Morphological characterisation of yeast colony growth on solid media using image processing

To rapidly determine the effect of environmental factors on yeast growth, a cell counting and colony sizing image analysis method was developed to characterise colony growth on solid media. A digitised microscopic image of the yeast was analysed using the Watershed algorithm for cell number determination and a morphological edge detection for colony size determination. The influence of temperature and physiological stress on yeast growth was then investigated over 12.5 h and data extracted by the image analysis method. © Rapid Science Ltd. 1998     

Screening of tannin acyl hydrolase (E.C.3.1.1.20) producing tannery effluent fungal isolates using simple agar plate and SmF process

Industrially important tannase producing fungi were isolated from tannery effluent using simple agar plate method. The isolates were screened by submerged fermentation using auto-controlled bioreactor. The colony diameter on the solid surface media shows high correlation with quantitative production of tannase. The isolate Aspergillus niger shows maximum production of both extracellular and intracellular enzyme.     

Macroscopic growth of filamentous fungi on solid substrate explained by a microscopic approach.

A quantitative model predicting biomass growth on solid media has been developed. The model takes into account steric interactions between hyphae and tips at the microscopic level (competition for substrate and tip-hypha collisions). These interactions effect a slowing down of the hyphal, population-averaged extension rate and are responsible, at the microscopic level, for the distribution of tip orientations observed at the colony border. At the macroscopic level, a limiting value of the colony radial extension rate is attained. A mathematical model that combines hyphal branching, tip diffusion, and biomass growth was proposed to explain such behavior. Experiments using Gibberella fujikuroi were performed to validate the model; good agreement between experiments and simulations was achieved. Most parameters can be measured by simple image analysis on the peripheral growth zone, and they have clear physical meaning; that is, they correspond to properties of single, leading hyphae. The model can be used to describe two-dimensional (2D) solid media fermentation experiments under varying culture conditions; the model can also be extended to consider growth in three-dimensional (3D), complex geometry substrates.     

Production of extracellular enzymes by isolates of Metarhizium anisopliae

Extracellular enzyme production in solid culture media was analyzed in order to determine the variability among different Metarhizium anisopliae isolates. Using specific substrates, amylase, lipase, chitinase, and protease production was tested in 11 isolates from different regions of Brazil. Enzyme production was determined by the formation of a halo around the colony, and the diameters of both halo and colony were measured. The enzymatic index was expressed by the colony diameter/halo diameter ratio. In general, the isolates from the same region had similar enzymatic indexes, although similar indexes were also found for isolates from geographically distinct regions. The different isolates were tentatively grouped according to index similarity.     

<Emphasis Type="Italic">ScreenMill</Emphasis>: A freely available software suite for growth measurement,analysis and visualization of high-throughput screen data

Background  

Many high-throughput genomic experiments, such as Synthetic Genetic Array and yeast two-hybrid, use colony growth on solid media as a screen metric. These experiments routinely generate over 100,000 data points, making data analysis a time consuming and painstaking process. Here we describe ScreenMill, a new software suite that automates image analysis and simplifies data review and analysis for high-throughput biological experiments.     

Extracellular enzyme production by haploids,heterocaryons and diploids of Aspergillus nidulans

Summary Extracellular amylase, lipase and protease produced by haploids, diploids and heterocaryons of Aspergillus nidulans were analysed. Three morphologically normal strains and 8 morphologic mutants as well as various genetic combinations of the 11 strains were examined in solid culture medium containing specific substrates. The enzyme production of each strain was determined by measuring the halo around the colony. It was observed that the colonies showing less growth also showed more alterations in enzyme production. The compact strains (BVIII and B6) and the slow-growing heterocaryons (pp+M32 and pp+M35) showed the highest enzymatic index for the three enzymes simultaneously. If colony growth is not considered, then for amylase and protease the highest values were reached by some diploid and heterocaryons and for lipase by one morphological strain. The results showed that morphological mutants and some combinations could be used for higher production of amylase, lipase and protease.     

Micro-colony array based high throughput platform for enzyme library screening

Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%.     

Determining specific growth rates in different regions of Salmonella typhimurium colonies

Computer-aided image analysis (IA) techniques were used to determine specific growth rates (μ) in different regions of Salmonella typhimurium colonies grown on solid medium at 30°C. Captured transmission images were converted to optical density values, allowing calculation of μ. Data show that growth was exponential throughout the colony during the period studied (between 13 and 21 h after inoculation), but that μ varied from 0·69 to 0·37 h⁻¹ at the periphery and centre, respectively. Results demonstrate regional variation in μ values within colonies, and support conceptual models of colony development. The technique described illustrates that kinetic growth data can be derived within bacterial colonies, and this may lead towards an improved understanding of the growth dynamics of these structures.     

