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 ...

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