correlated with yeast extract concentration. Additionally, identical viable cell concentration and colonies of similar morphologies were observed on both the colourless and LB agar; thus, suggesting that no inhibitory compounds were formed during agar preparation. Collectively, using commonly used buffer components, salts and nutrients, a colourless agar was prepared by segregating chromogenic compounds during heat sterilization; thereby, opening up its potential use in enhancing colour contrast between colonies and agar medium for revealing finer details of pure culture colonies, or more accurate automated colony identification and counting in screening and viable cell count assays.

scholarly journals Colourless agar for enhanced colour contrast between microbial colonies and solid medium

2013 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Yeast Extract ◽  
Agar Medium ◽  
Low Cost ◽  
Viable Cell ◽  
Automated Image Analysis ◽  
Cell Counting ◽  
Good Growth ◽  
Medium Components ◽  
Screening Experiments ◽  
Colour Contrast

Agar medium enables spatially-resolved simultaneous cultivation of different microbes in a simple and relatively low-cost format useful for preliminary screening of microbial diversity. Lack of colour contrast between colonies and agar, however, hampers colony identification by automated image analysis – thus, presenting a challenge for phenotype screening experiments and viable cell counting. Since microbes secrete pigments of myriad hues, this research sought to develop a colourless agar - which when placed on coloured paper of suitable hue – would enhance the colour contrast between agar and colonies of any colour. Nevertheless, the concept is confounded by formation of coloured compounds between medium components during autoclave sterilisation, which, in this study, was prevented by dissolving glucose and ammonium chloride in two separate solutions containing other medium components. Upon mixing the two solutions after heat sterilisation, a colourless agar was obtained – which remained colourless even after adding sterile yeast extract (max: 1 g/L) for providing essential vitamins to microbes unable to synthesise them. Culture experiments revealed good growth of Escherichia coli DH5α (ATCC 53868), Pseudomonas fluorescens Pf-5 (ATCC BAA-477), Pseudomonas aeruginosa (ATCC 15442), and Bacillus subtilis (ATCC 8473) with cell yield positively correlated with yeast extract concentration. Additionally, identical viable cell concentration and colonies of similar morphologies were observed on both the colourless and LB agar; thus, suggesting that no inhibitory compounds were formed during agar preparation. Collectively, using commonly used buffer components, salts and nutrients, a colourless agar was prepared by segregating chromogenic compounds during heat sterilisation; thereby, opening up its potential use in enhancing colour contrast between colonies and agar medium for revealing finer details of pure culture colonies, or more accurate automated colony identification and counting in screening and viable cell count assays.

scholarly journals Colorless agar for enhanced color contrast between microbe colonies and solid medium

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Yeast Extract ◽  
Solid Medium ◽  
Low Cost ◽  
Viable Cell ◽  
Automated Image Analysis ◽  
Cell Counting ◽  
Color Contrast ◽  
Good Growth ◽  
Medium Components ◽  
Screening Experiments

Solid medium enables spatially resolved simultaneous cultivation of different microbes in a simple and relatively low cost format useful for preliminary screening of microorganism diversity. Lack of color contrast between colonies and agar, however, hampers colony identification by automated image analysis – thus, presenting a challenge for phenotype screening experiments and viable cell counting. Since microbes secrete pigments of myriad hues, this research sought to develop a colorless agar - which when placed on colored paper of suitable hue – would enhance the color contrast between agar and colonies of any color. However, realization of the concept is hampered by heat-induced formation of colored compounds between medium components during autoclave sterilization, which in this study, was prevented by dissolving glucose and ammonium chloride in two separate solutions (each containing other medium components). Mixing the two sterilized solutions at 48 oC yielded a colorless agar – which remained colorless even after adding sterile yeast extract (max: 1 g/L) for providing essential vitamins to microbes unable to synthesize them. Culture experiments revealed good growth of Escherichia coli DH5α (ATCC 53868), Pseudomonas protegens Pf-5 (ATCC BAA-477), Pseudomonas aeruginosa (ATCC 15442), and Bacillus subtilis (ATCC 8473) with cell yield positively correlated with yeast extract concentration. In addition, identical viable cell concentration and colonies of similar morphologies were observed on both the colorless and LB agar; thus, suggesting that no inhibitory compounds were formed during agar preparation. Collectively, using commonly used buffer components as well as salts and nutrients, a colorless agar was prepared by segregating chromogenic compounds during heat sterilization; thus, opening up its potential use for enhancing color contrast between colonies and agar in revealing finer details of pure culture colonies, or more accurate automated colony identification and counting in screening and viable cell count assays.

scholarly journals Colorless agar for enhanced color contrast between microbial colonies and solid medium

