Name: quantitative analysis of aspergillus nidulans growth rate using live microscopy and open-source software
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This work was partly supported by the project &#34;A Greek Research Infrastructure for Visualizing and Monitoring Fundamental Biological Processes (BioImaging-GR)&#34; (MIS 5002755) which is implemented under the Action &#34;Reinforcement of the Research and Innovation Infrastructure&#34;, funded by the Operational Programme &#34;Competitiveness, Entrepreneurship and Innovation&#34; (NSRF 2014-2020) and co-financed by Greece and the E. U.

1. Inoculum preparation
NOTE: All steps should be performed under a laminar flow cabinet.
<ol>
	<li>Streak out fungal strain of interest, from a glycerol stock (-80 &#176;C) using a sterile inoculation loop, onto plates of minimal media (MM) supplemented with the appropriate nutritional requirements relevant to the strain examined&#160;[MM: 10.0 g/L glucose, 20 mL/L salt solution (salt solution: 26 g/L KCl, 26 g/L MgSO4&#183;7H2O, 76 g/L KH2PO4, 2....

<ol>
	<li>Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspergillus+nidulans:+A+Potential+Resource+of+the+Production+of+the+Native+and+Heterologous+Enzymes+for+Industrial+Applications.">Aspergillus nidulans: A Potential Resource of the Production of the Native and Heterologous Enzymes for Industrial Applications.</a> International Journal of Microbiology. 2020, 8894215 (2020).</li><li>K&#252;ck, U., Bloemendal, S., Teichert, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putting+Fungi+to+Work:+Harvesting+a+Cornucopia+of+Drugs,+Toxins,+and+Antibiotics.">Putting Fungi to Work: Harvesting a Cornucopia of Drugs, Toxins, and Antibiotics.</a> PLoS Pathogens. 10 (3), (2014).</li><li>Paterson, R. R. M., Lima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+Fungal+Human+Pathogens+from+Food+Emphasising+Aspergillus,+Fusarium+and+Mucor.">Filamentous Fungal Human Pathogens from Food Emphasising Aspergillus, Fusarium and Mucor.</a> Microoraganism. 5 (3), (2017).</li><li>Wang, C., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+Pathogenic+Fungi:+Genomics,+Molecular+Interactions,+and+Genetic+Improvements.">Insect Pathogenic Fungi: Genomics, Molecular Interactions, and Genetic Improvements.</a> Annual Review of Entomology. 62, 73-90 (2017).</li><li>Etxebeste, O., Espeso, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspergillus+nidulans+in+the+post-genomic+era:+a+top-model+filamentous+fungus+for+the+study+of+signaling+and+homeostasis+mechanisms.">Aspergillus nidulans in the post-genomic era: a top-model filamentous fungus for the study of signaling and homeostasis mechanisms.</a> International Microbiology. 23 (1), 5-22 (2020).</li><li>Riquelme, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+Morphogenesis,+from+the+Polarized+Growth+of+Hyphae+to+Complex+Reproduction+and+Infection+Structures.">Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures.</a> Microbiology and Molecular Biology Reviews MMBR. 82 (2), (2018).</li><li>Athanasopoulos, A., Andr&#233;, B., Sophianopoulou, V., Gournas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+plasma+membrane+domains.">Fungal plasma membrane domains.</a> FEMS Microbiology Reviews. , (2019).</li><li>Pantazopoulou, A., Pe&#241;alva, M. 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Monitoring fungal cell growth and phenotype by time-lapse microscopy is a powerful approach to assess cellular behavior in real-time and quantitatively and accurately determine whether a particular drug treatment and/or genetic intervention results in detectable cell growth or phenotypic differences over time.
In this study, a reliable live-cell imaging methodology was described to measure and quantitatively analyze fungal development, including the dynamics of germ tube and hyphal tip growth ...