Function of native OmtA in vivo and expression and distribution of this protein in colonies of Aspergillus parasiticus

The activities of two enzymes, a 168-kDa protein and a 40-kDa protein, OmtA, purified from the filamentous fungus Aspergillus parasiticus were reported to convert the aflatoxin pathway intermediate sterigmatocystin to O-methylsterigmatocystin in vitro. Our initial goal was to determine if OmtA is necessary and sufficient to catalyze this reaction in vivo and if this reaction is necessary for aflatoxin synthesis. We generated A. parasiticus omtA-null mutant LW1432 and a maltose binding protein-OmtA fusion protein expressed in Escherichia coli. Enzyme activity analysis of OmtA fusion protein in vitro confirmed the reported catalytic function of OmtA. Feeding studies conducted with LW1432 demonstrated a critical role for OmtA, and the reaction catalyzed by this enzyme in aflatoxin synthesis in vivo. Because of a close regulatory link between aflatoxin synthesis and asexual sporulation (conidiation), we hypothesized a spatial and temporal association between OmtA expression and conidiospore development. We developed a novel time-dependent colony fractionation protocol to analyze the accumulation and distribution of OmtA in fungal colonies grown on a solid medium that supports both toxin synthesis and conidiation. OmtA-specific polyclonal antibodies were purified by affinity chromatography using an LW1432 protein extract. OmtA was not detected in 24-h-old colonies but was detected in 48-h-old colonies using Western blot analysis; the protein accumulated in all fractions of a 72-h-old colony, including cells (0 to 24 h) in which little conidiophore development was observed. OmtA in older fractions of the colony (24 to 72 h) was partly degraded. Fluorescence-based immunohistochemical analysis conducted on thin sections of paraffin-embedded fungal cells from time-fractionated fungal colonies demonstrated that OmtA is evenly distributed among different cell types and is not concentrated in conidiophores. These data suggest that OmtA is present in newly formed fungal tissue and then is proteolytically cleaved as cells in that section of the colony age.     

Uptake of Glucose and Phosphorus by Growing Colonies of Fusarium oxysporum as Quantified by Image Analysis

Olsson, S. 1994. Uptake of glucose and phosphorus by growing colonies of Fusarium oxysporum as quantified by image analysis. Experimental Mycology 18, 33-47. The simplest of all heterogeneous environments for fungal colony growth is the petri dish with an agar medium. As the colony grows there will be a depression of nutrient concentrations under the colony caused by the uptake of nutrients by the growing colony. Image analysis methods have been developed for measuring medium concentrations of glucose and phosphorus with simultaneous biomass density determinations in agar systems. Maps of the concentrations in the agar medium under the colony and of colony biomass density were produced. A new method for weighing fungal colonies grown on agar is also presented. For Fusarium oxysporum phosphorus and glucose uptake from the medium was the same irrespective of the C/mineral ratios in the medium within the measured range of ratios. Even the concentration profiles of the nutrients under the colony were the same irrespective of nutrient ratios. Distribution of biomass density was affected by differences in glucose concentrations, being highest at the colony margin at the lower concentrations. The results indicate that the fungal colony is able to take up nutrients at the margin in excess of the local needs.     

Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing.

BACKGROUND: Clonogenic assays are used frequently to measure the cell killing and mutagenic effects of radiation and other agents. Clonogenic assays carried out manually are tedious and time-consuming and involve a significant element of subjectivity. However, several commercial automatic colony counters are available. Based on CCD video imaging and image analysis they are relatively expensive and can analyze only one petri dish at a time. METHOD: We have developed a cheaper and more efficient device, which employs a flat bed scanner to image 12 60-mm petri dishes at a time. Two major problems in automated colony counting are the clustering of colonies and edge effects. By using standard image analysis and implementing an inflection point algorithm, these problems were greatly diminished. The resulting system was compared with two manual colony counts, as well as with automated counts with the Oxford Optronix ColCount colony counter for cell lines V79 and HaCaT. RESULTS: Comparisons assuming the manual counts to be correct showed that our automatic counter was slightly more accurate than the commercial unit. CONCLUSIONS: As a whole, our automated colony counter performed significantly better than the commercial unit with regard to processing time, cost and accuracy.     