2017 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Yeast Extract ◽  
Solid Medium ◽  
Low Cost ◽  
Viable Cell ◽  
Automated Image Analysis ◽  
Cell Counting ◽  
Color Contrast ◽  
Good Growth ◽  
Medium Components ◽  
Screening Experiments

Solid medium enables spatially resolved simultaneous cultivation of different microbes in a simple and relatively low cost format useful for preliminary screening of microbial diversity. Lack of color contrast between colonies and agar, however, hampers colony identification by automated image analysis, which is a challenge for phenotype screening experiments and viable cell counting. Since microbes secrete pigments of myriad hues, this research sought to develop a colorless agar, which when placed on colored paper of suitable hue, would enhance the color contrast between agar and colonies of any color. However, practical realization of the concept is hampered by heat-induced formation of colored compounds between medium components during autoclave sterilization, which in this study, was prevented by dissolving glucose and ammonium chloride in two separate solutions (each containing other medium components). Mixing the two sterilized solutions at 48 oC yielded a colorless agar, which remained colorless even after adding sterile yeast extract (max: 1 g/L) for providing essential vitamins to microbes unable to synthesize them. Culture experiments revealed good growth of Escherichia coli DH5α (ATCC 53868), Pseudomonas protegens Pf-5 (ATCC BAA-477), Pseudomonas aeruginosa (ATCC 15442), and Bacillus subtilis (ATCC 8473) with cell yield positively correlated with yeast extract concentration. Additionally, identical viable cell concentration and colonies of similar morphologies were observed on both the colorless and LB agar; thus, suggesting that no inhibitory compounds were formed during agar preparation. Collectively, using commonly used buffer components as well as salts and nutrients, a colorless agar was prepared by segregating chromogenic compounds during heat sterilization; thus, suggesting its use for enhancing color contrast between colonies and agar in revealing finer details of pure culture colonies, or more accurate automated colony identification and counting in screening and viable cell count assays.

scholarly journals Colourless agar for enhancing colour contrast between microbial colonies and agar

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Yeast Extract ◽  
Agar Medium ◽  
Deionized Water ◽  
Good Growth ◽  
Automated Identification ◽  
E Coli ◽  
Colour Contrast ◽  
Varied Background ◽  
Ammonium Compounds ◽  
Standard Techniques

Lack of colour contrast hampers automated identification of colonies on agar, given that many microbial colonies are of the same colour as the background colour of most agar: beige colour. On the other hand, a colourless agar could increase the colour contrast between the agar background and microbial colonies. But, the challenge in preparing a colourless agar comes from the formation of coloured compounds when sugars and ammonium compounds are sterilized together in an autoclave. Hence, by separating glucose and ammonium compounds into different solutions for autoclave sterilization, a method was developed for preparing colourless agar that remained colourless even with 1 g/L of yeast extract supplementation. Specifically, three separate solutions were used in reconstituting the colourless agar at ~48 oC after individual sterilization. Solution A comprised glucose, MgSO4 and agar powder; Solution B contained NH4Cl, K2HPO4, KH2PO4, and NaCl; and Solution C was yeast extract solution for providing vitamins and trace elements needed by microorganisms unable to grow in minimal medium. Reconstituted colourless agar could be poured into agar plates using standard techniques and had a viscosity similar to many commercial agar. Composition of the colourless agar medium was [g/L]: D-Glucose, 2.0; NH4Cl, 0.5; K2HPO4, 0.5; KH2PO4, 0.1; NaCl, 0.5; MgSO4.7H2O, 1.0; Yeast extract, 1.0; Agar, 15.0. On observation against varied background images, the formulated colourless agar lacked the yellow tinge present in LB Lennox agar and had greater optical transparency. Good growth of common bacteria such as Escherichia coli DH5α (ATCC 53868), Bacillus subtilis NRS-762 (ATCC 8473) and Pseudomonas protegens Pf-5 (ATCC BAA-477) was observed in both liquid and solid versions of the formulated colourless agar medium. Specifically, colonies of E. coli DH5α, B. subtilis NRS-762 and P. protegens Pf-5 exhibited similar morphology and characteristics compared to growth on LB Lennox agar. Use of the formulated agar medium in cultivating microbial flora present in deionized water recovered many types of colonies, which suggested that the medium was able to support a variety of microbial species. Collectively, by separately sterilizing sugars and ammonium compounds, a method was developed for preparing colourless agar which helped improve colour contrast for colony identification and counting. The formulated colourless agar could find use in various microbial ecology studies seeking to profile microbes from different environmental niches.

scholarly journals Yacon-based Beverage as Non-dairy Vehicle for Bifidobacterium animalis ssp. lactis: Stability and In vitro Probiotic Viability