Filamentous fungi are of great socioeconomic and ecological importance, being both crucial as industrial/agricultural tools for enzyme and antibiotic production1,2 and as pathogens of crop plants3, pest insects4 and humans3. Moreover, filamentous fungi such as Aspergillus&#160;nidulans are widely used as model organisms for fundamental research, such as studies in genetics, cell and evolutionary biology as well as for the study of hyphal extension5. Filamentous fungi are highly polarized organisms that elongate through the continuous supply of membrane lipids/proteins and the de novo synthesis of cell wall at the extending tip6. A central role in the hyphal tip growth and polarity maintenance is a specialized structure named &#39;Spitzenkorper&#39; (SPK), a highly ordered structure consisting mostly of cytoskeletal components and the polarized distribution of the Golgi6,7,8.
Environmental stimuli/signals, such water-air interface, light, CO2 concentration, and the nutritional status are responsible for the developmental decisions made by these molds9. In submerged (liquid) cultures the differentiation of A. nidulans is repressed and growth occurs by hyphal tip elongation6. During vegetative growth, asexual spores (conidia) germinate by apical extension, forming an undifferentiated network of interconnected hyphal cells, the mycelium, which may continue to grow indefinitely as long as nutrients and space are available. On the other hand, on solid media hyphal tips elongate and after a defined period of vegetative growth (developmental competence), asexual reproduction is initiated and aerial conidiophore stalks extend from specialized foot cells of the mycelium6. These give rise to specialized developmental multicellular structures called conidiophores, which produce long chains of haploid conidia10 that can restart growth under favorable environmental conditions.
A widely used method for measuring filamentous fungal growth is to inoculate spores on nutrient agar contained in a Petri dish and macroscopically measure the diameter of the colony a few days later11. The diameter/area of the colony, most dependent on changes in mycelial growth rate and less on conidiophore density12, is then used as a value of growth. Although, measuring fungal population (colony) size growing on solid surfaces is quite adequate, it is by no means the most accurate measure of growth. Compared to population level averages (averages of fungal colony size), single cell&#160;measurements can capture the heterogeneity of a cell population and allow identification of novel sub-populations of cells, states13, dynamics, pathways as well as the biological mechanisms by which cells respond to endogenous and environmental changes14,15. Monitoring fungal cell growth and phenotype by time-lapse microscopy is arguably the most widely employed quantitative single cell observation approach.
Herein, we detail a label-free live imaging protocol using transmitted light microscopy techniques (such as phase-contrast, differential interference contrast (DIC), and polarized microscopy) to capture images, which independently of the combined use of fluorescence microscopy can be employed to analyze and quantify polar growth of A. nidulans strains in both submerged cultures and solid media.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-Slide 8 Well</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>Imaging slides</td>
		</tr>
		<tr>
			<td>4-Aminobenzoic acid</td>
			<td>Merck</td>
			<td>A9878</td>
			<td></td>
		</tr>
		<tr>
			<td>azhA&#916; ngnA&#916;</td>
			<td></td>
			<td></td>