Function of Native OmtA In Vivo and Expression and Distribution of This Protein in Colonies of Aspergillus parasiticus

The activities of two enzymes, a 168-kDa protein and a 40-kDa protein, OmtA, purified from the filamentous fungus Aspergillus parasiticus were reported to convert the aflatoxin pathway intermediate sterigmatocystin to O-methylsterigmatocystin in vitro. Our initial goal was to determine if OmtA is necessary and sufficient to catalyze this reaction in vivo and if this reaction is necessary for aflatoxin synthesis. We generated A. parasiticus omtA-null mutant LW1432 and a maltose binding protein-OmtA fusion protein expressed in Escherichia coli. Enzyme activity analysis of OmtA fusion protein in vitro confirmed the reported catalytic function of OmtA. Feeding studies conducted with LW1432 demonstrated a critical role for OmtA, and the reaction catalyzed by this enzyme in aflatoxin synthesis in vivo. Because of a close regulatory link between aflatoxin synthesis and asexual sporulation (conidiation), we hypothesized a spatial and temporal association between OmtA expression and conidiospore development. We developed a novel time-dependent colony fractionation protocol to analyze the accumulation and distribution of OmtA in fungal colonies grown on a solid medium that supports both toxin synthesis and conidiation. OmtA-specific polyclonal antibodies were purified by affinity chromatography using an LW1432 protein extract. OmtA was not detected in 24-h-old colonies but was detected in 48-h-old colonies using Western blot analysis; the protein accumulated in all fractions of a 72-h-old colony, including cells (0 to 24 h) in which little conidiophore development was observed. OmtA in older fractions of the colony (24 to 72 h) was partly degraded. Fluorescence-based immunohistochemical analysis conducted on thin sections of paraffin-embedded fungal cells from time-fractionated fungal colonies demonstrated that OmtA is evenly distributed among different cell types and is not concentrated in conidiophores. These data suggest that OmtA is present in newly formed fungal tissue and then is proteolytically cleaved as cells in that section of the colony age.     

Direct identification of pure Penicillium species using image analysis

This paper presents a method for direct identification of fungal species solely by means of digital image analysis of colonies as seen after growth on a standard medium. The method described is completely automated and hence objective once digital images of the reference fungi have been established. Using a digital image it is possible to extract precise information from the surface of the fungal colony. This includes color distribution, colony dimensions and texture measurements. For fungal identification, this is normally done by visual observation that often results in a very subjective data recording. Isolates of nine different species of the genus Penicillium have been selected for the purpose. After incubation for 7 days, the fungal colonies are digitized using a very accurate digital camera. Prior to the image analysis each image is corrected for self-illumination, thereby gaining a set of directly corresponding images with respect to illumination. A Windows application has been developed to locate the position and size of up to three colonies in the digitized image. Using the estimated positions and sizes of the colonies, a number of relevant features can be extracted for further analysis. The method used to determine the position of the colonies will be covered as well as the feature selection. The texture measurements of colonies of the nine species were analyzed and a clustering of the data into the correct species was confirmed. This indicates that it is indeed possible to identify a given colony merely by macromorphological features. A classifier (in the normal distribution) based on measurements of 151 colonies incubated on yeast extract sucrose agar (YES) was used to discriminate between the species. This resulted in a correct classification rate of 100% when used on the training set and 96% using cross-validation. The same methods applied to 194 colonies incubated on Czapek yeast extract agar (CYA) resulted in a correct classification rate of 98% on the training set and 71% using cross-validation.     

重组别藻蓝蛋白β亚基（His-β~（APC））表达菌株遗传稳定性的研究

工程菌株的遗传稳定性在目的产物的生产应用中至关重要。为了确定工程菌株的遗传稳定性,通过对重组别藻蓝蛋白β亚基（His-βAPC）生产菌株E.coliJM109（DE23）/pET28α-βAPC进行了菌体生长量的测定,平板划线,质粒酶切,产物鉴定等研究,检验了该工程菌质粒的稳定性。通过实验得到如下结果：该工程菌株在没有抗生素选择压力下传代,质粒丢失率为5代6%,10代8%,15代9%,20代15%;该菌株经固体平板连续划线传代20次后,菌落大小和形态基本不变;质粒经BamHⅠ和HindⅢ酶切后进行琼脂糖凝胶电泳结果显示该菌株携带的重组质粒目的片段在传代前后没有发生变化;经诱导培养后,His-βAPC在原代和第5、10、15和20代宿主菌中都可以表达,表达量没有明显差别,且表达产物在SDS-PAGE中的带型基本一致。以上结果表明,该工程菌质粒具有结构稳定性和分裂不稳定性。     