2020 ◽  
Vol 11 (4) ◽  
pp. 11458-11472
Keyword(s):  
Ascorbic Acid ◽  
Low Cost ◽  
Color Stability ◽  
Probiotic Strain ◽  
Viable Cell ◽  
Cell Counting ◽  
Common Source ◽  
Potential Health ◽  
Bifidobacterium Animalis

Probiotics are health-promoting microorganisms commonly applied to functional foods. Dairy products are the most common source of probiotics, which difficult probiotics’ consumption by lactose intolerants. In this work, a yacon-based (Smallanthus sonchifolius) beverage was evaluated as a non-dairy vehicle for probiotics (Bifidobacterium animalis ssp. lactis, BB-12®), concerning stability and probiotics viability, during 28 days. x The yacon-based beverage was properly obtained. To determine the better treatment to stabilize it during storage, combined treatments were optimized via experimental design, and the color stability evaluated the initial changes. The beverage was incubated with the probiotic strain Bifidobacterium animalis ssp. lactis and the fermentation were carried out at 45 °C and 35 °C for 10 h in anaerobic jars. The storage viability was evaluated for 28 days, and the viable cell counting was carried out every seven days. A combination of the immersion of yacon slices into ascorbic acid 0.5% (w/v), blanching (3 min), and the addition of 0.4% (w/v) of ascorbic acid as an antioxidant was the most promising treatment to maintain color stability. Moreover, the fermentation applying 35 °C maintained the beverage with probiotic status for 14 days. In this work, a probiotic beverage was proposed, with potential health benefits, based on an underused root with great productivity, which will result in a potential low-cost probiotic product compared to dairy-based probiotic beverages.

scholarly journals Colourless agar for enhancing colour contrast between microbial colonies and agar

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Yeast Extract ◽  
Agar Medium ◽  
Deionized Water ◽  
Good Growth ◽  
Automated Identification ◽  
E Coli ◽  
Colour Contrast ◽  
Varied Background ◽  
Ammonium Compounds ◽  
Standard Techniques

Lack of colour contrast hampers automated identification of colonies on agar, given that many microbial colonies are of the same colour as the background colour of most agar: beige colour. On the other hand, a colourless agar could increase the colour contrast between the agar background and microbial colonies. But, the challenge in preparing a colourless agar comes from the formation of coloured compounds when sugars and ammonium compounds are sterilized together in an autoclave. Hence, by separating glucose and ammonium compounds into different solutions for autoclave sterilization, a method was developed for preparing colourless agar that remained colourless even with 1 g/L of yeast extract supplementation. Specifically, three separate solutions were used in reconstituting the colourless agar at ~48 oC after individual sterilization. Solution A comprised glucose, MgSO4 and agar powder; Solution B contained NH4Cl, K2HPO4, KH2PO4, and NaCl; and Solution C was yeast extract solution for providing vitamins and trace elements needed by microorganisms unable to grow in minimal medium. Reconstituted colourless agar could be poured into agar plates using standard techniques and had a viscosity similar to many commercial agar. Composition of the colourless agar medium was [g/L]: D-Glucose, 2.0; NH4Cl, 0.5; K2HPO4, 0.5; KH2PO4, 0.1; NaCl, 0.5; MgSO4.7H2O, 1.0; Yeast extract, 1.0; Agar, 15.0. On observation against varied background images, the formulated colourless agar lacked the yellow tinge present in LB Lennox agar and had greater optical transparency. Good growth of common bacteria such as Escherichia coli DH5α (ATCC 53868), Bacillus subtilis NRS-762 (ATCC 8473) and Pseudomonas protegens Pf-5 (ATCC BAA-477) was observed in both liquid and solid versions of the formulated colourless agar medium. Specifically, colonies of E. coli DH5α, B. subtilis NRS-762 and P. protegens Pf-5 exhibited similar morphology and characteristics compared to growth on LB Lennox agar. Use of the formulated agar medium in cultivating microbial flora present in deionized water recovered many types of colonies, which suggested that the medium was able to support a variety of microbial species. Collectively, by separately sterilizing sugars and ammonium compounds, a method was developed for preparing colourless agar which helped improve colour contrast for colony identification and counting. The formulated colourless agar could find use in various microbial ecology studies seeking to profile microbes from different environmental niches.

scholarly journals Cyclooxygenase Inhibition Alters Proliferative, Migratory, and Invasive Properties of Human Glioblastoma Cells In Vitro