			<td>Genotype: zhA&#916;::pyrGAf; ngnA&#916;::pyrGAf; pyroA4 pantoB100 / References:Laboratory collection, Athanasopoulos et al., 2013</td>
		</tr>
		<tr>
			<td>Bacto Casamino Acids</td>
			<td>Gibco</td>
			<td>223030</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Merck</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>67-66-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) sulfate pentahydrate</td>
			<td>Merck</td>
			<td>C8027</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 8.0</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Statistical Software</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Image processing and analysis software</td>
		</tr>
		<tr>
			<td>Inoculating Loop</td>
			<td>Merck</td>
			<td>I8263-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(III) phosphate</td>
			<td>Merck</td>
			<td>1.03935</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite X</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Microscope software</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Merck</td>
			<td>63138</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese(II) sulfate monohydrate</td>
			<td>Merck</td>
			<td>M7899</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Leica TCS SP8</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide (Niacinamide)</td>
			<td>Supelco</td>
			<td>47865-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Millipore</td>
			<td>68971</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes for Microbiology Culture</td>
			<td>KISKER</td>
			<td>G090</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>P4504</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Merck</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxine hydrochloride</td>
			<td>Merck</td>
			<td>P6280</td>
			<td></td>
		</tr>
		<tr>
			<td>Quali - Microcentrifuge Tubes, 1,7 mL, DNase-, RNase and pyrogen free, sterile</td>
			<td>KISKER</td>
			<td>G052-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Quali - Microcentrifuge Tubes, 2.0 mL, sterile</td>
			<td>KISKER</td>
			<td>G053-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Quali - Standard Tips, Bevelled, 100-1000 &#181;L</td>
			<td>KISKER</td>
			<td>VL004G</td>
			<td></td>
		</tr>
		<tr>
			<td>Quali - Standard Tips, Bevelled, 1-200 &#181;L</td>
			<td>KISKER</td>
			<td>VL700G</td>
			<td></td>
		</tr>
		<tr>
			<td>Quali Microvolume Tips, DNase-, RNase free, 0,1-10 &#181;L/clear</td>
			<td>KISKER</td>
			<td>GC.TIPS.B</td>
			<td></td>
		</tr>
		<tr>
			<td>Riboflavin (B2)</td>
			<td>Supelco</td>
			<td>47861</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades NO. 11</td>
			<td>OdontoMed2011</td>
			<td>S2771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Merck</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tetraborate decahydrate</td>
			<td>Merck</td>
			<td>S9640</td>
			<td></td>
		</tr>
		<tr>
			<td>VS151 (PilA-GFP and H1-mRFP)</td>
			<td></td>
			<td></td>
			<td>Genotype: pyrG89; pilA::sgfp::AfpyrG+ argB2 nkuA&#916;::argB+&#160; pyroA4 hhoA::mrfp::Afribo+ riboB2 / References:Laboratory collection, Biratsi et al., 2021</td>
		</tr>
		<tr>
			<td>WT</td>
			<td></td>
			<td></td>
			<td>Genotype: nkuA&#916;::argB; pyrG89; pyroA4;pyrG89 / References: TN02A3 -FGSC A1149</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Millipore</td>
			<td>70161</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative analysis of aspergillus nidulans growth rate using live microscopy and open-source software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of&#160;the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