Image analysis for the automated estimation of clonal growth and its application to the growth of smooth muscle cells

Abstract. Image analysis was used for the automated measurement of colony frequency ( f ) and colony diameter ( d ) in cultures of smooth muscle cells, Initial studies with the inverted microscope showed that number of cells ( N ) in a colony varied directly with d : log N = 1.98 log d - 3.469 Image analysis generated the complement of a cumulative distribution for f as a function of d . The number of cells in each segment of the distribution function was calculated by multiplying f and the average N for the segment. These data were displayed as a cumulative distribution function. The total number of colonies ( f_T ) and the total number of cells ( N_T ) were used to calculate the average colony size ( N_A ). Population doublings (PD) were then expressed as log₂ N_A . Image analysis confirmed previous studies in which colonies were sized and counted with an inverted microscope. Thus, image analysis is a rapid and automated technique for the measurement of clonal growth.     

Preparation and crossing of basidiospore-derived monokaryons –- a useful tool for obtaining laccase and other ligninolytic enzyme higher-producing dikaryotic strains of Pleurotus ostreatus

Laccase and other ligninolytic enzyme higher-producing dikaryons of Pleurotus ostreatus were obtained after crossing of compatible basidiospore-derived monokaryons selected from the parental basidiospore population on the basis of exceptionality in enzyme production, mycelium extension rate and/or colony morphology. As all detected changes in enzyme activity, mycelium extension rate, colony appearance and degradation of the polymeric dye Poly B411 were relatively stable after repeated testing, the dikaryotic isolates prepared in this way seem to be useful for the future biotechnological exploitation. No correlation between the colony appearance or the mating type and the enzyme activity or other characteristics tested has been found.     

Synergetic microorganismic convection generated by Opercularia asymmetrica ciliates living in a colony as effective fluid transport on the micro-scale

Ciliates, being one of the main substrates in granular activated sludge (GAS) formation, are treated as a major factor in granulation process. Cilia beats of Opercularia asymmetrica provide a continuous nutrient flux, enhancing the colonization of bacteria on Peritrichia stalks. Given that the ciliates tend to live in colonies, the main focus of the present work was an analysis and comparison of the flow effects induced by a single ciliate and by a colony. Investigations of the flow generated by Opercularia asymmetrica were carried out using micro-particle image velocimetry with biocompatible seeding. The results obtained showed different flow structures for a single ciliate and a colony. Moreover, the synergetic work of Opercularia asymmetrica living in a colony is considered as effective fluid transport. Additionally, analysis of the shear and normal strain rates provided information on mixing phenomena within the fluid on the micro-scale. The influence of seeding substance concentration on the flow pattern was also studied.     

Automated image analysis with the potential for process quality control applications in stem cell maintenance and differentiation

The translation of laboratory processes into scaled production systems suitable for manufacture is a significant challenge for cell based therapies; in particular there is a lack of analytical methods that are informative and efficient for process control. Here the potential of image analysis as one part of the solution to this issue is explored, using pluripotent stem cell colonies as a valuable and challenging exemplar. The Cell‐IQ live cell imaging platform was used to build image libraries of morphological culture attributes such as colony “edge,” “core periphery” or “core” cells. Conventional biomarkers, such as Oct3/4, Nanog, and Sox‐2, were shown to correspond to specific morphologies using immunostaining and flow cytometry techniques. Quantitative monitoring of these morphological attributes in‐process using the reference image libraries showed rapid sensitivity to changes induced by different media exchange regimes or the addition of mesoderm lineage inducing cytokine BMP4. The imaging sample size to precision relationship was defined for each morphological attribute to show that this sensitivity could be achieved with a relatively low imaging sample. Further, the morphological state of single colonies could be correlated to individual colony outcomes; smaller colonies were identified as optimum for homogenous early mesoderm differentiation, while larger colonies maintained a morphologically pluripotent core. Finally, we show the potential of the same image libraries to assess cell number in culture with accuracy comparable to sacrificial digestion and counting. The data supports a potentially powerful role for quantitative image analysis in the setting of in‐process specifications, and also for screening the effects of process actions during development, which is highly complementary to current analysis in optimization and manufacture. © 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:215–223, 2016