2021 ◽  
Vol 22 (9) ◽  
pp. 4297
Author(s):  
Matthew Thomas Ferreira ◽  
Juliano Andreoli Miyake ◽  
Renata Nascimento Gomes ◽  
Fábio Feitoza ◽  
Pollyana Bulgarelli Stevannato ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Biology ◽  
Gelatin Zymography ◽  
Viable Cell ◽  
Cell Counting ◽  
Gelatinolytic Activity ◽  
Ep4 Receptor ◽  
And Migration ◽  
Proliferation And Migration

Prostaglandin E2 (PGE2) is known to increase glioblastoma (GBM) cell proliferation and migration while cyclooxygenase (COX) inhibition decreases proliferation and migration. The present study investigated the effects of COX inhibitors and PGE2 receptor antagonists on GBM cell biology. Cells were grown with inhibitors and dose response, viable cell counting, flow cytometry, cell migration, gene expression, Western blotting, and gelatin zymography studies were performed. The stimulatory effects of PGE2 and the inhibitory effects of ibuprofen (IBP) were confirmed in GBM cells. The EP2 and EP4 receptors were identified as important mediators of the actions of PGE2 in GBM cells. The concomitant inhibition of EP2 and EP4 caused a significant decrease in cell migration which was not reverted by exogenous PGE2. In T98G cells exogenous PGE2 increased latent MMP2 gelatinolytic activity. The inhibition of COX1 or COX2 caused significant alterations in MMP2 expression and gelatinolytic activity in GBM cells. These findings provide further evidence for the importance of PGE2 signalling through the EP2 and the EP4 receptor in the control of GBM cell biology. They also support the hypothesis that a relationship exists between COX1 and MMP2 in GBM cells which merits further investigation as a novel therapeutic target for drug development.

scholarly journals Optimization of Lipase Production in Solid-State Fermentation by Rhizopus Arrhizus in Nutrient Medium Containing Agroindustrial Wastes

2018 ◽  
Vol 12 (1) ◽  
pp. 189-203 ◽  
Author(s):  
Georgi Dobrev ◽  
Hristina Strinska ◽  
Anelia Hambarliiska ◽  
Boriana Zhekova ◽  
Valentina Dobreva
Keyword(s):  
Solid State ◽  
Moisture Content ◽  
Solid State Fermentation ◽  
Lipase Activity ◽  
Nutrient Medium ◽  
Solid Medium ◽  
Low Cost ◽  
Lipase Production ◽  
Rhizopus Arrhizus ◽  
Agroindustrial Wastes

Background: Rhizopus arrhizus is a potential microorganism for lipase production. Solid-state fermentation is used for microbial biosynthesis of enzymes, due to advantages, such as high productivity, utilization of abundant and low-cost raw materials, and production of enzymes with different catalytic properties. Objective: The objective of the research is optimization of the conditions for lipase production in solid-state fermentation by Rhizopus arrhizus in a nutrient medium, containing agroindustrial wastes. Method: Biosynthesis of lipase in solid-state fermentation by Rhizopus arrhizus was investigated. The effect of different solid substrates, additional carbon and nitrogen source, particles size and moisture content of the medium on enzyme production was studied. Response surface methodology was applied for determination of the optimal values of moisture content and tryptone concentration. A procedure for efficient lipase extraction from the fermented solids was developed. Results: Highest lipase activity was achieved when wheat bran was used as a solid substrate. The addition of 1% (w/w) glucose and 5% (w/w) tryptone to the solid medium significantly increased lipase activity. The structure of the solid medium including particles size and moisture content significantly influenced lipase production. A mathematical model for the effect of moisture content and tryptone concentration on lipase activity was developed. Highest enzyme activity was achieved at 66% moisture and 5% (w/w) tryptone. The addition of the non-ionic surfactant Disponyl NP 3070 in the eluent for enzyme extraction from the fermented solids increased lipase activity about three folds. Conclusion: After optimization of the solid-state fermentation the achieved 1021.80 U/g lipase activity from Rhizopus arrhizus was higher and comparable with the activity of lipases, produced by other fungal strains. The optimization of the conditions and the use of low cost components in solid-state fermentation makes the process economicaly effective for production of lipase from the investigated strain Rhizopus arrhizus.

scholarly journals An inexpensive, customizable microscopy system for the automated quantification and characterization of multiple adherent cell types

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4937 ◽  
Author(s):  
Vishwaratn Asthana ◽  
Yuqi Tang ◽  
Adam Ferguson ◽  
Pallavi Bugga ◽  
Anantratn Asthana ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Dynamic Range ◽  
Low Cost ◽  
Cell Types ◽  
Cell Counting ◽  
Adherent Cell ◽  
Essential Components ◽  
Automated Quantification ◽  
Automated Microscopy ◽  
Multiple Cell

Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate.

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