Following this protocol, we captured and analyzed various images corresponding to different growth/developmental stages of the filamentous fungus A. nidulans. The data presented in this study were processed and analyzed using the Fiji software. Measurements were saved as csv files, statistically analyzed and prepared as graphs using commercial statistical software and/or Python programming language using software libraries like pandas, numpy, statsmodels, matplotlib and seaborn. More details can be found in the ...

Watch this Scientific Journal Video about Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software at JoVE.com

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

We present a label-free live imaging protocol using transmitted light microscopy techniques to capture images, analyze and quantify growth kinetics of the filamentous fungus A. nidulans in both submerged cultures and solid media. This protocol can be used in conjunction with fluorescence microscopy.

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...

In this video, we describe a label free live imaging protocol to capture images of the model fungus aspergillus nidulans, using transmitted light microscopy techniques. We use this images to analyze and quantify growth kinetics of aspergillus nidulans in both liquid and solid media. A visual representation of this protocol allows users to become familiar with significant steps of image capturing and microscopy image processing.A common method used to measure filamentous fungal growth is two inoculate fungal spores in a Petri dish containing nutrient agar and to measure the diameter of the developing colony a few days later. However, this approach is not sensitive enough to quantify fine growth differences. Therefore, single cell measurements using time lapse microscopy were developed.This measurements are able not only to provide accurate and quantitative results, but also to reflect growth dynamics of different cells within a population. Moreover, this approach aims to better understanding of the molecular mechanisms involved in fungal growth responses to endogenous and environmental signals. Begin by pouring 15 milliliters of liquid nutrient agar into Petri dishes.Place the lid onto the top and allow it to cool. Use UV radiation to sterilize plates. Before streaking out the fungal strain of interest from a stock, heat the nodulating loop in the Bunsen burner until it is red hot, cool the loop by stabbing it into the agar.Vortex the tube and streak the loop across the surface of the agar minimal medium supplemented with the appropriate nutritional requirements. Incubate plates for two to three days at 37 degrees Celsius. Using a sterile toothpick, transfer a small number of conidia by gently touching a single colony to plates of complete media.Incubate plates for three to four days at 37 degrees Celsius. Conidia of aspergillus nidulans, are harvested in a sterile 1.5 milliliter vortex tube, with 1.0 milliliter of autoclave distilled water containing 0.05%volume per volume, Tween 80, for reducing the number of conidia clumps. A modified version of the inverted agar method is used for imaging filamentous fungi on agar medium surface.Initially spot 10 microliter aliquots of vigorously vortex conidial suspension, approximately two times 104 cells per milliliter onto Petri dishes of 15 milliliters minimal medium with one weight per volume agar. Incubate the experimental culture according to the developmental stage to be investigated. Here we incubate for three days at 30 degrees Celsius.After, slice out a block of agar containing the colony, using a sterile scalpel. Here we cut out a wedge of agar at the colony margin, invert and place the agar slice into an eight, well slide. Transfer 10 microliter aliquots of vigorously vortex conidial suspension of approximately two times 10 in the power of four in the wells of a well slide containing 200 microliters of minimal medium with the appropriate supplements.Incubate for the time and temperature to be examined each time. The choice of microscope depends upon the available equipment. In any case, the microscope set up should include an inverted stage, an environmental chamber, or at least a room with precise air temperature control.Initially preheat the thermostat and microscope chamber to stabilize the desired temperature. Here we set the temperature at 30 degrees Celsius. Turn on the microscope, the scanner power, the laser power and computer, and load the imaging software.Place the previously prepared well slide in the microscope stage and focus. Find fields of view that contain isolated, not overlapping cells, or at least not overcrowded, to facilitate growth measurements during image analysis. Select the transmitted light microscopy technique to be used and activate the transmitted light detector as well as detectors for fluorescence when necessary.Set the microscope to acquire images at desired time intervals and start time series acquisition. Loading, visualization and processing of images is accomplished with the open source imageJ Fiji software. Begin by importing the images to Fiji using plugins by a format.Select the default color mode and auto scale. In order to visualize a timestamp and a scale bar as overlay go to analyze, tools, scale bar. Set the desired parameters concerning width, height, color, and location of the scale bar.Go to image, properties, to display image properties in imageJ Fiji software. Observe frame interval information. Here we use a time interval of approximately 15 minutes and press okay.Then go to image, stacks, label, and set the correct information to interval. Set location of the label by modifying X and Y.Set the measurement unit in the field text and press preview. Use histogram matching for illumination correction between different frames by selecting image, adjust, bleach correction, histogram matching.A new window with corrected illumination appears. Use MtrackJ plugin at plugins, MtrackJ, to track growing hyphal tip To add the track, select the add button in the toolbar and place the first point at the hyphal tip using the left click of the mouse. The time series will automatically move to the next frame.To complete the tracking process, double click the mouse on the final point, or press the escape key. Scrolling the output table, the most important column for calculating hyphal tip growth, is the speed at any given frame. Safe track measurements, add file, save as, to the file format of your choice, for example, CSV, import the saved file into your spreadsheet program and compute the visualization and further tests.Press the movie button to generate a movie, RGB color type, containing the frames with the tracks drawn into them, which can be visualized in any standard multimedia player. Following this protocol, we captured and analyzed various images corresponding to different growth and developmental stages of the filamentous fungus aspergillus nidulans. Figure shows the comparison of wild type and azhAD Delta ngnA Delta growth rate measurements.The azhAD Delta ngnA Delta is a double deleted strain in two genes implicated in the detoxification and assimilation of the toxic fighter product L as added in two carboxylic acid. It shows statistically significant lower growth rate measurements compared to the wild type strain in submerged liquid cultures. This difference in growth is not detectable by measuring the colony area of these two strains in solid minimal medium.Single cell, long-term live imaging is of significant value in the effort to obtain spacial and temporal information of cellular proteins dynamics. This protocol is suitable also for carrying out long-term live cell imaging. Here we see germination of conidia co-expressing the GFP labeled core eisosomal protein PilA and the mRFP histonen H1.Here in we present a protocol for analyzing fungal growth kinetics in a reproducible and reliable manner without the need of any prior image analysis experience from the user.This protocol allows objective accurate quantification of fungal growth.

Research

JoVE Journal

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